After sorting Oct4GFP + out ( 5C - Oct4GFP + mES - Oct4GFP + ) , expression of markers of Xist ( 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

After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells), the expression of markers of Xist (C)


After sorting Oct4GFP + out ( 5C - Oct4GFP + mES - Oct4GFP + ) their abilities to form chimeras ( D ) were determined .

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

After sorting Oct4GFP+ cells out (5C-Oct4GFP+ and mES-Oct4GFP+ cells) their abilities to form chimeras (D) were determined.


( F ) isolated 5C - Oct4GFP + were cultured in naïve or primed medium for three days , changed towards R1 ESCs ( naïve PSCs ) R1 ESCs - primed ( primed PSCs ) , respectively . obtained were maintained for additional 20 passages . 5C - Oct4GFP + cultured in primed medium lost GFP . R1 ESCs - primed were primed PSCs generated from R1 ESCs . Scale bar , 100 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

(F) The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. The obtained cells were maintained for additional 20 passages. The 5C-Oct4GFP+ cells cultured in primed medium lost GFP fluorescence. R1 ESCs-primed were primed PSCs generated from R1 ESCs. Scale bar, 100 μM.


isolated 5C - Oct4GFP + were cultured in naïve or primed medium for three days , changed towards R1 ESCs ( naïve PSCs ) R1 ESCs - primed ( primed PSCs ) , respectively . ( G ) PSCs mentioned in ( F ) were used for - Seq . Similarity among gene expression profiles was determined .

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

The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days, and the morphology changed towards R1 ESCs (naïve PSCs) and R1 ESCs-primed (primed PSCs), respectively. (G) PSCs mentioned in (F) were used for RNA-Seq analysis. Similarity among gene expression profiles was determined.


isolated 5C - Oct4GFP + were cultured in naïve or primed medium for three days to generate 5C - Oct4GFP + ( naïve ) 5C - Oct4GFP + ( primed ) . expression of Xist ( H )

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

The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. The expression of Xist (H)


isolated 5C - Oct4GFP + were cultured in naïve or primed medium for three days to generate 5C - Oct4GFP + ( naïve ) 5C - Oct4GFP + ( primed ) . abilities to form chimeras ( I - J ) were determined .

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

The isolated 5C-Oct4GFP+ cells were cultured in naïve or primed medium for three days to generate 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) cells. the abilities to form chimeras (I-J) were determined.


expression Oct4GFP E - cadherin was determined with FACS on day 15 during reprogramming ( A ) .

The expression Oct4GFP and E-cadherin was determined with FACS on day 15 during reprogramming (A).


on FACS results , expression of Oct4GFP E - cadherin was summarized in Oct4GFP + E - cadherin + ( B ) in Oct4GFP - E - cadherin - ( 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

Based on the FACS results, the expression of Oct4GFP and E-cadherin was summarized in Oct4GFP+E-cadherin+ cells (B) and in Oct4GFP-E-cadherin- cells (C).


of E - cadherin + in Oct4GFP + was listed in ( D ) , while of Oct4GFP + in E - cadherin + was listed in ( E ) .

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

The percentage of E-cadherin+ cells in Oct4GFP+ cells was listed in (D), while the percentage of Oct4GFP+ cells in E-cadherin+ cells was listed in (E).


( F - G ) Energy metabolism was analyzed in 5C - Oct4GFP + mES - Oct4GFP + with Seahorse instrument .

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

(F-G) Energy metabolism was analyzed in 5C-Oct4GFP+ and mES-Oct4GFP+ cells with Seahorse instrument.


numbers of three types of Oct4GFP + colonies ( J )

The numbers of three types of Oct4GFP+ colonies (J)


expression of four epithelial markers in Oct4GFP + ( K ) were determined on day 15 . " Average " in ( K ) suggested average expression of four epithelia markers .

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

the expression of four epithelial markers in Oct4GFP+ cells (K) were determined on day 15. "Average" in (K) suggested the average expression of four epithelia markers.


( A - B ) Energy metabolism was analyzed with 5C - Oct4GFP + , R1 ESCs ( naïve PSCs ) , R1 ESCs - primed ( primed PSCs ) with Seahorse instrument .

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

(A-B) Energy metabolism was analyzed with 5C-Oct4GFP+, R1 ESCs (naïve PSCs), and R1 ESCs-primed (primed PSCs) with Seahorse instrument.


( C ) Expression of epithelial , mesenchymal , glycolysis oxidative phosphorylation ( OXPHOS ) markers ( listed in Dataset EV4 ) was summarized in R1 ESCs , 5C - Oct4GFP + ( naïve ) , 5C - Oct4GFP + , 5C - Oct4GFP + ( primed ) , R1 ESCs - primed , on current - Seq . For each marker , Log2 values of FKPM were normalized by subtracting average of five groups . Log2 values of FKPM of each group of markers were then averaged plotted . 5C - Oct4GFP + ( naïve ) 5C - Oct4GFP + ( primed ) were 5C - Oct4GFP + cultured in naïve primed medium , respectively , for three days .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of epithelial, mesenchymal, glycolysis and oxidative phosphorylation (OXPHOS) markers (listed in Dataset EV4) was summarized in R1 ESCs, 5C-Oct4GFP+ (naïve), 5C-Oct4GFP+, 5C-Oct4GFP+ (primed), and R1 ESCs-primed, based on the current RNA-Seq. For each marker, Log2 values of FKPM were normalized by subtracting the average of five groups. Log2 values of FKPM of each group of markers were then averaged and plotted. 5C-Oct4GFP+ (naïve) and 5C-Oct4GFP+ (primed) were 5C-Oct4GFP+ cultured in naïve and primed medium, respectively, for three days.


( D ) Genes with higher expression at different developmental stages were selected out from previously reported - seq ( Data ref : Bertone , 2015 ) . " Mor " , " ICM " , " pre - EPI " " post - EPI " represented genes that have high expression only in morula , inner mass , pre - implantation epiblast post - implantation epiblast , respectively . " Mor + ICM " , " ICM + pre " , " EPI " indicated genes that have high expression in both morula inner mass , in both inner mass pre - implantation epiblast , in both pre - implantation post - implantation epiblast , respectively . results of previously reported - seq were summarized in left panel , while expression of these genes in current - seq was summarized in right panel . Log2 values of FKPM were presented similarly as in ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Genes with higher expression at different developmental stages were selected out from previously reported RNA-seq (Data ref: Bertone, 2015). "Mor", "ICM", "pre-EPI" and "post-EPI" represented genes that have high expression only in morula, inner cell mass, pre-implantation epiblast and post-implantation epiblast, respectively. "Mor+ICM", "ICM+pre", and "EPI" indicated genes that have high expression in both morula and inner cell mass, in both inner cell mass and pre-implantation epiblast, and in both pre-implantation and post-implantation epiblast, respectively. The results of previously reported RNA-seq were summarized in the left panel, while the expression of these genes in current RNA-seq was summarized in the right panel. Log2 values of FKPM were presented similarly as in (C).


( F ) Markers of 5C , naïve primed states ( Dataset EV4 ) were used to analyze expression profiles of epiblast during early development of mouse ( Data ref : Mohammed et al , 2017a ) ( F ) . Single - was plotted on average expression of each group of markers .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Markers of 5C, naïve and primed states (Dataset EV4) were used to analyze expression profiles of epiblast during the early development of mouse embryos (Data ref: Mohammed et al, 2017a) (F). Single-cell was plotted based on the average expression of each group of markers.


( G ) average expression of both naïve primed markers was calculated on GSE100597 . top - 2 from E4 . 5 epiblast top - 5 from E5 . 5 epiblast were selected . expression of several naïve markers ( Dppa3 , Esrrb , Klf4 , Klf5 , Tbx3 , Zfp42 ) several mesenchymal markers ( Fgf5 , Lef1 , Nodal , Pou3f1 ) in these selected were listed ( upper part ) . average expression of these genes in E3 . 5 , 4 . 5 , 5 . 5 6 . 5 epiblast were also listed ( lower part ) . These results were normalized against average expression of these genes in tested .

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

(G) The average expression of both naïve and primed markers was calculated based on GSE100597. The top-2 cells from E4.5 epiblast and top-5 cells from E5.5 epiblast were selected. The expression of several naïve markers (Dppa3, Esrrb, Klf4, Klf5, Tbx3, and Zfp42) and several mesenchymal markers (Fgf5, Lef1, Nodal, and Pou3f1) in these selected cells were listed (upper part). The average expression of these genes in E3.5, 4.5, 5.5 and 6.5 epiblast were also listed (lower part). These results were all normalized against the average expression of these genes in all tested cells.


( H ) expression of representative markers of 5C , naïve primed states was summarized in E3 . 5 , 4 . 5 , 5 . 5 6 . 5 epiblast .

(H) The expression of representative markers of 5C, naïve and primed states was summarized in E3.5, 4.5, 5.5 and 6.5 epiblast.


from E5 . 5 epiblast were separated into 8 groups ( I - VIII ) basing on average expression of epithelial , mesenchymal , glycolysis markers ( H ) . average expression of three groups of markers were summarized in ( J ) .

