F Immunoblot of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h ( NEN ( low ) 0 . 6 µM or NEN ( High ) 1 . 2 µM , Domperidone ( Domp ) 30 µM ) Cells were transfected either with control ( siCtrl ) or UPP1 - targeting ( siUPP1 ) siRNAs 48h prior to treatment .

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

F Immunoblot of HCT116 whole cell lysate using antibodies against the indicated proteins upon treatment of the cells as indicated for 16h (NEN (low) 0.6 µM or NEN (High) 1.2 µM, Domperidone (Domp) 30 µM) Cells were transfected either with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 48h prior to treatment.


H Ratio of toxicity and viability of HCT116 spheroids ( 3D ) treated as indicated ( NEN 1 . 2 µM , Imipramine ( Imi ) , Desipramine ( Desi ) , Amitriptyline ( Ami ) , each 30 µM ) . Cells were transfected with control ( siCtrl ) or UPP1 - targeting ( siUPP1 ) siRNAs 24h prior to spheroid formation . Data information : data ( N = 3 ) are presented as mean ( SD ) and were analyzed by two - way ANOVA with Tukey post - hoc

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

H Ratio of toxicity and viability of HCT116 spheroids (3D) treated as indicated (NEN 1.2 µM, Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM). Cells were transfected with control (siCtrl) or UPP1-targeting (siUPP1) siRNAs 24h prior to spheroid formation. Data information: data (N=3) are presented as mean (SD) and were analyzed by two -way ANOVA with Tukey post-hoc test


A Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h ( NEN ( N ) 1 . 2 µM , Domperidone ( Domp , D ) , Imipramine ( Imi ) , Desipramine ( Desi ) , Amitriptyline ( Ami ) , each 30 µM , Clomipramine ( Clomi ) 20 µM ) . Data information : , data are presented as mean ( SD ) ( N = 3 ) and were analyzed by Kruskal - Wallis with Dunn ´ s post - hoc test ( A , left graph ) , one - way ANOVA with Tukey post - hoc test ( A , right graph ) In ( A ) , significance is indicated for the comparison of the different treatments to controls . * * p < 0 , 01 ; * * * * p < 0 , 0001 .

O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative mRNA expression levels of DHODH in HCT116 cells treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (Domp, D), Imipramine (Imi), Desipramine (Desi), Amitriptyline (Ami), each 30 µM, Clomipramine (Clomi) 20 µM). Data information: , data are presented as mean (SD) (N=3) and were analyzed by Kruskal-Wallis with Dunn´s post-hoc test (A, left graph), one-way ANOVA with Tukey post-hoc test (A, right graph) In (A), significance is indicated for the comparison of the different treatments to controls. ** p < 0,01; **** p < 0,0001.


D Caspase activity in HCT116 cells treated as indicated for 24h ( NEN 0 . 6 µM , Imipramine ( Imi ) , Desipramine ( Desi ) and Amitriptyline ( Ami ) , each 20 µM , Clomipramine ( Clomi ) 10 µM , A77126 ( A77 ) 50 µM ) . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by two - way ANOVA with Tukey post - hoc

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

D Caspase activity in HCT116 cells treated as indicated for 24h (NEN 0.6 µM, Imipramine (Imi), Desipramine (Desi) and Amitriptyline (Ami), each 20 µM, Clomipramine (Clomi) 10 µM, A77126 (A77) 50 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by two-way ANOVA with Tukey post-hoc test


A Immunofluorescence ( IF ) images of HCT116 cells treated as indicated for 12h ( Imipramine and Domperidone , each 30 µM ) and stained for cholesterol content using filipin III . B Immunofluorescence ( IF ) images of HCT116 cells treated with Domperidone and co - stained for cholesterol and Lamp1 .

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

A Immunofluorescence (IF) images of HCT116 cells treated as indicated for 12h (Imipramine and Domperidone, each 30 µM) and stained for cholesterol content using filipin III. B Immunofluorescence (IF) images of HCT116 cells treated with Domperidone and co-stained for cholesterol and Lamp1.


