Journal: JCI insight

Article Title: Prostate tumor-derived GDF11 accelerates androgen deprivation therapy-induced sarcopenia.

doi: 10.1172/jci.insight.127018

Figure Lengend Snippet: Figure 3. Castration increased active catabol- ic TGF-β family myokine proteins in skeletal muscles of tumor-bearing mice. (A–F) Protein expression in GAS muscle. (A) Representative immunoblots of soluble active myostatin (MSTN) C-terminal dimer and eukaryotic elongation factor 2 (EF2) expression in muscle from sets of 4 mice, castrated for the indicated times or sham castrat- ed. Lanes of immunoblots marked “C” contain identical control sample for interblot compari- son. (B) Quantification of relative MSTN levels for castrated mice (red) or sham-castrated mice (blue), from 3 determinations for each muscle (see Supplemental Figure 5 for additional immunoblots and supplemental materials for full, uncut gels). ELISA-determined protein levels of soluble active activin AA dimer (C), activin BB dimer (D), activin AB dimer (E), and soluble GDF11 (F), in muscle from 4 mice at each time point, measured 3 times each. (G–L) Protein expression in TRI muscle. (G) Representative immunoblot of MSTN and EF2 expression, as in A. (H) Quantification of MSTN levels, as in B. ELISA-determined protein levels of soluble active activin AA dimer (I), activin BB dimer (J), activin AB dimer (K), and soluble GDF11 (L), from 4 mice at each time point, measured 3 times each. Columns are sham-castrated normalized means at each time; bars are SEM. Individual mouse levels are indicated by open circles. *P < 0.05, **P < 0.01, and ***P < 0.001 versus sham-castrated group determined using 1-way ANOVA and Bonferroni’s correction (B and H) or Dunnett’s test (C–F and I–L).

Article Snippet: The lower membrane was incubated with rat anti-myostatin antibody (1:1000, clone 84214, R&D Systems, Bio-Techne MAB788), and the upper membrane was incubated with rabbit anti-EF2 antibody (1:2000, G270) (48), each diluted in 5% nonfat dried milk in Tris-buffered saline, overnight at 4°C and washed in Tris-buffered saline containing 0.1% Tween-20.

Techniques: Muscles, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: JCI insight

Article Title: Prostate tumor-derived GDF11 accelerates androgen deprivation therapy-induced sarcopenia.

doi: 10.1172/jci.insight.127018

Figure Lengend Snippet: Figure 5. Castration increased GDF11 and myostatin in tumor and serum. (A–C) GDF11 and myostatin protein expression in tumor. (A) ELISA-determined levels of GDF11 in prostate tumor tissue, from 4 mice at each time, mea- sured 3 times. (B) Representative immunoblots of soluble active myostatin C-terminal dimer (MSTN) and eukaryotic elongation factor 2 (EF2) expression in prostate tumor tissue from sets of 4 mice, castrated for the indicated times or sham castrated. Lanes of immunoblots marked “C” contain identical control sample for interblot comparison. (C) Quantification of MSTN levels in tumor tissue from castrated (red) or sham-castrated (blue) mice, from 3 determi- nations for each tumor (see Supplemental Figure 5 for additional immunoblots and supplemental materials for full, uncut gels). (D–F) GDF11 and myostatin protein expression in serum. (D) ELISA-determined levels of GDF11 in serum, from 4 mice at each time, measured 3 times. (E) Representative immunoblots of MSTN expression in equal quanti- ties of serum protein from the 4 mice in A. (F) Quantification of MSTN levels in serum, as in B. (G–I) Comparison of GDF11 and myostatin protein expression between muscle and tumor. (G) ELISA-determined levels of GDF11 in GAS muscle and prostate tumor from sets of 4 mice, sham castrated (blue) or 2 weeks after castration (red), measured 3 times. (H) Representative immunoblots of MSTN in GAS muscle and prostate tumor from sets of 4 mice, sham-cas- trated or 8 weeks after castration. (I) Quantification of relative levels of MSTN between GAS muscle and prostate tumor. Columns are normalized means; bars are SEM. n = 4/group, indicated by open circles. *P < 0.05, **P < 0.01, and ***P < 0.001 versus sham-castrated group (except G and I, GAS muscle vs. tumor tissue), determined using 1-way ANOVA and Dunnett’s test (A, D, and G) or 1-way ANOVA and Bonferroni’s correction (C, F, and I).

