( C ) viability ( TO - PRO - 3 FACS ) of GT1 - 7 neuronal treated with 1 , 3 or 10 µM of promethazine co - transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to GFP in GA or PG frame .

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(C) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells treated with 1, 3 or 10 µM of promethazine and co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame.


B Phos ‐ tag gel following complete conversion of Ub to phosphoUb , using GST ‐ PhPINK1 . A Ser65 mutant Ub is not modified .

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B Phos‐tag gel following complete conversion of Ub to phosphoUb, using GST‐PhPINK1. A Ser65 mutant Ub is not modified.


C ESI ‐ MS of pure Ub ( left , mass 8 , 565 . 65 Da , reference 8 , 565 . 80 Da ) purified phosphoUb ( right , mass 8 , 645 . 61 Da , reference 8 , 645 . 80 Da ) . difference of 80 Da corresponds to one phosphate group .

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C ESI‐MS analysis of pure Ub (left, mass 8,565.65 Da, reference 8,565.80 Da) and purified phosphoUb (right, mass 8,645.61 Da, reference 8,645.80 Da). The difference of 80 Da corresponds to one phosphate group.


D AQUA of PhPINK1 ‐ phosphorylated Lys63 ‐ linked hexaUb . Lys63 peptide ( K63 ) is modified by Ser65 phosphorylation during time course , as observed by rise of Lys63 / pSer65 peptide ( K63pS65 ) .

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D AQUA analysis of PhPINK1‐phosphorylated Lys63‐linked hexaUb. The Lys63 peptide (K63) is modified by Ser65 phosphorylation during the time course, as observed by rise of the Lys63/pSer65 peptide (K63pS65).


E AQUA after 180 min as in ( D ) with Lys6 ‐ , Lys11 ‐ , Lys33 ‐ , Lys48 ‐ , Lys63 ‐ , Met1 ‐ linked tetraUb ( K6 , K11 , K33 , K48 , K63 , M1 ) . For tetraUb chains , Ser65 is phosphorylated to greater than 80 % after incubation with PhPINK1 for 180 min .

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E AQUA analysis after 180 min as in (D) with Lys6‐, Lys11‐, Lys33‐, Lys48‐, Lys63‐, and Met1‐linked tetraUb (K6, K11, K33, K48, K63, M1). For all tetraUb chains, Ser65 is phosphorylated to greater than 80% after incubation with PhPINK1 for 180 min.


F Time course of autoubiquitination reactions with unphosphorylated full ‐ humanParkin ( hParkin ) Ub ( left ) , a 80 % Ub / 20 % phosphoUb mixture ( middle ) 100 % phosphoUb ( right ) .

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F Time course analysis of autoubiquitination reactions with unphosphorylated full‐length humanParkin (hParkin) and Ub (left), a 80% Ub/20% phosphoUb mixture (middle) and 100% phosphoUb (right).


G of different ratios of Ub : phosphoUb in a 120 ‐ min ubiquitination reaction of full ‐ hParkin . Parkin is activated by a mixture of Ub phosphoUb , is much less active in a reaction with no phosphoUb as well as with 100 % phosphoUb .

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G Analysis of different ratios of Ub:phosphoUb in a 120‐min ubiquitination reaction of full‐length hParkin. Parkin is activated by a mixture of Ub and phosphoUb, and is much less active in a reaction with no phosphoUb as well as with 100% phosphoUb.


A 1H , 15N ‐ BEST ‐ TROSY spectrum of phosphoUb ( shades of blue ) overlaid with wild ‐ type ( wt ) Ub ( orange ) . phosphoUb spectrum contains 130 non ‐ sidechain resonances that have been colored in blue for major species blue for minor species on assignment of each species in Supplementary Fig S2B - D . B - D Weighted chemical shift perturbation ( CSP ) graphs for ( B ) wt Ub versus major phosphoUb species , ( C ) wt Ub versus minor phosphoUb species , ( D ) major versus minor phosphoUb species .

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A 1H, 15N‐BEST‐TROSY spectrum of phosphoUb (shades of blue) overlaid with wild‐type (wt) Ub (orange). The phosphoUb spectrum contains 130 non‐sidechain resonances that have been colored in dark blue for the major species and light blue for the minor species based on the assignment of each species in Supplementary Fig S2B-D.B-D Weighted chemical shift perturbation (CSP) graphs for (B) wt Ub versus major phosphoUb species, (C) wt Ub versus minor phosphoUb species, (D) major versus minor phosphoUb species.


A Structure of phosphoUb in space group P1 with 8 molecules in asymmetric unit ( Table ) . Ub molecules are colored differently , atoms for pSer65 are shown as spheres with red oxygen purple phosphorous atoms .

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A Structure of phosphoUb in space group P1 with 8 molecules in the asymmetric unit (Table ). Ub molecules are colored differently, and atoms for pSer65 are shown as spheres with red oxygen and purple phosphorous atoms.


B Six of eight phosphoUb molecules form similar dimer interactions , where phosphoUb molecules interact via their Ile44 patch . This resembles crystal structure of Lys48 ‐ linked diUb [ pdb ‐ id 1aar ( Cook et al , ) ] . Atoms for pSer65 are shown as in ( A ) , Ile44 side chain is shown as blue sticks .

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B Six of eight phosphoUb molecules form similar dimer interactions, where phosphoUb molecules interact via their Ile44 patch. This resembles the crystal structure of Lys48‐linked diUb [pdb‐id 1aar (Cook et al, )]. Atoms for pSer65 are shown as in (A), and the Ile44 side chain is shown as blue sticks.


C Representative 2 | Fo | ‐ | Fc | electron density , contoured at 1 σ , covering phosphorylation site . pSer65 is shown in ball ‐ ‐ stick representation , neighboring side chains are shown in stick representation with red oxygen blue nitrogen atoms .

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C Representative 2|Fo|‐|Fc| electron density, contoured at 1 σ, covering the phosphorylation site. pSer65 is shown in ball‐and‐stick representation, and neighboring side chains are shown in stick representation with red oxygen and blue nitrogen atoms.


D In wt Ub ( left ) , Ser65 forms a hydrogen bond with backbone amide of Gln62 . Upon phosphorylation ( right ) , an oxygen atom from phosphate forms same backbone hydrogen bond .

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D In wt Ub (left), Ser65 forms a hydrogen bond with the backbone amide of Gln62. Upon phosphorylation (right), an oxygen atom from the phosphate forms the same backbone hydrogen bond.


E Cartoon representation electrostatic surface potential of Ub ( left ) phosphoUb ( right ) in identical orientations . Electrostatics were calculated using CHARMM ( www . charmm - gui . org ) are colored from red ( negative potential ) to blue ( positive potential ) .

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E Cartoon representation and electrostatic surface potential of Ub (left) and phosphoUb (right) in identical orientations. Electrostatics were calculated using CHARMM (www.charmm-gui.org) and are colored from red (negative potential) to blue (positive potential).


A Long ‐ range HNCO NMR experiments showing differences of hydrogen bonding patterns for major minor phosphoUb species . Through H bond correlations ( blue ) recorded for residue Ile44 in major minor species of phosphoUb with reference to a control HNCO spectrum ( red ) . In major form of phosphoUb ( left ) , HN of Ile44 is a H bond donor to His68 , as seen in wt Ub . In minor form of phosphoUb ( right ) , HN of Ile44 does not have a cross peak to His68 but instead has a correlation to Val70 .

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A Long‐range HNCO NMR experiments showing differences of hydrogen bonding patterns for major and minor phosphoUb species. Through H bond correlations (blue) recorded for residue Ile44 in the major and minor species of phosphoUb with reference to a control HNCO spectrum (red). In the major form of phosphoUb (left), the HN of Ile44 is a H bond donor to His68, as seen in wt Ub. In the minor form of phosphoUb (right), the HN of Ile44 does not have a cross peak to His68 but instead has a correlation to Val70.


C Structure of major form of phosphoUb , where phosphoUb residues 1 - 61 are shown under a surface , β5 strand is shown in stick representation with red oxygen , blue nitrogen purple phosphorus atoms . four Leu residues are shown in green . D Model of phosphoUb minor species as in ( C ) in which β5 strand is retracted into by two residues . This extends β4 - β5 loop significantly shortens C ‐ terminal tail of Ub .

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C Structure of the major form of phosphoUb, where phosphoUb residues 1-61 are shown under a surface, and the β5 strand is shown in stick representation with red oxygen, blue nitrogen and purple phosphorus atoms. The four core Leu residues are shown in green.D Model of the phosphoUb minor species as in (C) in which the β5 strand is retracted into the protein core by two residues. This extends the β4-β5 loop and significantly shortens the C‐terminal tail of Ub.


