Journal: STAR Protocols

Article Title: CyTOF mass cytometry analysis of human memory CD4 + T cells and memory B cells

doi: 10.1016/j.xpro.2022.101269

Figure Lengend Snippet:

Article Snippet: Maxpar nuclear antigen staining buffer set , Fluidigm , Cat#201063.

Techniques: Recombinant, Saline, Staining, Software, Cytometry

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Association of Serum HMGB2 Levels With In-Stent Restenosis

doi: 10.1161/atvbaha.116.308210

Figure Lengend Snippet: Figure 2. Hmgb2 deletion attenuates neointimal hyperplasia in injured femoral arteries of mice, whereas perivascular high-mobility group box (HMGB) 2 administration aggravates this pathology. A, Representative images of H&E staining of sham and wire-injured femoral artery sections from C57Bl/6 and Hmgb2–/– mice. Quantification of neointimal growth index (B) and intima-to-media (I/M) ratio (C) was performed in sham or injured arteries of C57Bl/6 and Hmgb2–/– mice. *P<0.05 vs injured arteries of C57Bl/6 mice; n=12. D, Immuno- fluorescent staining of α-smooth muscle actin (α-SMA) was performed in sections of injured arteries from C57Bl/6 and Hmgb2–/– mice. *P<0.05 vs injured arteries of C57Bl/6 mice; n=7. E, Expression of Col1a1, MMP2, and MMP9 in sham and injured arteries of C57Bl/6 and Hmgb2–/– mice. Data are quantified in Figure VI in the online-only Data Supplement. F, The mRNA levels of Col1a1, Col3a1, MMP2, and MMP9 in sham and injured arteries of C57Bl/6 and Hmgb2–/– mice as determined by reverse transcriptase polymerase chain reaction (RT- PCR). *P<0.05 vs injured arteries of C57Bl/6 mice; #P<0.05 vs sham arteries of C57Bl/6 or Hmgb2–/– mice; n=4. G, Representative images of H&E staining of sections of wire-injured arteries of PBS- or HMGB2-treated mice. H and I, Quantification of neointimal growth index (H) and intima-to-media (I/M) ratio (I) of wire-injured arteries of PBS- or HMGB2-treated mice. *P<0.05 vs PBS-treated group; n=10. J, Rep- resentative images of immunofluorescent staining for α-SMA performed in wire-injured arteries of PBS- or HMGB2-administrated mice (left). Staining was also quantified (right). *P<0.05 vs PBS-treated group; n=6 to 7. Masson’s trichrome staining of injury-induced femoral artery from C57Bl/6 and Hmgb2–/– mice (K) and PBS- or HMGB2-treated mice (L). Scale bar: 50 μm.

Article Snippet: The mice serum HMGB2 concentration was measured using a mouse HMGB2 ELISA kit (CSB-EL010560MO, CUSABIO Life Science) according to the manufacturer’s guidelines.

Techniques: Staining, Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Association of Serum HMGB2 Levels With In-Stent Restenosis

doi: 10.1161/atvbaha.116.308210

Figure Lengend Snippet: Figure 4. Reactive oxygen species (ROS) mediates high-mobility group box (HMGB) 2–promoted neointimal hyperplasia in mice. A, Rep- resentative images of dihydroethidium (DHE) staining in femoral arteries of C57Bl/6 or Hmgb2–/– mice with femoral artery wire injury or sham. B, Quantitative analysis of the DHE fluorescence intensity in arteries. #P<0.05 vs sham; *P<0.05 vs injured artery of C57Bl/6 mice; n=6. C, PF-127 gel containing PBS or HMGB2 was delivered around the femoral artery. Apocynin (APO) or vehicle was given in drinking water at a concentration of 1.5 mmol/L for 4 weeks. The sections of wire-injured arteries were stained with H&E, representative images are shown. Quantification of neointimal growth index (D) and intima-to-media (I/M) ratio (E). *P<0.05 vs PBS administration; #P<0.05 vs HMGB2 administration; n=10 to 11. F, Representative images of dihydroethidium (DHE) staining in wire-injured femoral arteries of C57Bl/6 mice with perivascular administration of HMGB2 or PBS, in the presence or absence of APO. G,: Quantitative analysis of the DHE fluores- cent intensity in arteries. *P<0.05 vs sham; #P<0.05 vs injured artery of C57Bl/6 mice; n=5. Scale bar: 50 μm.

