Fig. 6

SMO-193a.a. is a downstream target of HH signaling and in vivo effects of SMO-193a.a alteration. a Left, circ-SMO expression in 456 and 3691 CSCs treated with Shh. Right, IB of SMO-193a.a. and Gli1 in 456 and 3691 CSCs treated with Shh. b IB of SMO-193a.a. and Gli1 in 456 and 3691 CSCs with FUS KD. c Luciferase assay of wild type or Gli1 binding site mutated FUS promoter activities after Shh stimulation. d ChIP assay of FUS promoter sequences using Gli1 antibody in 456 and 3691 CSCs. Bcl2 was used as positive control. e IB and qPCR of FUS protein and mRNA levels in 456 and 3691 CSCs with Gli1 KD. f FUS and Gli1 protein level in 9 randomly selected GBM samples. g Upper, Representative images of in vivo tumorigenicity assay collected at day 25(456 CSC) and day 30 (3691 CSC) post-implantation using 456 and 3691 CSCs with indicated modifications. Each group contains 5 mice. Lower, Representative images of immunohistochemistry (IHC) of Gli1 expression in above mice. h Survival analysis of in vivo tumorigenicity assay using indicated cells. Each group contains 5 mice. i Correlation of Gli1 mRNA and circ-SMO in 86 GBM patients and correlation of Gli1 protein and SMO-193a.a. in 86 GBM patients. j Graphic abstract. Encoded by circular SMO (circ-SMO), SMO-193a.a. is required for Shh-induced SMO activation, via interacting with SMO, enhancing SMO cholesterol modification and releasing SMO from the inhibition of patched transmembrane receptors 1 (PTCH1). Moreover, circ-SMO/SMO-193a.a. is positively regulated by FUS, a direct transcriptional target of Gli1. Shh/Gli1/FUS/SMO-193a.a. form a positive feedback loop to sustain HH signaling activation in GBM. Lines show the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Data are representative from at least three experiments with similar results