A E1 ‐ mediated charging of E2 enzymes by Ub phosphoUb in a time course . Non ‐ reducing Coomassie ‐ stained 4 - 12 % SDS - PAGE gradient gels are shown , bands are labeled . " ˜ Ub " refers to generation of a thioester , while " Ub ‐ " refers to additional covalent ubiquitination of E2 enzyme . * contaminant from E2 purification .

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A E1‐mediated charging of E2 enzymes by Ub and phosphoUb in a time course analysis. Non‐reducing Coomassie‐stained 4-12% SDS-PAGE gradient gels are shown, and bands are labeled. "˜Ub" refers to generation of a thioester, while "Ub‐"refers to additional covalent ubiquitination of the E2 enzyme. * contaminant from E2 purification.


B SAXS ‐ derived ensemble of UBE2D3 ( Pruneda et al , ) , where E2 is shown under a blue surface Ub is shown as a green ribbon . Ser65 is shown in sphere representation . Ser65 does not contact E2 enzyme in any orientation of Ub at E2 enzyme . PDB files were downloaded from Klevit ‐ lab website ( http : / / depts . washington . edu / klvtlab / ) .

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B SAXS‐derived ensemble of UBE2D3 (Pruneda et al, ), where the E2 is shown under a blue surface and the Ub is shown as a green ribbon. Ser65 is shown in sphere representation. Ser65 does not contact the E2 enzyme in any orientation of the Ub at the E2 enzyme. PDB files were downloaded from the Klevit‐lab website (http://depts.washington.edu/klvtlab/).


C Quantitative mass spectrometry for UBE2T , UBE2E1 UBE2R1 with Ub phosphoUb using UbAQUA peptides reveals impaired chain formation with phosphoUb ( see also Supplementary Fig S10 ) .

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C Quantitative mass spectrometry analysis for UBE2T, UBE2E1 and UBE2R1 with Ub and phosphoUb using UbAQUA peptides reveals impaired chain formation with phosphoUb (see also Supplementary Fig S10).


D Charging of UBE2N , UBE2N / UBE2V1 ‐ mediated Lys63 chain assembly with Ub phosphoUb . Charging of UBE2N as in ( A ) proceeds identically with both Ub phosphoUb ( left 6 lanes ) , while UBE2V1 ‐ mediated Lys63 chain assembly only proceeds with Ub but not phosphoUb ( right 6 lanes Western blot ) .

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D Charging of UBE2N, and UBE2N/UBE2V1‐mediated Lys63 chain assembly with Ub and phosphoUb. Charging of UBE2N as in (A) proceeds identically with both Ub and phosphoUb (left 6 lanes), while UBE2V1‐mediated Lys63 chain assembly only proceeds with Ub but not phosphoUb (right 6 lanes and Western blot).


E Structure of Saccharomyces cerevisiae Ubc13 ‐ Mms2 ( blue / red , Mms2 is a homolog of UBE2V1 ) with acceptor Ub ( cyan ) bound non ‐ covalently to Mms2 [ pdb ‐ id 2gmi ( Eddins et al , ) ] . Ser65 of acceptor Ub interacts with Mms2 , Ser65 phosphorylation would prevent binding due to Mms2 Glu18 .

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E Structure of Saccharomyces cerevisiae Ubc13‐Mms2 (blue/red, Mms2 is a homolog of UBE2V1) with the acceptor Ub (cyan) bound non‐covalently to Mms2 [pdb‐id 2gmi (Eddins et al, )]. Ser65 of the acceptor Ub interacts with Mms2, and Ser65 phosphorylation would prevent binding due to Mms2 Glu18.


F PhosphoUb competition assays for UBE2N / UBE2V1 ‐ mediated Lys63 diUb formation . Time course with K63R Ub ( donor ) Ub ΔGG ( acceptor ) generates Lys63 diUb is not inhibited upon increasing concentrations of phosphoUb , revealing phosphoUb does not compete with Ub for UBE2N / UBE2V1 ‐ mediated Lys63 diUb formation .

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F PhosphoUb competition assays for UBE2N/UBE2V1‐mediated Lys63 diUb formation. Time course with K63R Ub (donor) and Ub ΔGG (acceptor) generates Lys63 diUb and is not inhibited upon increasing concentrations of phosphoUb, revealing phosphoUb does not compete with Ub for UBE2N/UBE2V1‐mediated Lys63 diUb formation.


A Time course for a ligase reaction with GST ‐ tagged cIAP1 ( aa 363 - 614 ) , UBE2D1 Ub or phosphoUb . See Supplementary Fig S11D for a reaction with UBE2D3 .

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A Time course for a ligase reaction with GST‐tagged cIAP1 (aa 363-614), UBE2D1 and Ub or phosphoUb. See Supplementary Fig S11D for a reaction with UBE2D3.


B Anti ‐ Ub Western blot of an identical reaction run for 3 h . C Comparison of number of Ub chain linkages in reaction in ( B ) . D Ub chain linkage profile for reaction in ( B ) .

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B Anti‐Ub Western blot of an identical reaction run for 3 h.C Comparison of the number of Ub chain linkages in the reaction in (B).D Ub chain linkage profile for the reaction in (B).


E Reaction of GST ‐ tagged TRAF6 ( aa 1 - 285 ) with UBE2D3 as in ( A ) . See Supplementary Fig S11E F for additional TRAF6 reactions .

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E Reaction of GST‐tagged TRAF6 (aa 1-285) with UBE2D3 as in (A). See Supplementary Fig S11E and F for additional TRAF6 reactions.


F Anti ‐ Ub Western blot of GST ‐ TRAF6 / UBE2D1 run for 3 h . G Comparison of number of Ub chain linkages in reaction in ( F ) . H Linkage composition in GST ‐ TRAF6 ‐ UBE2D1 reactions reveals 58 % Lys63 chains with wt Ub UBE2D1 . Significantly smaller quantities of linkages are assembled with phosphoUb .

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F Anti‐Ub Western blot of GST‐TRAF6/UBE2D1 run for 3 h.G Comparison of the number of Ub chain linkages in the reaction in (F).H Linkage composition in GST‐TRAF6‐UBE2D1 reactions reveals 58% Lys63 chains with wt Ub and UBE2D1. Significantly smaller quantities of linkages are assembled with phosphoUb.


I - K Chain assembly as in ( A ) with UBE2L3 ( I ) GST ‐ tagged NEDD4L ( aa 576 - 955 ) ( Mund Pelham , ) , ( J ) NleL ( aa 170 - 782 ) ( Hospenthal et al , ) ( K ) HOIP RBR ‐ LDD ( aa 699 - 1072 ) ( Smit et al , ) . While HECT E3 ligases NEDD4L NleL are active with Ub phosphoUb , HOIP is significantly less active with phosphoUb . See also Supplementary Fig S11G H .

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I-K Chain assembly as in (A) with UBE2L3 and (I) GST‐tagged NEDD4L (aa 576-955) (Mund Pelham, ), (J) NleL (aa 170-782) (Hospenthal et al, ) and (K) HOIP RBR‐LDD (aa 699-1072) (Smit et al, ). While the HECT E3 ligases NEDD4L and NleL are active with Ub and phosphoUb, HOIP is significantly less active with phosphoUb. See also Supplementary Fig S11G and H.


A Structure of TAB2 NZF ( red ) in with Lys63 diUb ( cyan ) [ pdb ‐ id 2wwz ( Kulathu et al , ) ] . Ser65 for both Ub moieties is shown as spheres highlighted with a blue circle .

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A Structure of TAB2 NZF (red) in complex with Lys63 diUb (cyan) [pdb‐id 2wwz (Kulathu et al, )]. Ser65 for both Ub moieties is shown as spheres and highlighted with a blue circle.


B Pull ‐ down assays with Lys63 ‐ specific TAB2 NZF against different tetraUb ( Ub4 ) species are shown . Phosphorylation of K63 Ub4 does not prevent binding to TAB2 NZF . Met1 ‐ linked tetraUb does not interact with TAB2 NZF , independent of Ser65 phosphorylation .

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B Pull‐down assays with the Lys63‐specific TAB2 NZF against different tetraUb (Ub4) species are shown. Phosphorylation of K63 Ub4 does not prevent binding to TAB2 NZF. Met1‐linked tetraUb does not interact with TAB2 NZF, independent of Ser65 phosphorylation.


A Structure of USP2 ( red ) bound to Ub ( cyan ) [ pdb ‐ id 2hd5 ( Renatus et al , ) ] , with a close ‐ up on Ub Ser65 in distal Ub . A negative charge at USP2 Asp345 position is conserved in most USP enzymes ( Ye et al , ) .

