MA3-019

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Experimental details: Western blot analysis of PDI was performed by loading 25 µg of HepG2 (Lane 1), Hela (Lane 2) and NIH-3T3 (Lane 3) cell lysates onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a PDI monoclonal antibody (Product # MA3-019) at a dilution of 1:1000 overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at approx. 57 kDa.

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Experimental details: Knockdown of PDI was achieved by transfecting A-431 cells with PDI specific siRNAs (Silencer® select Product # s441). Western blot analysis (Fig. a) was performed using membrane enriched extracts from the PDI knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-PDI Mouse Monoclonal Antibody (Product # MA3-019, 1:2000 dilution) and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Densitometric analysis of this Western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to PDI.

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Experimental details: Western blot was performed using Anti-PDI Mouse Monoclonal Antibody (Product # MA3-019) and a 57 kDa band corresponding to PDI was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2),NIH/3T3 (Lane 3), A-375 (Lane 4), Hep G2 (Lane 5), HL-60 (Lane 6), and PC-12 (Lane 7) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

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Experimental details: Immunofluorescent analysis of PDI (red) in MDCK cells. The cells were permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 3% BSA in PBS (Product # 37525) for 15 minutes at room temperature. Cells were stained with a PDI mouse monoclonal antibody (Product # MA3-019), at a concentration of 10 µg/mL in blocking buffer for at least 1 hour at room temperature, and then incubated with a Rabbit anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 594 conjugate (Product # A27027) at a dilution of 1:1000 for 30 minutes at room temperature (red). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

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Experimental details: Immunofluorescent analysis of PDI using anti-PDI monoclonal antibody (Product # MA3-019) shows staining in A549 Cells.

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Experimental details: Immunofluorescent analysis of PDI using anti-PDI monoclonal antibody (Product # MA3-019) shows staining in NS-1 Cells.

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Experimental details: Immunofluorescent analysis of PDI using anti-PDI monoclonal antibody (Product # MA3-019) shows staining in p19 Cells.

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Experimental details: Immunofluorescent analysis of PDI using anti-PDI monoclonal antibody (Product # MA3-019) shows staining in HMVEC Cells.

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Experimental details: Immunofluorescent analysis of Phalloidin (blue) and PDI (green) in NIH 3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA in PBS + 0.1% Triton X-100 (Product # 37525) for 30 minutes at room temperature. Cells were probed with a PDI monoclonal antibody (Product # MA3-019) at a dilution of 1:75 for at least 1 hour at room temperature, washed with PBS, and incubated with Dylight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:250 for 30 minutes at room temperature. Actin was stained with Dylight 350 Phalloidin (Product # 21830) at a dilution of 1:120 (2.5units/mL final concentration) and nuclei (red) were stained with DRAQ5 (Product # 62254) at a concentration of 1 µg/mL for 30 minutes. Images were taken on a Zeiss Axio Observer Z1 microscope at 20X magnification.

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Experimental details: Immunofluorescent analysis of Phalloidin (green) and PDI (red) in U2OS cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA (Product # 37525) in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were probed with a PDI monoclonal antibody (Product # MA3-019) at a dilution of 1:75 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 550 goat anti-mouse IgG secondary antibody (Product # 84540) at a dilution of 1:250 for 30 minutes at room temperature. Actin was stained with DyLight 488 Phalloidin (Product # 21833) at a dilution of 1:300 (1 unit/mL final concentration) for 30 minutes. Images were taken on a Thermo Scientific ArrayScan VTI at 20X magnification.

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Experimental details: Immunofluorescent analysis of Phalloidin (orange) and PDI (green) in NIH 3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA (Product # 37525) in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were probed with a PDI monoclonal antibody (Product # MA3-019) at a dilution of 1:75 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:250 for 30 minutes at room temperature. Actin was stained with DyLight 550 Phalloidin (Product # 21835) at a dilution of 1:120 (2.5 units/mL final concentration) and nuclei (blue) were stained with Hoechst (Product # 62249) at a concentration of 1 µg/mL for 30 minutes. Images were taken on a Zeiss Axio Observer Z1 microscope at 20X magnification.

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Experimental details: Immunofluorescent analysis of Phalloidin (purple) and PDI (green) in NIH 3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature and blocked with 2% BSA (Product # 37525) in PBS + 0.1% Triton X-100 for 30 minutes at room temperature. Cells were probed with a PDI monoclonal antibody (Product # MA3-019) at a dilution of 1:75 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:250 for 30 minutes at room temperature. Actin was stained with DyLight 650 Phalloidin (Product # 21838) at a dilution of 1:120 (2.5 units/mL final concentration) and nuclei (blue) were stained with Hoechst (Product # 62249) at a concentration of 1 µg/mL for 30 minutes. Images were taken on a Zeiss Axio Observer Z1 microscope at 20X magnification.

