Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Autophagy and the unfolded protein response promote profibrotic effects of TGF-β 1 in human lung fibroblasts

doi: 10.1152/ajplung.00372.2017

Figure Lengend Snippet: XBP splicing is required for TGF-β1-induced fibrosis in primary human IPF, but not non-IPF, fibroblasts. A: primary human non-IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of the IRE1α inhibitor MKC8866 (10 μM) for the indicated time points. The representative immunoblot shows the abundance of collagen 1α2, fibronectin, and sXBP in 4 different primary non-IPF fibroblasts. B and C: densitometry analysis (n = 4) confirmed that MKC8866 did not significantly reduce TGF-β1-induced collagen 1α2 or fibronectin biosynthesis in non-IPF human fibroblasts. Data are shown in dot plots of from 4 individual experiments using different cell lines. The horizontal line in each column represents the mean, with error bars showing SE. NSP > 0.05, no significant difference. D: primary human IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of IRE1 inhibitor (10 μM) for the indicated time points. Immunoblots are arranged to show expression of collagen 1α2, fibronectin and sXBP. The immunoblot is representative of experiments performed on 4 different primary IPF fibroblasts (n = 4). E and F: densitometry analysis confirmed that treatment of IPF fibroblasts with IRE1 inhibitor significantly reduced TGF-β1-induced collagen 1α2 and fibronectin biosynthesis. Data are shown in dot plots of from 4 individual experiments using different cell lines. The horizontal line in each column represents the mean, with error bars showing SE. ***P < 0.001; **P < 0.01. G: primary human IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of IRE1 inhibitor (10 μM) for 96 h. For a baseline control, cells were maintained in serum- and TGF-β1-deficient media. Quantitative PCR showed that MKC8866 significantly inhibited TGF-β1-induced fibronectin mRNA accumulation in IPF fibroblasts. The experiments were performed in duplicate on 2 different primary IPF fibroblast cell lines. Data are shown in dot plots from each experiment. The horizontal line in each column represents the mean, with error bars showing SE. ***P < 0.001.

Article Snippet: Bafilomycin-A1 (Baf-A1), rabbit anti-human/mouse/rat LC3βII (L8918, 1:3,000), anti-mouse IgG (A8924, 1:3,000), and anti-rabbit IgG (A6154, 1:5,000) were obtained from Sigma-Aldrich (Oakville, CA).The IRE1-specific inhibitor (MKC8866), which inhibits both basal and thapsigargin induced splicing of XBP1 mRNA ( 47 ), was provided by Mannkind (Westlake Village, CA).

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Autophagy and the unfolded protein response promote profibrotic effects of TGF-β 1 in human lung fibroblasts

doi: 10.1152/ajplung.00372.2017

Figure Lengend Snippet: Autophagy flux inhibition and IRE1α inhibition modulates UPR and autophagy in IPF fibroblasts. Primary human IPF fibroblasts were stimulated with TGF-β1 (2.5 ng/ml) in the presence or absence of bafilomycin-A1 (10 nM) or IRE1 inhibitor MKC8866 (10 μM) for 48 or 96 h. Immunoblots show abundance of autophagy (LC3βII, p62) and UPR (BIP) markers. The blot shown is representative of experiments performed on 2 different primary IPF fibroblast cell lines.

Article Snippet: Bafilomycin-A1 (Baf-A1), rabbit anti-human/mouse/rat LC3βII (L8918, 1:3,000), anti-mouse IgG (A8924, 1:3,000), and anti-rabbit IgG (A6154, 1:5,000) were obtained from Sigma-Aldrich (Oakville, CA).The IRE1-specific inhibitor (MKC8866), which inhibits both basal and thapsigargin induced splicing of XBP1 mRNA ( 47 ), was provided by Mannkind (Westlake Village, CA).

Techniques: Inhibition, Western Blot

Journal: eLife

Article Title: Cell-based HTS identifies a chemical chaperone for preventing ER protein aggregation and proteotoxicity

doi: 10.7554/eLife.43302

Figure Lengend Snippet: ( A ) Inhibition of the EUA-EGFP reporter in HEK293A cells treated with 0.2 μg/mL Tm in the presence of PERK inhibitor I (GSK2606414), IRE1 inhibitor I (STF083010) or Site-1-protease inhibitor (PF429242). ( B–C ) EUA-EGFP reporter activity and viability per well in the 1 st screen were counted. Figure 1—figure supplement 1—source data 1. Dataset for .

Article Snippet: IRE1 Inhibitor I (STF083010) was purchased from Toronto Research Chemicals (Ontario, Canada).

