figf5 ( a ) Immunoblot analysis of ASC immunoprecipitates ( IP ; left ) and total lysates ( right ) of differentiated THP - 1 cells left untransfected or transfected for 2 h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , probed for beclin - 1 and ASC ( below ) , then stripped and reprobed for p62 ( above ) . ( b ) Confocal microscopy of differentiated THP - 1 cells treated as in a , immunostained for p62 ( green ) and ASC ( red ) ; arrows indicate colocalized proteins . ( c ) Confocal microscopy of differentiated THP - 1 cells treated for 2 h with LPS ( 500 ng / ml ) and ATP ( 3mM ) , immunostained for p62 ( green ) and ASC ( red ) : left two images , merged channel volume and surface rendering of a three - dimensional reconstruction ; right four images , enlargement of images at left ( yellow arrow ) , with arrows indicating removal of part of the signal to reveal the red signal ( ASC ) inside the green - stained area ( p62 ) . ( d ) Immunoblot analysis of lysates of cells transfected twice with siRNA with scrambled sequence ( − ) or siRNA specific for beclin - 1 or p62 ; numbers below lanes indicate band intensity ( as in Fig . 1b ) . ( e ) Immunoblot analysis of the inflammasome and autophagosome fractions and total lysates of differentiated THP - 1 cells transfected as in d , with poly ( dA : dT ) included in the second transfection ; lysates were prepared 4 h after the final transfection . ( f ) Immunoblot analysis of supernatants and lysates of differentiated THP - 1 cells treated as in e ; supernatants were collected after culture for 6 h in serum - free medium . Scale bars ( b , c ) , 10 μm . Data are representative of at least two experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Immunoblot analysis of ASC immunoprecipitates (IP; left) and total lysates (right) of differentiated THP-1 cells left untransfected or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), probed for beclin-1 and ASC (below), then stripped and reprobed for p62 (above).(b) Confocal microscopy of differentiated THP-1 cells treated as in a, immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins.(c) Confocal microscopy of differentiated THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3mM), immunostained for p62 (green) and ASC (red): left two images, merged channel volume and surface rendering of a three-dimensional reconstruction; right four images, enlargement of images at left (yellow arrow), with arrows indicating removal of part of the signal to reveal the red signal (ASC) inside the green-stained area (p62).(d) Immunoblot analysis of lysates of cells transfected twice with siRNA with scrambled sequence (−) or siRNA specific for beclin-1 or p62; numbers below lanes indicate band intensity (as in Fig. 1b).(e) Immunoblot analysis of the inflammasome and autophagosome fractions and total lysates of differentiated THP-1 cells transfected as in d, with poly(dA:dT) included in the second transfection; lysates were prepared 4 h after the final transfection.(f) Immunoblot analysis of supernatants and lysates of differentiated THP-1 cells treated as in e; supernatants were collected after culture for 6 h in serum-free medium. Scale bars (b,c), 10 μm. Data are representative of at least two experiments.


figf6 ( a ) Immunoblot analysis of ASC immunoprecipitates of differentiated THP - 1 cells transfected for 2h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , probed for ubiquitin ( Ub ) , then stripped and reprobed for K63 - linked ubiquitin ( Ub - K63 ) , then stripped and reprobed for ASC . ( b ) Immunoassay of differentiated THP - 1 cells treated as in a with protein - dissociation buffer , followed by immunoprecipitation of ASC ( 24 kilodaltons ( kDa ) ) and immunoblot analysis . ( c ) Confocal microscopy of differentiated THP - 1 cells transfected for 2 h with poly ( dA : dT ) , immunostained for ASC ( red ) and polyubiquitin ( green ) . PolyUb , polyubiquitination . Scale bars , 10 μm . Brackets ( right margins in a , b ) indicate smear of ubiquitinated proteins . Data are representative of three ( a , c ) or two ( b ) experiments .

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figf6(a) Immunoblot analysis of ASC immunoprecipitates of differentiated THP-1 cells transfected for 2h with poly(dA:dT) (1.5 μg/ml), probed for ubiquitin (Ub), then stripped and reprobed for K63-linked ubiquitin (Ub-K63), then stripped and reprobed for ASC.(b) Immunoassay of differentiated THP-1 cells treated as in a with protein-dissociation buffer, followed by immunoprecipitation of ASC (24 kilodaltons (kDa)) and immunoblot analysis.(c) Confocal microscopy of differentiated THP-1 cells transfected for 2 h with poly(dA:dT), immunostained for ASC (red) and polyubiquitin (green). PolyUb, polyubiquitination. Scale bars, 10 μm. Brackets (right margins in a,b) indicate smear of ubiquitinated proteins. Data are representative of three (a,c) or two (b) experiments.


figf7 ( a ) Immunoblot analysis of lysates of human monocytes - macrophages primed with LPS ( 10 ng ) and then transfected with poly ( dA : dT ) ( 1 . 5 μg / ml ) or treated with nigericin ( 4 μM ) for 6 h ( numbers below lanes as in Fig . 1d ) . ( b ) Immunoblot analysis of processed caspase - 1 and IL - 1β ( top ) and enzyme - linked immunosorbent assay of IL - 1β ( middle ) in supernatants of monocytes - macrophages treated for 6 h with various combinations ( bottom ) of poly ( dA : dT ) ( b ) or nigericin ( c ) , plus 3 - MA and amino - acid starvation . * P 0 . 01 ( unpaired t test ) . ( d ) Confocal microscopy of LPS - primed human monocytes - macrophages transfected for 3 h with poly ( dA : dT ) ( top ) , treated for 3 h with nigericin ( middle ) or left unstimulated ( bottom ) , immunostained for p62 ( green ) and ASC ( red ) ; arrows indicate colocalized proteins . Second row , three - dimensional volume rendering reconstructed from confocal z - stack images . Scale bars , 10 μm . Data are representative of at least two experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) Immunoblot analysis of lysates of human monocytes-macrophages primed with LPS (10 ng) and then transfected with poly(dA:dT) (1.5 μg/ml) or treated with nigericin (4 μM) for 6 h (numbers below lanes as in Fig. 1d).(b) Immunoblot analysis of processed caspase-1 and IL-1β (top) and enzyme-linked immunosorbent assay of IL-1β (middle) in supernatants of monocytes-macrophages treated for 6 h with various combinations (bottom) of poly(dA:dT) (b) or nigericin (c), plus 3-MA and amino-acid starvation. *P 0.01 (unpaired t test).(d) Confocal microscopy of LPS-primed human monocytes-macrophages transfected for 3 h with poly(dA:dT) (top), treated for 3 h with nigericin (middle) or left unstimulated (bottom), immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins. Second row, three-dimensional volume rendering reconstructed from confocal z-stack images. Scale bars, 10 μm. Data are representative of at least two experiments.


figf8 ( a ) Immunoblot analysis of lysates of LPS ( 20 ng / ml ) - primed mouse BMDMs exposed to LPS ( 500 ng / ml ) and ATP ( 3 μM ) , poly ( dA : dT ) ( 1 . 5 μg / ml ) or uric acid crystals ( 50 μg / ml ) , detecting GTP - bound RalB ( collected by RaBP1 - agarose affinity purification ) and RalB in total lysates . ( b ) Immunoblot analysis of lysates and supernatants of mouse BMDMs transfected with control or RalB - specific siRNA on 2 sequential days , then primed with LPS and exposed for 6 h to poly ( dA : dT ) ( 1 . 5 μg / ml ) , probed for RalB , LC3 and IL - 1β ; supernatants were collected after overnight culture in serum - free medium ( numbers below lanes as in Fig . 1d ) . Nonsp , nonspecific band . ( c ) Immunoblot analysis of lysates of wild - type and Aim2 − / − mouse BMDMs transfected with control or AIM2 - specific siRNA on 2 sequential days , primed for 2 h with LPS and then exposed for 1 h to poly ( dA : dT ) ( numbers below lanes as in Fig . 1d ) . ( d ) Immunoblot analysis of ASC immunoprecipitates from LPS - primed mouse BMDMs left untreated or transfected for 1 h with poly ( dA : dT ) and treated for 30 min with LPS and ATP . Mock , immunoprecipitation with control antibody . ( e ) Confocal microscopy of LPS - primed mouse BMDMs left untransfected or transfected for 30 min with poly ( dA : dT ) , immunostained for RalB and AIM2 ( left three columns ) ; RalB and ASC ( middle columns ) ; and beclin - 1 and AIM2 ( right two columns ) . Bottom row , 3 × electronic enlargement of areas outlined above . Scale bars , 10 μm . Data are representative of at least two experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8(a) Immunoblot analysis of lysates of LPS (20 ng/ml)-primed mouse BMDMs exposed to LPS (500 ng/ml) and ATP (3 μM), poly(dA:dT) (1.5 μg/ml) or uric acid crystals (50 μg/ml), detecting GTP-bound RalB (collected by RaBP1-agarose affinity purification) and RalB in total lysates.(b) Immunoblot analysis of lysates and supernatants of mouse BMDMs transfected with control or RalB-specific siRNA on 2 sequential days, then primed with LPS and exposed for 6 h to poly(dA:dT) (1.5 μg/ml), probed for RalB, LC3 and IL-1β; supernatants were collected after overnight culture in serum-free medium (numbers below lanes as in Fig. 1d). Nonsp, nonspecific band.(c) Immunoblot analysis of lysates of wild-type and Aim2−/− mouse BMDMs transfected with control or AIM2-specific siRNA on 2 sequential days, primed for 2 h with LPS and then exposed for 1 h to poly(dA:dT) (numbers below lanes as in Fig. 1d).(d) Immunoblot analysis of ASC immunoprecipitates from LPS-primed mouse BMDMs left untreated or transfected for 1 h with poly(dA:dT) and treated for 30 min with LPS and ATP. Mock, immunoprecipitation with control antibody.(e) Confocal microscopy of LPS-primed mouse BMDMs left untransfected or transfected for 30 min with poly(dA:dT), immunostained for RalB and AIM2 (left three columns); RalB and ASC (middle columns); and beclin-1 and AIM2 (right two columns). Bottom row, 3× electronic enlargement of areas outlined above. Scale bars, 10 μm. Data are representative of at least two experiments.


