Journal: iScience

Article Title: Restored glyoxylate metabolism after AGXT gene correction and direct reprogramming of primary hyperoxaluria type 1 fibroblasts

doi: 10.1016/j.isci.2024.109530

Figure Lengend Snippet: Characterization of iHeps generated from PH1 patient-derived fibroblasts and AGXT gene corrected clones (A) Expression of hepatocyte specific genes in iHeps generated by direct cell reprogramming from PH1 patient-derived fibroblasts and AGXT gene corrected fibroblasts. The expression of genes involved in different liver functions was analyzed by RT-qPCR. Data from twenty-six independent experiments, conducted with two different PH1-donor fibroblasts (PH1-1 (n = 8) and PH1-2 (n = 4)), three AGXT point mutation corrected clones from the two PH1 donors (GC-1A (n = 4) and GC-1C (n = 1) derived from PH1-1. GC-2A (n = 2) derived from PH1-2) and four AGXT targeted knockin clones (pA-1A (n = 2), pB-1C (n = 2), and pD-1A (n = 2)), derived from PH1-1. pD-2A (n = 1) derived from PH1-2). Violin plot of data show median (bold dashed line) and interquartile range (light dashed line). Normal distribution of the data was assessed by a Kolmogorov-Smirnov test. Nonparametric Kruskal-Wallis with Dunn’s multiple comparisons test was used to compare, for each analyzed gene, iHeps with hepatocytes ($) or with fibroblasts (#). Significant differences between cell populations are marked as follows: $/#: p < 0.05; $$/##: p < 0.01; $$$/###: p < 0.001; $$$$/####: p < 0.0001. ABCC2: ATP binding cassette subfamily C member 2, CP: ceruloplasmin, FGL1: fibrinogen-like protein 1 and HP: haptoglobin. (B) Glycogen storage in the generated iHeps. Microscope images of PAS staining on starting human fibroblasts and generated iHeps, from healthy donor, PH1 patients and AGXT -corrected clones (GC-1A from PH1-1 and GC-2A from PH1-2). HepG2 cell line was used as positive control. All images were taken with 100× magnification. Scale bar: 75 μm.

Article Snippet: Three hundred micrograms of iHeps lysates and 5μg of HepG2 cell lysates were pre-incubated first with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG 1:5000 (Amersham, Cat#NA934), and second with the Protein A/G PLUS reagent (Santa Cruz Technologies, Cat#sc-2003).

Techniques: Generated, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Mutagenesis, Knock-In, Binding Assay, Microscopy, Staining, Positive Control

Journal: iScience

Article Title: Restored glyoxylate metabolism after AGXT gene correction and direct reprogramming of primary hyperoxaluria type 1 fibroblasts

doi: 10.1016/j.isci.2024.109530

Figure Lengend Snippet:

Article Snippet: Three hundred micrograms of iHeps lysates and 5μg of HepG2 cell lysates were pre-incubated first with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG 1:5000 (Amersham, Cat#NA934), and second with the Protein A/G PLUS reagent (Santa Cruz Technologies, Cat#sc-2003).

Techniques: Recombinant, Virus, Derivative Assay, Cell Culture, PCR Cloning, Reverse Transcription, SYBR Green Assay, Lactate Dehydrogenase Assay, Sequencing, Knock-In, Plasmid Preparation, Amplification, Gene Expression, Software, Extraction

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: 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: HepG2 cells were electroporated with GRP78/BiP plasmid (Origene, Cat. No. SC108086) using the Neon transfection system (Thermo Fisher Scientific, Cat. No. MPK5000) with slight modifications to the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Marker, Microscopy, Software, Western Blot, FLAG-tag, 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: 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: HepG2 cells were electroporated with GRP78/BiP plasmid (Origene, Cat. No. SC108086) using the Neon transfection system (Thermo Fisher Scientific, Cat. No. MPK5000) with slight modifications to the manufacturer’s instructions.

