Melanoma-induced loss of keratinocyte Dsg1 drives ERK1/2 dependent pro-migratory CXCL1 production. (A) Primary human keratinocytes were treated for 48 h with conditioned media from melanocytes (MC) or the melanoma cell lines WM1341D and 501mel and RNA or protein was collected. RT-PCR was performed for CXCL1 expression. Mean ± SEM depicted. n = 3. *P <0.05. One-way ANOVA. (B) Western blot was performed for Dsg1, ERK1/2, and phosphor-ERK1/2. Mean ± SEM depicted. n = 4, *P <0.05 **P < 0.01. One-way ANOVA. Increased ERK1/2 phosphorylation was observed in keratinocytes treated with melanoma-conditioned media. (C) Primary human keratinocytes were treated with melanocyte or melanoma-conditioned media with either DMSO or 5 μM U0126 (MEK1/2 inhibitor). Western blot was performed to confirm a decrease in ERK1/2 phosphorylation in U0126-treated cells. n = 3. (D) CXCL1 gene expression was no longer increased in keratinocytes treated with melanoma-conditioned media in the presence of U0126 n = 3. *P <0.05. One-way ANOVA. (E and F) Melanoma cells were treated with conditioned media from shNT, shDsg1, or shDsg1 plus a full-length (FL) wild-type Dsg1 rescue expressing keratinocytes for 24 h in the presence of DMSO or the CXCR2 inhibitor (500 nM SB22502) then plated for trans-well migration and collected after 24 h. Loss of keratinocyte Dsg1 no longer increased melanoma cell migration when the CXCL1 receptor, CXCR2, was inhibited. Mean ± SEM depicted. n = 3 *P < 0.05, **P < 0.01. One-way ANOVA. Source data are available for this figure: SourceData F6.
Figure 6.

Melanoma-induced loss of keratinocyte Dsg1 drives ERK1/2 dependent pro-migratory CXCL1 production. (A) Primary human keratinocytes were treated for 48 h with conditioned media from melanocytes (MC) or the melanoma cell lines WM1341D and 501mel and RNA or protein was collected. RT-PCR was performed for CXCL1 expression. Mean ± SEM depicted. n = 3. *P <0.05. One-way ANOVA. (B) Western blot was performed for Dsg1, ERK1/2, and phosphor-ERK1/2. Mean ± SEM depicted. n = 4, *P <0.05 **P < 0.01. One-way ANOVA. Increased ERK1/2 phosphorylation was observed in keratinocytes treated with melanoma-conditioned media. (C) Primary human keratinocytes were treated with melanocyte or melanoma-conditioned media with either DMSO or 5 μM U0126 (MEK1/2 inhibitor). Western blot was performed to confirm a decrease in ERK1/2 phosphorylation in U0126-treated cells. n = 3. (D) CXCL1 gene expression was no longer increased in keratinocytes treated with melanoma-conditioned media in the presence of U0126 n = 3. *P <0.05. One-way ANOVA. (E and F) Melanoma cells were treated with conditioned media from shNT, shDsg1, or shDsg1 plus a full-length (FL) wild-type Dsg1 rescue expressing keratinocytes for 24 h in the presence of DMSO or the CXCR2 inhibitor (500 nM SB22502) then plated for trans-well migration and collected after 24 h. Loss of keratinocyte Dsg1 no longer increased melanoma cell migration when the CXCL1 receptor, CXCR2, was inhibited. Mean ± SEM depicted. n = 3 *P < 0.05, **P < 0.01. One-way ANOVA. Source data are available for this figure: SourceData F6.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal