Journal: International journal of molecular sciences

Article Title: Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors.

doi: 10.3390/ijms23052780

Figure Lengend Snippet: Figure 2. ACVR1 (a,a’), BMPR1A (b,b’), BMPR1B (c,c’), and TGFBR1 (d,d’) mRNA and protein abundance in preantral (primordial, primary, and secondary follicles) and small antral follicles obtained from the control (CTR) and methoxychlor (MXC-) treated gilts. The mRNA expression (quantitative real-time PCR) is presented relative to GAPDH as mean ± SEM (a–d). Relative protein abundance was measured by the densitometric method and expressed as the ratio relative to β-actin abundance (mean ± SEM, a’–d’). The fragment of membranes with bands corresponding to predicted molecular weights is shown above graphs. Asterisks denote significant differences between the CTR and treated animals (for preantral follicles pool n = 5, for small antral follicle n = 15, * p < 0.05, ** p < 0.01, *** p < 0.001, Mann–Whitney U test).

Article Snippet: Next, the membranes were incubated for 1 h at room temperature with secondary anti-rabbit (in the case of AMH, AMHR2, BMP15, BMPR1A, BMPR1B, BMPR2, FSHR, GDF9, and TGFBR1) or anti-goat (for ACVR1) antibodies linked to horseradish peroxidase (Jackson ImmunoResearch, Cambridge, UK) at 1:10000 antibody dilution.

Techniques: Quantitative Proteomics, Control, Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: International journal of molecular sciences

Article Title: Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors.

doi: 10.3390/ijms23052780

Figure Lengend Snippet: Figure 3. Immunolocalization of ACVR1, BMPR1A, BMPR1B, and TGFBR1 in preantral (primordial, primary, and secondary follicle) (a) and small antral (b) follicles of the control (CTR) and methoxychlor (MXC-) treated gilts. ACVR1, BMPR1A, BMPR1B, and TGFBR1 positive staining was observed in oocytes (asterisks) and granulosa cells (arrows) of preantral follicles, as well as granulosa (arrows) and theca cells (arrowheads) of small antral follicles, in both examined groups. All sections were counterstained with hematoxylin QS. There was no positive staining observed in the negative control sections (a,b, insets). Prim—primordial follicles; SF—secondary follicles; AF—antral follicles. Bars = 50 µm.

Article Snippet: Next, the membranes were incubated for 1 h at room temperature with secondary anti-rabbit (in the case of AMH, AMHR2, BMP15, BMPR1A, BMPR1B, BMPR2, FSHR, GDF9, and TGFBR1) or anti-goat (for ACVR1) antibodies linked to horseradish peroxidase (Jackson ImmunoResearch, Cambridge, UK) at 1:10000 antibody dilution.

Techniques: Control, Staining, Negative Control

Journal: Journal of Clinical Investigation

Article Title: Anti-ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1

doi: 10.1172/jci153792

Figure Lengend Snippet: Figure 1. Anti-ACVR1 antibodies block BMP7 and activin A signaling in HEK293.ACVR1[R206H] cells but increase heterotopic bone formation in FOP mice. Activin A and BMP7 dose response was evaluated in stable pools of HEK293/BRE-luciferase reporter cells overexpressing ACVR1[R206H] (A). HEK293/ BRE-luciferase reporter cells overexpressing ACVR1[R206H] were treated with a fixed concentration (2 nM) of BMP7 (B) or activin A (C). Anti-ACVR1 anti- bodies inhibited Smad1/5/8 phosphorylation induced by BMP7 or activin A (B and C). Data show the mean (n = 4) ± SEM. Three biological replicates were performed for the in vitro signaling assays. (D) Acvr1[R206H]FlEx/+; GT(ROSA26)SorCreERT2/+ mice were injected with tamoxifen to initiate the model and concurrent- ly injected with anti-ACVR1 antibodies or isotype control antibody at 10 mg/kg weekly (n = 7–8/group). Total heterotopic bone lesion volume was measured 4 weeks after initiation. Data show the mean ± SD. *P < 0.05, **P < 0.01 by 1-way ANOVA with Dunnett’s multiple-comparison test. (E) Representative μCT images of FOP mice [Acvr1[R206H]FlEx/+; GT(ROSA26)SorCreERT2/+, after tamoxifen] treated with anti-ACVR1 antibody or isotype control antibody. Yellow arrows indicate the positions of heterotopic bone lesions.

