Figure 4
SBDS stabilizes microtubules in vivo.
(A and B) SDS patient cells are sensitive to nocodazole and resistant to taxol. Lymphoblast cell lines originating from SDS patients (DF259, DF250, CH127, and DF1227) or normal control lymphoblasts were treated with increasing concentrations of nocodazole for 24 h (A) or taxol for 72 h (B). Cell survival was determined using AqueousOne colorimetric assay reagent. Each cell survival assay was performed in triplicate for each experiment for a total of 3 independent experiments. SEM is denoted with vertical bars. (C and D) Nocodazole sensitivity and taxol resistance are SBDS dependent. DF259 lymphoblasts were transiently infected with a dual expression lentiviral vector containing either GFP and SBDS cDNA or GFP alone (vector). Two days following infection, cells were treated with 6.6 μM nocodazole (C) or 50 nM taxol (D) for 18 h. Cell death was assayed by flow cytometry for propidium iodide uptake. Three independent experiments were performed for each assay. P < 0.25 (C) and P < 0.05 (D). (E) SBDS–/– cells exhibit an increased mitotic arrest with nocodazole. Normal control or SDS patient primary BMSCs were treated with increasing doses of nocodazole for 18 h. Cells were stained with DAPI, and the percentage of cells in metaphase (expressed as the mitotic index) was visually scored for at least 100 cells in triplicate for 3 independent experiments. Samples were quantitated in a blinded fashion. The mitotic index was calculated from the total number of mitotic cells divided by the total cell number counted. BMSCs from 2 different SDS patients (DF250 and DF259) yielded similar results. *P < 0.01. (F) SBDS–/– cells are resistant to taxol-induced mitotic arrest. Normal control or SBDS–/– patient (DF250) primary BMSCs were treated with increasing doses of taxol for 10 h, and the mitotic index was quantitated as in E. BMSCs from 2 different SDS patients (DF250 and DF259) yielded similar results. #P = 0.11; ##P = 0.09. (G) SBDS–/– cells are resistant to micronuclei formation following taxol treatment. Normal control or SDS patient (DF250) primary BMSCs were treated with increasing concentrations of taxol for 10 h. Cells were stained with DAPI, and the percentage of cells with micronuclei was quantitated for at least 100 cells per experiment for 3 independent experiments. Samples were quantitated in a blinded fashion. Similar results were obtained with BMSCs from at least 2 different SDS patients. **P < 0.05.
