( G ) Validation of two candidate genes : IRF2BP1 IRF2BP2 were knocked down for 72 hours in HeLa , lysates were analyzed by immunoblotting with 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 - of EGFR surface expression : IRF2BP1 IRF2BP2 were knocked down for 48 hours in HeLa , were serum starved for 16 hours , stained with anti - EGFR antibodies analyzed by flow cytometry . Shown are means of 3 independent experiments + / - .

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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 FGF21 improve insulin resistance by countering inflammation in 3T3 - L1 adipocytes . were cultured in RAW - CM to induce inflammation insulin resistance , while vehicle , FGF21 or FGF21SS were added simultaneously . insulin sensitivity was indicated by insulin - induced AKT phosphorylation using western blotting . Error bars show of four independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - ) 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 . ( B ) was measured Error bars show of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ( Student ' s t - ) 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 of ten independent experiments . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ( Student ' s t - ) 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 U2OS were treated with indicated siRNA oligonucleotides for 72 h . extracts were resolved by SDS - PAGE , filters were probed with indicated antibodies . extent of biomarker modulation correlated with efficiency of silencing ( oligo 4 versus pool ) . VCP silencing determined stabilization of cyclin E ; induction of endoplasmic reticulum chaperone GRP78 , CHOP transcription factor its downstream target GADD - 34 ; conversion of autophagy regulator ATG8 / chain 3 ( LC3B ) from free to lipidated form ; activation of effector caspase - 3 ; cleavage of caspase target PARP - 1 . A graphical representation of cycle distribution ( of ) obtained from cycle ( Supplementary Fig . 3b ) is reported at bottom of each sample . C , control ; NT , nontargeting oligo ; P , oligonucleotide pool .