Article Snippet: The lower membrane was incubated with rat anti-myostatin antibody (1:1000, clone 84214, R&D Systems, Bio-Techne MAB788), and the upper membrane was incubated with rabbit anti-EF2 antibody (1:2000, G270) (48), each diluted in 5% nonfat dried milk in Tris-buffered saline, overnight at 4°C and washed in Tris-buffered saline containing 0.1% Tween-20.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Comparison

Journal: JCI insight

Article Title: Prostate tumor-derived GDF11 accelerates androgen deprivation therapy-induced sarcopenia.

doi: 10.1172/jci.insight.127018

Figure Lengend Snippet: Figure 6. Model of ADT-induced catabolic TGF-β family myokine endocrine signaling in PrCa tumor–bearing mice. Graphs of castration-induced changes in myokine concentrations (colored lines) in skeletal muscle and serum of mice without prostate cancer (left side) or in skeletal muscle, serum, and tumor of mice with prostate cancer (right side). Tumor lobes are represented by the green (left lobe) and red (right lobe) circles. In adult tumor-free mice myostatin and the activins increase after castration but prior to strength and muscle mass loss. In PrCa tumor–bearing mice, tumor secretes GDF11 (blue) and later myostatin (red) into serum. Endocrine-derived GDF11 and myostatin increase in muscle prior to strength loss (early myostatin also moves from muscle into serum). The tumor-derived catabolic TGF-β family myokines exacerbate castration-induced sarcopenia in tumor-bearing mice.

Article Snippet: The lower membrane was incubated with rat anti-myostatin antibody (1:1000, clone 84214, R&D Systems, Bio-Techne MAB788), and the upper membrane was incubated with rabbit anti-EF2 antibody (1:2000, G270) (48), each diluted in 5% nonfat dried milk in Tris-buffered saline, overnight at 4°C and washed in Tris-buffered saline containing 0.1% Tween-20.

Techniques: Derivative Assay

Journal: Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology

Article Title: Myostatin inhibitory region of fish (Paralichthys olivaceus) myostatin-1 propeptide.

doi: 10.1016/j.cbpb.2016.01.010

Figure Lengend Snippet: Fig. 2. SDS-PAGE of affinity purified, MBP-fused, truncated forms of flatfish MSTN1 propeptide and inhibition of MSTN activities by those proteins. A) Amylose resin affinity-purified Pro23–265, Pro45–100, Pro45–90, Pro45–80, Pro55–100, and Pro65– 100 were subjected to 12.5% SDS-PAGE, and proteins were visualized with Coomassie blue staining. B) HEK293 cells stably expressing (CAGA) 12-luciferase gene construct were used. Various concentrations of MBP-MSTN1pro proteins in combination with 1 nM MSTN were added to the HEK293 cells, followed by incubation for 24 h. Medium was removed, and luminescence substrate was added, followed by luminescence measurement. The error bars represent SEM (n = 3). The IC50 value means not sharing the same superscript are different at P b 0.05.

Article Snippet: The primary antibody was monoclonal anti-MSTN antibody (1:1000, MAB788, R&D Systems, MN, USA) for overnight at 4 °C.

Techniques: SDS Page, Inhibition, Staining, Stable Transfection, Expressing, Luciferase, Construct, Incubation

Journal: Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology

Article Title: Myostatin inhibitory region of fish (Paralichthys olivaceus) myostatin-1 propeptide.

doi: 10.1016/j.cbpb.2016.01.010

Figure Lengend Snippet: Fig. 3. SDS-PAGE analysis of the binding of Pro45–80 and Pro55–100 to MSTN in a pull-down assay. A) Amylose resin was mixed with either 10 μg of Pro45–80 plus 1 μg of MSTN (lane 1) or 10 μg of Pro55–100 plus 1 μg of MSTN (lane 2). After intensive washing, proteins bound to the amylose resin were eluted with reducing SDS-PAGE loading buffer, and eluted proteins were subjected to SDS-PAGE. Asterisk (*) indicates MSTN monomer. B) Amylose resin was mixed with 3 nM of MSTN plus various concentrations of Pro45–80 in separate tubes. After intensive washing, proteins bound to the resin were eluted, and elutions were subjected to Western blot analysis by blotting against anti-MSTN antibody. C) The band intensity was quantified by densitometry, and the intensity was expressed as a ratio to MSTN control (3 nM).