Cells from E5.5 epiblast were separated into 8 groups (I-VIII) basing on the average expression of epithelial, mesenchymal, and glycolysis markers (H). The average expression of three groups of markers were summarized in (J).


expression of six important markers of 5C state was summarized in these eight groups of ( K )

The expression of six important markers of 5C state was summarized in these eight groups of cells (K)


( B ) Samples from ( A ) were separated on a native agarose gel visualized by autoradiography Aberrant Replication Products ( ARPs ) are also indicated , as are different species of catenated dimers : nicked - nicked catenanes , ' n - nCats ' ; nicked - supercoiled catenanes , ' n - scCats ' ; supercoiled - supercoiled catenanes ' sc - scCats ' . ( C ) of total synthesis from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . Total lane signal ( AU ) was normalized to maximum recorded signal within each experiment . ( D ) of total synthesis from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . Total lane signal ( AU ) was normalized to maximum recorded signal within each experiment . ( E ) of supercoiled circular monomers ( ' scCMs ' ) from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . ( F ) of scCMs from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . ( G ) of linears ( ' Lins ' ) from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . ( H ) of Lins from ( B ) lanes 1 - 5 , 16 - 25 . Mean ± SD , n = 3 independent experiments . Data information : For ( E ) - ( H ) , abundance of indicated species was calculated as a of total lane signal ( % of total ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Samples from (A) were separated on a native agarose gel and visualized by autoradiography Aberrant Replication Products (ARPs) are also indicated, as are the different species of catenated dimers: nicked-nicked catenanes, 'n-nCats'; nicked-supercoiled catenanes, 'n-scCats'; supercoiled-supercoiled catenanes 'sc-scCats'. (C) Quantification of total DNA synthesis from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (D) Quantification of total DNA synthesis from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Total lane signal (AU) was normalized to the maximum recorded signal within each individual experiment. (E) Quantification of supercoiled circular monomers ('scCMs') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (F) Quantification of scCMs from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. (G) Quantification of linears ('Lins') from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. (H) Quantification of Lins from (B) lanes 1-5, 16-25. Mean ± SD, n=3 independent experiments. Data information: For (E)-(H), abundance of the indicated species was calculated as a percentage of total lane signal (% of total).


( A ) Plasmid was replicated in presence of [ α - 32P ] dATP vehicle control , etoposide , or doxorubicin . Purified intermediates were digested with XmnI , which cuts plasmid template once , to allow for identification of breaks . ( B ) Samples from ( A ) were separated on a native agarose gel visualized by autoradiography . ( C ) of breaks from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of breaks was calculated as a of total lane signal ( % of total ) . ( D ) of linears ( ' Lins ' ) from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of linears ( AU ) was normalized to maximum recorded signal within each experiment .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plasmid DNA was replicated in the presence of [α-32P]dATP and vehicle control, etoposide, or doxorubicin. Purified DNA intermediates were digested with XmnI, which cuts the plasmid template once, to allow for identification of DNA breaks. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. (C) Quantification of DNA breaks from (B). Mean ± SD, n=3 independent experiments. Abundance of breaks was calculated as a percentage of total lane signal (% of total). (D) Quantification of linears ('Lins') from (B). Mean ± SD, n=3 independent experiments. Abundance of linears (AU) was normalized to the maximum recorded signal within each individual experiment.


( F ) Samples from ( E ) were separated on native agarose gels visualized by autoradiography . ( G ) of DPC - containing molecules from ( F ) . Mean ± SD , n = 3 independent experiments . Abundance of DPC - containing molecules was calculated as a of total lane signal ( % of total ) . ( H ) of amount of supercoiled circular monomers ( ' scCMs ' ) from ' + ProK ' samples from ( F ) . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers ( AU ) was normalized to maximum recorded signal within each experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Samples from (E) were separated on native agarose gels and visualized by autoradiography. (G) Quantification of DPC-containing molecules from (F). Mean ± SD, n=3 independent experiments. Abundance of the DPC-containing molecules was calculated as a percentage of total lane signal (% of total). (H) Quantification of the amount of supercoiled circular monomers ('scCMs') from '+ProK' samples from (F). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers (AU) was normalized to the maximum recorded signal within each individual experiment.


( A ) Plasmid was replicated in Mock - or TOP2α - immunodepleted extracts in presence or absence of vehicle control , etoposide , or doxorubicin . In mock - depleted extracts ( ΔMock ) , final replication product are supercoiled circular monomers ( ' scCMs ' ) . In TOP2α - depleted extracts ( ΔTOP2α ) , final replication product are catenanes ( ' Cats ' ) because TOP2α is required for unlinking . ( B ) Samples from ( A ) were separated on a native agarose gel visualized by autoradiography . Linear molecules , arising from breaks , are indicated with a red arrow migrate at a similar molecular to a subset of catenanes . ( C ) of scCMs from ( B ) lanes 1 - 15 . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a of total lane signal ( % of total ) . ( D ) of Cats from ( B ) lanes 16 - 30 . Mean ± SD , n = 3 independent experiments . Abundance of catenanes was calculated as a of total lane signal ( % of total ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plasmid DNA was replicated in Mock- or TOP2α-immunodepleted extracts in the presence or absence of vehicle control, etoposide, or doxorubicin. In mock-depleted extracts (ΔMock), the final replication product are supercoiled circular monomers ('scCMs'). In TOP2α-depleted extracts (ΔTOP2α), the final replication product are catenanes ('Cats') because TOP2α is required for DNA unlinking. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. Linear molecules, arising from DNA breaks, are indicated with a red arrow migrate at a similar molecular weight to a subset of catenanes. (C) Quantification of scCMs from (B) lanes 1-15. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total). (D) Quantification of Cats from (B) lanes 16-30. Mean ± SD, n=3 independent experiments. Abundance of catenanes was calculated as a percentage of total lane signal (% of total).


( F ) Samples from ( E ) were separated on native agarose gels visualized by autoradiography . Linear molecules are indicated with a red arrow . ( G ) of scCMs from + ProK gel in ( F ) lanes 6 - 20 . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a of total lane signal ( % of total ) . ( H ) of DPC Containing Molecules from ( F ) lanes 6 - 20 . Mean ± SD , n = 3 independent experiments . Abundance of DPC - containing molecules was calculated as a of total lane signal ( % of total ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Samples from (E) were separated on native agarose gels and visualized by autoradiography. Linear molecules are indicated with a red arrow. (G) Quantification of scCMs from + ProK gel in (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total). (H) Quantification of DPC Containing Molecules from (F) lanes 6-20. Mean ± SD, n=3 independent experiments. Abundance of DPC-containing molecules was calculated as a percentage of total lane signal (% of total).


( A ) Plasmid was replicated in presence or absence of etoposide p97 - i , which inhibits removal of replisomes ( green ) from . At different timepoints - bound were recovered via plasmid pulldowns . ( B ) from ( A ) were recovered analyzed via Western blotting . Input corresponds to 1 % of total reaction . Modified TOP2α species are also indicated . ( C ) of TOP2α ( both modified unmodified species ) from ( B ) lanes 1 - 8 . ( D ) of TOP2α ( both modified unmodified species ) from ( B ) lanes 9 - 16 . ( E ) of CDC45 from ( B ) lanes 1 - 8 . ( F ) of CDC45 from ( B ) lanes 9 - 16 . Data information : ( C - F Mean ± SD , n = 3 independent experiments . abundance ( AU ) was normalized to maximum recorded signal within each experiment .

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

(A) Plasmid DNA was replicated in the presence or absence of etoposide and p97-i, which inhibits removal of replisomes (green) from DNA. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (B) Proteins from (A) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. Modified TOP2α species are also indicated. (C) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 1-8. (D) Quantification of TOP2α (both modified and unmodified species) from (B) lanes 9-16. (E) Quantification of CDC45 from (B) lanes 1-8. (F) Quantification of CDC45 from (B) lanes 9-16. Data information: (C-F Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.


( G ) Plasmid was replicated in presence or absence of etoposide aphidicolin ( Aph ) , which prevents synthesis but still permits unwinding by replisomes . At different timepoints - bound were recovered via plasmid pulldowns . ( H ) from ( G ) were recovered analyzed via Western blotting . Input corresponds to 1 % of total reaction . ( I ) of RPA30 from ( H ) lanes 9 - 16 . ( J ) of RPA30 from ( H ) lanes 1 - 8 . ( K ) of TOP2α ( both modified unmodified species ) from ( H ) lanes 1 - 4 9 - 12 . ( L ) of TOP2α ( both modified unmodified species ) from ( H ) lanes 5 - 8 13 - 16 . Data information : I - L ) Mean ± SD , n = 3 independent experiments . abundance ( AU ) was normalized to maximum recorded signal within each experiment .

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

(G) Plasmid DNA was replicated in the presence or absence of etoposide and aphidicolin (Aph), which prevents DNA synthesis but still permits unwinding by replisomes. At different timepoints chromatin-bound proteins were recovered via plasmid pulldowns. (H) Proteins from (G) were recovered and analyzed via Western blotting. Input corresponds to 1% of total reaction. (I) Quantification of RPA30 from (H) lanes 9-16. (J) Quantification of RPA30 from (H) lanes 1-8. (K) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 1-4 and 9-12. (L) Quantification of TOP2α (both modified and unmodified species) from (H) lanes 5-8 and 13-16. Data information: I-L) Mean ± SD, n=3 independent experiments. Protein abundance (AU) was normalized to the maximum recorded signal within each individual experiment.


( A ) Plasmid was replicated in presence of [ α - 32P ] dATP vehicle control , aclarubicin , or doxorubicin . ( B ) Samples from ( A ) were separated on a native agarose gel visualized by autoradiography . ( C ) of supercoiled circular monomers from ( B ) . Mean ± SD , n = 3 independent experiments . Abundance of supercoiled monomers was calculated as a of total lane signal ( % of total ) .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Plasmid DNA was replicated in the presence of [α-32P]dATP and vehicle control, aclarubicin, or doxorubicin. (B) Samples from (A) were separated on a native agarose gel and visualized by autoradiography. (C) Quantification of supercoiled circular monomers from (B). Mean ± SD, n=3 independent experiments. Abundance of supercoiled monomers was calculated as a percentage of total lane signal (% of total).


( E ) Samples from ( D ) were separated on a native agarose gel visualized by autoradiography . ( F ) of fork merger from ( E ) lanes 1 - 8 . Mean ± SD , n = 3 independent experiments . ( G ) of fork merger from ( E ) lanes 9 - 17 . Mean ± SD , n = 3 independent experiments . Fork merger was calculated as a of total lane signal ( % of total ) for ( F ) - ( G ) .

