The virus titer in the fluid was calculated based on the method of Reed and Muench

p38 kinase cascade and activated downstream SP1-dependent transcription of IL-10. We hypothesized that similar acetylated microtubule-dependent signal amplification c-Met inhibitor 2 site mechanisms may be involved in the induction of Myh10 during cell quiescenceinduced ciliogenesis but may depend on other kinases and transcription factors. Although further experiments would be required to determine how acetylated microtubules control Myh10 expression, the Mec-17-dependent Myh10 induction provides a potential mechanism for a coordinated regulation of acetylation of microtubules and delivery of ciliary membranes and proteins, two key elements for cilium assembly. Interestingly, Mec-17 mRNA level is also upregulated by serum starvation. These findings suggest a simple model for ciliogenesis where Mec17, upon cellular quiescence, is activated to catalyze microtubule acetylation for building ciliary axoneme. Microtubule acetylation also induces Myh10 expression, which increases the dynamics of the actin network by counteracting PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682342 Myh9, thereby assisting the assembly of PPC and enabling delivery of membrane and protein components for cilium growth. Recent genetic mouse models suggest that Mec-17 is dispensable for in vivo ciliogenesis, our data, however, indicates that Mec-17 regulates in the early steps of cilia formation and kinetics control. Whether a faster kinetics of ciliogenesis is physiological relevant in any in vivo context remains to be investigated. Nevertheless, our finding that increasing tubulin acetylation facilitates ciliogenesis may point to a potential intervention of various human diseases resulting from 14 / 21 A Mec17-Myosin II Axis Controls Ciliogenesis cilia defects and therefore may be worth further testing in animal models of ciliopathies. Materials and Methods Cell Lines, Constructs, Sirnas, Antibodies and Chemical Reagents Three cell lines were used in this study: RPE-Mchr1GFP cells were a kind gift from Dr. Peter Jackson’s laboratory at Genentech, Inc. IMCD3 cells were obtained from Dr. Nicholas Katsanis laboratory at Duke University Medical Center. ARPE-19 cells were purchased from Duke University Cell Culture Facility. All cells were maintained and passaged according to provider’s instructions. Myh9-GFP and Myh10-GFP constructs were purchased PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682619 from addgene and were originally constructed by Dr. Robert S. Adelstein’s laboratory. GFP-actin was obtained from Dr. Michael D. Ehlers’ laboratory. MSCV-Puro retroviral vector was a kind gift from Dr. Xiao-fan Wang’s lab at Duke University. Human Myh10 cDNA sequence was amplified by PCR reactions and cloned into a modified MSCV-puro vector to generate MSCV-HA-Flag-Myh10. Human pLKO.1 Mec-17 shRNA constructs were purchased from Sigma. pLKO.1 non-target control constructs was also a gift from Dr. Xiao-fan Wang’s lab at Duke University. Antibodies used in this study includes: rabbit anti-glu-tubulin, mouse anti-acetylated-tubulin, mouse anti-c-tubulin, rabbit anti-c-tubulin, mouse anti-Rab11, rabbit anti-Cep290, rabbit anti-PCM-1, mouse anti-Myh10, rabbit anti-Myh9. siRNA duplex sequences used in this study are listed in table 2. Chemical reagents used in this study includes: Blebbistatin, Latrunculin A, Tubastatin A, sodium butyrate. Cell Culture, Transfection and Cilia Formation Assay RPE-Mchr1GFP cells were maintained in DMEM containing 10% FBS in a 37 C humidified incubator with 5% CO2. ARPE-19 and IMCD3 cells were maintained in DMEM:F12 medium containing 10% FBS. Cells were passaged at 80%90% c