salubrinal 23-471-0 Search Results


salubrinal  (Tocris)
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( A ) Western blot analysis of human KRAS mutant H441 or EGFR mutant PC9 cells treated for 24 or 48 hours with 100uM or 200uM <t>Salubrinal</t> or DMSO vehicle 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 5uM thapsigargin (ER Ca+ ATPase pump inhibitor) to induce ER stress, or DMSO vehicle control. ( D ) Flow cytometric analysis of cell-surface CD155 and PD-L1 protein in DMSO vehicle control and thapsigargin-treated (5uM for 24 hours) H358 or HCC827 NSCLC cells. ( E ) Western blot analysis of H358 cells with control or UROD sgRNA. ( F ) Western blot analysis of H358 or HCC827 cells with control or UROD siRNA. ( G ) Western blot analysis in eIF2⍺ wildtype (S/S) or mutant (Ser51Ala A/A) mouse embryonic fibroblasts (MEFs) treated with DMSO vehicle control or 100uM 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 3 independent experiments.
Salubrinal, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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( A ) Western blot analysis of human KRAS mutant H441 or EGFR mutant PC9 cells treated for 24 or 48 hours with 100uM or 200uM Salubrinal or DMSO vehicle 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 5uM thapsigargin (ER Ca+ ATPase pump inhibitor) to induce ER stress, or DMSO vehicle control. ( D ) Flow cytometric analysis of cell-surface CD155 and PD-L1 protein in DMSO vehicle control and thapsigargin-treated (5uM for 24 hours) H358 or HCC827 NSCLC cells. ( E ) Western blot analysis of H358 cells with control or UROD sgRNA. ( F ) Western blot analysis of H358 or HCC827 cells with control or UROD siRNA. ( G ) Western blot analysis in eIF2⍺ wildtype (S/S) or mutant (Ser51Ala A/A) mouse embryonic fibroblasts (MEFs) treated with DMSO vehicle control or 100uM 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 3 independent experiments.

Journal: bioRxiv

Article Title: Coordinated translational control of multiple immune checkpoints by the integrated stress response pathway in lung cancer

doi: 10.1101/2024.10.23.619897

Figure Lengend Snippet: ( A ) Western blot analysis of human KRAS mutant H441 or EGFR mutant PC9 cells treated for 24 or 48 hours with 100uM or 200uM Salubrinal or DMSO vehicle 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 5uM thapsigargin (ER Ca+ ATPase pump inhibitor) to induce ER stress, or DMSO vehicle control. ( D ) Flow cytometric analysis of cell-surface CD155 and PD-L1 protein in DMSO vehicle control and thapsigargin-treated (5uM for 24 hours) H358 or HCC827 NSCLC cells. ( E ) Western blot analysis of H358 cells with control or UROD sgRNA. ( F ) Western blot analysis of H358 or HCC827 cells with control or UROD siRNA. ( G ) Western blot analysis in eIF2⍺ wildtype (S/S) or mutant (Ser51Ala A/A) mouse embryonic fibroblasts (MEFs) treated with DMSO vehicle control or 100uM 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 3 independent experiments.

Article Snippet: For ISR pathway activation and inhibition, cells were treated with 100uM or 200uM Salubrinal (Tocris, 23-471-0) and/or 500-800nM ISRIB (Fisher Scientific, 5284) for 24h or 48h.

Techniques: Western Blot, Mutagenesis, Control

( A ) Polysome profiling of H1944 Vehicle or Salubrinal-treated cells (100uM for 24 hours). ( B ) Quantitative real-time PCR analysis of PD-L1(CD274) and CD155(PVR) mRNA ( C ) 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 is available in Supplementary Figure 2. Fractions associated with <3 ribosomes were grouped to represent poorly translated mRNAs, 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 % of total in all fractions. Luciferase control mRNA was added to each fraction prior to RNA extraction to control for variability. Error bars represent standard deviation from the mean from three independent fractions (<3 or >3 ribosomes). ( D ) Diagram of the wildtype 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 co-transfected control Renilla luciferase. Luciferase activity was monitored after 48h. Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of three independent experiments. ( F ) qRT-PCR analysis of mean luciferase mRNA normalized to actin in MEFs shown in ( E ). Error bars represent standard deviation from the mean from n=3 biological replicates. ( G ) Dual luciferase assay of the CD155 5′ UTR in MEFs, Vehicle or Salubrinal-treated (100uM for 24 hours). Error bars represent standard deviation from the mean from n=3 biological replicates. ( H ) qRT-PCR analysis of mean luciferase mRNA normalized to actin in MEFs shown in ( G ). n=3 biological replicates. A Student’s t-test was used to determine statistical significance (* p<0.05, ** p<0.005, *** p<0.0005).

