Figure 4.
Smoke provoked formation of MUC1-C/galectin-3/EGFR complexes in polarized HBE cells. (A) HBE cells were treated with Ctrl or Smk medium at 23 (25%), 46 (50%) and 92 (100%) mg/m3 total suspension particles (TSP) for 4h. MUC1-C IP was analyzed by WB probed with galectin-3, EGFR and MUC1-C antibodies. Blots were stripped and reprobed with TyrP antibodies (4G10, PY20 and PY100). Bands recognized by 4G10 overlapped with EGFR (175kDa) and MUC1-C (20-25kDa) bands. Bands recognized by PY20 (20-25kDa) and PY100 (20-25kDa) overlapped with each other and with MUC1-C. Equal loading was confirmed with IgH bands. Quantitation of galectin-3, EGFR, EGFR-TyrP (4G10), MUC1-C-TyrP (4G10) and MUC1-C-TyrP (PY100) pulled down by MUC1-C after 100% Smk exposure was normalized to untreated control (designated as 1-fold) and reported as mean ± SEM fold change. **p < 0.01, Smk-treated cells versus untreated Ctrl. (B) HBE cells were treated with Ctrl or 100% Smk medium for 4h and immunostained with antibodies directed against MUC1-C (red, top panels), galectin-3 (green, middle panels of left two lanes) or EGFR (green, middle panels of right two lanes). Merged MUC1-C/galectin-3 signal (yellow, bottom panels of left two lanes) indicates smoke-induced MUC1-C/galectin-3 interaction (yellow arrowheads). Merged MUC1-C/EGFR signal (yellow, bottom panels of right two lanes) demonstrates smoke-induced formation of MUC1-C/EGFR complexes (yellow arrowheads). Cell nuclei were stained with DAPI (blue). Scale bar represents 50μm.