( b ) L4 larvae were fasted and RNA was extracted at the indicated times . Mean ddCts + s . e . m . show that the response mxl - 3 orchestrates is transient . Time 0 , n = 6 independent experiments ; time 3 - 12 h , n = 4 independent experiments ; time 18 - 24 h , n = 2 independent experiments .

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

(b) L4 larvae were fasted and RNA was extracted at the indicated times. Mean ddCts+s.e.m. show that the response mxl-3 orchestrates is transient. Time 0, n = 6 independent experiments; time 3-12 h, n = 4 independent experiments; time 18-24 h, n = 2 independent experiments.


( c ) Immunostainings of well - fed or 6 or 12 h fasted MXL - 3 : : GFP young adults are presented . Quantification of GFP - positive nuclei relative to total intestinal nuclei ( 4 , 6 - diamidino - 2 - phenylindole , DAPI ) of two independent experiments shows that MXL - 3 transiently delocalizes from the intestinal nuclei during fasting .

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

(c) Immunostainings of well-fed or 6 or 12 h fasted MXL-3::GFP young adults are presented. Quantification of GFP-positive nuclei relative to total intestinal nuclei (4,6-diamidino-2-phenylindole, DAPI) of two independent experiments shows that MXL-3 transiently delocalizes from the intestinal nuclei during fasting.


( d ) ChIP - quantitative PCR ( ChIP - qPCR ) analysis of well - fed and 6 h fasted mixed - stage worms expressing MXL - 3 : : GFP presented as Ct in αGFP immunoprecipitated DNA normalized to input DNA and relative to a mock promoter region ( CACTAT site − 88 of ama - 1 gene ) shows that MXL - 3 vacates the lipl promoters during early fasting . Three sets of primers surrounding CACGTG target sites found up to 500 bp of the ATG of the lipl - 1 and lipl - 3 genes were used . A representative experiment is presented ; see raw data of two independent experiments in Supplementary Table S9 .

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

(d) ChIP-quantitative PCR (ChIP-qPCR) analysis of well-fed and 6 h fasted mixed-stage worms expressing MXL-3::GFP presented as Ct in αGFP immunoprecipitated DNA normalized to input DNA and relative to a mock promoter region (CACTAT site −88 of ama-1 gene) shows that MXL-3 vacates the lipl promoters during early fasting. Three sets of primers surrounding CACGTG target sites found up to 500 bp of the ATG of the lipl-1 and lipl-3 genes were used. A representative experiment is presented; see raw data of two independent experiments in Supplementary Table S9.


( e ) Wild - type or lipl - 1 ( tm1954 ) lipl - 3 ( tm4498 ) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl - 3 RNAi plates , incubated for 12 h at 20 ° C , and processed for total FAMEs ; as a reference , an aliquot of wild - type young adults on control RNAi bacteria was fasted for 6 h . FAME quantification ( mean percentage of fed wild type ± s . e . m . ) shows that acute mxl - 3 inactivation , as fasting , reduces fat stores in a lipl - dependent manner . n = 3 independent experiments . NSD , P - value > 0 . 05 .

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

(e) Wild-type or lipl-1(tm1954) lipl-3(tm4498) double mutant animals grown on control RNAi plates were transferred as L4 larvae to control or mxl-3 RNAi plates, incubated for 12 h at 20 °C, and processed for total FAMEs; as a reference, an aliquot of wild-type young adults on control RNAi bacteria was fasted for 6 h. FAME quantification (mean percentage of fed wild type ±s.e.m.) shows that acute mxl-3 inactivation, as fasting, reduces fat stores in a lipl-dependent manner. n = 3 independent experiments. NSD, P-value>0.05.


( a ) Transcriptional levels of hlh - 30 and the mxl - 3 - dependent lipl genes measured in 5 h fasted wild - type ( WT ) or hlh - 30 ( tm1978 ) young - adult worms depicted as mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms show that hlh - 30 is induced following fasting and HLH - 30 induces lysosomal lipolysis . N = 4 independent experiments for lipl data , n = 2 independent experiments for hlh - 30 mRNA . HLH - 30 deficiency fully abrogates lipl - 2 , 3 and 5 ( P0 . 0001 ) , and impairs lipl - 1 ( P0 . 01 ) transcriptional activation following fasting .

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

(a) Transcriptional levels of hlh-30 and the mxl-3-dependent lipl genes measured in 5 h fasted wild-type (WT) or hlh-30(tm1978) young-adult worms depicted as mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms show that hlh-30 is induced following fasting and HLH-30 induces lysosomal lipolysis. N = 4 independent experiments for lipl data, n = 2 independent experiments for hlh-30 mRNA. HLH-30 deficiency fully abrogates lipl-2, 3 and 5 (P0.0001), and impairs lipl-1 (P0.01) transcriptional activation following fasting.


( b ) Animals treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults . Mean + s . e . m . of three independent experiments shows that inhibition of TOR is sufficient to induce hlh - 30 transcription .

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

(b) Animals treated from late L3 stage with RNAi against TOR or vector control were harvested as young adults. Mean+s.e.m. of three independent experiments shows that inhibition of TOR is sufficient to induce hlh-30 transcription.


( c ) L3 animals carrying the rescuing construct hlh - 30P : : HLH - 30 : : mGFP : : hlh - 30 3 ′ UTR ( mGFP , monomeric GFP , and UTR , untranslated region ) , well fed or fasted for 8 h show that HLH - 30 is enriched in intestinal nuclei of fasted worms ( exposure time : well fed , 500 ms ; fasted , 100 ms ) .

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

(c) L3 animals carrying the rescuing construct hlh-30P::HLH-30::mGFP::hlh-30 3′UTR (mGFP, monomeric GFP, and UTR, untranslated region), well fed or fasted for 8 h show that HLH-30 is enriched in intestinal nuclei of fasted worms (exposure time: well fed, 500 ms; fasted, 100 ms).


( d ) ORO staining of wild - type or hlh - 30 ( tm1978 ) young adults fasted for 8 h reveals that hlh - 30 is required for optimal lipid mobilization on fasting . Mean percentages + s . e . m . are shown below relative to well - fed worms treated in parallel , n = 3 independent experiments .

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

(d) ORO staining of wild-type or hlh-30(tm1978) young adults fasted for 8 h reveals that hlh-30 is required for optimal lipid mobilization on fasting. Mean percentages+s.e.m. are shown below relative to well-fed worms treated in parallel, n = 3 independent experiments.


( e ) mxl - 3 and hlh - 30 transcriptional levels in wild - type , hlh - 30 ( tm1978 ) and mxl - 3 ( ok1947 ) mutants in the basal and fasted states show that mxl - 3 transcription is hlh - 30 independent , and hlh - 30 transcription is mxl - 3 independent . Expression is presented relative to wild - type fed animals as mean ddCt + s . e . m . No significant differences relative to wild type in the same feeding state were observed , n = 3 independent experiments .

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

(e) mxl-3 and hlh-30 transcriptional levels in wild-type, hlh-30(tm1978) and mxl-3(ok1947) mutants in the basal and fasted states show that mxl-3 transcription is hlh-30 independent, and hlh-30 transcription is mxl-3 independent. Expression is presented relative to wild-type fed animals as mean ddCt+s.e.m. No significant differences relative to wild type in the same feeding state were observed, n = 3 independent experiments.


( f ) Representative ChIP - qPCR analysis of well - fed and 6 h fasted mixed stage worms expressing HLH - 30 : : GFP presented as in Fig . 3d shows that HLH - 30 occupies the lipl promoters during early fasting . See raw data of two independent experiments in Supplementary Table S9 .

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

(f) Representative ChIP-qPCR analysis of well-fed and 6 h fasted mixed stage worms expressing HLH-30::GFP presented as in Fig. 3d shows that HLH-30 occupies the lipl promoters during early fasting. See raw data of two independent experiments in Supplementary Table S9.


( g ) Transcriptional levels of the mxl - 3 - dependent lipl genes in wild - type , mxl - 3 ( ok1947 ) , hlh - 30 ( tm1978 ) or mxl - 3 ( ok1947 ) ; hlh - 30 ( tm1978 ) double mutant young - adult worms show that hlh - 30 suppresses the constitutive - induction - of - the - lipl - genes phenotype of mxl - 3 mutant animals ( mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms ) . n = 3 independent experiments .

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

(g) Transcriptional levels of the mxl-3-dependent lipl genes in wild-type, mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant young-adult worms show that hlh-30 suppresses the constitutive-induction-of-the-lipl-genes phenotype of mxl-3 mutant animals (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments.


