C ScV1 ' s catalytic hexamer contains one open two closed catalytic sites . Side view of three catalytic hexamer A / B pairs ( shown in surface representation ) . Note that two closed pairs ( AB ) 2 3 are non - equivalently closed .

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C ScV1's catalytic hexamer contains one open and two closed catalytic sites. Side view of the three catalytic hexamer A/B pairs (shown in surface representation). Note that the two closed pairs (AB)2 and 3 are non-equivalently closed.


D Alignment of A3B3DF from ScV1 nucleotide - free EhV1 by catalytic hexamers . A3B3DF from ScV1 is shown in same color scheme as in Fig 2 , EhV1 is in blue . alignment shows difference in position of central rotor subunit , D . Note that C - terminus of subunit D ( DCT ) is longer in eukaryotic V - ATPase ( see electron density contoured at 1 . 2 σ in box to right ) . E ScV1 EhV1are halted in different rotational positions . Alignment of A3B3DF of ScV1 nucleotide - free EhV1 ( blue , with dashed lines drawn through A subunits ) by central stalk subunit D , reveals that two catalytic cores are halted in different rotational positions . autoinhibited ScV1 requires 40 ° rotation to overlay its catalytic hexamer with that of EhV1 .

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D Alignment of the A3B3DF from ScV1 and nucleotide-free EhV1 by the catalytic hexamers. The A3B3DF from ScV1 is shown in the same color scheme as in Fig 2, EhV1 is in blue. The alignment shows the difference in position of the central rotor subunit, D. Note that the C-terminus of subunit D (DCT) is longer in eukaryotic V-ATPase (see electron density contoured at 1.2 σ in the box to the right).E ScV1 and EhV1are halted in different rotational positions. Alignment of the A3B3DF of ScV1 and nucleotide-free EhV1 (blue, with dashed lines drawn through the A subunits) by the central stalk subunit D, reveals that the two catalytic cores are halted in different rotational positions. The autoinhibited ScV1 requires 40° rotation to overlay its catalytic hexamer with that of the EhV1.


F Comparison of D subunits of ScV1 ( red ) nucleotide free EhV1 ( blue ) . HCT from ScV1 is shown in yellow A subunit from EhV1 is shown in blue .

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F Comparison of the D subunits of ScV1 (red) and nucleotide free EhV1 (blue). HCT from ScV1 is shown in yellow and A subunit from EhV1 is shown in dark blue.


A ScV1 - H subunit interactions . Region of electron density ( contoured at 1 . 2 σ ) outlined by box in side view of ScV1 shown to right . H subunit N - terminal domain ( HNT ) is bound to N - terminal α helices of peripheral stalk EG1 while its globular C - terminal domain ( HCT ) is bound at base of catalytic hexamer .

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A ScV1-H subunit interactions. Region of electron density (contoured at 1.2 σ) outlined by the box in the side view of ScV1 shown to the right. The H subunit N-terminal domain (HNT) is bound to the N-terminal α helices of peripheral stalk EG1 while its globular C-terminal domain (HCT) is bound at the base of the catalytic hexamer.


B Interaction between HNT peripheral stalk EG1 . View of HNT as in panel A rotated 110 ° along horizontal , showing α helical HEAT repeats of HNT forming a pocket for binding N - termini of peripheral stalk EG1 . HNT ' s α helices forming pocket are labeled as in ( Sagermann et al , 2001 ) . Pro197 on HNT ( red sphere ) has been previously shown to cross - link subunit E ( Jefferies Forgac , 2008 ) . C Interaction between HCT base of catalytic hexamer . H subunit inhibitory contacts at base of ScV1 are mediated by a loop in HCT ( highlighted in magenta ) that is seen to contact two short segments of C - terminal domain of subunit G from open catalytic site ( AB ) 1 ( highlighted in purple ) two turns of N - terminal a helix of central rotor subunit D ( highlighted in blue ) .

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B Interaction between HNT and peripheral stalk EG1. View of HNT as in panel A rotated 110° along the horizontal, showing the α helical HEAT repeats of HNT forming a pocket for binding the N-termini of peripheral stalk EG1. HNT's α helices forming the pocket are labeled as in (Sagermann et al, 2001). Pro197 on HNT (red sphere) has been previously shown to cross-link subunit E (Jefferies Forgac, 2008).C Interaction between HCT and the base of the catalytic hexamer. The H subunit inhibitory contacts at the base of the ScV1 are mediated by a loop in HCT (highlighted in magenta) that is seen to contact two short segments of the C-terminal domain of the subunit G from the open catalytic site (AB)1 (highlighted in purple) and two turns of the N-terminal a helix of the central rotor subunit D (highlighted in blue).


D HCT binds to open ( AB ) 1 pair . View of ScV1 looking along central rotor subunit D towards cytoplasm , showing position of H subunit binding at interface of open A / B pair ( AB ) 1 . catalytic hexamer is shown in surface representation central rotor , peripheral stalks subunit H are shown as ribbons .

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D HCT binds to the open (AB)1 pair. View of ScV1 looking along the central rotor subunit D towards the cytoplasm, showing the position of the H subunit binding at the interface of the open A/B pair (AB)1. The catalytic hexamer is shown in surface representation and the central rotor, peripheral stalks and subunit H are shown as ribbons.


C Growth phenotype of H subunit mutants . Deletion of V - ATPase subunits from yeast results in a conditional lethal phenotype ( Vma - ) characterized by an inability to grow at pH 7 . 5 in presence of 60 mM CaCl2 ( upper panel ) , but growth is observed at pH 5 ( lower panel ) . Subunit H mutations were introduced on a plasmid into a yeast deleted for H subunit their assessed . Both humanH subunit isoforms ( HsH1 , HsH2 ) , a chimeric H subunit ( HChim ) containing ScHNT HsHCT , mutations in yeast intended to mimic of human H loop accessibility ( Hloop ) , a point mutation of conserved aspartic acid residue in loop ( HD410A ) are shown compared to H subunit deletion ( ΔH ) wild type H on a plasmid ( H WTpl ) . grow on pH 5 medium , but human isoforms failed to complement deletion phenotype .

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C Growth phenotype of H subunit mutants. Deletion of V-ATPase subunits from yeast results in a conditional lethal phenotype (Vma-) characterized by an inability to grow at pH 7.5 in the presence of 60 mM CaCl2 (upper panel), but growth is observed at pH 5 (lower panel). Subunit H mutations were introduced on a plasmid into a yeast strain deleted for the H subunit and their phenotypes assessed. Both humanH subunit isoforms (HsH1, HsH2), a chimeric H subunit (HChim) containing ScHNT and HsHCT, mutations in yeast intended to mimic the length of the human H loop sequence and accessibility (Hloop), and a point mutation of the conserved aspartic acid residue in the loop sequence (HD410A) are shown compared to the H subunit deletion (ΔH) and wild type H on a plasmid (H WTpl). All grow on pH 5 medium, but the human isoforms failed to complement the deletion phenotype.


D ATPase activity of H subunit mutants . ScV1 sectors were purified from each that complemented phenotype as well as from ScV1ΔH , for comparison of MgATPase activity . Average specific activities are shown plotted + / - s . e . m from two independent purifications . ScV1 sector containing chromosomally encoded H subunit shows no detectable MgATPase activity ( Parra et al , 2000 ; Zhang et al , 2003 ) , whereas ScV1containing plasmid - borne wild type H ( H WTpl ) exhibits low of activity likely due to reduced expression from low copy plasmid . HChim HLoop mutants lead to a loss of activity silencing , displaying nearly same activity as ScV1ΔH .

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D ATPase activity of H subunit mutants. The ScV1 sectors were purified from each strain that complemented the phenotype as well as from ScV1ΔH, for comparison of MgATPase activity. Average specific activities are shown plotted +/- s.e.m from two independent purifications. The ScV1 sector containing chromosomally encoded H subunit shows no detectable MgATPase activity (Parra et al, 2000; Zhang et al, 2003), whereas ScV1containing plasmid-borne wild type H (H WTpl) exhibits low levels of activity likely due to reduced expression from the low copy plasmid. The HChim and HLoop mutants lead to a loss of activity silencing, displaying nearly the same activity as ScV1ΔH.


E SDS - PAGE Western Blot of ScV1 preparations . Left panel , 15 % SDS PAGE of ScV1 purified from subunit H mutant WT strains . Note that H WTch is purified from a yeast deleted for C subunit ( used for crystallization ) . remaining strains contain subunit C , which is co - purified with ScV1 in varying . Note that while H WTch ScV1 , which is expressed from a deleted for subunit C , displays no detectable MgATPase activity , H subunit mutants co - purified with most C subunit display least activity , suggesting that absence or presence of subunit C has no effect on MgATPase activity . Right panel , immunoblot probed with an antibody directed against N - terminal Myc tagged mutant H subunits expressed from a plasmid . Note that of mutant H expression does not appear to vary from to .

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E SDS-PAGE and Western Blot analysis of ScV1 preparations. Left panel, 15% SDS PAGE of ScV1 purified from the subunit H mutant and WT strains. Note that H WTch is purified from a yeast strain deleted for the C subunit (used for crystallization). The remaining strains contain subunit C, which is co-purified with ScV1 in varying levels. Note that while H WTch ScV1, which is expressed from a strain deleted for subunit C, displays no detectable MgATPase activity, the H subunit mutants co-purified with the most C subunit display the least activity, suggesting that absence or presence of subunit C has no effect on MgATPase activity. Right panel, immunoblot probed with an antibody directed against the N-terminal Myc tagged mutant H subunits expressed from a plasmid. Note that the levels of mutant H expression does not appear to vary from strain to strain.


A Overlay of ScV1 cryo - EM structure of V1Vo holoenzyme in " state 2 " ( Zhao et al , 2015 ) . structures of ScV1 ScV1Vo were overlaid by aligning A3B3 catalytic hexamers , thus highlighting structural differences in central rotor peripheral stator subunits . Coloring of ScV1Vo ( shown as semi - transparent ribbons ) is as used throughout for ScV1 for but A3B3F ( tan ) , subunit H ( green ) , subunit C Vo domain ( gray ) . EG1 - HNT interaction is boxed . Note shift in position of HNT EG1 between ScV1 ScV1Vo .

