A ) Representative images showing G1 localization of human M18BP1WT or human M18BP1SANTA in sfGFP - AID - M18BP1 DLD1 cells treated with 1 mM IAA for 24 h to remove endogenous M18BP1 . Centromeric localization is indicated by localization with ACA ( α - centromere autoantibody serum ) , early G1 cell cycle state is indicated by midbody staining in the tubulin channel . M18BP1 species indicated at left , immunolocalized protein indicated above . Scale bar , 10 μm . Insets are magnified 3X .

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A) Representative images showing G1 localization of human M18BP1WT or human M18BP1SANTA in sfGFP-AID-M18BP1 DLD1 cells treated with 1 mM IAA for 24 h to remove endogenous M18BP1. Centromeric localization is indicated by localization with ACA (α-centromere autoantibody serum), early G1 cell cycle state is indicated by midbody staining in the tubulin channel. M18BP1 species indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3X.


B ) Representative immunofluorescence images showing localization of full - length Flag - M18BP1 - 1 ( mutant species indicated at left ) without ( left panels ) and with ( right panels ) fusion to an SV40 NLS to sperm centromeres in interphase extract depleted of endogenous M18BP1 . Immunolocalized protein indicated above . Scale bar , 10 μm . Insets are magnified 3X . Quantification of immunofluorescence intensity at centromeres as total integrated fluorescence signal normalized to wild type levels . The values shown on each panel are represented as a percentage of wild - type Flag - M18BP1 - 1 ± SEM from three independent experiments .

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B) Representative immunofluorescence images showing localization of full-length Flag-M18BP1-1 (mutant species indicated at left) without (left panels) and with (right panels) fusion to an SV40 NLS to sperm centromeres in interphase extract depleted of endogenous M18BP1. Immunolocalized protein indicated above. Scale bar, 10 μm. Insets are magnified 3X. Quantification of immunofluorescence intensity at centromeres as total integrated fluorescence signal normalized to wild type levels. The values shown on each panel are represented as a percentage of wild-type Flag-M18BP1-1 ± SEM from three independent experiments.


C ) Quantification of Myc - CENP - A assembly in M18BP1 - depleted extract complemented with the indicated M18BP1 - 1 species . Graph shows mean immunofluorescence intensity at centromeres normalized to reactions complemented with WT M18BP1 - 1 . Error bars show SEM of at least three independent experiments .

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C) Quantification of Myc-CENP-A assembly in M18BP1-depleted extract complemented with the indicated M18BP1-1 species. Graph shows mean immunofluorescence intensity at centromeres normalized to reactions complemented with WT M18BP1-1. Error bars show SEM of at least three independent experiments.


D ) Representative Western blot of CENP - A assembly reactions in ( B ) . Efficient M18BP1 depletion is indicated by comparing lanes 1 and 10 . Add - back of wild - type or mutant M18BP1 - 1 is near endogenous levels .

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D) Representative Western blot of CENP-A assembly reactions in (B). Efficient M18BP1 depletion is indicated by comparing lanes 1 and 10. Add-back of wild-type or mutant M18BP1-1 is near endogenous levels.


b ) Volcano plot of contrast MPX vs healthy controls ; α < = 0 . 015 and | logFC | > = 1 . 35 were used for selection of regulated proteins .

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b) Volcano plot of contrast MPX vs healthy controls; α <= 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins.


d ) Boxplots illustrating key proteins that differ between patients with MPX and controls ( p - values and fdr for corresponding contrast MPX vs Control are provided in brackets ) : TTR ( p - value = 9E - 10 , fdr = 2E - 7 ) , LBP ( p - value = 2E - 4 , fdr = 2E - 3 ) , APOC1 ( p - value = 5E - 8 , fdr = 3E - 6 ) , and C9 ( p - value = 2E - 8 , fdr = 2E - 6 ) . Here , as usual , the central bar marks the median ( second quartile ) , the bottom edge of the box marks the first quartile , the top edge of the box marks the third quartile , and the bottom and top whiskers mark the minimum and maximum values that are not outliers . The specific values of the protein expressions are also shown . Provided p - values are obtained from moderated statistics implemented in limma , dfrs were calculated according to Benjamini - Hochberg .

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d) Boxplots illustrating key proteins that differ between patients with MPX and controls (p-values and fdr for corresponding contrast MPX vs Control are provided in brackets): TTR (p-value = 9E-10, fdr = 2E-7), LBP (p-value = 2E-4, fdr = 2E-3), APOC1 (p-value = 5E-8, fdr = 3E-6), and C9 (p-value = 2E-8, fdr = 2E-6). Here, as usual, the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided p-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg.


e ) Correlation between MPX severity ( NSkin lesions ) and protein expression ( y - axis ) . One MPX patient had an unclear additional skin condition ( not a pure case of MPX ) and therefore was excluded from the regression analysis that compares the number of skin lesions with the proteome ; however , the proteome of this patient was largely in agreement with those of the other MPX cases As a measure of MPX severity , the log2 ( 1 + NLesions / 15 ) was used . Here NLesions is the number of lesions . R2 shows squared correlation coefficient . MPX patients are colored orange , control patients green .

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e) Correlation between MPX severity (NSkin lesions) and protein expression (y-axis). One MPX patient had an unclear additional skin condition (not a pure case of MPX) and therefore was excluded from the regression analysis that compares the number of skin lesions with the proteome; however, the proteome of this patient was largely in agreement with those of the other MPX cases As a measure of MPX severity, the log2(1 + NLesions / 15) was used. Here NLesions is the number of lesions. R2 shows squared correlation coefficient. MPX patients are colored orange, control patients green.


a ) Heatmap displaying hierarchical clustering using differentially regulated proteins between patients with MPX , COVID - 19 , and controls .

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a) Heatmap displaying hierarchical clustering using differentially regulated proteins between patients with MPX, COVID-19, and controls.


b ) Scatterplot of log fold - change ( logFC ) for contrast MPX vs control ( C1 , x - axis ) and logFC for contrast COVID - 19 vs control ( C2 , y - axis ) . Only the central part of the cloud is shown here . Three truncated dots ( APOC1 , CRP , and SAA1 ) are shown in the lower left and upper right corner . Differentially abundant ( Regulated ; ' Reg ' ) proteins are color coded , with the red color corresponding to 37 proteins differentially abundant in both MPX vs control ( C1 ) and COVID - 19 vs control ( C2 ) , the orange color corresponding to proteins specifically changed in MPX vs control ( C1 ) only ( 16 proteins ) , and the green color corresponding to proteins responding to both MPX vs control ( C1 ) and MPX vs COVID - 19 ( C3 ) ( 3 proteins ) . There are no intersections between COVID - 19 vs control ( C2 ) and MPX vs COVID - 19 ( C3 ) . The blue color corresponds to proteins responding to MPX vs COVID - 19 ( C3 ) , but not in MPX vs control ( C1 ) ( 11 proteins ) , and the pink color to proteins responding to COVID - 19 vs control ( C2 ) only ( 19 proteins ) . The red dotted line shows a linear regression through the red dots , i . e . proteins differentially abundant in MPX vs control ( C1 ) and COVID - 19 vs control ( C2 ) . Note that orange and pink points have the same direction of regulation in both MPXV vs control ( C1 ) and COVID - 19 vs control ( C2 ) . Only green and blue dots ( except three proteins : ADIPOQ , GPLD1 , and IGHV1 - 2 ) have opposite directions in C1 and in C2 .

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b) Scatterplot of log fold-change (logFC) for contrast MPX vs control (C1, x-axis) and logFC for contrast COVID-19 vs control (C2, y- axis). Only the central part of the cloud is shown here. Three truncated dots (APOC1, CRP, and SAA1) are shown in the lower left and upper right corner. Differentially abundant (Regulated; 'Reg') proteins are color coded, with the red color corresponding to 37 proteins differentially abundant in both MPX vs control (C1) and COVID-19 vs control (C2), the orange color corresponding to proteins specifically changed in MPX vs control (C1) only (16 proteins), and the green color corresponding to proteins responding to both MPX vs control (C1) and MPX vs COVID-19 (C3) (3 proteins). There are no intersections between COVID-19 vs control (C2) and MPX vs COVID-19 (C3). The blue color corresponds to proteins responding to MPX vs COVID-19 (C3), but not in MPX vs control (C1) (11 proteins), and the pink color to proteins responding to COVID-19 vs control (C2) only (19 proteins). The red dotted line shows a linear regression through the red dots, i.e. proteins differentially abundant in MPX vs control (C1) and COVID-19 vs control (C2). Note that orange and pink points have the same direction of regulation in both MPXV vs control (C1) and COVID-19 vs control (C2). Only green and blue dots (except three proteins: ADIPOQ, GPLD1, and IGHV1-2) have opposite directions in C1 and in C2.


d ) Differentially regulated proteins of patients with MPX and COVID - 19 ( Volcano Plot ) ; α ≤ 0 . 015 and | logFC | > = 1 . 35 were used for selection of regulated proteins . The chosen significance level ensured that fdr for this contrast was below 22 % .

