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 .

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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 cells exemplify proteins 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 staining in U - 2 OS cells ( 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 the fibrillar center and nucleoplasm in GFP - tagged HeLa cells ( magenta ) , also supported by IF antibody staining 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 . The multilocalizing ribosomal protein RPL13 detected in the nucleoli , cytosol and ER in MCF - 7 cells ( 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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 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 .

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 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 .

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 : 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 and ACTA2 ) and proliferating cancer cells ( 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 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 ) .

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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 and perivascular cells .

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


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 .

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

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 assay with recombinant PTPIP51 _ ΔTM , PTPIP51 _ TPR , and 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 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 .

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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 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 .

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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 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 .

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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.


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 .

O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 ) 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 ) .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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 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 .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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 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 ) .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 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 ) .

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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).


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 .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

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.


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 ) .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 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 .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) 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 ) 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 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O

(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 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

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O

(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 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 .

O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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 ) 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 .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O

(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 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 .

O O O O O O O O O O O O O B-CONTROLLED_VAR B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) 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 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 ) .

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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 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 .

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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 ) 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 ) .

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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 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 ) .

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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 ) 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 . ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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

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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 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 .

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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 ) Heatmap representing the signal enrichment of JUN ChIP - seq reads in 2 kb regions centred on the 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 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 .

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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 and FOSL1 or JUN in MDA - MB - 231 cells shows co - precipitation of ZEB1 with FOSL1 and 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 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 .

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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 the closest TSS .

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