( a ) LRO ( lysosome - related organelle ) live Nile red - stained images of hlh - 30 ( tm1978 ) and wild - type ( WT ) L3 larvae starved for 60 h show that hlh - 30 is required for the expansion of the lysosomal compartment following fasting . Quantification as mean ± s . e . m . relative to fasted wild - type worms is also presented ; n = 3 independent experiments .

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

(a) LRO (lysosome-related organelle) live Nile red-stained images of hlh-30(tm1978) and wild-type (WT) L3 larvae starved for 60 h show that hlh-30 is required for the expansion of the lysosomal compartment following fasting. Quantification as mean±s.e.m. relative to fasted wild-type worms is also presented; n = 3 independent experiments.


( b ) Transcriptional levels of the autophagy genes lgg - 1 , lgg - 2 and atg - 16 . 2 in fasted wild - type and hlh - 30 ( tm1978 ) young - adult worms show that transcriptional activation of autophagy following fasting is hlh - 30 dependent ( mean ddCt + s . e . m . relative to wild - type ad libitum - fed worms ) . n = 3 independent

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

(b) Transcriptional levels of the autophagy genes lgg-1, lgg-2 and atg-16.2 in fasted wild-type and hlh-30(tm1978) young-adult worms show that transcriptional activation of autophagy following fasting is hlh-30 dependent (mean ddCt+s.e.m. relative to wild-type ad libitum-fed worms). n = 3 independent experiments


( c ) The mean percentage ( + s . d . ) of L1 larvae alive after 48 h fasting in minimum media shows that hlh - 30 ( tm1978 ) mutant animals are sensitive to starvation . n = 3 independent experiments . For L4 survival see Supplementary Fig . S6g .

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

(c) The mean percentage (+ s.d.) of L1 larvae alive after 48 h fasting in minimum media shows that hlh-30 (tm1978) mutant animals are sensitive to starvation. n = 3 independent experiments. For L4 survival see Supplementary Fig. S6g.


( a ) Expression analyses of the liver of C57BL / 6J 9 - week females fasted overnight ( ON ) for 10 h relative to siblings feeding ad libitum show that LipA ( mouse lysosomal acid lipase ) , Map1lc3a ( mammalian lgg - 1 / lgg - 2 ) and Tfeb ( mammalian hlh - 30 ) but not Max ( mammalian mxl - 3 ) are transcriptionally linked to nutrients in the mouse liver ( median + s . e . m . fold change ; ddCt ) . Levels of expression were normalized to ActB and Cog2 as internal controls ; all but Max differences are significant ( P0 . 05 ) , n = 3 independent experiments . Cyp4a14 is a positive 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 B-MEASURED_VAR 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

(a) Expression analyses of the liver of C57BL/6J 9-week females fasted overnight (ON) for 10 h relative to siblings feeding ad libitum show that LipA (mouse lysosomal acid lipase), Map1lc3a (mammalian lgg-1/lgg-2) and Tfeb (mammalian hlh-30) but not Max (mammalian mxl-3) are transcriptionally linked to nutrients in the mouse liver (median+s.e.m. fold change; ddCt). Levels of expression were normalized to ActB and Cog2 as internal controls; all but Max differences are significant (P0.05), n = 3 independent experiments. Cyp4a14 is a positive control.


( b ) Expression analyses of control or hTFEB siRNA treated HepG2 cells incubated in EBSS minimal medium for 2 , 4 or 8 h , compared with the expression of control or hTFEB siRNA treated hepatocytes in complete media , show that LAL ( human lysosomal acid lipase ) , MAP1LC3A and TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes , and that LAL and MAP1LC3A induction under nutrient deprivation are TFEB dependent ( median dCt + s . e . m . ) . Levels of expression were normalized to ACTB as internal control ; all butMAX differences are significant ( P0 . 05 ) , n = 3 independent experiments . IGFBP is a positive control . EBSS , Earles ' s balanced salt solution .

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

(b) Expression analyses of control or hTFEB siRNA treated HepG2 cells incubated in EBSS minimal medium for 2, 4 or 8 h, compared with the expression of control or hTFEB siRNA treated hepatocytes in complete media, show that LAL (human lysosomal acid lipase), MAP1LC3A and TFEB but not MAX are transcriptionally linked to nutrients in human hepatocytes, and that LAL and MAP1LC3A induction under nutrient deprivation are TFEB dependent (median dCt+s.e.m.). Levels of expression were normalized to ACTB as internal control; all butMAX differences are significant (P0.05), n = 3 independent experiments. IGFBP is a positive control. EBSS, Earles's balanced salt solution.


( a ) Wild - type ( WT ) , mxl - 3 ( ok1947 ) , hlh - 30 ( tm1978 ) or mxl - 3 ( ok1947 ) ; hlh - 30 ( tm1978 ) double mutant were used in longevity epistasis analyses , revealing that hlh - 30 suppresses the mxl - 3 extended lifespan phenotype . Animals were incubated at 20 ° C and transferred every other day until cessation of reproduction . The cumulative survival curve is depicted . Kaplan - Meier statistics , calculated using SSPS 17 , are also shown .

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

(a) Wild-type (WT), mxl-3(ok1947), hlh-30(tm1978) or mxl-3(ok1947);hlh-30(tm1978) double mutant were used in longevity epistasis analyses, revealing that hlh-30 suppresses the mxl-3 extended lifespan phenotype. Animals were incubated at 20°C and transferred every other day until cessation of reproduction. The cumulative survival curve is depicted. Kaplan-Meier statistics, calculated using SSPS 17, are also shown.


( b ) Live Nile red quantification of wild - type and lipl - 1lipl - 3 double mutant worms shows that lipl - 1 and lipl - 3 contribute to the clearance of lipids from the endocytic pathway . Mean + s . e . m . signal intensity is shown as percentage relative to wild type ; significant differences are indicated ; n = 4 independent experiments .

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

(b) Live Nile red quantification of wild-type and lipl-1lipl-3 double mutant worms shows that lipl-1 and lipl-3 contribute to the clearance of lipids from the endocytic pathway. Mean+s.e.m. signal intensity is shown as percentage relative to wild type; significant differences are indicated; n = 4 independent experiments.


( c , d ) Animals overexpressing LIPL - 1 or LIPL - 3 were incubated at 25 ° C and transferred every other day until cessation of reproduction . Data presented as in Fig . 7a show that activated lysosomal lipolysis extends C . elegans lifespan . a Survival presented as mean lifespan ( LS ) ± s . e . m . b Number of uncensored animals ( animals that crawled off the plate , bagged , exploded or became contaminated were censored ) .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Animals overexpressing LIPL-1 or LIPL-3 were incubated at 25 °C and transferred every other day until cessation of reproduction. Data presented as in Fig. 7a show that activated lysosomal lipolysis extends C. elegans lifespan. a Survival presented as mean lifespan (LS) ± s.e.m. b Number of uncensored animals (animals that crawled off the plate, bagged, exploded or became contaminated were censored).


( A ) EM analysis of cortical synapses derived from WT and MPS - IIIA mice at different ages . The size of synaptic vesicles was quantified from 400 - 500 vesicles ( taken from 5 mice for each genotype at each time point ) and expressed as the average of vesicle diameter ( nm ) . The number of synaptic vesicles per synapse was quantified from 20 different images ( taken from 5 mice for each genotype at each time point ) , normalized by the length of synaptic cleft and expressed as % of WT . The synaptic density was measured from 20 different images ( taken from 5 mice for each genotype at each time point ) as the number of synapses / area ( # / 500μm2 ) and expressed as % of WT . Arrows indicate the synaptic cleft , while asterisks indicate abnormal

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) EM analysis of cortical synapses derived from WT and MPS-IIIA mice at different ages. The size of synaptic vesicles was quantified from 400-500 vesicles (taken from 5 mice for each genotype at each time point) and expressed as the average of vesicle diameter (nm). The number of synaptic vesicles per synapse was quantified from 20 different images (taken from 5 mice for each genotype at each time point), normalized by the length of synaptic cleft and expressed as % of WT. The synaptic density was measured from 20 different images (taken from 5 mice for each genotype at each time point) as the number of synapses/area(#/500μm2) and expressed as % of WT.Arrows indicate the synaptic cleft, while asterisks indicate abnormal vacuoles


( B , C ) The size of the lysosomal compartment was evaluated by WB ( B ) analysis in the brain of MPS - IIIA mice at indicated ages . Quantitation of WB by densitometry analysis ( ImageJ ) was shown ( B ) .

O O O O O O O O O O O O O O O O O O O O O O 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, C) The size of the lysosomal compartment was evaluated by WB (B) analysis in the brain of MPS-IIIA mice at indicated ages. Quantitation of WB by densitometry analysis (ImageJ) was shown (B).


