Fig. 1.

Regulation of perforin expression in Dicer^−/− CTLs. (A) Perforin protein (Western blot) and mRNA (Northern blot) levels analyzed in effector CTLs differentiated in vitro from Dicer^fl/fl (WT) and Dicer^fl/fl:CD4-Cre (Dicer^−/−) mice. On day 6, CTLs were restimulated with PMA, ionomycin, or both, in the presence or absence of CsA. Results are representative of three independent experiments. (B) Scheme of Cre-mediated Dicer deletion in activated CD8⁺ T cells from P14⁺:Dicer^fl/fl mice. (C) Real-time PCR analysis of Dicer mRNA in control (P14⁺:Dicer^fl/+-Cre or P14⁺:Dicer^fl/fl-mock) and Dicer^−/− effector CTLs analyzed on day 6. Values are normalized to the P14⁺:Dicer^fl/+-Cre sample. Red letters on the x axis refer to Western blot lanes in D. Mean ± SE for three or four different experiments is shown. (D) Western blot analysis of perforin expression in resting control (P14⁺:Dicer^fl/+-Cre, P14⁺:Dicer^fl/+-mock–transduced, and P14⁺:Dicer^fl/fl-mock–transduced) and Dicer^−/− (P14⁺:Dicer^fl/fl-Cre) effector and memory CTLs. Lanes marked with a red letter represent samples subsequently used for RNA sequencing. Results are representative of two (IL-2 10 U) or four (IL-2 100 U) experiments. (E) Quantification of perforin protein and mRNA levels in samples corresponding to lanes marked by red letters in D. Results from three or four experiments are averaged. *P < 0.05; **P < 0.01; ns, not significant.