Article Snippet: The mice serum HMGB2 concentration was measured using a mouse HMGB2 ELISA kit (CSB-EL010560MO, CUSABIO Life Science) according to the manufacturer’s guidelines.

Techniques: Staining, Fluorescence, Concentration Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Association of Serum HMGB2 Levels With In-Stent Restenosis

doi: 10.1161/atvbaha.116.308210

Figure Lengend Snippet: Figure 5. P47phox phosphorylation–dependent reactive oxygen species (ROS) activation mediates high-mobility group box (HMGB) 2–induced effects in vivo and in vitro. A, Western blot analysis of phospho–p47phox levels in injured femoral arteries of C57Bl/6 and Hmgb2–/– mice and in sham arteries. B, Quantification of phospho–p47phox levels in A. #P<0.05 vs sham; *P<0.05 vs injured arteries of C57Bl/6; n=3. C, Immunofluorescent staining of phospho–p47phox (red) and α-smooth muscle actin (α-SMA; green) in sections of sham or wire injury fem- oral artery in C57Bl/6 and Hmgb2–/– mice. Representative images are shown. D, Quantification of phospho–p47phox levels in femoral arter- ies of C. #P<0.05 vs sham; *P<0.05 vs injured artery of C57Bl/6; n=5. E, Human aortic smooth muscle cells (hASMCs) were stimulated with HMGB2 for various times (0, 15, 30, and 60 minutes). Phospho–p47phox and total p47phox levels were determined using Western blot analy- sis, with quantification as given in Figure XIF in the online-only Data Supplement. F, H, I, and K, In hASMCs transfected with scramble-small interfering RNA (siRNA) or p47phox-siRNA for 24 h and then stimulated by HMGB2 or PBS in experiments, ROS, proliferation assay, Boyden chamber assay, and Wound-healing assay were performed, and respectively, quantified in G, J, and L. *P<0.05 vs scramble-siRNA with PBS stimulation; #P<0.05 vs scramble-siRNA with HMGB2 stimulation; n=3 to 6. Scale bar: 50 μm. DCF indicates 2′,7′-dichlorodihydrofluorescein.

Article Snippet: The mice serum HMGB2 concentration was measured using a mouse HMGB2 ELISA kit (CSB-EL010560MO, CUSABIO Life Science) according to the manufacturer’s guidelines.

Techniques: Phospho-proteomics, Activation Assay, In Vivo, In Vitro, Western Blot, Staining, Transfection, Small Interfering RNA, Proliferation Assay, Boyden Chamber Assay, Wound Healing Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Association of Serum HMGB2 Levels With In-Stent Restenosis

doi: 10.1161/atvbaha.116.308210

Figure Lengend Snippet: Figure 6. High-mobility group box (HMGB) 2 promotes p47phox phosphorylation and migration and proliferation of vascular smooth muscle cells (VSMCs) via receptor of advanced glycation end products (RAGE) but not NOX4. A, Human aortic smooth muscle cells (HASMCs) were transfected with scramble-small interfering RNA (siRNA), Toll-like receptor (TLR) 2-siRNA, TLR4-siRNA, or RAGE-siRNA for 24 h, followed by stimulation with PBS or HMGB2. The levels of phospho–p47phox and p47phox were determined by Western blot analysis. Quantification can be found in Figure XIII in the online-only Data Supplement. B and C, VSMCs derived from Rage–/– and Tlr4–/– mice were treated with PBS or HMGB2. The levels of phospho–p47phox and p47phox were determined by Western blot analysis. Quanti- fication can be found in Figure XIII in the online-only Data Supplement. D, F, and H, VSMCs derived from C57Bl/6 and Rage–/– mice were treated with PBS or HMGB2 in experiments. Reactive oxygen species (ROS), Boyden chamber, and proliferation assay were performed, and respectively, quantified in E and G. *P<0.05 vs C57Bl/6 mouse-derived VSMCs; #P<0.05 vs C57Bl/6 mouse-derived VSMCs with HMGB2 stimulation; n=3 to 5. I, Immunofluorescence confocal microscopy showed increased expression of HMGB2 and RAGE and colo- calization of these 2 proteins in neointima after vascular injury. n=4. Scale bar: 50 μm.

