Matography-mass spectrometry analysisSamples have been separated on a 10 SDS-PAGE gel, along with the gel stained with colloidal Coomassie blue. Protein bands of interest have been processed as described in [57].Supporting InformationFigure S1 Connected to Figure 1. HgfTg; Lkb1+/2 mice are highly prone to neonatal UVB-induced SCCs. (A) Table displaying tumor spectrum described in UVB -irradiated and Non UVB-irradiated mice. (B) Graph showing the number UVB-irradiated mice developing skin tumors. HgfTg (H), Lkb1+/2 (L) and HgfTg; Lkb1+/2 (HL). (C) Multiplicity of skin-SCC in neonatal UVBirradiated mice, HgfTg (H), Lkb1+/2 (L) and HgfTg; Lkb1+/2 (HL). UVB-Induced SCCs are extremely proliferative with low apoptosis rates and present undifferentiated regions. (D) Immunohistochemistry of UVB-induced SCCs showing representative staining of Cyclin D1, Involucrin, Keratin-14, p-c-MET, bCatenin (Bars 200 mm), and LKB1 (Bar 500 mm). Insets show a detail with the staining (Bars 50 mm). A panel of mouse SCCs showing differentiated (Diff.) and undifferentiated (Undiff.) regions. Immunohistochemistry shows staining for LKB1, bCatenin (Bars 500 mm),inset (Bars 100 mm), E-Cadherin (Bars 100 mm) and a6-Integrin (Bars 600 mm). Inset at show the loss of a6-Integrin expression in one of many keratinocyte nests (Bar 200 mm) (E) Ki67 and Triclabendazole sulfoxide Anti-infection cleaved caspase-3 staining of 3 unique tumors (Bars 200 mm). (F) Western-Blot showing the quantity of LKB1 and b-Actin in primary mouse SSC cell lines derived from tumors raised on HgfTg; Lkb1+/2 mice. HaCat cells total lysates are used as a manage. (G) Western-Blot displaying the quantity of LKB1and CDKN1A in skin extracts from indicated mice. GAPDH is showed as loading control. (TIF) Figure S2 Relate to Figure 1. (A) Keratynocytes differentiation is not compromised neither in the absence of LKB1 or overexpression of HGF. Mouse skin kind different genotypes have been stained for K14 (a ),E-Cadherin (e ), b-Catenin (i ), pErk1/2 (n ) and p-c-Met (q ). Representative photos are shown. Bars are 400 mm from a and j ; 200 mm e , n and r . (B) Lkb1+/2 and HgfTg; Lkb1+/2 mice showed an increased quantity of keratynocytes recruited in to the cell cycle upon UVB irradiation. Bars are 400 mm and one hundred mm for magnifications Graphs show quantification of Ki67 positive cells per field two hours and 48 hours after UVB irradiation (30 J/m2). No less than 30 field/ point had been evaluated. Error bars represent imply six SD. P-values had been calculated doing a student’s t-test. (TIF) Figure S3 Connected Figure 2. Lkb1 haploinsufficiency induces CDKN1A accumulation after UVB-mediated DNA harm. (A) Representative images of mouse skin stained with anti Ponceau S Autophagy p-CHK2 antibody 48 h right after UVB irradiation. Bars 100 mm. Graph shows quantification of p-CHK2 positive basal keratinocytes 48 h postirradiation. No less than fifty fields (206) from every single distinct mouse genotype (n = ten) have been quantified (WT, Lkb1+/2 (L), HgfTg (H) and HgfTg; Lkb1+/2 (HL)). P-values were calculated working with a student’s t-test. Error bars represent mean six SD. (B) Immunohistochemistry of CDKN1A staining showing representative images of mouse skin non-irradiated and 48 h after UVB irradiation. Bars 100 mm. Quantification of CDKN1A constructive cells in mouse skin 48 h post-irradiation. A minimum of two hundred and fifty fields (206) per mouse genotype (n = 10) had been quantified. Bars represent mean values. P-values had been calculated employing a student’s t-test. (C) Representative dot blot displaying a Worldwide genomicPLOS Genetics | plosgenetics.orgUVB-indu.
