Figure 8A Representative two - electrode voltage clamp measurements in Xenopus oocytes injected with TREK - 1 alone ( orange ) or TREK - 1 co - injected with cRNAs encoding the indicated proteins . Voltage was ramped from - 120 to + 40 mV . B Relative TREK - 1 current amplitudes at + 40 mV normalized to TREK - 1 injected alone . The number of oocytes is given within the bar graphs ( n = 37 - 97 ) . 3 - 5 batches of oocytes were used for each condition . C Relative currents normalized to TREK - 1 plus POPDC1 in the absence and presence of theophylline . Data are plotted as mean ± SEM . D , E Oocytes were treated with 0 . 5 mM theophylline prior to voltage - clamp measurements as in ( A , B ) .

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Figure 8A Representative two-electrode voltage clamp measurements in Xenopus oocytes injected with TREK-1 alone (orange) or TREK-1 co-injected with cRNAs encoding the indicated proteins. Voltage was ramped from -120 to +40 mV. B Relative TREK-1 current amplitudes at +40 mV normalized to TREK-1 injected alone. The number of oocytes is given within the bar graphs (n=37-97). 3-5 batches of oocytes were used for each condition.C Relative currents normalized to TREK-1 plus POPDC1 in the absence and presence of theophylline. Data are plotted as mean ± SEM.D, E Oocytes were treated with 0.5 mM theophylline prior to voltage-clamp measurements as in (A, B).


Figure 1Immunoblot ( IB ) analyses of the levels of β - catenin and RAS in DLD1 , WT - HCT116 , MT - HCT116 , HepG2 , and SW48 cells transfected with Myc - axin for 36 hrEffects of β - catenin knock - down on polyubiquitination - dependent degradation of RAS . IB analyses of MT - HCT116 cells transfected with control or two independent β - catenin siRNAs for 48 hrPolyubiquitination assays of MT - HCT116 cells cotransfected with two independent β - catenin siRNAs and HA - ubiquitin ( Ub ) for 72 hr in the presence of the proteasomal inhibitor MG132 , and WCLs were immunoprecipitated with an anti - pan - RAS antibodyIB analyses of HEK293 cells transfected with WT - or S33Y - Flag - β - catenin with or without HA - GSK3β for 36 hrIB analyses of HEK293 cells transfected with β - catenin siRNA or control siRNA for 48 h and then treated with L - cell - conditioned medium ( L - CM ) or Wnt3a - CM for 8 hrEffects of KYA1797K or XAV939 on degradation of β - catenin and RAS in isogenic HCT116 cells harboring WT - or MT - CTNNB1 . IB analyses of β - catenin and RAS levels in WT - or MT - HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24 hrICC analyses of WT - or MT - HCT116 cells treated with 20 µM of KYA1797K for 24 hr . Scale bars , 10 µmFor polyubiquitination assays , WT - or MT - HCT116 cells were transfected with FLAG - Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132 . WCLs were immunoprecipitated with β - catenin or pan - RAS antibody and subjected to IB analyses to detect each proteinThe 3 - ( 4 , 5 - dimethylthiazol - 2yl ) - 2 - 5 - diphenyltetrazolium bromide ( MTT ) assays were performed for measurements of growth of WT - or MT - HCT116 cells treated with the indicated concentrations of KYA1797K . Data are presented as the mean ± SD ( n = 3 )

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Figure 1Immunoblot (IB) analyses of the levels of β-catenin and RAS in DLD1, WT-HCT116, MT-HCT116, HepG2, and SW48 cells transfected with Myc-axin for 36 hrEffects of β-catenin knock-down on polyubiquitination-dependent degradation of RAS. IB analyses of MT-HCT116 cells transfected with control or two independent β-catenin siRNAs for 48 hrPolyubiquitination assays of MT-HCT116 cells cotransfected with two independent β-catenin siRNAs and HA-ubiquitin (Ub) for 72 hr in the presence of the proteasomal inhibitor MG132, and WCLs were immunoprecipitated with an anti-pan-RAS antibodyIB analyses of HEK293 cells transfected with WT- or S33Y-Flag-β-catenin with or without HA-GSK3β for 36 hrIB analyses of HEK293 cells transfected with β-catenin siRNA or control siRNA for 48 h and then treated with L-cell-conditioned medium (L-CM) or Wnt3a-CM for 8 hrEffects of KYA1797K or XAV939 on degradation of β-catenin and RAS in isogenic HCT116 cells harboring WT- or MT-CTNNB1. IB analyses of β-catenin and RAS levels in WT- or MT-HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24 hrICC analyses of WT- or MT-HCT116 cells treated with 20 µM of KYA1797K for 24 hr. Scale bars, 10 µmFor polyubiquitination assays, WT- or MT-HCT116 cells were transfected with FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132. WCLs were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analyses to detect each proteinThe 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) assays were performed for measurements of growth of WT- or MT-HCT116 cells treated with the indicated concentrations of KYA1797K. Data are presented as the mean ± SD (n = 3)


Figure 2Analyses of the interaction between β - catenin and RAS . WCLs of DLD1 cells were immunoprecipitated with β - catenin or pan - RAS antibody and subjected to IB analysesIB analyses of indicated proteins in the samples immunoprecipitated with anti - β - catenin antibody with WCLs of HEK293 cells treated with Wnt3a - CMLeft panel : MT - HCT116 cell extracts were fractionated as cytosolic and nuclear portions and subjected to IB analyses , respectively . Right panel : The cytosolic and nuclear fractions were immunoprecipitated with anti - β - catenin antibody and subjected to IB analysesβ - Catenin - RAS interaction was visualized by in situ proximity ligation assay in HEK293 cells treated with L - CM or Wnt3a - CM . Scale bars , 20 µm . The proximity ligation signal was quantified in 10 randomly distributed microscopic images that contained > 100 cellsIn vitro pulldown analyses to identify β - catenin binding region of RAS . Upper panel : Schematic of the HRAS full - length ( FL ) or two deletion mutants . Lower panel : Purified GST - HRAS - FL , - ΔHVR , or - Δ131 - 165 proteins were pulled - down with purified His - β - catenin - FL protein and subjected to IB analysesIB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG - β - catenin and Myc - HRAS - FL , - ΔHVR , or - Δ131 - 165 for 32 hrIB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG - β - catenin and Myc - HRAS - WT , - 2A , or - 2E for 32 hrUpper panel : Schematic of the β - catenin - FL or two deletion mutants . Lower panel : Purified His - β - catenin - FL , ΔN86 , or ΔC110 proteins were pull - down with purified GST - HRAS - FL protein and subjected to IB analysesIB analyses of IP with Myc antibody with WCLs of HEK293 cells cotransfected with Myc - HRAS and GFP - β - catenin - FL , - ΔN86 , or - ΔC110 for 32

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Figure 2Analyses of the interaction between β-catenin and RAS. WCLs of DLD1 cells were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analysesIB analyses of indicated proteins in the samples immunoprecipitated with anti-β-catenin antibody with WCLs of HEK293 cells treated with Wnt3a-CMLeft panel: MT-HCT116 cell extracts were fractionated as cytosolic and nuclear portions and subjected to IB analyses, respectively. Right panel: The cytosolic and nuclear fractions were immunoprecipitated with anti-β-catenin antibody and subjected to IB analysesβ-Catenin-RAS interaction was visualized by in situ proximity ligation assay in HEK293 cells treated with L-CM or Wnt3a-CM. Scale bars, 20 µm. The proximity ligation signal was quantified in 10 randomly distributed microscopic images that contained > 100 cellsIn vitro pulldown analyses to identify β-catenin binding region of RAS. Upper panel: Schematic of the HRAS full-length (FL) or two deletion mutants. Lower panel: Purified GST-HRAS-FL, -ΔHVR, or -Δ131-165 proteins were pulled-down with purified His-β-catenin-FL protein and subjected to IB analysesIB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-FL, -ΔHVR, or -Δ131-165 for 32 hrIB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-WT, -2A, or -2E for 32 hrUpper panel: Schematic of the β-catenin-FL or two deletion mutants. Lower panel: Purified His-β-catenin-FL, ΔN86, or ΔC110 proteins were pull-down with purified GST-HRAS-FL protein and subjected to IB analysesIB analyses of IP with Myc antibody with WCLs of HEK293 cells cotransfected with Myc-HRAS and GFP-β-catenin-FL, -ΔN86, or -ΔC110 for 32 hr


Figure 3Fluorescence spectroscopy analyses of the interaction between β - catenin and KRAS using purified recombinant proteins , intact β - catenin and KRAS . Molar ratios of β - catenin and KRAS are presented . A . U . , absorbance unitsNuclear magnetic resonance ( NMR ) titration for 15N - labeled KRAS ( full - length , 1 - 188 ) with β - catenin ( C - terminal fragment , 661 - 781 ) . The titration points are indicated in red ( 1 : 0 ) , green ( 1 : 5 ) , and blue ( 1 : 10 ) . A gradual perturbation in chemical shift is indicated by the

