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 .

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


Representative micrographs of a time - lapse microscopy of K and KM non - regressed tumour cells ( H2B - GFP green ) . Top : Mitotic cell with a chromatin bridge ( yellow arrow ) . Bottom : Mitotic cell with misalignment and cytokinesis failure resulting in binucleation ( yellow arrow ) .

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Representative micrographs of a time-lapse microscopy of K and KM non-regressed tumour cells (H2B-GFP green). Top: Mitotic cell with a chromatin bridge (yellow arrow). Bottom: Mitotic cell with misalignment and cytokinesis failure resulting in binucleation (yellow arrow).


Percentage of mitotic errors in K and KM primary tumours and in K and KM non - regressed tumours . Percentage of cells in K and KM primary tumours and in K and KM non - regressed tumours with the indicated mitotic errors . Scale bar 20 µm .

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Percentage of mitotic errors in K and KM primary tumours and in K and KM non-regressed tumours. Percentage of cells in K and KM primary tumours and in K and KM non-regressed tumours with the indicated mitotic errors. Scale bar 20 µm.


Somatic copy number alterations in each individual Kras and Kras / Mad2 non - regressed tumours . 7 K non - regressed and 16 KM non - regressed tumours were sequenced . Focal deletion ( DEL , green ) , Focal amplification ( AMP , light green ) , whole chromosome gain ( WCG , dark grey ) and loss ( WCL , light grey ) , partial chromosome gain ( PCG , dark blue ) and loss ( PCL , light blue ) and gross chromosomal rearrangement ( GCR , red ) .

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Somatic copy number alterations in each individual Kras and Kras/Mad2 non-regressed tumours. 7 K non-regressed and 16 KM non-regressed tumours were sequenced. Focal deletion (DEL, green), Focal amplification (AMP, light green), whole chromosome gain (WCG, dark grey) and loss (WCL, light grey), partial chromosome gain (PCG, dark blue) and loss (PCL, light blue) and gross chromosomal rearrangement (GCR, red).


Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene . Colour of the block corresponds to the segment mean ( the degree to which a genome segment is lost ( blue ) or gained ( red ) .

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Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene. Colour of the block corresponds to the segment mean (the degree to which a genome segment is lost (blue) or gained (red).


Immunostaining of phospho - cMet in non - regressed tumours carrying or not the amplification in chromosome 6 . Yellow numbers indicate the total number of cMet positive cells / total number of cells counted . Scale bar 100 µm .

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Immunostaining of phospho-cMet in non-regressed tumours carrying or not the amplification in chromosome 6. Yellow numbers indicate the total number of cMet positive cells/total number of cells counted. Scale bar 100 µm.


Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification . 0 indicates when doxycycline was removed . Each colour represents one tumour . Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth .

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Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification. 0 indicates when doxycycline was removed. Each colour represents one tumour. Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth.


Genome - wide log2 - ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non - regressed tumour showing no amplification in the primary tumour ( upper panels ) and a small amplification in the non - regressed tumours ( bottom panels , yellow arrow ) .

Genome-wide log2-ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non-regressed tumour showing no amplification in the primary tumour (upper panels) and a small amplification in the non-regressed tumours (bottom panels, yellow arrow).


Immunostaining of phospho - cMet in 3 biopsied primary tumours ( PT ) and their corresponding non - regressed tumours ( KM5 , KM7 and KM8 , which are also shown in Fig 2C ) . Scale bar 100 µm .

Immunostaining of phospho-cMet in 3 biopsied primary tumours (PT) and their corresponding non-regressed tumours (KM5, KM7 and KM8, which are also shown in Fig 2C). Scale bar 100 µm.


Representative two - dimensional scatter plots constructed with overlaid dPCR data of the reference ( VIC ) and cMet ( FAM ) from one tumour without cMet amplification and one with cMet amplified . Dots represent results of independent PCR reactions in the wells of a digital PCR chip . Reactions in the bottom left corner ( yellow ) are negative for both targets , while the ones in the top right corner ( green ) are double positives . Reactions in the top left ( blue ) and bottom right ( red ) corners are positive for cMet and the reference targets , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 two-dimensional scatter plots constructed with overlaid dPCR data of the reference (VIC) and cMet (FAM) from one tumour without cMet amplification and one with cMet amplified. Dots represent results of independent PCR reactions in the wells of a digital PCR chip. Reactions in the bottom left corner (yellow) are negative for both targets, while the ones in the top right corner (green) are double positives. Reactions in the top left (blue) and bottom right (red) corners are positive for cMet and the reference targets, respectively.


Representative photographs of FISH staining with a probe for Met ( red signal ) and a probe for a reference gene EML4 ( green signals ) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots ( white arrows ) . The lower panel shows an example of a tumour with cMet amplification ( several red dots ) and 2 green dots ( yellow arrow ) . Scale bar 10 µm .

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Representative photographs of FISH staining with a probe for Met (red signal) and a probe for a reference gene EML4 (green signals) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots (white arrows). The lower panel shows an example of a tumour with cMet amplification (several red dots) and 2 green dots (yellow arrow). Scale bar 10 µm.


Relative volume of tumours grown in 12 Rag2 - / - animals after treatment with Tepotinib or vehicle control ( 3K and 3KM tumours for each condition ) . No statistical significance was found by One - way ANOVA . Mean + / - SEM .

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Relative volume of tumours grown in 12 Rag2-/- animals after treatment with Tepotinib or vehicle control (3K and 3KM tumours for each condition). No statistical significance was found by One-way ANOVA. Mean +/- SEM.


Relative volume of tumours grown in Rag2 - / - animals after treatment with Tepotinib or vehicle control . One - way ANOVA , Tukey ' s multiple comparison . Ns : not significant ; * * p < 0 . 024 . Mean + / - SEM . A total of 19 Rag2 - / - mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls . For tumours with no cMet , 13 Rag2 - / - mice were treated with Tepotinib and 9 were used as control .

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 B-CONTROLLED_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 O O O O O O

Relative volume of tumours grown in Rag2-/- animals after treatment with Tepotinib or vehicle control. One-way ANOVA, Tukey's multiple comparison. Ns: not significant; ** p<0.024. Mean +/- SEM. A total of 19 Rag2-/- mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls. For tumours with no cMet, 13 Rag2-/- mice were treated with Tepotinib and 9 were used as control.


Immunostaining of phospho - cMet in the same tumours grown in Rag2 - / - mice after treatment with Tepotinib or vehicle control . Scale bar 200 µm .

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Immunostaining of phospho-cMet in the same tumours grown in Rag2-/- mice after treatment with Tepotinib or vehicle control. Scale bar 200 µm.


Quantification of pH3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control .

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Quantification of pH3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.


Quantification of caspase 3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control .

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

Quantification of caspase 3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.


( B - D ) Flow cytometric quantification of DHE > Eth . positive wild type yeast cells ( allowing the detection of both dead cells and cells exhibiting sub - lethal oxidative stress ) after 42 h ( B ) or 66 h ( C , D ) expressing Abeta constructs . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 5 - 12 biologically independent cultures . * * * , p < 0 . 001 , * * , p < 0 . 01 , * , p < 0 . 05 . ANOVA with Tukey ' s post - hoc test .

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(B-D) Flow cytometric quantification of DHE>Eth. positive wild type yeast cells (allowing the detection of both dead cells and cells exhibiting sub-lethal oxidative stress) after 42 h (B) or 66 h (C, D) expressing Abeta constructs. Dot plots show all data points along with the mean (bar) ± s.d.. n = 5-12 biologically independent cultures. ***, p < 0.001, **, p < 0.01, *, p < 0.05. ANOVA with Tukey's post-hoc test.


( E ) Annexin V ( AV ) / PI co - staining to assess cell death in yeast expressing A42 after 48 h . Data represent means ± s . d . n = 6 biologically independent cultures . * * * , p < 0 . 001 ( comparing PI positive populations ) . Unpaired , two - tailed t - test .

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(E) Annexin V (AV)/PI co-staining to assess cell death in yeast expressing A42 after 48 h. Data represent means ± s.d. n = 6 biologically independent cultures. ***, p < 0.001 (comparing PI positive populations). Unpaired, two-tailed t-test.


( F ) Immunoblot of whole cell extract ( WCE ) of wild type yeast cells after 16 h expression of Abeta constructs . For western blot ( WB ) Abeta - specific antibody ( Abeta ) 6E10 was used showing long ( Long exp . ) and short time exposure ( Short exp . ) . Ssc1 was used as a loading control .

