Supplementary Figure Legends

Suppl. Fig. 1

Normalised Gli1 mRNA levels in SAG-induced NIH3T3 cells as determined by QPCR. Cells were treated for 24 h with DMSO or 10 µM of the PKCδ inhibitor Rottlerin.

Suppl. Fig. 2

Influence of TPA (24 h treatment) on full-length GLI2 activity. NIH3T3 cells were transfected with a Hh reporter plasmid, renilla luciferase plasmid for normalisation and a GLI2 expression plasmid. As positive control, a SUFU expression plasmid was cotransfected.

Suppl. Fig. 3

Inhibition of the Hh signalling pathway by Mek1 blockade. NIH3T3 cells were transfected with a Hh reporter plasmid and a renilla luciferase plasmid for normalisation. Cells were then treated with the synthetic Smo agonist SAG in combination with DMSO or the Mek1 inhibitor PD98059 (50 µM).

Suppl. Fig. 4

Mek1-mediated superactivation of GLI1 is not inhibited by TPA. NIH3T3 cells were transfected with GLI1, together with EGFP or dominant-active Mek1, and Hh reporter plasmids. Subsequently, cells were treated with DMSO, TPA or the Mek1 inhibitor PD98059 (50 µM) for 24 h. Shown is a representative result of three independent experiments.

Suppl. Fig. 5

QPCR analysis of additional GLI target genes.

(A) PTCH expression. It should be noted that PTCH expression might be under control of TPA-regulated factors, which are Hh/GLI-independent (e.g. AP-1, as described in Brellier F et al., 2004). This could explain why TPA does not reduce GLI1 mRNA levels in 22Rv1 cells, but still reduces PTCH expression.

(B) HIP expression. Hedgehog-interacting protein (HIP) does not seem to be a reliable read-out for Gli activity in human tumour cells since neither FSK nor TPA can reduce HIP mRNA levels. Similar findings were reported previously (Olsen CL et al., 2004).

(C) NMYC expression. DU145 cells do not express NMYC. Furthermore, in analogy to PTCH regulation, there might be a Hh/GLI-independent influence of TPA on NMYC expression.

Suppl. Fig. 6

Simplified scheme of the Hh signalling pathway and the inhibitors used in this study. Please note that this scheme is based on genetic data and therefore Sufu is located between Smo and Gli2/3.