protein ctrp3  (Monobind)

Journal: Frontiers in Cardiovascular Medicine

Article Title: CTRP3 attenuates myocardial lipotoxicity via suppression of lipid accumulation, inflammation, apoptosis, and mitochondrial oxidative stress

doi: 10.3389/fcvm.2025.1575929

Figure Lengend Snippet: (A,B) Compared to the high-fat diet (HFD) group, supplementation with CTRP3 significantly improves cardiac function in mice, as evidenced by enhanced left ventricular ejection fraction. (C,D) Histological examination of myocardial tissue reveals substantial lipid droplet accumulation in the HFD group. However, CTRP3 supplementation reduces lipid droplet accumulation, improves myocardial tissue structure, and alleviates the degree of fibrosis. (E–M) Immunofluorescence results indicate that CTRP3 supplementation mitigates myocardial inflammation, apoptosis, and oxidative stress in mice. (N–Q) Serum ELISA assays demonstrate that levels of triglycerides (TG), total cholesterol (TCHO), inflammatory markers, apoptotic factors, and oxidative stress indicators are significantly improved in the CTRP3 group compared to the HFD group ( n ≥ 3, p < 0.05).

Article Snippet: The recombinant CTRP3 protein (rCTRP3, Catalog Number: CSB-EP883621HU) was commercially procured from Wuhan Cusabio Biotech Co., Ltd. (Hubei, China).

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: CTRP3 attenuates myocardial lipotoxicity via suppression of lipid accumulation, inflammation, apoptosis, and mitochondrial oxidative stress

doi: 10.3389/fcvm.2025.1575929

Figure Lengend Snippet: (A–F) Lipid testing results demonstrate that palmitic acid (PA) induces lipid accumulation in myocardial cells, while CTRP3 supplementation significantly reduces lipid droplet accumulation. (G) CTRP3 intervention improves the expression of genes related to fatty acid uptake, fatty acid oxidation, and lipid efflux in myocardial cells ( n ≥ 3, p < 0.05). (H) Quantitative PCR (q-PCR) results indicate that CTRP3 mitigates inflammation, apoptosis, and oxidative stress in myocardial cells stimulated by PA ( n ≥ 3, p < 0.05).

Article Snippet: The recombinant CTRP3 protein (rCTRP3, Catalog Number: CSB-EP883621HU) was commercially procured from Wuhan Cusabio Biotech Co., Ltd. (Hubei, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cardiovascular Medicine

Article Title: CTRP3 attenuates myocardial lipotoxicity via suppression of lipid accumulation, inflammation, apoptosis, and mitochondrial oxidative stress

doi: 10.3389/fcvm.2025.1575929

Figure Lengend Snippet: (A–E) RNA sequencing analysis of myocardial tissue from the control group, HFD group, and CTRP3 group indicates that CTRP3 likely exerts its effects within myocardial mitochondria, playing a role in the metabolic regulation of myocardial lipotoxicity.

Article Snippet: The recombinant CTRP3 protein (rCTRP3, Catalog Number: CSB-EP883621HU) was commercially procured from Wuhan Cusabio Biotech Co., Ltd. (Hubei, China).

Techniques: RNA Sequencing, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: CTRP3 attenuates myocardial lipotoxicity via suppression of lipid accumulation, inflammation, apoptosis, and mitochondrial oxidative stress

doi: 10.3389/fcvm.2025.1575929

Figure Lengend Snippet: (A,B) JC-1 staining results demonstrate a decrease in mitochondrial membrane potential in myocardial cells of the PA group. (C,D) Mito-Tracker staining results show a reduction in mitochondrial number in the PA group. (E) PA intervention in myocardial cells leads to decreased ATP production, while CTRP3 intervention improves ATP generation. (F) Transmission electron microscopy reveals increased mitochondrial damage and autophagy in myocardial cells following PA intervention. CTRP3 can mitigate mitochondrial damage and autophagy in myocardial cells.

Article Snippet: The recombinant CTRP3 protein (rCTRP3, Catalog Number: CSB-EP883621HU) was commercially procured from Wuhan Cusabio Biotech Co., Ltd. (Hubei, China).

