13-3900

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HeLa cells using PCNA Monoclonal Antibody, Mouse (Product # 13-3900). Lane 1: 1 µg/mL, Lane 2: 0.2 µg/mL, Lane 3: 0.05 µg/mL and Lane 4: 0.01 µg/mL.

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Experimental details: Knockdown of PCNA was achieved by transfecting HeLa cells with PCNA specific validated siRNAs (Silencer® select Product # S10135, S10133). Western blot analysis (Fig. a) was performed using membrane enriched extracts from the PCNA knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with PCNA Monoclonal Antibody (Product # 13-3900, 1 µg/mL) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25µg/mL, 1:4000 dilution). 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 PCNA .

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Experimental details: Western blot analysis of PCNA was performed by loading 20 µg of HCT 116 (lane1), U2OS (lane2), MCF7 (lane3), PC-3 (lane4), Caco-2 (lane5) and A549 (lane6) cell lysate using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0322BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. PCNA was detected at ~36 kDa using PCNA Mouse Monoclonal Antibody (Product # 13-3900) at 0.5-1 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: Immunohistochemical analysis of PCNA was performed using formalin-fixed paraffin-embedded human colon (neuro-endocrine tumor) tissue sections. To expose the target protein, heat-induced epitope retrieval (HIER) was performed on de-paraffinized sections using eBioscience™ IHC Antigen Retrieval Solution - High pH (10X) (Product # 00-4956-58) diluted to 1X solution in water in a decloaking chamber at 110 degree Celsius for 20 minutes. Following antigen retrieval, the sections were blocked with 2% normal goat serum in 1X PBS for 45 minutes at room temperature and then probed with or without PCNA Monoclonal Antibody (PC10) (Product # 13-3900) at 5 µg/mL in 0.1% normal goat serum overnight at 4 degree Celsius in a humidified chamber. Detection was performed using Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723) at a dilution of 1:2000 in 0.1% normal goat serum for 45 minutes at room temperature. Nuclei were stained with DAPI (Product # D1306) and the sections were mounted using ProLong™ Glass Antifade Mountant (Product # P36984). The images were captured on EVOS™ M7000 Imaging System (Product # AMF7000) at 20X magnification.

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Experimental details: Immunohistochemistry analysis of PCNA showing staining in the nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a PCNA monoclonal antibody (Product # 13-3900) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

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Experimental details: Immunohistochemistry analysis of PCNA showing staining in the nucleus of paraffin-embedded human colon carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a PCNA monoclonal antibody (Product # 13-3900) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

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Experimental details: Immunohistochemistry analysis of PCNA showing staining in the nucleus of paraffin-embedded mouse spleen tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a PCNA monoclonal antibody (Product # 13-3900) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

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Experimental details: Figure 2 p30 decreases the expression of proteins involved in S phase entry and progression . Hela cells were transduced for 48 h with GFP (control) or p30-myc expressing lentivirus. A ) p30-myc expression was detected by western blot (WB) using anti-myc antibody or B ) by indirect immunofluorescence (IF) staining. Nearly 100% of cells were transduced as shown with different magnifications. GFP-viral particles (HRCMV-GFP) were used as a control in the IF staining. C ) WB analysis of PCNA protein was performed in the same experimental conditions. D ) Quantification of E2F, cyclin E and p21waf mRNAs by real time PCR. E2F, Cyclin E and p21waf values were normalized to that of GAPDH which was used as an internal control. E ) Extracts of Hela cells transduced with GFP or p30-myc virus were used for WB analyses of the endogenous E2F, cyclin E and p21 proteins. F) 293T cells were transfected with 6xE2F-Luciferase reporter plasmid either with HTLV-1 p30 or HTLV-2 p28 expression vectors. Histograms represent the values of three different experiments with their standard deviations. WB analyses showing equal amounts of p30 and p28 are present in the cell extracts.

