Andro inhibits NSCLC cell proliferation by activating the mitochondrial apoptosis pathway. (A) Andro weakened ΔΨm in H1975 cells, as measured by JC-1 fluorescence. Scale bar: 20 µm; (B) Andro inhibited mitochondrial cyto C and cytoplasmic Bak expressions, and promoted cytoplasmic cyto C and mitochondrial Bak expressions in H1975 cells in a time-dependent manner, as evaluated through WB; (C) Andro enhanced Bax and Bak and inhibited Bcl-2 expression in H1975 cells in a dose-dependent manner, as evaluated through WB; (D) Transfection with Bak-siRNA significantly inhibited Bak mRNA and protein expressions in H1975 cells, as determined by qRT-PCR and WB, respectively; (E) Bak downregulation promoted H1975 cell proliferation, as measured through CCK-8 assay; (F) Bak downregulation enhanced ΔΨm in H1975 cells, as determined by the JC-1 fluorescence experiment. Scale bar: 20 µm; (G) Bak downregulation markedly inhibited apoptotic executive proteins (cleaved caspase 9, cleaved caspase 8, and cleaved caspase 3) expression in H1975 cells, as measured by WB. *, P<0.05; **, P<0.01; ***, P<0.001. Andro, andrographolide; NSCLC, non-small cell lung cancer; ΔΨm, mitochondrial membrane potential; JC-1: mitochondrial membrane depolarization sensor, 5, 50, 6, 60-tetrachloro-1, 10, 3, 30 tetraethyl benzimidazolo carbocyanine iodide; cyto C, cytochrome C; Bcl-2, B-cell leukemia/lymphoma 2; Bak, Bcl-2-antagonist/killer (Bak); Bax, Bcl2-associated X; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Mrna, messenger RNA; WB, western blotting; qRT-PCR, real-time quantitative reverse transcription-polymerase chain reaction; CCK-8, cell counting kit-8; OD, optical density; NC, negative control; si, small interfering.