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. 2008 Aug 15;295(4):L658–L669. doi: 10.1152/ajplung.90233.2008

Fig. 11.

Fig. 11.

Small interfering RNA (siRNA) knockdown of P63 expression reduced type II cell interactions with SP-A. Type II cells were transfected with nonspecific (NS or N-Sp siRNA) or P63-specific siRNA (P63 siRNA). [3H]choline was added to some dishes to label phosphatidylcholine (PC). A: Western blot for P63. The cells were cultured for 24 or 72 h and harvested, and lysates were subjected to Western blotting procedures using either P63 or β-actin Ab. The latter served as a loading control. No treatment, no siRNA added. B: P63-specific SP-A binding. At 72 h posttransfection, the cells were incubated (4°C, 1 h) with 0.5 μg/ml 125I SP-A without or with 25 μg/mg anti-P63 polyclonal Ab to block specific binding of SP-A to the cells. The cells were harvested with 0.2 N NaOH and counted. The data are the ng 125I-SP-A/mg cell protein bound to the cells that were inhibited by the anti-P63 Ab (SP-A binding with anti-P63 Ab subtracted from binding without anti-P63 Ab), thus representing P63-specific binding of SP-A. The total binding of SP-A to untreated cells (no siRNA) in the absence of Ab was low (15.3 ± 1.2 ng 125I-SP-A/mg cell protein, n = 3), possibly due to the fact that transdifferentiation of type II to type I cells after 72 h of culture (6, 15) may have affected the cell surface density of P63. Data are the means ± SE of triplicate determinations. *Statistically significant difference from no siRNA and nonspecific siRNA, P < 0.05. C: PC secretion. After 72 h, the cells that were previously incubated with [3H]choline were exposed to ATP (1 mM) ± SP-A (0.2 μg/ml) for 1 h. 3H-PC was extracted from cells and media and analyzed. Basal secretion was subtracted from all data = 0% maximum. Data are expressed as a percentage of PC secretion that occurred with nonspecific siRNA + ATP alone (1.75% ± 0.21% PC secretion, mean ± SE, n = 3), which was set equal to 100% (maximum). Data are means ± SE of duplicate or triplicate samples from 3 experiments. *Statistically significant difference from nonspecific siRNA (+ SP-A + ATP), P < 0.05; n = 3.