Characterization of the tumor suppressive potential of PRDM11. (A) Representative screening result showing focus formation assay of MEFs transduced with either scramble control shRNA or a pool of 4 shRNA constructs targeting Prdm11 and subsequently transduced with either control (LZRS-GPF-IRES-HA) or MYC (LZRS-GPF-IRES-HA-MYC). (B) Schematic structure of PRDM11 from human (NM_001256696) and the corresponding PRDM11 protein (NP_001243625.1). Protein coding exons are shown as vertical black lines and are indicated on the protein structures as E1 through E6. The positions of a zinc knuckle (dark gray), the PR domain (black), and a nuclear localization signal (white) are indicated. (C) Relative levels of Prdm11 expression measured by qRT-PCR in a panel of tissues from male and female C57BL/6 mice. Levels of Prdm11 were normalized to Gapdh. Error bars represent standard deviation (SD) of technical triplicates. (D) Colony formation assay of Prdm11 WT or Prdm11 KO MEFs transduced with either control (pBabe) or MYC (LZRS-GPF-IRES-HA-MYC). One representative experiment of 3 is shown. (E-F) MEFs were transduced with control (pBabe-GFP) or PRDM11 (pBabe-HA-PRDM11) and used for (E) immunoblotting (IB) of cleaved caspase 3. Middle panel confirms PRDM11 overexpression, and vinculin served as control for equal loading (n = 2). (F) Growth curves measured in quadruplicates by crystal violet staining; error bars denote SD; 1 representative experiment of 6 is shown. (G) Propidium iodide profiles of MEFs (n = 4) transduced with PRDM11 (red) or control vector (blue). (H-I) p53 KO MEFs and p53 WT MEFs were transduced with control (pBabe-GFP) or PRDM11 (pBabe-HA-PRDM11) and used for. (H) Growth curves measured in quadruplicates by using crystal violet staining. Error bars denote SDs; 1 representative experiment of 2 is shown. (I) IB analysis of p53 KO MEFs. One representative experiment of 2 is shown. LN, lymph node.