Western blot of Rap1 - YFP in indicated strains upon nmt1 promoter shutoff by thiamine addition . Membranes were probed with antibodies against GFP Act1 ( loading control ) . of Rap1 - YFP in experiments as in ( C ) . Rap1 - YFP are expressed as fold increase over time 0 after normalization through Act1 . Data points error bars are averages s . d . from at least four independent experiments . Statistical significance was assayed using unpaired , two - Student ' s t - . * P 0 . 05 , * * P 0 . 01 for cay1 + versus cay1 ∆ samples .

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

Western blot analysis of Rap1-YFP protein levels in the indicated strains upon nmt1 promoter shutoff by thiamine addition. Membranes were probed with antibodies against GFP and Act1 (loading control).Quantification of Rap1-YFP protein levels in experiments as in (C). Rap1-YFP levels are expressed as fold increase over time 0 after normalization through Act1. Data points and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two-tailed Student's t-test. *P 0.05, **P 0.01 for cay1+ versus cay1∆ samples.


Telomere of ApaI ‐ digested from cay1 ∆ trt1 ∆ harvested at increasing generation doublings ( gen ) .

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

Telomere length analysis of ApaI‐digested DNA from cay1∆trt1∆ cells harvested at increasing generation doublings (gen).


Telomere of ApaI ‐ digested from indicated strains . dh : centromeric dh repeats shown as loading control .

Telomere length analysis of ApaI‐digested DNA from the indicated strains. dh: centromeric dh repeats shown as loading control.


PFGE of telomeric fusions in strains grown to logarithmic phase ( log ) or G1 ‐ arrested by nitrogen starvation ( G1 ) . Genomic was digested with NotI hybridized to C , I , L , M probes detecting terminal fragments of chromosomes I II . Bands corresponding to chromosome end fusions are indicated ( fused ) .

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

PFGE analysis of telomeric fusions in strains grown to logarithmic phase (log) or G1‐arrested by nitrogen starvation (G1). Genomic DNA was digested with NotI and hybridized to C, I, L, and M probes detecting terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated (fused).


Northern blot of ARIA , ARRET , Tf2 retrotransposons , 18S rRNA ( loading control ) in indicated strains .

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

Northern blot analysis of ARIA, ARRET, Tf2 retrotransposons, and 18S rRNA (loading control) in the indicated strains.


qRT - PCR of TERRA expressed as fold increase over wt after normalization through act1 + mRNA . Bars error bars are averages s . d . from 3 independent experiments . Statistical significance was assayed using unpaired , two ‐ Student ' s t ‐ . * * P 0 . 01 relative to wt .

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

qRT-PCR quantification of TERRA levels expressed as fold increase over wt after normalization through act1+ mRNA. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. **P 0.01 relative to wt.


ChIP of H3K9ac total H3 for indicated strains . Immunoprecipitated is normalized to input expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only . Bars error bars are averages s . d . from at least four independent experiments . Statistical significance was assayed using unpaired , two ‐ Student ' s t ‐ . * * * P 0 . 001 relative to wt .

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 O O O O O O

ChIP analysis of H3K9ac and total H3 for the indicated strains. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from at least four independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to wt.


Serial dilutions of indicated strains were spotted on complete medium grown at 30 ° C or 20 ° C . Otrt1 ∆ Otrt1 ∆ cay1 ∆ are strains with circularized chromosomes lacking telomeric ( Supplementary Fig S7 ) .

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

Serial dilutions of the indicated strains were spotted on complete medium and grown at 30°C or 20°C. Otrt1∆ and Otrt1∆cay1∆ are strains with circularized chromosomes lacking telomeric sequence (Supplementary Fig S7).


Representative of wt cay1 ∆ grown for 24 h at 30 ° C or 20 ° C . Scale bar , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of wt and cay1∆ cells grown for 24 h at 30°C or 20°C. Scale bar, 20 μm.


of for strains grown in complete medium for 24 h at 30 ° C or 20 ° C . Each dot represents a single median ( m ) is indicated for each sample . Data are from three independent experiments at least 100 were scored per sample in each experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of cell length for strains grown in complete medium for 24 h at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from three independent experiments and at least 100 cells were scored per sample in each experiment.


Top : Examples of DAPI ‐ stained wt cay1 ∆ grown in complete medium for 24 h at 30 ° C or 20 ° C . arrowhead indicates a chromosome bridge . Scale bar , 10 μm . Bottom : of chromosome bridges . Bars error bars are averages s . d . from three independent experiments at least 200 were scored per sample in each experiment . Statistical significance was assayed using unpaired , two ‐ Student ' s t ‐ . * * * P 0 . 001 relative to 30 ° C .

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

Top: Examples of DAPI‐stained wt and cay1∆ cells grown in complete medium for 24 h at 30°C or 20°C. The white arrowhead indicates a chromosome bridge. Scale bar, 10 μm. Bottom: Quantification of chromosome bridges. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. ***P 0.001 relative to 30°C.


RT - PCR of pre ‐ mRNA splicing efficiencies of rap1 + poz1 + pre ‐ mRNAs in cay1 + cay1Δ expressing Cay1 ‐ YFP or Rap1 ‐ YFP . Amplification products corresponding to unspliced spliced mRNAs are indicated by asterisks gene names , respectively . rap1T indicates amplification products obtained with oligonucleotides spanning rap1 exon 3 thereby amplifying both spliced unspliced mRNA . Intronless act1 + was used as a loading control . Numbers at bottom of each panel are ratios between spliced unspliced forms ( s / u ) spliced act1 + ( s / act1 ) . Values are expressed as fold increase over wt .

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

RT-PCR analysis of pre‐mRNA splicing efficiencies of rap1+ and poz1+ pre‐mRNAs in cay1+ and cay1Δ cells expressing Cay1‐YFP or Rap1‐YFP. Amplification products corresponding to unspliced and spliced mRNAs are indicated by asterisks and gene names, respectively. rap1T indicates amplification products obtained with oligonucleotides spanning rap1 exon 3 and thereby amplifying both spliced and unspliced mRNA. Intronless act1+ was used as a loading control. Numbers at the bottom of each panel are ratios between spliced and unspliced forms (s/u) and spliced and act1+ (s/act1). Values are expressed as fold increase over wt.


Western blot of total from early generation cay1 + cay1Δ expressing Cay1 ‐ YFP , Rap1 ‐ YFP , Taz1 ‐ YFP using antibodies against Rap1 , Taz1 , Act1 ( loading control ) . Numbers at bottom are ratios between Rap1 ( endogenous plus ectopic ) Act1 ( Rap1 / Act1 ) . Values are expressed as fold increase over wt .

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

Western blot analysis of total proteins from early generation cay1+ and cay1Δ cells expressing Cay1‐YFP, Rap1‐YFP, and Taz1‐YFP using antibodies against Rap1, Taz1, and Act1 (loading control). Numbers at the bottom are ratios between Rap1 proteins (endogenous plus ectopic) and Act1 (Rap1/Act1). Values are expressed as fold increase over wt.


Left : Representative YFPfluorescence microscopy DIC of cay1 ∆ strains expressing Cay1 ‐ YFP . Scale bar , 10 μm . Right : Representative YFP RFP of Cay1 ‐ YFP ‐ Pot1 ‐ RFP ‐ expressing . Arrows point to YFP RFP foci within same nucleus demonstrating lack of co ‐ localization . Scale bar , 5 μm .

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 B-MEASURED_VAR 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

Left: Representative YFPfluorescence microscopy and DIC images of cay1∆ strains expressing Cay1‐YFP. Scale bar, 10 μm. Right: Representative YFP and RFP fluorescence images of Cay1‐YFP‐ and Pot1‐RFP‐expressing cells. Arrows point to YFP and RFP foci within the same nucleus demonstrating lack of co‐localization. Scale bar, 5 μm.


Representative YFPfluorescence microscopy DIC of cay1 + cay1 ∆ strains expressing Rap1 ‐ YFP . Scale bar , 5 μm .

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

Representative YFPfluorescence microscopy and DIC images of cay1+ and cay1∆ strains expressing Rap1‐YFP. Scale bar, 5 μm.


Telomere of ApaI ‐ digested from harvested at early ( left ) late generations ( right ) after expression of YFP ‐ tagged .

Telomere length analysis of ApaI‐digested DNA from cells harvested at early (left) and late generations (right) after expression of YFP‐tagged proteins.


Northern blot of ARIA , ARRET , 18S rRNA ( loading control ) at early generations after expression of YFP ‐ tagged . Numbers at bottom are ratios between ARRET 18S rRNA signals , expressed as fold increase over wt .

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

Northern blot analysis of ARIA, ARRET, and 18S rRNA (loading control) at early generations after expression of YFP‐tagged proteins. Numbers at the bottom are ratios between ARRET and 18S rRNA signals, expressed as fold increase over wt.


ChIP of subtelomeric H3K9ac , H3K9me3 , total H3 in indicated strains at early generations . Immunoprecipitated is normalized to input expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only . Bars error bars are averages s . d . from 3 independent experiments . Statistical significance was assayed using unpaired , two ‐ Student ' s t ‐ . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 relative to wt .

