Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: Extracellular GRP78 induces drug resistance through binding to cell surface ROR1, Cripto-1, and PD-L1. A, five-day cellular viability of U118, U87, and DIPG13 glioma cells with and without extracellular GRP78 (5 μg/ml) and under treatment at different concentrations of doxorubicin was measured with a live-cell CCK-8 assay. B, pediatric GBM SF9402, adult GBM U87, cell membrane proteins were biotinylated and solubilized using a nondenaturing membrane isolation kit and GRP78-bound proteins were eluted and analyzed by SDS-PAGE and mass spectrometry analysis. C, PAGE analysis of cell surface GRP78-binding proteins. Mass spectrometry analysis determined the bands were ROR1, PD-L1, and Cripto. MW. Molecular Weight Standards in kDa, GRP78 ppt = Surface proteins bound to GRP78. D, flow cytometry analysis of ROR1 expression on pediatric GBM SF9402 (43.4%) and adult GBM U87 (61.7%) cells. E, extracellular domains of ROR1, Cripto-1, PD-L1, and CD-44 were tested for GRP78 binding using a direct ELISA method. Kd = ROR1

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Binding Assay, CCK-8 Assay, Membrane, Isolation, SDS Page, Mass Spectrometry, Molecular Weight, Flow Cytometry, Expressing, Direct ELISA, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: Cell surface GRP78 colocalizes with ROR1, Cripto, and PD-L1 on late-stage patient GBM tissues but not on normal brain cerebrum tissues. A, three brain tissue microarrays with patient core tissue samples of GBM and normal tissues (#T174T from Biomax.us.) were stained for GBM markers. B, one microarray was stained with 4′,6-diamidino-2-phenylindole (DAPI) ( blue -DNA), and antibodies to cell surface GRP78 ( green -FITC), ROR1 ( red -PE). The last column is an overlay of all three stains to show colocalization. Cores B2 (stage 4 GBM) and C7 (normal cerebrum) are pictured. C, a second microarray was stained with DAPI ( blue -DNA), and antibodies to cell surface GRP78 ( green -FITC), PD-L1 ( red -PE). The last column is an overlay of all three stains to show colocalization. Cores B6 (stage 4 GBM) and C5 (normal cerebrum) are pictured. D, a third microarray was stained with DAPI ( blue -DNA), and antibodies to cell surface GRP78 ( green -FITC), PD-L1 ( red -PE). The last column is an overlay of all three stains to show colocalization. Cores B6 (stage 4 GBM) and C5 (normal cerebrum) are pictured. Scale bars represent 400 μm (10×). All images are from the same magnification of (10×) 400 μm. GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Staining, Microarray