( B , C ) The size of the lysosomal compartment was evaluated by IF ( C ) analysis in the brain of MPS - IIIA mice at indicated ages .

O O O O O O O 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, C) The size of the lysosomal compartment was evaluated by IF (C) analysis in the brain of MPS-IIIA mice at indicated ages.


( D ) Extracellular recordings ( fEPSPs ) in hippocampal brain slices from 6 months - old WT and MPS - IIIA mice . The left panel shows representative fEPSP traces . Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude . Data are the average of the values from 9 slices ( from 9 mice ) in the WT group and 6 slices ( from 5 mice ) in the MPS - IIIA . * p < 0 . 05 , repeated measure ANOVA .

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(D) Extracellular recordings (fEPSPs) in hippocampal brain slices from 6 months-old WT and MPS-IIIA mice. The left panel shows representative fEPSP traces. Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude. Data are the average of the values from 9 slices (from 9 mice) in the WT group and 6 slices (from 5 mice) in the MPS-IIIA. *p<0.05, repeated measure ANOVA.


( A ) Synaptic terminal endocytosis was analyzed in WT and MPS - IIIA DIV14 hippocampal neurons by quantification of incorporated FM1 - 43 dye fluorescence in ~ 300 individual boutons ( taken from 4 - 5 coverslips for each group ) . Fluorescence intensities were expressed as arbitrary units ( A . F . U . ) and displayed both as a distribution and as mean values ± s . e . m .

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(A) Synaptic terminal endocytosis was analyzed in WT and MPS-IIIA DIV14 hippocampal neurons by quantification of incorporated FM1-43 dye fluorescence in ~300 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensities were expressed as arbitrary units (A.F.U.) and displayed both as a distribution and as mean values ± s.e.m.


( B ) WT and MPS - IIIA hippocampal neurons ( DIV14 ) were transfected with v - Glut1 - pHluorin - mCherry plasmid and synaptic recycling was evaluated by the fluorescence change of the probe from 80 - 100 individual boutons ( taken from 4 - 5 coverslips for each group ) . Fluorescence intensity was quantified at each bouton during KCl perfusion , normalized to the fluorescence value obtained upon rapid alkalization ( NH4Cl perfusion ) and expressed as ∆ fluorescence ( normalized to baseline ; ΔFKCl / ΔF NH4Cl ) . Values were displayed both as fluorescence traces ( means ± s . e . m . at each time frame ) and maximum fluorescence after KCl perfusion ( means ± s . e . m . ) . Representative panels on the right show the fluorescence intensity change in control neurons ( WT ) . Pseudo - colour was applied to better reveal fluorescence changes . Note that NH4Cl alkalinizes all vesicles revealing the total ( recycling + resting ) pool in neuronal cells analyzed .

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(B) WT and MPS-IIIA hippocampal neurons (DIV14) were transfected with v-Glut1-pHluorin-mCherry plasmid and synaptic recycling was evaluated by the fluorescence change of the probe from 80-100 individual boutons (taken from 4-5 coverslips for each group). Fluorescence intensity was quantified at each bouton during KCl perfusion, normalized to the fluorescence value obtained upon rapid alkalization (NH4Cl perfusion) and expressed as ∆fluorescence (normalized to baseline; ΔFKCl/ΔF NH4Cl). Values were displayed both as fluorescence traces (means ± s.e.m. at each time frame) and maximum fluorescence after KCl perfusion (means ± s.e.m.). Representative panels on the right show the fluorescence intensity change in control neurons (WT). Pseudo-colour was applied to better reveal fluorescence changes.Note that NH4Cl alkalinizes all vesicles revealing the total (recycling + resting) pool in neuronal cells analyzed.


( C ) WT hippocampal neurons at DIV10 were treated with a lysosome inhibitor ' s cocktail for 3 days . The size of lysosomal compartment was evaluated in both treated and control untreated WT neurons by EM examination . Quantitation analysis was performed on 30 different images .

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(C) WT hippocampal neurons at DIV10 were treated with a lysosome inhibitor's cocktail for 3 days. The size of lysosomal compartment was evaluated in both treated and control untreated WT neurons by EM examination. Quantitation analysis was performed on 30 different images.


( D , E ) Endocytosis ( D ) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in ( A , B ) .

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(D, E) Endocytosis (D) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B).


( D , E ) exocytosis ( E ) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in ( A , B ) .

(D, E) exocytosis (E) in presynaptic boutons from treated and control untreated WT hippocampal neurons were monitored as in (A, B).


( F ) EM analysis of synaptic terminals was performed in treated and control untreated WT neurons . The number of synaptic vesicles per synapse was quantified from ~ 20 different images ( taken from 3 ultrathin sections for each group ) , normalized by the length of synaptic cleft and expressed as % of WT . Arrows indicate the synaptic cleft .

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(F) EM analysis of synaptic terminals was performed in treated and control untreated WT neurons. The number of synaptic vesicles per synapse was quantified from ~20 different images (taken from 3 ultrathin sections for each group), normalized by the length of synaptic cleft and expressed as % of WT. Arrows indicate the synaptic cleft.


( A ) VAMP2 , SNAP - 25 and Syntaxin - 1 SNAREs were immunoblotted in WT and MPS - IIIA synaptosomal brain samples at indicated ages . Synapsin - 1 was also blotted as a control . Quantitation of WB was shown .

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(A) VAMP2, SNAP-25 and Syntaxin-1 SNAREs were immunoblotted in WT and MPS-IIIA synaptosomal brain samples at indicated ages. Synapsin-1 was also blotted as a control. Quantitation of WB was shown.


( B ) Confocal microscopy images of WT and MPS - IIIA hippocampal neurons ( DIV14 ) double labeled with anti - synapsin - 1 ( presynaptic marker ; red ) and either anti - VAMP2 or anti - SNAP25 antibodies ( green ) . The merges ( yellow ) of confocal images are shown . SNAP25 - synapsin - 1 and VAMP2 - synapsin - 1 co - localizations were quantified using the Manders ' Colocalization Coefficients ( MCC ) ( ImageJ ) and displayed as % ( MCC x 100 ) of Synapsin - 1 co - localizing with either SNAP25 or VAMP2 ( means ± s . e . m . from 15 different images taken from 4 - 5 coverslips for each group ) .

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(B) Confocal microscopy images of WT and MPS-IIIA hippocampal neurons (DIV14) double labeled with anti-synapsin-1 (presynaptic marker; red) and either anti-VAMP2 or anti-SNAP25 antibodies (green). The merges (yellow) of confocal images are shown. SNAP25-synapsin-1 and VAMP2-synapsin-1 co-localizations were quantified using the Manders' Colocalization Coefficients (MCC) (ImageJ) and displayed as % (MCC x 100) of Synapsin-1 co-localizing with either SNAP25 or VAMP2 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group).


( C ) VAMP2 and SNAP - 25 protein levels were quantified by immunoblot analysis in WT and MPS - IIIA hippocampal neurons ( DIV14 ) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 ( 100 % ) . The proteasome was inhibited as indicated .

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(C) VAMP2 and SNAP-25 protein levels were quantified by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.


( D ) SDS - resistant complex levels were evaluated in WT and MPS - IIIA total brain samples at indicated ages by immunoblotting of non - boiled samples with VAMP2 or Syntaxin - 1 antibodies . Quantitation of WB was shown .

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(D) SDS-resistant complex levels were evaluated in WT and MPS-IIIA total brain samples at indicated ages by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.


( E ) Total brain lysates were immunoprecipitated with antibodies to Syntaxin - 1 and co - immunoprecipitated VAMP2 and SNAP25 proteins were revealed by WB analysis . The levels of immunoprecipitated proteins were quantified .

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(E) Total brain lysates were immunoprecipitated with antibodies to Syntaxin-1 and co-immunoprecipitated VAMP2 and SNAP25 proteins were revealed by WB analysis. The levels of immunoprecipitated proteins were quantified.


( F ) α - Synuclein and CSPα were immunoblotted in WT and MPS - IIIA total brain lysates at indicated ages Total protein levels were quantified .

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(F) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages Total protein levels were quantified.


( G ) α - Synuclein and CSPα were immunoblotted in WT and MPS - IIIA synaptosomal fractions at indicated ages . Hsc70 , an heat shock cognate protein , which forms together with CSPα and SGT ( small glutamine - rich tetratricopeptide repeat domain protein ) the " chaperon machine " was also blotted . Protein levels were quantified .

