Fig. 2: IFRD1 deletion enhances autophagy flux and promotes cell death under glutamine starvation.

a Western blot analysis of IFRD1 expression in WT and KO-IFRD1 PLC/PRF/5 cells cultured under normal or glutamine starvation conditions for 48 h. b Cell viability of WT and KO-IFRD1 PLC/PRF/5 cells under normal or glutamine starvation conditions. Arrow indicates the time point that glutamine starvation started. c Fluorescence microscopy images of WT and KO-IFRD1 PLC/PRF/5 cells cultured with glutamine-deprived medium for 48 h followed by double staining with propidium iodide (PI) and Hoechst 33342 (left). PI and Hoechst 33342 stained cells were counted, and the percentage of PI-positive cells was then quantified. Scale bar, 50 μm. d Quantification of percentage of PI-positive cells in WT and KO-IFRD1 PLC/PRF/5 cells under glutamine starvation conditions for 48 h with the addition of DMSO, Z-VAD (20 μM), Fer-1 (1 μM), and CQ (10 μM) for the last 24 h, respectively. e Cell viability of WT and KO-IFRD1 PLC/PRF/5 cells under glutamine starvation conditions with the addition of DMSO, Z-VAD (20 μM), Fer-1 (1 μM), and CQ (10 μM) for 5 days, respectively. f Representative immunoblots of LC3B and actin (loading control) in WT and KO-IFRD1 PLC/PRF/5 cells under glutamine starvation conditions for 36 h with or without the addition of Baf-A1 (400 nM) for the last 2 h (left). The ratio of (LC3B-II (+Baf-A1)/actin)/(LC3B-II (–Baf-A1)/actin) indicates autophagy flux (right). g Representative images of LC3B immunostaining in WT and KO-IFRD1 PLC/PRF/5 cells under glutamine starvation conditions for 36 h with the addition of DMSO or Baf-A1 (400 nM) for the last 2 h (left). DAPI staining indicates nucleus. Percentage of LC3B puncta per cell in WT and KO-IFRD1 PLC/PRF/5 cells (glutamine-starved for 36 h) with Baf-A1 was quantified (n = 5) (right). Scale bars, 20 μm. h Representative immunoblots of the indicated proteins in WT and KO-IFRD1 PLC/PRF/5 cells under glutamine starvation conditions for 36 h (left). Ratios of p62/actin were quantified (right). i Representative confocal images of WT and KO-IFRD1 PLC/PRF/5 cells expressing mRFP-GFP-LC3 under glutamine starvation conditions for 36 h with the addition of DMSO or Baf-A1 (400 nM) for the last 2 h (left). Insets: magnified views of the regions in the white boxes. Quantification of LC3B puncta representing autophagosomes (yellow) and autolysosomes (red) in cells (n = 6) (right). Scale bars, 10 μm. j Representative electronic micrographs of autophagosomes or autolysosomes of WT and KO-IFRD1 PLC/PRF/5 cells under glutamine starvation conditions for 36 h. Red arrows indicate autophagic structures. Insets: magnified views of the regions in the white boxes (left). The number of autophagic structures per area was quantified (n = 6) (right). Scale bars, 1 µm. Data shown are representative of three independent experiments. Data are mean ± SD. **P < 0.01, ***P < 0.001; ns, not significant by two-tailed unpaired Student’s t test or two-way ANOVA.