Journal: Chemmedchem

Article Title: 4‐(5‐Chloro‐3‐(3,4,5‐trimethoxybenzoyl)‐1 H ‐indol‐1‐yl)benzenesulfonamide: A Novel Polypharmacology Agent to Target Carbonic Anhydrase IX and XII With Improved Selectivity, Wnt/β‐Catenin Signaling Pathway, and P‐Glycoprotein

doi: 10.1002/cmdc.202500996

Figure Lengend Snippet: Compound 15 inhibits the β‐catenin/TCF‐4 interaction. A coimmunoprecipitation assay was performed in HEK293T cells transiently transfected with FLAG‐tagged β‐catenin (β‐catenin FLAG ) and MYC‐tagged TCF‐4 (TCF‐4 MYC ). Cells were treated with 50 mM LiCl and 60 μM 15 for 24 h. β‐Catenin FLAG was immunoprecipitated from total protein extracts using anti‐FLAG‐conjugated agarose beads (left panel; IP: immunoprecipitation). Coimmunoprecipitated proteins were analyzed by Western blotting using an anti‐MYC‐tag antibody to detect TCF‐4 MYC (left panel; IB: immunoblot). Input shows the levels of TCF‐4 MYC and β‐catenin FLAG in 5% whole‐cell lysates before immunoprecipitation (right panel).

Article Snippet: HEK293T cells were transfected with pcDNA/Myc TCF‐4 (Addgene #16512) and human β‐catenin pcDNA3 (#16828 Addgene).

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Apoptosis

Article Title: SREBP2 confers ferroptosis resistance by targeting GPX4 in colorectal cancer

doi: 10.1007/s10495-025-02209-7

Figure Lengend Snippet: β-catenin transcriptionally upregulated the expression of SREBP2. A, B. HT29 cells transfected with si-β-catenin or si-NC (as control). The levels of β-catenin, SREBP2 and GPX4 were examined by real-time PCR. B . The protein levels of total β-catenin, active-β-catenin, SREBP2 and GPX4 were determined by western blotting. C . The sequence logo of a potential TCF4 binding site (TBS) predicted by JASPAR database. The schematic diagram of the predicted binding site in the SREBP2 promoter sequence (from − 1628 to − 1621 bp) and the corresponding site-directed mutation were showed. D , E . ChIP-qPCR assay was performed using anti-β-catenin or control IgG antibody in HT29 cells. Agarose gel electrophoresis analysis of ChIP-qPCR products D . Analysis and visualization of ChIP-qPCR result ( E ). F . HT29 cells were transfected with luciferase reporter plasmids containing SREBP2 promoter (wild/mute type) and further transfected with si-β-catenin and si-NC (as control). The luciferase activity was measured and normalized. G, H . ChIP-qPCR assay was performed using anti-TCF4 or control IgG antibody in HT29 cells. Agarose gel electrophoresis analysis of ChIP-qPCR products G . Analysis and visualization of ChIP-qPCR result H . I . The interaction between β-catenin and TCF4 was examined by Co-IP. All the results represent mean ± SEM from at least three independent experiments. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The membrane was then blocked with 5% non-fat milk for one hour and incubated with the primary antibody, including anti-SREBP2 (1:2000; 28212-1-AP, Proteintech), anti-GPX4 (1:1000; sc-166570, santa), anti-ACSL4 (1:5000; 22401-1-AP, Proteintech), anti-SLC7A11 (1:2000; 26864-1-AP, Proteintech), anti-β-catenin (1:1000, #8480, CST), anti-active-β-catenin (1:1000, #19807, CST), anti-active-β-catenin (1:1000, #19807, CST), anti-β-ACTIN antibody (1:2000, sc-376421, Santa Cruz), anti-TCF4 antibody (1:2000, sc-166699, Santa Cruz), overnight at 4 °C.

Techniques: Expressing, Transfection, Control, Real-time Polymerase Chain Reaction, Western Blot, Sequencing, Binding Assay, Mutagenesis, ChIP-qPCR, Agarose Gel Electrophoresis, Luciferase, Activity Assay, Co-Immunoprecipitation Assay