Human whole blood transcriptomic signatures for timepoint across two observed phases of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . B , of expression in units of Z - scores per transcript for top 50 differentially expressed host transcripts between acute convalescent phase samples . Clinical variables are depicted for samples across top of ; convalescent post symptom onset anti - CHIKV antibody titer viral titer ( which was measured during acute phase ) are both in units of log10 dilutions . Hierarchical clustering ( using complete linkage ) was applied to both samples ( X axis ) transcripts ( Y axis ) . Two major clusters of samples ( largely separating acute convalescent samples ) are highlighted .

B-ORGANISM B-TISSUE I-TISSUE O O O O O O O O O O B-DISEASE O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY 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-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human whole blood transcriptomic signatures for timepoint across the two observed phases of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. B, heatmap of expression in units of Z-scores per transcript for the top 50 differentially expressed host transcripts between acute and convalescent phase samples. Clinical variables are depicted for all samples across the top of the heatmap; convalescent post symptom onset anti-CHIKV antibody titer and viral titer (which was measured during the acute phase) are both in units of log10 dilutions. Hierarchical clustering (using complete linkage) was applied to both samples (X axis) and transcripts (Y axis). Two major clusters of samples (largely separating acute and convalescent samples) are highlighted.


Human whole blood transcriptomic signatures for CHIKV viral titer at acute phase of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . C , volcano plot as in A but for differentially expressed host transcripts between samples with higher lower CHIKV viral titer . Transcripts to right of vertical dashed are associated with higher viral titer , while transcripts to left are associated with lower viral titer . Transcripts significant at FDR < 0 . 05 are colored red .

B-ORGANISM B-TISSUE I-TISSUE O O O B-DISEASE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-DISEASE O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O B-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O

Human whole blood transcriptomic signatures for CHIKV viral titer at the acute phase of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. C, volcano plot as in A but for differentially expressed host transcripts between samples with higher and lower CHIKV viral titer. Transcripts to the right of the vertical dashed line are associated with higher viral titer, while transcripts to the left are associated with lower viral titer. Transcripts significant at FDR < 0.05 are colored red.


Human whole blood transcriptomic signatures for symptom severity , across two observed phases of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . volcano plot for differentially expressed host transcripts between with more severe less severe acute - phase symptoms . Transcripts to right of vertical dashed were comparatively upregulated in more severe cases , while transcripts to left were upregulated in less severe cases .

B-ORGANISM B-TISSUE I-TISSUE O O O O O O O O O O O O B-DISEASE O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Human whole blood transcriptomic signatures for symptom severity, across the two observed phases of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. volcano plot for differentially expressed host transcripts between patients with more severe and less severe acute-phase symptoms. Transcripts to the right of the vertical dashed line were comparatively upregulated in more severe cases, while transcripts to the left were upregulated in less severe cases.


Human whole blood transcriptomic signatures for symptom severity across two observed phases of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . of expression for 20 differentially expressed host transcripts ( significant at FDR < 0 . 05 ) under a model that includes a severity - timepoint interaction . Transcripts have been filtered to those differentially expressed between timepoints in more severe cases but not in less severe cases .

B-ORGANISM B-TISSUE I-TISSUE O O O O O O O O O O O B-DISEASE O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O B-EXP_ASSAY O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O

Human whole blood transcriptomic signatures for symptom severity across the two observed phases of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. heatmap of expression for 20 differentially expressed host transcripts (all significant at FDR < 0.05) under a model that includes a severity-timepoint interaction. Transcripts have been filtered to those differentially expressed between the timepoints in more severe cases but not in less severe cases.


Human whole blood transcriptomic signatures for convalescent ( 15d post - symptom onset ) anti - CHIKV antibody ( Ab ) titer across two observed phases of natural CHIKV infection . Expression was measured with - seq quantified at transcript level , with models of differential expression adjusting for gender as covariates . For panels , 42 were sampled at 2 timepoints , each with 2 technical replicates ; q values are Benjamini - Hochberg adjusted P values from a moderated paired t - under mixed effects model . F , volcano plot for differentially expressed host transcripts between with higher lower convalescent ( 15d post symptom onset ) anti - CHIKV antibody ( Ab ) titers . Transcripts to right of vertical dashed were comparatively upregulated in with a higher convalescent anti - CHIKV antibody titer , while transcripts to left were upregulated in with a lower convalescent anti - CHIKV antibody titer .

B-ORGANISM B-TISSUE I-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-DISEASE O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O

Human whole blood transcriptomic signatures for convalescent (15d post-symptom onset) anti-CHIKV antibody (Ab) titer across the two observed phases of natural CHIKV infection. Expression was measured with RNA-seq and quantified at the transcript level, with all models of differential expression adjusting for patient age and gender as covariates. For all panels, 42 patients were sampled at 2 timepoints, each with 2 technical replicates; q values are Benjamini-Hochberg adjusted P values from a moderated paired t-test under the mixed effects model. F, volcano plot for differentially expressed host transcripts between patients with higher and lower convalescent (15d post symptom onset) anti-CHIKV antibody (Ab) titers. Transcripts to the right of the vertical dashed line were comparatively upregulated in patients with a higher convalescent anti-CHIKV antibody titer, while transcripts to the left were upregulated in patients with a lower convalescent anti-CHIKV antibody titer.


( A ) detected in Vps34 - TAP immunoprecipitations by MS are listed . number of identified peptides of each peptide coverage are also shown .

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(A) Proteins detected in Vps34-TAP immunoprecipitations by MS analysis are listed. The number of identified peptides of each protein and the peptide coverage are also shown.


( B ) expressing TAP - tagged Atg38 indicated HA - tagged were grown in YPD . extracts were prepared were immunoprecipitated by IgG - Dynabeads as described in Materials methods . whole - extract ( left , " input " ) precipitated ( right ) were analyzed by immunoblotting with indicated antibodies .

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(B) Cells expressing TAP-tagged Atg38 and the indicated HA-tagged proteins were grown in YPD. The extracts were prepared and were immunoprecipitated by IgG-Dynabeads as described in Materials and methods. The whole-cell extract (left, "input") and the precipitated proteins (right) were analyzed by immunoblotting with the indicated antibodies.


( C ) Relative abundances of identified by LC - MS / MS of Atg14 - TAP Atg38 - TAP purifications from YPD - grown . Relative abundances are defined as emPAIprey / emPAIbait are shown as mean ± SD of two independent experiments .

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(C) Relative abundances of proteins identified by LC-MS/MS of Atg14-TAP and Atg38-TAP purifications from YPD-grown cells. Relative abundances are defined as emPAIprey/emPAIbait and are shown as mean ± SD of two independent experiments.


( D ) extracts were prepared from wild - type atg14Δ expressing Atg38 - TAP as well as HA - tagged of I components . IgG Dynabead - precipitated ( bottom ) , together with whole - extracts ( top ) , were immunoblotted with indicated antibodies .

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(D) Cell extracts were prepared from wild-type and atg14Δ cells expressing Atg38-TAP as well as HA-tagged proteins of complex I components. The IgG Dynabead-precipitated proteins (bottom), together with the whole-cell extracts (top), were immunoblotted with the indicated antibodies.


( E ) ATG38 - 2GFP ATG17 - 2mCherry in wild - type atg14Δ were cultured at 30 ° C with or without rapamycin . After 180 min , were analyzed by microscopy . Bar , 5 µm .

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(E) ATG38-2GFP ATG17-2mCherry cells in wild-type and atg14Δ were cultured at 30°C with or without rapamycin. After 180 min, cells were analyzed by fluorescence microscopy. Bar, 5 µm.


