F - H RNA - FISH identifies linc - Myh in nuclei ( DAPI , blue ) of C2C12 myoblasts . ( G ) Adipor ( cytoplasmic ) and ( H ) Xist ( nuclear ) were used as controls .

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F-H RNA-FISH identifies linc-Myh in nuclei (DAPI, blue) of C2C12 myoblasts. (G) Adipor (cytoplasmic) and (H) Xist (nuclear) were used as controls.


( J ) Heat map of proteins pulled down in the same experiments as in I , demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc - MYH . Blue color indicates pull - down of the respective protein , white color indicates that the protein was not detected in the respective sample .

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(J) Heat map of proteins pulled down in the same experiments as in I, demonstrating that INO80 chromatin remodeler complex exclusively interacts with linc-MYH. Blue color indicates pull-down of the respective protein, white color indicates that the protein was not detected in the respective sample.


RNA - IP experiments using an anti - INO80 antibody to isolate the INO80 complex from murine C2C12 cells ( K ) INO80 in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR .

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RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from murine C2C12 cells (K) INO80 in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.


RNA - IP experiments using an anti - INO80 antibody to isolate the INO80 complex from human HSMM cells ( L ) . INO80 II in L refer to replicate INO80 precipitation experiments . Precipitated RNAs were detected by RT - PCR .

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RNA-IP experiments using an anti-INO80 antibody to isolate the INO80 complex from human HSMM cells (L). INO80 II in L refer to replicate INO80 precipitation experiments. Precipitated RNAs were detected by RT-PCR.


Body weight ( A ) of male linc - MYH KO and ctrl mice at 10 weeks ( n = 24 KO / 17 WT ; Student ' s t test , two - tailed , * * p < 0 . 01 )

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Body weight (A) of male linc-MYH KO and ctrl mice at 10 weeks (n = 24 KO/17 WT; Student's t test, two-tailed, **p < 0.01)


tibia length ( B ) of male linc - MYH KO and ctrl mice of 10 weeks ( n = 9 KO / 13 WT ; Student ' s t test , two - tailed , not significant ) . Data are mean ± S . E . M .

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tibia length (B) of male linc-MYH KO and ctrl mice of 10 weeks (n = 9 KO/13 WT; Student's t test, two-tailed, not significant). Data are mean ± S.E.M.


C , D Weight of m . tibialis anterior ( TA ) and m . digitorum longus ( EDL ) of male linc - MYH KO and ctrl mice at 10 weeks ( n = 9 KO / 10 WT for TA , n = 10 KO / 11 WT for EDL ; Student ' s t test , two - tailed , * * p < 0 . 01 , * * * p < 0 . 001 ) . Data are mean ± S . E . M .

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C, D Weight of m. tibialis anterior (TA) and m. digitorum longus (EDL) of male linc-MYH KO and ctrl mice at 10 weeks (n = 9 KO/10 WT for TA, n = 10 KO/11 WT for EDL; Student's t test, two-tailed, **p < 0.01, ***p<0.001). Data are mean ± S.E.M.


Size distribution of fibers in cross sections of TA ( E , F ) and EDL ( G , H ) muscle in male linc - MYH KO ( blue ) and ctrl ( grey ) animals at 10 weeks . A significant increase of fiber size was observed for TA and EDL muscles of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 fibers per animal were counted ) . Data are mean ± S . E . M .

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Size distribution of fibers in cross sections of TA (E, F) and EDL (G, H) muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.


Size distribution of fibers in cross sections of TA and EDL muscle in male linc - MYH KO ( blue ) and ctrl ( grey ) animals at 10 weeks . A significant increase of fiber size was observed for TA and EDL muscles of linc - MYH KO mice ( n = 5 KO / 4 WT for TA , n = 3 KO / 3 WT for EDL muscle , two - way ANOVA with Fisher ' s multiple comparisons ; * p < 0 . 05 , * * p < 0 . 01 ; > 250 fibers per animal were counted ) . Data are mean ± S . E . M .

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Size distribution of fibers in cross sections of TA and EDL muscle in male linc-MYH KO (blue) and ctrl (grey) animals at 10 weeks. A significant increase of fiber size was observed for TA and EDL muscles of linc-MYH KO mice (n = 5 KO/4 WT for TA, n = 3 KO/3 WT for EDL muscle, two-way ANOVA with Fisher's multiple comparisons; *p < 0.05, **p < 0.01; > 250 fibers per animal were counted). Data are mean ± S.E.M.


K Numbers of myonuclei in cross sections of TA and EDL muscle ( TA : n = 6 KO / 6 WT , EDL n = 3 KO / 3 WT animals , Mann - Whitney test one - tailed , * * p < 0 . 01 , * p = 0 . 05 ) Data are mean ± S . E . M .

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K Numbers of myonuclei in cross sections of TA and EDL muscle (TA: n = 6 KO/6 WT, EDL n = 3 KO/3 WT animals, Mann-Whitney test one-tailed, **p<0.01, *p = 0.05) Data are mean ± S.E.M.


L - O Number of myonuclei / fiber and of Pax7pos ( red ) MuSCs ( red arrows ) on myofibers isolated from flexor digitorum brevis muscle . Nuclei were stained using DAPI ( blue ) . Scale bar in L indicates 50 µm for L and M . ( N ) The number of myonuclei / isolated fiber is increased in linc - MYH KO compared to WT myofibers ( n = 4 KO / 4 WT , > 26 fibers / animal ; Mann - Whitney test two - tailed , * p < 0 . 05 ) . ( O ) Number of MuSCs on isolated fibers from linc - MYH KO and WT mice ( n = 4 KO / 4 WT animals , Mann - Whitney test two - tailed ; * * * * p < 0 . 0001 ) . Data are mean ± S . E . M .

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L-O Number of myonuclei / fiber and of Pax7pos (red) MuSCs (red arrows) on myofibers isolated from flexor digitorum brevis muscle. Nuclei were stained using DAPI (blue). Scale bar in L indicates 50 µm for L and M. (N) The number of myonuclei / isolated fiber is increased in linc-MYH KO compared to WT myofibers (n = 4 KO/4 WT, > 26 fibers/animal; Mann-Whitney test two-tailed, * p < 0.05). (O) Number of MuSCs on isolated fibers from linc-MYH KO and WT mice (n = 4 KO/4 WT animals, Mann-Whitney test two-tailed; ****p < 0.0001). Data are mean ± S.E.M.


P - S Number of Pax7 ( red ) positive MuSCs in cross sections of tibialis anterior muscle ( P - R ) ; the relative amount of quiescent MuSCs identified by double staining ( yellow arrows ) for Pax7 and CalcR ( green ) does not significantly differ between WT and linc - MYH KO animals ( S ) . Nuclei were stained with DAPI ( blue ) . Scale bar in P indicates 25 µm for P and Q . ( n = 3 KO / 3 WT animals , Mann - Whitney test one - tailed ; * p = 0 . 05 , ns : not significant ) . All data are mean ± S . E . M .

