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

(A) Western Blot for H3.3 comparing wild-type (WT), vector control (VC), hypomorph (HYPO), and double-knockout (DKO) RAW264.7 cell lines, membrane was stained with direct blue (DB) for equal loading. (B, C) RNAseq scatter (“volcano”)-plot analysis, log2 fold-change and -log10(FDR), of DKO compared to WT (B), and HYPO compared to WT (C) RAW264.7 at 120’. (D) Ratio of RNAseq fold change (log₂) for HYPO or DKO compared with WT at 60’ and 120’ LPS stimulation for all LPS-induced genes (top) and for the intersection of top H3.3S31ph genes and LPS-induced genes (bottom). ***<0.0001 by lower-tailed one-sample t-test (distribution below zero). (E) Heat map of fold change (log₂) for top H3.3S31ph genes among LPS-induced genes (left, 60’; right, 120’) with control constitutively expressed genes below. RNAseq was performed with two biological replicates per condition. (G) Time course plots of mean RNAseq expression (RPKM) from two experiments at time points 0’, 60’, and 120’ after LPS-stimulation for experiments performed in wild-type (WT), hypomorph (HYPO), and double-knockout (DKO) RAW247.6 cell lines at LPS-induced genes Myc, Ccl9, Slfn2, Tnfaip3, Ccl4, Plk2, and constitutively expressed genes Tubb5 and Tbp. (F) Time course plots of mean RNAseq expression (RPKM) from two experiments at time points 0’, 60’, and 120’ after LPS-stimulation for experiments performed in wild-type (WT), hypomorph (HYPO), and double-knockout (DKO) RAW264.7cell lines at LPS-induced genes and at top H3.3S31ph genes among LPS-induced genes. (H) RT-qPCR for the viral expression of H3.3 transgene, either WT, S31A, or S31E in RAW macrophages. (I) RT-qPCR for CCL4 expression in a time course of stimulated DKO RAW264.7 cells rescued with WT H3.3, S31A, or S31E.