( B ) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells . Gene architecture of human DUSP1 . The primers at - 243 / - 67 ( " - 67 " ) , - 473 / - 224 ( " - 224 " ) and - 1170 / - 961 ( " - 916 " ) relative to the TSS were used for ChIP experiments . IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides - 243 and - 67 .

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(B) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells. Gene architecture of human DUSP1. The primers at -243/-67 ("-67"), -473/-224 ("-224") and -1170/-961 ("-916") relative to the TSS were used for ChIP experiments. IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides -243 and -67.


( C ) ChIP / qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between - 243 and - 67 . Data show mean ( bar ) and individual data points from 2 biological replicates .

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(C) ChIP/qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between -243 and -67. Data show mean (bar) and individual data points from 2 biological replicates.


( D ) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF . Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked - down for endogenous IRF2BP1 , followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region - 243 and - 67 . Data show means + / - SEM from 3 biological replicates .

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(D) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF. Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked-down for endogenous IRF2BP1, followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region -243 and -67. Data show means +/- SEM from 3 biological replicates.


( E ) Q - PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1 . Data show means + / - SEM from 3 biological replicates .

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(E) Q-PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1. Data show means +/- SEM from 3 biological replicates.


( F ) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q - PCR ( left panel ) , data show means + / − SEM from 5 biological replicates ( left panel ) , and IRF2BP1 protein levels . As expected , IRF2BP1 V578A is not SUMOylated ( right panel ) . * refers to an unspecific band .

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(F) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q-PCR (left panel), data show means +/− SEM from 5 biological replicates (left panel), and IRF2BP1 protein levels. As expected, IRF2BP1 V578A is not SUMOylated (right panel). * refers to an unspecific band.


( G ) Q - PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 . Data show means + / − SEM from 4 biological replicates .

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(G) Q-PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1. Data show means +/− SEM from 4 biological replicates.


( A ) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1 . Uba2 and beta - actin serve as independent loading controls .

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(A) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1. Uba2 and beta-actin serve as independent loading controls.


( C ) Wound healing assay : HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days , grown to 90 % confluency and analysed for wound closure with or without addition of 100 ng / ml EGF . Data show means of the relative wound density + / - SEM from 4 biological replicates .

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(C) Wound healing assay: HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days, grown to 90% confluency and analysed for wound closure with or without addition of 100 ng/ml EGF. Data show means of the relative wound density +/- SEM from 4 biological replicates.


( D ) Proliferation assay : HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium . One representative biological experiment is shown with means + / - SEM from 5 technical replicates .

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(D) Proliferation assay: HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium. One representative biological experiment is shown with means +/-SEM from 5 technical replicates.


( E ) IRF2BP1 , IRF2BP2 , IRF2BP1 + IRF2BP2 were knocked - down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis ; non - targeting siRNAs were used as a control . IRF2BP1 and IRF2BP2 have distinct and overlapping functions . Experiments were done in triplicates .

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(E) IRF2BP1, IRF2BP2, IRF2BP1+IRF2BP2 were knocked-down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis; non-targeting siRNAs were used as a control. IRF2BP1 and IRF2BP2 have distinct and overlapping functions. Experiments were done in triplicates.


( G ) Validation of two candidate genes : IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells , and cell lysates were analyzed by immunoblotting with the indicated antibodies .

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(G) Validation of two candidate genes: IRF2BP1 and IRF2BP2 were knocked down for 72 hours in HeLa cells, and cell lysates were analyzed by immunoblotting with the indicated antibodies.


( H ) FACS - based analysis of EGFR surface expression : IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells , cells were serum starved for 16 hours , stained with fluorescent anti - EGFR antibodies and analyzed by flow cytometry . Shown are means of 3 independent experiments + / - SEM .

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(H) FACS-based analysis of EGFR surface expression: IRF2BP1 and IRF2BP2 were knocked down for 48 hours in HeLa cells, cells were serum starved for 16 hours, stained with fluorescent anti-EGFR antibodies and analyzed by flow cytometry. Shown are means of 3 independent experiments +/-SEM.


A . FGF21SS and FGF21 improve insulin resistance by countering the inflammation in 3T3 - L1 adipocytes . Cells were cultured in RAW - CM to induce inflammation and insulin resistance , while vehicle , FGF21 or FGF21SS were added simultaneously . The insulin sensitivity was indicated by insulin - induced AKT phosphorylation using western blotting analysis . Error bars show the SEM of four independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) vs RAW - CM treated only group .

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A. FGF21SS and FGF21 improve insulin resistance by countering the inflammation in 3T3-L1 adipocytes. Cells were cultured in RAW-CM to induce inflammation and insulin resistance, while vehicle, FGF21 or FGF21SS were added simultaneously. The insulin sensitivity was indicated by insulin-induced AKT phosphorylation using western blotting analysis. Error bars show the SEM of four independent experiments. *p < 0.05, **p < 0.01 (Student's t-test) vs RAW-CM treated only group.


Anti - diabetic effect evaluation in ob / ob mice . Mice were injected subcutaneously with vehicle , FGF21 or FGF21SS at indicated doses every day . Body weight ( B ) was measured Error bars show the SEM of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( Student ' s t - test ) vs vehicle .

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Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. Body weight (B) was measured Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.


Anti - diabetic effect evaluation in ob / ob mice . Mice were injected subcutaneously with vehicle , FGF21 or FGF21SS at indicated doses every day . plasma glucose ( D ) was measured every day . Plasma insulin ( C ) was analyzed at day 7 using insulin ELISA kit . Error bars show the SEM of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( Student ' s t - test ) vs vehicle .

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Anti-diabetic effect evaluation in ob/ob mice. Mice were injected subcutaneously with vehicle, FGF21 or FGF21SS at indicated doses every day. plasma glucose (D) was measured every day. Plasma insulin (C) was analyzed at day 7 using insulin ELISA kit. Error bars show the SEM of ten independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 (Student's t-test) vs vehicle.


( a ) Identification of biomarkers modulated upon VCP silencing . HeLa , HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h . Protein cell extracts were resolved by SDS - PAGE , and filters were probed with the indicated antibodies . The extent of biomarker modulation correlated with the efficiency of silencing ( oligo 4 versus pool ) . VCP silencing determined stabilization of cyclin E ; induction of the endoplasmic reticulum chaperone GRP78 , CHOP transcription factor and its downstream target GADD - 34 ; conversion of the autophagy regulator ATG8 / light chain 3 ( LC3B ) from the free to the lipidated form ; activation of the effector caspase - 3 ; and cleavage of the caspase target PARP - 1 . A graphical representation of the cell cycle distribution ( percentage of cells ) obtained from the cell cycle analysis ( Supplementary Fig . 3b ) is reported at the bottom of each sample . C , control ; NT , nontargeting oligo ; P , oligonucleotide pool .

O O O O O O O O B-GENEPROD O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a) Identification of biomarkers modulated upon VCP silencing. HeLa, HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h. Protein cell extracts were resolved by SDS-PAGE, and filters were probed with the indicated antibodies. The extent of biomarker modulation correlated with the efficiency of silencing (oligo 4 versus pool). VCP silencing determined stabilization of cyclin E; induction of the endoplasmic reticulum chaperone GRP78, CHOP transcription factor and its downstream target GADD-34; conversion of the autophagy regulator ATG8/light chain 3 (LC3B) from the free to the lipidated form; activation of the effector caspase-3; and cleavage of the caspase target PARP-1. A graphical representation of the cell cycle distribution (percentage of cells) obtained from the cell cycle analysis (Supplementary Fig. 3b) is reported at the bottom of each sample. C, control; NT, nontargeting oligo; P, oligonucleotide pool.