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P-S Number of Pax7 (red) positive MuSCs in cross sections of tibialis anterior muscle (P-R); the relative amount of quiescent MuSCs identified by double staining (yellow arrows) for Pax7 and CalcR (green) does not significantly differ between WT and linc-MYH KO animals (S). Nuclei were stained with DAPI (blue). Scale bar in P indicates 25 µm for P and Q. (n = 3 KO/3 WT animals, Mann-Whitney test one-tailed; *p = 0.05, ns: not significant). All data are mean ± S.E.M.


T The number of MuSC - related events in FACS experiments is significantly increased in linc - MYH - KO animals ( n = 7 KO / 7 WT , Student ' s t test , two - tailed , * p = 0 . 0126 ) . Data are mean ± S . E . M .

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T The number of MuSC-related events in FACS experiments is significantly increased in linc-MYH-KO animals (n = 7 KO/7 WT, Student's t test, two-tailed, *p = 0.0126). Data are mean ± S.E.M.


U - V Structure of MuSCs and the heterochromatin content of MuSC nuclei is not changed in linc - MYH KO ( V ) compared to WT mice ( U ) .

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U-V Structure of MuSCs and the heterochromatin content of MuSC nuclei is not changed in linc-MYH KO (V) compared to WT mice (U).


RNA in situ hybridization proximity ligation assay ( rISH - PLA ) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al . . ( B - C ) rISH - PLA confirms the proximity of linc - MYH to endogenous INO80 - V5 in the nucleus of proliferating WT MuSCs ( B ) . The specific signal is lost in linc - MYH deficient MuSCs ( C ) . ( n = 3 WT / 3 KO biological replicates ; Mann - Whitney test , * p = 0 . 05 ) . Data are mean ± S . E . M .

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RNA in situ hybridization proximity ligation assay (rISH-PLA) detects the close proximity of a specific RNA with proteins in situ modified from Roussis et al.. (B-C) rISH-PLA confirms the proximity of linc-MYH to endogenous INO80-V5 in the nucleus of proliferating WT MuSCs (B). The specific signal is lost in linc-MYH deficient MuSCs (C). (n = 3 WT/3 KO biological replicates; Mann-Whitney test, *p = 0.05). Data are mean ± S.E.M.


E - I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 ( green ) and EdU incorporation ( red ) . MuSCs without ( green arrows ) and with EdU - labeling ( red arrows ) are indicated . ( E , F ) Deletion of linc - MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU - positive MuSCs . ( G - I ) The increase in both the number of MuSCs and EdU positive MuSCs in linc - MYH mutant muscle is abolished by constitutive ( G : Pax7 - Crepos / INO80 - / - , Ino80 KO ; H : linc - MYH - / - / Pax7 - Crepos / INO80 - / - , dKO ) and induced ( I : linc - MYH - / - / Pax7 - CreERT2pos / INO80 - / - , dKO CreERT ) deletion of INO80 in Pax7 expressing cells .

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E-I Representative images of TA muscle sections of 10 weeks old mice stained for PAX7 (green) and EdU incorporation (red). MuSCs without (green arrows) and with EdU-labeling (red arrows) are indicated. (E, F) Deletion of linc-MYH results in increased numbers of muscle stem cells and pronounced increase of the ratio of EdU-positive MuSCs. (G-I) The increase in both the number of MuSCs and EdU positive MuSCs in linc-MYH mutant muscle is abolished by constitutive (G: Pax7-Crepos / INO80-/-, Ino80 KO; H: linc-MYH-/- / Pax7-Crepos / INO80-/-, dKO) and induced (I: linc-MYH-/- / Pax7-CreERT2pos / INO80-/-, dKO CreERT) deletion of INO80 in Pax7 expressing cells.


Statistical evaluation of the number of muscle stem cells observed in TA muscles ( J ) . ; ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

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Statistical evaluation of the number of muscle stem cells observed in TA muscles (J). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


Statistical evaluation of the ratio of EdU - incorporating MuSCs relative to all Pax7 - positive MuSCs ( K ) . ; ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

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Statistical evaluation of the ratio of EdU-incorporating MuSCs relative to all Pax7-positive MuSCs (K). ; (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes ( L ) ( n = 5 WT / 5 linc - MYH KO / 4 Ino80 KO / 5 dKO / 3 CreERT dKO animals in J / K , n = 6 WT / 6 linc - MYH KO / 5 Ino80 KO / 6 dKO / 3 CreERT dKO animals in L * * p < 0 . 01 , * * * * p < 0 . 0001 , > 31 MSCs / animal in K ; ANOVA test with multiple comparisons against WT . Sidak - Holm correction was used . Data are mean ± S . E . M . ) .

Statistical evaluation of the number of myonuclei in myofibers of TA muscles from different genotypes (L) (n = 5 WT/5 linc-MYH KO/4 Ino80 KO/5 dKO/3 CreERT dKO animals in J/K, n = 6 WT/6 linc-MYH KO/5 Ino80 KO/6 dKO/3 CreERT dKO animals in L **p < 0.01, ****p <0.0001, > 31 MSCs /animal in K; ANOVA test with multiple comparisons against WT. Sidak-Holm correction was used. Data are mean ± S.E.M.).


A - D Isolated linc - MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates . Deletion of INO80 eliminates the difference between Pax7 - Crepos / INO80 - / - ( Ino80 KO ) and linc - MYH - / - / linc - MYH - / - / Pax7 - Crepos / INO80 - / - ( dKO ) . Images are representative images for cultures after 13 days in culture .

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A-D Isolated linc-MYH KO MuSCs reach confluence much faster than WT MuSCs indicating higher proliferation rates. Deletion of INO80 eliminates the difference between Pax7-Crepos / INO80-/- (Ino80 KO) and linc-MYH-/- / linc-MYH-/-/ Pax7-Crepos / INO80-/- (dKO). Images are representative images for cultures after 13 days in culture.


E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs . Cell confluence was monitored by a live cell imaging analysis system for 13 days ( n = 7 WT / 6 linc - MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs ) .

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E Differences in proliferation rates are abolished by additional deletion of INO80 in MuSCs. Cell confluence was monitored by a live cell imaging analysis system for 13 days (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs).


F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation ( n = 7 WT / 6 linc - MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs , Student ' s t test , two - tailed ; * p < 0 . 05 , ns = not significant ) . Data are mean ± S . E . M .

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F Statistical analysis of cell confluence after 13 days of muscle stem cell proliferation (n = 7 WT / 6 linc-MYH KO / 3 Ino80 KO / 3 dKO independent preparations of MuSCs, Student's t test, two-tailed; *p < 0.05, ns = not significant). Data are mean ± S.E.M.


G - I Gene set enrichment analysis ( GSEA , broadinstitute . org ) of microarray data from proliferating MuSCs . Comparison of linc - MYH - / - vs . WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation ( G ) , enrichment of genes containing cis regulatory elements for YY1 ( H ) and p53 ( I ) . Enrichments are highly significant using the most conservative familywise - error rate method ( FWER ) . Enrichments were lost in linc - MYH - / - MuSCs after additional deletion of INO80 ( dKOs ) compared to Pax7 - Crepos / INO80 - / - MuSCs .

