Fig. 6.

MTR120 is required for maintaining normal diploid status. (A) Cells transfected with control siRNA (siCTL) or siRNA targeting MTR120 (siMTR120) were harvested 96 hours after transfection. Cell lysates were analyzed by western blotting for MTR120, acetylated α-tubulin, α-tubulin and β-actin. Asterisks indicate a degraded or smaller form of MTR120. (B) Quantification revealed the ratios of indicated protein levels in siMTR120-treated cells relative to those in siCTL-treated cells (three independent experiments). (C) Immunofluorescence images of cells transfected with siCTL or siMTR120. γ-tubulin and α-tubulin are the respective markers for centrosomes and MTs. DAPI was used to stain DNA. (D) Quantification revealed the mean percentages of multinucleated cells in the indicated groups; 100 cells from each group were counted from three independent experiments. (E) The bar graphs reveal the percentages of cells in different cell cycle phases. We analyzed 10,000 cells from each group (three independent experiments). (F) Parental cells and cells harboring siRNA-resistant wild-type MR120 or the D2 mutant were harvested and subjected to an immunoblotting analysis using anti-MTR120, anti-FLAG and anti-α-tubulin antibodies. (G,H) Cells that stably expressed siRNA-resistant wild-type MTR120 or the D2 mutant were transfected with siMTR120. The expression of siRNA-resistant wild-type or D2 mutant was confirmed by western blotting analysis (G). Cells were fixed and subjected to immunofluorescence staining with anti-α-tubulin and anti-FLAG (MTR120) antibodies (H). Scale bars: 10 µm. Error bars represent s.d.