Fig. 6: MRPL17 silencing impairs mitochondrial function and induces oxidative stress in NSCLC cells.

Mitochondrial function in primary NSCLC cells (pNSCLC-1), following transduction with control shRNA (shC), kdMRPL17-sh1, or kdMRPL17-sh2, was evaluated. Assessed parameters encompassed oxygen consumption rates (OCR) determined via Seahorse metabolic analysis (A), enzymatic activity of mitochondrial Complex I (B), quantification of total cellular ATP levels (C), and assessment of mitochondrial membrane potential based on JC-1 monomer fluorescence intensity (D). Corresponding indicators of oxidative stress measured in these pNSCLC-1 cells included the cellular glutathione redox state (GSH/GSSG ratio) (E), intracellular reactive oxygen species (ROS) levels detected using CellROX red (F) and DCF-DA blue (G) fluorescent probes, quantification of lipid peroxidation products via TBAR assay (H), and measurement of single-stranded DNA (ssDNA) content via ELISA as an index of DNA damage (I). The potential for functional rescue was subsequently investigated in pNSCLC-1 cells, comparing kdMRPL17-sh1 expressing cells to shC controls after pre-treatment with the antioxidant N-acetylcysteine (NAC, 0.4 mM) or cultivation in high-glucose medium (Hi-glu, adding 7.5 mM); evaluated endpoints included cell viability (J), cellular proliferation (nuclear EdU ratio, K), cell migration capacity (L), and overall cell death levels (M). The mitochondrial parameters were further examined in additional NSCLC models (pNSCLC-2/3 cells, A549 cell line) comparing kdMRPL17-sh1 effect relative to shC controls, including assessment of mitochondrial Complex I activity (N), total cellular ATP content (O), mitochondrial membrane potential via JC-1 staining (P), and intracellular ROS production utilizing the CellROX probe (Q). The primary human lung epithelial cells (pEpi1 or pEpi2) expressing kdMRPL17-sh1 to shC were cultivated for 36 h, total cellular ATP content (R), mitochondrial membrane potential via JC-1 staining (S), and intracellular ROS production utilizing the CellROX probe (T) were tested. “Ctrl” stands for the parental control cells. 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 group. # indicates P < 0.05 (J–M). “N.S.” stands for non-statistical difference. Scale bars represent 100 µm.