( A ) expressing Pho8Δ60 were grown in YPD shifted to SD ( - N ) medium for 3 h at 30 ° C . Lysates from each group of were tested for ALP activity . ALP activity is shown as mean ± SD of three independent experiments with activity of wild - type normalized to 1 .

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(A) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD(-N) medium for 3 h at 30°C. Lysates from each group of cells were tested for ALP activity. ALP activity is shown as mean ± SD of three independent experiments with the activity of wild-type cells normalized to 1.


( B ) with indicated genotypes were grown in YPD treated with rapamycin for 3 h at 30 ° C . Total was separated by SDS - PAGE detected by immunoblotting with anti - API .

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(B) Cells with the indicated genotypes were grown in YPD and treated with rapamycin for 3 h at 30°C. Total protein was separated by SDS-PAGE and detected by immunoblotting with anti-API.


( C ) were grown in YPD medium for 1 h at 30 ° C separated to intracellular ( I ; ) extracellular ( E ; medium ) fractions by centrifugation . fractions were treated as detailed in Materials methods aliquots of each were separated by SDS - PAGE . positions of precursor ( p2 - CPY ) mature ( m - CPY ) forms of CPY are indicated .

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(C) Cells were grown in YPD medium for 1 h at 30°C and separated to the intracellular (I; cells) and extracellular (E; medium) fractions by centrifugation. The fractions were treated as detailed in Materials and methods and aliquots of each were separated by SDS-PAGE. The positions of the precursor (p2-CPY) and mature (m-CPY) forms of CPY are indicated.


( A ) Wild - type atg38Δ expressing indicated GFP fusion Ape1 - mRFP were cultured at 30 ° C in presence of rapamycin . After 60 min , were analyzed by microscopy . Of total pool of Ape1 - mRFP signal , of Ape1 - mRFP colocalizing with GFP was calculated . This is shown as mean ± SD of two independent experiments , with number of counted indicated at bottom of graph . ( B ) Examples of wild - type atg38Δ expressing either Atg14 - 3 × GFP or Vps34 - 3 × GFP in A . Bar , 5 µm .

O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O

(A) Wild-type and atg38Δ cells expressing the indicated GFP fusion proteins and Ape1-mRFP were cultured at 30°C in the presence of rapamycin. After 60 min, cells were analyzed by fluorescence microscopy. Of the total pool of Ape1-mRFP signal, the percentage of Ape1-mRFP colocalizing with GFP was calculated. This percentage is shown as mean ± SD of two independent experiments, with the number of cells counted indicated at the bottom of the graph. (B) Examples of wild-type and atg38Δ cells expressing either Atg14-3 × GFP or Vps34-3 × GFP in A. Bar, 5 µm.


( C ) extracts were prepared from in A , then analyzed by immunoblotting with anti - GFP anti - Pgk1 antibodies .

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(C) Cell extracts were prepared from the cells in A, then analyzed by immunoblotting with anti-GFP and anti-Pgk1 antibodies.


( A ) extracts were prepared from wild - type atg38Δ strains expressing TAP - tagged Atg38 indicated HA . extracts were immunoprecipitated by IgG - Dynabeads . whole - extracts ( top , " input " ) precipitates ( bottom ) were analyzed by immunoblotting with indicated antibodies . Relative binding efficiency was determined as described in Materials methods . To compare results from multiple experiments , value for reference sample ( wild - type ) was normalized to 1 . 0 . Relative binding efficiency is shown as mean ± SD of three independent experiments .

O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell extracts were prepared from wild-type and atg38Δ strains expressing TAP-tagged Atg38 and the indicated HA proteins. The extracts were immunoprecipitated by IgG-Dynabeads. The whole-cell extracts (top, "input") and the precipitates (bottom) were analyzed by immunoblotting with the indicated antibodies. Relative binding efficiency was determined as described in Materials and methods. To compare results from multiple experiments, the value for the reference sample (wild-type cells) was normalized to 1.0. Relative binding efficiency is shown as mean ± SD of three independent experiments.


( B ) Relative abundances of identified in TAP purifications from indicated strains were analyzed as described in Fig . 1 C are shown as mean ± SD of two independent experiments .

O O O O O O O O B-GENEPROD 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) Relative abundances of proteins identified in TAP purifications from the indicated strains were analyzed as described in Fig. 1 C and are shown as mean ± SD of two independent experiments.


( C ) expressing Pho8Δ60 were grown in YPD shifted to SD ( - N ) medium for 3 h at 30 ° C . lysates from each group of were tested for ALP activity . ALP activity is shown as mean ± SD of three independent experiments with activity of wild - type normalized to 1 .

(C) Cells expressing Pho8Δ60 were grown in YPD and shifted to SD(-N) medium for 3 h at 30°C. Cell lysates from each group of cells were tested for ALP activity. ALP activity is shown as mean ± SD of three independent experiments with the activity of wild-type cells normalized to 1.


( D ) expressing Vps34 - 6HA , Vps30 - 9myc , Atg38 - 3FLAG , Atg14 - TAP were lysed in a buffer containing 150 mM NaCl . Atg14 - TAP immunoprecipitate with IgG - Dynabeads from this lysate was split into eight fractions washed with lysis buffer containing indicated concentrations of NaCl . resulting precipitates were analyzed by immunoblotting with indicated antibodies . Relative binding efficiency was determined as described in Materials methods , although a 150 - mM NaCl sample was used as a control normalized to 1 . 0 . Relative binding efficiency is shown as mean ± SD of three independent experiments .

O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O 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) Cells expressing Vps34-6HA, Vps30-9myc, Atg38-3FLAG, and Atg14-TAP were lysed in a buffer containing 150 mM NaCl. The Atg14-TAP immunoprecipitate with IgG-Dynabeads from this lysate was split into eight fractions and washed with lysis buffer containing the indicated concentrations of NaCl. The resulting precipitates were analyzed by immunoblotting with the indicated antibodies. Relative binding efficiency was determined as described in Materials and methods, although a 150-mM NaCl sample was used as a control and normalized to 1.0. Relative binding efficiency is shown as mean ± SD of three independent experiments.


( A ) AH109 containing indicated GAL4 activation domain fusions ( bait ) GAL4 - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . interaction of fusion was examined by plating on SD - Leu - Trp - His ( − LWH , middle ) SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d .

O O O B-ORGANISM O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(A) AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.


( B ) Interactions between indicated fusion were analyzed as in A . AH109 containing indicated GAL4 activation domain fusions ( bait ) GAL4 - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . interaction of fusion was examined by plating on SD - Leu - Trp - His ( − LWH , middle ) SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d .

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(B) Interactions between the indicated fusion proteins were analyzed as in A. AH109 cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.


( C ) TAP - tagged Vps34 was purified from vps30Δ atg14Δ vps38Δ . purified were separated on SDS - PAGE visualized by Coomassie . Recombinant GST - Atg38 ( WT ) , GST - Atg381 - 120 ( N ) , GST - Atg38121 - 226 ( C ) were incubated with Vps15 - Vps34 - TAP bound to IgG - Dynabeads . eluted were analyzed by immunoblotting .

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(C) The TAP-tagged Vps34 protein complex was purified from the vps30Δ atg14Δ vps38Δ cells. The purified proteins were separated on SDS-PAGE and visualized by Coomassie staining. Recombinant GST-Atg38 (WT), GST-Atg381-120 (N), and GST-Atg38121-226 (C) were incubated with Vps15-Vps34-TAP complex bound to IgG-Dynabeads. The eluted proteins were analyzed by immunoblotting.


( A ) Interactions between indicated fusion were analyzed as in Fig . 4 A . AH109cells containing indicated GAL4 activation domain fusions ( bait ) GAL4 - binding domain fusions ( prey ) were selected on SD - Leu - Trp plates at 30 ° C ( − LW , left ) . interaction of fusion was examined by plating on SD - Leu - Trp - His ( − LWH , middle ) SD - Leu - Trp - Ade ( − LWA , right ) plates at 30 ° C for 3 d .

