pd l1 promoter reporter plasmid  (Addgene inc)

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A ) Schematic of the deubiquitinase CRISPR knockout screening workflow. ( B ) Guides enriched in PD-L1–low and PD-L1–high populations with their fold enrichment. The guide code of each gene for further validation is indicated. ( C ) Guide hits described were validated by flow using individual guide knockouts. ( D ) Western blotting validation of ATXN3 knockout and PD-L1 expression with specific sgRNAs in LLC1 cells. WT, cells transfected with empty vector; KO, ATXN3-knockout stable cell strains. ( E and F ) Representative flow cytometry plots and quantification by MFI of cell-surface PD-L1 in LLC1 cells. ( G ) Western blotting analysis of ATXN3 and PD-L1 expression in A549 cells with knockout of ATXN3 . ( H and I ) Representative flow cytometry plots and quantification of cell-surface PD-L1 in A549 cells with knockout of ATXN3 . ( J and K ) Cd274 and Atxn3 mRNA levels were analyzed by reverse transcription quantitative PCR (RT-qPCR) in LLC1 cells. ( L and M ) Cd274 and Atxn3 mRNA levels were analyzed by RT-qPCR in B16 cells. ( N ) Correlation of CD274 mRNA levels with ATXN3 mRNA levels in lung cancer patients ( n = 22). ( O ) Correlation of CD274 with ATXN3 expression in multiple tumors based on TCGA data ( n = 40). C , F , and I – M : 2-tailed unpaired t test; N : Pearson’s correlation analysis. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: CRISPR, Knock-Out, Biomarker Discovery, Western Blot, Expressing, Transfection, Plasmid Preparation, Stable Transfection, Flow Cytometry, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A and B ) WT and ATXN3-KO cells were treated with IFN-γ (10 ng/mL) for 24 hours, and surface PD-L1 levels were analyzed. ( C ) ATXN3 interacts with IRF1 in transiently transfected HEK293T cells. ( D ) Interaction of endogenous ATXN3 and IRF1 in A549 cells. ( E ) HA-ubiquitin and FLAG-IRF1 expression plasmids were cotransfected with Myc-ATXN3 into HEK293T cells. IRF1 ubiquitination was determined by immunoprecipitation of IRF1 and immunoblotting with HA antibody. ( F and G ) FLAG-IRF1 was cotransfected with or without Myc-ATXN3 plasmids into HEK293T cells. The transfected cells were treated with cycloheximide (CHX) for different times. The protein levels of FLAG-IRF1 (top panel) and Myc-ATXN3 (middle panel) with β-actin control (bottom panel) were analyzed by Western blotting. Representative images ( F ) and quantification data from 3 independent experiments are shown ( G ). ( H and I ) Immunoblot analysis of IRF protein stability in WT and ATXN3-KO A549 cells as in F and G . ( J ) Interaction between ATXN3 and STAT3 in transfected HEK293T cells. ( K ) Endogenous interaction between ATXN3 and STAT3 in A549 cells. ( L ) The effect of ATXN3 on STAT3 ubiquitination was determined as in E . ( M and N ) The effects of ATXN3 on STAT3 protein stability were analyzed as in F and G . ( O and P ) Immunoblot analysis of STAT3 protein stability in WT and ATXN3-KO A549 cells as in H and I . ( Q ) The interaction between ATXN3 and STAT1 was tested in A549 cells. ( R ) ATXN3 enhances tumoral PD-L1 expression through protecting IRF1 and STAT3 from ubiquitination-induced protein degradation. B : Ordinary 1-way ANOVA; G , I , N , and P : 2-tailed unpaired t test; * P < 0.05, ** P < 0.01, *** P < 0.001. WCL, whole-cell lysate.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: Transfection, Ubiquitin Proteomics, Expressing, Immunoprecipitation, Western Blot, Control

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A and B ) A549 cells were cultured under normoxia and hypoxia (hyp) (1% pO 2 ) for 48 hours, and surface PD-L1 levels were analyzed by flow cytometry and quantification. ( C ) ATXN3 specifically interacts with HIF-2α. HA–HIF-2α expression plasmid was cotransfected with or without FLAG-ATXN3 into HEK293T cells. Their interactions were examined by co-IP with anti-FLAG antibodies and by Western blotting with anti-HA antibodies. ( D ) The interaction between ATXN3 and HIF-1α was tested in transfected HEK293T cells. ( E ) Endogenous interaction between ATXN3 and HIF-2α in A549 cells. ( F ) HA-ubiquitin, FLAG–HIF-2α, and Myc-ATXN3 plasmids were cotransfected into HEK293T cells. HIF-2α ubiquitination was determined by immunoprecipitation of HIF-2α with anti-FLAG antibodies and immunoblotting with anti-HA antibody. ( G and H ) HIF-2α was cotransfected with or without ATXN3 plasmids into HEK293T cells. The transfected cells were treated with CHX for different times. The protein levels of HIF-2α (top panel) and ATXN3 (middle panel) were analyzed by Western blotting. β-Actin was used as a loading control (bottom panel). ( I and J ) Immunoblot analysis of HIF-2α protein stability in WT and ATXN3-KO A549 cells. ( K ) ATXN3 enhances hypoxia-induced PD-L1 expression through protecting HIF-2α from ubiquitination-induced protein degradation. B : Ordinary 1-way ANOVA; H and J : 2-tailed unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: Cell Culture, Flow Cytometry, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Western Blot, Transfection, Ubiquitin Proteomics, Immunoprecipitation, Control

