MA5-11757

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of BCL-2 using monoclonal Antibody (Product # MA5-11757) on Human Lymphoma Tissue.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of BCL2 was performed with 10 µg of HeLa cells transfected with Transfection Reagent alone (Lane 1), 100nM Non-Targeting control siRNA (Lane 2), or 100nM siRNA against BCL2 (Lane 3). Proteins were resolved using a NuPAGE® Novex 4-12% Bis-Tris Gel (Product # NP0322BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), and a protein size ladder. Proteins were wet transferred to a Pierce Nitrocellulose Membrane (Product # 88025) OR Pierce PVDF Membrane (Product # 88518) and blocked with Pierce Starting Block T20 (PBS) Blocking Buffer (Product # 37539) for 1 hour at room temperature. BCL2 was detected at ~ 26 kDa using BCL2 Mouse monoclonal antibody (Product # MA5-11757) diluted in Pierce Starting Block T20 (PBS) Blocking Buffer 4°C overnight on a rocking platform. Pierce Goat Anti-Mouse (Product # 31437) HRP-Conjugated Antibodies at a 1:2500 dilution were used and chemiluminescent detection was performed using Pierce Supersignal West Dura Maximum Sensitivity Substrate (Product # 37071). Relative density of the bands normalized to GAPDH (36 kDa). BCL2 Antibody (Product # MA5-11757) confirms silencing of BCL2 expression.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of Bcl-2 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR946648_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Bcl-2 was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa CAS9 (Lane 2) and HeLa Bcl-2 KO (Lane 3) whole cell extracts. The samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Bcl-2 Monoclonal Antibody (100/D5) (Product # MA5-11757) using 1 µg/mL dilution and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Bcl-2. An uncharacterized band was observed at ~38 kDa in HeLa wild type and HeLa CAS9 samples.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of BCL-2 alpha (green) showing staining in the cytoplasm of A431 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a BCL-2 alpha monoclonal antibody (Product # MA5-11757) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of BCL-2 alpha (green) showing staining in the nucleus and cytoplasm of Hela cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a BCL-2 alpha monoclonal antibody (Product # MA5-11757) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of BCL-2 alpha (green) showing staining in the nucleus and cytoplasm of NIH-3T3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a BCL-2 alpha monoclonal antibody (Product # MA5-11757) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. TPG regulates apoptosis-associated factors in H9C2 cells. H9C2 cells were treated with TPG (10, 40 and 160 ug/ml) for 24 h and subjected to I/R injury. (A) Relative mRNA expression levels of caspase-3, PARP1, Bax and Bcl-2 were investigated by reverse transcription-quantitative polymerase chain reaction. (B) Relative protein expression levels of caspase-3, PARP1, Bax and Bcl-2 were evaluated by western blotting and normalized to beta-actin expression. (C) Blot results were semi-quantified. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 NuBCP-9 suppresses H460 paclitaxel resistant lung cancer cell growth in a xenograft zebrafish model (A) Effect of NuBCP-9, NuBCP-9/AA (inactive form) on H460 parental and paclitaxel resistant lung cancer cells after 72 hours of treatment in 1% serum. Two-way ANOVA with Dunnett's multi comparisons post-test, **** =P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 5 NEK6 inhibits apoptosis through the activation of STAT3. (A) Immunoblot analysis of the phosphorylated STAT1 (p-STAT1), STAT1, phosphorylated STAT3 (p-STAT3), and STAT3 in RAW 264.7 cells stimulated with 10 mug/mL of gamma-irradiated M. tuberculosis . (B) Immunoblot analysis of the p-STAT1, STAT1, p-STAT3, and STAT3 in RAW 264.7 cells and BMDMs transfected with Myc-NEK6. (C) BMDMs from WT and NEK6-deficient ( Nek6 -/- ) mice were stimulated with 10 mug/mL of gamma-irradiated M. tuberculosis for 24 h. The expressions of p-STAT1, STAT1, p-STAT3, STAT3, and NEK6 were analyzed by Western blotting. (D) Immunoblot analysis of the p-STAT3 and STAT3 in lungs, spleens, and livers from WT and Nek6 -/- mice at 7 dpi. (E and G) The expression levels of BCL-2, BCL-X L , BCL-W, and MCL-1 in 10 mug/mL of gamma-irradiated M. tuberculosis -stimulated BMDMs from WT and Nek6 -/- mice were calculated by qRT-PCR at the indicated times (E) and by Western blotting (G). (F and H) BMDMs from Nek6 -/- mice were pretreated with 20 ng/mL IL-6 for 4 h h, then cells were stimulated with 10 mug/mL of gamma-irradiated M. tuberculosis . The expression levels of BCL-2, BCL-X L , BCL-W, and MCL-1 were calculated by qRT-PCR (F) and Western blotting (H) at the indicated times. (I) BMDMs from WT and Nek6 -/- mice were stimulated with 10 mug/mL of gamma-irradiated M. tuberculosis for 24 h, and the apoptosis rates were detected by flow cytometry. (J) BMDMs from Nek6 -/- mice were pretreated with 20 ng/mL of I

