D . Rescue experiments of HeLaVPS41KO . Expression of VPS41WT - APEX2 - V5 , VPS41S285P - APEX2 - V5 or VPS41R662 * - APEX2 - V5 . Reintroduction of VPS41WT rescues TFE3 localization , whereas expression of mutant VPS41 has no effect ( quantified in D ' ) . > 83 per condition were quantified ( n = 3 ) . Scale bars , 10µm .

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

D. Rescue experiments of HeLaVPS41KO cells. Expression of VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5. Reintroduction of VPS41WT rescues TFE3 localization, whereas expression of mutant VPS41 has no effect (quantified in D'). >83 Cells per condition were quantified (n=3). Scale bars, 10µm.


B . Western blot on or secreted secretogranin II ( SgII ) of PC12 VPS41KO , or VPS41KO transduced with HA - VPS41WT or HA - VPS41S285P lentivirus . were washed incubated for 30 minutes in Tyrode ' s solution containing 2 . 5 mM K + ( basal ) or 90 mM K + ( stimulated ) . No difference in secreted sgII was observed between VPS41WT VPS41S285P .

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B. Western blot on cellular or secreted secretogranin II (SgII) of PC12 cells VPS41KO, or VPS41KO transduced with HA-VPS41WT or HA-VPS41S285P lentivirus. Cells were washed and incubated for 30 minutes in Tyrode's solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P.


C . Graph representing of with 6 normal dopaminergic neurons in anterior region of Pdat - 1 : : GFP ; Pdat - 1 : : α - syn ( UA44 ) with heterozygous expression of hVPS41 variants . Heterozygous expression of hVPS41WT with either variant ( strains UA389 or UA390 ) significantly rescues neurons from α - synuclein - induced degeneration at day 7 whereas compound heterozygous expression ( UA391 ) fails to rescue neurodegeneration . At day 10 , none of heterozygous backgrounds significantly rescues α - synuclein - induced neurodegeneration . n = 30 Adult worms for each of 3 independent experiments for α - synuclein ( α - syn ) ( total of 90 worms ) n = 90 for each independent transgenic ( 30 worms / trial x 3 independent transgenic lines = 270 worms ) for each of 3 independent experiments . D . Representative of DA neurons from C . elegans expressing Pdat - 1 : : GFP ; Pdat - 1 : : α - syn , with or without hVPS41 variants , as described in ( C ) . Intact DA neurons are indicated with arrowheads missing neurons are indicated with arrows . Scale bar 20µm .

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C. Graph representing the percentage of animals with 6 normal dopaminergic neurons in the anterior region of Pdat-1::GFP; Pdat-1:: α-syn (strain UA44) animals with heterozygous expression of hVPS41 variants. Heterozygous expression of hVPS41WT with either variant (strains UA389 or UA390) significantly rescues neurons from α-synuclein-induced degeneration at day 7 whereas compound heterozygous expression (strain UA391) fails to rescue neurodegeneration. At day 10, none of the heterozygous backgrounds significantly rescues α-synuclein-induced neurodegeneration. n=30 Adult worms for each of 3 independent experiments for the α-synuclein (α-syn) strain (total of 90 worms) and n=90 for each independent transgenic strain (30 worms/trial x 3 independent transgenic lines = 270 worms) for each of 3 independent experiments. D. Representative images of DA neurons from C. elegans expressing Pdat-1::GFP; Pdat-1:: α-syn, with or without hVPS41 variants, as described in (C). Intact DA neurons are indicated with arrowheads and missing neurons are indicated with arrows. Scale bar 20µm.


A Gene targeting strategy . Left : max genomic locus , targeting construct max targeted locus . In vitro Flipase ( Flip ) deletion of selectable markers lacZ neo results in a max targeted locus with exons 4 5 ( coding for bHLHLZip domain ) flanked by two loxP sites ( fl , flox allele ) . Arrows represent PCR primers used for genotyping MaxF9 MaxF10 . Right : genomic PCR of tail from F1 mice . PCR products of max wt ( 430 bp ) max flox ( 526 bp ) alleles . M , 1kb ladder .

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A Gene targeting strategy. Left: max genomic locus, targeting construct and max targeted locus. In vitro Flipase (Flip) deletion of the selectable markers lacZ and neo results in a max targeted locus with exons 4 and 5 (coding for bHLHLZip domain) flanked by two loxP sites (fl, flox allele). Arrows represent PCR primers used for genotyping MaxF9 and MaxF10. Right: genomic PCR analysis of tail DNA from F1 mice. PCR products of max wt (430 bp) and max flox (526 bp) alleles. M, 1kb ladder.


Flow cytometry analyses of B populations ( GFP + ) in Bone Marrow ( BM ) spleen . Single - suspensions were prepared from BM or spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against indicated markers , analyzed by flow cytometry . For BM , were isolated from femora tibiae . Pro - B ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B ( Ly - 6c - CD49b - B220 + IgM - CD43 - ) , immature B ( Ly - 6c - CD49b - B220loIgM + CD43 - ) , mature B ( Ly - 6c - CD49b - B220hiIgM + CD43 - ) To identify KO ( GFP + ) plots show gated GFP + B lymphocytes .

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Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) and spleen. Single-cell suspensions were prepared from BM or spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-), immature B cells (Ly-6c-CD49b-B220loIgM+CD43-), and mature B cells (Ly-6c-CD49b-B220hiIgM+CD43-) To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


Flow cytometry analyses of B populations ( GFP + ) in Bone Marrow ( BM ) Single - suspensions were prepared from BM of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against indicated markers , analyzed by flow cytometry . For BM , were isolated from femora tibiae . Pro - B ( Ly - 6c - CD49b - B220 + IgM - CD43 + ) , pre - B ( Ly - 6c - CD49b - B220 + IgM - CD43 - )

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Flow cytometry analyses of B cell populations (GFP+) in Bone Marrow (BM) Single-cell suspensions were prepared from BM of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. For BM analysis, cells were isolated from femora and tibiae. Pro-B cells (Ly-6c-CD49b-B220+IgM-CD43+), pre-B cells (Ly-6c-CD49b-B220+IgM-CD43-)


Flow cytometry analyses of B populations ( GFP + ) in spleen . Single - suspensions were prepared from spleen of MaxKO - mb1 , MycKO - mb1 , DKO - mb1 or heterozygous HET - mb1 control mice , stained with antibodies against indicated markers , analyzed by flow cytometry . spleen follicular B ( B220 + CD23 + CD21int ) , marginal zone B ( B220 + CD23 - CD21hi ) immature ( newly formed / transitional stage 1 ) B ( B220 + CD23 - CD21lo ) . To identify KO ( GFP + ) plots show gated GFP + B lymphocytes .

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Flow cytometry analyses of B cell populations (GFP+) in spleen. Single-cell suspensions were prepared from spleen of MaxKO-mb1, MycKO-mb1, DKO-mb1 or heterozygous HET-mb1 control mice, stained with antibodies against the indicated markers, and analyzed by flow cytometry. spleen follicular B cells (B220+CD23+CD21int), marginal zone B cells (B220+CD23-CD21hi) and immature (newly formed/transitional stage 1) B cells (B220+CD23-CD21lo). To identify KO cells (GFP+) all the plots show gated GFP+ B lymphocytes.


F , G , H Absolute numbers of B lymphocytes in Bone Marrow ( F , G ) spleen ( F , H ) from MaxKO - mb1 ( n = 6 ) , MycKO - mb - 1 ( n = 3 ) , DKO - mb1 ( n = 5 ) control HET - mb1 ( n = 4 ) mice . Columns represent mean values error bars standard deviations . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . different B populations were identified by flow cytometry Experiment representative of at least three independent experiments .

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F, G, H Absolute numbers of B lymphocytes in Bone Marrow (F, G) and spleen (F, H) from MaxKO-mb1 (n=6), MycKO-mb-1 (n=3), DKO-mb1 (n=5) and control HET-mb1 (n=4) mice. Columns represent mean values and error bars standard deviations. *p<0.05, **p<0.01, ***p<0.001. The different B cell populations were identified by flow cytometry Experiment representative of at least three independent experiments.


I cycle by flow cytometry of sorted GFP + pre - pro - B ( B220 + IgM - ) , immature ( B220loIgM + ) mature ( B220hiIgM + ) bone marrow B from MaxKO - mb1 , MycKO - mb1 , DKO - mb1 HET - mb1 control mice . Mice ( n = 5 ) were injected ( i . p . ) with 200 µg of EdU analyzed 4 h later . Single - suspensions were prepared , stained sorted . Sorted were subsequently stained with propidium iodide for content . Experiment representative of at least three independent experiments . Dotted is shown for EdU MFI comparison .

