Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 4. CHRDL1 functions as a general BMP-signaling amplifier but specifically antagonizes BMP7 in the presence of TWSG1. (A) P19 embryonal carcinoma cells transfected with the BMP transcriptional reporter pBRE-Luc were incubated overnight with BMP4 (5 ng/ml) or BMP7 (10 ng/ml) and increasing amounts of CHRDL1 (50 to 400 ng/ml) or Chordin (100 to 800 ng/ml). Both CHRDL1 and Chordin amplify BMP4 signaling; however, in contrast to Chordin, which antagonizes BMP7 signaling, CHRDL1 amplifies BMP7 signaling in a dosage-responsive manner. (B) In the presence of TWSG1 (100 to 400 ng/ml), CHRDL1 (200 ng/ml) continues to act as an amplifier of BMP4 signaling unlike Chordin (400 ng/ml), which becomes a potent BMP4 antagonist. In contrast, CHRDL1 amplification of BMP7 signaling is converted to antagonism by addition of TWSG1 in a dosage-dependent manner. (C) In the P19 pBRE-luc reporter assay, the addition of TWSG1, CHRDL1, or Chordin (400 ng/ml) alone does not affect transcriptional activation. (D) CHRDL1 binds BMP7 and TWSG1 only in a trimolecular complex. A co-immunoprecipitation experiment in which CHRDL1 (500 ng/ml) is incubated in the presence of TWSG1 (500 ng/ml) and/or BMP7 (250 ng/ml) demonstrates that CHRDL1 binds TWSG1 and BMP7 only when all three proteins are present. No evidence of binding is detected when TWSG1 or BMP7 alone is incubated with CHRDL1. Recombinant proteins were immunoprecipitated with a goat polyclonal antibody against CHRDL1, and blots of goat IgG serve as loading controls. The last panel, a CHRDL1 immunoblot of an identical immunoprecipitation substituting an irrelevant goat antibody, demonstrates the absence of nonspecific binding by CHRDL1 to either the goat antibody or Protein G beads used in the experiment. The first lane of all blots contains recombinant protein as a positive control. (E) TWSG1 is strongly expressed in tubule epithelia of the mouse and human kidney. (Top) Strong immunohistochemical staining for TWSG1 in tubule epithelia of adult mouse kidney but weak staining in the glomerulus (G) and interstitial cell population (inset). (Right) Negative control using an antibody of the same species specific for macrophage. (Bottom) Immunofluorescent micro- graphs of an adult human kidney showing TWSG1 expression (red) in both proximal tubules (PT), marked green with Lotus lectin, and other tubules. As in the mouse, glomerular expression of TWSG1 is much weaker than seen in tubular epithelia. (Right) Negative control for TWSG1.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Transfection, Incubation, Amplification, Reporter Assay, Activation Assay, Immunoprecipitation, Binding Assay, Recombinant, Western Blot, Positive Control, Immunohistochemical staining, Staining, Negative Control, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 5. CHRDL1 reduces BMP7-stimulated Smad activation and ID gene expression in the presence of TWSG1 but has no effect on BMP4 signaling. (A and B) In the presence of TWSG1, CHRDL1 inhibits Smad phosphorylation by BMP7 but not by BMP4. P19 cells were serum starved for 2 h and then incubated with BMPs (10 ng/ml) and antagonists for 2 h before lysis and Western blotting. Blots were probed for phosphorylated Smads 1, 5, and 8 and -tubulin. In lanes 3, 4, and 5, Noggin (200 ng/ml), TWSG1, and CHRDL1 alone (400 ng/ml) were added. In lanes 6 through 9 CHRDL1 (400 ng/ml) was added together with increasing concentrations of TWSG1 (50, 100, 200, and 400 ng/ml). BMPs were incubated at 37 C for 1 h with or without antagonists before application. Note that the arrow points to the band corresponding to pSmad1/5/8. The top band (arrowhead) is a contaminating band specific to P19 lysates and is not present in lysates from MDCK cells (compare C and D). (C and D) The effects of CHRDL1 and TWSG1 on BMP signaling can be reproduced in the MDCK kidney cell line. (E) RT-PCR showing expression of ID1, 2, and 3 genes in HK-2 cells stimulated with BMP4 or 7 (25 ng/ml) for 6 h in the presence of varying concentrations (100 to 400 ng/ml) of CHRDL1 and/or TWSG1. ID expression by HK-2 cells in response to BMP4 is unaffected by the presence of TWSG1. In cells incubated with BMP7, the addition of TWSG1 reduces ID gene expression.

