Fig. 4

Loss of SEMA5A induces EMT and activates Wnt signaling in PC cells. a-b Western blot analysis of whole cell lysates of T3M-4-Control and -shSEMA5A cells (a) and CD18/HPAF-Control and –shSEMA5A cells (b). The analysis shows a decrease in protein level of SEMA5A, E-cad, increase in N-cad, Snail and β-catenin in T3M-4-shSEMA5A cells or CD18/HPAF-shSEMA5A cells in comparison with T3M-4-Control cells (a) or CD18/HPAF-Control cells (b) β-actin or GAPDH was used as a loading control. The intensity of the bands in western blot analysis was quantified by using Image J software and was normalized with respect to the T3M-4-Control cells or CD18/HPAF-Control cells. c-e Immunofluorescence analysis of E-cad (c), N-cad (d), and β-catenin (e) in T3M-4-Control and T3M-4-shSEMA5A cells. Immunofluorescence analysis is showing lower E-cad expression on the plasma membrane in T3M-4-shSEMA5A cells in comparison with Control cells (c). Immunofluorescence analysis of T3M-4-Control and T3M-4-shSEMA5A cells showing an increase in N-cad expression on the plasma membrane as well as cytoplasm in T3M-4-shSEMA5A cells (d). Images (c-d) were acquired using LSM 710 Zeiss Confocal Microscope. E-cad/N-cad is stained in red, and the nucleus is stained with DAPI (blue). e Immunofluorescence analysis is showing an increase in intensity and transition of β-catenin localization from the plasma membrane to the cytoplasm in T3M-4-shSEMA5A cells. β-catenin is localized to the plasma membrane in T3M-4-Control cells. Images are taken using a Nikon Eclipse E800 fluorescent microscope. β-catenin staining is depicted in red, and the nucleus is stained with DAPI in blue. Scale bar represents 10 μm in length. f Bar graph showing an increase in fold expression of transcription factor-SNAIL (p = 0.035) in T3M-4-shSEMA5A in comparison with Control cells evaluated using RT-PCR. The Ct values in RT-PCR were normalized to HPRT. Values are mean Fold changes ± SEM (bars) of two experiments. g Bar graph showing an increase in Wnt activity T3M-4-shSEMA5A than T3M-4-Control cells. TOP-FLASH was utilized, and this assay and values were normalized to both FOP and Renilla vector illustrating increased Wnt activity upon SEMA5A knockdown. The bars in the graphs represent fold changes ± Standard deviation and * - represents statistical p-value less than 0.05 using Student t-test