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: American Journal of Cancer Research

Article Title: BMP signaling inhibition overcomes chemoresistance of prostate cancer

doi:

Figure Lengend Snippet: BMP type I receptors ALK2 and ALK3 expression is highly upregulated in chemoresistant prostate cancer cells. A and B. mRNA expression of all four BMP type I receptor members (ALK1, ALk2, ALK3 and ALK6) was examined by RT-PCT in both chemosensitive DU145 and PC3 cells and chemoresistant prostate cancer DU145-TxR and PC3-TxR cells (n=3, **P<0.01, ***P<0.001). C. Western blotting result confirmed that ALK2 and ALK3 expression levels were enhanced in DU145-TxR and PC3-TxR cells in comparison to DU145 and PC3 cells.

Article Snippet: The cells were transfected with Fugene HD transfection reagent (Promega, Madison, WI, USA) following the manufacturer’s instructions using ALK2 Human siRNA oligo Duplex (SR300056, OriGene, Rockville, MD, USA) or ALK3 human siRNA oligo Duplex (SR300454, OriGene, Rockville, MD, USA) at a concentration of 20 nM, respectively.

Techniques: Expressing, Western Blot, Comparison

Journal: American Journal of Cancer Research

Article Title: BMP signaling inhibition overcomes chemoresistance of prostate cancer

doi:

Figure Lengend Snippet: Knockdown of both ALK2 and ALK3 by siRNAs significantly sensitizes the chemoresistant DU145-TxR and PC3-TxR cells to docetaxel. A. siRNA knockdown of ALK2, ALK3 simultaneously sensitized the DU145-TxR and PC3-TxR cells to docetaxel (DTX in Figure). B. RT-PCR confirmed that siRNAs significantly knocked down both ALK2 and ALK3 simultaneously in DU145-TxR and PC3-TxR cells. All data are compared to control group (n=3, **P<0.01, ***P<0.001).

Article Snippet: The cells were transfected with Fugene HD transfection reagent (Promega, Madison, WI, USA) following the manufacturer’s instructions using ALK2 Human siRNA oligo Duplex (SR300056, OriGene, Rockville, MD, USA) or ALK3 human siRNA oligo Duplex (SR300454, OriGene, Rockville, MD, USA) at a concentration of 20 nM, respectively.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Control

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

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