Fig. 3: Induction of endogenous C15ORF48 increases autophagy.

a A549 cells were stimulated with IL-1α (10 ng/mL) or unstimulated for 24 h. After incubation, cells were fixed and stained with anti-C15ORF48 antibody and MitoTracker. Nuclei were counter stained with TO-PRO-3. Representative images are shown from three independent experiments. b, c A549 cells were transfected with the indicated siRNAs for 48 h and then stimulated with IL-1α (10 ng/mL) or unstimulated for 24 h. After incubation, median TMRM (b) or ATP amount in a cell (c) was analyzed and is shown as mean ± SD. Statistical significance was calculated using two-way ANOVA followed by Tukey’s multiple comparisons test (n = 3, biological replicates). d, e, f A549 cells were stimulated or unstimulated with IL-1α (10 ng/mL) for 20 h. After incubation, media were replaced with fresh media with or without IL-1α for an additional 4 h. Half the samples were treated with bafilomycin A1 (Baf A1, 200 μM) for the final 1 h. After incubation, cells were subjected to western blotting as in Fig. 1d (d). Alternatively, cells were fixed and analyzed for LC3 puncta by immunocytochemistry, as in Fig. 2b, c. Statistical significance was calculated using two-way ANOVA followed by Tukey’s multiple comparisons test (n = 70 cells from two independent experiments) (e, f). g, h, i As in Fig. 3d, e, f, except that cells were transfected with the indicated siRNAs for 48 before IL-1α stimulation. j, k, l A549 cells were stimulated or unstimulated with IL-1α (10 ng/mL) for 20 h. After incubation, media were replaced with fresh media together with or without IL-1α for an additional 4 h. Some samples were treated with DMSO (0.1%) or SBI-0206965 (SBI, 10 μM) for the final 4 h. Cells were used for western blotting (j) and LC3 puncta analysis by immunocytochemistry (k, l), as in Fig. 3d, e, f.