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Experimental details: Figure 3 p30 interacts with Cyclin E and CDK2 and reduces formation of the Cyclin E-CDK2 complex . A and B ) p30 disrupts interactions between Cyclin E and CDK2. 293T cells were tranfected with Cyclin E-myc, p30-flag and CDK2-HA. The interactions between Cyclin E and CDK2 in the presence or absence of p30 were assessed by either co-IP of Cyclin E or CDK2 and western blot for the corresponding partner. C ) Phosphorylation of Rb protein. 293T cells were transfected with empty control vector or p30 expressing vector, synchronized with ""HU"" and released for 0 h and 8 h. The cells extracts were then analyzed by WB to detect phosphorylated Rb. D ) Interactions between Cyclin E and p30 or ( E ) CDK2 and p30 were investigated after transient transfection in 293T cells and Co-IP/WB.

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Experimental details: Figure 1 Proliferative changes assessed by the proliferating cell nuclear antigen (PCNA) in Lupus Nephritis (LN). Superior panel stained by H and E. Inferior panel, immunohystochemistry for PCNA. a and d: Control biopses. b and e: Class III nephritis. c and f: Class IV nephritis (40x).

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Experimental details: Figure 3 Tissue self-repair after trauma indicated by immunohistochemical staining and semi-quantitative analysis of PCNA, Col I and OCN. (A, C, E) Immunohistochemical assays of the expression of PCNA (A), Col I (C) and OCN (E). (B, D, F) Bar graphs represent their expression density in unit area of bone marrow or unit area of bone trabeculae. (A) OBs; (C) a. OBs, b. osteocytes, c. trabeculae, d. VECs, e. OCs. The expression of PCNA decreased after trauma, and began to be detected in OBs 2 weeks after trauma and was maintained during the following time. Col I increased in the bone marrow for less than 1 week after trauma, and was significantly accumulated in the trabeculae by the end of the study. Both OBs and OCs also expressed increased levels of Col I following trauma. A significant decrease in OCN was detected 3 days after trauma, but it increased significantly 2 weeks later. High levels of OCN were consistently associated with peri-VECs and the fibrous medulla. N: normal group; 3 d, 1 w, 2 w, 3 w: 3 days, 1 week, 1 weeks, 3 weeks post-trauma, respectively. Quantification was based on at least 10 fields per section. * P

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Experimental details: Figure 4 Expression of cell cycle marker proteins was reduced in male SOD1 G93A rNPCs. ( A ) Western blot analysis was used to measure the protein expression of proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase (cdc2), two known cell proliferation markers. ( B ) Semi-quantitative densitometric analysis of PCNA protein expression. ( C and D ) Semi-quantitative densitometric analysis using Western blotting revealed that SOD1 G93A transgene altered the phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2) in male rNPCs. Three independent rNPC lines in each group were used for densitometric analysis. The data were expressed as means +- SEM.

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Experimental details: Figure 4 znf644a and znf644b Morphant Retinas Are Composed of Distinct Populations of Retinal Cells with Differing Characteristics (A) Immunostaining of retinal cross-sections monitoring BrdU incorporation and PCNA + cells at 48 hpf in WT, znf644a , and znf644b morphants. (B) Quantitation of BrdU + cells/mum 2 in central retina of WT, znf644a , or znf644b morphants at 48 hpf (n = 3 for each group). Error bars represent SD. * p < 0.05, Student's t test. (C-E) Immunostaining of pH3 expression in retinal cross-sections at 48 and 72 hpf in WT, and znf644a morphants (C). Quantitation of pH3 + cells/mum 2 in the central retina of WT, znf644a , or znf644b morphants at (D) 48 hpf (n = 5 for each group) and (E) 72 hpf (n = 2 for each group). Quantitation represented as mean +- SD. (F) Immunostaining monitoring marker protein expression in retinal cross-sections. At 72 hpf, amacrine cells express GABA, bipolar neurons express PKC, and cone photoreceptors express Zpr1. White arrows highlight populations of proliferative or differentiated cells.

