Fig 2: MINDY1 deubiquitylates ERa.A MCF-7 cells transfected with the indicated siRNA were treated with MG132 for 6 h before collection. ERa was immunoprecipitated with anti-ERa and immunoblotted with anti-HA. B Immunoblotting to detect the ubiquitination of ERa in HEK293 cells co-transfected with Flag- ERa, HA-Ubiquitin, and Myc-MINDY1 (wild type or C137A). C MINDY1 removed the ubiquitin chain of ERa in a dose-dependent manner. D ERa ubiquitylation was analyzed in cells transfected with E3 TRIM8 together with MINDY1 or not. E HA-WT, K6, K11, K27, K29, K33, K48, or K63 Ub was co-transfected with Flag-ERa and Myc- MINDY1 into HEK293 cells. After treatment with 10 µM MG132 for 6 h, cell lysates were subjected to ubiquitination assay and the ubiquitination level of ERa was detected by HA antibody. The experiment was independently repeated three times with three replicates. *P value < 0.05; **P value < 0.01; ***P value < 0.001.

Fig 3: MINDY1 depletion inhibits bladder cancer cell proliferation. A MINDY1 depletion inhibited cell proliferation in bladder cancer cells. B MINDY1 depletion induced G1 cell cycle arrest in bladder cancer cells. C MINDY1 depletion decreased clone formation capability of bladder cancer cells. D, E Representative images of EdU assay of bladder cancer cells. F MINDY1 depletion inhibits the tumor growth in vivo. T24 cells were stably transfected with lentivirus carrying a scrambled shRNA or MINDY1 shRNA. 1 × 106 T24 cells were injected to the right dorsal flank of each mouse (n = 6). Tumor sizes were measured every 5 days until the end of the experiment. G Representative images of immunohistochemical staining for Ki67, MINDY1 and YAP. *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001

Fig 4: MINDY1 increases YAP stability. A In the presence of the proteasome inhibitor MG132, depletion of MINDY1 did not further decrease the YAP protein level. Bladder cancer cells were transfected with siMINDY1 or siControl. After 48 h, cells were treated with 10 µM MG132 / vehicle for 6 h, cell lysates were prepared for western blot analysis. B T24 cells were transfected with MINDY1 (wild type or C137A) together with MINDY1 siRNA. The YAP protein levels were measured. C MINDY1 depletion decreased YAP half-life in bladder cancer cells. Bladder cancer cells were transfected with siMINDY1 or siControl. After 48 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for western blot analysis. D MINDY1C137A did not increase YAP half-life in HEK293 cells. HEK293 cells were transfected with YAP plasmid and Myc-tag, Myc-MINDY1 or Myc- MINDY1C137A plasmids. After 24 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for Western blot analysis

Fig 5: Increased ERα expression reverses the effect of MINDY1 depletion.A Cell proliferation assay of MCF-7. B Clone formation assay of MCF-7. C Representative images of EdU assay of breast cancer cells. D Wound-healing assay of MCF-7. E Overexpression of ERα in MINDY1-knockdown cells recovered tumor growth in vivo. The experiment was independently repeated three times with three replicates. *P value < 0.05; **P value < 0.01; ***P value < 0.001.