Journal: bioRxiv

Article Title: Endoplasmic reticulum stress signaling actively contributes to therapy resistance in colorectal cancer

doi: 10.1101/2024.01.07.574523

Figure Lengend Snippet: (A) Frequencies of ATF4, XBP1 and/or ATF6 gene amplification in patients with major solid cancer types (left top) and Kaplan-Meier analysis of disease-free survival among cancer patients with amplified ATF4, XBP1 or ATF6 compared to patients with no alterations from the public TCGA datasets. (B) Comparison of aneuploidy scores between CRC patients with ATF6 gene amplification (ATF6 AMP ) plus/minus TP53 mutation or homozygous deletion (TP53 MUT ) and patients with no alterations (TP53 WT and/or ATF6 WT ). * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. (C) Kaplan-Meier analysis of disease-free survival among CRC patients present with TP53 MUT and/or ATF6 AMP signatures, compared to patients with no alterations (TP53 WT and/or ATF6 WT ) from the public TCGA datasets. * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001. (D) The positivity of XBP1 and ATF6 was assessed by flow cytometry and compared between MSI group ( n = 3) and MSS group ( n = 3–4) in colon tumor and adjacent normal colon tissues. MSI, microsatellite instability. MSI, microsatellite instable; MSS, microsatellite stable or below the detection limit.

Article Snippet: HEK293 cells were cultured in 6-well plates and then transfected with 4 μg of previously described plasmid vectors encoding ER stress reporter genes ATF4-mS (#115970, Addgene, Cambridge, MA, USA), XBP1-mN (#115971), ATF6-GFP (#32955) or H2A-mS (#85051) by calcium phosphate precipitation.

Techniques: Amplification, Comparison, Mutagenesis, Flow Cytometry

Journal: bioRxiv

Article Title: Endoplasmic reticulum stress signaling actively contributes to therapy resistance in colorectal cancer

doi: 10.1101/2024.01.07.574523

Figure Lengend Snippet: (A) Comparison of basal gene expression levels of ATF4, (s/u)XBP1 and ATF6 between human cell lines. (B) Design of ER stress reporter genes encoded with fluorescently labelled ATF4, XBP1 or ATF6. (C) The reporter gene expression in transfected HEK293 cells was validated after treatment with Tg (1 μM, 24 h). Cut-off = 95% percentile of transfected cells without Tg treatment. (D) Total (endogenous plus exogenous) expression of ATF4, XBP1 and ATF6 in transfected HEK293 cells was analyzed by qRT-PCR after treatment with Tg (1 μM, 24 h). (E) Cross effect of the reporter gene expression on endogenous expression of the other ER stress transcription factors. Tg, Thapsigargin.

Article Snippet: HEK293 cells were cultured in 6-well plates and then transfected with 4 μg of previously described plasmid vectors encoding ER stress reporter genes ATF4-mS (#115970, Addgene, Cambridge, MA, USA), XBP1-mN (#115971), ATF6-GFP (#32955) or H2A-mS (#85051) by calcium phosphate precipitation.

Techniques: Comparison, Expressing, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Endoplasmic reticulum stress signaling actively contributes to therapy resistance in colorectal cancer

doi: 10.1101/2024.01.07.574523

Figure Lengend Snippet: (A) XTT assay of ER stress reporter cells to OxaPt (0–200 μM, 24 h). * p < 0.05, ** p < 0.01, compared to HEK293 WT cells. IC 50 values determined by the XTT assay ( n = 4– 5) were compared to HEK293 WT cells. (B) Total (endogenous plus exogenous) expression of ATF4, XBP1 and ATF6 in HEK293 reporter cells was analyzed by qRT-PCR after treatment with OxaPt (20 μM, 24 h). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, compared to untreated cells. OxaPt, Oxaliplatin.

Article Snippet: HEK293 cells were cultured in 6-well plates and then transfected with 4 μg of previously described plasmid vectors encoding ER stress reporter genes ATF4-mS (#115970, Addgene, Cambridge, MA, USA), XBP1-mN (#115971), ATF6-GFP (#32955) or H2A-mS (#85051) by calcium phosphate precipitation.