Techniques: Staining, Membrane, Transmission Assay, Electron Microscopy

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: SMN-depleted muscle cells secrete reduced levels of CTRP3 protein. Quantitative secretome analysis of metabolically labelled SMN-depleted muscle cells. a H-2K b -tsA58 cells were differentiated to multinucleated myotubes within 3 to 5 days (10x, 20x). b Representative Western blots and quantification showing knockdown efficiency. Negative control siRNA (Control) or siRNA against Smn (SmnKD) was transfected, and cells were differentiated for 72 h. GAPDH was used as a loading control ( n = 4). c Schematic drawing of quantitative secretome analysis. Simultaneous siRNA transfection and combined pulsed labelling of differentiating H-2K b -tsA58 muscle cells with azidohomoalanine (AHA) and stable isotope-labelled amino acids. Newly synthesized proteins were isolated by click chemistry and analyzed by mass spectrometry. d Volcano plot of secretome analysis; statistical significance (−log10, p -value) against fold change (FC, log2, Control/SmnKD). Normalized SILAC ratios of 4 replicates were used to calculate the log2 ratio between SmnKD (heavy) and control (intermediate) conditions, and p -values were determined using an unpaired two-sided t-test. Proteins with a p -value < 0.05 are highlighted in green/red and proteins with a FC > 50% are additionally labelled with name. e Representative Western blots and quantification of C2C12 cells transfected with negative control siRNA (Control) or siRNA against Smn (SmnKD), and differentiated for 5 days. ACTB was used as a loding control. f Representative Western blots and quantification of serum-free conditioned media of control and Smn KD C2C12 cells, differentiated for 5 days. CTRP3 levels were normalized to the total protein amount (stainfree gel). Dot graphs represent data from independent experiments and the bar graphs depict the mean ± s.d. Two-tailed unpaired student’s t-test was used to determine statistical signaficance. *** p < 0.001; ** p < 0.01

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Metabolic Labelling, Western Blot, Knockdown, Negative Control, Control, Transfection, Synthesized, Isolation, Mass Spectrometry, Multiplex sample analysis, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: CTRP3 treatment stimulates protein synthesis in motor neuron-like NSC-34 cells. Downstream pathway analysis of CTRP3 in motor neuron-like NSC-34 cells. a Schematic drawing showing whole proteome analysis of differentiated NSC-34 cells. b Volcano plot of whole proteome analysis; plotted statistical significance (−log10, p-value) against fold change (log2, CTRP3 treated/untreated). Cells were treated with 5 μg/ml recombinant mouse CTRP3 (mCTRP3). Three independent samples were used for analysis. P -values were determined using an unpaired two-sided t-test ( n = 3). Proteins with p < 0.05 are highlighted in green (upregulated)/ red (downregulated) and proteins with p < 0.05 and FC > 50% are additionally labelled with name. c Representative pathways changed by CTRP3 treatment. (proteins with p < 0.1 and pathways with p < 0.05, with g:Profiler). d Representative Western blots for PTEN and e quantification of PTEN levels in CTRP3 treated differentiated NSC-34 cells, Two-tailed unpaired student’s t-test was used to determine statistical signaficance, f Scheme of SUnSET assay. Differentiated NSC-34 cells were treated with mCTRP3 for 2 or 6 h. Cells were incubated with 1 μM puromycin for 1 h before analysis. g SUnSET assay shows that CTRP3 increases protein synthesis. h Dot plot bar graph summarizes SUnSET assay. SUnSET data were normalized to the total protein amount (ponceau). Each dot represents an independent experiment and the bar graph represents mean ± s.d. ( n = 16/10/10). One way ANOVA with Fisher’s LSD test was used to determine statistical significance; * p < 0.05, ** p < 0.01

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Recombinant, Western Blot, Two Tailed Test, Incubation

