Journal: The Journal of investigative dermatology

Article Title: Human mitochondrial NAD(P)(+)-dependent malic enzyme participates in cutaneous melanoma progression and invasion.

doi: 10.1038/jid.2014.385

Figure Lengend Snippet: Figure 4. AMP-activated protein kinase (AMPK) activation as the mechanism for melanoma cell growth inhibition in response to ME2 depletion. (a) Silencing ME2 diminished ATP production in melanoma cells. ATP levels in A2058 cells were measured and normalized to the respective protein concentration. (b) Increased reactive oxygen species levels in A2058 ME2-knockdown cells as determined by flow cytometry using 2’,7’- dichlorodihydrofluorescein diacetate (DCF). Each histogram is representative of three independent experiments. (c) Western blot analysis was used to determine the protein levels of p-AMPK (Thr172), total AMPK, acetyl Co-A carboxylase (ACC), p53, and p21. Phospho Ser-79 ACC is a biomarker of AMPK activity. (d) Western blot of p-AMPK, total AMPK, ACC, p53, and p21 in indicated melanoma cell lines transfected with control vector or with ME2- expressing vector. The level of ME2 protein after normalization with a-actinin is presented as fold change.

Article Snippet: After transferring the proteins to a polyvinylidine difluoride membrane (Millipore, Bedford, MA), the proteins were detected using antibodies against alpha-actinin (ACTN), p53, p21 (Santa Cruz Biotechnology, Santa Cruz, CA), ME2 (Sigma-Aldrich), and AMPK and acetyl Co-A carboxylase antibody sampler kit (Cell Signaling, Beverly, MA).

Techniques: Activation Assay, Inhibition, Protein Concentration, Knockdown, Cytometry, Western Blot, Biomarker Discovery, Activity Assay, Transfection, Control, Plasmid Preparation, Expressing

Journal: Nature Communications

Article Title: Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

doi: 10.1038/s41467-023-43054-z

Figure Lengend Snippet: a Localization of the putative pH-sensing residues selected for replacement on the three-dimensional structure of the AIP56 catalytic domain. Cartoon (left) and surface representation of AIP56 catalytic domain with (middle) or without (right) the middle domain. The catalytic residues (H165, E166 and H169) are shown in red, the putative pH-sensing residues in orange and the D209-K247 hairpin in marine blue. The cysteine residues (pink sticks) forming the disulfide bridge (yellow) are also shown. b Analysis of NF-kB p65 cleavage in mouse bone marrow-derived macrophages (mBMDM) by V5 plus His-tagged AIP56 variants. Cleavage of p65 was assessed by western blotting (upper panel; chromogenic detection) and protein loading by staining the membranes with Ponceau S (lower panel). The result shown is representative of six ( n = 6) independent experiments. c Peak ANS (8-Anilino-1-naphthalenesulfonic acid) fluorescence measured at 475 nm for the indicated pH, normalized by subtracting the corresponding values at pH 7 (fluorescence due to conformational changes caused by the mutation and not due to acidification). The measurement curves for each pH at different wavelengths are shown in Supplementary Fig. . The results shown are representative of at least three ( n = 3) independent experiments. AIP56, black; AIP56 E214K , blue; AIP56 E218K , purple; AIP56 H222K , green; AIP56 H231K , brown; AIP56 E234K , pink; AIP56 H231K/E234K , gray; AIP56 E214K/E218K/H222K , orange; d Coomassie Blue-stained SDS-PAGE gels from limited proteolysis of AIP56 and AIP56 H231K/E234K by Proteinase K. A and B mark the bands corresponding to the catalytic and receptor-binding domains, respectively. The results shown are representative of two ( n = 2) independent experiments. e Interaction of AIP56 or AIP56 variants with black lipid bilayers. Single-channel recordings of DiPhPC/n-decane membranes after addition of the indicated proteins to one side of the black lipid bilayer at a final concentration of 14 nM. Membrane activity was induced by acidification (pH 4.8; red arrows) of the aqueous phase at the cis-side of the chamber. Each result shown is representative of at least three ( n = 3) independent measurements. Source data for ( b ), ( c ) and ( d ) are provided in the Source data file.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (clone c-20) rabbit polyclonal antibody (sc-372, dilution 1:3000) was from Santa Cruz Biotechnology and the anti-V5 (R960-25, dilution 1:5000) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Derivative Assay, Western Blot, Staining, Fluorescence, Mutagenesis, SDS Page, Binding Assay, Concentration Assay, Membrane, Activity Assay