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(E) Samples from (D) were separated on a native agarose gel and visualized by autoradiography. (F) Quantification of fork merger from (E) lanes 1-8. Mean ± SD, n=3 independent experiments. (G) Quantification of fork merger from (E) lanes 9-17. Mean ± SD, n=3 independent experiments. Fork merger was calculated as a percentage of total lane signal (% of total) for (F)-(G).


B . DCC netrin - 1 expression ( FACS ) in B - lymphocytes isolated from DCC + / + DCCmut / mut mice spleen lymph nodes . Results are presented as expression ratios between means expression in mutant ( n = 3 ) control ones ( n = 3 ) . No significant difference was observed between wild - type mutant .

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B. DCC and netrin-1 expression (FACS analysis) in B-cells lymphocytes isolated from DCC+/+ and DCCmut/mut mice spleen and lymph nodes. Results are presented as expression ratios between means expression levels in mutant animals (n=3) and control ones (n=3). No significant difference was observed between wild-type and mutant animals.


C . DCCmut / mut mouse with splenomegaly systemic lymphadenopathy . Left panel : black arrows indicate respectively inguinal mesenteric lymph nodes . Right panel : enlarged spleen in a lymphoma - bearing mutant mouse ( left ) as compared to control organ ( right ) .

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C. DCCmut/mut mouse with splenomegaly and systemic lymphadenopathy. Left panel: black and white arrows indicate respectively the inguinal and mesenteric lymph nodes. Right panel: enlarged spleen in a lymphoma-bearing mutant mouse (left) as compared to control organ (right).


D . Incidence of lymphoid proliferations lymphoma in DCC + / + ( n = 19 ) DCCmut / mut ( n = 29 ) mice . Tumors classification was performed blinded to genotype according to anatomopathologists consensus ( 29 ) . * : p = 0 . 036 ; one - Fisher ' s exact .

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D. Incidence of lymphoid proliferations and lymphoma in DCC+/+ (n=19) and DCCmut/mut (n=29) mice. Tumors classification was performed blinded to genotype according to anatomopathologists consensus (29). *: p=0.036; one-tailed Fisher's exact test.


E . Histology immunophenotyping of lymphoid proliferations in DCC control mutant mice . Sections from control spleen , low - grade FL high - grade DLBCL were stained with hematoxylin - eosin - safran or with antibodies to B220 or Ki67 . w : pulp , r : red pulp , cr : compressed red pulp .

E. Histology analysis and immunophenotyping of lymphoid proliferations in DCC control and mutant mice. Sections from control spleen, low-grade FL and high-grade DLBCL were stained with hematoxylin-eosin-safran or with antibodies to B220 or Ki67. w: white pulp, r: red pulp, cr: compressed red pulp.


A - B . of DCC ( A ) netrin - 1 ( B ) expression in a cohort of normal tonsils ( n = 9 ) , Germinal - Center - B - - like - DLBCL ( n = 13 ) , Activated - B - - like - DLBCL ( n = 18 ) MCL ( n = 36 ) . of genes expression was performed by Q - RT - PCR , relatively to HPRT housekeeping gene . * * : p < 0 . 005 ; * * * * : p < 0 . 0001 ; two - sided Mann - Whitney U - .

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A-B. Analysis of DCC (A) and netrin-1 (B) expression in a cohort of normal tonsils (n=9), Germinal-Center-B-Cell-like-DLBCL (n=13), Activated-B-Cell-like-DLBCL (n=18) and MCL (n=36). Quantification of genes expression was performed by Q-RT-PCR, relatively to HPRT housekeeping gene. **: p<0.005; ****: p<0.0001; two-sided Mann-Whitney U-test.


C - D . of netrin - 1 expression in DLBCL ( C ) MCL ( D ) by immunohistochemistry . C . 21 DLBCL biopsies were used to build a TMA . Normal tissues ( normal tonsils , reactive lymph nodes , muscle , liver ) are included ( left - most spot , upper panel , corresponds to liver ) . Enlarged representative image of netrin - 1 expression in DLBCL is shown ( lower panel ) . D . Representative of netrin - 1 expression in paraffin - embedded biopsies of MCL ( n = 9 ) . Five tumors were positive for netrin - 1 ( left panel ) four were negative ( right panel ) .

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C-D. Analysis of netrin-1 expression in DLBCL (C) and MCL (D) by immunohistochemistry. C. 21 DLBCL patients biopsies were used to build a TMA. Normal tissues (normal tonsils, reactive lymph nodes, muscle, liver) are included (the left-most spot, upper panel, corresponds to liver). Enlarged representative image of netrin-1 expression in DLBCL is shown (lower panel). D. Representative images of netrin-1 expression in paraffin-embedded biopsies of MCL (n=9). Five tumors were positive for netrin-1 (left panel) and four were negative (right panel).


A . Expression of netrin - 1 DCC in lymphoma lines . was performed by Q - RT - PCR in 21 lymphoma lines . HPRT housekeeping gene was used as a standardization control . Netrin - 1 DCC are indicated as follow : − , not detectable ; + to + + + + , moderate to very high expression . ABC GC - DLBCL lines are highlighted respectively in blue green , MCL ones are in grey . Expression of netrin - 1 ( in green ) by immunofluorescence using netrin - 1 antibody on OCI - Ly3 Granta - 519 lines is shown on right panel . Nuclei are counterstained in blue by Hoechst .

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A. Expression of netrin-1 and DCC in lymphoma cell lines. Quantification was performed by Q-RT-PCR in 21 lymphoma cell lines. HPRT housekeeping gene was used as a standardization control. Netrin-1 and DCC levels are indicated as follow: −, not detectable; + to ++++, moderate to very high expression. ABC and GC-DLBCL cell lines are highlighted respectively in blue and green, MCL ones are in grey. Expression of netrin-1 (in green) by immunofluorescence using netrin-1 antibody on OCI-Ly3 and Granta-519 cell lines is shown on the right panel. Nuclei are counterstained in blue by Hoechst.


B . Caspase - 3 activity in Granta - 519 after transfection of scrambled siRNA ( si - Ctl ) or netrin - 1 siRNA ( si - netrin - 1 ) , with or without DCC siRNA ( si - DCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 03 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - .

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B. Caspase-3 activity in Granta-519 cells after transfection of scrambled siRNA (si-Ctl) or netrin-1 siRNA (si-netrin-1), with or without DCC siRNA (si-DCC). Results are means+/-std of four independent experiments. $: p=0.03; *: p=0.02; two-sided Mann-Whitney U-test.


D . Effect of net - 1 mAb on Granta - 519 ( left panel ) OCI - Ly3 ( right panel ) density . Results are means + / - std indexed to control of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 03 ; two - sided Mann - Whitney U - .

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D. Effect of net-1 mAb on Granta-519 (left panel) and OCI-Ly3 (right panel) cell density. Results are means+/-std indexed to control of four independent experiments. $: p=0.05; *: p=0.03; two-sided Mann-Whitney U-test.


E . Effect of net - 1 mAb on induction of Granta - 519 apoptosis , detected by TUNEL . Left panel : representative are shown . TUNEL positive are labelled in red . Nuclei are counterstained in blue by Hoechst . Right panel : of one representative experiment out of three performed is exposed . Results are presented as of TUNEL - positive per field . * : p = 0 . 04 ; two - sided Mann - Whitney U - .

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E. Effect of net-1 mAb on induction of Granta-519 cells apoptosis, detected by TUNEL staining. Left panel: representative images are shown. TUNEL positive cells are labelled in red. Nuclei are counterstained in blue by Hoechst staining. Right panel: quantification of one representative experiment out of three performed is exposed. Results are presented as percentage of TUNEL-positive cells per field. *: p=0.04; two-sided Mann-Whitney U-test.


F . Caspase - 3 activity in Granta - 519 treated with net - 1 mAb antibody or with an unrelated Ig - G1 antibody ( Ctl ) , with or without silencing of DCC via siRNA ( siDCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - .

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F. Caspase-3 activity in Granta-519 cells treated with net-1 mAb antibody or with an unrelated Ig-G1 antibody (Ctl), with or without silencing of DCC via siRNA (siDCC). Results are means+/-std of four independent experiments. $: p=0.05; *: p=0.02; two-sided Mann-Whitney U-test.


G - H . Effect of netrin - 1 interfering antibody net - 1 mAb on OCI - Ly3 ( G ) Granta - 519 ( H ) xenograft tumors growth . Results are presented as means + / - 95 % CIs of growth rates from beginning of treatment . * : p = 0 . 02 ; * * : p = 0 . 01 ; $ : p = 0 . 007 ; $ $ : p = 0 . 002 ; * * * : p = 0 . 0002 ; * * * * : p < 0 . 0001 ; two - sided Student t - ( Granta - 519 ) or Welch t - ( OCI - Ly3 ) .

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G-H. Effect of netrin-1 interfering antibody net-1 mAb on OCI-Ly3 (G) and Granta-519 (H) xenograft tumors growth. Results are presented as means+/-95% CIs of tumor growth rates from the beginning of treatment. *:p=0.02; **:p=0.01; $: p=0.007; $$:p=0.002; ***: p=0.0002; ****:p<0.0001; two-sided Student t-test (Granta-519) or Welch t-test (OCI-Ly3).


I . Caspase - 3 activity in tumors from control ( n = 4 ) or netrin - 1 - interfering antibody ( n = 4 ) treated mice . Tumors resection was performed at end of treatment . Results are means + / - std of caspase - 3 activity . * : p = 0 . 02 ; two - sided Mann - Whitney .

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I. Caspase-3 activity in tumors from control (n=4) or netrin-1-interfering antibody (n=4) treated mice. Tumors resection was performed at the end of the treatment. Results are means +/-std of caspase-3 activity. *:p=0.02; two-sided Mann-Whitney test.


