Journal: Cancer Research

Article Title: MYC Synergizes with Activated BRAFV600E in Mouse Lung Tumor Development by Suppressing Senescence

doi: 10.1158/0008-5472.can-13-3234

Figure Lengend Snippet: Figure 2. MYC activation suppresses BRAFV600E-induced senescence. A, analysis of p16INK4A expression by IHC in whole lung sections of control (n ¼ 2), vehicle-treated (VT) BMWT/KI (n ¼ 6), tamoxifen (TAM)-treated BMKI/WT (n ¼ 6), and BMKI/KI (n ¼ 3) mice. B, analysis of H3K9me3 and p16INK4A (top two) expression by immunofluorescence using confocal microscopy as indicated. DAPI was used to stain nuclei. Bottom, colocalization of H3K9me3 and p16INK4A

Article Snippet: The following antibodies were used for immunoblot analysis and immunostaining: c-MYC (sc-42), CC-10 (sc-9772), SP-C (sc7706), p16INK4A (sc-1662), p19Arf (sc-1066), p21CIP1 (sc-6246; all from Santa Cruz Biotechnology), ERK1/2 (4695s), and pERK1/2 (4370s) from Cell Signaling Technology, H3K9me3 (07-523, Millipore), Ki67 (SP6, Lab Vision), tubulin (T4026, Sigma), AlexaFluor488 goat anti-rat (A-1106), goat anti-rabbit (A11008) and rabbit anti-mouse (A-11059; all from Invitrogen), and biotinylated rabbit-anti-mouse (E046401-2) from DAKO.

Techniques: Activation Assay, Expressing, Control, Confocal Microscopy, Staining

Journal: Cancer Research

Article Title: MYC Synergizes with Activated BRAFV600E in Mouse Lung Tumor Development by Suppressing Senescence

doi: 10.1158/0008-5472.can-13-3234

Figure Lengend Snippet: Figure 3. Suppression of p16INK4A and p21CIP1 by MYC starts early coinciding with accelerated tumor onset and requires continuous MYC activity. A, schematic representation of the experimental setting to study the role of MYC activation in the early phase of tumorigenesis (top). Low-magnification images (middle) or images at 5 magnification (bottom) of H&E-stained lung sections showing neoplastic lesions and altered morphology in different genotypes and treatments. Representative photomicrographs of a total of 11 cases are presented; vehicle-treated (VT) BMWT/KI (n ¼ 2), tamoxifen (TAM)-treated BMKI/WT (n ¼ 2), and BMKI/KI (n ¼ 2) lungs 14 and 21 days post Ad-Cre inhalation. Vehicle or tamoxifen treatment started in all cases 7 days after Ad-Cre inhalation. B, immunoblot analyses of p16INK4A (top) and p21CIP1 (middle) in whole lung lysates in control, BMWT/KI þ VT, BMKI/WT þ tamoxifen, and BMKI/KI þ tamoxifen as indicated 14 days after Ad-Cre inhalation (7 days after vehicle or tamoxifen treatment). C, schematic representation of the time frame for MYC on/off experiments (top). Low-powered image of H&E-stained lung sections showing adenoma lesions in BMKI/WT mice exposed to tamoxifen for the first 7 days after tamoxifen withdrawal (bottom left) or mice exposed to tamoxifen continuously (bottom right). D, immunoblot analyses of p16INK4A (top) and p21CIP1 (middle) BMKI/WT in mice tamoxifen-treated for 7 days, then discontinued (lanes 1 and 2); BMKI/WT mice under continuous tamoxifen treatment (lanes 3 and 4).

Article Snippet: The following antibodies were used for immunoblot analysis and immunostaining: c-MYC (sc-42), CC-10 (sc-9772), SP-C (sc7706), p16INK4A (sc-1662), p19Arf (sc-1066), p21CIP1 (sc-6246; all from Santa Cruz Biotechnology), ERK1/2 (4695s), and pERK1/2 (4370s) from Cell Signaling Technology, H3K9me3 (07-523, Millipore), Ki67 (SP6, Lab Vision), tubulin (T4026, Sigma), AlexaFluor488 goat anti-rat (A-1106), goat anti-rabbit (A11008) and rabbit anti-mouse (A-11059; all from Invitrogen), and biotinylated rabbit-anti-mouse (E046401-2) from DAKO.

Techniques: Activity Assay, Activation Assay, Staining, Western Blot, Control

Journal: Pediatric surgery international

Article Title: Transglutaminases, involucrin, and loricrin as markers of epidermal differentiation in skin substitutes derived from human sweat gland cells.

doi: 10.1007/s00383-009-2517-5

Figure Lengend Snippet: Fig. 3 a Epidermal substitute derived from SG cells. Stratum spinosum and granulosum (between arrows), TG1 positive (green), fluorescence microscopy 940. b Epidermal substitute derived from SG cells. Stratum basale, spinosum, granulosum, and corneum (between arrows), TG3 positive (green), fluorescence microscopy 940. c Epidermal substitute derived from SG cells. Stratum basale, spinosum, and granulosum (between arrows), TG5 positive (green), fluorescence microscopy 940. d Epidermal substitute derived from SG cells. Stratum spinosum, granulosum, and corneum (between arrows), involucrin positive (red), fluorescence microscopy 940

Article Snippet: Antibodies For immunofluorescence, the following antibodies were used: From LabVision (P.H.Stehelin&CIE AG, Basel, Switzerland): Involucrin (clone SY5, 1:100); from Abcam (Cambridge, UK): Loricrin (polyclonal, 1:500); from Novus Biologicals (Littleton, CO, USA): transglutaminase 1 (polyclonal, 1:800), transglutaminase 3 (polyclonal, 1:50), transglutaminase 5 (polyclonal, 1:200).

