Fig. 3. mTOR-mediated autophagy defect in CDDP-resistant tumor cells.

a–c Autophagic flux analysis of H460^par and H460^res cells stably transfected with LC3-HiBiT reporter. a Cells were pre-treated with AZD8055 for 48 hours before adding the indicated doses of chloroquine for 6 hours. b, c Cells were pre-treated as indicated with chloroquine or AZD8055 for 48 hours before adding increasing doses of b 2DG or c DCA for 6 hours. Shown is reporter activity measured as LC3-HiBiT luminescence normalized to untreated of n = 2 replicates. d, e H460 cells were transfected with DsRed-LC3-GFP, which yields a diffuse cytoplasmic green fluorescence that, upon autophagy induction, shifts to red-fluorescent puncta marking autophagosomes²⁵. d Representative immunofluorescence images of DsRed-LC3-GFP-expressing cells following 48 hours treatment. Scale bars, 10 µm. e Quantification of d. Shown are mean ± SD, n = 3 independent experiments in each of which 100 cells were quantified, two-way ANOVA with Dunnet’s multiple comparisons test.