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: CBT300’s ROR1 kringle domain (Kr1) domain binds to GRP78 and inhibits tumor cell proliferation. A, secondary structure of ROR1 showing where GRP78 is estimated to bind to ROR1 kringle domain. B, binding of individual ROR1 Extra Cellular Domains (Ig, FZD, KRD) and other kringle GRP78 binding domains (Kr1, Kr1Fc, K5) to GRP78. C, in silico docking of K5 kringle domain or the ROR1 kringle(Kr1) domain with GRP78. The lowest free energy of binding is shown for each of the kringle domains to GRP78. ROR1 kringle (Kr1) shows strong binding with two salt bridges, six hydrogen bonds and 98 nonbonded contacts to GRP78. D, graphic depiction of CBT100 (K5Fc) or CBT300 (Kr1Fc). E, SDS-PAGE gels of CBT100 and CBT300 showing reduced (#1) and nonreduced (#2) lanes. For both SDS-PAGE gels, M = molecular weight standards, #1 = reduced CBT100 (K5Fc) or CBT300 (Kr1Fc) and #2 = nonreduced CBT100 (K5Fc) or CBT300 (Kr1Fc). Size-exclusion analysis of CBT300 showing > 99% purity after protein A purification. F, five-day cell viability assay of U87 GBM cells treated with various concentrations of K5, CDT300 (Kr1Fc) CBT200 (K5PEG), and CBT100 (K5Fc) in triplicate wells. G, cell viability in a 5-day assay of four pediatric DIPG stem cell lines and two pediatric GBM stem cell lines under treatment with various concentrations of CBT300. The number of live cells was measured with a CCK-8 reagent. All dose response curves were fitted with a 3-parameter curve fits calculating the IC50 value. CCK-8, Cell Counting Kit-8; GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; DIPG, diffuse intrinsic pontine glioma; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Binding Assay, In Silico, SDS Page, Molecular Weight, Purification, Viability Assay, CCK-8 Assay, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: CBT300’s anticancer efficacy increases cell death in 2D assays and regresses tumor brain tumor spheroids in 3D assays with patient derived adult and pediatric glioma stem cells. A, CBT300 significantly increased cell death of patient derived glioma stem cells (827) grown on laminin. Three replicates of 60 cell count each were used with trypan blue reagent to determine dead cells. B and C, cell viability assays with pediatric and adult glioma cells. Cells were tested in triplicate repeats with doxorubicin with or without GRP78 (5 μg/ml) and with or without CBT300 (0.1 nM). Extracellular GRP78 increases resistance to doxorubicin and CBT300 reverses this resistance in cell viability assays. D, Pediatric Diffuse Intrinsic Ponte Glioma stem cells were seeded at 10,000 cells per well in 96 U-bottom well ultra low attachment (ULA) plates and cultured as a spheroid for 14 days with either medium, doxorubicin (10 μM), CBT300 (550 nM), or CBT300 (550 nM) + doxorubicin (1 μM). Representative images of DIPG-50 and DIPG-24 spheroids are shown. E– H , pediatric and adult glioma cells were seeded in ULA plates in triplicate wells with the treatments listed. Pictures of spheroids were taken at various times, and the size of the spheroids was measured by Image J ( https://imagej.net ). Spheroid regression/growth curves points are a mean of four replicates with standard deviation shown. The final point of measurements with a representative spheroid picture was shown with treatment regime and percent inhibition compared to the Control. Standard p values (∗ p < 0.5, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001) with respect to control were calculated by two-tailed unpaired t test for final measurements. I , patient derived adult 827 glioma stem cells were plated at 30,000 cells per well in 96-flat well plates coated with laminin. Cells were treated with either PBS control or CBT300 (550 nM) for 7 days. Spheroids containing more than or equal to five cells were counted on the days shown. Percent inhibition of spheroid numbers was calculated at the final time point. Standard p value (∗∗∗ p < 0.001) with respect to control was calculated by two-tailed unpaired t test for final measurements. The data points are an average of 3–4 replicates. CCK-8, Cell Counting Kit-8; GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; DIPG, diffuse intrinsic pontine glioma; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Derivative Assay, Cell Characterization, Cell Culture, Standard Deviation, Inhibition, Control, Two Tailed Test, CCK-8 Assay, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: CBT300 reduces expression of cell surface GRP78, ROR1, Cripto, and PD-L1. A, pediatric SF9427 DIPG cells (50,000) were incubated with either media, media+extracellular GRP78 (5 μg/ml), or media+extracellular GRP78 (5 μg/ml)+CBT300 (100 nM) for 72 h. Cells were then stained with mAbs for cell surface GRP78(csGRP78 nonpermeabilized cells), ROR1, Cripto, and PD-L1. Flow cytometry analysis was used on a Guava PCA to determine surface expression of listed cell surface proteins. The average for the percent positive cells is reported as the mean with SD from three replicate assays. B, adult GBM cells (50,000) were incubated with either media, media+extracellular GRP78 (5 μg/ml), or media+extracellular GRP78 (5 μg/ml)+CBT300 (100 nM) for 72 h. Cells were then stained with mAbs for cell surface GRP78(csGRP78 nonpermeabilized cells), ROR1, Cripto, and PD-L1. Flow cytometry analysis was used on a Guava PCA to determine surface expression of listed cell surface proteins. The average for the percent positive cells is reported as the mean with SD from three replicate assays. Studies were performed with three independent replicates. The average mean and SD for each set and marker are shown. Significance was determined between groups by two-way ANOVA analysis with post hoc Tukey’s test using GraphPad Prism 10.1.2. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. CCK-8, Cell Counting Kit-8; GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; DIPG, diffuse intrinsic pontine glioma; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Expressing, Incubation, Staining, Flow Cytometry, Marker, CCK-8 Assay, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: CBT300 reduces expression of cell surface GRP78 leading to increased doxorubicin internalization in glioma cells. A, U87 cells (20,000) were seeded in quadruplicate wells of tissue coated 96-well plates with either media (negative control – no doxorubicin), media (positive control – doxorubicin (2 μm)), media+GRP78 (5 μg/ml), or media+GRP78 (5 μg/ml)+CBT300 (100 nM) for 48 h. Doxorubicin (2 μM) was then added to all the wells except for the negative control wells. 24 h after adding doxorubicin to the cells, cells were washed and flow cytometry analysis for red doxorubicin fluorescence was performed. B, U87 cells (3,000) were plated in four well chamber slides and allowed to attach overnight. Each chamber was treated with either media, media+GRP78 (5 μg/ml), or media+GRP78 (5 μg/ml)+CBT300 (100 nM) for 48 h. Doxorubicin (2 μM) was then added to each chamber and incubated for 24 h. Cells/chambers were then washed and stained with a mAb-FITC ( green ) to the C-terminal domain of GRP78, and 4′,6-diamidino-2-phenylindole ( blue ) to the DNA. Doxorubicin, which has a natural red fluorescence, was also measured for each treatment. A chart of three independent repeats was compiled for each marker, cell surface GRP78, and increase in doxorubicin internalized. Studies were performed with three independent replicates. The average mean and standard deviation for each set and marker are shown. Significance was determined between groups by two-way ANOVA analysis with post hoc Tukey’s test using GraphPad Prism 10.1.2. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. C, the proposed mechanism of action of CBT300’s inhibition of surface expressed GRP78 leading to tumor regression and apoptosis. Cell surface GRP78 promotes tumor growth through stabilization of checkpoint protein PD-L1, oncofetal proteins ROR1, and Cripto (tumor promotion). CBT300 and CBT200 remove the surface GRP78, eliminating its stabilization of PD-L1, ROR1, and Cripto which then induces tumor apoptosis and reducing drug and immune resistance (tumor apoptosis). GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Expressing, Negative Control, Positive Control, Flow Cytometry, Fluorescence, Incubation, Staining, Marker, Standard Deviation, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: Antiangiogenesis and GRP78 inhibitor K5 (kringle 5), regresses GBM tumors and significantly extends survival with durable complete regressions in an orthotopic glioma murine model. A, experimental setup of intracranial stereotactic injections with D54 adult GBM cells in the brains of nude mice and subsequent treatment with K5(3 X14-day osmotic mini pumps) and MRI analysis on days 28, 42, and 65 after tumor cell inoculation. B, MRI analysis of orthotopic adult GBM D54 tumors treated with and without K5. C, Kaplan–Meier curves showing survival of K5-treated mice versus controls. D, GBM D54 tumor volume at day 65 or at death as determined by MRI analysis. E–G, immunohistochemical analysis of GBM D54 tumor tissues were analyzed for vessel density (CD31) and apoptotic cells (TUNEL) at day 65. Five tissue samples were stained from each group and the average with SD were plotted. Survival curves in ( C ) were compared using log-rank test. Statistical analysis in ( D ) was performed using two-way ANOVA with post hoc Tukey’s test. Statistical analysis in ( E ) and ( F ) was performed using one-way ANOVA with post hoc Dunnett’s test. CCK-8, Cell Counting Kit-8; GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Immunohistochemical staining, TUNEL Assay, Staining, CCK-8 Assay, Cell Counting

Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: GRP78 inhibition by systemic dosing of CBT300 (Kr1Fc) improves survival in preclinical models of glioblastoma cancer. A, experimental setup of stereotactic intracranial injection of adult PDx CTG-2687-Luc cells in the brains of nude mice and subsequent treatment with CBT300 (Kr1Fc). B, tumor bioluminescence of treated mice at days 0 and 27 of Control and CBT300-treated mice. C, Kaplan–Meier survival curve showing a significant 60% survival of CBT300 (Kr1Fc)-treated mice versus controls upon intracranial injection of PDx GBM cells. D, individual mice tumor volume as determined by bioluminescence. Control treated mice in black lines . CBT300-treated mice in red lines. E, average tumor volume as determined by bioluminescence for the CBT300 and Control groups up to day 27. N = 5 mice per group. F, immunofluorescence staining of CTG-2687 tumors for CBT300 and Control treatments. The scale bar represents 400 μm (10×). G, statical analysis of Control and CBT300-treated PDx GBM tumors for tumor cell number (4′,6-diamidino-2-phenylindole), CBT300 (IgG1), cell surface GRP78, ROR1, and Cripto expression as determined by immunofluorescence. Three independent mice GBM tumor tissues were measured. H, statical analysis of Control and CBT300-treated PDx GBM tumors for tumor expression of ABCC1, ABCB1, AGCG2, PD-L1, and Ki-67. Three independent mice GBM tumor tissues were measured. I, circulation concentrations of CBT300 in CBT300 treated and Control mice blood on day 27 2 h after last dose. Statistical analysis was performed using Kaplan–Meier survival curves in (C) and (D-H) were compared with an unpaired t test analysis using a two-stage step-up method by Benjamini, Krieger, and Yekutieli. GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; DIPG, diffuse intrinsic pontine glioma; PDx, patient-derived xenograft; CBT, Creative BioTherapeutics.