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Figure 3Fluorescence spectroscopy analyses of the interaction between β-catenin and KRAS using purified recombinant proteins, intact β-catenin and KRAS. Molar ratios of β-catenin and KRAS are presented. A.U., absorbance unitsNuclear magnetic resonance (NMR) titration for 15N-labeled KRAS (full-length, 1-188) with β-catenin (C-terminal fragment, 661-781). The titration points are indicated in red (1:0), green (1:5), and blue (1:10). A gradual perturbation in chemical shift is indicated by the arrow


Figure 4Effects of KYA1797K on the interaction and phosphorylation of β - catenin and pan - RAS . WT - or MT - HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132 . WCLs were immunoprecipitated with pan - RAS antibody and subjected to IB analysesEffects of Wnt3a - CM treatment on the formation of β - catenin destruction complex and phosphorylation of β - catenin and RAS in HEK293 cells treated with L - CM or Wnt3a - CM in the presence of MG132 for 8 hr . WCLs were immunoprecipitated with β - catenin or pan - RAS antibodies and subjected to IB analysesIB analyses were performed to detect β - catenin and pan - RAS in DLD1 cells cotransfected with FLAG - β - catenin - WT or - MT and Myc - KRAS - WT or - 2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA - GSK3β for 24 hr ,

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Figure 4Effects of KYA1797K on the interaction and phosphorylation of β-catenin and pan-RAS. WT- or MT-HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analysesEffects of Wnt3a-CM treatment on the formation of β-catenin destruction complex and phosphorylation of β-catenin and RAS in HEK293 cells treated with L-CM or Wnt3a-CM in the presence of MG132 for 8 hr. WCLs were immunoprecipitated with β-catenin or pan-RAS antibodies and subjected to IB analysesIB analyses were performed to detect β-catenin and pan-RAS in DLD1 cells cotransfected with FLAG-β-catenin-WT or -MT and Myc-KRAS-WT or -2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA-GSK3β for 24 hr, respectively


Figure 5IB analyses to determine the interaction between β - catenin and BBR peptide by in vitro pull - down assays using purified His - β - catenin and GST - BBR proteinsIB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG - β - catenin and GFP - BBR or - Mock in the presence of proteasome inhibitor , ALLN , for 24 hrEffects of BBR overexpression on KYA1797K - induced RAS degradation . I analyses to detect levels of RAS in MT - HCT116 cells transfected with GFP - Mock or - BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hrEffects of BBR overexpression on KYA1797K - induced RAS degradation ICC ( D ; Scale bars , 10 µm ) analyses to detect levels of RAS in MT - HCT116 cells transfected with GFP - Mock or - BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hrMT - HCT116 cells were cotransfected with GFP - Mock or - BBR and FLAG - Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr , and WCLs were immunoprecipitated with pan - RAS antibodyIB analyses of IP with β - catenin antibody in MT - HCT116 cells transfected with GFP - BBR - WT , - 2E , or GFP - Mock in the presence of ALLN for 24 hrIB analyses of MT - HCT116 cells transfected with GFP - Mock , - BBR - WT , or - BBR - 2E for 24 hr and then treated with 20 µM of KYA1797K for 24 hrMT - HCT116 cells were transfected with GFP - Mock , - BBR - WT , or - BBR - 2E together with FLAG - Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells . WCLs were immunoprecipitated with pan - RAS antibody and subjected to IB

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Figure 5IB analyses to determine the interaction between β-catenin and BBR peptide by in vitro pull-down assays using purified His-β-catenin and GST-BBR proteinsIB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG-β-catenin and GFP-BBR or -Mock in the presence of proteasome inhibitor, ALLN, for 24 hrEffects of BBR overexpression on KYA1797K-induced RAS degradation. I analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hrEffects of BBR overexpression on KYA1797K-induced RAS degradation ICC (D; Scale bars, 10 µm) analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hrMT-HCT116 cells were cotransfected with GFP-Mock or -BBR and FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr, and WCLs were immunoprecipitated with pan-RAS antibodyIB analyses of IP with β-catenin antibody in MT-HCT116 cells transfected with GFP-BBR-WT, -2E, or GFP-Mock in the presence of ALLN for 24 hrIB analyses of MT-HCT116 cells transfected with GFP-Mock, -BBR-WT, or -BBR-2E for 24 hr and then treated with 20 µM of KYA1797K for 24 hrMT-HCT116 cells were transfected with GFP-Mock, -BBR-WT, or -BBR-2E together with FLAG-Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses


Figure 6Effects of KYA1797K and PTD - BBR on RAS degradation IB assay of MT - HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr , respectivelyEffects of KYA1797K and PTD - BBR o cell growth MTT assays of MT - HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr , respectively . Data are presented as the mean ± SD ( n = 3 ) Effects of overexpression of GFP - BBR - WT or - 2E mutant on KYA1797K - induced growth and transformation of CRC cells . MT - HCT116 or SW48 cells were transfected with GFP - BBR - WT , - 2E , or GFP - Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell growthEffects of overexpression of GFP - BBR - WT or - 2E mutant on KYA1797K - induced growth and transformation of CRC cells . MT - HCT116 or SW48 cells were transfected with GFP - BBR - WT , - 2E , or GFP - Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular transformationEffects of cotreatments with KYA1797K and PTD - BBR on xenografted tumor growth . MT - HCT116 cells were subcutaneously injected in nude mice with subsequent i . p . injection of vehicle , 20 mg / kg of KYA1797K , or a cotreatment of PTD - BBR ( 25 mg / kg ) and KYA1797K ( 20 mg / kg ) for 20 d . Tumor volumes ( upper panel ) were measured every 4 days , and tumor weights ( lower panel ) were measured at the times of sacrifice . Data are presented as the mean ± SD ( four mice per group ) WCLs prepared from tumor tissues were immunoprecipitated with anti - β - catenin antibody . WCLs or co - IP samples were subjected to IB analysesIHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI . Scale bar , 20

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Figure 6Effects of KYA1797K and PTD-BBR on RAS degradation IB assay of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectivelyEffects of KYA1797K and PTD-BBR o cell growth MTT assays of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively. Data are presented as the mean ± SD (n = 3)Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell growthEffects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular transformationEffects of cotreatments with KYA1797K and PTD-BBR on xenografted tumor growth. MT-HCT116 cells were subcutaneously injected in nude mice with subsequent i.p. injection of vehicle, 20 mg/kg of KYA1797K, or a cotreatment of PTD-BBR (25 mg/kg) and KYA1797K (20 mg/kg) for 20 d. Tumor volumes (upper panel) were measured every 4 days, and tumor weights (lower panel) were measured at the times of sacrifice. Data are presented as the mean ± SD (four mice per group)WCLs prepared from tumor tissues were immunoprecipitated with anti-β-catenin antibody. WCLs or co-IP samples were subjected to IB analysesIHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI. Scale bar, 20 µm


Figure 1 ( A ) smFISH targeting Bmal1 , Cry1 and Nr1d1 in wild - type NIH 3T3 cells at 17h , 29h and 25h after synchronisation with Dex , respectively . Nuclei are stained with DAPI ( blue ) . Each fluorescent dot ( white ) corresponds to a single transcript . Segmented cell boundaries are delineated in grey . The blue and white channels represent maximum z - projections . Scale bar : 20μm . ( B ) Number of Bmal1 , Cry1 , and Nr1d1 transcripts per cell as a function of time after treatment with Dex . These data combine all smFISH hybridizations from the 4h sampled Nr1d1 / Cry1 and Bmal1 / Cry1 experiment . Each dot shows the average over one replicate from an independent slide ( Methods ) . The solid lines represent fits using a two - harmonic cosinor model ( Equation ( 1 ) , Methods ) for each gene individually . ( D ) Distributions of Nr1d1 , Cry1 and Bmal1 transcripts at 21h , 29h and 37h after synchronisation with Dex . μ represents the mean of the distribution , CV represent the coefficient of variation ( standard deviation / mean ) , and N is the number of cells at the given time point . Total number of cells analysed for the Nr1d1 / Cry1 pair : 21h - 449 ; 25h - 414 ; 29h - 521 ; 33h - 463 ; 37h - 477 ; 41h - 429 . Total number of cells analysed for the Bmal1 / Cry1 pair : 17h - 465 ; 21h - 490 ; 25h - 504 ; 29h - 436 ; 33h - 407 ; 37h - 454 ; 41h - 404 .

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Figure 1(A) smFISH targeting Bmal1, Cry1 and Nr1d1 in wild-type NIH 3T3 cells at 17h, 29h and 25h after synchronisation with Dex, respectively. Nuclei are stained with DAPI (blue). Each fluorescent dot (white) corresponds to a single transcript. Segmented cell boundaries are delineated in grey. The blue and white channels represent maximum z-projections. Scale bar: 20μm.(B) Number of Bmal1, Cry1, and Nr1d1 transcripts per cell as a function of time after treatment with Dex. These data combine all smFISH hybridizations from the 4h sampled Nr1d1/Cry1 and Bmal1/Cry1 experiment. Each dot shows the average over one replicate from an independent slide (Methods). The solid lines represent fits using a two-harmonic cosinor model (Equation (1), Methods) for each gene individually.(D) Distributions of Nr1d1, Cry1 and Bmal1 transcripts at 21h, 29h and 37h after synchronisation with Dex. μ represents the mean of the distribution, CV represent the coefficient of variation (standard deviation / mean), and N is the number of cells at the given time point. Total number of cells analysed for the Nr1d1/Cry1 pair: 21h - 449; 25h - 414; 29h - 521; 33h - 463; 37h - 477; 41h - 429. Total number of cells analysed for the Bmal1/Cry1 pair: 17h - 465; 21h - 490; 25h - 504; 29h - 436; 33h - 407; 37h - 454; 41h - 404.


