plasmid pcmv6-grp78  (OriGene)

Journal: Heliyon

Article Title: eIF2α-mediated integrated stress response links multiple intracellular signaling pathways to reprogram vascular smooth muscle cell fate in carotid artery plaque

doi: 10.1016/j.heliyon.2024.e26904

Figure Lengend Snippet: eIF2α-mediated integrated stress responses verified in carotid stable and vulnerable plaques. (A) Representative specimens of stable and vulnerable plaques stained with H&E, and immunohistochemistry (IHC) of VSMCs with organelle markers, including α-SMA, PERK, XBP1, GRP78, cytochrome C (Cyt C), LAMP1, and eIF2α. (B) Quantitative analysis of the expression levels of α-SMA, PERK, XBP1, GRP78, Cyt C, LAMP1, and eIF2α (n = 15 cases in each group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 200 μm, 1 cm, 2 cm, respectively.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, blocked in 5% skin milk for 0.5 h, and incubated with the following primary antibodies: anti-PERK(D11A8; Cell Signaling Technology), anti-actin (BE0021-100; Easybio), anti-XBP1 (102256-T34; Sino Biological Inc.), anti-DRP1 (D6C7; Cell Signaling Technology), anti-phospho-DRP1 (Ser616) (pDrp1 (S616)) (D9A1; Cell Signaling Technology), anti-Tom 20 (D8T4N; Cell Signaling Technology), anti-light chain 3 (LC3; 14322-T44; Sino Biological Inc.), anti-LAMP1 (21997-1-AP; Proteintech), anti-GRP78 (HG12063-UT; Sino Biological Inc.), and anti-eIF2α (11170-1-AP; Proteintech) antibodies.

Techniques: Staining, Immunohistochemistry, Expressing

Journal: Heliyon

Article Title: eIF2α-mediated integrated stress response links multiple intracellular signaling pathways to reprogram vascular smooth muscle cell fate in carotid artery plaque

doi: 10.1016/j.heliyon.2024.e26904

Figure Lengend Snippet: Eukaryotic translation initiation factor (eIF)-2α integrates the stress responses of multiple organelles to regulate atherosclerotic plaque progression. (A) Representative image of ER labeled with GFP-Sec61β and mitochondria stained with deep red Mitotracker in HC-VSMCs treated with DMSO (Ctrl.), thapsigargin (Tg; 5 μM), Oligo (1 μM), and combined Tg and oligo for 24 h. (B) Quantitative analysis of the percentage of cells with ER whorls and the aspect ratio in HC-VSMCs treated with DMSO (Ctrl.), Tg, Oligo, and combined Tg and Oligo (n = 30 cells per group). (C) Representative image of ER labeled with GFP-Sec61β and mitochondria stained with deep red Mitotracker in HC-VSMCs treated with DMSO (Ctrl.), Tg (5 μM), ISRIB (100 nM), Oligo (1 μM), and their combination for 24 h. (D) Quantitative analysis of the percentage of cells with ER whorls and the aspect ratio in HC-VSMCs treated with DMSO, DTT, ISRIB, Oligo, and their combination (n = 30 cells per group). (E) Western blotting analysis of GRP78, XBP1-u, XBP1-s, p-DRP1(S616), LC3-I, and eIF2α in HC-VSMCs treated with DMSO (Cntrl.), Tg, Oligo, and combined Tg and Oligo (n = 3 in each group). (F) Western blotting analysis of GRP78, XBP1-u, XBP1-s, p-DRP1(S616), LC3-I, and eIF2α in HC-VSMCs treated with DMSO, Tg, ISRIB, Oligo, and their combination (n = 3 times each group). *p < 0.05, **p < 0.01, and ***p < 0.001. Scale bar = 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, blocked in 5% skin milk for 0.5 h, and incubated with the following primary antibodies: anti-PERK(D11A8; Cell Signaling Technology), anti-actin (BE0021-100; Easybio), anti-XBP1 (102256-T34; Sino Biological Inc.), anti-DRP1 (D6C7; Cell Signaling Technology), anti-phospho-DRP1 (Ser616) (pDrp1 (S616)) (D9A1; Cell Signaling Technology), anti-Tom 20 (D8T4N; Cell Signaling Technology), anti-light chain 3 (LC3; 14322-T44; Sino Biological Inc.), anti-LAMP1 (21997-1-AP; Proteintech), anti-GRP78 (HG12063-UT; Sino Biological Inc.), and anti-eIF2α (11170-1-AP; Proteintech) antibodies.

