As illustrated GSI-IX in Figure 1A, PDE6A, PDE6B, PDE6C, PDE6D, PDE6G and PDE6H mRNAs were expressed in the human lung. PDE6A, PDE6B, PDE6C and PDE6G showed no significant altera tions in the IPF lungs as compared to donor lungs. In con trast, PDE6D subunit was significantly down regulated in the IPF lungs as compared to the donor lungs and PDE6H showed a tendency of down regulation in the IPF lungs as compared to the donor lungs. In addition, the resultant PCR products were validated by direct sequencing, followed by BLAST analy sis that confirmed the similar sequence alignment for each subunit. Protein expression of the PDE6 enzyme subunits The protein content of the PDE6 subunits in whole lung tissue homogenates of donors and IPF patients was quan tified by immunoblotting.

As illustrated in Figure 2A, immunoreactivity was detected for PDE6A, PDE6B, PDE6D and PDE6G H subunits. PDE6A and PDE6B blocking peptide studies were carried out to reconfirm the specificity of PDE6A and PDE6B immunoreactivity. Additionally, pig retinal lysate served as a positive control for immunoreactivity and proper protein size. Notably, the PDE6D and PDE6G H subunits were significantly down regulated in the IPF lungs as compared to donor lungs, whereas PDE6A and PDE6B showed no significant alterations between donor and IPF derived lung tissues. Cellular localization of the PDE6 enzyme subunits The cellular localization of the PDE6 subunits was assessed by serial immunohistochemical stainings on tissue sections from donor and IPF lungs.

As shown in Figure 3A, PDE6A, PDE6B, PDE6D and PDE6G H were co stained with pro SPC, suggesting the presence of PDE6 subunits in ATII cells. PDE6A immunoreactivity was recognized in the cytoplasm and membrane of ATII cells, PDE6B immunoreactivity was recognized in the nuclei, PDE6D immunoreactivity in the cytoplasm and PDE6G H immunoreactivity in the membrane of ATII cells. PDE6 enzyme subunits expression in human AECs To confirm the AEC localization pattern, the PDE6 sub units were qRT PCR amplified from primary human donor and IPF derived ATII cells. All PDE6 subunits were found to be expressed by these cells. Notably, PDE6D mRNA levels were signifi cantly decreased in IPF derived ATII cells as compared to donor ATII cells. In contrast, PDE6A, PDE6B, PDE6G and PDE6H were not differentially regu lated in AECIIs from IPF versus control lungs.

TGF b1 down regulates PDE6D in A549 cells A549 cells were used as an in vitro AEC model. Firstly, the cells were characterized Anacetrapib for the expression of PDE6 subunits. mRNAs of all PDE6 subunits and the complete set of PDE6 pro teins were found to be expressed by these cells. Next, to explore whether TGF b1 promotes PDE6D down regulation in AECs, A549 cells were trea ted with two different concentrations of TGF b1 for 12 and 24 h.