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: 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