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Experimental details: Figure 6 The Retinal Functions of znf644a and znf644b Are Dependent on Functional and Physical Interactions with g9a (A) Immunostaining of cleaved Caspase3 (red) in retinal cross-sections of embryos injected with individual or combined subphenotypic doses of g9a-MO, znf644a-MO, or znf644b-MO (n = 3 in each group). Yellow arrows highlight apoptotic cells. (B) (Top) Immunostaining of PCNA (n = 6 in each group) in retinal cross-sections from embryos injected with individual and combined subphenotypic doses of MOs (n = 6 in each group). Yellow arrows denote the position of the PCNA+ population. (Bottom) WISH assays monitoring the expression of vsx2 in retinal cross-sections of embryos injected with individual subphenotypic doses (n = 10 in each group) and combined subphenotypic co-injection of g9a-MO and znf644a-MO (n = 10), g9a-MO and znf644b-MO (n = 13), and znf644a-MO and znf644b-MO (n = 10). Red arrows denote the position of the vsx2 expressing cells. (C) (Top) WISH assays in retinal cross-sections monitoring vsx2 expression at 48 hpf in znf644a morphant embryos rescued by co-injection of either WT human ZNF644 mRNA (n = 12) or C1263A mutant mRNA (n = 9). (Bottom) Immunostaining of retinal cross-sections monitoring PCNA expression at 48 hpf showing rescue of znf644b morphants by co-injection of WT human ZNF644 mRNA (n = 3) but not C1263A mutants (n = 3). (D) (Top) WISH assays in retinal cross-sections at 48 hpf monitoring vsx2 expression from znf644b morphant embryos rescued

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Experimental details: Figure 7 The Retinal Defects of g9a , znf644a , and znf644b Morphants Are Recapitulated in the Midbrain (A) WISH assays monitoring ccnd1 expression in WT, znf644a , or znf644b morphant midbrain cross-sections at 48 hpf. Arrows indicate mislocalized ccnd1 expression. (B) Immunostaining of cleaved Caspase3 in g9a or znf644b morphant midbrain cross-sections at 72 hpf and rescue by p53-MO (n = 3 in each group). Arrows denote the position of Caspase3+ cells. (C) Immunostaining of PCNA in WT or znf644a morphant midbrain cross-sections (n = 3 in each group). Arrows denote the position of PCNA+ cells.

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Experimental details: Figure 2 Proliferative index of wild-type and alpha1- embryonic dermal fibroblasts. ( A ) Mid cross-section of 16.5 embryos were stained with anti-PCNA antibodies, and subsequently counterstained with hematoxylin. Note the higher number of dermal PCNA-positive cells in the wild-type ( WT ) dermis, as compared with their alpha1- ( KO ) counterparts. ( B ) The dermal proliferative index (number of PCNA positive cells/total number of cells x 100) of three wild-type and three alpha1- 16.5 embryos was determined by random evaluation of 12 different 40x microscopic fields for each sample, counting a minimum of 900 cells around the entire circumference of embryonal dermis. Bars and errors indicate the mean and standard deviation. Differences between wild-type and alpha1- groups were significant with P < 0.05 by Student's t test. Bar, 30 mum.

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Experimental details: Figure 3 Proliferative index of wild-type and alpha1- adult fibroblasts in wounded dermis. ( A ) Sections of wild-type ( WT ) and alpha1-deficient ( KO ) wounded skin, 12 d after incision and primary closure, stained with anti-PCNA antibodies, and subsequently counterstained with hematoxylin. The proportion of PCNA-positive cells in the dermal wound (seen as dark nuclei in the micrograph) was similar in wild-type and alpha1- animals. ( B ) The dermal proliferative index (number of PCNA positive cells/total number of cells x 100) was determined by random evaluation of 3 different 40x microscopic fields for each sample, counting a minimum of 300 cells. Bars and errors indicate the mean and standard deviation. Bar, 100 mum.

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Experimental details: Figure 5 EGFR/AKT/mTOR and beta-catenin signaling in glioblastoma cells after treated with nanoparticles. Notes: ( A ) Confocal microscope images of U87 and U118 cells actin cytoskeleton. Cells were grown on extracellular matrix for 24 h and treated with diamond nanoparticles, graphite nanoparticles, or graphene oxide nanoparticles at a concentration of 20 mug/mL and incubated for 24 h. F-Actin was stained with phalloidin conjugated with Atto 633. ( B ) Western blot analysis of N-cadherin, pan-cadherin, vinculin, p-EGFR, and EGFR. GAPDH was used as a loading control. ( C ) Western blot analysis of nuclear and cytoplasmic protein fractions used for determination of beta-catenin protein level. PCNA and beta-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. ( D ) ELISA analysis of AKT and mTOR phosphorylation in comparison to control. Treatment with nanoparticles significantly reduced phospho-AKT ( P