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d) Differentially regulated proteins of patients with MPX and COVID-19 (Volcano Plot); α ≤ 0.015 and |logFC| >= 1.35 were used for selection of regulated proteins. The chosen significance level ensured that fdr for this contrast was below 22%.


e ) Key proteins that differ between patients with MPX and COVID - 19 ( Boxplots ) ( p - values and fdr for corresponding contrast MPX vs COVID - 19 are provided in brackets ) : LCP1 ( p - value = 3E - 4 , fdr = 3E - 2 ) , LDHB ( p - value = 2E - 3 , fdr = 6E - 2 ) , CFHR1 ( p - value = 2E - 4 , fdr = 3E - 2 ) . Here the central bar marks the median ( second quartile ) , the bottom edge of the box marks the first quartile , the top edge of the box marks the third quartile , and the bottom and top whiskers mark the minimum and maximum values that are not outliers . The specific values of the protein expressions are also shown . Provided p - values are obtained from moderated statistics implemented in limma , dfrs were calculated according to Benjamini - Hochberg .

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e) Key proteins that differ between patients with MPX and COVID-19 (Boxplots) (p-values and fdr for corresponding contrast MPX vs COVID-19 are provided in brackets): LCP1 (p-value = 3E-4, fdr = 3E-2), LDHB (p-value = 2E-3, fdr = 6E-2), CFHR1 (p-value = 2E-4, fdr = 3E-2). Here the central bar marks the median (second quartile), the bottom edge of the box marks the first quartile, the top edge of the box marks the third quartile, and the bottom and top whiskers mark the minimum and maximum values that are not outliers. The specific values of the protein expressions are also shown. Provided p-values are obtained from moderated statistics implemented in limma, dfrs were calculated according to Benjamini-Hochberg.


f ) Top 8 proteins of an SVM - trained model discriminating between healthy controls ( n = 15 ) and MPX cases ( n = 6 ) ( top ) or COVID - 19 ( n = 10 ) and MPX ( n = 6 ) cases ( bottom ) . Means of the relative coefficients over a 5 - fold cross - validation are shown . Error bars denote the standard deviations . Red denotes positive , blue denotes negative coefficients . The AUC was calculated based on withheld samples that were not used for training the model .

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f) Top 8 proteins of an SVM-trained model discriminating between healthy controls (n=15) and MPX cases (n=6) (top) or COVID-19 (n=10) and MPX (n=6) cases (bottom). Means of the relative coefficients over a 5-fold cross-validation are shown. Error bars denote the standard deviations. Red denotes positive, blue denotes negative coefficients. The AUC was calculated based on withheld samples that were not used for training the model.


b ) Hierarchical clustering using differentially regulated proteins between patients with MPX and controls ( Heatmap ) ; p < 0 . 05 with Mann - Whitney U test with FDR - based multiple testing correction .

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b) Hierarchical clustering using differentially regulated proteins between patients with MPX and controls (Heatmap); p < 0.05 with Mann-Whitney U test with FDR-based multiple testing correction.


c ) Key proteins that differ between patients with MPX and controls , and COVID - 19 ( Boxplots ) . Dashed blue lines indicate the lowest detected peptide concentration from calibration curves . Statistics based on Mann - Whitney U test with multiple testing corrections . For boxplots , median is indicated by a solid line , hinges show the 25th and 75th percentiles , whiskers show values that , at maximum , are within 1 . 5 times the interquartile range .

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c) Key proteins that differ between patients with MPX and controls, and COVID-19 (Boxplots). Dashed blue lines indicate the lowest detected peptide concentration from calibration curves. Statistics based on Mann-Whitney U test with multiple testing corrections. For boxplots, median is indicated by a solid line, hinges show the 25th and 75th percentiles, whiskers show values that, at maximum, are within 1.5 times the interquartile range.


d ) Top 15 peptides of an SVM - trained model discriminating between healthy controls ( n = 15 ) and MPX cases ( n = 6 ) . Means of the relative coefficients over a 5 - fold cross - validation are shown . Error bars denote the standard deviations . Red denotes positive , blue denotes negative coefficients . The AUC was calculated based on withheld samples that were not used for training the model .

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d) Top 15 peptides of an SVM-trained model discriminating between healthy controls (n=15) and MPX cases (n=6). Means of the relative coefficients over a 5-fold cross-validation are shown. Error bars denote the standard deviations. Red denotes positive, blue denotes negative coefficients. The AUC was calculated based on withheld samples that were not used for training the model.


HeLa cells were co - transfected with Myc empty vector ( Myc ) , Myc - tagged FUNDC1 ( F1 - Myc ) or Myc - tagged FUNDC1 - ΔLIR mutant ( ΔLIR - Myc ) and Mito - OM - cherry , which localizes to the mitochondrial outer membrane , and immunostained with fluorescent anti - p62 and anti - Myc antibodies . Scale bar = 10 μm .

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HeLa cells were co-transfected with Myc empty vector (Myc), Myc-tagged FUNDC1 (F1-Myc) or Myc-tagged FUNDC1-ΔLIR mutant (ΔLIR-Myc) and Mito-OM-cherry, which localizes to the mitochondrial outer membrane, and immunostained with fluorescent anti-p62 and anti-Myc antibodies. Scale bar=10 μm.


HeLa cells were co - transfected with Myc , F1 - Myc or ΔLIR - Myc and Mito - OM - cherry and immunostained with anti - Ubiquitin ( Ub ) and anti - Myc antibodies . Scale bar = 10 µm .

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HeLa cells were co-transfected with Myc, F1-Myc or ΔLIR-Myc and Mito-OM-cherry and immunostained with anti-Ubiquitin (Ub) and anti-Myc antibodies. Scale bar=10 µm.


Fractionation analysis of HeLa cells transfected with Myc , F1 - Myc or ΔLIR - Myc . The loading controls ( L . C . ) were α - Tubulin for total cell lysate ( TCL ) and cytosolic fraction ( Cyto ) , and HSP60 for mitochondrial fraction ( Mito ) .

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Fractionation analysis of HeLa cells transfected with Myc, F1-Myc or ΔLIR-Myc. The loading controls (L.C.) were α-Tubulin for total cell lysate (TCL) and cytosolic fraction (Cyto), and HSP60 for mitochondrial fraction (Mito).


HeLa cells transfected with Myc or Myc / His6 double - tagged F1 - Myc - His were lysed and precipitated with Ni - NTA beads . Samples were then subjected to SDS - PAGE . The differential band ( upper arrow ) was isolated and subjected to mass spectral analysis . The identified proteins with a score higher than 50 are listed .

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HeLa cells transfected with Myc or Myc/His6 double-tagged F1-Myc-His were lysed and precipitated with Ni-NTA beads. Samples were then subjected to SDS-PAGE. The differential band (upper arrow) was isolated and subjected to mass spectral analysis. The identified proteins with a score higher than 50 are listed.


Co - IP of endogenous HSC70 with F1 - Myc or ΔLIR - Myc . α - Myc , anti - Myc

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Co-IP of endogenous HSC70 with F1-Myc or ΔLIR-Myc. α-Myc, anti-Myc antibody


Co - IP of HSC70 with FUNDC1 . α - F1 , anti - FUNDC1 antibody . rIgG , control rabbit IgG .

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Co-IP of HSC70 with FUNDC1. α-F1, anti-FUNDC1 antibody. rIgG, control rabbit IgG.


Co - IP analysis of endogenous FUNDC1 with Flag - HSP70 or Flag - HSC70 . α - Flag , anti - Flag antibody . Flag ， empty vector .

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Co-IP analysis of endogenous FUNDC1 with Flag-HSP70 or Flag-HSC70. α-Flag, anti-Flag antibody. Flag，empty vector.


HeLa cells were co - transfected with Myc , F1 - Myc or ΔLIR - Myc and Mito - OM - cherry and immunostained with anti - HSC70 and anti - Myc antibodies . Scale bar = 10 µm .

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HeLa cells were co-transfected with Myc, F1-Myc or ΔLIR-Myc and Mito-OM-cherry and immunostained with anti-HSC70 and anti-Myc antibodies. Scale bar=10 µm.


Fractionation analysis of HeLa cells transfected with Myc or F1 - Myc .

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Fractionation analysis of HeLa cells transfected with Myc or F1-Myc.


HeLa cells were co - transfected with Ub - R - GFP and Myc or F1 - Myc and immunostained with fluorescent anti - Myc and anti - GFP ( α - GFP ) antibodies . The α - GFP signals in F1 - Myc transfected cells were lowered to avoid the overexposure of the mitochondrially localized R - GFP , making such signals of the equally distributed R - GFP invisible . Green arrowheads show the mitochondrially localized R - GFP proteins , which were more abundant than the equally distributed R - GFP and showed stronger R - GFP fluorescence , white arrowheads show the mitochondrially localized R - GFP proteins without stronger R - GFP fluorescence , although abundant . Scale bar = 10 µm .

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HeLa cells were co-transfected with Ub-R-GFP and Myc or F1-Myc and immunostained with fluorescent anti-Myc and anti-GFP (α-GFP) antibodies. The α-GFP signals in F1-Myc transfected cells were lowered to avoid the overexposure of the mitochondrially localized R-GFP, making such signals of the equally distributed R-GFP invisible. Green arrowheads show the mitochondrially localized R-GFP proteins, which were more abundant than the equally distributed R-GFP and showed stronger R-GFP fluorescence, white arrowheads show the mitochondrially localized R-GFP proteins without stronger R-GFP fluorescence, although abundant. Scale bar=10 µm.


Fractionation analysis of HeLa cells co - transfected with Ub - R - GFP and Myc or F1 - Myc .

