( E ) In vitro ubiquitination was performed using bacterially purified GST , GST tagged H2B its mutants as substrates . Recombinant E1 , E2 ( UBE2A ) ubiquitin were included in each reaction . Ubiquitination was detected by H2BUb antibody .

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(E) In vitro ubiquitination assay was performed using bacterially purified GST, GST tagged H2B and its mutants as substrates. Recombinant E1, E2(UBE2A) and ubiquitin were included in each reaction. Ubiquitination was detected by H2BUb antibody.


( F ) In vitro ubiquitination was performed using biotin tagged non - phospho phospho ( pY121 ) peptide of H2B as substrates in presence of E1 E2 . H2BUb was detected by immunoblotting with specific antibody .

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(F) In vitro ubiquitination was performed using biotin tagged non-phospho and phospho (pY121) peptide of H2B as substrates in the presence of E1 and E2. H2BUb was detected by immunoblotting with specific antibody.


( A ) In vitro phosphorylated GST H2B by Syk kinase was incubated with bacterially purified recombinant wild type SHP - 1 or elution buffer amount of released phosphate was assayed colorimetrically using malachite green reagent ( A620 nm ) . Data represent mean absorbance from four independent experiments . Error bar indicates SD ; * p < 0 . 05 , by Student ' s t .

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(A) In vitro phosphorylated GST H2B by Syk kinase was incubated with bacterially purified recombinant wild type SHP-1 or elution buffer and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represent mean absorbance from four independent experiments. Error bar indicates SD; *p<0.05, by Student's t test.


( B ) Bacterially purified GST H2B was subjected to in vitro kinase using HEK293T lysate , then phosphorylated were incubated with either bacterially purified MBP tagged constitutively active ( SHP - 1 ΔSH2 ) , or catalytically inactive SHP - 1 C / S mutant . phosphorylation status of recombinant H2B was detected by immunoblotting with phospho - tyrosine antibody .

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(B) Bacterially purified GST H2B was subjected to in vitro kinase assay using HEK293T cell lysate, and then phosphorylated proteins were incubated with either bacterially purified MBP tagged constitutively active (SHP-1 ΔSH2), or catalytically inactive SHP-1 C/S mutant. The phosphorylation status of recombinant H2B was detected by immunoblotting with phospho-tyrosine antibody.


( C ) Phospho ( pH2B ) Y121 non - phospho peptide of H2B was incubated with constitutively active ( SHP - 1 ΔSH2 ) or catalytically inactive mutant ( C / S ) of SHP - 1 released phosphate was assayed colorimetrically using malachite green reagent ( A620nm ) . n = 3 independent experiments , Error bars indicate SD ; * * * p < 0 . 001 ( one - way ANOVA , Tukey ' s multiple comparison ) .

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(C) Phospho (pH2B) Y121 and non-phospho peptide of H2B was incubated with constitutively active (SHP-1 ΔSH2) or catalytically inactive mutant (C/S) of SHP-1 and the released phosphate was assayed colorimetrically using the malachite green reagent (A620nm). n=3 independent experiments, Error bars indicate SD; ***p<0.001 (one-way ANOVA, Tukey's multiple comparison test).


( D ) 293T were transfected with vector , Myc tagged SHP - 1 WT or C / S mutant . phosphorylation status of endogenous H2B was detected by immunoblotting with pY121 - H2B antibody .

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(D) 293T cells were transfected with vector, Myc tagged SHP-1 WT or C/S mutant. The phosphorylation status of endogenous H2B was detected by immunoblotting with pY121-H2B antibody.


( E ) extracts of 293T WT SHP - 1 KO were made of H2B phosphorylation were detected using pY121 - H2B antibody .

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(E) Nuclear extracts of 293T WT and SHP-1 KO cells were made and the levels of H2B phosphorylation were detected using pY121-H2B antibody.


( F ) Wild type ( WT ) along with SHP - 1 KO 293T transfected with either Myc vector , Myc SHP - 1 or Myc SHP - 1 C / S mutant were lysed . of pY121 - H2B H2BUb were determined using specific antibodies .

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(F) Wild type (WT) along with SHP-1 KO 293T cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 C/S mutant were lysed. Levels of pY121-H2B and H2BUb were determined using specific antibodies.


( G ) 293T were treated with SHP - 1 inhibitor TPI ( 50nm ) for 1hr of pY121 - H2B was determined using pY121H2B antibody .

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(G) 293T Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr and levels of pY121-H2B was determined using pY121H2B antibody.


MCF7 were transduced with ( H ) CTR9 shRNA along with control shRNA . Post 72 h , were collected lysed to isolate soluble fractions . of phosphorylated ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies .

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MCF7 cells were transduced with (H) CTR9 shRNA along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.


MCF7 were transduced with ( I ) Paf1 shRNA , along with control shRNA . Post 72 h , were collected lysed to isolate soluble fractions . of phosphorylated ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies .

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MCF7 cells were transduced with (I) Paf1 shRNA, along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.


MCF7 were transduced with ( J ) RTF1 shRNA along with control shRNA . Post 72 h , were collected lysed to isolate soluble fractions . of phosphorylated ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies .

MCF7 cells were transduced with (J) RTF1 shRNA along with control shRNA. Post 72 h, cells were collected and lysed to isolate soluble and nuclear fractions. Levels of phosphorylated and ubiquitinated H2B were detected by immunoblotting with indicated specific antibodies.


( A ) lysates derived from 293T transfected with empty vector , SFB - tagged H2B , H2B Y121F or H2B Y121D constructs were pulled down with S - agarose beads . RNF20 interaction with H2B was detected by immunoblotting with specific antibody .

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(A) Cell lysates derived from 293T cells transfected with empty vector, SFB-tagged H2B, H2B Y121F or H2B Y121D constructs were pulled down with S-protein agarose beads. RNF20 interaction with H2B was detected by immunoblotting with specific antibody.


( B ) HEK293T were co - transfected with SFB - tagged H2B , H2B Y121F or H2B Y121D constructs along with Myc - tagged UBE2A . lysates were pulled down with S - beads , interaction was detected by immunoblotting with Myc antibody .

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(B) HEK293T cells were co-transfected with the SFB-tagged H2B, H2B Y121F or H2B Y121D constructs along with Myc-tagged UBE2A. Cell lysates were pulled down with S-protein beads, and interaction was detected by immunoblotting with Myc antibody.


( C ) Glutathione Sepharose beads bound with bacterially expressed recombinant GST - H2B , GST H2B Y121F GST - H2B Y121D were incubated with bacterially purified recombinant His UBE2A , interaction of UBE2A with H2B was detected by immunoblotting with His antibody . Recombinant expression pulldown was shown by Coomassie .

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(C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST-H2B, GST H2B Y121F and GST-H2B Y121D proteins were incubated with bacterially purified recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.


( D ) Bacterially purified GST , GST H2B GST H2B - p ( phosphorylated using Syk ) was incubated with recombinant His UBE2A , interaction of UBE2A with H2B was detected by immunoblotting with His antibody . Recombinant expression pulldown was shown by Coomassie .

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(D) Bacterially purified GST, GST H2B and GST H2B-p (phosphorylated using Syk) was incubated with recombinant His UBE2A, and interaction of UBE2A with H2B was detected by immunoblotting with His antibody. Recombinant protein expression and pulldown was shown by Coomassie staining.


( E ) WT 293T along with SHP - 1 KO were co - transfected with SFB vector or SFB H2B along with Myc UbE2A . lysates were pulldown using S - beads , interaction of UBE2A with H2B was determined by Western blotting with indicated antibodies .

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(E) WT 293T cells along with SHP-1 KO were co-transfected with SFB vector or SFB H2B along with Myc UbE2A. Cell lysates were pulldown using S-protein beads, and interaction of UBE2A with H2B was determined by Western blotting with the indicated antibodies.


( F ) HEK293T were transfected with SFB vector or SFB H2B along with Myc UBE2A . were treated with SHP - 1 inhibitor TPI ( 50nm ) for 1hr after 24hrs of transfection . lysates were subjected to pull - down with S - beads , western blotting was performed using specific antibodies .

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(F) HEK293T cells were transfected with SFB vector or SFB H2B along with Myc UBE2A.Cells were treated with SHP-1 inhibitor TPI (50nm) for 1hr after 24hrs of transfection. Cells lysates were subjected to pull-down with S-protein beads, and western blotting was performed using specific antibodies.


( A ) Representative super - resolution of MCF7 immunostained with SHP - 1 in combination with CDK9 , total Polymerse II , polymerase II Ser2P , Polymerase II Ser5P . Scale bar : 5µm . intensities along drawn in a representative section of were obtained using Zen software co - localization plots were made using GraphPad .

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(A) Representative super-resolution images of MCF7 cells immunostained with SHP-1 in combination with CDK9, total RNA Polymerse II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P. Scale bar: 5µm. Fluorescence intensities along the line drawn in a representative section of cell were obtained using Zen software and the co-localization plots were made using GraphPad.


( B ) Co - localization of SHP - 1 with CDK9 , total Polymerase II , Polymerase II S2P , Polymerase II S5P was measured by Pearson correlation coefficient using ImageJ . Values presented as mean ± SD , n = 10 ( from three independent experiments ) . * * * p value < 0 . 001 . ( One - way ANOVA ) .