Article Snippet: The mice serum HMGB2 concentration was measured using a mouse HMGB2 ELISA kit (CSB-EL010560MO, CUSABIO Life Science) according to the manufacturer’s guidelines.

Techniques: Phospho-proteomics, Migration, Transfection, Small Interfering RNA, Western Blot, Derivative Assay, Proliferation Assay, Immunofluorescence, Confocal Microscopy, Expressing

Journal: bioRxiv

Article Title: Replication-driven HBV cccDNA loss in chimeric mice with humanized livers

doi: 10.1101/2023.12.28.573542

Figure Lengend Snippet: A. and B. Kinetic serum HBsAg and HBV DNA levels. C. Intrahepatic HBsAg, rcDNA and cccDNA levels (copies/cell) were progressively increased during the spread of infection, then maintained at steady levels during persistent infection phase. Intracellular HBsAg was measured with ELISA. One mouse was sacrificed at each timepoint. D . Immunostaining of intracellular HBsAg shows the spread of infection by increasing the number of HBsAg positive cells and it appears that most cells were HBsAg positive around day 50 pi. Red arrows indicate two HBsAg positive cells on day 18 section. E . H&E staining shows severe liver parenchymal necrosis with infiltrates (green arrows) among HBV infected mice (orange: uninfected;: blue: untreated; green: treated). pi: post infection; cccDNA, covalently closed circular DNA; rcDNA, relaxed circular DNA. H&E, hematoxylin and eosin; HBsAg, hepatitis B surface antigen. Error bars were plotted with standard deviations.

Article Snippet: Blood was collected tri-weekly for quantification of serum HBV DNA (qPCR see below), HBeAg (CSB-E13557h, CUSABIO), HBsAg (GS HBsAg EIA 32591, Bio-Rad), anti-HBs antibody (MONOLISA Anti-HBs EIA 25200, Bio-Rad) with calibrators (MONOLISA Anti-HBs 20-Calibrator kit 25219, Bio-Rad) and human albumin (Human albumin ELISA kit E-80AL, Immunology Consultants Laboratory) levels by ELISA per instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Immunostaining, Staining

Journal: Nature Communications

Article Title: Skeletal muscle derived Musclin protects the heart during pathological overload

doi: 10.1038/s41467-021-27634-5

Figure Lengend Snippet: a Scheme depicting the experimental time line. Quantification of heart weight/tibia length (HW/TL) ratio, n = 7/group, **** p < 0.0001 ( b ), lung weight/tibia length (LW/TL) ratio, n = 7/group, *** p = 0.001 ( c ), left ventricular ejection fraction, n = 5/sham group and n = 8/TAC group, **** p < 0.0001 ( d ), quadriceps muscle weight, n = 7/group, **** p < 0.0001 ( e ), gastrocnemius muscle weight n = 7/group, *** p = 0.0003 ( f ), triceps brachii muscle weight, n = 7/group, ** p = 0.0051 ( g ), plantaris muscle weight, n = 7/group, * p = 0.0175 ( h ), soleus muscle weight, n = 7/group, * p = 0.0126 ( i ), and course of body weight (BW) during the experiment until 12 weeks after TAC or sham surgery, n = 4/sham group and n = 5/TAC group, *** p = 0.0001 ( j ). Inguinal fat weight, ** p = 0.0021 ( k ) in the group of mice that was analyzed by MRI to determine the amount of abdominal fat, * p = 0.0273 ( l ), psoas muscle, * p = 0.0499 ( m ) and autochthonal muscle, * p = 0.0412 ( n ) 12 week after sham or TAC surgery, n = 4/group. Example MRI cross-sections are shown in ( o ). The red arrows indicate abdominal fat. Scale bar: 5 mm. Cross-sectional fiber area type IIb, ** p = 0.0028 for 1000–2000 µm 2 ; * p = 0.0254 for 2000–3000 µm 2 and * p = 0.0125 for 3000–4000 µm 2 of muscle fiber area from sham vs. TAC group ( p ), and type IId, **** p < 0.0001 of fiber area <1000 µm 2 ( q ) of quadriceps muscles 12 weeks after sham or TAC surgery, n = 4/group, analyzed from microscopic pictures with ATPase staining (pH 4.2) as shown in ( r ). Scale bar: 300 μm. s Ostn (Musclin) mRNA levels in different organs 12 weeks after sham or TAC surgery, n = 3/group. t Immunoblot (the size of the proteins is indicated in kDa) and immunofluorescence staining ( u ) showing Musclin protein levels in the quadriceps muscle of sham and TAC mice. Scale bar: 100 μm. v Musclin plasma levels in mice 12 weeks after sham or TAC surgery, n = 10 for sham and n = 9 for TAC, * p = 0.0115. w Time course (2, 6 and 12 weeks after TAC or sham surgery), n = 4/group, showing the decline of Ostn mRNA levels in the gastrocnemius, triceps, *** p = 0.0007 6 weeks and * p = 0.0241 12 weeks, in quadriceps muscles, ** p = 0.002 by 12 weeks after TAC compared to sham mice. Data are shown as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as determined by Kruskal–Wallis test with Dunn’s multiple comparisons test (for s ) or two-tailed Student’s t test (all other numerical data containing panels) or p value determined by one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test ( p , q ). Source data are provided as a source data file.

