Representative images of synchronised HeLa stained with an anti - geminin to confirm the indicated phase of mitosis ( scale bar 15 µm ) .

Representative images of synchronised HeLa stained with an anti-geminin to confirm the indicated phase of mitosis (scale bar 15 µm).


Nucleus height was measured by immunofluorescence using Hoechst staining


Representative images of synchronised MEFs stained with an anti - geminin to confirm the indicated phase of

Representative images of synchronised MEFs stained with an anti-geminin to confirm the indicated phase of mitosis


Nucleus height of synchronised MEFs ( black dot ) and synchronised MRC - 5 ( blue dot ) were measured by immunofluorescence using Hoechst staining . Data are presented as mean ± s . e . m .

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Nucleus height of synchronised MEFs (black dot) and synchronised MRC-5 (blue dot) were measured by immunofluorescence using Hoechst staining. Data are presented as mean ± s.e.m.


Nucleus height was measured by immunofluorescence using lamin A / C staining .

Nucleus height was measured by immunofluorescence using lamin A/C staining.


Representative HeLa - FUCCI cells in S - G2 - M ( green ) and G1 phase ( red ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 31 µm .

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Representative HeLa-FUCCI cells in S-G2-M (green) and G1 phase (red) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 31 µm.


Corresponding percentage of HeLa - FUCCI in mitosis .

Corresponding percentage of HeLa-FUCCI in mitosis.


Representative flow cytometry histograms for EdU positive cells in Ctrl , Blebb and Blebb + AP conditions . Corresponding percentage of EdU positive cells .

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Representative flow cytometry histograms for EdU positive cells in Ctrl, Blebb and Blebb + AP conditions. Corresponding percentage of EdU positive cells.


Analysis of transcription factor activity ( Affymetrix combo protein - DNA array ) for Ctrl , Blebb and Blebb + AP conditions . Each square corresponds to a specific transcription factor ( TF ) activity and is color coded to show the relative activity . ① To identify TFs regulated by nuclear shape , we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation - treated cells ( Blebb ) .

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Analysis of transcription factor activity (Affymetrix combo protein-DNA array) for Ctrl, Blebb and Blebb + AP conditions. Each square corresponds to a specific transcription factor (TF) activity and is color coded to show the relative activity. ① To identify TFs regulated by nuclear shape, we selected TFs that were more active in Ctrl and Blebb + AP compared to blebbistation-treated cells (Blebb).


TF ' s target genes and GAPDH mRNA levels were analyzed by real - time qPCR . Results are expressed as relative mRNA expression levels .

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TF's target genes and GAPDH mRNA levels were analyzed by real-time qPCR. Results are expressed as relative mRNA expression levels.


TEAD activity was analyzed in HeLa cells co - transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD - responsive promoter , and with a renilla plasmid as a gene reporter ; HeLa cells were cultured on 1 and 50 kPa ( n = 3 , t - test * * * p < 0 . 01 ) or treated with blebbistatin and blebbistatin + AP ( n = 6 ; t - test * p < 0 . 5 ) .

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

TEAD activity was analyzed in HeLa cells co-transfected with a renilla plasmid as a luciferase reporter plasmid controlled by the TEAD-responsive promoter, and with a renilla plasmid as a gene reporter; HeLa cells were cultured on 1 and 50 kPa (n = 3, t-test ***p<0.01) or treated with blebbistatin and blebbistatin + AP (n = 6; t-test *p<0.5).


AP1 activity was assessed in the indicated conditions using AP1 reporter assay .

AP1 activity was assessed in the indicated conditions using AP1 reporter assay.


Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1 .

Heatmap of mRNA expression profiles of genes regulated by TEAD and AP1.


Representative cells stained for p - Jun Ser63 ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . ( b ) Corresponding quantifications of p - Jun Ser63 nuclear intensity .

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Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. (b) Corresponding quantifications of p-Jun Ser63 nuclear intensity.


Representative cells stained for YAP ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . Corresponding quantifications of YAP activity ( ratio nucleus / cytosol ) .

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Representative cells stained for YAP (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (ratio nucleus/cytosol).


Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in Ctrl , Blebb , Blebb + AP condition and in Ctrl , Y27632 and Y27632 + AP .

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Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Blebb, Blebb + AP condition and in Ctrl, Y27632 and Y27632 + AP.


Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in Ctrl , Cytochalasin D ( CytoD ) , CytoD + AP condition .

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Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in Ctrl, Cytochalasin D (CytoD), CytoD + AP condition.


Quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2 .

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Quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity in cells depleted or not for SUN1 and SUN2.


Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p - Jun Ser63 ( magenta ) , YAP ( cyan ) and for DNA ( green ) . Additionally , cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei . Scale bar = 10 µm . Nuclear heights were measured using Hoechst staining . Corresponding quantifications of YAP activity ( nucleo - cytoplasmic ratio ) and p - Jun Ser63 nuclear intensity .

O O O O O O 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 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

Representative cells cultured on circular micropatterns with surfaces of 1500 and 500 µm2 and stained for p-Jun Ser63 (magenta), YAP (cyan) and for DNA (green). Additionally, cells were cultured on the micropatterns of 500 µm2 and an agarose pad was used to flatten their nuclei. Scale bar = 10 µm. Nuclear heights were measured using Hoechst staining. Corresponding quantifications of YAP activity (nucleo-cytoplasmic ratio) and p-Jun Ser63 nuclear intensity.


Quantifications of YAP activity in Ctrl , Blebb and Blebb + AP conditions , treated or not with importazol ( nucleo - cytoplasmic ratio ) .

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

Quantifications of YAP activity in Ctrl, Blebb and Blebb + AP conditions, treated or not with importazol (nucleo-cytoplasmic ratio).


Immunoblots of p - YAP ( ser127 ) , YAP , p - LATS ( Thr1079 ) , LATS , p - MST ( Thr1083 ) , MST , Kibra , PTPN14 and GADPH for Ctrl , Blebb and Blebb + AP conditions .

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Immunoblots of p-YAP (ser127), YAP, p-LATS (Thr1079), LATS, p-MST (Thr1083), MST, Kibra, PTPN14 and GADPH for Ctrl, Blebb and Blebb + AP conditions.


Corresponding quantification of p - YAP ( Ser125 ) relative to Ctrl and normalized to GAPDH . Corresponding quantification of p - LATS ( thr1079 ) relative to Ctrl and normalized to GAPDH .

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Corresponding quantification of p-YAP (Ser125) relative to Ctrl and normalized to GAPDH. Corresponding quantification of p-LATS (thr1079) relative to Ctrl and normalized to GAPDH.


Quantifications of p - Jun Ser63 nuclear intensity in Ctrl , Blebb , and Blebb + AP conditions for cells treated with DMSO , SB203580 , a MAP Kinase inhibitor and SP600125 , a JNK inhibitor .

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Quantifications of p-Jun Ser63 nuclear intensity in Ctrl, Blebb, and Blebb + AP conditions for cells treated with DMSO, SB203580, a MAP Kinase inhibitor and SP600125, a JNK inhibitor.


Representative cells stained for p - Jun Ser63 ( magenta ) and for DNA ( cyan ) in Ctrl , Blebb and Blebb + AP conditions for cells depleted or not for lamin - A / C . Scale bar = 10 µm . Corresponding quantification of p - Jun Ser63 nuclear intensity .

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Representative cells stained for p-Jun Ser63 (magenta) and for DNA (cyan) in Ctrl, Blebb and Blebb + AP conditions for cells depleted or not for lamin-A/C. Scale bar = 10 µm. Corresponding quantification of p-Jun Ser63 nuclear intensity.


A ) Representative immunofluorescence images showing localization of Myc - tagged M18BP1 - 1 truncations schematized in ( B ) to metaphase sperm centromeres in M18BP1 - depleted extract . Truncations analyzed are indicated on the left , immunolocalized proteins are indicated above . Scale bar , 10 μm . Insets are magnified 3X . B ) Schematic of M18BP1 - 1 truncations used to define the metaphase targeting domain in ( A ) and ( C ) . C ) Quantification of the average Myc intensity at centromeres from ( A ) . Values are normalized to centromeric signal of full - length M18BP1 - 1 . Graph shows the mean ± SEM of at least three experiments . Significance determined by Welch ' s unpaired two - tailed t - test , * p < 0 . 05 , * * p < 0 . 005 .

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

A) Representative immunofluorescence images showing localization of Myc-tagged M18BP1-1 truncations schematized in (B) to metaphase sperm centromeres in M18BP1-depleted extract. Truncations analyzed are indicated on the left, immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3X. B) Schematic of M18BP1-1 truncations used to define the metaphase targeting domain in (A) and (C). C) Quantification of the average Myc intensity at centromeres from (A). Values are normalized to centromeric signal of full-length M18BP1-1. Graph shows the mean ± SEM of at least three experiments. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005.


