Figure 4

The effects of Zbp1 knockout (KO) on the osteogenic differentiation of murine periodontal ligament (PDL) cells. (A) Quantitative reverse transcription-polymerase chain reaction analysis of Zbp1 mRNA expression is shown (n = 3 wells). (B) Western blot analysis using anti-Zbp1 and anti-β-actin antibodies was performed. The original image is included in Supplementary Figure S6. The quantification of the band volume was performed. Each value was normalized to the value of the control sample. (C) Alkaline phosphatase (ALP) enzymatic activity during osteogenic differentiation was measured. High, low, and KO clones were cultured in osteogenic medium, and their ALP activity was measured on days 0, 3, 6, and 9 (n = 3 wells). The results of the analysis are shown as the mean ± standard error. (D) High and KO clones were cultured in osteogenic medium and stained with Alizarin Red S on days 8 and 16 of differentiation induction. The experiments were performed in triplicate (n = 3 wells) for each group. E. The quantification of Alizarin staining area performed. Each value was normalized to the value of osteogenic induction on day 16 of the High clone. Statistical analysis was performed using GraphPad Prism v9.0.2 (www.graphpad.com). The area analysis was performed by ImageJ software v1.53c (imagej.net).