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(F) Immunoblot of whole cell extract (WCE) of wild type yeast cells after 16 h expression of Abeta constructs. For western blot (WB) Abeta-specific antibody (Abeta) 6E10 was used showing long (Long exp.) and short time exposure (Short exp.). Ssc1 was used as a loading control.


( G ) Confocal microscopy of wild type yeast cells after 18 h of Abeta expression . Colors indicate fluorescence intensity .

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(G) Confocal microscopy of wild type yeast cells after 18 h of Abeta expression. Colors indicate fluorescence intensity.


( A ) Immunoblot of total cytoplasmic post - nuclear supernatant ( PNS ) , mitochondrial , microsomal and cytosolic fractions of wild type yeast cells after 18 h expression of EGFP - Abeta42 ( A42 ) , EGFP - Abeta40 ( A40 ) and EGFP empty vector ( ev ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) and short time exposure ( Short exp . ) . Purity of fractions was tested with antibodies against Cyc1 ( mitochondria ) , Sec61 ( microsomes ) and Pgk1 ( cytosol ) .

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

(A) Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial, microsomal and cytosolic fractions of wild type yeast cells after 18 h expression of EGFP-Abeta42 (A42), EGFP-Abeta40 (A40) and EGFP empty vector (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Purity of fractions was tested with antibodies against Cyc1 (mitochondria), Sec61 (microsomes) and Pgk1 (cytosol).


( B ) Cellular ATP content of wild type yeast cells after 42 h expression of EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 16 biologically independent cultures . * * , p < 0 . 01 . Unpaired , two - tailed t - test .

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(B) Cellular ATP content of wild type yeast cells after 42 h expression of EGFP-A42 (A42) or EGFP empty vector (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 16 biologically independent cultures. **, p < 0.01. Unpaired, two-tailed t-test.


( C ) Relative protein abundance of EGFP - Abeta42 ( A42 ) versus empty vector ( ev ) expressing wild type yeast cells . The significant subset of cytosolic heat shock proteins ( HSP ) detected in a proteomics analysis of isolated mitochondria is depicted . Log2 SILAC ratios of A42 / ev of two independent proteome measurements are shown . Dot plots show all data points along with the mean ( line ) . Significance was determined using an outlier test ( Significance A , p < 0 . 003 ) .

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

(C) Relative protein abundance of EGFP-Abeta42 (A42) versus empty vector (ev) expressing wild type yeast cells. The significant subset of cytosolic heat shock proteins (HSP) detected in a proteomics analysis of isolated mitochondria is depicted. Log2 SILAC ratios of A42/ev of two independent proteome measurements are shown. Dot plots show all data points along with the mean (line). Significance was determined using an outlier test (Significance A, p <0.003).


( D ) Screen of 123 deletion strains assessing Abeta42 ( A42 ) - induced toxicity ( assessed by DHE > Eth . positive cells indicative of the sum of sub - lethal oxidative stress and cell death ) in yeast . Relative A42 - toxicity normalized to wild type ( WT ) cells is depicted . Potential hits ( mutants that reduce A42 toxicity ) are shown in green . Data represent means ± s . d . . n ≥ 3 biologically independent cultures .

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(D) Screen of 123 deletion strains assessing Abeta42 (A42)-induced toxicity (assessed by DHE>Eth. positive cells indicative of the sum of sub-lethal oxidative stress and cell death) in yeast. Relative A42-toxicity normalized to wild type (WT) cells is depicted. Potential hits (mutants that reduce A42 toxicity) are shown in green. Data represent means ± s.d.. n ≥ 3 biologically independent cultures.


Quantification of cells positive for PI indicating cell death at indicated time points after start of expression of EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) of wild type ( WT ) ( A ) and YDJ1 deleted ( Δydj1 ) cells ( B ) . Mean ± s . d . . n = 5 biologically independent cultures . Comparisons by two - way ANOVA ( mixed - design ) followed by simple main effects ( * * * , p < 0 . 001 , vs . control ) .

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Quantification of cells positive for PI indicating cell death at indicated time points after start of expression of EGFP-A42 (A42) or EGFP empty vector (ev) of wild type (WT) (A) and YDJ1 deleted (Δydj1) cells (B). Mean ± s.d.. n = 5 biologically independent cultures. Comparisons by two-way ANOVA (mixed-design) followed by simple main effects (***, p < 0.001, vs. control).


Immunoblot of whole cell extract ( Input ) and eluate of FLAG - tagged Ydj1 . Immunoprecipitation ( IP : FLAG ) of YDJ1 deletion strain ( Δydj1 ) cells expressing EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) and co - overexpressing Ydj1 - FLAG using Abeta - specific antibody ( 6E10 ) and FLAG antibody ( FLAG ) .

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Immunoblot of whole cell extract (Input) and eluate of FLAG-tagged Ydj1. Immunoprecipitation (IP: FLAG) of YDJ1 deletion strain (Δydj1) cells expressing EGFP-A42 (A42) or EGFP empty vector (ev) and co-overexpressing Ydj1-FLAG using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).


Confocal microscopy of wild type ( WT ) yeast cells expressing EGFP - A42 ( A42 ) only and co - overexpressing Ydj1 - FLAG or with the corresponding vector control after 18 h of expression . Colors indicate fluorescence intensity .

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Confocal microscopy of wild type (WT) yeast cells expressing EGFP-A42 (A42) only and co-overexpressing Ydj1-FLAG or with the corresponding vector control after 18 h of expression. Colors indicate fluorescence intensity.


Confocal microscopy of wild type ( WT ) yeast cells expression of EGFP - A42 ( A42 ) and co - overexpressing Ydj1 - FLAG after 18 h of expression . Mitochondria were visualized with MitoTracker Red ( magenta ) . See also Fig EV2J , EV1C .

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Confocal microscopy of wild type (WT) yeast cells expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG after 18 h of expression. Mitochondria were visualized with MitoTracker Red (magenta). See also Fig EV2J, EV1C.


Immunoblot of total cytoplasmic post - nuclear supernatant ( PNS ) , mitochondrial ( Mito . ) , microsomal ( Micro . ) and cytosolic ( Cyt . ) fractions of wild type ( WT ) and YDJ1 deletion strain ( Δydj1 ) cells after 18 h of expression of EGFP - A42 ( A42 ) and co - overexpressing Ydj1 - FLAG or corresponding empty vector controls ( ev ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) and short time exposure ( Short exp . ) . Tom22 - specific antibody is a marker for mitochondria , Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction .

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

Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.) and cytosolic (Cyt.) fractions of wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Tom22-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.


Ratio of full length EGFP - A42 in the mitochondrial fraction / cytosolic fraction , using densitometry quantification of the immunoblot representatively shown in Fig 3F and Appendix Fig S1C from three / 4 independent experiments . Dot plots show all data points along with the mean ( line / bar ) ± s . d . . n = 3 - 4 biologically independent cultures . * , p < 0 . 05 . Unpaired , two - tailed t - test .

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Ratio of full length EGFP-A42 in the mitochondrial fraction / cytosolic fraction, using densitometry quantification of the immunoblot representatively shown in Fig 3F and Appendix Fig S1C from three/4 independent experiments. Dot plots show all data points along with the mean (line/bar) ± s.d.. n = 3-4 biologically independent cultures. *, p < 0.05. Unpaired, two-tailed t-test.


Assay for protein degradation using cycloheximide ( CH ) to stall protein translation . EGFP fluorescence intensity was measured at two time points ( t0 and t2 , 2 h after CH administration ) and normalized to t0 in wild type ( WT ) and YDJ1 deletion strain ( Δydj1 ) cells after 18 h of expression of EGFP - A42 ( A42 ) and co - overexpressing Ydj1 - FLAG or corresponding empty vector controls ( ev ) . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 8 biologically independent cultures . * * * , p < 0 . 001 . ANOVA with Tukey ' s post - hoc . See also Appendix Fig S2D .

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

Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity was measured at two time points (t0 and t2, 2 h after CH administration) and normalized to t0 in wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 8 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc. See also Appendix Fig S2D.


Quantification of the ratio between EGFP - A42 tetramer and monomer in wild type ( WT ) and YDJ1 deletion strain ( Δydj1 ) expressing EGFP - A42 ( J ) as well as between WT expressing EGFP - A42 only and co - overexpressing Ydj1 - FLAG ( K ) at indicated time points . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 8 ( J ) n = 12 ( K ) biologically independent cultures . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 . ANOVA with Tukey ' s post - hoc test . See also Appendix Fig S2A - B .