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(G) α-Synuclein and CSPα were immunoblotted in WT and MPS-IIIA synaptosomal fractions at indicated ages. Hsc70, an heat shock cognate protein, which forms together with CSPα and SGT (small glutamine-rich tetratricopeptide repeat domain protein) the "chaperon machine" was also blotted. Protein levels were quantified.


( A ) WB analysis of LC3 and p62 ( an autophagy substrate ) was performed on WT and MPS - IIIA brain samples at indicated ages . WB quantitation was shown .

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(A) WB analysis of LC3 and p62 (an autophagy substrate) was performed on WT and MPS-IIIA brain samples at indicated ages. WB quantitation was shown.


( B ) α - Synuclein was immunoblotted in WT and MPS - IIIA both total and synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength . Soluble ( Sol . ) , lowly insoluble ( L . Insol . ) and highly insoluble ( H . Insol . ) forms correspond to the protein solubilized respectively in Triton - X100 , SDS 10 % and 8M UREA .

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(B) α-Synuclein was immunoblotted in WT and MPS-IIIA both total and synaptosomal brain fractions at indicated ages after sequential extraction with detergents with increased strength. Soluble (Sol.), lowly insoluble (L. Insol.) and highly insoluble (H. Insol.) forms correspond to the protein solubilized respectively in Triton-X100, SDS 10% and 8M UREA.


( C ) Co - immunofluorescence analysis of α - synuclein with SMI - 32 in WT and MPS - IIIA hippocampal neurons ( DIV14 ) . α - Synuclein synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image . Quantification of α - synuclein synaptic puncta was calculated from 30 different enlarged images c .

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(C) Co-immunofluorescence analysis of α-synuclein with SMI-32 in WT and MPS-IIIA hippocampal neurons (DIV14). α-Synuclein synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of α-synuclein synaptic puncta was calculated from 30 different enlarged images c.


( D ) Confocal analysis of α - synuclein ( green ) and LAMP1 ( red ) in WT and MPS - IIIA hippocampal neurons ( DIV14 ) . Enlarged merge images are also shown . Co - localization was quantified using the MCC coefficient ( ImageJ ) and displayed as % ( MCC x 100 ) of α - synuclein co - localizing with LAMP1 ( means ± s . e . m . from 15 different images taken from 4 - 5 coverslips for each group ) .

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(D) Confocal analysis of α-synuclein (green) and LAMP1 (red) in WT and MPS-IIIA hippocampal neurons (DIV14). Enlarged merge images are also shown. Co-localization was quantified using the MCC coefficient (ImageJ) and displayed as % (MCC x 100) of α-synuclein co-localizing with LAMP1 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group).


( E ) CSPα was immunoblotted in WT and MPS - IIIA total brain lysates at indicated ages after sequential extraction with detergents with increased strength as in ( B ) .

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(E) CSPα was immunoblotted in WT and MPS-IIIA total brain lysates at indicated ages after sequential extraction with detergents with increased strength as in (B).


( F ) Co - immunofluorescence analysis of CSPα and SMI - 32 in DIV14 hippocampal neurons . CSPα - synaptic puncta were quantified as in ( C ) .

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(F) Co-immunofluorescence analysis of CSPα and SMI-32 in DIV14 hippocampal neurons. CSPα- synaptic puncta were quantified as in (C).


( G ) CSPα protein levels were evaluated by immunoblot analysis in WT and MPS - IIIA hippocampal neurons ( DIV14 ) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 ( 100 % ) . The proteasome was inhibited as indicated .

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(G) CSPα protein levels were evaluated by immunoblot analysis in WT and MPS-IIIA hippocampal neurons (DIV14) at different times after cycloheximide treatment and expressed as % of remaining protein at T0 (100 %). The proteasome was inhibited as indicated.


( H ) Palmitoylation - dependent shift in the molecular weight of CSPα was evaluated in WT and MPS - IIIA brain samples at indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulphydryl agents ( beta - mercaptoethanol or dithiothreitol ) .

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(H) Palmitoylation-dependent shift in the molecular weight of CSPα was evaluated in WT and MPS-IIIA brain samples at indicated ages by immunoblotting CSPα in boiled samples prepared without exposure to sulphydryl agents (beta-mercaptoethanol or dithiothreitol).


( I ) The protein levels of RPN10 ( the regulatory subunit of 26S proteasome ) and ubiquitinated proteins formed by Lys - 48 ( K48 ) residue linkage ( involved in protein degradation via the proteasome ) were evaluated in WT and MPS - IIIA brain samples at indicated ages by WB analysis . WB quantitation was shown .

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(I) The protein levels of RPN10 (the regulatory subunit of 26S proteasome) and ubiquitinated proteins formed by Lys-48 (K48) residue linkage (involved in protein degradation via the proteasome) were evaluated in WT and MPS-IIIA brain samples at indicated ages by WB analysis. WB quantitation was shown.


( L ) Proteasome activity was evaluated by measuring the chymotrypsin - like activity in WT and MPS - IIIA mouse brain samples at different ages . Proteasome activity was expressed as % of WT

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(L) Proteasome activity was evaluated by measuring the chymotrypsin-like activity in WT and MPS-IIIA mouse brain samples at different ages. Proteasome activity was expressed as % of WT activity


( A ) Autophagy was monitored in WT hippocampal neurons treated with the lysosome inhibitor ' s cocktail for 3 days by immunoblot analysis with anti - LC3 antibodies . As control DIV10 WT neurons were left untreated and monitored by LC3 blot over the three days period . The LC3 - II levels quantitation was shown .

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(A) Autophagy was monitored in WT hippocampal neurons treated with the lysosome inhibitor's cocktail for 3 days by immunoblot analysis with anti-LC3 antibodies. As control DIV10 WT neurons were left untreated and monitored by LC3 blot over the three days period. The LC3-II levels quantitation was shown.


( B ) Proteasome activity was measured in treated and control untreated WT neurons using pZsProSensor - 1 vector ( green ) . Cells were co - stained with anti SMI - 32 ( blu ) . Proteasome activity was quantified by measuring the green fluorescence ( inversely correlated with proteasome activity ) in 10 different cells ( taken from 4 - 5 coverslips for each group ) and expressed as fold to WT .

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(B) Proteasome activity was measured in treated and control untreated WT neurons using pZsProSensor-1 vector (green). Cells were co-stained with anti SMI-32 (blu). Proteasome activity was quantified by measuring the green fluorescence (inversely correlated with proteasome activity) in 10 different cells (taken from 4-5 coverslips for each group) and expressed as fold to WT.


( C ) Activation of calpain 2 and caspase - 3 proteolytic systems was evaluated by WB measurement of the protein levels of calpain 2 and caspase - 3 ( both full - length and activated cleaved forms of ~ 17 KDa ) in treated and control untreated WT neurons . Arrow indicated the full - length caspase - 3 protein .

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(C) Activation of calpain 2 and caspase-3 proteolytic systems was evaluated by WB measurement of the protein levels of calpain 2 and caspase-3 (both full-length and activated cleaved forms of ~17 KDa) in treated and control untreated WT neurons. Arrow indicated the full-length caspase-3 protein.


( D ) After 3 days - treatment cell lysates from treated and control untreated WT neurons were immunoblotted with α - synuclein and CSPα and protein levels were quantified .

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(D) After 3 days-treatment cell lysates from treated and control untreated WT neurons were immunoblotted with α-synuclein and CSPα and protein levels were quantified.


( E ) Treated and control untreated WT neurons were subjected to α - synuclein ( green ) and LAMP1 ( red ) confocal analysis . Enlarged merge images are also shown . Co - localization was quantified using the MCC coefficient ( ImageJ ) and displayed as % ( MCC x 100 ) of α - synuclein co - localizing with LAMP1 ( means ± s . e . m . from 15 different images taken from 4 - 5 coverslips for each group ) . Cells were also co - stained with anti SMI - 32 .

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(E) Treated and control untreated WT neurons were subjected to α-synuclein (green) and LAMP1 (red) confocal analysis. Enlarged merge images are also shown. Co-localization was quantified using the MCC coefficient (ImageJ) and displayed as % (MCC x 100) of α-synuclein co-localizing with LAMP1 (means ± s.e.m. from 15 different images taken from 4-5 coverslips for each group). Cells were also co-stained with anti SMI-32.


( F ) Treated and control untreated WT neurons were subjected to CSPα / SMI - 32 co - immunofluorescence . CSPα synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image . Quantification of CSPα synaptic puncta was calculated from 30 different enlarged images taken from 4 - 5 coverslips for each group .

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(F) Treated and control untreated WT neurons were subjected to CSPα/SMI-32 co-immunofluorescence. CSPα synaptic puncta present in a neurite tract of 10µm is showed in a representative enlarged image. Quantification of CSPα synaptic puncta was calculated from 30 different enlarged images taken from 4-5 coverslips for each group.


