Journal: International Journal of Molecular Sciences

Article Title: Tip60/KAT5 Histone Acetyltransferase Is Required for Maintenance and Neurogenesis of Embryonic Neural Stem Cells

doi: 10.3390/ijms24032113

Figure Lengend Snippet: Tip60- deficient brains show proliferation defects and cell cycle dysregulation. ( A ) Representative images of brains at E14.5 stained for BrdU and Tbr2 (markers for BPs) by immunofluorescence. DAPI was used for nuclear staining. Scale bars, 100 µm. The ratio of BrdU-positive and -negative cells in Tbr2-positive cells is shown. At least 10 different sections were counted. The graph shows means ± SD. Two independent experiments were performed. ( B ) Representative images of brains at E14.5 stained for BrdU and Pax6 (marker for APs) by immunofluorescence. DAPI was used for nuclear staining. Scale bars, 100 µm. The ratio of BrdU-positive and -negative cells in Pax6-positive cells is shown. At least 10 different sections were counted. The graph shows means ± SD. Two independent experiments were performed. ( C ) Representative images of brains at E16.5 stained for H3S10P (marker for mitosis) and Pax6 by immunofluorescence. DAPI was used for nuclear staining. Scale bars, 200 µm. The number of H3S10P-positive cells in SVZ is counted. At least 10 different sections were counted. The graph shows means ± SD. At least two independent experiments were performed.

Article Snippet: The antibodies used were as follows: MAP2 (1:250, Proteintech, Rosemont, IL, USA, 17490-1-AP), GFAP (1:2000 Sigma, St. Louis, MI, USA, G3893), Tbr1 (1:500 Abcam, Cambridge, UK, ab31940), Ctip2 (1:1000 Abcam ab18465), Cux1 (1:500 Santa Cruz sc-13024), Tbr2 (1:1000 Abcam ab183991), Pax6 (1:1000 MBL PD022), histone H3S10P (1:1000 Millipore, Burlington, MA, USA, 05-806), activated caspase 3 (1:2000 CST 9665), Iba1 (1:1000 Abcam ab178846), GLP-1 (1:250 Santa Cruz sc-57166), H4K8Ac (1:1000 MBL MABI0408), H4K12Ac (1:500 Abcam ab1761), γH2AX (1:500 Upstate 05-636), γH2AX (1:500 Cell Signaling, Danvers, MA, USA, 2577), p21 (1:500 BD Sciences, Franklin Lakes, NJ, USA, 556431), and 53BP1 (1:1000 BETHYL A300-273A).

Techniques: Staining, Immunofluorescence, Marker

Journal: International Journal of Molecular Sciences

Article Title: The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies

doi: 10.3390/ijms252212456

Figure Lengend Snippet: A disrupted expression of ectoderm-associated genes in embryonic bodies spontaneously differentiated from IFNB-iPSCs. IFNB-iPSC lines LA8 and LC8 and parental K7-iPSCs were cultured in low-adhesive conditions to stimulate the formation of embryoid bodies (EBs). On day 20, RNA was isolated and gene expressions were analyzed using RT-PCR. Whole-cell lysates of EBs were also analyzed by a Western blot. ( a – c ) Relative expressions of endoderm- ( a ), mesoderm- ( b ), and ectoderm- ( c ) associated markers in iPSCs and 20-day EBs. Data are shown as boxes and whiskers with minimal and maximal values (summarized results of at least 2 independent experiments). The significance of the differences was determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. Figures indicate the FDRs for the main comparisons, irrespectively of their significance (i.e., inter-line comparisons on differentiation day 20 and intra-line comparisons between iPSCs and 20-day EBs). ( d ) The Western blot analysis of PAX6 protein in IFNB-EBs and K7-EBs (one experiment). The graph shows the relative densitometric values of PAX6 normalized to HSP90.

Article Snippet: The membrane was blocked with a 5% non-fat dry milk (Cell Signaling Technology, Danvers, MA, USA) in a TNT buffer (10 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 0.1% Tween-20) and incubated with the following primary antibodies (4 °C, overnight, 1% milk): rabbit anti-human-IFN-β polyclonal antibodies (1:500, FNab10475, FineTest Biotech, Wuhan, Hubei, China), rabbit anti-human-PAX6 polyclonal antibodies (1:500, PAH446Ra01, Cloud-Clone Corp.), mouse anti-human-OCT-4 monoclonal antibody (1:1000, ab184665, Abcam, Cambridge, UK), mouse anti-human-actin-β antibodies (1:10,000, Abcam); and rabbit anti-human-HSP90 polyclonal antibodies (1:10,000, Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Adhesive, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: Derivation of induced pluripotent stem cells in Japanese macaque ( Macaca fuscata )

doi: 10.1038/s41598-018-30734-w

Figure Lengend Snippet: Directed differentiation of jm-iPSCs (J5F1) into neural cells ( a ). Schematic design of neural differentiation of jm-iPSCs. ( b ) Neurospheres of jm-iPSCs (J5F1) after 1-week floating culture. Scale bar; 100 µm. ( c ) Neuronal differentiation of neurospheres after 2-week adherent culture. Scale bar; 200 µm. ( d ) Immunofluorescence analyses of neural stem cell (NESTIN, PAX6) and neuron markers (TUJ1, MAP2, DREBRIN) in 2-week neuronal differentiation culture of neurospheres. Nuclei were counterstained with DAPI. Scale bar; 100 µm. ( e ) RT-PCR analysis of neural marker genes. β-ACTIN was examined as an internal control, and water was used as a negative control. NS, neurospheres after 2-week neuronal differentiation culture. Full-length gels are presented in Supplementary Figure .

Article Snippet: The following primary antibodies were used: mouse anti-OCT4 (BD Biosciences, 611202), rabbit anti-NANOG (REPROCELL, RCAB0003P), goat anti-LIN28A (R&D systems, AF3757), rabbit anti-DPPA4 (Abcam, ab154642), mouse anti-SOX2 (R&D systems, MAB2018), rabbit anti-SALL4 (Abcam, ab29112), mouse anti-SSEA4 (Millipore, MAB4304), mouse anti-TRA-1-81 (Millipore, MAB4381), mouse anti-E-CADHERIN (BD Biosciences, 610182), mouse anti-Keratan sulfate (R-10G) (Wako, 011-25811), mouse anti-TUBULIN β3 (BioLegend, MMS-435P), mouse anti-α-SMA (Abcam, ab7817), rabbit anti-VIMENTIN (Abcam, ab92547), goat anti-SOX17 (R&D systems, AF1924), mouse anti-AFP (R&D systems, MAB1369), mouse anti-NESTIN (Millipore, MAB5326), rabbit anti-PAX6 (Wako, 019-27291), rabbit anti-MAP2 (Millipore, AB5622), mouse anti-DREBRIN (Wako, 015-27271) antibodies.

Techniques: Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Marker, Negative Control