Article Snippet: Doxorubicin hydrochloride (Sigma-Aldrich Cat# 44583), GRP78 (StressMarq Cat# His-SPR-107C, SPR-119C, ATPase active domain containing no ADP or ATP), ROR1 ECD (ACRO Biosystems Cat# RO1-522y, Gln30- Glu403), ROR1 Ig-like domain (ACRO Biosystems Cat# RO1-H5221, Glu39–Gly151), ROR1 kringle domain (ACRO Biosystems Cat# RO1-H5223, Asn308–Asp 395), ROR1 Frizzled domain (ACRO Biosystems Cat# RO1-H5222, Glu165–Asp305), Cripto (Human TDGF1,fc ACRO Biosystems Cat# CRO-H5253), CD44 ECD (Sino Biological Cat# P16070-1, Met1-Pro220) were used per manufacturer’s instructions.

Techniques: Inhibition, Injection, Control, Immunofluorescence, Staining, Expressing, Derivative Assay

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: 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

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: Pro-inflammatory diet inhibits endoplasmic reticulum stress signaling involving ATF4/HSPA5 pathway. (A) RNA-seq analysis on principal component analysis of all genes to evaluate the differences among CN group (in black, n = 4) and PI group (in red, n = 5). (B) Number of differentially expressed colonic genes between two groups. (C) Heatmap of gene set variation analysis showing several downregulated genes in PI group belonging to heat shock protein 70 family. (D) Quantitative RT–PCR analysis showing the down-regulation of Hspa1b , Hspa1a , Hspa5 and Hsph1 after pro-inflammatory diet treatment. (E) Comparisons of the relative mRNA level of Perk , Atf4 , Atf6 and Chop between mice treated with or without pro-inflammatory diet. (F) Heatmap on the expression of Perk , Atf4 , Hspa5 and GPX4 between ND group and PD group. (G-I) Western blotting and immunohistochemical results demonstrating decreased expression of ATF4 and HSPA5 in PD group. Corresponding animal experiments had been repeated for 3 or more times. Data are represented as the mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemical staining

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: The involvement of ferroptosis in intestinal inflammation of CD patients with pro-inflammatory diet pattern. (A-C) Results from Dataset Accession number GSE6731 showing the downregulation of GPX4 and the upregulation of HSPA5 and ATF4 in the disease affected colon from patients with IBD. CD-un, disease unaffected tissues from CD patients; CD-aff, disease affected tissues from CD patients; UC-un, disease unaffected tissues from UC patients; UC-aff, disease affected tissues from UC patients. (D-F) Relative expression of PERK , ATF4 , HSPA5 and GPX4 in colon from CD patients of our hospital in mRNA and protein level. HC, health control group; CD, patients with CD group; (G) The DII scores of 32CD patients included in this study were compared between High-DII group (patients with higher DII group) and Low-DII group (patients with lower DII group). (H) Representative immunohistochemical staining of GPX4 and HSPA5 in CD colon tissues from High-DII group and Low-DII group. (I-J) Quantification of GPX4 and HSPA5 expression using integrated optical density/specimen area (IOD/area). Data are represented as the mean ± SD. ns, no significance.