Figure 3D : KD - values of wild - type , mutant and phosphorylated peptides binding to clathrin NTD ( HURP832 - 846 ) , RANBP2 RanBD4 ( DHX38260 - 275 ) and SNX27 PDZ ( CDC25C30 - 44 ) . Significant differences indicated by the Mann - Whitney Test ( * * : p ≤ 0 . 01 ) . Measurements are from two independent protein preparations with two or three technical replicates each . E : Displacement curves of the KPNA4 ARM domain with the RanBP3114 - 129 peptides ( wild - type , phosphomimetic and phosphorylated ) sampling the effects of two distinct phosphosites ( T124 and S125 ) . IC50 - values for the different peptides are indicated in µM and with SEM . Measurements were obtained in technical triplicates .

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Figure 3D: KD-values of wild-type, mutant and phosphorylated peptides binding to clathrin NTD (HURP832-846), RANBP2 RanBD4 (DHX38260-275) and SNX27 PDZ (CDC25C30-44). Significant differences indicated by the Mann-Whitney Test (**: p≤0.01). Measurements are from two independent protein preparations with two or three technical replicates each.E: Displacement curves of the KPNA4 ARM domain with the RanBP3114-129 peptides (wild-type, phosphomimetic and phosphorylated) sampling the effects of two distinct phosphosites (T124 and S125). IC50-values for the different peptides are indicated in µM and with SEM. Measurements were obtained in technical triplicates.


Figure 4B : Displacement curves of the three different bait domains and the indicated peptides using wild - type , phosphomimetic and phosphorylated peptide . IC50 values of the different peptides are indicated in µM and with SEM . Measurements were in technical triplicates .

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Figure 4B: Displacement curves of the three different bait domains and the indicated peptides using wild-type, phosphomimetic and phosphorylated peptide. IC50 values of the different peptides are indicated in µM and with SEM. Measurements were in technical triplicates.


Figure 6A : Displacement curves of the FP experiment using clathrin NTD and wild - type , phosphomimetic and phosphorylated HURP peptides . IC50 values are indicated in in µM and with SEM . Measurements were in four to five technical replicates . F : Co - IP of clathrin with Venus - tagged HURPWT and HURP ∆ MUT constructs in asynchronous and mitotic HeLa cells . G Co - IP of clathrin with Venus - tagged HURPWT , HURP ∆ MUT and HURPS839A constructs in mitotic HeLa cells . H : Representative images of HURPWT , HURP ∆ MUT and HURPS839A in the different mitotic stages . I - J : RNAi rescue experiments ( n = 2 ) , which included the knockdown of endogenous HURP and re - expressing either RNAi resistant HURPWT , HURP ∆ MUT and HURPS839A . In order to assess mitotic behaviour of the cells , the time required of control , HURP knockdown , HURPWT , HURP ∆ MUT and HURPS839A HeLa cells to progress through mitosis ( NEBD - anaphase ) was established using time - lapse microscopy . Every circle represents one analysed cell . The number of cells analysed is indicated as n in I . Mann - Whitney test was performed to assess significant differences in the median time required for mitotic progression ( * : p - value ≤ 0 . 05 , * * * : p - value : ≤ 0 . 001 , * * * * : p - value ≤ 0 . 0001 ; ns : non - significant )

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Figure 6A: Displacement curves of the FP experiment using clathrin NTD and wild-type, phosphomimetic and phosphorylated HURP peptides. IC50 values are indicated in in µM and with SEM. Measurements were in four to five technical replicates.F: Co-IP of clathrin with Venus-tagged HURPWT and HURP∆MUT constructs in asynchronous and mitotic HeLa cells.G Co-IP of clathrin with Venus-tagged HURPWT, HURP∆MUT and HURPS839A constructs in mitotic HeLa cells.H: Representative images of HURPWT, HURP∆MUT and HURPS839A in the different mitotic stages.I-J: RNAi rescue experiments (n=2), which included the knockdown of endogenous HURP and re-expressing either RNAi resistant HURPWT, HURP∆MUT and HURPS839A. In order to assess mitotic behaviour of the cells, the time required of control, HURP knockdown, HURPWT, HURP∆MUT and HURPS839A HeLa cells to progress through mitosis (NEBD-anaphase) was established using time-lapse microscopy. Every circle represents one analysed cell. The number of cells analysed is indicated as n in I. Mann-Whitney test was performed to assess significant differences in the median time required for mitotic progression (*: p-value≤0.05, ***: p-value: ≤0.001, ****: p-value ≤0.0001; ns: non-significant)


Figure 2 ( A ) Plot from flow cytometry analysis of neutrophils in forepaw skin from 10 - wk - old WT and RDEB mice . ( B ) Bar graph of flow cytometry analyses as in A of neutrophils in forepaw skin of newborn , 4 - wk - old and 10 - wk - old WT and RDEB mice . Plotted as fold increase compared to mean of age - matched WT paws ; replicates per age = 5 - 6 . ( Data information : Individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction P values < 0 . 05 are considered significant . ( C ) Plot from flow cytometry analysis of CD38 + and Egr2 + macrophages in forepaw skin from 10 - wk - old WT and RDEB mice . ( D ) Bar graph of flow cytometry analyses as in C of inflammatory macrophages ( CD38 + Egr2 - ) or tissue - repair macrophages ( CD38 - Egr2 + ) macrophages in forepaw skin of newborn , 4 - wk - old and 10 - wk - old WT and RDEB mice . Plotted as fold increase compared to mean of age - matched WT paws ; replicates per age ; n = 5 - 6 ( CD38 + Egr2 - ) ; n = 4 - 5 ( CD38 - Egr2 + ) . ( Data information : Individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction P values < 0 . 05 are considered significant . ( F - G ) The mean fluorescence intensity ( MFI ) of MHC II staining on ( F ) neutrophils ( n = 3 - 4 biological replicates ) and ( G ) macrophages ( n = 6 - 7 biological replicates ) . Data information : Individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction P values < 0 . 05 are considered significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Plot from flow cytometry analysis of neutrophils in forepaw skin from 10-wk-old WT and RDEB mice. (B) Bar graph of flow cytometry analyses as in A of neutrophils in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age = 5-6. ( Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.(C) Plot from flow cytometry analysis of CD38+ and Egr2+ macrophages in forepaw skin from 10-wk-old WT and RDEB mice. (D) Bar graph of flow cytometry analyses as in C of inflammatory macrophages (CD38+Egr2-) or tissue-repair macrophages (CD38-Egr2+) macrophages in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age; n = 5-6 (CD38+Egr2-); n = 4-5 (CD38-Egr2+). ( Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.(F-G) The mean fluorescence intensity (MFI) of MHC II staining on (F) neutrophils (n = 3-4 biological replicates) and (G) macrophages (n = 6-7 biological replicates). Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.


Figure 3 ( A - D ) Skin from healthy young ( 3 - 8 years old ) and adult donors ( > 25 years old ) and from donors with RDEB with different stages of fibrosis - early ( 0 - 3 years old ) , mild ( 6 - 10 years old ) and advanced ( > 15 years old ) - stained for CD68 ( green ) , CD3ε ( green ) , periostin ( green ) and tenascin - C ( red ) . Nuclei were counterstained with DAPI ( blue ) . A and B , scale bar = 50 μm ; C and D , scale bar = 100 μm . The bar graphs at the right to the stained sections , represent for A and B quantification of the number positive cells per mm2 analyzed from 3 donors for each group and for C and D , the mean intensity of staining from 3 - 6 donors per group . Individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction . P values < 0 . 05 are considered significant .

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Figure 3(A-D) Skin from healthy young (3-8 years old) and adult donors ( > 25 years old) and from donors with RDEB with different stages of fibrosis - early (0-3 years old), mild (6-10 years old) and advanced ( > 15 years old) - stained for CD68 (green), CD3ε (green), periostin (green) and tenascin-C (red). Nuclei were counterstained with DAPI (blue). A and B, scale bar = 50 μm; C and D, scale bar = 100 μm. The bar graphs at the right to the stained sections, represent for A and B quantification of the number positive cells per mm2 analyzed from 3 donors for each group and for C and D, the mean intensity of staining from 3-6 donors per group. Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction. P values < 0.05 are considered significant.