Article Snippet: Human serum Musclin concentrations were determined by the human Musclin ELISA kit (Cusabio, # CSB-E12021h) according to manufacturer’s instructions in male healthy blood donors or male patients suffering from severe aortic stenosis before aortic valve replacement was conducted (the patient characteristics are shown in Table ).

Techniques: Muscles, Staining, Western Blot, Immunofluorescence, Clinical Proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: Skeletal muscle derived Musclin protects the heart during pathological overload

doi: 10.1038/s41467-021-27634-5

Figure Lengend Snippet: a OSTN mRNA levels in vastus lateralis muscle biopsy samples from control individuals ( n = 9) and from patients with sarcopenia ( n = 6) or cachexia ( n = 5), * p = 0.0236. b Concentrations of Musclin protein in the serum of healthy control individuals ( n = 56) and of patients with severe aortic stenosis ( n = 26), * p = 0.0403 ( c ) and of the same patients excluding the ones with diabetes mellitus ( n = 18), ** p = 0.0044. Data are shown as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 as determined by ANOVA followed by the Holms–Sidak’s multiple comparisons test ( a ) and the two-tailed Mann–Whitney test ( b , c ). Source data are provided as a source data file.

Article Snippet: Human serum Musclin concentrations were determined by the human Musclin ELISA kit (Cusabio, # CSB-E12021h) according to manufacturer’s instructions in male healthy blood donors or male patients suffering from severe aortic stenosis before aortic valve replacement was conducted (the patient characteristics are shown in Table ).

Techniques: Control, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Skeletal muscle derived Musclin protects the heart during pathological overload

doi: 10.1038/s41467-021-27634-5

Figure Lengend Snippet: Clinical characteristics of the patients (all male) with aortic stenosis.

Article Snippet: Human serum Musclin concentrations were determined by the human Musclin ELISA kit (Cusabio, # CSB-E12021h) according to manufacturer’s instructions in male healthy blood donors or male patients suffering from severe aortic stenosis before aortic valve replacement was conducted (the patient characteristics are shown in Table ).

Techniques:

Journal: Nature Communications

Article Title: Skeletal muscle derived Musclin protects the heart during pathological overload

doi: 10.1038/s41467-021-27634-5

Figure Lengend Snippet: a Scheme depicting the experimental time line. b mRNA level of Ostn (Musclin) 9 weeks after TAC and intramuscular application of AAV6 control (Co) or AAV6 Musclin (Mu) vector ( n = 12/group), *p = 0.036 ( c ) Immunoblot for Musclin 9 weeks after TAC and intramuscular application of AAV6 Co or AAV6 Mu. GAPDH, Actin and Tubulin are loading controls. The size of the proteins is indicated in kDa. d Musclin plasma levels in AAV6 Co ( n = 21) or AAV6 Mu ( n = 22) treated mice, ** p = 0.0029. e Heart weight/tibia length (HW/TL) ratio in AAV6 Co (sham n = 5, TAC n = 7) or AAV6 Mu (sham n = 5, TAC n = 6) treated mice 9 weeks after sham or TAC surgery, *** p = 0.0004, * p = 0.0316. f – h Echocardiographic ejection fraction 3 weeks (all sham n = 5/group, TAC AAV6 Co n = 17, TAC AAV6 Mu n = 15), **** p < 0.0001, ** p = 00015 and * p = 0.0293 ( f ), and 6 weeks after surgery, **** p < 0.0001 in AAV6 Control and AAV6 Musclin groups for sham vs. TAC surgery, ** p = 0.0021 ( g ), and 9 weeks after surgery, **** p < 0.0001, *** p = 0.0006 and ** p = 0.0051 ( h ). Cardiac systolic contractility (dp/dt max, i ), * p = 0.0122 and diastolic relaxation (dp/dt min, j ), * p = 0.0164 determined by left ventricular catheterization in the indicated mice (all sham n = 5/group, TAC AAV6 Co n = 9, AAV6 Mu n = 8) 9 weeks after sham or TAC surgery. Representative Sirius red-stained heart sections ( k ) and quantified myocardial fibrotic area ( l ) and of mice treated as shown (all sham n = 5/group, all TAC n = 8/group, 9 weeks after surgery), ** p = 0.0093 and * p = 0.0206. Scale bar: 500 µm. m – p qPCR analysis of the indicated fibrosis genes 9 weeks after sham or TAC surgery (sham AAV6 Co n = 5, sham AAV6 Mu n = 4, TAC AAV6 Co n = 7, TAC AAV6 Mu n = 8), ** p = 0.0064 ( m ), ** p = 0.0081 ( n ), * p = 0.0248 ( o ), * p = 0.0369 ( p ). q Mature CNP plasma levels in AAV6 Co or AAV6 Mu treated mice 3 weeks after TAC surgery ( n = 4/group), * p = 0.0462. Data are shown as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as determined by two-tailed Student’s t test ( d , q ) or by one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test (other bar graphs). Source data are provided as a source data file.

Article Snippet: Human serum Musclin concentrations were determined by the human Musclin ELISA kit (Cusabio, # CSB-E12021h) according to manufacturer’s instructions in male healthy blood donors or male patients suffering from severe aortic stenosis before aortic valve replacement was conducted (the patient characteristics are shown in Table ).

Techniques: Control, Plasmid Preparation, Western Blot, Clinical Proteomics, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Skeletal muscle derived Musclin protects the heart during pathological overload

doi: 10.1038/s41467-021-27634-5

Figure Lengend Snippet: a Scheme depicting the Musclin ( Ostn ) knockout (KO) targeting strategy. The exon 3 of the Ostn gene is floxed and b deleted through the crossing with double transgenic mice that express Cre recombinase selectively in skeletal muscle and only following doxycycline treatment. c Scheme depicting the experimental time line. d qPCR analysis of Ostn mRNA in the quadriceps muscle and the heart 2 weeks after TAC or sham surgery in KO vs. littermate control (WT) mice (all sham n = 8/group, all TAC n = 12/group), * p = 0.0359. e Immunoblot for Musclin in the quadriceps muscle and the heart 2 weeks after TAC or sham surgery in WT vs. KO mice. GAPDH and Tubulin are loading controls. The size of the proteins is indicated in kDa. f Immunofluorescence staining for Musclin in the quadriceps muscle of WT and KO mice. Scale bar: 100 μm. g Musclin plasma levels of WT ( n = 21) and KO mice ( n = 16), ** p = 0.0014. h Ostn mRNA levels in the indicated organs of the mice as shown (n = 7/group), ** p = 0.0027. Quantification of the heart weight/tibia length ratio (HW/TL, **** p < 0.0001) ( i ), echocardiographic ejection fraction, ** p = 0.0018 and **** p < 0.0001, * p = 0.0445 ( j ), systolic contractility ( k , by LV-catheter, * p = 0.0334) and diastolic relaxation ( l , by LV-catheter, ** p = 0.0094) in mice treated as indicated in ( c ), (for HW/TL ratio and for echocardiography: all sham n = 6/group, TAC WT n = 18, TAC KO n = 14; for LV-catheter: n = 8/group). m , n Myocardial fibrotic area with representative Sirius red-stained myocardial sections of WT and KO mice treated as indicated (all sham n = 8/group, TAC WT n = 9, TAC KO n = 10, ** p = 0.0033 in WT and **** p < 0.0001 in KO mice after sham vs. TAC surgery, ** p = 0.0033 in WT vs. KO mice after TAC). Scale bar: 500 µm. o Mature CNP peptide plasma levels in the indicated mice ( n = 8/group, * p = 0.0312). Data are shown as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, **** p < 0.0001 determined by two-tailed Student’s t test ( g , h , o ) or one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test (other bar graphs). Source data are provided as a source data file.