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 MiR-203-3p and Sema3A regulated the apoptosis of HG-induced podocytes. HG-induced podocytes were transfected with miR-con, miR-203-3p, miR-203-3p + pcDNA or miR-203-3p + Sema3A. (a) Podocyte apoptosis was evaluated using flow cytometry. (b) The activity of caspase-3 was measured by the Caspase-3 Activity Detection Kit. (c and d) The WB analysis determined the protein levels of apoptosis-associated markers Bax and Bcl-2. * P < 0.05.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 2 Circ_CLASP2 overexpression promoted proliferation and inhibited apoptosis in HUVECs under HG conditions. (A) Circ_CLASP2 expression was assessed by qRT-PCR in HUVECs transfected with Vector or circ_CLASP2. HUVECs were transfected with Vector or circ_CLASP2 and then treated with HG, followed by the measurement of cell colony formation using a colony formation assay (B) , cell proliferation by MTT assay (C) , Ki67 level by Western blot (D) , cell apoptosis by flow cytometry (E) , Bcl-2, Bax and Cleaved PARP levels (F), and VEGF expression (G) by Western blot. Vector: negative control vector, circ_CLASP2: circ_CLASP2 overexpression vector. n = 3 independent biological replicates; data were presented as mean +- SD; * p < 0.05 by a two-tailed Student''s t -test or ANOVA followed by Tukey-Kramer post hoc test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 4 Overexpression of circ_CLASP2 regulated proliferation and apoptosis of HG-treated HUVECs by miR-140-5p. (A) MiR-140-5p expression was tested by qRT-PCR in HUVECs transfected with Vector, circ_CLASP2, circ_CLASP2+miR-NC mimic, or circ_CLASP2+miR-140-5p mimic. HUVECs were transfected with Vector, circ_CLASP2, circ_CLASP2+miR-NC mimic, or circ_CLASP2+miR-140-5p mimic before HG treatment, followed by the determination of cell colony formation using a colony formation assay (B) , cell proliferation by MTT assay (C) , Ki67 level by Western blot (D) , cell apoptosis by flow cytometry (E) , Bcl-2, Bax and Cleaved PARP levels (F) , and VEGF expression (G) by Western blot. Vector: negative control vector, circ_CLASP2: circ_CLASP2 overexpression vector. n = 3 independent biological replicates; data were presented as mean +- SD; * p < 0.05 by ANOVA followed by Tukey-Kramer post hoc test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 6 MiR-140-5p regulated HUVEC proliferation and apoptosis under HG conditions by FBXW7. (A,B) FBXW7 mRNA and protein levels were measured by qRT-PCR or Western blot assay in HUVECs transfected with anti-miR-NC, anti-miR-140-5p, anti-miR-140-5p + si-NC, or anti-miR-140-5p + si-FBXW7. HUVECs were transfected with anti-miR-NC, anti-miR-140-5p, anti-miR-140-5p + si-NC, or anti-miR-140-5p + si-FBXW7 before HG treatment, followed by the determination of cell colony formation using a colony formation assay (C) , cell proliferation by MTT assay (D) , Ki67 level by Western blot (E) , cell apoptosis by flow cytometry (F) , Bcl-2, Bax and Cleaved PARP levels (G) , and VEGF expression (H) by Western blot. * p < 0.05. n = 3 independent biological replicates; data were presented as mean +- SD; * p < 0.05 by ANOVA followed by Tukey-Kramer post hoc test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Overexpression of circ_0000629 inhibits the growth of BC cells in vitro. Overexpression plasmid of circ_0000629 was transfected into T24 and SW780 cells. ( A ) circ_0000629 expression in the cells examined by RT-qPCR; ( B ) CCK-8 assay to detect the activity of T24 and SW780 cells; ( C ) EdU staining for proliferative capacity of T24 and SW780 cells; ( D ) flow cytometric analysis of apoptotic T24 and SW780 cells; ( E ) Caspase-3/7 kits detection of caspase-3/7 activity in T24 and SW780 cells; ( F ) expression of apoptosis-related proteins PUMA, Bax, and Bcl-2 in cells examined by Western blot. Date are mean +- SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA and Tukey''s multiple comparison test).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 miR-1290 mimic promotes the growth and aggressiveness of BC cells in vitro. T24 and SW780 cells were co-transfected with oe-circ_0000629 + miR-1290 mimic or oe-circ_0000629 + mimic NC. ( A ) expression of miR-1290 in cells after co-transfection by RT-qPCR; ( B ) EdU staining for proliferative capacity of T24 and SW780 cells; ( C ) flow cytometric analysis of apoptotic T24 and SW780 cells; ( D ) expression of apoptosis-related proteins PUMA, Bax, and Bcl-2 in cells examined by Western blot; ( E ) the migration capacity of T24 and SW780 cells examined by Transwell assay; ( F ) the invasion capacity of T24 and SW780 cells examined by Transwell assay. Date are mean +- SD, n = 3. ** p < 0.01 (two-way ANOVA and Tukey''s multiple comparison test).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8 Knockdown of CDC73 attenuates the inhibitory effect of oe-circ_0000629 on BC cells. T24 and SW780 cells were co-transfected with oe-circ_0000629 + shScr, oe-circ_0000629 + shCDC73-#1 or oe-circ_0000629 + shCDC73-#2. ( A ) mRNA expression of CDC73 in cells after co-transfection by RT-qPCR; ( B ) protein expression of CDC73 in cells after co-transfection by Western blot; ( C ) EdU staining for proliferative capacity of T24 and SW780 cells; ( D ) flow cytometric analysis of apoptotic T24 and SW780 cells; ( E ) expression of apoptosis-related proteins PUMA, Bax, and Bcl-2 in cells examined by Western blot; ( F ) the migration capacity of T24 and SW780 cells examined by Transwell assay; ( G ) the invasion capacity of T24 and SW780 cells examined by Transwell assay. Date are mean +- SD, n = 3. ** p < 0.01 (two-way ANOVA and Tukey''s multiple comparison test).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 5 Representative photomicrographs of immunohistochemical expression of BCL-2 in cardiac tissue of rats (magnification x400). (A) Normal control, (B) valsartan (VAL) 5 mg/kg/day, (C) amlodipine (ADP) 0.5 mg/kg/day, (D) lisinopril (LSP) 0.035 mg/kg/day, (E) trastuzumab ( TZM ) 2.25 mg/kg/day, (F) TZM + valsartan 5 mg/kg/day, (G) TZM + amlodipine 0.25 mg/kg/day, (H) TZM + lisinopril 0.035 mg/kg/day, (I) TZM + amlodipine + lisinopril, (J) TZM + valsartan + lisinopril, and (K) intensity score of BCL-2 expression, mean +- SEM ( n = 3), and significant difference denoted by # p < 0.05 vs. normal control or * p < 0.05 vs . TZM by one-way ANOVA followed by Turkey''s post hoc test.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 TP73-AS1 knockdown inhibits tumor growth and metastasis in nude mice. A: Images of resected tumors from nude mice from the indicated groups. n = 5 mice per group; B: Tumor growth curves established by measuring tumor volume after injection at the indicated time points; C: Weights of resected tumors from nude mice from the indicated groups; D and E: Quantitative real-time polymerase chain reaction to detect the expression of TP73-AS1 (D) and miR-128-3p (E) in xenograft tumor tissues; F: Ki67 expression in resected tumor tissues evaluated by immunohistochemistry analysis; G: Representative images and quantification of hematoxylin and eosin staining of lungs isolated from mice; H: Apoptosis markers detected by Western blot. b P < 0.01. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 3 Desoxyrhapontigenin ameliorates neuronal apoptosis in the brain tissues of rats with anaesthesia-induced neuronal injury. a TUNEL-positive cells; b Protein expression of caspase-2, Bcl-2, and Bax by Western blotting. Mean +- SEM (n = 15); ## p < 0.01 vs. controls; ** p < 0.01 vs. ISF group