Techniques: XTT Assay, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Endoplasmic reticulum stress signaling actively contributes to therapy resistance in colorectal cancer

doi: 10.1101/2024.01.07.574523

Figure Lengend Snippet: (A) XTT cell viability assay of HCT116 and HEK293 cells treated with inhibitors for ER-stress receptors and OxaPt. HCT116 and HEK293 cells were treated with GSK2656157 (GSK’157, 10 μM, 1 h), STF083010 (STF’010, 50 μM, 1 h) or Ceapin-A7 (Ceapin, 5 or 10 μM, 6 h) prior to the treatment with OxaPt (0–200 μM, 24 h). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001. (B) Flow cytometric analysis of apoptosis in HCT116 and HEK293 cells treated with Ceapin-A7 (ATF6 inh., 5 μM, 1 h) prior to the treatment with OxaPt (20 μM, 24 h). (C) Patient-derived organoids (PODs) were treated with Ceapin-A7 (5 μM, 6 h) prior to the treatment with OxaPt (0–20 μM, 48 h), followed by co-staining with SYTOX and Hoechst. SYTOX/Hoechst ratios (each point represent one organoid) were compared. Organoids were isolated from adjacent normal colon tissues from a stage IV CRC patient. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, compared to the vehicle control (DMSO). OxaPt, Oxaliplatin.

Article Snippet: HEK293 cells were cultured in 6-well plates and then transfected with 4 μg of previously described plasmid vectors encoding ER stress reporter genes ATF4-mS (#115970, Addgene, Cambridge, MA, USA), XBP1-mN (#115971), ATF6-GFP (#32955) or H2A-mS (#85051) by calcium phosphate precipitation.

Techniques: Viability Assay, Derivative Assay, Staining, Isolation

Journal: bioRxiv

Article Title: Endoplasmic reticulum stress signaling actively contributes to therapy resistance in colorectal cancer

doi: 10.1101/2024.01.07.574523

Figure Lengend Snippet: Time-lapse analysis of the ER stress inducible markers in HEK293 cells treated with OxaPt (20 μM, 0–96 h) or Tg (1 μM, 0–24 h) by (A) Western blotting and (B) XBP1 mRNA splicing assay. (C) Optical section images of HEK293 ATF6-GFP reporter and H2A-mS control cells after treatment with OxaPt (20 μM, 0–6 h) or Tg (1 μM, 0–6 h). The cells were co-stained with DAPI and anti-Golgin-97 antibody. Scale bar = 10 nm. (D) Quantification of ATF6-GFP reporter localization to nucleus or Golgi regions in HEK293 cells was evaluated and compared that of H2A-mS reporter in respective cells based on the optical section images (workflows are depicted in Fig. S5). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, compared to the 0 h treatment. OxaPt, Oxaliplatin; Tg, Thapsigargin.

Article Snippet: HEK293 cells were cultured in 6-well plates and then transfected with 4 μg of previously described plasmid vectors encoding ER stress reporter genes ATF4-mS (#115970, Addgene, Cambridge, MA, USA), XBP1-mN (#115971), ATF6-GFP (#32955) or H2A-mS (#85051) by calcium phosphate precipitation.

Techniques: Western Blot, Splicing Assay, Staining

Journal: bioRxiv

Article Title: Endoplasmic reticulum stress signaling actively contributes to therapy resistance in colorectal cancer

doi: 10.1101/2024.01.07.574523

Figure Lengend Snippet: (A) The numbers of significantly down/upregulated proteins ( q ≤ 0.05) in HEK293 WT, XBP1-mN and ATF6-GFP reporter cells treated with OxaPt (20 μM, 24 h), compared to untreated condition, and overlaps of those for each cell line. (B) The numbers of significantly down/upregulated proteins ( q ≤ 0.05) in XBP1-mN and ATF6-GFP reporter cells treated with OxaPt (20 μM, 24 h) or Tg (1 μM, 24 h), compared to untreated condition, and overlaps of those for each treatment within each cell line. (C, D) Volcano plots of down/upregulated proteins in (C) XBP1-mN or (D) ATF6-GFP cells, compared to untreated conditions (left panels) and enrichment pathway analysis based on gene ontology terms performed for upregulated proteins (right panels, q ≤ 0.05). OxaPt, Oxaliplatin.

