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, Representative images of H&E coloration of paraffin-embedded sections of SCC12 in response to control hDF transfected with control siRNA (siLuc) or p300 (sip300) prior to control (veh.) or LIF stimulation. Scale bar, 100 mm (n ¼ 3; mean ± s.d.; ***Po0.001). (b) Immunoblot of p300, AcSTAT3 and pSTAT3 in hDF transfected with RNAi control (siLuc) or targeting p300 (siP300#1 and #2) before LIF stimulation. Immunoblot of STAT3 and tubulin as control. (c) Immunoblot of p300, AcSTAT3, pSTAT3 and STAT3 in hDF following immunoprecitation of STAT3 on 24 h LIF stimulation. Immunblot of p300 and tubulin in total lysate as control. (d) Immunoblot of pJAK1, AcSTAT3 and pSTAT3 in hDF in presence or absence of C646 inhibitor followed by long-term control (veh.) or LIF stimulation, Figure 2 | P300 acetylates STAT3 for proinvasive CAF activity. (a)

, Representative images of H&E colouration of paraffin-embedded sections of SCC12 in response to control hDF (veh.) and hDF_LIF overexpressing a control (empty-GFP) or a wild-type STAT3 (STAT3-GFP) or acetylated-deficient STAT3 mutant (STAT3-K685R-GFP). Scale bar, p.100

, n ¼ 3; mean ± s.d.; ***Po0.001). (g) Immunoblot of pJAK1, pSTAT3 and AcSTAT3 in hDF overexpressing a control vector

, STAT3 (STAT3-GFP) or acetylated-deficient STAT3 mutant (STAT3-K685R-GFP) in response to long-term LIF stimulation

, Representative images of H&E colouration of paraffin-embedded sections of SCC12 in response to CAF in the absence (Veh.) or presence of 5 0 -Aza inhibitor (n ¼ 3, I.I., invasion index, mean±s.d., ***Po0.001)

, Scale bar 100 mm. (d) Quantification of mRNA level of DNMT1, DNMT3a and DNMT3b in hDF following 48 h stimulation of LIF relative to control hDF (red dotted line) (n ¼ 3 in triplicate, mean þ s.d., ***Po0.001). (e) Immunoblot of AcSTAT3, DNMT3b and STAT3 in hDF cells overexpressing a control or a wild type STAT3 or acetylated deficient STAT3 mutant following STAT3 immunoprecipitation upon LIF stimulation, Percentage of gel contraction by CAF transfected with siRNA control (siLuc) or targeting DNMT1 (siDNMT1#1, #2, #3, #4) (n ¼ 3 in triplicate, mean þ s.d., ***Po0.001)

, Immunoblot of JAK1, STAT3 and tubulin as control. (e) Schematic illustration of the PTPN6 gene structure and the regions analysed by bisulfite sequencing. The PTPN6 gene has two alternative promoters. We restricted our analysis to Promoter 1 since Promoter 2 is mostly active in the haematopoietic lineage. Vertical lines in Region 1 and Region 2 indicate the position of the CpG dinucleotides. (f) Bisulfite-sequencing results in two regions of the PTPN6 promoter (R1 and R2) in hDF transfected with control RNAi (siLuc) and RNAi targeting DNMT3b (siDNMT3b#1 and #2) and subsequently long-term control ( À ) or LIF-activated ( þ ), | Epigenetic-dependent sustained JAK1/STAT3 signalling in CAF. (a) Immunoblot of pJAK1, pSTAT3 and AcSTAT3 in CAF after 7-days treatment in the presence or absence of 5 0 -Aza inhibitor at 10 and 3 mM final concentration. Immunoblot of JAK1, STAT3 and tubulin as control. (b)

, Representative images of H&E coloration of paraffin-embedded sections of SCC12 in response to CAF overexpressing control vector (GFP) or SHP-1

, 001). (h) Immunoblot of SHP-1, pJAK1, pSTAT3 and AcSTAT3 in CAF overexpressing control vector (GFP) or SHP-1 gene (SHP-1_GFP)

I. ,

I. ,

I. ,

I. I. ,

, After inhibitors treatment for 7 or 21 days, cells are embedded in matrix for a 50-day gel contraction assay, assayed every day for a period of 5 days for molecular analysis, and assayed at day 5 after inhibitors removal for proinvasive ability. (See also Supplementary Fig. 5). (b) Percentage of gel contraction of hDF and CAF for a period of 50 days following a 7-day treatment of either Ruxolitinib or 5 0 -Aza or both inhibitors together. (c) Representative images of H&E coloration of paraffin-embedded sections of SCC12 in response to CAF, hDF_LIF and hDF_TGFb1, Figure 5 | Phenotypic and molecular reversion of CAF. (a), vol.5

, Methods Cell culture. Human primary dermal fibroblasts (hDF), MDA-MB-468 and human HEK293 Phoenix cells were maintained in DMEM supplemented with 10% FCS (fetal calf serum). Human CAF isolated from patients with head and neck, lung and breast cancers were cultured in DMEM

C. Invitrogen, SCC12 cells were cultured in FAD media, as described in Gaggioli et al. 9 . Long-term LIF and TGFb1-activated fibroblasts

, ) and was used at 2 ng ml À 1, Cytokines and neutralizing antibodies and inhibitors. TGFb1 was purchased from Peprotech, pp.100-121

M. #lif1005 and M. ). Billerica, and was used at a concentration of 2 ng ml À 1 . LIF neutralizing antibody (AB-250-NA, R&D, Minneapolis, MN) this study: Pyridone 6 (#42009, Calbiochem, Los Angeles, CA) was used at 5 mM, Ruxolitinib (#1598, Axon medchem, recombinant human LIF was purchased from Millipore, vol.10, pp.25-28, 2385.

