Extended Data Fig. 10: Characterization of H3.3 mutant RAW264.7 cell lines.

a, Western blot for H3.3 comparing wild-type, vector control (VC), HYPO and DKO RAW264.7 cell lines, membrane was stained with direct blue (DB) for equal loading. b, c, RNA-seq scatter (volcano) plot analysis, log₂-transformed fold change and −log₁₀(FDR) of DKO compared to wild-type (b), and HYPO compared to wild-type (c) RAW264.7 cells at 120 min. d, Ratio of RNA-seq fold change (log₂-transformed) for HYPO or DKO compared with wild-type cells after LPS stimulation (60 and 120 min) for all LPS-induced genes (top) and for the intersection of top H3.3S31ph genes and LPS-induced genes (bottom). ***P < 0.0001, lower-tailed one-sample t-test (distribution below zero). e, Heat map of fold change (log₂-transformed) for top H3.3S31ph genes among LPS-induced genes (left, 60 min; right, 120 min) with control constitutively expressed genes below. RNA-seq analysis was performed with two biological replicates per condition. f, Time course plots of mean RNA-seq expression (RPKM) from two experiments at time points 0, 60 and 120 min after LPS-stimulation for experiments performed in wild-type, HYPO and DKO RAW264.7cell lines at LPS-induced genes and at top H3.3S31ph genes among LPS-induced genes. g, Time course plots of mean RNA-seq expression (RPKM) from two experiments at time points 0, 60 and 120 min after LPS-stimulation for experiments performed in wild-type, HYPO and DKO RAW247.6 cell lines at LPS-induced genes Myc, Ccl9, Slfn2, Tnfaip3, Ccl4, Plk2 and constitutively expressed genes Tubb5 and Tbp. h, RT–qPCR for the viral expression of H3.3 transgene, wild-type, S31A or S31E in RAW macrophages. i, RT–qPCR for Ccl4 expression in a time course of stimulated DKO RAW264.7 cells rescued with wild-type H3.3 or S31A or S31E mutants.