Fig. 6

USP11-mediated cell density-dependent PTEN regulation. a Representative images of sparse- and dense-confluent primary MEFs (left). Immunoblotting (IB) of sparse- and dense-confluent primary (middle) or transformed MEFs (right). Scale bars, 50μm. b Total RNAs from (a) were subjected to RT-qPCR. n = 3. c Lysates from sparse- and dense-confluent MEFs treated with cycloheximide (CHX, 100 μg ml⁻¹) for the indicated times were subjected to IB (top). PTEN protein levels were quantified by normalizing to the intensity of the actin band (bottom). n = 3, p value was determined by ANOVA. d Lysates from sparse- and dense-confluent MEFs treated with 10 μM MG132 for 4 h were immunoprecipitated (IP) with anti-PTEN, and the resulting IP were subjected to IB. # indicates the heavy chain of IgG. e, f Lysates and total RNAs from sparse- and dense-confluent MEFs were subjected to IB for the indicated proteins (e) and RT-qPCR (f). n = 3. g Lysates from sparse- and dense-confluent MEFs expressing Usp11 shRNA were subjected to IB. h Lysates from sparse- and dense-confluent MEFs expressing two independent Foxo1 shRNAs were subjected to IB. i Luciferase reporter analysis of the USP11 promoter in NIH-3T3 cells expressing Foxo1 shRNAs. n = 3. j Luciferase reporter analysis of two potent FOXO-binding site mutants (Mut) of the USP11 promoter in NIH-3T3 cells expressing FOXO1. n = 3. k Chromatin levels of FOXO1 at the proximal promoters of mouse Usp11 were compared between sparse- and dense-confluent MEFs by quantitative ChIP assays. Enrichment of FOXO1 was analyzed with respect to the input control (before IP) and normalized to IgG control. TSS, transcription start site. n = 3. l Lysates from sparse- and dense-confluent Foxo1^+/+ and Foxo1^-/- MEFs were subjected to IB. Error bars represent  ±  SEM. p Value was determined by Student’s t test (n.s., non-significant; *p < 0.05, **p < 0.01, ***p < 0.001). ChIP, chromatin immunoprecipitation; MEFs, mouse embryonic fibroblasts; RT-qPCR, quantitative reverse transcription PCR