Fig. 3: POLRMT silencing or KO disrupts mitochondrial functions and provokes apoptosis in primary skin SCC cells.

Stable C1 primary human skin SCC cells, expressing the lentiviral POLRMT shRNA (“shPOLRMT”), the CRISPR/Cas9-POLRMT-KO construct (“koPOLRMT”) or scramble control shRNA plus Cas9 control construct (“c-sh+Cas9”) were established and cultivated for the designated time periods; The cellular ROS contents were analyzed by measuring CellROX fluorescence intensity (A), Mitochondrial depolarization and lipid peroxidation were examined by JC-1 dye (B) and TBAR activity (C) assays, respectively; The cellular ATP contents were measured as well (D). Caspase-apoptosis activation was tested by the listed assays (E, F). The C1 primary skin SCC cells were treated with the POLRMT inhibitor IMT1 (500 nM) or the vehicle control (0.1% DMSO, “Veh”) for applied time periods, cell proliferation, migration, depolarization of mitochondria and apoptosis were measured by nuclear EdU/DAPI staining (G), “Transwell” (H), JC-1 staining (I) and TUNEL/DAPI staining (J) assays, respectively. “Pare” stands for parental control C1 primary cells. *P < 0.05 versus “Pare” cells/“Veh” treatment. Scale bar = 100 μm.