O O O B-MEASURED_VAR O B-MEASURED_VAR 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

ChIP analysis of subtelomeric H3K9ac, H3K9me3, and total H3 in the indicated strains at early generations. Immunoprecipitated DNA is normalized to input DNA and expressed as fold increase over wt after subtraction of values obtained for negative control immunoprecipitations performed with beads only. Bars and error bars are averages and s.d. from 3 independent experiments. Statistical significance was assayed using the unpaired, two‐tailed Student's t‐test. *P 0.05, **P 0.01, ***P 0.001 relative to wt.


Serial dilutions of early generation cay1 + cay1 ∆ expressing Cay1 ‐ YFP Rap1 ‐ YFP were spotted on complete medium grown at 30 ° C 20 ° C .

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

Serial dilutions of early generation cay1+ and cay1∆ cells expressing Cay1‐YFP and Rap1‐YFP were spotted on complete medium and grown at 30°C and 20°C.


of in strains grown for 24 h in complete medium at 30 ° C or 20 ° C . Each dot represents a single median ( m ) is indicated for each sample . Data are from 3 independent experiments at least 100 were scored per sample in each experiment .

Quantification of cell length in strains grown for 24 h in complete medium at 30°C or 20°C. Each dot represents a single cell and median cell length (m) is indicated for each sample. Data are from 3 independent experiments and at least 100 cells were scored per sample in each experiment.


of chromosome bridges of strains grown for 24 h in complete medium at 30 ° C or 20 ° C . Bars error bars are averages s . d . from three independent experiments at least 200 were scored per sample in each experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of chromosome bridges of strains grown for 24 h in complete medium at 30°C or 20°C. Bars and error bars are averages and s.d. from three independent experiments and at least 200 cells were scored per sample in each experiment.


Northern blot of Tf2 retrotransposons , U6 snRNA , act1 + mRNA ( loading control ) in wt , cay1Δ , prp1 ‐ 1 . prp1 ‐ 1 were grown at 36 ° C for 4 h before harvesting . Black arrowhead indicates unprocessed Tf2 transcripts . asterisk indicates unspliced U6 . Numbers at bottom are ratios between Tf2 act1 + signals ( Tf2 / Act1 ) expressed as fold increase over wt .

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

Northern blot analysis of Tf2 retrotransposons, U6 snRNA, and act1+ mRNA (loading control) in wt, cay1Δ, and prp1‐1 cells. prp1‐1 cells were grown at 36°C for 4 h before harvesting. Black arrowhead indicates unprocessed Tf2 transcripts. The asterisk indicates unspliced U6. Numbers at the bottom are the ratios between Tf2 and act1+ signals (Tf2/Act1) and expressed as fold increase over wt.


Northern blot as in ( A ) in mpn1 + mpn1 ∆ strains carrying a stably integrated extra copy of snu6 + gene or empty vector control plasmids ( ev ) .

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Northern blot analysis as in (A) in mpn1+ and mpn1∆ strains carrying a stably integrated extra copy of the snu6+ gene or empty vector control plasmids (ev).


A Dose - response viability curves in presence of increasing concentrations of IMT1 for one - week , viable were counted normalized to DMSO - treated controls . Data are expressed as mean ± SD of n = 3 independent experiments with four technical replicates . IC50 values were calculated with non - linear least square fit , RKO : 521 . 8 nM , MiaPaCa - 2 : 291 . 4 nM HeLa : 29 . 9 nM .

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

A Dose-response viability curves in presence of increasing concentrations of IMT1 for one-week, viable cells were counted and normalized to DMSO-treated controls. Data are expressed as mean ± SD of n=3 independent experiments with four technical replicates. IC50 values were calculated with non-linear least square fit, RKO: 521.8 nM, MiaPaCa-2: 291.4 nM and HeLa: 29.9 nM.


B Mitochondrial transcript measured by qRT - PCR in RKO after 96 hours of 1 μM IMT1 treatment . Data are relative to DMSO - treated controls are expressed as mean ± SD of n = 4 independent experiments . Ordinary one - way ANOVA was used for comparisons to DMSO - treated controls ( RNR1 , RNR2 , MT - COX2 , MT - ATP6 , MT - ND1 : * * * p < 0 , 0001 ) .

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

B Mitochondrial transcript levels measured by qRT-PCR in RKO cells after 96 hours of 1 μM IMT1 treatment. Data are relative to DMSO-treated controls and are expressed as mean ± SD of n=4 independent experiments. Ordinary one-way ANOVA was used for comparisons to DMSO-treated controls (RNR1, RNR2, MT-COX2, MT-ATP6, MT-ND1: ***p<0,0001).


D Volcano plot showing proteomic changes of RKO treated with 1 μM IMT1 for 96 hours . Data are plotted as average log2 - fold change versus log10 of adjusted p - value of DMSO - treated controls ; mitochondrial are highlighted in red .

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

D Volcano plot showing proteomic changes of RKO cells treated with 1 μM IMT1 for 96 hours. Data are plotted as average log2-fold change versus log10 of adjusted p-value of DMSO-treated controls; mitochondrial protein are highlighted in red.


F CRISPR guides enriched ( positive hits ) in IMT1 - treated RKO plotted as log2 fold change ( LFC ) versus log10 of adjusted p - value . mTORC1 VHL pathway - related genes are reported in green blue , respectively .

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

F CRISPR guides enriched (positive hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value. mTORC1 and VHL pathway-related genes are reported in green and blue, respectively.


B - D Viable in presence of DMSO , IMT1 alone or in combination with rapamycin in RKO ( B ) , MiaPaCa - 2 ( C ) HeLa ( D ) . Data are expressed as mean values ± SD of n = 6 independent experiments each including four technical intra - plate replicates . Statistical significance was calculated with one - way ANOVA . Paired IMT1 vs . IMT1 + RAPA comparisons : RKO : * * * p < 0 , 0001 , MiaPaCa - 2 : * * p = 0 , 0014 , HeLa : * p = 0 , 0238 .

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

B-D Viable cells count in presence of DMSO, IMT1 alone or in combination with rapamycin in RKO (B), MiaPaCa-2 (C) and HeLa (D) cells. Data are expressed as mean values ± SD of n=6 independent experiments each including four technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Paired IMT1 vs. IMT1 + RAPA comparisons: RKO: ***p<0,0001, MiaPaCa-2: **p= 0,0014, HeLa: *p= 0,0238.


F - H Viability assessment of RKO ( F ) , MiaPaCa - 2 ( G ) HeLa ( H ) treated with DMSO , IMT1 alone in combination with FG4592 . Data are expressed as mean values ± SD of n = 6 , 5 , 6 independent experiments for RKO , HeLa MiaPaCa - 2 , respectively . Statistical significance was calculated with one - way ANOVA . Paired IMT1 vs IMT1 + FG4592 comparisons : RKO : * * p < 0 , 0028 , MiaPaCa - 2 : * * p = 0 , 0011 , HeLa : * p = 0 , 0029 .

O 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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

F-H Viability assessment of RKO (F), MiaPaCa-2 (G) and HeLa (H) cells treated with DMSO, IMT1 alone and in combination with FG4592. Data are expressed as mean values ± SD of n=6, 5, 6 independent experiments for RKO, HeLa and MiaPaCa-2, respectively. Statistical significance was calculated with one-way ANOVA. Paired IMT1 vs IMT1 + FG4592 comparisons: RKO: **p<0,0028, MiaPaCa-2: **p= 0,0011, HeLa: *p= 0,0029.


C Western blot analyses of OXPHOS steady state of RKO after treatment with DMSO , IMT1 , rapamycin , rapamycin with IMT1 , FG4592 FG4592 with IMT1 . ACTINB is shown as loading control .

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C Western blot analyses of OXPHOS protein steady state levels of RKO cells after treatment with DMSO, IMT1, rapamycin, rapamycin with IMT1, FG4592 and FG4592 with IMT1. ACTINB is shown as loading control.


F , G OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function . RKO were treated with DMSO , rapamycin ( F ) or FG4592 ( G ) in presence or absence of IMT1 . Data are expressed as mean ± of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA . DMSO - treated controls vs : IMT1 : p = 0 , 0008 ; RAPA : p = 0 , 1578 ; RAPA + IMT1 : p = 0 , 0034 ; FG4592 : p = 0 , 872 ; FG4592 + IMT1 : p = 0 , 0029 . Paired t - of IMT1 vs RAPA + IMT1 : p = 0 , 034 ; IMT1 vs FG4592 + IMT1 : non - significant .

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

F, G Cellular OCR measured with Seahorse extracellular flux analyzer after sequential addition of different modulators of mitochondrial function. RKO cells were treated with DMSO, rapamycin (F) or FG4592 (G) in presence or absence of IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. DMSO-treated controls vs: IMT1: p=0,0008; RAPA: p=0,1578; RAPA + IMT1: p= 0,0034; FG4592: p=0,872; FG4592 + IMT1: p=0,0029. Paired t-test of IMT1 vs RAPA + IMT1: p= 0,034; IMT1 vs FG4592 + IMT1: non-significant.