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(B) Co-localization of SHP-1 with CDK9, total RNA Polymerase II, RNA Polymerase II S2P, and RNA Polymerase II S5P was measured by Pearson correlation coefficient using ImageJ. Values presented as the mean ± SD, n=10 (from three independent experiments). ***p value < 0.001. (One-way ANOVA).


( C ) Endogenous interaction of SHP - 1 with Polymerase II , polymerase II Ser2P , Polymerase II Ser5P CDK9 was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP - 1 antibody using TurboNuclease treated lysate .

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(C) Endogenous interaction of SHP-1 with RNA Polymerase II, RNA polymerase II Ser2P, and RNA Polymerase II Ser5P and CDK9 was detected by immunoblotting after performing immunoprecipitation with either IgG or SHP-1 antibody using TurboNuclease treated cell lysate.


( D ) extracts of MCF - 7 WT SHP - 1 knockout were made of Polymerase II , Polymerase II Ser2P Polymerase II Ser5P were detected by immunoblotting using specific antibodies .

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(D) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P and RNA Polymerase II Ser5P were detected by immunoblotting using specific antibodies.


( E ) extracts of treated with either DMSO or SHP - 1 inhibitor TPI ( 50nm ) were used to detect total Polymerase II its modifications by immunoblotting using specific antibodies .

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(E) Nuclear extracts of cells treated with either DMSO or SHP-1 inhibitor TPI (50nm) were used to detect total RNA Polymerase II and its modifications by immunoblotting using specific antibodies.


( F ) extracts of MCF - 7 WT SHP - 1 knockout transfected with Myc vector , H2B Y121F or H2B Y121D constructs were made of Polymerase II , Polymerase II Ser2P , Polymerase II Ser5P Lamin B1 were detected by immunoblotting using specific antibodies .

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(F) Nuclear extracts of MCF-7 WT and SHP-1 knockout cells transfected with Myc vector, H2B Y121F or H2B Y121D constructs were made and the levels of RNA Polymerase II, RNA Polymerase II Ser2P, RNA Polymerase II Ser5P and Lamin B1 were detected by immunoblotting using specific antibodies.


( G ) Immunofluorescence of WT or SHP - 1 knockout MCF7 labelled with 5 - Ethynyl Uridine ( EU ) for 60 minutes to capture nascent synthesis . Scale bars 10μm . ( H ) EU incorporation in MCF - 7 WT SHP - 1 KO was quantified in three independent experiments . Values presented as mean ± SD , * * * indicates p < 0 . 001 ( One - way ANOVA ) .

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(G) Immunofluorescence of WT or SHP-1 knockout MCF7 cells labelled with the 5-Ethynyl Uridine (EU) for 60 minutes to capture nascent RNA synthesis. Scale bars 10μm. (H) The EU incorporation in MCF-7 WT and SHP-1 KO cells was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).


( I ) Representative of immunofluorescence of DMSO or TPI treated ( 50nm ) MCF7 labelled with EU for 60 minutes was shown . Scale bar : 40µm . ( J ) EU incorporation in MCF7 treated with DMSO or TPI was quantified in three independent experiments . Values presented as mean ± SD , * * * indicates p < 0 . 001 ( One - way ANOVA ) .

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(I) Representative images of immunofluorescence of DMSO or TPI treated (50nm) MCF7 cells labelled with EU for 60 minutes was shown. Scale bar: 40µm. (J) The EU incorporation in MCF7 cells treated with DMSO or TPI was quantified in three independent experiments. Values presented as the mean ± SD, ***indicates p<0.001 (One-way ANOVA).


( C , D ) MCF - 7 WT along with SHP - 1 KO transfected with either vector , H2B Y121F or H2B Y121D mutant were used for ChIP q - PCR of H2BUb at indicated genes . data shown is derived from two independent experiments . Error bars indicate mean ± SD .

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(C, D) MCF-7 WT cells along with SHP-1 KO cells transfected with either vector, H2B Y121F or H2B Y121D mutant were used for ChIP q-PCR analysis of the H2BUb at indicated genes. The data shown is derived from two independent experiments. Error bars indicate the mean ± SD.


( A ) Lysates of WT SHP - 1 knockout MCF7 were made of LC3 - I II were detected by immunoblotting . ( B ) of LC3 II were normalized against total LC3 ( LC3 I + LC3 II ) . data shown is derived from three independent experiments . Error bars indicate mean ± SD . * p value < 0 . 05 , Student ' s t - .

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(A) Lysates of WT and SHP-1 knockout MCF7 cells were made and the levels of LC3-I and II were detected by immunoblotting. (B) Levels of LC3 II were normalized against total LC3 (LC3 I+LC3 II). The data shown is derived from three independent experiments. Error bars indicate the mean ± SD. *p value < 0.05, Student's t-test.


( C ) Representative super - resolution of WT SHP - 1 KO MCF7 immunostained with LC3 antibody , ( Scale bar : 5µm ) . ( D ) of LC3 accumulation per done by using ImageJ was shown . Bars represents mean ± SD . ( N = 60 ) * * * p value < 0 . 001 , Student ' s t - .

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(C) Representative super- resolution images of WT and SHP-1 KO MCF7 cells immunostained with LC3 antibody, (Scale bar: 5µm). (D) Quantification of LC3 puncta accumulation per cell done by using ImageJ was shown. Bars represents mean ± SD.(N=60) ***p value < 0.001, Student's t-test.


( E ) WT along with SHP - 1 KO MCF7 transfected with either Myc vector , Myc SHP - 1 or Myc SHP - 1 NLS mutant were lysed . of LC3 actin were determined using specific antibodies .

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(E) WT along with SHP-1 KO MCF7 cells transfected with either Myc vector, Myc SHP-1 or Myc SHP-1 NLS mutant were lysed. Levels of LC3 and actin were determined using specific antibodies.


( F ) WT SHP - 1 knockout MCF7 transfected with Myc vector or Myc - tagged H2B , H2B Y121F or H2B Y121D constructs were immunostained with endogenous p62 Myc antibody . Image acquisition was done using LSM 700 , ( Scale bar : 20µm ) . ( G ) Accumulation of p62 was quantified by counting p62 positive per . ( N = 25 ) ; values presented as mean ± SD . * * * p value < 0 . 001 ( One - way ANOVA ) .

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(F) WT and SHP-1 knockout MCF7 cells transfected with the Myc vector or Myc-tagged H2B, H2B Y121F or H2B Y121D constructs were immunostained with endogenous p62 and Myc antibody. Image acquisition was done using LSM 700, (Scale bar: 20µm). (G) Accumulation of p62 was quantified by counting p62 positive puncta per cell. (N= 25); values presented as the mean ± SD. ***p value < 0.001 (One-way ANOVA).


( H ) Representative super - resolution of LC3 LAMP2 immunostaining in WT SHP - 1 KO MCF7 . ( Scale bar : 20 µm for merge & 5 µm for Zoom ) . ( I ) LC3 co - localizing with LAMP2 per were quantified using ImageJ . ( N = 45 ) ; values presented as mean ± SD . * * * p value < 0 . 001 , Student ' s t - .

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(H) Representative super-resolution images of LC3 and LAMP2 immunostaining in WT and SHP-1 KO MCF7 cells. (Scale bar: 20 µm for merge & 5 µm for Zoom). (I) LC3 puncta co-localizing with LAMP2 per cell were quantified using ImageJ. (N=45); values presented as the mean ± SD. ***p value < 0.001, Student's t-test.


B . UTP6 in U - 2 OS exemplify localized to whole nucleoli ( HPA025936 ) .

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B. UTP6 in U-2 OS cells exemplify proteins localized to whole nucleoli (HPA025936).


C . Fibrillar center localization shown by a UBTF IF in U - 2 OS ( HPA006385 ) .

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C. Fibrillar center localization shown by a UBTF IF staining in U-2 OS cells (HPA006385).


E . Dual localization of LEO1 to both fibrillar center nucleoplasm in GFP - tagged HeLa ( magenta ) , also supported by IF antibody using HPA040741 ( green ) .

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E. Dual localization of LEO1 to both the fibrillar center and nucleoplasm in GFP-tagged HeLa cells (magenta), also supported by IF antibody staining using HPA040741 (green).


F . multilocalizing ribosomal RPL13 detected in nucleoli , cytosol ER in MCF - 7 ( HPA051702 ) .

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F. The multilocalizing ribosomal protein RPL13 detected in the nucleoli, cytosol and ER in MCF-7 cells (HPA051702).


A . KRI1 localized to nucleoli in MCF - 7 ( HPA043574 ) . Data information : of interest is shown in green , marker DAPI in blue reference marker of microtubules in red . Scale bar 10 µm .

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A. KRI1 localized to the nucleoli in MCF-7 cells (HPA043574). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


B . GPATCH4 localized to nucleoli in U - 2 OS ( HPA054319 ) . Data information : of interest is shown in green , marker DAPI in blue reference marker of microtubules in red . Scale bar 10 µm .

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B. GPATCH4 localized to the nucleoli in U-2 OS cells (HPA054319). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


C . FOXL2NB localized to fibrillar center in SiHa ( HPA061017 ) . Data information : of interest is shown in green , marker DAPI in blue reference marker of microtubules in red . Scale bar 10 µm .

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C. FOXL2NB localized to the fibrillar center in SiHa cells (HPA061017). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


D . FAM71E1 localized to fibrillar center in U - 2 OS ( HPA048111 ) . Data information : of interest is shown in green , marker DAPI in blue reference marker of microtubules in red . Scale bar 10 µm .

