Therefore, ROS may be involved in the induction of HO-1 in CE exposed epithelial cells

alytic activity. Control, 1 and 3 Mec-17 knockdown ARPE19 cells were infected with pBabe retrovirus expressing HA-tagged Mec-17 wild type or Mec-17 catalytic dead mutant. Myh10 protein expression was examined by western blot. Acetylated tubulin was used as a positive control to indicate the presence of Mec-17 catalytic activity. b-actin was used as an internal protein loading control. HA blot showed expression of Mec-17-HA fusion protein. Inhibiting tubulin deacetylase activity upregulates Myh10 expression. RPE-Mchr1GFP cells were treated with different deacetylase PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682730 inhibitors, lane 1: DMSO; lane 2: 5 mM tubastatin; lane 3: 5 mM sodium butyrate and analyzed for Myh9 and Myh10 expression. b-actin was used as an internal loading control. Inhibiting HDAC6 activity enhanced spontaneous ciliogenesis. RPE-Mchr1GFP cells cultured in DMEM/20% FBS were switched to 5% FBS containing DMSO or 5 mM tubastatin A for 24 hours. Cells were fixed by methanol and visualized under a confocal microscopy. Mchr1-GFP was used to identify and quantify cilia. buy 2883-98-9 Yellow inset shows magnified image of representative cilium. Results from 3 biological replicates were averaged and plotted. DAPI was used to label the nuclei. , t-test p,0.001. Scale bar: 10 mm. Mec-17 is required for centrosomal PCM-1 cluster organization. Mec-17 1 shRNA transduced cells were methanol fixed and stained with rabbit PCM-1 antibody to examine the intracelluar distribution of PCM-1 proteins. Blebbistatin accelerated ciliogenesis in Mec-17 KD cells. Non-target control and Mec-17 1 KD cells were treated with DMSO or 25 mM blebbistatin in DMEM with 0.2% FBS and quantified for percent of ciliated cells at 0, 12 and 24 hours. Error bars represent standard deviation from triple biological replicates. Reduced expression of Myh9 during late stage of ciliogenesis compensates for Myh10 loss to promote cilium formation in Mec-17-knockdown cells. Non-target control and Mec-17 KD 1 cells were serum starved and collected at indicated time points to monitor Myh9 and Myh10 expression. Acetylated tubulin was used to show the efficient knockdown of Mec-17. b-actin was used an internal protein loading control. doi:10.1371/journal.pone.0114087.g006 12 / 21 A Mec17-Myosin II Axis Controls Ciliogenesis experiments support PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682619 the conclusion that Mec-17 promotes Myh10 expression by increasing microtubule acetylation. If Mec-17 is required for Myh10 expression, one would expect defective ciliogenesis in Mec-17 knockdown cells. Indeed, we found that Mec-17 knockdown caused PCM-1 dispersion and ciliogenesis was significantly reduced in the early phase. Similar to Myh10 knockdown cells, the ciliogenesis defect in Mec-17 KD cells can be corrected by Blebbistatin treatment that inactivates Myh9. Intriguingly, cilium formation in Mec-17 knockdown cells eventually recovered at later time points, as it was previously reported. This later phase recovery of ciliogenesis is also consistent with normal cilia formation observed in Mec-17-deficient mice. Analysis of Myh9 protein in Mec-17-knockdown cells revealed that it was decreased in later time points after serum starvation. Given the inhibitory activity of Myh9 on cilium formation, the loss of Myh9 could explain the eventual recovery of cilium formation in Mec-17 knockdown cells. Our data reveal that Mec17 regulates ciliogenesis, at least in part, through Myh10 induction. Discussion The formation of primary cilium requires polarized growth of the membrane material delivered by th