Journal: Signal Transduction and Targeted Therapy

Article Title: Modulation of innate immune response to viruses including SARS-CoV-2 by progesterone

doi: 10.1038/s41392-022-00981-5

Figure Lengend Snippet: The progesterone-PGR axis promotes innate antiviral signaling. a Effects of progesterone on transcription of antiviral genes. BMDCs or T-47D cells were treated with the indicated doses of P4 for 1 h and then infected with SeV for 6 h before qPCR analysis of mRNA levels of the indicated antiviral genes. b Effects of progesterone on IFN-γ-induced transcription of IRF1 gene. BMDCs or T-47D cells were treated with the indicated doses of P4 for 1 h and then stimulated with IFN-γ (100 ng/ml) for 6 h before qPCR analysis of IRF1 mRNA level. c Effects of progesterone on transcription of antiviral genes in Pgr +/+ and Pgr − / − BMDCs. Pgr +/+ and Pgr − /− BMDCs were treated with P4 (1 μM) for 1 h and then infected with SeV for 6 h before qPCR analysis of mRNA levels of the indicated genes. d Effects of progesterone on transcription of antiviral genes in PGR-knockout cells. The control and PGR-knockout T-47D cells were treated with P4 (1 μM) for 1 h and then infected with SeV for 6 h before qPCR analysis of mRNA levels of the indicated genes. e Effects of progesterone treatment on virus-induced activation of IRF3 in control and PGR-knockout T-47D cells. The control and PGR-knockout T-47D cells were treated with P4 (1 μM) for 1 h, and then left uninfected or infected with SeV for the indicated times before immunoblotting analysis with the indicated antibodies. Data shown in a-d are mean ± SD ( n = 3) from one representative experiment, which was repeated for at least two times with similar results. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: Progesterone (HY-N0437) and Dasatinib (HY-10181) (MedChemExpress); IFN-γ (300-02, Peptech); Anti-mouse IFNAR-1-in vivo (clone MAR1-5A3; Selleck A2121); ELISA kits for progesterone (582601, Cayman Chemical), GnRH (MM-0506M1) and LH (MM-44039M1) (MEIMAIN), murine IFN-β(42400, PBL), murine CXCL10 (EMC121, Neobioscience), murine TNFα (430904, BioLegend), murine IL-6 (431304, BioLegend); Mouse monoclonal antibodies against HA (TA180128, OriGene), Flag (F3165) and β-actin (A2228) (Sigma), IRF3 (sc-33641, Santa Cruz Biotechnology); Rabbit polyclonal antibodies against PGR (8757), SRC (2109), p-SRC Y419 (2101) and p-Tyrosine (9411S) (Cell signaling technology), TBK1 (ab40676), p-TBK1 S172 (ab109272) and p-IRF3 S386 (ab76493) (Abcam) were purchased from the indicated companies.

Techniques: Infection, Knock-Out, Activation Assay, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Modulation of innate immune response to viruses including SARS-CoV-2 by progesterone

doi: 10.1038/s41392-022-00981-5

Figure Lengend Snippet: The progesterone-PGR axis promotes innate antiviral response via activation of SRC. a Effects of progesterone or viral infection on PR activation. The PGR-overexpressed HEK293 cells were transfected with PR reporter for 24 h, and then left untreated or treated with increased doses of progesterone (0.01, 0.1, 1 μM) or infected with different doses of SeV for 10 h before luciferase assays. b Effects of progesterone and viral infection on transcription of nuclear PGR-targeted genes. T-47D cells were treated with DMSO or P4 (1 μM) for 1 h and then left uninfected or infected with SeV for 6 h before qPCR analysis of mRNA levels of the indicated genes. c Effects of progesterone on virus-induced transcription of antiviral genes in SRC-knockdown cells. The control and SRC-knockdown T-47D cells were treated with P4 (1 μM) for 1 h and then left uninfected or infected with SeV for 6 h before qPCR analysis of mRNA levels of the indicated genes. The knockdown efficiency of SRC was shown by qPCR analysis of mRNA level in the left panels. d Effects of progesterone on IFN-γ-induced transcription of IRF1 genes in SRC-knockdown cells. The control and SRC-knockdown T-47D cells were treated with P4 (1 μM) for 1 h and then left untreated or treated with IFN-γ (100 ng/ml) for 6 h before qPCR analysis of IRF1 mRNA level. e Effects of progesterone on virus-induced phosphorylation events in SRC-knockdown T-47D cells. The control and SRC-knockdown T-47D cells were treated with P4 (1 μM) for 1 h, and then left uninfected or infected with SeV for the indicated times before immunoblotting analysis with the indicated antibodies. The relative intensities of p-IRF3 S386 and SRC (normalized to β-actin) were analyzed by ImageJ. f Effects of SRC inhibitor on progesterone-potentiated transcription of antiviral genes. BMDCs were pretreated with DMSO or Dasatinib (SRC inhibitor, 10 μM) and together with the indicated doses of P4 for 1 h. The cells were then left uninfected or infected with SeV for 6 h before qPCR analysis of the mRNA levels of Ifnb1 and Cxcl10 genes. g Effects of progesterone on virus-induced activation of SRC, TBK1 and IRF3. T-47D cells were treated with P4 (1 μM) for 1 h, and then left un-infected or infected with SeV for the indicated times before immunoblotting analysis with the indicated antibodies. h Effects of PGR-deficiency on SeV-induced activation of SRC, TBK1, and IRF3. The control and PGR-KO T-47D cells were cultured in P4-containing medium, and then left uninfected or infected with SeV for the indicated times before immunoblotting analysis with the indicated antibodies. Data shown in a-d, f are mean ± SD ( n = 3) from one representative experiment, which was repeated for at least two times with similar results. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant

Article Snippet: Progesterone (HY-N0437) and Dasatinib (HY-10181) (MedChemExpress); IFN-γ (300-02, Peptech); Anti-mouse IFNAR-1-in vivo (clone MAR1-5A3; Selleck A2121); ELISA kits for progesterone (582601, Cayman Chemical), GnRH (MM-0506M1) and LH (MM-44039M1) (MEIMAIN), murine IFN-β(42400, PBL), murine CXCL10 (EMC121, Neobioscience), murine TNFα (430904, BioLegend), murine IL-6 (431304, BioLegend); Mouse monoclonal antibodies against HA (TA180128, OriGene), Flag (F3165) and β-actin (A2228) (Sigma), IRF3 (sc-33641, Santa Cruz Biotechnology); Rabbit polyclonal antibodies against PGR (8757), SRC (2109), p-SRC Y419 (2101) and p-Tyrosine (9411S) (Cell signaling technology), TBK1 (ab40676), p-TBK1 S172 (ab109272) and p-IRF3 S386 (ab76493) (Abcam) were purchased from the indicated companies.

Techniques: Activation Assay, Infection, Transfection, Luciferase, Western Blot, Cell Culture

Journal:

Article Title: RhoC is dispensable for embryogenesis and tumor initiation but essential for metastasis

doi: 10.1101/gad.1310805

Figure Lengend Snippet: RhoC is not essential in T- or B-cell development, activation, apoptosis, or migration. (A) Flow cytometric analyses of thymic, lymph node, and splenic total T- and B-lymphocyte populations in wild-type (upper panels) or RhoC-/- (lower panels) mice. Results representative of seven independent experiments are shown. (B) Proliferative responses of T and B lymphocytes to stimuli. Purified T and B cells from wild-type (Wt; black bars) and RhoC-/- (white bars) mice were stimulated for 48 h with anti-CD3 with or without costimulation by anti-CD28 and/or IL-2, or PMA plus ionomycine (Iono) for T cells; or anti-IgM, anti-CD40, anti-IgM plus anti-CD40, LPS, or CpG for B cells. [3H]-thymidine incorporation was then assessed. (C) Thymocyte sensitivity to apoptosis. Triplicate cultures of thymocytes from wild-type (black bars) or RhoC-/- (white bars) mice were treated with dexamethasone (DEX) to induce mitochondria-mediated apoptosis, or with anti-CD95 to induce death-receptor-mediated apoptosis. Cell viability was determined 24 h later by Annexin V FITC/PI staining. (UT) Untreated control. (D) Cell migration. T and B cells and thymocytes (∼106 cells) from wild-type (black bars) or RhoC-/- (white bars) mice were placed in the upper chamber of Transwell motility plates. Data shown are the mean percentages of duplicate sample of cells that migrated to the lower chamber after 24 h. For B, C and D, data are presented as the mean ± SD and are representative of three independent experiments.

Article Snippet: T cells were stimulated in triplicate with soluble anti-CD3 (5 μg/mL, Pharmingen) with or without anti-CD28 (2 μg/mL, Pharmingen), or IL-2 (100 U/mL, Pharmingen).

Techniques: Activation Assay, Migration, Purification, Staining