Inhibition of the RhoA GTPase Activity Increases Sensitivity of Melanoma Cells to UV Radiation Effects

Figure 1

Immunofluorescence analysis was performed on the parental MeWo clone and the MeWo-RhoA-N19 and MeWo-RhoA-V14 mutant clones to evaluate the profile of stress fiber formation and RhoA distribution after damage induced by 50 KJ/m² UVA, 80 J/m² UVB, or 4 J/m² UVC irradiation. A total of 200,000 cells were seeded on 35 mm culture dishes at 24 h before treatment. At 1 h after the given radiation treatment, the cells were fixed in 3% paraformaldehyde/2% sucrose/PBS buffer (a) or 10% TCA/PBS (b) and permeabilized with 0.5% Triton X-100/6.84% sucrose/3 mM MgCl₂/PBS buffer. Subsequently, the cells were blocked in 3% BSA/PBS for 30 min and incubated in 1 : 500 Alexa Fluor 488 Phalloidin (Invitrogen, Carlsbad, CA, USA) for 1 h at 4°C (a) or in 1 : 250 mouse anti-RhoA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h followed by 1 : 15000 Alexa Fluor 680 secondary antibody (Invitrogen, Carlsbad, CA, USA) (b). After washing with PBS, the cells were mounted on coverslips in VECTASHIELD medium containing DAPI, and images were acquired using a Zeiss LSM 510 laser confocal microscope. The photomicrographs are representative of three different fields in two independent experiments.

(a)

(b)