Journal: Cell Death & Disease

Article Title: Human embryonic stem cell neural differentiation and enhanced cell survival promoted by hypoxic preconditioning

doi: 10.1038/cddis.2009.22

Figure Lengend Snippet: Hypoxic preconditioning induced gene regulation. ( a ) The HIF1- α mRNA level in hESC neurospheres was upregulated after 3–24 h under the 0.1% oxygen condition, revealed by RT-PCR measurements. The last column shows that the HIF1- α level remained high after finishing a 12-h hypoxia plus 12-h back to 21% oxygen (reoxygenation). No change in the expression of the proapoptotic gene BNIP3 was observed. β -Actin was used as a loading control. ( b ) Western blot analysis shows the upregulation of the protein levels of HIF1- α and pAkt induced by the hypoxia treatment. The Akt activation (phosphorylation of S473) was inhibited by the PI3K inhibitor Wortmannin that also partially inhibited hypoxic stabilization of HIF1- α . ( c ) Western blot analysis showed significant upregulation of several survival genes, including HIF1- α , HIF2- α , pAkt, EPO and Bcl-2 after the 12-h 0.1% oxygen treatment. α -Tubulin was used as a loading control. ( d ) Gene regulation after the termination of hypoxic treatment. Different time points after hypoxia, cells were returned to 21% oxygen. The protective gene bcl-2 decreased and proapoptotic gene Bax increased after reoxygenation. However, a significant upregulation of EPO, mature VEGF and Bcl-2, as well as a downregulation of Bax, were evident after 72-h reoxygenation. * P <0.05 between normoxic controls and 12-h hypoxic culture; ** P <0.05 between normoxic controls and 72-h reoxygenation

Article Snippet: Membranes were blocked for 1 h with 5% BSA containing phosphatase inhibitors (50 mM NaF, 200 μ M Na 3 VO 4 ) before overnight incubation with primary antibodies: VEGF (sc-507; 1 : 1000), Flk-1 (sc-6251; 1 : 1000), EPOR (sc-697; 1 : 1000), Akt (sc-8312; 1 : 1000), Bcl-2 (sc-7382; 1 : 500), Bcl-Xl (sc-8392; 1 : 500), BDNF (sc-546; 1 : 1000), α -tubulin (sc-5286; 1 : 5000), EPAS-1 (sc-28706; 1 : 2000), EPO (sc-7956; 1 : 2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-Akt (S473) (#9271; 1 : 1000), NF-ÊB (#3035; 1 : 2000; Cell Signaling Technology, Danvers, MA, USA), GLUT-1 (AB1340; 1 : 7500), 17 kDa Caspase 3 (AB3623; 1 : 2000), FGF-2 (AB1458; 1 : 500; Chemicon/Millipore), HIF1- α (NB100-479; 1 : 2000; Novus Biologicals, Littleton, CO, USA), Bax (no. 54179; 1 : 500; Anaspec, Fremont, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Western Blot, Activation Assay, Phospho-proteomics

Journal: eLife

Article Title: Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9

doi: 10.7554/eLife.51662

Figure Lengend Snippet:

Article Snippet: The membranes were blocked in Odyssey Blocking Buffer (LI-COR) then incubated with antibodies specific for HSV-1 ICP8 (1:5000, rabbit serum 3–83 [ ], HSV-1 ICP4 (1:2000, monoclonal mouse (mAb) 58S [ ], purified from hybridoma cell line 58S (ATCC HB8183)), ICP27 (1:5000, mAb, Eastcoast Bio), or GAPDH (1:10000, mAb, Abcam).

Techniques: Recombinant, Purification, Clone Assay, Plasmid Preparation, Software

Journal: mBio

Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

doi: 10.1128/mBio.00345-15

Figure Lengend Snippet: Effect of BAF on viral transcript levels. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. (A) Knockdown efficiency was confirmed by quantitative RT-PCR. (B to D) Cells were harvested at the postinfection times indicated, total RNAs were prepared, and IE ( ICP4 [B] and ICP27 [C]) and E ( ICP8 [D]) gene mRNA levels were measured by quantitative RT-PCR. The mRNA levels were normalized to 18S rRNA. Results shown are means and standard deviations from three independent experiments.

Article Snippet: The membrane was then incubated with antibodies specific for HSV ICP8 (1:5,000, rabbit serum 3-83 [ ]), HSV ICP4 (1:4,000, mouse monoclonal antibody [MAb] 58S [ ]), ICP27 (1:5,000, MAb; Eastcoast Bio), gD (1:5,000, MAb; Eastcoast Bio), BAF (1:2,000, rabbit serum; Millipore), GFP (1:2,000, rabbit serum; Abcam), lamin B1 (1:10,000, rabbit serum; Abcam), GAPDH (1:10,000, MAb; Abcam), and FLAG M2 (1:2,000, MAb; Sigma).

Techniques: Transfection, Infection, Quantitative RT-PCR

Journal: mBio

Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

doi: 10.1128/mBio.00345-15

Figure Lengend Snippet: Knockdown of BAF reduces the size and numbers of viral RCs, but heterochromatin is excluded from the RCs. HFF cells were transfected with NT control or BAF-specific siRNA and infected with HSV-1 at an MOI of 10. At 8 hpi, the cells were fixed and processed for indirect immunofluorescence assays with antibodies specific for BAF (red in top row), ICP8 (red in bottom row), and H3K9me3 (green). Arrows indicate ICP8-stained RCs.

Article Snippet: The membrane was then incubated with antibodies specific for HSV ICP8 (1:5,000, rabbit serum 3-83 [ ]), HSV ICP4 (1:4,000, mouse monoclonal antibody [MAb] 58S [ ]), ICP27 (1:5,000, MAb; Eastcoast Bio), gD (1:5,000, MAb; Eastcoast Bio), BAF (1:2,000, rabbit serum; Millipore), GFP (1:2,000, rabbit serum; Abcam), lamin B1 (1:10,000, rabbit serum; Abcam), GAPDH (1:10,000, MAb; Abcam), and FLAG M2 (1:2,000, MAb; Sigma).

Techniques: Transfection, Infection, Immunofluorescence, Staining

Journal: mBio

Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

doi: 10.1128/mBio.00345-15

Figure Lengend Snippet: BAF localizes to viral RCs. (A) HFF cells were infected with HSV-1 at an MOI of 10, and an indirect immunofluorescence assay was performed with antibodies specific for BAF and ICP8 at 8 hpi. Images were deconvoluted by the inverse filter algorithm in the AxioVision 4.8 image acquisition software. (B) HeLa cell expressing FLAG-BAF were infected with HSV-1 DG1 at an MOI of 50, harvested at 4 hpi, and immunoprecipitated (IP) with antibodies specific for FLAG or ICP8. ICP4, ICP8, VP16-GFP, and FLAG-BAF were detected with antibodies specific for ICP4, ICP8, GFP, and FLAG.

Article Snippet: The membrane was then incubated with antibodies specific for HSV ICP8 (1:5,000, rabbit serum 3-83 [ ]), HSV ICP4 (1:4,000, mouse monoclonal antibody [MAb] 58S [ ]), ICP27 (1:5,000, MAb; Eastcoast Bio), gD (1:5,000, MAb; Eastcoast Bio), BAF (1:2,000, rabbit serum; Millipore), GFP (1:2,000, rabbit serum; Abcam), lamin B1 (1:10,000, rabbit serum; Abcam), GAPDH (1:10,000, MAb; Abcam), and FLAG M2 (1:2,000, MAb; Sigma).

Techniques: Infection, Immunofluorescence, Software, Expressing, Immunoprecipitation