Journal: Nature Communications

Article Title: Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation

doi: 10.1038/ncomms10830

Figure Lengend Snippet: ( a , b ) SMCs were serum-restricted for 48 h followed by stimulation with vehicle control or 20 ng ml −1 PDGF-BB for 24 h ( a ) or 48 h ( b ). ( a ) SMCs were fixed, immunofluorescently stained for PTEN (green) and analysed for PTEN localization using confocal microscopy; nuclei were stained for DAPI (blue). ( b ) PTEN was immunoprecipitated (IP) from cytoplasmic (cyto) and nuclear (nuc) fractions of vehicle- or PDGF-stimulated SMCs. Co-immunoprecipitating SRF was detected by immunoblotting (IB). Representative western blot from three separate experiments. ( c ) SMCs were transfected with a construct expressing SRF–GFP, maintained in serum-restricted conditions or stimulated with 20 ng ml −1 PDGF-BB, fixed and analysed for GFP localization; nuclei were stained for DAPI (blue). Shown are representative images (two serum-restricted and four PDGF-stimulated cells are shown); arrows indicate cytoplasmic localized SRF–GFP; nuclei are outlined with white lines. ( d ) SMCs were transfected with HA-tagged wild-type PTEN (WT), nuclear localized PTEN (NLS) or nuclear excluded PTEN (NES). SMCs were maintained in serum-restricted conditions or stimulated with 20 ng ml −1 PDGF-BB, fixed, immunofluorescently stained for HA (red) and analysed for PTEN localization; nuclei were stained for DAPI (blue). Arrowheads, HA–PTEN-transfected SMCs. ( e ) SMCs were transfected with GFP–SRF (ctrl) or co-transfected with GFP–SRF and WT PTEN or nuclear localized PTEN (NLS) then maintained in serum-restricted conditions or stimulated with 20 ng ml −1 PDGF-BB. Transfected SMCs exhibiting cytoplasmic GFP expression were scored as described in Methods. Data represent average per cent positive±s.e.m. N =3 independent experiments; * P <0.01 versus control 0.1% CS; ** P <0.01 versus control PDGF and WT PTEN PDGF; *** P <0.01 versus control PDGF. ( f ) Non-transfected (Ctrl) and HA–PTEN-transfected SMCs were co-transfected with an Acta2 promoter-Luciferase reporter construct. SMCs were serum-restricted for 48 h followed by stimulation with vehicle control or 20 ng ml −1 PDGF-BB for an additional 24 h. Luciferase activity normalized to β-galactosidase was determined; shown are fold changes from Ctrl serum-restricted (0.1% CS) SMCs. Data represent average fold changes±s.e.m. N =3 independent experiments; * P <0.01 versus Ctrl 0.1% CS; ** P <0.01 versus Ctrl PDGF. Molecular weight markers were cropped out for final SRF blot; please see . Scale bars for all images, 20 μm.

Article Snippet: Plasmid-encoding human SRF tagged with GFP (RG208596) was obtained from OriGene.

Techniques: Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing, Luciferase, Activity Assay, Molecular Weight

Journal: The Journal of Physiological Sciences : JPS

Article Title: Exercise preconditioning-induced late phase of cardioprotection against exhaustive exercise: possible role of protein kinase C delta

doi: 10.1007/s12576-014-0323-x

Figure Lengend Snippet: Alteration of PKCδ expression in the late cardiac effect of EP. a Immunohistochemistry staining of PKCδ in cardiomyocytes. PKCδ demonstrated diffuse cytoplasmic staining in cardiomyocytes in all the groups (original magnification, ×400). C–N demonstrated the negative result of none treated with PKCδ primary antibody in control group. b Semi-quantitative analysis of PKCδ immunostaining. The positive area and IOD of the immunostaining in group EE and group LEP were significantly higher than those in group C (P < 0.05). Compared with group LEP + EE, the two values in group CHE + LEP + EE significantly increased (P < 0.05). c Myocardial PKCδ levels determined by western blotting. Consistent with the results of immunostaining, total PKCδ levels in group EE and group LEP were also significantly higher than those in group C (P < 0.05). Compared with group LEP + EE, PKCδ levels in group CHE + LEP + EE significantly increased (P < 0.05). Values are mean ± SD, significant differences (P < 0.05) are indicated as follows: from group C (*), from group LEP + EE (&)

Article Snippet: In situ hybridization In situ hybridization was performed according to previously described approaches [ 23 – 25 ], and an in situ hybridization detection kit for PKCδ mRNA (Boster Bio-engineering, Wuhan, China) was used.

