Representative electron micrographs of homofibrils formed by M451 - 90 , RIPK1497 ‑ 583 , His - Ub - RIPK3387 ‑ 518 or ZBP1170 - 355 individually , and heterofibrils formed by indicated mixtures . Samples prepared twice from independent protein

O O O O O O O O O O O O O O O O O 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 electron micrographs of homofibrils formed by M451-90, RIPK1497‑583, His-Ub-RIPK3387‑518 or ZBP1170-355 individually, and heterofibrils formed by indicated mixtures.Samples prepared twice from independent protein preparations


Heterofibrils containing mCHERRY - M451 - 90 and YPet ‑ RIPK1497 ‑ 583 C Heterofibrils containing mCHERRY - M451 - 90 and YPet - RIPK3387 ‑ 518 D Heterofibrils containing mCHERRY - M451 - 90 and YPet ‑ ZBP1170 ‑ 355 E Heterofibrils containing mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK3387 ‑

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

Heterofibrils containing mCHERRY-M451-90 and YPet‑RIPK1497‑583 C Heterofibrils containing mCHERRY-M451-90 and YPet-RIPK3387‑518 D Heterofibrils containing mCHERRY-M451-90 and YPet‑ZBP1170‑355 E Heterofibrils containing mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑51


Heterofibrils containing mCHERRY - RIPK3387 ‑ 518 and YPet ‑ ZBP1170 ‑ 355 Pearson ' s correlation coefficients measured from two confocal images of the indicated protein mixtures .

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

Heterofibrils containing mCHERRY- RIPK3387‑518 and YPet‑ZBP1170‑355 Pearson's correlation coefficients measured from two confocal images of the indicated protein mixtures.


Heterofibrils containing ECFP-M451-90 with mCHERRY- RIPK1497‑583 and YPet-RIPK3387‑518


STED images of heteromeric fibrils formed by co - assembly of mCHERRY - M451 - 90 and YPet - RIPK3387 ‑

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

STED images of heteromeric fibrils formed by co-assembly of mCHERRY-M451-90 and YPet-RIPK3387‑518


SDS AGE analysis of monomeric or assembled forms of YPet - RIPK3387 ‑ 518 and mCHERRY - M451 - 90WT or mCHERRY - M451 - 90AAAA . Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis , as indicated . Assembled samples incubated with 0 or 2 % SDS at RT for 10 min before electrophoresis . Protein components as indicated above each lane .

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

SDS AGE analysis of monomeric or assembled forms of YPet-RIPK3387‑518 and mCHERRY-M451-90WT or mCHERRY-M451-90AAAA. Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis, as indicated. Assembled samples incubated with 0 or 2% SDS at RT for 10 min before electrophoresis. Protein components as indicated above each lane.


SDS AGE analysis of assemblies of mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK3387 ‑ 518 with or without four times molar equivalents of His - Ub - M45 .

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

SDS AGE analysis of assemblies of mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 with or without four times molar equivalents of His-Ub-M45.


SDS AGE analysis of assemblies of YPet - RIPK3387 ‑ 518 and mCHERRY - ZBP1170 - 355 with or without four times molar equivalents of His - Ub - M45 .

SDS AGE analysis of assemblies of YPet-RIPK3387‑518 and mCHERRY-ZBP1170-355 with or without four times molar equivalents of His-Ub-M45.


Confocal microscopy image of a single fibril composed of YPet - M451 -

Confocal microscopy image of a single fibril composed of YPet-M451-90


( A ) incorporation of 14C - labeled glycerol 3 - phosphate ( G3P ) into various lipids in the presence of palmitoyl - CoA ( 10 µM ) in microsomal membranes prepared from HEK293 - AT1 cells . After incubations at room temperature for 30 min , lipids were extracted and separated by TLC before quantification using autoradiography and phosphorimaging . Lipid species corresponding to the major spots visualized are indicated . Note the appearance of phosphatidylcholine ( PC ) and cytidine diphosphate - diacylglycerol ( CDP - DAG ) in the presence of cytidine trisphosphate ( CTP ) , as well as the production of phosphatidylinositol ( PI ) only when both CTP and myo - inositol are present . Also , there is a relatively small effect of the DAG kinase inhibitor ( DGKi ) on CDP - DAG and PI formation in spite of the fact that plenty of phosphatidic acid ( PA ) is available from de novo synthesis ( although it should be noted that no ATP was added during these reactions ) . A representative experiment is shown , but these results have been reproduced in other replicates using different treatment combinations . The insert shows the effect of the DGK inhibitor on PI labeling .

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

(A) incorporation of 14C-labeled glycerol 3-phosphate (G3P) into various lipids in the presence of palmitoyl-CoA (10 µM) in microsomal membranes prepared from HEK293-AT1 cells. After incubations at room temperature for 30 min, lipids were extracted and separated by TLC before quantification using autoradiography and phosphorimaging. Lipid species corresponding to the major spots visualized are indicated. Note the appearance of phosphatidylcholine (PC) and cytidine diphosphate-diacylglycerol (CDP-DAG) in the presence of cytidine trisphosphate (CTP), as well as the production of phosphatidylinositol (PI) only when both CTP and myo-inositol are present. Also, there is a relatively small effect of the DAG kinase inhibitor (DGKi) on CDP-DAG and PI formation in spite of the fact that plenty of phosphatidic acid (PA) is available from de novo synthesis (although it should be noted that no ATP was added during these reactions). A representative experiment is shown, but these results have been reproduced in other replicates using different treatment combinations. The insert shows the effect of the DGK inhibitor on PI labeling.


( B ) Effect of lipin - 1γ overexpression on the metabolic fate of de novo synthesized PA . Experiments were performed as in panel A , except that the membranes were also prepared from cells expressing a GFP - tagged lipin 1γ , as indicated . Note the large amount of TG and increased production of PC under these conditions . Results of a representative experiment are shown , and these experiments were repeated with identical results .

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 B-MEASURED_VAR 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

(B) Effect of lipin-1γ overexpression on the metabolic fate of de novo synthesized PA. Experiments were performed as in panel A, except that the membranes were also prepared from cells expressing a GFP-tagged lipin 1γ, as indicated. Note the large amount of TG and increased production of PC under these conditions. Results of a representative experiment are shown, and these experiments were repeated with identical results.


Effect of different fatty - acyl - CoA analogs ( each used in 10 µM ) on the metabolic fate of de novo synthesized PA . Note the preferential conversion of oleoyl - CoA containing PA into TG and the very poor incorporation of stearoyl - CoA into TG . Arachidonoyl - CoA is a relatively poor substrate for the generation of de novo synthesized PA ; however , this substrate has the highest conversion rate into PI , which was calculated as the ratio of the PI and PA mass shown in panel D . , while quantified values ( means ± range ) from two such experiments are shown in ( D ) and ( E ) .

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

Effect of different fatty-acyl-CoA analogs (each used in 10 µM) on the metabolic fate of de novo synthesized PA. Note the preferential conversion of oleoyl-CoA containing PA into TG and the very poor incorporation of stearoyl-CoA into TG. Arachidonoyl-CoA is a relatively poor substrate for the generation of de novo synthesized PA; however, this substrate has the highest conversion rate into PI, which was calculated as the ratio of the PI and PA mass shown in panel D. , while quantified values (means ± range) from two such experiments are shown in (D) and (E).


Live imaging of histone - GFP ( green ) and ParB - mCherry ( magenta ) expressed in larval salivary gland nuclei using 1151 - Gal4 . H3 . 3 - GFP levels are increased at the E ( spl ) - C in the presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ ( A ) . ParB - mCherry binds to its cognate int DNA sequence inserted within the E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C and yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( white ) = 5 µm . Quantifications of relative fluorescence intensity of histone - GFP and ParB - mCherry across the E ( spl ) - C in Notch - OFF ( upper ) and Notch - ON ( lower ) conditions . Mean + / - SEM ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 and nglands = 5 , 5 , 5 , 5 , 5 , 4 where each gland represents a biological replicate ( from top to bottom ) .

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

Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. H3.3-GFP levels are increased at the E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Live imaging of histone - GFP ( green ) and ParB - mCherry ( magenta ) expressed in larval salivary gland nuclei using 1151 - Gal4 . E ( spl ) - C in the presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ but there is little change in H3 - GFP between Notch - OFF and Notch - ON nuclei ParB - mCherry binds to its cognate int DNA sequence inserted within the E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C and yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( white ) = 5 µm . Quantifications of relative fluorescence intensity of histone - GFP and ParB - mCherry across the E ( spl ) - C in Notch - OFF ( upper ) and Notch - ON ( lower ) conditions . Mean + / - SEM ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 and nglands = 5 , 5 , 5 , 5 , 5 , 4 where each gland represents a biological replicate ( from top to bottom ) .

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

Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ but there is little change in H3-GFP between Notch-OFF and Notch-ON nuclei ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Live imaging of histone - GFP ( green ) and ParB - mCherry ( magenta ) expressed in larval salivary gland nuclei using 1151 - Gal4 . E ( spl ) - C in the presence of constitutively active Notch , NΔECD ( Notch - ON ) , compared to control Notch - OFF nuclei expressing LacZ ( A ) . with H3 . 3core - GFP ParB - mCherry binds to its cognate int DNA sequence inserted within the E ( spl ) - C [ 8 , 64 ] . Yellow dotted box contains E ( spl ) - C and yellow arrow indicates position of E ( spl ) - C on chromosome . Scale bars ( white ) = 5 µm . Quantifications of relative fluorescence intensity of histone - GFP and ParB - mCherry across the E ( spl ) - C in Notch - OFF ( upper ) and Notch - ON ( lower ) conditions . Mean + / - SEM ; nnuclei = 7 , 6 , 5 , 8 , 9 , 11 and nglands = 5 , 5 , 5 , 5 , 5 , 4 where each gland represents a biological replicate ( from top to bottom ) .