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(A) Interactions between the indicated fusion proteins were analyzed as in Fig. 4 A. AH109cells containing the indicated GAL4 activation domain fusions (bait) and GAL4 DNA-binding domain fusions (prey) were selected on SD-Leu-Trp plates at 30°C (−LW, left). The interaction of fusion proteins was examined by plating cells on SD-Leu-Trp-His (−LWH, middle) and SD-Leu-Trp-Ade (−LWA, right) plates at 30°C for 3 d.


( B ) of Atg38 by gel filtration . Recombinant Atg38 was fractionated over a gel filtration column . Each fraction was subjected to SDS - PAGE visualized by Coomassie .

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(B) Analysis of Atg38 by gel filtration. Recombinant Atg38 protein was fractionated over a gel filtration column. Each fraction was subjected to SDS-PAGE and visualized by Coomassie staining.


( A ) extracts were prepared from atg38Δ strains expressing Atg14 - TAP either Vps34 - 6HA or Vps15 - 6HA with Atg381 - 120 fused with GFP either GBP or GBP3A . extracts were immunoprecipitated with IgG - Dynabeads . precipitated , together with whole - extracts , were immunoblotted with indicated antibodies ( left ) . Relative binding efficiency was determined as described in Fig . 3 A was shown as mean ± SD of three independent experiments .

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(A) Cell extracts were prepared from atg38Δ strains expressing Atg14-TAP and either Vps34-6HA or Vps15-6HA with Atg381-120 fused with GFP and either GBP or GBP3A. The cell extracts were immunoprecipitated with IgG-Dynabeads. The precipitated proteins, together with the whole-cell extracts, were immunoblotted with the indicated antibodies (left). Relative binding efficiency was determined as described in Fig. 3 A and was shown as mean ± SD of three independent experiments.


B . Endogenous CUL4A CUL4B complexes were isolated from 293T lysates by immunoprecipitation ( IP ) associated were detected by immunoblotting ( IB ) . Mock includes beads only control .

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B. Endogenous CUL4A and CUL4B complexes were isolated from 293T cell lysates by immunoprecipitation (IP) and associated proteins were detected by immunoblotting (IB). Mock includes beads only control.


C . Flag - tagged WT mutant WDTC1proteins were transiently expressed in 293T . Flag - WDTC1 complexes were immunoprecipitated with anti - FLAG their associated were detected by immunoblotting as indicated ; EV , empty vector control .

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C. Flag-tagged WT and mutant WDTC1proteins were transiently expressed in 293T cells. Flag-WDTC1 complexes were immunoprecipitated with anti-FLAG and their associated proteins were detected by immunoblotting as indicated; EV, empty vector control.


E . 293T were transfected with HA - ubiquitin along with various combinations of plasmid siRNA as indicated . Transfected were lysed under denaturing conditions to obtain whole extracts ( WCE ) immunoprecipitated with anti - FLAG . WDTC1 ubiquitylation was evaluated by immunoblotting as indicated .

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E. 293T cells were transfected with HA-ubiquitin along with various combinations of plasmid and siRNA as indicated. Transfected cells were lysed under denaturing conditions to obtain whole cell extracts (WCE) and immunoprecipitated with anti-FLAG. WDTC1 ubiquitylation was evaluated by immunoblotting as indicated.


F . 293T transiently expressing Flag - WDTC1 were transfected with scramble ( scrm ) siRNA or siRNAs against DDB1 , CUL4A CUL4B . Flag - WDTC1 knockdown efficiency were assessed by immunoblotting as indicated .

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F. 293T cells transiently expressing Flag-WDTC1 were transfected with scramble (scrm) siRNA or siRNAs against DDB1, CUL4A and CUL4B. Flag-WDTC1 levels and knockdown efficiency were assessed by immunoblotting as indicated.


A . 3T3 - L1preadipocytes were adipogenically induced lysates were prepared from collected at indicated days . Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti - WDTC1 followed by immunoblot analyses with indicated antibodies . Fatty acid synthase ( FAS ) were monitored to evaluate induction .

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A. 3T3-L1preadipocytes were adipogenically induced and lysates were prepared from cells collected at indicated days. Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti-WDTC1 followed by immunoblot analyses with indicated antibodies. Fatty acid synthase (FAS) levels were monitored to evaluate induction.


B - D . 3T3 - L1 stable expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 were adipogenically induced . Adipogenesis was assessed by Oil Red O ( ORO ) ( B ) ; plate ( top ) microscopic ( bottom ) views , 0 . 1 mm scale bar .

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B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenesis was assessed by Oil Red O staining (ORO) (B); plate (top) and microscopic (bottom) views, 0.1 mm scale bar.


B - D . 3T3 - L1 stable expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 were adipogenically induced . Triglyceride were quantified by an enzymatic ( C ) .

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B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Triglyceride levels were quantified by an enzymatic assay (C).


B - D . 3T3 - L1 stable expressing vector control ( EV ) , Flag - tagged WT or mutant WDTC1 were adipogenically induced . Adipogenic gene expression was evaluated by RT - qPCR ( D ) .

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B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenic gene expression was evaluated by RT-qPCR analysis (D).


A . Schematic of WDTC1 knockdown - rescue lentiviral vector ( top ) . 3T3 - L1preadipocytes were lentivirally transduced to generate stable expressing a non - specific ( NS ) shRNA control , shWDTC1 targeting endogenous Wdtc1mRNA , or simultaneously expressing shWDTC1 shRNA - resistant WDTC1 ; bottom , confirmation of knockdown WDTC1 ectopic expression .

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A. Schematic of WDTC1 knockdown-rescue lentiviral vector (top). 3T3-L1preadipocytes were lentivirally transduced to generate stable cells expressing a non-specific (NS) shRNA control, shWDTC1 targeting endogenous Wdtc1mRNA, or cells simultaneously expressing shWDTC1 and shRNA-resistant WDTC1; bottom, confirmation of knockdown and WDTC1 ectopic expression.


B - D . 3T3 - L1 stable described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by ORO ( B )

O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O

B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by ORO (B)


B - D . 3T3 - L1 stable described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by triglyceride quantitation ( C )

O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O

B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by triglyceride quantitation (C)


B - D . 3T3 - L1 stable described in ( A ) were adipogenically induced . Their adipogenic potential was assessed by adipogenic gene expression ( D ) .

O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O

B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by adipogenic gene expression (D).


A . Induced 3T3 - L1 stably expressing Flag - WDTC1 were subjected to subcellular fractionation . Aliquots of total extract ( TCE ) subcellular fractions were analyzed by immunoblot as indicated ; fraction purity was assessed by α - Tubulin histone H3 antibodies .

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A. Induced 3T3-L1 cells stably expressing Flag-WDTC1 were subjected to subcellular fractionation. Aliquots of total cell extract (TCE) and subcellular fractions were analyzed by immunoblot as indicated; fraction purity was assessed by α-Tubulin and histone H3 antibodies.


B . enriched extracts from 293T cotransfected with Flag - WDTC1 HA tagged histones were immunoprecipitated ( IP ) with anti - FLAG immunoblotted ( IB ) as indicated . probable H2AK119ub1 band detected in extract is indicated by an asterisk .

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B. Chromatin enriched nuclear extracts from 293T cells cotransfected with Flag-WDTC1 and HA tagged core histones were immunoprecipitated (IP) with anti-FLAG and immunoblotted (IB) as indicated. The probable H2AK119ub1 band detected in the nuclear extract is indicated by an asterisk.