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Experimental details: Figure 4 PCNA-positive cells in the epithelium and stroma of the rat ventral prostate. (A) SC, standard chow diet, (B) HF-S , high-fat diet rich in saturated fatty acid (lard), (C) HF-P , high-fat diet rich in polyunsaturated fatty acid (canola oil) and (D) HF-SP, high-fat diet rich in saturated and polyunsaturated fatty acids. The symbol [a] indicates a result that is different from the SC group (one-way ANOVA and Bonferroni's post hoc test, p

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Experimental details: 5 FIGURE Effect of iNOS inhibition on 3-nitrotyrosine expression, iNOS gene expression and proliferation. (a) Representative images and (b) quantification of an immunofluorescence staining targeting nitrotyrosine in control (control + placebo), water-treated (porcine pancreatic elastase (PPE )+ placebo) and N(6)-(1-iminoethyl)- l -lysine (L-NIL)-treated (PPE + L-NIL) mice. The fluorescence intensity was normalized to DAPI, n = 5 per group. Scale bar indicates 150 mum. (c) Expression of iNOS in lung homogenate of control ( n = 8), water-treated (PPE + placebo, n = 7) and L-NIL-treated (PPE + L-NIL, n = 7) mice. (d,e) Quantification of immunofluorescence staining for the proliferation marker PCNA in mouse lungs treated with either water (PPE + placebo, n = 6) or L-NIL (PPE + L-NIL, n = 6) as well as healthy controls (control + placebo, n = 6) for (d) surfactant protein C (SPC)-positive cells and (e) smooth muscle actin (SMA)-positive cells

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Experimental details: Fig. 5 Proliferation and apoptosis. a Immunohistochemical staining for proliferation marker Ki67 in kidney tissue (left), alongside quantification of the percentage of Ki67-positive cells (right). b Western blot on kidney tissue measuring proliferation markers PCNA, VEGF, survivin, and beta-actin (left) alongside quantification (right). c Immunohistochemical staining for apoptosis marker Caspase-3 in kidney tissue. d Western blot on kidney tissue measuring apoptosis markers BAX, Bcl-2, and Caspase-3 (left) alongside quantification (right). Each group has n = 3 mice. Significant difference a p < 0.05: relative to untreated control; b p < 0.05: relative to AKI; c p < 0.05: relative to AKI-EV; d p < 0.05: relative to AKI-EV-pFUS

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Experimental details: Fig. 6 Signaling pathway analysis. a Western blot and protein quantification showing the expression of p-ERK, ERK, p-MEK, MEK, and beta-actin in kidney tissue. b Western blot and protein quantification showing the expression of p-Akt, Akt, and beta-actin in kidney tissue. c Western blot and protein quantification showing the expression of SIRT3, eNOS, and beta-actin in kidney tissue. Each group has n = 3 mice. Significant difference a p < 0.05: relative to untreated control; b p < 0.05: relative to AKI; c p < 0.05: relative to AKI-EV; d p < 0.05: relative to AKI-EV-pFUS

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Experimental details: Figure 3 Dose- and time-dependent effect of vaspin on luteal cell proliferation. The cells were treated with 0.1, 1, and 10 ng/mL of vaspin for 24 h to 72 h, after which cell proliferation ( A ) was analyzed using the alamarBlue assay. Additionally, cells were incubated for 24 h with 0.1, 1, and 10 ng/mL of vaspin and then real-time PCR and western blot analysis were performed to determine PCNA (proliferating cells nuclear antigen) mRNA ( B ) and PCNA or cyclin A protein expression ( C ). Gene expression levels were normalized to cyclophylin A (PPIA), while proteins to actin. Experiments were independently performed and repeated four times ( n = 4). The data were plotted as the means +- SEM. Significance between the control and vaspin treatments is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.01; Control (C), Relative Fluorescence Units (RFU).