( G ) SDS - resistant complex levels in treated and control untreated WT neurons were evaluated by immunoblotting analysis of non - boiled samples with VAMP2 or SNAP - 25 antibodies . The amounts of SNARE complexes were quantified .

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(G) SDS-resistant complex levels in treated and control untreated WT neurons were evaluated by immunoblotting analysis of non-boiled samples with VAMP2 or SNAP-25 antibodies. The amounts of SNARE complexes were quantified.


( A ) α - Synuclein , CSPα , VAMP2 and SNAP - 25 were immunoblotted in total brain homogenates and / or synaptosomal fractions of 1 . 5 month - old WT ( CSPα + / + / α - syn + / + ) , CSPα - / - , CSPα + / - , α - syn - / - , α - syn + / - and CSPα + / - / α - syn + / - . Synapsin - 1 was also blotted as a control . Quantitation of WB in synaptosomal samples was shown .

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(A) α-Synuclein, CSPα, VAMP2 and SNAP-25 were immunoblotted in total brain homogenates and/or synaptosomal fractions of 1.5 month-old WT (CSPα+/+/α-syn+/+), CSPα-/-, CSPα+/-, α-syn-/-, α-syn+/- and CSPα+/-/α-syn+/-. Synapsin-1 was also blotted as a control. Quantitation of WB in synaptosomal samples was shown.


( B ) SDS - resistant complex levels were evaluated in brain homogenates derived from mice with indicated genotypes by immunoblotting of non - boiled samples with VAMP2 or Syntaxin - 1 antibodies . Quantitation of WB was shown .

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(B) SDS-resistant complex levels were evaluated in brain homogenates derived from mice with indicated genotypes by immunoblotting of non-boiled samples with VAMP2 or Syntaxin-1 antibodies. Quantitation of WB was shown.


( C ) EM analysis of cortical synapses derived from mice with indicated genotypes . The number of synaptic vesicles per synapse was quantified from 40 different images ( taken from 5 different mice for each genotype ) , normalized by the length of synaptic cleft and expressed as % of WT . The size of synaptic vesicles was quantified from 400 - 500 vesicles ( taken from 5 different mice for each genotype ) and expressed as the average of vesicle diameter ( nm ) Arrows indicate the synaptic cleft .

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(C) EM analysis of cortical synapses derived from mice with indicated genotypes. The number of synaptic vesicles per synapse was quantified from 40 different images (taken from 5 different mice for each genotype), normalized by the length of synaptic cleft and expressed as % of WT. The size of synaptic vesicles was quantified from 400-500 vesicles (taken from 5 different mice for each genotype) and expressed as the average of vesicle diameter (nm) Arrows indicate the synaptic cleft.


( D ) Extracellular recordings ( fEPSPs ) in hippocampal brain slices from 1 . 5 months - old WT ( CSPα + / + / α - syn + / + ) and CSPα + / - / α - syn + / - mice . The left panel shows representative fEPSP traces . Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude . Data are the average of the values from 5 slices ( from 4 mice ) in the WT group and 5 slices ( from 4 mice ) in the CSPα + / - / α - syn + / - . * p < 0 . 05 , repeated measure ANOVA .

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(D) Extracellular recordings (fEPSPs) in hippocampal brain slices from 1.5 months-old WT (CSPα+/+/α-syn+/+) and CSPα+/-/α-syn+/- mice. The left panel shows representative fEPSP traces. Summary graphs show the fEPSPs slope as a function of either the applied stimulus intensity or the prevolley amplitude. Data are the average of the values from 5 slices (from 4 mice) in the WT group and 5 slices (from 4 mice) in the CSPα+/-/α-syn+/-. *p<0.05, repeated measure ANOVA.


( A ) Anti - myc immunostaining in different brain regions derived from 10 - months - old MPS - IIIA mice intraventricularly injected with AAV2 / 9 vectors encoding CSPα under Synapsin - 1 promoter ( MPS - IIIA - myc - CSPα experimental group ) . As a control immunostaining was performed in brain sections from 10 - months - old MPS - IIIA mice injected with empty AAV2 / 9 vectors ( MPS - IIIA - empty experimental groups ) . To evaluate co - localization between exogenous CSPα and presynaptic compartment brain sections were also co - stained with anti - myc and anti - synapsin1 antibodies ( confocal images are also shown ) .

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(A) Anti-myc immunostaining in different brain regions derived from 10-months-old MPS-IIIA mice intraventricularly injected with AAV2/9 vectors encoding CSPα under Synapsin-1 promoter (MPS-IIIA-myc-CSPα experimental group). As a control immunostaining was performed in brain sections from 10-months-old MPS-IIIA mice injected with empty AAV2/9 vectors (MPS-IIIA-empty experimental groups). To evaluate co-localization between exogenous CSPα and presynaptic compartment brain sections were also co-stained with anti-myc and anti-synapsin1 antibodies (confocal images are also shown).


( B ) Anti - myc WB in synaptosomal fractions derived from MPS - IIIA - myc - CSPα mice . As a control WB was performed in samples from 10 - months - old WT and MPS - IIIA mice injected with empty AAV2 / 9 vectors ( WT - empty and MPS - IIIA - empty experimental groups ) . Synaptosomal fractions were also blotted with anti - CSPα .

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(B) Anti-myc WB in synaptosomal fractions derived from MPS-IIIA-myc-CSPα mice. As a control WB was performed in samples from 10-months-old WT and MPS-IIIA mice injected with empty AAV2/9 vectors (WT-empty and MPS-IIIA-empty experimental groups). Synaptosomal fractions were also blotted with anti-CSPα.


( C ) VAMP2 , SNAP - 25 and Syntaxin - 1 were immunoblotted in synaptosomal samples derived from the three experimental groups of mice . Protein levels were quantified .

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(C) VAMP2, SNAP-25 and Syntaxin-1 were immunoblotted in synaptosomal samples derived from the three experimental groups of mice. Protein levels were quantified.


( D ) The amount of SNARE - complexes was detected in synaptosomal brain samples derived from the three experimental groups of mice by immunoblotting analysis of non - boiled samples with VAMP2 , SNAP - 25 or Syntaxin - 1 antibodies . The amounts of SNARE complexes were quantified .

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(D) The amount of SNARE-complexes was detected in synaptosomal brain samples derived from the three experimental groups of mice by immunoblotting analysis of non-boiled samples with VAMP2, SNAP-25 or Syntaxin-1 antibodies. The amounts of SNARE complexes were quantified.


( E ) Total brain lysates derived from the three experimental groups of mice were immunoprecipitated with Syntaxin - 1 antibodies and co - immunoprecipitated VAMP2 and SNAP - 25 proteins were revealed by WB .

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(E) Total brain lysates derived from the three experimental groups of mice were immunoprecipitated with Syntaxin-1 antibodies and co-immunoprecipitated VAMP2 and SNAP-25 proteins were revealed by WB.


( A ) EM analysis of cortical and hippocampal synapsis derived from the three experimental groups of mice . Synaptic vesicles number per synapse was quantified from ~ 40 different images ( taken from 5 mice for each group ) , normalized by the length of synaptic cleft and expressed as % of WT . The synaptic density was measured from 20 different images ( taken from 5 mice for each group ) as the number of synapses / area ( # / 500μm2 ) and expressed as % of WT . Arrows indicate the synaptic cleft while asterisks indicate abnormal vacuoles . Values are means ± s . e . m . * P < 0 . 05 , Student ' s t - test : Either MPS - IIIA - empty or MPS - IIIA - myc - CSPα vs WT - empty . Scale bar : 0 . 2

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(A) EM analysis of cortical and hippocampal synapsis derived from the three experimental groups of mice. Synaptic vesicles number per synapse was quantified from ~40 different images (taken from 5 mice for each group), normalized by the length of synaptic cleft and expressed as % of WT. The synaptic density was measured from 20 different images (taken from 5 mice for each group) as the number of synapses/area(#/500μm2) and expressed as % of WT. Arrows indicate the synaptic cleft while asterisks indicate abnormal vacuoles. Values are means ± s.e.m. *P<0.05, Student's t-test: Either MPS-IIIA-empty or MPS-IIIA-myc-CSPα vs WT-empty. Scale bar: 0.2 µm


( B ) Extracellular recordings ( fEPSPs ) in hippocampal brain slices from WT - empty ( n = 8 ) , MPS - IIIA - empty ( n = 5 ) and MPS - IIIA - myc - CSPα mice ( n = 5 ) . The upper panel shows representative fEPSP traces . Summary graph shows the fEPSPs slope as a function of the applied stimulus intensity . Data are means ± s . e . m . ; * P < 0 . 05 ( WT - empty vs MPS - IIIA - empty ) , # P < 0 . 05 ( MPS - IIIA - myc - CSPα vs . MPS - IIIA - empty ) , Kruskal - Wallis ANOVA followed by Dunn ' s test .

