Fig 1: Systemic inflammatory response syndrome drives apoptosis and not necroptosis in intestinal epithelial cells.Co-housed 10–12 week old female C57Bl6/N wt mice were injected with 0.75 µg/g or 1 µg/g of ice-cold recombinant, LPS-free mouse TNF intravenously. Control mice received a corresponding volume of ice-cold LPS-free PBS. Mice were sacrificed 6 h post-injection. Untreated wt mice were additionally analysed (n = 3 in each group). A Representative images of small intestine and colon sections (Swiss-rolls) of wt mice as indicated were stained with H&E. Slides were digitalised in a digital slide scanner and pictures were acquired in QuPath. Scale bars: 50 µm. B Graph showing histology score in whole intestine. Data are presented as mean + SEM and each dot represents one mouse. P values were calculated via one-way Anova, Tukey’s multiple comparisons test. **P ≤ 0.01, ns not significant. C Representative images of ileum and colon sections (Swiss-rolls) of wt mice as indicated immunostained with pMLKL-S345 and c-Casp3. Slides were digitalised in a digital slide scanner and pictures were acquired in QuPath. Scale bars: 50 µm. Arrowheads indicate pMLKL-S345-positive cells and c-Casp3-positive areas. D Graph showing quantification of illustrated pictures for each immunostaining obtained via QuPath after slides were digitalised in a digital slide scanner as described in the Supplementary Methods section. Total numbers of cells were obtained to calculate the percentage of positive cells over the total amount of cells detected. Data are presented as mean + SEM and each dot represents one mouse. P values were calculated via two-way Anova, Tukey’s multiple comparisons test. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns not significant.
Fig 2: Calhm6 −/− macrophages and NK cells are capable of normal responses to activating signals when stimulated in isolation, in vitro A, B WT and Calhm6 −/− mice were injected i.p. with Poly(I:C) (200 μg/mouse) or PBS. On day 3 spleens were collected, stained with antibodies against NK cell‐maturation markers and analysed by flow cytometry. Representative contour plots (A) and pooled flow cytometry results (B) are shown (results from one experiment, Poly(I:C) WT mice = 5, Calhm6 −/− mice = 5, control WT mice = 2, Calhm6 −/− mice = 2 mice, One‐way ANOVA with multiple comparisons).C CD3−NK1.1+ cells were isolated from naïve mouse spleens and stimulated in vitro with PMA/ionomycin, IL‐12 (50 ng/ml), TNF (50 ng/ml), or IL‐18 (50 ng/ml). After 6 h stimulation, supernatant was collected and IFN‐γ was measured by ELISA (pooled results from two independent experiments, Untreated and PMA stimulation WT mice = 6, Calhm6 −/− mice = 6, TNF + IL‐12 stimulation: WT mice = 3, Calhm6 −/− mice = 3, otherwise WT mice = 3, Calhm6 −/− mice = 2, One‐way ANOVA with multiple comparisons).D, E BMDM with or without 24 h IFN‐γ priming were grown in antibiotic‐free medium, washed and cultured with Listeria monocytogenes (MOI 1) for 30 min, at which time gentamicin (5 ng/ml) was added to the medium to kill extracellular bacteria. Supernatant from BMDM infected with L. monocytogenes was collected after 6 h. IL‐1β (D, pooled results from five independent experiments, WT mice = 5, Calhm6 −/− mice = 5, Kruskal–Wallis test) and IL‐18 concentration (E, pooled results from three independent experiments, WT mice = 3, Calhm6 −/− mice = 3, Kruskal–Wallis test) were quantified by ELISA, and normalised to WT set to 100%, to allow pooling of independent experiments.F NO production in WT and Calhm6 −/− BMDM treated overnight with LPS or Poly(I:C) quantified with a Griess test on cell supernatant (results from one experiment, WT mice = 3, Calhm6 −/− mice = 3, Kruskal–Wallis test with multiple comparisons).G BMDM with or without IFN‐γ priming were grown overnight in antibiotic‐free medium. Cells were infected with L. monocytogenes (MOI 1) for 30 min. Gentamicin (5 ng/ml) was then added to the medium to kill extracellular bacteria. At 0, 4 and 8 h cells were harvested, lysed with 0.1% Triton X‐100 and the lysate was plated on antibiotic‐free BHI plates for 24 h, and CFU were quantified (pooled results from three independent experiments, WT mice = 3, Calhm6 −/− mice = 3, Two‐way ANOVA test). Data information: Error bars represent SD. Source data are available online for this figure.