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Experimental details: Immunohistochemistry was performed on normal deparaffinized Human colon tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing PDI (Product # MA3-019) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on cancer biopsies of deparaffinized Human lung adenocarcinoma tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing PDI (Product # MA3-019) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Immunohistochemistry was performed on normal deparaffinized Human tonsil tissue tissues. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing PDI (Product # MA3-019) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

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Experimental details: Flow cytometry analysis of PDI in NIH/3T3 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a PDI monoclonal antibody (Product # MA3-019) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

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Experimental details: Flow cytometry analysis of PDI in Hela cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a PDI monoclonal antibody (Product # MA3-019) at a dilution of 1 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

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Experimental details: Flow cytometry analysis of PDI in K562 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a PDI monoclonal antibody (Product # MA3-019) at a dilution of 1 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

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Supportive validation

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Experimental details: Figure 4 Search for potential mechanisms involved in Brucella abortus -induced mitochondrial fragmentation ( a ). TOM20 immunostaining in HeLa cells pre-incubated for 30 min with the indicated concentration of ruthenium red and infected with B. abortus 2308 mCherry with or without the inhibitor - 48 h PI (Representative of n = 3). Green: TOM20 (Alexa488)/Red: B. abortus 2308 (mCherry)/Turquoise: Nuclei (Hoechst) ( b , c ). Aspect ratio ( b ) and end point/branch point ratio ( c ) of HeLa cells pre-incubated for 30 min with the indicated concentration of ruthenium red and infected (white) or not (black) with B. abortus 2308 mCherry with or without the inhibitor - 48 h PI. Results represent means +- SD for three independent experiments (n = 3). Statistical analysis: two-way ANOVA on Box Cox transformed data. P value for interaction: 0.446 and 0.051 respectively. The numbers indicated in the columns represent the number of cells analysed for each condition. ( d ) PDI and TOM20 co-immunostaining in HeLa cells infected or not (control) with B. abortus 2308 mCherry - 48 h PI (Representative of n = 3). Green: PDI (Alexa488)/Blue: TOM20 (Alexa633)/Red: B. abortus 2308 (mCherry)/Turquoise: Nuclei (Hoechst). ( e ) Western blot analysis of P-IRE1 and IRE1 abundance in RAW 264.7 macrophages infected or not with B. abortus 2308 mCherry - 48 h PI (Representative of n = 3 in different conditions). RAW 264.7 cells treated for 6 h with 10 uM tunicamycin and then left for 18 h for

Supportive validation

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Experimental details: Fig. 1 Western blot analysis of Cyp3a2, Sec61alpha, Oatp1a1, Ntcp, Mrp2, and PDI in SER and RER isolated from Wistar and TR - rat livers. 5 mug (Cyp3a2, Mrp2), 50 mug (Sec61alpha) or 100 mug (Oatp1a1, Ntcp) protein was loaded per lane and separated by SDS-PAGE as given in section 2.4. Samples were probed against Cyp3a2, Sec61alpha, Oatp1a1, Ntcp, Mrp2, and PDI as described in 2.4. PDI was used as loading control in SER and RER fractions. Basolateral membrane fraction (BLM) isolated from Sprague Dawley rat liver was used as positive control for Oatp1a1 and Ntcp expression. Canalicular (CM) membrane fraction isolated from Sprague Dawley rat liver was used as positive control for Mrp2 expression. The figure shows a representative Western blot of at least two independent SER and RER isolations.

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Experimental details: Figure 4 Subcellular localization of DP4 and DP5 in the presence or absence of Ii. ( a - c ) HEK293 cells were transfected with the indicated combinations of genes. Fixed cells were permeabilized and stained for DP (red) in conjunction with an ER marker, PDI (green, left panels), an early endosomal marker, EEA1 (green, middle panels) or a late endosomal/lysosomal marker, LAMP-1 (green, right panels) and analysed by confocal microscopy. Inset boxes indicate the areas depicted at higher magnification. Scale bar in all images, 10 mum. Quantification of co-localized spots represents means+-s.d. of three counted cells in each condition. ns, not significant; * P