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A Structure of USP2 (red) bound to Ub (cyan) [pdb‐id 2hd5 (Renatus et al, )], with a close‐up on Ub Ser65 in the distal Ub. A negative charge at the USP2 Asp345 position is conserved in most USP enzymes (Ye et al, ).


B - H Ubiquitination reactions from Fig A were used as input for deubiquitinase ( DUB ) . Silver stained time course gels are shown . DUBs tested ( USP2 , USP8 , USP15 , USP30 , Ataxin ‐ 3 , USP21 ) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains , with exception of vOTU USP21 .

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B-H Ubiquitination reactions from Fig A were used as input for deubiquitinase (DUB) analysis. Silver stained time course gels are shown. All DUBs tested (USP2, USP8, USP15, USP30, Ataxin‐3, USP21) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains, with the exception of vOTU and USP21.


B - H Ubiquitination reactions from Fig A were used as input for deubiquitinase ( DUB ) . Silver ‐ stained time ‐ course gels are shown . DUBs tested ( USP2 , USP8 , USP15 , USP30 , Ataxin ‐ 3 , USP21 ) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains , with exception of vOTU USP21 .

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B-H Ubiquitination reactions from Fig A were used as input for deubiquitinase (DUB) analysis. Silver‐stained time‐course gels are shown. All DUBs tested (USP2, USP8, USP15, USP30, Ataxin‐3, USP21) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains, with the exception of vOTU and USP21.


I USP30 activity was significantly lower for cIAP polyUb as compared to other USP DUBs . Cleavage assays against diUb panel reveals preference for Lys6 ‐ linked chains .

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I USP30 activity was significantly lower for cIAP polyUb as compared to other USP DUBs. Cleavage assays against the diUb panel reveals preference for Lys6‐linked chains.


J USP30 activity is decreased against phosphorylated Lys6 diUb .

J USP30 activity is decreased against phosphorylated Lys6 diUb.


K - N Deubiquitinase assays against phosphorylated tetraUb . activity of AMSH ( K ) , TRABID ( L ) , OTUB1 ( M ) OTULIN ( N ) are inhibited with Lys63 , Lys33 , Lys48 Met1 phosphorylated tetraUb , respectively .

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K-N Deubiquitinase assays against phosphorylated tetraUb. The activity of AMSH (K), TRABID (L), OTUB1 (M) and OTULIN (N) are inhibited with Lys63, Lys33, Lys48 and Met1 phosphorylated tetraUb, respectively.


qRT - PCR for INHBA relative to RPL27 using from SCC13 transduced with a lentiviral vector allowing expression of INHBA in a doxycycline ( DOX ) - inducible manner ( SCC13 Act clone 1 2 ) or empty vector ( EV ) ( N = 3 ) . SCC13 Act EV lines were generated from lentivirally transduced upon single clonal expansion .

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qRT-PCR for INHBA relative to RPL27 using RNA from SCC13 cells transduced with a lentiviral vector allowing expression of INHBA in a doxycycline (DOX)-inducible manner (SCC13 Act clone 1 and 2) or empty vector (EV) (N=3). SCC13 Act and EV cell lines were generated from lentivirally transduced cells upon single cell clonal expansion.


Western blot of lysate conditioned media ( CM ; bottom ) of transduced SCC13 for activin βA subunit GAPDH ( loading control for lysate ) under reducing conditions . higher molecular of recombinant activin βA results from HA - epitope tag . Ponceau S of was used as a loading control for CM .

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Western blot of cell lysate and conditioned media (CM; bottom) of transduced SCC13 cells for the activin βA subunit and GAPDH (loading control for cell lysate) under reducing conditions. The higher molecular weight of recombinant activin βA results from the HA-epitope tag. Ponceau S staining of the membrane was used as a loading control for the CM.


Representative pictures of 5 week - old tumors ( indicated by arrows ) formed in NOD / SCID mice upon intradermal injection of SCC13 Act ( clone 2 ) or SCC13 EV volume at different time points of development . N = 3 - 6 tumors .

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Representative pictures of 5 week-old tumors (indicated by arrows) formed in NOD/SCID mice upon intradermal injection of SCC13 Act (clone 2) or SCC13 EV cells and tumor volume at different time points of tumor development. N=3-6 tumors.


Representative of H & E stainings of tumors formed by SCC13 EV Act ( clone 2 ) at day 35 ( asterisk indicates cartilage , arrow indicates site of invasion ) . An inset panel indicates where invade into cartilage . sections were evaluated for invasion through basement . N = 6 ( EV ) , N = 6 Act ( 3 tumors from clone 1 3 tumors from clone 2 ) . Tumors from Act mice were pooled in graph ) .

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

Representative images of H&E stainings of tumors formed by SCC13 EV and Act cells (clone 2) at day 35 (asterisk indicates cartilage, arrow indicates site of invasion). An inset panel indicates the area where tumor cells invade into the cartilage. Tumor sections were evaluated for invasion through the basement membrane. N=6 (EV), N=6 Act (3 tumors from clone 1 and 3 tumors from clone 2). Tumors from Act mice were pooled in the graph).


at endpoint ( 5 after injection ) . N = 3 - 6 tumors .

Tumor weight at endpoint (5 weeks after injection). N=3-6 tumors.


Herovici of of collagen - positive ( blue ) per field of view . N = 3 tumors , n = 3 - 4 histological sections .

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Herovici staining and quantification of percentage of collagen-positive (dark blue) area per field of view. N=3 tumors, n=3-4 histological sections.


Representative of sections from tumors formed by SCC13 EV SCC13 Act ( clone 2 ) stained for Ki67 ( red ) , PDGFR - α ( green ) counterstained with Hoechst ( blue ) . of Ki67 / PDGFR - α positive is shown in graph . N = 3 tumors , n = 3 - 4 histological sections .

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Representative images of sections from tumors formed by SCC13 EV and SCC13 Act (clone 2) stained for Ki67 (red), PDGFR-α (green) and counterstained with Hoechst (blue). Quantification of Ki67/PDGFR-α positive cells is shown in the graph. N=3 tumors, n=3-4 histological sections.


Representative of sections from 3D cultures of human skin fibroblasts with either SCC13 EV or SCC13 Act ( clones 1 2 ) stained with either H & E or Herovici .

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Representative images of sections from 3D cultures of primary human skin fibroblasts with either SCC13 EV or SCC13 Act (clones 1 and 2) cells stained with either H&E or Herovici.


of Ki67 - positive in sections from SCC13 EV SCC13 Act ( clone 2 ) organotypic cultures ( co - stained with E - cadherin to indicate epithelial ) . N = 3 independent cultures , n = 3 histological sections .

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Quantification of Ki67-positive cells in sections from SCC13 EV and SCC13 Act (clone 2) organotypic cultures (co-stained with E-cadherin to indicate epithelial cells). N=3 independent cultures, n=3 histological sections.


Representative of sections from SCC13 EV SCC13 Act ( clone 2 ) organotypic cultures stained for K14 ( red ) collagen IV ( green ) combined with Hoechst ( blue ) of K14 - positive below basement ( BM ) . N = 3 independent cultures , n = 4 histological sections . indicated with a rectangle in left picture is shown at higher magnification in right picture .

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

Representative images of sections from SCC13 EV and SCC13 Act (clone 2) organotypic cultures stained for K14 (red) and collagen IV (green) combined with Hoechst staining (blue) and quantification of the K14-positive area below the basement membrane (BM). N=3 independent cultures, n=4 histological sections. The area indicated with a white rectangle in the left picture is shown at higher magnification in the right picture.


murine dermal fibroblasts , which had been treated with recombinant activin A at different concentrations , were analyzed for BrdU incorporation . N = 3 - 4 .

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Primary murine dermal fibroblasts, which had been treated with recombinant activin A at different concentrations, were analyzed for BrdU incorporation. N=3-4.


Representative of collagen gels of extent of gel contraction ( of initial gel ) in presence or absence of activin A ( 20 ng / ml ) / or TGF - β1 ( 1 ng / ml ) . N = 3 - 4 .

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

Representative images of collagen gels and quantification of the extent of gel contraction (percentage of initial gel size) in the presence or absence of activin A (20 ng/ml) and/or TGF-β1 (1 ng/ml). N=3-4.


qRT - PCR Western blot analyses of ACTA2 / αSMA expression using total / lysates of fibroblasts treated with activin A or TGF - β1 for 6 h . N = 3 . was re - probed with GAPDH mDia2 antibodies .

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qRT-PCR and Western blot analyses of ACTA2/αSMA expression using total RNA/lysates of fibroblasts treated with activin A or TGF-β1 for 6 h. N=3. The membrane was re-probed with GAPDH and mDia2 antibodies.