E Relative mRNA expression levels of the specified genes in HCT116 cells , treated as indicated for 16h ( NEN ( N ) 1 . 2 µM , Domperidone ( D ) 30 µM , Cyclodextrin ( CyD ) 0 . 75 % ) . F Immunoblot analysis of HCT116 whole cell lysates using antibodies against gammaH2AX and treated as indicated for 16h ( NEN 1 . 2 µM , Domperidone ( Domp ) 30 µM , Cyclodextrin ( CD ) 0 . 75 % ) . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by a one - way ANOVA with Tukey post - hoc test * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O B-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative mRNA expression levels of the specified genes in HCT116 cells, treated as indicated for 16h (NEN (N) 1.2 µM, Domperidone (D) 30 µM, Cyclodextrin (CyD) 0.75%). F Immunoblot analysis of HCT116 whole cell lysates using antibodies against gammaH2AX and treated as indicated for 16h (NEN 1.2 µM, Domperidone (Domp) 30 µM, Cyclodextrin (CD) 0.75%). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test * p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


A Ratio of toxicity and viability determined in pancreatic cancer - derived organoids from two patients ( ( left ) PDO - 42 and ( right ) PDO - 48 ) treated as indicated for 3 ( PDO - 42 ) or 5 ( PDO - 48 ) days . Significant differences are shown for the comparison between single and combined drug treatments as indicated ( NEN 1 . 2 µM or 2 . 5 µM , Amitriptyline ( Ami , A ) , Imipramine ( Imi , I ) , Desipramine ( Desi , D ) , each 30 µM , Clomipramine ( Clomi , C ) 20 µM ) . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by a one - way ANOVA with Tukey post - hoc test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-DISEASE I-DISEASE O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between single and combined drug treatments as indicated (NEN 1.2 µM or 2.5 µM, Amitriptyline (Ami, A), Imipramine (Imi, I), Desipramine (Desi, D), each 30 µM, Clomipramine (Clomi, C) 20 µM). Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


B Ratio of toxicity and viability determined in pancreatic cancer - derived organoids from two patients ( ( left ) PDO - 42 and ( right ) PDO - 48 ) treated as indicated for 3 ( PDO - 42 ) or 5 ( PDO - 48 ) days . Significant differences are shown for the comparison between drug treatments as indicated to the control condition ( NEN ( N ) 1 . 2 µM or 2 . 5 µM , Amitriptyline ( A ) , Imipramine ( I ) 20 µM , Paclitaxel ( Pacli ) , 2 , 20 or 200nM ) . As references for sensitization effects to standard chemotherapy , the bars showing single Paclitaxel treatments using the indicated concentrations are highlighted in blue . Bars for controls ( Ctrl ) , Paclitaxel ( Pacli ) , NEN ( N ) alone and combined NEN + Paclitaxel treatment are identical in the individual graphs for comparison to combinations with the respective TCA drugs as indicated . Data information : data are presented as mean ( SD ) ( N = 3 ) and were analyzed by a one - way ANOVA with Tukey post - hoc test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-DISEASE I-DISEASE O O B-TISSUE O O B-ORGANISM O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE 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 B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 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 O O O O O O O O O O O O O O O O O O O O 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 Ratio of toxicity and viability determined in pancreatic cancer-derived organoids from two patients ((left) PDO-42 and (right) PDO-48) treated as indicated for 3 (PDO-42) or 5 (PDO-48) days. Significant differences are shown for the comparison between drug treatments as indicated to the control condition (NEN (N) 1.2 µM or 2.5 µM, Amitriptyline (A), Imipramine (I) 20 µM, Paclitaxel (Pacli), 2, 20 or 200nM). As references for sensitization effects to standard chemotherapy, the bars showing single Paclitaxel treatments using the indicated concentrations are highlighted in blue. Bars for controls (Ctrl), Paclitaxel (Pacli), NEN (N) alone and combined NEN+Paclitaxel treatment are identical in the individual graphs for comparison to combinations with the respective TCA drugs as indicated. Data information: data are presented as mean (SD) (N=3) and were analyzed by a one-way ANOVA with Tukey post-hoc test.* p< 0.05; ** p< 0.01;*** p< 0.001; **** p < 0.0001.


