Journal: iScience

Article Title: Functional characterization of CASP, a CUX1 isoform, reveals its tumor-promoting role in colorectal cancer via TRIM21-mediated signaling

doi: 10.1016/j.isci.2026.114783

Figure Lengend Snippet: CASP impedes MAPK signaling by negatively regulating TRIM21 protein stability (A) Immunoblot analysis of TRIM21 immunoprecipitated using an FLAG tag fused with CASP in 293T cells. (B) Immunoblot analysis of CASP immunoprecipitated using a His tag fused with TRIM21 in 293T cells. (C) Immunoblot validation of endogenous co-precipitation of TRIM21 with CASP antibody in DLD-1and LoVo cells. (D) Co-IP analysis with an anti-His antibody was performed in DLD-1 cells 48h after transfection with constructs expressing the His-tagged full-length, N-terminal cytoplasmic, or C-terminal Golgi domain of CASP (or an empty vector), to assess their respective interactions with TRIM21. (E and F) qPCR (E) and WB (F) analyses of TRIM21 expression at the mRNA and protein levels, respectively, in DLD-1 cells following CASP knockdown or overexpression compared to their respective controls. (G) Quantification of TRIM21 protein expression from the WB shown ( C), which displays TRIM21 and CASP levels in protein extracts from subcutaneous xenograft tumors of nude mice. Band intensities for TRIM21 were normalized to β-actin (loading control) using ImageJ software. (H) 293T cells were co-transfected with plasmids encoding HA-Ub, His-TRIM21, and control or shCASP-1. Cells were treated with MG132 for 6 h prior to lysis. His-TRIM21 was immunoprecipitated from cell lysates using Ni-NTA beads, and its ubiquitination level was detected by immunoblotting with anti-HA antibody. (I and J) Turnover of TRIM21 protein was assessed in scrambled and CASP-KD DLD-1 cells treated with 60 μg/mL CHX for the indicated times. Representative blots (I) and quantification (J) demonstrating that CASP depletion significantly slows the degradation rate of TRIM21. (K and L) c-Myc turnover was analyzed in parallel under the similar conditions. Representative blots (K) and quantification (L) revealing that CASP knockdown accelerates the degradation kinetics of c-Myc. Protein levels were normalized to GAPDH and are presented as the percentage remaining relative to time 0. (M) DLD-1 cells were treated with the proteasome inhibitor MG132 (20 μM) for the indicated times (0, 1, 2, 4, 6, and 8 h). WB analysis was performed to monitor the stabilization of TRIM21 protein. (N) Alterations in the proliferative capacity of CASP-KD DLD-1 cells and their control counterparts after transient transfection with TRIM21 siRNA. Immunoblot analysis showing the knockdown efficiency of TRIM21(up-left); Cell viability measured on days 1, 3, 5, and 7 using the CCK-8 assay(up-right); Schematic representation of the cell colony formation assay (down-left); Statistical graph showing the number of colonies for each group (down-right). (O) Alterations in key molecules of the MAPK pathway in control and CASP-KD DLD-1 cells following TRIM21 downregulation. Data are expressed as mean ± SD. Statistical analyses were performed on GraphPad Prism. (E, G, and N) Unpaired t test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns = no significant difference.

Article Snippet: Ni-NTA Magnetic Agarose Beads , MedChemExpress , Cat# HY-K0241.

Techniques: Western Blot, Immunoprecipitation, FLAG-tag, Biomarker Discovery, Co-Immunoprecipitation Assay, Transfection, Construct, Expressing, Plasmid Preparation, Knockdown, Over Expression, Control, Software, Lysis, Ubiquitin Proteomics, CCK-8 Assay, Colony Assay