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Activation Assay, Gene Expression, Phospho-proteomics, Incubation, Lysis, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of the American Society of Nephrology

Article Title: Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

doi: 10.1681/asn.2008070768

Figure Lengend Snippet: Figure 6. Overexpression of mouse Chrdl1 in the collecting duct substan- tially reduces BMP signaling in vivo. (A) Diagram of mouse Chrdl1 transgene construct driven by a collecting duct–specific enhancer element from intron 1 of the Bmp7 gene; quantitative PCR assay shows Chrdl1 overexpression in kidneys of two embryonic day 17.5 transgenic embryos (150 and 151) com- pared with wild-type. (B through D) Immunolocalization of phosphorylated Smads (red) in the embryonic day 17.5 wild-type kidney (B), and kidneys from transgenic embryos 150 and 151 (C and D) show that BMP signaling is substantially reduced by expression of the Chrdl1 transgene. Collecting ducts (CD) were localized by staining with the lectin Dolichos Biflorus Agglu- tinin (green), and nuclei were counterstained with DAPI (blue).

Article Snippet: BMP7 was detected with mouse anti- BMP7 (R&D Systems) and a sheep anti-mouse HRP conjugate (Am- ersham).

Techniques: Over Expression, In Vivo, Construct, Real-time Polymerase Chain Reaction, Transgenic Assay, Expressing, Staining

Journal: bioRxiv

Article Title: NG2 + /Nestin + mesenchymal stem cells dictate DTC dormancy in the bone marrow through TGFβ2

doi: 10.1101/2020.10.22.349514

Figure Lengend Snippet: a . qPCR of sorted CD45 − CD31 − Nestin-GFP − and Nestin-GFP bright MSCs from Nestin-GFP mice (4 independent experiments, mean and SEM, 2-tailed Mann–Whitney tests, *p<0.05). b. TGFβ1, 2 and 3 and BMP7 levels in BM supernatant of WT and NG2-Cre ER -iDTR mice 2 weeks after TAM and DT treatments (2 independent experiments, n=24, median and interquartile range, 2-tailed Mann– Whitney tests, *p<0.05). c. Imaging of Nestin-GFP + MSCs in Nestin-GFP mice using IF. Dormancy factors TGFβ2 (white) and BMP7 (red) are expressed near MSCs. Scale bar 100um. Dotted rectangles, high-magnification inserts. d. TGFβ2 and BMP7 levels from BM plasma samples from ER + BC patients with (M1) or without (M0) evidence of systemic recurrence (data from SATT clinical study , Fisher’s exact test, *p<0.05). e. Metastasis-free survival analysis of the subgroup of patients who did not receive any secondary chemotherapy, excluding treatment-related interpretation bias, separated by detectable BMP7 levels compared to patients with no BMP7.

Article Snippet: ELISA assays were performed using human TGFb2 (R & D System, DY302) and BMP7 (R & D System, DY354) kits following manufacture’s recommendation.

Techniques: MANN-WHITNEY, Imaging, Clinical Proteomics

Journal: bioRxiv

Article Title: NG2 + /Nestin + mesenchymal stem cells dictate DTC dormancy in the bone marrow through TGFβ2

doi: 10.1101/2020.10.22.349514

Figure Lengend Snippet: a. 3D Matrigel assay used to track solitary cell to cluster growth. Single cells were plated on top of Matrigel in low density and the percentage of single cells, doublets and clusters was quantified 4 days after. b-c. Co-culture of human HNSCC PDX-derived T-HEp3 ( b ) and mouse BC MMTV-PyMT ( c ) cells with sorted Nestin-GFP − and Nestin-GFP + MSCs for 4 days. d-k. E0771 cells were treated every day for 4 days with TGFβ2, BMP7 or bone-marrow conditioned media (BM-CM) of TGFβ2 +/+ or TGFβ2 +/− mice. d and h. Percentage of cancer cells in a single cell, doublet or cluster state with the indicated treatments. e-g and i-k. Quantifications of c-Cas-3, p-ATF2 and p27 with the indicated treatments. l. Western blots for the indicated antigens detected in E0771 cells treated for 24 hours with the TGFβ2, BMP7 and different BM-CM preparations. m-p. T-HEp3 cells with ERK, p38 and p27 activity biosensors were reversed transfected with control siRNA or siRNAs for TGFBRIII and BMPRII followed by 24-hour treatments with TGFβ2 and BMP7. m. TGFBRIII and BMPRII mRNA levels 48 hours after transfection with the indicated siRNAs. n-p. Quantification of the T-HEp3-biosensors activity. k. qPCR of TGFβ1, TGFβ2 and BMP7 from Control and rMSCs. All graphs: 3-5 independent experiments, mean and SEM, 2-tailed Mann–Whitney tests, *p<0.05.