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I Cell cycle analysis by flow cytometry of sorted GFP+ pre- and pro-B (B220+IgM-), immature (B220loIgM+) and mature (B220hiIgM+) bone marrow B cells from MaxKO-mb1, MycKO-mb1, DKO-mb1 and HET-mb1 control mice. Mice (n=5) were injected (i.p.) with 200 µg of EdU and analyzed 4 h later. Single-cell suspensions were prepared, stained and sorted. Sorted cells were subsequently stained with propidium iodide for DNA content. Experiment representative of at least three independent experiments. Dotted line is shown for EdU MFI comparison.


J of annexin - V + GFP + in BM of MaxKO - mb1 ( n = 5 ) , MycKO - mb1 ( n = 3 ) , DKO - mb1 ( n = 3 ) control ( n = 4 ) mice . Pro - B , pre - B , immature mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E . * p < 0 . 05 , * * * p < 0 . 001 . Experiment representative of at least three independent experiments .

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J Percentage of annexin-V+GFP+ cells in the BM of MaxKO-mb1(n=5), MycKO-mb1(n=3), DKO-mb1 (n=3) and control (n=4) mice. Pro-B, pre-B, immature and mature B lymphocyte subpopulations were identified by flow cytometry as indicated in E. *p<0.05, ***p<0.001. Experiment representative of at least three independent experiments.


Flow cytometry absolute numbers of B220loCD138 + ( plasmablasts ) ( A , B ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted activated in vitro with LPS plus IL - 4 for 72 h . were stained with monoclonal antibodies against indicated markers analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values error bars standard deviations . Experiment representative of at least three independent experiments .

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Flow cytometry analysis and absolute numbers of B220loCD138+ (plasmablasts) (A, B) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.


Flow cytometry absolute numbers of B220 + IgG1 + switched B ( C , D ) from HET - cd19 ( n = 6 ) , MaxKO - cd19 ( n = 8 ) , MycKO - cd19 ( n = 4 ) , DKO - cd19 ( n = 5 ) mice . Spleen B220 + GFP + B lymphocytes were sorted activated in vitro with LPS plus IL - 4 for 72 h . were stained with monoclonal antibodies against indicated markers analyzed by flow cytometry . * p < 0 . 05 , * * p < 0 . 01 . Columns represent mean values error bars standard deviations . Experiment representative of at least three independent experiments .

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Flow cytometry analysis and absolute numbers of B220+IgG1+ switched B cells (C, D) from HET-cd19 (n=6), MaxKO-cd19 (n=8), MycKO-cd19 (n=4), and DKO-cd19 (n=5) mice. Spleen B220+GFP+ B lymphocytes were sorted and activated in vitro with LPS plus IL-4 for 72 h. Cells were stained with monoclonal antibodies against the indicated markers and analyzed by flow cytometry. *p<0.05, **p<0.01. Columns represent mean values and error bars standard deviations. Experiment representative of at least three independent experiments.


E Spleen B220 + GFP + B lymphocytes were stained with 7AAD annexin - V to monitor apoptosis . 7 - AAD - annexin - v - ( alive ) , 7 - AAD - annexin - v + ( early apoptotic ) , 7 - AAD + annexin - v + ( late apoptotic ) , 7 - AAD + annexin - v - ( necrosis ) . F Relative numbers of live , early apoptotic , late apoptotic necrotic from E .

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E Spleen B220+GFP+ B lymphocytes were stained with 7AAD and annexin-V to monitor apoptosis. 7-AAD- annexin-v- (alive), 7-AAD- annexin-v+ (early apoptotic), 7-AAD+ annexin-v+ (late apoptotic), 7-AAD+ annexin-v- (necrosis). F Relative numbers of live, early apoptotic, late apoptotic and necrotic cells from E.


G CD69 surface expression in purified B220 + GFP + mature spleen . Sorted B were activated in vitro with LPS plus IL - 4 for 72 h , stained analyzed by flow cytometry . solid grey peak represents expression of CD69 marker in non - activated mature B lymphocytes at day 0 . H Relative numbers of CD69 + B lymphocytes in cultures from G ( n = 4 ) . Experiment representative of at least three independent experiments .

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G CD69 surface expression in purified B220+GFP+ mature spleen cells. Sorted B cells were activated in vitro with LPS plus IL-4 for 72 h, stained and analyzed by flow cytometry. The solid grey peak represents the expression levels of CD69 marker in non-activated mature B lymphocytes at day 0. H Relative numbers of CD69+ B lymphocytes in the cultures from G (n=4). Experiment representative of at least three independent experiments.


I of germinal center ( GC ) formation in spleen of MaxKO - cd19 , Myc - KO - cd19 , DKO - cd19 heterozygous control mice immunized with TNP - KLH . Representative of frozen spleen sections stained with IgM ( grey / blue ) , PNA ( GC marker ; red ) , GFP ( Max - , c - Myc - or c - Myc / Max - deficient B ; green ) . Scale bar , 80µm .

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I Analysis of germinal center (GC) formation in the spleen of MaxKO-cd19, Myc-KO-cd19, DKO-cd19 and heterozygous control mice immunized with TNP-KLH. Representative images of frozen spleen sections stained with IgM (grey/blue), PNA (GC marker; red), and GFP (Max-, c-Myc- or c-Myc/Max-deficient B cells; green). Scale bar, 80µm.


J Upper , Number of GCs per mouse in spleen sections of immunized MaxKO - cd19 ( n = 3 mice , n = 186 follicles analyzed ) , MycKO - cd19 ( n = 3 mice , n = 178 follicles analyzed ) , DKO - cd19 ( n = 3 mice , n = 191 follicles analyzed ) heterozygous control mice ( n = 3 mice , n = 203 follicles analyzed ) . Lower , Frequencies of GC ( PNA + ) GFP + ( deleted ) or GFP - ( non - deleted ) in immunized MaxKO - cd19 ( n = 13 GCs ) , MycKO - cd19 ( n = 21 GCs ) , DKO - cd19 ( n = 27 GCs ) heterozygous control ( n = 62 GCs ) mice . was performed at 13 days post - immunization . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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J Upper, Number of GCs per mouse in spleen sections of immunized MaxKO-cd19 (n=3 mice, n=186 follicles analyzed), MycKO-cd19 (n=3 mice, n=178 follicles analyzed), DKO-cd19 (n=3 mice, n=191 follicles analyzed) and heterozygous control mice (n=3 mice, n=203 follicles analyzed). Lower, Frequencies of GC (PNA+) GFP+ (deleted cells) or GFP- (non-deleted cells) in immunized MaxKO-cd19 (n=13 GCs), MycKO-cd19 (n=21 GCs), DKO-cd19 (n=27 GCs) and heterozygous control (n=62 GCs) mice. Analysis was performed at 13 days post-immunization. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


A Number of divisions in activated mature B lymphocytes . Sorted B220 + GFP + spleen from HET - cd19 ( n = 5 ) , MaxKO - cd19 ( n = 5 ) , MycKO - cd19 ( n = 3 ) , DKO - cd19 ( n = 3 ) mice were stained with a tracer , activated in vitro with LPS plus IL - 4 for 72 h analyzed by flow cytometry . Experiment representative of at least three independent experiments B from cultures C of B cultures Scale bar , 500 µm . 4 × magnification . D Forward scatter ( FSC ) histograms of activated B lymphocytes An arbitrary dashed is shown for comparison .

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A Number of cell divisions in activated mature B lymphocytes. Sorted B220+GFP+ spleen cells from HET-cd19 (n=5), MaxKO-cd19 (n=5), MycKO-cd19 (n=3), and DKO-cd19 (n=3) mice were stained with a cell tracer, activated in vitro with LPS plus IL-4 for 72 h and analyzed by flow cytometry. Experiment representative of at least three independent experiments B Cell counts from the cultures C Photographs of B cell cultures Scale bar, 500 µm. 4× magnification. D Forward scatter (FSC) histograms of activated B lymphocytes An arbitrary dashed line is shown for comparison.


cycle of sorted B220 + GFP + B lymphocytes activated in vitro . were activated EdU was added 4 h before harvesting . were stained with propidium iodide to measure content . Experiment representative of at least three independent experiments . An arbitrary dashed shows difference of EdU MFI . S phase mean ( MFI ) : 7871 HET , 6512 MaxKO , 5860 MycKO , 6420

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Cell cycle analysis of sorted B220+GFP+ B lymphocytes activated in vitro. Cells were activated and EdU was added 4 h before harvesting. Cells were stained with propidium iodide to measure DNA content. Experiment representative of at least three independent experiments. An arbitrary dashed line shows the difference of EdU MFI. S phase mean fluorescence intensity (MFI): 7871 HET, 6512 MaxKO, 5860 MycKO, 6420 DKO


Representation of labeling with nucleotide analogues CldU ( red ) IdU ( green ) of from sorted B220 + GFP + B lymphocytes activated for 48 h . Scale bar , 10 µm .

