Figure 2

Differential display and Northern blot analysis of R5020 and epidermal growth factor (EGF) regulation of desmoplakin I (DPI) and II (DPII) in T47D cells. (a) Autoradiography of a representative differential display RT-PCR acrylamide gel. ³³P-labeled DNA fragments generated by RT-PCR from RNAs of control T47D cells (lane 1), 10 nmol/l R5020 treatment (lane 2), 10 nmol/l EGF treatment (lane 3), 10 nmol/l R5020 plus 10 nmol/l EGF treatment (lane 4). Each lane contains 5 μL PCR mixture. (A) 1:20 dilution of RT-PCR cDNA. (B) 1:40 dilution of RT-PCR cDNA. The arrow indicates desmoplakin (DP). Molecular weight standards of 0.65, 0.42, and 0.41 kb are indicated in lane 5. (b) Photograph of a representative Northern blot analysis of DPI and DPII, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in T47D cells. Lane 1: control T47D cells were left untreated for 48 hours. Matched sister colonies of T47D cells received 10nmol/l R5020 (lane 2), 10 nmol/l EGF (lane 3), or 10 nmol/l R5020 plus 10 nmol/l EGF for 48 hours (lane 4). Ten micrograms of total RNA was subjected to Northern blot analysis as described in the Methods section. (c) The Northern blot films were scanned. The graphs indicate the fold difference of desmoplakin expression compared vehicle (set at a value of 1) following measurement of the band intensities. In each independent experiment, the desmoplakin band for each treatment condition was normalized to the GAPDH signal. The experiment was repeated three times and the mean values from these experiments ± SEM are reported. Statistical significance was determined using t-test. *P < 0.05, **P < 0.01, versus control.