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: Figure 2 Radiotherapy (RT) enhanced cytosolic dsRNA accumulation for TLR3-mediated type I IFN production. (A) HCT116 cells were irradiated for 24 hours, and then RNA was extracted for electrophoresis (7.5% polyacrylamide gel electrophoresis). HCT116 cells were irradiated for 24 and 48 hours, and the content of cytosolic dsRNA was measured by dsRNA ELISA kit (n=3). *p<0.05, **p<0.01. One-way ANOVA test. (B) SW480 cells were irradiated with 5 Gy for 24 hours, and the level of dsRNA was observed by immunofluorescence staining. *p<0.05 Unpaired t-test. (C) SW480 cells were treated with conditioned medium for 24 hours and then examined by qRT-PCR (n=3). The conditioned medium was collected from irradiated cells and then incubated with RNase A and RNase III for 2 hours. The level of IFNβ1 was analyzed by qRT-PCR (n=3). ***p<0.001. One-way ANOVA test. (D) SW480 cells were treated with conditioned medium for 24 hours and then examined by qRT-PCR (n=3). The conditioned medium was collected from irradiated cells and then incubated with RNase A and RNase III for 2 hours. The level of CXCL10 was analyzed by qRT-PCR (n=3). **p<0.01. One-way ANOVA test. (E) Representative images of cytosolic dsRNA in pre- neoCRT biopsies and post-neoCRT surgical tissues. (F) The correlation between cytosolic dsRNA and tumor IFNβ expression was measured (non-linear regression model, p=0.0012, r=0.2794, n=131). (G) SW480 cells were infected with lentivirus carrying shRNA against RIG1, MDA5 and MAVS. The knockdown efficacy was examined by western blotting. (H) SW480shNC, SW480shRIG1, SW480shTLR3, SW480shMDA5 and SW480shMAVS cells were irradiated with 5 Gy. After 24 hours, the cells were harvested, and the mRNA levels of IFNβ1 and CXCL10 were examined by qRT-PCR (n=3). *p<0.05, **p<0.01. One-way ANOVA test. (I) HCT116 cells were infected with lentivirus carrying shRNA against TLR3. The knockdown efficacy was measured by immunoblotting. HCT116shNC, HCT116shTLR3#1 and HCT116shTLR3#2 cells were irradiated with 5 and 10 Gy. After 24 hours, the cells were harvested, and the mRNA levels of IFNβ1 and CXCL10 were examined by qRT-PCR (n=3). **p<0.01, ***p<0.001. One-way ANOVA test. (J) HCT116shNC and HCT116shTLR3 cells were with or without irradiation with 5 Gy. After 24 hours, CFSE-labeled SupT1 T cells were seeded in the upper wells, and the migrated T cells were examined by flow cytometry (n=3). **p<0.01, ***p<0.001. One-way ANOVA test. (K) HCT116 isogenic TLR3 knockout cells (HCT116-TLR3KO) constructed with TLR3 gRNA and HCT115-WT cells constructed with non-targeting (NT) gRNA. HCT116-WT and HCT116-TLR3KO cells were irradiated with 5 Gy and harvested after 24 hours. The levels of IFNβ1 were analyzed by qRT-PCR (n=3). *p<0.05. One-way ANOVA test. ANOVA, analysis of variance; CFSE, Carboxyfluorescein succinimidyl ester; 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), 3Huang KC- Y, et al. J Immunother Cancer 2024;12:e008515. doi:10.1136/jitc-2023-008515 RIG1 (A0550, Abclonal, Massachusetts, USA), TLR3 (ab62566, Abcam), MDA5 (A2203, Abclonal), MAVS (A5764, Abclonal), beta- actin (IR2- 7, iREAL Biotech.), p- IRF3S388 (AP1333 and AP0263, Abclonal, IRF3 (#41075, Cell Signaling Tech.), p- STAT1T701 (bs- 1657R, Bioss antibodies and AP0054, Abclonal) and STAT1 (sc- 464, Santa Cruz, California, USA).

