Fig 2: ABLIM1 is aberrantly overexpressed in CRCs and augmented expression correlates with poor disease-free survival.A Representative ABLIM1 immunohistochemistry staining images of the CRC samples and adjacent normal counterparts in a tissue microarray. B Semiquantitative analysis results of immunohistochemistry staining images based on the H-score method. Data were shown as Mean ± SEM. ***P < 0.001 by paired student’s t-test. C Relative mRNA levels of ABLIM1 in 44 cases of freshly frozen CRC samples and their paired normal control mucosae. P = 0.007 determined by paired t-test. D ABLIM1 mRNA expressions in two datasets (GSE20916 and GSE5206) of the GEO database. Data were shown as Mean ± SEM. Unpaired student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001. E Relative mRNA levels of ABLIM1 in a normal human colon epithelial cell line, HCoEpic, and 5 human CRC cell lines, including HCT116, RKO, SW480, SW620, and Lovo. N = 3 for each cell line. Kruskal–Wallis test. F ABLIM1 protein levels in HCoEpic and 5 human CRC cell lines were assayed by immunoblotting. Kaplan-Meier disease-free survival curves stratified by ABLIM1 expression levels using data downloaded from the CBioPortal database (G, TCGA PanCancer Atlas; H, TCGA Firehose Legacy). G P = 0.034, hazard ratio = 2.34 [1.04–5.33, 95% confidence interval (CI)]. H, P = 0.024, hazard ratio = 1.65 [1.08–2.50, 95% CI].

Fig 3: IκBα/NF-κB p65 intervention reversed the phenotypes induced by ABLIM1 alteration.A Western blots for p65, p-P65, IκBα, p-IκBα, and ABLIM1 in HCT116 cells overexpressed with ABLIM1 or empty vector. Cells were treated with BAY11-7082 (10 μM) or DMSO control for 8 h before harvest. B Effect of BAY11-7082 treatment (10 μM, 24 h) on cell viabilities of HCT116 cells overexpressed with ABLIM1 or empty vector was detected by CCK-8 kit. n = 3 for each group. Kruskal–Wallis test. Influence of BAY11-7082 treatment on cell migration C and invasion D capabilities of HCT116 cells. E Western blots of P65 and ABLIM1 for validation of RELA overexpression efficiency in stable shABLIM1 or shVec HCT116 cells. F Cell viabilities assayed by CCK-8 kit revealed the effects of RELA overexpression on proliferation of stable shABLIM1 or shVec HCT116 cells. n = 3 for each group. Kruskal–Wallis test. Effect of RELA overexpression on HCT116 cell migration G and invasion H in Transwells. I RTCA monitored the influence of RELA overexpression on CRC cell proliferation, migration, and invasion abilities in HCT116 cells with or without ABLIM1 knockdown. n = 4 for each group. ***P < 0.001, indicated the statistical significances between shVec+Vec group and shABLIM1+Vec group at the end of assays. ###P < 0.01, indicated the statistical significances between shABLIM1+RELAOE group and shABLIM1+Vec group at the end of assays. Paired t test. J 48 h after transfection of IκBα S32/36 A phosphorylation mutant, full length ABLIM1, or both, NF-κB transcriptional activities in HCT116 and SW620 cells were detected by dual-luciferase assay. n = 3 for each group. Kruskal–Wallis test.

Fig 4: ABLIM1 interacts with IκBα and facilitates its ubiquitination.A Relative mRNA levels of NFKBIA and ABLIM1 in HCT116, RKO, and SW620 cells assayed by qRT-PCR. n = 3 for each group. Kruskal–Wallis test: *P < 0.05. B Immunoblotting of IκBα and ABLIM1 in HCT116 cells treated with CHX (10 μM) for indicated time. Quantification of the IκBα levels relative to GAPDH expression was displayed at the right. C Immunoblotting results of IκBα and ABLIM1 in ABLIM1 knockdown or overexpression HCT116 cells treated with PS341 (100 nM). D Co-immunoprecipitation assays revealed the interaction between ABLIM1 and IκBα in HCT116 and RKO cells. Cells were transfected with NFKBIA in combination with ABLIM1 or empty vector for 48 h. In control IP, IgG was used instead of ABLIM1 antibody. E Co-immunoprecipitation assayed the effect of ABLIM1 overexpression on IκBα ubiquitination in HCT116 and 293 T cells. Cells were transfected with NFKBIA, UBB, and ABLIM1 or empty vector for 48 h and treated with MG132 (20 μM) for 4 h before harvest. F In vivo ubiquitination assay using Ni-NTA pull-down confirmed that ABLIM1 promotes the poly-ubiquitination of IκBα.

Fig 5: ABLIM1 is a novel E3 ligase, which interacts with IκBα via HP domain and ubiquitinates it depending on 402-778aa.A E3 ligase activity of ABLIM1 was determined by in vitro ubiquitination assay. HCT116 cells overexpressed with ABLIM1 or vector underwent IP using ABLIM1 antibody and magnetic beads, and then the beads were incubated with E1, E2, ATP, and recombinant IκBα, followed by immunoblotting examination. B Auto-ubiquitination assay revealed that recombinant ABLIM1 376-778aa rather than 1-401aa harbored E3 ligase activity. Ubiquitinated protein was detected by immunoblotting with an anti-ubiquitin antibody. C 402-778aa region of ABLIM1 was required for the interaction between ABLIM1 and IκBα. A diagram shows the structures of ABLIM1 (top). HCT116 cells were transfected with Flag-ABLIM1 constructs (full length, 1-216aa, 1-401aa, 402-778aa) or empty vector, respectively. D Co-IP assay revealed LIM domains were required for the ubiquitination activity of ABLIM1. E NF-κB transcriptional activities in RKO and SW620 cells were detected by dual-luciferase assay after transfection of different ABLIM1 mutant constructs (full length, 1-401aa, 402-778aa). n = 3 for each group. Kruskal–Wallis test: *P < 0.05; **P < 0.01. F Immunoblotting images of Flag, p-IκBα, CCL20, and GAPDH in HCT116 cells after transfection of different ABLIM1 mutant constructs. G In RKO, co-IP assay using ABLIM1 402-778aa mutant constructs (coiled-coil deleted, HP domain deleted) confirmed HP domain was responsible for the interaction between ABLIM1 and IκBα. H Western blot images of co-IPs performed on lysates of HCT116 cells transfected with ABLIM1 and indicated HA-IκBα constructs.