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

Representation of labeling with nucleotide analogues CldU (red) and IdU (green) and photographs of DNA fibers from sorted B220+GFP+ B lymphocytes activated for 48 h. Scale bar, 10 µm.


H Fork rate of fibres from B lymphocytes sorted activated Data is pooled from three replicate experiments . Horizontal bars represent median values . 150 - 250 forks were measured per condition . * * * , p < 0 . 001 ; * * , p < 0 .

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

H Fork rate of DNA fibres from primary B lymphocytes sorted and activated Data is pooled from three replicate experiments. Horizontal bars represent median values. 150-250 forks were measured per condition. ***, p<0.001; **, p<0.01


I Western blotting of replication in activated B lymphocytes Sorted B220 + GFP + spleen were activated in vitro with LPS plus IL - 4 for 72 h . MEK2 , SMC1 are shown as loading controls . blots shown are representative of three independent experiments .

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I Western blotting of DNA replication proteins in activated B lymphocytes Sorted B220+GFP+ spleen cells were activated in vitro with LPS plus IL-4 for 72 h. MEK2, SMC1 are shown as loading controls. The blots shown are representative of three independent experiments.


A Bar plot showing number of DEGs ( adj . p - value < 0 . 01 , FC + 1 . 5 in red , 1 . 5 < FC > - 1 . 5 in , FC < - 1 . 5 in blue ) . Sorted B220 + GFP + spleen B from MaxKO - cd19 , MycKO - cd19 , DKO - cd19 control HET - cd19 mice were activated with LPS plus IL - 4 for 72 h analyzed . Two samples for each condition were used .

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A Bar plot showing the number of DEGs (adj. p-value <0.01, FC +1.5 in red, 1.5 < FC >-1.5 in white, FC< -1.5 in blue). Sorted B220+GFP+ spleen B cells from MaxKO-cd19, MycKO-cd19, DKO-cd19 and control HET-cd19 mice were activated with LPS plus IL-4 for 72 h and analyzed. Two samples for each condition were used.


C Hierarchical clustering heat map of DEGs in any comparison ( two samples per condition ) . blue in left panel indicates significance for that contrast column . Colour code : red represents upregulation , , unchanged , blue downregulation of expression . Gender : colour code for genetic condition . Pink are blue colour mice .

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C Hierarchical clustering heat map of DEGs in any comparison (two samples per condition). Dark blue in left panel indicates significance for that contrast column. Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression. Gender: colour code for genetic condition. Pink are female and blue colour male mice.


D Venn diagrams of up - ( left ) down - regulated genes ( right ) for three conditions ( + / - 1 . 5 - FC p < 0 . 01 ) . Heatmaps of genes only deregulated in MaxKO - cd19 , MycKO - cd19 , or DKO - cd19 mice are shown .

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D Venn diagrams of up- (left) and down-regulated genes (right) for all three conditions (+/-1.5-FC and p<0.01). Heatmaps of genes only deregulated in MaxKO-cd19, MycKO-cd19, or DKO-cd19 mice are shown.


B Heatmaps of genes involved in relevant pathways Colour code : red represents upregulation , , unchanged , blue downregulation of expression .

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B Heatmaps of genes involved in the relevant pathways Colour code: red represents upregulation, white, unchanged, and blue downregulation of expression.


C qPCR of up - down - regulated genes Pooled was prepared from sorted B220 + GFP + spleen B of MaxKO - cd19 ( n = 4 ) , MycKO - cd19 ( n = 4 ) , or DKO - cd19 ( n = 4 ) control HET - cd19 ( n = 4 ) mice activated Genes were randomly selected qPCR was performed using specific primers for cd72 , med13l , card11 , ssh2 , cdc7 , mrpl12 , rpl39 , cysc . max c - myc were included as controls actin was used for normalization .

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C qPCR analysis of up- and down-regulated genes Pooled RNA was prepared from sorted B220+GFP+ spleen B cells of MaxKO-cd19 (n=4), MycKO-cd19 (n=4), or DKO-cd19 (n=4) and control HET-cd19 (n=4) mice activated Genes were randomly selected and qPCR was performed using specific primers for cd72, med13l, card11, ssh2, cdc7, mrpl12, rpl39, and cysc. max and c-myc were included as controls and actin was used for normalization.


growth assessed by color change due to medium acidification for both undefined ATCC 1161 semi - defined CSY media with or without sucrose , 4 % 1 % respectively . ( + ) , inoculated wells ; ( - ) , non - inoculated control . picture was taken after a 24 - hour incubation period .

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Bacterial growth assessed by color change due to medium acidification for both the undefined ATCC 1161 and semi-defined CSY media with or without sucrose, 4% and 1% respectively. (+), inoculated wells; (-), non-inoculated control. The picture was taken after a 24-hour incubation period.


Impact of horse serum ( HS ) yeast extract ( YE ) dilution on M . florum doubling time . green red dotted lines indicate HS YE concentrations found in ATCC 1161 medium ( 20 % HS , 1 . 35 % YE ) CSY medium ( 0 . 313 % HS , 0 . 02 % YE ) , respectively . Doubling times were measured in a CMRL - 1066 base medium using colorimetric assays . Dots error bars indicate mean standard deviation calculated from three biological replicates .

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Impact of horse serum (HS) and yeast extract (YE) dilution on M. florum doubling time. The green and red dotted lines indicate HS and YE concentrations found in ATCC 1161 medium (20% HS, 1.35% YE) and CSY medium (0.313% HS, 0.02% YE), respectively. Doubling times were measured in a CMRL-1066 base medium using colorimetric assays. Dots and error bars indicate the mean and standard deviation calculated from three biological replicates.


maximal biomass observed for M . florum cultures growing in CSY medium with varying initial concentrations of sucrose . Biomass was measured using colony forming units ( CFU / ml ; left axis ) converted to grams of dry ( gDW / L ; right axis ) . A rectangular hyperbola fit is shown ( yellow ) , dotted represents maximal biomass value predicted by fit ( 1e8 x 5 . 95 CFU / ml ; 0 . 013 gDW / L ) .

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The maximal biomass concentration observed for M. florum cultures growing in CSY medium with varying initial concentrations of sucrose. Biomass was measured using colony forming units (CFU/ml; left axis) and converted to grams of dry weight (gDW/L; right axis). A rectangular hyperbola fit is shown (yellow), and the dotted line represents the maximal biomass value predicted by the fit (1e8 x 5.95 CFU/ml; 0.013 gDW/L).


Sucrose specific uptake rate ( e ) combined lactate / acetate specific secretion rates ( f ) at varying initial sucrose concentrations in CSY medium . Boxplots represent median interquartile range of uptake or secretion rate values calculated at different time intervals during exponential growth phase of M . florum cultures . Whiskers indicate minimal maximal values . Dotted lines indicate selected uptake ( - 5 . 26 mmol ∙ gDW - 1 ∙ hr - 1 ) secretion rate ( 8 . 69 mmol ∙ gDW - 1 ∙ hr - 1 ) values used for modeling . Sucrose quantifications were performed in technical duplicate , whereas lactate acetate quantifications were performed in single replicates . See Appendix Figure S5 for further details .

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

Sucrose specific uptake rate (e) and combined lactate/acetate specific secretion rates (f) at varying initial sucrose concentrations in CSY medium. Boxplots represent the median and interquartile range of uptake or secretion rate values calculated at different time intervals during the exponential growth phase of M. florum cultures. Whiskers indicate minimal and maximal values. Dotted lines indicate the selected uptake (-5.26 mmol∙gDW-1∙hr-1) and secretion rate (8.69 mmol∙gDW-1∙hr-1) values used for modeling. Sucrose quantifications were performed in technical duplicate, whereas lactate and acetate quantifications were performed in single replicates. See Appendix Figure S5 for further details.


Week 10 COs stained for ventricular radial glia ( vRGs ) marker CRYAB , together with WWOX pan - radial glia marker SOX2 . on right are enlargements of boxed . dashed defines ventricular space . Scale = 50µm ( left ) , 25µm ( right ) ( WT : n = 8 from 3 batches , KO : n = 8 from 3 batches ) .

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Week 10 COs stained for the ventricular radial glia (vRGs) marker CRYAB, together with WWOX and the pan-radial glia marker SOX2. The images on the right are enlargements of the boxed area. The dashed line defines the ventricular space. Scale= 50µm (left), 25µm (right) (WT: n=8 from 3 batches, KO: n=8 from 3 batches).


Immunofluorescent ( IF ) for glutamatergic neurons marker VGLUT1 GABAergic neurons marker GAD67 ( GAD1 ) in week 10 COs ( WT : n = 8 from 3 batches , KO : n = 8 from 3 batches , W - AAV : n = 4 from 1 batch ) . Scale = 100µm . of represented in figure 1E . VGLUT1 GAD67 were quantified as surface covered by were normalized to number of nuclei in each image . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . As samples were not normally distributed , statistical significance was determined using Kruskal - Wallis with Dunn ' s multiple comparisons . Outliers were removed using ROUT ( Q = 1 % ) . Data information : n . s ( non - significant ) , * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 .

