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. 2014 Oct 27;124(12):5352–5367. doi: 10.1172/JCI76561

Figure 5. TCR and TGF-β signaling converge on cMYC to differentially regulate miR-23a expression in CTLs.

Figure 5

(A) TCR activation, but not stimulation strength, suppresses miR-23a expression in CTLs. miR-23a expression in purified naive CTLs activated in vitro with the indicated concentrations of anti-CD3 (μg/ml) and 5 μg/ml anti-CD28 for 3 days. Data represent mean ± SEM; n = 4. *P < 0.05 by 1-way ANOVA and Bonferroni post-test. (B–D) TGF-β promotes miR-23a expression in CTLs. (B) Mature miR-23a and (C) Prdm1 expression in purified pMel-1 CTLs activated in vitro with varying concentrations of TGF-β for 72 hours. *P < 0.05 by 1-way ANOVA and Bonferroni post-test. (D) miR-23a expression in CTLs activated in vitro with tumor cell–conditioned media (25% in total medium) and neutralizing anti–TGF-β antibody. Data shown in B–D represent mean ± SEM; n = 3. Sup, supernatant from tumor cell–conditioned medium. (E–J) cMYC transcriptionally represses miR-23a in activated CTLs. (E) cMYC protein induction in purified CTLs upon 24 hours of TCR activation. (F) Pri-miR-23a and (I) mature miR-23a expression in purified pMel-1 CTLs treated with or without the cMyc inhibitor 10058-F4. Data in F and I represent mean ± SEM; n = 3 and n = 5, respectively. (G and H) mRNA expression of (G) cMYC and (H) regulators of cMYC activity in activated CTLs upon TGF-β treatment. Data shown in G and H represent mean ± SEM; n = 3. (J) Pri-miR-23a expression in Dicer-deficient CTLs upon cMyc inhibition, expressed as mean ± SEM; n = 3, and this represents 2 independent experiments. Red dashed lines represent expression levels in activated CTLs (control groups) that were set as 1.0, from which relative expression in experimental groups was calculated.