tiRNA and 5 ' - tiRNAAla production in HCT116KO following different treatments as indicated . ANGR66A , ANG receptor binding site variant ; ANGK40I , ANG enzymatic activity null variant ; BAY , inhibitor of TNF - α - induced κB kinase ( IKK )

O O O O O O O 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

tiRNA and 5'-tiRNAAla production in HCT116KO following different treatments as indicated. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylation


The effect of 5 ' - tiRNA knockdown or overexpression on TNF - α - induced apoptosis in ANGWT - or ANGK40I - treated HCT116KO . si5 ' - tiRNA : tiRNA - targeting

O O O O O O O O O O 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 effect of 5'-tiRNA knockdown or overexpression on TNF-α-induced apoptosis in ANGWT- or ANGK40I-treated HCT116KO. si5'-tiRNA: tiRNA-targeting siRNA


The interaction between 5 ' - tiRNA and cytochrome c ( Cyt c ) in HCT116 cells as detected by RIP .

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

The interaction between 5'-tiRNA and cytochrome c (Cyt c) in HCT116 cells as detected by RIP.


Caspase activity of HCT116KO following different treatments as indicated . ANGR66A , ANG receptor binding site variant ; ANGK40I , ANG enzymatic activity null variant ; BAY , inhibitor of TNF - α - induced κB kinase ( IKK )

O O 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

Caspase activity of HCT116KO following different treatments as indicated. ANGR66A, ANG receptor binding site variant; ANGK40I, ANG enzymatic activity null variant; BAY, inhibitor of TNF-α-induced κB kinase (IKK) phosphorylation


Cell viability of HCT116KO following different treatments as indicated . siCtrl : control siRNA ; siPLXNB2 - 1 and siPLXNB2 - 2 : PLXNB2

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

Cell viability of HCT116KO following different treatments as indicated. siCtrl: control siRNA; siPLXNB2-1 and siPLXNB2-2: PLXNB2 siRNAs


Detection of 47S pre - rRNA transcription level with northern blotting in HCT116KO following different treatments as indicated . siPLXNB2 - 1 and siPLXNB2 - 2 : PLXNB2

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

Detection of 47S pre-rRNA transcription level with northern blotting in HCT116KO following different treatments as indicated. siPLXNB2-1 and siPLXNB2-2: PLXNB2 siRNAs


tiRNA and 5 ' - tiRNAAla production in IECs isolated from WT and Ang - / - colitis mice at indicated time point .

tiRNA and 5'-tiRNAAla production in IECs isolated from WT and Ang-/- colitis mice at indicated time point.


47S pre - rRNA transcription level in IECs from WT and Ang - / - colitis mice at indicated time point .

47S pre-rRNA transcription level in IECs from WT and Ang-/- colitis mice at indicated time point.


Western blotting of ANG in colonic tissue from WT mice ( n = 2 ) at indicated time point in the ANG prevention model . The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment ( untreated ) is arbitrarily set as 1 .

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

Western blotting of ANG in colonic tissue from WT mice (n = 2) at indicated time point in the ANG prevention model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1.


Body weight loss of the pre - treated WT colitis mice ( n = 8 ) .

Body weight loss of the pre-treated WT colitis mice (n = 8).


DAI of the pre - treated WT colitis mice ( n = 8 ) .

DAI of the pre-treated WT colitis mice (n = 8).


Colon length of colon section from the pre - treated WT colitis mice on day 8 ( n = 8 ) .

Colon length of colon section from the pre-treated WT colitis mice on day 8 (n = 8).


serum FITC - dextran level of colon section from the pre - treated WT colitis mice on day 8 ( n = 8 ) .

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

serum FITC-dextran level of colon section from the pre-treated WT colitis mice on day 8 (n = 8).


representative HE staining image , and ( H ) histopathological score of colon section from the pre - treated WT colitis mice on day 8 ( n = 8 ) .

O O O O O O O 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 HE staining image, and (H) histopathological score of colon section from the pre-treated WT colitis mice on day 8 (n = 8).


Western blotting of ANG in colonic tissue from WT ( n = 2 ) mice at indicated time point in the ANG treatment model . The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment ( untreated ) is arbitrarily set as 1 .

Western blotting of ANG in colonic tissue from WT (n = 2) mice at indicated time point in the ANG treatment model. The grayscale of ANG band in WT mice with no recombinant ANG and DSS treatment (untreated) is arbitrarily set as 1.


Body weight loss of the treated WT colitis mice ( n = 8 ) .

Body weight loss of the treated WT colitis mice (n = 8).


DAI of the treated WT colitis mice ( n = 8 ) .

DAI of the treated WT colitis mice (n = 8).


Colon length of colon sections from the treated WT colitis mice on day 8 ( n = 8 ) .

Colon length of colon sections from the treated WT colitis mice on day 8 (n = 8).


serum FITC - dextran level of colon sections from the treated WT colitis mice on day 8 ( n = 8 ) .

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

serum FITC-dextran level of colon sections from the treated WT colitis mice on day 8 (n = 8).


representative HE staining image , and ( P ) histopathological score of colon sections from the treated WT colitis mice on day 8 ( n = 8 ) .

O O O O O 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 HE staining image, and (P) histopathological score of colon sections from the treated WT colitis mice on day 8 (n = 8).


( H , I ) . Secretory vesicles of primary chromaffin cells were examined by transmission electron microscopy after 100 nM AngII stimulation , with or without pre - incubation with 400 nM Compound 7 . Scale bars : ( H , I ) , 150 nm . Red arrows indicate morphologically docked LDCVs and green arrows stands for undocked LDCVs . PM represents plasma membrane . ( J ) . Vesicle numbers according to different distances from the plasma membrane were calculated in the presence or absence of 100 nM AngII or 400 nM Compound 7 . PM represents plasma membrane . Data information : Data were analyzed using one - way ANOVA and displayed as the mean ± s . e . m . # , P < 0 . 05 ; # # , P < 0 . 01 ; # # # , P < 0 . 001 : cells pre - incubated with PTP - MEG2 inhibitors compared with control vehicles . N . S . means no significant difference . Data were from 6 independent experiments . # indicates P < 0 . 05 and # # # indicates P < 0 . 001 compared with control group .

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

(H, I). Secretory vesicles of primary chromaffin cells were examined by transmission electron microscopy after 100 nM AngII stimulation, with or without pre-incubation with 400 nM Compound 7. Scale bars: (H, I), 150 nm. Red arrows indicate morphologically docked LDCVs and green arrows stands for undocked LDCVs. PM represents plasma membrane. (J). Vesicle numbers according to different distances from the plasma membrane were calculated in the presence or absence of 100 nM AngII or 400 nM Compound 7. PM represents plasma membrane. Data information: Data were analyzed using one-way ANOVA and displayed as the mean ± s.e.m. #, P<0.05; ##, P<0.01; ###, P<0.001: cells pre-incubated with PTP-MEG2 inhibitors compared with control vehicles. N.S. means no significant difference. Data were from 6 independent experiments. # indicates P<0.05 and ### indicates P<0.001 compared with control group.


( D ) . Phosphorylated NSF interacted with PTP - MEG2 . PC12 cells were transfected with FLAG - NSF . After stimulation of the cells with 100nM AngII in the presence or absence of 100μM H2O2 for 5 minutes ( H2O2 was exploited to increase the overall tyrosine phosphorylation of the cellular proteins ) , the potential PTP - MEG2 substrate in cell lysates was pulled down with GST - PTP - MEG2 - D470A or GST control .

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

(D). Phosphorylated NSF interacted with PTP-MEG2. PC12 cells were transfected with FLAG-NSF. After stimulation of the cells with 100nM AngII in the presence or absence of 100μM H2O2 for 5 minutes (H2O2 was exploited to increase the overall tyrosine phosphorylation of the cellular proteins), the potential PTP-MEG2 substrate in cell lysates was pulled down with GST-PTP-MEG2-D470A or GST control.


( B ) . Relative values of the decreased phosphatase activities of different PTP - MEG2 mutants towards pNPP ( p - Nitrophenyl phosphate ) compared with wild - type PTP - MEG2 . Data information : * * * P < 0 . 001 : PTP - MEG2 mutants compared with the control group . Data were obtained from 3 independent experiments . N . S . means no significant difference . All the data were analyzed using one - way ANOVA , and displayed as the mean ± s . e . m . In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho - NSF segement than the pNPP . The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho - segement but not pNPP . The white column stands for the mutants that have similar effects on pNPP and NSF phospho - segement .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Relative values of the decreased phosphatase activities of different PTP-MEG2 mutants towards pNPP (p-Nitrophenyl phosphate) compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement.


( C ) . Fold - change decreases in the phosphatase activities of different PTP - MEG2 mutants towards the NSF - pY83 phospho - segment compared with wild - type PTP - MEG2 . Data information : * * * P < 0 . 001 : PTP - MEG2 mutants compared with the control group . Data were obtained from 3 independent experiments . N . S . means no significant difference . All the data were analyzed using one - way ANOVA , and displayed as the mean ± s . e . m . In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho - NSF segement than the pNPP . The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho - segement but not pNPP . The white column stands for the mutants that have similar effects on pNPP and NSF phospho - segement .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Fold-change decreases in the phosphatase activities of different PTP-MEG2 mutants towards the NSF-pY83 phospho-segment compared with wild-type PTP-MEG2. Data information: *** P<0.001: PTP-MEG2 mutants compared with the control group. Data were obtained from 3 independent experiments. N.S. means no significant difference. All the data were analyzed using one-way ANOVA, and displayed as the mean ± s.e.m. In the red column indicates that the mutation sites have greater effects on the enzyme activities toward phospho-NSF segement than the pNPP. The green column indicates that the mutation sites only have significant effects on the enzyme activity toward NSF phospho-segement but not pNPP. The white column stands for the mutants that have similar effects on pNPP and NSF phospho-segement.


