Name / Acession number
7H3 / NM_033025
ARHGAP1 / NM_004308
ARHGAP10 / NM_024605
ARHGAP11A / NM_014783
ARHGAP12 / NM_018287
ARHGAP15 / NM_018460
ARHGAP17 / NM_018054
ARHGAP18 / NM_033515
ARHGAP19 / NM_032900
ARHGAP20 / NM_020809
ARHGAP21 / NM_020824
ARHGAP22 / NM_021226
ARHGAP23 / XM_290799
ARHGAP24 / NM_031305
ARHGAP25 / NM_014882
ARHGAP26 / NM_015071
ARHGAP28 / NM_030672
ARHGAP4 / NM_001666
ARHGAP5 / NM_001173
ARHGAP6 / NM_001174
ARHGAP8 / NM_001017526
ARHGAP9 / NM_032496
BNIP2 / NM_004330
C5ORF5 / NM_016603
CDGAP / NM_020754
CENTD1 / NM_015230
CENTD2 / NM_015242
CENTD3 / NM_022481
CHN1 / NM_001822
CHN2 / NM_004067
DEPDC1 / NM_017779
DEPDC1B / NM_018369
DLC1 / NM_006094
FKSG42 / NM_032032
FLJ13815 / XM_086186
FLJ30058 / NM_144967
FLJ32810 / XM_370651
GMIP / NM_016573
GRLF1 / NM_004491
HA-1 / NM_012292
INPP5B / NM_005540
KIAA0672 / NM_014859
KIAA1688 / NM_025251
LOC201176 / NM_199282
LOC257106 / NM_181720
LOC285101 / XM_210411
LOC343578 / XM_293123
LOC389211 / XM_371697
MYO9A / NM_006901
MYO9B / NM_004145
OCRL / NM_000276
OPHN1 / NM_002547
PARG1 / NM_004815
PIK3R1 / NM_181504
PIK3R2 / NM_005027
RACGAP1 / NM_013277
RALBP1 / NM_006788
RICS / NM_014715
SH3BP1 / NM_018957
SNX26 / NM_052948
SRGAP1 / NM_020762
SRGAP2 / NM_015326
SRGAP3 / NM_014850
STARD13 / NM_052851
STARD8 / NM_014725
TAGAP / NM_054114

Supplementary Data Table S1

Cell line / Chromo-some count / srGAP3 BAC FISH copy per cell / Number of apparently normal chr 3 / Comments
HMEC / 46 / 2 R/G / 2 / Apparently diploid cell line with occasional tetraploidy.
MDA-MB-231 / 57~64 / 3 R/G / 2 / Hypotriploid cell line with occasional genomic doubling and some chromosomal instability.
MCF-7 / 78~86 / 6 R/G / 3~4 / Hypotetraploid cell line with occasional genomic doubling and some chromosomal instability.
T47D / 64~65
(61~66) / 3 R/G / 2 / Hypotriploid cell line with some instability.
Hs 578T / 55~58/ 102~111 / ~3 R/G/
5~6 R/G / 2~3/
4~6 / Hypotriploid cell line with substantial population with genomic doubling (~50% of cells) and some chromosomal instability.
BT549 / 71~78 / ~5 R/G / 3 / Hypertriploid cell line with occasional genomic doubling and some chromosomal instability.
SK-BR-3 / 78 (76~81) / 3 R/G / 3 / Hypertriploid cell line with some instability.
SK-BR-7 / 43 (42~45) / 2 R/G / 2 / Near diploid cell line.
BT-474 / ~102 (100~108) / 5 R/G / 3 / Hypertetraploid cell line with some chromosomal instability.

Supplementary Data Table S2

Supplementary data legends

Table S1 The 66 human Rho GTPase activating proteins (Rho GAPs).