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A Overlay of ScV1 and the cryo-EM structure of the V1Vo holoenzyme in "state 2" (Zhao et al, 2015). The structures of ScV1 and ScV1Vo were overlaid by aligning the A3B3 catalytic hexamers, thus highlighting structural differences in the central rotor and peripheral stator subunits. Coloring of ScV1Vo (shown as semi-transparent ribbons) is as used throughout for ScV1 for all but the A3B3F (tan), subunit H (dark green), subunit C and Vo domain (gray). The EG1-HNT interaction is boxed. Note the shift in position of HNT and EG1 between ScV1 and ScV1Vo.


B Comparison of peripheral stalk EG1 HNT in ScV1 versus ScV1Vo . boxed view in A is shown enlarged . N - terminal α helices of peripheral stalk EG1 HNT change in conformation , likely induced by movement of HCT when going from holo enzyme to autoinhibited conformation .

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B Comparison of peripheral stalk EG1 and HNT in ScV1 versus ScV1Vo. The boxed view in A is shown enlarged. The N-terminal α helices of peripheral stalk EG1 and HNT change in conformation, likely induced by the movement of HCT when going from the holo enzyme to the autoinhibited conformation.


C View as in A rotated 150º around vertical . Comparisons of peripheral stalk EG3 HCT are boxed . D Comparison of peripheral stalk EG3 in ScV1 versus ScV1Vo . In ScV1Vo , EG3 binds to C subunit head domain ( Chead ; gray ) whereas in ScV1 , EG3 is bent towards C - terminal domain of a catalytic A subunit ( enlarged view of box labeled EG3 in panel C ) . E Comparison of HCT ' s conformation in ScV1 versus ScV1Vo . Enlarged view of box labeled HCT in panel C highlighting conformational changes binding interactions of HCT ; for clarity only aNT , HCT central rotor ( DF ) are shown . In ScV1Vo , most C - terminal α helix ( green ) of HCT ( green ) binds to aNT ( gray ) , with inhibitory loop ( green spheres ) rotated away inaccessible for binding B subunit central stalk ( transparent red ) . In ScV1 , HCT ( yellow ) inhibitory loop ( yellow spheres ) binds to central stalk subunit D ( red ) B subunit of open ( AB ) 1 catalytic site . difference in conformation between ScV1Vo ScV1 involves a ~ 150 ° rotation of HCT . Note that HCT loop ( spheres ) is rotated away from central stalk in ScV1Vo whereas in ScV1 , C - terminal α helix ( orange ) involved in binding aNT is rotated away unavailable for rebinding to .

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C View as in A rotated 150º around the vertical. Comparisons of peripheral stalk EG3 and HCT are boxed.D Comparison of peripheral stalk EG3 in ScV1 versus ScV1Vo. In ScV1Vo, EG3 binds to the C subunit head domain (Chead; gray) whereas in ScV1, EG3 is bent towards the C-terminal domain of a catalytic A subunit (enlarged view of box labeled EG3 in panel C).E Comparison of HCT's conformation in ScV1 versus ScV1Vo. Enlarged view of box labeled HCT in panel C highlighting the conformational changes and binding interactions of HCT; for clarity only aNT, HCT and the central rotor (DF) are shown. In ScV1Vo, the most C-terminal α helix (dark green) of HCT (green) binds to aNT (light gray), with the inhibitory loop (green spheres) rotated away and inaccessible for binding the B subunit and the central stalk (transparent red). In ScV1, the HCT (yellow) inhibitory loop (yellow spheres) binds to the central stalk subunit D (red) and the B subunit of the open (AB)1 catalytic site. The difference in conformation between ScV1Vo and ScV1 involves a ~150° rotation of HCT. Note that the HCT loop (spheres) is rotated away from the central stalk in ScV1Vo whereas in ScV1, the C-terminal α helix (orange) involved in binding aNT is rotated away and unavailable for rebinding to the membrane.


F HCT conformational changes . Available subunit H structures as obtained for isolated H ( Sagermann et al , 2001 ) ( purple ) , in ScV1Vo ( Zhao et al , 2015 ) ( green ) in ScV1 ( yellow ) are shown aligned by their N - terminal domains ( HNT ) illustrating conformational flexibility of HCT . loop involved in inhibition of ScV1 is shown as red spheres , highlighting 180º rotation of HCT when going from conformation of isolated H ( HCT 1ho8 ) to inhibitory conformation in ScV1 ( HCT ScV1 ) .

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F HCT conformational changes. Available subunit H structures as obtained for isolated H (Sagermann et al, 2001) (purple), in ScV1Vo (Zhao et al, 2015) (green) and in ScV1 (yellow) are shown aligned by their N-terminal domains (HNT) illustrating the conformational flexibility of HCT. The loop involved in inhibition of ScV1 is shown as red spheres, highlighting the 180º rotation of HCT when going from the conformation of isolated H (HCT 1ho8) to the inhibitory conformation in ScV1(HCT ScV1).


rapZ mutants were expressed from plasmids under control of PBAD promoter ( induction with arabinose , repression by glucose ) tested for ability to complement a lacking chromosomal rapZ gene in ( b ) Northern blotting experiment detecting GlmZ ( top panel ) 5S rRNA ( bottom panel ; loading control ) to assess degree of GlmZ cleavage triggered by RapZ variants . Processed GlmZ is indicated with an asterisk . Strains Z8 ( wild - type , lanes 1 , 2 ) Z28 ( ∆ rapZ , other lanes were used . Z28 carried plasmid pBAD33 ( lanes 3 , 4 ; empty vector control = VC ) or derivatives triggering synthesis of indicated RapZ

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The rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (b) Northern blotting experiment detecting GlmZ (top panel) and 5S rRNA (bottom panel; loading control) to assess the degree of GlmZ cleavage triggered by the RapZ variants. Processed GlmZ is indicated with an asterisk. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variants


rapZ mutants were expressed from plasmids under control of PBAD promoter ( induction with arabinose , repression by glucose ) tested for ability to complement a lacking chromosomal rapZ gene in ( c ) GlmS ( MW = 66 . 9 kDa ) in same cultures as revealed by SDS - PAGE of total extracts followed by Coomassie blue . Strains Z8 ( wild - type , lanes 1 , 2 ) Z28 ( ∆ rapZ , other lanes were used . Z28 carried plasmid pBAD33 ( lanes 3 , 4 ; empty vector control = VC ) or derivatives triggering synthesis of indicated RapZ

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The rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (c) GlmS protein (MW = 66.9 kDa) levels in the same cultures as revealed by SDS-PAGE of total protein extracts followed by Coomassie blue staining. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variants


rapZ mutants were expressed from plasmids under control of PBAD promoter ( induction with arabinose , repression by glucose ) tested for ability to complement a lacking chromosomal rapZ gene in ( d ) β - Galactosidase activities produced by same cultures replicates ( mean values standard deviations are shown ) . carry a glmS ' - lacZ fusion on chromosome , which requires base - pairing with sRNA GlmZ for high expression . Strains Z8 ( wild - type columns 1 , 2 ) Z28 other columns ) were used . Z28 carried plasmid pBAD33 columns 3 , 4 ; empty vector control = VC ) or derivatives triggering synthesis of indicated RapZ

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The rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (d) β-Galactosidase activities produced by the same cultures and replicates (mean values and standard deviations are shown). Cells carry a glmS'-lacZ fusion on the chromosome, which requires base-pairing with sRNA GlmZ for high expression. Strains Z8 (wild-type columns 1, 2) and Z28 all other columns) were used. Strain Z28 carried plasmid pBAD33 columns 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variants


C ) MXE saturation . Whereas increasing amounts of - seq reads should lead to confirmation of further MXE candidates , more - seq reads might also result in rejection of previously validated MXEs . green curves show number of validated MXEs in relation to of total - seq reads used for validation . orange curves indicate number of initially ' validated MXEs ' that were rejected with increasing amounts of reads . Grey dashed lines indicate point of saturation , which is defined as point where a two - fold increase in reads leads to rejection of less than 1 % of validated MXEs . Of note , whereas rejection of validated MXEs saturates with 20 % of data amount of novel MXE validations is still rapidly increasing .

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C) MXE saturation analysis. Whereas increasing amounts of RNA-seq reads should lead to the confirmation of further MXE candidates, more RNA-seq reads might also result in the rejection of previously validated MXEs. The green curves show the number of validated MXEs in relation to the percentage of total RNA-seq reads used for validation. The orange curves indicate the number of initially 'validated MXEs' that were rejected with increasing amounts of reads. Grey dashed lines indicate the point of saturation, which is defined as the point where a two-fold increase in reads leads to rejection of less than 1% of the validated MXEs. Of note, whereas the rejection of validated MXEs saturates with 20% of the data the amount of novel MXE validations is still rapidly increasing.


E ) distribution of multi - cluster MXEs .

E) Size and distribution of multi-cluster MXEs.


A ) showing differentially expressed MXE clusters with at least 3 RPKM . Here , we used Gini coefficient , which is a measure of inequality among values of a frequency distribution ( Ceriani & Verme , 2012 ) has successfully been used to determine tissue - enriched gene sets ( Zhang et al , 2017 ) , to determine highly tissue - specific MXEs ( maximum normalized Gini index of cluster ) MXEs with a broad tissue expression distribution ( minimum Gini index ) . For each MXE cluster - spliced - in ( PSI ) value of ubiquitous MXE ( minimum Gini index ) is subtracted from PSI value of specific MXE ( maximum Gini index of cluster ) ( delta PSI value ) scaled between - 1 ( broad tissue distribution ) 1 ( highly tissue specific ) . Each column represents an MXE pair each row represents MXE expression in a tissue , - type , or at a developmental time point . bar graph summarizes where specific MXE is 1 . 5 fold more spliced in than ubiquitous MXE .

