Fluoxetine induces cytotoxic endoplasmic reticulum stress and autophagy in triple negative breast cancer

Figure 4 Key proteins along the autophagy, unfolded protein response, and apoptosis pathways were examined across cell lines after 10 μmol/L fluoxetine treatment at various times. A, D: Critical effector of autophagy, AMPK, was activated after 2 h and 24 h of treatment in SUM149PT. Autophagy in cell lines was detected by the presence of cleaved LC3 (LC3-II) bands; B, C: The balance, level, and duration of ER stress sensors (B) and effectors of UPR (C) may ultimately dictate whether a cell lives or dies; D: protein levels were monitored for up to 48 h in SUM149PT after fluoxetine treatment, showing concurrent induction of UPR and autophagy by 24 h. Blots shown were representative of at least 3 different experiments. The amounts of total cell extracts loaded for each cell line were indicated in the bottom panels in (A) for cleaved LC3 detection, while total loading in all cell lines for AMPKα detection was 100 μg. In panels B, C, and D, the amount of cell extracts loaded in each lane was 30 μg, 100 μg and 50 μg, respectively. Since the Abcam antibody for cleaved LC3 detection was very sensitive in panel A, we chose another manufacturer (cell signaling technology) to detect the same cleaved protein in panel D without the problem of overexposure during chemiluminescence. UPR: Unfolded protein response; ER: Endoplasmic reticulum; AMPK: Adenosine monophosphate kinase.