B ) Representative Western blot showing disruption of the interaction between M18BP1 - 1 and CENP - C by mutation of the SANTA domain by co - immunoprecipitation . Extract depleted of endogenous M18BP1 was supplemented with full - length Flag - M18BP1 - 1WT or Flag - M18BP1 - 1SANTA . The M18BP1 - 1 species added to each reaction and the cell cycle state of the extract is indicated above . The immunoblotted species is indicated at left . Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot showing disruption of the interaction between M18BP1-1 and CENP-C by mutation of the SANTA domain by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Flag-M18BP1-1WT or Flag-M18BP1-1SANTA. The M18BP1-1 species added to each reaction and the cell cycle state of the extract is indicated above. The immunoblotted species is indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.


C ) Representative images showing that mutation of the SANTA domain in M18BP1 - 1 causes decreased localization to sperm centromeres . Metaphase extracts were depleted of endogenous M18BP1 and either mock - depleted or depleted of CENP - C . The extracts were then complemented with WT or SANTA mutant M18BP1 - 1 . M18BP1 - 1 species and depletion conditions are indicated at left , immunolocalized proteins are indicated above . Scale bar , 10 μm . Insets are magnified 3X . D ) Quantification of the average Flag - M18BP1 - 1 intensity at centromeres from ( C ) . Values are normalized to centromeric signal of M18BP1 - 1WT in mock - depleted extract . Graph shows the mean ± SEM of three experiments . Significance determined by Welch ' s unpaired two - tailed t - test , * * p < 0 . 005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images showing that mutation of the SANTA domain in M18BP1-1 causes decreased localization to sperm centromeres. Metaphase extracts were depleted of endogenous M18BP1 and either mock-depleted or depleted of CENP-C. The extracts were then complemented with WT or SANTA mutant M18BP1-1. M18BP1-1 species and depletion conditions are indicated at left, immunolocalized proteins are indicated above. Scale bar, 10 μm. Insets are magnified 3X. D) Quantification of the average Flag-M18BP1-1 intensity at centromeres from (C). Values are normalized to centromeric signal of M18BP1-1WT in mock-depleted extract. Graph shows the mean ± SEM of three experiments. Significance determined by Welch's unpaired two-tailed t-test, ** p<0.005.


A ) A representative autoradiograph from a GST - pulldown of CENP - C to map the CENP - C binding domain of M18BP1 - 1 . Radiolabeled Myc - M18BP1 - 1 truncations ( amino acids indicated at the top ) were mixed with recombinant GST - CENP - C1191 - 1400 . Material bound to glutathione agarose was resolved by SDS - PAGE and visualized by autoradiography C ) Quantification of ( A ) . Bound material as a fraction of the input was calculated from autoradiographs . The graph shows mean fraction bound ± SD of three independent experiments normalized to M18BP1 - 1161 - 580 , the CENP - C binding domain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A representative autoradiograph from a GST-pulldown of CENP-C to map the CENP-C binding domain of M18BP1-1. Radiolabeled Myc-M18BP1-1 truncations (amino acids indicated at the top) were mixed with recombinant GST-CENP-C1191-1400. Material bound to glutathione agarose was resolved by SDS-PAGE and visualized by autoradiography C) Quantification of (A). Bound material as a fraction of the input was calculated from autoradiographs. The graph shows mean fraction bound ± SD of three independent experiments normalized to M18BP1-1161-580, the CENP-C binding domain.


D ) Representative Western blot showing metaphase - specific binding of M18BP1 - 1161 - 580 to CENP - C by co - immunoprecipitation . Extract depleted of endogenous M18BP1 was supplemented with full - length Myc - M18BP1 - 1 or Myc - M18BP1 - 1161 - 580 . M18BP1 - 1 species added to each reaction and cell cycle state of the extract is indicated above . Immunoblotted species indicated at left . Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control .

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

D) Representative Western blot showing metaphase-specific binding of M18BP1-1161-580 to CENP-C by co-immunoprecipitation. Extract depleted of endogenous M18BP1 was supplemented with full-length Myc-M18BP1-1 or Myc-M18BP1-1161-580. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated above. Immunoblotted species indicated at left. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.


