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Tocris salubrinal 23-471-0

<t>ISR</t> pathway activation induces PD-L1 and CD155 protein. A, Western blot analysis of human KRAS -mutant H441 or EGFR -mutant PC9 cells treated for 24 or 48 hours with 100 or <t>200</t> <t>μmol/L</t> salubrinal or DMSO vehicle (Veh) control. Vinculin served as a loading control for this and subsequent Western blots. B, Western blot analysis of human H441, H358, PC9, and HCC827 cells after 24 hours in RPMI 1640 media supplemented with or without amino acids. C, Western blot analysis of human H441, H358, PC9, and HCC827 cells treated for 24 hours with 5 μmol/L thapsigargin (ER Ca+ ATPase pump inhibitor) to induce ER stress or DMSO vehicle control. D, Western blot analysis of H358 cells with control or UROD sgRNA. E, Western blot analysis of H358 or HCC827 cells with control or UROD siRNA. F, Flow cytometric analysis of cell-surface CD155 and PD-L1 protein in DMSO vehicle control and thapsigargin-treated (5 μmol/L for 24 hours) H358 or HCC827 NSCLC cells. G, Western blot analysis in eIF2α WT (S/S) or mutant (Ser51Ala A/A) MEFs treated with DMSO vehicle control or 100 μmol/L salubrinal for 24 hours. H, Western blot analysis of thapsigargin- and ISRIB-treated human NSCLC cells. Data from a single experiment are shown and are representative of at least three independent experiments. p-eIF2α, phosphorylated eIF2α.

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1) Product Images from "The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer"

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

Journal: Cancer Research

doi: 10.1158/0008-5472.CAN-24-3844

ISR pathway activation induces PD-L1 and CD155 protein. A, Western blot analysis of human KRAS -mutant H441 or EGFR -mutant PC9 cells treated for 24 or 48 hours with 100 or 200 μmol/L salubrinal or DMSO vehicle (Veh) control. Vinculin served as a loading control for this and subsequent Western blots. B, Western blot analysis of human H441, H358, PC9, and HCC827 cells after 24 hours in RPMI 1640 media supplemented with or without amino acids. C, Western blot analysis of human H441, H358, PC9, and HCC827 cells treated for 24 hours with 5 μmol/L thapsigargin (ER Ca+ ATPase pump inhibitor) to induce ER stress or DMSO vehicle control. D, Western blot analysis of H358 cells with control or UROD sgRNA. E, Western blot analysis of H358 or HCC827 cells with control or UROD siRNA. F, Flow cytometric analysis of cell-surface CD155 and PD-L1 protein in DMSO vehicle control and thapsigargin-treated (5 μmol/L for 24 hours) H358 or HCC827 NSCLC cells. G, Western blot analysis in eIF2α WT (S/S) or mutant (Ser51Ala A/A) MEFs treated with DMSO vehicle control or 100 μmol/L salubrinal for 24 hours. H, Western blot analysis of thapsigargin- and ISRIB-treated human NSCLC cells. Data from a single experiment are shown and are representative of at least three independent experiments. p-eIF2α, phosphorylated eIF2α.

Figure Legend Snippet: ISR pathway activation induces PD-L1 and CD155 protein. A, Western blot analysis of human KRAS -mutant H441 or EGFR -mutant PC9 cells treated for 24 or 48 hours with 100 or 200 μmol/L salubrinal or DMSO vehicle (Veh) control. Vinculin served as a loading control for this and subsequent Western blots. B, Western blot analysis of human H441, H358, PC9, and HCC827 cells after 24 hours in RPMI 1640 media supplemented with or without amino acids. C, Western blot analysis of human H441, H358, PC9, and HCC827 cells treated for 24 hours with 5 μmol/L thapsigargin (ER Ca+ ATPase pump inhibitor) to induce ER stress or DMSO vehicle control. D, Western blot analysis of H358 cells with control or UROD sgRNA. E, Western blot analysis of H358 or HCC827 cells with control or UROD siRNA. F, Flow cytometric analysis of cell-surface CD155 and PD-L1 protein in DMSO vehicle control and thapsigargin-treated (5 μmol/L for 24 hours) H358 or HCC827 NSCLC cells. G, Western blot analysis in eIF2α WT (S/S) or mutant (Ser51Ala A/A) MEFs treated with DMSO vehicle control or 100 μmol/L salubrinal for 24 hours. H, Western blot analysis of thapsigargin- and ISRIB-treated human NSCLC cells. Data from a single experiment are shown and are representative of at least three independent experiments. p-eIF2α, phosphorylated eIF2α.

