Fig. 2: ACTL6A reprograms GSH metabolism to maintain GC malignant progression.

a Gene categories significantly (P ≤ 0.05) enriched for genes deregulated in KEGG pathways, owing to knockdown of ACTL6A in SNU638 cells. b Venn diagram illustrating the overlap among the top 35 enriched pathways of RNA array and two GC databases. c An enrichment analysis of gene sets available from RNA array and two GC databases revealed that ACTL6A expression is positively correlated with Glutathione Metabolism. d Schematic of GSH/GSSG cycle that affects oxidative stress and NADPH/NADP+ ratio. e Measurement of relative GSH/GSSG ratio in SNU638 cells treated with ACTL6A shRNA and scrambled shRNA. The data are presented as the means ± SD, n = 3 biologically independent experiments. f Measurement of relative NADP + /NADPH ratio in SNU638 cells treated with ACTL6A shRNA and scrambled shRNA. The data are presented as the means ± SD, n = 3 biologically independent experiments. g Relative DCFH-DA fluorescence intensity measured by flow cytometry, SNU638 cells are treated with ACTL6A shRNA and scrambled shRNA and cultured with or without 50 μM H₂O₂ for 24 h. Data are presented as the means ± SD, n = 3 biologically independent experiments. h, i Relative cell growth rate of SNU638 cells treated with ACTL6A shRNA or scrambled shRNA and cultured with or without 100 μM NAC. The data are presented as the means ± SEM, n = 3 biologically independent experiments. j PDOs were treated with ACTL6A shRNA or scrambled shRNA and cultured with or without 100 μM NAC for 9 days. Representative pictures at day 0, day 3, and day 9 after being treated with NAC were shown. Scale bars represent 25 μm. The size of organoids on the 9th day was calculated, presented as the means ± SD, n = 8 for each group. k–n SNU638 cells (1 × 10⁶) treated with ACTL6A shRNA or scrambled shRNA were transplanted to nude mice (n = 6 for each group). Mice were treated with or without NAC (k). Images (l), tumor volumes (m) and tumor weight (n) are shown. The data are presented as the means ± SD. P values were determined by unpaired two-tailed T test for panels e–g, j, n, and two-way ANOVA followed by Tukey test for panels h, i, m.