A . Immunoblot of cell extracts following depletion of WDR26 , RanBP9 , ARMC8 , and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells . Endogenous levels of the indicated proteins were monitored by western blotting ( n = 3 ) .

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

A. Immunoblot of cell extracts following depletion of WDR26, RanBP9, ARMC8, and GID4 using pools of siRNAs for 72 hr in HeLa Kyoto cells. Endogenous levels of the indicated proteins were monitored by western blotting (n=3).


Western blotting of samples after ectopic overexpression of HBP1 ( B ) alone , or together with WDR26 or GID4 in HEK - 293T cells . HBP1 levels were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) .

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

Western blotting of samples after ectopic overexpression of HBP1 (B) alone, or together with WDR26 or GID4 in HEK-293T cells. HBP1 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).


Western blotting of samples after ectopic overexpression of ZMYND19 ( C ) alone , or together with WDR26 or GID4 in HEK - 293T cells . ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10 - 12 h ( n = 3 ) .

Western blotting of samples after ectopic overexpression of ZMYND19 (C) alone, or together with WDR26 or GID4 in HEK-293T cells. ZMYND19 levels were monitored after treatment of MG132 or DMSO for 10-12 h (n=3).


C . , D . Western blot analysis of in vitro ubiquitinated HBP1 , which was performed by mixing purified HBP1 with ubiquitin E1 , UBCH5a , and ubiquitin in the presence of the indicated hGID sub - complexes ( n = 3 ) .

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

C., D. Western blot analysis of in vitro ubiquitinated HBP1, which was performed by mixing purified HBP1 with ubiquitin E1, UBCH5a, and ubiquitin in the presence of the indicated hGID sub-complexes (n=3).


E . Immunoblots of in vitro ubiquitinated ZMYND19 , which was performed by mixing purified ZMYND19 with ubiquitin E1 , UBE2H , ubiquitin and the 6 - subunit hGID complex ( ARCM8 , RanBP9 , WDR26 , MAEA , RMND5a and TWA1 ) in the presence or absence of GID4 and a 10 - fold excess of the PGLV GID4 - specific peptide ( n = 2 )

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

E. Immunoblots of in vitro ubiquitinated ZMYND19, which was performed by mixing purified ZMYND19 with ubiquitin E1, UBE2H, ubiquitin and the 6-subunit hGID complex (ARCM8, RanBP9, WDR26, MAEA, RMND5a and TWA1) in the presence or absence of GID4 and a 10-fold excess of the PGLV GID4-specific peptide (n=2)


D . Western blotting of samples following ectopic overexpression of HBP1 either alone , or with full - length ( FL ) or WD40 - truncated WDR26 ( ΔWD40 ) in HEK - 293T cells . HBP1 levels were monitored in cells treated with MG132 or DMSO for 12 - 14 h ( n = 3 ) .

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

D. Western blotting of samples following ectopic overexpression of HBP1 either alone, or with full-length (FL) or WD40-truncated WDR26 (ΔWD40) in HEK-293T cells. HBP1 levels were monitored in cells treated with MG132 or DMSO for 12-14 h (n=3).


E . Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 ( WT ) or the WDR26 ( ΔWD40 ) mutant ( n = 2 ) .

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

E. Western blot analysis of in vitro ubiquitinated HBP1 in the presence of wild type WDR26 (WT) or the WDR26 (ΔWD40) mutant (n=2).


C . Transiently expressed FLAG - GID4 and HSS - tagged ARMC8 isoforms ( α or β ) in HEK293T cells . The presence of GID4 in isoform - specific ARMC8 immunoprecipitates was visualized by immunoblotting ( left panels ) . The right panel shows a western blot of transiently expressed and immunoprecipitated HSS - WDR26 from HEK - 293T cells and the presence of endogenous ARMC8 isoforms ( α or β ) was probed by immunoblotting ( n = 2 ) .