Techniques Used: Activation Assay, Western Blot, Mutagenesis, Control

$ISR pathway activation enhances PD-L1 and CD155 translation. A, Polysome profiling of H1944 vehicle- or salubrinal-treated cells (100 μmol/L for 24 hours). B and C, qRT-PCR analysis of PD-L1(CD274) ( B ) and CD155(PVR) ( C ) mRNA in ribosomal fractions from A . qRT-PCR analysis for each gene shown was performed with 1 primer set spanning an exon–exon junction (primer set 1). Data for primers set 2 are available in Supplementary Fig. S2. Fractions associated with <3 ribosomes were grouped to represent poorly translated mRNAs, and fractions associated with >3 ribosomes were grouped as efficiently translated mRNAs. PD-L1 and CD155 mRNA expression in each fraction was normalized to luciferase , and mRNA abundance was calculated as the percent of total in all fractions. Luciferase control mRNA was added to each fraction prior to RNA extraction to control for variability. Error bars represent the SD from the mean from three independent fractions (<3 or >3 ribosomes). D, Diagram of the WT human CD155 5′ UTR with 6 CTGs and mutant constructs with CTGs mutated to CTCs cloned upstream of a luciferase reporter. E, Dual luciferase assay of MEFs transfected with indicated CD155 -5′ UTR firefly luciferase reporter constructs normalized to cotransfected control Renilla luciferase. Luciferase activity was monitored after 48 hours. Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. F, qRT-PCR analysis of the mean luciferase mRNA normalized to actin in MEFs shown in E . Error bars represent the SD from the mean from n = 3 biological replicates. G, Dual luciferase assay of the CD155 5′ UTR in MEF vehicle- or salubrinal-treated cells (100 μmol/L for 24 hours). Error bars represent the SD from the mean from n = 3 biological replicates. H, qRT-PCR analysis of the mean luciferase mRNA normalized to actin in MEFs shown in G . n = 3 biological replicates. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005.$
Figure Legend Snippet: ISR pathway activation enhances PD-L1 and CD155 translation. A, Polysome profiling of H1944 vehicle- or salubrinal-treated cells (100 μmol/L for 24 hours). B and C, qRT-PCR analysis of PD-L1(CD274) ( B ) and CD155(PVR) ( C ) mRNA in ribosomal fractions from A . qRT-PCR analysis for each gene shown was performed with 1 primer set spanning an exon–exon junction (primer set 1). Data for primers set 2 are available in Supplementary Fig. S2. Fractions associated with <3 ribosomes were grouped to represent poorly translated mRNAs, and fractions associated with >3 ribosomes were grouped as efficiently translated mRNAs. PD-L1 and CD155 mRNA expression in each fraction was normalized to luciferase , and mRNA abundance was calculated as the percent of total in all fractions. Luciferase control mRNA was added to each fraction prior to RNA extraction to control for variability. Error bars represent the SD from the mean from three independent fractions (<3 or >3 ribosomes). D, Diagram of the WT human CD155 5′ UTR with 6 CTGs and mutant constructs with CTGs mutated to CTCs cloned upstream of a luciferase reporter. E, Dual luciferase assay of MEFs transfected with indicated CD155 -5′ UTR firefly luciferase reporter constructs normalized to cotransfected control Renilla luciferase. Luciferase activity was monitored after 48 hours. Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. F, qRT-PCR analysis of the mean luciferase mRNA normalized to actin in MEFs shown in E . Error bars represent the SD from the mean from n = 3 biological replicates. G, Dual luciferase assay of the CD155 5′ UTR in MEF vehicle- or salubrinal-treated cells (100 μmol/L for 24 hours). Error bars represent the SD from the mean from n = 3 biological replicates. H, qRT-PCR analysis of the mean luciferase mRNA normalized to actin in MEFs shown in G . n = 3 biological replicates. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005.

