Journal: Circulation research

Article Title: Is Growth Differentiation Factor 11 a Realistic Therapeutic for Aging-Dependent Muscle Defects?

doi: 10.1161/CIRCRESAHA.116.307962

Figure Lengend Snippet: Functional activity of rGDF11 was determined by measuring GDF11 dose-dependent activation of Smad2/3 signaling in HepG2 reporter cells using a luciferase assay as reported in Smith et al. A. Similar activity was observed for both PeproTech and R&D Systems rGDF11. B. Biological activity of rGDF11 (R&D) was maintained for up to 4 weeks when stored in NaAcetate Buffer at 37°, pH 4.5 at a concentration of 1mg/ml.

Article Snippet: Recombinant GDF11 [rGDF11: 0.1mg/kg, Peprotech] was administered daily over 4 weeks to restore normal GDF11 levels in old mice.

Techniques: Functional Assay, Activity Assay, Activation Assay, Luciferase, Concentration Assay

Journal: Cell Proliferation

Article Title: Growth differentiation factor 11 inhibits adipogenic differentiation by activating TGF‐beta/Smad signalling pathway

doi: 10.1111/cpr.12631

Figure Lengend Snippet: GDF11 impairs adipogenic differentiation of human MSCs. A, MTS assay of hMSCs treated with different concentrations of rGDF11. B, Oil Red O staining 21 d after adipogenic differentiation. Scale bar, 25 μm. C, Quantification of lipid accumulation in hMSCs supplemented with rGDF11. Triglyceride content was measured at 500 nm after extracting Oil Red O. D, qRT‐PCR analysis revealed reduced mRNA expressions of adipocyte‐specific molecular markers PPARG , CEBPA , LPL , PLIN1 , CD36 and ADIPOQ 7 d after differentiation in high concentrations (50 and 100 ng mL −1 ) of rGDF11‐treated hMSCs. E, qRT‐PCR analysis revealed reduced mRNA expressions of adipocyte‐specific molecular markers PPARG , CEBPA , LPL , PLIN1 , CD36 and ADIPOQ 14 d after differentiation in high concentrations (50 and 100 ng mL −1 ) of rGDF11‐treated hMSCs. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. Results were shown as mean ± SEM, ANOVA

Article Snippet: To induce adipogenic differentiation, hMSCs were seeded in tissue culture plates, and confluent cells were induced by the aforementioned culture medium in the presence of MDI (0.5 mmol L −1 IBMX, 1 μmol L −1 dexamethasone and 10 μg mL −1 insulin, all from Sigma) supplemented with or without recombinant GDF11 (rGDF11, PeproTech).

Techniques: MTS Assay, Staining, Quantitative RT-PCR

Journal: Cell Proliferation

Article Title: Growth differentiation factor 11 inhibits adipogenic differentiation by activating TGF‐beta/Smad signalling pathway

doi: 10.1111/cpr.12631

Figure Lengend Snippet: GDF11 inhibits adipogenic differentiation of 3T3‐L1 pre‐adipocytes. A, MTS assay of 3T3‐L1 pre‐adipocytes treated with different concentrations of rGDF11. B, Oil Red O staining of 3T3‐L1 pre‐adipocytes 7 d after adipogenic differentiation. Scale bar, 25 μm. C, Quantification of lipid accumulation in of 3T3‐L1 pre‐adipocytes supplemented with rGDF11. Triglyceride content, measured at 500 nm after extracting Oil Red O, was diminished in 50 and 100 ng mL −1 rGDF11‐treated groups. D, qRT‐PCR results. The relative mRNA expressions of adipocyte‐specific molecular markers Pparg, Cebpa , Lpl, Plin1 , Cd36 and Adipoq were analysed on Day 3 after differentiation. E, qRT‐PCR results. The relative mRNA expressions of adipocyte‐specific molecular markers Pparg, Cebpa , Lpl, Plin1 , Cd36 and Adipoq were analysed on Day 5 after the differentiation. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001. Results were shown as mean ± SEM, ANOVA

Article Snippet: To induce adipogenic differentiation, hMSCs were seeded in tissue culture plates, and confluent cells were induced by the aforementioned culture medium in the presence of MDI (0.5 mmol L −1 IBMX, 1 μmol L −1 dexamethasone and 10 μg mL −1 insulin, all from Sigma) supplemented with or without recombinant GDF11 (rGDF11, PeproTech).