Techniques: Derivative Assay, Microscopy

Journal: Pediatric surgery international

Article Title: Transglutaminases, involucrin, and loricrin as markers of epidermal differentiation in skin substitutes derived from human sweat gland cells.

doi: 10.1007/s00383-009-2517-5

Figure Lengend Snippet: Fig. 2 a Normal human skin. Stratum spinosum and granulosum (between arrows), TG1 positive (green), confocal microscopy 940. b Normal human skin. Stratum granulosum and corneum (between arrows), TG3 positive (green), confocal microscopy 940. The remaining areas with seemingly positive signals are background signals. c Normal human skin. Stratum basale, spinosum, and granulosum (between arrows), TG5 positive (green), confocal microscopy 940. d Normal human skin. Stratum granulosum and corneum (between arrows), involucrin positive (red), fluorescence microscopy 940

Article Snippet: Antibodies For immunofluorescence, the following antibodies were used: From LabVision (P.H.Stehelin&CIE AG, Basel, Switzerland): Involucrin (clone SY5, 1:100); from Abcam (Cambridge, UK): Loricrin (polyclonal, 1:500); from Novus Biologicals (Littleton, CO, USA): transglutaminase 1 (polyclonal, 1:800), transglutaminase 3 (polyclonal, 1:50), transglutaminase 5 (polyclonal, 1:200).

Techniques: Confocal Microscopy, Microscopy

Journal: Redox Biology

Article Title: PER1 interaction with GPX1 regulates metabolic homeostasis under oxidative stress

doi: 10.1016/j.redox.2020.101694

Figure Lengend Snippet: PER1 Binding to GPX1 Mediates Diurnal GPx Activity . A, B) Representative co-immunoprecipitation results of PER1 and GPX1 interaction from cytoplasmic extracts from WT (A) and Per1 −/− (B) mouse liver or small intestine at ZT1 or ZT13. β-actin as an input control. C) Molecular docking of GPX1 to PER1 and the backbone of PER1 and GPX-1 are shown in blue and red (C, top), respectively. Location of key residues was identified by mutagenesis (C, bottom). D-E) Representative immunoprecipitation analysis of PER1 and GPX1 interaction from lysates from NIH-3T3 cells. Lysates from NIH-3T3 cells expressing HA-tagged PER1 or mutant forms of PER1 (178–324 aa deletion and 1056–1177 aa deletion as CK) were immunoprecipitated with an anti-HA antibody (D). Lysates from NIH-3T3 cells coexpressing HA-tagged fragments of GPX1 and a vector expressing PER1 were immunoprecipitated with an anti-HA antibody (E). *GPX1 residues 1–136 are not detected because the UGA stop codon terminates translation in advance while recoded as selenocysteine (46 aa) in full-length conditions. F) In vitro a supplement of the fragment PER1 208–414 increases Gpx activity in Per1 −/− mice (n = 4 per group, *p < 0.05, **p < 0.01 versus β-actin group). β-actin, fragment PER1975-1290 (4 μg/ml) and Proteinase K (PK) digested the fragment PER1 208–414(4 μg/ml) products were performed as negative controls. G, H) Representative immunofluorescence images of GPX1 (Alexa Fluor 568-labeled) and Per1-GFP in NIH-3T3 cells (G). MitoTracker Red CMXRos-labeled mitochondria and Per1-GFP staining in NIH-3T3 cells (H). Images were obtained using a Delta Vision OMX system (GE Healthcare) and processed using GE SoftWoRx Imaging Analysis software. Bars indicate 1 μm in length. Throughout, male mice for all experiments were maintained on standard chow. Data are presented as the mean ± SEM and analyses were performed using one-way ANOVA for F. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Then, samples were imaged using a Delta Vision OMX system (GE Healthcare), and processed using GE SoftWoRx Imaging Analysis software.

Techniques: Binding Assay, Activity Assay, Immunoprecipitation, Mutagenesis, Expressing, Plasmid Preparation, In Vitro, Immunofluorescence, Labeling, Staining, Imaging, Software

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

Article Title: Expression of CD86 on human islet endothelial cells facilitates T cell adhesion and migration.

doi: 10.4049/jimmunol.181.9.6109

Figure Lengend Snippet: FIGURE 1. Phenotypic characterization of primary cultures of human islet ECs. A and B, The morphology of islet ECs at confluence observed by light microscopy (phase contrast) (A) and the expression of endothelial surface phenotypic markers on cultured human islet ECs including CD105, CD31, CD146 and E-selectin (B). C and D, The expression of MHC class I (HLA-ABC), MHC class II (HLA-DR), ICAM-1, and VCAM-1 adhesion molecules on resting islet ECs (C) and the basal expression of costimulatory molecules including CD40, CD80, CD86, and ICOS-L on resting islet ECs (D). Empty histograms represent staining with isotype-matched control mAbs. Study data are representative of at least three separate flow cytometric analyses conducted on islet ECs from all three islet donors.

Article Snippet: Analysis of the immune phenotype of islet endothelial cells For the analysis of the surface expression of immune phenotypic markers, confluent EC monolayers were detached from wells with Accutase (TCS CellWorks), washed in PBS/2% FCS, and stained for 30 min at 4°C with fluorochrome-conjugated mouse anti-human CD40, CD54, CD62E, and CD86 (all from Serotec); mouse anti-human CD80, CD106, HLA-DR, and HLA-ABC (all BD Biosciences and BD PharMingen); and mouse antihuman ICOS-L (Lab Vision) or appropriate isotype control mAbs.

Techniques: Light Microscopy, Expressing, Cell Culture, Staining, Control