Journal: The Journal of Biological Chemistry

Article Title: Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression *

doi: 10.1074/jbc.M116.714733

Figure Lengend Snippet: Characterization of anti-DCT and anti-CAV1 antibodies in Western blot and immunofluorescence microscopy. MJS (72 h) and SK28 (96 h) melanoma cell lines transfected with siRNA control (si-ct), si-DCT (si-DCT), or si-CAV1(si-CAV1) were assessed for DCT and CAV1 expression in Western blot (WB) and immunofluorescence microscopy (IF) with D18 or α-hDCT and α-CAV1-CS or α-CAV1-sc, respectively. WBs were assessed with ECL or SuperSignal West Femto chemiluminescent substrate (Femto) detection systems. Both anti-DCT and anti-CAV1 antibodies recognize specifically in the two cell lines the products encoded by the DCT and CAV1 gene, respectively, different CAV1 constituents (monomers, oligomers, in Nonidet P-40-soluble (sol) and Nonidet P-40-insoluble (insol) fractions). Importantly, in both cell lines, an insoluble oligomeric CAV1 pool detected with both anti-CAV1 antibodies was resistant to CAV1 down-regulation. Unlike in MJS, the soluble oligomeric CAV1 detected preponderantly with α-CAV1-sc in SK28 cells was also resistant to si-CAV1 indicating that CAV1 aggregation is different in the two cell lines. The nonspecific DCT and CAV1 bands are indicated by stars. Calnexin (Clx) was used as loading control for Nonidet P-40-soluble fractions. The IF images were acquired using ×40 objective. Scale bar represents 10 μm. Each experiment is a representative one of three.

Article Snippet: Glycolytic Treatments Approximately 25 or 10 μg of total protein from Nonidet P-40-soluble fractions (determined by BCA assay) were set in reactions with either endoglycosidase H (EndoH) or peptide: N -glycosidase F (PNGase F) (New England Biolabs), according to a protocol described elsewhere ( 28 ).

Techniques: Western Blot, Immunofluorescence, Microscopy, Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression *

doi: 10.1074/jbc.M116.714733

Figure Lengend Snippet: DCT silencing affects CAV1 stability, assembly, and subcellular distribution in amelanotic melanoma cell phenotypes. A and B, CAV1 expression and assembly in si-DCT cells. MJS (A) and SK28 (B) sub-confluent (48 and 72 h), semi-confluent (72 and 96 h), and confluent (96 and 120 h) cultures in si-ct and si-DCT experiments analyzed for CAV1 MOs and OGs in Nonidet P-40-soluble and Nonidet P-40-insoluble fractions in thermally treated (+) or not (−) samples by WB. CAV1 was assessed with α-CAV1-CS and DCT with D18. Actin was used as loading control. C and D, impact of DCT gene silencing on CAV1 mRNA expression. DCT and CAV1 mRNA in si-ct and si-DCT MJS and SK28 cells were determined by real time RT-qPCR. Graphs show average of experiments (n = 6 replicates for each cell line and time point); error bars represent S.E. E, subcellular distribution of DCT (red), CAV1 (green), and Cavin-1 (blue) in MJS (72 h) and SK28 (96 h) si-ct and si-DCT cells assessed by immunofluorescence confocal microscopy. Last columns represent enlarged details of merged images. Scale bar, 10 μm in merged images and 5 μm in enlarged details. Images were acquired using ×63 oil immersion objective. Each experiment is a representative one of three.

Article Snippet: Glycolytic Treatments Approximately 25 or 10 μg of total protein from Nonidet P-40-soluble fractions (determined by BCA assay) were set in reactions with either endoglycosidase H (EndoH) or peptide: N -glycosidase F (PNGase F) (New England Biolabs), according to a protocol described elsewhere ( 28 ).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression *

doi: 10.1074/jbc.M116.714733

Figure Lengend Snippet: DCT and CAV1 expressions are oppositely modulated by environmental and cellular factors in MJS phenotype. A and B, MJS cells were plated and cultured for 48, 72, or 96 h, and in one experiment the culture medium was replenished with a fresh one every 24 h (MR+) or not changed during the indicated time periods (MR−). C, MJS cells cultured for 24 h were incubated with fresh culture medium (fresh) or with medium resulting from a 96-h (old) culture for an additional 24 h (left panel); MJS cells were cultured for 72 h in culture medium with 10 or 1% FCS (right panel). D, MJS and MNT cells were cultured and treated for the indicated time periods as described for A and B. For all experiments, the Nonidet P-40- soluble and Nonidet P-40-insoluble fractions of the MJS cell lysates were analyzed by WB for the expression of CAV1 (with α-CAV1-CS), DCT (with D18 or α-hDCT), TYR (with α-Pep7h), and TRP-1 (with αPep1h). Actin was used as loading control. Each experiment is a representative one of three. E, DCT (red) and CAV1 (green) distribution in cell populations during sub-confluent (48 h) to semi-confluent (72 h) and confluent (96 h) transition assessed by immunofluorescence microscopy. Last columns represent enlarged details of separate images. The antibody combinations for DCT and CAV1 immunostaining are indicated in each panel. Images were acquired using ×40 objective. Scale bar represents 20 μm. Each experiment is a representative one of three. F, image cytometry analysis of triple labeled (DCT/CAV1/nuclei with the two antibody combinations and DAPI) samples performed using the TF system. MFI and MSI were determined for DCT and for CAV1 expression, respectively. For each staining, DCT expression scattergrams for 48 h (upper left), 72 h (middle left), and 96 h (lower left) are gated to select cells with high marker expression (upper right quadrant represents cells positive for DCT, and the rectangle delineates the gated DCThigh cell subpopulation, shown as red dots). For the 96-h time point, CAV1 scattergrams of all cells (bottom middle) as compared with DCThigh cell subpopulation (bottom right) were generated. Values representing mean expression for DCT and CAV1 as well as % of cells in DCThigh subset were derived from Tissue Quest data statistics and are represented as graphs. One representative experiment of two performed is shown.

Article Snippet: Glycolytic Treatments Approximately 25 or 10 μg of total protein from Nonidet P-40-soluble fractions (determined by BCA assay) were set in reactions with either endoglycosidase H (EndoH) or peptide: N -glycosidase F (PNGase F) (New England Biolabs), according to a protocol described elsewhere ( 28 ).

Techniques: Cell Culture, Incubation, Expressing, Immunofluorescence, Microscopy, Immunostaining, Cytometry, Labeling, Staining, Marker, Generated, Derivative Assay