Techniques: MTS Assay, Staining, Quantitative RT-PCR

Journal: Cell Proliferation

Article Title: Growth differentiation factor 11 inhibits adipogenic differentiation by activating TGF‐beta/Smad signalling pathway

doi: 10.1111/cpr.12631

Figure Lengend Snippet: GDF11 activates Smad2/3‐dependent TGF‐beta signalling pathway. A, Western blot analysis revealed that GDF11 stimulated the phosphorylation of Smad2 and Smad3. B, Immunofluorescence staining of 3T3‐L1 pre‐adipocytes indicated that GDF11 activated the phosphorylation of Smad2 in 30 min. Scale bar, 1 mm. C, Cell immunofluorescence analysis demonstrated that GDF11 increased pSmad3‐positive cells in 3T3‐L1 pre‐adipocytes cultured under adipogenic conditions. Scale bar, 1 mm. D, Quantification of the pSmad2‐positive cell in (B). E, Quantification of the pSmad3‐positive cell in (C). F, ChIP assay revealed that rGDF11 reduced the abundance of HDAC1 at the CEBP binding site of the PPARγ promoter. n = 3. * P < 0.05. *** P < 0.001. Results were shown as mean ± SEM, t test

Article Snippet: To induce adipogenic differentiation, hMSCs were seeded in tissue culture plates, and confluent cells were induced by the aforementioned culture medium in the presence of MDI (0.5 mmol L −1 IBMX, 1 μmol L −1 dexamethasone and 10 μg mL −1 insulin, all from Sigma) supplemented with or without recombinant GDF11 (rGDF11, PeproTech).

Techniques: Western Blot, Phospho-proteomics, Immunofluorescence, Staining, Cell Culture, Binding Assay

Journal: Cell Proliferation

Article Title: Growth differentiation factor 11 inhibits adipogenic differentiation by activating TGF‐beta/Smad signalling pathway

doi: 10.1111/cpr.12631

Figure Lengend Snippet: ALK4,5 inhibitors attenuate the effect of GDF11 on adipogenic differentiation. A, 3T3‐L1 cells were fixed and stained with Oil Red O 7 d after differentiation. The presence of SB431542 recovered the number of red‐stained cells which was inhibited by rGDF11. Scale bar, 25 μm. B, Triglyceride content was measured at 500 nm after extracting Oil Red O. Lipid accumulation diminished by rGDF11 was fully restored by SB431542. C, qRT‐PCR results. The relative mRNA expressions of adipocyte‐specific molecular markers Pparg, Cebpa , Lpl, Plin1 , Cd36 and Adipoq were analysed on 3 d after differentiation. n = 3. * P < 0.05 vs GDF11(−)/SB431542(−), ** P < 0.01 vs GDF11(−)/SB431542(−), *** P < 0.001 vs GDF11(−)/SB431542(−). # P < 0.05 vs GDF11(+)/SB431542(−), ## P < 0.01 vs GDF11(+)/SB431542(−), ### P < 0.001 vs GDF11(+)/SB431542(−). Results were shown as mean ± SEM, ANOVA

Article Snippet: To induce adipogenic differentiation, hMSCs were seeded in tissue culture plates, and confluent cells were induced by the aforementioned culture medium in the presence of MDI (0.5 mmol L −1 IBMX, 1 μmol L −1 dexamethasone and 10 μg mL −1 insulin, all from Sigma) supplemented with or without recombinant GDF11 (rGDF11, PeproTech).

