Extended Data Fig. 9. SIRT1 inhibition rescues the DSCC1 cohesion defect independent of p53 and not via direct SMC3 deacetylation.
a, Representative images of immunoblots showing the effect of increasing concentrations of SIRT1 inhibitor on the p53-K382 acetylation levels in the RPE-1 DSCC1Δ/flox CREtam cell line upon gamma irradiation in the presence of the HDAC1 inhibitor, vorinostat. Uncropped western images are presented in Supplementary Fig. 2. This experiment was performed once. b, DSCC1 mRNA quantification by RT-qPCR in the RPE-1 DSCC1Δ/flox CREtam cell line after the indicated treatments show that DSCC1 can be depleted by the addition of 100 nM 4-OHT. Note that SIRT1 inhibition (SIRT1i) does not significantly affect DSCC1 levels. n = 3 independent experiments/biological replicates with n = 5 technical replicates each. Bars represent mean with s.d. Significance was assessed by a Student’s two-tailed t-test (NS, not significant; P > 0.05). c, Independent experiment in the RPE-1 DSCC1Δ/flox CREtam cell line in the presence of SIRT1 inhibitor upon DSCC1 depletion by 4-OHT treatment shows that SIRT1i can significantly rescue cell viability. Significance calculated using a two-tailed Student’s t-test. The experiment was repeated three independent times (biological replicates) with three technical replicates each. Mean is plotted with the error bars denoting the s.d. d, Quantification of the western blots for which representative images are presented in Fig. 4d showing SMC3 acetylation at K105 is significantly restored in the DSCC1Δ/flox CREtam cells upon SIRT1i. Statistical analysis was performed using a two-tailed Student’s t-test; bars represent mean with s.d. The experiment was performed three times independently. e, Representative immunoprecipitation (IP) followed by immunoblotting from a SIRT1 in vitro deacetylation assay performed by using recombinant SIRT1 protein (rSIRT1). The rSIRT1 can deacetylate p53 at K382 (upper panels) but cannot deacetylate SMC3 even in absence of HDAC8 (lower panels). n = 3 independent repeats (biological replicates). f, On the left, representative images of metaphase chromosomes from three independent experiments/biological replicates illustrating normal, railroad (RR) chromosomes as well as chromosomes with premature sister chromatid separation (PCS) in different stages from TERT-RPE-1-p53 KO cells as compared to TERT-RPE-1 p53 KO DSCC1 KO with and without SIRT1i. Size bar 5 µm. Below is represented the timeline for the experimental setup. On the right, quantification of the different RR and PCS events in the metaphasis from RPE-1 p53 KO vs. RPE-1 p53 KO DSCC1 KO with and without SIRT1i. The experiment was repeated n = 3 independent times (biological replicates). More than 50 metaphases/genotype were analysed. Statistical analysis comparing the proportions of normal cell metaphases and cell-defect metaphases was performed with a logistic regression model90; NS, not significant; P > 0.05.