Figure 6. Mec-17 Controls Myh10 Expression to Regulate Ciliogenesis Kinetics.

(A) Mec-17 knockdown reduced Myh10 protein expression. RPE-Mchr1^GFP 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 (left panal). 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 (right panel). (B) Myh10 protein expression depends on Mec-17 catalytic activity. Control, #1 and #3 Mec-17 knockdown ARPE-19 cells were infected with pBabe retrovirus expressing HA-tagged Mec-17 wild type (WT) or Mec-17 catalytic dead mutant (CD). 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. β-actin was used as an internal protein loading control. HA blot showed expression of Mec-17-HA fusion protein. (C) Inhibiting tubulin deacetylase activity upregulates Myh10 expression. RPE-Mchr1^GFP cells were treated with different deacetylase inhibitors, lane 1: DMSO; lane 2: 5 µM tubastatin (TBSA); lane 3: 5 mM sodium butyrate(NaB) and analyzed for Myh9 and Myh10 expression. β-actin was used as an internal loading control. (D) Inhibiting HDAC6 activity enhanced spontaneous ciliogenesis. RPE-Mchr1^GFP cells cultured in DMEM/20% FBS were switched to 5% FBS containing DMSO or 5 µM tubastatin A for 24 hours. Cells were fixed by methanol and visualized under a confocal microscopy. Mchr1-GFP (pseudo-colored red) was used to identify and quantify cilia. Yellow inset shows magnified image of representative cilium. Results from 3 biological replicates were averaged and plotted (right panel). DAPI (blue) was used to label the nuclei. ***, t-test p<0.001. Scale bar: 10 µm. (E) 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 (lower panel, green). (F) Blebbistatin accelerated ciliogenesis in Mec-17 KD cells. Non-target control (NT, black line) and Mec-17 #1 KD cells were treated with DMSO (green line) or 25 µM blebbistatin (red line) 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. (G) 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. β-actin was used an internal protein loading control.