PMC

Titration of Ba-L and HXB2 pseudotyped HIV-1 on HeLa P4P cells. 10⁴ HeLa P4P cells/well/100 μl were seeded in 96 flat-bottomed well plates. Infections were performed 24 hours later. Cells were lysed 72 hours post infection with 100 μl of lysis buffer (Promega, Madison USA). 100 μl of commercially available luciferase substrate (Promega, Madison, USA) were added to 20 μl of each lysate and then luciferase activity read with a luminometer (Lumat LB 9501, Berthold) as relative light units (RLU) per minute. A: Ba-L pseudotyped HIV-1; B: HXB2 pseudotyped HIV-1.

Minigenome assay. (a) Negative-strand minigenomic RNA containing the ORF of the luciferase gene was transfected into HEK 293 cells expressing wt or mutant N protein, together with P and L proteins. Luciferase activity of cell extracts was measured using a Mini Lumat LB 9506 (Berthold Technologies). The data represent means±sd for triplicate samples. Statistical analysis was performed using Student’s t-test; *P<0.05 compared with NiV-N-wt. (b) Protein expression was detected by Western blotting with NiV-N-specific antibody and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody.

Functional analysis of the TTV promoter in the viral UTR. (A) Schematic representation of the 1.0-kb UTR sequence. The TATA box element and putative transcription factor-binding sites are shown. Transcription factor-binding sites were identified using the TRASFAC database and a search program (http://motif.genome.ad.jp/). The transcription initiation site (+1) is indicated and corresponds to nt 121 (AB025946). The numbers at the bottom of vertical dotted lines indicate the start points of the full-length promoter construct and deletion mutants of the promoter constructs used in Fig. and . (B) Cell type specificity of the TTV promoter activities. Cells were transfected with p(−890/+115) (+) or promoter-less pGL3-Basic (−) together with pRL-TK (Renilla luciferase). Cell extracts were prepared 16 h after transfection, and luciferase activities in the extracts were determined using a dual-luciferase reporter assay system (Promega) with the Lumat LB9501 luminometer (Berthold). All values were normalized to Renilla luciferase activities and are shown as means ± standard deviations (error bars) of three independent samples.

UHRF1 is essential for maintaining calcium homeostasis in oocytes. A) Distribution of mitochondria with high membrane potential (red) and low membrane potential (green) in oocytes with the indicated genotypes, respectively. Scale bar, 20 µm. B) Relative fluorescence intensity of ratio of red/green fluorescence analysis for each oocyte was conducted using Image J software. Significant difference between control and Uhrf1^fl/△;SKO oocytes was observed. Data are expressed asmean±SEM of at least three independent experiments (n = 3). **p <0.001. C) The adenosine triphosphate (ATP) content of mouse oocytes with the indicated genotypes, respectively. ATP was measured using a Berthold Lumat LB 9501 luminometer and a commercial assay kit. Data are expressed as mean±SEM of at least three independent experiments(n = 3). **p <0.01. D) [Ca²⁺]i oscillation patterns after parthenogenetic activation of oocytes with indicated genotypes, respectively. E) control and Uhr^f1fl/△;SKO MII oocytes were parthenogenetically activated in an activation medium for 6 h, then cultured in KSOM. Representative images of parthenogenetic activated embryos with the indicated genotypes at day 2, respectively.