( D ) . Statistical diagram of the quantal size in Fig . 5A and Appendix Figure S6A - D . The secretion amount of each group was standardized with respect to the control group . Data information : Green color represents the amino acids which are found to modulate foot probability based on structure analysis , and red color means amino acids which does not regulate foot probability . , * indicates PTP - MEG2 mutants overexpression group compared with the WT overexpression group . # indicates the overexpression group compared with the control group . * * , P < 0 . 01 ; * * * P < 0 . 001 and # , P < 0 . 05 ; # # , P < 0 . 01 ; # # # P < 0 . 001 . N . S . means no significant difference . Data were from 6 independent experiments . All the data were analyzed using one - way ANOVA and showed as the mean ± s . e . m .

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

(D). Statistical diagram of the quantal size in Fig. 5A and Appendix Figure S6A-D. The secretion amount of each group was standardized with respect to the control group. Data information: Green color represents the amino acids which are found to modulate foot probability based on structure analysis, and red color means amino acids which does not regulate foot probability. , * indicates PTP-MEG2 mutants overexpression group compared with the WT overexpression group. # indicates the overexpression group compared with the control group. **, P<0.01; *** P<0.001 and #, P<0.05; ##, P<0.01; ### P<0.001. N.S. means no significant difference. Data were from 6 independent experiments. All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m.


( C ) . The GST pull - down assay suggested that PACSIN1 , MUNC18 - 1 , VAMP7 , SNAP25 , DYNAMIN1 and DYNAMIN2 directly interact with PTP - MEG2 . PC12 cells were transfected with plasmids of candidate substrates , including SYN1 , MUNC18 - 3 , PACSIN1 , SCAMP1 , MUNC18 - 1 , PPP3CA , STX17 , VAMP7 , SYT7 , SYT11 , SNAP25 , DYNAMIN1 , and DYNAMIN2 stimulated with 100 nM AngII . The tyrosine phosphorylation of these proteins was verified by specific anti - pY antibodies ( Appendix Figure S8A - E ) . The potential substrates of PTP - MEG2 in cell lysates were pulled down with a GST - PTP - MEG2 - D470A trapping mutant and then detected by Western blot . White arrow stands for Western blot band consistent with the predicted molecular weight of the potential PTP - MEG2 substrate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). The GST pull-down assay suggested that PACSIN1, MUNC18-1, VAMP7, SNAP25, DYNAMIN1 and DYNAMIN2 directly interact with PTP-MEG2. PC12 cells were transfected with plasmids of candidate substrates, including SYN1, MUNC18-3, PACSIN1, SCAMP1, MUNC18-1, PPP3CA, STX17, VAMP7, SYT7, SYT11, SNAP25, DYNAMIN1, and DYNAMIN2 stimulated with 100 nM AngII. The tyrosine phosphorylation of these proteins was verified by specific anti-pY antibodies (Appendix Figure S8A-E). The potential substrates of PTP-MEG2 in cell lysates were pulled down with a GST-PTP-MEG2-D470A trapping mutant and then detected by Western blot. White arrow stands for Western blot band consistent with the predicted molecular weight of the potential PTP-MEG2 substrate.


( D ) . Interactions of the PTP - MEG2 - trapping mutants with the MUNC18 - 1 - Y145 mutants and DYNAMIN2 - Y125 mutants . PC12 cells were transfected with FLAG - MUNC18 - 1 - Y145 , FLAG - DYNAMIN2 - Y125 and different mutations of the FLAG - MUNC18 - 1 - Y145A , Y145H , Y145E or Y145F , FLAG - DYNAMIN2 - Y125A , Y125E , Y125F , 24 hours before stimulation with 100 nM AngII , respectively . The cell lysates were then incubated with GST - beads - PTP - MEG2 - D470A complex for 2 hours with constant rotation . The potential PTP - MEG2 substrates were pulled down by GST - beads , and their levels were examined by the FLAG antibody with Western blot .

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

(D). Interactions of the PTP-MEG2-trapping mutants with the MUNC18-1-Y145 mutants and DYNAMIN2-Y125 mutants. PC12 cells were transfected with FLAG-MUNC18-1-Y145, FLAG-DYNAMIN2-Y125 and different mutations of the FLAG-MUNC18-1-Y145A, Y145H, Y145E or Y145F, FLAG-DYNAMIN2-Y125A, Y125E, Y125F, 24 hours before stimulation with 100 nM AngII, respectively. The cell lysates were then incubated with GST-beads-PTP-MEG2-D470A complex for 2 hours with constant rotation. The potential PTP-MEG2 substrates were pulled down by GST-beads, and their levels were examined by the FLAG antibody with Western blot.


( H ) . The MUNC18 - 1 - Y145 mutations decreased the interaction between MUNC18 - 1 and SYNTAXIN1 . PC12 cells were transfected with plasmid encoding SYNTAXIN - 1 . The proteins in cell lysates were pulled down with purified GST - beads - MUNC18 - 1 - Y145 complex and the GST - MUNC18 - 1 - Y145H / E / F , and detected with SYNTAXIN1 antibody . The right histogram shows the quantified protein levels . Data information * indicates MUNC18 - 1 , mutants overexpression group compared with the WT overexpression group . # indicates MUNC18 - 1 , overexpression group compared with the control group . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 N . S . means no significant difference . Data were from 3 ( H ) independent experiments . All the data were analyzed using one - way ANOVA , and showed as the mean ± s . e . m .

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

(H). The MUNC18-1-Y145 mutations decreased the interaction between MUNC18-1 and SYNTAXIN1. PC12 cells were transfected with plasmid encoding SYNTAXIN-1. The proteins in cell lysates were pulled down with purified GST-beads-MUNC18-1-Y145 complex and the GST-MUNC18-1-Y145H/E/F, and detected with SYNTAXIN1 antibody. The right histogram shows the quantified protein levels. Data information * indicates MUNC18-1, mutants overexpression group compared with the WT overexpression group. # indicates MUNC18-1, overexpression group compared with the control group. *, P<0.05; **, P<0.01; *** P<0.001 N.S. means no significant difference. Data were from 3 (H) independent experiments. All the data were analyzed using one-way ANOVA, and showed as the mean ± s.e.m.


( E ) . Relative phosphatase activities of different PTP - MEG2 mutants towards the MUNC18 - 1 - pY145 phospho - segment compared with wild - type PTP - MEG2 . ( F ) . Relative phosphatase activities of different PTP - MEG2 mutants towards the DYNAMIN 2 - pY125 phospho - segment compared with wild - type PTP - MEG2 . Data information : ( E , F ) Residues are coloured according to Figure 8B . ( E , F ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 : PTP - MEG2 mutants compared with the control . N . S . means no significant difference . Data were obtained from 3 independent experiments . * * , P < 0 . 01 ; * * * P < 0 . 001 and # # P < 0 . 01 ; # # # P < 0 . 001 . N . S . means no significant difference . Data were obtained from 10 cells in each group of 6 independent experiments . . All the data were analyzed using one - way ANOVA and showed as the mean ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Relative phosphatase activities of different PTP-MEG2 mutants towards the MUNC18-1-pY145 phospho-segment compared with wild-type PTP-MEG2. (F). Relative phosphatase activities of different PTP-MEG2 mutants towards the DYNAMIN 2-pY125 phospho-segment compared with wild-type PTP-MEG2.Data information: (E, F) Residues are coloured according to Figure 8B. (E, F), * P<0.05, ** P<0.01, *** P<0.001: PTP-MEG2 mutants compared with the control. N.S. means no significant difference. Data were obtained from 3 independent experiments. **, P<0.01; *** P<0.001 and ## P<0.01; ### P<0.001. N.S. means no significant difference. Data were obtained from 10 cells in each group of 6 independent experiments. . All the data were analyzed using one-way ANOVA and showed as the mean ± s.e.m.


Representative images of changes in intracellular calcium , as measured in fluorescence ratio images ( F340 / 380 ) ( A ) Examples show a TRPM8 - expressing DRG neuron ( left in A responding to both the TRPM8 agonist menthol ( 300 μM ) and to a cold stimulus from 32 ° C to 4 ° C a neuron that responds to cold ( right in A but does not express any of TRPM8 , TRPA1 or TRPC5 , as shown by lack of response to the TRPM8 agonist menthol ( 300 μM ) Calcium increase is due to influx from extracellular

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

Representative images of changes in intracellular calcium, as measured in fluorescence ratio images (F340/380) (A) Examples show a TRPM8-expressing DRG neuron (left in A responding to both the TRPM8 agonist menthol (300 μM) and to a cold stimulus from 32°C to 4°C a neuron that responds to cold (right in A but does not express any of TRPM8, TRPA1 or TRPC5, as shown by lack of response to the TRPM8 agonist menthol (300 μM) Calcium increase is due to influx from extracellular solution


experiments showing an SCG neuron responding to the TRPA1 agonist AITC ( left in E and a novel cold - sensitive SCG neuron that responds to cold but does not express any of TRPM8 , TRPA1 or TRPC5 ( right in E Many glial cells are also visibly activated by AITC but not by cold .

