Fig. 3. Overexpression of mitochondria-targeting ERβ inhibits TNBC cell proliferation. A and B. MDA-MB-231 (A) and MDA-MB-436 (B) cells were transfected with pHA-mito-ERβ or control plasmid, and cell proliferation over 72 hrs was assessed by CCK-8 assays. Mitochondrial ERβ expression was analyzed by immunoblotting after subcellular fractionation. C and D. MDA-MB-231 and MDA-MB-436 cells were transfected with pHA-mito-ERβ or control plasmid, followed by assessment of cell cycle distribution. Data were analyzed by FACS (C), and the cell proliferation index (PI) was calculated and reported as described in the Materials and Methods section (D). E. MDA-MB-231 cells were transfected with pHA-ERβ, pHA-ER△N, or pHA-ER△C plasmid, and cell cycle distribution was analyzed using a FACScanto II.