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: Expressing, Control, Immunohistochemical staining, Staining

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: Bacteroides uniformis ameliorates DSS-induced acute colitis. (A) Experimental design for antibiotics and DSS treatment, as well as the microbiota transplantation. BU group (oral administration of B. uniformis , n = 5), UA group (oral S. mutans , n = 5), and PBS group ( n = 5). (B) Culture of the fecal bacteria before and after antibiotic cocktails treatment using the blood agar plate. (C-D) Measurement of body weight lost and colon length at sacrifice of three groups. (E-F) H&E staining of colon section and the pathological score for each group (Scale bars: 625um). (G) Comparison of colonic MPO activity among three groups. (H-I) Detection of the relative mRNA level of Mucin-2 , tight juncture proteins ( Zo-1 and Cldn8 ) and inflammatory cytokines ( Tnfα , Il17a , Il6 , Il1β , Il12 , Il1α , Il10 and Tgfβ1 ) in colon. (J-L) Higher expression of GPX4, HSPA5 and ATF4 in both RNA and protein level after administrated with B. uniformis. Corresponding animal experiments had been repeated for 3 or more times. Data are represented as the mean ± SD.

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: Transplantation Assay, Bacteria, Staining, Comparison, Activity Assay, Expressing

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: Bacteroides uniformis ameliorates Pro-inflammatory diet-induced colitis in DSS model by reversing ferroptosis. (A) Experimental design for dietary and DSS treatment. Mice fed on the pro-inflammatory diet were divided into three groups: BU group (oral B. uniformis , n = 5), UA group (oral S. mutans , n = 5), PBS group ( n = 5). (B-D) Comparisons of body weight lost during DSS treatment, the colon length and the colonic MPO activity at sacrifice among three groups. (E-F) Quantitative RT–PCR detection of inflammatory cytokines ( Tnfα , Il17a , Il6 , Il1β , Il12 Il1α , Il10 and Tgfβ1 ), tight junction proteins ( Cldn1 , Cldn2 , Jam and Cldn14 ) and Mucin-2 expression in colon. (G-H) Representative images with H&E staining and the pathological score to estimate intestinal inflammation (Scale bars: 625um). (I-K) Measurement of Fe, GSH and MDA content in colon. (L) Relative mRNA expression of ferroptosis related genes ( Acsl4 , Tfr1 , Nox1 , Cox1 , Gpx4 , Fpn , Fth1 , Ftl , Tf , Ireb2 , Slc7a11 and Slc3a2 ). (M) Representative images of transmission electron microscopy for distal colon. Arrows head to mitochondria. (N-O) The recovery of GPX4, HSPA5, ATF4 and PERK expression in both RNA and protein level after administrated with B. uniformis. Corresponding animal experiments had been repeated for 3 or more times. Data are represented as the mean ± SD.

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Staining, Transmission Assay, Electron Microscopy

Journal: Journal of Advanced Research

Article Title: Bacteroides uniformis ameliorates pro-inflammatory diet-exacerbated colitis by targeting endoplasmic reticulum stress-mediated ferroptosis

doi: 10.1016/j.jare.2024.11.025

Figure Lengend Snippet: Co-cultured with Bacteroides uniformis inhibits ER stress pathway-mediated ferroptosis in vitro . (A-B) Improvement of cell viability in a concentration- and time-dependent pattern when co-culture with B. uniformis . (C-D) Decreased level of LDH level in cell coculture supernatant. Relative expression of PERK, ATF4, HSPA5 and GPX4 in RNA level (E, F) and in protein level (G H) after treated cells with B. uniformis . A, C, E and G showed the experimental results in Caco-2 cell. B, D, F and H showed the experimental results in NCM460 cell. Experiments had been repeated for 3 or more times. Data are represented as the mean ± SD.

Article Snippet: Upon reaching 70–80 % confluence, the cell were treated with HSPA5 inhibitor (1 uM, HY-100437, MCE, Shanghai, China), HSPA5 recombined protein (10 ng / mL, HY-P71742, MCE, Shanghai, China) or PERK inhibitor (1uM or 5uM, GSK2606414, MCE, Shanghai, China) for two days and were then harvested for RT-qPCR and Western blot analysis.