Journal: Nature Communications

Article Title: Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

doi: 10.1038/s41467-023-43054-z

Figure Lengend Snippet: a Schematic representation of chimera Bla L19-W286 AIP56 P210-N497 . Bla, β-lactamase. b FRET-based assay to access the effect of Hsp90 inhibition on Bla delivery. The cleaved/uncleaved CCF4-AM ratios were determined by quantifying the indicated number of microscopic fields per condition. Results shown represent one out of three ( n = 3) independent experiments. Statistical significance was tested by Kruskal–Wallis nonparametric test and the adjusted p values for individual comparisons were obtained by Bonferroni correction. Data are presented as mean values ± SD. Actual p values from left-to-right and upwards: p < 0.0001, p < 0.0001, p = 0.0993, p = 0.8533, p < 0.0001, p = 0.0051; ns non-significant, CCF4 Fluorescence Resonance Energy Transfer (FRET) substrate, DMAG 17-(dimethylaminoethylamino)−17-demethoxygeldanamycin (Hsp90 inhibitor), Bla β-lactamase. c Control of 17-DMAG activity by confirming its inhibitory effect on NF-kB p65 (nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65) cleavage upon AIP56 intoxication of mBMDM. A representative blot of three ( n = 3) independent experiments is shown. Loading correction was achieved by dividing the density of p65 by the respective density of the Ponceau S staining. Data are presented as mean values ± SD. Different symbols represent independent experiments. DMAG 17-(dimethylaminoethylamino)−17-demethoxygeldanamycin (Hsp90 inhibitor). Source data for ( b ) and ( c ) are provided in the Source data file.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (clone c-20) rabbit polyclonal antibody (sc-372, dilution 1:3000) was from Santa Cruz Biotechnology and the anti-V5 (R960-25, dilution 1:5000) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Inhibition, Fluorescence, Förster Resonance Energy Transfer, Control, Activity Assay, Staining

Journal: Nature Communications

Article Title: Unconventional structure and mechanisms for membrane interaction and translocation of the NF-κB-targeting toxin AIP56

doi: 10.1038/s41467-023-43054-z

Figure Lengend Snippet: a V5 plus His-tagged AIP56 modified in the aspartate-rich motif (AIP56 D274S/D276-278S and AIP56 D274N/D276-278N ) is unable to cleave p65 in intact cells. Cleavage of p65 was assessed by western blotting and protein loading by Ponceau S staining. The result shown is representative of six ( n = 6) independent experiments. b V5 plus His-tagged AIP56 D274S/D276-278S and AIP56 D274N/D276-278N were unable to translocate across the host cell membrane in response to acidification. In all experiments, mock-treated cells were used as controls. NF-kB p65 cleavage was analyzed by western blotting. The result shown is representative of five ( n = 5) independent experiments. The plot shows the quantification of intact NF-kB p65 normalized for Ponceau S. Statistical significance was tested by one-way ANOVA and p values for the individual comparisons were calculated using Tukey’s HSD test. Data are presented as mean values ± SD. Actual p values from left-to-right and upwards for pH 7.0: p < 0.001, p = 0.91, p > 0.99, p = 0.005, p = 0.90, p = 0.81; for pH 4.5: p = 0.02, p > 0.99, p > 0.99, p > 0.99, p = 0.78, p = 0.36; ns non-significant. Open (without concanamycin A) or closed (with concanamycin A) symbols as well as color coding have been added to facilitate the reading of the experimental conditions, as specified below the graph. Samples were derived from the same experiment and the blots processed in parallel. NF-kB p65, nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65; ConcA, concanamycin A (black); AIP56, purple, AIP56 D274S/D276-278S , red; AIP56 D274N/D276-278N , green. c AIP56 D274S/D276-278S and AIP56 D274N/D276-278N retained the ability to interact with black lipid bilayers. Proteins were used at a final concentration of 14 nM. Membrane activity was induced by acidification (pH 4.8; red arrows) of the aqueous phase at the cis-side of the chamber. Each result shown is representative of at least three ( n = 3) independent measurements. Source data for ( b ) and ( c ) are provided in the Source data file.

Article Snippet: The anti-human NF-κB p65 C-terminal domain (clone c-20) rabbit polyclonal antibody (sc-372, dilution 1:3000) was from Santa Cruz Biotechnology and the anti-V5 (R960-25, dilution 1:5000) mouse monoclonal antibody was purchased from Invitrogen.

