Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: IL-6 predicts a poor response to ICI therapy in patients with PD-L1-high NSCLC. ( A, B ) Tumor tissues from 234 patients with NSCLC before ICI therapy were subjected to RNA-seq. The patients were divided into low- CD274 and high- CD274 (PD-L1) groups based on the median value. Differences in pathway enrichment between responders with a partial response (PR) and non-responders with stable disease (SD) or progressive disease (PD) in each group were analyzed using GSEA. ( C ) The heatmap shows the relative mRNA expression levels of the genes in core enrichment sites of the IL-6/Jak/Stat3 gene set. ( D ) Differences in IL6 expression (as presented as z-scores) among patients with PR, SD, and PD or between those with PR and SD+PD in the low- CD274 and high- CD274 groups. ( E ) ICI responsiveness among patients divided into low-expression and high-expression groups for CD274 and IL6 , based on the median value for CD274 and a TPM cut-off of 1.805 for IL6 , as determined using Cutoff Finder software. Differences in the response rate were compared using Pearson’s χ2 test. ( F ) Kaplan-Meier analysis of progression-free survival (PFS) after ICI therapy was performed according to CD274 (left upper), IL6 (left lower), and combined CD274 and IL6 (right) expression. Survival differences were compared using a log-rank test. ( G ) The heatmap shows the relative immune cell abundance of each group. ( H ) Differences in CTL, M2 macrophage, Treg, and MDSC scores between patients with PR and SD+PD in the low- CD274 and high- CD274 groups. Correlations between MDSC and Treg scores and IL-6 expression among patients with PR and SD+PD in the low-PD-L1 and high-PD-L1 groups. ( I ) Correlations between serum IL-6 levels and PD-L1 expression determined by the TPS of PD-L1 (22C3) IHC in the ICI-serum cohort (n=57). ( J ) The proportions of patients with PD and non-PD in the low-serum and high-serum IL-6 groups (cut-off value estimated using Cutoff Finder software) were compared using Pearson’s chi-squared test. ( K ) Kaplan-Meier analysis of PFS and overall survival (OS) after ICI therapy according to serum IL-6 levels. Differences in survival were analyzed using a log-rank test. The data in the histogram are presented as means±SEM. Correlations were calculated using Spearman’s correlation test. *p<0.05, **p<0.01. ADC, adenocarcinoma; ICI, immune checkpoint inhibitor; MNSCLC, non-small-cell lung cancer; DSC, myeloid-derived suppressor cell; SqCC, squamous cell carcinoma.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: RNA Sequencing, Expressing, Software, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: PD-L1 activates the IL-6/Jak2/Stat3 pathway in lung cancer cells. ( A, B ) RNA-seq was performed in A549 cells transfected with PD-L1 vector or empty vector and in H460 cells transfected with PD-L1 siRNA or scrambled (sc) siRNA. DEGs in PD-L1-overexpressing A549 cells and PD-L1-knockdown H460 cells are displayed in a volcano plot. ( C ) Venn diagram illustrating the numbers of up-regulated genes in PD-L1-overexpressing A549 cells and down-regulated genes in PD-L1-knockdown H460 cells. Of the 359 shared genes, the expression of representative genes as determined in triplicate RNA-seq experiments with both cell lines is displayed in a heatmap. ( D, E ) GSEA was performed in each cell line using DEGs and the Hallmark gene set. ( F ) Expression of representative cytokines and chemokines included in the IL-6/Jak/Stat3 gene set as determined in triplicate RNA-seq experiments with both cell lines (molecules validated by further in vitro analyses