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A) Heatmap showing all differentially expressed MXE clusters with at least 3 RPKM. Here, we used the Gini coefficient, which is a measure of the inequality among values of a frequency distribution (Ceriani & Verme, 2012) and has successfully been used to determine tissue-enriched gene sets (Zhang et al, 2017), to determine highly tissue-specific MXEs (maximum normalized Gini index of cluster) and MXEs with a broad tissue expression distribution (minimum Gini index). For each MXE cluster the percent-spliced-in (PSI) value of the ubiquitous MXE (minimum Gini index) is subtracted from the PSI value of the specific MXE (maximum Gini index of cluster) (delta PSI value) and scaled between -1 (broad tissue distribution) and 1 (highly tissue specific). Each column represents an MXE pair and each row represents MXE expression in a tissue, cell-type, or at a developmental time point. The bar graph summarizes counts where the specific MXE is 1.5 fold more spliced in than the ubiquitous MXE.


C ) showing delta PSI values ( PSI value of non - SNP - containing MXE subtracted from PSI value of SNP - containing MXE ) of MXE clusters containing pathogenic SNPs scaled between - 1 1 ( blue = high expression non - SNP - containing MXE , red = high expression SNP - containing MXE ) . Columns represent MXE clusters rows tissues , types , developmental stages . column bar graph summarizes where SNP - containing MXE is 1 . 5 fold more expressed than non - SNP - containing MXE , whereas row bar graph shows this for each tissue , type , developmental stage .

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C) Heatmap showing the the delta PSI values (PSI value of the non-SNP-containing MXE subtracted from the PSI value of the SNP-containing MXE) of MXE clusters containing pathogenic SNPs scaled between -1 and 1 (blue = high expression non-SNP-containing MXE, red = high expression SNP-containing MXE). Columns represent MXE clusters and rows tissues, cell types, and developmental stages. The column bar graph summarizes counts where the SNP-containing MXE is 1.5 fold more expressed than the non-SNP-containing MXE, whereas the row bar graph shows this for each tissue, cell type, and developmental stage.


B atg19Δ containing HTB ‐ Atg19 were grown to mid ‐ log phase . Atg19 was affinity purified subjected to mass spectrometric phosphorylation mapping . Phosphorylation sites : enlarged ; lysine substitutions : gray ; Atg11 binding region : green .

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B atg19Δ cells containing HTB‐Atg19 were grown to mid‐log phase. Atg19 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; lysine substitutions: gray; Atg11 binding region: green.


C atg19Δ containing GFP ‐ Atg19 wild ‐ type or mutants as indicated were grown to mid ‐ log phase . Processing of endogenous Ape1 was analyzed by Western blotting quantified by calculating of versus uncleaved Ape1 normalized to wild ‐ type . Atg19 expression was assessed by anti ‐ GFP Western blotting .

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C atg19Δ cells containing GFP‐Atg19 wild‐type or mutants as indicated were grown to mid‐log phase. Processing of endogenous Ape1 was analyzed by Western blotting and quantified by calculating the ratio of cleaved versus uncleaved Ape1 normalized to the wild‐type. Atg19 expression was assessed by anti‐GFP Western blotting.


D atg19Δ GFP ‐ Atg11 Atg1 ‐ TAP containing myc ‐ Atg19 as indicated were grown to log phase . Atg11 was immunoprecipitated , bound Atg19 was analyzed by Western blotting .

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D atg19Δ GFP‐Atg11 Atg1‐TAP cells containing myc‐Atg19 as indicated were grown to log phase. Atg11 was immunoprecipitated, and bound Atg19 was analyzed by Western blotting.


E GST ‐ fused propeptide of Ape1 , Atg19 as indicated , a His ‐ tagged fragment of Atg11 ( 685 - 1178 ) were purified from Escherichia coli , mixed , propeptide ‐ bound were isolated analyzed by Coomassie . of three experiments is shown . Error bars represent standard deviation .

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E GST‐fused propeptide of Ape1, Atg19 as indicated, and a His‐tagged fragment of Atg11 (amino acids 685-1178) were purified from Escherichia coli, mixed, and propeptide‐bound proteins were isolated and analyzed by Coomassie staining. Quantification of three individual experiments is shown. Error bars represent standard deviation.


A GFP ‐ Atg11 Ape1 ‐ mRuby atg19Δ containing myc ‐ Atg19 as indicated Cup1 ‐ Ape1 were grown to log phase . Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h , autophagy was induced by treating for 1 h with rapamycin . Scale bar , 5 μm .

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A GFP‐Atg11 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h, and autophagy was induced by treating cells for 1 h with rapamycin. Scale bar, 5 μm.


B Ape1 ‐ mRuby atg19Δ containing GFP ‐ Atg19 wild ‐ type or GFP ‐ Atg19 ‐ 3A were analyzed in log phase . Scale bar , 5 μm .

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B Ape1‐mRuby atg19Δ cells containing GFP‐Atg19 wild‐type or GFP‐Atg19‐3A were analyzed in log phase. Scale bar, 5 μm.


C GFP ‐ Atg8 Ape1 ‐ mRuby atg19Δ containing myc ‐ Atg19 as indicated Cup1 ‐ Ape1 were analyzed as in ( A ) . Scale bar , 5 μm .

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C GFP‐Atg8 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were analyzed as in (A). Scale bar, 5 μm.


B atg34Δ yeast containing HTB ‐ Atg34 were grown to mid ‐ log phase treated with rapamycin . Atg34 was affinity purified subjected to mass spectrometric phosphorylation mapping . Phosphorylation sites : enlarged ; Atg11 binding region : green ; lysine substitution : gray .

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B atg34Δ yeast cells containing HTB‐Atg34 were grown to mid‐log phase and treated with rapamycin. Atg34 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; Atg11 binding region: green; lysine substitution: gray.


C Ams1 ‐ GFP atg19Δatg34Δ containing A ( Stag ) ‐ tagged Atg34 as indicated were starved for 4 h . Processing of endogenous Ams1 ‐ GFP was analyzed by Western blotting quantified by calculating of free GFP versus uncleaved Ams1 ‐ GFP normalized to wild ‐ type . Expression of Atg34 was assessed by anti ‐ A Western blotting .

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C Ams1‐GFP atg19Δatg34Δ cells containing protein A (Stag)‐tagged Atg34 as indicated were starved for 4 h. Processing of endogenous Ams1‐GFP was analyzed by Western blotting and quantified by calculating the ratio of free GFP versus uncleaved Ams1‐GFP normalized to the wild‐type. Expression of Atg34 proteins was assessed by anti‐protein A Western blotting.


E GST , GST ‐ Atg19 , GST ‐ Atg34 were purified from Escherichia coli in vitro phosphorylated with immunoprecipitated Atg1 ‐ TAP bound to IgG magnetic beads .

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E GST, GST‐Atg19, and GST‐Atg34 were purified from Escherichia coli and in vitro phosphorylated with immunoprecipitated Atg1‐TAP bound to IgG magnetic beads.


F In vitro phosphorylation of recombinant Atg19 , Atg19 ‐ 3A , Atg19 ‐ 3D using wild ‐ type kinase ‐ dead Atg1 ‐ D211A as described in ( E ) .

F In vitro phosphorylation of recombinant Atg19, Atg19‐3A, and Atg19‐3D using wild‐type and kinase‐dead Atg1‐D211A as described in (E).


A Wild ‐ type , hrr25 ‐ ts , hrr25 ‐ ts containing HRR25 on a centromeric plasmid were grown to mid ‐ log phase at 18 ° C followed by 2 4 h of log ‐ phase growth at 37 ° C . Ape1 processing was analyzed by Western blotting .

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A Wild‐type, hrr25‐ts, and hrr25‐ts cells containing HRR25 on a centromeric plasmid were grown to mid‐log phase at 18°C followed by 2 and 4 h of log‐phase growth at 37°C. Ape1 processing was analyzed by Western blotting.


B atg19Δ atg19Δ hrr25 ‐ ts containing GFP ‐ Atg19 as indicated were analyzed for Ape1 processing in log phase after 2 h at 37 ° C .

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B atg19Δ and atg19Δ hrr25‐ts cells containing GFP‐Atg19 as indicated were analyzed for Ape1 processing in log phase after 2 h at 37°C.


C GFP ‐ Atg11 , GFP ‐ Atg11 hrr25 ‐ ts , GFP ‐ Atg11 atg1 ‐ D211A were analyzed for GFP ‐ Atg11 dot formation . of at least 70 in three experiments is shown . Error bars represent standard deviation . Scale bar , 3 μm .

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C GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg1‐D211A cells were analyzed for GFP‐Atg11 dot formation. Quantification of at least 70 cells in three individual experiments is shown. Error bars represent standard deviation. Scale bar, 3 μm.


D GFP ‐ Atg11 , GFP ‐ Atg11 hrr25 ‐ ts , GFP ‐ Atg11 atg19Δ containing BFP ‐ Ape1 Cup1 ‐ Ape1 were grown to log phase . Overexpression of Ape1 was induced by addition of 250 μM CuSO4 for 3 h . coloc . : with GFP ‐ Atg11 / BFP ‐ Ape1 co ‐ localization . n = 50 ( wt ) , 84 ( hrr25 ‐ ts ) , 127 ( atg19Δ ) . Scale bar , 3 μm .

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D GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg19Δ cells containing BFP‐Ape1 and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM CuSO4 for 3 h. coloc.: Cells with GFP‐Atg11/BFP‐Ape1 co‐localization. n = 50 (wt), 84 (hrr25‐ts), 127 (atg19Δ). Scale bar, 3 μm.


E Hrr25 was immunoprecipitated from Hrr25 ‐ TAP as a control from a non ‐ tagged wild ‐ type . In vitro phosphorylation of recombinant Atg19 , Atg19 ‐ 3A , Atg34 , GST using kinase ‐ bound beads was performed as described in Fig E . Both Atg19 panels are from same gel with same exposure .

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E Hrr25 was immunoprecipitated from Hrr25‐TAP cells and as a control from a non‐tagged wild‐type strain. In vitro phosphorylation of recombinant Atg19, Atg19‐3A, Atg34, and GST using the kinase‐bound beads was performed as described in Fig E. Both Atg19 panels are from the same gel with the same exposure.


