Figure 3B . 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 . 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 . 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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Figure 3B. 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.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.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.


Figure 4A . 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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Figure 4A. 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.


Figure 5C - 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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Figure 5C-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.


Figure 6A . 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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Figure 6A. 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.


Figure 7A - 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 . 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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Figure 7A-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.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.


Figure 1c , 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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Figure 1c, 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).


Figure 2c , Log expression of parenchymal gene markers commonly associated with CAFs and perivascular cells .

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Figure 2c, Log expression of parenchymal gene markers commonly associated with CAFs and perivascular cells.


Figure 3b , 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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Figure 3b, 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.


Figure 4c - 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 ) . , 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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Figure 4c-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)., 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.


Figure 5c - 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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Figure 5c-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.


Figure 6b , 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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Figure 6b, 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.


Figure 3PIP 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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Figure 3PIP 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.


Figure 4Results 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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Figure 4Results 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.


Figure 5CL 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5CL 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.


Figure 1 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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).(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.


Figure 2 ( 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 ) . ( 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 ) . 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 ) .

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Figure 2(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).(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).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).


Figure 3 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.(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.(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.


Figure 4 ( 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 . ( 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 ) . ( 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 . ) .

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

Figure 4(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.(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).(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.).


Figure 5 ( 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 . ( 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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Figure 5(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.(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.


Figure 1 ( 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 . ( 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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Figure 1(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.(E) Heatmap representing the signal enrichment of JUN ChIP-seq reads in 2 kb regions centred on the ZEB1 peak summits.


Figure 2 ( 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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Figure 2(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.


Figure 3 ( 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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Figure 3(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.


Figure 4 ( 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 . ( B ) Overlap of ZEB1 , YAP and JUN peaks at the promoter of CYR61 , CTGF and ANKRD1 and in a known enhancer region of the DOCK9 gene . ( C ) ZEB1 - only peak at the promoter of LLGL2 , a well - known ZEB1 repressed target . ( D ) Luciferase reporter assay in MDA - MB - 231 cells comparing control and transient siRNA - mediated knockdown of ZEB1 . Reporter constructs of regulatory regions of genes For reporter assays , firefly luciferase activity was normalized to co - transfected renilla luciferase . ( E ) Luciferase reporter assays with the ANKRD1 promoter and DOCK9 enhancer constructs in MCF7 cells upon overexpression of ZEB1 , YAP and JUN / FOSL1 ( AP - 1 ) or different combinations of the factors . n = 5 ( ANKRD1 ) and n = 3 ( DOCK9 ) ; shown is mean ± s . e . m . ; * P ≤ 0 . 05 ; ratio paired t - test . For reporter assays , firefly luciferase activity was normalized to co - transfected renilla luciferase .

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

Figure 4(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.(B) Overlap of ZEB1, YAP and JUN peaks at the promoter of CYR61, CTGF and ANKRD1 and in a known enhancer region of the DOCK9 gene.(C) ZEB1-only peak at the promoter of LLGL2, a well-known ZEB1 repressed target.(D) Luciferase reporter assay in MDA-MB-231 cells comparing control and transient siRNA-mediated knockdown of ZEB1. Reporter constructs of regulatory regions of genes For reporter assays, firefly luciferase activity was normalized to co-transfected renilla luciferase.(E) Luciferase reporter assays with the ANKRD1 promoter and DOCK9 enhancer constructs in MCF7 cells upon overexpression of ZEB1, YAP and JUN/FOSL1 (AP-1) or different combinations of the factors. n = 5 (ANKRD1) and n = 3 (DOCK9); shown is mean ± s.e.m.; *P ≤ 0.05; ratio paired t-test. For reporter assays, firefly luciferase activity was normalized to co-transfected renilla luciferase.


