recombinant gdf11 protein rgdf11  (PeproTech)

Journal: Frontiers in Physiology

Article Title: Loss of Growth Differentiation Factor 11 Shortens Telomere Length by Downregulating Telomerase Activity

doi: 10.3389/fphys.2021.726345

Figure Lengend Snippet: In vitro loss of growth differentiation factor 11 (GDF11) shortens telomere length in Neuro 2a cells. Representative images of telomere quantitative fluorescence in situ hybridization (Telo-FISH, A–D ) in single clone-derived GDF11 KO or wild type (WT) Neuro 2a cells; young (Y, 10 days in culture), old (O, 65 days in culture). Quantification of the average telomere length (E) (two-way ANOVA, interaction: F (1, 217) = 1.513, p = 0.2201; WT vs. GDF11 KO : F (1, 217) = 37.64, p < 0.0001; young vs. old: F (1, 217) = 1.183, p = 0.2779; Sidak’s test, young: WT vs. GDF11 KO , p < 0.0001; old: WT vs. GDF11 KO , p < 0.0018; Y-WT: n = 50, Y-GDF11 KO : n = 68, O-WT: n = 66, O-GDF11 KO : n = 48), and the average relative fluorescence of short telomeres (F) (two-way ANOVA, interaction: F (1, 37) = 6.364, p = 0.0161; WT vs. GDF11 KO : F (1, 37) = 4.643, p = 0.0378; young vs. old: F (1, 37) = 5.642, p = 0.0228; Y-WT: n = 3, Y-GDF11 KO : n = 18, O-WT: n = 8, O-GDF11 KO : n = 12). Data are represented as mean ± SEM, * p < 0.05, ** p < 0.01. Scale bars, 10 μm.

Article Snippet: Recombinant GDF11 protein (rGDF11, Peprotech, Cat#120-11) of 100 ng/ml was added into the DMEM medium (Gibco, Carlsbad, CA, United States) without fetal bovine serum of WT and GDF11 KO Neuro 2a cells at their appropriate cell confluence (>50%), and the treatment lasted for 24 h. Then, the cells were collected for detecting the transcription of TERT and TERC, and telomerase activity.

Techniques: In Vitro, Fluorescence, In Situ Hybridization, Derivative Assay

Journal: Frontiers in Physiology

Article Title: Loss of Growth Differentiation Factor 11 Shortens Telomere Length by Downregulating Telomerase Activity

doi: 10.3389/fphys.2021.726345

Figure Lengend Snippet: Loss of GDF11 regulates telomere maintenance-related genes. Heatmap of upregulated (3, red) and downregulated (4, blue) (A) and bar graphs of 4 downregulated (B) telomere maintenance-related genes caused by deletion of GDF11 in single clone derived Neuro2a cells that were cultured for 65 days (two-tailed unpaired t -test, Tert: t = 7.262, df = 4, p = 0.0019; Rpa1: t = 24.58, df = 4, p < 0.0001; RPA2: t = 15.17, df = 4, p = 0.0001; DCLRE1b: t = 10.63, df = 4, p = 0.0004, n = 3 per group). Data are represented as mean ± SEM. * p < 0.05, ** p < 0.01.

Article Snippet: Recombinant GDF11 protein (rGDF11, Peprotech, Cat#120-11) of 100 ng/ml was added into the DMEM medium (Gibco, Carlsbad, CA, United States) without fetal bovine serum of WT and GDF11 KO Neuro 2a cells at their appropriate cell confluence (>50%), and the treatment lasted for 24 h. Then, the cells were collected for detecting the transcription of TERT and TERC, and telomerase activity.

Techniques: Derivative Assay, Cell Culture, Two Tailed Test

Journal: Frontiers in Physiology

Article Title: Loss of Growth Differentiation Factor 11 Shortens Telomere Length by Downregulating Telomerase Activity

