Journal: iScience

Article Title: MFAP5 + synovial fibroblasts drive LOX upregulation to promote osteoarthritis progression

doi: 10.1016/j.isci.2026.116286

Figure Lengend Snippet: MFAP5 + SFs promoted collagen deposition in synovium (A) Dendrogram showing the hierarchy organization of MEGENA network modules in GSE176308 . The top layer represented the root and largest parent modules, which branched hierarchically into smaller child modules. (B) Density plots displaying c1_9 module scores across SF clusters, with color-coded density values; red indicated higher density, and blue indicated lower density. (C) Network of pathways and genes involved in the c1_9 module, where a two-point line indicated that a gene was part of a specific pathway. (D) Raincloud plot showing c1_9 score in GSE152805 . (E and F) Gene set enrichment analysis showed collagen pathways were upregulated in MFAP5 + SFs in both GSE176308 (E) and GSE152805 (F). (G) Correlation coefficients between the expression of MFAP5 and one ECM pathway score across three datasets. (H) Violin plots of ECM binding (left) and ECM organization (right) scores across SF clusters in GSE176308 . Color-coded by SF clusters. (I) Density plots of ECM binding and ECM organization scores across SF clusters in SCP469 and GSE152805 . (J) Comparison of significant ligand-receptor pairs from SF clusters to T cells in GSE152805 . (K) Immunofluorescence images of MFAP5, CD4, and vimentin colocalization in synovial tissues. Scale bars, 200 or 20 μm. Statistical significance was calculated using two-tailed Wilcoxon rank-sum test (D and H) and Spearman's rank correlation analysis (G). ∗∗∗ p < 0.001.

Article Snippet: Sections were then incubated overnight at 4 °C with primary antibodies, including vimentin (GB11192, Servicebio, China), FBN1 (860327, Zenbio, China), MFAP5 (15727-1-AP, Proteintech, China), and CD4 (GB150062-50, Servicebio, China).

Techniques: Expressing, Binding Assay, Comparison, Immunofluorescence, Two Tailed Test

Journal: Molecular Therapy Oncology

Article Title: Engineered Salmonella -mediated c-di-AMP delivery activates STING to remodel the tumor microenvironment

doi: 10.1016/j.omton.2026.201185

Figure Lengend Snippet: Effect of SL disA therapy on T cells and macrophage from tumor samples (A, B) Flow Cytometry analysis of CD3 and CD8 surface markers(A) and CD3 + CD8 + cells statistical graph (B). (C, D) Flow cytometry analysis of CD3 and CD4 surface markers (C) and CD3 + CD4 + cells statistical graph (D). (E, F) Flow cytometry analysis of F4/80 and CD86 surface markers (E) and F4/80 + CD86 + cells statistical graph (F). Data are expressed as mean ± SEM, n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons tests.

Article Snippet: The following antibodies were used: FITC anti-mouse F4/80 (clone CI: A3-1), APC anti-mouse CD86 (clone GL-1), FITC anti-mouse CD3 (clone 17A2), APC anti-mouse CD4 (clone GK1.5), and APC anti-mouse CD8 (clone YTS-169), all purchased from Elabscience Biotechnology Co., Ltd.

Techniques: Flow Cytometry

Journal: Science Advances

Article Title: SRSF3 determines T reg cell fate in antitumor immunity and autoimmunity