Electron microscopy showing two adjacent mitochondria ( one intact one ruptured ) in a DM platelet . a . Lower powered view of single DM platelet . b . enlarged inset highlights ruptured mitochondria ( with loss of cristae ) next to intact mitochondria . c . Black dash indicates mitochondria red dash indicate phagophore formation . ( M - mitochondria )

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Electron microscopy showing two adjacent mitochondria (one intact and one ruptured) in a DM platelet. a. Lower powered view of the single DM platelet. b. The enlarged inset highlights the ruptured mitochondria (with loss of cristae morphology) next to the intact mitochondria. c. Black dash line indicates mitochondria membrane and red dash line indicate phagophore formation. (M- mitochondria)


Immunoprecipitation ( Meng et al ) of MsrB2 in HC DM human platelets followed by MsrB2 LC3I / II western .

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Immunoprecipitation (Meng et al) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I/II western.


LC3 MsrB2 immuno - EM of HC DM platelets . 15 nm dots indicate immunogold - labeled LC3 clusters 5nm dots indicate immunogold - labeled MsrB2 clusters . No significant clusters were found in HC ( a ) platelets . Representative areas of clusters of gold labeling in DM ( b - d ) are presented . nonparametric t was performed for comparisons of 2 groups . was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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LC3 and MsrB2 immuno-EM analysis of HC and DM platelets. 15 nm dots indicate immunogold-labeled LC3 clusters and 5nm dots indicate immunogold-labeled MsrB2 clusters. No significant clusters were found in HC (a) platelets. Representative areas of clusters of gold labeling in DM patients (b-d) are presented. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Western blot of MsrB2 LC3 I / II in Meg - 01 after shMsrB2 transfection ( 72 hrs ) . were then treated with H2O2 ( 1 mM for 1 hr ) alone or with NAC ( 100 μM for 30 min ) . GAPDH was used as loading control . of groups . GAPDH served as loading control . ( shMsrB2 vs . shCon ; * p = 0 . 024 , shMsrB2 / H2O2 vs . shCon / H2O2 ; * p = 0 . 0295 , shMsrB2 / H2O2 / NAC vs . shCon / H2O2 / NAC * * p = 0 . 0100 , n = 3 )

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Western blot analysis of MsrB2 and LC3 I/II in Meg-01 cell after shMsrB2 transfection (72 hrs). Cells were then treated with H2O2 (1 mM for 1 hr) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control. Quantification and analysis of individual groups. GAPDH served as the loading control. (shMsrB2 vs. shCon; *p=0.024, shMsrB2/H2O2 vs. shCon/H2O2; *p=0.0295, shMsrB2/H2O2/NAC vs. shCon/H2O2/NAC **p=0.0100, n=3)


Western blot of MsrB2 , pp53 ( S15 ) LC3 I / II in Meg - 01 after 25 mM HG treatment for 24 hrs . GAPDH was used as loading control . on groups . GAPDH served as loading control . ( MsrB2 : shCon / HG vs . shCon ; * * p = 0 . 0017 , shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0189 , pp53 : shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0317 , LC3II : shCon / HG vs . shCon ; * p = 0 . 0215 , shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0158 , n = 3 ) nonparametric t was performed for comparisons of 2 groups . was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Western blot analysis of MsrB2, pp53(S15) and LC3 I/II in Meg-01 cell after 25 mM HG treatment for 24 hrs. GAPDH was used as the loading control. Quantification analysis on individual groups. GAPDH served as the loading control. (MsrB2: shCon/HG vs. shCon; **p=0.0017, shMsrB2/HG vs. shCon/HG; *p=0.0189, pp53: shMsrB2/HG vs. shCon/HG; *p=0.0317, LC3II: shCon/HG vs. shCon; *p=0.0215, shMsrB2/HG vs. shCon/HG; *p=0.0158, n=3) The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


of reactive oxygen species ( ROS ) quantified in platelets using dye DCFH - DA . ( ROS ; * p = 0 . 0139 , MsrB2 fl / fl n = 5 , MsrB2 - / - n = 6 )

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Levels of reactive oxygen species (ROS) quantified in the platelets using the fluorescent dye DCFH-DA. (ROS; *p=0.0139, MsrB2 fl/fl n=5, MsrB2 -/- n=6)


C . Representative image of mitochondrial apoptosis , measured in freshly isolated platelets from MsrB2 knockouts matched floxed mice . Results are expressed as % apoptotic . ( TMRE ; * p = 0 . 0118 , MsrB2 fl / fl n = 5 , MsrB2 - / - n = 5 )

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C. Representative image and quantification of mitochondrial apoptosis, measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice. Results are expressed as % apoptotic cells. (TMRE; *p=0.0118, MsrB2 fl/fl n=5, MsrB2 -/- n=5)


Representative showing intraplatelet expression of LC3 active caspase - 3 in CD41 stained platelets from KO mice using labelled antibodies against respective of interest . Magnification : 100X . Rightmost panel represents zoom in on an platelet showing colocalization expression of LC3 active caspase - 3 .

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Representative images showing the intraplatelet expression of LC3 and active caspase-3 in CD41 stained platelets from the KO mice using fluorescent labelled antibodies against the respective proteins of interest. Magnification: 100X. Rightmost panel represents zoom in on an individual platelet showing the colocalization and expression of LC3 and active caspase-3.


Graph indicates platelets positive for active caspase - 3 signal . y ‐ axis indicates of caspase - 3 positive from total calculated from n = 10 independent per sample . ( active caspase - 3 signal ; * * p = 0 .

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Graph indicates platelets positive for active caspase-3 signal. The y‐axis indicates percentage of caspase-3 positive cells from total cells calculated from n = 10 independent images per sample. (active caspase-3 signal ; **p=0.0003


Intracellular caspase - 3 activity measured in freshly isolated platelets using chromophore Ac - DEVD - pNA . Experiments performed in a minimum of n = 6 samples per group . Values expressed as Mean ± . ( caspase - 3 activity ; * p = 0 . 0157 )

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Intracellular caspase-3 activity measured in freshly isolated platelets using the chromophore Ac-DEVD-pNA. Experiments performed in a minimum of n=6 samples per group. Values expressed as Mean ± SEM. (caspase-3 activity ; *p=0.0157)


H . Western blot of Parkin , LC3I / II , MsrB2 active caspasee3 in platelet after penetration peptide injection in chow HFD mice . Control peptide ( CP ) , LIF peptide ( Foroud et al ) . of band . ( Parkin ; * p = 0 . 02964 , LC3II ; * p = 0 . 03200 , MsrB2 ; * * p = 0 . 0059 , Active caspase3 ; * p = 0 . 0368 vs . chow or HFD group , each group n = 3 ) . Actin served as loading control . nonparametric t was performed for comparisons of 2 groups . was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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and H. Western blot analysis of Parkin, LC3I/II, MsrB2 and active caspasee3 in platelet after cell penetration peptide injection in chow and HFD mice. Control peptide (CP), LIF peptide (Foroud et al). Quantification analysis of individual band intensity. (Parkin; *p=0.02964, LC3II; *p=0.03200, MsrB2; **p=0.0059, Active caspase3; *p=0.0368 vs. chow or HFD group, each group n=3). Actin served as the loading control. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Immunoprecipitation ( Meng et al ) of MsrB2 in HC DM platelets , followed by western blot of precipitated Parkin modified Parkin .

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Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets, followed by western blot analysis of precipitated Parkin and modified Parkin.


Immunoprecipitation ( Meng et al ) of Parkin in HC DM platelets , followed by Western blot of precipitated Parkin MsrB2 .

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Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets, followed by Western blot analysis of precipitated Parkin and MsrB2.


Confocal microscopy was used to corroborate Western using triple for Parkin , LC3 MsrB2 in HC DM platelets . Arrows indicate sites of colocalization of Parkin , LC3 MsrB2 as determined by Volocity software ( PerkinElmer , USA ) . Graphical of colocalization between Parkin , LC3 MsrB2 signal .

Confocal microscopy was used to corroborate the Western analysis using triple staining for Parkin, LC3 and MsrB2 in HC and DM platelets. Arrows indicate sites of colocalization of Parkin, LC3 and MsrB2 as determined by Volocity software (PerkinElmer, USA). Graphical quantification of colocalization between Parkin, LC3 and MsrB2 signal.


Signal of each group was converted to fold compared with HC values ( colocalization of parkin , LC3 MsrB2 ; * * p = 0 . 0006 ) .

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Signal intensity of each group was converted to fold and compared with HC values (colocalization of parkin, LC3 and MsrB2; **p=0.0006).


Immunoprecipitation ( Meng et al ) of Parkin in HC DM platelets . Western blot of MetO Parkin using specific Parkin MetO antibodies . detection of MetO Parkin , was performed w / o β - mercaptoethanol ( NonRe ) SDS sample buffer .

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Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets. Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies. The detection of MetO Parkin, was performed w/o β-mercaptoethanol (NonRe) SDS sample buffer.


In vitro . Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 at 37ºC for 2hrs . Western blot of Parkin MsrB2 was performed . ( red arrow , low MsrB2 expression ; blue arrow , high MsrB2 expression ) . nonparametric t was performed for comparisons of 2 groups . was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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In vitro assay. Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs. Western blot analysis of Parkin and MsrB2 was performed. (red arrow, low MsrB2 expression; blue arrow, high MsrB2 expression). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Western blot of Methionine Sulfoxidation ( MetO ) , Methionine sulfide reductase B2 ( MsrB2 ) Parkin in human Healthy Control ( HC ) Diabetic Mellitus ( DM ) platelets . P . C . is a BSA - MetO positive control ( Cayman Chemical Co . ) . Quantitation of western blot shown in Fig 1C . Graphical representation statistical of HC ( n = 3 ) DM ( n = 12 ) individuals . ( MetO ; * * p = 0 . 0003 , MsrB2 ; * p = 0 . 0186 , Parkin ; * * p = 0 . 0003 vs . HC ) .

