L Adoptive transfer of ACE2 - containing exosomes facilitates SARS - CoV - 2 pseudovirus infection . Serum exosomes ( exo ) isolated from wild - type ( WT ) mice and hACE2 KI / activated GLK transgenic ( PolII - GLK E351K Tg ) mice were adoptively transferred into wild - type recipient mice every 3 days for 12 days , followed by SARS - CoV - 2 pseudovirus infection . hACE2 - containing exosomes in the lung tissues of exosome - recipient wild - type mice were determined by PLA using anti - human ACE2 antibody plus anti - CD9 antibody ( L ) . Scale bars , 10 μm ( L ) . Data information : WT , wild - type mice ; KI , hACE2 knockin mice . Tg , transgenic mice .

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

L Adoptive transfer of ACE2-containing exosomes facilitates SARS-CoV-2 pseudovirus infection. Serum exosomes (exo) isolated from wild-type (WT) mice and hACE2 KI/activated GLK transgenic (PolII-GLK E351K Tg) mice were adoptively transferred into wild-type recipient mice every 3 days for 12 days, followed by SARS-CoV-2 pseudovirus infection. hACE2-containing exosomes in the lung tissues of exosome-recipient wild-type mice were determined by PLA using anti-human ACE2 antibody plus anti-CD9 antibody (L). Scale bars, 10 μm (L). Data information: WT, wild-type mice; KI, hACE2 knockin mice. Tg, transgenic mice.


B . Flow cytometry analyses of recipient blood 2 months post - transplant . Bi . Transplanted cells detected by GFP fluorescence . Bii . CD11b lebelling shows a large proportion of monocytes expressing GFP .

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B. Flow cytometry analyses of recipient blood 2 months post-transplant. Bi. Transplanted cells detected by GFP fluorescence. Bii. CD11b lebelling shows a large proportion of monocytes expressing GFP.


C . Demyelinating lesions in chimeric mice at 3 dpl . Ci . Demyelination was induced by injecting LPC in the spinal cord white matter . Cii - Civ . Co - immunolabeling for GFP , MBP , and a mixture of CD11b / CD68 , showing graft - derived macrophages in the lesion ( identified by lack of MBP staining ) . White lines indicate lesion borders . Scale bar 20 µm . MBP - myelin basic protein ; GFP - green fluorescent protein .

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C. Demyelinating lesions in chimeric mice at 3 dpl. Ci. Demyelination was induced by injecting LPC in the spinal cord white matter. Cii-Civ. Co-immunolabeling for GFP, MBP, and a mixture of CD11b/CD68, showing graft-derived macrophages in the lesion (identified by lack of MBP staining). White lines indicate lesion borders. Scale bar 20 µm. MBP-myelin basic protein; GFP-green fluorescent protein.


Transduction of HSCs . B . Lower numbers of GFP + cells , verified using flow cytometry , after transduction with Sema3F vector , * * * p < 0 . 0001 , Student t test , n = 8 independent experiments .

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Transduction of HSCs. B. Lower numbers of GFP+ cells, verified using flow cytometry, after transduction with Sema3F vector, ***p<0.0001, Student t test, n=8 independent experiments.


Transduction of HSCs . C . Western blot showing GFP expression in cells transduced with both vectors and Sema3F expression in the supernatant of Sema3F - transduced cells only .

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Transduction of HSCs. C. Western blot showing GFP expression in cells transduced with both vectors and Sema3F expression in the supernatant of Sema3F-transduced cells only.


E . Fold increase in cells that crossed the membrane insert compared to NT . * * * p ‹ 0 . 0001 . One - way ANOVA followed by Bonferroni ' s Multiple Comparison test . n = 1 - 2 normalized replicates from 3 - 5 independent experiments . Mean ± SEM . * * * p < 0 . 0001 . R . = recombinant . Super = supernatant . Anti - Nrp2 = Neuropilin2 function blocking antibody .

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E. Fold increase in cells that crossed the membrane insert compared to NT. ***p‹0.0001. One-way ANOVA followed by Bonferroni's Multiple Comparison test. n= 1-2 normalized replicates from 3-5 independent experiments. Mean±SEM. ***p<0.0001. R.=recombinant. Super=supernatant. Anti-Nrp2=Neuropilin2 function blocking antibody.


Expression of Nrp2 gene by OPCs is maintained in middle - age ( 12 month ( m ) old ) mice . A . Microarray analyses results for Nrp2 expression by OPCs purified using FACS from the brains of 2 months versus 12 months old PDGFRα − GFP mice ( n = 3 - 4 independent experiments ) .

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Expression of Nrp2 gene by OPCs is maintained in middle-age (12 month (m) old) mice. A. Microarray analyses results for Nrp2 expression by OPCs purified using FACS from the brains of 2 months versus 12 months old PDGFRα−GFP mice (n=3-4 independent experiments).


Expression of Nrp2 gene by OPCs is maintained in middle - age ( 12 month ( m ) old ) mice . B . RNA sequencing results for Nrp2 expression by oligodendroglia - enriched preparations isolated from the spinal cords of 2 month versus 12 month old mice . ( n = 3 - 4 independent experiments ) .

Expression of Nrp2 gene by OPCs is maintained in middle-age (12 month (m) old) mice. B. RNA sequencing results for Nrp2 expression by oligodendroglia-enriched preparations isolated from the spinal cords of 2 month versus 12 month old mice. (n=3-4 independent experiments).


C - G . Sema 3F expression in demyelinating lesions at 4 dpl ( onset of OPC recruitment ) is decreased in middle - aged ( 12 m old ) and old ( 18m old ) mice compared to young mice ( 2m old ) . C - E . Colabellings for Sema3F and Iba1 ( macrophage marker ) in demyelinating lesions at 4 dpl in 2m ( C ) , 12m ( D ) , and 18m old mice ( E ) . Arrowheads point to double - labelled cells . F . Quantification of Sema3F expressing cells in the lesion . G . Percentage of Iba1 + cells positive for Sema3F . NWM - normal white matter . dpl - days post lesion . n = 4 - 8 mice / group .

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C-G. Sema 3F expression in demyelinating lesions at 4 dpl (onset of OPC recruitment) is decreased in middle-aged (12 m old) and old (18m old) mice compared to young mice (2m old). C-E. Colabellings for Sema3F and Iba1 (macrophage marker) in demyelinating lesions at 4 dpl in 2m (C), 12m (D),and 18m old mice (E). Arrowheads point to double-labelled cells. F. Quantification of Sema3F expressing cells in the lesion. G. Percentage of Iba1+ cells positive for Sema3F. NWM-normal white matter. dpl-days post lesion. n=4-8 mice/group.


H . Transwell chamber assay was used to evaluate migration of young versus middle - age OPCs in response to recombinant Sema3F and supernatant from PGK - GFP - T2A - Sema3F transduced cells . OPCs of both ages migrate in response to recombinant Sema3F and PGK - GFP - T2A - Sema3F - transduced cell supernatant . n = 1 - 2 normalized replicates from 2 - 5 independent experiments . Mean ± SEM . One - way ANOVA followed by Bonferroni ' s Multiple Comparison test . * p < 0 . 05 , * * p < 0 . 01 . Scale bar 50 µm .

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H. Transwell chamber assay was used to evaluate migration of young versus middle-age OPCs in response to recombinant Sema3F and supernatant from PGK-GFP-T2A-Sema3F transduced cells. OPCs of both ages migrate in response to recombinant Sema3F and PGK-GFP-T2A-Sema3F-transduced cell supernatant. n= 1-2 normalized replicates from 2-5 independent experiments. Mean±SEM. One-way ANOVA followed by Bonferroni's Multiple Comparison test. *p<0.05, ** p<0.01. Scale bar 50 µm.


B . Percentage of transplanted ( female ) cells in the blood . n = 18 - 19 mice / group .

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B. Percentage of transplanted (female) cells in the blood. n=18-19 mice/group.


C . Percentage of transduced ( GFP + ) cells in the blood . Mean ± SEM . * * * p < 0 . 0001 . n = 22 - 25 mice / group .

C. Percentage of transduced (GFP+) cells in the blood . Mean±SEM. ***p<0.0001. n=22-25 mice/group.


D . Percentages of GFP + cells labeled with CD11b , CD19 , and CD3 . The proportion of GFP + cells labeled with CD11b is increased in Sema3F mice ( p = 0 . 01 ) , while that of CD19 - labeled cells is decreased ( p = 0 . 0003 ) . Mean ± SEM . Unpaired Student t - test . n = 9 mice / group . * p < 0 . 05 , * * * p < 0 . 001 .

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D. Percentages of GFP+ cells labeled with CD11b, CD19, and CD3. The proportion of GFP+ cells labeled with CD11b is increased in Sema3F mice (p=0.01), while that of CD19-labeled cells is decreased (p=0.0003). Mean±SEM. Unpaired Student t-test. n=9 mice/group. *p<0.05,***p<0.001.


E . Percentages of total blood cells expressing CD11b , CD19 , and CD3 . Red dashed lines indicate the percentage expression of these antigens in blood cells of control mice . n = 9 mice / group . Mean ± SEM .