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Experimental details: 3 (A) Western blot analysis of HEK293 cells transfected with mDC-STAMP-GFP constructs. (Lane 1) HEK293 cells; (lane 2) HEK293/GFP; (lane 3) HEK293/mDC-STAMP-GFP; and (lane 4) HEK293/mDC-STAMP-GFP plus tunicamycin. Western blots were stained with anti-GFP mAb. The arrow indicates unglycosylated mDC-STAMP-GFP. (B) HEK293 cells, stably transfected with mDC-STAMP-GFP or hDC-STAMP-GFP constructs, respectively, were stained with antibodies against beta1 integrins, against the protein present in the ER (anti-PDI), and the intermediate compartment (anti-ERGIC-53). Cells adherent to the fibronectin-coated slides were visualized by CLSM for GFP autofluorescence (green) and the markers (blue) as indicated. (C) Immunoelectron microscopy of HEK293 cells stably transfected with hDC-STAMP-GFP. Sections were stained with the antibody against GFP and incubated with the 10-nm gold beads. The arrows point to the gold beads at the nuclear membrane (left) and in the ER (right). The other gold particles in the left panel are located on ER structures, as is more clearly shown in the right panel. No staining was visible at the mitochondria (M) or in the nucleus (N). Original magnification, 20,000x.

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Experimental details: 4 DC-STAMP-GFP localizes to the ER in DC. Mo-IDC transduced with the adenovirus Ad5fib35 encoding hDC-STAMP-GFP (green) were stained with a beta2-integrin antibody (red), an ER marker (anti-PDI, blue), and an intermediate compartment marker (anti-ERGIC-53, blue), and fluorescence was visualized by CLSM.

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Experimental details: Figure 2 WNV NS3 is distributed throughout the cytoplasm, while NS2B remains in the ER in transfected cells. ( A ) NS3-GFP transfected ( a - c ) or NS3-GFP and IKKepsilon-FLAG co-transfected ( d ), HEK293T cells were fixed at 24 h post-transfection and immunostained with antibodies against protein disulfide isomerase (PDI), an ER marker, (red; a ), anti-GM130, a Golgi apparatus marker, (red; b ), anti-tubulin, a cytoskeletal marker, (red; c ), or anti-IKKepsilon, a soluble cytoplasmic protein marker, (red; d ). ( B ) NS2B-V5/His transfected ( a - c ) or NS2B-V5/His and IKKepsilon-FLAG co-transfected ( d ) HEK293T cells were fixed 24 h after transfection and immunolabeled with rabbit anti-PDI (green; a ), anti-giantin (green; b ), anti-tubulin (green; c ) or anti-IKKepsilon (green; d ) antibodies to visualize the ER, Golgi apparatus, cytoskeleton or the cytoplasmic protein kinase IKKepsilon protein, respectively. Visualized viral and host proteins are listed on the top right corner of each panel. Nuclear DNA was labeled with DAPI. Confocal microscopy images were of optical slice thickness ~1 mum. Scale bar, 10 mum. The main image depicts the merged image from three channels separately shown on the side. White arrows indicate colocalization between the green and red channels. The transfection efficiency (TE) for cells transfected with one construct or the co-transfection efficiency (Co-TE) for cells co-expressing the viral protein and IKKepsilon are listed at the bottom of each

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Experimental details: Figure 3 NS3 localizes to the ER when NS2B is provided in trans and in cis. ( A ) HEK293T cells were co-transfected with NS3-GFP and NS2B-V5 expressing plasmids ( a - c ). At 24 h post-transfection, cells were fixed and co-stained with anti-GFP and antibodies against PDI (red; a ), giantin (red; b ) or tubulin (red; c ). HEK293T cells were also triple-transfected with NS3-GFP, NS2B-V5/His and IKKepsilon-FLAG expressing plasmids ( d ) and triple stained with anti-GFP (green; d ), anti-V5/His (red; d ) and anti-IKKepsilon (blue; d ) antibodies. ( B ) HEK293T cells were transfected with NS2B-NS3-GFP ( a - c ) or co-transfected with NS2B-NS3-GFP and IKKepsilon-FLAG expressing plasmids ( d ) and processed for immunofluorescence at 24 h after transfection using antibodies against PDI (red; a ), giantin (red; b ), tubulin (red; c ) and IKKepsilon (red; d ). The detected viral and host proteins are listed on the top right corner of each panel. White arrows indicate colocalization between the GFP-tagged viral protein and the ER marker. Nuclear DNA was labeled with DAPI. Scale bar represents 10 mum. The main panels depict merged images with the side panels showing the individual channels. The transfection efficiency (TE), the co-transfection efficiency (Co-TE) and the triple-transfection efficiency (Triple-TE) for cells expressing one, two, or three gene constructs, respectively, are listed at the bottom of the panel. Images are representative of two independent transfection experiment

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Experimental details: 3 Increased binding of vitronectin and LM609 to Mn 2+ -stimulated endothelial cells requires free thiols in alpha V beta 3 and active PDI. Endothelial cells were stimulated with Mn 2+ , and their binding of LM609 (A, B) and vitronectin (C, D) was evaluated by flow cytometry in the absence or presence of the membrane-impermeant sulfhydryl group reagent PCMBS, a PDI-specific inhibitor (A, C), bacitracin (B), or blocking antibody to PDI (mAb 019) (D).

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Experimental details: FIGURE 3 Confocal analysis of the subcellular localization of FAM46A in transfected K562 cells. Cells were transfected with an expression construct encoding the FAM46A-EGFP protein, which was detected in the nuclear (A) and cytoplasmic (B) compartments. Within the nucleus (A) , FAM46A was predominantly confined in the chromatin domains more accessible to the transcription complexes (as indicated by the acK9-Histone H3 staining) and were mostly excluded from the regions with condensed chromatin structures (as indicated by the meK9-Histone H3 staining). In addition, significant co-localization of FAM46A with NuMA and lamin A/C was observed. DAPI staining defined the morphology of nuclei. Scale bar: 5 mum. Within the cytoplasm (B) , the majority of FAM46A was found to co-localize with the endoplasmic reticulum marker, protein disulfide-isomerase (PDI). By contrast, co-localization of FAM46A with trans-Golgi network marker gamma-adaptin (AP-1) and early endosome marker transferrin receptor (TrfR) is relatively low. DAPI staining defines the morphology of nuclei. Scale bar: 5 mum.

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Experimental details: 10.1371/journal.pone.0242960.g003 Fig 3 Endoplasmic reticulum chaperones are downregulated in CTEPH-EC. (A) Representative blot of the GRP94 expression in static, low, and high magnitude shear stress (0, 5, and 15 dynes/cm 2 ). Data are presented as the mean +- standard error: (*) HPAEC vs CTEPH-EC, (&) HPAEC 15 vs CTEPH-EC 15. (n = 5-7). (B) GRP78 and PDI expression before and after shear stress exposure as described in methods. Data are presented as the mean +- standard error: (*) HPAEC vs HPAEC 15, (#) CTEPH-EC vs CTEPH-EC 5 (&) HPAEC 15 vs CTEPH-EC 15, P < 0.05. (n = 6-7). All of the samples were normalized to ss-actin and run in the same gel.

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Experimental details: FIGURE 5: RUSH assay reveals mislocalization of B4GalT1 to endolysosomes in GARP-KO RPE1 cells. RPE1 cells were transfected with plasmids encoding Str-KDEL_flB4GalT1-SBP-mCherry in biotin-free medium. Following overnight incubation, biotin (40 uM) and cycloheximide (50 uM) were added for 0, 2, and 6 h. (A) Colocalization of B4GalT1 with ER marker PDI at time 0. (B, C) Colocalization of B4GalT1 with Golgi protein GM130 at 2 h (B) and 6 h (C). The graph on the right side of C shows the quantification of B4GalT1 and GM130 colocalization at 6 h of biotin/cycloheximide addition. Values in the bar graph represent the mean +- SD of the colocalization between B4GalT1 and GM130 in approximately 40 cells in WT and VPS54-KOs. Colocalization in VPS53-KO cells was measured using each field in approximately 20 cells. Statistical significance was calculated using one-way ANOVA. **** P

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Experimental details: Fig. 6 Co-immunostaining of CypB and cell organelles in HCoV-229E-infected Huh7 cells . For the identification of the intense cyclophilin B bleb-like structures infected cells (MOI = 1; 48 h p.i.) were co-stained with anti-CypB and antibodies directed against markers of the ER (anti-PDI), cis-GOLGI (anti-GM130), autophagosomes (anti-SQSTM1), anti-P-bodies (anti-hDcp1a) and stress granules (anti-PABP). Cyclophilin B normally distributes within the ER. In the presence of HCoV-229E, it intriguingly concentrates at bleb-like structures of the ER. Fig. 6

