Fig. 4

NS-398 effects on morphology and neuron specific and epithelial cell specific protein expression. C4-2b cells were cultured with NS-398 (10 µM) for 72 h, samples were obtained every 24 h. Cells were photographed and total cellular protein isolated. (A) Morphological changes in C4-2b: phase contrast photomicrograph of 72 h C4-2b cultures (400X magnification), (left) 0.1% DMSO treated control C4-2b cells and (right) 10 µM NS-398 treated cells. A low cell density area was photographed in the NS-398 cultures to visualize dendrite-like cytoplasmic processes. (B) Cytokeratin 18 in C4-2b cell lysate protein: Cytokeratin 18 was detected by Western blot analysis. 300 ng of total protein was loaded per well. Lane 1, 0.1% DMSO control; lane 2, 10 µM NS-398. Cytokeratin-18 was quantified by image intensity analysis of Western blots using a digital imaging system. Cytokeratin-18 levels are expressed as arbitrary area intensity units (255 equivalent to black, 0 equivalent to white). Band intensity analysis determined a 2-fold increase in cytokeratin 18 concentration in NS-398 treated cells (8516 ± 1558, DMSO control; 20680 ± 1760, NS-398 treatment). Blot is representative of data from two independent experiments.