( a ) Brains of the ' rescued ' mice ( Becn1 − / − ; Becn1EGFP / + ) and of control Becn1 + / + littermates were lysed and subjected to immune isolation using an anti - GFP antibody . Proteins bound to anti - GFP antibody were isolated , detected and identified by SDS - PAGE , Coomassie brilliant blue staining and mass spectrometry . Seven bands pulled down by anti - GFP antibody specifically from ' rescued ' mice lysate are numbered ( this is a copy of image from our previous report with modification ) .

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

(a) Brains of the 'rescued' mice (Becn1−/−; Becn1EGFP/+) and of control Becn1+/+ littermates were lysed and subjected to immune isolation using an anti-GFP antibody. Proteins bound to anti-GFP antibody were isolated, detected and identified by SDS-PAGE, Coomassie brilliant blue staining and mass spectrometry. Seven bands pulled down by anti-GFP antibody specifically from 'rescued' mice lysate are numbered (this is a copy of image from our previous report with modification).


( b ) Mouse brain lysates were subjected to co - immunoprecipitation ( co - IP ) followed by WB analysis using the indicated antibodies .

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(b) Mouse brain lysates were subjected to co-immunoprecipitation (co-IP) followed by WB analysis using the indicated antibodies.


( c ) Mouse brain lysates were subjected to co - IP by NRBF2 antibody and followed by WB analysis using the indicated antibodies .

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(c) Mouse brain lysates were subjected to co-IP by NRBF2 antibody and followed by WB analysis using the indicated antibodies.


( d ) Gel filtration analysis . The supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10 / 30 column and each fraction was immunoblotted with the indicated antibodies . Relative amounts of each fraction determined by Odyssey software are plotted .

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(d) Gel filtration analysis. The supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10/30 column and each fraction was immunoblotted with the indicated antibodies. Relative amounts of each fraction determined by Odyssey software are plotted.


( e ) The deficiency of Atg14L impairs the protein interaction between Beclin 1 and NRBF2 . NIH3T3 cells were transfected with Atg14L or NRBF2 RNAi for 72 h , respectively , and then were lysed and subjected to IP using Beclin 1 antibody . The immunoprecipitants were immunoblotted using the indicated antibodies .

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(e) The deficiency of Atg14L impairs the protein interaction between Beclin 1 and NRBF2. NIH3T3 cells were transfected with Atg14L or NRBF2 RNAi for 72 h, respectively, and then were lysed and subjected to IP using Beclin 1 antibody. The immunoprecipitants were immunoblotted using the indicated antibodies.


( f ) Direct binding between Atg14L and NRBF2 . Flag - tagged Atg14L purified from HEK293T cells overexpressing Flag - Atg14L was incubated with GST or NRBF2 - GST purified from Escherichia coli . The GST pull - down assay was performed to examine the direct binding between Flag - Atg14L and NRBF2 - GST .

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(f) Direct binding between Atg14L and NRBF2. Flag-tagged Atg14L purified from HEK293T cells overexpressing Flag-Atg14L was incubated with GST or NRBF2-GST purified from Escherichia coli. The GST pull-down assay was performed to examine the direct binding between Flag-Atg14L and NRBF2-GST.


( b ) NRBF2 KO MEFs were transfected with CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP for 48 h . Atg14L - linked Vps34 was pulled down by an anti - Atg14L antibody and subjected to KA . IP products and inputs were immunoblotted using the antibodies indicated . ( c ) Quantification of IPed Vps34 kinase activity ( PI ( 3 ) P ) , Vps34 and Vps15 protein versus IPed Atg14L protein . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , multiple t - test are followed by Bonferroni correction , n = 4 ( otherwise indicated , n means number of independent experiments ; NS , not significant ) ) .

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(b) NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 48 h. Atg14L-linked Vps34 was pulled down by an anti-Atg14L antibody and subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (c) Quantification of IPed Vps34 kinase activity (PI(3)P), Vps34 and Vps15 protein versus IPed Atg14L protein. (Data are shown as mean±s.e.m., P values are indicated on the figures, multiple t-test are followed by Bonferroni correction, n=4 (otherwise indicated, n means number of independent experiments; NS, not significant)).


( d ) Overexpression of NRBF2 enhances Atg14L - linked Vps34kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and Flag - Atg14L in the presence of CFP or NRBF2 - CFP . Atg14L - linked Vps34 is pulled down by an anti - Flag antibody and subjected to KA ( upper panel ) . IP products and inputs were immunoblotted using the antibodies indicated ( upper panel ) . ( e ) Quantification of Vps34kinase activity ( lower panel ) shows the significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on the figures , one sample t - test versus hypothetical mean 1 , n = 5 ) .

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(d) Overexpression of NRBF2 enhances Atg14L-linked Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and Flag-Atg14L in the presence of CFP or NRBF2-CFP. Atg14L-linked Vps34 is pulled down by an anti-Flag antibody and subjected to KA (upper panel). IP products and inputs were immunoblotted using the antibodies indicated (upper panel). (e) Quantification of Vps34kinase activity (lower panel) shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


( a ) Starvation - induced long - lived protein degradation is impaired after NRBF2 knockdown . Compared with control ( con ) siRNA , NRBF2 siRNA decreases long - lived protein degradation ( proteolysis rate ) in NIH3T3 cells under starvation ( ST ) condition . This difference is diminished when the starved cells are co - treated with wortmannin ( WM , 100 nM ) . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , one - way analysis of variance with Dunnett as post hoc test , n = 4 ; NS , not significant ) .

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(a) Starvation-induced long-lived protein degradation is impaired after NRBF2 knockdown. Compared with control (con) siRNA, NRBF2 siRNA decreases long-lived protein degradation (proteolysis rate) in NIH3T3 cells under starvation (ST) condition. This difference is diminished when the starved cells are co-treated with wortmannin (WM, 100 nM). (Data are shown as mean±s.e.m., P values are indicated on the figures, one-way analysis of variance with Dunnett as post hoc test, n=4; NS, not significant).


( b ) Rapamycin ( Rap ) - induced autophagy is impaired in the NRBF2 KO cells . The MEFs were treated with ( dimethyl sulfoxide ) DMSO or 50 μg ml − 1 of Rap for 4 h then the cell lysates were immunoprobed with anti - LC3 and anti - p62 antibodies . ( c ) LC3 - II and p62 levels were quantified and normalized with β - actin . Quantification results show that Rap - induced LC3 lipidation ( upper ) and p62 degradation ( lower ) were markedly impaired in the NRBF2 KO MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , one sample t - test ( WT con versus KO con ) or unpaired t - test ( WT Rap versus KO Rap ) ) . p62 blots quantification , n = 6 ; LC3 blots quantification , n = 3 ) .

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(b) Rapamycin (Rap)-induced autophagy is impaired in the NRBF2 KO cells. The MEFs were treated with (dimethyl sulfoxide) DMSO or 50 μg ml−1 of Rap for 4 h then the cell lysates were immunoprobed with anti-LC3 and anti-p62 antibodies. (c) LC3-II and p62 levels were quantified and normalized with β-actin. Quantification results show that Rap-induced LC3 lipidation (upper) and p62 degradation (lower) were markedly impaired in the NRBF2 KO MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figures, one sample t-test (WT con versus KO con) or unpaired t-test (WT Rap versus KO Rap)). p62 blots quantification, n=6; LC3 blots quantification, n=3).


( d , e ) The MEFs were subjected to Rap ( 50 μg ml − 1 ) or starvation ( HBSS ) treatment then stained with anti - LC3 ( d ) or anti - WIPI2 ( e ) antibody . ( f ) The LC3 and WIPI2 puncta per cell were counted and quantified . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , independent samples Kruskal - Wallis test followed by pairwise comparisons , n = 30 randomly selected cells , a representative result from three independent repeats ) .

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(d,e) The MEFs were subjected to Rap (50 μg ml−1) or starvation (HBSS) treatment then stained with anti-LC3 (d) or anti-WIPI2 (e) antibody. (f) The LC3 and WIPI2 puncta per cell were counted and quantified. (Data are shown as mean±s.e.m., P values are indicated on the figures, independent samples Kruskal-Wallis test followed by pairwise comparisons, n=30 randomly selected cells, a representative result from three independent repeats).