doi: 10.1126/sciadv.aeh1671

Figure Lengend Snippet: ( A ) RNA splicing of human FOXP3 and mouse Foxp3 pre-mRNA. ( B ) Alignment of human FOXP3 (hFOXP3) and mouse Foxp3 (mFoxp3) exon 2 and nearby 50-bp intron sequences. ( C ) Mutations in mouse intron according to corresponding human sequences. Minigene plasmids were transfected into 293 cells. Exon 2 splicing was analyzed by RT-PCR. ( D ) A minigene system contains human genomic sequence from exon 1 to exon 3 of FOXP3 gene (wt). The corresponding sequence of mt4 was mutated to mouse sequence (mmt). Exon 2 splicing was analyzed by RT-PCR. ( E ) The Foxp3 humanized mouse (Foxp3-Hu) was generated by replacing C57BL/6N mouse Foxp3 exon 2 and adjacent 50-bp intron sequences with human corresponding sequences. ( F ) Foxp3 exon 2 skipping in Foxp3-Hu T reg cells was confirmed by RT-PCR and DNA sequencing. ( G ) Total FOXP3 (all-Foxp3) or full-length FOXP3 (full-Foxp3) expression in T reg cells was analyzed by flow cytometry. Full-length FOXP3 protein is recognized by FJK-16s antibody. Total FOXP3 protein is recognized by NRRF-30 antibody. The histograms showed quantification of T reg cells and the mean fluorescent intensity (MFI) of total FOXP3 or full-length FOXP3 ( n = 3). ( H to J ) A total of 0.5 × 10 5 MC38 cells were injected subcutaneously into left axilla of humanized or WT mice. Tumor sizes were measured every 2 to 3 days. * P < 0.05 and ** P < 0.01. [(I) and (J)] Mice were euthanized at day 28, and tumors were isolated and weighted. ( K and L ) A total of 2 × 10 5 MC38 cells were injected subcutaneously into humanized or WT mice. Mice were euthanized at the humane endpoints. ( L ) The intratumoral populations of CD4 + , CD8 + , or T reg cells were analyzed with flow cytometry. Histogram summarized amounts of intratumoral CD8 + and T reg cells and total FOXP3 MFI in T reg cells. Statistical significance was determined by an unpaired t test or a Mann-Whitney test. Survival analysis was performed with a log-rank test. n.s., not significant.

Article Snippet: Mouse cells were stained with eFluor 660–conjugated rat anti-mouse Foxp3 (FJK-16s; 50-5773-80; eBioscience, recognizing full-length Foxp3 protein), PE-conjugated rat anti-mouse Foxp3 (NRRF-30; 12-4771-82; eBioscience, recognizing all Foxp3 proteins), fluorescein isothiocyanate (FITC)–conjugated rat anti-mouse CD4 (RM4-5; 11-0042-85; Invitrogen), allophycocyanin (APC)-conjugated rat anti-mouse CD8a (53-6.7; 100711; BioLegend), Alexa Fluor 700–conjugated hamster anti-mouse TCRβ (H57-597; 109224; BioLegend), eFluor 660–conjugated rat IgG2a isotype antibody (eBR2a; 50-4321-82; eBioscience), FITC-conjugated rat IgG2a isotype antibody (eBR2a; 11-4321-82; eBioscience), Alexa Fluor 700–conjugated hamster IgG isotype (HTK888; 400926; BioLegend), APC-conjugated rat IgG2a isotype antibody (RTK2758; 400511; BioLegend), PE-conjugated rat IgG2a isotype antibody (eBR2a; 12-4321-80; eBioscience), APC-conjugated rat anti-mouse CD44 (IM7; 17-0441-81; eBioscience), APC-conjugated rat IgG2b isotype antibody (eB149/10H5; 17-4031-82; eBioscience), and PE-conjugated rat anti-mouse CD62L (MEL-14; 12-0621-81; eBioscience).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Sequencing, Generated, DNA Sequencing, Expressing, Flow Cytometry, Injection, Isolation, MANN-WHITNEY

Journal: Science Advances

Article Title: SRSF3 determines T reg cell fate in antitumor immunity and autoimmunity

