Figure 1
Stable microtubule levels are rapidly diminished, but also acetylated, upon FQI1 treatment. (a,e) Representative immunoblots from lysates harvested at the indicated timepoints, derived from FH-B (a) and RPE cells (e) treated with 4 µM FQI1 (“F”) or vehicle (“D”, 0.01% DMSO) and separated into soluble and insoluble tubulin fractions using a microtubule sedimentation assay. Blots were probed for α-tubulin (upper blots), then reprobed for K40 acetyl- α-tubulin (lower blots). Microtubule-stabilizing taxol (“T”, 1 µM) and microtubule-destabilizing nocodazole (“N”, 1 µM) were added to the cells for 30 min and served as positive and negative controls, respectively. (b,f) Quantitation of the percentage of the total α-tubulin in the insoluble fractions at the indicated time-points after addition of FQI1 or vehicle in FH-B cells (b) and RPE cells (f). Three to four independent experiments were performed for each cell line; p values were generated using an unpaired two-sample t-test. Bars and corresponding error bars represent mean ± s.e.m. Numbers above brackets represent p-values. (c) FH-B cells were pretreated with 2 mM thymidine for 18 h, followed by incubation in fresh media containing 2 mM thymidine and either 4 µM FQI1, 1 µM nocodazole, or vehicle (0.01% DMSO) for the indicated times. Whole cell lysates were blotted for ⍺-tubulin and acetyl-⍺-tubulin. This experiment was performed once. (d,g) Quantitation of the amount of insoluble acetylated α-tubulin relative to the total insoluble α-tubulin at the indicated time-points after addition of FQI1 or vehicle in FH-B cells (d) and RPE cells (g). P-values were calculated using an unpaired two-sample t-test. Bars and error bars represent the mean ± s.e.m. from 2 to 4 biological replicates. Numbers above brackets represent p-values. For full immunoblots of (a,c,e), see Supplementary Fig. S7 online.