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G-I Gene set enrichment analysis (GSEA, broadinstitute.org) of microarray data from proliferating MuSCs. Comparison of linc-MYH-/- vs. WT proliferating MuSC transcriptomes shows enrichment of genes related to cell proliferation (G), enrichment of genes containing cis regulatory elements for YY1 (H) and p53 (I). Enrichments are highly significant using the most conservative familywise-error rate method (FWER). Enrichments were lost in linc-MYH-/- MuSCs after additional deletion of INO80 (dKOs) compared to Pax7-Crepos / INO80-/- MuSCs.


Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G - I in linc - MYH - / - vs . WT MuSCs and in linc - MYH - / - / Pax7 - Crepos / INO80 - / - dKO vs . Pax7 - Crepos / INO80 - / - MuSCs . Expression of up - regulated genes in linc - MYH - / - was normalized after additional deletion of INO80 ( dKOs ) , indicating that effects of linc - MYH completely depend on INO80 ( WT n = 4 , linc - MYH - / - n = 4 , Pax7 - Crepos / INO80 - / - n = 4 , Pax7 - Cre dKO n = 3 animals used for independent isolations of MuSCs ) .

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

Heat maps of the 10 most upregulated genes of the respective gene sets depicted in G-I in linc-MYH-/- vs. WT MuSCs and in linc-MYH-/-/ Pax7-Crepos / INO80-/- dKO vs. Pax7-Crepos / INO80-/- MuSCs. Expression of up-regulated genes in linc-MYH-/- was normalized after additional deletion of INO80 (dKOs), indicating that effects of linc-MYH completely depend on INO80 (WT n = 4, linc-MYH-/- n = 4, Pax7-Crepos / INO80-/- n = 4, Pax7-Cre dKO n = 3 animals used for independent isolations of MuSCs).


A - D γ - H2AX staining of proliferating MuSCs from WT ( A ) , linc - MYH deficient ( B ) , Pax7 - Crepos / INO80 KO ( C ) , and linc - MYH / INO80 dKO ( D ) mice . Deletion of INO80 results in increased γ - H2AX signals while deletion of linc - MYH has no effects ( asterisk : low γ - H2AX signals , arrow : γ - H2AX - foci detected in nuclei , arrowhead : pan - nuclear γ - H2AX signal .

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

A-D γ-H2AX staining of proliferating MuSCs from WT (A), linc-MYH deficient (B), Pax7-Crepos / INO80 KO (C), and linc-MYH/INO80 dKO (D) mice. Deletion of INO80 results in increased γ-H2AX signals while deletion of linc-MYH has no effects (asterisk: low γ-H2AX signals, arrow: γ-H2AX-foci detected in nuclei, arrowhead: pan-nuclear γ-H2AX signal.


E - G Statistical evaluation of the presence of γ - H2AX signals in MuSCs of different genotypes ( n = 3 / 3 / 3 / 3 animals used for independent isolations of MuSCs , two technical replicates for each animal and at least 164 cells were counted , Mann - Whitney test , * * p < 0 . 01 . Data are mean ± s . e . m . ) .

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

E-G Statistical evaluation of the presence of γ-H2AX signals in MuSCs of different genotypes (n = 3/3/3/3 animals used for independent isolations of MuSCs, two technical replicates for each animal and at least 164 cells were counted, Mann-Whitney test, **p<0.01. Data are mean ± s.e.m.).


H - I Gene set enrichment analysis of microarray data obtained for proliferating MuSCs shows enrichment of genes related to activity of ATR in response to replication stress recovery ( H ) and enrichment of genes involved in chromosome maintenance ( I ) in WT compared to Pax7 - Crepos INO80 - / - MuSCs . Enrichment of these or similar gene sets was not observed when comparing WT to linc - MYH - / - MuSCs .

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

H-I Gene set enrichment analysis of microarray data obtained for proliferating MuSCs shows enrichment of genes related to activity of ATR in response to replication stress recovery (H) and enrichment of genes involved in chromosome maintenance (I) in WT compared to Pax7-Crepos INO80-/- MuSCs. Enrichment of these or similar gene sets was not observed when comparing WT to linc-MYH-/- MuSCs.


A , B Co - Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc - MYH KO mice followed by Western Blot analysis . ( B ) Statistical analysis of Co - IP / Western Blot data . The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc - MYH , while the amounts of YY1 , WDR5 and TFPT pulled down by INO80 increase ( n = 3 WT / 3 linc - MYH KO animals ; one - way ANOVA , multiple comparisons by Fishers LSD test , * p < 0 . 05 ) . Data are mean ± S . E . M .

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

A, B Co-Immunoprecipitation of INO80 complex subunits from TA muscles of WT and linc-MYH KO mice followed by Western Blot analysis. (B) Statistical analysis of Co-IP/Western Blot data. The amount of RUVBL1 and RUVBL2 in the INO80 complex is not changed after deletion of linc-MYH, while the amounts of YY1, WDR5 and TFPT pulled down by INO80 increase (n = 3 WT / 3 linc-MYH KO animals; one-way ANOVA, multiple comparisons by Fishers LSD test, *p<0.05). Data are mean ± S.E.M.


C - K Proximity ligation assays between INO80 and YY1 ( C , D ) , INO80 and WDR5 ( F , G ) , and INO80 and RuvBl2 ( I , J ) in proliferating MuSCs . Red signals indicate close proximity of INO80 - V5 and the respective interacting protein . Nuclei were stained using DAPI ( blue ) . Statistical evaluation of proximity ligation assays indicating increased interactions of INO80 - V5 and YY1 ( E , n = 3 / 3 animals , 3 independent wells per animal , two - tailed students t - test , * p < 0 . 05 ) and of INO80 - V5 to WDR5 ( H , n = 2 ( WT ) / 2 ( KO ) animals , 3 independent wells per animal , two - tailed students t - test , * * p < 0 . 01 ) . No increase in proximity was detected between Ino80 - V5 and Ruvbl2 ( K , n = 2 ( WT ) / 2 ( KO ) animals , 3 independent wells per animal , two - tailed students t - test , ns : not significant ) . Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays . No signals were detected for these controls . Data are mean ± S . E . M .

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

C-K Proximity ligation assays between INO80 and YY1 (C, D), INO80 and WDR5 (F, G), and INO80 and RuvBl2 (I, J) in proliferating MuSCs. Red signals indicate close proximity of INO80-V5 and the respective interacting protein. Nuclei were stained using DAPI (blue). Statistical evaluation of proximity ligation assays indicating increased interactions of INO80-V5 and YY1 (E, n = 3/3 animals, 3 independent wells per animal, two-tailed students t-test, *p<0.05) and of INO80-V5 to WDR5 (H, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, **p<0.01). No increase in proximity was detected between Ino80-V5 and Ruvbl2 (K, n = 2 (WT)/2 (KO) animals, 3 independent wells per animal, two-tailed students t-test, ns: not significant). Specificity of all analyzed signals was analyzed using single primary or single secondary antibodies in PLA assays. No signals were detected for these controls. Data are mean ± S.E.M.