( g ) NRBF2 - CFP stable cells were starved in HBSS for 4 h and then subjected to WIPI2 staining . Under starved condition , NRBF2 - CFP forms puncta structures that co - localized with WIPI2 . This is a representative image from three independent experiments .

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(g) NRBF2-CFP stable cells were starved in HBSS for 4 h and then subjected to WIPI2 staining. Under starved condition, NRBF2-CFP forms puncta structures that co-localized with WIPI2. This is a representative image from three independent experiments.


( h ) The NRBF2 KO MEFs were transfected with CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP for 24 h and then followed by co - immunostaining with GFP and WIPI2 antibodies . ( i ) The WIPI2 puncta number per cell in GFP - positive cells were counted and quantified . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , independent samples Kruskal - Wallis test followed by pairwise comparisons , n = 30 randomly selected cells , a representative result from two independent repeats ) . Scale bars , 20 μm , or length indicated on the figure . CM , culture medium .

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(h) The NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 24 h and then followed by co-immunostaining with GFP and WIPI2 antibodies. (i) The WIPI2 puncta number per cell in GFP-positive cells were counted and quantified. (Data are shown as mean±s.e.m., P values are indicated on the figures, independent samples Kruskal-Wallis test followed by pairwise comparisons, n=30 randomly selected cells, a representative result from two independent repeats). Scale bars, 20 μm, or length indicated on the figure. CM, culture medium.


( a ) NRBF2 KO MEFs display impaired autophagosome acidification . WT and NRBF2 KO MEFs are transiently transfected with mCherry - GFP - LC3B . WT MEFs expressing mCherry - GFP - LC3B contain many red - only puncta along with yellow ( presence of both red and green ) puncta . The number of both red - only puncta and yellow puncta markedly increases after rapamycin ( Rap ; 50 μg ml − 1 , 4 h ) or starvation ( HBSS , 4 h ) treatment , suggesting the increased autophagolysosomes and nascent autophagosomes . In contrast , NRBF2 KO MEFs expressing mCherry - GFP - LC3B contain quite fewer red - only puncta and yellow puncta , even after Rap or starvation treatment . ( b ) The quantification results show that both the numbers of red - only puncta and percentage of red - only puncta ( verses total puncta ) in KO MEFs were markedly reduced . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , Mann - Whitney U - test , n = 20 randomly selected cells , a representative result from three independent repeats ) , indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells . Scale bars , 20 μm or 10 μm ( amplified regions ) .

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(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry-GFP-LC3B. WT MEFs expressing mCherry-GFP-LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta markedly increases after rapamycin (Rap; 50 μg ml−1, 4 h) or starvation (HBSS, 4 h) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry-GFP-LC3B contain quite fewer red-only puncta and yellow puncta, even after Rap or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were markedly reduced. (Data are shown as mean±s.e.m., P values are indicated on the figures, Mann-Whitney U-test, n=20 randomly selected cells, a representative result from three independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bars, 20 μm or 10 μm (amplified regions).


( c ) Autophagolysome formation is impaired in NRBF2 KO MEFs . WT and NRBF2 KO MEFs were transiently transfected with RFP - LC3 then stained with LAMP2 antibody . The co - localization between RFP - LC3 and LAMP2 are illustrated by line profile . RFP - LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs ( as indicated by white arrows ) , suggesting the impairment of autophagolysosome formation in KO cells . Scale bars , 20 μm or 10 μm ( amplified regions ) . This is a representative image from three independent experiments .

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(c) Autophagolysome formation is impaired in NRBF2 KO MEFs. WT and NRBF2 KO MEFs were transiently transfected with RFP-LC3 then stained with LAMP2 antibody. The co-localization between RFP-LC3 and LAMP2 are illustrated by line profile. RFP-LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs (as indicated by white arrows), suggesting the impairment of autophagolysosome formation in KO cells. Scale bars, 20 μm or 10 μm (amplified regions). This is a representative image from three independent experiments.


( d ) Overexpression of NRBF2 enhances UVRAG - linked Vps34 kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and FLAG - UVRAG , in the presence of CFP or NRBF2 - CFP . UVRAG - linked Vps34 was pulled down by an anti - FLAG antibody and subjected to KA . IP products and inputs are immunoblotted using the antibodies indicated . ( e ) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on the figures , one sample t - test versus hypothetical mean 1 , n = 5 ) .

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(d) Overexpression of NRBF2 enhances UVRAG-linked Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and FLAG-UVRAG, in the presence of CFP or NRBF2-CFP. UVRAG-linked Vps34 was pulled down by an anti-FLAG antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


( a ) The gross anatomical views of representative 4 % paraformaldehyde - fixed livers and quantification of liver index . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test , n = 3 mice each group ) . Scale bar , 1 cm .

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(a) The gross anatomical views of representative 4% paraformaldehyde-fixed livers and quantification of liver index. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=3 mice each group). Scale bar, 1 cm.


( b ) The haematoxylin and eosin ( HE ) and CD45 staining of 10 - month - old WT and NRBF2 KO mice livers . The NRBF2 KO liver shows mild increase of ductular reaction ( yellow arrow ) and isolated necrosis ( red arrows ) . The necrotic foci were further confirmed by CD45 staining ( black arrow ) . This is a representative image from four mice per genotype . Scale bar , 50 μm . ( c ) The number of necrotic foci and ductular reaction scores were recorded under bright - field microscope . At least 10 randomly selected fields ( 10 × ) from each mouse and four mice per genotype were analysed . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures . Ductular reaction scores , unpaired Student ' s t - test ; necrotic foci , Mann - Whitney U - test , n = 4 mice each group ) . BD , bile duct ; CV , central vein ; PV , portal vein .

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(b) The haematoxylin and eosin (HE) and CD45 staining of 10-month-old WT and NRBF2 KO mice livers. The NRBF2 KO liver shows mild increase of ductular reaction (yellow arrow) and isolated necrosis (red arrows). The necrotic foci were further confirmed by CD45 staining (black arrow). This is a representative image from four mice per genotype. Scale bar, 50 μm. (c) The number of necrotic foci and ductular reaction scores were recorded under bright-field microscope. At least 10 randomly selected fields (10 × ) from each mouse and four mice per genotype were analysed. (Data are shown as mean±s.e.m., P values are indicated on the figures. Ductular reaction scores, unpaired Student's t-test; necrotic foci, Mann-Whitney U-test, n=4 mice each group). BD, bile duct; CV, central vein; PV, portal vein.


( a ) WB analysis shows accumulation of p62 in the liver of NRBF2 KO mice . Liver lysates were immunoprobed with antibodies indicated . p62 protein levels are upregulated in the KO mouse liver . ( b ) The quantification result shows that the p62 level increased more than threefold in the NRBF2 KO miceliver compared with WT miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test ; WT , n = 5 mice ; KO , n = 4 mice ) .

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(a) WB analysis shows accumulation of p62 in the liver of NRBF2 KO mice. Liver lysates were immunoprobed with antibodies indicated. p62 protein levels are upregulated in the KO mouse liver. (b) The quantification result shows that the p62 level increased more than threefold in the NRBF2 KO miceliver compared with WT miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test; WT, n=5 mice; KO, n=4 mice).


( c ) WB analysis shows accumulation of high - molecular weight ( HMW ) ubiquitin in the liver of NRBF2 KO mice . Insoluble fractions of liver lysates were immunoprobed with an anti - ubiquitin antibody , n = 4 mice per genotype .

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(c) WB analysis shows accumulation of high-molecular weight (HMW) ubiquitin in the liver of NRBF2 KO mice. Insoluble fractions of liver lysates were immunoprobed with an anti-ubiquitin antibody, n=4 mice per genotype.


( d ) Atg14L - linked Vps34kinase activity is impaired in the mice of NRBF2 KO mice . Atg14L - linked Vps34 is pulled down by an anti - Atg14L antibody and is subjected to KA . IP products and inputs were immunoblotted using the antibodies indicated . ( e ) Quantification of IPed Vps34kinase activity versus IPed Atg14L shows the significant difference between WT and NRBF2 KO miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 4 mice each group ) .