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. Pristimerin ameliorates neuronal cell apoptosis in brain tissue homogenates of mice with sepsis-induced brain injuries. (A) Effects of pristimerin on expression levels of Bax, Bcl-2 and caspase-3, as revealed by Western blotting. (B) Effect of pristimerin on the apoptosis index, as revealed by the TUNEL assay. TUNEL: terminal deoxynucleotidyl transferase dUTP nick end-labelling. Means +- SEMs ( n = 10); ## p < 0.01 compared with positive controls; ** p < 0.01 compared with negative controls.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Overexpression of ASAP1-IT1 blocked miR-509-3p-mediated cell functions in cancer stem cells both in vitro and in vivo . A Number of A549 cell tumor spheres after expression of Mock, pcDNA, miR-509-3p, and ASAP1-IT1, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA, F p < 0.01 compared with miR-509-3p + ASAP1-IT1. B qRT-PCR analysis on stemness-related genes (SOX2, CD44, and CD133) in A549 stem cells, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01compared with miR-509-3p + pcDNA. C Annexin V-FITC/PI staining of A549 stem cells with Mock, pcDNA, miR-509-3p, and ASAP1-IT1, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. D Western blotting analysis on apoptosis-associated genes (Bax, Bcl-2, and cleaved-caspase-3) in A549 stem cells, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. E Colony formation assay on cell growth after 15 days culture, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. F qRT-PCR analysis of growth-associated genes (Cyclin A1, Cyclin B1, and PCNA) in A549 stem cells, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. G Tumor volume in nude mice at the indicated time after inoculation, * p < 0.01 compared with Mock + pcDNA, phi p < 0.01 compared with miR-509-3p + pcDNA. H Photo of harvested tumors at the 24th day after inoculation. I Tumor weight at the 24th day after

