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: Molecular Oncology

Article Title: The synthetic oleanane triterpenoid CDDO‐2P‐Im binds GRP78 / BiP to induce unfolded protein response‐mediated apoptosis in myeloma

doi: 10.1002/1878-0261.13447

Figure Lengend Snippet: CDDO‐2P‐Im binds GRP78 and activates the PERK and IRE1α branches of the UPR in a dose‐dependent manner. (A) Cell lysate of RPMI‐8226 was incubated with DMSO or CDDO‐2P‐Im for 30 min. Lysates were then subjected to 0.025 mg·mL −1 pronase for 30 min at 4 °C, followed by SDS/PAGE and western blot using antibodies against GRP78 and GAPDH. CDDO‐2P‐Im protects GRP78 from pronase cleavage compared with DMSO control. (B) Thermal shift assay was performed on recombinant GRP78 treated with control (DMSO) or CDDO‐2P‐Im. Relative fluorescent units (RFU) were measured by a SpectraMax Paradigm plate reader. The melting temperature (Tm) has a shift of −1.9 °C with the treatment of CDDO‐2P‐Im. (C, D) ARH‐77 and RPMI‐8226 cells were incubated with CDDO‐2P‐Im for 6 h. Cells were lysed and samples were prepared for SDS/PAGE. Western blot of UPR signaling proteins and downstream targets were performed. (E) 5T33 tumors were extracted from mice 15 days after tumor injection. Mice were treated once with either vehicle or 24 mg·kg −1 CDDO‐2P‐Im for 12 h prior to extraction. Samples were immunoblotted for UPR proteins. (F) RNA‐Sequencing data for ATF6 regulated genes showed no differences between Control, 0.1 μ m CDDO‐2P‐Im, and 0.4 μ m CDDO‐2P‐Im in RPMI‐8226 cells. (G, H) Western blot of ATF6 in ARH‐77 and RPMI‐8226 show no changes at 6 h with the treatment of CDDO‐2P‐Im. Values were given as mean ± SD. Fragments Per Kilobase of transcript per Million mapped reads is abbreviated as FPKM. Western blot and DARTS data are representatives of at least three independent experiments for ARH‐77 cells and RPMI‐8226 cells. Thermal shift assay was performed three independent times and representative data was shown. Western blot of 5T33 tumors was performed once. RNA‐Sequencing was performed once. Student t ‐tests were performed to calculate P value. 2P‐Im, CDDO‐2P‐Im; CDDO, CDDO‐2P‐Im.

Article Snippet: 10 μ m of recombinant GRP78 protein (PROTP11021; Boster Biological Technology, Pleasanton, CA, USA) was incubated with 150 μ m 2P‐Im controlled with 1% DMSO, and thermal shift assay was performed as previously described [ ].

Techniques: Incubation, SDS Page, Western Blot, Control, Thermal Shift Assay, Recombinant, Injection, Extraction, RNA Sequencing

Journal: Molecular Oncology

Article Title: The synthetic oleanane triterpenoid CDDO‐2P‐Im binds GRP78 / BiP to induce unfolded protein response‐mediated apoptosis in myeloma

doi: 10.1002/1878-0261.13447

Figure Lengend Snippet: Inhibition of the PERK‐ATF4‐CHOP arm of the UPR partially rescues CDDO‐2P‐Im‐induced apoptosis. (A) Cell lysate of WT and PERK KO RPMI‐8226 were probed for PERK protein by western blot. (B) WT and DDIT3 (CHOP) KO RPMI‐8226 were incubated with DMSO or CDDO‐2P‐Im for 6 h and lysed for protein analysis. Western blot of CHOP was performed to investigate the knockout of CHOP protein. (C) Cells were preincubated with DMSO or CDDO‐2P‐Im for 24 h and evaluated for cell viability by CellTiter‐Glo™. (D) Cells were preincubated with DMSO or 0.25 μ m ISRIB for 3 h before treating with DMSO or CDDO‐2P‐Im for an additional 16 h. Cells were then measured for cell viability by CellTiter‐Glo™. Controls were cells that were not treated with CDDO‐2P‐im but were treated with DMSO or ISRIB where appropriate. (E–H) PERK KO and WT RPMI‐8226 cells or ARH‐77 cells pretreated with ISRIB for 3 h were incubated with DMSO control or 0.4 μ m CDDO‐2P‐Im for 6 h. (E, F) Cells were extracted for RNA and qRT‐PCR was performed to investigate changes in the UPR. (G, H) In another round of experiments, cells were also extracted for protein to confirm such changes in the UPR. Values were given as mean ± SD. Student t ‐tests were performed to calculate P value. * P < 0.05 and ** P < 0.01, compared with control. Cell viability data are representative of three independent experiments. Western blots and qRT‐PCR data are representatives of two independent experiments. (I) We provide a working model of the actions of CDDO‐2P‐Im in cancer cells. At low concentrations (panel A), 2P‐Im binds and inhibits KEAP1, an adaptor protein for ubiquitin ligase that negatively regulates Nrf2 levels. Nrf2 will translocate to the nucleus and dimerize with small Maf proteins (sMaf) to activate the transcription of Nrf2 target genes. At higher concentrations (panel B), 2P‐Im binds GRP78/BiP, resulting in the activation of the UPR. GRP78 dissociates from its binding partners leading to the phosphorylation of PERK and IRE1α and activation of these branches of the UPR. The transcription factors, CHOP and XBP1, will cause UPR‐associated gene expression changes which when prolonged will lead to apoptosis. Interestingly, XBP1 is known to increase the expression of HRD1, a negative regulator of Nrf2, and independent of KEAP1. 2P/2P‐Im, CDDO‐2P‐Im; ISRIB, integrated stress response inhibitor; WT, wild‐type; KO, knockout.

Article Snippet: 10 μ m of recombinant GRP78 protein (PROTP11021; Boster Biological Technology, Pleasanton, CA, USA) was incubated with 150 μ m 2P‐Im controlled with 1% DMSO, and thermal shift assay was performed as previously described [ ].

Techniques: Inhibition, Western Blot, Incubation, Knock-Out, Control, Quantitative RT-PCR, Ubiquitin Proteomics, Activation Assay, Binding Assay, Phospho-proteomics, Gene Expression, Expressing