Article Snippet: ELISA assays were performed using human TGFb2 (R & D System, DY302) and BMP7 (R & D System, DY354) kits following manufacture’s recommendation.

Techniques: Matrigel Assay, Co-Culture Assay, Derivative Assay, Western Blot, Activity Assay, Transfection, Control, MANN-WHITNEY

Journal: Stem Cells International

Article Title: Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries

doi: 10.1155/2016/7403890

Figure Lengend Snippet: ELISA. (a) Analysis of the protein BMP2 and (b) BMP7 by ELISA of MSCs transduced with the vectors AdBMP2, AdBMP7, the combination AdBMP2/AdBMP7, and the positive (OSTEO) and negative (DMEM) controls on days 8, 16, and 32 after induction.

Article Snippet: Production of BMP2 and BMP7 proteins was verified by ELISA in ADMSCs transduced with AdBMP2, AdBMP7, the combination AdBMP2/AdBMP7, and the positive and negative controls on days 8, 16, and 32 after induction, using the commercial kits Quantikine-ELISA Human BMP2 and Quantikine-ELISA Human BMP7 (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Transduction

Journal: International Journal of Molecular Medicine

Article Title: Bone morphogenetic protein 7 enhances the osteogenic differentiation of human dermal-derived CD105 + fibroblast cells through the Smad and MAPK pathways

doi: 10.3892/ijmm.2018.3938

Figure Lengend Snippet: BMP7 promotes the osteogenic differentiation of CD105 + hDDFCs in vitro . CD105 + hDDFCs were infected with recombinant adenovirus expressing human BMP7 for 48 h and cultured in BM or OM. Detection of BMP7 and osteogenesis-associated gene expression by (A) reverse transcription-quantitative polymerase chain reaction and (B) western blot analyses at 7 days. Densitometric quantification of the immunoblot normalized to GAPDH. * P<0.05 and ** P<0.01, vs. BM; # P<0.05 and ## P<0.01, vs. OM+Ad-LacZ. Osteogenic differentiation of CD105 + hDDFCs infected with or without recombinant adenovirus in the presence of BM or OM was determined by (C) Alizarin Red S at 21 days, (D) ALP staining at 7 days, and an (E) ALP activity assay at 7 days. Scale bar=200 µ m. * P<0.05, vs. BM; # P<0.05, vs. OM+Ad-LacZ. hDDFCs, human dermal-derived fibroblast cells; BM, basal medium; OM, osteogenic medium; BMP7, bone morphogenetic protein 7; RUNX2, runt related transcription factor 2; OSX, osterix; OCN, osteocalcin; OPN, osteopontin; ALP, alkaline phosphatase.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST overnight, and incubated with primary antibodies against p-Smad 1/5/8 (1:1,000; cat. no. 9511), Smad1 (1:1,000; cat. no. 9743), extracellular signal-regulated kinase (ERK; 1:2,000; cat. no. 4696), phosphorylated (p)-ERK (1:1,000; cat. no. 9101), c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. 9252), and p-JNK (1:1,000; cat. no. 9251) from Cell Signaling Technology, Inc. (Beverly, MA, USA); p-p38 (1:500; cat. no. sc-7973), p38 (1:1,000; cat. no. sc-7972), BMP7 (1:200; cat. no. sc-9305), runt-related transcription factor 2 (RUNX2; 1:1,000; cat. no. sc-12488), and ALP (1:200; cat. no. sc-15065) from Santa Cruz Biotechnology, Inc.); and OSX (1:500; cat. no. ab94744), OPN (1:1,000; cat. no. ab8448), OCN (1:500; cat. no. ab93876) and GAPDH (1:2,000; cat. no. ab22555) from Abcam (Cambridge, UK) at 4°C overnight.

Techniques: In Vitro, Infection, Recombinant, Expressing, Cell Culture, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Staining, ALP Activity Assay, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Bone morphogenetic protein 7 enhances the osteogenic differentiation of human dermal-derived CD105 + fibroblast cells through the Smad and MAPK pathways