Article Snippet: The primary antibody was monoclonal anti-MSTN antibody (1:1000, MAB788, R&D Systems, MN, USA) for overnight at 4 °C.

Techniques: SDS Page, Binding Assay, Pull Down Assay, Western Blot, Control

Journal: Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology

Article Title: Myostatin inhibitory region of fish (Paralichthys olivaceus) myostatin-1 propeptide.

doi: 10.1016/j.cbpb.2016.01.010

Figure Lengend Snippet: Fig. 4. Inhibition of MSTN activity by synthetic flatfish MSTN1pro peptides of various regions. HEK293 cells stably expressing (CAGA) 12-luciferase gene construct were used. Various concentrations of synthetic flatfish MSTN1pro peptides in combination with 1 nM MSTN were added to the HEK293 cells, followed by incubation for 24 h. Medium was removed, and luminescence substrate was added, followed by luminescence measurement. The error bars represent SEM (n = 3). The IC50 value means not sharing the same superscript are different at P b 0.05.

Article Snippet: The primary antibody was monoclonal anti-MSTN antibody (1:1000, MAB788, R&D Systems, MN, USA) for overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Stable Transfection, Expressing, Luciferase, Construct, Incubation

Journal: Journal of Neuroinflammation

Article Title: Stabilizing histamine release in gut mast cells mitigates peripheral and central inflammation after stroke

doi: 10.1186/s12974-023-02887-7

Figure Lengend Snippet: Information of antibodies

Article Snippet: Recombinant Human GDF11 protein (ab50159) , abcam ab50159 , 1:1000.

Techniques: Recombinant

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Inhibitory effects of growth differentiation factor 11 on autophagy deficiency-induced dedifferentiation of arterial smooth muscle cells

doi: 10.1152/ajpheart.00342.2018

Figure Lengend Snippet: Exogenous growth differentiation factor (GDF)11 promoted carotid arterial smooth muscle cell (CASMC) differentiation and phenotypic transition. In vitro, primary CASMCs isolated from the carotid artery in normal mice were used. A: mRNA expression of differentiation markers [smooth muscle (SM)22α, myosin heavy chain (MyHC), myogenin, and myogenic differentiation (MyoD)] and dedifferentiation markers [vimentin and proliferating cell nuclear antigen (PCNA)] after GDF11 treatment (100 ng/ml, 24 h) detected by RT-PCR. B: representative Western blot analysis showing the effects of GDF11 on the protein expression of differentiation markers (SM22α and calponin) and dedifferentiation markers (vimentin and PCNA) in CASMCs treated with different doses of GDF11 (0–100 ng/ml, 24 h). C–F: summarized data showing the effects of GDF11 on the protein expression of differentiation markers (SM22α and calponin) and dedifferentiation markers (vimentin and PCNA) in CASMCs treated with different doses of GDF11. Data are expressed as means ± SD; n = 5. *P < 0.05 vs. the control (Ctrl) group.

Article Snippet: Incubation with primary antibodies [SM22α (1:1500, Abcam), vimentin (1:1500, Abcam), and GDF11 (1:100, BD Biosciences)] was performed overnight at 4°C.

Techniques: In Vitro, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Inhibitory effects of growth differentiation factor 11 on autophagy deficiency-induced dedifferentiation of arterial smooth muscle cells

doi: 10.1152/ajpheart.00342.2018

Figure Lengend Snippet: Endogenous growth differentiation factor (GDF)11 induced carotid arterial smooth muscle cell (CASMC) differentiation and phenotypic transition. A: effect of GDF11-specific CRISPR-cas9 activating plasmids on GDF11 RNA expression by RT-PCR (n = 5). B: effects of GDF11-specific CRISPR-cas9 activating plasmids on GDF11 protein expression by Western blot analysis (n = 3). C and D: time-dependent changes of vimentin and smooth muscle (SM)22α RNA expression induced by GDF11-specific CRISPR-cas9 activating plasmids (n = 5). Data are expressed as means ± SD. *P < 0.05 vs. the CRISPR control (Ctrl) group.

Article Snippet: Incubation with primary antibodies [SM22α (1:1500, Abcam), vimentin (1:1500, Abcam), and GDF11 (1:100, BD Biosciences)] was performed overnight at 4°C.

