Equivalent point mutations replacing Arg11 in Mif2 or Arg10 in Ame1 with Alanine prevent binding to the MN complex . Mif2 and Ame1 wild - type N - termini serve as a control , protein bands are highlighted in the indicated color .

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

Equivalent point mutations replacing Arg11 in Mif2 or Arg10 in Ame1 with Alanine prevent binding to the MN complex. Mif2 and Ame1 wild-type N-termini serve as a control, protein bands are highlighted in the indicated color.


Analysis of these Mtw1 mutants in vivo using the anchor - away technique . Mtw1 - FRB strains containing RPL13 - FKBP12 for cytoplasmic anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 ° C .

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

Analysis of these Mtw1 mutants in vivo using the anchor-away technique. Mtw1-FRB strains containing RPL13-FKBP12 for cytoplasmic anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.


Tetrad analysis of Mtw1 - wt or mutants . Surviving spores in which the Mtw1 - wt or mutant rescue construct was the only source of Mtw1 are indicated in pink circles .

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

Tetrad analysis of Mtw1-wt or mutants. Surviving spores in which the Mtw1-wt or mutant rescue construct was the only source of Mtw1 are indicated in pink circles.


Western blotting confirming the expression of Mtw1 - 6x - Flag wt or mutant proteins in the Mtw1 - FRB strain background .

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

Western blotting confirming the expression of Mtw1-6x-Flag wt or mutant proteins in the Mtw1-FRB strain background.


Analytical SEC runs and accompanying SDS - PAGE of Mtw1c - wt or - 5A mutant ( blue ) , AO ( grey ) or a stoichiometric combination of both complexes ( green ) at 10 µM each . Boxes indicate corresponding fractions in left and right panel . Note that the same Ame1 - Okp1 elution profile and SDS - PAGE ( middle panel ) is displayed in both sets to improve clarity .

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

Analytical SEC runs and accompanying SDS-PAGE of Mtw1c-wt or -5A mutant (blue), AO (grey) or a stoichiometric combination of both complexes (green) at 10 µM each. Boxes indicate corresponding fractions in left and right panel. Note that the same Ame1-Okp1 elution profile and SDS-PAGE (middle panel) is displayed in both sets to improve clarity.


Analysis of Ame1 N - terminal deletion and swap mutant using the anchor - away technique . Ame1 - FRB strains containing RPL13 - FKBP12 for ribosome anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 ° C .

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

Analysis of Ame1 N-terminal deletion and swap mutant using the anchor-away technique. Ame1-FRB strains containing RPL13-FKBP12 for ribosome anchoring and additionally harboring the indicated rescue alleles were plated in serial dilution on YEPD or YEPD + Rapamycin plates incubated at 30 °C.


Expression levels of 3xHA tagged Ame1 rescue constructs from ( B ) analyzed by western blotting .

Expression levels of 3xHA tagged Ame1 rescue constructs from (B) analyzed by western blotting.


Tetrad dissection of strains where one endogenous copy of Ame1 was replaced with the indicated construct .

Tetrad dissection of strains where one endogenous copy of Ame1 was replaced with the indicated construct.


Phenotypic analysis of different Ame1 rescue constructs using fluorescence microscopy . Images of representative large budded cells immediately and 4 h after Rapamycin addition are shown , indicated as t = 0 h and t = 4 h . Brightfield images show the morphology of the corresponding yeast cell . Only large budded cells were included in the analysis . One kinetochore cluster is enlarged in the white box . Scale bar represents 5 µm .

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

Phenotypic analysis of different Ame1 rescue constructs using fluorescence microscopy. Images of representative large budded cells immediately and 4 h after Rapamycin addition are shown, indicated as t = 0 h and t = 4 h. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. One kinetochore cluster is enlarged in the white box. Scale bar represents 5 µm.


Quantification of ( E ) in which large budded cells were classified according to their Mtw1 - GFP signal into the three indicated groups . Scale bar represents 5 µm . A total of 100 cells was quantified for every strain . Mean values of the three indicated groups were calculated and plotted in a bar chart . Error bars indicate the SEM of 3 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

Quantification of (E) in which large budded cells were classified according to their Mtw1-GFP signal into the three indicated groups. Scale bar represents 5 µm. A total of 100 cells was quantified for every strain. Mean values of the three indicated groups were calculated and plotted in a bar chart. Error bars indicate the SEM of 3 independent experiments.


Analysis of Mif2 N - terminal and swap mutant using the anchor - away technique as for ( B ) . Phenotypes of the indicated Mif2 constructs were observed in an otherwise wild - type or ctf19 ∆ background .

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

Analysis of Mif2 N-terminal and swap mutant using the anchor-away technique as for (B). Phenotypes of the indicated Mif2 constructs were observed in an otherwise wild-type or ctf19∆ background.


Western blotting showing 6xFlag tagged Mif2 expression levels of strains from ( G ) .

Western blotting showing 6xFlag tagged Mif2 expression levels of strains from (G).


Co - purification of Strep - Mtw1c through Flag - Mif2 after co - expression in insect cells . Proteins were detected by western blotting using their respective tags .

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

Co-purification of Strep-Mtw1c through Flag-Mif2 after co-expression in insect cells. Proteins were detected by western blotting using their respective tags.


Western blotting of elution fractions from an SEC experiment of Mif2 - wt or Mif2Ame1 - N swap in the absence ( upper panel ) or presence ( lower panel ) of Mtw1c .

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

Western blotting of elution fractions from an SEC experiment of Mif2-wt or Mif2Ame1-N swap in the absence (upper panel) or presence (lower panel) of Mtw1c.


ITC analysis of Mif21 - 41 or Ame11 - 20 binding to MN . Experiments were performed 3 or 4 times , respectively . One representative binding curve is shown , whereas the KD value represents the average of all 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

ITC analysis of Mif21-41 or Ame11-20 binding to MN. Experiments were performed 3 or 4 times, respectively. One representative binding curve is shown, whereas the KD value represents the average of all experiments.


Cross - link ( XL ) MS analysis of the Mif2wt protein in isolation or bound to Cse4n using the cross - linker Bis [ sulfosuccinimidyl ] glutarate . Mif2wt alone or in combination with Cse4n were subjected to analytical size exclusion chromatography at 10 µM concentration . The corresponding Coomassie - stained SDS - PAGE gels of Mif2 - wt alone or in combination with Cse4n are shown . Fractions used for cross - linking are highlighted . Topological map of Mif2 based on the identified intra - molecular cross - links . Common cross links that are found in both samples are shown in red , whereas unique crosslinks that can be only found in one of the samples are highlighted in blue . Conserved regions and their described functions are indicated . A list of cross - links that link N - terminal residues to C - terminal regions of Mif2 is shown for Mif2 alone and bound to Cse4n leading to a proposed conformation of Mif2 .

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

Cross-link (XL) MS analysis of the Mif2wt protein in isolation or bound to Cse4n using the cross-linker Bis[sulfosuccinimidyl] glutarate. Mif2wt alone or in combination with Cse4n were subjected to analytical size exclusion chromatography at 10 µM concentration. The corresponding Coomassie-stained SDS-PAGE gels of Mif2-wt alone or in combination with Cse4n are shown. Fractions used for cross-linking are highlighted. Topological map of Mif2 based on the identified intra-molecular cross-links. Common cross links that are found in both samples are shown in red, whereas unique crosslinks that can be only found in one of the samples are highlighted in blue. Conserved regions and their described functions are indicated. A list of cross-links that link N-terminal residues to C-terminal regions of Mif2 is shown for Mif2 alone and bound to Cse4n leading to a proposed conformation of Mif2.


Mif2Flag - wt or - ∆ sigAT directly from insect cell lysate was immobilized on M2 Flag Agarose , washed and incubated with Mtw1 - Nnf1 complex ( MN ) . After washing , bound complexes were eluted by adding 3xFlag peptide . Western blotting against the His - tag on Nnf1 confirmed increased binding of MN to Mif2 - ∆ sigAT , already visible in the Coomassie stained SDS - PAGE gel .

