Anti-GAPDH antibody - Loading Control

• WB

Supplier Data

Western blot - Anti-GAPDH antibody - Loading Control (AB22555)

Lane 1:

Western blot - Anti-GAPDH antibody - Loading Control (ab22555)

Lanes 2 - 4:

Western blot - Anti-GAPDH antibody - Loading Control (ab22555) at 1/1000 dilution

Lane 1:

HeLa cell lysate at 25 µg

Lane 2:

293 cell lysate at 25 µg

Lane 3:

HUVEC cell lysate at 25 µg

Lane 4:

PC12 cell lysate at 25 µg

Predicted band size: 36 kDa

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• WB

Supplier Data

Western blot - Anti-GAPDH antibody - Loading Control (AB22555)

All lanes:

Western blot - Anti-GAPDH antibody - Loading Control (ab22555) at 1/1000 dilution

Lane 1:

A431 cell lysate at 30 µg

Lane 2:

COS-7 cell lysate at 30 µg

Lane 3:

MDCK cell lysate at 30 µg

Lane 4:

PC-3 cell lysate at 30 µg

Lane 5:

Mouse brain tissue lysate at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L) Superclonal™ HRP conjugate at 1/4000 dilution

Predicted band size: 36 kDa

Observed band size: 37 kDa

false

• WB

CiteAb

Western blot - Anti-GAPDH antibody - Loading Control (AB22555)

Western Blotting using Anti-GAPDH antibody - Loading Control, ab22555. Publication image from Gil, J. et al., 2018, Cancer Cell, 29990503. Legend direct from paper.

Regulation of Alternative Splicing by PTBP1 Controls the SASP(A) Distribution of the five types of AS events detected in senescent cells compared with proliferating cells by RNA-seq (see Figure 4A).(B) PTBP1 RNA binding motifs across alternative exons upon PTBP1 knockdown. Top : scheme. Motifs are mapped to potential regulatory sequences around the target alternatively spliced exon (dark-gray box). The yellow peak represents the area of predicted enrichment of PTBP1 binding responsible for exon splicing repression (red line), with no role known for PTBP1 in exon splicing enhancement (dashed blue line). Middle : motif density for exons with inclusion increasing (putatively repressed, red), decreasing (putatively enhanced, blue), or not altered (not regulated, gray) upon PTBP1 knockdown. Bottom : statistical significance for local motif enrichment in putatively repressed (red) and enhanced (blue) exons.(C) Exon-skipping events and δPSI cutoffs used for shortlisting events changing due to loss of PTBP1. A stricter cutoff was used for events changing upon PTBP1 loss but not affected upon senescence.(D) Strategy to link PTBP1-driven alternative splicing and SASP regulation.(E) Ninety-five PTBP1-spliced genes were targeted with four siRNAs and screened for IL-8 and IL-6 regulators as described in Figure 1. NPI shown as mean of three replicates and cutoffs for hit selection (dotted lines). Hit siRNAs represent siRNAs targeting genes scoring with ≥2 siRNAs in both readouts.(F) Experimental design of (G).(G) IMR90 ER : RAS cells were transfected with AONs either not targeting (NC) or targeting the indicated exons. IF analysis of IL-6 (left) and IL-8 (right). Data represent mean ± SD (n = 4). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Comparisons with NC, si_PTBP1_5 + 4OHT, one-way ANOVA (Dunnett's test).(H and I) Effect of AONs targeting EXOC7 exon 7 splicing on the SASP downregulation caused by PTBP1 knockdown. Timeline as in (F). (H) Immunoblot of protein extracts of IMR90 ER : RAS cells 5 days after 4OHT induction. (I) Representative IF images of IL-8 8 days after 4OHT induction. Scale bar, 100 µm.See also Figure S7 and Tables S2 and S3.

false

• WB

CiteAb

Western blot - Anti-GAPDH antibody - Loading Control (AB22555)

Western Blotting using Anti-GAPDH antibody - Loading Control, ab22555. Publication image from Gil, J. et al., 2018, Cancer Cell, 29990503. Legend direct from paper.