E. Percentages of total blood cells expressing CD11b, CD19, and CD3. Red dashed lines indicate the percentage expression of these antigens in blood cells of control mice. n=9 mice/group. Mean±SEM.


A - F . Co - labeling for GFP and MBP on spinal cord sections of GFP ( A - C ) and Sema3F - GFP chimeras ( D - F ) sacrificed at 7 ( A , D ) , 10 ( B , E ) , and 60 ( C , F ) days post lesion ( dpl ) . Absence of MBP labeling in A - B , D - E indicates the lesion ( delimited by yellow lines ) . Areas faintly labeled with MBP in C - F likely represent remyelinated areas .

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A-F. Co-labeling for GFP and MBP on spinal cord sections of GFP (A-C) and Sema3F-GFP chimeras (D-F) sacrificed at 7 (A,D), 10 (B,E), and 60 (C,F) days post lesion (dpl). Absence of MBP labeling in A-B,D-E indicates the lesion (delimited by yellow lines). Areas faintly labeled with MBP in C-F likely represent remyelinated areas.


G . Quantification of GFP + cells . Kruskal - Wallis test p = 0 . 02 . No significant differences between GFP and Sema3F at any of the time points ( Dunn ' s post test ; Mann - Whitney test ) . Decrease in GFP + cells between 7 and 60 dpl for both GFP and Sema3F mice 2m .

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G. Quantification of GFP+ cells. Kruskal-Wallis test p=0.02. No significant differences between GFP and Sema3F at any of the time points (Dunn's post test; Mann-Whitney test). Decrease in GFP+ cells between 7 and 60 dpl for both GFP and Sema3F mice 2m.


H - M . Co - labeling for GFP / MBP / Iba1 ( macrophage / microglia marker ) shows colocalization of GFP with Iba1 . Arrows indicate double - labeled cells .

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H-M. Co-labeling for GFP/MBP/Iba1 (macrophage/microglia marker) shows colocalization of GFP with Iba1. Arrows indicate double-labeled cells.


N - P Co - labeling for GFP and T cell marker CD3 . Arrows indicate CD3 - labeled cells .

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N-P Co-labeling for GFP and T cell marker CD3. Arrows indicate CD3-labeled cells.


Q - S . Co - labeling for GFP and B cell marker CD45R / B220 . Arrows indicate CD45R / B220 - labeled cells .

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Q-S. Co-labeling for GFP and B cell marker CD45R/B220. Arrows indicate CD45R/B220-labeled cells.


T . Percentages of GFP + cells that are Iba1 + , CD3 + , and CD45R / B220 + . The absolute majority of GFP + cells are Iba1 + .

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T. Percentages of GFP+ cells that are Iba1+, CD3+, and CD45R/B220+. The absolute majority of GFP+ cells are Iba1+.


U . Quantification of Iba1 + , CD3 + , and B220 + cells . For all 3 populations , no significant differences in cell numbers were observed between GFP and Sema3F mice , and a general decrease was observed at 60 dpl compared to earlier time points .

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U. Quantification of Iba1+, CD3+, and B220+ cells. For all 3 populations, no significant differences in cell numbers were observed between GFP and Sema3F mice, and a general decrease was observed at 60 dpl compared to earlier time points.


V - Aa . Co - labeling for GFP and microglial marker P2Y12 . Purple arrows indicate P2Y12 + cells . Exclusion between the two markers is obvious in both groups . Ab . Quantification of P2Y12 + cells . No significant changes are observed between the groups or across the time .

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V-Aa. Co-labeling for GFP and microglial marker P2Y12. Purple arrows indicate P2Y12+ cells. Exclusion between the two markers is obvious in both groups. Ab. Quantification of P2Y12+ cells. No significant changes are observed between the groups or across the time.


B - G . Lesions at 7 dpl in GFP ( B , E ) and Sema3F ( C , F ) mice . B - C . Co - labeling for PDGFRα , MBP , and DAPI . Arrows indicate OPCs in the lesion . Bi and Ci - insets of white dotted squares in B and C . D . Higher numbers of PDGFRα + ( OPCs ) cells in Sema3F mice at 7 dpl ( Mann Whitney test : p = 0 . 0031 , n = 6 - 8 mice / group ) . E - F . Co - labeling for Olig2 and MBP . E ' - F ' . Co - labeling for Olig2 and GFP of the lesions shown in E - F . 3D reconstructions . Proximity of OPCs and Sema 3F - carrying cells . E ' i - E ' iii and F ' i - F ' iii - insets of E ' - F ' . G . Higher numbers of Olig2 + ( oligodendroglial ) cells in Sema3F - GFP mice at 7 dpl ( Mann Whitney test : p = 0 . 0047 , n = 5 - 8 mice / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G. Lesions at 7 dpl in GFP (B,E) and Sema3F (C,F) mice. B-C. Co-labeling for PDGFRα, MBP, and DAPI. Arrows indicate OPCs in the lesion. Bi and Ci-insets of white dotted squares in B and C. D. Higher numbers of PDGFRα+ (OPCs) cells in Sema3F mice at 7 dpl (Mann Whitney test: p=0.0031, n=6-8 mice/group). E-F. Co-labeling for Olig2 and MBP. E'-F'. Co-labeling for Olig2 and GFP of the lesions shown in E-F. 3D reconstructions. Proximity of OPCs and Sema 3F-carrying cells. E'i-E'iii and F'i-F'iii-insets of E'-F'. G. Higher numbers of Olig2+ (oligodendroglial) cells in Sema3F-GFP mice at 7 dpl (Mann Whitney test: p=0.0047, n=5-8 mice/group).


H - I . Immunolabelling for activated OPC marker Nkx2 . 2 . J . Numbers of Nkx2 . 2 + cells are increased in lesions of Sema3F - GFP mice at 7dpl ( Mann Whitney test : p = 0 . 014 , n = 6 - 7 mice / group ) . Arrows indicate Nkx2 . 2 + cells in close proximity of GFP + cells .

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H-I. Immunolabelling for activated OPC marker Nkx2.2. J. Numbers of Nkx2.2+ cells are increased in lesions of Sema3F-GFP mice at 7dpl (Mann Whitney test: p=0.014, n=6-7 mice/group). Arrows indicate Nkx2.2+ cells in close proximity of GFP+ cells.


K - P . colabelling for Nkx2 . 2 and proliferation marker Ki67 . Arrows indicate double labeled cells . Q . Numbers of Nkx2 . 2 + Ki67 + cells are increased at 7 dpl in Sema3F - GFP mice . Mean ± SEM . n = 6 - 7 mice / group . R . The proportion of Nkx2 . 2 + cells co - expressing Ki67 is unchanged . Mean ± SEM . n = 6 - 7 mice / group .

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K-P. colabelling for Nkx2.2 and proliferation marker Ki67. Arrows indicate double labeled cells. Q. Numbers of Nkx2.2+Ki67+ cells are increased at 7 dpl in Sema3F-GFP mice. Mean±SEM. n=6-7 mice/group. R. The proportion of Nkx2.2+ cells co-expressing Ki67 is unchanged. Mean±SEM. n=6-7 mice/group.


S - X . Colabelling for Olig2 and cleaved caspase 3 , marker of apoptosis indicates occasional apoptotic cells in both groups of mice that are Olig2 - . Arrows indicate cleaved caspase 3 + cells . Y . Numbers of cleaved caspase 3 + cells are the same in two groups . Mean ± SEM . n = 6 mice / group .

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S-X. Colabelling for Olig2 and cleaved caspase 3, marker of apoptosis indicates occasional apoptotic cells in both groups of mice that are Olig2-. Arrows indicate cleaved caspase 3+ cells.Y. Numbers of cleaved caspase 3+ cells are the same in two groups. Mean±SEM. n=6 mice/group.


A - B . Labelling for APC / CC1 , a marker of oligodendrocytes . Dotted lines indicate the lesion border . APC + cells are scarce in the lesions of GFP mice ( A ) . B . APC + cells in the lesion of Sema3F mice . C . Quantification of APC + cells ( Mann Whitney test ; p = 0 . 0061 for 7 dpl , p = 0 . 0081 for 10 dpl ; n = 6 - 7 mice / group ) .

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A-B. Labelling for APC/CC1, a marker of oligodendrocytes. Dotted lines indicate the lesion border. APC+ cells are scarce in the lesions of GFP mice (A). B. APC+ cells in the lesion of Sema3F mice. C. Quantification of APC+ cells (Mann Whitney test; p=0.0061 for 7 dpl, p=0.0081 for 10 dpl; n= 6-7 mice/group).


D - E . EM images of the lesion in GFP ( D ) and Sema3F ( E ) chimeras at 14 dpl . Thin myelin sheaths in Sema 3F mice ( yellow arrows ) . F . Higher power image of a new myelin sheath in a Sema3F mouse . Fi . Inset of yellow square in F . G . Quantification of remyelinated axons ( Mann Whitney test : p = 0 . 03 , n = 6 mice / group ) . * p < 0 . 05 . H . In Sema3F mice , oligodendroglial cells ( O ) and early remyelination ( * ) are frequently observed in macrophage ( M ) vicinity . Hi . Inset of yellow dashed square in H .