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

Live imaging of histone-GFP (green) and ParB-mCherry (magenta) expressed in larval salivary gland nuclei using 1151-Gal4. E(spl)-C in the presence of constitutively active Notch, NΔECD (Notch-ON), compared to control Notch-OFF nuclei expressing LacZ (A). with H3.3core-GFP ParB-mCherry binds to its cognate int DNA sequence inserted within the E(spl)-C [8,64]. Yellow dotted box contains E(spl)-C and yellow arrow indicates position of E(spl)-C on chromosome. Scale bars (white) = 5 µm. Quantifications of relative fluorescence intensity of histone-GFP and ParB-mCherry across the E(spl)-C in Notch-OFF (upper) and Notch-ON (lower) conditions. Mean+/-SEM; nnuclei = 7, 6, 5, 8, 9, 11 and nglands = 5, 5, 5, 5, 5, 4 where each gland represents a biological replicate (from top to bottom).


Effects from depleting chromatin remodelers and histone chaperones , as indicated ( wide range shown in A , on recruitment of Su ( H ) - GFP in Notch - ON nuclei ( expressing NΔECD ) of larval salivary glands . Different OSA RNAi stocks used in C are denoted by ( 1 ) and ( 2 ) . In all conditions except Moira RNAi ( A ) nuclei exhibit a bright accumulation of Su ( H ) - GFP at a single locus when imaged live . Scale bars ( yellow ) = 5 µm . Percentage of Notch - ON nuclei retaining a single clear band of Su ( H ) - GFP when the indicated RNAi is co - expressed with NΔECD . For each genotype , 5 nuclei from each of 10 glands were scored ( 50 nuclei total ) . * * * * A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed ; p < 0 . 0001 , two - tailed Fisher ' s exact test calculated using the raw ( non - percentage ) scoring data .

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

Effects from depleting chromatin remodelers and histone chaperones, as indicated (wide range shown in A, on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. Different OSA RNAi stocks used in C are denoted by (1) and (2). In all conditions except Moira RNAi (A) nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.


Effects from depleting chromatin remodelers and histone chaperones , BRM complex components shown in C ) , on recruitment of Su ( H ) - GFP in Notch - ON nuclei ( expressing NΔECD ) of larval salivary glands . w RNAi is a control and BrmK804R is expression of dominant - negative Brm . Different OSA RNAi stocks used in C are denoted by ( 1 ) and ( 2 ) . , Snr1 RNAi and BrmK804R expression ( C ) , nuclei exhibit a bright accumulation of Su ( H ) - GFP at a single locus when imaged live . Scale bars ( yellow ) = 5 µm . Percentage of Notch - ON nuclei retaining a single clear band of Su ( H ) - GFP when the indicated RNAi is co - expressed with NΔECD . For each genotype , 5 nuclei from each of 10 glands were scored ( 50 nuclei total ) . * * * * A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed ; p < 0 . 0001 , two - tailed Fisher ' s exact test calculated using the raw ( non - percentage ) scoring data .

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

Effects from depleting chromatin remodelers and histone chaperones , BRM complex components shown in C), on recruitment of Su(H)-GFP in Notch-ON nuclei (expressing NΔECD) of larval salivary glands. w RNAi is a control and BrmK804R is expression of dominant-negative Brm. Different OSA RNAi stocks used in C are denoted by (1) and (2). , Snr1 RNAi and BrmK804R expression (C), nuclei exhibit a bright accumulation of Su(H)-GFP at a single locus when imaged live. Scale bars (yellow) = 5 µm. Percentage of Notch-ON nuclei retaining a single clear band of Su(H)-GFP when the indicated RNAi is co-expressed with NΔECD. For each genotype, 5 nuclei from each of 10 glands were scored (50 nuclei total). **** A significant fraction of nuclei lost the fluorescent band when core components of the BRM complex were perturbed; p<0.0001, two-tailed Fisher's exact test calculated using the raw (non-percentage) scoring data.


Chromatin accessibility in Notch - OFF ( B ) and Notch - ON ( NΔECD expression , C ) salivary gland nuclei measured by ATAC - qPCR ; fold enrichment at the indicated regions compared to a " closed ctrl " region . Expression of dominant - negative Brm , BrmK804R , led to reduced accessibility of E ( spl ) mβ - HLH and E ( spl ) m3 - HLH enhancer regions in Notch - OFF conditions , and to a more widespread reduction in accessibility in Notch - ON conditions . " Eip78C enh " corresponds to the ecdysone receptor - binding region of the Eip78C enhancer , which is highly accessible but not Notch - responsive ; " Rab11 tr " and " Mst87F tr " represent highly and lowly - expressed control genes respectively . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with two - tailed Welch ' s t - test comparing LacZ and BrmK804R samples .

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

Chromatin accessibility in Notch-OFF (B) and Notch-ON (NΔECD expression, C) salivary gland nuclei measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Expression of dominant-negative Brm, BrmK804R, led to reduced accessibility of E(spl)mβ-HLH and E(spl)m3-HLH enhancer regions in Notch-OFF conditions, and to a more widespread reduction in accessibility in Notch-ON conditions. "Eip78C enh" corresponds to the ecdysone receptor-binding region of the Eip78C enhancer, which is highly accessible but not Notch-responsive; "Rab11 tr" and "Mst87F tr" represent highly and lowly-expressed control genes respectively. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test comparing LacZ and BrmK804R samples.


Chromatin accessibility in salivary gland nuclei depleted for Su ( H ) by RNAi , measured by ATAC - qPCR ; fold enrichment at the indicated regions compared to a " closed ctrl " region . Accessibility is increased across most of the E ( spl ) - C compared to controls expressing LacZ . Control primer regions are as in B and C . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with two - tailed Welch ' s t - test compared to LacZ controls .

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

Chromatin accessibility in salivary gland nuclei depleted for Su(H) by RNAi, measured by ATAC-qPCR; fold enrichment at the indicated regions compared to a "closed ctrl" region. Accessibility is increased across most of the E(spl)-C compared to controls expressing LacZ. Control primer regions are as in B and C. Mean +/- SEM; n = 3; * p<0.05 with two-tailed Welch's t-test compared to LacZ controls.


Effect of brm and Snr1 RNAi on brm and Snr1 cDNA levels respectively , measured by reverse transcription - qPCR in Kc167 cells ; percentage cDNA compared to GFP RNAi . The knockdowns are highly effective , with only 1 - 2 % of brm and Snr1 cDNA remaining detectable . Mean + / - SEM ; 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

Effect of brm and Snr1 RNAi on brm and Snr1 cDNA levels respectively, measured by reverse transcription-qPCR in Kc167 cells; percentage cDNA compared to GFP RNAi. The knockdowns are highly effective, with only 1-2% of brm and Snr1 cDNA remaining detectable. Mean +/- SEM; n = 3.


Knockdown of components of the BRM complex reduces Su ( H ) recruitment both in Notch - OFF ( B ) and Notch - ON ( C ) conditions . Fold enrichment of Su ( H ) occupancy at the indicated positions detected by ChIP , relative to input , in Kc167 cells treated with brm , Snr1 or GFP RNAi as a control . Notch - ON conditions ( C ) were induced by 30 minutes of EGTA treatment . Mean + / - SEM , n = 3 ( B ) ; Mean , n = 2 ( C ) ; * p < 0 . 05 with one - tailed student ' s t - test compared to GFP RNAi 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Knockdown of components of the BRM complex reduces Su(H) recruitment both in Notch-OFF (B) and Notch-ON (C) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with brm, Snr1 or GFP RNAi as a control. Notch-ON conditions (C) were induced by 30 minutes of EGTA treatment. Mean +/- SEM, n = 3 (B); Mean, n = 2 (C); * p<0.05 with one-tailed student's t-test compared to GFP RNAi control.


Effect of brm RNAi on E ( spl ) mβ - HLH ( mβ ) and E ( spl ) m3 - HLH ( m3 ) induction by Notch activation ( EGTA treatment ) measured by reverse transcription - qPCR ; shown as fold difference to lacZ RNAi control . Mean + / - SEM ; 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

Effect of brm RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) induction by Notch activation (EGTA treatment) measured by reverse transcription-qPCR; shown as fold difference to lacZ RNAi control. Mean +/- SEM; n = 3.


Effect of Brm dominant - negative on expression of E ( spl ) mβ - HLH ( mβ ) and E ( spl ) m3 - HLH ( m3 ) measured by reverse transcription - qPCR . Expression was analyzed in stable cell lines containing pMT - inducible BrmWT or BrmK804R in the absence ( left , uninduced ) or presence of copper sulfate ( right , Cu2 + induced ) . The response of E ( spl ) mβ - HLH and E ( spl ) m3 - HLH to Notch activation ( " N - On " = EGTA treatment vs . " N - Off " = PBS control ) was reduced in the BrmK804R - expressing cells compared to BrmWT - expressing cells , only when induced with copper ( right graph ) . Mean , n = 2 ( left ) ; Mean + / - SEM , n = 3 ( right ) ; * p < 0 . 05 with one - tailed student ' s t - test comparing BrmWT and BrmK804R .

