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. 2011 Jan 6;286(11):9268–9279. doi: 10.1074/jbc.M110.179903

FIGURE 7.

FIGURE 7.

A, TNF-α activation of NFκB is independent of Src and EGFR. LLC-PK1 cells grown on coverslips were pretreated with 100 nm AG1478 or PP2 for 30 min followed by 10 ng/ml TNF-α for 30 min. The cells were fixed and permeabilized, and p65 NFκB was visualized by immunostaining with anti-p65 and CY3-coupled secondary antibody. The nuclei were counterstained using DAPI. The pictures show p65 (red) and DAPI (purple) staining of the same field as well as the merged image. B and C, TNF-α enhances proliferation through the EGFR. MDCK cells were plated in 96-well plates, serum-depleted overnight, and then exposed to 10 ng/ml TNF-α or 100 ng/ml EGF for 24 h. DNA synthesis was assessed using a BrdU incorporation assay as described under “Experimental Procedures.” In each experiment the BrdU signal in the control cells were taken as 1, and-fold changes were calculated. B shows data from n = 4 independent experiments performed in triplicate. C shows data of a typical experiment performed in 6 parallel determinations. In C, the asterisk indicates that the column is significant versus control (p < 0.05). D–F, shown is the effect of TNF-α on epithelial cell growth measured using ECIS. Changes in the capacitance (D) and impedance (E) were followed using ECIS as described under “Experimental Procedures.” Curves represent the averages of the duplicate measurements. D and E show capacitance and resistance data of the same measurement, respectively. All curves were normalized to the first obtained point. The red dashed lines on D and E indicate the 2 and 8 h time points, and the blue dashed lines indicate the 0.25 capacitance point. F summarizes data from capacitance measurements. In each measurement the time required for a 75% drop in the capacitance (0.25 value on the normalized capacitance curve) was determined and expressed as -fold change compared with the control in the same experiment taken as 1. The graph shows data from n = 8 curves obtained in 4 independent experiments (mean ± S.E., n = 8). The asterisk indicates p < 0.01 versus control.