Fig. 6

ADAM12 knockdown reduces the basal level of EGFR activation. a SUM159PT_shADAM12 or SUM159PT_shControl cells were incubated for 4 days in the presence or absence of 1 μg/ml of doxycycline. Alternatively, SUM159PT cells were transfected with a pool of four control siRNAs (siControl) or a pool of four ADAM12 siRNAs (siADAM12) and analyzed three days later by Western blotting. b Hs578T_shADAM12, Hs578T _shControl, or parental Hs578T cells were treated and analyzed as in panel a. c The effect of batimastat (BB-94) on the basal activation level of EGFR. SUM159PT cells were incubated for 24 h with 0, 10 μM, or 50 μM BB-94, in the absence or presence of serum, as indicated. d The effect of ADAM12 knockdown on the activation of EGFR is abolished by BB-94 or exogenous EGF. SUM159PT cells were transfected with control siRNAs or with two individual ADAM12 siRNAs. Two days after transfection, cells were incubated in complete media supplemented with 10 μM BB-94 for additional 24 h. Alternatively, three days after transfection, cells were incubated for 30 min with 20 ng/ml EGF. In a-d, representative blots are shown and relative changes in pEGFR/EGFR (means ± SEM; n = 3) are indicated; values significantly lower than 1 (P < 0.05) are shown in red. e qRT-PCR analysis of the indicated transcripts in SUM159PT_shADAM12 or SUM159PT_shControl cells incubated for 4 days in the presence or absence of 1 μg/ml of doxycycline. Expression was normalized to \( \beta \)-ACTIN and is shown as fold change after doxycycline treatment; 3≤n≤5