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D. FAM71E1 localized to the fibrillar center in U-2 OS cells (HPA048111). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


E . LRRC23 localized to nucleoli in U - 2 OS ( HPA057533 ) . Data information : of interest is shown in green , marker DAPI in blue reference marker of microtubules in red . Scale bar 10 µm .

E. LRRC23 localized to the nucleoli in U-2 OS cells (HPA057533). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


F . ZNF853 localized to nucleoli in U - 2 OS ( HPA067690 ) . Data information : of interest is shown in green , marker DAPI in blue reference marker of microtubules in red . Scale bar 10 µm .

F. ZNF853 localized to the nucleoli in U-2 OS cells (HPA067690). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


G . METTL5 localized to nucleoli in U - 2 OS ( HPA038223 ) . Data information : of interest is shown in green , marker DAPI in blue reference marker of microtubules in red . Scale bar 10 µm .

G. METTL5 localized to the nucleoli in U-2 OS cells (HPA038223). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


B . IF of MKI67 shows localization to nucleoli rim in U - 251 ( CAB000058 ) . Data information : of interest is shown in green , marker DAPI / Hoechst in blue microtubules reference marker in red . Scale bar 10 µm .

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B. IF staining of MKI67 shows localization to nucleoli rim in U-251 cells (CAB000058). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


C . IF of GNL3 shows localization to nucleoli rim in U - 2 OS ( HPA036743 ) . Data information : of interest is shown in green , marker DAPI / Hoechst in blue microtubules reference marker in red . Scale bar 10 µm .

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C. IF staining of GNL3 shows localization to nucleoli rim in U-2 OS cells (HPA036743). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


D . HEK 293T expressing endogenous of N - terminus mNG - MKI67 show a faint nucleoli rim localization , although still visible . arrows indicate where rim could be seen . Data information : of interest is shown in green , marker DAPI / Hoechst in blue microtubules reference marker in red . Scale bar 10 µm .

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D. HEK 293T cells expressing endogenous levels of N-terminus mNG-MKI67 show a faint nucleoli rim localization, although still visible. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


E . Fixed HEK 293T expressing mNG - tagged MKI67 show a clear nucleoli rim localization . Data information : of interest is shown in green , marker DAPI / Hoechst in blue microtubules reference marker in red . Scale bar 10 µm .

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E. Fixed HEK 293T cells expressing mNG-tagged MKI67 show a clear nucleoli rim localization. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


F . HEK 293T expressing endogenous of C - terminus tagged GNL3 show nucleoli rim localization . arrows indicate where rim could be seen . Data information : of interest is shown in green , marker DAPI / Hoechst in blue microtubules reference marker in red . Scale bar 10 µm .

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F. HEK 293T cells expressing endogenous levels of C-terminus tagged GNL3 show nucleoli rim localization. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


A . MKI67 ( HPA000451 ) . B . NOC2L ( HPA044258 ) . C . EMG1 ( HPA039304 ) . D . BMS1 ( HPA043081 ) . E . BRIX1 ( HPA039614 ) . F . RSL1D1 ( HPA043483 ) . Data information : of interest is shown in green , microtubules in red DAPI in blue . of interphase were acquired from a different experiment intensities cannot be compared between interphase mitotic . Scale bar 10 μm .

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A. MKI67 (HPA000451). B. NOC2L (HPA044258). C. EMG1 (HPA039304). D. BMS1 (HPA043081). E. BRIX1 (HPA039614). F. RSL1D1 (HPA043483). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.


C - E . IF stainings of previously uncharacterized mitotic chromosome predicted to be highly disordered . of interest shown in green DAPI in blue . Scale bar 10 µm . C . CCDC86 ( HPA041117 ) , 100 % predicted disorder level . D . RRP15 ( HPA024639 ) , 87 % predicted disorder level . E . PRR19 ( HPA070350 ) , 67 % predicted disorder level .

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C-E. IF stainings of previously uncharacterized mitotic chromosome proteins predicted to be highly disordered. Protein of interest shown in green and DAPI in blue. Scale bar 10 µm. C. CCDC86 (HPA041117), 100% predicted disorder level. D. RRP15 (HPA024639), 87% predicted disorder level. E. PRR19 (HPA070350), 67% predicted disorder level.


A . MKI67 is detected on chromosomes throughout mitotic phases exemplifies in early recruitment category . B - F . IF stainings of where localization to mitotic chromosomes is seen after metaphase , showing a typical late recruitment expression pattern . B . NOL12 ( HPA003547 ) . C . TIGD1 ( HPA041717 ) . D . BYSL ( HPA031219 ) . E . ACTL6B ( HPA045126 ) . F . ZNF202 ( HPA059229 ) . Data information : of interest is shown in green , microtubules in red DAPI in blue . of interphase were acquired from a different experiment intensities cannot be compared between interphase mitotic . Scale bar 10 μm .

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A. MKI67 is detected on the chromosomes throughout all mitotic phases and exemplifies proteins in the early recruitment category. B-F. IF stainings of proteins where localization to mitotic chromosomes is seen after metaphase, showing a typical late recruitment expression pattern. B. NOL12 (HPA003547). C. TIGD1 (HPA041717). D. BYSL (HPA031219). E. ACTL6B (HPA045126). F. ZNF202 (HPA059229). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.


A - B . GNL3 PES1 are localizing to around chromosomes in MKI67 KO . A . GNL3 ( HPA036743 ) . Median overlapKO = 0 . 539 , median overlapWT = 0 . 795 ( P = 9 . 34x10 - 9 ) median Pearson correlationKO = - 0 . 335 , median Pearson correlationWT = 0 . 134 ( P = 3 . 58x10 - 6 ) . B . PES1 ( HPA040210 ) . Median overlapKO = 0 . 516 , median overlapWT = 0 . 755 ( P = 1 . 77x10 - 13 ) median Pearson correlationKO = - 0 . 293 , median Pearson correlationWT = 0 . 129 ( P = 4 . 06x10 - 13 ) . Data information : of interest is shown in green DAPI in blue . Scale bar 10 μm . For colocalization data ; biological replicate 1 highlighted in grey biological replicate 2 in green . Both overlap Pearson correlation between DAPI / was measured a two - unpaired Wilcoxon was used .

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A-B. GNL3 and PES1 are localizing to protein aggregates around the chromosomes in the MKI67 KO cells. A. GNL3 (HPA036743). Median overlapKO = 0.539, median overlapWT = 0.795 (P = 9.34x10-9) and median Pearson correlationKO = -0.335, median Pearson correlationWT = 0.134 (P = 3.58x10-6). B. PES1 (HPA040210). Median overlapKO = 0.516, median overlapWT = 0.755 (P = 1.77x10-13) and median Pearson correlationKO = -0.293, median Pearson correlationWT = 0.129 (P = 4.06x10-13). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used.


C . No were observed in MKI67 KO for PWP1 ( HPA038708 ) , despite being localized to mitotic chromosomes in WT . Median overlapKO = 0 . 523 , median overlapWT = 0 . 861 ( P = 1 . 05x10 - 13 ) median Pearson correlationKO = - 0 . 190 , median Pearson correlationWT = 0 . 515 ( P = 8 . 71x10 - 15 ) . Data information : of interest is shown in green DAPI in blue . Scale bar 10 μm . For colocalization data ; biological replicate 1 highlighted in grey biological replicate 2 in green . Both overlap Pearson correlation between DAPI / was measured a two - unpaired Wilcoxon was used . Box limits mark 1st 3rd quantile , whiskers 1 . 5x interquartile range center median .

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C. No protein aggregates were observed in MKI67 KO cells for PWP1 (HPA038708), despite being localized to mitotic chromosomes in WT cells. Median overlapKO = 0.523, median overlapWT = 0.861 (P = 1.05x10-13) and median Pearson correlationKO = -0.190, median Pearson correlationWT = 0.515 (P = 8.71x10-15). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.


D - E . Late recruited do not follow same recruitment pattern in MKI67 KO compared to WT HeLa . arrows indicate where can be seen . D . DENND4A ( HPA065343 ) . Median overlapKO = 0 . 671 , median overlapWT = 0 . 803 ( P = 1 . 02x10 - 4 ) median Pearson correlationKO = - 0 . 224 , median Pearson correlationWT = 0 . 236 ( P = 1 . 91x10 - 5 ) . E . NOL12 ( HPA003547 ) . Median overlapKO = 0 . 468 , median overlapWT = 0 . 820 ( P = 1 . 01x10 - 17 ) median Pearson correlationKO = - 0 . 318 , median Pearson correlationWT = 0 . 381 ( P = 1 . 81x10 - 10 ) . arrow indicates metaphase . Data information : of interest is shown in green DAPI in blue . Scale bar 10 μm . For colocalization data ; biological replicate 1 highlighted in grey biological replicate 2 in green . Both overlap Pearson correlation between DAPI / was measured a two - unpaired Wilcoxon was used . Box limits mark 1st 3rd quantile , whiskers 1 . 5x interquartile range center median .