Techniques: Inhibition, Protease Inhibitor, Activity Assay

Journal: eLife

Article Title: Cell-based HTS identifies a chemical chaperone for preventing ER protein aggregation and proteotoxicity

doi: 10.7554/eLife.43302

Figure Lengend Snippet: ( A–C ) Inhibition of the 25X ERSE2-EGFP ( A ), 25X UPRE-EGFP ( B ) or 25X AARE-EGFP ( C ) reporters in HEK293A cells treated with 0.2 μg/mL Tm in the presence or absence of IBT21. Error bars show the mean ± SD ( n = 4). ( D ) Representative immunoblots of ATF4, GADD34, CHOP, DNAJB9, SEL1L and HERPUD1 in HEK293A cells treated with 0.2 μg/mL Tm overnight in the presence or absence of IBT21 or AZO. β-actin was used as a loading control. ( E ) Representative immunoblots of ATF4, GADD34, CHOP, DNAJB9, SEL1L and HERPUD1 in HEK293A cells treated with 0.2 μg/mL Tm overnight in the presence or absence of 10 μM IBT21, 1 μM PERK inhibitor I (GSK2606414), 200 μM IRE1 inhibitor I (STF083010) or 2 μM Site-1-protease inhibitor (PF429242). β-actin was used as a loading control. Figure 4—source data 1. Dataset for .

Article Snippet: IRE1 Inhibitor I (STF083010) was purchased from Toronto Research Chemicals (Ontario, Canada).

Techniques: Inhibition, Western Blot, Control, Protease Inhibitor

Journal: Frontiers in Cellular Neuroscience

Article Title: Homer1a Attenuates Endoplasmic Reticulum Stress-Induced Mitochondrial Stress After Ischemic Reperfusion Injury by Inhibiting the PERK Pathway

doi: 10.3389/fncel.2019.00101

Figure Lengend Snippet: Homer1a preserves mitochondrial function by reducing ER stress mainly via the PERK pathway after I/R injury. TM was used to activate the ER stress whereas the GSK2656157 and 3,5-dibromosalicylaldehyde (DBSA) were used to inhibit the PERK and IRE1 respectively. Different DBSA concentrations were tested in cortical neurons and detected by Western blot (A) as well as GSK2656157 (B) . Then 20 μM DBSA and 5 nM GSK2656157 were chosen to apply to follow experiments. The cytochrome c release to cytoplasm in each group was detected through calculating the ratio of cytoplasmic/mitochondrial cytochrome c by Western blot (B) . The intracellular ATP level was measured by ATP assay (C) and the intracellular ROS level was investigated through H 2 -DCFDA (D) . The data are represented as means ± SEM. $ p < 0.05, * p < 0.05 vs. Control, # p < 0.05 vs. OGD, & p < 0.05 vs. OGD+LV-Homer1a.

Article Snippet: The p-IRE1 inhibitor 3,5-dibromosalicylaldehyde (DBSA) was obtained from Tokyo Chemical Industry (TCI, Tokyo, Japan).

Techniques: Western Blot, ATP Assay, Control

Journal: Journal of Cell Science

Article Title: Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells

doi: 10.1242/jcs.258685

Figure Lengend Snippet: KRB incubation activates IRE1. (A) Western blot visualization of IRE1-p (using the anti IRE1-p antibody, Genentech) in cells in Schneider's medium (Sch), Sch+DTT (5 mM), KRB, KRB+amino acids (AAs) (5 mM) and KRB+AMG18 (10 µM) for 4 h after blotting. Note that KRB incubation elicits IRE1-p more strongly than Sch+DTT, and that addition of AAs to KRB partially reverses this phosphorylation. Addition of the IRE1 kinase attenuator AMG18 (10 µM) strongly inhibits IRE1-p formation. Quantification underneath is the ratio of IRE1-p (middle band) to α-tubulin for the blot shown. (B) Visualization of the PCR products of ire1 and h2a mRNAs from cells incubated in Sch, KRB and SCH100+DTT for 4 h at 26°C. (C) Visualization of spliced ( xbp1s ) and unspliced ( xbp1u ) PCR products of xbp1 upon conditions indicated on the panel. (D,D′) Immunofluorescence visualization (D) of the protein Bip in cells incubated in the conditions indicated on the panel. Quantification is in D′. Note that the UPR is stimulated in KRB and many other conditions. (E) Quantification of Sec body formation (marked by Sec16) in cells incubated in KRB, SCH150 or SCH100+DTT with or without 4u8C (30 µM), AMG18 (10 µM), HG (5 µM) or AMG18+HG. ‘d’ indicates the mean±s.e.m. decrease in Sec body formation when compared to the absence of inhibitors. P -value (SCH150 and SCH150+AMG18) is 0.104 and the P -value (KRB and KRB+HG) is 0.0019. The other differences are highly significant (<10 −4 ). Errors bars: s.e.m. Scale bar: 10 µm.

Article Snippet: Drugs were used at the concentrations mentioned in Table S2 , including the IRE1 kinase inhibitor amgen small molecule 18 (AMG18) ( ).

