Fig 2: USP16 regulates c-Myc stability at the post-transcriptional level. a and b Expression of USP16 and c-Myc proteins were measured by Western blot, and mRNA levels of USP16 and c-Myc were detected by qRT-PCR. c and d PC3 and DU145 cells were treated with 10 µmol/L of MG132 for 6 h and then lysed. e and f PC3 cells were transfected, treated with 50 µg/ml of CHX, and harvested at the indicated times. The plot illustrates c-Myc band intensities quantified by densitometry

Fig 3: EP4 receptor and HIF1a-related gene expression in human colon and rectal cancer tissues analyzed using the TCGA database. (A, B, C) Comparison of the mRNA expression of EP4 receptors (PTGER4), c-Myc (MYC), Sp-1 (SP1), and HIF-1a (HIF1A) (A), HIF-1a target mRNAs such as VEGF-A165 (VEGFA), VEGFR-1 (FLT1), and GLUT-1 (SLC2A1) (B), and the factors involved in the stabilization of HIF-1a such as von Hippel-Lindau (VHL), prolyl hydroxylase domain-containing enzymes (PHDs; also known as Egl-9 family hypoxia-inducible factors [EGLNs]), factor-inhibiting HIF-1a (FIH1; also known as HIF-1a inhibitor [HIF1AN]), copper metabolism domain-containing 1 (COMMD1), heat shock protein (HSP) 70 proteins, such as HSPA1A, and cAMP response element-binding protein-binding protein (CBP)/p300-interacting transactivator 2 (CITED2) (C), between cancer tissues (gray boxes) and paired noncancer tissues (white boxes). & P < 0.05, the Mann-Whitney U-test, significantly lower than noncancer tissues. # P < 0.05, the Mann-Whitney U-test, significantly higher than noncancer tissues. n.s.; not significant (D) Schematic models depict the c-Myc-Sp-1 complex-mediated transcriptional activation of EP4 receptors in low cellular density-cultured HCA-7 cells. In high cellular density-cultured HCA-7 cells, increased HIF-1a competes with and displaces c-Myc for Sp-1 binding and followed by pulling Sp-1 out from its binding site, resulting in the down-regulation of EP4 receptor transcriptional activation

Fig 4: USP16 co-localizes and interacts with c-Myc. a and b Indicated plasmids were transfected into HEK293T cells alone or in combination, and an anti-Flag antibody was used for immunoprecipitation. IP, immunoprecipitation; IB immunoblotting; WCE, whole-cell extract. c and d Exogenous USP16 or c-Myc was immunoprecipitated from PC3 cells using an anti-Flag antibody. Endogenous c-Myc or USP16 was analysed by Western blot. e Interaction between endogenous c-Myc and USP16 in PC3 cells was analysed by Western blot. f Immunofluorescent staining of USP16 and c-Myc in PC3 and DU145 cells. Scale bar, 10 µm. g Flag-c-Myc and HA-ubiquitin were co-transfected with v5-USP16 or v5-C205s, and the ubiquitination of c-Myc was measured using an in vivo ubiquitination assay. Precipitates and WCEs were analysed by Western blot. h Control siRNA (siNC) or siUSP16 was transfected into HEK293T cells. After 48 h, cells were transduced with Flag-c-Myc and HA-Ub for 24 h and then harvested. i HEK293T cells were transfected with v5-USP16, Flag-c-Myc, and different forms of HA-ubiquitins. Cell lysates were immunoprecipitated with an anti-Flag antibody and ubiquitination levels were measured using an anti-HA antibody

Fig 5: KDM4B and c-Myc are co-recruited at the LDHA locus. (A-B) Transactivation activity of C4-2B cells (LKO, sh4B#1, or sh4B#2) transfected with a LDHA reporter construct (A) or C4-2B co-transfected with a LDHA reporter construct, a KDM4B vector [KDM4B wild-type (WT), inactive KDM4B mutant (H189A)], and/or c-Myc. (B) The Beta-Glo/One-Glo Assay System was applied to assess the luciferase activity. ß-galactosidase was an internal control. (C-E) Chromatin immunoprecipitation (ChIP) analysis of KDM4B and c-Myc occupancy on the LDHA (C), ENO1 (D), and PFK (E) promoter loci. ChIP was performed with specific antibodies against KDM4B, c-Myc, and H3K9me3, respectively in control (LKO) and KDM4B-knockdown (sh4B#1 and sh4B#2) C4-2B cells. ChIP DNAs were analyzed by qPCR with primers that amplify genomic regions. Data were presented as the average of fold enrichment normalized to IgG ± SD from three independent experiments. Statistical analysis was assessed by Student's t-test.