Figure 2 ( A ) Log conditional mean mRNA count for a given area as a function of the log area . Each cell is sorted by area by placing it in one of 20 bins that range from the 5th to the 95th percentile . The conditional mean given the area E [ m | A ] is then the mean mRNA count of each bin . The area is normalised such that the average area is equal to one . Solid lines represent a least - squares linear fit to the data . ( B ) Top image : Dual - channel smFISH targeting Bmal1 and Cry1 simultaneously , taken at 17h after Dex synchronisation . Blue , nuclei stained with DAPI ; green dots , Bmal1 transcripts ; red , Cry1 transcripts . Bottom image : Dual - channel smFISH targeting Nr1d1 and Cry1 simultaneously , taken at 25h after Dex synchronisation . Blue , nuclei stained with DAPI ; green dots , Nr1d1 transcripts ; red , Cry1 transcripts . Scale bar : 20μm . ( C ) Bivariate distributions of mRNA counts per cell for dual - channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1 . Each dot corresponds to a single cell , and the contours represent KDE estimates of the density . Time represents the number of hours after Dex synchronisation , and R represents the Pearson correlation coefficient .

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Figure 2(A) Log conditional mean mRNA count for a given area as a function of the log area. Each cell is sorted by area by placing it in one of 20 bins that range from the 5th to the 95th percentile. The conditional mean given the area E[m|A] is then the mean mRNA count of each bin. The area is normalised such that the average area is equal to one. Solid lines represent a least-squares linear fit to the data.(B) Top image: Dual-channel smFISH targeting Bmal1 and Cry1 simultaneously, taken at 17h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Bmal1 transcripts; red, Cry1 transcripts. Bottom image: Dual-channel smFISH targeting Nr1d1 and Cry1 simultaneously, taken at 25h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Nr1d1 transcripts; red, Cry1 transcripts. Scale bar: 20μm.(C) Bivariate distributions of mRNA counts per cell for dual-channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1. Each dot corresponds to a single cell, and the contours represent KDE estimates of the density. Time represents the number of hours after Dex synchronisation, and R represents the Pearson correlation coefficient.


Figure 3 ( C ) Posterior parameter estimates . Burst frequency is plotted as a function of time , where the solid line represents the posterior mean and the shaded area represents the 90 % confidence interval . Posterior probability densities are shown for the average burst size , β ( which controls the dependence between burst size and cell area ) and ρ ( representing the correlation in burst size between genes ( in log space ) ) , where β is inferred from model M2 and the average burst size and ρ are calculated from model M4 . ( D ) Comparison of the probability density of the data ( kernel density estimates ) with the model ( using model M4 ) . To estimate the probability from the model , the data set was simulated 15 times using the posterior mean parameter values combined with the measured cell areas .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(C) Posterior parameter estimates. Burst frequency is plotted as a function of time, where the solid line represents the posterior mean and the shaded area represents the 90% confidence interval. Posterior probability densities are shown for the average burst size, β (which controls the dependence between burst size and cell area) and ρ (representing the correlation in burst size between genes (in log space)), where β is inferred from model M2 and the average burst size and ρ are calculated from model M4.(D) Comparison of the probability density of the data (kernel density estimates) with the model (using model M4). To estimate the probability from the model, the data set was simulated 15 times using the posterior mean parameter values combined with the measured cell areas.


Figure 1D Heatmaps showing pairwise comparison of MPRA activity for all constructs ( mRNA / DNA ) between donors - left panel : resting CD4 T cells ; right panel : stimulated CD4 T cells .

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Figure 1D Heatmaps showing pairwise comparison of MPRA activity for all constructs (mRNA/DNA) between donors - left panel: resting CD4 T cells; right panel: stimulated CD4 T cells.


Figure 2B Activity of each allele at 10 positive control SNPs and 1 negative control SNP in stimulated T cells . GATA1 , NF - κB , and RUNX1 constructs were designed to include a binding site for the indicated transcription factors ( + ) or with that site disrupted ( - ) . Fixed effects meta - analysis P value is shown : * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 . Box and whisker plots represent median and IQR ( box ) and min - to - max ( whiskers ) . D Expression - modulating effect of each SNP [ log2 ( OR ) ] as measured in MPRA and validation experiments . OR were calculated using the median activity of allelic constructs and are presented with respect to the risk allele ( luciferase : n = 5 , MPRA : n = 12 ) .

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Figure 2B Activity of each allele at 10 positive control SNPs and 1 negative control SNP in stimulated T cells. GATA1, NF-κB, and RUNX1 constructs were designed to include a binding site for the indicated transcription factors (+) or with that site disrupted (-). Fixed effects meta-analysis P value is shown: *<0.05; **<0.01, ***<0.001, ****<0.0001. Box and whisker plots represent median and IQR (box) and min-to-max (whiskers).D Expression-modulating effect of each SNP [log2(OR)] as measured in MPRA and validation experiments. OR were calculated using the median activity of allelic constructs and are presented with respect to the risk allele (luciferase: n=5, MPRA: n=12).


Figure 3 A Pie chart depicting fine - mapping results ( Huang et al , 2017 ) ( posterior probabilities ) for an IBD - associated locus on 21q21 ( left panel ) . MPRA results in resting ( centre panel ) and stimulated CD4 T cells ( right panel ) showing that the putative causal variant has significant expression - modulating effect . B Pie chart depicting Bayesian fine - mapping results for an AS - associated locus on 2p15 ( left panel ) . MPRA results in stimulated T cells ( right panel ) showing that rs6759298 has a significant expression - modulating effect ( the strongest of any variant at this locus ) while the other candidate SNPs have negligible effects . C GWAS results ( Liu et al , 2015 ) at a Crohn ' s disease and multiple sclerosis - associated locus on 6p23 ( left panel ) . MPRA for candidate SNPs in stimulated T cells ( right panel ) identifying a SNP ( rs34421390 ) with by far the greatest expression - modulating effect at this locus ( blue , risk allele reduces expression ; red , risk allele increases expression ) . Dotted horizontal line represents significance threshold ( corrected for multiple - testing ) . D GWAS results at a Type 1 Diabetes - associated locus on 14q32 ( Onengut - Gumuscu et al , 2015 ; left panel ) . MPRA for the candidate SNPs in stimulated T cells ( right panel ) . The construct with the largest and most significant effect contains the risk alleles for 2 SNPs ( rs1988588 and rs3902659 ) , each of which has a smaller effect when tested individually ( position indicated by vertical dotted line ) . Dotted horizontal line represents significance threshold ( corrected for multiple - testing ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 A Pie chart depicting fine-mapping results (Huang et al, 2017) (posterior probabilities) for an IBD-associated locus on 21q21 (left panel). MPRA results in resting (centre panel) and stimulated CD4 T cells (right panel) showing that the putative causal variant has significant expression-modulating effect.  B Pie chart depicting Bayesian fine-mapping results for an AS-associated locus on 2p15 (left panel). MPRA results in stimulated T cells (right panel) showing that rs6759298 has a significant expression-modulating effect (the strongest of any variant at this locus) while the other candidate SNPs have negligible effects.  C GWAS results (Liu et al, 2015) at a Crohn's disease and multiple sclerosis-associated locus on 6p23 (left panel). MPRA for candidate SNPs in stimulated T cells (right panel) identifying a SNP (rs34421390) with by far the greatest expression-modulating effect at this locus (blue, risk allele reduces expression; red, risk allele increases expression). Dotted horizontal line represents significance threshold (corrected for multiple-testing). D GWAS results at a Type 1 Diabetes-associated locus on 14q32 (Onengut-Gumuscu et al, 2015; left panel). MPRA for the candidate SNPs in stimulated T cells (right panel). The construct with the largest and most significant effect contains the risk alleles for 2 SNPs (rs1988588 and rs3902659), each of which has a smaller effect when tested individually (position indicated by vertical dotted line). Dotted horizontal line represents significance threshold (corrected for multiple-testing).


Figure 4 A IBD GWAS results ( Liu et al , 2015 ) at a multi - disease - associated locus on chromosome 6q23 . C A single variant ( rs6927172 ) has the largest and most significant expression - modulating activity in resting ( left panel ) and stimulated CD4 T cells ( right panel ) with the risk allele reducing transcription . Plots represent median and IQR ( box ) and min - to - max ( whiskers ) . FDR - corrected meta - analysis P value shown . E Allele - specific NF - κB binding in CD4 T cells from rs6927172 heterozygotes , demonstrating reduced NF - κB binding to the risk allele following stimulation ( n = 8 ; one - sample t - test , two - tailed ) . F Allele - specific expression of enhancer RNA in heterozygous CD4 T cells . DNA used for technical control ( n = 6 ; paired t - test ; two - tailed ) . G Genome - wide H3K27ac ChIP - seq in stimulated CD4 T cells from major - and minor - allele homozygotes at rs6927172 ( n = 6 ) . Upper panels show input - normalised H3K27ac signals plotted against enhancer rank . Super - enhancers are defined above the inflection point of the curve . Lower panels show H3K27ac reads from a major ( left ) and a minor ( risk ) allele homozygote ( right ) in a 9kb window around rs6927172 . I Expression of genes on 6q23 in CD4 T cells from 131 patients with active IBD , stratified by rs6927172 genotype ( qPCR ; one - way ANOVA ) . Error bars represent SD . Expression of IL20RA and IL22RAR2 not detected .