A Differences in sensitivity to increasing IMT1 doses between RKO resistant RKO . graph shows mean values ± of n = 3 independent experiments .

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

A Differences in sensitivity to increasing IMT1 doses between RKO cells and resistant RKO cells. The graph shows mean values ± SEM of n=3 independent experiments.


B Western blot analyses of OXPHOS steady state at increasing concentrations of IMT1 of RKO ( left ) IMT1 - resistant RKO ( right ) . samples were collected analyzed after eight of chronic IMT1 treatment of resistant ; GAPDH is shown as loading control .

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B Western blot analyses of OXPHOS protein steady state levels at increasing concentrations of IMT1 of RKO (left) and IMT1-resistant RKO cells (right). The samples were collected and analyzed after eight weeks of chronic IMT1 treatment of the resistant line; GAPDH is shown as loading control.


G Relative mtDNA copy number measured in RKO IMT1 - resistant treated with IMT1 for 96 hours . Data are expressed as average from n = 3 experiments ± . Statistical significance was calculated with one - way ANOVA . RKO + IMT1 vs Resistant + IMT1 : MT - ND1 * * p = 0 , 0035 , MT - ATP6 * * * p = 0 , 0002 , MT - CYTB * * p = 0 , 0028 .

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

G Relative mtDNA copy number measured in RKO and IMT1-resistant cells treated with IMT1 for 96 hours. Data are expressed as average from n=3 experiments ± SEM. Statistical significance was calculated with one-way ANOVA test. RKO + IMT1 vs Resistant + IMT1: MT-ND1 **p=0,0035, MT-ATP6 ***p=0,0002, MT-CYTB **p=0,0028.


H OCR measured by Seahorse extracellular flux after sequential addition of different modulators of mitochondrial function in RKO IMT1 - resistant RKO treated with DMSO or IMT1 . Data are expressed as mean ± of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA . RKO DMSO vs RKO + IMT1 : p = 0 , 0008 ; RKO DMSO vs resistant DMSO : p = 0 , 0343 ; RKO DMSO vs resistant + IMT1 : p = 0 , 0092 . Paired t - of RKO + IMT1 vs resistant + IMT1 : p = 0 , 135 .

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H Cellular OCR measured by Seahorse extracellular flux analysis after sequential addition of different modulators of mitochondrial function in RKO and IMT1-resistant RKO cells treated with DMSO or IMT1. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test. RKO DMSO vs RKO + IMT1: p=0,0008; RKO DMSO vs resistant DMSO: p=0,0343; RKO DMSO vs resistant + IMT1: p= 0,0092. Paired t-test of RKO + IMT1 vs resistant + IMT1: p= 0,135.


A ECAR measured by Seahorse extracellular flux in DMSO IMT1 - treated RKO IMT1 - resistant . Data are expressed as mean ± of n = 3 independent experiments with six technical replicates . Statistical significance was calculated with one - way ANOVA for multiple comparisons . RKO DMSO vs RKO + IMT1 : * * p = 0 , 0019 ; RKO + IMT1 vs resistant + IMT : * p = 0 , 0214 ; RKO DMSO vs resistant DMSO : non - significant .

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A ECAR measured by Seahorse extracellular flux analysis in DMSO and IMT1-treated RKO and IMT1-resistant cells. Data are expressed as the mean ± SEM of n=3 independent experiments with six technical replicates. Statistical significance was calculated with one-way ANOVA test for multiple comparisons. RKO DMSO vs RKO + IMT1: **p=0,0019; RKO + IMT1 vs resistant + IMT: *p=0,0214; RKO DMSO vs resistant DMSO: non-significant.


D , E IMT1 extracellular ( D ) extracellular ( E ) concentrations measured in medium at 0 , 2 24 hours of IMT1 treatment in RKO IMT1 - resistant RKO . Mean values ± of n = 3 experiments .

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D, E IMT1 extracellular (D) and extracellular (E) concentrations measured in the medium at 0, 2 and 24 hours of IMT1 treatment in RKO and IMT1-resistant RKO cells. Mean values ± SEM of n=3 experiments.


B Comparison between proteomic analyses of parental RKO resistant treated with 1 μM IMT1 for 96 hours ( r : sample correlation coefficient ) . Data are expressed as average log2 - fold change of controls ( DMSO - treated RKO ) . Mitochondrial are reported in red .

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B Comparison between proteomic analyses of parental RKO and resistant cells treated with 1 μM IMT1 for 96 hours (r: sample correlation coefficient). Data are expressed as average log2-fold change of controls (DMSO-treated RKO cells). Mitochondrial proteins are reported in red.


A Depleted CRISPR guides ( negative hits ) in IMT1 - treated RKO plotted as log2 fold change ( LFC ) versus log10 of adjusted p - value .

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A Depleted CRISPR guides (negative hits) in IMT1-treated RKO cells plotted as log2 fold change (LFC) versus log10 of adjusted p-value.


C Representative related plot of IMT1 - resistant RKO spheroid areas in presence of aforementioned compounds for two ( IMT1 : 1 μM , CAP : 1 μg / ml ) ; Scale bar : 1mm . Data show mean values ± SD of n = 4 experiments . Statistical significance was calculated with paired t - : IMT1 vs IMT1 + CAP : * p = 0 , 0242 .

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C Representative images and related plot of IMT1-resistant RKO spheroid areas in presence of the aforementioned compounds for two weeks (IMT1: 1 μM, CAP: 1 μg/ml); Scale bar: 1mm. Data show the mean values ± SD of n=4 experiments. Statistical significance was calculated with paired t-test: IMT1 vs IMT1+ CAP: *p=0,0242.


G Viable of IMT1 - resistant RKO treated for one - week with either DMSO or IMT1 in presence of controls ( control # 1 , # 2 ) or TFAM ( TFAM # 1 , TFAM # 2 ) siRNAs . Data are expressed as mean values ± SD of n = 5 independent experiments each including six technical intra - plate replicates . Statistical significance was calculated with one - way ANOVA . Controls + IMT1 vs TFAM # 1 + IMT1 : p = 0 , 2148 ; Controls + IMT1 vs TFAM # 2 + IMT1 : * p = 0 , 0415 .

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

G Viable cells counts of IMT1-resistant RKO cells treated for one-week with either DMSO or IMT1 in presence of controls (control #1, #2) or TFAM (TFAM #1, TFAM #2) siRNAs. Data are expressed as mean values ± SD of n=5 independent experiments each including six technical intra-plate replicates. Statistical significance was calculated with one-way ANOVA test. Controls + IMT1 vs TFAM #1 + IMT1: p= 0,2148; Controls + IMT1 vs TFAM #2 + IMT1: *p=0,0415.


( B ) Detection of soluble , NrCAM ( sNrCAM ) full - , mature NrCAM ( mNrCAM ) soluble APPα ( sAPPα ) in neuronal supernatants lysates ( prepared from E16 neurons ) , treated with GI254023x ( 5 µM ) , or solvent for 48h . Densitometric quantifications of Western Blots are shown ( * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 , two - sided students t - n = 6 - 8 ) .

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(B) Detection of soluble, cleaved NrCAM (sNrCAM) and full-length, mature NrCAM (mNrCAM) and soluble APPα (sAPPα) in neuronal supernatants and lysates (prepared from E16 neurons), treated with GI254023x (5 µM), or solvent for 48h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8).


( C ) Detection of sAPPα , sNrCAM mNrCAM in ADAM10 cKO neuronal supernatants lysates at 7 days in vitro ( DIV7 ) . neurons prepared from floxed ADAM10 ( ADAM10 fl / fl ) mice were infected with a lentivirus encoding improved Cre recombinase ( iCre ) or a control GFP lentivirus at DIV2 . Conditioned media were collected for 48 h . Densitometric quantifications of Western Blots are shown ( * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 , two - sided students t - n = 6 - 8 ) .

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(C) Detection of sAPPα, sNrCAM and mNrCAM in ADAM10 cKO neuronal supernatants and lysates at 7 days in vitro (DIV7). The neurons prepared from floxed ADAM10 (ADAM10 fl/fl) mice were infected with a lentivirus encoding improved Cre recombinase (iCre) or a control GFP lentivirus at DIV2. Conditioned media were collected for 48 h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8).


HEK 293 were transfected with a C - terminally VSV - tagged NrCAM construct or empty vector . were then treated with DAPT ( 1 µM ) , GI254023x ( 5 µM ) , either substances or solvent for 24 h . C - terminal NrCAM - fragment was detected with an antibody against VSV - tag . Representative Western Blots are shown .

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HEK 293 cells were transfected with a C-terminally VSV-tagged NrCAM construct or empty vector. Cells were then treated with DAPT (1 µM), GI254023x (5 µM), either substances or solvent for 24 h. The C-terminal NrCAM-fragment was detected with an antibody against the VSV-tag. Representative Western Blots are shown.


