Fig. 2

A-D Upregulation of ER stress response along all three arms of the UPR in HepG2 cells expressing FLAG-tagged-WT/missense variants of LDLR. A Relative mRNA expression of the three arms of ER stress sensors and their downstream targets, namely (i) GRP78/BiP (ii) XBP-1(s) (iii) ATF6 (iv) ATF4 (v) CHOP (vi) P58IPK and (vii) EDEM1. Comparisons were done between non-transduced HepG2 and transduced HepG2 and represented as mean + SD. Dunnett’s 1-way ANOVA; p < 0.05, p < 0.01 = **, n = 3 replicates. B Western blotting with antibodies to (iii) the ER chaperone-BiP, and UPR arms confirm the activation of ER stress response along the (i, ii, and vi) IRE1Alpha/spliced XBP-1-, (iv and v) PERK/eIF2A-, and (vii) ATF6 branches, along with (viii) GAPDH. The inactive, ER- membrane-bound ATF6 protein of 90 kDa is represented as pATF6 (90). pATF6(50) represents the activated and cleaved nuclear fragment, whereas ‘**’ denotes the intermediate isoforms of ATF6 ((pATF6(intermediates)) detected on Western blots. The corresponding histograms represent the relative fold change of phosphorylated to Total protein or GAPDH, the loading control. Data are represented as mean + SD. Statistical analyses were carried out using Students’ unpaired, two-tailed t- test or 2-way ANOVA (Turkey’s post hoc), P < 0.05 = * or #, P < 0.01 = **, P < 0.001 = ***, ns = not significant; n = 2 replicates. C Immunofluorescence image panel illustrating ATF6 translocation from ER to Golgi complex and nucleus in mock, WT-, D482H- and C667F-expressing HepG2 cells. The white horizontal lines at the bottom right corner of each merged image represents the scale bar = 20 µm. D Merged panels zoomed to 50% magnification. The fluorescent images were captured using the 100-X oil immersion objective of a Nikon Eclipse 2000 Confocal Microscope and color-enhanced using ImageJ software. Color images were obtained by merging red (ATF6) and green (Calnexin, Golgin-97, or H3) channels. ER marker- Calnexin (CNX), Golgi complex marker- Golgin-97, Nuclear marker- H3 (Histone3). The thin blue arrows in the merged panels are used to highlight regions emitting signals from the green channel alone and represent regions where ATF6 does not express with the respective organelle marker. The thick white arrows in these panels highlight yellow fluorescence indicating overlapping signals from the green and red channels and denote regions where ATF6 co-expresses with the respective organelle markers recorded from both the green