Figure 3

Downregulation of PAK4 inhibits anchorage-independent growth, migration and modulated gene expression profile in glioma xenograft cells. (a) Quantitative RT-PCR showing the relative PAK4 mRNA levels normalized to internal GAPDH controls. Mean±S.E. values obtained from three replicates were presented (*P<0.01). (b) Whole-cell lysates were subjected to immunoblotting in mock-, pSV- and PAK4si-treated cells after 48 h of transfection. Blots were re-probed with GAPDH to monitor equal loading. Relative PAK4 protein levels were estimated using ImageJ 1.42 (NIH) and plotted as mean±S.E. values obtained from three independent experiments (*P<0.01). (c) The 4910 and 5310 cells cultured in adhered (Adh) and suspended (Sus) conditions in 96-well plates were subjected and to pSV and PAK4si treatments for 48 h. The percentage of cell viability was estimated as described in Materials and Methods and plotted as mean±S.E. values obtained from three repetitive experiments (*P<0.05 and **P<0.01). (d) Percentage of cell adhesion on vitronectin and fibronectin (5 μg/ml each) was determined and graphically represented as mean±S.E. obtained from three experimental replicates. (e) Phase contrast microscopic pictures of pSV- and PAK4si-treated 4910 and 5310 cells (left). The mean±S.E. values of percentage of rounded cells were plotted in the bar diagram and significance was represented by * at P<0.01 (right). (f) Transwell matrigel invasion assay depicts the invaded cells in pSV- and PAK4si-treated cells after Hema-3 staining (top). Percentage cell invasion was represented as mean±S.E. obtained from three experimental replicates, *P<0.01 (bottom). (g) Wound-healing migration assay was performed as described in Materials and Methods and representative images from three independent experiments were presented (top). The average migration distances were measured using a light microscope with an ocular micrometer and plotted in the form of mean±S.E. values obtained from three replicates. Significant difference was represented by * at P<0.01 (bottom). (h) Modulation of gene expression profile in PAK4si-treated 4910 cells in comparison with pSV-treated controls. The cDNA PCR arrays were performed using RT² Profiler PCR Array kits (SA Biosciences) for ECM and adhesion; MAPK, PI3K/AKT and JAK/STAT signaling pathways as described in Materials and Methods and relative fold change values obtained from three experimental replicates were represented in the form of heat maps