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

Journal:

Article Title: Role of Cholesterol Pathways in Norovirus Replication ▿

doi: 10.1128/JVI.00005-09

Figure Lengend Snippet: Expression of RL after transfection of pCI, pNV-GFP, pNV-RL, or pNV-RLΔGDD alone or with pCI-SHP or pCMV6-LDLR into MVA-T7-infected Vero cells

Article Snippet: To examine the role of LDLR expression in NV replication, the cytomegalovirus (CMV) promoter-based vector expressing LDLR (pCMV6-LDLR) was purchased from OriGene (Rockville, MD).

Techniques: Expressing, Transfection, Luciferase

Journal:

Article Title: Role of Cholesterol Pathways in Norovirus Replication ▿

doi: 10.1128/JVI.00005-09

Figure Lengend Snippet: Correlation of the expression levels of LDLR and NV replication (A) and promotion of NV replication by the expression of LDLR (B). (A) The levels of LDLR RNA and NV genome versus treatment with various agents, including statins, ACAT inhibitors, and lipoprotein-free FBS, or the transfection of pCI-SHP or pCI-LC were significantly correlated (R2 = 0.89) by a simple linear regression model. These data are from both collated results from the other figures and additional experiments. (B) Transfection of pNV-GFP alone, cotransfection of pNV-GFP with pCMV6-LDLR, or cotransfection of pNV-GFPΔGDD with pCI-SHP into MVA-T7-infected BHK cells. GFP-positive cells were observed under a fluorescent (upper panel) or a light microscope (lower panel). Cotransfection of pNV-GFPΔGDD and pCI-SHP served as a negative control.

Article Snippet: To examine the role of LDLR expression in NV replication, the cytomegalovirus (CMV) promoter-based vector expressing LDLR (pCMV6-LDLR) was purchased from OriGene (Rockville, MD).

Techniques: Expressing, Transfection, Cotransfection, Infection, Light Microscopy, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Hypercholesterolemia-Induced HDL Dysfunction Can Be Reversed: The Impact of Diet and Statin Treatment in a Preclinical Animal Model

doi: 10.3390/ijms23158596

Figure Lengend Snippet: Effects of diet and rosuvastatin intervention on HDL antioxidant capacity. Changes in HDL antioxidant capacity are presented as percent antioxidant index (( A ), % AOI; measure for capacity to reverse LDL oxidation) and levels of conjugated dienes (( B – E ) product of lipid oxidation). Data are shown as mean ± SEM (( A ); for visual clarity of the graph) or SD (whiskers) over the period of 40 days. ( A ) includes all four groups, whereas ( B – E ) represent each group individually. Shapiro–Wilk test confirmed normality (alpha = 0.05), and data were analyzed by one-way ANOVA with Tukey’s multicomparison test and considered significant with a p -value < 0.05. For ( A ): * p < 0.05 in comparison to day 0; † p < 0.05 in comparison to day 10; # p < 0.05 compared to HC; § p < 0.05 in comparison to NC. For ( B – E ): * p < 0.05, ** p < 0.0001 in comparison to day 0; † p < 0.05 in comparison to day 10. d0–40: day 0–40; HC: hypercholesterolemic diet; NC: normocholesterolemic diet; +R/+rosu: + rosuvastatin; LDL: low-density lipoprotein.

Article Snippet: HDL AOI is based on the capacity of HDL particles to actively reverse LDL oxidation using 2,7-dichlorofluoresceindiacetate (H2DCFDA) (Invitrogen Inc., Carlsbad, CA, USA) and copper-oxidized LDL particles (Kalen Biomedical Inc., Savage, MD, USA).

Techniques: