Journal: Frontiers in Oncology

Article Title: RhoC Modulates Cell Junctions and Type I Interferon Response in Aggressive Breast Cancers

doi: 10.3389/fonc.2021.712041

Figure Lengend Snippet: RhoC expression influences gene and protein expression of Type I interferon signaling response. (A) Type I interferon signaling pathway and the genes significantly downregulated in crRhoC knockout cells compared to wild-type, as measured in RNAseq. Genes in blue circles were downregulated in crRhoC compared to wild-type; genes in grey circles were not significantly differentially expressed between crRhoC and wild-type. (B) Western blot of interferon signaling markers in MDA 231 wild-type and crRhoC cells—crRhoC cells have increased p-STAT2 and IRF9 compared to wild type. (C) Western blot of interferon signaling markers in SUM 149 wild-type and crRhoC cells—crRhoC cells have decreased p-STAT2 and STAT2 compared to wild type. (D) Short-term and long-term signaling through type I interferon signaling pathways. Short-term interferon signaling is driven by phosphorylated STAT1 and STAT2 complexed with IRF9 that translocates to the nucleus, binds to interferon stimulated response elements (ISREs), and promotes transcription of interferon stimulated genes (ISGs); phosphorylation of STAT1 and STAT2 peak about 2 hours after treatment with a type I interferon. Long-term signaling is driven by unphosphorylated STAT1 and STAT2 complexed with IRF9, and peaks around 72 hours after treatment with type I interferons. Genes listed in order of decreasing magnitude of log fold-change.

Article Snippet: The following primary antibodies were used: anti-E-cadherin rabbit polyclonal antibody (ThermoFisher #PA5-32178) at 1:500 dilution, anti-β-catenin mouse monoclonal antibody (Invitrogen #MA1-300) at 1:500 dilution, anti-ZO-1 mouse monoclonal antibody (Invitrogen #33-9100) at 1:150 dilution for immunofluorescent staining and 1:200 for Western Blot, anti-Occludin rabbit polyclonal antibody (Zymed #71-1500) at 1:300 dilution for immunofluorescent staining and Western Blot, anti-p-STAT1 rabbit monoclonal antibody (CST #9167) at 1:1000 dilution, anti-STAT1 rabbit monoclonal antibody (CST #9172) at 1:1000 dilution, anti-p-STAT2 rabbit antibody (CST #4441) at 1:500 dilution, anti-STAT2 rabbit antibody (CST #4594) at 1:500 dilution, anti-IRF9 rabbit monoclonal antibody (CST #76684) at 1:500 dilution, anti-IFI27 rabbit polyclonal antibody (ThermoFisher #PA5-68038) at 1:1000 dilution, anti-IFITM1 mouse monoclonal antibody (Proteintech #60074-1-IG) at 1:20,000 dilution, anti-MX1 rabbit polyclonal antibody (Proteintech #13750-1-AP) at 1:1000, anti-ISG15 rabbit polyclonal antibody (Proteintech #15981-1-AP) at 1:1000, and anti-actin antibody (Sigma #A3854) at 1:15,000 dilution.

Techniques: Expressing, Knock-Out, Western Blot, Protein-Protein interactions, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP

doi: 10.1016/j.jbc.2024.105779

Figure Lengend Snippet: Nsp1 inhibited the IFN response. A–D , HEK-293T cells were cotransfected with pCAGGS-2×HA-STAT1/pCAGGS-2×HA-STAT2/pCAGGS-2×HA-IRF9/ISGF3 (pCAGGS-2×HA-STAT1, pCAGGS-2×HA-STAT2, and pCAGGS-2×HA-IRF9), pIFN-β-luc, and pRL-TK at a ratio of 1:0.2:0.05. After 12 h, the cells were transfected with pCAGGS-3×Flag-nsp1. After 24 h, the cells were collected to detect ISRE promoter activity through the dual luciferase reporter assay. E , HEK-293T cells were co-transfected with pCAGGS-3×Flag-nsp1, pIFN-β-luc, and pRL-TK at a ratio of 1:0.2:0.05. After 24 h, the cells were stimulated by human IFN-β for 4 h. Then, the cells were collected to detect ISRE promoter activity through the dual luciferase reporter assay. F , HEK-293T cells were transfected with pCAGGS-2×HA-STAT1/pCAGGS-2×HA-STAT2/pCAGGS-2×HA-IRF9/ISGF3 (pCAGGS-2×HA-STAT1, pCAGGS-2×HA-STAT2, and pCAGGS-2×HA-IRF9). After 12 h, the cells were transfected with pCAGGS-3×Flag-nsp1. The cells were then collected to detect the nsp1, STAT1, STAT2, IRF9, and ISGF3 expression levels via Western blotting. G–J , HEK-293T cells were transfected with pCAGGS-2×HA-STAT1/pCAGGS-2×HA-STAT2/pCAGGS-2×HA-IRF9/ISGF3 (pCAGGS-2×HA-STAT1, pCAGGS-2×HA-STAT2, and pCAGGS-2×HA-IRF9). After 12 h, the cells were transfected with pCAGGS-3×Flag-nsp1. Then, the cells were collected to detect the OAS1, ISG15, and ISG56 mRNA levels using RT-qPCR. J , HEK-293T cells were transfected with pCAGGS-3×Flag-nsp1 or pCAGGS-3×Flag-nsp1-mutant. After 24 h, the cells were stimulated by SeV. Then, the cells were collected to detect the OAS1, ISG15, and ISG56 mRNA levels using RT-qPCR. Data are the mean ± SD. The p -value was calculated using the t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. HEK, human embryonic kidney cell line; IFN, interferon; IRF9, interferon regulatory factor-9; ISG, interferon-stimulated gene; ISGF3, interferon-stimulated gene factor 3; ISRE, interferon-stimulated response elements; nsp1, nonstructure protein 1; OAS1, 2′-5′-oligoadenylate synthetase 1; SeV, Sendai virus; STAT, signal transducer and activator of transcription.

