PIM genetic silencing in PTCL by shRNA.

<p>(A) MyLa cells were infected with lentiviral particles containing the pLKO.1-puro vector with a non-targeting shRNA, the Turbo-GFP gene, or shRNAs for each of the PIM family genes. Cells were maintained under Puromycin selection for 3 weeks. Images were obtained 8 days post-infection with a Nikon Ti Epi-Fluorescence microscope (10X magnification). Green fluorescence was also assessed in the negative (NT sh) and the positive controls by flow cytometry, and percentages of green cells are indicated in the histograms. (B) Triple knockdown efficiency was measured by RT-qPCR and compared with the Non-Targeting shRNA (NTsh). Graphs show the silencing at the mRNA level 15 days after the lentiviral infection. RQ, relative quantification, has been calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112148#s2" target="_blank">Methods</a> section (RQ = 2^−ΔΔCt). (C) Western blots showing the effect of the triple lentiviral infection on PIM protein levels 15 days post-infection (NI, non-infected cells; NTsh, cells infected with the non-targeting shRNA; GFP, cells infected with Turbo-GFP; Triple sh, cells infected with shPIM1+ shPIM2+ shPIM3). (D) These knockdown conditions did not affect cell viability. The percentage of non-viable cells was calculated as Annexin V+/7AAD− plus Annexin V+/7AAD+ cells: (NI, non-infected cells; NTsh, cells infected with the non-targeting shRNA; GFP, cells infected with Turbo-GFP; Triple sh, cells infected with shPIM1+ shPIM2+ shPIM3).