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Quantification of the ratio between EGFP-A42 tetramer and monomer in wild type (WT) and YDJ1 deletion strain (Δydj1) expressing EGFP-A42 (J) as well as between WT expressing EGFP-A42 only and co-overexpressing Ydj1-FLAG (K) at indicated time points. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8(J) n=12(K) biologically independent cultures. ***, p < 0.001; **, p < 0.01; *, p < 0.05. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2A-B.


Quantification of propidium iodide ( PI ) - staining positive cells indicating cell death at indicated time points of expression of EGFP - A42 ( A42 ) Δydj1 cells with co - expression of DnaJA1 - FLAG ( DnaJA1 ) ( B ) or corresponding empty vector controls ( ev ) ( A ) . Mean ± SD n = 4 - 6 biologically independent cultures . Comparisons by two - way ANOVA ( mixed design ) followed by simple main effects ( * P < 0 . 05 , versus control ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 propidium iodide (PI)-staining positive cells indicating cell death at indicated time points of expression of EGFP-A42 (A42) Δydj1 cells with co-expression of DnaJA1-FLAG (DnaJA1) (B) or corresponding empty vector controls (ev) (A). Mean ± SD n = 4-6 biologically independent cultures. Comparisons by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus control).


( C ) Immunoblot ( WB ) of whole - cell extract ( Input ) and eluate of FLAG - tagged DnaJA1 immunoprecipitation ( IP : FLAG ) of wild - type strain ( WT ) expressing EGFP - A42 ( A42 ) and co - expressing DnaJA1 - FLAG ( A1 ) using Abeta - specific antibody ( 6E10 ) and FLAG antibody ( FLAG ) .

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

(C)Immunoblot (WB) of whole-cell extract (Input) and eluate of FLAG-tagged DnaJA1 immunoprecipitation (IP: FLAG) of wild-type strain (WT) expressing EGFP-A42 (A42) and co-expressing DnaJA1-FLAG (A1) using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).


Assay for protein degradation using cycloheximide ( CH ) to stall protein translation . EGFP fluorescence intensity of YDJ1 deletion strain ( Δydj1 ) and wild type ( WT ) cells expressing EGFP - A42 ( A42 ) only or co - expressing DnaJA1 was measured at two time points ( t0 and t2 ) and normalized to t0 . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 6 biologically independent cultures . * * * , p < 0 . 001 . ANOVA with Tukey ' s post - hoc test . See also Appendix Fig S2C .

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

Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity of YDJ1 deletion strain (Δydj1) and wild type (WT) cells expressing EGFP-A42 (A42) only or co-expressing DnaJA1 was measured at two time points (t0 and t2) and normalized to t0. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2C.


Immunoblot ( WB ) of total cytoplasmic post - nuclear supernatant ( PNS ) , mitochondrial ( Mito . ) , microsomal ( Micro . ) , and cytosolic ( Cyt . ) fractions of YDJ1 - deleted cells ( Δydj1 ) after 18 h expression of EGFP - A42 ( A42 ) only or co - expressing DnaJA1 - FLAG ( DnaJA1 ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) and short time ( Short exp . ) exposure ( two sections from one immunoblot ) . Cox4 - specific antibody is a marker for mitochondria , Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction .

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

Immunoblot (WB) of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.), and cytosolic (Cyt.) fractions of YDJ1-deleted cells (Δydj1) after 18 h expression of EGFP-A42 (A42) only or co-expressing DnaJA1-FLAG (DnaJA1) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time (Short exp.) exposure (two sections from one immunoblot). Cox4-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.


Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 ( A1 ) and quantified by densitometry in ( G ) .

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

Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 (A1) and quantified by densitometry in (G).


Quantification of the oligomer - to - monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in ( F ) . Dot plots show all data points along with the mean ( line ) ± SD n = 3 biologically independent cultures . P - values by two - way ANOVA ( mixed design ) followed by simple main effects ( * P < 0 . 05 , versus A42 control ) . See also Appendix Fig S2D .

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

Quantification of the oligomer-to-monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in (F). Dot plots show all data points along with the mean (line) ± SD n = 3 biologically independent cultures. P-values by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus A42 control). See also Appendix Fig S2D.


A42 beta - sheet - rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ . Data represent means of at least eight measurements . See also Appendix Fig S1E .

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

A42 beta-sheet-rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ. Data represent means of at least eight measurements. See also Appendix Fig S1E.


( A ) Immunoblot of cytosol - enriched cerebral tissue homogenate ( Input ) and eluate of Abeta42 or DnaJA1 using Abeta - specific antibody ( 6E10 ) and DnaJA1 - specific antibody ( DnaJA1 ) , respectively , showing optimal and long exposure ( Long exp . ) . Immunoprecipitation using magnetic beads without antibody , DnaJA1 or 6E10 antibody of female ( 15 months old ) wild type ( WT ) or 3xTg ( PS1M146V / APPSwe / tauP301L ) mice . Sections showing monomer and low - n oligomers , dodecamer and full - length amyloid precursor protein ( APP ) are from 1 immunoblot .

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

(A) Immunoblot of cytosol-enriched cerebral tissue homogenate (Input) and eluate of Abeta42 or DnaJA1 using Abeta-specific antibody (6E10) and DnaJA1-specific antibody (DnaJA1), respectively, showing optimal and long exposure (Long exp.). Immunoprecipitation using magnetic beads without antibody, DnaJA1 or 6E10 antibody of female (15 months old) wild type (WT) or 3xTg (PS1M146V/APPSwe/tauP301L) mice. Sections showing monomer and low-n oligomers, dodecamer and full-length amyloid precursor protein (APP) are from 1 immunoblot.


( B ) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients ( AD ) and aged non - demented controls ( ctrl ) . PonceauS serves as a loading control . ( C ) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients ( AD ) and aged non - demented controls ( ctrl ) . Representative immunoblot is shown in ( B ) . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 4 - 5 independent patients . * , p < 0 . 05 . Unpaired , two - tailed t - test .

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

(B) Representative immunoblot of DnaJA1 levels from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). PonceauS serves as a loading control. (C) Quantification by densitometry of DnaJA1 normalized to PonceauS from the hippocampi of AD patients (AD) and aged non-demented controls (ctrl). Representative immunoblot is shown in (B). Dot plots show all data points along with the mean (bar) ± s.d.. n = 4-5 independent patients. *, p < 0.05. Unpaired, two-tailed t-test.


( A ) Representative confocal microscopy of 10 - day - old male fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) and reference nuclei staining with DAPI ( blue ) of Droj2 knockdown flies ( Droj2 + / - ) flies and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) .

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

(A) Representative confocal microscopy of 10-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).


( B ) Representative confocal and gSTED deconvolved ( decon ) images of Kenyon cells in 18 - day - old male fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) and reference nuclei staining with DAPI ( blue ) of Droj2 knockdown flies ( Droj2 + / - ) flies and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) .

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

(B) Representative confocal and gSTED deconvolved (decon) images of Kenyon cells in 18-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and reference nuclei staining with DAPI (blue) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).


( C - D ) Average intensity ( C ) and total area ( D ) of Abeta ( 6E10 ) signal from confocal images representatively shown in ( A ) from 12 brains of w1118 wild type ( Droj2 + / + ) and knockdown ( Droj2 + / - ) 10 - day - old male flies . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 12 . * , p < 0 . 05 . Unpaired , two - tailed t - test .

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

(C-D) Average intensity (C) and total area (D) of Abeta (6E10) signal from confocal images representatively shown in (A) from 12 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 10-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 12. *, p < 0.05. Unpaired, two-tailed t-test.


( E - F ) qPCR analysis of Droj2 - mRNA levels of 3 - 6 days old female ( E ) and male ( F ) flies expressing human Abeta42 ( UAS - A42 ) of w1118 wild type flies ( Droj2 + / + ) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression ( Droj2 + / + ctrl ) . Reference gene is Rpl32 . Dot plots show all data points along with the mean ( line ) ± s . d . . n = 3 . * * , p < 0 . 01 . One sample t - test against 1 .

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

(E-F) qPCR analysis of Droj2-mRNA levels of 3-6 days old female (E) and male (F) flies expressing human Abeta42 (UAS-A42) of w1118 wild type flies (Droj2+/+) normalized to corresponding isogenic w1118 wild type flies without Abeta42 expression (Droj2+/+ ctrl). Reference gene is Rpl32. Dot plots show all data points along with the mean (line) ± s.d.. n = 3. **, p < 0.01. One sample t-test against 1.