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D-E. Late recruited proteins do not follow the same recruitment pattern in the MKI67 KO cells compared to WT HeLa cells. White arrows indicate where protein aggregates can be seen. D. DENND4A (HPA065343). Median overlapKO = 0.671, median overlapWT = 0.803 (P = 1.02x10-4) and median Pearson correlationKO = -0.224, median Pearson correlationWT = 0.236 (P = 1.91x10-5). E. NOL12 (HPA003547). Median overlapKO = 0.468, median overlapWT = 0.820 (P = 1.01x10-17) and median Pearson correlationKO = -0.318, median Pearson correlationWT = 0.381 (P = 1.81x10-10). White arrow indicates metaphase cell. Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.


c , Log normalised expression of markers for epithelial ( EPCAM ) , mature luminal epithelial ( ESR1 ) , myoepithelial ( KRT5 , KRT14 ACTA2 ) proliferating cancer ( MKI67 ) .

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c, Log normalised expression of markers for epithelial (EPCAM), mature luminal epithelial (ESR1), myoepithelial (KRT5, KRT14 and ACTA2) and proliferating cancer cells (MKI67).


e , Log normalised expression of markers for fibroblasts ( PDGFRB , THY1 , COL1A1 , ITGB1 S100A4 ) , endothelial ( PECAM1 ) , T - ( CD3D ) , CD8 T ( CD8A ) , T - regulatory ( FOXP3 ) , B - ( MS4A1 ) , myeloid ( CD68 ) plasma ( JCHAIN ) .

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e, Log normalised expression of markers for fibroblasts (PDGFRB, THY1, COL1A1, ITGB1 and S100A4), endothelial cells (PECAM1), T-cells (CD3D), CD8 T cells (CD8A), T-regulatory cells (FOXP3), B-cells (MS4A1), myeloid cells (CD68) and plasma cells (JCHAIN).


c , Log expression of parenchymal gene markers commonly associated with CAFs perivascular .

c, Log expression of parenchymal gene markers commonly associated with CAFs and perivascular cells.


b , Candidate transcriptional drivers of each CAF PVL subset . Violin plots showing log normalised gene expression ( left ) of TF its respective AUC regulon activity ( right ) . TFs ZEB1 FOXP1 enriched in myofibroblast - like CAFs , EGR2 TCF7L2 enriched in inflammatory - like CAFs , MEF2C enriched in PVL NR2F2 enriched in immature - PVL .

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b, Candidate transcriptional drivers of each CAF and PVL subset. Violin plots showing the log normalised gene expression (left) of the TF and its respective AUC regulon activity (right). TFs ZEB1 and FOXP1 enriched in myofibroblast-like CAFs, EGR2 and TCF7L2 enriched in inflammatory-like CAFs, MEF2C enriched in PVL cells and NR2F2 enriched in immature-PVL cells.


c - d , Immunofluorescence of cultured human CAFs ( c ) PVL ( d ) from passage 8 , for CD34 ( CAFs ) , ⍺ - SMA ( myCAFs PVL ) , CD146 ( PVL ) CD36 ( imPVL ) .

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c-d, Immunofluorescence of cultured human CAFs (c) and PVL cells (d) from passage 8, staining for CD34 (CAFs), ⍺-SMA (myCAFs and PVL cells), CD146 (PVL cells) and CD36 (imPVL cells).


e - f , Quantitative of collagen abundance ( e ) orientation ( f ) using second harmonic generation ( SHG ) from derived matrices from stromal subsets representative multiphoton SHG ( n = 3 biological replicates ) . SHG values within each replicate were normalised to SHG of myCAFs . Error bars represent standard deviation . Statistical significance for collagen abundance ( e ) was determined using unpaired two - Student ' s t with equal standard deviation with p ‐ values denoted by asterisks : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 . After normalization of orientation peak distributions ( f ) , statistical significant was determined using a Kruskal - Wallis with Dunn ' s post - hoc multiple comparisons ( p value < 0 . 0001 ) .

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e-f, Quantitative analysis of collagen abundance (e) and orientation (f) using second harmonic generation (SHG) from cellular derived matrices from stromal subsets and representative images multiphoton SHG images (n = 3 biological replicates). All SHG intensity values within each replicate were normalised to the SHG intensity of the myCAFs. Error bars represent standard deviation. Statistical significance for collagen abundance (e) was determined using unpaired two-tailed Student's t test with equal standard deviation with p‐values denoted by asterisks: *p < 0.05, **p < 0.01 and ***p < 0.001. After normalization of the orientation peak distributions (f), statistical significant was determined using a Kruskal-Wallis test with Dunn's post-hoc multiple comparisons test (p value < 0.0001).


, Immunohistochemical of PDGFRβ , ⍺ - SMA , CD34 CD146 in serial sections cut 4 μm apart from matched cases ; - 2 ( g ) were aligned using FIJI . Tumour ( T ) regions are annotated by solid yellow . Co - localisation of CD34 CD146 was used to distinguish blood vessels ( BV ) , where their differential was used to identify CAFs PVL . g , MyCAFs were found to be localised at invasive stromal interface ( insert A ) , whilst iCAFs were located at distal regions ( insert B ) with a high abundance of tumour infiltrating lymphocytes ( TILs ) .

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, Immunohistochemical staining of PDGFRβ, ⍺-SMA, CD34 and CD146 in serial sections cut 4 μm apart from matched cases; Patient-2 (g) Images were aligned using FIJI. Tumour (T) regions are annotated by the solid yellow line. Co-localisation of CD34 and CD146 was used to distinguish blood vessels (BV), where their differential staining was used to identify CAFs and PVL cells. g, MyCAFs were found to be localised at the invasive stromal interface (insert A), whilst iCAFs were located at distal regions (insert B) with a high abundance of tumour infiltrating lymphocytes (TILs).


Immunohistochemical of PDGFRβ , ⍺ - SMA , CD34 CD146 in serial sections cut 4 μm apart from matched cases ; - 4 ( h ) . were aligned using FIJI . Tumour ( T ) regions are annotated by solid yellow . Co - localisation of CD34 CD146 was used to distinguish blood vessels ( BV ) , where their differential was used to identify CAFs PVL . h , Case with a high abundance of perivascular - like ( PVL ) in regions surrounded by blood vessels .

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Immunohistochemical staining of PDGFRβ, ⍺-SMA, CD34 and CD146 in serial sections cut 4 μm apart from matched cases; Patient-4 (h). Images were aligned using FIJI. Tumour (T) regions are annotated by the solid yellow line. Co-localisation of CD34 and CD146 was used to distinguish blood vessels (BV), where their differential staining was used to identify CAFs and PVL cells. h, Case with a high abundance of perivascular-like (PVL) cells in regions surrounded by blood vessels.


c - e , Imputed gene expression of selected candidate signalling molecules identified between four stromal subsets malignant ( c ) epithelial , ( d ) myeloid ( e ) T - . Expression of ligands in stromal clusters are represented on left , with cognate receptors on target on right .

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c-e, Imputed gene expression of selected candidate signalling molecules identified between the four stromal subsets and malignant (c) epithelial, (d) myeloid and (e) T-cells. Expression of ligands in stromal clusters are represented on the left, with cognate receptors on target cells on the right.


b , Representative cohort ( METABRIC ) showing prognostic value of iCAF T - dysfunction signature in context of CTLs for a total of 233 . Kaplan - Meier ' s present two groups of , ' low CTL ' ( blue ) ' high CTL ' ( red ) , as estimated according to average expression of CTL - specific genes stratified as compared to mean . Tumours with low iCAF T - dysfunction signatures ( top ) show with high CTL have a better survival outcome . In contrast , this survival benefit is lost in tumours with a high iCAF T - dysfunction signature ( bottom ) . P - values were defined from Cox proportional hazard ( Cox - PH ) model .

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b, Representative cohort (METABRIC) showing the prognostic value of iCAF T-cell dysfunction signature in the context of CTLs for a total of 233 patients. Kaplan-Meier's present two groups of patients, 'low CTL' (blue line) and 'high CTL' (red line), as estimated according to the average expression of CTL-specific genes and stratified as compared to the mean. Tumours with low iCAF T-cell dysfunction signatures (top) show patients with high CTL levels have a better survival outcome. In contrast, this survival benefit is lost in tumours with a high iCAF T-cell dysfunction signature (bottom). P-values were defined from the Cox proportional hazard (Cox-PH) model.


dPVL profiles CTL exclusion consistent in our study . g - h , Accurate of CTLs ( g ) representative immunohistochemistry for CD8 on matched tumour sections ( h ) . n = 5 stromal 1 mm2 regions were counted per tumour . central band , boxes whiskers represent median , lower / upper quartile min / max CTL per 1mm2 , respectively . P3 is shown as an example of a low dPVL profile with high CTLs . In contrast , P4 has a high dPVL profile with low CTLs . Statistical significance was determined using pairwise comparison with Student ' s t with p ‐ values denoted by asterisks : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 .

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dPVL profiles and CTL exclusion consistent in our study. g-h, Accurate quantification of CTLs (g) and representative immunohistochemistry staining for CD8 on matched patient tumour sections (h). n = 5 stromal 1 mm2 regions were counted per tumour. The central band, boxes and whiskers represent the median, lower/upper quartile and min/max CTL counts per 1mm2, respectively. P3 is shown as an example of a low dPVL profile with high CTLs. In contrast, P4 has a high dPVL profile with low CTLs. Statistical significance was determined using pairwise comparison with Student's t test with p‐values denoted by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.


PIP strip lipid binding with recombinant PTPIP51 _ ΔTM , PTPIP51 _ TPR , PTPIP51 _ ΔTPR .

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PIP strip lipid binding assay with recombinant PTPIP51_ΔTM, PTPIP51_TPR, and PTPIP51_ΔTPR.