Techniques: Modification, Western Blot, Staining, Membrane, Derivative Assay, Concentration Assay, Activity Assay

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: BioMed Research International

Article Title: MicroRNA-205-5p Targets HMGB1 to Suppress Inflammatory Responses during Lung Injury after Hip Fracture

doi: 10.1155/2019/7304895

Figure Lengend Snippet: Determination of successful hip fracture in a Sprague-Dawley (SD) rat and of HMGB1 as the target of miR-205-5p. (a) Representative X-ray image of a SD rat model of hip fracture and control (1 : 1). (b) Representative hematoxylin and eosin-stained lung tissue sections and representative tissue sections stained immunohistochemically to detect HMGB1 are shown in the top and bottom panels, respectively (magnification, 40×). (c) Representative fluorescent images of tissues subjected to the TUNEL assay. Green and blue areas represent apoptotic cells and cell nuclei, respectively (magnification, 40×). (d) The relative expression levels of miR-205-5p and HMGB1 and IL-6 mRNA in the hip fracture and control groups were determined using qPCR. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the control. (e) Western blot analysis of the levels of HMGB1, p65, p-IkB α /IkB α , and RAGE. Equal protein loading was verified using GAPDH or Lamin A as an internal control. (f) Relative levels of miR-205-5p and HMGB1 in serum samples from human patients relative to controls. ∗∗ P < 0.01; ∗∗∗ P < 0.001. (g) Correlation analysis between the levels of miR-205-5p and HMGB1 in human serum samples. (h) Predicted conserved miR-205-5p binding sites in HMGB1 mRNA. (i) Luciferase activity analysis of cells transfected with constructs encoding wild-type or mutated HMGB1 in the presence of miR-205-5p or NC. WT-3′UTR (+) indicates wild-type HMGB 3′UTR. WT-3′UTR (-) indicates mutant-3′UTR HMGB1. Mimics indicates miR-205-5p mimics. ∗∗ P < 0.01 vs. the control.

Article Snippet: Equal concentrations of protein per sample were prepared in 20 μ L of loading buffer and separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA) at a constant voltage of 100 V for 90 min and then blocked in a 5% skim milk solution for 2 h. Then the band was cut according to the size of target band and incubated with primary rabbit antibodies specific for RAGE (1 : 1000, ab228861, Abcam), HMGB1 (1 : 4000, ab79823, Abcam), p65 (1 : 1000, ab16502, Abcam), and p-IkB α /IKB α (1 : 1000, ab133462/ab32518, Abcam) in Tris-buffered saline containing Tween-20 (TBS-T) overnight at 4°C.

Techniques: Staining, TUNEL Assay, Expressing, Western Blot, Binding Assay, Luciferase, Activity Assay, Transfection, Construct, Mutagenesis

Journal: BioMed Research International

Article Title: MicroRNA-205-5p Targets HMGB1 to Suppress Inflammatory Responses during Lung Injury after Hip Fracture

doi: 10.1155/2019/7304895

Figure Lengend Snippet: Effects of a transfected miR-205-5p mimic or inhibitor in HPAEpiC cells. (a) The relative expression levels of miR-205-5p and HMGB1 were determined by qPCR. ∗ P < 0.05, ∗∗ P < 0.01, ## P < 0.01 and vs. the other two groups. (b) CCK8 assay of cell proliferation. The results represent the means of three independent experiments. ∗∗ P < 0.01 and # P < 0.05 vs. the other two groups. (c) Western blot analysis of the levels of HMGB1, p65, p-IkB α /IkB α , and RAGE. Equal protein loading was verified using GAPDH or Lamin A as internal controls. (d) The levels of HMGB1 and p65 were determined via immunofluorescence analysis (magnification, LSM 120×). (e) Representative micrographs of EdU-labeled samples from three groups (magnification, 10×) and (f) corresponding fluorescence images of samples stained with Hoechst 33258 (magnification, 20×). (g) Flow cytometric detection of apoptotic cells in samples double-stained with Annexin V-FITC and PI. ∗∗∗ P < 0.001 vs. the other groups. (h) Images of a cell cycle analysis as determined by PI staining and flow cytometry. ∗∗ P < 0.01 vs. the other groups. (i) The concentrations of HMGB1, IL-6, and TNF- α in cell supernatants were determined by ELISA. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the other groups.

Article Snippet: Equal concentrations of protein per sample were prepared in 20 μ L of loading buffer and separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA) at a constant voltage of 100 V for 90 min and then blocked in a 5% skim milk solution for 2 h. Then the band was cut according to the size of target band and incubated with primary rabbit antibodies specific for RAGE (1 : 1000, ab228861, Abcam), HMGB1 (1 : 4000, ab79823, Abcam), p65 (1 : 1000, ab16502, Abcam), and p-IkB α /IKB α (1 : 1000, ab133462/ab32518, Abcam) in Tris-buffered saline containing Tween-20 (TBS-T) overnight at 4°C.