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(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 growth . HeLa , HCT116 U2OS were treated for 72 h with different VCP siRNA oligonucleotides 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 were treated for 24 h with siRNA to VCP then were subjected to dose - response 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 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 addition of NMS - 859 lacking 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 NMS - 873 on wild - type VCP 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 EDA - ADP - ATTO 590 probe to wild - type VCP in 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 EDA - ADP - ATTO 590 probe to wild - type ( WT ) VCP K615V N616F mutants in presence of NMS - 249 . Minor differences in mP values are due to 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 EDA - ADP - ATTO 590 probe to wild - type VCP Walker A mutants in presence of NMS - 249 . Data shown are representative results of one of three independent experiments : each determination was done in triplicate , data points denote average value , 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 of biomarker modulation in HCT116 . HCT116 were treated with indicated doses of inhibitors for 8 h ( top ) or 24 h ( bottom ) , extracts were resolved on SDS - PAGE gel . Filters were probed with 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 . Top , confocal time - lapse of live stable , inducible T - REx - 293 expressing a bicistronic construct of mCherry - BimS ( 2A ) ΔC ( red ) with eGFP - Mcl - 1 ( green ) after doxycycline treatment . were treated with cycloheximide ( CHX , 50 μg ml − 1 ) ± 2 . 5 μM of NMS - 873 for 6 h . Bottom , total of eGFP - Mcl - 1 mCherry - BimS ( 2A ) ΔC was measured for time - lapse series shown at top . A decrease in eGFP - Mcl - 1 mCherry - BimS ( 2A ) ΔC total was calculated for two different concentrations of indicated compounds 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 of poly - Ub CHOP induction upon treatment with VCP inhibitors . HeLa were treated with increasing doses of indicated compounds . were then fixed immunostained with indicated antibodies . numbers mean ( MFI ) were determined using ArrayScan platform . Data were collected in triplicate ( n = 3 ) , 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 of BAL from Csf2ra - / - recipients 8 after neonatal i . n . transfer of YS EMP from CD45 . 1 + E10 . 5 , 8 - 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 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 ( circle ) numbers of EMP - derived AM ( red circles ) in BAL lung of adult Csf2ra - / - recipients 8 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 BAL ( left ) lung ( right ) ( C ) in BAL of adult Csf2ra - / - recipients 8 after transfer ( n = 5 mice ) . - matched Csf2ra - / - ( KO ) ( n = 3 mice ) WT ( n = 3 mice ) were included as negative 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 in BAL of adult Csf2ra - / - recipients 8 after transfer ( n = 5 mice ) . - matched Csf2ra - / - ( KO ) ( n = 3 mice ) WT ( n = 3 mice ) were included as negative 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 BAL of adult Csf2ra - / - recipients 8 after transfer ( n = 5 mice ) . - matched Csf2ra - / - ( KO ) ( n = 3 mice ) WT ( n = 3 mice ) were included as negative 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 ( left ) of donor - derived AM ( right ) in BAL lung from Csf2ra - / - recipients 8 after neonatal transfer of 1 : 1 : 1 mixture with E17 . 5 liver ( FeLi ) monocytes ( Mo ) ( CD45 . 1 + ) , E14 . 5 FeLi Mo ( CD45 . 1 + CD45 . 2 + ) , E10 . 5 YS EMP ( CD45 . 2 + ) ( F ) or E17 . 5 lung ( FeLu ) Mo ( CD45 . 1 + ) , E14 . 5 FeLu Mo ( CD45 . 1 + CD45 . 2 + ) , 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 ( left ) of donor - derived AM ( right ) in BAL ( top ) lung ( bottom ) from Csf2ra - / - recipients 10 after neonatal transfer of 1 : 1 mixture with CD45 . 1 + E14 . 5 FeLi Mo CD45 . 2 + E14 . 5 FeLi pMΦ ( Group 1 ) or CD45 . 1 + E14 . 5 FeLi pMΦ CD45 . 2 + FeLi Mo ( Group 2 ) ( n = 3 mice ) . expansion fold of donor in Csf2ra - / - recipients 10 after neonatal transfer of 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 ( left ) of donor - derived AM ( right ) of BAL lung from Csf2ra - / - recipients 8 after neonatal transfer of 1 : 1 : 1 mixture with CD45 . 1 + E17 . 5 FeLi pMΦ , CD45 . 1 + CD45 . 2 + E14 . 5 FeLi pMΦ , CD45 . 2 + E10 . 5 YS pMΦ ( n = 3 mice ) . Total numbers of donor - derived AM in BAL lung of adult Csf2ra - / - recipients 8 after transfer of described above in C ( n = 3 mice ) . - matched Csf2ra - / - ( KO ) ( n = 3 mice ) WT ( n = 3 mice ) were included as negative 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 ( left ) of donor - derived AM ( right ) of BAL lung from Csf2ra - / - recipients 8 after neonatal transfer of 1 : 1 : 1 mixture with CD45 . 1 + E17 . 5 FeLu pMΦ , CD45 . 1 + CD45 . 2 + E14 . 5 FeLu pMΦ , CD45 . 2 + E10 . 5 YS pMΦ ( n = 3 mice ) . Total numbers of donor - derived AM in BAL lung of adult Csf2ra - / - recipients 8 after transfer of described above in E ( n = 3 mice ) . - matched Csf2ra - / - ( KO ) ( n = 3 mice ) WT ( n = 3 mice ) were included as negative 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 ( left ) of donor - derived AM ( right ) in BAL lung from Csf2ra - / - recipients 7 after neonatal transfer of 1 : 1 mixture with CD45 . 1 + E14 . 5 FeLi Mo from WT CD45 . 2 + E14 . 5 FeLi pMΦ from Myb - / - ( G ) or Myb + / + ( H ) ( 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 of BAL from Csf2ra - / - recipients 7 after neonatal i . n . transfer of pMΦ or Mo from CD45 . 1 + E14 . 5 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 BAL ( left ) lung ( right ) of adult Csf2ra - / - recipients 7 ( B ) or 1 year ( C ) after neonatal transfer of described in A ( n = 7 mice ) . - matched Csf2ra - / - ( KO ) ( n = 5 mice ) WT ( n = 4 mice ) were included as negative 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 in BAL of adult Csf2ra - / - recipients 7 after neonatal transfer of described in A ( n = 7 mice ) . - matched Csf2ra - / - ( KO ) ( n = 5 mice ) WT ( n = 4 mice ) were included as negative 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 BAL of adult Csf2ra - / - recipients 7 after neonatal transfer of described in A ( n = 7 mice ) . - matched Csf2ra - / - ( KO ) ( n = 5 mice ) WT ( n = 4 mice ) were included as negative 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 in BAL of adult Csf2ra - / - recipients 1 year ( F , G ) after neonatal transfer of described in A ( n = 7 mice ) . - matched Csf2ra - / - ( KO ) ( n = 5 mice ) WT ( n = 4 mice ) were included as negative 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 BAL of adult Csf2ra - / - recipients 1 year ( F , G ) after neonatal transfer of described in A ( n = 7 mice ) . - matched Csf2ra - / - ( KO ) ( n = 5 mice ) WT ( n = 4 mice ) were included as negative 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.