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A – C ) WT or ATXN3-KO LLC1 cells were injected subcutaneously into C57BL/6 mice ( n = 10). Tumor growth curve ( A ), photograph ( B ), and weight ( C ) are shown. ( D ) MFI of surface PD-L1 on LLC1 tumors ( n = 5). ( E – G ) Quantification of CD4 + ( E ) and CD8 + T cell ( F ) and Treg ( G ) percentages ( n = 5–10). ( H and I ) Quantification of cell-surface PD-1 ( H ) and PD-L1 ( I ) MFI on CD8 + T cells ( n = 5). ( J and K ) Quantification of cell-surface CTLA-4 MFI ( J ) and Tim3 percentage ( K ) in CD8 + T cells ( n = 5). ( L ) MFI of cell-surface LAG3 and percentage in CD8 + T cells ( n = 5). ( M – O ) Intracellular staining of Blimp1 + CD8 + T cell ( M ), EOMES + CD8 + T cell ( N ), and T-bet + CD8 + T cell ( O ) percentage in LLC1 tumors ( n = 5-7). ( P ) Quantification of cell-surface CD44 + CD8 + T cell percentage from LLC1 tumors ( n = 5–10). ( Q ) Apoptotic CD8 + T cells in the tumors were analyzed ( n = 5–7). ( R and S ) Representative flow staining and quantification of intracellular cytokine staining of granzyme B + CD8 + and IFN-γ + CD8 + in CD45 + T cell populations from LLC1 tumors ( n = 5). ( T ) Tumor growth curve and tumor photograph of C57BL/6 mice injected subcutaneously with WT and ATXN3-KO LLC1 cells with or without treatment of anti-CD8 depleting antibodies ( n = 5). ( U ) Left: Tumor cell-surface PD-L1 expression. Right: Tumor growth of WT or ATXN3-KO LLC1 cells stably expressing PD-L1 (as shown in the left plot) in C57BL/6 mice ( n = 5). A and C – S : 2-tailed unpaired t test; T and U : ordinary 1-way ANOVA. * P < 0.05, ** P < 0.01,*** P < 0.001.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: Injection, Staining, Expressing, Stable Transfection

Journal: The Journal of Clinical Investigation

Article Title: CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

doi: 10.1172/JCI167728

Figure Lengend Snippet: ( A ) Representative images from immunohistochemical staining of PD-L1, ATXN3, IRF1, and HIF-2α in human lung adenocarcinoma (LUAD) and melanoma patients. Scale bar: 50 μm. ( B ) PD-L1, ATXN3, IRF1, and HIF-2α protein levels in tumor tissues compared with normal tissues in LUAD patients ( n = 61); “PD-L1%” means the percentage of PD-L1–positive area versus all tissue area. ( C ) PD-L1, ATXN3, IRF1, and HIF-2α protein levels in tumor tissues compared with normal tissues in patients with melanoma ( n = 48). ( D ) Correlation analysis of ATXN3 expression with PD-L1, IRF1, and HIF-2α expression in LUAD patients ( n = 61). ( E ) Correlation analysis of ATXN3 expression with PD-L1, IRF1, and HIF-2α expression in melanoma patients ( n = 48). ( F ) ATXN3 is a positive regulator for PD-L1 transcription through stabilizing multiple transcription factors including HIF-2α, IFR1, STAT3, and JunB and enhances tumor evasion. B and C : 2-tailed unpaired t test; D and E : Pearson’s correlation analysis. ** P < 0.01, *** P < 0.001.

Article Snippet: HEK293T cells were plated in 96-well plates and, the following day, cotransfected with 0.01 μg TK control ( Renilla luciferase), 0.05 μg ATXN3 plasmid, and 0.05 μg CD274 promoter reporter (firefly luciferase, Addgene, 107007) constructs using Lipofectamine 3000 (Invitrogen, catalog L3000150).

Techniques: Immunohistochemical staining, Staining, Expressing