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

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: A - B Effect of oxLDL on UPR in HepG2 cells stably expressing WT /variants of LDLR A Western blots showing the expression pattern of ER chaperone-BiP (vi) and ER stress sensors (iii-v and vii-ix), apoptotic and inflammatory markers (xi-xvii) after 24 h of oxLDL treatment. NTC-Non treated control, TM-Tunicamycin (2 µg per ml, 24 h), DMSO-vehicle control, oxLDL-oxidized LDL (100 µg per ml, 24 h). GAPDH and Alpha tubulin were used as loading controls. Image panels were created using the QuickFigures plugin of ImageJ software, the contrast was enhanced uniformly, and confirmed that there were no significant changes with respect to proteins that exhibited minimal basal expression. ‘**’ represents the intermediate isoforms of ATF6. B (i-xi))-Histograms representing the change in expression of all the target proteins evaluated. For each of the four types of HepG2 cells studied (HepG2 mock , HepG2 WT−LDLR , HepG2 D482H−LDLR , and HepG2 C667F−LDLR ), comparisons and statistical analyses were carried out between the respective non-treated versus treated conditions and represented as mean + SD. Statistical analyses were carried out using Dunnett’s 1-way ANOVA, or 2-way ANOVA for ATF6 (Turkey’s post hoc test), where P < 0.05 = *, P < 0. 01 = **, P < 0.001 = ***, P < 0.0001 = ****, ns = not significant, n = 3 replicates

Article Snippet: HepG2 cells were electroporated with GRP78/BiP plasmid (Origene, Cat. No. SC108086) using the Neon transfection system (Thermo Fisher Scientific, Cat. No. MPK5000) with slight modifications to the manufacturer’s instructions.

Techniques: Stable Transfection, Expressing, Western Blot, Software

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: 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. HepG2 mock , HepG2 WT−LDLR , HepG2 D482H−LDLR , and HepG2 C667F−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

Article Snippet: HepG2 cells were electroporated with GRP78/BiP plasmid (Origene, Cat. No. SC108086) using the Neon transfection system (Thermo Fisher Scientific, Cat. No. MPK5000) with slight modifications to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Lactate Dehydrogenase Assay, Software, Negative Control

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: A - C BiP expression in oxLDL treated cells at various time points. Western blots depicting GRP78/BiP expression in HepG2 cells overexpressing empty vector, WT/variants of LDLR treated with 100 µg per ml of oxLDL at ( A ) 16 h, ( B ) 24 h, and ( C ) 48 h. Non- transduced HepG2 cells were used as controls to monitor the expression pattern of endogenous BiP expression at various time points. Non-treated or oxLDL-treated conditions for each cell type evaluated are marked by ‘-’ and ‘ + ’. Data are represented as mean + SD. 2-way ANOVA using Turkey’s post hoc test was used to compare BiP expression in non-treated (-) or treated ( +) conditions of each cell type with the non-treated and non-transduced HepG2 control cells (HepG2 − ). P < 0.05 = *, P < 0.01 = **, P < 0.001 = ***, P < 0.0001 = **** is represented for the comparisons between non-transduced HepG2 control cells (HepG2 − ) versus non-treated or treated HepG2 WT−LDLR , HepG2 D482H−LDLR , and HepG2 C667F−LDLR . P < 0.05 = # when the treated cells were compared with the respective non-treated cell types. ns = not significant, n = 3 replicates

Article Snippet: HepG2 cells were electroporated with GRP78/BiP plasmid (Origene, Cat. No. SC108086) using the Neon transfection system (Thermo Fisher Scientific, Cat. No. MPK5000) with slight modifications to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Plasmid Preparation

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: 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: For the generation of stably transfected HepG2 cell lines by lentiviral transduction, pLenti C-Myc-DDK-P2A Puro Lentiviral empty vector (Origene, Cat. No. PS100092), wild type-, D482H-, and C667F-LDLR plasmids were sequentially digested with MluI (NEB, Cat. No. R0198S) and AsiS/SgfI (NEB, Cat. No. R0630S).

Techniques: Expressing, Immunofluorescence, Marker, Microscopy, Software, Western Blot, FLAG-tag, 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: 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: For the generation of stably transfected HepG2 cell lines by lentiviral transduction, pLenti C-Myc-DDK-P2A Puro Lentiviral empty vector (Origene, Cat. No. PS100092), wild type-, D482H-, and C667F-LDLR plasmids were sequentially digested with MluI (NEB, Cat. No. R0198S) and AsiS/SgfI (NEB, Cat. No. R0630S).

