Journal: PLoS ONE

Article Title: OBATOCLAX and ABT-737 Induce ER Stress Responses in Human Melanoma Cells that Limit Induction of Apoptosis

doi: 10.1371/journal.pone.0084073

Figure Lengend Snippet: A. Following treatment of melanoma cell lines Mel-RM and MM200 for indicated periods with Obatoclax (1nM) or ABT-737 (10µM), whole cell lysates were subjected to Western blot analysis for the ER stress markers GRP78 and phosphorylated eIF2α. Data are representative of three individual experiments. B. Expression of the active form of XBP-1 was determined by agarose gel electrophoresis following Apa-LI digestion of XBP-1 mRNA. Indicated is the longer (active) fragment and the two shorter (inactive) bands. Data are representative of three individual experiments.

Article Snippet: The mouse mAb against Bcl-2 and Mcl-1, and the rabbit polyclonal Abs against Bcl-XL and GRP78, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Expressing, Agarose Gel Electrophoresis

Journal: Science translational medicine

Article Title: Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica.

doi: 10.1126/scitranslmed.aai9111

Figure Lengend Snippet: Fig. 4. Identification of GRP78 as the target antigen of NMO-rAbs ON-12-2-46 and ON-07-5-31. (A) Proteins isolated from crude membrane lysates of U87MG (glioblastoma) and OL (oligodendroglia) cells were separated by polyacrylamide gel electrophoresis and analyzed by Western blot. Multiple protein bands reacted with the NMO-rAbs. Unlabeled lanes contain molecular weight standards. (B) Schematic detailing the purification scheme (X-linker, cross-linker; DSS, disuccinimidyl suber- ate). (C) The protein eluted with DTT from rAb cross-linked to the U87MG cells using DTSSP/disuccinimidyl suberate (DSS) was subjected to polyacrylamide gel elec- trophoresis and immunoblot analysis (lane 1, molecular weight standards; lane 2, protein bound to ON-07-5-31 rAb; lane 3, protein bound to ON-12-2-46 rAb). The blots were probed with the indicated NMO-rAb. (D) Immunofluorescence labeling of U87MG glioblastoma cells with ON-07-5-31 rAb (10 mg/ml) and rabbit anti-GRP78 antisera shows colocalization of the two proteins (merge in yellow). Nuclei are counterstained with DAPI (blue). (E) ON-12-2-46 rAb (20 mg/ml; green channel) and rabbit anti-GRP78 antiserum (red channel) immunoreactivity on U87MG glioblastoma cells after 24 hours of treatment with 0.5 mM thapsigargin (TG) or DMSO (control). The merged image shows colocalization of the two proteins (yellow). (F) Immunoblot analysis of commercial recombinant GRP78 protein purified from transfected human embryonic kidney (HEK) 293 cells (lane 1) or bacteria (lane 2) and probed with rAb or rabbit anti-GRP78 serum demonstrates that NMO-rAbs ON-12-2-46 and ON-07-5-31 recognize GRP78. (G) Binding of ON-12-2-46 rAb (2 mg/ml) (green channel) to the U87MG glioblastoma cells in competition immunofluorescence assay is blocked in the presence of commercial recombinant GRP78 protein (5 mg). DAPI-stained nuclei are depicted in blue.

Article Snippet: For Western blot verification, purified GRP78 protein [from OriGene (100 ng per well) and Novus Biologicals (0.5 mg per well)] was 9 of 12 SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on A pril 01, 2024 subjected to SDS–polyacrylamide gel electrophoresis, transferred to PVDF, incubated with 0.5% glutaraldehyde in PBS (15 min), with 0.25 M tris (pH 7.5) in PBS (15 min), washed three times in TBS (10 min), blocked, and then processed as described above.

Techniques: Isolation, Membrane, Polyacrylamide Gel Electrophoresis, Western Blot, Molecular Weight, Purification, Immunofluorescence, Labeling, Control, Recombinant, Transfection, Bacteria, Binding Assay, Staining

Journal: Science translational medicine

Article Title: Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica.

doi: 10.1126/scitranslmed.aai9111

Figure Lengend Snippet: Fig. 5. Removal of GRP78-specific IgG from pooled NMO-IgG reduces NF-kB p65 nuclear translocation in BMECs. Numbers of cells positive for nuclear NF-kB p65 translocation were compared in cultures exposed to pooled NMO-IgG (200 and 150 mg/ml) immunoadsorbed against GRP78 or control SNAP25 protein. Data are normalized to control SNAP25-absorbed NMO IgG. Data are means ± SEM from five experiments in triplicate. Statistical significance was assessed by paired two-tailed t test (*P < 0.05).