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(d) Atg14L-linked Vps34kinase activity is impaired in the mice of NRBF2 KO mice. Atg14L-linked Vps34 is pulled down by an anti-Atg14L antibody and is subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity versus IPed Atg14L shows the significant difference between WT and NRBF2 KO miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=4 mice each group).


( f ) Focal accumulation of p62 in the liver of NRBF2 KO mice . Liver frozen sections ( 15 μm in thickness ) were stained with an anti - p62 antibody . Focal accumulation of p62 is observed in the KO mice liver but not in the WT mice liver . Scale bars , 100 μm or 20 μm ( amplified regions ) . This is a representative image from four mice per genotype .

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(f) Focal accumulation of p62 in the liver of NRBF2 KO mice. Liver frozen sections (15 μm in thickness) were stained with an anti-p62 antibody. Focal accumulation of p62 is observed in the KO mice liver but not in the WT mice liver. Scale bars, 100 μm or 20 μm (amplified regions). This is a representative image from four mice per genotype.


( g ) Accumulated p62 colocalizes with NQO1 . Liver frozen sections were double stained with an anti - p62 antibody and an anti - NQO1 antibody . This is a representative image from four mice per genotype . Scale bars , 20 μm .

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(g) Accumulated p62 colocalizes with NQO1. Liver frozen sections were double stained with an anti-p62 antibody and an anti-NQO1 antibody. This is a representative image from four mice per genotype. Scale bars, 20 μm.


WT and NRBF2 KO MEFs were treated with DMSO , 1 μM thapsigargin ( TP ) or 1 μM tunicamycin ( TN ) for 24 or 48 h . ( a ) Then cells were either imaged under phase - contrast microscope or ( b ) stained with propidium iodide ( PI ) and subjected to cell death rate analysis by flow cytometry . The representative images of cell morphology after treatments and quantitative analysis of PI - positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 5 ) . Scale bars , 100 μm ; Con , control .

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WT and NRBF2 KO MEFs were treated with DMSO, 1 μM thapsigargin(TP) or 1 μM tunicamycin (TN) for 24 or 48 h. (a) Then cells were either imaged under phase-contrast microscope or (b) stained with propidium iodide (PI) and subjected to cell death rate analysis by flow cytometry. The representative images of cell morphology after treatments and quantitative analysis of PI-positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=5). Scale bars, 100 μm; Con, control.


( c ) Re - introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress . NRBF2 - CFP stably expressing cells were established in the NRBF2 KO background . Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry . The cell death rate in CFP - positive ( + ) population ( NRBF2 - CFP expression ) and negative ( − ) population ( no NRBF2 - CFP expression ) were recorded and analysed , respectively . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 3 ) .

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(c) Re-introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress. NRBF2-CFP stably expressing cells were established in the NRBF2 KO background. Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry. The cell death rate in CFP-positive (+) population (NRBF2-CFP expression) and negative (−) population (no NRBF2-CFP expression) were recorded and analysed, respectively. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=3).


( d ) ER stress inducers trigger caspase - 8 and caspase - 3 activation in NRBF2 KO MEFs . MEFs were treated with DMSO , 1 μM TP or 1 μM TN for 24 h , and then immunoprobed with indicated antibodies . Marked accumulation of cleaved caspase - 8 ( C - Cas 8 ; p43 and p18 units ) and cleaved caspase - 3 ( C - Cas 3 ) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN . This is a representative image from three independent experiments .

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(d) ER stress inducers trigger caspase-8 and caspase-3 activation in NRBF2 KO MEFs. MEFs were treated with DMSO, 1 μM TP or 1 μM TN for 24 h, and then immunoprobed with indicated antibodies. Marked accumulation of cleaved caspase-8 (C-Cas 8; p43 and p18 units) and cleaved caspase-3 (C-Cas 3) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN. This is a representative image from three independent experiments.


( e ) C - Cas 8 preferentially localizes at p62 aggregation foci . MEFs were treated with DMSO or 1 μM TP for 24 h , and then co - stained with an anti - p62 antibody and an anti - cleaved caspase - 8 antibody . The C - Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci . Scale bars , 20 μm . This is a representative image from three independent experiments .

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(e) C-Cas 8 preferentially localizes at p62 aggregation foci. MEFs were treated with DMSO or 1 μM TP for 24 h, and then co-stained with an anti-p62 antibody and an anti-cleaved caspase-8 antibody. The C-Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci. Scale bars, 20 μm. This is a representative image from three independent experiments.


( a ) NRBF2 is critical for the assembly of Vps34 - Vps15 and Atg14L - Beclin 1 complex . Brain lysate from WT and NRBF2 KO mice were subjected to co - IP by Vps34 antibody and Atg14L antibody . The IP products were subjected to in vitro lipid KA and WB analysis using the indicated antibodies . ( b , c ) Quantification results shows that Vps34 - Vps15 and Atg14L - Beclin 1 complex interactions as well as Atg14L - linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test , n = 4 mice in each group ; NS , not significant ) .

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(a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid KA and WB analysis using the indicated antibodies. (b,c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=4 mice in each group; NS, not significant).


( d ) Overexpression of NRBF2 enhances overall Vps34kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP . Total Vps34 was pulled down by an anti - myc antibody and subjected to KA . IP products and inputs are immunoblotted using the antibodies indicated . ( e ) Quantification of IPed Vps34kinase activity , Vps15 protein normalized with IPed Vps34 shows that NRBF2 - CFP , but not CFP , dMIT - CFP or dCCD - CFP significantly enhance Vps34kinase activity and Vps34 - Vps15 interaction . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures . IPed Vps15 / IPed Vps34 , one sample t - test versus hypothetical mean 1 , Bonferroni correction , n = 3 ; IPed Vps34kinase activity / IPed Vps34 , multiple t - test followed by Bonferroni correction , n = 3 ) .

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(d) Overexpression of NRBF2 enhances overall Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34kinase activity and Vps34-Vps15 interaction. (Data are shown as mean±s.e.m., P values are indicated on the figures. IPed Vps15/IPed Vps34, one sample t-test versus hypothetical mean 1, Bonferroni correction, n=3; IPed Vps34kinase activity/IPed Vps34, multiple t-test followed by Bonferroni correction, n=3).


b - d ) Comparative characterization of CHO cell bulk transfection cultures , selected for stable expression of the UnaG - or EGFP - encoding reporter constructs . b ) Flow cytometry revealed efficient induction of green fluorescence , after treatment of both UnaG and EGFP - expressing CHO bulk cultures with CoCl2 , while growth under hypoxia ( 1 % oxygen ) for 12 hours , selectively induced green fluorescence only in dUnaG expressing cells . Under normoxia ( 21 % oxygen ) only background fluorescence was observed . c ) Assessment of the mean fluorescence intensity ( MFI ) to determine the activity of the 5xHRE - CMV promoter after 12 and 24hrs . Fluorescence intensity increased between 12 and 24hrs .

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b - d) Comparative characterization of CHO cell bulk transfection cultures, selected for stable expression of the UnaG- or EGFP-encoding reporter constructs.b) Flow cytometry revealed efficient induction of green fluorescence, after treatment of both UnaG and EGFP-expressing CHO bulk cultures with CoCl2, while growth under hypoxia (1% oxygen) for 12 hours, selectively induced green fluorescence only in dUnaG expressing cells. Under normoxia (21% oxygen) only background fluorescence was observed.c) Assessment of the mean fluorescence intensity (MFI) to determine the activity of the 5xHRE-CMV promoter after 12 and 24hrs. Fluorescence intensity increased between 12 and 24hrs.


d ) Representative images of dUnaG - or dEGFP - expressing CHO cells grown either under normoxia ( 21 % oxygen ) , CoCl2 - treatment or hypoxia ( 1 % oxygen ) for 12 hours . Scale bars =

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d) Representative images of dUnaG- or dEGFP-expressing CHO cells grown either under normoxia (21% oxygen), CoCl2-treatment or hypoxia (1% oxygen) for 12 hours. Scale bars= 50µm


e ) Fluorescence intensity in dUnaG - or dEGFP - expressing CHO cell bulks grown under increasingly hypoxic conditions of 10 % , 5 % and 1 % oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5 % .