Fig 3: IRE1 kinase activity does not affect insulin signaling through Akt. Phosphorylation of Akt (pAkt) (Ser473) relative to total Akt (tAkt) was measured by immunoblotting in 3T3-L1 adipocytes in the absence or presence of 1 μM KirA6. Cells were pretreated with or without KirA6 for 1 h before addition of ligand for 48 h. Cells were treated with or without 100 nM insulin for the final 10 min after cells were exposed for 48 h to: A) PGN (10 μg/ml), B) thapsigargin (1 μM), C) isoproterenol (2 μM), D) LPS (500 ng/ml), or E) TNF (10 ng/ml). Control (C), FK565 (FK), KirA6 (K), thapsigargin (Th or Thap), isoproterenol (Iso), lipopolysaccharide (LPS), tumour necrosis factor (TNF). Values are mean ± SEM (N = 6–8). Statistical significance was measured as p < 0.05 using two-way ANOVA. Post hoc analysis was performed using Tukey’s multiple comparisons test. Conditions with different letters (a, b) denote a statistical difference compared with all other conditions without the same letter.
Fig 4: L-WRN CM activity is maintained over several weeks of storage following thaw.(A-E) L-WRN CM was stored at 4C for 0WK, 1WK, 2WK, 3WK or subjected to a second freeze-thaw cycle (2XFT). An aliquot of the 2XFT sample was removed prior to the second freeze-thaw cycle to serve as a direct control (2XFT Cont). Spheroids cultured in differentiation medium with EP4 inhibitor (DM + EP4i), treated with cycloheximide and tumor necrosis factor (CHX + TNF), or treated with butyrate served as negative controls. (A) Schematic of experimental time line for assays in (B) and (C). (B) Graph of CellTiter-Glo data presented as fold change (mean ± s.e.m.) relative to DM + EP4i; n = 3 independent experiments. ****P < 0.0001 by 1-way ANOVA and Dunnett’s post test relative to 0WK. (C) Graphs of mRNA gene expression for indicated genes as determined by qPCR. Data are presented as fold change (mean ± s.e.m.) relative to 0WK; n = 3 independent experiments. (D) Schematic of experimental time line for (E). (E) Graph of Cdc25A-CBRluc data normalized to the average 0 h value of all samples; n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA (B, C) or by 2-way repeated measures ANOVA (E) using Dunnett’s post test relative to 0WK (B, C, E).
Fig 5: IFT88 is acting independent to the ciliary axoneme in fibroblast-like chondrocytes. (A) Confocal immunofluorescence microscopy of chondrocytes, for acetylated α-tubulin (ACαTUB; green), IFT88 (purple) and primary cilia (highlighted by arrowheads). Scale bar: 2 µm. (B) TIRF microscopy of cilia, ACαTUB (green) and IFT88 (purple). Scale bar: 2 µm. (C) NFκB P65 (orange) in a ciliated (top image) and a non-ciliated (bottom image) cell (cilia identifiable by ACαTUB staining in green), 30 min after cytokine stimulation. Scale bar: 5 µm. Graphs on the right show the mean±s.e.m. nuclear P65 fluorescent intensity over a 30 min time course for ciliated (solid) and non-ciliated (dashed) cells in response to either 10 ng/ml IL-1β (red) or TNF (blue, n>50 nuclei per condition, per time point). **P=0.005 and 0.002 for IL-1β and TNF, respectively (two-way ANOVA). (D) KIF3AsiRNA cells cultured with or without 10 ng/ml IL-1β for 24 h. Protein levels quantified from western blot analysis (Fig. S5B,C) and the data presented as a mean±s.d. fold change from mean iNOS/β-actin or COX2/β-actin levels for the no IL-1β NT condition (iNOS n=6; COX2 n=9). Not significant (P>0.05, Student's t-test) for iNOS; *P=0.0188 for COX2 (Mann–Whitney test. U=14). (E) Nitric oxide (NO) analysis (mean±s.d.) of conditioned medium from KIF3AsiRNA and IFT88siRNA cells cultured with or without 10 ng/ml IL–1β for 24 h compared with NT (n=36 and 27, respectively). ***P=0.002, ****P<0.0001 (Mann–Whitney test).
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