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

C. Transiently expressed FLAG-GID4 and HSS-tagged ARMC8 isoforms (α or β) in HEK293T cells. The presence of GID4 in isoform-specific ARMC8 immunoprecipitates was visualized by immunoblotting (left panels). The right panel shows a western blot of transiently expressed and immunoprecipitated HSS-WDR26 from HEK-293T cells and the presence of endogenous ARMC8 isoforms (α or β) was probed by immunoblotting (n=2).


D . Baculoviral co - expression in Sf9 cells of the 5 - subunit hGID complex ( 5mer ; Strep - RanBP9 , His - WDR26 , FLAG - MAEA , His - RMND5a and His - TWA1 ) along with His - GID4 in the presence of ARMC8α or ARMC8β . Strep - or His - pulldowns revealed the presence of GID4 in ARMC8α , but not in ARMC8β , complexes ( n = 3 ) .

O O B-ORGANISM O O O O B-CELL_LINE O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O

D. Baculoviral co-expression in Sf9 cells of the 5-subunit hGID complex (5mer; Strep-RanBP9, His-WDR26, FLAG-MAEA, His-RMND5a and His-TWA1) along with His-GID4 in the presence of ARMC8α or ARMC8β. Strep- or His-pulldowns revealed the presence of GID4 in ARMC8α, but not in ARMC8β, complexes (n=3).


E . Immunoblot analysis of in vitro ubiquitinated GID4 - hGID complexes containing either ARMC8α or ARMC8β ( 6mer ; ARMC8 , RanBP9 , WDR26 , MAEA , RMND5a and TWA1 ) . Where indicated , the reaction was carried out in the presence of 20 - fold molar excess of the PGLV GID4 - binding peptide ( n = 3 ) .

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

E. Immunoblot analysis of in vitro ubiquitinated GID4-hGID complexes containing either ARMC8α or ARMC8β (6mer; ARMC8, RanBP9, WDR26, MAEA, RMND5a and TWA1) . Where indicated, the reaction was carried out in the presence of 20-fold molar excess of the PGLV GID4-binding peptide (n=3).


F . Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5 - subunit complex in the presence of the different ARMC8 isoforms ( α or β ) ( n = 2 ) .

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

F. Western blot analysis of in vitro ubiquitinated HBP1 by the hGID 5-subunit complex in the presence of the different ARMC8 isoforms (α or β) (n=2).


B , heatmap of log10 scaled PBMC community frequencies for all samples ( n = 42 patients × 2 timepoints = 84 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 communities ( Y axis ) . Four major clusters of communities and two major clusters of samples ( largely separating acute and convalescent samples ) are highlighted .

O O O O O O B-CELL_TYPE O B-EXP_ASSAY 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 O O O O O O O B-ORGANISM O 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 O O O O O O O O O

B, heatmap of log10 scaled PBMC community frequencies for all samples (n = 42 patients × 2 timepoints = 84 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 communities (Y axis). Four major clusters of communities and two major clusters of samples (largely separating acute and convalescent samples) are highlighted.


C , fold changes in log10 frequencies for PBMC communities between acute and convalescent phase samples , filtered to a significance threshold of FDR < 0 . 05 ( moderated paired t - test , mixed effects model ) . Error bars represent the 95 % confidence interval . The acute phase frequency for each community is depicted with the purple heatmap . * , FDR - adjusted P ( FDR P ) < 0 . 05 ; * * , FDR P < 0 . 01 , * * * , FDR P < 0 . 001 .

O O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O

C, fold changes in log10 frequencies for PBMC communities between acute and convalescent phase samples, filtered to a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model). Error bars represent the 95% confidence interval. The acute phase frequency for each community is depicted with the purple heatmap. *, FDR-adjusted P (FDR P) < 0.05; **, FDR P < 0.01, ***, FDR P < 0.001.