Article Snippet: For Western blot verification, purified GRP78 protein [from OriGene (100 ng per well) and Novus Biologicals (0.5 mg per well)] was 9 of 12 SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on A pril 01, 2024 subjected to SDS–polyacrylamide gel electrophoresis, transferred to PVDF, incubated with 0.5% glutaraldehyde in PBS (15 min), with 0.25 M tris (pH 7.5) in PBS (15 min), washed three times in TBS (10 min), blocked, and then processed as described above.

Techniques: Translocation Assay, Control, Two Tailed Test

Journal: Science translational medicine

Article Title: Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica.

doi: 10.1126/scitranslmed.aai9111

Figure Lengend Snippet: Fig. 6. In vivo effects of ON-12-2-46 on mouse brain vasculature. (A) Experimental setup. IV, intravenously; IP, intraperitoneally. 3D images of 20-mm mouse coronal brain sections immunostained for human IgG (rAb ON-7-5-53) and AQP4 (B), human IgG (rAb ON-7-5-53) and mouse fibrinogen (C), or human IgG, mouse albumin, and AQP4 (D) after administration of murinized GRP78-specific NMO rAb ON-12-2-46 or control rAb in combination with human AQP4-specific rAb ON-7-5-53. (E) Mea- surement of the average diameter of vessels (mean ± SEM) in mice treated with GRP78-specific NMO rAb ON-12-2-46 (six mice) and control rAb (four mice) in two separate experiments. Vessels were quantified from control-rAb–injected (n = 35) and ON-12-2-46–injected (n = 31) 20× high-power fields, and the diameters were measured using AxioVision LE. P values were calculated by Mann-Whitney U test (***P < 0.0001).

Article Snippet: For Western blot verification, purified GRP78 protein [from OriGene (100 ng per well) and Novus Biologicals (0.5 mg per well)] was 9 of 12 SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on A pril 01, 2024 subjected to SDS–polyacrylamide gel electrophoresis, transferred to PVDF, incubated with 0.5% glutaraldehyde in PBS (15 min), with 0.25 M tris (pH 7.5) in PBS (15 min), washed three times in TBS (10 min), blocked, and then processed as described above.

Techniques: In Vivo, Control, Injection, MANN-WHITNEY

Journal: Cancer Research

Article Title: Glucose-Regulated Protein 78 Controls Cross-talk between Apoptosis and Autophagy to Determine Antiestrogen Responsiveness

doi: 10.1158/0008-5472.can-12-0269

Figure Lengend Snippet: Figure 1. GRP78 is elevated in antiestrogen-resistant breast cancer. A, immunohistochemical staining of human ERþ, triple- negative, and HER2-amplified breast tumors and their surrounding normal tissue. Sections were stained using a GRP78-specific antibody; tissue sections incubated with a nonspecific mouse IgG were used as a negative control. B, quantification of GRP78 expression in human ERþ, triple- negative (TN), and HER2-amplified breast tumors. , P < 0.05; n ¼ 2–4, average of 3 fields for each section was quantified. C, proteins were isolated from LCC1, LCC9, MCF7, and MCF7-RR human breast cancer cell lines and Western blotting hybridization conducted to measure GRP78 protein expression. Immunohistochemical staining of LCC1 and LCC9 orthotopic tumor sections using a GRP78-specific antibody, n ¼ 4. D, representative tumor sections from tamoxifen (TAM)-treated and control rat mammary tumors stained with GRP78-specific antibody.