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Experimental details: FIG 6 The LTM-1 and LTM-2 motifs of NTPase have the potential to direct fusion proteins to the ER. (A) Schematic diagram of various NTPase(1-179) deletion mutants. The ability and degree of NTPase(1-179)-GFP and its deletion mutants for LD localization and for ER localization are summarized. (B) Colocalization of NTPase(1-179) deletion constructs with LDs or with the ER in cells. A7 cells were transfected with the indicated expression plasmids for 24 h, and then the transfected cells were dually stained with LipidTOX and anti-PDI antibody; scale bars, 10 mum. The quantitative results are plotted as mean +- SEM ( n = 15 cells obtained from two independent experiments); *, P < 0.05 for results compared to those from the empty vector group (GFP); #, P < 0.05 for results compared to those from the NTPase(1-179)-GFP-transfected group. (C) Confocal microscopic analysis of the localization of NTPase(1-179)-GFP in cells at different time points (6, 18, and 24 h) after transfection. A7 cells were transfected with the NTPase(1-179)-GFP expression plasmid for 6, 18, or 24 h, and then the subcellular localization of this protein to LDs or the ER in transfected cells was analyzed by confocal microscopy; scale bars, 10 mum. The quantitative results are plotted as mean +- SEM ( n = 15 cells obtained from two independent experiments); *, P < 0.05 for results compared to those from the 6 h group.

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Experimental details: FIGURE 4 The intracellular localization of CatD and enzyme activity in No. 1002 cells. (a) The immunofluorescence cell localization of wild-type and mutant CatD expressed in No. 1002 cells. After transfecting vector, wild-type, and mutant plasmids into No. 1002 cells, the co-localization of the CatD protein (green) and lysosome (red), as well as the co-localization of the CatD protein (green) and endoplasmic reticulum (red) were observed. (b) The ratio of mature-CatD and pro-CatD in No. 1002 cells transfected with the wild-type and mutant of the CTSD gene, respectively. (c) The CatD activity of wild-type and mutant No. 1002 cells. CatD activity expressed as relative fluorescence units (RFU) per million cells

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Experimental details: Figure 1. EN1 expression is up-regulated in fibrotic skin. (A) Representative immunofluorescence stainings for EN1 (green) and costainings with P4H (red) and CD45 (magenta) in the dermis of healthy donors and SSc patients at 1,000-fold magnification (scale bars = 20 um). Hematoxylin and eosin stainings (200-fold magnification, scale bars = 100 um), Voronoi diagrams, and percentages of EN1-positive fibroblasts from all fibroblasts (defined as P4H + CD45 - cells) are included. Data are shown from one experiment ( n >= 4). (B and C) EN1 mRNA (B) and EN1 intracellular or secreted protein levels (C) in cultured fibroblasts or their supernatants from healthy donors and SSc patients. Representative Western blot images and quantifications are included (C). Data are shown from one experiment ( n = 3). (D and E) En1 protein levels in the skin of mice challenged with bleomycin (D) or Tsk-1 mice (E) and their respective controls. Representative Western blot images and quantifications are included. Data are compiled from two independent experiments ( n >= 4). (F and G) Representative immunofluorescence stainings for EN1 (green) and costainings with Vimentin (red) and CD45 (magenta) in the dermis of mice challenged with bleomycin and their controls at 1,000-fold magnification (F; scale bars = 20 um) and in the hypodermis of Tsk-1 mice and their controls, respectively, at 1,000-fold magnification (G; scale bars = 20 um). Hematoxylin and eosin stainings (100-fold magnification, scale bars =

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Experimental details: Figure S1. EN1 expression in healthy and SSc skin. (A-C) Representative immunofluorescence stainings for EN1 (green) and costainings with CD31 (A) and CD45 (B; both magenta) in the dermis of healthy donors and SSc patients at 1,000-fold magnification (scale bars = 20 um). Hematoxylin and eosin stainings (200-fold magnification, scale bars = 100 um) are included. Data for A-C were obtained from n >= 4 biological samples from two independent experiments. (D) Composition of clusters from the segmented IMC images in ventral and dorsal skin biopsies from SSc patients. (E) tSNE plot showing distribution of cells from ventral and dorsal SSc skin across clusters. (F) tSNE plot showing expression of EN1 across the ventral and dorsal SSc clusters. Data for D-F were obtained from n = 8 biological samples from four SSc patients (paired ventral and dorsal biopsies).