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Figure 4 A IBD GWAS results (Liu et al, 2015) at a multi-disease-associated locus on chromosome 6q23. C A single variant (rs6927172) has the largest and most significant expression-modulating activity in resting (left panel) and stimulated CD4 T cells (right panel) with the risk allele reducing transcription. Plots represent median and IQR (box) and min-to-max (whiskers). FDR-corrected meta-analysis P value shown. E Allele-specific NF-κB binding in CD4 T cells from rs6927172 heterozygotes, demonstrating reduced NF-κB binding to the risk allele following stimulation (n=8; one-sample t-test, two-tailed). F Allele-specific expression of enhancer RNA in heterozygous CD4 T cells. DNA used for technical control (n=6; paired t-test; two-tailed). G Genome-wide H3K27ac ChIP-seq in stimulated CD4 T cells from major- and minor-allele homozygotes at rs6927172 (n=6). Upper panels show input-normalised H3K27ac signals plotted against enhancer rank. Super-enhancers are defined above the inflection point of the curve. Lower panels show H3K27ac reads from a major (left) and a minor (risk) allele homozygote (right) in a 9kb window around rs6927172. I Expression of genes on 6q23 in CD4 T cells from 131 patients with active IBD, stratified by rs6927172 genotype (qPCR; one-way ANOVA). Error bars represent SD. Expression of IL20RA and IL22RAR2 not detected.


Figure 5B Primary CD4 T cell editing efficiency for indicated combinations of 5 ' and 3 ' gRNAs ( n = 6 for DB , DH , FH , and n = 2 for FB ) . Distribution of indels assessed using ICE . D Expression of genes on 6q23 in EU - containing mRNA ( EU added at time of stimulation ) showing that deletion of the NF - κB binding site specifically reduces transcription of TNFAIP3 , but not other genes at this locus ( n = 6 ; one sample t - test ) . Representative data from the DH gRNA combination . E Expression of CD69 , an activation marker , following CRISPR editing with indicated gRNA combinations or the non - targeting ( negative ) control ( NTC ) - data shown for ATTO - 550 positive ( RNP - containing ) and negative cells ( n = 6 ; paired t - test , one - tailed ) . Inset flow cytometry plot depicting representative gating of ATTO - 550 positive and negative cells . F Correlation between editing efficiency ( total indel rate ) and levels of phosphorylated IκBα in CD4 T cells ( normalised to the mean fluorescence intensity in NTC ; n = 9 , linear regression ) . G Secretion of effector cytokines following deletion of the NF - κB binding site - Th1 ( left panel , IFNγ ) , Th17 ( centre panel , IL - 17A ) and Th2 subsets ( right panel , IL - 4 ) ( n = 6 , paired t - test , one - tailed ) .

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Figure 5B Primary CD4 T cell editing efficiency for indicated combinations of 5' and 3' gRNAs (n=6 for DB, DH, FH, and n=2 for FB). Distribution of indels assessed using ICE.D Expression of genes on 6q23 in EU-containing mRNA (EU added at time of stimulation) showing that deletion of the NF-κB binding site specifically reduces transcription of TNFAIP3, but not other genes at this locus (n=6; one sample t-test). Representative data from the DH gRNA combination.E Expression of CD69, an activation marker, following CRISPR editing with indicated gRNA combinations or the non-targeting (negative) control (NTC) - data shown for ATTO-550 positive (RNP-containing) and negative cells (n=6; paired t-test, one-tailed). Inset flow cytometry plot depicting representative gating of ATTO-550 positive and negative cells.F Correlation between editing efficiency (total indel rate) and levels of phosphorylated IκBα in CD4 T cells (normalised to the mean fluorescence intensity in NTC; n=9, linear regression). G Secretion of effector cytokines following deletion of the NF-κB binding site - Th1 (left panel, IFNγ), Th17 (centre panel, IL-17A) and Th2 subsets (right panel, IL-4) (n=6, paired t-test, one-tailed).


Figure 1 ( A ) MCF10A - eGFP - LC3 cells were left untreated ( Control ) or treated with 5 μM rapamycin ( Rapa ) , 500 ng / ml TRAIL or 10 ng / ml TNF for 24 h . When indicated , 10 mM 3 ‐ MA or 5 μg / ml TRAIL ‐ R2 antagonist antibody was added 1 h before the drugs . Representative confocal images ( 20 μm scale bars ) and the percentages of cells with LC3 translocation are shown . ( B ) MCF10A - eGFP - LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting ( left ) , or stimulated with 500 ng / ml TRAIL for 24 h and analysed for LC3 translocation ( right ) . ( C ) MCF10A cells left untreated ( Control ) or treated with 500 ng / ml TRAIL or 5 μM rapamycin ( Rapa ) for 24 h were analysed for LC3 ‐ I , LC3 ‐ II and tubulin by immunoblotting . Pepstatin A and E64d were added to the cells 4 h before harvesting . Similar results were obtained in two independent experiments . ( D ) MCF10A cells were treated with 5 μM rapamycin ( Rapa ) or indicated concentrations of TRAIL for 24 h , and the increase in the degradation of long ‐ lived proteins as compared with the untreated cultures ( Control ) was measured . ( E ) MCF10A - eGFP - LC3 cells transfected with the indicated siRNAs were left untreated 48 h later ( Control ) or treated with 500 ng / ml TRAIL for 24 h and analysed for the DNA content by flow cytometry . The percentage of apoptotic cells with sub ‐ G1 DNA content is shown . The values represent mean ± s . d . for three ( A , D ) or four ( B , E ) independent experiments . * P ‐ value 0 . 05 , * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with control sample ( A - left panel and D ) , with TRAIL ‐ treated cells without anti ‐ TRAIL ‐ R2 ( A - right panel ) or with TRAIL ‐ treated cells without siRNA ( B , E ) .

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Figure 1(A) MCF10A-eGFP-LC3 cells were left untreated (Control) or treated with 5 μM rapamycin (Rapa), 500 ng/ml TRAIL or 10 ng/ml TNF for 24 h. When indicated, 10 mM 3‐MA or 5 μg/ml TRAIL‐R2 antagonist antibody was added 1 h before the drugs. Representative confocal images (20 μm scale bars) and the percentages of cells with LC3 translocation are shown.(B) MCF10A-eGFP-LC3 cells were transfected with the indicated siRNAs for 48 h and analysed by immunoblotting (left), or stimulated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (right).(C) MCF10A cells left untreated (Control) or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa) for 24 h were analysed for LC3‐I, LC3‐II and tubulin by immunoblotting. Pepstatin A and E64d were added to the cells 4 h before harvesting. Similar results were obtained in two independent experiments.(D) MCF10A cells were treated with 5 μM rapamycin (Rapa) or indicated concentrations of TRAIL for 24 h, and the increase in the degradation of long‐lived proteins as compared with the untreated cultures (Control) was measured.(E) MCF10A-eGFP-LC3 cells transfected with the indicated siRNAs were left untreated 48 h later (Control) or treated with 500 ng/ml TRAIL for 24 h and analysed for the DNA content by flow cytometry. The percentage of apoptotic cells with sub‐G1 DNA content is shown. The values represent mean±s.d. for three (A, D) or four (B, E) independent experiments. *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with control sample (A-left panel and D), with TRAIL‐treated cells without anti‐TRAIL‐R2 (A-right panel) or with TRAIL‐treated cells without siRNA (B, E).


Figure 2 ( A ) Protein lysates from MCF10A cells left untreated or treated with 500 ng / ml TRAIL or 5 μM rapamycin ( Rapa , positive control ) for 24 h were analysed by immunoblotting for phosphorylated p70S6K ( P ‐ p70S6K ) , total p70S6K and GAPDH ( loading control ) . ( B ) Immunoblot analysis of the levels of P ‐ ACC and tubulin ( loading control ) in lysates from MCF10A cells left untreated or treated with 500 ng / ml TRAIL for 24 h , 1 mM AICAR for 4 h ( positive control ) or 5 μM rapamycin for 24 h ( negative control ) . ( C ) Immunoblot analysis of the level of P ‐ ACC and GAPDH ( loading control ) in lysates from MCF10A cells treated with 500 ng / ml TRAIL for the indicated times . ( D ) MCF10A cells were transfected with indicated siRNAs for 48 h and analysed by immunoblotting for the indicated proteins immediately ( upper panel ) or after additional 24 h ( middle panel ) or 2 h ( lower panel ) incubation with ( + ) or without ( − ) 500 ng / ml TRAIL . ( E ) MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h and incubated for an additional 24 h with or without 500 ng / ml TRAIL were analysed for LC3 translocation ( top ) and the DNA content ( bottom ) . The values represent mean ± s . d . for four independent experiments . * P ‐ value 0 . 05 and * * * P ‐ value 0 . 001 as compared with TRAIL ‐ treated cells without siRNA . Similar results were obtained in two ( A , B and D ) or three ( C , D ) independent experiments .