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

experiments showing an SCG neuron responding to the TRPA1 agonist AITC (left in E and a novel cold-sensitive SCG neuron that responds to cold but does not express any of TRPM8, TRPA1 or TRPC5 (right in E Many glial cells are also visibly activated by AITC but not by cold.


( G ) Mean current voltage relation of 10 SCG neurons at 32 ° C ( dashed line ) , 4 ° C ( blue ) and 32 ° C ( red dots ) . ( H ) Difference trace from ( G ) .

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

(G) Mean current voltage relation of 10 SCG neurons at 32°C (dashed line), 4°C (blue) and 32°C (red dots). (H) Difference trace from (G).


( C ) Mean cold - sensitive increase in calcium - dependent fluorescence ratio F340 / 380 in SCG neurons is significantly smaller in neurons recorded in voltage - clamp at - 60mV ( 0 . 56 ± 0 . 08 , mean ± SEM , n = 21 neurons ) than in neurons recorded in current - clamp mode ( 1 . 16 ± 0 . 29 , n = 11 neurons , p = 0 . 0165 , unpaired t - test ) .

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

(C) Mean cold-sensitive increase in calcium-dependent fluorescence ratio F340/380 in SCG neurons is significantly smaller in neurons recorded in voltage-clamp at -60mV (0.56 ±0.08, mean ± SEM, n=21 neurons) than in neurons recorded in current-clamp mode (1.16 ±0.29, n=11 neurons, p=0.0165, unpaired t-test).


( F ) Summary of results of experiments on SCG neurons ( mean ± SEM , n = 115 neurons , 4 separate cultures ) . YM58483 caused a significant decrease in cold - response amplitude ( p < 0 . 0001 , RM one - way ANOVA + Dunnett ' s test ) , with only partial recovery . Temperature trace below . ( G ) In neurons with a small response to cold ( F340 / 380 increase < 0 . 2 ; n = 24 neurons ) , many responses are potentiated by verapamil ( 31 % of cold - insensitive neurons ) and are fully blocked by ORAI channel antagonist YM58483 ( p < 0 . 0001 , RM one - way ANOVA + Dunnett ' s test ) .

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

(F) Summary of results of experiments on SCG neurons (mean ±SEM, n=115 neurons, 4 separate cultures). YM58483 caused a significant decrease in cold-response amplitude (p<0.0001, RM one-way ANOVA + Dunnett's test), with only partial recovery. Temperature trace below. (G) In neurons with a small response to cold (F340/380 increase <0.2; n=24 neurons), many responses are potentiated by verapamil (31% of cold-insensitive neurons) and are fully blocked by ORAI channel antagonist YM58483 (p<0.0001, RM one-way ANOVA + Dunnett's test).


( D ) Representative fluorescence images of SCG neurons from neonatal mice ( age P5 - 7 ) electroporated with plasmids coding for mCherry and for siRNA targeting STIM1 . Neurons were cooled from 35 ° C to 7 ° C in the presence of 100μM verapamil . Electroporation was indicated by expression of mCherry ( # in left - hand panel ) ; in these neurons a cold response was absent ( # in right - hand panel ) while in neurons not expressing mCherry ( * ) cold responses were still observed . Scale bar 20μm .

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

(D) Representative fluorescence images of SCG neurons from neonatal mice (age P5-7) electroporated with plasmids coding for mCherry and for siRNA targeting STIM1. Neurons were cooled from 35°C to 7°C in the presence of 100μM verapamil. Electroporation was indicated by expression of mCherry (# in left-hand panel); in these neurons a cold response was absent (# in right-hand panel) while in neurons not expressing mCherry (*) cold responses were still observed. Scale bar 20μm.


( F ) Cold response amplitudes ( F340 / 380 , mean ± SEM ) . Successfully electroporated neurons ( mCherry positive , red bar , n = 111 neurons from four separate cultures ) had a significantly lower cold response amplitude than neurons not electroporated ( mCherry negative , black bar , n = 104 neurons from same cultures , p = 0 . 0082 , unpaired t - test ) . ( G ) Percentage of neurons that responded to cold ramp with F340 / 380 increase > 0 . 2 . Only 5 % of mCherry - positive neurons ( successfully electroporated , red bar ) responded to cold , compared to 19 % of mCherry - negative neurons in same cultures ( non - electroporated , black bar , p < 0 . 001 , chi - square test ) .

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

(F) Cold response amplitudes (F340/380, mean ±SEM). Successfully electroporated neurons (mCherry positive, red bar, n=111 neurons from four separate cultures) had a significantly lower cold response amplitude than neurons not electroporated (mCherry negative, black bar, n=104 neurons from same cultures, p=0.0082, unpaired t-test). (G) Percentage of neurons that responded to cold ramp with F340/380 increase >0.2. Only 5% of mCherry-positive neurons (successfully electroporated, red bar) responded to cold, compared to 19% of mCherry-negative neurons in same cultures (non-electroporated, black bar, p<0.001, chi-square test).


( A ) Representative TIRF microscopy images ( imaging depth 100nm ) showing that cooling from 35 ° C to 13 ° C increases ORAI1 puncta formation and colocalization of ORAI1 with STIM1 in HEK293 cells transfected with ORAI1 - CFP and STIM1 - YFP . Cooling to 13 ° C causes the appearance of new ORAI1 puncta ( examples shown by * ) that are not present in the 35 ° C image and are colocalised with STIM1 . Cooling also causes STIM1 to move towards the cell surface in edge - on views ( arrows ) . To reduce cell motility , the experiment was conducted in nominal 0 [ Ca ] e , which potentially caused partial formation of STIM1 puncta before the images shown . Scale bar = 5μm . Temperature trace shown below . Mean fluorescence intensity adjusted to compensate for increase in intrinsic fluorescence of CFP and YFP caused by cooling ( see Fig . S12 ) .

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

(A) Representative TIRF microscopy images (imaging depth 100nm) showing that cooling from 35°C to 13°C increases ORAI1 puncta formation and colocalization of ORAI1 with STIM1 in HEK293 cells transfected with ORAI1-CFP and STIM1-YFP. Cooling to 13°C causes the appearance of new ORAI1 puncta (examples shown by *) that are not present in the 35°C image and are colocalised with STIM1. Cooling also causes STIM1 to move towards the cell surface in edge-on views (arrows). To reduce cell motility, the experiment was conducted in nominal 0[Ca]e, which potentially caused partial formation of STIM1 puncta before the images shown. Scale bar = 5μm. Temperature trace shown below. Mean fluorescence intensity adjusted to compensate for increase in intrinsic fluorescence of CFP and YFP caused by cooling (see Fig. S12).


( A ) Representative immunofluorescence images showing adult mouse stellate ganglion ( SG ) sections stained for nNOS ( red ) , to label nitrergic neurons , βIII Tubulin ( green ) to label all neurons , and DAPI to label nuclei . nNOS is widely expressed in neurons of the stellate ganglion , but is absent from glial cells ( glial nuclei identifiable in merged image , similar results in n = 5 animals ) . Antibody against nNOS from Abcam ; similar results obtained with antibodies from ECM and Millipore . All three antibodies show nNOS widely expressed in SG cell bodies and neurites .

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

(A) Representative immunofluorescence images showing adult mouse stellate ganglion (SG) sections stained for nNOS (red), to label nitrergic neurons, βIII Tubulin (green) to label all neurons, and DAPI to label nuclei. nNOS is widely expressed in neurons of the stellate ganglion, but is absent from glial cells (glial nuclei identifiable in merged image, similar results in n=5 animals). Antibody against nNOS from Abcam; similar results obtained with antibodies from ECM and Millipore. All three antibodies show nNOS widely expressed in SG cell bodies and neurites.


( B ) Adult mouse SCG ( n = 2 animals , upper ) and SG ( n = 5 animals , lower ) sections stained for nNOS ( red ) , tyrosine hydroxylase ( TH , green ) and DAPI ( blue ) to label nitrergic neurons , noradrenergic neurons and nuclei respectively . Many noradrenergic cells also express nNOS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Adult mouse SCG (n=2 animals, upper) and SG (n=5 animals, lower) sections stained for nNOS (red), tyrosine hydroxylase (TH, green) and DAPI (blue) to label nitrergic neurons, noradrenergic neurons and nuclei respectively. Many noradrenergic cells also express nNOS.


E Quantification of mean fluorescence intensity ( MFI ) for CD80 , CD86 , CD206 , CD163 , CCR2 , and MHC - Ⅱ in TAMEMs and typical macrophages subtypes via flow cytometry ( * P < 0 . 05 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; n = 6 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using ordinary one - way ANOVA with Tukey ' s multiple comparison test in ( E ) in Graph Pad Prism 8 .

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

E Quantification of mean fluorescence intensity (MFI) for CD80, CD86, CD206, CD163, CCR2, and MHC-Ⅱ in TAMEMs and typical macrophages subtypes via flow cytometry (*P < 0.05, ***P < 0.001, and ****P < 0.0001; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (E) in Graph Pad Prism 8.


F Representative immunofluorescent ( IF ) staining images for CD86 and CD206 in TAMEMs and typical macrophages subtypes ( scale bar : 10 μm ; n = 6 , biological replicates ) .

