Journal: PLoS ONE

Article Title: Lack of Sik1 in Mouse Embryonic Stem Cells Impairs Cardiomyogenesis by Down-Regulating the Cyclin-Dependent Kinase Inhibitor p57 kip2

doi: 10.1371/journal.pone.0009029

Figure Lengend Snippet: ( A ) Expression profile of genes encoding for CDK inhibitors belonging to the KIP/CIP family ( Cdkn1a , Cdkn1b and Cdkn1c ) measured by quantitative real-time PCR at different time points during cardiomyocyte differentiation of wild-type and sik1 flpflp ES cells, n = 3, *, P<0.05, **, P<0.001 vs. control cells. P values were calculated using a two-tailed, unpaired t test. ( B ) Western blots analysis of CDKIs protein level during in vitro cardiomyocyte differentiation of wild-type and sik1 flpflp ES cells. ( C ) p21, p27 and p57 protein levels were expressed as the ratio between their arbitrary densitometric units (ADU) and b-actin. Densitometry analyses were performed using the ImageQuant 5.2 software (GE Healthcare). ( D ) Expression profile of Cdkn1c gene measured by quantitative real-time PCR at different time points during neuronal differentiation of wild-type and sik1 flpflp ES cells.

Article Snippet: Western blots were performed using antibodies against β-Galactosidase (dilution 1∶2000; Chemicon), p21 (dilution 1∶200; Abcam), p27 (M-197) (dilution 1∶200; Santa Cruz), p57 (H-91) (dilution 1∶200; Santa Cruz) and actin (dilution 1∶10000; Sigma-Aldrich).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Two Tailed Test, Western Blot, In Vitro, Software

Journal: PLoS ONE

Article Title: Lack of Sik1 in Mouse Embryonic Stem Cells Impairs Cardiomyogenesis by Down-Regulating the Cyclin-Dependent Kinase Inhibitor p57 kip2

doi: 10.1371/journal.pone.0009029

Figure Lengend Snippet: (A) Western blot analysis with the anti-p57 antibodies to detect the expression of transgene in three different clones in sik1 flpflp ES cells. (B) Measurement of myosin positive cells in wild-type, sik1 flp/flp and sik1 flp/flp overexpressing p57 EBs by FACS, analyzed at days 0, 5, 6 and 7 of differentiation with the antibody MF20. Data represent results of one out of two independent experiments (C) Q-PCR analysis of terminal cardiac markers ( α-MyHC , Mlc2V , αCach and c TnI ).

Article Snippet: Western blots were performed using antibodies against β-Galactosidase (dilution 1∶2000; Chemicon), p21 (dilution 1∶200; Abcam), p27 (M-197) (dilution 1∶200; Santa Cruz), p57 (H-91) (dilution 1∶200; Santa Cruz) and actin (dilution 1∶10000; Sigma-Aldrich).

Techniques: Western Blot, Expressing, Clone Assay

Journal: Burns & Trauma

Article Title: Adipose-derived stem cell peptide 5 alleviates hypertrophic scarring through targeting pyruvate carboxylase or p50 to coordinate PI3K/AKT/mTOR-autophagy and NF-κB/IL-6 signaling

doi: 10.1093/burnst/tkag040

Figure Lengend Snippet: Effects of ADSCP5 on ROS levels, mitochondrial membrane potential, autophagic flux, and autophagy-related protein expression in HSFs. ( a ) Measurement of intracellular ROS levels. The mean fluorescence intensity was quantified using ImageJ software. Scale bar: 100 μm. ( b ) JC-1 staining observed by Leica STELLARIS STED confocal microscopy: JC-1 aggregates (red fluorescence) indicate normal mitochondrial membrane potential, while JC-1 monomers (green fluorescence) indicate mitochondrial membrane depolarization. The data are presented as the mean ± SD from the mean fluorescence intensity per cell from 5 random fields and three independent experiments ( n = 3). Scale bar: 50 μm. ( c ) Flow cytometry analysis of JC-1 staining. Statistical analysis of fluorescence signal percentages. ( d ) Fluorescence imaging using a Leica STELLARIS STED confocal microscopy system. Autophagic vesicles were characterized by merged fluorescence signals: mCherry single-positive/EGFP-negative puncta (red fluorescence) indicate autolysosomes, while mCherry/EGFP double-positive puncta (yellow fluorescence) indicate autophagosomes. Statistical analysis of autophagic flux. Changes were quantified by measuring the relative abundances of autolysosomes (red) and autophagosomes (yellow). Scale bar: 10 μm. ( e ) Western blot analysis of autophagy-related proteins. Grayscale analysis of the western blot bands was performed using ImageJ software. ( f ) Western blot analysis of the effects of NAC (a ROS scavenger). The data are presented as the mean ± SD from three or four independent experiments ( n = 3 or 4). The mean fluorescence intensity in the control group for ROS detection or JC-1 staining was normalized to 1. The fluorescence intensity in the control group for autophagic flux was normalized to 100%, and the protein expression levels in the control group were normalized to 1. One-way ANOVA followed by Tukey’s multiple comparisons test was used. * P < 0.05, ** P < 0.01. ADSCP5 adipose-derived stem cell peptide 5, Control solvent control, Sc scrambled peptide control

Article Snippet: Cells were treated with ADSCP5 or Sc (25 μM) for 24–48 h. For rescue experiments, an NF-κB inhibitor (10 nM; BAY 11–7082, MCE, USA) or an AKT activator (SC79, 40 μM, prepared in DMSO; T2274, TargetMOI, USA) was applied with or without ADSCP5 for 36 h. The cells were pretreated with the ROS scavenger NAC (N-acetylcysteine; HY-B0215, MedChemExpress) at 1 mM (dissolved in DMSO) for 2 h with or without ADSCP5 for another 36 h.

Techniques: Membrane, Expressing, Fluorescence, Software, Staining, Confocal Microscopy, Flow Cytometry, Imaging, Western Blot, Control, Derivative Assay, Solvent

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: ER β inhibits oxalate-induced oxidative stress via modulating NOX2 expression. (a) The qRT-PCR analysis of NADPH oxidase subunits after knocking down ER β in HK-2 and HKC-8 cells. Cells were transduced with shER β or with shLuc as control after 72 hr incubation, and then treated with 0.75 mM oxalate for 6 hr. (b) Western blot analysis of NOX2 expression in renal cells with/without shER β after 6 hr exposure to 0.75 mM oxalate. (c) Rescue assay using HK-2 and HKC-8 cells with/without shER β . shNOX2 showed partially reversed knockdown of ER β - (shER β -) induced ROS production. All cells were exposed to 0.75 mM oxalate for 6 hr prior to collection for data analysis. Upper panels show representative images of DHE staining, and quantification is shown in the lower left panel. Western blot in the lower right panel shows NOX2 knockdown efficiency. (d) After treating with 0.75 mM oxalate for 6 hr, shNOX2 can partly reverse the silenced ER β -induced H 2 O 2 production in conditioned media. (e) Rescue assay using HK-2 and HKC-8 cells with/wthout shNOX2 showed partially reversed shER β -induced LDH release. All the cells in Figures – were exposed to 0.75 mM oxalate for 6 hr prior to collection for data analysis. For (a), (c), (d), and (e), data are presented as mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: Expressing, Quantitative RT-PCR, Transduction, Control, Incubation, Western Blot, Rescue Assay, Knockdown, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: Mechanism dissection on how ER β regulates NOX2 expression. (a) NOX2 mRNA levels were regulated by E2 or ER β antagonists in HK-2 and HKC-8 cells. Cells were treated for 6 hr with vehicle (control), 0.75 mM oxalate, 0.75 mM oxalate + 10 nM E2, 0.75 mM oxalate + 10 nM E2 + 10 μ M ICI, or 0.75 mM oxalate + 10 nM E2 + 10 μ M PHTPP for q-PCR of NOX2 mRNA levels. Bonferroni's test was applied for statistical analysis of results. (b) Predicted 3 potential EREs located in the 3 kb-NOX2 promoter region. (c) ChIP was performed in HK-2 cells, and only ERE I of the NOX2 promoter could be bound by ER β . (d) ERE I wild-type or mutant of NOX2 promoter reporter constructs were cotransfected with pRL-TK at a ratio of 1000 : 1 into HK-2-shLuc and HK-2-shER β cells. Luciferase reporter assay was performed after 48 hr incubation. For (a) and (d), data shown are mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01. n.s. = not significant.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: Dissection, Expressing, Control, Mutagenesis, Construct, Luciferase, Reporter Assay, Incubation

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: In vivo mouse model data confirm that ER β deficiency promotes renal CaOx crystal deposition via increasing the hepatic oxalate biosynthesis and renal oxidative stress. (a) Diagram describing the injection schedule for mock control, a selective ER β antagonist PHTPP, and glyoxylate (upper panel). Crystal staining (lower panels) showing the deposition of CaOx crystals in kidney tissues of the PHTPP-treated and mock-control mice. CaOx crystal formation was detected by Pizzolato staining. Crystallization in each kidney section was quantified by calculating the ratio (percent) of the area containing crystals to the entire kidney section using ImageJ software. (b) Staining (left panels) and quantification (right panels) results for CaOx crystal deposition as detected by Pizzolato staining in kidney tissues of the ER β KO and their WT female littermate mice. (c) Detection of 24 hr oxalate excretion in urine samples collected from the ER β KO and WT female mice prior to sacrifice. (d) Western blot of 60 μ g liver protein from ER β KO and WT mice probed with affinity-purified rabbit antibody raised against recombinant mouse AGT1 shows lower expression of AGT1 in the ER β KO mice. (e) Fresh-frozen renal tissues were stained for 30 min with DHE. Representative sections from the ER β KO and their WT female littermate mice are shown (left panels). Right bar graphs show quantification of superoxide generation. (f) H 2 O 2 concentration in urine sample from ER β KO and WT female mice. (g) Representative micrographs of NOX2 immunostaining in renal tissue from the ER β KO and their WT littermate mice. The NOX2 protein was mainly located in renal tubular cells. Quantification of NOX2 is shown in the right panel. Scores were classified as 0 to 3, based on the intensity of staining and the percentage of positive cells. (h) Inhibitory effect of apocynin treatment in mice. CaOx crystal formation in ER β KO mice was significantly decreased after in vivo apocynin treatment. (i) Apocynin treatment reduced H 2 O 2 concentration in urine samples from ER β KO mice. For (a)–(c) and (e)–(i), data are presented as mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: In Vivo, Injection, Control, Staining, Crystallization Assay, Software, Western Blot, Affinity Purification, Recombinant, Expressing, Concentration Assay, Immunostaining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: Suppressing ER β activity could increase renal CaOx crystal formation in HLP-induced rat model. (a) A diagram describing the injection schedule for PHTPP/DMSO treatment. (b) Crystal staining showing the deposition area of CaOx crystals in kidney tissues of the rats treated with PHTPP or vehicle control. CaOx crystal formation was detected by Pizzolato staining and imaged by polarized light optical microphotography. (c) The PHTPP-treated rats showed increased urine oxalate excretion compared to vehicle-treated control rats. Prior to sacrifice, we collected and detected 24 hr oxalate excretion in urine of rats treated with PHTPP or vehicle control. (d) IHC staining of AGT1 in liver tissues of the rats treated with PHTPP or vehicle. The PHTPP-treated rats showed decreased AGT1 expression. (e) Detection of 24 hr urine H 2 O 2 levels in the rats treated with PHTPP or vehicle. The PHTPP-treated rats showed higher H 2 O 2 levels. (f) IHC staining of 8-OHdG in kidney tissues of the rats treated with PHTPP or vehicle. The PHTPP-treated rats showed increased 8-OHdG expression. (g) IHC staining of NOX2 in kidney tissues of the rats treated with PHTPP or vehicle. The PHTPP-treated rats showed increased NOX2 expression as compared to control rats. For (b)–(g), data are presented as mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: Activity Assay, Injection, Staining, Control, Immunohistochemistry, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

doi: 10.1155/2019/5305014

Figure Lengend Snippet: Scheme. The schematic illustration of the ER β signal regulation of CaOx crystal formation in the liver and kidney. For the liver part, ER β signaling promotes activity of AGT1, which decreases the biosynthesis of oxalate by converting glyoxylate into glycine decreasing CaOx crystal formation. For the kidney part, ER β has a protective effect in human renal tubular cells against oxalate-induced oxidative stress via suppression of NOX2 (a NADPH oxidase), which finally leads to decreasing CaOx crystal deposition on the damaged cell surface.

Article Snippet: NOX2 antibody was purchased from BosterBio (PA1667, Pleasanton, CA).

Techniques: Activity Assay