Article Snippet: The following materials were obtained as indicated: 4- hydroxytamoxifen (Sigma-Aldrich); ICI 182,780 (Tocris Bioscience); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL); FBS and bovine calf charcoal-stripped serum (CCS; Equitech-Bio Inc.); Lipofectamine RNAiMAX reagent (Invitrogen); GRP78 and AMPK siRNA (OnTarget plus SMART pool; consisting of 3 different siRNA for same target; ThermoScientific Dharmacon); GRP78 plasmid (HSPA5 Trueclone cDNA; OriGene); ATG5 and TSC2 siRNA (Cell Signaling Technology); mouse IgG negative control antibody (Dako); crystal violet (Fisher Scientific); and caspase inhibitor Z-VAD-FMK (Tocris Bioscience).

Techniques: Immunohistochemical staining, Staining, Incubation, Negative Control, Expressing, Isolation, Western Blot, Hybridization, Control

Journal: Cancer Research

Article Title: Glucose-Regulated Protein 78 Controls Cross-talk between Apoptosis and Autophagy to Determine Antiestrogen Responsiveness

doi: 10.1158/0008-5472.can-12-0269

Figure Lengend Snippet: Figure 2. Modulation of GRP78 in human breast cancer cells alters antiestrogen responsiveness. LCC9 and MCF7-RR cells were transfected with control or GRP78 siRNA, and LCC1 and MCF7 cells were transfected with control pcDNA or GRP78(þ); protein homogenates were isolated to determine GRP78 protein knockdown by Western blotting hybridization. LCC9 transfected with control or GRP78 siRNA, treated with ICI (A) or tamoxifen (TAM; B; 0.1% v/v ethanol vehicle, 10, 100, 1,000 nmol/L for 6 days), and cell density measured by crystal violet. C, MCF7-RR cells transfected with either control or GRP78 siRNA, treated with TAM (0.1% v/v ethanol vehicle, 10, 100, 1,000 nmol/L for 6 days), and cell density measured by crystal violet. LCC1 (D) or MCF7 (E) cells transfected with either control pcDNA or GRP78(þ), treated with ICI or TAM (0.1% v/v ethanol vehicle, 10, 100, 1,000 nmol/L for 6 days), and cell density measure following crystal violet staining (n ¼ 3–4; one-way ANOVA with Dunnett post hoc analysis; , P < 0.05 compared with vehicle-treated control).

Article Snippet: The following materials were obtained as indicated: 4- hydroxytamoxifen (Sigma-Aldrich); ICI 182,780 (Tocris Bioscience); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL); FBS and bovine calf charcoal-stripped serum (CCS; Equitech-Bio Inc.); Lipofectamine RNAiMAX reagent (Invitrogen); GRP78 and AMPK siRNA (OnTarget plus SMART pool; consisting of 3 different siRNA for same target; ThermoScientific Dharmacon); GRP78 plasmid (HSPA5 Trueclone cDNA; OriGene); ATG5 and TSC2 siRNA (Cell Signaling Technology); mouse IgG negative control antibody (Dako); crystal violet (Fisher Scientific); and caspase inhibitor Z-VAD-FMK (Tocris Bioscience).

Techniques: Transfection, Control, Isolation, Knockdown, Western Blot, Hybridization, Staining

Journal: Cancer Research

Article Title: Glucose-Regulated Protein 78 Controls Cross-talk between Apoptosis and Autophagy to Determine Antiestrogen Responsiveness

doi: 10.1158/0008-5472.can-12-0269

Figure Lengend Snippet: Figure 3. GRP78 regulates apoptosis. LCC9 and MCF7-RR breast cancer cells were transfected with control or GRP78 siRNA, and LCC1 and MCF7 human breast cancer cells were transfected with control pcDNA or GRP78 expression vector and treated with either 0.1% v/v ethanol vehicle, 100 nmol/L ICI (LCC9/ LCC1), or 100 nmol/L tamoxifen (TAM; MCF7-RR/MCF7) for 6 days. Western blotting hybridization of protein homogenates was used to measure cleaved caspase-7 (A þ D) or cleaved PARP (B þ E) levels. C þ F, Annexin V-FITC–stained cells counted using flow cytometry. All studies, n ¼ 3–5; one-way ANOVA with Dunnett post hoc analysis; , P < 0.05 compared with vehicle control. FITC, fluorescein isothiocyanate.