C . Soluble extracts were prepared from preadipocyte or induced 3T3 - L1 stably expressing control ( EV ) Flag - WDTC1 . H2A monoubiquitylation was detected by H2AK119ub1 - specific antibody total H2A served as loading control . Relative represents of H2AK119ub1 over total H2A signal normalized to EV control .

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C. Soluble chromatin extracts were prepared from preadipocyte or induced 3T3-L1 cells stably expressing control (EV) and Flag-WDTC1 proteins. H2A monoubiquitylation was detected by H2AK119ub1-specific antibody and total H2A served as loading control. Relative intensity represents ratio of H2AK119ub1 over total H2A signal normalized to EV control.


D . Affinity purified Flag - WDTC1 wildtype mutant ΔH complexes from 293T were source of E3 ligase in in vitro ubiquitylation assays . Copurification of CRL4 E3 ligase was confirmed by immunoblotting Coomassie Blue ( left panel ) . In vitro ubiquitylation assays of recombinant H2A were performed in presence of indicated . Reaction products were resolved by SDS - PAGE , ubiquitylatedH2A was detected by anti - H2AK119ub1 ( right panel ) .

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D. Affinity purified Flag-WDTC1 wildtype and mutant ΔH complexes from 293T cells were the source of E3 ligase in in vitro ubiquitylation assays. Copurification of CRL4 E3 ligase proteins was confirmed by immunoblotting and Coomassie Blue staining (left panel). In vitro ubiquitylation assays of recombinant H2A were performed in the presence of indicated proteins. Reaction products were resolved by SDS-PAGE, and ubiquitylatedH2A was detected by anti-H2AK119ub1 (right panel).


E . In vitro ubiquitylation assays were performed with purified wild - type mutant histone as substrates . H2A ubiquitylation was assessed by immunoblotting with anti - ubiquitin various antibodies as indicated .

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E. In vitro ubiquitylation assays were performed with purified wild-type and mutant histone proteins as substrates. H2A ubiquitylation was assessed by immunoblotting with anti-ubiquitin and various antibodies as indicated.


B , C Effects of H2A mutations ( B ) H2A . B mutations ( C ) on nucleosome resistance to MNase digestion . MNase digested nucleosomal are analyzed by 10 % Native - PAGE . MNase digestion experiments are repeated twice .

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B,C Effects of H2A mutations (B) and H2A.B mutations (C) on nucleosome resistance to MNase digestion. MNase digested nucleosomal DNA are analyzed by 10% Native-PAGE. All MNase digestion experiments are repeated twice.


D , E Graph presentation of effects of H2A mutations ( D ) H2A . B mutations ( E ) on nucleosome resistance to MNase digestion . MNase digestion efficiency for each sample is calculated from quantitation of digested against total . experiments are repeated twice . Data are mean ± SD , n = 2 .

O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O 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,E Graph presentation of effects of H2A mutations (D) and H2A.B mutations (E) on nucleosome resistance to MNase digestion. MNase digestion efficiency for each sample is calculated from quantitation of the digested DNA against total DNA. All experiments are repeated twice. Data are mean ± SD, n = 2.


B Effect of canonical noncanonical H2A ROF on histone octamer assembly . Left : Gel filtration chromatography profiles of octamers assembled by histones including various H2A family members or mutants . Highlighted in blue shadow are chromatography fractions subject to SDS - PAGE . Right : SDS - PAGE of histone octamer integrity by visualizing histone stoichiometry . stars indicate lnkH2B - H2A containing different H2A family members or mutants present in chromatography fractions . triangles indicate chromatography fractions containing majority of lnkH2B - H2A or their mutants .

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B Effect of the canonical and noncanonical H2A ROF on histone octamer assembly. Left: Gel filtration chromatography profiles of octamers assembled by core histones including various H2A family members or mutants. Highlighted in blue shadow are chromatography fractions subject to SDS-PAGE analysis. Right: SDS-PAGE analysis of histone octamer integrity by visualizing histone stoichiometry. The stars indicate lnkH2B-H2A containing different H2A family members or mutants present in chromatography fractions. The triangles indicate the chromatography fractions containing the majority of the lnkH2B-H2A or their mutants.


D , E Effects of canonical noncanonical H2A ROF on nucleosome resistance to MNase digestion . Displayed are Native - PAGE results ( D ) graph of MNase digestion efficiency calculated from quantitation of digested against total ( E ) . experiments are repeated twice . Data are mean ± SD , n = 2 .

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D,E Effects of the canonical and noncanonical H2A ROF on nucleosome resistance to MNase digestion. Displayed are Native-PAGE results (D) and graph of MNase digestion efficiency calculated from quantitation of the digested DNA against total DNA (E). All experiments are repeated twice. Data are mean ± SD, n = 2.


D SDS - PAGE of SWR1 purified from S . cerevisiae .

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D SDS-PAGE analysis of SWR1 complex purified from S. cerevisiae.


E Effect of H2A . Z . 2 . 2 ROF on facilitating histone replacement . Incorporations of yeast H2A . Z - H2B dimers containing either wild type H2A . Z or Z . 2 . 2 - like H2A . Z were analyzed by SWR1 - catalyzed histone replacement . H2A . Z Z . 2 . 2 - like H2A . Z incorporated into nucleosome were resolved by 6 % Native - PAGE detected by SYBR Green western blotting .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O

E Effect of H2A.Z.2.2 ROF on facilitating histone replacement. Incorporations of yeast H2A.Z-H2B dimers containing either the wild type H2A.Z or Z.2.2-like H2A.Z were analyzed by SWR1-catalyzed histone replacement assay. H2A.Z and Z.2.2-like H2A.Z incorporated into nucleosome were resolved by 6% Native-PAGE and detected by SYBR Green staining and western blotting.


A . expression PV incorporation of spike were assessed by Western blotting using an anti - S2 mAb . Lentiviral capsid components were detected using anti - p24 / 55 .

O O O B-ORGANISM O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-ORGANISM B-SUBCELLULAR O O O O O O B-GENEPROD O O O

A. Cellular expression and PV incorporation of spike were assessed by Western blotting using an anti-S2 mAb. Lentiviral capsid components were detected using anti-p24/55.


F . Western blot detection of ACE2 ( rabbit anti - ACE2 ) in lysates of stated . ACE2 was detected in HEK 293T samples after loading 3X more lysate increasing signal exposure time ( labelled in boldface ) .

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F. Western blot detection of ACE2 (rabbit anti-ACE2) in cell lysates of the stated cells. ACE2 was detected in HEK 293T samples after loading 3X more lysate and increasing signal exposure time (labelled in boldface).


J . Over - expression confirmed by Western blotting , actin CD81 were used as loading controls for ACE2 TMPRSS2 respectively .

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J. Over-expression confirmed by Western blotting, actin and CD81 were used as loading controls for ACE2 and TMPRSS2 respectively.


L . expression PV incorporation of stated spike were assessed by Western blotting . displayed samples are taken from same image have been cropped to remove irrelevant lanes / bands .

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L. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting. The displayed samples are taken from the same image and have been cropped to remove irrelevant lanes/bands.


N . expression PV incorporation of stated spike were assessed by Western blotting , note change in sample order relative to panel M .

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N. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting, note the change in sample order relative to panel M.


E . expression PV incorporation of stated spike were assessed by Western blotting .

O O B-EXP_ASSAY B-ORGANISM O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O

E. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting.


J . Expression PV incorporation of stated spike were assessed by Western blotting .

J. Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting.


D . Expression PV incorporation of stated spike were assessed by Western blotting .

D. Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting.


( B ) Relative TAG in different genetic backgrounds were measured by glyceride kit . For , TAG were normalized to . Error bars represent ± . * * * : P < 0 . 001 ; NS : non - significant .

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(B) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. Error bars represent ± SEM. ***: P < 0.001; NS: non-significant.


