Fig 2: Caspase-10 represses ACLY promoted malignant phenotype a, b A549 control (control), caspase-10 knockdown (CASP10kd), caspase-10/ACLY double knockdown (CASP10kd/ACLYkd), caspase-10/GCN5 double knockdown (CASP10kd/GCN5kd), and caspase-10/ACLY/GCN5 triple knockdown (CASP10kd/ACLYkd/GCN5kd) cells were subjected to glucose starvation for 48 h. The cells were treated with sodium acetate (5 mM), palmitic acid (0.2 mM) or both for the last 24 h of starvation period as indicated. a In vitro invasion, and (b) migration potential of the cells was then measured. Error bars are means ± SD of three biological replicates. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). ***P < 0.001. c A549 control, A549 CASP10kd, A549 CASP10kd/ACLYkd, A549 CASP10kd/GCN5kd, and A549 CASP10kd/ACLYkd/GCN5kd cells were seeded in soft agar and maintained in glucose starvation (5 mM) conditions, and treated with sodium acetate (5 mM), palmitic acid (0.2 mM) or both as indicated. Scale bars: 200μm. Representative images of colonies are shown. d A549 control (control), A549 CASP10kd, A549 CASP10kd/ACLYkd, and A549 CASP10kd/GCN5kd cells were injected subcutaneously into nude mice. Post-one week of injection, mice were treated with metformin (5 mg/ml in drinking water). Tumor volume was measured on the indicated days. The data shown are representative of three biological replicates (n = 5 mice/independent experiment). Error bars represent mean ± SD from five individual mice. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). ****P < 0.0001. e At the end of 30 days, tumors were excised and weighed. The data shown are representative of three biological replicates (n = 5 mice/independent experiment). Error bars represent mean ± SD from five individual mice. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). *P < 0.05; ***P < 0.001. f Immunofluorescence staining for Ki67 was performed on tumor sections (panel d above). Results shown is the representative of staining in five individual biological samples performed in triplicates. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar: 10 μm. g At the end of 30 days, lysates of tumors (panel d above) were analyzed by immunoblotting. The data shown are representative of three independent experiments. Source data are provided as a Source Data file

Fig 3: Caspase-10 downregulates ACLY-promoted aggressive tumor phenotype a A549Luc2 control (control), caspase-10 knockdown (CASP10kd), caspase-10/ACLY double knockdown (CASP10kd/ACLYkd), and caspase-10/GCN5 double knockdown (CASP10kd/GCN5kd) cells were orthotopically injected into the lung of nude mice. Post-one week of injection, mice were administered metformin (5 mg/ml in drinking water). Bioluminescence imaging was performed weekly and representative images are shown. The data shown are representative of three independent experiments using five individual mice per group. b Bioluminescence quantification (panel a above) was performed at indicated time points. The data shown are representative of three independent experiments using five individual mice per group. Error bars are mean ± SD from five individual mice (n = 5 mice per group). Statistical analyses were done using two-way ANOVA (Tukey’s post hoc test). ***P < 0.001. c At the end of 4 weeks, blood from mice in (panel a above), was used to isolate genomic DNA for examining circulating tumor cells. The data shown are representative of three independent experiments using five individual mice per group. Error bars are mean ± SD from five individual mice (n = 5 mice per group). Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). ***P < 0.001. d At the end of 5 weeks, tumor lysates were prepared (panel a above) and analyzed by immunoblotting for the indicated proteins. The data shown are representative of three independent experiments using five individual mice per group. e Representative image of immunostaining of ACLY, caspase-10 and H3K9Ac in different grades of human lung adenocarcinoma and matched normal adjacent tissue (NAT). DAPI was used to counter stain nucleus. Scale bar: 20 μm. f Quantitation of ACLY, caspase-10 and H3K9Ac levels in different grades of human lung adenocarcinoma with respect to matched normal adjacent tissue. The data shown are representative of three independent experiments. Error bars are mean ± SD. Statistical analyses were done using one-way ANOVA (Dunn’s multiple comparison test). *P < 0.05, ***P < 0.001. Source data are provided as a Source Data file

