Journal: mBio

Article Title: PCV2 infection induces the differentiation of Treg cells via the TGF-β/Smad3 pathway

doi: 10.1128/mbio.01366-25

Figure Lengend Snippet: ( A ) PCV2 infection induced Treg cell differentiation through the TGF-β/Smad3 pathway. Detection of the mRNA levels of IL-2 and TGF-β. PBMCs were isolated from peripheral blood at multiple time points post-challenge, and the mRNA expression levels were quantified by qPCR. ( B ) Detection of the expression levels of IL-2 and TGF-β. All animals were euthanized at 35 dpi, and cytokine levels were measured by ELISA. ( C ) The frequency of Treg cells. T cells were stimulated with various cytokines and analyzed for Treg cell frequency by flow cytometry. Unless otherwise specified, T cell stimulations lasted five days in vitro . ( D ) The MFI of Foxp3 in CD4 + Foxp3 + double-positive cells. Foxp3 expression in Treg cells was quantified by calculating the arithmetic MFI using flow cytometry. ( E ) The subcellular localization of Smad3 in Treg cells. ( F ) Detection of phosphorylation level of Smad3. Total proteins were extracted from T cells and analyzed by Western blot for target protein expression. ( G ) pGL3-promoter-Foxp3 schematic diagram. ( H ) The double luciferase assay. The assay was used to investigate the effect of Smad3 on the activity of the Foxp3 enhancer. ( I ) Quantification of Foxp3 mRNA expression levels. ( J ) The frequency of Treg cells. ( K ) The MFI of Foxp3 in CD4 + Foxp3 + double-positive cells.

Article Snippet: SIS3, a specific Smad3 phosphorylation inhibitor (HY-13013), and SB-431542, a TGF-β receptor inhibitor (HY-10431), were purchased from MedChemExpress.

Techniques: Infection, Cell Differentiation, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, In Vitro, Phospho-proteomics, Western Blot, Luciferase, Activity Assay

Journal: mBio

Article Title: PCV2 infection induces the differentiation of Treg cells via the TGF-β/Smad3 pathway

doi: 10.1128/mbio.01366-25

Figure Lengend Snippet: The schematic diagram indicates that PCV2 infection induces the differentiation of Treg cells via the TGF-β/Smad3 pathway. PCV2 infection markedly enhances TGF-β secretion from host cells, subsequently activating the Smad3 signaling pathway in T cells. Phosphorylated Smad3 translocates into the nucleus, specifically binds to the Foxp3 gene enhancer region, and significantly increases Foxp3 transcription, ultimately promoting Treg cell differentiation. Notably, treatment with the Smad3-specific inhibitor SIS3 and TGF-β receptor inhibitor SB-431542 effectively inhibits Treg differentiation, confirming the crucial role of this pathway in PCV2-induced Treg cell differentiation.

Article Snippet: SIS3, a specific Smad3 phosphorylation inhibitor (HY-13013), and SB-431542, a TGF-β receptor inhibitor (HY-10431), were purchased from MedChemExpress.

Techniques: Infection, Cell Differentiation

Journal: Investigative ophthalmology & visual science

Article Title: The TCF4 Gene Regulates Apoptosis of Corneal Endothelial Cells in Fuchs Endothelial Corneal Dystrophy.

doi: 10.1167/iovs.66.3.16

Figure Lengend Snippet: FIGURE 6. Effect of TCF4 knockout on TGF-β2–mediated ECM production and apoptosis. (A) The bHLH region or 20 bases in exon 9 of TCF4 in iFECD were knocked out using CRISPR/Cas9 (iFECD TCF4−/−, iFECD TCF4bHLH). Cells were cultured in serum-free medium for 24 hours, then treated with or without TGF-β2 (10 ng/mL) for 24 hours. Phase-contrast images show that iFECD forms a monolayer with polygonal morphology. TGF-β2–induced cell death in iFECD but not in iFECD TCF4−/−or iFECD TCF4bHLH. Scale bar: 200 μm. (B) Sanger sequencing confirmed the deletion of 20 bases in exon 9 of TCF4 in iFECD TCF4−/−. Red lines indicate the deleted bases. (C) Western blot- ting showed TGF-β2–induced cleavage of caspase-3 and PARP in iFECD, which was suppressed in iFECD TCF4−/−and iFECD TCF4bHLH. (D) Flow cytometric analysis of Annexin V–positive apoptotic cells in response to TGF-β2 treatment. TGF-β2 treatment substantially increased the percentage of Annexin V–positive cells to 31.4% ± 2.0% in iFECD cells. Both iFECD TCF4−/−and iFECD TCF4bHLH cells demonstrated resistance to TGF-β2–induced apoptosis, showing lower percentages of Annexin V–positive cells (19.8% ± 1.3% and 18.0% ± 1.6%, respec- tively; P = 5.28 × 10−2 and P = 3.02 × 10−2, compared to TGF-β2–treated iFECD). Data are presented as mean ± SEM from three independent experiments. (E) Western blotting confirmed suppression of TCF4 in both iFECD TCF4−/−and iFECD TCF4bHLH. TGF-β2 upregulated Snail1 in iFECD, but this upregulation was suppressed in both mutant cell lines. ZEB1 expression was unaffected by TGF-β2 in all cell lines. Fibronectin levels increased in iFECD but not in either iFECD TCF4−/−or iFECD TCF4bHLH with TGF-β2 treatment. (F) Phosphorylation of Smad2 and Smad3 by TGF-β2 was confirmed in both iFECD and iFECD TCF4−/−, while this phosphorylation was suppressed in iFECD TCF4bHLH. All experiments were performed independently at least three times with reproducible results.

Article Snippet: The proteins were then separated by SDS-PAGE and transferred onto PVDF membranes, which were then blocked with 3% nonfat dry milk for 1 hour at room temperature and incubated overnight at 4°C with primary antibodies against cleaved caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), cleaved poly (ADP-ribose) polymerase (cleaved PARP) (1:1000; Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3000; Medical & Biological Laboratories Co., Ltd., Tokyo, Japan), TCF455 (1:500), Snail1 (1:1000; Cell Signaling Technology), ZEB1 (1:1000; Cell Signaling Technology), fibronectin (1:20,000; BD Biosciences), phosphorylated Smad3 (p-Smad3) (1:1000; Cell Signaling Technology), Smad2 (1:1000; Cell Signaling Technology), phosphorylated Smad2 (p-Smad2) (1:1000; Cell Signaling Technology), and Smad3 (1:1000; Cell Signaling Technology).

Techniques: Knock-Out, CRISPR, Cell Culture, Sequencing, Western Blot, Mutagenesis, Expressing, Phospho-proteomics