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Immunofluorescent (IF) staining for the glutamatergic neurons marker VGLUT1 and GABAergic neurons marker GAD67 (GAD1) in week 10 COs (WT: n=8 from 3 batches, KO: n=8 from 3 batches, W-AAV: n=4 from 1 batch). Scale = 100µm.   Quantification of the images represented in figure 1E. VGLUT1 and GAD67 were quantified as the surface area covered by the staining and were normalized to the number of nuclei in each image. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. As the samples were not normally distributed, statistical significance was determined using Kruskal-Wallis test with Dunn's multiple comparisons test. Outliers were removed using the ROUT test (Q=1%). Data information: n.s (non-significant), *p ≤ 0.05, ****p ≤ 0.0001.


Sample recordings from 7 - week - old hESC - derived Cerebral Organoids ( COs ) . Sample traces show visible differences in local field potential , with WWOX - KO COs ( Red ) showing increased activity compared to WT ( Blue ) in baseline condition ( Left ) in presence of 100µM 4AP ( Right ) .

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Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). Sample traces show visible differences in local field potential, with WWOX-KO COs (Red) showing increased activity compared to WT (Blue) in baseline condition (Left) and in the presence of 100µM 4AP (Right).


Sample recordings from 7 - week - old hESC - derived Cerebral Organoids ( COs ) . WWOX ' s coding was re - introduced into week 6 WWOX - KO COs using lentiviral transduction ( lenti - WWOX ) ( F ) Immunofluorescent showing WWOX expression in different populations in WWOX - KO organoids following infection with lentivirus . NT = non - treated . Scale = 50µm .

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Sample recordings from 7-week-old hESC-derived Cerebral Organoids (COs). WWOX's coding sequence was re-introduced into week 6 WWOX-KO COs using lentiviral transduction (lenti-WWOX) (F) Immunofluorescent staining showing WWOX expression in different populations in WWOX-KO organoids following infection with lentivirus. NT= non-treated. Scale = 50µm .


Week 15 week 24 COs stained for astrocytic radial glia marker GFAP , astrocyte - specific marker S100β ( WT W15 : n = 9 from 3 batches , KO W15 : n = 16 from 3 batches , W - AAV W15 : n = 4 organoids from 1 batch . WT W24 : n = 10 from 4 batches , KO W24 : n = 9 from 3 batches , W - AAV W24 : n = 4 from 1 batch ) . Scale =

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Week 15 and week 24 COs stained for the astrocytic and radial glia marker GFAP, and the astrocyte-specific marker S100β (WT W15: n=9 from 3 batches, KO W15: n=16 from 3 batches, W-AAV W15: n=4 organoids from 1 batch. WT W24: n=10 from 4 batches, KO W24: n=9 from 3 batches, W-AAV W24: n=4 from 1 batch). Scale = 100µm


IF of week 6 COs for astrocytic markers in surrounding of VZs ( WT : n = 6 from 2 batches , KO : n = 6 from 2 batches ) . Scale = 100µm ( left ) , 50µm ( right ) . for damage markers γH2AX 53BP1 in nuclei of in VZ of week 6 COs at physiological conditions , together with pan - radial glia marker SOX2 ( WT : n = 8 from 3 batches , KO : n = 12 from 3 batches , W - AAV : n = 4 from 1 batch ) . Scale = 50µm ( left ) , 25µm ( right ) . of γH2AX ( top ) 53BP1 foci ( bottom ) in nuclei of composing innermost layer of VZ , normalized to total number of nuclei in this layer . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WT : n = 8 organoids from 3 batches , KO : n = 12 organoids from 3 batches , W - AAV : n = 4 from 1 batch ) . Data information : n . s ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 .

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IF staining of week 6 COs for astrocytic markers in the surrounding of VZs (WT: n=6 from 2 batches, KO: n=6 from 2 batches). Scale = 100µm (left) , 50µm (right).   Staining for the DNA damage markers γH2AX and 53BP1 in the nuclei of cells in the VZ of week 6 COs at physiological conditions, together with the pan-radial glia marker SOX2 (WT: n=8 from 3 individual batches, KO: n=12 from 3 individual batches, W-AAV: n=4 from 1 batch). Scale = 50µm (left), 25µm (right).   Quantification of γH2AX (top) and 53BP1 foci (bottom) in the nuclei of cells composing the innermost layer of the VZ, normalized to the total number of nuclei in this layer. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=8 organoids from 3 batches, KO: n=12 organoids from 3 batches, W-AAV: n=4 from 1 batch).  Data information: n.s (non-significant), *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001.


sequencing ( - seq ) of week 15 COs transcriptome ( WT : n = 2 , KO : n = 4 ) . qPCR for selected Wnt target genes validating results of - seq . Y - axis indicates relative expression fold change . Data are represented as mean ± . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WT : n = 4 from 1 batch , KO : n = 4 from 1 batch , W - AAV : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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RNA sequencing (RNA-seq) of week 15 COs and transcriptome analysis (WT: n=2, KO: n=4). qPCR analysis for selected Wnt target genes validating the results of the RNA- seq. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=4 from 1 batch, KO: n=4 from 1 batch, W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Week 16 COs were sub - fractionated into a ( C ) ( N ) fractions . experiment was run twice with a total of 2 WT organoids 4 KOs organoids ( 2 for each KO ) . KAP - 1 marks nucleus HSP90 marks cytoplasm . numbers at bottom are a of fraction of β - catenin band intensities normalized to fraction [ β - C ( N / C ) ] .

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Week 16 COs were sub-fractionated into a cytoplasmic (C) and nuclear (N) fractions. The experiment was run twice with a total of 2 WT organoids and 4 KOs organoids (2 for each KO line). KAP-1 marks the nucleus and HSP90 marks the cytoplasm. The numbers at the bottom are a quantification of the nuclear fraction of β-catenin band intensities normalized to the cytosolic fraction [β-C (N/C)].


IF in week 15 COs validating decreased of deep - layer cortical markers CTIP2 ( BCL11B ) TBR1 , superficial - layer marker SATB2 ( WT : n = 3 , KO : n = 4 , W - AAV : n = 4 ) . Scale = 50µm . of cortical markers seen in G , normalized to total number of nuclei . Y - axis indicates fold change compared to average of WT COs . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WT : n = 9 from 3 batches , KO : n = 16 from 3 batches , W - AAV : n = 4 organoids from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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IF staining in week 15 COs validating the decreased levels of the deep-layer cortical markers CTIP2 (BCL11B) and TBR1, and superficial-layer marker SATB2 (WT: n=3, KO: n=4, W-AAV: n=4). Scale = 50µm.   Quantification of the cortical markers seen in G, normalized to the total number of nuclei. Y-axis indicates the fold change compared to the average of the WT COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WT: n=9 from 3 batches, KO: n=16 from 3 batches, W-AAV: n=4 organoids from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Peripheral blood mononuclear ( PBMCs ) were isolated from a WOREE from his healthy parents were reprogrammed into iPSCs , subsequently were differentiated into COs . Week 10 WSM COs stained for progenitor marker SOX2 , neuronal marker β3 - Tubulin WWOX ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm .

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Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs.   Week 10 WSM COs stained for the progenitor marker SOX2, neuronal marker β3-Tubulin and WWOX (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.


Peripheral blood mononuclear ( PBMCs ) were isolated from a WOREE from his healthy parents were reprogrammed into iPSCs , subsequently were differentiated into COs . Average firing rate over 24 neurons from WSM P COs ( 4 organoids ) , 41 neurons from WSM S COs ( 3 organoids ) 40 neurons from WSM S W - AAV organoids ( 3 organoids ) . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons . Bars represents mean ± . Data information : ns ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. Average firing rate over 24 neurons from WSM P COs (4 organoids), 41 neurons from WSM S COs (3 organoids) and 40 neurons from WSM S W-AAV organoids (3 organoids). Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test. Bars represents the mean ± SEM. Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Peripheral blood mononuclear ( PBMCs ) were isolated from a WOREE from his healthy parents were reprogrammed into iPSCs , subsequently were differentiated into COs . GAD67 VGLUT1 immunostaining in week 10 WSM COs ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . of data shown in fig 5D . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 2 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Data information : ns ( non - significant ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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Peripheral blood mononuclear cells (PBMCs) were isolated from a WOREE patient and from his healthy parents and were reprogrammed into iPSCs, and subsequently were differentiated into COs. GAD67 and VGLUT1 immunostaining in week 10 WSM COs (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the data shown in fig 5D. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=2 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: ns (non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Week 15 WSM COs stained for astrocytic markers GFAP S100β . ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . of damage foci , marked by γH2AX ( left ) 53BP1 ( right ) , in nuclei of in VZs of week 6 WSM COs . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WSM F1 : n = 4 from 1 batch , WSM M2 : n = 4 from 1 batch , WSM S2 : n = 4 from 1 batch , WSM S5 : n = 4 from 1 batch , WSM S5 W - AAV3 : n = 4 from 1 batch , WSM S5 W - AAV6 : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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