are shown in red). ( G ) Cytokine mRNA expression and secretion were measured by qRT-PCR and ELISA, respectively, in PD-L1-overexpressing (PDL1 OE ) A549 cells and PD-L1-knockdown (PDL1 KD ) H460 cells (EV, empty vector; Con, scrambled siRNA). ( H ) Cytokine secretion determined by ELISA in the culture supernatant of patient-derived primary human lung tumor (cancer) cells with low-PD-L1 expression (TPS<1, n=5) and high-PD-L1 expression (TPS≥50, n=3) (left). Primary lung cancer cells with low and high PD-L1 expression were transfected with PD-L1-overexpressing vector and PD-L1-knockdown siRNA, respectively, and cytokine secretion was measured by ELISA (right). ( I, J ) Phosphorylated Stat3 and Jak2 and IL-6 expression were detected using western blotting and immunofluorescence staining in PD-L1-overexpressing A549 and H522 cells and in PD-L1-knockdown H460 and H596 cells (scale bar=20 µm). The data in the histogram are presented as means ±SEM. *p<0.05, **p<0.01, ***p<0.001. DEGs, differentially expressed genes.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: RNA Sequencing, Transfection, Plasmid Preparation, Knockdown, Expressing, In Vitro, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot, Immunofluorescence, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: PD-L1 binds to p-Stat3 in the nucleus and enhances its efficiency in IL-6 transcription. ( A ) Three-dimensional visualization of nuclear PD-L1. Images corresponding to x-z sections reconstructed along the red lines are displayed at the top of each x-y section. Images corresponding to y-z sections reconstructed along the green solid lines are displayed at the right of each x-y section (scale bar=10 µm). ( B ) Nuclear localization of PD-L1 and p-Y705-Stat3 assessed by western blotting after cellular fractionation into nuclear and non-nuclear fractions, in PD-L1-overexpressing A549 cells and PD-L1-knockdown H460 cells. ( C ) Co-immunoprecipitation assay for p-Y705-Stat3 and PD-L1 using whole cell lysates (upper panels) and nuclear extracts (lower panels) of PD-L1-overexpressing A549 cells and PD-L1-knockdown H460 cells. ( D ) Interaction between p-Y705-Stat3 and PD-L1 in the cytoplasm and nucleus visualized in a Duolink assay of PD-L1-overexpressing A549 cells and PD-L1-knockdown H460 cells (scale bar=10 µm). Red dots indicate the interaction between p-Y705-Stat3 and PD-L1. Fluorescence intensity across dotted lines is depicted in the red lines of the right graphs (DAPI in blue lines). ( E ) A density heatmap of PD-L1 CUT&Tag sequencing data using H460 cells shows PD-L1 enrichment within 3 kb around the transcription start site (TSS) (Rep, replica). ( F ) Integrative genomics viewer (IGV) tracks for IL6 and CXCL1 promoters from the PD-L1 and p-Stat3 CUT&Tag analyses. ( G ) The density heatmap of the p-Stat3 CUT&Tag data shows compromised p-Stat3 enrichment in PD-L1-knockdown H460 cells vs control cells within 3 kb around the TSS. ( H ) IGV tracks for IL6 and CXCL1 promoters from the p-STAT3 CUT&Tag analysis of PD-L1-knockdown H460 cells and control cells. ( I ) Chromatin immunoprecipitation (ChIP) was performed using anti-PD-L1 and anti-Y705-Stat3 antibodies in PD-L1-knockdown H460 cells. The binding of PD-L1 and p-Y705-Stat3 to the IL6 and CXCL1 promoters was then assessed by qPCR (sequential ChIP-qPCR). The data are presented as the mean±SEM of three independent experiments. ***p<0.001.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: Western Blot, Cell Fractionation, Knockdown, Co-Immunoprecipitation Assay, Fluorescence, Sequencing, Control, Chromatin Immunoprecipitation, Binding Assay, ChIP-qPCR

