Journal: JCI Insight

Article Title: Stem cell–associated osteogenic deficiency causes craniofacial deformities with progeroid accumulation of prelamin A

doi: 10.1172/jci.insight.196932

Figure Lengend Snippet: ( A ) Limiting dilution analysis of stem cell–mediated bone formation with renal capsule transplantation. Representative images of whole-mount von Kossa staining detecting ectopic bone formation in the mouse recipients transplanted by the indicated number of suture cells into the renal capsule. Arrowheads indicate the ectopic bones. ( B ) Representative images showing the analysis of Axin2-expressing cells using the Axin2 mGFP allele in the indicated 1-month-old (1M) suture. ( C ) Representative images examining the BMPR1A + and GLI1 + cell population within the indicated 1-month-old (1M) suture. ( D ) Graphs indicate the quantitation of the average percentage of BMPR1A + and GLI1 + cells in 3 independent experiments ( P < 0.005 or 0.05, n = 3, mean ± SEM, 2-tailed Student’s t test). SAG, sagittal; COR, coronal; AF, anterior frontal. Scale bars: 1 mm ( A ) and 50 μm ( B and C ).

Article Snippet: Mouse monoclonal antibodies against Bmpr1a (NBP2-37421, 1:75, Novus Biologicals), fascin1 (SC-21743, 1:100, Santa Cruz), and Alexa Fluor 568 phalloidin (A12380, 1:400, Invitrogen); rabbit polyclonal antibodies Osx (ab22552, 1:800, Abcam), OCN (23418-1-AP, 1:50, Proteintech), Gli1 (NBP1-78259, 1:100, Novus Biologicals), Myl2 (3671, 1:100, Cell Signaling Technology), and GM130 (2296, 1:200, Cell Signaling Technology); rabbit monoclonal antibodies lamin A/C (MA5-35284, 1:200, Invitrogen) and SUN2(EPR6557, 1:100, Abcam); anti-rabbit secondary antibody (BA-1000, 1:200, Vector laboratories); and anti-mouse IgG secondary antibodies (PK-2200, 1:250, Vector laboratories), were used in the immunostaining studies.

Techniques: Transplantation Assay, Staining, Expressing, Quantitation Assay

Journal: BioMed Research International

Article Title: Next-Generation Sequencing Panel Analysis of Clinically Relevant Mutations in Circulating Cell-Free DNA from Patients with Gestational Trophoblastic Neoplasia: A Pilot Study

doi: 10.1155/2020/1314967

Figure Lengend Snippet: Details of overlapping mutations.

Article Snippet: Reagents included antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (cat. no. KC-5G4; Aksomics, Shanghai, China), antibone morphogenetic protein receptor type 1A (BMPR1A) antibody (cat. no. 12702-1-AP; Proteintech, IL, USA), antimitogen-activated protein kinase kinase kinase 1 (MAP3K1) antibody (cat. no. 19970-1-AP; Proteintech, IL, USA), Magnetic Serum/Plasma DNA Maxi Kit (Tiangen, Beijing, China), TRIzol reagent (Invitrogen, CA, USA), PrimeScript RT kit, and SYBR Premix Ex Taq II (Takara, Shiga, Japan).

Techniques:

Journal: BioMed Research International

Article Title: Next-Generation Sequencing Panel Analysis of Clinically Relevant Mutations in Circulating Cell-Free DNA from Patients with Gestational Trophoblastic Neoplasia: A Pilot Study

doi: 10.1155/2020/1314967

Figure Lengend Snippet: Protein expression of BMPR1A and MAP3K1 in three cell lines. (a) Western blotting gel image for BMPR1A and MAP3K1, and GAPDH was used as loading control. (b) Values of mean ± S.D. of triplicate experiments were plotted ( ∗ p < 0.05, ∗∗ p < 0.001).

Article Snippet: Reagents included antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (cat. no. KC-5G4; Aksomics, Shanghai, China), antibone morphogenetic protein receptor type 1A (BMPR1A) antibody (cat. no. 12702-1-AP; Proteintech, IL, USA), antimitogen-activated protein kinase kinase kinase 1 (MAP3K1) antibody (cat. no. 19970-1-AP; Proteintech, IL, USA), Magnetic Serum/Plasma DNA Maxi Kit (Tiangen, Beijing, China), TRIzol reagent (Invitrogen, CA, USA), PrimeScript RT kit, and SYBR Premix Ex Taq II (Takara, Shiga, Japan).