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Experimental details: 3 FIGURE ER retention of VWF in ECFCs with VWF propeptide variants. A, Ex 4-5del; B, p.Cys633Arg; C, p.Arg273Trp; D, p.Pro293Glnfs*164 and p.Gln419* ECFCs. Co-staining of ECFCs for VWF (green) and the ER marker PDI (red) shows co-localization (yellow) indicating ER retention of the variant (diffuse) VWF in heterozygous and homozygous VWF propeptide ECFCs. In normal ECFCs (Unaffected III-5, III-6), VWF is in WPBs and not associated with the ER marker. Images were obtained using a Leica TCS SP8 microscope at 63x magnification, upgraded with Hyvolution software (scale bars = 50 mum; and 10 mum for zoomed in panels). White boxes show the magnified area. The optical zoom is provided at the top right corner of each zoomed in image. C. Het, compound heterozygous; ECFCs, endothelial colony-forming cells; ER, endoplasmic reticulum; Het, heterozygous; Hom, homozygous; PDI, protein disulfide-isomerase; VWF, von Willebrand factor; WPBs, Weibel-Palade bodies

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Experimental details: FIG 3 N-terminal 179-aa region of GII-NTPase is required for vesicle formation and ER colocalization. (A) Schematic diagram of the full-length and deletion constructs of F-NTPase. The abilities of F-NTPase and its deletions to promote vesicle formation and ER colocalization are summarized in the diagram. (B) Western blot analysis of F-NTPase and its deletions expressed in 293T cells. (C) Representative confocal immunofluorescence images of F-NTPase constructs (green) and PDI (red) in A7 cells. White arrows indicate the positions of vesicle-like structures formed by F-NTPase or its deletions. Yellow arrows represent the colocalization between the indicated F-NTPase constructs and PDI in the merged images. Scale bars, 10 mum.

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Experimental details: Fig. 3 Caveolin-1 accumulates in mitochondria-associated ER membranes, precluding their remodelling upon early-phase ER stress. a Wild-type HeLa cells were labelled by immunogold staining with antibodies against CAV1. Blue arrowheads indicate CAV1 signals at the plasma membrane; red arrowheads indicate CAV1 signals at ER cisternae; yellow arrowheads indicate CAV1 at the ER-mitochondria interface. In the lower right panel, the ER is highlighted in red and mitochondria (mito) in green. Scale bar: 1 um. b Cells lysates were fractionated by differential sedimentation. HM heavy membranes (crude mitochondria), LM light membranes (microsomes). c HeLa mock and CAV1 cells in control condition (con) or under early ER stress with tunicamycin (tun) were processed and fractionated as in b . CAV1 levels were analysed by western blotting using PDI as a loading control. d Quantification of membrane-bound CAV1 analysed in c ( n = 3). e HM fraction obtained as in ( b ) was separated into MAM and pure mitochondria (mito) fractions by Percoll density gradient centrifugation. f Markers of cell fractionation procedures shown in b and e : FACL4 short isoform (mito), long isoform (MAM); COX IV (mito), Calnexin (CNX) (LM and MAM), SERCA2b (LM) and PDI (C, LM and HM). g Experimental groups as in c were processed and fractionated as in e . CAV1 levels were analysed by western blotting using PDI as a loading control. h Quantification of CAV1 in MAM analysed in g ( n = 3). i Experimental groups as in c w

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Experimental details: Fig. 6 Caveolin-1 expression precludes DRP1 phosphorylation and redistribution during the early phase of ER stress. a Mock and CAV1 cells in control condition (con) or under early ER stress with tunicamycin (tun) were fractionated and processed for detection of phospho-DRP1 and DRP1 by western blotting using PDI as a loading control. C cytosol, LM light membranes (microsomes), HM heavy membranes (crude mitochondria). b Quantification of DRP1 phosphorylation analysed in ( a ) ( n = 3). c Quantification of membrane-bound DRP1 distribution analysed in a ( n = 3). d Experimental groups as in ( a ) were processed for immunofluorescence analysis. CAV1, mtHSP70 (mitochondrial marker, Mito) and DRP1 were stained with respective antibodies and then imaged using confocal microscopy. Merge between DRP1 and CAV1 images shows colocalized areas in yellow. Merge between DRP1 and Mito images shows colocalized areas in cyan. In merge images of Mito, DRP1 and CAV1, triple colocalization areas appear as white pixels. e CAV1 fluorescence per cell was quantified from images obtained in d ( n = 3). f DRP1-CAV1 colocalization was quantified as Manders' coefficients of images obtained in ( d ) ( n = 3). g DRP1-Mito colocalization was quantified as Manders' coefficients of images obtained in ( d ) ( n = 3). h From images obtained in d , thresholded CAV1 (C) and Mito (M) images were generated. The intersection between M and DRP1 corresponds to mitochondrial DRP1 (DRP1 mito ). Merge between M and C ima