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Western blot analysis of Methionine Sulfoxidation (MetO), Methionine sulfide reductase B2 (MsrB2) and Parkin in human Healthy Control (HC) and Diabetic Mellitus (DM) platelets. P.C. is a BSA-MetO positive control (Cayman Chemical Co.). Quantitation of western blot analysis shown in Fig 1C. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (MetO; **p=0.0003, MsrB2; *p=0.0186, Parkin; **p=0.0003 vs. HC).


Western blot of Parkin Parkin aggregation in human Healthy Control ( HC ) ( n = 3 ) Diabetic Mellitus ( DM ) ( n = 12 ) platelets . Graphical representation statistical of HC ( n = 3 ) DM ( n = 12 ) individuals . ( modified parkin * p = 0 . 035 vs . HC ) . Actin served as loading control .

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Western blot analysis of Parkin and Parkin aggregation in human Healthy Control (HC) (n=3) and Diabetic Mellitus (DM) (n=12) platelets. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (modified parkin *p=0.035 vs. HC). Actin served as the loading control.


Parkin IP was performed in HC , HC plus H2O2 , DM .

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Parkin IP was performed in HC, HC plus H2O2, and DM patients.


MetO sites were analyzed by mass spectrometry using Mascot program Representative mass spectrometry demonstrating identification of MetO192 peptide on Parkin . nonparametric t was performed for comparisons of 2 groups . was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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MetO sites were analyzed by mass spectrometry using the Mascot program Representative mass spectrometry analysis demonstrating identification of MetO192 peptide on Parkin. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Immunoprecipitation ( Meng et al ) of MsrB2 in HC DM platelets . Western blot of precipitated MsrB2 , Parkin ubiquitinated MsrB2 ( UB ) .

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Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets. Western blot analysis of precipitated MsrB2, Parkin and ubiquitinated MsrB2 (UB).


Confocal microscopy in platelets in HC vs DM assessing for CoxIV ( mitochondria marker ) , ubiquitin MsrB2 . Colocalization is demomstrated by arrows quantitated . ( colocalization ubiquitin , MsrB2 ; * * p = 0 . 0003 vs . HC ) .

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Confocal microscopy in individual platelets in HC vs DM assessing for CoxIV (mitochondria marker), ubiquitin and MsrB2. Colocalization is demomstrated by arrows and quantitated. (colocalization ubiquitin, MsrB2; **p=0.0003 vs. HC).


MsrB2 ubiquitination after transient transfection of MsrB2 - GFP ( 1µg ) with RFP empty vector ( E . V . ) or RFP - Parkin ( 3µg ) in HEK293 . After transfection ( 48hrs ) , an IP was performed using GFP - Trap bead , followed by Western blot using UB MsrB2 Parkin antibodies . nonparametric t was performed for comparisons of 2 groups . was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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MsrB2 ubiquitination assay after transient transfection of MsrB2-GFP (1µg) with RFP empty vector (E.V.) or RFP-Parkin (3µg) in HEK293. After transfection (48hrs), an IP was performed using GFP-Trap bead, followed by Western blot analysis using UB and MsrB2 and Parkin antibodies. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Western blot of MsrB2 Cytochrome C in isolated mitochondria after incubation w / o Cyclosporin A ( CsA ; 2 μM for 2 hrs . ) or w / CsA . ATPB served as loading negative control ( mitochondrial matrix ) .

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Western blot analysis of MsrB2 and Cytochrome C in isolated mitochondria after incubation w/o Cyclosporin A (CsA; 2 μM for 2 hrs.) or w/ CsA. ATPB served as the loading and negative control (mitochondrial matrix protein).


Western blot of Parkin , LC3I / II , ubiquitinated MsrB2 in chow ( n = 4 ) HFD ( n = 6 ) mouse platelets .

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Western blot analysis of Parkin, LC3I/II, ubiquitinated MsrB2 in chow (n=4) and HFD (n=6) mouse platelets.


Confocal microscopy was initially stained with Tubulin antibody ( Panel a . , Chow diet , or b . High Fat Diet ) to assess platelet architecture . This was followed by using ATPB ( mitochondria marker - green ) MsrB2 antibodies ( Blue ) ( Panel c - e ) . There was loss of colocalization in HFD mice supporting release ( blue ) from ruptured mitochondria ( green ) ( Panel d & e ) . nonparametric t was performed for comparisons of 2 groups . was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Confocal microscopy was initially stained with Tubulin antibody (Panel a., Chow diet, or b. High Fat Diet) to assess platelet architecture. This was followed by staining using ATPB (mitochondria marker - green) and MsrB2 antibodies (Blue) (Panel c-e). There was loss of colocalization in the HFD mice supporting release (blue) from ruptured mitochondria (green) (Panel d & e). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Representative western blot of MsrB2 , LC3I / II parkin using Parkinson ' s disease ( PD ) platelets . of band . ( MsrB2 ; * p = 0 . 0255 , LC3II ; * p = 0 . 0101 vs . HC ( n = 5 ) PD ( n = 12 ) ) . GAPDH served as loading control .

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Representative western blot analysis of MsrB2, LC3I/II and parkin using Parkinson's disease (PD) platelets. Quantification analysis of individual band intensity. (MsrB2; *p=0.0255, LC3II; *p=0.0101 vs. HC (n=5) and PD (n=12)). GAPDH served as the loading control.


Platelet apoptosis ( Annexin V ) were measured by flow cytometry in HC ( n = 9 ) PD ( n = 13 ) platelets . ( * * p < 0 . 01 vs . HC ) . nonparametric t was performed for comparisons of 2 groups . was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Platelet apoptosis (Annexin V) were measured by flow cytometry analysis in HC (n=9) and PD (n=13) platelets. (**p<0.01 vs. HC). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


A - F Indirect calorimetry was performed using Oxymax / CLAMS system in Cdk4 + / + ( n = 6 ) Cdk4 - / - ( n = 5 ) mice . Whole - oxygen consumption rate ( VO2 ) ( A - B ) , energy expenditure ( EE ) ( C - D ) respiratory exchange ( RER ) ( E - F ) were measured at 24 ° C during a cold challenge at 6 ° C during both ( rectangle ) ( black rectangle ) phases over a 2 - day period .

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A-F Indirect calorimetry was performed using the Oxymax/CLAMS system in Cdk4+/+ (n=6) and Cdk4-/- (n=5) mice. Whole-body oxygen consumption rate (VO2) (A-B), energy expenditure (EE) (C-D) and respiratory exchange ratio (RER) (E-F) were measured at 24°C and during a cold challenge at 6°C during both the light (white rectangle) and dark (black rectangle) phases over a 2-day period.


CalR was used to GLM - regression plot for each group corresponding to association between energy balance total mass at 24 ° C ( G ) association between energy balance total mass at 6 ° C ( H ) .

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CalR was used to GLM-regression plot for each group corresponding to the association between energy balance and total body mass at 24°C (G) and the association between energy balance and total body mass at 6°C (H).


J - K Basal rectal was measured at 24 ° C ( J ) , rectal was monitored during acute cold exposure at 4 ° C for 6 hours ( K ) in Cdk4 + / + ( n = 10 ) Cdk4 - / - ( n = 8 ) mice .

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J-K Basal rectal temperature was measured at 24°C (J), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (K) in Cdk4+/+ (n=10) and Cdk4-/- (n=8) mice.


A Gross of iBAT from Cdk4 + / + Cdk4 - / - mice .

A Gross morphology of iBAT from Cdk4+/+ and Cdk4-/- mice.


C Hematoxylin - eosin UCP1 immunohistochemical ( ) of iBAT sections ( scale bar 100 μm ) from Cdk4 + / + Cdk4 - / - mice .

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C Hematoxylin-eosin staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm) from Cdk4+/+ and Cdk4-/- mice.


D - E Western blot of UCP1 CDK4 expression ( D ) quantified of UCP1 ( E ) ( n = 4 biological replicates ) in iBAT . HSP90 was used as loading control .

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D-E Western blot analysis of UCP1 and CDK4 protein expression (D) and quantified protein levels of UCP1 (E) (n=4 biological replicates) in iBAT. HSP90 was used as loading control.


F Expression of thermogenic genes brown adipocyte markers in iBAT of Cdk4 + / + ( n = 6 ) Cdk4 - / - ( n = 6 ) mice as assessed by RT - PCR .

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F Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4+/+ (n=6) and Cdk4-/- (n=6) mice as assessed by RT-PCR.


G - H Transmission electron microscopy ( ) of iBAT ( G ) ( scale bar 5 μm ( upper panel ) 1 μm ( lower panel , LD indicates lipid droplet M mitochondria ) ) of lipid droplet number ( LD ) mitochondrial volume ( Mito ) ( H ) ( Cdk4 + / + ( n = 4 ) Cdk4 - / - ( n = 4 ) ) .

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G-H Transmission electron microscopy (TEM) images of iBAT (G) (scale bar 5 μm (upper panel) and 1 μm (lower panel, LD indicates lipid droplet and M mitochondria)) and quantification of lipid droplet number (LD) and mitochondrial volume (Mito) (H) (Cdk4+/+ (n=4) and Cdk4-/- (n=4)).


I Respirometry of iBAT as measured by Oroboros system ( Cdk4 + / + ( n = 6 ) Cdk4 - / - ( n = 6 ) ) .

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I Respirometry analysis of iBAT as measured by the Oroboros system (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).


A - B Rectal was measured at 24 ° C ( A ) , rectal was monitored for 6 hours during acute cold exposure at 4 ° C ( B ) in Cdk4flox / flox ( n = 9 ) Cdk4flox / flox Ucp1 - Cre mice ( n = 9 ) .

