Fig. 6
From: ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP
ERAP1 inhibition impairs Hh-dependent tumor growth in vivo. a–f NSG mice were grafted with spontaneous primary MB from Math1-cre/PtcC/C mice. Tumor masses (150 mm3) were intratumorally injected with Leu-SH. a Tumor growth was monitored. b Representative flank allograft average volumes (lower panel) and quantification of tumor explants (upper panel). c, d Ki67, NeuN, and cleaved Caspase-3 (Cl.Casp-3) immunohistochemical stainings of allograft tumor samples. d Quantification of immunohistochemical stainings shown in c. Scale bar 100 μm. e mRNA and f protein expression levels of Hh targets from tumors assayed in b. g Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor in NSG mice after i.p. injection of Leu-SH. Scale bars, 500 μm and 200 μm (upper and lower panels, respectively). h Representative average volume of orthotopic tumor. i–j NSG mice were grafted with spontaneous primary MB from Math1-cre/PtcC/C mice genetically silenced for ERAP1 expression. i Representative images of mice and the explanted tumor masses. j Quantification of the flank allograft average tumor volume. ERAP1 protein expression is shown below. In f, j, actin was used as loading control. k H&E and representative Masson’s trichrome staining of tumors. Scale bar 100 μm. l mRNA levels of the indicated Hh target genes. m Representative H&E images (low and high magnification) of a murine MB cell-derived orthotopic tumor genetically interfered for ERAP1 before the injection in NSG mice cerebella. Scale bars, 500 and 200 μm (upper and lower panels, respectively). n–p ERAP1 accelerates Hh-MB formation. n Tumor volume of mice subcutaneously transplanted with GCPs from tumor-prone Math1-cre/PtcC/C animals overexpressing ERAP1. o Representative flank allograft average volumes (lower panel) and quantification of the explanted tumor masses (upper panel). p mRNA expression of Hh target genes from the tumor masses assayed in o. q Survival curves of Math-cre/PtcC/C mice treated with Leu-SH or vehicle. Results in e, l, p were normalized to endogenous GAPDH and HPRT controls and expressed as in Fig. 1. All data represent the mean of three independent experiments. Mean ± S.D. of tumor (n = 6) for each treatment. *P < 0.05, **P < 0.01, *** P < 0.001 calculated by two-sided Student’s t-test
