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Fig. 7 CXCL8 links PKC-δ to the focal adhesion complex (A) E4-HUVEC and parental HUVEC cells were treated with rhCXCL8 (10 ng/mL) for the indicated times. The black triangle indicates the ∼130-kDa form of p-PKC-δ and the white triangle indicates the ∼78-kDa form. (B) Immunoprecipitation (IP) for p-PKC-δ with immunoblotting for p-paxillin and actin is shown. Input lysate is indicated by (+); (−) indicates beads-only control. Treatment was with 10 ng/mL rhCXCL8 or PBS for 30 min. (C) E4-HUVEC or parental HUVEC cells were treated with 10 ng/mL rhCXCL8 for the indicated times and immunoblotting for p-ERK1/2 and ERK1/2 was performed. (D) E4-HUVEC cells were transduced with lentivirus expressing shRNA against PRKCD or scramble control. Immunoblotting for p-ERK1/2 and ERK1/2 was performed. (E) shRNA knockdown of PKC-δ expression. (F) Bulk RNA sequencing was performed in E4-HUVEC cells treated with rhCXCL8 for 6 h or PBS as a control. Significantly differentially regulated genes are shown with key hematopoietic factors highlighted. Each row indicates a biological replicate. (G) Proposed model for HSPC remodeling of the niche via CXCL8 and PKC-δ. HSPCs produce CXCL8 upon encountering the vascular niche, which signals via CXCR1, releases DAG, and activates PKC family members. Transcription of PKC-δ is normally tightly controlled in order to maintain a reserve capacity to support HSPCs. CXCL8 signaling induces association of PKC-δ with the focal adhesion complex, activating niche functions such as HSPC cuddling and growth factor production. See also Figure S7 and Table S5 and S6.