Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) Immunofluorescence of SPARC and immunohistochemistry of TNC and S100A4 in mouse uteri on D4 (n=5), D4.5 (n=5), D5 (n=5), and D5.5 (n=5) of pregnancy. LE, luminal epithelium; St, stroma; * Embryo. Scale bar, 50 μm. ( B ) Western blot analysis of α-SMA, SPARC, TNC protein level under in vitro decidualization (EP) for 24 hr. *, p<0.05; **, p<0.01; ***, p<0.001. Figure 1—source data 1. Raw data of all western blots from . Figure 1—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Immunofluorescence, Immunohistochemistry, Western Blot, In Vitro

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) Western blot analysis on the effects of TNC on decidualization markers (BMP2, WNT4, E2F8 and CYCLIN D3) after stromal cells were treatment with TNC for 72 hr. ( B ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with TNC for 72 hr. ( C ) Western blot analysisof the effects of S100A4 on decidualization markers after stromal cells were treated with S100A4 for 72 hr. ( D ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with S100A4 for 72 hr. ( E ) Western blot analysis on the effects after stromal cells were treated with SPARC for 72 hr. ( F ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated SPARC for 72 hr. ( G ) Western blot analysis on ACTIVIN A protein levels in mouse uteri on D4, D4.5, PD4, and PD4.5, respectively. ( H ) Western blot analysis on the effects of ACTIVIN A on decidualization markers after stromal cells were treated with ACTIVIN A for 72 hr. ( I ) QPCR analysis of Prl8a2 mRNA level after mouse stromal cells were treated with ACTIVIN A for 72 hr. ( J ) Western blot analysis on the effects of ACTIVIN A on decidualization markers after stromal cells were treated with ACTIVIN A for 48 hr under in vitro decidualization. EP, 17β-estradiol+progesterone. All data were is presented as means ± SD. *, p<0.05; **, p<0.01; ***, p<0.001. CYC D3: CYCLIN D3; ACT-A: ACTIVIN A. Figure 2—source data 1. Raw data of all western blots from . Figure 2—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Western Blot, In Vitro

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet: ( A ) AA concentration in uterine luminal fluid flushed on D3 (n=20 mice), D4 (n=20 mice), and D4.5 (n=20 mice) of pregnancy. ( B ) P-CPLA2α immunofluorescence in mouse uteri on D4 (n=6) and D4.5 (n=6). * Embryo. Scale bar = 50 μm. ( C ) P-CPLA2α immunofluorescence in mouse uteri at implantation sites and inter-implantation sites on D5 (n=6 mice). * Embryo. NI, inter-implantation site; IS, implantation site. Scale bar = 50 μm. ( D ) p-CPLA 2α immunofluorescence of in mouse uteri 12 and 24 h after delayed implantation was activated by estrogen treatment, respectively (n=4 mice). * Embryo. Scale bar = 0 μm. ( E ) Western blot analysis of CPLA 2α and P-CPLA 2α protein levels in mouse uteri on D4, D4.5 PD4 and PD4.5 (n=4 mice), respectively. ( F ) Western blot analysis of CPLA 2α and P-CPLA 2α protein levels in mouse uteri 12 and 24 hr after delayed implantation was activated by estrogen treatment (n=4 mice). ( G ) Immunostaining of TNC and S100A4 in mouse uteri 12, 24, 36, and 48 hr after delayed implantation was activated by estrogen treatment (n=4 mice). * Embryo. ( H ) Western blot analysis α-SMA, TNC, and SPARC protein levels in mouse uteri on D4 and PD4 (n=4 mice). *, p<0.05; **, p<0.01; ***, p<0.001. Figure 5—source data 1. Raw data of all western blots from . Figure 5—source data 2. Complete and uncropped membranes of all western blots from .

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Concentration Assay, Immunofluorescence, Western Blot, Immunostaining

Journal: eLife

Article Title: Embryo-derive TNF promotes decidualization via fibroblast activation

doi: 10.7554/eLife.82970

Figure Lengend Snippet:

Article Snippet: Stromal cells were treated with TNC (5, 50 and 500 ng/ml, 3358-TC-050, R&D systems), S100A4 (50 and 500 ng/ml, HY-P71084, MedChemExpress), SPARC (1 and 10 μM, HY-P71086, MedChemExpress), AA (0.2, 2 and 20 μM, A3611, Sigma-Aldrich), PGI analogue ILOPROST (0.1, 1 and 10 μM, HY-A0096, MedChemExpress), SELEXIPAG (0.1, 1 and 10 μM, HY-14870, MedChemExpress), PPARδ agonist GW501516 (0.1, 1 and 10 μg/ml, 317318-70-0, Cayman Chemical, Ann Arbor, MI), COX-2 antagonist NS 398 (20 and 40 μM, S8433, Selleck, Shanghai, China), PPARδ antagonist GSK0660 (40 μM, 1014691-61-2, Selleck), and ACTIVIN A (1, 10 and 100 ng/ml, HY-P70311, MedChemExpress) in DMEM/F12 containing 2% cFBS, respectively.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Software

Journal: The Journal of Neuroscience

Article Title: Blockade of Astrocytic Calcineurin/NFAT Signaling Helps to Normalize Hippocampal Synaptic Function and Plasticity in a Rat Model of Traumatic Brain Injury

doi: 10.1523/jneurosci.1930-15.2016

Figure Lengend Snippet: Figure7. AAV-Gfa2-VIVITdoesnotalterGFAPorIba1levels,butcausesanincreaseinhevin levels.RepresentativeWesternblots(A,C)andmean SD.GFAPandIba1proteinlevels(B,D) in the contralateral and ipsilateral hippocampus of AAV-treated rats at 7 d after CCI. Note that both glial markers showed a significant increase in the hippocampus of the ipsilateral hemi- sphere, but were not significantly altered by pretreatment with AAV-Gfa2-VIVIT. #p 0.001 ipsilateralversuscontralateral,Fisher’sPLSD.n5–6rats,group.E–H,RepresentativeWest- ern blots (E, G) and mean SD SPARC and hevin protein levels (F, H) in the contralateral and ipsilateralhippocampusofAAV-treatedratsat7dafterCCI.Novirusorinjury-dependenteffects wereobservedforSPARC.Incontrast,hevinwassensitivetobothinjuryandAAVtreatment.In bothAAVgroups,hevinwaselevatedintheipsilateralrelativetothecontralateralhemisphere. OverallhevinlevelsweregreaterintheVIVIT-treatedgroupregardlessofhemisphere,butwere highest in the injured hemisphere. *p 0.05; #p 0.001 ipsilateral versus contralateral, Fisher’s LSD, n 5–6 rats.

Article Snippet: Westerns were performed with the following primary antibodies: GFAP (Cell Signaling Technology, catalog #3670S), Iba1 (Wako, catalog #019-19741), PSD-95 (Cell Signaling Technology, catalog #3450S), synapsin-1 (Cell Signaling Technology, catalog #6710S), GluR1 (Millipore, catalog #AB1504), NR2A (Millipore 07-632), NR2B (Millipore, catalog #05-920), CNA (Millipore, catalog #07-1492), GAPDH (Abcam, catalog #ab9484), hevin (R&D Systems, catalog #MAB2836), and SPARC (R&D Systems, catalog #MAB942).

Techniques:

Journal: Febs Letters

Article Title: miRNA ‐29 regulates epidermal and mesenchymal functions in skin repair

doi: 10.1002/1873-3468.70051

Figure Lengend Snippet: miRNA‐29 regulates ECM, proliferation, and adhesion through SPARC, FERMT2, and COL4. (A) Selected miRNA‐29 target genes represented in ECM and cell–cell adhesion cluster were confirmed by qPCR in human primary DF following inhibition of miRNA‐29 (magenta) vs. control (blue). (B) Expression of SPARC and FERMT2 was assessed by the western blot with tubulin as a loading control in DF transfected with miRNA‐29 inhibitors (abc) or control oligo (nsa). (C) Representative images for FERMT2 (green), miRNA‐29 inhibitors (abc) or control (nsa) antisense oligos in red (ASO) and nuclei (DAPI, blue). Scale bar = 25 μm. (D) Quantification of FERMT2 expression from western blots at 5, 48, and 72 h post‐transfection, N = 3, unpaired t ‐test. * P < 0.05 or ** P < 0.01. (E and F) Representative images (E) and quantification of collagen IV (F) using 10 images per condition. Unpaired t ‐test, ** P < 0.01. Scale bar = 35 μm. (G) Soluble SPARC was quantified by ELISA in the medium conditioned by miRNA‐29 inhibitors (abc) and control (nsa) inhibitors. Graph shows direct ELISA results where medium samples were diluted for precise quantification. The actual levels of SPARC are shown as numbers next to the graph. Both one‐way and two‐way ANOVA followed by Šídák's multiple comparison tests were performed, showing a highly significant increase in secreted SPARC, **** P < 0.0001. (H) Adhesion of keratinocytes was quantified on the increasing concentrations of conditioned media from miRNA‐29 inhibitors (abc) or control (nsa)‐treated fibroblasts. N = 3, unpaired t ‐test, * P < 0.05 or ** P < 0.01. The absorbance value coming from the PrestoBlue reagent corresponds to the number of alive adherent cells. (I) Interaction between proposed molecular players of enhanced keratinocyte adhesion regulated through miRNA‐29. BK – basal keratinocyte, BM – basal membrane, ECM – extracellular matrix, f – fibroblasts, and miRNA‐29 inhibitors – antisense oligonucleotides (ASO). Error bars indicate standard deviation of the mean.