Figure 5 ( C ) The upper panel shows a schematic representation of the ANKRD1 luciferase reporter constructs . The lower panel shows luciferase reporter assays with these different ANKRD1 promoter constructs in MCF7 cells upon overexpression of ZEB1 , YAP and JUN / FOSL1 or different combinations of the factors . n = 4 - 5 ; shown is mean ± s . e . m . ; * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; ratio paired t - test . Firefly luciferase activity was normalized to co - transfected renilla luciferase . ( D ) Left panel : EMSA using nuclear extracts from MDA - MB - 231 and labelled TEAD binding side as probe ( black arrow ) . Competition of specific binding complex ( read arrow ) with cold probe ( c ) , anti - JUN , - panTEAD , and - ZEB1 antisera . Right panel : recombinant GST - ZEB1 , but not GST - Ctrl , only binds to Z - box .

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

Figure 5(C) The upper panel shows a schematic representation of the ANKRD1 luciferase reporter constructs. The lower panel shows luciferase reporter assays with these different ANKRD1 promoter constructs in MCF7 cells upon overexpression of ZEB1, YAP and JUN/FOSL1 or different combinations of the factors. n = 4-5; shown is mean ± s.e.m.; *P ≤ 0.05; **P ≤ 0.01; ratio paired t-test. Firefly luciferase activity was normalized to co-transfected renilla luciferase.(D) Left panel: EMSA using nuclear extracts from MDA-MB-231 and labelled TEAD binding side as probe (black arrow). Competition of specific binding complex (read arrow) with cold probe (c), anti-JUN, -panTEAD, and -ZEB1 antisera. Right panel: recombinant GST-ZEB1, but not GST-Ctrl, only binds to Z-box.


Figure 6 ( B ) Comparison of ZEB1 ChIP - seq peaks with ZEB1 - dependent changes in chromatin activation status identified by ATAC - seq on control and ZEB1 knock - down MDA - MB - 231 cells . Percentage of ZEB1 - only ( n = 5 , 963 ) and ZEB1 / YAP / JUN peaks ( n = 1 , 993 ) falling into ZEB1 repressed or activated genomic regions are shown . ( C ) Genome browser tracks of ZEB1 , YAP and JUN ChIP - seq signal intensity in wild type cells overlaid to ATAC - seq signal intensity in wild type , control ( shCtrl ) and ZEB1 ( shZEB1 ) knock - down MDA - MB - 231 cells . One known ZEB1 repressed target ( CDH1 ) and one activated target ( ANKRD1 ) are shown .

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

Figure 6(B) Comparison of ZEB1 ChIP-seq peaks with ZEB1-dependent changes in chromatin activation status identified by ATAC-seq on control and ZEB1 knock-down MDA-MB-231 cells. Percentage of ZEB1-only (n=5,963) and ZEB1/YAP/JUN peaks (n=1,993) falling into ZEB1 repressed or activated genomic regions are shown.(C) Genome browser tracks of ZEB1, YAP and JUN ChIP-seq signal intensity in wild type cells overlaid to ATAC-seq signal intensity in wild type, control (shCtrl) and ZEB1 (shZEB1) knock-down MDA-MB-231 cells. One known ZEB1 repressed target (CDH1) and one activated target (ANKRD1) are shown.


Figure 7 ( A ) Correlation of ZEB1 mRNA expression with the expression of YAP , FOSL1 and JUN across all breast cancer cell lines included in the cancer cell line encyclopedia ( CCLE ) . rs : Spearman ' s rank correlation coefficient . ( B ) Heatmap of RNA - seq expression data of all CCLE breast cancer cell lines .

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

Figure 7(A) Correlation of ZEB1 mRNA expression with the expression of YAP, FOSL1 and JUN across all breast cancer cell lines included in the cancer cell line encyclopedia (CCLE). rs: Spearman's rank correlation coefficient.(B) Heatmap of RNA-seq expression data of all CCLE breast cancer cell lines.