( G ) of lipid droplet in ( C F ) . ND : normal diet ; HFD : high - fat diet . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison . Each point represents data from one fat , at least 30 were analyzed for each fat . Error bars represent ± . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

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(G) Quantification of the lipid droplet size in (C and F). ND: normal diet; HFD: high-fat diet. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were analyzed for each fat body. Error bars represent ±SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


( B 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Scale bars represent 25 µm . ( B ) RDH / CG2064 RNAi suppresses enlarged lipid droplet phenotype in CSN2 RNAi under DGAT1 overexpression ( ppl > DGAT1 ) or high - fat diet conditions . ( C ) of lipid droplet in ( B ) . HFD : high - fat diet . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison . Each point represents data from one fat , at least 30 were examined in each fat . Error bars represent ± . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O 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-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (B) RDH/CG2064 RNAi suppresses the enlarged lipid droplet phenotype in CSN2 RNAi under DGAT1 overexpression (ppl>DGAT1) or high-fat diet conditions. (C) Quantification of the lipid droplet size in (B). HFD: high-fat diet. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


, F 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Scale bars represent 25 µm . ( F ) RDH / CG2064 overexpression increases lipid droplet in ppl > DGAT1 larvae . ( G ) of lipid droplet in ( F ) . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison . Each point represents data from one fat , at least 30 were examined in each fat . Error bars represent ± . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

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, F 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (F) RDH/CG2064 overexpression increases lipid droplet size in ppl>DGAT1 larvae. (G) Quantification of the lipid droplet size in (F). data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Scale bars represent 25 µm . ( H ) Relative TAG in different genetic backgrounds were measured by glyceride kit . For , TAG were normalized to . ( I ) RDH / CG2064 CG2065 deletion does not affect lipid droplet under normal or high - fat diet conditions . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison . Each point represents data from one fat , at least 30 were examined in each fat . Error bars represent ± . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O 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-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (H) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. (I) RDH/CG2064 and CG2065 deletion does not affect lipid droplet size under normal or high-fat diet conditions. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


( B ) 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Overexpressing wild - type or site - mutated RDH / CG2064 increases lipid droplet in larvae with DGAT1 overexpression ( ppl > DGAT1 ) . Scale bar represents 25 µm .

O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O B-ORGANISM O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O

(B) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Overexpressing wild-type or site-mutated RDH/CG2064 increases the lipid droplet size in larvae with DGAT1 overexpression (ppl>DGAT1). Scale bar represents 25 µm.


( E ) Immunofluorescence imaging of RDH / CG2064 - GFP , detected with anti - GFP antibody , ER marker RFP - KDEL in 3rd instar larval fat . RDH / CG2064 co - localizes with RFP - KDEL . Scale bar represents 10 µm .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-SUBCELLULAR O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O

(E) Immunofluorescence imaging of RDH/CG2064-GFP, detected with anti-GFP antibody, and the ER protein marker RFP-KDEL in 3rd instar larval fat body. RDH/CG2064 co-localizes with RFP-KDEL. Scale bar represents 10 µm.


Lysates of 3rd instar larval fat samples of different genetic backgrounds were immunoprecipitated with anti - Flag antibody then probed by anti - GFP or anti - Flag antibody in a western blot . Plin2 , but not Plin1 or Bmm , is specifically coimmunoprecipitated with RDH / CG2064 ( A ) .

O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O O O

Lysates of 3rd instar larval fat body samples of different genetic backgrounds were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin2, but not Plin1 or Bmm, is specifically coimmunoprecipitated with RDH/CG2064 (A).


( E ) Lysates of Drosophila S2 expressing full - Plin1 or Plin1 with a C - terminal deletion ( Plin1ΔC ) were immunoprecipitated with anti - Flag antibody then probed by anti - GFP or anti - Flag antibody in a western blot . Plin1ΔC coimmunoprecipitated with RDH / CG2064 .

O O O O O B-ORGANISM B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O

(E) Lysates of Drosophila S2 cells expressing full-length Plin1 or Plin1 with a C-terminal deletion (Plin1ΔC) were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin1ΔC coimmunoprecipitated with RDH/CG2064.


( F ) 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Under high - fat conditions , RDH / CG2064 overexpression increases lipid droplet , Plin2 RNAi suppresses enlargement induced by RDH / CG2064 overexpression . Scale bar represents 25 µm .

O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O

(F) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Under high-fat conditions, RDH/CG2064 overexpression increases the lipid droplet size, and Plin2 RNAi suppresses the enlargement induced by RDH/CG2064 overexpression. Scale bar represents 25 µm.


( A , B of Plin2 - GFP in 3rd instar larval fat of different genotypes . Scale bar represents 25 µm . ( A ) CSN2 RNAi or RDH / CG2064 overexpression reduces peripheral localization of Plin2 - GFP increases localization of Plin2 - GFP on small peri - lipid droplets ( boxed ) . DAPI ( blue ) stains nuclei . arrows indicate peripheral lipid droplet . Red arrows indicate medial lipid droplets . ( B ) peripheral localization of Plin2 - GFP is greatly reduced in fat from larvae with CSN2 RNAi or RDH / CG2064 overexpression . were taken by focusing on periphery of fat .

O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O B-ORGANISM O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-CELL_TYPE O

(A, B Images of Plin2-GFP in 3rd instar larval fat body cells of different genotypes. Scale bar represents 25 µm. (A) CSN2 RNAi or RDH/CG2064 overexpression reduces the peripheral localization of Plin2-GFP and increases the localization of Plin2-GFP on small peri-nuclear lipid droplets (boxed). DAPI (blue) stains nuclei. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. (B) The peripheral localization of Plin2-GFP is greatly reduced in fat body cells from larvae with CSN2 RNAi or RDH/CG2064 overexpression. Images were taken by focusing on the periphery of fat cells.


C , of Plin2 - GFP in 3rd instar larval fat of different genotypes . Scale bar represents 25 µm . ( C ) In fat from ppl > DGAT1 larvae with CSN2 RNAi or RDH / CG2064 overexpression , localization of Plin2 - GFP is greatly reduced at periphery is dramatically increased on large medial lipid droplets . arrows indicate peripheral lipid droplet . Red arrows indicate medial lipid droplets . ( D ) plots of in ( C ) . GFP along across a lipid droplet was measured by ImageJ . In ppl > DGAT1 larvae with CSN2 RNAi or RDH / CG2064 overexpression , Plin2 - GFP localization on large medial lipid droplets is represented by two peaks . More than 30 lipid droplets were measured for each genotype . plot shown is a typical image curve for indicated genotype .

O O O B-GENEPROD O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-ORGANISM O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O

C, Images of Plin2-GFP in 3rd instar larval fat body cells of different genotypes. Scale bar represents 25 µm. (C) In fat body cells from ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, the localization of Plin2-GFP is greatly reduced at the periphery and is dramatically increased on large medial lipid droplets. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. (D) Fluorescence intensity line plots of the images in (C). GFP intensity along the line across a lipid droplet was measured by ImageJ. In ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, Plin2-GFP localization on large medial lipid droplets is represented by two peaks. More than 30 lipid droplets were measured for each genotype. The plot shown is a typical image curve for the indicated genotype.


( G ) peripheral lipid droplets in larval fat from different genetic backgrounds were stained by BODIPY ( green ) . CSN3 RNAi or RDH / CG2064 overexpression reduces number of peripheral lipid droplets in both wild - type ppl > DGAT1 larvae .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-ORGANISM B-CELL_TYPE O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O

(G) The peripheral lipid droplets in larval fat body cells from different genetic backgrounds were stained by BODIPY (green). CSN3 RNAi or RDH/CG2064 overexpression reduces the number of peripheral lipid droplets in both wild-type and ppl>DGAT1 larvae.


