I Effects of IL - 1β and active form of Caspase - 1 reconstitution on Lamtor1 KO THP - 1 cells . ELISA assay shows IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , and Lamtor1 KO cells reconstituted with IL - 1β , active form of Caspase - 1 , all of which were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) and LPS ( 200 ng / mL , 2 h ) .

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I Effects of IL-1β and active form of Caspase-1 reconstitution on Lamtor1 KO THP-1 cells. ELISA assay shows IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with IL-1β, active form of Caspase-1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/mL, 2 h).


A Representative images of mutant Lamtor1 . PMA - primed Lamtor1 KO THP - 1 macrophages reconstituted with full - length or truncated Lamtor1 .

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A Representative images of mutant Lamtor1. PMA-primed Lamtor1 KO THP-1 macrophages reconstituted with full-length or truncated Lamtor1.


B Effects of the Lamtor1 point mutation on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assays show IL - 1β secretion in THP - 1 cell supernatants from WT cells , Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full - length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine ( G2A ) ; all cell types were then treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

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B Effects of the Lamtor1 point mutation on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion in THP-1 cell supernatants from WT cells, Lamtor1 KO cells and Lamtor1 KO cells reconstituted with full-length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine (G2A); all cell types were then treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


C Effects of Lamtor1 deficiency on mTORC1 activity . Immunoblot analysis of Lamtor1flox / flox and Lamtor1flox / flox LysM - Cre BMDMs treated with LPS ( 200 ng / mL , 4 h ) .

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C Effects of Lamtor1 deficiency on mTORC1 activity. Immunoblot analysis of Lamtor1flox/flox and Lamtor1flox/flox LysM-Cre BMDMs treated with LPS (200 ng/mL, 4 h).


D Effect of the rapamycin treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in supernatants from PMA ( 50 nM , overnight ) primed WT THP - 1 cells treated with rapamycin at indicated concentration for 2 h , then treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) .

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D Effect of the rapamycin treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from PMA (50 nM, overnight) primed WT THP-1 cells treated with rapamycin at indicated concentration for 2 h, then treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h).


E Effect of rapamycin treatment on Caspase - 1 processing . Western blot shows caspase - 1 / p20 . LPS ( 200 ng / mL , 4 h ) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin ( 15 μM , 1 h ) .

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E Effect of rapamycin treatment on Caspase-1 processing. Western blot shows caspase-1/p20. LPS (200 ng/mL, 4 h) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin (15 μM, 1 h).


F Effect of Torin - 1 treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assay shows IL - 1β secretion in supernatants from WT BMDMs pretreated with Torin - 1 ( 0 , 1 , 10 , 100 , or 250 nM ) for 2 h before LPS ( 200 ng / ml , 4 h ) and nigericin ( 15 μM , 1 h ) treatment ( left panel ) . Western blot shows phospho - p70 - S6K from BMDMs pretreated with Torin - 1 at the indicated concentrations for 2 h before LPS and nigericin treatment ( right panel ) .

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F Effect of Torin-1 treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL-1β secretion in supernatants from WT BMDMs pretreated with Torin-1 (0, 1, 10, 100, or 250 nM) for 2 h before LPS (200 ng/ml, 4 h) and nigericin (15 μM, 1 h) treatment (left panel). Western blot shows phospho-p70-S6K from BMDMs pretreated with Torin-1 at the indicated concentrations for 2 h before LPS and nigericin treatment (right panel).


A Effects of the HDAC6 inhibitor treatment on IL - 1β secretion after NLRP3 inflammasome activation . ELISA assays show IL - 1β secretion and caspase - 1 processing ( p10 ) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin ( left to right : 10 and 20 mM ) or rocilinostat ( left to right : 30 and 60 mM ) .

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A Effects of the HDAC6 inhibitor treatment on IL-1β secretion after NLRP3 inflammasome activation. ELISA assays show IL-1β secretion and caspase-1 processing (p10) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin (left to right: 10 and 20 mM) or rocilinostat (left to right: 30 and 60 mM).