Representative of fibroblasts treated with activin A , TGF - β1 or vehicle ( control ) as in ( C ) , stained for αSMA ( green ) , counterstained with rhodamine - coupled phalloidin ( red ) Hoechst ( blue ) . Bar graph shows of fibroblasts with stress . N = 3 .

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

Representative images of fibroblasts treated with activin A, TGF-β1 or vehicle (control) as in (C), stained for αSMA (green), counterstained with rhodamine-coupled phalloidin (red) and Hoechst (blue). Bar graph shows percentage of fibroblasts with stress fibers. N=3.


epresentative of control activin A - treated fibroblasts stained with rhodamine - coupled phalloidin ; scatter plot shows of filopodium in control , activin A - TGF - β1 - treated fibroblasts . N = 27 - 70 filopodia .

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epresentative images of control and activin A-treated fibroblasts stained with rhodamine-coupled phalloidin; scatter plot shows quantification of filopodium length in control, activin A- and TGF-β1-treated fibroblasts. N=27-70 filopodia.


Scratch of fibroblasts treated with activin A or vehicle for 24 h . N = 3 . covered by in original scratch was quantified . Migration in one control culture was set to 1 .

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Scratch assay of fibroblasts treated with activin A or vehicle for 24 h. N=3. The area covered by cells in the original scratch was quantified. Migration in one control culture was set to 1.


Chemotactic migration of mouse fibroblasts from wt mice for 24 h in a transwell towards increasing concentrations of activin A or vehicle . N = 3 .

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Chemotactic migration of primary mouse fibroblasts from wt mice for 24 h in a transwell assay towards increasing concentrations of activin A or vehicle. N=3.


qRT - PCR of CAF marker genes relative to Rps29 using from fibroblasts treated with activin A for 6 h . N = 3 .

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qRT-PCR analysis of CAF marker genes relative to Rps29 using RNA from fibroblasts treated with activin A for 6 h. N=3.


qRT - PCR for INHBA relative to RPL27 using from human fibroblasts transduced with a lentiviral vector allowing expression of INHBA in an inducible manner after treatment with DOX for 24 h ( Fb Act , clones 1 2 ) or with empty vector ( Fb EV ) ( N = 3 ) . Fb Act Fb EV cultures were generated from lentivirally - transduced upon clonal expansion of resistant single .

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qRT-PCR for INHBA relative to RPL27 using RNA from primary human fibroblasts transduced with a lentiviral vector allowing expression of INHBA in an inducible manner after treatment with DOX for 24 h (Fb Act, clones 1 and 2) or with empty vector (Fb EV) (N=3). Fb Act and Fb EV cultures were generated from lentivirally-transduced cells upon clonal expansion of resistant single cells.


Western blot of conditioned media ( CM ) of transduced fibroblasts showing mature activin βA . Ponceau S of was used as a loading control .

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Western blot of conditioned media (CM) of transduced fibroblasts showing mature activin βA. Ponceau S staining of the membrane was used as a loading control.


Chemotactic migration of SCC13 for 24 h in a transwell containing CM from Fb Act ( clone 2 ) or Fb EV . N = 4 . Migration in one control culture was set to 1 .

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Chemotactic migration of SCC13 cells for 24 h in a transwell assay containing CM from Fb Act (clone 2) or Fb EV. N=4. Migration in one control culture was set to 1.


Relative colony of SCC13 upon culture in CM from Fb Act ( clone 2 ) or EV . N = 3 .

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Relative colony area of SCC13 cells upon culture in CM from Fb Act (clone 2) or EV. N=3.


Representative immunofluorescence of de - cellularized matrix from Fb Act ( clone 2 ) Fb EV stained for fibronectin or collagen I .

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Representative immunofluorescence images of de-cellularized matrix from Fb Act (clone 2) and Fb EV stained for fibronectin or collagen I.


Relative colony of SCC13 plated on either de - cellularized matrix from Fb Act ( clone 2 ) or Fb EV . N = 3 .

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Relative colony area of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=3.


Migration of SCC13 plated on either de - cellularized matrix from Fb Act ( clone 2 ) or Fb EV . N = 6 . experimental setup is shown schematically above graph .

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Migration of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=6. The experimental setup is shown schematically above the graph.


Fb Act ( clone 2 ) or Fb EV were cultured together in 2D with SCC13 . After 7 day of culture SCC13 were identified by K14 number of K14 - positive colony was quantified . N = 6 .

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Fb Act (clone 2) or Fb EV cells were cultured together in 2D with SCC13 cells. After 7 day of culture SCC13 cells were identified by K14 and the number of K14-positive colony area was quantified. N=6.


of SCC13 spheroid in single hanging drop including CM from Fb EV or Fb Act ( clone 2 ) ( including 1 % FBS ) , DMEM / 1 % FBS , or DMEM / 1 % FBS plus 20 ng / mL recombinant activin A . of spheroid formed in one Fb EV sample was set to 1 . N = 9 - 10 hanging drops .

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Quantification of SCC13 tumor spheroid area in single hanging drop including CM from Fb EV or Fb Act (clone 2) (including 1% FBS), DMEM/1% FBS, or DMEM/1% FBS plus 20 ng/mL recombinant activin A. The area of the spheroid formed in one Fb EV sample was set to 1. N=9-10 hanging drops.


Representative of K14 - stained areas from 3D cultures of SCC13 seeded on top of either Fb Act ( clone 2 ) or Fb EV . N = 3 independent cultures , n = 3 histological sections .

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Representative images and quantification of K14-stained areas from 3D cultures of SCC13 cells seeded on top of either Fb Act (clone 2) or Fb EV. N=3 independent cultures, n=3 histological sections.


Representative pictures of tumors formed in NOD / SCID mice 6 - 7 after intradermal injection of SCC13 with Fb Act ( clone 2 ) or Fb EV . volume at different time points of development ( 6 - 7 after injection ) . N = 5 tumors per group .

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Representative pictures of tumors formed in NOD/SCID mice 6-7 weeks after intradermal injection of SCC13 cells with Fb Act (clone 2) or Fb EV. Tumor volume at different time points of tumor development (6-7 weeks after injection). N=5 tumors per group.


mDia2 ( Diap3 ) expression quantified by sequencing of fibroblasts isolated from ear skin . N = 3 pools of 3 - 6 mice .

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mDia2 (Diap3) expression quantified by RNA sequencing of fibroblasts isolated from ear skin. N=3 pools of 3-6 mice.


mouse fibroblasts were treated with activin A or TGF - β1 or vehicle for 6h analyzed by qRT - PCR ( N = 3 ) Western blot for mDia2 ( Diap3 ) mDia2 expression , respectively . GAPDH loading control is identical to one in Fig 2C , since same was probed for mDia2 , αSMA GAPDH .

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Primary mouse fibroblasts were treated with activin A or TGF-β1 or vehicle for 6h and analyzed by qRT-PCR (N=3) and Western blot for mDia2 (Diap3) and mDia2 expression, respectively. The GAPDH loading control is identical to the one in Fig 2C, since the same membrane was probed for mDia2, αSMA and GAPDH.


Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX - inducible dominant - negative ActRIB mutant ( dnActRIB ) or an empty lentivirus ( EV ) , treated with activin A for 2 . 5 h analyzed by Western blot for mDia2 GAPDH .

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Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX-inducible dominant-negative ActRIB mutant (dnActRIB) or an empty lentivirus (EV), treated with activin A for 2.5 h and analyzed by Western blot for mDia2 and GAPDH.


Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act ( clone 2 ) stained for mDia2 or K14 ( green ) ( top ) , mDia2 ( green ) or MECA32 / LYVE1 ( red , bottom ) counterstained with Hoechst ( blue ) .

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Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act cells (clone 2) stained for mDia2 or K14 (green) (top), mDia2 (green) or MECA32/LYVE1 (red, bottom) and counterstained with Hoechst (blue).


qRT - PCR for mDia2 / DIAPH3 relative to RPL27 using from normal human skin ( N = 11 ) , SCCs ( N = 9 ) BCCs ( N = 7 ) .

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qRT-PCR for mDia2/DIAPH3 relative to RPL27 using RNA from normal human skin (N=11), SCCs (N=9) and BCCs (N=7).


Representative skin / sections from normal human skin , SCC BCC stained for mDia2 / DIAPH3 . Note strong mDia2 / DIAPH3 - positivity in stroma . Scale bars : 100 μm . D : Dermis , E : Epidermis : T : .

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Representative skin/tumor sections from normal human skin, SCC and BCC patients stained for mDia2/DIAPH3. Note the strong mDia2/DIAPH3-positivity in the tumor stroma. Scale bars: 100 μm. D: Dermis, E: Epidermis: T: Tumor.