( A , Confocal of Bmm - GFP ( green ) in 3rd instar larval fat bodies of different genotypes . DAPI ( blue ) stains nuclei . Scale bar represents 25 µm . ( A ) Knocking down CSN2 or overexpressing RDH / CG2064 in with DGAT1 overexpression ( ppl > DGAT1 ) decreases level lipid droplet localization of Bmm - GFP . ( B ) GFP along across a lipid droplet in ( A ) was measured by ImageJ . lipid droplet localization of Bmm - GFP , represented by two peaks , is clearly visible in fat from ppl > DGAT1 larvae , but it is lost in fat from ppl > DGAT1 larvae with CSN2 RNAi or overexpression of RDH / CG2064 . More than 30 lipid droplets of each genotype were measured . One typical image curve is shown for each genotype .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-ORGANISM O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O

(A, Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (A) Knocking down CSN2 or overexpressing RDH/CG2064 in animals with DGAT1 overexpression (ppl>DGAT1) decreases the level and lipid droplet localization of Bmm-GFP. (B) The GFP intensity along the line across a lipid droplet in (A) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, is clearly visible in fat cells from ppl>DGAT1 larvae, but it is lost in fat cells from ppl>DGAT1 larvae with CSN2 RNAi or overexpression of RDH/CG2064. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.


F ) Confocal of Bmm - GFP ( green ) in 3rd instar larval fat bodies of different genotypes . DAPI ( blue ) stains nuclei . Scale bar represents 25 µm . ( F ) Overexpression of Plin2 decreases level lipid droplet localization of Bmm - GFP in fat from larvae with DGAT1 overexpression . ( G ) GFP along across a lipid droplet from ( F ) was measured by ImageJ . lipid droplet localization of Bmm - GFP , represented by two peaks , was evident in fat from ppl > DGAT1 larvae , but is lost in ppl > DGAT1 larvae with overexpression of Plin2 . More than 30 lipid droplets of each genotype were measured . One typical image curve is shown for each genotype .

O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-ORGANISM O B-GENEPROD O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

F) Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (F) Overexpression of Plin2 decreases the level and lipid droplet localization of Bmm-GFP in fat cells from larvae with DGAT1 overexpression. (G) GFP intensity along the line across a lipid droplet from (F) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, was evident in fat cells from ppl>DGAT1 larvae, but is lost in ppl>DGAT1 larvae with overexpression of Plin2. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.


( H ) 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . bmm RNAi increases lipid droplet in fat from larvae with DGAT1 overexpression . Scale bar represents 25 µm .

O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O B-ORGANISM O B-GENEPROD O O O O O O O O

(H) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. bmm RNAi increases the lipid droplet size in fat cells from larvae with DGAT1 overexpression. Scale bar represents 25 µm.


( A ) S2 expressing RDH / CH2064 - GFP were treated with synthesis inhibitor cycloheximide ( CHX ) for indicated time ( h : hours ) . level of RDH / CH2064 - GFP was determined by western blotting using anti - GFP antibody . Tubulin was used as loading control . ( B ) of relative RDH / CH2064 - GFP in ( A ) . Error bars represent ± .

O O O B-CELL_LINE O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(A) S2 cells expressing RDH/CH2064-GFP were treated with the protein synthesis inhibitor cycloheximide (CHX) for the indicated time (h: hours). The level of RDH/CH2064-GFP was determined by western blotting using anti-GFP antibody. Tubulin was used as loading control. (B) Quantification of the relative RDH/CH2064-GFP protein levels in (A). Error bars represent ± SEM.


( C ) S2 expressing Bmm - GFP were treated with synthesis inhibitor cycloheximide ( CHX ) for indicated time ( h : hours ) . level of Bmm - GFP was determined by western blotting using anti - GFP antibody . Tubulin was used as loading control . ( D ) of relative Bmm - GFP in ( C ) . Error bars represent ± .

O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(C) S2 cells expressing Bmm-GFP were treated with the protein synthesis inhibitor cycloheximide (CHX) for the indicated time (h: hours). The level of Bmm-GFP was determined by western blotting using anti-GFP antibody. Tubulin was used as loading control. (D) Quantification of the relative Bmm-GFP protein levels in (C). Error bars represent ± SEM.


( F ) Oil red of 3rd instar larval oenocytes in different genetic backgrounds under fed or starvation conditions .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-TISSUE I-TISSUE I-TISSUE B-CELL_TYPE O O O O O O O O O O

(F) Oil red staining of 3rd instar larval oenocytes in different genetic backgrounds under fed or starvation conditions.


B , C , Representative of pPLT3 : : erCFP ( cyan ) expressing PI - stained ( red ) Arabidopsis roots in Col or wox5 background , respectively . D , Mean intensities of pPLT3 : : erCFP roots summarized in box scatter plots . mean of CFP signal in Col roots was to set to 100 % . D , Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . data was statistically analyzed by one - way ANOVA Holm - Sidak post - hoc multiple comparisons . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype results from two technical replicates . B , C , Scale bars represent 10 µm . PI = propidium iodide CFP = cyan .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE B-GENEPROD O B-GENEPROD O

B, C, Representative images of pPLT3::erCFP (cyan) expressing and PI-stained (red) Arabidopsis roots in Col or wox5 background, respectively. D, Mean fluorescence intensities of the pPLT3::erCFP roots summarized in box and scatter plots. The mean fluorescence intensity of the CFP signal in Col roots was to set to 100 %. D, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. B, C, Scale bars represent 10 µm. PI = propidium iodide CFP = cyan fluorescent protein.


E , F , Representative of pPLT3 : : PLT3 - YFP ( yellow ) expressing FM4 - 64 - stained ( red ) Arabidopsis roots in Col or wox5 mutant background , respectively . g , Mean intensities of pPLT3 : : PLT3 - YFP expressing roots summarized in box scatter plots . mean of YFP signal in Col roots was to set to 100 % . G , Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . data was statistically analyzed by one - way ANOVA Holm - Sidak post - hoc multiple comparisons . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype results from two technical replicates . , E , F , Scale bars represent 10 µm . YFP = yellow

O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O B-ORGANISM B-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY B-TISSUE O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD

E, F, Representative images of pPLT3::PLT3-YFP (yellow) expressing and FM4-64-stained (red) Arabidopsis roots in Col or wox5 mutant background, respectively. g, Mean fluorescence intensities of the pPLT3::PLT3-YFP expressing roots summarized in box and scatter plots. The mean fluorescence intensity of the YFP signal in Col roots was to set to 100%. G, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. , E, F, Scale bars represent 10 µm. YFP = yellow fluorescent protein


A - F , Representative FM4 - 64 - stained Arabidopsis roots ( grey ) expressing pWOX5 : : mVenus - NLS ( green ) in Col , plt2 , plt3 plt2 , plt3 double mutant background in longitudinal ( A - D ) , or transversal ( E - F ) optical sections . E ' , F ' , of representative in ( E ) ( F ) in Imaris to detect expressing nuclei . E ' ' , F ' ' , Overlay of 10 roots ( biological replicates ) showing of detected ( high in red , low in blue ) in Col plt2 , plt3 double mutant roots . G , Number of nuclei ( biological replicates ) expressing pWOX5 : : mVenus - NLS in Col plt2 , plt3 double mutant roots summarized in box scatter plots . H , of WOX5 expression in µm2 in Col plt2 , plt3 double mutant roots summarized in box scatter plots . G , H Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . ( G , H ) Kruskal - Wallis ANOVA with subsequent Dunns ( G ) or one - way ANOVA post - hoc Holm - Sidak multiple comparisons was used to for statistical significance ( H ) . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype results from three technical replicates per genotype . Scale bars represent 10 µm ; NLS = localisation signal .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY B-ORGANISM B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-F, Representative FM4-64-stained Arabidopsis roots (grey) expressing pWOX5::mVenus-NLS (green) in Col, plt2, plt3 and plt2, plt3 double mutant background in longitudinal (A-D), or transversal (E-F) optical sections. E', F', Analysis of representative images in (E) and (F) in Imaris to detect and count individual expressing nuclei. E'', F'', Overlay of 10 roots (biological replicates) showing the area of detected fluorescence (high levels in red, low levels in blue) in Col and plt2, plt3 double mutant roots. G, Number of nuclei (biological replicates) expressing pWOX5::mVenus-NLS in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. H, Area of WOX5 expression in µm2 in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. G, H Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. (G, H) Kruskal-Wallis ANOVA analysis with subsequent Dunns test (G) or one-way ANOVA and post-hoc Holm-Sidak multiple comparisons test was used to test for statistical significance (H). Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from three technical replicates per genotype. Scale bars represent 10 µm; NLS = nuclear localisation signal.