Figure 4 ( A ) Human dermal RDEBF treated daily with 10 μM LY2109761 ( LY ) ( TGF - β Receptor I / II inhibitor ) , 10 μM 1D11 ( neutralizing antibody to all TGF - βs ) 100 nM Ang - ( 1 - 7 ) , or 10 μM losartan ( AT1R antagonist ) . After five days of treatment cell and matrix lysates were extracted and analyzed by western blotting for the indicated proteins , THBS - 1 , thrombospondin - 1 , β - tubulin was used as loading control . ( B ) Densitometric quantification of blots as in A . The abundance was normalized to β - tubulin and then expressed as % of untreated ( - ) . P values are shown ; the data were analyzed with one - way ANOVA with Dunnett ' s correction ( n = 5 biological replicates ) . ( Data information : P values < 0 . 05 are considered significant and shown , ns = not significant . individual data points , mean ± S . E . M are shown . ( F ) Western blots of fibroblast lysates from co - cultures as described in E . The blots were probed with antibodies detecting fibronectin , thrombospondin - 1 ( THBS - 1 ) and pSMAD - 2 / 3 . β - tubulin was used as loading control . ( G ) Densitometric quantification of western blots of fibroblast cell and matrix lysates as shown in F from multiple independent experiments , n = 4 - 5 using cells from 4 donors with RDEB . The abundance was normalized to β - tubulin then and then expressed as % of both untreated . The data were analyzed by one - way ANOVA with Tukey ' s correction . Data information : P values < 0 . 05 are considered significant and shown , ns = not significant . For B , D and G individual data points , mean ± S . E . M are shown .

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Figure 4(A) Human dermal RDEBF treated daily with 10 μM LY2109761 (LY) (TGF-β Receptor I/II inhibitor), 10 μM 1D11 (neutralizing antibody to all TGF-βs) 100 nM Ang-(1-7), or 10 μM losartan (AT1R antagonist). After five days of treatment cell and matrix lysates were extracted and analyzed by western blotting for the indicated proteins, THBS-1, thrombospondin-1, β-tubulin was used as loading control. (B) Densitometric quantification of blots as in A. The abundance was normalized to β-tubulin and then expressed as % of untreated (-). P values are shown; the data were analyzed with one-way ANOVA with Dunnett's correction (n = 5 biological replicates). ( Data information: P values < 0.05 are considered significant and shown, ns = not significant. individual data points, mean ± S.E.M are shown.(F) Western blots of fibroblast lysates from co-cultures as described in E. The blots were probed with antibodies detecting fibronectin, thrombospondin-1 (THBS-1) and pSMAD-2/3. β-tubulin was used as loading control. (G) Densitometric quantification of western blots of fibroblast cell and matrix lysates as shown in F from multiple independent experiments, n = 4 -5 using cells from 4 donors with RDEB. The abundance was normalized to β-tubulin then and then expressed as % of both untreated. The data were analyzed by one-way ANOVA with Tukey's correction. Data information: P values < 0.05 are considered significant and shown, ns = not significant. For B, D and G individual data points, mean ± S.E.M are shown.


Figure 5 ( B ) Kaplan Meier survival curve of mice treated as in A , P values as indicated , the data were tested with log - rank test - PBS treated vs . 0 . 1 mg / kg Ang - ( 1 - 7 ) treated and PBS treated vs . 1 . 0 mg / kg Ang - ( 1 - 7 ) treated ; n = 12 - 15 mice as indicated in the figure . ( C ) Photos of left and right forepaws of RDEB mice before and after 7 weeks of daily injections with 0 . 1 , 1 . 0 mg / kg Ang - ( 1 - 7 ) or PBS . ( D ) Plot of numbers of toes lost during the 7 - week observation period , data tested with Kruskal - Wallis test , P values indicated , n = 20 - 24 paws as indicated in the figure . Median and range are shown . (

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Figure 5(B) Kaplan Meier survival curve of mice treated as in A, P values as indicated, the data were tested with log-rank test - PBS treated vs. 0.1 mg/kg Ang-(1-7) treated and PBS treated vs. 1.0 mg/kg Ang-(1-7) treated; n = 12-15 mice as indicated in the figure.(C) Photos of left and right forepaws of RDEB mice before and after 7 weeks of daily injections with 0.1, 1.0 mg/kg Ang-(1-7) or PBS. (D) Plot of numbers of toes lost during the 7-week observation period, data tested with Kruskal-Wallis test, P values indicated, n= 20-24 paws as indicated in the figure. Median and range are shown. (


Figure 6 ( A ) Haemotoxylin and Eosin ( H & E ) staining of toes from forepaws from WT and RDEB mice treated with daily injections of 0 . 1 , 1 . 0 mg / kg Ang - ( 1 - 7 ) or PBS for seven weeks . Inflammatory cells are elevated in the toes from the PBS and the two Ang - ( 1 - 7 ) treated RDEB mice compared to the WT counterpart . Scale bar = 100 μm . Data information scale bar = 100 μm . ( B - D ) Flow cytometry analysis of the number of CD45 positive cells ( B ) , percentage of neutrophils ( C ) and of inflammatory macrophages ( CD38 + Egr2 - ) and tissue - repair macrophages ( CD38 - Egr2 + ) ( D ) in forepaw skin of RDEB mice treated as in A . Data information : individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction , P values < 0 . 05 are considered significant and shown ; n = 5 - 14 biological replicates . ( F ) Staining for IgG ( red ) in forepaws , nuclei counterstained with DAPI . ( G ) Quantification of fluorescence staining intensity of samples as in F . Data information : scale bar = 100 μm . , individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction , P values < 0 . 05 are considered significant and shown ; n = 5 - 14 biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Haemotoxylin and Eosin (H&E) staining of toes from forepaws from WT and RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Inflammatory cells are elevated in the toes from the PBS and the two Ang-(1-7) treated RDEB mice compared to the WT counterpart. Scale bar = 100 μm. Data information scale bar = 100 μm.(B-D) Flow cytometry analysis of the number of CD45 positive cells (B), percentage of neutrophils (C) and of inflammatory macrophages (CD38+Egr2-) and tissue-repair macrophages (CD38-Egr2+) (D) in forepaw skin of RDEB mice treated as in A. Data information: individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates.(F) Staining for IgG (red) in forepaws, nuclei counterstained with DAPI. (G) Quantification of fluorescence staining intensity of samples as in F. Data information: scale bar = 100 μm. , individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates.


Figure 7 ( A ) Picrosirius red staining visualized under polarized light and Elastica van Gieson ( EvG ) staining ) of forepaw toes from 12 - wk - old WT or RDEB mice treated with daily injections of 0 . 1 , 1 . 0 mg / kg Ang - ( 1 - 7 ) or PBS for seven weeks . Note the reduced signal for picrosirus red in RDEB mice treated with 0 . 1 mg / kg Ang - ( 1 - 7 ) indicating less parallel alignment of and / or thinner collagen fibrils , and additionally the more organized and less disrupted appearance of elastic fibers ( black in the EvG staining ) . ( B ) Quantification of picrosirius red stained forepaws visualized as in A . A digital threshold was applied to the red channel before quantification . The data were analyzed by one - way ANOVA with Tukey ' s correction , P values < 0 . 05 are considered significant ; n = 10 - 15 . Individual data points , mean ± S . E . M are shown . ( Data information scale bar = 100 μm ( C ) Western blotting for the indicated proteins on tissue lysates from forepaws from RDEB mice treated as in A . α - SMA , α - smooth muscle actin ; Thbs - 1 , thrombospondin - 1 . Data information : β - actin or β - tubulin was used as loading control ( D ) Dermal β - arrestin - 1 / 2 ( red ) and NFκB ( red ) staining in forepaws from WT and RDEB mice treated as in A . Nuclei counterstained with DAPI ( blue ) . Data information scale bar = 20 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Picrosirius red staining visualized under polarized light and Elastica van Gieson (EvG) staining) of forepaw toes from 12-wk-old WT or RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Note the reduced signal for picrosirus red in RDEB mice treated with 0.1 mg/kg Ang-(1-7) indicating less parallel alignment of and/or thinner collagen fibrils, and additionally the more organized and less disrupted appearance of elastic fibers (black in the EvG staining). (B) Quantification of picrosirius red stained forepaws visualized as in A. A digital threshold was applied to the red channel before quantification. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant; n = 10-15. Individual data points, mean ± S.E.M are shown. ( Data information scale bar = 100 μm(C) Western blotting for the indicated proteins on tissue lysates from forepaws from RDEB mice treated as in A. α-SMA, α-smooth muscle actin; Thbs-1, thrombospondin-1. Data information: β- actin or β-tubulin was used as loading control(D) Dermal β-arrestin-1/2 (red) and NFκB (red) staining in forepaws from WT and RDEB mice treated as in A. Nuclei counterstained with DAPI (blue). Data information scale bar = 20 μm.