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

Effect of Brm dominant-negative on expression of E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) measured by reverse transcription-qPCR. Expression was analyzed in stable cell lines containing pMT-inducible BrmWT or BrmK804R in the absence (left, uninduced) or presence of copper sulfate (right, Cu2+ induced). The response of E(spl)mβ-HLH and E(spl)m3-HLH to Notch activation ("N-On" = EGTA treatment vs. "N-Off" = PBS control) was reduced in the BrmK804R-expressing cells compared to BrmWT-expressing cells, only when induced with copper (right graph). Mean, n = 2 (left); Mean +/- SEM, n = 3 (right); * p<0.05 with one-tailed student's t-test comparing BrmWT and BrmK804R.


Knockdown of actin - related subunit , BAP55 , reduces Su ( H ) recruitment in both Notch - OFF ( PBS treatment ) and Notch - ON ( EGTA treatment ) conditions . Fold enrichment of Su ( H ) occupancy at the indicated positions detected by ChIP , relative to input , in Kc167 cells treated with Bap55 or lacZ RNAi as a control . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with one - tailed student ' s t - test compared to lacZ RNAi 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O

Knockdown of actin-related subunit, BAP55, reduces Su(H) recruitment in both Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Fold enrichment of Su(H) occupancy at the indicated positions detected by ChIP, relative to input, in Kc167 cells treated with Bap55 or lacZ RNAi as a control. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.


Effect of Bap55 RNAi on E ( spl ) mβ - HLH ( mβ ) and E ( spl ) m3 - HLH ( m3 ) expression levels measured by reverse transcription - qPCR in Notch - OFF ( PBS treatment ) and Notch - ON ( EGTA treatment ) conditions . Expression level of RpII215 is shown as a control gene . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with one - tailed student ' s t - test compared to lacZ RNAi 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 O O O O O O O O O O O O O O O O O O O O O O O

Effect of Bap55 RNAi on E(spl)mβ-HLH (mβ) and E(spl)m3-HLH (m3) expression levels measured by reverse transcription-qPCR in Notch-OFF (PBS treatment) and Notch-ON (EGTA treatment) conditions. Expression level of RpII215 is shown as a control gene. Mean +/- SEM; n = 3; * p<0.05 with one-tailed student's t-test compared to lacZ RNAi control.


Nucleosome turnover measured by CATCH - IT - qPCR ; fold enrichment over input samples compared to Sec15 tr control region . Su ( H ) - bound enhancers show increased nucleosome turnover in response to Notch signaling . Notch signaling is activated in Kc167 cells by 6 hours of copper induction of pMT - NICD with copper excluded in the control . Positions of E ( spl ) - C primers are shown in Fig 3A ; the remaining primers are control non - Notch - responsive regions . Mean , n = 2 .

Nucleosome turnover measured by CATCH-IT-qPCR; fold enrichment over input samples compared to Sec15 tr control region. Su(H)-bound enhancers show increased nucleosome turnover in response to Notch signaling. Notch signaling is activated in Kc167 cells by 6 hours of copper induction of pMT-NICD with copper excluded in the control. Positions of E(spl)-C primers are shown in Fig 3A; the remaining primers are control non-Notch-responsive regions. Mean, n = 2.


brm RNAi reduces nucleosome turnover at Notch - responsive regions . CATCH - IT - qPCR results as in A after brm or lacZ RNAi as a control . Mean + / - SEM ; n = 5 ; * p < 0 . 05 with one - tailed student ' s t - test comparing brm and lacZ RNAi .

brm RNAi reduces nucleosome turnover at Notch-responsive regions. CATCH-IT-qPCR results as in A after brm or lacZ RNAi as a control. Mean +/- SEM; n = 5; * p<0.05 with one-tailed student's t-test comparing brm and lacZ RNAi.


Brm is required for Notch - responsive nucleosome turnover . CATCH - IT - qPCR results after brm or lacZ RNAi as in B and pMT - NICD expression as in A . Mean + / - SEM ; n = 3 ; * p < 0 . 05 with two - tailed student ' s t - test compared to control ( lacZ RNAi Notch - ON bars are compared to lacZ RNAi Notch - OFF bars and brm RNAi bars are compared to their respective lacZ RNAi control bars ) .

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

Brm is required for Notch-responsive nucleosome turnover. CATCH-IT-qPCR results after brm or lacZ RNAi as in B and pMT-NICD expression as in A. Mean +/- SEM; n=3; * p<0.05 with two-tailed student's t-test compared to control (lacZ RNAi Notch-ON bars are compared to lacZ RNAi Notch-OFF bars and brm RNAi bars are compared to their respective lacZ RNAi control bars).


brm RNAi reduces incorporation of histone H3 . 3 . V5 ChIP - qPCR in Kc167 cells after lacZ or brm RNAi treatment in cells with H3 - V5 or H3 . 3 - V5 expression induced from the pMT promoter by 3 hours of copper treatment , shown as fold enrichment over input samples . Mean + / - SEM ; n = 3 . * p < 0 . 05 with two - tailed Welch ' s t - test compared to lacZ RNAi 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 O O O O O O O O O O O O O O O O

brm RNAi reduces incorporation of histone H3.3. V5 ChIP-qPCR in Kc167 cells after lacZ or brm RNAi treatment in cells with H3-V5 or H3.3-V5 expression induced from the pMT promoter by 3 hours of copper treatment, shown as fold enrichment over input samples. Mean +/- SEM; n = 3. * p<0.05 with two-tailed Welch's t-test compared to lacZ RNAi control.


Chaining indices of the indicated genotypes . Statistical significance was compared with control . ; n = 9 , 6 , 7 ( E ) groups ( 10 flies per group ) . Courtship indices for one target ( Canton - S ) male and one tester male of the indicated genotypes . Changes were determined by comparing the data with control . ; n = 9 , 8 , 9 ( F ) flies . Sexual preference of single male of indicated genotypes toward a decapitated virgin female and a decapitated naïve male . Statistical significance was compared with the other gender . ; n = 10 , 10 , 10 ( F ) flies .

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

Chaining indices of the indicated genotypes. Statistical significance was compared with control. ; n = 9, 6, 7 (E) groups (10 flies per group). Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data with control. ; n = 9, 8, 9 (F) flies. Sexual preference of single male of indicated genotypes toward a decapitated virgin female and a decapitated naïve male. Statistical significance was compared with the other gender. ; n = 10, 10, 10 (F) flies.


Courtship indices for one target ( Canton - S ) male and one tester male of the indicated genotypes . Changes were determined by comparing the data with relative control . n = 8 , 13 , 13 ( D )

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

Courtship indices for one target (Canton-S) male and one tester male of the indicated genotypes. Changes were determined by comparing the data with relative control. n = 8, 13, 13 (D)


( A - C ) DA level ( pictograms per head ) in males of the indicated genotypes . n = 3 , 3 , 3 ( A ) ; n = 4 , 5 , 3 , 3 ( B ) ; n = 3 , 3 , 3 ( C ) times ( 100 heads per genotype ) .

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

(A-C) DA level (pictograms per head) in males of the indicated genotypes. n = 3, 3, 3 (A); n = 4, 5, 3, 3 (B); n = 3, 3, 3 (C) times (100 heads per genotype).


( D ) Posterior confocal sections of UAS - mCD8 - GFP ; Ddc - Gal4 adult male brain in combination with UAS - LacZ or UAS - Myc . RNAi - 1 stained with antibody to nc82 ( pan - neuropil marker ; magenta ) . Scale bars represent 50 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

(D) Posterior confocal sections of UAS-mCD8-GFP; Ddc-Gal4 adult male brain in combination with UAS-LacZ or UAS-Myc.RNAi-1 stained with antibody to nc82 (pan-neuropil marker; magenta). Scale bars represent 50 μm.


( F , G ) Western blot of proteins extracted from adult heads of indicated genotypes . Statistical analysis showing the protein level of GFP ( F ) or Ddc ( G ) to that of β - actin in 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

(F, G) Western blot of proteins extracted from adult heads of indicated genotypes. Statistical analysis showing the protein level of GFP (F) or Ddc (G) to that of β-actin in Western blot.


( B ) Drosophila S2 cells transfected with the pUAST - HA empty vector or the pUAST - HA - Myc plasmid were co - transfected with each of the firefly luciferase reporter vector bearing the fragment shown in ( A ) . After 48 hours , the luciferase activity was detected in each cell lysate ( n = 5 times ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Drosophila S2 cells transfected with the pUAST-HA empty vector or the pUAST-HA-Myc plasmid were co-transfected with each of the firefly luciferase reporter vector bearing the fragment shown in (A). After 48 hours, the luciferase activity was detected in each cell lysate (n = 5 times).


( C ) Drosophila S2 cells transfected with the pUAST - HA - Myc plasmid or the empty vector were used for quantification of ChIP - PCRs ( n = 4 times ) .

O O O O O O O O O O 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) Drosophila S2 cells transfected with the pUAST-HA-Myc plasmid or the empty vector were used for quantification of ChIP-PCRs (n = 4 times).


( D ) RNA from adult male heads ( n = 50 heads per genotype ) of indicated genotypes was subjected to real - time qPCR .

O O O 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) RNA from adult male heads (n = 50 heads per genotype) of indicated genotypes was subjected to real-time qPCR.


( E ) DA level ( pictograms per head ) in the head of males of the indicated genotypes . n = 4 ( 100 heads per genotype ) .

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

(E) DA level (pictograms per head) in the head of males of the indicated genotypes. n = 4 (100 heads per genotype).