( b ) Effect of VCP silencing on cell growth . HeLa , HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter ( data represent mean values of two experiments ) .

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(b) Effect of VCP silencing on cell growth. HeLa, HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter (data represent mean values of two experiments).


( c ) Combination of VCP siRNA with standard - of - care therapeutics . HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose - response analysis with a variety of chemotherapeutic agents .

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(c) Combination of VCP siRNA with standard-of-care therapeutics. HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose-response analysis with a variety of chemotherapeutic agents.


( b ) LC / MS analysis of VCP after 1 h incubation at 20 ° C with 1 % DMSO ( top ) or 100 μM NMS - 859 ( bottom ) , showing a 313 - Da increase corresponding to the addition of NMS - 859 lacking the Cl atom .

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(b) LC/MS analysis of VCP after 1 h incubation at 20 °C with 1% DMSO (top) or 100 μM NMS-859 (bottom), showing a 313-Da increase corresponding to the addition of NMS-859 lacking the Cl atom.


( a ) Activity of VCP inhibitors NMS - 249 and NMS - 873 on wild - type VCP and mutants . Data represent mean values of at least three independent experiments .

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(a) Activity of VCP inhibitors NMS-249 and NMS-873 on wild-type VCP and mutants. Data represent mean values of at least three independent experiments.


( b ) Binding of the EDA - ADP - ATTO 590 probe to wild - type VCP in the presence of different allosteric inhibitors or ADP .

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(b) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP in the presence of different allosteric inhibitors or ADP.


( c ) Binding of the EDA - ADP - ATTO 590 probe to wild - type ( WT ) VCP and the K615V and N616F mutants in the presence of NMS - 249 . Minor differences in the mP values are due to the use of different enzyme batches .

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(c) Binding of the EDA-ADP-ATTO 590 probe to wild-type (WT) VCP and the K615V and N616F mutants in the presence of NMS-249. Minor differences in the mP values are due to the use of different enzyme batches.


( d ) Binding of the EDA - ADP - ATTO 590 probe to wild - type VCP and Walker A mutants in the presence of NMS - 249 . Data shown are representative results of one of three independent experiments : each determination was done in triplicate , data points denote the average value , and error bars represent s . d .

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(d) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP and Walker A mutants in the presence of NMS-249. Data shown are representative results of one of three independent experiments: each determination was done in triplicate, data points denote the average value, and error bars represent s.d.


( a ) Western blot analysis of biomarker modulation in the HCT116 cell line . HCT116 cells were treated with the indicated doses of inhibitors for 8 h ( top ) or 24 h ( bottom ) , and protein cell extracts were resolved on SDS - PAGE gel . Filters were probed with the indicated antibodies . C , control ; B , bortezomib ( 0 . 01 μM ) ; T , thapsigargin ( 0 . 1 μM ) ; Mg , MG132 ( 0 . 1 μM ) .

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(a) Western blot analysis of biomarker modulation in the HCT116 cell line. HCT116 cells were treated with the indicated doses of inhibitors for 8 h (top) or 24 h (bottom), and protein cell extracts were resolved on SDS-PAGE gel. Filters were probed with the indicated antibodies. C, control; B, bortezomib (0.01 μM); T, thapsigargin (0.1 μM); Mg, MG132 (0.1 μM).


( b ) NMS - 873 compound - mediated stabilization of Mcl - 1 in live cells . Top , confocal time - lapse images of live stable , inducible T - REx - 293 cells expressing a bicistronic construct of mCherry - BimS ( 2A ) ΔC ( red ) with eGFP - Mcl - 1 ( green ) after doxycycline treatment . Cells were treated with cycloheximide ( CHX , 50 μg ml − 1 ) ± 2 . 5 μM of NMS - 873 for 6 h . Bottom , total intensity of eGFP - Mcl - 1 and mCherry - BimS ( 2A ) ΔC was measured for time - lapse series shown at top . A decrease in eGFP - Mcl - 1 and mCherry - BimS ( 2A ) ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control . WT , wild type . Scale bar , 10 μm .

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(b) NMS-873 compound-mediated stabilization of Mcl-1 in live cells. Top, confocal time-lapse images of live stable, inducible T-REx-293 cells expressing a bicistronic construct of mCherry-BimS(2A)ΔC (red) with eGFP-Mcl-1 (green) after doxycycline treatment. Cells were treated with cycloheximide (CHX, 50 μg ml−1) ± 2.5 μM of NMS-873 for 6 h. Bottom, total intensity of eGFP-Mcl-1 and mCherry-BimS(2A)ΔC was measured for time-lapse series shown at top. A decrease in eGFP-Mcl-1 and mCherry-BimS(2A)ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control. WT, wild type. Scale bar, 10 μm.


( c ) High - content analysis of poly - Ub and CHOP induction upon treatment with VCP inhibitors . HeLa cells were treated with increasing doses of the indicated compounds . Cells were then fixed and immunostained with the indicated antibodies . Cell numbers and mean fluorescence intensity ( MFI ) were determined using the ArrayScan platform . Data were collected in triplicate ( n = 3 ) , and error bars represent s . d . AU , arbitrary units .

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(c) High-content analysis of poly-Ub and CHOP induction upon treatment with VCP inhibitors. HeLa cells were treated with increasing doses of the indicated compounds. Cells were then fixed and immunostained with the indicated antibodies. Cell numbers and mean fluorescence intensity (MFI) were determined using the ArrayScan platform. Data were collected in triplicate (n = 3), and error bars represent s.d. AU, arbitrary units.


Representative flow cytometric analysis of BAL from Csf2ra - / - recipients 8 weeks after neonatal i . n . transfer of YS EMP from CD45 . 1 + E10 . 5 embryos , 8 - week old un - transferred Csf2ra - / - , and WT ( CD45 . 2 ) mice . Pre - gated viable CD45 + singlets .

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Representative flow cytometric analysis of BAL from Csf2ra-/- recipients 8 weeks after neonatal i.n. transfer of YS EMP from CD45.1+ E10.5 embryos, 8-week old un-transferred Csf2ra-/-, and WT (CD45.2) mice. Pre-gated viable CD45+ singlets.


Numbers of EMP effectively transferred in neonates ( white circle ) and numbers of EMP - derived AM ( red circles ) in BAL and lung of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) .

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Numbers of EMP effectively transferred in neonates (white circle) and numbers of EMP-derived AM (red circles) in BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice).


Numbers of EMP - derived AM in the BAL ( left ) and lung ( right ) ( C ) in the BAL of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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Numbers of EMP-derived AM in the BAL (left) and lung (right) (C) in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


total protein in the BAL of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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total protein in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


total eFluor780 + events ( E ) in the BAL of adult Csf2ra - / - recipients 8 weeks after transfer ( n = 5 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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total eFluor780+ events (E) in the BAL of adult Csf2ra-/- recipients 8 weeks after transfer (n = 5 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) in the BAL and lung from Csf2ra - / - recipients 8 weeks after neonatal transfer of 1 : 1 : 1 mixture with E17 . 5 fetal liver ( FeLi ) monocytes ( Mo ) ( CD45 . 1 + ) , E14 . 5 FeLi Mo ( CD45 . 1 + CD45 . 2 + ) , and E10 . 5 YS EMP ( CD45 . 2 + ) ( F ) or E17 . 5 fetal lung ( FeLu ) Mo ( CD45 . 1 + ) , E14 . 5 FeLu Mo ( CD45 . 1 + CD45 . 2 + ) , and E10 . 5 YS EMP ( CD45 . 2 + ) ( G ) ( n = 3 mice ) .

