Supplemental data (sup. data)
Sup. data 1: Mono- and co-culture experiments with H2 cells: FDC-LCs or BMSCs were cultured in 6
well- plates until approximately 70% of confluency in IMDM containing 10% FCS, 2 mM sodium pyruvate,
penicillin and streptomycin (100 U/ml each) (Life Technologies, CA). Cell monolayers were washed twice in
PBS, and 2.5 ml/well of IMDM containing 1% BSA was added on adherent cell monocultures. In co-culture
conditions, 2.5 ml of IMDM containing 1.5x106 H2 cells were added to cell monolayers. As a control, 2.5 ml
of IMDM containing 1.5x106 H2 cells were incubated without FDC-LCs or BMSCs. Where indicated, TNF
(10 ng/ml) was added to the cell culture. After 2 days, cell culture supernatants were harvested, clarified by
centrifugation (200g, 5 min and 15,000 rpm, 15 min) and treated with NaOH/HCl to remove TGF from
inactivating complexes (protocol R&D system, Minneapolis, MN). For each supernatant, 500 µl was kept
for TGF ELISA determination (see below). The remaining volume (2ml) was divided in two and incubated
with or without 1µg/ml of TGF blocking Ab (MAB2401, R&D Systems) for 60 min at 37 oC. This Ab
neutralizes TGF (R&D system). Mono- or co-culture cell supernatants were then used as a source of
TGF to stimulate H2 cells. Serum-starved H2 cells (30x106) were resuspended in 1 ml of undiluted culture
cell supernatants prepared as described above. Negative and positive controls included H2 stimulated by
recombinant TGF in fresh RPMI or IMDM alone or containing TNF (10 ng/ml). At indicated time H2 cells
were lysed and Smad immunoprecipitations and Western-blots were performed as described below.
Sup. data 2: Production of Wild-type (wt) and mutated (mut) Smad1 constructs.
Wt-Smad1 and mut-Smad1 sequences were cloned into a murine leukemia virus-based retroviral vector
pMMP-f2-IRES-GFP. To generate recombinant retroviruses, the Smad1 plasmids were co-transfected with
expression plasmids encoding MLV gag-pol and VSV-G proteins into 293T by Calcium Phosphate
method.51 The viruses were concentrated by ultracentrifugation and titrated on NIH3T3 cells by flow
cytometric analysis.
Sup. data 3: IHC tissue samples and procedure.
Tissues were obtained after informed consent and IRB approval. All lymphoma cases had been classified
according to the WHO classification for lymphoid neoplasms.[Harris, 1999 #468] The series included 20
cases of FL grades 1 or 2, and 7 biopsies of either FL with concomitant development of DLBCL in the same
1
biopsy or DLBCL with a confirmed antecedent diagnosis of FL, indicating histologic transformation. Slides
were deparaffinized and antigen retrieval was performed by microwave heating in the antigen retrieval
solution as per the manufacturer’s recommendations (Dako, Carpinteria, CA). After blocking with goat
serum, slides were incubated with the primary antibody (dilution 1:50) overnight. Detection was performed
with the Vectastain ABC kit anti-rabbit (Vector Laboratories, Burlingame, CA) according to the instructions
of the manufacturer.
Sup. data 4: no cross-reactivity between Smad1 and Smad2 antibodies (see figure).
Panel a: protein immunoprecipitation was performed incubating H2 cell lysates with anti-Smad2 Ab
(sc-6200), irrelevant goat IgG (negative control), or anti-Smad1 #06-053 (positive control). The membrane
was probed with anti-Smad1 Ab (#06-053) and developed using protein A-HRP and ECL (upper gel).
Membranes were stripped and reprobed with anti-Smad2 Ab (# 06-054) and protein A-HRP (lower gel).
Conclusion: the anti-Smad2 Ab used in immunoprecipitation detected Smad2 but not Smad1, although
Smad1 is present in H2 cell lysates. Panel B: Test of a potential cross-eactivity of anti-Smad1 Ab (#06-053)
with Smad2. Cell lysates from Jurkat or DHL16 known to contain Smad2 but not Smad1 were incubated in
the presence of anti-Smad1 Ab (# 06-053). Immunoprecipitated material has been separated by
SDS-PAGE and western-blotted with anti-Smad2 Ab (#06-054). Immunoprecipitation of Smad2 in Jurkat
cell lysates is shown as a positive control. No band appeared in the Smad1 immunoprecipitation, whereas
Smad2 was clearely present in the Jurkat cell lysates. This excludes cross-reactivity of Smad1 Ab with
Smad2.
Sup. data 5: activation of Smad1 in lymphoma B cell (see figure).
Panel a: purified primary FL cells were stimulated with TGF (10 ng/ml) for 15 and 30 min and lysed in 1%
NP40 lysis buffer.