I. Dharmacon and . Lafayette, CO) at 100 nM final concentration of RNAi. For long-term activation listed below: p300

. Dnmt3b,

. Dnmt3b,

. Dnmt3b,

. Dnmt3b,

. Antibodies, Antibodies against STAT3 (#9139; 1/1,000), pp.705-708

, ac-K685 STAT3 (#2523000), pY1022/1023-JAK1 (#3331Signalling (Cell SignalingTechnology, Beverly MA), a-tubulin from sigma (T4026

, SHP-1 (#sc-7289, 1/ 500 for western blot analysis), vol.1, pp.3-130740

, CA) and SHP-1 (#ab2020; 1/200 for immunohistochemical staining) from Abcam (#ab2020, /1,000) from Santa Cruz Biotechnology

M. #wbkls0500 and M. ). Billerica, Uncropped western blots are shown in the Supplementary Information section and labelled as Supplementary Fig. 7. For co-immunoprecipitation analysis, cells were lysed on ice in modified RIPA buffer (50 mM Tris pH 7.4 150 mM NaCl, 1%NP-40, 0.1% SDS, 0.5% SD Dexoxycholate, 5 mM NaF, 2.5 mM Nappi, and protease inhibitor (#04693159001, Roche)) for 30 min and isolated by centrifugation, For immunoblotting analysis, cells were lysed on ice in lysis buffer (25 mM Tris, vol.6

. Rt-qpcr-analysis, RNA isolation was performed using RNeasy Mini kit (#217004, Qiagen, Turnberry Ln, Valencia, CA) according to the manufacturer's instructions. Reverse transcription (RT) of 500 ng RNA by Superscript II reverse transcriptase (#18064-014, ) was followed by real-time PCR using Fast SYBR Green Master Mix, pp.18064-18078

, Primers sequences are listed as follows: DNMT1: sense, 5 0 -CCAAAGCCCGAGAGAGTGCCTCAG-3 0 and antisense, Step One Plus Real-Time PCR system (Applied Biosystems, vol.5, pp.0-3

, DNMT3a: sense, 5 0 -CTGAAGGACTTGGGCATTCAG-3 0 and antisense, vol.5, pp.0-3

, DNMT3b: sense, 5 0 -TACACAGACGTGTCCAACATGGGC-3 0 and antisense

, GAPDH: sense, 5 0 -ACCCAGAAGACTGTGGATGG-3 0 , and antisense

, Sections were washed three times in PBS (5 min per wash) before endogenous peroxidase activity was blocked in 1% H2O2 for 10 min and washed three times in PBS. After incubation in blocking buffer for 2 h, SHP-1 staining, and in EDTA buffer (1 mM

. Vector, CA) diluted 1:400 in PBS for 30 min and washed three times in PBS. Samples were then processed using Vectastain ABC kit (#PK4001, Vector, Burlingame, CA) and DAB peroxidase substrate kit (#SK4100, Vector, Burlingame, CA) according to manufacturer's instructions. Sections were next counterstained with haematoxylin for 5 s, rinsed in water, blued 10 s in 0,08% ammonia water, dehydrated, cleared and mounted with cover clips. Two authors, blinded to each other's assessment, scored the slides using the Quick Score method 16 to determine pSTAT3, AcSTAT3, DNMT3b and SHP-1 status within the tumour bulk and stroma. The protocol was approved by the local ethic committee of the Nice University Hospital, 0.3% Triton X100 in PBS), sections were incubated with primary antibody diluted 1:50 in blocking buffer overnight at 4°C. After three washes in PBS, sections were incubated with biotinylated secondary antibody (#BA-1000 Vector

, Microscope conditions (lamp brightness, condenser opening, objective, zoom, exposure time and gain parameters) were constant throughout the imaging of all samples. A minimal threshold was set on appropriate control sections for each experiment in which only the light passing through the orthogonally oriented polarizers representing fibrous structures (that is, excluding residual light from the black background) was included. The threshold was maintained for all images across all conditions within each experiment. The area of the, Picrosirius Red stain and quantification. Picrosirius Red stain was achieved through the use of 5-mm paraffin sections stained with 0.1% Picrosirius Red (Direct Red80

, The sequence of the oligonucleotide primers used are: STAT3-K685R-Forward: 5 0 -GAGGAGGCATTTGGAAGGTACTGTAGGCCC GAG-3 0 ; and STAT3 K685R-Reverse: 5 0 -CTCGGGCCTACAGTACCTTCCAAAT GCCTCCTC-3 0 and constructs were then coupled to GFP. Following constructs verification by nucleotide sequencing, the STAT3 (wild-type and K685R) fragments were excised from pcDNA5 by Pme1 and BamH1 digestion and directionally cloned into pBabe retroviruses expression vectors (containing the puromycin resistance gene as a selectable marker, Orthotopic syngenic tumours xenograft in BALB/c mice. Eight-week-old female BALB/c mice were anaesthetized using ketamine and xylazine by peritoneal injection, p.1401, 113467.

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