Assessment of mtDNA damage by quantitative PCR . HEK293 cells infected with control ( empty vector ) or shATG5 for 5 days . Cells were then incubated with DMSO or 4 mM 3 - NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h . Cells with or without washout were used for the extraction of total DNA . All DNA samples were used for amplification of 8 . 9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment . PCR products were quantitated by PicoGreen staining using Micro Plate Reader . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . The data in ( A ) were further calculated for the frequency of mtDNA damage . The equation was seen in " Materials and Methods " . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

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

Assessment of mtDNA damage by quantitative PCR. HEK293 cells infected with control (empty vector) or shATG5 for 5 days. Cells were then incubated with DMSO or 4 mM 3-NPA for 2 h and either immediately harvested or washed with fresh medium and incubated for another 1 h. Cells with or without washout were used for the extraction of total DNA. All DNA samples were used for amplification of 8.9 kb mtDNA fragment using quantitative PCR and were normalized to amplification of a 221 bp mtDNA fragment. PCR products were quantitated by PicoGreen staining using Micro Plate Reader. Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. The data in (A) were further calculated for the frequency of mtDNA damage. The equation was seen in "Materials and Methods". Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, **p < 0.01.


Representative images show 8 - oxo - dG staining . Control or shATG5 HeLa cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , and then either fixed immediately or washed with fresh medium and incubated for another 1 h . Cells were immunostained with DAPI and anti - 8 - oxo - dG antibody and analyzed by confocal microscopy . Scale bar , 10 µm . Quantification of the relative 8 - oxo - dG fluorescence intensity in ( C ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by a two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 .

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

Representative images show 8-oxo-dG staining. Control or shATG5 HeLa cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and then either fixed immediately or washed with fresh medium and incubated for another 1 h. Cells were immunostained with DAPI and anti-8-oxo-dG antibody and analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in (C). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01.


Control or shATG5 HEK293 cells stably expressing mito - Keima . Control cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h , and shATG5 cells were treated with 200 µM H2O2 as a negative control . Cells were then imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Right panels show the pixel intensity of red ( mitochondria within lysosomes ) and green ( mitochondria in the cytoplasm ) from a line . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561 nm / 458 nm ) of the cells described in ( E ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by a two - way ANOVA , N . S . , not significant , * * p < 0 . 01 .

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

Control or shATG5 HEK293 cells stably expressing mito-Keima. Control cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h, and shATG5 cells were treated with 200 µM H2O2 as a negative control. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Right panels show the pixel intensity of red (mitochondria within lysosomes) and green (mitochondria in the cytoplasm) from a line. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (E). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by a two-way ANOVA, N.S., not significant, **p < 0.01.


HeLa cells expressing GFP - LC3 were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h . Cells were stained with anti - Tom20 and anti - DNA antibodies , then analyzed by confocal microscopy . The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA / Tom20 . Scale bar , 10 µm . Quantification of the LC3 punta colocalizing or contacting with mtDNA / Tom20 in a cell . n = 30 cells from 3 coverslips , Data are presented as mean ± SD ( n = 30 ) , statistical significance was assessed by a one - way ANOVA with Tukey ' s multiple comparisons test , * * * p < 0 . 001 .

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

HeLa cells expressing GFP-LC3 were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were stained with anti-Tom20 and anti-DNA antibodies, then analyzed by confocal microscopy. The white arrows were indicated the LC3 punta colocalizing or contacting with mtDNA/Tom20. Scale bar, 10 µm. Quantification of the LC3 punta colocalizing or contacting with mtDNA/Tom20 in a cell. n = 30 cells from 3 coverslips, Data are presented as mean ± SD (n = 30), statistical significance was assessed by a one-way ANOVA with Tukey's multiple comparisons test, ***p < 0.001.


Screening of mitophagy regulators for removing damaged mtDNA . HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors . Five days later , cells were treated with 200 µM H2O2 for 2 h . Cells were then imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima . ShATG5 was used as a negative control . Right panels for each image show the FACS - based mito - Keima dot plots . The y - axis represents the fluorescence emission of mito - Keima at pH 4 . 0 ( lysosome ) , while the x - axis indicates mito - Keima at pH 7 . 0 ( mitochondria ) . The percentages of cells within the different regions are indicated . Scale bar , 10 µm .

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

Screening of mitophagy regulators for removing damaged mtDNA. HeLa cells were infected by lentiviral particles containing the indicated knockdown vectors. Five days later, cells were treated with 200 µM H2O2 for 2 h. Cells were then imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima. ShATG5 was used as a negative control. Right panels for each image show the FACS-based mito-Keima dot plots. The y-axis represents the fluorescence emission of mito-Keima at pH 4.0 (lysosome), while the x-axis indicates mito-Keima at pH 7.0 (mitochondria). The percentages of cells within the different regions are indicated. Scale bar, 10 µm.


Control or ATAD3B KO HeLa cells stably expressing mito - Keima were treated with 4 mM 3 - NPA for 2 h and imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561 nm / 458 nm ) of the cells described in ( B ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

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

Control or ATAD3B KO HeLa cells stably expressing mito-Keima were treated with 4 mM 3-NPA for 2 h and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561 nm/458 nm) of the cells described in (B). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.


Control or KO ATAD3B HeLa cells were treated with 4 mM 3 - NPA for 2 h . Cells were washed with fresh medium and incubated for another 1 h . Cells were then fixed and immunostained with DAPI and anti - 8 - oxo - dG antibodies and were analyzed by confocal microscopy . Scale bar , 10 µm . Quantification of the relative 8 - oxo - dG fluorescence intensity in cells described in ( D ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

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

Control or KO ATAD3B HeLa cells were treated with 4 mM 3-NPA for 2 h. Cells were washed with fresh medium and incubated for another 1 h. Cells were then fixed and immunostained with DAPI and anti-8-oxo-dG antibodies and were analyzed by confocal microscopy. Scale bar, 10 µm. Quantification of the relative 8-oxo-dG fluorescence intensity in cells described in (D). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test , N.S., not significant, **p < 0.01.


PINK1 KO HeLa cells stably expressing mito - Keima were infected with control or shATAD3B . Five days later , cells were treated with DMSO , 200 µM H2O2 , or 4 mM 3 - NPA for 2 h . Cells were then analyzed and imaged with 458 nm ( measuring mitochondria with a neutral pH ) and 561 nm ( measuring mitochondria with an acidic pH ) laser excitation for mito - Keima by confocal microscopy . Scale bar , 10 µm . Quantification of the relative ratio of red to green fluorescence intensity ( 561nm / 458nm ) of the cells described in ( A ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , N . S . , not significant , * * p < 0 . 01 .

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

PINK1 KO HeLa cells stably expressing mito-Keima were infected with control or shATAD3B. Five days later, cells were treated with DMSO, 200 µM H2O2, or 4 mM 3-NPA for 2 h. Cells were then analyzed and imaged with 458 nm (measuring mitochondria with a neutral pH) and 561 nm (measuring mitochondria with an acidic pH) laser excitation for mito-Keima by confocal microscopy. Scale bar, 10 µm. Quantification of the relative ratio of red to green fluorescence intensity (561nm/458nm) of the cells described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, N.S., not significant, **p < 0.01.


HeLa cells were treated with DMSO , 4 mM 3 - NPA , 8 mM 3 - NPA , or 10 µM CCCP for 2 h , and cell lysates were then analyzed by Western blotting using the indicated antibodies .