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e) Fluorescence intensity in dUnaG- or dEGFP-expressing CHO cell bulks grown under increasingly hypoxic conditions of 10%, 5% and 1% oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5%.


f ) Maximal fluorescence of the UnaG - based hypoxia reporter in CHO cells grown at 1 % oxygen for 24 hrs hours was only observed at serum concentrations of 5 % FCS or higher , reflecting the dependence of UnaG on its serum cofactor bilirubin .

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f) Maximal fluorescence of the UnaG-based hypoxia reporter in CHO cells grown at 1% oxygen for 24 hrs hours was only observed at serum concentrations of 5% FCS or higher, reflecting the dependence of UnaG on its serum cofactor bilirubin.


b ) Microscopic assessment of the averaged fluorescence intensity ( AFI ) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP . Expression under hypoxia was induced by incubation in 1 % oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia . Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly - destabilized UnaG - variants , while the doubly destabilized doUnaG displayed the most rapid switching behavior , but the least brightness . To reduce phototoxicity , illumination intensity was kept minimal . Data points represent one viewfield of 425 µm2 ± SEM .

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b) Microscopic assessment of the averaged fluorescence intensity (AFI) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP. Expression under hypoxia was induced by incubation in 1% oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia. Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly-destabilized UnaG-variants, while the doubly destabilized doUnaG displayed the most rapid switching behavior, but the least brightness. To reduce phototoxicity, illumination intensity was kept minimal. Data points represent one viewfield of 425 µm2 ± SEM.


c ) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG . Open bars represent control cultures grown under normoxia . Solid bars indicate culture for 4 , 8 , 12 , 16 , 20 or 24 hrs under hypoxia ( 1 % oxygen ) . To assess deactivation , cultures previously kept under hypoxia ( 1 % oxygen ) for 24hrs were shifted to normoxia ( 21 % oxygen ) for the indicated times . Error bars represent standard errors .

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c) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG. Open bars represent control cultures grown under normoxia. Solid bars indicate culture for 4, 8, 12, 16, 20 or 24 hrs under hypoxia (1% oxygen). To assess deactivation, cultures previously kept under hypoxia (1% oxygen) for 24hrs were shifted to normoxia (21% oxygen) for the indicated times. Error bars represent standard errors.


d ) Representative maximum intensity projections ( MIPs ) from the life cell cultures depicted in b . Cells were grown for the indicated times at hypoxic ( 1 % oxygen ) conditions and subsequently switched for the indicated times to normoxia ( 21 % oxygen ) ( red arrows in b ) to illustrate the dynamics of the different UnaG - based hypoxia sensors . Increasing destabilization is associated with reduced brightness but , also reduced background and improved switching behavior . Scale bars = 50 µm .

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d) Representative maximum intensity projections (MIPs) from the life cell cultures depicted in b. Cells were grown for the indicated times at hypoxic (1% oxygen) conditions and subsequently switched for the indicated times to normoxia (21% oxygen)(red arrows in b) to illustrate the dynamics of the different UnaG-based hypoxia sensors. Increasing destabilization is associated with reduced brightness but, also reduced background and improved switching behavior. Scale bars = 50 µm.


a ) UnaG - based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model . 500 Gli36 glioblastoma cells , constitutively expressing mCherry and stably transfected with the HRE - dUnaG sensor construct , were stereotactically transplanted into the cortex of a SCIDmouse . Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation . Tumor cells are distinguished from the surrounding cortex by mCherry expression . Blood vessels were contrasted by immunostaining against PECAM - 1 . Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density ( outlined by white line in the composite panel ( bottom right ) . b ) Representative area from the tumor shown in a ) , which is located outside the viewfield in a ) positioned more closely to the tumor border . Also at the tumor border UnaG - expressing cells are preferentially observed at a distance to PECAM - 1 + vessels .

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a) UnaG-based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model. 500 Gli36 glioblastoma cells, constitutively expressing mCherry and stably transfected with the HRE-dUnaG sensor construct, were stereotactically transplanted into the cortex of a SCIDmouse. Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation. Tumor cells are distinguished from the surrounding cortex by mCherry expression. Blood vessels were contrasted by immunostaining against PECAM-1. Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density (outlined by white line in the composite panel (bottom right).b) Representative area from the tumor shown in a), which is located outside the viewfield in a) positioned more closely to the tumor border. Also at the tumor border UnaG-expressing cells are preferentially observed at a distance to PECAM-1+ vessels.


c ) Areas of dUnaG expression correlate well with HIF - 1α stabilization . Immunostaining for HIF - 1α ( cyan ) revealed the predominantly nuclear localization of stabilized HIF - 1α in cells that were also dUnaG - positive ( green ) . d ) Analysis of dUnaG expressing cells in c ) for mCherry fluorescence and HIF - 1α stabilization . dUnaG - positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF - 1α The threshold was set in either case to channel 70 of 256 intensity channels . Using this classification > 60 % of UnaG positive cells displayed only background level mCherry fluorescence . On the other hand , > 98 % of the UnaG expressing cells also expressed HIF - 1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo .

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c) Areas of dUnaG expression correlate well with HIF-1α stabilization. Immunostaining for HIF-1α (cyan) revealed the predominantly nuclear localization of stabilized HIF-1α in cells that were also dUnaG-positive (green).d) Analysis of dUnaG expressing cells in c) for mCherry fluorescence and HIF-1α stabilization. dUnaG-positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF-1α The threshold was set in either case to channel 70 of 256 intensity channels. Using this classification >60% of UnaG positive cells displayed only background level mCherry fluorescence. On the other hand, >98% of the UnaG expressing cells also expressed HIF-1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo.


e ) The dUnaG - based senor was efficiently applied in intravital microscopy . 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG - sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy . We noted two distinct areas of intense UnaG fluorescence ( red lines ) . Shown is a MIP of a 60 µm z stack . The vasculature was contrasted by intravenous injection of non - functionalized Qdots . f ) Depth color coding of the MIP shown in e ) revealed the spatial relation between the vasculature , ( contrasted by Qdots ) , areas of increased dUnaG expression ( bordered by green lines ) and tumor cells expressing residual levels of mCherry . The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner ( red asterisk ) and the smaller vessels with a more axial orientation ( white arrows ) . Scale bars =

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e) The dUnaG-based senor was efficiently applied in intravital microscopy. 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG-sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy. We noted two distinct areas of intense UnaG fluorescence (red lines). Shown is a MIP of a 60 µm z stack. The vasculature was contrasted by intravenous injection of non-functionalized Qdots.f) Depth color coding of the MIP shown in e) revealed the spatial relation between the vasculature, (contrasted by Qdots), areas of increased dUnaG expression (bordered by green lines) and tumor cells expressing residual levels of mCherry. The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner (red asterisk) and the smaller vessels with a more axial orientation (white arrows). Scale bars = 100µm


b ) Microscopic assessment of the averaged fluorescence intensity ( AFI ) in CHO cells stably transfected with the dUnOHR sensor . Hypoxia was induced by incubation in 1 % oxygen for 18 hrs , then the culture was switched to normoxia for 24 hrs , followed by another 14 hrs hypoxia and finally normoxia again . As expected UnaG fluorescence is efficiently induced under hypoxia , while mOrange fluorescence only appears after the switch to normoxia only . The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE - mCMV promoter activity . This behavior is repeated in subsequent hypoxia / normoxia cycles . The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period . Plotted is the average of the mean ± SEM . c ) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b . ) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange . Scale bars = 100 µm .

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b) Microscopic assessment of the averaged fluorescence intensity (AFI) in CHO cells stably transfected with the dUnOHR sensor. Hypoxia was induced by incubation in 1% oxygen for 18 hrs, then the culture was switched to normoxia for 24 hrs, followed by another 14 hrs hypoxia and finally normoxia again. As expected UnaG fluorescence is efficiently induced under hypoxia, while mOrange fluorescence only appears after the switch to normoxia only. The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE-mCMV promoter activity. This behavior is repeated in subsequent hypoxia / normoxia cycles. The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period. Plotted is the average of the mean ± SEM.c) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b.) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange. Scale bars = 100 µm.


a ) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct . The perimeter of the developing tumor ( white line in the merged picture , lower right panel ) was delineated by Hoechst 33342 staining . As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density . Vascular endothelium was identified by PECAM - 1 staining . 30 µm cryosection . Scale bar = 500µm . b ) Details of the tumor shown in a ) . Sparse , distinct cells are characterized by intense green and orange fluorescence ( shown as orange ) and are located preferentially to the edge of the hypoxic phalanx ( white arrows ) . A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence ( asterisks ) , indicative of previously hypoxic environment and subsequent reoxygenation . Scale bar = 50 µm .

