Fig 2: Proposed model for the role of Rac1 signalling in the observed transcriptional switch. On receptor activation, exchange factors (GEF) promote GTP binding of Rac1 that stimulates two independent pathways. Active Rac1 binds and activates protein kinase PAK1 that migrates into the nucleus and phosphorylates chromatin-bound BCL-6, leading to its inactivation and loss of promoter occupancy. In parallel, a protein complex is formed between active Rac1, MgcRacGAP and STAT5, promoting phosphorylation by a tyrosine kinase (TK) and translocation into the nucleus. Here, MgcRacGAP stimulates GTP hydrolysis by Rac1 and phospho-STAT5 is released and activates gene transcription following binding to the vacant promoter sites previously repressed by BCL-6.

Fig 3: Mutated active Rac1 plasmid (Q61L) recuses the function in DEPDC1B-knockdown cells. (A) The levels of active Rac1, total Rac1, phosphorylated PAK1, and total PAK1 protein were detected by Western blotting in DEPDC1B-knockdown cells combined with Q61L. (B and C) Representative images of cell migration (B) and invasion (C) were analyzed using DEPDC1B-knockdown or control cells combined with Q61L (left panels), and a quantification analysis of the migrated or invaded cell counts (right panels) is shown. (D) Representative images of wound-healing assays using DEPDC1B-knockdown or control cells combined with Q61L in DU145 (left panels) and PC3 (right panels) cells, showing reversing cell motility after using Q61L in DEPDC1B-overexpressing cells. (E) A quantification analysis of cell migration index is shown. (F) Representative images of three-dimensional (3D) cell culture using DEPDC1B-knockdown or control cells combined with Q61L showing rescued cell motility after using Q61L in DEPDC1B-knockdown cells. (G) Cell viability was rescued by using Q61L in DEPDC1B-knockdown or control cells. (H) Colony formation assays were constructed in DEPDC1B-knockdown or control cells combined with NSC23766. (I) A quantification analysis of the colony formation number was shown. Unpaired t-test was used to analyze two groups of data; *P < .05, **P < .01, and ***P<.001. Two-way ANOVA was performed to analyze factorial designed data, # P < .05, ##P < .01, and ###P<.001. Scale bars: 25 µm

Fig 4: Tiam1 and Pak modulate responses to Eg5 inhibitors.(a–e) MDCK II cells were transiently transfected with two siRNAs against Pak2 or non-targeting control (NT) as indicated, and 2 days later treated with 25 µM monastrol for 16 h. (a) Cells were lysed and analysed for knockdown by immunoblotting with the indicated antibodies. (b) Cells were fixed and stained for FACS analysis with propidium iodide and MPM2 antibody to detect % of mitotic cells (n?3, 10,000 cells/rep). (c–e) Cells were fixed and then stained by IF with antibodies against a-tubulin, ?-tubulin and DAPI. (c) Example mitotic spindles: a-tubulin=green, ?-tubulin=red and DAPI=blue, scale bars, 5 µm. (d) Quantification of the proportion of bipolar spindles (n?3). (e) Quantification of the proportion of mitotic cells in anaphase and telophase (n?3). (b–e) All graphs show mean+s.e.m. Unpaired two-sided t-test: **P<0.01. (f) MDCK II cells expressing histone-2B-GFP (H2B-GFP) and a-tubulin-RFP were transiently transfected with siRNAs against Tiam1, Pak1, Pak2 or non-targeting control (NT) as indicated for 2 days then treated with 25 µm monastrol before being imaged using time-lapse confocal microscopy as described in Methods. Graph shows % of cells which reached metaphase as a function of time after the start of prophase (first frame with evidence of condensed chromosomes). Small vertical lines indicate cells at the end of imaging time. Gehan-Breslow-Wilcoxon test: **P<0.001 (compared with NT control).

Fig 5: Rac1 signalling switches promoter occupancy from BCL-6 to STAT5. (A) DLD-1 cells were transfected with Myc-Rac1-Q61L or control empty vector and 24 h later analyzed by Western blot for the subcellular distribution of STAT5 between a soluble (S) and a chromatin-bound non-soluble (NS) fraction (detection of a-tubulin and histone 2B served as controls). Note that active Rac1 promotes retention of STAT5 in the non-soluble chromatin fraction. (B) Subcellular localization of STAT5 determined by confocal fluorescence microscopy in DLD-1 cells co-transfected with DsRed-Rac1-Q61L and GFP-STAT5A. The overlay image of the DAPI, GFP and DsRed channels is shown. A microscopic field was chosen that contained side by side untransfected cells (blue nuclei), cells that transfected only with GFP-STAT5 (green cells) and cells that co-transfected with both GFP-STAT5 and DsRed-Rac1-Q61L (red cells). Note the nuclear STAT5 signal in Rac1-expressing red cells. In addition, two plots are given showing the DAPI and GFP signal intensities measured along the indicated regions of interest (ROI, white lines). The signal intensity of GFP did not increase across the nuclear DAPI region when cells expressed only GFP-STAT5 (green cells, ROI 1), whereas nuclear GFP signal clearly increased when cells co-expressed active Rac1 (red cells, ROI 2), confirming nuclear translocation of GFP-STAT5. (C) DLD-1 cells were co-transfected with the reporter and the indicated expression vectors, as described for Figure 1B. Note that STAT5 activates the BCL-6 luciferase reporter and, when combined with PAK1, reaches the stimulation levels normally induced by active Rac1, *P < 0.05 and #P > 0.05. (D) Chromatin immunoprecipitation (ChIP) of the reporter vector with anti-(a)-BCL-6, a-STAT5 or a non-specific antibody (NS immunoglobulin G) from lysates of DLD-1 cells transfected with the indicated expression vectors. A representative semi-qPCR of the precipitated promoter fragment quantities with a graphical representation of the respective band intensities quantified by densitometry from digital images obtained in 3 independent transfection experiments, *P < 0.05, is shown. Two serial dilutions of input DNA were co-amplified to guarantee semi-qPCR conditions and allow product quantity extrapolation from band intensities.