Fig. 5.
ERAP1 impinges Hh-dependent tumor cell growth in vitro. a–f Primary cell cultures from Math1-cre/PtcC/C mice MBs were treated with different amounts of Leu-SH. a, b Cells were counted with trypan blue at the indicated time points to evaluate the growth rate of viable cells (a) and the percentage of cell death (b). c Cleaved Caspase-3 protein levels in cells treated with Leu-SH at the indicated concentration for 24 h. d–f Percentage of BrdU uptake (d) and Gli1 mRNA (e), and protein (f) expression in MB cells treated with Leu-SH at the indicated concentrations for 24 h. g MB Stem-Like Cells (MB-SLCs) from Math1-cre/PtcC/C mice were treated with Leu-SH as in (a) and counted with trypan blue at the indicated time points. h MB-SLCs were dissociated and treated with the indicated concentrations of Leu-SH or DTT as control. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was evaluated. The self-renewal MB-SLCs capability is expressed as percentage of neurosphere-forming cells (right). Representative bright field images of tumor neurospheres after Leu-SH treatment are shown (left). Scale bar 100 µM. i, j mRNA and protein expression levels of Hh target genes of MB-SLCs treated with the indicated concentrations of Leu-SH for 24 h. Actin was used as loading control. Results in e, i were normalized to endogenous GAPDH and HPRT controls and expressed as described in Fig. 1 legend. All data are representative of three independent experiments. Mean ± S.D. *P < 0.05; **P < 0.01 calculated using two-tailed Student’s t-test