Article Snippet: Human serum Musclin concentrations were determined by the human Musclin ELISA kit (Cusabio, # CSB-E12021h) according to manufacturer’s instructions in male healthy blood donors or male patients suffering from severe aortic stenosis before aortic valve replacement was conducted (the patient characteristics are shown in Table ).

Techniques: Knock-Out, Transgenic Assay, Control, Western Blot, Immunofluorescence, Staining, Clinical Proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: Skeletal muscle derived Musclin protects the heart during pathological overload

doi: 10.1038/s41467-021-27634-5

Figure Lengend Snippet: a Scheme depicting the proposed mechanism of augmented cardiomyocyte contractility by Musclin. Scheme ( b ) and results ( c ) of the NPR-C/Musclin/CNP competition assay. Increasing levels of recombinant CNP led to Musclin displacement from NPR-C ( n = 2 per CNP concentration, mean values are shown). d Representative fluorescence images from Hek293 cells with and without transfection of the NPR-C-GFP construct. Scale bar 100 µm. e CNP concentrations (determined by ELISA) in the supernatant of transfected and untransfected Hek293 cells (as shown in d ) 1 h after treatment as indicated ( n = 4/condition, * p = 0.0104). f Representatives traces of sarcomere length and g quantification of cell shortening (from traces as shown in f ) in isolated wild-type mouse cardiomyocytes treated with recombinant CNP or Musclin as indicated ( n = 13 cardiomyocytes/group, ** p = 0.0016 for Vehicle vs. CNP (10 nM), ** p = 0.0012 for CNP (10 nM) vs. CNP (100 nM) and ** p = 0.0026 for CNP (100 nM) vs. CNP (100 nM) plus Musclin (10 nM)). h Representative Fura-2 Ca 2+ traces and quantitative analysis ( i ) in cardiomyocytes treated as described for ( f ) ( n = 12 cells/group, * p = 0.0168 for Vehicle vs. CNP and * p = 0.0169 for cells stimulated with CNP vs. Musclin). j – l Cell shortening in cardiomyocytes treated as indicated from wild-typ (WT) (vehicle cardiomyocytes n = 46, after CNP stimulation n = 24, treated with Musclin n = 21, cardiomyocytes treated with CNP and Musclin n = 16 cells), **** p < 0.0001, * p = 0.0278 and ** p = 0.0091 ( j ), cardiomyocyte-specific Npr1 knockout (vehicle-treated cardiomyocytes n = 25, after CNP stimulation n = 27, cardiomyocytes treated with Musclin n = 25, cardiomyocytes treated with CNP and Musclin n = 22, * p = 0.01 and *** p = 0.0003 ( k ), and global Npr2 knockout mice (vehicle-treated cardiomyocytes n = 40, after CNP stimulation n = 39, treated with Musclin n = 31, cardiomyocytes treated with CNP and Musclin n = 28 cells) ( l ). Data in bar graphs are shown as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 determined by Kruskal–Wallis with Dunn’s multiple comparisons test ( e ) or by one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test (all other panels). Source data are provided as a source data file.

Article Snippet: Human serum Musclin concentrations were determined by the human Musclin ELISA kit (Cusabio, # CSB-E12021h) according to manufacturer’s instructions in male healthy blood donors or male patients suffering from severe aortic stenosis before aortic valve replacement was conducted (the patient characteristics are shown in Table ).

Techniques: Competitive Binding Assay, Recombinant, Concentration Assay, Fluorescence, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Isolation, Knock-Out