( A ) Bone Marrow Derived Macrophages ( BMDMs ) were treated with either the AMPK activator 991 , Dex or a combination for 1 h before analysis by western blot . ( B , C ) Phospho - AMPK ( B ) and phospho - ACC ( C ) were quantified . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 6 ( B , C ) , biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using student ' s t - test ( B , C )

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 O O O O O O O

(A) Bone Marrow Derived Macrophages (BMDMs) were treated with either the AMPK activator 991, Dex or a combination for 1 h before analysis by western blot. (B, C) Phospho-AMPK (B) and phospho-ACC (C) were quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 6 (B,C), biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using student's t-test (B, C)


( D ) WT and AMPKα1 - / - BMDMs were treated with Dex for 1 h before analysis by western blot and GR phosphorylation at S211 was quantified . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 3 ( D ) biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using ANOVA tests ( 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

(D) WT and AMPKα1-/- BMDMs were treated with Dex for 1 h before analysis by western blot and GR phosphorylation at S211 was quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3 (D) biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests (D,


( E ) WT and AMPKα1 - / - BMDMs were treated with Dex for 24 h and analyzed by RNA - seq . Differentially expressed genes between any condition were clustered using c - means clustering .

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

(E) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and analyzed by RNA-seq. Differentially expressed genes between any condition were clustered using c-means clustering.


( F ) WT and AMPKα1 - / - BMDMs were treated with Dex for 24 h and anti - / pro - inflammatory markers assessed using immunofluorescence . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 3 - 4 ( F ) biological

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

(F) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and anti-/pro-inflammatory markers assessed using immunofluorescence. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3-4 (F) biological replicates


( G WT and AMPKα1 - / - BMDMs were treated with Dex for 72 h , washed and conditioned medium was recovered after 24 h used to treat muscle stem cells . Proliferation ( Ki67 immunostaining ) ( G ) were assessed in muscle stem cells . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 4 biological replicates ( G * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using ANOVA tests Bar = 25 µm (

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

(G WT and AMPKα1-/- BMDMs were treated with Dex for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. Proliferation (Ki67 immunostaining) (G) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates (G * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests Bar=25 µm (G


H ) WT and AMPKα1 - / - BMDMs were treated with Dex for 72 h , washed and conditioned medium was recovered after 24 h used to treat muscle stem cells . differentiation ( desmin immunostaining and counting the percentage of muscle cells with 2 nuclei or more ) ( H ) were assessed in muscle stem cells . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 4 biological replicates H ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using ANOVA tests , H ) . Bar = 25 µm H ) .

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 O O O

H) WT and AMPKα1-/- BMDMs were treated with Dex for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. differentiation (desmin immunostaining and counting the percentage of muscle cells with 2 nuclei or more) (H) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates H). * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests ,H). Bar=25 µm H).


After cardiotoxin injection to damage the muscle , AMPKα1fl / fl ( WT ) and LysM - α1 - / - mice were treated with a single dose of Dexamethasone ( Dex ) intra - peritoneal ( i . p . ) ( 0 . 1 mg / kg ) at day 3 ( D3 ) and Tibialis Anterior ( TA ) muscles were harvested at day 8 ( D8 ) and 14 ( D14 ) after injury . ( A ) Representative embryonic myosin heavy chain ( EmbMHC ) immunostaining on whole TA muscle section at D8 and D14 after injury . ( B ) Quantification of ( A ) , results are expressed as percentage of positive myofibers expressing the EmbMHC on the whole muscle section . Data information : results are displayed with box and whiskers in which the central band represents the median ( B N = 7 - 12 muscles ( B * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 using ANOVA tests . Bars = 500 µm ( A ) ,

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

After cardiotoxin injection to damage the muscle, AMPKα1fl/fl (WT) and LysM-α1-/- mice were treated with a single dose of Dexamethasone (Dex) intra-peritoneal (i.p.) (0.1 mg/kg) at day 3 (D3) and Tibialis Anterior (TA) muscles were harvested at day 8 (D8) and 14 (D14) after injury. (A) Representative embryonic myosin heavy chain (EmbMHC) immunostaining on whole TA muscle section at D8 and D14 after injury. (B) Quantification of (A), results are expressed as percentage of positive myofibers expressing the EmbMHC on the whole muscle section. Data information: results are displayed with box and whiskers in which the central band represents the median (B N= 7-12 muscles (B * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 500 µm (A),


( F ) Macrophage polarization was determined by immunofluorescence in vehicle , DAMPs and DAMPs + Dex treated WT and AMPKα1 - / - BMDMs . Data information : results are displayed with box and whiskers in which the central band represents the means ± SEM ( F ) . N = 4 - 5 biological replicates ( F ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 using ANOVA tests . Bars = 25 µm ( F ) .

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

(F) Macrophage polarization was determined by immunofluorescence in vehicle, DAMPs and DAMPs+Dex treated WT and AMPKα1-/- BMDMs. Data information: results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 4-5 biological replicates (F). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 25 µm (F).


( F ) Macrophage polarization was determined by immunofluorescence in vehicle , LPS and LPS + Dex treated WT and AMPKα1 - / - BMDMs , data are expressed as percentage positive cells . Data information : Results are displayed with box and whiskers in which the central band represents the means ± SEM ( F ) . N = 3 ( F ) biological replicates . Statistical analysis by ANOVA F ) * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bar = 25 µm ( F ) .

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

(F) Macrophage polarization was determined by immunofluorescence in vehicle, LPS and LPS+Dex treated WT and AMPKα1-/- BMDMs, data are expressed as percentage positive cells. Data information: Results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 3 (F) biological replicates. Statistical analysis by ANOVA F) * p<0.05, ** p<0.01, *** p<0.001. Bar = 25 µm (F).


( F ) WT and AMPKα1 - / - Bone Marrow Derived Macrophages ( BMDMs ) were treated with either Dex or the AMPK activator 991 and protein phosphorylated on serine were immunoprecipitated . Westen - blot for FOXO3 , AMPKα1 , actin and the phosphorylated form of FOXO3 were done . Quantification of the band intensity is provided normalized to those of actin and total FOXO3 , a representative example is shown . Data information : results are box and whiskers in which the central band represents the median F , N = 4 - 5 ( F biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 using ANOVA and Kruskal - Wallis

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT and AMPKα1-/- Bone Marrow Derived Macrophages (BMDMs) were treated with either Dex or the AMPK activator 991 and protein phosphorylated on serine were immunoprecipitated. Westen-blot for FOXO3, AMPKα1, actin and the phosphorylated form of FOXO3 were done. Quantification of the band intensity is provided normalized to those of actin and total FOXO3, a representative example is shown. Data information: results are box and whiskers in which the central band represents the median F, N= 4-5 (F biological replicates. * p<0.05, ** p<0.01, *** p<0.001 **** p<0.0001 using ANOVA and Kruskal-Wallis tests


( A ) Confocal micrographs of adult brain of a young ( 2 d old ) wild - type fly . Positive staining for Ref ( 2 ) P and ubiquitin is not evident . ( B - D ) Confocal micrographs of adult brain of an old ( 8 wk old ) wild - type fly . Ref ( 2 ) P accumulates in protein aggregates that often colocalize with ubiquitin ( arrows ) . The white rectangle in C indicates the area shown in D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal micrographs of adult brain of a young (2 d old) wild-type fly. Positive staining for Ref(2)P and ubiquitin is not evident. (B-D) Confocal micrographs of adult brain of an old (8 wk old) wild-type fly. Ref(2)P accumulates in protein aggregates that often colocalize with ubiquitin (arrows). The white rectangle in C indicates the area shown in D.


( E and F ) Western blot analysis of total cell lysates ( E ) and insoluble protein fraction ( F ) of wild - type young and old adult heads probed with anti - ref ( 2 ) P and anti - ubiquitin antibodies , demonstrating a significant increase in Ref ( 2 ) P and insoluble ubiquitinated protein levels in old flies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F) Western blot analysis of total cell lysates (E) and insoluble protein fraction (F) of wild-type young and old adult heads probed with anti-ref(2)P and anti-ubiquitin antibodies, demonstrating a significant increase in Ref(2)P and insoluble ubiquitinated protein levels in old flies.


( A - D ) Electron micrographs of adult brain of old wild - type flies . Ref ( 2 ) P localizes in electron - dense structures ranging from 50 nm to 1 μm in diameter that are surrounded ( arrows ) or not ( arrowhead ) by a limiting membrane . ( E and F ) Electron micrographs of wild - type brain of old flies demonstrating that Ref ( 2 ) P forms shell - like structures that surround aggregated filamentous material ( arrows ) . Bars : ( A - C ) 50 nm ; ( D ) 100 nm ; ( E and F ) 200 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Electron micrographs of adult brain of old wild-type flies. Ref(2)P localizes in electron-dense structures ranging from 50 nm to 1 μm in diameter that are surrounded (arrows) or not (arrowhead) by a limiting membrane. (E and F) Electron micrographs of wild-type brain of old flies demonstrating that Ref(2)P forms shell-like structures that surround aggregated filamentous material (arrows). Bars: (A-C) 50 nm; (D) 100 nm; (E and F) 200 nm.


