Journal: International Journal of Molecular Sciences

Article Title: Ocular Albinism Type 1 Regulates Melanogenesis in Mouse Melanocytes

doi: 10.3390/ijms17101596

Figure Lengend Snippet: The overexpression of OA1 affects the melanin content in an MITF-dependent fashion. Mice OA1 was PCR amplified and then subcloned into the pMSCV PIG vector with Xho I and EcoR I restriction sites. The pMSCV-OA1-GFP plasmid or empty vector was transfected into the mouse melanocytes. The cells were harvested to extract total RNA and total protein. The expression levels of OA1 were qualitatively and quantitatively analyzed by qRT-PCR and Western blot. ( A ) Melanin contents in melanocytes transfected with OA1 , as well as the control; ( B – D ) transfection efficiency of OA1 in mouse melanocytes; ( E – G ) OA1 overexpression influences microphthalmia-associated transcription factor ( MITF ) mRNA and protein levels in mouse melanocytes. Data are shown as the mean ± standard errors ( n = 3 each), * p < 0.05, ** p < 0.01.

Article Snippet: The mice OA1 gene was PCR amplified and then was cloned into the pMSCV PIG vector (Addgene, Cambridge, MA, USA) with Xho I and EcoR I restriction sites.

Techniques: Over Expression, Amplification, Plasmid Preparation, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: Development & Reproduction

Article Title: Enhancement of Transgene Expression by HDAC Inhibitors in Mouse Embryonic Stem Cells

doi: 10.12717/DR.2013.17.4.379

Figure Lengend Snippet: Selection of G418 resistant cells and characterization of the transgenic ES cells. (A) Mouse ES cells were selected with G418 after transfection with pMSCV-GFP using Lipofectamine™ 2000 and NaB; high percentage of cells expressed GFP. (B) Stem cell marker expression in GFP positive mouse ES cells was determined by FACS analysis.

Article Snippet: The pMSCV-GFP vector was isolated using Qiagen plasmid MIDI-prep kit (Qiagen, West Sussex, UK).

Techniques: Selection, Transgenic Assay, Transfection, Marker, Expressing

Journal: Cancer discovery

Article Title: EGFR kinase domain duplication (EGFR-KDD) is a novel oncogenic driver in lung cancer that is clinically responsive to afatinib

doi: 10.1158/2159-8290.CD-15-0654

Figure Lengend Snippet: (a) Schematic representation of EGFR-KDD depicting the genetic and protein domain structures. ECD = extracellular domain. TM = transmembrane domain. Blue = EGFR exons 18-25 #1. Green = EGFR exons 18-25 #2. KD1 = first kinase domain. KD2 = second kinase domain. C-term = carboxyl terminus. (b) Representative western blot of NR6 cells stably expressing indicated EGFR constructs. EGFR-KDD-dead is a kinase dead version of EGFR-KDD. (c) NR6 cells stably expressing the indicated constructs (pMSCV = vector only) were plated in triplicate in soft agar, grown for 15 days, and quantified for colony formation. (d) Representative western blot of BA/F3 cells expressing indicated EGFR constructs. (e) BA/F3 cells transfected with indicated constructs (pMSCV = vector only) were grown in the absence of IL-3 and counted every 24 hours. (f) Ribbon diagram and space-filling model of the EGFR-KDD kinase domains (GLY 696 - PRO 1370) illustrating the proposed mechanism of auto-activation. Blue = first kinase domain; green = second kinase domain; red = linker; yellow asterisks = active sites.

Article Snippet: The empty pMSCV-puro retroviral vector or pMSCV-puro vectors encoding EGFR (either EGFR-WT, EGFR-L858R, EGFR-KDD, or EGFR-KDD-dead) were transfected, along with the envelope plasmid pCMV-VSV-G (CellBioLabs), into cells Plat-GP packaging cells (CellBioLabs).