PA binding of PTPIP51 ( PTPIP51 _ ΔTM PTPIP51 _ TPR ) was monitored by a PA precipitation a liposome flotation . POPC : POPE : POPA ratios in each liposome were 50 : 50 : 0 , 50 : 40 : 10 , 50 : 30 : 20 , 50 : 20 : 30 . Diagrams of liposome precipitation flotation assays are illustrated separately .

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PA binding of PTPIP51 (PTPIP51_ΔTM and PTPIP51_TPR) was monitored by a PA precipitation assay and a liposome flotation assay. The POPC:POPE:POPA ratios in each liposome were 50:50:0, 50:40:10, 50:30:20, and 50:20:30. Diagrams of the liposome precipitation and flotation assays are illustrated separately.


Results of polarization ( FP ) experiments to monitor interaction between FFAT - like motif - containing peptide of PTPIP51 VAPB _ ΔTM . Kd value of interaction determined in FP experiment was ~ 53 µM . Peptides with mutation of conserved phenylalanine tyrosine residues in FFAT motif were also tested . data are presented as mean ± SD of technical quadruplicate experiments .

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Results of fluorescence polarization (FP) experiments to monitor the interaction between the FFAT-like motif-containing peptide of PTPIP51 and VAPB_ΔTM. The Kd value of the interaction determined in the FP experiment was ~53 µM. Peptides with mutation of conserved phenylalanine and tyrosine residues in the FFAT motif were also tested. The data are presented as the mean ± SD of technical quadruplicate experiments.


CL after PTPIP51 depletion reconstitution were monitored . A doxycycline - induced shRNA expression system was used to deplete PTPIP51 in HeLa . Left , western blot of HeLa in which PTPIP51 was depleted restored . β - ACTIN was used as loading control . Right , mitochondrial CL level ( nmol / mg ) according to PTPIP51 expression . To reconstitute PTPIP51 , full - PTPIP51 gene was transiently expressed . data are presented as mean ± SD of technical triplicate experiments from one representative experiment ( n = 3 ) , p - values calculated using Student ' s t - are shown .

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CL levels after PTPIP51 depletion and reconstitution were monitored. A doxycycline-induced shRNA expression system was used to deplete the PTPIP51 protein in HeLa cells. Left, western blot analysis of HeLa cells in which PTPIP51 was depleted and restored. β-ACTIN was used as the loading control. Right, the mitochondrial CL level (nmol/mg) according to PTPIP51 expression. To reconstitute PTPIP51, the full-length PTPIP51 gene was transiently expressed. The data are presented as the mean ± SD of technical triplicate experiments from one representative experiment (n = 3), and p-values calculated using Student's t-test are shown.


of CL in mitochondria using lipidomic after PTPIP51 depletion reconstitution . Color codes for each sample were presented in lower right subpanel . Experiments were technically repeated three times , results are represented as means ± SD of technical replicates ( n = 3 ) . * p < 0 . 05 ; * * p < 0 . 01 ( Student ' s t - ) . Quality control ( QC ) samples , which were pooled identical aliquots of mitochondria samples , were measured four times throughout run for data reproducibility . Upper right , western blot of HeLa in which PTPIP51 WT or ∆ FFAT mutant were depleted restored . β - Actin was used as a loading control .

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Quantification of CL in mitochondria using lipidomic analysis after PTPIP51 depletion and reconstitution. Color codes for each sample were presented in the lower right subpanel. Experiments were technically repeated three times, and the results are represented as the means ± SD of technical replicates (n = 3). *p < 0.05; **p < 0.01 (Student's t-test). Quality control (QC) samples, which were pooled identical aliquots of the mitochondria samples, were measured four times throughout the run for data reproducibility. Upper right, western blot analysis of HeLa cells in which PTPIP51 WT or ∆FFAT mutant were depleted and restored. β-Actin was used as a loading control.


( A ) Transfer of K18 - ATTO 594 fibrils from donor to H2B - GFP - expressing acceptor . Representative confocal of each population are in upper panels , below are pictures after 24 hours of co - culture of two populations ( left ) , of acceptor treated with conditioned medium from donor for 24 hours ( SN , right ) . In is labelling with WGA ( wheat germ agglutinin ) coupled to Alexa 647 . arrows point to acceptor containing fibrils , scale bars are 10 μm .

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(A) Transfer of K18-ATTO 594 fibrils from donor cells to H2B-GFP-expressing acceptor cells. Representative confocal images of each population are in the upper panels, below are pictures after 24 hours of co-culture of the two populations (left), and of acceptor cells treated with conditioned medium from donor cells for 24 hours (SN, right). In white is the cell membrane labelling with WGA (wheat germ agglutinin) coupled to Alexa 647. The arrows point to acceptor cells containing fibrils, scale bars are 10 μm.


( B ) by flow cytometry of of K18 - ATTO 594 - positive acceptor after co - culturing donor acceptor ( total ) , or culturing acceptor with donor - conditioned medium for 24 hours ( secretion ) . total transfer is arbitrarily set at 100 % , - contact transfer is calculated by subtracting secretion transfer from total transfer . Data represent means ( + SD ) of 4 independent experiments , with statistical by two - unpaired t - ( * * * * P = 4 . 64E - 08 ) .

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(B) Quantification by flow cytometry of the percentage of K18-ATTO 594-positive acceptor cells after co-culturing donor and acceptor cells (total), or culturing acceptor cells with donor-conditioned medium for 24 hours (secretion). The total transfer is arbitrarily set at 100%, and cell-cell contact transfer is calculated by subtracting secretion transfer from total transfer. Data represent the means (+SD) of 4 independent experiments, with statistical analysis by two-tailed unpaired t-test (****P = 4.64E-08).


( C ) by flow cytometry of relative of K18 - ATTO 594 - positive acceptor upon treatment with CK666 during co - culture . Left , data represent means ( 132 % ) + SD , normalized to non - treated co - culture arbitrarily set at 100 % , of 3 independent experiments , with statistical by two - unpaired t - ( * P = 0 . 015 ) . hatched of each bar represents part due to secretion ( respectively 4 . 8 4 . 3 % in absence presence of CK666 ) . Right is same when were cultured in sparse conditions , not allowing - to - contacts ( mean = 47 . 7 % , 3 independent experiments , * P = 0 . 020 ) .

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(C) Quantification by flow cytometry of the relative percentage of K18-ATTO 594-positive acceptor cells upon treatment with CK666 during the co-culture. Left, data represent the means (132%) +SD, normalized to non-treated co-culture arbitrarily set at 100%, of 3 independent experiments, with statistical analysis by two-tailed unpaired t-test (*P = 0.015). The hatched area of each bar represents the part due to secretion (respectively 4.8 and 4.3% in the absence and presence of CK666). Right is the same analysis when the cells were cultured in sparse conditions, not allowing cell-to-cell contacts (mean= 47.7%, 3 independent experiments, *P = 0.020).


( D ) Representative confocal ( 40x ) of CAD treated with 1 µM K18 - ATTO 594 fibrils , 24h after fibrils addition , fixed stained with WGA - Alexa 488 ( green ) DAPI ( blue in merge panels ) . Bottom panels are a bottom slice corresponding to substrate - attached surface of ( z3 ) , upper panels correspond to slice 10 of same picture , ( z10 ) , not attached to substrate ) . On right below z10 pictures , are orthogonal views ( xz yz ) of same region covering 27 slices over 11 μm in total . arrows point to red fibrils into a WGA - positive TNT . Scale bars are 10 μm .

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(D) Representative confocal images (40x) of CAD cells treated with 1 µM K18-ATTO 594 fibrils, 24h after fibrils addition, fixed and stained with WGA-Alexa 488 (green) and DAPI (blue in the merge panels). Bottom panels are a bottom slice corresponding to the substrate-attached surface of cells (z3), upper panels correspond to slice 10 of the same picture, (z10), not attached to the substrate). On the right and below z10 pictures, are the orthogonal views (xz and yz) of the same region covering 27 slices over 11 μm in total. The arrows point to red fibrils into a WGA-positive TNT. Scale bars are 10 μm.


( A ) Representative confocal of DS1 ( expressing soluble Tau RD - YFP ) , non - treated treated with increasing concentrations of non - labelled K18 fibrils , fixed after two days stained with WGA - Alexa 594 ( red ) . arrows point to examples of aggregate - containing , scale bars are 40 μm . On right is of mean + of where green were detected ( total number of analyzed over 3 independent experiments were 1188 for non - treated , 1552 for 0 . 3 μM , 1506 for 1 μM ) , with statistical by one - way ANOVA Bonferroni post hoc ( * * * * P = 6 . 20E - 06 6 . 74E - 05 for 0 to 0 . 3 0 to 1 comparisons respectively ) .

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(A) Representative confocal images of DS1 cells (expressing soluble Tau RD-YFP), non-treated and treated with increasing concentrations of non-labelled K18 fibrils, fixed after two days and stained with WGA-Alexa 594 (red). The arrows point to examples of aggregate-containing cells, scale bars are 40 μm. On the right is the quantification of the mean percentage +SEM of cells where green aggregates were detected (the total number of cells analyzed over 3 independent experiments were 1188 for non-treated, 1552 for 0.3 μM, and 1506 for 1 μM), with statistical analysis by one-way ANOVA and Bonferroni post hoc test (****P = 6.20E-06 and 6.74E-05 for 0 to 0.3 and 0 to 1 comparisons respectively).