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Experimental details: Fig. 7 In situ confirmation of the abundance of major microglial subsets. a RNA expression levels of markers enriched in different subsets of microglia in the scRNA-seq dataset: ISG15 for interferon cluster 4, CD83 for the cytokine signaling enriched clusters 5 and 6, CD74 for antigen presentation related cluster 7 and PCNA for the proliferative cluster 9. The size of the circles represent the percentage of cells per cluster in which the given gene was detected, while the color coding represent the normalized z -scores. The z -score matrix is available in Supplementary Data 16 . b Box-and-whisker plot representing the normalized gene expression of CD74 (in TPMs) among the different microglia clusters. Note that CD74 gene expression is ~2-fold higher in cluster 7 when compared to the expression in other clusters (see Source Data file). The boxes represent the 25th percentile, median, and 75th percentile. The whiskers extend to the furthest value that is no more than 1.5 times the inter-quartile range (default parameter for R's boxplot function). The number of cells in each microglial cluster is also shown. c Distribution of the expression levels of CD74 on microglia in situ as measured by immunofluorescence and CellProfiler analysis. Note the second small peak at high expression values that we highlight with a red box. Source data are provided in the Source Data file. d Black symbols represent the quantification of the ISG15+, CD83+, and PCNA+ and CD74 high microglia in the do

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Experimental details: Figure 4 Analysis of B16F1 cell proliferation on collagen type I-coated stiffness plates. B16F1 melanoma cells were cultured for 48 h on culture plates coated with different concentrations of collagen type I and fibronectin as control. ( a ) Phase contrast images of cultures and average densitometry values of PCNA (Proliferating cell nuclear antigen) in immunoblot normalized to actin in three independently performed experiments were averaged and are expressed in the graph. ( b ) BrdU (5-bromo-2''-deoxyuridine) incorporation measurements in the same culture conditions. Mean +/- SEM. In the graph, the average size of n = 5-8 biological replicates of each condition is shown. Note: * p < 0.05; *** p < 0.001; scale: 100 um.

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Experimental details: Figure 1. Immunohistochemistry of NTN4 expression within NB. Representative light microscopy images of a neuroblastoma sample from a female patient (age > 18M); tumor with retroperotineal location and in disseminated stage. (a) Hematoxylin and eosin staining reveals presence of ganglionar-differentiated cells (yellow arrowhead) and primitive neuroblasts (yellow asterisk). (b) Immunohistochemistry of NTN4 with its corresponding negative control (inset). (c) PCNA staining. (d) NTN4 is expressed preferentially in the endothelium (yellow arrows), as confirmed by CD31 staining (inset). (e) Detail of ganglionar-differentiated cells with strong NTN4 cytoplasmic expression in a characteristic punctuated pattern (yellow arrowhead). (f) Close up image of (b) as indicated, highlighting NTN4 expression within the stroma (yellow asterisk). A, B, C; Scale bar = 40 mum. D, E, F; Scale bar = 10 mum; inset = 10 mum.

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Experimental details: Figure 3 Loss of Tnmd results in blood vessel ingrowth, macrophage infiltration, and increased cell apoptosis in the OAF. (a and b) Immunofluorescence staining with anti-CD31 antibody reveals increased vessel number in the OAF of Tnmd -/- than WT. (c and d) Higher number of F4/80-positive macrophages was detected in the OAF of Tnmd -/- versus WT. (e and f) TUNEL staining demonstrates increased number of apoptotic cells in Tnmd -/- OAF than WT. (g and h) Reduced number of proliferating cells was observed in Tnmd -/- compared to WT mice by PCNA immunofluorescence staining. All quantitative histomorphometry data were assessed by two-tailed nonparametric Mann-Whitney test; n = 5 animals. * p < .05. EP, endplate; IAF, inner annulus fibrous; OAF, outer annulus fibrous; TUNEL, transferase-mediated dUTP-biotin nick end labeling; WT, wild-type; white dotted line, OAF-IAF boundary. Scale bar, 200 mum

