Fig 2: KIF26B enhances nuclear localization of ZC3H13/CBLL1. A-B.CBLL1 and ZC3H13 mRNA expression in MDA-MB-157 (A) and NMFH-1 (B) cells with lentiviral mediated KIF26B knockdown. C–F. KIF26B, ZC3H13, and CBLL1 protein expression in the cytoplasmic (C), and nuclear (N) fractions from MDA-MB-157 (panel C and E) and NMFH-1 (panel D and F) cells with KIF26B knockdown alone (C-D) or in combination with WT-KIF26B overexpression (E-F). G. Immunofluorescence analysis of CBLL1 (green), ZC3H13 (green), and DAPI (blue, cell nuclei) in KIF26B knockdown and control MDA-MB-157 and NMFH-1 cells. Scale bar, 20 μm. H–I. METTL3, METTL14, VIRILIZER, and WTAP protein expression in cytoplasmic (C), and nuclear (N) fractions from MDA-MB-157 (H) and NMFH-1 (I) cells with or without KIF26B knockdown. J-K. Interactions between WTAP and METTL3, METLL14, VIRILIZER, CBLL1, and ZC3H13 were determined by co-IP using cytoplasmic fractions from MDA-MB-157 (J) and NMFH-1 (K) cells with or without KIF26B knockdown. n.s., not significant. ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Fig 3: KIF26B interacts with ZC3H13 and CBLL1 in the cytoplasmic fraction of multiple cancer cell lines. A. IF staining of KIF26 (red), CBLL1 (green), and ZC3H13 (green) in MDA-MB-157, NMFH-1 and TT cells. B. Left panel, Co-IP was conducted using the cytoplasmic fraction MDA-MB-157 (top), NMFH-1 (middle), and TT (bottom) cells. Right panel, reciprocal IP assay was performed to explore the interaction of ZC3H13 and CBLL1 with KIF26B. KIF26B was immunoprecipitated by anti-CBLL1 and anti-C3H13. C and D. The structure of the wild-type full length (FL) and mutation constructs of Flag-tag labeled ZC3H13 (C) and CBLL1 (D). E and F. Flag-tagged FL or truncated mutation constructs were co-transfected with HA-KIF26B into NMFH-1 cells. The KIF26B/Flag-tag complexes were immunoprecipitated by anti-HA antibodies. Flag-ZC3H13 (E) and Flag-CBLL1 (F) proteins were detected using anti-Flag antibodies.

Fig 4: Semi-quantitation of nuclear/cytoplasma protein ratio from western blotting in Fig. 2. A-D. Semi-quantitation of nuclear/cytoplasma (N/C) CBLL1 and ZC3H13 ratio matched to each group in Fig. 2C (A), Fig. 2D (B), Fig. 2E (C), and Fig. 2F (D). E-F. Semi-quantitation of N/C METTL3, METTL13, VIRILIZER, and WTAP ratio matched to each group in Fig. 2H (E) and Fig. 2I (F). ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Fig 5: Survival analysis of two genes (CBLL1 and LGR5) in separated CRC cohorts using the expression level to divide each cohort into two populations of high and low expression. (a) Boxplots presenting the expression of CBLL1 in CRC tumour samples separated in two groups by expression level (high 491 samples, red, and low 362 samples, green); and the corresponding Kaplan–Meier curve presenting the survival analysis in these two groups of samples. This corresponds to the survival analysis using CBLL1 expression in all 843 patients (i.e., all subtypes CMS1, 2, 3 and 4); (b) boxplot presenting the expression of CBLL1 in CRC tumour samples separated by high expression (90 samples, red) and low expression (214 samples, green); and the corresponding Kaplan–Meier curve presenting the survival analysis in these two groups of samples. The second survival analysis is conducted only on the CRC samples of CMS2 subtype. For the other figures: (c) is the same as (a) and (d) is the same as (b) but using the expression signal of gene LGR5.