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

Mif2Flag-wt or -∆sigAT directly from insect cell lysate was immobilized on M2 Flag Agarose, washed and incubated with Mtw1-Nnf1 complex (MN). After washing, bound complexes were eluted by adding 3xFlag peptide. Western blotting against the His-tag on Nnf1 confirmed increased binding of MN to Mif2-∆sigAT, already visible in the Coomassie stained SDS-PAGE gel.


Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mif2 - wt or Mif2 - ∆ sigAT and MN alone and in combination at 10 µM concentration . All combinations were incubated for 1 h at 4 ° C prior to the run . MN ( blue ) , Mif2 ( red ) , combination ( green ) . Note that the same MN elution profile and SDS - PAGE ( upper panel ) is displayed in both sets to improve clarity . Dashed boxes highlighting corresponding fractions were included to improve comparability .

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

Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mif2-wt or Mif2-∆sigAT and MN alone and in combination at 10 µM concentration. All combinations were incubated for 1 h at 4°C prior to the run. MN (blue), Mif2 (red), combination (green). Note that the same MN elution profile and SDS-PAGE (upper panel) is displayed in both sets to improve clarity. Dashed boxes highlighting corresponding fractions were included to improve comparability.


Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Cse4n ( light blue ) , Mif2 - wt ( red ) , combination ( green ) . Note that the same Cse4n ( upper panel ) and Mif2 ( middle panel ) elution profiles and SDS - PAGEs are also displayed as reference in B and C ( Mif2 ) or C ( Cse4n ) to improve clarity .

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

Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Cse4n (light blue), Mif2-wt (red), combination (green). Note that the same Cse4n (upper panel) and Mif2 (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in B and C (Mif2) or C (Cse4n) to improve clarity.


Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Mif2 - wt ( red ) , combination ( green ) . Note that the same Mtw1c ( upper panel ) elution profile and SDS - PAGE is also displayed as reference in C to improve clarity .

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

Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Mif2-wt (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in C to improve clarity.


Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Cse4n ( light blue ) , Mif2 - wt ( red ) , combination ( 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

Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Cse4n (light blue), Mif2-wt (red), combination (green).


Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Cse4n ( light blue ) , Mif2Ame - 1N swap ( red ) , combination ( green ) . Note that the same Cse4n ( upper panel ) and Mif2Ame - 1N swap ( middle panel ) elution profiles and SDS - PAGEs are also displayed as reference in E and F ( Mif2Ame - 1N swap ) or F ( Cse4n ) to improve clarity .

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

Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Cse4n (light blue), Mif2Ame-1N swap (red), combination (green). Note that the same Cse4n (upper panel) and Mif2Ame-1N swap (middle panel) elution profiles and SDS-PAGEs are also displayed as reference in E and F (Mif2Ame-1N swap) or F (Cse4n) to improve clarity.


Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Mif2Ame - 1N swap ( red ) , combination ( green ) . Note that the same Mtw1c ( upper panel ) elution profile and SDS - PAGE is also displayed as reference in F to improve clarity .

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

Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Mif2Ame-1N swap (red), combination (green). Note that the same Mtw1c (upper panel) elution profile and SDS-PAGE is also displayed as reference in F to improve clarity.


Analytical SEC chromatograms and corresponding Coomassie stained SDS - PAGE gels of Mtw1c ( blue ) , Cse4n ( light blue ) , Mif2Ame - 1N swap ( red ) , combination ( 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

Analytical SEC chromatograms and corresponding Coomassie stained SDS-PAGE gels of Mtw1c (blue), Cse4n (light blue), Mif2Ame-1N swap (red), combination (green).


Phenotypic analysis of Mif2 mutants using a galactose inducible overexpression system . Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS - Glucose or doHIS - Raffinose / Galactose plates and incubated at 30 ° C .

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

Phenotypic analysis of Mif2 mutants using a galactose inducible overexpression system. Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.


Serial dilution assay of chromosomally integrated Mif2 variants under the control of the pGAL promoter . Strains were plated on YEP - Glucose and YEP - Raffinose / Galactose plates .

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

Serial dilution assay of chromosomally integrated Mif2 variants under the control of the pGAL promoter. Strains were plated on YEP-Glucose and YEP-Raffinose/Galactose plates.


Analysis of chromosome segregation in strains from ( B ) . Distribution of fluorescently labeled Chromosome III was analyzed in large - budded cells 16 hours after switching the medium to Galactose . Scale bar represents 2 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

Analysis of chromosome segregation in strains from (B). Distribution of fluorescently labeled Chromosome III was analyzed in large-budded cells 16 hours after switching the medium to Galactose. Scale bar represents 2 μm.


Microscopic analysis of kinetochores in Mif2 overexpressing cells visualizing Mtw1 - GFP and Spc42 - mCherry . Images of representative large budded cells after 16h in Galactose are shown . One kinetochore cluster is enlarged in the white box for each strain . Scale bar represents 5 µm . Brightfield images show the morphology of the corresponding yeast cell . Only large budded cells were included in the analysis . Quantification of ( D ) in which large budded cells were classified according to their Mtw1 - GFP signal into the four indicated groups . A total of 100 cells was quantified for every strain . Mean values of the three indicated groups were calculated and plotted in a bar chart . Error bars indicate the SEM of 2 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

Microscopic analysis of kinetochores in Mif2 overexpressing cells visualizing Mtw1-GFP and Spc42-mCherry. Images of representative large budded cells after 16h in Galactose are shown. One kinetochore cluster is enlarged in the white box for each strain. Scale bar represents 5 µm. Brightfield images show the morphology of the corresponding yeast cell. Only large budded cells were included in the analysis. Quantification of (D) in which large budded cells were classified according to their Mtw1-GFP signal into the four indicated groups. A total of 100 cells was quantified for every strain. Mean values of the three indicated groups were calculated and plotted in a bar chart. Error bars indicate the SEM of 2 independent experiments.


Co - IP experiment of indicated Mif2 - Flag variants and Mtw1 - Myc from yeast cell extracts .

Co-IP experiment of indicated Mif2-Flag variants and Mtw1-Myc from yeast cell extracts.


Phenotypic analysis of Mif2 - ∆ sigAT mutant using the same galactose inducible overexpression system as in ( A ) . Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS - Glucose or doHIS - Raffinose / Galactose plates and incubated at 30 ° C .

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

Phenotypic analysis of Mif2-∆sigAT mutant using the same galactose inducible overexpression system as in (A). Strains harboring the indicated Mif2 expression constructs on pESC plasmids were plated in serial dilution on doHIS-Glucose or doHIS-Raffinose/Galactose plates and incubated at 30 °C.


( A and B ) Immunoblot of 9 - mo - old quadricepsmuscle lysates from control ( cont ) or VCP - WT , - RH9 , or - RH12 mutant transgenic lines with p62 ( A ) or LC3 ( B ) antibodies . Note the increase in p62 and LC3II isoforms in mutant animals . Densitometric quantification is from more than six 9 - mo - old animals / group . LC3 and p62 levels are normalized to loading control . Error bars represent the standard error from six independent experiments . * * , P < 0 . 001 . AU , arbitrary unit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Immunoblot of 9-mo-old quadricepsmuscle lysates from control (cont) or VCP-WT, -RH9, or -RH12 mutant transgenic lines with p62 (A) or LC3 (B) antibodies. Note the increase in p62 and LC3II isoforms in mutant animals. Densitometric quantification is from more than six 9-mo-old animals/group. LC3 and p62 levels are normalized to loading control. Error bars represent the standard error from six independent experiments. **, P < 0.001. AU, arbitrary unit.


( C ) p62 immunostaining of tibialis anterior ( TA ) or quadriceps ( Quad ) muscle from 9 - mo - old control , VCP - WT , or one of two VCP - RH transgenic lines ( RH9 or RH12 ) . Note accumulations of p62 in the middle of myofibers or along subsarcolemmal regions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) p62 immunostaining of tibialis anterior (TA) or quadriceps (Quad) muscle from 9-mo-old control, VCP-WT, or one of two VCP-RH transgenic lines (RH9 or RH12). Note accumulations of p62 in the middle of myofibers or along subsarcolemmal regions.