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Fractionation analysis of HeLa cells co-transfected with Ub-R-GFP and Myc or F1-Myc.


HeLa cells were co - transfected with Ub - R - GFP and F1 - Myc . Mitochondria were isolated and then treated with or without proteinase K ( Pro . K ) , or proteinase K in combination with digitonin or Triton X - 100 ( TX - 100 ) . Samples were then subjected to Immunoblot analysis . HSP60 , TIM23 and TOM20 are representatives of mitochondrial matrix - , mitochondrial inner membrane - and mitochondrial outer membrane - localized proteins , respectively .

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HeLa cells were co-transfected with Ub-R-GFP and F1-Myc. Mitochondria were isolated and then treated with or without proteinase K (Pro.K), or proteinase K in combination with digitonin or Triton X-100 (TX-100). Samples were then subjected to Immunoblot analysis. HSP60, TIM23 and TOM20 are representatives of mitochondrial matrix-, mitochondrial inner membrane- and mitochondrial outer membrane-localized proteins, respectively.


Fractionation analysis of HeLa cells stably transfected with control ( Ctrl ) shRNA vector or HSC70 shRNA and transiently co - transfected with Ub - R - GFP , Flag or Flag - HSC70 ( shRNA - resistant ) and Myc , or F1 - Myc , as indicated .

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Fractionation analysis of HeLa cells stably transfected with control (Ctrl) shRNA vector or HSC70 shRNA and transiently co-transfected with Ub-R-GFP, Flag or Flag-HSC70 (shRNA-resistant) and Myc, or F1-Myc, as indicated.


Fractionation analysis of the HeLa cells transfected with Ub - R - GFP and treated with DMSO ( control vehicle ) or 10 μM MG132 for 4 hours .

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Fractionation analysis of the HeLa cells transfected with Ub-R-GFP and treated with DMSO (control vehicle) or 10 μM MG132 for 4 hours.


Proteinase K protection analysis of the mitochondria isolated from the HeLa cells transfected with Ub - R - GFP and treated with MG132 .

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Proteinase K protection analysis of the mitochondria isolated from the HeLa cells transfected with Ub-R-GFP and treated with MG132.


Proteinase K protection analysis of the mitochondria isolated from HeLa cells co - transfected with Ub - R - GFP and Ctrl , TOM20 , TOM22 or TOM70 shRNA and treated with MG132 .

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Proteinase K protection analysis of the mitochondria isolated from HeLa cells co-transfected with Ub-R-GFP and Ctrl, TOM20, TOM22 or TOM70 shRNA and treated with MG132.


Fractionation analysis of HeLa cells stably transfected with Ctrl or LONP1 shRNA and transiently co - transfected with Ub - R - GFP and Myc or LONP1 ( shRNA - resistant ) and then treated with DMSO or MG132 .

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Fractionation analysis of HeLa cells stably transfected with Ctrl or LONP1 shRNA and transiently co-transfected with Ub-R-GFP and Myc or LONP1 (shRNA-resistant) and then treated with DMSO or MG132.


Fractionation analysis of HeLa cells stably transfected with Ctrl shRNA , F1 shRNA , or F1 shRNA together with shRNA - resistant F1 - Myc or ΔLIR - Myc , and transiently transfected with Ub - R - GFP and then treated with DMSO or MG132 .

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Fractionation analysis of HeLa cells stably transfected with Ctrl shRNA, F1 shRNA, or F1 shRNA together with shRNA-resistant F1-Myc or ΔLIR-Myc, and transiently transfected with Ub-R-GFP and then treated with DMSO or MG132.


Fractionation analysis of HeLa cells stably transfected with Ctrl or HSC70 shRNA and transiently co - transfected with Ub - R - GFP and Flag or shRNA - resistant Flag - HSC70 and then treated with DMSO or MG132 .

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Fractionation analysis of HeLa cells stably transfected with Ctrl or HSC70 shRNA and transiently co-transfected with Ub-R-GFP and Flag or shRNA-resistant Flag-HSC70 and then treated with DMSO or MG132.


Fractionation analysis of HeLa cells stably transfected with Ctrl or HSP70 shRNA and transiently co - transfected with Ub - R - GFP and Flag or shRNA - resistant Flag - HSP70 and then treated with DMSO or MG132 .

Fractionation analysis of HeLa cells stably transfected with Ctrl or HSP70 shRNA and transiently co-transfected with Ub-R-GFP and Flag or shRNA-resistant Flag-HSP70 and then treated with DMSO or MG132.


Mitochondria were isolated from the untransfected cells , cytosols were isolated from the cells transfected with GFP or Ub - R - GFP and treated with DMSO or MG132 for 8 hours , the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis .

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Mitochondria were isolated from the untransfected cells, cytosols were isolated from the cells transfected with GFP or Ub-R-GFP and treated with DMSO or MG132 for 8 hours, the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis.


Mitochondria isolated from the untransfected cells were resuspended in the cytosol isolated from the cells transfected with Ub - R - GFP and incubated at RT for I hour , the mitochondria were then isolated from the in vitro system , then treated with or without proteinase K and subjected to immunoblot analysis .

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Mitochondria isolated from the untransfected cells were resuspended in the cytosol isolated from the cells transfected with Ub-R-GFP and incubated at RT for I hour, the mitochondria were then isolated from the in vitro system, then treated with or without proteinase K and subjected to immunoblot analysis.


Mitochondria were isolated from the cells transfected with ctrl shRNA , F1 shRNA or F1 shRNA together with F1 - Myc , cytosols were isolated from the cells transfected with Ub - R - GFP , the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis .

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Mitochondria were isolated from the cells transfected with ctrl shRNA, F1 shRNA or F1 shRNA together with F1-Myc, cytosols were isolated from the cells transfected with Ub-R-GFP, the isolated mitochondria and cytosols were then subjected to in vitro protein import analysis.


Cytosol isolated from the cells transfected with Ub - R - GFP were mixed with the cytosols isolated from the cells transfected with ctrl shRNA , HSC70 shRNA or HSC70 shRNA together with Flag - HSC70 , mitochondria were isolated from the untransfected cells , the isolated mitochondria and cytosol mixtures were then subjected to in vitro protein import analysis .

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Cytosol isolated from the cells transfected with Ub-R-GFP were mixed with the cytosols isolated from the cells transfected with ctrl shRNA, HSC70 shRNA or HSC70 shRNA together with Flag-HSC70, mitochondria were isolated from the untransfected cells, the isolated mitochondria and cytosol mixtures were then subjected to in vitro protein import analysis.


Co - IP of HSC70 with FUNDC1 in HeLa cells treated with MG132 as indicated .

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Co-IP of HSC70 with FUNDC1 in HeLa cells treated with MG132 as indicated.


Immunoblot analysis of the HeLa cells transfected with or without HA - Bcl - xL and treated with DMSO or MG132 for 6 hours .

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Immunoblot analysis of the HeLa cells transfected with or without HA-Bcl-xL and treated with DMSO or MG132 for 6 hours.


Ni2 + - NTA precipitation of HeLa cells transfected with indicated vectors . S13A - Myc - His , Myc / His6 double - tagged FUNDC1 - S13A mutant .

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Ni2+-NTA precipitation of HeLa cells transfected with indicated vectors. S13A-Myc-His, Myc/His6 double-tagged FUNDC1-S13A mutant.


D , E . Fractionation analysis of HeLa cells transfected with indicated vectors and treated with DMSO or MG132 . Quantification is shown in ( E ) .

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D, E. Fractionation analysis of HeLa cells transfected with indicated vectors and treated with DMSO or MG132. Quantification is shown in (E).


HeLa cells were transfected with GFP - p62 , treated with DMSO or 10 μM MG132 for 8 hours and then immunostained with anti - Ub and anti - γ - Tubulin antibodies . Note that the aggresome ( blue arrowhead ) , rather than the MAPAs ( white arrowheads ) , colocalizes with γ - Tubulin . Scale bar = 10 µm .

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HeLa cells were transfected with GFP-p62, treated with DMSO or 10 μM MG132 for 8 hours and then immunostained with anti-Ub and anti-γ-Tubulin antibodies. Note that the aggresome (blue arrowhead), rather than the MAPAs (white arrowheads), colocalizes with γ-Tubulin. Scale bar=10 µm.


B , C . HeLa cells were stably transfected with the indicated vectors , treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm .

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B, C. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm.


B , C . HeLa cells were stably transfected with the indicated vectors , treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . Quantification is show in ( C ) .

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B, C. HeLa cells were stably transfected with the indicated vectors, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (C).


D , E . HeLa cells were transfected with Ctrl , TOM20 , TOM22 or TOM70 shRNA vectors including nuclear localized GFP ( N - GFP ) expressing elements , then treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . Quantification is show in ( E ) .

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D, E. HeLa cells were transfected with Ctrl, TOM20, TOM22 or TOM70 shRNA vectors including nuclear localized GFP (N-GFP) expressing elements, then treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (E).


F , G . HeLa cells were transfected with GFP or GFP - Bcl - xL , then treated with MG132 and immunostained with anti - p62 antibodies . Scale bar = 10 µm . Quantification is show in ( G ) .

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F, G. HeLa cells were transfected with GFP or GFP-Bcl-xL, then treated with MG132 and immunostained with anti-p62 antibodies. Scale bar=10 µm. Quantification is show in (G).


