Journal: bioRxiv

Article Title: Targeting the canonical ER stress IRE1α/XBP1 pathway counteracts pancreatic cancer-induced skeletal muscle wasting

doi: 10.1101/2025.05.05.652304

Figure Lengend Snippet: Orthotopic model of pancreatic cancer was established by injecting 2 x 10 5 KPC cells into the pancreas of mice. PBS injected mice served as controls. Quantification of (A) tumor-free body weight (BW), (B) change in BW relative to day 0, and (C) four-paw grip strength normalized by tumor-free BW on day 21 after injection of KPC cells. (D) Quantitative RT-PCR (qRT-PCR) analysis of proteolysis markers (MAFbx, MuRF1, and MUSA1, Beclin1, LC3b, Atg12) in gastrocnemius (GA) muscle of KPC tumor-bearing mice compared to corresponding muscle of control mice. (E) The Volcano plot presented here shows significant changes in the mRNA levels of various genes (Log2FC ≥ 0.25 and p-value < 0.05) in GA muscle of KPC tumor-bearing mice compared to corresponding muscle of control mice in RNA-Seq dataset. (F) Bar graph shows pathways associated with enriched genes in cachectic GA muscle of KPC tumor bearing mice. (G) Heatmap showing relative mRNA levels of various components involved in protein processing in the ER in GA muscle of control and KPC tumor-bearing mice. (H) Relative mRNA levels of IRE1α ( Ern1 ), Xbp1 , Dnajb9 , Edem1 , CHOP ( Ddit3 ) and PERK ( Eif2ak3 ) in control and tumor-bearing mice assayed by performing qRT-PCR analysis. n=3 mice per group. (I) Immunoblots and (J) quantification of levels of IRE1α, sXBP1, CHOP, and PERK protein in GA muscle of control and KPC tumor-bearing mice. (K) qRT-PCR analysis showing mRNA levels of known RIDD substrates Bloc1s1 , Pdgfr , Scara3 , and Sparc in GA muscle of control and KPC tumor-bearing mice. n=3-4 mice per group. All data are presented as mean ± SEM. * p < 0.05, values significantly different from GA muscle of PBS-injected mice; analyzed by unpaired Student t test.

Article Snippet: For myotube formation, primary myoblasts were incubated in DM for 48 h. Myotubes were transfected with control, IRE1α, XBP1, or STAT3 siRNA (Santa Cruz Biotechnology) using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific) for 24 h. For pharmacological treatments, myotube cultures were treated with 4μ8C or IXA4.

Techniques: Injection, Quantitative RT-PCR, Control, RNA Sequencing, Western Blot

Journal: Phytotherapy research : PTR

Article Title: A Marine-Derived Small Molecule Inhibits Prostate Cancer Growth by Promoting Endoplasmic Reticulum Stress Induced Apoptosis and Autophagy.

doi: 10.1002/ptr.8354

Figure Lengend Snippet: FIGURE 3 | MHO7 upregulated ER stress-related genes in PCa cells in vitro. (A) The top Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was analyzed by RNA-seq. (B) Gene set enrichment analysis of UPR genes was performed between vehicle and MHO7 (4 μM)- treated 22Rv1 cells (n = 3 per group). (C) The heat map of MHO7 (4 μM) related genes to ER stress (n = 3 per group). (D, E) RT-qPCR determined the modulation of the MHO7 related to ER stress genes in vehicle and MHO7 (4 μM)-treated 22Rv1 and Du145 cells (n = 5 per group). (F, G) Western blotting assays were performed to detect the expression of IRE1α, p-IRE1α, XBP-1 s, PERK, p-eIF2α, ATF6, CHOP and β-actin in 22Rv1, and Du145 cells after treatment with different concentrations (0, 2, 4, 8 μM) of MHO7 for 48 h (n = 3 per group). Each assay was performed in triplicate and the data are shown as the means ± SD. p values were calculated by t-test or χ2 test (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: The IRE1α (sc40705- SH) shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: In Vitro, RNA Sequencing, Quantitative RT-PCR, Western Blot, Expressing