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Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with E17.5 fetal liver (FeLi) monocytes (Mo) (CD45.1+), E14.5 FeLi Mo (CD45.1+CD45.2+), and E10.5 YS EMP (CD45.2+) (F) or E17.5 fetal lung (FeLu) Mo (CD45.1+), E14.5 FeLu Mo (CD45.1+CD45.2+), and E10.5 YS EMP (CD45.2+) (G) (n = 3 mice).


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) in the BAL ( top ) and lung ( bottom ) from Csf2ra - / - recipients 10 weeks after neonatal transfer of 1 : 1 mixture with CD45 . 1 + E14 . 5 FeLi Mo and CD45 . 2 + E14 . 5 FeLi pMΦ ( Group 1 ) or CD45 . 1 + E14 . 5 FeLi pMΦ and CD45 . 2 + FeLi Mo ( Group 2 ) ( n = 3 mice ) . The expansion fold of donor cells in Csf2ra - / - recipients 10 weeks after neonatal transfer of cells described above in A ( n = 3 mice ) .

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Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL (top) and lung (bottom) from Csf2ra-/- recipients 10 weeks after neonatal transfer of 1:1 mixture with CD45.1+ E14.5 FeLi Mo and CD45.2+ E14.5 FeLi pMΦ (Group 1) or CD45.1+ E14.5 FeLi pMΦ and CD45.2+ FeLi Mo (Group 2) (n = 3 mice). The expansion fold of donor cells in Csf2ra-/- recipients 10 weeks after neonatal transfer of cells described above in A (n = 3 mice).


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) of BAL and lung from Csf2ra - / - recipients 8 weeks after neonatal transfer of 1 : 1 : 1 mixture with CD45 . 1 + E17 . 5 FeLi pMΦ , CD45 . 1 + CD45 . 2 + E14 . 5 FeLi pMΦ , and CD45 . 2 + E10 . 5 YS pMΦ ( n = 3 mice ) . Total numbers of donor - derived AM in the BAL and lung of adult Csf2ra - / - recipients 8 weeks after transfer of cells described above in C ( n = 3 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

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Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) of BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with CD45.1+ E17.5 FeLi pMΦ, CD45.1+CD45.2+ E14.5 FeLi pMΦ, and CD45.2+ E10.5 YS pMΦ (n = 3 mice). Total numbers of donor-derived AM in the BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer of cells described above in C (n = 3 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) of BAL and lung from Csf2ra - / - recipients 8 weeks after neonatal transfer of 1 : 1 : 1 mixture with CD45 . 1 + E17 . 5 FeLu pMΦ , CD45 . 1 + CD45 . 2 + E14 . 5 FeLu pMΦ , and CD45 . 2 + E10 . 5 YS pMΦ ( n = 3 mice ) . Total numbers of donor - derived AM in the BAL and lung of adult Csf2ra - / - recipients 8 weeks after transfer of cells described above in E ( n = 3 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 3 mice ) and WT ( n = 3 mice ) were included as negative and positive controls .

Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) of BAL and lung from Csf2ra-/- recipients 8 weeks after neonatal transfer of 1:1:1 mixture with CD45.1+ E17.5 FeLu pMΦ, CD45.1+CD45.2+ E14.5 FeLu pMΦ, and CD45.2+ E10.5 YS pMΦ (n = 3 mice). Total numbers of donor-derived AM in the BAL and lung of adult Csf2ra-/- recipients 8 weeks after transfer of cells described above in E (n = 3 mice). Age-matched Csf2ra-/- (KO) (n = 3 mice) and WT (n = 3 mice) were included as negative and positive controls.


Representative flow cytometric analysis ( left ) and percentage of donor - derived AM ( right ) in the BAL and lung from Csf2ra - / - recipients 7 weeks after neonatal transfer of 1 : 1 mixture with CD45 . 1 + E14 . 5 FeLi Mo from WT embryos and CD45 . 2 + E14 . 5 FeLi pMΦ from Myb - / - ( G ) or Myb + / + ( H ) embryos ( n = 3 mice ) .

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Representative flow cytometric analysis (left) and percentage of donor-derived AM (right) in the BAL and lung from Csf2ra-/- recipients 7 weeks after neonatal transfer of 1:1 mixture with CD45.1+ E14.5 FeLi Mo from WT embryos and CD45.2+ E14.5 FeLi pMΦ from Myb-/- (G) or Myb+/+ (H) embryos (n = 3 mice).


Representative flow cytometric analysis of BAL from Csf2ra - / - recipients 7 weeks after neonatal i . n . transfer of pMΦ or fetal Mo from CD45 . 1 + E14 . 5 fetal livers , 7 - week old un - transferred Csf2ra - / - , WT ( CD45 . 2 ) mice . Pre - gated viable CD45 + singlets .

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Representative flow cytometric analysis of BAL from Csf2ra-/- recipients 7 weeks after neonatal i.n. transfer of pMΦ or fetal Mo from CD45.1+ E14.5 fetal livers, 7-week old un-transferred Csf2ra-/-, WT (CD45.2) mice. Pre-gated viable CD45+ singlets.


Numbers of donor - derived AM in the BAL ( left ) and lung ( right ) of adult Csf2ra - / - recipients 7 weeks ( B ) or 1 year ( C ) after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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Numbers of donor-derived AM in the BAL (left) and lung (right) of adult Csf2ra-/- recipients 7 weeks (B) or 1 year (C) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


Total protein in the BAL of adult Csf2ra - / - recipients 7 weeks after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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Total protein in the BAL of adult Csf2ra-/- recipients 7 weeks after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


total eFluor780 + events in the BAL of adult Csf2ra - / - recipients 7 weeks after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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total eFluor780+ events in the BAL of adult Csf2ra-/- recipients 7 weeks after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


Total protein in the BAL of adult Csf2ra - / - recipients 1 year ( F , G ) after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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Total protein in the BAL of adult Csf2ra-/- recipients 1 year (F, G) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


total eFluor780 + events in the BAL of adult Csf2ra - / - recipients 1 year ( F , G ) after neonatal transfer of cells described in A ( n = 7 mice ) . Age - matched Csf2ra - / - ( KO ) ( n = 5 mice ) and WT ( n = 4 mice ) were included as negative and positive controls .

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total eFluor780+ events in the BAL of adult Csf2ra-/- recipients 1 year (F, G) after neonatal transfer of cells described in A (n = 7 mice). Age-matched Csf2ra-/- (KO) (n = 5 mice) and WT (n = 4 mice) were included as negative and positive controls.


Body weight measurement of influenza virus ( 20 pfu of PR8 strain ) - infected Csf2ra - / - recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14 . 5 fetal livers ( n = 7 mice from each transferred group ) , 24 - week old un - transferred Csf2ra - / - ( n = 5 ) , and WT ( n = 8 ) .

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Body weight measurement of influenza virus (20 pfu of PR8 strain)-infected Csf2ra-/- recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14.5 fetal livers (n = 7 mice from each transferred group), 24-week old un-transferred Csf2ra-/- (n = 5), and WT (n = 8).


temperature ( B ) measurement of influenza virus ( 20 pfu of PR8 strain ) - infected Csf2ra - / - recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14 . 5 fetal livers ( n = 7 mice from each transferred group ) , 24 - week old un - transferred Csf2ra - / - ( n = 5 ) , and WT ( n = 8 ) .

