Fig 2: Identification of PDK1, KDR, and ZNF367 as the Targets for PAK1 in Human SSCs(A and B) Electropherogram showed the concentrations and nucleotides (nt) of RNA isolated from the human SSC line with the control siRNA (A) and PAK1-siRNA 2 (B). (C and D) Scatterplots (C) and hierarchical clustering (D) illustrated the differentially expressed genes (DEGs) between PAK1-siRNA 2 and control siRNA. Red dots and green dots in (C) represented upregulated and downregulated genes, respectively. (E) Real-time PCR revealed the changes of PDK1, KDR, ZNF367, GPX3, OIP5, THAP10, DBP, and TET1 mRNA by PAK1-siRNA 2 compared to the control siRNA. *Statistically significant differences (p < 0.05) between PAK1-siRNA 2-treated cells and the control siRNA.

Fig 3: STAT5S725 and STAT5S779 phosphorylations are independent of BCR-ABLp185 kinase activity and STAT5Y694 phosphorylation. (a) BCR-ABLp185+ cells were treated with imatinib (2 µM) and STAT5 phosphorylation on residues Y694, S725 and S779 monitored by immunoblotting. (b) Immunoblotting of K562 cells after treatment with 2 µM imatinib for indicated time points. Levels of active versions of PAK1 and PAK2 (pPAK1T423; pPAK2T402) do not alter upon inhibition of BCR-ABL kinase activity. (c) Immunoblotting of Stat5fl/fl Mx-1Cre+ BCR-ABLp185+ cells expressing indicated STAT5 mutants. Endogenous Stat5 was deleted via interferon-ß (IFN-ß) treatment 4 weeks before use. STAT5 proteins harboring a Y694 mutation maintain phosphorylations on S725 and S779. (d) Dose–response curves of PAK2 shRNA- or random shRNA-expressing BCR-ABLp185+ cells toward PF-3758309 (left panel) or imatinib (right panel). (e) Scheme summarizing activation of STAT5 via PAK1/2 kinases independently of BCR-ABL kinase activity.

Fig 4: Rac1 expression activates macropinocytosis in H1299 cells in a PAK dependent manner and is necessary for glucose independence. (A) Immunoblot analysis comparing Rac signaling in Rac1 WT and Rac1 KO H1299 cells generated using CRISPR-Cas9 technology. The two knockout clones were generated using two independent guides against Rac1. (B) Quantification of the levels of FITC-dextran uptake in glucose starved H1299 Rac1 WT and H1299 Rac1 KO cells. Data is normalized to Rac1 WT cells, error bars indicate ± SD of 10 fields scored containing at least 10 cells. (C) Relative density of Rac1 WT and H1299 Rac1 KO cells cultured in GFM for 48 h as determined by crystal violet staining. Data is normalized to density of Rac1 WT cells at 48 h, error bars indicate ± SEM of three independent experiments. For (B,C), significance was calculated using Student’s t-test. (D) Immunoblot analysis of Pak signaling in Rac1 KO H1299 control cells or cells stably overexpressing the constitutive active Pak1 mutant T423E. (E) Quantification of the levels of FITC-dextran uptake in glucose starved Rac1 WT, Rac1 KO, and Rac1 KO/Pak1 (T423E) cells. Data is normalized to Rac1 WT cells; error bars indicate ± SD of 10 fields scored containing at least 10 cells. Significance was calculated using ANOVA with Holm-Sidak multiple comparisons across cell lines, * p < 0.05, ** p < 0.005, *** p < 0.0001.

Fig 5: PI3K regulates Rac-driven macropinocytosis. (A) Immunoblot analysis of phospho-Akt (Thr308) in glucose independent (red) and glucose addicted (blue) cells following 6 h of glucose starvation. (B) Immunoblot analysis of Pak and Akt phosphorylation following treatment with vehicle or 1 µm BKM120 for 1 h. (C) Quantification of FITC-dextran uptake in glucose starved cells treated with either vehicle or 1 µm BKM120 for 1 h prior to incubation with FITC-dextran. Data is normalized to vehicle treated cells. Error bars indicate ± SD of 10 fields scored containing at least 10 cells, and statistics was calculated using Student’s t-test. (D) Immunoblot analysis of Pak signaling in Rac1 KO H1299 control cells or cells stably overexpressing the constitutive active Pak1 mutant T423E following treatment with vehicle or 1 µm BKM120 for 1 h. Densitometry was used to quantify P-Pak band intensities normalized to T-Pak1. (E) Quantification of FITC-dextran uptake in glucose starved Rac1 WT, Rac1 KO, and Rac1 KO/Pak1 (T423E) cells treated with vehicle or 1 µm BKM120 1 h prior to incubation with FITC-dextran. Data is normalized to Rac1 WT cells, error bars indicate ± SD of 10 fields scored containing at least 10 cells. Significance was calculated using ANOVA with Holm-Sidak multiple comparisons between control and BKM120 treated cell lines, ** p < 0.005, *** p < 0.0001.