H , I . HeLa cells were stably transfected with Ctrl or LONP1 shRNA , treated with MG132 and immunostained with the indicated antibodies . Scale bar = 10 µm . Quantification is show in ( I ) .

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H, I. HeLa cells were stably transfected with Ctrl or LONP1 shRNA, treated with MG132 and immunostained with the indicated antibodies. Scale bar=10 µm. Quantification is show in (I).


csCLEM analysis of cells transfected with GFP - p62 and treated with MG132 . Red arrowheads show the dark red fluorescent beads used to correlate the images from light microscopy ( LM ) and transmission electron microscopy ( TEM ) . The blue arrowhead shows a MAPA observed under LM and TEM .

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csCLEM analysis of cells transfected with GFP-p62 and treated with MG132. Red arrowheads show the dark red fluorescent beads used to correlate the images from light microscopy (LM) and transmission electron microscopy (TEM). The blue arrowhead shows a MAPA observed under LM and TEM.


TEM analysis of HeLa cells treated with DMSO or MG132 . Note the layered structures ( red arrowheads ) in the mitochondrial matrix ( ii , iii ) , budding from mitochondria ( iv ) , attached to mitochondria ( v - vi ) , away from mitochondria ( vii ) or inside the MAPAs ( viii , ix ) in MG132 - treated cells . Scale bar = 500 nm . M , mitochondrion . N , nucleus .

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TEM analysis of HeLa cells treated with DMSO or MG132. Note the layered structures (red arrowheads) in the mitochondrial matrix (ii, iii), budding from mitochondria (iv), attached to mitochondria (v-vi), away from mitochondria (vii) or inside the MAPAs (viii, ix) in MG132-treated cells. Scale bar=500 nm. M, mitochondrion. N, nucleus.


Immunoblot analysis of the TX - 100 - soluble and - insoluble fractions of HeLa cells treated with MG132 as indicated .

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Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells treated with MG132 as indicated.


Immunoblot analysis of the TX - 100 - soluble and - insoluble fractions of HeLa cells transfected with Ub - R - GFP and treated with MG132 as indicated .

Immunoblot analysis of the TX-100-soluble and -insoluble fractions of HeLa cells transfected with Ub-R-GFP and treated with MG132 as indicated.


HeLa cells were co - transfected with Mito - OM - cherry and Mito - MTX - GFP , then treated with DMSO or MG132 and immunostained with anti - p62 antibody . Note the colocalization of a p62 punctum with a structure that is positive for Mito - OM - cherry but not for Mito - MTX - GFP ( white arrowheads ) in MG132 - treated cells . Scale bar = 10 µm .

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HeLa cells were co-transfected with Mito-OM-cherry and Mito-MTX-GFP, then treated with DMSO or MG132 and immunostained with anti-p62 antibody. Note the colocalization of a p62 punctum with a structure that is positive for Mito-OM-cherry but not for Mito-MTX-GFP (white arrowheads) in MG132-treated cells. Scale bar=10 µm.


HeLa cells were transfected with Mito - MTX - GFP , then treated with DMSO or MG132 and immunostained with anti - p62 and anti - TIM23 antibodies . Note the colocalization of a p62 punctum with a structure that is positive for TIM3 but not for Mito - MTX - GFP ( white arrowhead ) in MG132 - treated cells . Scale bar = 10 µm .

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HeLa cells were transfected with Mito-MTX-GFP, then treated with DMSO or MG132 and immunostained with anti-p62 and anti-TIM23 antibodies. Note the colocalization of a p62 punctum with a structure that is positive for TIM3 but not for Mito-MTX-GFP (white arrowhead) in MG132-treated cells. Scale bar=10 µm.


HeLa cells were co - transfected with Mito - MTX - GFP and OTC - Myc or OTC - Δ - Myc , treated with DMSO or MG132 and immunostained with anti - Myc and anti - p62 antibodies . Note that the p62 - labled MAPAs were colocalized with OTC - ∆ ( red arrowheads ) , but not with OTC ( white arrowheads ) in the MG132 treated cells . Scale bar = 10 µm .

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HeLa cells were co-transfected with Mito-MTX-GFP and OTC-Myc or OTC-Δ-Myc, treated with DMSO or MG132 and immunostained with anti-Myc and anti-p62 antibodies. Note that the p62-labled MAPAs were colocalized with OTC-∆ (red arrowheads), but not with OTC (white arrowheads) in the MG132 treated cells. Scale bar=10 µm.


TEM analysis of HeLa cells transfected with indicated vectors , treated with MG132 and stained with DAB . Blue arrowheads show MAPAs , red arrowheads show electron - dense Apex signals . Scale bar = 500 nm . M , mitochondrion .

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TEM analysis of HeLa cells transfected with indicated vectors, treated with MG132 and stained with DAB. Blue arrowheads show MAPAs, red arrowheads show electron-dense Apex signals. Scale bar=500 nm. M, mitochondrion.


Time - lapse analysis of live HeLa cells transfected with OTC - Δ - GFP and Mito - MTX - DsRed and treated with MG132 . White arrowheads show a OTC - Δ - GFP - positive Mito - MTX - DsRed - negative structure that eventually segregated from the mitochondrial network .

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Time-lapse analysis of live HeLa cells transfected with OTC-Δ-GFP and Mito-MTX-DsRed and treated with MG132. White arrowheads show a OTC-Δ-GFP-positive Mito-MTX-DsRed-negative structure that eventually segregated from the mitochondrial network.


Immunoblot analysis of the MG132 - induced deposition of FUNDC1 , TIM23 and p62 in HeLa cells transfected with Ctrl or DLP1 shRNA .

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Immunoblot analysis of the MG132-induced deposition of FUNDC1, TIM23 and p62 in HeLa cells transfected with Ctrl or DLP1 shRNA.


C , D . Immunoblot analysis of the MG132 - induced deposition of FUNDC1 in cells transfected with ctrl shRNA , FIS1 shRNA or FIS1 shRNA together with shRNA - resistant Flag - FIS1 . Quantification is shown in ( D ) .

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C, D. Immunoblot analysis of the MG132-induced deposition of FUNDC1 in cells transfected with ctrl shRNA, FIS1 shRNA or FIS1 shRNA together with shRNA-resistant Flag-FIS1. Quantification is shown in (D).


E , F . HeLa cells were stably transfected with Ctrl or FIS1 shRNA , treated with MG132 and immunostained with indicated antibodies . Scale bar = 10 µm . Quantification is shown in ( F ) .

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E, F. HeLa cells were stably transfected with Ctrl or FIS1 shRNA, treated with MG132 and immunostained with indicated antibodies. Scale bar=10 µm. Quantification is shown in (F).


G , H . Fractionation analysis of HeLa cells stably transfected with Ctrl or FIS1 shRNA , then transiently co - transfected with Ub - R - GFP and Flag or Flag - FIS1 and treated with DMSO or MG132 . Quantification is shown in ( H ) .

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G, H. Fractionation analysis of HeLa cells stably transfected with Ctrl or FIS1 shRNA, then transiently co-transfected with Ub-R-GFP and Flag or Flag-FIS1 and treated with DMSO or MG132. Quantification is shown in (H).


HeLa cells were transfected with GFP - LC3 , treated with DMSO or 10 μM MG132 for 8 hours and immunostained with anti - p62 antibody . Scale bar = 10 µm .

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HeLa cells were transfected with GFP-LC3, treated with DMSO or 10 μM MG132 for 8 hours and immunostained with anti-p62 antibody. Scale bar=10 µm.


Immunoblot analysis of the TCL of HeLa cells treated with MG132 as indicated .

Immunoblot analysis of the TCL of HeLa cells treated with MG132 as indicated.


Immunoblot analysis of the TCL of HeLa cells stably transfected with Ctrl or ATG5 shRNA and treated with MG132 as indicated .

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Immunoblot analysis of the TCL of HeLa cells stably transfected with Ctrl or ATG5 shRNA and treated with MG132 as indicated.


Immunoblot analysis of the TCL of HeLa cells stably transfected with the indicated vectors and treated with MG132 as indicated . Quantification of the results from panels ( D ) and ( E ) . 3 replicates were analyzed using Student ' s 2 - tailed t - test and are presented as the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Immunoblot analysis of the TCL of HeLa cells stably transfected with the indicated vectors and treated with MG132 as indicated.   Quantification of the results from panels (D) and (E). 3 replicates were analyzed using Student's 2-tailed t-test and are presented as the means ± SEM. * p<0.05, ** p<0.01, *** p<0.001.


HeLa cells were stably transfected with the indicated vectors , transiently transfected with GFP - LC3 , then treated with 10 μM MG132 for 8 hours and immunostained with anti - p62 antibody . Scale bar = 10 µm .

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HeLa cells were stably transfected with the indicated vectors, transiently transfected with GFP-LC3, then treated with 10 μM MG132 for 8 hours and immunostained with anti-p62 antibody. Scale bar=10 µm.


TEM analysis of F1 shRNA / F1 - Myc or F1 shRNA / ΔLIR - Myc cells treated with MG132 for 8 hours . Yellow arrowheads show the MAPAs . Blue arrows show the places where membrane is absent . White arrowheads show the layered aggregates budding from mitochondria . Scale bar = 500 nm ; M , mitochondrion .