doi: 10.1126/sciadv.aeh1671

Figure Lengend Snippet: ( A to H ) Single cells were isolated from fresh cancer or adjacent normal tissues. Cells were labeled with anti-CD4 and anti-TCR antibodies, followed by intracellular anti-FOXP3 and anti-SRSF3 labeling. [(A) and (E)] Gating strategy to identify TCR + CD4 + FOXP3 + T reg cells and representative fluorescence-activated cell sorting (FACS) plots showing the expression levels of FOXP3 and SRSF3 in T reg cells isolated from oral squamous cell carcinoma (A) and breast cancer (E) or their adjacent normal tissues, respectively. [(B) and (F)] Summary of SRSF3-positive population percentage of T reg cells in oral squamous cell carcinoma (A) or breast cancer (E) tissues. [(C), (D), (G), and (H)] Summary of FOXP3 and SRSF3 MFI of T reg cells in oral squamous cell carcinoma [(C) and (D)] ( n = 5) or breast cancer [(G) and (H)] ( n = 8) tissues. Data are mean ± SEM. ( I ) Human T reg cells were purified from PBMCs and then transfected with siRNA [anti-SRSF3 or nonspecific (NS)]. PBMCs from the same donor were labeled by CFSE and mixed with T reg cells as the indicated ratio. Cells were cultured for 4 days in the presence of anti-human CD3 antibody (0.5 μg/ml). Then, cells were stained with an anti-CD8 antibody. The proliferation of CD8 + cells was measured by FACS. Data are mean ± SEM, n = 3. ( J ) Down-regulation of SRSF3 released the inhibition of T reg cell on the expression of TNF-α, IFN-γ, and IL-2 by CD8 + T cells. The expression levels of TNF-α, IFN-γ, and IL-2 in CD8 + T cells after in vitro suppression assay were analyzed by intracellular cytokine staining and FACS. Data are mean ± SEM, n = 5. P values are from a two-sided unpaired t test [(I) and (J)] or a paired t test [(B), (C), (D), (F), (G), and (H)].

Article Snippet: Mouse cells were stained with eFluor 660–conjugated rat anti-mouse Foxp3 (FJK-16s; 50-5773-80; eBioscience, recognizing full-length Foxp3 protein), PE-conjugated rat anti-mouse Foxp3 (NRRF-30; 12-4771-82; eBioscience, recognizing all Foxp3 proteins), fluorescein isothiocyanate (FITC)–conjugated rat anti-mouse CD4 (RM4-5; 11-0042-85; Invitrogen), allophycocyanin (APC)-conjugated rat anti-mouse CD8a (53-6.7; 100711; BioLegend), Alexa Fluor 700–conjugated hamster anti-mouse TCRβ (H57-597; 109224; BioLegend), eFluor 660–conjugated rat IgG2a isotype antibody (eBR2a; 50-4321-82; eBioscience), FITC-conjugated rat IgG2a isotype antibody (eBR2a; 11-4321-82; eBioscience), Alexa Fluor 700–conjugated hamster IgG isotype (HTK888; 400926; BioLegend), APC-conjugated rat IgG2a isotype antibody (RTK2758; 400511; BioLegend), PE-conjugated rat IgG2a isotype antibody (eBR2a; 12-4321-80; eBioscience), APC-conjugated rat anti-mouse CD44 (IM7; 17-0441-81; eBioscience), APC-conjugated rat IgG2b isotype antibody (eB149/10H5; 17-4031-82; eBioscience), and PE-conjugated rat anti-mouse CD62L (MEL-14; 12-0621-81; eBioscience).

Techniques: Isolation, Labeling, Fluorescence, FACS, Expressing, Purification, Transfection, Cell Culture, Staining, Inhibition, In Vitro, Suppression Assay

Journal: Science Advances

Article Title: SRSF3 determines T reg cell fate in antitumor immunity and autoimmunity