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. ASMTL-AS1 downregulation inhibits HCC proliferation and migration in vitro. a. qRT-PCR analysis of ASMTL-AS1 expression in HCC cell lines and THLE2 cells. b. qRT-PCR analysis of ASMTL-AS1 expression in HCC tissues and normal tissues. c. ASMTL-AS1 mainly located in cytoplasm. d. The effects of transfection of siASMTL-AS1 and si-NC in HCC cell lines (HCCLM3 and Huh7 cells) were detected by PCR analysis. e. CCK-8 assay was performed to test the cell viability and proliferation in downregulated CASMTL-AS1 groups (si-ASMTL-AS1) and negative control group (si-NC). f. Western blotting analysis was performed to detect the Bax and Bcl-2 expression in HCCLM3 and Huh7 cells. G. Transwell assay was performed the migrated and invaded abilities in HCC cell lines. * P < 0.05, ** P < 0.001, vs. Si-NC. H. ASMTL-AS1 downregulation inhibited HCC tumor growth in vivo. ** P < 0.001, vs. Sh-NC.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4. MiR-1343-3p inhibitor abrogates the deceleration of HCC cell proliferation and migration caused by ASMTL-AS1 silence. HCCLM3 and Huh cells transfected with si-NC, si-ASMTL-AS1, miR-1343-3p inhibitor, inhibitor NC, si-ASMTL-AS1+ miR-1343-3p inhibitor. a. Proliferation test by CCK8 assay. b. Western blot detection of Bcl-2 and Bax. d. Migration was assessed using transwell migration assays. * P < 0.05,** P < 0.001,vs.Si-NC; #P < 0.05, ## P < 0.001, vs.inhibitor-NC; DeltaP

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. LAMC1 silence attenuates the accelerated proliferation and migration of HCC cells caused by miR-1343-3p inhibitor. HCCLM3 and Huh7 were transfected with si-NC, si-LAMC1, miR-1343-3p inhibitor, inhibitor NC and si-LAMC1+ miR-1343-3p inhibitor. After 48 h transfection, a. CCK8 to detect cell proliferation change. b. Western blot and Western blot analysis of Bax and Bcl-2 protein expression. c. Migration was assessed using transwell migration assays. *P < 0.05, **P < 0.001, vs.Si-NC; # P < 0.05, ## P < 0.001, vs. inhibitor-NC; DeltaP