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TEM analysis of F1 shRNA/F1-Myc or F1 shRNA/ΔLIR-Myc cells treated with MG132 for 8 hours. Yellow arrowheads show the MAPAs. Blue arrows show the places where membrane is absent. White arrowheads show the layered aggregates budding from mitochondria. Scale bar=500 nm; M, mitochondrion.


A , B . HeLa cells were treated with DMSO or 10 μM MG132 for 8 hours , then washed with fresh medium three times and cultured for another 3 days . Cells were then fixed and subjected to SA - β - gal staining . The senescent cells show SA - β - gal - positive staining ( Blue ) . Quantification is shown in ( B ) .

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A, B. HeLa cells were treated with DMSO or 10 μM MG132 for 8 hours, then washed with fresh medium three times and cultured for another 3 days. Cells were then fixed and subjected to SA-β-gal staining. The senescent cells show SA-β-gal-positive staining (Blue). Quantification is shown in (B).


C , D . Immunoblot analysis of HeLa cells treated with DMSO or MG132 for 8 hours , then washed and cultured for the indicated times . Quantification is shown in ( D ) .

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C, D. Immunoblot analysis of HeLa cells treated with DMSO or MG132 for 8 hours, then washed and cultured for the indicated times. Quantification is shown in (D).


E , Quantification of SA - β - gal staining in HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C ( C . C ) for another 3 days .

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E, Quantification of SA-β-gal staining in HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C (C.C) for another 3 days.


F . HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C ( C . C ) for another 3 days . Immunoblot analysis of the cells is shown in ( F ) .

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F. HeLa cells treated with DMSO or MG132 for 8 hours then washed and incubated in fresh medium containing DMSO or Compound C (C.C) for another 3 days. Immunoblot analysis of the cells is shown in (F).


Quantification of SA - β - gal staining of HeLa cells stably transfected with the indicated vectors , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1 - Myc or ΔLIR - Myc , although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells .

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Quantification of SA-β-gal staining of HeLa cells stably transfected with the indicated vectors, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1-Myc or ΔLIR-Myc, although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells.


H . HeLa cells stably transfected with the indicated vectors , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( H ) . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1 - Myc or ΔLIR - Myc , although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells .

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H. HeLa cells stably transfected with the indicated vectors, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (H). The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by FUNDC1 knockdown and restored by reintroduction of F1-Myc or ΔLIR-Myc, although the basal level of p53 in FUNDC1 knockdown cells is higher than that in control cells.


Quantification of MG132 - induced SA - β - gal staining in HeLa cells stably transfected with Ctrl , HSP70 or HSC70 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown , although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells . Both p53 and p21 were up - regulated in HSP70 knockdown cells treated with or without MG132 .

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Quantification of MG132-induced SA-β-gal staining in HeLa cells stably transfected with Ctrl, HSP70 or HSC70 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown, although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells. Both p53 and p21 were up-regulated in HSP70 knockdown cells treated with or without MG132.


J . HeLa cells stably transfected with Ctrl , HSP70 or HSC70 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( J ) . The up - regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown , although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells . Both p53 and p21 were up - regulated in HSP70 knockdown cells treated with or without MG132 .

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

J. HeLa cells stably transfected with Ctrl, HSP70 or HSC70 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (J). The up-regulation of p53 and p21 at 3 days after pulse treatment with MG132 was alleviated by HSC70 knockdown, although the basal level of p53 in HSC70 knockdown cells is higher than that in control cells. Both p53 and p21 were up-regulated in HSP70 knockdown cells treated with or without MG132.


HeLa cells stably transfected with tetracycline ( Tet ) - inducible Bcl - xL expression vector were incubated in DMEM containing Tet or not for 12 hours , then treated with DMSO or 10 μM MG132 for 8 hours , then washed and incubated in fresh medium for another 3 days , Cells were then subjected to SA - β - gal staining . The induced Bcl - xL was detectable even 3 days after treatment withdrawal , because this protein is stable .

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

HeLa cells stably transfected with tetracycline (Tet)-inducible Bcl-xL expression vector were incubated in DMEM containing Tet or not for 12 hours, then treated with DMSO or 10 μM MG132 for 8 hours, then washed and incubated in fresh medium for another 3 days, Cells were then subjected to SA-β-gal staining. The induced Bcl-xL was detectable even 3 days after treatment withdrawal, because this protein is stable.


L . HeLa cells stably transfected with tetracycline ( Tet ) - inducible Bcl - xL expression vector were incubated in DMEM containing Tet or not for 12 hours , then treated with DMSO or 10 μM MG132 for 8 hours , then washed and incubated in fresh medium for another 3 days , Immunoblot analysis of the cells is shown in ( L ) . The induced Bcl - xL was detectable even 3 days after treatment withdrawal , because this protein is stable .

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

L. HeLa cells stably transfected with tetracycline (Tet)-inducible Bcl-xL expression vector were incubated in DMEM containing Tet or not for 12 hours, then treated with DMSO or 10 μM MG132 for 8 hours, then washed and incubated in fresh medium for another 3 days, Immunoblot analysis of the cells is shown in (L). The induced Bcl-xL was detectable even 3 days after treatment withdrawal, because this protein is stable.


Quantification of the SA - β - gal staining of HeLa cells stably transfected with Ctrl or LONP1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days .

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Quantification of the SA-β-gal staining of HeLa cells stably transfected with Ctrl or LONP1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days.


, N . HeLa cells stably transfected with Ctrl or LONP1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( N ) .

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

, N. HeLa cells stably transfected with Ctrl or LONP1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (N).


Quantification of the SA - β - gal staining of HeLa cells stably transfected with Ctrl or FIS1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days .

Quantification of the SA-β-gal staining of HeLa cells stably transfected with Ctrl or FIS1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days.


P . HeLa cells stably transfected with Ctrl or FIS1 shRNA , treated with DMSO or MG132 for 8 hours , then washed and cultured for 3 days . Immunoblot analysis of the cells is shown in ( P ) .

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P. HeLa cells stably transfected with Ctrl or FIS1 shRNA, treated with DMSO or MG132 for 8 hours, then washed and cultured for 3 days. Immunoblot analysis of the cells is shown in (P).


Western blot analysis of Shp2 in control and Shp2 ko cells .

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Western blot analysis of Shp2 in control and Shp2 ko cells.


Immunofluorescence analysis of Ki67 on control and Shp2 ko cells at day 7 . Scale bar , 50 μm . Quantification of the numbers of Ki67 + cells shown in ( C ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 .

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Immunofluorescence analysis of Ki67 on control and Shp2 ko cells at day 7. Scale bar, 50 μm. Quantification of the numbers of Ki67+cells shown in (C). Error bars represent SEM (n = 3). **P 0.01.


phere formation by control and Shp2 ko cells at day 7 . Scale bar , 200 μm . Quantification of the numbers of spheres formed by control and Shp2 ko cells shown in ( E ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 .

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phere formation by control and Shp2 ko cells at day 7. Scale bar, 200 μm. Quantification of the numbers of spheres formed by control and Shp2 ko cells shown in (E). Error bars represent SEM (n = 3). **P 0.01.


Growth kinetics of spheres formed by control and Shp2 ko cells by counting sphere cells at day 4 , 7 , and 12 . Error bars represent SEM ( n = 3 ) .

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Growth kinetics of spheres formed by control and Shp2 ko cells by counting sphere cells at day 4, 7, and 12. Error bars represent SEM (n = 3).


SA ‐ β ‐ gal staining on spheres formed by control and Shp2 ko cells at day 7 . Scale bar , 100 μm . Quantification of the numbers of senescent cells in the spheres shown in ( H ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 .

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SA‐β‐gal staining on spheres formed by control and Shp2 ko cells at day 7. Scale bar, 100 μm. Quantification of the numbers of senescent cells in the spheres shown in (H). Error bars represent SEM (n = 3). **P 0.01.


Western blot analysis of H3K9me3 , p27 , p53 pS18 ( Ser18 ‐ phosphorylated p53 ) , total p53 , Shp2 , and α ‐ tubulin in control and Shp2 ko cells .

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Western blot analysis of H3K9me3, p27, p53 pS18 (Ser18‐phosphorylated p53), total p53, Shp2, and α‐tubulin in control and Shp2 ko cells.


SA ‐ β ‐ gal staining on spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM at day 7 . Scale bar , 100 μm . Quantification of the numbers of senescent cells shown in ( K ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . Quantification of the numbers of spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . Statistical significance was assessed by Student ' s unpaired t ‐ test .

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

SA‐β‐gal staining on spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM at day 7. Scale bar, 100 μm. Quantification of the numbers of senescent cells shown in (K). Error bars represent SEM (n = 3). **P 0.01. Quantification of the numbers of spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM. Error bars represent SEM (n = 3). **P 0.01. Statistical significance was assessed by Student's unpaired t‐test.


qRT - PCR analysis of mRNA levels of Skp2 , Aurka , Dll1 , and Hey1 in control , Shp2 ko , and Shp2 ‐ inhibited cells . Error bars represent SEM ( n = 3 ) .

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qRT-PCR analysis of mRNA levels of Skp2, Aurka, Dll1, and Hey1 in control, Shp2 ko, and Shp2‐inhibited cells. Error bars represent SEM (n = 3).