Article Snippet: HEK293 cells were cultured in 6-well plates and then transfected with 4 μg of previously described plasmid vectors encoding ER stress reporter genes ATF4-mS (#115970, Addgene, Cambridge, MA, USA), XBP1-mN (#115971), ATF6-GFP (#32955) or H2A-mS (#85051) by calcium phosphate precipitation.

Techniques:

Journal: bioRxiv

Article Title: Endoplasmic reticulum stress signaling actively contributes to therapy resistance in colorectal cancer

doi: 10.1101/2024.01.07.574523

Figure Lengend Snippet: (A) qRT-PCR analysis of the total (endogenous plus exogenous) expression of ATF4, XBP1 and ATF6 in HEK293 reporter cells 24 h after irradiation at 2 Gy. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, compared to that of 0 h after treatment. (B) Flow cytometric detection of the reporter gene expression in HEK293 reporter cells 30 h after irradiation at 0–8 Gy. (C) Flow cytometric analysis of apoptosis in HEK293 cells 48 h after irradiation at 0–8 Gy. (D) Colony formation assay of HEK293 reporter cells irradiated at 0–4 Gy and comparison of the survival rates between the cells irradiated at 2 Gy.

Article Snippet: HEK293 cells were cultured in 6-well plates and then transfected with 4 μg of previously described plasmid vectors encoding ER stress reporter genes ATF4-mS (#115970, Addgene, Cambridge, MA, USA), XBP1-mN (#115971), ATF6-GFP (#32955) or H2A-mS (#85051) by calcium phosphate precipitation.

Techniques: Quantitative RT-PCR, Expressing, Irradiation, Colony Assay, Comparison

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) A representative immunoblot of CD44 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. (B) Cell survival rate of shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment (n = 10). (C) Functional enrichment analyses of genes that were up- or downregulated (q value <0.05) by both shCD44–1 and shCD44–2 expression in IMR90-hTert cells. (D) Real-time qPCR data of representative UPR genes in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells (n = 3). Data were normalized to GAPDH . (E) A representative immunoblot of HSPA5 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. (F) Relative density of HSPA5/b-Actin analyzed by ImageJ (n = 3). (G) The boxplot shows the effects of CD44 overexpression on the expression levels of the indicated genes in IMR90 cells. The effects are shown for the genes that are upregulated by ATF6 activating molecule AA147 and the genes co-expressed with ATF6, ATF4, or XBP1 (retrieved from Enrichr). (H) Percentages of overlap between CD44 co-expressing genes (retrieved from COXPRESdb v7) and the indicated genes. (I) Relative cell survival rate of shCD44–1 and shCD44–2 IMR90-hTert cells compared with shLuc IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment. Vehicle (DMSO) or 10 μM HA15 was added to the medium during tunicamycin treatment (n = 10). All immunoblots were repeated once with similar results. Error bars are presented as mean ± SD values. *p < 0.05; one-way ANOVA with post hoc Dunnett’s test for (B and F), two-way ANOVA with post hoc Dunnett’s test for (D), Wilcoxon test for (G), Fisher’s exact test for (H), and two-tailed t test with Bonferroni-Dunn correction for (I).

