Fig. 6: The O-GlcNAcylation of LARS1 controls leucine-derived ATP production and protects cells under glucose starvation.
a, c, d, e SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of glucose and leucine for 4 h then leucine was added for 4 h. a cells were exposed to 2 μM oligomycin, 0.5 μM FCCP, and 0.5 μM/0.5 μM rotenone/antimycin, and the OCR was measured. Left: OCR over time; right: bar graph of basal OCR and FCCP-treated maximal OCR from left (mean ± SEM, n = 3 independent experiments). b Summary of the leucine catabolism pathway. c, d, e cells were harvested and analyzed with c NADH/NAD+ assay kit and d ATP assay kit. e cells were incubated with CellToxTM Green dye and dead cells were detected by a live-cell imaging analyzer. (mean ± SEM, n = 3 independent experiments). f SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of glucose and leucine for 4 h then 0.4 mM leucine was added for 4 h with vehicle or indicated compounds: KIC, 200 μM α-ketoisocaproic acid; Rapa, 10 nM rapamycin; CHX, 20 μM cycloheximide. g SW620 control or S1042A knock-in cells were starved of glucose and leucine for 4 h then 0.4 mM leucine was added for 4 h. f, g 1 μM puromycin was added to the medium of the cells for 30 min. h, i, j SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of glucose and leucine for 4 h then leucine was added for 4 h. Each sample was harvested and analyzed with h ATP assay kit and i CellToxTM Green dye signals (mean ± SEM, n = 3 independent experiments). j During leucine re-supplementation, vehicle or 1 μM puromycin was also added and incubated for 30 min. P-value was determined by two-tailed unpaired Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Representative data of three experiments with similar results. Source data are provided as a Source Data file.