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D-E. EM images of the lesion in GFP (D) and Sema3F (E) chimeras at 14 dpl. Thin myelin sheaths in Sema 3F mice (yellow arrows). F. Higher power image of a new myelin sheath in a Sema3F mouse. Fi. Inset of yellow square in F. G. Quantification of remyelinated axons (Mann Whitney test: p=0.03, n=6 mice/group). *p<0.05. H. In Sema3F mice, oligodendroglial cells (O) and early remyelination (*) are frequently observed in macrophage (M) vicinity. Hi. Inset of yellow dashed square in H.


Identification of Rtt105 - TAP associated proteins . Purified proteins were resolved on a 4 - 12 % gel , revealed by silver staining , and identified by mass spectrometry ( Table EV1 ) . * Non - specific band also in control .

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Identification of Rtt105-TAP associated proteins. Purified proteins were resolved on a 4-12% gel, revealed by silver staining, and identified by mass spectrometry (Table EV1). *Non-specific band also in control.


Cells containing Rfa1 - TAP were synchronized at G1 , S , and G2 / M phase and used for the TAP purification . Rfa1 - TAP associated protein complexes were analyzed by Western blot using CBP , Rfa2 , and Rtt105 antibodies ( left panel ) . DNA content was monitored by flow cytometry ( right panel ) .

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Cells containing Rfa1-TAP were synchronized at G1, S, and G2/M phase and used for the TAP purification. Rfa1-TAP associated protein complexes were analyzed by Western blot using CBP, Rfa2, and Rtt105 antibodies (left panel). DNA content was monitored by flow cytometry (right panel).


MBP - Rtt105 pulled down recombinant RPA , resolved on SDS - PAGE gels and visualized by Coomassie Brilliant Blue ( CBB ) staining . MBP was used as a negative control .

MBP-Rtt105 pulled down recombinant RPA, resolved on SDS-PAGE gels and visualized by Coomassie Brilliant Blue (CBB) staining. MBP was used as a negative control.


GST - tagged full length ( FL ) and Rtt105 deletion mutants ( Schematic of the Rtt105 truncations is shown in the upper panel ) were purified and used to pull down Rfa1 protein . GST proteins were used as a negative control . Isolated protein complexes were resolved on 15 % SDS - PAGE gels and visualized by CBB staining .

GST-tagged full length (FL) and Rtt105 deletion mutants (Schematic of the Rtt105 truncations is shown in the upper panel) were purified and used to pull down Rfa1 protein. GST proteins were used as a negative control. Isolated protein complexes were resolved on 15% SDS-PAGE gels and visualized by CBB staining.


Rfa1 - TAP purification was performed using Rfa1 - TAP rtt105 ∆ strains expressing WT and indicated Rtt105 mutant forms . pRS313 serves as vector control ( Vc ) .

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Rfa1-TAP purification was performed using Rfa1-TAP rtt105∆ strains expressing WT and indicated Rtt105 mutant forms. pRS313 serves as vector control (Vc).


Snapshots of Rfa1 ChIP - Seq peak at ARS305 from WT and rtt105 ∆ cells released into HU - medium . The average Rfa1 ChIP - Seq read density from cells released into HU - medium around ACS sites . ACS : ARS consensus sequence .

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Snapshots of Rfa1 ChIP-Seq peak at ARS305 from WT and rtt105∆ cells released into HU-medium. The average Rfa1 ChIP-Seq read density from cells released into HU-medium around ACS sites. ACS: ARS consensus sequence.


Snapshots of Rfa2 ChIP - Seq peaks at ARS305 from cells released at 16 ° C for 72 min . The average Rfa2 ChIP - Seq read density surrounding ACS sites from cells released at 16 ° C for 72 min .

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Snapshots of Rfa2 ChIP-Seq peaks at ARS305 from cells released at 16°C for 72 min. The average Rfa2 ChIP-Seq read density surrounding ACS sites from cells released at 16°C for 72 min.


Rfa1 occupancy analysis of cells progression through S phase . G1 - arrested cells were released into fresh YPD medium to allow cell progression through S phase at standard growth temperature ( 25 ° C ) . Equal amounts of cells were collected at each time point . Rfa1 ChIP were performed using IgG beads for TAP - tag . ARS305 and ARS607 , early fired replication origins ; ARS607 + 14kb , a corresponding 14kb away region of ARS607 . The mean and standard deviation ( SD ) of three biological replicates were shown .

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Rfa1 occupancy analysis of cells progression through S phase. G1-arrested cells were released into fresh YPD medium to allow cell progression through S phase at standard growth temperature (25°C). Equal amounts of cells were collected at each time point. Rfa1 ChIP were performed using IgG beads for TAP-tag. ARS305 and ARS607, early fired replication origins; ARS607+14kb, a corresponding 14kb away region of ARS607. The mean and standard deviation (SD) of three biological replicates were shown.


The nuclear localization of Rfa1 proteins is altered in rtt105 ∆ mutant cells . Rfa1 tagged with GFP at its C terminus was used to analyze the localization of Rfa1 - GFP fusion protein ( Green ) and the nuclear envelop is visualized with the Nup49 - mCherry fusion protein ( Red ) using fluorescence microscopy . DIC : differential interference contrast . Scale bar : 5μm .

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The nuclear localization of Rfa1 proteins is altered in rtt105∆ mutant cells. Rfa1 tagged with GFP at its C terminus was used to analyze the localization of Rfa1-GFP fusion protein (Green) and the nuclear envelop is visualized with the Nup49-mCherry fusion protein (Red) using fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5μm.


NLS - RFA1 - GFP rescues the nuclear localization of Rfa1 in rtt105 ∆ mutant cells . The wild type RFA1 gene was fused with an SV40 large T Antigen nuclear localization sequence ( NLS ) at its 5 ' - end and a GFP gene at its 3 ' end to obtain a construct , driven by the RFA1 promoter to express NLS - RFA1 - GFP fusion protein . The engineered construct was then transformed into WT or rtt105 ∆ mutant yeast cells to replace its endogenous RFA1 expression . The resulting yeast cells were then visualized under microscope , and GFP signals enriched in the nuclei were scored . RFA1 - GFP lacking the NLS sequence was transformed into rtt105 ∆ rfa1 ∆ mutant cells as a control . DAPI staining indicates nuclear DNA .

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NLS-RFA1-GFP rescues the nuclear localization of Rfa1 in rtt105∆ mutant cells. The wild type RFA1 gene was fused with an SV40 large T Antigen nuclear localization sequence (NLS) at its 5'-end and a GFP gene at its 3' end to obtain a construct, driven by the RFA1 promoter to express NLS-RFA1-GFP fusion protein. The engineered construct was then transformed into WT or rtt105∆ mutant yeast cells to replace its endogenous RFA1 expression. The resulting yeast cells were then visualized under microscope, and GFP signals enriched in the nuclei were scored. RFA1-GFP lacking the NLS sequence was transformed into rtt105∆ rfa1∆ mutant cells as a control. DAPI staining indicates nuclear DNA.


An increase in nuclear localization of Rfa1 could not rescue the RPA binding defects at the replication regions in rtt105 ∆ cells . The percentage of Rfa2 ChIP - DNA over the total input DNA was calculated . The mean and standard error ( SE ) of three biological replicates were shown . Statistical significance was evaluated based on Student ' s t - tests ( * : 0 . 01 ≤ P value < 0 . 05 ) .

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An increase in nuclear localization of Rfa1 could not rescue the RPA binding defects at the replication regions in rtt105∆ cells. The percentage of Rfa2 ChIP-DNA over the total input DNA was calculated. The mean and standard error (SE) of three biological replicates were shown. Statistical significance was evaluated based on Student's t-tests (*: 0.01≤P value<0.05).


Purified RPA and Rtt105 - RPA complex were resolved on 15 % SDS - PAGE gels and analyzed by silver staining .

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Purified RPA and Rtt105-RPA complex were resolved on 15% SDS-PAGE gels and analyzed by silver staining.


EMSA assay was performed to analyze ssDNA binding ability of RPA or Rtt105 - RPA complex . Cy3 - labeled 30nt ssDNA was used as the substrate , and reaction mixtures were resolved on native PAGE gels . Quantification of the RPA - bound ssDNA is shown in ( C ) .

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EMSA assay was performed to analyze ssDNA binding ability of RPA or Rtt105-RPA complex. Cy3-labeled 30nt ssDNA was used as the substrate, and reaction mixtures were resolved on native PAGE gels. Quantification of the RPA-bound ssDNA is shown in (C).


Recombinant Rtt105 - RPA complex was mixed with a 30nt - ssDNA in a 2 : 1 ratio , and the reaction product were subjected to gel filtration chromatography . Equal volume of fraction 5 - 10 were resolved on SDS - PAGE for detecting proteins by Ruby staining ( upper panel ) or were resolved by native agarose gel for detection of Cy3 - ssDNA signal ( lower panel ) .

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Recombinant Rtt105-RPA complex was mixed with a 30nt-ssDNA in a 2:1 ratio, and the reaction product were subjected to gel filtration chromatography. Equal volume of fraction 5-10 were resolved on SDS-PAGE for detecting proteins by Ruby staining (upper panel) or were resolved by native agarose gel for detection of Cy3-ssDNA signal (lower panel).


