33-6400

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of BAX in A549 whole cell lysate (lane 1) and Daudi whole cell lysate (lane 2) using a BAX monoclonal antibody (Product # 33-6400) at a dilution of 1:100. Secondary detection was performed using an HRP-Goat anti-Mouse IgG, IgM (H+L) cross-adsorbed secondary antibody (Product # 31446) at a dilution of 1:5000. Data provided courtesy of Antibody Resource.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of BAX in A549 whole cell lysate (lane 1) and Daudi whole cell lysate (lane 2) using a BAX monoclonal antibody (Product # 33-6400) at a dilution of 1:100. Secondary detection was performed using an HRP-Goat anti-Mouse IgG, IgM (H+L) cross-adsorbed secondary antibody (Product # 31446) at a dilution of 1:5000. Data provided courtesy of Antibody Resource.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of BAX was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR612938_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of BAX was performed by loading 50 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHeLa BAX KO (Lane 3) whole cell extracts. The samples were electrophoresed using Novex™ 16%, Tricine, 1.0 mm, Mini Protein Gel (Product # EC66952BOX). 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-Bax Monoclonal Antibody (2D2) (Product # 33-6400, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to BAX.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Bax Monoclonal Antibody (2D2)(Product # 33-6400) and a 19kDa band corresponding to Bax was observed across cell lines tested. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), MCF7 treated with Etoposide (50uM, 16 Hrs.) (Lane 2), A549 (Lane 3), A549 treated with Etoposide (50uM, 16 Hrs.) (Lane 4) 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 (1:1000 Dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).Expression of BAX was found to be up regulated upon etoposide treatment in A549 and MCF-7 cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Classification of 56 patients by prognosis based on 108 protein expression values. (A) Boxplot of protein expression values before (left) and after (right) quantile normalization. The plot shows the quantile normalized distribution of protein expression values for each patient. Horizontal axis represents individual patients. Vertical axis represents H-score. (B) Heatmap and dendrogram as a result of hierarchical clustering of GBM samples. Top dendrogram represents clustering of patients. Left dendrogram represents clustering of proteins. Of two patient branches, samples in the left branch represent cluster 1 and samples in the right branch represent cluster 2, consisting of 19 and 37 patients, respectively. (C) Univariate survival analysis of overall survival by Kaplan-Meier method. Kaplan-Meier survival plot of the two clusters of patients defined by the hierarchical clustering. Cluster 1 is the poor survival group. The log-rank test shows that the difference between two curves is significant (p

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 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: 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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. Circ_0000285 silence lessens tumorigenic potency both in vitro and in vivo. A. RT-qPCR analysis of circ_0000285 expression in SHG-44 and U251 cells transfected with si-circ_0000285 and si-NC, vs. si-NC, **P < 0.001. B. CCK8 assays assessing the proliferation of SHG-44 and U251 cells upon circ_0000285 silence or not, vs. si-NC, **P < 0.001. C. The quantification of the invasion assay is done by counting 3 different random fields and plotted average cell number per field. D. Western blotting analysis of Bax and Bcl-2 expression in SHG-44 and U251 cells transfected with si-circ_0000285 and si-NC, vs. si-NC, **P < 0.001. E. Subcutaneous tumor growth, xenograft tumor images and tumor weights of xenografts from SHG-44 cells with or without circ_0000285 silence in nude mice, vs. si-NC, **P < 0.001. si-circ, circ_0000285 siRNA; si-NC, si-circ negative control. N = 3, repetition = 3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Cell malignant phenotypes suppressed by circ_0000285 silence is restored upon miR-197-3p inhibitor. SHG-44 and U251 cells were transfected with si-circ_0000285, si-NC, miR-197-3p inhibitor, inhibitor NC, si-circ_0000285+ miR-197-3p inhibitor. 48 h posttransfection. A. RT-qPCR analysis of miR-197-3p expression. B. Cell viability was measured with CCK8. C. Cell invasion was detected by Transwell invasion assay. D. Western blotting analysis of Bax and Bcl-2 expression. Vs.si-NC,**P < 0.001; vs.inhibitor-NC, ++ P < 0.001; vs,si-circ+inhibitor, ## P < 0.001. si-circ, circ_0000285 siRNA; si-NC, si-circ negative control; inhibitor, miR-197-3p inhibitor; inhibitor-NC, inhibitor negative control. N = 3, repetition = 3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7. Silencing CKS1B also compromises the malignant behaviors of glioma cells induced by miR-197-3p inhibitor. SHG-44 and U251 cells were cotransfected with si-CKS1B, si-NC, miR-197-3p inhibitor, inhibitor NC, si-CKS1B+ inhibitor. A. Western blotting analysis of CKS1B expression. B. Cell viability was measured with CCK8. C. Cell invasion was detected by Transwell invasion assay. D. Western blotting analysis of Bax and Bcl-2 expression, vs. si-NC, **P < 0.001; vs. inhibitor-NC, ++ P < 0.001; vs. si-CKS1B+inhibitor, # P < 0.05, ## P < 0.001. si-CKS1B, CKS1B siRNA; si-NC, si-CKS1B negative control; inhibitor, miR-197-3p inhibitor; inhibitor-NC, inhibitor negative control. N = 3, repetition = 3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8. CKS1B overexpression reversed the malignant behavior of glioma cells inhibited by circ_0000285 knockdown. SHG-44 and U251 cells were cotransfected with si-circ, si-NC, si-circ+empty vector, si-circ+OE-CKS1B. A. Western blotting analysis of CKS1B expression. B. Cell viability was measured with CCK8. C. Cell invasion was detected by Transwell invasion assay. D. Western blotting analysis of Bax and Bcl-2 expression, vs. si-NC, *P < 0.05, **P < 0.001; vs. si-circ+empty vector, #P < 0.05, ##P < 0.001. si-circ, circ_0000285 siRNA; si-NC, si-circ negative control; OE-CKS1B, CKS1B overexoression. N = 3, repetition = 3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 5 Apoptosis is produced by activation of P73. A) Impairment of the expression of the anti-apoptotic proteins BCL-2 and XIAP is shown. B ) The decreased expression of the anti-apoptotic proteins was caspase independent, as shown by the use of Z-VAD-FMK pancaspase inhibitor. Samples were treated with 2 pulses of 40 muM of pan-caspase inhibitor, Z-VAD-FMK (R&D, Systems), to 12 and 15 h of curcumin treatment. K562 cells were harvested after 18 h, lysed and total protein extracts were obtained for western blot analysis, using specific antibodies against of Caspase-3, PARP, BCL-2 and XIAP. C ) The diminution of the expression of the antiapoptotic proteins was consistent with decreases in the IkappaBalpha and D ) activation and nuclear translocation of the P73 protein, as consequence of the E) diminished expression of the P73 promoter repressor protein C/EBPalpha; M = medium, D = DMSO.