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.

Fig 3: GLI1 enhances tissue identification and discriminatory power of Merlin.(A and B) Breast tumor tissues showed significantly decreased staining intensity for Merlin (A) and significantly elevated staining intensity for GLI1 (B). (C and D) Logistic regression model applied to a binary variable of normal and tumor tissue to the data demonstrates the discriminatory power of Merlin (C) and GLI1 (D). (E) GLI1 mitigates the discriminatory power of Merlin.

Fig 4: Loss of Merlin prompts mesenchymal attributes through modulation of Hh signaling.(A) Merlin-deficient MCF10AT cells (KD) demonstrate significantly decreased (p < 0.01) invasion when treated with GANT61. (B) Loss of Merlin corroborates with loss of integrity of a basement membrane as evidenced by laminin-V staining that was rescued by GANT61 indicating that blocking Hh signaling attenuates invasive phenotype manifested upon loss of Merlin. Acinar structures formed by Merlin-silenced cells in a three-dimensional matrix were stained with anti-laminin-V antibody (red) to visualize the basement membrane. Nuclei are stained with DAPI (blue). (C) Merlin expression in SUM159 cells blunted mesenchymal attributes (N-Cadherin [CDH2], MMP-2, MMP-9, SNAI1, TWIST1, Vimentin, ZEB1 and ZEB2); abrogation of Merlin blunted the epithelial attributes (E-cadherin [CDH1] and Keratin 18) concomitant with a gain in the mesenchymal phenotype in MCF10AT cells. The graphs represent data analyzed to compare SUM159 (vector control) cells versus the SUM159 cells engineered to express Merlin and the MCF10AT (non-targeting) control cells versus the MCF10AT cells silenced for Merlin. Data was acquired by quantitative RT-PCR data and normalized to ß-actin expression relative to vector control. (D) Immunocytochemistry of MCF7 cells knocked down for Merlin shows an increase in Vimentin expression with a concomitant decrease in E-Cadherin expression. Nuclei are stained with DAPI (blue). The bar indicates 10 µm. (E) The GLI inhibitor GANT61 antagonized the effects of menadione on increase in mesenchymal attributes. MCF7 cells treated with Menadione showed an increase in EMT markers that were thwarted by the GLI transcription inhibitor GANT61.