Article Snippet: ELISA for human SPARC was performed using a ready‐to‐go sandwich assay kit with positive controls purchased from R&D Systems and following the manufacturer's guidelines.

Techniques: Inhibition, Control, Expressing, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Direct ELISA, Comparison, Membrane, Standard Deviation

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 1. Quantification of TSP-1, TSP-2, TNC, and SPARC mRNA expression in human fetal and adult adrenal glands. A, qRT-PCR was performed on RNA isolated from human fetal (14–23 wk, n 7) and adult adrenals (n 4). TSP-1 (open circle), TSP-2 (black triangle), TNC (X), and SPARC (black square) mRNA expression normalized to GUS, an endogenous control. The human adult brain serves as a positive control for expression of the matricellular proteins (values are shown on log scale). B, SPARC mRNA levels in fetal and adult adrenals. *, P 0.005. C, Western blot analysis of SPARC protein in human fetal and adult adrenals. Immunoblot was performed using tissue lysate of a 20-wk human fetal adrenal and human adult adrenal tissue lysate from a commercial source (Pierce). Protein (10 g/lane) was loaded in right two lanes. Human SPARC protein (10 ng) was used as a positive control (PC). Equal loading of protein was confirmed by probing for actin. Densitometric analysis showed 2.8-fold higher expression of SPARC protein in the fetal adrenal than the adult. The blot shown is representative of two experiments with similar results.

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Control, Positive Control, Western Blot

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 4. Effects of forskolin and 8-Br-cAMP on SPARC mRNA levels. FZ cells isolated from human fetal adrenals were exposed to forskolin (F; 1 M) or 8-Br-cAMP (Br; 1 mM) for 48 h. GUS-normalized data (mean SE) are from four independent experiments using different fetal adrenals. *, P 0.05, **, P 0.01 vs. unstimulated, time- matched controls (Co).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Isolation

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 3. A, Dose-dependent effect of ACTH on SPARC and P450c17 mRNA levels. Isolated human fetal adrenal cortical cells (FZ cells) were treated with various concentrations of ACTH for 48 h. Total RNA was extracted and analyzed by qRT-PCR. Constitutively expressed GUS mRNA levels served as normalization controls. Data shown are mean SE of three independent experiments on cells derived from different fetuses. SPARC and P450c17 mRNA levels were increased after exposure to ACTH for 48 h in a dose-dependent manner (P 0.05 and P 0.01, respectively, based on Friedman’s ANOVA). B, Time- dependent effect of ACTH on SPARC mRNA levels. Isolated FZ cells (left panel) or DZ cells (right panel) were treated with ACTH (1 nM) for 24 or 48 h. Each bar represents mean SE of four independent experiments using different fetal adrenals. Black and white bars indicate ACTH-treated and time-matched, unstimulated control cells, respectively. *, P 0.05 vs. control (without ACTH treatment).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Isolation, Quantitative RT-PCR, Derivative Assay, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 2. SPARC protein expression in the human fetal adrenal gland. A, Immunofluorescence of a 22-wk gestation human fetal adrenal gland showing SPARC staining restricted to the FZ. Note the lack of staining in the narrow band corresponding to the DZ. B, Labeling for SPARC (red) and CD56 (green), a DZ cell marker, in a 21-wk human fetal adrenal gland, illustrating the lack of SPARC immunoreactivity in the DZ. C and D, Labeling for SPARC and LDL-R, a FZ cell marker, on an 18-wk human fetal adrenal gland, illustrating FZ-specific lo- calization of SPARC. Note costaining for LDL-R on the membrane (red) and SPARC protein (green) in the cytoplasm in FZ cells. Original magnification, 100 (A and C) and 200 (B and D).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Marker, Membrane

