p stat1 t701  (Cell Signaling Technology Inc)

Journal: Journal for Immunotherapy of Cancer

Article Title: Colorectal cancer-specific IFNβ delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy

doi: 10.1136/jitc-2023-008515

Figure Lengend Snippet: TLR3 and IFNAR1 are indispensable for irradiation-induced type I IFN and CXCL10 production. (A) SW480 cells were irradiated and treated with 2.5 µg/mL poly (I:C) and 10 µM TLR3 inhibitor together for 24 hours. The mRNA levels of TLR3 , IFNβ1, CXCL10 and MX1 were analyzed by qRT-PCR (n=3). *p<0.05, **p<0.01 and ***p<0.001. One-way ANOVA test. (B) SW480 cells were irradiated and treated with 2.5 µg/mL poly (I:C) and 10 µM TLR3 inhibitor together for 24 hours. The protein levels of p-IRF3, IRF3, p-STAT1, and STAT1 were examined by immunoblotting. *p<0.05, **p<0.01, ***p<0.001. One-way ANOVA test. (C) SW480 cells were transfected with pCMV6-vector, pCMV-TLR3-WT, or pCMV-TLR3-L412F for 24 hours and then irradiated with 5 Gy. After 24 hours, we analyzed the protein level by immunoblotting. (D) The quantification of p-IRF3/IRF3 and p-STAT1/STAT1 is shown. *p<0.05, **p<0.01. One-way ANOVA test. (E) HCT116 cells were infected with lentivirus carrying shRNA against IFNAR. The knockdown efficacy was measured by immunoblotting. HCT116 shNC , HCT116 shIFNAR1#1 and HCT116 shIFNAR1#2 cells were irradiated with 5 and 10 Gy. After 24 hours, the cells were harvested, and the mRNA level of CXCL10 was examined by qRT-PCR. ***p<0.001. One-way ANOVA test. (F) HCT116 shNC and HCT116 shIFNAR1#2 cells were irradiated with 5 Gy. After 24 hours, conditioned medium was harvested to analyze the level of CXCL10 by ELISA. ***p<0.001. One-way ANOVA test. ANOVA, analysis of variance; qRT-PCR, quantitated by real-time PCR.

Article Snippet: The following antibodies were used: cGAS (ab224144, Abcam, Cambridge, UK), STING (#13647, Cell Signaling Tech., California, USA), GAPDH (Ab9485, Abcam), RIG1 (A0550, Abclonal, Massachusetts, USA), TLR3 (ab62566, Abcam), MDA5 (A2203, Abclonal), MAVS (A5764, Abclonal), beta-actin (IR2-7, iREAL Biotech.), p-IRF3 S388 (AP1333 and AP0263, Abclonal, IRF3 (#41075, Cell Signaling Tech.), p-STAT1 T701 (bs-1657R, Bioss antibodies and AP0054, Abclonal) and STAT1 (sc-464, Santa Cruz, California, USA).

Techniques: Irradiation, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Infection, shRNA, Knockdown, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: In vivo CRISPR screen identifies KRAS-induced COX-2 as a driver of immune evasion and immunotherapy resistance in lung cancer

doi: 10.1101/2023.04.13.536740

Figure Lengend Snippet: (A) Flow cytometry analysis showing surface expression of IFN-β receptor subunit β on KPAR cells and Ifngr2 -/- cells. (B) Immunoblot for pSTAT1 and STAT1 in KPAR cells and Ifngr2 -/- cells treated for 24h with 100ng/ml IFNγ. (C) Incucyte analysis showing growth rate of KPAR cells and Ifngr2 -/- cells in vitro . (D) Kaplan-Meier survival of immune-competent or Rag2 -/- ; Il2rg -/- mice following orthotopic transplantation with KPAR cells or Ifngr2 -/- (clone 2), n=5-10 per group. Analysis of survival curves was carried out using log-rank (Mantel-Cox) test; * P<0.05. (E) Incucyte analysis showing growth of KPAR cells in the presence of 200ng/ml IFNα, 200ng/ml IFN′3 or 100ng/ml IFNγ. (F) Heatmap showing mRNA expression of IFN-response genes in KPAR cells and Ifngr2 -/- cells treated with 100ng/ml IFNγ. (G) Flow cytometry analysis showing surface expression of H2-Db (left) and PD-L1 (right) on KPAR cells and Ifngr2 -/- cells treated for 24h with either 200ng/ml IFNα, 200ng/ml IFN′3 or 100ng/ml IFNγ. (H) Surface expression of the IFNγ-receptor ′3 chain on KPAR cells treated with 10nM trametinib, 1µM linsitinib and 40nM everolimus for 24h.

Article Snippet: Proteins were detected by Western blotting using the following primary antibodies against: Flag (M2, Sigma), ERK1/2 (3A7, Cell Signalling), p-ERK1/2 (Thr202/Tyr204) (9101, Cell Signalling), Myc (Y69, Abcam), STAT1 (#9172, Cell Signaling), p-STAT1 (T701) (58D6, Cell Signaling) STAT2 (D9J7L, Cell Signaling) COX-2 (D5H5, Cell Signalling), Vinculin (VIN-11-5, Sigma) and β-Actin (8H10D10, Cell Signaling).

Techniques: Flow Cytometry, Expressing, Western Blot, In Vitro, Transplantation Assay