Techniques Used: Activation Assay, Quantitative RT-PCR, Expressing, Luciferase, Control, RNA Extraction, Mutagenesis, Construct, Clone Assay, Transfection, Activity Assay

ISR pathway activation diminishes immune cell function in vitro . A, ELISAs for IL2 and granzyme B of Jurkat T cells cocultured with H358 cells. H358 cells were pretreated for 24 hours with salubrinal and 800 nmol/L ISRIB and then washed and cocultured with Jurkat T cells for an additional 24 hours with αCD3 and αCD28 activating antibodies (4 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. B, ELISAs for IL2 and granzyme B of Jurkat T cells cocultured with H358 cells with control or UROD sgRNA and 800 nmol/L ISRIB. H358 cells were cocultured with Jurkat T cells for 24 hours with αCD3 and αCD28 activating antibodies (4 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of two independent experiments. C, ELISAs for IL2 and granzyme B of primary human PBMCs cocultured with H358 cells. H358 cells were pretreated for 24 hours with salubrinal and 800 nmol/L ISRIB and then washed and cocultured with PBMCs for an additional 24 hours with αCD3 and αCD28 activating antibodies (1 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. D, ELISAs for IL2 and granzyme B of PBMCs cocultured with H358 cells with control or UROD sgRNA and 800 nmol/L ISRIB. H358 cells were cocultured with PBMCs for 24 hours with αCD3 and αCD28 activating antibodies (1 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of two independent experiments. E, ELISAs for IL2 and granzyme B of OT-1 T cells cocultured with murine KP cells (KdP67-1) expressing OVA and crystal violet assay of KP cells after coculture with OT-1 T cells. KP cells were pretreated with 100 μmol/L salubrinal and/or 500 nmol/L ISRIB for 24 hours and then cocultured for 24 hours with activated CD8 + OT-1 T cells. Error bars represent the SD from the mean from n = 3–4 biological replicates. Data from a single experiment are shown and are representative of two independent experiments. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005. sg NS , nonspecific (control) sgRNA; sg UROD , UROD sgRNA.

Figure Legend Snippet: ISR pathway activation diminishes immune cell function in vitro . A, ELISAs for IL2 and granzyme B of Jurkat T cells cocultured with H358 cells. H358 cells were pretreated for 24 hours with salubrinal and 800 nmol/L ISRIB and then washed and cocultured with Jurkat T cells for an additional 24 hours with αCD3 and αCD28 activating antibodies (4 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. B, ELISAs for IL2 and granzyme B of Jurkat T cells cocultured with H358 cells with control or UROD sgRNA and 800 nmol/L ISRIB. H358 cells were cocultured with Jurkat T cells for 24 hours with αCD3 and αCD28 activating antibodies (4 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of two independent experiments. C, ELISAs for IL2 and granzyme B of primary human PBMCs cocultured with H358 cells. H358 cells were pretreated for 24 hours with salubrinal and 800 nmol/L ISRIB and then washed and cocultured with PBMCs for an additional 24 hours with αCD3 and αCD28 activating antibodies (1 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. D, ELISAs for IL2 and granzyme B of PBMCs cocultured with H358 cells with control or UROD sgRNA and 800 nmol/L ISRIB. H358 cells were cocultured with PBMCs for 24 hours with αCD3 and αCD28 activating antibodies (1 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of two independent experiments. E, ELISAs for IL2 and granzyme B of OT-1 T cells cocultured with murine KP cells (KdP67-1) expressing OVA and crystal violet assay of KP cells after coculture with OT-1 T cells. KP cells were pretreated with 100 μmol/L salubrinal and/or 500 nmol/L ISRIB for 24 hours and then cocultured for 24 hours with activated CD8 + OT-1 T cells. Error bars represent the SD from the mean from n = 3–4 biological replicates. Data from a single experiment are shown and are representative of two independent experiments. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005. sg NS , nonspecific (control) sgRNA; sg UROD , UROD sgRNA.

Techniques Used: Activation Assay, Cell Function Assay, In Vitro, Control, Expressing, Crystal Violet Assay

ISR activation enhances tumorigenesis and reduces immune cell infiltration in vivo . A, Western blot analysis of KRAS -mutant murine CMT167 cells treated for 24 or 48 hours with 100 or 200 μmol/L salubrinal or DMSO vehicle control. p-eIF2α, phosphorylated eIF2α; Veh, vehicle. B, Quantification of tumor volumes of CMT167 cells transplanted in C57BL/6J mice ( n = 11 mice per group for vehicle-treated mice; n = 10 mice per group for salubrinal-treated mice). The graph represents mean tumor volumes, and error bars represent the SEM. C and D, Endpoint tumor volumes ( C ) and tumor mass ( D ) of resected tumors shown in B . Horizontal bars represent mean values. E, Multiplexed IHC-F was performed on tumors from B . Representative images (10×) are shown. Scale bar, 200 μm. F–H, Quantification of CD3 + , CD4 + , and CD8 + tumor-infiltrating lymphocytes (Rexpressed as counts/mm 3 ). Five fields were quantified per mouse from five mice. Graphs represent mean counts, and error bars represent the SD from the mean. A Student t test was used to determine statistical significance. **, P < 0.005; ***, P < 0.0005.

