HDAC6 Regulates EGFR Trafficking Figure 10. Inhibition of HDAC6 activity decreased the phosphorylation of ERK1/2 and normalized the localization of EGFR from apical to basolateral. (A) Knockdown HDAC6 with shRNA increases the degradation of EGFR and decreases the phosphorylation of ERK1/2. Serum- starved PN24 cells transduced with lentiviral plasmid pGIPZ-shHDAC6 or control empty vector pGIPZ-NS, were pretreated with 10 mg/ml cycloheximide for 2 hours and then stimulated with 100 ng/ml EGF for indicated times. (B) Inhibition of HDAC6 activity with TSA decreases the activation of EGFR and ERK1/2 . PN24 cells were pretreated with 100 ng/ml TSA for 12 hours and then starved for another 12 hours with TSA. After that, the cells were stimulated with 100 ng/ml EGF in the presence of 10 mg/ml cycloheximide and TSA for indicated times. (C) and (D) Treatment with TSA normalized the mislocalized EGFR from apical to basolateral in cystic epithelia in postnatal day 7 (P7) kidneys from Pkd1flox/flox:ksp-Cre pups. Immunohistochemical staining of EGFR in P7 kidney sections from Pkd1flox/flox:ksp-Cre neonates treated with DMSO (C) or TSA (D). Arrows pointed to apical (C) and basolateral (D) regions, respectively. Scale bar, 20 mm. doi:10.1371/journal.pone.0049418.g010 icycler iQTM Real-time PCR detection system. Genes were amplified using the following primers. EGFR-F: 59-AT- CAAAGTTCTGGGTTCGGG-39; EGFR-R: 59- GTTGGCTTTTGGAGATGTGG-39; Actin-F 59-AAGAGC- TATGAGCTGCCTGA-39; Actin-R: 59-TACGGATGT- CAACGTCACAC-39. The complete reactions were subjected to the following program of thermal cycling: 40 cycles of 10s at 95uC and 20s at 63uC, a melting curve was run after the PCR cycles, followed by a cooling step. Each sample was run in triplicated in every experiment, and each experiment was repeated three times. The expression level of EGFR was normalized to the expression level of actin. Mouse Strain and Treatment Pkd1nl mice were generated as described.[23] Homozygous Pkd1nl mice, with only 13–20% normally spliced Pkd1 transcripts caused by an intronic neomycin-selectable marker, are viable, showing bilaterally enlarged polycystic kidneys. Pkd1-floxed:Ksp-cre mice were generated as described.[28] Each Pkd1 flox/flox:Ksp-cre pup was injected with 0.5 mg/g TSA or DMSO (control) daily from postnatal day 3 (P3) to P6. All the kidneys were harvested at postnatal day 7 (P7) for further analysis. Immunohistochemistry For HDAC6 staining, kidneys were fixed with 4% para- formaldehyde overnight. Polyclonal goat anti-HDAC6 antibody (1:40, Santa Cruz, CA), a peroxidase conjugate secondary antibody (1:200, Sigma, MO), and DAB (3,39-diaminabenzidine tetrahydrochloride) substrate system were used. For EGFR staining, kidneys were fixed with 4% paraformaldehyde for 60 min at 4uC, then stepwise dehydrated in acetone, and embedded in resin blocks using a Immuno-Bed Kit (Polyscience Inc., Warrington, PA). 3 mM sections were then cut, and immunohistochemical staining of the EGFR was conducted using the rabbit polyclonal anti-EGFR antibody (Abcam, Cambridge, UK) (1:50). Images were analyzed with a NIKON ECLIPSE 80i Microscope. HDAC6 Enzymatic Activity Assay The measurement of HDAC6 activity from cell lysates of Pkd1 wild type, heterozygous and homozygous renal epithelial cell lines PLOS ONE | www.plosone.org 11 November 2012 | Volume 7 | Issue 11 | e49418