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. Author manuscript; available in PMC: 2021 May 1.
Published in final edited form as: Cancer Res. 2020 Aug 27;80(21):4791–4804. doi: 10.1158/0008-5472.CAN-20-1459

Figure 2. TRIM37-catalyzed H2Aub is required for chemoresistance in TNBC.

Figure 2.

(A) Top, Tail moment in DMSO or Dox-treated MDA MB 468 cells expressing non-silencer (NS) or TRIM37 shRNA (#1, #2). Bottom, Representative images of the tails for each group are shown. Scale bars, 100 μm. (B) Top, Quantification of γ-H2AX foci in MDA MB 468 cells expressing NS or TRIM37 shRNA (#1, #2) following treatment with Dox. Bottom, Representative immunofluorescence images of γ-H2AX foci (Green) in Dox-treated MDA MB 468 cells expressing NS or TRIM37 shRNA (#1, #2). DAPI (Blue) stains the nucleus. Scale bars, 50 μm. (C) Caspase 3 activity assay (Top) and immunoblot for PARP and cleaved PARP (c-PARP) (Middle) in DMSO or Dox-treated MDA MB 468, MDA MB 231, and MCF7 cells expressing NS or TRIM37 shRNA (#1, #2). Quantification of PARP cleavage relative to total PARP is shown (Bottom). (D) Quantification of the fold change in chemotherapeutic drug-resistant colonies obtained for MDA MB 468 cells expressing either NS or TRIM37 shRNA (#1, #2) by a clonogenic assay. Cells were treated with DMSO, Dox, temozolomide (TMZ), etoposide (EPO), daunorubicin (Daun), and cisplatin (CPN). Results were normalized to the colony forming unit (cfu) for DMSO. (E) ChIP monitoring TRIM37 and H2Aub binding at FAR3B and Actin in MCF10AT cells expressing vector control (VC), TRIM37, or mutant TRIM37 (TRIM37(C18R)). (F) Top, Tail moment in DMSO or Dox-treated VC-, TRIM37-, or TRIM37(C18R)-expressing MCF10AT cells. Bottom, Representative images of the tail moment for each group are shown. Scale bars, 100 μm. (G-H) Caspase 3 activity assay (G) and immunoblot for PARP and c-PARP (H, Left) in DMSO or Dox-treated VC, TRIM37 or TRIM37(C18R) expressing MCF10AT cells. Quantification of PARP cleavage relative to total PARP is shown (Right). (I) Quantification of the fold change in chemotherapeutic drug resistant colonies obtained for VC, TRIM37, or TRIM37(C18R) by clonogenic assay. Cells were treated with DMSO, Dox, temozolomide (TMZ), etoposide (EPO), daunorubicin (Daun), and cisplatin (CPN). Results were normalized to the cfu for DMSO. Error bars indicate standard deviation and range of at least three biological replicates. * p<0.05; ** p<0.01; *** p<0.001.