Figure Legend Snippet: ISR activation enhances tumorigenesis and reduces immune cell infiltration in vivo . A, Western blot analysis of KRAS -mutant murine CMT167 cells treated for 24 or 48 hours with 100 or 200 μmol/L salubrinal or DMSO vehicle control. p-eIF2α, phosphorylated eIF2α; Veh, vehicle. B, Quantification of tumor volumes of CMT167 cells transplanted in C57BL/6J mice ( n = 11 mice per group for vehicle-treated mice; n = 10 mice per group for salubrinal-treated mice). The graph represents mean tumor volumes, and error bars represent the SEM. C and D, Endpoint tumor volumes ( C ) and tumor mass ( D ) of resected tumors shown in B . Horizontal bars represent mean values. E, Multiplexed IHC-F was performed on tumors from B . Representative images (10×) are shown. Scale bar, 200 μm. F–H, Quantification of CD3 + , CD4 + , and CD8 + tumor-infiltrating lymphocytes (Rexpressed as counts/mm 3 ). Five fields were quantified per mouse from five mice. Graphs represent mean counts, and error bars represent the SD from the mean. A Student t test was used to determine statistical significance. **, P < 0.005; ***, P < 0.0005.

Techniques Used: Activation Assay, In Vivo, Western Blot, Mutagenesis, Control

ISR pathway inhibition improves response to PD-1 blockade in a syngeneic mouse model. A, Western blot analysis of Kras -mutant murine CMT167 cells expressing doxycycline-inducible control shRNA or two independent shRNAs targeting Urod . Ab, antibody. B, Schematic illustration of CMT167 syngeneic experiment. C, Quantification of tumor volumes of CMT167 cells expressing the indicated shRNA sequence transplanted in C57BL/6J mice ( n = 15 mice per group; scrambled shRNA data are shown in Supplementary Fig. S4C and S4D). Graph represents mean tumor volumes. Error bars, SEM. D, Uniform manifold approximation and projection (UMAP) analysis of tumor-infiltrating lymphocytes, colored by cell types. E, Quantification of tumor-infiltrating lymphocytes (expressed as a percentage of CD45 + cells) from flow mass cytometry (mass CyTOF). n = 5 scrambled mice; n = 5 Urod shRNA mice; n = 5 Urod shRNA + ISRIB; n = 4 Urod shRNA + αPD-1; n = 5 Urod shRNA + ISRIB +αPD-1. Graphs represent mean values, and error bars represent the SD from the mean. F, Quantification of tumor volumes of CMT167 cells expressing the indicated shRNAs transplanted in C57BL/6J mice ( n = 11–12 mice per group; scrambled shRNA data are shown in Supplementary Fig. S5C). Graph represents mean tumor volumes. Error bars, SEM. G, Quantification of CD8 + T cells (expressed as a percentage of CD45 + cells) from mice in F . n = 5 scrambled mice; n = 4 Urod shRNA mice; n = 5 Urod shRNA + ISRIB; n = 5 Urod shRNA + ISRIB+ αPD-1; n = 5 Urod shRNA + ISRIB +αTIGIT; n = 5 Urod shRNA + ISRIB + αPD-1+ αTIGIT. Graph represent mean values. Error bars, SD from the mean. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ns, not significant. p-eIF2α, phosphorylated eIF2α; sh Urod , short-hairpin Urod ; Tregs, regulatory T cells. B, Created in BioRender. O’Donnell, K. (2025) https://BioRender.com/vphxffz .

