INKA1-OE alters PAK4 localization and shifts CD34⁺ CD38⁻cells into a quiescence cluster. Transduced 8227 AML cells (n = 3) were sorted into 4 BFP⁺ fractions according to CD34/CD38 expression, fixated on slides, and subjected to confocal imaging of PAK4, CDK6, H4K16ac, and 4′,6-diamidino-2-phenylindole (DAPI). Images were processed by ImageJ/Fijji, generating a combined .csv file comprised of multiple parameter measurements per cell and stain (total cell, nuclear, and nonnuclear Integrated Density [IntDen], percent nuclear area, 403 cells). The resulting file was imported as .fcs file into FlowJo10. (A) Shown are the 2 CD34⁺ fractions per condition, combined with nuclear PAK4 levels (IntDen) plotted against nonnuclear PAK4 levels. (B) Example images of CTRL and INKA1-OE CD34⁺CD38⁻ and CD34⁺CD38⁺ cells captured by a Zeiss LSM700 Confocal (oil, 63×/1.4 NA, inverted, Zen 2012 software). Downstream analysis was performed with ImageJ/Fiji. Bars in merge image represent 2 μm. (C) Transduced 8227 AML cells were subjected to CD34 surface staining before intracellular staining for H4K16ac and Ki67 (n = 4-5). Student t test *P < .05. (D) The FlowJo10 TSne plugin was used for multidimensional reduction of the entire immunofluorescence data set (2 conditions with each 4 populations, 12 input parameters: total, nuclear, and cytoplasmic IntDen for CDK6 and PAK4, nuclear IntDen for H4K16ac and 4′,6-diamidino-2-phenylindole, percent nuclear area for all 4 stains; perplexity: 20, iterations: 1000, η: 200, Θ: 0.5). The resulting t-SNE plot revealed 2 major clusters (Cluster1, Cluster2). (E) Comparative visualization of Cluster1 (gray) and Cluster2 (violet) for nuclear (nucl) PAK4, CDK6, and H4K16ac protein expression levels (IntDen). (F) The same t-SNE x and y parameters were used to plot CTRL and INKA1 transduced cells individually. Split in 2 panels, the same data are shown as in panel D, with the individual 8227 subpopulations highlighted in different colors: CD34⁺CD38⁻(red), CD34⁺CD38⁺(orange); CD34⁻CD38⁺(green); CD34⁻CD38⁻(blue). (G) Cluster1 and Cluster2 gates were separately applied to the 2 CD34⁺ populations of CTRL and INKA1 to estimate their relative abundance in these clusters. (H-J) Analog analysis was performed with shCTRL and shINKA1 transduced and CD34⁺CD38⁻ sorted cells (n = 3). (H) Example images of CTRL and INKA1-KD CD34⁺CD38⁻ cells. (I) Shown are nuclear PAK4 levels (IntDen) plotted against nonnuclear PAK4 levels for all replicates. (J) Protein expression levels (IntDen) of CDK6, PAK4, and H4K16ac upon INKA1-KD (immunostaining). Student t test. *P < .05; ***P < .001.