Table S2srGAP3 FISH analyses were performed by the Molecular Cytogenetics core facility at the Memorial Sloan-Kettering Cancer Center.FISH was performed using probes derived from BAC clones RP11-614E19 (R; red) and RP11-963B11 (G; green) spanning respectively the 3’ and 5’ ends of srGAP3 in chromosome band 3p25. The clones were obtained from BACPAC Resources, Children’s Hospital of Oakland Research Center, CA. Each clone was labeled by nick translation with Green dUTP or Red dUTP (Abbott Molecular) as indicated. A minimum of 10 DAPI-banded metaphases was imaged. Full karyotype analysis was not performed. There is no evidence of amplification or translocation affecting srGAP3 in any of the cell lines analyzed.

Figure S1 (A) Phosphorylation levels of AKT and total AKT in HMEC/hTERT/LT/c-myc after srGAP3 and PTEN depletion (Phospho-Akt Thr308 antibody, cell signaling 9275; Phospho-Akt Ser473 antibody, cell signaling 9271; Akt antibody, Cell signaling 9272). Cells were transiently transfected with 60nM of siRNA and lysed four days later with RIPA buffer plus 100mM PMSF and 1 tablet of Complete mini Protease Inhibitor (Roche11836153001). (B) HMEC/hTERT/LT/c-myc were infected with pQCXIP and pQCXIP-L61Rac1 and selected with 1g/ml puromycin for 4 days. Images were taken 8 days after infection. Bar: 100m

Figure S2 DNA methylation inhibitor, 5-azacytidine (5-AzaC, Sigma) and histone deacetylation inhibitor, trichostatin A, (TSA, Sigma) treatment of SKBR7, SKBR3, MDA-MB-231 and Hs578t breast cancer cells. 1 x 105 cells were plated the day before treatment in a 6 well plate and 2.5 µM or 5 µM 5-AzaC was added for 96 hours or 0.5µM TSA was added for 20-24 hours. Protein lysates and mRNA were analyzed by western blot and Q-PCR respectively, after treatment. Data are represented as fold inductions relative to HMECs. , p<0,05. , p<0.01.

Figure S3 (A) srGAP3 and srGAP3GAP protein levels in MDA-MB-231, MCF7 and BT549. Lysates were made 8-14 days after infection in MDA-MB-231 and BT549, and 3 days after nucleofection in MCF7 using RIPA buffer plus 100mM PMSF and 1 tablet of Complete mini Protease Inhibitor (Roche11836153001). (B) Images from anchorage independent growth assays of MDA-MB-231 and MCF7 expressing the control plasmid, srGAP3 and srGAP3GAP, 3 weeks after plating.

Figure S4 The upper panel shows phase contrast microscopy images of MDA-MB-231 stellate colonies growing in 3D matrigel after 4 days and expressing the indicated plasmids. The lower pannel shows colonies growing in matrigelafter 4 days and stained with 1:300 rhodamine-conjugated phalloidin (Invitrogen R415) and 1:10000 Hoechst (10mg/ml) (Sigma B2261). Bar=100µm.

Figure S5 The upper panel shows aphase contrast microscopy image of BT549 stellate colonies growing in 3D matrigel after 4 days and expressing the indicated plasmids. The lower pannel shows colonies growing in matrigel after 4 days and stained with 1:300 rhodamine-conjugated phalloidin (Invitrogen R415) and 1:10000 Hoechst (10mg/ml) (Sigma B2261). Bar=100µm.

Movie S1 MDA-MB-231 expressing srGAP3HisV5 (right panel) or the empty vector (left panel)in 3D matrigel cultures.

Movie S2MDA-MB-231 expressing srGAP3HisV5 (right panel) or the empty vector (left panel)in 3D matrigel culturesin presence of 10µM of Y27623.

Cells for the movies were grown in a 4 well ThermoFisher coverglass bottom Scientific Chamber (155383). The bottom of the wells was coated with 15µl of a mix of 80% matrigel (BD Biosciences) and 20% collagen I (Cultrex 3440-100-01). 2 x104 cells were resuspended in 5% matrigel and plated within the coated chamber (adapted from Kenny et al 2007). Images were acquired every 10 min for 72 hours starting 20 hours after plating the cells in the chamber. An Axiovert 200M microscope equipped with a 5× 0.16Ph1 objective lens, a motorized stage and an Orca-ER-1394 camera (Hamamatsu Photonics) controlled by Axiovision software (Carl Zeiss, Inc.) was used for the acquisition.