doi: 10.3389/fphys.2021.726345

Figure Lengend Snippet: In vitro loss of GDF11 downregulates TERT, TERC, and telomerase activity. Quantification of the mRNA level of telomerase reverse transcriptase (TERT, A ) (two-way ANOVA, interaction: F (4, 20) = 0.4232, p = 0.7901; WT vs. GDF11 KO : F (1, 20) = 153.1, p < 0.0001; time: F (4, 20) = 3.983, p = 0.0155; Sidak’s test, 1 day: WT vs. GDF11 KO , p = 0.0007; 10 days: WT vs. GDF11 KO , p = 0.0003; 20 days: WT vs. GDF11 KO , p < 0.0001; 40 days: WT vs. GDF11 KO , p < 0.0001; 65 days: WT vs. GDF11 KO , p < 0.0001; n = 3 per group) and telomerase RNA component (TERC, B ) (two-way ANOVA, interaction: F (4, 20) = 43.49, p < 0.0001; WT vs. GDF11 KO : F (1, 20) = 185.5, p < 0.0001; Time: F (4, 12) = 39.47, p < 0.0001; Sidak’s test, 40 days: WT vs. GDF11 KO , p < 0.0001; 65 days: WT vs. GDF11 KO , p < 0.0001; WT: 1 vs. 40 days, p < 0.0001; WT: 1 vs. 65 days, p < 0.0001; n = 3 per group) as well as the telomerase activity (C) (two-way ANOVA, interaction: F (2, 12) = 5.662, p = 0.0186; WT vs. GDF11 KO : F (1, 12) = 80.51, p < 0.0001; time: F (2, 12) = 9.197, p = 0.0038; Sidak’s test, 1 day: WT vs. GDF11 KO , p = 0.0017; 40 days: WT vs. GDF11 KO , p = 0.0265; 65 days: WT vs. GDF11 KO , p < 0.0001; WT: 1 vs. 65 days, p = 0.0067; GDF11 KO : 1 vs. 40 days, p = 0.0104; n = 3 per group) in single clone-derived GDF11 KO and WT Neuro 2a cells which were cultured for different periods of time. (D) Effect of rGDF11 on the transcription of TERT in WT or GDF11 KO Neuro 2a cells cultured for 65 days (Brown-Forsythe ANOVA test, Brown-Forsythe ANOVA test: F * (DFn, DFd) = 29.64 (2.000, 4.350), p = 0.0029; Welch’s ANOVA test: W (DFn, DFd) = 23.73 (2.000, 3.723), p = 0.0076; Dunnett’s T3 test, WT + BSA vs. GDF11 KO + BSA, p = 0.0109, GDF11 KO + BSA vs. GDF11 KO + rGDF11, p = 0.0159; n = 3 per group). (E) Effect of rGDF11 on the transcription of telomeric RNA component (TERC) in WT or GDF11 KO Neuro 2a cells cultured for 65 days (Brown-Forsythe ANOVA test, Brown-Forsythe ANOVA test: F * (DFn, DFd) = 29.57 (2.000, 3.365), p = 0.0073; Welch’s ANOVA test: W (DFn, DFd) = 18.08 (2.000, 2.697), p = 0.0274; Dunnett’s T3 test, WT + BSA vs. GDF11 KO + BSA, p = 0.0326; GDF11 KO + BSA vs. GDF11 KO + rGDF11, p = 0.7786; n = 3 per group). (F) Effect of rGDF11 on the telomerase activity of WT or GDF11 KO Neuro 2a cells cultured for 65 days (Brown-Forsythe ANOVA test, Brown-Forsythe ANOVA test: F * (DFn, DFd) = 55.10 (2.000, 3.288), p = 0.0030; Welch’s ANOVA test: W (DFn, DFd) = 34.01 (2.000, 3.400), p = 0.0056; Dunnett’s T3 test, WT + BSA vs. GDF11 KO + BSA, p = 0.0151; GDF11 KO + BSA vs. GDF11 KO + rGDF11, p = 0.1590; n = 3 per group). Effects of overexpression of GDF11 (G) on transcription of TERT (H) and TERC (I) in GDF11 KO Neuro 2a cells cultured for 65 days. For statistical analysis details, please see . Data are represented as mean ± SEM, * p < 0.05, ** p < 0.01.

Article Snippet: Recombinant GDF11 protein (rGDF11, Peprotech, Cat#120-11) of 100 ng/ml was added into the DMEM medium (Gibco, Carlsbad, CA, United States) without fetal bovine serum of WT and GDF11 KO Neuro 2a cells at their appropriate cell confluence (>50%), and the treatment lasted for 24 h. Then, the cells were collected for detecting the transcription of TERT and TERC, and telomerase activity.

