Figure 1. SPARC controls SLUG expression and other markers of the EMT-like transition in melanoma cells and primary melanocytes.

(A) Analysis of EMT regulators mRNA levels: RNAs were prepared from 501mel cells infected with control adenovirus (AdCTRL) or adenovirus-expressing SPARC (AdSPARC) for 3 days. Relative gene expression levels of the indicated E-cadherin repressors were analyzed by SYBR green-based real-time Q-PCR. Data are expressed in arbitrary units as fold change between AdSPARC-infected and control cells. Columns, mean of three independent amplifications performed in duplicate; error bars, SD. *P<0.05; NS, not significant (Student’s test). (B) Expression levels of EMT markers in SPARC overexpressing melanoma cells: immunoblots of 501mel cells infected with control adenovirus (AdCTRL) or adenovirus-expressing SPARC (AdSPARC) for the indicated times, and of 501mel cell clones expressing SPARC (cl. SPARC #1 and #2) or control vector (cl. CTRL). Total protein lysates and cell supernatants were analyzed for expression of SPARC-Myc transgene, endogenous SPARC, SLUG, E-cadherin, P-cadherin and Fibronectin. HSP60 and Ponceau S-stained bands were used as loading controls in lysates and culture media, respectively. (C) SPARC induces an EMT-like phenotype: F-actin and immunofluorescent staining of 501mel cell clones expressing SPARC (cl.SPARC) or control vector (cl. CTRL). Cells were co-stained with Texas Red-X phalloidin, anti-SLUG (green) and anti-E-cadherin (cyan) and imaged with confocal microscopy. Merged images of the corresponding three channels are presented. In separate panels, fluorescence signal specific to Fibronectin antibody was visualized as blue. Bars, 10 µm. (D) SPARC promotes EMT-like transition in melanocytes: immunoblots of normal human melanocytes (NHM) infected with control adenovirus (AdCTRL) or adenovirus-expressing SPARC (AdSPARC) for 4 days. Total protein lysates and cell supernatants were analyzed for expression of the indicated proteins.