( A ) ADAM10 fl / fl neurons were treated with an iCre ( to induce an ADAM10 KO ) , or a control lentivirus at DIV2 . At DIV7 , surface were labeled with biotin enriched by streptavidin pull - down . biotinylated were detected by immunoblotting . Total lysates were analyzed to compare mNrCAM ' s surface / lysate . Densitometric quantifications of Western Blots are shown . Two - sided students t - ( * * * * p < 0 . 0001 , n = 7 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 .

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(A) ADAM10 fl/fl neurons were treated with an iCre (to induce an ADAM10 KO), or a control lentivirus at DIV2. At DIV7, cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAM's surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1.


( B ) murine neurons were treated with GI254023x ( 5 µM ) or solvent , for 48h . surface were enriched like in A . Densitometric quantifications of Western Blots are shown . Two - sided students t - ( * * * * p < 0 . 0001 , n = 11 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 . Representative Western Blots are shown .

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(B) Primary murine neurons were treated with GI254023x (5 µM) or solvent, for 48h. The cell surface proteins were enriched like in A. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 11). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.


( C ) Workflow of neurite outgrowth . ( D ) Representative pictures of single neurites at time - point 0h ( DIV3 ) 24h ( DIV4 ) . Neurons were infected 4h after plating with lentiviruses encoding GFP ( to visualize neurons ) together with shRNA expression cassettes ( sh1 sh2 ) targeting either NrCAM or carrying a scrambled ( scr ) control construct . of neurites were taken at three days in vitro ( DIV3 ) 24h later at DIV4 . In order to study effect of ADAM10 on neurite outgrowth , neurons were treated with ADAM10 inhibitor GI254023x , or vehicle ( control ) , at DIV3 , after taking first pictures with an epifluorescent microscope . differences in neurite were calculated as absolute values ( neurite at 24h minus neurite of 0h ) for neurites passing through middle channels of chambers . Only neurites that had already entered main channel at 0h had not yet left those channels at 0h were considered . red arrows indicate start end of respective measurements . scale bar indicates 40 µm . ( E ) One - way ANOVA with post hoc Dunnett ' s . Given are mean + / - standard error of them mean ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 , n - numbers for each condition are shown in graph in 3E ) .

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(C) Workflow of the neurite outgrowth assay. (D) Representative pictures of single neurites at time-point 0h (DIV3) and 24h (DIV4). Neurons were infected 4h after plating with lentiviruses encoding GFP (to visualize the neurons) together with shRNA expression cassettes (sh1 and sh2) targeting either NrCAM or carrying a scrambled (scr) control construct. Images of neurites were taken at three days in vitro (DIV3) and 24h later at DIV4. In order to study the effect of ADAM10 on neurite outgrowth, neurons were treated with the ADAM10 inhibitor GI254023x, or vehicle (control), at DIV3, after taking the first pictures with an epifluorescent microscope. The differences in neurite length were calculated as absolute values (neurite length at 24h minus neurite length of 0h) for individual neurites passing through the middle channels of the chambers. Only neurites that had already entered the main channel at 0h and had not yet left those channels at 0h were considered. The red arrows indicate the start and the end of the respective length measurements. The scale bar indicates 40 µm. (E) One-way ANOVA with post hoc Dunnett's test. Given are mean +/- the standard error of them mean (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n-numbers for each condition are shown in the graph in 3E).


( A ) Wt neurons were kept in culture until DIV5 , then were treated with acitretin ( 4 µM ) or vehicle control for 48h , as described earlier ( Tippmann et al , 2009 ) . ( B ) Densitometric quantifications of Western Blots are shown . Given are mean + / - standard error of mean ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , two - sided students t - n = 9 - 20 ) . mean of solvent treated were set to 1 .

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(A) Wt neurons were kept in culture until DIV5, then the cells were treated with acitretin (4 µM) or vehicle control for 48h, as described earlier (Tippmann et al, 2009). (B) Densitometric quantifications of the Western Blots are shown. Given are mean +/- the standard error of the mean (*** p < 0.001; **** p < 0. 0001, two-sided students t-test n = 9-20). The mean levels of solvent treated cells were set to 1.


( C ) Detection of sNrCAM human serum albumin ( hSA ) in AD CSF that had been treated with acitretin ( 30 mg / d ) or placebo for 30 days ( n = 9 ) ( Endres et al , 2014 ) . ( D ) Densitometric of Western Blots in ( C ) . sNrCAM / hSA ratios were calculated , then treatment to baseline ratios ( t / b ) for every were calculated . Two - sided student ' s t - ( n = 9 ) . Given are mean + / - standard error of them mean . Representative Western Blots are shown .

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(C) Detection of sNrCAM and human serum albumin (hSA) in AD patients CSF that had been treated with acitretin (30 mg/d) or placebo for 30 days (n = 9) (Endres et al, 2014). (D) Densitometric quantification of the Western Blots in (C). sNrCAM/hSA ratios were calculated, and then the treatment to baseline ratios (t/b) for every patient were calculated. Two-sided student's t-test (n = 9). Given are mean +/- the standard error of them mean. Representative Western Blots are shown.


( A ) To ensure proper synapse formation , neurons were kept in culture for ten days prior to treatment ( Wan et al , 2012 ) . Then , were treated with NMDA ( 50 µM ) , NMDA ( 50 µM ) GI254023x ( 5 µM ) , GI254023x ( 5 µM ) alone or vehicle for 30 minutes . Total mADAM10 remained unchanged by treatment , which is in with NMDA altering mainly intracellular localization of ADAM10 by driving to synaptic membranes ( Marcello et al , 2007 ) . Densitometric quantifications of Western Blots are shown . One - way ANOVA with post hoc Dunnett ' s ( * * * p < 0 . 001 , n = 7 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 . was reprobed with different indicated antibodies .

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(A) To ensure proper synapse formation, primary neurons were kept in culture for ten days prior to the treatment (Wan et al, 2012). Then, cells were treated with NMDA (50 µM), NMDA (50 µM) and GI254023x (5 µM), GI254023x (5 µM) alone or vehicle for 30 minutes. Total cellular mADAM10 levels remained unchanged by the treatment, which is in line with NMDA altering mainly the intracellular localization of ADAM10 by driving the protein to the synaptic membranes (Marcello et al, 2007). Densitometric quantifications of the Western Blots are shown. One-way ANOVA with post hoc Dunnett's test (*** p < 0.001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. The membrane was reprobed with the different indicated antibodies.


( B ) Wt neurons were kept in culture until DIV10 , then were treated with NMDA ( 50 µM ) , or vehicle for 30 minutes . surface were labeled with biotin enriched by streptavidin pull - down . biotinylated were detected by immunoblotting . Total lysates were analyzed to compare mNrCAMs surface / lysate . Densitometric quantifications of Western Blots are shown . Two - sided students t - ( * * p < 0 . 01 , n = 6 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 . Representative Western Blots are shown .

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(B) Wt neurons were kept in culture until DIV10, then the cells were treated with NMDA (50 µM), or vehicle for 30 minutes. Cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAMs surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (** p < 0.01, n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.


Volcano plot of proteomic of CSF from AD treated with acitretin or placebo for 30 days . Every circle represents one . log10 transformed t - p - values ( again treatment / baseline for every ) are plotted against log2 transformed label - free ratios of acitretin placebo CSF . ADAM10 substrates are indicated in bold writing . In total , more than 50 ADAM10 substrates were identified , but only significantly altered substrates as well as NrCAM APP are indicated .

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Volcano plot of the proteomic analysis of CSF from AD patients treated with acitretin or placebo for 30 days. Every circle represents one protein. The log10 transformed t-test p-values (again treatment/baseline for every patient) are plotted against the log2 transformed label-free quantification intensity ratios of acitretin and placebo CSF. ADAM10 substrates are indicated in bold writing. In total, more than 50 ADAM10 substrates were identified, but only the significantly altered substrates as well as NrCAM and APP are indicated.


HEK293 were transiently transfected with hNrCAM , FLAG - tagged hTSPAN15 , either plasmids or empty vector . Detection of sAPPα sNrCAM in conditioned media ADAM10 in lysate . Densitometric of Western Blots are shown ( * * p < 0 . 01 ) , ( * * * p < 0 . 001 ) , ( * * * * p < 0 . 0001 ) , two - sided students t - , or one - way ANOVA with post hoc Dunnett ' s ( n = 6 ) . Given are mean + / - standard error of them mean . mean of solvent treated were set to 1 . Representative Western Blots are shown .

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HEK293 cells were transiently transfected with hNrCAM, FLAG-tagged hTSPAN15, either plasmids or the empty vector. Detection of sAPPα and sNrCAM in the conditioned media and ADAM10 in the cell lysate. Densitometric quantification of the Western Blots are shown (** p < 0.01), (*** p < 0.001), (**** p < 0.0001), two-sided students t-test, or one-way ANOVA with post hoc Dunnett's test (n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.


( A ) Time series of TIRF microscopyimages showing GFP - dynein ( green in merge ) localising to plus ends of dynamic Alexa568 - microtubules ( Alexa568 - MT , magenta in merge ) . concentrations were 10 nM GFP - dynein , 20 nM human dynactin , 20 nM EB1 17 . 5 µM Alexa568 - tubulin ( 5 % labelling ) .

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(A) Time series of TIRF microscopyimages showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).


( B ) dual colour kymographs showing GFP - dynein ( green in merge ) localising to plus ends of dynamic Alexa568 - microtubules ( Alexa568 - MT , magenta in merge ) . concentrations were 10 nM GFP - dynein , 20 nM human dynactin , 20 nM EB1 17 . 5 µM Alexa568 - tubulin ( 5 % labelling ) .

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(B) individual and dual colour kymographs showing GFP-dynein (green in merge) localising to the plus ends of dynamic Alexa568-microtubules (Alexa568-MT, magenta in merge). Protein concentrations were 10 nM GFP-dynein, 20 nM human dynactin, 20 nM EB1 and 17.5 µM Alexa568-tubulin (5% labelling ratio).


( C ) Merged triple colour single channel kymographs showing microtubule end tracking of GFP - dynein ( green in merge ) Alexa647 - EB3 ( red in merge ) on dynamic Alexa568 - microtubules ( blue in merge ) . Concentrations were 10 nM GFP - dynein , 10 nM human dynactin , 10 nM Alexa647 - EB3 , 17 . 5 µM tubulin .

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(C) Merged triple colour and single fluorescence channel kymographs showing microtubule end tracking of GFP-dynein (green in merge) and Alexa647-EB3 (red in merge) on dynamic Alexa568-microtubules (blue in merge). Concentrations were 10 nM GFP-dynein, 10 nM human dynactin, 10 nM Alexa647-EB3, and 17.5 µM tubulin.


( D , E ) Dual single colour kymographs of GFP - dynein ( green ) on dynamic Alexa568 - microtubules ( magenta ) in absence of either dynactin ( D ) . Concentrations of present as in A .

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(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of either dynactin (D) . Concentrations of the proteins present as in A.


( D , E ) Dual single colour kymographs of GFP - dynein ( green ) on dynamic Alexa568 - microtubules ( magenta ) in absence of EB ( E ) . Concentrations of present as in A .

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(D, E) Dual and single colour kymographs of GFP-dynein (green) on dynamic Alexa568-microtubules (magenta) in the absence of EB proteins (E). Concentrations of the proteins present as in A.


( F ) Averaged profiles of GFP - dynein at growing microtubule plus ends in presence of both human dynactin EB1 ( blue squares , as in A ) , in absence of dynactin ( red circles , as in D ) , or in absence of an EB ( black triangles , as in E ) . Mean values from at least two separate experiments per condition ( with a total of at least 30 kymographs ) are shown ; error bars are s . e .

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

(F) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of both human dynactin and EB1 (blue squares, as in A), in the absence of dynactin (red circles, as in D), or in the absence of an EB protein (black triangles, as in E). Mean values from at least two separate experiments per condition (with a total of at least 30 kymographs) are shown; error bars are s. e.


( G ) Kymographs of GFP - dyneinmicrotubule end tracking in absence of ATP ( in contrast to other experiments that contain ATP ) . Other conditions as in A . For merged kymographs , microtubule plus minus ends are labelled by ( + ) ( − ) . Experiments were performed at 30 ° C .

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(G) Kymographs of GFP-dyneinmicrotubule end tracking in the absence of ATP (in contrast to all other experiments that contain ATP). Other conditions as in A. For merged kymographs, microtubule plus and minus ends are labelled by (+) and (−). Experiments were performed at 30°C.


( A ) Merged triple colour single channel TIRF microscopy kymographs of GFP - dynein showing end tracking , processive motility diffusive static binding in presence of DDB components EB1 . concentrations are 10 nM GFP - dynein ( green in merge ) , 20 nM human dynactin , 20 nM EB1 , 200 nM Alexa647 - BicD2 - N ( magenta in merge ) , 17 . 5 µM Alexa568 - tubulin ( blue in merge ) . Alexa647 - BicD2 - N often co - localises with processively moving occasionally with statically bound GFP - dynein , but not with plus end - tracking GFP - dynein .

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 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 B-MEASURED_VAR 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 B-MEASURED_VAR O

(A) Merged triple colour and single fluorescence channel TIRF microscopy kymographs of GFP-dynein showing end tracking, processive motility and diffusive and static binding in the presence of all DDB components and EB1. Protein concentrations are 10 nM GFP-dynein (green in merge), 20 nM human dynactin, 20 nM EB1, 200 nM Alexa647-BicD2-N (magenta in merge), and 17.5 µM Alexa568-tubulin (blue in merge). Alexa647-BicD2-N often co-localises with processively moving and occasionally with statically bound GFP-dynein, but not with plus end-tracking GFP-dynein.


( B ) Averaged profiles of GFP - dynein ( green squares ) Alexa647 - BicD2 - N ( magenta circles ) at growing microtubule plus ends . Mean values from three separate experiments per condition ( with a total of at least 75 kymographs ) are shown ; error bars are s . e .

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(B) Averaged fluorescence intensity profiles of GFP-dynein (green squares) and Alexa647-BicD2-N (magenta circles) at growing microtubule plus ends. Mean values from three separate experiments per condition (with a total of at least 75 kymographs) are shown; error bars are s. e.


( C , D ) Dual single colour kymographs of an Atto565 - microtubule ( magenta ) in presence of GFP - dynein ( green ) , human dynactin EB1 , either ( C ) in absence or ( D ) presence of 5 µM BicD2 - N ; other conditions as in A . increased BicD2 - N in D compared to A strongly reduces microtubule plus end tracking of GFP - dynein .

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(C, D) Dual and single colour kymographs of an Atto565-microtubule (magenta) in the presence of GFP-dynein (green), human dynactin and EB1, and either (C) in the absence or (D) presence of 5 µM BicD2-N; other conditions as in A. The increased BicD2-N concentration in D compared to A strongly reduces microtubule plus end tracking of GFP-dynein.


( E ) Averaged profiles of GFP - dynein at growing microtubule plus ends for condition without BicD2 - N ( red circles , as in C ) or with 5 µM BicD2 - N ( black squares , as in D ) . Mean values from three separate experiments per condition ( with a total of at least 106 kymographs ) are shown ; error bars are s . e . Microtubule plus minus ends in merged kymographs are labelled by ( + ) ( − ) . Experiments were performed at 30 ° C .

O O O O O O O O B-MEASURED_VAR 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 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

(E) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the condition without BicD2-N (red circles, as in C) or with 5 µM BicD2-N (black squares, as in D). Mean values from three separate experiments per condition (with a total of at least 106 kymographs) are shown; error bars are s. e. Microtubule plus and minus ends in merged kymographs are labelled by (+) and (−). Experiments were performed at 30°C.


( A ) Example dual colour kymograph a schematic showing how of initiation of processive runs was measured . concentrations were 10 nM GFP - dynein , 20 nM pig dynactin , 5 µM BicD2 - N , 20 nM EB1 17 . 5 µM Atto647 - tubulin .

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(A) Example dual colour kymograph and a schematic showing how the distance of initiation of processive runs was measured. The protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 5 µM BicD2-N, 20 nM EB1 and 17.5 µM Atto647-tubulin.


( B ) Histograms of spatial initiation probabilities of processive runs within first 7 . 2 µm from growing microtubule plus ends in absence ( black bars ) , or presence of 10 nM EB1 ( red bars ) . 199 221 initiation positions were determined for two conditions ( from three experiments each ) .

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(B) Histograms of the spatial initiation probabilities of processive runs within the first 7.2 µm from growing microtubule plus ends in the absence (black bars), or presence of 10 nM EB1 (red bars). 199 and 221 initiation positions were determined for the two conditions (from three experiments each).


( C ) Averaged profiles of GFP - dynein at growing microtubule plus ends for conditions as in B . Mean values from three separate experiments per condition ( with a total of at least 69 kymographs ) are shown ; error bars are s . e . Experiments were performed at 30 ° C .

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(C) Averaged fluorescence intensity profiles of GFP-dynein at growing microtubule plus ends for the conditions as in B. Mean values from three separate experiments per condition (with a total of at least 69 kymographs) are shown; error bars are s. e. Experiments were performed at 30°C.


( D ) Histogram of spatial initiation probabilities of DDB on static GMPCPP - microtubules . 294 initiation positions were detemined ( from three experiments ) . Concentrations of DDB components as in B .

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(D) Histogram of spatial initiation probabilities of DDB on static GMPCPP-microtubules. 294 initiation positions were detemined (from three experiments). Concentrations of DDB components as in B.


A , B ) Dual single colour kymographs showing under certain conditions p150 or dynactin - dependent microtubule plus end tracking of dynein in absence of EB : ( A ) 10 nM GFP - dynein ( green ) tracking growing end of a Atto565 - microtubule ( magenta ) in presence of pig dynactin at an elevated of 80 nM . Experiments in ( A ) ( B ) were performed in standard buffer ( BRB20 supplemented with 50 mM KCl , for details see Methods ) .

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 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

A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (A) 10 nM GFP-dynein (green) tracking the growing end of a Atto565-microtubule (magenta) in the presence of pig dynactin at an elevated concentration of 80 nM. Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).