( B ) Immunoblot of immunoprecipitated HA - SOX9 or FLAG - FBW7α their pull - down products . corresponding Western blotting from whole extract ( WCE ) of transfected HEK293 showed of exogenous HA - SOX9 or FLAG - FBW7α in total input . were treated with 10 µM MG132 for 4 hours prior to harvesting for immunoprecipitation . Blots are representative from 3 independent experiments .

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(B) Immunoblot analysis of the immunoprecipitated HA-SOX9 or FLAG- FBW7α and their pull-down products. The corresponding Western blotting from the whole cell extract (WCE) of the transfected HEK293 showed the levels of exogenous HA-SOX9 or FLAG-FBW7α proteins in the total input. Cells were treated with 10 µM MG132 for 4 hours prior to harvesting for the immunoprecipitation. Blots are representative from 3 independent experiments.


( C ) FBW7α immunoblot eluted from agarose bead - bound peptide , which had been incubated with recombinant SCF - FBW7α for 1 hour at 37 ◦ C . agarose bead - bound peptide contains either non - phosphorylated SOX9 acid spanning from residue number 231 to 245 ( SOX9 peptide ) , or those with threonine corresponding to residue number 236 240 being phosphorylated ( pSOX9 peptide ) . input ( 10 % ) showed level of supplemented recombinant SCF - FBW7α in in vitro binding reaction . Shown blot is representative from 2 independent experiments .

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(C) FBW7α immunoblot eluted from the agarose bead-bound peptide, which had been incubated with the recombinant SCF-FBW7α for 1 hour at 37◦C. The agarose bead-bound peptide contains either the non-phosphorylated SOX9 amino acid sequence spanning from residue number 231 to 245 (SOX9 peptide), or those with the threonine corresponding to residue number 236 and 240 being phosphorylated (pSOX9 peptide). The input (10%) showed the level of the supplemented recombinant SCF-FBW7α in the in vitro binding reaction. Shown blot is representative from 2 independent experiments.


( D ) Representative Western blotting set from 3 independent repeats of pT236 - SOX9 from lysates of HEK293 transfected with either HA - EV or HA - SOX9 . Each lysate were immediately divided left untreated or subjected to lambda phosphatase ( λ - PPase ) treatment for 1 hour at 37 ◦ C prior to gel electrophoresis . SOX9 blot showed presence of SOX9 in both untreated phosphatase - treated SOX9 - transfected lysates .

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(D) Representative Western blotting set from 3 independent repeats of pT236-SOX9 from lysates of HEK293 transfected with either HA-EV or HA-SOX9. Each lysate were immediately divided and left untreated or subjected to lambda phosphatase (λ-PPase) treatment for 1 hour at 37◦C prior to gel electrophoresis. SOX9 blot showed the presence of SOX9 protein in both the untreated and the phosphatase-treated SOX9-transfected cell lysates.


( E ) Bead - immobilized in vitro translated ( IVT ) HA - SOX9 wild type ( WT ) or T236 / 240 mutant were either untreated or subjected to in vitro GSK3 kinase reaction for 90 minutes at 37 ◦ C prior to elution gel electrophoresis . SOX9 blot showed total SOX9 eluted from beads from each reaction . Shown immunoblots are single representative experiment from 3 independent repeats .

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(E) Bead-immobilized in vitro translated (IVT) HA-SOX9 wild type (WT) or T236/240 mutant were either untreated or subjected to in vitro GSK3 kinase reaction for 90 minutes at 37◦C prior to elution and gel electrophoresis. The SOX9 blot showed total SOX9 protein eluted from the beads from each reaction. Shown immunoblots are single representative experiment from 3 independent repeats.


( F ) GSK3 - mediated phosphorylation of threonine 236 promoted SOX9 interaction with recombinant SCF - FBW7α in vitro . Bead - immobilized IVT HA - SOX9 were either untreated or phosphorylated with GSK3 as described in ( E ) prior to further incubation with recombinant SCF - FBW7α for in vitro binding . Immunoblot showed FBW7α in in vitro binding reaction ( input ) as well as those which had been eluted from untreated GSK3 - treated immobilized SOX9 . set of blots shown is representative from 3 independent experiments .

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(F) GSK3-mediated phosphorylation of threonine 236 promoted SOX9 interaction with recombinant SCF-FBW7α in vitro. Bead-immobilized IVT HA-SOX9 were either untreated or phosphorylated with GSK3 as described in (E) prior to further incubation with recombinant SCF-FBW7α for in vitro binding assay. Immunoblot showed FBW7α protein in the in vitro binding reaction (input) as well as those which had been eluted from the untreated and GSK3-treated immobilized SOX9. The set of blots shown is representative from 3 independent experiments.


( A ) Endogenous SOX9 turnover in presence of cycloheximide following RNAi depletion of FBW7α , β or γ . HEK293 were transfected with 20 nM of non - targeting scramble or those specifically targeting FBW7α , β or γ for 72 hours prior to chase with addition of 100 ng / mL cycloheximide . Immunoblot of Cyclin E , an established SCF - FBW7 substrate , was used to assess efficacy of RNAi - mediated depletion of FBW7 with GAPDH served as loading control .

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(A) Endogenous SOX9 protein turnover in the presence of cycloheximide following RNAi depletion of FBW7α, β or γ. HEK293 cells were transfected with 20 nM of non-targeting scramble RNA or those specifically targeting FBW7α, β or γ for 72 hours prior to chase with addition of 100 ng /mL cycloheximide. Immunoblot of Cyclin E, an established SCF-FBW7 substrate, was used to assess the efficacy of RNAi-mediated depletion of FBW7 protein with GAPDH served as protein loading control.


( B ) Immunofluorescence of D324MED medulloblastoma showing co - localization of HA - SOX9 WT ( AlexaFluor 568 ; Red ) FLAG - FBW7α ( AlexaFluor 488 ; Green ) . nuclei were counter - stained with DAPI ( blue ) . Image set is representative for multiple image fields taken at 40x objective magnification . Scale bar indicate 20 µm .

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(B) Immunofluorescence staining of D324MED medulloblastoma cells showing nuclear co-localization of HA-SOX9 WT (AlexaFluor 568; Red) and FLAG-FBW7α (AlexaFluor 488; Green). The cell nuclei were counter-stained with DAPI (blue). Image set is representative for multiple image fields taken at 40x objective magnification. Scale bar indicate 20 µm.


( C ) Western blotting of HEK293 endogenous SOX9 24 hours following forced expression of 100 , 250 , 500 , 1000 ng of plasmid expressing FLAG - FBW7α . Changes in SOX9 level in representative blots were analysed relative to GAPDH using ImageJ . set of blots shown is representative from 3 independent experiments .

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(C) Western blotting of HEK293 endogenous SOX9 proteins 24 hours following forced expression of 100, 250, 500, and 1000 ng of plasmid expressing FLAG-FBW7α. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.


( D ) Expression of FLAG - FBW7α enhanced HA - SOX9 - WT turnover , which can be attenuated with addition of 10 µM MG - 132 ( MG ) in HEK293 . By contrast , forced expression of FLAG - FBW7α did not enhance HA - SOX9 - T236A - T236A / T240A turnover over course of 4 hours chase . loading for each sample was indicated by level of β - actin . set of blots shown is representative from 4 independent experiments .

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(D) Expression of FLAG-FBW7α enhanced HA-SOX9-WT protein turnover, which can be attenuated with addition of 10 µM MG-132 (MG) in HEK293 cells. By contrast, forced expression of FLAG-FBW7α did not enhance HA-SOX9-T236A and -T236A/T240A protein turnover over the course of 4 hours protein chase. Protein loading for each sample was indicated by the level of β-actin protein. The set of blots shown is representative from 4 independent experiments.


( E ) Treatment of HEK293 with increasing of BIO , a GSK3α / β inhibitor , increased HA - SOX9 - WT level in dose - dependent manner . HA - SOX9 - WT FLAG - FBW7α were co - expressed in HEK293 overnight prior to treatment with various of BIO for 4 hours . Whole lysates were collected for Western blotting with loading for each sample adjusted to GAPDH level . set of blots shown is representative from 2 independent experiments .

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(E) Treatment of HEK293 cells with increasing concentration of BIO, a GSK3α/β inhibitor, increased HA-SOX9-WT protein level in dose-dependent manner. HA-SOX9-WT and FLAG-FBW7α were co-expressed in HEK293 overnight prior to treatment with various concentration of BIO for 4 hours. Whole cell lysates were collected for Western blotting with protein loading for each sample adjusted to GAPDH protein level. The set of blots shown is representative from 2 independent experiments.


( F ) RNAi depletion of GSK3ß decreased FBW7α - induced HA - SOX9 - WT turnover in HEK293 . HA - SOX9 - WT turnover was examined in presence of 100 ng / mL cycloheximide , 48 hours following si - GSK3ß ( 20 nM ) transfection , 24 hours after co - expression of HA - SOX9 - WT FLAG - FBW7α . Immunoblots of GSK3ß demonstrated depletion of GSK3ß with siRNA . Changes in SOX9 level in representative blots were analysed relative to GAPDH using ImageJ . set of blots shown is representative from 3 independent experiments .

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(F) The RNAi depletion of GSK3ß decreased FBW7α-induced HA-SOX9-WT turnover in HEK293 cells. The HA-SOX9-WT protein turnover was examined in the presence of 100 ng/mL cycloheximide, 48 hours following si-GSK3ß (20 nM) transfection, 24 hours after co-expression of HA-SOX9-WT and FLAG-FBW7α. Immunoblots of GSK3ß demonstrated depletion of GSK3ß protein with the siRNA. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.


( G ) Forced FLAG - FBW7α - WT expression promoted poly - ubiquitylation of endogenous SOX9 in HEK293 , which accumulated further in presence of 10 µM MG132 . Expression of non - functional FBW7α lacking F - box domain ( ΔF ) , or those containing R465A mutation did not induce SOX9 poly - ubiquitylation in vivo . Ubiquitylation was performed under denaturing condition to disrupt non - covalently linked ubiquitin as described in Materials Methods . Expression of various FLAG - FBW7α constructs endogenous SOX9 were examined in whole extract ( WCE ) . GAPDH was used to indicate total loading with loading . set of blots shown is representative from 4 independent experiments .