( B ) Representative confocal ( 40x ) of RD - YFP SH , non - treated treated with 1 μM K18 - ATTO 594 fibrils , 48h after fibrils addition . Right insets are threefold enlargements of boxed region of treated , merge single channel pictures ; arrows point to partial colocalization . Scale bars are 10 μm , 2 μm in insets . On right is of mean ( 24 % ) + SD of where green were detected ( 2874 treated analyzed over 6 independent experiments ) , with statistical by two - unpaired t - ( * * * * P = 3 . 75E - 07 ) .

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(B) Representative confocal images (40x) of RD-YFP SH cells, non-treated and treated with 1 μM K18-ATTO 594 fibrils, 48h after fibrils addition. Right insets are threefold enlargements of the boxed region of treated cells, merge and single channel pictures; white arrows point to partial colocalization. Scale bars are 10 μm, 2 μm in the insets. On the right is the quantification of the mean percentage (24%) +SD of cells where green aggregates were detected (2874 treated cells analyzed over 6 independent experiments), with statistical analysis by two-tailed unpaired t-test (****P = 3.75E-07).


( C ) Relative proportion of DS9 expressing of Tau RD - YFP over a 50 - hour period monitored by IncuCyte . DS9 were left untreated , graph represents means of 3 independent experiments ± SD .

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(C) Relative proportion of DS9 cells expressing aggregates of Tau RD-YFP over a 50-hour period monitored by IncuCyte. DS9 cells were left untreated, the graph represents means of 3 independent experiments ±SD.


( D ) Monitoring conversion of DS1 into inclusion - containing over a 70 - hour period upon treatment with K18 fibrils . were plated , treated or not with K18 - ATTO 594 fibrils at 1 μM ( time 0 ) placed into IncuCyte incubator for six hours next kept in culture for an additional 60 hours after medium change . scatter plot represents means of 5 independent experiments ( ± SD for green black curves ) .

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(D) Monitoring the conversion of DS1 cells into inclusion-containing cells over a 70-hour period upon treatment with K18 fibrils. Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. The scatter plot represents means of 5 independent experiments (± SD for green and black curves).


were plated , treated or not with K18 - ATTO 594 fibrils at 1 μM ( time 0 ) placed into IncuCyte incubator for six hours next kept in culture for an additional 60 hours after medium change . ( E ) Monitoring conversion of RD - YFP - expressing SH - SY5Y into inclusion - containing over a 70 - hour period upon treatment with K18 fibrils . scatter plot shows means of 3 independent experiments ± SD for green black curves .

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Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. (E) Monitoring the conversion of RD-YFP-expressing SH-SY5Y cells into inclusion-containing cells over a 70-hour period upon treatment with K18 fibrils. the scatter plot shows means of 3 independent experiments ±SD for the green and black curves.


were plated , treated or not with K18 - ATTO 594 fibrils at 1 μM ( time 0 ) placed into IncuCyte incubator for six hours next kept in culture for an additional 60 hours after medium change . ( F ) Monitoring conversion of RD - YFP - expressing SH - SY5Y into inclusion - containing over a 70 - hour period upon treatment with a cortex crude extracts from a AD - deceased . , scatter plot shows means of 2 independent experiments ± SD for green curve . IncuCyte ( 20x objective ) was set to acquire every 30 minutes , phase - contrast green channel ( Excitation 440 - 480 nm , 400 ms ) were acquired ( 9 / well ) . was performed with IncuCyte software to give relative proportion of being converted to inclusion - containing ( end point of treated was arbitrarily set at 100 % , black curve : non - treated , green curve : treated ) .

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Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. (F) Monitoring the conversion of RD-YFP-expressing SH-SY5Y cells into inclusion-containing cells over a 70-hour period upon treatment with a cortex crude extracts from a AD-deceased patient. , the scatter plot shows means of 2 independent experiments ±SD for the green curve. IncuCyte (20x objective) was set to acquire images every 30 minutes, phase-contrast and green channel (Excitation 440-480 nm, 400 ms) were acquired (9 images/well). Analysis was performed with IncuCyte software to give the relative proportion of cells being converted to inclusion-containing cells (the end point of treated cells was arbitrarily set at 100%, black curve: non-treated cells, green curve: treated cells).


( A ) RD - YFP SH were challenged with non - labelled K18 fibrils , incubated 2 days later with Bafilomycin A1 ( second of panels ) , Bortezomide ( bottom panels ) or left non - treated ( NT , upper panels ) for 4 hours before fixation , saponin - permeabilization with antibody recognizing p62 ( red ) WGA ( in left panels ) . are confocal pictures after deconvolution ( 63x , zoom 1 . 8 , px = 60 nm , slice of 0 . 43 μm ) representative of three independent experiments . Insets are threefold enlargements of boxed regions of second column ; arrows point to co - labelling ; scale bars are 10 μm , 2 μm in insets .

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(A) RD-YFP SH cells were challenged with non-labelled K18 fibrils, incubated 2 days later with Bafilomycin A1 (second line of panels), Bortezomide (bottom panels) or left non-treated (NT, upper panels) for 4 hours before fixation, saponin-permeabilization and staining with antibody recognizing p62 (red) and WGA (white in left panels). Images are confocal pictures after deconvolution (63x, zoom 1.8, px size = 60 nm, slice of 0.43 μm) representative of three independent experiments. Insets are threefold enlargements of the boxed regions of second column; white arrows point to co-labelling; scale bars are 10 μm, 2 μm in insets.


( B ) of colocalization of RD - YFP material with p62 - positive structures ( left graph ) or LAMP1 - positive vesicles ( right ) upon K18 fibril - induced aggregation . Confocal pictures were analyzed in 3D with Imaris software from pictures obtained as in A , graphs represent mean ( + ) of green material overlapping with p62 or LAMP1 : if have been converted ( agg ) or soluble material otherwise . Below each graph , corresponding Pearson correlation coefficient ( PCC ) is indicated . number of analyzed over 2 independent experiments is 40 , 51 , 63 , 34 for each respective condition for p62 , 36 , 59 , 44 , 27 for LAMP1 .

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(B) Quantification of colocalization of RD-YFP material with p62-positive structures (left graph) or LAMP1-positive vesicles (right) upon K18 fibril-induced aggregation. Confocal pictures were analyzed in 3D with Imaris software from pictures obtained as in A, the graphs represent the mean percentage (+ SEM) of green material overlapping with p62 or LAMP1: aggregates if cells have been converted (agg) or soluble material otherwise. Below each graph, the corresponding Pearson correlation coefficient (PCC) is indicated. The number of cells analyzed over 2 independent experiments is 40, 51, 63, 34 for each respective condition for p62, 36, 59, 44, 27 for LAMP1.


( A ) RD - YFP SH were challenged with non - labelled K18 fibrils , incubated 2 days later with Bafilomycin A1 ( second of panels ) , Bortezomide ( bottom panels ) or left non - treated ( NT , upper panels ) for 4 hours before fixation , saponin - permeabilization with antibody WGA ( in left panels ) . are confocal pictures after deconvolution ( 63x , zoom 1 . 8 , px = 60 nm , slice of 0 . 43 μm ) representative of three independent experiments . Insets are threefold enlargements of boxed regions of second column ; arrows point to co - labelling ; scale bars are 10 μm , 2 μm in insets . anti - LAMP1 antibody was

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(A) RD-YFP SH cells were challenged with non-labelled K18 fibrils, incubated 2 days later with Bafilomycin A1 (second line of panels), Bortezomide (bottom panels) or left non-treated (NT, upper panels) for 4 hours before fixation, saponin-permeabilization and staining with antibody and WGA (white in left panels). Images are confocal pictures after deconvolution (63x, zoom 1.8, px size = 60 nm, slice of 0.43 μm) representative of three independent experiments. Insets are threefold enlargements of the boxed regions of second column; white arrows point to co-labelling; scale bars are 10 μm, 2 μm in insets. anti-LAMP1 antibody was used


( D ) RD - YFP SH were challenged with AD - derived brain extracts , trypsinized replated 3 days later for an additional 24h before fixation , saponin - permeabilization with antibody recognizing p62 or LAMP1 ( in red ) . are confocal pictures ( 63x , zoom 1 . 6 , px = 60 nm , slice of 0 . 43 μm ) representative of two independent experiments . Insets are threefold enlargements of boxed regions ; arrows point to co - labelling ; scale bars are 10 μm , 2 μm in insets .

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(D) RD-YFP SH cells were challenged with AD-derived brain extracts, trypsinized and replated 3 days later for an additional 24h before fixation, saponin-permeabilization and staining with antibody recognizing p62 or LAMP1 (in red). Images are confocal pictures (63x, zoom 1.6, px size = 60 nm, slice of 0.43 μm) representative of two independent experiments. Insets are threefold enlargements of the boxed regions; white arrows point to co-labelling; scale bars are 10 μm, 2 μm in the insets.


( E ) of colocalization of RD - YFP material with p62 - positive structures ( left graph ) or LAMP1 - positive vesicles ( right ) upon AD extract - induced aggregation . Confocal pictures were analyzed in 3D with Imaris software from pictures obtained as in D , graphs represent mean ( + ) of green material overlapping with p62 or LAMP1 : if have been converted ( agg ) or soluble material otherwise . Below each graph , corresponding Pearson correlation coefficient ( PCC ) is indicated . number of analyzed over 2 independent experiments is 21 ( sol ) , 27 ( agg ) for p62 , 12 ( sol ) , 17 ( agg ) for LAMP1 .

