Fig. 1.
mRNA levels in murine (m) inner medullary collecting duct (IMCD) 3 cell lines with stable knockdown of nephronophthisis Nphp3, Nphp6, or Nphp8 by real-time q-PCR analysis. Stable knockdown of Nphp3 (A), Nphp6 (B), and Nphp8 (C) was performed by retrovirus-mediated short-hairpin RNA (shRNA) expression in mIMCD3 cells. Three different duplexes for Nphp3 (Nphp3.1, Nphp3.2, and Nphp3.3), Nphp6 (Nphp6.1, Nphp6.2, and Nphp6.3), and Nphp8 (Nphp8.1, Nphp8.2, and Nphp8.3) were used to stably knock down the genes. Relative mRNA levels were evaluated by real-time PCR analysis. Triplicate sample for each mRNA was used to evaluate the level of mRNA expression. Data represented in the diagram are %means ± SD for each mRNA sample after normalization with GAPDH mRNA abundance. shRNA sequences used for stable knockdowns are available from the authors. D: knockdown of specific Nphp gene in IMCD3 cells does not affect other Nphp subtypes. Real-time PCR analysis using cDNA from mIMCD3 cells with stable knockdown with Nphp3, Nphp6, and Nphp8 and specific primers for corresponding genes (Nphp3, Nphp6, and Nphp8) revealed that lower mRNA level was detected only in the Nphp3 knockdown cells but not in the Nphp6 or Nphp8 cell lines. Similarly, knock down of Nphp6 was detected only in the Nphp6 cell lines but not in either Nphp3 or Nphp8 cell lines. Analysis of Nphp8 mRNA revealed the specific knockdown of Nphp8 cell line but not in the Nphp3 or Nphp6 cell lines. mRNA from unrelated shRNA-derived cells (sh-neg) were considered to be 100% for all three knockdown analyses. Relative mRNA expression data from triplicate samples were determined using the 2−ΔΔCT method and represented as means ± SD. *Significance level at P < 0.001.