A-B Rectal temperature was measured at 24°C (A), and rectal temperature was monitored for 6 hours during acute cold exposure at 4°C (B) in Cdk4flox/flox (n=9) and Cdk4flox/flox Ucp1-Cre mice (n=9).


C Gross of iBAT from Cdk4flox / flox Cdk4flox / flox Ucp1 - Cre mice D iBAT mass compared to of Cdk4flox / flox ( n = 8 ) Cdk4flox / flox Ucp1 - Cre ( n = 7 ) mice .

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C Gross morphology of iBAT from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice D iBAT mass compared to body weight of Cdk4flox/flox (n=8) and Cdk4flox/flox Ucp1-Cre (n=7) mice.


E Hematoxylin - eosin ( H & E ) UCP1 immunohistochemical ( ) of iBAT sections ( scale bar 100 μm ( upper panel ) 50 μm ( lower panel ) ) from Cdk4flox / flox Cdk4flox / flox Ucp1 - Cre mice .

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E Hematoxylin-eosin (H&E) staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm (upper panel) and 50 μm (lower panel)) from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice.


F - G Western blot of UCP1 CDK4 expression ( F ) quantified of UCP1 ( G ) ( n = 4 biological replicates ) in iBAT . HSP90 was used as a loading control .

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F-G Western blot analysis of UCP1 and CDK4 protein expression (F) and quantified protein levels of UCP1(G) (n=4 biological replicates) in iBAT. HSP90 was used as a loading control.


H Expression of thermogenic genes brown adipocyte markers in iBAT of Cdk4flox / flox ( n = 5 ) Cdk4flox / flox Ucp1 - Cre ( n = 6 ) mice as assessed by RT - PCR .

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H Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4flox/flox (n=5) and Cdk4flox/flox Ucp1-Cre (n=6) mice as assessed by RT-PCR.


A - B TH immunohistochemical in iBAT sections ( A ) ( scale bar 20 μm , arrows indicate TH parenchymal ) corresponding of number of TH relative to 100 adipocytes ( B ) ( Cdk4 + / + ( n = 6 ) Cdk4 - / - ( n = 6 ) ) .

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A-B TH immunohistochemical staining in iBAT sections (A) (scale bar 20 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (B) (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).


C Hematoxylin - eosin in scWAT showing multilocular adipocytes in Cdk4 - / - ( scale bar 50 μm ) .

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C Hematoxylin-eosin staining in scWAT showing multilocular adipocytes in Cdk4-/- (scale bar 50 μm).


D Expression of thermogenic genes brown adipocyte markers in scWAT of Cdk4 + / + ( n = 5 ) Cdk4 - / - ( n = 6 ) mice as assessed by RT - PCR .

D Expression of thermogenic genes and brown adipocyte markers in scWAT of Cdk4+/+ (n=5) and Cdk4-/- (n=6) mice as assessed by RT-PCR.


E - F Acute cold ( 4 ° C ) after treatment with β - adrenergic antagonist propranolol ( Prop ) in Cdk4 + / + ( n = 7 ) Cdk4 - / - ( n = 6 ) or with vehicle ( NaCl ) ( Cdk4 + / + ( n = 7 ) Cdk4 - / - ( n = 6 ) ) ( E ) corresponding of under curve ( AUC ) .

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E-F Acute cold test (4°C) after treatment with the β-adrenergic antagonist propranolol (Prop) in Cdk4+/+ (n=7) and Cdk4-/- (n=6) animals or with vehicle (NaCl) (Cdk4+/+ (n=7) and Cdk4-/- (n=6)) (E) and corresponding quantification of the area under the curve (AUC).


A Cdk4 mRNA expression in dissected VMH of Cdk4flox / flox ( n = 5 ) Cdk4flox / flox Sf1 - Cre ( n = 6 )

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A Cdk4 mRNA expression in the dissected VMH of Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre (n=6) mice


B - C Rectal was measured at 24 ° C ( B ) , rectal was monitored during acute cold exposure at 4 ° C for 6 hours ( C ) in Cdk4flox / flox ( n = 15 ) Cdk4flox / flox Sf1 - Cre mice ( n = 15 ) .

B-C Rectal temperature was measured at 24°C (B), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (C) in Cdk4flox/flox (n=15) and Cdk4flox/flox Sf1-Cre mice (n=15).


D Hematoxylin - eosin ( H & E ) of iBAT sections ( scale bar 100 μm ) from Cdk4flox / flox Cdk4flox / flox Sf1 - Cre mice .

D Hematoxylin-eosin (H&E) staining of iBAT sections (scale bar 100 μm) from Cdk4flox/flox and Cdk4flox/flox Sf1-Cre mice.


E Expression of thermogenic genes brown adipocyte markers expression in iBAT Cdk4flox / flox ( n = 9 ) Cdk4flox / flox Sf1 - Cre mice ( n = 9 ) mice as assessed by RT - PCR .

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E Expression of thermogenic genes and brown adipocyte markers expression in iBAT Cdk4flox/flox (n=9) and Cdk4flox/flox Sf1-Cre mice (n=9) mice as assessed by RT-PCR.


F - G TH immunohistochemical ( ) in iBAT sections ( F ) ( scale bar 40 μm , arrows indicate TH parenchymal ) corresponding of number of TH relative to 100 adipocytes ( G ) ( Cdk4flox / flox ( n = 5 ) Cdk4flox / flox Sf1 - Cre mice ( n = 5 ) ) .

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F-G TH immunohistochemical (IHC) staining in iBAT sections (F) (scale bar 40 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (G) (Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre mice (n=5)).


H - I Western blot ( H ) of p - CREBS133 expression following 6 hours of cold exposure ( 4 ° C ) ( I ) ( n = 3 biological replicates ) . HSP90 was used as a loading control .

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H-I Western blot analysis (H) and quantification of p-CREBS133 protein expression following 6 hours of cold exposure (4°C) (I) (n=3 biological replicates). HSP90 was used as a loading control.


A - D Confocal illustrations of frontal hypothalamic sections from Cdk4 + / + mice ( A , C , N = 3 ) Cdk4 - / - mice ( B , D , N = 3 ) . Low magnification showing nuclei ( Hoechst , left pannel ) c - Fos activated - ( , right pannel ) in mPOA ( A1 - 2 , B1 - 2 ) ARC , VMH , DMH , LH ( C1 - 2 , D1 - 2 ) . High magnifications corresponding to frame in A1 - 2 , B1 - 2 , C2 , D2 showing c - Fos - IR ( A3 - 4 , B3 - 4 , C3 - 6 , D3 - 6 ) . Scale bars ( A1 - 2 , B1 - 2 , C1 - 2 , D1 - 2 ) : 200μm , ( A3 - 4 , B3 - 4 , C3 - 4 , D3 - 4 ) : 50μm . E of c - Fos IR in brain sections of Cdk4 + / + Cdk4 - / - mice ( n = 15 - 24 slices according hypothalamic nucleus genotype ) .

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A-D Confocal illustrations of frontal hypothalamic sections from Cdk4+/+ mice (A, C, N=3) and Cdk4-/- mice (B, D, N=3). Low magnification showing nuclei (Hoechst, left pannel) and c-Fos activated-cells (white, right pannel) in the mPOA (A1-2, B1-2) and the ARC, the VMH, the DMH, the LH (C1-2, D1-2). High magnifications corresponding to the frame area in A1-2, B1-2, C2, D2 and showing c-Fos-IR cells (A3-4, B3-4, C3-6, D3-6). Scale bars (A1-2, B1-2, C1-2, D1-2): 200μm, (A3-4, B3-4, C3-4, D3-4): 50μm. E Quantification of c-Fos IR cells in brain sections of Cdk4+/+ and Cdk4-/- mice (n=15-24 slices according hypothalamic nucleus and genotype).


A , B . A fraction of OTULIN is localized at mitochondria where it suppresses M1 - linked ubiquitination . HEK293T were transfected with control or OTULIN siRNA . were harvested 72 h ( A ) or 48 h ( B ) after transfection or 15 min after TNF treatment ( 25 ng / ml ) . Mitochondria were isolated by differential centrifugation purified by ultracentrifugation using an OptiPrepTM density gradient . 38 % ( A ) or 20 % ( B ) of mitochondrial fractions 2 % ( A , B ) of whole lysates were analyzed by immunoblotting using antibodies indicated . For detection of M1 - linked ubiquitin chains , either 1E3 ( A ) or 1F11 / 3F5 / Y102L ( B ) antibody was used .

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A, B. A fraction of OTULIN is localized at mitochondria where it suppresses M1-linked ubiquitination. HEK293T cells were transfected with control or OTULIN siRNA. Cells were harvested 72 h (A) or 48 h (B) after transfection or 15 min after TNF treatment (25 ng/ml). Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrepTM density gradient. 38% (A) or 20% (B) of the mitochondrial fractions and 2% (A, B) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated. For the detection of M1-linked ubiquitin chains, either the 1E3 (A) or the 1F11/3F5/Y102L (B) antibody was used.


C . TNF induces M1 - ubiquitination at mitochondria in various types . indicated types were treated with TNF ( 25 ng / ml , 15 min ) purified mitochondrial fractions analyzed as described in A .

C. TNF induces M1-ubiquitination at mitochondria in various cell types. The indicated cell types were treated with TNF (25 ng/ml, 15 min) and purified mitochondrial fractions analyzed as described in A.


D . TNF - induced mitochondrial M1 - ubiquitination does not occur in HOIP - deficient . Wildtype ( WT ) HOIP - KO HAP were treated with TNF ( 25 ng / ml , 15 min ) analyzed as described in A .

D. TNF-induced mitochondrial M1-ubiquitination does not occur in HOIP-deficient cells. Wildtype (WT) and HOIP-KO HAP cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.


E . Mitochondria M1 - linked ubiquitin co - localize after TNF treatment . SH - SY5Y were treated with TNF ( 25 ng / ml , 15 min ) , fixed , stained with antibodies against HSP60 ( green ) M1 - ubiquitin ( 1E3 , red ) analyzed by SR - SIM .

