Fig 1: NKX2-8 silencing-induced PTHrP promotes osteoclastogenesis in vitro. (A) Left: schematic illustration of osteoclastogenesis under treatment with CM from breast cancer cells or from osteoblasts pretreated with CM from breast cancer cells. Middle: osteoclast differentiation assays assessed using TRAP staining cultured CM obtained from the indicated cells. Right: Quantification of the number of TRAP-positive multinuclear osteoclasts and TRAP activity. (B) ELISA analysis of the RANKL/OPG ratio in CM from osteoblast precursor cells MC3T3-E1 cells cultured in the CM obtained from the indicated breast cancer cells. (C) Real-time PCR analysis indicating osteoclastogenesis regulator expression in the indicated cells. The pseudocolor represents the intensity scale of genes in the indicated cells generated by a log2 transformation. GAPDH serves as the loading control. (D) ELISA analysis of secreted PTHrP levels in CM from the indicated cells. (E) Real-time PCR analysis of PTHrP levels in sh-vector- and PTHrP-shRNA(s)-transduced MDA-MB-231/NKX2-8 sh#1 cells. GAPDH served as the loading control. (F) Left: schematic illustration of osteoclastogenesis under treatment with CM from osteoblasts pretreated with CM from breast cancer cells. Right: ELISA analysis of the RANKL/OPG ratio in CM from osteoblast precursor cells MC3T3-E1 cells in the presence of CM from the indicated breast cancer cells, and quantification of the TRAP-positive multinuclear osteoclasts number and TRAP activity cultured in the CM obtained from the indicated cells. (G) Left: schematic illustration of breast cancer CM-induced “vicious cycle” between the indicated cells. Middle: The TGF-ß1 levels analyzed using ELISA assay in CM from RAW 264.7 cells cultured onto the bone slice in CM obtained from the breast cancer cells. Right: MTT assay analysis of proliferation rate of indicated cells from experiment in left panel. (H) Schematic illustration of PTHrP-induced “vicious cycle” between the indicated cells. BC: Breast Cancer. N.S. means not significant (P > 0.05), *** means P < 0.001.
Fig 2: NKX2-8 interacts with HDAC1 in NKX2-8-inhibited PTHrP transcription. (A) Schematic diagram of interaction between NKX2-8 and the HDAC1 in the PTHrP promoter using CAPATURE approach. (B) ChIP analysis of the enrichment of HDAC1 in the PTHrP promoter in the indicated breast cancer cells. (C) ChIP analysis of the enrichment of H3K27ac in the PTHrP promoter in the indicated breast cancer cells. (D) Real-time PCR analysis of PTHrP mRNA (left) and ELISA analysis of serum PTHrP (right) expression in the indicated cells. GAPDH was used as the loading control. (E) The relative expression of HDAC1 in the indicated cells using real-time PCR analysis. GAPDH served as the loading control. (F) ChIP analysis of the association of NKX2-8 with the promoter of PTHrP gene in the indicated breast cancer cells. (G) The relative enrichment of H3K27ac on the PTHrP promoter determined by ChIP analysis in the indicated breast cancer cells. N.S. means not significant (P > 0.05), *** means P < 0.001.
Fig 3: Targeting PTHrP inhibits NKX2-8 silencing-induced bone metastasis. (A) Schematic illustration of osteoclastogenesis in the indicated condition. (B) Osteoclast differentiation assays analyzed as shown by TRAP staining of the indicated CM-treated pre-osteoclasts with addition of PBS or PTHrP7-34, IgG or anti-PTHrP antibody, or vehicle or 6-TG. (C) Quantification of the number of TRAP-positive multinuclear cells (upper) and examination of TRAP activity (lower). (D) µCT images of bone lesions from mice injected with MDA-MB-231/NKX2-8 sh#1, and treated with PBS or PTHrP7-34 or IgG or anti-PTHrP antibody or vehicle or 6-TG. (E, F) Normalized BLI signals of bone metastases quantification of the µCT osteolytic lesion area (E), and Kaplan-Meier bone metastasis-free survival curve (F) of the indicated mice in the experimental metastasis phase (n = 8/group). *** means P < 0.001.
Fig 4: NKX2-8 transcriptionally represses PTHrP. (A) ChIP analysis of the enrichment of NKX2-8 on promoter of PTHrP gene in MDA-MB-231 cells. (B) Relative luciferase activities of the PTHrP promoter in the indicated breast cancer cells. (C) NKX2-8 levels were negatively associated with PTHrP expression in breast cancer tissues (n = 304). The representative IHC staining images (left) and expression correlation (right) of NKX2-8 and PTHrP protein in breast cancer tissues. Scale bars, 20 µm. (D) Relative expression of NKX2-8 and PTHrP in primary breast cancer tissues (n = 20) with bone metastasis (n = 10; P < 0.001). ** means P < 0.01., *** means P < 0.001 N.S. means not significant (P > 0.05).
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