GST - RPA pull down Rtt105 with increased amount of 30nt ssDNA , and the bound proteins were resolved on SDS - PAGE and visualized by silver staining .

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GST-RPA pull down Rtt105 with increased amount of 30nt ssDNA, and the bound proteins were resolved on SDS-PAGE and visualized by silver staining.


Kymograms showing most ssDNA molecules two distinct stretching patterns of RPA - ssDNA molecules . Pattern 1 , slow stretching ; Pattern 2 , fast stretching . The counts of the two patterns in each reaction were plotted in ( C ) . Please note a fraction of individual ssDNA shows both patterns . Rtt105 increases the rate of ssDNA stretching upon RPA binding . Stretching rate was defined as the length change of ssDNA upon binding to RPA - mCherry in unit time as shown in ( B , white arrow ) . Rtt105 increases the extent of ssDNA stretching upon RPA binding . Length increment = DNA length at 8 minutes / DNA length at 3 minutes .

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Kymograms showing most ssDNA molecules two distinct stretching patterns of RPA-ssDNA molecules. Pattern 1, slow stretching; Pattern 2, fast stretching. The counts of the two patterns in each reaction were plotted in (C). Please note a fraction of individual ssDNA shows both patterns. Rtt105 increases the rate of ssDNA stretching upon RPA binding. Stretching rate was defined as the length change of ssDNA upon binding to RPA-mCherry in unit time as shown in (B, white arrow). Rtt105 increases the extent of ssDNA stretching upon RPA binding. Length increment = DNA length at 8 minutes / DNA length at 3 minutes.


Snapshots of BrdU IP - Seq peaks at ARS305 from cells released into YPD medium containing HU for 45 min . The average BrdU IP - Seq read density around fired ACS from cells released into YPD medium containing HU for 45 min sites . Snapshots of BrdU IP - Seq peaks at ARS305 from cells released into YPD medium at 16 ° C for 72 min . The average BrdU IP - Seq read density around ACS sites from cells released into 16 ° C for 72 min .

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Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium containing HU for 45 min. The average BrdU IP-Seq read density around fired ACS from cells released into YPD medium containing HU for 45 min sites. Snapshots of BrdU IP-Seq peaks at ARS305 from cells released into YPD medium at 16°C for 72 min. The average BrdU IP-Seq read density around ACS sites from cells released into 16°C for 72 min.


Rad52 - GFP foci were counted in WT and rtt105 ∆ cells , the percentage of cells with Rad52 - foci is reported . Error bars indicate one standard deviation from three independent experiments with at least 150 cells counted in each replicate .

Rad52-GFP foci were counted in WT and rtt105∆ cells, the percentage of cells with Rad52-foci is reported. Error bars indicate one standard deviation from three independent experiments with at least 150 cells counted in each replicate.


Rad53 phosphorylation was analyzed in WT and rtt105 ∆ cells by immunoblotting of protein extracts with anti - Rad53 antibodies . Ponceau S ( Pon S ) staining was applied as a loading control .

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Rad53 phosphorylation was analyzed in WT and rtt105∆ cells by immunoblotting of protein extracts with anti-Rad53 antibodies. Ponceau S (Pon S) staining was applied as a loading control.


Deletion of RTT105 leads to an increased rate of gross chromosomal rearrangements ( GCRs ) . Wild - type ( WT ) and rtt105 ∆ mutant strains integrated with the yWSS439 - 5ori ∆ yeast artificial chromosomes ( YACs ) . GCRs rates in the evaluation of telomere marker loss were

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Deletion of RTT105 leads to an increased rate of gross chromosomal rearrangements (GCRs). Wild-type (WT) and rtt105∆ mutant strains integrated with the yWSS439-5ori∆ yeast artificial chromosomes (YACs). GCRs rates in the evaluation of telomere marker loss were assayed


Ten - fold serial dilutions WT or rtt105 ∆ cells in two different backgrounds were assayed on normal growth media ( YPD ) and on media containing the indicated DNA damaging agents , methyl - methane sulfonate ( MMS ) , camptothecin ( CPT ) , bleomycin ( Bleo ) , and hydroxyurea ( HU ) .

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Ten-fold serial dilutions WT or rtt105∆ cells in two different backgrounds were assayed on normal growth media (YPD) and on media containing the indicated DNA damaging agents, methyl-methane sulfonate (MMS), camptothecin (CPT), bleomycin (Bleo), and hydroxyurea (HU).


( A ) HeLa cells stably expressing GFP - Parkin ( HeLa - GFP - Parkin cells ) were treated with 1 µM valinomycin and immunostained with anti - NDP52 and anti - Tom20 antibodies . Boxed areas in the images are shown in the next panels on the right . Colocalization of GFP - Parkin , NDP52 , and Tom20 were determined using Line scan . Fluorescence intensities of each channels were measured along the dotted white arrow . Scale bars , 10 µm . Images are representative of three independent

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(A) HeLa cells stably expressing GFP-Parkin (HeLa-GFP-Parkin cells) were treated with 1 µM valinomycin and immunostained with anti-NDP52 and anti-Tom20 antibodies. Boxed areas in the images are shown in the next panels on the right. Colocalization of GFP-Parkin, NDP52, and Tom20 were determined using Line scan. Fluorescence intensities of each channels were measured along the dotted white arrow. Scale bars, 10 µm. Images are representative of three independent experiments


( B ) HeLa - GFP - Parkin cells were transfected with siNDP52 or scrambled siRNA . Seventy - two hours after transfection , cells were incubated with 1 µM valinomycin for the indicated times . Western blotting was performed using the indicated antibodies . Data are representative of three independent experiments

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(B) HeLa-GFP-Parkin cells were transfected with siNDP52 or scrambled siRNA. Seventy-two hours after transfection, cells were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments


( C ) Mitophagy flux assay using Mtphagy dye . Increase in Mtphagy dye fluorescence intensity indicates the recruitment of mitochondria to lysosomes during valinomycin treatment . Results are a summary of three independent experiments . Values are the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 compared with scrambled oligo - treated cells , determined with one - way ANOVA followed by the Student ' s t test

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(C) Mitophagy flux assay using Mtphagy dye. Increase in Mtphagy dye fluorescence intensity indicates the recruitment of mitochondria to lysosomes during valinomycin treatment. Results are a summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t test


( D ) Fluorescence images of NDP52 KD and control cells expressing mCherry - LC3 and MTS - TagBFP . Arrows indicate mCherry - LC3 puncta colocalized with MTS - TagBFP . Scale bars , 10 µm . Boxed areas in images are shown in the next panels on the right . Images are representative of four independent experiments

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(D) Fluorescence images of NDP52 KD and control cells expressing mCherry-LC3 and MTS-TagBFP. Arrows indicate mCherry-LC3 puncta colocalized with MTS-TagBFP. Scale bars, 10 µm. Boxed areas in images are shown in the next panels on the right. Images are representative of four independent experiments


( E ) The number of mCherry - LC3 puncta colocalized with MTS - TagBFP ( representing mitophagosomes ) per cell was counted . Results were from at least six microscopic fields of three independent experiments . Values are the means ± SEM . * * p < 0 . 01 compared with scrambled oligo - treated cells , determined with one - way ANOVA followed by the Student ' s t test

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(E) The number of mCherry-LC3 puncta colocalized with MTS-TagBFP (representing mitophagosomes) per cell was counted. Results were from at least six microscopic fields of three independent experiments. Values are the means ± SEM. **p < 0.01 compared with scrambled oligo-treated cells, determined with one-way ANOVA followed by the Student's t test


( F , G ) Electron micrographs of NDP52 KD and control cells . Asterisks indicate mitochondria sequestered by autophagosomes ( mitophagosomes , yellow dotted line ) . The ratios of mitophagosomes per mitochondrial area are shown in ( G ) . Ten cells from three independent experiments were counted . Values are the means ± SD . * p < 0 . 05 compared with scrambled - oligo treated cells , determined with one - way ANOVA followed by the Student ' s t

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(F, G) Electron micrographs of NDP52 KD and control cells. Asterisks indicate mitochondria sequestered by autophagosomes (mitophagosomes, yellow dotted line). The ratios of mitophagosomes per mitochondrial area are shown in (G). Ten cells from three independent experiments were counted. Values are the means ± SD. *p < 0.05 compared with scrambled-oligo treated cells, determined with one-way ANOVA followed by the Student's t test


( Images of HeLa GFP - Parkin cells expressing mCherry - NDP52 ( A under valinomycin treatment . The images are from Movie EV 2 and 3 , respectively . Cyan , GFP - Parkin ; magenta , mCherry - NDP5 Scale bars , 10 µm . Images are representative of five independent

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( Images of HeLa GFP-Parkin cells expressing mCherry-NDP52 (A under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta, mCherry-NDP5 Scale bars, 10 µm. Images are representative of five independent experiments


( B ) Time - lapse images of HeLa GFP - Parkin cells expressing mCherry - NDP52 under valinomycin treatment . The images are representative of five independent experiments and correspond to the boxed regions of Figure 2A and Movie EV 2 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . The white arrowheads indicate colocalization of GFP - Parkin and mCherry - NDP52 . The yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring

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(B) Time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin treatment. The images are representative of five independent experiments and correspond to the boxed regions of Figure 2A and Movie EV 2. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures


Images o HeLa GFP - Parkin cells expressin mCherry - OPTN ( C ) under valinomycin treatment . The images are from Movie EV 2 and 3 , respectively . Cyan , GFP - Parkin ; magenta OPTN . Scale bars , 10 µm . Images are representative of five independent

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Images o HeLa GFP-Parkin cells expressin mCherry-OPTN (C) under valinomycin treatment. The images are from Movie EV 2 and 3, respectively. Cyan, GFP-Parkin; magenta OPTN. Scale bars, 10 µm. Images are representative of five independent experiments


( D ) mCherry - NDP52 or OPTN signals colocalized with Parkin - ring structures and existing inside of Parkin - ring structures ( irrupted ) were quantified . Data were from five independent experiments in cells incubated with valinomycin for 2 h . Values are the means ± SEM . * * p < 0 . 01 compared with mCherry - OPTN expressing cells , determined with one - way ANOVA followed by the Student " s

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(D) mCherry-NDP52 or OPTN signals colocalized with Parkin-ring structures and existing inside of Parkin-ring structures (irrupted) were quantified. Data were from five independent experiments in cells incubated with valinomycin for 2 h. Values are the means ± SEM. **p < 0.01 compared with mCherry-OPTN expressing cells, determined with one-way ANOVA followed by the Student"s t


( E ) Mitophagic flux in NDP52 KD cells expressing siRNA - resistant NDP52 or NDP52 mutants . Cells were incubated with valinomycin for 3 h . Results are the summary of three independent experiments . Values are the means ± SEM . * p < 0 . 05 , * * p < 0 . 01 vs scrambled oligo - transfected cells ; † p < 0 . 05 , † † p < 0 . 01 vs empty vector - transfected cells , determined with one - way ANOVA followed by the Tukey - Kramer post hoc

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(E) Mitophagic flux in NDP52 KD cells expressing siRNA-resistant NDP52 or NDP52 mutants. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. *p < 0.05, **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05, ††p < 0.01 vs empty vector-transfected cells, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test


( F ) HeLa - GFP - Parkin cells expressing mCherry - NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h . Cell lysates were subjected to immunoprecipitation using anti - RFP magnetic beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

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(F) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


( G ) Images of HeLa GFP - Parkin cells expressing mCherry - NDP52 ∆ LIM - L or mCherry - NDP52 ∆ SKICH treated with valinomycin for 2h . The images are from Movie EV 4 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 mutant . Scale bars , 10 µm . Images are representative of three independent

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(G) Images of HeLa GFP-Parkin cells expressing mCherry-NDP52∆LIM-L or mCherry-NDP52∆SKICH treated with valinomycin for 2h. The images are from Movie EV 4. Cyan, GFP-Parkin; magenta, mCherry-NDP52 mutant. Scale bars, 10 µm. Images are representative of three independent experiments


( A ) Mitophagic flux in cells transfected with siNDP52 , siTBK1 , siOPTN or a combination of siRNAs . Results are the summary of three independent experiments . Values are the means ± SEM . * * p < 0 . 01 vs scrambled oligo - transfected cells ; † p < 0 . 05 vs siNDP52 - transfected cells ; § p < 0 . 05 , § § p < 0 . 01 vs siTBK1 ; NS : not significant , determined with one - way ANOVA followed by the Tukey - Kramer post hoc

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(A) Mitophagic flux in cells transfected with siNDP52, siTBK1, siOPTN or a combination of siRNAs. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; †p < 0.05 vs siNDP52-transfected cells; §p < 0.05, §§p < 0.01 vs siTBK1; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test


( B ) HeLa - GFP - Parkin cells transfected with siNDP52 , siOPTN , siTBK1 or scrambled siRNA were incubated with 1 µM valinomycin for the indicated times . Cell lysates were subjected to SDS - PAGE or Phos - tag - PAGE prior to western blotting using the indicated antibodies . Data are representative of two independent experiments . The position of phosphorylated forms of NDP52 or OPTN are indicated by

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(B) HeLa-GFP-Parkin cells transfected with siNDP52, siOPTN, siTBK1 or scrambled siRNA were incubated with 1 µM valinomycin for the indicated times. Cell lysates were subjected to SDS-PAGE or Phos-tag-PAGE prior to western blotting using the indicated antibodies. Data are representative of two independent experiments. The position of phosphorylated forms of NDP52 or OPTN are indicated by arrowheads


( C ) Images of TBK1 KD cells expressing mCherry - NDP52 or mCherry - OPTN under valinomycin treatment from Movie EV 5 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . Images are representative of two independent experiments . Scale bars , 10

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(C) Images of TBK1 KD cells expressing mCherry-NDP52 or mCherry-OPTN under valinomycin treatment from Movie EV 5. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm


( D ) TBK1 KD and control cells expressed mCherry - NDP52 . Lysates were subjected to immunoprecipitation using anti - RFP beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

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(D) TBK1 KD and control cells expressed mCherry-NDP52. Lysates were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


Anti - RFP immunoprecipitates from mCherry - NDP52 and mCherry - NDP52 ∆ SKICH - transfected cells were subjected to SDS - PAGE followed by silver staining . Four bands specific for mCherry - NDP52 are numbered and were identified by mass spectrometric analysis . Asterisk ( * ) and double asterisk ( * * ) indicate mCherry - NDP52 and mCherry - NDP52 ∆ SKICH bands ,

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Anti-RFP immunoprecipitates from mCherry-NDP52 and mCherry-NDP52 ∆SKICH-transfected cells were subjected to SDS-PAGE followed by silver staining. Four bands specific for mCherry-NDP52 are numbered and were identified by mass spectrometric analysis. Asterisk (*) and double asterisk (**) indicate mCherry-NDP52 and mCherry-NDP52∆SKICH bands, respectively


( C ) HeLa - GFP - Parkin cells expressing mCherry - NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h . Cell lysates were subjected to immunoprecipitation using anti - RFP magnetic beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

(C) HeLa-GFP-Parkin cells expressing mCherry-NDP52 mutants were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


( D ) HeLa - GFP - Parkin cells transfected with siMTPAP were incubated with 1 µM valinomycin for the indicated times . Western blotting was performed using the indicated antibodies . Data are representative of three independent

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(D) HeLa-GFP-Parkin cells transfected with siMTPAP were incubated with 1 µM valinomycin for the indicated times. Western blotting was performed using the indicated antibodies. Data are representative of three independent experiments


( E ) Mitophagic flux in cells transfected with siNDP52 , siMTPAP and combinations of siRNAs . Cells were incubated with valinomycin for 3 h . Results are the summary of three independent experiments . Values are the means ± SEM . * * p < 0 . 01 vs scrambled oligo - transfected cells ; NS : not significant , determined with one - way ANOVA followed by the Tukey - Kramer post hoc

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(E) Mitophagic flux in cells transfected with siNDP52, siMTPAP and combinations of siRNAs. Cells were incubated with valinomycin for 3 h. Results are the summary of three independent experiments. Values are the means ± SEM. **p < 0.01 vs scrambled oligo-transfected cells; NS: not significant, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test


( F ) Images of MTPAP KD cells expressing mCherry - NDP52 under valinomycin treatment . The images are from Movie EV 7 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . Images are representative of two independent experiments . Scale bars , 10

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(F) Images of MTPAP KD cells expressing mCherry-NDP52 under valinomycin treatment. The images are from Movie EV 7. Cyan, GFP-Parkin; magenta, mCherry-NDP52. Images are representative of two independent experiments. Scale bars, 10 µm


( A ) HeLa - GFP - Parkin cells were incubated with valinomycin for 0 or 2 h . Immunostaining was performed with anti - MTPAP and anti - Tom20 antibodies . Images are representative of two independent experiments . Scale bar , 10

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(A) HeLa-GFP-Parkin cells were incubated with valinomycin for 0 or 2 h. Immunostaining was performed with anti-MTPAP and anti-Tom20 antibodies. Images are representative of two independent experiments. Scale bar, 10 µm


( B ) Mitochondrial - rich fraction prepared from HeLa - GFP - Parkin cells treated with or without valinomycin and epoxomicin for 2 h , was incubated with various concentrations of trypsin ( 0 - 1000 µg / ml ) for 30 min on ice . Protease protection was analyzed by western blotting using antibodies for mitochondrial marker proteins ( Tom20 for OMM , Tim23 for intermembrane space , UQCRC1 and TFAM for mitochondrial matrix ) . Data are representative of five independent

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(B) Mitochondrial-rich fraction prepared from HeLa-GFP-Parkin cells treated with or without valinomycin and epoxomicin for 2 h, was incubated with various concentrations of trypsin (0-1000 µg/ml) for 30 min on ice. Protease protection was analyzed by western blotting using antibodies for mitochondrial marker proteins (Tom20 for OMM, Tim23 for intermembrane space, UQCRC1 and TFAM for mitochondrial matrix). Data are representative of five independent experiments


