Figure 3.

KDM6B demethylase activity dependent loss of H3K27me3 upon Salmonella infection requires release of SPI1 effectors: [A] Immunoblot representing H3K27me3 levels in RAW264.7 macrophages 4h p.i. with different strains of SL. [B] Expression analysis of KDM6B at RNA analyzed by qPCR (HPRT was used for normalization) and [C] immunoblot representing corresponding H3K27me3 levels 4h post treatment with Salmonella LPS (100 ɳg/ml), heat killed Salmonella (HKS), SL and mutants of Salmonella SPI1 (∆SPI1) and SPI2 (∆SPI2) in RAW264.7 macrophages. [D] Immunoblot representing H3K27me3 levels at 4h p.i. with SL, SPI1 effector regulator mutants ΔHilA and ΔHilD. H3 was used as loading control. [E] Fold Change in KDM6B expression in RAW264.7 4h upon infection with SL, SPI1 effector regulator mutants ΔHilA and ΔHilD (HPRT was used for normalization). [F] Immunoblot representing H3K27me3 levels at 4h p.i. with SL, SPI1 translocon protein SipC mutant ΔSipC and 3XΔSipC (3 times of MOI used for SL and SipC). H3 was used as loading control. [G] Fold change in KDM6B expression in RAW264.7 4h upon infection with SL, SPI1 translocon protein SipC mutant ΔSipC and 3XΔSipC(3 times of MOI used for SL and SipC)(HPRT was used for normalization)Statistical significance was analyzed using Student unpaired t-test. (‘***’- p-value <.001; ‘**’- p-value <.01, ‘*’ p-value <.05, ns- not significant)