Journal: Human Cell

Article Title: Ethambutol induces optic neuropathy through SDHB-mediated ferroptosis in retinal ganglion cells via Smad4 pathway

doi: 10.1007/s13577-025-01342-4

Figure Lengend Snippet: EMB modulates SDHB transcriptional activity via Smad4 in RGC-5 cells. A WB analyses were conducted to assess the NFκB, Smad2/3, Smad4, STAT4 levels, and their phosphorylation levels in RGC-5 cells treated with 2 mM EMB. N = 3. * p < 0.05, ** p < 0.01. B Utilizing the JASPAR database, a prediction was made regarding the binding of Smad4 to the SDHB promoter. C Detection of SLC7A11 and GPX4 protein expression in Smad4-overexpressing RGC-5 cells by WB. N = 3. * p < 0.05. D WB assay was conducted to verify the Smad4 overexpression in RGC-5 cells. N = 3. * p < 0.05. E , F RT-qPCR and WB assays were performed to quantify SDHB mRNA ( E ) and protein ( F ) levels in Smad4-overexpressing RGC-5 cells. N = 3. * p < 0.05. G Dual-luciferase reporter assays were employed to demonstrate EMB’s regulation of SDHB promoter activity via Smad4. SDHB-Mut: mutating the Smad4 binding sites on SDHB promoters. N = 3. * p < 0.05. H CHIP assay to detect DNA enriched fragments of SDHB promoter was conducted using Smad4-specific antibodies in EMB-treated RGC-5 cells. N = 3. ** p < 0.01. I WB analysis of Smad4 knockdown efficiency in RGC-5 cells. N = 3. * p < 0.05. J In SDHB-overexpressing RGC-5 cells with Smad4 knockdown, the protein expression levels of SDHB, SLC7A11, and GPX4 were detected by WB. Data shown are representative of three independent experimental replicates

Article Snippet: STAT4 , Proteintech , 67568-2-Ig.

Techniques: Activity Assay, Phospho-proteomics, Binding Assay, Expressing, Over Expression, Quantitative RT-PCR, Luciferase, Knockdown

Journal: Cancer Cell

Article Title: Single-cell analysis defines a pancreatic fibroblast lineage that supports anti-tumor immunity

doi: 10.1016/j.ccell.2021.06.017

Figure Lengend Snippet:

Article Snippet: Anti-human/mouse pSTAT4 [Y693] 148Nd clone 38/p-Stat4 , Fluidigm , 3148006A.

Techniques: Blocking Assay, Virus, Recombinant, Plasmid Preparation, Saline, Lysis, Staining, Reverse Transcription, In Vivo, Electroporation, Mass Cytometry, Conjugation Assay, Illumina Sequencing, Library Quantification, In Vitro, Quantitative RT-PCR, Software, Real-time Polymerase Chain Reaction, Cytometry, Microscopy, Imaging, Spectrophotometry

Journal: The Journal of Biological Chemistry

Article Title: Mycobacterium tuberculosis Directs T Helper 2 Cell Differentiation by Inducing Interleukin-1β Production in Dendritic Cells

doi: 10.1074/jbc.M112.375154

Figure Lengend Snippet: ESAT-6 induces Th2 cell differentiation via IL-1β production. A , IL-4 production in DCs co-cultured with naïve OT-II CD4 + T cells after infection with H37Rv or BCG in the presence or absence of anti-IL-1β. B , IL-4 production in DCs co-cultured with naïve OT-II CD4 + T cells after infection with H37Rv or mutant strains (H37RvΔRD1, H37RvΔESAT-6, and Comp ESAT-6) in the presence or absence of anti-IL-1β. IL-4 levels were higher in cultures with H37Rv or Comp ESAT-6 compared with cultures with BCG or other mutant strains ( p < 0.003). C , Western blot analysis of STAT6, pSTAT6, STAT4, and pSTAT4 proteins from DCs infected with H37Rv or H37RvΔESAT-6 and co-cultured with OT-II CD4 + T cells, to show modulated Th1 and Th2 responses in the presence or absence of anti-IL-1β. The results shown are representative of at least four independent experiments. Error bars , ±S.D.

Article Snippet: STAT6, pSTAT6, STAT4, and pSTAT4 proteins were detected with anti-STAT6 (Sc-621), -pSTAT6 (Sc-11762-R), -STAT4 (Sc-486) and -pSTAT4 (Sc-16317-R) polyclonal antibodies, respectively, at a dilution of 1:250 and as recommended by the manufacturer (Santa Cruz Biotechnology).