Techniques: Staining, Quantitative RT-PCR

Journal: Cell Proliferation

Article Title: Growth differentiation factor 11 inhibits adipogenic differentiation by activating TGF‐beta/Smad signalling pathway

doi: 10.1111/cpr.12631

Figure Lengend Snippet: ALK4,5 inhibitors eliminate GDF11‐induced phosphorylation of Smad2/3. A, Western blot analysis indicated that the phosphorylation of Smad2/3 by rGDF11 was totally blocked by SB431542. B, Cell immunofluorescence analysis showed that rGDF11 failed to increase the number of pSmad2‐positive cells under the treatment of SB431542. Scale bar, 1 mm. C, Cell immunofluorescence analysis demonstrated that SB431542 diminished the rGDF11‐activated Smad3 phosphorylation. Scale bar, 1 mm. D, The positive cell rate in (B). E, The positive cell rate in (C). n = 3. *** P < 0.001 vs GDF11(−)/SB431542(−). ### P < 0.001 vs GDF11(+)/SB431542(−). Results were shown as mean ± SEM, ANOVA

Article Snippet: To induce adipogenic differentiation, hMSCs were seeded in tissue culture plates, and confluent cells were induced by the aforementioned culture medium in the presence of MDI (0.5 mmol L −1 IBMX, 1 μmol L −1 dexamethasone and 10 μg mL −1 insulin, all from Sigma) supplemented with or without recombinant GDF11 (rGDF11, PeproTech).

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence

Journal: Skeletal Muscle

Article Title: A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice

doi: 10.1186/s13395-019-0197-y

Figure Lengend Snippet: GDF11PRO-Fc associates with GDF11 and MSTN. Protein-protein interactions between GDF11PRO-Fc or MPRO-Fc and rGDF11, rMSTN, or rActivin A were determined by a pull-down assay. GDF11PRO-Fc or MPRO-Fc was incubated with rGDF11, rMSTN, or rActivin A for 1 h at 4 °C. Fc-fused protein complexes were separated on a protein A/G-coated agarose resin and eluates were run on a 12% SDS-PAGE gel under reducing conditions and probed by western blot. Input control was 5% of the input material. WB western blot

Article Snippet: To assess complex formation, rGDF11 (1958-GD-010; R&D Systems; Minneapolis, MN), recombinant MSTN (rMSTN; 788-G8–010; R&D Systems; Minneapolis, MN) or recombinant activin A (rActivinA; 338-AC-010; R&D Systems; Minneapolis, MN) were added to cell lysates to a final concentration of 100–500 ng/ml.

Techniques: Protein-Protein interactions, Pull Down Assay, Incubation, SDS Page, Western Blot, Control

Journal: Skeletal Muscle

Article Title: A GDF11/myostatin inhibitor, GDF11 propeptide-Fc, increases skeletal muscle mass and improves muscle strength in dystrophic mdx mice

doi: 10.1186/s13395-019-0197-y

Figure Lengend Snippet: GDF11PRO-Fc blocks GDF11/MSTN-induced myotube atrophy in C2C12 cells. a Schematic detailing experimental timeline in C2C12 myotubes. AAV6-EGFP or AAV6-GDF11PRO-Fc was added to C2C12 myotubes at a MOI of 10 on day 5 post-differentiation and 100 ng/ml rGDF11 or rMSTN was added on day 7. Myotubes were stained and analyzed on day 10. b EGFP expression was evident at 48–72 h in C2C12 myotubes treated with AAV6-EGFP (MOI 10 ). Scale bars represents 50 μm. c Vector genome copy number per diploid genome in C2C12 myotubes 72 h after addition of AAV6-EGFP or AAV6-GDF11PRO-Fc (MOI 10 ). d Representative immunofluorescence images of C2C12 myotubes. C2C12 myotube membranes were visualized by staining with an anti-dystrophin antibody (red). Nuclei were stained with DAPI (blue). Inset shows a zoomed-in region. Scale bars represent 50 μm (main panel) and 25 μm (panel inset). e The fraction of nuclei incorporated into myotubes (differentiation index) was calculated and presented as a percentage of control. f Average myotube diameter relative to control and ( g ) distribution of diameter measurements. For myotube diameter measurements, each myotube was measured at three points along the length of the myotube and averaged. h Number of nuclei incorporated per myotube. A minimum of 50 myotubes were analyzed per experimental condition. i pSMAD2/3 relative to tSMAD2/3 was assessed by western blot. Equal protein loading was verified by Ponceau S staining and GAPDH was used as a loading control. Data represents results from three separate experiments. All error bars represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant; compared to AAV6-EGFP-treated control. † p < 0.05; †† p < 0.01; ††† p < 0.001; compared to AAV6-EGFP + ligand-treated. pSMAD2/3: phosphorylated SMAD2/3; tSMAD2/3: total SMAD2/3