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Western Blot, Functional Assay, Expressing, Over Expression, Two Tailed Test

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) Representative immunoblots of IRE1, PERK, and ATF6 in IMR90 cells 3 days after transfection with siLuc, siIRE1, siPERK, or siATF6. The experiment was repeated once with similar results. (B and D) Relative cell survival rate of shCD44–1 and shCD44–2 IMR90-hTert cells compared with shLuc IMR90-hTert cells after 6 days of 0.5 μg/mL tunicamycin treatment. (B) Cells were transfected with siLuc, siIRE1, siPERK, or siATF6 2 days before starting tunicamycin treatment (n = 10). (D) Vehicle (DMSO), 50 μM 4μ8C, 20 nM GSK2606414, 10 μM PF429242, 5 nM SCH772984, or 20 μM SB202190 was added to the medium during tunicamycin treatment. (C) Relative expression levels of representative UPR genes in IMR90 cells 24 h after 0.5 μg/mL tunicamycin treatment. Vehicle (DMSO), 50 μM 4μ8C, 20 nM GSK2606414, or 10 μM PF429242 was supplemented to the medium during tunicamycin treatment (n = 3). Expression levels were measured by real-time qPCR and normalized to GAPDH . Error bars are presented as mean ± SD values. *p < 0.05; two-way ANOVA with post hoc Dunnett’s test.

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Western Blot, Transfection, Expressing

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) Protein-protein interactions among the 41 CD44-associated proteins plus IRE1, PERK, and ATF6 were mapped using the STRING software. Only highest confidence interactions (interaction score ≥0.9) that are experimentally determined (purple line) or stored in curated databases (blue line) were included in the analysis. IRE1, PERK, and ATF6 are shown as red nodes and the 16 CD44-associated ER proteins are shown as blue nodes. CD44-associated proteins are defined here as proteins that are detected by cross-linked immunoprecipitation-mass spectrometry of IMR90 cells using anti-CD44 antibody with at least 10-fold greater abundance than in control experiment using normal rabbit IgG antibody. (B) Representative immunoblots of CALR, HSP47, and CD44 in whole cell lysate and in CD44-immunoprecipitates prepared from IMR90 cells. The experiment was repeated once with similar results. (C) Representative confocal images of IMR90 cells co-stained with antibodies against CD44 and organelle markers (mitochondrial marker mtTFA and ER markers ERp57, HSP47, and RPN2). (D) The dot plot shows Pearson correlation coefficients between immunofluorescence signals of CD44 and organelle markers. Each dot represents a single cell (n = 30). Error bars are presented as mean ± SD values. Scale bars, 10 μm.

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Software, Immunoprecipitation, Mass Spectrometry, Western Blot, Staining, Marker, Immunofluorescence

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: (A) The heatmap shows relative abundances of ER-associated proteins detected by mass spectrometry of ER fractions isolated from shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Cells were collected 24 h after starting 0.5 μg/mL tunicamycin treatment. ER-associated proteins were defined here as the proteins whose ER localization have been confirmed by the Human Protein Atlas or associated with the GO term “endoplasmic reticulum lumen.” (B) Representative immunoblots of COL6A3, SEC62, and HSPA5 in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Whole cell lysates and culture supernatants were collected 24 h after starting 0.5 μg/mL tunicamycin treatment. (C) Representative immunoblots of ATF6, IRE1, PERK, CALR, EGFR, and Vinculin in shLuc, shCD44–1, and shCD44–2 IMR90-hTert cells. Cells were lysed in buffer containing either 1% Triton or 2% SDS. (D) A representative immunoblot of ATF6 in U2OS cells and in ATF6 KO U2OS cells infected with empty vector or vector expressing wild-type ATF6, ATF6 Y392C, or ATF6 Y567N. (E) Cell survival rate of ATF6 KO U2OS cells after 5 days of 2 μg/mL tunicamycin treatment (n = 8). Cells were infected with vector expressing wild-type ATF6, ATF6 Y392C, or ATF6 Y567N and selected with 100 μg/mL hygromycin prior to the experiment. (F) Relative expression levels of HSP90B1 and HSP5A in CD44 KO U2OS cells (n = 3). Cells were infected with empty vector or vector encoding CD44-ectodomain-KDEL 3 days before sample collection. Data were normalized to GAPDH . (G) Cell survival rate of CD44 KO U2OS cells after 5 days of 2 μg/mL tunicamycin treatment. Cells were infected with empty vector or vector encoding CD44-ectodomain-KDEL 3 days before starting tunicamycin treatment (n = 8). All immunoblots were repeated at least once with similar results. Error bars are presented as mean ± SD values. *p < 0.05; two-way ANOVA with post hoc Dunnett’s test (E) and two-tailed t test with (F) or without (G) Bonferroni-Dunn correction.