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Experimental details: Fig. 1 Role of PDIA1 in oxidant generation by colon carcinoma cells with distinct levels of KRas activation. a KRas expression and activity in Caco2, HKE3, and HCT116 cells. Active KRas was pulled down with GST-RBD beads from lysates of serum-starved cells treated with EGF 25 ng/mL for 10 min. Aliquots of the lysates were blotted for total KRas and GAPDH as loading control. b RNAseq analysis of P4HB (PDIA1) gene in Caco2, HCT116, and HCT15 cells, showing mean expression in FPKM (Fragments Per Kilobase Million), P4HB fold-change expression in HCT15 vs. Caco2 (q < 0.000001) and HCT116 vs. Caco2 (q < 0.000001). c PDIA1 basal protein expression by western analysis, normalized for GAPDH ( n = 3) and quantified using Odyssey software. d Intracellular PDIA1 titration, using Human P4HB ELISA Pair Set (SinoBiological). Test t ** p < 0.01, ( n = 4); e-g ROS production after 72 h of PDIA1 silencing, measured by HPLC analysis of DHE oxidation products. DHE oxidation produces, among many others, two major products: 2-hidroxyethidium (EOH), which is representative of superoxide species and ethidium, representative of other oxidant species. Ctrl negative, si-RNA control; si-PDI, si-RNA against PDIA1 protein. Representative immunoblots of PDI silencing for each cell type Test t * p < 0.05; ** p < 0.01. For Caco2 cells, n = 3; HKE3 cells, n = 4; HCT116 cells, n = 5

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Experimental details: Fig. 2 Nox NADPH oxidase expression and superoxide generation by HCT116 cells with overactivated KRas. a Nox1, NoxA1, p67phox, NoxO1, and p47phox basal protein expression by western analysis normalized for GADPH. Nox1 immunoblot densities were quantified using Odyssey software. * P < 0.05, ANOVA plus Tukey's multiple comparisons test ( n = 3). b RhoGDIalpha, RhoA, and Rac1 basal protein expression by western analysis normalized for GAPDH. Rac1 immunoblot densities were quantified using Odyssey software. ** P < 0.01; *** P < 0.005, ANOVA plus Tukey's multiple comparisons test ( n = 3). c Nox1 complex scheme. d Rac1 basal activity, using Rac1 G-LISA activation assay (Cytoskeleton,inc). Test t * p < 0.05, ( n = 2). e Rac1 protein expression by western analysis after 72 h of PDIA1 silencing. Rac1 Immunoblot densities were quantified using Odyssey software ** P < 0.01; ns: not significant, Student's t -test ( n = 3). f Western analysis of NoxO1 and RhoGDIalpha protein expression before and after PDIA1 silencing, normalized for GAPDH. RhoGDIalpha immunoblot densities were quantified using Odyssey software, **** P < 0.001; ns: not significant, Student's t -test ( n = 3). g Superoxide production after 72h of PDIA1 silencing in HCT116 cells treated or not with 10 uM of NoxA1ds Nox1's peptide inhibitor. EOH 2-hidroxyethidiium relative superoxide production, si-PDI, si-RNA against PDIA1 protein; Scrmb NoxA1ds, NoxA1ds negative control peptide; NoxA1ds, Nox1 peptide inhibitor. * P < 0.05

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Experimental details: Fig. 5 Effects of PDIA1 silencing in signaling targets and EMT marker expression. PathScan array analysis. All array's spots were quantified and analyzed using ImageJ software. PDIA1 silencing was checked by immunoblot analysis. Ctrl, si-RNA negative control; si-PDI, si-RNA against PDIA1; ERK1/2, Thr202/Tyr204 phosphorylation; Stat1, Tyr701 phosphorylation; Stat3, Tyr705 phosphorylation; Akt, Thr308 phosphorylation; Akt, Ser473 phosphorylation; AMPKa, Thr172 phosphorylation, s6 ribosomal protein, Ser235/236 phosphorylation; mTOR, Ser2448 phosphorylation; HSP27, Ser78 phosphorylation; Bad, Ser112 phosphorylation; p70 S6 kinase, Thr389 phosphorylation; PRAS40, Thr246 phosphorylation; p53, Ser15 phosphorylation; p38, Thr180/Tyr182 phosphorylation; SAPK/JNK, Thr183/Tyr185 phosphorylation; PARP, Asp214 cleavage; Caspase-3, Asp175 Cleavage; GSK-3beta, Ser9 phosphorylation. b GSK-3beta, Ser9 phosphorylation plot. c Stat3, Tyr705 phosphorylation. d Basal E-cadherin (E-cad) protein expression by immunoblot normalized for GAPDH ( n = 3). e Immunoblot for E-cadherin before and after PDIA1 silencing in Caco2 cells ( n = 2). f Immunoblot for E-cadherin before and after PDIA1 silencing in HKE3 cells.; ns: not significant, Student's t test ( n = 3). g Immunoblot for E-cadherin before and after PDIA1 silencing in HCT116 cells * P < 0.05, Student's t test ( n = 3). Immunoblot densities were quantified using Odyssey software