Figure 1 ( A ) Mouse embryos were immunostained with anti - neurofilament antibody ( blue ) , showing mesencephalic projection . Dispersed axon projections ( arrows point to the area of the superior colliculus ) and decreased axon fibers ( arrowheads point to the area of the pretectal commissure ) were observed in Plcg1f / f ; Nes - Cre mice at E11 . 5 ( n = 20 ) . The areas of the superior colliculus is shown magnified in each inset ( scale bar = 200 μm ) ( B ) Representative images between the posterior and anterior borderlines of the superior colliculus ( a and b ) . The red boxes were drawn as rostrocaudal region of interest ( ROI ) based on the upper part of the pretectal commissure in the embryonic mesencephalon ( scale bar = 100 μm ) ( C and D ) The axons passing through the ROI was quantified by measuring the fluorescence intensity in the ROI , and the distribution of axons was statistically analyzed ( n = 5 ; t - test ; * * P < 0 . 005 ) ( E ) Whole - mount immunostaining images show neurofilament ( red ) , netrin - 1 ( purple ) , and DCC ( green ) , respectively . An E11 . 5 Plcg1f / f ; Nes - Cre embryo showed shortened dorsal ramus and opthalmic nerves ( white and yellow boxes ) . Scale bar : 200 μm ( F - G ) Enlarged views of the opthalmic nerve ( yellow arrow heads ) and dorsal ramus ( white arrow heads ) ( scale bar : 50 μm ) ( H ) Nerve length measurements ; Op : ophthalmic nerve ; DR : dorsal ramus ( n = 3 ; t - test ; * * P < 0 . 005 ) . Data are presented as the mean ± s . e . m ( I ) Double immunostaining images . The pattern of expression and colocalization of DCC ( green ) - and TH ( red ) - positive axon bundles extending toward the alar thalamus . Scale bar : 100

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Mouse embryos were immunostained with anti-neurofilament antibody (blue), showing mesencephalic projection. Dispersed axon projections (arrows point to the area of the superior colliculus) and decreased axon fibers (arrowheads point to the area of the pretectal commissure) were observed in Plcg1f/f;Nes-Cre mice at E11.5 (n = 20). The areas of the superior colliculus is shown magnified in each inset (scale bar = 200 μm)(B) Representative images between the posterior and anterior borderlines of the superior colliculus (a and b). The red boxes were drawn as rostrocaudal region of interest (ROI) based on the upper part of the pretectal commissure in the embryonic mesencephalon (scale bar = 100 μm)(C and D) The axons passing through the ROI was quantified by measuring the fluorescence intensity in the ROI, and the distribution of axons was statistically analyzed (n = 5; t-test; **P < 0.005)(E) Whole-mount immunostaining images show neurofilament (red), netrin-1 (purple), and DCC (green), respectively. An E11.5 Plcg1f/f;Nes-Cre embryo showed shortened dorsal ramus and opthalmic nerves (white and yellow boxes). Scale bar: 200 μm(F - G) Enlarged views of the opthalmic nerve (yellow arrow heads) and dorsal ramus (white arrow heads) (scale bar: 50 μm)(H) Nerve length measurements; Op: ophthalmic nerve; DR: dorsal ramus (n = 3; t-test; **P < 0.005). Data are presented as the mean ± s.e.m(I) Double immunostaining images. The pattern of expression and colocalization of DCC (green)- and TH (red)-positive axon bundles extending toward the alar thalamus. Scale bar: 100 μm


Figure 2 ( A ) In primary mesencephalic neurons , the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin - 1 concentration . The lower graph presents quantification of the band intensities in ( A ) ( n = 3 ) ( B ) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA ( siDcc ) ( N = 3 ) ( C ) Western blot for the NTN1 secretion test . Lane # 1 : transfection - free lysate , # 2 : transfection - free supernatant , # 3 : NTN1 - transfected cell lyasate , # 3 and # 4 : NTN - 1 - transfected supernatant ( D ) Ventral mesencephalic ( A9 ) cultures ( daggers ) derived from control or Plcg1f / f ; Nes - Cre mice , were co - cultured with netrin - 1 - releasing 293T cell aggregates ( asterisks ; scale bar = 100 μm ) . The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets ( n = 4 ) ( F ) Mean angle of the axon extension ( G ) Quantification of the ventral mesencephalic axons . The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin - 1 - releasing cell aggregates ( n = 200 ) . Each experimental set was performed in quadruplicate ( H ) Anti - tyrosine hydroxylase ( TH ) immunostaining in ventral mesencephalic ( VM ) tissue . ( scale bar = 200 μm ) ( I ) Primary mesencephalic neuron culture in the absence or presence of NTN1 - conditioned media ( n = 100 ; scale bar : 500μm ) ( K ) Mean length of neurites ( F - value : 1 . 82 , t - test , P * * < 0 . 005 )

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Figure 2(A) In primary mesencephalic neurons, the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin-1 concentration. The lower graph presents quantification of the band intensities in (A) (n = 3)(B) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA (siDcc) (N = 3)(C) Western blot for the NTN1 secretion test. Lane #1: transfection-free lysate, #2: transfection-free supernatant, #3: NTN1-transfected cell lyasate, #3 and #4: NTN-1- transfected supernatant(D) Ventral mesencephalic (A9) cultures (daggers) derived from control or Plcg1f/f;Nes-Cre mice, were co-cultured with netrin-1-releasing 293T cell aggregates (asterisks; scale bar = 100 μm). The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets (n = 4)(F) Mean angle of the axon extension(G) Quantification of the ventral mesencephalic axons. The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin-1-releasing cell aggregates (n = 200). Each experimental set was performed in quadruplicate(H) Anti-tyrosine hydroxylase (TH) immunostaining in ventral mesencephalic (VM) tissue. (scale bar = 200 μm)(I) Primary mesencephalic neuron culture in the absence or presence of NTN1-conditioned media (n = 100; scale bar: 500μm)(K) Mean length of neurites (F-value:1.82, t-test, P**<0.005)


Figure 3 ( A ) Co - immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor ( n = 2 , N = 3 ) . ( B ) Immunoprecipitation assay showed that Src kinase interacted with PLCγ1 . Rac1 is a positive interacting control for PLCγ1 ( N = 3 ) . ( C ) In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1 . ( D ) Quantification of the band intensities in ( C ) ( N = 3 ) . ( E ) Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 ( arrow ; Src I IC50 conc = 44 nM ; N = 3 ) . ( F ) Rac1 activity assay ( N = 3 ) . ( G ) Quantification of Rac1 activity levels in ( F ) . ( H ) Representative immunoblot of cultured Plcg1f / f ( n = 2 , N = 3 ) and Plcg1f / f ; Nes - Cre ( n = 2 , N = 3 ) mesencephalic neurons stimulated with conditioned media containing Netrin - 1 .

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Figure 3(A) Co-immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor (n = 2, N = 3).(B) Immunoprecipitation assay showed that Src kinase interacted with PLCγ1. Rac1 is a positive interacting control for PLCγ1 (N = 3).(C) In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1. (D) Quantification of the band intensities in (C) (N = 3).(E) Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 (arrow; Src I IC50 conc = 44 nM; N = 3).(F) Rac1 activity assay (N = 3). (G) Quantification of Rac1 activity levels in (F).(H) Representative immunoblot of cultured Plcg1f/f (n = 2, N = 3) and Plcg1f/f;Nes-Cre (n = 2, N = 3) mesencephalic neurons stimulated with conditioned media containing Netrin-1.


