Figure 6A . Mitochondria isolated from tumors ( T ) or adjacent normal ( N ) tissues of WT and OMA1 - / - mice were subjected to blue native PAGE ( BN - PAGE ) , and respiratory chain complexes were analyzed by Western blotting with anti - NDUFB8 ( Complex I ) , anti - SDHA ( Complex II ) , anti - COX4L1 ( Complex IV ) , and anti - ATP5A1 ( Complex V , ATP synthase ) antibodies . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative protein levels were further evaluated by densitometry analysis using ImageJ software . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B . Digitonin - solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia ( 1 % O2 ) for 12 hours , and then analyzed by BN - PAGE and immunoblotting using the indicated antibodies against NDUFB8 ( Complex I ) , SDHA ( Complex II ) , NDUFA4 ( Complex IV ) , and ATP5A1 ( Complex V ) . SDS - PAGE with antibody against HSPD1 was used as a loading control . Relative protein levels were further evaluated by densitometry analysis using ImageJ software . Error bars are presented as mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . N . S . , not significant , * p < 0 . 05 . C . The lysates of adjacent normal tissues ( N ) or tumors ( T ) from WT and OMA1 - / - mice were analyzed by Western blotting with antibodies against NDUFA4 , COX4L1 , NDUFB5 , DNUFB6 , UQCRC2 , or Tubulin . Relative protein levels were further evaluated using ImageJ software . Error bars indicate the mean ± SD by a two - way ANOVA ( n = 3 independent experiments ) . * p < 0 . 05 , * * * p < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Mitochondria isolated from tumors (T) or adjacent normal (N) tissues of WT and OMA1-/- mice were subjected to blue native PAGE (BN-PAGE), and respiratory chain complexes were analyzed by Western blotting with anti-NDUFB8 (Complex I), anti-SDHA (Complex II), anti-COX4L1 (Complex IV), and anti-ATP5A1 (Complex V, ATP synthase) antibodies. SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, **p < 0.01, ***p < 0.001.B. Digitonin-solubilized mitochondria were isolated from control and OMA1 KO HCT116 cells cultured in normoxia or hypoxia (1% O2) for 12 hours, and then analyzed by BN-PAGE and immunoblotting using the indicated antibodies against NDUFB8 (Complex I), SDHA (Complex II), NDUFA4 (Complex IV), and ATP5A1 (Complex V). SDS-PAGE with antibody against HSPD1 was used as a loading control. Relative protein levels were further evaluated by densitometry analysis using ImageJ software. Error bars are presented as mean ± SD by a two-way ANOVA (n = 3 independent experiments). N.S., not significant, *p<0.05.C. The lysates of adjacent normal tissues (N) or tumors (T) from WT and OMA1-/- mice were analyzed by Western blotting with antibodies against NDUFA4, COX4L1, NDUFB5, DNUFB6, UQCRC2, or Tubulin. Relative protein levels were further evaluated using ImageJ software. Error bars indicate the mean ± SD by a two-way ANOVA (n = 3 independent experiments). *p<0.05, ***p < 0.001.


figf1 ( a ) Densitometric analysis relative to actin of A53T α - synuclein clearance in stable inducible PC12 cell line expressing A53T α - synuclein . Transgene expression was induced with doxycycline for 48 h , and then switched off ( by removing doxycycline ) , with drug ( all 1 μM ) or DMSO ( control ) treatment for 24 h . Control condition is set to 100 % . Error bars show s . e . m . ( b ) Densitometric analysis relative to actin of soluble EGFP - HDQ74 clearance in stable inducible PC12 cell line expressing EGFP - HDQ74 . Transgene expression was induced with doxycycline for 8 h , and then switched off ( by removing doxycycline ) with drug ( all 1 μM ) or DMSO ( control ) treatment for 96 h . Control condition is set to 100 % . Error bars show s . e . m . ( c ) SK - N - SH cells transfected with EGFP - HDQ74 construct for 4 h were treated with drugs ( all 1 μM ) or DMSO ( control ) for 48 h post - transfection . The proportions of EGFP - positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1 . Error bars show 95 % confidence interval . ( d ) PC12 cells were treated with clonidine , minoxidil and verapamil ( all 1 μM ) or DMSO ( control ) for 24 h . Endogenous LC3 - II levels were detected with anti - LC3 antibody and quantified relative to actin . Rapamycin ( 0 . 2 μM ) was positive control . Error bars show s . e . m . ( e ) The proportions of EGFP - positive cells with EGFP - HDQ74 aggregates in wild - type ( Atg5 + / + ) and knockout ( Atg5 − / − ) Atg5 MEFs , transfected with EGFP - HDQ74 for 4 h and then treated for 48 h with drugs ( all 1 μM ) or DMSO ( control ) . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant . ( a ) Densitometric analysis relative to actin of A53T α - synuclein clearance in stable inducible PC12 cell line expressing A53T α - synuclein . Transgene expression was induced with doxycycline for 48 h , and then switched off ( by removing doxycycline ) , with drug ( all 1 μM ) or DMSO ( control ) treatment for 24 h . Control condition is set to 100 % . Error bars show s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m.(b) Densitometric analysis relative to actin of soluble EGFP-HDQ74 clearance in stable inducible PC12 cell line expressing EGFP-HDQ74. Transgene expression was induced with doxycycline for 8 h, and then switched off (by removing doxycycline) with drug (all 1 μM) or DMSO (control) treatment for 96 h. Control condition is set to 100%. Error bars show s.e.m.(c) SK-N-SH cells transfected with EGFP-HDQ74 construct for 4 h were treated with drugs (all 1 μM) or DMSO (control) for 48 h post-transfection. The proportions of EGFP-positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1. Error bars show 95% confidence interval.(d) PC12 cells were treated with clonidine, minoxidil and verapamil (all 1 μM) or DMSO (control) for 24 h. Endogenous LC3-II levels were detected with anti-LC3 antibody and quantified relative to actin. Rapamycin (0.2 μM) was positive control. Error bars show s.e.m.(e) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs, transfected with EGFP-HDQ74 for 4 h and then treated for 48 h with drugs (all 1 μM) or DMSO (control). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.(a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m.