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 6. Zonal expression of mRNAs encoding SPARC, ACTH-R, LDL-R, and P450c17. Outer, DZ and inner, FZ cells in the midges- tation HFA (18–22 wk) were collected using laser capture microdis- section. Total RNA was extracted from cells of the respective zones and analyzed by qRT-PCR as described in Materials and Methods. GUS-normalized data are shown. SPARC, ACTH-R, LDL-R, and P450c17 mRNA levels were observed primarily in the FZ (black bars), compared with those of the DZ (white bars).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Expressing, Quantitative RT-PCR

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Differential zonal expression and adrenocorticotropin regulation of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, in the midgestation human fetal adrenal gland: implications for adrenal development.

doi: 10.1210/jc.2005-2514

Figure Lengend Snippet: FIG. 5. Western blot analysis of SPARC protein in cultured human fetal adrenal cortical cells (FZ cells) and conditioned medium. A, The cells were incubated with basal media or in the presence of different concentrations of ACTH (0.001–10 nM) for 48 h. Total cell protein was analyzed by immunoblot. Fifty micrograms of protein were loaded per lane. Data (mean SE) are the densitometric units of SPARC relative to actin from three experiments using different fetal adrenals. The relative ratio of unstimulated cells (control) is arbitrarily presented as 1. SPARC protein levels in cell lysates were increased after expo- sure to ACTH for 48 h in a dose-dependent manner (P 0.05, based on Kruskal-Wallis ANOVA). A representative blot from a 21-wk-old human fetal adrenal is shown above. B, Exposure to 8-Br-cAMP (Br; 1 mM) for 48 h increased abundance of SPARC protein in cell lysates (50 g protein/lane), mimicking the effects of ACTH (Ac; 1 nM). The summary of densitometric evaluation of three independent experi- ments is shown below. Data (mean SE) are the densitometric units of SPARC relative to actin, and the relative ratio of the control (Co; unstimulated cells) is arbitrarily presented as 1. A representative blot from a 19-wk-old human fetal adrenal is shown above. C, Exposure to 8-Br-cAMP (Br; 1 mM) for 48 h increased SPARC secretion into the conditioned medium, mimicking the effects of ACTH (Ac; 1 nM). Total protein (150 g protein) was loaded in each lane, and equal loading was confirmed by the Ponceau S staining. Densitometry is shown for three experiments using different fetal adrenals, with a representa- tive blot from a 22-wk-old human fetal adrenal. The relative ratio of the control (Co; unstimulated cells) is arbitrarily presented as 1. *, P 0.05, **, P 0.01, based on Mann-Whitney U test in comparison with respective control (unstimulated cells).

Article Snippet: Primary antibody incubation was performed with a 1:50 dilution of goat antihuman SPARC polyclonal antibody (R&D Systems, Minneapolis, MN), a combination of the anti-SPARC antibody and a 1:30 dilution of rabbit antihuman LDL-R (Research Diagnostics, Flanders, NJ), or a combination of the anti-SPARC antibody and 1:10 dilution of mouse anti-CD56 monoclonal antibody (Leu-19; BD Biosciences, San Jose, CA), for 1 h at room temperature.

Techniques: Western Blot, Cell Culture, Incubation, Control, Staining, MANN-WHITNEY, Comparison

Journal: Journal of cell science

Article Title: Autocrine control of glioma cells adhesion and migration through IRE1α-mediated cleavage of SPARC mRNA.

doi: 10.1242/jcs.099291

Figure Lengend Snippet: Fig. 7. IRE1a-mediated Sparc mRNA controls U87 architectural structure. (A) EV and DN_IRE1 cells were subjected to SPARC silencing by siRNA or non-target luciferase (GL2) silencing as a control. SPARC protein levels and FAK phosphorylation were evaluated by western blotting. Tubulin (Tub) was used as a loading control. (B,C). Relative quantification of SPARC protein levels and FAK phosphorylation. Values were normalized to tubulin levels. (D) EV and DN_IRE1 cells were subjected to SPARC silencing by siRNA or non-target luciferase (GL2) silencing as a control and were assessed for RhoA activation. (*P,0.05; **P,0.01).

Article Snippet: Antibodies against P-FAK and fibronectin were purchased from BD Transduction Laboratory (Oxford, UK), alpha-tubulin from Sigma (St Louis, MO, USA), RhoA from Santa Cruz Biotechnology (Santa Cruz, CA) and SPARC from Cell Signaling Technology (Danvers, MA).

Techniques: Luciferase, Control, Phospho-proteomics, Western Blot, Quantitative Proteomics, Activation Assay

Journal: Journal of cell science

Article Title: Autocrine control of glioma cells adhesion and migration through IRE1α-mediated cleavage of SPARC mRNA.

doi: 10.1242/jcs.099291

Figure Lengend Snippet: Fig. 9. Schematic representation of the mode of action of IRE1a signaling in gliomas leading to control of cell proliferation and migration. U87 wild-type cancer cells can still proliferate under challenging conditions through enhanced adaptability. In contrast, when IRE1a signaling is impaired, cell proliferation capacity is decreased, which is associated to the post- transcriptional derepression of Sparc mRNA expression (1 and 2). Secreted SPARC (3) will in turn, interact with the extracellular matrix (4) and consecutively enhanced cell migration, stress fiber formation and focal adhesion number through RhoA-dependent mechanisms (5). ECM, extracellular matrix; ER, endoplasmic reticulum; FA, focal adhesion.

Article Snippet: Antibodies against P-FAK and fibronectin were purchased from BD Transduction Laboratory (Oxford, UK), alpha-tubulin from Sigma (St Louis, MO, USA), RhoA from Santa Cruz Biotechnology (Santa Cruz, CA) and SPARC from Cell Signaling Technology (Danvers, MA).

Techniques: Control, Migration, Expressing

Journal: Aging Cell

Article Title: Hevin Promotes Aging‐Related Cardiac Dysfunction via Facilitating Cardiac Inflammation in Male Mice

doi: 10.1111/acel.70369

Figure Lengend Snippet: Hevin elevates in aging mouse circulation and correlates with cardiac function. (A) Human circulating Hevin levels were measured from young adults to nonagenarians with the SomaScan aptamer technology and analyzed using a public database. (B) Serum Hevin levels were measured in young and aging mice using a commercial kit ( n = 18). (C, D) Pearson's correlation between serum Hevin levels and serum NT‐proBNP levels or FS in aging mice ( n = 18). (E) Human circulating Hevin levels were measured from young participants (< 60 years old) and old participants (> 60 years old) using a commercial kit ( n = 18). (F, G) Pearson's correlation between serum Hevin levels and serum NT‐proBNP levels or ejection fraction in old participants ( n = 18). (H) mRNA level of Hevin in different organs in mice ( n = 6). (I) Hevin expression in 3 T3‐L1 cells during differentiation ( n = 6). All data are expressed as the mean ± S.D. and analyzed using one‐way ANOVA followed by Tukey post hoc test. * p < 0.05 versus the matched group.

Article Snippet: Recombinant mouse Hevin (#4547‐SL) was purchased from R&D system (Minneapolis, Minnesota, USA).

Techniques: Expressing

Journal: Aging Cell

Article Title: Hevin Promotes Aging‐Related Cardiac Dysfunction via Facilitating Cardiac Inflammation in Male Mice

doi: 10.1111/acel.70369

Figure Lengend Snippet: Exposure to Hevin promotes aging‐related cardiac inflammation and dysfunction. (A) Representative images and quantitative results of SA‐β gal staining in hearts from young and aging mice with or without Hevin administration ( n = 6). (B) Relative telomere length in hearts ( n = 6). (C) Relative lipofuscin accumulation in hearts ( n = 6). (D‐E) The immunofluorescent staining and quantitative results of CD68 and F4/80 in murine hearts ( n = 6). (F‐G) Echocardiographic and hemodynamic parameters of cardiac function in mice, including FS, LVIDd and LVIDs in mice ( n = 6). (H) Tissue Doppler imaging was employed to measure E/A and E/E' to evaluate the diastolic function ( n = 6). (I) Representative images and quantitative results of WGA staining in hearts ( n = 6). (J) Heart weight‐to‐Tibia length (HW/TL) in mice ( n = 6). (K) Representative images and quantitative results of PSR staining in hearts ( n = 6). All data are expressed as the mean ± S.D., and analyzed using one‐way ANOVA followed by Tukey post hoc test. * p < 0.05 versus the matched group.

Article Snippet: Recombinant mouse Hevin (#4547‐SL) was purchased from R&D system (Minneapolis, Minnesota, USA).

Techniques: Staining, Imaging

Journal: Aging Cell

Article Title: Hevin Promotes Aging‐Related Cardiac Dysfunction via Facilitating Cardiac Inflammation in Male Mice

doi: 10.1111/acel.70369

Figure Lengend Snippet: Hevin knockout alleviates aging‐related cardiac inflammation and dysfunction. (A) Representative images and quantitative results of SA‐β gal staining in hearts from young and aging mice ( n = 6). (B) Relative telomere length in hearts ( n = 6). (C) Relative lipofuscin accumulation in hearts ( n = 6). (D‐E) The immunofluorescent staining and quantitative results of CD68 and F4/80 in murine hearts ( n = 6). (F‐G) Echocardiographic and hemodynamic parameters of cardiac function in mice, including FS, LVIDd and LVIDs in mice ( n = 6). (H) Tissue Doppler imaging was employed to measure E/A and E/E' to evaluate the diastolic function ( n = 6). (I) Representative images and quantitative results of WGA staining in hearts ( n = 6). (J) HW/TL ratio in mice ( n = 6). (K) Representative images and quantitative results of PSR staining in hearts ( n = 6). All data are expressed as the mean ± S.D., and analyzed using one‐way ANOVA followed by Tukey post hoc test. * p < 0.05 versus the matched group.

Article Snippet: Recombinant mouse Hevin (#4547‐SL) was purchased from R&D system (Minneapolis, Minnesota, USA).

Techniques: Knock-Out, Staining, Imaging

Journal: Aging Cell

Article Title: Hevin Promotes Aging‐Related Cardiac Dysfunction via Facilitating Cardiac Inflammation in Male Mice

doi: 10.1111/acel.70369

Figure Lengend Snippet: Hevin administration stimulates CCL5 expression. (A) Heart samples from aging mice with Hevin or saline administration based on RNA‐seq analysis. (B) KEGG pathway analysis of RNA‐seq. (C) GSEA of TNF signaling pathway. (D) Volcano map of significantly different genes in the heart tissues from mice in 20 M with Hevin or saline administration based on RNA‐seq analysis. (E) Cardiac CCL5 levels were measured in mice hearts using a commercial kit ( n = 6). (F) Representative images and quantitative results of CCL5 staining in hearts ( n = 6). (G) Pearson's correlation between serum Hevin levels and cardiac CCL5 levels in aging mice with Hevin administration ( n = 12). (H) Pearson's correlation between serum NT‐proBNP levels and cardiac CCL5 levels in aging mice with Hevin administration ( n = 12). (I) Pearson's correlation between FS and cardiac CCL5 levels in aging mice with Hevin administration ( n = 12). All data are expressed as the mean ± S.D., and analyzed using one‐way ANOVA followed by Tukey post hoc test. * p < 0.05 versus the matched group.

Article Snippet: Recombinant mouse Hevin (#4547‐SL) was purchased from R&D system (Minneapolis, Minnesota, USA).

Techniques: Expressing, Saline, RNA Sequencing, Staining

Journal: Aging Cell

Article Title: Hevin Promotes Aging‐Related Cardiac Dysfunction via Facilitating Cardiac Inflammation in Male Mice

doi: 10.1111/acel.70369

Figure Lengend Snippet: Hevin exacerbates age‐related cardiac inflammation and dysfunction by inducing CCL5. (A) Quantitative results of SA‐β gal staining in hearts from aging mice ( n = 6). (B) Cardiac CXCL2 levels were measured in mice hearts using a commercial kit ( n = 6). (C) Cardiac CXCL1 levels were measured in mice hearts using a commercial kit ( n = 6). (D) Cardiac CCL2 levels were measured in mice hearts using a commercial kit ( n = 6). (E‐G) Echocardiographic and hemodynamic parameters of cardiac function in mice, including FS, LVIDd, and LVIDs in mice ( n = 6). (H) Tissue Doppler imaging was employed to measure E/A to evaluate the diastolic function ( n = 6). (I‐K) Representative images and quantitative results of WGA and PSR staining in hearts ( n = 6, differences between saline groups were not statistically evaluated). All data are expressed as the mean ± S.D., and analyzed using one‐way ANOVA followed by Tukey post hoc test. * p < 0.05 versus the matched group.

Article Snippet: Recombinant mouse Hevin (#4547‐SL) was purchased from R&D system (Minneapolis, Minnesota, USA).

Techniques: Staining, Imaging, Saline

Journal: Aging Cell

Article Title: Hevin Promotes Aging‐Related Cardiac Dysfunction via Facilitating Cardiac Inflammation in Male Mice

doi: 10.1111/acel.70369

Figure Lengend Snippet: CCL5 promotes macrophage polarization in the aging heart. (A) Schematic protocol for Hevin treatment in vitro. (B) Representative pictures of SA‐β gal‐stained cell ( n = 6). (C) Western blot images of p16, p19, and p21 in NRCMs ( n = 6). (D) Relative Ccl5 and Mmp9 mRNA levels in RAW264.7 ( n = 6). (E‐G) TNF‐α, IL‐6, and IL‐1β levels were measured in RAW264.7 using a commercial kit ( n = 6). (H) Representative gating scheme for identification of M1‐like (CD86 + CD206−) and M2‐like (CD86 − CD206+) macrophages in RAW264.7 ( n = 6). (I) Representative image of CD86 and CD68 staining in RAW264.7 ( n = 6). (J) Representative image of CD68 and F4/80 staining in hearts ( n = 6). (K) Cardiac CCL5 levels were measured in mice hearts using a commercial kit ( n = 6). (L) The immunofluorescent staining and quantitative results of CD68 + CCL5+ cells in murine hearts ( n = 6). All data are expressed as the mean ± S.D., and analyzed using one‐way ANOVA followed by Tukey post hoc test. * p < 0.05 versus the matched group.

Article Snippet: Recombinant mouse Hevin (#4547‐SL) was purchased from R&D system (Minneapolis, Minnesota, USA).

Techniques: In Vitro, Staining, Western Blot

Journal: Aging Cell

Article Title: Hevin Promotes Aging‐Related Cardiac Dysfunction via Facilitating Cardiac Inflammation in Male Mice

doi: 10.1111/acel.70369

Figure Lengend Snippet: Hevin regulates CCL5 expression through binding to TLR4 and activation of the p65. (A) Representative pictures and quantitative results of SA‐β gal‐stained cells ( n = 6). (B) Western blot images of p16, p19, and p21 in NRCMs ( n = 6). (C) Cardiac CCL5 levels were measured in mice hearts using a commercial kit ( n = 6). (D) Western blot images of p65 and P‐p65 in hearts ( n = 6). (E) Quantitative results of SA‐β gal staining in hearts ( n = 6). (F) Tissue doppler imaging was employed to measure E/A to evaluate the diastolic function ( n = 6). (G‐H) Echocardiographic and hemodynamic parameters of cardiac function in mice, including FS, LVIDd, and LVIDs in mice ( n = 6). (I, J) Quantitative results of WGA and PSR staining in hearts ( n = 6). All data are expressed as the mean ± S.D., and analyzed using one‐way ANOVA followed by Tukey post hoc test. * p < 0.05 versus the matched group.

Article Snippet: Recombinant mouse Hevin (#4547‐SL) was purchased from R&D system (Minneapolis, Minnesota, USA).

Techniques: Expressing, Binding Assay, Activation Assay, Staining, Western Blot, Imaging

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Secreted protein acidic and rich in cysteine (SPARC) improves glucose tolerance via AMP-activated protein kinase activation.

doi: 10.1096/fj.201900453R

Figure Lengend Snippet: Figure 1. Glucose metabolism is impaired in skeletal muscle derived from SPARC-knockout mice. The panels display data concerning 4-h fasting blood glucose and plasma insulin (A), whole-body respiration (B), endurance capacity (C), 2-deoxy- glucose (2-DG) uptake in the absence or presence of insulin (50 nM) in soleus muscle (D), and phosphorylation of AMPK-aThr172

Article Snippet: Separately, recombinant mouse SPARC (R&D Systems, Minneapolis, MN, USA) was intraperitoneally injected at 15 mg/kg bodyweight.

Techniques: Derivative Assay, Knock-Out, Clinical Proteomics, Phospho-proteomics

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Secreted protein acidic and rich in cysteine (SPARC) improves glucose tolerance via AMP-activated protein kinase activation.

doi: 10.1096/fj.201900453R

Figure Lengend Snippet: Figure 3. Effect of exogenous SPARC on glucose uptake (3-OMG) in skeletal muscle from wild-type and AMPK-g3–knockout mice. Glucose uptake (A) and AMPK-aThr172 and ACC2Ser212 phosphorylation (B) in EDL muscle from 4-h unfed wild-type (WT) or AMPK-g3–knockout (g3KO) mice incubated in the absence or presence of SPARC (1 mg/ml). Values of immunoblotting data are expressed as normalized ratio to the basal value of WT mice. Results are presented as means 6 SE, with n = 5–7/group. *P , 0.05 vs. basal.

Article Snippet: Separately, recombinant mouse SPARC (R&D Systems, Minneapolis, MN, USA) was intraperitoneally injected at 15 mg/kg bodyweight.

