Fig. 1

Confirmation of SEMA5A knockdown in PC cells and resulting mesenchymal morphology. a. Bar graph showing relative fold decrease in mRNA expression of SEMA5A. HPRT is used as a control by RT-PCR analysis. The values are mean relative fold changes ± Standard Error of Mean (SEM, bars). b-c. Western blot analysis of cell lysates of T3M-4-Control and T3M-4-shSEMA5A cells (b) and Control and CD18/HPAF-shSEMA5A cells (c) showing decreased SEMA5A protein expression with GAPDH as a loading control. Quantification of the protein of interest by the intensity of the bands with respect to their loading control was performed by Image J software. Bands were normalized on the T3M-4-Control cells or CD18/HPAF-Control cells. d-e Morphological changes upon SEMA5A knockdown. Cobblestone-like T3M-4-Control cells change to spindle-shaped cells in T3M-4-shSEMA5A cells. Furthermore, on expressing Flag-tagged mouse sema5A in T3M-4-shSEMA5A cells, the cells reverted back to their cobblestone appearance. Scale bar represents 100 μm in length (d). The transition of overlapping colonies of CD18/HPAF-Control cells to flat spindle-shape cells in CD18/HPAF-shSEMA5A cells. Scale bar represents 10 μm in length (e)