( G ) Immunoprecipitation ( IP : 6E10 ) of w1118 wild type flies ( Droj2 + / + ) using synthetic Abeta42 added to the fly head extract ( Input ) . Abeta - specific antibody 6E10 was used for Abeta42 and DnaJA1 - specific antibody for Droj2 immunoblot detection . Showing input , supernatant , supernatant after washing ( WS ) and eluate .

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(G) Immunoprecipitation (IP: 6E10) of w1118 wild type flies (Droj2+/+) using synthetic Abeta42 added to the fly head extract (Input). Abeta-specific antibody 6E10 was used for Abeta42 and DnaJA1-specific antibody for Droj2 immunoblot detection. Showing input, supernatant, supernatant after washing (WS) and eluate.


( A - B ) Survival of female ( A ) and male ( B ) w1118 wild type flies ( Droj2 + / + ) and Droj2 knockdown flies ( Droj2 + / - ) with expression of human Abeta42 ( UAS - A42 ) or control flies without expression ( ctrl ) , upon supplementation of sugar ( 10 % sucrose ) with 20 mM MnCl2 . Survival was determined at indicated time points . n = 6 with 100 - 120 flies per experiment . The indicated p - value refers to the interaction ( int . ) term of a Cox Proportional Hazards model comparing Abeta42 toxicity ( UAS - A42 vs . ctrl ) and Droj2 expression ( Droj2 + / + vs . Droj2 + / - ) as main factors . The following pairwise comparisons of the indicated groups survival were done by Log Rank test ( * * * * , p < 0 . 0001 ; ns , p > 0 . 05 ) .

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

(A-B) Survival of female (A) and male (B) w1118 wild type flies (Droj2+/+) and Droj2 knockdown flies (Droj2+/-) with expression of human Abeta42 (UAS-A42) or control flies without expression (ctrl), upon supplementation of sugar (10% sucrose) with 20 mM MnCl2. Survival was determined at indicated time points. n = 6 with 100-120 flies per experiment. The indicated p-value refers to the interaction (int.) term of a Cox Proportional Hazards model comparing Abeta42 toxicity (UAS-A42 vs. ctrl) and Droj2 expression (Droj2+/+ vs. Droj2+/-) as main factors. The following pairwise comparisons of the indicated groups survival were done by Log Rank test (****, p < 0.0001; ns, p > 0.05).


( C ) Aversive associative memory performance 2 min after training of aged ( 18 days old ) male Droj2 knockdown flies ( Droj2 + / - ) and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) both expressing human Abeta42 ( UAS - A42 ) of six independent biological replicates . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 6 . * , p < 0 . 05 . Unpaired , two - tailed t - test .

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

(C) Aversive associative memory performance 2 min after training of aged (18 days old) male Droj2 knockdown flies (Droj2+/-) and corresponding isogenic w1118 wild type flies (Droj2+/+) both expressing human Abeta42 (UAS-A42) of six independent biological replicates. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6. *, p < 0.05. Unpaired, two-tailed t-test.


( D ) Representative confocal and gSTED deconvolved images of Kenyon cells in 15 - day - old male fly brains immunostained with Abeta - specific antibody ( Abeta ) 6E10 ( magenta ) and mitochondrial marker ATP5A - specific antibody ( ATP5A , green ) of Droj2 knockdown flies ( Droj2 + / - ) flies and corresponding isogenic w1118 wild type flies ( Droj2 + / + ) expressing human Abeta42 ( UAS - A42 ) .

(D) Representative confocal and gSTED deconvolved images of Kenyon cells in 15-day-old male fly brains immunostained with Abeta-specific antibody (Abeta) 6E10 (magenta) and mitochondrial marker ATP5A-specific antibody (ATP5A, green) of Droj2 knockdown flies (Droj2+/-) flies and corresponding isogenic w1118 wild type flies (Droj2+/+) expressing human Abeta42 (UAS-A42).


( E - F ) Solidity ( E ) and circularity ( F ) of ATP5A - stained mitochondria from fly brain gSTED deconvolved images representatively shown in ( D ) from 8 - 10 brains of w1118 wild type ( Droj2 + / + ) and knockdown ( Droj2 + / - ) 15 - day - old male flies . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 8 - 10 . * , p < 0 . 05 . Unpaired , two - tailed t - test .

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(E-F) Solidity (E) and circularity (F) of ATP5A-stained mitochondria from fly brain gSTED deconvolved images representatively shown in (D) from 8-10 brains of w1118 wild type (Droj2+/+) and knockdown (Droj2+/-) 15-day-old male flies. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8-10. *, p < 0.05. Unpaired, two-tailed t-test.


A WT and Ccnc - / - MEF cultures were treated with 0 . 4 mM H2O2 as indicated , extracts prepared and immunoprecipitated with antibody recognizing the active conformation of Bax . The immunoprecipitates were subjected to Western blot analysis and probed for total Bax or cyclin C . Whole cell extracts ( WCE ) were subjected to Western blot analysis as indicated to control for protein concentrations in the extracts . Molecular weight markers ( kDa ) are indicated on the left .

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

A WT and Ccnc-/- MEF cultures were treated with 0.4 mM H2O2 as indicated, extracts prepared and immunoprecipitated with antibody recognizing the active conformation of Bax. The immunoprecipitates were subjected to Western blot analysis and probed for total Bax or cyclin C. Whole cell extracts (WCE) were subjected to Western blot analysis as indicated to control for protein concentrations in the extracts. Molecular weight markers (kDa) are indicated on the left.


B Cyclin C and Bax directly interact . Recombinant Bax and SUMO - His6 - cyclin C were either incubated together or separately at room temperature then passed over Co + + resin . The elutions were subjected to Western blot analysis probing for Bax and cyclin C ( lanes 4 - 6 ) . Input controls for each sample ( 1 / 10 ) are included in lanes 1 - 3 . Molecular weight markers ( kDa ) are indicated on the left .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Cyclin C and Bax directly interact. Recombinant Bax and SUMO-His6-cyclin C were either incubated together or separately at room temperature then passed over Co++ resin. The elutions were subjected to Western blot analysis probing for Bax and cyclin C (lanes 4-6). Input controls for each sample (1/10) are included in lanes 1-3. Molecular weight markers (kDa) are indicated on the left.


C Mitochondrial relocalization of cyclin C is required for Bax activation . The experiment described in ( A ) was repeated except that H2O2 treated cells were treated with 1 µM pifithrin - µ ( PFT ) for 24 h as indicated . Molecular weight markers ( kDa ) are indicated on the left .

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C Mitochondrial relocalization of cyclin C is required for Bax activation. The experiment described in (A) was repeated except that H2O2 treated cells were treated with 1 µM pifithrin-µ (PFT) for 24 h as indicated. Molecular weight markers (kDa) are indicated on the left.


D Cyclin C is required for efficient Bax - Bcl - XL disassociation . Extracts prepared from MEF cells with the indicated genotypes following treatment with H2O2 ( 0 . 4 mM , 4 h ) were immunoprecipitated with active conformation Bax antibodies . The immunoprecipitates were subjected to Western blot analysis probing with Bcl - XL antibodies . Western blot analysis of WCE was performed as input concentration controls . Molecular weight markers ( kDa ) are indicated on the left .

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

D Cyclin C is required for efficient Bax-Bcl-XL disassociation. Extracts prepared from MEF cells with the indicated genotypes following treatment with H2O2 (0.4 mM, 4 h) were immunoprecipitated with active conformation Bax antibodies. The immunoprecipitates were subjected to Western blot analysis probing with Bcl-XL antibodies. Western blot analysis of WCE was performed as input concentration controls. Molecular weight markers (kDa) are indicated on the left.


E The mitochondrial fission complex is required for cyclin C - dependent Bax activation . Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 ( 0 . 4 mM , 4 h ) . The extracts were treated as in A . Molecular weight markers ( kDa ) are indicated on the left .

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

E The mitochondrial fission complex is required for cyclin C-dependent Bax activation. Extracts were prepared MEF cultures with the indicated genotypes treated with H2O2 (0.4 mM, 4 h). The extracts were treated as in A. Molecular weight markers (kDa) are indicated on the left.


F Drp1 is required for cyclin C - Bax interaction . Extracts were prepared from wild type or Drp1 - / - MEF cultures treated or not with H2O2 and analyzed for the presence of Bax and cyclin C as described in A . Molecular weight markers ( kDa ) are indicated on the left .