Figure Legend Snippet: ISR pathway inhibition improves response to PD-1 blockade in a syngeneic mouse model. A, Western blot analysis of Kras -mutant murine CMT167 cells expressing doxycycline-inducible control shRNA or two independent shRNAs targeting Urod . Ab, antibody. B, Schematic illustration of CMT167 syngeneic experiment. C, Quantification of tumor volumes of CMT167 cells expressing the indicated shRNA sequence transplanted in C57BL/6J mice ( n = 15 mice per group; scrambled shRNA data are shown in Supplementary Fig. S4C and S4D). Graph represents mean tumor volumes. Error bars, SEM. D, Uniform manifold approximation and projection (UMAP) analysis of tumor-infiltrating lymphocytes, colored by cell types. E, Quantification of tumor-infiltrating lymphocytes (expressed as a percentage of CD45 + cells) from flow mass cytometry (mass CyTOF). n = 5 scrambled mice; n = 5 Urod shRNA mice; n = 5 Urod shRNA + ISRIB; n = 4 Urod shRNA + αPD-1; n = 5 Urod shRNA + ISRIB +αPD-1. Graphs represent mean values, and error bars represent the SD from the mean. F, Quantification of tumor volumes of CMT167 cells expressing the indicated shRNAs transplanted in C57BL/6J mice ( n = 11–12 mice per group; scrambled shRNA data are shown in Supplementary Fig. S5C). Graph represents mean tumor volumes. Error bars, SEM. G, Quantification of CD8 + T cells (expressed as a percentage of CD45 + cells) from mice in F . n = 5 scrambled mice; n = 4 Urod shRNA mice; n = 5 Urod shRNA + ISRIB; n = 5 Urod shRNA + ISRIB+ αPD-1; n = 5 Urod shRNA + ISRIB +αTIGIT; n = 5 Urod shRNA + ISRIB + αPD-1+ αTIGIT. Graph represent mean values. Error bars, SD from the mean. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ns, not significant. p-eIF2α, phosphorylated eIF2α; sh Urod , short-hairpin Urod ; Tregs, regulatory T cells. B, Created in BioRender. O’Donnell, K. (2025) https://BioRender.com/vphxffz .

Techniques Used: Inhibition, Western Blot, Mutagenesis, Expressing, Control, shRNA, Sequencing, Mass Cytometry

Model of translational control of CD155 and PD-L1 in response to ISR activation. Stresses commonly present in the TME activate the ISR pathway in tumor cells. This results in enhanced translation of CD155 and PD-L1 , which suppresses T-cell function by binding the inhibitory T-cell receptors PD-1 and TIGIT, respectively, thereby leading to enhanced tumorigenesis. Created in BioRender. O’Donnell, K. (2025) https://BioRender.com/vphxffz .

Figure Legend Snippet: Model of translational control of CD155 and PD-L1 in response to ISR activation. Stresses commonly present in the TME activate the ISR pathway in tumor cells. This results in enhanced translation of CD155 and PD-L1 , which suppresses T-cell function by binding the inhibitory T-cell receptors PD-1 and TIGIT, respectively, thereby leading to enhanced tumorigenesis. Created in BioRender. O’Donnell, K. (2025) https://BioRender.com/vphxffz .

Techniques Used: Control, Activation Assay, Cell Function Assay, Binding Assay

Image Search Results

OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

Journal: Neural Regeneration Research

Article Title: Neuroserpin alleviates cerebral ischemia-reperfusion injury by suppressing ischemia-induced endoplasmic reticulum stress

doi: 10.4103/NRR.NRR-D-24-00044

Figure Lengend Snippet: OGD/R induces early activation of ER stress sensors followed by apoptosis in cultured cortical neurons, which is ameliorated by ER stress inhibitors. (A) Schematic representation of the timeline of the experimental procedures. Cortical neurons (7 DIV) were cultured in normal conditions (Control group) or exposed to oxygen/glucose deprivation for 4 hours followed by reperfusion (OGD/R group). Cell lysates were collected at various time points for Western blotting, and cell viability was evaluated after 24 hours reperfusion. (B) The cell viability of primary cortical neurons after OGD/R 24 hours was detected by MTT assay. Cortical neurons (7 DIV) under standard culture conditions were used as the control group ( n = 3). ** P < 0.01, vs. control, unpaired t -test. (C) Cell lysates from the OGD/R group were collected at different time points (0–24 hours) after reperfusion. For the control group, cell lysates were collected at the same time corresponding to 0 hours reperfusion of OGD/R group. Levels of ER stress sensors, protein synthesis, and apoptosis-related proteins were examined by western blot analysis. (D–K) Quantifications of the normalized levels of p-PERK/PERK (D, n = 4), p-eIF2α/eIF2α (E, n = 3), p-IRE1/IRE1 (F, n = 3), ATF6 (G, n = 3), puromycin (H, n = 3), ATF4 (I, n = 3), CHOP (J, n = 3), and cleaved-caspase-3 (K, n = 3). GAPDH and α-tubulin served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control, one-way analysis of variance followed by Dunnett’s multiple comparison test. (L) A schematic illustration of the application of ER stress inhibitors, sodium 4-PBA or Sal, to neurons 1 hour before OGD and during OGD (−5 to 0 hours). (M, N) MTT assay was performed to evaluate cell viability of neurons treated with 4-PBA or Sal ( n = 3). **** P < 0.0001, OGD/R vs. control; # P < 0.05, ## P < 0.01, 4-PBA (50 or 100 µM) or Sal (25 or 100 µM) vs. OGD/R, unpaired t -test. Data are shown as mean ± SEM. 4-PBA: Sodium 4-phenylbutyrate; ATF: activating transcription factor; CHOP: CCAAT/enhancer binding protein homologous protein; C-Casp-3: cleaved-caspase-3; DIV: day in vitro ; eIF2α: eukaryotic translation initiation factor 2α; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IRE1: inositol requiring enzyme 1; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; OGD: oxygen-glucose deprivation; OGD/R: oxygen-glucose deprivation/reperfusion; p-: phosphorylated-; PERK: double-stranded RNA-activated protein kinase-like ER kinase; Sal: salubrinal.