Figure 8 ( A ) Kaplan Meier plots from survival analyses of human ER - / PR - breast cancers showing distant metastasis free survival ( DMFS ) based on the expression of the indicated genes or gene combinations . HR : hazard ratio . p - value from log - rank test . ( B ) Hypergeometric testing showed a significant association of nuclear ZEB1 and FOSL1 expression on the protein level as determined by immunohistochemistry on 81 human breast cancer samples . The upper panel shows staining of a serial section of a negative and a positive case . Note that stromal cells ( asterisks ) are negative for FOSL1 , and partially positive for ZEB1 and YAP , and that expression of ZEB1 in tumour cells ( arrows ) of positive cases is weaker than in stromal cells . Odds ratio ( OR ) : measure of association between categorical variables with OR > 1 indicating a positive association . ( C ) Heatmap of mRNA expression data of ZEB1 , YAP , JUN and 9 selected activated ZEB1 / YAP / AP - 1 target genes in breast cancer patient samples ( GSE18229 ) . Genes were selected for known roles in tumour promoting properties like cell migration . Tumours in which ≥ 6 out of 9 genes of the selected ZEB1 / YAP / AP - 1 target gene set were expressed higher than the 60th percentile of expression were defined as " tumour promoting gene set high " ; when expression of ≥ 6 out of the 9 genes was below the 40th percentile , tumours were defined as " tumour promoting gene set low " . Tumours were annotated according to their molecular subtype . ( E ) Kaplan Meier plots from survival analyses of human breast cancers patient samples ( GSE18229 ) showing relapse free survival ( RFS ) based on the combined expression of the 9 selected ZEB1 / YAP / AP - 1 activated targets HR : hazard ratio . p - value from log - rank test . ( F ) Kaplan Meier plots from survival analyses of human ER - / PR - breast cancers showing distant metastasis free survival ( DMFS ) based on the combined expression of the 9 selected ZEB1 / YAP / AP - 1 activated targets HR : hazard ratio . p - value from log - rank test .

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

Figure 8(A) Kaplan Meier plots from survival analyses of human ER-/PR- breast cancers showing distant metastasis free survival (DMFS) based on the expression of the indicated genes or gene combinations. HR: hazard ratio. p-value from log-rank test.(B) Hypergeometric testing showed a significant association of nuclear ZEB1 and FOSL1 expression on the protein level as determined by immunohistochemistry on 81 human breast cancer samples. The upper panel shows staining of a serial section of a negative and a positive case. Note that stromal cells (asterisks) are negative for FOSL1, and partially positive for ZEB1 and YAP, and that expression of ZEB1 in tumour cells (arrows) of positive cases is weaker than in stromal cells. Odds ratio (OR): measure of association between categorical variables with OR > 1 indicating a positive association.(C) Heatmap of mRNA expression data of ZEB1, YAP, JUN and 9 selected activated ZEB1/YAP/AP-1 target genes in breast cancer patient samples (GSE18229). Genes were selected for known roles in tumour promoting properties like cell migration. Tumours in which ≥ 6 out of 9 genes of the selected ZEB1/YAP/AP-1 target gene set were expressed higher than the 60th percentile of expression were defined as "tumour promoting gene set high"; when expression of ≥ 6 out of the 9 genes was below the 40th percentile, tumours were defined as "tumour promoting gene set low". Tumours were annotated according to their molecular subtype.(E) Kaplan Meier plots from survival analyses of human breast cancers patient samples (GSE18229) showing relapse free survival (RFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets HR: hazard ratio. p-value from log-rank test. (F) Kaplan Meier plots from survival analyses of human ER-/PR- breast cancers showing distant metastasis free survival (DMFS) based on the combined expression of the 9 selected ZEB1/YAP/AP-1 activated targets HR: hazard ratio. p-value from log-rank test.