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

F Drp1 is required for cyclin C-Bax interaction. Extracts were prepared from wild type or Drp1-/- MEF cultures treated or not with H2O2 and analyzed for the presence of Bax and cyclin C as described in A. Molecular weight markers (kDa) are indicated on the left.


A Wild type MEF cells were fixed before ( control ) and following S - HAD treatment ( 10 µM , 4 h ) then nuclei ( DAPI ) , cyclin C ( indirect immunofluorescence ) and mitochondria ( MitoTracker Red ) were visualized by fluorescence microscopy . Merge panels present DAPI staining ( blue ) only . Zoom panels ( 4X ) are indicated by the boxes in the merge panels . Green arrows indicate cyclin C and mitochondrial co - localization . Bar indicates 10 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Wild type MEF cells were fixed before (control) and following S-HAD treatment (10 µM, 4 h) then nuclei (DAPI), cyclin C (indirect immunofluorescence) and mitochondria (MitoTracker Red) were visualized by fluorescence microscopy. Merge panels present DAPI staining (blue) only. Zoom panels (4X) are indicated by the boxes in the merge panels. Green arrows indicate cyclin C and mitochondrial co-localization. Bar indicates 10 µM.


B Timecourse experiment with wild type and Ccnc - / - MEF cells treated with S - HAD ( 10 µM ) for the times indicated ( n = 3 ) . Error bars indicate SD . Asterisks indicate p values < 0 . 05 from pretreatment control ( Student ' s T - Test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Timecourse experiment with wild type and Ccnc-/- MEF cells treated with S-HAD (10 µM) for the times indicated (n=3). Error bars indicate SD. Asterisks indicate p values <0.05 from pretreatment control (Student's T-Test).


MEF cells were fixed before ( control ) and following S - HAD treatment ( 10 µM , 4 h ) then nuclei ( DAPI ) , cyclin C ( indirect immunofluorescence ) and mitochondria ( MitoTracker Red ) were visualized by fluorescence microscopy . Merge panels present DAPI staining ( blue ) only . S - HAD peptide activity requires cyclin C . The experiment described in A was repeated with Ccnc - / - MEF cultures .

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

MEF cells were fixed before (control) and following S-HAD treatment (10 µM, 4 h) then nuclei (DAPI), cyclin C (indirect immunofluorescence) and mitochondria (MitoTracker Red) were visualized by fluorescence microscopy. Merge panels present DAPI staining (blue) only. S-HAD peptide activity requires cyclin C. The experiment described in A was repeated with Ccnc-/- MEF cultures.


D S - HAD treatment increases Bax mitochondrial localization . Western blot analysis of the indicated proteins in nuclear and mitochondrial fractions before and following S - HAD treatment ( 10 µM , 4 h ) . Atp5A and fibrillarin levels controlled for mitochondrial and nuclear loadings , respectively . Molecular weight markers ( kDa ) are indicated on the left .

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D S-HAD treatment increases Bax mitochondrial localization. Western blot analysis of the indicated proteins in nuclear and mitochondrial fractions before and following S-HAD treatment (10 µM, 4 h). Atp5A and fibrillarin levels controlled for mitochondrial and nuclear loadings, respectively. Molecular weight markers (kDa) are indicated on the left.


E Bax activation was monitored in wild - type MEF cultures treated with S - HAD ( 10 µM ) for the times indicated . The anti - oxidant N - acetyl cysteine ( NAC ) was added ( 1 mM ) 2 h prior to S - HAD treatment . Active Bax immunoprecipitates were subjected to Western blot analysis for Bax and cyclin C as indicated . Bax , cyclin C and ß - actin levels were monitored in extract preparations as input controls . Molecular weight markers ( kDa ) are indicated on the left .

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E Bax activation was monitored in wild-type MEF cultures treated with S-HAD (10 µM) for the times indicated. The anti-oxidant N-acetyl cysteine (NAC) was added (1 mM) 2 h prior to S-HAD treatment. Active Bax immunoprecipitates were subjected to Western blot analysis for Bax and cyclin C as indicated. Bax, cyclin C and ß-actin levels were monitored in extract preparations as input controls. Molecular weight markers (kDa) are indicated on the left.


A Caspase activation was measured by fluorescent cell analysis in wild - type MEF cells treated with S - HAD ( 10 µM , 24 h ) or cisplatin ( CP , 30 µM , 16 h ) . n = 3 . Error bars indicate SD with * and * * indicate p < 0 . 05 and p < 0 . 01 , respectively using Student ' s T test .

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A Caspase activation was measured by fluorescent cell analysis in wild-type MEF cells treated with S-HAD (10 µM, 24 h) or cisplatin (CP, 30 µM, 16 h). n = 3. Error bars indicate SD with * and ** indicate p <0.05 and p<0.01, respectively using Student's T test.


B PARP cleavage was not induced by S - HAD treatment . PARP cleavage was monitored by Western blot analysis in Hela cells treated with S - HAD or cisplatin ( CP ) as described in ( A ) . The blot was stripped and reprobed with ß - actin antibodies as a loading control . Molecular weight markers ( kDa ) are indicated on the left .

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B PARP cleavage was not induced by S-HAD treatment. PARP cleavage was monitored by Western blot analysis in Hela cells treated with S-HAD or cisplatin (CP) as described in (A). The blot was stripped and reprobed with ß-actin antibodies as a loading control. Molecular weight markers (kDa) are indicated on the left.


C Wild type and Ccnc - / - MEF cultures were treated with S - HAD and cisplatin ( CP ) as just described . Cells were imaged and examined for a " rounding up " phenotype diagnostic for death .

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C Wild type and Ccnc-/- MEF cultures were treated with S-HAD and cisplatin (CP) as just described. Cells were imaged and examined for a "rounding up" phenotype diagnostic for death.


D Cyclin C is required for normal Bax oligomerization in response to cisplatin . Whole cell extracts prepared from DSP crosslinked wild type and CCNC - / - MEF cells exposed to cisplatin ( 30 µM , 16 h ) were probed for total Bax . Bax multimers are indicated . Molecular weight markers ( kDa ) are indicated on the left .

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D Cyclin C is required for normal Bax oligomerization in response to cisplatin. Whole cell extracts prepared from DSP crosslinked wild type and CCNC-/- MEF cells exposed to cisplatin (30 µM, 16 h) were probed for total Bax. Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left.


E S - HAD does not induce Bax oligomerization . Experiments described in D were repeated with cultures treated with S - HAD ( 10 µM , 24 h ) . Bax multimers are indicated . Molecular weight markers ( kDa ) are indicated on the left .

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E S-HAD does not induce Bax oligomerization. Experiments described in D were repeated with cultures treated with S-HAD (10 µM, 24 h). Bax multimers are indicated. Molecular weight markers (kDa) are indicated on the left.


F S - HAD treatment sensitizes Hela cells to cisplatin . Hela cultures were treated with cisplatin ( 30 µM ) , S - HAD ( 10 µM ) and N - acetyl cysteine ( NAC , 1 mM ) as indicated . Fluorescent cell analysis was used to quantitate the apoptotic ( Annexin V positive ) and necrotic ( PI positive ) population percentages , respectively . n = 3 independent cultures . Error bars indicate SD . Asterisk indicates p < 0 . 05 ( Student ' s T - test ) .

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F S-HAD treatment sensitizes Hela cells to cisplatin. Hela cultures were treated with cisplatin (30 µM), S-HAD (10 µM) and N-acetyl cysteine (NAC, 1 mM) as indicated. Fluorescent cell analysis was used to quantitate the apoptotic (Annexin V positive) and necrotic (PI positive) population percentages, respectively. n = 3 independent cultures. Error bars indicate SD. Asterisk indicates p <0.05 (Student's T-test).


A S - HAD treatment induces mitochondrial ROS . Wild type and Ccnc - / - MEF cultures were treated with S - HAD ( 10 µM , 24 h ) , rotenone or vehicle control as indicated . Mitochondrial ROS was measured by MitoSox staining ( 4 µM , 15 min ) and quantitative confocal imaging . Error bars indicate SD . n = 3 , asterisk indicates p < 0 . 05 ( Student ' s T - test ) , n . s . , not significantly different .

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A S-HAD treatment induces mitochondrial ROS. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 24 h), rotenone or vehicle control as indicated. Mitochondrial ROS was measured by MitoSox staining (4 µM, 15 min) and quantitative confocal imaging. Error bars indicate SD. n=3, asterisk indicates p<0.05 (Student's T-test), n.s., not significantly different.