Techniques: Expressing, Western Blot, Control

Journal: mAbs

Article Title: Discovery of ginisortamab, a potent and novel anti-gremlin-1 antibody in clinical development for the treatment of cancer

doi: 10.1080/19420862.2023.2289681

Figure Lengend Snippet: Ginisortamab inhibits gremlin-1 antagonism of BMP signaling pathways in human CRC cell lines. (a) CRC cell lines HCT 116, DLD-1, and LS174T show decreased pSMAD 1/8 in the presence of recombinant human gremlin-1, which can be inhibited by ginisortamab. Gremlin-1 (15 nM) and ginisortamab or hIgG4 control (150 nM) were pre-incubated together for 30 min before adding to CRC cells for 16 h before pSMAD 1/8 analysis by phospho-flow cytometry. Graph shows mean and individual data points of normalized % pSMAD 1/8 signal from n = 2/3 independent experiments. (b) Ginisortamab can reverse recombinant human gremlin-1 suppression of pSMAD 1/8 in HCT 116 CRC cells. HCT 116 were incubated with 15 nM gremlin-1 for 16 h before addition of 150 nM ginisortamab for 30 to 120 min. Graph shows mean and individual data points of normalized % pSMAD 1/8 signal from n = 3 independent experiments. (c) Ginisortamab exhibits dose-dependent reversal of pSMAD 1/8 inhibition. HCT 116 cells were incubated with 15 nM gremlin-1 for 16 h before addition of a titration of ginisortamab for the final 120 min before pSMAD 1/8 analysis. Graph shows individual data points of normalized % pSMAD 1/8 signal from n = 3 independent experiments and non-linear fit curve; IC50 for ginisortamab = 1.578 nM. (d) quantitative RT-PCR analysis of ID1 mRNA level in CRC cell lines stimulated with gremlin-1 in the presence of hIgG4 or ginisortamab. Fold changes are shown relative to hIgG4. Graph shows mean and individual data points from n = 3 independent experiments.

Article Snippet: The surface BMP receptor expression profile of CRC cell lines was measured by flow cytometry using biotin-labeled anti-BMP receptor antibodies obtained from R&D Systems: BMPR1A (BAF820), BMPR1B (BAF505), BMPR2 (BAF811), ACVR1 (BAF637), ACVR2A (BAF340), ACVR2B (BAF339), and goat IgG isotype control (BAF108).

Techniques: Protein-Protein interactions, Recombinant, Control, Incubation, Flow Cytometry, Inhibition, Titration, Quantitative RT-PCR

Journal: mAbs

Article Title: Discovery of ginisortamab, a potent and novel anti-gremlin-1 antibody in clinical development for the treatment of cancer

doi: 10.1080/19420862.2023.2289681

Figure Lengend Snippet: Ginisortamab inhibits endogenous gremlin-1-mediated antagonism of BMP target gene expression in human CRC cells and fibroblasts. (a) Representative immunofluorescence images of CellTracker Deep red-labeled and CellTracker green CMFDA-labeled HCT 116 cells cultured alone or in direct co-culture. Cultures were imaged 72 h after seeding. Representative flow cytometry dot plots showing the gating strategy to isolate CellTracker green CMFDA-labeled CRC cells and CellTrace violet-labeled fibroblasts from co-cultures. TO-PRO3 was added to discriminate between viable and dead cells. (b) quantitative RT-PCR analysis of ID1 mRNA level in CRC cell lines and fibroblasts cultured on their own or in direct co-culture in presence of 150 nM hIgG4 or ginisortamab. CRC cell lines and fibroblast co-cultures were incubated for 72 h and sorted prior to mRNA analysis to isolate pure populations of CRC cell lines (sorted CRC cells) and fibroblasts (sorted ‘Fbs). Fold changes in CRC cell lines and fibroblasts are shown relative to either CRC cell lines or fibroblasts alone treated with hIgG4, respectively. The left y-axes depict gene expression change in CRC cells, and the right y-axes depict gene expression change in fibroblasts. Graphs show mean and individual data points from n = 3 independent experiments.