( A and B ) atg8a mutants exhibiting a striking positive staining for Ref ( 2 ) P and ubiquitin staining in all the regions of the adult brain . As it is shown in high magnification of the optic lobe ( medulla region ; B ) , Ref ( 2 ) P - and ubiquitin - positive aggregates are observed ( arrows ) . Genotypes : ( A and B ) atg8aKG07569 /

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) atg8a mutants exhibiting a striking positive staining for Ref(2)P and ubiquitin staining in all the regions of the adult brain. As it is shown in high magnification of the optic lobe (medulla region; B), Ref(2)P- and ubiquitin-positive aggregates are observed (arrows). Genotypes: (A and B) atg8aKG07569/Y


( C ) Normal phenotype is observed in β2 and 6 dominant - negative temperature - sensitive mutants at 25 ° C . ( D ) Accumulation of Ref ( 2 ) P - and ubiquitin - positive aggregates are observed in β2 and 6 mutants at 29 ° C ( arrows ) . ( C and D ) elavGal4 / + ; UAS - β6 / + ; UAS - β2 / +

(C) Normal phenotype is observed in β2 and 6 dominant-negative temperature-sensitive mutants at 25°C. (D) Accumulation of Ref(2)P- and ubiquitin-positive aggregates are observed in β2 and 6 mutants at 29°C (arrows). (C and D) elavGal4/+; UAS-β6/+; UAS-β2/+


( E and F ) double mutants for atg8a and ref ( 2 ) P loss - of - function alleles lacking the PB1 ( E ) or UBA ( F ) domain do not form ubiquitinated protein aggregates . Ubiquitin exhibits a cytoplasmic staining pattern ( arrows ) . Genotypes : ( E ) atg8aKG07569 / Y ; ref ( 2 ) Pod2 / ref ( 2 ) Pod2 , and ( F ) atg8aKG07569 / Y ; ref ( 2 ) Pod3 / ref ( 2 ) Pod3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F) double mutants for atg8a and ref(2)P loss-of-function alleles lacking the PB1 (E) or UBA (F) domain do not form ubiquitinated protein aggregates. Ubiquitin exhibits a cytoplasmic staining pattern (arrows). Genotypes: (E) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, and (F) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3.


( A and B ) Protein aggregates in atg8aKG07569 mutants exhibit sequestosome morphology and are positively labeled for Ref ( 2 ) P ( A ) and ubiquitin ( B ) . Protein aggregates are surrounded by small electron - dense vesicles ( arrows ) .

O O O O O O O O O O O O O O O O O O O O O O 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 and B) Protein aggregates in atg8aKG07569 mutants exhibit sequestosome morphology and are positively labeled for Ref(2)P (A) and ubiquitin (B). Protein aggregates are surrounded by small electron-dense vesicles (arrows).


( C ) Western blot analysis of cell lysates of wild type and atg8aKG07569 young adult heads demonstrating a significant increase of Ref ( 2 ) P protein levels in atg8aKG07569 flies .

O O O O O O O O O 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) Western blot analysis of cell lysates of wild type and atg8aKG07569 young adult heads demonstrating a significant increase of Ref(2)P protein levels in atg8aKG07569 flies.


( D , left ) Ref ( 2 ) P protein migration profile in ref ( 2 ) P loss - of - function alleles . Ref ( 2 ) P mutant protein for the PB1 domain has an apparent molecular mass of 85 kD ( ref ( 2 ) Pod2 / ref ( 2 ) Pod2 ) and the Ref ( 2 ) P mutant protein for the UBA domain has an apparent molecular mass of 69 kD ( ref ( 2 ) Pod3 / ref ( 2 ) Pod3 ) . ( D , right ) Western blot analysis of insoluble protein fractions of adult heads of double mutants for atg8a and ref ( 2 ) P probed for ubiquitin . Genotypes : ( 1 ) atg8aKG07569 / Y ; ref ( 2 ) Pod2 / Cyo , ( 2 ) atg8aKG07569 / Y ; ref ( 2 ) Pod2 / ref ( 2 ) Pod2 , ( 3 ) atg8aKG07569 / Y ; ref ( 2 ) Pod3 / CyO , and ( 4 ) atg8aKG07569 / Y ; ref ( 2 ) Pod3 / ref ( 2 ) Pod3 . The insoluble ubiquitinated protein profile of the double mutants is diminished compared with the one of the respective control heterozygous for ref ( 2 ) P .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, left) Ref(2)P protein migration profile in ref(2)P loss-of-function alleles. Ref(2)P mutant protein for the PB1 domain has an apparent molecular mass of 85 kD (ref(2)Pod2/ref(2)Pod2) and the Ref(2)P mutant protein for the UBA domain has an apparent molecular mass of 69 kD (ref(2)Pod3/ref(2)Pod3). (D, right) Western blot analysis of insoluble protein fractions of adult heads of double mutants for atg8a and ref(2)P probed for ubiquitin. Genotypes: (1) atg8aKG07569/Y; ref(2)Pod2/Cyo, (2) atg8aKG07569/Y; ref(2)Pod2/ref(2)Pod2, (3) atg8aKG07569/Y; ref(2)Pod3/CyO, and (4) atg8aKG07569/Y; ref(2)Pod3/ref(2)Pod3. The insoluble ubiquitinated protein profile of the double mutants is diminished compared with the one of the respective control heterozygous for ref(2)P.


( E and F ) Neuronal cells mutant for atg1 ( green ) accumulate Ref ( 2 ) P - ( E ) and ubiquitin - positive ( F ) structures ( arrows ) , whereas the normal surrounding cells do not exhibit positive staining . Genotypes : ( E and F ) yw / UAS - CD8 - GFP , hs - flp ; tubGal4 / + ; atg1Δ3D , FRT80 / tubGal80 ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F) Neuronal cells mutant for atg1 (green) accumulate Ref(2)P- (E) and ubiquitin-positive (F) structures (arrows), whereas the normal surrounding cells do not exhibit positive staining. Genotypes: (E and F) yw/UAS-CD8-GFP, hs-flp; tubGal4/+; atg1Δ3D, FRT80/tubGal80, FRT80


( G ) Confocal micrographs of the optic lobe ( medulla region ) of young flies expressing a mutated form of human tau protein exhibiting Ref ( 2 ) P - and tau - positive aggregates ( arrows ) . Genotypes : ( G ) elav / + ; UAS - E14 / + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 micrographs of the optic lobe (medulla region) of young flies expressing a mutated form of human tau protein exhibiting Ref(2)P- and tau-positive aggregates (arrows). Genotypes: (G) elav/+;UAS-E14/+.


( D ) Fragment length distributions of human - ( pink ) and mouse - ( blue ) specific reads from a DBS sample . Two vertical lines indicate 146 and 166 bp , the observed peaks for ctDNA and cfDNA , respectively .

O O O O O O O O O O O O O O O O O O O O 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) Fragment length distributions of human- (pink) and mouse- (blue) specific reads from a DBS sample. Two vertical lines indicate 146 and 166 bp, the observed peaks for ctDNA and cfDNA, respectively.


( E ) Example of an absolute copy number ( ACN ) profile successfully generated from human - specific reads from a DBS collected from a PDX mouse of patient line 828 . ( F ) Matching ACN profile generated from sWGS of PDX tumour tissue .

O O O O O O O O O O O O O O O O O O O O O O O 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) Example of an absolute copy number (ACN) profile successfully generated from human-specific reads from a DBS collected from a PDX mouse of patient line 828. (F) Matching ACN profile generated from sWGS of PDX tumour tissue.


( B ) xTF change from baseline during the first 29 days since treatment start for each PDX patient line . xTFs were normalised to baseline ( day 1 ) xTF values for each mouse ( dashed lines ) . Carboplatin - treated mice are shown in purple , control mice are shown in teal . Bold lines show the linear - model fitted line across all mice within the same treatment and patient group . Horizontal dashed lines at y = 1 indicate normalised baseline .

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

(B) xTF change from baseline during the first 29 days since treatment start for each PDX patient line. xTFs were normalised to baseline (day 1) xTF values for each mouse (dashed lines). Carboplatin-treated mice are shown in purple, control mice are shown in teal. Bold lines show the linear-model fitted line across all mice within the same treatment and patient group. Horizontal dashed lines at y=1 indicate normalised baseline.


( C ) Fraction of blood spot samples in which putative driver amplifications were detected over time . The fraction of samples with detected gene amplifications decreases in carboplatin treated group , while increasing in the control group over time .

O O O O 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

(C) Fraction of blood spot samples in which putative driver amplifications were detected over time. The fraction of samples with detected gene amplifications decreases in carboplatin treated group, while increasing in the control group over time.


( D ) Correlation between xTF values and tumour volumes of the nearest matched time - point for both untreated ( Pearson ' s R = 0 . 45 , p = 0 . 00018 ) , and carboplatin - treated ( Pearson ' s R = 0 . 056 , p = 0 . 78 ) PDX 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(D) Correlation between xTF values and tumour volumes of the nearest matched time-point for both untreated (Pearson's R = 0.45, p = 0.00018), and carboplatin-treated (Pearson's R = 0.056, p = 0.78) PDX mice.


( A - D ) Weekly measured tumour volumes ( mm3 ) for PDX mice over time . Treatment start is indicated by grey solid vertical line . Solid coloured lines show modelled tumour growth curves using heteroscedastic point - wise random intercept linear mixed models ( see Methods ) . Growth curve inflection points were determined ( see Methods ) to estimate the start of treatment effect and tumour regrowth ( dashed vertical purple lines labelled t1 and t2 , respectively ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Weekly measured tumour volumes (mm3) for PDX mice over time. Treatment start is indicated by grey solid vertical line. Solid coloured lines show modelled tumour growth curves using heteroscedastic point-wise random intercept linear mixed models (see Methods). Growth curve inflection points were determined (see Methods) to estimate the start of treatment effect and tumour regrowth (dashed vertical purple lines labelled t1 and t2, respectively).


