Figure 3: KPT-6566 interferes with PIN1 oncogenic functions.
From: A covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action
(a) Immunoblotting of PIN1 client proteins expressed in MDA-MB-231 breast cancer cells treated with 5 μM KPT-6566 (+) or DMSO (−) for 48 h. (b) Quantitative RT-PCR analysis of mut-p53 and NOTCH1 target genes (BUB1, DEPDC1 and HEY2, BIRC5, respectively) in MDA-MB-231 cells treated with indicated concentrations of KPT-6566 or DMSO (−) for 48 h. (c) Left, representative pictures of MDA-MB-231 colonies in the indicated experimental conditions. Right, histogram showing colony formation efficiency of MDA-MB-231 cells in the indicated experimental conditions. Cells were transfected with control siRNA (siRNA Ctrl) or with PIN1 siRNA#1. After 24 h cells were trypsinized, plated for colony forming assay and treated with 5 μM PiB, 1 μM KPT-6566 or DMSO every two days. Colonies ≥50 pixels were counted 10 days after seeding using ImageJ software. (d) Histogram showing invasive ability and proliferation (cell number) of MDA-MB-231 breast cancer cells plated in Matrigel-coated Boyden chambers in the indicated experimental conditions for 20 h. (e) Histogram showing percentage of secondary mammosphere formation efficiency (%M2FE) of MDA-MB-231 cells in the indicated experimental conditions. (f) Immunoblotting of the indicated proteins expressed in MDA-MB-231 breast cancer cells treated with 5 μM KPT-6566 (+) or DMSO (−) for 48 h. (g) %M2FE of MCF10AT1 cells with stable control- (shRNA Ctrl) or PIN1 silencing (shRNA PIN1), treated as indicated. (h) %M2FE of MCF10A cells transduced with empty- (Vec) or HA-PIN1-expressing vectors (PIN1), treated as indicated. (a,f) Actin levels are reported as loading control; size markers are indicated. Data shown in b–e,g,h are the means±s.d. of n=3 independent experiments, *P<0.05, **P<0.01, n.s. not significant; two-tailed Student’s t-test.