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

F Representative immunofluorescent (IF) staining images for CD86 and CD206 in TAMEMs and typical macrophages subtypes (scale bar: 10 μm; n=6, biological replicates).


A Quantification of concentrations of 22 soluble proteins from the cytokine and angiogenesis array analysis ( * P < 0 . 05 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; ns : not significan ; n = 3 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison test in ( A , in Graph Pad Prism 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Quantification of concentrations of 22 soluble proteins from the cytokine and angiogenesis array analysis (*P < 0.05, ***P < 0.001, and ****P < 0.0001; ns: not significan; n=3, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in (A, in Graph Pad Prism 8.


F , G Western Blotting analysis of phosphorylated IκB ( F ) and quantification of gray value ( G ) in fibroblasts ( L929 ) that pre - treated with LPS and then cultured with CMs from TAMEMs and other controls for 24 h ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , and ns : not significant ( P > 0 . 05 ) vs . the LPS - treated group ; n = 3 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison test in G , in Graph Pad Prism 8 .

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

F, G Western Blotting analysis of phosphorylated IκB (F) and quantification of gray value (G) in fibroblasts (L929) that pre-treated with LPS and then cultured with CMs from TAMEMs and other controls for 24 h (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the LPS-treated group; n=3, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in G, in Graph Pad Prism 8.


H Representative images and quantification of the Matrigel - based angiogenesis assay in mouse vascular endothelial cell ( SVEC4 - 10 ) treated with CMs from TAMEMs and other controls , VEGF treatment as a positive control ( * * * * P < 0 . 0001 and ns : not significant ( P > 0 . 05 ) vs . the normal medium group ; scale bar : 100 μm ; n = 6 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using ordinary one - way ANOVA with Tukey ' s multiple comparison test in H ) in Graph Pad Prism 8 .

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

H Representative images and quantification of the Matrigel-based angiogenesis assay in mouse vascular endothelial cell (SVEC4-10) treated with CMs from TAMEMs and other controls, VEGF treatment as a positive control (****P < 0.0001 and ns: not significant (P > 0.05) vs. the normal medium group; scale bar: 100 μm; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in H) in Graph Pad Prism 8.


E Representative images of the wound closure process were captured during the twenty - one - day in vivo experiments ( scale bar : 500 μm ; n = 8 , biological replicates ) .

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

E Representative images of the wound closure process were captured during the twenty-one-day in vivo experiments (scale bar: 500 μm; n=8, biological replicates).


G H & E ( day 7 and 14 ) and Masson ' s ( day 14 ) staining for full - thickness skin samples containing the entire wound area ( scale bar : 500 μm ; n = 8 ) , the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 ( scale bar : 100 μm ; n = 8 , biological replicates ) , ( gt : granulation tissue , he : hyperproliferative epithelium , black arrow : wound edges ) .

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

G H&E (day 7 and 14) and Masson's (day 14) staining for full-thickness skin samples containing the entire wound area (scale bar: 500 μm; n=8), the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 (scale bar: 100 μm; n=8, biological replicates), (gt: granulation tissue, he: hyperproliferative epithelium, black arrow: wound edges).


H - J Quantitative analysis of regenerated granulation ( H ) on day 7 and Re - epithelialization of wound bed ( I ) and Masson ' s trichrome - stained tissue ( J ) on day 14 ( blue for collagen ) ( * * * P < 0 . 001 , * * * * P < 0 . 0001 , and ns : not significant ( P > 0 . 05 ) vs . the blank control group ; n = 8 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using ordinary one - way ANOVA with Tukey ' s multiple comparison test in ( H , I , J , in Graph Pad Prism 8 .

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

H- J Quantitative analysis of regenerated granulation (H) on day 7 and Re-epithelialization of wound bed (I) and Masson's trichrome-stained tissue (J) on day 14 (blue for collagen) (***P < 0.001, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (H, I, J, in Graph Pad Prism 8.


E IF staining for CD31 ( red ; endothelial marker , for new capillary formation ) at day 7 and α - SMA ( green ; smooth muscle marker , for vascular maturation ) at day 14 , with both the entire wound region and magnified yellow - box zone ( scale bar : 100 μm ; n = 8 , biological replicates ) ( gt : granulation tissue , he : hyperproliferative epithelium ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 IF staining for CD31 (red; endothelial marker, for new capillary formation) at day 7 and α-SMA (green; smooth muscle marker, for vascular maturation) at day 14, with both the entire wound region and magnified yellow-box zone (scale bar: 100 μm; n=8, biological replicates) (gt: granulation tissue, he: hyperproliferative epithelium).


F Quantification of the CD31 + vessels at day 7 and CD31 + α - SMA + vessels and CD31 + α - SMA - vessels at day 14 ( * * * * P < 0 . 0001 and ns : not significant ( P > 0 . 05 ) vs . the blank control group ; n = 8 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using ordinary one - way ANOVA with Tukey ' s multiple comparison test in ( F , in Graph Pad Prism 8 .

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

F Quantification of the CD31+ vessels at day 7 and CD31+ α-SMA+ vessels and CD31+ α-SMA- vessels at day 14 (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparison test in (F, in Graph Pad Prism 8.


G Representative multiphoton projection images of blood vessels ( lectin ) in healed skin wound tissue after the administration of different treatments at day 21 post - injury , uninjured skin was analyzed as positive control . Up : the images of the entire sampling area ( yellow circled ) containing the healed wound ( green circled ) ; Down : magnified zone ( blue boxed in the upper images ; scale bar : 100 μm ; n = 3 , biological replicates ) .

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

G Representative multiphoton projection images of blood vessels (lectin) in healed skin wound tissue after the administration of different treatments at day 21 post-injury, uninjured skin was analyzed as positive control. Up: the images of the entire sampling area (yellow circled) containing the healed wound (green circled); Down: magnified zone (blue boxed in the upper images; scale bar: 100 μm; n=3, biological replicates).


G The proliferation rate of L929 cells pretreated with different CMs was analyzed using the CCK - 8 kit ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , and ns : not significant ( P > 0 . 05 ) vs . the normal medium group ; n = 6 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison test in G ) in Graph Pad Prism 8 .

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

G The proliferation rate of L929 cells pretreated with different CMs was analyzed using the CCK-8 kit (*P < 0.05, **P < 0.01, ****P < 0.0001, and ns: not significant (P > 0.05) vs. the normal medium group; n=6, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in G) in Graph Pad Prism 8.


H Immunofluorescent staining for α - SMA and type I Collagen in L929 cells after 48 h treatment with different CMs ( scale bar : 10 μm ; n = 6 , biological replicates ) .

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

H Immunofluorescent staining for α-SMA and type I Collagen in L929 cells after 48 h treatment with different CMs (scale bar: 10 μm; n=6, biological replicates).


B Gross view of the wound closure process was captured during the twenty - one - day in vivo experiments ( scale bar : 500 μm ; n = 8 , biological replicates ) . C The percentage of wound closure determined at each time point ( * * * * P < 0 . 0001 and ns : not significant ( P > 0 . 05 ) vs . the blank control group ; n = 8 , biological replicates ) . Data information : Data represent means ± SD . The differences between groups were analyzed using two - way ANOVA with Tukey ' s multiple comparison test in ( C ) in Graph Pad Prism 8 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Gross view of the wound closure process was captured during the twenty-one-day in vivo experiments (scale bar: 500 μm; n=8, biological replicates). C The percentage of wound closure determined at each time point (****P < 0.0001 and ns: not significant (P > 0.05) vs. the blank control group; n=8, biological replicates). Data information: Data represent means ± SD. The differences between groups were analyzed using two-way ANOVA with Tukey's multiple comparison test in (C) in Graph Pad Prism 8.


D Full - thickness skin samples containing entire wound sites were stained with H & E ( day 7 and day 14 ) and Masson ( day 14 ) ( scale bar : 500 μm , n = 8 , biological replicates ) , the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 ( scale bar : 100 μm ; n = 8 , biological replicates ) ( gt : granulation tissue , he : hyperproliferative epithelium , black arrow : wound edges ) .

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

D Full-thickness skin samples containing entire wound sites were stained with H&E (day 7 and day 14) and Masson (day 14) (scale bar: 500 μm, n=8, biological replicates), the under images are the higher magnification images that indicates an area in the red boxed region in day 7 and day 14 (scale bar: 100 μm; n=8, biological replicates) (gt: granulation tissue, he: hyperproliferative epithelium, black arrow: wound edges).


F Representative multiphoton projection images of blood vessels ( lectin ) in healed skin wound tissue after the administration of different treatments at day 21 postinjury , uninjured skin was analyzed as positive control ( Up : the images of entire sampling sites ( the yellow circular region ) containing healed wound sites ( the green circular region ) ; Down : the images of higher magnification that indicates an area in the blue boxed region in the upper images ; scale bar : 100 μm ; n = 3 , biological replicates ) .

F Representative multiphoton projection images of blood vessels (lectin) in healed skin wound tissue after the administration of different treatments at day 21 postinjury, uninjured skin was analyzed as positive control (Up: the images of entire sampling sites (the yellow circular region) containing healed wound sites (the green circular region); Down: the images of higher magnification that indicates an area in the blue boxed region in the upper images; scale bar: 100 μm; n=3, biological replicates).