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

a) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct. The perimeter of the developing tumor (white line in the merged picture, lower right panel) was delineated by Hoechst 33342 staining. As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density. Vascular endothelium was identified by PECAM-1 staining. 30 µm cryosection. Scale bar = 500µm.b) Details of the tumor shown in a). Sparse, distinct cells are characterized by intense green and orange fluorescence (shown as orange) and are located preferentially to the edge of the hypoxic phalanx (white arrows). A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence (asterisks), indicative of previously hypoxic environment and subsequent reoxygenation. Scale bar = 50 µm.


c ) Recently reoxygenated cells ( arrows ) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber ( double arrows ) . Scale bar = 100 µm .

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c) Recently reoxygenated cells (arrows) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber (double arrows). Scale bar = 100 µm.


d ) Previously hypoxic cells , frequently undergo apoptosis after reoxygenation . We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas . Scale bar = 50µm .

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d) Previously hypoxic cells, frequently undergo apoptosis after reoxygenation. We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas. Scale bar = 50µm.


e ) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis . Scale bar = 50µm .

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e) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis. Scale bar = 50µm.


Ookinete formation in vitro . After 12 h of culture , the ookinetes were Giemsa - stained and analyzed for ookinete morphology . The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes .

Ookinete formation in vitro. After 12 h of culture, the ookinetes were Giemsa-stained and analyzed for ookinete morphology. The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes.


Oocyst counts in the mosquitoes at day 7 post blood - feeding . x / y on the top is the number of mosquitoes containing oocyst / the number of mosquitoes dissected ; the percentage number is the mosquito infection prevalence .

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Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence.


Co - staining of P28 and GAP45 ( IMC protein ) in female gametes ( P28 + only ) and zygotes ( P28 + / GAP45 + ) of the 17XNL , Δap2 - o3 and Δcdpk4 strains . Percentages of female gametes and zygotes were indicated respectively . n is the number of cells counted . Scale bars = 5 μm . Note : the signal of P28 and GAP45 is reduced similarly in all Δap2 - o3 female gametes and zygotes compared to that of 17XNL .

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Co-staining of P28 and GAP45 (IMC protein) in female gametes (P28+ only) and zygotes (P28+/GAP45+) of the 17XNL, Δap2-o3 and Δcdpk4 strains. Percentages of female gametes and zygotes were indicated respectively. n is the number of cells counted. Scale bars = 5 μm. Note: the signal of P28 and GAP45 is reduced similarly in all Δap2-o3 female gametes and zygotes compared to that of 17XNL.


Day 7 midgut oocyst counts from mosquitoes infected with parasites , including 17XNL , Δap2 - o3 , Δmap2 , or Δnek4 strain alone as well as mixtures of Δmap2 / Δnek4 , Δap2 - o3 / Δmap2 , and Δap2 - o3 / Δnek4 . Δnek4 and Δmap2 are female and male gamete - defect parasites , respectively . n is the number of mosquitoes dissected , Mann - Whitney test applied , two experiments repeated .

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Day 7 midgut oocyst counts from mosquitoes infected with parasites, including 17XNL, Δap2-o3, Δmap2, or Δnek4 strain alone as well as mixtures of Δmap2/Δnek4, Δap2-o3/Δmap2, and Δap2-o3/Δnek4. Δnek4 and Δmap2 are female and male gamete-defect parasites, respectively. n is the number of mosquitoes dissected, Mann-Whitney test applied, two experiments repeated.


Transcript expression heatmap by RNA - seq of 26 genes involving DNA replication and DNA repair between DFsc7 ( WT ) and DFsc7 ; Δap2 - o3 ( KO ) strains .

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Transcript expression heatmap by RNA-seq of 26 genes involving DNA replication and DNA repair between DFsc7 (WT) and DFsc7;Δap2-o3 (KO) strains.


Representative fluorescence microscopy images of mCherry and DPOD2 : : GFP expression in female and male gametocytes of the DTS1 ( ccp2 : : mCherry ; dpod2 : : gfp ) and DTS1 ; Δap2 - o3 strains . mCherry is specifically expressed in female gametocytes . Flow cytometry detection of mCherry and DPOD2 : : GFP expression in female and male gametocytes of the DTS1 and DTS1 ; Δap2 - o3 strains . The female and male gametocyte populations are circled by solid and dashed line respectively . E , F Similar analysis ( as in C , D ) in DTS2 ( ccp2 : : mCherry ; dpod1 : : gfp ) and DTS2 ; Δap2 - o3 strains . G , H Similar analysis ( as in C , D ) in DTS3 ( ccp2 : : mCherry ; rpa1 : : gfp ) and DTS3 ; Δap2 - o3 strains . I , J Similar analysis ( as in C , D ) in DTS4 ( ccp2 : : mCherry ; mcm7 : : gfp ) and DTS4 ; Δap2 - o3 strains .

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Representative fluorescence microscopy images of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 (ccp2::mCherry;dpod2::gfp) and DTS1;Δap2-o3 strains. mCherry is specifically expressed in female gametocytes. Flow cytometry detection of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 and DTS1;Δap2-o3 strains. The female and male gametocyte populations are circled by solid and dashed line respectively. E,F Similar analysis (as in C, D) in DTS2 (ccp2::mCherry;dpod1::gfp) and DTS2;Δap2-o3 strains. G,H Similar analysis (as in C, D) in DTS3 (ccp2::mCherry;rpa1::gfp) and DTS3;Δap2-o3 strains. I,J Similar analysis (as in C, D) in DTS4 (ccp2::mCherry;mcm7::gfp) and DTS4;Δap2-o3 strains.


EMSA using the recombinant GST - fused AP2 domain of AP2 - O3 and a synthesized DNA probe containing three repeats of predicted motif . GST was used as a negative control . An arrowhead indicates the shifted band .

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EMSA using the recombinant GST-fused AP2 domain of AP2-O3 and a synthesized DNA probe containing three repeats of predicted motif. GST was used as a negative control. An arrowhead indicates the shifted band.


ChIP - qPCR validation of the binding between AP2 - O3 and the upstream region of four male genes The regions detected via ChIP - qPCR are indicated by pink line ( in the peak ) and purple line ( out of the peak ) . mean ± SEM from three independent experiments .

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ChIP-qPCR validation of the binding between AP2-O3 and the upstream region of four male genes The regions detected via ChIP-qPCR are indicated by pink line (in the peak) and purple line (out of the peak). mean±SEM from three independent experiments.


qRT - PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID .

qRT-PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID.


Western blot of GAP45 protein expression in non - activated ( NAG ) and activated gametocyte ( AG ) of 17XNL and Δap2 - o3 strains . BiP as loading control .

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Western blot of GAP45 protein expression in non-activated (NAG) and activated gametocyte (AG) of 17XNL and Δap2-o3 strains. BiP as loading control.


IFA of AP2 - O3 protein expression in activated gametocytes of Pccp2 strains .

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IFA of AP2-O3 protein expression in activated gametocytes of Pccp2 strains.


52 - day - old ( A ) plants of the genotypes indicated grown in LDs . Scale bars : 5 cm . B - C The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A the numbers indicate independent transgenic lines . The letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes . Data information : In ( B - C data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD test ) .

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52-day-old (A) plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B-C The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B-C data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).


D - E qPCR results showing mRNA expression of CIB1 , FT in the genotypes indicated . The numbers indicate independent transgenic lines . The transcript levels of CIB1 or FT in Col were set to 1 . 0 . Samples were collected from 7 - day - old LD grown seedlings at ZT16 . Data information : error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed .

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D-E qPCR results showing mRNA expression of CIB1, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CIB1 or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.


F . 25 - day - old plants ( F ) of the genotypes indicated grown in LDs . Scale bars : 5 cm . , G - H . The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in the numbers indicate independent transgenic lines . The letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes . Data information : In G - H ) , data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD test ) .