doi: 10.3892/ijmm.2018.3938

Figure Lengend Snippet: BMP7 activates Smad and MAPK pathways in CD105 + hDDFCs cells. CD105 + hDDFCs or BMP7-expressing adenovirus-infected CD105 + hDDFCs were treated with BM or OM, and the phosphorylation of (A) Smad- and (B) MAPK-related proteins was analyzed by western blot analysis following 24 h of culture. Densitometric quantification of the immunoblot normalized to total protein. * P<0.05, vs. BM; # P<0.05, vs. OM+Ad-LacZ. hDDFCs, human dermal-derived fibroblast cells; BMP7, bone morphogenetic protein 7; BM, basal medium; OM, osteogenic medium; Smad, small mothers against decapentaplegic; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; p-, phosphorylated.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST overnight, and incubated with primary antibodies against p-Smad 1/5/8 (1:1,000; cat. no. 9511), Smad1 (1:1,000; cat. no. 9743), extracellular signal-regulated kinase (ERK; 1:2,000; cat. no. 4696), phosphorylated (p)-ERK (1:1,000; cat. no. 9101), c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. 9252), and p-JNK (1:1,000; cat. no. 9251) from Cell Signaling Technology, Inc. (Beverly, MA, USA); p-p38 (1:500; cat. no. sc-7973), p38 (1:1,000; cat. no. sc-7972), BMP7 (1:200; cat. no. sc-9305), runt-related transcription factor 2 (RUNX2; 1:1,000; cat. no. sc-12488), and ALP (1:200; cat. no. sc-15065) from Santa Cruz Biotechnology, Inc.); and OSX (1:500; cat. no. ab94744), OPN (1:1,000; cat. no. ab8448), OCN (1:500; cat. no. ab93876) and GAPDH (1:2,000; cat. no. ab22555) from Abcam (Cambridge, UK) at 4°C overnight.

Techniques: Expressing, Infection, Phospho-proteomics, Western Blot, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Bone morphogenetic protein 7 enhances the osteogenic differentiation of human dermal-derived CD105 + fibroblast cells through the Smad and MAPK pathways

doi: 10.3892/ijmm.2018.3938

Figure Lengend Snippet: SMAD4 knockdown attenuates the BMP7-induced promotion of CD105 + hDDFCs osteogenic differentiation. (A) Recombinant adenovirus-infected CD105 + hDDFCs transfected with siRNA for Smad4 or control were subjected to western blot analysis for the detection of Smad/p-Smad and osteogenesis- associated gene expression 7 days following osteogenic induction. Densitometric quantification of p-Smad was normalized to total Smad; densitometric quantification of other proteins was normalized to GAPDH. * P<0.05, vs. Ad-LacZ; # P<0.05, vs. Ad-BMP7+siR-Scr. Osteogenic differentiation of CD105 + hDDFCs infected with or without recombinant adenovirus in presence of osteogenic medium was determined by (B) Alizarin Red S and ALP staining and an (C) ALP activity assay 7 or 21 days following osteogenic induction. Scale bar=200 µ m. * P<0.05. hDDFCs, human dermal-derived fibroblast cells; Smad, small mothers against decapentaplegic; siR, small interfering RNA; Scr, scramble; BMP7, bone morphogenetic protein 7; RUNX2, runt related transcription factor 2; OSX, osterix; OCN, osteocalcin; OPN, osteopontin; ALP, alkaline phosphatase; p-, phosphorylated.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST overnight, and incubated with primary antibodies against p-Smad 1/5/8 (1:1,000; cat. no. 9511), Smad1 (1:1,000; cat. no. 9743), extracellular signal-regulated kinase (ERK; 1:2,000; cat. no. 4696), phosphorylated (p)-ERK (1:1,000; cat. no. 9101), c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. 9252), and p-JNK (1:1,000; cat. no. 9251) from Cell Signaling Technology, Inc. (Beverly, MA, USA); p-p38 (1:500; cat. no. sc-7973), p38 (1:1,000; cat. no. sc-7972), BMP7 (1:200; cat. no. sc-9305), runt-related transcription factor 2 (RUNX2; 1:1,000; cat. no. sc-12488), and ALP (1:200; cat. no. sc-15065) from Santa Cruz Biotechnology, Inc.); and OSX (1:500; cat. no. ab94744), OPN (1:1,000; cat. no. ab8448), OCN (1:500; cat. no. ab93876) and GAPDH (1:2,000; cat. no. ab22555) from Abcam (Cambridge, UK) at 4°C overnight.