Article Snippet: The ER stress inhibitors sodium 4-phenylbutyrate (4-PBA, 50 and 100 μM, Selleck, Houston, TX, USA) and salubrinal (25 and 100 μM, Selleck) were added for 1 hour before OGD or after reperfusion.

Techniques: Activation Assay, Cell Culture, Control, Western Blot, MTT Assay, Comparison, Binding Assay, In Vitro

Journal: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: ISR pathway activation induces PD-L1 and CD155 protein. A, Western blot analysis of human KRAS -mutant H441 or EGFR -mutant PC9 cells treated for 24 or 48 hours with 100 or 200 μmol/L salubrinal or DMSO vehicle (Veh) control. Vinculin served as a loading control for this and subsequent Western blots. B, Western blot analysis of human H441, H358, PC9, and HCC827 cells after 24 hours in RPMI 1640 media supplemented with or without amino acids. C, Western blot analysis of human H441, H358, PC9, and HCC827 cells treated for 24 hours with 5 μmol/L thapsigargin (ER Ca+ ATPase pump inhibitor) to induce ER stress or DMSO vehicle control. D, Western blot analysis of H358 cells with control or UROD sgRNA. E, Western blot analysis of H358 or HCC827 cells with control or UROD siRNA. F, Flow cytometric analysis of cell-surface CD155 and PD-L1 protein in DMSO vehicle control and thapsigargin-treated (5 μmol/L for 24 hours) H358 or HCC827 NSCLC cells. G, Western blot analysis in eIF2α WT (S/S) or mutant (Ser51Ala A/A) MEFs treated with DMSO vehicle control or 100 μmol/L salubrinal for 24 hours. H, Western blot analysis of thapsigargin- and ISRIB-treated human NSCLC cells. Data from a single experiment are shown and are representative of at least three independent experiments. p-eIF2α, phosphorylated eIF2α.

Article Snippet: For ISR pathway activation and inhibition, cells were treated with 100 or 200 μmol/L salubrinal (Tocris, 23-471-0) and/or 500 to 800 nmol/L ISRIB (Thermo Fisher Scientific, 5284) for 24 or 48 hours.

Techniques: Activation Assay, Western Blot, Mutagenesis, Control

Journal: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: ISR pathway activation enhances PD-L1 and CD155 translation. A, Polysome profiling of H1944 vehicle- or salubrinal-treated cells (100 μmol/L for 24 hours). B and C, qRT-PCR analysis of PD-L1(CD274) ( B ) and CD155(PVR) ( C ) mRNA in ribosomal fractions from A . qRT-PCR analysis for each gene shown was performed with 1 primer set spanning an exon–exon junction (primer set 1). Data for primers set 2 are available in Supplementary Fig. S2. Fractions associated with <3 ribosomes were grouped to represent poorly translated mRNAs, and fractions associated with >3 ribosomes were grouped as efficiently translated mRNAs. PD-L1 and CD155 mRNA expression in each fraction was normalized to luciferase , and mRNA abundance was calculated as the percent of total in all fractions. Luciferase control mRNA was added to each fraction prior to RNA extraction to control for variability. Error bars represent the SD from the mean from three independent fractions (<3 or >3 ribosomes). D, Diagram of the WT human CD155 5′ UTR with 6 CTGs and mutant constructs with CTGs mutated to CTCs cloned upstream of a luciferase reporter. E, Dual luciferase assay of MEFs transfected with indicated CD155 -5′ UTR firefly luciferase reporter constructs normalized to cotransfected control Renilla luciferase. Luciferase activity was monitored after 48 hours. Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. F, qRT-PCR analysis of the mean luciferase mRNA normalized to actin in MEFs shown in E . Error bars represent the SD from the mean from n = 3 biological replicates. G, Dual luciferase assay of the CD155 5′ UTR in MEF vehicle- or salubrinal-treated cells (100 μmol/L for 24 hours). Error bars represent the SD from the mean from n = 3 biological replicates. H, qRT-PCR analysis of the mean luciferase mRNA normalized to actin in MEFs shown in G . n = 3 biological replicates. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Luciferase, Control, RNA Extraction, Mutagenesis, Construct, Clone Assay, Transfection, Activity Assay