Techniques: CRISPR, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Inhibitory effects of growth differentiation factor 11 on autophagy deficiency-induced dedifferentiation of arterial smooth muscle cells

doi: 10.1152/ajpheart.00342.2018

Figure Lengend Snippet: Endogenously produced growth differentiation factor (GDF)11 (GDF11) by trichostatin A (TSA)-induced carotid arterial smooth muscle cell (CASMC) differentiation and phenotypic transition. A: dose-dependent effects of TSA on GDF11 mRNA expression by RT-PCR (n = 5). B: dose-dependent effects of TSA on the ratio of vimentin to smooth muscle (SM)22α by RT-PCR (n = 5). C: representative Western blot analysis showing the dose-dependent effects of TSA on GDF11, vimentin, and SM22α protein expression. D: summarized data showing the dose-dependent effects of TSA on GDF11 protein expression (n = 5). E: summarized results showing the dose-dependent effects of TSA on expression of vimentin. F: summarized data showing the dose-dependent effects on the expression of SM22α (n = 5). Data are expressed as means ± SD. *P < 0.05 vs. the control (Ctrl) group.

Article Snippet: Incubation with primary antibodies [SM22α (1:1500, Abcam), vimentin (1:1500, Abcam), and GDF11 (1:100, BD Biosciences)] was performed overnight at 4°C.

Techniques: Produced, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Inhibitory effects of growth differentiation factor 11 on autophagy deficiency-induced dedifferentiation of arterial smooth muscle cells

doi: 10.1152/ajpheart.00342.2018

Figure Lengend Snippet: Growth differentiation factor (GDF)11 reversed carotid arterial smooth muscle cell (CASMC) dedifferentiation and autophagosome accumulation induced by autophagy inducer or lysosome function inhibitor. GDF11 (100 ng/ml), 7-ketocholesterol (7-Ket; 5 μg/ml), and bafilomycin (Baf; 5 nM) were used to treat CASMCs for 24 h. A: representative Western blot analysis showing the expression of vimentin, smooth muscle (SM)22α, and p62. B: summarized data showing the expression of vimentin. C: summarized data showing the expression of SM22α. D: summarized data showing the expression of p62. Data are expressed as means ± SD; n = 5. *P < 0.05 vs. the vehicle (Vehl)-control (Ctrl)-DMSO group; #P < 0.05 vs. the Ctrl-DMSO group; &P < 0.05 vs. the Ctrl-Baf group.

Article Snippet: Incubation with primary antibodies [SM22α (1:1500, Abcam), vimentin (1:1500, Abcam), and GDF11 (1:100, BD Biosciences)] was performed overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Inhibitory effects of growth differentiation factor 11 on autophagy deficiency-induced dedifferentiation of arterial smooth muscle cells

doi: 10.1152/ajpheart.00342.2018

Figure Lengend Snippet: Effects of trichostatin A (TSA) on neointima formation and growth differentiation factor (GDF)11 expression in the partial ligated carotid artery (PLCA) mouse model. A: representative hematoxylin and eosin staining showing neointima formation in the PLCA wall. Black arrowheads represent the medial area; white arrowheads represent the intima area. B: quantification analysis of the ratio between intima and media of the arteries in PLCA. C: representative immunohistochemistry staining showing the expression of GDF11 in the carotid artery of PLCA CD38 wild-type (CD38+/+) and CD38 gene-deficient (CD38−/−) mice treated with a normal diet (ND) or Western diet (WD). D: summarized data showing the quantification of GDF11 relative expression by the calculation of the percentage of positive staining in the artery wall. L, lumen side. Data are expressed as means ± SD; n = 5. *P < 0.05 vs. the ND-control (Ctrl)-CD38+/+ group; #P < 0.05 vs. the Ctrl-CD38+/+ group; &P < 0.05 vs. Ctrl-CD38−/− group.

Article Snippet: Incubation with primary antibodies [SM22α (1:1500, Abcam), vimentin (1:1500, Abcam), and GDF11 (1:100, BD Biosciences)] was performed overnight at 4°C.

Techniques: Expressing, Staining, Immunohistochemistry, Western Blot

Journal: Science translational medicine

Article Title: ACTRIIA-Fc rebalances activin/GDF versus BMP signaling in pulmonary hypertension

doi: 10.1126/scitranslmed.aaz5660

Figure Lengend Snippet: Immunohistochemical staining for activin A, GDF8, and GDF11 along with α-SMA costain performed on paraffin-embedded lung sections from healthy controls and patients with IPAH or HPAH (n = 5 each) and lung sections from control, MCT-exposed, or SU-Hx–exposed rats (n = 3 each). Horseradish peroxidase (HRP)–conjugated secondary antibody (3,3′-diaminobenzidine staining, as indicated by label HRP in brown) and α-smooth muscle actin (α-SMA) AP-conjugated primary antibody (as indicated by label SMA in blue; right) are shown. Scale bars, 50 μm.