Article Snippet: To assess complex formation, rGDF11 (1958-GD-010; R&D Systems; Minneapolis, MN), recombinant MSTN (rMSTN; 788-G8–010; R&D Systems; Minneapolis, MN) or recombinant activin A (rActivinA; 338-AC-010; R&D Systems; Minneapolis, MN) were added to cell lysates to a final concentration of 100–500 ng/ml.

Techniques: Staining, Expressing, Plasmid Preparation, Immunofluorescence, Control, Western Blot

Journal: Stem Cells Translational Medicine

Article Title: GDF11 enhances therapeutic efficacy of mesenchymal stem cells for myocardial infarction via YME1L ‐mediated OPA1 processing

doi: 10.1002/sctm.20-0005

Figure Lengend Snippet: GDF11 protected MSCs against apoptosis under hypoxia condition in vitro. A, GDF11 expression in MSCs were detected by Western blot under normoxia and hypoxia condition at specified times, and β‐actin served as a loading control. B, Quantification of relative GDF11 protein level (n = 3). C, MSCs were pretreated with rGDF11 or overexpressed GDF11 by viral transduction (LV‐GDF11). Representative TUNEL staining images of Control (Ctrl), rGDF11, LV (vector control) and LV‐GDF11 were captured. Scale bar = 50 μm. Quantification of apoptotic cells was presented as ratio of TUNEL‐positive nuclei over the total nuclei from 8 to 10 randomly selected fields in each sample. D, Cleaved caspase 3 (Cl‐cas3) and cleaved caspase 9 (Cl‐cas9) proteins in MSCs pretreated with rGDF11 were assessed by Western blot and quantified by densitometry (n = 3). E, Representative TUNEL staining images of MSCs transfected with siRNA GDF11 (si‐GDF11) or siRNA control (si‐NC). Scale bar = 50 μm. Quantification of apoptotic cells was presented as ratio of TUNEL‐positive nuclei over the total nuclei from 8 to 10 randomly selected fields in each sample. F, Cleaved caspase 3 and 9 proteins of MSCs after transfected with si‐NC and si‐GDF11 were assessed by Western blot; and protein expression levels were quantified by densitometry (n = 4). Data are shown as mean ± SD. * P < .05 vs Ctrl/si‐NC

Article Snippet: MSCs were also cultured with recombinant GDF11 (rGDF11, 50 ng/mL) (Peprotech, Rocky Hill, Connecticut) for 24 hours (MSC rGDF11 ) for the specified analysis.

Techniques: In Vitro, Expressing, Western Blot, Control, Transduction, TUNEL Assay, Staining, Plasmid Preparation, Transfection

Journal: Stem Cells Translational Medicine

Article Title: GDF11 enhances therapeutic efficacy of mesenchymal stem cells for myocardial infarction via YME1L ‐mediated OPA1 processing

