Extended Data Fig. 4: Shigella flexneri uses IpaH1.4 to degrade RNF213.
From: Shigella flexneri evades LPS ubiquitylation through IpaH1.4-mediated degradation of RNF213
(a) Complementation of S. flexneri ΔipaH1.4 with plasmid-encoded ipaH1.4 rescues degradation of RNF213 in infected host cells. Left: Flow cytometry of MEFs expressing GFP-RNF213 under control of a Doxycycline-inducible promoter, induced with Doxycycline, and infected with the indicated strains of S. flexneri harboring plasmids encoding constitutively expressed mRuby or TagRFP-T and anhydrotetracycline-inducible ipaH1.4, induced with anhydrotetracycline (ATc) as indicated (off – not induced, on – induced). Cells were analyzed by flow cytometry at 5 h post infection. Right: histogram plots of GFP-RNF213 intensity in infected cells. (b) Analysis of endogenous RNF213 levels in S. flexneri-infected HeLa cells by flow cytometry. Wild type or RNF213 KO HeLa cells infected with the indicated strains of S. flexneri expressing mRuby were stained for endogenous RNF213. Levels of RNF213 are depicted as histograms from the indicated cell populations, with bar heights in the accompanying bar graph denoting the mean and error bars denoting the standard deviations from n = 3 experiments; ** denotes the following p-values, in this order: p = 0.005, p = 0.003, p = 0.0003, p = 0.007, p = 0.0005, * denotes p = 0.048 (all derived from two-sided unpaired t-tests). The difference in staining between RNF213 WT and KO cells is indicative of the specific contribution of RNF213 to the assay. Infection with either S. flexneri WT or ΔipaH2.5, but not ΔipaH1.4, causes loss of RNF213 protein. (c) Left: Gating strategy for fluorescence-activated cell sorting (FACS) of HeLa cells. HeLa cells were infected with the indicated mRuby-expressing S. flexneri strains. At 4 h post infection the three indicated populations were isolated by FACS. Right: Immunoblot analysis of RNF213 in the indicated cell populations. HSP60 – loading control.
