1. Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression
Cell Physiol Biochem 2018;45:1759– DOI: /
Fig. 1. Expression of BMPR1a of breast cancer cells at the tumor-bone interface is significantly high. A, Immunofluorescent analysis for BMPR1a (red, left panel) in sectioned bone tissue of breast cancer bone metastasis demonstrates a prominently stronger staining of BMPR1a of human breast cancer MDA-MB-231-luc cells at the tumor-bone (TB) interface compared with breast cancer cells distant from the interface. Nuclei of cells were stained by DAPI (blue, middle panel). Merged image, right panel. B, Immunohistochemical analysis for BMPR1a in sectioned bone tissue of breast cancer bone metastasis demonstrates a higher expression of BMPR1a of breast cancer cells in TB interface compared with that of MDA-MB-231-luc cells or bone tissue alone. B, bone. T, tumor. C, Quantification of IHC staining for BMPR1a with ImageJ software. *, P<0.05.
© 2018 The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

2. Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression
Cell Physiol Biochem 2018;45:1759– DOI: /
Fig. 2. Knockdown of BMPR1a inhibits tumor growth in vivo. A, RT-PCR analysis of BMPR1a in MDA-MB-231-luc cells transduced with nonsense shRNA (shCTL) or three different shRNAs (shRNAs 1, 2 and 3) targeted against human BMPR1a gene. B, Western blotting analysis of BMPR1a in MDA-MB-231-luc cells transduced with shCTL or shRNAs targeted against BMPR1a gene. β-Actin was used as a control for equal loading. C, Graphs show intensities of BMPR1a protein in shRNA groups compared with control or shCTL. D, Bioluminescence (BLI) imaging of a representative mouse at 5 weeks following intratibial injection of CTL, shCTL or shBMPR1a MDA-MB-231-luc cells. E, The average radiant efficiency of the BLI signal measured at each region of interest (ROI) per mouse at 5 weeks after inoculation. Intensity of BLI was measured by Living Image software Version 4.5 (n = 3). F, Dissected tumors generated 6 weeks after intratibial injection of CTL, shCTL or shBMPR1a cells. G&H, Quantification of tumor weight (G) and tumor size (H) of CTL, shCTL and shBMPR1a group (n = 5). CTL, MDA-MB-231-luc cells; shCTL, MDAMB-231-luc cells transduced with a scrambled shRNA; shRNA-1, shRNA-2, shRNA-3, three different shRNAs targeted against human BMPR1a gene. *, P<0.05, **, P<0.01.
© 2018 The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

3. Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression
Cell Physiol Biochem 2018;45:1759– DOI: /
Fig. 3. Knockdown of BMPR1a attenuates breast cancer-induced osteolytic lesion in vivo. A, Two-dimensional (2D) microCT slices of the sagittal plane of tibia indicates a decrease in lytic bone lesion in mice inoculated with shBMPR1a MDAMB-231-luc cells compared with shCTL or CTL mice. Scale bar, 1mm. B, Measurement of bone volume fraction (Bone volume/Total volume). C, Three-dimensional (3D) microCT reconstruction of lytic tibia from Fig. 3A. Scale bar, 1mm. *, P<0.05, **, P<0.01.
© 2018 The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

4. Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression
Cell Physiol Biochem 2018;45:1759– DOI: /
Fig. 4. Knockdown of BMPR1a decreases breast cancer-induced osteoclast formation. A, Top, H&E staining of metastatic bone sections of tumor-bearing nude mice inoculated with MDAMB-231-luc transduced with shBMPR1a or a scrambled shRNA (40 ×). Middle, TRAP staining of consecutive bone tissue sections (40 ×). Bottom, Amplified area of middle panel of Fig. 4A. Osteoclasts are stained red. All images were obtained from different mice at 6 weeks after cancer cells inoculation. B, Quantification of areas of tumor relative to tibia in histological sections (n = 5). C, Quantification of percentage of TRAP+area relative to total bone area from histological bone sections (n = 5). **, P<0.01. B, bone. T, tumor.
© 2018 The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

5. Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression
Cell Physiol Biochem 2018;45:1759– DOI: /
Fig. 5. Expression of Cathepsin-K was reduced at the tumor-bone interface in shBMPR1a-treated mice. Immunofluorescent staining for Cathepsin-K (green, left panel) in sectioned legs of mice injected with shBMPR1a MDA-MB-231-luc cells indicates a reduced expression of Cathepsin-K at the tumor-bone interface compared with CTL or shCTL mice. Nuclei of cells were stained by DAPI (blue, middle panel). Merged image, right panel. B, bone. T, tumor.
© 2018 The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

6. Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression
Cell Physiol Biochem 2018;45:1759– DOI: /
Fig. 6. Conditioned media of shBMPR1a breast cancer cells inhibits osteoclast differentiation in vitro. A, TRAP staining of bone marrow mononuclear cells (BMMCs) treated with conditioned media of human breast cancer MDA-MB-231-luc cells transduced with shBMPR1a. Arrows indicate mature osteoclasts after differentiation. B, Quantification of TRAP+ osteoclasts in (A). C, TRAP staining of BMMCs treated with conditioned media of human breast cancer MCF-7 cells transduced with shBMPR1a. Arrows indicate mature osteoclasts after differentiation. D, Quantification of TRAP+ osteoclasts in (C). *, P<0.05.
© 2018 The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

7. Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression
Cell Physiol Biochem 2018;45:1759– DOI: /
Fig. 7. Attenuated non-canonical BMP signaling of breast cancer cells suppresses production of RANKL. A, Top, Western blotting analysis of RANKL and OPG of MDA-MB-231-luc cells from CTL, shCTL or shBMPR1a group. Bottom, Relative ratio of RANKL to OPG calculated from top panel. B, Top, Western blotting analysis of RANKL and OPG of MCF-7 cells from CTL, shCTL or shBMPR1a group. Bottom, Relative ratio of RANKL to OPG calculated from top panel. C, Quantification of RANKL protein level in conditioned media of MDAMB-231-luc cells from CTL, shCTL or shBMPR1a group. D, Quantification of RANKL protein level in conditioned media of MCF-7 cells from CTL, shCTL or shBMPR1a group. E, Western blotting analysis of p-p38, p-Erk1/2 and p-JNK of MDA-MB-231-luc cells from CTL, shCTL or shBMPR1a group. F, Western blotting analysis of p-p38, p-Erk1/2 and p-JNK of MCF-7 cells from CTL, shCTL or shBMPR1a group. G, Left panel, Western blotting analysis of RANKL or OPG of MDA-MB-231-luc cells treated with SP600125, SB203580, or U0126, respectively. Right panel, Quantification of intensities of RANKL protein in left panel. H. Left panel, Western blotting analysis of RANKL or OPG of MCF-7 cells treated with SP600125, SB203580, or U0126, respectively. Right panel, Quantification of intensities of RANKL protein in left panel. β-Actin was used as a control for equal loading. *, P<0.05, **, P<0.01.
© 2018 The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0

8. Knockdown of Bone Morphogenetic Proteins Type 1a Receptor (BMPR1a) in Breast Cancer Cells Protects Bone from Breast Cancer-Induced Osteolysis by Suppressing RANKL Expression
Cell Physiol Biochem 2018;45:1759– DOI: /
Fig. 8. Conditioned media of sip38 breast cancer cells inhibits osteoclast differentiation in vitro and in vivo. A, Left panel, TRAP staining of RAW264.7 cells treated with conditioned media of MDA-MB-231-luc cells transduced with sip38 or a scrambled siRNA (siCTL). Right panel, Quantification of TRAP+ osteoclasts in left panel. B, Left panel, TRAP staining of RAW264.7 cells treated with conditioned media of human breast cancer MCF-7 cells transduced with sip38 or siCTL. Right panel, Quantification of TRAP+ osteoclasts in left panel. C, Left panel, TRAP staining of metastatic bone sections of tumor-bearing nude mice inoculated with MDA-MB-231-luc cells transduced with sip38 or a scrambled siRNA. Osteoclasts are stained red. All images were obtained from different mice at 6 weeks after cancer cells inoculation. Right panel, Quantification of percentage of TRAP+ area relative to total bone area from histological bone sections. D, Left panel, TRAP staining of metastatic bone sections of tumor-bearing nude mice inoculated with MCF-7 cells transduced with sip38 or a scrambled siRNA. Right panel, Quantification of percentage of TRAP+ area relative to total bone area from histological bone sections. *, P<0.05.
© 2018 The Author(s). Published by S. Karger AG, Basel - CC BY-NC-ND 4.0