Techniques: Cell Differentiation, Cell Culture, Infection, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The long noncoding RNA LOC105374325 causes podocyte injury in individuals with focal segmental glomerulosclerosis

doi: 10.1074/jbc.RA118.005579

Figure Lengend Snippet: Role of C/EBPβ in the expression of LOC105374325 in podocytes. A, prediction of transcription factors binding to LOC105374325 promoter region; B, level of LOC105374325 in podocytes treated with ADR, C/EBPβ siRNA, c-JUN siRNA, ERα siRNA, p53 siRNA, STAT1 siRNA, STAT4 siRNA, and XBP-1 siRNA (n = 3); C, level of C/EBPβ and p-C/EBPβ protein in podocytes treated with ADR and SB203580 (n = 3); D, ChIP analysis of the binding between C/EBPβ and LOC105374325 promoter in podocytes treated with ADR (n = 3); E, schematic of the constructed LOC105374325 promoter-luciferase reporter plasmids; F, normalized luciferase activity of reporter constructs in podocytes cotransfected with C/EBPβ plasmid (n = 5); G, level of LOC105374325 in podocytes transfected with C/EBPβ plasmid (n = 5); H and I, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes transfected with C/EBPβ plasmid (n = 5); J, level of Bax and Bak protein in podocytes transfected with C/EBPβ plasmid (n = 3); K, flow cytometric analysis of apoptotic cells in podocytes transfected with C/EBPβ plasmid (n = 5); L and M, RNA pulldown and RT-PCR analysis of the LOC105374325–miR-34c and LOC105374325–miR-196a/b complexes in podocytes treated with ADR and C/EBPβ siRNA (n = 5); N, level of Bax and Bak protein in podocytes treated with ADR and C/EBPβ siRNA (n = 3); O, flow cytometric analysis of apoptotic cells in podocytes treated with ADR and C/EBPβ siRNA (n = 5). For statistical analysis, a two-tailed Student's t test was used for F, G, and K, and one-way ANOVA with Tukey's post hoc test was used for B, H, I, L, M, and O. *, p < 0.05 compared with control; #, p < 0.05 compared with podocytes treated with ADR.

Article Snippet: BAX siRNA (sc-29212), BAK siRNA (sc-29786), C/EBPβ siRNA (sc-44251), c-JUN siRNA (sc-29223), ERα siRNA (sc-29305), p53 siRNA (sc-29435), STAT1 siRNA (sc-44123), STAT4 siRNA (sc-36568), and XBP-1 siRNA (sc-38627) were bought from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Expressing, Binding Assay, Construct, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Interleukin-12p35 Deletion Promotes CD4 T-Cell–Dependent Macrophage Differentiation and Enhances Angiotensin II–Induced Cardiac Fibrosis

doi: 10.1161/atvbaha.112.249706

Figure Lengend Snippet: Figure 3. Phenotypic analysis of cardiac- infiltrated macrophages in wild-type (WT) and p35-knockout (KO) mice with angio tensin II (Ang II) infusion. A, Flow cytometry of F4/80CD206M2 macrophages gated on CD45 cells in WT and p35-KO mouse hearts with saline or Ang II infusion (n=5 per group). *P<0.05 vs WTAng II. B, Immunohistochemistry of expression of CD206 in hearts with saline or Ang II infu sion. Bars=50 m. C, Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of mRNA expression of inducible NO synthase (iNOS), Arg-1, and Fizz1 in hearts with saline or Ang II infusion (n=5 per group). *P<0.05 vs WTAng II. D, Immuno histochemistry of expression of iNOS and Arg-1 in hearts. Bars=100 m. E, Western blot analysis of phosphorylated p65, signal transduction and activator of transcription (STAT) 3, and STAT4 protein in cardiac tis sue with saline or Ang II infusion (left) and quantitative analysis (right, n=3). *P<0.05 vs WTsaline, #P<0.05 vs WTAng II. F, Immunofluorescence analysis of phospho- p65 expression in F4/80macrophages in hearts.

Article Snippet: In total, 50 g protein lysates were separated by 10% SDS-PAGE before transfer to nitrocellulose membranes (Bio-Rad), which were incubated with the primary antibodies anti-α-SMA (1:3000), anti-IL12p40 (1:500), anti-IL23p19 (1:500), anti-IL12p70 (1:500), anti-total or phospho-STAT3, STAT4, or NF-κB p65 (1:1000), anti-TGF-β (1:500), and anti-glyceraldehyde-3-phosphate dehydrogenase (1:5000) at 4°C overnight, and then with infrared Dye-conjugated secondary antibodies (1:5000; Rockland Immunochemicals, Gilbertsville, PA) for 1 hour at room temperature.

Techniques: Knock-Out, Flow Cytometry, Saline, Immunohistochemistry, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transduction, Immunofluorescence

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