RT-PCR Confirmation of gene expression in Lent-clones.

<p>Total RNAs were isolated from Lent-clones and un-transduced Hela cells and a two-step RT-PCR was performed using Phusion RT-PCR kit (Thermo Scientific). Corresponding pairs of primers were used to amply different fragments. House-keeping gene β-actin expression was also included as controls.</p

IHC staining of Lent-clones.

<p>Cells from Lent-clones and Hela cells were harvested and cytospun onto glass slides. After fixation, the cells were stained with anti-mouse IL-12 antibody or isotype control antibody followed by anti-mouse IgG secondary antibody conjugated with HRP. The cells were then treated with substrate DAB and observed under microscope.</p

Lentiviral titer determination.

<p>Each virally transduced 293-Lent clones (Lent-IF/Lent-IL12/Lent-Fas/Lent-LacZ) were collected for EmGFP-based cytometry analysis. Viral titer of each lent-clone was calculated based on cytometry reading and represented.</p

Induction of apoptosis in tumor cells.

<p>A total of 2x10^5 cells of Hela cells and lent-clones in triplicates were co-cultured with human NK cells for 24 hours. After the removal of suspension NK92 cells, the caspase 3 activities in the remaining tumor cells were detected using a Caspase 3 Assay Kit (abcam). The statistical analyses were conducted between the corresponding un-induced and induced groups using one-way analysis of variance (ANOVA) with the Tukey’s post test (***p<0.001).</p

Activation of human NK92 cells.

<p>(a) IFN-γ ELISA. Cells of Lent-clones and Hela cells in triplicates were co-cultured with human NK92 cells and the supernatants were collected. IFN-γ activity in the supernatants was detected using Human IFN-γ ELISA Kit. The statistical analyses were conducted between the control (Hela+NK) and each Lent-clone (as the bars represented in the figure) using one-way analysis of variance (ANOVA) with Tukey’s post test. (*p<0.05; **p<0.01) (b) Cytotoxicity of NK92 cells. Cells of each lent-clone and Hela in triplicates were co-cultured with human NK92 cells. After co-culture, NK92 cells were removed and remaining tumor cells’ proliferation was measured with Promega’s CellTiter 96ueous nonRadioactive cell proliferation assay kit. The statistical analyses were conducted between the control (Hela+NK) and each virally transduced lent-clone using one-way analysis of variance (ANOVA) with the Tukey’s post test (*p<0.05; **p<0.01).</p

Scatterplot of enriched KEGG pathways.

<p>The ordinate represents the enriched pathways, and the abscissa represents the rich factor of corresponding pathways; the size of the spots represents the number of genes related to DMRs enriched in each pathway, while the color of the spot represents the corrected <i>p</i> value of each pathway. The rich factors indicate the ratio of the number of DMGs mapped to a certain pathway to the total number of genes mapped to this pathway. Greater rich factor means greater enrichment. DMRs: differentially methylated regions; DMGs: differentially methylated genes.</p

DMR distribution among different genetic elements in Japanese Black cattle and Chinese Red Steppes.

<p>DMR distribution among different genetic elements in Japanese Black cattle and Chinese Red Steppes.</p

Top 20 DMRs based on the ratio of methylation levels between Japanese Black cattle and Chinese Red Steppes.

<p>Top 20 DMRs based on the ratio of methylation levels between Japanese Black cattle and Chinese Red Steppes.</p

Numbers of DMRs, hyper-methylated genes, and hypo-methylated genes in Japanese black cattle (Wagyu) and Chinese Red Steppes.

<p>(A) Numbers of DMRs; (B) Numbers of genes related to DMRs. DMRs: differentially methylated regions.</p

DNA methylation levels across genomic elements in Japanese black cattle (Wagyu) and Chinese Red Steppes.

<p>Abscissa represents different genomic elements, the ordinate represents the level of methylation and different colors represents different sequence contexts (CpG, CHG, CHH). WC_LM1, WC_LM2, WC_LM3: longissimus dorsi muscle samples of Japanese black cattle (Wagyu); RC_LM1, RC_LM2, RC_LM3: longissimus dorsi muscle samples of Chinese Red Steppes.</p