Fig. 8: PSMD14 inhibitor Thiolutin restrains breast cancer progression.

A Immunoblot analysis showing the PSMD14 inhibitor Thiolutin decreases ERα protein stability. MCF-7 cells were treated with different concentrations of Thiolutin. Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as internal control. B qRT-PCR analysis showed that the PSMD14 inhibitor Thiolutin decreases the expression of ERα target genes (GREB1, TFF1, IL20). C Luciferase assays showing Thiolutin affects ERE-luciferase activity in MCF-7 cells. D PSMD14 inhibitor Thiolutin inhibits the proliferation of ER POSITIVE breast cancer cells. MCF-7 cells were treated with different concentrations of Thiolutin. After 24 h, a CCK-8 assay was used to determine the cellular metabolic activity at the indicated time points after Thiolutin treated. Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparisons. E, F PSMD14 inhibitor Thiolutin reduced the number of EdU-positive ER POSITIVE breast cancer cells. MCF-7 cells were treated with different concentrations of Thiolutin. After 24 h, EdU was added to the medium for 2 h of incubation. The absolute cell number was determined to indicate cell proliferation activity (E). Right panel shows quantification of Edu results by ImageJ software (F). Scale bar 100 μm. N = 3, *P < 0.05; **P < 0.01; ***P < 0.001 for cell growth comparisons. G, H Cell-cycle analysis by flow cytometry of MCF-7 cells were treated with different concentrations of Thiolutin. After 24 h, the cells were harvested, fixed with 70% ethanol, and stained with propidium iodide. The cells were subjected to FACS analysis. Experiments were performed in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 for cell proportion comparisons. The PSMD14 inhibitor Thiolutin inhibits the tumor growth of MCF-7 cells in a xenograft model. Harvested and photographed tumors in the Thiolutin (1 mg/kg) and the Vehicle group (I), tumor volume (J) and weight (K) growth in each mouse from the Thiolition group and the Vehicle group in vivo. *P < 0.05; **P < 0.01; ***P < 0.001. L The levels of PSMD14, ERα and Ki67 in xenografts treated with Thiolutin by using H&E and IHC staining. Scale bar,100 µm. M–O Thiolutin inhibited cell proliferation of ER-positive breast cancer patient samples in patient-derived explant (PDEx) assay. The patient-derived tumor samples were cultured ex vivo on gelatin sponges for 48 h with 10% FBS in the presence of 2 μM Thiolutin or vehicle. The tumor samples were fixed and stained with PSMD14, ERα, and Ki67 via IHC analysis (M). The dynamic change of ERα positive and Ki67 positive cells were counted and shown (N, O). Scale bars are 100 μm.