Article Snippet: After 15 minutes of blocking (Thermo Fisher Scientific, 14-9161-73), cells were stained with anti-ACVR1 primary antibody (R&D Systems, MAB637) for 1 hour followed by staining with Alexa Fluor 647–conjugated secondary antibody (Thermo Fisher Scientific, A-21236) for 30 minutes.

Techniques: Blocking Assay, Luciferase, Concentration Assay, Phospho-proteomics, In Vitro, Injection, Control, Comparison

Journal: Journal of Clinical Investigation

Article Title: Anti-ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1

doi: 10.1172/jci153792

Figure Lengend Snippet: Figure 2. Anti-ACVR1 antibody–induced changes in hepcidin and iron levels are consistent with inhibition of WT ACVR1 and activation of ACVR1[R206H] in vivo. (A and C) In WT mice (n = 8/group), anti-ACVR1 mAb 1 decreased serum hepcidin (A) and increased serum iron (C). (B and D) In FOP mice [Acvr1[R206H]FlEx/+; GT(ROSA26)SorCreERT2/+, after tamoxifen] (n = 5–6/ group), anti-ACVR1 mAb 1 increased serum hepcidin (B) and decreased serum iron (D). ***P < 0.001 by Student’s t test.

Article Snippet: After 15 minutes of blocking (Thermo Fisher Scientific, 14-9161-73), cells were stained with anti-ACVR1 primary antibody (R&D Systems, MAB637) for 1 hour followed by staining with Alexa Fluor 647–conjugated secondary antibody (Thermo Fisher Scientific, A-21236) for 30 minutes.

Techniques: Inhibition, Activation Assay, In Vivo

Journal: Journal of Clinical Investigation

Article Title: Anti-ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1

doi: 10.1172/jci153792

Figure Lengend Snippet: Figure 3. Ligand-independent dimerization of ACVR1[R206H], but not WT ACVR1, induces Smad1/5/8 signaling. HEK293 cells harboring p-Smad1/5/8– responsive luciferase reporter (BRE) were transfected with hACVR1-DmrB (A) or hACVR1[R206H]-DmrB (B). Homodimerization of C-terminally DmrB- tagged ACVR1 was induced with 20 nM B/B homodimerizer for 16 hours. Activin A activated Smad1/5/8 signaling only in hACVR1[R206H]-DmrB cells, but BMP6 activated Smad1/5/8 signaling both in hACVR1-DmrB and hACVR1[R206H]-DmrB cells (A and B). Intracellular homodimerization of hACVR1[R206H] activated Smad1/5/8 signaling in the absence of exogenous ligands (C) as well as in the presence of 300 nM ACVR2B-Fc ligand trap (D). Data show the mean (n = 4) ± SEM. Three biological replicates were performed for the in vitro signaling assays.

Article Snippet: After 15 minutes of blocking (Thermo Fisher Scientific, 14-9161-73), cells were stained with anti-ACVR1 primary antibody (R&D Systems, MAB637) for 1 hour followed by staining with Alexa Fluor 647–conjugated secondary antibody (Thermo Fisher Scientific, A-21236) for 30 minutes.

