Representative gel electrophoresis of PCR amplicon using sequencing primers from different DNA preparations.

<p>(A) PCR amplicon generated from genomic DNA purified from peripheral blood cells. (B) PCR amplicon generated from Guthrie blood spot. (C) PCR amplicon generated from a crude lysate of peripheral blood cells (C). M: DNA markers; the arrow indicates the PCR amplicon with the size of 307 bp. W: indicates water blank as a negative control.</p

Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood - Fig 1

<p>(A) Representative melting curve-based genotyping of rs429358 at codon 112 of APOE. Black color indicates TT homozygote; red color indicates CC homozygote while blue color indicates TC heterozygote. (B) Representative melting curve-based genotyping of rs7412 at codon 158 of APOE. Black color indicates TT homozygote; red color indicates CC homozygote while blue color indicates TC heterozygote.</p

Sequences of primers, annealing temperatures and the sizes of PCR product in this study.

<p>Sequences of primers, annealing temperatures and the sizes of PCR product in this study.</p

Traces of Sanger sequencing of APOE genotyping of codon 112 (rs429358) and codon 158 (rs7412).

<p>TT indicates homozygote of TT; TC indicates TC heterozygote, and CC indicates homozygote of CC. The arrow indicates the position of the single nucleotide polymorphism.</p

Genotype and allele frequencies of APOE2, E3 and E4 in this study.

<p>Genotype and allele frequencies of APOE2, E3 and E4 in this study.</p

Meth reduces susceptibility to influenza A virus infections in human lung epithelial A549 cells.

<p>(A) A549 cells grown on glass coverslips were left un-treated, or treated with chloroquine (Clq.; 10 µM; as positive control) or meth at indicated concentrations, followed by the infection with influenza A/WSN/33 (H1N1) virus at an MOI of 1 PFU/cell in the absence of trypsin (single-cycle growth) and presence of the corresponding drugs at indicated concentrations. At 24 h post-infection, cells were fixed with formaldehyde and subjected to immunofluorenscence staining for detecting the infected cells by using an antibody against viral nucleoprotein (NP; green); cellular nuclei were located by DAPI staining (blue). A representative result from three independent experiments is shown. Mock: cells were neither exposed to the drugs nor infected with the virus. Scale bar: 100 µm. (B) NP-positive cells were counted from ten microscopic fields with >90% cell confluence. Data are expressed as mean values ± SD from a representative result of three independent experiments. Significant differences from the drug-untreated infected group (control) were indicated (***: p < 0.0001).</p

Meth reduces influenza A virus propagation in human lung epithelial A549 cells.

<p>A549 cells were left untreated or treated with meth at indicated concentrations for 24 h, followed by infection with human influenza virus strain A/WSN/33 (H1N1) at an MOI of 0.001 PFU/cell (trypsin present) in the presence of meth at respective concentrations. Virus progenies were collected at indicated time points, and subjected to plaque assays in MDCK cells to determine virus titers. Values are means ± SD of three replicates from a representative result of three independent experiments. Significant differences from the meth-untreated control were indicated (**: p < 0.01, ***: p < 0.0001).</p

Meth reduces synthesis of influenza viral proteins in human lung epithelial A549 cells.

<p>A549 cells were left untreated (control) or treated with meth at indicated concentrations for 24 h, and then infected with human influenza virus strain A/WSN/33 (H1N1) at an MOI of 0.001 PFU/cell in the presence of trypsin (multi-cycle growth) and meth at respective concentrations. Whole cell lysates were prepared at 48 h post-infection and subjected to Western blot analysis using antibodies against viral matrix protein-1 [M1] (A), viral nonstructural protein-1 [NS1] (C), and cellular actin. Mock: meth-untreated cells without influenza infection. A representative result from three independent experiments is shown. The expression levels of detected proteins were measured by densitometric analysis. M1 (B), and NS1 (D) levels were normalized by actin levels, and the relative optical density values were expressed as percentages of control. The results represent mean values ± SD of three replicates from a representative result of three independent experiments. Significant differences from the control were indicated (**: p < 0.01, ***: p < 0.0001).</p

The cytotoxic effect of meth on human lung epithelial A549 cells.

<p>A549 cells were treated with meth at indicated concentrations at 37°C for 72 h. (A) The trypan blue dye exclusion assay was performed to count the total, viable, and dead cells. (B) The cell survival rate was calculated, and data were expressed as percentages of viable counts in meth-treated groups relative to that in the meth-untreated group (control). The results are means ± SD of five replicates from a representative result of three independent experiments. Significant differences were indicated (***: p < 0.0001 versus control).</p

Effects of meth on influenza infection-induced IFN responses in human lung epithelial A549 cells.

<p>A549 cells were left untreated (control) or treated with meth at indicated concentrations for 24 h, followed by infection with human influenza virus strain A/WSN/33 (H1N1) at an MOI of 0.001 PFU/cell in the presence of trypsin (multi-cycle growth) and meth at respective concentrations. At 48 h post-infection, whole cell lysates were prepared and subjected to Western blot analysis using antibodies against cellular STAT1 (A), phospho-STAT1 [Tyr701] (C), MxA (E), and actin. Mock: meth-untreated cells without influenza infection. A representative result from three independent experiments is shown. The levels of detected proteins were measured by densitometric analysis. The expression levels of STAT1 (B), phospho-STAT1 [Tyr701] (D), and MxA (F) were normalized by actin levels, and the relative optical density values are expressed as percentage of control. The results represent mean values ± SD of three replicates from a representative result of three independent experiments. Significant differences from the control were indicated (*: p < 0.05, **: p < 0.01, ***: p < 0.0001).</p