Figure 4 ( B ) Immunohistochemical staining of control and Plcg1 conditional knockout ( cKO ) sagittal brain sections . The deletion of Plcg1 caused structural changes in SI and OT projections in the Plcg1f / f ; Nes - Cre ( d ) mice compared to the controls ( a ) ( n = 4 ; scale bar = 1 mm ) . The areas delineated by the white boxes ( insets ) are magnified images of the OT ( b and e ) , and the yellow boxes are magnified images of the SI ( c and f ) . ( C ) Quantification of the axon bundle distribution . Green boxes in ( b ) and ( e ) were selected as tegions of interest ( ROIs ) using the ZEN software ( n = 4 ) ( one - way ANOVA , F - value : 118 . 5745 , P * * < 0 . 005 ) . ( D ) Representative images of tyrosine hydroxylase ( TH ) - positive neurons ( scale bar = 1 mm ) . The areas delineated by the white boxes are magnified in the insets . The yellow arrow indicated misoriented TH - positive axons ( magenta : TH ) . ( E ) Fluorescence intensities in the ROIs were measured according to distance ( n = 4 ) ( one - way ANOVA , F - value : 271 . 8 , * * P < 0 . 005 ) . Cumulative values of fluorescence intensities per section are shown in the lower graph ( S : section ) . ( F ) In situ hybridization of PLCγ1 in mDA neurons ( scale bar = 50 μm ) ( green : TH , violet : DIG - labeled probe ) . ( G ) Representative images of the CC axon bundles in control ( left ) and a Plcg1f / f ; Nes - Cre ( right ) mice ( n = 4 ) . The tip of the CC was triangular in the control mouse , but oval in the Plcg1f / f ; Nes - Cre mouse . The areas delineated by the white boxes are magnified in the insets ( red : neurofilament , green : glial fibrillary acidic protein , blue : Hoechst ; scale bar = 1 mm ) . ( H ) The area of the CC in the Plcg1f / f ; Nes - Cre mouse was 36 % smaller than that in the control mouse ( scale bar = 500 μm ) ( I ) Quantitative comparison of the CC areas in the control and Plcg1f / f ; Nes - Cre mice ( n = 4 ; t - test ; * * p < 0 . 005 ) . Data are presented as the mean ± s . e . m .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Immunohistochemical staining of control and Plcg1 conditional knockout (cKO) sagittal brain sections. The deletion of Plcg1 caused structural changes in SI and OT projections in the Plcg1f/f;Nes-Cre (d) mice compared to the controls (a) (n = 4; scale bar = 1 mm). The areas delineated by the white boxes (insets) are magnified images of the OT (b and e), and the yellow boxes are magnified images of the SI (c and f). (C) Quantification of the axon bundle distribution. Green boxes in (b) and (e) were selected as tegions of interest (ROIs) using the ZEN software (n = 4) (one-way ANOVA, F-value: 118.5745, P**<0.005).(D) Representative images of tyrosine hydroxylase (TH)- positive neurons (scale bar = 1 mm). The areas delineated by the white boxes are magnified in the insets. The yellow arrow indicated misoriented TH-positive axons (magenta: TH).(E) Fluorescence intensities in the ROIs were measured according to distance (n = 4) (one-way ANOVA, F-value: 271.8, **P<0.005). Cumulative values of fluorescence intensities per section are shown in the lower graph (S: section).(F) In situ hybridization of PLCγ1 in mDA neurons (scale bar = 50 μm) (green: TH, violet: DIG-labeled probe).(G) Representative images of the CC axon bundles in control (left) and a Plcg1f/f;Nes-Cre (right) mice (n = 4). The tip of the CC was triangular in the control mouse, but oval in the Plcg1f/f;Nes-Cre mouse. The areas delineated by the white boxes are magnified in the insets (red: neurofilament, green: glial fibrillary acidic protein, blue: Hoechst; scale bar = 1 mm).(H) The area of the CC in the Plcg1f/f;Nes-Cre mouse was 36% smaller than that in the control mouse (scale bar = 500 μm) (I) Quantitative comparison of the CC areas in the control and Plcg1f/f;Nes-Cre mice (n = 4; t-test; **p < 0.005). Data are presented as the mean ± s.e.m.


Figure 5 ( A ) Cumulative histograms based on fractional anisotropy ( FA ) values . ( B ) , show the CC volume relative to FA values . As FA values increased , the structural ( CC volume differences increased between the control and Plcg1f / f ; Nes - Cre mice . ( n = 4 ; Shapiro - Wilk and t - test ; * p < 0 . 05 ) . Data are presented as the mean ± s . e . m . ( C ) , Cumulative histograms based on fractional anisotropy ( FA ) values . ( D ) , show the number of tensors relative to FA values . As FA values increased , the structural number of tensors ) differences increased between the control and Plcg1f / f ; Nes - Cre mice . ( n = 4 ; Shapiro - Wilk and t - test ; * p < 0 . 05 ) . Data are presented as the mean ± s . e . m . ( E ) Cumulative histograms based on fractional anisotropy ( FA ) values . ( F ) show the CC volume and the number of tensors relative to FA values . As FA values increased , the structural ( CC volume and the number of tensors ) differences increased between the control and Plcg1f / f ; Nes - Cre mice . Depending on the number of tensors , the length in the CC appeared to be shortened ( E , F ) . ( n = 4 ; Shapiro - Wilk and t - test ; * p < 0 . 05 ) . Data are presented as the mean ± s . e . m .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Cumulative histograms based on fractional anisotropy (FA) values. (B), show the CC volume relative to FA values. As FA values increased, the structural (CC volume differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.(C), Cumulative histograms based on fractional anisotropy (FA) values. (D), show the number of tensors relative to FA values. As FA values increased, the structural number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.(E) Cumulative histograms based on fractional anisotropy (FA) values. (F) show the CC volume and the number of tensors relative to FA values. As FA values increased, the structural (CC volume and the number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. Depending on the number of tensors, the length in the CC appeared to be shortened (E, F). (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.


Figure 1B Induction of NF - κB targets in Jurkat T cells in response to 20 ng / ml TNF treatment for 1 , 2 , and 4 hours as measured by RT - qPCR . Target values were normalized to GAPDH and are reported as fold change relative to basal expression . Data are presented as mean ± standard deviation ( SD ) of 3 biological replicates . D Maximum intensity projections of smFISH fluorescence microscopy z - stacks of basal Jurkat T cells stained for the indicated genes . Nfkbia , Tnfaip3 , Tnf , and Il6 were labeled with Quasar 670 , and Il8 and Csf2 were labeled with fluorescein . All images were filtered as described in Materials and Methods . Brightness and contrast were enhanced for visualization . Scale bars : 10 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B Induction of NF-κB targets in Jurkat T cells in response to 20 ng/ml TNF treatment for 1, 2, and 4 hours as measured by RT-qPCR. Target values were normalized to GAPDH and are reported as fold change relative to basal expression. Data are presented as mean ± standard deviation (SD) of 3 biological replicates.D Maximum intensity projections of smFISH fluorescence microscopy z-stacks of basal Jurkat T cells stained for the indicated genes. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm.


Figure 2A Maximum intensity projections of smFISH fluorescence microscopy z - stacks of Jurkat T cells stained for the indicated genes after 1 - hour ( Nfkbia , Tnfaip3 , Tnf , Il8 ) or 4 - hour ( Il6 , Csf2 ) treatment with 20 ng / mL TNF . Nfkbia , Tnfaip3 , Tnf , and Il6 were labeled with Quasar 670 , and Il8 and Csf2 were labeled with fluorescein . All images were filtered as described in Materials and Methods . Brightness and contrast were enhanced for visualization . Scale bars : 10 μm . B Bar graphs of mean of smFISH distributions before and after TNF treatment for the indicated genes . Cells were combined from three replicates ( Nfkbia 1h ; Tnfaip3 1h ; Tnf 1h , 2h ; and Il6 2h , 4h ) two replicates ( Nfkbia 2h ) or one replicate ( Tnfaip3 2h ; Il8 1h , 2h ; Csf2 2h , 4h ) . Basal data are same as in Fig . 1E . Error bars indicate bootstrapped 95 % CIs for the samples in ( C ) . Significant differences indicated by non - overlapping CIs . C

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Maximum intensity projections of smFISH fluorescence microscopy z-stacks of Jurkat T cells stained for the indicated genes after 1-hour (Nfkbia, Tnfaip3, Tnf, Il8) or 4-hour (Il6, Csf2) treatment with 20 ng/mL TNF. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm. B Bar graphs of mean of smFISH distributions before and after TNF treatment for the indicated genes. Cells were combined from three replicates (Nfkbia 1h; Tnfaip3 1h; Tnf 1h, 2h; and Il6 2h, 4h) two replicates (Nfkbia 2h) or one replicate (Tnfaip3 2h; Il8 1h, 2h; Csf2 2h, 4h). Basal data are same as in Fig. 1E. Error bars indicate bootstrapped 95% CIs for the samples in (C). Significant differences indicated by non-overlapping CIs. C


Figure 3B - C Burst size ( B ) and burst frequency ( C ) parameter fits from the two - state model in the basal state and after treatment with 20 ng / mL TNF for 1 , 2 or 4 hours . Error bars indicate bootstrapped 95 % CIs . Significant differences indicated by non - overlapping CIs . The fit for Nfkbia ( 1h TNF ) was unstable and thus is not

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Figure 3B-C Burst size (B) and burst frequency (C) parameter fits from the two-state model in the basal state and after treatment with 20 ng/mL TNF for 1, 2 or 4 hours. Error bars indicate bootstrapped 95% CIs. Significant differences indicated by non-overlapping CIs. The fit for Nfkbia (1h TNF) was unstable and thus is not reported


Figure 4A Enrichment of histone H3 , AcH3 , total RNPII , ser5 - p RNPII , ser2 - p RNPII , and NELF - E in the basal state ( 0 hours ) and after treatment ( 2 and 4 hours ) with 20 ng / ml TNF quantified using ChIP and shown as % input ( non - IP control ) . Data are presented as mean ± standard error of the mean ( s . e . m . ) of three biological replicates . Significance calculated by Dunnett ' s multiple comparison test ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) .

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Figure 4A Enrichment of histone H3, AcH3, total RNPII, ser5-p RNPII, ser2-p RNPII, and NELF-E in the basal state (0 hours) and after treatment (2 and 4 hours) with 20 ng/ml TNF quantified using ChIP and shown as % input (non-IP control). Data are presented as mean ± standard error of the mean (s.e.m.) of three biological replicates. Significance calculated by Dunnett's multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).