( A , B ) Dual single colour kymographs showing under certain conditions p150 or dynactin - dependent microtubule plus end tracking of dynein in absence of EB : ( B ) 10 nM GFP - dynein ( green ) tracking plus end of an Atto565 - microtubule ( blue ) in presence of 10 nM Alexa647 - p150 ( a fragment containing first 517 of p150 ; called p150 - N in text ) ( red ) . Experiments in ( A ) ( B ) were performed in standard buffer ( BRB20 supplemented with 50 mM KCl , for details see Methods ) .

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 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

(A, B) Dual and single colour kymographs showing under certain conditions p150 or dynactin-dependent microtubule plus end tracking of dynein in the absence of EB proteins: (B) 10 nM GFP-dynein (green) tracking the plus end of an Atto565-microtubule (blue) in the presence of 10 nM Alexa647-p150 (a fragment containing the first 517 amino acids of p150; called p150-N in the text) (red). Experiments in (A) and (B) were performed in standard assay buffer (BRB20 supplemented with 50 mM KCl, for details see Methods).


( C , D ) Dual single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength : ( C ) In standard buffer , 10 nM Alexa647 - p150 ( green ) accumulates at plus end on GMPCPP - stabilised segment of a growing Atto565 - microtubule ( magenta ) .

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(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (C) In standard assay buffer, 10 nM Alexa647-p150 (green) accumulates at the plus end and on the GMPCPP-stabilised segment of a growing Atto565-microtubule (magenta).


( C , D ) Dual single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength : ( D ) At higher ionic strength ( BRB80 supplemented with 60 mM KCl , see Methods ) , Alexa647 - p150 ( green ) binds only weakly to microtubule without a detectable plus end preference . concentrations as in C . Atto565 - tubulin was always 17 . 5 µM tubulin . was 30 ° C .

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(C, D) Dual and single colour kymographs showing that microtubule binding behaviour of p150 depends strongly on ionic strength: (D) At higher ionic strength (BRB80 supplemented with 60 mM KCl, see Methods), Alexa647-p150 (green) binds only weakly to the microtubule without a detectable plus end preference. Protein concentrations as in C. The Atto565-tubulin concentration was always 17.5 µM tubulin. The temperature was 30°C.


( A - C ) Kymographs showing GFP - dynein ( green ) on dynamic Atto647N - microtubules ( magenta ) in presence of regulators at different Lis1 concentrations . concentrations were 10 nM GFP - dynein , 20 nM pig dynactin , 20 nM EB1 , 5 µM BicD2 - N , ( A ) either no Lis1 , ( B ) 1 μM mCherry - Lis1 , or ( C ) 5 μM mCherry - Lis1 . Together with BicD2 , Lis1 increases number of processive dynein runs high concentrations of Lis1 restore plus - end localisation of dynein in presence of BicD2 - N . Microtubule orientation as indicated .

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(A-C) Kymographs showing GFP-dynein (green) on dynamic Atto647N-microtubules (magenta) in the presence of all the regulators at different Lis1 concentrations. Protein concentrations were 10 nM GFP-dynein, 20 nM pig dynactin, 20 nM EB1, 5 µM BicD2-N, and (A) either no Lis1, (B) 1 μM mCherry-Lis1, or (C) 5 μM mCherry-Lis1. Together with BicD2, Lis1 increases the number of processive dynein runs and high concentrations of Lis1 restore plus-end localisation of dynein in the presence of BicD2-N. Microtubule orientation as indicated.


( D ) Averaged profiles of GFP - dynein at growing microtubule plus ends in presence of EB1 , dynactin 5 µM BicD2 - N ( black squares , as in A ) , in presence of 1 µM mCherry - Lis1 ( blue circles , as in B ) 5 µM mCherry - Lis1 ( red triangles , as in C ) . Mean values from three separate experiments per condition ( with a total of at least 49 kymographs ) are shown ; error bars are s . e .

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 B-CONTROLLED_VAR 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

(D) Averaged intensity profiles of GFP-dynein at growing microtubule plus ends in the presence of EB1, dynactin and 5 µM BicD2-N (black squares, as in A), in the presence of 1 µM mCherry-Lis1 (blue circles, as in B) and 5 µM mCherry-Lis1 (red triangles, as in C). Mean values from three separate experiments per condition (with a total of at least 49 kymographs) are shown; error bars are s. e.


( E ) average number of processive DDB runs per µm microtubule for three different Lis1 conditions ( as in A , B , C ) . Error bars are s . d . * p = 0 . 001 , * * p = 0 . 003 , * * * p = 0 . 001 ( unpaired t - ) . 218 , 969 , 646 processive DDB events were analysed for three conditions , respectively ( from two experiments each ) .

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(E) The average number of processive DDB runs per µm microtubule length for the three different Lis1 conditions (as in A, B and, C). Error bars are s. d. *p = 0.001, **p = 0.003, ***p = 0.001 (unpaired t-test). 218, 969, and 646 processive DDB events were analysed for the three conditions, respectively (from two experiments each).


( F ) Bar graphs showing mean velocity mean run of processive DDB events for three Lis1 conditions ( as in A , B C ) . Error bars are s . e . m . Mean velocities were 0 . 37 ± 0 . 02 µm / s ( A ) , 0 . 38 ± 0 . 03 µm / s ( B ) , 0 . 35 ± 0 . 03 µm / s ( C ) . Mean run lengths were 3 . 1 ± 0 . 4 µm ( A ) , 3 . 2 ± 0 . 5 µm ( B ) , 2 . 9 ± 0 . 3 µm ( C ) . For each condition over 300 complexes were analysed from three different data sets .

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(F) Bar graphs showing the mean velocity and the mean run length of processive DDB events for the three Lis1 conditions (as in A, B and C). Error bars are the s.e.m. Mean velocities were 0.37 ± 0.02 µm/s (A), 0.38 ± 0.03 µm/s (B), and 0.35 ± 0.03 µm/s (C). Mean run lengths were 3.1 ± 0.4 µm (A), 3.2 ± 0.5 µm (B), and 2.9 ± 0.3 µm (C). For each condition over 300 complexes were analysed from three different data sets.


( G ) Histogram of spatial initiation probabilities of DDB runs in presence of EB1 Lis1 . 381 initiation positions were determined ( from two experiments ) . Experiments were performed at 30 ° C .

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(G) Histogram of spatial initiation probabilities of DDB runs in the presence of EB1 and Lis1. 381 initiation positions were determined (from two experiments). Experiments were performed at 30°C.


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , WDR74 HAUS5 promoters for IgG DVL3 in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) .

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(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, WDR74 and HAUS5 promoters for IgG and DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) ChIP - qPCRs at RFX5 - GBAT2 , COX14 , HLA - E PSMB8 promoters for IgG DVL3 in MCF7 ( NTC , shA DVL3 , shB DVL3 ) .

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(D) ChIP-qPCRs at RFX5-GBAT2, COX14, HLA-E and PSMB8 promoters for IgG and DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3).


( E ) RT - qPCR - of expression changes of RFX5 , GBAT2 , WDR74 , HCG15 , POU2F3 , SNORD3B , APOC1P1 , HLA - A , TAP1 , PSMD8 , PDG1 JUNB genes in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) . ( F ) RT - qPCR - of expression changes of RFX5 , GBAT2 , PCAT6 , ZNF672 , SSC5D , APOC1P1 , HLA - A , HLA - F TAP1 genes in MCF7 ( NTC , shA DVL3 , shB DVL3 ) . D

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

(E) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, WDR74, HCG15, POU2F3, SNORD3B, APOC1P1, HLA-A, TAP1, PSMD8, PDG1 and JUNB genes in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of RFX5, GBAT2, PCAT6, ZNF672, SSC5D, APOC1P1, HLA-A, HLA-F and TAP1 genes in MCF7 (NTC, shA DVL3, and shB DVL3) cells. D


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , KDM5B , APOC1P1 , PSMB8 promoters for IgG H3K4me3 in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) .

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(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, KDM5B, APOC1P1, and PSMB8 promoters for IgG and H3K4me3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) ChIP - qPCRs at RFX5 - GBAT2 , PCAT6 , APOC1P1 HAUS5 promoters for IgG H3K4me3 in MCF7 ( NTC , shA DVL3 , shB DVL3 ) .

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

(D) ChIP-qPCRs at RFX5-GBAT2, PCAT6, APOC1P1 and HAUS5 promoters for IgG and H3K4me3 in MCF7 (NTC, shA DVL3, and shB DVL3).


( C ) ChIP - qPCRs at ARAP1 , RFX5 - GBAT2 , PSMB8 promoters for IgG KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) .

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

(C) ChIP-qPCRs at ARAP1, RFX5-GBAT2, and PSMB8 promoters for IgG and KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3).


( D ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , H4K8ac peak ( fourth row ) ChIP - Seq data in MDA - MB - 468 for PRDM10 , TRIM26 , BTN2A1 , PNRC2 , MR1 genes , visualized by IGV . green rectangular bars are locations of statistically - qualified peak of H3K27ac or H4K8ac in MDA - MB - 468 .