Expression of mDia2 / DIAPH3 in normal tissues on TCGA cancer data ( TCGA data version 20141017 , http : / / firebrowse . org / ) . Gene expression were presented as normalized log2 RSEM ( - Seq by Expectation - Maximization ) index from database . BLCA : Bladder Urothelial Carcinoma ( N = 408 & 19 normal tissue samples , respectively ) , BRCA : Breast Invasive Carcinoma ( N = 1100 & 112 ) , CESC : Cervical SCC ( N = 306 & 3 ) , ESCA : Esophageal SCC ( N = 185 & 11 ) , HNSC : Head - Neck SCC ( N = 522 & 44 ) , LIHC : Liver Hepatocellular Carcinoma ( N = 373 & 47 ) , LUSC : Lung SCC ( N = 501 & 51 ) , PAAD : Pancreatic Adenocarcinoma ( N = 179 & 4 ) , PRAD : Prostate Adenocarcinoma ( N = 496 & 50 ) , READ : Rectum Adenocarcinoma ( N = 167 & 10 ) , STES : Stomach Esophageal Carcinoma ( N = 600 & 46 ) , UCEC : Uterine Corpus Endometrial Carcinoma ( N = 546 & 35 ) . Box plot shows relative mDia2 / DIAPH3 expression in human ( red ) normal ( blue ) tissue . Boxes indicate interquartile range , whiskers indicate data points within 1 . 5 times interquartile range , dots outside whiskers indicate outliers , central indicates median .

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Expression levels of mDia2/DIAPH3 in tumor and normal tissues based on TCGA cancer data (TCGA data version 20141017, http://firebrowse.org/). Gene expression levels were presented as normalized log2 RSEM (RNA-Seq by Expectation-Maximization) index from the database. BLCA: Bladder Urothelial Carcinoma (N = 408 & 19 tumor and normal tissue samples, respectively), BRCA: Breast Invasive Carcinoma (N = 1100 & 112), CESC: Cervical SCC (N = 306 & 3), ESCA: Esophageal SCC (N = 185 & 11), HNSC: Head-Neck SCC (N = 522 & 44), LIHC: Liver Hepatocellular Carcinoma (N = 373 & 47), LUSC: Lung SCC (N = 501 & 51), PAAD: Pancreatic Adenocarcinoma (N = 179 & 4), PRAD: Prostate Adenocarcinoma (N = 496 & 50), READ: Rectum Adenocarcinoma (N = 167 & 10), STES: Stomach and Esophageal Carcinoma (N = 600 & 46), UCEC: Uterine Corpus Endometrial Carcinoma (N = 546 & 35). Box plot shows relative mDia2/DIAPH3 expression in human tumor (red) and normal (blue) tissue. Boxes indicate the interquartile range, whiskers indicate data points within 1.5 times interquartile range, dots outside the whiskers indicate outliers, and the central line indicates median.


Relative gene expression of mDia2 / DIAPH3 INHBA in stroma of intrahepatic cholangiocarcinoma , breast colorectal cancers vs . stroma of respective normal tissues on datasets GSE45001 ( D ) ( N = 10 normal tissue & 10 cancer samples , GSE9014 ( E ) ( N = 4 & 13 ) , GSE35602 ( F ) ( N = 6 & 53 ) , in CAFs from breast cancer samples vs . normal breast fibroblasts ( GSE29270 dataset ) ( G ) ( N = 5 & 23 ) . NS : Normal stroma , CS : Cancer stroma , NF : Normal fibroblasts , CAF : Cancer - associated fibroblasts ) . expression values of datasets were obtained using GEO2R tool in GEO database .

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Relative gene expression of mDia2/DIAPH3 and INHBA in the stroma of intrahepatic cholangiocarcinoma, breast and colorectal cancers vs. stroma of respective normal tissues based on datasets GSE45001 (D) (N = 10 normal tissue & 10 cancer samples, GSE9014 (E) (N = 4 & 13), and GSE35602 (F) (N = 6 & 53), and in CAFs from breast cancer samples vs. normal breast fibroblasts (GSE29270 dataset) (G) (N = 5 & 23). NS: Normal stroma, CS: Cancer stroma, NF: Normal fibroblasts, CAF: Cancer-associated fibroblasts). The expression values of the datasets were obtained using GEO2R tool in the GEO database.


Kaplan - Meier survival curves on datasets from GEO , EGA TCGA , demonstrating that high expression of both mDia2 / DIAPH3 INHBA in with liver , stomach breast cancer correlates with poor overall survival . Data statistical analyses were collected from KMPlotter .

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Kaplan-Meier survival curves based on datasets from GEO, EGA and TCGA, demonstrating that high expression of both mDia2/DIAPH3 and INHBA in patients with liver, stomach and breast cancer correlates with poor overall survival. Data and statistical analyses were collected from KMPlotter.


Immortalized mouse fibroblasts were transduced with a lentiviral vector expressing different mDia2 - targeting shRNAs ( shmDia2 # 1 - 3 ) or empty vector ( sh - EV ) . knock - down was confirmed by qRT - PCR ( N = 3 ) , Western blot immunofluorescence .

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Immortalized mouse fibroblasts were transduced with a lentiviral vector expressing different mDia2-targeting shRNAs (shmDia2 #1-3) or empty vector (sh-EV). The knock-down was confirmed by qRT-PCR (N=3), Western blot and immunofluorescence.


Ki67 of mDia2 knock - down control mouse fibroblasts . N = 3 .

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Ki67 staining of mDia2 knock-down and control mouse fibroblasts. N=3.


migration in scratch assays of mDia2 knock - down control mouse fibroblasts . N = 3 .

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migration in scratch assays of mDia2 knock-down and control mouse fibroblasts. N=3.


qRT - PCR for mDia1 , mDia3 , Fmn1 Fmn2 several CAF genes relative to Rps29 using from control ( sh - EV ) mDia2 knock - down ( sh - mDia2 ) fibroblasts . N = 3 .

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qRT-PCR for mDia1, mDia3, Fmn1 and Fmn2 and several CAF genes relative to Rps29 using RNA from control (sh-EV) and mDia2 knock-down (sh-mDia2) fibroblasts. N=3.


Representative of mDia2 knockdown control mouse fibroblasts stained for p53 ( left ) , of showing accumulation of p53 in nucleus ( right ) . N = 3 .

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Representative images of mDia2 knockdown and control mouse fibroblasts stained for p53 (left), and quantification of cells showing accumulation of p53 in the nucleus (right). N=3.


Representative Western blots for p53 , vinculin histone H1 using fractions total lysate of mDia2 knock - down ( F ) or activin A - treated fibroblasts ( 6h , 20 ng / ml ) ( G ) control mouse fibroblasts of p53 / histone H1 . Vinculin histone H1 were used as loading controls for fractions , respectively . N = 6 .

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Representative Western blots for p53, vinculin and histone H1 using nuclear and cytoplasmic fractions and total lysate of mDia2 knock-down (F) or activin A-treated fibroblasts (6h, 20 ng/ml) (G) and control mouse fibroblasts and quantification of the nuclear p53/histone H1 ratio. Vinculin and histone H1 were used as loading controls for the cytoplasmic and nuclear fractions, respectively. N=6.


Representative Western blots for p53 , mDia2 GAPDH using total lysates of mDia2 knock - down control mouse fibroblasts treated for 24 h with pifithrin - α ( PFT - α ) ( 15 μM ) .

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Representative Western blots for p53, mDia2 and GAPDH using total lysates of mDia2 knock-down and control mouse fibroblasts treated for 24 h with pifithrin-α (PFT-α) (15 μM).


Representative p53 immunofluorescence of mDia2 knock - down mouse fibroblasts treated with PFT - α or vehicle ( DMSO ) ( top ) of showing accumulation of p53 in nucleus ( bottom ) . N = 3 .

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Representative p53 immunofluorescence images of mDia2 knock-down mouse fibroblasts treated with PFT-α or vehicle (DMSO) (top) and quantification of cells showing accumulation of p53 in the nucleus (bottom). N=3.


qRT - PCR for mDia2 , Postn , Mmp13 , Fn1 , Spp1 Acta2 relative to Rps29 using from mDia2 knock - down control fibroblasts treated with PFT - α or vehicle . N = 3 .

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qRT-PCR for mDia2, Postn, Mmp13, Fn1, Spp1 and Acta2 relative to Rps29 using RNA from mDia2 knock-down and control fibroblasts treated with PFT-α or vehicle. N=3.


Colony of SCC13 Act ( clone 2 ) after co - culture with either immortalized mDia2 knockdown ( sh - mDia2 ) or control ( sh - EV ) mouse fibroblasts . N = 4 .