A , Schematic representation of a longitudinal section of an Arabidopsis RM . QC are marked in red , CSCs are marked in blue , CCs in blue . Combined mPSPI ( grey ) EdU ( red ) for 24 hours ( SCN ) to analyse CSC ( A - I ) QC division phenotype ( J - R ) within same roots are shown . B - H , Representative of SCN in Col , indicated single , double , triple mutant roots . QC positions are marked by yellow arrowhead . I , Analyses of SCN for CSC . Frequencies of roots showing 0 , 1 , 2 , or 3 CSC layers are plotted as bar graphs . J , Schematic representation of a transversal section of an Arabidopsis RM . QC are marked in red , CEI initials are marked in turquoise . K - Q , Representative of transversal sections with QC outlined in yellow . R , Analyses of SCN for QC division . Frequencies of roots showing 0 , 1 , 2 , 3 or ≥ 4 dividing QC are plotted as bar graphs . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype results from 2 - 5 technical replicates per genotype . QC = quiescent center , CSC = columella , CEI = cortex endodermis initial , SCN = niche , mPSPI = modified pseudo - Schiff propidium iodide , EdU = 5 - ethynyl - 2 ' - deoxyuridine , scale bars represent 5 µm .

O O O O O O O O O O B-ORGANISM B-TISSUE O B-TISSUE O O O O O B-TISSUE O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

A, Schematic representation of a longitudinal section of an Arabidopsis RM. QC cells are marked in red, CSCs are marked in dark blue, CCs in light blue. Combined mPSPI (grey) and EdU (red) staining for 24 hours (SCN staining) to analyse the CSC (A-I) and QC division phenotype (J-R) within the same roots are shown. B-H, Representative images of the SCN staining in Col, and the indicated single, double, and triple mutant roots. QC positions are marked by yellow arrowhead. I, Analyses of the SCN staining for CSC phenotypes. Frequencies of roots showing 0, 1, 2, or 3 CSC layers are plotted as bar graphs. J, Schematic representation of a transversal section of an Arabidopsis RM. QC cells are marked in red, CEI initials are marked in turquoise. K-Q, Representative images of transversal sections with QC cells outlined in yellow. R, Analyses of the SCN staining for QC division phenotypes. Frequencies of roots showing 0, 1, 2, 3 or ≥4 dividing QC cells are plotted as bar graphs. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype. QC = quiescent center, CSC = columella stem cell, CEI = cortex endodermis initial, SCN = stem cell niche, mPSPI = modified pseudo-Schiff propidium iodide, EdU = 5-ethynyl-2'-deoxyuridine, scale bars represent 5 µm.


combined results of SCN 3 are shown as 2D plots to visualise correlation of CSC layer QC division . Number of CSC layers are shown on y axis QC division phenotype is shown on x axis . darker colour , more roots show respective phenotype ( see colour gradient top right indicating frequencies in ) . Col wild - type roots show one layer of CSCs no EdU stained ( no QC division ) after 24 h EdU . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype results from 2 - 5 technical replicates per genotype .

O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE O B-SMALL_MOLECULE B-EXP_ASSAY O O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O

The combined results of the SCN staining 3 are shown as 2D plots to visualise the correlation of the CSC layer and QC division phenotypes. Number of CSC layers are shown on the y axis and the QC division phenotype is shown on the x axis. The darker the colour, the more roots show the respective phenotype (see colour gradient top right indicating the frequencies in percent). Col wild-type roots show one layer of CSCs and no EdU stained cells (no QC division) after 24 h EdU staining. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype.


A - H , Lifetime Imaging ( FLIM ) results of transiently expressing N . benthamiana leaf epidermal . A - H , Representative FLIM of WOX5 - mVenus ( donor only ) plus indicated acceptors after a pixel - wise mono - exponential fit of mVenus signal . lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of two high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE B-CELL_TYPE O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

A-H, Fluorescence Lifetime Imaging (FLIM) results of transiently expressing N. benthamiana leaf epidermal cells. A-H, Representative FLIM images of WOX5-mVenus (donor only) plus the indicated acceptors after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm.


I , lifetimes in ns are summarized in combined jitter box plots . dashed represents lifetime mean value of WOX5 - mV co - expressed with free mCh as negative control . one - way ANOVA Holm - Sidak post - hoc multiple comparisons was used to for statistical significance . Samples with identical letters do not show significant differences ( α = 0 . 01 ) . Number of nuclei analyzed ( n ) ( biological replicates ) is indicated results from 2 to 9 technical replicates . Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , ▪ = mean value .

O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I, Fluorescence lifetimes in ns are summarized in combined jitter and box plots. The dashed line represents the fluorescence lifetime mean value of the WOX5-mV co-expressed with free mCh as negative control. The one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test was used to test for statistical significance. Samples with identical letters do not show significant differences (α = 0.01). Number of nuclei analyzed (n) (biological replicates) is indicated and results from 2 to 9 technical replicates. Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, ▪ = mean value.


J , Representative FLIM of WOX5 - mVenus ( donor only ) after a pixel - wise mono - exponential fit of mVenus signal . lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of two high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

J, Representative FLIM images of WOX5-mVenus (donor only) after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm.


K , nuclei showing bodies during co - expression of WOX5 - mV PLT3 - mCh were analysed for WOX5 - mV lifetime in bodies or nucleoplasm separately ( n = 7 ( biological replicates ) , from four technical replicates . mCh = mCherry . mV = mVenus .

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K, Individual nuclei showing nuclear bodies during co-expression of WOX5-mV and PLT3-mCh were analysed for WOX5-mV lifetime in the nuclear bodies or nucleoplasm separately (n = 7 (biological replicates), from four technical replicates. mCh = mCherry. mV = mVenus.


( a ) was analysed after activation of pathways in presence absence of enforced Atg5 expression ( 18 h cultures ) . Values are means ± s . e . m . of three independent experiments .

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(a) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression (18 h cultures). Values are means ± s.e.m. of three independent experiments.


( b ) regulated by Atg5 is apoptosis . data presented are from 5 h cultures . Caspase - 3 - like activity analysed in HeLa ( graph ) . of Jurkat is also shown . numbers in lower right corner indicate of , which was determined in parallel experiments using flow

O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY

(b) The cell death regulated by Atg5 is apoptosis. All data presented are from 5 h cultures. Caspase-3-like activity analysed in HeLa cells (graph). Morphology of Jurkat cells is also shown. The numbers in the lower right corner indicate the percentage of cell death, which was determined in parallel experiments using flow cytometry


c ) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat blocked by z - VAD - fmk . Numbers indicate results of quantitative ( ) of each dot blot .