Article Snippet: The surface BMP receptor expression profile of CRC cell lines was measured by flow cytometry using biotin-labeled anti-BMP receptor antibodies obtained from R&D Systems: BMPR1A (BAF820), BMPR1B (BAF505), BMPR2 (BAF811), ACVR1 (BAF637), ACVR2A (BAF340), ACVR2B (BAF339), and goat IgG isotype control (BAF108).

Techniques: Targeted Gene Expression, Immunofluorescence, Labeling, Cell Culture, Co-Culture Assay, Flow Cytometry, Quantitative RT-PCR, Incubation, Gene Expression

Journal: mAbs

Article Title: Discovery of ginisortamab, a potent and novel anti-gremlin-1 antibody in clinical development for the treatment of cancer

doi: 10.1080/19420862.2023.2289681

Figure Lengend Snippet: Taqman probe ID list.

Article Snippet: The surface BMP receptor expression profile of CRC cell lines was measured by flow cytometry using biotin-labeled anti-BMP receptor antibodies obtained from R&D Systems: BMPR1A (BAF820), BMPR1B (BAF505), BMPR2 (BAF811), ACVR1 (BAF637), ACVR2A (BAF340), ACVR2B (BAF339), and goat IgG isotype control (BAF108).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Insight into Molecular Mechanism for Activin A-Induced Bone Morphogenetic Protein Signaling

doi: 10.3390/ijms21186498

Figure Lengend Snippet: Only BMP type I receptor subtype ALK2, but not ALK1/3/6, is able to mediate activin A-induced BMP signaling upon FKBP12 dissociation. ( A ) RT-PCR confirmed that ALK2 is the only predominately expressed BMP type I receptor subtype in HepG2BRA cells and only a trace amount of ALK3 and ALK6 can be detected (note: the y axis is in log scale). The expression level of ALK1 was set as 1, and all the other numbers were normalized with ALK1. ( B ) The specific siRNA effectively knocked down ALK2 in HepG2BRA cells. ( C ) None of the three BMP type I receptor subtypes (ALK1/3/6) could rescue the luciferase downregulation caused by ALK2 knockdown in the presence of activin A and FK506, suggesting that ALK1/3/6 cannot signal activinA-induced BMP signaling upon FKBP12 dissociation, and ( D ) this result was confirmed by Western blotting. Results of luciferase assay and the RT-PCR assay were represented as a mean relative luciferase units (RLU) ± SEM and relative quantity (RQ) ± SEM ( n = 3, * p < 0.05, ** p < 0.01).

Article Snippet: The following plasmids were used in the current study: pCDNA3 plasmids harboring ALK2 WT , ALK3 WT and ALK6 WT were purchased from Addgene (#80870, #80873 and #80882). pCMV3 plasmids harboring activin A receptor type 2A (ActR2A) and human activin A receptor type 2B (ActR2B) were obtained from Sino Biological Inc.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Luciferase, Knockdown, Western Blot

Journal: American Journal of Hematology

Article Title: A Recombinant Antibody Against ALK2 Promotes Tissue Iron Redistribution and Contributes to Anemia Resolution in a Mouse Model of Anemia of Inflammation

