Fig 2: PAK2 affects proliferation of head and neck cancer cells.PAK2 expression was depleted in two types of HNC cell lines (H157 and H413), and differential proliferation status of these cell lines was analyzed. a, e Knockdown of PAK2 expression was analyzed at mRNA and protein levels (n = 3). b, f Relative cell proliferation was analyzed through MTT assay (n = 3). c, g mRNA and d, h protein expression status of cell proliferation marker Ki67 (left) was analyzed through qPCR and flow cytometer, respectively. (Right) Mean fluorescence intensity (MFI) of Alexa Fluor 488-Ki67 was measured using software FlowJo 10 (n = 3). i, k (Left) Cell-cycle stages were analyzed using flow cytometer (representative image of 3 independent experiment) (right) bar graph shows average of three independent experiments and j, l cyclin D1 (CCND1) protein expression level was analyzed through immunoblotting (n = 3). Abs absorbance. Samples in (a–c) and (e–g) were analyzed in duplicate. Error bars shows mean values ± SD. Differences were considered statistically significant with *P < 0.05, **P < 0.01 and ***P < 0.001, ns non-significant difference (P > 0.05)

Fig 3: Detection and validation of PAK2 overexpression in head and neck tumor samples.a Heat map of top 10 overexpressed histone modifiers in head and neck normal and tumor samples (generated using DNA Chip analyzer). b Quantitative mRNA expression analysis of KDM1A, PAK2 and DAPK3 normalized to ß-actin in10 normal and tumor samples of HNC patients. c Expression pattern of PAK2 gene in independent cohorts of head and neck cancer gene expression profiles. Box plot shows the elevated expression of PAK2 in head and neck tumor tissues compared to normal tissue. d Analysis of PAK2 expression in four different microarray profiles using Oncomine database. The y-axis represents log2-median centered intensity. e Analysis of PAK2 transcript per million (TPM) in normal and head and neck tumor samples through MiPanda (http://www.mipanda.org) cancer RNA-seq database. f Analysis of PAK2 protein expression in 26 normal and tumor tissue samples of HNC patients by PAK2 immunoblotting (also see Supplementary Figure S1g). N normal, T tumor, GSE Genomic Spatial Event. Error bars shows mean values ± SD. Differences were considered statistically significant with *P < 0.05, **P < 0.01 and ***P < 0.001, ns non-significant difference (P > 0.05)

Fig 4: Effect of anti-PAK siRNAs on JURKAT cell adhesion.Cells were nucleofected with 2 µl esiRNA against PAK1 or PAK2, with the combination of both esiRNAs (PAK1+PAK2) or with non-targeting siRNA (ctrl) and the cell adhesion to fibronectin (ACF- adherent cell fraction) was tested 22 h post nucleofection. The level of PAK1, PAK2 and pPAK2 (Ser141) was assessed at the same time by western-bloting. (A) The experiment with the highest silencing efficacy obtained. The graph of cell adhesivity shows means and standard deviations of sample quadruplicates. (B) Summary of results for all adhesion experiments (N = 7). The adherent cell fraction was expressed as relative to the adhesivity of controls and the resulting values were compared using paired Student’s t-test.

Fig 5: Expression of PAK kinases in the studied cell lines.Cell proteins were separated using gel electrophoresis and the expression levels of PAK1, PAK2, phospho-PAK (Ser 144/Ser141) and phospho-PAK2 (Ser20) were determined using western-blotting. (A) Lanes 1–8 correspond to the individual cell lines: (1) JURL-MK1, (2) MOLM-7, (3) K562, (4) CML-T1, (5) HL60, (6) Karpas-299, (7) Jurkat, (8) HEL. (B) CML-T1 cells were treated with IPA-3 at the indicated concetrations for 2 h, then lysed and subjected to electrophoresis and western-blotting. (C) Relative expression levels of phospho-PAK2 (Ser141) and PAK2 in cell lines treated for 2 h with IPA-3 at different concentrations. The band intensity from western-blots with anti-PAK antibodies was corrected for small differencies in protein loads using actin bands and the resulting values were expressed as relative to controls (without treatment). JURL-MK1:closed circles, MOLM-7:closed triangles, CML-T1:closed squares, K562:open circles, HL60:open squares, JURKAT:open triangles, Karpas-299:open diamonds, HEL:closed diamonds.