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. 2013 Dec;133(12):2809-2812.
doi: 10.1038/jid.2013.218. Epub 2013 May 7.

Myosin-Va contributes to manifestation of malignant-related properties in melanoma cells

Cleidson P Alves  1 , Milene H Moraes  1 , Josane F Sousa  1 , Carmen Lucia S Pontes  1 , Anelisa Ramão  1 , Satoru Yokoyama  2 , Daniel M Trindade  1 , David E Fisher  3 , Enilza M Espreafico  4
Affiliations

Myosin-Va contributes to manifestation of malignant-related properties in melanoma cells

Cleidson P Alves et al. J Invest Dermatol. 2013 Dec.
No abstract available

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Figures

Figure 1
Figure 1. Myosin-Va is highly expressed in melanoma cells and its knockdown impairs cell adhesion and spreading on fibronectin-coated surface
(a) Relative MYO5A mRNA expression detected by qPCR in melanocytes (pMel1 to 4) versus melanoma cells, using β-actin for normalization and mean of all melanocytes as reference value. (b) Western-blot of myosin-Va in a panel of melanoma cell lines of radial growth phase (RGP), vertical growth phase (VGP) and metastasis (M), including two genetic pairs (WM793 and 1205Lu; WM278 and WM1617). (c-e) Western-blots for myosin-Va in (c, d) WM1617 and (e) UACC-257. WM1617 cells were lysed 3 days after transduction with lentiviral vectors carrying shRNAs targeted to bacterial Lac-Z (shControl) or MYO5A (shMYO5A#1), or after stable selection with antibiotics for about 2-3 weeks in the case of shMYO5A#2-3 and respective shControl (Figure S3). UACC-257 cells were lysed 3 days after transfection with siRNA against myosin-Va or control. (f) Confocal images of F-actin stained cells adhered to fibronectin-coated coverslips for the indicated times. Arrows indicate transduced cells visualized by GFP expression (inserts). Scale bar = 20μm. (g) Cells allowed to adhere on fibronectin-coated surface for the indicated times were counted and data were plotted as mean ± SD from 3 independent experiments. (h) Cell spreading. Imaged as in (f) and the areas for 60 cells/time point were measured using ImageJ.
Figure 2
Figure 2. Ablation of myosin-Va inhibits colony formation, migration and invasiveness of metastatic melanoma cells without affecting cell proliferation
(a-c) Lentiviral transduced WM1617 cells, using three independent shMYO5A or shControls, were used to assess: (a) Colony formation in soft-agar after 30 days of growth. Scale bar = 500μm; (b) Proliferation rates by absorbance measurement of crystal violet staining; (c) Migration in transwell and invasion in transwell-matrigel assay. Cells were kept in starvation conditions 24 hour prior the assay and were then allowed to migrate/invade for 24 hours. Scale bar = 50μm. (d) Migration in 3D collagen. After 24 or 48 hours of incubation - distance from spheroid edge to invasive front was measured and the data from three independent experiments were plotted as a percentage of control. Scale bar = 100μm. (e-f) Transwell invasion and proliferation rates of UACC257 cells transiently transfected with duplex siRNA targeted to MYO5A and irrelevant siRNA. Invasion assay was done as described in c, and proliferation rates were estimated based on ATP measurements. Data were plotted as mean ± SD from 3 independent experiments. Scale bar = 50μm.
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