Techniques: Expressing, Immunohistochemistry, Staining, Control, Immunostaining, Western Blot

Journal: The Journal of Physiological Sciences : JPS

Article Title: Exercise preconditioning-induced late phase of cardioprotection against exhaustive exercise: possible role of protein kinase C delta

doi: 10.1007/s12576-014-0323-x

Figure Lengend Snippet: Alteration of p-PKCδThr507 expression in the late cardiac effect of EP. a Immunohistochemistry staining of PKCδ in cardiomyocytes. p-PKCδThr507 demonstrated a granular, scattered cytoplasmic staining pattern in group C and group EE while the pattern changed in group LEP such that p-PKCδThr507 was preferentially localized to intercalated disks (arrow), but not in group LEP + EE and group CHE + LEP + EE. C–N demonstrated the negative result of none treated with p-PKCδThr507 primary antibody in the control group, original magnification, ×1,000. b Semiquantative analysis of p-PKCδThr507 immunostaining. The positive area and IOD of the immunostaining in group LEP + EE were significantly lower than those in group EE (P < 0.05). Compared with group LEP + EE, the two values in group CHE + LEP + EE were increased but not significantly (P < 0.05). c Myocardial p-PKCδThr507 levels determined by western blotting. Consistent with the results of immunostaining, total p-PKCδThr507 levels in group EE were significantly higher than those in group C (P < 0.05). However, compared with group EE, total p-PKCδThr507 levels in group LEP + EE were significantly decreased (P < 0.05). Compared with group LEP + EE, p-PKCδThr507 levels in group CHE + LEP + EE were significantly increased. Values are mean ± SD, significant differences (P < 0.05) are indicated as follows: from group C (*), from group EE (#), and from group LEP + EE (&)

Article Snippet: In situ hybridization In situ hybridization was performed according to previously described approaches [ 23 – 25 ], and an in situ hybridization detection kit for PKCδ mRNA (Boster Bio-engineering, Wuhan, China) was used.

Techniques: Expressing, Immunohistochemistry, Staining, Control, Immunostaining, Western Blot

Journal: The Journal of Physiological Sciences : JPS

Article Title: Exercise preconditioning-induced late phase of cardioprotection against exhaustive exercise: possible role of protein kinase C delta

doi: 10.1007/s12576-014-0323-x

Figure Lengend Snippet: Alteration of PKCδ mRNA expression in the late cardiac effect of EP. a Distribution of PKCδ mRNA in cardiomyocytes by in situ hybridization. The positive signal of PKCδ mRNA demonstrated a granular, diffuse cytoplasmic distribution pattern in cardiomyocytes. The intensity of the hybridization signal was markedly strong in group LEP. C–N, the negative result of none treated with PKCδ mRNA probes in the control group. b Semiquantative analysis of the positive signal of PKCδ mRNA. The area and IOD of the positive signal of PKCδ mRNA in group LEP were significantly higher than those in group C. The c PKCδ mRNA levels determined by quantitative real-time PCR. PKCδ mRNA levels in group LEP were significantly higher than those in group C (P < 0.05). Values are mean ± SD, significant differences (P < 0.05) are indicated as follows: from group C (*), from group EE (#)

Article Snippet: In situ hybridization In situ hybridization was performed according to previously described approaches [ 23 – 25 ], and an in situ hybridization detection kit for PKCδ mRNA (Boster Bio-engineering, Wuhan, China) was used.

Techniques: Expressing, In Situ Hybridization, Hybridization, Control, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Targeting AGGF 1 (angiogenic factor with G patch and FHA domains 1) for Blocking Neointimal Formation After Vascular Injury

doi: 10.1161/JAHA.117.005889

Figure Lengend Snippet: AGGF 1 regulates expression of phenotypic switching markers of vascular smooth muscle cells ( VSMC s). A, The platelet‐derived growth factor subunit B homodimer ( PDGF ‐ BB ) decreases the expression levels of α‐ SMA (α smooth muscle actin), SM 22 (smooth muscle protein 22‐α or transgelin), and MYH 11 (myosin heavy polypeptide 11, smooth muscle) at the protein level. PDGF ‐ BB does not affect the expression level of AGGF 1. B, AGGF 1 blocks PDGF ‐induced downregulation of contractile markers at the protein level. C, AGGF 1 blocks PDGF ‐induced downregulation of contractile markers at the mRNA level. NC indicates negative control. D, AGGF 1 increases the expression levels of α‐ SMA , SM 22, and MYH 11 in mouse VSMC line MOVAS ‐1 VSMC s. E, AGGF 1 increases the expression levels of α‐ SMA , SM 22, and MYH 11 in primary VSMC s isolated from mouse aortas. F, The expression levels of α‐ SMA , SM 22, and MYH 11 in MOVAS ‐1 VSMC s are significantly less than in primary mouse aortic VSMC s. G, Knockdown of SRF encoding the serum response factor by si RNA (si SRF ) abolishes the effect of AGGF 1 on PDGF at the protein level. H, Knockdown of SRF by si RNA (si SRF ) abolishes the effect of AGGF 1 on PDGF at the mRNA level. * P <0.05 (n=3/group). NS indicates not significant.

