(A, B) Cytotoxicity assay of differentiated CTLs from P14+wild type, P14+let-7Tg, and P14+Lin28Tg lymph nodes (A) or from P14+wild-type (WT), or P14+let-7Tg+Lin28Tg+Rag2−/− (4Tg) lymphocytes co-cultured with either LCMV gp33 or LCMV np396 peptide-pulsed splenocytes for 4–5 hr, either in the presence or absence of doxycycline (B). (C) Analysis of the internal complexity (FSC, forward scatter; SSC, side scatter) of effector (5 days after anti-CD3 mAb stimulation) CD8 T cells generated from wildtype, let-7Tg, and Lin28Tg lymphocytes (left) and quantification of SSC of CD8 T cells normalized to wild-type. (D) Quantification of Granzyme A and Granzyme B- positive granules in wild-type, let-7Tg, and Lin28Tg CTLs via MilliPore Amnis ImageStream. (E) Quantitative RT-PCR analysis of effector molecule mRNA expression: Gzma (Granzyme A), Gzmb (Granzyme B), Prf1 (Perforin) in naive and effector CD8 T cells from wild-type, let-7Tg, and Lin28Tg lymph nodes, presented relative to expression of the ribosomal protein Rpl13a. (F) Staining (top, middle) and MFI (bottom) of Granzyme B, Interferon-γ, and Granzyme A in wild-type, let-7Tg, and Lin28Tg effector CD8 T cells normalized to wild type. (G) Western blot analysis of lysates of wild-type, let-7Tg, and Lin28Tg effector CD8 T cells, probed with monoclonal antibodies against Perforin and actin. n.s., not significant (p>0.05), *p<0.05, **p<0.01, and ***p<0.001, compared with wild-type using two-tailed Student’s t-test. Data are from one experiment representative of three experiments (a, b, e; mean and s.e.m. of technical triplicates, f; mean and s.e.m of three experiments).