Techniques: In Vitro, Activity Assay, Reverse Transcription, Derivative Assay, Cell Culture, Over Expression

Journal: Frontiers in Physiology

Article Title: Loss of Growth Differentiation Factor 11 Shortens Telomere Length by Downregulating Telomerase Activity

doi: 10.3389/fphys.2021.726345

Figure Lengend Snippet: Loss of GDF11 enhances SMAD2 binding to the promoters of TERT and TERC. CHIP-qPCR assessment of the enrichment of SMAD2 at the promoters of TERT (A) (two-way ANOVA, interaction: F (1, 12) = 3,595, p < 0.001; WT vs. GDF11 KO : F (1, 12) = 3,176, p < 0.001; IgG vs. SMAD2: F (1, 12) = 4,788, p < 0.001; Sidak’s test, SMAD2: WT vs. GDF11 KO , p < 0.001; four samples per group) and TERC (B) (two-way ANOVA, interaction: F (1, 12) = 101, p < 0.001; WT vs. GDF11 KO : F (1, 12) = 83.6, p < 0.001; IgG vs. SMAD2: F (1, 12) = 157, p < 0.001; Sidak’s test, SMAD2: WT vs. GDF11 KO , p < 0.001; four samples per group) in the single clone-derived GDF11 KO and WT Neuro 2a cells that were cultured for 40 days. Data are represented as mean ± SEM, ** p < 0.01.

Article Snippet: Recombinant GDF11 protein (rGDF11, Peprotech, Cat#120-11) of 100 ng/ml was added into the DMEM medium (Gibco, Carlsbad, CA, United States) without fetal bovine serum of WT and GDF11 KO Neuro 2a cells at their appropriate cell confluence (>50%), and the treatment lasted for 24 h. Then, the cells were collected for detecting the transcription of TERT and TERC, and telomerase activity.

Techniques: Binding Assay, ChIP-qPCR, Derivative Assay, Cell Culture

Journal: Scientific Reports

Article Title: Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus

doi: 10.1038/s41598-019-54766-y

Figure Lengend Snippet: Generation of Gdf11 -IRES-GFP knock-in reporter mice using CRISPR/Cas9. ( A) Schematic of Gdf11 -IRES-GFP targeting to the Gdf11 locus. Blue underlined text indicates the protospacer adjacent motif (PAM) for sgRNA3. Red text indicates the target sequence for sgRNA3. Black arrowhead indicates the predicted cut site for sgRNA3. Primers used for PCR-based screening are designated as A, B, C, D, and E above each allele, and predicted amplicon sizes are listed beneath each allele. The location of NcoI restriction sites and Southern blot probe sequences are indicated in red and blue text, respectively. HA-L: Left homology arm. HA-R: Right homology arm. ( B ) PCR screening of 5 founder mice from Round #1 of injections using primer pair A–C. Expected size: WT = 3.1 kb; KI = 4.3 kb. Gel image is uncropped with the entirety of the captured image shown. ( C) Chromatogram illustrating sequence of boundaries between top: left homology arm (HA-L) and IRES-GFP, and bottom: IRES-GFP and right homology arm (HA-R). ( D) PCR screening of 36 founder mice from Rounds #3 and #4 of using primer pair B-C. Expected size: WT = 0.8 kb; KI = 2.1 kb; *Non-specific band. Green boxes indicate founder animals harboring the Gdf11 -IRES-GFP knock-in allele. Red boxes indicate founder animals harboring large deletions in Gdf11 . Gel image is uncropped. Positive and negative control reactions for PCR amplification were run on a separate gel, which is presented in Supplementary Fig. .

Article Snippet: Increasing volumes of conditioned media (5 μL, 10 μL, or 20 μL) or 10 ng of recombinant GDF11 (rGDF11) protein (Peprotech) were processed under non-reducing or reducing conditions.

Techniques: Knock-In, CRISPR, Sequencing, Amplification, Southern Blot, Negative Control