( E ) The mean xTF slope was estimated for each treatment group across the four patient lines ( Fig . 2B ) and compared to the mean time to endpoint ( Pearson ' s R = - 0 . 73 , p = 0 . 039 ) and tumour regrowth ( Pearson ' s R = - 0 . 97 , p = 0 . 025 ) . The four different patient lines are indicated by different colours .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mean xTF slope was estimated for each treatment group across the four patient lines (Fig. 2B) and compared to the mean time to endpoint (Pearson's R = -0.73, p = 0.039) and tumour regrowth (Pearson's R = -0.97, p = 0.025). The four different patient lines are indicated by different colours.


( A ) Solubilized lysosomes cross ‐ linked with DSP were subjected to affinity chromatography with the antibody against the cytosolic tail of lamp2a ( lanes 1 and 2 ) . Eluted proteins were subjected to SDS - PAGE and silver stained ( lane 1 ) , or immunoblotted for lamp2a ( lane 2 ) . The sequence of the N ‐ terminus of the 32 kDa band is shown . Part of the solubilized lysosomes were directly subjected to non ‐ reducing SDS - PAGE ( 5 % ) and immunoblotted for lamp2a ( lane 3 ) or PPCA ( lane 4 ) . Arrowheads indicate lamp2a multimeric complexes . The 600 kDa ( * ) and 200 kDa ( * * ) molecular weight complexes of lamp2a were excised and subjected to reducing SDS - PAGE ( 12 % ) and silver staining ( lanes 5 and 6 ) . Molecular weight ( kDa ) is indicated 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Solubilized lysosomes cross‐linked with DSP were subjected to affinity chromatography with the antibody against the cytosolic tail of lamp2a (lanes 1 and 2). Eluted proteins were subjected to SDS-PAGE and silver stained (lane 1), or immunoblotted for lamp2a (lane 2). The sequence of the N‐terminus of the 32 kDa band is shown. Part of the solubilized lysosomes were directly subjected to non‐reducing SDS-PAGE (5%) and immunoblotted for lamp2a (lane 3) or PPCA (lane 4). Arrowheads indicate lamp2a multimeric complexes. The 600 kDa (*) and 200 kDa (**) molecular weight complexes of lamp2a were excised and subjected to reducing SDS-PAGE (12%) and silver staining (lanes 5 and 6). Molecular weight (kDa) is indicated on the left.


( B ) Immunoblot analysis of PPCA in rat liver lysosomes , their membranes ( MB ) and matrices ( MTX ) ( 100 μg of protein ) incubated alone ( None ) or with 5 mM MgCl2 . Arrowheads indicate the precursor ( 54 kDa ) and heavy chain ( 32 kDa ) of the mature PPCA .

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

(B) Immunoblot analysis of PPCA in rat liver lysosomes, their membranes (MB) and matrices (MTX) (100 μg of protein) incubated alone (None) or with 5 mM MgCl2. Arrowheads indicate the precursor (54 kDa) and heavy chain (32 kDa) of the mature PPCA.


( C ) Immunoblot analysis of hsc73 ( upper ) and PPCA ( lower ) in membranes from rat liver lysosomes incubated alone ( None ) or with 5 mM MgCl2 , CaCl2 , MnCl2 or 2 mM EDTA .

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

(C) Immunoblot analysis of hsc73 (upper) and PPCA (lower) in membranes from rat liver lysosomes incubated alone (None) or with 5 mM MgCl2, CaCl2, MnCl2 or 2 mM EDTA.


( D ) Immunoblot analysis of lamp2a co ‐ immunoprecipitated with PPCA in the absence of chemical cross ‐ linkers from membranes of liver lysosomes incubated as in ( B ) . Light and heavy chains of IgG are shown . Lane 1 contains 1 / 10 of the total amount of lysosomal membranes used for immunoprecipitation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of lamp2a co‐immunoprecipitated with PPCA in the absence of chemical cross‐linkers from membranes of liver lysosomes incubated as in (B). Light and heavy chains of IgG are shown. Lane 1 contains 1/10 of the total amount of lysosomal membranes used for immunoprecipitation.


( E ) Membranes from lysosomes treated as in ( D ) were subjected to immunoprecipitation with an antibody specific for lamp2a or with a pre ‐ immune serum . Carboxypeptidase activity in total lysosomes and in the immunoprecipitates was measured as described under Materials and methods . Values are expressed as percentage of total carboxypeptidase activity in lysosomes present in the immunoprecipitates and are the mean ± SE of three different 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 O O O O O O O O O O O

(E) Membranes from lysosomes treated as in (D) were subjected to immunoprecipitation with an antibody specific for lamp2a or with a pre‐immune serum. Carboxypeptidase activity in total lysosomes and in the immunoprecipitates was measured as described under Materials and methods. Values are expressed as percentage of total carboxypeptidase activity in lysosomes present in the immunoprecipitates and are the mean ± SE of three different experiments.


Levels of PPCA associated with the lysosomal membrane correlate with rates of CMA . Membranes of lysosomes incubated with increasing concentrations of GAPDH ( A ) or MgCl2 ( B , top panel ) as labeled , were analyzed by immunoblot for all forms of lamp2 ( upper ) , lamp2a ( middle ) or PPCA ( lower ) . The arrowhead indicates a previously identified truncated form of lamp2 lacking the cytosolic / transmembrane region ( Cuervo and Dice , 2000b ) . ( B , bottom panel ) Chart showing the effect of increasing concentrations of MgCl2 , CaCl2 or EDTA on the degradation of [ 14C ] GAPDH by intact ratliver lysosomes . Values are means ± SE of six different experiments . Differences from the control value were significant at P 0 . 001 ( * * ) or P 0 . 01 ( * ) .

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

Levels of PPCA associated with the lysosomal membrane correlate with rates of CMA. Membranes of lysosomes incubated with increasing concentrations of GAPDH (A) or MgCl2 (B, top panel) as labeled, were analyzed by immunoblot for all forms of lamp2 (upper), lamp2a (middle) or PPCA (lower). The arrowhead indicates a previously identified truncated form of lamp2 lacking the cytosolic/transmembrane region ( Cuervo and Dice, 2000b). (B, bottom panel) Chart showing the effect of increasing concentrations of MgCl2, CaCl2 or EDTA on the degradation of [14C]GAPDH by intact ratliver lysosomes. Values are means ± SE of six different experiments. Differences from the control value were significant at P 0.001 (**) or P 0.01 (*).


( C ) Immunoblot analysis for PPCA ( upper ) and cathepsin L ( lower ) of rat liver lysosomes ( 100 μg of protein ) with high ( H + ) and low ( H − ) activity for CMA and of the lysosomal membranes ( L . Memb ) and matrices ( L . Mtx ) from both groups of lysosomes ( 200 μg of protein ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis for PPCA (upper) and cathepsin L (lower) of rat liver lysosomes (100 μg of protein) with high (H+) and low (H−) activity for CMA and of the lysosomal membranes (L.Memb) and matrices (L.Mtx) from both groups of lysosomes (200 μg of protein).


( D ) Immunoblot analysis for lamp2a , PPCA and cathepsin D , as labeled , of lysosomes ( 100 μg of protein ) from fed or 48 h starved rats and the membranes ( L . Memb ) and matrices ( L . Mtx ) of both goups of lysosomes ( 200 μg of protein ) . Only mature forms of the cathepsins are

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis for lamp2a, PPCA and cathepsin D, as labeled, of lysosomes (100 μg of protein) from fed or 48 h starved rats and the membranes (L. Memb) and matrices (L. Mtx) of both goups of lysosomes (200 μg of protein). Only mature forms of the cathepsins are shown


( E ) Correlation between CMA activity , measured as in ( B , bottom panel ) and levels of PPCA at the lysosomal membrane determined by denstometric quantification of immunoblots similar to those shown in Figures 1 and 2 in lysosomes from fed or 20 h starved rats ( Starv 20h ) , highly active ( H + ) or less active ( H − ) lysosomes , or lysosomes incubated with 1 mM CaCl2 , MgCl2 , EDTA , or increasing amounts ( 2 , 5 , 10 μg ) of GAPDH . Values are expressed as times the value in fed rats ( control ) and are means ± SE of 3 - 6 different 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 O O O O O O O O O O O O B-CONTROLLED_VAR 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

(E) Correlation between CMA activity, measured as in (B, bottom panel) and levels of PPCA at the lysosomal membrane determined by denstometric quantification of immunoblots similar to those shown in Figures 1 and 2 in lysosomes from fed or 20 h starved rats (Starv 20h), highly active (H+) or less active (H−) lysosomes, or lysosomes incubated with 1 mM CaCl2, MgCl2, EDTA, or increasing amounts (2, 5, 10 μg) of GAPDH. Values are expressed as times the value in fed rats (control) and are means ± SE of 3-6 different experiments.


( A ) Rates of protein degradation in skin fibroblasts from wild ‐ type ( control ) or PPCA ( − / − ) mice maintained in the presence ( serum + ) or absence of serum ( serum − ) . Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of four different 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 O O O

(A) Rates of protein degradation in skin fibroblasts from wild‐type (control) or PPCA(−/−) mice maintained in the presence (serum +) or absence of serum (serum −). Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of four different experiments.


( B ) Degradation of [ 14C ] GAPDH after 30 min incubation with lysosomes isolated from wild ‐ type and PPCA ( − / − ) mice skin fibroblasts supplemented or deprived of serum was measured as described in Material and methods . Where indicated , 25 μg of RNase A were added . Values are means ± SE of four different experiments . Differences from the control value were significant to P 0 . 001 ( * * ) .