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Mass Spectrometry, Isolation, Western Blot, Infection, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: CD44 correlates with longevity and enhances basal ATF6 activity and ER stress resistance

doi: 10.1016/j.celrep.2023.113130

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Vectors encoding human ATF6 and S1P site-mutated GFP-ATF6 were obtained from Addgene (#11974 and #32956)., Mouse standard form CD44, mouse LDLR, and NMR standard form CD44 were cloned in pBabe-hygro vector (Addgene #1765).

Techniques: Recombinant, Transfection, Protease Inhibitor, CyQUANT Assay, Proliferation Assay, Expressing, Mass Spectrometry, Software

Journal: PNAS Nexus

Article Title: Gradual ER calcium depletion induces a progressive and reversible UPR signaling

doi: 10.1093/pnasnexus/pgae229

Figure Lengend Snippet: Incremental activation of the UPR sensors upon calcium depletion in the ER. a) HeLa cells were treated with the indicated concentrations of tBu. PERK and IRE1 phosphorylation status was monitored using Phos-tag SDS-PAGE and Western blot. b) Quantification of results presented in (a) reported in percentage of phosphorylated protein over the total PERK/IRE1 protein quantity (mean ± SEM of N = 3). c) ATF6 subcellular colocalization with Giantin (Golgi) and DAPI (nucleus) following treatment with 30 µM tBuBHQ for the indicated time. Scale bar = 10 µm. d) Quantification of ATF6 translocation to the Golgi following treatment with the indicated doses of tBuBHQ. Values of Golgi/reticular intensity ratio were normalized to basal values observed before the addition of tBuBHQ. Mean of n = 45 (control), n = 43 (3 µM), n = 39 (5 µM), n = 22 (10 µM), n = 51 (30 µM) ( N = 3). SEM was omitted for the sake of clarity (data are provided in Fig. ).

Article Snippet: 535 nm for GFP-ATF6 driven by Simple 32 Software from Compix Incorporated.

Techniques: Activation Assay, SDS Page, Western Blot, Translocation Assay, Control

Journal: PNAS Nexus

Article Title: Gradual ER calcium depletion induces a progressive and reversible UPR signaling

doi: 10.1093/pnasnexus/pgae229

Figure Lengend Snippet: Data-driven computational simulation of IRE1, PERK, and ATF6 activation. a) Schematic representation of the model of activation of IRE1 and PERK based on the dissociation of BiP from the sensors upon accumulation of UP. The modes of activation are assumed to be similar for IRE1and PERK, although the two differ by the values of the kinetic constants. The model assumes the existence of preformed oligomers of IRE1 and PERK. Equations of the model are given in the Materials and methods section. b) Schematic representation of the model of activation of ATF6 based on the dissociation of BiP upon accumulation of UP. When freed from BiP, ATF6 translocates to the Golgi complex where it is cleaved by S1P and S2P. Equations of the model are given in the Materials and methods section. c) Computational simulations of the activation of the 3 branches of the UPR after treatment with different concentrations of tBu. Blue lines correspond to the results of the simulations of the models schematized in a and b, with the ), , –( and the parameter values listed in Table . Blue squares represent experimental data and the grey shaded region, the curve interpolated between those data ± SEM. d) Model prediction of the evolution of ER Ca 2+ concentration after washout of tBu 30 µM. In the model ) and ( , tBu concentration is multiplied by 0.05 at time 120 min. e) Model prediction of the reversion of IRE1 and PERK activation after washout of tBu 30 µM, corresponding to the evolution of [Ca 2+ ] ER shown in (d). Results were obtained by integration of ), , –( .

Article Snippet: 535 nm for GFP-ATF6 driven by Simple 32 Software from Compix Incorporated.

Techniques: Activation Assay, Concentration Assay