<i>P</i>. <i>aeruginosa</i> secreted virulence factors induce ER stress in primary bronchial epithelial cells.

<p>A. Time-dependent induction of ER stress in primary bronchial epithelial cells, as assessed by <i>XBP1</i> splicing, <i>CHOP</i> and <i>GADD34</i> mRNA after treatment with CM-PAO1 (n = 5; mean ± SEM). B. Dose-response of spliced <i>XBP1</i>, <i>CHOP</i> and <i>GADD34</i> mRNA in primary bronchial epithelial cells treated with CM-PAO1 for 12 hours (n = 5; mean ± SEM). C. Time-dependent phosphorylation of eIF2α (p-EIF2α) and synthesis of GADD34 and GRP78 (visualised with anti-KDEL antibody). Relative quantifications for each protein are shown within; representative of n = 3). D. Time-dependent decrease of puromycin incorporation in nascent proteins. Total eIF2α and β-actin serve as loading controls. * p<0.05, ** p<0.01, ***p<0.001 versus control (ctrl) with two-way repeated-measurements ANOVA (Bonferroni <i>post-hoc</i>) or untreated (-) with a one-way repeated-measurements ANOVA (Bonferroni <i>post-hoc</i>).</p

Conditioned medium of <i>P</i>. <i>aeruginosa</i> induces ER stress via TAK1-p38 MAP kinase (MAPK).

<p>A. Time-dependent phosphorylation of p38 MAPK in 16HBE after treatment with CM-PAO1. Total p38 MAPK and β-actin serve as loading controls. Numbers display the fold increase of phosphorylated p38 MAPK to total p38 MAPK compared to phosphorylated p38 MAPK at t = 0 (representative of n = 3). B. Western blot of phosphorylated p38 MAPK from 16HBE after pre-treatment for 30 min with the TAK-1 inhibitor LL-Z1640-2 (LL) or p38 MAPK inhibitor SB203580 (SB), followed by CM-PAO1 stimulation for 6 hours. Total p38 MAPK and β-actin serve as loading controls. Numbers display the fold increase of phosphorylated p38 MAPK to total p38 MAPK compared to phosphorylated p38 MAPK at t = 0 (representative of n = 3). C. IL-8 release of 16HBE cells treated as in B (n = 3; mean ± SEM). D. Normalised mRNA levels of <i>spliced XBP1</i>, <i>CHOP</i>, <i>GADD34</i> and <i>GRP78</i> in 16HBE cells, treated as B (n = 3; mean ± SEM). All values are normalised to the housekeeping genes <i>RPL13A</i> and <i>ATP5B</i>. * p<0.05, ** p<0.01, ***p<0.001 versus untreated (-) with a one-way repeated-measurements ANOVA (Bonferroni <i>post-hoc</i>).</p

Overlap between the +1 interactome for the products of genes in the GWAS white+grey zone and those of genes in the +1 interactome of the worm RNAi screen.

<p>The human AD GWAS white+grey zone genes, and their +1 interactors, overlap with the human orthologues of worm-screen hits, and their +1 interactors. Worm screen genes that have human orthologues with a significantly non-random +1 interactome overlap with the GWAS list are shown in bold. Worm screen genes that have human orthologues that interact directly with GWAS genes are marked with an asterisk.</p

Induction of GADD34 protects against <i>P</i>. <i>aeruginosa</i> mediated cell cytotoxicity.

<p>A. LDH release of <i>Gadd34</i>^<i>+/+</i> and <i>Gadd34</i>^{<i>ΔC/ΔC</i>} MEFs after stimulation with CM-PAO1 for 16 and 24 hours (n = 3; mean ± SEM). B. MTT assay assessing cell viability of HeLa cells conditionally expressing GADD34 (± dox) after treatment with CM-PAO1 (n = 3; mean ± SEM). C. LDH release (<i>left</i>) and cell viability assessed with a MTT assay (<i>right</i>) of wild-type MEFs treated with CM-PAO1 after repleting the cell culture medium with iron (Fe³⁺) (n = 3; mean ± SEM). * p<0.05, ** p<0.01, *** p<0.001 versus untreated (-) with a two-way repeated-measurements ANOVA (Bonferroni <i>post-hoc</i>).</p

Splicing of XBP1 mRNA is dependent on the ER stress responsive kinase IRE1α.