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Colony size of SCC13 Act cells (clone 2) after co-culture with either immortalized mDia2 knockdown (sh-mDia2) or control (sh-EV) mouse fibroblasts. N=4.


Representative pictures of 5 - week - old tumors formed upon intradermal co - injection of SCC13 Act ( clone 2 ) with sh - EV or sh - mDia2 knock - down immortalized mouse fibroblasts . Graph shows volume at different timepoints of development . N = 3 - 5 tumors per group .

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Representative pictures of 5-week-old tumors formed upon intradermal co-injection of SCC13 Act cells (clone 2) with sh-EV or sh-mDia2 knock-down immortalized mouse fibroblasts. Graph shows tumor volume at different timepoints of tumor development. N=3-5 tumors per group.


Representative immunofluorescence of sections from ear skin tumors formed by SCC13 Act ( clone 2 ) co - injected with sh - EV or sh - mDia2 fibroblasts , stained for E - cadherin ( green ) periostin or p53 ( red ) , counterstained with Hoechst ( blue ) .

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Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 Act (clone 2) co-injected with sh-EV or sh-mDia2 fibroblasts, stained for E-cadherin (green) and periostin or p53 (red), counterstained with Hoechst (blue).


volume at different time points after co - injection of SCC13 with EV or dnActRIB fibroblasts . N = 5 tumors .

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Tumor volume at different time points after co-injection of SCC13 cells with EV or dnActRIB fibroblasts. N=5 tumors.


Representative immunofluorescence of sections from ear skin tumors formed by SCC13 co - injected with EV or dnActRIB fibroblasts stained for K14 ( left ) or PDGFRα ( right ) ( green ) mDia2 ( red ) , counterstained with Hoechst ( blue ) . Bar graph shows of mDia2 - positive . N = 3 .

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Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 cells co-injected with EV or dnActRIB fibroblasts stained for K14 (left) or PDGFRα (right) (green) and mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.


SCC13 EV or SCC13 Fst were injected into ear skin of NOD / SCID mice was determined 12 after injection . N = 9 - 10 tumors .

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SCC13 EV or SCC13 Fst cells were injected into the ear skin of NOD/SCID mice and tumor weight was determined 12 weeks after injection. N=9-10 tumors.


Representative immunofluorescence of sections from ear skin tumors formed by SCC13 EV or SSC13 Fst stained for K14 or mDia2 ( red ) , counterstained with Hoechst ( blue ) . Bar graph shows of mDia2 - positive . N = 3 .

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Representative immunofluorescence images of sections from ear skin tumors formed by SCC13 EV or SSC13 Fst cells stained for K14 or mDia2 (red), counterstained with Hoechst (blue). Bar graph shows the percentage of mDia2-positive area. N=3.


Tumors formed by SCC13 Act were treated ex vivo for 6h with follistatin ( FST ) ( 50 ng / ml ) / or SMIFH2 ( 10 μM ) , or with activin A ( 20 ng / ml ) stained for Ki67 . N = 3 explants , n = 4 histological sections .

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Tumors formed by SCC13 Act cells were treated ex vivo for 6h with follistatin (FST) (50 ng/ml) and/or SMIFH2 (10 μM), or with activin A (20 ng/ml) and stained for Ki67. N=3 tumor explants, n=4 histological sections.


Representative of spheroids formed by SCC13 Act labeled with PKH67 ( green ) with human fibroblasts embedded in collagen gels graph showing increase in spheroid between day 0 day 3 . Spheroids were either treated with FST ( 50 ng / ml ) or SMIFH2 ( 10 μM ) or left untreated . N = 6 - 8 spheroids .

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Representative images of spheroids formed by SCC13 Act cells labeled with PKH67 (green) with primary human fibroblasts embedded in collagen gels and graph showing increase in the spheroid area between day 0 and day 3. Spheroids were either treated with FST (50 ng/ml) or SMIFH2 (10 μM) or left untreated. N=6-8 tumor spheroids.


Increase in A431 volume ( mm3 ) compared to day 0 ( time when tumors were first injected with FST , approximately 3 - 4 after injection of ) upon treatment of tumors with FST ( 100 ng per injection ) or vehicle ( PBS ) . N = 3 - 5 tumors per group . Representative immunofluorescence FST or vehicle - treated tumors , stained for mDia2 ( green ) K14 ( red ) , counterstained with Hoechst ( blue ) .

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Increase in A431 tumor volume (mm3) compared to day 0 (time when the tumors were first injected with FST, approximately 3-4 weeks after injection of the cells) upon treatment of the tumors with FST (100 ng per injection) or vehicle (PBS). N=3-5 tumors per group. Representative immunofluorescence images the FST or vehicle-treated tumors, stained for mDia2 (green) and K14 (red), counterstained with Hoechst (blue).


( A ) C57BL / 6 mice were inoculated with 6x105 B16Bl6 parental or dnTNF - R1 on day 0 treated daily with 7µg mTNF p . l . from day 10 . growth is shown as mean index ( TSI ) + ( n = 6 ) . under graph represents treatment period .

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(A) C57BL/6 mice were inoculated with 6x105 B16Bl6 parental or dnTNF-R1 cells on day 0 and treated daily with 7µg mTNF p.l. from day 10. Tumor growth is shown as mean tumor size index (TSI) + SEM (n = 6). The line under the graph represents the treatment period.


( B ) B16Bl6 growth after daily p . l . treatment with 7µg mTNF in C57BL / 6J wild - type ( WT ) , TNF - R1 - / - , conditional TNF - R1 reactivation knockout ( p55cneo / cneo ) , or p55cneo / cneo mice with Cre expression in endothelium ( Flk1Cre ) or ( DelCre ) . growth is shown as mean TSI + ( n = 7 for WT Flk1Cre , 8 for DelCre , 9 for TNF - R1 - / - 10 for p55cneo / cneo ) . under graph represents treatment period .

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(B) B16Bl6 tumor growth after daily p.l. treatment with 7µg mTNF in C57BL/6J wild-type (WT), TNF-R1-/-, conditional TNF-R1 reactivation knockout (p55cneo/cneo), or p55cneo/cneo mice with Cre expression in endothelium (Flk1Cre) or all cells (DelCre). Tumor growth is shown as mean TSI + SEM (n = 7 for WT and Flk1Cre, 8 for DelCre, 9 for TNF-R1-/- and 10 for p55cneo/cneo). The line under the graph represents the treatment period.


( C ) PCR for detection of wild - type , conditional knockout ( cneo ) or reactivated ( lox ) TNF - R1 allele in whole tissues , lung endothelial ( LECs ) lung single - suspension depleted of CD31 + .

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(C) PCR analysis for the detection of wild-type, conditional knockout (cneo) or reactivated (lox) TNF-R1 allele in whole tissues, lung endothelial cells (LECs) and lung single-cell suspension depleted of CD31+ cells.


( D ) Toxicity of a single i . v . bolus injection of indicated dose of mTNF . Mean rectal + cumulative survival rates are shown ( n = 4 for Flk1Cre 10µg ; 6 for DelCre 10µg , Flk1Cre 10µg p55cneo / cneo 15µg ; 7 for WT 10µg ; 9 for WT 8µg , 10 for DelCre 20µg ; 5 for other groups ) . For continuity of graphs , dead mice were included with a of 20 ° C .

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(D) Toxicity of a single i.v. bolus injection of the indicated dose of mTNF. Mean rectal body temperature + SEM and cumulative survival rates are shown (n = 4 for Flk1Cre 10µg; 6 for DelCre 10µg, Flk1Cre 10µg and p55cneo/cneo 15µg; 7 for WT 10µg; 9 for WT 8µg, 10 for DelCre 20µg; and 5 for all other groups). For continuity of the temperature graphs, dead mice were included with a temperature of 20°C.


( B , C ) Viability of L929 parental or mCD20 - expressing after 72h stimulation with mTNF or sc mTNF mutant Y86F fused to a BcII10 or mCD20 VHH ( B ) viability of MCF7 parental or hCD20 - expressing after 72h stimulation with hTNF or sc hTNF mutant Y87F fused to a BcII10 or hCD20 VHH ( C ) . viability was measured via an ATP luminescence . Each point is mean of three replicates error bars are .

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(B,C) Viability of L929 parental or mCD20-expressing cells after 72h stimulation with mTNF or sc mTNF mutant Y86F fused to a BcII10 or mCD20 VHH (B) and viability of MCF7 parental or hCD20-expressing cells after 72h stimulation with hTNF or sc hTNF mutant Y87F fused to a BcII10 or hCD20 VHH (C). Cell viability was measured via an ATP luminescence assay. Each point is the mean of three replicates and error bars are SEM.


( D ) IL - 8 in HUVEC supernatant after 24h stimulation with indicated of stimulus , measured via ELISA .

