Figure 6.
Efficacy of 5-Aza in CIMP-positive NK-LCLs. (A) Number of live cells in CIMP-positive NK-LCLs (n = 2), CIMP-negative NK-LCLs (n = 1), and normal NK cells from healthy volunteers (n = 2) treated in vitro with 4 different concentrations of 5-Aza ranging from 8.0 × 10–3 (×1/125) to 1 μg/mL (×1) for 7 days relative to those treated with vehicle (DMSO). Three wells per concentration were used in each experiment (mean ± standard deviation [SD]), and the experiment was repeated twice for validation. The overall P value was calculated using the Kruskal-Wallis test. The P values for pairwise comparisons were calculated using the Mann-Whitney U test. (B) M values were calculated for CIMP-positive NK-LCLs (S_C_10) and NK cells from a healthy volunteer (Control 4) at probes where silencing by DNA methylation was observed in CIMP-positive CAEBV. The ΔM values, representing the difference between the average M values from 3 wells of 5-Aza (1×) treatment and 3 wells of the vehicle control (DMSO), were plotted for each probe (mean ± 95% CI; n = 855 probes). Significance was assessed using the Mann-Whitney U test. (C) Hypomethylated (ΔM < –0.5 and adj P < .05) and upregulated genes in CIMP-positive NK-LCLs (S_C_10) after 7 days of in vitro 5-Aza treatment vs vehicle. Significantly upregulated genes (log2 fold change ≥ 1 and adj P < .05) are highlighted in red. (D) Pathway analysis of DNA hypomethylated and upregulated genes in CIMP-positive NK-LCLs after in vitro 5-Aza treatment was performed using Metascape software. (E) Schematic showing the protocol for the in vivo study of 5-Aza using NOG-IL2 mice injected with CIMP-positive NK-LCLs (S_C_10). (F) Percentage of hCD45+ cells in the peripheral blood of 5-Aza vs vehicle-treated NOG-IL2 mice at 42 days after transplantation of the CIMP-positive NK-LCLs (S_C_10) (mean ± SD; n = 5 mice per group). Differences were evaluated using 2-way analysis of variance. (G-H) Spleen weight (G) and percentage of hCD45+ cells in the spleen (H) on day 42 in mice treated with 5-Aza or vehicle (mean ± SD; n = 5 mice per group). The difference was evaluated using the Mann-Whitney U test. DMSO, dimethyl sulfoxide; dsRNA, double-stranded RNA; GO, gene ontology; R-HSA, reactome-Homo sapiens.

Efficacy of 5-Aza in CIMP-positive NK-LCLs. (A) Number of live cells in CIMP-positive NK-LCLs (n = 2), CIMP-negative NK-LCLs (n = 1), and normal NK cells from healthy volunteers (n = 2) treated in vitro with 4 different concentrations of 5-Aza ranging from 8.0 × 10–3 (×1/125) to 1 μg/mL (×1) for 7 days relative to those treated with vehicle (DMSO). Three wells per concentration were used in each experiment (mean ± standard deviation [SD]), and the experiment was repeated twice for validation. The overall P value was calculated using the Kruskal-Wallis test. The P values for pairwise comparisons were calculated using the Mann-Whitney U test. (B) M values were calculated for CIMP-positive NK-LCLs (S_C_10) and NK cells from a healthy volunteer (Control 4) at probes where silencing by DNA methylation was observed in CIMP-positive CAEBV. The ΔM values, representing the difference between the average M values from 3 wells of 5-Aza (1×) treatment and 3 wells of the vehicle control (DMSO), were plotted for each probe (mean ± 95% CI; n = 855 probes). Significance was assessed using the Mann-Whitney U test. (C) Hypomethylated (ΔM < –0.5 and adj P < .05) and upregulated genes in CIMP-positive NK-LCLs (S_C_10) after 7 days of in vitro 5-Aza treatment vs vehicle. Significantly upregulated genes (log2 fold change ≥ 1 and adj P < .05) are highlighted in red. (D) Pathway analysis of DNA hypomethylated and upregulated genes in CIMP-positive NK-LCLs after in vitro 5-Aza treatment was performed using Metascape software. (E) Schematic showing the protocol for the in vivo study of 5-Aza using NOG-IL2 mice injected with CIMP-positive NK-LCLs (S_C_10). (F) Percentage of hCD45+ cells in the peripheral blood of 5-Aza vs vehicle-treated NOG-IL2 mice at 42 days after transplantation of the CIMP-positive NK-LCLs (S_C_10) (mean ± SD; n = 5 mice per group). Differences were evaluated using 2-way analysis of variance. (G-H) Spleen weight (G) and percentage of hCD45+ cells in the spleen (H) on day 42 in mice treated with 5-Aza or vehicle (mean ± SD; n = 5 mice per group). The difference was evaluated using the Mann-Whitney U test. DMSO, dimethyl sulfoxide; dsRNA, double-stranded RNA; GO, gene ontology; R-HSA, reactome-Homo sapiens.

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