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: PD-L1 promotes CXCL1- and IL-6-dependent migration and activation of MDSCs. ( A ) Heatmap showing the expression of 40 MDSC signature genes in TCGA-LUAD (lung adenocarcinoma) and TCGA-LUSC (lung squamous cell carcinoma) according to PD-L1 low vs high expression (cut-off median). ( B ) The correlation between the MDSC score and the expression of each cytokine in TCGA-LUAD and TCGA-LUSC was calculated using Spearman’s correlation test. ( C ) M-MDSCs and PMN-MDSCs infiltrating the tumor tissues of patients with NSCLC were analyzed by flow cytometry, and the correlation between the MDSC population and PD-L1 TPS was calculated using Spearman’s correlation test. ( D–M ) After Ficoll gradient separation, HLA-DR lo CD11b + CD33 + CD14 + cells/HLA-DR lo CD11b + CD33 + CD15 + cells/and CD15 + low density neutrophils were isolated from PBMCs of healthy donors, and a mixture of these cells (“human MDSCs in figure hereafter”) was submitted to in vitro assays. ( D ) A migration assay for human MDSCs was performed using PD-L1-overexpressing and PD-L1-knockdown NSCLC cells in an 8 μm pore Transwell system. ( E ) Primary lung cancer cells with low and high PD-L1 expression were transfected with PD-L1-overexpressing vector and PD-L1-knockdown siRNA, respectively. MDSC migration assays using CM from PD-L1-overexpressing and PD-L1-knockdown primary lung cancer cells were then performed. ( F, G ) Migration assays of MDSCs with PD-L1-overexpressing A549 cells or CM from PD-L1-overexpressing primary lung cancer cells were performed in the presence of the indicated chemokine-neutralizing antibodies (scale bar=1000 µm). ( H ) MDSCs were co-cultured with PD-L1-overexpressing A549 cells in a 0.4 μm pore Transwell system in the presence of the indicated cytokine-neutralizing antibodies (1 µg/mL each). Arg1 , iNOS , and IDO1 expression in MDSCs was then assessed by qRT-PCR. ( I ) MDSCs and PD-L1-overexpressing A549 cells were co-cultured in a 0.4 μm pore Transwell system in the presence of the indicated cytokine-neutralizing antibodies. MDSCs were then co-cultured in a 0.4 μm pore Transwell system with CFSE-labeled anti-CD3/CD28 bead-stimulated CD4 + or CD8 + T-cells; T-cell proliferation was then analyzed by flow cytometry. ( J ) MDSCs were cultured with CM from PD-L1-low (TPS<1) and PD-L1-high (TPS≥50) primary lung cancer cells or CM from PD-L1-overexpressing or PD-L1-knockdown primary lung cancer cells, then analyzed for IDO1 , Arg1 , and iNOS expression by qRT-PCR. ( K, L ) MDSCs were cultured with CM from PD-L1-overexpressing primary lung cancer cells in the presence of IL-6-neutralizing antibodies and then assessed for IDO1 , Arg1 , and iNOS expression by qRT-PCR. iNOS expression in MDSCs was detected by immunofluorescence staining (scale bar=10 µm). ( M ) MDSCs were cultured in CM from PD-L1-overexpressing primary lung cancer cells or co-cultured with PD-L1-overexpressing A549 cells and then co-cultured with CFSE-labeled anti-CD3/CD28 bead-stimulated CD8 + T-cells in the presence of the indicated inhibitors; T-cell proliferation was analyzed by flow cytometry. The data are presented as the mean±SEM of 4–6 independent experiments. *p<0.05, **p<0.01, ***p<0.001. MDSC, myeloid-derived suppressor cell; PMN, polymorphonuclear; TCGA, The Cancer Genome Atlas.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: Migration, Activation Assay, Expressing, Flow Cytometry, Isolation, In Vitro, Knockdown, Transfection, Plasmid Preparation, Cell Culture, Quantitative RT-PCR, Labeling, Immunofluorescence, Staining, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: PD-L1 promotes tumor growth in vivo via IL-6-induced immune suppression in a PD-1-independent manner. Stable PD-L1-overexpressing LLC cells and control cells were injected subcutaneously into the flanks of C57BL/6 mice. (A−C) For MDSC depletion, mice were intraperitoneally injected with 150 µg of anti-Ly6C/Ly6G (Gr-1) antibody five times, once every 2 days, starting from 1 day before cancer cell injection. Tumor size