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


( B ) measurement of influenza ( 20 pfu of PR8 ) - infected Csf2ra - / - recipients 24 after neonatal transfer of pMΦ or Mo from E14 . 5 livers ( n = 7 mice from each transferred group ) , 24 - week old un - transferred Csf2ra - / - ( n = 5 ) , 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 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 of Csf2ra ( CD116 ) expression on different precursors from WT . solid gray histogram represents minus one ( FMO ) controls , open black histogram represents stained samples . gray number represents mean ( MFI ) of FMO controls , black number represents MFI of stained samples . E10 . 5 YS were pooled from 8 - 10 , E14 . 5 lung were pooled from 3 - 4 , E14 . 5 liver were from .

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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 of Csf2ra expressions on pMΦ - Mo - derived AM in Csf2ra - / - recipients 4 after transfer of pMΦ or Mo from E14 . 5 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.


of AM signature - up genes ( D ) , AM signature - down genes ( E ) , AM - specific transcription factors ( F ) in precursors mature AM described above in A . data are from one - 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 ) of Myb , Maf , Mafb , Pparg expression in different precursors from WT 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 Mo from E14 . 5 WT liver at baseline after treatment with oligomycin ( Oligo ) , FCCP or rotenone ( Rot ) 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 Mo from E14 . 5 WT liver at baseline 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).


- seq ( C , n = 2 biological replicates ) of Eno1 , Slc2a6 , Txn1 , Txnip gene expression in pMΦ Mo from E14 . 5 WT 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 ) of Eno1 , Slc2a6 , Txn1 , Txnip gene expression in pMΦ Mo from E14 . 5 WT 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 for CCNA , CCNB , Cdk1 GAPDH ( loading control ) expression in lysates from control ( si - GFP ) p53 - depleted ( si - p53 ; si - p53 _ 1 ) ( C ) MDA - MB - 231 CAL27 lines . Representative are shown .

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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 for CCNA , CCNB , Cdk1 GAPDH ( loading control ) expression in lysates from control ( si - GFP ) YAP - depleted ( si - YAP ; si - YAP _ 1 ) ( D ) MDA - MB - 231 CAL27 lines . Representative 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 for CCNA , CCNB , Cdk1 GAPDH ( loading control ) expression in lysates from control ( si - GFP ) TAZ - depleted ( si - TAZ ) ( E ) MDA - MB - 231 CAL27 lines . Representative are shown .

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

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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 YAP / TAZ activity signature of Zhang et al . ( 2009 ) ( p < 0 . 0001 , n = 701 ) .

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


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

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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 MDA - MB - 468 knocked - down as indicated in 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 .

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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 cdk1 mRNA in SKBr3 upon transduction with si - GFP si - YAP ( left graph ) or si - p53 ( right graph ) . Values are means + s . d . of three replicates from three independent experiments .

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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 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 transfections presented in right panel . Values are means + s . d . of three replicates from three independent experiments .

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

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

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

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

O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY 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 . extracts from H1299 transiently transfected with 2 μg of each indicated plasmids were immunoprecipitated with rabbit polyclonal YAP antibody . lysates derived from transfected with empty vector were used as negative control of immunoprecipitation .

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

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

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

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

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

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

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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 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 - t - .

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

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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 treated with 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 figures ; p - values were calculated with two - t - .

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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 Cdk1 expression transcripts were analyzed by RT - qPCR in cDNA derived from MDA - MB - 231 ( left panel ) SKBr3 ( right panel ) 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 - t - . P - value < 0 . 01 .