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temperature (B) measurement of influenza virus (20 pfu of PR8 strain)-infected Csf2ra-/- recipients 24 weeks after neonatal transfer of pMΦ or fetal Mo from E14.5 fetal livers (n = 7 mice from each transferred group), 24-week old un-transferred Csf2ra-/- (n = 5), and WT (n = 8).


Oxygen ( O2 ) saturation in influenza - infected mice described in A , B 7 days post - infection ( p . i . ) ( n = 5 mice from Csf2ra - / - group ; n = 7 mice from each transferred group ; n = 8 from WT group ) .

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Oxygen (O2) saturation in influenza-infected mice described in A, B 7 days post-infection (p.i.) (n = 5 mice from Csf2ra-/- group; n = 7 mice from each transferred group; n=8 from WT group).


Survival curve of influenza - infected mice described in A , B ( n = 5 mice from Csf2ra - / - group ; n = 7 mice from each transferred group ; n = 8 from WT group ) .

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Survival curve of influenza-infected mice described in A, B (n = 5 mice from Csf2ra-/- group; n = 7 mice from each transferred group; n=8 from WT group).


Total eFluor780 + events in the BAL of influenza - infected mice described in A , B 10 days p . i . ( n = 3 mice from each group ) .

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Total eFluor780+ events in the BAL of influenza-infected mice described in A, B 10 days p.i. (n = 3 mice from each group).


Representative flow cytometric analysis of Csf2ra ( CD116 ) expression on different fetal precursors from WT embryos . The solid gray histogram represents the fluorescence minus one ( FMO ) controls , the open black histogram represents stained samples . The gray number represents mean fluorescence intensity ( MFI ) of FMO controls , the black number represents MFI of stained samples . E10 . 5 YS cells were pooled from 8 - 10 embryos , E14 . 5 lung cells were pooled from 3 - 4 embryos , and E14 . 5 liver cells were from individual embryo .

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Representative flow cytometric analysis of Csf2ra (CD116) expression on different fetal precursors from WT embryos. The solid gray histogram represents the fluorescence minus one (FMO) controls, the open black histogram represents stained samples. The gray number represents mean fluorescence intensity (MFI) of FMO controls, the black number represents MFI of stained samples. E10.5 YS cells were pooled from 8-10 embryos, E14.5 lung cells were pooled from 3-4 embryos, and E14.5 liver cells were from individual embryo.


Representative flow cytometric analysis of Csf2ra expressions on pMΦ - and fetal Mo - derived AM in Csf2ra - / - recipients 4 weeks after transfer of pMΦ or fetal Mo from E14 . 5 fetal livers . AM from 4 - week old WT mice were included as controls .

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Representative flow cytometric analysis of Csf2ra expressions on pMΦ- and fetal Mo-derived AM in Csf2ra-/- recipients 4 weeks after transfer of pMΦ or fetal Mo from E14.5 fetal livers. AM from 4-week old WT mice were included as controls.


Heatmap of AM signature - up genes ( D ) , AM signature - down genes ( E ) , and AM - specific transcription factors ( F ) in the precursors and mature AM described above in A . the data are from one RNA - seq experiment ( 1 - 2 biological replicates per group ) .

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Heatmap of AM signature-up genes (D), AM signature-down genes (E), and AM-specific transcription factors (F) in the precursors and mature AM described above in A. the data are from one RNA-seq experiment (1 - 2 biological replicates per group).


qPCR ( n = 3 biological replicates ) analysis of Myb , Maf , Mafb , and Pparg expression in different fetal precursors from WT embryos and mature AM from WT adult mice .

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qPCR (n = 3 biological replicates) analysis of Myb, Maf, Mafb, and Pparg expression in different fetal precursors from WT embryos and mature AM from WT adult mice.


Oxygen consumption rate ( OCR ) of pMΦ and fetal Mo from E14 . 5 WT fetal liver at baseline and after treatment with oligomycin ( Oligo ) , FCCP or rotenone ( Rot ) and antimycin A ( AA ) .

Oxygen consumption rate (OCR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with oligomycin (Oligo), FCCP or rotenone (Rot) and antimycin A (AA).


Extracellular acidification rate ( ECAR ) of pMΦ and fetal Mo from E14 . 5 WT fetal liver at baseline and after treatment with glucose , Oligo , or 2 - Deoxy - D - glucose ( 2 - DG ) .

Extracellular acidification rate (ECAR) of pMΦ and fetal Mo from E14.5 WT fetal liver at baseline and after treatment with glucose, Oligo, or 2-Deoxy-D-glucose (2-DG).


RNA - seq ( C , n = 2 biological replicates ) analysis of Eno1 , Slc2a6 , Txn1 , and Txnip gene expression in pMΦ and fetal Mo from E14 . 5 WT fetal liver .

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RNA-seq (C, n = 2 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.


qPCR ( D , n = 3 biological replicates ) analysis of Eno1 , Slc2a6 , Txn1 , and Txnip gene expression in pMΦ and fetal Mo from E14 . 5 WT fetal liver .

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qPCR (D, n = 3 biological replicates) analysis of Eno1, Slc2a6, Txn1, and Txnip gene expression in pMΦ and fetal Mo from E14.5 WT fetal liver.


C - E . Western blot analysis for CCNA , CCNB , Cdk1 and GAPDH ( loading control ) protein expression in lysates from control ( si - GFP ) and p53 - depleted ( si - p53 ; si - p53 _ 1 ) ( C ) MDA - MB - 231 and CAL27 cell lines . Representative images are shown .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O

C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and p53-depleted (si-p53; si-p53_1) (C) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


C - E . Western blot analysis for CCNA , CCNB , Cdk1 and GAPDH ( loading control ) protein expression in lysates from control ( si - GFP ) and YAP - depleted ( si - YAP ; si - YAP _ 1 ) ( D ) MDA - MB - 231 and CAL27 cell lines . Representative images are shown .

C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and YAP-depleted (si-YAP; si-YAP_1) (D) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


C - E . Western blot analysis for CCNA , CCNB , Cdk1 and GAPDH ( loading control ) protein expression in lysates from control ( si - GFP ) and TAZ - depleted ( si - TAZ ) ( E ) MDA - MB - 231 and CAL27 cell lines . Representative images are shown .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O

C-E. Western blot analysis for CCNA, CCNB, Cdk1 and GAPDH (loading control) protein expression in lysates from control (si-GFP) and TAZ-depleted (si-TAZ) (E) MDA-MB-231 and CAL27 cell lines. Representative images are shown.


A . Primary human breast cancers of the METABRIC dataset were stratified according to high or low YAP activity signature [ 47 ] and by TP53 mutational status , and then the levels of the cycline signature score were determined in the four groups . Cyclin activity is significantly higher in mut - p53 tumors with high levels of the YAP signature , as visualized by box - plot . Signature scores have been obtained summarizing the standardized expression levels of signature genes into a combined score with zero mean [ 7 ] . The values shown in graphs are thus adimensional . The bottom and top of the box are the first and third quartiles , and the band inside the box is the median ; whiskers represent 1st and 99th percentiles ; values lower and greater are shown as circles ( p < 0 . 0001 , n = 701 ) .

