Fig. 5
A-C GRP78/BiP mediated rescue of oxLDL and ER stress-induced hepatotoxicity. A-1(i-x))-Western blot panel representing the alleviation of ER stress response, apoptotic and inflammatory markers in HepG2 cells overexpressing GRP78/BiP. HepG2mock, HepG2WT−LDLR, HepG2D482H−LDLR, and HepG2C667F−LDLR that transiently overexpress BiP are marked ‘ + ’, while cells expressing endogenous BiP are denoted with ‘-’ signs. oxLDL was administered at a dose of 100 µg per ml for 48 h and non-treated and treated conditions are denoted by ‘-’ and ‘ + ’, respectively. NTC-non treated control, TM-tunicamycin. A-2(i-vii))-The band intensities of GAPDH normalized target proteins were quantitated and data are represented as mean + SD. Statistical analysis was performed using One-way ANOVA (Turkey’s multiple comparison test). P < 0.05 = *, P < 0.01 = **, P < 0.001 = ***, P < 0.0001 = ****, ns = not significant. n = 3 replicates. B (i-iv))- Bar diagrams representing LDH release assay for (i) Mock (ii) WT-LDLR-Fg (iii) D482H-LDLR-Fg and (iv) C667F-LDLR-Fg-expressing HepG2 cells under various treatment conditions. Two-way ANOVA (Turkey’s post hoc test) was used to calculate statistical significance using GraphPad Prism6 software. P < 0.5 *, P < 0.001 **, n = 3 replicates. 5C (i-xxiv)- Representative FACS plots of non-treated control (NTC) cells used for gating (i, vii, xiii and xix); NTC as negative control (ii, xviii, xiv and xx); CCCP-treated positive controls for JC-1 assay (iii, ix, xv and xxi); oxLDL treated (iv, x, xvi and xxii); non-oxLDL treated, BiP/GRP78- overexpressing cells (v, xi, xvii and xxiii); BiP/GRP78- overexpressing, oxLDL- treated cells (vi, xii, xviii, xxiv). The histogram represents the ratio of cells exhibiting JC-1 aggregates (Q2) to monomers (Q4) as a measure of the mitochondrial membrane potential C (xxv)). Data represent the mean ± SD of three independent experiments. Statistical significance was determined using two-way ANOVA and Turkey’s post hoc test. P < 0.05, * P < 0.01 ** and P < 0.001, ***n = 3 replicates