siRNA-mediated depletion of hMps1 perturbs centrosome duplication. (A) At 72 h posttransfection with siRNAs, asynchronously grown HeLa cells were analyzed by immunoblot with antibodies against hMps1 (SCB540) and α-tubulin as indicated. –, Mock transfection; L, 33 nM Lamin-siRNA; M*, 16.5 nM hMps1 siRNA; M, 33 nM hMps1-siRNA. The percentage of cells in each transfection with one centrosome is indicated below the blots [1cen. (%)]. (B) HeLa cells were transfected with siRNAs, then enriched in G₁ with a 48-h serum starvation. At 8 h after serum stimulation, cells were analyzed by immunoblot as described in A. –, Mock transfection; L, 33 nM Lamin-siRNA; M, 33 nM hMps1-siRNA. The percentage of cells in each transfection with one centrosome is indicated below the blots [1cen. (%)]. (C) At 72 h posttransfection with siRNAs, HeLa cells were analyzed by IIF as described in Fig. 1. Representative HeLa cells transfected with 33 nM hMps1-siRNA (a) or 33 nM Lamin-siRNA (b) showing hMps1 (green), γ-tubulin (red), and DNA (blue). (D) HeLa cells expressing GFP-centrin were enriched in G₁ as described in B, and BrdUrd was included during the 8-h serum stimulation. Representative HeLa cells transfected with 33 nM hMps1-siRNA (a) or Lamin-siRNA (b) showing GFP-centrin (green), DNA (blue), and BrdUrd (red). Insets in C and D show 4-fold magnification of centrosomes. (Scale bar, 5 μm.)

hMps1 regulates centrosome duplication. (A and B) HeLa cells expressing GFP or GFP-hMps1KD were arrested in S phase with a 24-h HU treatment and analyzed by IIF. (A) Representative HeLa cells expressing GFP alone (a) (green) or GFP-hMps1KD (b) (green) showing γ-tubulin (red) and DNA (blue). (Scale bar, 5 μm.) (B) Centrosome number was determined in HeLa cells expressing GFP or GFP-hMps1KD. (C) U2OS cells expressing GFP, GFP-hMps1, or GFP-hMps1KD were arrested in S phase with a 24-h HU treatment. Centrosome number was determined after an additional 24 h of S-phase arrest.

hMps1 localizes to centrosomes in human cells. (A–C) The localization of hMps1 was determined by IIF in asynchronously grown RPE1 (A) and U2OS (B and C) cells with the hMps1Ag3 antibody. (C) The specificity of the hMps1Ag3 antibody was verified in U2OS cells by preincubation with the GST-hMps1400-507 fusion protein. hMps1, green; γ-tubulin, red; DNA, blue. (D) Asynchronously grown HeLa cells expressing GFP-hMps1were analyzed by IIF. GFP-hMps1 epifluorescence, green; γ-tubulin, red; DNA, blue. (Scale bar, 5 μm.)

Specificity of hMps1 antibodies. (A) Lysates from HU-arrested HeLa cells were analyzed by immunoblot with the hMps1Ag3 antibody before (–) or after (+) its preincubation with the GST-hMps1400-507 fusion protein. The arrow indicates the position of hMps1, and numbers represent the position of molecular mass markers in kDa. (B) Recombinant GST-hMps1-ha and GST-hMps1KD-ha were treated with λ-phosphatase (+) or buffer alone (–), then simultaneously analyzed by immunoblot with the hMps1Ag3 (Ag3) and hMps1N1 (N1) antibodies.

siRNA-mediated depletion of hMps1 perturbs normal mitotic progression. (A and B) HeLa cells were analyzed for mitotic abnormalities by IIF 72 h after transfection with hMps1- or Lamin-siRNAs. (A) Cells were scored for the presence (abnormal) or absence (normal) of chromosome alignment and segregation defects. (B) Representative chromosome alignment and segregation defects in hMps1-siRNA-transfected HeLa cells. (a–c) Kinetochores (green) and γ-tubulin (red). (d–f) Kinetochores (green) and α-tubulin (red). (g–i) γ-tubulin (green). (a, d, and e) Representative chromosome alignment defects; arrows indicate unaligned and/or unattached chromosomes. (b, c, f, and g) Representative chromosome segregation defects; carets indicate unsegregated chromosomes. (g) A representative cell attempting to divide through unsegregated chromosomes. (h) A representative micronucleated cell with elongated nuclear morphology. (i) A binucleated prophase cell with two centrosomes. (a–i) DNA is shown in blue. (Scale bar, 5 μm.) (C) HeLa cells were analyzed by IIF to determine the distribution of Mad2 72 h after transfection with siRNAs. Representative HeLa cells transfected with 33 nM hMps1-siRNAs (a) or Lamin-siRNAs (b) showing kinetochores (green), Mad2 (red), and DNA (blue). (Scale bar, 5 μm.) (D) HeLa cells were analyzed by IIF to determine the distribution of CENP-E 72 h after transfection with siRNAs. Representative HeLa cells transfected with 33 nM hMps1-siRNAs (a and b) or Lamin-siRNAs (c and d) showing kinetochores (green), CENP-E (red), and DNA (blue). (a) Decreased binding of CENP-E to kinetochores at metaphase. (b) Failure of CENP-E to redistribute to the spindle midzone during anaphase. (c) Normal kinetochore binding of CENP-E at metaphase. (d) Normal redistribution of CENP-E to the spindle midzone during anaphase. (Scale bar, 5 μm.)