SA ‐ β ‐ gal staining on control , Shp2 ko , and Shp2 ko cells with overexpression of Skp2 , Aurka , or N3IC cDNA . Scale bar , 100 μm . Quantification of the numbers of senescent cells shown in ( C ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 ; * * * P 0 . 001 . Quantification of the numbers of spheres formed by cells shown in ( C ) . Error bars represent SEM ( n = 3 ) . * P 0 . 05 ; * * P 0 . 01 ; and * * * P 0 . 001 . Statistical significance was assessed by Student ' s unpaired t ‐ test .

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

SA‐β‐gal staining on control, Shp2 ko, and Shp2 ko cells with overexpression of Skp2, Aurka, or N3IC cDNA. Scale bar, 100 μm. Quantification of the numbers of senescent cells shown in (C). Error bars represent SEM (n = 3). **P 0.01; ***P 0.001. Quantification of the numbers of spheres formed by cells shown in (C). Error bars represent SEM (n = 3). *P 0.05; **P 0.01; and ***P 0.001. Statistical significance was assessed by Student's unpaired t‐test.


SA ‐ β ‐ gal staining on control , Shp2 ko , and Shp2 ko cells with shRNA knockdown of p27 or p53 . Scale bar , 50 μm . Quantification of the numbers of senescent cells shown in ( A ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . Quantification of the numbers of spheres formed by cells shown in ( A ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 .

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

SA‐β‐gal staining on control, Shp2 ko, and Shp2 ko cells with shRNA knockdown of p27 or p53. Scale bar, 50 μm. Quantification of the numbers of senescent cells shown in (A). Error bars represent SEM (n = 3). **P 0.01. Quantification of the numbers of spheres formed by cells shown in (A). Error bars represent SEM (n = 3). **P 0.01.


Western blot analysis of p27 , p53 , and α ‐ tubulin in control , Shp2 ko , and Shp2 ko cells with overexpression of Skp2 , Aurka , or N3IC cDNAs . The numbers below indicate the relative ratios of band intensities between p27 and α ‐ tubulin or between p53 and α ‐ tubulin .

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

Western blot analysis of p27, p53, and α‐tubulin in control, Shp2 ko, and Shp2 ko cells with overexpression of Skp2, Aurka, or N3IC cDNAs. The numbers below indicate the relative ratios of band intensities between p27 and α‐tubulin or between p53 and α‐tubulin.


Western blot analysis of p27 , p53 , and α ‐ tubulin in PyMT cells treated with control ( DMSO ) or inhibitors against Skp2 ( MLN4924 ) , Aurora A ( VX ‐ 680 ) , or Notch ( DAPT ) at the indicated concentrations . The numbers below indicate the relative ratios of band intensities between p27 and α ‐ tubulin or between p53 and α ‐ tubulin .

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Western blot analysis of p27, p53, and α‐tubulin in PyMT cells treated with control (DMSO) or inhibitors against Skp2 (MLN4924), Aurora A (VX‐680), or Notch (DAPT) at the indicated concentrations. The numbers below indicate the relative ratios of band intensities between p27 and α‐tubulin or between p53 and α‐tubulin.


Quantification of the numbers of senescent cells in PyMT cells treated with control , MLN4924 , MLN4924 plus p27 knockdown or MLN4924 plus p53 knockdown . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 ; NS , not significant .

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Quantification of the numbers of senescent cells in PyMT cells treated with control, MLN4924, MLN4924 plus p27 knockdown or MLN4924 plus p53 knockdown. Error bars represent SEM (n = 3). **P 0.01; NS, not significant.


Quantification of the numbers of senescent cells in PyMT cells treated with control , VX ‐ 680 , VX ‐ 680 plus p27 knockdown , or VX ‐ 680 plus p53 knockdown . Error bars represent SEM ( n = 3 ) . * P 0 . 05 ; NS , not significant .

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Quantification of the numbers of senescent cells in PyMT cells treated with control, VX‐680, VX‐680 plus p27 knockdown, or VX‐680 plus p53 knockdown. Error bars represent SEM (n = 3). *P 0.05; NS, not significant.


Quantification of the numbers of senescent cells in PyMT cells treated with control , DAPT , DAPT plus p27 knockdown , or DAPT plus p53 knockdown . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 ; NS , not significant . Statistical significance was assessed by Student ' s unpaired t ‐ test .

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Quantification of the numbers of senescent cells in PyMT cells treated with control, DAPT, DAPT plus p27 knockdown, or DAPT plus p53 knockdown. Error bars represent SEM (n = 3). **P 0.01; NS, not significant. Statistical significance was assessed by Student's unpaired t‐test.


SA ‐ β ‐ gal staining on MLN4924 tumorcells treated with control , Shp2 inhibitor GS493 ( 15 μM ) , Src inhibitor PP2 ( 2 . 5 μM ) , Fak inhibitor TAE226 ( 0 . 5 μM ) , or Mek1 inhibitor U0126 ( 20 μM ) . Scale bar , 100 μm .

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SA‐β‐gal staining on MLN4924 tumorcells treated with control, Shp2 inhibitor GS493 (15 μM), Src inhibitor PP2 (2.5 μM), Fak inhibitor TAE226 (0.5 μM), or Mek1 inhibitor U0126 (20 μM). Scale bar, 100 μm.


Quantification of the numbers of senescent cells in the presence or absence of the inhibitors , as shown in ( A ) . Error bars represent SEM ( n = 3 ) . * P 0 . 05 ; * * P 0 . 01 . Quantification of the numbers of spheres in the presence or absence of the inhibitors , as in ( A ) . Error bars represent SEM ( n = 3 ) . * P 0 . 05 ; * * P 0 . 01

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the numbers of senescent cells in the presence or absence of the inhibitors, as shown in (A). Error bars represent SEM (n = 3). *P 0.05; **P 0.01. Quantification of the numbers of spheres in the presence or absence of the inhibitors, as in (A). Error bars represent SEM (n = 3). *P 0.05; **P 0.01


Western blot analysis of pY527 , pY416 and total Src , pY925 and total Fak , pT202 / pT204 and total Erk1 / 2 , and α ‐ tubulin in MLN4924 tumorcells with or without treatment with 5 μM GS493 .

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Western blot analysis of pY527, pY416 and total Src, pY925 and total Fak, pT202/pT204 and total Erk1/2, and α‐tubulin in MLN4924 tumorcells with or without treatment with 5 μM GS493.


qRT - PCR analysis of mRNA levels of Skp2 , Aurka , Dll1 , and Hey1 in control cells and those treated with the indicated inhibitors . Error bars represent SEM ( n = 3 ) .

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qRT-PCR analysis of mRNA levels of Skp2, Aurka, Dll1, and Hey1 in control cells and those treated with the indicated inhibitors. Error bars represent SEM (n = 3).


A Kaplan - Meier survival analysis of humanpatients ( n = 3 , 935 ) with invasive breast cancers divided into two groups based on the gene expression level of Shp2 ( see ) .

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A Kaplan-Meier survival analysis of humanpatients (n = 3,935) with invasive breast cancers divided into two groups based on the gene expression level of Shp2 (see ).


B - D Kaplan - Meier survival analyses of the humanpatients ( n = 3 , 935 ) divided into two groups based on the expression level of the genes of each overrepresented class determined in Fig A : cell cycle genes , DNA replication genes , or p53 target genes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Kaplan-Meier survival analyses of the humanpatients (n = 3,935) divided into two groups based on the expression level of the genes of each overrepresented class determined in Fig A: cell cycle genes, DNA replication genes, or p53 target genes.


E , F Kaplan - Meier survival analyses of humanpatients ( n = 3 , 935 ) divided into two groups based on the gene expression level of Skp2 or Aurka . HR , hazard ratio .

O O O O O O O O O O O O O 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 Kaplan-Meier survival analyses of humanpatients (n = 3,935) divided into two groups based on the gene expression level of Skp2 or Aurka. HR, hazard ratio.


Mammary gland whole ‐ mounts from control MLN4924 and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice at indicated ages . The characteristic lymph node of the mammary glandfat pad is marked by white arrowheads ; premalignant lesions are marked by red arrowheads . Scale bar , 4 mm . Quantification of premalignant lesions in mammary glands of control MLN4924 and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice , as shown in ( B ) . Error bars represent SEM ( n = 6 ) . * P 0 . 05 ( Student ' s unpaired t ‐ test ) .

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Mammary gland whole‐mounts from control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice at indicated ages. The characteristic lymph node of the mammary glandfat pad is marked by white arrowheads; premalignant lesions are marked by red arrowheads. Scale bar, 4 mm. Quantification of premalignant lesions in mammary glands of control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice, as shown in (B). Error bars represent SEM (n = 6). *P 0.05 (Student's unpaired t‐test).


Immunostaining of EYFP and CK8 on mammary gland sections from MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ; EYFP − / fl and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice at hyperplasia , adenoma , and carcinoma stages . Scale bar , 100 μm . n = 8 .

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Immunostaining of EYFP and CK8 on mammary gland sections from MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice at hyperplasia, adenoma, and carcinoma stages. Scale bar, 100 μm. n = 8.


Immunohistochemistry analysis of Cre recombinase on paraffin sections of primary tumors ( top ) and metastases ( bottom ) from MLN4924 ; Shp2fl / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice . Scale bar , 100 μm . n = 8 .