Journal: Phytotherapy research : PTR

Article Title: A Marine-Derived Small Molecule Inhibits Prostate Cancer Growth by Promoting Endoplasmic Reticulum Stress Induced Apoptosis and Autophagy.

doi: 10.1002/ptr.8354

Figure Lengend Snippet: FIGURE 6 | MHO7 can bind to IRE1α protein and enhance the stability of IRE1α protein. (A) Flexible docking analysis of MHO7 with IRE1α. Compounds are shown in yellow sticks while amino acids are in blue sticks and protein is in light blue cartoon. (B) The molecular dynamics simulations package was used to explore IRE1α and MHO7 binding behavior. The root mean square deviation (RMSD) values and root mean square fluctuation (RMSF) values of IRE1α and MHO7 were calculated. (C) The molecular mechanics Poisson–Boltzmann surface area analysis was used to calculate the energy of the IRE1α-MHO7 complex. (D) The cellular thermal shift assay (CETSA) showed that the interaction between MHO7 and IRE1 improved the thermal stability of IRE1α protein (n = 3 per group). (E) Drug affinity responsive target stability (DARTS) assay showed that interaction between MHO7 and IRE1α decreased in the protease sensitivity of IRE1α protein. Each sample was subjected to protease digestion with varying dilution concentrations (0, 1:104, 1:103, 1:102) of pronase (1 mg per 4000 μg of total protein) in the lysate (n = 3 per group). (F) The immunoblot analysis of IRE1α levels in the presence of CHX, with or without MHO7 (4 μM), showed that interaction between MHO7 and IRE1α attenuated the degradation of IRE1α (n = 3 per group). Each assay was performed in triplicate and the data are shown as the means ± SD. p values were calculated by t-test (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: The IRE1α (sc40705- SH) shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Binding Assay, Thermal Shift Assay, Western Blot

Journal: Phytotherapy research : PTR

Article Title: A Marine-Derived Small Molecule Inhibits Prostate Cancer Growth by Promoting Endoplasmic Reticulum Stress Induced Apoptosis and Autophagy.

doi: 10.1002/ptr.8354

Figure Lengend Snippet: FIGURE 7 | MHO7 suppressed tumor proliferation and induced autophagy and apoptosis in vivo. (A) Diagram of the procedure for tumor model. 22RV1 cells were injected subcutaneously into the axillae of nude mice (n = 5 per group). (B) Hematoxylin and Eosin staining of the heart, liver, spleen, lung, kidney, and testicle in mice treated with vehicle or 50 mg/kg MHO7 (n = 5 per group). Scale bar: 20 μm. (C) Nude mice were sacrificed and their tumors were photographed after 5 weeks (n = 5 per group). (D) Xenograft tumors were measured every 7 days. V tumor = 0.5 × L × W2 (n = 5 per group). (E) Mice were sacrificed after 5 weeks and their tumor were excised and weighed (n = 5 per group). (F) Immunohistochemical (IHC) staining of the xenograft tumor samples were subjected to Ki67, IRE1α, LC3I/II, and cleaved-caspase3 staining (n = 5 per group). Scale bar: 20 μm.

Article Snippet: The IRE1α (sc40705- SH) shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: In Vivo, Injection, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: Phytotherapy research : PTR

Article Title: A Marine-Derived Small Molecule Inhibits Prostate Cancer Growth by Promoting Endoplasmic Reticulum Stress Induced Apoptosis and Autophagy.

doi: 10.1002/ptr.8354

Figure Lengend Snippet: FIGURE 8 | The predicted mechanism of MHO7 treatment in prostate cancer. MHO7 is a compound derived from the mangrove fungus Aspergillus ustus, and it exhibits remarkable antitumor activity against prostate cancer both in vivo and in vitro. In a dose- and time-dependent manner, MHO7 effectively inhibits the growth of prostate cancer cells. This inhibition occurs through the accumulation of reactive oxygen species (ROS), resulting in endoplasmic reticulum (ER) stress, and subsequently activating autophagy via the IRE1α-XBP-1s signaling pathways. In addition, MHO7 may promote apoptosis in prostate cancer cells through IRE1α and PERK pathway. Notably, the interaction between MHO7 and the IRE1α protein enhances the stability and protein level of IRE1α, leading to the induction of autophagy and apoptosis. Collectively, our findings suggest that MHO7 holds great promise as a therapeutic agent for prostate cancer.