Techniques: Luciferase, Transfection, In Vitro

Journal: Journal of Clinical Investigation

Article Title: Anti-ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1

doi: 10.1172/jci153792

Figure Lengend Snippet: Figure 4. Dimeric anti-ACVR1 antibodies activate, whereas monomeric anti-ACVR1 Fabs block, ACVR1[R206H]. (A) Acvr1[R206H]FlEx/+; GT(ROSA26)SorCreERT2/+ mice (n = 7–9/group) received plasmids expressing anti-ACVR1 Fabs or a plasmid encoding a control mAb by hydrodynamic delivery (HDD) 5 days after initiation of the model with tamoxifen. HO was triggered in the hind limb by muscle pinch 7 days after HDD and total heterotopic bone volume was mea- sured 6 weeks after injury. FOP mice [Acvr1[R206H]FlEx/+; GT(ROSA26)SorCreERT2/+, after tamoxifen] expressing anti-ACVR1 Fab showed reduced HO compared with control mice. Data show the mean ± SD. *P < 0.05 by 1-way ANOVA with Dunnett’s multiple-comparison test. (B) Representative μCT images of FOP mice expressing either anti-ACVR1 Fab or an isotype control antibody. (C) Acvr1[R206H]/+; GT(ROSA26)SorCreERT2/+ ([R206H]/+) mES cells (mESC) were treated with activin A, anti-ACVR1 mAb 2, anti-ACVR1 Fab 2, or anti-activin A mAb (REGN2476) in various combinations for 1 hour. Activin A and anti-ACVR1 mAb 2 but not anti-ACVR1 Fab 2 induced Smad1/5/8 phosphorylation. Anti-ACVR1 Fab 2 significantly reduced activin A–induced Smad1/5/8 phosphorylation, whereas anti-ACVR1 mAb 2 only slightly reduced activin A–induced Smad1/5/8 phosphorylation. (D) Anti-ACVR1 antibody activation of ACVR1[R206H] is independent of activin A. Acvr1[R206H]FlEx/+; GT(ROSA26)SorCreERT2/+ mice (n = 6–8/group) were injected with tamoxifen to initiate the model and concurrently injected with antibodies at 10 mg/kg weekly. Total heterotopic bone volume was measured 3 weeks after initiation. Data show the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: After 15 minutes of blocking (Thermo Fisher Scientific, 14-9161-73), cells were stained with anti-ACVR1 primary antibody (R&D Systems, MAB637) for 1 hour followed by staining with Alexa Fluor 647–conjugated secondary antibody (Thermo Fisher Scientific, A-21236) for 30 minutes.

Techniques: Blocking Assay, Expressing, Plasmid Preparation, Control, Comparison, Phospho-proteomics, Activation Assay, Injection

Journal: Journal of Clinical Investigation

Article Title: Anti-ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1

doi: 10.1172/jci153792

Figure Lengend Snippet: Figure 5. Anti-ACVR1 antibody activation of ACVR1[R206H] is type II receptor dependent. (A) Acvr1[R206H]/+; GT(ROSA26)SorCreERT2/+ ([R206H]/+) mES cells lacking Acvr2a plus Acvr2b, or Bmpr2 or all 3 of these type II receptor genes were treated with 10 nM activin A, BMP7, BMP2, BMP10, or anti-ACVR1 mAb 1 for 1 hour. Activin A, BMP7, BMP2, BMP10, and anti-ACVR1 mAb 1 induced Smad1/5/8 phosphorylation in cells that lack Bmpr2 but retain Acvr2a and Acvr2b, but not in cells where Acvr2a and Acvr2b or all 3 type II receptors have been knocked out. (B) ACVR2B coimmunoprecipitates with both ACVR1 and ACVR1[R206H] from W20 cells expressing Myc-tagged ACVR1 and/or HA-tagged ACVR2B. Myc-ACVR1 was immunoprecipi- tated using an anti-Myc antibody. ACVR1 and ACVR2B were detected using an anti-ACVR1 or anti-HA antibody, respectively.

Article Snippet: After 15 minutes of blocking (Thermo Fisher Scientific, 14-9161-73), cells were stained with anti-ACVR1 primary antibody (R&D Systems, MAB637) for 1 hour followed by staining with Alexa Fluor 647–conjugated secondary antibody (Thermo Fisher Scientific, A-21236) for 30 minutes.