Fig 4: Tau is hypoacetylated, a result of impaired production of acetyl-CoA upon Elongator depletion. (A) Western blotting to detect and quantify Acly, and ß-Actin in control and Elp1KD cells extracts. Histograms of proportion of Acly expression to ß-Actin. (B,C) Analysis of Acly activity by malate dehydrogenase coupled method performed in control and Elp1KD cells lysates. Histogram of relative Acly activity over assay (B) and of slopes (C) from the linear phase of the reaction. (D) Immunoprecipitation followed by Western blotting to detect and quantify Acetylated Tau, in control and Elp1KD cells extracts. Histograms of proportion of acetylated lysine expression to Tau. (E) Immunoprecipitation followed by Western blotting to detect and quantify acetylated Tau, in extracts from HEK293 cells transfected with Tau, Elp3 or empty vector. Histograms of proportion of acetylated lysine expression to Tau. (F) Western blotting to detect and quantify Hdac6, and ß-Actin in control and Elp1KD cells extracts. Histograms of proportion of Hdac6 expression to ß-Actin. (G) In vitro deacetylation assay of endogenously acetylated bovine brain tubulin incubated for 4 h with extracts of WT and Elp1KD cells, or without cells extract (control). (H) Immunoblotting to detect p300, ß-Actin in control and Elp1KD cells extracts and histograms of proportion of p300 expression to ß-Actin. (I) LC-MS quantification of Acetyl-CoA levels in control and Elp1KD cells lysates. All graphs show values of means ± SEM. Significance was determined by: (A–I) two-sided t test, Specifically, [(A) p = 0.0059; (C) p < 0.0001; (D) p = 0.0256; (E) p = 0.0019; (F) p = 0.9833; (H) p = 0.7225; (I) p = 0.0456; ] (G) two-sided Kruskal Wallis one-way ANOVA, Specifically, [p = 0.0003, K = 9.346; ] In addition, the post hoc multiple comparisons, to analyze statistical difference of each condition compared to control by Dunnett test, **p < 0.01. n = Number of experimental repeats: (A) Control n = 8; Elp1 KD n = 9; (C) Control n = 3; Elp1 KD n = 3; (D) Control n = 3; Elp1 KD n = 3; (E) Control n = 4; Elp1 KD n = 4; (F) Control n = 3; Elp1 KD n = 3; (G) Control n = 6; WT n = 3; Elp1 KD n = 3; (H) Control n = 4; Elp1 KD n = 4; (I) Control n = 3; Elp1 KD n = 3.

Fig 5: Caspase-10 represses proliferative and metastatic genes a A549 control (control), caspase-10 knockdown (CASP10kd), caspase-10/GCN5 double knockdown (CASP10kd/GCN5kd), caspase-10/p300 double knockdown (CASP10kd/p300kd) and caspase-10/PCAF double knockdown (CASP10kd/PCAFkd) cells were subjected to glucose starvation for 48 h. The cells were then harvested and sandwich ELISA was performed for acetylated H3 and acetylated H4. Error bars are means ± SD of biological replicates. Statistical analyses were done using one-way ANOVA (Tukey’s post hoc test). **P < 0.01; ***P < 0.001. b A549 control (A549), caspase-10 knockdown (A549 CASP10kd) and caspase-10/GCN5 double knockdown (A549 CASP10kd/GCN5kd) cells were subjected to glucose starvation for the indicated time points. These cells were harvested and western blotting was performed for the indicated proteins. c A549 control (control), caspase-10 knockdown (CASP10kd), caspase-10/ACLY double knockdown (CASP10kd/ACLYkd) and caspase-10/GCN5 double knockdown (CASP10kd/GCN5kd) cells were subjected to glucose starvation for the indicated time points. Total RNA was then isolated and relative mRNA levels were analyzed by RT-qPCR for the indicated genes. Heat map comparing relative mRNA levels (mean fold change from three independent experiments with triplicate samples) is shown. Blue and red indicate upregulation or downregulation respectively. d A549 control (A549), caspase-10 knockdown (A549 CASP10kd), caspase-10/ACLY double knockdown (A549 CASP10kd/ACLYkd) and caspase-10/GCN5 double knockdown (A549 CASP10kd/GCN5kd) cells were subjected to glucose starvation for the indicated time points. ChIP assay was then performed with control IgG or acetylated-H3K9 antibody. Error bars are means ± SD of three biological replicates. Statistical analyses were done using two-way ANOVA (Tukey’s post hoc test). *P < 0.05; **P < 0.01; ***P < 0.001. Source data are provided as a Source Data file