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(B) Extracellular recordings (fEPSPs) in hippocampal brain slices from WT-empty (n=8), MPS-IIIA-empty (n=5) and MPS-IIIA-myc-CSPα mice (n=5). The upper panel shows representative fEPSP traces. Summary graph shows the fEPSPs slope as a function of the applied stimulus intensity. Data are means ± s.e.m.; *P < 0.05 (WT-empty vs MPS-IIIA-empty), #P < 0.05 (MPS-IIIA-myc-CSPα vs. MPS-IIIA-empty), Kruskal-Wallis ANOVA followed by Dunn's test.


( C ) Mean distance travelled , maximal speed , immobility time and line crossing during 10 min testing in the open field , divided into 5 minute intervals in WT - empty , MPS - IIIA - empty and MPS - IIIA - myc - CSPα female mice ( n = 9 , n = 9 , n = 7 respectively ) . Distance , percentage open entries and open time in the elevated plus maze and latency to fall off the wire ( * body weight ) in the wire hanging test in WT - empty , MPS - IIIA - empty and MPS - IIIA - myc - CSPα male mice ( n = 10 , n = 8 , n = 9 respectively ) . Values are means ± s . e . m ; * P < 0 . 05 ( MPS - IIIA - empty vs . WT - empty ) , # P < 0 . 05 ( MPS - IIIA - myc - CSPα vs . MPS - IIIA - empty ) , two - way ANOVA for repeated measures for open field measures and t - test for the plus maze and the wire hanging followed by Duncan post - hoc test . Animals ' average age was 28 ± 2 weeks .

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(C) Mean distance travelled, maximal speed, immobility time and line crossing during 10 min testing in the open field, divided into 5 minute intervals in WT-empty, MPS-IIIA-empty and MPS-IIIA-myc-CSPα female mice (n=9, n=9, n=7 respectively). Distance, percentage open entries and open time in the elevated plus maze and latency to fall off the wire (*body weight) in the wire hanging test in WT-empty, MPS-IIIA-empty and MPS-IIIA-myc-CSPα male mice (n=10, n=8, n=9 respectively). Values are means ± s.e.m; *P < 0.05 (MPS-IIIA-empty vs. WT-empty), #P < 0.05 (MPS-IIIA-myc-CSPα vs. MPS-IIIA-empty), two-way ANOVA for repeated measures for open field measures and t-test for the plus maze and the wire hanging followed by Duncan post-hoc test. Animals' average age was 28±2 weeks.


( D ) Neuronal cell death was evaluated in the cerebellum ( Calbindin immunostaining ) and in different layers of cortex ( NeuN immunostaining of frontal cortex ) of 10 months - old mice belonging to each experimental group of mice . Number of cells was quantified from 20 different images ( taken from 5 mice for each group ) and expressed as % of WT . Values are means ± s . e . m . * P < 0 . 05 , * * P < 0 . 001 Student ' s t - test . Scale bar : 50 µm .

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(D) Neuronal cell death was evaluated in the cerebellum (Calbindin immunostaining) and in different layers of cortex (NeuN immunostaining of frontal cortex) of 10 months-old mice belonging to each experimental group of mice. Number of cells was quantified from 20 different images (taken from 5 mice for each group) and expressed as % of WT. Values are means ± s.e.m. *P<0.05, **P<0.001 Student's t-test. Scale bar: 50 µm.


( E ) Kaplan - Meier survival analysis in WT - empty ( n = 13 ) , MPS - IIIA - empty ( n = 13 ) and MPS - IIIA - myc - CSPα ( n = 13 ) male mice . The Kaplan - Meier survival curve was analyzed with the Chi - square test . A P value < 0 . 05 was considered to be statistically significant . P = 0 . 00003 ( MPS - IIIA - empty vs WT - empty ) , P = 0 . 000776 ( MPS - IIIA - myc - CSPα vs MPS - IIIA - empty ) .

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(E) Kaplan-Meier survival analysis in WT-empty (n=13), MPS-IIIA-empty (n=13) and MPS-IIIA-myc-CSPα (n=13) male mice. The Kaplan-Meier survival curve was analyzed with the Chi-square test. A P value <0.05 was considered to be statistically significant. P= 0.00003 (MPS-IIIA-empty vs WT-empty), P= 0.000776 (MPS-IIIA-myc-CSPα vs MPS-IIIA-empty).


A Number of differentially up - and down - regulated genes ( DEG ) in RNAi - ASCO1 seedlings as compared to wild type ( WT ) according to the RNA - seq data ( FDR < 0 . 01 , | log2FC | > = 0 . 75 ) . B Fraction of DEG found in each transcript class as defined in the Araport11 gene annotation . AS stands for antisense .

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A Number of differentially up- and down-regulated genes (DEG) in RNAi-ASCO1 seedlings as compared to wild type (WT) according to the RNA-seq data (FDR < 0.01, |log2FC| >= 0.75). B Fraction of DEG found in each transcript class as defined in the Araport11 gene annotation. AS stands for antisense.


D Representative picture of 14 - day - old plants grown 9 days in liquid 1 / 2MS supplemented with 1 µM flg22 . The scale bar representing 0 . 6cm is included in the picture . E Lateral root density of WT and two independent RNAi - ASCO lines 9 days after transfer in 1 / 2MS supplemented with 0 . 1 µM or 1 µM flg22 .

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D Representative picture of 14-day-old plants grown 9 days in liquid 1/2MS supplemented with 1 µM flg22. The scale bar representing 0.6cm is included in the picture. E Lateral root density of WT and two independent RNAi-ASCO lines 9 days after transfer in 1/2MS supplemented with 0.1 µM or 1 µM flg22.


D Representative picture of 14 - day - old plants grown 9 days in liquid 1 / 2MS supplemented with 1 µM flg22 . The scale bar representing 0 . 6cm is included in the picture .

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D Representative picture of 14-day-old plants grown 9 days in liquid 1/2MS supplemented with 1 µM flg22. The scale bar representing 0.6cm is included in the picture.


F Representative picture of root apical meristems after cell wall staining , in response to flg22 . TZ : Transition Zone ; QC : Quiescent Center . G Root apical meristem size of WT and RNAi - ASCO1 ( eg distance from QC to TZ in µm ) .

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F Representative picture of root apical meristems after cell wall staining, in response to flg22. TZ: Transition Zone; QC: Quiescent Center. G Root apical meristem size of WT and RNAi-ASCO1 (eg distance from QC to TZ in µm).


F Representative picture of root apical meristems after cell wall staining , in response to flg22 . TZ : Transition Zone ; QC : Quiescent Center .

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F Representative picture of root apical meristems after cell wall staining, in response to flg22. TZ: Transition Zone; QC: Quiescent Center.


B Number of genes containing at least one differential RNA processing event ( as defined by RNAprof p . adj < 0 . 001 ) in CDS , introns , 5 ' UTR and 3 ' UTR between RNAi - ASCO1 and WT . Up and Down fractions correspond to increase or decrease , respectively , of RNA - seq coverage in RNAi - ASCO1 for each specified gene feature .

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B Number of genes containing at least one differential RNA processing event (as defined by RNAprof p.adj < 0.001) in CDS, introns, 5'UTR and 3'UTR between RNAi-ASCO1 and WT. Up and Down fractions correspond to increase or decrease, respectively, of RNA-seq coverage in RNAi-ASCO1 for each specified gene feature.


B Scatter plot showing the respective gene expression fold change in RNAi - ASCO and 35S : ASCO lines as compared to WT . Genes showing significant changes in RNAi - ASCO and 35S : ASCO are highlighted as yellow and blue dots , respectively .

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B Scatter plot showing the respective gene expression fold change in RNAi-ASCO and 35S:ASCO lines as compared to WT. Genes showing significant changes in RNAi-ASCO and 35S:ASCO are highlighted as yellow and blue dots, respectively.


C Scatter plots showing the respective Percent Spliced In difference ( dPSI ) in RNAi - ASCO and 35S : ASCO lines as compared to WT . Genes showing significant changes in RNAi - ASCO , 35S : ASCO or in both lines are highlighted as red , green or blue dot , respectively . Gray dots represent all AS events .