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(D) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MDA-MB-468 for the PRDM10, TRIM26, BTN2A1, PNRC2, and MR1 genes, visualized by IGV. The green rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MDA-MB-468.


( E ) An assembly of IgG ( first row ) , DVL3 ( second row ) , H3K27ac ( third row ) , H4K8ac peak ( fourth row ) ChIP - Seq data in MCF7 for WDR74 , YWHAQ , CDNF / HSPA14 , BTN2A1 , MR1 genes , visualized by IGV . turquoise purple rectangular bars are locations of statistically - qualified peak of H3K27ac or H4K8ac in MCF7 , respectively .

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(E) An assembly of IgG (first row), DVL3 (second row), H3K27ac (third row), and H4K8ac peak (fourth row) ChIP-Seq data in MCF7 for the WDR74, YWHAQ, CDNF/HSPA14, BTN2A1, and MR1 genes, visualized by IGV. The turquoise and purple rectangular bars are the locations of the statistically-qualified peak of H3K27ac or H4K8ac in MCF7, respectively.


( D ) RT - qPCR - of expression changes of DVL3 in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) . ( E ) RT - qPCR - of expression changes of KMT2D in MDA - MB - 468 ( NTC , shA DVL3 , shB DVL3 ) . ( F ) RT - qPCR - of expression changes of DVL3 in MCF7 ( NTC , shA DVL3 , shB DVL3 ) . ( G ) RT - qPCR - of expression changes of KMT2D in MCF7 ( NTC , shA DVL3 , shB DVL3 ) . ( H

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(D) RT-qPCR-based analysis of expression changes of DVL3 in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (E) RT-qPCR-based analysis of expression changes of KMT2D in MDA-MB-468 (NTC, shA DVL3, and shB DVL3) cells. (F) RT-qPCR-based analysis of expression changes of DVL3 in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (G) RT-qPCR-based analysis of expression changes of KMT2D in MCF7 (NTC, shA DVL3, and shB DVL3) cells. (H


( H ) Immunofluorescence of KMT2D localization in MDA - MB - 468 . were probed with KMT2D antibody ( red ) . nucleus was stained with DAPI ( blue ) actin filaments ( green ) were stained with Phalloidin . Merge of KMT2D ( red ) ( blue ) is shown as KMT2D / DAPI , merge of KMT2D ( red ) actin ( green ) is shown as KMT2D / ACTIN , , merge of KMT2D ( red ) , ( blue ) actin ( green ) is shown as MERGE for NTC , shA DVL3 shB DVL3 . Scale bar :

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(H) Immunofluorescence of KMT2D localization in MDA-MB-468 cells. The cells were probed with KMT2D antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of KMT2D (red) and nuclear staining (blue) is shown as KMT2D/DAPI, merge of KMT2D (red) and actin (green) is shown as KMT2D/ACTIN, and, merge of KMT2D (red), nuclear staining (blue) and actin (green) is shown as MERGE for NTC, shA DVL3 and shB DVL3 cells. Scale bar: 10µm


( C ) IVIS showing representative tumors generated by injecting luciferase - expressing MDA - MB - 468 ( NTC , shA DVL3 or shB DVL3 ) in mammary fat pad of SCID mice . Color scale ( luminescent signal ) : blue , least intense signal ; red , most intense signal . ( D ) of luminescence changes in tumors generated by injecting luciferase - expressing MDA - MB - 468 ( NTC , shA DVL3 or shB DVL3 ) in mammary fat pad of SCID mice , Average luminescence was used to measure volume of ; n ≥ 9 . (

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(C) IVIS images showing representative tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (D) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MDA-MB-468 cells (NTC, shA DVL3 or shB DVL3) in the mammary fat pad of SCID mice, Average luminescence was used to measure volume of tumor; n ≥ 9. (


( G ) IVIS showing representative tumors generated by injecting luciferase - expressing MCF7 ( NTC , shA DVL3 ) in mammary fat pad of SCID mice . Color scale ( luminescent signal ) : blue , least intense signal ; red , most intense signal . ( H ) of luminescence changes in tumors generated by injecting luciferase - expressing MCF7 ( NTC , shA DVL3 ) in mammary fat pad of SCID mice . Average luminescence was used to measure volume of . n = 7 .

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(G) IVIS images showing representative tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Color scale (luminescent signal intensity): blue, least intense signal; red, most intense signal. (H) Quantification of luminescence changes in tumors generated by injecting luciferase-expressing MCF7 cells (NTC, shA DVL3) in the mammary fat pad of SCID mice. Average luminescence was used to measure volume of tumor. n=7.


( A ) Immunofluorescence of HA - tagged DVL3 localization in MDA - MB - 468 . were probed with an HA antibody ( red ) . nucleus was stained with DAPI ( blue ) actin filaments ( green ) were stained with Phalloidin . Merge of actin ( green ) ( blue ) is shown as ACTIN / DAPI , merge of DVL3 ( red ) ( blue ) is shown as HA / DAPI , merge of DVL3 ( red ) actin ( green ) is shown as HA / ACTIN , , merge of DVL3 ( red ) , ( blue ) actin ( green ) is shown as MERGE for empty vector ( EV ) , HA - DVL3 - WT HA - DVL3 - NESm transient transfected . Scale bar : 10µm .

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(A) Immunofluorescence of HA-tagged DVL3 localization in MDA-MB-468 cells. The cells were probed with an HA antibody (red). The nucleus was stained with DAPI (blue) and the actin filaments (green) were stained with Phalloidin. Merge of actin (green) and nuclear staining (blue) is shown as ACTIN/DAPI, merge of DVL3 (red) and nuclear staining (blue) is shown as HA/DAPI, merge of DVL3 (red) and actin (green) is shown as HA/ACTIN, and, merge of DVL3 (red), nuclear staining (blue) and actin (green) is shown as MERGE for empty vector (EV), HA-DVL3-WT and HA-DVL3-NESm transient transfected cells. Scale bar: 10µm.


( B ) RT - qPCR - of expression changes of DVL3 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) . ( C ) RT - qPCR - of expression changes of KMT2D in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) . ( D ) RT - qPCR - of expression changes of ARAP1 , RFX5 , GBAT2 , WDR74 , SNORD3D , HAUS5 , TAP1 , HLA - A , PDG1 , , MR1 in stable MDA - MB - 468 ( EV , HA - DVL3 - NESm ) . ( E

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

(B) RT-qPCR-based analysis of expression changes of DVL3 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (C) RT-qPCR-based analysis of expression changes of KMT2D in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (D) RT-qPCR-based analysis of expression changes of ARAP1 , RFX5, GBAT2, WDR74, SNORD3D, HAUS5, TAP1, HLA-A, PDG1, and, MR1 in stable MDA-MB-468 (EV, HA-DVL3-NESm) cells. (E


( A ) Representative clones screened for CD24 expression under regulation of tetracycline . were exposed to 1 µg / ml tetracycline for 48 h . 20 µg from each sample were subjected to Western blot for CD24 analyzed with anti - CD24 SWA11 mAb . was reprobed with anti - tubulin antibody to assess uniformity of sample loading .

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(A) Representative clones screened for CD24 expression under regulation of tetracycline. The cells were exposed to 1 µg/ml tetracycline for 48 h. 20 µg from each sample were subjected to Western blot analysis for CD24 and analyzed with anti-CD24 SWA11 mAb. The membrane was reprobed with anti-tubulin antibody to assess the uniformity of sample loading.


( D ) Western blot of EXO - CD24 exosomes . Tetracycline ( Tet ) was used to induce CD24 expression in from which exosomes are derived . HSP70 was used as an exosomal marker .

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(D) Western blot analysis of EXO-CD24 exosomes. Tetracycline (Tet) was used to induce CD24 expression in the cell line from which exosomes are derived. HSP70 was used as an exosomal marker.


( C ) Western blot of EXO - mCD24 exosomes . HSP70 was used as an exosomal marker .

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(C) Western blot analysis of EXO-mCD24 exosomes. HSP70 was used as an exosomal marker.


( B ) respiratory rate ( C ) Blood oxygen ( SpO2 ) before treatment ( Day 1 , measured before administration of first dose ) after treatment ( Day 7 ) . Descriptive statistics ( minimum , maximum , median , first quartile , third quartile ) were calculated for Blood oxygen ( SpO2 ) respiratory rate . these are presented graphically via box plots before after treatment .

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(B) respiratory rate and (C) Blood oxygen (SpO2) and before treatment (Day 1, measured before administration of first dose) and after treatment (Day 7). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for Blood oxygen (SpO2) and respiratory rate. these are presented graphically via box plots before and after treatment.


Systemic CRP . Values are normalized against baseline value ( Day 0 ) . Descriptive statistics ( minimum , maximum , median , first quartile , third quartile ) were calculated for CRP . these are presented graphically via box plots before after treatment ( n = 35 )

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Systemic CRP levels. Values are normalized against baseline value (Day 0). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for CRP levels. these are presented graphically via box plots before and after treatment (n=35 patients)


( B ) Systemic cytokine . Cytokine were measured during ( Day 3 / 4 ) or after ( Day 7 / 8 ) treatment with EXO - CD24 using Quantibody Multiplex ELISA Array . Values are normalized against baseline value ( Day 0 ) . Samples from 11 were analysed . Results from 8 are shown ( results for 3 are not shown because one or more baseline results were below detection level ) . Box plot of Systemic cytokine by days after treatment ( minimum , maximum , median , first quartile , third quartile ) .

