Fig. 3: SKP2 depletion induces p27^Kip1-dependent cell cycle arrest reducing growth.

a Representative western blot on cells transfected with Scrambled (siSCR) or SKP2 siRNA (siSKP2). α-TUBULIN is the loading control. b mRNA levels of SKP2, CDKN1A and CDKN1B on cells treated as in (a) were reported as fold increase over siSCR (1 arbitrary unit, not reported). Data presented as mean values ± SD, Student’s two-tailed t-test. c Representative western blot of co-Immunoprecipitation of endogenous SKP2. (*) indicates IgG. d Representative confocal pictures of Proximal Ligation Assay (PLA) for SKP2 and p27^Kip1. Proximity/interactions are visualized as red dots (upper). DAPI (blue) stained nuclei and WGA (green) membrane glycoproteins. p27^Kip1 (middle) or SKP2 (lower) antibodies alone were PLA negative controls. Scale Bar = 10 μm. e Representative flow cytometry cell cycle diagrams of cells treated as in (a). f Histogram depicts fold changes of the percentage of siSKP2-cells in cell cycle phases over siSCR-cells (1 arbitrary unit, not reported). Data presented as mean values ± SD, Student’s two-tailed t-test. g Representative western blot on cells transfected with SCR, SKP2, p27^Kip1 or SKP2 + p27^Kip1 siRNA 72 h post-transfection. Vinculin is the loading control. h Growth curve analysis of cells treated as in (g). Data presented as mean values ± SD, two-way ANOVA (upper siSKP2+sip27^Kip1 vs siSCR, middle siSKP2+sip27^Kip1 vs siSKP2, lower siSKP2 vs siSCR). i Dose-response curves of cells treated with increasing concentration of SMIP004. Data presented as mean values ± SEM. j Representative western blot on cells treated with SMIP004 20 µM for 48 h. Vinculin is the loading control. k mRNA levels of SKP2, CDKN1A and CDKN1B on cells treated as in (j) were reported as fold increase over DMSO (1 arbitrary unit, not reported). Data presented as mean values ± SD, Student’s two-tailed t-test. l Growth curve analysis of cells treated as in (j). Data presented as mean values ± SD, Student’s two-tailed t-test. m Histogram depicts fold changes of the percentage of SMIP004-treated cells in cell cycle phases over DMSO cells (1 arbitrary unit, not reported). Data presented as mean values ± SD, Student’s two-tailed t-test. All the presented data derive from n = 3 independent experiments. Source data are provided as a Source Data file.