figf2 ( a ) A53T α - synuclein clearance in stable PC12 cells as in Figure 1a , treated with or without 1 μM rilmenidine , 1 mM dibutyl - cAMP ( db - cAMP ) , 24 μM forskolin or 500 μM 2 ′ 5 ′ - dideoxyadenosine ( 2 ′ 5 ′ ddA ) for 24 h . Note that control clearance levels for a given substrate may vary from figure to figure , due to different exposures of blots , which aid illustration of agents that either increase or retard clearance . ( b ) The proportions of EGFP - positive cells with EGFP - HDQ74 aggregates in SK - N - SH ( for rilmenidine ) and COS - 7 ( for db - cAMP , forskolin and 2 ′ 5 ′ ddA ) cells as in Figure 1c , treated with concentrations used in a for 48 h post - transfection . Error bars show 95 % confidence interval . ( c ) Endogenous LC3 - II levels in PC12 cells treated with 1 μM rilmenidine or 500 μM 2 ′ 5 ′ ddA for 24 h . ( d ) A53T α - synuclein clearance in stable PC12 cells as in Figure 1a , treated with or without 8 - CPT - 2Me - cAMP or 6 - Bnz - cAMP ( 1 μM or 10 μM ) . ( e ) The proportions of EGFP - positive COS - 7 cells with aggregates as in Figure 1c , treated with or without 10 μM 8 - CPT - 2Me - cAMP or 6 - Bnz - cAMP for 48 h post - transfection . Error bars show 95 % confidence interval . ( f ) COS - 7 cells , transfected for 4 h with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) , dominant - negative S17N Rap2B ( DN Rap2B ) or wild - type PLC - ε ( 1 : 3 ratio ) , were assessed for the proportions of EGFP - positive cells with aggregates after 48 h . Error bars show 95 % confidence interval . ( g ) Endogenous LC3 - II levels in COS - 7 cells transfected with a dominant - negative Rap2B ( DN Rap2B ) for 4 h were assessed after 48 h expression . ( h ) The proportions of EGFP - positive COS - 7 cells with aggregates , transfected with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) or dominant - negative Rap2B ( 1 : 3 ratio ) for 4 h , were treated with or without 10 μM 8 - CPT - 2Me - cAMP for 48 h . Error bars show 95 % confidence interval . ( i ) COS - 7 cells , transfected with EGFP - HDQ74 and dsRed2 or cytosolic dsRed2 - IP3 kinase A ( 1 : 3 ratio ) for 4 h , were assessed at 48 h post - transfection for aggregation and cell death ( as in Fig . 1c ) . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 1 μM rilmenidine, 1 mM dibutyl-cAMP (db-cAMP), 24 μM forskolin or 500 μM 2′5′-dideoxyadenosine (2′5′ddA) for 24 h. Note that control clearance levels for a given substrate may vary from figure to figure, due to different exposures of blots, which aid illustration of agents that either increase or retard clearance.(b) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in SK-N-SH (for rilmenidine) and COS-7 (for db-cAMP, forskolin and 2′5′ddA) cells as in Figure 1c, treated with concentrations used in a for 48 h post-transfection. Error bars show 95% confidence interval.(c) Endogenous LC3-II levels in PC12 cells treated with 1 μM rilmenidine or 500 μM 2′5′ddA for 24 h.(d) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 8-CPT-2Me-cAMP or 6-Bnz-cAMP (1 μM or 10 μM).(e) The proportions of EGFP-positive COS-7 cells with aggregates as in Figure 1c, treated with or without 10 μM 8-CPT-2Me-cAMP or 6-Bnz-cAMP for 48 h post-transfection. Error bars show 95% confidence interval.(f) COS-7 cells, transfected for 4 h with EGFP-HDQ74 along with empty vector (pcDNA3.1), dominant-negative S17N Rap2B (DN Rap2B) or wild-type PLC-ε (1:3 ratio), were assessed for the proportions of EGFP-positive cells with aggregates after 48 h. Error bars show 95% confidence interval.(g) Endogenous LC3-II levels in COS-7 cells transfected with a dominant-negative Rap2B (DN Rap2B) for 4 h were assessed after 48 h expression.(h) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or dominant-negative Rap2B (1:3 ratio) for 4 h, were treated with or without 10 μM 8-CPT-2Me-cAMP for 48 h. Error bars show 95% confidence interval.(i) COS-7 cells, transfected with EGFP-HDQ74 and dsRed2 or cytosolic dsRed2-IP3 kinase A (1:3 ratio) for 4 h, were assessed at 48 h post-transfection for aggregation and cell death (as in Fig. 1c). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf3 ( a ) SK - N - SH cells were pretreated with or without 10 , 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM ( ± ) - Bay K8644 for 4 h . Calpain activity was detected by immunoblotting with anti - calpain small subunit antibody . Densitometry analysis is relative to actin . Error bars show s . e . m . ( b ) SK - N - SH cells transfected with EGFP - HDQ74 for 4 h , then pretreated with or without 10 , 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM ( ± ) - Bay K8644 for 48 h post - transfection , were assessed for the proportion of EGFP - positive cells with EGFP - HDQ74 aggregates . Error bars show 95 % confidence interval . ( c ) SK - N - SH cells were pretreated with or without 10 , 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM ( ± ) - Bay K8644 for 4 h . Endogenous LC3 - II levels were detected by immunoblotting with anti - LC3 antibody . ( d ) Comparison between calpain activity , EGFP - HDQ74 aggregation and autophagosomes as seen in a - c . The control condition for all the assessments was set at 100 % . Calpastatin lowered the increased calpain activity and EGFP - HDQ74 aggregation caused by ( ± ) - Bay K8644 in a dose - dependent manner with a simultaneous increase in LC3 - II levels ( autophagosomes ) . ( e ) COS - 7 cells transfected with empty vector ( pcDNA3 . 1 ) or wild - type PLC - ε for 4 h and treated with or without 50 μM calpeptin for 48 h post - transfection were assessed for the proportion of EGFP - positive cells with EGFP - HDQ74 aggregates . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Calpain activity was detected by immunoblotting with anti-calpain small subunit antibody. Densitometry analysis is relative to actin. Error bars show s.e.m.(b) SK-N-SH cells transfected with EGFP-HDQ74 for 4 h, then pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 48 h post-transfection, were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval.(c) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Endogenous LC3-II levels were detected by immunoblotting with anti-LC3 antibody.(d) Comparison between calpain activity, EGFP-HDQ74 aggregation and autophagosomes as seen in a-c. The control condition for all the assessments was set at 100%. Calpastatin lowered the increased calpain activity and EGFP-HDQ74 aggregation caused by (±)-Bay K8644 in a dose-dependent manner with a simultaneous increase in LC3-II levels (autophagosomes).(e) COS-7 cells transfected with empty vector (pcDNA3.1) or wild-type PLC-ε for 4 h and treated with or without 50 μM calpeptin for 48 h post-transfection were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf4 ( a ) The proportions of EGFP - positive SK - N - SH cells with EGFP - HDQ74 aggregates or cell death as in Figure 1c , treated with or without 10 μM calpastatin , 50 μM N - acetyl - Leu - Leu - methional ( ALLM ) or 50 μM calpeptin for 48 h post - transfection . DMSO was control for ALLM and calpeptin . Error bars show 95 % confidence interval . ( b ) HeLa cells were transfected with EGFP - HDQ74 construct along with siGLO ( control siRNA ) in presence or absence of calpain 1 or calpain 2 siRNA ( 1 : 3 ratio ) for 96 h . EGFP - and siGLO - positive cells were assessed for aggregation and cell death as in Figure 1c . Error bars show 95 % confidence interval . ( c ) HeLa cells , transfected with siGLO or siRNAs for calpain 1 or calpain 2 for 96 h , were analyzed with anti - calpain 1 or anti - calpain 2 domain III antibodies . ( d ) Clearance of soluble EGFP - HDQ74 in stable PC12 cells as in Figure 1b , treated with drugs ( concentrations in a ) for 96 h . Densitometry is relative to actin . Error bars show s . e . m . ( e ) A53T α - synuclein clearance in undifferentiated or nonmitotic neuronally differentiated ( with 100 ng ml - 1 nerve growth factor for 5 d ) stable PC12 cells as in Figure 1a , treated with or without 10 μM calpastatin for 24 h . Densitometry is relative to actin . Error bars show s . e . m . ( f ) COS - 7 cells , transfected with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) or constitutive active ( CA ) m - calpain ( 1 : 3 ratio ) for 48 h , were assessed for aggregation and cell death as in Figure 1c . Error bars show 95 % confidence interval . ( g ) SK - N - SH cells , transfected with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) or constitutive active m - calpain ( 1 : 3 ratio ) for 4 h , were subsequently treated with DMSO ( control ) , 1 μM verapamil or 1 μM clonidine for 48 h . The proportions of EGFP - positive cells with aggregates were assessed as in Figure 1c . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) The proportions of EGFP-positive SK-N-SH cells with EGFP-HDQ74 aggregates or cell death as in Figure 1c, treated with or without 10 μM calpastatin, 50 μM N-acetyl-Leu-Leu-methional (ALLM) or 50 μM calpeptin for 48 h post-transfection. DMSO was control for ALLM and calpeptin. Error bars show 95% confidence interval.(b) HeLa cells were transfected with EGFP-HDQ74 construct along with siGLO (control siRNA) in presence or absence of calpain 1 or calpain 2 siRNA (1:3 ratio) for 96 h. EGFP- and siGLO-positive cells were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval.(c) HeLa cells, transfected with siGLO or siRNAs for calpain 1 or calpain 2 for 96 h, were analyzed with anti-calpain 1 or anti-calpain 2 domain III antibodies.(d) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with drugs (concentrations in a) for 96 h. Densitometry is relative to actin. Error bars show s.e.m.(e) A53T α-synuclein clearance in undifferentiated or nonmitotic neuronally differentiated (with 100 ng ml-1 nerve growth factor for 5 d) stable PC12 cells as in Figure 1a, treated with or without 10 μM calpastatin for 24 h. Densitometry is relative to actin. Error bars show s.e.m.(f) COS-7 cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active (CA) m-calpain (1:3 ratio) for 48 h, were assessed for aggregation and cell death as in Figure 1c. Error bars show 95% confidence interval.(g) SK-N-SH cells, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or constitutive active m-calpain (1:3 ratio) for 4 h, were subsequently treated with DMSO (control), 1 μM verapamil or 1 μM clonidine for 48 h. The proportions of EGFP-positive cells with aggregates were assessed as in Figure 1c. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05.