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 2 FIGURE Circ_0001946 expression affects cell proliferation, invasion, migration and apoptosis in vitro. (a-h) ECA109 and KYSE450 cells were introduced with vector control or a recombinant plasmid expressing circ_0001946 (oe-circ_0001946). (a) qRT-PCR validating the circ_0001946 upregulation efficacy of oe-circ_0001946 transfection in ECA109 and KYSE450 cells. (b) CCK-8 assay with transfected ECA109 and KYSE450 cells to assess cell proliferation. (c) Representative EDU assay showing cell proliferation ability performed in transfected ECA109 and KYSE450 cells. (d) Representative images showing a cell apoptosis assay and flow cytometry with transfected ECA109 and KYSE450 cells. (e) Representative immunoblotting analysis showing the levels of Bax and Bcl-2 in transfected ECA109 and KYSE450 cells. (f and g) Representative transwell assay presenting cell migration and invasion rates performed in transfected ECA109 and KYSE450 cells. (h) Representative pictures depicting a cell migration assay performed by wound-healing assay with transfected ECA109 and KYSE450 cells. * p < 0.05

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 4 FIGURE Reduction of miR-1290 underlies circ_0001946-dependent phenotypes in vitro. (a) qRT-PCR revealing the miR-1290 increase efficacy of miRNA mimic introduction in ECA109 and KYSE450 cells. (b) qRT-PCR for miR-1290 expression in circ_0001946-expressing or control ECA109 and KYSE450 cells transfected with or without miR-1290 mimic or mimic NC. (c) Proliferation analysis by CCK-8 assay with circ_0001946-expressing or control ECA109 and KYSE450 cells transfected as indicated. (d) Proliferation analysis by EDU assay with circ_0001946-expressing or control ECA109 and KYSE450 cells transfected as indicated. (e) Apoptosis assessment by flow cytometry with circ_0001946-expressing or control ECA109 and KYSE450 cells transfected as indicated. (f) Representative immunoblotting analysis showing Bax and Bcl-2 levels in circ_0001946-expressing or control ECA109 and KYSE450 cells transfected as indicated. (g and h) Representative migration (g) and invasion (h) analyses by transwell assay with circ_0001946-expressing or control ECA109 and KYSE450 cells transfected as indicated. (i) Migration evaluation by wound-healing assay with circ_0001946-expressing or control ECA109 and KYSE450 cells transfected as indicated. * p < 0.05

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 6 FIGURE SOX6 is a downstream effector of miR-1290. (a) qRT-PCR showing the miR-1290 downregulation efficacy of miR-1290 inhibitor transfection. (b) Representative immunoblotting analysis revealing the transfection efficiency of si-SOX6 in suppressing SOX6. (c-j) ECA109 and KYSE450 cells were transiently introduced with si-SOX6 + miR-1290 inhibitor, si-con+miR-1290 inhibitor, miR-1290 inhibitor or inhibitor NC. (c) Representative immunoblotting analysis showing SOX6 protein level in transfected ECA109 and KYSE450 cells. (d) Proliferation analysis by CCK-8 assay with transfected ECA109 and KYSE450 cells. (e) Proliferation analysis by EDU assay with transfected ECA109 and KYSE450 cells. (f) Apoptosis assessment by flow cytometry with transfected ECA109 and KYSE450 cells. (g) Representative immunoblotting analysis showing Bax and Bcl-2 levels in transfected ECA109 and KYSE450 cells. (h and i) Representative migration (h) and invasion (i) analyses by transwell assay with transfected ECA109 and KYSE450 cells. (j) Migration evaluation by wound-healing assay with transfected ECA109 and KYSE450 cells. * p < 0.05

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 7 FIGURE Circ_0001946 affects tumor growth in vivo. (a-e) ECA109 cells were infected by circ_0001946-expressing lentivirus (lenti-circ_0001946) or control lentivirus (vector). 5 x 10 6 infected cells were hypodermically implanted into the left flanks of the BALB/c nude mice ( n = 5 per group). (a) Growth curves of the xenografts. Tumor growth was monitored for 4 weeks. (b) Images and average weight of the xenografts. (c) qRT-PCR showing the expression of circ_0001946, miR-1290 and SOX6 mRNA in the xenografts. (d) Representative immunoblotting analysis presenting SOX6 protein level in the xenografts. (e) Representative immunohistochemistry images showing ki-67, Bax, Bcl-2, MMP2, and MMP9 staining of sections of the xenografts. * p < 0.05