was measured every 2–3 days using calipers. Tumor volume was measured using the IVIS luminescence imaging system before the mice were euthanized. ( D ) Immune cell populations in the tumors were assessed using flow cytometry. (E−G) Mice were injected intraperitoneally with 200 µg of anti-mouse IL-6 antibodies every 3 days (a total of five times). Tumor size was measured every 2–3 days using calipers. Tumor volumes were measured using the IVIS luminescence imaging system. (H−J) Immune cell populations in tumors were assessed using flow cytometry. ( K ) MDSCs isolated from the mouse tumors were cultured with CFSE-labeled anti-CD3/CD28 bead-stimulated CD8 + T-cells isolated from the spleen of tumor-free C57BL/6 mice. T-cell proliferation was then assessed using flow cytometry. ( L ) IHC staining of CD8 + , GzmB + , and Ly6C/Ly6G + cells in the tumors (scale bar=200 µm). (M−O) Stable PD-L1-overexpressing LLC cells and control cells were injected subcutaneously into the flanks of PDCD1 (PD-1)-knockout C57BL/6 mice. Tumor size was measured once every 2–3 days using calipers. Tumor volumes were measured using the IVIS luminescence imaging system. (P−R) Immune cell populations in tumors were assessed using flow cytometry. The data are presented as the mean±SEM of five independent experiments. *p<0.05, **p<0.01, ***p<0.001. LLC, Lewis lung carcinoma; MDSC, myeloid-derived suppressor cell.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: In Vivo, Control, Injection, Imaging, Flow Cytometry, Isolation, Cell Culture, Labeling, Immunohistochemistry, Knock-Out, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: IL-6 secreted by PD-L1-overexpressing cells promotes tumor growth in vivo via myeloid cell activation and an immunosuppressive TME. ( A ) Stable PD-L1-overexpressing LLC cells and control cells were cultured for 48 hours. CD11b + Ly6G + and CD11b + Ly6C + myeloid cells isolated from tumor-free mouse spleens were then cultured for 48 hours in CM from PD-L1-overexpressing LLC cells (LLC-PDL1 OE -CM) in the presence or absence of IL-6-neutralizing antibodies and then injected with fresh wild-type LLC cells (at an equivalent ratio of 1:3) into the flanks of mice (EV, empty vector). ( B, C ) Tumor size was measured once every 2–3 days using calipers. Tumor volume was measured using the IVIS Spectrum imaging system, after which the mice were euthanized and tumor weight was determined. (D−F) Immune cell populations in the tumors were assessed using flow cytometry. (G−I) CD11b + Ly6G + and CD11b + Ly6C + myeloid cells isolated from tumor-free mouse spleens were cultured for 48 hours in CM from PD-L1-knockdown LLC cells (LLC-PDL1 KD -CM) and then injected with fresh wild-type LLC cells (at an equivalent ratio of 1:3) into the flanks of mice (Con, control). For CD8 + T-cell depletion, the mice were intraperitoneally injected with 100 µg of anti-CD8 antibody five times, once every 2 days starting from 1 day before cancer cell injection. Tumor size was measured once every 2–3 days using calipers. Tumor size was measured using the IVIS Spectrum imaging system, after which the mice were euthanized and tumor weight was determined. (J−L) CD11b + Ly6G + and CD11b + Ly6C + myeloid cells isolated from tumor-free mouse spleens were cultured for 48 hours in CM from PD-L1-overexpressing LLC cells (LLC-PDL1 OE -CM) and then injected with fresh wild-type LLC cells (at an equivalent ratio of 1:3) into the flanks of mice. For Treg depletion, the mice were intraperitoneally injected with 100 µg of anti-CD25 antibody five times, once every 2 days starting from 1 day before cancer cell injection. Tumor size was measured once every 2–3 days using calipers. Tumor size was measured using the IVIS Spectrum imaging system, after which the mice were euthanized and tumor weight was determined. *p<0.05, **p<0.01, ***p<0.001. LLC, Lewis lung carcinoma.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: In Vivo, Activation Assay, Control, Cell Culture, Isolation, Injection, Plasmid Preparation, Imaging, Flow Cytometry, Knockdown