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

(B) Degradation of [14C]GAPDH after 30 min incubation with lysosomes isolated from wild‐type and PPCA(−/−) mice skin fibroblasts supplemented or deprived of serum was measured as described in Material and methods. Where indicated, 25 μg of RNase A were added. Values are means ± SE of four different experiments. Differences from the control value were significant to P 0.001 (**).


( C ) Immunofluorescent staining for lamp2a and lamp1 of skin fibroblasts from wild ‐ type and PPCA ( − / − ) mice . Gain for lamp2a images ( top ) was 1 / 8 of the one for lamp1 ( bottom ) to visualize the punctate pattern of lamp2a in PPCA ( − / − ) mice . Inset : detail of the vesicular distribution of lamp2a . The gain in wild type was eight times the gain in PPCA ( − / − ) mice ) . Bars : 100 μm ( top ) , 50 μm ( bottom ) , 10 μm ( inset ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunofluorescent staining for lamp2a and lamp1 of skin fibroblasts from wild‐type and PPCA(−/−) mice. Gain for lamp2a images (top) was 1/8 of the one for lamp1 (bottom) to visualize the punctate pattern of lamp2a in PPCA(−/−) mice. Inset: detail of the vesicular distribution of lamp2a. The gain in wild type was eight times the gain in PPCA(−/−) mice). Bars: 100 μm (top), 50 μm (bottom), 10 μm (inset).


( D ) Immunoblot analysis for lamp2a , all lamp2s , lamp1 or cathepsin D ( cath D ) of homogenates ( 75 μg of protein ) , lysosomes ( 10 μg of protein ) and lysosomal membranes ( L . MB ) and matrices ( L . MTX ) ( 5 μg of protein ) isolated from wild ‐ type ( WT ) and PPCA ( − / − ) skin fibroblasts . The bottom chart shows the densitometric quantification of the membrane proteins ( means + SE ) from four immunoblots similar to those shown here . Differences from the control value were significant to P 0 . 001 ( * * ) and P 0 . 05 ( * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis for lamp2a, all lamp2s, lamp1 or cathepsin D (cath D) of homogenates (75 μg of protein), lysosomes (10 μg of protein) and lysosomal membranes (L.MB) and matrices (L.MTX) (5 μg of protein) isolated from wild‐type (WT) and PPCA(−/−) skin fibroblasts. The bottom chart shows the densitometric quantification of the membrane proteins (means + SE) from four immunoblots similar to those shown here. Differences from the control value were significant to P 0.001 (**) and P 0.05 (*).


( A ) Immunoblot analysis for lamp2a , all lamp2s , lamp1 , transferrin receptor ( TFR ) or cathepsin D ( cath D ) of liver homogenates ( 100 μg of protein ) from wild ‐ type ( WT ) , PPCA ( − / − ) and β ‐ galactosidase ( − / − ) ( GM ) mice . Right : densitometric quantification of three immunoblots ( means + SE ) similar to the ones shown here . Differences from the control value were significant to P 0 . 001 ( * * ) and P 0 . 05 ( * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis for lamp2a, all lamp2s, lamp1, transferrin receptor (TFR) or cathepsin D (cath D) of liver homogenates (100 μg of protein) from wild‐type (WT), PPCA(−/−) and β‐galactosidase(−/−) (GM) mice. Right: densitometric quantification of three immunoblots (means + SE) similar to the ones shown here. Differences from the control value were significant to P 0.001 (**) and P 0.05 (*).


( B ) Immunocytochemistry for lamp2a ( left ) , lamp1 ( center ) or lamp2s ( right ) in hematoxylin and eosin ‐ stained sections from the same livers . Bar : 50 μm . Black arrows : hepatocytes ; white arrows : Kupffer cells .

O O O O O O O O O O O O O O O O O O O O O O O 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) Immunocytochemistry for lamp2a (left), lamp1 (center) or lamp2s (right) in hematoxylin and eosin‐stained sections from the same livers. Bar: 50 μm. Black arrows: hepatocytes; white arrows: Kupffer cells.


( C ) Immunofluorescence for lamp2a ( top ) and P ‐ glycoprotein ( bottom ) in mouse liver sections . The right panel shows merged images . Bar : 100 μm . Inset shows detailed area at higher magnification . Bar : 50 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

(C) Immunofluorescence for lamp2a (top) and P‐glycoprotein (bottom) in mouse liver sections. The right panel shows merged images. Bar: 100 μm. Inset shows detailed area at higher magnification. Bar: 50 μm.


( A ) Autofluorography of the sequential immunoprecipitation of lamp2a first ( left ) and all the remaining isoforms of lamp2 second ( right ) from metabolically labeled wild ‐ type and PPCA ( − / − ) mouse skin fibroblasts . Values are means ± SE of three different experiments similar to the ones shown in the upper panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Autofluorography of the sequential immunoprecipitation of lamp2a first (left) and all the remaining isoforms of lamp2 second (right) from metabolically labeled wild‐type and PPCA(−/−) mouse skin fibroblasts. Values are means ± SE of three different experiments similar to the ones shown in the upper panels.


( B ) Immunoblot analysis for lamp2a ( top ) and all lamp2 isoforms ( bottom ) in lysosomalmembranes from wild ‐ type ( WT ) or PPCA ( − / − ) mouseskinfibroblasts at different times after incubation at 37 ° C . The open arrowhead indicates the truncated form of lamp2 lacking the cytosolic / transmembrane region ( Cuervo and Dice , 2000b ) . Right shows the densitometric quantification ( mean ± SE ) of four different experiments similar to the one shown here . Values are expressed as percentage of remaining lamp2a , and are means ± 1 SE of three different 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 O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis for lamp2a (top) and all lamp2 isoforms (bottom) in lysosomalmembranes from wild‐type (WT) or PPCA(−/−) mouseskinfibroblasts at different times after incubation at 37°C. The open arrowhead indicates the truncated form of lamp2 lacking the cytosolic/transmembrane region ( Cuervo and Dice, 2000b). Right shows the densitometric quantification (mean ± SE) of four different experiments similar to the one shown here. Values are expressed as percentage of remaining lamp2a, and are means ± 1 SE of three different experiments.


( A ) Rates of total protein degradation in the presence and absence of serum of normal skin fibroblasts ( CTR ) , GS cells ( None ) and GS cells supplemented with the precursor forms of native PPCA ( + PPCA ) , a catalytic mutant of PPCA ( + PPCA S / A ) or cathepsin L ( Cath L ) . Values are means ± SE of six different experiments . Differences from the control value were significant for P 0 . 001 ( * * ) and P 0 . 05 ( * ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Rates of total protein degradation in the presence and absence of serum of normal skin fibroblasts (CTR), GS cells (None) and GS cells supplemented with the precursor forms of native PPCA (+PPCA), a catalytic mutant of PPCA (+PPCA S/A) or cathepsin L (Cath L). Values are means ± SE of six different experiments. Differences from the control value were significant for P 0.001 (**) and P 0.05 (*).


( B ) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37 ° C of lysosomes isolated from PPCA ( − / − ) cells ( None ) and PPCA ( − / − ) cells supplemented as in ( A ) . At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a ( open arrowhead ) . Right : densitometric quantification for lamp2a ( means + range of values ) of two different experiments similar to the one shown . Values are expressed as percentage of the lamp2a present in non ‐ supplemented cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37°C of lysosomes isolated from PPCA(−/−) cells (None) and PPCA(−/−) cells supplemented as in (A). At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a (open arrowhead). Right: densitometric quantification for lamp2a (means + range of values) of two different experiments similar to the one shown. Values are expressed as percentage of the lamp2a present in non‐supplemented cells.


( C ) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37 ° C of lysosomes isolated from PPCA ( − / − ) cells ( None ) and PPCA ( − / − ) cells supplemented as in ( A ) . At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a ( open arrowhead ) . Right : densitometric quantification for lamp2a ( means + range of values ) of two different experiments similar to the one shown . Values are expressed as percentage of the lamp2a present in non ‐ supplemented cells .

(C) Immunoblot analysis for lamp2a at time 0 or after 40 min incubation at 37°C of lysosomes isolated from PPCA(−/−) cells (None) and PPCA(−/−) cells supplemented as in (A). At 40 min immunoblot with the antibody against the luminal region of lamp2a is depicted to show the truncated form of lamp2a (open arrowhead). Right: densitometric quantification for lamp2a (means + range of values) of two different experiments similar to the one shown. Values are expressed as percentage of the lamp2a present in non‐supplemented cells.


( D ) Immunocytochemistry for lamp2a of liver ( top ) and kidney ( bottom ) sections of age ‐ matched wild ‐ type ( WT ) , PPCA ( − / − ) and bone marrow transplanted ( BMT ) mice . Bar , 50 μm ( liver ) or 100 μm ( kidney ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunocytochemistry for lamp2a of liver (top) and kidney (bottom) sections of age‐matched wild‐type (WT), PPCA(−/−) and bone marrow transplanted (BMT) mice. Bar, 50 μm (liver) or 100 μm (kidney).


( A ) Left : amino acid sequences of the C ‐ terminal regions of lamp2a from different species and of other lamp2 isoforms and lamp1 from mouse . The proposed cleavage site for PPCA in lamp2a is highlighted . Right : immunoblot analysis for HA of lysosomal membranes isolated from two different clones of HEK293 ( mouse ; upper panel ) and NIH 3T3 cells ( human ; lower panel ) stably transfected with wild ‐ type ( FL ) and mutated ( GL ) HA ‐ lamp2a . The arrowhead indicates the truncated form of lamp2 lacking the cytosolic / transmembrane region .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: amino acid sequences of the C‐terminal regions of lamp2a from different species and of other lamp2 isoforms and lamp1 from mouse. The proposed cleavage site for PPCA in lamp2a is highlighted. Right: immunoblot analysis for HA of lysosomal membranes isolated from two different clones of HEK293 (mouse; upper panel) and NIH 3T3 cells (human; lower panel) stably transfected with wild‐type (FL) and mutated (GL) HA‐lamp2a. The arrowhead indicates the truncated form of lamp2 lacking the cytosolic/transmembrane region.