Journal: Scientific Reports

Article Title: Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus

doi: 10.1038/s41598-019-54766-y

Figure Lengend Snippet: Validation of Gdf11 -IRES-GFP knock-in reporter mouse lines. ( A , B ) Southern blot analysis of ( A ), Gdf11- IRES-GFP targeted founder mice and ( B ), Gdf11- IRES-GFP F1 progeny. Nco1-digested genomic DNA was hybridized with the internal probe. Expected fragment size: WT = n/a; T (targeted) = 3.5 kb. AI: Additional integration. Blot images were cropped to focus on the target bands. Uncropped blots are presented in Supplementary Fig. . ( C ) TLA sequencing coverage and analysis plots from line 1B using outward facing primers residing in the GFP transgene. ( D ) Flow cytometry analysis of GFP expression in live (7AAD − ) peripheral blood cells in left : mice exhibiting correct targeting (lines 1B, 11 and 12) and right: mice exhibiting incorrect targeting (lines 1A and 13). ( E,F ) Real time PCR analysis of Gdf11 levels in FACS-purified GFP high and GFP low splenocytes from line 1B using ( E ), primers spanning exons 1-2 and ( F ), primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in GFP low splenocytes. N = 4 males (blue), 4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( G,H ) Real time PCR analysis of Gdf11 levels in whole spleen from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. Relative Gdf11 expression levels were assayed using ( G ), primers spanning exons 1–2 and ( H) , primers spanning exons 2–3. β - actin was used as a housekeeping gene. Transcript levels were normalized to levels in C57BL/6J mice. N = 3–4 males (blue), 3–4 females (red). Data are presented as individual data points overlaid with mean ± SD. ( I,J ), Quantification of ( I ), GDF11 protein levels, and ( J) , GDF8 protein levels, in serum from correctly targeted lines (1B, 11 and 12) and age- and sex-matched C57BL/6J mice. ( K,L ) Whole mount in situ hybridization for Gdf11 (top) and Gfp (bottom) in E10.5 Gdf11 +/+ and Gdf11 KI/+ embryos from line 1B. For each embryo, the right-most images show the dissected forelimb. mb: midbrain, fb: forebrain, psm: pre-somitic mesoderm, fl: forelimb, hl: hindlimb, s: somite. Scale bar: 0.5 mm.

Article Snippet: Increasing volumes of conditioned media (5 μL, 10 μL, or 20 μL) or 10 ng of recombinant GDF11 (rGDF11) protein (Peprotech) were processed under non-reducing or reducing conditions.

Techniques: Knock-In, Southern Blot, Sequencing, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Purification, In Situ Hybridization

Journal: Scientific Reports

Article Title: Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus

doi: 10.1038/s41598-019-54766-y

Figure Lengend Snippet: Gdf11 -IRES-GFP expression is primarily detected within T and B lymphocytes of the peripheral blood. ( A ) Representative flow cytometry analysis of GFP expression within CD3 + T cells, CD19 + B cells, CD11b + /Ly6G − monocytes and CD11b + /Ly6G + neutrophils from peripheral blood. ( B ) Quantification of GFP+ T cells, B cells, monocytes and neutrophils in 2 month old mice from lines 1B, 11 and 12 and WT controls. N = 3–8 males and 3–8 females per genotype. Circles: males. Triangles: Females. Individual data points overlaid with mean ± SD. ( C ) Real time PCR analysis of Gdf11 levels in CD19+ and CD19- splenic cells from young (2-month old) and aged (24-month old) mice. Hprt was used as a housekeeping gene. ( D,E ) Quantification of ( D ), GFP + peripheral blood T cells and ( E ), GFP + peripheral blood B cells within heterozygous mice from line 1B during aging. ( F,G ) Quantification of GFP mean fluorescence intensity within F , peripheral blood T cells and ( G ), peripheral blood B cells in heterozygous mice from line 1B during aging. Mean fluorescence intensity (MFI) values normalized to wild type mice for each timepoint. ( H ) Quantification of total T cell frequency (red) and B cell frequency (blue) out of live peripheral blood cells during aging. N = 25 males and 19 females. Data points represent mean with error bars denoting SEM.

Article Snippet: Increasing volumes of conditioned media (5 μL, 10 μL, or 20 μL) or 10 ng of recombinant GDF11 (rGDF11) protein (Peprotech) were processed under non-reducing or reducing conditions.