Journal: Journal for Immunotherapy of Cancer

Article Title: Cell-intrinsic PD-L1 signaling drives immunosuppression by myeloid-derived suppressor cells through IL-6/Jak/Stat3 in PD-L1-high lung cancer

doi: 10.1136/jitc-2024-010612

Figure Lengend Snippet: Combined blockade of IL-6 and PD-1 efficiently controls tumor growth and elicits antitumor immune response. ( A ) Wild-type LLC or TC-1 cells were injected subcutaneously into the flanks of C57BL/6 mice. For anti-PD-1 immunotherapy, the mice were injected intraperitoneally with 200 µg of anti-PD-1 antibody every 5 days a total of three times. For anti-IL-6 antibody (Ab) treatment, the mice were injected intraperitoneally with 200 µg of anti-mouse IL-6 Ab every 3 days for a total of five times. ( B, C, E, F ) Tumor size was measured every 2–3 days using calipers. Tumor volume was measured using the IVIS Spectrum imaging system, after which the mice were euthanized and tumor weight was determined. ( D, G ) Mice were observed for survival every 2–3 days. Differences in survival were compared using a log-rank test. (H−L) The immune cell populations of LLC tumors were assessed using flow cytometry. ( M ) IHC staining of CD8 + , GzmB + , and Ly6C/Ly6G + cells in LLC tumors (scale bar=200 µm). ( N ) MDSCs isolated from LLC tumors were cultured with CFSE-labeled anti-CD3/CD28 bead-stimulated CD8 + T-cells isolated from tumor-free C57BL/6 mice; the proliferation of CD8 + T-cells was then assessed using flow cytometry. ( O ) CD8 + T-cells isolated from LLC tumors were cultured with CFSE-labeled wild-type LLC cells for 24 hours. The cells were then stained with 7-AAD and Annexin V, and LLC viability was determined by flow cytometry. ( P ) Schematic diagram showing that tumor-cell-intrinsic PD-L1 activates Jak2/Stat3 signaling and contributes to IL-6 production, which drives MDSC-mediated immunosuppression in PD-L1-high lung cancer. Combined therapy targeting PD-1 and IL-6 may thus be effective in tumor control by restoring antitumor immunity. The data are presented as the mean±SEM of five independent experiments. *p<0.05, **p<0.01, ***p<0.001. CFSE, carboxyfluorescein succinimidyl ester; LLC, Lewis lung carcinoma; MDSC, myeloid-derived suppressor cell.

Article Snippet: Human PD-L1-expressing plasmids (HG10084-UT), PD-L1-Flag-tagged plasmids (HG10084-NF), control vectors (CV020), and human protein tyrosine phosphatase 1B (PTP1B)-Myc-tagged plasmids (HG10304-CM) were purchased from Sino Biological (China). siRNAs targeting human PD-L1 (NM 014143.2) were designed and synthesized by Bioneer (Republic of Korea) with the following sequences: sense 5′-CUG AGA AUC AAC ACA ACA A (dTdT)-3′ and antisense 5′-UUG UUG UGU UGA UUC UCA G (dTdT)-3′.

Techniques: Injection, Imaging, Flow Cytometry, Immunohistochemistry, Isolation, Cell Culture, Labeling, Staining, Control, Derivative Assay

Journal: Nature Communications

Article Title: Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression

doi: 10.1038/s41467-020-17093-9

Figure Lengend Snippet: Candidate genes for TANGO proof of concept.