( B ) Total rates of protein degradation in the presence and absence of serum in non ‐ transfected HEK293 cells ( WT ) and in the transfected HEK293 clones described in ( A ) . Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of three different 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

(B) Total rates of protein degradation in the presence and absence of serum in non‐transfected HEK293 cells (WT) and in the transfected HEK293 clones described in (A). Values are expressed as the percentage of initially radiolabeled proteins remaining at each time and are means ± SE of three different experiments.


( C ) Top : immunoblot analysis with an anti ‐ HA antibody of purified wild ‐ type ( FL ) and mutated ( GL ) mouse HA ‐ lamp2a incubated in an acidic pH buffer alone , or with PPCA . Bottom : wild ‐ type HA ‐ lamp2a was incubated as in the top panel without additions ( None ) or in the presence of 2 mM EDTA , 10 μM L ‐ trans ‐ epoxysuccinyl ‐ leucylamide ‐ ( 4 ‐ guanido ) ‐ butane ( E ‐ 64 ) , 2 μM pepstatin A ( Pepst ) or 10 μM ABSF . The open arrowhead indicates the truncated form of lamp2a .

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

(C) Top: immunoblot analysis with an anti‐HA antibody of purified wild‐type (FL) and mutated (GL) mouse HA‐lamp2a incubated in an acidic pH buffer alone, or with PPCA. Bottom: wild‐type HA‐lamp2a was incubated as in the top panel without additions (None) or in the presence of 2 mM EDTA, 10 μM L‐trans‐epoxysuccinyl‐leucylamide‐(4‐guanido)‐butane (E‐64), 2 μM pepstatin A (Pepst) or 10 μM ABSF. The open arrowhead indicates the truncated form of lamp2a.


( A ) siRNA knockdown of Tollip in SH - SY5Y cells was performed over 72 hours , with cells reverse transfected with siRNA oligos specific for Tollip ( or mock ) for 24 hours before a second transfection was performed for a further 48 hours . Knockdown was confirmed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) siRNA knockdown of Tollip in SH-SY5Y cells was performed over 72 hours, with cells reverse transfected with siRNA oligos specific for Tollip (or mock) for 24 hours before a second transfection was performed for a further 48 hours. Knockdown was confirmed by western blotting.


( B ) After knockdown of Tollip , SH - SY5Y cells were treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or 25 μM Antimycin A ( AA ) alone for 2 hours , then fixed and stained for PDH E2 / E3bp ( green ) , TOM20 ( red ) and nuclei ( blue ) . Images were captured using widefield immunofluorescence microscopy . Zoom insets represent 4x magnification and arrowheads indicate TOM20 - positive MDVs .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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

(B) After knockdown of Tollip, SH-SY5Y cells were treated with 5 μM antimycin A/10 μM oligomycin B (AO) or 25 μM Antimycin A (AA) alone for 2 hours, then fixed and stained for PDH E2/E3bp (green), TOM20 (red) and nuclei (blue). Images were captured using widefield immunofluorescence microscopy. Zoom insets represent 4x magnification and arrowheads indicate TOM20-positive MDVs.


( C , D ) The number of TOM20 + ve / PDH - ve MDVs ( C ) or PDH + ve / TOM20 - ve MDVs ( D ) per cell were quantified from immunofluorescence images

O O O O O O O O O O O O O O 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) The number of TOM20+ve/PDH-ve MDVs (C) or PDH+ve/TOM20-ve MDVs (D) per cell were quantified from immunofluorescence images captured


( E ) SH - SY5Y cells were transfected with GFP - labelled Tollip for 24 hours then treated with AO , AO / 100 μM bafilomycin A1 ( AO / BfnA1 ) or AA alone for 2 hours prior to fixation . Cells were then immunostained for GFP ( green ) , PDH E2 / E3bp ( red ) and TOM20 ( blue ) and imaged by widefield immunofluorescence microscopy . GFP - Tollip was observed on TOM20 + ve / PDH - ve MDVs ( arrowheads ) . Zoom insets represent 7x magnification .

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

(E) SH-SY5Y cells were transfected with GFP-labelled Tollip for 24 hours then treated with AO, AO/100 μM bafilomycin A1 (AO/BfnA1) or AA alone for 2 hours prior to fixation. Cells were then immunostained for GFP (green), PDH E2/E3bp (red) and TOM20 (blue) and imaged by widefield immunofluorescence microscopy. GFP-Tollip was observed on TOM20+ve/PDH-ve MDVs (arrowheads). Zoom insets represent 7x magnification.


( F ) Images captured using super - resolution radial fluctuations ( SRRF ) microscopy showing both TOM20 ( arrowheads ) and PDH ( arrows ) MDVs in SH - SY5Y cells after Tollip siRNA knockdown . Left side images are the first in each of the original widefield image stacks . Scale bar for zoom images is 0 . 5 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

(F) Images captured using super-resolution radial fluctuations (SRRF) microscopy showing both TOM20 (arrowheads) and PDH (arrows) MDVs in SH-SY5Y cells after Tollip siRNA knockdown. Left side images are the first in each of the original widefield image stacks. Scale bar for zoom images is 0.5 μm.


( A ) TOM20 - positive MDV formation was induced in SH - SY5Y by siRNA knockdown of Tollip . MDVs were defined as TOM20 + ve / Cytochrome c ( Cyt c ) - ve via immunofluorescence microscopy , which indicated some colocalisation with Parkin ( arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TOM20-positive MDV formation was induced in SH-SY5Y by siRNA knockdown of Tollip. MDVs were defined as TOM20+ve/Cytochrome c (Cyt c)-ve via immunofluorescence microscopy, which indicated some colocalisation with Parkin (arrowheads).


( B ) SH - SY5Y cells were transfected with GFP - Tollip for 24 hours , then treated with 5 μM antimycin A / 10 μM oligomycin B ( AO ) or AO / 100 μM bafilomycin A1 ( BfnA1 ) for 2 hours prior to fixation and immunostaining . Antibodies specific to GFP ( green ) , Parkin ( red ) and ubiquitin ( blue ) were used .

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

(B) SH-SY5Y cells were transfected with GFP-Tollip for 24 hours, then treated with 5 μM antimycin A/10 μM oligomycin B (AO) or AO/100 μM bafilomycin A1 (BfnA1) for 2 hours prior to fixation and immunostaining. Antibodies specific to GFP (green), Parkin (red) and ubiquitin (blue) were used.


C ) GFP - Tollip / Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO / BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell , across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ) . Results are represented as a percentage of Tollip puncta positive for Parkin .

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

C) GFP-Tollip/Parkin colocalisation was quantified from cells under steady state conditions or treated with AO and AO/BfnA1 by counting overall GFP puncta and GFP puncta positive for Parkin per cell, across 3-6 GFP-transfected cells/condition, per experiment (n = 3). Results are represented as a percentage of Tollip puncta positive for Parkin.


( D ) SH - SY5Y cells expressing GFP - Tollip were immunostained for GFP ( green ) , Parkin ( red ) , and Rab7a ( blue ) . Arrowheads indicate areas of colocalisation between all three markers .

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

(D) SH-SY5Y cells expressing GFP-Tollip were immunostained for GFP (green), Parkin (red), and Rab7a (blue). Arrowheads indicate areas of colocalisation between all three markers.


( E - F ) SH - SY5Y cells were transfected with GFP - Tollip R78A or CUE mutant for 24 hours , then treated with AO or AO / BfnA1 for 2 hours prior to fixation and

O O O O O O 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

(E-F) SH-SY5Y cells were transfected with GFP-Tollip R78A or CUE mutant for 24 hours, then treated with AO or AO/BfnA1 for 2 hours prior to fixation and immunostaining


( G ) The percentage of GFP - Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0162 , * * p = 0 . 0034 , * * * * 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

(G) The percentage of GFP-Tollip puncta positive for Parkin was quantified from immunofluorescence images immunostained for GFP and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/ condition, per experiment (n = 3; *p=0.0162, **p=0.0034, ****p<0.0001).


( H ) The percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0382 , * * p = 0 . 0068 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 percentage of ubiquitin puncta positive for Tollip was quantitated from immunuofluorescence images immunostained for ubiquitin and GFP from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0382, **p=0.0068).


( I ) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * p = 0 . 0232 , * * p = 0 . 0062 ) .

(I) The percentage of ubiquitin puncta positive for Parkin was quantitated from immunuofluorescence images immunostained for ubiquitin and Parkin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; *p=0.0232, **p=0.0062).


( J ) The percentage of GFP - Tollip / Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP , Parkin , and ubiquitin from WT , R78A , and CUE mutant expressing cells across 3 - 6 GFP - transfected cells / condition , per experiment ( n = 3 ; * * p = 0 . 0070 ) .

(J) The percentage of GFP-Tollip/Parkin vesicles positive for ubiquitin were quantitated from immunofluorescence images immunostained for GFP, Parkin, and ubiquitin from WT, R78A, and CUE mutant expressing cells across 3-6 GFP-transfected cells/condition, per experiment (n = 3; **p=0.0070).