figf1 ( a ) Representative images of rat liver ( FAO ) cells immunostained with TSC2 , TSC1 or Rheb ( green ) and PMP70 ( peroxisome marker ) or LAMP1 ( lysosome marker ) ( red ) antibodies . Scale bars , 10 μm . ( b ) Pearson ' s correlation coefficient for TSC1 or TSC2 co - localization with PMP70 or LAMP1 calculated using Imaris software . Quantification was performed on 8 - 12 cells from each of the 4 independent experiments , giving rise to a total of 40 cells . All error bars represent s . e . m . , * * * P0 . 001 . ( c ) Representative images using HeLa cells transfected with Myc - TSC1 and Flag - TSC2 wild type ( WT ) and stained with anti - Flag ( green ) and anti - Myc ( red ) antibodies . Scale bar , 10 μm . ( d ) Representative images of HepG2 cells transfected with control or TSC2 siRNA immunostained for TSC2 ( green ) and PMP70 ( red ) . Individual boundaries are shown to identify cells with TSC2 knockdown ( white ) versus cells that retain TSC2 ( yellow ) . Scale bars , 10 μm . ( e ) Corresponding immunoblots for HepG2 cells transfected with control or TSC2 siRNA showing the extent of knockdown ( average over population of cells ) . ( f ) HepG2 cells from d were analysed for Pearson ' s correlation coefficient of TSC2 co - localization with PMP70 using Imaris software . Quantification was performed on 8 - 12 cells from each of the 4 independent experiments , giving rise to a total of 40 cells . All error bars represent s . e . m . , * * * P0 . 001 . Uncropped images of western blots are shown in Supplementary Fig . S6 . Source data for the statistical analysis are shown in Supplementary Table S1 .

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

figf1(a) Representative images of rat liver (FAO) cells immunostained with TSC2, TSC1 or Rheb (green) and PMP70 (peroxisome marker) or LAMP1 (lysosome marker) (red) antibodies. Scale bars, 10 μm. (b) Pearson's correlation coefficient for TSC1 or TSC2 co-localization with PMP70 or LAMP1 calculated using Imaris software. Quantification was performed on 8-12 cells from each of the 4 independent experiments, giving rise to a total of 40 cells. All error bars represent s.e.m., ***P0.001.(c) Representative images using HeLa cells transfected with Myc-TSC1 and Flag-TSC2 wild type (WT) and stained with anti-Flag (green) and anti-Myc (red) antibodies. Scale bar, 10 μm.(d) Representative images of HepG2 cells transfected with control or TSC2 siRNA immunostained for TSC2 (green) and PMP70 (red). Individual boundaries are shown to identify cells with TSC2 knockdown (white) versus cells that retain TSC2 (yellow). Scale bars, 10 μm. (e) Corresponding immunoblots for HepG2 cells transfected with control or TSC2 siRNA showing the extent of knockdown (average over population of cells). (f) HepG2 cells from d were analysed for Pearson's correlation coefficient of TSC2 co-localization with PMP70 using Imaris software. Quantification was performed on 8-12 cells from each of the 4 independent experiments, giving rise to a total of 40 cells. All error bars represent s.e.m., ***P0.001. Uncropped images of western blots are shown in Supplementary Fig. S6. Source data for the statistical analysis are shown in Supplementary Table S1.


figf2 ( a , b ) Subcellular fractionation of FAO ( a ) cells demonstrated the localization of TSC2 , TSC1 , Rheb , TBC1D7 ( FAO , a ) and AKT in various subcellular compartments . Catalase , PMP70 and lamin A / C were used as subcellular markers for the peroxisome ( P ) and nuclear ( N ) fractions , respectively . EEA1 , LAMP1 and VDAC were used as markers for endosomes , lysosomes and mitochondria , respectively . WCE , whole - cell extracts ; M , membrane ; C , cytosol . ( a , b ) Subcellular fractionation of HepG2 ( b ) cells demonstrated the localization of TSC2 , TSC1 , Rheb , TBC1D7 ( FAO , a ) and AKT in various subcellular compartments . Catalase , PMP70 and lamin A / C were used as subcellular markers for the peroxisome ( P ) and nuclear ( N ) fractions , respectively . EEA1 , LAMP1 and VDAC were used as markers for endosomes , lysosomes and mitochondria , respectively . WCE , whole - cell extracts ; M , membrane ; C , cytosol . ( c ) Subcellular fractionation of TSC2 + / + and TSC2 − / − mouse embryonic fibroblasts ( MEFs ) demonstrating the localization of TSC2 , TSC1 , Rheb and AKT . PMP70 , catalase ( peroxisome fraction , P ) , LDH ( cytosolic fraction , C ) , β - integrin ( membrane fraction , M ) and lamin A / C ( nuclear fraction , N ) were used as subcellular markers . Uncropped images of western blots are shown in Supplementary Fig . S6 .

