Supplementary Figure Legends
Supplementary Figure 1. Expression of wild type and mutant forms of ezrin alters
DLBCL cell growth. (a) Lysates of peripheral blood B cells (PB) and GC B cells (GCB)
from 3 healthy individuals were subjected to immunoblotting with pThrERM and ezrin
antibodies. (b) Schematic representation of wild type ezrin (Ez-WT), and its
phosphomimetic (Ez-TD), non-phosphorylatable (Ez-TA), and truncation (Ez-DN)
mutants. (c) OCI-LY-10 cells were mock-transfected (MT) or transfected with Ez-DN (T)
for 24 h and cell lysates probed with antibodies to pThrERM, VSVG and ezrin. (d)
Expression of YFP-tagged ezrin in lysates of transiently transfected OCI-LY-10 cell line
was assessed by immunoblotting with antibodies to ezrin and actin. (e) OCI-LY10, SUDHL-6 and OCI-LY-3 cells were mock-transfected (red histograms) or transfected with
Ez-WT-YFP construct (blue histograms), and expression assessed by flow cytometry.
(f) OCI-LY-10 cells were transfected with pEYFP vector (Vector), or constructs encoding
Ez-WT, Ez-TA or Ez-TD for 24 h, and viable cell count obtained by trypan blue
exclusion. Mean ± SEM of three independent experiments is shown.
Supplementary Figure 2. Pharmacological inhibition of ERM proteins decreases
their phosphorylation and inhibit cell growth. OCI-LY-10 cells were treated with
DMSO or indicated concentrations of NSC668394 (a) or NSC305787 (b) for 1 h and cell
lysates were probed with antibodies to pThrERM and ezrin. OCI-LY-10 cells were
treated with DMSO or indicated concentrations of NSC668394 (c) or NSC305787 (d) for
24, 48 and 72 h and viable cell counts determined by trypan blue exclusion.
Supplementary Figure 3. Effect of IKK inhibition on OCI-LY-3 cell growth. OCI-LY3 cells were incubated with indicated concentrations of PS-1145 and viable cells were
counted every day for 72 h.
Supplementary Figure 4. Effect of ERM knockdown on DLBCL cell growth. OCILY-10 and SU-DHL-6 cells were incubated with scrambled siRNAs or those targeting
ezrin, radixin and moesin for 6 days. (a) Cell lysates were collected at 96 h and probed
with antibodies to ERM, pThrERM and actin. (b) Viable cells were counted every day for
6 days and cell counts for indicated days are shown. Data are representative of two
independent experiments.
Supplementary Figure 5. BCR distribution in DLBCL cell lines. The indicated ABCand GCB-DLBCL cell lines were stained with AF488-conjugated antibodies to surface
IgM (a) or IgG (b) and imaged by TIRF microscopy. Scale bar, 1 µm. Data are
representative of two independent experiments.
Supplementary Figure 6. Wild type ezrin restores BCR organization in ezrin
inhibitor-treated cells. (a) OCI-LY-10 cells were transfected with vector or Ez-WT,
followed by treatment with DMSO or 5 µM Ez-Inh for 48 h and imaging. Scale bar 1 µm.
(b) Quantification of the number and area of BCR microclusters from two experiments.
Twenty-five cells were imaged per experiment and 40–50 individual clusters/experiment
analyzed for quantification (*P < 0.05; **P < 0.01; ***P < 0.001). For imaging
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experiments shown in Figure 5, representative cell surface profiles of IgM for OCI-LY-10
(c) and OCI-LY-3 (d) and IgG for SU-DHL-6 (e) are shown. Numbers shown within the
plots indicate median fluorescence intensity of BCR expression. Indicated DLBCL cell
lines were either incubated with DMSO or NSC668394 (5 µM) (left panels in c-e) or
mock transfected (MT) or transfected with Ez-DN (T) (right panels in c-e).
Supplementary Figure 7. Pro-survival signaling is impaired upon ERM inhibition
by NSC668394 and siRNA knockdown. TMD8 or SU-DHL-6 cells were transfected
with Ez-DN (a), or treated with indicated concentrations of NSC668394 (b) and cell
lysates probed with antibodies to pIκB, IκB, pAkt, Akt. OCI-LY-10 cells were transfected
with Ez-WT for 24 h, followed by treatment with 5 μM of NSC668394 (Ez-Inh) for 120 h.
Lysates of LY-10 cells treated for 0, 48 and 120 h were probed with antibodies to pY
and actin (c), or pIκB, pAkt, Akt and Bcl2 (d). (e) OCI-LY-10 and SU-DHL-6 cells were
incubated with scrambled siRNAs or those targeting ezrin, radixin and moesin, and after
96 h, cell lysates were probed with antibodies to pIκB, IκB, pAkt, Akt. Data are
representative of two independent experiments.
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