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Immunohistochemistry analysis of Cre recombinase on paraffin sections of primary tumors (top) and metastases (bottom) from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice. Scale bar, 100 μm. n = 8.


qRT - PCR analyses of Shp2mRNA levels in tumor tissues from MLN4924 ; Shp2fl / fl ( n = 8 ) , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ( n = 6 ) , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ( n = 6 ) mice . Error bars represent upper and lower quartiles .

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qRT-PCR analyses of Shp2mRNA levels in tumor tissues from MLN4924;Shp2fl/fl (n = 8), MLN4924;MMTV‐Cre;Shp2+/fl (n = 6), and MLN4924;MMTV‐Cre;Shp2fl/fl (n = 6) mice. Error bars represent upper and lower quartiles.


Western blot analysis of Shp2protein levels in tumor tissues from MLN4924 ; Shp2fl / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice .

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Western blot analysis of Shp2protein levels in tumor tissues from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice.


Immunohistochemistry analysis of EYFP ( top ) and SA ‐ β ‐ gal staining ( bottom ) on cryosections of mammary glandtumors from control MLN4924 ; Shp2fl / fl ; EYFP − / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ; EYFP − / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice . Note that SA ‐ β ‐ gal activity was present in the EYFP ‐ positive ( Shp2 ‐ negative ) areas ( right , arrows ) and was absent in the EYFP ‐ negative ( Shp2 ‐ positive ) areas ( right , arrowhead ) of mammary glandtumors of MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice . Consecutive sections of EYFP and SA ‐ β ‐ gal staining for each group are shown . Scale bar : 100 μm . n = 5 .

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Immunohistochemistry analysis of EYFP (top) and SA‐β‐gal staining (bottom) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Note that SA‐β‐gal activity was present in the EYFP‐positive (Shp2‐negative) areas (right, arrows) and was absent in the EYFP‐negative (Shp2‐positive) areas (right, arrowhead) of mammary glandtumors of MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Consecutive sections of EYFP and SA‐β‐gal staining for each group are shown. Scale bar: 100 μm. n = 5.


Immunofluorescence analysis of Ki67 , EYFP and CK8 ( pan ‐ epithelial marker ) ( top panel ) , p27 and EYFP ( middle panel ) , and p53 and EYFP ( bottom panel ) on cryosections of mammary glandtumors from control MLN4924 ; Shp2fl / fl ; EYFP − / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ; EYFP − / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice . DAPI was used for nuclear counterstain . Ki67 was absent in EYFP ‐ positive cells ( top right , arrows ) and was present in EYFP ‐ negative cells ( top right , arrowheads ) ; p27 was present in EYFP ‐ positive cells ( middle right , arrows ) and was absent in EYFP ‐ negative cells ( middle right , arrowhead ) ; and p53 was present in EYFP ‐ positive cells ( bottom right , arrows ) and was absent in EYFP ‐ negative cells ( bottom right , arrowhead ) . Scale bar : top panel , 100 μm ; middle and bottom panel , 50 μm . n = 5 .

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

Immunofluorescence analysis of Ki67, EYFP and CK8 (pan‐epithelial marker) (top panel), p27 and EYFP (middle panel), and p53 and EYFP (bottom panel) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. DAPI was used for nuclear counterstain. Ki67 was absent in EYFP‐positive cells (top right, arrows) and was present in EYFP‐negative cells (top right, arrowheads); p27 was present in EYFP‐positive cells (middle right, arrows) and was absent in EYFP‐negative cells (middle right, arrowhead); and p53 was present in EYFP‐positive cells (bottom right, arrows) and was absent in EYFP‐negative cells (bottom right, arrowhead). Scale bar: top panel, 100 μm; middle and bottom panel, 50 μm. n = 5.


qRT - PCR analysis of mRNA levels of Shp2 , Skp2 , Aurka , Dll1 , and Hey1 in EYFP + and EYFP − cells isolated by FACS from MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / fltumors . Error bars represent SEM ( n = 5 ) .

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qRT-PCR analysis of mRNA levels of Shp2, Skp2, Aurka, Dll1, and Hey1 in EYFP+ and EYFP−cells isolated by FACS from MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/fltumors. Error bars represent SEM (n = 5).


Kaplan - Meier analysis of tumor formation in control ( blue , n = 8 ) and GS493 ‐ pretreated ( red , n = 8 ) MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice . The bar indicates the duration of treatment .

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Kaplan-Meier analysis of tumor formation in control (blue, n = 8) and GS493‐pretreated (red, n = 8) MLN4924;MMTV‐Cre;Shp2fl/flmice. The bar indicates the duration of treatment.


Tumorgrowth curves for control ( blue , n = 5 ) and GS493 ‐ treated ( red , n = 5 ) MLN4924 mice . Note that after day 24 , all mice in the control group were sacrificed due to the tumor size . Error bars represent SEM ( n = 5 ) .

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Tumorgrowth curves for control (blue, n = 5) and GS493‐treated (red, n = 5) MLN4924 mice. Note that after day 24, all mice in the control group were sacrificed due to the tumor size. Error bars represent SEM (n = 5).


( A ) SDS - PAGE gels stained with coomassie showing the purity of the Chlamydomonas IFT - B2 components , over - expressed in E . coli or insect cells , after the last purification step by SEC . IFT54 / 20 and IFT57 / 38 were purified as hetero - dimeric complexes whereas IFT80 , IFT172 and an N - terminal IFT172 ( 1 - 968 ) ( IFT172ΔC ) constructs were purified as single components . A cartoon representation of the predicted domain architecture of IFT - B2 components using HHPRED ( Söding et al , 2005 ) is shown at the center .

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(A) SDS-PAGE gels stained with coomassie showing the purity of the Chlamydomonas IFT-B2 components, over-expressed in E. coli or insect cells, after the last purification step by SEC. IFT54/20 and IFT57/38 were purified as hetero-dimeric complexes whereas IFT80, IFT172 and an N-terminal IFT172(1-968) (IFT172ΔC) constructs were purified as single components. A cartoon representation of the predicted domain architecture of IFT-B2 components using HHPRED (Söding et al, 2005) is shown at the center.


( B ) Pull - down analysis for the hexameric IFT - B2 complex . A mixture of IFT172ΔC , IFT80 , and the IFT57 / 38 complex ( ' input ' ) shows only weak non - specific binding to GSH - resin ( ' empty ' ) , but all proteins are pulled down by IFT54 / 20 - GST complex . Note that the IFT20 - GST and IFT38 proteins co - migrate on the gel and cannot be properly resolved . However , the presence of the IFT57 protein in the pull - down sample implies that its binding partner IFT38 is also there .

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(B)Pull-down analysis for the hexameric IFT-B2 complex. A mixture of IFT172ΔC, IFT80, and the IFT57/38 complex ('input') shows only weak non-specific binding to GSH-resin ('empty'), but all proteins are pulled down by IFT54/20-GST complex. Note that the IFT20-GST and IFT38 proteins co-migrate on the gel and cannot be properly resolved. However, the presence of the IFT57 protein in the pull-down sample implies that its binding partner IFT38 is also there.


( C ) ( left ) SEC elution profiles of individual IFT - B2 components and of the assembled hexameric IFT - B2 complex . The elution peak of IFT - B2 is shifted compared to all of the individual components . Note that the peak heights were adjusted for better visualization of the result . ( right ) Coomassie stained SDS PAGE gel of the six main fractions of the IFT - B2 SEC elution peak demonstrating the presence of all six IFT - B2 subunits . Weak contaminating bands are marked with asterisks .

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(C) (left) SEC elution profiles of individual IFT-B2 components and of the assembled hexameric IFT-B2 complex. The elution peak of IFT-B2 is shifted compared to all of the individual components. Note that the peak heights were adjusted for better visualization of the result. (right) Coomassie stained SDS PAGE gel of the six main fractions of the IFT-B2 SEC elution peak demonstrating the presence of all six IFT-B2 subunits. Weak contaminating bands are marked with asterisks.


( A - C ) SEC elution profiles and corresponding SDS PAGE gels of IFT172ΔC , IFT57 / 38 , IFT80 , IFT54 / 20 as well as mixtures of IFT172ΔC / 57 / 38 , IFT80 / 57 / 38 and IFT172ΔC / 80 / 57 / 38 demonstrating that IFT57 / 38 associates with both IFT80 and IFT172ΔC . Only the central peak fraction from each SEC experiment is shown on the SDS PAGE gel . Note that the presence of both IFT172ΔC and IFT80 in a single peak in ( c ) ( peak 3 ) is due to the fact that these two proteins elute in the same position , and not because they physically interact ( see also Appendix Fig . S2B ) .

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(A-C) SEC elution profiles and corresponding SDS PAGE gels of IFT172ΔC, IFT57/38, IFT80, IFT54/20 as well as mixtures of IFT172ΔC/57/38, IFT80/57/38 and IFT172ΔC/80/57/38 demonstrating that IFT57/38 associates with both IFT80 and IFT172ΔC. Only the central peak fraction from each SEC experiment is shown on the SDS PAGE gel. Note that the presence of both IFT172ΔC and IFT80 in a single peak in (c) (peak 3) is due to the fact that these two proteins elute in the same position, and not because they physically interact (see also Appendix Fig. S2B).


