recombinant ctrp3 protein  (Cusabio)

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: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: CTRP3 Regulates Endochondral Ossification and Bone Remodeling During Fracture Healing

doi: 10.1002/jor.24553

Figure Lengend Snippet: (A) Representative whole-femur μCT isosurface renderings at 5, 10 and 20dpf for CTRP3 KO, WT and TG mice. Femurs were scanned after removal of intramedullary pins. Apparent bends in 5dpf bones are artifacts of this removal: fractures are straight in vivo. Quantitative callus μCT data for (B) bone volume fraction (BV/TV), (C) bone mineral content (BMC) and (D) bone mineral density (BMD). Solid lines represent p<0.05 by genotype. Maroon up-arrows represent female mice and navy down-arrows represent male mice. For additional quantitative measures of calluses, see Fig S1.

Article Snippet: The fracture procedure was then repeated in WT mice aged 34±3 weeks to test a systemic protein delivery strategy; animals in this cohort received either phosphate buffered saline (PBS) vehicle (Vh) or 5.0μg recombinant human CTRP3 (rhCTRP3) (R&D Systems, produced in mouse NS0 cells) in PBS and harvested at 21dpf.

Techniques: In Vivo

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: CTRP3 Regulates Endochondral Ossification and Bone Remodeling During Fracture Healing

doi: 10.1002/jor.24553

Figure Lengend Snippet: Safranin-O micrographs from CTRP3 KO, WT and TG mice at (A) 5dpf, (B) 10dpf and (C) 20dpf. Images are oriented to show one side of the callus in longitudinal section with cortical bone at the bottom (light blue staining). Three selected areas of callus (white squares) are magnified on the right of each panel. For associated histomorphometry, see Fig S2.

Article Snippet: The fracture procedure was then repeated in WT mice aged 34±3 weeks to test a systemic protein delivery strategy; animals in this cohort received either phosphate buffered saline (PBS) vehicle (Vh) or 5.0μg recombinant human CTRP3 (rhCTRP3) (R&D Systems, produced in mouse NS0 cells) in PBS and harvested at 21dpf.

Techniques: Staining

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: CTRP3 Regulates Endochondral Ossification and Bone Remodeling During Fracture Healing

doi: 10.1002/jor.24553

Figure Lengend Snippet: Whole-callus mRNA expression of (A) CTRP3, (B) α-actin-2 (ACTA2), (C) osterix (SP7), (D) collagen-II (COL2A1) and (E) tartrate-resistant acid phosphatase (TRAP) vary based on CTRP3 genotype. Relative expression is presented at 2−dCt with ACTB as a housekeeping gene. Statistics were run on dCt values. Solid lines represent p<0.05 by genotype. Maroon up-arrows represent female mice and navy down-arrows represent male mice.

Article Snippet: The fracture procedure was then repeated in WT mice aged 34±3 weeks to test a systemic protein delivery strategy; animals in this cohort received either phosphate buffered saline (PBS) vehicle (Vh) or 5.0μg recombinant human CTRP3 (rhCTRP3) (R&D Systems, produced in mouse NS0 cells) in PBS and harvested at 21dpf.

Techniques: Expressing

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: CTRP3 Regulates Endochondral Ossification and Bone Remodeling During Fracture Healing

doi: 10.1002/jor.24553

Figure Lengend Snippet: μCT (A) quantitation and (B) isosurface renderings at 21dpf in mice that received either subcutaneous Vh or 5μg rhCTRP3 protein at time of surgery. Outputs include total volume (TV), bone volume (BV), bone volume fraction (BV/TV), bone mineral content (BMC), bone mineral density (BMD), tissue mineral content (TMC) and tissue mineral density (TMD). Maroon up-arrows represent female mice and navy down-arrows represent male mice.

Article Snippet: The fracture procedure was then repeated in WT mice aged 34±3 weeks to test a systemic protein delivery strategy; animals in this cohort received either phosphate buffered saline (PBS) vehicle (Vh) or 5.0μg recombinant human CTRP3 (rhCTRP3) (R&D Systems, produced in mouse NS0 cells) in PBS and harvested at 21dpf.