O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Primary human breast cancers of the METABRIC dataset were stratified according to high or low YAP activity signature [47] and by TP53 mutational status, and then the levels of the cycline signature score were determined in the four groups. Cyclin activity is significantly higher in mut-p53 tumors with high levels of the YAP signature, as visualized by box-plot. Signature scores have been obtained summarizing the standardized expression levels of signature genes into a combined score with zero mean [7]. The values shown in graphs are thus adimensional. The bottom and top of the box are the first and third quartiles, and the band inside the box is the median; whiskers represent 1st and 99th percentiles; values lower and greater are shown as circles (p<0.0001, n=701).


B . Same as in A ) using the YAP / TAZ activity signature of Zhang et al . ( 2009 ) ( p < 0 . 0001 , n = 701 ) .

O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

B. Same as in A) using the YAP/TAZ activity signature of Zhang et al. (2009) (p<0.0001, n=701).


C . Kaplan - Meier analysis representing the probability of disease - specific survival in mutant and wild - type p53 breast cancer patients from the METABRIC dataset stratified according high or low YAP / TAZ signature score . The log - rank test p - value reflects the significance of the association between high levels of the YAP / TAZ signature score and shorter survival in in mutant - p53 as compared to wild - type p53 patients ( p < 0 . 0001 , n = 251 ) . All p - values were calculated with two - tailed Student ' s t - test .

O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD B-TISSUE O B-ORGANISM O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Kaplan-Meier analysis representing the probability of disease-specific survival in mutant and wild-type p53 breast cancer patients from the METABRIC dataset stratified according high or low YAP/TAZ signature score. The log-rank test p-value reflects the significance of the association between high levels of the YAP/TAZ signature score and shorter survival in in mutant-p53 as compared to wild-type p53 patients (p<0.0001, n=251). All p-values were calculated with two-tailed Student's t-test.


A . SKBr3 and MDA - MB - 468 cells knocked - down as indicated in the figure , were transiently transfected with pCCAAT - B2LUC ( 100 ng ) or pmutCCAAT - B2LUC ( 100 ng ) luciferase reporter vectors . Values are means + s . d . of three replicates from three independent experiments .

O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. SKBr3 and MDA-MB-468 cells knocked-down as indicated in the figure, were transiently transfected with pCCAAT-B2LUC (100 ng) or pmutCCAAT-B2LUC (100 ng) luciferase reporter vectors. Values are means + s.d. of three replicates from three independent experiments.


B . RT - qPCR of CCNA , CCNB2 and cdk1 mRNA levels in SKBr3 cells upon transduction with si - GFP and si - YAP ( left graph ) or si - p53 ( right graph ) . Values are means + s . d . of three replicates from three independent experiments .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

B. RT-qPCR of CCNA, CCNB2 and cdk1 mRNA levels in SKBr3 cells upon transduction with si-GFP and si-YAP (left graph) or si-p53 (right graph). Values are means + s.d. of three replicates from three independent experiments.


C . H1299 transfected with the indicated siRNAs , were transiently transfected with pCCAAT - B2LUC ( 100 ng ) luciferase reporter vector together with empty pcDNA3 or mutant p53R175H . Representative western blotting to control the transfections presented in the right panel . Values are means + s . d . of three replicates from three independent experiments .

O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O

C. H1299 transfected with the indicated siRNAs, were transiently transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector together with empty pcDNA3 or mutant p53R175H. Representative western blotting to control the transfections presented in the right panel. Values are means + s.d. of three replicates from three independent experiments.


D . RT - qPCR of CCNA and cdk1 mRNA levels in H1299 cells upon transfection with si - GFP or si - YAP oligos and empty pCDNA3 or mutp53R280H expression vectors as indicated in the figure . Error bars represent mean + s . d . , from three biological replicates . P - values are indicated in the figures ; two - tailed Student ' s t - test was applied throughout .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. RT-qPCR of CCNA and cdk1 mRNA levels in H1299 cells upon transfection with si-GFP or si-YAP oligos and empty pCDNA3 or mutp53R280H expression vectors as indicated in the figure. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures; two-tailed Student's t-test was applied throughout.


A and B . Coimmunoprecipitation and western blot analysis from H1299 cell lysates showing endogenous YAP ( A ) and overexpressed GFP - YAP ( B ) bound to mutp53R273H ( A ) or to mutp53R175H ( B ) . In ( A ) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and rabbit IgG was used as negative control of IP . In ( B ) p53 protein was immunoprecipitated with a sheep polyclonal antibody and the total lysate from H1299 transfected with empty pCDNA3 vector was used in as negative control of IP .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O

A and B. Coimmunoprecipitation and western blot analysis from H1299 cell lysates showing endogenous YAP (A) and overexpressed GFP-YAP (B) bound to mutp53R273H (A) or to mutp53R175H (B). In (A) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and rabbit IgG was used as negative control of IP. In (B) p53 protein was immunoprecipitated with a sheep polyclonal antibody and the total lysate from H1299 transfected with empty pCDNA3 vector was used in as negative control of IP.


C and D . Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins ( described in Materials and Methods ) ( C ) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP .

O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O

C and D. Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins (described in Materials and Methods) (C) YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP.


C and D . Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins ( described in Materials and Methods ) ( C ) and from HCT116 cells expressing wild type p53 protein ( D ) . YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP .

O O O O B-EXP_ASSAY O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O

C and D. Immunoprecipitations of YAP protein and western blot analysis for p53 binding are performed from lysates of cancer cell lines expressing different mutant p53 proteins (described in Materials and Methods) (C) and from HCT116 cells expressing wild type p53 protein (D). YAP protein was immunoprecipitated with a rabbit polyclonal antibody and the same amount of rabbit IgG was used as negative control of IP.


E . Cellular extracts from H1299 cells transiently transfected with 2 μg of each indicated plasmids were immunoprecipitated with rabbit polyclonal YAP antibody . Cell lysates derived from cells transfected with the empty vector were used as negative control of immunoprecipitation .

O O O O O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

E. Cellular extracts from H1299 cells transiently transfected with 2 μg of each indicated plasmids were immunoprecipitated with rabbit polyclonal YAP antibody. Cell lysates derived from cells transfected with the empty vector were used as negative control of immunoprecipitation.


F . Cellular extracts from MDA - MB - 468 were immunoprecipitated with rabbit polyclonal YAP , sheep polyclonal p53 and rabbit polyclonal NF - YB antibodies . Rabbit ( for YAP and NF - YB IPs ) and sheep ( for p53 IP ) IgGs were used as negative control of IPs .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O

F. Cellular extracts from MDA-MB-468 were immunoprecipitated with rabbit polyclonal YAP, sheep polyclonal p53 and rabbit polyclonal NF-YB antibodies. Rabbit (for YAP and NF-YB IPs) and sheep (for p53 IP) IgGs were used as negative control of IPs.


G . Cell lysates from CAL27 cells transfected with si - GFP and si - NF - YB oligonucleotides were immunoprecipitated with rabbit polyclonal YAP . As specificity control , immunoprecipitations were performed with rabbit IgG .

O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O

G. Cell lysates from CAL27 cells transfected with si-GFP and si-NF-YB oligonucleotides were immunoprecipitated with rabbit polyclonal YAP. As specificity control, immunoprecipitations were performed with rabbit IgG.