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C Scatter plots showing the respective Percent Spliced In difference (dPSI) in RNAi-ASCO and 35S:ASCO lines as compared to WT. Genes showing significant changes in RNAi-ASCO, 35S:ASCO or in both lines are highlighted as red, green or blue dot, respectively. Gray dots represent all AS events.


Analyses of RT - PCR products of SR34 transcripts on 8 % acrylamide gel . Quantification of the ratio of SR34 isoforms detected in the gel in ( B ) RNAs were extracted from WT and RNAi - ASCO1 14 - day - old plants . The asterisk ( * ) indicates a significant difference as determined by Student ' s T test ( p < 0 . 05 , n = 3 biological replicates ) . Error bars show mean + / - standard deviation .

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Analyses of RT-PCR products of SR34 transcripts on 8% acrylamide gel. Quantification of the ratio of SR34 isoforms detected in the gel in (B) RNAs were extracted from WT and RNAi-ASCO1 14-day-old plants. The asterisk (*) indicates a significant difference as determined by Student's T test (p < 0.05, n = 3 biological replicates). Error bars show mean +/- standard deviation.


Analyses of RT - PCR products of NUDT7 transcripts on 8 % acrylamide gel . Quantification of the ratio of NUDT7 isoforms detected in the gel in ( E ) respectively . RNAs were extracted from WT and RNAi - ASCO1 14 - day - old plants . The asterisk ( * ) indicates a significant difference as determined by Student ' s T test ( p < 0 . 05 , n = 3 biological replicates ) . Error bars show mean + / - standard deviation .

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Analyses of RT-PCR products of NUDT7 transcripts on 8% acrylamide gel. Quantification of the ratio of NUDT7 isoforms detected in the gel in (E) respectively. RNAs were extracted from WT and RNAi-ASCO1 14-day-old plants. The asterisk (*) indicates a significant difference as determined by Student's T test (p < 0.05, n = 3 biological replicates). Error bars show mean +/- standard deviation.


A Analysis of ASCO enrichment by ChIRP using two sets of independent biotinylated probes ODD and EVEN compared to negative control with probes designed against the LacZ RNA . The fold enrichment was calculated between ODD or EVEN samples against LacZ . These samples were used for protein precipitation and Mass Spectrometry analyses ( ChIRP - MS ) , from which PRP8a was identified as a potential ASCO partner .

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A Analysis of ASCO enrichment by ChIRP using two sets of independent biotinylated probes ODD and EVEN compared to negative control with probes designed against the LacZ RNA. The fold enrichment was calculated between ODD or EVEN samples against LacZ. These samples were used for protein precipitation and Mass Spectrometry analyses (ChIRP-MS), from which PRP8a was identified as a potential ASCO partner.


B Validation of PRP8a - ASCO interaction by PRP8a - RIP . U5 RNA was used as a positive control . Data information : In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT - qPCR

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B Validation of PRP8a-ASCO interaction by PRP8a-RIP. U5 RNA was used as a positive control. Data information: In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT-qPCR


B Validation of PRP8a - ASCO interaction by PRP8a - RIP . U5 RNA was used as a positive control . Data information : In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT -

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C Immunoblot analysis was performed during PRP8a immunoprecipitation ( IP ) . The same volume of input , unbound fraction was loaded as well as 20 % of the eluted IP fraction . α - PRP8a and α - IgG : IP performed with anti - PRP8a or control rabbit IgG antibody , respectively .

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C Immunoblot analysis was performed during PRP8a immunoprecipitation (IP). The same volume of input, unbound fraction was loaded as well as 20 % of the eluted IP fraction. α-PRP8a and α-IgG : IP performed with anti-PRP8a or control rabbit IgG antibody, respectively.


: In D , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT - qPCR , and IgG RIP was used as a negative control .

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: In D, the results are expressed as a percentage of the Input for PRP8a RIP followed by RT-qPCR, and IgG RIP was used as a negative control.


A ASCO transcript levels in WT and prp8 - 7 mutants .

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A ASCO transcript levels in WT and prp8-7 mutants.


prp8 - 7 leaky mutant displays similar AS events as observed in RNAi - ASCO . Quantification of SR34 isoforms splicing index by RT - qPCR .

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prp8-7 leaky mutant displays similar AS events as observed in RNAi-ASCO. Quantification of SR34 isoforms splicing index by RT-qPCR.


C prp8 - 7 leaky mutant displays similar AS events as observed in RNAi - ASCO . Quantification of ESP isoforms splicing index by RT - qPCR .

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C prp8-7 leaky mutant displays similar AS events as observed in RNAi-ASCO. Quantification of ESP isoforms splicing index by RT-qPCR.


D ASCO transcript levels in smd1b mutant . In A and D RNAs were extracted from WT , prp8 - 7 and smd1b 14 - day - old plants .

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D ASCO transcript levels in smd1b mutant. In A and D RNAs were extracted from WT, prp8-7 and smd1b 14-day-old plants.


E SmD1b can bind ASCO in vivo . U6 RNA was used as a positive control and a housekeeping gene ( HKG2 , AT4G26410 ) RNA as a negative control .

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E SmD1b can bind ASCO in vivo. U6 RNA was used as a positive control and a housekeeping gene (HKG2, AT4G26410) RNA as a negative control.


F SmD1b recognizes in vivo the RNAs of 4 genes regulated by ASCO . In E and F the results were expressed as % INPUT in SmD1b - GFP RIP and IgG RIP used as a negative control .

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F SmD1b recognizes in vivo the RNAs of 4 genes regulated by ASCO. In E and F the results were expressed as % INPUT in SmD1b-GFP RIP and IgG RIP used as a negative control.


smd1b mutant displays similar AS events as observed in RNAi - ASCO . Quantification of SR34 isoforms splicing index by RT - qPCR .

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smd1b mutant displays similar AS events as observed in RNAi-ASCO. Quantification of SR34 isoforms splicing index by RT-qPCR.


H smd1b mutant displays similar AS events as observed in RNAi - ASCO . Quantification of ESP isoforms splicing index by RT - qPCR .

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H smd1b mutant displays similar AS events as observed in RNAi-ASCO. Quantification of ESP isoforms splicing index by RT-qPCR.


( B ) Phenotypes of affected individuals . Camptodactyly , absent flexion crease , and limited forearm supination were observed .

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(B) Phenotypes of affected individuals. Camptodactyly, absent flexion crease, and limited forearm supination were observed.


( C - E ) T1 weighted MRI scan on upper limbs of subject IV : 7 . ( C ) The pronator quadratus absence of affected side was indicated by a red arrow . ( D ) No difference was found in palmar muscles . ( E ) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow . ( F - H ) T1 weighted MRI scan on upper limbs of subject IV : 8 . ( F ) Increased epimysial fat was indicated by red arrow . ( G ) Completely loss of thenar eminences of both hands were indicated by red arrows . ( H ) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows .

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(C-E) T1 weighted MRI scan on upper limbs of subject IV:7. (C) The pronator quadratus absence of affected side was indicated by a red arrow. (D) No difference was found in palmar muscles. (E) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow. (F-H) T1 weighted MRI scan on upper limbs of subject IV:8. (F) Increased epimysial fat was indicated by red arrow. (G) Completely loss of thenar eminences of both hands were indicated by red arrows. (H) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows.


( I ) The MET variants by Sanger sequencing was indicated by red arrow .

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(I) The MET variants by Sanger sequencing was indicated by red arrow.


( J ) 293T cells were transfected with FLAG - tagged MET / METMut / Vector plasmids , and 48 h post transfection , cells were treated with 10 ng / mL recombinant human HGF for 1 h . Then MET / METMut protein purification and tyrosine kinase assay were conducted . Western Blot pictures representative of n = 3 experiments . METMut means p . Y1234C mutant MET . EGFR means epidermal growth factor receptor and serves as a positive control . NC means negative control .

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(J) 293T cells were transfected with FLAG-tagged MET/METMut/Vector plasmids, and 48 h post transfection, cells were treated with 10 ng/mL recombinant human HGF for 1 h. Then MET/METMut protein purification and tyrosine kinase assay were conducted. Western Blot pictures representative of n = 3 experiments. METMut means p.Y1234C mutant MET. EGFR means epidermal growth factor receptor and serves as a positive control. NC means negative control.


( A ) Gross appearance of wild - type and heterozygous newborns . ( B ) Graph showing weight of P0 mice . Sample size : WT ( n = 4 ) and Hetero ( n = 5 ) . * P < 0 . 05 , by one - way ANOVA and follow by Dunnett ' s post hoc test .

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(A) Gross appearance of wild-type and heterozygous newborns. (B) Graph showing weight of P0 mice. Sample size: WT (n = 4) and Hetero (n = 5). *P < 0.05, by one-way ANOVA and follow by Dunnett's post hoc test.