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

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

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

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

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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 were subcutaneously inoculated into C57BL / 6 mice when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( C ) Kaplan - Meier survival curves of mice treated with indicated therapies ( n = 6 ) . Log rank ( Mantel - Cox ) statistical 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 B-ORGANISM 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 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 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 were subcutaneously inoculated into C57BL / 6 mice when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( F ) of collagen thickness ( n = 6 for control , BIBF BRAFi / MEKi groups n = 5 for BRAFi / MEKi + BIBF group ) . Two - way ANOVA statistical was used for statistical of mature collagen thickness . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Significance was calculated against 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 B-ORGANISM 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 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 B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE B-GENEPROD O B-GENEPROD 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 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 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 were subcutaneously inoculated into C57BL / 6 mice when tumors reached 100 mm3 mice were treated with vehicle ( Ctrl ) , Nintedanib / BIBF1120 ( BIBF ) , MAPKi ( BRAFi , Vemurafenib MEKi , Trametinib ) or BRAFi / MEKi plus BIBF ( n = 6 ) . ( G ) showing differential expression of ECM myofibroblast / CAF genes in mice treated with MAPK - targeted therapies with or without BIBF compared to control mice ( log2 , n = 5 ) .

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE O B-CELL_TYPE O O O O B-ORGANISM B-CELL_LINE O O O B-CELL_LINE 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 B-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE O B-CELL_TYPE O O O O B-ORGANISM B-CELL_LINE 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 were quantified in M238P treated for 72 h with BIBF1120 ( BIBF , 2 μM ) , MAPKi ( BRAFi , Vemurafenib MEKi , Trametinib ) ( 1µM ) , or with MAPKi ( 1 μM ) plus BIBF ( 2 µM ) by RT - qPCR normalized to miR - 16 - 5p . Data is represented as mean ± from a triplicate representative of 3 independent experiments . One - way ANOVA was used for statistical . * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . Significance was calculated against control group . Statistical significance of BRAFi / MEKi vs BRAFi / MEKi + BIBF was also calculated . ( C ) Expression of miR - 143 - 3p miR - 145 - 5p in control mice mice treated with indicated therapies was quantified by RT - qPCR . Data is represented as mean ± from 2 independent experiments performed on 6 mice , with 2 sites of injections . One - way ANOVA was used for statistical . * P ≤ 0 . 05 , * * * * P ≤ 0 . 0001 . Significance was calculated against 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 B-CELL_LINE 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 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 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 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 I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM I-ORGANISM 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 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 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 was quantified in M238P stimulated for 48 h with TGF - β ( 10 ng / mL ) or PDGF - BB ( 20 ng / mL ) by RT - qPCR normalized to miR - 16 - 5p . Data is represented as mean ± from a triplicate representative of 3 independent experiments . P - values were calculated using Paired Student t - . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . ( E ) Relative miRNA expression was quantified in M238R treated for 48 h with triple kinase inhibitor Nintedanib / BIBF1120 ( BIBF , 2 μM ) , TGF - β receptor kinase inhibitor SB431542 ( SB , 10 µM ) , pan - AKT inhibitor GSK690693 ( GSK , 10 µM ) by RT - qPCR . Data is represented as mean ± from a triplicate representative of 3 independent experiments . P - values were calculated using Paired Student t - . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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(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 ) resistant ( R ) PDX samples . log2 fold change or of fold change R vs N is shown for each couple of samples .

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(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 were treated 72 h with BRAFi ( Vemurafenib , 3 μM ) in presence or absence of LNA - anti - miR - 143 ( LNA - 143 ) or anti - miR - 145 ( LNA - 145 ) ( 50 nM ) or a combination of 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 . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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 B-SMALL_MOLECULE 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 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

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

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

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

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

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

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

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

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

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

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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 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 of M238R stably overexpressing FSCN1 during 6 days with indicated doses of BRAFi Vemurafenib . Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . Paired Student t - was used for statistical . * * * * P ≤ 0 . 0001 .

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

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

O O O B-CELL_LINE 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 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 B-GENEPROD O B-SMALL_MOLECULE 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 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

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

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

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

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

(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.


E ) M238P were transfected with miR - 143 - 3p , miR - 145 - 5p or a control mimic ( miR - neg ) ( 72 h , 30 nM ) . ( E ) Effect of miR - 143 - 3p or miR - 145 - 5p overexpression on expression of YAP / MRTF target genes assessed by RT - qPCR . Data are normalized to expression in control . Data is represented as mean ± SD from a triplicate representative of at least 3 independent experiments . Paired Student t - was used for statistical . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 .