Figure 5C Bar graphs of mean mRNA level for basal condition , 1h TNF , 1h TNF + 4h pretreatment with 300nM A - 485 , and 1h TNF + 62 . 5nM JQ1 cotreatment measured by smFISH for Tnfaip3 ( left ) and Tnf ( right ) . Cells were combined from two replicates ( Tnfaip3 A - 485 , JQ1 ; Tnf A - 485 ) or one replicate ( Tnf JQ1 ) . Error bars indicated bootstrapped 95 % CIs . Samples with non - overlapping CIs are significant . D Change in enrichment of total and Serine - 5 - phosphorylated RNAPII after 20ng / mL TNF treatment for 1 hour with 62 . 5nM JQ1 measured by ChIP - qPCR and shown as % input ( non - IP control ) normalized to the uninhibited control for each gene . Data are presented as mean for two biological replicates . E

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5C Bar graphs of mean mRNA level for basal condition, 1h TNF, 1h TNF + 4h pretreatment with 300nM A-485, and 1h TNF+62.5nM JQ1 cotreatment measured by smFISH for Tnfaip3 (left) and Tnf (right). Cells were combined from two replicates (Tnfaip3 A-485, JQ1; Tnf A-485) or one replicate (Tnf JQ1). Error bars indicated bootstrapped 95% CIs. Samples with non-overlapping CIs are significant. D Change in enrichment of total and Serine-5-phosphorylated RNAPII after 20ng/mL TNF treatment for 1 hour with 62.5nM JQ1 measured by ChIP-qPCR and shown as % input (non-IP control) normalized to the uninhibited control for each gene. Data are presented as mean for two biological replicates. E


Figure 1Ratiometric and mTFP1 donor images of EKAR2G in an EGF ‐ stimulated PC ‐ 12 cell at the indicated time points , with t = 0 ' corresponding to EGF application . Upper panel : FRET ratio image is color ‐ coded for ERK activity . Lower panel : raw mTFP1 donor image in black / white contrast . Scale bar = 20 μm . Population average of ERK activation dynamics measured by Western blot using a phosphoERK antibody . Population average of ERK activation dynamics cell ‐ averaged EKAR2G emission ratios ( ERs ) from n = at least 111 cells . StDev are shown . Selected EKAR2G ratio time series illustrating , from top to bottom , sustained , oscillatory , or transient ERK activity dynamics . ER is color ‐ coded as in bar . Scale bar = 20 μm . Single ‐ cell ERK activity trajectories . Cell ‐ averaged ERs for n = 10 cells , standard deviation range ( StDev ) , population average for the indicated GFs and dosages . Experimental time courses were normalized to the mean of 5 time points immediately preceding GF application . Waterfall plots of single ‐ cell ERK activity trajectories . Cell ‐ averaged ER trajectories are color ‐ coded ( n = 78 cells ) , population average ( bottom ) . Vertical dotted line indicates GF application .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Ratiometric and mTFP1 donor images of EKAR2G in an EGF‐stimulated PC‐12 cell at the indicated time points, with t = 0' corresponding to EGF application. Upper panel: FRET ratio image is color‐coded for ERK activity. Lower panel: raw mTFP1 donor image in black/white contrast. Scale bar = 20 μm.Population average of ERK activation dynamics measured by Western blot using a phosphoERK antibody.Population average of ERK activation dynamics cell‐averaged EKAR2G emission ratios (ERs) from n= at least 111 cells. StDev are shown.Selected EKAR2G ratio time series illustrating, from top to bottom, sustained, oscillatory, or transient ERK activity dynamics. ER is color‐coded as in bar. Scale bar = 20 μm.Single‐cell ERK activity trajectories. Cell‐averaged ERs for n = 10 cells, standard deviation range (StDev), population average for the indicated GFs and dosages. Experimental time courses were normalized to the mean of 5 time points immediately preceding GF application.Waterfall plots of single‐cell ERK activity trajectories. Cell‐averaged ER trajectories are color‐coded (n = 78 cells), population average (bottom). Vertical dotted line indicates GF application.


Figure 2Trajectories from all sustained GF stimulation experiments ( n = 307 cells , same ERK activation trajectories as in Fig 1 ) were pooled and five representative trajectories were identified using k ‐ means clustering with squared Euclidean distance . Raw ( color ‐ coded by cluster ) and cluster representative trajectories ( black ) . Overlaid cluster representative trajectories . Population distribution of representative ERK activity trajectories in response to different GF dosages . Data representative of n = 3 experiments . Peak emission ratio intensity for each cluster . Boxplots with median , interquartile ( box ) and 1 . 5 IQR ( whiskers ) range , and raw datasets are shown . Boxplot notches extend 1 . 58 IQR / √ Nobs , which gives approximately 95 % confidence interval for comparing medians .

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Figure 2Trajectories from all sustained GF stimulation experiments (n = 307 cells, same ERK activation trajectories as in Fig 1) were pooled and five representative trajectories were identified using k‐means clustering with squared Euclidean distance. Raw (color‐coded by cluster) and cluster representative trajectories (black).Overlaid cluster representative trajectories.Population distribution of representative ERK activity trajectories in response to different GF dosages. Data representative of n = 3 experiments.Peak emission ratio intensity for each cluster. Boxplots with median, interquartile (box) and 1.5 IQR (whiskers) range, and raw datasets are shown. Boxplot notches extend 1.58 IQR / √Nobs, which gives approximately 95% confidence interval for comparing medians.


Figure 3A - DSingle GF pulse experiments . Cell ‐ averaged ERs , population average , and StDev range for n = 10 cells . Pulse application indicated by black bars . 3 ′ ( A , C ) and 10 ′ ( B , D ) pulse . High ( A , B ) and low ( C , D ) GF concentrations . ERationale for measuring amplitude and duration of 1st ERK activity peak in response to GF stimulation . Peak amplitude was measured as the ER change from when the GF stimulation starts until the highest ERK activity before adaptation occurs . Peak duration was estimated as the time between the first point before reaching the half ‐ maximum of the peak in the ascending phase , and the first point after the half ‐ maximum in the descending phase . FAmplitude of 1st ERK activity peak in response to different sustained or pulsed GF stimulation experiments . Notched boxplots of 1st ERK activity peak amplitude with median , interquartile range ( box ) , and data within 1 . 5 IQR range of the lower and upper quartiles ( whiskers ) are shown ( n = 20 cells per experiment ) . GDuration of 1st ERK activity peak in response to sustained and pulsed GF stimulation . Boxplots of 1st ERK activity peak duration with median , interquartile range ( box ) , and data within 1 . 5 IQR range of the lower and upper quartiles ( whiskers ) are shown ( n = 20 cells per experiment ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-DSingle GF pulse experiments. Cell‐averaged ERs, population average, and StDev range for n = 10 cells. Pulse application indicated by black bars. 3′ (A, C) and 10′ (B, D) pulse. High (A, B) and low (C, D) GF concentrations.ERationale for measuring amplitude and duration of 1st ERK activity peak in response to GF stimulation. Peak amplitude was measured as the ER change from when the GF stimulation starts until the highest ERK activity before adaptation occurs. Peak duration was estimated as the time between the first point before reaching the half‐maximum of the peak in the ascending phase, and the first point after the half‐maximum in the descending phase.FAmplitude of 1st ERK activity peak in response to different sustained or pulsed GF stimulation experiments. Notched boxplots of 1st ERK activity peak amplitude with median, interquartile range (box), and data within 1.5 IQR range of the lower and upper quartiles (whiskers) are shown (n = 20 cells per experiment).GDuration of 1st ERK activity peak in response to sustained and pulsed GF stimulation. Boxplots of 1st ERK activity peak duration with median, interquartile range (box), and data within 1.5 IQR range of the lower and upper quartiles (whiskers) are shown (n = 20 cells per experiment).


Figure 4A - CDouble ‐ pulse GF experiments . ( A ) 3 ′ GF / 3 ′ pause / 3 ′ GF . ( B ) 3 ′ GF / 10 ′ pause / 3 ′ GF . ( C ) 3 ′ GF / 20 ′ pause / 3 ′ GF . Cell ‐ averaged ERs , population average , and StDev range for n = 10 cells . D - GMulti ‐ pulse GF experiments . Population average ERs ( n = at least 30 cells ) for different EGF / NGF dosages . ( D ) 3 ′ GF / 3 ′ pause . ( E ) 3 ′ GF / 10 ′ pause . ( F ) 3 ′ GF / 20 ′ pause . ( G ) 3 ′ GF / 60 ′ pause . HDecay kinetics of ERK activity maxima from ( D - G ) . Boxplots with median , interquartile ( box ) , and 1 . 5 IQR ( whiskers ) range are shown for at least n = 30 cells .

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Figure 4A-CDouble‐pulse GF experiments. (A) 3′ GF/3′ pause/3′ GF. (B) 3′ GF/10′ pause/3′ GF. (C) 3′ GF/20′ pause/3′ GF. Cell‐averaged ERs, population average, and StDev range for n = 10 cells.D-GMulti‐pulse GF experiments. Population average ERs (n= at least 30 cells) for different EGF/NGF dosages. (D) 3′ GF/3′pause. (E) 3′ GF/10′ pause. (F) 3′ GF/20′ pause. (G) 3′ GF/60′ pause.HDecay kinetics of ERK activity maxima from (D-G). Boxplots with median, interquartile (box), and 1.5 IQR (whiskers) range are shown for at least n = 30 cells.


Figure 7ARepresentative micrographs of tubulin ‐ stained PC ‐ 12 NS ‐ 1 subclone cells stimulated with sustained and multi ‐ pulse GF regimes . Scale bar = 20 μm . B , CQuantification of neuronal differentiation fate . ( B ) Total neurite outgrowth per cell normalized to the major axis length of cell soma for sustained and multi ‐ pulse GF regimes . ( C ) Differentiation ratio calculated as the fraction of cells with normalized total neurite outgrowth per cell larger than 1 . At least 5 , 000 cells quantified in at least 2 independent experiments for each condition . Boxplots with median , interquartile ( Box ) , and 1 . 5 IQR ( Whiskers ) are shown .