Article Snippet: This study used the following antibodies and reagents: mouse anti-FLAG-tag monoclonal antibody (mAb), mouse anti-HA-tag mAb, horseradish peroxidase (HRP)-conjugated goat anti-mouse (H+L), HRP-conjugated goat anti-rabbit (H+L), HRP-conjugated mouse anti-rabbit (L), HRP-conjugated goat anti-mouse (L), p-STAT1 rabbit mAb, STAT1 rabbit mAb, p-STAT2 rabbit mAb, STAT2 rabbit mAb, IRF9 rabbit mAb, p-JAK1 rabbit mAb, JAK1 rabbit mAb, TYK2 rabbit mAb, and p-TYK2 rabbit mAb (ABclonal); CREB-BP rabbit mAb (Affinity); beta-actin rabbit antibody (Proteintech); MG132 and Z-VAD-FMK (Beyotime); CHX710 (MedChemExpress); human IFN-β (InvivoGen); Lipofectamine 3000 transfection reagent (Sigma); and Dylight-conjugated 488 goat anti-mouse IgG (Abbkine).

Techniques: Transfection, Activity Assay, Luciferase, Reporter Assay, Expressing, Western Blot, Quantitative RT-PCR, Mutagenesis, Virus

Journal: The Journal of Biological Chemistry

Article Title: SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP

doi: 10.1016/j.jbc.2024.105779

Figure Lengend Snippet: Nsp1 inhibited STAT1 phosphorylation. A and B , HEK-293T cells and LLC-PK1 cells were transfected with pCAGGS-3×Flag-nsp1. After 24 h, HEK-293T cells were incubated with human IFN-β for 4 h, and LLC-PK1 cells were stimulated by SeV for 8 h. STAT1, p-STAT1, STAT2, and p-STAT2 expression was detected by Western blotting. All protein levels were analyzed using ImageJ. Western blotting assay was repeated in two independent experiments. C , HEK-293T cells were transfected with pCAGGS-3×Flag-nsp1-mutant. After 24 h, HEK-293T cells were incubated with human IFN-β for 4 h. STAT1 and p-STAT1 expression was detected by Western blotting. All protein levels were analyzed using ImageJ. Western blotting assay was repeated in two independent experiments. The data are the means ± SD. The p -value was calculated using the t test. ∗∗∗ p < 0.001. HEK, human embryonic kidney cell line; IFN-β, interferon-β; nsp1, nonstructure protein 1; SeV, Sendai virus; STAT, signal transducer and activator of transcription.

Article Snippet: This study used the following antibodies and reagents: mouse anti-FLAG-tag monoclonal antibody (mAb), mouse anti-HA-tag mAb, horseradish peroxidase (HRP)-conjugated goat anti-mouse (H+L), HRP-conjugated goat anti-rabbit (H+L), HRP-conjugated mouse anti-rabbit (L), HRP-conjugated goat anti-mouse (L), p-STAT1 rabbit mAb, STAT1 rabbit mAb, p-STAT2 rabbit mAb, STAT2 rabbit mAb, IRF9 rabbit mAb, p-JAK1 rabbit mAb, JAK1 rabbit mAb, TYK2 rabbit mAb, and p-TYK2 rabbit mAb (ABclonal); CREB-BP rabbit mAb (Affinity); beta-actin rabbit antibody (Proteintech); MG132 and Z-VAD-FMK (Beyotime); CHX710 (MedChemExpress); human IFN-β (InvivoGen); Lipofectamine 3000 transfection reagent (Sigma); and Dylight-conjugated 488 goat anti-mouse IgG (Abbkine).

Techniques: Phospho-proteomics, Transfection, Incubation, Expressing, Western Blot, Mutagenesis, Virus