Fig 2: Kdm5a represses Notch2 and Notch signaling in SCLC in vivo. (A) Representative H&E and IHC for Kdm5a and Notch2 in SCLC lung tumors from LSL-Cas9 mice injected with either sgKdm5a RPP or sgControl RPP adenoviruses. Scale bar, 100 µm. (B) Quantification of percent Notch2-positive tumor cells by IHC from A. n = 11 lung tumors (sgKdm5a RPP) or 19 lung tumors (sgControl RPP). (C) Gene set enrichment analysis (GSEA) using the hallmarks gene sets of NOTCH signaling (left), epithelial mesenchymal transition (middle), and KRAS signaling up (right), from the RNA-seq data obtained from lung tumors arising in the LSL-Cas9 mice injected with sgKdm5a RPP compared with sgControl RPP adenoviruses. n = 8 lung tumors for each group. q-values are indicated. (D) Immunoblot analysis of cell lines maintained on J2 fibroblast feeder layers (see Materials and Methods) from SCLC tumors that grew in LSL-Cas9 mice injected with sgKdm5a RPP or sgControl RPP adenoviruses. (E) Volcano plot of the RNA-seq data showing the log2fold change in gene expression in sgKdm5a RPP tumors compared with sgControl RPP tumors. Dlk1 is a canonical Notch inhibitory ligand. (F,G) Dlk1 (F) and Nell1 (G) mRNA levels, as determined by qRT-PCR, in samples used for the RNA-seq experiment above. n = 8 for sgKdm5a RPP, n = 8 for sgControl RPP. For all experiments, data are represented as ±SEM. (*) P < 0.05. (**) P < 0.01.

Fig 3: Downregulation of KDM5A promoted osteogenic differentiation of hPDLSCs with periodontitis. KDM5A shRNA lentivirus was transfected into hPDLSCs in the LPS+OI group and hPDLSCs transfected with NC shRNA were set as the control. Then, samples of hPDLSCs were divided into the LPS+OI group, LPS+OI+sh-NC group, and LPS+OI+sh-KDM5A group. A: RT-qPCR detected the interference efficiency of KDM5A shRNA; B: ALP staining and ALP activity determination were conducted after 7 days of OI; C: Alizarin red staining was performed to detect mineralization of hPDLSCs after 14 days of OI; RT-qPCR (D) and Western blot (E) detected the level of Runx2, OCN and OPN in hPDLSCs. Each cell experiment was performed 3 times and data were represented as mean ± SD; Comparisons among multiple groups in panels A–C were analyzed using one-way ANOVA; Comparisons among multiple groups in panels D–E were analyzed using two-way ANOVA. All data were checked by Tukey's multiple comparisons test. **P < 0.01.

Fig 4: miR-495-3p knockdown weakened the effect of sh-KDM5A to promote osteogenic differentiation in hPDLSCs with periodontitis. miR-495-3p inhibitor-lentivirus was transfected into hPDLSCs with NC transfection as the control. A: RT-qPCR detected the interference efficiency of miR-495-3p inhibitor; B: ALP staining and ALP activity determination were performed after 7 d of OI; C: Alizarin red staining and detection of mineralized nodules were performed after 14 d of OI; D–E: RT-qPCR and Western blot detected the expression patterns of Runx2, OCN and OPN after 14 d of OI; F: RT-qPCR detected the HOXC8 mRNA expression. Each cell experiment was performed 3 times and data were represented as mean ± SD; Comparisons among multiple groups were used one-way ANOVA in panels A–C and F; Comparisons among multiple groups were used two-way ANOVA in panel E. All data were checked by Tukey's multiple comparisons test. *P < 0.05, **P < 0.01.

Fig 5: Mechanism of histone demethylase KDM5A regulating hPDLSCs in periodontitis condition. KDM5A could bind to the miR-495-3p promoter and inhibit miR-495-3p level via demethylation of H3K4me3, leading to upregulation of HOXC8 mRNA and inhibition in OD, proliferation, and migration of hPDLSCs with periodontitis.