FIGURE 3:

Gle1 is required for the PCNT, NIN, and MT organization at the centrosome. (A) Total cell lysates of scrambled control siRNA– or GLE1 siRNA no. 7 (Hs_GLE1L_7 FlexiTube siRNA)–transfected cells were analyzed by immunoblotting for Gle1. Actin served as a loading control. A 30-μg amount of total protein was loaded per lane. (B) Human RPE-1 cells transfected with scrambled control siRNA or GLE1 siRNA no. 7 were processed for indirect immunofluorescence microscopy with antibodies against Gle1, as well as against PCNT (top), NIN (middle), or CETN (bottom). (C) Human RPE-1 cells transfected with scrambled control siRNA, GLE1 siRNA no. 7, or NXF1 siRNA were subjected to a MT regrowth assay, fixed at the indicated time points, and stained with antibodies to α-tubulin (α-Tub), followed by in situ hybridization using Cy3-labeled oligo-dT probes to label poly(A)-containing RNA. In GLE1-knockdown cells, increased ectopic cytoplasmic MT nucleation (6 min, GLE1 siRNA, arrowheads), and few detectable MTs anchored at the centrosome (12 min, GLE1 siRNA) were observed. (D) Quantification of the MT nucleation events in the MT regrowth assay between RPE-1 cells transfected with scrambled control siRNA, GLE1 siRNA no. 7, or NXF1 siRNA. Values are mean ± SEM, and n is the number of cells analyzed in each condition. Scale bar, 1 μm (B), 10 μm (C). Using a second GLE1 siRNA (i.e., GLE1 siRNA no. 4, Hs_GLE1L_4 FlexiTube siRNA) recapitulated the phenotypes (see Supplemental Figure S2).