Techniques: Transfection, Expressing, CCK-8 Assay, Western Blot, Immunofluorescence, Labeling, Fluorescence, Staining, Cell Cycle Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: BioMed Research International

Article Title: MicroRNA-205-5p Targets HMGB1 to Suppress Inflammatory Responses during Lung Injury after Hip Fracture

doi: 10.1155/2019/7304895

Figure Lengend Snippet: Effects of HMGB1 administration and siRNA-mediated inhibition in HPAEpiC cells. (a) The relative expression levels of miR-205-5p and HMGB1 were determined by qPCR. The control and siRNA control (siRNA-NC) groups are indicated by black and green bars, respectively. (b) Results of a CCK8 proliferation assay. The results are presented as the means of three independent experiments. ∗∗ P < 0.05 and ## P < 0.05 vs. the other groups. (c) Western blot analysis of the levels of HMGB1, p65, p-IkB α /IkB α , and RAGE. Equivalent protein loading was verified using GAPDH or Lamin A as internal controls. (d) Representative micrographs of EdU-labeled samples from four groups and (e) corresponding fluorescence images of samples stained with Hoechst 33258. (f, h) Apoptotic cells were detected by flow cytometry and double-staining with Annexin V-FITC and PI. ∗∗ P < 0.01 vs. the other groups. (g, i) Images of a flow cytometry-based cell cycle analysis of PI-labeled cells. ∗∗ P < 0.01 and ## P < 0.01 vs. the other groups. (j) ELISA analysis of the levels of HMGB1, IL-6, and TNF- α in culture supernatants. ∗ P < 0.05, ∗∗ P < 0.01, and ## P < 0.01 vs. the other groups.

Article Snippet: Equal concentrations of protein per sample were prepared in 20 μ L of loading buffer and separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA) at a constant voltage of 100 V for 90 min and then blocked in a 5% skim milk solution for 2 h. Then the band was cut according to the size of target band and incubated with primary rabbit antibodies specific for RAGE (1 : 1000, ab228861, Abcam), HMGB1 (1 : 4000, ab79823, Abcam), p65 (1 : 1000, ab16502, Abcam), and p-IkB α /IKB α (1 : 1000, ab133462/ab32518, Abcam) in Tris-buffered saline containing Tween-20 (TBS-T) overnight at 4°C.

Techniques: Inhibition, Expressing, Proliferation Assay, Western Blot, Labeling, Fluorescence, Staining, Flow Cytometry, Double Staining, Cell Cycle Assay, Enzyme-linked Immunosorbent Assay

Journal: BioMed Research International

Article Title: MicroRNA-205-5p Targets HMGB1 to Suppress Inflammatory Responses during Lung Injury after Hip Fracture

doi: 10.1155/2019/7304895

Figure Lengend Snippet: Effects of a transfected miR-205-5p mimic and HMGB1 treatment in HPAEpiC cells. (a) Fluorescence images of cells labeled with Hoechst 33258. (b) Apoptotic cells were detected by flow cytometry after double-staining with Annexin V-FITC and PI. ∗ P < 0.05 vs. the other groups. (c) Images of a flow cytometric cell cycle analysis of PI-labeled cells. ∗∗ P < 0.01 and # P < 0.05 vs. the other groups. (d) Western blot analysis of the levels of HMGB1, p65, p-IkB α /IkB α , and RAGE in cultured cells. (e) ELISA analysis of the concentrations of HMGB1, IL-6, and TNF- α in the culture supernatants. ∗ P < 0.05 and ∗∗ P < 0.01 vs. the other groups.

Article Snippet: Equal concentrations of protein per sample were prepared in 20 μ L of loading buffer and separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at a constant voltage of 80 V. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA) at a constant voltage of 100 V for 90 min and then blocked in a 5% skim milk solution for 2 h. Then the band was cut according to the size of target band and incubated with primary rabbit antibodies specific for RAGE (1 : 1000, ab228861, Abcam), HMGB1 (1 : 4000, ab79823, Abcam), p65 (1 : 1000, ab16502, Abcam), and p-IkB α /IKB α (1 : 1000, ab133462/ab32518, Abcam) in Tris-buffered saline containing Tween-20 (TBS-T) overnight at 4°C.

Techniques: Transfection, Fluorescence, Labeling, Flow Cytometry, Double Staining, Cell Cycle Assay, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Nature Structural & Molecular Biology

Article Title: Telomeric 8-oxo-guanine drives rapid premature senescence in the absence of telomere shortening