Article Snippet: Measurement of GDF8 in human serum was performed using a monospecific anti-human GDF8 antibody lacking cross-reactivity with GDF11 on a multiplex ELISA system (Meso Scale Discovery) using the standard manufacturer’s protocol.

Techniques: Immunohistochemical staining, Staining, Control

Journal: Science translational medicine

Article Title: ACTRIIA-Fc rebalances activin/GDF versus BMP signaling in pulmonary hypertension

doi: 10.1126/scitranslmed.aaz5660

Figure Lengend Snippet: (A) Expression of phosphorylated SMAD1/5/9 and SMAD2/3 in human pulmonary micro-vascular endothelial cells (PMVECs) exposed to BMP9 (100 pg/ml), GDF8 (50 ng/ml), GDF11 (50 ng/ml), activin A (50 ng/ml), or activin B (50 ng/ ml) for 30 min with or without ACTRIIA-Fc (2500 ng/ml), analyzed by Western blot. n = 3 replicates; N = 3 independent experiments. (B) Luciferase activity in telomerase immortalized human microvascular endothelial (TIME) cells transfected with BMP-responsive element transcriptional reporter (BRE-Luc) and exposed to varying concentrations of BMP9 with or without ACTRIIA-Fc (n = 3; N = 3; RLU, relative luminescence units). (C) Activation of SMAD1/5 in human PMVECs treated with BMP9 (below the EC50 250 ng/ml) with or without varying concentrations of ACTRIIA-Fc, analyzed by In-Cell Western. Data represent means ± SEM. *P < 0.05, **P < 0.01, †P < 0.001, and #P < 0.0001 compared to untreated controls in each group, two-way ANOVA with Dunnett’s test for multiple comparisons (n = 3; N = 3). (D and E) BMP-mediated transcriptional activity in TIME cells treated with BMP9, GDF8, GDF11, and activin A with or without ACTRIIA-Fc (n = 6; N = 3). (F and G) Proliferation of control- and PAH-derived PMVECs in response to BMP9, activin A, and GDF11 with or without ACTRIIA-Fc. (H) Expression of phosphorylated SMAD1/5/9 and SMAD2/3 in human pulmonary artery smooth muscle cells (PASMCs) exposed to TGFβ1 (1 ng/ml), GDF8 (50 ng/ml), GDF11 (50 ng/ml), activin A (50 ng/ml), or BMP4 (50 ng/ml) for 30 min with or without ACTRIIA-Fc (2500 ng/ml), analyzed by Western blot. (I and J) Expression of α-SMA and calponin mRNA at 24 hours after stimulation with TGFβ1 (1 ng/ml), GDF8 (50 ng/ml), GDF11 (50 ng/ml), or activin A (50 ng/ml) in human PASMCs with or without ACTRIIA-Fc (n = 3 to 6). (K and L) Expression of α-SMA and calponin protein in PASMCs at 72 hours after stimulation with TGFβ1, GDF8, GDF11, activin A, or activin B with or without ACTRIIA-Fc, as assessed by In-Cell Western (n = 4; N = 3). (M) Proliferation in PASMCs exposed to GDF8 (100 ng/ml), GDF11 (100 ng/ml), activin A (100 ng/ml), activin B (100 ng/ml), PDGF-BB (1 ng/ml), or complete media for 24 hours with or without ACTRIIA-Fc (10,000 ng/ml) as measured by 3H-thymidine incorporation. (n = 5; N = 3). Data represent means ± SEM. *P < 0.05, **P < 0.01, †P < 0.001, and #P < 0.0001 compared to vehicle controls, one-way ANOVA with Sidak’s test for multiple comparisons.

Article Snippet: Measurement of GDF8 in human serum was performed using a monospecific anti-human GDF8 antibody lacking cross-reactivity with GDF11 on a multiplex ELISA system (Meso Scale Discovery) using the standard manufacturer’s protocol.

Techniques: Expressing, Western Blot, Luciferase, Activity Assay, Transfection, Activation Assay, In-Cell ELISA, Control, Derivative Assay