Fig 1: Inflammatory ligands stimulate lipolysis via IRE1 kinase not RNase activity.A–C, glycerol release rates (μM/h) in 3T3-L1 adipocytes treated with increasing concentrations of the IRE1 inhibitor KirA6 (A; N = 4), the IRE1 RNase inhibitor KirA8 (B; N = 16), or the IRE1 RNase inhibitor 4μ8C (C; N = 12–16). D, expression of spliced Xbp1 in 3T3-L1 adipocytes treated with 1 μM Thapsigargin for 3 h with and without 10 μM KirA8. E, glycerol release rates in differentiated 3T3-L1 adipocytes treated with or without 1 μM KirA6 (IRE1 inhibitor) and one of vehicle (mock treatment), 10 μg/ml FK565 (PGN), 1 μM thapsigargin (ER stress activator), 2 μM isoproterenol (β-adrenergic agonist), 500 ng/ml LPS (TLR4 activator), or 10 ng/ml TNF, as indicated (N = 12–14). F, glycerol release rates in 3T3-L1 adipocytes treated with 1 μM KirA6 and 0.5 mM 8-Br-cAMP (N = 10–12). G, phosphorylation of IRE1 was measured in 3T3-L1 adipocytes after stimulation with vehicle, PGN (10 μg/ml), or thapsigargin (1 μM) for 3 h, with or without 1 μM KirA6 (N = 4–6). Blots were stripped and reprobed for total IRE1. H, quantification of western blots for the ratio between phosphorylated IRE1 and total IRE1, expressed relative to the basal control sample. I–K, RNA isolated from 3T3-L1 adipocytes was treated for 3 h with vehicle, FK565 (PGN, 10 μg/ml), thapsigargin (1 μM), or isoproterenol (2 μM) in the absence or presence of KirA6 (1 μM). Expression of BiP (I), CHOP (J), and spliced Xbp1(K) (N = 12). Values are mean ± SEM. 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 2: Tumor necrosis factor (TNF) increases locomotor activity and exploratory behavior in pups, but has no impact on USV communication, body temperature regulation and olfactory orientation. Timeline (A). USV call rates at P6 in pups injected with TNF (20 mg/Kg) or vehicle, presented as means ± SEM, Mann-Whitney’s U-test: p > 0.05 (B). Change in pup body temperature during USV recording, presented as means ± SEM, Mann-Whitney’s U-test: p > 0.05 (C). Olfactory orientation at P9: latency to the first entry in the maternal bedding zone, presented as means ± SEM, Mann-Whitney’s U-test: p > 0.05 (D); time spent in maternal bedding zone, center zone and clean bedding zone, presented as means ± SEM, 2-way ANOVA: p(Treatment) = 0.9602, p(Time) < 0.0001, p(Interaction) = 0.7046 (E). Exploratory behavior at P13: time spent mobile in pups aged P13, by 2-min time segments, presented as means ± SEM (F). GEE estimates, 95% CI and p-values associated with the effects of treatment, time and cohort on time spent mobile: p(Treatment) = 0.0014, p(Time) = 0.1632, p(Cohort) = 0.6345 (G). Total distance traveled, presented as means ± SEM, Mann-Whitney’s U-test: p(Treatment) = 0.0157 (H). Exploratory index, presented as means ± SEM, Mann-Whitney’s U-test: p(Treatment) = 0.0266 (I). n = 29–30 Veh, n = 29–32 TNF depending on the test; *p < 0.05, **p < 0.01. Only statistically significant differences are presented.
Fig 3: IFT88 is expressed at low levels in non-ciliated bone marrow-derived macrophages, where its removal inhibits inflammatory response. (A) Western blot analysis of IFT88 in BMDMs from either IFT88fl/fl control or IFT88fl/fl /ROSA26ERT2 mice, all treated with the ethanol vehicle with or without 4-hydroxytamoxifen (4-OH-T). β-actin was used as a loading control. Cells cultured for 7 days after isolation before lysate collection. (B) Western blot analysis using alternative IFT88 antibody in BMDMs from either IFT88fl/fl control or IFT88fl/fl /ROSA26ERT2 mice, all treated with the ethanol vehicle with or without 4-OH-T. β-actin was usedas a loading control. Cells cultured for 7 days after isolation before lysate collection. Blot also shows expression in chondrocyte line with and without siRNA to IFT88 and the ORPK mutation (note less protein was loaded for these samples). (C) qPCR analysis probing for Ift88 in mRNA from BMDMs treated with or without 4-OH-T and stimulated as indicated of not stimulated (NS). Data presented as mean±s.d. mRNA expression as a fold change from the paired negative 4-OH-T (n=9). ****P<0.0001 (two-way ANOVA). (D) Ift88 mRNA expression in all stimulation conditions at point of stimulation shown as mean±s.d. (n=9). **P<0.01 (one-way ANOVA). (E) qPCR analysis probing for Nos2 in mRNA from BMDMs treated with or without 4-OH-T. Data presented as mean±s.d. mRNA expression as a fold change from the paired negative 4-OH-T condition (n=9). *P=0.0110 (Student's t-test, t=2.873 d.f.=16). (F) qPCR analysis probing for Nos2 in mRNA from BMDMs treated with or without 4-OH-T, stimulated with LPS (left), TNF (middle) and IL-1β (right). Data presented as mean±s.d. mRNA expression as a fold change from the respective with or without 4-OH-T paired NS condition (n=9). ****P<0.0001 (two-way ANOVA).