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F. 25-day-old plants (F) of the genotypes indicated grown in LDs. Scale bars: 5 cm. , G-H. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in the numbers indicate independent transgenic lines. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In G-H), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).


, I - J . qPCR results showing mRNA expression of CO , FT in the genotypes indicated . The numbers indicate independent transgenic lines . The transcript levels of CO or FT in Col were set to 1 . 0 . Samples were collected from 7 - day - old LD grown seedlings at ZT16 . Data information : error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed .

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, I-J. qPCR results showing mRNA expression of CO, FT in the genotypes indicated. The numbers indicate independent transgenic lines. The transcript levels of CO or FT in Col were set to 1.0. Samples were collected from 7-day-old LD grown seedlings at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.


Immunoblots showing CIB1 ( A ) protein levels in WT and co - 1 backgrounds ( A ) over the course of 20 hours under LDs . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , stripped and re - probed with the anti - actin antibody ( ACT ) .

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Immunoblots showing CIB1 (A) protein levels in WT and co-1 backgrounds (A) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).


B . Immunoblots showing CO ( B ) protein levels in WT and cibh backgrounds ( B ) over the course of 20 hours under LDs . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , stripped and re - probed with the anti - actin antibody ( ACT ) .

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B. Immunoblots showing CO (B) protein levels in WT and cibh backgrounds (B) over the course of 20 hours under LDs. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-actin antibody (ACT).


The relative levels of CIB1 in WT and co - 1 backgrounds over the course of 20 hours under LDs were calculated Data information : Error bars represent the standard deviation of three biological replicates of the Western blots .

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The relative levels of CIB1 in WT and co-1 backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.


D . The relative levels of CO proteins in WT and cibh backgrounds over the course of 20 hours under LDs were calculated Data information : Error bars represent the standard deviation of three biological replicates of the Western blots .

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D. The relative levels of CO proteins in WT and cibh backgrounds over the course of 20 hours under LDs were calculated Data information: Error bars represent the standard deviation of three biological replicates of the Western blots.


A . Fluorescent microscopy images showing that CIB1 co - localizes with CO in nuclear speckles . Scale bars : 2 μm .

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A. Fluorescent microscopy images showing that CIB1 co-localizes with CO in nuclear speckles. Scale bars: 2 μm.


B . BiFC assays to show in vivo protein interactions . Epidermal cells of N . benthamiana leaves were co - transformated with cCFP - CO and nYFP - CIB1 , with cCFP - CO and nYFP , or with cCFP and nYFP - CIB1 . BF , bright field . Merge , overlay of the YFP and bright field images . Scale bars : 50 μm . Data information : For each experiment , two or more biological replicates were performed .

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B. BiFC assays to show in vivo protein interactions. Epidermal cells of N. benthamiana leaves were co-transformated with cCFP-CO and nYFP-CIB1, with cCFP-CO and nYFP, or with cCFP and nYFP-CIB1. BF, bright field. Merge, overlay of the YFP and bright field images. Scale bars: 50 μm. Data information: For each experiment, two or more biological replicates were performed.


C . Split - LUC assay showing that CIB1 interacts with CO . Leaf epidermal cells of N . benthamiana were co - transformed with CO - nLUC and cLUC - CIB1 or cLUC or nLUC with cLUC - CIB or cLUC . Data information : For each experiment , two or more biological replicates were performed .

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C. Split-LUC assay showing that CIB1 interacts with CO. Leaf epidermal cells of N. benthamiana were co-transformed with CO-nLUC and cLUC-CIB1 or cLUC or nLUC with cLUC-CIB or cLUC. Data information: For each experiment, two or more biological replicates were performed.


D . Co - IP assays of samples prepared from 10 - day - old 35S : Myc - CIB1 , 35S : CO - Flag or 35S : Myc - CIB1 / 35S : CO - Flag seedlings grown in LDs . Data information : For each experiment , two or more biological replicates were performed .

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D. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1, 35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag seedlings grown in LDs. Data information: For each experiment, two or more biological replicates were performed.


F . In vitro pull - down assays showing the interaction between CIBC and CO , or CIBC and COΔN and the lack of interaction between CIB1C and COΔC . His - TF tagged CO , COΔN , COΔC or His - TF tagged UVR8 were mixed with GST - CIB1C purified from E . coli , and His antibody was used for the in vitro pull - down assay . The products were analyzed by immunoblots probed with the anti - CIB1C antibody or the anti - His antibody ( UVR8 , CO , COΔC , COΔN ) . Data information : For each experiment , two or more biological replicates were performed .

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F. In vitro pull-down assays showing the interaction between CIBC and CO, or CIBC and COΔN and the lack of interaction between CIB1C and COΔC. His-TF tagged CO, COΔN, COΔC or His-TF tagged UVR8 were mixed with GST-CIB1C purified from E. coli, and His antibody was used for the in vitro pull-down assay. The products were analyzed by immunoblots probed with the anti-CIB1C antibody or the anti-His antibody (UVR8, CO, COΔC, COΔN). Data information: For each experiment, two or more biological replicates were performed.


A . 28 - day - old plants of the genotypes indicated grown in LDs . Scale bars : 5 cm . B , C . The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A . The letters " a " to " c " indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes . Data information : In ( B , C ) , data are presented as mean ± SD , n ≥ 15 per genotypes indicated . P ≤ 0 . 05 ( Tukey ' s HSD test ) .

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A. 28-day-old plants of the genotypes indicated grown in LDs. Scale bars: 5 cm. B, C. The number of rosette leaves and the time to flowering at the time of flowering of the genotypes shown in A. The letters "a" to "c" indicate statistically significant differences between flowering time or leaf numbers of the indicated genotypes. Data information: In (B, C), data are presented as mean ± SD, n ≥ 15 per genotypes indicated. P≤0.05 (Tukey's HSD test).


D . Immunoblots showing Myc - CIB1 and CO - Flag protein levels in the genotypes indicated . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , stripped and re - probed with the anti - Flag antibody .

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D. Immunoblots showing Myc-CIB1 and CO-Flag protein levels in the genotypes indicated. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, stripped and re-probed with the anti-Flag antibody.


E . qPCR results showing mRNA expression of FT in the genotypes indicated . The transcript levels in Col are set to 1 . 0 . Samples were collected from 7 - day - old seedlings of the genotypes indicated at ZT16 . Data information : error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed .

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E. qPCR results showing mRNA expression of FT in the genotypes indicated. The transcript levels in Col are set to 1.0. Samples were collected from 7-day-old seedlings of the genotypes indicated at ZT16. Data information: error bars indicate standard error within three biological replicates and for each of these two technical replicates were performed.


G . Box plots ( the minimum , first quartile , median , third quartile , and maximum are shown , with data > 1 . 5 interquartile ranges denoted with circles ) for relative reporter activity ( LUC / REN ) in plants expressing different reportors and effectors . The relative LUC activities normalized to the REN activity are shown . The LUC / REN in plants expressing ProFT alone is set to 1 . 0 . Data information : data are presented as mean ± SD , n = 4 . The letters " a " to " g " indicate statistically significant differences determined by Tukey ' s HSD test ( P ≤ 0 . 05 ) . Two biological replicates were performed .

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

G. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The relative LUC activities normalized to the REN activity are shown. The LUC/REN in plants expressing ProFT alone is set to 1.0. Data information: data are presented as mean ± SD, n=4. The letters "a" to "g" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.


A . Fluorescent microscopy images showing that CRY2 , CIB1 and CO co - localized to the nucleus . Scale bars : 2

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A. Fluorescent microscopy images showing that CRY2, CIB1 and CO co-localized to the nucleus. Scale bars: 2 μ


B . Co - IP assays showing that CRY2 , CIB1 and CO form a protein complex . Samples were prepared from 10 - day - old WT or 35S : Myc - CIB1 / 35S : CO - Flag or 35S : Myc - CIB1 / 35S : CO - Flag / cry2 - 1 seedlings grown in LDs ( 16L / 8D ) , and samples were collected at ZT16 .

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B. Co-IP assays showing that CRY2, CIB1 and CO form a protein complex. Samples were prepared from 10-day-old WT or 35S:Myc-CIB1/35S:CO-Flag or 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D), and samples were collected at ZT16.