B Quenching mitochondrial ROS reduces cyclin C - Bax interaction . Wild type and Ccnc - / - MEF cultures were treated with S - HAD ( 10 µM , 16 h ) and 100 nM mitochondrial - specific reactive oxygen scavenger MitoTempol . Western blot analysis was conducted as described in Fig . 1 . Molecular weight markers ( kDa ) are indicated on the left .

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B Quenching mitochondrial ROS reduces cyclin C-Bax interaction. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 16 h) and 100 nM mitochondrial-specific reactive oxygen scavenger MitoTempol. Western blot analysis was conducted as described in Fig. 1. Molecular weight markers (kDa) are indicated on the left.


C Insulin treatment stimulates S - HAD induced Bax activation . Wild type and Ccnc - / - MEF cultures were grown with or without insulin ( 10 µg / ml ) then treated with S - HAD ( 10 µM , 2 h ) . Mitochondrial and cytosolic fractions were prepared and probed for the indicated proteins . Atp5A and ß - actin levels control for mitochondrial and cytosolic loading , respectively . The two blots were prepared , processed and developed identically . Molecular weight markers ( kDa ) are indicated on the left .

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C Insulin treatment stimulates S-HAD induced Bax activation. Wild type and Ccnc-/- MEF cultures were grown with or without insulin (10 µg/ml) then treated with S-HAD (10 µM, 2 h). Mitochondrial and cytosolic fractions were prepared and probed for the indicated proteins. Atp5A and ß-actin levels control for mitochondrial and cytosolic loading, respectively. The two blots were prepared, processed and developed identically. Molecular weight markers (kDa) are indicated on the left.


A , Primary HBMEC were challenged with type I ( RH ) or type II ( ME49 ) strains of T . gondii for the indicated time points . Cell lysates were used to probe for total Akt and phospho - Ser473 Akt by immunoblot .

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A, Primary HBMEC were challenged with type I (RH) or type II (ME49) strains of T. gondii for the indicated time points. Cell lysates were used to probe for total Akt and phospho-Ser473 Akt by immunoblot.


B , C , Mouse endothelial cells ( mHEVc ; B ) and human retinal pigment epithelial cells ( RPE , C ) were challenged with RH T . gondii followed by assessment of Akt phosphorylation .

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B, C, Mouse endothelial cells (mHEVc; B) and human retinal pigment epithelial cells (RPE, C) were challenged with RH T. gondii followed by assessment of Akt phosphorylation.


D , HBMEC were challenged with either live or killed T . gondii . Cell lysates were subject to immunoblotting as indicated .

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D, HBMEC were challenged with either live or killed T. gondii. Cell lysates were subject to immunoblotting as indicated.


E , HBMEC were transiently transfected with a plasmid encoding PH - Akt - GFP . Cells treated with or without LY294002 ( 20 µM ) for 1 h prior to challenge with T . gondii - RFP . Cells were examined by immunofluorescence at 5 min post - challenge to examine accumulation of PH - Akt - GFP around the parasites .

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E, HBMEC were transiently transfected with a plasmid encoding PH-Akt-GFP. Cells treated with or without LY294002 (20 µM) for 1 h prior to challenge with T. gondii-RFP. Cells were examined by immunofluorescence at 5 min post-challenge to examine accumulation of PH-Akt-GFP around the parasites.


F , HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T . gondii . Densitometry data represent means ± SEM of 4 experiments . A vertical line was inserted between densitometry data from control and LY294002 - treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 .

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F, HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T. gondii. Densitometry data represent means ± SEM of 4 experiments. A vertical line was inserted between densitometry data from control and LY294002-treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1.


G , HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA . Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T . gondii 48 h after transfection . Cell lysates were subject to immunoblotting as indicated . Densitometry data are shown as above and represent means ± SEM of 3 experiments . Results shown are representative of 3 - 4 independent experiments .

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G, HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA. Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T. gondii 48 h after transfection. Cell lysates were subject to immunoblotting as indicated. Densitometry data are shown as above and represent means ± SEM of 3 experiments. Results shown are representative of 3-4 independent experiments.


A , HBMEC , mHEVc and human RPE cells were incubated with or without Akt inhibitor IV ( 1 . 25 µM ) for 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy 2 h and 24 h post - challenge .

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A, HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.


B , HBMEC were transfected with control siRNA or Akt siRNA . Cells were then challenged with T . gondii 48 h after transfection . Monolayers were examined microscopically 24 h post - challenge .

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B, HBMEC were transfected with control siRNA or Akt siRNA. Cells were then challenged with T. gondii 48 h after transfection. Monolayers were examined microscopically 24 h post-challenge.


C , RAW 264 . 7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy 2 h and 24 h post - challenge .

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C, RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.


D , mHEVc - LC3 - EGFP cells were incubated with or without Akt inhibitor IVfollowed by challenge with T . gondii - RFP . Monolayers were examined by fluorescence microscopy 5 h post - challenge . Arrowheads indicate accumulation of LC3 around the parasite .

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D, mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IVfollowed by challenge with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite.


E , HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii ( T ) and then processed for electron microscopy at 5 h post - challenge . Images at the bottom represent magnification of the areas within the boxes . Arrow indicates the PVM ; arrowhead indicates the double membrane structure around the vacuole .

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E, HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole.


F , Control or Akt inhibitor IV - treated HBMEC were challenged with T . gondii - YFP . Expression of LAMP - 1 was examined by fluorescent microscopy 8 h post - challenge . Arrowheads indicate accumulation of LAMP - 1 around the

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F, Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite


G , H , mHEVc cells were transfected with Beclin 1 siRNA ( G ) , Atg7 siRNA ( H ) or control siRNA . After 48 h , cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy at 24 h .

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G, H, mHEVc cells were transfected with Beclin 1 siRNA (G), Atg7 siRNA (H) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 24 h.


I , mHEVc were treated with or without Akt inhibitor IV and infected with T . gondii . 1 h post infection cells were treated with or without leupeptin plus pepstatin ( Lys inhibitors ) . Monolayers were examined microscopically 24 h post - challenge .

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I, mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii. 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge.


J , Mouse microglia were incubated with or without Akt inhibitor IV . 3 - methyl adenine ( 3MA ; 10 mM ) or vehicle were added 2 h post - challenge with T . gondii . Monolayers were examined microscopically 24 h post - challenge . Results are shown as the mean ± SEM and are representative of 3 independent experiments .

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J, Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii. Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


A , HBMEC were treated with or without pertussis toxin ( PTx ; 100 ng / mL ) for 4 h prior to challenge with T . gondii . Cell lysates were obtained at 5 , 15 or 30 min post - incubation with T . gondii and used to examine total Akt and phospho - Akt serine 473 by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control and PTx - treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . HBMEC were also treated with or without LPA ( 10 µM ) in the presence or absence of PTx . Cell lysates were obtained at 5 min and subjected to

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A, HBMEC were treated with or without pertussis toxin (PTx; 100 ng/mL) for 4 h prior to challenge with T. gondii. Cell lysates were obtained at 5, 15 or 30 min post-incubation with T. gondii and used to examine total Akt and phospho-Akt serine 473 by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and PTx-treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. HBMEC were also treated with or without LPA (10 µM) in the presence or absence of PTx. Cell lysates were obtained at 5 min and subjected to immunoblotting


B , HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h . Cell lysates were used to examine total Akt and phospho - serine 473 Akt by immunoblot . Results shown are representative of 3 independent experiments .

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B, HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h. Cell lysates were used to examine total Akt and phospho-serine 473 Akt by immunoblot. Results shown are representative of 3 independent experiments.


A , HBMEC cells were challenged with RH T . gondii and cell lysates were obtained to probe for total EGFR , phospho - tyrosine 1068 EGFR and phospho - tyrosine 1148 EGFR by immunoblot .

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A, HBMEC cells were challenged with RH T. gondii and cell lysates were obtained to probe for total EGFR, phospho-tyrosine 1068 EGFR and phospho- tyrosine 1148 EGFR by immunoblot.


B , HBMEC were challenged with live vs killed tachyzoites . Total EGFR and phospho - tyrosine 1068 EGFR expression was examined by immunoblot .

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B, HBMEC were challenged with live vs killed tachyzoites. Total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot.


C , D , Human RPE cells ( C ) and mouse microglia ( D ) were challenged with RH T . gondii and total EGFR and phospho - tyrosine 1068 EGFR expression was examined by immunoblot . Results shown are representative of 3 independent experiments .

C, D, Human RPE cells (C) and mouse microglia (D) were challenged with RH T. gondii and total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot. Results shown are representative of 3 independent experiments.


