Fig 2: USP22 directly deubiquitylates CCND1 protein independent of phosphorylation at T286. (A) H1299 cell lysates subjected to an endogenous CCND1 IP under nondenaturing conditions using A/G beads. Precipitates probed for CCND1 to detect high molecular-weight species, presumably ubiquitylated CCND1. (B) Sf9 cells were infected with baculovirus-expressing Flag-Fbxo4, Flag-cyclin D1, CDK4, HA-α-B-crystallin, HA-Cul1, HA-Skp1, and HA-Rbx1. SCFFbxo4 complex and CCND1/CDK4 complex then purified using anti-FLAG M2 and combined with E1/E2 ligases, ATP, and ubiquitin for 30 min at 37 °C to ubiquitylate CCND1 protein. Postubiquitylation assay, the CCND1/CDK4 complex was isolated and incubated with or without USP22 at 37 °C for indicated times in deubiquitylation buffer. Protein levels detected by IB; high molecular-weight CCND1 species indicative of polyubiquitylated CCND1. (C) HCT116 cell lysates subjected to in vitro analysis of CCND1 ubiquitylation status following USP22 depletion using UbiTest, as described in the Materials and Methods. Cells treated with MG132 before harvest and lysates generated using buffer containing protease mixture inhibitor, pan DUB inhibitor PR619, and the conventional chelator o-phenanthroline. Eluates then either left undigested (lanes 1 and 2) or subsequently digested with a global-DUB to strip polyubiquitin (lanes 3 and 4). Protein levels were detected by IB. The black arrowheads indicate unit length CCND1 (nonubiquitylated) and the green arrowheads indicate high molecular-weight species presumably representing ubiquitylated CCND1. (D) Schematic representing UbiTest experimental design from C. (E) H1299 cells made to express HA-tagged CCND1 WT or mutant with lysines from K33-K123. USP22 depleted in cells with shRNA-mediated lentivirus and CCND1 protein levels were assessed via anti-HA by IB. (F) HA-tagged CCND1 was expressed in H1299 cells as either WT or with phosphorylation site mutation T286A. Following USP22 depletion with two distinct shRNA constructs, cells were lysed and CCND1 protein levels were assessed by IB. (G) H1299 cells expressing the WT or T286A isoforms of HA-tagged CCND1 were subjected to USP22 depletion and subsequent analysis by IB in the presence or absence of proteasome inhibition (EPX). (H) Phosphorylation-dependence of USP22-depletion induced CCND1 degradation was assessed by inhibition of the T286 kinase GSK3β via treatment with TSW-119. Following depletion of USP22, CCND1 protein levels were assessed by IB. The established GSK3β pathway substrate β-catenin was included as control for TSW119 efficacy.

Fig 3: USP22 protects CCND1 protein from proteasome-mediated degradation. (A) CCND1 protein levels assessed in the presence of the proteasome inhibitors MG132 or EPX or the Calpain I inhibitor ALLN in H1299 cells. (B) USP22 depletion in H1299, MCF7, and PC3 cells accomplished using two distinct shRNA constructs. CCND1 protein levels assessed in the presence or absence of the proteasome inhibitor EPX. (C) The effect of USP22 depletion on the levels of ectopically expressed CCND1 assessed in H1299 cells. The impact of proteasome inhibition examined following MG132 treatment. (D) CCND1 protein half-life evaluated by treatment of cells with CHX for the indicated times. (E) CCND1 protein levels quantified for three CHX time-course experiments, followed by normalization to actin. Initial CCND1 levels set to 100%. (F) Bar graph representing the average and SD of CCND1 protein half-life for the experiments plotted in E. Error bars indicate SD based on three independent experiments. **P < 0.02.

Fig 4: miR-195 inhibits cell proliferation by reducing CDC42 and CCND1 expression levels in OVCAR-3 cells in vitro. mRNA expression levels of (A) CDC42 and (B) CCND1 in OVCAR-3 cells transfected with miR-195 inhibitor, inhibitor-NC, miR-195 inhibitor + NC and miR-195 inhibitor + CDC42 or CCND1 siRNAs. mRNA expression levels of (C) CDC42 and (D) CCND1 in OVCAR-3 cells transfected with miR-195 mimic, mimic-NC, miR-195 mimic + NC-vector and miR-195 mimic + CDC42 or CCND1 pcDNA. (E) Cell proliferation was assessed using BrdU immunostaining at 24 h after transfection. Red, OVCAR-3 cells labeled with BrdU; blue, nuclei counterstained by BrdU. Scale bar, 50 µm. (F) Evaluation of cell proliferation via colony formation assays at 48 h after transfection. Data were analyzed using one-way ANOVA. *P<0.05 vs. Inh-NC or Mim-NC groups; #P<0.05 vs. miR Inh-siNC or miR Mim + NC-Vector groups. BrdU, 5-bromo-2-deoxyuridine; miR, miR-195; Inh, inhibitor; NC, negative control, siRNA, short interfering RNA; Mim, mimic; CDC42, Cell division cycle 42; CCND1, cyclin D1.

Fig 5: miR-195 suppresses cell migration and invasion in OVCAR-3 cells by inhibiting CDC42 and CCND1 expression levels in vitro. Effects of miR-195 (A) inhibitor or (B) mimic, CDC42 and CCND1 on OVCAR-3 cell migration using wound healing assay at 0 and 24 h after transfection. Scale bar, 200 µm. (C) Effects of miR-195, CDC42 and CCND1 on OVCAR-3 cell invasion via Transwell invasion assay at 48 h after transfection. Scale bar, 50 µm. Data were analyzed using one-way ANOVA. *P<0.05 vs. Inh-NC or Mim-NC groups; #P<0.05 vs. miR Inh-siNC or miR Mim + NC-Vector groups. miR, miR-195; Inh, inhibitor; NC, negative control, siRNA, short interfering RNA; Mim, mimic; CDC42, Cell division cycle 42; CCND1, cyclin D1.