Figure 6.
Adenovirus-delivered shRNA knockdown of GALNT6 in polarized HBE cells. (A) GALNT6 colocalized with MUC1-N and Golgi marker 58K. HBE cells were treated with Ctrl or Smk medium for 4h and immunostained with antibodies against MUC1-N (red, top panels of left two lanes), 58K (red, top panels of right two lanes), GALNT6 (green, middle panels). Merged MUC1-N/GALNT6 images (yellow signals, bottom panels of left two lanes) indicate MUC1-N colocalized with GALNT6 (yellow arrowheads) before and after smoke treatment. Merged 58K/GALNT6 images (yellow signals, bottom panels of right two lanes) demonstrate localization of GALNT6 in Golgi before and after smoke exposure (yellow arrowheads). Cell nuclei were stained with DAPI (blue). Scale bar represents 50μm. (B) Pseudostratified HBE cells were infected with 1*MOI (multiplicity of infectivity) adenovirus-delivered scrambled shRNA or GALNT6 shRNA overnight, followed by 4 days in culture. Immunostaining of MUC1-N (red, top row), GALNT6 (green, 2nd row), and merged MUC1-N/GALNT6 (yellow, 3rd row) indicate colocalization. Nuclei were visualized with DAPI (blue). Scar bar represents 50μm. Monochrome images of GALNT6 were inverted to black signals (red circles, bottom panels) and quantitated with ImageJ software. Knocking down GALNT6 in GALNT6 shRNA-treated cells was compared to scrambled shRNA-treated control (designated as 1-fold) and graphed as mean ± SEM fold change. **p < 0.01, GALNT6 shRNA-treated cells versus scrambled shRNA control. (C) Polarized HBE cells were infected with 1*MOI or 2*MOI of adenovirus-delivered scrambled shRNA or GALNT6 shRNA overnight and cultured for another 4 days. Cells treated with Smk or Ctrl medium for 4h were IP’d with MUC1-C and probed with MUC1-N and MUC1-C antibodies. Densitometric quantitation of the 400kDa MUC1-N band (mean ± SEM, **p < 0.01) compared to scrambled shRNA/Ctrl exposed cells (designated as 1-fold),