Journal: International journal of andrology

Article Title: TCam-2 seminoma cell line exhibits characteristic foetal germ cell responses to TGF-beta ligands and retinoic acid.

doi: 10.1111/j.1365-2605.2011.01170.x

Figure Lengend Snippet: Figure 1 Foetal germ cell and seminoma markers in TCam-2 cells. (a) The presence of transcripts characteristic of seminoma and foetal germ cells assessed by RT-PCR and Q-PCR in TCam-2 cells. For the RT-PCR (left panel) TCam-2 (T), HeLa cells (H) and human placenta (P) are indicated, alongside a control lacking reverse transcriptase (-). Amplification of b-actin was used as a loading control. Lower bands (arrowheads) correspond to unincorporated primers. OCT3 ⁄ 4, AP2g, KIT and low level KIT ligand (KITL) transcripts were detected in TCam-2 cells; two independent samples were tested, of which a representative sample is shown. Q-PCR (right panel) analysis of TCam-2 (T) and HeLa (H) cells. OCT3 ⁄ 4, AP2c, KIT and low level KITL transcripts were detected in TCam-2 cells. Three independent samples were analysed for each transcript, and data averaged with error bars indicating SEM. (b) Presence and subcellular localization of BLIMP1, OCT3 ⁄ 4, NANOG, KIT, VASA and DAZL1 proteins assayed in TCam-2 cells by immunofluorescence. Confocal imaging was performed at 40· magnification. The control images (no primary antibody control) are representative of all control immunofluorescence samples, in which no background signal was detected with the secondary antibodies alone. (c) A representative flow cytometric profile of TCam-2 and HeLa (KIT negative) cells stained for KIT (CD117). The top two panels are negative controls (no primary antibody present), whereas the lower two panels show intensity profiles representing KIT positive cells. No KIT-positive HeLa cells were detected. The mean fluorescence intensity of the whole cell population is indicated for each sample.

Article Snippet: Antibodies against human proteins: BLIMP1, STELLAR, VASA, DAZL1 (Abcam, Cambridge, MA, USA), OCT3 ⁄ 4, ACTRIB (ALK4), ACTRIIA, ACTRIIB, KIT (CD117) (Santa Cruz Biotechnology, Santa Cruz, CA), and NANOG (R&D systems, Inc) were verified to detect proteins of the appropriate size by Western blot (not shown) prior to use in immunofluorescence.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription, Imaging, Staining

Journal: Journal of molecular cell biology

Article Title: Dazl promotes germ cell differentiation from embryonic stem cells.

doi: 10.1093/jmcb/mjp026

Figure Lengend Snippet: Figure 5 Suppressed Nanog expression and induced GCNA expression in Dazl-transfected mES cells. Mouse ES cells were transiently trans- fected with plenti6Ubc-Dazl-V5, by which Dazl is expressed as a fusion protein with the V5 tag, and cultured under standard ES medium with LIF for 24 h. (A, B) Dual-immunofluorescence staining of V5 and Nanog shows that Nanog expression was suppressed in Dazl-overexpressing mES cells. (C and D) Dual-immunofluorescence staining of GCNA and Nanog. (E–H) Dual-immunofluorescence staining of V5 and GCNA, G is the overlap of E and F, H is the overlap of V5, GCNA and DAPI; this result indicates that the expression of GCNA was induced by Dazl.

Article Snippet: For immunofluorescence with cells, the following antibodies were used: FE-J1 antibody (The Developmental Studies Hybridoma Bank, University of Iowa); acetyl-a-tubulin antibody (Sigma-Aldrich Corp, St. Louis, MO, USA); Anti-CDF-9 antibody (R&D Systems Inc., Minneapolis, MN 55413, USA); Oct4 antibody (c-10 monoclonal ab from Santa Cruz Biotech, CA, USA); V5 antibody (Invitrogen); Nanog antibody (R&D Systems); GCNA antibody (Rat IgG, kindly provided by George C. Enders, The University of Kansas); Stra8 antibody (Abcam, Cambridge, MA, USA); b-Tubulin (Sigma, Clone Tub2.1).

Techniques: Expressing, Transfection, Cell Culture, Staining

Journal: Journal of molecular cell biology

Article Title: Dazl promotes germ cell differentiation from embryonic stem cells.

doi: 10.1093/jmcb/mjp026

Figure Lengend Snippet: Figure 6 Dazl regulates the expression of germ cell specific genes. (A) Mouse ES cells were infected by plenti6Ubc-Dazl-V5 lentivirus medium for the overexpression of Dazl. Western blotting analyses shows that Dazl was overexpressed and that overexpression of Dazl induced the expression of GCNA (top panel, control was of mES cells without transfection), whereas the expression of Nanog was suppressed by Dazl (bottom panel). (B) Western blotting analysis shows that Dazl was knocked down by siRNA in mES cells; Dazl siRNA2 was more efficient than Dazl siRNA1, thus, the cells trans- fected with Dazl siRNA2 were used for further experiments. (C) RT– PCR analyses were performed to test the expression of early germ cell markers including Stella, MVH and Prdm1, in the Dazl knockdown mES cells. (D) RT–PCR analyses show that germ cell markers includ- ing Prdm1 for PGCs, Stra8 for premeiotic germ cells and Protamine1 for postmeiotic cells, were activated temporally in Dazl-transfected mES cells cultured under DMEM/F12 medium containing 15% FBS without LIF. (Con/Ve is for mixed total RNAs collected from non- infected cells and vector-infected cells cultured over the same period of culture time as did for pLenti-UBC-Dazl-V5 infected cells, Day 1 is for 1 day after infection, Day 2 is for 2 days after infection and so on).

Article Snippet: For immunofluorescence with cells, the following antibodies were used: FE-J1 antibody (The Developmental Studies Hybridoma Bank, University of Iowa); acetyl-a-tubulin antibody (Sigma-Aldrich Corp, St. Louis, MO, USA); Anti-CDF-9 antibody (R&D Systems Inc., Minneapolis, MN 55413, USA); Oct4 antibody (c-10 monoclonal ab from Santa Cruz Biotech, CA, USA); V5 antibody (Invitrogen); Nanog antibody (R&D Systems); GCNA antibody (Rat IgG, kindly provided by George C. Enders, The University of Kansas); Stra8 antibody (Abcam, Cambridge, MA, USA); b-Tubulin (Sigma, Clone Tub2.1).

Techniques: Expressing, Infection, Over Expression, Western Blot, Control, Transfection, Reverse Transcription Polymerase Chain Reaction, Knockdown, Cell Culture, Plasmid Preparation

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