Journal: bioRxiv

Article Title: Coordinated translational control of multiple immune checkpoints by the integrated stress response pathway in lung cancer

doi: 10.1101/2024.10.23.619897

Figure Lengend Snippet: ( A ) Polysome profiling of H1944 Vehicle or Salubrinal-treated cells (100uM for 24 hours). ( B ) Quantitative real-time PCR analysis of PD-L1(CD274) and CD155(PVR) mRNA ( C ) 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 is available in Supplementary Figure 2. Fractions associated with <3 ribosomes were grouped to represent poorly translated mRNAs, 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 % of total in all fractions. Luciferase control mRNA was added to each fraction prior to RNA extraction to control for variability. Error bars represent standard deviation from the mean from three independent fractions (<3 or >3 ribosomes). ( D ) Diagram of the wildtype 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 co-transfected control Renilla luciferase. Luciferase activity was monitored after 48h. Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of three independent experiments. ( F ) qRT-PCR analysis of mean luciferase mRNA normalized to actin in MEFs shown in ( E ). Error bars represent standard deviation from the mean from n=3 biological replicates. ( G ) Dual luciferase assay of the CD155 5′ UTR in MEFs, Vehicle or Salubrinal-treated (100uM for 24 hours). Error bars represent standard deviation from the mean from n=3 biological replicates. ( H ) qRT-PCR analysis of mean luciferase mRNA normalized to actin in MEFs shown in ( G ). n=3 biological replicates. A Student’s t-test was used to determine statistical significance (* p<0.05, ** p<0.005, *** p<0.0005).

Article Snippet: For ISR pathway activation and inhibition, cells were treated with 100uM or 200uM Salubrinal (Tocris, 23-471-0) and/or 500-800nM ISRIB (Fisher Scientific, 5284) for 24h or 48h.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Luciferase, Control, RNA Extraction, Standard Deviation, Mutagenesis, Construct, Clone Assay, Transfection, Activity Assay

( A ) ELISAs for IL-2 and Granzyme B of Jurkat T cells co-cultured with H358 cells. H358 cells were pre-treated for 24 hours with Salubrinal and 800nM ISRIB, then washed and co-cultured with Jurkat T cells for an additional 24 hours with α-CD3 and α-CD28 activating antibodies (4ug/ml each). Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of three independent experiments. ( B ) ELISAs for IL-2 and Granzyme B of Jurkat T cells co-cultured with H358 cells with control or UROD sgRNA and 800nM ISRIB. H358 cells were co-cultured with Jurkat T cells for 24 hours with α-CD3 and α-CD28 activating antibodies (4ug/ml each). Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of two independent experiments. ( C ) ELISAs for IL-2 and Granzyme B of primary human PBMCs co-cultured with H358 cells. H358 cells were pre-treated for 24 hours with Salubrinal and 800nM ISRIB, then washed and co-cultured with PBMCs for an additional 24 hours with α-CD3 and α-CD28 activating antibodies (1ug/ml each). Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of three independent experiments. ( D ) ELISAs for IL-2 and Granzyme B of PBMCs co-cultured with H358 cells with control or UROD sgRNA and 800nM ISRIB. H358 cells were co-cultured with PBMCs for 24 hours with α-CD3 and α-CD28 activating antibodies (1ug/ml each). Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of two independent experiments. A Student’s t-test was used to determine statistical significance (* p<0.05, ** p<0.005, *** p<0.0005).