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E. Mitochondria and M1-linked ubiquitin co-localize after TNF treatment. SH-SY5Y cells were treated with TNF (25 ng/ml, 15 min), fixed, stained with antibodies against HSP60 (green) and M1-ubiquitin (1E3, red) and analyzed by SR-SIM.


F . TNF induces recruitment of LUBAC components to mitochondria . HEK293T were treated with TNF ( 25 ng / ml , 15 min ) analyzed as described in A .

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F. TNF induces recruitment of LUBAC components to mitochondria. HEK293T cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.


G . TNF induces a fast transient increase in M1 - K63 - specific ubiquitination at mitochondria . HEK 293T were treated with TNF ( 25 ng / ml ) for indicated time mitochondrial fractions were analyzed by immunoblotting using M1 - , K63 - , K48 - specific ubiquitin antibodies .

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G. TNF induces a fast and transient increase in M1- and K63-specific ubiquitination at mitochondria. HEK 293T cells were treated with TNF (25 ng/ml) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1-, K63-, and K48-specific ubiquitin antibodies.


A , B . PINK1 is stabilized by TNF treatment . HEK293T were treated with TNF ( 25 ng / ml , 15 min ) or CCCP ( 10 µM , 90 min ) before harvesting or incubated with OTULIN - specific siRNA for 48 h ( A ) . Purified mitochondrial fractions were analyzed by immunoblotting using indicated antibodies . of PINK1 - specific signals normalized to TIM23 is shown in right panel . Data represent mean values with standard deviations of 4 independent experiments . * p < 0 . 05 . A two - non - parametric Mann - Whitney U - was used to analyze statistical significance . In ( A ) , * unprocessed PINK1 ; * * processed PINK1 .

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A, B. PINK1 is stabilized by TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) before harvesting or incubated with OTULIN-specific siRNA for 48 h (A). Purified mitochondrial fractions were analyzed by immunoblotting using the indicated antibodies. Quantification of PINK1-specific signals normalized to TIM23 is shown in the right panel. Data represent the mean values with standard deviations of 4 independent experiments. *p < 0.05. A two-tailed non-parametric Mann-Whitney U-test was used to analyze statistical significance. In (A), *unprocessed PINK1; **processed PINK1.


C . TNF - induced PINK1 stabilization does not induce mitophagy . HeLa expressing mt - mKeima were treated with TNF ( 25 ng / ml ) for 30 min or 16 h . As a control , mt - mKeima - expressing Hela were treated with antimycin A oligomycin ( A / O , 10 µM each ) for 1 h . was done by flow cytometry gating of lysosomal neutral mt - mKeima .

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C. TNF-induced PINK1 stabilization does not induce mitophagy. HeLa cells expressing mt-mKeima were treated with TNF (25 ng/ml) for 30 min or 16 h. As a control, mt-mKeima -expressing Hela cells were treated with antimycin A and oligomycin (A/O, 10 µM each) for 1 h. The analysis was done by flow cytometry gating of lysosomal and neutral mt-mKeima.


D . Catalytically active HOIP ubiquitinates overexpressed PINK1 . HEK293T were transfected with plasmids indicated . After 24 h , were harvested under denaturing conditions PINK1 was immunoprecipitated via V5 tag followed by immunoblotting against ubiquitin .

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D. Catalytically active HOIP ubiquitinates overexpressed PINK1. HEK293T cells were transfected with the plasmids indicated. After 24 h, the cells were harvested under denaturing conditions and PINK1 was immunoprecipitated via the V5 tag followed by immunoblotting against ubiquitin.


E . Recombinant HOIP ubiquitinates PINK1 in vitro . V5 - tagged - PINK1 immunoprecipitated from transiently transfected HEK293T via V5 tag was incubated with recombinant mouse Ube1 , UBE2L3 , C - terminal HOIP ubiquitin for in vitro ubiquitination . samples were then analyzed by immunoblotting using V5 antibodies .

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E. Recombinant HOIP ubiquitinates PINK1 in vitro. V5-tagged-PINK1 immunoprecipitated from transiently transfected HEK293T cells via the V5 tag was incubated with recombinant mouse Ube1, UBE2L3, C-terminal HOIP and ubiquitin for in vitro ubiquitination. The samples were then analyzed by immunoblotting using V5 antibodies.


F . Endogenous PINK1 is modified with M1 ubiquitin chains after TNF treatment . HEK293T were treated with TNF ( 25 ng / ml , 15 min ) , then endogenous PINK1 was immunoprecipitated from mitochondrial fractions . mildly overexpressing PINK1 - V5 were also included ( lanes 4 , 5 , 8 ) , to make sure that immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1 . As a control for presence of M1 - linked ubiquitin chains , immunoprecipitated PINK1 was treated with recombinant OTULIN . As controls for specificity of immunoprecipitation , beads only ( lanes 6 - 8 ) beads plus IgG ( lane 9 ) were included . samples were analyzed by immunoblotting using antibodies against PINK1 , M1 - ubiquitin , phosphorylated ubiquitin . For immunoprecipitation of overexpressed PINK1 , only 50 % of were used in comparison to immunoprecipitation of endogenous PINK1 .

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F. Endogenous PINK1 is modified with M1 ubiquitin chains after TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min), then endogenous PINK1 was immunoprecipitated from mitochondrial fractions. Cells mildly overexpressing PINK1-V5 were also included (lanes 4, 5, 8), to make sure that the immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1. As a control for the presence of M1-linked ubiquitin chains, immunoprecipitated PINK1 was treated with recombinant OTULIN. As controls for the specificity of the immunoprecipitation, beads only (lanes 6 - 8) and beads plus IgG (lane 9) were included. The samples were analyzed by immunoblotting using antibodies against PINK1, M1-ubiquitin, and phosphorylated ubiquitin. For immunoprecipitation of overexpressed PINK1, only 50% of cells were used in comparison to the immunoprecipitation of endogenous PINK1.


A . PINK1 antagonizes OTULIN activity in . HEK293T expressing HOIP HOIL - 1L , were transfected with PINK1 , WT OTULIN or inactive OTULIN mutant W96A , as indicated . were lysed 24 h later under denaturing conditions , lysates were subjected to affinity purification using Strep - tagged UBAN domain of NEMO to enrich modified with M1 - linked ubiquitin . affinity - purified by Strep - Tactin beads were immunoblotted against ubiquitin .

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A. PINK1 antagonizes OTULIN activity in cells. HEK293T cells expressing HOIP and HOIL-1L, were transfected with PINK1, WT OTULIN or the inactive OTULIN mutant W96A, as indicated. The cells were lysed 24 h later under denaturing conditions, and lysates were subjected to affinity purification using the Strep-tagged UBAN domain of NEMO to enrich proteins modified with M1-linked ubiquitin. Proteins affinity-purified by Strep-Tactin beads were immunoblotted against ubiquitin.


B . Catalytically active PINK1 increases p - S65 - ubiquitin at mitochondria . HEK293T were transfected with wildtype PINK1 or kinase - dead ( K / D ) PINK1 . 48 h after transfection , were treated with TNF ( 25 ng / ml , 15 min ) lysed . Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p - S65 - ubiquitin , PINK1 , VDAC ( loading control ) .

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B. Catalytically active PINK1 increases p-S65-ubiquitin at mitochondria. HEK293T cells were transfected with wildtype PINK1 or kinase-dead (K/D) PINK1. 48 h after transfection, the cells were treated with TNF (25 ng/ml, 15 min) and lysed. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p-S65-ubiquitin, PINK1, and VDAC (loading control).


C . TNF - induced increase in p - S65 - ubiquitin is abolished in PINK1 - deficient . HEK293T were transfected with control or PINK1 - specific siRNA treated with TNF ( 25 ng / ml , 15 min ) or CCCP ( 10 µM , 90 min ) 48 h after transfection . whole lysates were analyzed by immunoblotting using indicated antibodies .

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C. The TNF-induced increase in p-S65-ubiquitin is abolished in PINK1-deficient cells. HEK293T cells were transfected with control or PINK1-specific siRNA and treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) 48 h after transfection. The whole cell lysates were analyzed by immunoblotting using the indicated antibodies.


D . TNF - induced M1 ubiquitination is reduced in PINK1 - deficient . WT PINK1 - KO MEFs were treated with TNF ( 25 ng / ml , 15 min ) then harvested . Purified mitochondrial fractions were subjected to affinity purification using Strep - tagged UBAN domain , as described in A , followed by immunoblotting using M1 - ubiquitin - specifc antibodies , Strep - Tactin conjugated to horse radish peroxidase ( to control UBAN pulldown efficiency ) , TIM23 ( input control ) . of M1 - ubiquitin - positive signal intensities is shown in lower panel . Data represent mean values with standard deviations of 3 independent experiments ; n . s . not significant , * * * p < 0 . 001 . A two - way ANOVA was used to analyze statistical significance .

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D. TNF-induced M1 ubiquitination is reduced in PINK1-deficient cells. WT and PINK1-KO MEFs were treated with TNF (25 ng/ml, 15 min) and then harvested. Purified mitochondrial fractions were subjected to affinity purification using the Strep-tagged UBAN domain, as described in A, followed by immunoblotting using M1-ubiquitin-specifc antibodies, Strep-Tactin conjugated to horse radish peroxidase (to control the UBAN pulldown efficiency), and TIM23 (input control). Quantification of the M1-ubiquitin-positive signal intensities is shown in the lower panel. Data represent the mean values with standard deviations of 3 independent experiments; n.s. not significant, ***p < 0.001. A two-way ANOVA test was used to analyze statistical significance.


A . STS - induced mitochondrial Bax recruitment is reduced by TNF . HeLa were treated with STS ( 1 μM , 1 h ) with or without a 15 min pretreatment with TNF ( 25 ng / ml ) then harvested . Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax M1 - ubiquitin . input was immunoblotted for TIM23 ( upper panel ) . Bax - specific signal intensities were quantified normalized to TIM23 - specific signals ( lower panel ) .