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Figure 2(A) Protein lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL or 5 μM rapamycin (Rapa, positive control) for 24 h were analysed by immunoblotting for phosphorylated p70S6K (P‐p70S6K), total p70S6K and GAPDH (loading control).(B) Immunoblot analysis of the levels of P‐ACC and tubulin (loading control) in lysates from MCF10A cells left untreated or treated with 500 ng/ml TRAIL for 24 h, 1 mM AICAR for 4 h (positive control) or 5 μM rapamycin for 24 h (negative control).(C) Immunoblot analysis of the level of P‐ACC and GAPDH (loading control) in lysates from MCF10A cells treated with 500 ng/ml TRAIL for the indicated times.(D) MCF10A cells were transfected with indicated siRNAs for 48 h and analysed by immunoblotting for the indicated proteins immediately (upper panel) or after additional 24 h (middle panel) or 2 h (lower panel) incubation with (+) or without (−) 500 ng/ml TRAIL.(E) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h and incubated for an additional 24 h with or without 500 ng/ml TRAIL were analysed for LC3 translocation (top) and the DNA content (bottom). The values represent mean±s.d. for four independent experiments. *P‐value 0.05 and ***P‐value 0.001 as compared with TRAIL‐treated cells without siRNA. Similar results were obtained in two (A, B and D) or three (C, D) independent experiments.


Figure 3 ( A ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs ( top ) and proteins ( bottom by RT - PCR and immunoblotting , respectively . Similar results were obtained in three independent experiments . ( B ) MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h , and left untreated or treated with 500 ng / ml TRAIL for 2 h , 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h ( starvation ) were analysed for P ‐ ACC and tubulin ( loading control ) expression by immunoblotting . Similar results were obtained in two independent experiments . ( C ) Cells transfected as in ( B ) and left untreated or treated with 500 ng / ml TRAIL , 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation . The values represent mean ± s . d . of three independent experiments . * * * P ‐ value 0 . 001 as compared with cells treated in a same way , but transfected without siRNA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top) and proteins (bottom by RT-PCR and immunoblotting, respectively. Similar results were obtained in three independent experiments.(B) MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h, and left untreated or treated with 500 ng/ml TRAIL for 2 h, 10 μM ionomycin for 24 h or starved for amino acids and glucose for 24 h (starvation) were analysed for P‐ACC and tubulin (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments.(C) Cells transfected as in (B) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation. The values represent mean±s.d. of three independent experiments. ***P‐value 0.001 as compared with cells treated in a same way, but transfected without siRNA.


Figure 4 ( A ) The lysates of MCF10A cells treated with 500 ng / ml TRAIL for the indicated times were analysed by immunoblotting . The control lane ( 0 min TRAIL ) originates from the same blot and has been treated and exposed identically . Similar results were obtained in three independent experiments . ( B , C ) The lysates of MCF10A - eGFP - LC3 cells transfected with indicated siRNAs for 48 h and left untreated ( − ) or treated with 500 ng / ml TRAIL for 2 h were analysed by immunoblotting . ( C , lower panel ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT - PCR . Similar results were obtained in two independent experiments . ( D , E ) MCF10A - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h , treated as in Figure 3C and analysed for LC3 translocation ( left ) and sub ‐ G1 DNA content ( right ) . The values represent mean ± s . d . of a minimum of three independent experiments except for the right panel in E ( two experiments ) . * P ‐ value 0 . 05 and * * P ‐ value 0 . 01 as compared with cells treated in a same way , but transfected without siRNA .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) The lysates of MCF10A cells treated with 500 ng/ml TRAIL for the indicated times were analysed by immunoblotting. The control lane (0 min TRAIL) originates from the same blot and has been treated and exposed identically. Similar results were obtained in three independent experiments.(B, C) The lysates of MCF10A-eGFP-LC3 cells transfected with indicated siRNAs for 48 h and left untreated (−) or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting. (C, lower panel) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs by RT-PCR. Similar results were obtained in two independent experiments.(D, E) MCF10A-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h, treated as in Figure 3C and analysed for LC3 translocation (left) and sub‐G1 DNA content (right). The values represent mean±s.d. of a minimum of three independent experiments except for the right panel in E (two experiments). *P‐value 0.05 and **P‐value 0.01 as compared with cells treated in a same way, but transfected without siRNA.


Figure 5 ( A ) Protein lysates from MCF10A cells left untreated ( 0 ) or treated with 50 ng / ml TNF , 500 ng / ml TRAIL ( positive control ) or 10 ng / ml IL ‐ 1β for indicated times were analysed by immunoblotting for phosphorylated ACC ( P ‐ ACC ) , phosphorylated IκB ( P ‐ IκB ) and GAPDH ( loading control ) . The values show the P ‐ ACC / GAPDH and P ‐ IkB / GAPDH ratios as percentages of the ratios in untreated cells ( left lanes ) and are representative of two independent experiments . ( B , C ) MCF10A - eGFP - LC3 cells left untreated or treated with 500 ng / ml TRAIL , 50 ng / ml TNF or 10 ng / ml IL ‐ 1β for 24 h were analysed for LC3 translocation ( B ) and the DNA content ( C ) . The values represent mean ± s . d . for two independent experiments . * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with untreated cells .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Protein lysates from MCF10A cells left untreated (0) or treated with 50 ng/ml TNF, 500 ng/ml TRAIL (positive control) or 10 ng/ml IL‐1β for indicated times were analysed by immunoblotting for phosphorylated ACC (P‐ACC), phosphorylated IκB (P‐IκB) and GAPDH (loading control). The values show the P‐ACC/GAPDH and P‐IkB/GAPDH ratios as percentages of the ratios in untreated cells (left lanes) and are representative of two independent experiments.(B, C) MCF10A-eGFP-LC3 cells left untreated or treated with 500 ng/ml TRAIL, 50 ng/ml TNF or 10 ng/ml IL‐1β for 24 h were analysed for LC3 translocation (B) and the DNA content (C). The values represent mean±s.d. for two independent experiments. **P‐value 0.01 and ***P‐value 0.001 as compared with untreated cells.


Figure 6 ( A ) The lysates of hTERT - RPE1 cells transfected with indicated siRNAs for 48 h and left untreated or treated with 500 ng / ml TRAIL for 2 h were analysed by immunoblotting . ( B ) hTERT - RPE1 - eGFP - LC3 cells transfected with indicated siRNAs for 48 h were left untreated or treated with 500 ng / ml TRAIL for 24 h and analysed for LC3 translocation ( left ) and sub ‐ G1 DNA content ( middle ) and AMPKα expression ( right ) . ( C ) hTERT - RPE1 - eGFP - LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs ( top ) . After 48 h , cells were left untreated or treated with 500 ng / ml TRAIL for 2 h , and analysed for P ‐ ACC and GAPDH ( loading control ) expression by immunoblotting . Similar results were obtained in two independent experiments . ( D ) Cells transfected as described in ( C ) and left untreated or treated with 500 ng / ml TRAIL , 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation . ( E ) Lysates of wild ‐ type ( WT ) MEFs and MEFs with an inactive TAK1 knock ‐ in ( TAK1Δ ) treated with 500 ng / ml TRAIL were analysed by immunoblotting . Similar results were obtained in two independent experiments . ( F ) WT and TAK1Δ MEFs left untreated and treated with 250 or 500 ng / ml TRAIL were analysed for cell death by the LDH release assay . Similar results were obtained in two independent experiments ( A , C and E ) . The values represent mean ± s . d . for two ( D ) or a minimum of three independent experiments ( B , F ) . * P ‐ value 0 . 05 , * * P ‐ value 0 . 01 and * * * P ‐ value 0 . 001 as compared with cells treated in the same way , but transfected without siRNA ( B , D ) or the wild ‐ type cells ( F ) .

O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) The lysates of hTERT-RPE1 cells transfected with indicated siRNAs for 48 h and left untreated or treated with 500 ng/ml TRAIL for 2 h were analysed by immunoblotting.(B) hTERT-RPE1-eGFP-LC3 cells transfected with indicated siRNAs for 48 h were left untreated or treated with 500 ng/ml TRAIL for 24 h and analysed for LC3 translocation (left) and sub‐G1 DNA content (middle) and AMPKα expression (right).(C) hTERT-RPE1-eGFP-LC3 cells were transfected with indicated siRNAs for 48 h and analysed for the indicated mRNAs (top). After 48 h, cells were left untreated or treated with 500 ng/ml TRAIL for 2 h, and analysed for P‐ACC and GAPDH (loading control) expression by immunoblotting. Similar results were obtained in two independent experiments.(D) Cells transfected as described in (C) and left untreated or treated with 500 ng/ml TRAIL, 10 μM ionomycin or starved for amino acids and glucose for 24 h were analysed for LC3 translocation.(E) Lysates of wild‐type (WT) MEFs and MEFs with an inactive TAK1 knock‐in (TAK1Δ) treated with 500 ng/ml TRAIL were analysed by immunoblotting. Similar results were obtained in two independent experiments.(F) WT and TAK1Δ MEFs left untreated and treated with 250 or 500 ng/ml TRAIL were analysed for cell death by the LDH release assay. Similar results were obtained in two independent experiments (A, C and E). The values represent mean±s.d. for two (D) or a minimum of three independent experiments (B, F). *P‐value 0.05, **P‐value 0.01 and ***P‐value 0.001 as compared with cells treated in the same way, but transfected without siRNA (B, D) or the wild‐type cells (F).