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(G) Forced FLAG-FBW7α-WT expression promoted poly-ubiquitylation of endogenous SOX9 in HEK293, which accumulated further in the presence of 10 µM MG132. Expression of non-functional FBW7α lacking the F-box domain (ΔF), or those containing R465A mutation did not induce SOX9 poly-ubiquitylation in vivo. Ubiquitylation assay was performed under denaturing condition to disrupt non-covalently linked ubiquitin as described in the Materials Methods. Expression of various FLAG-FBW7α constructs and endogenous SOX9 protein were examined in the whole cell extract (WCE). GAPDH protein was used to indicate total protein loading with protein loading. The set of blots shown is representative from 4 independent experiments.


( H ) Reconstitution of SOX9 ubiquitylation reaction by FLAG - FBW7α in vitro . Immobilized - IVT HA - SOX9 WT were incubated with complete ubiquitylation reaction mixture containing either eluted FLAG - FBW7α - WT or ΔF mutant for 60 minutes at 37 ◦ C . Reaction mixture lacking UbE1 , an E1 ubiquitin - activating enzyme , or UbcH3 , an E2 ubiquiting - conjugating enzyme , served as negative control for reaction . SOX9 ubiquitylation was assessed following elution of from immobilized beads under denaturing condition as described in Materials Methods . set of blots shown is representative from 3 independent experiments .

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(H) Reconstitution of SOX9 ubiquitylation reaction by FLAG-FBW7α in vitro. Immobilized-IVT HA-SOX9 WT were incubated with complete ubiquitylation reaction mixture containing either the eluted FLAG-FBW7α-WT or the ΔF mutant for 60 minutes at 37◦C. Reaction mixture lacking the UbE1, an E1 ubiquitin-activating enzyme, or UbcH3, an E2 ubiquiting-conjugating enzyme, served as negative control for the reaction. SOX9 ubiquitylation was assessed following elution of the protein from the immobilized beads under denaturing condition as described in Materials Methods. The set of blots shown is representative from 3 independent experiments.


( B ) Immunoblotting of HA - SOX9 - WT following expression of various FLAG - FBW7α constructs including wild type ( WT ) , or those with R465A or R479A mutation in Daoy . were transfected with equal amount of HA - SOX9 - WT FLAG - FBW7α whole lysates were collected 24 hours following transfection . GAPDH were shown to indicate total loading for each sample . set of blots shown is representative from 2 independent experiments .

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(B) Immunoblotting of HA-SOX9-WT protein following expression of various FLAG-FBW7α constructs including wild type (WT), or those with R465A or R479A mutation in Daoy. The cells were transfected with equal amount of HA-SOX9-WT and FLAG-FBW7α and the whole cell lysates were collected 24 hours following the transfection. GAPDH proteins were shown to indicate total protein loading for each sample. The set of blots shown is representative from 2 independent experiments.


( C ) Co - immunoprecipitation of HA - SOX9 with either wild type ( WT ) or R465A - FLAG - FBW7α . Unlike wild type , FLAG - FBW7α containing arginine 465 mutation did not significantly co - immunoprecipitate with HA - SOX9 ( IP with anti HA antibody ) vice versa ( IP with anti FLAG antibody ) . expressions of HA - SOX9 FLAG - FBW7α - WT or R465A mutant were assessed in whole extract ( WCE ) of transfected HEK293 with GAPDH used to indicate total loading .

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(C) Co-immunoprecipitation of HA-SOX9 with either wild type (WT) or R465A- FLAG-FBW7α. Unlike the wild type protein, FLAG-FBW7α containing arginine 465 mutation did not significantly co-immunoprecipitate with HA-SOX9 (IP with anti HA antibody) and vice versa (IP with anti FLAG antibody). The expressions of HA-SOX9 and FLAG- FBW7α- WT or R465A mutant were assessed in the whole cell extract (WCE) of the transfected HEK293 with GAPDH protein used to indicate total protein loading.


( D ) Comparison of FBW7 mRNA expression across medulloblastoma subgroups ( WNT , SHH , Group 3 , 4 ) in a cohort containing 423 medulloblastoma cases . Expression of FBW7 in both normal ( n = 5 ) adult cerebellum ( n = 13 ) were used for comparison against medulloblastoma .

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(D) Comparison of FBW7 mRNA expression across the medulloblastoma subgroups (WNT, SHH, Group 3, and 4) in a cohort containing 423 medulloblastoma cases. Expression of FBW7 in both normal fetal (n= 5) and adult cerebellum (n = 13) were used for comparison against the medulloblastoma.


( E ) Overall survival of medulloblastoma upon FBW7 expression level . was performed on 383 out of 423 cases from which survival data were available . Cohort was divided into high - ( blue ) low - FBW7 ( red ) expressing subgroup using FBW7 median expression as group classifier . Statistical was determined by using a LogRank .

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(E) Overall survival analysis of medulloblastoma patient based upon FBW7 expression level. Analysis was performed on 383 out of the 423 cases from which the survival data were available. Cohort was divided into high- (blue line) and low- FBW7 (red line) expressing subgroup using FBW7 median expression as group classifier. Statistical analysis was determined by using a LogRank test.


( F ) Correlation between SOX9 / mRNA FBW7 mRNA expression level from medulloblastoma tissue microarray consisting of 142 tissue samples . SOX9 level was scored following immunohistochemistry , while SOX9 FBW7 mRNAs expression by Scope as described in Materials Methods . A modest negative correlation was determined by Kendall ' s correlation ( τ = - 0 . 223 , p0 . 001 ) between SOX9 / mRNA FBW7 expression . Affiliation of samples with medulloblastoma subgroups was indicated by different colored circle ( blue = WNT , red = SHH , yellow = Group 3 , green = Group 4 , grey = Undetermined ) .

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(F) Correlation analysis between SOX9 protein/mRNA and FBW7 mRNA expression level from medulloblastoma tissue microarray consisting of 142 tissue samples. SOX9 protein level was scored following immunohistochemistry staining, while SOX9 and FBW7 mRNAs expression by the RNA Scope as described in Materials Methods. A modest negative correlation was determined by Kendall's correlation test (τ = - 0.223, p0.001) between SOX9 protein/mRNA and FBW7 expression. Affiliation of samples with medulloblastoma subgroups was indicated by different colored circle (blue=WNT, red=SHH, yellow=Group 3, green=Group 4, grey=Undetermined).


( A ) Overall survival of mice bearing Daoy expressing SOX9 with doxycycline ( dox ) - inducible FBW7α ( Daoy - SOX9 / T - FBW7α ) . dox - preconditioned or control ( 105cells ) were orthotopically xenografted to cerebellum of 6 - old Athymic Nude - Foxn1nu mice , which were continuously fed with either dox - containing chow ( red ) or regular chow food ( green ) , respectively . Micexenografted with dox - preconditioned Daoy - SOX9 / T - FBW7α fed continuously with dox - containing chow ( red ) survived longer ( p = 0 . 0285 ) than control group ( green )

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(A) Overall survival analysis of mice bearing Daoy cell line expressing SOX9 with doxycycline (dox) -inducible FBW7α ( Daoy-SOX9/T-FBW7α). The dox-preconditioned or the control cells (105cells) were orthotopically xenografted to the cerebellum of 6-weeks old Athymic Nude-Foxn1nu mice, which were continuously fed with either the dox-containing chow (red line) or the regular chow food (green line), respectively. Micexenografted with dox-preconditioned Daoy-SOX9/T-FBW7α and fed continuously with dox-containing chow (red line) survived longer (p= 0.0285) than the control group (green line)


( B ) Representative of Haemotoxylin Eosin ( Hamp ; amp ; E ) of medulloblastoma tumors arising from orthotopic xenograft experiment described in ( A ) . Both control ( no dox - treated ) dox - treated mice developed intracerebellar tumors ( green red arrows , respectively ) . SOX9 - expressing Daoy tumors disseminated to spinal cord ( 3 / 4 ; green asterisks ) , while group continuously receiving dox - containing chow showed no metastatic spread ( 0 / 4 ) .

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(B) Representative of Haemotoxylin Eosin (Hamp;amp;E) staining of medulloblastoma tumors arising from the orthotopic xenograft experiment described in (A). Both the control (no dox-treated) and the dox-treated mice developed primary intracerebellar tumors (green and red arrows, respectively). SOX9-expressing Daoy tumors disseminated to the spinal cord (3/4; green asterisks), while the group continuously receiving dox-containing chow showed no metastatic spread (0/4).


( C ) Representative bright field of migrating Daoy - SOX9 / T - FBW7α in absence or presence of Dox . Daoy - SOX9 / T - FBW7α were either maintain in regular culture or pre - conditioned with dox prior to experiments . ( 2 x 105cells / insert ) were seeded let to migrate across 5 μm filter pore toward laminin - coated trans - well for 4 hours prior to fixation with crystal violet . Histogram of migration were performed by measuring crystal violet absorbance of stained - migrating presented as mean + standard deviation from 2 independent experiments each containing 2 technical replicates .

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(C) Representative bright field images of migrating Daoy-SOX9/T-FBW7α in the absence or presence of Dox. Daoy-SOX9/T-FBW7α cells were either maintain in regular culture or pre-conditioned with dox prior to experiments. The cells (2 x 105cells / insert) were seeded and let to migrate across 5 μm filter pore toward the laminin-coated trans-well for 4 hours prior to fixation and staining with crystal violet. Histogram quantification of cell migration were performed by measuring the crystal violet absorbance of the stained-migrating cells and presented as mean + standard deviation from 2 independent experiments each containing 2 technical replicates.


( D ) Representative H & amp ; amp ; E of ( arrow ) disseminated tumors ( asterisk ) arising from orthotopic xenograft of parental SOX9 - expressing medulloblastoma initiating ( MIC MIC - Sox9 , respectively ) . of metastatic tumors arising from both of MIC ( n = 16 ) MIC - SOX9 ( n = 8 ) is shown in histogram ( p = 0 . 0000136 ) .

