Loss of Grhl1 is associated with melanoma-mediated Dsg1 loss. (A) Model depicting the proposed signaling pathway leading to Dsg1 loss in keratinocytes. (B and C) Primary human keratinocytes were treated for 24 h with conditioned media from melanocytes (MC) or the melanoma cell lines WM1341D and 501mel and RNA or protein was collected. RT-PCR and Western blots were performed for Grhl1. Significantly lower mRNA and protein expression of Grhl1 was observed in the keratinocytes treated with melanoma-conditioned media when compared with melanocyte control–conditioned media. RNA levels and quantification for blots represent average fold change. Mean ± SEM depicted. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA. (D) Melanocytes or WM1341D melanoma cells were seeded with normal primary keratinocytes and grown as 3D organotypic raft cocultures for 6 d. Paraffin sections were costained for Grhl1 and Mel-A. Pixel intensities were determined and the ratio of proximal to distant intensities plotted. A significant decrease in Grhl1 intensity proximal to the WM1341D lesions was observed. Mean ± SEM *P < 0.05. Student’s t test. (E) Paraffin-embedded sections of benign nevi and melanomas were costained for Grhl1 and Mel-A and nuclear and cytoplasmic pixel intensities were measured in cells proximal to Mel-A stained cells and plotted as nuclear/cytoplasmic ratios. A significant decrease in keratinocyte Grhl1 intensity was observed in keratinocytes adjacent to melanoma compared with those in benign nevi. Mean ± SEM depicted. n > 3. **P < 0.01. Student’s t test. (F) Melanocytes or WM1341D melanoma cells were seeded with primary keratinocytes and grown as 3D organotypic raft cocultures for 6–10 d. Paraffin sections were costained for Slug and Mel-A. Pixel intensities were determined, and the ratio of proximal to distant intensities was plotted. A significant increase in Slug intensity proximal to the WM1341D lesions was observed. n = 3 ±SEM; ***P < 0.001. Student’s t test. (G) Paraffin-embedded sections of benign nevi and melanomas were costained for Slug and Mel-A. Nuclear and cytoplasmic pixel intensities were measured in cells proximal to Mel-A stained cells and the nuclear/cytoplasmic ratios were plotted. A significant increase in keratinocyte Slug intensity was observed in keratinocytes adjacent to melanoma compared with those in benign nevi. Mean ± SEM depicted. n > 3. *P < 0.05. Student’s t test. (H) Primary human keratinocytes exogenously expressing GFP or Grhl1 were treated for 24 h with conditioned media from melanocytes (MC) or the melanoma cell lines WM1341D, and 501mel and RNA was collected. RT-PCR was performed for Dsg1. Introduction of Grhl1 into the keratinocytes rescued Dsg1 loss caused by melanoma conditioned media. n = 3. Mean ± SEM depicted. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA. (I and J) Similarly, primary human keratinocytes exogenously expressing siCTL or siSNAI2 were treated for 24 h with conditioned media from melanocytes (MC) or the melanoma cell lines WM1341D, and 501mel and RNA was collected. RT-PCR was performed for Dsg1 and Grhl1. siSNAI2 transfected keratinocytes rescue Dsg1 and Grhl1 loss caused by melanoma-conditioned media. n = 3. Mean ± SEM depicted. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA. Source data are available for this figure: SourceData F3.
Figure 3.

Loss of Grhl1 is associated with melanoma-mediated Dsg1 loss. (A) Model depicting the proposed signaling pathway leading to Dsg1 loss in keratinocytes. (B and C) Primary human keratinocytes were treated for 24 h with conditioned media from melanocytes (MC) or the melanoma cell lines WM1341D and 501mel and RNA or protein was collected. RT-PCR and Western blots were performed for Grhl1. Significantly lower mRNA and protein expression of Grhl1 was observed in the keratinocytes treated with melanoma-conditioned media when compared with melanocyte control–conditioned media. RNA levels and quantification for blots represent average fold change. Mean ± SEM depicted. n = 3. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA. (D) Melanocytes or WM1341D melanoma cells were seeded with normal primary keratinocytes and grown as 3D organotypic raft cocultures for 6 d. Paraffin sections were costained for Grhl1 and Mel-A. Pixel intensities were determined and the ratio of proximal to distant intensities plotted. A significant decrease in Grhl1 intensity proximal to the WM1341D lesions was observed. Mean ± SEM *P < 0.05. Student’s t test. (E) Paraffin-embedded sections of benign nevi and melanomas were costained for Grhl1 and Mel-A and nuclear and cytoplasmic pixel intensities were measured in cells proximal to Mel-A stained cells and plotted as nuclear/cytoplasmic ratios. A significant decrease in keratinocyte Grhl1 intensity was observed in keratinocytes adjacent to melanoma compared with those in benign nevi. Mean ± SEM depicted. n > 3. **P < 0.01. Student’s t test. (F) Melanocytes or WM1341D melanoma cells were seeded with primary keratinocytes and grown as 3D organotypic raft cocultures for 6–10 d. Paraffin sections were costained for Slug and Mel-A. Pixel intensities were determined, and the ratio of proximal to distant intensities was plotted. A significant increase in Slug intensity proximal to the WM1341D lesions was observed. n = 3 ±SEM; ***P < 0.001. Student’s t test. (G) Paraffin-embedded sections of benign nevi and melanomas were costained for Slug and Mel-A. Nuclear and cytoplasmic pixel intensities were measured in cells proximal to Mel-A stained cells and the nuclear/cytoplasmic ratios were plotted. A significant increase in keratinocyte Slug intensity was observed in keratinocytes adjacent to melanoma compared with those in benign nevi. Mean ± SEM depicted. n > 3. *P < 0.05. Student’s t test. (H) Primary human keratinocytes exogenously expressing GFP or Grhl1 were treated for 24 h with conditioned media from melanocytes (MC) or the melanoma cell lines WM1341D, and 501mel and RNA was collected. RT-PCR was performed for Dsg1. Introduction of Grhl1 into the keratinocytes rescued Dsg1 loss caused by melanoma conditioned media. n = 3. Mean ± SEM depicted. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA. (I and J) Similarly, primary human keratinocytes exogenously expressing siCTL or siSNAI2 were treated for 24 h with conditioned media from melanocytes (MC) or the melanoma cell lines WM1341D, and 501mel and RNA was collected. RT-PCR was performed for Dsg1 and Grhl1. siSNAI2 transfected keratinocytes rescue Dsg1 and Grhl1 loss caused by melanoma-conditioned media. n = 3. Mean ± SEM depicted. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA. Source data are available for this figure: SourceData F3.

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