Research Paper

Figure 4. CircRABL2B silencing reduces autophagy in autophagy-induced NSCLC cell lines. (A) NSCLC cell lines were transfected with siCtrl, siCircRABL2B, siRABL2B, and siCom for 48 h. A549 and H838 cell transfection with siCircRABL2B or siCom downregulated conversion of LC3B-I to II; no significant effect was observed in H1299 cells. siRABL2B transfection had no effect on conversion of LC3B-I to II in all cell lines. (B–D) NSCLC cell lines were subjected to siRNA transfection for 48 h and infected for 24 h with Premo Autophagy Tandem RFP-GFP-LC3B. (Top) Fluorescence microscopy was used for puncta visualization. The yellow puncta (RFP+GFP+) in the merged images indicate autophagosomes, while the red-only puncta (RFP+GFP-) indicate autolysosomes. Scale bar: 10 μm. (Bottom) Quantification of autophagosome (yellow) puncta per cell and autolysosome (red-only) puncta per cell. Puncta were counted in each 200× field; a minimum of 100 cells were analyzed per siRNA and cell line. *p<0.05, **p<0.01 vs. siCtrl [one-way ANOVA]. Results are shown as means ± SDs. All in vitro experiments: 3 biological replicates × 3 technical replicates unless otherwise specified.