Fig 1: β-Catenin stabilization and translocation. (A,B) Schematic presentation of the experiment using siRNAs and rhWNTs. To explore the dynamic of crucial Wnt ligands and the relevance of Wnt/β-catenin-dependent pathway, the effect of IL-6 (20 ng/mL) on expression of WNT2B, WNT10B, and WNT5A along with stabilization and translocation of β-catenin in hPDLSCs during in vitro osteogenic induction upon treatment with siRNAs or rhWNTs was analyzed. Sets of siRNAs (siWNT2B, siWNT10B, or siWNT5A) and rhWNTs (rhWNT2B, rhWNT10B, or rhWNT5A) were used. RT-qPCR analysis of the mRNA expression levels of crucial Wnt ligands, including (C) WNT2B, (D) WNT10B, and (E) WNT5A, at days 1, 3, 7, 14, and 21. Superscript letters indicate significant difference vs. the undifferentiated control (a), osteogenic control (b), and osteogenic induction (IL-6 at 20 ng/mL) (c) (p < 0.05). Subcellular stabilization and translocation of β-catenin at (F) 6, (G) 24, and (H) 48 h, respectively. Quantitative expression of β-catenin in the nuclear fraction at (I) 6, (J) 24, and (K) 48 h along with expression in the cytoplasmic fraction at (L) 6, (M) 24, and (N) 48 h, as determine with the QuanTL program. Lamin B1 and β-ACTIN were used as reference proteins in the nuclear and cytoplasmic fractions, respectively (n = 4). Bars indicate significant difference (p < 0.05). Original blots are presented in Supplementary Figs. 1A–3C.
Fig 2: Proliferation and viability of hPDLSCs upon IL-6 treatment. (A) Schematic presentation of the experiment. (B) Proliferation of hPDLSCs upon IL-6 treatment (10 and 20 ng/mL) at days 1, 5, and 7, as determined by alamarBlue™ staining. (C) Viability of hPDLSCs upon IL-6 treatment (10 and 20 ng/mL) at days 1, 5, and 7 (calcien AM/propidium iodide) (n = 4). Bars indicate significant differences (p < 0.05).
Fig 3: Effect of IL-6 on osteogenic differentiation potential of hPDLSCs in vitro upon silencing of WNT2B, WNT10B, or WNT5A. (A) Schematic presentation of the experiment. To determine the relevance of potential Wnt ligands, the effect of IL-6 (20 ng/mL) on the osteogenic differentiation potential of hPDLSCs in vitro upon silencing of WNT2B, WNT10B, or WNT5A was analyzed. siWNT2B, siWNT10B, or siWNT5A was used to knockdown the related Wnt ligand. (B) ALP activity was analyzed at days 14 and 21. Bars indicate significant difference (p < 0.05). RT-qPCR was conducted to quantify mRNA expression levels of osteogenic markers (RUNX2, OSX, COL1, ALP, OCN, and OPN) upon silencing of (C) WNT2B, (D) WNT10B, or (E) WNT5A at days 1, 3, 7, 14, and 21. Superscript letters indicate significant vs. the undifferentiated control (a), osteogenic control (b), and osteogenic induction (IL-6 at 20 ng/mL) upon siRNA treatment (c) (p < 0.05). (F) Matrix mineralization as determined by Von Kossa and Alizarin Red S staining at days 14 and 21 (n = 4).
Fig 4: Effect of IL-6 on osteogenic differentiation potential of hPDLSCs in vitro. (A) Schematic presentation of the experiment. (B) Effect of IL-6 (10 and 20 ng/mL) on the in vitro osteogenic differentiation potential of hPDLSCs, as determined by ALP activity at days 14 and 21. Bars indicated significant differences (p < 0.05) (n = 4). (C) RT-qPCR analysis of osteogenic mRNA marker expression (RUNX2, OSX, COL1, ALP, OCN, and OPN) at days 1, 3, 7, 14, and 21 (n = 4). Superscript letters indicate significant differences vs. the undifferentiated control (a), osteogenic control (b), and osteogenic induction upon IL-6 treatment at 10 ng/mL (c) (p < 0.05). (D) Matrix mineralization as determined by Von Kossa and Alizarin Red S staining at days 14 and 21.
Fig 5: Effect of IL-6 on osteogenic differentiation potential of hPDLSCs in vitro upon the inhibition of crucial osteogenic-regulating pathways. (A) Schematic presentation of the experiment. The potential regulating pathways related to the effect of IL-6 (20 ng/mL) on osteogenic differentiation potential of hPDLSCs in vitro upon inhibition of crucial osteogenic-regulating pathways (canonical Wnt, non-canonical Wnt, TGF-β1, and Notch pathways) were investigated. Dkk-1, SP600125, SB431542, and DAPT were used to block the canonical Wnt, non-canonical Wnt, TGF-β1, and Notch signaling pathways, respectively. (B) ALP activity was analyzed at days 14 and 21. Bars indicated significant differences (p < 0.05). Osteogenic mRNA marker expression (RUNX2, OSX, COL1, ALP, OCN, and OPN) upon the inhibition of the (C) canonical Wnt, (D) non-canonical Wnt, (E) TGF-β1, and (F) Notch signaling pathways, as determined by RT-qPCR at days 1, 3, 7, 14, and 21. Superscript letters indicate significant difference vs. the undifferentiated control (a), osteogenic control (b), and osteogenic induction (with 20 ng/mL IL-6) upon specific inhibitor treatment (c) (p < 0.05). (G) Matrix mineralization as determined by Von Kossa and (H) Alizarin Red S staining at days 14 and 21 (n = 4).
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