Sections of spine from wild types and heterozygotes at P0 , were conducted with HE staining and immunofluorescence staining using anti - myosin heavy chain antibody . Scale bars , 100 μm .

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Sections of spine from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.


Sections of forelimbs , hindlimbs , hand , and foot from wild types and heterozygotes at P0 , were conducted with HE staining and immunofluorescence staining using anti - myosin heavy chain antibody . Scale bars , 100 μm .

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Sections of forelimbs, hindlimbs, hand, and foot from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.


( H ) The mean number of myofibers in the muscles of the spine , forelimb , hindlimb , hand and foot was qualified . NS means no statistic significance . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed independent student ' s t test . In ( C - H ) , n = 3 .

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(H) The mean number of myofibers in the muscles of the spine, forelimb, hindlimb, hand and foot was qualified. NS means no statistic significance. *P < 0.05, **P < 0.01, by two-tailed independent student's t test. In (C-H), n = 3.


( I ) Transmission electron microscope analysis of gastrocnemius from wild - type and heterozygous newborns . Black arrows denote mitochodria . Scale bars , 2 μm . WT means wild types , Hetero means heterozygotes .

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(I) Transmission electron microscope analysis of gastrocnemius from wild-type and heterozygous newborns. Black arrows denote mitochodria. Scale bars, 2 μm. WT means wild types, Hetero means heterozygotes.


( A ) In situ hybridization of E10 . 5 embryos using Pax3 probe . Pax3 expression ( brown signal ) was observed in limb bud and demomyotome ( DM ) , respectively . Cells labeled with Pax3 were indicated by red arrow . Scale bars , 200 μm .

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(A) In situ hybridization of E10.5 embryos using Pax3 probe. Pax3 expression (brown signal) was observed in limb bud and demomyotome (DM), respectively. Cells labeled with Pax3 were indicated by red arrow. Scale bars, 200 μm.


( B ) Gross appearance of E14 . 5 embryos of indicated genotype . Scale bars , 2 . 5 mm .

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(B) Gross appearance of E14.5 embryos of indicated genotype. Scale bars, 2.5 mm.


( C ) HE staining of forelimb , hindlimb , and paraspinal muscle of E14 . 5 embryos . Scale bars , 100 μm .

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(C) HE staining of forelimb, hindlimb, and paraspinal muscle of E14.5 embryos. Scale bars, 100 μm.


( D ) Gross appearance of E16 . 5 embryos of indicated genotype . Scale bars , 2 . 5 mm .

(D) Gross appearance of E16.5 embryos of indicated genotype. Scale bars, 2.5 mm.


( E ) HE staining of forelimb , hindlimb , and paraspinal muscle of E16 . 5 embryos . n = 3 , scale bars , 200 μm .

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(E) HE staining of forelimb, hindlimb, and paraspinal muscle of E16.5 embryos. n = 3, scale bars, 200 μm.


( F ) Anti - Ki67 antibody was used to label proliferative myoblasts ( red fluorescence ) with DAPI labeled nucleuses ( blue fluorescence ) in paraspinal muscle . Scale bar , 25 μm . ( G ) Bar graph showing statistical analysis of positive rate of Ki67 - labeled nuclei , n = 3 with more than 150 cells analyzed per n , * * P < 0 . 01 , by chi - square test ( χ2 test ) . WT means wild types , Hetero means heterozygotes , and Homo means homozygotes .

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(F) Anti-Ki67 antibody was used to label proliferative myoblasts (red fluorescence) with DAPI labeled nucleuses (blue fluorescence) in paraspinal muscle. Scale bar, 25 μm. (G) Bar graph showing statistical analysis of positive rate of Ki67-labeled nuclei, n = 3 with more than 150 cells analyzed per n, **P < 0.01, by chi-square test (χ2 test). WT means wild types, Hetero means heterozygotes, and Homo means homozygotes.


( C Upper part , schematic illustration of the experimental procedure . Western blot analysis of S6 Ribosomal Protein and phospho - S6 Ribosomal Protein ( Ser235 / 236 ) from whole - liver protein extracts of female and male at P8 ( C ) treated mice . Mice were sampled after 4 days of Et - OH or rapamycin treatment .

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(C Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P8 (C) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.


D ) Upper part , schematic illustration of the experimental procedure . Western blot analysis of S6 Ribosomal Protein and phospho - S6 Ribosomal Protein ( Ser235 / 236 ) from whole - liver protein extracts of female and male at P34 ( D ) treated mice . Mice were sampled after 4 days of Et - OH or rapamycin treatment .

D) Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P34 (D) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.


Keratinocytes were isolated from the skin of a 4 - year - old patient with severe - generalised RDEB linked to homozygous insertion - deletion in COL7A1 ( Hilal et al , 1993 ) . Cultured RDEB cells ( blue line ) were serially passaged for more than 4 months , displaying a growth potential similar to non - diseased control cells ( YF29 ) isolated from the foreskin of a newborn ( black line ) . To calculate the percentage of growing colonies , 100 to 1 , 000 cells were plated into indicator dishes at each passage . Cells were grown for 12 days , fixed and stained with rhodamine B . Colonies were scored as growing or aborted ( Barrandon Green , 1987 ) .

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Keratinocytes were isolated from the skin of a 4-year-old patient with severe-generalised RDEB linked to homozygous insertion-deletion in COL7A1 (Hilal et al, 1993). Cultured RDEB cells (blue line) were serially passaged for more than 4 months, displaying a growth potential similar to non-diseased control cells (YF29) isolated from the foreskin of a newborn (black line). To calculate the percentage of growing colonies, 100 to 1,000 cells were plated into indicator dishes at each passage. Cells were grown for 12 days, fixed and stained with rhodamine B. Colonies were scored as growing or aborted (Barrandon Green, 1987).


Clonal analysis demonstrated the presence of stem cells ( holoclones ) in a passage VII RDEB culture ( 95 % of growing colonies ) .

Clonal analysis demonstrated the presence of stem cells (holoclones) in a passage VII RDEB culture (95% of growing colonies).


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . COLVII detection in clones by immunostaining . COLVII expression ( green ) was detectable in some clones ( 6 , 17 , 22 , 58 and 61 ) and not in others ( 3 , 24 and 54 ) ; nuclei were stained with Hoechst 33342 ( blue ) . Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3 - J2 feeder cells . Scale bar : 50 μm .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation.COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm.


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . Quantitative RT - PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes . All clones shown in ( A ) were transduced but expressed different levels of COL7A1 transcripts . Clones 6 , 17 , 22 , 54 , 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors ( YF29 and OR - CA , control 1 and 2 , respectively ) . The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1 .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Quantitative RT-PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1.


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . Determination of proviral rearrangements in transduced clones . A Southern blot was performed using genomic DNA of RDEB cells , clones and the infected mass culture from which the clones were isolated . Genomic DNA was digested with EcoRV and SpeI that cut at the 3 ′ and 5 ′ end of the provirus ( Supplementary Fig S2 ) and hybridised with a 907 - bp COL7A1 probe radiolabelled with 32P isotope . The upper band corresponded to the endogenous signal . The retroviral producer line Flp293A - E1aColVII1 was used as a control for the digested 9 . 6 - kb provirus ( proviral signal ) . Smaller bands corresponded to rearranged proviruses marked with an asterisk .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk.


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . Identification of stem cells producing COLVII . Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant , while clone 54 and surprisingly clone 22 did not ( see A ) . RDEB cells were used as a negative control and healthy donor cells as a positive control . The secreted matrix metalloproteinase 2 ( MMP2 ) was used as a loading control .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.


Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII . Non - COLVII - producing holoclone 54 ( black lines ) and COLVII - producing holoclone 6 ( red lines ) were serially transferred once a week until exhaustion ( Rochat et al , 1994 ) . Theoretical number of epidermal cells available for characterisation and CEA production from corrected ( clone 6 ) and uncorrected stem cells ( clone 54 ) calculated from the day of cloning . The colony - forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling , the generation number and the total progeny of isolated stem cells . Both corrected and uncorrected epidermal stem cells show high growth potential in vitro .

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Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII. Non-COLVII-producing holoclone 54 (black lines) and COLVII-producing holoclone 6 (red lines) were serially transferred once a week until exhaustion (Rochat et al, 1994).Theoretical number of epidermal cells available for characterisation and CEA production from corrected (clone 6) and uncorrected stem cells (clone 54) calculated from the day of cloning. The colony-forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling, the generation number and the total progeny of isolated stem cells. Both corrected and uncorrected epidermal stem cells show high growth potential in vitro.