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. 2025 May 6;14(5):e70083. doi: 10.1002/jev2.70083

FIGURE 4.

FIGURE 4

m6A levels in EVs regulate inflammatory responses via TLR8 in macrophages. Differentiated THP‐1 cells (dTHP‐1) were pretreated with or without CU‐CPT‐9a and CU‐CPT‐4a. (A) Whole‐cell lysates obtained from dTHP‐1 cells were subjected to Western blot analysis using an anti‐phospho p65 antibody, anti‐p65 antibody and anti‐β‐actin antibody. Representative images from three independent experiments are shown. Conditioned medium from dTHP‐1 cells treated with CCD‐841‐CoN or HT29 small EVs was used for ELISA. TNF‐α (B, left and C, left), and IL‐6 (B, right and C, right). Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant. dTHP‐1 cells were pre‐transfected with or without TLR8 siRNA. (D) Whole‐cell lysates obtained from dTHP‐1 cells treated with CCD‐841‐CoN small EVs or HT29 small EVs were subjected to Western blot analysis using an anti‐phospho p65 antibody, anti‐p65 antibody and anti‐β‐actin antibody. Representative images from three independent experiments are shown. Conditioned medium was used for the ELISA. TNF‐α (E, left) and IL‐6 (E, right). Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; **p < 0.01, ***p < 0.001, ****p < 0.0001. dTHP‐1 cells were pre‐transfected with or without TLR3 siRNA. (F) Whole‐cell lysates obtained from dTHP‐1 cells treated with CCD‐841‐CoN small EVs or HT29 small EVs were subjected to Western blot analysis using an anti‐phospho p65 antibody, anti‐p65 antibody and anti‐β‐actin antibody. Representative images from three independent experiments are shown. Conditioned medium was used for ELISA. TNF‐α (G, left) and IL‐6 (G, right). Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; ***p < 0.001, ****p < 0.0001; ns, not significant. (H) HT29 cells were cultured in a conditioned medium from dTHP‐1 cells transfected with or without TLR8 siRNA and CCD‐841‐CoN small EVs or HT29 small EVs. Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; *p < 0.05, **p < 0.01. HT29‐derived EVs‐RNA were pretreated with or without rALKBH5 and introduced into dTHP‐1 cells transfected with or without TLR8 siRNA. TNF‐α (I) and IL‐6 (J) concentrations were measured via ELISA using conditioned medium from dTHP‐1 cells. Values are presented as the mean ± SEM for each group. One‐way ANOVA post hoc Tukey's test; *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, not significant. See also Figure S7.