Journal: Nature Communications

Article Title: Skeletal muscle derived Musclin protects the heart during pathological overload

doi: 10.1038/s41467-021-27634-5

Figure Lengend Snippet: Cardiomyocytes were isolated from either cGES-DE5 transgenic (for cGMP measurements) or from Epac2-camps (for cAMP measurements) transgenic mice. a , b Fluorescence resonance energy transfer (FRET)-based measurements of cGMP in single cultured cardiomyocytes ( n = 7 cells). The stimulation with CNP and Musclin was conducted as indicated. Representative traces ( a ) and a quantitative analysis ( b ) are shown, * p = 0.0342. c , d FRET-based measurements of cAMP in single cardiomyocytes treated as indicated ( n = 13 cells). The cells were stimulated with Isoproterenol (Iso), CNP and subsequently Musclin as indicated. The graph illustrates the FRET-responses to CNP in % of the maximal Iso effect. Representative traces ( c ) and a quantitative analysis ( d ) are shown, ** p = 0.0019. e – h ELISA-based measurements of cGMP and cAMP in cultured cardiomyocytes under the indicated conditions from wild-typ (WT) ( e , g ), ** p = 0.0012 for vehicle-treated cells vs. stimulated with CNP (100 nM), **** p < 0.0001 for cells stimulated with Musclin vs. with CNP (100 nM) and Musclin, ** p = 0.0013 for cells treated with CNP (100 nM) vs. cells stimulated with Musclin and CNP (100 nM) ( e ), * p = 0.034 ( g ) and global Npr2 knockout mice ( f , h ) (for cGMP measurement n = 4/condition and for cAMP n = 3/condition). i , j FRET-based measurements of cAMP in single WT cardiomyocytes treated as indicated. Representative traces ( i ) and a quantitative analysis ( j ) are shown ( n = 6 cells without Musclin treatment and n = 8 cells with Musclin). k Representatives traces of sarcomere length and l quantification of cell shortening (from traces as shown in k ) in isolated wild-type mouse cardiomyocytes treated as indicated ( n = 9 cardiomyocytes per group, * p = 0.017). Cilostamide was used as PDE3 inhibitor. Data in bar graphs are shown as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, **** p < 0.0001 determined by two-tailed Student’s t test ( b , d , j ), one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test ( e , f , l ) or by Kruskal–Wallis with Dunn’s multiple comparisons test ( g , h ). n.s. denotes “not significant”. Source data are provided as a source data file.

Article Snippet: Human serum Musclin concentrations were determined by the human Musclin ELISA kit (Cusabio, # CSB-E12021h) according to manufacturer’s instructions in male healthy blood donors or male patients suffering from severe aortic stenosis before aortic valve replacement was conducted (the patient characteristics are shown in Table ).

Techniques: Isolation, Transgenic Assay, Fluorescence, Förster Resonance Energy Transfer, Cell Culture, Enzyme-linked Immunosorbent Assay, Knock-Out, Two Tailed Test

Journal: Nature Communications

Article Title: Skeletal muscle derived Musclin protects the heart during pathological overload

doi: 10.1038/s41467-021-27634-5

Figure Lengend Snippet: Cardiomyocyte cGMP ( a , * p = 0.0268), ( c , * p = 0.048) and cAMP ( b , * p = 0.029), ( d , * p = 0.025) levels (determined by ELISA) after sham and TAC surgery in control (WT) and Musclin knockout (KO) mice after TAC, as well as in Sham and TAC operated mice treated either with AAV6 Control (Co) or AAV6 Musclin (Mu) (WT sham n = 5, WT TAC n = 9, KO TAC n = 10, sham AAV6 Co n = 4, sham AAV6 Mu n = 3, TAC AAV6 Co n = 6, TAC AAV6 Mu n = 7). Immunoblots for the indicated proteins (GAPDH as loading control) from cardiac protein lysate 3 days ( e ), cardiomyocyte protein lysate 14 days ( f ) or cardiac protein lysate 9 weeks ( g ) after TAC or sham surgery in WT or KO mice or in mice treated with AAV6 Co or AAV6 Mu as shown. The size of the proteins is indicated in kDa. Data in bar graphs are shown as mean ± standard error of the mean (SEM). * p < 0.05, determined by one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test. Source data are provided as a source data file.