Smad1 immunoprecipitation as well as Smad1-P or Smad1 Western blots were
performed as in figure 1c. Panel b: H2 were incubated for 1h with increasing concentrations of TGFas
indicated. Smad1 immunoprecipitations and anti-Smad1/Smad1-P Western-blots were performed as in
figure 1c. Panel c: TAT-1 cells were incubated with TGF, BMP2 or BMP7 at indicated concentrations for
1h. Smad1 was immunoprecipitated from cell lysates, analyzed for phosphorylation and quantified as
described in figure 1c. Note: IpC = negative control for Smad immunoprecipitation with isotype-match
antibody. (*) = non-specific band. These results are representative of 3 different experiments.
2
Sup. data 6: Effect of Smad1-specific iRNA on TGF response (see figure).
Panel a: H2 cells were transfected by nucleofection with eGFP and iRNA (random sequence or four
different Smad1 specific iRNA labeled A, B, C or D). Random iRNA: Smad1 iRNA sequences: iRNA A:
sense CCAAGAAUUUGCUCAGUUAUU, antisense 5’-P.UAACUGAGCAAAUUCUUGGUU; iRNA B:
sense CAAAUGGGUUCACCUCAUAUU, antisense 5’-P.UAUGAGGUGAACCCAUUUGUU. iRNA C:
sense AAACACUGGUGCUCUAUUGUU, antisense 5’P.CAAUAGAGCACCAGUGUUUUU; iRNA D: sense
ACUAUGAGCUCAACAAUCGUU, antisense 5’P.CGAUUGUUGAGCUCAUAGUUU. After cell sorting of
the cells by cytometry and overnight recovery, Smad1 expression was analyzed 48h later by western-blot of
75 µg of proteins from H2 cell lysates obtained as previously described (panel a and see legend of figure 1
for detailed protocol). Panel B: Smad1 material was quantified by densitoimetry of the upper gel and
expressed as a percent of the control (random iRNA=100%). Panel c: Effect of Smad1 iRNA on
TGF-induced inhibition of cell proliferation: H2 cells were transfected with random or Smad1 specific iRNA
(A or B) and sorted as above. After overnight recovery, cells were treated with different concentrations of
TGF for 48h as indicated in the plot. Overnight thymidine incorporation was measured and plotted as a
percent of the total cpm in the control without TGF. This is representative of two independent experiments.
Asterix mark the inhibitions.
Sup. data 7: TNF increased TGF released by H2 cells but not by FDCs (see figure).
H2 and FDCs were incubated with or without TNF. After 48, TGF concentration was measured by ELISA
as in Figure 7 panel a. As can be seen, TNF increased TGF concentration in supernatants from H2 cells,
but not from FDCs.
Sup. data 8: expression of TGF family member receptors in lymphoma cells (see figure).
cDNA were prepared from total RNA of indicated cell lines. HUVEC and placenta are used as a positive
control, as well as amplification of actin. The expected size of each amplicon is indicated on the left. (S):
endoglin short form; (L): endoglin long form. Primers are indicated in the table below. For ALK-1, a two-step
PCR has been performed (labeled “out” and “in” in the table) to exclude low expression of the gene in
lymphoma cells.
Primer table:
3
GENE
ActRIIA
ActRIIB
BMPRII
TGFRII out
TGFRII in
ALK-1 out
ALK-1 in
ALK-2 (Ac tRI)
ALK-3 (BMPR IA)
ALK-5 (TGF RI)
ALK-6 (BMPR IB)
glycan (TGFR III)
Endoglin, S and L
(CD105)
FORWARD
ttggctggatgatatcaactg
tgctggcctttggatgtacc
cagtagaagcaactggacagca
tgtggccgctgcagatcgt
ctgtgagaagccacaggaa
tctgatgctgctgatggcct
aaccacaacgtgtccctggt
tggatcattcgtgtacatcagg
acctcggaggagaaact
aattcctcgagataggccgt
aagttacgcccctcattc
ctcacacaggggagacagcagg
ccaagaccgggtctcaagac
REVERSE
tccccggaattaacataagtg
tctgccacgactgcttgtc
agacaccatggttgtagcagtg
aagatcttgactgatggcaca
atccttccaggatgatggcaca
ccacgatgccattcacgatg
caggatgagactggactctc
tcaatttgtcgagggaattatc
ctgctgcgctcatttatc
tgcggttgtggagatatag
tgatgtcttttgctctgc
gaattctagtgtggtacagaagccc
ctggggtcgagtggaggactg
annealing
. Temp.
55
55
48
55
55
55
55
48
55
55
58
58
54
Mg2+
(mM)
1.5
2.5
1.5
3.5
3.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
4