H and I . ChIP analysis of mutant p53 , YAP and H4 acetylated histone - bound chromatin from MDA - MB - 468 cells on transduction with siRNAs oligos targeting endogenous YAP ( siYAP ) ( H ) , NF - Y ( siNF - YB ) ( I ) , or siGFP as a negative control ( H and I ) . The experiment was performed in biological triplicates . The CCNA , CCNB2 and Cdk1 promoter occupancy was analyzed by RT - qPCR . Normalization was performed to the amount of input chromatin . The ChIP results were further normalised on the RT - qPCR of a region resulted negative for the recruitment of mutant p53 ( Appendix Table S5 ) . P - values were calculated with two tailed Student ' s t - test . Statistically significant results were referred with p - value < 0 . 05 .

O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H and I. ChIP analysis of mutant p53, YAP and H4 acetylated histone-bound chromatin from MDA-MB-468 cells on transduction with siRNAs oligos targeting endogenous YAP (siYAP) (H), NF-Y (siNF-YB) (I), or siGFP as a negative control (H and I). The experiment was performed in biological triplicates. The CCNA, CCNB2 and Cdk1 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The ChIP results were further normalised on the RT-qPCR of a region resulted negative for the recruitment of mutant p53 (Appendix Table S5). P-values were calculated with two tailed Student's t-test. Statistically significant results were referred with p-value<0.05.


A and B . Proliferation curves of SKBr3 cell line knocked - down for YAP ( A ) or p53 ( B ) . si - GFP oligos are used as negative control . Cell viability analysis in A and B was determined by trypan blue dye exclusion staining , in C was determined by ATPlite luminescence analysis . All the values are means + s . d . of six replicates from three independent experiments .

O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A and B. Proliferation curves of SKBr3 cell line knocked-down for YAP (A) or p53 (B). si-GFP oligos are used as negative control.Cell viability analysis in A and B was determined by trypan blue dye exclusion staining, in C was determined by ATPlite luminescence analysis. All the values are means + s.d. of six replicates from three independent experiments.


C . Viability of CAL27 cell line transfected with si - GFP or si - p53 oligos and empty vector or GFP - YAP construct , as indicated in the legend to the graph . Cell viability analysis in A and B was determined by trypan blue dye exclusion staining , in C was determined by ATPlite luminescence analysis . All the values are means + s . d . of six replicates from three independent experiments .

O O B-EXP_ASSAY O B-CELL_LINE O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

C. Viability of CAL27 cell line transfected with si-GFP or si-p53 oligos and empty vector or GFP-YAP construct, as indicated in the legend to the graph.Cell viability analysis in A and B was determined by trypan blue dye exclusion staining, in C was determined by ATPlite luminescence analysis. All the values are means + s.d. of six replicates from three independent experiments.


A . CCNA and CCNB2 expression transcripts were analyzed by RT - qPCR in cDNA derived from tumours of control ( saline ) or zoledronic acid ( ZA ) - treated mice ( ref . 31 ) . N = 3 mice per group . P - values were calculated with two - tailed t - test .

O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O

A. CCNA and CCNB2 expression transcripts were analyzed by RT-qPCR in cDNA derived from tumours of control (saline) or zoledronic acid (ZA)-treated mice (ref. 31). N=3 mice per group. P-values were calculated with two-tailed t-test.


B . ChIP analysis of mutant p53 - bound chromatin from MDA - MB - 231 cells treated or not with 1 µM cerivastatin ( Cer ) for 24h . The CCNB2 promoter occupancy was analyzed by RT - qPCR . Normalization was performed to the amount of input chromatin . The experiment was performed in biological triplicate . P - values were calculated with two - tailed t - test .

O O B-EXP_ASSAY O O O B-GENEPROD O O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

B. ChIP analysis of mutant p53 -bound chromatin from MDA-MB-231 cells treated or not with 1 µM cerivastatin (Cer) for 24h. The CCNB2 promoter occupancy was analyzed by RT-qPCR. Normalization was performed to the amount of input chromatin. The experiment was performed in biological triplicate. P-values were calculated with two-tailed t-test.


C . MDA - MB - 231 cells treated with the indicated concentrations of Cer for 24h were transfected with pCCAAT - B2LUC ( 100 ng ) luciferase reporter vector . Error bars represent mean + s . d . , from three biological replicates . P - values are indicated in the figures ; p - values were calculated with two - tailed t - test .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. MDA-MB-231 cells treated with the indicated concentrations of Cer for 24h were transfected with pCCAAT-B2LUC (100 ng) luciferase reporter vector. Error bars represent mean + s.d., from three biological replicates. P-values are indicated in the figures;p-values were calculated with two-tailed t-test.


D . CCNA , CCNB2 and Cdk1 expression transcripts were analyzed by RT - qPCR in cDNA derived from MDA - MB - 231 ( left panel ) and SKBr3 ( right panel ) cell lines treated or not with 1 µM Cer for 48h . Error bars represent mean + s . d . , from three biological replicates . P - values were calculated with two - tailed t - test . P - value < 0 . 01 .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-CELL_LINE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231 (left panel) and SKBr3 (right panel) cell lines treated or not with 1 µM Cer for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.


E . Western blot analysis for CCNA , CCNB and Actin ( loading control ) expression in lysates from MDA - MB - 231 cells treated or not with 1 µM Cer .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O

E. Western blot analysis for CCNA, CCNB and Actin (loading control) expression in lysates from MDA-MB-231 cells treated or not with 1 µM Cer.


F . CCNA , CCNB2 and Cdk1 expression transcripts were analyzed by RT - qPCR in cDNA derived from MDA - MB - 231cell line treated or not with 30µM ZA for 48h . Error bars represent mean + s . d . , from three biological replicates . P - values were calculated with two - tailed t - test . P - value < 0 . 01 .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. CCNA, CCNB2 and Cdk1 expression transcripts were analyzed by RT-qPCR in cDNA derived from MDA-MB-231cell line treated or not with 30µM ZA for 48h. Error bars represent mean + s.d., from three biological replicates. P-values were calculated with two-tailed t-test. P-value<0.01.


G . Viability assay of siGFP and sip53 - MDA - MB - 231 cells after treatment with increasing amounts of cerivastatin ( 0 , 0 . 01 , 0 . 1 , 1 and 10 µM ) for 48 h . Data are normalized to untreated . Error bars represent mean + s . d . , from three biological replicates .

O O B-EXP_ASSAY O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Viability assay of siGFP and sip53 -MDA-MB-231 cells after treatment with increasing amounts of cerivastatin (0, 0.01, 0.1, 1 and 10 µM) for 48 h. Data are normalized to untreated. Error bars represent mean + s.d., from three biological replicates.


Mouse YUMM1 . 7 melanoma cells were subcutaneously inoculated into C57BL / 6 mice and when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib and MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( C ) Kaplan - Meier survival curves of mice treated with the indicated therapies ( n = 6 ) . Log rank ( Mantel - Cox ) statistical test was used for MAPKi vs MAPKi / BIBF1120 . * * * * P ≤ 0 . 0001 .

B-ORGANISM B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O

Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (C) Kaplan-Meier survival curves of mice treated with the indicated therapies (n = 6). Log rank (Mantel-Cox) statistical test was used for MAPKi vs MAPKi/BIBF1120. ****P≤0.0001.


Mouse YUMM1 . 7 melanoma cells were subcutaneously inoculated into C57BL / 6 mice and when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib and MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( F ) Quantification of collagen fibers thickness ( n = 6 for control , BIBF and BRAFi / MEKi groups and n = 5 for BRAFi / MEKi + BIBF group ) . Two - way ANOVA statistical test was used for statistical analysis of mature collagen fibers thickness quantification . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Significance was calculated against the control group . Statistical significance of BIBF vs BIBF + BRAFi / MEKi was also calculated .