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

HeLa cells were treated with DMSO, 4 mM 3-NPA, 8 mM 3-NPA, or 10 µM CCCP for 2 h, and cell lysates were then analyzed by Western blotting using the indicated antibodies.


PINK1 KO HeLa cells stably expressing ATAD3A - Flag ( 3A - Flag ) or ATAD3B - Flag ( 3B - Flag ) were mixed with control PINK1 KO cells respectively , and incubated for 24 h . Cells were treated with 4 mM 3 - NPA ( D ) or 200 µM H2O2 ( F ) for 2 h , and then washed with fresh medium and incubated for another 1 h . Cells were fixed and immunostained with anti - 8 - oxo - dG and anti - Flag antibodies and analyzed by confocal microscopy . 8 - oxo - dG fluorescence intensity of cells treated with 3 - NPA ( E ) or H2O2 ( G ) was quantified by ImageJ software . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by individual two - tailed student ' s t - test , N . S . , not significant , * p < 0 . 05 , * * p < 0 . 01 . Scale bar , 10 µm .

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

PINK1 KO HeLa cells stably expressing ATAD3A-Flag (3A-Flag) or ATAD3B-Flag (3B-Flag) were mixed with control PINK1 KO cells respectively, and incubated for 24 h. Cells were treated with 4 mM 3-NPA (D) or 200 µM H2O2 (F) for 2 h, and then washed with fresh medium and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and anti-Flag antibodies and analyzed by confocal microscopy. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (E) or H2O2 (G) was quantified by ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by individual two-tailed student's t-test, N.S., not significant, *p < 0.05, **p < 0.01. Scale bar, 10 µm.


Control ( GFP + ) and shATAD3B ( GFP - ) PINK1 KO HeLa cells were mixed and incubated for 24 h . Cells were then treated with 4 mM 3 - NPA ( H ) or 200 µM H2O2 ( J ) for 2 h , and washed with fresh medium , and incubated for another 1 h . Cells were fixed and immunostained with anti - 8 - oxo - dG and DAPI , then analyzed by confocal microscopy . Control or shATAD3B cells are circled by white dashed lines . 8 - oxo - dG fluorescence intensity of cells treated with 3 - NPA ( I ) or H2O2 ( K ) was quantified using ImageJ software . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by two - tailed student ' s t - test , * * p < 0 . 01 . Scale bar , 10 µm .

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

Control (GFP+) and shATAD3B (GFP-) PINK1 KO HeLa cells were mixed and incubated for 24 h. Cells were then treated with 4 mM 3-NPA (H) or 200 µM H2O2 (J) for 2 h, and washed with fresh medium, and incubated for another 1 h. Cells were fixed and immunostained with anti-8-oxo-dG and DAPI, then analyzed by confocal microscopy. Control or shATAD3B cells are circled by white dashed lines. 8-oxo-dG fluorescence intensity of cells treated with 3-NPA (I) or H2O2 (K) was quantified using ImageJ software. Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by two-tailed student's t-test, **p < 0.01. Scale bar, 10 µm.


293T cells were treated with H2O2 for 2 h , and cell lysates were then immunoprecipitated with Dynabeads Protein G pre - coupled with rabbit IgG ( control ) or anti - ATAD3 antibody , followed by Western blotting with anti - LC3B or anti - ATAD3 antibodies .

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293T cells were treated with H2O2 for 2 h, and cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with rabbit IgG (control) or anti-ATAD3 antibody, followed by Western blotting with anti-LC3B or anti-ATAD3 antibodies.


ATAD3 DKO HeLa cells expressing GFP - LC3B were transiently transfected with control , ATAD3A - Flag or ATAD3B - Flag . 48 h after transfection , cell lysates were immunoprecipitated ( IP ) with anti - Flag M2 affinity gel , and analyzed by Western blotting using anti - Flag or anti - GFP antibodies .

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

ATAD3 DKO HeLa cells expressing GFP-LC3B were transiently transfected with control, ATAD3A-Flag or ATAD3B-Flag. 48 h after transfection, cell lysates were immunoprecipitated (IP) with anti-Flag M2 affinity gel, and analyzed by Western blotting using anti-Flag or anti-GFP antibodies.


HeLa cells were treated with 200 µM H2O2 for 2 h , and cell lysates were incubated with the indicated GST - ATAD3A or GST - ATAD3B protein fragments ( expressed in E . coli ) coupled to glutathione agarose beads ( Pierce ) for GST pull - down assay . Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B .

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

HeLa cells were treated with 200 µM H2O2 for 2 h, and cell lysates were incubated with the indicated GST-ATAD3A or GST-ATAD3B protein fragments (expressed in E.coli) coupled to glutathione agarose beads (Pierce) for GST pull-down assay. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.


The indicated GST - ATAD3A or GST - ATAD3B protein fragments and His - LC3B were expressed in E . coli and purified . GST pull - down assay was performed using glutathione agarose beads . Eluted protein samples were analyzed by Western blotting using anti - GST or anti - His antibodies .

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

The indicated GST-ATAD3A or GST-ATAD3B protein fragments and His-LC3B were expressed in E.coli and purified. GST pull-down assay was performed using glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using anti-GST or anti-His antibodies.


The indicated GST - ATAD3B ( WT ) or GST - ATAD3B ( LIR mutations ) and His - LC3 were expressed in E . coli and purified . GST pull - down assay was performed using the glutathione agarose beads . Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B .

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

The indicated GST-ATAD3B(WT) or GST-ATAD3B (LIR mutations) and His-LC3 were expressed in E.coli and purified. GST pull-down assay was performed using the glutathione agarose beads. Eluted protein samples were analyzed by Western blotting using antibodies against GST or LC3B.


HeLa expressing GFP - LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B . Five days later , cells were treated with DMSO , or 200 µM H2O2 for 2 h . Cells were fixed and immunostained with anti - Tom20 and imaged by confocal microscopy . The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 ( mitochondria ) . Scale bar , 10 µm .

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HeLa expressing GFP-LC3 monoclonal cell line was infected with lentivirus particles containing control or shATAD3B. Five days later, cells were treated with DMSO, or 200 µM H2O2 for 2 h. Cells were fixed and immunostained with anti-Tom20 and imaged by confocal microscopy. The white arrows indicate the LC3 puncta colocalizing or contacting with Tom20 (mitochondria). Scale bar, 10 µm.


Quantification of the GFP - LC3 puncta colocalized or contacted with Tom20 ( mitochondria ) in cells described in ( G ) . Data are presented as mean ± SD ( n = 30 cells from 3 coverslips ) , statistical significance was assessed by a two - way ANOVA , N . S . , not significant , * * * p < 0 . 001 .

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Quantification of the GFP-LC3 puncta colocalized or contacted with Tom20 (mitochondria) in cells described in (G). Data are presented as mean ± SD (n =30 cells from 3 coverslips), statistical significance was assessed by a two-way ANOVA, N.S., not significant, ***p < 0.001.


