RAC1 GTPASE-DEFICIENT HELA CELLS PRESENT REDUCED DNA
REPAIR, PROLIFERATION AND SURVIVAL UNDER UV OR GAMMA
IRRADIATION
Gisele Espinha, Juliana H. Osaki, Yuli T. Magalhaes and Fabio L. Forti*
Laboratory of Signaling in Biomolecular Systems, Department of Biochemistry,
Institute of Chemistry, University of Sao Paulo, Sao Paulo-SP, Brazil
Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922
CEP: 05508-900 - Cidade Universitária - São Paulo-SP, Brazil
*Corresponding author:
Fabio Luis Forti, PhD
Av. Prof. Lineu Prestes, 748 - Bl.09i, Sl.922
CEP: 05508-900 - Cidade Universitária - São Paulo-SP, Brazil
Phone: 55-11-3091-9905 / Fax: 55-11-3091-2186
E-mail address: flforti@iq.usp.br
ELECTRONIC SUPPLEMENTARY INFORMATION
Supplementary Figure 1 Constitutively active HeLa-Rac1 clones behave in
an opposite manner to HeLa-Rac1-N17 deficient clones. A) HeLa-Rac1-V12
clones exhibit high basal levels of Rac1-GTP that are still slightly increased by
gamma or UV radiation. B) Gamma and UV treatments are able to increase
stress fiber formation in HeLa cells but not in the dominant-negative HeLaRac1-N17 clones, which exhibit higher levels of stress fibers. Quantification of
fluorescence intensity was performed by the LSM Software (Zeiss) after
normalization of all parameters (laser intensity, detector gain, pinhole, etc.) to
the control condition and after maintenance for all other treatments, in
approximately 100 cells (see the exact number over the bars) from three
independent experiments.
Supplementary Figure 2 Constitutively active HeLa-Rac1-V12 clones
exhibit high basal levels of Rac1-GTP that can be directly correlated to a fast
and efficient DDR, comparable to in HeLa cells, by measuring H2AX protein
phosphorylation for both treatments, either by immunofluorescence at 1 h (A) or
by western blot in kinetic experiments up to 6 h after gamma or UV irradiation,
using a specific antibody against phospho-H2AX-Ser139 (B).