( D ) Histochemistry and immunohistochemistry of quadriceps muscle from VCP - RH - expressing transgenic mice . Hematoxylin and eosin ( H & amp ; amp ; E ) staining of a 15 - mo - old animal with an RV , p62 immunostaining of an RV from a 12 - mo - old animal , LC3 immunostaining of an RV , and subsarcolemmal accumulations of LC3 from a 12 - mo - old animal . The bracket highlights one LC3 - positive myofiber . A single muscle fiber is outlined in white . ( C and D ) Arrows denote vacuoles or accumulations of p62 or LC3 . Bars , 30 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histochemistry and immunohistochemistry of quadriceps muscle from VCP-RH-expressing transgenic mice. Hematoxylin and eosin (H&amp;E) staining of a 15-mo-old animal with an RV, p62 immunostaining of an RV from a 12-mo-old animal, LC3 immunostaining of an RV, and subsarcolemmal accumulations of LC3 from a 12-mo-old animal. The bracket highlights one LC3-positive myofiber. A single muscle fiber is outlined in white. (C and D) Arrows denote vacuoles or accumulations of p62 or LC3. Bars, 30 µm.


( A ) Lysates from scrambled siRNA or VCP - targeted siRNA - treated U20S cells immunoblotted with antibodies to VCP , LC3 , p62 , or α - tubulin . Densitometric analysis is graphically represented from four independent experiments . LC3 and p62 levels are normalized to loading 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

(A) Lysates from scrambled siRNA or VCP-targeted siRNA-treated U20S cells immunoblotted with antibodies to VCP, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control.


( B ) Fluorescent microscopy images of siRNA scrambled control ( Scr ) or VCP - targeted siRNA KD cells expressing GFP - LC3 . Basal numbers of GFP - LC3 puncta / cell were counted .

O O O O O O O O O O O O O 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) Fluorescent microscopy images of siRNA scrambled control (Scr) or VCP-targeted siRNA KD cells expressing GFP-LC3. Basal numbers of GFP-LC3 puncta/cell were counted.


( C ) Lysates from control or myc - tagged VCP - WT , ATPase - inactive VCP - EQ , or one of two IBMPFD mutants , VCP - RH or - AE , expressed for 16 h from stably transfected tetracycline - inducible U20S cells and immunoblotted with antibodies to myc , LC3 , p62 , or α - tubulin . Densitometric analysis is graphically represented from four independent experiments . LC3 and p62 levels are normalized to loading control . Note the increase in LC3II and p62 in VCP siRNA KD and mutant - expressing cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysates from control or myc-tagged VCP-WT, ATPase-inactive VCP-EQ, or one of two IBMPFD mutants, VCP-RH or -AE, expressed for 16 h from stably transfected tetracycline-inducible U20S cells and immunoblotted with antibodies to myc, LC3, p62, or α-tubulin. Densitometric analysis is graphically represented from four independent experiments. LC3 and p62 levels are normalized to loading control. Note the increase in LC3II and p62 in VCP siRNA KD and mutant-expressing cells.


( D ) Fluorescent microscopy images of tetracycline - inducible U20S cells expressing GFP - LC3 treated with and without Baf for 4 h ( DMSO control [ left ] or Baf + [ right ] ) . Cells are control U20S cells and VCP - WT , - RH , - AE , and - EQ . ( E ) The number of GFP - LC3 puncta / cell was counted for control U20S and VCP - WT , - EQ , - RH , and - AE with and without Baf for 4 h ( DMSO or Baf ) . Note the increase in basal GFP - LC3 puncta in mutant - expressing cells . ( A - C and E ) Error bars represent the standard error from four independent experiments . * , P < 0 . 01 ; and * * , P < 0 . 001 . Bars , 25 µm .

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

(D) Fluorescent microscopy images of tetracycline-inducible U20S cells expressing GFP-LC3 treated with and without Baf for 4 h (DMSO control [left] or Baf+ [right]). Cells are control U20S cells and VCP-WT, -RH, -AE, and -EQ. (E) The number of GFP-LC3 puncta/cell was counted for control U20S and VCP-WT, -EQ, -RH, and -AE with and without Baf for 4 h (DMSO or Baf). Note the increase in basal GFP-LC3 puncta in mutant-expressing cells. (A-C and E) Error bars represent the standard error from four independent experiments. *, P < 0.01; and **, P < 0.001. Bars, 25 µm.


( A ) Epifluorescent images for GFP and mRFP in U20S or VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells transfected with mRFP - GFP - LC3 ( tfLC3 ) and treated with rapamycin for 2 h to induce autolysosome formation . ( B ) siRNA control ( Scr ) or VCP - KD - or Baf - treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation . ( A and B ) Open arrows denote autophagosomes ( both GFP and mRFP fluorescence ) , whereas closed arrows highlight autolysosomes ( mRFP only fluorescence ) . ( C ) The graph represents Pearson ' s coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments . Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 001 .

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

(A) Epifluorescent images for GFP and mRFP in U20S or VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with mRFP-GFP-LC3 (tfLC3) and treated with rapamycin for 2 h to induce autolysosome formation. (B) siRNA control (Scr) or VCP-KD- or Baf-treated control U20S cells transfected with tfLC3 and treated with rapamycin for 2 h to induce autolysosome formation. (A and B) Open arrows denote autophagosomes (both GFP and mRFP fluorescence), whereas closed arrows highlight autolysosomes (mRFP only fluorescence). (C) The graph represents Pearson's coefficient of GFP and mRFP colocalization from 10 independent fields of cells in two different experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.


( D ) Lysates from U20S or tetracycline - inducible VCP - WT , - RH , or - AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α - tubulin . Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant ( RH and AE ) - expressing cells . Bars , 15 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(D) Lysates from U20S or tetracycline-inducible VCP-WT, -RH, or -AE cells treated with vehicle or Baf for 4 h and immunoblotted for LC3 and α-tubulin. Note that Baf treatment does not increase the LC3II levels in IBMPFD mutant (RH and AE)-expressing cells. Bars, 15 µm.


( A ) Lysates from siRNA - treated U20S cells ( scramble control [ Scr ] or VCP siRNA KD ) after autophagic induction via nutrient deprivation for 0 , 2 , or 4 h and immunoblotting for LC3 and α - tubulin . LC3II degrades over time in control but not in KD cells . One of two experiments is shown .

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

(A) Lysates from siRNA-treated U20S cells (scramble control [Scr] or VCP siRNA KD) after autophagic induction via nutrient deprivation for 0, 2, or 4 h and immunoblotting for LC3 and α-tubulin. LC3II degrades over time in control but not in KD cells. One of two experiments is shown.


( B ) Lysates from U20S or tetracycline - inducible VCP - WT , - EQ , - RH , or - AE cells after autophagic induction via nutrient deprivation for 0 , 2 , 4 , or 6 h and immunoblotting for LC3 and α - tubulin . LC3II degrades in control and VCP - WT but degrades less efficiently in mutant ( EQ , RH , or AE ) - expressing cells . ( C ) Graphical representation of densitometric evaluation of LC3II and α - tubulin at each time point 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Lysates from U20S or tetracycline-inducible VCP-WT, -EQ, -RH, or -AE cells after autophagic induction via nutrient deprivation for 0, 2, 4, or 6 h and immunoblotting for LC3 and α-tubulin. LC3II degrades in control and VCP-WT but degrades less efficiently in mutant (EQ, RH, or AE)-expressing cells. (C) Graphical representation of densitometric evaluation of LC3II and α-tubulin at each time point from three independent experiments.


( A ) Epifluorescent images for GFP - LC3 ( LC3 ) and LTR of VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells transfected with GFP - LC3 and treated with rapamycin for 2 h to induce autolysosome formation . Open arrows highlight autolysosomes ( GFP and LTR colocalized ) , and closed arrows show autophagosomes ( GFP only ) . ( B ) Pearson ' s coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different 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

(A) Epifluorescent images for GFP-LC3 (LC3) and LTR of VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Open arrows highlight autolysosomes (GFP and LTR colocalized), and closed arrows show autophagosomes (GFP only). (B) Pearson's coefficient of GFP and LTR colocalization from 10 independent fields of cells in two different experiments.