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

E) M238P cells were transfected with miR-143-3p, miR-145-5p or a control mimic (miR-neg) (72 h, 30 nM). (E) Effect of miR-143-3p or miR-145-5p overexpression on the expression of YAP/MRTF target genes assessed by RT-qPCR. Data are normalized to the expression in control cells. 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≤0.05, **P≤0.01, ****P≤0.0001.


M238P were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . Effect of FSCN1 downregulation on MRTFA ( F ) YAP1 ( G ) translocation assessed by immunofluorescence in M238P . Data are represented as scatter plot with mean ± SD ( n ≥ 30 per condition ) . Each point represents / cytoplasm . Mann - Whitney U was used for statistical . * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . Scale bar 40 μm .

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M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). Effect of FSCN1 downregulation on MRTFA (F) and YAP1 (G) nuclear translocation assessed by immunofluorescence in M238P. Data are represented as scatter plot with mean ± SD (n≥30 cells per condition). Each point represents the nuclear/cytoplasm ratio. Mann-Whitney U test was used for statistical analysis. ***P≤0.001, ****P≤0.0001. Scale bar 40 μm.


M238P were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA ( 72 h , 100 nM ) . ( H ) RT - qPCR for expression of MRTFA / YAP target genes in M238P transfected with indicated siRNAs . Data are normalized to expression in parental . Data is represented as mean ± SE from a triplicate representative of at least 3 independent experiments . Paired Student t - was used for statistical . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . Scale bar 40 μm .

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M238P cells were transfected with two different sequences of siRNAs vs FSCN1 or with a control siRNA (72 h, 100 nM). (H) RT-qPCR analysis for the expression of MRTFA/YAP target genes in M238P cells transfected with the indicated siRNAs. Data are normalized to the expression in parental cells. 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≤0.05, **P≤0.01, ***P≤0.001. Scale bar 40 μm.


( B - C ) Coimmunoprecipitation of Ams1 ( B ) Ape4 ( C ) with Nbr1 . Endogenously TAP - tagged Nbr1 was immunoprecipitated ( IP ) using IgG Sepharose beads . lysates immunoprecipitates were examined by immunoblotting ( IB ) .

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(B-C) Coimmunoprecipitation of Ams1 (B) and Ape4 (C) with Nbr1. Endogenously TAP-tagged Nbr1 was immunoprecipitated (IP) using IgG Sepharose beads. Cell lysates and immunoprecipitates were examined by immunoblotting (IB).


( D - E ) Subcellular localization of Ams1 ( D ) Ape4 ( E ) . Wild - type ( WT ) , nbr1Δ , sst4Δ , atg1Δ were grown to mid - log phase in EMM medium examined by live imaging . In ( D ) , Ams1 was endogenously tagged at its C - terminus with mCherry . In ( E ) , Ape4 was endogenously tagged at its C - terminus with GFP in same GFP - binding - ( GBP ) fused 3xUb was expressed under P41nmt1 promoter . Cpy1 is a vacuole lumen marker . Scale bar , 3 μm .

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(D-E) Subcellular localization of Ams1 (D) and Ape4 (E). Wild-type (WT), nbr1Δ, sst4Δ, and atg1Δ cells were grown to mid-log phase in EMM medium and examined by live cell imaging. In (D), Ams1 was endogenously tagged at its C-terminus with mCherry. In (E), Ape4 was endogenously tagged at its C-terminus with GFP and in the same cells GFP-binding-protein (GBP) fused 3xUb was expressed under the P41nmt1 promoter. Cpy1 is a vacuole lumen marker. Scale bar, 3 μm.


Localization of Ams1 in WT , lap2Δ , ape2Δ , ape4Δ . Scale bar , 3 μm . Localization of Ape4 in WT , lap2Δ , ape2Δ , ams1Δ . Scale bar , 3 μm .

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Localization of Ams1 in WT, lap2Δ, ape2Δ, and ape4Δ cells. Scale bar, 3 μm.   Localization of Ape4 in WT, lap2Δ, ape2Δ, and ams1Δ cells. Scale bar, 3 μm.