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: CTRP3 increases SMN and VEGF protein levels in primary WT and SMA motor neurons. Representative Western blots and quantification of SMN and VEGF protein levels in CTRP3 treated motor neurons isolated from spinal cord of E13 mice. Embryonic motor neurons from WT ( a - c ) and SMA ( d - f ) mice were cultured for 5 days in vitro (5DIV) and treated with 5 μg/ml mCTRP3 protein for 6 or 24 h. b , c , e and f Dot plot bar graphs represent quantification of VEGF and SMN levels. ACTB was used as a loading control. Each dot represents independent experiment and the bar graph represents mean ± s.d. (WT-SMN: n = 11, N = 5; WT-VEGF: n = 7, N = 3; SMA-SMN: n = 8; N = 3; SMA-VEGF: n = 11, N = 5, n: number of independent experiments, N: number of independent neuron cultures) One way ANOVA with Fisher’s LSD test was used to determine statistical significance; * p < 0.05, ** p < 0.01

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Western Blot, Isolation, Cell Culture, In Vitro, Control

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: CTRP3 activates the PI3K/mTOR and MAPK/ERK pathway in motor neurons. a - e Representative Western blots and quantification of AKT and ERK activity in NSC-34 cells treated with 5 μg/ml mCTRP3 for 5, 15, 30 or 60 min. Activity of PI3K and MAPK/ERK pathways were measured using anti-p-ERK (Thr202/Tyr204) and anti-p-AKT (S473) antibodies. ( n = 3) f - j WT and k - o SMA motor neurons treated with 5 μg/ml mCTRP3 for 5, 15, 30 or 60 min. Activity of PI3K/mTOR and MAPK/ERK pathways were measured using anti-p-S6K (Thr389) and anti-p-ERK (Thr202/Tyr204) antibodies. ACTB was used as a loading control. g - j , l - o Dot plot bar graphs summarize the repeated experiments. Each dot represents an independent experiment and bar graphs depict the mean ± s.d. (WT: n = 11 for p-ERK and n = 9 for p-S6K, SMA: n = 6 for p-ERK n = 8 for p-S6K) Kruskal Wallis and Uncorrected Dunn’s test was used to determine statistical significance; ** p < 0.01 and * p < 0.05. p Differentiated NSC-34 cells were treated with 100 nM of water-soluble mTOR inhibitor WYE-687 dihydrochloride and/or 5 μg/ml human CTRP3 (hCTRP3) for 2 h. q Representative Western blots of NSC-34 cells with anti-p-S6 (Ser235/236), anti-S6 and anti-SMN antibodies show that elevated SMN levels by CTRP3 treatment were mTOR dependent. ACTB was used as a loading control. r Dot plot bar graphs summarize the repeated experiments. Each dot represents an independent experiment and bar graphs depict the mean ± s.d. Kruskal Wallis and Uncorrected Dunn’s test was used to determine statistical significance; ** p < 0.01 and * p < 0.05

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Western Blot, Activity Assay, Control