doi: 10.1002/sctm.20-0005

Figure Lengend Snippet: GDF11 enhanced paracrine effects of MSCs in vitro. A, Schematic working showed the experiments for evaluating the paracrine effect of MSCs with different treatment. B‐D, The VEGFA levels in conditioned medium from MSCs (Ctrl, rGDF11) (B) or (LV, LV‐GDF11) (C) and (si‐NC, si‐GDF11) (D) were determined by ELISA assay. E, Representative images of tube formation assay of HUVECs/GFP under fluorescent microscopy. HUVECs were cultured with DMEM or conditioned medium of MSCs that had been cultured in the absence (Control) or presence of rGDF11. Scale bar = 100 μm. Quantification of tube formation was shown in bar graphs (n = 8). F, Representative images of tube formation assay of HUVECs/GFP that were cultured with DMEM or conditioned medium of MSCs that had been transfected with siRNAs (si‐GDF11, and NC as negative control). Quantification of tube formation was shown in bar graphs (n = 13). Scale bar = 100 μm. G, TUNEL assay of apoptosis of H9C2 cells that were cultured with DMEM medium or conditioned medium of MSCs that had been cultured in the absence (Control) or presence of rGDF11 (n = 8). Scale bar =50 μm. H, Western blot analysis of apoptosis‐related proteins in H9C2 cells cultured as in G. β‐actin was used as internal control. The quantification was shown in bar graphs (n = 3). The conditioned medium had been normalized by an equivalent number of MSCs (1 × 10 6 cells). Data are shown as mean ± SD. * P < .05 vs DMEM/Ctrl/si‐NC, # P < .05 vs Ctrl

Article Snippet: MSCs were also cultured with recombinant GDF11 (rGDF11, 50 ng/mL) (Peprotech, Rocky Hill, Connecticut) for 24 hours (MSC rGDF11 ) for the specified analysis.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Tube Formation Assay, Microscopy, Cell Culture, Control, Transfection, Negative Control, TUNEL Assay, Western Blot

Journal: Stem Cells Translational Medicine

Article Title: GDF11 enhances therapeutic efficacy of mesenchymal stem cells for myocardial infarction via YME1L ‐mediated OPA1 processing

doi: 10.1002/sctm.20-0005

Figure Lengend Snippet: GDF11 protected mitochondrial morphology and function under hypoxic condition. A, Representative images of MSCs under hypoxia condition were taken by electron microscopy (×10 000). Scale bar =1 μm. B, Mitochondrial morphology was analyzed by quantification of area and longitudinal length of mitochondria in MSCs Ctrl and MSCs rGDF11 (n = 77 for MSCs Ctrl , and n = 95 for MSCs rGDF11 ). C, D, Mitochondrial respiration reflected by OCR was detected in MSCs alone, MSCs treated with rGDF11 under normoxic (C) or hypoxic (D) conditions. OCRs were assayed under both basal and maximal conditions (n = 3). E. Cellular ATP levels of MSCs Ctrl and MSCs rGDF11 under normoxic or hypoxic conditions were measured through luciferin/luciferase‐based assay, and the data were calibrated with protein content. F, Images of MSCs stained with TMRM under fluorescence microscope were used to measure mitochondrial membrane potential. MSCs were treated with rGDF11 (50 ng/mL) for 24 hours then exposed to hypoxia conditions for 48 hours. MSCs treated with either FCCP (50 μmol/L) or oligomycin (10 μmol/L) were served as negative and positive controls, respectively. Scale bar = 100 μm. Bar graph shows relative mean of fluorescence intensity (MFI) of Control or rGDF11 divided by the difference of MFI between Oligomycin and FCCP (n = 8). Data were shown as mean ± SD. * P < .05 vs Ctrl, # P < .05 vs Hypoxia Ctrl

Article Snippet: MSCs were also cultured with recombinant GDF11 (rGDF11, 50 ng/mL) (Peprotech, Rocky Hill, Connecticut) for 24 hours (MSC rGDF11 ) for the specified analysis.

Techniques: Electron Microscopy, Luciferase, Staining, Fluorescence, Microscopy, Membrane, Control

Journal: Stem Cells Translational Medicine

Article Title: GDF11 enhances therapeutic efficacy of mesenchymal stem cells for myocardial infarction via YME1L ‐mediated OPA1 processing