Article Snippet: The IRE1α (sc40705- SH) shRNA lentiviral particles were purchased from Santa Cruz Biotechnology (Santa Cruz, USA).

Techniques: Derivative Assay, Activity Assay, In Vivo, In Vitro, Inhibition, Protein-Protein interactions

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) mRNA was extracted from WT or IRE1 DN-expressing U87 cells and from empty vector (EV), IRE1WT- or IRE1Q780*-expressing RADH85 and RADH87 cells. CD95 mRNA was quantified by RT-qPCR and normalized to GAPDH. Mean ± SEM, n = 3–5. ( B ) CD95 cell surface level expression was evaluated by flow cytometry. Mean of MFI ratio ± SEM, n = 3–4. Unpaired t -test for U87 (** p = 0.001126); one-way ANOVA with Tukey multiple comparison correction for RADH85 (* p = 0.018416 and ** p = 0.003549) and RADH87 (* p = 0.0263 and ** p = 0.0054). ( C , D ) U87 cells pre-treated for 2 h with MKC-8866 (30 μM) as indicated were further treated with 500 ng/mL tunicamycin for the indicated times. Lysates were analysed using western blot. ( C ) One representative experiment out of three independent ones is shown. ( D ) Quantification for three independent experiments is depicted. Mean ± SEM. ( E , F ) U87 cells pre-treated for 2 h with MKC-8866 (30 μM) as indicated were further treated with 50 nM thapsigargin for the indicated times. Lysates were analysed using western blot. ( E ) One representative experiment out of three independent ones is shown. ( F ) Quantification for three independent experiments is depicted. Mean ± SEM. .

Article Snippet: Specifically, 30 nM siRNA (Dharmacon TM On target plus SMART pool Human XBP1 (L-009552-00-0010); Dharmacon TM On target plus SMART pool Human EIF2AK3 (L-004883-00-0005); Dharmacon TM On-target plus TM Control pool Non-targeting pool (D-001810-10-05); Santa Cruz Biotechnology, IRE1ɑ siRNA (h) (sc-40705) were used.

Techniques: Expressing, Plasmid Preparation, Quantitative RT-PCR, Flow Cytometry, Comparison, Western Blot

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) RNA (2 μg) extracted from U87 cells was incubated with the indicated amounts of recombinant IRE1 for 1 h. CD95 mRNA was then quantified by RT-qPCR and normalized to GAPDH. Mean ± SEM, n = 3. One-way ANOVA with Dunnett multiple comparison correction, *** p = 0.0003 (0 vs 0.5 μg IRE1 groups), *** p = 0.0007 (0 vs 1 μg IRE1 groups) ( B ) Predicted folded structure of CD95 mRNA. The two predicted cleavage sites within hairpin loops are highlighted. ( C , D ) RNA (2 μg) extracted from U87 cells was incubated with the indicated amounts of recombinant IRE1 for 1 h. 136-bp ( C ) and 121-bp ( D ) parts of CD95 mRNA including the indicated potential cleavage sites were then quantified by RT-qPCR and normalized to GAPDH. Mean ± SEM, n = 3. One-way ANOVA with Dunnett multiple comparison correction, ( D ) * p = 0.0331, ** p = 0.0096. .

Article Snippet: Specifically, 30 nM siRNA (Dharmacon TM On target plus SMART pool Human XBP1 (L-009552-00-0010); Dharmacon TM On target plus SMART pool Human EIF2AK3 (L-004883-00-0005); Dharmacon TM On-target plus TM Control pool Non-targeting pool (D-001810-10-05); Santa Cruz Biotechnology, IRE1ɑ siRNA (h) (sc-40705) were used.