Techniques: Labeling, Staining, Western Blot

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, Membrane, Control, 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 - 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, Control, Comparison, Lactate Dehydrogenase Assay, Software, Negative Control, Membrane

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, 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 graphical abstract summarizing the study model and major findings. The thin black arrows and red lightning symbols represent ER stress-caused by ER retention of LDLR, and oxLDL induced stress- caused by oxidation of excess levels of LDL. The brown, maroon and blue colored arrows represent ER stress dissipated along the ATF6, PERK and IRE1A branches, respectively. The pacman symbol denotes S1P/S2P protease in the Golgi Complex. The red inhibitory arrows denote the proteins that inhibit/are inhibited during the process. The scissors symbolize splicing of XBP-1 transcripts. The blue dots and red arrows in the mitochondria denote release of cytochrome C caused by disrupted mitochondrial membrane potential. Purple arrows-direction of LDH release from cells due to cytotoxicity. The continuous low-medium dial denotes restored cellular responses brought about by neon transfection of GRP78/BiP plasmid along ( A ) ER, ( B ) mitochondria and ( C ) plasma membrane. The downward arrows denote lowering of target proteins evaluated

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: Membrane, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Discovery of a Novel Target for the Dysglycemic Chromogranin A Fragment Pancreastatin: Interaction with the Chaperone GRP78 to Influence Metabolism

doi: 10.1371/journal.pone.0084132

Figure Lengend Snippet: ( 1A ) and ( 1B ) Affinity chromatography followed by LC-tandem-MS/MS . ( 1A ): SDS-PAGE separation, followed by trypsin digestion and C-18 liquid chromatography . The mouse liver homogenate was subjected to ligand affinity chromatography using a biotinylated human pancreastatin (hCgA 273–301 -amide) peptide as “bait”. Triton-solubilized liver membranes (as “prey”) were incubated with bio-PST, which was pre-incubated with streptavidin agarose. Bound proteins were eluted by lowering pH to 5.5, and analyzed by SDS-PAGE. Gel lanes: 1 = Protein size standards; 2–4 = serial fractions during elution at lower pH = 5.5. The major ∼75 kDa band was excised from the gel and subjected to trypsin digestion, and the resulting peptides were separated by liquid chromatography (chromatogram shown above). Selection (arrow) of a peak for further analysis is also shown. ( 1B ): First and second dimensions of MS, with sequence identification . The first dimension of MS is displayed above. In the second dimension of MS, one ion is chosen (arrow), and a representative MS/MS spectrum of mouse GRP78 354–368 is shown. ( 1C ): Identification of GRP78 (HSPA5) after PST affinity elution: Peptide coverage during LC-MS/MS . The map was generated from target MS/MS fragment sequences in Protein Pilot 2.0 (Life Technologies).; Peptides identified with at least 95% confidence are in bold type. Template: UniProtKB P20029 (GRP78_MOUSE).

Article Snippet: The pCMV promoter-driven human GRP78 (HSPA5) expression plasmid was from Origene < www.origene.com/ >. pSV-Beta-Gal (a transfection efficiency control plasmid, in which the SV40 early promoter drives expression of beta-galactosidase) was obtained from Promega < www.promega.com >.

Techniques: Affinity Chromatography, Tandem Mass Spectroscopy, SDS Page, Liquid Chromatography, Incubation, Selection, Sequencing, Liquid Chromatography with Mass Spectroscopy, Generated

Journal: PLoS ONE

Article Title: Discovery of a Novel Target for the Dysglycemic Chromogranin A Fragment Pancreastatin: Interaction with the Chaperone GRP78 to Influence Metabolism

doi: 10.1371/journal.pone.0084132

Figure Lengend Snippet: ( A ) Liver and adrenal gland expression of GRP78 in CHGA(−/−) mice compared to wild-type (WT, +/+). ( B ) Expression of UPR pathway genes in liver (upper panel) and adrenal gland (lower panel).

