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. 2023 Jan 9;9:4. doi: 10.1038/s41420-023-01301-8

Fig. 6. TOFA and palmitoylation-deficient tubulin alter MT dynamic instability.

Fig. 6

a, b TOFA hampered cold-induced MT disassembly at the SP. a Representative images of cells subjected to cold exposure. Cells were untreated (−) or pretreated for 1 h with 100 μM TOFA, followed by 5 min cold exposure before fixation and staining for centrin2 (green), α-tubulin (red), and DAPI (blue). Cells fixed without cold exposure (No cold) served as the control. The white arrow indicates the site of MT disassembly at the SP, and the yellow arrow indicates the site where MT remnants were observed at the SP after cold exposure. b Percentages of untreated or TOFA-pretreated CGL2 cells with MT remnants at the SP after 5 min cold exposure. Mean ± SD from three independent experiments is shown. *P < 0.05 by Student’s t test. c–e C376A mutation on α-tubulin renders spindle MTs resistant to cold-induced disassembly. c Western blots show the expression efficiency of EYFP-tubulin-WT and -C376A in HeLaS3 cells. Cells transduced with empty vector pLAS5w.Pneo served as the control. d Representative images of EYFP-tubulin-WT- or C376A-expressing cells subjected to 10 min cold exposure, fixation, and staining for CEP152 (red) and DAPI (blue). MT fibers of EYFP-tubulin are displayed in green. Cells fixed without cold exposure (No cold) served as the control. e Percentages of the cells described in (d) that exhibit MT fibers with EYFP-tubulin-WT and -C376A. Mean ± SD from two independent experiments is shown. *P < 0.05 by Student’s t test. f Representative image of the normal and abnormal mitotic spindles. Cells were stained for CEP152 (green), α-tubulin (red) and DAPI (blue). g TOFA and 2-BrPA enhanced taxol- and nocodazole-induced spindle abnormalities. CGL2 cells were treated for 4 h with TOFA or 2-BrPA, alone or in combination with 0.5-h treatments of taxol or nocodazole as indicated. Treated cells were fixed and stained for spindle analysis. Percentages of cells with abnormal spindles (as in f) are shown as mean ± SD of at least 600 cells from two independent experiments. *P < 0.05 by Student’s t test. h TOFA enhanced taxol-induced cytotoxicity. MDA-MB-231 cells were treated with TOFA and taxol for 24 h, alone or in combination as indicated. Treated cells were subjected to colony-formation assay. Percentages of colonies formed compared to the vehicle control are shown as mean ± SD from six independent experiments. *P < 0.05 by Student’s t test.