( C ) Epifluorescent images for GFP - LC3 and endogenous Lamp1 immunohistochemistry of U20S , VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells or U20S cells cotreated with Baf transfected with GFP - LC3 and treated with rapamycin for 2 h to induce autolysosome formation . Arrows highlight autolysosomes ( GFP and Lamp1 colocalized ) . ( A and C ) The boxed regions in the merge field are enlarged in the adjacent panels . ( D ) Pearson ' s coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments . ( B and D ) Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Epifluorescent images for GFP-LC3 and endogenous Lamp1 immunohistochemistry of U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells or U20S cells cotreated with Baf transfected with GFP-LC3 and treated with rapamycin for 2 h to induce autolysosome formation. Arrows highlight autolysosomes (GFP and Lamp1 colocalized). (A and C) The boxed regions in the merge field are enlarged in the adjacent panels. (D) Pearson's coefficient of GFP and Lamp1 colocalization from 10 independent fields of cells in two different experiments. (B and D) Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.001.


( E ) Electron microscopy of tetracycline - inducible control or VCP - WT - , VCP - RH - , or VCP - AE - expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h . Note the accumulation of autophagic structures in the mutant - expressing cell lines . Arrows denote autophagic structures .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electron microscopy of tetracycline-inducible control or VCP-WT-, VCP-RH-, or VCP-AE-expressing U20S cells induced for 16 h and then treated with rapamycin for 2 h. Note the accumulation of autophagic structures in the mutant-expressing cell lines. Arrows denote autophagic structures.


( A ) Immunofluorescence images of quadriceps muscle using p62 and Lamp1 or - 2 antibodies . Quadriceps section from a 9 - mo - oldVCP - WT mouse , age - matched VCP - RH transgenic , 12 - mo - oldVCP - RH transgenic , and IBMPFD patientmuscle biopsy are shown . Open arrows highlight p62 and Lamp1 / 2 colocalization , and closed arrows show regions of only p62immunofluorescence . Single myofibers containing RVs are outlined in the merge panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescence images of quadriceps muscle using p62 and Lamp1 or -2 antibodies. Quadriceps section from a 9-mo-oldVCP-WT mouse, age-matched VCP-RH transgenic, 12-mo-oldVCP-RH transgenic, and IBMPFD patientmuscle biopsy are shown. Open arrows highlight p62 and Lamp1/2 colocalization, and closed arrows show regions of only p62immunofluorescence. Single myofibers containing RVs are outlined in the merge panels.


( B and C ) Transmission electron microscopy of tibialis anterior muscle from one of two independent lines ( B shows RH12 , and C shows RH9 lines ) of 12 - mo - old VCP - RH - expressing transgenic mice . Note the large vacuolated structures that are perinuclear or subsarcolemmal . Bars : ( A ) 30 µm ; ( B and C ) 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C) Transmission electron microscopy of tibialis anterior muscle from one of two independent lines (B shows RH12, and C shows RH9 lines) of 12-mo-old VCP-RH-expressing transgenic mice. Note the large vacuolated structures that are perinuclear or subsarcolemmal. Bars: (A) 30 µm; (B and C) 500 nm.


( A ) Representative images using in vivo bioluminescence of control or IBMPFD mutant RH - expressing mice ( RH12 ) 2 d after electroporation ( baseline ) or after 24 h of nutrient deprivation ( starvation ) of polyQ80 ( Q80 ) - or polyQ19 - luciferase ( Q19 ) in the right and left tibialis anterior , respectively . The ratio of polyQ80 - luciferase / polyQ19 - luciferase is indicated below each image set . Values are in × 104 photons per second per square centimeter per steradian . ( B ) Box and whisker plot of the change ( Δ ) in the ratio of polyQ80 - luciferase / polyQ19 - luciferase activity in the left and right tibialis anterior muscle of control , VCP - WT , or one of two VCP - RH ( RH12 or RH9 ) transgenic mouse lines after 24 h of starvation . The graph is representative of three animals per group . The p - value for RH12 was 0 . 05 and 0 . 06 when compared with control animals or VCP - WT transgenic . The p - value for RH9 was 0 . 03 when compared with either control or VCP - WT transgenic . The p - value was 0 . 01 and 0 . 02 when combined RH12 and RH9 animals were compared with control or VCP - WT groups , respectively . There was no statistical difference between control or VCP - WT groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images using in vivo bioluminescence of control or IBMPFD mutant RH-expressing mice (RH12) 2 d after electroporation (baseline) or after 24 h of nutrient deprivation (starvation) of polyQ80 (Q80)- or polyQ19-luciferase (Q19) in the right and left tibialis anterior, respectively. The ratio of polyQ80-luciferase/polyQ19-luciferase is indicated below each image set. Values are in ×104 photons per second per square centimeter per steradian. (B) Box and whisker plot of the change (Δ) in the ratio of polyQ80-luciferase/polyQ19-luciferase activity in the left and right tibialis anterior muscle of control, VCP-WT, or one of two VCP-RH (RH12 or RH9) transgenic mouse lines after 24 h of starvation. The graph is representative of three animals per group. The p-value for RH12 was 0.05 and 0.06 when compared with control animals or VCP-WT transgenic. The p-value for RH9 was 0.03 when compared with either control or VCP-WT transgenic. The p-value was 0.01 and 0.02 when combined RH12 and RH9 animals were compared with control or VCP-WT groups, respectively. There was no statistical difference between control or VCP-WT groups.


( A ) Quantitation of mCherry - TDP - 43 distribution ( nuclear only or cytoplasmic ) in control U20S , VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells , or control U20S cells treated with 10 µM Baf or 50 µM chloroquine ( Chlq ) for 4 h from 10 different fields from two independent experiments . Error bars represent the standard error from 20 fields in two independent experiments . * , P < 0 . 02 when compared with VCP - WT - expressing cells . ( B ) Epifluorescent images for mCherry - TDP - 43 ( red ) and DAPI ( blue ) in control U20S , VCP - WT - , VCP - RH - , or VCP - AE - expressing cells , or control U20S cells treated with Baf or chloroquine for 4 h . Arrows denote cytosolic TDP - 43 and perinuclear TDP - 43 inclusions .

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

(A) Quantitation of mCherry-TDP-43 distribution (nuclear only or cytoplasmic) in control U20S, VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells, or control U20S cells treated with 10 µM Baf or 50 µM chloroquine (Chlq) for 4 h from 10 different fields from two independent experiments. Error bars represent the standard error from 20 fields in two independent experiments. *, P < 0.02 when compared with VCP-WT-expressing cells. (B) Epifluorescent images for mCherry-TDP-43 (red) and DAPI (blue) in control U20S, VCP-WT-, VCP-RH-, or VCP-AE-expressing cells, or control U20S cells treated with Baf or chloroquine for 4 h. Arrows denote cytosolic TDP-43 and perinuclear TDP-43 inclusions.


( C , top ) Immunoblot for TDP - 43 , lamin A / C , and actin from nuclear or cytosolic lysate fractions of mCherry - TDP - 43 - transfected VCP - WT - , VCP - RH - , VCP - AE - , or VCP - EQ - expressing cells . Note the increase in cytosolic TDP - 43 from IBMPFD mutant - and EQ - expressing cells . ( bottom ) Immunoblot for TDP - 43 , lamin A / C , and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry - TDP - 43 or similarly transfected cells treated with 30 µg / ml ( cq1 ) or 120 µg / ml ( cq2 ) chloroquine diphosphate or 200 ng / ml Baf . Data are representative of three independent experiments . Bar , 15 µm .

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

(C, top) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of mCherry-TDP-43-transfected VCP-WT-, VCP-RH-, VCP-AE-, or VCP-EQ-expressing cells. Note the increase in cytosolic TDP-43 from IBMPFD mutant- and EQ-expressing cells. (bottom) Immunoblot for TDP-43, lamin A/C, and actin from nuclear or cytosolic lysate fractions of untreated U20S cells transfected with mCherry-TDP-43 or similarly transfected cells treated with 30 µg/ml (cq1) or 120 µg/ml (cq2) chloroquine diphosphate or 200 ng/ml Baf. Data are representative of three independent experiments. Bar, 15 µm.