Article Snippet: The 6xHis‐tagged AGGF1 protein was purified as described by us previously., , Antibodies against AGGF1, SM22, α‐SMA, MYH11, serum response factor (SRF), myocardin, and Enhanced Green Fluorescent Protein (EGFP) were from Proteintech.

Techniques: Expressing, Derivative Assay, Negative Control, Isolation, Knockdown

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Targeting AGGF 1 (angiogenic factor with G patch and FHA domains 1) for Blocking Neointimal Formation After Vascular Injury

doi: 10.1161/JAHA.117.005889

Figure Lengend Snippet: AGGF 1 regulates transcriptional activation of vascular smooth muscle cells ( VSMC s) phenotypic switching markers. A, Luciferase assays showing that AGGF 1 increases transcriptional activation of VSMC s contractile marker genes encoding α‐ SMA (α smooth muscle actin), SM 22 (smooth muscle protein 22‐α or transgelin), and MYH 11 (myosin heavy polypeptide 11, smooth muscle) in the presence of SRF (serum response factor) ( SRF vs SRF +rh AGGF 1). NC indicates negative control. B, Luciferase assays showing that the platelet‐derived growth factor subunit B homodimer ( PDGF ‐ BB ) represses SRF ‐induced transcriptional activation of VSMC s contractile marker genes encoding α‐ SMA , SM 22, and MYH 11, but the effects are abolished by AGGF 1 protein. C, Chromatin immunoprecipitation assays to detect protein– DNA interaction between SRF and the CA rG elements at the promoter/regulatory regions of VSMC s contractile marker genes. PDGF reduces the SRF binding to CA rG elements, but the effects are abolished by AGGF 1 protein. D, Co‐immunoprecipitation assays showing that the AGGF 1 protein increases the interaction between SRF and myocardin in MOVAS ‐1 VSMC s with overexpression of both myocardin and SRF . An anti‐myocardin antibody was used for immunoprecipitation, and an anti‐ SRF antibody was used for immunoblotting. E, Co‐immunoprecipitation assays showing that PDGF reduced the interaction between SRF and myocardin, but the effect was reversed by AGGF 1. * P <0.05 and ** P <0.01 (n=3/group).

Article Snippet: The 6xHis‐tagged AGGF1 protein was purified as described by us previously., , Antibodies against AGGF1, SM22, α‐SMA, MYH11, serum response factor (SRF), myocardin, and Enhanced Green Fluorescent Protein (EGFP) were from Proteintech.

Techniques: Activation Assay, Luciferase, Marker, Negative Control, Derivative Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Over Expression, 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 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

Journal: Nature genetics

Article Title: Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease

doi: 10.1038/s41588-024-01669-y

Figure Lengend Snippet: a . The CHD MPRA library included 6590 REF-ALT pairs. After pooled library synthesis of barcoded oligos, the oligos were PCR amplified and cloned into lentivirus genome backbone. A minimal promoter (miniP)-GFP cassette was then inserted into the cloned oligo library. b . Summary of activity of CHD MPRA library. Plot on bottom indicates the occurrence of the indicated annotation with a vertical line. Enrichment score represents enrichment of the indicated set of annotations at either end of the list of all regions, ranked by activity. Enrichment p-value was determined by 1-sided permutation test, with Bonferroni correction. Active enhancers had barcodes overrepresented in RNA compared to DNA (DESeq2 P adj < 0.05). c . Pearson correlation (PCC) between regions shared between the Mutagenesis MPRA and the CHD MPRA. The same genomic sequences had different barcodes in the two assays. d . Validation of the effect of variants on transcription factor binding. EMSA assay was used to test the binding of SRF or TBX20 to REF or ALT variant sequences. For the GLB1L3 CRE, ALT disrupted the SRF motif and reduced SRF binding in the EMSA assay. For the PIP4K2A CRE, ALT generated a TBX20 motif and increased TBX20 binding in the EMSA assay. Representative of three independent experiments. Two-tailed t-test. n = 3 per group. Graph shows mean ± SD.

Article Snippet: Recombinant human proteins used in this study included SMAD2 (Abcam, ab85329), SRF (OriGene, TP308596), TBX20 (OriGene, TP762422), HIC2 (OriGene, TP760963), SOX9 (OriGene, TP308944) and GATA4 (OriGene, TP310945).