Co - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated

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Co-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids


Co - immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6 . Lamtor1 - Flag co - immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1 - Flag knocked - in THP - 1 cells . ( D )

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Co-immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Lamtor1-Flag co-immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1-Flag knocked-in THP-1 cells. (D)


Endogenous Lamtor1 co - immunoprecipitated with endogenous HDAC6 from lysates of WT THP - 1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6 . Co - immunoprecipitation assay shows interactions between Lamtor1 and HDAC6 .

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Endogenous Lamtor1 co-immunoprecipitated with endogenous HDAC6 from lysates of WT THP-1 cells Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co-immunoprecipitation assay shows interactions between Lamtor1 and HDAC6.


G PLA to confirm endogenous binding between Lamtor1 and HDAC6 . Lamtor1 - HDAC6 complexes are shown in red , and nuclei are depicted in blue . In situ PLA signals were quantified over nine images in each experiment .

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G PLA to confirm endogenous binding between Lamtor1 and HDAC6. Lamtor1-HDAC6 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over nine images in each experiment.


H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells . HEK293T cells were transiently transfected with NanoLuc - fused Lamtor1 and Halo - tag - fused HDAC6 using Lipofectamine 2000 . Luminescence was measured 48 h after transfection following the addition of Nano - Glo Luciferase Assay Substrate ( Promega ) .

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H NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells. HEK293T cells were transiently transfected with NanoLuc-fused Lamtor1 and Halo-tag-fused HDAC6 using Lipofectamine 2000. Luminescence was measured 48 h after transfection following the addition of Nano-Glo Luciferase Assay Substrate (Promega).


I Immunoblot analysis of the truncated form of Flag - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - 144 ; ∆ 95 - 161 , Met1 - Val94 ) co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids .

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I Immunoblot analysis of the truncated form of Flag-tagged Lamtor1 mutant (∆145-161, Met1-144; ∆95-161, Met1-Val94) co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids.


Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP - 1 cells . ELISA assay showing IL - 1β secretion in supernatants from WT cells , Lamtor1 KO cells , Lamtor1 KO cells reconstituted with full - length Lamtor1 , and Met1 - Val94 Lamtor1 cells ; all cell types were treated with nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

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Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP-1 cells. ELISA assay showing IL-1β secretion in supernatants from WT cells, Lamtor1 KO cells, Lamtor1 KO cells reconstituted with full-length Lamtor1, and Met1-Val94 Lamtor1 cells; all cell types were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


A Yeast two hybrid ( Y2H ) assays for Lamtor1 interactions with NLRP3 . Positive Y2H interaction between Lamtor1 as prey , and NLRP3 as bait . Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control .

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A Yeast two hybrid (Y2H) assays for Lamtor1 interactions with NLRP3. Positive Y2H interaction between Lamtor1 as prey, and NLRP3 as bait. Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control.


B PLA to confirm endogenous binding between Lamtor1 and NLRP3 . Lamtor1 - NLRP3 complexes are shown in red , and nuclei are depicted in blue . In situ PLA signals were quantified over 10 images in each experiment .

B PLA to confirm endogenous binding between Lamtor1 and NLRP3. Lamtor1-NLRP3 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over 10 images in each experiment.


C , D Immunoblot analysis of Lamtor1 - V5 co - immunoprecipitated with NLRP3 - Flag from lysates of HEK293T cells transfected with the indicated plasmids .

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C, D Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.


Immunoblot analysis of the truncated form of V5 - tagged Lamtor1 mutant ( ∆ 145 - 161 , Met1 - Ser144 ) co - immunoprecipitated with NLRP3 - flag from lysates of HEK293T cells transfected with the indicated plasmids .