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(E) Quantification of colocalization of RD-YFP material with p62-positive structures (left graph) or LAMP1-positive vesicles (right) upon AD extract-induced aggregation. Confocal pictures were analyzed in 3D with Imaris software from pictures obtained as in D, the graphs represent the mean percentage (+ SEM) of green material overlapping with p62 or LAMP1: aggregates if cells have been converted (agg) or soluble material otherwise. Below each graph, the corresponding Pearson correlation coefficient (PCC) is indicated. The number of cells analyzed over 2 independent experiments is 21 (sol), 27 (agg) for p62, 12 (sol), 17 (agg) for LAMP1.


( F ) Confocal pictures of RD - YFP , treated with Bortezomide phalloidin - Texas red was incubated together with secondary Alexa 647 antibody . Upper panels are bottom slice corresponding to substrate - attached surface of ( z1 ) , below are projections of slices 6 to 9 of same pictures ( z6 - z9 , not attached to substrate ) . Below is a two - fold magnification of framed in merged projection , showing a TNT . arrows point to overlapping between RD - YFP p62 , arrowheads to not colocalizing with p62 inside TNT ; scale bars are 10 μm , 5 μm in insets . Bottom is a 3D reconstruction ( using Imaris software ) of same region .

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(F) Confocal pictures of RD-YFP cells, treated with Bortezomide phalloidin-Texas red was incubated together with the secondary Alexa 647 antibody. Upper panels are the bottom slice corresponding to the substrate-attached surface of cells (z1), below are projections of slices 6 to 9 of the same pictures (z6-z9, not attached to the substrate). Below is a two-fold magnification of the area framed in the merged projection, showing a TNT. White arrows point to overlapping between RD-YFP aggregates and p62, and arrowheads to aggregates not colocalizing with p62 inside the TNT; scale bars are 10 μm, 5 μm in the insets. Bottom is a 3D reconstruction (using Imaris software) of the same region.


( A ) Below schematic representation of experiment are representative confocal of two - day co - cultures of RD - YFP SH ( acceptor ) with K18 - ATTO 594 fibrils - treated SH - SY5Y ( donor , upper panel ) . Bottom panels are acceptor cultured with supernatant of K18 - ATTO 594 fibrils - treated SH - SY5Y ( donor SN ) . WGA ( ) labels DAPI nuclei in merge panels . Arrows point to with green red , scale bars are 20 μm . ( B ) of seeding after transfer in RD - YFP SH in experiments described in ( A ) , depending on condition ( donor or donor SN ) . was performed using ICY software , data represent number of aggregate - containing over total number of green ± SD ( total number of RD - YFP analyzed over 3 independent experiments were 1011 for co - culture 1265 for SN ) with statistical by two - unpaired t - ( * * * * P = 4 . 18E - 07 ) .

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(A) Below the schematic representation of the experiment are representative confocal images of two-day co-cultures of RD-YFP SH cells (acceptor cells) with K18-ATTO 594 fibrils-treated SH-SY5Y cells (donor cells, upper panel). Bottom panels are acceptor cells cultured with the supernatant of K18-ATTO 594 fibrils-treated SH-SY5Y cells (donor SN). WGA (white) labels cell membrane and DAPI the nuclei in the merge panels. Arrows point to cells with green and red aggregates, scale bars are 20 μm. (B) Quantification of seeding after transfer in RD-YFP SH cells in the experiments described in (A), depending on the condition (donor cells or donor SN). Analysis was performed using ICY software, data represent the number of aggregate-containing cells over the total number of green cells ±SD (the total number of RD-YFP cells analyzed over 3 independent experiments were 1011 for co-culture and 1265 for SN) with statistical analysis by two-tailed unpaired t-test (****P =4.18E-07).


( C ) Below schematic representation of experiment are representative confocal showing maximal projections of 6 z - slices ( covering 2 μm of thickness ) of 24 - hour co - culture of RD - YFP SH ( acceptor ) with RD - YFP SH expressing nls - Red challenged with K18 - ATTO 594 fibrils ( donor ) . WGA ( ) labels DAPI nuclei in merge panel . Stars label donor containing green red , arrows indicate an acceptor with green red , arrowheads show an acceptor with green but devoid of red fibrils . Below are two - fold enlargement of respective framed . apparent discontinuity in picture corresponds to boundary between two adjacent tiles . Scale bars are 10 μm , 5 μm in enlargements .

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(C) Below the schematic representation of the experiment are representative confocal images showing maximal intensity projections of 6 z-slices (covering 2 μm of thickness) of 24-hour co-culture of RD-YFP SH cells (acceptor cells) with RD-YFP SH cells expressing nls-Red and challenged with K18-ATTO 594 fibrils (donor cells). WGA (white) labels cell membrane and DAPI the nuclei in the merge panel. Stars label donor cells containing green and red aggregates, arrows indicate an acceptor cell with green and red aggregates, and the arrowheads show an acceptor cell with green aggregates but devoid of red fibrils. Below are two-fold enlargement of the respective framed cells. The apparent discontinuity in the picture corresponds to the boundary between two adjacent tiles. Scale bars are 10 μm, 5 μm in the enlargements.


( D ) using ICY software of of containing red fibrils among population of containing green . graph shows mean percentages with , respectively 66 . 3 % 36 . 9 % for donor acceptor , statistical was performed by two - unpaired t - ( * * * * p = 3 . 65E - 05 ) .

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(D) Analysis using ICY software of the percentage of cells containing red fibrils among the population of cells containing green aggregates. The graph shows the mean percentages with SEM, respectively 66.3% and 36.9% for donor and acceptor cells, statistical analysis was performed by two-tailed unpaired t-test (**** p =3.65E-05).


( E ) Dot plot showing number of red fibrils per with among population of containing green red . Each analyzed is represented ( note that because of chosen scale , 6 data points are outside axis limits ) , bars indicate means ± ( respectively 3 . 9 1 . 9 for donor acceptor ) , statistical was performed by two - unpaired t - ( * * p = 0 . 0015 ) .

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(E) Dot plot showing the number of red fibrils per cell with SEM among the population of cells containing green and red aggregates. Each analyzed cell is represented (note that because of the chosen scale, 6 data points are outside the axis limits), the bars indicate the means ± SEM (respectively 3.9 and 1.9 for donor and acceptor cells), statistical analysis was performed by two-tailed unpaired t-test (** p=0.0015).


( F ) of number of green dots per among population of containing green . Each analyzed is represented on dot plot , bars indicate means ± ( respectively 30 . 2 , 26 . 1 , 32 . 0 , 32 . 2 ) . Statistical was performed by one - way ANOVA Tukey post - hoc pair - wise comparisons were not significant ( n . s . ) .

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(F) Analysis of the number of green dots per cell among the population of cells containing green aggregates. Each analyzed cell is represented on the dot plot, the bars indicate the means ± SEM (respectively 30.2, 26.1, 32.0, and 32.2). Statistical analysis was performed by one-way ANOVA and Tukey post-hoc test and all the pair-wise comparisons were not significant (n.s.).


( A ) Below schematics of experiment are representative confocal of donor neuron - like ( CAD labelled with Tracker Green ( CTG ) ) acceptor neurons after 24 hours in co - culture . upper panels show control conditions ( non - challenged CAD , CTL ) , bottom panels show donor CTG - CADs that were loaded with K18 - ATTO 594 fibrils prior to co - culture with cortical neurons . are representative Z - stack projections covering whole of donor acceptor . In merged , show neurons labelled with MAP - 2 , green are CTG - labelled donor CAD , red corresponds to K18 - ATTO 594 nuclei are stained in blue . single channel pictures are gray scale . Yellow arrowheads point to K18 inside acceptor . MAP - 2 K18 - ATTO 594 panels are threefold enlargements of boxed regions in merged pictures . On right are orthogonal views of same regions covering 14 slices . Scale bars are 20 μm in merge panels , 5 μm in insets orthogonal views .

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(A) Below the schematics of the experiment are representative confocal images of donor neuron-like cells (CAD cells labelled with Cell Tracker Green (CTG)) and acceptor primary neurons after 24 hours in co-culture. The upper panels show control conditions (non-challenged CAD cells, CTL), and the bottom panels show donor CTG-CADs that were loaded with K18-ATTO 594 fibrils prior to co-culture with primary cortical neurons. The images are representative Z-stack projections covering the whole cell body of donor and acceptor cells. In the merged images, white show neurons labelled with MAP-2, green are CTG-labelled donor CAD cells, red corresponds to K18-ATTO 594 puncta and nuclei are stained in blue. The single channel pictures are gray scale images. Yellow arrowheads point to K18 puncta inside acceptor cells. MAP-2 and K18-ATTO 594 panels are threefold enlargements of the boxed regions in the merged pictures. On the right are the orthogonal views of the same regions covering 14 slices. Scale bars are 20 μm in the merge panels, and 5 μm in the insets and orthogonal views.


( B ) Below schematics of experiment are representative confocal of acceptor neurons ( labelled by MAP - 2 ) cultured with donor , consisting in RD - YFP SH , first K18 - challenged for 2 days , therefore expressing RD - YFP ( upper panels ) or with supernatant ( SN ) of donor ( lower panels ) after 24 hours . single channel pictures are gray scale . Yellow arrowheads point to Tau RD - YFP inside acceptor , red arrowheads point to inside donor . Scale bars are 20 μm .