( C ) HeLa - GFP - Parkin cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 0 - 3 h in the presence or absence of 10 µM epoxomicin . Cell lysates were subjected to immunoprecipitation using anti - NDP52 antibody or control IgG . Immunoprecipitates were analyzed by western blotting . Data are representative of five independent experiments . ( D ) Quantification of MTPAP co - immunoprecipitated with NDP52 in ( C ) . Results are a summary of five independent experiments . Values are the means ± SEM . * * p < 0 . 01 compared to cells treated with valinomycin and bafilomycin A1 for 3 h , determined with one - way ANOVA followed by the Tukey - Kramer post hoc

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(C) HeLa-GFP-Parkin cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 0-3 h in the presence or absence of 10 µM epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of five independent experiments. (D) Quantification of MTPAP co-immunoprecipitated with NDP52 in (C). Results are a summary of five independent experiments. Values are the means ± SEM. **p < 0.01 compared to cells treated with valinomycin and bafilomycin A1 for 3 h, determined with one-way ANOVA followed by the Tukey-Kramer post hoc test


( E ) HeLa - GFP - Parkin cells were incubated with 100 µM DFO and 100 nM bafilomycin A1 for 0 , 4 and 8 h in the presence or absence of epoxomicin . Cell lysates were subjected to immunoprecipitation using anti - NDP52 antibody . Immunoprecipitates were analyzed by western blotting . Data are representative of two independent

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(E) HeLa-GFP-Parkin cells were incubated with 100 µM DFO and 100 nM bafilomycin A1 for 0, 4 and 8 h in the presence or absence of epoxomicin. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments


( F ) HeLa cells and HEK293T cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h or 100 µM DFO and 100 nM bafilomycin A1 for 8 h . Cell lysates were subjected to immunoprecipitation using anti - NDP52 antibody . Immunoprecipitates were analyzed by western blotting . Data are representative of two independent

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(F) HeLa cells and HEK293T cells were incubated with 1 µM valinomycin and 100 nM bafilomycin A1 for 3 h or 100 µM DFO and 100 nM bafilomycin A1 for 8 h. Cell lysates were subjected to immunoprecipitation using anti-NDP52 antibody. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments


( G ) Immunoelectron micrographs of HeLa - GFP - Parkin cells expressing FLAG - NDP52 . Cells treated for 2 h with valinomycin and bafilomycin A1 with or without epoxomicin , were processed for immune - electron microscopy with anti - FLAG antibody . Images are representative of ten cells . Arrow , immunogold labeled FLAG - NDP52 . Mito , mitochondrion ; MP , mitophagosome . Scale bars , 200

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(G) Immunoelectron micrographs of HeLa-GFP-Parkin cells expressing FLAG-NDP52. Cells treated for 2 h with valinomycin and bafilomycin A1 with or without epoxomicin, were processed for immune-electron microscopy with anti-FLAG antibody. Images are representative of ten cells. Arrow, immunogold labeled FLAG-NDP52. Mito, mitochondrion; MP, mitophagosome. Scale bars, 200 nm


( H ) Selected frames from time - lapse images of HeLa GFP - Parkin cells expressing mCherry - NDP52 under valinomycin and bafilomycin A1 treatment . The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 6 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 . The white arrowheads indicate colocalization of GFP - Parkin and mCherry - NDP52 . The yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring structures . Scale bars , 1

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(H) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 6. Cyan, GFP-Parkin; magenta, mCherry-NDP52. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm


( A , B ) mCherry - LC3 and MTS - TagBFP were expressed in MTPAP KD and control cells . Cells were incubated with valinomycin for the indicated times and immunostained with anti - NDP52 antibody . Images are representative of two independent experiments . Yellow arrows indicate co - localization of MTS - TagBFP , mCherry - LC3 and NDP52 . White arrows indicate co - localization of MTS - TagBFP and NDP52 ( not mCherry - LC3 ) . Scale bars , 10 µm . Co - localization of mCherry - LC3 and NDP52 on mitochondria is quantified in ( B ) . At least ten cells from two independent experiments were quantified . Values are the means ± SEM . * * p < 0 . 01 vs control , determined with one - way ANOVA followed by the Student " s

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(A, B) mCherry-LC3 and MTS-TagBFP were expressed in MTPAP KD and control cells. Cells were incubated with valinomycin for the indicated times and immunostained with anti-NDP52 antibody. Images are representative of two independent experiments. Yellow arrows indicate co-localization of MTS-TagBFP, mCherry-LC3 and NDP52. White arrows indicate co-localization of MTS-TagBFP and NDP52 (not mCherry-LC3). Scale bars, 10 µm. Co-localization of mCherry-LC3 and NDP52 on mitochondria is quantified in (B). At least ten cells from two independent experiments were quantified. Values are the means ± SEM. **p < 0.01 vs control, determined with one-way ANOVA followed by the Student"s t


MTPAP KD and control cells expressed mCherry - NDP52 or empty vector ( C Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti - RFP beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

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MTPAP KD and control cells expressed mCherry-NDP52 or empty vector (C Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


MTPAP KD and control cells expresse mCherry - LC3 ( D ) . Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti - RFP beads . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

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MTPAP KD and control cells expresse mCherry-LC3 (D). Lysates from cells treated with 1 µM valinomycin and 100 nM bafilomycin A1 for 2 h were subjected to immunoprecipitation using anti-RFP beads. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


( E ) MTPAP KD and control cells were incubated with valinomycin and bafilomycin A1 for 2 h . Cell lysates were subjected to immunoprecipitation using an anti - NDP52 antibody or control IgG . Immunoprecipitates were analyzed by western blotting . Data are representative of three independent

(E) MTPAP KD and control cells were incubated with valinomycin and bafilomycin A1 for 2 h. Cell lysates were subjected to immunoprecipitation using an anti-NDP52 antibody or control IgG. Immunoprecipitates were analyzed by western blotting. Data are representative of three independent experiments


( F ) GST - NDP52 conjugated with glutathione - Sepharose resin was incubated with purified recombinant LC3 and MTPAP ∆ MTS ∆ C . The pulled down complex was subjected to western blotting . Data are representative of three independent

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(F) GST-NDP52 conjugated with glutathione-Sepharose resin was incubated with purified recombinant LC3 and MTPAP∆MTS∆C. The pulled down complex was subjected to western blotting. Data are representative of three independent experiments


( A ) HeLa - GFP - Parkin cells expressing mCherry - NDP52 D439R / C443K mutant or WT NDP52 were incubated with valinomycin and nM bafilomycin A1 for 3 h . Cell lysates were subjected to immunoprecipitation using anti - RFP magnetic beads . Immunoprecipitates were analyzed by western blotting . Data are representative of two independent experiments

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(A) HeLa-GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant or WT NDP52 were incubated with valinomycin and nM bafilomycin A1 for 3 h. Cell lysates were subjected to immunoprecipitation using anti-RFP magnetic beads. Immunoprecipitates were analyzed by western blotting. Data are representative of two independent experiments


( B ) Selected frames from time - lapse images of HeLa GFP - Parkin cells expressing mCherry - NDP52 D439R / C443K mutant under valinomycin and bafilomycin A1 treatment . The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 9 . Cyan , GFP - Parkin ; magenta , mCherry - NDP52 D439R / C443K . The white arrowheads indicate colocalization of GFP - Parkin and mCherry - NDP52 . The yellow arrowheads indicate mCherry - NDP existing inside GFP - Parkin ring structures . Scale bars , 1

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(B) Selected frames from time-lapse images of HeLa GFP-Parkin cells expressing mCherry-NDP52 D439R/C443K mutant under valinomycin and bafilomycin A1 treatment. The images are representative of two independent experiments and correspond to the boxed regions of Movie EV 9. Cyan, GFP-Parkin; magenta, mCherry-NDP52 D439R/C443K. The white arrowheads indicate colocalization of GFP-Parkin and mCherry-NDP52. The yellow arrowheads indicate mCherry-NDP existing inside GFP-Parkin ring structures. Scale bars, 1 µm


A - B HT ‑ 29 cells were transfected with non - silencing control siRNA ( sictr ) or siRNAs against USP22 ( siUSP22 ) for 48 h at 20 nM . After transfection , cells were treated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα either for 6 h and analyzed by Western blotting ( A ) or for 18 h , and the percentage of PI - positive cells was assessed by fluorescence - based PI staining ( B ) . Vinculin served as a loading control .

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A-B HT‑29 cells were transfected with non-silencing control siRNA (sictr) or siRNAs against USP22 (siUSP22) for 48 h at 20 nM. After transfection, cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα either for 6 h and analyzed by Western blotting (A) or for 18 h, and the percentage of PI-positive cells was assessed by fluorescence-based PI staining (B). Vinculin served as a loading control.


C HT ‑ 29 CRISPR / Cas9 control ( ctr ) and USP22 KO cells were analyzed by Western blotting for USP22 expression . GAPDH served as loading control .

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C HT‑29 CRISPR/Cas9 control (ctr) and USP22 KO cells were analyzed by Western blotting for USP22 expression. GAPDH served as loading control.