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(D) Representative H&amp;E staining of primary (arrow) and disseminated tumors (asterisk) arising from orthotopic xenograft of parental and SOX9-expressing medulloblastoma initiating cells (MIC and MIC-Sox9, respectively). Quantification of primary and metastatic tumors arising from both of MIC (n=16) and MIC-SOX9 (n=8) is shown in the histogram (p=0.0000136).


( E ) Quantitative comparison of MIC MIC - SOX9 to migrate across 5 μm filter pore toward laminin - coated trans - well for 4 hours . of migration were performed as described in ( C ) presented as mean + standard error mean from 6 replicates .

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(E) Quantitative comparison of MIC and MIC-SOX9 cells to migrate across 5 μm filter pore toward the laminin-coated trans-well for 4 hours. Quantification of cell migration were performed as described in (C) and presented as mean + standard error mean from 6 replicates.


( F ) Transwell migration of SOX9 - expressing MIC with dox - inducible FBW7α or dominant negative ∆ F FBW7α . were either maintain in regular culture or pre - conditioned with dox prior to assessment of in vitro transwell migration as described in ( C ) . Data are mean + standard deviation from 2 independent experiments each containing 2 technical replicates .

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(F) Transwell migration of SOX9-expressing MIC with dox-inducible FBW7α or dominant negative ∆F FBW7α. The cells were either maintain in regular culture or pre-conditioned with dox prior to assessment of in vitro transwell migration as described in (C). Data are mean + standard deviation from 2 independent experiments each containing 2 technical replicates.


( G ) Representative bright field of ( F )

(G) Representative bright field images of (F)


( H ) Correlation between clinical metastatic staging SOX9 across TMA of 142 cases of human medulloblastoma . SOX9 were determined by in Figure 3F . Tumors with SOX9 level frequently presented ( p = 0 . 038 , Fisher ' s exact , ) with M3 metastatic staging , spread to spinal cord ( red bars ) . was carried out by comparing with high SOX9 rank ( Rank 5 - 7 ) versus with low SOX9 rank ( Rank 1 - 3 ) .

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(H) Correlation between clinical metastatic staging and SOX9 protein across the TMA of 142 cases of human medulloblastoma. The SOX9 protein levels were determined by IHC in Figure 3F. Tumors with SOX9 protein level frequently presented (p=0.038, Fisher's exact test,) with the M3 metastatic staging, spread to the spinal cord (red bars). Analysis was carried out by comparing patients with high SOX9 protein rank (Rank 5-7) versus patients with low SOX9 protein rank (Rank 1-3).


( A ) Scatter plot depicting relationship between transcriptional changes measured as log2 FC between EV to SOX9 - WT ( x - axis ) EV to SOX9 - T236 / T240A ( y - axis ) . Each dot represents one transcript , with colored dots indicating transcripts upregulated ( log2 FC > 0 . 585 ; p < 0 . 05 ; red dots ) or downregulated ( log2 FC < - 0 . 585 ; p < 0 . 05 ; blue dots ) in both comparisons . transcript corresponding to SOX9 gene is highlighted as a red filled dot . Indicated r - value indicates Spearman ' s rank correlation coefficient between two FC profiles .

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(A) Scatter plot depicting the relationship between transcriptional changes measured as log2 FC between EV to SOX9-WT (x-axis) and EV to SOX9-T236/T240A (y-axis). Each dot represents one transcript, with colored dots indicating transcripts upregulated (log2 FC>0.585; p<0.05; red dots) or downregulated (log2 FC<-0.585; p<0.05; blue dots) in both comparisons. The transcript corresponding to the SOX9 gene is highlighted as a red filled dot. Indicated r-value indicates Spearman's rank correlation coefficient between the two FC profiles.


( B ) Box - - Whisker plot displaying sample specific expression of three FBW7 isoforms ; FBW7α ( NM _ 033632 ) , FBW7β ( NM _ 018315 ) FBW7γ ( NM _ 001013415 ' ) . Each box represents expression distribution over three replicates .

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(B) Box-and-Whisker plot displaying the sample specific expression of three FBW7 isoforms; FBW7α (NM_033632), FBW7β (NM_018315) and FBW7γ(NM_001013415'). Each box represents the expression distribution over three replicates.


( D ) Heat map of row - wise z - scores of 16 EMT hallmark genes in SOX9 - WT - T236 / T240A samples . Heat map was generated using GenePattern software ( Reich et al . , 2006 ) .

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(D) Heat map of the row-wise z-scores of 16 EMT hallmark genes in SOX9-WT and -T236/T240A samples. Heat map was generated using the GenePattern software (Reich et al., 2006).


( E ) Quantitative PCR ( qPCR ) of SNAI2 ( SLUG ) , VIM POU3F1 ( OCT6 ) ( normalized to GAPDH ) in MB002 stably transduced with EV ( set to 1 ) , SOX9 - WT SOX9 - T236 / 240A . were induced for 8 or 24 hours with doxycycline .

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(E) Quantitative PCR (qPCR) of SNAI2 (SLUG), VIM and POU3F1 (OCT6) (normalized to GAPDH levels) in MB002 cells stably transduced with EV (set to 1), SOX9-WT and SOX9-T236/240A. Cells were induced for 8 or 24 hours with doxycycline.


( F ) Western blot of SOX9 , HA - SOX9 , pSOX9 - T236 , SNAI2 ( SLUG ) , VIM POU3F1 ( OCT6 ) in MB002 stably transduced with EV , SOX9 - WT SOX9 - T236 / 240A . were induced for 24 48 hours with doxycycline GAPDH were used as a loading control .

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(F) Western blot of SOX9, HA-SOX9, pSOX9-T236, SNAI2 (SLUG), VIM and POU3F1 (OCT6) in MB002 cells stably transduced with EV, SOX9-WT and SOX9-T236/240A. Cells were induced for 24 and 48 hours with doxycycline and GAPDH were used as a loading control.


( G ) Heat map of row - wise z - scores of 14 pro - metastasis genes differentially expressed between SOX9 - WT - T236 / T240A samples previously identified as SOX9 target genes ( Kadaja et al . , 2014 , Larsimont et al . , 2015 , Oh et al . , 2014 ) . Heat map was generated using GenePattern software ( Reich et al . , 2006 ) .

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(G) Heat map of the row-wise z-scores of 14 pro-metastasis genes differentially expressed between SOX9-WT and -T236/T240A samples and previously identified as SOX9 target genes (Kadaja et al., 2014, Larsimont et al., 2015, Oh et al., 2014). Heat map was generated using the GenePattern software (Reich et al., 2006).


( A ) Cisplatin dose response curves of ( i ) MB002 , ( ii ) Daoy , ( iii ) MIC in absence ( EV ) or presence of SOX9 by Alamar blue . were pre - conditioned with doxycycline to induce expression of SOX9 ( or EV ) prior to treatment with increasing concentrations of cisplatin . IC50 were calculated following 5 ( MB002 MIC ) or 3 days ( Daoy ) of treatment . Data are mean + standard deviation from 3 independent repeats , each containing 5 technical replicates .

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(A) Cisplatin dose response curves of (i) MB002, (ii) Daoy, and (iii) MIC in the absence (EV) or presence of SOX9 by Alamar blue. Cells were pre-conditioned with doxycycline to induce expression of SOX9 (or EV) prior to treatment with increasing concentrations of cisplatin. The IC50 were calculated following 5 (MB002 and MIC) or 3 days (Daoy) of treatment. Data are mean + standard deviation from 3 independent repeats, each containing 5 technical replicates.


( B ) Cisplatin dose response curves of SOX9 - expressing ( i ) Daoy ( ii ) MIC in absence or presence of FBW7α . Experiments data were performed as described in ( A )

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(B) Cisplatin dose response curves of SOX9-expressing (i) Daoy and (ii) MIC in the absence or presence of FBW7α. Experiments and data analysis were performed as described in (A)


( C ) Overall survival of mice bearing Daoy or Daoy - expressing dox - inducible SOX9 treated with cisplatin . dox - preconditioned ( 105 ) were orthotopically xenografted to Nude - Foxn1nu mice left for 1 week to prior to being treated with vehicle control or cisplatin ( 2mg / kg ) intraperitoneally for every other day for a total of 6 doses .

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(C) Overall survival analysis of mice bearing Daoy or Daoy-expressing dox-inducible SOX9 treated with cisplatin. The dox-preconditioned cells (105 cells) were orthotopically xenografted to Nude-Foxn1nu mice and left for 1 week to prior to being treated with vehicle control or cisplatin (2mg/kg) intraperitoneally for every other day for a total of 6 doses.


( D ) Heat map of row - wise z - scores of 11 genes associated with cisplatin resistance in MB002 expressing Sox9 - WT or Sox9 - T236 / T240A . Heat map was generated using GenePattern software .

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(D) Heat map of the row-wise z-scores of 11 genes associated with cisplatin resistance in MB002 expressing Sox9-WT or Sox9-T236/T240A. Heat map was generated using the GenePattern software.


( E ) Quantitative of ATP7A , DUSP2 , TTK mRNAs in MB002 following expression of SOX9 - WT or SOX9 - T236 / 240A . Total were collected 24 hours following doxycycline treatment , from which cDNA were generated for qPCR . Data are mean mRNA level ( normalized to B2M transcript ) + standard deviation from 3 independent experiments with statistical significance were determined by Multiple comparisons 2 - way ANOVA with Bonferroni ' s post - .

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(E) Quantitative analysis of ATP7A, DUSP2, and TTK mRNAs in MB002 following expression of SOX9-WT or SOX9-T236/240A. Total RNA were collected 24 hours following doxycycline treatment, from which cDNA were generated for qPCR. Data are mean mRNA level (normalized to B2M transcript) + standard deviation from 3 independent experiments with statistical significance were determined by Multiple comparisons 2-way ANOVA with Bonferroni's post-test.


( F ) Time course western blotting of HA - SOX9 , ATP7A , DUSP2 , ERK1 / 2 pThr202 / Tyr204 total ERK1 / 2 in MB002 following doxycycline induction of either EV , SOX9 - WT or SOX9 - T236 / 240A . GAPDH was used as a loading control .