Techniques: Knock-Out, Phospho-proteomics, Incubation, Western Blot

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Secreted protein acidic and rich in cysteine (SPARC) improves glucose tolerance via AMP-activated protein kinase activation.

doi: 10.1096/fj.201900453R

Figure Lengend Snippet: Figure 2. Acute SPARC treatment improves glucose tolerance in mice. A) Blood glucose concentration after an oral GTT or an ITT. B, C) Respiratory metabolic performance (B) and phosphorylation of AMPK-aThr172 and ACC2Ser212 in gastrocnemius muscle (C) isolated from wild-type (WT) or SPARC-knockout (SPKO) mice treated with saline or recombinant SPARC (15 mg/kg body weight). D) Immunohistochemical staining for biotinylated SPARC in gastrocnemius muscle isolated from wild-type mice treated with saline or biotin-labeled SPARC. Each cryosection was immunohistochemically stained for biotinylated SPARC and dystrophin. White arrows denote identical positions of SPARC immunoreactivity. Scale bars, 50 mm. Values of immunoblotting data are expressed as normalized ratio to the saline condition. Results are presented as means 6 SE, with n = 4–7/group. *P , 0.05, **P , 0.01 vs. saline-treatment. ##P , 0.01 vs. wild-type mice.

Article Snippet: Separately, recombinant mouse SPARC (R&D Systems, Minneapolis, MN, USA) was intraperitoneally injected at 15 mg/kg bodyweight.

Techniques: Concentration Assay, Phospho-proteomics, Isolation, Knock-Out, Saline, Recombinant, Immunohistochemical staining, Staining, Labeling, Western Blot

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Secreted protein acidic and rich in cysteine (SPARC) improves glucose tolerance via AMP-activated protein kinase activation.

doi: 10.1096/fj.201900453R

Figure Lengend Snippet: Figure 4. SPARC-induced improvement in glucose metabolism is mediated via activated AMPK in cultured muscle cells. Glycogen synthesis in hSkMCs incubated in the absence or presence of SPARC (0.2–2 mg/ml) for 60 min (A) or incubated in the presence of SPARC (1 mg/ml) with or without STO-609 and siRNA against AMPK-a1 or AMPK-a2 or CACNB1 (a gene encoding for a subunit of VDCC) (D, E, H). Phosphorylation of AMPK-aThr172 and ACC2Ser222 in hSkMCs incubated in the absence or presence of SPARC (1 mg/ml) for 10 min with or without STO-609 and siRNA against AMPK (siAMPK) and VDCC (siVDCC) (B, F, H). mRNA of GLUT4 and PGC1-a1 in hSkMCs incubated in the absence or presence of SPARC (1 mg/ml) for 60 min (C). Time-dependent changes in intracellular calcium concentrations in C2C12 cells in response to ionomycin or SPARC stimulation (1 mg/ml) with or without EDTA using Fura-2 (G). hSkMCs were prepared using skeletal muscle from 3 healthy volunteers. Values of immunoblotting and quantitative PCR data are expressed as normalized ratio to the basal condition. BS, basal; Ctrl, control; Scr, scramble; SP, SPARC; Sub, subject. Results are presented as means 6 SE, with n = 3 for hSkMCs and n = 6 for C2C12. *P , 0.05, **P , 0.01 vs. basal.

Article Snippet: Separately, recombinant mouse SPARC (R&D Systems, Minneapolis, MN, USA) was intraperitoneally injected at 15 mg/kg bodyweight.

Techniques: Cell Culture, Incubation, Phospho-proteomics, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: Secreted protein acidic and rich in cysteine (SPARC) improves glucose tolerance via AMP-activated protein kinase activation.

doi: 10.1096/fj.201900453R

Figure Lengend Snippet: Figure 5. Chronic SPARC treatment improves glucose tolerance in high-fat (HF) diet–fed mice. Body and tissue weights (A), blood chemistry (B), blood glucose concentration during an oral GTT or an ITT (C), and phosphorylation level of AMPK- aThr172, ACC2Ser212, and AS160Thr642 in gastrocnemius muscle (D) with or without SPARC treatment in mice fed chow or high-fat diet. Values of immunoblotting data are expressed as normalized ratio to the data from chow-fed mice with saline. Results are presented as means 6 SE, with n = 6–9/group. *P , 0.05, **P , 0.01, ***P , 0.001 vs. saline.

Article Snippet: Separately, recombinant mouse SPARC (R&D Systems, Minneapolis, MN, USA) was intraperitoneally injected at 15 mg/kg bodyweight.

Techniques: Concentration Assay, Phospho-proteomics, Western Blot, Saline