Fig 2: Therapeutic effects of miR-3140 for tumor growth in vivo. (a) The experimental schedule for miR-3140 treatment in nude mice which were subcutaneously inoculated with MIAPaCa2 cells. (b) The representative image of tumor-bearing nude mice at 23 days after the inoculation of MIAPaCa2 cells. Tumors are denoted by arrowheads. (c) Tumor growth curves of xenograft mouse models treated with miR-NC or miR-3140 (n = 5, each). Tumor volume was calculated using the following formula: (shortest diameter) 2 × (longest diameter) × 0.5. Bar, SD for 5 mice; *P < 0.05. (d) Expression analysis of miR-3140 in resected tumors. The expression level of miR-3140 was measured by qRT-PCR using the relative ratio normalized based on the expression of RNU6B. Each experiment was performed in duplicate. Bar, SD. (e) Representative images of immunohistochemical staining for BRD4, BRD3, CDK2 and EGFR in resected tumors. Scale bar, 50 μm \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$.$$\end{document}. (f) Schematic models for the mechanism by which miR-3140 suppresses tumor growth.

Fig 3: BRD4 depletion inhibits DUX4 expression. a–c Expression of BET genes (a), BET proteins (b), and DUX4 and DUX4 targets (c) after BRD2, BRD3, or BRD4 siRNA knockdown in 54-2 FSHD1 myoblasts. Error bars indicate the standard deviation from the mean of three biological replicates. GAPDH serves as a loading control. p values were calculated using a one-way analysis of variance with Dunnett’s post test. *p < 0.05

Fig 4: Endometrial and prostate cancer SPOP mutants induce opposing effects on BET protein levels.(a) Outer surface of the SPOP substrate recognition domain with recurrently mutated amino acid residues highlighted in blue for prostate cancer and red for endometrial cancer, respectively7. Substrate in green in the substrate binding cleft. (b) Scatter plot of protein expression changes of SPOP mutants (MTs) vs. SPOP wild type (WT) in Ishikawa endometrial cancer cells, dotted red line = 2 s.d. (c) Representative Western blot (WB) validation for indicated proteins in Ishikawa cells stably expressing vector control, SPOP-WT, or endometrial cancer SPOP-MTs (n=5). (d) Representative WB for indicated proteins in Ishikawa endometrial cancer cells expressing prostate cancer SPOP-MTs (n=3). (e) Representative WB for indicated proteins in 22Rv1 prostate cancer cells stably expressing prostate cancer SPOP-MTs (n=3). (f) Representative images of primary human endometrial cancer tissues stained for BRD2, BRD3 and BRD4 with corresponding expression analysis on primary tumors stratified accordingly SPOP mutation status. Scale bars, 20µm. (g) BRD2, BRD3 and BRD4 expression analysis of primary human prostate cancer tissues stratified accordingly SPOP mutation status (R correlation coefficient and p values are derived from Kendall’s tau-b). N indicates the number of independent experiments performed.

Fig 5: BET proteins are differentially ubiquitylated and degraded by endometrial and prostate SPOP mutants.(a) Representative WB (n=4) of BET proteins and SPOP in Ishikawa and EN human endometrial cell lines. Statistical significance was determined by unpaired, two-tailed Student’s t-test (n.s., non-signicant). (b) Representative WB of indicated proteins in Ishikawa and EN cells with or without MG132 treatment (n=3). (c) Representative WB of indicated proteins after cycloheximide (CHX) treatment in Ishikawa and EN cells (n=3). (d) Interaction between HA-BRD3 and SPOP-WT, endometrial cancer mutants (SPOP-E50K, -R121Q), and one prostate cancer mutant (SPOP-W131G). HA-IP and WCE of transiently transfected 293T cells overexpressing HA-BRD3 and indicated SPOP constructs (n=3). (e) Effects of SPOP-WT and SPOP mutants on in vivo ubiquitylation of HA-BRD3 (n=3). (f) In vivo ubiquitylation of HA-BRD3-WT or HA-BRD3-Degron-MT by SPOP-E50K (n=3). Representative WBs are shown. N indicates the number of independent experiments performed.