figf5 ( a ) The proportions of EGFP - positive MEFs with aggregates , transfected with EGFP - HDQ74 and pcDNA3 . 1 ( empty vector ) in Atg5 + / + cells , or transfected with EGFP - HDQ74 and pcDNA3 . 1 , WT Atg5 or K130R Atg5 constructs in Atg5 - / - cells for 4 h ( 1 : 3 ratios ) , were treated for next 48 h with or without 10 μM calpastatin . All data are from the same experiment , but odds ratios for reference conditions in specific experiments have been set to 1 , to facilitate comparisons . Atg5 - / - cells had increased EGFP - HDQ74 aggregates compared to Atg5 + / + cells . Error bars show 95 % confidence interval . ( b ) HeLa cells were transfected with siGLO ( control ) with or without calpain 1 siRNA or calpain 2 siRNA ( 1 : 3 ratio ) for 96 h , followed by transfection with EGFP - LC3 construct for 4 h . Cells were fixed after a further 2 h . The proportions of transfected cells with > 5 EGFP - LC3 vesicles were assessed . Error bars show 95 % confidence interval . ( c ) Endogenous LC3 - II levels in SK - N - SH cells treated with or without 10 μM or 30 μM calpastatin for 24 h . ( d ) HeLa cells stably expressing EGFP - LC3 were treated for 4 h with 400 nM bafilomycin A1 in presence or absence of 10 μM calpastatin , 50 μM ALLM or 50 μM calpeptin . Calpain inhibitors were treated for 24 h before bafilomycin A1 addition . EGFP - LC3 - II was detected with anti - EGFP antibody . Data from different calpain inhibitors are from different blots . ( e ) COS - 7 cells , transfected with myc - LC3 and EGFP - C1 with pcDNA3 . 1 ( empty vector ) or constitutively active ( CA ) m - calpain ( 1 : 1 : 3 ratio ) for 4 h , were analyzed after 24 h post - transfection for LC3 - II levels by immunoblotting with anti - myc antibody . Error bars show s . e . m . ( f ) COS - 7 cells , transfected with EGFP - LC3 and pcDNA3 . 1 ( empty vector ) or CA m - calpain ( 1 : 3 ratio ) for 4 h and fixed 24 h post - transfection , were assessed for proportions of EGFP - positive cells with > 5 EGFP - LC3 vesicles . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) The proportions of EGFP-positive MEFs with aggregates, transfected with EGFP-HDQ74 and pcDNA3.1 (empty vector) in Atg5+/+ cells, or transfected with EGFP-HDQ74 and pcDNA3.1, WT Atg5 or K130R Atg5 constructs in Atg5-/- cells for 4 h (1:3 ratios), were treated for next 48 h with or without 10 μM calpastatin. All data are from the same experiment, but odds ratios for reference conditions in specific experiments have been set to 1, to facilitate comparisons. Atg5-/- cells had increased EGFP-HDQ74 aggregates compared to Atg5+/+ cells. Error bars show 95% confidence interval.(b) HeLa cells were transfected with siGLO (control) with or without calpain 1 siRNA or calpain 2 siRNA (1:3 ratio) for 96 h, followed by transfection with EGFP-LC3 construct for 4 h. Cells were fixed after a further 2 h. The proportions of transfected cells with >5 EGFP-LC3 vesicles were assessed. Error bars show 95% confidence interval.(c) Endogenous LC3-II levels in SK-N-SH cells treated with or without 10 μM or 30 μM calpastatin for 24 h.(d) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with 400 nM bafilomycin A1 in presence or absence of 10 μM calpastatin, 50 μM ALLM or 50 μM calpeptin. Calpain inhibitors were treated for 24 h before bafilomycin A1 addition. EGFP-LC3-II was detected with anti-EGFP antibody. Data from different calpain inhibitors are from different blots.(e) COS-7 cells, transfected with myc-LC3 and EGFP-C1 with pcDNA3.1 (empty vector) or constitutively active (CA) m-calpain (1:1:3 ratio) for 4 h, were analyzed after 24 h post-transfection for LC3-II levels by immunoblotting with anti-myc antibody. Error bars show s.e.m.(f) COS-7 cells, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or CA m-calpain (1:3 ratio) for 4 h and fixed 24 h post-transfection, were assessed for proportions of EGFP-positive cells with >5 EGFP-LC3 vesicles. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf6 ( a ) A53T α - synuclein clearance in stable PC12 cells as in Figure 1a , treated with or without 100 nM PACAP , with or without 10 μM calpastatin or 500 μM 2 ′ 5 ′ ddA for 24 h . ( b ) The proportion of EGFP - positive cells with aggregates in SK - N - SH cells as in Figure 1c , treated for 48 h with 1 μM PACAP or 200 μM NF449 . Error bars show 95 % confidence interval . ( c ) Aggregation in COS - 7 cells transfected with either pcDNA3 : 1 ( empty vector ) or constitutively active ( CA ) m - calpain and EGFP - HDQ74 ( 3 : 1 ratio ) for 4 h and then treated with or without 500 μM 2 ′ 5 ′ ddA for 48 h . Error bars show 95 % confidence interval . ( d ) HeLa cells transfected with control or Gsα siRNA for 72 h were analyzed for Gsα levels by immunoblotting with anti - Gsα antibody . Aggregation was assessed in HeLa cells transfected with control or Gsα siRNA and EGFP - HDQ74 for 72 h . Error bars show 95 % confidence interval . ( e ) Endogenous LC3 - II levels in HeLa cells transfected with control or Gsα siRNA for 72 h . ( f ) Endogenous LC3 - II levels in PC12 cells treated with or without 200 μM NF449 for 24 h . ( g ) Endogenous LC3 - II levels in HeLa cells transfected with control or Gsα siRNA for 72 h and treated with or without 400 nM bafilomycin A1 for the last 4 h . Densitometric analysis is relative to actin . Error bars show s . e . m . ( h ) The proportions of EGFP - positive HeLa cells with aggregates , transfected with control or Gsα siRNA for 48 h , and then retransfected together with pcDNA 3 . 1 ( empty vector ) or CA m - calpain and EGFP - HDQ74 ( 3 : 1 ratio ) for a further 48 h . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 100 nM PACAP, with or without 10 μM calpastatin or 500 μM 2′5′ddA for 24 h.(b) The proportion of EGFP-positive cells with aggregates in SK-N-SH cells as in Figure 1c, treated for 48 h with 1 μM PACAP or 200 μM NF449. Error bars show 95% confidence interval.(c) Aggregation in COS-7 cells transfected with either pcDNA3:1 (empty vector) or constitutively active (CA) m-calpain and EGFP-HDQ74 (3:1 ratio) for 4 h and then treated with or without 500 μM 2′5′ddA for 48 h. Error bars show 95% confidence interval.(d) HeLa cells transfected with control or Gsα siRNA for 72 h were analyzed for Gsα levels by immunoblotting with anti-Gsα antibody. Aggregation was assessed in HeLa cells transfected with control or Gsα siRNA and EGFP-HDQ74 for 72 h. Error bars show 95% confidence interval.(e) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h.(f) Endogenous LC3-II levels in PC12 cells treated with or without 200 μM NF449 for 24 h.(g) Endogenous LC3-II levels in HeLa cells transfected with control or Gsα siRNA for 72 h and treated with or without 400 nM bafilomycin A1 for the last 4 h. Densitometric analysis is relative to actin. Error bars show s.e.m.(h) The proportions of EGFP-positive HeLa cells with aggregates, transfected with control or Gsα siRNA for 48 h, and then retransfected together with pcDNA 3.1 (empty vector) or CA m-calpain and EGFP-HDQ74 (3:1 ratio) for a further 48 h. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf7 ( a ) The proportions of EGFP - positive COS - 7 cells with aggregates or cell death , after transfection with EGFP - HDQ74 and rheb or pcDNA3 . 1 ( empty vector ) ( 1 : 3 ratio ) for 4 h and treatment with or without 10 μM calpastatin or 50 μM calpeptin for 48 h post - transfection . The control condition for assessing the effect of calpain inhibitors in rheb - transfected cells was taken as 1 . Error bars show 95 % confidence interval . ( b ) The proportions of EGFP - positive COS - 7 cells with > 5 EGFP - LC3 vesicles , transfected with EGFP - LC3 and pcDNA3 . 1 ( empty vector ) or rheb ( 1 : 3 ratio ) for 4 h and treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post - transfection . Error bars show 95 % confidence interval . ( c ) The proportions of EGFP - positive COS - 7 cells with aggregates , transfected with EGFP - HDQ74 and constitutively active ( CA ) m - calpain or pcDNA3 . 1 ( empty vector ) ( 1 : 3 ratio ) for 4 h and treated with or without 0 . 2 μM rapamycin for 48 h post - transfection . Error bars show 95 % confidence interval . ( d ) The proportions of EGFP - positive COS - 7 cells with > 5 EGFP - LC3 vesicles , transfected with EGFP - LC3 and CA m - calpain or pcDNA3 . 1 ( empty vector ) ( 1 : 3 ratio ) for 4 h and treated with or without 0 . 2 μM rapamycin for 24 h post - transfection . Error bars show 95 % confidence interval . ( e , f ) The proportions of EGFP - positive COS - 7 cells with aggregates ( e ) and cell death ( f ) as in Figure 1c , treated with or without 0 . 2 μM rapamycin , 10 μM calpastatin or both for 48 h . Error bars show 95 % confidence interval . ( g ) Clearance of soluble EGFP - HDQ74 in stable PC12 cells as in Figure 1b , treated with or without 0 . 2 μM rapamycin , 10 μM calpastatin or both for the 48 h switch - off period . Error bars show s . e . m . ( h ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1a , treated with or without 0 . 2 μM rapamycin , 10 μM calpastatin or both for the 8 h switch - off period . Error bars show s . e . m . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) The proportions of EGFP-positive COS-7 cells with aggregates or cell death, after transfection with EGFP-HDQ74 and rheb or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treatment with or without 10 μM calpastatin or 50 μM calpeptin for 48 h post-transfection. The control condition for assessing the effect of calpain inhibitors in rheb-transfected cells was taken as 1. Error bars show 95% confidence interval.(b) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and pcDNA3.1 (empty vector) or rheb (1:3 ratio) for 4 h and treated with or without 10 μM calpastatin or 50 μM calpeptin for 24 h post-transfection. Error bars show 95% confidence interval.(c) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 and constitutively active (CA) m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 48 h post-transfection. Error bars show 95% confidence interval.(d) The proportions of EGFP-positive COS-7 cells with >5 EGFP-LC3 vesicles, transfected with EGFP-LC3 and CA m-calpain or pcDNA3.1 (empty vector) (1:3 ratio) for 4 h and treated with or without 0.2 μM rapamycin for 24 h post-transfection. Error bars show 95% confidence interval.(e,f) The proportions of EGFP-positive COS-7 cells with aggregates (e) and cell death (f) as in Figure 1c, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for 48 h. Error bars show 95% confidence interval.(g) Clearance of soluble EGFP-HDQ74 in stable PC12 cells as in Figure 1b, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 48 h switch-off period. Error bars show s.e.m.(h) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1a, treated with or without 0.2 μM rapamycin, 10 μM calpastatin or both for the 8 h switch-off period. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf8 ( a ) Western blot showing EGFP - HDQ71 - induced degeneration of rod photoreceptors at 9 d . p . f . as indicated by the decrease in the abundance of of monomeric ( ∼ 36 kDa ) and dimeric ( ∼ 72 kDa ) rhodopsin . Rhodopsin expression is further reduced by 3 μM ( ± ) - Bay K8644 , and recovered upon treatment with 3 μM verapamil , 100 μM 2 ′ 5 ′ ddA , 3 μM clonidine and 1 μM calpastatin . No general toxicity to the zebrafish at these concentrations was observed . ( b ) Expression of EGFP - HDQ71 significantly reduces the levels of rhodopsin expression when compared to wild - type ( WT ) nontransgenic controls . This effect is further abrogated by 3 μM ( ± ) - Bay K8644 , whereas 3 μM verapamil , 100 μM 2 ′ 5 ′ ddA , 3 μM clonidine and 1 μM calpastatin all protected against rod photoreceptor degeneration . Error bars show s . e . m . ( c ) Magnified bright field and EGFP images of zebrafish eye sections , taken at the ventral marginal zone for each of the indicated treatments ( with concentrations as in b ) , are shown . Scale bar , 10 μm . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 . ( d ) The total number of aggregates across 8 × 12 μm sections was counted and compared to the EGFP - HDQ71 DMSO - treated control . 3 μM ( ± ) - Bay K8644 was found to significantly increase the aggregate burden , whereas 3 μM verapamil , 1 μM calpastatin , 3 μM clonidine and 100 μM 2 ′ 5 ′ ddA decreased the aggregate load . As in our cell models , these aggregates were insoluble in 4 % triton / SDS . Error bars show s . e . m . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8(a) Western blot showing EGFP-HDQ71-induced degeneration of rod photoreceptors at 9 d.p.f. as indicated by the decrease in the abundance of of monomeric (∼36 kDa) and dimeric (∼72 kDa) rhodopsin. Rhodopsin expression is further reduced by 3 μM (±)-Bay K8644, and recovered upon treatment with 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin. No general toxicity to the zebrafish at these concentrations was observed. (b) Expression of EGFP-HDQ71 significantly reduces the levels of rhodopsin expression when compared to wild-type (WT) nontransgenic controls. This effect is further abrogated by 3 μM (±)-Bay K8644, whereas 3 μM verapamil, 100 μM 2′5′ddA, 3 μM clonidine and 1 μM calpastatin all protected against rod photoreceptor degeneration. Error bars show s.e.m.(c) Magnified bright field and EGFP images of zebrafish eye sections, taken at the ventral marginal zone for each of the indicated treatments (with concentrations as in b), are shown. Scale bar, 10 μm. ***P 0.001; **P 0.01; *P 0.05. (d) The total number of aggregates across 8 × 12 μm sections was counted and compared to the EGFP-HDQ71 DMSO-treated control. 3 μM (±)-Bay K8644 was found to significantly increase the aggregate burden, whereas 3 μM verapamil, 1 μM calpastatin, 3 μM clonidine and 100 μM 2′5′ddA decreased the aggregate load. As in our cell models, these aggregates were insoluble in 4% triton/SDS. Error bars show s.e.m. ***P 0.001; **P 0.01; *P 0.05.


