Fig 1: Heme scavenging increases mechanical pain threshold in a rodent model of hemolysis. Phenylhydrazine hydrochloride (PHZ, 50 mg/kg, IP) or saline was administered to adult male C57BL/6 mice on two consecutive days. Six hours following the second injection, subset of mice were given purified human hemopexin (Hx) (4 mg/kg, IP) or saline. Mechanical hypersensitivity was assessed at baseline and on day two by measuring paw withdrawal thresholds to application of von Frey filaments. PHZ-challenged mice exhibited decreased paw withdrawal thresholds compared to mice that received Hx post-PHZ (n = 9–10) (A). On day two, mice exposed to PHZ had elevated plasma levels of cell-free heme (n = 10–13) (B), higher levels of the ER stress marker, GrpP78/BiP, in peripheral leukocytes (n = 5–10) (C) and lower levels of β-endorphin in both leukocytes (n = 5–10) (D) and plasma (n = 14–16) (E). Hx treatment abrogated the effects of PHZ. Individual values and means ± SEM. *P < 0.05 vs. groups at the end of individual lines; unpaired t-tests (A) or one-way ANOVA followed by Tukey post hoc testing (B–E).
Fig 2: ER stress inhibition restores palmitate-dependent impairment of autophagy and induction of UPR in macro- and microvascular endothelial cells. (a) Immunofluorescence of the ER morphology in HUVEC treated with 150 μm of oleate (OA) or palmitate (PA) in combination with DMSO or 2.5 mM 4-PBA. Prior to treatment the cells were transfected with the ER-scarlet probe. Lipid droplets were stained using BODIPY 493/503. The samples were imaged using Zeiss AxioObserver. Scale bar represents 15 μm. (b) Immunoblot analysis of HUVEC treated for 16 h with 150 μm PA in combination with DMSO or 2.5 mM 4-PBA. Cells were lysed in 2x sample buffer and analysed for expression of LC3B, p62, CHOP and Grp78. GAPDH immunblot was used as a loading control. (c) Quantification of immunoblot results from (b). 20 images per condition were analyzed in total in 3 independent experiments. (d) Immunoflourescence analysis of CHOP and p62 expression in HUVEC. The cells were treated as in (b), fixed and stained with anti-CHOP and anti-p62 antibodies and DAPI. Scale bar represents 50 μm. (e) Quantification of immunofluorescence analysis depicted in (d). For CHOP quantification percentage of CHOP positive cells is displayed and for p62 mean fluorescence intensity (MFI) normalized to control sample. 5 images per condition were analyzed in each of 3 independent experiments. (f) Immunofluorescence analysis of CHOP and p62 expression in HMVEC. Sample preparation and imaging was performed as in (d). (g) Quantification of CHOP and p62 staining in HMVEC depicted in (f) was performed as described for HUVEC in (e). (i) FITC labelled dextran leakage assay was performed in HUVEC. Cells were seeded on Transwell inserts and treated for 16 h with BSA + DMSO as a control, 150 µM PA + DMSO or 150 µM PA + 2.5 mM 4-PBA, 150 µM OA or 10 ng/ml TNFa as positive control. The leakage of FITC-labelled 75 kDa Dextran into lower compartment was measured by reading of mean fluorescence intensity at 488 nm using the microplate reader. 3 technical replicates were measured for each condition in 3 independent experiments. GraphPad Prism software and one-way ANOVA with Tukey’s multiple comparison test were used for statistical analysis. Error bars represent standard deviation (SD)
Fig 3: p62 levels are increased in serum of obese individuals. (a) Age of selected CARLA Study participants grouped in normal weight (BMI 18.5–24.99 kg/m2, n = 20) and obese individuals (BMI 30.00–34.99 kg/m2, n = 20). Waist circumference (cm) (b) and waist-to-hip-ratio (c) are depicted for normal weight and obese participants. (d) Cholesterol/HDL ratio in normal weight and obese sample groups is shown. Cholesterol and HDL concentrations were measured as described previously (Greiser et al.,.). Results of ELISA-based quantification of p62 (e) and Grp78 (f) in human serum. Each sample was measured in duplicate. Outliers were identified using GraphPad Prism and excluded from graphical representation and statistical analysis. Student’s t-test was performed to test for statistical difference between sample groups. Error bars represent standard deviation (SD)
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