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. 2018 Oct 9;42(6):3181–3192. doi: 10.3892/ijmm.2018.3921

Figure 3.

Figure 3

HY promotes YAP activation in a HIF-1α-dependent manner. (A) Protein expression of HIF-1α and VEGF in growth plate chondrocytes cultured under HY (1% O2) for 0, 2, 4 and 8 h. β-actin served as an internal control. (B) The density of the bands were quantified and normalized to the control group (0 h). (C) mRNA expression of HIF-1α and VEGF in chondrocytes cultured under HY (1% O2) for 0, 2, 4 and 8 h. (D) Immunofluorescence imaging of HIF-1α (red) in chondrocytes cultured under NO (21% O2) or HY (1% O2) for 4 h. DAPI (blue) was used for nuclear staining. Scale bar=200 μm. (E) Immunofluorescence imaging of VEGF (red) in chondrocytes cultured under NO (21% O2) or HY (1% O2) for 4 h. DAPI (blue) was used for nuclear staining. Scale bar=200 μm. (F) Protein expression of HIF-1α in growth plate chondrocytes following siRNA silencing of HIF-1α and then culture under HY (1% O2) for 4 h to detect the effect of HIF-1α silencing. β-actin served as an internal control. (G) The density of the bands was quantified and normalized to the NC group. Data are presented as mean ± SD (n=3). *P<0.05 and **P<0.01 vs. the NC group. (H) Protein expression of YAP in growth plate chondrocytes cultured under NO or under HY with siRNA negative control (HY+NC group) or under HY with siHIF2 siRNA (HY+siHIF2 group) for 4 h. β-actin served as an internal control. (I) The density of the bands was quantified and normalized to the NO group. (J) Protein expression of SOX9 in growth plate chondrocytes cultured under NO (NO group) or under HY with siRNA negative control (HY+NC group) or under HY with siRNA siHIF2 (HY+siHIF2 group) for 4 h. β-actin served as an internal control. (K) The density of the bands was quantified and normalized to the NO group. **P<0.01 vs. the NO group. (L) Immunofluorescence imaging of YAP (red) in chondrocytes cultured under NO (21% O2) or HY (1% O2) with or without siHIF-1α for 4 h. DAPI (blue) was used for nuclear staining. Scale bar=200 μm. Data are presented as mean ± standard deviation (n=3), and normalized to the control (0 h)/NC groups. *P<0.05, **P<0.01 and ***P<0.001 vs. the control (0 h) or NC groups. ###P<0.001 vs. the HY+NC group. YAP, yes-associated protein; HIF-1α, hypoxia-inducible factor 1α; SOX9, sex-determining region-box 9 protein; siRNA, small interfering RNA; NO, normoxia; HY, hypoxia; RFP, red fluorescent protein; VEGF, vascular endothelial growth factor; NC, negative control.