Techniques: Incubation, Recombinant, Quantitative RT-PCR, Comparison

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) U87 cells were transfected with siRNA control or targeting XBP1, IRE1 or PERK as indicated. 16 h later, cells were treated with DMSO (D) or 250 nM thapsigargin (TG) for 8 h. Lysates were analysed using western blot. One experiment representative of at least three independent ones is shown. ( B ) U87 cells were transfected with a plasmid coding for FLAG-XBP1s (XBP1s) or an empty vector (EV). 48 h later, cells were treated with the indicated concentrations of CD95L for 48 h. Viability was assessed using MTT assay and normalized to untreated cell values. Mean ± SEM of three independent experiments. Inset: western blot analyses of cell lysates 48 h post-transfection. ( C ) U87 cells were treated with DMSO or MKC-8866 (30 mM) for the indicated times. Lysates were analysed using western blot. One experiment representative of three independent ones is shown. ( D ) U87 cells treated with 30μM MKC-8866 or DMSO were further treated with 1 µg/mL CD95L for the indicated times. Cell death was evaluated using Cytotox red positivity. Mean ± SEM, n = 3. ( E – G ) U87 were transfected with siRNA control or targeting XBP1, IRE1 or PERK as indicated. 72 h later, cell lysates were analysed using western blot. One experiment representative of at least three independent ones is shown. ( H , I ) CD95 expression z-scores of 45 GB and 62 TNBC tumours were plotted according to the RIDD activity score ( H ) and according to the XBP1s activity score ( I ). The distribution of z-score is represented as violin plots. For GB n = 45; for TNBC n = 62. Statistical difference of expression between groups was calculated using Mann–Whitney tests and the p-value is indicated ( H GB *** p = 4.3e−05, TNBC *** p = 4.1e−06; I GB** p = 0.0025, * TNBC p = 0.015). .

Article Snippet: Specifically, 30 nM siRNA (Dharmacon TM On target plus SMART pool Human XBP1 (L-009552-00-0010); Dharmacon TM On target plus SMART pool Human EIF2AK3 (L-004883-00-0005); Dharmacon TM On-target plus TM Control pool Non-targeting pool (D-001810-10-05); Santa Cruz Biotechnology, IRE1ɑ siRNA (h) (sc-40705) were used.

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, MTT Assay, Expressing, Activity Assay, MANN-WHITNEY

Journal: EMBO Reports

Article Title: IRE1 RNase controls CD95-mediated cell death

doi: 10.1038/s44319-024-00095-9

Figure Lengend Snippet: ( A ) U87 cells were transfected with siRNA control or targeting XBP1, IRE1 or PERK as indicated. 16 h later, cells were treated with 2.5 μg/mL thapsigargin for 8 h. Lysates were analysed using western blot. One experiment representative of at least three independent ones is shown. ( B ) SUM159 cells were transfected with a plasmid coding for FLAG-XBP1s (XBP1s) or an empty vector (EV). 48 h later, cells were treated with the indicated concentrations of CD95L for 48 h. Viability was assessed using MTT assay and normalized to untreated cell values. Mean ± SEM of three independent experiments. Inset: western blot analyses of cell lysates 48 h post-transfection. ( C ) U87 cells were treated with DMSO or Z4 (25 μM) for the indicated times. Lysates were analysed using western blot. One experiment representative of three independent ones is shown. ( D ) U87 were transfected with siRNA control or targeting IRE1 as indicated. 72 h later, cells were treated with 100 ng/mL CD95L. % of cell death was defined as the % of Cytotox red-positive cells as detected by the Incucyte. Two independent experiments are shown. .

Article Snippet: Specifically, 30 nM siRNA (Dharmacon TM On target plus SMART pool Human XBP1 (L-009552-00-0010); Dharmacon TM On target plus SMART pool Human EIF2AK3 (L-004883-00-0005); Dharmacon TM On-target plus TM Control pool Non-targeting pool (D-001810-10-05); Santa Cruz Biotechnology, IRE1ɑ siRNA (h) (sc-40705) were used.

