Journal: Cell Communication and Signaling : CCS

Article Title: Aurantio-obtusin modulates Wilms Tumour 1 within the breast tumour microenvironment reducing immunosuppression and tumour growth

doi: 10.1186/s12964-025-02292-y

Figure Lengend Snippet: WT1 regulates the STAT1/3 pathway in CAFs. A Protein levels of WT1, STAT1, STAT3, PD-L1 and the TGFβ receptor were determined in WT1 knockdown ( A ) and WT1 overexpressed ( B ) CAFs by Western blotting. Actin was used as a loading control. IDO release was determined by ELISA. RNA levels of PD-L1 was determined by qRT-PCR. TBP was used as a loading control. Representative experiment is shown of n = 3–5 biological replicates. Data represents mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: The blots were probed with primary antibodies against WT1 (Invitrogen, MA5-38,406; 1:500), STAT1 (Invitrogen, AHO0832; 1:500), STAT3 (Invitrogen, PA5-85,199; 1:500), PD-L1 (R&D systems, AF156; 1:100), TGF-β RI (R&D systems, AF3025; 1:500), p53 (R&D systems, AF1355; 1:1,000), β-actin (Sigma Aldrich, A5441; 1:20,000), and GAPDH (Cell Signaling Technology, 2118S; 1:1,000) overnight at 4 °C and detected by chemiluminescence using Goat anti-Rabbit IgG secondary antibody (Invitrogen, 31,460; 1:5,000), Rabbit anti-Goat IgG secondary antibody (Invitrogen, 31,402; 1:5,000), Goat anti-mouse IgG secondary antibody (Invitrogen, 31,430; 1:5,000).

Techniques: Knockdown, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Cell Communication and Signaling : CCS

Article Title: Aurantio-obtusin modulates Wilms Tumour 1 within the breast tumour microenvironment reducing immunosuppression and tumour growth

doi: 10.1186/s12964-025-02292-y

Figure Lengend Snippet: Chemotherapeutic drugs DOX and PTX increase WT1 levels in CAFs and enhance their ability to inhibit T cell proliferation. A Schematic overview of drug treatment, co-culture of CAFs and PBMCs followed by T cell proliferation assay. B Protein expression of WT1 and p53 in CAFs following DOX and PTX treatment was determined by Western blotting. GAPDH was used as a loading control. C Protein levels of STAT1, STAT3, PD-L1 levels. IDO release from DOX and PTX treated CAFs was determined by ELISA. D DOX treated CAFs were co-cultured with PBMCs and CFSE-labelled CD4 + and CD8 + T cell proliferation was determined by flow cytometry ( E ) PTX treated CAFs were co-cultured with PBMCs and CFSE-labelled CD4 + and CD8. + T cell proliferation was determined by flow cytometry. Representative experiment is shown of n = 3 biological replicates. Data are mean ± SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: The blots were probed with primary antibodies against WT1 (Invitrogen, MA5-38,406; 1:500), STAT1 (Invitrogen, AHO0832; 1:500), STAT3 (Invitrogen, PA5-85,199; 1:500), PD-L1 (R&D systems, AF156; 1:100), TGF-β RI (R&D systems, AF3025; 1:500), p53 (R&D systems, AF1355; 1:1,000), β-actin (Sigma Aldrich, A5441; 1:20,000), and GAPDH (Cell Signaling Technology, 2118S; 1:1,000) overnight at 4 °C and detected by chemiluminescence using Goat anti-Rabbit IgG secondary antibody (Invitrogen, 31,460; 1:5,000), Rabbit anti-Goat IgG secondary antibody (Invitrogen, 31,402; 1:5,000), Goat anti-mouse IgG secondary antibody (Invitrogen, 31,430; 1:5,000).

Techniques: Co-Culture Assay, Proliferation Assay, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Aurantio-obtusin modulates Wilms Tumour 1 within the breast tumour microenvironment reducing immunosuppression and tumour growth

doi: 10.1186/s12964-025-02292-y

Figure Lengend Snippet: AO reduces WT1 levels in CAFs and reduces the ability of CAFs to suppress T cell proliferation. A Comparison of chemical structures of DOX and AO. B Protein levels of WT1, STAT1, STAT3, and PD-L1 after AO treatment of CAFs were determined by Western blotting. Actin was used as a loading control. C Dose–response curves of DOX, PTX and AO treatment of CAFs. P1 and P3 (Luminal A), P2 (TNBC). Curves were generated by non-linear regression analysis by GraphPad. D AO treated CAFs were co-cultured with PBMCs and CFSE-labelled CD4 + and CD8. + T cell proliferation was determined by flow cytometry. Representative experiment is shown of n = 3 biological replicates. Data represent mean ± SEM; * P < 0.05, and ** P < 0.01

Article Snippet: The blots were probed with primary antibodies against WT1 (Invitrogen, MA5-38,406; 1:500), STAT1 (Invitrogen, AHO0832; 1:500), STAT3 (Invitrogen, PA5-85,199; 1:500), PD-L1 (R&D systems, AF156; 1:100), TGF-β RI (R&D systems, AF3025; 1:500), p53 (R&D systems, AF1355; 1:1,000), β-actin (Sigma Aldrich, A5441; 1:20,000), and GAPDH (Cell Signaling Technology, 2118S; 1:1,000) overnight at 4 °C and detected by chemiluminescence using Goat anti-Rabbit IgG secondary antibody (Invitrogen, 31,460; 1:5,000), Rabbit anti-Goat IgG secondary antibody (Invitrogen, 31,402; 1:5,000), Goat anti-mouse IgG secondary antibody (Invitrogen, 31,430; 1:5,000).

Techniques: Comparison, Western Blot, Control, Generated, Cell Culture, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Aurantio-obtusin modulates Wilms Tumour 1 within the breast tumour microenvironment reducing immunosuppression and tumour growth

doi: 10.1186/s12964-025-02292-y

Figure Lengend Snippet: Proposed model highlighting how WT1 in CAFs modulates immune evasion and tumour growth in the breast TME. WT1 is upregulated in breast patient-derived CAFs. WT1 is a transcription factor, regulating expression of STAT1/STAT3 in CAFs. STAT1/STAT3 regulate PD-L1 expression and IDO release in CAFs. Both PD-L1 and IDO inhibit T cell activity and cytotoxic GZMB levels within the TME, contributing to increased tumour growth. Targeting WT1 in CAFs reduces the release of immunosuppressive factors into the TME. T cells are more active and are able to limit tumour growth

Article Snippet: The blots were probed with primary antibodies against WT1 (Invitrogen, MA5-38,406; 1:500), STAT1 (Invitrogen, AHO0832; 1:500), STAT3 (Invitrogen, PA5-85,199; 1:500), PD-L1 (R&D systems, AF156; 1:100), TGF-β RI (R&D systems, AF3025; 1:500), p53 (R&D systems, AF1355; 1:1,000), β-actin (Sigma Aldrich, A5441; 1:20,000), and GAPDH (Cell Signaling Technology, 2118S; 1:1,000) overnight at 4 °C and detected by chemiluminescence using Goat anti-Rabbit IgG secondary antibody (Invitrogen, 31,460; 1:5,000), Rabbit anti-Goat IgG secondary antibody (Invitrogen, 31,402; 1:5,000), Goat anti-mouse IgG secondary antibody (Invitrogen, 31,430; 1:5,000).

Techniques: Derivative Assay, Expressing, Activity Assay