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: 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

Journal: Cancer cell international

Article Title: Dysregulation of the miR-30a/BiP axis by cigarette smoking accelerates oral cancer progression.

doi: 10.1186/s12935-021-02276-1

Figure Lengend Snippet: Fig. 4 CSC induced BiP expression in OSCC cells by downregulating miR-30a. A YD38 and SCC25 cells were treated with either vehicle control or 120 μg/ml CSC for 48 h. The expression of miR-30a was evaluated using qRT-PCR. B Sequence alignment of miR-30a with the target sequence on the 3-UTR of BiP transcript. C The direct target relationship between miR-30a and BiP was confirmed using a luciferase assay. OSCC cells transfected with negative control (miR-NC) or miR-30a mimics were further transfected with pBiP-3’UTR plasmids followed by CSC treatment. Luciferase activity was detected using a luciferase assay. All data are presented as the mean ± SEM. SEM, error bar. *P < 0.05 by Student’s t test or one-way ANOVA followed by Bonferroni’s post hoc test

Article Snippet: Luciferase reporter gene assay The 3′UTR of BiP containing the putative miR-30a binding site was cloned into the pMirTarget 3′UTR assay vector (SC216266, OriGene, Rockville, MD, USA).

Techniques: Expressing, Control, Quantitative RT-PCR, Sequencing, Luciferase, Transfection, Negative Control, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Licorice attenuates cisplatin-induced hepatotoxicity by alleviating endoplasmic reticulum stress and apoptosis

doi: 10.3389/fphar.2025.1557125

Figure Lengend Snippet: Gene primer sequences.

Article Snippet: WB analysis was performed as previously described ( ) using specific primary antibodies against CHOP (1:1000, 15204-1-AP, Proteintech), ATF4 (1:1000, BM5179, Boster), ATF6 (1:1000, A00655, Boster), GRP78 (1:1000, BA 2042, Boster), p-IRE1α (1:1000, NB100-2323, Novus), p-eIF2α (1:1000, BM3942, Boster), Bcl-2 (1:1000, A00040-1, Boster), Bax (1:1000, A00183, Boster), cleaved caspase-3 (1:1000, #9664T, Cell Signaling Technology), caspase-12 (1:1000, BA3142, Boster), cleaved caspase-8 (1:5000, ab108333, Abcam), p-PERK (1:1000, abs137056, Absin) and β-actin (1:5000, 20536-1-AP, Proteintech).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Licorice attenuates cisplatin-induced hepatotoxicity by alleviating endoplasmic reticulum stress and apoptosis

doi: 10.3389/fphar.2025.1557125

Figure Lengend Snippet: Effects of GC on CP-induced ER stress. (A) Effect of GC on the morphological changes of ER in liver cells induced by CP. Red arrows show the morphology of the endoplasmic reticulum and ribosomes. (B, C) Effect of GC on CP-induced ER stress-related indicators. GRP78, ATF6, and p-IRE1α protein expression were detected by WB analysis. GRP78 and ATF6 gene expression were detected by qRT-PCR analysis. All data are presented as the mean ± SD. ## p < 0.01 compared with control group; * p < 0.05, ** p < 0.01 compared with CP group.

Article Snippet: WB analysis was performed as previously described ( ) using specific primary antibodies against CHOP (1:1000, 15204-1-AP, Proteintech), ATF4 (1:1000, BM5179, Boster), ATF6 (1:1000, A00655, Boster), GRP78 (1:1000, BA 2042, Boster), p-IRE1α (1:1000, NB100-2323, Novus), p-eIF2α (1:1000, BM3942, Boster), Bcl-2 (1:1000, A00040-1, Boster), Bax (1:1000, A00183, Boster), cleaved caspase-3 (1:1000, #9664T, Cell Signaling Technology), caspase-12 (1:1000, BA3142, Boster), cleaved caspase-8 (1:5000, ab108333, Abcam), p-PERK (1:1000, abs137056, Absin) and β-actin (1:5000, 20536-1-AP, Proteintech).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control

Journal: Frontiers in Pharmacology

Article Title: Licorice attenuates cisplatin-induced hepatotoxicity by alleviating endoplasmic reticulum stress and apoptosis

doi: 10.3389/fphar.2025.1557125

Figure Lengend Snippet: (A-C) MTT assay for determining cell viability. (D-E) GC reduced the expression of ER stress-related indicators. Expression levels of GRP78, ATF6, and p-IRE1α protein were tested by WB analysis. Expression levels of GRP78 and ATF6 mRNA were tested by qRT-PCR analysis.

Article Snippet: WB analysis was performed as previously described ( ) using specific primary antibodies against CHOP (1:1000, 15204-1-AP, Proteintech), ATF4 (1:1000, BM5179, Boster), ATF6 (1:1000, A00655, Boster), GRP78 (1:1000, BA 2042, Boster), p-IRE1α (1:1000, NB100-2323, Novus), p-eIF2α (1:1000, BM3942, Boster), Bcl-2 (1:1000, A00040-1, Boster), Bax (1:1000, A00183, Boster), cleaved caspase-3 (1:1000, #9664T, Cell Signaling Technology), caspase-12 (1:1000, BA3142, Boster), cleaved caspase-8 (1:5000, ab108333, Abcam), p-PERK (1:1000, abs137056, Absin) and β-actin (1:5000, 20536-1-AP, Proteintech).

Techniques: MTT Assay, Expressing, Quantitative RT-PCR