( a ) Morphological analysis . Jurkat T cells were cultured in the presence and absence of the indicated anticancer drugs for 48 h . Drug - treated cells were classified as normal , apoptotic or abnormal , with enlarged and irregular nuclei , with multipolar mitoses or multinucleated . Cultures treated with etoposide ( 0 . 25 μM ) , cisplatin ( 3 μM ) , taxol ( 4 nM ) and nocodazole ( 10 nM ) exhibited high levels of cells with abnormal nuclei . None of the treated populations showed a significant fraction of apoptosis ( see also Supplementary Fig . S1 ) . Values are means ± s . d . for three independent experiments . Right : representative examples of these morphological categories are shown . Scale bar , 10 μM . Similar abnormalities were also seen after treating NB4 and MDA - MA - 231 cells with etoposide or cisplatin ( Supplementary Fig . S2 ) .

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

(a) Morphological analysis. Jurkat T cells were cultured in the presence and absence of the indicated anticancer drugs for 48 h. Drug-treated cells were classified as normal, apoptotic or abnormal, with enlarged and irregular nuclei, with multipolar mitoses or multinucleated. Cultures treated with etoposide (0.25 μM), cisplatin (3 μM), taxol (4 nM) and nocodazole (10 nM) exhibited high levels of cells with abnormal nuclei. None of the treated populations showed a significant fraction of apoptosis (see also Supplementary Fig. S1). Values are means±s.d. for three independent experiments. Right: representative examples of these morphological categories are shown. Scale bar, 10 μM. Similar abnormalities were also seen after treating NB4 and MDA-MA-231 cells with etoposide or cisplatin (Supplementary Fig. S2).


( b ) Immunoblotting . Jurkat T cells were treated as indicated and ATG5 ( monomeric and ATG12 - conjugated ) , ATG12 ( conjugated with ATG5 ) , LC3 ( 18 and 16 kDa ) , Beclin 1 and GAPDH detected . Chloroquine ( CQ ) was employed to block lysosomal proteases . Each immunoblot is representative of at least three independent experiments . The same assays were performed using NB4 and MDA - MA - 231 cells ( Supplementary Fig . S2 ) . Moreover , as a marker for autophagy onset , increased LC3 - II formation following etoposide or cisplatin treatment was also documented using fluorescence microscopy ( Supplementary Fig . S2 ) . Full - length immunoblots are provided in Supplementary Fig . S8 .

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

(b) Immunoblotting. Jurkat T cells were treated as indicated and ATG5 (monomeric and ATG12-conjugated), ATG12 (conjugated with ATG5), LC3 (18 and 16 kDa), Beclin 1 and GAPDH detected. Chloroquine (CQ) was employed to block lysosomal proteases. Each immunoblot is representative of at least three independent experiments. The same assays were performed using NB4 and MDA-MA-231 cells (Supplementary Fig. S2). Moreover, as a marker for autophagy onset, increased LC3-II formation following etoposide or cisplatin treatment was also documented using fluorescence microscopy (Supplementary Fig. S2). Full-length immunoblots are provided in Supplementary Fig. S8.


( a ) Time - lapse microscopy . HeLa H2B - mCherry - α - tubulin - EGFP cells were cultured in the presence and absence of etoposide and cisplatin , respectively . Treated cells frequently developed multiple nuclei , including micronuclei ( upper panel ) , and demonstrated evidence of multipolar spindles and misaligned chromosomes ( lower panel ) . Cells were also much larger than untreated cells ( see also Supplementary Fig . S3 ) . Scale bar , 10 μm . ( b ) Statistical analysis of the data shown above . At least 100 cells were counted for each drug exposure time according to the classification used in Fig . 1 . Values are means ± s . d . ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time-lapse microscopy. HeLa H2B-mCherry-α-tubulin-EGFP cells were cultured in the presence and absence of etoposide and cisplatin, respectively. Treated cells frequently developed multiple nuclei, including micronuclei (upper panel), and demonstrated evidence of multipolar spindles and misaligned chromosomes (lower panel). Cells were also much larger than untreated cells (see also Supplementary Fig. S3). Scale bar, 10 μm. (b) Statistical analysis of the data shown above. At least 100 cells were counted for each drug exposure time according to the classification used in Fig. 1. Values are means±s.d. (n=3).


( a ) Immunoblotting . Jurkat T cells were cultured as indicated for 48 h . Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained . Monomeric ATG5 ( shown under the dotted line ) was detected after a longer exposure time compared with the ATG5 conjugated with ATG12 . Results are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblotting. Jurkat T cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction was evidence of increased autophagic activity obtained. Monomeric ATG5 (shown under the dotted line) was detected after a longer exposure time compared with the ATG5 conjugated with ATG12. Results are representative of three independent experiments.


( b ) Morphological analysis . Jurkat T cells were cultured as in a . Values are means ± s . d . of three independent experiments .

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

(b) Morphological analysis. Jurkat T cells were cultured as in a. Values are means±s.d. of three independent experiments.


c ) Morphological analysis . Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5 , ATG5 - K130R , Beclin 1 or control constructs for 48 h . Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5 - K130R lentiviral gene transfer or of etoposide treatment . Increased Beclin 1 levels or rapamycin treatment had no such effect . Representative examples of morphology are shown . Scale bar , 10 μm . Right : statistical analysis of the data . At least 100 cells were counted for each condition . Values are means ± s . d . ( n = 3 ) .

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

c) Morphological analysis. Jurkat T cells were treated with the indicated drugs or transduced with lentivirus ATG5, ATG5-K130R, Beclin 1 or control constructs for 48 h. Enlarged and multinucleated cells were seen as a consequence of ATG5 or ATG5-K130R lentiviral gene transfer or of etoposide treatment. Increased Beclin 1 levels or rapamycin treatment had no such effect. Representative examples of morphology are shown. Scale bar, 10 μm. Right: statistical analysis of the data. At least 100 cells were counted for each condition. Values are means±s.d. (n=3).


( d ) Immunoblotting . Enforced expression of ATG5 , Beclin 1 , or ATG5 - K130R following lentiviral gene transfer .

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

(d) Immunoblotting. Enforced expression of ATG5, Beclin 1, or ATG5-K130R following lentiviral gene transfer.


( e ) Time - lapse microscopy . HeLa H2B - mCherry - α - tubulin - EGFP cells were transduced by lentiviral gene transfer as indicated . Only ATG5 - overexpressing cells demonstrated evidence for mitotic catastrophe ( see also Supplementary Movies ) . As with the GFP control , the Beclin 1 construct also included GFP , hence the intensity in green was very high . 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

(e) Time-lapse microscopy. HeLa H2B-mCherry-α-tubulin-EGFP cells were transduced by lentiviral gene transfer as indicated. Only ATG5-overexpressing cells demonstrated evidence for mitotic catastrophe (see also Supplementary Movies). As with the GFP control, the Beclin 1 construct also included GFP, hence the intensity in green was very high. Scale bar, 10 μm.


( a ) Cell cycle analysis . After lentivirus gene transfer , Jurkat T cells overexpressing ATG5 or GFP were followed for the indicated times and analysed for DNA content by flow cytometry . Untransduced cells were left untreated or treated with etoposide for comparison . Values are means ± s . d . ( n = 3 ) . Right : representative flow cytometry diagrams for the 48 h time point .

O O O O 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 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

(a) Cell cycle analysis. After lentivirus gene transfer, Jurkat T cells overexpressing ATG5 or GFP were followed for the indicated times and analysed for DNA content by flow cytometry. Untransduced cells were left untreated or treated with etoposide for comparison. Values are means±s.d. (n=3). Right: representative flow cytometry diagrams for the 48 h time point.


( b ) Cell cycle analysis . Jurkat cells were cultured as indicated for 48 h . Only under conditions of ATG5 induction , we observed an accumulation of cells in G2 - or M - phase arrest . Results are representative of three independent experiments .

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

(b) Cell cycle analysis. Jurkat cells were cultured as indicated for 48 h. Only under conditions of ATG5 induction, we observed an accumulation of cells in G2- or M-phase arrest. Results are representative of three independent experiments.


( c ) Immunoblotting . Jurkat T cells overexpressing ATG5 or GFP were cultured for the indicated times after transduction and analysed . As controls , cells were treated with etoposide or rapamycin . Although rapamycin , similar to etoposide , induced autophagy , no increase in ATG5 expression and no cell cycle arrest were observed . ATG5 overexpression and etoposide treatment resulted in a similar expression pattern for all proteins investigated . Each immunoblot is representative of at least three independent experiments .

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

(c) Immunoblotting. Jurkat T cells overexpressing ATG5 or GFP were cultured for the indicated times after transduction and analysed. As controls, cells were treated with etoposide or rapamycin. Although rapamycin, similar to etoposide, induced autophagy, no increase in ATG5 expression and no cell cycle arrest were observed. ATG5 overexpression and etoposide treatment resulted in a similar expression pattern for all proteins investigated. Each immunoblot is representative of at least three independent experiments.


( a ) Flow cytometry . Untreated Jurkat T cells , cells treated with etoposide continuously ( 0 . 25 μM ) , or transduced with lentivirus ATG5 or GFP were cultured for 48 h , fixed , permeabilized and exposed to antiphospho - H2AX ( Ser139 ) antibody , then stained with antimouse - APC - conjugated antibody , and analysed using a flow cytometer .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometry. Untreated Jurkat T cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 or GFP were cultured for 48 h, fixed, permeabilized and exposed to antiphospho-H2AX (Ser139) antibody, then stained with antimouse-APC-conjugated antibody, and analysed using a flow cytometer.