doi: 10.1038/s41594-022-00790-y

Figure Lengend Snippet: a , YFP-XRCC1 localization to telomeres indicated by FAP-mCer-TRF1 after 10 min dye + light (DL) treatment of RPE FAP-TRF1 cells. b , Percent YFP-XRCC1 positive telomeres per nucleus after no treatment (UT) or 10 min DL in wild-type or OGG1ko RPE FAP-TRF1 cells. Error bars represent the mean ± s.d. from the indicated number n of nuclei analyzed from a representative experiment. Statistical analysis by one-way ANOVA (*** P < 0.001). Immunoblot for FAP-TRF1 and OGG1 in extracts from RPE FAP-TRF1 wild-type and OGG1ko cells. Arrow indicates nonspecific band stained by anti-OGG1. c – e , Cell counts of BJ ( c ), RPE ( d ) or primary BJ ( e ) FAP-TRF1 cells obtained 4 days after recovery from 5 or 20 min dye (D) and light (L) alone, or in combination (DL) as indicated, relative to untreated cells. f , RPE FAP-TRF1 cell cycle analysis 24 h after no treatment or 5 min D, L, DL, 20 J m –2 UVC, or 1 h with 2.5 or 10 mM KBrO 3 determined by flow cytometry. g , RPE FAP-TRF1 colony formation efficiency 7–10 days after indicted treatment. h , i , Percent β-galactosidase-positive BJ FAP-TRF1 cells obtained 4 days after the indicated treatments; 2.5 mM KBrO 3 and 50 μM ETP treatments were for 1 h. In c – i , error bars represent the mean ± s.d. from the number of independent experiments indicated by the black circles. Statistical significance was determined by one-way ANOVA (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). j , Representative image of 5 min DL-treated BJ FAP-TRF1 β-galactosidase-positive cells. Arrows mark positive cells (turquoise). k , Mitochondrial respiration was examined 24, 48 and 96 h after 5 min D, L or DL. Data are means and error bars are ±95% CI from two independent experiments with seven to eight technical replicates each for BJ and RPE FAP-TRF1 cells.

Article Snippet: Protein concentrations were determined with the BCA assay (Pierce) and 10–30 μg of protein was electrophoresed on 4–12% (or 12% for OGG1ko blot) Bis-Tris gels (Thermo) before transferring to polyvinylidene difluoride membranes (GE Healthcare).

Techniques: Western Blot, Staining, Cell Cycle Assay, Flow Cytometry

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

doi: 10.1164/rccm.201202-0302OC

Figure Lengend Snippet: Laser capture microdissection (LCM) reveals matrix metalloproteinase (MMP)19 overexpression in hyperplastic epithelial cells of idiopathic pulmonary fibrosis (IPF) lungs. (A) Representative histological structures for LCM. Dense fibrotic regions, “Changed” epithelial cells adjacent to fibrotic regions, and “Normal looking” alveolar epithelial cells in nonfibrotic region. (B) Gene expression patterns in different regions with the same patient. The number denotes sample number and indicates samples from all three regions from four patients. (C) MMP gene expression patterns in different regions within the same patient. The number denotes sample number and indicates samples from all three regions from all four patients. Yellow indicates up-regulated genes, purple indicates down-regulated genes, gray indicates unchanged. C = hyperplastic epithelial cells adjacent to fibrotic regions; F = dense fibrotic regions; N = normal-looking alveolar epithelial cells in nonfibrotic regions.

Article Snippet: MMP19 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Laser Capture Microdissection, Over Expression, Gene Expression

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

doi: 10.1164/rccm.201202-0302OC

Figure Lengend Snippet: Up-regulation of matrix metalloproteinase (MMP)19 in idiopathic pulmonary fibrosis (IPF) lungs. (A, B) Intense intracytoplasmic MMP19 labeling is observed in alveolar epithelial cells in IPF lungs (A, 40×; B, 10×). (C) Normal histology lungs showing no staining (40×). (D) IPF lung negative control omitting the primary antibody (40×). (E) ELISA assay of MMP19 protein in bronchoalveolar lavage of IPF lungs and normal volunteer lungs. (F) Western blot analysis of MMP19 in lung homogenates from IPF and normal lungs. (G) Densitometry of Western blot of MMP19 relative to β-actin. Data in E and G represent the mean ± SEM. NL = normal lungs.

Article Snippet: MMP19 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Labeling, Staining, Negative Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

doi: 10.1164/rccm.201202-0302OC

Figure Lengend Snippet: Effect of matrix metalloproteinase (MMP)19 on A549 cell migration. (A) Reverse transcriptase–polymerase chain reaction (qRT-PCR) from normal A549 epithelial cells (N), control SiRNA (C), and cells transfected with MMP19 SiRNA (M). Cells were harvested at 24 hours after transfection, and RNA was extracted and reverse transcribed. (B) Western blot data from parallel experiment as in A. (C, D) Quantification of RT-PCR and Western blot data. *P < 0.05. (E) Photomicrograph of scratch assay. Cells were seeded in triplicate in collagen IV–coated culture dishes at 1 × 105 cells/dish. A scratch through the center axis of the plate was gently made using pipette tip. Migration of the cells into the scratch was observed at time point 0 (upper panels) and 48 hours (bottom panels) after scratch. (F) Induction of A549 cell migration/invasion with MMP19 expression in Matrigel. Five high-power fields were counted for each treatment. Assays were performed in triplicate. Data represent mean ± SEM of five experiments.