Week 15 WSM COs stained for astrocytic markers GFAP and S100β. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of DNA damage foci, marked by γH2AX (left) and 53BP1 (right), in the nuclei of the cells in the VZs of week 6 WSM COs. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=4 from 1 batch, WSM M2: n=4 from 1 batch, WSM S2: n=4 from 1 batch, WSM S5: n=4 from 1 batch, WSM S5 W-AAV3: n=4 from 1 batch, WSM S5 W-AAV6: n=4 from 1 batch).   Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


qPCR for selected Wnt target genes in week 10 WSM COs . Y - axis indicates relative expression fold change . Data are represented as mean ± . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WSM P : n = 6 from 1 batch , WSM S : n = 4 from 1 batch , WSM S W - AAV : n = 4 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

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qPCR analysis for selected Wnt target genes in week 10 WSM COs. Y-axis indicates relative expression fold change. Data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM P: n=6 from 1 batch, WSM S: n=4 from 1 batch, WSM S W-AAV: n=4 from 1 batch). Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.


Week 15 WSM COs stained for cortical layers ' markers CTIP2 SATB2 . ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Scale = 50µm . of presented in E . boxplot represents 1st 3rd quartile , with its whiskers showing minimum maximum points a central band representing median . Statistical significance was determined using one - way ANOVA with Tukey ' s multiple comparisons ( WSM F1 : n = 2 from 1 batch , WSM M2 : n = 3 from 1 batch , WSM S2 : n = 2 from 1 batch , WSM S5 : n = 2 from 1 batch , WSM S5 W - AAV3 : n = 2 from 1 batch , WSM S5 W - AAV6 : n = 2 from 1 batch ) . Data information : * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . E

O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Week 15 WSM COs stained for the cortical layers' markers CTIP2 and SATB2. (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch). Scale = 50µm.   Quantification of the staining presented in E. The boxplot represents the 1st and 3rd quartile, with its whiskers showing the minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparisons test (WSM F1: n=2 from 1 batch, WSM M2: n=3 from 1 batch, WSM S2: n=2 from 1 batch, WSM S5: n=2 from 1 batch, WSM S5 W-AAV3: n=2 from 1 batch, WSM S5 W-AAV6: n=2 from 1 batch).  Data information: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. E


C Calculated BRET ratios for indicated pairs . positive control PA - mCit - NL interacting fusion NL - BCL2L1 PA - mCit - BAD show high BRET ratios .

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C Calculated BRET ratios for the indicated protein pairs. The positive control protein PA-mCit-NL and the interacting fusion proteins NL-BCL2L1 and PA-mCit-BAD show high BRET ratios.


D Calculated LuC ratios for indicated pairs . positive control PA - NL PA - mCit - NL interacting NL - BCL2L1 PA - mCit - BAD show high LuC ratios .

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D Calculated LuC ratios for the indicated protein pairs. The positive control proteins PA-NL and PA-mCit-NL and the interacting proteins NL-BCL2L1 and PA-mCit-BAD show high LuC ratios.


Reproducibility of LuTHy PPI mapping experiments with positive negative reference sets . scatter plots show mean cBRET ( A ) ratios from two independent experiments ( Exp 1 Exp 2 ) , calculated two - Pearson correlation coefficient is indicated ; * * * p < 0 . 001 .

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Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cBRET (A) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.


Reproducibility of LuTHy PPI mapping experiments with positive negative reference sets . scatter plots show mean cLuC ( B ) ratios from two independent experiments ( Exp 1 Exp 2 ) , calculated two - Pearson correlation coefficient is indicated ; * * * p < 0 . 001 .

Reproducibility of LuTHy PPI mapping experiments with positive and negative reference sets. The scatter plots show the mean cLuC (B) ratios from two independent experiments (Exp 1 and Exp 2), the calculated two-tailed Pearson correlation coefficient is indicated; ***p<0.001.


C Receiver operating characteristic ( ROC ) of cBRET ( AUC = 0 . 80 ± 0 . 04 ) cLuC ( AUC = 0 . 72 ± 0 . 04 ) data determines thresholds to define true positive binary interactions .

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C Receiver operating characteristic (ROC) analysis of cBRET (AUC=0.80±0.04) and cLuC (AUC=0.72±0.04) data determines the thresholds to define true positive binary interactions.


D Performance of BRET LuC assays in benchmarking studies with hPRS hRRS .

D Performance of BRET and LuC assays in benchmarking studies with hPRS and hRRS.


C Recovery rates of PPIs from affinity - sub - groups with BRET LuC assays . Significance was calculated by two - sided Fisher ' s exact ; * p < 0 . 05 . D Success of PPI detection from AIRS sub - groups considering single double LuTHy readouts .

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C Recovery rates of PPIs from affinity-based sub-groups with BRET and LuC assays. Significance was calculated by two-sided Fisher's exact test; *p<0.05. D Success of PPI detection from AIRS sub-groups considering single and double LuTHy readouts.


E Scatter plot depicting relationships between published binding affinities ( KD ) in - BRET50 measurements for 25 interactions in AIRS ; BRET50 values are mean from two independent experiments . Significance was calculated by two - Spearman correlation ; * * * p < 0 . 001 .

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E Scatter plot depicting the relationships between published binding affinities (KD) and in-cell BRET50 measurements for 25 interactions in AIRS; BRET50 values are the mean from two independent experiments. Significance was calculated by two-tailed Spearman correlation; ***p<0.001.


A Live - bioluminescence imaging ( BLI ) of HEK293 co - producing indicated fusion . mCitrine was excited at 500 nm , emitted was detected at 535 nm . After substrate addition , short ( 460 ) long ( 535 ) band - pass ( BP ) filters in a dual - view adapter were used to detect emitted luminescence simultaneously at respective wavelengths . BRET were calculated by dividing 535 BP by 460 BP using ImageJ . Scale bar = 20 μm .

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A Live-cell bioluminescence imaging (BLI) of HEK293 cells co-producing the indicated fusion proteins. mCitrine was excited at 500 nm, emitted fluorescence was detected at 535 nm. After substrate addition, short (460) and long (535) band-pass (BP) filters in a dual-view adapter were used to detect the emitted luminescence simultaneously at the respective wavelengths. BRET images were calculated by dividing the 535 BP by the 460 BP images using ImageJ. Scale bar = 20 μm.


B of rapamycin - induced interaction between NL - FKBP12 PA - mCit - FRB . Plasmids encoding fusion were co - transfected in HEK293 24 h later treated with indicated concentrations of rapamycin . After an additional incubation for 4 h BRET was quantified upon substrate addition . After lysis in buffer with indicated rapamycin concentrations , LuC ratios were determined . EC50 values were obtained with non - linear curve fitting .

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B Quantification of the rapamycin-induced interaction between NL-FKBP12 and PA-mCit-FRB. Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and 24 h later treated with the indicated concentrations of rapamycin. After an additional incubation for 4 h BRET was quantified upon substrate addition. After cell lysis in buffer with indicated rapamycin concentrations, LuC ratios were determined. EC50 values were obtained with non-linear curve fitting.


Plasmids encoding fusion were co - transfected in HEK293 After an additional incubation for 4 h BRET was quantified upon substrate addition . LuC ratios were determined . EC50 values were obtained with non - linear curve fitting . C Effects of Nutlin - 3 on interaction between NL - MDM2 PA - mCit - p53 . for 6 h treatment with Nutlin - 3 .

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Plasmids encoding the fusion proteins were co-transfected in HEK293 cells and After an additional incubation for 4 h BRET was quantified upon substrate addition. LuC ratios were determined. EC50 values were obtained with non-linear curve fitting. C Effects of Nutlin-3 on the interaction between NL-MDM2 and PA-mCit-p53. for 6 h treatment with Nutlin-3.


D Time resolved HSF1 oligomerization by Hsp90 inhibition . HEK293 co - producing NL - HSF1 PA - mCit - HSF1 for 24 h were transferred to 384 - well plates containing Ganetespib or Geldanamycin to reach a final of 1 µM . Luminescence was measured at indicated time points calculated BRET ratios normalized to respective untreated control . Data are mean of two biological replicates . values are mean ± . Significance was calculated by two - way ANOVA followed by Dunnett ' s multiple comparison ' s post - hoc .

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

D Time resolved HSF1 oligomerization by Hsp90 inhibition. HEK293 cells co-producing NL-HSF1 and PA-mCit-HSF1 for 24 h were transferred to 384-well plates containing Ganetespib or Geldanamycin to reach a final concentration of 1 µM. Luminescence was measured at the indicated time points and the calculated BRET ratios normalized to the respective untreated control. Data are the mean of two biological replicates. All values are mean ± sem. Significance was calculated by two-way ANOVA followed by Dunnett's multiple comparison's post-hoc test.