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: CTRP3 stimulates axonal growth and protein synthesis of motor neurons. a Axon outgrowth assay with WT and SMA motor neurons treated with 5 μg/ml hCTRP3 for 24 h (2DIV-3DIV). Representative images of motor neurons stained with anti-Tau (green), anti-ChAT (red) and DAPI (blue). Scale bars: 20 μm. b Dot plot bar graph summarizes axon outgrowth analysis. Each dot represents the average axon length of 20–25 neurons in a coverslip. Bar graphs depict the mean ± s.d. (WT: n = 9, N = 3; SMA: n = 17, N = 4; n = number of coverslips, N = number of independent cultures). Two way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance; ** p < 0.01, **** p < 0.0001. c Scheme of SUnSET assay to investigate the effect of CTRP3 on axonal protein synthesis. Motor neurons (3DIV) were treated with 5 μg/ml hCTRP3 for 2 h and newly synthesized proteins were labelled with 1 μM puromycin for 30 min before analysis. Data are compared with non-CTRP3 treated cells. As a negative control, protein synthesis was blocked with 50 μM anisomycin for 1 h before analysis. d Representative images of WT and SMA motor neurons immunologically stained with anti-puromycin antibody. Rainbow scale heatmap indicating the intensity of SUnSET signals. e Dot plot bar graph represents the mean intensity of the SUnSET signal in soma of WT motor neurons. Each dot represents the average value of 24–28 neurons from an independent culture and bar graphs depict the mean ± s.d. (Untreated n = 8/ N = 8, CTRP3 treated n = 8/ N = 8, anisomycin treated n = 5/ N = 5, n = number of coverslips, N = number of independent cultures, 25 neurons per coverslip). Ordinary one-way ANOVA and Uncorrected Fisher’s LSD were used to determine statistical significance. **** p < 0.0001, f - g Quantification of the mean intensity of the SUnSET signal in axons (per 20 μm). f SUnSET signal in axon (WT N = 8, SMA N = 8, N = number of independent cultures). Each dot represents average data from one independent neuron culture. Therefore, data were only compared in the same culture with and without CTRP3 treatment. Two-tailed paired student’s t-test was used to determine statistical significance, ** p < 0.01. To compare WT and SMA, unpaired t-test was used to determine statistical significance, and it was not significant. g Each dot represents a value from a neuron, WT untreated n = 204, WT CTRP3 treated n = 293, SMA untreated n = 200, SMA CTRP3 treated n = 200. Two way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance; * p < 0.05, ** p < 0.01, h - i Quantification of the ratio of the SUnSET signal in axons (per 20 μm) compared to average signal in soma. h Ratio between soma and axons were increased by CTRP3 treatment, WT N = 8, SMA N = 8. Each dot represents average data from one independent neuron culture. Therefore, data were only compared in the same culture with and without CTRP3 treatment. Two-tailed paired student’s t-test was used to determine statistical significance. *** p < 0.001 and ** p < 0.01. To compare WT and SMA, unpaired t-test was used to determine statistical significance. * p < 0.05. i Ratio between soma and axons in individual neurons, each dot represents a value from a neuron, WT untreated n = 204, WT CTRP3 treated n = 293, SMA untreated n = 200, SMA CTRP3 treated n = 200. Two way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Staining, Comparison, Synthesized, Negative Control, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: SMN-depleted muscle cells secrete reduced levels of CTRP3 protein. Quantitative secretome analysis of metabolically labelled SMN-depleted muscle cells. a H-2K b -tsA58 cells were differentiated to multinucleated myotubes within 3 to 5 days (10x, 20x). b Representative Western blots and quantification showing knockdown efficiency. Negative control siRNA (Control) or siRNA against Smn (SmnKD) was transfected, and cells were differentiated for 72 h. GAPDH was used as a loading control ( n = 4). c Schematic drawing of quantitative secretome analysis. Simultaneous siRNA transfection and combined pulsed labelling of differentiating H-2K b -tsA58 muscle cells with azidohomoalanine (AHA) and stable isotope-labelled amino acids. Newly synthesized proteins were isolated by click chemistry and analyzed by mass spectrometry. d Volcano plot of secretome analysis; statistical significance (−log10, p -value) against fold change (FC, log2, Control/SmnKD). Normalized SILAC ratios of 4 replicates were used to calculate the log2 ratio between SmnKD (heavy) and control (intermediate) conditions, and p -values were determined using an unpaired two-sided t-test. Proteins with a p -value < 0.05 are highlighted in green/red and proteins with a FC > 50% are additionally labelled with name. e Representative Western blots and quantification of C2C12 cells transfected with negative control siRNA (Control) or siRNA against Smn (SmnKD), and differentiated for 5 days. ACTB was used as a loding control. f Representative Western blots and quantification of serum-free conditioned media of control and Smn KD C2C12 cells, differentiated for 5 days. CTRP3 levels were normalized to the total protein amount (stainfree gel). Dot graphs represent data from independent experiments and the bar graphs depict the mean ± s.d. Two-tailed unpaired student’s t-test was used to determine statistical signaficance. *** p < 0.001; ** p < 0.01