Techniques: Activation Assay, Phospho-proteomics, Expressing

Journal: Journal of Clinical Investigation

Article Title: Anti-ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1

doi: 10.1172/jci153792

Figure Lengend Snippet: Figure 6. ACVR1[R206H;S330P] is activated by anti-ACVR1 antibodies but to a lesser degree than ACVR1[R206H]. (A and B) Acvr1[R206H]FlEx/+; GT(ROSA26) SorCreERT2/+ mice or Acvr1huecto;[R206H]FlEx;[S330P]/+; GT(ROSA26)SorCreERT2/+ (FOP[S330P]) mice were injected with tamoxifen to initiate the model and concurrently injected with anti-ACVR1 mAb 1 or isotype control antibody at 10 mg/kg weekly (n = 8/group). Total heterotopic bone volume was measured 3 weeks after initiation of the model. ACVR1 mAb 1 increased HO compared with isotype control in both mouse models, though to a lesser degree in FOP[S330P] mice. Data show the mean ± SD. *P < 0.05, ***P < 0.001 by 1-way ANOVA with Bonferroni’s multiple-comparison test. (C) Acvr1huecto;[R206H;S330P]/+; GT(ROSA26)SorCreERT2/+ ([R206H;S330P]/+) mES cells were treated with 10 nM activin A, anti-ACVR1 mAb 1, or anti-hACVR1 antibody and assessed for phosphorylated Smad1/5/8. Anti-hACVR1 mAb (that only binds ACVR1[huecto;R206H;S330P] and not WT mouse ACVR1) induced Smad1/5/8 phosphorylation, whereas mAb 1 (which recognizes both human and mouse ACVR1) did not drive an appreciable level of Smad1/5/8 phosphorylation.

Article Snippet: After 15 minutes of blocking (Thermo Fisher Scientific, 14-9161-73), cells were stained with anti-ACVR1 primary antibody (R&D Systems, MAB637) for 1 hour followed by staining with Alexa Fluor 647–conjugated secondary antibody (Thermo Fisher Scientific, A-21236) for 30 minutes.

Techniques: Injection, Control, Comparison, Phospho-proteomics

Journal: International Journal of Oncology

Article Title: Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival

doi: 10.3892/ijo.2021.5223

Figure Lengend Snippet: Involvement of ALK2, ALK3 and ALK6 in the effects of AMH in COV434-AMHRII and SKOV3-AMHRII cells. (A) pSMAD1/5 levels were evaluated following incubation with 25 nM LR-AMH for 6 h starting 24 (COV434-AMHRII cells) or 48 (SKOV3-AMHRII cells) h post-transfection with si-ALK2, si-ALK3 and si-ALK6. Data are presented as the mean + max of pSMAD1/5:GAPDH ratios. n=2. (B) Caspase-3/7 activity and cleaved caspase-3 and PARP levels were analyzed following incubation with 25 nM AMH for 6 h (24 and 48 h post-transfection). * P<0.05; ** P<0.01. AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; si, small interfering RNA; p, phosphorylated; C., cleaved.

Article Snippet: Anti-ALK2 and anti-ALK3 IgG1 and BsAbs were produced in CHO (Evitria AG) and 293T cells (ATCC ® CRL-1573) For antibody production in 293T cells, the cells were cultured in 150 mm 2 dishes to 70% confluence.

Techniques: Incubation, Transfection, Activity Assay, Recombinant, Small Interfering RNA

Journal: International Journal of Oncology

Article Title: Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival

doi: 10.3892/ijo.2021.5223

Figure Lengend Snippet: Incubation with recombinant LR-AMH modulates ALK2 and ALK3 expression in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN ovarian cancer cells. Immunofluorescence analysis of AMHRII, ALK2, ALK3 and ALK6 expression in basal conditions and after incubation with 25 nM LR-AMH for 90 min. Data are presented as the ratio of receptor (green) and nucleus (blue) labeling. * P<0.05, ** P<0.01 and *** P<0.001 n=6. AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; IR, irrelevant antibody; NT, non-treated.

Article Snippet: Anti-ALK2 and anti-ALK3 IgG1 and BsAbs were produced in CHO (Evitria AG) and 293T cells (ATCC ® CRL-1573) For antibody production in 293T cells, the cells were cultured in 150 mm 2 dishes to 70% confluence.

