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: Genes

Article Title: JCHAIN : A Prognostic Marker Based on Pan-Cancer Analysis to Inhibit Breast Cancer Progression

doi: 10.3390/genes16091070

Figure Lengend Snippet: The JCHAIN inhibits breast cancer progression through IL-2 and STAT4. ( A ) JCHAIN protein expression in the Control group as well as in the OE-JCHAIN group. ( B ) Colony generation experiment showed that JCHAIN inhibited breast cancer colony generation. ( C ) The CCK-8 experiment showed that JCHAIN inhibits cell proliferation. ( D ) Cell scratch experiment showed that JCHAIN inhibited breast cancer cell migration. ( E ) Transwell experiment showed that JCHAIN inhibited breast cancer cell migration and invasive ability. ( F ) Immunoblot analysis of STAT4, p-JAK3, JAK3, and IL-2 protein levels after JCHAIN overexpression in breast cancer cells. ( G ) JCHAIN mRNA expression in normal tissues as well as breast cancer tumour tissues. Student’s t -test. Ns denotes no statistically significant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The following primary antibodies were used: STAT4 (1:1000, item number A4523), p-JAK3 (1:1000, item number AF8160), JAK3 (1:2000, item number AF0008), JCHAIN (1:1000, item number ab269855), IL-2 (1:5000, item number 83558-5RR), β- actin (1:100,000, stock number 81115-1-RR), where β-actin and IL-2 were from Proteintech Group, Inc. (Wuhan, China), STAT4 and JCHAIN were from ABclonal Technology Co., Ltd. (Wuhan, China) and abcam (Shanghai, China), respectively, and p-JAK3 and JAK3 from Affinity Biosciences (Jiangsu, China).

Techniques: Expressing, Control, CCK-8 Assay, Migration, Western Blot, Over Expression