Journal: Frontiers in Immunology

Article Title: Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission

doi: 10.3389/fimmu.2021.785072

Figure Lengend Snippet: CGRP and SAX, but not CGRP 1–8 , increase STAT4 expression in MDLCs. (A, B) PHA/IL2-activated PBMCs were serum-starved overnight at 37°C, and left untreated (Un) or stimulated for 30 min with either IL12 or IFNα. Shown are representative Western blots (of n = 4 independent experiments using PBMCs from different individuals) of total STAT4 (A) and pSTAT4 (B) expression. (C, D) MDLCs were cytokine-starved overnight at 37°C, and treated with CGRP (0.1 μM), SAX (0.1 μM), CGRP 1–8 (10 μM) or LPS (10 μg/ml) as positive control. In panel (C) , shown is a representative Western blot (of n = 4 independent experiments using MDLCs from different individuals) of total STAT4 expression. In panel (D) , shown are mean ± SEM folds expression of total STAT4, normalized to that of beta actin. *p < 0.0500, **p < 0.0050, two-sided Student’s t-test. (E) MDLCs were cytokine-starved overnight at 37°C and treated with CGRP (0.1 μM) or SAX (0.1 μM). The CGRP receptor antagonist BIBN4096 (BIBN, 1 μM) was added 15 min before addition of agonists. Shown are mean ± SEM (of n = 4 independent experiments using MDLCs from different individuals) folds expression of total STAT4, normalized to that of beta actin. (F) MDLCs were treated as described in panels (C, D) above, and further stimulated for 30 min with combination of IL12 + IFNα. Shown is a representative Western blot (of n = 4 independent experiments using MDLCs from different individuals) of pSTAT4 expression.

Article Snippet: The blots were next incubated overnight at 4°C with commercial rabbit polyclonal Abs suitable for WB, directed against human STAT4 (Proteintech #13028-1AP, 0.5 μg/ml) or phosphorylated STAT4 (pSTAT4; R&D systems, #AF4319, 1 μg/ml), followed by 1:1,000 dilution of HRP-conjugated donkey-anti-rabbit IgG Ab (Southern Biotech) for 1 h at room temperature.

Techniques: Expressing, Western Blot, Positive Control

Journal: Frontiers in Immunology

Article Title: Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission

doi: 10.3389/fimmu.2021.785072

Figure Lengend Snippet: Summary of the requirements of CGRP receptor activation for inhibition of mucosal HIV-1 transmission. (1) In LCs, HIV-1 binding to langerin induces viral internalization and subsequent degradation, while virions escaping degradation trans-infect CD4+ T-cells. (2) We previously showed that CGRP activates its cognate receptor expressed by LCs and affects a multitude of cellular and molecular process (not shown), resulting in significant inhibition of mucosal HIV-1 trans-infection in-vitro and ex-vivo . (3) We show in the present study that SAX, a long-acting metabolically stable analogue of CGRP, also activates the CGRP receptor. (4) Both CGRP and SAX increase expression of langerin (not shown) and STAT4 (that can be readily phosphorylated upon subsequent cytokine stimulation), which result in inhibition of HIV-1 trans-infection in-vitro and ex-vivo . (5) In contrast, several CGRP peptide fragments fail to activate the CGRP receptor and to increase langerin/STAT4 expression, and accordingly lack anti-HIV-1 activity. (6) CGRP-mediated inhibition of HIV-1 dissemination from LCs to CD4+ T-cells might permit their long-term maintenance in the BLT model of mucosal HIV-1 infection in-vivo .

Article Snippet: The blots were next incubated overnight at 4°C with commercial rabbit polyclonal Abs suitable for WB, directed against human STAT4 (Proteintech #13028-1AP, 0.5 μg/ml) or phosphorylated STAT4 (pSTAT4; R&D systems, #AF4319, 1 μg/ml), followed by 1:1,000 dilution of HRP-conjugated donkey-anti-rabbit IgG Ab (Southern Biotech) for 1 h at room temperature.

Techniques: Activation Assay, Inhibition, Transmission Assay, Binding Assay, Infection, In Vitro, Ex Vivo, Metabolic Labelling, Expressing, Activity Assay, In Vivo

Journal: Cell reports

Article Title: ZEB1 promotes pathogenic Th1 and Th17 cell differentiation in multiple sclerosis

doi: 10.1016/j.celrep.2021.109602

Figure Lengend Snippet: (A and B) Heatmap for JAK family gene expression in Th1 and Th17 conditions from siCTL and siZEB1 cohort (human, (A) or WT and CD4 Cre ZEB1 L/L mice (B). (C) Western blot showing the expression of JAK family protein in human CD4 + T cells in Th17 conditions from siCTL and siZEB1 cohort at 12, 24, 48, and 72 h after nucleofection. (D) Western blot showing JAK2 expression in Th17 cells from WT and CD4 Cre ZEB1 L/L mice. (E and F) Western blot showing JAK2, total STAT4, and phosphorylated-STAT4 [pSTAT4 (pTyr693)] expression in Th1 cells from human siCTL and siZEB1 cohorts (E) or WT and CD4 Cre ZEB1 L/L mice (F). (G) Flow cytometry showing pSTAT4 expression in Th1 cells from WT and CD4 Cre ZEB1 L/L mice. (H and I) GSEA plot demonstrating the effect of ZEB1 loss on the expression of an IL12-STAT4 gene signature in (H) human and (I) mouse CD4 + Th1 cells. NES, normalized enrichment score. (J and K) Transcription level of TBX21 and IFNG in (J) human siCTL- and siZEB1-treated cells or (K) mouse WT and CD4 Cre ZEB1 L/L cells in Th1 conditions. (L and M) Flow cytometry evaluating JAK2 re-expression on Th17 differentiation in siZEB1 nucleofected CD4 + naive T cells. The contour plots (gated by GFP + ) are representative 3 different human donors, summarized in (M). Representative results from at least two individual experiments are shown in (C)–(F). Statistical differences in (G) were determined using unpaired Student’s t test (two-tailed). Statistical differences in (M) were tested using paired Student’s t test (two-tailed). NS, not significant; *p < 0.05. p values in (H) and (I) were calculated using Wald test and adjusted using the Benjamini-Hochberg method.