( A ) TDP - 43 immunostaining of quadriceps skeletal muscle from 15 - mo - old control ( Cont ) or VCP - WT , - RH9 , or - RH12 transgenic mice . A single myofiber is outlined in white .

O O O O O O O O O O O O O O O O O O O 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) TDP-43 immunostaining of quadriceps skeletal muscle from 15-mo-old control (Cont) or VCP-WT, -RH9, or -RH12 transgenic mice. A single myofiber is outlined in white.


( B ) TDP - 43 immunostaining of quadriceps skeletal muscle from 3 - mo - old mice treated with saline or 50 mg / kg / d intraperitoneal chloroquine ( Chlq ) for 4 wk . ( A and B ) Insets show one myonuclei from each field . Bars , 30 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(B) TDP-43 immunostaining of quadriceps skeletal muscle from 3-mo-old mice treated with saline or 50 mg/kg/d intraperitoneal chloroquine (Chlq) for 4 wk. (A and B) Insets show one myonuclei from each field. Bars, 30 µm.


E Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function in GA of mice receiving PBS or indicated EVs . Data information : Western blot signals are quantified and normalized to Gapdh .

O O O O O O O O O O O O O O O O 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 Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function in GA of mice receiving PBS or indicated EVs. Data information: Western blot signals are quantified and normalized to Gapdh.


F RT - qPCR showing relative levels of miR - 122 and indicated mRNAs in GA of indicated groups of mice . Data were normalized to U6 ( for miR - 122 ) or Ppib ( for all mRNAs ) and compared to control group receiving PBS ( one - way ANOVA , n = 4 or 5 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

F RT-qPCR showing relative levels of miR-122 and indicated mRNAs in GA of indicated groups of mice. Data were normalized to U6 (for miR-122) or Ppib (for all mRNAs) and compared to control group receiving PBS (one-way ANOVA, n=4 or 5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


H ROS levels in GA from indicated groups of mice detected by DHE staining ( red ) . DAPI ( blue ) shows the nuclei . Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis ( one - way ANOVA , n = 4 mice per group ) . Scale bar : 50 µm . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

H ROS levels in GA from indicated groups of mice detected by DHE staining (red). DAPI (blue) shows the nuclei. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (one-way ANOVA, n=4 mice per group). Scale bar: 50 µm. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


I ROS levels in GA from mice that had received indicated EV treatment for 5 weeks . Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis ( one - way ANOVA , n = 4 mice per group ) . Scale bar : 50 µm . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

I ROS levels in GA from mice that had received indicated EV treatment for 5 weeks. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (one-way ANOVA, n=4 mice per group). Scale bar: 50 µm. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


J RT - qPCR showing relative levels of indicated mRNAs in GA of indicated groups of mice ( one - way ANOVA , n = 5 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

J RT-qPCR showing relative levels of indicated mRNAs in GA of indicated groups of mice (one-way ANOVA, n=5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


K Relative mtDNA / nDNA ratio in GA determined by PCR ( one - way ANOVA , n = 5 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

K Relative mtDNA/nDNA ratio in GA determined by PCR (one-way ANOVA, n=5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


C Responsiveness of the reporters to miR - 122 mimic in transfected C2C12 ( t - test , n = 3 biological replicates ) . Data information : In bar graph , values are shown as mean ± SD . * * P < 0 . 01 , ns : not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Responsiveness of the reporters to miR-122 mimic in transfected C2C12 (t-test, n=3 biological replicates). Data information: In bar graph, values are shown as mean ± SD. **P<0.01, ns: not significant.


D CFSE signals in C2C12 myotubes treated with indicated CFSE - labeled Evs for 24 h , indicating EV uptake . Scale bar : 100 µm . Data information : In bar graph , values are shown as mean ± SD . * * P < 0 . 01 , ns : not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CFSE signals in C2C12 myotubes treated with indicated CFSE-labeled Evs for 24 h, indicating EV uptake. Scale bar: 100 µm. Data information: In bar graph, values are shown as mean ± SD. **P<0.01, ns: not significant.


A Western blot showing levels of indicated proteins in C2C12 treated with indicated EVs , or transfected as indicated with miR - 122 mimic or TP53 expression plasmid carrying silent mutation of the miR - 122 binding site . Data information : Western blot signals are quantified and normalized to Gapdh .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot showing levels of indicated proteins in C2C12 treated with indicated EVs, or transfected as indicated with miR-122 mimic or TP53 expression plasmid carrying silent mutation of the miR-122 binding site. Data information: Western blot signals are quantified and normalized to Gapdh.


C Relative ATP levels in C2C12 myotubes treated as indicated ( one - way ANOVA , n = 3 biological replicates ) . Data information : In bar graphs , values are shown as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

C Relative ATP levels in C2C12 myotubes treated as indicated (one-way ANOVA, n=3 biological replicates). Data information: In bar graphs, values are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


D Relative ROS levels in C2C12 myotubes treated as indicated ( one - way ANOVA , n = 3 - 5 biological replicates ) . Data information : In bar graphs , values are shown as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

D Relative ROS levels in C2C12 myotubes treated as indicated (one-way ANOVA, n=3-5 biological replicates). Data information: In bar graphs, values are shown as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


B Indicated MDA - MB - 231 - derived BC cells were injected into the mammary fat pad of female NSG mice to form tumors . Tumor - bearing and age - matched tumor - free mice were sacrificed for muscle collection at week 6 . Western blots show protein levels in GA . Western blot signals are quantified and normalized to Gapdh .

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B Indicated MDA-MB-231-derived BC cells were injected into the mammary fat pad of female NSG mice to form tumors. Tumor-bearing and age-matched tumor-free mice were sacrificed for muscle collection at week 6. Western blots show protein levels in GA. Western blot signals are quantified and normalized to Gapdh.


D RT - qPCR indicated mRNAs ( D ) in GA of tumor - free and tumor - bearing mice . Data were normalized to Ppib ( for all mRNAs ) and compared to the tumor - free group ( one - way ANOVA , n = 4 or 5 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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D RT-qPCR indicated mRNAs (D) in GA of tumor-free and tumor-bearing mice. Data were normalized to Ppib (for all mRNAs) and compared to the tumor-free group (one-way ANOVA, n=4 or 5 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


F Representative TEM images showing SS and IMF mitochondria and their quantification in GA ( one - way ANOVA , n = 3 mice per group for the left subpanel and n = 154 - 164 mitochondria analyzed per group for the right subpanel ) . Scale bar : 1 µm . Yellow bars : depths of SS mitochondrial areas ; arrows : IMF mitochondrial area . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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F Representative TEM images showing SS and IMF mitochondria and their quantification in GA (one-way ANOVA, n=3 mice per group for the left subpanel and n=154-164 mitochondria analyzed per group for the right subpanel). Scale bar: 1 µm. Yellow bars: depths of SS mitochondrial areas; arrows: IMF mitochondrial area. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


H Western blots of GA from female BALB / c mice bearing 4T1 - derived tumors with tumor - free mice as controls . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant . Western blot signals are quantified and normalized to Gapdh .

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

H Western blots of GA from female BALB/c mice bearing 4T1-derived tumors with tumor-free mice as controls. Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant. Western blot signals are quantified and normalized to Gapdh.


I RT - qPCR showing levels of indicated genes in of GA from indicated BALB / c mice ( one - way ANOVA , n = 4 or 5 mice ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

I RT-qPCR showing levels of indicated genes in of GA from indicated BALB/c mice (one-way ANOVA, n=4 or 5 mice). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


A GA were collected from female NSG mice that had received indicated EVs or PBS for 5 weeks . Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function . Data information : Western blot signals are quantified and normalized to Gapdh .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 GA were collected from female NSG mice that had received indicated EVs or PBS for 5 weeks. Western blot analysis showing protein levels of p53 and genes related to mitochondrial homeostasis and function. Data information: Western blot signals are quantified and normalized to Gapdh.


B Relative mtDNA / nDNA ratio in GA determined by PCR ( one - way ANOVA , n = 4 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , ns : not significant .

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B Relative mtDNA/nDNA ratio in GA determined by PCR (one-way ANOVA, n=4 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ns: not significant.


C RT - qPCR showing indicated mRNAs in GA of indicated groups of mice . Data were normalized to Ppib and compared to control group receiving PBS ( one - way ANOVA , n = 4 mice per group ) . Data information : In bar graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , ns : not significant .