( b ) Confocal microscopy . Untreated HeLa cells , cells treated with etoposide continuously ( 0 . 25 μM ) , or transduced with lentivirus ATG5 were cultured for 48 h . Additional DAPI nuclear staining was used . Scale bar , 10 μm . Right : phospho - H2AX was quantified by measuring the mean fluorescence intensity ( MFI ) in five high - power fields of each condition using IMARIS software . Values are means ± s . d .

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

(b) Confocal microscopy. Untreated HeLa cells, cells treated with etoposide continuously (0.25 μM), or transduced with lentivirus ATG5 were cultured for 48 h. Additional DAPI nuclear staining was used. Scale bar, 10 μm. Right: phospho-H2AX was quantified by measuring the mean fluorescence intensity (MFI) in five high-power fields of each condition using IMARIS software. Values are means±s.d.


( c ) Jurkat T cells were treated as in a and analysed by immunoblotting . Besides phospho - H2AX ( Ser139 ) , we also detected phospho - ATM ( Ser1981 ) and phospho - ATR ( Ser428 ) , two additional markers of DNA damage response . Intensity of the protein bands were quantified with a Licor / Odyssey scanner and software ( shown beneath each immunoblot ) . The fold change in phosphorylation levels compared with no treatment ( untreated ) are presented . The data in all panels are representative of at least three independent experiments . Full - length immunoblots are provided in Supplementary Fig . S9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Jurkat T cells were treated as in a and analysed by immunoblotting. Besides phospho-H2AX (Ser139), we also detected phospho-ATM (Ser1981) and phospho- ATR (Ser428), two additional markers of DNA damage response. Intensity of the protein bands were quantified with a Licor/Odyssey scanner and software (shown beneath each immunoblot). The fold change in phosphorylation levels compared with no treatment (untreated) are presented. The data in all panels are representative of at least three independent experiments. Full-length immunoblots are provided in Supplementary Fig. S9.


( a ) Immunoblotting . Jurkat T cells overexpressing ATG5 or control cells were cultured in the presence and absence of 3 - MA for 48 h . 3 - MA blocked the formation of LC3 - II and p62 degradation , indicating that it indeed inhibited ATG5 - induced autophagy . Results are representative of three independent experiments .

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 I-CONTROLLED_VAR 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

(a) Immunoblotting. Jurkat T cells overexpressing ATG5 or control cells were cultured in the presence and absence of 3-MA for 48 h. 3-MA blocked the formation of LC3-II and p62 degradation, indicating that it indeed inhibited ATG5-induced autophagy. Results are representative of three independent experiments.


( b ) Cell cycle analysis . Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3 - MA . Induction of G2 - or M - phase arrest was seen as a consequence of ATG5 overexpression independent of treatment with 3 - MA , as normal Jurkat T cells exhibited no G2 / M arrest following 3 - MA treatment . Representative flow cytometry diagrams are shown ( n = 3 ) . Moreover , induction of autophagy by Beclin 1 overexpression or starvation failed to produce cell cycle arrest ( Supplementary Fig . S4 ) .

O O 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 I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell cycle analysis. Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3-MA. Induction of G2- or M-phase arrest was seen as a consequence of ATG5 overexpression independent of treatment with 3-MA, as normal Jurkat T cells exhibited no G2/M arrest following 3-MA treatment. Representative flow cytometry diagrams are shown (n=3). Moreover, induction of autophagy by Beclin 1 overexpression or starvation failed to produce cell cycle arrest (Supplementary Fig. S4).


( c ) Morphological analysis . Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3 - MA and analysed . Representative examples of morphology are shown ( n = 3 ) ; large multinucleated cells and cells with abnormal nuclei were seen as a consequence of ATG5 overexpression independent of 3 - MA . Neither induction of autophagy by Beclin 1 overexpression nor starvation resulted in such abnormal cells ( Supplementary Fig . S4 ) . 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 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 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

(c) Morphological analysis. Normal Jurkat T cells and cells overexpressing ATG5 were cultured for 48 h in the presence and absence of 3-MA and analysed. Representative examples of morphology are shown (n=3); large multinucleated cells and cells with abnormal nuclei were seen as a consequence of ATG5 overexpression independent of 3-MA. Neither induction of autophagy by Beclin 1 overexpression nor starvation resulted in such abnormal cells (Supplementary Fig. S4). Scale bar, 10 μm. (


( d ) Viability assays . Jurkat T cells either overexpressing ATG5 following lentiviral gene transfer or treated with etoposide were cultured in the presence and absence of the indicated pharmacological inhibitors for the indicated times to block autophagy and caspase activity . All values are means ± s . d . ( n = 3 ) .

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

(d) Viability assays. Jurkat T cells either overexpressing ATG5 following lentiviral gene transfer or treated with etoposide were cultured in the presence and absence of the indicated pharmacological inhibitors for the indicated times to block autophagy and caspase activity. All values are means±s.d. (n=3).


( a ) Subcellular fractionation and immunoblotting . Jurkat T cells were cultured in the presence of etoposide for the indicated times . Following etoposide treatment , the ATG5 monomer was newly present in both cytoplasm and nucleus . The exposure of the ATG5 monomer ( shown under the dotted line ) was longer than that shown above for the ATG5 conjugated to ATG12 . Full - length immunoblots are provided in Supplementary Fig . S10 .

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

(a) Subcellular fractionation and immunoblotting. Jurkat T cells were cultured in the presence of etoposide for the indicated times. Following etoposide treatment, the ATG5 monomer was newly present in both cytoplasm and nucleus. The exposure of the ATG5 monomer (shown under the dotted line) was longer than that shown above for the ATG5 conjugated to ATG12. Full-length immunoblots are provided in Supplementary Fig. S10.


b ) A comparison of ATG5 sequences in different species demonstrates the presence of a conserved putative NES , similar to that present in Beclin 1 . The sequence alignment was performed with Clustal W using sequence data obtained from GenBank . The characteristic leucines are indicated in red and the resulting sequence of ATG5 - ΔNES , following mutation of three leucines within this motif , is shown . ( c )

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

b) A comparison of ATG5 sequences in different species demonstrates the presence of a conserved putative NES, similar to that present in Beclin 1. The sequence alignment was performed with Clustal W using sequence data obtained from GenBank. The characteristic leucines are indicated in red and the resulting sequence of ATG5-ΔNES, following mutation of three leucines within this motif, is shown. (c)


( c ) Subcellular fractionation and immunoblotting of the nuclear fraction . Jurkat T cells , either treated with etoposide , or overexpressing ATG5 following lentiviral gene transfer , were cultured in the presence and absence of leptomycin B for 72 h . Leptomycin B increased relative ATG5 and Beclin 1 levels in the nucleus .

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

(c) Subcellular fractionation and immunoblotting of the nuclear fraction. Jurkat T cells, either treated with etoposide, or overexpressing ATG5 following lentiviral gene transfer, were cultured in the presence and absence of leptomycin B for 72 h. Leptomycin B increased relative ATG5 and Beclin 1 levels in the nucleus.


( d ) Subcellular fractionation and immunoblotting . Jurkat T cells were transduced to express ATG5 - ΔNES , ATG5 , or GFP . Although increased levels of ATG5 were observed in both cytoplasm and nucleus , ATG5 - ΔNES accumulated following lentiviral gene transfer in the cytoplasm only . Full - length immunoblots are provided in Supplementary Fig . S11 .

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

(d) Subcellular fractionation and immunoblotting. Jurkat T cells were transduced to express ATG5-ΔNES, ATG5, or GFP. Although increased levels of ATG5 were observed in both cytoplasm and nucleus, ATG5-ΔNES accumulated following lentiviral gene transfer in the cytoplasm only. Full-length immunoblots are provided in Supplementary Fig. S11.


( e ) Immunoblotting . ATG5 - ΔNES overexpression induced LC3 - II formation and p62 degradation , indicating increased autophagic flux .

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

(e) Immunoblotting. ATG5-ΔNES overexpression induced LC3-II formation and p62 degradation, indicating increased autophagic flux.


( f ) Cell cycle analysis . Jurkat T cells overexpressing ATG5 or ATG5 - ΔNES , as well as control cells were cultured for 48 h . Induction of G2 and M phase arrest was seen as a consequence of ATG5 , but not ATG5 - ΔNES overexpression . As an additional control , we transduced cells with GFP , which , similar to ATG5 - ΔNES - overexpressing cells , failed to exhibit G2 - and M - phase arrest . Representative flow cytometry diagrams are shown ( n = 3 ) .

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

(f) Cell cycle analysis. Jurkat T cells overexpressing ATG5 or ATG5-ΔNES, as well as control cells were cultured for 48 h. Induction of G2 and M phase arrest was seen as a consequence of ATG5, but not ATG5-ΔNES overexpression. As an additional control, we transduced cells with GFP, which, similar to ATG5-ΔNES-overexpressing cells, failed to exhibit G2- and M-phase arrest. Representative flow cytometry diagrams are shown (n=3).


( g ) Morphological analysis . Non - transduced Jurkat T cells and cells overexpressing ATG5 or ATG5 - ΔNES were cultured for 48 h . Large , multinucleated cells were seen as a consequence of ATG5 , but not ATG5 - ΔNES overexpression . Representative examples are shown ( n = 3 ) . 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

(g) Morphological analysis. Non-transduced Jurkat T cells and cells overexpressing ATG5 or ATG5-ΔNES were cultured for 48 h. Large, multinucleated cells were seen as a consequence of ATG5, but not ATG5-ΔNES overexpression. Representative examples are shown (n=3). Scale bar, 10 μm.