Techniques: Incubation, Western Blot, Phospho-proteomics, Immunofluorescence

Journal: Journal of Cell Science

Article Title: Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells

doi: 10.1242/jcs.258685

Figure Lengend Snippet: KRB incubation is mimicked by a moderate salt stress combined with activation of IRE1 and PERK. (A–C) Immunofluorescence (IF) micrographs of Sec16 in cells in Schneider's medium (Sch), KRB, Sch supplemented with DTT (5 mM) and thapsigargin (Thapsi, 2 µM) for 4 h at 26°C (A). Overexpression of the constitutively active (CA) IRE1 mutant tagged by V5 (in green) in cells incubated in Sch and Schneider's buffer (C). A quantification of the percentage of cells with Sec bodies is shown in B. Trans, transfected. (D) Western blot visualization of eIF2α-p in cells in Sch, Sch+DTT (5 mM) and KRB. (E) Quantification of Sec body formation (marked by Sec16) upon PERK depletion, PERK inhibition (5 µM), combined inhibition of PERK and IRE1 kinase (AMG18), and PERK and IRE1 nuclease (4u8C), as well as ATF6 depletion upon KRB incubation for 4 h at 26°C. ‘d’ indicates the mean±s.e.m. decrease in Sec body formation. (F,F′) Visualization (F) of Sec body formation (marked by Sec16) in cells incubated in SCH100 and SCH100+DTT (5 mM) for 4 h at 26°C. Quantification in F′. Errors bars: s.e.m. Scale bars: 10 µm.

Article Snippet: Drugs were used at the concentrations mentioned in Table S2 , including the IRE1 kinase inhibitor amgen small molecule 18 (AMG18) ( ).

Techniques: Incubation, Activation Assay, Immunofluorescence, Over Expression, Mutagenesis, Transfection, Western Blot, Inhibition

Journal: Journal of Cell Science

Article Title: Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells

doi: 10.1242/jcs.258685

Figure Lengend Snippet: Activation of SIK, IRE1 and PERK and SIK to form Sec bodies. Blue pathway, Sec bodies can form upon high-salt stress (SCH150) through SIK activation, which, when strong is sufficient. A moderate salt stress also activates SIKs, but it is not enough to stimulate Sec body formation. Red pathway: amino acid starvation in KRB leads to IRE1, PERK and SIK activation leading to Sec body formation (mimicked by SCH100+DTT). IRE1 and PERK activation are necessary but not sufficient. In green are features appearing upon KRB incubation, such as cytoplasm acidification, a decrease in the cytoplasmic ATP concentration and the stimulation of the UPR that we propose to be a key factor in Sec body formation.

Article Snippet: Drugs were used at the concentrations mentioned in Table S2 , including the IRE1 kinase inhibitor amgen small molecule 18 (AMG18) ( ).

Techniques: Activation Assay, Incubation, Concentration Assay

Journal: bioRxiv

Article Title: HNRNPA2B1 controls an unfolded protein response-related prognostic gene signature in prostate cancer

doi: 10.1101/2022.06.21.495112

Figure Lengend Snippet: (A) Venn diagram of protein-coding genes differentially-expressed and co-regulated by XBP1, IRE1 and HNRNPA2B1 with Log 2 fold change <-0.5 and p<0.05 in RNA-Seq datasets from LNCaP cells treated with siRNA to XBP1 or IRE1 inhibitor MKC8866 or PC3M cells treated with siRNA to HNRNPA2B1. (B) Derivation of prognostic biomarker panel by elastic net selection of 20 HNRNPA2B1, IRE1, and XBP1 co-regulated protein-coding genes in the TCGA cohort to generate a single four gene panel as the best predictors of disease relapse. (B, left panel) Cross-validation curve (red dots) with standard deviation. Left vertical dashed line is the value of λ that gives minimum mean cross-validated error (lambda.min), right vertical dashed line is the value of λ that gives the most regularized model such that the cross-validated error is within one standard error of the minimum (lambda.1se). (B, right panel) Heat map displaying the log 2 fold change expression of the four HIX signature genes following treatment of LNCaPs with the IRE1 inhibitor MKC886, or XBP1 or HNRNPA2B1 depletion in LNCaP and PC3M cells respectively. (C-D, top panels) Distribution plot of risk scores for (C) derivation (TCGA) and (D) validation (MSKCC) cohorts. Vertical red lines represent mean of low and high percentile risk scores. (C-D, bottom panels) Kaplan-Meier plots of disease-free survival probabilities for patients from (C) derivation (TCGA) and (D) validation (MSKCC) datasets stratified by risk groups. The number of patients at risk for each group are presented in the table below each X-axis time point. Univariable Cox PH-dervied hazard ratios with 95% confidence intervals (CI) and two-tailed log-rank test p-values are shown. (E) PC3M cells were transfected with 3μg plasmid DNA (72 hours) encoding HNRNPA2 or vector only (VO) control. (E, top panel) Western blot shows HNRNPA2 protein expression compared to Beta Actin loading control. (E, bottom panel) PC3M cell viability was measured by MTT assay following transfection with 300 ng of plasmid DNA vector encoding HNRNPA2 or VO control. Cells were simultaneously treated with either 50 or 100 μM STF083010 or vehicle control (DMSO). Three biological replicates were used, and Two-tailed T-test was used to compare treatment groups. * = p<0.05

Article Snippet: The IRE1 inhibitor STF083010 was purchased from Merck Life Science, UK (SML0409).

Techniques: RNA Sequencing Assay, Biomarker Assay, Selection, Standard Deviation, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Western Blot, MTT Assay