Extended Data Fig. 2: Histone acetylation and cell proliferation capability induced by SRR knockout and rescued by pyruvate or acetate supplementation.
From: Serine racemase enhances growth of colorectal cancer by producing pyruvate from serine
a. Relationships between the cell number and the relative SRR mRNA level obtained from Fig. 2b, d. b. pyruvate concentration in DMEM without pyruvate with 10% FBS used in all the experiments unless otherwise mentioned. Data are means ± SD of n = 3 biologically independent samples and are representative of two independent experiments. c. The levels of M + 3-labeled D-serine in EV and SRR-KO HCT116 cells cultured with 13C3-L-serine for 6 hours. Data are means ± SD of n = 3 biologically independent samples. d. Intracellular lactate levels in EV and SRR-KO DLD-1 and HCT116 cells. Data are means ± SD of n = 4 biologically independent samples and are representative of two independent experiments. e. Immunoblotting of SRR in DLD-1 EV and SRR-KO cells transfected with control empty vector (pVec), Wild-type (WT)-SRR and Q155D-SRR. Blots are representative of n = 3 biologically independent experiments. f. Relative number of cells at 5 days after seeding of 2 × 104 cells. Data are means ± SD of n = 3 biologically independent samples and are representative of three independent experiments. g. Relative number of EV and SRR-KO DLD-1 and HCT116 cells cultured in medium containing 5 mM acetate or vehicle (DDW) for 6 days. Data are means ± SD of n = 3 biologically independent samples and are representative of two independent experiments. h. Immunoblotting of acetylated histone H3, acetylated histone H3K27, and histone H3 in DLD-1 EV and SRR-KO cells treated with 1 mM pyruvate or vehicle (DDW) for 72 hours. Blots are representative of n = 2 biologically independent experiments. i. Immunoblotting of acetylated histone H3 and histone H3 in DLD-1 EV and SRR-KO cells transfected with control empty vector (pVec), Wild-type (WT)-SRR and Q155D-SRR. Blots are representative of n = 2 biologically independent experiments. j. Representative images of two independent experiments of immunohistochemistry of acetyl-histone H3 in xenograft tumors of DLD-1 EV and SRR-KO cells with high (score 3), intermediate (score 2) and low (score 1) signal intensity, which were used to calculate H-scores. Scale bars, 100 μm. P-values were determined by two-tailed Student’s t-test.
