Nek4 participates in mRNA splicing regulation. HEK293T cells were methanol fixed and immunofluorescence against endogenous Nek4 and SC35 (a nuclear speckle marker) was performed. (A) Confocal microscopy shows the dotted staining of Nek4 in the nucleus, related to nuclear speckles (colocalization with SC-35). (B-D) To verify the involvement of Nek4 in RNA splicing, HEK293 Flp-In T-REx cells stably expressing empty vector (Flag), Nek4.1 F, Nek4.2 F or Nek4.1 KDF were transfected together with the E1A minigene encoding the pMTE1A plasmid. After 48 h, total RNA was extracted and the cDNA synthesized. Minigene splicing generates 3 main variants (represented in B), depending on the splicing site selection. The E1A isoforms generated in Nek4.1, Nek4.2 and Nek4.1 KD expression can be visualized in the agarose gel, depicted in (D). The band intensities for each isoform were measured and the percentage of 9S, 12S and 13S isoform in relation to the total isoforms generated is depicted in (C). Vertical bars in the graphs indicate ± standard deviation. Wherever it exists, the significance of the difference relative to the control (empty vector - Flag) is indicated by *p < 0.05.**p < 0.01. # represents the significant difference relative to Nek4.1. # p< 0.05 and ## p < 0.01; n = 3 (t – Test).