Figure 1Uncoupled ( LEAK ) mitochondrial respiration of soleus ( SOL ) and extensor digitorum longus ( EDL ) muscle of wild type ( WT ) vs . Ucp1 - TG ( TG ) mice ( WT n = 9 , TG n = 5 ) . Relative mRNA expression in quadriceps ( Quad ) of ISR components ( n = 8 per genotype ) . Representative immunoblots of ISR component eIF2α and quantification of phospho - eIF2α ( p - eIF2aSer51 ) relative protein expression in quadriceps skeletal muscle ( WT n = 3 , TG n = 4 ) . Multi - tissue transcriptomic profiling of Gdf15 gene expression . Heatmap is shown as raw ct expression values ( n = 4 per genotype ) . Quantification of Gdf15 mRNA expression in TG mice is shown as fold change compared to WT littermates ( WT n = 5 , TG n = 4 ) . Skeletal muscle ( Quad and Gastroc ) GDF15 protein content normalized to total protein content ( WT n = 9 , TG n = 18 ) . Ex vivo secretion of GDF15 from SOL and EDL muscle of WT vs . TG mice ( n = 6 per genotype ) after 2h incubation normalized to muscle wet weight ( mg ) . Mouse GDF15 plasma levels in post - absorptive state of male and female WT vs . TG mice at 20wks of age ( male WT n = 8 , TG n = 9 ; female WT n = 5 , TG n = 6 ) . Mouse GDF15 plasma levels in post - absorptive state of male WT vs . TG mice fed low - fat diet ( LFD ) or high - fat diet ( HFD ) at 24wks of age ( LFD WT n = 6 , TG n = 5 ; HFD WT n = 5 , TG n = 5 ) . Mouse GDF15 plasma levels in post - absorptive state of young ( 10wks ) vs . old ( 95wks ) male WT and TG mice ( young WT n = 10 , TG n = 13 ; old WT n = 13 , TG n = 13 ) . Gdf15 gene expression of differentiated C2C12 muscle cells treated with vehicle control ( Ctrl ) or chemical mitochondrial uncoupler ( FCCP , 1µM vs . 5µM ) for 5hrs ( n = 3 biological replicates )

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Figure 1Uncoupled (LEAK) mitochondrial respiration of soleus (SOL) and extensor digitorum longus (EDL) muscle of wild type (WT) vs. Ucp1-TG (TG) mice (WT n=9, TG n=5).Relative mRNA expression in quadriceps (Quad) of ISR components (n=8 per genotype).Representative immunoblots of ISR component eIF2α and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression in quadriceps skeletal muscle (WT n=3, TG n=4).Multi-tissue transcriptomic profiling of Gdf15 gene expression. Heatmap is shown as raw ct expression values (n=4 per genotype). Quantification of Gdf15 mRNA expression in TG mice is shown as fold change compared to WT littermates (WT n=5, TG n=4).Skeletal muscle (Quad and Gastroc) GDF15 protein content normalized to total protein content (WT n=9, TG n=18).Ex vivo secretion of GDF15 from SOL and EDL muscle of WT vs. TG mice (n=6 per genotype) after 2h incubation normalized to muscle wet weight (mg).Mouse GDF15 plasma levels in post-absorptive state of male and female WT vs. TG mice at 20wks of age (male WT n=8, TG n=9; female WT n=5, TG n=6).Mouse GDF15 plasma levels in post-absorptive state of male WT vs. TG mice fed low-fat diet (LFD) or high-fat diet (HFD) at 24wks of age (LFD WT n=6, TG n=5; HFD WT n=5, TG n=5).Mouse GDF15 plasma levels in post-absorptive state of young (10wks) vs. old (95wks) male WT and TG mice (young WT n=10, TG n=13; old WT n=13, TG n=13).Gdf15 gene expression of differentiated C2C12 muscle cells treated with vehicle control (Ctrl) or chemical mitochondrial uncoupler (FCCP, 1µM vs. 5µM) for 5hrs (n=3 biological replicates)


Figure 2Genotyping PCR panel of Gdf15 and HSA - Ucp1 loci shown for wild type ( WT ) , Gdf15 - KO ( KO ) , Ucp1 - TG ( TG ) and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice . Relative mRNA expression in quadriceps ( Quad ) of Ucp1 and Gdf15 in 20wks male WT ( n = 8 ) , TG ( n = 7 ) and TGxKO ( n = 6 ) mice . Plasma mGDF15 levels at 20wks of age ( WT n = 9 , TG n = 10 , TGxKO n = 9 ) . Voluntary wheel running ( VWR ) activity shown hourly over 24 - hrs at 15 wks of age ( n = 4 per genotype ) . Grip strength at 10 - 20wks of age ( WT n = 15 , TG n = 14 , TGxKO n = 11 ) . Skeletal muscle mass relative to body lean mass of quadriceps ( Quad ) and gastrocnemius ( Gastroc ) from mice at 95wk of age ( WT n = 8 , TG n = 5 , TGxKO n = 5 ) . Representative H & E histological staining of tibialis anterior ( TA ) muscle ( G ) ( scale bars represent 50 µm ) , and cross - sectional area ( CSA ) of myofibers ( H ) at 95wks ( WT n = 12 , TG n = 8 , TGxKO n = 8 ) . Representative traces of oxygen consumption rate ( OCR ) during substrate - uncoupler - inhibitor - titration ( SUIT ) protocol for mitochondrial respiratory capacity in permeabilized mouse soleus muscle fibers , PM ( Pyruvate + Malate ; LEAK respiration ) , ADP ( OXPHOS capacity ) , Cyt c ( cytochrome c , integrity of outer mt - membrane ) , G ( glutamate ; ) , S ( succinate ) , U ( Uncoupler , FCCP ) , Rot ( rotenone ) , Ama ( antimycin A ; less than 2 % residual oxygen consumption , ROX ) . Mitochondrial respiratory capacity ( oxygen consumption rate , OCR ) from oxidative soleus ( SOL ) ( J ) and glycolytic extensor digitorum longus ( EDL ) muscle fibers ( K ) , and NetOXPHOS control ratio of 95wks old mice ( L ) ( n = 5per genotype ) . Quadriceps ( Quad ) relative mRNA expression of ISR components at 95wks of age ( WT n = 8 , TG n = 5 , TGxKO n = 5 ) . Representative immunoblots of ISR component eIF2α in quadriceps ( Quad ) muscle of 95wks old mice . Gastrocnemius enzyme activity of NQO1 and GPX in 95wks old male mice ( WT n = 8 , TG n = 5 , TGxKO n = 5 ) .

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Figure 2Genotyping PCR panel of Gdf15 and HSA-Ucp1 loci shown for wild type (WT), Gdf15-KO (KO), Ucp1-TG (TG) and Ucp1-TGxGdf15-KO (TGxKO) mice.Relative mRNA expression in quadriceps (Quad) of Ucp1 and Gdf15 in 20wks male WT (n=8), TG (n=7) and TGxKO (n=6) mice.Plasma mGDF15 levels at 20wks of age (WT n=9, TG n=10, TGxKO n=9).Voluntary wheel running (VWR) activity shown hourly over 24-hrs at 15 wks of age (n=4 per genotype).Grip strength at 10 - 20wks of age (WT n=15, TG n=14, TGxKO n=11).Skeletal muscle mass relative to body lean mass of quadriceps (Quad) and gastrocnemius (Gastroc) from mice at 95wk of age (WT n=8, TG n=5, TGxKO n=5).Representative H&E histological staining of tibialis anterior (TA) muscle (G) (scale bars represent 50 µm), and cross-sectional area (CSA) of myofibers (H) at 95wks (WT n=12, TG n=8, TGxKO n=8).Representative traces of oxygen consumption rate (OCR) during substrate-uncoupler-inhibitor-titration (SUIT) protocol for mitochondrial respiratory capacity in permeabilized mouse soleus muscle fibers, PM (Pyruvate+Malate; LEAK respiration), ADP (OXPHOS capacity), Cyt c (cytochrome c, integrity of outer mt-membrane), G (glutamate;), S (succinate), U (Uncoupler, FCCP), Rot (rotenone), Ama (antimycin A; less than 2% residual oxygen consumption, ROX).Mitochondrial respiratory capacity (oxygen consumption rate, OCR) from oxidative soleus (SOL) (J) and glycolytic extensor digitorum longus (EDL) muscle fibers (K), and NetOXPHOS control ratio of 95wks old mice (L) (n=5per genotype).Quadriceps (Quad) relative mRNA expression of ISR components at 95wks of age (WT n=8, TG n=5, TGxKO n=5).Representative immunoblots of ISR component eIF2α in quadriceps (Quad) muscle of 95wks old mice.Gastrocnemius enzyme activity of NQO1 and GPX in 95wks old male mice (WT n=8, TG n=5, TGxKO n=5).


Figure 3Body mass ( A ) , body lean mass ( B ) and body fat mass ( C ) development . Data are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) miceBody mass ( D ) , body lean mass ( E ) and body fat mass ( F ) during aging at 20wks , 45wks and 95wks of age . Data are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) miceSubcutaneous white adipose tissue ( sWAT ) ( G ) and epididymal white adipose tissue ( eWAT ) ( H ) mass development at 10wks , 20wks , 45wks and 95wks of age . Data are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 10wks ( WT n = 6 , TG n = 7 , TGxKO n = 5 ) , 20wks ( WT n = 10 , TG n = 10 , TGxKO n = 10 ) , 45wks ( WT n = 5 , TG n = 6 , TGxKO n = 7 ) and 95wks ( WT n = 8 , TG n = 5 , TGxKO n = 5 ) .

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Figure 3Body mass (A), body lean mass (B) and body fat mass (C) development. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) miceBody mass (D), body lean mass (E) and body fat mass (F) during aging at 20wks, 45wks and 95wks of age. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) miceSubcutaneous white adipose tissue (sWAT) (G) and epididymal white adipose tissue (eWAT) (H) mass development at 10wks, 20wks, 45wks and 95wks of age. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 10wks (WT n=6, TG n=7, TGxKO n=5), 20wks (WT n=10, TG n=10, TGxKO n=10), 45wks (WT n=5, TG n=6, TGxKO n=7) and 95wks (WT n=8, TG n=5, TGxKO n=5).


Figure 4Representative H & E histological staining of sWAT at 20wks of age ( scale bars represent 50 μm ) Data are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice . relative mRNA expression profile in sWAT of male mice ( WT n = 7 , TG n = 8 , TGxKO n = 8 ) ( B ) . Data are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice . Representative immunoblots ( C ) and quantification ( D ) of UCP1 protein expression in sWAT of male mice at 20wks of age ( WT n = 4 , TG n = 6 , TGxKO n = 4 ) . Data are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice . Relative mRNA expression profile in eWAT ( WT n = 7 , TG n = 8 , TGxKO n = 8 ) . Data are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice . Plasma FGF21 levels from male mice at 20wks of age ( n = 10 per genotype ) . Plasma Leptin levels from male mice at 20wks of age ( n = 5 per genotype ) . Post - absorptive plasma insulin levels at 20wks of age ( WT n = 8 , TG n = 9 , TG n = 9 ) . : Data are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice . Blood glucose ( I ) and insulin levels ( J ) with total area under the curve ( AUC ) of insulin ( K ) during oral glucose tolerance test ( oGTT ) at 17wks of age ( WT n = 8 , TG n = 11 , TG n = 11 ) . Data are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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Figure 4Representative H&E histological staining of sWAT at 20wks of age (scale bars represent 50 μm) Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.relative mRNA expression profile in sWAT of male mice (WT n=7, TG n=8, TGxKO n=8) (B). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.Representative immunoblots (C) and quantification (D) of UCP1 protein expression in sWAT of male mice at 20wks of age (WT n=4, TG n=6, TGxKO n=4). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.Relative mRNA expression profile in eWAT (WT n=7, TG n=8, TGxKO n=8). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.Plasma FGF21 levels from male mice at 20wks of age (n=10 per genotype). Plasma Leptin levels from male mice at 20wks of age (n=5 per genotype). Post-absorptive plasma insulin levels at 20wks of age (WT n=8, TG n=9, TG n=9). : Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.Blood glucose (I) and insulin levels (J) with total area under the curve (AUC) of insulin (K) during oral glucose tolerance test (oGTT) at 17wks of age (WT n=8, TG n=11, TG n=11). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Figure 5Physical activity of male mice shown hourly over 24 - hrs and as day and night time . Data shown are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of age ( WT n = 12 , TG n = 11 , TG n = 10 ) and shown as means + SEMenergy expenditure of male mice shown hourly over 24 - hrs and as day and night time . Data shown are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of age ( WT n = 12 , TG n = 11 , TG n = 10 ) and shown as means + SEM , total assimilated energy of male mice shown hourly over 24 - hrs and as day and night time . Table showing mean values of assimilated energy ( kJ ) at day / night time and per 24 - hrs . Energy balance calculated as delta of assimilated energy ( AE ) and energy expenditure ( EE ) and energy balance ( J ) per 24 - hrs . Data shown are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of age ( WT n = 12 , TG n = 11 , TG n = 10 ) and shown as means + SEMRespiratory quotient ( RQ ) shown hourly over 24 - hrs ( J ) , RQ amplitude ( K ) and metabolic flexibility via percentage relative cumulative frequency ( PRCF ) ( L ) . Data shown are from male wild type ( WT ) , Ucp1 - TG ( TG ) , and Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of age ( WT n = 12 , TG n = 11 , TG n = 10 ) and shown as means +

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Physical activity of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEMenergy expenditure of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM, total assimilated energy of male mice shown hourly over 24-hrs and as day and night time. Table showing mean values of assimilated energy (kJ) at day/night time and per 24-hrs. Energy balance calculated as delta of assimilated energy (AE) and energy expenditure (EE) and energy balance (J) per 24-hrs. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEMRespiratory quotient (RQ) shown hourly over 24-hrs (J), RQ amplitude (K) and metabolic flexibility via percentage relative cumulative frequency (PRCF) (L). Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM


Figure 6Relative quadriceps ( Quad ) mRNA expression of the ISR components Atf4 , Atf5 , Atf6 and Chop ( WT day n = 9 , WT night n = 5 , TG day n = 20 , TG night n = 12 ) . Data shown are from male wild type ( WT ) vs . Ucp1 - TG ( TG ) mice sacrificed at day ( 10am ) or night ( 10pm ) . Representative immunoblots of ISR component eIF2α ( B ) and quantification of phospho - eIF2α ( p - eIF2aSer51 ) relative protein expression ( C ) in skeletal muscle ( Gastroc , WT day n = 4 , WT night n = 5 , TG day n = 9 , TG night n = 9 ) . Data shown are from male wild type ( WT ) vs . Ucp1 - TG ( TG ) mice sacrificed at day ( 10am ) or night ( 10pm ) . Relative mRNA expression of Rev - erba ( D ) and Gdf15 ( E ) in quadriceps , liver and eWAT ( WT day n = 9 , WT night n = 5 , TG day n = 20 , TG night n = 12 ) . Data shown are from male wild type ( WT ) vs . Ucp1 - TG ( TG ) mice sacrificed at day ( 10am ) or night ( 10pm ) . Skeletal muscle ( Quad ) GDF15 protein content normalized to total protein content ( WT day n = 4 , WT night n = 5 , TG day n = 14 , TG night n = 12 ) Circulating GDF15 plasma levels ( WT day n = 9 , WT night n = 5 , TG day n = 18 , TG night n = 12 ) . Data shown are from male wild type ( WT ) vs . Ucp1 - TG ( TG ) mice sacrificed at day ( 10am ) or night ( 10pm ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Relative quadriceps (Quad) mRNA expression of the ISR components Atf4, Atf5, Atf6 and Chop (WT day n=9, WT night n=5, TG day n=20, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).Representative immunoblots of ISR component eIF2α (B) and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression (C) in skeletal muscle (Gastroc, WT day n=4, WT night n=5, TG day n=9, TG night n=9). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).Relative mRNA expression of Rev-erba (D) and Gdf15 (E) in quadriceps, liver and eWAT (WT day n=9, WT night n=5, TG day n=20, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).Skeletal muscle (Quad) GDF15 protein content normalized to total protein content (WT day n=4, WT night n=5, TG day n=14, TG night n=12) Circulating GDF15 plasma levels (WT day n=9, WT night n=5, TG day n=18, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).