A , mHEVc cells were transfected with control siRNA or EGFR siRNA . Expression of EGFR and actin were assessed by immunoblot 48 h post - transfection . mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T . gondii . Cell lysates were used to examine the expression total Akt or phospho - Ser473 Akt by immunoblot .

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A, mHEVc cells were transfected with control siRNA or EGFR siRNA. Expression of EGFR and actin were assessed by immunoblot 48 h post-transfection. mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot.


B , Mouse microglia were treated with or without AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . Cell lysates were used to examine the expression total Akt or phospho - Ser473 Akt by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478 - treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments .

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B, Mouse microglia were treated with or without AG1478 (1 µM) 1 h prior to challenge with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478-treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


A , HBMEC were incubated with AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . Monolayers were examined by light microscopy at 2 and 24 h .

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A, HBMEC were incubated with AG1478 (1 µM) 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.


B , Human RPE cells transfected with either EGFR or control siRNA were challenged with T . gondii followed by examination of monolayers by light microscopy at 24 h .

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B, Human RPE cells transfected with either EGFR or control siRNA were challenged with T. gondii followed by examination of monolayers by light microscopy at 24 h.


C , Mouse bone marrow - derived macrophages were incubated with AG1478 and challenged with T . gondii . Monolayers were examined by light microscopy at 2 and 24 h .

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C, Mouse bone marrow-derived macrophages were incubated with AG1478 and challenged with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.


D , Parental CHO and CHO - EGFR cells were challenged with RH T . gondii . Monolayers were examined microscopically 2 h or 24 h post - challenge .

D, Parental CHO and CHO-EGFR cells were challenged with RH T. gondii. Monolayers were examined microscopically 2 h or 24 h post-challenge.


E , mHEVc - LC3 - EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T . gondii - RFP . Monolayers were examined by fluorescence microscopy 5 h post - challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite .

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E, mHEVc-LC3-EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite.


F , HBMEC cells treated with or without AG1478 were challenged with either T . gondii - YFP . Expression of LAMP - 1 was examined by fluorescent microscopy 8 h post - challenge . The percentages of endothelial cells with LAMP - 1 accumulation of around the parasite were determined .

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F, HBMEC cells treated with or without AG1478 were challenged with either T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. The percentages of endothelial cells with LAMP-1 accumulation of around the parasite were determined.


G , H , mHEVc cells transfected with Beclin1 siRNA ( G ) or Atg7 siRNA ( H ) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T . gondii . Monolayers were examined by light microscopy 24 h post - challenge . Results are shown as the mean ± SEM and are representative of 3 independent experiments .

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G, H, mHEVc cells transfected with Beclin1 siRNA (G) or Atg7 siRNA (H) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T. gondii. Monolayers were examined by light microscopy 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


HBMEC were treated with or without GM6001 ( 10 µM ) for 1 h prior to challenge with T . gondii ( A ) or with Pertussis Toxin ( PTx ; 100 ng / ml ) for 4 h prior to parasite challenge ( B ) . Cell lysates were used to examine total EGFR and phospho - tyrosine 1068 EGFR by immunoblot . Densitometry data represent means ± SEM of 3 experiments . A vertical line was inserted between densitometry data from control and GM6001 - or PTX - treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells , which were given an arbitrary number of 1 . Results shown are representative of 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HBMEC were treated with or without GM6001 (10 µM) for 1 h prior to challenge with T. gondii (A) or with Pertussis Toxin (PTx; 100 ng/ml) for 4 h prior to parasite challenge (B). Cell lysates were used to examine total EGFR and phospho-tyrosine 1068 EGFR by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and GM6001- or PTX-treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


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A , B , C HBMEC were challenged with ΔHx ( WT ) , MIC1 ko , MIC3 ko , MIC1 - 3 ko T . gondii at MOIs that yielded similar percentages of infected cells ( A ) .

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A, B, C HBMEC were challenged with ΔHx (WT), MIC1 ko, MIC3 ko, MIC1-3 ko T. gondii at MOIs that yielded similar percentages of infected cells (A).


Cell lysates were obtained and used to examine total EGFR and phospho - tyrosine 1068 EGFR ( B ) or total Akt and phospho - Ser 473 Akt ( C ) by immunoblot .

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Cell lysates were obtained and used to examine total EGFR and phospho-tyrosine 1068 EGFR (B) or total Akt and phospho-Ser 473 Akt (C) by immunoblot.


HBMEC were incubated with 10 nM of recombinant MIC4 , MIC3 , M2AP or MIC6 for 15 minutes followed by examination of total EGFR and phospho - tyrosine 1068 EGFR ( D ) or total Akt and phospho - Ser473 ( E ) by immunoblot . Densitometry data represent means ± SEM of 4 experiments . Band densities from infected cells or cells treated with MICs were compared to bands from uninfected or untreated cells ( Un ) , which were given an arbitrary number of 1 . Results shown are representative of 4 independent experiments .

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HBMEC were incubated with 10 nM of recombinant MIC4, MIC3, M2AP or MIC6 for 15 minutes followed by examination of total EGFR and phospho-tyrosine 1068 EGFR (D) or total Akt and phospho-Ser473 (E) by immunoblot. Densitometry data represent means ± SEM of 4 experiments. Band densities from infected cells or cells treated with MICs were compared to bands from uninfected or untreated cells (Un), which were given an arbitrary number of 1. Results shown are representative of 4 independent experiments.


A , hmCD40 mHEVc expressing LC3 - EGFP were treated with or without CD154 followed by challenge with T . gondii - RFP in the presence or absence of EGF ( 50 ng / ml ) or AG1478 ( 1 µM ) . LC3 accumulation around T . gondii was assessed by immunofluorescence . B , mHEVc - LC3 - EGFP cells were infected with T . gondii - T . gondii . Cells were treated with vehicle or rapamycin ( 1 µM ) 2 h after challenge with T . gondii in the presence or absent of EGF or AG1478 . Recruitment of LC3 around the parasite was examined at 5 h after challenge with T . gondii .

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A, hmCD40 mHEVc expressing LC3-EGFP were treated with or without CD154 followed by challenge with T. gondii-RFP in the presence or absence of EGF (50 ng/ml) or AG1478 (1 µM). LC3 accumulation around T. gondii was assessed by immunofluorescence. B, mHEVc-LC3-EGFP cells were infected with T. gondii-T. gondii. Cells were treated with vehicle or rapamycin (1 µM) 2 h after challenge with T. gondii in the presence or absent of EGF or AG1478. Recruitment of LC3 around the parasite was examined at 5 h after challenge with T. gondii.


. C , hmCD40 mHEVc - LC3 - EGFP cells were treated with or without CD154 followed by challenged with WT , MIC1 ko , MIC1 ko + MIC1 , MIC3 ko , MIC3 ko + MIC3 , MIC1 - 3 ko , MIC1 - 3 ko + MIC1 - 3 tachyzoites . LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post - challenge . D , hmCD40 mHEVc - LC3 - EGFP treated with or without CD154 were challenged with WT , MIC1 ko , MIC1 ko + MIC1 , MIC3 ko , MIC3 ko + MIC3 , MIC1 - 3 ko or MIC1 - 3 ko + MIC1 - 3 tachyzoites . Parasite load was examined 24 h post - challenge .

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. C, hmCD40 mHEVc-LC3-EGFP cells were treated with or without CD154 followed by challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko, MIC1-3 ko+MIC1-3 tachyzoites. LC3 accumulation around the parasite was examined by fluorescence microscopy 5 h post-challenge. D, hmCD40 mHEVc-LC3-EGFP treated with or without CD154 were challenged with WT, MIC1 ko, MIC1 ko+MIC1, MIC3 ko, MIC3 ko+MIC3, MIC1-3 ko or MIC1-3 ko+MIC1-3 tachyzoites. Parasite load was examined 24 h post-challenge.


E , HBMEC cells were challenged with WT , MIC1 ko , MIC3 ko , MIC1 - 3 ko tachyzoites followed by treatment with vehicle or rapamycin . Parasite load was examined 24 h post - challenge . F , HBMEC treated with or without CD154 or IFN - γ / TNF - α were challenged with WT , MIC1 ko , MIC3 ko , MIC1 - 3 ko tachyzoites . Monolayers were examined microscopically at 24 h post - challenge .