Supportive validation

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Experimental details: Immunocytochemical staining of cortical cell culture with antibodies against BCL-2 ( A , B ) and BAX ( D , E ), regulators of apoptosis, after 24-h incubation with 3 mug/mL of different-sized SeNPs without OGD/R ( A , D ) and 24 h after OGD/R ( B , E ). ( C , F )--Intensity levels of BCL-2 ( C ) and BAX ( F ) were determined by confocal imaging. We analyzed individual cells that had fluorescence of secondary antibodies. The quantative data reflecting the level of BCL-2 or BAX expression are presented as fluorescence intensity values in summary bar charts (mean +- SEM). The values were averaged by 150 cells for each column. The results obtained after immunostaining agree with the data of fluorescence presented in panels ( A , B , D , E ). Each value is the mean +- SE ( n >= 3, p < 0.05). Statistical significance was assessed using paired t -test. Black asterisks represent comparison of baseline expression levels after incubation with different sized nanoparticles versus control. Red asterisks represent comparison of expression level after incubation with different-sized SeNPs versus OGD/R-induced levels; n/s--data not significant ( p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001.

Supportive validation

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Experimental details: Figure 2 NSC-exosomes attenuates H 2 O 2 -induced neuronal apoptosis by activating autophagy in vitro. Cell apoptosis was examined by TUNEL staining (A), flow cytometry assay (B), and western blot of apoptosis markers (C). The autophagy flux was examined by immunofluorescence staining (D) and western blot of autophagy flux markers (E). The band intensities were analyzed using the gray values in Image J software.

Supportive validation

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Experimental details: Figure 7 Simvastatin treatment alters the expression of anti-apoptotic proteins in the Bcl-2 family and is cell line-specific. Daoy ( A , D - F ), D283 ( B , G - I ), and D341 ( C , J - L ) cells were treated with 10 uM simvastatin and cells were collected every 24 h until 72 h, at which point Western blots for the anti-apoptotic proteins were performed. The anti-apoptotic proteins McL-1 ( A - D , G , J ), Bcl-xl ( A - E , H , K ), and Bcl-2 ( A - C , F , I , L ) were probed. Western blots were done to measure the expression of these anti-apoptotic proteins. GAPDH was used as the loading control. Western blot signals showing the expression of these pro-apoptotic proteins were quantified by ImageJ and Excel. Western blot signals were first normalized to the control at 24 h and these values were then normalized to their own GAPDH, which had also been normalized to its own control at 24 h. The simvastatin treatment values were then compared to their respective time-point control values and presented as a percentage. Data was collected in three independent biological experiments ( n = 3) and expressed as means +- SEM. Statistical differences are evaluated by one-way ANOVA, using GraphPad Prism 6.0. p -value is reported as *** p < 0.001, ** p < 0.01, or * p < 0.05. For the continuous full length-image of Western blot signals in Figure 7 A-C, please refer to Supplementary Figure S3 .

Supportive validation

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Experimental details: Figure 1 Expression of connexin 43 (Cx43) and B-cell lymphoma-2 (Bcl-2) in head and neck squamous cell carcinoma (HNSCC) cell lines. ( A ) Cells were subjected to western blot analysis with antibodies against Cx43, Bcl-2 and the loading control, alpha-tubulin. ( B ) Densitometry analysis of Cx43 and Bcl-2 protein expression in Detroit 562 (metastatic pharyngeal carcinoma), FaDu (hypopharynx squamous cell carcinoma) and SCC25 (tongue squamous cell carcinoma) cells. Quantitative PCR (qPCR) analysis of Cx43 and Bcl-2 mRNA expression in HNSCC cell lines. Densitometry analysis and qPCR analysis show the results of three independent experiments. The expressions of all proteins and mRNAs were normalized to the expression of alpha-tubulin. Data are presented as mean +- SD (standard deviation). Statistical analysis was performed by Student's t -test, the Cx43 and Bcl-2 expression of the cell lines were compared to each other. * p < 0.05 ( C ) Representative immunofluorescence images of Cx43 and Bcl-2 expression in Detroit 562, FaDu and SCC25 cell lines. Cx43 and Bcl-2 were marked with Alexa Fluor 488 (green), nuclei were stained with DRAQ5 (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Silencing of HSPB1 increases the radiosensitivity of non-small cell lung carcinoma cells by regulating the expression of cell cycle- and apoptosis-associated proteins. Representative western blot and protein expression of cyclin B1, cyclin G1, Bax, Bcl-2, pro-caspase-3 and cleaved caspase-3 in A549 cells following irradiation with 0 or 6 Gy, and transfection with control, NC or si-HSPB1. Data are presented as the mean +- standard deviation. # P