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MCR-14-0420 Revision Version 1
RhoC is an Unexpected Target of RhoGDI2 and
Prevents Lung Colonization of Bladder Cancer Cells
SUPPLEMENTARY FIGURE AND TABLE LEGENDS
Supplementary Figure 1. Co-immuoprecipitation of RhoGDI2 with RhoC in nonbladder cell types
Endogenous RhoGDI2 from MDA-MB-231 breast cancer cells (Left), Jurkat lymphocyte
cells (Center), or OVCAR-4 ovarian cancer cells (Right) was immunoprecipitated and
analyzed
by
western
blot
for
co-immunoprecipitation
of
Rac1
and
RhoC.
Representative western blots of 3 independent experiments shown.
Supplementary Figure 2. Effect of RhoC knock down in LuL2 bladder cancer cells
A. Expression of RhoA and RhoC in LuL2 cells following stable knock down of RhoC by
shRNA. Representative western blots shown.
B. Invasion of LuL2 cells expressing shCont or shRhoC-1 through matrigel was
determined using a transwell assay. Results expressed as number of invaded cells per
well presented as the mean± S.E. *P<0.05. Independent experiments (n=3) were
performed in triplicate.
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C. Colony formation in soft agar was assessed in LuL2 cells expressing shCont or
shRhoC-1. Bargraph indicates the number of colonies formed and is presented as the
mean± S.E. *P<0.05. Independent experiments (n=3) were performed in triplicate.
Supplementary Figure 3. Depletion of RhoC with a second independent shRNA
A. Doubling time for T24T cells expressing shCont or shRhoC-2 was determined by
counting cells at 24 h intervals for 96 h. Bargraph shows the mean doubling time±s.e.
(n=3). *P<0.05.
B. Colony formation in soft agar was assessed in T24T cells expressing shCont or
shRhoC-2. Bargraph shows the number of colonies formed and is presented as the
mean±s.e. *P<0.05. 3 independent experiments were performed in triplicate.
C. Graph representing incidence of lung colonization upon tail vein injection of T24T
cells expressing shControl or shRhoC-2. p=0.66, Fisher’s exact test.
D. Scatter plot comparing lung burden in animals injected with T24T cells expressing
shControl or shRhoC-2 as measured by human DNA-specific qPCR amplification of
lung genomic DNA. Mean is indicated. p=0.016, analysis of non-zero values only, Mann
Whitney test.
Supplementary Table 1. List of proteins co-immunoprecipitating with RhoGDI2 in RT4
cells identified by mass spectrometry. MW, molecular weight; Xcorr, cross-correlation
score for the SEQUEST search.
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Supplementary Table 2. Differential expression of RhoC/RhoGDI2 signature genes
between normal urothelial and bladder tumor samples in Sanchez-Carbayo et al (n =
129) and Kim et al (n = 255) patient cohorts. Values in the table are in the form fold
change (p-value), where only genes with significant differential expression (p < 0.05) are
shown, and '-' indicates a non-significant result. A blank indicates that the gene was not
profiled on the microarray platform for that cohort. A fold change above 1 indicates that
expression is higher in tumor samples compared to normal. Genes with fold changes in
the expected direction (>1 for down-regulated genes and <1 for up-regulated genes) are
highlighted in yellow.
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