Fig 2: Knockdown of IL-23 in P. endodontalis LPS-treated SH-9 cells inhibits osteoclastogenesis. (A) SH-9 cells were transfected with siCont or siIL-23 RNA. The mRNA expression levels of IL-23 were examined by reverse transcription-quantitative polymerase chain reaction. IL-23 mRNA expression levels were reduced by siIL-23, compared with siCont transfection. The results are presented as the mean ± standard error. (B) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated siCont or siIL-23 cells for 48 h. The cells were fixed and subjected to TRAP staining. Representative fields are presented and TRAP-positive cells are indicated by arrows (scale bar, 50 µm; magnification, ×20). (C) TRAP-positive multinuclear cells with >3 nuclei were counted. The number of TRAP-positive multinuclear cells was reduced in cells treated with conditioned medium from siIL-23, compared with siCont cells. The results are presented as the mean ± standard error. (D) RAW264.7 cells were treated with conditioned medium from P. endodontalis LPS-treated siCont and siIL-23 cells for 24 h. Protein expression was examined by western blot analysis. Treatment with conditioned medium from siIL-23 cells reduced NFATc1 and c-Fos expression compared with treatment with conditioned medium from siCont cells. **P<0.01 vs. siCont group. LPS, lipopolysaccharide; IL-23, interleukin 23; P. endodontalis, Porphyromonas endodontalis; TRAP, tartrate-resistant acid phosphatase; siCont, negative control siRNA; siIL-23, IL-23 siRNA; siRNA, small interfering RNA; NFATc1, nuclear factor of activated T cells, cytoplasmic 1.

Fig 3: Representative immunofluorescence examinations and EMG recording trace for in vivo OFUS stimulation.a The schematic of OFUS stimulation in vivo. b Representative images of c-Fos and DAPI staining within the stimulation and control area. Red: c-Fos. Blue: DAPI. Orange outline: OFUS stimulated area. Green outline: control group at the contralateral area. Scale bar: middle and lower panel, 50 μm. c Statistic analysis of the percentage of c-Fos positive neurons. ***p < 0.001, two-sample t-test. d Representative EMG recordings of 2 s OFUS stimulation and control group targeting the somatosensory cortex. Orange box: laser on. e EMG signals after the band-pass filter and full-wave rectifier and envelope. f Histology results after stimulation in vivo

Fig 4: RSK2-mediated ELK3 transactivation activity regulates c-fos promoter activity-mediated AP-1 transactivation activity. (a) Left graph, ELK3 activates c-fos promoter activity. The c-fos-luciferase reporter plasmids were introduced into HEK293T cells with indicated doses of ELK3 and 80 pg of phRL-luciferase expression vector. Right graph, ELK3 transactivation activity is regulated by RSK2. The c-fos-luciferase reporter plasmids and ELK3 expression vectors were introduced into HEK293T cells with indicated doses of RSK2 expression vector and 80 pg of phRL-luciferase expression vector. (b) ELK3 transactivation activity was inhibited by inhibition of MEKs-ERKs-RSK2 signaling. The c-fos-luciferase reporter plasmid and ELK3 expression vector were introduced into HEK293T cells as indicated. Inhibitory effects of kaempferol, an RSK2 inhibitor, and U0126, a MEK inhibitor, on the transactivation activity of ELK3 induced by EGF were analyzed by examining luciferase activity. (c,d) Blockage of RSK2 suppresses ELK3-mediated c-fos promoter activity and AP-1 transactivation activity. (c) RSK2 knockdown suppressed EGF-induced ELK3 transactivation activity. The c-fos-promoter luciferase reporter plasmids and ELK3 expression vectors were introduced into sh-mock and sh-RSK2 stable cells and stimulated with EGF as indicated. (d) RSK2 knockout suppressed c-fos promoter activity and AP-1 transactivation activity. The c-fos- and AP-1-luciferase reporter plasmids were introduced into RSK2+/+ and RSK2−/− mouse embryonic fibroblasts and stimulated with EGF as indicated. (e) Representative images showing the effects of RSK2-mediated ELK3 transactivation activity and RasG12V-mediated cell transformation. Constitutive active Ras (RasG12V), RSK2, ELK3, and si-RSK2 were introduced into NIH3T3 cells for assaying of foci formation as indicated. The cells were cultured for 10–14 days to form foci by abrogation of cell-cell contact inhibition. The formed foci were visualized by crystal violet staining. Formed foci numbers were counted using the NIH ImageJ computer program (Version 1.6). Fold induction is expressed relative to that of NIH3T3-mock. (a–d) The firefly luciferase activity was normalized to Renilla luciferase activity. Data were obtained from three independent experiments, and the values presented are means ± SEM. * p < 0.05; ** p < 0.01 versus an indicated control group (Student’s t-test).

Fig 5: Npnt gene down-regulation by PMA regulated via c-Jun and c-Fos transcription factors. MC3T3-E1 cells were pretreated with or without c-Jun siRNA (20 nM) or c-Fos siRNA (20 nM) for 24 h, and then treated with PMA (100 nM) alone or in combination for 24 h with c-Jun or for three hours with c-Fos. (A) Proteins were extracted and subjected to western blotting to detect c-Jun and actin. (B) Total cellular RNA was extracted, and mRNAs for Npnt and Gapdh were examined using real-time PCR analysis. (C) Proteins were extracted using the same procedures shown in (A) and (B), and subjected to western blotting to detect c-Fos and Actin. (D) Total cellular RNA was extracted, and mRNAs for Npnt and Gapdh were examined using real-time PCR analysis. Results are shown as the mean ± SD of three samples. *P < 0.05, **P < 0.01, Student’s t-test, as compared to presence or absence of PMA, c-Jun siRNA, and c-Fos siRNA.