The Splicing Factor PTBP1 Regulates the SASP without Affecting Growth Arrest(A) Immunoblot of protein extracts 6 days after 4OHT induction of IMR90 ER : RAS cells infected with indicated pGIPz shRNA vectors targeting PTBP1. Vec, empty vector.(B) Quantification of cells positive for BrdU incorporation at indicated days after 4OHT treatment. Data represent mean ± SD (n = 3).(C) Crystal violet-stained 6-well dishes of cells fixed 12 days following 4OHT treatment.(D) Quantification of BrdU incorporation 8 days after 4OHT treatment, 15 days after empty vector or PTBP1 shRNA infection. Data represent mean ± SD (n = 3); ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. Comparisons with Vec + 4OHT. One-way ANOVA (Dunnett's test).(E) Quantification of cells positive for the senescence markers p16, p21, p53, and γH2AX 6 days after 4OHT and β-galactosidase 8 days after 4OHT by IF analysis. Data represent mean ± SD (n = 3). ∗∗∗p < 0.001; ns, not significant. Comparisons with Vector + 4OHT, two-way ANOVA (Bonferroni’s test).(F) Expression levels of the indicated SASP genes assessed by qRT-PCR 6 days after 4OHT induction normalized and compared with Vector + 4OHT. Data represent mean ± SD (n = 3); ∗∗∗p < 0.001, two-way ANOVA (Dunnett's test).(G) IMR90 WT cells were infected with indicated pGIPZ empty vector or PTBP1 shRNAs and treated with doxorubicin to induce senescence. Left : IF analysis of the indicated senescence markers 6 days after doxorubicin induction. Right : mRNA analysis of the indicated genes by qRT-PCR (right) 8 days after doxorubicin induction normalized to the Vector + doxycycline (Doxo) condition. Data represent mean ± SD (n = 3). ∗p < 0.05, ∗∗∗p < 0.001; ns, not significant. Comparisons with Vector + Doxo, two-way ANOVA (Dunnett's test).(H) IMR90 ER : RAS cells were transfected with two independent siRNAs targeting PTBP1 at day 5 after senescence induction as indicated in the scheme (left). Senescence establishment at day 6 was monitored by IF analysis (middle). Knockdown of PTBP1 and the effect on the indicated genes was assessed by qRT-PCR 5 days after siRNA transfection, and 10 days after senescence induction (right), normalized to the si_Scramble + 4OHT condition. Data represent mean ± SD (n = 3). ∗∗∗p < 0.001; ns, not significant. Comparisons with si_Scramble + 4OHT, two-way ANOVA (Dunnett's test).See also Figure S3.

false

• WB

CiteAb

Western blot - Anti-GAPDH antibody - Loading Control (AB22555)

Western Blotting using Anti-GAPDH antibody - Loading Control, ab22555. Publication image from Gil, J. et al., 2018, Cancer Cell, 29990503. Legend direct from paper.

PTBP1 Regulates Alternative Splicing of EXOC7 to Control the SASP(A) SASP expression and EXOC7 isoform switching following PTBP1 overexpression in IMR90 cells. Immunoblot of protein extracts (left) and mRNA analysis by qRT-PCR (right) 2 days after induction of PTBP1 expression with doxycycline (Dox). Normalized and compared with Vec − Dox. Data represent mean ± SD (n = 5). ∗∗∗p < 0.001; ns, not significant; one-way ANOVA (Dunnett's test).(B) Comparison of SASP production following overexpression of EXOC7-S (S) and EXOC7-L (L) 4 days after 4OHT and doxycycline treatment of IMR90 ER : RAS cells by immunoblot analysis. v, empty vector.(C) Effect of EXOC7-S on the SASP downregulation caused by PTBP1 knockdown. Representative IF images of IL-8 of IMR90 ER : RAS cells without (−) and with doxycycline treatment (EXOC7 S) 8 days after 4OHT induction. Scale bar, 100 µm.(D and E) Effect of EXOC7 depletion on the SASP (D). Left : experimental design. Right : mean expression (average of the normalized read counts for 3 replicates) in relation to log2(FC) for the indicated comparison. Significantly changing genes are highlighted in red. (E) Correlation between the expression of SASP genes upon PTBP1 and EXOC7 siRNA-mediated knockdown.(F) Comparison of EXOC7-S and EXOC7-L phosphorylation assessed by EXOC7 immunoprecipitation followed by immunoblotting. Experimental details as in (B).(G) Comparison of EXOC7-S and EXOC7-L localization to the plasma membrane in proliferating and senescent cells. Quantification of cells showing the diffuse EXOC7 pattern. Data represent mean ± SD (n = 3). p < 0.01, comparing EXOC7-S + DMSO with either Vector + DMSO or EXOC7-L + DMSO; p < 0.05, comparing EXOC7-S + 4OHT with either Vector + 4OHT or EXOC7-L + 4OHT; two-way ANOVA (Bonferroni’s test). Experimental details as in (B). Scale bar, 100 µm.(H) PTBP1 expression versus EXOC7 exon 7 inclusion in data from the Genotype-Tissue Expression (GTEx) project.(I) Top 11 hallmarks with normalized enrichment score >2 and false discovery rate <0.05 in genes with expression positively correlating with EXOC7 exon 7 skipping in GTEx samples.(J) Effect of EXOC7 knockdown on the immune surveillance response. Top : experimental design. Bottom : quantification of NRAS+ mouse hepatocytes, CXCL5 expression in NRAS+ hepatocytes, and infiltrated MHC II+ and CD3+ cells 6 days after transposon delivery of NRASG12V_shRenilla (n = 5), NRASG12V_shPTBP1 (n = 4), or NRASG12V_EXOC7 (n = 4). Data represent mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Comparisons with NRASG12V_shRenilla, one-way ANOVA (Bonferroni’s test).See also Figure S8.