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Experimental details: Fig. 1 Obese hyperleptinemic state associates with decreased effectiveness of tamoxifen. A Mice were fed high-fat-diet (HFD) or control normal-diet (ND) to achieve obese/lean phenotype. Body weights of mice were measured after 6, 12, 18, and 24 weeks of diet intervention ( n = 24). * p < 0.05, compared with ND mice. B MCF7 cells-derived tumors were developed in obese and lean mice and treated with 4-hydroxytamoxifen (T) or vehicle (C). * p < 0.05. C Representative tumor pictures and average tumor weights from obese and lean mice treated with tamoxifen (T) or vehicle (C) are shown. D Expression of PCNA was examined using immunohistochemistry. Representative images are included. Scale bar: 50 um. E Serum samples from HFD/obese or ND/lean mice were subjected to ELISA to quantify leptin levels at indicated time intervals. * p < 0.05, compared with untreated controls. F Kaplan-Meier analyses for overall survival of the cohort of patients with ER-positivity, receiving tamoxifen treatment with chemotherapy in relation to mean leptin (LEP) expression ( p = 0.003). G Upregulated expression of a set of cytokine pathway genes was observed in MCF7 cells treated with leptin. H MCF7 and T47D cells were treated with 100 ng/ml leptin (L) and/or 1 muM 4-hydroxytamoxifen (T) as indicated and anchorage-dependent growth was examined by clonogenicity assay. Vehicle-treated cells are denoted with ( C ). I , J Soft-agar colony-formation of MCF7 and T47D cells treated with leptin (L) and/or 4-hydrox

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Experimental details: Figure 2. miR-125a-5p inhibits proliferation and promotes apoptosis via tafazzin phospholipid-lysophospholipid transacylases signaling. Cellular inhibition rates were determined using CCK-8 assays. MCF-7/Adr cells were treated with various concentrations of Adr or Doc for 72 h. Inhibitory effects were increased with miR-125a-5p transfection. The IC 50 was reduced from 5.99 uM (NC) to 2.28 uM (miR) in the MCF-7/Adr cells (A) treated with Adr, and from 5.01 uM(NC) to 1.63 uM (miR) in the cells (B) treated with Doc. MCF-7/Adr cells were transfected with miR-125a-5p and treated with (C) Adr (2.28 uM) or (D) Doc (1.63 uM) for 24, 48, 72, 96 and 120 h. The CCK-8 assay results indicated that this combination inhibited MCF-7/Adr cell viability. (E and G) Apoptotic rate of MCF-7/Adr breast cancer cells treated with Adr, as detected via flow cytometry. (F and H) Apoptotic rate of MCF-7/Adr breast cancer cells treated with Doc as detected via flow cytometry. a, NC group; b, drug group; c, miR (miR-125a-5p) group; d, miR/drug group. Caspase-3 activation and increased Bax expression were observed in MCF7/Adr cells transfected with miR-125a-5p and treated with (I) Adr or (J) Doc; the combination of miR-125a-5p and Adr or Doc kept Bcl-2 and PCNA expression at the lowest level, as determined via western blot analysis. GAPDH served as an internal control. (K and L) Semi-quantification of protein expression levels in panel (I). (M and N) Semi-quantification of protein expression levels in pane

