HDAC6 Regulates EGFR Trafficking Figure 7. HDAC6 regulates EGFR intracellular transport. (A) mIMCD3 cells were serum-starved and then stimulated with 100 ng/ml EGF for indicated periods of time. The cells were then processed for double-labeling immunostaining with anti-EEA1 and anti-EGFR antibodies. Localization of EGFR to the EEA1-containing vesicles was analyzed. (B) PN24 cells were serum-starved and then stimulated with 100 ng/ml EGF for indicated periods of time. The cells were then processed for immunostaining as in (A). Localization of EGFR to the EEA1-containing vesicles was analyzed. (C) mIMCD3 cells were processed for double-labeling immunostaining with anti-LAMP2 and anti-EGFR antibodies. Co-localization of EGFR to LAMP-2-containing vesicles was examined. (D) PN24 cells were processed for double-labeling immunostaining with anti-LAMP2 and anti-EGFR antibodies. Co-localization of EGFR to LAMP-2-containing vesicles was examined. * p , 0.05. doi:10.1371/journal.pone.0049418.g007 Figure 8. Microtubule stability affected EGF induced EGFR degradation. (A) and (B) Influence of nocodazole on EGF-induced EGFR degradation. Pkd1 wild-type (WT) MEK cells (A) and Pkd1null/null (Null) MEK cells (B) were pretreated with 10 mg/ml cycloheximide for 2 hours. Pretreated cells were treated with 0.2 mg/ml nocodazole and/or 100 ng/ml EGF for another 2 hours. EGF-induced EGFR degradation was strikingly attenuated after co-treatment with EGF and nocodazole, which disturbed the stability of microtubule. * p , 0.05. ** p , 0.01. doi:10.1371/journal.pone.0049418.g008 PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49418