Journal: Autophagy

Article Title: Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

doi: 10.1080/15548627.2020.1725378

Figure Lengend Snippet: Snell dwarf mice have a reduction in the levels of CMA substrate CIP2A in the liver and kidney. (A) Representative western blots of whole liver lysate, showing relative protein levels of CIP2A, MYC, and the three PPP2 subunits (the antibody for subunit A recognizes PPP2R1A and PPP2R1B; the antibody for subunit B recognizes PPP2R2A, PPP2R2B, PPP2R2G, PPP2R2D, and the antibody subunit C recognizes PPP2CA and PPP2CB). (B) Western blot of isolated lysosomes from the liver of fed Snell and sibling control mice and ghr KO and sibling control mice injected with leupeptin or PBS control, showing accumulation of CIP2A in lysosomes of Snell and ghr KO mice. (C) Quantification of relative protein levels of CIP2A, MYC, and the PPP2 subunits from the liver, as shown in (A), normalized to the sibling controls. (D) Quantification of relative protein levels of CIP2A, MYC, and the PPP2 subunits from kidney, normalized to the sibling controls. (E & F) Relative mRNA levels of Myc and Cip2a mRNA from liver and kidney, respectively. Gapdh is shown as a control. Error bars are S.E.M. Where applicable, significant t-test results are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following antibodies were used: ACTB (Cell Signaling Technologies; CST, 4967L), PPP2 subunit A (CST, 2039S; recognizes PPP2R1A and PPP2R1B), PPP2 subunit B (CST, 2290S; recognizes PPP2R2A, PPP2R2B, PPP2R2G, PPP2R2D), PPP2 subunit C (CST, 2259S; recognizes PPP2CA and PPP2CB), ENO1 (CST, 3810S), GAPDH (CST, 2118S), ACADL (AbCam, ab196655), MAP1LC3B (CST, 2775S), CTSD (AbCam, ab75852), LAMP1 (AbCam, ab24170), LAMP2A (AbCam, ab125068), CIP2A (Novus Biologicals, NB110-59722 & NB100-68264), MYC (AbCam, ab32072), GFAP (AbCam, ab7260), pS8 GFAP (ThermoFisher, PA5-12991), EEF1A (AbCam, ab37969), MAPT (CST, 46687S).

Techniques: Western Blot, Isolation, Control, Injection

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Acute effects of AβOs on total levels of proteins that regulate mitochondrial dynamics. (A) Mfn1 and (B) DRP1 western blot of lysates from PC12 cells treated with FCCP (10 μM, 2 h) and AβOs (0.5 μM, 1 and 2 h). A dotted line on the western blot indicates different regions of the same gel. (C, D) Quantification of Mfn1 and DRP1 levels normalized to beta actin. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 compared between the control group. One-way ANOVA with the Dunnett's multiple comparisons test was used for all statistical analyses. Mfn1, mitofusin 1; DRP1, dynamin-related protein 1. (n = 4–5 for each group) (original gel blot are provided on ).

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Acute effects of AβOs on immunoreactivity of proteins that regulate mitochondrial dynamics. Representative epifluorescence images of (A) Mfn1 and (B) DRP1 immunoreactivity in PC-12 cells control and treated with AβOs (0.5 μM) for 1 and 2 h. Quantification of (C) Mfn1 and (D) DRP1 immunoreactivity (intensity), under the same experimental conditions. Scale bars: 20 μm. Data are represented as mean ± SEM. *p < 0.05, ***p < 0.001 compared between the control group. One-way ANOVA with the Dunn's multiple comparisons test was used for all statistical analyses. Mfn1, mitofusin 1; DRP1, dynamin-related protein 1. (n=3–6 for each group, N= 56–103) (entire inmunocytochemistry for Mfn1 and DRP1 with control are provided on ).

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: Changes in PGC‐1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model

doi: 10.3389/fphar.2020.00709

Figure Lengend Snippet: Model of alterations induced on SIRT1/PGC-1α pathway by AβOs. (A) . Healthy neuronal conditions were energy depletion and/or decreased catabolic rates can be sensed by SIRT1 promoting the PGC-1α deacetylation (1). Transcription and co-activates of factors like NRF- 1/2 for the expression of nuclear-encoded mitochondrial genes and dynamic mitochondrial proteins, requires of PGC-1α translocation (2). Coordinated mitochondrial dynamics (3, fission/fusion), depends of adequate expression of Mfn1, Drp1. (B) . In AβOs treated neurons, the PGC-1α is unable to be deacetylated and to translocate to the nucleus (4). The expression of key genes is loss (5), and imbalance between fusion and fission to promote the granular mitochondrial phenotype (6) and neuronal death.

Article Snippet: Samples were incubated for 1 h at RT with the following primary antibodies: SIRT1 (mouse 1:300, Novus Biologicals, IF3), PGC-1α (rabbit 1:400, Novus Biologicals, NBP1-04676), Ser-46 SIRT1 (Sigma 1:200, SAB4301426), DRP1 (rabbit 1:200, Novus Biologicals, NB 110-55288), and Mfn1 (rabbit 1:200, Novus Biologicals, NBP1-51841).

