Fig. 3

HSP70 and GRP78 induce fat accumulation in PBMCs and HepG2. ^*P < 0.05, ^**P < 0.004, ^***P < 0.0009. Number of samples = 5. Data are mean ± s.e.m. or are expressed as median plus minimum and maximum values for whisker plots. a–d PBMCs from 5 different healthy controls (a, c) or 5 batches of HepG2 (b, d) were incubated with 50–800 pg mL⁻¹ of each HSP and the expression of Plin2 measured by flow cytometry. (a, b) Refer to HSP70, while (c, d) refer to GRP78. The data are reported in Supplementary Table 1. e, f Plin2 protein expression (MFI) in PBMCs from healthy controls (e) and in HepG2 cells (f) is significantly increased after stimulation with serum from obese IR, NAFLD subjects before metabolic surgery. Sera from subjects after metabolic surgery determined effects similar to those of healthy controls (PBMCs: after surgery 57.82 ± 8.48, healthy controls 56.50 ± 10.09, P = NS; HepG2: after surgery 74.40 ± 0.93, healthy controls 70.60 ± 2.09, P = NS). g, h, i,j To exclude that Plin2 increase can be an unspecific effect, PBMCs and HepG2 were stimulated with IgG and with a combination of immunoprecipitation buffer and HSP70/GRP78 antibody (negative control). No effect on Plin2 expression was observed after stimulation with IgG or negative control. PBMCs: serum depleted (immunoprecipitation, IP) of HSP70 72.63 ± 1.52 vs. complete serum 104.8 ± 2.62, P = 0.0009 (g); HepG2: serum depleted of HSP70 20.21 ± 2.24 vs. complete serum 27.29 ± 1.49, P = 0.00235 (h); PBMCs: serum depleted of GRP78 69.00 ± 9.74 vs. complete serum 133.5 ± 8.02, P = 0.012 (i); HepG2: serum depleted of GRP78 7.53 ± 1.17 vs. complete serum 10.01 ± 1.12, P = 0.0014 (j). k Representative images of Nile Red staining in HepG2 untreated, treated with HSP70, GRP78, serum from an obese IR, NAFLD subject before and after metabolic surgery and after immune depletion of HSP70 or GRP78. Scale bar is 50 µm