( D ) GSH resin pull - down of a mixture of IFT80 and IFT172ΔC ( input , lane 1 ) with GST - tagged CH - domains of IFT57 or IFT38 ( IFT57CH or IFT38CH ) or with the GST - tagged coiled - coil ( CC ) region of IFT57 mixed with His - tagged coiled - coil region of IFT38 . The SDS - PAGE gel shows interaction between IFT57CH - IFT172ΔC , IFT38CH - IFT80 and IFT57CC - IFT38CC .

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(D) GSH resin pull-down of a mixture of IFT80 and IFT172ΔC (input, lane 1) with GST-tagged CH-domains of IFT57 or IFT38 (IFT57CH or IFT38CH) or with the GST-tagged coiled-coil (CC) region of IFT57 mixed with His-tagged coiled-coil region of IFT38. The SDS-PAGE gel shows interaction between IFT57CH-IFT172ΔC, IFT38CH-IFT80 and IFT57CC-IFT38CC.


( E ) Ni - NTA resin pull - down of untagged IFT54 / 20 with His - tagged IFT38 / 38 ( both subunits His - tagged ) . The two bands for IFT38 on the SDS PAGE gel correspond to His - tagged ( upper band ) and proteolytically cleaved untagged IFT38 ( lower band ) .

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(E) Ni-NTA resin pull-down of untagged IFT54/20 with His-tagged IFT38/38 (both subunits His-tagged). The two bands for IFT38 on the SDS PAGE gel correspond to His-tagged (upper band) and proteolytically cleaved untagged IFT38 (lower band).


( F ) Ni - NTA resin pull - down of untagged IFT54 / 20 with His - tagged IFT80 .

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(F) Ni-NTA resin pull-down of untagged IFT54/20 with His-tagged IFT80.


( A ) Ni2 + - NTA affinity pull - down of unpolymerised bovine αβ - tubulin with His - tagged IFT - B2 ( sub ) - complexes . A Coomassie stained SDS PAGE gel shows the IFT - B2 complexes used for pull - downs ( top ) and a western blot for anti - α - tubulin shows co - precipitated αβ - tubulin ( bottom ) .

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(A) Ni2+-NTA affinity pull-down of unpolymerised bovine αβ-tubulin with His-tagged IFT-B2 (sub)-complexes. A Coomassie stained SDS PAGE gel shows the IFT-B2 complexes used for pull-downs (top) and a western blot for anti-α-tubulin shows co-precipitated αβ-tubulin (bottom).


( F ) Ni2 + - NTA affinity pull - down of unpolymerised bovine αβ - tubulin with WT and mutant MmIFT54CH . ( top ) Coomassie stained SDS PAGE gel of input ( I ) and pulled down ( PD ) proteins and the corresponding western blot to visualize αβ - tubulin .

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(F) Ni2+-NTA affinity pull-down of unpolymerised bovine αβ-tubulin with WT and mutant MmIFT54CH. (top) Coomassie stained SDS PAGE gel of input (I) and pulled down (PD) proteins and the corresponding western blot to visualize αβ-tubulin.


( A ) Coomassie - stained SDS - PAGE gel showing GST - pulldown experiments between the IFT - B2 complex and an IFT88 / 52N subcomplex . The pre - assembled IFT - B2 complex ( lane 1 ) shows no non - specific interaction with empty GSH - beads ( lane 2 ) , but is efficiently pulled down by IFT88 / 52N - GST ( with an IFT52 ( 1 - 335 ) construct comprising the N - terminal domain as well as the IFT88 - binding region ) ( lane 3 ) . Both IFT88 and IFT52N are required for the interaction with IFT - B2 because neither the N - terminal IFT52 domain ( residues 1 - 275 ; lane 4 ) alone , nor an IFT88 / 52 complex lacking the N - terminal IFT52 domain ( with an IFT52 ( 281 - 335 ) construct containing only the IFT88 - binding region ) ( lane 5 ) show efficient pull - down of IFT - B2 . Contaminants from the purification of the complex shown in lane 5 are marked with asterisks .

O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR 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 O O O 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) Coomassie-stained SDS-PAGE gel showing GST-pulldown experiments between the IFT-B2 complex and an IFT88/52N subcomplex. The pre-assembled IFT-B2 complex (lane 1) shows no non-specific interaction with empty GSH-beads (lane 2), but is efficiently pulled down by IFT88/52N-GST (with an IFT52(1-335) construct comprising the N-terminal domain as well as the IFT88-binding region) (lane 3). Both IFT88 and IFT52N are required for the interaction with IFT-B2 because neither the N-terminal IFT52 domain (residues 1-275; lane 4) alone, nor an IFT88/52 complex lacking the N-terminal IFT52 domain (with an IFT52(281-335) construct containing only the IFT88-binding region) (lane 5) show efficient pull-down of IFT-B2. Contaminants from the purification of the complex shown in lane 5 are marked with asterisks.


( B ) Coomassie - stained SDS - PAGE gel showing a salt - stability test of the ( IFT88 / 52N ) - ( IFT - B2 ) interaction . Pre - assembled IFT - B2 complex was bound to IFT88 / 52N - GST immobilized on GSH - beads . The beads were then divided into five aliquots which were washed with buffers containing increasing concentrations of NaCl . After these washes the bound material was eluted and analyzed . IFT172ΔC is the only salt - labile component in this complex and is already washed off at a NaCl - concentration of 150 - 200 mM , whereas all the other IFT - B2 proteins remain associated even after washing with 1 M NaCl .

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(B) Coomassie-stained SDS-PAGE gel showing a salt-stability test of the (IFT88/52N)-(IFT-B2) interaction. Pre-assembled IFT-B2 complex was bound to IFT88/52N-GST immobilized on GSH-beads. The beads were then divided into five aliquots which were washed with buffers containing increasing concentrations of NaCl. After these washes the bound material was eluted and analyzed. IFT172ΔC is the only salt-labile component in this complex and is already washed off at a NaCl-concentration of 150-200 mM, whereas all the other IFT-B2 proteins remain associated even after washing with 1 M NaCl.


( C ) Coomassie - stained SDS - PAGE gels showing GST - pulldown experiments between IFT88 / 52N - GST complex immobilized on GSH beads and various combinations of IFT - B2 proteins . The left gel shows the IFT - B2 input mixtures , the right gel shows the bound material after washing and elution from the beads . A pentameric IFT - B2 complex ( IFT80 / 57 / 54 / 38 / 20 ) is efficiently pulled down by IFT88 / 52N - GST , but not by empty beads ( compare lanes 1 and 2 ) . Omission of IFT54 / 20 ( lane 4 ) , or IFT80 ( lane 5 ) does not influence the binding of any of the other components , but a lack of IFT57 / 38 in the mixture abolishes detectable pull - downs of IFT54 / 20 and IFT80 ( lane 4 ) , indicating that IFT57 / 38 is the direct interaction partner of IFT88 / 52N . Indeed , IFT57 / 38 alone ( lane 7 ) , but not IFT54 / 20 ( lane 6 ) or IFT80 ( lane 8 ) , is sufficient to be pulled down in this assay . Contaminating bands from the IFT88 / 52N sample are marked with asterisks .

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

(C) Coomassie-stained SDS-PAGE gels showing GST-pulldown experiments between IFT88/52N-GST complex immobilized on GSH beads and various combinations of IFT-B2 proteins. The left gel shows the IFT-B2 input mixtures, the right gel shows the bound material after washing and elution from the beads. A pentameric IFT-B2 complex (IFT80/57/54/38/20) is efficiently pulled down by IFT88/52N-GST, but not by empty beads (compare lanes 1 and 2). Omission of IFT54/20 (lane 4), or IFT80 (lane 5) does not influence the binding of any of the other components, but a lack of IFT57/38 in the mixture abolishes detectable pull-downs of IFT54/20 and IFT80 (lane 4), indicating that IFT57/38 is the direct interaction partner of IFT88/52N. Indeed, IFT57/38 alone (lane 7), but not IFT54/20 (lane 6) or IFT80 (lane 8), is sufficient to be pulled down in this assay. Contaminating bands from the IFT88/52N sample are marked with asterisks.


( B ) Coomassie stained SDS PAGE gel of a pull - down of IFT - B2 using WT or mutant IFT88 / 521 - 335 - GST . Whereas single point mutations slightly reduced the amounts of pulled down IFT - B2 , the K130E / R204E double mutation reduced it to background levels ( compare lanes 2 and 6 ) .

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(B) Coomassie stained SDS PAGE gel of a pull-down of IFT-B2 using WT or mutant IFT88/521-335-GST. Whereas single point mutations slightly reduced the amounts of pulled down IFT-B2, the K130E/R204E double mutation reduced it to background levels (compare lanes 2 and 6).


( A ) SEC - profiles of the IFT - B1 and IFT - B2 complexes as well as the 15 - subunit reconstituted IFT - B complex . IFT - B1 and IFT - B2 elute at similar volumes , but after mixing the resulting high - molecular weight complex is clearly shifted towards a smaller elution volume . The Coomassie - stained SDS - PAGE gel on the right shows the peak fractions of the individual runs .

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(A) SEC-profiles of the IFT-B1 and IFT-B2 complexes as well as the 15-subunit reconstituted IFT-B complex. IFT-B1 and IFT-B2 elute at similar volumes, but after mixing the resulting high-molecular weight complex is clearly shifted towards a smaller elution volume. The Coomassie-stained SDS-PAGE gel on the right shows the peak fractions of the individual runs.