E , differences in CHIKV E2 protein expression between acute - phase and convalescent - phase samples per MetaHybridLouvain sub - community . Sub - communities are ordered by largest to smallest difference and filtered to sub - communities where the median of the channel means per sample was higher in the acute - phase samples at a significance threshold of FDR < 0 . 05 . Sub - communities are named by their parent canonical community name plus an arbitrary

O O O O B-ORGANISM 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 O O O O O O O O O O O O O O O O O O O O O O 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, differences in CHIKV E2 protein expression between acute-phase and convalescent-phase samples per MetaHybridLouvain sub-community. Sub-communities are ordered by largest to smallest difference and filtered to sub-communities where the median of the channel means per sample was higher in the acute-phase samples at a significance threshold of FDR < 0.05. Sub-communities are named by their parent canonical community name plus an arbitrary number


F , heatmap of log10 scaled PBMC sub - community frequencies for all samples ( n = 42 patients × 2 timepoints = 84 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 sub - communities ( Y axis ) . Four major clusters of sub - communities and two major clusters of samples ( largely separating acute and convalescent samples ) are highlighted .

O O O O O O B-CELL_TYPE O O O B-EXP_ASSAY 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 O O O O O O O B-ORGANISM O 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 O O O O O O O O O O O O O

F, heatmap of log10 scaled PBMC sub-community frequencies for all samples (n = 42 patients × 2 timepoints = 84 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 sub-communities (Y axis). Four major clusters of sub-communities and two major clusters of samples (largely separating acute and convalescent samples) are highlighted.


, fold changes in log10 frequencies for PBMC sub - communities contrasted between acute and convalescent phase samples , filtered to a significance threshold of FDR < 0 . 05 ( moderated paired t - test , mixed effects model ) . Error bars represent the 95 % confidence interval . The acute phase frequency for each sub - community is depicted with the purple heatmap . * , FDR - adjusted P ( FDR P ) < 0 . 05 ; * * , FDR P < 0 . 01 , * * * , FDR P < 0 . 001 .

O O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O

, fold changes in log10 frequencies for PBMC sub-communities contrasted between acute and convalescent phase samples, filtered to a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model). Error bars represent the 95% confidence interval. The acute phase frequency for each sub-community is depicted with the purple heatmap. *, FDR-adjusted P (FDR P) < 0.05; **, FDR P < 0.01, ***, FDR P < 0.001.


A , relative expression of CD14 + and CD16 + in CD14 + CD16 + sub - communities , depicted as boxplots of the mean expression levels for all samples ( n = 42 patients × 2 timepoints = 84 samples ) , indicates that sub - community 1 has a CD14 + CD16 + + ( aka " non - classical " ) phenotype , while sub - community 2 has a CD14 + + CD16 + ( aka " intermediate " ) phenotype . Differences shown here are significant at FDR < 0 . 05 ( moderated paired t - test , mixed effects model ) ; for a view of all differences significant at this

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

A, relative expression of CD14+ and CD16+ in CD14+CD16+ sub-communities, depicted as boxplots of the mean expression levels for all samples (n = 42 patients × 2 timepoints = 84 samples), indicates that sub-community 1 has a CD14+CD16++ (aka "non-classical") phenotype, while sub-community 2 has a CD14++CD16+ (aka "intermediate") phenotype. Differences shown here are significant at FDR < 0.05 (moderated paired t-test, mixed effects model); for a view of all differences significant at this threshold


B , heatmap of relative expression of markers that most differentiate the sub - communities of CD14 + monocytes by difference in mean expression levels for all samples ( n = 42 patients × 2 timepoints = 84 samples ) . Markers are filtered to those different at a significance threshold of FDR < 0 . 05 ( moderated paired t - test , mixed effects model ) and fold change > 1 . 3 ; for a view of all differences significant at FDR < 0 . 05 The mean intensity of each channel across all sub - communities is shown by the adjacent purple heatmap .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, heatmap of relative expression of markers that most differentiate the sub-communities of CD14+ monocytes by difference in mean expression levels for all samples (n = 42 patients × 2 timepoints = 84 samples). Markers are filtered to those different at a significance threshold of FDR < 0.05 (moderated paired t-test, mixed effects model) and fold change > 1.3; for a view of all differences significant at FDR < 0.05 The mean intensity of each channel across all sub-communities is shown by the adjacent purple heatmap.


( n = 42 pairs of samples , 1 pair per patient ) . Wilcoxon signed - rank test was used to determine statistical significance , with a Benjamini - Hochberg adjustment for FDR ( 40 comparisons ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . A , CXC chemokines . B , CC chemokines . C , interferons . D , TNF superfamily cytokines . E , interleukins . F , colony - stimulating factors .