figf1a , Internalization of beads with PAM3CSK , LPS , or Alexa - Fluor - 555 - labelled BSA ( Red - BSA ; yellow arrows ) and association with GFP - LC3 in RAW cells was followed by time - lapse video for 3 h , 1 frame / 2 min ( representative frames are shown ) . GFP - LC3 in RAW cells translocates to phagosomes containing zymosan ( red ) , assessed as in ac , Time course of translocation of GFP - LC3 to a phagosome containing zymosan ( red ) ( see Supplementary Movie 2 ) . Time ( minutes ) is indicated in the top left of each panel . d , LC3 translocation to the phagosome is induced by TLR signalling . GFP - LC3 was transiently transfected into wild - type or Tlr2 - / - macrophages , which were exposed to zymosan for 45 min . e , The percentage of phagosomes ( n ≥ 50 per group ) with GFP - LC3 in wild - type and Tlr2 - / - macrophages . Means and ranges are shown ( 4 mice per group were used ) .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1a, Internalization of beads with PAM3CSK, LPS, or Alexa-Fluor-555-labelled BSA (Red-BSA; yellow arrows) and association with GFP-LC3 in RAW cells was followed by time-lapse video for 3 h, 1 frame/2 min (representative frames are shown).GFP-LC3 in RAW cells translocates to phagosomes containing zymosan (red), assessed as in ac, Time course of translocation of GFP-LC3 to a phagosome containing zymosan (red) (see Supplementary Movie 2). Time (minutes) is indicated in the top left of each panel.d, LC3 translocation to the phagosome is induced by TLR signalling. GFP-LC3 was transiently transfected into wild-type or Tlr2-/- macrophages, which were exposed to zymosan for 45 min.e, The percentage of phagosomes (n ≥ 50 per group) with GFP-LC3 in wild-type and Tlr2-/- macrophages. Means and ranges are shown (4 mice per group were used).


figf2e , The percentage of phagosomes ( n ≥ 50 per group ) with GFP - LC3 in wild - type and Tlr2 - / - macrophages . Means and ranges are shown ( 4 mice per group were used ) .

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figf2e, The percentage of phagosomes (n ≥ 50 per group) with GFP-LC3 in wild-type and Tlr2-/- macrophages. Means and ranges are shown (4 mice per group were used).


figf3a , RAW cells expressing GFP - LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides . At 24 h after transfection , cells were fed with zymosan ( yellow arrows ) and the internalization process was followed by time - lapse video microscopy . Images obtained at 5 and 30 min after zymosan engulfment are shown . b , The percentage of GFP - LC3 - associated phagosomes ( n ≥ 100 per / group ) was obtained from three independent time - lapse videos ( 2 h each ) of macrophages transfected with control or Atg5 siRNA ( means and ranges are shown ) . e , The percentage of phagosomes ( n ≥ 50 per group ) with GFP - LC3 in wild - type and Tlr2 - / - macrophages . Means and ranges are shown ( 4 mice per group were used ) . e , Ly294002 ( Ly29 , 10 µM ) was added 10 min before zymosan , and wortmannin was added after engulfment but before GFP - LC3 translocation in RAW - GFP - LC3 cells . Representative images are shown . f , Percentage of GFP - LC3 - associated phagosomes ( n ≥ 50 per group ) with P ( I ) 3K inhibitors assessed by time - lapse video ( means and ranges are shown ; three independent 3 h videos ) . g , RAW cells expressing GFP - LC3 and the PX domain of p40 ( phox ) fused to mCherry ( red ) were fed with zymosan ( yellow arrows ) or with beads ( Supplementary Fig . 6 ) . Internalization was followed at 1 - min intervals ( Supplementary Movie 5 ) , representative images are shown . Single cells were followed for at least 2 h ( n ≥ 20 per group ) ( Supplementary Movies 6 , 7 and Supplementary Fig . 6 ) . Mean ± s . d . duration of zymosan - PX translocation to the zymosan for beads ( B ) and zymosan ( Z ) are shown . h , Internalization of zymosan ( yellow arrows ) was followed at 1 . 5 - min intervals for at least 2 h in RAW cells transiently transfected with GFP - BECN1 . Representative images are shown . Translocation of GFP - BECN1 to the phagosome for beads ( B ) and zymosan ( Z ) was quantified ( mean ± s . d . ) using time - lapse movies ( Supplementary Movie 8 and 9 ; n ≥ 20 cells per group ) . For g and h , time ( in minutes ) is indicated at the top left of each panel .

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figf3a, RAW cells expressing GFP-LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides. At 24 h after transfection, cells were fed with zymosan (yellow arrows) and the internalization process was followed by time-lapse video microscopy. Images obtained at 5 and 30 min after zymosan engulfment are shown. b, The percentage of GFP-LC3-associated phagosomes (n ≥ 100 per /group) was obtained from three independent time-lapse videos (2 h each) of macrophages transfected with control or Atg5 siRNA (means and ranges are shown).e, The percentage of phagosomes (n ≥ 50 per group) with GFP-LC3 in wild-type and Tlr2-/- macrophages. Means and ranges are shown (4 mice per group were used).e, Ly294002 (Ly29, 10 µM) was added 10 min before zymosan, and wortmannin was added after engulfment but before GFP-LC3 translocation in RAW-GFP-LC3 cells. Representative images are shown. f, Percentage of GFP-LC3-associated phagosomes (n ≥ 50 per group) with P(I)3K inhibitors assessed by time-lapse video (means and ranges are shown; three independent 3 h videos).g, RAW cells expressing GFP-LC3 and the PX domain of p40(phox) fused to mCherry (red) were fed with zymosan (yellow arrows) or with beads (Supplementary Fig. 6). Internalization was followed at 1-min intervals (Supplementary Movie 5), representative images are shown. Single cells were followed for at least 2 h (n ≥ 20 per group) (Supplementary Movies 6, 7 and Supplementary Fig. 6). Mean ± s.d. duration of zymosan-PX translocation to the zymosan for beads (B) and zymosan (Z) are shown.h, Internalization of zymosan (yellow arrows) was followed at 1.5-min intervals for at least 2 h in RAW cells transiently transfected with GFP-BECN1. Representative images are shown. Translocation of GFP-BECN1 to the phagosome for beads (B) and zymosan (Z) was quantified (mean ± s.d.) using time-lapse movies (Supplementary Movie 8 and 9; n ≥ 20 cells per group). For g and h, time (in minutes) is indicated at the top left of each panel.


figf4a , Internalization of control or PAM3CSK4 - covered beads was followed in RAW - GFP - LC3 cells pre - loaded with lysotracker ( red ) by time - lapse video for 3 h . Time ( in minutes ) is indicated at the top left of each panel . b , RAW cells pre - loaded with lysotracker were exposed to control or PAM3CSK - covered beads for 6 h . Acidification of the beads ( n ≥ 20 per group ) , measured as maximum lysotracker relative light units ( RLU ) on a horizontal cross - section of each bead represented in the x axis ( mean ± s . d . ; Wilcoxon - Mann - Whitney test , P 0 . 0005 ) , is shown . c , d , Knockdown of ATG5 reduced zymosan - induced GFP - LC3 translocation and lysosomal fusion . e , Primary macrophages from wild - type ( white bars ) and Atg7 - null mice ( grey bars ) were fed live yeast for 1 h and washed . Internalized yeast were extracted from the macrophages at 4 h and 8 h after re - plating . Means and ranges from six different measurements using two mice per group are shown . f , Initiation and duration of translocation of PX - mCherry to phagosomes ( yellow bar ) , GFP - BECN1 translocation ( blue bar ) , GFP - LC3 translocation ( green bar ) and Lysotracker recruitment ( acidification ) were determined during the first 2 h after internalization ( 10 cells per group , error bars indicate s . d . )

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figf4a, Internalization of control or PAM3CSK4-covered beads was followed in RAW-GFP-LC3 cells pre-loaded with lysotracker (red) by time-lapse video for 3 h. Time (in minutes) is indicated at the top left of each panel.b, RAW cells pre-loaded with lysotracker were exposed to control or PAM3CSK-covered beads for 6 h. Acidification of the beads (n ≥ 20 per group), measured as maximum lysotracker relative light units (RLU) on a horizontal cross-section of each bead represented in the x axis (mean ± s.d.; Wilcoxon-Mann-Whitney test, P 0.0005), is shown.c, d, Knockdown of ATG5 reduced zymosan-induced GFP-LC3 translocation and lysosomal fusion.e, Primary macrophages from wild-type (white bars) and Atg7-null mice (grey bars) were fed live yeast for 1 h and washed. Internalized yeast were extracted from the macrophages at 4 h and 8 h after re-plating. Means and ranges from six different measurements using two mice per group are shown.f, Initiation and duration of translocation of PX-mCherry to phagosomes (yellow bar), GFP-BECN1 translocation (blue bar), GFP-LC3 translocation (green bar) and Lysotracker recruitment (acidification) were determined during the first 2 h after internalization (10 cells per group, error bars indicate s.d.)