false

• WB

CiteAb

Western blot - Anti-GAPDH antibody - Loading Control (AB22555)

Western Blotting using Anti-GAPDH antibody - Loading Control, ab22555. Publication image from Gil, J. et al., 2018, Cancer Cell, 29990503. Legend direct from paper.

PTBP1 Regulates Alternative Splicing of EXOC7 to Control the SASP(A) SASP expression and EXOC7 isoform switching following PTBP1 overexpression in IMR90 cells. Immunoblot of protein extracts (left) and mRNA analysis by qRT-PCR (right) 2 days after induction of PTBP1 expression with doxycycline (Dox). Normalized and compared with Vec − Dox. Data represent mean ± SD (n = 5). ∗∗∗p < 0.001; ns, not significant; one-way ANOVA (Dunnett's test).(B) Comparison of SASP production following overexpression of EXOC7-S (S) and EXOC7-L (L) 4 days after 4OHT and doxycycline treatment of IMR90 ER : RAS cells by immunoblot analysis. v, empty vector.(C) Effect of EXOC7-S on the SASP downregulation caused by PTBP1 knockdown. Representative IF images of IL-8 of IMR90 ER : RAS cells without (−) and with doxycycline treatment (EXOC7 S) 8 days after 4OHT induction. Scale bar, 100 µm.(D and E) Effect of EXOC7 depletion on the SASP (D). Left : experimental design. Right : mean expression (average of the normalized read counts for 3 replicates) in relation to log2(FC) for the indicated comparison. Significantly changing genes are highlighted in red. (E) Correlation between the expression of SASP genes upon PTBP1 and EXOC7 siRNA-mediated knockdown.(F) Comparison of EXOC7-S and EXOC7-L phosphorylation assessed by EXOC7 immunoprecipitation followed by immunoblotting. Experimental details as in (B).(G) Comparison of EXOC7-S and EXOC7-L localization to the plasma membrane in proliferating and senescent cells. Quantification of cells showing the diffuse EXOC7 pattern. Data represent mean ± SD (n = 3). p < 0.01, comparing EXOC7-S + DMSO with either Vector + DMSO or EXOC7-L + DMSO; p < 0.05, comparing EXOC7-S + 4OHT with either Vector + 4OHT or EXOC7-L + 4OHT; two-way ANOVA (Bonferroni’s test). Experimental details as in (B). Scale bar, 100 µm.(H) PTBP1 expression versus EXOC7 exon 7 inclusion in data from the Genotype-Tissue Expression (GTEx) project.(I) Top 11 hallmarks with normalized enrichment score >2 and false discovery rate <0.05 in genes with expression positively correlating with EXOC7 exon 7 skipping in GTEx samples.(J) Effect of EXOC7 knockdown on the immune surveillance response. Top : experimental design. Bottom : quantification of NRAS+ mouse hepatocytes, CXCL5 expression in NRAS+ hepatocytes, and infiltrated MHC II+ and CD3+ cells 6 days after transposon delivery of NRASG12V_shRenilla (n = 5), NRASG12V_shPTBP1 (n = 4), or NRASG12V_EXOC7 (n = 4). Data represent mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Comparisons with NRASG12V_shRenilla, one-way ANOVA (Bonferroni’s test).See also Figure S8.

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