Article Snippet: Human serum Musclin concentrations were determined by the human Musclin ELISA kit (Cusabio, # CSB-E12021h) according to manufacturer’s instructions in male healthy blood donors or male patients suffering from severe aortic stenosis before aortic valve replacement was conducted (the patient characteristics are shown in Table ).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Knock-Out, Western Blot

Journal: Nature Communications

Article Title: Skeletal muscle derived Musclin protects the heart during pathological overload

doi: 10.1038/s41467-021-27634-5

Figure Lengend Snippet: a Measurement of cardiac fibroblast proliferation by BrDU incorporation ELISA with addition of CNP and/or Musclin ( n = 5/condition, ** p = 0.0039 for vehicle vs. CNP, ** p = 0.0019 for vehicle vs. musclin and *** p = 0.0002). b Assessment of cardiac fibroblast migration through detection of recovery of a scratch wound after 4 h during stimulation as indicated ( n = 6/condition, ** p = 0.0023, *** p = 0.0003 for vehicle-treated cells vs. stimulated with Musclin, and *** p = 0.0005 vs. cells stimulated with CNP and Musclin). c – e Measurement of cardiac fibroblast proliferation by BrDU incorporation ELISA under the indicated conditions ( c – e ) ( n = 6/condition, except siNPR2 treatment n = 7); * p = 0.0103 for siControl cells, vehicle treated vs. stimulated with CNP, ** p = 0.0016 vs. stimulated with Musclin and *** p = 0.0001 vs. stimulated with CNP and Musclin, * p = 0.0103 for siNPR1 cells, vehicle treated vs. stimulated with CNP, **** p < 0.0001 vs. stimulated with Musclin and vs. stimulated with CNP and Musclin ( c ); * p = 0.0243, ** p = 0.0055 and *** p = 0.0003 ( d ); ** p = 0.0021 for siControl cells, vehicle treated vs. stimulated with CNP, ***p = 0.0002 vs. stimulated with Musclin, and **** p < 0.0001 vs. treated with both CNP and Musclin, *** p = 0.0002 for siNPR3 cells, vehicle treated vs. stimulated with CNP, **** p < 0.0001 vs. treated with Musclin and vs. cells treated with both CNP and Musclin ( e ). Assessment of cardiac fibroblast migration by scratch assay in cardiac fibroblasts ( n = 6/condition) treated with siRNA, CNP and Musclin as indicated ( f – h ), * p = 0.036 for siControl cells, vehicle treated vs. treated with CNP, *** p < 0.0001 vs. Musclin and vs. stimulated with CNP and Musclin, * p = 0.046 for siNPR1 cells, vehicle treated vs. stimulated with CNP, ** p = 0.0013 vs. Musclin and **** p < 0.0001 vs. cells treated with both CNP and Musclin ( f ); * p = 0.0148 for siControl cells, vehicle treated vs. treated with CNP, ** p = 0.0013, and * p = 0.0148 between siControl and siNPR2 cells treated with both CNP and Musclin ( g ); * p = 0.0162 for siControl cells, vehicle treated vs. cells stimulated with CNP, ** p = 0.0042 vs. cells treated with Musclin and *** p = 0.0004 vs. cells stimulated with both CNP and Musclin, * p = 0.0162 for siNPR3 cells, vehicle treated vs. stimulated with CNP or Musclin and *** p = 0.0002 vs. stimulated with Musclin and CNP, *** p = 0.0005 for siControl cells treated with Musclin und CNP vs. siNPR3 cells stimulated with Musclin and CNP, *** p = 0.0008 for vehicle-treated siControl cells vs. vehicle-treated siNPR3 cells ( h ). i , j Assessment of cardiac fibroblast proliferation and migration after stimulation with Musclin, CNP or the PKG inhibitor DT3 as indicated (for proliferation n = 7/condition, ** p = 0.0018 for vehicle vs. Musclin treated cells and * p = 0.016 for cells stimulated with Musclin vs. cells treated with Musclin and DT3 ( i ), and for migration n = 4/condition, ** p = 0.0043 for vehicle-treated cells vs. stimulated with Musclin, *** p = 0.0005 for cells treated with Musclin vs. treated with DT3 ( j )). k Immunoblot from cardiac fibroblasts’ protein lysate for the indicated proteins after stimulation as indicated. GAPDH was used as loading control. The size of the proteins is indicated in kDa. Data in bar graphs are shown as mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 determined by one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test. Mu stands for Musclin. Source data are provided as a source data file.

Article Snippet: Human serum Musclin concentrations were determined by the human Musclin ELISA kit (Cusabio, # CSB-E12021h) according to manufacturer’s instructions in male healthy blood donors or male patients suffering from severe aortic stenosis before aortic valve replacement was conducted (the patient characteristics are shown in Table ).

Techniques: BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay, Migration, Wound Healing Assay, Western Blot, Control