E Schematic depiction of a heat shock experiment . HEK293 co - producing NL - HSF1 / PA - mCit - HSF1 for 48h were subjected to a heat shock at 42 ° C for 6 h , after which were recovered at 37 ° C for 24h . Luminescence was measured at indicated time points : immediately before heat shock , during heat shock at 90 , 180 360 min after a recovery phase of 24 h . Calculated cBRET ratios were normalized to a control plate that was not heat treated . values are mean ± from three independent experiments . Significance was calculated by one - way ANOVA followed by Dunnett ' s multiple comparisons post - hoc ; * p < 0 . 05 .

O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Schematic depiction of a heat shock experiment. HEK293 cells co-producing NL-HSF1/PA-mCit-HSF1 for 48h were subjected to a heat shock at 42°C for 6 h, after which the cells were recovered at 37°C for 24h. Luminescence was measured at the indicated time points: immediately before the heat shock, during the heat shock at 90, 180 and 360 min and after a recovery phase of 24 h. Calculated cBRET ratios were normalized to a control plate that was not heat treated. All values are mean ± sem from three independent experiments. Significance was calculated by one-way ANOVA followed by Dunnett's multiple comparisons post-hoc test; *p<0.05.


B , C , D VCP UBX domain - containing were co - expressed as N - C - terminal NL PA - mCit fusion in eight different orientations for which cBRET ( B ) cLuC ( C ) ratios were generated . cBRET cLuC values over threshold of ≥ 0 . 01 ≥ 0 . 03 , respectively , are colored in cyan . D cBRET cLuC ratios were normalized to highest of obtained eight values to easily identify orientation among eight tested interactions with highest cBRET cLuC value .

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B,C,D VCP and the UBX domain-containing proteins were co-expressed as N- and C-terminal NL and PA-mCit fusion proteins in eight different orientations for which cBRET (B) and cLuC (C) ratios were generated. cBRET and cLuC values over the threshold of ≥0.01 and ≥0.03, respectively, are colored in cyan. D The cBRET and cLuC ratios were normalized to the highest of the obtained eight values to easily identify the orientation among the eight tested interactions with the highest cBRET and cLuC value.


Tukey box plots of mean cBRET ( B ) ratios obtained from PPI experiments with wild - type CSPα - mCit - PA its mutant variants .

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Tukey box plots of the mean cBRET (B) ratios obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.


Tukey box plots of mean cLuC ratios ( C ) obtained from PPI experiments with wild - type CSPα - mCit - PA its mutant variants .

Tukey box plots of the mean cLuC ratios (C) obtained from PPI experiments with wild-type CSPα-mCit-PA and its mutant variants.


D Number of screened presynaptic manually annotated according to their function localization in presynaptic terminal . Number of identified with wild - type ( wt ) CSPα - mCit - PA or its mutant variants ( ΔL116 L115R ) are indicated .

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D Number of screened presynaptic proteins manually annotated according to their function and localization in the presynaptic terminal. Number of proteins identified with wild-type (wt) CSPα-mCit-PA or its mutant variants (ΔL116 and L115R) are indicated.


E Influence of mutations in CSPα ( ΔL116 , L115R ) on interactions with ZDHHC17 in hippocampal neurons . BRET LuC ratios were systematically quantified for interactions with wild - type mutant CSPα fusion .

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E Influence of mutations in CSPα (ΔL116, L115R) on the interactions with ZDHHC17 in primary hippocampal neurons. BRET and LuC ratios were systematically quantified for interactions with wild-type and mutant CSPα fusion proteins.


F Immunoblot of SHEP transfected with plasmids encoding wild - type CSPα - mCit - PA mutant variants . Blot was developed with an anti - CSP antibody ; loading control : anti - Histone - H3 antibody . Arrowhead bracket indicate monomeric CSPα - mCit - PA SDS - insoluble , respectively .

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F Immunoblot analysis of SHEP cells transfected with plasmids encoding wild-type CSPα-mCit-PA and mutant variants. Blot was developed with an anti-CSP antibody; loading control: anti-Histone-H3 antibody. Arrowhead and bracket indicate monomeric CSPα-mCit-PA and SDS-insoluble protein aggregates, respectively.


G Immunofluorescence image of SHEP co - transfected with NL - ZDHHC17 CSPα - mCit - wt , - ΔL116 or - L115R constructs lacking PA - tag . NL - ZDHHC17 was stained with an anti NanoLuc antibody . Scale bar : 20 µm .

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G Immunofluorescence image of SHEP cells co-transfected with NL-ZDHHC17 and CSPα-mCit-wt, -ΔL116 or -L115R constructs lacking the PA-tag. NL-ZDHHC17 was stained with an anti NanoLuc antibody. Scale bar: 20 µm.


A Flow cytometric of lymphocytes , monocytes , B - , natural killer ( NK ) , dendritic ( DCs ) granulocytes in peripheral venous blood samples from non - diabetic ( ND ) type - 2 diabetic ( T2D ) with COVID - 19 .

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A Flow cytometric quantification of lymphocytes, monocytes, B-cells, natural killer (NK) cells, dendritic cells (DCs) and granulocytes in peripheral venous blood samples from non-diabetic (ND) and type-2 diabetic (T2D) patients with COVID-19.


B of monocyte subpopulation as classical ( CD14Hi CD16 - ) , intermediate ( CD14Hi CD16 + ) or non - classical ( CD14Lo CD16 + ) .

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B Quantification of monocyte subpopulation phenotypes as classical (CD14Hi CD16-), intermediate (CD14Hi CD16+) or non-classical (CD14Lo CD16+).


C Principle component of lymphocyte monocyte population frequencies in ND T2D COVID - 19 in T2D without COVID - 19 .

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C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID-19 patients and in T2D patients without COVID-19.


A of CD14 expression ( FSC ) in monocyte populations from non diabetic ( ND ) type - 2 diabetic ( T2D ) COVID - 19 .

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A Quantification of CD14 expression and size (FSC) in monocyte populations from non diabetic (ND) and type-2 diabetic (T2D) COVID-19 patients.


B Frequency of conventional ( FSCLo ) large ( FSCHi ) monocytes in ND T2D COVID - 19 .

B Frequency of conventional (FSCLo) and large (FSCHi) monocytes in ND and T2D COVID-19 patients.


C Expression of CD16 , CD14 HLA - DR in FSCLo FSCHi monocytes from COVID - 19 .

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C Expression of CD16, CD14 and HLA-DR in FSCLo and FSCHi monocytes from COVID-19 patients.


D Proportions of classical , intermediate non - classical monocytes within FSCLo FSCHi monocytes from COVID - 19 .

D Proportions of classical, intermediate and non-classical monocytes within FSCLo and FSCHi monocytes from COVID-19 patients.


E Correlative analyses of IL8 expression in peripheral blood mononuclear ( PBMCs ) to monocyte FSC .

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E Correlative analyses of IL8 expression in peripheral blood mononuclear cells (PBMCs) to monocyte FSC.


F IL8 , IL6 CCL2 mRNA expression in PBMCs from ND T2D COVID - 19 .

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F IL8, IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID-19 patients.


G Monocyte quantified in ND T2D COVID - 19 treated in general wards ( Gen ) or in intensive care unit ( ICU ) .

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G Monocyte size quantified in ND and T2D COVID-19 patients treated in general wards (Gen) or in intensive care unit (ICU).


A of IRF5 IFNB1 mRNA expression in peripheral blood mononuclear of non - diabetic ( ND ) type 2 diabetic ( T2D ) COVID - 19 ( A ) .

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A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non-diabetic (ND) and type 2 diabetic (T2D) COVID-19 patients (A).


B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples .

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B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples.


C IRF5 median ( MFI ) in monocytes of ND T2D with COVID - 19 .

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C IRF5 median fluorescence intensity (MFI) in monocytes of ND and T2D patients with COVID-19.


D IRF5 expression overlaid onto monocyte phenotypic gating .

D IRF5 expression overlaid onto monocyte phenotypic gating.


E IRF5 expression ( MFI ) in monocyte subtypes from ND T2D COVID - 19 .

E IRF5 expression (MFI) in monocyte subtypes from ND and T2D COVID-19 patients.


F HLA - DR IRF5 expression in monocytes from COVID - 19 .

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F HLA-DR and IRF5 expression in monocytes from COVID-19 patients.


G IRF5 expression in monocytes of ND or T2D COVID - 19 admitted to intensive care unit ( ICU ) or treated exclusively in general wards ( Gen ) .

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G IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted to intensive care unit (ICU) or treated exclusively in general wards (Gen) .