Article Snippet: MMP19 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Migration, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Wound Healing Assay, Transferring, Expressing

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

doi: 10.1164/rccm.201202-0302OC

Figure Lengend Snippet: Total and differential cell count and myeloperoxidase activity in bronchoalveolar lavage (BAL). (A) Total cell counts from wild-type (WT) and matrix metalloproteinase (MMP)19-deficient mice of control and bleomycin instilled at 7 days (n = 7). (B) BAL cell profile. E = eosinophils; L = lymphocytes; M = macrophages; N = neutrophils. (n = 7). (C) BAL myeloperoxidase activity of Mmp19−/− and WT mice (n = 4). Results are shown as mean ± SD of two independent experiments. *P < 0.01; **P < 0.02.

Article Snippet: MMP19 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Cell Counting, Activity Assay, Control

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

doi: 10.1164/rccm.201202-0302OC

Figure Lengend Snippet: Bleomycin induced severe pulmonary fibrosis in matrix metalloproteinase (Mmp)19-deficient mice. (A–F) Light micrographs in the lung sections from Mmp19−/− and WT mice. (A, B) Wild-type (WT) mice, 21 days after saline or bleomycin instillation, stained with hematoxylin and eosin (10×). (C) WT mice, 21 days after bleomycin (40×). (D, E) MMP19−/− mice, 21 days after saline or bleomycin instillation, stained with hematoxylin and eosin (10×). (F) Mmp19−/− mice, 21 days after bleomycin (40×). (G, H) Immunostaining of αSMA in the lung sections from Mmp19−/− and WT mice; samples were counterstained with hematoxylin (10×). Inset: positive fibroblasts in a fibroblast focus (40×). Bottom: lung hydroxyproline levels in Mmp19−/− and their WT littermates after bleomycin; results represent mean ± SD from six knockout and WT mice; *P < 0.05.

Article Snippet: MMP19 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Saline, Staining, Immunostaining, Knock-Out

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

doi: 10.1164/rccm.201202-0302OC

Figure Lengend Snippet: Tenascin C is increased in bleomycin-exposed matrix metalloproteinase (Mmp)19-deficient mice. (A) Lungs from control mice and 21-day bleomycin-instilled mice were homogenized in RIPA lysis buffer, and tenascin C was analyzed by Western blot. (B) Densitometric analysis of the bands of tenascin C normalized with β-tubulin. Results are presented as mean ± SD from two independent experiments; *P < 0.01, **P < 0.02. (C, D) Immunohistochemical evaluation of tenascin C in Mmp19+/+ (C) and Mmp19−/− (D) mice. Pictures are representative of three WT and three MMP-19–deficient mice.

Article Snippet: MMP19 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Lysis, Western Blot, Immunohistochemical staining

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

doi: 10.1164/rccm.201202-0302OC

Figure Lengend Snippet: Matrix metalloproteinase (MMP)19 regulation of PTGS2 (prostaglandin–endoperoxide synthase 2) expression in A549 cells. A549 cells were transfected with full-length cDNA or siRNA of MMP19 and harvested 24 hours after the treatment. (A) MMP19 and PTGS2 mRNA levels were measured by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) (MMP19: MMP19+ vs. control, *P = 6.23 × 10−6; MMP19− vs. control, *P = 0.0004; PTGS2: MMP19+ vs. control, *P = 0.0002; MMP19− vs. control, P = 0.04). (B) MMP19 and PTGS2 protein by Western blot analysis in homogenates of A549 cells. (C) Densitometric analysis of the Western blot shown in B. MMP19 versus scramble, *P = 0.03; MMP19+ versus mock, *P = 0.004; PTGS2 (MMP19+) versus mock, *P < 0.007. (D) PTGS2 mRNA levels quantified by qRT-PCR. RNA was extracted from A549 cells 24 hours after transiently transfected with MMP19 that were pretreated with PBS, MMP inhibitors (GM6001 and MMP inhibitors IV), and MMP19 siRNA transfection for 6 hours. Y-axis: fold expression over mock transfection. Data represent mean ± SEM of three experiments. (E) A549 cell migration was attenuated by the MMP inhibitor GM6001 as well as the specific PTGS2 inhibitor, COX-2 inhibitor II, a cell-permeable isoxazolyl-benzenesulfonamide compound; *P < 0.05; **P < 0.01. DMSO = dimethyl sulfoxide.

Article Snippet: MMP19 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Transfection, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control, Western Blot, Migration

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

doi: 10.1164/rccm.201202-0302OC

Figure Lengend Snippet: Matrix metalloproteinase (Mmp)19 is required for PTGS2 (prostaglandin–endoperoxide synthase 2) expression in mice. (A) Western blot analysis of PTGS2 protein in bronchoalveolar lavage from the lungs of Mmp19+/+ and Mmp19−/− mice treated with bleomycin or saline control. (B) Western blot analysis of PTGS2 protein in the lungs of Mmp19+/+ and Mmp19−/− mice treated with bleomycin or saline control. (C) Densitometry analysis of the Western blot of PTGS2 protein in the lungs of Mmp19+/+ and Mmp19−/− mice treated with bleomycin or saline control. Y-axis: the ratio of PTGS2 to β-actin. Data represent mean of three experiments with SEM (P < 0.01).