Techniques: Quantitation Assay

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

Journal: Oncotarget

Article Title: C1q/Tumor necrosis factor-related protein-3 protects macrophages against LPS-induced lipid accumulation, inflammation and phenotype transition via PPARγ and TLR4-mediated pathways

doi: 10.18632/oncotarget.19657

Figure Lengend Snippet: (A) Impacts of increasing CTRP3 concentration on the viability of THP-1 differentiated macrophages and mouse peritoneal macrophages. Cells were suspended into 1 × 10 4 cells /100μL/well; THP-1 cells were induced to differentiate into macrophages by culture with 100 ng/mL PMA for 48 h. Mouse peritoneal macrophages were extracted and cultured as mentioned above, after which increasing doses of CTRP3 (0-10 μg/mL) were added; 24 h later, the CCK-8 assay was used to detect viability (mean ± SD). (B-C) Impacts of CTRP3 on LPS-triggered macrophage lipid deposition and foam cell formation in both kinds of macrophages (1 × 10 6 cells/mL/well). The cholesterol content was detected using a cholesterol quantification kit to evaluate lipid deposition in LPS-triggered macrophages (B, mean ± SD, * : compared to the control group, P < 0.05; # : compared to the ox-LDL and LPS treatment group, P < 0.05). Oil red O staining was performed to analyze the effect of CTRP3 on foam cell formation (C, THP-1 cells: 200×, mouse peritoneal macrophages: 400×). At least three independent experiments were performed.

Article Snippet: Human recombinant CTRP3 protein obtained using an in vitro wheat germ expression system was purchased from Abnova (Taipei, Taiwan).

Techniques: Concentration Assay, Cell Culture, CCK-8 Assay, Control, Staining

Journal: Oncotarget

Article Title: C1q/Tumor necrosis factor-related protein-3 protects macrophages against LPS-induced lipid accumulation, inflammation and phenotype transition via PPARγ and TLR4-mediated pathways

doi: 10.18632/oncotarget.19657

Figure Lengend Snippet: THP-1 cells and mouse peritoneal macrophages (1 × 10 6 cells/mL/well) were preincubated with different concentrations of CTRP3 (0-1.0 μg/ mL) for 30 min. After LPS and ox-LDL stimulation, the supernatant protein levels of TNFα, IL-6, MCP-1, IL-1β and MMP-9 were evaluated by ELISA. At least three independent experiments were performed (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS and ox-LDL treatment group, P < 0.05).

Article Snippet: Human recombinant CTRP3 protein obtained using an in vitro wheat germ expression system was purchased from Abnova (Taipei, Taiwan).

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: Oncotarget

Article Title: C1q/Tumor necrosis factor-related protein-3 protects macrophages against LPS-induced lipid accumulation, inflammation and phenotype transition via PPARγ and TLR4-mediated pathways

doi: 10.18632/oncotarget.19657

Figure Lengend Snippet: Cells (1 × 10 6 cells /mL/well) were treated, and expression of specific M1 and M2 markers on the cell surface was detected by FCM to observe macrophages polarization. For THP-1 cells, cells expressing CD68 but not CD206/MR (CD68 + CD206 − ) were considered to be M1 macrophages; cells co-expressing CD68 and CD206 (CD68 + CD206 + ) were considered to be M2 macrophages. For mouse peritoneal macrophages, cells expressing CCR7 and CD86 were considered to be M1 macrophages; cells expressing CD163 and CD206/MR were considered to be M2 macrophages. The macrophage phenotype ratio was calculated as (CCR7 plus CD86 expression)/(CD163 plus CD206 expression). At least three independent experiments were performed (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS but without CTRP3 treatment group, P < 0.05).

Article Snippet: Human recombinant CTRP3 protein obtained using an in vitro wheat germ expression system was purchased from Abnova (Taipei, Taiwan).