D HT ‑ 29 control and USP22 KO cells were treated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα after pre - incubation with 30 µM Nec1 - s and 20 µM GSK ' 872 for 1 h and incubated for 18 h before fluorescence - based quantification of PI - positive cells .

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D HT‑29 control and USP22 KO cells were treated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα after pre-incubation with 30 µM Nec1-s and 20 µM GSK'872 for 1 h and incubated for 18 h before fluorescence-based quantification of PI-positive cells.


E HT ‑ 29 control and USP22 KO cells , generated with 2 guide RNAs ( 2g ) , expressing empty vector ( EV ) or PAM mutated 3xFLAG - HA - USP22 WT ( USP22 PAM ) , C185S ( USP22 PAM C185S ) or C185A ( USP22 PAM C185A ) were analyzed by Western blotting for USP22 expression levels . β - Actin was used as a loading control .

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E HT‑29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were analyzed by Western blotting for USP22 expression levels. β-Actin was used as a loading control.


F HT - 29 control and USP22 KO cells , generated with 2 guide RNAs ( 2g ) , expressing empty vector ( EV ) or PAM mutated 3xFLAG - HA - USP22 WT ( USP22 PAM ) , C185S ( USP22 PAM C185S ) or C185A ( USP22 PAM C185A ) were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 , 1 ng / ml TNFα for 18 h . The percentage of PI - positive cells was assessed by fluorescence - based PI staining .

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F HT-29 control and USP22 KO cells, generated with 2 guide RNAs (2g), expressing empty vector (EV) or PAM mutated 3xFLAG-HA-USP22 WT (USP22 PAM), C185S (USP22 PAM C185S) or C185A (USP22 PAM C185A) were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6, 1 ng/ml TNFα for 18 h. The percentage of PI-positive cells was assessed by fluorescence-based PI staining.


A HT ‑ 29 control and USP22 KO cells were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for the indicated time points . Detection of indicated proteins was carried out by Western blotting . GAPDH served as a loading control .

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A HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for the indicated time points. Detection of indicated proteins was carried out by Western blotting. GAPDH served as a loading control.


B HT ‑ 29 control and USP22 KO cells were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for 4 h . Detection of indicated proteins was carried out by Western blotting . β - Actin served as a loading control .

B HT‑29 control and USP22 KO cells were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Detection of indicated proteins was carried out by Western blotting. β-Actin served as a loading control.


C HT ‑ 29 control and USP22 KO cells were incubated with 30 µM Nec1 - s or 20 µM Dab for 18 h , as indicated . Cell were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for 5 h . 100 μg of each lysate were incubated with 400 U / μl λ - phosphatase for 30 min at 30 ° C . Protein expression of RIPK3 was monitored by Western blotting . β - Actin was used as loading control . High molecular weight RIPK3 ' smears ' were quantified after λ - phosphatase treatment and normalized to total RIPK3 and β - Actin levels .

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C HT‑29 control and USP22 KO cells were incubated with 30 µM Nec1-s or 20 µM Dab for 18 h, as indicated. Cell were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 5 h. 100 μg of each lysate were incubated with 400 U/μl λ-phosphatase for 30 min at 30 °C. Protein expression of RIPK3 was monitored by Western blotting. β-Actin was used as loading control. High molecular weight RIPK3 'smears' were quantified after λ-phosphatase treatment and normalized to total RIPK3 and β-Actin levels.


A HeLa TRex RIPK3 CRISPR / Cas9 control ( ctr ) and USP22 KO cells were treated with 1 µg / ml Dox overnight . Protein expression of induced Strep - RIPK3 was analyzed by Western blotting . GAPDH served as loading control . The asterisk marks an unspecific band .

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A HeLa TRex RIPK3 CRISPR/Cas9 control (ctr) and USP22 KO cells were treated with 1 µg/ml Dox overnight. Protein expression of induced Strep-RIPK3 was analyzed by Western blotting. GAPDH served as loading control. The asterisk marks an unspecific band.


B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg / ml Dox for 18 h before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI - positive nuclei .

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B HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox for 18 h before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added and cell death was measured after 4 and 5 h by analysis of PI-positive nuclei.


C HeLa TRex RIPK3 control and USP22 KO cells were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 , 2 , 3 , 4 and 5 h . Protein expression of phosphorylated RIPK1 , total RIPK1 , total RIPK3 , phosphorylated MLKL , total MLKL and USP22 , without ( left ) or with ( right ) 1 µg / ml Dox treatment overnight , was monitored by Western blotting . GAPDH was used as a loading control .

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C HeLa TRex RIPK3 control and USP22 KO cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3, 4 and 5 h. Protein expression of phosphorylated RIPK1, total RIPK1, total RIPK3, phosphorylated MLKL, total MLKL and USP22, without (left) or with (right) 1 µg/ml Dox treatment overnight, was monitored by Western blotting. GAPDH was used as a loading control.


D HT - 29 control cells and RIPK3 KO cells re - expressing PAM - mutated Dox - inducible RIPK3 WT were incubated overnight with 1 µg / ml Dox . Cells were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h , as indicated . Strep - RIPK3 was immunoprecipitated using anti ‑ Strep - beads and the indicated co - immunoprecipitated proteins were analyzed by Western blotting . β - Actin served as a loading control .

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D HT-29 control cells and RIPK3 KO cells re-expressing PAM-mutated Dox-inducible RIPK3 WT were incubated overnight with 1 µg/ml Dox. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h, as indicated. Strep-RIPK3 was immunoprecipitated using anti‑Strep-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.


E USP22 KO HT - 29 cells and USP22 KO cells re - expressing PAM - mutated 3xFLAG - HA - USP22 were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα was added for 2 h , as indicated . 3xFLAG - HA - USP22 was immunoprecipitated using anti - HA - beads and the indicated co - immunoprecipitated proteins were analyzed by Western blotting . β - Actin served as a loading control .

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E USP22 KO HT-29 cells and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα was added for 2 h, as indicated. 3xFLAG-HA-USP22 was immunoprecipitated using anti-HA-beads and the indicated co-immunoprecipitated proteins were analyzed by Western blotting. β-Actin served as a loading control.


A HT - 29 control , USP22 KO and USP22 KO cells re - expressing PAM - mutated 3xFLAG - HA - USP22 WT or C185S were stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 and 1 ng / ml TNFα for 4 h . Poly - ubiquitinated proteins were enriched by GST - TUBE pull - down , followed by incubation with the catalytic domain of USP2 , as indicated . RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies . β - Actin served as loading control . Ponceau - staining was used to confirm equal loading of GST - TUBE .

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A HT-29 control, USP22 KO and USP22 KO cells re-expressing PAM-mutated 3xFLAG-HA-USP22 WT or C185S were stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 and 1 ng/ml TNFα for 4 h. Poly-ubiquitinated proteins were enriched by GST-TUBE pull-down, followed by incubation with the catalytic domain of USP2, as indicated. RIPK3 and USP22 expression and levels of ubiquitinated RIPK3 were monitored using Western blotting with the indicated antibodies. β-Actin served as loading control. Ponceau-staining was used to confirm equal loading of GST-TUBE.


B HT - 29 control and USP22 KO cells were transfected with His - ubiquitin for 24 h , as indicated . Cells were pre ‑ stimulated with 20 µM zVAD . fmk , 0 . 5 µM BV6 for 1 h . Following pre - treatment , 1 ng / ml TNFα was added for 4 h . His - ubiquitin was immunoprecipitated using Ni - NTA beads and detection of indicated proteins was performed by Western blotting . β - Actin served as loading control for the input , whereas His - ubiquitin levels served as loading control for immunoprecipitated ubiquitin .

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B HT-29 control and USP22 KO cells were transfected with His-ubiquitin for 24 h, as indicated. Cells were pre‑stimulated with 20 µM zVAD.fmk, 0.5 µM BV6 for 1 h. Following pre-treatment, 1 ng/ml TNFα was added for 4 h. His-ubiquitin was immunoprecipitated using Ni-NTA beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas His-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.


C HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg / ml Dox and transfected with HA - ubiquitin for 24 h , as indicated . Cells were pre - stimulated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . Following pre - treatment , 10 ng / ml TNFα were added for 3 h . HA - ubiquitin was immunoprecipitated using anti - HA - beads and detection of indicated proteins was performed by Western blotting . β - Actin served as loading control for the input , whereas HA - levels served as loading control for immunoprecipitated ubiquitin .

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C HeLa TRex RIPK3 control and USP22 KO cells were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-stimulated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. Following pre-treatment, 10 ng/ml TNFα were added for 3 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-levels served as loading control for immunoprecipitated ubiquitin.


B Bar graphs demonstrating the number of up ‐ , non ‐ , and downregulated ubiquitination sites in USP22 KO HT - 29 cells versus control under TBZ - treatment .

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B Bar graphs demonstrating the number of up‐, non‐, and downregulated ubiquitination sites in USP22 KO HT-29 cells versus control under TBZ-treatment.


D HeLa cells expressing Dox - inducible RIPK3 WT , D160N , K42R , K351 , K518R , 2xKR or 3xKR were pre ‑ incubated with 1 µg / ml Dox , 20 µM Dab , or 10 µM NSA overnight , followed by pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 4 h . Cell death was measured by quantification of PI - positive nuclei .

