Extended Data Fig. 8: Global histone acetylation and chromatin accessibility are not consistently changed upon CPT1a and/or KAT2a loss.
a. KAT2a expression in 3D spheroid 4T1 cells growing for 5 d in the absence or presence of additional palmitate, oleate and stearate. A representative image of n = 3 experiments is shown. b. Intracellular levels of acetyl-CoA in 4T1 cells growing in 3D for 5 d in medium containing only 10% FBS, or supplemented with palmitate (75 mM), oleate (116 μm) or acetate (5 mM). Data are presented as mean ± SEM (n = 4 biological replicates). c. Heatmap of the signal intensity of H3K9ac-targeted gene loci in non-targeting RNA control, CPT1a and KAT2a knockout 4T1 3D spheroids cultured for 5 d in medium containing extra palmitate (75 μm) (n = 3 biological replicates). d. Correlation plot of H3K9 acetylation in 4T1 3D spheroids cultured in the presence of palmitate upon CPT1a inhibition with and without acetate (5 d). e. Heatmap and hierarchical clustering of top-scored downregulated genes of the NF-κB signaling pathway upon CPT1a deletion in 4T1 3D spheroids cultured in the presence of palmitate for 5 d, represented together with the expression status of the same genes upon acetate rescue and KAT2a deletion non-targeting sgRNA is used in control transfected samples (n = 3 biological replicates). f. Proliferation of 4T1 cells upon genetic inhibition of either Cpt1a or Kat2a in 2D culture measured using incucyte. Mean of growth rate ± SEM is shown (n = 6 biological replicates). One-way ANOVA with Dunnett’s multiple comparison test. g. 3D spheroids in 4T1 cells upon pharmacologic inhibition of either KAT2a using the inhibitor CPTH2 (2 μm) or CPT1A using etomoxir (50 μm) cultured for 5 d in medium with or without extra palmitate supplementation. Size quantification is represented by the average spheroids area of >100 spheroids. Data are presented as mean ± SEM (n = 3 biological replicates). One-way ANOVA with Tukey’s multiple comparison test. h. 3D spheroids number per well of 4T1 cells upon palmitate supplementation (75 μm), CPT1a or KAT2a genetic inhibition performed by CRISPR (sgCpt1a and sgKat2a) compared to non-targeting sgScrambled as a control, and upon metabolic rescue by acetate (5mM). Data are presented as mean ± SEM (n = 4 and 5 biological replicates). One-way ANOVA with Tukey’s multiple comparison test. i. Dose-response of 3D spheroid growth to the pharmacologic inhibition of KAT2a using CPTH2 inhibitor with or without extra supplementation of palmitate. Left panel, representative pictures. Right panel, spheroid size quantification is represented by the average spheroids area of >100 spheroids (n = 4 biological replicates). Two-way ANOVA with Tukey’s multiple comparison test. j. Total area and number of metastases in the lung of mice after 14 d of intravenous (i.v.) injections with 4T1 Kat2a knockout (sgKat2a, n = 8 mice) or non-targeting sgScrambled control cells (n = 10 mice) analyzed by H&E staining. Unpaired two-tailed t-tests with Welch correction.