E ) Representative immunofluorescence images showing Myc - M18BP1 - 1161 - 580 localization at sperm centromeres in extract depleted of endogenous M18BP1 . In addition , extract was mock - depleted or immunodepleted of CENP - C ( indicated at left ) . Cell cycle state indicated at left , immunolocalized protein indicated above . Scale bar , 10 μm . Insets magnified 3X . F ) Quantification of ( E ) . Graph shows mean Myc - M18BP1 - 1161 - 580 intensity at centromeres ± SEM of two independent experiments normalized to the mock - depleted , metaphase condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunofluorescence images showing Myc-M18BP1-1161-580 localization at sperm centromeres in extract depleted of endogenous M18BP1. In addition, extract was mock-depleted or immunodepleted of CENP-C (indicated at left). Cell cycle state indicated at left, immunolocalized protein indicated above. Scale bar, 10 μm. Insets magnified 3X. F) Quantification of (E). Graph shows mean Myc-M18BP1-1161-580 intensity at centromeres ± SEM of two independent experiments normalized to the mock-depleted, metaphase condition.


A , B ) Metaphase binding of M18BP1 - 1 to CENP - C requires phosphorylation . Either full - length Myc - M18BP1 - 1 ( A ) or Myc - M18BP1 - 1161 - 580 ( B ) was immunoprecipitated from metaphase extract . Half of the immunoprecipitate was treated with λ - protein phosphatase ( + LPPase ) to assess whether binding to CENP - C required phosphorylation . Immunoprecipitation with an equivalent amount of mouse IgG served as a negative control . Immunoblotted species is indicated at left .

A, B) Metaphase binding of M18BP1-1 to CENP-C requires phosphorylation. Either full-length Myc-M18BP1-1 (A) or Myc-M18BP1-1161-580 (B) was immunoprecipitated from metaphase extract. Half of the immunoprecipitate was treated with λ-protein phosphatase (+ LPPase) to assess whether binding to CENP-C required phosphorylation. Immunoprecipitation with an equivalent amount of mouse IgG served as a negative control. Immunoblotted species is indicated at left.


C ) Cdk inhibition with flavopiridol prevents M18BP1 - 1161 - 580 localization at metaphase centromeres . Representative images showing Myc - M18BP1 - 1161 - 580 localization at sperm centromeres in metaphase extract depleted of endogenous M18BP1 following treatment with various mitotic kinase inhibitors . Inhibitor treatment is indicated at left , immunolocalized protein is indicated above . Scale bar , 10 μm . Insets are magnified 3X .

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

C) Cdk inhibition with flavopiridol prevents M18BP1-1161-580 localization at metaphase centromeres. Representative images showing Myc-M18BP1-1161-580 localization at sperm centromeres in metaphase extract depleted of endogenous M18BP1 following treatment with various mitotic kinase inhibitors. Inhibitor treatment is indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X.


E ) Quantification of immunofluorescence experiments examining localization of M18BP1 - 1161 - 580 phosphorylation site mutants to sperm centromeres in metaphase extract depleted of endogenous M18BP1 . Graph shows mean centromere intensity ± SEM of three independent experiments normalized to the WT condition . Significance determined by Welch ' s unpaired two - tailed t - test , * p < 0 . 05 , * * p < 0 . 005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of immunofluorescence experiments examining localization of M18BP1-1161-580 phosphorylation site mutants to sperm centromeres in metaphase extract depleted of endogenous M18BP1. Graph shows mean centromere intensity ± SEM of three independent experiments normalized to the WT condition. Significance determined by Welch's unpaired two-tailed t-test, * p<0.05, ** p<0.005.


A ) Representative immunofluorescence images showing M18BP1 - 1 mutant localization . M18BP1 - depleted metaphase extract was supplemented with in vitro translated full - length Flag - M18BP1 - 1 mutants and sperm chromatin . In addition , extract was either mock - depleted or CENP - C depleted to assess CENP - C dependent metaphase localization . M18BP1 - 1 species and CENP - C depletion status are indicated at left , immunolocalized protein is indicated above . Scale bar , 10 μm . Insets are magnified 3X .

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

A) Representative immunofluorescence images showing M18BP1-1 mutant localization. M18BP1-depleted metaphase extract was supplemented with in vitro translated full-length Flag-M18BP1-1 mutants and sperm chromatin. In addition, extract was either mock-depleted or CENP-C depleted to assess CENP-C dependent metaphase localization. M18BP1-1 species and CENP-C depletion status are indicated at left, immunolocalized protein is indicated above. Scale bar, 10 μm. Insets are magnified 3X.