Fig 4: Hypomorphic and siRNA-mediated disruption of IFT88 in fibroblast-like chondrocytes inhibits select responses to inflammatory cytokines and alters NFκB activation dynamics before ciliary elongation. (A) Western blot analysis probing for IFT88 (left) in non-targeting (NT) control and IFT88siRNA cell lysates, from cells cultured with or without 10 ng/ml IL-1β for 24 h. IFT88 was quantified (middle) and the data presented as a mean±s.d. fold change from mean IFT88/β-actin levels of the no IL-1β NT condition (set at 1) (n=15). ****P<0.0001 (Student's t-test. t=14 d.f.=28). Effect of siRNA-mediated IFT88 depletion on cilia (right), shown as mean±s.d. percentage ciliated cells in NT and IFT88siRNA cell cultures (n=6 fields from two repeats). ****P<0.0001 (Fisher's exact test on contingency data). (B) iNOS protein expression in NT and IFT88siRNA cells cultured with or without 10 ng/ml IL-1β for 24 h. Protein levels were quantified and the data presented as a mean±s.d. fold change from mean iNOS/β-actin levels of the no IL-1β NT condition (set at 1). (n=9). **P<0.0001 (Student's t-test. t=3.215. d.f.=16. Mann–Whitney test. U=18). (C) Western blot analysis of conditioned medium from WT, NT and IFT88siRNA cells cultured with or without 10 ng/ml IL-1β for 24 h, probing for AGEG, with a medium only/no cell control in the first lane. (D) Quantification of the mean±s.d. amount of AGEG in the conditioned medium from WT, NT and IFT88siRNA cells cultured with or without 10 ng/ml IL-1β for 24 h (n=3 for each condition). ns, not significant between conditions (P=0.1625, two-way ANOVA). (E) Representative images used to calculate cilia length for the WT cell line (left), and primary porcine chondrocytes (right) with or without 10 ng/ml IL-1β or TNF for 24 h. Scale bars: 1 μm. (F) Data from E presented as box-and-whisker plots, where the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles (n>90 cilia for each condition for the WT cell line; n>260 cilia for each condition for the primary cells). *P=0.0163, ****P<0.0001 (one-way ANOVA with Tukey's multiple comparisons test, WT cell line); ****P<0.0001 (one-way ANOVA with Dunn's multiple comparisons test, primary cells). (G) Representative image of P65 localization in unstimulated (lower left) and 30 min cytokine-stimulated (lower right) WT chondrocytes. Nuclear P65 intensity was quantified by defining the nucleus as a region of interest using DAPI signal (top), and measuring the P65 signal within that region. Scale bars: 5 µm. (H) Data from G presented as mean±s.e.m. for WT (solid lines) and IFT88ORPK (dashed lines) cells, normalized to their own 0 h starting intensity. Experiments had a 30 min time course of 10 ng/ml IL-1β (red lines) or TNF (blue lines), with 10, 20 and 30 min cytokine time points and an unstimulated medium only 0 h time point (n=140 to 200 nuclei per condition).
Fig 5: Tumor necrosis factor (TNF) impacts reflex acquisitions in pups but not overall growth and developmental milestones. Timeline of the experiment (A). P1 to P16 body mass (BM) of pups injected with TNF (20 mg/Kg) or vehicle, presented as means ± SEM, note overlapping curves and reduced SEM (B). GEE estimates, 95% CI and p-values associated with the effects of treatment, time and cohort on BM: p(Treatment) = 0.2183, p(Time) < 0.0001, p(Cohort) < 0.0001 (C). Ambulation abilities scored from 0 to 3 in pups aged P6–P12, presented as means ± SEM, note overlapping curves and reduced SEM (D). GEE estimates, 95% CI and p-values associated with the effects of treatment, time and cohort on ambulation abilities: p(Treatment) = 0.5604, p(Time) < 0.0001, and p(Cohort) = 0.0216 (E). Ear development of pups aged P3–P5: a score of 0, 1 or 2 was set according to the number of ears everted/animal, presented as means ± SEM (F). GEE estimates, 95% CI and p-values associated with the effects of treatment, time and cohort on ear development: p(Treatment) = 0.729, p(Time) < 0.0001, p(Cohort) < 0.0001 (G). Eye opening of pups aged P12–P16: score of 0, 1 or 2 was set according to the number of eyes opened/animal, presented as means ± SEM (H). GEE estimates, 95% CI and p-values associated with the effects of treatment, time and cohort on eye opening: p(Treatment) = 0.212, p(Time) < 0.0001, p(Cohort) < 0.0001 (I). Righting reflex latency to turn of pups aged P2–P8, presented as means ± SEM (J). GEE estimates, 95% CI and p-values associated with the effects of treatment, time and cohort on righting reflex acquisition: p(Treatment) = 0.0308, p(Time) < 0.0001, p(Cohort) = 0.840 (K). Acoustic startle reflex of pups aged P10–P14: a score of either 0 or 1, where 1 is given to pups with startle and 0, to pups without startle, presented as means ± SEM (L). GEE estimates, 95% CI and p-values associated with the effects of treatment, time and cohort on the acoustic startle acquisition: p(Treatment) = 0.0332, p(Time) < 0.0001, p(Cohort) = 0.0101 (M). n = 29–30 PBS, n = 29–32 TNF depending on the test. *p < 0.05, ****p < 0.0001. Only statistically significant differences are presented.
Supplier Page from BioLegend for Recombinant Mouse TNF-α (carrier-free)