Figure 1 ( D ) Using the dual color labeling system ( esg - Gal4 > UASp - FRT - H3 - eGFP - FRT - H3 - mCherry ) , old and new H3 distribution in prophase and prometaphase ISCs shows large , separable domains of H3 - eGFP ( old ) and H3 - mCherry ( new ) . Note : all figure panels in this work are maximum intensity projection images to show signals from all Z - stacks . ( E ) Using the UASp - FRT - histone - eGFP - T2A - histone - mCherry - PolyA transgene that co - expresses H3 - eGFP and H3 - mCherry , significant overlap between the two signals were detected in ISCs during prophase and prometaphase . ( F ) Quantification of the colocalization between old and new H3 , as well as between the co - expressed eGFP - and mCherry - tagged H3 in prophase and prometaphase ISCs . Pearson ' s correlation coefficients were measured , where 1 represents complete colocalization and 0 stands for no colocalization . Old and new H3 showed significantly less colocalization ( Pearson ' s correlation coefficient for H3 = 0 . 44 + 0 . 02 , n = 81 ISCs ) when compared to the co - expressed eGFP - and mCherry - tagged H3 ( Pearson ' s correlation coefficient for H3 co - expressed = 0 . 86 + 0 . 01 , n = 19 ISCs ) . Individual data points and mean values are shown . Error bars represent SEM . * * * * p < 0 . 0001 ; unpaired t test to compare two individual datasets to each other . Data information : n for individual ISCs . Scale bar in ( D ) , ( E ) : 5 µm .

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Figure 1(D) Using the dual color labeling system (esg-Gal4>UASp-FRT-H3-eGFP-FRT-H3-mCherry), old and new H3 distribution in prophase and prometaphase ISCs shows large, separable domains of H3-eGFP (old) and H3-mCherry (new). Note: all figure panels in this work are maximum intensity projection images to show signals from all Z-stacks. (E) Using the UASp-FRT-histone-eGFP-T2A-histone-mCherry-PolyA transgene that co-expresses H3-eGFP and H3-mCherry, significant overlap between the two signals were detected in ISCs during prophase and prometaphase. (F) Quantification of the colocalization between old and new H3, as well as between the co-expressed eGFP- and mCherry-tagged H3 in prophase and prometaphase ISCs. Pearson's correlation coefficients were measured, where 1 represents complete colocalization and 0 stands for no colocalization. Old and new H3 showed significantly less colocalization (Pearson's correlation coefficient for H3 = 0.44 + 0.02, n = 81 ISCs) when compared to the co-expressed eGFP- and mCherry-tagged H3 (Pearson's correlation coefficient for H3 co-expressed = 0.86 + 0.01, n = 19 ISCs). Individual data points and mean values are shown. Error bars represent SEM. **** p < 0.0001; unpaired t test to compare two individual datasets to each other. Data information: n for individual ISCs. Scale bar in (D), (E): 5 µm.


Figure 2 ( A ) Old and new H4 distribution in prophase and prometaphase ISCs ( esg - Gal4 > UASp - FRT - H4 - eGFP - FRT - H4 - mCherry ) . Separable domains of H4 - eGFP ( old ) and H4 - mCherry ( new ) were observed ( B ) Old and new H2A distribution in prophase and prometaphase ISCs ( esg - Gal4 > UASp - FRT - H2A - eGFP - FRT - H2A - mCherry ) , showing largely overlapping signals between H2A - eGFP ( old ) and H2A - mCherry ( new ) . Data information : n for individual ISCs . Scale bar in ( A ) , ( B ) 5 µm . ( D ) Late - stage mitotic ISC showing asymmetric segregation of H3 between the two sets of sister chromatids at the opposite poles of the ISC . ( E ) Late - stage mitotic ISC showing less asymmetric segregation of H4 , compared to H3 , between the two sets of sister chromatids at the opposite poles of the ISC . ( F ) Late - stage mitotic ISC showing symmetric segregation of H2A between the two sets of sister chromatids at the opposite poles of the ISC . Data information : n for individual ISCs . Scale bar in ( D ) , ( E ) , and ( F ) : 5 µm .

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Figure 2(A) Old and new H4 distribution in prophase and prometaphase ISCs (esg-Gal4>UASp-FRT-H4-eGFP-FRT-H4-mCherry). Separable domains of H4-eGFP (old) and H4-mCherry (new) were observed (B) Old and new H2A distribution in prophase and prometaphase ISCs (esg-Gal4>UASp-FRT-H2A-eGFP-FRT-H2A-mCherry), showing largely overlapping signals between H2A-eGFP (old) and H2A-mCherry (new). Data information: n for individual ISCs. Scale bar in (A), (B) 5 µm.(D) Late-stage mitotic ISC showing asymmetric segregation of H3 between the two sets of sister chromatids at the opposite poles of the ISC. (E) Late-stage mitotic ISC showing less asymmetric segregation of H4, compared to H3, between the two sets of sister chromatids at the opposite poles of the ISC. (F) Late-stage mitotic ISC showing symmetric segregation of H2A between the two sets of sister chromatids at the opposite poles of the ISC. Data information: n for individual ISCs. Scale bar in (D), (E), and (F): 5 µm.


Figure 3 ( A ) Old and new H3 distribution in a post - mitotic pair of cells with asymmetric Dl - nLacZ labeling , showing that H3 - eGFP ( old ) is asymmetrically inherited by the Delta - high cell , while H3 - mCherry ( new ) is enriched in the Delta - low cell ( esg - Gal4 > UASp - FRT - H3 - eGFP - FRT - H3 - mCherry ) . ( B ) Old and new H3 distribution in a post - mitotic pair of cells with symmetric Dl - nLacZ labeling , showing that both H3 - eGFP ( old ) and H3 - mCherry ( new ) are more symmetrically distributed between the two cells . Scale bar in ( A ) , ( B ) , 5 µm ; asterisk , ISC side . ( E ) Old and new H4 distribution in a post - mitotic pair of cells with asymmetric Dl - nLacZ labeling , showing that H4 - eGFP ( old ) is asymmetrically inherited by the Delta - high cell , while H4 - mCherry ( new ) is enriched toward the Delta - low cell ( esg - Gal4 > UASp - FRT - H4 - eGFP - FRT - H4 - mCherry ) . ( F ) Old and new H4 distribution in a post - mitotic pair with symmetric Dl - nLacZ labeling , showing that H4 - eGFP ( old ) and H4 - mCherry ( new ) are symmetrically distributed between the two cellss . Scale bar in ( E ) , ( F ) , 5 µm ; asterisk , ISC side . ( I ) Old and new H2A distribution in a post - mitotic pair of cells with asymmetric Dl - nLacZ labeling , showing that H2A - eGFP ( old ) and H2A - mCherry ( new ) are symmetrically inherited by the two cells ( esg - Gal4 > UASp - FRT - H2A - eGFP - FRT - H2A - mCherry ) . ( J ) Old and new H2A distribution in a post - mitotic pair of cells with symmetric Dl - nLacZ labeling , showing that H2A - eGFP ( old ) and H2A - mCherry ( new ) are symmetrically distributed between the two cells . Scale bar in ( I ) , ( J ) , 5 µm ; asterisk , ISC side . ( M ) The ISC - specific combination of drivers esg - Gal4 , Su ( H ) - Gal80 drive H3 transgene [ esg - Gal4 , Su ( H ) - Gal80 > UASp - FRT - H3 - eGFP - FRT - H3 - mCherry ] with heat shock treatment and recovery ( see Materials and Methods ) in a Delta - asymmetric pair of cells . Old and new H3 distribution in a post - mitotic pair of cells with asymmetric Dl - nLacZ labeling , showing that H3 - eGFP ( old ) is asymmetrically inherited by the Delta - high cell , while H3 - mCherry ( new ) is enriched in the Delta - low cell . ( N ) ISC specific drivers express the H3 transgene in a Delta - symmetric pair of cells . Old and new H3 distribution in a post - mitotic pair of cells with symmetric Dl - nLacZ labeling , showing that both H3 - eGFP ( old ) and H3 - mCherry ( new ) are more symmetrically distributed between the two cells . Scale bar in ( M ) , and ( N ) : 5 µm ; asterisk , ISC side .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Old and new H3 distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H3-eGFP (old) is asymmetrically inherited by the Delta-high cell, while H3-mCherry (new) is enriched in the Delta-low cell (esg-Gal4>UASp-FRT-H3-eGFP-FRT-H3-mCherry). (B) Old and new H3 distribution in a post-mitotic pair of cells with symmetric Dl-nLacZ labeling, showing that both H3-eGFP (old) and H3-mCherry (new) are more symmetrically distributed between the two cells. Scale bar in (A), (B), 5 µm; asterisk, ISC side.(E) Old and new H4 distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H4-eGFP (old) is asymmetrically inherited by the Delta-high cell, while H4-mCherry (new) is enriched toward the Delta-low cell (esg-Gal4>UASp-FRT-H4-eGFP-FRT-H4-mCherry). (F) Old and new H4 distribution in a post-mitotic pair with symmetric Dl-nLacZ labeling, showing that H4-eGFP (old) and H4-mCherry (new) are symmetrically distributed between the two cellss. Scale bar in (E), (F), 5 µm; asterisk, ISC side.(I) Old and new H2A distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H2A-eGFP (old) and H2A-mCherry (new) are symmetrically inherited by the two cells (esg-Gal4>UASp-FRT-H2A-eGFP-FRT-H2A-mCherry). (J) Old and new H2A distribution in a post-mitotic pair of cells with symmetric Dl-nLacZ labeling, showing that H2A-eGFP (old) and H2A-mCherry (new) are symmetrically distributed between the two cells. Scale bar in (I), (J), 5 µm; asterisk, ISC side.(M) The ISC-specific combination of drivers esg-Gal4, Su(H)-Gal80 drive H3 transgene [esg-Gal4, Su(H)-Gal80>UASp-FRT-H3-eGFP-FRT-H3-mCherry] with heat shock treatment and recovery (see Materials and Methods) in a Delta-asymmetric pair of cells. Old and new H3 distribution in a post-mitotic pair of cells with asymmetric Dl-nLacZ labeling, showing that H3-eGFP (old) is asymmetrically inherited by the Delta-high cell, while H3-mCherry (new) is enriched in the Delta-low cell. (N) ISC specific drivers express the H3 transgene in a Delta-symmetric pair of cells. Old and new H3 distribution in a post-mitotic pair of cells with symmetric Dl-nLacZ labeling, showing that both H3-eGFP (old) and H3-mCherry (new) are more symmetrically distributed between the two cells. Scale bar in (M), and (N): 5 µm; asterisk, ISC side.