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

figf2(a,b) Subcellular fractionation of FAO (a) cells demonstrated the localization of TSC2, TSC1, Rheb, TBC1D7 (FAO, a) and AKT in various subcellular compartments. Catalase, PMP70 and lamin A/C were used as subcellular markers for the peroxisome (P) and nuclear (N) fractions, respectively. EEA1, LAMP1 and VDAC were used as markers for endosomes, lysosomes and mitochondria, respectively. WCE, whole-cell extracts; M, membrane; C, cytosol.(a,b) Subcellular fractionation of HepG2 (b) cells demonstrated the localization of TSC2, TSC1, Rheb, TBC1D7 (FAO, a) and AKT in various subcellular compartments. Catalase, PMP70 and lamin A/C were used as subcellular markers for the peroxisome (P) and nuclear (N) fractions, respectively. EEA1, LAMP1 and VDAC were used as markers for endosomes, lysosomes and mitochondria, respectively. WCE, whole-cell extracts; M, membrane; C, cytosol.(c) Subcellular fractionation of TSC2+/+ and TSC2−/− mouse embryonic fibroblasts (MEFs) demonstrating the localization of TSC2, TSC1, Rheb and AKT. PMP70, catalase (peroxisome fraction, P), LDH (cytosolic fraction, C), β-integrin (membrane fraction, M) and lamin A/C (nuclear fraction, N) were used as subcellular markers. Uncropped images of western blots are shown in Supplementary Fig. S6.


figf3 ( a ) Western analysis of whole - cell extracts ( WCE ) , membrane ( M ) , cytosolic ( C ) and peroxisome ( P ) fractions from parental and MCF - 7 cells stably expressing constitutively active myristoylated AKT ( myr - AKT ) immunoblotted for TSC2 , LDH , catalase and PMP70 . Light and dark represent short and long autoradiographic exposures , respectively . ( b ) Western analysis of whole - cell extracts , membrane , cytosolic and peroxisome fractions from MCF - 7 cells stimulated with insulin ( 200 nM ) for 30 min after 1 h of serum starvation and immunoblotted for TSC2 , LDH , catalase and PMP70 . Light and dark represent short and long autoradiographic exposures , respectively . ( c ) Representative western analysis of HEK293 whole - cell extracts , cytosolic and peroxisome fractions , immunoblotted for phospho - TSC2 ( S939 ; inactive ) , TSC2 and phospho - AKT ( S473 and T308 ; activated ) . LDH and catalase were used as markers for the cytosolic and peroxisome fractions , respectively . ( d ) Proteinase K protection assays performed in the presence or absence of Triton X - 100 to disrupt peroxisomes on equal masses of peroxisome fractions ( HEK293 cells ) collected at the indicated time points . The lysates were immunoblotted for TSC2 , TSC1 , Rheb , catalase and PMP70 . Uncropped images of western blots are shown in Supplementary Fig . S6 .

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

figf3(a) Western analysis of whole-cell extracts (WCE), membrane (M), cytosolic (C) and peroxisome (P) fractions from parental and MCF-7 cells stably expressing constitutively active myristoylated AKT (myr-AKT) immunoblotted for TSC2, LDH, catalase and PMP70. Light and dark represent short and long autoradiographic exposures, respectively.(b) Western analysis of whole-cell extracts, membrane, cytosolic and peroxisome fractions from MCF-7 cells stimulated with insulin (200 nM) for 30 min after 1 h of serum starvation and immunoblotted for TSC2, LDH, catalase and PMP70. Light and dark represent short and long autoradiographic exposures, respectively.(c) Representative western analysis of HEK293 whole-cell extracts, cytosolic and peroxisome fractions, immunoblotted for phospho-TSC2 (S939; inactive), TSC2 and phospho-AKT (S473 and T308; activated). LDH and catalase were used as markers for the cytosolic and peroxisome fractions, respectively.(d) Proteinase K protection assays performed in the presence or absence of Triton X-100 to disrupt peroxisomes on equal masses of peroxisome fractions (HEK293 cells) collected at the indicated time points. The lysates were immunoblotted for TSC2, TSC1, Rheb, catalase and PMP70. Uncropped images of western blots are shown in Supplementary Fig. S6.