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(n = 42 pairs of samples, 1 pair per patient). Wilcoxon signed-rank test was used to determine statistical significance, with a Benjamini-Hochberg adjustment for FDR (40 comparisons). *P < 0.05, **P < 0.01, ***P < 0.001. A, CXC chemokines. B, CC chemokines. C, interferons. D, TNF superfamily cytokines. E, interleukins. F, colony-stimulating factors.


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 . A , volcano plot of differentially expressed host transcripts between acute - and convalescent - phase samples , with negative log10 - scaled q values on the Y axis , and the fitted β coefficient for each transcript ( corresponding to modeled log2 fold change ) on the X axis . Transcripts to the right of the vertical dashed line were comparatively upregulated in acute - phase samples , while transcripts to the left were upregulated during the convalescent phase . Transcripts that pass FDR < 0 . 05 and fold change > 2 are colored salmon and turquoise for acute - associated and convalescent - associated transcripts , respectively . Top transcripts by q value or fold change are individually labeled by their corresponding gene symbol .

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 I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A, volcano plot of differentially expressed host transcripts between acute- and convalescent-phase samples, with negative log10-scaled q values on the Y axis, and the fitted β coefficient for each transcript (corresponding to modeled log2 fold change) on the X axis. Transcripts to the right of the vertical dashed line were comparatively upregulated in acute-phase samples, while transcripts to the left were upregulated during the convalescent phase. Transcripts that pass FDR < 0.05 and fold change > 2 are colored salmon and turquoise for acute-associated and convalescent-associated transcripts, respectively. Top transcripts by q value or fold change are individually labeled by their corresponding gene symbol.


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 .

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 I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM 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 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 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-ORGANISM O 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 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 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 .

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

B-ORGANISM B-TISSUE I-TISSUE O 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 I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM 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 O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 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 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 .

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 I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM 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 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 O B-EXP_ASSAY 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. 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 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 .

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 O B-DISEASE O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-ORGANISM 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 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-ORGANISM 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 B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM 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 ) 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 .

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

(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 ) 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 .

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

(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 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 .

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

(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 ) 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 .

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

(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 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 .

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

(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 ) 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 .

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 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

(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 ) 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 .

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

(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 ) 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 .

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

(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 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 .

O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O 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 O O O O O O B-GENEPROD O 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 ) Cell extracts were prepared from the cells in A , then analyzed by immunoblotting with anti - GFP and 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 ) 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 .

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(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 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 .

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(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 ) 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 .

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(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 ) 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 .

O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O 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 O O B-SMALL_MOLECULE 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 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 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 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 .

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

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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 ) 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 .

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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 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 .

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

(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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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

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 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 ) .

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

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 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 .

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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 cells 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 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 cells 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 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 cells 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 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 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 .

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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 . 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 .

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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 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 .

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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 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 ) .

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

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 and mutant histone proteins as substrates . H2A ubiquitylation was assessed by immunoblotting with anti - ubiquitin and 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 ) 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 .

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

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 ) 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 .

O O O O O O O O B-GENEPROD O 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 B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 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 .

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

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 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 .

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

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 analysis of SWR1 complex 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 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 .

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 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 O B-GENEPROD I-GENEPROD I-GENEPROD O 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 O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O 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 . 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 .

O O 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 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 ) .

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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 and CD81 were used as loading controls for ACE2 and 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 . 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 .

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

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 . 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 .

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

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

O O O B-EXP_ASSAY O B-ORGANISM O O O O B-GENEPROD O 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 . Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting .

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


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

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


( 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 .

O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 O O

(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 ) 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 .

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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 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 .

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 O 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 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 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 O O B-TISSUE I-TISSUE O O O O O O O O O O B-TISSUE I-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

(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 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 .

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

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

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 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 .

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 O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O 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 , 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 .

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 O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-TISSUE I-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 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 ) .

O O B-TISSUE I-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 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 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 .

O O O O O B-ORGANISM B-CELL_LINE O O 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 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 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 .

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 O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O 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 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 .

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

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