Techniques: Western Blot, Stable Transfection, Expressing, Construct, Plasmid Preparation, Transfection, Activation Assay

Journal: Cancer discovery

Article Title: EGFR kinase domain duplication (EGFR-KDD) is a novel oncogenic driver in lung cancer that is clinically responsive to afatinib

doi: 10.1158/2159-8290.CD-15-0654

Figure Lengend Snippet: (a) BA/F3 lines stably expressing EGFR-WT, -L858R or -KDD were treated with increasing doses of erlotinib, afatinib, or AZD9291 for 2 hours and lysed for western blot analysis with the indicated antibodies. (b) BA/F3 lines stably expressing EGFR-L858R or EGFR-KDD were treated with increasing doses of erlotinib, afatinib, or AZD9291 for 72 hours. Cell titer blue assays were performed to assess cell viability. Each point represents quadruplicate replicates. Data are presented as the mean percentage of viable cells compared to vehicle control ± s.d.

Article Snippet: The empty pMSCV-puro retroviral vector or pMSCV-puro vectors encoding EGFR (either EGFR-WT, EGFR-L858R, EGFR-KDD, or EGFR-KDD-dead) were transfected, along with the envelope plasmid pCMV-VSV-G (CellBioLabs), into cells Plat-GP packaging cells (CellBioLabs).

Techniques: Stable Transfection, Expressing, Western Blot

Journal: Molecular and Cellular Biology

Article Title: CD99-Derived Agonist Ligands Inhibit Fibronectin-Induced Activation of β1 Integrin through the Protein Kinase A/SHP2/Extracellular Signal-Regulated Kinase/PTPN12/Focal Adhesion Kinase Signaling Pathway

doi: 10.1128/MCB.00675-16

Figure Lengend Snippet: Functional analysis of highly conserved regions of CD99 in the regulation of β1 integrin activity. (A, B) A 7-mer peptide was shortened by either 4-mer or 3-mer. Cells were seeded onto PLL- or fibronectin (FN)-coated coverslips and treated with three different sizes of peptides for 1 h as indicated. In situ PLA was performed to determine the interaction of talin or kindlin with β1 integrin. (C) Cells were treated with three different 3-mer peptides (20 μM) that originated from three different conserved regions, as well as mutated forms of CD99CRIII3. The interaction between talin and β1 integrin was confirmed by in situ PLA. (D) To determine the receptor-specific binding of CD99CRIII3, wild-type, CD99 knockdown, or β1 integrin knockout MCF-7 cells were stained with FITC-conjugated CD99CRIII3 and then observed under a confocal microscope. Unconjugated peptides (control, CD99CRIII3, and CD99CRIII3mut1) were used as competitors. (E) Cells were treated with CD99CRIII3 for 1 h, and cell lysates were subjected to SDS-PAGE to analyze β1 integrin activity. (F) CD99-deficient human keratinocytes, HaCaT cells, were infected with a retroviral vector carrying wild-type CD99 cDNA and seeded into 35-mm dishes coated with fibronectin. After treatment with CD99CRIII3, β1 integrin activity was analyzed by flow cytometry with a BD Accuri C6 system. (G, H) Wild-type CD99 and a series of variant cDNA constructs of CD99. Each of the cDNA constructs was inserted into a pMSCV retroviral vector containing a neomycin resistance gene. The β1 integrin-talin interaction was examined by in situ PLA. (A to C, H) Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Red spots indicate the physical connection of the target molecules. PLA signals in cell populations (n = 8) were quantified by NIS Elements analysis. The average number of rolling-circle products (RCPs) per cell ± the standard error is shown. Asterisks represent statistically significant differences from untreated cells as follows: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Magnification, ×600. Scale bars = 10 μm. wt, wild type.

Article Snippet: His-tagged CD99 variant cDNAs were subcloned into EcoRI and XhoI sites of pMSCV-neo vectors (BD Biosciences Clontech, San Jose, CA).

Techniques: Functional Assay, Activity Assay, In Situ, Binding Assay, Knock-Out, Staining, Microscopy, SDS Page, Infection, Plasmid Preparation, Flow Cytometry, Variant Assay, Construct