Article Snippet: MMP19 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Western Blot, Saline, Control

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Matrix Metalloproteinase-19 Is a Key Regulator of Lung Fibrosis in Mice and Humans

doi: 10.1164/rccm.201202-0302OC

Figure Lengend Snippet: PTGS2 (prostaglandin–endoperoxide synthase 2) is increased in patients with idiopathic pulmonary fibrosis (IPF). (A) Western blot analysis of PTGS2 protein in bronchoalveolar lavage from normal volunteers and patients with IPF. (B) Western blot analysis of PTGS2 protein in the lungs from normal volunteers and patients with IPF. (C) Densitometric analysis of the Western blot shown in E. Data represent mean ± SEM of three experiments (P < 0.01). (D, E) Immunostaining of PTGS2 showing strong staining in epithelial cells of IPF lungs (D, 10×; E, 40×). (F) Control lung. (G) Representative images of colocalization of PTGS2 with matrix metalloproteinase (MMP)19 in IPF lungs. Red indicates PTGS2, green indicates MMP19, blue indicates nucleus.

Article Snippet: MMP19 siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Immunostaining, Staining, Control

Journal: Tumour Biology

Article Title: PI3K and MAPK pathways mediate the BDNF/TrkB-increased metastasis in neuroblastoma

doi: 10.1007/s13277-016-5433-z

Figure Lengend Snippet: The molecular targets that mediate BDNF/TrkB effects on TB3 cell migration and invasion in vitro. a TB3 cells cultured in the absence of TET were scratched with a 200-μl pipette tip across the center of the well; then cells were pre-treated with Akt inhibitor perifosine, or mTOR inhibitor rapamycin for 1 h followed by the BDNF treatment for 30 h. Gap closing was photographed. The cell migration rate was calculated as described in “ ” section. Bars , SD. ** P < 0.01, BDNF-treated vs. control. ## P < 0.01, Akt or mTOR pre-treated vs. BDNF-treated. b Migration assay was performed as described in the “ ” section. Representative fields of migrating cells under microscope were shown upper of the figure. The cells that migrated to the underside of the inserts were counted, and Student’s t test was done. Bars , SD. ** P < 0.01, BDNF-treated vs. control. ## P < 0.01, Akt or mTOR pre-treated vs. BDNF-treated. c Invasion assay was performed as described in the “ ” section. Representative fields of invading cells under microscope were shown upper of the figure. The cells that invaded to the underside of the inserts were counted, and Student’s t test was done. Bars , SD. ** P < 0.01, BDNF-treated vs. control. ## P < 0.01, Akt or mTOR pre-treated vs. BDNF-treated. d TB3 cells were pre-treated with Akt inhibitor perifosine, or mTOR inhibitor rapamycin for 1 h followed by the BDNF treatment for 1 h, and then harvested. Total protein was extracted for Western blotting

Article Snippet: Akt inhibitor perifosine was obtained from Selleckchem (Houston, Texas, USA).

Techniques: Migration, In Vitro, Cell Culture, Transferring, Control, Microscopy, Invasion Assay, Western Blot

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) expression increased in human umbilical artery endothelial cells (HUAECs), human umbilical vein endothelial cells (HUVECs), and rat brain microvascular endothelial cells (RBMECs) stimulated with VEGF. (A,B) Endothelial cell (EC) migration distance was evaluated by scratch assay. ECs were treated with a dose curve of RGMa (500–3000 ng/ml), and images were taken at the beginning and 12 h. (C,D) ECs were treated with RGMa (2 µg/ml), and lysates were collected over a time course of 50 min. The amount of phosphorylated focal adhesion kinase (p-FAK) protein was visualized with western blot analysis. (E,F) ECs were treated with VEGF (50 ng/ml), and lysates were collected over a course of 120 min. p-FAK protein levels were visualized with western blot analysis. (G–I) Quantitative real-time polymerase chain reaction showed RGMa mRNA level was upregulated in HUAECs, HUVECs, and RBMECs exposed to VEGF (50 ng/ml) for 30 min. (J–L) RGMa protein levels were visualized in HUAECs, HUVECs, and RBMECs exposed to VEGF at 30 min by western blot analysis. Data in bar graphs represent the means ± SD of ≥4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, # VS 1500 ng/ml, ### P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Migration, Wound Healing Assay, Western Blot, Real-time Polymerase Chain Reaction