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(D) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of stimulus, measured via ELISA.


( A ) Toxicity of wt sc mTNF mutant Y86F after injection ( ↑ ) in naïve C56BL / 6 mice . First injection was i . v . , other injections were i . p . Mean rectal ± cumulative survival rates are shown ( n = 4 ) . For continuity of graphs , dead mice were included with a of 20 ° C .

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(A) Toxicity of wt sc mTNF and mutant Y86F after injection (↑) in naïve C56BL/6 mice. First injection was i.v., other injections were i.p. Mean rectal body temperature ± SEM and cumulative survival rates are shown (n = 4). For continuity of the temperature graphs, dead mice were included with a temperature of 20°C.


( B ) Plasma IL - 6 concentrations measured via ELISA . Blood samples were taken 6 hours after i . v . injection of wt sc mTNF or mutant Y86F .

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

(B) Plasma IL-6 concentrations measured via ELISA. Blood samples were taken 6 hours after i.v. injection of wt sc mTNF or mutant Y86F.


( C ) Immunohistochemical for PECAM - 1 ( red ) , ICAM - 1 ( green ) ( blue ) of B16Bl6 tumors after p . l . treatment with 7µg wt mTNF or 50µg of mCD13 - AFR . Scale bar is 50µm .

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

(C) Immunohistochemical staining for PECAM-1 (red), ICAM-1 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 7µg wt mTNF or 50µg of mCD13-AFR. Scale bar is 50µm.


( D ) Expression of ICAM - 1 , E - selectin VEGF - R2 in B16Bl6 6h after treatment , by qPCR of whole . Error bars are . ns , non - significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 by one - way ANOVA with Bonferroni ' s multiple comparison .

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

(D) Expression of ICAM-1, E-selectin and VEGF-R2 in B16Bl6 tumor 6h after treatment, by qPCR analysis of whole tumor RNA. Error bars are SEM. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by one-way ANOVA with Bonferroni's multiple comparison test.


( E ) growth change after daily p . l . injection of indicated ( equimolar ) doses of mCD13 - AFR , non - targeted AFR ( BcII10 - AFR ) or mCD13 VHH control ( CD13 - hIFNα2 ) in B16Bl6 melanoma model . under graph represents treatment period . growth is shown as mean TSI , error bars are ( n = 5 ) . * * * , p < 0 . 001 by two - way ANOVA with Bonferroni ' s multiple comparison .

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

(E) Tumor growth and body weight change after daily p.l. injection of indicated (equimolar) doses of mCD13-AFR, non-targeted AFR (BcII10-AFR) or mCD13 VHH control (CD13-hIFNα2) in the B16Bl6 melanoma model. The line under the graph represents the treatment period. Tumor growth is shown as mean TSI, error bars are SEM (n = 5). ***, p < 0.001 by two-way ANOVA with Bonferroni's multiple comparison test.


( F ) B16Bl6 growth after daily p . l . treatment with indicated doses of mCD13 - AFR , Wortmannin ( Wm ) , Birinapant ( Bir ) or combinations thereof . Wortmannin Birinapant were given 1 hour before CD13 - AFR . growth is shown as mean TSI + ( n = 4 for Wm Bir , 5 for other groups ) . under graph represents treatment period . TSI of tumors of indicated treatment groups at day 17 after inoculation .

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

(F) B16Bl6 tumor growth after daily p.l. treatment with indicated doses of mCD13-AFR, Wortmannin (Wm), Birinapant (Bir) or combinations thereof. Wortmannin and Birinapant were given 1 hour before CD13-AFR. Tumor growth is shown as mean TSI + SEM (n = 4 for Wm and Bir, 5 for other groups). The line under the graph represents the treatment period. TSI of individual tumors of the indicated treatment groups at day 17 after tumor inoculation.


( A ) Schematic representation of CAR construct used , consisting of N - terminal anti - hCD70 VHH , hCD8α hinge transmembrane ( TM ) domain , CD137 co - stimulatory domain C - terminal CD3ζ activation domain . ( B , C ) SKOV3 growth in NSG mice after i . v . injection of 6 x 106 hCD70 CAR T - on day 12 p . l . treatment with 50µg mCD13 - AFR ( ↑ ) . growth is shown as mean TSI , error bars are ( n = 5 ) ( B ) or as TSI of mice ( C ) .

O O O O O O O O O O O O O O O O O O O O O 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-CELL_LINE B-EXP_ASSAY O O B-ORGANISM 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 O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM O O O O

(A) Schematic representation of the CAR construct used, consisting of N-terminal anti-hCD70 VHH, hCD8α hinge and transmembrane (TM) domain, CD137 co-stimulatory domain and C-terminal CD3ζ activation domain. (B,C) SKOV3 tumor growth in NSG mice after i.v. injection of 6 x 106 hCD70 CAR T-cells on day 12 and p.l. treatment with 50µg mCD13-AFR (↑). Tumor growth is shown as mean TSI, error bars are SEM (n = 5) (B) or as TSI of individual mice (C).


( D ) Flow cytometric of hCD45 + hCD3 + eGFP + CAR T - in SKOV3 tumors on day 18 after inoculation . Mice were injected with 6 x 106 CAR T - on day 12 treated with PBS or CD13 -

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(D) Flow cytometric analysis of hCD45+hCD3+eGFP+ CAR T-cells in SKOV3 tumors on day 18 after tumor inoculation. Mice were injected with 6 x 106 CAR T-cells on day 12 and treated with PBS or CD13-AFR


( E ) B16Bl6 growth change after daily p . l . injection of 30µg mCD8 - AFN , 50µg mCD13 - AFR or a combination thereof . growth is shown as mean TSI + ( n = 6 ) . under graph represents treatment period . ( F ) TSI of tumors of indicated treatment groups at day 19 after inoculation .

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

(E) B16Bl6 tumor growth and body weight change after daily p.l. injection of 30µg mCD8-AFN, 50µg mCD13-AFR or a combination thereof. Tumor growth is shown as mean TSI + SEM (n = 6). The line under the graph represents the treatment period. (F) TSI of individual tumors of the indicated treatment groups at day 19 after tumor inoculation.


( A , B ) B16Bl6 growth after daily p . l . treatment with 7µg mTNF , 30µg hTNF alone or in combination with 10000 IU mIFN - γ in wt C57BL / 6 mice ( A ) or homozygous Flk1 dnIFN - γR1 transgenic mice ( B ) . under graph represents treatment period . growth is depicted as mean TSI , error bars are ( n = 5 ) .

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

(A,B) B16Bl6 tumor growth after daily p.l. treatment with 7µg mTNF, 30µg hTNF alone or in combination with 10000 IU mIFN-γ in wt C57BL/6 mice (A) or homozygous Flk1 dnIFN-γR1 transgenic mice (B). The line under the graph represents the treatment period. Tumor growth is depicted as mean TSI, error bars are SEM (n = 5).


( C ) IL - 8 in HUVEC supernatant after 24h stimulation with indicated of sc hTNF mutant Y87F ( Y87F ) , hCD13 - AFR or wt hTNF , alone or in combination with 200ng / ml hIFN - γ , measured via ELISA .

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

(C) IL-8 concentration in HUVEC supernatant after 24h stimulation with the indicated concentration of sc hTNF mutant Y87F (Y87F), hCD13-AFR or wt hTNF, alone or in combination with 200ng/ml hIFN-γ, measured via ELISA.


( E ) Viability of B16Bl6 parental or mCD20 - expressing after 72h stimulation with wt mIFN - γ or del8 mutant fused to a BcII10 or mCD20 VHH , in presence of 60ng / ml mTNF . viability was measured via an ATP luminescence .

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

(E) Viability of B16Bl6 parental or mCD20-expressing cells after 72h stimulation with wt mIFN-γ or del8 mutant fused to a BcII10 or mCD20 VHH, in the presence of 60ng/ml mTNF. Cell viability was measured via an ATP luminescence assay.


( A , B ) growth , change rectal after daily p . l . treatment with mCD13 - AFR , mCD13 - AFN - II or combination thereof , in B16Bl6 model in C57BL / 6 mice ( A ) or RL model in NSG mice ( B ) . growth is shown as mean TSI , error bars are ( n = 5 ) ( upper panel ) , or as TSI of mice ( lower panel ) .

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

(A,B) Tumor growth, body weight change and rectal body temperature after daily p.l. treatment with mCD13-AFR, mCD13-AFN-II or combination thereof, in the B16Bl6 model in C57BL/6 mice (A) or RL model in NSG mice (B). Tumor growth is shown as mean TSI, error bars are SEM (n = 5) (upper panel), or as TSI of individual mice (lower panel).