( a ) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide ( 48 - h cultures ) were immunoprecipitated with anti - ATG5 or antisurvivin antibodies . A physical interaction between ATG5 and survivin , absent in untreated or GFP - expressing control cells , was detected reciprocally . Full - length immunoblots are provided in Supplementary Fig . S12 .

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

(a) Lysates of Jurkat T cells overexpressing ATG5 or treated with etoposide (48-h cultures) were immunoprecipitated with anti-ATG5 or antisurvivin antibodies. A physical interaction between ATG5 and survivin, absent in untreated or GFP-expressing control cells, was detected reciprocally. Full-length immunoblots are provided in Supplementary Fig. S12.


( b ) Jurkat T cells overexpressing ATG5 - ΔNES exhibited no ATG5 / survivin molecular interaction . ( c ) The ATG5 - K130R mutant did show coprecipitation of survivin with ATG5 .

O O O O O O O O O O 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) Jurkat T cells overexpressing ATG5-ΔNES exhibited no ATG5/survivin molecular interaction. (c) The ATG5-K130R mutant did show coprecipitation of survivin with ATG5.


( d ) Calpain - mediated , truncated ATG5 ( tATG5 ) , that is , the N - terminal part of ATG5 , failed to bind survivin . The ATG5 - K130R mutant was the control .

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

(d) Calpain-mediated, truncated ATG5 (tATG5), that is, the N-terminal part of ATG5, failed to bind survivin. The ATG5-K130R mutant was the control.


( e ) Confocal microscopy . MDA - MA - 231 cells were etoposide treated or transduced as indicated ( 48 - h cultures ) and stained . In untreated cells , survivin and ATG5 were in the cytosol with no colocalization . ATG5 - transduced cells exhibited ATG5 / survivin colocalization in the nucleus . ATG5 - ΔNES - transduced cells showed survivin , but not ATG5 , in the nuclei of cycling cells . Numerical analysis was performed and Pearson ' s correlation coefficients are indicated in the images . Scale bar , 10 μm . Right : Statistical analysis ( analysis of variance ) is presented in which the results of ten representative cells within each group were integrated . Values are means ± s . d .

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

(e) Confocal microscopy. MDA-MA-231 cells were etoposide treated or transduced as indicated (48-h cultures) and stained. In untreated cells, survivin and ATG5 were in the cytosol with no colocalization. ATG5-transduced cells exhibited ATG5/survivin colocalization in the nucleus. ATG5-ΔNES-transduced cells showed survivin, but not ATG5, in the nuclei of cycling cells. Numerical analysis was performed and Pearson's correlation coefficients are indicated in the images. Scale bar, 10 μm. Right: Statistical analysis (analysis of variance) is presented in which the results of ten representative cells within each group were integrated. Values are means±s.d.


( f ) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5 . Neither ATG5 nor HSP90 were detectable in the Aurora B - containing immune precipitate , but ATG5 molecularly interacted with survivin . Results representative of three independent experiments .

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

(f) The physical interaction between Aurora B and survivin was analysed reciprocally and reduced in cells expressing high levels of nuclear ATG5. Neither ATG5 nor HSP90 were detectable in the Aurora B-containing immune precipitate, but ATG5 molecularly interacted with survivin. Results representative of three independent experiments.


( g ) Confocal microscopy . HeLa cells treated with etoposide or transduced as indicated ( 48 - h cultures ) were stained with centromere - specific antiserum and anti - Aurora B ( top panels ) or antisurvivin antibodies ( lower panels ) . In untreated or ATG5 - ΔNES transduced cells , both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres . In contrast , etoposide - treated or ATG5 - transduced cells recruited much less Aurora B to prometaphase centromeres . Such recruitment , however , was observed during anaphase . Compared with cells in normal mitosis , survivin , although reduced , was recruited to centromeres in prometaphase , but , similar to Aurora B , remained attached to centromeres during anaphase . Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects . Colocalization images were prepared using Imaris software . Numerical analysis was performed and correlation coefficients are indicated . Scale bar , 2 μm .

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

(g) Confocal microscopy. HeLa cells treated with etoposide or transduced as indicated (48-h cultures) were stained with centromere-specific antiserum and anti-Aurora B (top panels) or antisurvivin antibodies (lower panels). In untreated or ATG5-ΔNES transduced cells, both Aurora B and survivin were recruited to prometaphase centromeres and located at the central spindle during anaphase where they no longer colocalized with centromeres. In contrast, etoposide-treated or ATG5-transduced cells recruited much less Aurora B to prometaphase centromeres. Such recruitment, however, was observed during anaphase. Compared with cells in normal mitosis, survivin, although reduced, was recruited to centromeres in prometaphase, but, similar to Aurora B, remained attached to centromeres during anaphase. Such mislocation of Aurora B and survivin was associated with severe chromosome alignment and segregation defects. Colocalization images were prepared using Imaris software. Numerical analysis was performed and correlation coefficients are indicated. Scale bar, 2 μm.


( h ) Confocal microscopy . Sections of lung ( non - small - cell lung cancer ) and oesophageal carcinoma stained for ATG5 and survivin expression . In untreated patients , ATG5 expression in cancer cells was slight and mainly cytosolic . Following radio - and / or chemotherapy , ATG5 was preferentially expressed in the nucleus and colocalized with survivin . Overlay images present the average Pearson ' s correlation coefficient as calculated with Imaris . Results are representative of three independent experiments . 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

(h) Confocal microscopy. Sections of lung (non-small-cell lung cancer) and oesophageal carcinoma stained for ATG5 and survivin expression. In untreated patients, ATG5 expression in cancer cells was slight and mainly cytosolic. Following radio- and/or chemotherapy, ATG5 was preferentially expressed in the nucleus and colocalized with survivin. Overlay images present the average Pearson's correlation coefficient as calculated with Imaris. Results are representative of three independent experiments. Scale bar, 10 μm.


( A ) Kaplan - Meier survival curves ( n = 12 - 16 ) .

(A) Kaplan-Meier survival curves (n = 12 - 16).


( C ) Body weight ; ( D ) Heart weight and ( E ) Heart - to - body weight ratio ( n = 28 - 40 ) .

O O O O O O 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) Body weight; (D) Heart weight and (E) Heart-to-body weight ratio (n = 28-40).


( F ) Relative expression levels of cardiac hypertrophy markers ( Nppa and Nppb ) ( n = 5 ) . Bars represent mean ± S . E . M . ( Student ' s t test ; * * p < 0 . 01 , * * * * p < 0 . 0001 ) .

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

(F) Relative expression levels of cardiac hypertrophy markers (Nppa and Nppb) (n = 5). Bars represent mean ± S.E.M. (Student's t test; **p < 0.01, ****p < 0.0001).


( G ) Enzyme histochemistry double staining for COX and SDH activities . ( n = 4 ) .

(G) Enzyme histochemistry double staining for COX and SDH activities. (n = 4).


( H ) Assessment of cardiac fibrosis by Masson ' s trichrome staining ( n = 4 ) . White scale bars , 100μm ; black scale bars , 1 mm .

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

(H) Assessment of cardiac fibrosis by Masson's trichrome staining (n = 4). White scale bars, 100μm; black scale bars, 1 mm.


( A ) Western blot analysis and ( B ) Relative quantification of UPRmt markers in heart extracts . HSC70 is used as a loading control ( CTRL ) , ( n = 3 ) .

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

(A) Western blot analysis and (B) Relative quantification of UPRmt markers in heart extracts. HSC70 is used as a loading control (CTRL), (n = 3).


( C and D ) Relative expression levels normalized to WT of ( C ) established UPRmt markers in 3 - 4 week old mice ( n = 3 - 4 ) .

O O O O O O O O O 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 and D) Relative expression levels normalized to WT of (C) established UPRmt markers in 3 - 4 week old mice (n = 3 - 4).


( C and D ) Relative expression levels normalized to WT of ( D ) mitochondrial chaperones , proteases and TFAM in 3 - 4 week old mice ( n = 3 - 4 ) .

(C and D) Relative expression levels normalized to WT of (D) mitochondrial chaperones, proteases and TFAM in 3 - 4 week old mice (n = 3 - 4).


( E ) Western blot analysis of cellular stress markers . HSC70 is used as a loading control ( CTRL ) , ( n = 3 )

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

(E) Western blot analysis of cellular stress markers. HSC70 is used as a loading control (CTRL), (n = 3)


( F ) Relative expression levels of transcription factors involved in UPRmt and ISR ( left ) and Fgf21 ( right ) ( n = 5 ) . Bars represent mean ± S . E . M . ( Student ' s t test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) .

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

(F) Relative expression levels of transcription factors involved in UPRmt and ISR (left) and Fgf21 (right) (n = 5). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).


( A ) Western blot analysis of individual OXPHOS subunits in heart extracts . HSC70 and Calnexin are used as loading controls , ( n = 3 ) . Antibodies against individual OXPHOS subunits ( on the right ) were used to detect OXPHOS complexes ( on the left ) .

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

(A) Western blot analysis of individual OXPHOS subunits in heart extracts. HSC70 and Calnexin are used as loading controls, (n = 3). Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left).