Figure 1B . Yeast two - hybrid analysis of the interactions between FREE1 and core microprocessor . SD - 2 : SD / - Leu - Trp . SD - 4 : SD / - Ade - His - Leu - Trp . Three dots represent three yeast colonies randomly picked out in each sample . C . BiFC assay to verify the interaction between FREE1 and CPL1 . YC - FREE1 and YN - CPL1 , were co - expressed with mCherry - HYL1 , SE - mCherry , or DCL1 - mCherry in Arabidopsis protoplasts , respectively , followed by confocal observation . D . Co - immunoprecipitation analysis of interactions between FREE1 and CPL1 and CPL2 . Arabidopsis protoplasts co - expressing mCherry - FREE1 with YFP , YFP - CPL1 or YFP - CPL2 were subjected to protein extraction and IP with GFP - trap followed by immunoblotting with mCherry and GFP antibodies . E . Co - immunoprecipitation analysis of the associations between FREE1 and microprocessor components . Transgenic plants expressing GFP - FREE1 were subjected to protein extraction and IP with GFP - trap followed by immunoblotting with indicated antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Yeast two-hybrid analysis of the interactions between FREE1 and core microprocessor. SD-2: SD/-Leu-Trp. SD-4: SD/-Ade-His-Leu-Trp. Three dots represent three yeast colonies randomly picked out in each sample.C. BiFC assay to verify the interaction between FREE1 and CPL1. YC-FREE1 and YN-CPL1, were co-expressed with mCherry-HYL1, SE-mCherry, or DCL1-mCherry in Arabidopsis protoplasts, respectively, followed by confocal observation.D. Co-immunoprecipitation analysis of interactions between FREE1 and CPL1 and CPL2. Arabidopsis protoplasts co-expressing mCherry-FREE1 with YFP, YFP-CPL1 or YFP-CPL2 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with mCherry and GFP antibodies.E. Co-immunoprecipitation analysis of the associations between FREE1 and microprocessor components. Transgenic plants expressing GFP-FREE1 were subjected to protein extraction and IP with GFP-trap followed by immunoblotting with indicated antibodies.


Figure 2B . Small RNA gel blots for 7 - day - old seedlings Col - 0 and free1 - ctmut plants . U6 was used as a loading control . The numbers indicate the relative abundance of miR168 , miR166 , miR162 , siRNA1003 , tasiRNA255 , IR71 , miR156 and miR172 in the plants ( the intensity in WT was arbitrarily annotated as 1 . 00 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. Small RNA gel blots for 7-day-old seedlings Col-0 and free1-ctmut plants. U6 was used as a loading control. The numbers indicate the relative abundance of miR168, miR166, miR162, siRNA1003, tasiRNA255, IR71, miR156 and miR172 in the plants (the intensity in WT was arbitrarily annotated as 1.00).


Figure 3A . Detection of HYL1 phosphorylation in Col - 0 , free1 - ctmut or cpl1 - 8 . Proteins extracted from 7 - day - old Arabidopsis seedlings were separated by Phos - tag polyacrylamide gels or SDS polyacrylamide gels followed by immunoblotting with indicated antibodies . The numbers in the first row indicate the relative grayscale intensities of total HYL1 , the numbers in the second row indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1 , and the intensity in WT was arbitrarily set to 1 . 00 . B . Transgenic plants expressing pHYL1 : : YFP - HYL1 in Col - 0 , free1 - ctmut or cpl1 - 8 backgrounds were subjected to protein extraction and IP with GFP - trap , followed by detection with phos - tag BTL - 111 or GFP antibody . The numbers indicate the relative intensities of each band detected with phos - tag BTL - 111 and anti - GFP , and the first lane was arbitrarily set to 1 . 00 . C . Confocal observation of the YFP - HYL1 - containing D - bodies in Col - 0 and free1 - ctmut roots . Arrows indicated the YFP - HYL1 - containing D - bodies . D . Statistically analysis of the number of YFP - HYL1 - containing D - bodies as shown in Fig 4C . The X - axis represents the number of D - bodies per cell , and the Y - axis represents the percentage of cells with the corresponding numbers . The quantification was performed by observing more than 400 cells from 20 roots for each genotypeE . Protein abundance of core components of miRNA processing complex in Col - 0 and free1 - ctmut . Proteins extracted from 7 - day - old Arabidopsis seedlings were used for immunoblotting with indicated antibodies . The numbers represent the intensities of each band detected with indicated antibody relative to anti - UGPase , and the first lane was arbitrarily set to 1 . 00 . F . Proteins extracted from 7 - day - old Arabidopsis seedlings treated with CHX ( 0 . 5 mM ) in dark for 0h , 2h , 4h were used for immunoblotting analysis with indicated antibodies . The numbers represent the relative intensities of each band detected with anti - HYL1 and anti - Actin , and the first lane was arbitrarily set to 1 . 00 . G . Proteins extracted from 7 - day - old Arabidopsis seedlings with 3 - d continuous light or dark treatment were used for immunoblotting analysis with indicated antibodies .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Detection of HYL1 phosphorylation in Col-0, free1-ctmut or cpl1-8. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels or SDS polyacrylamide gels followed by immunoblotting with indicated antibodies. The numbers in the first row indicate the relative grayscale intensities of total HYL1, the numbers in the second row indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the intensity in WT was arbitrarily set to 1.00.B. Transgenic plants expressing pHYL1:: YFP-HYL1 in Col-0, free1-ctmut or cpl1-8 backgrounds were subjected to protein extraction and IP with GFP-trap, followed by detection with phos-tag BTL-111 or GFP antibody. The numbers indicate the relative intensities of each band detected with phos-tag BTL-111 and anti-GFP, and the first lane was arbitrarily set to 1.00.C. Confocal observation of the YFP-HYL1-containing D-bodies in Col-0 and free1-ctmut roots. Arrows indicated the YFP-HYL1-containing D-bodies. D. Statistically analysis of the number of YFP-HYL1-containing D-bodies as shown in Fig 4C. The X-axis represents the number of D-bodies per cell, and the Y-axis represents the percentage of cells with the corresponding numbers. The quantification was performed by observing more than 400 cells from 20 roots for each genotypeE. Protein abundance of core components of miRNA processing complex in Col-0 and free1-ctmut. Proteins extracted from 7-day-old Arabidopsis seedlings were used for immunoblotting with indicated antibodies. The numbers represent the intensities of each band detected with indicated antibody relative to anti-UGPase, and the first lane was arbitrarily set to 1.00.F. Proteins extracted from 7-day-old Arabidopsis seedlings treated with CHX (0.5 mM) in dark for 0h, 2h, 4h were used for immunoblotting analysis with indicated antibodies. The numbers represent the relative intensities of each band detected with anti-HYL1 and anti-Actin, and the first lane was arbitrarily set to 1.00.G. Proteins extracted from 7-day-old Arabidopsis seedlings with 3-d continuous light or dark treatment were used for immunoblotting analysis with indicated antibodies.


Figure 4A . Proteins extracted from Arabidopsis protoplasts co - expressing YFP - CPL1 and different amount of 3myc - FREE1 were used for immunoprecipitation with GFP - Trap followed by detection with indicated antibodies . B . Proteins extracted from transgenic plants expressing YFP - CPL1 in Col - 0 or free1 - ctmut background were used for IP with GFP - trap followed by immunoblotting analysis with indicated antibodies . C . Statistically analysis of the relative intensity of FREE1 , HYL1 and SE in Col - 0 or free1 - ctmut background ( IP / Input ) . Data are presented as means ± s . d . ( 3 biological replicates ) , the asterisk indicates significant difference between the samples ( * * P < 0 . 01 by Student ' s t - test , compared with WT ) . D . Transgenic plants of YFP - HYL1 / WT , YFP - HYL1 / free1 - ctmut , YFP - HYL1 / cpl1 - 8 and GFP / WT were subjected to protein extraction and IP with GFP - trap followed by immunoblotting analysis with indicated antibodies . E . Statistically analyzed the relative intensity of FREE1 and HYL1 ( IP / Input ) . Data are presented as means ± s . d . ( 3 biological replicates ) , the asterisk indicates significant difference between the samples ( * * P < 0 . 01 by Student ' s t - test , compared with WT ) . F . Proteins extracted from 7 - day - old Arabidopsis seedlings were separated by Phos - tag polyacrylamide gels followed by immunoblotting analysis with HYL1 antibody . The numbers indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1 , and the first lane was arbitrarily set to 1 . 00 .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Proteins extracted from Arabidopsis protoplasts co-expressing YFP-CPL1 and different amount of 3myc-FREE1 were used for immunoprecipitation with GFP-Trap followed by detection with indicated antibodies.B. Proteins extracted from transgenic plants expressing YFP-CPL1 in Col-0 or free1-ctmut background were used for IP with GFP-trap followed by immunoblotting analysis with indicated antibodies.C. Statistically analysis of the relative intensity of FREE1, HYL1 and SE in Col-0 or free1-ctmut background (IP/Input). Data are presented as means ± s.d. (3 biological replicates), the asterisk indicates significant difference between the samples (**P < 0.01 by Student's t-test, compared with WT).D. Transgenic plants of YFP-HYL1 / WT, YFP-HYL1 / free1-ctmut, YFP-HYL1 / cpl1-8 and GFP / WT were subjected to protein extraction and IP with GFP-trap followed by immunoblotting analysis with indicated antibodies.E. Statistically analyzed the relative intensity of FREE1 and HYL1(IP/Input). Data are presented as means ± s.d. (3 biological replicates), the asterisk indicates significant difference between the samples (**P < 0.01 by Student's t-test, compared with WT).F. Proteins extracted from 7-day-old Arabidopsis seedlings were separated by Phos-tag polyacrylamide gels followed by immunoblotting analysis with HYL1 antibody. The numbers indicate the relative intensities of dephosphorylated HYL1 and phosphorylated HYL1, and the first lane was arbitrarily set to 1.00.


