A BAC transgene containing mTPX2-LAP rescues the phenotype of TPX2-RNAi. (a) Western blot of cell lines with anti-mTPX2 antibody, which recognizes both human TPX2 and mTPX2-LAP, and anti–α-tubulin, after TPX2 or CON RNAi. hTPX2 is efficiently depleted in all lines after TPX2 RNAi, whereas mTPX2-LAP transgenes remain. The asterisk represents the position of the TPX2^ΔN protein, which runs faster than the full length, as expected. (b) Immunofluorescence of U2OS cells after CON or TPX2 RNAi and TPX2^WT cells after TPX2 RNAi, stained for α-tubulin (green), Cep135 (red), and DNA (blue). U2OS cells without a rescuing transgene show a characteristic phenotype after hTPX2 depletion of collapsed poles and lack of a bipolar spindle, whereas TPX2^WT cells containing mTPX2-LAP after hTPX2 depletion have normal spindle morphology. Bar,10 μm. (c) Quantification of the TPX2 depletion phenotype and mTPX2-LAP rescue displayed in b. The percent of mitotic cells (n = >1,000 cells per experiment; three to five experiments for each condition) and percent bipolar spindles (n > 50) mitotic cells per experiment; three to five experiments per condition) after CON or TPX2 RNAi in U2OS or TPX2^WT cells were determined. Error bars represent standard deviation. The mTPX2-LAP construct is able to rescue depletion of endogenous TPX2.