H IRF5 expression in monocytes of ND or T2D COVID - 19 admitted ICU or treated exclusively in Gen .

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H IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted ICU or treated exclusively in Gen.


B Proportions of lymphocytes monocytes in peripheral venous blood from T2D with COVID - 19 either treated in Gen or requiring ICU admission .

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B Proportions of lymphocytes and monocytes in peripheral venous blood from T2D patients with COVID-19 either treated in Gen or requiring ICU admission.


C Phenotypic of lymphocyte monocyte subpopulations in peripheral venous blood from T2D COVID - 19 treated in Gen or requiring ICU treatment .

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C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID-19 patients treated in Gen or requiring ICU treatment.


a ) QF - FRL of unilamellar liposomes containing 95 mol % of phosphatidylcholine 5 mol % of a phosphoinositide , labelled with GST - PX . Only liposomes containing PtdIns ( 3 ) P were labelled intensely . GST - PXR58A , a PX - domain mutant that does not bind PtdIns ( 3 ) P , yielded virtually no labelling in PtdIns ( 3 ) P - containing liposomes . Scale bar , 0 . 2 μm .

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a) QF-FRL of unilamellar liposomes containing 95 mol% of phosphatidylcholine and 5 mol% of a phosphoinositide, labelled with GST-PX. Only liposomes containing PtdIns(3)P were labelled intensely. GST-PXR58A, a PX-domain mutant that does not bind PtdIns(3)P, yielded virtually no labelling in PtdIns(3)P-containing liposomes. Scale bar, 0.2 μm.


( b ) QF - FRL of unilamellar liposomes containing 0 - 5 mol % of PtdIns ( 3 ) P . Scale bar , 0 . 2 μm . labelling density increased roughly in proportion to PtdIns ( 3 ) P content of liposomes . experiment was repeated twice number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes . Values for concentrations are statistically different from those in adjacent conditions ( for example , 1 versus 2 mol % ; mean ± s . e . m . ; Student ' s t - ; P0 . 05 ) .

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(b) QF-FRL of unilamellar liposomes containing 0-5 mol % of PtdIns(3)P. Scale bar, 0.2 μm. The labelling density increased roughly in proportion to the PtdIns(3)P content of the liposomes. The experiment was repeated twice and the number of colloidal gold particles per μm2 was measured in more than 75 randomly selected liposomes. Values for all concentrations are statistically different from those in the adjacent conditions (for example, 1 versus 2 mol%; mean±s.e.m.; Student's t-test; P0.05).


( b ) QF - FRL in wild - type yeast in log phase . PtdIns ( 3 ) P was labelled with GFP - PX marked with colloidal gold ( 10 nm ) . vacuolar PF was labelled intensely , whereas other organellar membranes did not exhibit significant labelling . Scale bar , 0 . 5 μm .

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(b) QF-FRL in wild-type yeast in log phase. PtdIns(3)P was labelled with GFP-PX and marked with colloidal gold (10 nm). The vacuolar membrane PF was labelled intensely, whereas other organellar membranes did not exhibit significant labelling. Scale bar, 0.5 μm.


c ) of labelling of PtdIns ( 3 ) P ( mean ± s . e . m . ) . experiment was repeated three times number of colloidal gold particles per μm2 was measured in more than ten randomly selected .

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c) Quantification of the labelling intensity of PtdIns(3)P (mean±s.e.m.). The experiment was repeated three times and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.


( d , e ) Negative controls . Both wild - type yeast incubated with GST - PXR58A ( d ) vps34Δ yeast incubated with GST - PX ( e ) were virtually unlabelled . Scale bar , 0 . 5 μm .

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(d,e) Negative controls. Both wild-type yeast incubated with GST-PXR58A (d) and vps34Δ yeast incubated with GST-PX (e) were virtually unlabelled. Scale bar, 0.5 μm.


( a ) Wild - type yeast cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled intensely in EF of autophagosomal membranes . A lower but still significant amount of labelling was also seen in PF of autophagosome membranes as well as in EF of vacuolar . Scale bar , 0 . 5 μm .

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(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagosomal membranes. A lower but still significant amount of labelling was also seen in the PF of autophagosome membranes as well as in the EF of the vacuolar membrane. Scale bar, 0.5 μm.


( b ) Autophagosomes in wild - type yeast cultured in S ( - NC ) for 4 h . autophagosome was identified as a smooth - surfaced double - structure , seen in two different patterns , as shown in diagrams in c . Upper panel : convex structures displaying outer EF inner PF . Lower panel : concave structures displaying outer PF inner EF . In both cases , PtdIns ( 3 ) P was labelled more intensely in EF than in PF . Scale bars , 0 . 2 μm .

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(b) Autophagosomes in wild-type yeast cultured in S(-NC) for 4 h. The autophagosome was identified as a smooth-surfaced double-membrane structure, and seen in two different patterns, as shown in the diagrams in c. Upper panel: convex structures displaying the outer membrane EF and the inner membrane PF. Lower panel: concave structures displaying the outer membrane PF and the inner membrane EF. In both cases, PtdIns(3)P was labelled more intensely in the EF than in the PF. Scale bars, 0.2 μm.


( a ) Wild - type yeast cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled intensely in EF of autophagic membranes in vacuolar lumen . A lower amount of labelling was also seen in PF of autophagic membranes . Scale bar , 0 . 5 μm . (

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(a) Wild-type yeast cultured in S(-NC) for 4 h. PtdIns(3)P was labelled intensely in the EF of autophagic body membranes in the vacuolar lumen. A lower amount of labelling was also seen in the PF of autophagic body membranes. Scale bar, 0.5 μm. (


( c ) Labelling of PtdIns ( 3 ) P in wild - type yeast cultured in S ( - NC ) for 4 h ( average ± s . e . m . ) . PtdIns ( 3 ) P was found in both leaflets of autophagosome autophagic , labelling was much stronger in EF than in PF . In vacuolar , significant labelling was found in EF , albeit at much lower than in PF . experiment was repeated three times , number of colloidal gold particles per μm2 was measured in more than ten randomly selected .

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

(c) Labelling intensity of PtdIns(3)P in wild-type yeast cultured in S(-NC) for 4 h (average±s.e.m.). PtdIns(3)P was found in both leaflets of the autophagosome and autophagic body membrane, and the labelling was much stronger in the EF than in the PF. In the vacuolar membrane, significant labelling was found in the EF, albeit at much lower levels than in the PF. The experiment was repeated three times, and the number of colloidal gold particles per μm2 was measured in more than ten randomly selected images.


( a ) Huh7 expressing GFP - LC3 treated with 0 . 25 μM Torin1 0 . 4 μM bafilomycin A1 for 1 h . autophagosome was identified as IMP - poor double - structure labelled with anti - GFP antibody ( large gold , 10 nm ) . Both PtdIns ( 3 ) P ( small gold , 6 nm ) GFP - LC3 were seen exclusively in PF in both convex concave structures . small large gold particles are shown as green red dots , respectively , in figures at right . Scale bars , 0 . 1 μm . Figures displaying wider areas , as well as additional examples , are presented in Supplementary Fig . 4c . (

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(a) Huh7 cells expressing GFP-LC3 treated with 0.25 μM Torin1 and 0.4 μM bafilomycin A1 for 1 h. The autophagosome was identified as the IMP-poor double-membrane structure labelled with anti-GFP antibody (large gold, 10 nm). Both PtdIns(3)P (small gold, 6 nm) and GFP-LC3 were seen exclusively in the PF in both convex and concave structures. The small and large gold particles are shown as green and red dots, respectively, in the figures at right. Scale bars, 0.1 μm. Figures displaying wider areas, as well as additional examples, are presented in Supplementary Fig. 4c. (


( b ) NIH3T3 expressing Atg4C78A were incubated in Earle ' s balanced salt solution for 1 h . isolation was observed as parallel membranes with few IMPs . PtdIns ( 3 ) P was labelled only in PF . Scale bar , 0 . 1 μm . Other examples are presented in Supplementary Fig . 5 .

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(b) NIH3T3 cells expressing Atg4C78A were incubated in Earle's balanced salt solution for 1 h. The isolation membrane was observed as parallel membranes with few IMPs. PtdIns(3)P was labelled only in the PF. Scale bar, 0.1 μm. Other examples are presented in Supplementary Fig. 5.


( c ) Labelling density of PtdIns ( 3 ) P in mammalian autophagosomal ( mean ± s . e . m . ) . experiment was repeated three times number of colloidal goldparticles per μm2 was measured in more than ten randomly selected . Some labelling was observed in EF but majority of gold particles in EF occurred near EF - PF boundary ( circles ; see Supplementary Fig . 6c for more examples ) , suggesting that many of them actually represent PtdIns ( 3 ) P in PF .