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(F) Time course western blotting of HA-SOX9, ATP7A, DUSP2, ERK1/2 pThr202/Tyr204 and total ERK1/2 in MB002 cells following doxycycline induction of either EV, SOX9-WT or SOX9-T236/240A. GAPDH was used as a loading control.


( A ) Western blotting of pT236 - total SOX9 following treatment of Daoy with a panel of inhibitors targeting PI3K / AKT / mTOR pathway . whole lysates were collected for gel electrophoresis following 6 hours treatment with 1 μM of AZD5363 , 8186 , or 2014 . set of blots shown is a representative from 3 independent repeats .

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(A) Western blotting of pT236- and total SOX9 following treatment of Daoy with a panel of inhibitors targeting PI3K/AKT/mTOR pathway. The whole cell lysates were collected for gel electrophoresis following 6 hours treatment with 1 μM of AZD5363, 8186, or 2014. The set of blots shown is a representative from 3 independent repeats.


( B ) Western blotting of endogenous SOX9 turnover in presence of PI3K / mTOR dual inhibitor , AZD2014 . Daoy were transfected with either siScr or siFBW7 for 48 hours prior to further treatment with 1 µM AZD2014 . cycloheximide chase of SOX9 turnover was examined 2 hours following inhibitor treatment . Immunobloting of Cyclin E was used to indicate efficacy of siFBW7 transfection . Changes in SOX9 level in representative blots were analysed relative to GAPDH using ImageJ . set of blots shown is representative from 3 independent experiments .

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(B) Western blotting of endogenous SOX9 protein turnover in the presence of PI3K/mTOR dual inhibitor, AZD2014. Daoy were transfected with either siScr or siFBW7 for 48 hours prior to further treatment with 1 µM AZD2014. The cycloheximide chase of SOX9 protein turnover was examined 2 hours following the inhibitor treatment. Immunobloting of Cyclin E protein was used to indicate the efficacy of siFBW7 transfection. Changes in SOX9 protein level in the representative blots were analysed relative to GAPDH using ImageJ. The set of blots shown is representative from 3 independent experiments.


( C ) of resazurin - Daoy MB002 viability following treatment with Cisplatin ( blue bars ) , AZD2014 ( red bars ) , or their combination ( green bars ) . Daoy were treated for 72 hours , while MB002 were subjected to 5 day course of treatment . cellviability were calculated relative to vehicle - treated . synthetic lethality combination index ( CI ) for each treatment was calculated from mean of 3 independent experiments using Compusyn software . Error bars indicate standard deviation .

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(C) Quantification of resazurin-based Daoy and MB002 cell viability following treatment with Cisplatin (blue bars), AZD2014 (red bars), or their combination (green bars). Daoy were treated for 72 hours, while MB002 were subjected to 5 day course of treatment. The cellviability were calculated relative to the vehicle-treated cells. The synthetic lethality combination index (CI) for each treatment was calculated from the mean of 3 independent experiments using the Compusyn software. Error bars indicate standard deviation.


( D ) Time course western blotting of endogenous SOX9 apoptotic markers including PARP caspase 3 following MB002 treatment with cisplatin , AZD2014 , or their combination . β - actin were used as a loading control . set of blots shown is representative from 3 independent experiments .

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(D) Time course western blotting of endogenous SOX9 and apoptotic markers including cleaved PARP and caspase 3 following MB002 treatment with cisplatin, AZD2014, or their combination. β-actin were used as a loading control. The set of blots shown is representative from 3 independent experiments.


( E ) Western blotting profiling of MB002 expressing EV , SOX9 - WT , or SOX9 - T236 / 240A following 48 hours treatment with cisplatin , AZD2014 , their combination . set of blots shown is representative from 3 independent experiments .

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(E) Western blotting profiling of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 48 hours treatment with cisplatin, AZD2014, and their combination. The set of blots shown is representative from 3 independent experiments.


( F ) of viability of MB002 expressing EV , SOX9 - WT , or SOX9 - T236 / 240A following 5 days treatment with either of 10 μM cisplatin ( black bars ) , 0 . 5 µM AZD2014 ( blue bars ) , or their combination ( red bars ) . Data are mean + standard deviation from 3 independent experiments , from which statistical significance were analysed by 2 - way ANOVA multiple comparisons with Bonferroni ' s post - .

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(F) Quantification of cell viability of MB002 expressing EV, SOX9-WT, or SOX9-T236/240A following 5 days treatment with either of 10 μM cisplatin (black bars), 0.5 µM AZD2014 (blue bars), or their combination (red bars). Data are mean + standard deviation from 3 independent experiments, from which statistical significance were analysed by 2-way ANOVA multiple comparisons with Bonferroni's post-test.


( G ) Immunoblots of ATP7A DUSP2 following doxycycline treatment of Daoy - SOX9 / T - FBW7α . were either untreated or incubated with 5 µg / mL doxycycline for 72 hours prior to being harvested for gel electrophoresis . set of blots shown is representative from 2 independent experiments .

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(G) Immunoblots of ATP7A and DUSP2 protein following doxycycline treatment of Daoy-SOX9/T-FBW7α. The cells were either untreated or incubated with 5 µg/mL doxycycline for 72 hours prior to being harvested for gel electrophoresis. The set of blots shown is representative from 2 independent experiments.


( A ) HeLa expressing GFP ‐ tagged UBQLN1 or UBQLN2 ( or GFP for controls ) were transferred to starvation medium ( PBS ) for indicated time . Where specified , only serum was removed from culture medium . Viability of transfected was assessed by PI analysed by flow cytometry . graph represents mean of three experiments ± s . e .

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(A) HeLa cells expressing GFP‐tagged UBQLN1 or UBQLN2 (or GFP for controls) were transferred to starvation medium (PBS) for the indicated time. Where specified, only serum was removed from the culture medium. Viability of transfected cells was assessed by PI staining and analysed by flow cytometry. The graph represents the mean of three experiments±s.e.


( B ) HeLa were depleted of UBQLN1 or UBQLN2 by siRNA transfection ; depletion was verified by Western blot . were starved for increasing periods of time , their viability was analysed as in ( A ) . graph represents mean of three experiments ± s . e ; * P0 . 05 between control UBQLN2 ‐ KD at 6 h ( paired Student ' s t ‐ ) . GAPDH , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ; GFP , green ; KD , knockdown ; PI , propidium iodide ; siRNA , short interfering ; UBQLN , ubiquilin .

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(B) HeLa cells were depleted of UBQLN1 or UBQLN2 by siRNA transfection; depletion was verified by Western blot. Cells were starved for increasing periods of time, and their viability was analysed as in (A). The graph represents the mean of three experiments±s.e; *P0.05 between control and UBQLN2‐KD at 6 h (paired Student's t‐test). GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; GFP, green fluorescent protein; KD, knockdown; PI, propidium iodide; siRNA, short interfering RNA; UBQLN, ubiquilin.


( A ) HeLa were transfected with siRNAs for autophagy - related gene ATG5 ( siATG5 ) or with a non - silencing siRNA ( siC ) . Depletion of ATG5 , which decreased of ATG5 - ATG12 covalent , was confirmed by Western blot . Loading control : transferrin receptor ( TfR ) .

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(A) HeLa cells were transfected with siRNAs for autophagy-related gene ATG5 (siATG5) or with a non-silencing siRNA (siC). Depletion of ATG5, which decreased the levels of the ATG5-ATG12 covalent complex, was confirmed by Western blot. Loading control: transferrin receptor (TfR).


( B ) Control or ATG5 ‐ KD transfected with UBQLN1 were starved , viability was assessed as in Fig 1 ; graph represents mean of three experiments ± s . e .

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(B) Control or ATG5‐KD cells transfected with UBQLN1 were starved, and the cell viability was assessed as in Fig 1; the graph represents the mean of three experiments±s.e.


( C ) HeLa were stably transfected with control ( shC ) or ATG7 ( shATG7 ) shRNA ; depletion was verified by Western blot ( loading control : GAPDH ) .

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(C) HeLa cells were stably transfected with control (shC) or ATG7 (shATG7) shRNA; depletion was verified by Western blot (loading control: GAPDH).


( D ) Control or ATG7 ‐ KD transfected with UBQLN1 were starved for 3 or 6 h assessed for their viability . graph represents mean of three experiments ± s . e . GAPDH , glyceraldehyde ‐ 3 ‐ phosphate dehydrogenase ; KD , knockdown ; Pl , propidium iodide ; shRNA , short hairpin ; siRNA , short interfering ; UBQLN , ubiquilin .

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(D) Control or ATG7‐KD cells transfected with UBQLN1 were starved for 3 or 6 h and assessed for their viability. The graph represents the mean of three experiments±s.e. GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; KD, knockdown; Pl, propidium iodide; shRNA, short hairpin RNA; siRNA, short interfering RNA; UBQLN, ubiquilin.


( A ) Immunolocalization of transfected Myc ‐ UBQLN ( 1 or 2 ) GFP ‐ tagged microtubule ‐ associated 1 chain 3 ( GFP ‐ LC3 ) was analysed by microscopy . Inset : magnification of UBQLN1 vesicular ( arrow ) , which , after acquisition of Z ‐ stacks , was found in UBQLN structures analysed .

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(A) Immunolocalization of transfected Myc‐UBQLN (1 or 2) and GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) was analysed by microscopy. Inset: magnification of UBQLN1 vesicular staining (arrow), which, after acquisition of Z‐stacks, was found in all UBQLN structures analysed.


( B ) were starved for 2 h , with ( top row ) or without BAF A1 ( bottom row ) . were stained for endogenous UBQLN2 ( magenta ) LC3 ( green ) .

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(B) Cells were starved for 2 h, with (top row) or without BAF A1 (bottom row). Cells were stained for endogenous UBQLN2 (magenta) and LC3 (green).


( C ) Control ( untransfected , right panel ) or overexpressing Myc ‐ UBQLN2 GFP ‐ LC3 ( left panel ) were transferred into starvation medium for 2 h . Localization of Myc ‐ UBQLN2 GFP ‐ LC3 was analysed by immunoelectron microscopy using Myc ( arrow ) GFP ( arrowhead ) antibodies coupled to 10 15 nm gold particles , respectively . Double ‐ vacuoles ( autophagosomes ) in transfected ( left panel ) show increased by both antibodies compared with autophagosomes in untransfected ( right panel ) . Scale bar , 0 . 5 μm . BAF A , bafilomycin A1 ; GFP , green ; UBQLN , ubiquilin .