Techniques: Expressing, Translocation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: LAD2 mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against MrgX2. (A) Western blotting was performed to determine MrgX2 expression in control and MrgX2 knockdown (KD) cells. (B) shRNA control and MrgX2 KD cells were stimulated with hBD2, hBD3, cortistatin (CST) or C3a and percent degranulation (β-hexosaminidase release) was determined. Data are mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Stable Transfection, Transduction, shRNA, Control, Western Blot, Expressing, Knockdown

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: (A) RBL-2H3 cells stably expressing MrgX2 were stimulated with buffer, hBD2, hBD3 or cortistatin (CST) for 30 min and β-hexosaminidase release was measured. Data shown are representative of 3 similar experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001. RBL-2H3 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca2+ mobilization in response to (B) hBD2, (C) hBD3 or (D) CST was determined. HEK293 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca2+ mobilization in response to (E) hBD3 or (F) CST was determined. Traces shown are representative of 3 individual experiments.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Stable Transfection, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: (A) BMMCs were transiently transfected with HA tagged MrgX2 (solid line) or control plasmid vector (broken line) and MrgX2 receptor expression level was analyzed using flow cytometry. A representative histogram is shown. (B) Control and MrgX2 expressing BMMCs were incubated with DNP specific mouse IgE (1 μg/mL, 16 h). Cells were exposed to buffer (control), CST, hBD3, mCRAMP or DNP-BSA (10 ng/mL) for 30 minutes and β-hexosaminidase release was measured. LAD2 cells were stimulated with mCRAMP and (C) intracellular Ca2+ mobilization or (D) degranulation was determined. Traces are representative of 3 independent experiments. Bar graphs represent mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Flow Cytometry, Incubation

Journal: PLoS ONE

Article Title: The Acute Neutrophil Response Mediated by S100 Alarmins during Vaginal Candida Infections Is Independent of the Th17-Pathway

doi: 10.1371/journal.pone.0046311

Figure Lengend Snippet: Vaginal lavage fluid from uninoculated or inoculated wild-type, IL-23p19 −/− , IL-17RA −/− and IL-22 −/− mice with high PMNs were evaluated for (A) S100A8 and (B) S100A9 concentrations by ELISA. The results are cumulative data of 1 to 3 repeat experiment(s) testing lavage samples collected on day 7 post-inoculation. LF, lavage fluid. SEM, standard error of the mean.

Article Snippet: Briefly, tissues were treated with peroxidase, goat serum, avidin and biotin blocking buffers and then incubated with monoclonal rat anti-mouse S100A8 or S100A9 antibody (10 μg/ml; R&D Systems), monoclonal mouse anti-human AE1/AE3 antibody (epithelial cytokeratin markers, 5 μg/ml; MP Biomedicals, Solon, OH), or isotype controls (rat IgG2a, rat IgG2b and mouse IgG1) overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The Acute Neutrophil Response Mediated by S100 Alarmins during Vaginal Candida Infections Is Independent of the Th17-Pathway

doi: 10.1371/journal.pone.0046311

Figure Lengend Snippet: Vaginal tissue sections from uninoculated or inoculated mice with high PMNs were stained with anti-S100A8 or S100A9 antibodies. Images are shown at ×400 magnification. Arrows represent epithelium positively stained for S100A8 or S100A9. Images show representative results of 15 uninoculated and 30 inoculated animals on day 7 post-inoculation.

Article Snippet: Briefly, tissues were treated with peroxidase, goat serum, avidin and biotin blocking buffers and then incubated with monoclonal rat anti-mouse S100A8 or S100A9 antibody (10 μg/ml; R&D Systems), monoclonal mouse anti-human AE1/AE3 antibody (epithelial cytokeratin markers, 5 μg/ml; MP Biomedicals, Solon, OH), or isotype controls (rat IgG2a, rat IgG2b and mouse IgG1) overnight at 4°C.

Techniques: Staining

Journal: World Journal of Surgical Oncology

Article Title: LAIR-1 overexpression inhibits epithelial–mesenchymal transition in osteosarcoma via GLUT1-related energy metabolism

doi: 10.1186/s12957-020-01896-7

Figure Lengend Snippet: LAIR-1 inhibits Glut1-related glucose uptake in OS cells. a Heatmap showing the levels of differentially expressed mRNAs. b Top 20 KEGG pathway annotation categories for target gene functions of predicted mRNAs. c Selected significantly differentially expressed mRNA-related to EMT in RNA-seq data between two groups, *** P < 0.001. d qPCR validation of differentially expressed EMT-related genes in LV-NC and LV-LAIR-1-overexpressing OS cells, ** P < 0.01. e Glut1 expression analyzed by western blotting. f Immunofluorescence staining of Glut1 in the LV-LAIR-1-overexpressing OS cells. Scale bar = 50 μm. Data were obtained from at least two independent experiments.

Article Snippet: Total protein was extracted using a routine procedure and blotted with the following primary antibodies: LAIR-1 (sc-398141; Santa Cruz Biotechnology), phospho-Foxo1 (Ser256) (84192; Cell Signaling Technology, Danvers, MA, USA), Foxo1 (2880; Cell Signaling Technology), phospho-Akt (Ser473) (AF8355; Affinity Biosciences, Cincinnati, OH, USA), Akt (9272; Cell Signaling Technology), proliferating cell nuclear antigen (PCNA; BM0104; Boster Biotech Co., Ltd., Wuhan, China), Twist1 (ab50581; Abcam, Cambridge, UK), Glut1 (NB110-39113, Novus Biologicals, Littleton, CO, USA), and β-actin (30101ES50; Yeasen Biotech Co., Ltd., Shanghai, China).

Techniques: RNA Sequencing, Biomarker Discovery, Expressing, Western Blot, Immunofluorescence, Staining