Fig. 9: COX8A serves as a downstream effector of MRPL17, mediating its pro-cancerous effects in pNSCLC-1 cells.

Expression of COX8A mRNA and protein levels in pNSCLC-1 cells with shC (scramble control shRNA), kdMRPL17-sh1, nonsense sgRNA (koC) or MRPL17 knockout using koMRPL17-sg1, empty vector control (Vec) or two independent stable cell colonies overexpressing MRPL17 (oeMRPL17-Sl1, oeMRPL17-Sl2) were tested (A–D); In MRPL17-silenced cells (shMRPL17-sh1), the expression of COX8A and endogenous MRPL17 was examined subsequent to ectopic overexpression of COX8A (oeCOX8A) (E). Functional characterization encompassed assessment of mitochondrial membrane potential based on JC-1 monomer fluorescence intensity (F), intracellular reactive oxygen species (ROS) levels detected using CellROX red (G) and DCF-DA blue (H) fluorescent probes, and quantification of total cellular ATP levels (I), as well as assessment of cellular proliferation via nuclear EdU incorporation assays (J) and evaluation of cell migration using Transwell assays (K). COX8A mRNA and protein levels were determined in pNSCLC-1 cells stably transduced with shC or COX8A specific shRNA (shCOX8A) (L, M). Cellular proliferation (N) and migration (O) were evaluated as well. Chromatin immunoprecipitation (ChIP) assays measured the binding of the transcription factor Sp1 to the COX8B promoter in pNSCLC-1 cells with altered MRPL17 expression (shRNA-mediated knockdown, CRISRP/Cas9-induced knockout, or overexpression). The data were normalized (P, Q). Additionally, the total protein levels of Sp1 were analyzed from cell lysates to confirm its expression across all experimental conditions (P, Q). All quantitative data are presented as mean ± standard deviation (SD) from five independent experiments (n = 5). Asterisks indicate statistical significance (*P < 0.05) relative to the shC or Vec group. # indicates P < 0.05 (E–K). “N.S.” stands for non-statistical difference. Scale bars represent 100 µm.