Figure 6

Binding of FoxA2 to Muc5b promoter depends on STAT6 pathway. (A) Chromatin Immunoprecipitation (ChIP) method shows that specific binding of FoxA2 to Muc5b promoter (in gray squares) is less in Pneumocystis-infected rats compared to non-infected rats without or with Kaempferol (^*P = 0.0159 and ^***P = 0.0079). These significant decreases are lost with addition of Kaempferol to Pneumocystis-infected rats suggesting reversal. Controls (IgG) are shown in blue. Results are expressed as percentage of input. n = 5. (B) mRNA levels of FoxA2 are not significantly different in different experimental groups suggesting decrease in FoxA2 binding to Muc5b promoter is not influenced by gene expression. Results are expressed as mean fold change in gene expression using 2^−ddCt method and the group of non-infected, saline treated, rats (Pc−S) as reference (n = 4). (C) Plot of whole cell protein levels of FoxA2 in lung samples of each animal group as detected by WB (n = 4). Lack of differences suggests that changes in FoxA2 are compartmentalized within the cell. Results are indicated as mean +/− SD and were normalized by Actin. (D,E) Representative western blot of nuclear protein levels of FoxA2 in lung samples of each experimental group (n = 4) (E) suggesting a decrease in FoxA2 nuclear localization in infected rats that reverts with addition of Kaempferol as shown in (F) Plot of the WB shown in D. Results are expressed as mean +/− SD and were normalized by H1 levels. ^* indicates P = 0.0286. ns indicates no significance. Statistical comparisons were made using Mann-Whitney test.