Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: Generation of iPSCs and NPCs from human skin fibroblasts (NB1RGB). ( A ) To identify the PSC status of the established iPSC clone 2 (C2) from human skin fibroblasts (NB1RGB), cells were immunostained using PSC markers Nanog, SSEA4, OCT4 and KLF4. The cell nucleus was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). ( B ) iPSC-like colonies were stained with alkaline phosphatase staining. ( C ) To confirm pluripotency, iPSCs were differentiated into three germ layers and stained with germ-layer markers Otx2, Brachury and Sox17 for the ectoderm, mesoderm and endoderm, respectively. ( D ) iPSCs were differentiated to neural progenitor cells (NPCs) and stained with neural stem cell markers GFAP, Nestin, Pax6, Sox1 and Sox2 and the differentiated neuron marker β-III Tubulin (Tuj1). NPCs were positive for all the NPC markers except Tuj1. Scale bar represents 25 μm or 50 μm, as indicated. iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, PSC, pluripotent stem cell = DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Staining, Marker

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: DDR after IR in iPSCs and NPCs. ( A ) Fibroblasts (NB1RGB), iPSCs (NB1RGB C2 and 201B7) and NPCs (C2) were irradiated at 2 Gy and fixed with 4% PFA at designated times. Cells were immunostained with 53BP1 (green) and γ-H2AX (red) antibodies. The cell nucleus was counterstained with DAPI. ( B ) Immunostained cells containing >10 53BP1- and γ-H2AX-positive foci (dot foci) were counted and graphed. ( C ) γ-H2AX apoptotic foci (pan staining) were also counted. At least 200 cells were counted and all experiments were performed three times. Scale bar represents 25 μm. Error bars represent the standard error of the mean (SEM). Welch’s (one-tailed) t -test was performed and statistical significance is indicated by asterisks ( * P < 0.05; ** P < 0.01; *** P < 0.001). DDR = DNA damage response, IR = ionizing radiation, iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, PFA = paraformaldehyde, 53BP1 = p53 binding protein 1, γ-H2AX = γ-H2A histone family member X, DAPI = 4′,6-diamidino-2-phenylindole.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Irradiation, Staining, One-tailed Test, Binding Assay

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: Apoptosis detection by TUNEL assay in iPSCs. ( A ) Fibroblasts (NB1RGB) and iPSCs (NB1RGB C2) were irradiated at 2 Gy and fixed at designated times after IR. Apoptosis was detected via the TUNEL assay. ( B ) At least 200 cells were counted and all experiments were performed three times and graphed. Scale bar represents 25 μm. Error bars represent the SEM. Welch’s (one-tailed) t -test was performed and statistical significance is indicated by asterisks ( * P < 0.05). iPSCs = induced pluripotent stem cells, TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling, SEM = standard error of the mean.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: TUNEL Assay, Irradiation, One-tailed Test

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: RNA-Seq analysis revealed transcriptional alteration in fibroblasts, iPSCs and NPCs. ( A ) Fibroblasts (NB1RGB), iPSCs (NB1RGB C2 and 201B7) and NPCs (C2) were irradiated at 5 Gy. One hour after IR, RNA samples were extracted and analyzed using the Illumina HiSeq 2500 next-generation sequencer for RNA-Seq. Raw data were quality controlled and aligned to the reference hg19. Heat maps classed as HR repair, NHEJ repair and apoptosis were obtained using R software. Red, upregulated; green, downregulated. ( B ) Differential expression patterns of each gene were represented by FPKM. Expression differences were classified according to molecular machinery components: DNA repair, cell cycle checkpoints and apoptosis. ( C ) Differential expression patterns by RNA-Seq were confirmed by immunoblotting. ATM, NBS1, CHK1, p53 and p21 antibodies were used for immunoblotting. GAPDH antibody was used as loading control. Immunoblotting data were obtained using C-digit as digital data. Full scan data of each band are provided in and . RNA-Seq = RNA sequencing, iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, HR = homologous recombination, NHEJ = nonhomologous end joining, FPKM = fragments per kilobase of exon per million reads mapped.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Irradiation, Software, Quantitative Proteomics, Expressing, Western Blot, Control, Homologous Recombination

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: IR sensitivity and persistent activation of DDR after IR in iPSCs . ( A ) To determine cell sensitivity to IR exposure, colony formation assay was performed. Fibroblasts (NB1RGB) and iPSCs (NB1RGBC2 and 201B7) were used. Compared with fibroblasts, iPSCs showed hypersensitivity to IR exposure. Error bars represent the SEM. ( B ) After IR exposure, cell extracts were immunoblotted with ATM, p53, serine 15 phospho-p53, KAP1 and serine 824 phospho-KAP1 antibodies. GAPDH antibody was used as a loading control. Immunoblotting data were obtained using C-digit as digital data. Full scan data of each band are provided in . DDR hyperactivation increased in a time-dependent manner in iPSCs but not in fibroblasts and NPCs. IR = ionizing radiation, DDR = DNA damage response, iPSCs = induced pluripotent stem cells, SEM = standard error of the mean, ATM = ataxia-telangiectasia-mutated, KAP1 = Kruppel-associated box domain (KRAB)-associated protein-1, GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Colony Assay, Control, Western Blot