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

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: A - B Effect of oxLDL on UPR in HepG2 cells stably expressing WT /variants of LDLR A Western blots showing the expression pattern of ER chaperone-BiP (vi) and ER stress sensors (iii-v and vii-ix), apoptotic and inflammatory markers (xi-xvii) after 24 h of oxLDL treatment. NTC-Non treated control, TM-Tunicamycin (2 µg per ml, 24 h), DMSO-vehicle control, oxLDL-oxidized LDL (100 µg per ml, 24 h). GAPDH and Alpha tubulin were used as loading controls. Image panels were created using the QuickFigures plugin of ImageJ software, the contrast was enhanced uniformly, and confirmed that there were no significant changes with respect to proteins that exhibited minimal basal expression. ‘**’ represents the intermediate isoforms of ATF6. B (i-xi))-Histograms representing the change in expression of all the target proteins evaluated. For each of the four types of HepG2 cells studied (HepG2 mock , HepG2 WT−LDLR , HepG2 D482H−LDLR , and HepG2 C667F−LDLR ), comparisons and statistical analyses were carried out between the respective non-treated versus treated conditions and represented as mean + SD. Statistical analyses were carried out using Dunnett’s 1-way ANOVA, or 2-way ANOVA for ATF6 (Turkey’s post hoc test), where P < 0.05 = *, P < 0. 01 = **, P < 0.001 = ***, P < 0.0001 = ****, ns = not significant, n = 3 replicates

Article Snippet: For the generation of stably transfected HepG2 cell lines by lentiviral transduction, pLenti C-Myc-DDK-P2A Puro Lentiviral empty vector (Origene, Cat. No. PS100092), wild type-, D482H-, and C667F-LDLR plasmids were sequentially digested with MluI (NEB, Cat. No. R0198S) and AsiS/SgfI (NEB, Cat. No. R0630S).

Techniques: Stable Transfection, Expressing, Western Blot, Software

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: 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. HepG2 mock , HepG2 WT−LDLR , HepG2 D482H−LDLR , and HepG2 C667F−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

Article Snippet: For the generation of stably transfected HepG2 cell lines by lentiviral transduction, pLenti C-Myc-DDK-P2A Puro Lentiviral empty vector (Origene, Cat. No. PS100092), wild type-, D482H-, and C667F-LDLR plasmids were sequentially digested with MluI (NEB, Cat. No. R0198S) and AsiS/SgfI (NEB, Cat. No. R0630S).

Techniques: Western Blot, Expressing, Lactate Dehydrogenase Assay, Software, Negative Control

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: A - C BiP expression in oxLDL treated cells at various time points. Western blots depicting GRP78/BiP expression in HepG2 cells overexpressing empty vector, WT/variants of LDLR treated with 100 µg per ml of oxLDL at ( A ) 16 h, ( B ) 24 h, and ( C ) 48 h. Non- transduced HepG2 cells were used as controls to monitor the expression pattern of endogenous BiP expression at various time points. Non-treated or oxLDL-treated conditions for each cell type evaluated are marked by ‘-’ and ‘ + ’. Data are represented as mean + SD. 2-way ANOVA using Turkey’s post hoc test was used to compare BiP expression in non-treated (-) or treated ( +) conditions of each cell type with the non-treated and non-transduced HepG2 control cells (HepG2 − ). P < 0.05 = *, P < 0.01 = **, P < 0.001 = ***, P < 0.0001 = **** is represented for the comparisons between non-transduced HepG2 control cells (HepG2 − ) versus non-treated or treated HepG2 WT−LDLR , HepG2 D482H−LDLR , and HepG2 C667F−LDLR . P < 0.05 = # when the treated cells were compared with the respective non-treated cell types. ns = not significant, n = 3 replicates

Article Snippet: For the generation of stably transfected HepG2 cell lines by lentiviral transduction, pLenti C-Myc-DDK-P2A Puro Lentiviral empty vector (Origene, Cat. No. PS100092), wild type-, D482H-, and C667F-LDLR plasmids were sequentially digested with MluI (NEB, Cat. No. R0198S) and AsiS/SgfI (NEB, Cat. No. R0630S).

Techniques: Expressing, Western Blot, Plasmid Preparation