Fig 2: Iron nanoparticle-mediated isolation of EpH4 cell lysosomes. A, TEM images of EpH4 cells showing iron nanoparticles residing in degradative lysosomal vacuoles (arrowheads) or in lysosomal vacuoles devoid of degradative material (arrows). Scale bars: 500 nm. B, bright-field microscopy and quantification of iron nanoparticle–induced cell death of EpH4 cells observed solely in the presence of hydrogen peroxide (H2O2). EpH4 cells were treated with H2O2 for 19 h. Scale bars: 25 µm. C, schematic of the magnetic iron nanoparticle lysosomal purification protocol. Adapted from Sargeant et al. (20). D, TEM images of lysosomes (top panel) and negatively stained lysosomes (bottom panel) isolated using the magnetic iron nanoparticle purification protocol. Arrowheads mark areas containing iron nanoparticles. Arrows indicate unidentifiable membranous fragments, which may be endolysosomal tubules or remnants from the ER or Golgi apparatus. Scale bars: 500 nm. E, isolated EpH4 lysosomes are predominantly clear of mitochondria. EpH4 cells were labeled with 10 kDa FITC-dextran (2.5 mg/ml (green)) with (Mag+) or without (Mag-) iron nanoparticle–containing media for 2 h and subsequently stained with MitoTrackerTM (500 nm (red)) for 30 min prior to isolation. Representative images of the PNS, post-magnetic SN, and magnetic lysosomal pellet (LP) of the two conditions are shown. Scale bar: 10 µm. F, isolated EpH4 lysosomes are highly pure with undetectable contamination from other organelles as observed by immunoblotting. N, nuclear lysate. Organelle marker proteins: LAMP2, lysosomes; ERp57, endoplasmic reticulum; GAPDH, cytoplasm; Rab5, early endosomes; Cox IV, mitochondria; Histone H3, nucleus.

Fig 3: LCWE induces IL-1β and caspase-1 secretion through IRE1.(A) WT BMDMs were treated with indicated doses of LCWE for 16 hours. The ER stress inducer tunicamycin (TM) was used as positive control. Protein lysates were analyzed by Western blotting using specific antibodies against p-IRE1 (S747) and ERp57. (B) BMDMs were pretreated with IRE1 kinase inhibitors AMG-18 (3 μM) and KIRA6 (15 μM) and RNase inhibitor 4μ8c (100 μM) for 1 hour and then treated with LCWE (75 μg/mL) for 14 hours along with the inhibitors. The supernatants were analyzed by Western blotting for cleaved caspase-1 (p10) and mature IL-1β. Whole-cell lysates were analyzed by Western blotting using specific antibodies against p-IRE1 (S747), IRE1, and β-actin (n = 3). (C) BMDMs derived from LysMCre+ Ern1Δ/Δ and littermate control Ern1fl/fl mice were treated with indicated doses of LCWE for 16 hours to activate the inflammasome. Supernatants were collected and analyzed by Western blotting for cleaved caspase-1 (p10) and mature IL-1β. Whole-cell lysates were prepared from the cells and analyzed by Western blotting using specific antibodies against IRE1, p-IRE1 (S747), and β-actin (n = 3). (D) Quantitative real-time PCR (qRT-PCR) of sXbp1, Il1b, Il6, and Tnfa transcripts in BMDMs derived from LysMCre+ Ern1Δ/Δ and littermate control Ern1fl/fl mice treated with either PBS or LCWE. **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA with Tukey’s multiple-comparison test. Casp-1, cleaved caspase-1.

Fig 4: Increased expression of IRE1 pathway–related proteins during LCWE-induced KD vasculitis.(A) Experimental schema. WT mice were injected with either PBS or LCWE, and 1 week later, heart tissues and abdominal aortas were collected. Heart tissue sections were used for immunofluorescence staining and abdominal aortas for immunoprecipitation and Western blot analysis. (B) H&E and immunofluorescence staining of ERp57 (red) and DAPI (blue) in heart sections of PBS- or LCWE-injected mice, 1 week after injection (n = 4–5/group). Scale bars, 100 μm. Quantification of ERp57 fluorescence intensity (right panel). (C) Western blot analysis of p-IRE1 (S747) and β-actin in abdominal aorta tissues from PBS-injected (n = 3 tissues pooled/lane) and LCWE-injected mice (n = 2 tissues pooled/lane) at 1 week postinjection. (D) Abdominal aortas were collected from PBS- and LCWE-injected mice, and tissue lysates (n = 3/condition) were immunoprecipitated using anti-IRE1 antibody or IgG rabbit as a control. Western blot was performed using anti–phospho-IRE1 (S747) antibody. Lysates from HEK293T cells treated with DMSO and thapsigargine (TG) were also immunoprecipitated with anti-IRE1 and used as positive controls. *P < 0.05 by Mann-Whitney test. CTCF, corrected total cell fluorescence.

Fig 5: B. cenocepacia invasion is specifically inhibited by PDI inhibitors targeting ERp57.(a) Effect of 30 μM rutin on B. cenocepacia ability to adhere and invade 9HTEo- cells. (b,c). Effect of 20 and 40 μM EGCG on the invasion (panel b) or on the total number of bacteria (panel c) interacting with 9HTEo-cells. (d) Differential effects of epicathechin (EC) and epigallocatechin gallate (EGCG) on B. cenocepacia ability to adhere and invade 9HTEo- cells. (e) EGCG impairs B. cenocepacia invasivity in different epithelial cell lines. The reported data are the mean ± SD of, respectively, 3 (panels a,b,d,e) or 8 (panel c) independent experiments. Asterisks denote statistically significant results: **p < 0.001.