doi: 10.1002/sctm.20-0005

Figure Lengend Snippet: OPA1 was indispensable for the protective effects of GDF11 in response to hypoxic condition. A, Quantitative analysis of mRNAs of genes involved in mitochondrial homeostasis for MSCs Ctrl and MSCs rGDF11 under normoxic or hypoxic condition. Relative mRNA levels were all compared with 18 seconds RNA. B, Western blot analysis of mitochondrial proteins: PGC‐1α, OPA1, Mfn1/2 and DRP1. Monoclonal antibody for OPA1 was used. Quantification of the proteins relative to control β‐actin was shown in right (n = 3). C, Western blot analysis of OPA1, YME1L and OMA1 in MSCs Ctrl and MSCs rGDF11 under hypoxic condition. OPA1 detected by a polyclonal antibody. Quantification of the proteins relative to control β‐actin was shown in the bar graph (n = 3). D, Western blot analysis of OPA1 and YME1L in MSCs si‐NC and MSCs si‐GDF11 in the same way as in C (n = 4). E, Western blot analysis of Cyto‐C in cytoplasm and mitochondrial. The values of Cyto‐C in mitochondrial were normalized with TOM20. And the values of Cyto‐C in cytoplasm were normalized with tubulin. Quantification was shown in the bar graph (n = 4). F, TUNEL staining of MSCs that were transfected with siRNAs (si‐NC and si‐OPA1), followed by treatment with rGDF11 for 24 hours and then exposed to hypoxia condition for 48 hours. Scale bar = 50 μm. Apoptotic cells were quantified by counting TUNEL‐positive nuclei out of total cells (n = 9). G, Western blot analysis of OPA1, cleaved caspase 3 and 9. Quantification of the proteins relative to control β‐actin was shown in the bar graph (n = 3). Data were shown as mean ± SD. * P < .05 vs si‐NC/Ctrl

Article Snippet: MSCs were also cultured with recombinant GDF11 (rGDF11, 50 ng/mL) (Peprotech, Rocky Hill, Connecticut) for 24 hours (MSC rGDF11 ) for the specified analysis.

Techniques: Western Blot, Control, TUNEL Assay, Staining, Transfection

Journal: Stem Cells Translational Medicine

Article Title: GDF11 enhances therapeutic efficacy of mesenchymal stem cells for myocardial infarction via YME1L ‐mediated OPA1 processing

doi: 10.1002/sctm.20-0005

Figure Lengend Snippet: GDF11‐mediated OPA1 processing depends on YME1L. A, Representative TEM images of MSCs transfected with siRNA‐OPA1 or siRNA‐YME1L for 48 hours and then incubated with rGDF11 (50 ng/mL) for 24 hours and then exposed to hypoxia conditions for 48 hours (×10 000). Scale bars: 1 μm. B, C, Quantification of mitochondrial area, longitudinal length and size distribution according to their length: long tubules (>0.65 μm), intermediate (≤0.65 μm, ≥0.32 μm), and fragmented (<0.32 μm) (n = 105 for MSC si‐NC , n = 64 for MSCs si‐NC + rGDF11 , n = 123 for MSC si‐OPA1 , n = 68 for MSC si‐OPA1 + rGDF11 , n = 93 for MSC si‐YME1L , and n = 96 for MSC si‐YME1L + rGDF11 ). D, Representative immunoblots and densitometric quantification for the expression of YME1L and OMA1 under normoxic and hypoxic conditions (n = 4). E, mRNA levels of YME1L and OMA1 in MSCs Ctrl and MSCs rGDF11 , and 18 seconds served as control. F, Western blot analysis of YME1L and OPA1. YME1L and L‐OPA1 were quantified and presented as relative level by comparing with β‐actin control (n = 3). G, Intracellular ATP levels in MSCs at specified conditions were determined. ATP levels was calibrated with protein content (n = 3). Data were shown as mean ± SD. * P < .05 vs si‐NC

Article Snippet: MSCs were also cultured with recombinant GDF11 (rGDF11, 50 ng/mL) (Peprotech, Rocky Hill, Connecticut) for 24 hours (MSC rGDF11 ) for the specified analysis.

Techniques: Transfection, Incubation, Western Blot, Expressing, Control

Journal: Stem Cells Translational Medicine

Article Title: GDF11 enhances therapeutic efficacy of mesenchymal stem cells for myocardial infarction via YME1L ‐mediated OPA1 processing

doi: 10.1002/sctm.20-0005

Figure Lengend Snippet: GDF11 protected MSCs from hypoxia‐induced apoptosis through ALK5‐Smad2/3 pathway. A, Localization of p‐Smad3 in MSCs. DAPI were stained with for nuclear (blue) and fluorescence‐labeled Ab against phosphorylated Smad3 (red). Scale bar = 50 μm. Fluorescence intensity was quantified. B, C, Western blot analysis of p‐Smad3, cleaved caspase 3 and 9 in MSCs which were treated with TGFβR1 inhibitor SB4431542 (B) or p‐Smad3 inhibitor SIS3 (C) for 30 minutes, and incubated with rGDF11 (50 ng/mL) for 24 hours and then exposed to hypoxia condition for 48 hours (n = 3 in B and C). D, Representative TEM images of MSCs treated as described in (B, C) (×10 000). Scale bar =1 μm. Mitochondria were visually scored for their area and longitudinal length. n = 149 for MSC Ctrl , n = 63 for MSCs rGDF11 , n = 153 for MSC Ctrl + SB , n = 160 for MSC rGDF11 + SB , n = 151 for MSC Ctrl + SIS and n = 139 for MSC rGDF11 + SIS . E, F, Immunoblot analysis of L‐OPA1 and YME1L in MSCs treated as specified. Relative proteins were presented by comparing each band density with that of β‐actin (n = 3 in E and F). G, H, The abilities of Smad2/3 binding to YME1L promoter sites at 473 to 485 and 2550 to 2562 were analyzed by ChIP assay. The purified DNA and input genomic DNA were analyzed by real‐time PCR (G). The PCR products were analyzed by gel electrophoresis (H). Each in vitro experiment was repeated three times. Data were shown as mean ± SD. * P < .05 vs IgG, # P < .05 vs Ctrl

Article Snippet: MSCs were also cultured with recombinant GDF11 (rGDF11, 50 ng/mL) (Peprotech, Rocky Hill, Connecticut) for 24 hours (MSC rGDF11 ) for the specified analysis.

Techniques: Staining, Fluorescence, Labeling, Western Blot, Incubation, Binding Assay, Purification, Real-time Polymerase Chain Reaction, Nucleic Acid Electrophoresis, In Vitro

Journal: Stem Cells Translational Medicine

Article Title: GDF11 enhances therapeutic efficacy of mesenchymal stem cells for myocardial infarction via YME1L ‐mediated OPA1 processing

doi: 10.1002/sctm.20-0005

Figure Lengend Snippet: Delivery of GDF11 overexpressing MSCs improved cardiac function after MI in vivo. A, Representative echocardiographic M‐mode images showed the changes of cardiac function in each group at 28 days after MI. B, Left ventricular ejection fraction and left ventricular fractional shortening were quantified at 3, 7, 14, and 28 days post MI (n = 5 for Sham group, n = 6 for DMEM group, n = 6 for MSCs LV group, and n = 6 for MSCs LV‐GDF11 group, respectively). C, MSCs retention at day 3 post‐MI (n = 6 for each group). Representative images of GFP staining were assayed by fluorescence microscopy, wherein GFP‐positive cells (in green) were specifically counted for MSCs retention, with nuclei stained with DAPI. Scale bar = 100 μm. D, Representative immunofluorescence image of CD31 staining for capillaries (red) and α‐SMA staining for arterioles (green) with nuclei stained with DAPI in the remote zone of ischemic heart from DMEM, MSCs LV , and MSCs LV‐GDF11 group mice at day 28 post‐MI. The capillary and arteriolar densities in the remote zones are quantified by 6‐8 HPFs per section in the bar graphs. Scale bar =100 μm. Data are shown as mean ± SD. * P < .05 vs DMEM/MSCs LV , # P < .05 vs MSCs LV

Article Snippet: MSCs were also cultured with recombinant GDF11 (rGDF11, 50 ng/mL) (Peprotech, Rocky Hill, Connecticut) for 24 hours (MSC rGDF11 ) for the specified analysis.

Techniques: In Vivo, Staining, Fluorescence, Microscopy, Immunofluorescence