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D HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K42R, K351, K518R, 2xKR or 3xKR were pre‑incubated with 1 µg/ml Dox, 20 µM Dab, or 10 µM NSA overnight, followed by pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 4 h. Cell death was measured by quantification of PI-positive nuclei.


E HeLa cells expressing Dox - inducible RIPK3 mutants were treated with 1 µg / ml Dox overnight . Protein levels of inducible RIPK3 expression were analyzed by Western blotting . GAPDH served as loading control .

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E HeLa cells expressing Dox-inducible RIPK3 mutants were treated with 1 µg/ml Dox overnight. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. GAPDH served as loading control.


A HeLa cells expressing Dox - inducible RIPK3 WT , K518R or 3xKR were treated with 1 µg / ml Dox and / or 10 µM NSA and 20 µM Dab overnight . Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting . β - Actin was used as a loading control .

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A HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox and/or 10 µM NSA and 20 µM Dab overnight. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were examined by Western blotting. β-Actin was used as a loading control.


B HeLa cells expressing Dox - inducible RIPK3 WT or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 , 2 , 3 and 4 h . Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting . β - Actin served as loading control .

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B HeLa cells expressing Dox-inducible RIPK3 WT or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1, 2, 3 and 4 h. Protein levels of inducible RIPK3 expression and phosphorylated MLKL were analyzed by Western blotting. β-Actin served as loading control.


C HeLa cells expressing Dox - inducible RIPK3 WT , 2xKR or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . Protein levels of inducible RIPK3 expression were analyzed by Western blotting . β - Actin served as loading control . D HeLa cells expressing Dox - inducible RIPK3 WT , K518R or 3xKR were treated with 1 µg / ml Dox overnight before pre - treatment with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . Whole cell lysates were generated using RIPA lysis buffer containing 2 % SDS . Protein levels of inducible RIPK3 expression were analyzed by Western blotting . β - Actin served as loading control .

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C HeLa cells expressing Dox-inducible RIPK3 WT, 2xKR or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control. D HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were treated with 1 µg/ml Dox overnight before pre-treatment with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. Whole cell lysates were generated using RIPA lysis buffer containing 2% SDS. Protein levels of inducible RIPK3 expression were analyzed by Western blotting. β-Actin served as loading control.


E HeLa cells expressing Dox - inducible RIPK3 WT , D160N , K518R or 3xKR were incubated overnight with 1 µg / ml Dox and pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 1 and 2 h . Strep - RIPK3 was immunoprecipitated by using anti - Strep - beads . Co - immunoprecipitated phosphorylated MLKL and RIPK1 , as well as protein expression of indicated proteins were analyzed by Western blotting . β - Actin served as a loading control .

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E HeLa cells expressing Dox-inducible RIPK3 WT, D160N, K518R or 3xKR were incubated overnight with 1 µg/ml Dox and pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 1 and 2 h. Strep-RIPK3 was immunoprecipitated by using anti-Strep-beads. Co-immunoprecipitated phosphorylated MLKL and RIPK1, as well as protein expression of indicated proteins were analyzed by Western blotting. β-Actin served as a loading control.


F HeLa cells expressing Dox - inducible RIPK3 WT , K518R or 3xKR were incubated with 1 µg / ml Dox and transfected with HA - ubiquitin for 24 h , as indicated . Cells were pre - treated with 20 µM zVAD . fmk , 5 µM BV6 for 1 h . After pre - treatment , 10 ng / ml TNFα were added for 2 h . HA - ubiquitin was immunoprecipitated using anti - HA - beads and detection of indicated proteins was performed by Western blotting . β - Actin served as loading control for the input , whereas HA - ubiquitin levels served as loading control for immunoprecipitated ubiquitin .

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F HeLa cells expressing Dox-inducible RIPK3 WT, K518R or 3xKR were incubated with 1 µg/ml Dox and transfected with HA-ubiquitin for 24 h, as indicated. Cells were pre-treated with 20 µM zVAD.fmk, 5 µM BV6 for 1 h. After pre-treatment, 10 ng/ml TNFα were added for 2 h. HA-ubiquitin was immunoprecipitated using anti-HA-beads and detection of indicated proteins was performed by Western blotting. β-Actin served as loading control for the input, whereas HA-ubiquitin levels served as loading control for immunoprecipitated ubiquitin.


A HT ‑ 29 RIPK3 KO cells re - expressing Dox - inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg / ml Dox overnight . Cells were treated with 20 µM zVAD . fmk , 5 µM BV6 and 10 ng / ml TNFα for 4 h . Cell death was measured by analysis of PI - positive nuclei .

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A HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 4 h. Cell death was measured by analysis of PI-positive nuclei.


B HT ‑ 29 RIPK3 KO cells re - expressing Dox - inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg / ml Dox overnight , as indicated . Cells were treated with 20 µM zVAD . fmk , 5 µM BV6 and 10 ng / ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels . β - Actin served as a loading control .

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B HT‑29 RIPK3 KO cells re-expressing Dox-inducible WT RIPK3 or the indicated RIPK3 mutants were incubated with 1 µg/ml Dox overnight, as indicated. Cells were treated with 20 µM zVAD.fmk, 5 µM BV6 and 10 ng/ml TNFα for 2 h and analyzed by Western blotting for RIPK3 and phosphorylated MLKL expression levels. β-Actin served as a loading control.


A - D Proportional distribution of chymotrypsin - like activity of the proteasome across nine tissues from female ( n = 9 ) , and male ( n = 10 ) mice . Proportional distribution of trypsin - like activity of the proteasome across nine tissues from female ( n = 9 ) , and male ( n = 10 ) mice . Graphical representation of color code of tissues analyzed .

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A-D Proportional distribution of chymotrypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Proportional distribution of trypsin-like activity of the proteasome across nine tissues from female (n=9), and male (n=10) mice. Graphical representation of color code of tissues analyzed.


Chymotrypsin - like activity of the proteasome across nine tissues in individual mice . Each data point ( n = 9 females and n = 10 males ) indicates data from a single mouse performed in triplicate . Each central line and error bar indicate mean + / - SEM . * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples .

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Chymotrypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


trypsin - like activity of the proteasome across nine tissues in individual mice . Each data point ( n = 9 females and n = 10 males ) indicates data from a single mouse performed in triplicate . Each central line and error bar indicate mean + / - SEM . * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples .

trypsin-like activity of the proteasome across nine tissues in individual mice. Each data point (n=9 females and n=10 males)indicates data from a single mouse performed in triplicate. Each central line and error bar indicate mean +/- SEM . * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


A - D Western blot analysis for K48Ub - linked proteins in spinal cord ( A ) and intestine ( C ) lysates from male and female mice . ( B , D ) Quantification of panels A and C , respectively . Values were normalized to actin as a loading control . Each central line and error bar indicate mean + / - SEM of a biological replicates . Data information : * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples .

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A-D Western blot analysis for K48Ub-linked proteins in spinal cord (A) and intestine (C) lysates from male and female mice. (B,D) Quantification of panels A and C, respectively. Values were normalized to actin as a loading control. Each central line and error bar indicate mean +/- SEM of a biological replicates. Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


E ) Curve of correlation between chymotrypsin - like activity and Ub - K48 linked proteins in the intestine , where variability between mice is observed .

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E) Curve of correlation between chymotrypsin-like activity and Ub-K48 linked proteins in the intestine, where variability between mice is observed.


F ) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core ( CP ) or assembled with regulatory core ( RP ) in male ( n = 5 ) and females ( n = 5 ) mice in intestine .

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F) Native gel followed by western of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and females (n=5) mice in intestine.


G ) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core ( CP ) or assembled with regulatory core ( RP ) in male ( n = 5 ) and female ( n = 5 ) spinal cord .

G) Native gel followed by western blot of β5 subunit to distinguish unassembled catalytic core (CP) or assembled with regulatory core (RP) in male (n=5) and female (n=5) spinal cord.


H ) Quantification of the ratio of assembled ( RP - CP ) versus unassembled ( CP ) proteasome in the intestine of males and females . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 per group ) . I ) Quantification of the ratio of RP - CP versus CP proteasome in the spinal cord of males and females . Each central line and error bar indicate mean + / - SEM of a biological replicates ( n = 5 per group ) . Data information : * indicates p < 0 . 05 by unpaired two - tailed students t - test comparing male and female samples .

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H) Quantification of the ratio of assembled (RP-CP) versus unassembled (CP) proteasome in the intestine of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). I) Quantification of the ratio of RP-CP versus CP proteasome in the spinal cord of males and females. Each central line and error bar indicate mean +/- SEM of a biological replicates (n=5 per group). Data information: * indicates p<0.05 by unpaired two-tailed students t-test comparing male and female samples.


J ) Correlation curve between chymotrypsin - like activity and RP - CP / CP ratio in individual female ( purple ) and male ( blue ) intestine . K ) Correlation curve between chymotrypsin - like activity and RP - CP / CP ratio in individual female ( purple ) and male ( blue ) spinal cord .

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J) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) intestine. K) Correlation curve between chymotrypsin-like activity and RP-CP/CP ratio in individual female (purple) and male (blue) spinal cord.