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. 2021 Jan 15;129(1):017007. doi: 10.1289/EHP7699

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EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted.

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Figure 8A is a flowchart depicting effects of treatment on infected mice having three steps. Step 1: Developmentally Exposed weanlings is divided into two parts vehicle and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Step 2: Step 1 infused with water the effects of treatment with control and Zebularine lead to D N A methylation. Step 3: The D N A methylation in control vehicle and 2,3,7,8-tetrachlorodibenzo-p-dioxin is maintained and the D N A methylation in Zebularine vehicle and 2,3,7,8-tetrachlorodibenzo-p-dioxin is decreased. Figures 8B, 8C, 8D, 8E, 8F, and 8G are clustered bar graphs titled Total, Virus-specific, Activated, Naïve, T helper, and T follicular helper plotting number (10 begin superscript 5 end superscript), ranging from 0 to 15 in increments of 5; number (10 begin superscript 4 end superscript) ranging from 0.0 to 1.5 in increments of 0.5, percentage of clusters of differentiation 4, ranging from 0 to 25 in increments of 5; percentage of clusters of differentiation 4, ranging from 0 to 80 in increments of 20; percentage of clusters of differentiation 4, ranging from 0 to 25 in increments of 5; and percentage of clusters of differentiation 4, ranging from 0 to 5 in unit increments (y-axis) for control and Zebularine, including vehicle and 2,3,7,8-tetrachlorodibenzo-p-dioxin (x-axis), respectively. — Effects of Zebularine (Zeb) on $CD 4^{+}$ T cells from developmentally exposed mice after infection with IAV. (A) Mice were developmentally exposed to vehicle or TCDD. At weaning (21 d of age), mice were randomly selected be given normal vivarium water to maintain DNA methylation levels or water containing Zeb ( $0.2 mg / mL$ ) to decrease DNA methylation levels. At 6–8 wk of age, mice developmentally exposed to vehicle (white bar) and TCDD (orange bar) were infected with IAV. Mediastinal lymph nodes (MLNs) were harvested 9 d after infection, and cells stained for flow cytometry. (B) The number of $CD 4^{+}$ T cells or (C) virus-specific ${(I - A {}^{b}{NP}_{311 - 325})}^{+}$ $CD 4^{+}$ T cells were quantified from MLNs of mice on control (black outline) or Zeb (green outline) water. The percentage of (D) activated ( $CD 44^{hi} CD 62 L^{-}$ ) or (E) naïve ( $CD 44^{lo} CD 62 L^{+}$ ) $CD 4^{+}$ T cells from control or Zeb mice. The percentage of (F) Th1 ( ${Tbet}^{+} CD 4^{+}$ ) or (G) Tfh ( $CXCR 5^{+} PD 1^{+} CD 44^{hi} CD 4^{+}$ ) isolated from control or Zeb mice. For each group, 5–9 developmentally exposed offspring were used: control water vehicle (8), control water TCDD (5), Zeb water vehicle (9), and Zeb water TCDD (6). Due to a limited number of offspring, individual offspring were defined as the statistical unit, rather than the dam. All data shown are $mean \pm SEM$ . *, $p \leq 0.05$ , using a two-way ANOVA with Tukey’s HSD and Student’s $t$ -test. #, $p \leq 0.05$ , using Student’s $t$ -test. The numerical data in the graphs and $p$ -values are provided in Table S7. Note: ANOVA, analysis of variance; HSD, honestly significant difference; $H_{2} O$ , water; IAV, influenza A virus; SEM, standard error of the mean; T, TCDD; Tbet, T-box transcription factor; TCDD, 2,3,7,8-tetrachlorodibenzo- $p$ -dioxin; Tfh, T follicular helper; Th, T helper; V, vehicle.