Fig 2: Proteomic analysis (UbiScan) identifies CCND1 as a candidate substrate of USP22. (A) USP22-dependent regulation of CCND1 ubiquitylation status identified by UbiScan. This unbiased proteomic analysis was performed in HCT116 cells following USP22 depletion and proteasome inhibition. Ubiquitylated lysine residues and their reported functional roles in CCND1 are listed, along with potential links to cancer mutations. (B) Efficient knockdown of USP22 shown by IB for samples subjected to UbiScan analysis. (C) Schematic representation of CCND1 indicating known posttranslational modifications and ubiquitylated residues identified by UbiScan. (D) Levels of CCND1 protein following USP22 depletion determined by IB in HCT116, H1299, MCF7, and PC3 cells. (E) CCND1 protein levels following loss of USP22 evaluated in nonmalignant, human fibroblast 2091 cells. (F) FLAG-tagged USP22 was ectopically expressed in HCT116 cells via a stably integrated tetracycline-inducible vector. CCND1 and CDK4/6 protein levels assessed following USP22 induction for the indicated time points. (G) Protein levels of phosphorylated pRB, RB, CCND1, CCND2, CCND3 CDK4, and CDK6 assessed by IB in H1299 cells. (H) CCND1 mRNA levels measured by qRT-PCR following USP22 depletion in H1299, MCF7, and PC3 cells.

Fig 3: siRNA-mediated YAP1 knockdown suppressed GAC cell proliferation in vitro and in vivo . (A) YAP1 knockdown suppressed cell proliferation in MGC-803, NCI-N87 and SGC-7901 cells (**, P < 0.001). The 575 nm absorbance readings in Day 6 were calculated for the P-Value. (B) siYAP1-MGC-803 formed smaller xenografts than siScramble-MGC-803 control in nude mice (**, P < 0.001). The tumor sizes in the Day 28 were calculated for the P-Value. (C) YAP1 knowdown decreased the expression of CCND3, CDK6 and p-Rb, but increased the expression of p21 and p27. The cleaved-PARP showed activated expression in AGS and MGC-803 cells upon YAP1 knockdown.

Fig 4: The protein expression level of activating transcription factor 2 (Atf2), Regulatory Associated Protein Of MTOR Complex 1 (Rptor), inhibitor of nuclear factor kappa B kinase regulatory subunit ? (Ikbkg), and Cyclin D3 (Ccnd3) genes in satellite cells (SCs) from young, MID, and MID+ T groups by western blot assay. The seven-day cultured SCs from each group were lysed and the lysate was subjected to western blot assay with the indicated antibodies. a-Tubulin served as an internal control for amounts of protein loaded on the gel.

Fig 5: GLA induces MM cell cycle G2/M arrest. A MM cell lines (RPMI-8226 and LP1) were treated with various indicated concentrations of GLA for 12 h. And the cells were then stained with PI and analyzed by flow cytometer. B The cell cycle-related proteins CCNB1, CCND1, CCND2 and CCND3 in MM cell lines (RPMI-8226 and LP1) were measured by western blot assay