Skip to main content
. 2018 Oct 24;78(1):74–82. doi: 10.1136/annrheumdis-2018-213532

Figure 1.

Figure 1

Salmonella exhibits enhanced replication in the presence of HLA-B27 and can activate XBP-1 and ATF6. (A) Two-dimensional isoelectric focusing of lysates from B27.HC cells and immunoblotted with anti-V5 pK demonstrates dimer formation (arrow) (top panel), while B35.HC does not form dimers. (B) HLA.B27.HC-expressing cells, transfected with the XBP-1ΔDBDv reporter construct (UPR leads to a 26nt intronic sequence excision, leading to the expression of the venus fluorescent protein) and treated with increasing concentrations of TPG exhibit a reduced UPR threshold. (C) The fold increase in ΔDBDXBP-1v activation following infection of HeLa cells with ST.mCherry at MOI 3:1, 50:1 and 100:1. (D) The fold increase in ΔDBDXBP-1v activation following infection of HeLa cells with ST.mCherry. Non-infected cells (mCherry-negative) from the same well expressing ΔDBDXBP-1v were used as non-infected controls to calculate the fold increase in XBP-1 activation in the infected cells (mCherry-positive). No induction of XBP-1v activation was seen in cells treated with heat-killed Salmonella enterica Typhimurium (HTST), Lipopolysaccharide (LPS) and flagellin (FGN). (E) Activation of endogenous XBP-1s was demonstrated by qPCR. All cycle threshold values were normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and data plotted as a fold induction of XBP-1s mRNA compared with levels detected at baseline. (F) The fold increase in ATF6 activation as measured by the cleaved cytosolic-FLAG tagged domain, following infection of HeLa cells with ST.mCherry. (G) Immunoblot with anti-FLAG tag antibody of AMP dependent transcription factor 6 (ATF6) following infection in two separate experiments (INF1 and INF2) compared with non-infected (NI) cells. Inactivated (i) and activated (a) ATF6 are denoted by arrows. For FACS analysis of ΔDBDXBP-1v activation (n=3) and densitometry analysis ofATF6 activation (n=3) in infected cells mean values ±SEM are shown and ANOVA was performed (p <0.0001 for both figures) with Tukey’smultiple comparison post-test. Significant differences between groups areindicated P< 0.05 (*), P< 0.01 (**) and P< 0.001 (***) (H) Relative fold increase of bacteria recovered from four individual experiments. Fold increases in Salmonella recovered were calculated based on cfu recovered from control E84. There in an average 4.2-fold increase in bacteria recovered from Salmonella-infected HeLa.B27.HC cells relative to the E84 and HLA-B35.HC control lines 24 hours post infection. The Mann-Whitney test was used to compare cfu recoveries between HLA-B35-expressing and HLA-B27-expressing cell lines and the exact p values were calculated (*p=0.0286). ATF6, AMP dependent transcription factor 6;cfu, colony-forming unit; E84, empty 84; HC, heavy chain; HeLa, Henrietta Lacks; HLA, human leucocyte antigen; LPS, Lipopolysaccharide; MOI, multiplicity of infections; TPG, thapsigargin; UPR, unfolded protein response; v, venus.