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

Journal: PLoS ONE

Article Title: Disassembly of Shank and Homer Synaptic Clusters Is Driven by Soluble β-Amyloid 1-40 through Divergent NMDAR-Dependent Signalling Pathways

doi: 10.1371/journal.pone.0006011

Figure Lengend Snippet: Cultured cortical neurons were pre-treated for 45 min with the indicated pharmacological inhibitors before being treated for 1 h with Aβ (1 µM). Inhibitors were present in the medium during Aβ treatment. (A) PI-3K/mTOR pathway is required for Homer1b declustering. Pre-treatment with wortmannin (Wort, 2 µM) abolished the ability of Aβ to disperse Homer1b clusters (101.1±15.3%, cf. Aβ+Wort and Wort alone). Likewise, inhibition of the PI-3K downstream kinases PKB (with API-2, 30 µM) or mTor (with rapamycin, Rapa, 5 µM) prevented Aβ-induced declustering of Homer1b (90.6±8.4%, cf. Aβ+AP2 and API-2 alone; 95±8.2%, cf. Aβ+Rapa and rapamycin alone). Either the blockade of MEK/ERK pathway by pre-treatment with UO126 (10 µM; 70.9±4.8% UO126+Aβ vs UO126 alone) or cdk-5 inhibition by roscovitine (10 µM;63.7±5.8%, cf. Aβ+Rosco and Rosco alone) pre-treatment did affect Aβ-induced Homer declustering. (B) MEK/ERK pathway, but not PI-3K pathway, is required for Aβ-induced dispersal of Shank1. Pre-treatment with the PI-3K inhibitor wortmannin before Aβ treatment did not affect Aβ-induced Shank cluster dispersal (64.7±10.3%, cf. Wort+Aβ and Wort alone; p<0.05). Notably, wortmannin itself decreased Shank1 (but not Homer1b) cluster size (74.3±10.3%, cf. Wort and vehicle; p<0.05). Two structurally unrelated MEK inhibitors, UO126 (10 µM) and PD98059 (25 µM) prevented Aβ-induced declustering of Shank1 (96.8±12.1%, cf. PD98059+Aβ and PD98059 alone; 92.2±15.4%, cf. UO126+Aβ and UO126 alone). Likewise, the blockade of the Erk targeted kinase RSK by pre-treatment with SL0101-1 blocked Aβ effects (100.4±9.8%, p>0.05). Pre-treatment of neurons with the specific cdk-5 inhibitor roscovitine (Rosco, 10 µM) did not interfere with the ability of Aβ to reduce the size of Shank1 (72.6±10.8%, cf. Rosco+Aβ and Aβ alone) clusters. (C,D) PP2B is required for the dispersal of Homer1b, but not Shank1, clusters by Aβ. Pre-treatment with structurally unrelated PP2B inhibitors FK506 (10 µM) or cyclosporin (Cyclo, 100 µM) significantly blocked (p<0.05) Aβ-induced Homer1b declustering (98.8±10.4%, comparing FK506+Aβ vs. Aβ alone; 83.4±5.8%, comparing Aβ+Cyclo and Cyclo alone; and 61±7.9% in vehicle+Aβ-treated cells; see panel C ); however, all PP2B inhibitors were ineffective in preventing Shank1 declustering after Aβ exposure (61.3±4.5%, cf. FK506+Aβ and FK506 alone; 90.8±12.4%, cf. Cyclo+Aβ and Cyclo alone, see panel D ). For each condition, 8–12 neurons from 3–4 replicate experiments were considered (N = 8–12), and at least 50 synapses on each neuron were evaluated (on average, n = 500 puncta/condition).

Article Snippet: Nifedipine, (±)-verapamil, roscovitine, NiCl 2 , cycloheximide, cyclosporin and FK506 were purchased from Sigma Chemicals (Deisenhofen, Germany); NMDA, Bay-K4688, SL0101-1, MK801, UO126, PD98059, API-2, wortmannin, cantharidine were from Tocris (Bristol, UK); and sodium orthovanadate, rapamycin, TDZT, SU6656 and MG132 were from Calbiochem (La Jolla, CA).

Techniques: Cell Culture, Inhibition