Techniques: Knockdown, Recombinant, Infection, Transfection, Control, Western Blot, Gene Expression, Staining, ALP Activity Assay, Derivative Assay, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: Bone morphogenetic protein 7 enhances the osteogenic differentiation of human dermal-derived CD105 + fibroblast cells through the Smad and MAPK pathways

doi: 10.3892/ijmm.2018.3938

Figure Lengend Snippet: Inhibition of p38 MAPK attenuates the BMP7-induced promotion of CD105 + hDDFCs osteogenic differentiation. Recombinant adenovirus infected CD105 + hDDFCs were incubated with OM in the presence or absence of the p38 inhibitor (SB203580, 10 µ M). (A) Western blot detection of p38 MAPK following 24 h of culture in OM, and osteogenesis-associated gene expression 7 days following osteogenic induction. Densitometric quantification of p-p38 was normalized to total p38; densitometric quantification of other proteins was normalized to GAPDH. * P<0.05, vs. Ad-LacZ; # P<0.05, vs. Ad-BMP7. Osteogenic differentiation of CD105 + hDDFCs infected with or without recombinant adenovirus in the presence of BM or OM was determined by (B) Alizarin Red S and ALP staining, and an (C) ALP activity assay 7 or 21 days following osteogenic induction. Scale bar=200 µ m. * P<0.05. hDDFCs, human dermal-derived fibroblast cells; BMP7, bone morphogenetic protein 7; BM, basal medium; OM, osteogenic medium; Smad, small mothers against decapentaplegic; BMP7, bone morphogenetic protein 7; RUNX2, runt related transcription factor 2; OSX, osterix; OCN, osteocalcin; OPN, osteopontin; ALP, alkaline phosphatase; p-, phosphorylated.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST overnight, and incubated with primary antibodies against p-Smad 1/5/8 (1:1,000; cat. no. 9511), Smad1 (1:1,000; cat. no. 9743), extracellular signal-regulated kinase (ERK; 1:2,000; cat. no. 4696), phosphorylated (p)-ERK (1:1,000; cat. no. 9101), c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. 9252), and p-JNK (1:1,000; cat. no. 9251) from Cell Signaling Technology, Inc. (Beverly, MA, USA); p-p38 (1:500; cat. no. sc-7973), p38 (1:1,000; cat. no. sc-7972), BMP7 (1:200; cat. no. sc-9305), runt-related transcription factor 2 (RUNX2; 1:1,000; cat. no. sc-12488), and ALP (1:200; cat. no. sc-15065) from Santa Cruz Biotechnology, Inc.); and OSX (1:500; cat. no. ab94744), OPN (1:1,000; cat. no. ab8448), OCN (1:500; cat. no. ab93876) and GAPDH (1:2,000; cat. no. ab22555) from Abcam (Cambridge, UK) at 4°C overnight.

Techniques: Inhibition, Recombinant, Infection, Incubation, Western Blot, Gene Expression, Staining, ALP Activity Assay, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Bone morphogenetic protein 7 enhances the osteogenic differentiation of human dermal-derived CD105 + fibroblast cells through the Smad and MAPK pathways

doi: 10.3892/ijmm.2018.3938

Figure Lengend Snippet: BMP7 induces bone formation of CD105 + hDDFCs cells in vivo . Adenovirus-infected CD105 + hDDFCs with calcium alginate were injected intramuscularly into nude mice (n=5). (A) X-ray images of opaque tissue were captured and bone volumes were measured at 12 weeks post-injection. (B) Hematoxylin and eosin staining of tissue sections. Scale bar=50 µ m. Green arrows indicate trabecular bone generated by osteocyte-like cells; black arrows indicate undegraded alginate material. hDDFCs, human dermal-derived fibroblast cells; BMP7, bone morphogenetic protein 7.

Article Snippet: The membranes were blocked with 5% nonfat milk in TBST overnight, and incubated with primary antibodies against p-Smad 1/5/8 (1:1,000; cat. no. 9511), Smad1 (1:1,000; cat. no. 9743), extracellular signal-regulated kinase (ERK; 1:2,000; cat. no. 4696), phosphorylated (p)-ERK (1:1,000; cat. no. 9101), c-Jun N-terminal kinase (JNK; 1:1,000; cat. no. 9252), and p-JNK (1:1,000; cat. no. 9251) from Cell Signaling Technology, Inc. (Beverly, MA, USA); p-p38 (1:500; cat. no. sc-7973), p38 (1:1,000; cat. no. sc-7972), BMP7 (1:200; cat. no. sc-9305), runt-related transcription factor 2 (RUNX2; 1:1,000; cat. no. sc-12488), and ALP (1:200; cat. no. sc-15065) from Santa Cruz Biotechnology, Inc.); and OSX (1:500; cat. no. ab94744), OPN (1:1,000; cat. no. ab8448), OCN (1:500; cat. no. ab93876) and GAPDH (1:2,000; cat. no. ab22555) from Abcam (Cambridge, UK) at 4°C overnight.

Techniques: In Vivo, Infection, Injection, Staining, Generated, Derivative Assay