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Figure 7ARepresentative micrographs of tubulin‐stained PC‐12 NS‐1 subclone cells stimulated with sustained and multi‐pulse GF regimes. Scale bar = 20 μm.B, CQuantification of neuronal differentiation fate. (B) Total neurite outgrowth per cell normalized to the major axis length of cell soma for sustained and multi‐pulse GF regimes. (C) Differentiation ratio calculated as the fraction of cells with normalized total neurite outgrowth per cell larger than 1. At least 5,000 cells quantified in at least 2 independent experiments for each condition. Boxplots with median, interquartile (Box), and 1.5 IQR (Whiskers) are shown.


Figure 1 ( B ) The concentration - effect relationships of PARP1 inhibition by thioparib , talazoparib , and olaparib , as assayed by histone - based ELISA . Data are obtained from three biological replicates and depicted as mean ± SEM . ( C ) Concentration - dependent increase in PARP1 - DNA binding by three PARP inhibitors using DSB FA assays . FA was measured 60 min after adding NAD + , and the ΔFA values represent the changes in PARP1 - DNA binding . Data from four biological replicates are presented as mean ± SEM . ( ( F ) and ( G ) Effects of thioparib on BRCA1 - deficient MDA - MB - 436 ( F ) and BRCA2 - deficient Capan - 1 ( G ) xenografts . Mice bearing MDA - MB - 436 tumors were dosed orally with 10 mg / kg thioparib , 100 mg / kg olaparib , or vehicle ( Veh ) once daily for 3 weeks ; and mice bearing Capan - 1 tumors were dosed orally with 5 or 25 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( Veh ) once daily for 3 weeks ( n = 6 ) . Data are shown as mean ± SEM . Statistical analysis was performed by two - way ANOVA . * * * P < 0 . 0001 . Representative images of xenograft tumors are shown , and mice tails represent complete regression of tumors .

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Figure 1(B) The concentration-effect relationships of PARP1 inhibition by thioparib, talazoparib, and olaparib, as assayed by histone-based ELISA. Data are obtained from three biological replicates and depicted as mean ± SEM. (C) Concentration-dependent increase in PARP1-DNA binding by three PARP inhibitors using DSB FA assays. FA was measured 60 min after adding NAD+, and the ΔFA values represent the changes in PARP1-DNA binding. Data from four biological replicates are presented as mean ± SEM. ((F) and (G) Effects of thioparib on BRCA1-deficient MDA-MB-436 (F) and BRCA2-deficient Capan-1 (G) xenografts. Mice bearing MDA-MB-436 tumors were dosed orally with 10 mg/kg thioparib, 100 mg/kg olaparib, or vehicle (Veh) once daily for 3 weeks; and mice bearing Capan-1 tumors were dosed orally with 5 or 25 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (Veh) once daily for 3 weeks (n = 6). Data are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA. ***P < 0.0001. Representative images of xenograft tumors are shown, and mice tails represent complete regression of tumors.


Figure 2 ( C ) The IC50 values of thioparib in six acquired PARPi - resistant cell lines . Numbers 1 - 6 represent the PARPi - resistant Capan - 1 / OP , Capan - 1 / TP , MDA - MB - 436 / OP , MDA - MB - 436 / TP , U251 / OP , and U251 / TP cells , respectively . Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay . The IC50 values are expressed as the mean ± SD from three separate experiments . ( E ) Efficacy of thioparib in the PDX model BR - 05 - 0028 ( BRCA1 - deficient ) . Mice bearing BR - 05 - 0028 tumors derived from a patient with breast cancer ( n = 6 ) were treated with 10 or 30 mg / kg thioparib or 100 mg / kg olaparib for 6 weeks . Data are depicted as mean ± SEM . * * * P < 0 . 0001 .

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Figure 2(C) The IC50 values of thioparib in six acquired PARPi-resistant cell lines. Numbers 1-6 represent the PARPi-resistant Capan-1/OP, Capan-1/TP, MDA-MB-436/OP, MDA-MB-436/TP, U251/OP, and U251/TP cells, respectively. Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay. The IC50 values are expressed as the mean ± SD from three separate experiments.(E) Efficacy of thioparib in the PDX model BR-05-0028 (BRCA1-deficient). Mice bearing BR-05-0028 tumors derived from a patient with breast cancer (n = 6) were treated with 10 or 30 mg/kg thioparib or 100 mg/kg olaparib for 6 weeks. Data are depicted as mean ± SEM. ***P < 0.0001.


Figure 3 ( B ) Concentration - dependent effects of thioparib on the viability of malignant hematologic cells . Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay . Data from three independent experiments were presented as mean ± SEM . ( D ) Kaplan - Meier survival curve of M - NSG mice transplanted with JeKo - 1 cells treated with 10 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( n = 10 ) . Statistical analysis was performed by log - rank ( Mantel - Cox ) test . * * * P < 0 . 0001 , ns : P = 0 . 3526 .

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Figure 3(B) Concentration-dependent effects of thioparib on the viability of malignant hematologic cells. Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay. Data from three independent experiments were presented as mean ± SEM.(D) Kaplan-Meier survival curve of M-NSG mice transplanted with JeKo-1 cells treated with 10 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (n = 10). Statistical analysis was performed by log-rank (Mantel-Cox) test. ***P < 0.0001, ns: P = 0.3526.


Figure 4 ( B ) Western blotting of γH2AX and phosphor - RPA32 in cells exposed to thioparib , talazoparib , or olaparib for 12 h . ( E ) and ( F ) . Thioparib caused changes in apoptosis - related proteins in JeKo - 1 or THP - 1 cells , as determined by western blotting . Cells were exposed to thioparib , talazoparib , olaparib , or Cpd 391 for 24 h and then subjected to western blotting . ( G ) and ( H ) . Depletion of PARP1 in HT - 29 and Capan - 1 / TP cells resulted in a decrease in DNA damage and thioparib resistance . The upper panel shows the changes in γH2AX and phospho - RPA32 in HT - 29 PARP1 knockout ( KO ) clones ( G ) or Capan - 1 / TP PARP1 KO clones ( H ) and their parental cells after exposure to thioparib for 24 h . The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells . Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments . Data are presented as the mean ± SD . ThP : Thioparib , OP : Olaparib , TP : Talazoparib , 391 : Cpd 391 .

O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Western blotting of γH2AX and phosphor-RPA32 in cells exposed to thioparib, talazoparib, or olaparib for 12 h.(E) and (F). Thioparib caused changes in apoptosis-related proteins in JeKo-1 or THP-1 cells, as determined by western blotting. Cells were exposed to thioparib, talazoparib, olaparib, or Cpd 391 for 24 h and then subjected to western blotting.(G) and (H). Depletion of PARP1 in HT-29 and Capan-1/TP cells resulted in a decrease in DNA damage and thioparib resistance. The upper panel shows the changes in γH2AX and phospho-RPA32 in HT-29 PARP1 knockout (KO) clones (G) or Capan-1/TP PARP1 KO clones (H) and their parental cells after exposure to thioparib for 24 h. The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells. Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments. Data are presented as the mean ± SD. ThP: Thioparib, OP: Olaparib, TP: Talazoparib, 391: Cpd 391.


Figure 5 ( A ) HR repair assays in U2OS - DR - GFP cells treated with increasing concentrations of thioparib . Cells were incubated with the indicated drugs at the time of I - SceI introduction for 48 h . GFP - positive cells were analyzed after I - SceI transfection for 72 h by flow cytometry . The ATR inhibitor VE - 821 was used as a positive control . Three independent experiments were performed and data are expressed as mean ± SD . Statistical analysis was performed by one - way ANOVA , and all groups were compared with control . * * * P < 0 . 0001 , * P = 0 . 0205 , 0 . 0101 ( from left to right ) . ( C ) Representative images of RAD51 foci in U2OS - DR - GFP cells with or without radiation . Graphs ( right panel ) show quantification of cells with ≥ 5 RAD51 foci in vehicle - and drug - treated cells . Scale bar : 5 μm . Data are obtained from three biological replicates and presented as mean ± SD . Statistical analysis was performed by one - way ANOVA . Left to right : P < 0 . 0001 , = 0 . 0033 , < 0 . 0001 , = 0 . 0006 , = 0 . 0031 , = 0 . 0015 . ( H ) HR repair assay . U2OS - DR - GFP ( WT ) and PARP1 KO cells ( KO1 and KO2 ) were treated with the indicated drugs for 48 h and collected after 72 h of I - SceI transfection as in ( A ) . VE - 821 served as a positive control . PARP1 knockout efficiency was evaluated by western blotting . Data from three biological replicates are presented as mean ± SD . Statistical analysis was performed by one - way ANOVA , and each group was compared with the control . * * * P < 0 . 0001 , * P = 0 . 0143 , 0 . 0483 , ns : P = 0 . 3061 , 0 . 8269 , 0 . 9997 , 0 . 3906 ( from left to right ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) HR repair assays in U2OS-DR-GFP cells treated with increasing concentrations of thioparib. Cells were incubated with the indicated drugs at the time of I-SceI introduction for 48 h. GFP-positive cells were analyzed after I-SceI transfection for 72 h by flow cytometry. The ATR inhibitor VE-821 was used as a positive control. Three independent experiments were performed and data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA, and all groups were compared with control. ***P < 0.0001, *P = 0.0205, 0.0101 (from left to right).(C) Representative images of RAD51 foci in U2OS-DR-GFP cells with or without radiation. Graphs (right panel) show quantification of cells with ≥ 5 RAD51 foci in vehicle- and drug-treated cells. Scale bar: 5 μm. Data are obtained from three biological replicates and presented as mean ± SD. Statistical analysis was performed by one-way ANOVA. Left to right: P < 0.0001, = 0.0033, < 0.0001, = 0.0006, = 0.0031, = 0.0015.(H) HR repair assay. U2OS-DR-GFP (WT) and PARP1 KO cells (KO1 and KO2) were treated with the indicated drugs for 48 h and collected after 72 h of I-SceI transfection as in (A). VE-821 served as a positive control. PARP1 knockout efficiency was evaluated by western blotting. Data from three biological replicates are presented as mean ± SD. Statistical analysis was performed by one-way ANOVA, and each group was compared with the control. ***P < 0.0001, *P = 0.0143, 0.0483, ns: P = 0.3061, 0.8269, 0.9997, 0.3906 (from left to right).


