TRAP1 promotes cell proliferation through LUC7L3 regulation. (A–C) Effects of TRAP1 or LUC7L3 depletion in proliferation of PEA1 cells. TRAP1 or LUC7L3 was depleted for 72 h. Then, siCTRL and siTRAP1/siLUC7L3 cells were seeded in 96-well plates, and cell proliferation was monitored every 24 h using almarBlue assay. Significance was assessed by nonlinear fit regression analysis (n = 7). Statistical significance is represented as follows: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Error bars represent SEM. (B–D) Effects of TRAP1 or LUC7L3 depletion in clonogenicity of PEA1 cells. TRAP1 or LUC7L3 was depleted for 72 h. Then, siCTRL and siTRAP1/siLUC7L3 cells were seeded in 6-well plates and cultured for 10–14 days, until the appearance of visible colonies. At the end point, cells were fixed and stained with 25% methanol, 0.5% crystal violet. The area covered by the colonies was measured using the ColonyArea ImageJ plug-in. Significance was assessed by unpaired Student's t-test (n = 3). Statistical significance is represented as follows: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Error bars represent SEM. (E,F) PEA1 cells expressing shCTRL or shTRAP1 were transfected with FLAG-tagged LUC7L3 or FLAG-HA-tagged TRAP1 constructs. Twenty-four hours post-transfection, cells were seeded and assessed for proliferation (n = 5) and clonogenic (n = 3) assays as in A–D. Statistical significance is represented as follows: (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. Error bars represent SEM. (G) Immunohistochemical analysis of HGSOC tissues. TRAP1: cytoplasmic staining; please note the focal membranous reinforcement. Stromal cells are negative. LUC7L3: nuclear staining. Stromal cells are positive. Images were taken at 200× magnification.