B-ORGANISM B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O

Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (F) Quantification of collagen fibers thickness (n = 6 for control, BIBF and BRAFi/MEKi groups and n = 5 for BRAFi/MEKi + BIBF group). Two-way ANOVA statistical test was used for statistical analysis of mature collagen fibers thickness quantification. **P≤0.01, ***P≤0.001, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BIBF vs BIBF + BRAFi/MEKi was also calculated.


Mouse YUMM1 . 7 melanoma cells were subcutaneously inoculated into C57BL / 6 mice and when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib and MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( G ) Heatmap showing the differential expression of ECM and myofibroblast / CAF genes in mice treated with MAPK - targeted therapies with or without BIBF compared to control mice ( log2 ratio , n = 5 ) .

B-ORGANISM B-CELL_LINE I-CELL_LINE I-CELL_LINE B-DISEASE O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE O B-CELL_TYPE O O B-ORGANISM O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O O O O O O O O

Mouse YUMM1.7 melanoma cells were subcutaneously inoculated into C57BL/6 mice and when tumors reached 100 mm3 mice were treated with vehicle (Ctrl), Nintedanib/BIBF1120 (BIBF), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) or BRAFi/MEKi plus BIBF (n=6). (G) Heatmap showing the differential expression of ECM and myofibroblast/CAF genes in mice treated with MAPK-targeted therapies with or without BIBF compared to control mice (log2 ratio, n = 5).


( H ) Heatmap and Western Blot showing the expression of ECM , myofibroblast / CAF and phenotype switch markers in human M238R cells compared to the parental M238P cells . Heatmap represents the mean of expression of 3 independent experiments by RT - qPCR . ( I ) Heatmap showing the expression of ECM , myofibroblast / CAF and phenotype switch markers in human M238R cells treated with BIBF ( 2 µM , 72 h ) or vehicle alone by RT - qPCR ( n = 3 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE O B-CELL_TYPE O O O O O B-ORGANISM B-CELL_LINE O O O O O B-CELL_LINE O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE O B-CELL_TYPE O O O O O B-ORGANISM B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

(H) Heatmap and Western Blot showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells compared to the parental M238P cells. Heatmap represents the mean of expression of 3 independent experiments by RT-qPCR. (I) Heatmap showing the expression of ECM, myofibroblast/CAF and phenotype switch markers in human M238R cells treated with BIBF (2 µM, 72 h) or vehicle alone by RT-qPCR (n = 3).


( B ) Relative miRNA expression levels were quantified in M238P cells treated for 72 h with BIBF1120 ( BIBF , 2 μM ) , MAPKi ( BRAFi , Vemurafenib and MEKi , Trametinib ) ( 1µM ) , or with MAPKi ( 1 μM ) plus BIBF ( 2 µM ) by RT - qPCR and normalized to miR - 16 - 5p . Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments . One - way ANOVA was used for statistical analysis . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . Significance was calculated against the control group . Statistical significance of BRAFi / MEKi vs BRAFi / MEKi + BIBF was also calculated . ( C ) Expression of miR - 143 - 3p and miR - 145 - 5p in control mice and mice treated with the indicated therapies was quantified by RT - qPCR . Data is represented as mean ± SEM from 2 independent experiments performed on 6 mice , with 2 sites of injections . One - way ANOVA was used for statistical analysis . * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 . Significance was calculated against the control group . Statistical significance of BRAFi / MEKi vs BRAFi / MEKi + BIBF was also calculated .

O O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O

(B) Relative miRNA expression levels were quantified in M238P cells treated for 72 h with BIBF1120 (BIBF, 2 μM), MAPKi (BRAFi, Vemurafenib and MEKi, Trametinib) (1µM), or with MAPKi (1 μM) plus BIBF (2 µM) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. One-way ANOVA was used for statistical analysis. **P≤0.01, ***P≤0.001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated. (C) Expression of miR-143-3p and miR-145-5p in control mice and mice treated with the indicated therapies was quantified by RT-qPCR. Data is represented as mean ± SEM from 2 independent experiments performed on 6 mice, with 2 sites of injections. One-way ANOVA was used for statistical analysis. *P≤0.05, ****P≤0.0001. Significance was calculated against the control group. Statistical significance of BRAFi/MEKi vs BRAFi/MEKi + BIBF was also calculated.


( D ) Relative miRNA expression levels was quantified in M238P cells stimulated for 48 h with TGF - β ( 10 ng / mL ) or PDGF - BB ( 20 ng / mL ) by RT - qPCR and normalized to miR - 16 - 5p . Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments . P - values were calculated using Paired Student t - test . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . ( E ) Relative miRNA expression levels was quantified in M238R cells treated for 48 h with the triple kinase inhibitor Nintedanib / BIBF1120 ( BIBF , 2 μM ) , the TGF - β receptor kinase inhibitor SB431542 ( SB , 10 µM ) , and the pan - AKT inhibitor GSK690693 ( GSK , 10 µM ) by RT - qPCR . Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments . P - values were calculated using Paired Student t - test . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

O O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Relative miRNA expression levels was quantified in M238P cells stimulated for 48 h with TGF-β (10 ng/mL) or PDGF-BB (20 ng/mL) by RT-qPCR and normalized to miR-16-5p. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, ***P≤0.001. (E) Relative miRNA expression levels was quantified in M238R cells treated for 48 h with the triple kinase inhibitor Nintedanib/BIBF1120 (BIBF, 2 μM), the TGF-β receptor kinase inhibitor SB431542 (SB, 10 µM), and the pan-AKT inhibitor GSK690693 (GSK, 10 µM) by RT-qPCR. Data is represented as mean ± SEM from a triplicate representative of 3 independent experiments. P-values were calculated using Paired Student t-test. *P≤0.05, **P≤0.01, ***P≤0.001.


( F , Phenotype switch / invasive / ECM markers were quantified in therapy - naïve ( N ) and resistant ( R ) PDX samples . The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples .

O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, Phenotype switch/invasive/ECM markers were quantified in therapy-naïve (N) and resistant (R) PDX samples. The log2 fold change or the ratio of the fold change R vs N is shown for each couple of samples.


( B ) M238P cells were treated 72 h with BRAFi ( Vemurafenib , 3 μM ) in the presence or the absence of LNA - based anti - miR - 143 ( LNA - 143 ) or anti - miR - 145 ( LNA - 145 ) ( 50 nM ) or a combination of the two . ECM markers RT - qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . One - way ANOVA was used for statistical analysis . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. ECM markers RT-qPCR data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. One-way ANOVA was used for statistical analysis. *P≤0.05, **P≤0.01, ***P≤0.001.


( C ) Western Blot analysis of ECM remodeling markers on total cell lysates from M238P cells transfected as in ( A ) . ( D ) Western Blot analysis of ECM remodeling markers on total cell lysates from cells treated with or without BRAFi and the indicated combination of

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O B-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O

(C) Western Blot analysis of ECM remodeling markers on total cell lysates from M238P cells transfected as in (A). (D) Western Blot analysis of ECM remodeling markers on total cell lysates from cells treated with or without BRAFi and the indicated combination of inhibitors


( F ) Migration assay performed in Boyden chambers . Representative images showing migrating cells in the different conditions . The histogram represents the quantitative determination of mean ± SD from 6 independent fields representative of 3 independent experiments , using ImageJ software . Paired Student t - test was used for statistical analysis . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Migration assay performed in Boyden chambers. Representative images showing migrating cells in the different conditions. The histogram represents the quantitative determination of mean ± SD from 6 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. * P≤0.05, ***P≤0.001.