C . Co - IP assays of samples prepared from 10 - day - old 35S : Myc - CIB1 / 35S : CO - Flag 35S : Myc - CIB1 / 35S : CO - Flag / cry2 - 1 seedlings grown in LDs ( 16L / 8D ) . Samples were collected at the times indicated over the course of one day .

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C. Co-IP assays of samples prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag 35S:Myc-CIB1/35S:CO-Flag /cry2-1 seedlings grown in LDs (16L/8D). Samples were collected at the times indicated over the course of one day.


E . Representative results of the ChIP - qPCR assays . Chromatin fragments ( ~ 500 bp ) were prepared from 10 - day - old 35S : Myc - CIB1 / 35S : CO - Flag seedlings , immunoprecipitated by the anti - CRY2 , anti - Flag ( CO - Flag ) , anti - Myc ( Myc - CIB1 ) antibody or anti - preimmune IgG , and the precipitated DNA was analyzed by qPCR using the primer pairs indicated . The IP / input ratio of anti - preimmune is set to 1 . 0 . Data information : the relative IP / input ratios are shown with their standard deviation ( n = 3 ) . Two biological replicates were performed .

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

E. Representative results of the ChIP-qPCR assays. Chromatin fragments (~500 bp) were prepared from 10-day-old 35S:Myc-CIB1/35S:CO-Flag seedlings, immunoprecipitated by the anti-CRY2, anti-Flag (CO-Flag) , anti-Myc (Myc-CIB1) antibody or anti-preimmune IgG, and the precipitated DNA was analyzed by qPCR using the primer pairs indicated. The IP/input ratio of anti-preimmune is set to 1.0. Data information: the relative IP/input ratios are shown with their standard deviation (n = 3). Two biological replicates were performed.


F . Box plots ( the minimum , first quartile , median , third quartile , and maximum are shown , with data > 1 . 5 interquartile ranges denoted with circles ) for relative reporter activity ( LUC / REN ) in plants expressing different reportors and effectors . The LUC / REN in plants expressing ProFT alone is set to 1 . 0 . Immunoblots were done to show GFP - CRY2W374A , Myc - CIB1 and Myc - CO protein levels in the transient Dual - LUC assay . Samples were separated on 10 % SDS - PAGE , blotted , probed with the anti - Myc antibody , and with the anti - GFP antibody . Data information : the relative LUC activities normalized to the REN activity are shown ( LUC / REN ) with their standard deviation ( n = 6 ) . The letters " a " to " f " indicate statistically significant differences determined by Tukey ' s HSD test ( P ≤ 0 . 05 ) . Two biological replicates were performed .

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

F. Box plots (the minimum, first quartile, median, third quartile, and maximum are shown, with data >1.5 interquartile ranges denoted with circles) for relative reporter activity (LUC/REN) in plants expressing different reportors and effectors. The LUC/REN in plants expressing ProFT alone is set to 1.0. Immunoblots were done to show GFP-CRY2W374A, Myc-CIB1 and Myc-CO protein levels in the transient Dual-LUC assay. Samples were separated on 10% SDS-PAGE, blotted, probed with the anti-Myc antibody, and with the anti-GFP antibody. Data information: the relative LUC activities normalized to the REN activity are shown (LUC/REN) with their standard deviation (n =6). The letters "a" to "f" indicate statistically significant differences determined by Tukey's HSD test (P≤0.05). Two biological replicates were performed.


Confocal z - projections of the indicated genotypes and age , labeled with membrane bound GFP ( mCD8 - GFP ; CD8 ) driven by the γ - specific Gal4 driver GMR71G10 - Gal4 ( γ - Gal4 ) . While γ - axons of control flies project through the entire lobe ( C is the RNAi control ; n = 12 / 12 , E ; is the tsCRISPR control ; n = 14 / 14 ) , knockdown of dpr12 by RNAi ( D ; n = 12 / 12 ) or knockout by tsCRISPR ( F ; n = 14 / 14 ) resulted in short axons . At L3 , γ - axons in dpr12 ∆ 50 - 81 homozygotus mutant animals ( K ; n = 20 / 20 ) resemble WT γ - axons ( G ; n = 20 / 20 ) . At 48hr APF , γ - axons normally re - extend to form the adult lobe ( H ; n = 12 / 12 ) . dpr12 ∆ 50 - 81 γ - axons ( L ; n = 14 / 14 ) fail to extend to the end of the lobe . This defect persists to adult ( I ; n = 11 / 11 vs . M ; n = 18 / 18 ) . Expressing a UAS - Dpr12 transgene within γ - KCs in dpr12 ∆ 50 - 81 homozygote mutant animals rescued the axon regrowth defect ( N ; n = 23 / 24 , J ; n = 14 / 14 ) . The adult γ - lobe and α / β lobes are outlined in ( C , D ) in yellow and orange , respectively , for clarity . Asterisks demarcate the distal part of the lobe . Green and white indicate mCD8 - GFP . Magenta represents FasII staining . Scale bar is 20 µm .

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

Confocal z-projections of the indicated genotypes and age, labeled with membrane bound GFP (mCD8-GFP; CD8) driven by the γ-specific Gal4 driver GMR71G10-Gal4 (γ-Gal4). While γ-axons of control flies project through the entire lobe (C is the RNAi control; n=12/12, E; is the tsCRISPR control; n=14/14), knockdown of dpr12 by RNAi (D; n=12/12) or knockout by tsCRISPR (F; n=14/14) resulted in short axons. At L3, γ-axons in dpr12∆50-81 homozygotus mutant animals (K; n=20/20) resemble WT γ-axons (G; n=20/20). At 48hr APF, γ-axons normally re-extend to form the adult lobe (H; n=12/12). dpr12∆50-81 γ-axons (L; n=14/14) fail to extend to the end of the lobe. This defect persists to adult (I; n=11/11 vs. M; n=18/18). Expressing a UAS-Dpr12 transgene within γ-KCs in dpr12∆50-81 homozygote mutant animals rescued the axon regrowth defect (N; n=23/24, J; n=14/14). The adult γ-lobe and α/β lobes are outlined in (C, D) in yellow and orange, respectively, for clarity. Asterisks demarcate the distal part of the lobe. Green and white indicate mCD8-GFP. Magenta represents FasII staining. Scale bar is 20 µm.


Confocal z - projections of MARCM neuroblast ( NB , A - F ) and single - cell ( SC , G - L ) clones labeled with membrane bound GFP ( mCD8 - GFP ; CD8 ) driven by the γ specific Gal4 driver GMR71G10 - Gal4 ( γ - Gal4 ) . At L3 , NB and SC clones expressing dpr12 RNAi are similar to equivalent WT clones ( A ; n = 20 / 20 , D ; n = 15 / 15 , G ; n = 15 / 15 and J ; n = 17 / 17 ) . At 48hr APF and adult stage , WT NB ( B ; n = 15 / 15 , C ; n = 10 / 10 ) and SC ( H ; n = 16 / 16 , I ; n = 13 / 13 ) clones extend their axons to form the full adult lobe . In contrast , clones expressing dpr12 RNAi ( E ; n = 14 / 14 , F ; n = 22 / 2 , K ; n = 18 / 24 , L ; n = 19 / 27 ) fail to extend their axons to the distal part of the medial lobe ( asterisks ) . I ' and L ' are traces of multiple single - cell clones depicting each cell in a different color .

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

Confocal z-projections of MARCM neuroblast (NB, A-F) and single-cell (SC, G-L) clones labeled with membrane bound GFP (mCD8-GFP; CD8) driven by the γ specific Gal4 driver GMR71G10-Gal4 (γ-Gal4). At L3, NB and SC clones expressing dpr12 RNAi are similar to equivalent WT clones (A; n=20/20, D; n=15/15, G; n=15/15 and J; n=17/17). At 48hr APF and adult stage, WT NB (B; n=15/15, C; n=10/10) and SC (H; n=16/16, I; n=13/13) clones extend their axons to form the full adult lobe. In contrast, clones expressing dpr12 RNAi (E; n=14/14, F; n=22/2, K; n=18/24, L; n=19/27) fail to extend their axons to the distal part of the medial lobe (asterisks). I' and L' are traces of multiple single-cell clones depicting each cell in a different color.