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A. STS-induced mitochondrial Bax recruitment is reduced by TNF. HeLa cells were treated with STS (1 μM, 1 h) with or without a 15 min pretreatment with TNF (25 ng/ml) and then harvested. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1-ubiquitin. The input was immunoblotted for TIM23 (upper panel). Bax-specific signal intensities were quantified and normalized to TIM23-specific signals (lower panel).


B . STS - induced cytochrome C release is decreased by TNF . HeLa were treated as described in A . fractions were analyzed by immunoblotting using cytochrome c antibodies . GAPDH was used as a reference . of 5 biological replicates is shown in lower panel . Signal intensities were quantified normalized to that of GAPDH . * p < 0 . 05 . Kolmogorow - Smirnov normality , paired t - , two - , n = 5 .

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B. STS-induced cytochrome C release is decreased by TNF. HeLa cells were treated as described in A. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. GAPDH was used as a reference. Quantification of 5 biological replicates is shown in the lower panel. Signal intensities were quantified and normalized to that of GAPDH. *p < 0.05. Kolmogorow-Smirnov normality test, paired t-test, two-tailed, n=5.


C , D . TNF treatment prevents damage of mitochondrial cristae under pro - apoptotic conditions . C . SH - SY5Y were treated as described in A , fixed , embedded mitochondrial ultrastructure was imaged by electron microscopy . Scale bar : 400 nm . D . Cristae abundance ( average cristae number per mitochondrial ) cristae ( average cristae per mitochondrium ) were analyzed by Imaris 9 . 8 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 01 . Cristae abundance : Shapiro - Wilk normality , One - way ANOVA followed by Tukey ' s multiple comparison , n = 27 . Cristae : Kruskal - Wallis followed by Dunn ' s multiple comparison , n = 24 - 27 . Bars represent mean ±

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C, D. TNF treatment prevents damage of mitochondrial cristae under pro-apoptotic conditions. C. SH-SY5Y cells were treated as described in A, fixed, embedded and the mitochondrial ultrastructure was imaged by electron microscopy. Scale bar: 400 nm. D. Cristae abundance (average cristae number per mitochondrial area) and cristae length (average cristae length per mitochondrium) were analyzed by Imaris 9.8. *p < 0.05, **p < 0.01, ***p < 0.01. Cristae abundance: Shapiro-Wilk normality test, One-way ANOVA followed by Tukey's multiple comparison test, n = 27. Cristae length: Kruskal-Wallis test followed by Dunn's multiple comparison test, n=24-27. Bars represent mean ± SEM


E . fast anti - apoptotic effect of TNF is not affected by NF - κΒ inhibitor IκΒα . SH - SY5Y were transiently transfected with NF - κΒ super - repressor IᴋBα - 2S or luciferase as a control . 24 h later , were treated with STS ( 5 µM , 2 h ) with or without a 15 min pretreatment with TNF ( 25 ng / ml ) . were fixed stained by antibodies against active caspase - 3 . Signal intensities were quantified by immunocytochemistry microscopy . Expression of IᴋBα - 2S was tested by immunoblotting using antibodies against HA tag . Data represent standard deviation of three independent experiments ; at least 300 were counted per experiment . * p < 0 . 05 , * * p < 0 . 01 . Student ' s t - , two - , n = 3 .

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E. The fast anti-apoptotic effect of TNF is not affected by the NF-κΒ inhibitor IκΒα. SH-SY5Y cells were transiently transfected with the NF-κΒ super-repressor IᴋBα-2S or luciferase as a control. 24 h later, the cells were treated with STS (5 µM, 2 h) with or without a 15 min pretreatment with TNF (25 ng/ml). Cells were fixed and stained by antibodies against active caspase-3. Signal intensities were quantified by immunocytochemistry and fluorescence microscopy. Expression of IᴋBα-2S was tested by immunoblotting using antibodies against the HA tag. Data represent the standard deviation of three independent experiments; at least 300 cells were counted per experiment. *p < 0.05, **p < 0.01. Student's t-test, two-tailed, n=3.


F . protective effect of TNF is dependent on HOIP expression . HeLa were transiently transfected with control or HOIP siRNA . 48 h after transfection , were treated with STS ( 1 μM , 1 h ) with or without a 15 min TNF pretreatment ( 25 ng / ml ) then harvested . fractions were analyzed by immunoblotting using cytochrome c antibodies . HOIP silencing efficiency was analyzed in whole lysates using antibodies against HOIP . GAPDH actin were immunoblotted as input controls .

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F. The protective effect of TNF is dependent on HOIP expression. HeLa cells were transiently transfected with control or HOIP siRNA. 48 h after transfection, cells were treated with STS (1 μM, 1 h) with or without a 15 min TNF pretreatment (25 ng/ml) and then harvested. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP. GAPDH and actin were immunoblotted as input controls.


G . protective effect of TNF is dependent on PINK1 expression . HeLa were transiently transfected with control or PINK1 siRNA treated as described in F . fractions were analyzed by immunoblotting using cytochrome c PINK1 antibodies . GAPDH was immunoblotted as input control .

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G. The protective effect of TNF is dependent on PINK1 expression. HeLa cells were transiently transfected with control or PINK1 siRNA and treated as described in F. The cytosolic fractions were analyzed by immunoblotting using cytochrome c and PINK1 antibodies. GAPDH was immunoblotted as input control.


A . NF - κB signaling components are recruited to mitochondria upon TNF treatment . A . HEK293T were treated with TNF ( 25 ng / ml , 15 min ) , harvested purified mitochondrial fractions were analyzed by immunoblotting using antibodies indicated ; p - p65 : phospho - S536 - p65 ; p - TBK1 : phospho - S172 - TBK1 .

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A. NF-κB signaling components are recruited to mitochondria upon TNF treatment. A. HEK293T cells were treated with TNF (25 ng/ml, 15 min), harvested and purified mitochondrial fractions were analyzed by immunoblotting using the antibodies indicated; p-p65: phospho-S536-p65; p-TBK1: phospho-S172-TBK1.


B . Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment . Representative immunofluorescence of SH - SY5Y using proximity ligation ( PLA ) between phospho - S536 - p65 ( rabbit ) TOM20 ( mouse ) couple antibodies . One set of were treated with TNF ( 25 ng / ml , 15 min ) before fixation . Nuclei were stained with DAPI , mitochondria were stained with MitoTrackerTM Red CMXRos ( red ) , PLA amplification reaction was visualized by green foci . As a positive control , fixed were incubated with TOM20 ( mouse ) TOM70 ( rabbit ) antibodies subjected to PLA . As negative controls , fixed were incubated with either TOM20 or p - p65 antibodies prior to PLA .

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B. Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment. Representative immunofluorescence images of SH-SY5Y cells using the proximity ligation assay (PLA) between phospho-S536-p65 (rabbit) and TOM20 (mouse) couple antibodies. One set of cells were treated with TNF (25 ng/ml, 15 min) before fixation. Nuclei were stained with DAPI, mitochondria were stained with MitoTrackerTM Red CMXRos (red), and the PLA amplification reaction was visualized by green foci. As a positive control, fixed cells were incubated with primary TOM20 (mouse) and TOM70 (rabbit) antibodies and subjected to the PLA assay. As negative controls, fixed cells were incubated with either TOM20 or p-p65 antibodies prior to the PLA assay.


C . of phospho - S536 - p65 / TOM20 PLA foci per . Data represent number of PLA foci per ( mean ± SD ) ; at least 84 in total were analyzed per condition . * * * * p < 0 . 0001 . A two - non - parametric Mann - Whitney was used to analyze statistical significance .

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C. Quantification of the phospho-S536-p65/TOM20 PLA foci per cells. Data represent the number of PLA foci per cell (mean ±SD); at least 84 cells in total were analyzed per condition. ****p < 0.0001. A two-tailed non-parametric Mann-Whitney test was used to analyze statistical significance.


A . translocation of p65 is impaired in silenced for PINK1 expression . SH - SY5Y were transfected with control or PINK1 - specific siRNAs . For rescue experiments , were co - transfected with wildtype PINK1 , PINK1Δ77 , or kinase - dead PINK1 - K / D . Two days after transfection were treated with TNF ( 25 ng / ml , 25 min ) , fixed stained with p65 antibodies . fraction of showing translocation of p65 was determined for each condition . Data represent mean ± of 3 independent experiments . experiment was performed in triplicates more than 300 were quantified per experiment condition .

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A. Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression. SH-SY5Y cells were transfected with control or PINK1-specific siRNAs. For rescue experiments, cells were co-transfected with wildtype PINK1, PINK1Δ77, or kinase-dead PINK1-K/D. Two days after transfection the cells were treated with TNF (25 ng/ml, 25 min), fixed and stained with p65 antibodies. The fraction of cells showing nuclear translocation of p65 was determined for each condition. Data represent the mean ± SEM of 3 independent experiments. The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition.


B . PINK1 phosphorylates ubiquitinated NEMO . HEK293T were co - transfected with V5 - tagged wildtype PINK1 or kinase - dead PINK1 - K / D HA - tagged NEMO . One day after transfection were lysed subjected to immunoprecipitation using antibodies against HA . Precipitated were then detected by immunoblotting using p - S65 - ubiquitin antibodies . input was immunoblotted for NEMO , PINK1 , p - S65 - ubiquitin , GAPDH .

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B. PINK1 phosphorylates ubiquitinated NEMO. HEK293T cells were co-transfected with V5-tagged wildtype PINK1 or kinase-dead PINK1-K/D and HA-tagged NEMO. One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using p-S65-ubiquitin antibodies. The input was immunoblotted for NEMO, PINK1, p-S65-ubiquitin, and GAPDH.