<p>Splicing of <i>XBP1</i> mRNA in 16HBE cells after treatment with CM-PAO1 in the presence of 30 μM 4μ8C, a selective inhibitor of the ER stress responsive kinase IRE1α (n = 3; mean ± SEM). All values are normalised to the housekeeping genes <i>RPL13A</i> and <i>ATP5B</i>. * p<0.05, ** p<0.01, *** p<0.001 versus untreated (-) with a two-way repeated-measurements ANOVA (Bonferroni <i>post-hoc</i>).</p

<i>GADD34</i> mRNA is regulated via the activation of the integrated stress response by <i>P</i>. <i>aeruginosa</i>.

<p>B-G. <i>Gadd34</i> mRNA normalised expression in <i>Perk</i>^<i>-/-</i>, <i>eIF2α</i>^<i>AA</i>, <i>Atf4</i>^<i>-/-</i>, <i>Pkr</i>^<i>-/-</i>, <i>Gcn2</i>^<i>-/-</i> and <i>Hri</i>^<i>-/-</i> mouse embryonic fibroblasts (MEFs) exposed to CM-PAO1 for 8, 16 or 24 hours or tunicamycin (Tm) for 6 hours as a positive control (n = 3; mean ± SEM). All values are normalised to the housekeeping genes <i>Actb</i> and <i>Sdha</i>. H. <i>GADD34</i> mRNA levels in HeLa cells upon exposure to CM-PAO1 after knock-down of GCN2 or HRI with siRNA (n = 3; mean ± SEM). All values are normalised to the housekeeping genes <i>RPL13A</i> and <i>ATP5B</i>. I. Normalised expression values of <i>spliced XBP1</i>, <i>CHOP</i>, <i>GADD34</i> and <i>GRP78</i> mRNA in 16HBE cells after stimulation with 1–100 nM deferoxamine (DFO). All values are normalised to the housekeeping genes <i>RPL13A</i> and <i>ATP5B</i>. J. <i>Gadd34</i> mRNA levels in wild-type MEFs after repletion of the cell culture medium with iron (Fe³⁺) when treated with CM-PAO1 (n = 3; mean ± SEM). The first column (- Fe³⁺,—CM-PAO1) reflects medium control cells, without adding or depleting iron from the cell culture medium. All values are normalised to the housekeeping genes <i>Actb</i> and <i>Sdha</i>. * p<0.05, ** p<0.01, *** p<0.001 versus untreated (-) with a two-way repeated-measurements ANOVA (Bonferroni <i>post-hoc</i>).</p

Enhancers of the Aβ-paralysis phenotype.

<p>The list consists of the human orthologues of worm genes that, when targeted by RNAi, enhance the paralysis phenotype in Aβ-expressing worms (n = 3 genes).</p

Distribution of rankings of worm genes.

<p>The x-axis represents the log of the gene ranking bin boundaries, arranged in decreasing gene-product connectedness from left to right. The y-axis represents the log of the fraction of genes in each bin (where 100% is 60 genes). The dashed line shows the linear regression for the worm screen results. The results of the screens are shown as black diamonds and results of random simulations are shown as empty triangles.</p

Worm genes +1 interactome.

<p>The +1 interaction network for the human orthologues of the 60 worm genes that were highlighted in our worm RNAi screen. For the 60 worm-screen genes with human orthologues, there were 3191 interactions in the +1 interactome.</p

Bacterial strains.

<p>Bacterial strains.</p