Article Snippet: The pCMV promoter-driven human GRP78 (HSPA5) expression plasmid was from Origene < www.origene.com/ >. pSV-Beta-Gal (a transfection efficiency control plasmid, in which the SV40 early promoter drives expression of beta-galactosidase) was obtained from Promega < www.promega.com >.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Discovery of a Novel Target for the Dysglycemic Chromogranin A Fragment Pancreastatin: Interaction with the Chaperone GRP78 to Influence Metabolism

doi: 10.1371/journal.pone.0084132

Figure Lengend Snippet: ( A ) ATPase enzymatic activity of GRP78 (concentration-dependent, steady-state) was measured as an increase in A 620 (liberated as free phosphate, Pi) using 0.063, 0.125, 0.25 or 0.5 µM GRP78 in 50 µl of assay buffer, as described in . ( B ) PST inhibition of the ATPase enzymatic activity of GRP78 . ATPase activity was measured in the presence of 0, 1, or 10 µM PST: IC 50 of PST inhibition (by interpolation). ( C ) GRP78 gene expression: Inhibition by PST . Human HepG2 hepatocytes were transfected with GRP78 promoter driving a luciferase reporter along with beta Gal reporter plasmid and 5 hrs after transfection cells were treated with tunicamycin (5 µg/mL) in the presence or absence of PST (100 nM). Cells were harvested after 18–20 hrs for measurement of luciferase activity, beta gal activity and protein.

Article Snippet: The pCMV promoter-driven human GRP78 (HSPA5) expression plasmid was from Origene < www.origene.com/ >. pSV-Beta-Gal (a transfection efficiency control plasmid, in which the SV40 early promoter drives expression of beta-galactosidase) was obtained from Promega < www.promega.com >.

Techniques: Activity Assay, Concentration Assay, Inhibition, Expressing, Transfection, Luciferase, Plasmid Preparation

Journal: PLoS ONE

Article Title: Discovery of a Novel Target for the Dysglycemic Chromogranin A Fragment Pancreastatin: Interaction with the Chaperone GRP78 to Influence Metabolism

doi: 10.1371/journal.pone.0084132

Figure Lengend Snippet: ( A ) G6Pase expression, PST, and GRP78 . Human HepG2 hepatocytes were transfected with G6P-ase→luciferase, along with pCMV-GRP78 or an empty vector (pCMV promoter without insert). 5 hrs after transfection, cells were treated with PST (100 nM) itself (versus mock), or by UPR activation (tunicamycin, 5 µg/mL; or thapsigargin, 0.3 µM). Cells were harvested at 18–20 hrs to measure luciferase activity and protein. ( B ) G6Pase expression during ATPase enzymatic activity inhibition of GRP78 . Human HepG2 hepatocytes were transfected as in (A), and 5 hrs later were treated with the GRP78 ATPase inhibitors VER-155008 (1 µM), ADP (10 µM), or PST (100 nM). Cells were harvested at 18–20 hrs to measure luciferase activity and protein. ( C ) GRP78 expression: Visualization by immunoblot . Changes in GRP78 expression are shown by anti-GRP78 immunoblot (control: anti-actin), during either UPR activation (by thapsigargin 0.3 µM or tunicamycin 5 µg/mL), or exogenous over-expression (transfection of pCMV-GRP78).

Article Snippet: The pCMV promoter-driven human GRP78 (HSPA5) expression plasmid was from Origene < www.origene.com/ >. pSV-Beta-Gal (a transfection efficiency control plasmid, in which the SV40 early promoter drives expression of beta-galactosidase) was obtained from Promega < www.promega.com >.

Techniques: Expressing, Transfection, Plasmid Preparation, Activation Assay, Luciferase, Activity Assay, Inhibition, Western Blot, Over Expression