Supplemental Data - Figure Legends

Supplemental Data - Figure Legends

Essential, non-redundant roles of B-Raf and Raf-1 in Ras-driven skin tumorigenesis

Florian Kern, Eszter Doma, Christian Rupp, Theodora Niault, and Manuela Baccarini

Supplementary Figure 1-Raf ablation does not induce apoptosis (linked to Figures 1, 3, and 5)

Supplementary Figure 2- Efficient conversion of B-Raff/f alleles to Δ/Δep in tamoxifen injected animals (linked to Figure 3)

Supplementary Figure 3- B-Raf ablation correlates with reduced ERK activation and a partial rescue of cofilin phosphorylation in vitro (linked to Figure 4).

Supplementary Figure 4 - Concomitant ablation of B-Raf and Raf-1 decreases the phosphorylation of STAT3 and the expression of Myc in K5-SOS-F-driven tumors (linked to Figure 5)

Supplementary Figure 5- B-Raf/Raf-1 ablated continuously growing keratinocytes show a decrease in pERK comparable to that of B-Raf-deficient cells and an increase in pCofilin comparable to that of Raf-1-deficient cells (linked to Figure 5).

Supplementary Figure 1 - Raf ablation does not induce apoptosis.

Apoptotic cells were detected by TUNEL staining as previously described (Ehrenreiter et al., 2009). Only few TUNEL positive cells could be detected in size-matched chemically induced (a) or K5SOS-F+ (b) f/f and Δ/Δep tumors, and (c) in established tumors 6 days after B-Raf (Δ/ΔepTX) or compound B-Raf and Raf-1 ablation (Δ/Δep2TX). The scale bars represent 100µm. The plots next to the Figures represent the results of the analysis of at least three animals/ genotype (± SD). The number of apoptotic cells remained constant throughout the observation period (10 weeks for Δ/ΔepTX, 3 weeks for Δ/Δep2TX animals).

Supplementary Figure 2 - Efficient conversion of B-Raff/f alleles to Δ/Δep in tamoxifen injected animals. 50.000-100.000µm2 of tumor tissue of tamoxifen injected animals were dissected using laser microdissection and genotyped by PCR.

Supplementary Figure 3 - B-Raf ablation correlates with reduced ERK activation and a partial rescue of cofilin phosphorylation in vitro. Quantification of immunoblot results obtained with EGF-stimulated or CaCl2-treated treated primary keratinocytes of different genotypes. pERK (a) and pCofilin (b) levels were normalized to a loading control (GAPDH) and are shown as fold changes relative to levels of continuously growing wild type cells (± SD). Expression of K5SOS-F+ significantly (p < 0.01) increases phosphorylation of ERK and significantly decreases phosphorylation of cofilin in all genotypes and under all conditions, with the exception of pCofilin in B-Raf-deficient keratinocytes cultured in CaCl2 for 48 hrs. Biological replicates ≥ 3. *p < 0.05 and **p < 0.01 according to Student's t test.

Supplementary Figure 4 - Concomitant ablation of B-Raf and Raf-1 decreases the phosphorylation of STAT3 and the expression of Myc in K5-SOS-F-driven tumors.

STAT3 phosphorylation and Myc expression (lower panel) are dramatically reduced in regressing K5-SOS-F;Δ/Δep2TX tumors (day 6 after tamoxifen treatment). STAT3 phosphorylation and Myc expression were determined by immunohistochemistry. Positive cells are stained in brown. The scale bar represents 100 μm.

Supplementary Figure 5 - B-Raf/Raf-1 ablated continuously growing keratinocytes show a decrease in pERK comparable to that of B-Raf-deficient cells and an increase in pCofilin comparable to that of Raf-1-deficient cells. Quantification of immunoblot results obtained with continuously growing primary keratinocytes of different genotypes. pERK (a) and pCofilin (b) levels were normalized to a loading control (GAPDH) and are shown as fold changes relative to levels of continuously growing wild type cells (± SD). Biological replicates ≥ 3. *p < 0.05 and **p < 0.01 according to Student's t test.

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