Techniques: Amplification, Clone Assay, Activity Assay, Mutagenesis, Genomic Sequencing, Binding Assay, Variant Assay, Generated, Two Tailed Test

Journal: Nature genetics

Article Title: Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease

doi: 10.1038/s41588-024-01669-y

Figure Lengend Snippet: a . BCOR downregulation in SMAD2 Het and KO iPSC-CMs. Gene expression was measured by RNA-seq. One-way ANOVA with Dunnett’s multiple comparison test versus WT. n = 3. b . Effect of ncDNVs on binding of transcription factors to CREs near CHD genes. 39 bp duplexes centered on ncDNVs neighboring 4 CHD genes were synthesized. Binding of purified, recombinant proteins to the REF or ALT sequence was measured by electrophoretic mobility shift assay (EMSA). SMAD2 and HIC2 bound CREs near BCOR and ACVRL1 more strongly for REF compared to ALT. In contrast, SRF and TBX20 bound CREs near ADAMTS6 and MYOCD more strongly for ALT compared to REF. Note lower free probe in MYOCD -ALT compared to REF. Results are representative of at least three independent experiments. Quantification of TBX20 EMSA: mean ± SD; n = 3; two-sided t-test. Graphs in a and b show mean ± SD.

Article Snippet: Recombinant human proteins used in this study included SMAD2 (Abcam, ab85329), SRF (OriGene, TP308596), TBX20 (OriGene, TP762422), HIC2 (OriGene, TP760963), SOX9 (OriGene, TP308944) and GATA4 (OriGene, TP310945).

Techniques: Expressing, RNA Sequencing Assay, Comparison, Binding Assay, Synthesized, Purification, Recombinant, Sequencing, Electrophoretic Mobility Shift Assay

Journal: Stem Cells International

Article Title: Resveratrol Enhances Cardiomyocyte Differentiation of Human Induced Pluripotent Stem Cells through Inhibiting Canonical WNT Signal Pathway and Enhancing Serum Response Factor-miR-1 Axis

doi: 10.1155/2016/2524092

Figure Lengend Snippet: Serum response factor- (SRF-) miRNA-1 axis also involves the CMs differentiation enhanced by RSV. (a) Transcription levels of SRF in cultured EBs treated with RSV (50 μ M) were detected by qRT-PCR on day 4. (b, c) Western Blot was further carried out to determine the protein levels of RSV (50 μ M) treated EBs after 4 days. (d) miRNA-1 expression level was also detected using qRT-PCR 4 days after RSV (50 μ M) administration. (e, f) Knockdown of SRF using produced Lentivirus expressing shRNA targeting SRF was confirmed by Western Blot analysis. (g) SRF-miRNA-1 signal axis was confirmed in EBs treated with RSV (50 μ M) on day 4. (h) Inhibition of endogenous miRNA-1 in floating cultured EBs 4 days after transfection of Lentivirus within anti-miRNA-1. (i) The effects of modulation of SRF or/and miRNA-1 on the ratio of beating EBs were evaluated on day 24 ( n = 100). Data represent the mean ± s.d. of three biological replicates. ∗ P < 0.05 compared with control; # P < 0.05 compared with RSV alone.

Article Snippet: Human SRF shRNA lentiviral particles and the control shRNA lentiviral particles were purchased from OriGene (TL320530).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Produced, shRNA, Inhibition, Transfection

Journal: Journal of Molecular Neuroscience

Article Title: Interactions of Antibodies to the Gram-Negative Gastric Bacterium Helicobacter pylori with the Synaptic Calcium Sensor Synaptotagmin 5, Correlate to Impaired Vesicle Recycling in SiMa Human Neuroblastoma Cells

doi: 10.1007/s12031-020-01670-0

Figure Lengend Snippet: Western blot analysis of the cross-reactivity of antibodies directed to Helicobacter pylori (α- HPy ) and Campylobacter jejuni (α- CJe ) with different protein samples as provided by commercial HEK-293 overexpression lysates. a Cross-reactivity of α- HPy as revealed by a distinct immunopositive band can be observed for Syt5 and Vglut1 (Slc17a7), whereas Stmn4 and Ncan reveal no such band. Also a control lysate of non-transfected HEK293 cells, as well as an overexpression lysate of Srf is negative. b For α- CJe cross-reactivity as revealed by a distinct immunopositive band can be observed for Syt5 and Vglut1, whereas also in this case Stmn4 and Ncan reveal no such band. Again a control lysate of non-transfected HEK293 cells, as well as an overexpression lysate of Srf is negative. A corresponding negative control incubated with secondary antibodies only is shown in Supplementary Fig.

Article Snippet: Human SRF -transfected HEK293 overexpression lysate (Origene, cat. no. LY418874).

Techniques: Western Blot, Over Expression, Transfection, Negative Control, Incubation