Journal: Cell Transplantation

Article Title: Establishment of Immortalized Human Amniotic Mesenchymal Stem Cells

doi: 10.3727/096368912x655055

Figure Lengend Snippet: Figure 3. Immunohistochemical analysis of fHAM cells and iHAM cells. The expressions of stem cell markers on the fHAM cells and iHAM cells were detected by immunofluorescence. Most of the upper layer was fHAM cells, including some octamer binding transcription protein 3/4 (Oct3/4)-positive cells. From the third layer, they were iHAM cells. The nuclei of most cells were stained by Oct3/4, sex-determining region Y box 2 (Sox2), Lin28, and Krüppel-like factor 4 (Klf4). Some nuclei were stained strongly by Nanog, nestin, and stage-specific embryonic antigen-4 (SSEA-1). Vimentin and anti-a-smooth muscle (a-SM) actin were clearly expressed in the cytoplasm. No cytokeratin (CK)-positive cells were observed. Scale bar: 50.0 mm.

Article Snippet: After blocking for nonspecific reaction with BLOCK ACE (Dainippon Pharmaceutical, Osaka, Japan) for 45 min, cells were incubated in the following primary antibodies diluted 1:200 in PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 24 h at 4°C: octamer binding transcription factor 3/4 (Oct3/4), Nanog, Krüppel-like factor 4 (KLF4), stage-specific embryonic antigen 4 (SSEA4), a-smooth muscle (a-SM) actin, nestin, c-myc (all from Santa Cruz Biotechnology, Inc., CA, USA), sex-determining region Y box 2 (Sox2), anti-human Lin28 (R&D System, Inc., Minneapolis, MN, USA), vimentin (Dako, Glostrup, Denmark), and cytokeratin 18 (CK18; Progen, Heidelberg, Germany).

Techniques: Immunohistochemical staining, Immunofluorescence, Binding Assay, Staining

Journal: Cell Transplantation

Article Title: Establishment of Immortalized Human Amniotic Mesenchymal Stem Cells

doi: 10.3727/096368912x655055

Figure Lengend Snippet: Figure 4. (A) Expression of representative stem cell marker genes by RT-PCR. Samples are as follows: lane -RT, reagent control; lane 1, freshly isolated HAM cells (fHAM cells); lane 2, HAM cells cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at passage 2 (HAM cells P2); lanes 3–6, iHAM cells lines (iHAM3 542, iHAM3 542-344, iHAM4 136-344, and iHAM4 344-96). All iHAM cells lines showed stronger reactions than fHAM cells and HAM cells P2 for Oct-4, Klf4, c-myc, breast cancer resistance gene (BCRP), and nestin (neural progenitor cell marker). However, expression was weaker than fHAM cells and HAM cells P2 for Sox2 and Nanog. b-Actin mRNA was used as a positive control. (B) Formation of spheres. iHAM cells made spheres easily after they were cultured on the ultralow attachment dish. There were various sizes of spheres. Notice that they indicated alkaline phosphatase (ALP) activity (scale bar: 500 mm) and Oct3/4 reaction (scale bar: 200 mm).

Article Snippet: After blocking for nonspecific reaction with BLOCK ACE (Dainippon Pharmaceutical, Osaka, Japan) for 45 min, cells were incubated in the following primary antibodies diluted 1:200 in PBS containing 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 24 h at 4°C: octamer binding transcription factor 3/4 (Oct3/4), Nanog, Krüppel-like factor 4 (KLF4), stage-specific embryonic antigen 4 (SSEA4), a-smooth muscle (a-SM) actin, nestin, c-myc (all from Santa Cruz Biotechnology, Inc., CA, USA), sex-determining region Y box 2 (Sox2), anti-human Lin28 (R&D System, Inc., Minneapolis, MN, USA), vimentin (Dako, Glostrup, Denmark), and cytokeratin 18 (CK18; Progen, Heidelberg, Germany).

Techniques: Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Control, Isolation, Cell Culture, Modification, Positive Control, Activity Assay

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 2 Vps26a knockdown prolongs the maintenance of stemness under ND conditions in P19 ECCs. a, b The effect of Vps26a knockdown on the expression of ESC stemness genes were determined by semi-qPCR (a) and western blot (b) analyses of Oct3/4 and Nanog using shCTL- and shVps26a-ECCs. c Morphological changes of shCTL (shC)- and shVps26a (shV)-ECCs during retinoic acid-induced neurogenesis (RA-ND) for the indicated time periods. The yellow arrowheads indicate cells with differentiated morphologies. d–f Expression kinetics of ESC stemness and neuronal markers examined by semi-qPCR (d), qPCR (e), and western blotting (f)

Article Snippet: Subsequently, antibodies against Oct3/4 (SC-5279, Santa Cruz Biotechnology), Nanog (NBP2-19469, Novus Biological), MAP2 (AB5622, Millipore), Tubb3 (MAB1637, Millipore), pERK1/2 (4370, Cell Signaling), Hif2α (NB100-122, Novus Biological), Nox4 (PA1-46014, Thermo Fisher Scientific), and Vps26a (AB23892, Abcam) were incubated with the prepared cells at 4 °C overnight.

Techniques: Knockdown, Expressing, Western Blot

Journal: Oncotarget

Article Title: Cytokine-induced killer cells efficiently kill stem-like cancer cells of nasopharyngeal carcinoma via the NKG2D-ligands recognition

doi:

Figure Lengend Snippet: A. Schematic diagram of lentiviral vector pLV-P Nanog -GFP-T2A-Luc, in which GFP and Luc expression was controlled by human Nanog promoter. The construct map is not drawn to the scale. Abbreviations: Luc : firefly luciferase; GFP : green fluorescent protein. B–C. Representative GFP expression was measured in NPC cell lines (e.g., CNE2 and SUNE1) carrying P Nanog -GFP-T2A-Luc transgene by inverted fluorescence microscope (B) and by flow cytometry (C). D. CNE2 cells harboring Luc have robust reporter gene expression as shown by bioluminescence imaging (BLI). E. A strong correlation exists between BLI signal and CNE2 cell number. F–G. GFP+ and GFP- fractions sorted from CNE2 and SUNE1 cells carrying P Nanog -GFP-T2A-Luc transgene (shown in Fig. 3B, 3C) by fluorescence-activated cell sorting (FACS) were subjected to Western blotting for the detection of Nanog, Oct4, Sox2 and Klf4 expression (F), and tumor spheroid formation assay (G). H–I. The growth (H), migration (I) and invasion (I) of GFP+ and GFP- NPC cells were evaluated by colony formation assay, transwell migration assay and matrigel-coated Boyden chamber assay, respectively. GFP+ and GFP- fractions were sorted from CNE2 and SUNE1 cells carrying P Nanog -GFP-T2A-Luc transgene (shown in Fig. 3B, 3C) by FACS. J. GFP+ and GFP- fractions sorted from CNE2 and SUNE1 cells carrying P Nanog -GFP-T2A-Luc transgene (shown in Fig. 3B, 3C) by FACS were subjected to Western blotting for the detection of E-cadherin, α-catenin, vimentin and N-cadherin expression.

Article Snippet: The plasmid of pL-SIN-P Nanog -EGFP, carrying human Nanog promoter, was obtained from Addgene (plasmid 21321).

Techniques: Plasmid Preparation, Expressing, Construct, Luciferase, Fluorescence, Microscopy, Flow Cytometry, Gene Expression, Imaging, FACS, Western Blot, Tube Formation Assay, Migration, Colony Assay, Transwell Migration Assay, Boyden Chamber Assay