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Metabolic Labelling, Western Blot, Knockdown, Negative Control, Control, Transfection, Synthesized, Isolation, Mass Spectrometry, Multiplex sample analysis, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: CTRP3 treatment stimulates protein synthesis in motor neuron-like NSC-34 cells. Downstream pathway analysis of CTRP3 in motor neuron-like NSC-34 cells. a Schematic drawing showing whole proteome analysis of differentiated NSC-34 cells. b Volcano plot of whole proteome analysis; plotted statistical significance (−log10, p-value) against fold change (log2, CTRP3 treated/untreated). Cells were treated with 5 μg/ml recombinant mouse CTRP3 (mCTRP3). Three independent samples were used for analysis. P -values were determined using an unpaired two-sided t-test ( n = 3). Proteins with p < 0.05 are highlighted in green (upregulated)/ red (downregulated) and proteins with p < 0.05 and FC > 50% are additionally labelled with name. c Representative pathways changed by CTRP3 treatment. (proteins with p < 0.1 and pathways with p < 0.05, with g:Profiler). d Representative Western blots for PTEN and e quantification of PTEN levels in CTRP3 treated differentiated NSC-34 cells, Two-tailed unpaired student’s t-test was used to determine statistical signaficance, f Scheme of SUnSET assay. Differentiated NSC-34 cells were treated with mCTRP3 for 2 or 6 h. Cells were incubated with 1 μM puromycin for 1 h before analysis. g SUnSET assay shows that CTRP3 increases protein synthesis. h Dot plot bar graph summarizes SUnSET assay. SUnSET data were normalized to the total protein amount (ponceau). Each dot represents an independent experiment and the bar graph represents mean ± s.d. ( n = 16/10/10). One way ANOVA with Fisher’s LSD test was used to determine statistical significance; * p < 0.05, ** p < 0.01

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Recombinant, Western Blot, Two Tailed Test, Incubation

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: CTRP3 increases SMN and VEGF protein levels in primary WT and SMA motor neurons. Representative Western blots and quantification of SMN and VEGF protein levels in CTRP3 treated motor neurons isolated from spinal cord of E13 mice. Embryonic motor neurons from WT ( a - c ) and SMA ( d - f ) mice were cultured for 5 days in vitro (5DIV) and treated with 5 μg/ml mCTRP3 protein for 6 or 24 h. b , c , e and f Dot plot bar graphs represent quantification of VEGF and SMN levels. ACTB was used as a loading control. Each dot represents independent experiment and the bar graph represents mean ± s.d. (WT-SMN: n = 11, N = 5; WT-VEGF: n = 7, N = 3; SMA-SMN: n = 8; N = 3; SMA-VEGF: n = 11, N = 5, n: number of independent experiments, N: number of independent neuron cultures) One way ANOVA with Fisher’s LSD test was used to determine statistical significance; * p < 0.05, ** p < 0.01

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Western Blot, Isolation, Cell Culture, In Vitro, Control

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: CTRP3 activates the PI3K/mTOR and MAPK/ERK pathway in motor neurons. a - e Representative Western blots and quantification of AKT and ERK activity in NSC-34 cells treated with 5 μg/ml mCTRP3 for 5, 15, 30 or 60 min. Activity of PI3K and MAPK/ERK pathways were measured using anti-p-ERK (Thr202/Tyr204) and anti-p-AKT (S473) antibodies. ( n = 3) f - j WT and k - o SMA motor neurons treated with 5 μg/ml mCTRP3 for 5, 15, 30 or 60 min. Activity of PI3K/mTOR and MAPK/ERK pathways were measured using anti-p-S6K (Thr389) and anti-p-ERK (Thr202/Tyr204) antibodies. ACTB was used as a loading control. g - j , l - o Dot plot bar graphs summarize the repeated experiments. Each dot represents an independent experiment and bar graphs depict the mean ± s.d. (WT: n = 11 for p-ERK and n = 9 for p-S6K, SMA: n = 6 for p-ERK n = 8 for p-S6K) Kruskal Wallis and Uncorrected Dunn’s test was used to determine statistical significance; ** p < 0.01 and * p < 0.05. p Differentiated NSC-34 cells were treated with 100 nM of water-soluble mTOR inhibitor WYE-687 dihydrochloride and/or 5 μg/ml human CTRP3 (hCTRP3) for 2 h. q Representative Western blots of NSC-34 cells with anti-p-S6 (Ser235/236), anti-S6 and anti-SMN antibodies show that elevated SMN levels by CTRP3 treatment were mTOR dependent. ACTB was used as a loading control. r Dot plot bar graphs summarize the repeated experiments. Each dot represents an independent experiment and bar graphs depict the mean ± s.d. Kruskal Wallis and Uncorrected Dunn’s test was used to determine statistical significance; ** p < 0.01 and * p < 0.05