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E, HBMEC cells were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites followed by treatment with vehicle or rapamycin. Parasite load was examined 24 h post-challenge. F, HBMEC treated with or without CD154 or IFN-γ/TNF-α were challenged with WT, MIC1 ko, MIC3 ko, MIC1-3 ko tachyzoites. Monolayers were examined microscopically at 24 h post-challenge.


G , hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1 - 3 ko tachyzoites in the presence of complete medium ( CM ) alone or medium plus MIC3 , MIC4 , M2AP or MIC6 ( all 10 nM ) . Parasite load was determined microscopically 24 h post challenge . Results shown are representative of 3 - 4 independent experiments .

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G, hmCD40 mHEVc cells treated with or without CD154 were infected with either WT or MIC1-3 ko tachyzoites in the presence of complete medium (CM) alone or medium plus MIC3, MIC4, M2AP or MIC6 (all 10 nM). Parasite load was determined microscopically 24 h post challenge. Results shown are representative of 3-4 independent experiments.


a , Flow chart of an in vivo screen to identify active DUBs by using biotin - Ub - VME pull - down assay ( left panel ) , in which the USP8 peptide was identified by mass - spectrometry analysis ( right panel ) . Silver staining and immunoblot ( middle panel ) of precipitants pulled down by biotin - Ub - VME probe of MDA - MB - 231 cells .

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a, Flow chart of an in vivo screen to identify active DUBs by using biotin-Ub-VME pull-down assay (left panel), in which the USP8 peptide was identified by mass-spectrometry analysis (right panel). Silver staining and immunoblot (middle panel) of precipitants pulled down by biotin-Ub-VME probe of MDA-MB-231 cells.


b , Experimental procedure in vivo : flow chart and pictures of an in vivo screen to identify DUBs that promote breast cancer metastasis in mice ( left panel ) . Low metastatic MDA - MB - 231 - Luciferase / GFP breast cancer cells were infected with viral vectors expressing DUB cDNAs and subsequently intracardially injected into nude mice . The mice were monitored for 4 weeks by in vivo BLI : of the 30 mice injected with cells expressing DUB cDNA pool ; 3 had metastases in the brain and bone ( 3 / 30 ) , 4 had metastases on the back and in the bone ( 4 / 30 ) , and 9 had micro - metastasis ( 9 / 30 ) ; the 30 mice injected with control cells showed no detectable metastasis ( 30 / 30 ) . Right panel : hints of the identified DUBs were shown .

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b, Experimental procedure in vivo: flow chart and pictures of an in vivo screen to identify DUBs that promote breast cancer metastasis in mice (left panel). Low metastatic MDA-MB-231-Luciferase/GFP breast cancer cells were infected with viral vectors expressing DUB cDNAs and subsequently intracardially injected into nude mice. The mice were monitored for 4 weeks by in vivo BLI: of the 30 mice injected with cells expressing DUB cDNA pool; 3 had metastases in the brain and bone (3/30), 4 had metastases on the back and in the bone (4/30), and 9 had micro-metastasis (9/30); the 30 mice injected with control cells showed no detectable metastasis (30/30). Right panel: hints of the identified DUBs were shown.


c , The expression distribution of USP8 gene in breast cancer tumor tissues ( n = 1097 ) and normal tissues ( n = 572 ) , where the horizontal axis represents different groups of samples , the vertical axis represents the gene expression distribution ( left panel ) . The box - and - whisker plots represent the medians ( middle line ) , first quartiles ( lower bound line ) , third quartiles ( upper bound line ) , and the ± 1 . 5 × interquartile ranges ( whisker lines ) . Kaplan - Meier curves showing the metastasis - free survival of individuals was negatively correlated with USP8 expression by log - rank test ( right panel ) .

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c, The expression distribution of USP8 gene in breast cancer tumor tissues (n = 1097) and normal tissues (n = 572), where the horizontal axis represents different groups of samples, the vertical axis represents the gene expression distribution (left panel). The box-and-whisker plots represent the medians (middle line), first quartiles (lower bound line), third quartiles (upper bound line), and the ±1.5× interquartile ranges (whisker lines). Kaplan-Meier curves showing the metastasis-free survival of individuals was negatively correlated with USP8 expression by log-rank test (right panel).


d , MCF10A - RAS cells infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 and # 2 ) were analyzed in a tumor sphere assay . The number of the mammospheres with diameter > 60 μm was quantified . The graph shows the number ( No . ) of tumor spheres per 5 × 103 cells seeded . Scale bar 50

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d, MCF10A-RAS cells infected with control (Co.sh) or USP8 shRNA lentivirus (# 1 and # 2) were analyzed in a tumor sphere assay. The number of the mammospheres with diameter > 60 μm was quantified. The graph shows the number (No.) of tumor spheres per 5 × 103 cells seeded. Scale bar 50 μm


e , MCF10A - RAS cells infected with control ( Co . sh ) or USP8 shRNA lentivirus ( # 1 and # 2 ) were subcutaneously injected into nude mice at the indicated numbers . Mean of tumor volumes at week 5 ( n = 10 mice for each group ) .

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e, MCF10A-RAS cells infected with control (Co.sh) or USP8 shRNA lentivirus (# 1 and # 2) were subcutaneously injected into nude mice at the indicated numbers. Mean of tumor volumes at week 5 (n = 10 mice for each group).


f , Microscopy pictures of representative images of zebrafish tail fin injected with control or USP8 stably depleted - MDA - MB - 231 cells at 5 days post injection , n = 20 for each group . g , The final invasive areas of zebrafish embryos displaying invasion and experimental metastasis at 5 dpi ( left panel ) ; Percentage of embryos injected with control or USP8 stably depleted MDA - MB - 231 cells ( # 1 and # 2 ) displaying metastasis at 1 , 3 and 5 days post injection ( right panel ) .

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f, Microscopy pictures of representative images of zebrafish tail fin injected with control or USP8 stably depleted-MDA-MB-231 cells at 5 days post injection, n = 20 for each group. g, The final invasive areas of zebrafish embryos displaying invasion and experimental metastasis at 5 dpi (left panel); Percentage of embryos injected with control or USP8 stably depleted MDA-MB-231 cells (# 1 and # 2) displaying metastasis at 1, 3 and 5 days post injection (right panel).


h , Bioluminescent images of three representative mice from each group ( n = 6 ) at week 6 injected into left heart ventricle with control or MDA - MB - 231 cells stably depleted of endogenous USP8 with two independent shRNA ( # 1 and # 2 ) . i , BLI signal ( left panel ) , number of bone metastasis of every mouse ( middle panel ) and the percentage of bone metastasis free mice in each experimental group ( n = 6 mice per group ) followed in time as in h ( right panel ) .

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h, Bioluminescent images of three representative mice from each group (n = 6) at week 6 injected into left heart ventricle with control or MDA-MB-231 cells stably depleted of endogenous USP8 with two independent shRNA (# 1 and # 2). i, BLI signal (left panel), number of bone metastasis of every mouse (middle panel) and the percentage of bone metastasis free mice in each experimental group (n = 6 mice per group) followed in time as in h (right panel).


a , Heatmap of USP8 - correlated genes in a comparison of breast cancer patients with USP8 - high ( n = 38 ) versus with USP8 - low ( n = 39 ) .

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a, Heatmap of USP8-correlated genes in a comparison of breast cancer patients with USP8-high (n = 38) versus with USP8-low (n = 39).


b , Pre - ranked gene set enrichment analysis ( GSEA ) in USP8 - high ( n = 38 ) versus USP8 - low ( n = 39 ) breast cancer patients .

b, Pre-ranked gene set enrichment analysis (GSEA) in USP8-high (n = 38) versus USP8-low (n = 39) breast cancer patients.


c , Experimental procedure in vivo : MDA - MB - 231 cells were intracardially injected into nude mice ( n = 10 ) . qRT - PCR analysis of bone metastatic cells showing the correlations between USP8 and Snai1 or Snai2 ( Slug ) in cells isolated from mutiple metastatic nodules ( n = 21 ) from mice .

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c, Experimental procedure in vivo: MDA-MB-231 cells were intracardially injected into nude mice (n = 10). qRT-PCR analysis of bone metastatic cells showing the correlations between USP8 and Snai1 or Snai2 (Slug) in cells isolated from mutiple metastatic nodules (n = 21) from mice.


d , 3D spheroid invasion assay of MCF10A - Ras cells showing USP8 wt increased TGF - β - induced invasion . Relative invasion was quantified and shown in the right panel .

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d, 3D spheroid invasion assay of MCF10A-Ras cells showing USP8 wt increased TGF-β-induced invasion. Relative invasion was quantified and shown in the right panel.