( F ) Luciferase assay in HEK cells overexpressing miR - 143 or miR - 145 transfected with a plasmid harboring the WT or muted sequence of the miR - 143 and miR - 145 binding sites present in FSCN1 3 ' UTR . Each bar represents the mean ± SE of experiments performed at least in triplicate . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . P - values were calculated using Paired Student t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Luciferase assay in HEK cells overexpressing miR-143 or miR-145 transfected with a plasmid harboring the WT or muted sequence of the miR-143 and miR-145 binding sites present in FSCN1 3'UTR. Each bar represents the mean ± SE of experiments performed at least in triplicate. ***P≤0.001, ****P≤0.0001. P-values were calculated using Paired Student t-test.


( G ) RT - qPCR analysis of FSCN1 expression in M238P cells transfected with the indicated mimics . Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments . Paired Student t - test was used for statistical analysis . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) RT-qPCR analysis of FSCN1 expression in M238P cells transfected with the indicated mimics. Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ** P≤0.01, ***P≤0.001.


( B ) FSCN1 immunofluorescent staining and quantification of fluorescence intensity in M238P cells treated or not with BRAFi ( Vemurafenib , 3 μM ) in the presence or the absence of LNA - based anti - miR - 143 ( LNA - 143 ) or anti - miR - 145 ( LNA - 145 ) ( 50 nM ) or a combination of the two . Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments , Mann - Whitney U test was used for statistical analysis . * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) FSCN1 immunofluorescent staining and quantification of fluorescence intensity in M238P cells treated or not with BRAFi (Vemurafenib, 3 μM) in the presence or the absence of LNA-based anti-miR-143 (LNA-143) or anti-miR-145 (LNA-145) (50 nM) or a combination of the two. Data are represented as scatter plots with mean ± SD from 10 independent fields representative of 3 independent experiments, Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.


M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( D ) Migration assay performed in Boyden chambers . Representative images showing migration of M238P cells treated with the indicated siRNAs . The bar graph represents the quantitative determination of data obtained of mean ± SD from 5 independent fields representative of 3 independent experiments , using ImageJ software . Paired Student t - test was used for statistical analysis . * P ≤ 0 . 05 , * * * P ≤ 0 .

B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (D) Migration assay performed in Boyden chambers. Representative images showing migration of M238P cells treated with the indicated siRNAs. The bar graph represents the quantitative determination of data obtained of mean ± SD from 5 independent fields representative of 3 independent experiments, using ImageJ software. Paired Student t-test was used for statistical analysis. *P≤0.05, ***P≤0.001


M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( E ) Western Blot analysis of phenotype - switch markers on cell lysates from M238P cells transfected with the indicated siRNAs .

B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_LINE O O O O O O O

M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Western Blot analysis of phenotype-switch markers on cell lysates from M238P cells transfected with the indicated siRNAs.


BRAFi - resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct . M238R transduced with a Ctrl lentivirus were used as control . ( G ) Western Blot analysis of FSCN1 , phenotype switch markers and ECM remodeling markers on cell lysates from control and FSCN1 overexpressing cells .

B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD B-ORGANISM O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O

BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (G) Western Blot analysis of FSCN1, phenotype switch markers and ECM remodeling markers on cell lysates from control and FSCN1 overexpressing cells.


BRAFi - resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct . M238R transduced with a Ctrl lentivirus were used as control . ( H ) Crystal violet viability assay of M238R cells stably overexpressing FSCN1 during 6 days with the indicated doses of the BRAFi Vemurafenib . Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . Paired Student t - test was used for statistical analysis . * * * * P ≤ 0 . 0001 .

B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD B-ORGANISM O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

BRAFi-resistant M238R cells overexpressing FSCN1 were obtained after transduction with a FSCN1 lentiviral construct. M238R transduced with a Ctrl lentivirus were used as control. (H) Crystal violet viability assay of M238R cells stably overexpressing FSCN1 during 6 days with the indicated doses of the BRAFi Vemurafenib. Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments. Paired Student t-test was used for statistical analysis. ****P≤0.0001.


( A , M238P cells were transfected with miR - 143 - 3p , miR - 145 - 5p or a control mimic ( miR - neg ) ( 72 h , 30 nM ) . ( B , M238P cells were treated 72 h with BRAFi ( Vemurafenib , 3 µM ) or a combination of BRAFi ( Vemurafenib , 0 . 5 µM ) and MEKi ( Trametinib , 1 µM ) . ( A - B ) Images and quantification of cell area in cells stained for F - actin ( red ) and nuclei ( blue ) . Data is represented as scatter plot with mean ± SD ( n ≥ 30 cells per condition ) . Mann - Whitney U test was used for statistical analysis . * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (B, M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (A-B) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.


C ) M238P cells were transfected with miR - 143 - 3p , miR - 145 - 5p or a control mimic ( miR - neg ) ( 72 h , 30 nM ) . D ) M238P cells were treated 72 h with BRAFi ( Vemurafenib , 3 µM ) or a combination of BRAFi ( Vemurafenib , 0 . 5 µM ) and MEKi ( Trametinib , 1 µM ) . ( C - D ) Images and quantification of focal adhesions number in cells stained for pPaxillin ( green ) and nuclei ( blue ) . Focal adhesions ( FA ) number is represented as mean ± SD ( n ≥ 30 cells per condition ) . Each point represents the average number of focal adhesions per cell calculated for each field . Paired Student t - test has been used for statistical analysis . * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P . ≤ 0 . 0001 . Scale bar 40 μm .

O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). D) M238P cells were treated 72 h with BRAFi (Vemurafenib, 3 µM) or a combination of BRAFi (Vemurafenib, 0.5 µM) and MEKi (Trametinib, 1 µM). (C-D) Images and quantification of focal adhesions number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions (FA) number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test has been used for statistical analysis. *P≤0.01, ***P≤0.001, ****P. ≤0.0001. Scale bar 40 μm.


( E - F ) M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( E ) Images and quantification of cell area in cells stained for F - actin ( red ) and nuclei ( blue ) . Data is represented as scatter plot with mean ± SD ( n ≥ 30 cells per condition ) . Mann - Whitney U test was used for statistical analysis . * * * * P ≤ 0 . 0001 . Scale bar 40 μm . ( F ) Images and quantification of focal adhesions ( FA ) number in cells stained for pPaxillin ( green ) and nuclei ( blue ) . Focal adhesions number is represented as mean ± SD ( n ≥ 30 cells per condition ) . Each point represents the average number of focal adhesions per cell calculated for each field . Paired Student t - test was used for statistical analysis . * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

O O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-F) M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (E) Images and quantification of cell area in cells stained for F-actin (red) and nuclei (blue). Data is represented as scatter plot with mean ± SD (n≥30 cells per condition). Mann-Whitney U test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm. (F) Images and quantification of focal adhesions (FA) number in cells stained for pPaxillin (green) and nuclei (blue). Focal adhesions number is represented as mean ± SD (n≥30 cells per condition). Each point represents the average number of focal adhesions per cell calculated for each field. Paired Student t-test was used for statistical analysis. ****P≤0.0001. Scale bar 40 μm.