( B ) BN - PAGE and subsequent Western blot analysis for OXPHOS complexes . Antibodies against individual OXPHOS subunits ( on the right ) were used to detect OXPHOS complexes ( on the left ) .

O O O O O O O O O O O O O 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) BN-PAGE and subsequent Western blot analysis for OXPHOS complexes. Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left).


( C , D ) In gel activity of complexes I ( C ) and IV ( D ) performed after BN - PAGE .

O 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, D) In gel activity of complexes I (C) and IV (D) performed after BN-PAGE.


( E ) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate - glutamate - malate ( C I ) and pyruvate - glutamate - malate + succinate ( C I + C II ) as substrates . State 3 ( substrates + ADP ) ; State 4 ( + oligomycin ) ; Uncoupled ( + FCCP ) . ( n = 3 ) . Bars represent mean ± S . E . M . ( Student ' s t test , * p < 0 . 05 , * * p < 0 . 01 , * * * 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 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate-glutamate-malate (C I) and pyruvate-glutamate-malate + succinate (C I + C II) as substrates. State 3 (substrates+ADP); State 4 (+oligomycin); Uncoupled (+FCCP). (n = 3). Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001).


( A ) Representative gel of the In organello translation analysis of heart mitochondria . De novo synthetized proteins are isolated after labeling with 35S - met ( 1 hour Pulse ) or after cold chase ( 3h Chase ) . Positions of individual proteins are indicated on the left . Position of full - length proteins ( Proficient ) and low molecular weight polypeptides ( Abortive ) are indicated on the right . Note that in WT and ClpP KO low molecular weight polypeptides include ATP8 / ND4L proteins .

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

(A) Representative gel of the In organello translation analysis of heart mitochondria. De novo synthetized proteins are isolated after labeling with 35S-met (1 hour Pulse) or after cold chase (3h Chase). Positions of individual proteins are indicated on the left. Position of full-length proteins (Proficient) and low molecular weight polypeptides (Abortive) are indicated on the right. Note that in WT and ClpP KO low molecular weight polypeptides include ATP8/ND4L proteins.


( B ) Relative overall protein synthesis and turnover rate .

(B) Relative overall protein synthesis and turnover rate.


( C ) Western blot analysis of DARS2 levels . HSC70 is used as a loading control ( CTRL ) .

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

(C) Western blot analysis of DARS2 levels. HSC70 is used as a loading control (CTRL).


( D ) Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level .

(D) Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level.


( E ) Relative levels of protein synthesis of Proficient and Abortive polypeptides . ( F ) Quantification of the turnover rate of full - length ( Proficient ) de novo synthesized proteins . Bars represent mean ± S . E . M . ( Student ' s t test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ) , ( n = 3 - 4 ) .

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 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

(E) Relative levels of protein synthesis of Proficient and Abortive polypeptides. (F) Quantification of the turnover rate of full-length (Proficient) de novo synthesized proteins. Bars represent mean ± S.E.M. (Student's t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), (n=3-4).


Three sex chromosome axes could be detected in the 2 - month - old XUsp26 + XUsp26 - Y spermatocytes . Immunofluorescence analysis of SYCP3 ( red ) and SYCP1 ( green ) in ( J ) , ATR ( red ) and SYCP3 ( green ) in ( K ) was performed in 2 - month - old WT and XUsp26 + XUsp26 - Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrows indicate the Y chromosome . The arrowheads indicate the X chromosome .

Three sex chromosome axes could be detected in the 2-month-old XUsp26+XUsp26-Y spermatocytes. Immunofluorescence analysis of SYCP3 (red) and SYCP1 (green) in (J), ATR (red) and SYCP3 (green) in (K) was performed in 2-month-old WT and XUsp26+XUsp26-Y spermatocytes. Nuclei were stained with DAPI (blue).The arrows indicate the Y chromosome. The arrowheads indicate the X chromosome.


D Histological analysis of the seminiferous tubules and caudal epididymis of the 6 - month - old Usp26 + / Y and Usp26 - / Y 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

D Histological analysis of the seminiferous tubules and caudal epididymis of the 6-month-old Usp26+/Y and Usp26-/Y mice.


F The deletion of Usp26 causes pachytene and meiotic division arrest . Representative PAS - hematoxylin staining in 6 - month - old Usp26 + / Y and Usp26 - / Y seminiferous tubules . Parafﬁn sections from Usp26 + / Y and Usp26 - / Y testes were stained with PAS - hematoxylin . P : pachytene spermatocyte , D : diplotene spermatocyte , rST : round spermatid , eST : elongating spermatid , M : meiotic spermatocyte , aP : apoptotic pachytene spermatocytes , aM : abnormal meiotic divisions .

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

F The deletion of Usp26 causes pachytene and meiotic division arrest. Representative PAS-hematoxylin staining in 6-month-old Usp26+/Y and Usp26-/Y seminiferous tubules. Parafﬁn sections from Usp26+/Y and Usp26-/Y testes were stained with PAS-hematoxylin. P: pachytene spermatocyte, D: diplotene spermatocyte, rST: round spermatid, eST: elongating spermatid, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions.


E The X and Y chromosomes were unpaired in Usp26 - / Y spermatocytes . Immunofluorescence analysis of Chr X - FISH ( green ) , Chr Y - FISH ( red ) , and SYCP3 ( white ) was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . The arrowheads indicate the X chromosome . F Quantification of unpaired X and Y chromosomes in 6 - month - old Usp26 + / Y and Usp26 - / Y mice ( n = 5 independent experiments ) . Red dots indicate Usp26 + / Y mice and green dots indicate Usp26 - / Y mice .

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

E The X and Y chromosomes were unpaired in Usp26-/Y spermatocytes. Immunofluorescence analysis of Chr X-FISH (green), Chr Y-FISH (red), and SYCP3 (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. The arrowheads indicate the X chromosome. F Quantification of unpaired X and Y chromosomes in 6-month-old Usp26+/Y and Usp26-/Y mice (n= 5 independent experiments). Red dots indicate Usp26+/Y mice and green dots indicate Usp26-/Y mice.


Sex chromosome recombination was perturbed in 6 - month - old Usp26 - / Y mice . Immunofluorescence analysis of SYCP3 ( green ) , MRE11 ( red ) ( G ) ; was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrows indicate the sex chromosomes .

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

Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MRE11 (red) (G); was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.


Sex chromosome recombination was perturbed in 6 - month - old Usp26 - / Y mice . Immunofluorescence analysis of ; SYCP3 ( green ) , γH2AX ( red ) ( H ) ; SYCP3 ( green ) , ATR ( red ) , p - ATM ( pink ) ( I ) was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) . The arrows indicate the sex chromosomes .

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

Sex chromosome recombination was perturbed in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of ; SYCP3 (green), γH2AX (red) (H); SYCP3 (green), ATR (red), p-ATM (pink) (I) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue). The arrows indicate the sex chromosomes.


L Crossover on sex chromosomes was impaired in 6 - month - old Usp26 - / Y mice . Immunofluorescence analysis of SYCP3 ( green ) , MLH1 ( red ) , and ATR ( white ) was performed in 6 - month - old Usp26 + / Y and Usp26 - / Y spermatocytes . Nuclei were stained with DAPI ( blue ) .

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

L Crossover on sex chromosomes was impaired in 6-month-old Usp26-/Y mice. Immunofluorescence analysis of SYCP3 (green), MLH1 (red), and ATR (white) was performed in 6-month-old Usp26+/Y and Usp26-/Y spermatocytes. Nuclei were stained with DAPI (blue).


D Representative TUNEL results in Usp26 + / Y and Usp26 - / Y testes . Parafﬁn sections from 6 - month - old Usp26 + / Y and Usp26 - / Y testes were stained with TUNEL ( green ) and DAPI ( blue ) to show dead cells in stage IV , VIII and XII tubules with pachytene and metaphase spermatocytes , respectively . The arrowheads indicate the laggard chromosomes . P : pachytene spermatocytes , rSt : round spermatid , spz : spermatozoa , M : meiotic spermatocyte , aP : apoptotic pachytene spermatocytes , aM : abnormal meiotic divisions , arSt : apoptotic round spermatid .

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

D Representative TUNEL results in Usp26+/Y and Usp26-/Y testes. Parafﬁn sections from 6-month-old Usp26+/Y and Usp26-/Y testes were stained with TUNEL (green) and DAPI (blue) to show dead cells in stage IV, VIII and XII tubules with pachytene and metaphase spermatocytes, respectively. The arrowheads indicate the laggard chromosomes. P: pachytene spermatocytes, rSt: round spermatid, spz: spermatozoa, M: meiotic spermatocyte, aP: apoptotic pachytene spermatocytes, aM: abnormal meiotic divisions, arSt: apoptotic round spermatid.


E The protein level of three known SAC proteins was reduced in 2 - month - old and 6 - month - old Usp26 − / Y mouse testes . Immunoblotting of MAD2 , BUBR1 , PLK1 and USP26 was performed in 2 - month - old and 6 - month - old Usp26 + / Y and Usp26 - / Y testes . H3Ser10p and Tub served as loading control .

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

E The protein level of three known SAC proteins was reduced in 2-month-old and 6-month-old Usp26−/Y mouse testes. Immunoblotting of MAD2, BUBR1, PLK1 and USP26 was performed in 2-month-old and 6-month-old Usp26+/Y and Usp26-/Y testes. H3Ser10p and Tub served as loading control.