Ams1 , Lap2 , Ape2 did not co - immunoprecipitate with Ape4 . Endogenously TAP - tagged Ape4 was immunoprecipitated . Ams1 was endogenously tagged with an mCherry tag .

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Ams1, Lap2, and Ape2 did not co-immunoprecipitate with Ape4. Endogenously TAP-tagged Ape4 was immunoprecipitated. Ams1 was endogenously tagged with an mCherry tag.


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The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ams1.   The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ape4.


ZZ1 region but not ZZ2 - ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ams1 . Scale bar , 3 μm . ZZ1 region but not ZZ2 - ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ape4 . Scale bar , 3 μm . ZZ2 - ZZ3 region but not ZZ1 region in Nbr1 is necessary for vacuolar targeting of Ape2 . Scale bar , 3 μm . ZZ2 - ZZ3 region but not ZZ1 region in Nbr1 is necessary for vacuolar targeting of Lap2 . Scale bar , 3 μm .

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The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ams1. Scale bar, 3 μm.   The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ape4. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Ape2. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Lap2. Scale bar, 3 μm.


Surface plasmon resonance ( SPR ) of Nbr1 - Ams1 interaction . GST - tagged ZZ1 - L1 fragment of Nbr1 purified from E . coli was captured on a CM5 sensor chip using GST Capture Kit . Ams1 purified from S . pombe ( two - fold serial dilutions from 1000 nM to 62 . 5 nM ) was flowed through chip surface .

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Surface plasmon resonance (SPR) analysis of the Nbr1-Ams1 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ams1 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.


SPR of Nbr1 - Ape4 interaction . GST - tagged ZZ1 - L1 fragment of Nbr1 purified from E . coli was captured on a CM5 sensor chip using GST Capture Kit . Ape4 purified from S . pombe ( two - fold serial dilutions from 1000 nM to 62 . 5 nM ) was flowed through chip surface .

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SPR analysis of the Nbr1-Ape4 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ape4 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.


effects of individually mutating first two residues of Ams1 on its Nbr1 - binding ability . Nbr1 - Ams1 interactions were examined by Pil1 co - tethering . Scale bar , 3 μm .

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The effects of individually mutating the first two residues of Ams1 on its Nbr1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.


effects of individually mutating third residue other residues in U - shaped loop of Ape4 on its Nbr1 - binding ability . Nbr1 - Ape4 interactions were examined by Pil1 co - tethering . Scale bar , 3 μm .

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The effects of individually mutating the third residue and other residues in the U-shaped loop of Ape4 on its Nbr1-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.


( A - C ) . Mice gain ( A ) , lean mass fat mass ( B ) gWAT index ( C ) . At 8 of , C57BL / 6 mice were switched to HFD . After 12 of HFD feeding , mice were divided into three groups receiving non - exercise , endurance exercise or resistance exercise for 14 days . ( n = 8 per group ) .

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(A-C). Mice body weight gain (A), the lean mass and fat mass (B) and gWAT index (C). At 8 weeks of age, male C57BL/6 mice were switched to HFD. After 12 weeks of HFD feeding, mice were divided into three groups receiving non-exercise, endurance exercise or resistance exercise for 14 days. (n = 8 per group).


( D ) . Relative changes in metabolites in response to resistance exercise . Heat maps show changes of metabolites in serums from mice receiving resistance exercise or non - exercise . C57BL / 6 mice ( 10 ) fed with normal chow were divided into two groups receiving either non - exercise or resistance exercise for 40 min ( n = 6 per group ) . Shades of yellow blue represent fold increase fold decrease of a metabolite , respectively ( see color scale ) .

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(D). Relative changes in metabolites in response to resistance exercise. Heat maps show changes of metabolites in the serums from mice receiving resistance exercise or non-exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group). Shades of yellow and blue represent fold increase and fold decrease of a metabolite, respectively (see color scale).


( G ) . Two Pearson ' s correlation coefficient of plasma AKG level mass index ( BMI ) in Chinese adults ( 10 35 ) .

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(G). Two tailed Pearson's correlation coefficient analysis of plasma AKG level and body mass index (BMI) in Chinese adults (10 males and 35 females).