293T cells transiently transfected with control or ATAD3B - Flag were treated with or without 200 µM H2O2 for 2 h . Cell lysates were then immunoprecipitated with Dynabeads Protein G pre - coupled with anti - Flag antibody , followed by Western blotting with anti - Flag or anti - LC3B antibodies . Relative protein levels of LC3B and ATAD3B - Flag were further evaluated by

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

293T cells transiently transfected with control or ATAD3B-Flag were treated with or without 200 µM H2O2 for 2 h. Cell lysates were then immunoprecipitated with Dynabeads Protein G pre-coupled with anti-Flag antibody, followed by Western blotting with anti-Flag or anti-LC3B antibodies. Relative protein levels of LC3B and ATAD3B-Flag were further evaluated by densitometry


Mic10 KO COS7 cells stably expressing ATAD3A - Flag or ATAD3B - Flag were treated with DMSO or 200 µM H2O2 for 2 h , and then fixed and immunostained with anti - Tom20 and anti - Flag antibodies , and analyzed by confocal microscopy . Scale bar , 10 µm .

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Mic10 KO COS7 cells stably expressing ATAD3A-Flag or ATAD3B-Flag were treated with DMSO or 200 µM H2O2 for 2 h, and then fixed and immunostained with anti-Tom20 and anti-Flag antibodies, and analyzed by confocal microscopy. Scale bar, 10 µm.


293T cells treated with DMSO ( B ) or H2O2 ( 200 µM , 2 h ) ( D ) , were harvested for mitochondrial isolation . Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice , and then were analyzed by Western blotting with anti - ATAD3 , anti - Tom20 ( OMM ) , anti - Tim23 ( IMS ) , and anti - HSP60 ( matrix ) . Relative protein levels of proteins in ( B ) or ( D ) were further evaluated by densitometry analysis using ImageJ software . Relative trends of proteolysis of indicated mitochondrial proteins from DMSO - or H2O2 - treated cells were shown ( C and E ) . Data are presented as mean ± SD ( n = 3 independent experiments ) .

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

293T cells treated with DMSO (B) or H2O2 (200 µM, 2 h) (D), were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice, and then were analyzed by Western blotting with anti-ATAD3, anti-Tom20 (OMM), anti-Tim23 (IMS), and anti-HSP60 (matrix). Relative protein levels of proteins in (B) or (D) were further evaluated by densitometry analysis using ImageJ software. Relative trends of proteolysis of indicated mitochondrial proteins from DMSO- or H2O2-treated cells were shown (C and E). Data are presented as mean ± SD (n = 3 independent experiments).


Control , ATAD3A KO , ATAD3B KO , or ATAD3 DKO HeLa cells were harvested for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min , and then were analyzed by Western blotting with the indicated antibodies .

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Control, ATAD3A KO, ATAD3B KO, or ATAD3 DKO HeLa cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min, and then were analyzed by Western blotting with the indicated antibodies.


293T cells were treated with DMSO , 200 µM H2O2 , 10 mM NAC , or 200 µM H2O2 plus 10 mM NAC for 2 h . Cells were then harvested and used for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min at 37 ℃ , and then were analyzed by Western blotting with the indicated antibodies . Quantification of the relative protein level of ATAD3 oligomers to HSP60 described in ( F ) . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 .

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

293T cells were treated with DMSO, 200 µM H2O2, 10 mM NAC, or 200 µM H2O2 plus 10 mM NAC for 2 h. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies. Quantification of the relative protein level of ATAD3 oligomers to HSP60 described in (F). Data are presented as mean ± SD (n = 3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.


143B , 143B ρ0 cells were harvested for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min at 37 ℃ , and then were analyzed by Western blotting with the indicated antibodies .

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

143B, 143B ρ0 cells were harvested for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37 ℃, and then were analyzed by Western blotting with the indicated antibodies.


293T cells were treated with DMSO or ddC ( 50 µM ) plus EB ( 0 . 5 µg / ml ) for 10 days . Cells were then harvested and used for mitochondrial isolation . Purified mitochondria were treated with DMSO or EDC ( 20 mM ) for 30 min at 37 ℃ , and then were analyzed by Western blotting with the indicated antibodies .

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

293T cells were treated with DMSO or ddC (50 µM) plus EB (0.5 µg/ml) for 10 days. Cells were then harvested and used for mitochondrial isolation. Purified mitochondria were treated with DMSO or EDC (20 mM) for 30 min at 37℃, and then were analyzed by Western blotting with the indicated antibodies.


ATAD3A KO HeLa cells were harvested for mitochondrial isolation . Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice , and then were analyzed by Western blotting with anti - ATAD3 , anti - Tom20 ( OMM ) , anti - Tim23 ( IMS ) , and anti - HSP60 ( matrix ) ( K ) . Relative protein levels of proteins in ( K ) were further evaluated by densitometry analysis using ImageJ software . Relative trends of proteolysis of indicated mitochondrial proteins from ATAD3A KO HeLa cells were shown ( L ) . Data are presented as mean ± SD ( n = 3 independent experiments ) .

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

ATAD3A KO HeLa cells were harvested for mitochondrial isolation. Purified mitochondria were treated with the indicated gradient concentration of proteinase K for 20 min on ice, and then were analyzed by Western blotting with anti-ATAD3, anti-Tom20 (OMM), anti-Tim23 (IMS), and anti-HSP60 (matrix) (K). Relative protein levels of proteins in (K) were further evaluated by densitometry analysis using ImageJ software. Relative trends of proteolysis of indicated mitochondrial proteins from ATAD3A KO HeLa cells were shown (L). Data are presented as mean ± SD (n = 3 independent experiments).


ρ0206 _ A and ρ0206 _ B cells were stained with mitoSOX and analyzed by confocal microscopy . DIC , Differential Interference Contrast . Scale bar , 25 µm . Quantification of the MitoSOX fluorescence intensity described in ( A ) . Data are presented as mean ± SD ( n = 3 independent experiments , 20 cells per experiment ) , statistical significance was assessed by student ' s t - test , * * p < 0 . 01 .

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

ρ0206_A and ρ0206_B cells were stained with mitoSOX and analyzed by confocal microscopy. DIC, Differential Interference Contrast. Scale bar, 25 µm. Quantification of the MitoSOX fluorescence intensity described in (A). Data are presented as mean ± SD (n = 3 independent experiments, 20 cells per experiment), statistical significance was assessed by student's t-test, **p < 0.01.


Mitochondria carrying the 3243A > G mutation in the tRNA Leu ( UUR ) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206 _ A ( 100 % wild - type mtDNA ) and ρ0206 _ B ( ~ 90 % m . 3243A > G mtDNA ) cell lines . ρ0206 _ A and ρ0206 _ B cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - Tubulin antibodies . Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software . Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by student ' s t - test , * * * p < 0 . 001 .

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

Mitochondria carrying the 3243A>G mutation in the tRNA Leu(UUR) gene from the same MELAS myoblasts were transferred into ρ0 206 cell line to generate ρ0206_A (100% wild-type mtDNA) and ρ0206_B (~90% m.3243A>G mtDNA) cell lines. ρ0206_A and ρ0206_B cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.


Normal or MELAS patient fibroblasts cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - Tubulin antibodies . Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software . Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by student ' s t - test , * * * p < 0 . 001 .

