Journal: Immunology

Article Title: DNA methylation‐mediated Siglec‐7 regulation in natural killer cells via two 5′ promoter CpG sites

doi: 10.1111/imm.13179

Figure Lengend Snippet: Correlation in the cellular expression between FoxO1 and Siglec‐7 in three NK‐92MI cell lines, and peripheral Siglec‐7+ and Siglec‐7− NK cells. (a) Representative staining of peripheral blood from a healthy donor for the gating strategy to identify CD56+ natural killer (NK) cells and levels of Siglec‐7 expression (left), and the distribution of NK population for the Siglec‐7 subset is shown as a percentage of positive cells (right). ***P < 0·005, paired Student's t‐test. (b) Real‐time polymerase chain reaction (PCR) analysis of Siglec‐7 transcripts in sorted Siglec‐7−CD56+ and Siglec‐7+CD56+ cells from four healthy donors. Siglec‐7 expression in Siglec‐7+ subset was related to that in Siglec‐7− cells, and all expressions were normalized to GAPDH. ***P < 0·005, paired Student's t‐test. (c) Relative expression of FoxO1 among three NK cell lines, and the results were compared with those of parental cells (arbitrarily set as 1·0). The results were representative of three experiments. Data are presented as mean ± SD of triplicates. ***P < 0·005, Student's t‐test. (d) Western blot comparing nuclear and cytoplasmic FoxO1 levels among three NK cells, and the results were normalized with LaminA and GAPDH, respectively, as indicated at the bottom. These representative data were from one of three independent experiments. (e) Comparison of nuclear FoxO1 expression between sorted Siglec‐7−CD56+ and Siglec‐7+CD56+ cells from five healthy donors by performing flow cytometric analysis. The shaded area represented the results from cells incubated with isotype control, and the blue and red lines represented the results from indicated cells (left). The representative of mean fluorescence intensity (MFI) of NK subsets (right). ns, not significant, paired Student's t‐test.

Article Snippet: Human NK cell line, NK‐92MI, was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan), and the NK‐92MI‐S and ‐S7N cells were established in our laboratory as described previously.

Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Fluorescence

Journal: Immunology

Article Title: DNA methylation‐mediated Siglec‐7 regulation in natural killer cells via two 5′ promoter CpG sites

doi: 10.1111/imm.13179

Figure Lengend Snippet: Inhibition of the DNA methyltransferase (DNMT) activity promoted Siglec‐7 expression in natural killer (NK) cells. (a) Three NK cells were examined by immunoblotting for the protein expression of DNMT1, DNMT3A and DNMT3B, with GAPDH as a loading control. Relative amount was used to compare the protein level from each group, and was shown at the bottom by setting the reference protein at 1. Representative data were from one of three independent experiments. (b) Representative flow cytometric staining pattern along a time course (as indicated) for the NK cell surface Siglec‐7 comparison in expression after the inhibition of methylation with 5‐azacytidine (5azaC) treatments in three NK‐92MI cell lines. The shaded area represented the results obtained from cells incubated with isotype control, and the blue and red lines indicated control and treated groups, respectively. (c) Column chart of relative MFI of Siglec‐7 expression. For each cell line, data were first normalized to the isotype control, and presented in the time course manner for with or without 5azaC treatment. *P < 0·05; ***P < 0·005, Student's t‐test. The result shown was representative of three independent experiments. (d) Column chart presenting real‐time polymerase chain reaction (PCR) analysis of Siglec‐7 transcripts in NK cells, as indicated, with or without 5azaC treatments from the indicated time point. All the expression was first normalized to GAPDH and, for each cell, the expression level of Siglec‐7 transcript from different time points was compared with that of untreated DMSO control. ***P < 0·005 comparison within the same cell type; ### P < 0·005 comparison between parental, NK‐92MI‐S and ‐S7N, Student's t‐test.

Article Snippet: Human NK cell line, NK‐92MI, was purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan), and the NK‐92MI‐S and ‐S7N cells were established in our laboratory as described previously.

Techniques: Inhibition, Activity Assay, Expressing, Western Blot, Staining, Methylation, Incubation, Real-time Polymerase Chain Reaction