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(B) Systemic cytokine levels. Cytokine levels were measured during (Day 3/4) or after (Day 7/8) treatment with EXO-CD24 using Quantibody Multiplex ELISA Array. Values are normalized against baseline value (Day 0). Samples from 11 patients were analysed. Results from 8 patients are shown (results for 3 patients are not shown because one or more baseline results were below detection level). Box plot of Systemic cytokine levels by days after treatment (minimum, maximum, median, first quartile, third quartile).


( C ) During course of EXO - CD24 treatment follow - up , cytokine were measured for of ( n = 24 ) .

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(C) During the course of EXO-CD24 treatment and follow-up, cytokine levels were measured for the rest of the patients (n=24 patients).


( D ) Monitoring systemic IL - 6 cytokine level following EXO - CD24 treatment . Samples from 17 , representing 4 experimental groups , were analysed using BD ™ Cytometric Bead Array ( CBA ) .

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(D) Monitoring systemic IL-6 cytokine level following EXO-CD24 treatment. Samples from 17 patients, representing the 4 experimental groups, were analysed using the BD™ Cytometric Bead Array (CBA).


A . GFP - Snc1 - PEM accumulates in atg1Δ , atg8Δ , atg11Δ mutant . Lysates were prepared from wild type ( WT ) , ypt1 - 1 ( for comparison ) , atg1Δ , atg8Δ , atg11Δ mutant transformed with a 2μ plasmid expressing GFP - Snc1 - PEM from TPI promoter . level of GFP - Snc1 - PEM was determined using immunoblot with anti - GFP antibodies . bands were quantified increase in level in mutant versus WT is shown under blot adjusted to G6PDH loading control .

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A. GFP-Snc1-PEM protein accumulates in atg1Δ, atg8Δ, and atg11Δ mutant cells. Lysates were prepared from wild type (WT), ypt1-1 (for comparison), atg1Δ, atg8Δ, and atg11Δ mutant cells transformed with a 2μ plasmid expressing GFP-Snc1-PEM from the TPI promoter. The level of GFP-Snc1-PEM was determined using immunoblot analysis with anti-GFP antibodies. The bands were quantified and increase in the protein level in mutant versus the WT cells is shown under the blot and adjusted to the G6PDH loading control.


B . GFP - Snc1 - PEM accumulates in aberrant ER structures in atg1Δ , atg8Δ , atg11Δ mutant . ER - marker Sec61 was tagged with mCherry in strains from panel A , were examined by live - microscopy . Shown from left to right : DIC , GFP , mCherry , merge , % with intracellular GFP - Snc1 - PEM ( number of with internal GFP - Snc1 - PEM / number of visualized ) , % in which intra - Snc1 - PEM co - localizes with Sec61 .

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B. GFP-Snc1-PEM protein accumulates in aberrant ER structures in atg1Δ, atg8Δ, and atg11Δ mutant cells. The ER-marker Sec61 was tagged with mCherry in strains from panel A, and the cells were examined by live-cell microscopy. Shown from left to right: DIC, GFP, mCherry, merge, % cells with intracellular GFP-Snc1-PEM (number of cells with internal GFP-Snc1-PEM / number of cells visualized), and % cells in which intra-cellular Snc1-PEM co-localizes with Sec61.


C . UPR is induced in atg1Δ , atg8Δ , atg11Δ mutant . from panel A were transformed with a second plasmid that expresses β - gal from a UPR promoter . UPR was determined expressed as % of WT response .

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C. UPR is induced in atg1Δ, atg8Δ, and atg11Δ mutant cells. Cells from panel A were transformed with a second plasmid that expresses β-gal from a UPR promoter. UPR was determined and expressed as % of the WT response.


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM level (

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D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM level ( D ) , intra - accumulation of GFP - Snc1 - PEM , ( E ) ,

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

D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E),


D - E . Unlike deletion of Atg11 , deletion of other known selective Atgs required for CVT pathway ( Atg19 ) , mitophagy ( Atg32 ) pexophagy ( Atg36 ) , does not result in increase of GFP - Snc1 - PEM level ( D ) , intra - accumulation of GFP - Snc1 - PEM , ( E ) , induction of UPR response ( F ) . Wild type ( WT ) , atg19Δ , atg11Δ , atg32Δ , atg36Δ mutant overexpressing GFP - Snc1 - PEM were analyzed as described for panels A - C , respectively . E . Shown from left to right : DIC , GFP , % with intracellular Snc1 - PEM structures . + / - error bars represent STDEV . Results in this figure represent at least two independent experiments .

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

D-E. Unlike deletion of Atg11, deletion of other known selective Atgs required for the CVT pathway (Atg19), mitophagy (Atg32) and pexophagy (Atg36), does not result in increase of GFP-Snc1-PEM protein level (D), intra-cellular accumulation of GFP-Snc1-PEM, (E), and induction of the UPR response (F). Wild type (WT), atg19Δ, atg11Δ, atg32Δ, and atg36Δ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for panels A-C, respectively. E. Shown from left to right: DIC, GFP, and % cells with intracellular Snc1-PEM structures. +/- and error bars represent STDEV. Results in this figure represent at least two independent experiments.


A - C . While level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ atg2 ∆ , but not atg18 ∆ , mutant , it does not accumulate in aberrant structures does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , atg2 ∆ mutant overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . tested : level of GFP - Snc1 - PEM ( A ) ,

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A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A),


A - C . While level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ atg2 ∆ , but not atg18 ∆ , mutant , it does not accumulate in aberrant structures does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , atg2 ∆ mutant overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . tested : level of GFP - Snc1 - PEM ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B )

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

A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B)


A - C . While level of overexpressed GFP - Snc1 - PEM is increased in atg9 ∆ atg2 ∆ , but not atg18 ∆ , mutant , it does not accumulate in aberrant structures does not induce UPR . Wild type ( WT ) , atg9 ∆ , atg18 ∆ , atg2 ∆ mutant overexpressing GFP - Snc1 - PEM were analyzed as described for Fig 1A - 1C , respectively . tested : level of GFP - Snc1 - PEM ( A ) , accumulation of GFP - Snc1 - PEM in aberrant structures ( B ) , induction of UPR response ( C , atg1 ∆ is shown as a positive control ) .

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

A-C. While the level of overexpressed GFP-Snc1-PEM is increased in atg9∆ and atg2∆, but not atg18∆, mutant cells, it does not accumulate in aberrant structures and does not induce UPR. Wild type (WT), atg9∆, atg18∆, and atg2∆ mutant cells overexpressing GFP-Snc1-PEM were analyzed as described for Fig 1A-1C, respectively. The tested phenotypes: the level of GFP-Snc1-PEM protein (A), accumulation of GFP-Snc1-PEM in aberrant structures (B), and induction of the UPR response (C,atg1∆ is shown as a positive control).


D - G . Atg9 is epistatic to Atg11 in macro - ER - phagy . ATG9 was deleted in wild type atg11 ∆ mutant effects of overexpression of GFP - Snc1 - PEM were determined in single double mutants as described in Fig 1 legend . D . Deletion of ATG9 in wild type ( WT ) or atg11 ∆ mutant results in an increase of GFP - Snc1 - PEM level similar to increase in atg11 ∆ mutant .

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

D-G. Atg9 is epistatic to Atg11 in macro-ER-phagy. ATG9 was deleted in wild type and atg11∆ mutant cells and the effects of overexpression of GFP-Snc1-PEM were determined in the single and double mutants as described in Fig 1 legend. D. Deletion of ATG9 in wild type (WT) or atg11∆ mutant cells results in an increase of GFP-Snc1-PEM protein level similar to the increase in atg11∆ mutant cells.


E . Deletion of ATG9 in wild type or atg11 ∆ mutant results in an increase of intracellular GFP - Snc1 - PEM . However , only ~ 20 % of atg9 ∆ single - , atg9 ∆ atg11 ∆ double - mutant accumulate GFP - Snc1 - PEM in aberrant structures , as compared with ~ 75 % of atg11 ∆ mutant . Shown from top to bottom : DIC , GFP , % with aberrant intracellular GFP - Snc1 - PEM structures , of GFP - Snc1 - PEM inside / PM ( 30 were analyzed for each ) .

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

E. Deletion of ATG9 in wild type or atg11∆ mutant cells results in an increase of intracellular GFP-Snc1-PEM fluorescence. However, only ~20% of the atg9∆ single-, and atg9∆ atg11∆ double-mutant cells accumulate GFP-Snc1-PEM in aberrant structures, as compared with ~75% of atg11∆ mutant cells. Shown from top to bottom: DIC, GFP, % cells with aberrant intracellular GFP-Snc1-PEM structures, ratio of GFP-Snc1-PEM fluorescence inside/PM (30 cells were analyzed for each strain).