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Experimental details: Figure 3. TAFAZZIN is a target of miR-125a-5p in MCF-7/Adr cells. (A) Binding site of miR-125a-5p and TAFAZZIN 3'-UTR according to online bioinformatics analysis. (B) Luciferase activity was decreased for wild-type-TAFAZZIN, but not for the mutant. (C) RNA immunoprecipitation assay indicated that miR-125a-5p and TAFAZZIN were conjunct in MCF-7/Adr cell lines. (D) TAFAZZIN mRNA expression in miR-125a-5p and control groups as detected via reverse transcription-quantitative PCR. (E) Western blot analysis of TAFAZZIN protein expression in miR-125a-5p and control groups. (F) Semi-quantification of TAFAZZIN protein expression in panel (E). MCF-7/Adr cells were treated with siRNA-TAFAZZIN, and the expression levels of (G) TAFAZZIN, TG2, p-AKT, t-AKT, E-cadherin, N-cadherin, vimentin, beta-catenin, (H) cleaved caspase-3, caspase-3, pro-apoptotic protein (Bax) and PCNA were detected via western blotting. Knockdown of TAFAZZIN expression could significantly reduce TAFAZZIN, TG2, p-AKT, N-cadherin, vimentin, beta-catenin, Bcl-2 and PCNA protein expression and increase those of E-cadherin and Bax, as well as activate caspase-3. (I-L) Semi-quantification of protein levels in panels (G and H). GAPDH was used as an internal reference. Data are shown as the mean +- SD. Similar results were obtained from three independent experiments. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 1 Matrine inhibited the proliferation of cancer cells in vitro and in vivo. A Human non-small cell lung cancer cell A549, human pancreatic cancer cell BxPC-3, and human ovarian cancer cell SKOV3 were treated with matrine at the concentrations of 0, 0.5, 1.0, 2.0, and 3.0 mg/mL for 0, 24, 48, and 72 h. MTT assay was conducted to examine the effect of matrine on the proliferation of cancer cells. Data are presented as mean +- SD ( *P < 0.05, **P < 0.01). B-D Xenograft models were established by subcutaneous injection of A549 ( B ), BxPC-3 ( C ), and SKOV3 ( D ) cells in BALB/c nude mice, respectively. Tumor volume, size, and weight in matrine-treated mice were significantly smaller than those in normal saline-treated mice (150 mg/kg in A549 and BxPC-3 cells, 100 mg/kg in SKOV3 cells, i.p.). Data are presented as mean +- SD ( n = 4, *P < 0.05). E The representative histological examinations of dissected tumors of mice inoculated with A549, BxPC-3, and SKOV3 cells after matrine or normal saline treatment. Scale bar: 50 mum. Results are representative of three experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 4 hsa_circ_0009035 silencing regulated CC cell migration, invasion, and radiosensitivity in vitro . HeLa and Siha cells were stably transduced with sh-NC, sh-hsa_circ_0009035, or sh-hsa_circ_0009035#2. (A and B) Cell migration and invasion by transwell assay. (C and D) Survival analysis by colony formation assay in transduced cells upon radiation (0, 2, 4, 6, and 8 Gy) exposure. (E and F) Protein levels of PCNA, c-caspase 3, procaspase 3, and MMP3 determined by Western blotting in transduced cells. * , P < 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 8 The effects of miR-889-3p overexpression on CC progression and radiosensitivity in vitro were mediated by HOXB7. (A) Relative HOXB7 protein level determined by Western blotting in cells transfected with a negative-control plasmid (pcDNA) or a HOXB7-overexpressing plasmid (HOXB7). HeLa and Siha cells were transfected with an miR-NC mimic, an miR-899-3p mimic, an miR-899-3p mimic plus pcDNA, or an miR-899-3p mimic plus HOXB7, followed by the determination of HOXB7 protein level by Western blotting (B), cell proliferation by CCK-8 assay (C and D), analysis of cell colony formation by colony formation assay (E), analysis of cell cycle progression and apoptosis by flow cytometry (F to H), analysis of cell migration and invasion by transwell assay (I to L), determination of cell survival fraction by colony formation upon radiation exposure (M and N), and measurement of the levels of PCNA, c-caspase 3, procaspase 3, and MMP3 by Western blotting (O and P). * , P < 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Simultaneous pharmacologic inhibition of GLS1 and YAP1/TAZ in monocrotaline-exposed rats decreases pulmonary vascular cell proliferation and pulmonary vascular remodeling. ( A ) Representative images of small pulmonary arterioles (0.05). ( C ) The wall thickness of pulmonary arterioles (diameter

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Propidium iodide (PI) 488 assay analysis. Notes: Effect of C 60 on the number (percentage) of ( A ) HS-5, ( B ) HepG2 or ( C ) C3A cells in the cycle phases. ( D ) Relative HS-5, HepG2 and C3A cell population in G2/M cycle (%). ( E ) Western blot analysis of beta-catenin, N-cadherin, vinculin and PCNA. GAPDH was used as a loading control. Abbreviations: C 60 , fullerenes; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 CRISPR/Cas9 editing of GPRC6A decreased prostate cancer tumor growth in xenograft models. Tumor development was monitored over a 28-day period by measuring whole-body luciferase activity. Mice were then sacrificed at day 29. a Editing GPRC6A in PC-3 cells inhibits growth of tumor xenograft in nude mice. PC-3 cells xenograft were detected by in vivo bioluminescence imaging. BLI ratio of intensity (ROI) was shown in right bar graft. b Gross appearance and tumor weight of each group. c GPRC6A, RUNX2 and PCNA protein expression in tumor tissues, analyzed by immunohistochemistry. Bar graphs on the right quantify GPRC6A, RUNX2, and PCNA expression by staining intensity after specific antibody, secondary antibody and histochemical color development. ** Significant difference from control group and stimulated group at p < 0.01 ( n = 3)