Dulbecco’s Modified Eagle’s Medium and fetal bovine serum were purchased from Gibco

Myh10 protein abundance was not due to protein degradation as inhibiting proteasome function by MG132 did not restore Myh10 level. Indeed, quantitative PCR detected a significant reduction of Myh10 mRNA in Mec-17-knockdown cells, suggesting that Mec-17 regulates Myh10 protein expression via a transcriptional mechanism. A modest reduction of Myh9 mRNA was also observed although its protein level was not affected. Importantly, Myh10 was not induced by serum starvation in Mec-17 knockdown cells. Collectively, these results showed that Mec-17 is required for Myh10 upregulation in response to serum starvation. The tubulin acetyltransferase Mec-17 has been indicated to possess both catalytic-dependent and independent functions. To determine if Myh10 expression depends on Mec-17 catalytic activity, we expressed wild type and catalytic-dead D157N mutant of mouse Mec-17. As shown in Fig. 6B, wild type Mec17 restored Myh10 expression to different degrees in two Mec-17 KD 10 / PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1968231 21 A Mec17-Myosin II Axis Controls Ciliogenesis Fig. 5. Myh10 Is Upregulated During Ciliogenesis. EW-7197 chemical information Schematic illustration of selected gene expression analysis during ciliogenesis in time course. Myh10 protein level is upregulated during ciliogenesis. RPE-Mchr1GFP cells serum starved for 0, 1, 2, 6, 12 and 24 hours were collected for western blot analysis of Myh10 and Myh9 protein level. b-actin was used an internal loading control. Relative protein bands from were quantified using Fiji gel analysis function. Results from three independent experiments were quantified, normalized to b-actin loading control and averaged. , t-test p,0.01. Myh10 mRNA is upregulated during ciliogenesis. mRNA samples collected at indicated serum starvation time points were used as template for real-time PCR to measure Myh9 and Myh10 mRNA expression levels. Error bars represent standard deviations from triple biological replicates. doi:10.1371/journal.pone.0114087.g005 lines that correlated with the extent of tublin acetylation whereas catalytic-dead D157N mutant was unable to restore tubulin acetylation or Myh10 expression. Of interest, over-expression of Mec-17 in control KD cells increased tublin acetylation and Myh10 levels. These results indicate that Mec-17 induces Myh10 via its acetyltransferase activity, likely by acetylating microtubules. Indeed, treatment with tubastatin A, a specific inhibitor for tubulin deacetylase HDAC6 significantly increased tubulin acetylation and Myh10 expression in RPE-Mchr1GFP cells, whereas sodium butyrate, a HDAC inhibitor that does not target HDAC6, had no PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682064 effect. Importantly, TBSA treatment significantly increased spontaneously formed cilia in the presence of serum, which further supports the critical role of Myh10 protein expression in cilium formation. Altogether, these 11 / 21 A Mec17-Myosin II Axis Controls Ciliogenesis Fig. 6. Mec-17 Controls Myh10 Expression to Regulate Ciliogenesis Kinetics. Mec-17 knockdown reduced Myh10 protein expression. RPEMchr1GFP cells were transduced with 4 different shRNA lentiviruses targeting Mec-17. Knockdown efficiency was assessed by acetylated tubulin level. Myh10 and Myh9 protein level was analyzed by western blotting. GAPDH was used as an internal loading control. Intensity of protein bands in left panel were quantified using FIJI gel analysis tools and normalized to GAPDH level. Relative bands intensity of each protein were further normalized to the NT control and plotted. Myh10 protein expression depends on Mec-17 cat