Techniques: Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Scientific Reports

Article Title: Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus

doi: 10.1038/s41598-019-54766-y

Figure Lengend Snippet: Novel Gdf11 deletion alleles recapitulate skeletal defects observed in Gdf11 knockout mice. ( A ) Schematic of Gdf11 deletion alleles recovered from Rounds #3 and #4 of CRISPR injections. Dotted lines denote deleted region of the DNA sequence. Size of deletion noted above dotted line. ( B ) Schematic of exogenous amino acids added (underlined) to the C-terminal domain of GDF11 in the four Gdf11 deletion alleles. Native amino acids are not underlined. *Stop codon. ( C ) Images of E17.5 or E18.5 embryos from lines 4A, 4B and 11 depicting loss of the tail in homozygous animals. ( D ) Skeletal preparation analysis of Gdf11 +/+ , Gdf11 4B/+ and Gdf11 4B/4B embryos stained with Alcian Blue (to mark cartilage) and Alizarin Red (to mark bone). Numbers indicate thoracic vertebrae. Black arrow denotes tail, which is visibly shortened in the homozygote. hl: hindlimb. ( E ) Top: Skeletal preparations of skulls from Gdf11 +/+ , Gdf11 4B/+ and Gdf11 4B/4B embryos stained with Alcian Blue and Alizarin Red. oc: otic capsule. White arrow denotes oc. Insets depict isolated oc. Bottom: Isolated oc from Gdf11 +/− and Gdf11 −/− and embryos. Scale bar: 1 mm. ( F,G ) Quantification of total number of vertebrae within embryos from F , each individual deletion allele, and ( G ), all deletion alleles pooled together. ( H ) Schematic of vertebrae depicting quantification of cervical (C), thoracic (T) and lumbar (L) vertebrae. ( I ) Quantification of the number of cervical, thoracic and lumbar vertebrae among the genotypes indicated.

Article Snippet: Increasing volumes of conditioned media (5 μL, 10 μL, or 20 μL) or 10 ng of recombinant GDF11 (rGDF11) protein (Peprotech) were processed under non-reducing or reducing conditions.

Techniques: Knock-Out, CRISPR, Sequencing, Staining, Isolation

Journal: Scientific Reports

Article Title: Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus

doi: 10.1038/s41598-019-54766-y

Figure Lengend Snippet: Mice homozygous for Gdf11 deletion allele 7 ( Gdf11 WE ) lack skeletal phenotypes seen in Gdf11 loss-of-function mutants and are viable with no substantial alterations in body parameters. ( A ) Images of E18.5 embryos from line 7. Gdf11 +/+ , Gdf11 WE/+ and Gdf11 WE/WE embryos all exhibit an external tail. ( B ) Skeletal analysis of Gdf11 WE / + and Gdf11 WE/WE embryos stained with Alcian Blue (to mark cartilage) and Alizarin Red (to mark bone). Numbers indicate thoracic vertebrae. hl: hindlimb. Scale bar: 1 mm. ( C ) Skeletal preparations of skulls from Gdf11 WE/+ and Gdf11 WE/WE embryos stained with Alcian Blue and Alizarin Red. oc: otic capsule. White arrow denotes oc. Inset depicts isolated oc. Scale bar: 1 mm. ( D ) Representative images of lumbar vertebrae within Gdf11 +/+ , Gdf11 WE/+ and Gdf11 WE/WE embryos. Arrows denote L5 and L6/S1 vertebrae. ( E ) Quantification of the number of cervical, thoracic and lumbar vertebrae among the genotypes indicated. ( F ) PCR analysis of Gdf11 locus in Gdf11 +/+ , Gdf11 WE/+ and Gdf11 WE/WE weanlings using primer pair B-C. NTC: no template control. Gel image was cropped to focus on the amplicons. Full length gel is presented in Supplementary Fig. . ( G – J ) Quantification of ( G ), Body weight, ( H ), Normalized heart weight (relative to tibia length), I , Raw spleen weight, and ( J ), Normalized muscle weight (relative to tibia length) in 5–8 month old mice. N = 5–8 males (blue) and N = 7–14 females (red) per genotype. ( K,L ) Quantification of ( K ), raw and ( L ), normalized grip strength measurements (relative to body weight) in 5–8 month old mice. N = 5–8 males (blue) and N = 7–14 females (red) per genotype. Each data point represents the average of two technical replicates. ( M,N ) Quantification of ( M ) GDF11 serum levels and N , GDF8 serum levels in 6–8-week old mice. N = 8–11 males (blue) and N = 8–14 females (red) per genotype. Individual data points overlaid with mean ± SD.

Article Snippet: Increasing volumes of conditioned media (5 μL, 10 μL, or 20 μL) or 10 ng of recombinant GDF11 (rGDF11) protein (Peprotech) were processed under non-reducing or reducing conditions.

Techniques: Staining, Isolation