TPX2 and HURP cooperate in promoting spindle assembly in cells. (A) Immunofluorescence staining of HURP, α-tubulin, and DNA (DAPI) in control and HURP KO HeLa cells. (B) Following cold treatment at 0°C for 5 min, the control and HURP KO HeLa cells were fixed and immunostained for HURP, α-tubulin, and DNA (DAPI). (C and D) Quantification of the intensity of spindle MTs (C) and pole-to-pole distance (D) in control and HURP KO HeLa cells. The values in C were normalized to the intensity of control HeLa cells. n = 20–23 cells. (E and F) Quantification of the intensity of cold-stable MTs (E) and pole-to-pole distance (F) in control and HURP KO HeLa cells after cold treatment. The values in E were normalized to the intensity of control HeLa cells. n = 22–25 cells. (G) Immunofluorescence staining of HURP, α-tubulin, and DNA (DAPI) in control and HURP KO cells expressing TPX2 WT (upper panels) or FL89A mutant (lower panels). (H–J) Quantification of pole-to-pole distance (H), normalized spindle MT intensity (I), and the length-to-width ratio of metaphase plate (J) in control and HURP KO cells expressing TPX2 WT or FL89A mutant. The values in I were normalized to the intensity of control cells expressing TPX2 WT. n = 21–52 cells. Scale bars, 2 μm. Data represent mean ± SD. ***P < 0.001; n.s., not significant, two-tailed t test (unpaired).