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Experimental details: Fig. 6 Disruption of ER-Golgi trafficking disassembles vRNP hotspots. A549 cells were infected or mock-infected with PR8 virus at an MOI of 3. a At 90 min p.i., 2 mug mL -1 of brefeldin A (BFA) or 10 mug mL -1 nocodazole (NOC) were added and incubated for 10 h until the supernatant was collected and viral titres determined or cells were harvested in Laemmli's and NP levels detected by western blotting. Statistical analysis of data was performed using a non-parametric one-way ANOVA, followed by Friedman's multiple comparisons test (* p < 0.05). b - d At 8 hpi, cells were also treated or mock-treated with 2 mug mL -1 of BFA for 1 h. b Cells were immunostained for the ER marker PDI (in green), the viral protein NP (in red) and the cis -Golgi marker GM130 (in gray) and imaged by confocal microscopy. Selected areas of the cytoplasm are marked by white boxes and displayed on the top left corner of the images. Bar = 10 mum. c The frequency distribution of NP inclusions within the three size categories (in mum 2 ), the number of inclusions per mum 2 , and the percentage of NP staining that is inside inclusions were plotted for each condition. Statistical analysis of data was performed using a non-parametric Kruskal-Wallis test, followed by Dunn's multiple comparisons test (*** p < 0.001). An average of 60 cells from 2 independent experiments was analyzed per condition. d Infected cells were stained for the host protein Rab11 (in green) and the viral proteins NP (in re

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Experimental details: Fig. 7 Disruption of ER exit sites dissolves viral inclusions. HeLa cells were transfected with plasmids encoding GFP, SAR1 WT-GFP (SAR1) or SAR1-GTP-GFP (SAR1-GTP) and, 24 h later, infected or mock-infected with PR8 virus, at an MOI of 10. At 16 hpi, cells were fixed and processed for immunofluorescence. a Cells were stained for the viral protein NP (in red) and for the ER protein PDI (in gray). Areas highlighted by the white box are shown on the right of each panel. b The number of inclusions per mum 2 , the percentage of NP staining that is inside inclusions and the frequency distribution of NP inclusions within the three area categories (in mum 2 ) were plotted for each condition. Statistical analysis of data was performed using a non-parametric Kruskal-Wallis test, followed by Dunn's multiple comparisons test (*** p < 0.001). More than 40 cells from 2 independent experiments were analyzed per condition. c Infected cells were stained for the viral protein HA and imaged by confocal microscopy. Bar = 10 mum

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Experimental details: FIGURE 9 Immunohistochemical localization of m2R in neuronal compartments involved in endocytosis, synthesis, maturation, and degradation in fixed hippocampal neurons. Hippocampal neurons were transfected with m2R-WT. Neurons were stimulated with CCh at 30 muM for 6, 20 min and 1 h fixed, and processed for visualization of m2R together with markers of intraneuronal compartments and observed by confocal microscopy. ( A-C) 6 min after CCh stimulation (30 muM), some m2R immunopositive punta colocalize with CHC in clathrin-coated pits, EEA1 in early endosomes and Rab9 in late endosomes (arrow heads). (D-E) Twenty minutes after CCh stimulation (30 muM), we failed to detect no colocalization of m2R with PDI, a marker of endoplasmic reticulum and GM130, a marker of Golgi apparatus. (F-F) One hour after CCh stimulation (30 muM), some m2R immunopositive puncta colocalize with CathD, a marker of lysosomes (arrow heads). The quantitative analysis of the colocalization of m2R and markers of subcellular compartment in neurons was performed using the Jacop Plugin of ImageJ and statistical data are reported from the Costes's randomization-based colocalization module (see Materials and Methods). Data are expressed as a Pearson's coefficient (pc) and pc were compared using the Mann-Whitney U -test. Our analysis shows that the colocalization of the immunofluorescent signals for m2R with CHC, EEA1, Rab9, and CathD is higher after treatment with CCh compared to untreated neurons (CHC, Rab9