Fig 2: Increase in cellular ROS prompted by loss of Merlin results in activated Hh signaling.(A) Merlin knockdown in MCF10AT and MCF7 cells increases whereas Merlin expression in SUM159 decreases GLI luciferase activity. The promoter activity was normalized to total protein and represented as percentage of activity compared to vehicle control. *indicates statistically significant. (B) Silencing Merlin in MCF7 and MCF10AT cells (KD) results in a significant increase in transcript levels of GLI1 and GLI2. The expression was assessed by qRT-PCR and normalized to ß-Actin. (*p < 0.05). (C) Merlin knockdown in MCF10AT cells increases GLI1 and GLI2 expression and facilitates nuclear accumulation of GLI1 and GLI2. Nuclear and cytosolic fractions were immunoblotted for GLI1 and GLI2. HDAC and a-tubulin were used to ascertain enrichment of the nuclear and cytosolic fractions, respectively. Shown are cropped images of the blots. (D) Increase in cellular ROS in Merlin-silenced cells activates Hh signaling. MCF7 cells silenced for Merlin were treated with MnTBaP or MnTBaP + menadione. Transcript levels of bona fide Hh transcription targets were assessed by real-time qRT-PCR. *p < 0.05 compared to vehicle control cells. (E) Addition of exogenous GSH counteracts the effect of Merlin deficiency on 8XGLI activity in MCF10AT KD cells (in a dose dependent manner). MCF10AT KD cells were transiently transfected with pGL3-8XGLI-Luc and treated with different concentrations of GSH. *indicates p < 0.05 compared to vehicle control cells. (F) GSH treatment of MCF10AT KD cells reinstates the inhibitory effect on Hh/GLI transcriptional targets. Cells were treated with different concentrations of GSH for 24 hours. Untreated MCF10AT KD cells were used as control. All experiments were done in triplicates. *p < 0.05 compared to vehicle control cells. (G) Merlin deficiency enforces dependence on Hh/GLI signaling. Merlin-deficient MCF10AT KD cells and non-targeting control transfected cells (MCF10AT NT) were xenografted into female athymic, nude mice and treated with either vehicle control or GANT61. Cells silenced for Merlin form tumors that grow rapidly (p < 0.05) compared to control tumors. Cells deficient for Merlin expression are effectively inhibited (p < 0.01) in their growth as tumors relative to the vehicle-treated cells. *p < 0.05 compared to vehicle control cells.