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(c) Labelling density of PtdIns(3)P in mammalian autophagosomal membrane (mean±s.e.m.). The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images. Some labelling was observed in the EF but the majority of gold particles in the EF occurred near the EF-PF boundary (white circles; see Supplementary Fig. 6c for more examples), suggesting that many of them actually represent PtdIns(3)P in the PF.


( a ) ymr1Δsjl3Δ cultured in S ( - NC ) for 4 h . PtdIns ( 3 ) P was labelled in comparable densities in PF EF of autophagosomal membranes . Scale bar , 0 . 2 μm .

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(a) ymr1Δsjl3Δ cells cultured in S(-NC) for 4 h. PtdIns(3)P was labelled in comparable densities in PF and EF of autophagosomal membranes. Scale bar, 0.2 μm.


( b ) PF / EF of PtdIns ( 3 ) P labelling density in autophagosomal ( mean ± s . e . m . ) . was small ( that is , PF ) in WT , but close to 1 ( that is , PF ≅ EF ) in ymr1Δsjl3Δ . experiment was repeated three times number of colloidal goldparticles per μm2 was measured in more than ten randomly selected .

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(b) The PF/EF ratio of the PtdIns(3)P labelling density in autophagosomal membrane (mean±s.e.m.). The ratio was small (that is, PF) in WT, but close to 1 (that is, PF≅EF) in ymr1Δsjl3Δ. The experiment was repeated three times and the number of colloidal goldparticles per μm2 was measured in more than ten randomly selected images.


( A - B ) Live third instar discs incubated in DiBAC . DiBAC is observed in wing imaginal disc in both apical ( A - A ' ) , basal ( B - B ' ) optical sections . Increased is observed in center of pouch ( arrow , B ' ) , at dorsoventral ( D - V ) compartment boundary in anterior compartment ( yellow arrow , B ' , inset , D ) .

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(A-B) Live third instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A-A'), and basal (B-B') optical sections. Increased fluorescence is observed in the center of the pouch (white arrow, B'), and at the dorsoventral (D-V) compartment boundary in the anterior compartment (yellow arrow, B', and inset, D).


( C - E ) Comparison of DiBAC with voltage - insensitive dye FM4 - 64 . Incubation of live discs in FM4 - 64 ( E ) shows more uniform when compared to DiBAC ( D ) . Quantitative comparison of in two boxes in each panel is shown in ( C ) . N = 7 discs for each treatment , data compared using an unpaired t , * * indicates p < 0 . 001 , error bars are standard deviations . region boxed in yellow in ( D ) is shown at higher magnification to show DiBAC at D - V compartment boundary .

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(C-E) Comparison of DiBAC with the voltage-insensitive dye FM4-64. Incubation of live discs in FM4-64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N=7 discs for each treatment, data compared using an unpaired t test, ** indicates p<0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D-V compartment boundary.


( F , G ) Incubation in ouabain results in brighter more uniform DiBAC .

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(F, G) Incubation in ouabain results in brighter and more uniform DiBAC fluorescence.


( H - H ' ' ) Live wing discs expressing UAS - RFP anterior to compartment boundary , under control of ptc - Gal4 , were incubated in DiBAC , showing that stripe of increased DiBAC coincides with posterior edge of ptc expression . arrowhead in ( H ) indicates stripe in wing pouch ; yellow arrowheads indicate stripe in dorsal ventral hinge .

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(H-H'') Live wing discs expressing UAS-RFP anterior to the compartment boundary, under the control of ptc-Gal4, were incubated in DiBAC, showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression. White arrowhead in (H) indicates the stripe in the wing pouch; yellow arrowheads indicate the stripe in the dorsal and ventral hinge.


( I , I ' ) Early L3 discs incubated in DiBAC . Patterned depolarization is evident throughout third larval instar , with stripe of increased becoming narrower in more mature discs with developmental time ( Compare I with A , bracket in I indicates width of increased DiBAC ) .

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(I, I') Early L3 discs incubated in DiBAC. Patterned depolarization is evident throughout the third larval instar, with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time (Compare I with A, bracket in I indicates width of increased DiBAC fluorescence).


Expression of Rpk is increased anterior to A - P compartment boundary , in two rows of flanking D - V compartment boundary in A compartment .

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Expression of Rpk is increased anterior to the A-P compartment boundary, and in two rows of cells flanking the D-V compartment boundary in the A compartment.


( B - B ' ' ) Blockade of DEG / ENaC channels by incubation in amiloride abolishes stripe of increased DiBAC along A - P compartment boundary ( arrowhead ) . boxes indicate regions used to calculate average DiBAC across compartment boundary = 0 . 98 , standard deviation = 0 . 05 , n = 5 discs . Increased at D - V boundary in A compartment is still observable ( yellow arrowhead ) .

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(B-B'') Blockade of DEG/ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A-P compartment boundary (white arrowhead). White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0.98, standard deviation = 0.05, n = 5 discs. Increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).


( C , C ' ) Amiloride incubation does not diminish Rpk expression . arrowhead indicates approximate position of A - P compartment boundary , yellow arrowhead indicates approximate position of D - V compartment boundary .

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(C, C') Amiloride incubation does not diminish Rpk expression. White arrowhead indicates approximate position of the A-P compartment boundary, yellow arrowhead indicates approximate position of the D-V compartment boundary.


( D , E ) Expression of rpk RNAi using dpp - Gal4 results in diminished DiBAC along A - P compartment boundary ( arrowhead ) , while increased at D - V boundary in A compartment is still observable ( yellow arrowhead ) .

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(D, E) Expression of rpk RNAi using dpp-Gal4 results in diminished DiBAC fluorescence along the A-P compartment boundary (white arrowhead), while increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).


( A - A ' ' ' ) Immunostaining of discs expressing ptc > RFP with antibodies to Rpk ATPα showing elevated of both anterior to compartment boundary . arrowheads indicate approximate position of A - P compartment boundary .

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(A-A''') Immunostaining of discs expressing ptc>RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary. White arrowheads indicate approximate position of the A-P compartment boundary.


( B - C ' ) L3 discs that are - sensitive for hh following shift to restrictive for 12 h show loss of increased expression of Rpk ( B , B ' ) ATPα ( C , C ' ) anterior to compartment boundary . Controls shown in Figure EV2 . arrowheads indicate approximate position of A - P compartment boundary .

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(B-C') L3 discs that are temperature-sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk (B, B') and ATPα (C, C') anterior to the compartment boundary. Controls shown in Figure EV2. White arrowheads indicate approximate position of the A-P compartment boundary.


( D - E ' ' ' ) Clones of expressing a constitutively activated form of transcription factor Ci show elevated Rpk ATPα expression in anterior compartment ( box ) as well as posterior compartment ( yellow arrowhead ) . ( E - E ' ' ' ) A single clone in A compartment is shown at higher magnification .

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(D-E''') Clones of cells expressing a constitutively activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment (white box) as well as the posterior compartment (yellow arrowhead). (E-E''') A single clone in the A compartment is shown at higher magnification.


( F - F ' ) A clone of in anterior compartment expressing ci3M following incubation in DiBAC .

(F-F') A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC.


( G - G ' ) A clone of in anterior compartment expressing ptc RNAi following incubation in DiBAC . clones also express RFP .

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(G-G') A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC. The clones also express RFP.


( A - A ' ' ' ) Immunostaining of Smo ( red ) full - Ci ( blue ) in discs expressing en - Venus . Smo is expressed at higher in P compartment in a stripe 5 - 10 wide just anterior to A - P compartment boundary . arrowhead indicates just anterior to A - P compartment boundary .

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(A-A''') Immunostaining of Smo protein (red) and full-length Ci (light blue) in discs expressing en-Venus. Smo is expressed at higher levels in the P compartment and in a stripe 5-10 cells wide just anterior to the A-P compartment boundary. White arrowhead indicates cells just anterior to the A-P compartment boundary.


( B - C ) Effect of expressing an RNAi against ENaC channel rpk in dorsal compartment of disc using ap - Gal4 . Knockdown of Rpk in dorsal compartment results in decreased accumulation of Hh signal transducer Smo ( B , B ' ) , activator form of Ci ( B , B ' ' ) . expression of ptc , visualized using an anti - Ptc antibody ( C ) , a downstream target gene of Hh signaling , is also diminished . Rpk RNAi BL : 39053 is used in panels shown , but phenotype was validated with a second RNAi ( BL : 25847 ) .

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(B-C) Effect of expressing an RNAi against the ENaC channel rpk in the dorsal compartment of the disc using ap-Gal4. Knockdown of Rpk in the dorsal compartment results in decreased accumulation of the Hh signal transducer Smo (B, B'), and the activator form of Ci (B, B''). The expression of ptc, visualized using an anti-Ptc antibody (C), a downstream target gene of Hh signaling, is also diminished. The Rpk RNAi line BL:39053 is used in all panels shown, but the phenotype was validated with a second RNAi line (BL:25847).