Figure 6 ( C ) Effects of thioparib on CXCL9 , CXCL10 , and IL15 mRNA in JeKo - 1 cells . Data from three independent experiments are shown as the mean ± SD . Statistical analysis was performed by two - way ANOVA . Left panel : * P = 0 . 0367 , * * P = 0 . 0011 ; median panel : * * * P = 0 . 0002 , < 0 . 0001 , < 0 . 0001 ; right panel : * * * P = 0 . 0005 , 0 . 0009 , 0 . 0001 ( from left to right ) . ( D ) The protein levels of p - STAT1 , p - TBK1 , γH2AX , and phospho - RPA32 in HT - 29 parent , PARP1 − / − ( # KO1 ) , and PARP7 − / − cell lines after 24 or 48 h thioparib treatment . ( F ) Western blot analysis of p - STAT1 , p - TBK1 , and γH2AX in HT - 29 parent , STING − / − , and TBK1 − / − cells after 24 or 48 h thioparib treatment . ( H ) Immunohistochemistry staining of CD4 , CD8 , and CD45 expression in MC38 tumor tissue sections . Quantification of cells positive of CD4 , CD8 , CD45 were shown . Scale bar : 25 μm . Data from ten technical replicates from each of the two mice are presented as mean ± SD . Statistical analysis was performed by unpaired t - test . * * * P < 0 . 0001 , ns : P = 0 . 1958 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(C) Effects of thioparib on CXCL9, CXCL10, and IL15 mRNA in JeKo-1 cells. Data from three independent experiments are shown as the mean ± SD. Statistical analysis was performed by two-way ANOVA. Left panel: *P = 0.0367, **P = 0.0011; median panel: ***P = 0.0002, < 0.0001, < 0.0001; right panel: ***P = 0.0005, 0.0009, 0.0001 (from left to right).(D) The protein levels of p-STAT1, p-TBK1, γH2AX, and phospho-RPA32 in HT-29 parent, PARP1−/− (#KO1), and PARP7−/− cell lines after 24 or 48 h thioparib treatment.(F) Western blot analysis of p-STAT1, p-TBK1, and γH2AX in HT-29 parent, STING−/−, and TBK1−/−cells after 24 or 48 h thioparib treatment.(H) Immunohistochemistry staining of CD4, CD8, and CD45 expression in MC38 tumor tissue sections. Quantification of cells positive of CD4, CD8, CD45 were shown. Scale bar: 25 μm. Data from ten technical replicates from each of the two mice are presented as mean ± SD. Statistical analysis was performed by unpaired t-test. ***P < 0.0001, ns: P = 0.1958.


Figure 1B Representative pictures of different CTCs from the initial CellSearch ® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line . The detected tumor cells display clear keratin and DAPI staining , CD45 - negativity as well as lack of , or very weak ( 4 , 8 ) , ERBB2 expression . Cells of small ( about 5 µm in diameter , 1 , 2 ) and large size ( larger than 10µm in diameter , 2 , 3 ) were detected . While some CTCs displayed dot - like perinuclear keratin signals ( 1 , 2 ) , the majority showed diffuse keratin staining . Additionally , CTC clusters of 4 - 8 cells were present ( 5 , 6 ) . Some CTCs showed multiple / lobed nuclei ( 4 , 7 , 8 ) . C Bright field images of CTC - ITB - 01 cells growing adherently . D Bright field images of CTC - ITB - 01 cells growing non - adherently ( relation between adherent and non - adherent cells : 80 / 20 % ) .

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Figure 1B Representative pictures of different CTCs from the initial CellSearch® analysis of the metastatic breast cancer patient who gave rise to the breast CTC line. The detected tumor cells display clear keratin and DAPI staining, CD45-negativity as well as lack of, or very weak (4, 8), ERBB2 expression. Cells of small (about 5 µm in diameter, 1, 2) and large size (larger than 10µm in diameter, 2,3) were detected. While some CTCs displayed dot-like perinuclear keratin signals (1, 2), the majority showed diffuse keratin staining. Additionally, CTC clusters of 4-8 cells were present (5, 6). Some CTCs showed multiple/lobed nuclei (4, 7, 8).C Bright field images of CTC-ITB-01 cells growing adherently. D Bright field images of CTC-ITB-01 cells growing non-adherently (relation between adherent and non-adherent cells: 80/20 %).


Figure 2Unsupervised hierarchical clustering ( Ward linkage with Euclidean distance ) of CNA profiles generated from patient primary tumors , the vaginal metastasis , isolated single and pooled CTCs from the primary blood collection and CTC - ITB - 01 ( early and later passage ) and leukocytes . " CL " represents the cell line , followed by the passage analyzed ( p04 / p30 ) . " CTC " marks circulating tumor cells isolated from the initial blood draw , followed by 01 / 05 / 10 indicating single ( 01 ) or pooled cells ( 5 or 10 , respectively ) . Light and dark grey bars to the left signify chromosomes , starting from the top with chromosome 1 . Red color represents chromosome loss , blue represents gain . All copy number aberrations were calculated using the patient ' s own germline DNA extracted from leukocytes as reference to eliminate copy number variations ( CNVs ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Unsupervised hierarchical clustering (Ward linkage with Euclidean distance) of CNA profiles generated from patient primary tumors, the vaginal metastasis, isolated single and pooled CTCs from the primary blood collection and CTC-ITB-01 (early and later passage) and leukocytes. "CL" represents the cell line, followed by the passage analyzed (p04/p30). "CTC" marks circulating tumor cells isolated from the initial blood draw, followed by 01/05/10 indicating single (01) or pooled cells (5 or 10, respectively). Light and dark grey bars to the left signify chromosomes, starting from the top with chromosome 1. Red color represents chromosome loss, blue represents gain. All copy number aberrations were calculated using the patient's own germline DNA extracted from leukocytes as reference to eliminate copy number variations (CNVs).


Figure 3Direct comparisons of CNA profiles . Percentages of gene copy number gains and losses across the chromosomes for different sample types . Copy number gains are displayed in green , losses in red . A CNAs generated from CTCs from the primary blood collection ( upper panel ) and CTC - ITB - 01 , indicated as " CL " ( medium panel ) . Significant distinctions between both samples were calculated using Fisher ' s exact test ( Joosse et al , 2018 ) and are indicated in lowest panel ( blue ) . Increased blue color intensity represents lower p - value and therefore higher statistical significance . Direct comparisons of CNA profiles . Percentages of gene copy number gains and losses across the chromosomes for different sample types . Copy number gains are displayed in green , losses in red . B CNA comparison between early ( p04 ) and late ( p30 ) CTC - ITB - 01 passages ( upper and medium panel ) . Significant distinctions between both samples were calculated using Fisher ' s exact test ( Joosse et al , 2018 ) and are indicated in lowest panel ( in blue ) . Increased blue color intensity represents lower p - value and therefore higher statistical significance .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Direct comparisons of CNA profiles. Percentages of gene copy number gains and losses across the chromosomes for different sample types. Copy number gains are displayed in green, losses in red. A CNAs generated from CTCs from the primary blood collection (upper panel) and CTC-ITB-01, indicated as "CL" (medium panel). Significant distinctions between both samples were calculated using Fisher's exact test (Joosse et al, 2018) and are indicated in lowest panel (blue). Increased blue color intensity represents lower p-value and therefore higher statistical significance.Direct comparisons of CNA profiles. Percentages of gene copy number gains and losses across the chromosomes for different sample types. Copy number gains are displayed in green, losses in red. B CNA comparison between early (p04) and late (p30) CTC-ITB-01 passages (upper and medium panel). Significant distinctions between both samples were calculated using Fisher's exact test (Joosse et al, 2018) and are indicated in lowest panel (in blue). Increased blue color intensity represents lower p-value and therefore higher statistical significance.