C RT-qPCR showing indicated mRNAs in GA of indicated groups of mice. Data were normalized to Ppib and compared to control group receiving PBS (one-way ANOVA, n=4 mice per group). Data information: In bar graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ns: not significant.


AAV8 - TP53 virus or AAV8 - eGFP control virus were injected into GA of both sides twice during the entire experiments , first immediately following implantation of MDA - MB - 231 cells into the mammary fat pad , and then one month later . Muscles were collected at 6 weeks after tumor implantation . A Western blot showing indicated protein levels in the GA . Western blot signals are quantified and normalized to Gapdh .

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

AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. A Western blot showing indicated protein levels in the GA. Western blot signals are quantified and normalized to Gapdh.


AAV8 - TP53 virus or AAV8 - eGFP control virus were injected into GA of both sides twice during the entire experiments , first immediately following implantation of MDA - MB - 231 cells into the mammary fat pad , and then one month later . Muscles were collected at 6 weeks after tumor implantation . C Relative mtDNA / nDNA ratio in GA determined by PCR ( two - tailed Student ' s t - test , n = 8 mice per group ) . Data information : In all bar and line graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. C Relative mtDNA/nDNA ratio in GA determined by PCR (two-tailed Student's t-test, n=8 mice per group). Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


AAV8 - TP53 virus or AAV8 - eGFP control virus were injected into GA of both sides twice during the entire experiments , first immediately following implantation of MDA - MB - 231 cells into the mammary fat pad , and then one month later . Muscles were collected at 6 weeks after tumor implantation . D Representative TEM images showing SS and IMF mitochondria and their quantification in GA ( two - tailed Student ' s t - test , n = 3 mice per group for the left subpanel and n = 166 or 181 mitochondria analyzed per group for the right subpanel ) . Scale bar : 1 µm . Yellow bars : depths of SS mitochondrial areas ; arrows : IMF mitochondrial area . Data information : In all bar and line graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. D Representative TEM images showing SS and IMF mitochondria and their quantification in GA (two-tailed Student's t-test, n=3 mice per group for the left subpanel and n=166 or 181 mitochondria analyzed per group for the right subpanel). Scale bar: 1 µm. Yellow bars: depths of SS mitochondrial areas; arrows: IMF mitochondrial area. Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


AAV8 - TP53 virus or AAV8 - eGFP control virus were injected into GA of both sides twice during the entire experiments , first immediately following implantation of MDA - MB - 231 cells into the mammary fat pad , and then one month later . Muscles were collected at 6 weeks after tumor implantation . E ROS levels in GA from indicated groups of mice . Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis ( two - tailed Student ' s t - test , n = 4 mice per group ) . Scale bar : 50 µm . Data information : In all bar and line graphs , values are shown as mean ± SD . The boxes in the box - and - whiskers plots show the median ( center line ) and the quartile range ( 25 - 75 % ) , and the whiskers extend from the quartile to the minimum and maximum values . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : not significant .

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

AAV8-TP53 virus or AAV8-eGFP control virus were injected into GA of both sides twice during the entire experiments, first immediately following implantation of MDA-MB-231 cells into the mammary fat pad, and then one month later. Muscles were collected at 6 weeks after tumor implantation. E ROS levels in GA from indicated groups of mice. Quantified DHE signals were normalized to DAPI signals on the same section and used in analysis (two-tailed Student's t-test, n=4 mice per group). Scale bar: 50 µm. Data information: In all bar and line graphs, values are shown as mean ± SD. The boxes in the box-and-whiskers plots show the median (center line) and the quartile range (25-75%), and the whiskers extend from the quartile to the minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.


Fluorescent microscopy of cultured Gfp - Lc3 GMPs ( a ) and HSCs ( b )

Fluorescent microscopy of cultured Gfp-Lc3 GMPs (a) and HSCs (b)


Electron microscopy images of WT HSCs . Arrowheads indicate autophagic vesicles .

Electron microscopy images of WT HSCs. Arrowheads indicate autophagic vesicles.


d , GFP - LC3 loss in cultured Gfp - Lc3 HSCs ( n = 3 ) . Results are expressed as percentage of GFP - LC3 MFI in - BafA compared to + BafA conditions .

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

d, GFP-LC3 loss in cultured Gfp-Lc3 HSCs (n = 3). Results are expressed as percentage of GFP-LC3 MFI in -BafA compared to +BafA conditions.


GFP - LC3 levels in HSCs of fed and starved Gfp - Lc3 mice ( n = 3 ) . Results are expressed as percent of GFP - LC3 MFI in fed mice . Data are means ± s . d . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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GFP-LC3 levels in HSCs of fed and starved Gfp-Lc3 mice (n = 3). Results are expressed as percent of GFP-LC3 MFI in fed mice. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


Apoptosis levels in cultured WT HSCs ( n = 3 - 5 ) . Results are expressed as percent caspase activation compared to + cytokines conditions .

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Apoptosis levels in cultured WT HSCs (n = 3-5). Results are expressed as percent caspase activation compared to +cytokines conditions.


Apoptosis levels in cultured control ( Cnt ) and Atg12cKO HSCs and GMPs ( n = 3 ) . Results are expressed as percent caspase activation compared to + cytokines Cnt HSCs ( 100 % ) .

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Apoptosis levels in cultured control (Cnt) and Atg12cKO HSCs and GMPs (n = 3). Results are expressed as percent caspase activation compared to +cytokines Cnt HSCs (100%).


Absolute number of HSCs in fed and starved Cnt and Atg12cKO mice ( n = 4 - 8 mice per group ) . Results are expressed as percent of HSCs in fed mice . Data are means ± s . d . Hatching indicates starved conditions . * P ≤ 0 . 05 , * * P ≤ 0 . 01 .

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Absolute number of HSCs in fed and starved Cnt and Atg12cKO mice (n = 4-8 mice per group). Results are expressed as percent of HSCs in fed mice. Data are means ± s.d. Hatching indicates starved conditions. *P ≤ 0.05, **P ≤ 0.01.


Status of the autophagy machinery in WT HSCs ( n = 3 ) . Results are expressed as log2 fold expression compared to WT GMPs ( set to 0 ) .

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Status of the autophagy machinery in WT HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (set to 0).


b , c , qRT - PCR analyses of pro - autophagic genes in WT ( b ) and FoxO3a − / − ( c ) HSCs ( n = 3 ) . Results are expressed as log2 fold expression compared to WT GMPs ( b ) or FoxO3a + / + HSCs ( c ) ( set to 0 ) .

b, c, qRT-PCR analyses of pro-autophagic genes in WT (b) and FoxO3a−/− (c) HSCs (n = 3). Results are expressed as log2 fold expression compared to WT GMPs (b) or FoxO3a+/+ HSCs (c) (set to 0).


Autophagy flux in cultured FoxO3a − / − : : Gfp - Lc3 HSCs ( n = 3 ) . Results are expressed as percent GFP - LC3 MFI in - BafA vs . + BafA conditions , and normalized to FoxO3a + / + − : : Gfp - Lc3 HSCs . Data are means ± s . d . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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Autophagy flux in cultured FoxO3a−/−::Gfp-Lc3 HSCs (n = 3). Results are expressed as percent GFP-LC3 MFI in -BafA vs. +BafA conditions, and normalized to FoxO3a+/+−::Gfp-Lc3 HSCs. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


Ongoing autophagy in old HSCsElectron microscopy images of young and old HSCs . Arrowheads indicate autophagic vesicles .

Ongoing autophagy in old HSCsElectron microscopy images of young and old HSCs. Arrowheads indicate autophagic vesicles.


a , qRT - PCR analyses of pro - autophagic genes in old HSCs ( n = 3 - 5 ) . Results are expressed as log2 fold expression compared to young HSCs ( set to 0 ) .

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a, qRT-PCR analyses of pro-autophagic genes in old HSCs (n = 3-5). Results are expressed as log2 fold expression compared to young HSCs (set to 0).


Apoptosis levels in cultured young and old HSCs ( n = 3 ) . Results are expressed as percent caspase activation compared to + cytokines young HSCs . Hatching indicates - cytokines conditions .

