Journal: Lipids in health and disease

Article Title: GRP78/BiP alleviates oxLDL-induced hepatotoxicity in familial hypercholesterolemia caused by missense variants of LDLR in a HepG2 cellular model.

doi: 10.1186/s12944-023-01835-x

Figure Lengend Snippet: Fig. 1 A-B Generation of lentiviral mediated stable HepG2 lines expressing Wild Type/missense variants of LDLR. A Immunofluorescence with anti-FLAG (Fg), anti-Calnexin (CNX), and anti- Na+K+ ATPase antibodies to understand the subcellular localization of FLAG-tagged (Fg) WT-LDLR, D482H-LDLR, and C667F-LDLR in stable HepG2 cells. Wild-type LDLR is expressed predominantly on the plasma membrane, whereas D482H-LDLR and C667F-LDLR are retained in the Endoplasmic Reticulum (ER). Calnexin (CNX) was used as the ER marker and Na+K+ ATPase as the marker of the plasma membrane. The color images represent merged images of red (Fg) and green (Calnexin or Na+K+ ATPase) channels. 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. Scale bar = 20 µm. B (i)) Western blotting with anti-FLAG antibody confirms the expression of mature (M) and precursor (P) LDLR protein of 160 kDa and 120 kDa, respectively in WT-LDLR expressing HepG2. The missense variants D482H-LDLR and C667F-LDLR fail to express the mature 160 kDa protein and confirm the ER retention by the prominent expression of the immature 120 kDa protein. The FLAG tag of 25 amino acids is not expressed in Non-transduced HepG2 control cells and not detected in mock transduced HepG2. Alpha tubulin represents the loading control. (B(ii)). The fold change of Alpha tubulin normalized mature and immature forms of FLAG-tagged LDLR is represented as mean + SD. n = 3. Unpaired students’ t-test, two-tailed, P < 0.05 = *, P < 0.01 = **

Article Snippet: The ORF of wildtype and missense variants of LDLR was ligated into pLenti C-Myc-DDK-P2A Puro Lentiviral empty vector (Origene, Cat. No. PS100092), treated with Antarctic phosphatase (NEB, Cat No. M0289S), using T4DNA ligase (NEB, Cat. No. M020L).

Techniques: Expressing, Immunofluorescence, Clinical Proteomics, Membrane, Marker, Microscopy, Software, Western Blot, FLAG-tag, Control, Two Tailed Test

Journal: Lipids in health and disease

Article Title: GRP78/BiP alleviates oxLDL-induced hepatotoxicity in familial hypercholesterolemia caused by missense variants of LDLR in a HepG2 cellular model.

doi: 10.1186/s12944-023-01835-x

Figure Lengend Snippet: 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

Article Snippet: The ORF of wildtype and missense variants of LDLR was ligated into pLenti C-Myc-DDK-P2A Puro Lentiviral empty vector (Origene, Cat. No. PS100092), treated with Antarctic phosphatase (NEB, Cat No. M0289S), using T4DNA ligase (NEB, Cat. No. M020L).

Techniques: Expressing, Western Blot, Activation Assay, Membrane, Control, Two Tailed Test, Immunofluorescence, Translocation Assay, Microscopy, Software, Marker, Fluorescence