doi: 10.1002/ajh.27578

Figure Lengend Snippet: RKER‐216 decreases hepcidin transcription in vitro and controls iron availability by lowering hepcidin secretion in vivo. (A) Hep3B cells were serum starved with 1% FBS overnight and incubated with an ascending dose of RKER‐216 (0.02–1 μg/mL) in the absence or presence of 5 ng/mL of BMP6, BMP2/6, or BMP2 for 6 h ( n = 4–5 per group). (B) Characterization of human ACVR1 knockout in HepG2 and Huh7 cells by quantifying ACVR1 and BMPR1A transcript copy number ( n = 3 per group). (C) ACVR1 KO and the negative control cells were treated with RKER‐216 at 1–30 μg/mL for 6 h ( n = 3 per group). Relative HAMP mRNA levels were determined by qRT‐PCR and transcripts were normalized to an internal control RPL19 . The average of PBS control without ligand stimulation or ACVR1 wildtype without RKER‐216 was set to 1. For ligand preference, hepcidin stimulated by each ligand was set to 100%. Values represent mean ± SEM. Results were compared across RKER‐216 and BMP ligands by one‐ or two‐way ANOVA with Tukey's post hoc test. Means without a common superscript differ significantly ( p < 0.05) or * p < 0.05, *** p < 0.001 relative to untreated cells of the same genotype. (D‐G) B6N male mice at 8 weeks were treated with a single SC dose of isotype control or RKER‐216 at 3 mg/kg for times as indicated ( n = 5 per group). Serum was collected to quantify (D) RKER‐216 exposure and (E) serum hepcidin by ELISA, and (F) serum iron and (G) serum transferrin saturation (TSAT) by colorimetric assays. Values represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 relative to the isotype control mice of the same time point by Student's t ‐test.

Article Snippet: Human cDNA clones for ACVR1 (RC207486; Origene) and BMPR1A (RC206048; Origene) were used to generate standard curve in quantifying transcript copy number.

Techniques: In Vitro, In Vivo, Incubation, Knock-Out, Negative Control, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Hepcidin expression is associated with increased γ-secretase–mediated cleavage of neogenin in the liver

doi: 10.1016/j.jbc.2024.107927

Figure Lengend Snippet: Hjv prevents Netrin-1–induced Neo1 degradation, and Alk3 facilitates the generation of γ-secretase–cleaved Neo1-ECD/TMD in Hep3B cells. A , diagram of fNeo1, mouse Hjv with an N-terminal 3xFLAG epitope (fHjv), mouse Alk3 with an N-terminal FLAG/MYC epitope (fAlk3), and mouse Netrin-1. B , co-expression with fHjv does not inhibit α-secretase cleavage of fNeo1. At 56 h after transfection, cell surface proteins were biotinylated. The eluted cell surface proteins, ∼10% of input lysate, and a fraction of concentrated CM were subjected to SDS-PAGE and immunodetection (IB). C , co-expression with fAlk3 increases fNeo1, fNeo1-ECD/TMD, and fNeo1-ECD. Experiments were performed as described above in ( B ) except that LY450139 (10 μM) was used to inhibit γ-secretase proteolysis. Each panel was cropped from the same image. D , quantification of cell surface fNeo1 and fNeo1-ECD/TMD bands in ( C ) (panel-1, lane 7/9), as well as fNeo1-ECD bands in ( C ) (lowest panel, lane 2/4) (n = 3). E , co-expression with fAlk3 increases γ-secretase–cleaved fNeo1-ICD. Experiments were performed as described in the legend to Fig. 3 B . Each panel was cropped from the same image. Two sets of images with different exposure for anti-FLAG antibody were presented. F , quantification of fNeo1-ICD bands in ( E ) (panel-2; lane 5/6) (n = 3). G , incubation with Netrin-1 decreases fNeo1 levels in Hep3B cells. fNeo1-transfected Hep3B cells were incubated with mouse Netrin-1 at 0, 0.1, 0.25, 0.5, or 1.0 μg/ml for ∼16 h. Cell surface proteins were subjected to biotinylation and immunodetection. H , quantification of total cell surface fNeo1 in ( G ) (panel-4) (n = 3). I , co-expression of Hjv and Neo1 prevents Netrin-1–mediated degradation of Neo1. Cells were transfected as described above in ( B ) and incubated with mouse Netrin-1 at 0 and 0.5 μg/ml for ∼16 h, followed by biotinylation and immunodetection. All experiments were repeated at least three times with consistent results. All quantification data shown are means ± SD. ∗, p < 0.05; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001.

Article Snippet: Mouse Neo1 ORF (NM_008684) with a C-terminal FLAG/MYC epitope (fNeo1) in pCMV6 vector (#MR226235) and mouse Alk3 ORF (NM_009758.3) with a C-terminal FLAG/MYC epitope (fAlk3) in pCMV6 vector (#MR227586) were obtained from OriGene Technologies Inc. We obtained pEGFP-N1 plasmid from Clontech.

Techniques: Expressing, Transfection, SDS Page, Immunodetection, Incubation