Journal: bioRxiv

Article Title: Coordinated translational control of multiple immune checkpoints by the integrated stress response pathway in lung cancer

doi: 10.1101/2024.10.23.619897

Figure Lengend Snippet: ( A ) ELISAs for IL-2 and Granzyme B of Jurkat T cells co-cultured with H358 cells. H358 cells were pre-treated for 24 hours with Salubrinal and 800nM ISRIB, then washed and co-cultured with Jurkat T cells for an additional 24 hours with α-CD3 and α-CD28 activating antibodies (4ug/ml each). Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of three independent experiments. ( B ) ELISAs for IL-2 and Granzyme B of Jurkat T cells co-cultured with H358 cells with control or UROD sgRNA and 800nM ISRIB. H358 cells were co-cultured with Jurkat T cells for 24 hours with α-CD3 and α-CD28 activating antibodies (4ug/ml each). Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of two independent experiments. ( C ) ELISAs for IL-2 and Granzyme B of primary human PBMCs co-cultured with H358 cells. H358 cells were pre-treated for 24 hours with Salubrinal and 800nM ISRIB, then washed and co-cultured with PBMCs for an additional 24 hours with α-CD3 and α-CD28 activating antibodies (1ug/ml each). Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of three independent experiments. ( D ) ELISAs for IL-2 and Granzyme B of PBMCs co-cultured with H358 cells with control or UROD sgRNA and 800nM ISRIB. H358 cells were co-cultured with PBMCs for 24 hours with α-CD3 and α-CD28 activating antibodies (1ug/ml each). Error bars represent standard deviation from the mean from n=3 biological replicates. Data from a single experiment are shown and representative of two independent experiments. A Student’s t-test was used to determine statistical significance (* p<0.05, ** p<0.005, *** p<0.0005).

Article Snippet: For ISR pathway activation and inhibition, cells were treated with 100uM or 200uM Salubrinal (Tocris, 23-471-0) and/or 500-800nM ISRIB (Fisher Scientific, 5284) for 24h or 48h.

Techniques: Cell Culture, Standard Deviation, Control

( A ) Western blot analysis of KRAS mutant murine CMT167 cells treated for 24 or 48 hours with 100uM or 200uM Salubrinal or DMSO vehicle control. ( 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). Graph represents mean tumor volumes and error bars represent standard error of the mean. ( C ) End-point tumor volumes and ( D ) tumor mass of resected tumors shown in ( B ). Horizontal bars represent mean values. ( E ) Multiplexed IHC-F was performed on tumors from ( B ), 10X representative images shown, scale bar=200μm. ( F,G,H ) Quantification of CD3 + , CD4 + , and CD8 + tumor infiltrating lymphocytes (TILs, expressed as counts/mm 3 ). 5 fields were quantified per mouse from 5 mice, graphs represent mean counts and error bars represent standard deviation from the mean. A Student’s t-test was used to determine statistical significance (* p<0.05, ** p<0.005, *** p<0.0005).

Journal: bioRxiv

Article Title: Coordinated translational control of multiple immune checkpoints by the integrated stress response pathway in lung cancer

doi: 10.1101/2024.10.23.619897

Figure Lengend Snippet: ( A ) Western blot analysis of KRAS mutant murine CMT167 cells treated for 24 or 48 hours with 100uM or 200uM Salubrinal or DMSO vehicle control. ( 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). Graph represents mean tumor volumes and error bars represent standard error of the mean. ( C ) End-point tumor volumes and ( D ) tumor mass of resected tumors shown in ( B ). Horizontal bars represent mean values. ( E ) Multiplexed IHC-F was performed on tumors from ( B ), 10X representative images shown, scale bar=200μm. ( F,G,H ) Quantification of CD3 + , CD4 + , and CD8 + tumor infiltrating lymphocytes (TILs, expressed as counts/mm 3 ). 5 fields were quantified per mouse from 5 mice, graphs represent mean counts and error bars represent standard deviation from the mean. A Student’s t-test was used to determine statistical significance (* p<0.05, ** p<0.005, *** p<0.0005).

Article Snippet: For ISR pathway activation and inhibition, cells were treated with 100uM or 200uM Salubrinal (Tocris, 23-471-0) and/or 500-800nM ISRIB (Fisher Scientific, 5284) for 24h or 48h.

Techniques: Western Blot, Mutagenesis, Control, Standard Deviation