Journal: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: ISR pathway activation diminishes immune cell function in vitro . A, ELISAs for IL2 and granzyme B of Jurkat T cells cocultured with H358 cells. H358 cells were pretreated for 24 hours with salubrinal and 800 nmol/L ISRIB and then washed and cocultured with Jurkat T cells for an additional 24 hours with αCD3 and αCD28 activating antibodies (4 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. B, ELISAs for IL2 and granzyme B of Jurkat T cells cocultured with H358 cells with control or UROD sgRNA and 800 nmol/L ISRIB. H358 cells were cocultured with Jurkat T cells for 24 hours with αCD3 and αCD28 activating antibodies (4 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of two independent experiments. C, ELISAs for IL2 and granzyme B of primary human PBMCs cocultured with H358 cells. H358 cells were pretreated for 24 hours with salubrinal and 800 nmol/L ISRIB and then washed and cocultured with PBMCs for an additional 24 hours with αCD3 and αCD28 activating antibodies (1 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. D, ELISAs for IL2 and granzyme B of PBMCs cocultured with H358 cells with control or UROD sgRNA and 800 nmol/L ISRIB. H358 cells were cocultured with PBMCs for 24 hours with αCD3 and αCD28 activating antibodies (1 μg/mL each). Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of two independent experiments. E, ELISAs for IL2 and granzyme B of OT-1 T cells cocultured with murine KP cells (KdP67-1) expressing OVA and crystal violet assay of KP cells after coculture with OT-1 T cells. KP cells were pretreated with 100 μmol/L salubrinal and/or 500 nmol/L ISRIB for 24 hours and then cocultured for 24 hours with activated CD8 + OT-1 T cells. Error bars represent the SD from the mean from n = 3–4 biological replicates. Data from a single experiment are shown and are representative of two independent experiments. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005. sg NS , nonspecific (control) sgRNA; sg UROD , UROD sgRNA.

Techniques: Activation Assay, Cell Function Assay, In Vitro, Control, Expressing, Crystal Violet Assay

Journal: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: ISR activation enhances tumorigenesis and reduces immune cell infiltration in vivo . A, Western blot analysis of KRAS -mutant murine CMT167 cells treated for 24 or 48 hours with 100 or 200 μmol/L salubrinal or DMSO vehicle control. p-eIF2α, phosphorylated eIF2α; Veh, vehicle. B, Quantification of tumor volumes of CMT167 cells transplanted in C57BL/6J mice ( n = 11 mice per group for vehicle-treated mice; n = 10 mice per group for salubrinal-treated mice). The graph represents mean tumor volumes, and error bars represent the SEM. C and D, Endpoint tumor volumes ( C ) and tumor mass ( D ) of resected tumors shown in B . Horizontal bars represent mean values. E, Multiplexed IHC-F was performed on tumors from B . Representative images (10×) are shown. Scale bar, 200 μm. F–H, Quantification of CD3 + , CD4 + , and CD8 + tumor-infiltrating lymphocytes (Rexpressed as counts/mm 3 ). Five fields were quantified per mouse from five mice. Graphs represent mean counts, and error bars represent the SD from the mean. A Student t test was used to determine statistical significance. **, P < 0.005; ***, P < 0.0005.

