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. 2021 Jul 6;10:e64872. doi: 10.7554/eLife.64872

Figure 2. 7,8-Dihydroxyflavone (7,8-DHF) promoted osteogenesis via osteoblast-related signaling pathways.

MC3T3-E1 cells were treated with or without 7,8-DHF for 3 days. The mRNA level was evaluated by quantitative real-time PCR (qRT-PCR) and the protein level was detected by western blot. GAPDH was used as an internal control. (A) The protein levels of p-GSK3β and GSK3β. (B) Quantification of the p-GSK3β band intensities normalized to total GSK3β band intensities in each case. (C-E) The mRNA levels of β-catenin, Runx2, and Osterix. (F-I) The protein levels of β-catenin, Runx2, and Osterix. The expression levels of target proteins in the 0 μM group were normalized to 1. (J-K) β-Catenin knockdown by siRNA was performed in MC3T3-E1 cells with or without 7,8-DHF treatment. The protein levels of β-catenin, Runx2, and Osterix. The expression levels of target proteins in the 0 μM of negative control group were normalized to 1. Representative images from three independent experiments are shown in (A, F, J). Source files of the full raw unedited blots and blots with the relevant bands labeled were provided in Figure 2—source data 1. (N) The mRNA levels of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL). All results were expressed as mean ± SD (A-N: n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant; A-I, N: one-way analysis of variance [ANOVA]; J-M: two-way ANOVA).

Figure 2—source data 1. The original files of the full raw unedited blots and blots with the relevant bands labeled in Figure 2A, F and J.
The folders named ‘Figure 2A’, ‘Figure 2F ’, and ‘Figure 2J ’ contain the original images in Figure 2A, Figure 2F, and Figure 2J, respectively (the individual file name containing ‘(labeled)’ is blot with the relevant bands labeled by a red outline).

Figure 2.

Figure 2—figure supplement 1. β-Catenin knockdown by siRNA was performed in MC3T3-E1 cells with or without 7,8-dihydroxyflavone (7,8-DHF) treatment.

Figure 2—figure supplement 1.

The mRNA levels of β-catenin (A), Runx2 (B), and Osterix were evaluated by quantitative real-time PCR (qRT-PCR) and GAPDH was used as an internal control. All results were expressed as mean ± SD (A-C: n = 3; *p < 0.05, ns: not significant; two-way analysis of variance [ANOVA]).
Figure 2—figure supplement 2. 7,8-Dihydroxyflavone (7,8-DHF) had no obvious influence on Smad2 and TAK1.

Figure 2—figure supplement 2.

The protein level was detected by western blot and GAPDH was used as an internal control. (A) Representative images from three independent experiments were shown. (B) Quantification of the p-Smad2 band intensities normalized to total Smad2 band intensities in each case. (C) The protein levels of TAK1. All results were expressed as mean ± SD (A-C: n = 3; one-way analysis of variance [ANOVA]). Source files of the full raw unedited blots and blots with the relevant bands labeled were provided in Figure 2—figure supplement 2—source data 1.
Figure 2—figure supplement 2—source data 1. The original files of the full raw unedited blots and blots with the relevant bands labeled in Figure 2—figure supplement 2A.
The individual file name containing ‘(labeled)’ is blot with the relevant bands labeled by a red outline.