.Supplementary Figure Legend
Supplementary Figure 1. Expression status of FGFRs in CLL B-cells. (A) CLL Bcells express low levels of FGFR1/FGFR2/FGFR4. Lysates from CLL B- or normal Bcells were analyzed for the expression of FGFR1, FGFR2 and FGFR4 by Western blots
using specific antibodies. Actin was used as a loading control. Densitometric analyses
suggest that expression levels of FGFR1/FGFR2/FGFR4 in CLL B-cells are no different
than those detected in normal B-cells. (B) CLL B-cells overexpress FGFR3. Lysates
from CLL B- or normal B-cells used above were further analyzed for the expression of
FGFR3 by Western blot using FGFR3 antibody from Cell Signaling Technologies. Actin
was used as a loading control. Normal subjects (N1, N2) or CLL patients (P1 – P8) are
indicated by arbitrary numbers.
Supplementary Figure 2. CLL B-cells express FGFR3. Reverse-transcribed cDNAs
obtained from total RNA of purified CLL B-cells were amplified to detect FGFR3
transcripts by polymerase-chain reaction using FGFR3-exon specific primers as
indicated (also see Supplementary Methods). GAPDH was used as loading control. We
detected FGFR3 amplification only when reverse primer for exon-11 and forward primer
at exon-6 were used for the PCR. CLL patients (P1 – P10) are indicated by arbitrary
numbers. Primer dimer bands are indicated by arrows.
Supplementary Figure 3. Role of bFGF on activating FGFRs in CLL B-cells. (A)
Effect of exogenous bFGF on FGFR phosphorylation. Purified CLL B-cells from
previously untreated CLL patients (n=3) or MDA-MB-231 cells (used as a positive
control) were treated with recombinant bFGF (100ng/ml) for 0, 5, and 10 min. Levels of
phosphorylation on FGFRs were analyzed in the cell lysates by Western blots using a
phospho-FGFR specific antibody. While FGFRs in MDA-MB-231 cells were activated by
bFGF-ligation within 5 min, we did not find any significant activation of FGFRs in CLL Bcells from the basal levels in response to bFGF-ligation. (B) Impact of a neutralizing
antibody to bFGF on FGFR phosphorylation. Purified CLL B-cells used in the above
experiment (panel A) or MDA-MB-231 cells were treated with a neutralizing antibody to
bFGF for 24 hours. Cell lysates were then analyzed for the phosphorylation status of
FGFRs at Y653/654 using a specific antibody in Western blots. Actin was used as
loading control. Results suggest no significant effect of the bFGF-neutralizing antibody
on basal level phosphorylation on FGFRs in both the cell types. However, an earlier
report demonstrates that MDA-MB-231 do not express FGF2 (bFGF).
Supplementary Figure 4. Targeting Axl in CLL B-cells by RNA interference
reduces FGFR phosphorylation. Purified CLL B-cells from previously untreated CLL
patients (n=3) were transfected with the control sc-siRNA (indicated by “-“) or Axlspecific siRNA using Amaxa human B-cell nucleofaction reagents for 48 hours. Cell
lysates were analyzed for the status of Axl expression and P-FGFR (Y653/654) level by
Western blots using specific antibodies. Actin was used as loading control. Results from
a representative CLL sample are shown.
Supplementary Figure 5. Effect of exogenous stimulants on Axl phosphorylation
in CLL B-cells. (A). Gas6-ligation could not alter Axl phosphorylation levels in
CLL B-cells. Axl was immunoprecipitated from lysates of CLL B-cells treated with the
recombinant-Gas6 (200ng/ml) for 0, 5 and 10 min, followed by Western blot analysis
using a P-Tyr (4G10) antibody. The blot was stripped and reprobed to detect
immunoprecipitated Axl. IgG HC was used as loading control. CLL patients (P1 – P3)
are indicated by arbitrary numbers. Gas6-ligation did not alter significantly the
phosphorylation levels on Axl while the Fig 1Q shows a significant increase of Axl
phosphorylation within 5 min of Gas6-ligation in MDA-MB-231 cells. (B). BCR
stimulation augments Axl phosphorylation. Axl was immunoprecipitated from lysates
of CLL B-cells treated with an anti-human IgM (10µg/ml) for 6 hour or left untreated,
followed by Western blot analysis using 4G10 antibody. The blot was stripped and
reprobed to detect immunoprecipitated Axl. IgG HC was used as loading control. CLL
patients (P1 – P3) are indicated by arbitrary numbers. While BCR stimulation increases
Axl phosphorylation in CLL B-cells with low basal P-Axl levels (P1, P3), it did not show
any effect on Axl phosphorylation in CLL B-cells (P2) with high basal P-Axl level.
Supplementary Figure 6. Impact of TP-0903 treatment on Axl and FGFR3
association in CLL B-cells. Axl was immunoprecipitated from lysates of CLL B-cells
treated with a sub-lethal dose of a high-affinity Axl inhibitor TP-0903, followed by
Western blot analysis using a P-Tyr (4G10) or FGFR3 antibody. The blot was stripped
and reprobed to detect immunoprecipitated Axl. IgG HC was used as loading control.
CLL patients (P1 – P2) are indicated by arbitrary numbers.
.