Techniques: Irradiation, Electrophoresis, Polyacrylamide Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Incubation, Expressing, Infection, shRNA, Knockdown, Western Blot, Labeling, Flow Cytometry, Knock-Out, Construct, Real-time Polymerase Chain Reaction

Journal: Gut Microbes

Article Title: cGAS-STING effectively restricts murine norovirus infection but antagonizes the antiviral action of N-terminus of RIG-I in mouse macrophages

doi: 10.1080/19490976.2021.1959839

Figure Lengend Snippet: cGAS and STING are necessary for MNV infection-induced IFN response . (a) RAW264.7 cells were uninfected or infected with MNV-1 for 1 h, then untreated or treated with H151 (2 µg/ml) for 20 h. The mRNA levels of STAT1, ISG15 and IFIT1 were analyzed by qRT-PCR (n = 5). RAW264.7 WT, cGas -/- and Sting -/- cells were uninfected or infected with MNV-1 for 20 h. The mRNA levels of (b) IFN-β, and (c) ISG15, IFIT1, MX1 and MX2 were analyzed by qRT-PCR (n = 4), respectively. RAW264.7 WT, cGas -/- and Sting -/- cells were uninfected or infected with MNV CW3 or MNV CR6 for 20 h. The mRNA levels of (d) IFN-β, and (e) ISG15 and IFIT1 were analyzed by qRT-PCR (n = 4), respectively. Data were normalized to the uninfected WT control (set as 1). *P < .05; **P < .01; ***P < .001

Article Snippet: Antibodies against STAT1 (#9172), pSTAT1 (Ser727, #9177), RIG-I (D14G6, #3743), MDA5 (D74E4, #5321), cGAS (D3O8O, #31659), STING (D2P2F, #13647), Myc-tag (71D10, #2278) and Myc-Tag (9B11, #2276) were purchased from Cell Signaling Technology.

Techniques: Infection, Quantitative RT-PCR, Control

Journal: Gut Microbes

Article Title: cGAS-STING effectively restricts murine norovirus infection but antagonizes the antiviral action of N-terminus of RIG-I in mouse macrophages

doi: 10.1080/19490976.2021.1959839

Figure Lengend Snippet: STING interacts with RIG-I . (a) HEK293T cells were transfected with pFlag-RIG-I_WT, pFlag-RIG-I_N, or the empty vectors (1 µg/each) for 24 h. Expression of transfected vectors were examined by western blotting. HEK293T cells were transfected with pMyc-STING, and pFlag-RIG-I_WT, pFlag-RIG-I_N or empty vectors (1 µg/each for confocal assay for 24 h; 1.5 µg/each for co-IP assay for 48 h). (b) Expression and co-localization of STING with RIG-I_WT and RIG-I_N were analyzed by confocal assay. (c) Co-IP assay was performed using anti-Flag MAb (1:1000). The precipitated proteins were analyzed by western blotting using anti-Flag and anti-Myc antibodies. HEK293T cells were transfected with pMyc-cGAS, and pFlag-RIG-I_WT, pFlag-RIG-I_N or empty vectors (1 µg/each for confocal assay for 24 h; 1.5 µg/each for co-IP assay for 48 h). (d) Expression and co-localization of cGAS with RIG-I_WT and RIG-I_N was analyzed by confocal assay. (e) Co-IP assay was performed using anti-Flag MAb (1:1000). The precipitated proteins were analyzed by western blotting using anti-Flag and anti-Myc antibodies. β-actin was used as a loading control

Article Snippet: Antibodies against STAT1 (#9172), pSTAT1 (Ser727, #9177), RIG-I (D14G6, #3743), MDA5 (D74E4, #5321), cGAS (D3O8O, #31659), STING (D2P2F, #13647), Myc-tag (71D10, #2278) and Myc-Tag (9B11, #2276) were purchased from Cell Signaling Technology.

