Fig 2: Overactivated Pparα Causes Cytotoxicity(A) HepG2 cells and LO2 cells was treated with a high dose (HD) of WY-14643 (400 μM) or vehicle (Veh) DMSO for 48 hr, and then the protein levels of Pparα and Lmnb1 from HepG2 cells nuclear protein or Prkcd and β-actin from LO2 cells lysate were detected by western blotting (upper) and calculated by ImageJ (lower). (B) HepG2 cells were treated with a high concentration of WY-14643 (400 μM) or vehicle DMSO for 24 hr, and then flow cytometry was performed to analyze the ROS levels in HepG2 cells. Left, representative flow cytometry analysis showing the intensities of ROS (CM-H2DCFDA) in HepG2 cells. Right, histogram summarizes the ROS levels in HepG2 cells. (C) HepG2 cells were treated with a high concentration of WY-14643 (400 μM) or vehicle DMSO for 48 hr, and then the expression levels of Cyclind1 and Il1a were assayed using quantitative real-time PCR. (D) LO2 cells and HepG2 cells were treated with a high concentration of WY-14643 (400 μM) or vehicle DMSO for 48 hr, and then cells were collected. The protein expression of Rad51 and γ-H2a.x were measured by western blotting (upper) and calculated by ImageJ (lower); the increased Rad51 protein expression and γ-H2a.x were observed in WY-14643-treated LO2 cells and HepG2 cells, respectively. B-actin was used as an internal control for the examination of Rad51, while Lmnb1 was used as an internal control for the detection of γ-H2a.x. (E) HepG2 cells were treated with a high concentration of WY-14643 (400 μM) or vehicle DMSO for 48 hr, and then cells were subjected to the TUNEL assay. The data are shown as means ± SDs, *p < 0.05, **p < 0.01, determined by ANOVA.

Fig 3: Exogenous Expression of miR-181a-5p Reduces the Cytotoxicity Induced by Pparα Overactivation(A) HepG2 cells and LO2 cells were transfected with the miR-181a-5p mimic or control mimic for 6 hr and then were treated with a high concentration of WY-14643 (400 μM) for 48 hr, and then the protein levels of Pparα and Lmnb1 from HepG2 cells nuclear protein and Prkcd and Gapdh from LO2 cells lysate were detected by western blotting (upper) and calculated by ImageJ (lower). (B) HepG2 cells were transfected with the miR-181a-5p mimic or control mimic for 6 hr and then treated with a high concentration of WY-14643 (400 μM) for 24 hr, followed by flow cytometry analysis of the ROS levels in HepG2 cells. Left, representative flow cytometry analysis showing the intensities of ROS (CM-H2DCFDA) in HepG2 cells. Right, histogram summarizes the ROS levels in HepG2 cells. (C) HepG2 cells were transfected with the miR-181a-5p mimic or control mimic for 6 hr and then were treated with a high concentration of WY-14643 (400 μM) for 48 hr, followed by assay of the expression of Cyclind1 and Il1a using quantitative real-time PCR. (D) LO2 cells and HepG2 cells were transfected with the miR-181a-5p mimic or control mimic for 6 hr and then were treated with a high concentration of WY-14643 (400 μM) for 48 hr, and then cells were collected. The protein expression of Rad51 and γ-H2a.x were measured by western blotting (upper) and calculated by ImageJ (lower); the decreased Rad51 protein expression and γ-H2a.x were observed in WY-14643-treated LO2 cells and HepG2 cells, respectively. B-actin was used as an internal control for the examination of Rad51, while Lmnb1 was used as an internal control for the detection of γ-H2a.x. (E) HepG2 cells were transfected with the miR-181a-5p mimic or control mimic for 6 hr and then were treated with a high concentration of WY-14643 (400 μM) for 48 hr, and then cells were subjected to the TUNEL assay. The data are shown as means ± SDs, *p < 0.05, **p < 0.01, determined by ANOVA.

Fig 4: Correlation between B7-H4 and p-PKCδ levels. A TCGA dataset analysis was performed on the GEPIA website (http://gepia.cancer-pku.cn/). This analysis showed that PRKCD (PKCδ) mRNA expression was positively correlated with VTCN1 (encoding B7-H4) mRNA expression. B The protein levels of B7-H4 and p-PKCδ in the NCM460, SW480, HCT116, SW620 and RKO cell lines were determined by Western blot analysis. The data are expressed as the mean ± SD values; n = 3. C Serial p-PKCδ+ tumor sections were examined for B7-H4 expression, and positive staining (400 ×) of B7-H4 and p-PKCδ in CRC samples is shown. Double IF staining of CRC samples was performed. CRC tissue samples were stained for p-PKCδ (green, 200 ×) and B7-H4 (red, 200 ×). B7-H4+/p- PKCδ+ cells were identified in CRC tissue specimens (dark yellow; original magnification). D TCGA COAD dataset analysis was performed on the XENA website. This analysis showed that the B7-H4 mRNA level was positively correlated with lymph node metastasis

Fig 5: 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.