Article Snippet: The following materials were obtained as indicated: 4- hydroxytamoxifen (Sigma-Aldrich); ICI 182,780 (Tocris Bioscience); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL); FBS and bovine calf charcoal-stripped serum (CCS; Equitech-Bio Inc.); Lipofectamine RNAiMAX reagent (Invitrogen); GRP78 and AMPK siRNA (OnTarget plus SMART pool; consisting of 3 different siRNA for same target; ThermoScientific Dharmacon); GRP78 plasmid (HSPA5 Trueclone cDNA; OriGene); ATG5 and TSC2 siRNA (Cell Signaling Technology); mouse IgG negative control antibody (Dako); crystal violet (Fisher Scientific); and caspase inhibitor Z-VAD-FMK (Tocris Bioscience).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Hybridization, Staining, Cytometry

Journal: Cancer Research

Article Title: Glucose-Regulated Protein 78 Controls Cross-talk between Apoptosis and Autophagy to Determine Antiestrogen Responsiveness

doi: 10.1158/0008-5472.can-12-0269

Figure Lengend Snippet: Figure 4. GRP78 regulates autophagy. LCC9 and MCF7-RR cells transfected with control or GRP78 siRNA and LCC1 and MCF7 cells transfected with control pcDNA or GRP78 expression vector and treated with 0.1% v/v ethanol vehicle, 100 nmol/L ICI (LCC9/LCC1), or 100 nmol/L tamoxifen (TAM; MCF7-RR/MCF7) for 6 days. Western blotting hybridization of protein homogenates was used to measure LC3-II (A þ D) or p62 (B þ E) protein expression. One-way ANOVA with Dunnett post hoc analysis; , P < 0.05 compared with vehicle-treated controls. C, LCC9 cells transfected with GFP-LC3 and control or GRP78 siRNA and LCC1 cells were transfected with GFP-LC3 and control pcDNA or GRP78(þ) (F), and treated with 0.1% v/v ethanol vehicle or 500 nmol/L ICI for 72 hours. LC3- positive punctate pattern was observed by confocal microscopy.

Article Snippet: The following materials were obtained as indicated: 4- hydroxytamoxifen (Sigma-Aldrich); ICI 182,780 (Tocris Bioscience); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL); FBS and bovine calf charcoal-stripped serum (CCS; Equitech-Bio Inc.); Lipofectamine RNAiMAX reagent (Invitrogen); GRP78 and AMPK siRNA (OnTarget plus SMART pool; consisting of 3 different siRNA for same target; ThermoScientific Dharmacon); GRP78 plasmid (HSPA5 Trueclone cDNA; OriGene); ATG5 and TSC2 siRNA (Cell Signaling Technology); mouse IgG negative control antibody (Dako); crystal violet (Fisher Scientific); and caspase inhibitor Z-VAD-FMK (Tocris Bioscience).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Hybridization, Confocal Microscopy

Journal: Cancer Research

Article Title: Glucose-Regulated Protein 78 Controls Cross-talk between Apoptosis and Autophagy to Determine Antiestrogen Responsiveness

doi: 10.1158/0008-5472.can-12-0269

Figure Lengend Snippet: Figure 5. GRP78 modulates mTOR activity to promote autophagy. A, LCC1 cells transfected with GRP78(þ) cDNA or control pcDNA, and LCC9 cells transfected with control or GRP78 siRNA and treated with either 0.1% v/v ethanol vehicle or 100 nmol/L ICI for 6 days. Protein homogenates were isolated and Western blotting hybridization used to measure phospho-mTOR, mTOR, phospho-TSC2, TSC1, phospho- AMPK, and b-actin expression. B, LCC1 cells transfected with control, TSC2, and/or AMPK siRNA and with control pcDNA or GRP78(þ) cDNA. Protein homogenates were isolated and Western blotting hybridization was used to measure GRP78, phospho-TSC2, phospho-AMPK, TORC1, p62, LC3, and b-actin expression.

Article Snippet: The following materials were obtained as indicated: 4- hydroxytamoxifen (Sigma-Aldrich); ICI 182,780 (Tocris Bioscience); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL); FBS and bovine calf charcoal-stripped serum (CCS; Equitech-Bio Inc.); Lipofectamine RNAiMAX reagent (Invitrogen); GRP78 and AMPK siRNA (OnTarget plus SMART pool; consisting of 3 different siRNA for same target; ThermoScientific Dharmacon); GRP78 plasmid (HSPA5 Trueclone cDNA; OriGene); ATG5 and TSC2 siRNA (Cell Signaling Technology); mouse IgG negative control antibody (Dako); crystal violet (Fisher Scientific); and caspase inhibitor Z-VAD-FMK (Tocris Bioscience).