Figure 4 ( A ) Old and new H3T3A distribution in prophase ISC , showing separable domains between H3T3A - GFP ( old ) and H3T3A - mKO ( new ) ( esg - Gal4 > UASp - FRT - H3T3A - eGFP - FRT - H3T3A - mCherry ) . Data information : Scale bar in ( A ) , ( C ) , ( E ) , ( F ) : 5 µm , ( B ) Quantification of colocalization between H3T3A - GFP ( old ) and H3T3A - mKO ( new ) in prophase and prometaphase ISCs using Pearson ' s correlation coefficients . The H3 and H2A data are from Figure 2C for direct comparison . Old and new H3T3A distribution is more similar to that of H3 ( Pearson ' s correlation coefficient for H3T3A = 0 . 49 + 0 . 02 , n = 55 ISCs ) . Individual data points and mean values are shown . Error bars represent SEM . * * * * p < 0 . 0001 , * p < 0 . 05 ; unpaired t test to compare two individual datasets to each other . ( E ) In a post - mitotic pair of cells with comparable Dl - nLacZ , both H3T3A - GFP ( old ) and H3T3A - mKO ( new ) are symmetrically distributed between the two cells . ( F ) In a post - mitotic pair of cells with asymmetric Dl - nLacZ expression , H3T3A - GFP ( old ) is enriched in the Delta - high cell , while H3T3A - mKO ( new ) is more toward the Delta - low cell . Data information : Scale bar in ( E ) , ( F ) : 5 µm , asterisk , ISC side . ( H ) Distribution of the Dl - nLacZ - positive ( magenta ) cells in an H3 - expressing midgut , which are well spaced and interspersed within the intestinal epithelium . ( I ) Distribution of the Dl - nLacZ - positive ( magenta ) cells in an H3T3A - expressing midgut , which are unevenly distributed in clusters with two or more Delta - positive cells . DAPI : white in ( H - I ) . Data information : Scale bar in ( H ) and ( I ) : 50 µm ( K ) Percentages of clusters with 1 - , 2 - , or > 3 - Delta - positive cell clusters for H3 - expressing midguts ( avg . percentage of 1 - Dl + cells = 87 . 7 % + 1 . 1 % , avg . percentage of 2 - Dl + cells = 11 . 9 % + 0 . 9 % , avg . percentage of > 3 - Dl + cells = 0 . 4 % + 0 . 2 % , N = 4 midguts , n = 1 , 482 clusters ) and H3T3A - expressing midguts ( avg . percentage of 1 - Dl + cells = 68 . 2 % + 1 . 2 % , avg . percentage of 2 - Dl + cells = 28 . 5 % + 0 . 9 % , avg . percentage of > 3 - Dl + cells = 3 . 3 % + 0 . 4 % , N = 5 midguts , n = 1 , 862 clusters ) . Bars represent mean proportions of cell clusters with 1 , 2 , or 3 + Dl + cells out of all cell clusters counted . Error bars represent SEM . Comparing H3 - expressing midguts with H3T3A - expressing midguts , there was a statistically significant difference in the distributions of Delta - positive cells per cluster , determined by a Chi - Square test ( X2 ( 2 , n = 1862 ) = 908 . 75 , * * * * p < 0 . 0001 ) . As shown , there are significant increases in 2 and > 3 - Delta - positive cells per cluster in the H3T3A - expressing intestines when compared to the H3 - expressing intestines . ( L ) Percentages of single Delta - positive cells out of total Delta - positive cells for H3 - expressing midguts ( avg . single Dl + cells = 77 . 9 % + 1 . 9 % , N = 4 midguts , n = 1 , 674 Dl + cells ) and H3T3A - expressing midguts ( avg . single Dl + cells = 50 . 4 % + 1 . 5 % , N = 5 midguts , n = 2 , 522 Dl + cells ) . Bars represent mean proportion of single Dl + ISCs out of all ISCs counted . Error bars represent SEM . There are fewer single Delta - positive cells in H3T3A - expressing midguts compared to H3 - expressing midguts ( two sample t test , * * * p < 0 . 001 ) . ( M ) Comparison of the mitotic index in H3 - expressing ( N = 8 midguts ) and H3T3A - expressing ( N = 8 midguts ) midguts . Total number of mitotic ISCs were quantified per midgut by the presence of H3S10 phosphorylation mark . H3T3A - expressing midguts had a significantly higher mitotic index when compared to H3 - expressing midguts ( two sample t test , * p < 0 . 05 ) . Bars represent mean number of mitotic cells per midgut . Error bars represent SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Old and new H3T3A distribution in prophase ISC, showing separable domains between H3T3A-GFP (old) and H3T3A-mKO (new) (esg-Gal4>UASp-FRT-H3T3A-eGFP-FRT-H3T3A-mCherry). Data information: Scale bar in (A), (C), (E), (F): 5 µm,(B) Quantification of colocalization between H3T3A-GFP (old) and H3T3A-mKO (new) in prophase and prometaphase ISCs using Pearson's correlation coefficients. The H3 and H2A data are from Figure 2C for direct comparison. Old and new H3T3A distribution is more similar to that of H3 (Pearson's correlation coefficient for H3T3A= 0.49 + 0.02, n = 55 ISCs). Individual data points and mean values are shown. Error bars represent SEM. **** p < 0.0001, * p < 0.05; unpaired t test to compare two individual datasets to each other.(E) In a post-mitotic pair of cells with comparable Dl-nLacZ, both H3T3A-GFP (old) and H3T3A-mKO (new) are symmetrically distributed between the two cells. (F) In a post-mitotic pair of cells with asymmetric Dl-nLacZ expression, H3T3A-GFP (old) is enriched in the Delta-high cell, while H3T3A-mKO (new) is more toward the Delta-low cell. Data information: Scale bar in (E), (F): 5 µm, asterisk, ISC side.(H) Distribution of the Dl-nLacZ-positive (magenta) cells in an H3-expressing midgut, which are well spaced and interspersed within the intestinal epithelium. (I) Distribution of the Dl-nLacZ-positive (magenta) cells in an H3T3A-expressing midgut, which are unevenly distributed in clusters with two or more Delta-positive cells. DAPI: white in (H-I). Data information: Scale bar in (H) and (I): 50 µm(K) Percentages of clusters with 1-, 2-, or >3-Delta-positive cell clusters for H3-expressing midguts (avg. percentage of 1-Dl+ cells = 87.7% + 1.1%, avg. percentage of 2-Dl+ cells = 11.9% + 0.9%, avg. percentage of >3-Dl+ cells = 0.4% + 0.2%, N = 4 midguts, n = 1,482 clusters) and H3T3A-expressing midguts (avg. percentage of 1-Dl+ cells = 68.2% + 1.2%, avg. percentage of 2-Dl+ cells = 28.5% + 0.9%, avg. percentage of >3-Dl+ cells = 3.3% + 0.4%, N = 5 midguts, n = 1,862 clusters). Bars represent mean proportions of cell clusters with 1, 2, or 3+ Dl+ cells out of all cell clusters counted. Error bars represent SEM. Comparing H3-expressing midguts with H3T3A-expressing midguts, there was a statistically significant difference in the distributions of Delta-positive cells per cluster, determined by a Chi-Square test (X2(2, n = 1862) = 908.75, **** p < 0.0001). As shown, there are significant increases in 2 and >3-Delta-positive cells per cluster in the H3T3A-expressing intestines when compared to the H3-expressing intestines.(L) Percentages of single Delta-positive cells out of total Delta-positive cells for H3-expressing midguts (avg. single Dl+ cells = 77.9% + 1.9%, N = 4 midguts, n = 1,674 Dl+ cells) and H3T3A-expressing midguts (avg. single Dl+ cells = 50.4% + 1.5%, N = 5 midguts, n = 2,522 Dl+ cells). Bars represent mean proportion of single Dl+ ISCs out of all ISCs counted. Error bars represent SEM. There are fewer single Delta-positive cells in H3T3A-expressing midguts compared to H3-expressing midguts (two sample t test, *** p < 0.001).(M) Comparison of the mitotic index in H3-expressing (N = 8 midguts) and H3T3A-expressing (N = 8 midguts) midguts. Total number of mitotic ISCs were quantified per midgut by the presence of H3S10 phosphorylation mark. H3T3A-expressing midguts had a significantly higher mitotic index when compared to H3-expressing midguts (two sample t test, * p < 0.05). Bars represent mean number of mitotic cells per midgut. Error bars represent SEM.