Figure 1Fluorescent images ( left panels ) and tracings ( right panels ) of PVD neurons of the indicated genotypes . PVD is visualized by the wdIs52 transgene . Primary ( 1º ) , secondary ( 2º ) , tertiary ( 3º ) , and quaternary ( 4º ) dendrites are indicated , and the cell body is marked with an asterisk . Anterior is to the left and dorsal is up in all panels . Scale bars represent 20μm . Quantification of the number of quaternary branches ( indicated in schematic ) in different aman - 2 alleles and in wild type control animals . All loss of function alleles of aman - 2 display a significant decrease in PVD quaternary branch number . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and is indicated ( * * * * p ≤ 0 . 0001 ) . n = 15 for all genotypes and are biological replicates . Quantification of the number of full tertiaries ( marked by secondary and tertiary branches as shown in schematic ) in the genotypes indicated . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and is indicated ( * * * * p ≤ 0 . 0001 ; ns = not significant ) . n = 15 for all genotypes and are biological replicates . Fluorescent images and tracings of PVD in mnr - 1 ( dz213 ) hypomorphic animals alone and in combination with an aman - 2 ( gk248486 ) null mutant . The dz213 introduces a L135F missense mutation in the DUF2181 domain of MNR - 1 / Menorin . PVD is visualized by the dzIs53 transgene . The cell body is marked with an asterisk . Scale bars represent 20μm . Quantification of the number of full tertiaries in genotypes indicated and traced in ( F ) . The loss of aman - 2 severely enhances the mnr - 1 ( dz213 ) phenotype . Control indicates wild type control animals as shown in ( A ) . Data are represented as mean ± SEM . Statistical significance was calculated using the Mann - Whitney test and is indicated ( * * * * p ≤ 0 . 0001 ; ns = not significant ) . n > 23 for all genotypes and are biological replicates . Fluorescent images and tracings of PVD in kpc - 1 ( dz254 ) hypomorph animals alone and in combination with an aman - 2 ( gk248486 ) null mutant . PVD is visualized by the dzIs53 transgene . White arrows indicate self - avoidance defects in tertiary branches . Red arrows show gaps between tertiary branches ( no self - avoidance defects ) . The cell body is marked with an asterisk . Scale bars represent 20μm . Quantification of the percent of self - avoidance defects in genotypes indicated and traced in ( G ) . Control indicates wild type control animals as shown in ( A ) . The loss of aman - 2 suppresses defects in the kpc - 1 ( dz254 ) phenotype . Data are represented as mean . Statistical significance was calculated using the Z - test and is indicated ( * * * * p ≤ 0 . 0001 ) . n > 15 for all genotypes and are biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Fluorescent images (left panels) and tracings (right panels) of PVD neurons of the indicated genotypes. PVD is visualized by the wdIs52 transgene. Primary (1º), secondary (2º), tertiary (3º), and quaternary (4º) dendrites are indicated, and the cell body is marked with an asterisk. Anterior is to the left and dorsal is up in all panels. Scale bars represent 20μm.Quantification of the number of quaternary branches (indicated in schematic) in different aman-2 alleles and in wild type control animals. All loss of function alleles of aman-2 display a significant decrease in PVD quaternary branch number. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (****p ≤ 0.0001). n = 15 for all genotypes and are biological replicates.Quantification of the number of full tertiaries (marked by secondary and tertiary branches as shown in schematic) in the genotypes indicated. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (****p ≤ 0.0001; ns = not significant). n = 15 for all genotypes and are biological replicates.Fluorescent images and tracings of PVD in mnr-1(dz213) hypomorphic animals alone and in combination with an aman-2(gk248486) null mutant. The dz213 introduces a L135F missense mutation in the DUF2181 domain of MNR-1/Menorin. PVD is visualized by the dzIs53 transgene. The cell body is marked with an asterisk. Scale bars represent 20μm. Quantification of the number of full tertiaries in genotypes indicated and traced in (F). The loss of aman-2 severely enhances the mnr-1(dz213) phenotype. Control indicates wild type control animals as shown in (A). Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n >23 for all genotypes and are biological replicates.Fluorescent images and tracings of PVD in kpc-1(dz254) hypomorph animals alone and in combination with an aman-2(gk248486) null mutant. PVD is visualized by the dzIs53 transgene. White arrows indicate self-avoidance defects in tertiary branches. Red arrows show gaps between tertiary branches (no self-avoidance defects). The cell body is marked with an asterisk. Scale bars represent 20μm. Quantification of the percent of self-avoidance defects in genotypes indicated and traced in (G). Control indicates wild type control animals as shown in (A). The loss of aman-2 suppresses defects in the kpc-1(dz254) phenotype. Data are represented as mean. Statistical significance was calculated using the Z-test and is indicated (****p ≤ 0.0001). n >15 for all genotypes and are biological replicates.


Figure 2Fluorescent images and tracings of wild type control ( top ) and lect - 2 ( gk846764 ) hypomorphic animals ( bottom ) fed on plates with 300µm swainsonine vs a DMSO control . PVD is visualized by the wyIs581 transgene . The cell body is denoted with an asterisk . Anterior is to the left and dorsal is up in all panels . Vesicular gut autofluorescence is visible as white circular staining . Scale bars represent 20μm . Quantification of the number of full tertiaries in denoted genetic backgrounds ( aman - 2 null is gk248486 ) . Black data points indicate DMSO and orange data points show swainsonine treated animals . Data are represented as mean ± SEM . Statistical significance was calculated using the Mann - Whitney test and is indicated ( * * * * p ≤ 0 . 0001 ; ns = not significant ) . n = 12 for all genotypes and are biological replicates . Quantification of the number of quaternary dendrites in wild type control animals fed on plates with and without 300µm swainsonine . Animals treated with swainsonine show a significant decrease in quaternary branch number , Data are represented as mean ± SEM . Statistical significance was calculated using the Mann - Whitney test and is indicated ( * * * * p ≤ 0 . 0001 ) . n = 12 for each experiment and are biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Fluorescent images and tracings of wild type control (top) and lect-2(gk846764) hypomorphic animals (bottom) fed on plates with 300µm swainsonine vs a DMSO control. PVD is visualized by the wyIs581 transgene. The cell body is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Vesicular gut autofluorescence is visible as white circular staining. Scale bars represent 20μm. Quantification of the number of full tertiaries in denoted genetic backgrounds (aman-2 null is gk248486). Black data points indicate DMSO and orange data points show swainsonine treated animals. Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001; ns= not significant). n = 12 for all genotypes and are biological replicates. Quantification of the number of quaternary dendrites in wild type control animals fed on plates with and without 300µm swainsonine. Animals treated with swainsonine show a significant decrease in quaternary branch number, Data are represented as mean ± SEM. Statistical significance was calculated using the Mann-Whitney test and is indicated (****p ≤ 0.0001). n = 12 for each experiment and are biological replicates.