Confocal z - projections of brains expressing MiMIC mediated Dpr12GFSTF ( Dpr12 - GFP ; A - D ) and DIP - δGFSTF ( DIP - δ - GFP ; E - H ) fusion proteins at the indicated time points . See Figures EV 1 and 4 for more details on the fusion protein structure . ( A - D ) Dpr12 - GFP is localized to the distal part of the γ - lobe at L3 ( A ; n = 10 / 10 ) , 48hr APF ( C ; n = 12 / 12 ) and the adult stage ( D ; n = 20 / 20 ) , where it colocalizes with the γ4 > γ1γ2 MBON ( γ4 ; labeled by GMR18H09 - Gal4 driving the expression of CD4 - tdT ) . At 24hr APF ( B ; n = 10 / 10 ) , Dpr12 - GFP appears diffuse . ( E - H ) DIP - δ - GFP is localized to the distal part of the γ - lobe throughout development : L3 ( E ; n = 16 / 16 ) , 24hr APF ( F ; n = 10 / 10 ) , 48hr APF ( G ; n = 12 / 12 ) and adult ( H ; n = 24 / 24 ) . At the adult stage , DIP - δ - GFP is colocalized with the γ4 > γ1γ2 MBON ( γ4 ) .

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Confocal z-projections of brains expressing MiMIC mediated Dpr12GFSTF (Dpr12-GFP; A-D) and DIP-δGFSTF (DIP-δ-GFP; E-H) fusion proteins at the indicated time points. See Figures EV 1 and 4 for more details on the fusion protein structure. (A-D) Dpr12-GFP is localized to the distal part of the γ-lobe at L3 (A; n=10/10), 48hr APF (C; n=12/12) and the adult stage (D; n=20/20), where it colocalizes with the γ4>γ1γ2 MBON (γ4; labeled by GMR18H09-Gal4 driving the expression of CD4-tdT). At 24hr APF (B; n=10/10), Dpr12-GFP appears diffuse. (E-H) DIP-δ-GFP is localized to the distal part of the γ-lobe throughout development: L3 (E; n=16/16), 24hr APF (F; n=10/10), 48hr APF (G; n=12/12) and adult (H; n=24/24). At the adult stage, DIP-δ-GFP is colocalized with the γ4>γ1γ2 MBON (γ4).


Confocal z - projections DIP - δ hetero - and homozygous brains in which γ - KCs were labeled by expressing membrane bound tandem tomato ( mtdT - HA ) driven by the γ specific QF2 driver R71G10 - QF2 ( γ - QF2 ) . Larval ( L3 ) γ - axons grow normally in DIP - δT2A - Gal4 heterozygotes ( A ; n = 16 / 16 ) and homozygotes ( E ; n = 24 / 24 ) . In contrast , at 48hr APF and adult , γ - axons within DIP - δT2A - Gal4 homozygotes do not enter the distal part of the lobe ( asterisks ; F ; n = 8 / 8 , G ; n = 20 / 20 ) , while they grow normally in heterozygotes ( B ; n = 12 / 12 , C ; n = 12 / 12 ) . Overexpression of a DIP - δ transgene driven by the Gal4 activity of DIP - δT2A - Gal4 ( see also Figure S4 ) does not affect normal growth ( D ; n = 10 / 10 ) and rescues mutant phenotypes ( H ; n = 20 / 20 ) . Note that while the R71G10 driver is consistently expressed in γ - KCs , it is also expressed in α / β - KCs in a stochastic manner . The adult γ - lobe and α / β lobes are outlined in C - D in yellow and orange , respectively , for clarity .

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

Confocal z-projections DIP-δ hetero- and homozygous brains in which γ-KCs were labeled by expressing membrane bound tandem tomato (mtdT-HA) driven by the γ specific QF2 driver R71G10-QF2 (γ-QF2). Larval (L3) γ-axons grow normally in DIP-δT2A-Gal4 heterozygotes (A; n=16/16) and homozygotes (E; n=24/24). In contrast, at 48hr APF and adult, γ-axons within DIP-δT2A-Gal4 homozygotes do not enter the distal part of the lobe (asterisks; F; n=8/8, G; n=20/20), while they grow normally in heterozygotes (B; n=12/12, C; n=12/12). Overexpression of a DIP-δ transgene driven by the Gal4 activity of DIP-δT2A-Gal4 (see also Figure S4) does not affect normal growth (D; n=10/10) and rescues mutant phenotypes (H; n=20/20). Note that while the R71G10 driver is consistently expressed in γ-KCs, it is also expressed in α/β-KCs in a stochastic manner. The adult γ-lobe and α/β lobes are outlined in C-D in yellow and orange, respectively, for clarity.


Confocal z - projections of brains expressing DIP - δ - RNAi driven by the indicated Gal4 . γ - KCs are labeled by mtdT - HA driven by R71G10 - QF2 ( γ - QF2 ) . Expression of DIP - δ - RNAi in all glia ( Repo - Gal4 , J ; n = 28 / 28 ) or all KCs ( OK107 - Gal4 , K ; n = 12 / 12 ) did not affect γ - neuron regrowth . In contrast , expression of DIP - δ - RNAi in all postmitotic neurons ( C155 - Gal4 , L ; n = 9 / 12 ) or DIP - δ expressing neurons ( DIP - δT2A - Gal4 , M ; n = 21 / 22 ) induced a defect in γ4 / 5 innervation by γ - axons .

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Confocal z-projections of brains expressing DIP-δ-RNAi driven by the indicated Gal4. γ-KCs are labeled by mtdT-HA driven by R71G10-QF2 (γ-QF2). Expression of DIP-δ-RNAi in all glia (Repo-Gal4, J; n=28/28) or all KCs (OK107-Gal4, K; n=12/12) did not affect γ-neuron regrowth. In contrast, expression of DIP-δ-RNAi in all postmitotic neurons (C155-Gal4, L; n=9/12) or DIP-δ expressing neurons (DIP-δT2A-Gal4, M; n=21/22) induced a defect in γ4/5 innervation by γ-axons.


( A - D ) Confocal z - projections of brains expressing DIP - δGFSTF ( DIP - δ - GFP ) together with the indicated Gal4s and transgenes . Expressing diphtheria toxin ( DTi ) and the membrane bound RFP ( mCD8 - RFP ; CD8 ) driven by the γ4 > γ1γ2 MBON driver MB294B - Gal4 ( MBONγ4 > γ1γ2 - Gal4 ) did not affect DIP - δ - GFP expression ( A , n = 16 / 16 ; B , n = 14 / 14 ) . In contrast , similar expression of DTi and membrane bound Tomato ( CD4 - tdT ; CD4 ) in PAM - DANs ( using the GMR58E02 - Gal4 ; PAM - DAN - Gal4 ) abolished the normal DIP - δ - GFP expression in the γ4 / 5 zone ( compare D , n = 18 / 18 , to C , n = 16 / 16 ) and within the PAM - DAN cell bodies ( compare D ' to C ' ) . Magenta is CD8 - RFP ( A - B ) , CD4 - mtdT ( C - D ) . Green is GFP , grayscale depict individual channels as labeled . Scale bar is 20 µm . Yellow dashed line demarcates the γ - lobe based on FasII staining ( not shown ) .

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(A-D) Confocal z-projections of brains expressing DIP-δGFSTF (DIP-δ-GFP) together with the indicated Gal4s and transgenes. Expressing diphtheria toxin (DTi) and the membrane bound RFP (mCD8-RFP; CD8) driven by the γ4>γ1γ2 MBON driver MB294B-Gal4 (MBONγ4>γ1γ2-Gal4) did not affect DIP-δ-GFP expression (A, n=16/16; B, n=14/14). In contrast, similar expression of DTi and membrane bound Tomato (CD4-tdT; CD4) in PAM-DANs (using the GMR58E02-Gal4; PAM-DAN-Gal4) abolished the normal DIP-δ-GFP expression in the γ4/5 zone (compare D, n=18/18, to C, n=16/16) and within the PAM-DAN cell bodies (compare D' to C'). Magenta is CD8-RFP (A-B), CD4-mtdT (C-D). Green is GFP, grayscale depict individual channels as labeled. Scale bar is 20 µm. Yellow dashed line demarcates the γ-lobe based on FasII staining (not shown).