( C ) Immunohistochemical for PECAM - 1 ( red ) , Caspase - 3 ( green ) ( blue ) of B16Bl6 tumors after p . l . treatment with 50µg mCD13 - AFR 23 . 8µg mCD13 - AFN - II . Scale bar is 100µm .

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

(C) Immunohistochemical staining for PECAM-1 (red), cleaved Caspase-3 (green) and DNA (blue) of B16Bl6 tumors after p.l. treatment with 50µg mCD13-AFR and 23.8µg mCD13-AFN-II. Scale bar is 100µm.


A : ( PAS , upper panels ) of glomerular lesions ( lower panel ) of kidneys from young aged mice . Original magnification X400 . Scale bar = 20 μm . n = 6 for young old mice , respectively . B : WT1 immunohistochemistry ( upper panels ) of WT1 - positive glomerular ( lower panel ) in kidneys from young aged mice . Original magnification X400 . Scale bar = 20 μm . n = 6 for young old mice , respectively . C : TUNEL ( upper panels ) of TUNEL - positive tubular ( upper panel ) in glomeruli from young aged mice . Panels are representative of 6 young old mice , respectively . Scale bar = 20 μm . D : Senescence - associated β - galactosidase ( upper panels ) of β - galactosidase - positive glomeruli ( lower panel ) in kidneys from young aged mice . Original magnification X400 . Scale bar = 20 μm . Panels are representative of 4 young old mice , respectively . Data information : Data are means ± . Statistical : t - student : young versus old mice .

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

A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively. B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive tubular cells (upper panel) in glomeruli from young and aged mice. Panels are representative images of 6 young and old mice, respectively. Scale bar = 20 μm. D: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. Panels are representative images of 4 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


E : p21 / griffonia simplicifolia / nephrin coimmunostaining in kidneys from young aged mice ( left panels ) ( right panel ) of p21 - positive endothelial per glomeruli . Original magnification X630 . Scale bar = 10 μm . Panels are representative of 6 young old mice , respectively . Data information : Data are means ± . Statistical : t - student : young versus old mice .

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

E: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys from young and aged mice (left panels) and quantification (right panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


F : 53BP1 / CD34 co - immunostaining in kidneys from young aged mice ( left panels ) ( right panels ) of 53BP1 foci per glomerular endothelial . Original magnification X630 . Scale bar = 10 μm . Panels are representative of 6 young old mice , respectively . Data information : Data are means ± . Statistical : t - student : young versus old mice .

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

F: 53BP1/CD34 co-immunostaining in kidneys from young and aged mice (left panels) and quantification (right panels) of 53BP1 foci per glomerular endothelial cell. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


I : PAI - 1 immunohistochemistry ( left panels ) of PAI - 1 - positive glomeruli ( right panel ) in kidneys from young aged mice . Original magnification X400 . Scale bar = 20 μm . n = 5 for young old mice , respectively . Data information : Data are means ± . Statistical : t - student : young versus old mice .

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

I: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 5 for young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


A : ( PAS , upper panels ) of glomerular lesions ( lower panel ) of kidneys from irradiated ( RT + ) non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 4 n = 5 for RT + RT - , respectively . B : WT1 immunohistochemistry ( upper panels ) of WT1 - positive glomerular ( lower panel ) in kidneys from irradiated ( RT + ) non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 5 for RT + RT - mice , respectively . C : Senescence - associated β - galactosidase ( upper panels ) of β - galactosidase - positive glomeruli ( lower panel ) in kidneys from irradiated ( RT + ) non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 5 for RT + RT - mice , respectively . Data information : Data are means ± . Statistical : t - student : irradiated versus non - irradiated mice .

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

A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 5 for RT+ and RT-, respectively. B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively. C: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


E : p21 / griffonia simplicifolia / nephrin coimmunostaining ( left panels ) of endothelial p21 - positive ( right panel ) in glomeruli of kidneys from irradiated ( RT + ) non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X630 . Scale bar = 10 μm . n = 4 for RT + RT - mice , respectively . Data information : Data are means ± . Statistical : t - student : irradiated versus non - irradiated mice .

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

E: p21/griffonia simplicifolia/nephrin coimmunostaining (left panels) and quantification of endothelial p21-positive cells (right panel) in glomeruli of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


F : 53BP1 / CD34 coimmunostaining ( left panels ) of glomerular endothelial 53BP1 foci ( right panel ) in kidneys from irradiated ( RT + ) non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X630 . Scale bar = 10 μm . n = 4 for RT + RT - mice , respectively . Arrows show 53BP1 foci . Data information : Data are means ± . Statistical : t - student : irradiated versus non - irradiated mice .

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

F: 53BP1/CD34 coimmunostaining (left panels) and quantification of glomerular endothelial cell 53BP1 foci (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively. Arrows show 53BP1 foci. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


H : PAI - 1 immunohistochemistry ( left panels ) of PAI - 1 - positive glomeruli ( right panel ) in kidneys from irradiated ( RT + ) non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . n = 4 for RT + RT - mice , respectively . Data information : Data are means ± . Statistical : t - student : irradiated versus non - irradiated mice .

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

H: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. n = 4 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


A : WT1 ( left upper panels ) p21 ( left middle lower panels ) immunostaining in 4 - , 12 - , 18 - 24 - month - old mice . of WT1 - positive ( right upper panels ) , glomerular tubular p21 - postive ( right lower panels ) . Original magnification X400 . Scale bar = 20 μm . n = 4 for each group . Arrows show positive p21 nuclei , either in glomeruli or in tubules . B : WT1 ( left upper panels ) p21 ( left middle lower panels ) immunostaining in 4 - 12 - month - old irradiated ( RT + ) non - irradiated ( RT - ) mice . of WT1 - positive ( right upper panels ) , glomerular tubular p21 - postive ( right lower panels ) . Original magnification X400 . Scale bar = 20 μm . n = 4 for each group . Arrows show positive p21 nuclei , either in glomeruli or in tubules . Data information : Data are means ± . Statistical : ANOVA followed by Tukey - Kramer .

O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O B-CELL_TYPE 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 B-GENEPROD B-SUBCELLULAR O O O B-TISSUE O O B-TISSUE O O O B-GENEPROD O O O O O B-GENEPROD 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 B-ORGANISM O O B-GENEPROD O O O O O O O O B-CELL_TYPE 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 B-GENEPROD B-SUBCELLULAR O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O

A: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4-, 12-, 18- and 24-month-old mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. B: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4- and 12-month-old irradiated (RT+) and non-irradiated (RT-) mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test.


A : ( PAS , upper panels ) of glomerular lesions ( lower panel ) of kidneys from 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . B : WT1 immunostaining ( upper panels ) of WT1 - positive glomerular ( lower panel ) in 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . C : PAI - 1 immunostaining ( upper panels ) of PAI - 1 - positive glomeruli ( lower panel ) in 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . Data information : Data are means ± . Statistical : t - student : AP + versus AP - mice .

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

A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. B: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. C: PAI-1 immunostaining (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. Data information: Data are means ± SEM. Statistical analysis: t-student test: AP+ versus AP- mice.


A : PAI - 1 immunohistochemistry ( upper panels ) of PAI - 1 - positive glomeruli ( lower panel ) in kidneys of PAI - 1flox PAI - 1 ∆ endo mice at 22 months of . Original magnification X400 . Scale bar = 20 μm . n = 4 for PAI - 1flox PAI - 1 ∆ endo mice , respectively . B : ( PAS , upper panels ) of glomerular lesions ( lower panel ) of kidneys from PAI - 1flox PAI - 1 ∆ endo mice at 22 months of . Original magnification X400 . Scale bar = 20 μm . n = 12 for PAI - 1flox PAI - 1 ∆ endo mice , respectively . C : TUNEL ( upper panels ) of TUNEL - positive glomeruli ( lower panel ) in kidneys from PAI - 1flox PAI - 1 ∆ endo mice at 22 months of . Original magnification X400 . Scale bar = 20 μm . n = 6 for PAI - 1flox PAI - 1 ∆ endo mice , respectively . D : WT1 immunostaining ( upper panels ) of WT1 - positive glomerular ( lower panel ) in kidneys from PAI - 1flox PAI - 1 ∆ endo mice at 22 months of . Original magnification X400 . Scale bar = 20 μm . n = 4 n = 6 for PAI - 1flox PAI - 1 ∆ endo mice , respectively . Data information : Data are means ± . Statistical : t - student : PAI - 1 ∆ endo versus PAI - 1flox mice .

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

A: PAI-1 immunohistochemistry (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in kidneys of PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 for PAI-1flox and PAI-1∆endo mice, respectively. B: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 12 for PAI-1flox and PAI-1∆endo mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive glomeruli (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. D: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.