O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O

c) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z-VAD-fmk. Numbers indicate the results of the quantitative analysis (percentage) of each dot blot.


d ) Reduction of drug - induced by silencing of Atg5 . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) . effects of Atg5 siRNA on Atg5 GAPDH were analysed by immunoblotting . asterisks indicate P 0 . 05 . n . s . , no significant difference .

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d) Reduction of drug-induced cell death by silencing of Atg5. Values are means ± s.e.m. of three independent experiments (18 h cultures). The effects of the Atg5 siRNA on Atg5 and GAPDH levels were analysed by immunoblotting. The asterisks indicate P 0.05. n.s., no significant difference.


a ) Spontaneous neutrophil apoptosis . Apoptosis was associated with appearance of a 24K anti - Atg5 - reactive . 33K Atg5 disappeared during apoptosis in a time - dependent manner . HeLa overexpressing Atg5 served as positive control .

O O O B-CELL_TYPE O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-CELL_LINE O B-GENEPROD O O O O O

a) Spontaneous neutrophil apoptosis. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein. 33K Atg5 disappeared during apoptosis in a time-dependent manner. HeLa cells overexpressing Atg5 served as positive control.


b ) Anti - CD95 - induced apoptosis of Jurkat . Apoptosis was associated with appearance of a 24K anti - Atg5 - reactive a reduction of 33K Atg5 in a time - dependent manner . 24K anti - Atg5 - reactive was also visible in Jurkat after treatment with actinomycin D ( Act . D ) or cycloheximide ( CHX ; 5 h treatment in both cases ) . GAPDH expression indicates equal loading in each panel . immmunoblots are representative of at least three independent experiments .

O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O

b) Anti-CD95-induced apoptosis of Jurkat cells. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein and a reduction of 33K Atg5 in a time-dependent manner. The 24K anti-Atg5-reactive protein was also visible in Jurkat cells after treatment with actinomycin D (Act. D) or cycloheximide (CHX; 5 h treatment in both cases). GAPDH expression indicates equal loading in each panel. All immmunoblots are representative of at least three independent experiments.


e ) Recombinant Atg5 was digested with calpain products sequenced by Edman degradation . Calpain Atg5 at Thr 193 . Comparison of Atg5 sequences of different species demonstrates presence of same calpain cleavage site in mice . alignment was perfromed with Clustal W using data obtained from GenBank . position of six - - acid deletion causing calpain resistance is indicated in red .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Recombinant Atg5 was digested with calpain and the products sequenced by Edman degradation. Calpain cleaved Atg5 at Thr 193. Comparison of Atg5 sequences of different species demonstrates the presence of the same calpain cleavage site in mice. The sequence alignment was perfromed with Clustal W using sequence data obtained from GenBank. The position of the six-amino-acid deletion causing calpain resistance is indicated in red.


( d ) Following calpain - mediated cleavage of recombinant Atg5 in vitro , sample was fractionated by preparative reversed - phase HPLC analysed by mass spectrometry . exact relative molecular masses for full - truncated Atg5 are indicated .

O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O

(d) Following calpain-mediated cleavage of recombinant Atg5 in vitro, the sample was fractionated by preparative reversed-phase HPLC and analysed by mass spectrometry. The exact relative molecular masses for full-length and truncated Atg5 are indicated.


( f ) A six - - acid deletion surrounding cleavage site ( 35S - Atg5Δ191 - 196 ) led to a loss of calpain cleavage fragment .

O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O

(f) A six-amino-acid deletion surrounding the cleavage site (35S-Atg5Δ191-196) led to a loss of the calpain cleavage fragment.


( g ) Calpain inhibition by ALLN blocked both cleavage of Bax Atg5 in spontaneous Fas receptor - mediated apoptosis of neutrophils . In contrast , Atg6 did not show evidence for cleavage its remained unchanged in this system . Results of 7 h cultures are shown .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O O O O O O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(g) Calpain inhibition by ALLN blocked both cleavage of Bax and Atg5 in spontaneous and Fas receptor-mediated apoptosis of neutrophils. In contrast, Atg6 did not show evidence for cleavage and its levels remained unchanged in this system. Results of 7 h cultures are shown.


( h ) Atg5 is not in apoptotic HeLa ( 5 h staurosporine treatment ) lacking small subunit of calpain , which was silenced by using specific siRNA .

O O O B-GENEPROD O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O

(h) Atg5 is not cleaved in apoptotic HeLa cells (5 h staurosporine treatment) lacking the small subunit of calpain, which was silenced by using specific siRNA.


( i ) Calpain resistant Atg5Δ191 - 196 was unable to sensitize HeLa lacking endogeneous Atg5 ( due to gene silencing , see Fig . 1c ) to staurosporine - induced . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) .

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(i) Calpain resistant Atg5Δ191-196 was unable to sensitize HeLa cells lacking endogeneous Atg5 (due to gene silencing, see Fig. 1c) to staurosporine-induced cell death. Values are means ± s.e.m. of three independent experiments (18 h cultures).


( a ) HeLa were transiently transfected with 24K 33K Atg5 for 24 h subsequently analysed by Hoechst . Values are means ± s . e . m . of three independent experiments . Bax transfection was used as a positive control . ( b ) Representative after Hoechst demonstrating fragmentation of HeLa that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h . Quantitative of these experiments is shown in a . Control transfected were loaded with Mitotracker orange subsequently processed for cytochrome c immunostaining . Enforced expression of 24K Atg5 resulted in cytochrome c release into cytosol apoptosis ( condensation , shrinkage ; approximately 80 % of ) .

O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O

(a) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining. Values are means ± s.e.m. of three independent experiments. Bax transfection was used as a positive control. (b) Representative images after Hoechst staining demonstrating nuclear fragmentation of HeLa cells that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h. Quantitative analysis of these experiments is shown in a. Control and transfected cells were loaded with Mitotracker orange and subsequently processed for cytochrome c immunostaining. Enforced expression of 24K Atg5 resulted in cytochrome c release into the cytosol and apoptosis (nuclear condensation, cell shrinkage; approximately 80% of the cells).


( c ) Jurkat were transfected with 24K 33K Atg5 using lentiviral gene transfer . was observed as a consequence of enforced expression of truncated Atg5 within two days . Morphological ( Diff - Quik ) is also shown revealed that type of was apoptosis ( condensation , shrinkage ) .

O O O B-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O 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) Jurkat cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer. Cell death was observed as a consequence of enforced expression of truncated Atg5 within two days. Morphological analysis (Diff-Quik) is also shown and revealed that the type of death was apoptosis (nuclear condensation, cell shrinkage).


( d ) CEM that either lacked Bcl - 2 or expressed high of Bcl - 2 received truncated Atg5 or GFP by lentiviral gene transfer . In absence of Bcl - 2 , induction of apoptosis as assessed by fragmentation was observed due to enforced expression of truncated Atg5 within two days . Apoptosis was largely blocked in expressing high of Bcl - 2 . GFP transfection did not affect viability of in this system .

O O O B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O

(d) CEM cells that either lacked Bcl-2 or expressed high levels of Bcl-2 received truncated Atg5 or GFP by lentiviral gene transfer. In the absence of Bcl-2, induction of apoptosis as assessed by DNA fragmentation was observed due to enforced expression of truncated Atg5 within two days. Apoptosis was largely blocked in cells expressing high levels of Bcl-2. GFP transfection did not affect viability of the cells in this system.


( e ) Staurosporine - induced Atg5 cleavage ( 5 h treatment ) in CEM independent of Bcl - 2 expression . Blots were stripped reprobed with anti - GAPDH antibody .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O

(e) Staurosporine-induced Atg5 cleavage (5 h treatment) in CEM cells independent of Bcl-2 expression. Blots were stripped and reprobed with anti-GAPDH antibody.