Fig. 4: SLC13A3 knockdown induces autophagic ferroptosis.

a Growth curves and colony formation of HepG2 and SNU398 cells. Stably control (shNC, gray) and SLC13A3-knockdown (sh#1, blue) cells were established using shRNA lentivirus. For re-expression study, cells were transfected with EV- (orange) or SLC13A3-overexpressing (purple) plasmid for 24 h. Cell viability was determined at the indicated time. Colony formation was measured by incubating cells in 12-well plates for 2 weeks. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA test. b Cell viability of HepG2 and SNU398 cells. Stably control (shNC, gray bar) and SLC13A3-knockdown (sh#1, blue bar and sh#2, dark blue bar) cells were treated with 1 μM ferrostatin-1 (Fer-1) or 10 μM liproxstatin-1 (Lip-1) for 48 h. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA test. c Intracellular fluorescence of C11-BODIPY probe and malonyl dialdehyde (MDA) levels in HepG2 and SNU398 cells. Stably control (shNC, gray) and SLC13A3-knockdown (sh#1, blue and sh#2, orange) cells were incubated with 2% FBS-PBS containing 5 μM C11-BODIPY for 30 min, and the fluorescence was determined using flow cytometric analysis. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA test. d Representative western blots of HepG2 and SNU398 cells (n = 3 independent experiments). Stably control (shNC) and SLC13A3-knockdown (sh#1) cells were established using shRNA lentivirus. e ROS levels in HepG2 and SNU398 cells. Stably control (shNC, gray bar) and SLC13A3-knockdown (sh#1, blue bar and sh#2, dark blue bar) cells were incubated with 10 μM DCFH-DA for 30 min, and the fluorescence was measured. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA test. f Cell viability of SLC13A3-knockdown (sh#1, blue curve and sh#2, dark blue curve) HepG2 and SNU398 cells in the presence of sorafenib at the indicated concentrations (0, 1.25, 2.5, 5, 10 μM) for 48 h. Data are presented as the mean ± SEM (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA test. g Autophagosome formation was measured using mRFP-GFP-LC3 puncta in HepG2 and SNU398 cells (n = 3 independent experiments). Stably control (shNC) and SLC13A3-knockdown (sh#1) cells were transfected with 1 μg mRFP-GFP-LC3 plasmid for 24 h, and imaged using confocal microscopy (GFP, green; RFP, red; merge, yellow). Source data are provided as a Source Data file.