Supplemental Material

This article contains the following supporting material:

• Figure S1 - UXT interacts specifically with Cdc14A in the yeast two-hybrid system. Yeast strain AH109 was transformed with a combination of plasmids, as indicated and plated on selective medium (-Ade, -His, -Leu, -Trp) containing X-alpha-gal. The image was taken after 3 days. The plasmids pGBKT7:Cdc14A and pGBTK7:LamC contain the Gal4 DNA-binding domain (Gal4-BD) fused with Cdc14A and human Lamin C, respectively. pGBKT7 is the parental empty vector. The plasmids pGADT7:UXT and pGADT7:T encode the transcription activation domain (TAD) fused with UXT and SV40 large T-antigen, respectively. Note that only yeast cells with co-expression of Gal4-BD:Cdc14A and TAD:UXT can grow robustly on the selective medium and turn blue, which indicates the activation of the HIS and Gal reporters harbored in this strain. In comparison, the yeast colonies transformed with the control plasmids grow slowly and turn red due to lack of adenine in the medium.
• Figure S2 - Centrosome localization of UXT mutant proteins. U2OS cells were transiently transfected with EGFP-tagged UXT point mutants or deletion mutants (green) and immunostained with the anti-gamma-tubulin antibody (red). The arrows indicate the centrsomes.
• Figure S3 - Effects on microtubule network by overexpression of UXT. U2OS cells were transiently transfected with EGFP-tagged UXT (green) and immunostained with anti-beta-tubulin antibody (red). The arrows indicate the location whereby microtubule network irradiates from.
• Figure S4 - UXT siRNA1 knockdown causes cell death. A. Efficacy of siRNA knockdown of the endogenous UXT protein. U2OS cells were treated with transfection reagent (NT), non-specific siRNA (NS siRNA) or siRNA for UXT (UXT siRNA1), respectively. The cell lysates were subject to Western blot using either the anti-UXT antibody 1B2 (top panel) or the anti-beta-tubulin antibody (bottom panel). The protein levels of UXT were specifically reduced after 72 hours treatment with siRNA for UXT. B. UXT knockdown leads to cell death. U2OS cells were treated with non-specific RNA or the UXT siRNA1 oligos. 72 hours later, all the cells were collected and used for PI staining of DNA content by FACS. C. p53 is not required for cell death caused by UXT knockdown. The HCT116 (p53+/+) or p53-negative HCT116 (p53-/-) cells were treated with either the control siRNA or the UXT siRNA1. 72 hours after transfection, the percentage of cell death was assessed by the trypan blue assay. The diagram shows the representative results of three experiments.