LEGEND of SUPPLEMENTAL FIGURES

Figure S1 & S2: Syk regulates MMP-9 expression and invasiveness in Oci-Ly8 (S1) and WSU-FSCCL (S2) cells

A/ MMP-9 protein expression level was evaluated by western-blot in FL cell lines treated or not (DMSO) for 48 hours with 2.5 or 5 µM R406 and 24 hours post-second transfection with non-targeting (control) or Syk siRNA. -actin expression was used as a control of protein expression.Data shown are representative of three independent experiments. Insert, Syk protein expression was evaluated in FL cells by western-blot 24 hours after two rounds of non-targeting (control) or Syk siRNA transfection. -actin expression was used as a control of protein expression. Results are representative of three independent experiments. B/ FL cell lines were treated or not (DMSO) with R406 at the indicated dose for 1 hour or transfected by non-targeting (control) or Syk siRNA (analysis 24 hours after the second round of transfection). Activation of Akt and p70S6K were evaluated by western-blot analysis. -actin expression was used as a control of protein expression. Results are representative of at least three independent experiments. C/ FL cells were treated or not (DMSO) with 2.5 µM BiPS (2 hours) or 2.5 µM R406 (48 hours) and 3D invasion was quantified by confocal microscopy as described in the Methods. Histograms are expressed as a percentage of cells invading more than 10 µm relative to the DMSO-treated control cells. Results are the mean of three independent fields + SD and are representative of at least three independent experiments. * p< 0.05.

Figure S3: MMP-9 contributes to the invasive potential of RL cells

A/ MMP-9 protein expression was evaluated in RL cells by western-blot 24 hours after two rounds of non-targeting (control) or MMP-9 siRNA transfection. -actin expression was used as a control of protein expression. Results are representative of three independent experiments. B/ 3D invasion was quantified by confocal microscopy as described in the Methods 24 hours post-second transfection. Histograms are expressed as a percentage of cells invading more than 10 µm relative to the non-targeting transfected cells. Results are the mean of three independent fields + SD and are representative of at least three independent experiments. * p< 0.05.

Figure S4: PI3K is implicated in the regulation of MMP-9 expression

RL (Fig S4A1), Oci-LY8 (Fig. S4B1) and WSU-FSCCL (Fig. S4C1) cells were treated or not (DMSO) with 100, 250 or 500 nM GDC-0941 for 1 hour and the activation status of Akt and p70S6K were evaluated by western-blot analysis. -actin expression was used as a control of protein expression. Results are representative of at least three independent experiments. MMP-9 protein levels were analyzed by western-blot analysis after 48 hours of GDC-0941 treatment in RL (A2), Oci-LY8 (B2) and WSU-FSCCL (C2) respectively. -actin expression was used as a control of protein expression. Results are representative of at least three independent experiments.

Figure S5: MMP-9 staining in FL patients

Evaluation of MMP-9 protein expression by immunohistochemistry (x 25) on lymph node tumor samples from FL patients (Pt). Images represent examples of weak (patient # 3), bright (patient # 1) or no MMP-9 staining (patient # 12).

Figure S6: Involvement of Syk in VEGF expression in Oci-Ly8 and WSU-FSCLL cells

Oci-Ly8 (A1) and WSU-FSCCLL (B1) cells were treated or not (DMSO) with 2.5 µM or 5 µM R406 for 48 hours and VEGF mRNA levels were quantified by Q-PCR. Histograms represent relative VEGF expression (N-fold regulation) and are the mean of three independent experiments + SD. * p< 0.05. Oci-Ly8 (A2) and WSU-FSCCL (B2) cells were transfected by non-targeting (control) or Syk siRNA and VEGF mRNA levels were measured 24 hours post-second transfection by Q-PCR. Results are expressed as relative expression and are the mean of three independent experiments + SD. * p< 0.05.

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