doi: 10.1126/sciadv.aeh1671

Figure Lengend Snippet: ( A ) Srsf3-flox mice were crossed with the Foxp3 YFP-cre mice to produce T reg cell–specific Srsf3-KO mice, including both Foxp3 YFP-Cre Srsf3 flox/flox homozygous (Srsf3-cKO) and Foxp3 YFP-cre Srsf3 flox/+ heterozygous (Srsf3 +/− ) KO mice. ( B ) Genotyping of Srsf3 gene KO in Srsf3-cKO mice. ( C ) T reg cells of Srsf3-cKO mice were isolated and checked for genomic deletion of Srsf3 gene by PCR (the presence of cleaved DNA fragment). CD8 + T cells were used as the non-KO control. WT are WT mice control. CD4 gene was used as DNA template control. ( D ) Srsf3-cKO and Srsf3 +/− mice at postnatal day 28. ( E ) Srsf3-cKO mice showed significant lower body weight than Srsf3 +/− mice at day 21. ( F ) Survival analysis of Srsf3-cKO and Srsf3 +/− mice. ( G ) FACS analyses of Srsf3 expression and population of T reg cells in the thymuses of Srsf3-cKO and Srsf3 +/− mice. ( H ) Serum anti-dsDNA antibody levels in Srsf3 cKO or Srsf3 +/− mice were analyzed by enzyme-linked immunosorbent assay. ( I ) Representative images (specimens and/or hematoxylin and eosin staining) of skin, thymus, spleen, lymph node, and liver from Srsf3-cKO and Srsf3 +/− mice. Histograms show thymus weight ( n = 4), spleen weight/body weight ( n = 4), and lymph node weight ( n = 4). Scale bar, 20 μm. ( J ) T reg cells in spleens or lymph nodes from Srsf3-cKO and Srsf3 +/− mice ( n = 4 or 5). ( K ) The expression levels of CD62L and CD44 in CD8 + T cells from the spleens or lymph nodes of Srsf3-cKO and Srsf3 +/− mice ( n = 3). Statistical significance was determined by a two-sided unpaired t test. Survival analysis was performed with a log-rank test.

Article Snippet: Mouse cells were stained with eFluor 660–conjugated rat anti-mouse Foxp3 (FJK-16s; 50-5773-80; eBioscience, recognizing full-length Foxp3 protein), PE-conjugated rat anti-mouse Foxp3 (NRRF-30; 12-4771-82; eBioscience, recognizing all Foxp3 proteins), fluorescein isothiocyanate (FITC)–conjugated rat anti-mouse CD4 (RM4-5; 11-0042-85; Invitrogen), allophycocyanin (APC)-conjugated rat anti-mouse CD8a (53-6.7; 100711; BioLegend), Alexa Fluor 700–conjugated hamster anti-mouse TCRβ (H57-597; 109224; BioLegend), eFluor 660–conjugated rat IgG2a isotype antibody (eBR2a; 50-4321-82; eBioscience), FITC-conjugated rat IgG2a isotype antibody (eBR2a; 11-4321-82; eBioscience), Alexa Fluor 700–conjugated hamster IgG isotype (HTK888; 400926; BioLegend), APC-conjugated rat IgG2a isotype antibody (RTK2758; 400511; BioLegend), PE-conjugated rat IgG2a isotype antibody (eBR2a; 12-4321-80; eBioscience), APC-conjugated rat anti-mouse CD44 (IM7; 17-0441-81; eBioscience), APC-conjugated rat IgG2b isotype antibody (eB149/10H5; 17-4031-82; eBioscience), and PE-conjugated rat anti-mouse CD62L (MEL-14; 12-0621-81; eBioscience).

Techniques: Isolation, Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining

Journal: Dose-Response

Article Title: The Effects of Tibetan Medicine Renqing Changjue Extracts on Cyclophosphamide-Induced Immunosuppression in a Mouse Model

doi: 10.1177/15593258261454535

Figure Lengend Snippet: The effect of RQCJ on the proportion of CD4 + T and CD8 + T cells. (A) CD4 + /CD8 + T ratio in the different groups; (B-F) The flow cytometric analysis of CD4 + T and CD8 + T cells. Compared with the control group: ## p < 0.01. Compared with the CTX group: * p < 0.05, * * p < 0.01. (n = 12, one-way ANOVA was used for data analysis).

Article Snippet: We transferred 100 μL cell suspensions into flow cytometry tubes and added 10 μL of anti-mouse CD4 + T antibodies (FITC anti-mouse CD4, Clone: RM4-5, Concentration: 0.5 mg/mL) and CD8 + T antibodies (PE anti-mouse CD8a, Clone: 53-6.7, Concentration: 0.2 mg/mL) labeled with different fluorochromes (Dakewe, China).

Techniques: Control