Download Table and Suppl Figure legends

Supplementary Tables and Figure legends
Table S1: Comparison of the amino acid concentrations of the media/versus
human plasma. Amino acid concentrations for each of the culture media used in this
study are described. These concentrations are compared with physiological
concentration determined by Miyagi et al. (PLoS One. 2011)
Table S2: Analysis of the mutations generated by ZFN in each LS174T-derived
cell line used.
Figure S1: Quantification of respective mRNA and protein expression of each
member of the CD98/LAT1 complex and β1 integrin in the various cell lines and
knockout derivatives. A: Quantification of LAT1 and CD98 total expression in
LS174T WT, LAT1KO and CD98KO cells. Quantifications represent the average of
three independent immunoblotting experiments. B: The LAT1 and CD98 mRNA
expression were analyzed by RT-qPCR in LS174T WT, LAT1KO and CD98KO cells
demonstrating a transcriptional regulation of CD98 in LAT1KO cells. 2 independent
clonal cell lines of LAT1 and CD98 knockout are shown (#1, #2). RPLP0 was used as
the control gene of reference. WT cells were used as the control sample reference.
These results represent the average three independent experiments. C: LAT1 and
CD98 protein expression were analyzed by immunoblotting in LS174T WT, LAT1 KD
and CD98KD cells. ERK 1/2 was used as a loading control. D: LAT1 and CD98 protein
expression were analyzed by immunoblotting in A549 WT and LAT1KO cells
demonstrating an interdependence of protein expression for CD98. Tubulin was used
as a loading control. E: Plasma membrane expression of CD98 was analyzed by
flow cytometry in A549 WT and LAT1KO cells. Relative membrane expression of
CD98 is noted on each panel as a percentage (%) compared to WT cells. F: Total β1
Integrin expression was analyzed by immunoblotting (RD systems, MAB1778) in
LS174T WT and CD98KO cells with ARD1 used as a loading control. G: Plasma
membrane expression of β1 integrin was analyzed by flow cytometry in LS174T WT
dans CD98KO cells. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; n.s: not significant.
Figure S2: Phenotypic analysis of the genetic disruption of LAT1 in the lung
adenocarcinoma cell line: A549
A: A549 WT and LAT1KO cells were cultivated for 24h in 0.3X DMEM. Changes in
phosphorylation status and protein abundance of members of the two major amino
acid sensing pathways GCN2 (p-GCN2/p-EIF2a/ATF4) and mTORC1 (p-p70-S6K
and p-RPS6) were analyzed by Western blot. Tubulin was used as a loading control.
B: Cell proliferation of A549 WT (black) and LAT1KO (grey) cells. Cells were cultivated
for 5 days in 0.3X DMEM and media was replaced at day 3. Proliferation rates are
presented as fold increase (see methods for detailed description). C: Clonal growth of
A549 WT and LAT1KO cells. Cells were cultivated 15 days in 0.3X DMEM and colored
for visualization using Geimsa. Media was replaced every 3 days. * = P < 0.05; ** = P
< 0.01; *** = P < 0.001; n.s: not significant.
Figure S3: Disruption of CD98 in A549 cell line has not impact on AA sensing
pathways and proliferation.
A: The LAT1 and CD98 mRNA expression were analyzed by RT-qPCR in A549
shCTRL and CD98KD cells demonstrating a transcriptional downregulation of CD98
and LAT1 mRNA in CD98KD cells. RPLP0 was used as the control gene of reference
and WT cells acted as the control reference. These results represent the average of
three independent experiments. B: A549 shCTRL and CD98KD cells were cultivated
for 24 hours in 0.3X media. Changes in phosphorylation status and protein
abundance of members of the two major amino acid sensing pathways GCN2 (pGCN2/p-EIF2a/ATF4) and mTORC1 (p-p70-S6K and p-RPS6) were analyzed by
Western blot. Tubulin was used as a loading control. C: Cell proliferation of A549
shCTRL (black) and CD98KD (grey) cells. Cells were cultivated for 5 days in 0.3X
DMEM and media was replaced at day 3. Proliferation rates are presented as fold
increase (see methods for detailed description). D: Clonal growth of A549 shCTRL
and CD98KD cells cultivated for 15 days in 0.3X DMEM and treated with DMSO or
different concentrations of JPH203 (10 μM or 30 μM) with visualization achieved via
Giemsa staining. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; n.s: not significant.
Figure S4: LAT1 activity is a key limiting step for AA homeostasis and
proliferation in multiple cancer types.
JPH203 is a LAT1 competitive inhibitor and its effect is directly linked to the AA
concentration in the media and expression level of the transporter. Therefore in order
to observe a magnified effect of the JPH203 the following experiments were
performed using the commercial F12 media that contains low EAA concentration (see
Table S2) and a rather high JPH203 concentration (30μM).
A: 6 independent cancer cell lines (Colon adenocarcinoma: LS174T and HT29, Lung
adenocarcinoma: A549 and H1975, Kidney carcinoma: 786-O and A498) cells were
cultivated for 48 hours with DMSO (-) or JPH203 (+) (30μM). Changes in
phosphorylation status and protein abundance of members of the two major amino
acid sensing pathways GCN2 (p-GCN2/p-EIF2a/ATF4) and mTORC1 (p-p70-S6K
and p-RPS6) were analyzed by Western blot. Actin was used as a loading control. B:
Proliferation assays of the 6 cell lines were conducted in presence of vehicle (DMSO)
or JPH203 (30μM). Cells were counted after 72 hours and data are presented as fold
increase (see methods for detailed description).
Figure S5: Rescue of CD98 expression in A549 LAT1KO cells
A: Plasma membrane expression of CD98 was analyzed by flow cytometry in A549
WT, LAT1KO cells and LAT1KO cells transiently expressing either the xCT or LAT1
cDNA to observe rescue of CD98 expression. B: Cell proliferation analysis of A549
LAT1KO and LAT1KO cells transiently expressing either xCT or LAT1. Cells were
counted after 3 days of culture in 0.3X DMEM. These results represent the average
of two independent experiments. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; n.s: not
significant.
Figure S6: Tumorigenicity and mTORC1 activity of WT and double CD98KO/
LAT1KO cells.
A: Tumor volumes of nude mice injected subcutaneously with LS174T WT (black) or
LAT1KO / CD98KO (dKO, grey) cells revealed dramatic inhibition of tumor growth with
dKO cells. B: Protein levels of LAT1, CD98 and mTORC1 (p-RPS6, RPS6) were
analyzed by immunoblotting in WT and dKO tumors. Tubulin acted as a protein
loading control. SE: Short exposition, LE: Long exposition *** = P < 0.001