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) suppressed VEGF expression, phosphorylation of focal adhesion kinase (FAK), proliferation, migration, and tube formation in ECs. (A,B) Lysate was collected, and VEGF was detected by western blot in human umbilical artery endothelial cells (HUAECs) treated with RGMa (2 µg/ml); (C–E) ELISA kit assay showed VEGFA decreased in endothelial cell (EC)-culture supernatant exposed to RGMa compared with cell-culture supernatant from control group. (F–H) Cell proliferation was evaluated with cell-counting kit-8 and 5-ethynyl-2′-deoxyuridine (EdU) assays (I,J) . RGMa decreased proliferation of ECs stimulated and unstimulated with VEGF. (K,L) FAK (Tyr397) phosphorylation was measured with western blot in HUAECs treated with vehicle, RGMa (2 µg/ml), VEGF (50 ng/ml), or VEGF plus RGMa. (M–P) ECs were grown to 100% confluence, serum-starved overnight, wounded with a sterile pipette tip to remove cells, and treated with control, RGMa, VEGF, or VEGF plus RGMa. Photographs (40×) were taken at 12 h after injury. Wound closure of ≥3 wells was quantified and reported as mean ± SD. (Q–T) Migration activity of ECs treated with RGMa, VEGF, VEGF plus RGMa, or control was measured with transwell assay. Photographs (200×) were taken 18 h after treatment. (U–X) HUAECs were starved overnight, treated as indicated, and seeded into 96-well plates coated with Matrigel. Photographs (40×) were taken at 3 h after treatment. The number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Expressing, Phospho-proteomics, Migration, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Cell Counting, Sterility, Transferring, Activity Assay, Transwell Assay

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited angiogenesis in vitro via neogenin. (A) Human umbilical artery endothelial cells (HUAECs) were transfected with CRISPR/Cas9 neogenin knockout kit and purified with puromycin, then the result of neogenin knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or neogenin gRNA were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa. The focal adhesion kinase (FAK) (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or neogenin gRNA were determined by scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length were analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: In Vitro, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Phospho-proteomics, Migration

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Unc5b is involved in the effect of repulsive guidance molecule a (RGMa) on phosphorylation of focal adhesion kinase (FAK), migration, and tube formation in human umbilical artery endothelial cells (HUAECs). (A) HUAECs were transfected with CRISPR/Cas9 Unc5b knockout kits and purified with puromycin, then the effect of Unc5b knockout was validated with western blot. (B,C) HUAECs transfected with SgRNA or Unc5b gRNA were treated with control, RGMa, VEGF, or VEGF plus RGMa. FAK (Tyr397) phosphorylation was measured with western blot. (D–G) Migration and (H–K) tube formation of HUAECs transfected with SgRNA or Unc5b gRNA were determined with scratch, transwell, and Matrigel tube-formation assays. The relative number of tubes, tube area, and tube length was analyzed with Image J. Scale bar, 100 µm. Data shown are representative of experimental and quantitative results. N ≥ 4 independent experiments. Bars represent mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Phospho-proteomics, Migration, Transfection, CRISPR, Knock-Out, Purification, Western Blot, Control

Journal: Frontiers in Neurology

Article Title: Repulsive Guidance Molecule a Inhibits Angiogenesis by Downregulating VEGF and Phosphorylated Focal Adhesion Kinase In Vitro

doi: 10.3389/fneur.2017.00504

Figure Lengend Snippet: Repulsive guidance molecule a (RGMa) inhibited cytoskeleton reassembly, filopodia, and lamellipodia formation in human umbilical artery endothelial cells (HUAECs) via neogenin and Unc5b. Before the immunofluorescence experiment, HUAECs were treated with vehicle, RGMa, VEGF, or VEGF plus RGMa for 40 min. F-actin was stained with phalloidin conjugated with FITC and phosphorylated focal adhesion kinase (p-FAK) connected with primary antibody was labeled with Alexa Fluor 555 donkey anti-rabbit (H + L) secondary antibody. (A) Immunofluorescence showed the cytoskeleton (green) change and p-FAK (red) distribution. (B) Immunofluorescence showing the cytoskeleton change of HUAECs transfected with SgRNA or neogenin gRNA. (C) Immunofluorescence showed the cytoskeleton change of HUAECs transfected with SgRNA or Unc5b gRNA. The filopodia are indicated as sharp spikes (arrowhead), and lamellipodia (arrow) are indicated as flat intensive staining. The merged images are shown in the upper panels, and the amplified indicated areas are shown in the lower panel for different groups. Photographs were obtained with laser scanning confocal microscopy (Nikon, A1 + R, magnification 400×). Results shown are representative images of ≥4 independent experiments.

Article Snippet: Recombinant human RGMa, recombinant rat RGMa, recombinant human VEGF, and recombinant rat VEGF were obtained from R&D Systems (MN, USA).

Techniques: Immunofluorescence, Staining, Labeling, Transfection, Amplification, Confocal Microscopy