Article Snippet: PD-L1 antibody was validated via overexpression using cDNA from the PD-L1/TCR Activator Mammalian Expression Kit purchased from BPS Bioscience (Cat# 60610). cDNA and TCR activator were specially requested not to be mixed.

Techniques:

Journal: Nature Communications

Article Title: Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression

doi: 10.1038/s41467-020-17093-9

Figure Lengend Snippet: a RT-PCR (left panel) and qPCR (right panel) of a selected PCCA ASO (ASO-29) targeting the exon inclusion event transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0008 and 0.0001, respectively. b RT-PCR (left panel) and qPCR (right panel) of a selected SYNGAP1 ASO (ASO-71) targeting the alternative 3′ss transfected in HEK293 cells at increasing concentrations for 24 h. Exact p -values for bars with asterisks are 0.0001 and 0.0003, 6.99e-5 and 4.25e-5, respectively. c RT-PCR (left panel) and qPCR (right panel) of a selected CD274 ASO (ASO-125) targeting the alternative intron transfected in Huh7 cells at increasing concentrations for 24 h. Cell were treated with 50 μg/mL of CHX for 3 h prior to harvesting to visualize and quantify the non-productive mRNA. Exact p -values for bars with asterisks are 6.38e-6 and 9.21e-5, respectively. d RT-PCR (left panel) and qPCR (right panel) of two selected SCN1A ASOs (ASO-135 and ASO-136) targeting the exon inclusion event delivered by free uptake into ReNCell VM cells at increasing concentrations for 72 h. RT-PCR results (bar graphs on the left) show dose-dependent reductions of the non-productive mRNA and qPCR results (bar graphs on the right) show dose-dependent increases of productive mRNA. Exact p-values for bars with asterisks are 1.69e-10, 2.50e-11, and 1.49e-11; 2.91e-8, 2.05e-10, and 2.59e-11; 4.21e-7, 1.10e-8, and 1.35e-10; and 7.26e-5, 4.01e-8, and 9.08e-12, respectively. No-ASO (−), scramble (SC), and mismatch (MM) controls were included in each experiment at the same increasing concentrations as the respective targeting ASOs. All experiments were performed in three biological replicates. Data are presented as mean values ±SD. P -values were calculated using two-sided t -test. Asterisks denote ASOs that are statistically significant ( p < 0.001) in both RT-PCR and qPCR analyses. Red and gray rectangles denote NMD-inducing event and protein-coding exons, respectively. Source data are provided as a Source Data .

Article Snippet: PD-L1 antibody was validated via overexpression using cDNA from the PD-L1/TCR Activator Mammalian Expression Kit purchased from BPS Bioscience (Cat# 60610). cDNA and TCR activator were specially requested not to be mixed.

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Nature Communications

Article Title: Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression

doi: 10.1038/s41467-020-17093-9

Figure Lengend Snippet: a Bar graph plotting the quantification of PCCA western blot analysis from HEK293 cells treated with hit ASO at increasing concentrations for 48 h. Equal protein loading was confirmed with Ponceau staining (Supplementary Fig. ). b Same for SynGAP ( p = 4.62e-6). c Bar graph plotting flow cytometry derived fold change of the Mean Fluorescent Intensity of PD-L1 from Huh7 cells treated with the hit ASO at increasing concentrations for 120 h. No-ASO (−), scramble (SC), and mismatch (MM) controls were included in each experiment at the same increasing concentrations as the respective targeting ASOs ( p = 7.70e-5). All experiments were performed in three biological replicates. Data are presented as mean values ±SD. P -values were calculated based on two-sided t -test. Asterisks indicate p < 0.001. Source data are provided as a Source Data .

Article Snippet: PD-L1 antibody was validated via overexpression using cDNA from the PD-L1/TCR Activator Mammalian Expression Kit purchased from BPS Bioscience (Cat# 60610). cDNA and TCR activator were specially requested not to be mixed.

Techniques: Western Blot, Staining, Flow Cytometry, Derivative Assay