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

Normal or MELAS patient fibroblasts cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-Tubulin antibodies. Relative protein levels of proteins were further evaluated by densitometry analysis using ImageJ software. Quantification of the relative protein level of ATAD3B to Tubulin was shown at right panels. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by student's t-test, ***p < 0.001.


ρ0206 _ A , ρ0206 _ B , and ρ0206 - B cells pretreated with ddC ( 50µM ) plus EB ( 0 . 5µg / ml ) for 10 days , and then harvested and lysed . Cell lysates were analyzed by Western blotting with anti - ATAD3 or anti - GAPDH antibodies .

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ρ0206_A, ρ0206_B, and ρ0206-B cells pretreated with ddC (50µM) plus EB (0.5µg/ml) for 10 days, and then harvested and lysed. Cell lysates were analyzed by Western blotting with anti-ATAD3 or anti-GAPDH antibodies.


ρ0206 _ B cells were infected with control , ATAD3B - Flag or ATAD3B ( mLIR ) - Flag . After two weeks , total DNA from cells was isolated and used to calculate the mutation rate of 3243A > G by quantitative real - time PCR using the TaqMan Probe . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 .

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ρ0206_B cells were infected with control, ATAD3B-Flag or ATAD3B (mLIR)-Flag. After two weeks, total DNA from cells was isolated and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.


ρ0206 _ B cells were infected with control , ATAD3A - Flag , ATAD3B - Flag , or ATAD3B ( mLIR ) - Flag . After two weeks , the cellular ATP levels were measured . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * p < 0 . 05 .

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ρ0206_B cells were infected with control, ATAD3A-Flag, ATAD3B-Flag, or ATAD3B (mLIR)-Flag. After two weeks, the cellular ATP levels were measured. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, *p < 0.05.


MELAS patient fibroblasts were infected with control , ATAD3A - Flag , ATAD3B - Flag or ATAD3B ( mLIR ) - Flag . After two weeks , total DNA was isolated from fibroblasts and used to calculate the mutation rate of 3243A > G by quantitative real - time PCR using the TaqMan Probe . Data are presented as mean ± SD ( n = 3 independent experiments ) , statistical significance was assessed by a one - way ANOVA , N . S . , not significant , * * p < 0 . 01 .

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

MELAS patient fibroblasts were infected with control, ATAD3A-Flag, ATAD3B-Flag or ATAD3B(mLIR)-Flag. After two weeks, total DNA was isolated from fibroblasts and used to calculate the mutation rate of 3243A>G by quantitative real-time PCR using the TaqMan Probe. Data are presented as mean ± SD (n =3 independent experiments), statistical significance was assessed by a one-way ANOVA, N.S., not significant, **p < 0.01.


Comparison of bacterial colony forming unit ( CFU ) in feces from vehicle - and ABX - treated mice ( n = 5 mice / group ) . n . d . , non - detectable .

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

Comparison of bacterial colony forming unit (CFU) in feces from vehicle- and ABX-treated mice (n = 5 mice/group). n.d., non-detectable.


Representative image and measurements of lengths for CD31 + / LYVE - 1 + lacteal , CD31 + / LYVE - 1 - capillary plexus and DAPI + villus .

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Representative image and measurements of lengths for CD31+/LYVE-1+ lacteal, CD31+/LYVE-1- capillary plexus and DAPI+ villus.


Images and comparisons of villi lengths and absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of vehicle - or ABX - treated mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 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

Images and comparisons of villi lengths and absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of vehicle- or ABX-treated mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group).


Images and comparisons of the number of Prox1 + LECs within initial 100 μm portion ( F ) presented as relative fluorescence intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit .

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

Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (F) presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.


Images and comparisons of the VEGFR3 expression ( G ) , presented as relative fluorescence intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit .

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

Images and comparisons of the VEGFR3 expression (G), presented as relative fluorescence intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit.


Images and comparison of VE - cadherin LEC junctions in lacteals of jejunum between vehicle - and ABX - treated mice . Each boxed portion is highly magnified for visualization of detailed distributions of VE - cadherin to distinguish button - like ( red arrowheads ) , zipper - like ( open arrowheads ) and mixed LEC junctions in lacteals ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 50 μm .

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

Images and comparison of VE-cadherin LEC junctions in lacteals of jejunum between vehicle- and ABX-treated mice. Each boxed portion is highly magnified for visualization of detailed distributions of VE-cadherin to distinguish button-like (red arrowheads), zipper-like (open arrowheads) and mixed LEC junctions in lacteals (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 50 μm.


Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals . Asterisks indicate the site in lamina propria where fluorescence intensity ( FI ) was measured . Relative FI divided by mean FI of controls at 16 min ( left ) and normalized FI divided by FI at 16 min in each villus ( right ) after initial loading of BODIPY - FA were quantified . For normalization of FI , villi that showed peak intensity earlier or later than 16 min were excluded ( n = 5 - 7 mice / group , 5 - 10 villi / mouse ) . Scale bars , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative intravital imaging and quantification of lipid clearance from lamina propria via lacteals. Asterisks indicate the site in lamina propria where fluorescence intensity (FI) was measured. Relative FI divided by mean FI of controls at 16 min (left) and normalized FI divided by FI at 16 min in each villus (right) after initial loading of BODIPY-FA were quantified. For normalization of FI, villi that showed peak intensity earlier or later than 16 min were excluded (n = 5-7 mice/group, 5-10 villi/mouse). Scale bars, 100 μm.


Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions ( n = 4 mice / group , 4 - 6 images / mouse ) . Jejunum of vehicle - and ABX - treated mice were harvested 2 hours after oral lipid loading . Note that the junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated mice . CM , chylomicron ; IS , interstitium of lamina propria ; LC , large caveola ; Lu , lacteal lumen ; Ve , vesicle containing lipoproteins . Scale bars , 1 μm .

Representative images from transmission electron microscopic examination of lacteals and quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Jejunum of vehicle- and ABX-treated mice were harvested 2 hours after oral lipid loading. Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice. CM, chylomicron; IS, interstitium of lamina propria; LC, large caveola; Lu, lacteal lumen; Ve, vesicle containing lipoproteins. Scale bars, 1 μm.


quantification of frequency of open junctions ( n = 4 mice / group , 4 - 6 images / mouse ) . Note that the junction between LECs ( red colored bidirectional arrows ) is open in vehicle - treated mice , but not in ABX - treated 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

quantification of frequency of open junctions (n = 4 mice/group, 4-6 images/mouse). Note that the junction between LECs (red colored bidirectional arrows) is open in vehicle-treated mice, but not in ABX-treated mice.


Comparisons of serum triglyceride ( TG ) and free fatty acids ( FFA ) at indicated time points in vehicle - and ABX - treated mice ( n = 5 - 7 mice / group ) .

Comparisons of serum triglyceride (TG) and free fatty acids (FFA) at indicated time points in vehicle- and ABX- treated mice (n = 5-7 mice/group).