B ) Representative Western blot showing co - immunoprecipitation of CENP - C with phosphorylation site mutants of M18BP1 - 1 . M18BP1 - depleted metaphase or interphase extract was supplemented with in vitro translated Flag - M18BP1 - 1 mutants . M18BP1 - 1 mutation and cell cycle state indicated above , immunoblotted species indicated at right . Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot showing co-immunoprecipitation of CENP-C with phosphorylation site mutants of M18BP1-1. M18BP1-depleted metaphase or interphase extract was supplemented with in vitro translated Flag-M18BP1-1 mutants. M18BP1-1 mutation and cell cycle state indicated above, immunoblotted species indicated at right. Mock precipitations using rabbit reticulocyte lysate without any in vitro translated protein served as a negative control.


C ) Quantification of ( A ) . Flag - M18BP1 - 1 mutant centromere intensity normalized to WT localization in the mock - depleted condition . Error bars represent SEM from three independent experiments .

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

C) Quantification of (A). Flag-M18BP1-1 mutant centromere intensity normalized to WT localization in the mock-depleted condition. Error bars represent SEM from three independent experiments.


A ) Representative immunofluorescence images showing incorporation of exogenous Myc - CENP - A at centromeres . Sperm nuclei were incubated in M18BP1 - depleted interphase Xenopus egg extracts complemented with the indicated M18BP1 - 1 protein . Extracts were supplemented with RNA encoding Myc - CENP - A to track new CENP - A assembly and in vitro translated HJURP . Scale bar , 10 μm . Insets are magnified 3X . B ) Quantification of Myc - CENP - A loading at Xenopus sperm centromeres in ( A ) . Values are normalized to the centromere signals in mock - depleted extract . Dashed lines indicate the Myc - CENP - A assembly signal observed upon M18BP1 depletion ( bottom ) and Flag - M18BP1 - 1WT add - back ( top ) as points of reference for mutant rescue . Graph shows the mean ± SEM of four independent experiments .

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

A) Representative immunofluorescence images showing incorporation of exogenous Myc-CENP-A at centromeres. Sperm nuclei were incubated in M18BP1-depleted interphase Xenopus egg extracts complemented with the indicated M18BP1-1 protein. Extracts were supplemented with RNA encoding Myc-CENP-A to track new CENP-A assembly and in vitro translated HJURP. Scale bar, 10 μm. Insets are magnified 3X. B) Quantification of Myc-CENP-A loading at Xenopus sperm centromeres in (A). Values are normalized to the centromere signals in mock-depleted extract. Dashed lines indicate the Myc-CENP-A assembly signal observed upon M18BP1 depletion (bottom) and Flag-M18BP1-1WT add-back (top) as points of reference for mutant rescue. Graph shows the mean ± SEM of four independent experiments.


C ) Interphase Mis18 complex formation is unaffected by M18BP1 - 1 mutations that prevent binding to CENP - C . Extract depleted of endogenous M18BP1 was supplemented with Myc - Mis18α , Myc - Mis18β , and Flag - M18BP1 - 1 . M18BP1 - 1 species added to each reaction and cell cycle state of the extract is indicated at the top . Co - immunoprecipitation of Myc - Mis18α / β was assessed by α - Myc immunoblot following Flag precipitation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Interphase Mis18 complex formation is unaffected by M18BP1-1 mutations that prevent binding to CENP-C. Extract depleted of endogenous M18BP1 was supplemented with Myc-Mis18α, Myc-Mis18β, and Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract is indicated at the top. Co-immunoprecipitation of Myc-Mis18α/β was assessed by α-Myc immunoblot following Flag precipitation.


D ) HJURP association with M18BP1 - 1 is unaffected by mutations that prevent CENP - C binding . Extract depleted of endogenous M18BP1 was supplemented with Flag - M18BP1 - 1 . M18BP1 - 1 species added to each reaction and cell cycle state of the extract are indicated at the top . Coimmunoprecipitation of Flag - M18BP1 - 1 was assessed by α - Flag immunoblot following HJURP precipitation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HJURP association with M18BP1-1 is unaffected by mutations that prevent CENP-C binding. Extract depleted of endogenous M18BP1 was supplemented with Flag-M18BP1-1. M18BP1-1 species added to each reaction and cell cycle state of the extract are indicated at the top. Coimmunoprecipitation of Flag-M18BP1-1 was assessed by α-Flag immunoblot following HJURP precipitation.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

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

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 .

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

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 .

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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 .

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

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 .

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

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 .

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

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 .

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 .

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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 ) .

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 .

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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 .

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 .

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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 .

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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 .

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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 ) .

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, 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 .

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 .

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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 .

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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 .

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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 .

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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

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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 .

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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 .

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.