Fig. 7: Knockout of TBD in HURP increases vinorelbine sensitivity and reduces K-fiber in the hepatoma cell.
From: HURP binding to the vinca domain of β-tubulin accounts for cancer drug resistance
Cell cycle phases (G1, S, G2/M) represented as blue, purple, and green bars. Data are mean ± SD from five independent experiments. One-tailed Student’s t-test; Statistical differences: *p < 0.05 for the G2/M phase in the wild-type and mutant cell lines. b–d, Growth curves for (b) wild-type and (c, d) two homozygous mutant Hep3B cell lines in the absence and presence of 0.005 μM vinorelbine. Data are mean ± SD from five independent experiments. The cell number differences at the day 10 between untreated and treated with vinorelbine (0.005 μM) are indicated. e, f Representative immunofluorescence staining images of (e) wild-type and (f) HURPΔTBS-1 mutant cell lines using α-tubulin (green, left panels) and CENP-A (red, middle panels) antibodies. The merged fluorescent images are shown in the right panels. Scale bar: 5 μm. g, h Quantification plots (arb. units) of (g) total microtubule and (h) K-fiber (α-tubulin fluorescence intensities) in wild-type and mutant cells at metaphase. Data are mean ± SD from three independent experiments (n = 21 (WT) and n = 19 (mutant) cells). Two-tailed Student’s t-test; Statistical differences: *p < 0.05; ***, P < 0.001; ****, P < 0.0001. i, j Representative images of (i) wild-type and (j) HURPΔTBS-1 mutant cells after cold treatment and immunofluorescently stained using antibodies against α-tubulin (green, left panels) and CENP-A (red, middle panels). The merged fluorescence images are shown in the panels at right. Scale bar: 5 μm. k The quantification plot (arb. units) of cold stable K-fiber (α-tubulin fluorescence intensities) in wild-type and mutant cells at metaphase. Data are mean ± SD from three independent experiments (n = 24 cells). Two-tailed Student’s t-test; Statistical differences: ***, P < 0.001; ****, P < 0.0001. l A schematic model: Importin-β suppresses HURP’s microtubule-binding activity during mitosis, preventing spindle assembly. Ran-GTP releases HURP, promoting tubulin recruitment and microtubule assembly. m, n In the dividing cell, (m) vinorelbine decreases the microtubule polymerization rate, blocking mitotic spindle assembly. However, (n) elevated HURP counters vinorelbine’s effects by stabilizing K-fibers.