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: C1q/Tumor necrosis factor-related protein-3 protects macrophages against LPS-induced lipid accumulation, inflammation and phenotype transition via PPARγ and TLR4-mediated pathways

doi: 10.18632/oncotarget.19657

Figure Lengend Snippet: PMA-induced THP-1 macrophages (1 × 10 6 cells/mL/well) were divided into three groups: the control group was treated with PBS for 24 h; the LPS group was treated with 100 ng/mL LPS for 24 h; the LPS+CTRP3 group was treated with 1 μg/mL CTRP3 for 30 min before LPS stimulation for 24 h. mRNA expression of specific M1 marker and M2 markers was assessed by qRT-PCR. Relative mRNA concentrations were calculated using the ΔΔCT method, and the expression level was normalized to that of endogenous β-actin. At least three independent experiments were performed (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS group, P < 0.05).

Article Snippet: Human recombinant CTRP3 protein obtained using an in vitro wheat germ expression system was purchased from Abnova (Taipei, Taiwan).

Techniques: Control, Expressing, Marker, Quantitative RT-PCR

Journal: Oncotarget

Article Title: C1q/Tumor necrosis factor-related protein-3 protects macrophages against LPS-induced lipid accumulation, inflammation and phenotype transition via PPARγ and TLR4-mediated pathways

doi: 10.18632/oncotarget.19657

Figure Lengend Snippet: Different concentrations of CTRP3 were added before cells (1 × 10 6 cells/mL/well) were exposed to LPS and ox-LDL, and protein expression of TLR4, MyD88, p-NF-κB p65 and NF-κB p65 was assessed by WB (A-D) . Protein expression of PPARγ, LXRα, ABCA1 and ABCG1 was assessed by WB (E-I) (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS and ox-LDL treatment group, P < 0.05).

Article Snippet: Human recombinant CTRP3 protein obtained using an in vitro wheat germ expression system was purchased from Abnova (Taipei, Taiwan).

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: C1q/Tumor necrosis factor-related protein-3 protects macrophages against LPS-induced lipid accumulation, inflammation and phenotype transition via PPARγ and TLR4-mediated pathways

doi: 10.18632/oncotarget.19657

Figure Lengend Snippet: PMA-induced THP-1 macrophages (1 × 10 6 cells/mL/well) were divided into five groups:the control group was treated with PBS; the LPS group was treated with 100 ng/mL LPS for 24 h prior to 100 μg/mL ox-LDL for 24 h; the LPS+CTRP3 (CT3) group was pretreated with 1 μg/mL CTRP3 for 30 min before LPS and ox-LDL stimulation; the LPS+PDTC group was pretreated with 100 μM PDTC for 1 h and then stimulated with LPS and ox-LDL; the LPS+PDTC+CT3 group was treated with 100 μM PDTC for 1 h and then 1 μg/mL CTRP3 for 30 min before LPS and ox-LDL stimulation. Expression of TLR4, MyD88, NF-κB p-p65, t-p65 and β-actin was detected by WB (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS group, P < 0.05, $ : compared to the LPS+CT3 group, P < 0.05).

Article Snippet: Human recombinant CTRP3 protein obtained using an in vitro wheat germ expression system was purchased from Abnova (Taipei, Taiwan).

Techniques: Control, Expressing

Journal: Oncotarget

Article Title: C1q/Tumor necrosis factor-related protein-3 protects macrophages against LPS-induced lipid accumulation, inflammation and phenotype transition via PPARγ and TLR4-mediated pathways

doi: 10.18632/oncotarget.19657

Figure Lengend Snippet: PMA-induced THP-1 macrophages (1 × 10 6 cells/mL/well) were divided into six groups: the first three group were treated as mentioned above, and the other three groups were transfected with si-ctrl, si-PPARγ or si-LXRα for 48 h, and then treated with 1 μg/mL CTRP3 for 30 min, and finally stimulated with LPS and ox-LDL. Expression of PPARγ, LXRα, ABCA1, ABCG1 and β-actin was detected by WB (mean ± SD, * : compared to the control group, P < 0.05, # : compared to the LPS group, P < 0.05, $ : compared to the LPS+CT3 group, P < 0.05).

Article Snippet: Human recombinant CTRP3 protein obtained using an in vitro wheat germ expression system was purchased from Abnova (Taipei, Taiwan).

Techniques: Transfection, Expressing, Control