Fig. 2.

Constitutive Pin1 downregulation suppresses oncogenic biological functions and signaling in vitro. a To establish stable-shPin1 cell lines, HL-60 or U937 or KG-1a cells were infected with lentivirus expressing scramble (Vec) or Pin1 shRNA (shPin1) with Puro^r. After puromycin selection for 1 week, Pin1 levels were validated by immunoblotting analysis. b Pin1 downregulation inhibits cell proliferation in indicated AML cell lines. Cell growth was monitored for 1 week by cell counting. p values were derived from the cell numbers for each group at the end point. c–e Cells were cultured in normal medium supplemented with methylcellulose for 1 or 2 weeks. When colonies became visible, cells were stained with p-iodonitrotetrazolium violet for counting. The number (d) and area (e) of colonies was measured and counted using ImageJ. Results present the mean ± SD of three independent experiments. f Cell lysates were subjected to western blot analysis with antibodies against the downstream oncogenic proteins of Pin1. Statistically significant differences using Student’s t test are indicated by p values. (*p < 0.05, **p < 0.01, ***p < 0.001)