Images and comparisons of absolute and relative lacteal lengths in jejunum between vehicle - or ABX - treated mice at P7 , P14 , and P28 . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 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

Images and comparisons of absolute and relative lacteal lengths in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from vehicle - or ABX - treated mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from vehicle- or ABX-treated mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between vehicle - or ABX - treated mice at P7 , P14 , and P28 . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in right ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparisons of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between vehicle- or ABX-treated mice at P7, P14, and P28. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in right (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of 8 - week old SPF , GF or CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of 8-week old SPF, GF or CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of the number of Prox1 + LECs within initial 100 μm portion ( C ) and VEGFR3 expression ( D , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from SPF , GF and CONV mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparisons of the number of Prox1+ LECs within initial 100 μm portion (C) and VEGFR3 expression (D, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from SPF, GF and CONV mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum among SPF , GF and CONV mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum among SPF, GF and CONV mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Images and comparison of VEGFR3 expression ( presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum between WT and VR3iΔLEC mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparison of VEGFR3 expression (presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum between WT and VR3iΔLEC mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and VR3iΔLEC mice . Note that , in addition to the alterations in the lacteal lengths in VR3iΔLEC mice , diameters of the lacteal also shrank by VEGFR3 ablation . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and VR3iΔLEC mice. Note that, in addition to the alterations in the lacteal lengths in VR3iΔLEC mice, diameters of the lacteal also shrank by VEGFR3 ablation. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion in CD31 + / LYVE - 1 + lacteals of jejunum from WT and VR3iΔLEC mice . Dotted lines outline villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion in CD31+/LYVE-1+ lacteals of jejunum from WT and VR3iΔLEC mice. Dotted lines outline villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum from WT and VR3iΔLEC mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum from WT and VR3iΔLEC mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Comparisons of serum triglyceride ( TG ) at indicated time points in WT and VR3iΔLEC mice ( n = 6 mice / group ) .

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

Comparisons of serum triglyceride (TG) at indicated time points in WT and VR3iΔLEC mice (n = 6 mice/group).


Comparison of protein levels of VEGF - C in the whole tissue of jejunum and ileum from SPF , GF , and CONV mice ( n = 3 - 5 mice / group ) .

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

Comparison of protein levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA levels of VEGF - C in the whole tissue of jejunum and ileum from SPF , GF , and CONV mice ( n = 3 - 5 mice / group ) .

Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA levels of VEGF - C in sorted CD45 - stromal cells and CD45 + MHCII + F4 / 80 + macrophages from jejunum and ileum of vehicle - treated mice . The mean of transcription levels of VEGF - C in CD45 - stromal cells was normalized to 1 , and the relative levels in macrophages were presented as fold change ( n = 4 mice / group ) .

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

Comparison of mRNA levels of VEGF-C in sorted CD45- stromal cells and CD45+ MHCII+ F4/80+ macrophages from jejunum and ileum of vehicle-treated mice. The mean of transcription levels of VEGF-C in CD45- stromal cells was normalized to 1, and the relative levels in macrophages were presented as fold change (n = 4 mice/group).


Comparisons of VEGF - C transcription level in CD45 - stromal cells ( left ) and CD45 + MHCII + F4 / 80 + macrophages ( right ) in jejunum and ileum of vehicle - and ABX - treated mice . The mean of transcription levels of VEGF - C in vehicle - treated mice was normalized to 1 , and the relative levels in ABX - treated mice were presented as fold change ( n = 5 - 6 mice / group ) .

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

Comparisons of VEGF-C transcription level in CD45- stromal cells (left) and CD45+ MHCII+ F4/80+ macrophages (right) in jejunum and ileum of vehicle- and ABX-treated mice. The mean of transcription levels of VEGF-C in vehicle-treated mice was normalized to 1, and the relative levels in ABX-treated mice were presented as fold change (n = 5 -6 mice/group).


Images and comparison of F4 / 80 + macrophages in the villi of duodenum ( DD ) , jejunum ( JJ ) , and ileum ( IL ) from vehicle - or ABX - treated mice . Each dot indicates mean value of 5 sites in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparison of F4/80+ macrophages in the villi of duodenum (DD), jejunum (JJ), and ileum (IL) from vehicle- or ABX-treated mice. Each dot indicates mean value of 5 sites in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells ( E ) macrophage gated on DAPI - CD45 + cells from jejunum and ileum of vehicle - and ABX - treated mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells (E) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric analysis and comparison of CX3CR1 + F4 / 80 + macrophage gated on DAPI - CD45 + MHCII + cells ( F ) macrophage gated on DAPI - CD45 + cells from jejunum and ileum of vehicle - and ABX - treated mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of CX3CR1+ F4/80+ macrophage gated on DAPI- CD45+ MHCII+ cells (F) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells from jejunum and ileum of WT and DTR mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group).


Comparison of mRNA levels of VEGF - C in the whole tissue of jejunum and ileum from WT and DTR mice ( n = 5 mice / group ) .

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

Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from WT and DTR mice (n = 5 mice/group).


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) and VEGFR3 expression ( lower , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT and DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT and DTR mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and DTR mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells from jejunum and ileum of WT and MyD88ΔMP mice ( n = 5 mice / group ) .

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

Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and MyD88ΔMP mice (n = 5 mice/group).


Comparisons of mRNA levels of VEGF - C in sorted MHCII + F4 / 80 + macrophages from jejunum and ileum of WT and MyD88ΔMP mice . The mean of transcription levels of VEGF - C in WT mice was normalized to 1 , and the relative level in MyD88ΔMP mice were presented as fold change ( n = 5 mice / group ) .

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

Comparisons of mRNA levels of VEGF-C in sorted MHCII+ F4/80+ macrophages from jejunum and ileum of WT and MyD88ΔMP mice. The mean of transcription levels of VEGF-C in WT mice was normalized to 1, and the relative level in MyD88ΔMP mice were presented as fold change (n = 5 mice/group).


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 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 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

Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) and VEGFR3 expression ( lower , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT and MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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

Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT and MyD88ΔMP mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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

Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and MyD88ΔMP mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative Southern blots showing DSB repair products in WT , rad59 ∆ , rad52 - R70A and rad59 ∆ rad52 - R70A cells . DNA was digested with Bsp1286I and probed with a MAT - distal sequence ( yellow box in panel B ) .

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

Representative Southern blots showing DSB repair products in WT, rad59∆, rad52-R70A and rad59∆ rad52-R70A cells. DNA was digested with Bsp1286I and probed with a MAT-distal sequence (yellow box in panel B).


Viability of indicated strains ( mean ± SD ; n = 3 ) . Data information : Welch ' s unpaired t - test was used to determine the p -

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

Viability of indicated strains (mean ± SD; n = 3). Data information: Welch's unpaired t-test was used to determine the p-value


Representative Southern blots showing DSB repair products in WT , rad59 ∆ , and rad52 - R70A . DNA was digested with XhoI and EcoRI and probed with a Z sequence ( yellow box ) .

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

Representative Southern blots showing DSB repair products in WT, rad59∆, and rad52-R70A. DNA was digested with XhoI and EcoRI and probed with a Z sequence (yellow box).


Percentage of chromosome loss of indicated mutants . Data information : χ2 test is used to determine the p - value and number of colonies tested per mutant is indicated in Appendix Table S1 .

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

Percentage of chromosome loss of indicated mutants. Data information: χ2 test is used to determine the p-value and number of colonies tested per mutant is indicated in Appendix Table S1.