Techniques: Transfection, Expressing, Western Blot, Confocal Assay, Co-Immunoprecipitation Assay, Control

Journal: Gut Microbes

Article Title: cGAS-STING effectively restricts murine norovirus infection but antagonizes the antiviral action of N-terminus of RIG-I in mouse macrophages

doi: 10.1080/19490976.2021.1959839

Figure Lengend Snippet: The N-terminus of RIG-I exerts antiviral effect, but is antagonized by STING expression . (a) Analysis of ISRE related firefly luciferase activity in Huh7-ISRE-Luc cells transfected with pFlag-RIG-I_WT (1 µg), pFlag-RIG-I_N (1 µg) or empty vectors (1 µg) for 48 h (n = 6). (b) HEK293T cells were transfected with pFlag-RIG-I_WT (1 µg), pFlag-RIG-I_N (1 µg) or empty vectors (1 µg) for 48 h. The mRNA levels of IFN-β, ISG15 and IFIT1 were analyzed by qRT-PCR (n = 5). (c) HEK293T cells were transfected with pFlag-CD300lf (0.5 µg), pFlag-RIG-I_N (1 µg) or empty vectors (1 µg) for 24 h, then infected with MNV for 20 h. The viral RNA, NS1/2 protein and expression of transfected vectors were analyzed by qRT-PCR (n = 5) and western blotting, respectively. (d) HEK293T cells were transfected with pFlag-CD300lf (0.5 µg), pFlag-RIG-I_N (1 µg) or empty vectors (1 µg) for 24 h, then infected with MNV for 20 h. The titers of produced viruses in the supernatant were analyzed by TCID50 assay (n = 4). (e) HEK293T cells were transfected with pFlag-RIG-I_N (1 µg) or empty vectors (1 µg) for 24 h, then untreated or treated with JAK inhibitor 1 (5 µg/ml) for 20 h. The mRNA levels of IFN-β, ISG15 and IFIT1, and STAT1 expression and phosphorylation were analyzed by qRT-PCR (n = 4) and western blotting, respectively. (f) HEK293T cells were transfected with pFlag-CD300lf (0.5 µg), pFlag-RIG-I_N (1 µg) or empty vectors (1 µg) for 24 h, then infected with MNV for 1 h, refreshed medium with or without JAK inhibitor 1 (5 µg/ml) for 20 h. The viral RNA, NS1/2 protein and expression of transfected vectors were analyzed by qRT-PCR (n = 4) and western blotting, respectively. (g) J774A.1 and RAW264.7 cells were transfected with pFlag-RIG-I_N (1 µg) or empty vectors (1 µg) for 24 h, then infected with MNV for 20 h. The viral RNA levels were analyzed in J774A.1 (n = 4) and RAW264.7 (n = 7) cells by qRT-PCR. (h) RAW264.7 cGas -/- or Sting -/- cells were transfected with pFlag-RIG-I_N (1 µg) or empty vectors (1 µg) for 24 h, then infected with MNV for 20 h. The viral RNA levels were analyzed by qRT-PCR (n = 4). (i) RAW264.7 cGas -/- or Sting -/- cells were transfected with pFlag-RIG-I_N (1 µg), pMyc-cGAS (1 µg), pMyc-STING (1 µg), or empty vectors (1 µg) for 24 h, then infected with MNV for 20 h. The viral RNA levels in cGas -/- (n = 6) or Sting -/- (n = 6) RAW264.7 cells were analyzed by qRT-PCR. Data were normalized to the EV control (set as 1). *P < .05; **P < .01; ***P < .001; ns, not significant. β-actin was used as a loading control

Article Snippet: Antibodies against STAT1 (#9172), pSTAT1 (Ser727, #9177), RIG-I (D14G6, #3743), MDA5 (D74E4, #5321), cGAS (D3O8O, #31659), STING (D2P2F, #13647), Myc-tag (71D10, #2278) and Myc-Tag (9B11, #2276) were purchased from Cell Signaling Technology.

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Infection, Western Blot, Produced, TCID50 Assay, Phospho-proteomics, Control

Journal: Gut Microbes

Article Title: cGAS-STING effectively restricts murine norovirus infection but antagonizes the antiviral action of N-terminus of RIG-I in mouse macrophages

doi: 10.1080/19490976.2021.1959839

Figure Lengend Snippet: A proposed model for host immune response upon MNV infection . Upon infection, MNV is recognized by the pattern recognition receptor MDA5. This activates the downstream cascades, including NF-ĸB and IRF3, inducing production of IFNs. Then, IFNs bind to the receptors and further activate JAK/STAT signaling pathway, leading to transcription of hundreds of ISGs. RIG-I, cGAS and STING can restrict MNV replication in mouse macrophages, but whether they are involved in MNV recognition needs further studies. Also, whether the interaction between RIG-I and STING involving the participation of other host factors needs to be further explored. STING agonists DMXAA and 2ʹ3ʹ-cGAMP inhibit MNV replication through induction of antiviral ISGs, and this effect is partially reversed by JAK inhibitor treatment. The red line with a blunt end indicates the inhibitory effect

Article Snippet: Antibodies against STAT1 (#9172), pSTAT1 (Ser727, #9177), RIG-I (D14G6, #3743), MDA5 (D74E4, #5321), cGAS (D3O8O, #31659), STING (D2P2F, #13647), Myc-tag (71D10, #2278) and Myc-Tag (9B11, #2276) were purchased from Cell Signaling Technology.

Techniques: Infection

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: Radiotherapy (RT) enhanced cytosolic dsRNA accumulation for TLR3-mediated type I IFN production. (A) HCT116 cells were irradiated for 24 hours, and then RNA was extracted for electrophoresis (7.5% polyacrylamide gel electrophoresis). HCT116 cells were irradiated for 24 and 48 hours, and the content of cytosolic dsRNA was measured by dsRNA ELISA kit (n=3). *p<0.05, **p<0.01. One-way ANOVA test. (B) SW480 cells were irradiated with 5 Gy for 24 hours, and the level of dsRNA was observed by immunofluorescence staining. *p<0.05 Unpaired t-test. (C) SW480 cells were treated with conditioned medium for 24 hours and then examined by qRT-PCR (n=3). The conditioned medium was collected from irradiated cells and then incubated with RNase A and RNase III for 2 hours. The level of IFNβ1 was analyzed by qRT-PCR (n=3). ***p<0.001. One-way ANOVA test. (D) SW480 cells were treated with conditioned medium for 24 hours and then examined by qRT-PCR (n=3). The conditioned medium was collected from irradiated cells and then incubated with RNase A and RNase III for 2 hours. The level of CXCL10 was analyzed by qRT-PCR (n=3). **p<0.01. One-way ANOVA test. (E) Representative images of cytosolic dsRNA in pre-neoCRT biopsies and post-neoCRT surgical tissues. (F) The correlation between cytosolic dsRNA and tumor IFNβ expression was measured (non-linear regression model, p=0.0012, r =0.2794, n=131). (G) SW480 cells were infected with lentivirus carrying shRNA against RIG1, MDA5 and MAVS. The knockdown efficacy was examined by western blotting. (H) SW480 shNC , SW480 shRIG1 , SW480 shTLR3 , SW480 shMDA5 and SW480 shMAVS cells were irradiated with 5 Gy. After 24 hours, the cells were harvested, and the mRNA levels of IFNβ1 and CXCL10 were examined by qRT-PCR (n=3). *p<0.05, **p<0.01. One-way ANOVA test. (I) HCT116 cells were infected with lentivirus carrying shRNA against TLR3. The knockdown efficacy was measured by immunoblotting. HCT116 shNC , HCT116 shTLR3#1 and HCT116 shTLR3#2 cells were irradiated with 5 and 10 Gy. After 24 hours, the cells were harvested, and the mRNA levels of IFNβ1 and CXCL10 were examined by qRT-PCR (n=3). **p<0.01, ***p<0.001. One-way ANOVA test. (J) HCT116 shNC and HCT116 shTLR3 cells were with or without irradiation with 5 Gy. After 24 hours, CFSE-labeled SupT1 T cells were seeded in the upper wells, and the migrated T cells were examined by flow cytometry (n=3). **p<0.01, ***p<0.001. One-way ANOVA test. (K) HCT116 isogenic TLR3 knockout cells (HCT116-TLR3 KO ) constructed with TLR3 gRNA and HCT115-WT cells constructed with non-targeting (NT) gRNA. HCT116-WT and HCT116-TLR3 KO cells were irradiated with 5 Gy and harvested after 24 hours. The levels of IFNβ1 were analyzed by qRT-PCR (n=3). *p<0.05. One-way ANOVA test. ANOVA, analysis of variance; CFSE, Carboxyfluorescein succinimidyl ester; 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, Electrophoresis, Polyacrylamide Gel Electrophoresis, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Incubation, Expressing, Infection, shRNA, Knockdown, Western Blot, Labeling, Flow Cytometry, Knock-Out, Construct, Real-time Polymerase Chain Reaction