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Apoptosis levels in cultured young and old HSCs (n = 3). Results are expressed as percent caspase activation compared to +cytokines young HSCs. Hatching indicates -cytokines conditions.


2 - NBD glucose uptake in cultured young and old HSCs ( n = 3 ) .

2-NBD glucose uptake in cultured young and old HSCs (n = 3).


Percent colony formation in young and old WT HSCs cultured for 32h ± BafA and methylpyruvate ( M - Pyr ) ( n = 3 ) . Colonies were counted at day 10 and normalized to - BafA conditions . Data are means ± s . d . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 .

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Percent colony formation in young and old WT HSCs cultured for 32h ± BafA and methylpyruvate (M-Pyr) (n = 3). Colonies were counted at day 10 and normalized to -BafA conditions. Data are means ± s.d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.


Recovery of infectious recombinant MHV - GFP . Cell culture supernatants containing viruses produced after virus rescue from two MHV - GFP YAC clones ( Clone1 , Clone 2 ) were used to infect 17Cl - 1 cells . At 48 hours post - infection , infected cells were visualised for GFP expression ( left panels ) and by bright field microscopy ( right panels ) . Mock represents 17Cl - 1 cells inoculated with supernatant from BHK - MHV - N cells electroporated without viral RNAs .

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Recovery of infectious recombinant MHV-GFP. Cell culture supernatants containing viruses produced after virus rescue from two MHV-GFP YAC clones (Clone1, Clone 2) were used to infect 17Cl-1 cells. At 48 hours post-infection, infected cells were visualised for GFP expression (left panels) and by bright field microscopy (right panels). Mock represents 17Cl-1 cells inoculated with supernatant from BHK-MHV-N cells electroporated without viral RNAs.


Replication kinetics of parental MHV - GFP and recombinant MHV - GFP Clone 1 and Clone 2 . L929 cells were infected at an MOI of 0 . 1 . Cell culture supernatants were harvested at indicated time points post - infection and titrated by plaque assay . Data are representative of three independent experiments with two replicates per virus in each experiment . Error bars show standard deviation ( SD ) from the mean . pfu / ml , plaque forming units per millilitre .

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Replication kinetics of parental MHV-GFP and recombinant MHV-GFP Clone 1 and Clone 2. L929 cells were infected at an MOI of 0.1. Cell culture supernatants were harvested at indicated time points post-infection and titrated by plaque assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. pfu/ml, plaque forming units per millilitre.


Rescue of recombinant MERS - CoV and MERS - CoV - GFP . Following delivery of viral RNAs into BHK - 21 cells via electroporation the cells were co - cultivated with VeroB4 cells and supernatants containing recombinant viruses produced were used to infect new VeroB4 cells . Infected cells were visualised by bright field microscopy for recombinant MERS - CoV ( upper panel ; 5 days post infection ) , and for GFP expression for recombinant MERS - CoV - GFP ( lower panel , 3 days post infection ) . Mock represents VeroB4 cells inoculated with supernatant from BHK - 21 cells electroporated without viral RNAs .

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Rescue of recombinant MERS-CoV and MERS-CoV-GFP. Following delivery of viral RNAs into BHK-21 cells via electroporation the cells were co-cultivated with VeroB4 cells and supernatants containing recombinant viruses produced were used to infect new VeroB4 cells. Infected cells were visualised by bright field microscopy for recombinant MERS-CoV (upper panel; 5 days post infection), and for GFP expression for recombinant MERS-CoV-GFP (lower panel, 3 days post infection). Mock represents VeroB4 cells inoculated with supernatant from BHK-21 cells electroporated without viral RNAs.


Replication kinetics of the MERS - CoV - EMC ( laboratory adapted isolate ) , rMERS - CoV and rMERS - CoV - GFP . VeroB4 cells were infected at an MOI of 0 . 01 . Cell culture supernatants were harvested at indicated time points post infection and titrated by TCID50 assay . Data are representative of three independent experiments with two replicates per virus in each experiment . Error bars show standard deviation ( SD ) from the mean . TCID50 / ml , 50 % tissue culture infectious dose per millilitre .

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Replication kinetics of the MERS-CoV-EMC (laboratory adapted isolate), rMERS-CoV and rMERS-CoV-GFP. VeroB4 cells were infected at an MOI of 0.01. Cell culture supernatants were harvested at indicated time points post infection and titrated by TCID50 assay. Data are representative of three independent experiments with two replicates per virus in each experiment. Error bars show standard deviation (SD) from the mean. TCID50/ml, 50% tissue culture infectious dose per millilitre.


Rescue of recombinant rSARS - CoV - 2 and rSARS - CoV - 2 - GFP . The experimental setting is illustrated in the upper panel . Two clones of each construct 1 - 6 were used to prepare YAC DNAs and in - vitro transcribed viral genome RNAs were electroporated into BHK - 21 cells and / or BHK - SARS - N together with an mRNA encoding the SARS - CoV - 2 N protein . Electroporated cells were co - cultivated with VeroE6 cells in a T25 flask and cell culture supernatants were transferred to 12 - well plates to perform plaque assay for rSARS - CoV - 2 constructs 1 - 3 and fluorescence microscopy for rSARS - CoV - 2 - GFP constructs 4 - 6 . Plaque assays are shown in the lower panel for SARS - CoV - 2 clones 1 . 1 , 2 . 2 , and 3 . 1 . VeroE6 cells were infected with 10 − 1 , 10 − 2 , and 10 − 3 dilutions of 1 ml supernatant from the individual rescue experiments and are compared to non - infected cells ( Mock ) . The expression of GFP in SARS - CoV - 2 - GFP infected VeroE6 cells is shown for clones 4 . 1 , 5 . 2 , and 6 . 1 . Mock represents non - infected VeroE6 cells .

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Rescue of recombinant rSARS-CoV-2 and rSARS-CoV-2-GFP. The experimental setting is illustrated in the upper panel. Two clones of each construct 1-6 were used to prepare YAC DNAs and in-vitro transcribed viral genome RNAs were electroporated into BHK-21 cells and/or BHK-SARS-N together with an mRNA encoding the SARS-CoV-2 N protein. Electroporated cells were co-cultivated with VeroE6 cells in a T25 flask and cell culture supernatants were transferred to 12-well plates to perform plaque assay for rSARS-CoV-2 constructs 1-3 and fluorescence microscopy for rSARS-CoV-2-GFP constructs 4-6. Plaque assays are shown in the lower panel for SARS-CoV-2 clones 1.1, 2.2, and 3.1. VeroE6 cells were infected with 10−1, 10−2, and 10−3 dilutions of 1 ml supernatant from the individual rescue experiments and are compared to non-infected cells (Mock). The expression of GFP in SARS-CoV-2-GFP infected VeroE6 cells is shown for clones 4.1, 5.2, and 6.1. Mock represents non-infected VeroE6 cells.


( D ) Kaplan - Meier survival curves . Statistics : log rank Gehan Breslow Wilcoxon test , p < 0 . 0001 , n = 20 per group .

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(D) Kaplan-Meier survival curves. Statistics: log rank Gehan Breslow Wilcoxon test, p < 0.0001, n=20 per group.


( C ) Kaplan - Meier survival curves of the 4 groups of mice shown in panel B . Statistics : Gehan - Breslow - Wilcoxon test .

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(C) Kaplan-Meier survival curves of the 4 groups of mice shown in panel B. Statistics: Gehan-Breslow-Wilcoxon test.


( B ) Representative immunohistochemical staining for calreticulin in tumor masses of control ( PBS ) and BoxA - treated mice . Scale bar 50 μm . The distribution of calreticulin positive pixels describes the location of calreticulin ( intracellular vs ecto - calreticulin ) . Statistics : Mann - Whitney test , n = 4 .

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(B) Representative immunohistochemical staining for calreticulin in tumor masses of control (PBS) and BoxA-treated mice. Scale bar 50 μm. The distribution of calreticulin positive pixels describes the location of calreticulin (intracellular vs ecto-calreticulin). Statistics: Mann-Whitney test, n=4.