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Western Blot, Activity Assay, Control

Journal: Acta Neuropathologica Communications

Article Title: Muscle regulates mTOR dependent axonal local translation in motor neurons via CTRP3 secretion: implications for a neuromuscular disorder, spinal muscular atrophy

doi: 10.1186/s40478-019-0806-3

Figure Lengend Snippet: CTRP3 stimulates axonal growth and protein synthesis of motor neurons. a Axon outgrowth assay with WT and SMA motor neurons treated with 5 μg/ml hCTRP3 for 24 h (2DIV-3DIV). Representative images of motor neurons stained with anti-Tau (green), anti-ChAT (red) and DAPI (blue). Scale bars: 20 μm. b Dot plot bar graph summarizes axon outgrowth analysis. Each dot represents the average axon length of 20–25 neurons in a coverslip. Bar graphs depict the mean ± s.d. (WT: n = 9, N = 3; SMA: n = 17, N = 4; n = number of coverslips, N = number of independent cultures). Two way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance; ** p < 0.01, **** p < 0.0001. c Scheme of SUnSET assay to investigate the effect of CTRP3 on axonal protein synthesis. Motor neurons (3DIV) were treated with 5 μg/ml hCTRP3 for 2 h and newly synthesized proteins were labelled with 1 μM puromycin for 30 min before analysis. Data are compared with non-CTRP3 treated cells. As a negative control, protein synthesis was blocked with 50 μM anisomycin for 1 h before analysis. d Representative images of WT and SMA motor neurons immunologically stained with anti-puromycin antibody. Rainbow scale heatmap indicating the intensity of SUnSET signals. e Dot plot bar graph represents the mean intensity of the SUnSET signal in soma of WT motor neurons. Each dot represents the average value of 24–28 neurons from an independent culture and bar graphs depict the mean ± s.d. (Untreated n = 8/ N = 8, CTRP3 treated n = 8/ N = 8, anisomycin treated n = 5/ N = 5, n = number of coverslips, N = number of independent cultures, 25 neurons per coverslip). Ordinary one-way ANOVA and Uncorrected Fisher’s LSD were used to determine statistical significance. **** p < 0.0001, f - g Quantification of the mean intensity of the SUnSET signal in axons (per 20 μm). f SUnSET signal in axon (WT N = 8, SMA N = 8, N = number of independent cultures). Each dot represents average data from one independent neuron culture. Therefore, data were only compared in the same culture with and without CTRP3 treatment. Two-tailed paired student’s t-test was used to determine statistical significance, ** p < 0.01. To compare WT and SMA, unpaired t-test was used to determine statistical significance, and it was not significant. g Each dot represents a value from a neuron, WT untreated n = 204, WT CTRP3 treated n = 293, SMA untreated n = 200, SMA CTRP3 treated n = 200. Two way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance; * p < 0.05, ** p < 0.01, h - i Quantification of the ratio of the SUnSET signal in axons (per 20 μm) compared to average signal in soma. h Ratio between soma and axons were increased by CTRP3 treatment, WT N = 8, SMA N = 8. Each dot represents average data from one independent neuron culture. Therefore, data were only compared in the same culture with and without CTRP3 treatment. Two-tailed paired student’s t-test was used to determine statistical significance. *** p < 0.001 and ** p < 0.01. To compare WT and SMA, unpaired t-test was used to determine statistical significance. * p < 0.05. i Ratio between soma and axons in individual neurons, each dot represents a value from a neuron, WT untreated n = 204, WT CTRP3 treated n = 293, SMA untreated n = 200, SMA CTRP3 treated n = 200. Two way ANOVA with Sidak’s multiple comparison test was used to determine statistical significance; * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Full-length recombinant proteins were purchased from Cusabio (mouse CTRP3: CSB-EP875360MO; human CTRP3: CSB-EP883621HU) and MyBioSource (human CTRP3: #MBS1265203).

Techniques: Staining, Comparison, Synthesized, Negative Control, Two Tailed Test