Figure 3 ( B ) Fluorescent composite images ( top ) and tracings ( bottom ) of mnr - 1 ( dz213 ) in an aman - 2 ( gk248486 ) and an aman - 2 ( gk248486 ) ; MGAT - 1 ( null ) background . An MGAT null mutant lacks the three C . elegans paralogs : gly - 12 , gly - 13 , and gly - 14 . PVD is visualized by the wyIs581 transgene . The cell body is denoted with an asterisk . Anterior is to the left and dorsal is up in all panels . Scale bars represent 20μm . ( C ) Quantification of full tertiaries of denoted genotypes . Data are represented as mean ± SEM . Statistical comparisons were performed using the Kruskal - Wallis test . Statistical significance is indicated ( * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , ns = not significant ) . n = 15 for all genotypes and are biological replicates . ( D ) Quantification of the number of quaternary branches in wild type control , aman - 2 ( gk248486 ) null , and MGAT1 ( null ) animals . Loss of MGAT1 suppresses the decrease in quaternary branch number in the aman - 2 ( null ) background . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and is indicated ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . n = 15 for all genotypes and are biological replicates . Western blot against GFP in C . elegans lysate expressing no transgenes ( N2 ) and expressing DMA - 1 : : GFP ( qyIs369 ) , after precipitating with anti - GFP antibody . The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase . The downwards size shift reveals that N - glycan structures are present on DMA - 1 . Ladder is marked in kilodaltons ( kDa ) . The GFP tag contains no N - glycosylation sites . The experiment was repeated four times with biological replicates . Western blot against GFP in C . elegans lysate DMA - 1 : : GFP ( qyIs369 ) , after precipitating with anti - GFP antibody . Control indicates an otherwise wild type background as opposed to an aman - 2 ( gk248486 ) null background . The upward size shift in the mutant reveals that the loss of aman - 2 alters the identity of N - glycan structures on DMA - 1 . The experiment was repeated four times with biological replicates . Western blot against GFP in C . elegans lysate DMA - 1 : : GFP ( qyIs369 ) , after precipitating with anti - GFP antibody . Control indicates an otherwise wild type background as opposed to an aman - 2 ( gk248486 ) null background . The red boxed + F indicates that the lysate is treated with the PNGase F glycosidase , while the green boxed + H corresponds to the Endo H glycosidase , which cleaves high - mannose and hybrid type N - glycans . For complementary experiments using the Endo D glycosidase , which cleaves paucimannose type N - glycans , Size shifts indicate that some hybrid / high - mannose structures are present on DMA - 1 ( left ) , and that the aman - 2 mutant results in only hybrid / high - mannose structures on DMA - 1 ( right ) . The experiment was repeated four times with biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Fluorescent composite images (top) and tracings (bottom) of mnr-1(dz213) in an aman-2(gk248486) and an aman-2(gk248486); MGAT-1(null) background. An MGAT null mutant lacks the three C. elegans paralogs: gly-12, gly-13, and gly-14. PVD is visualized by the wyIs581 transgene. The cell body is denoted with an asterisk. Anterior is to the left and dorsal is up in all panels. Scale bars represent 20μm. (C) Quantification of full tertiaries of denoted genotypes. Data are represented as mean ± SEM. Statistical comparisons were performed using the Kruskal-Wallis test. Statistical significance is indicated (***p ≤ 0.001, ****p ≤ 0.0001, ns=not significant). n=15 for all genotypes and are biological replicates. (D) Quantification of the number of quaternary branches in wild type control, aman-2(gk248486) null ,and MGAT1(null) animals. Loss of MGAT1 suppresses the decrease in quaternary branch number in the aman-2(null) background. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and is indicated (**p ≤ 0.01, ****p ≤ 0.0001). n = 15 for all genotypes and are biological replicates.Western blot against GFP in C. elegans lysate expressing no transgenes (N2) and expressing DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. The red boxed plus sign indicates that the lysate is treated with the PNGase F glycosidase. The downwards size shift reveals that N-glycan structures are present on DMA-1. Ladder is marked in kilodaltons (kDa). The GFP tag contains no N-glycosylation sites. The experiment was repeated four times with biological replicates.Western blot against GFP in C. elegans lysate DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The upward size shift in the mutant reveals that the loss of aman-2 alters the identity of N-glycan structures on DMA-1. The experiment was repeated four times with biological replicates.Western blot against GFP in C. elegans lysate DMA-1::GFP (qyIs369), after precipitating with anti-GFP antibody. Control indicates an otherwise wild type background as opposed to an aman-2(gk248486) null background. The red boxed +F indicates that the lysate is treated with the PNGase F glycosidase, while the green boxed +H corresponds to the Endo H glycosidase, which cleaves high-mannose and hybrid type N-glycans. For complementary experiments using the Endo D glycosidase, which cleaves paucimannose type N-glycans, Size shifts indicate that some hybrid/high-mannose structures are present on DMA-1 (left), and that the aman-2 mutant results in only hybrid/high-mannose structures on DMA-1 (right). The experiment was repeated four times with biological replicates.


Figure 4Quantification of the number of quaternary branches in DMA - 1 : : 2xFLAG control animals and animals with combinations of DMA - 1 : : 2xFLAG N - glycan attachment sites mutated . Note that DMA - 1 : : 2xFLAG control animals display a slightly reduced number of quaternary dendrites compared to wild type animals Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and Mann - Whitney between individual comparisons , and is indicated ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , ns = not significant ) . n = 15 for all genotypes and are biological replicates . Quantification of the number of quaternary branches in DMA - 1 : : 2xFLAG control animals alone and in combination with an aman - 2 ( gk248486 ) null mutant ( shaded in red ) . Control data is identical as in ( B ) and shown for comparison only . Data are represented as mean ± SEM . Statistical significance was calculated using the Kruskal - Wallis test and Mann - Whitney between individual comparisons , and is indicated ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 , ns = not significant ) . n = 15 for all genotypes and are biological replicates . Fluorescent images ( left ) and tracings ( center ) of PVD in animals of denoted backgrounds ( aman - 2 ( null ) is gk248486 ) . S4 corresponds to the endogenous alteration of N - glycan attachment site N386 of DMA - 1 . PVD is visualized by the dzIs117 transgene . The cell body is marked with an asterisk . Scale bars represent 20μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals and animals with combinations of DMA-1::2xFLAG N-glycan attachment sites mutated. Note that DMA-1::2xFLAG control animals display a slightly reduced number of quaternary dendrites compared to wild type animals Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.Quantification of the number of quaternary branches in DMA-1::2xFLAG control animals alone and in combination with an aman-2(gk248486) null mutant (shaded in red). Control data is identical as in (B) and shown for comparison only. Data are represented as mean ± SEM. Statistical significance was calculated using the Kruskal-Wallis test and Mann-Whitney between individual comparisons, and is indicated (**p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns=not significant). n = 15 for all genotypes and are biological replicates.Fluorescent images (left) and tracings (center) of PVD in animals of denoted backgrounds (aman-2(null) is gk248486). S4 corresponds to the endogenous alteration of N-glycan attachment site N386 of DMA-1. PVD is visualized by the dzIs117 transgene. The cell body is marked with an asterisk. Scale bars represent 20μm.


Figure 1 ( A ) Exomer components localize to different extent to the TGN and to the cytoplasm . Fluorescence images of cells expressing GFP - tagged exomer components and TGN marker Sec7 - DsRed . The brightness and contrast were adjusted differently in the GFP and merge images . Bar , 5 µm . ( B ) Quantification of the relative TGN to cytoplasm concentration of the GFP - tagged ChAPs in the cell . The mean fluorescence at the TGN and cytoplasm was determined using the Icy imaging software . 20 - 30 individual cells were analysed . For statistical significance testing the Student ' s T test was used . ( C ) FCS measures the in vivo mobility of exomer components in the cytoplasm . FCS analysis was performed on the GFP , Chs5 - GFP and GFP - tagged ChAPs in the wild - type background ( top ) and on GFP - tagged ChAPs in the Δchs5 background ( bottom ) . 10 - 20 FCS measurements were performed for each condition using different cells . The ACF of free GFP is fitted with a one - component anomalous diffusion model . The ACF of Chs5 in WT and ChAPs in WT and Δchs5 background is fitted with a two - component diffusion model . Representative ACF curves ( thin full lines ) and fits ( thick dashed lines ) are shown . Estimated diffusion correlation times , diffusion coefficients and fractions are shown in Table 1 . ( D ) Quantification of the Chs5 independent fraction of ChAPs present in the cytoplasm based on the measurements shown in ( C ) . ( E ) Comparison of the residuals of the one - component and two - component fit ( arrows ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Exomer components localize to different extent to the TGN and to the cytoplasm. Fluorescence images of cells expressing GFP-tagged exomer components and TGN marker Sec7-DsRed. The brightness and contrast were adjusted differently in the GFP and merge images. Bar, 5 µm.(B) Quantification of the relative TGN to cytoplasm concentration of the GFP-tagged ChAPs in the cell. The mean fluorescence at the TGN and cytoplasm was determined using the Icy imaging software. 20-30 individual cells were analysed. For statistical significance testing the Student's T test was used.(C) FCS measures the in vivo mobility of exomer components in the cytoplasm. FCS analysis was performed on the GFP, Chs5-GFP and GFP-tagged ChAPs in the wild-type background (top) and on GFP-tagged ChAPs in the Δchs5 background (bottom). 10-20 FCS measurements were performed for each condition using different cells. The ACF of free GFP is fitted with a one-component anomalous diffusion model. The ACF of Chs5 in WT and ChAPs in WT and Δchs5 background is fitted with a two-component diffusion model. Representative ACF curves (thin full lines) and fits (thick dashed lines) are shown. Estimated diffusion correlation times, diffusion coefficients and fractions are shown in Table 1.(D) Quantification of the Chs5 independent fraction of ChAPs present in the cytoplasm based on the measurements shown in (C).(E) Comparison of the residuals of the one-component and two-component fit (arrows).