Techniques: Activity Assay, Transfection, Control, Isolation, Western Blot, Hybridization, Expressing

Journal: Cancer Research

Article Title: Glucose-Regulated Protein 78 Controls Cross-talk between Apoptosis and Autophagy to Determine Antiestrogen Responsiveness

doi: 10.1158/0008-5472.can-12-0269

Figure Lengend Snippet: Figure 6. GRP78 facilitates cross- talk between apoptosis and autophagy to affect cell survival and proliferation. A, LCC9 cells were pretreated with 50 mmol/L pan-caspase inhibitor (Z-VAD- FMK, FMK) for 30 minutes before transfection with control or GRP78 siRNA. These cells were then treated with ICI and/or Z-VAD- FMK for 6 days and cell density measured following crystal violet staining. B, protein homogenates from these treatment groups (100 nmol/L ICI for 6 days) were subjected to Western blotting hybridization to measure GRP78, cleaved caspase-7, LC3, and b-actin expression. C, LCC1 cells transfected with control or ATG5 siRNA and/or control or GRP78 cDNA and treated with 0.1% v/v ethanol vehicle or ICI. D, protein homogenates from these treatment groups (100 nmol/L ICI for 6 days) were subjected to Western blotting hybridization to measure GRP78, ATG5, cleaved caspase-7, p62, LC3, and b-actin. E, LCC9 cells constitutively expressing control shRNA or beclin-1 shRNA were transfected with control or GRP78 siRNA, treated with 0.1% v/v ethanol vehicle or ICI for 6 days, and cell density measured following crystal violet staining. F, protein homogenates from these treatment groups (100 nmol/L ICI for 6 days) were subjected to Western blotting hybridization to measure GRP78, beclin-1, cleaved caspase-7, p62, LC3, and b-actin. For all experiments, n ¼ 3–4; one- way ANOVA with Bonferroni post hoc analysis; , P < 0.05. Synergy is determined by RI [(survival A survival B)/(survival AþB)] < 2.0.

Article Snippet: The following materials were obtained as indicated: 4- hydroxytamoxifen (Sigma-Aldrich); ICI 182,780 (Tocris Bioscience); penicillin and Improved Minimal Essential Medium (IMEM; Gibco Invitrogen BRL); FBS and bovine calf charcoal-stripped serum (CCS; Equitech-Bio Inc.); Lipofectamine RNAiMAX reagent (Invitrogen); GRP78 and AMPK siRNA (OnTarget plus SMART pool; consisting of 3 different siRNA for same target; ThermoScientific Dharmacon); GRP78 plasmid (HSPA5 Trueclone cDNA; OriGene); ATG5 and TSC2 siRNA (Cell Signaling Technology); mouse IgG negative control antibody (Dako); crystal violet (Fisher Scientific); and caspase inhibitor Z-VAD-FMK (Tocris Bioscience).

Techniques: Transfection, Control, Staining, Western Blot, Hybridization, Expressing, shRNA

Journal: bioRxiv

Article Title: TAZ ( Wwtr1 ) deficiency leads to ER stress and mitochondrial dysfunction in a mouse model of Fuchs’ endothelial corneal dystrophy

doi: 10.64898/2026.02.17.706456

Figure Lengend Snippet: (A) Immunofluorescence staining of GRP78 in corneal endothelial wholemounts from wildtype (WT) and TAZ KO mice (scale bar = 20 µm). The arrow indicates nuclear translocation of GRP78. (B) TAZ KO mice showed significantly greater GRP78 expression in corneal endothelium compared to WT mice (unpaired t-test). Triangles, ≤ 2 months of age; rectangles, > 10 months of age; circles: 3-10 months of age. (C) Violin plots of ER stress-related genes in WT and TAZ KO CEnC at 2 and 11 months of age.

Article Snippet: For GRP78 staining, a primary antibody against GRP78 (1:50, Proteintech, 11587-1-AP) was used.

Techniques: Immunofluorescence, Staining, Translocation Assay, Expressing