Microtubules are more stable and more highly acetylated in ethanol-treated hepatic cells  George T. Kannarkat, Dean J. Tuma, Pamela L. Tuma  Journal of Hepatology  Volume 44, Issue 5, Pages 963-970 (May 2006) DOI: 10.1016/j.jhep.2005.07.007 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Ethanol impairs microtubule polymerization. WIF-B cells were treated with 50mM ethanol (EtOH) for 72h. In the continued absence or presence of ethanol, 5μM nocodazole was added for 1h at 37°C to depolymerize microtubules. To allow repolymerization, the nocodazole was removed and the cells reincubated at 37°C for the indicated times. Cells were extracted in a microtubule-stabilizing buffer containing 0.15% Triton X-100 for 1min at 37°C. Cells were then lysed in a microtubule stabilizing buffer containing 0.15% Triton X-100. Soluble tubulin was released into the supernatant while polymeric tubulin remained cell-associated. Samples were immunoblotted for α-tubulin (A and B) or acetylated α-tubulin (C). A, The percent of polymeric tubulin at each time point is plotted. B, From the values in A, the percent of total tubulin that repolymerized was calculated. C, The percent of polymeric, acetylated α-tubulin at each time point is plotted. In A and C, values are the average ±SEM from four independent experiments. *P<0.004, **P<0.04. Journal of Hepatology 2006 44, 963-970DOI: (10.1016/j.jhep.2005.07.007) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Microtubules are shorter and gnarled in ethanol-treated cells. WIF-B cells were treated in the absence or presence of 50mM ethanol (EtOH) for 72h. Phase images (a and b) and α-tubulin labeling are shown (c and d). Asterisks are marking selected BCs. Journal of Hepatology 2006 44, 963-970DOI: (10.1016/j.jhep.2005.07.007) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Microtubules are more highly acetylated in ethanol-treated cells. A, WIF-B cells were treated in the absence or presence of 50mM ethanol (EtOH) for 72h. Cells were labeled for acetylated α-tubulin. Asterisks are marking selected BCs. B, Cell extracts from control or ethanol-treated WIF-B cells and liver homogenates from control and ethanol pair-fed rats were immunoblotted for α-tubulin and acetylated α-tubulin. Journal of Hepatology 2006 44, 963-970DOI: (10.1016/j.jhep.2005.07.007) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Microtubules are more stable in ethanol-treated cells. Control (a, c, e and g) or ethanol-treated cells (b, d, f and h) were incubated with 33μM nocodazole for the indicated times and labeled for α-tubulin. Asterisks mark selected BCs. Journal of Hepatology 2006 44, 963-970DOI: (10.1016/j.jhep.2005.07.007) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Microtubules are more stable in ethanol-treated cells. WIF-B cells were treated with nocodazole for the indicated times then extracted in a microtubule-stabilizing buffer containing 0.15% Triton X-100 for 1min at 37°C. Supernatants (S) containing the released soluble tubulin were collected and cells containing intact polymers (P) were lysed. Samples were immunoblotted for acetylated α-tubulin. The immunoblots shown are representative of three independent experiments. Journal of Hepatology 2006 44, 963-970DOI: (10.1016/j.jhep.2005.07.007) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Taxol induces more extensive microtubule bundling in ethanol-treated cells. Control (a, c and e) or ethanol-treated cells (b, d, and f) were incubated with 10μM taxol for the indicated times and labeled for α-tubulin. Journal of Hepatology 2006 44, 963-970DOI: (10.1016/j.jhep.2005.07.007) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Increased microtubule acetylation and stability are time- and dose-dependent. A, WIF-B cells were treated for 0–4 days with 50mM ethanol. B, Cells were treated with the indicated ethanol concentrations for 72h. Samples in A and B were immunoblotted for α-tubulin and acetylated α-tubulin. Acetylated α-tubulin values were normalized to the total α-tubulin present in each sample and their relative levels calculated. The graph in A is representative of four independent experiments. Values in B are the average±SEM from five independent experiments. Journal of Hepatology 2006 44, 963-970DOI: (10.1016/j.jhep.2005.07.007) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 8 Increased microtubule acetylation and stability are dependent on ethanol metabolism. Microtubule acetylation was monitored by immunoblotting WIF-B cell extracts after the following treatments: A, incubation in the absence or presence of 4-MP (1–3mM) or 50mM ethanol for 72h or B, incubation with 5mM ethanol and increasing cyanamide concentrations (as indicated) for 72h. In A, values are the average±SEM from three independent experiments. In B, a representative immunoblot is shown from three independent experiments. *P<0.03 (4-MP vs. EtOH); **P<0.025 (EtOH vs. EtOH +4-MP). Journal of Hepatology 2006 44, 963-970DOI: (10.1016/j.jhep.2005.07.007) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions