Expression of Muc5ac and Gob5 in mouse lungs after intratracheal instillation of IL-13. Balb/c female mice were given intratracheal injections of IL-13 at 500 ng/50 μl. The lungs were isolated 24 h later for total RNA isolation. A two-step real-time RT-PCR protocol was then performed with primers specific for Muc5ac and Gob5. Copy numbers of each gene were normalized to GAPDH. (A) Fold of induction of Muc5ac as normalized to GAPDH in wild-type mice (filled bar) and Stat6-deficient (striped bar) Balb/c mice after intratracheal instillation of PBS and IL-13. (B) Fold of induction of Gob5 expression as normalized to GAPDH of Gob5 in wild-type (filled bar) and Stat6-deficient (striped bar) Balb/c mice after intratracheal instillation. Y axis is in logarithmic scale. n = 3; *P = 0.002; **P = 0.003. Data are presented as mean ± 1 SEM.

Dose-dependent effects of IL-13 on MUC5AC and hCLCA1 expression by primary human TBE cells in vitro. Well differentiated primary human TBE cells were prepared as described in the text, and treated with IL-13 at doses from 2 to 50 ng/ml for 24 h as shown. (A) Effects of IL-13 doses on MUC5AC expression by primary human TBE cells. (B) Effects of IL-13 doses on hCLCA1 expression by primary human TBE cells. n = 4; *P < 0.018; **P < 0.001; ***P < 0.007.

Effects of JAK inhibitors on IL-13–induced Gob5 expression by mouse primary TBE cells in culture. Mouse TBE cell cultures were performed as described in Figure 2. Before IL-13 (20 ng/ml) treatment, cultured cells were pretreated for 30 min with the JAK inhibitors, JAK inhibitor I (100 nM) and AG490 (10 μM), and RNA was isolated 24 h after IL-13 treatment. Relative fold increase of Gob5 message that is normalized with GAPDH is shown in the figure. n = 3; *P = 0.029; **P = 0.047.

Effects of peptidoglycan on the expression of MUC5AC and hCLCA1 by primary human TBE cells. Well differentiated primary human TBE cells were treated with different amounts of peptidoglycan (PGN) from Staphylococcus Aureus (Invivogen) for 24 h. (A) Fold induction of MUC5AC (normalized with GAPDH). (B) Fold induction of hCLCA1 (normalized with GAPDH). n = 2; *P = 0.041.

Double immunofluorescent staining of primary human TBE cells treated with IL-13. Paraffin blocks were prepared from primary TBE cultures treated with IL-13 (10 ng/ml) for 1 wk on both the basolateral and apical areas, and paraffin sections (5 μm) were prepared. After deparaffinization, double antibodies specific for hCLCA1 and MUC5AC were used to stain these sections, which were further stained with Alexa Fluor (red)-conjugated or FITC (green)-conjugated secondary antibody, respectively. Nuclei were further stained with DAPI, yielding a blue color. The slides were examined using a Zeiss Confocal microscope under the ×63 lens (A, B) and a Zeiss fluorescent microscope (C, D). Z-Stacking was performed at 0.5-μm cuts and 10–12 images were obtained through the thickness of the sections. The images in (A) and (B) are single 0.5-μm slices from the Z-Stacked cuts. (A) A 0.5-μm slice showing the colocalization of hCLCA1 and MUC5AC. Arrow indicates a cell that was stained by both antibodies, and the merged color gives off a yellowish glow. Blue fluorescent stains are nuclei stained with DAPI. (B) Another region of the same slide that showed different cells stained for hCLCA1 and MUC5AC with no overlap seen. (C and D) Fluorescent views of another paraffin section that was examined with a Zeiss fluorescent microscope under the filter specific for FITC (green [C], 45M1) or Alexa Fluor (red, (D), anti-hCLCA1), with a 40× lens.

In vitro effects of IL-13 on Muc5ac and Gob5 gene expression in primary mouse TBE cells. Mouse TBE cells were obtained from wild-type and Stat6 knockout Balb/c mice, as described in the text. TBE cells were cultured in an air-liquid interface (ALI) condition, with either vehicle (PBS) or IL-13 at 20 ng/ml for 24 h, and then total RNA was isolated and real-time RT-PCR was performed as described in Figure 1. (A) Fold induction of Muc5ac (normalized with GAPDH) in these mouse lungs after IL-13 instillation. (B) Fold induction of Gob5 expression (normalized with GAPDH) in mouse TBE cultures after IL-13 treatment. n = 3; *P = 0.069; **P = 0.029.

Effect of JAK inhibitors on IL-13–induced hCLCA1 expression by human primary TBE cells. The experiment was performed the same as described in Figure 2. Before IL-13 treatment, cells were treated with different amounts of JAK inhibitors; JAK inhibitor I (A) and AG490 (B) for 30 min. The doses of JAK Inhibitor I were 10, 100, and 300 nM. The doses of AG490 were 2, 10, and 50 μM. Twenty-four hrs after IL-13 treatment, the cells were harvested for measuring the relative fold induction of hCLCA1 expression (normalized with GAPDH). The control study, treated with an equal amount of these inhibitors showed no quantitative change of hCLCA1 message in these cultures. For this reason, only one data point with “no treatment” was used in the figure. n = 3; *P = 0.005; **P = 0.009; ***P = 0.014.

Western blot analysis of STAT6 phosphorylation in primary humanTBE cells after IL-13 treatment. Primary human TBE cells were treated with IL-13 at 20 ng/ml, as described in Figure 2. At various times after the treatment, cultures were harvested for protein extraction and Western blot analysis with both anti–phosphorylated STAT6 (upper blot) and anti–total STAT6 protein (lower blot) antibodies. A similar result has been repeated in another primary human TBE culture derived from a different donor (data not shown).

Western blot analysis of hCLCA1 expression in human TBE cells after IL-13 treatment. Well-differentiated primary TBE cultures (at Day 21) were treated with IL-13 (10 ng/ml) as described in Figure 3. At 24, 48, and 96 h after IL-13 treatment, both untreated and treated cultures were harvested for protein preparations. Equal amount of proteins (30 μg/lane) were loaded on to the gel. Immunoblot preparation and analysis were performed as described in the text. Anti–β-tubulin staining was used to further verify the equal protein loading (data not shown). This figure is taken from one of three experiments, all of which showed similar findings.