Techniques: Cell Culture, In Vitro, Concentration Assay, Co-Culture Assay, Expressing

Journal: Journal of clinical immunology

Article Title: Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

doi: 10.1007/s10875-012-9841-6

Figure Lengend Snippet: Fig. 2 Effect of ACPAs and different protein molecules on the ratio of p-ERK1/2/ERK1/2, p-JNK/JNK and p-p38/p38 MAPKs in U937 cells. The concentration of different proteins were: non-specific human IgG (6 μg/ml), ACPAs (100 IU/ml), ACPA-depleted [ACPA(−) RA] sera (6 μg/ml), ACPAs (100 IU/ml)+anti-Toll-like receptor 4 blocking antibody (anti-TLR4, 10 μg/ml), ACPAs (100 IU/ml)+cit-GRP78 (10 μg/ml) or ACPAs (100 IU/ml)+GRP78 (10 μg/ml) for 24 h. The cells were lysed and both phosphorylated and native forms of three MAPKs were analyzed by Western blotting. a pERK1/2/ERK1/2 ratio, b p-JNK/JNK ratio, c p-p38/p38 ratio, and d A representative example to show the expression of phosphorylated and native forms of MAPKs after incubation with different proteins by Western blot; * denote p<0.05 compared to the non-specific human IgG

Article Snippet: Recombinant GRP78 (Novus Biologicals, Littleton, CO, USA) was successfully citrullinated (cit-GRP78) after incubation of the molecules with 10 mM CaCl2 and peptidylarginine deiminase (Sigma-Aldrich) at a concentration of 20 IU per 1 mg GRP78 protein for 2 h.

Techniques: Concentration Assay, Blocking Assay, Western Blot, Expressing, Incubation

Journal: Journal of clinical immunology

Article Title: Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

doi: 10.1007/s10875-012-9841-6

Figure Lengend Snippet: Fig. 4 Effect of ACPAs and different protein molecules on p-Akt/Akt and p-IKK-α/IKK-α ratio of U937 cells. U937 cells (1×106 cells/ml) were incubat- ed with non-specific human IgG (5 μg/ml), ACPAs (80 IU/ ml), ACPA(−) RA sera (5 μg/ ml), ACPAs (80 IU/ml)+anti- TLR4 (10 μg/ml), ACPAs (80 IU/ml)+cit-GRP78 (10 μg/ ml) for 24 h. The cells were lysed and both phosphorylated and native form of a Akt and b IKK-α were analyzed by West- ern blotting. * denote p<0.05 compared to the non-specific human IgG

Article Snippet: Recombinant GRP78 (Novus Biologicals, Littleton, CO, USA) was successfully citrullinated (cit-GRP78) after incubation of the molecules with 10 mM CaCl2 and peptidylarginine deiminase (Sigma-Aldrich) at a concentration of 20 IU per 1 mg GRP78 protein for 2 h.

Techniques:

Journal: Journal of clinical immunology

Article Title: Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

doi: 10.1007/s10875-012-9841-6

Figure Lengend Snippet: Fig. 5 Comparison of citrullinated (cit-GRP78) and native forms (GRP78) GRP78 molecules on the surface mem- brane extracts (150 μg) among U937 cells, normal human PBMCs, and RA PBMCs by Western blotting. The expres- sion of transferrin receptor (TfR) was used as an internal control. a The expression ratio of cit-GRP78/TfR among the three groups b The expression of GRP78/TfR among three groups c The expression ratio of cit-GRP78/GRP78 among three groups. d A representative example is shown

Article Snippet: Recombinant GRP78 (Novus Biologicals, Littleton, CO, USA) was successfully citrullinated (cit-GRP78) after incubation of the molecules with 10 mM CaCl2 and peptidylarginine deiminase (Sigma-Aldrich) at a concentration of 20 IU per 1 mg GRP78 protein for 2 h.

Techniques: Comparison, Western Blot, Control, Expressing

Journal: Journal of clinical immunology

Article Title: Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

doi: 10.1007/s10875-012-9841-6

Figure Lengend Snippet: Fig. 6 A schematic diagram to indicate that the MAPK signaling pathways are transduced by ACPAs after binding to surface- expressed cit-GRP78 on RA PBMCs that leads to TNF-α production

Article Snippet: Recombinant GRP78 (Novus Biologicals, Littleton, CO, USA) was successfully citrullinated (cit-GRP78) after incubation of the molecules with 10 mM CaCl2 and peptidylarginine deiminase (Sigma-Aldrich) at a concentration of 20 IU per 1 mg GRP78 protein for 2 h.

Techniques: Protein-Protein interactions, Binding Assay