Techniques: Incubation, Recombinant, Expressing, Immunofluorescence, Labeling

Journal: International Journal of Oncology

Article Title: Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival

doi: 10.3892/ijo.2021.5223

Figure Lengend Snippet: Differential expression of ALK2 and ALK3 following LR-AMH treatment is associated with the expression of cell survival and apoptosis markers in COV434-AMHRII, SKOV3-AMHRII, OVCAR8 and KGN ovarian cancer cells. Quantification of the western blot analysis of AMH signaling (pSMAD1/5), ALK2 and ALK3 expression, apoptosis induction (cleaved caspase-3 and PARP) and pAKT levels following incubation with 1.6 and 25 nM LR-AMH for 6 h. Data are presented as the fold-change relative to control (no LR-AMH) by bars corresponding from left to right to 0, 1.6 and 25 nM for each protein expression. n>3. Raw data are presented in . AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; p, phosphorylated; c, cleaved; PARP, poly(ADP-ribose) polymerase.

Article Snippet: Anti-ALK2 and anti-ALK3 IgG1 and BsAbs were produced in CHO (Evitria AG) and 293T cells (ATCC ® CRL-1573) For antibody production in 293T cells, the cells were cultured in 150 mm 2 dishes to 70% confluence.

Techniques: Expressing, Western Blot, Incubation, Recombinant

Journal: International Journal of Oncology

Article Title: Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival

doi: 10.3892/ijo.2021.5223

Figure Lengend Snippet: Differential expression of ALK2 and ALK3 following LR-AMH treatment is associated with the expression of cell survival and apoptosis markers in primary ovarian cancer cells. Quantification of the western blot analysis of AMH signaling (pSMAD1/5), ALK2 and ALK3 expression, apoptosis induction (cleaved caspase-3 and PARP) and pAKT levels following incubation of cells isolated from ascites of three patients with ovarian cancer with 1.6 and 25 nM LR-AMH for 6 h. Data are presented as the fold change relative to control (no LR-AMH) by bars corresponding from left to right to 0, 1.6 and 25 nM for each protein expression. n=1 for each patient. Raw data are presented in . AMH, anti-Müllerian hormone; LR-AMH, active recombinant AMH; AMHRII, AMH type II receptor; ALK, activin receptor-like kinase; P, phosphorylated; c, cleaved; PARP, poly(ADP-ribose) polymerase.

Article Snippet: Anti-ALK2 and anti-ALK3 IgG1 and BsAbs were produced in CHO (Evitria AG) and 293T cells (ATCC ® CRL-1573) For antibody production in 293T cells, the cells were cultured in 150 mm 2 dishes to 70% confluence.

Techniques: Expressing, Western Blot, Incubation, Isolation, Recombinant

Journal: International Journal of Oncology

Article Title: Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival

doi: 10.3892/ijo.2021.5223

Figure Lengend Snippet: Affinity of the MAbs and BsAbs for their targets estimated by assessing the EC 50 by ELISA.

Article Snippet: Anti-ALK2 and anti-ALK3 IgG1 and BsAbs were produced in CHO (Evitria AG) and 293T cells (ATCC ® CRL-1573) For antibody production in 293T cells, the cells were cultured in 150 mm 2 dishes to 70% confluence.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Oncology

Article Title: Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival

doi: 10.3892/ijo.2021.5223

Figure Lengend Snippet: The anti-AMHRII-ALK2 BsAb 12G4-2F9 inhibits the growth of COV434-AMHRII cell xenografts in vivo . Nude mice bearing COV434-MISRII cell-derived tumors were treated with 17 mg/kg of anti-AMHRII-CD5 (control BsAb targeting AMHRII and CD5), anti-AMHRII-ALK2 (12G4-2C1 and 12G4-2F9), anti-AMHRII-ALK3 (12G4-3D7 and 12G4-3H6) BsAbs or vehicle (NaCl) twice per week for 4 weeks. Data are presented as the mean ± SEM. * P<0.05 and ** P<0.01. AMHRII, anti-Müllerian hormone type II receptor; ALK, activin receptor-like kinase; BsAbs, bispecific antibodies.

Article Snippet: Anti-ALK2 and anti-ALK3 IgG1 and BsAbs were produced in CHO (Evitria AG) and 293T cells (ATCC ® CRL-1573) For antibody production in 293T cells, the cells were cultured in 150 mm 2 dishes to 70% confluence.

Techniques: In Vivo, Derivative Assay