Article Snippet: Antibodies were from cell signaling technology unless otherwise noted and included ZEB1 (clone E2G6Y), JAK2 (clone D2E12), STAT3 (clone 124H6), pSTAT3 Y705 (clone D3A7), pSTAT3 S727 (clone D8C2Z), STAT4 clone (C46B10), pSTAT4 Y693 (clone D2E4), JAK1 (clone D1T6W), JAK3 (clone D7B12), TYK2 (clone D4I5T), and β-actin (clone C4, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Xanthatin synergizes with cisplatin to suppress homologous recombination through JAK2/STAT4/BARD1 axis in human NSCLC cells

doi: 10.1111/jcmm.16271

Figure Lengend Snippet: Xa inhibites HR pathway by down‐regulating BARD1 via JAK2‐STAT4 pathway. (A) WB analysis of γH2AX, BARD1, BRCA1 and RAD51, showing that BARD1 was successfully overexpressed, and overexpression of BARD1 (BARD1 OE) partially reversed the down‐regulation of BRCA1 and RAD51 caused by Xa. (B) CCK‐8 assay, displaying the effect of Xa was partially reversed by BARD1 OE. (C) WB analysis demonstrated that Xa reduces the protein level of phosphorylation of JAK2 (p‐JAK2) and p‐STAT4 and BARD1, which behaves similar to JAK2 inhibitor TG101348. Experiments were repeated four times. *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: The membranes with proteins were subsequently blocked in 5% BSA and then incubated with antibodies specific to FAS (13098‐1‐AP; Proteintech), ALDH1A3 (25167‐1‐AP; Proteintech), ZMAT3 (10504‐1‐AP; Proteintech), IGFBP3 (10189‐2‐AP; Proteintech), CCNB3(AB44527; Absci), BBC3 (55120‐1‐AP; Proteintech), SESN1 (21668‐1‐AP; Proteintech), NTRK3 (A14033; ABclonal), DUSP6 (A3171; ABclonal), HMOX1(10701‐1‐AP; Proteintech), CDKN1A(10355‐1‐AP; Proteintech), BARD1 (A1685; ABclonal), BRCA1 (A0212; ABclonal), RAD51 (14961‐1‐AP; Proteintech), γH2AX (AP0099; ABclonal), NTRK3 (A14033; ABclonal), JAK2 (A19629; ABclonal), Phospho‐JAK2‐Y1007/1008 (p‐JAK2; AP0531; ABclonal), STAT4 (A4523; ABclonal), Phospho‐STAT4‐Y693 (p‐STAT4; AP0137; ABclonal).

Techniques: Over Expression, CCK-8 Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Xanthatin synergizes with cisplatin to suppress homologous recombination through JAK2/STAT4/BARD1 axis in human NSCLC cells

doi: 10.1111/jcmm.16271

Figure Lengend Snippet: A schematic model of the molecular mechanism underlying the role of Xa in NSCLC cells. Left panel shows drug resistant to Cis of NSCLC due to the abnormal enhancement of DNA repair capacity in cancer cells, leading to cell survival. Right panel shows Xa suppresses HR and synergizes with Cis. Mechanistically, Xa inhibites the conversion of JAK2 to p‐JAK2, making STAT4 unable to form its active form p‐STAT4. Then, the expression of BARD1 is decreased due to the lack of the transcription factor p‐STAT4, leading to reduction of the BARD1 protein. As a result, assembling of BARD1‐BRCA1 complexes and the recruitment of RAD51 at double‐strand breaks (DSBs) sites were decreased, resulted in degradation of BRCA1 and RAD51. Eventually, the down‐regulation of BARD1 triggered by Xa decreases the ability of cells against DSBs and increases the NSCLC cells sensitivity to Cis, leading to cell apoptosis

Article Snippet: The membranes with proteins were subsequently blocked in 5% BSA and then incubated with antibodies specific to FAS (13098‐1‐AP; Proteintech), ALDH1A3 (25167‐1‐AP; Proteintech), ZMAT3 (10504‐1‐AP; Proteintech), IGFBP3 (10189‐2‐AP; Proteintech), CCNB3(AB44527; Absci), BBC3 (55120‐1‐AP; Proteintech), SESN1 (21668‐1‐AP; Proteintech), NTRK3 (A14033; ABclonal), DUSP6 (A3171; ABclonal), HMOX1(10701‐1‐AP; Proteintech), CDKN1A(10355‐1‐AP; Proteintech), BARD1 (A1685; ABclonal), BRCA1 (A0212; ABclonal), RAD51 (14961‐1‐AP; Proteintech), γH2AX (AP0099; ABclonal), NTRK3 (A14033; ABclonal), JAK2 (A19629; ABclonal), Phospho‐JAK2‐Y1007/1008 (p‐JAK2; AP0531; ABclonal), STAT4 (A4523; ABclonal), Phospho‐STAT4‐Y693 (p‐STAT4; AP0137; ABclonal).

Techniques: Expressing