Supplementary figures legends

Sup. Figure 1: Generation of Fos phosphorylation knock-in mice

A. Nucleotide sequence determination of the region encoding the FosC-terminus of wild type and Fos AA homozygotes. PCR fragments were amplified from tail DNA and both strands were sequenced. Red boxes indicate the serines 362 and 374 codons. Only the sequence of the coding strand is shown. B. Immunohistological analysis of brain (hippocampus) sections of kainate treated (30mg/kg, 3 hours) wild type and FosAA mice. Total Fos (top) and phosphorylated serine 362 (P-Fos, bottom) immunoreactivity are shown, Magnification 10x.

Sup. Figure 2: Bone and bone-related blood parameters in Fos phosphorylation knock-in mice

Volumetric tissue mineral density (vTMD) determined by micro-computed tomography in the tibiae of 4, 12 and 25-week-old Fos AA (A) and 12-week-old Fos DD, Fos362A and Fos374A (B) mice and their corresponding control littermates. n≥5/5, n≥2 independent litters. C. Blood alkaline phosphatase (ALP, U/L, left) and Osteocalcin (Oc, pg/ml, right) in 12-week-old Fos AA, Fos DD, Fos362A and Fos374A mice and their corresponding control littermates. n≥4/4, n≥2 independent litters. D. Blood calcium (mg/dl, left) and phosphate (mg/dl, right) in 12-week-old Fos AA, Fos DD, Fos362A and Fos374A mice and their corresponding control littermates. n≥7/7, n≥3 independent litters. E. Blood OPG/RANKL ratio in 4- (left) and 12- (right) week-old Fos AA, Fos DD, Fos362A and Fos374A mice and their corresponding control littermates. n≥6/6, n≥3 independent litters. All values are presented as mean ± standard deviation.

Sup. Figure 3: In vitro osteoblast cultures from Fos AA knock-in mice

A.Cumulative cell number (proliferation) of wild type and Fos AA osteoblasts isolated from the calvariae of newborn mice and cultured in triplicate. Results are presented as mean ± standard deviation.B.In vitro osteoblast differentiation of wt and Fos AA calvarial osteoblasts. Bone nodules are visualized by alizarin red staining (red) at day 21 of culture. qRT-PCR quantification of runx2, alkaline phosphatase (alp) and osteocalcin (oc) mRNA expression at day 15 of culture. Results are presented as mean ± standard deviation of one representative culture with expression in control culture from wt littermates set to 1.

Sup. Figure 4: Relative TNF(A), IL-6 (B), and IL-10 (C) protein levels in culture supernatants from Fos AA, Fos DD, Fos362A, Fos374A and Fos-deficient (Fos KO) M-CSF-dependent bone marrow macrophages, stimulated with 0.1 ng/ml LPS for 48 hours. Values are presented as mean ± standard deviation of at least 2 independent experiments with protein expression in the respective culture from control littermate set to 1. * p<0.01, Student t-test.

Sup. Figure 5: Fos phosphorylation modulates SOS-induced papilloma formation

A. Immunohistological analysis of papilloma sections (tail) from tumour-prone K5-SOS-F (SoS+) mice in a Fos wt or Fos AA mutant background at one week of age. Total Fos (top) and phosphorylated serine 362 (P-Fos, bottom) immunoreactivity are shown, arrows point to positive nuclei.B. Quantification of the relative tumour volume (%) in 1 week old SoS+ Fos wt and SoS+ Fos AA animals. The number of analysed mice per genotype is indicated below each bar. Values are presented as mean ± standard error of the mean. C. Hematoxylin and Eosin (HE, top) staining and Keratin 5 (middle) and Loricrin (bottom) immunohistochemistry in papilloma sections from 4 weeks old SoS+ Fos wt and SoS+ Fos AA littermates. Magnification 10x.D. Immunohistological analysis of Fos in papilloma sections (tail) from tumour-prone K5-SOS-F (SoS+) mice in a wild type or Rsk2-deficient background at one week of age, arrows point to positive nuclei. E. mRNA expression (qRT-PCR) of egfr in tail papillomas from 4 week old SoS+ FosAA, FosDD, Fos362A and Rsk2-/y mice compared to their respective control littermates. Values are presented as mean ± standard deviation with expression in control set to 1 (n=2/2).F. Immunohistological analysis of Fra1 expression in papilloma sections (tail) from 4 week old SoS+ in a wild-type, FosAA, FosDD, Fos362A and Rsk2-/y background. Bar=25M.G. mRNA expression (qRT-PCR) of pdpn and klk12 in tail papillomas from 4 week old SoS+ Fos362A and Rsk2-/y mice compared to their respective control littermates. Values are presented as mean ± standard deviation with expression in control set to 1 (n≥2/2).

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