Fig 2: PECAM1 and CXCR4 are highly expressed in LPS-treated HDPFs. (A) Morphology of the HDPFs observed under the microscope. (B) Expression of the HDPF-specific biomarkers determined by immunofluorescence staining. (C) Positive expression of HDPF-specific biomarkers examined by flow cytometry. (D–F) mRNA and protein expression of MEF2C. (D) PECAM1 (E), and CXCR4 (F) in cells examined by RT-qPCR and immunofluorescence staining, respectively. (G–I), secretion of TNF-α, IL-6, IL-8 in cells determined using ELISA kits. Data were collected from three individual experiments and expressed as mean ± SD. Data were analyzed by unpaired t-test. **p < 0.01 vs. PBS.

Fig 3: Overexpression of MEF2C reverses the functions of shPECAM1 and shCXCR4 in cells. (A,B) MEF2C overexpression vector was further administrated into HDPFs in the presence of shPECAM1 and shCXCR4. (A,B) mRNA and protein expression of MEF2C, PECAM1, and CXCR4 in cells determined by RT-qPCR and western blot analysis, respectively. (C–F) Levels of PGE2, TNF-α, IL-6, and IL-8 in cells determined by RT-qPCR and ELISA kits, respectively. (G) apoptosis of HDPFs determined by TUNEL assay. Data were collected from three individual experiments and expressed as mean ± SD. Data were analyzed by one-way (C–G) or two-way ANOVA (A,B) followed by Tukey’s multiple comparison test. **p < 0.01 vs. shPECAM1 + oe-NC; ##p < 0.01 vs. shCXCR4 + oe-NC.

Fig 4: Knockdown of MEF2C reduces LPS-induced inflammation and apoptosis in HDPFs. (A) mRNA expression of MEF2C, PECAM1, and CXCR4 in cells after sh-MEF2C transfection examined by RT-qPCR. (B–E) mRNA and protein levels of inflammatory cytokines PGE2, TNF-α, IL-6, and IL-8 in cells examined by RT-qPCR and ELISA kits. (F) Apoptosis of HDPFs after sh-MEF2C transfection examined by TUNEL assay. Data were collected from three individual experiments and expressed as mean ± SD. Data were analyzed by the unpaired t-test (B–F) or two-way ANOVA (A) followed by Tukey’s multiple comparison test. **p < 0.01 vs. LPS + sh-NC.

Fig 5: PEACM1 binds to CXCR4 to activate the NF-κB signaling pathway. (A,B) Co-localization of PECAM1 and CXCR4 in rat dental pulp tissues (A) and in HDPFs (B) determined by double immunofluorescence labeling. (C–D) Binding relationship between PECAM1 and CXCR4 determined by IP assays. (E–F) Phosphorylation of NF-κB p65 in rat dental pulp tissues determined by western blot analysis (E) and IHC staining, respectively. (G) Phosphorylation of NF-κB p65 in HDPFs measured by western blot analysis. (H) Sub-cellular localization of phosphorylated p65 in cells determined by immunofluorescence staining. Data were collected from three individual experiments and expressed as mean ± SD. Data were analyzed by one-way (F,H) or two-way ANOVA (E,G) followed by Tukey’s multiple comparison test. **p < 0.01.