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(B) Below the schematics of the experiment are representative confocal images of acceptor primary neurons (labelled by MAP-2) cultured with donor cells, consisting in RD-YFP SH cells, first K18-challenged for 2 days, therefore expressing RD-YFP aggregates (upper panels) or with supernatant (SN) of donor cells (lower panels) after 24 hours. The single channel pictures are gray scale images. Yellow arrowheads point to Tau RD-YFP puncta inside acceptor cells, red arrowheads point to aggregates inside donor cells. Scale bars are 20 μm.


( C ) Below schematics of co - culture experiment are representative confocal of neuron - like ( CTG - CADs growing on top of organotypic slices ( n = 6 ) , in which astrocytes were labelled with GFAP . upper panels show control CTG - CADs middle panels show donor CTG - CADs loaded with K18 - ATTO 594 fibrils , co - cultured with an acceptor hippocampal slice . are representative Z - stack projections covering whole of donor . In merged , are acceptor astrocytes ( GFAP positive ) , green are donor CTG - CADs , red are K18 - ATTO 594 nuclei are stained in blue . Insets are threefold enlargements of boxed region in merged picture . orthogonal views of bottom panels show 25 slices of K18 - ATTO 594 fibrils in donor ( 1 ) , or transfer of K18 - ATTO 594 fibrils from donor CTG - CADs to hippocampal astrocytes of organotypic slice ( 2 to 5 ) . Yellow arrowheads point to Tau inside acceptor astrocytes . Orthogonal views show each of five K18 - ATTO 594 positive - contained inside acceptor as indicated in inset of middle panel . Scale bars are 100 μm in merged panel 10 μm in insets .

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(C) Below the schematics of the co-culture experiment are representative confocal images of neuron-like cells (CTG-CADs growing on top of organotypic slices (n=6), in which the astrocytes were labelled with GFAP. The upper panels show control CTG-CADs and the middle panels show donor CTG-CADs loaded with K18-ATTO 594 fibrils, co-cultured with an acceptor hippocampal slice. The images are representative Z-stack projections covering the whole cell body of donor cells. In the merged images, white are acceptor astrocytes (GFAP positive), green are donor CTG-CADs, red are K18-ATTO 594 puncta and nuclei are stained in blue. Insets are threefold enlargements of the boxed region in the merged picture. The orthogonal views of the bottom panels show 25 slices of K18-ATTO 594 fibrils in donor cells (1), or transfer of K18-ATTO 594 fibrils from donor CTG-CADs to hippocampal astrocytes of the organotypic slice (2 to 5). Yellow arrowheads point to Tau puncta inside acceptor astrocytes. Orthogonal views show each of the five K18-ATTO 594 positive-puncta contained inside acceptor cells as indicated in the inset of the middle panel. Scale bars are 100 μm in the merged panel and 10 μm in the insets.


( C ) Below schematics of co - culture experiment are representative confocal of neuron - like ( CTG - CADs as in A ) growing on top of organotypic slices ( n = 6 ) , in which astrocytes were labelled with upper panels show control CTG - CADs middle panels show donor CTG - CADs loaded with K18 - ATTO 594 fibrils , co - cultured with an acceptor hippocampal slice . are representative Z - stack projections covering whole of donor . In merged , are acceptor astrocytes , green are donor CTG - CADs , red are K18 - ATTO 594 nuclei are stained in blue . Insets are threefold enlargements of boxed region in merged picture . of K18 - ATTO 594 fibrils in donor ( 1 ) , or transfer of K18 - ATTO 594 fibrils from donor CTG - CADs to hippocampal astrocytes of organotypic slice ( 2 to 5 ) . Yellow arrowheads point to Tau inside acceptor astrocytes . Orthogonal views show each of five K18 - ATTO 594 positive - contained inside acceptor as indicated in inset of middle panel . Scale bars are 100 μm in merged panel 10 μm in insets . neurons in slices were labelled with dendrite marker MAP - 2 axonal marker β - III - tubulin ( ) , orthogonal views show 26 slices .

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(C) Below the schematics of the co-culture experiment are representative confocal images of neuron-like cells (CTG-CADs as in A) growing on top of organotypic slices (n=6), in which the astrocytes were labelled with The upper panels show control CTG-CADs and the middle panels show donor CTG-CADs loaded with K18-ATTO 594 fibrils, co-cultured with an acceptor hippocampal slice. The images are representative Z-stack projections covering the whole cell body of donor cells. In the merged images, white are acceptor astrocytes , green are donor CTG-CADs, red are K18-ATTO 594 puncta and nuclei are stained in blue. Insets are threefold enlargements of the boxed region in the merged picture. of K18-ATTO 594 fibrils in donor cells (1), or transfer of K18-ATTO 594 fibrils from donor CTG-CADs to hippocampal astrocytes of the organotypic slice (2 to 5). Yellow arrowheads point to Tau puncta inside acceptor astrocytes. Orthogonal views show each of the five K18-ATTO 594 positive-puncta contained inside acceptor cells as indicated in the inset of the middle panel. Scale bars are 100 μm in the merged panel and 10 μm in the insets. the neurons in the slices were labelled with the dendrite marker MAP-2 and the axonal marker β-III-tubulin (white), and the orthogonal views show 26 slices.


( B ) Enrichment profiles of TEAD4 AP - 1 consensus motifs in 600 bp regions centred on ZEB1 peak summits . two grey dashed lines highlight 200 bp regions around ZEB1 peak summits which were used for HOMER known motif

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(B) Enrichment profiles of TEAD4 and AP-1 consensus motifs in 600 bp regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the 200 bp regions around the ZEB1 peak summits which were used for HOMER known motif analysis


( D ) Enrichment profile of JUN peak summit positions in 1 kb regions centred on ZEB1 peak summits . two grey dashed lines highlight region of + / - 200 bp around ZEB1 peak summit positions where JUN peaks are counted as overlapping with ZEB1 peaks .

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(D) Enrichment profile of JUN peak summit positions in 1 kb regions centred on the ZEB1 peak summits. The two grey dashed lines highlight the region of +/- 200 bp around the ZEB1 peak summit positions where JUN peaks are counted as overlapping with ZEB1 peaks.


( E ) representing signal enrichment of JUN ChIP - seq reads in 2 kb regions centred on ZEB1 peak summits .

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(E) Heatmap representing the signal enrichment of JUN ChIP-seq reads in 2 kb regions centred on the ZEB1 peak summits.


( A ) In situ proximity ligation ( PLA ) of ZEB1 with JUN , FOSL1 TAZ shows close proximity of ZEB1 with JUN FOSL1 but not TAZ in nucleus of MDA - MB - 231 indicated by red dots . As negative control ZEB1 was transiently knocked down by siRNA . In left panel representative microscopic are shown . Scale bar = 20 µm . of PLA is shown on right .

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(A) In situ proximity ligation assay (PLA) of ZEB1 with JUN, FOSL1 and TAZ shows close proximity of ZEB1 with JUN and FOSL1 but not TAZ in the nucleus of MDA-MB-231 cells indicated by red fluorescent dots. As negative control ZEB1 was transiently knocked down by siRNA. In the left panel representative microscopic images are shown. Scale bar = 20 µm. Quantification of the PLA is shown on the right.


( B ) Co - immunoprecipitation ( Co - IP ) of endogenous ZEB1 FOSL1 or JUN in MDA - MB - 231 shows co - precipitation of ZEB1 with FOSL1 JUN .

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(B) Co-immunoprecipitation (Co-IP) of endogenous ZEB1 and FOSL1 or JUN in MDA-MB-231 cells shows co-precipitation of ZEB1 with FOSL1 and JUN.


( A ) Definition of two different ZEB1 peak subsets on presence or absence of overlapping YAP JUN peaks . two illustrative genome browser show one example of a ZEB1 - only peak one example of a ZEB1 / YAP / JUN peak ( at EFEMP1 gene within SHROOM3 gene , respectively ) . Control ( ctrl ) is input for ZEB1 IgG for YAP JUN ChIP - seqs . ChIP - seqs respective controls were scaled accordingly .

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(A) Definition of two different ZEB1 peak subsets based on the presence or absence of overlapping YAP and JUN peaks. The two illustrative genome browser images show one example of a ZEB1-only peak and one example of a ZEB1/YAP/JUN peak (at the EFEMP1 gene and within the SHROOM3 gene, respectively). Control (ctrl) is the input for ZEB1 and IgG for the YAP and JUN ChIP-seqs. ChIP-seqs and respective controls were scaled accordingly.


( C ) Localisation of ZEB1 peak summits relative to closest TSS .

(C) Localisation of ZEB1 peak summits relative to the closest TSS.


( A ) qRT - PCR in TGFß treated MCF10A after siRNA mediated knockdown of ZEB1 , FOSL1 or YAP compared to control knockdown . Knockdown efficiency of three factors is shown on left . right panel shows their effects on 11 ZEB1 / YAP / AP - 1 activated target genes .

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(A) qRT-PCR in TGFß treated MCF10A cells after siRNA mediated knockdown of ZEB1, FOSL1 or YAP compared to control knockdown cells. Knockdown efficiency of all three factors is shown on the left. The right panel shows their effects on 11 ZEB1/YAP/AP-1 activated target genes.