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, MTT Assay

Journal: iScience

Article Title: BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1

doi: 10.1016/j.isci.2022.105626

Figure Lengend Snippet: BRCA1 protein interacts with and ubiquitinates PERK and IRE1 (A–D) Total protein extracts were isolated from (A) control or BRCA1-depleted MCF7, (B) control or BRCA1-depleted MDA-MB-231cells, (C) MDA-MB-436 cells (+ or def), and (D) control, BRCA1 or BARD1 over-expressing MDA-MB-436 BRCA1-def cells. (E and F) Immunoprecipitation (IP) of BRCA1 protein pull-downs of PERK and IRE1 from MCF7 (E) or MDA-MB-231 (F) cytoplasmic protein extract. BRCA1 IB: ∗ = hyperphosphorylated BRCA1, ∗∗ = truncated BRCA1. ∗∗∗ = delta11q isoform. IRE1 IB: ∗ = possible ubiquitinated IRE1. (G and H) Ubiquitination analysis of PERK and IRE1 in (G) MCF7 cells or (H) MDA-MB-231 cells. Cells were transfected with control or BRCA1 siRNA. BRCA1 siRNA transfected cells were either untreated or treated with DMSO or Bortezomib overnight before harvesting for protein analysis. (I and J) In vitro ubiquitination analysis of (I) PERK or (J) IRE1 with E1, UBE2J1, BRCA1, BARD1, and/or ubiquitin. Ubiquitinated PERK or IRE1 was detected with an anti-Ub antibody. Ub = Ubiquitin. Bortz = Bortezomib. Data are represented as mean ± SD. P value was calculated by Student’s two-tailed, unpaired t -test. ∗ <0.05. See also Figures S4–S6 .

Article Snippet: IRE1 siRNA , Santa Cruz Biotech , Cat# sc-40705.

Techniques: Isolation, Control, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Transfection, In Vitro, Two Tailed Test

Journal: iScience

Article Title: BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1

doi: 10.1016/j.isci.2022.105626

Figure Lengend Snippet: Depleting UPR components is lethal to the BRCA1-def cancer cells (A) MDA-MB-436 (BRCA1-def or +), HCC1937 BRCA1-def and SUMPT149 BRCA1-def breast cancer cells were transfected with control, CypB, IRE1, GRP94, HSP70, BiP, PERK or DERL1 siRNA and assessed for cell survival. (B) HCC1937 BRCA1+, MDA-MB-231, MCF7, and MCF10A cells were transfected with CypB siRNA and assessed for cell survival. (C) MDA-MB-436 (+ or def) and HCC1937 (+ or def) breast cancer cells were transfected with control, CypB-A or CypB-B siRNA. Top, western blot analysis. Bottom, clonal cell survival analysis. (D) MDA-MB-436 BRCA1-def cells were transfected with either (i) pCMV vector and siRNA control, (ii) PPIB/pCMV and siRNA control, (iii) pCMV control and CypB 3′UTR siRNA or (iv) PPIB/pCMV and CypB 3′UTR siRNA for cell survival and Western blot analysis. N.S. = not significant. F-CypB = Flag-tagged cyclophilin B. Bar charts are the summary of the quantitative analysis of the colony forming units (CFU) from each siRNA transfection experiment. Data are displayed as mean ± SD. P values were calculated by Student’s two-tailed, unpaired t -test. See also Figures S7 and .

Article Snippet: IRE1 siRNA , Santa Cruz Biotech , Cat# sc-40705.

Techniques: Transfection, Control, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: iScience

Article Title: BRCA1 mediates protein homeostasis through the ubiquitination of PERK and IRE1

doi: 10.1016/j.isci.2022.105626

Figure Lengend Snippet:

Article Snippet: IRE1 siRNA , Santa Cruz Biotech , Cat# sc-40705.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Extraction, SYBR Green Assay, Isolation, Control, Plasmid Preparation, Software