Techniques: Activation Assay, In Vivo, Western Blot, Mutagenesis, Control

Journal: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: ISR pathway inhibition improves response to PD-1 blockade in a syngeneic mouse model. A, Western blot analysis of Kras -mutant murine CMT167 cells expressing doxycycline-inducible control shRNA or two independent shRNAs targeting Urod . Ab, antibody. B, Schematic illustration of CMT167 syngeneic experiment. C, Quantification of tumor volumes of CMT167 cells expressing the indicated shRNA sequence transplanted in C57BL/6J mice ( n = 15 mice per group; scrambled shRNA data are shown in Supplementary Fig. S4C and S4D). Graph represents mean tumor volumes. Error bars, SEM. D, Uniform manifold approximation and projection (UMAP) analysis of tumor-infiltrating lymphocytes, colored by cell types. E, Quantification of tumor-infiltrating lymphocytes (expressed as a percentage of CD45 + cells) from flow mass cytometry (mass CyTOF). n = 5 scrambled mice; n = 5 Urod shRNA mice; n = 5 Urod shRNA + ISRIB; n = 4 Urod shRNA + αPD-1; n = 5 Urod shRNA + ISRIB +αPD-1. Graphs represent mean values, and error bars represent the SD from the mean. F, Quantification of tumor volumes of CMT167 cells expressing the indicated shRNAs transplanted in C57BL/6J mice ( n = 11–12 mice per group; scrambled shRNA data are shown in Supplementary Fig. S5C). Graph represents mean tumor volumes. Error bars, SEM. G, Quantification of CD8 + T cells (expressed as a percentage of CD45 + cells) from mice in F . n = 5 scrambled mice; n = 4 Urod shRNA mice; n = 5 Urod shRNA + ISRIB; n = 5 Urod shRNA + ISRIB+ αPD-1; n = 5 Urod shRNA + ISRIB +αTIGIT; n = 5 Urod shRNA + ISRIB + αPD-1+ αTIGIT. Graph represent mean values. Error bars, SD from the mean. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ns, not significant. p-eIF2α, phosphorylated eIF2α; sh Urod , short-hairpin Urod ; Tregs, regulatory T cells. B, Created in BioRender. O’Donnell, K. (2025) https://BioRender.com/vphxffz .

Techniques: Inhibition, Western Blot, Mutagenesis, Expressing, Control, shRNA, Sequencing, Mass Cytometry

Journal: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: Model of translational control of CD155 and PD-L1 in response to ISR activation. Stresses commonly present in the TME activate the ISR pathway in tumor cells. This results in enhanced translation of CD155 and PD-L1 , which suppresses T-cell function by binding the inhibitory T-cell receptors PD-1 and TIGIT, respectively, thereby leading to enhanced tumorigenesis. Created in BioRender. O’Donnell, K. (2025) https://BioRender.com/vphxffz .

Techniques: Control, Activation Assay, Cell Function Assay, Binding Assay

The effect of HA15, a specific inhibitor of BiP, on raphin‐1's downstream targets. (A, B) SU‐DIPG‐VI and KNS‐42 cells were plated in triplicates and treated with HA15 as described in Section . Values are mean survival (%) or cell death (%) ± SD of two independent experiments. The significance of the differences between treatments and control were determined using an unpaired Student t ‐test—** P < 0.005. (C, D) SU‐DIPG‐VI and KNS‐42 cells were treated with the indicated concentrations of HA15 for 24 h and processed for western blot analysis as described in Section . Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The number of determinations for each experimental condition were as follows: C: n = 2, D: n = 2.

Journal: Molecular Oncology

Article Title: Raphin‐1 mediates the survival and sensitivity to radiation of pediatric‐type diffuse high‐grade glioma via phosphorylated eukaryotic initiation factor 2α‐dependent and ‐independent processes

doi: 10.1002/1878-0261.70081

Figure Lengend Snippet: The effect of HA15, a specific inhibitor of BiP, on raphin‐1's downstream targets. (A, B) SU‐DIPG‐VI and KNS‐42 cells were plated in triplicates and treated with HA15 as described in Section . Values are mean survival (%) or cell death (%) ± SD of two independent experiments. The significance of the differences between treatments and control were determined using an unpaired Student t ‐test—** P < 0.005. (C, D) SU‐DIPG‐VI and KNS‐42 cells were treated with the indicated concentrations of HA15 for 24 h and processed for western blot analysis as described in Section . Numbers at the bottom of the autoradiograms indicate treatment‐dependent changes in the level of the proteins and equal loading control (Ponceau). The number of determinations for each experimental condition were as follows: C: n = 2, D: n = 2.

Article Snippet: Salubrinal (#HY‐15486), CB‐5083 (#HY‐12861), and HA15 (#HY‐100437) were from MedChem Express (NJ).

Techniques: Control, Western Blot

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