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(C) Control cells (untransfected, right panel) or cells overexpressing Myc‐UBQLN2 and GFP‐LC3 (left panel) were transferred into starvation medium for 2 h. Localization of Myc‐UBQLN2 and GFP‐LC3 was analysed by immunoelectron microscopy using Myc (arrow) and GFP (arrowhead) antibodies coupled to 10 and 15 nm gold particles, respectively. Double‐membrane vacuoles (autophagosomes) in transfected cells (left panel) show increased staining by both antibodies compared with autophagosomes in untransfected cells (right panel). Scale bar, 0.5 μm. BAF A, bafilomycin A1; GFP, green fluorescent protein; UBQLN, ubiquilin.


( B ) Each mutant was co ‐ transfected with GFP ‐ tagged microtubule ‐ associated 1 chain 3 ( GFP ‐ LC3 ) localization of each was analysed by microscopy .

(B) Each mutant was co‐transfected with GFP‐tagged microtubule‐associated protein 1 light chain 3 (GFP‐LC3) and the localization of each protein was analysed by microscopy.


( C ) Starvation ‐ induced was assessed in expressing UBQLN1 or UBQLN1ΔUBA . graph represents mean of three experiments ± s . e . , with * P ⩽ 0 . 05 ( P = 0 . 0017 by one ‐ way ANOVA , comparing control , UBQLN1 UBQLN1ΔUBA ) . No difference was found between control UBQLN1ΔUBA at any time point . ANOVA , of variance ; GFP , green ; Pl , propidium iodide ; UBA , ubiquitin ‐ associated ; UBQLN , ubiquilin .

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(C) Starvation‐induced cell death was assessed in cells expressing UBQLN1 or UBQLN1ΔUBA. The graph represents the mean of three experiments±s.e., with *P⩽0.05 (P=0.0017 by one‐way ANOVA test, comparing control, UBQLN1 and UBQLN1ΔUBA). No difference was found between control and UBQLN1ΔUBA at any time point. ANOVA, analysis of variance; GFP, green fluorescent protein; Pl, propidium iodide; UBA, ubiquitin‐associated; UBQLN, ubiquilin.


( A ) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN ‐ or autophagy ‐ related gene ATG5 ‐ KD expressing GFP ‐ tagged microtubule associated 1 chain 3 ( GFP ‐ LC3 ) .

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(A) Autophagosome subcellular localization during starvation was analysed by microscopy in UBQLN‐ or autophagy‐related gene ATG5‐KD cells expressing GFP‐tagged microtubule associated protein 1 light chain 3 (GFP‐LC3).


( B ) Autophagosome spatial distribution was quantified by measuring of each LC3 vesicle from nucleus ( in pixels ) by analysing frequency distribution .

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(B) Autophagosome spatial distribution was quantified by measuring the distance of each LC3 vesicle from the nucleus (in pixels) and by analysing the frequency distribution.


( C ) Control or UBQLN ‐ KD expressing GFP ‐ LC3 were starved stained with mitochondria dye Mitotracker ™ . Dual of LC3 ( green ) with Mitotracker ( magenta ) was analysed by microscopy .

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(C) Control or UBQLN‐KD cells expressing GFP‐LC3 were starved and stained with the mitochondria dye Mitotracker™. Dual staining of LC3 (green) with Mitotracker (magenta) was analysed by microscopy.


( D ) Control UBQLN ‐ KD stained with Mitotracker were starved for 6 h ; BAF A was added where indicated . Mitotracker was measured by flow cytometry . Unless specified otherwise , values for UBQLN ‐ KD control + BAF A were compared with that of controls at same time points , with * * * P0 . 001 , * * P0 . 01 * P0 . 05 ( paired Student ' s t ‐ ) .

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(D) Control and UBQLN‐KD cells stained with Mitotracker were starved for 6 h; BAF A was added where indicated. Mitotracker fluorescence was measured by flow cytometry. Unless specified otherwise, values for UBQLN‐KD and control+BAF A were compared with that of controls at the same time points, with ***P0.001, **P0.01 and *P0.05 (paired Student's t‐test).


( E ) were transfected with pShooter ™ together with control or UBQLN ( 1 + 2 ) siRNA . Expression of pShooter was comparable in control UBQLN ‐ KD ( top ) . were starved for 3 or 6 h , of GFP was quantified by flow cytometry ( bottom ) . value obtained with controls ( no starvation ) was considered as 100 % , value obtained for 3 ‐ 6 ‐ h time points was normalized to controls . graph represents mean ± s . e . of four assessments .

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(E) Cells were transfected with pShooter™ together with control or UBQLN (1+2) siRNA. Expression of pShooter was comparable in control and UBQLN‐KD cells (top). Cells were starved for 3 or 6 h, and the intensity of GFP was quantified by flow cytometry (bottom). The fluorescence value obtained with controls (no starvation) was considered as 100%, and the value obtained for the 3‐ and 6‐h time points was normalized to controls. The graph represents the mean±s.e. of four assessments.


( F ) expressing a chimaeric LC3 fused to both GFP mCherry were transfected with control or UBQLN siRNA , starvation was induced for indicated times . from 25 were scored analysed by microscopy for their colour . graph represents mean of three experiments ± s . e . number of mCherry single ‐ positive in UBQLN ‐ KD was compared with that of controls for each time point ; * * P0 . 01 * P0 . 05 ( paired Student ' s t ‐ ) .

(F) Cells expressing a chimaeric LC3 fused to both GFP and mCherry were transfected with control or UBQLN siRNA, and starvation was induced for the indicated times. Puncta from 25 cells were scored and analysed by microscopy for their fluorescence colour. The graph represents the mean of three experiments±s.e. The number of mCherry single‐positive puncta in UBQLN‐KD cells was compared with that of controls for each time point; **P0.01 and *P0.05 (paired Student's t‐test).


( G ) Control or UBQLN ‐ KD were starved for increasing periods . Where indicated , BAF A was added to starvation medium ( + B ) . lysates were analysed by Western blot for their content in LC3 ‐ II . Loading control : transferrin receptor ( TfR ) . BAF A , bafilomycin A1 ; GFP , green ; KD , knockdown ; siRNA , short interfering ; UBQLN , ubiquilin .

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(G) Control or UBQLN‐KD cells were starved for increasing periods. Where indicated, BAF A was added to the starvation medium (+B). Cell lysates were analysed by Western blot for their content in LC3‐II. Loading control: transferrin receptor (TfR). BAF A, bafilomycin A1; GFP, green fluorescent protein; KD, knockdown; siRNA, short interfering RNA; UBQLN, ubiquilin.


( I ) Representative high - resolution confocal of C . elegans expressing mitochondria - targeted GFP in wall muscle ( scale bar = 10 μm ) . of mitochondrial ( elongation ) is reported in right panel ( one - way ANOVA with Holm - Sidak ' s correction , n = 37 - 47 from 3 independent experiments ) . Data information : , middle represents median , upper lower lines represent upper lower quartiles , respectively . Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(I) Representative high-resolution confocal images of C. elegans expressing mitochondria-targeted GFP in the body wall muscle cells (scale bar= 10 μm). Quantification of mitochondrial morphology (elongation) is reported in the right panel (one-way ANOVA with Holm-Sidak's correction, n=37-47 animals from 3 independent experiments). Data information: , middle line represents the median, and the upper and lower lines represent the upper and lower quartiles, respectively. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( J ) Representative western blot densitometry of zcIs13 [ hsp - 6p : : GFP ] expression in control , - 1 ( fc21 ) - 1 ( fc21 ) ; cest - 2 . 2 O / E ( one - way ANOVA with Holm - Sidak ' s correction , n = 3 biological replicates ) . Data information : , bars represent mean ± Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(J) Representative western blot analysis and densitometry of zcIs13[hsp-6p::GFP] expression in control, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E animals (one-way ANOVA with Holm-Sidak's correction, n=3 biological replicates). Data information: , bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( M ) Immunoblot statistical of zcIs13 [ hsp - 6p : : GFP ] expression in control nuo - 6 ( qm200 ) fed with empty vector ( C RNAi ) cest - 2 . 2 RNAi ( unpaired t - , n = 3 biological replicates ) . Data information : bars represent mean ± Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(M) Immunoblot and statistical analysis of zcIs13[hsp-6p::GFP] expression in control and nuo-6(qm200) animals fed with empty vector (C RNAi) and cest-2.2 RNAi bacteria (unpaired t-test, n=3 biological replicates). Data information: bars represent mean± SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( A ) Representative of 5 - day - old wt , - 1 ( fc21 ) - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant stained with Oil Red O ( ORO ) ( scale bar = full image - 100 μm ; hindgut magnification - 20 μm ) . ( B ) of hindgut ( left panel ) , foregut ( center panel ) whole ( right panel ) of 5 - day - old wt , - 1 ( fc21 ) - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes stained with ORO ( one - way ANOVA with Tukey ' s multiple comparison , n = 18 - 26 ) . ( C ) of mean of ORO in hindgut of wt , - 1 ( fc21 ) - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes at different time points , starting from L4 stage ( one - way ANOVA with Dunnett ' s post hoc correction , n = 18 - 26 ) . ( D ) Mean of ORO in whole of 5 - day - old wt , - 1 ( fc21 ) - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes plotted against . Data information : , bars represent mean ± points represent mean ± . Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(A) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with Oil Red O (ORO) (scale bar= full image-100 μm; hindgut magnification-20 μm). (B) Quantification of hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes stained with ORO (one-way ANOVA with Tukey's multiple comparison test, n=18-26 animals). (C) Quantification of mean intensity of ORO staining in the hindgut of wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes at different time points, starting from L4 stage (one-way ANOVA with Dunnett's post hoc correction, n=18-26 animals). (D) Mean intensity of ORO staining in the whole body of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes plotted against body area. Data information: , bars represent mean ±SEM points represent mean ±SEM. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.