Figure 6.

Deficiency of Scribble restores fitness of Yap/Taz^Δ/Δ HSC and associates with Rac activation. (A) Number of colony-forming units (CFU) from BM cells. (B) Cell-cycle analysis of Lin⁻ CD48⁻ CD150⁺ HSC harvested from the BM of Mx1Cre Wt, Taz^Δ/Δ;Yap1^Δ/Δ, and Scribble^Δ/Δ;Taz^Δ/Δ;Yap1^Δ/Δ mice 1 week after poly I:C. Stages of cell cycle were assessed by FACS analysis with incorporation of DNA binding Hoechst 33342 (2 mg/mL) and nucleotide binding PyroninY (0.25 mg/mL). (C) Kaplan-Meier survival analysis after serial myeloablation with 5-fluorouracil (150 mg/kg) 5 days apart. (D) Immunofluorescence of HSC showing Cdc42 expression and localization along with active Cdc42-GTP expression and localization. Nuclei are counterstained with DAPI and merged images are shown in the right micrographs. Scale bar 5 µm. (E) Quantification of Cdc42 expression measured by MFI. (F) Quantification of Cdc42-GTP expression measured by MFI. (G) Heat map depicting genes within the Rho guanyl nucleotide exchange factor activity and small GTPase activity gene ontology pathways showing differential regulation between Mx1Cre Wt, Taz^Δ/Δ;Yap1^Δ/Δ, and Scribble^Δ/Δ;Taz^Δ/Δ;Yap1^Δ/Δ HSC. (H) Rac1/Cdc42 activation PAK pulldown on lineage-depleted (Lin⁻) BM cells. (I) Cdc42 effector PAK pull-down from on Lin⁻ BM cells. (J) Cartoon depicting a complete picture of the Scribble polarity complex and its components in relation with HSC self-renewal and cell-cycle entry. *P < .05 and ***P < .001.