( C ) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours . Statistics : One - way Anova with Dunnett ' s post - test ; n = 3 . Data information : columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(C) HMGB1 levels in the medium of AB1 cells treated with BoxA or 1 μM MTX for 24 hours. Statistics: One-way Anova with Dunnett's post-test; n=3. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( D ) Ratio of phosphorylated eIF2α ( p - eIF2α ) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours ; β - actin is shown as loading control . Statistics : One - way Anova with Dunnett ' s post - test ; n = 2 . Data information : columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(D) Ratio of phosphorylated eIF2α (p-eIF2α) over eIF2α in MM cells treated with increasing concentrations of BoxA or 1 μM MTX for 24 hours; β-actin is shown as loading control. Statistics: One-way Anova with Dunnett's post-test; n=2. Data information: columns represent the average and bars standard deviation of a representative experiment out of at least 2 performed. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( G ) Confocal immunofluorescence microscopy of cleaved caspase 3 ( red ) in MM cells treated for 24 hours with MTX ( 1 μM ) or increasing concentrations of BoxA . Nuclei were stained with Hoechst ( blue ) and cytosol with phalloidin ( green ) . Scale bar 50 μm .

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(G) Confocal immunofluorescence microscopy of cleaved caspase 3 (red) in MM cells treated for 24 hours with MTX (1 μM) or increasing concentrations of BoxA. Nuclei were stained with Hoechst (blue) and cytosol with phalloidin (green). Scale bar 50 μm.


( I ) Kaplan - Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H ( Log rank Gehan Breslow Wilcoxon test , p = 0 . 59 ) .

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(I) Kaplan-Meier survival curve of mice injected with MM cells pretreated with PBS or BoxA shown in panel H (Log rank Gehan Breslow Wilcoxon test, p=0.59).


( E ) Representative immunofluorescence staining of CD47 ( red ) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours . Nuclei are counterstained with DAPI . Scale bar 50 μm .

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(E) Representative immunofluorescence staining of CD47 (red) in unpermeabilized and permeabilized MM cells treated or not with 800 nM BoxA for 24 hours. Nuclei are counterstained with DAPI. Scale bar 50 μm.


( A ) Ecto - calreticulin exposure ( expressed as MFI ) in CT26 cells treated with 800 nM BoxA or 1 μM MTX for 2 and 4 hours . Error bars indicate standard deviation . Data were pooled from two different experiments . Data information : bars and error bars represent mean ± SD ; statistics : One - way Anova with Dunnett ' s post - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(A) Ecto-calreticulin exposure (expressed as MFI) in CT26 cells treated with 800 nM BoxA or 1 μM MTX for 2 and 4 hours. Error bars indicate standard deviation. Data were pooled from two different experiments. Data information: bars and error bars represent mean ± SD; statistics: One-way Anova with Dunnett's post-test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( I ) Kaplan - Meier survival curves of mice shown in panel H . Statistics : Gehan - Breslow - Wilcoxon test .

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(I) Kaplan-Meier survival curves of mice shown in panel H. Statistics: Gehan-Breslow-Wilcoxon test.


( A ) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4 , CD47 and RAGE , CD47 and TLR4 . Representative images from one experiment out of 3 performed are shown . MM cells were incubated overnight with 400 nM BoxA or PBS . Nuclei are in blue ( DAPI ) , phalloidin is in green . Red dots represent physical contact of CD47 with CXCR4 or RAGE or TLR4 . Scale bar , 20 μm . The intensity of red signal in PLA was quantified as described in Materials and Methods in individual cells ( n as indicated ) . Insets are enlargements of the y axis . Mean and SD are indicated . Statistics : Kolmogorov - Smirnov test .

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(A) Confocal images of Proximity Ligation Assays performed on CD47 and CXCR4, CD47 and RAGE, CD47 and TLR4. Representative images from one experiment out of 3 performed are shown. MM cells were incubated overnight with 400 nM BoxA or PBS. Nuclei are in blue (DAPI), phalloidin is in green. Red dots represent physical contact of CD47 with CXCR4 or RAGE or TLR4. Scale bar, 20 μm. The intensity of red signal in PLA was quantified as described in Materials and Methods in individual cells (n as indicated). Insets are enlargements of the y axis. Mean and SD are indicated. Statistics: Kolmogorov-Smirnov test.


( C ) Surface CD47 on MM cells after incubation for 24 hours with the indicated concentrations of CXCL12 . The experiment shown is representative of 2 performed , in biological triplicate . Data information : bars and error bars represent mean ± SD ; statistics : one - way Anova plus Dunnett ' s post - test . In all panels , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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(C) Surface CD47 on MM cells after incubation for 24 hours with the indicated concentrations of CXCL12. The experiment shown is representative of 2 performed, in biological triplicate. Data information: bars and error bars represent mean ± SD; statistics: one-way Anova plus Dunnett's post-test. In all panels, *p<0.05, **p<0.01, ****p<0.0001.


A - B : Percentage of pDCs ( A ) and mDCs ( B ) from total PBMCs collected at D1 was quantified by flow cytometry and grouped to self - reported duration of symptoms prior to D1 ( A - B ; 0 - 4 n = 22 ; 5 - 8 n = 39 ; 9 - 12 n = 39 ; ≥ 13 n = 16 ) . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Kruskal - Wallis test * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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A-B: Percentage of pDCs (A) and mDCs (B) from total PBMCs collected at D1 was quantified by flow cytometry and grouped to self-reported duration of symptoms prior to D1 (A-B; 0-4 n= 22; 5-8 n=39; 9-12 n= 39; ≥13 n=16). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Kruskal-Wallis test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.


PBMCs and plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) and after 5 days ( D5 ) . C - D : Percentage of pDCs ( C ) and mDCs ( D ) at D1 and D5 matched samples from all patients independent of symptom duration ( n = 93 ) . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). C-D: Percentage of pDCs (C) and mDCs (D) at D1 and D5 matched samples from all patients independent of symptom duration (n=93). Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.


PBMCs and plasma samples were collected from SARS - CoV - 2 infected individuals at time of hospitalization ( D1 ) and after 5 days ( D5 ) . E - J : Matched D1 and D5 plasma samples were available from 55 donors and protein levels were determined for IFNα2a ( E , n = 30 ) , IL - 3 ( F , n = 38 ) , IL - 8 ( G , n = 44 ) , IL - 6 ( H , n = 51 ) , TNFα ( I , n = 42 ) and CXCL10 ( J , n = 46 ) , note that some samples could not be quantified and were excluded from the analysis . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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PBMCs and plasma samples were collected from SARS-CoV-2 infected individuals at time of hospitalization (D1) and after 5 days (D5). E-J: Matched D1 and D5 plasma samples were available from 55 donors and protein levels were determined for IFNα2a (E, n=30), IL-3 (F, n=38), IL-8 (G, n=44), IL-6 (H, n=51), TNFα (I, n=42) and CXCL10 (J, n=46), note that some samples could not be quantified and were excluded from the analysis. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges Statistical significance was determined using the Wilcoxon matched pairs signed rank test *<p0.05, **<p0.01 ***<p0.001, ns= not significant.


K - L : Percentage of pDCs grouped by COVID - 19 severity , as determined by oxygen supplementation ( no oxygen n = 43 , nasal oxygen n = 65 and high flow oxygen n = 5 ) . Percentage of pDCs ( K ) was correlated to COVID - 19 disease severity ( L ) using simple linear regression . Data information : Each dot represents a patient , lines with error bars show the median values with interquartile ranges mean values with the standard error of the mean Statistical significance was determined using the Kruskal - Wallis test and simple linear regression * < p0 . 05 , * * < p0 . 01 * * * < p0 . 001 , ns = not significant .

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K-L: Percentage of pDCs grouped by COVID-19 severity, as determined by oxygen supplementation (no oxygen n=43, nasal oxygen n=65 and high flow oxygen n=5). Percentage of pDCs (K) was correlated to COVID-19 disease severity (L) using simple linear regression. Data information: Each dot represents a patient, lines with error bars show the median values with interquartile ranges mean values with the standard error of the mean Statistical significance was determined using the Kruskal-Wallis test and simple linear regression *<p0.05, **<p0.01 ***<p0.001, ns= not significant.