Fig 2: Runx2 target genes are downregulated in AOB cells. (A) Runx2 target genes are downregulated in AOB cells. The relative mRNA levels of Runx2 target genes, including OSC, ALPL, SPP1, COL1A1, IBSP and MMP13, in HOB and AOB cells were determined by qRT-PCR. CUL4A was used as a control. *P<0.05. (B) The expression of Runx2 target genes was dependent on the CtBP2 level. The HOB-1, HOB-2, AOB-1 and AOB-2 cells were transfected with control-siRNA or CtBP2-siRNA, respectively. After 48 hours, the cells were subjected to RNA isolation, followed by measuring the expression of Runx2 target genes by qRT-PCR. CUL4A was used as a control. **P<0.001.

Fig 3: NDAH levels determine CtBP2 dimerization and regulate Runx2 target gene levels. (A) The elevated NADH levels increased CtBP2 dimerization. The HOB-1 cells were transfected with a pCDNA3-2xFlag (control), pCDNA3-2xFlag-CtBP2 or pCDNA3-2xFlag-CtBP2-G189A plasmid. After 48 hours, cells were treated with CoCl2 or CoCl2+HQNO. The treated cells were then subjected to IP analysis with anti-Flag-agarose, and the pull-down products were then subjected to western blot with anti-Flag, anti-CtBP2 and p300. GAPDH was used as an input loading control. (B) The elevated NADH levels increased Runx2 target gene expression. The mRNAs from cells used in (A) were subjected to qRT-PCR analysis to determine the expression of Runx2 target genes, including OSC, ALPL, SPP1, COL1A1, IBSP and MMP13. CUL4A was used a control. **P<0.001.

Fig 4: FO promotes osteogenic activity in prednisolone (PDS)-pretreated MC3T3-E1 cells. Mouse preosteoblast MC3T3-E1 cells (1 × 104 cells/mL) were pretreated with 10 μM PDS for two days prior to treatment with FO (0–200 μg/mL) for five days. Fresh media with FO and/or PDS were replenished every two days. At day 7, (A) bone mineralization was evaluated by alizarin red staining, and (B) ALP activity was evaluated using a TRACP & ALP Double-Staining Kit. (C) In a parallel experiment, total mRNA was extracted, and RT-PCR was performed to evaluate the gene expressions of ALP, RUNX2, and OSX. GAPDH was used as the internal control. (D) Total proteins were extracted, and Western blotting was performed to evaluate the expression of ALP, RUNX2 and OSX proteins. β-Actin was used as the internal control. All data are presented as means ± standard error of the mean (### p < 0.001 vs. untreated MC3T3-E1 cells; * p < 0.05 and *** p < 0.001 vs. PDS-treated MC3T3-E1 cells). ALP: alkaline phosphatase; RUNX2: runt-related transcription factor 2; OSX: osterix; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Fig 5: LaCl3 application mitigates vascular calcification and suppresses the osteoinduction signal transduction in the context of CRF. (A) Typical Scatter plots, Western blots as well as arithmetic means ± SEM (n = 3) of normalized cytoplasmic TRAF6/β-actin, NF-κB/β-actin, BMP-2/β-actin, and SM22α/β-actin protein ratio in VSMCs with control or with high phosphorus without or with additional treatment with LaCl3. (B) Quantification of Western blots to represent the amount of cytoplasmic protein expression (n = 3). (C) Typical Scatter plots, Western blots as well as arithmetic means ± SEM (n = 3) for the standardized nuclear NF-κB/Lamin A and Runx2/Lamin A protein ratio in VSMCs with control or with high phosphorus without or with additional treatment with LaCl3. (D) Quantification of Western blots to represent the amount of nuclear protein expression (n = 3). (E) Scatter plots and arithmetic means ± SEM (n = 3) of TRAF6, BMP-2, SM22α, and Runx2, relative mRNA expression in VSMCs with Pi or control, with or without LaCl3 treatment. ##P < 0.01; ###P < 0.001 compared with control VSMCs; *P < 0.05; **P < 0.01; ***P < 0.001 compared with Pi-treated VSMCs. &P < 0.05; &⁣&P < 0.01 compared with VSMCs treated with Pi and LaCl3 (60 μM).