HDAC6 Regulates EGFR Trafficking Figure 3. Knockdown HDAC6 with shRNA increases EGFR degradation. (A) and (B) Western blotting analysis of the expression of HDAC6, EGFR and acetyl-a-tubulin in Pkd1 wild-type (WT) MEK cells (A) and Pkd1null/null (Null) MEK cells (B) transduced with lentiviral vector pGIPZ-shHDAC6 mediated HDAC6 shRNA or control lentiviral empty vector. The levels of EGFR were reduced and the levels of acetyl-a-tubulin were increased in HDAC6 knockdown cells. (C) EGFR expression at mRNA level in Pkd1 mutant (Null) MEK cells with HDAC6 shRNA or vector (control) was analyzed by real-time PCR. Knockdown HDAC6 with shRNA did not affect the transcription of EGFR. Average values from triplicated experiments were shown. doi:10.1371/journal.pone.0049418.g003 of HDAC6 activity by pharmacological and shRNA mediated inhibition increased EGFR endocytic trafficking from early endosomes to later endosomes and increased the degradation of EGFR (Fig. 3 and 7); 2) treatment with nocodazole, which depolymerized microtubules, decreased the degradation of EGFR in Pkd1 mutant MEK cells stimulated with EGF (Fig. 8) and EGFR endocytic trafficking from early endosomes to later endosomes and (Fig. 9); 3) pharmacological and shRNA mediated inhibition of HDAC6 also decreased the expression of phospho-ERK1/2, a EGFR axis mediated downstream target (Fig. 10A and 10B). In addition, inhibition of HDAC activity with TSA normalized the localization of EGFR from apical to basolateral of the cystic epithelial cells in Pkd1 conditional knockout mouse kidneys (Fig. 10C and 10D). Thus, targeting HDAC6 to downregulate EGFR and its downstream targets as well as to normalize its PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49418