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Immunoblot analysis of the truncated form of V5-tagged Lamtor1 mutant (∆145-161, Met1-Ser144) co-immunoprecipitated with NLRP3-flag from lysates of HEK293T cells transfected with the indicated plasmids.


G Effect of the HDAC6 knockdown on NLRP3 - Lamtor1 interactions . Immunoblot analysis of Lamtor1 - V5 co - immunoprecipitated with NLRP3 - Flag from lysates of HEK293T cells transfected with the indicated plasmids .

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G Effect of the HDAC6 knockdown on NLRP3-Lamtor1 interactions. Immunoblot analysis of Lamtor1-V5 co-immunoprecipitated with NLRP3-Flag from lysates of HEK293T cells transfected with the indicated plasmids.


A , B Representative images of Lamtor1 and ASC . PMA - treated THP - 1 cells stably expressing GFP - tagged ASC and td - Tomato - tagged Lamtor1 were primed with LPS ( 200 ng / mL , 2 h ) and then stimulated with nigericin for the indicated duration . Time shows after nigericin stimulation ( min : s ) .

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A, B Representative images of Lamtor1 and ASC. PMA-treated THP-1 cells stably expressing GFP-tagged ASC and td-Tomato-tagged Lamtor1 were primed with LPS (200 ng/mL, 2 h) and then stimulated with nigericin for the indicated duration. Time shows after nigericin stimulation (min:s).


C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation . Representative images of PMA - primed THP - 1 macrophages stably expressing GFP - tagged ASC treated with or without LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) . Scale bars = 10 μm .

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C Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation. Representative images of PMA-primed THP-1 macrophages stably expressing GFP-tagged ASC treated with or without LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h). Scale bars = 10 μm.


Effects of DL - all - rac - α - tocopherol on the interactions between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL - all - rac - α - tocopherol ( B )

Effects of DL-all-rac-α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL-all-rac-α-tocopherol (B)


IL - 1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL - all - rac - α - tocopherol ( C ) at the indicated concentrations . ELISA assay showing IL - 1β secretion in supernatants from WT THP - 1 cells treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

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IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL-all-rac-α-tocopherol (C) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


D ) IL - 6 secretion after overnight pretreatment with DL - all - rac - α - tocopherol . ELISA assay showing IL - 6 secretion in supernatants from WT BMDMs treated with LPS ( 200 ng / mL , overnight ) and DL - all - rac - α - tocopherol at the indicated concentrations ( overnight ) .

D) IL-6 secretion after overnight pretreatment with DL-all-rac-α-tocopherol. ELISA assay showing IL-6 secretion in supernatants from WT BMDMs treated with LPS (200 ng/mL, overnight) and DL-all-rac-α-tocopherol at the indicated concentrations (overnight).


Effects of α - tocopherol on the interactions between Lamtor1 and HDAC6 . Immunoblot analysis of Lamtor1 - Flag co - immunoprecipitated with HDAC6 - myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the D - α - tocopherol ( E )

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Effects of α-tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1-Flag co-immunoprecipitated with HDAC6-myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the D -α-tocopherol (E)


IL - 1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with D - α - tocopherol ( F ) at the indicated concentrations . ELISA assay showing IL - 1β secretion in supernatants from WT THP - 1 cells treated with LPS ( 200 ng / mL , 2 h ) and nigericin ( 15 μM , 1 h ) after priming with PMA ( 50 nM , overnight ) .

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IL-1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with D -α-tocopherol (F) at the indicated concentrations. ELISA assay showing IL-1β secretion in supernatants from WT THP-1 cells treated with LPS (200 ng/mL, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight).


G Effects of DL - all - rac - α - tocopherol in an acute gouty arthritis model . MSU crystals ( 0 . 5 mg ) suspended in 20 μL endotoxin - free PBS or PBS control were injected intra - articularly into the tibia - tarsal joint ( ankle ) of C57BL / 6 WT mice with or without DL - all - rac - α - tocopherol . Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin - stained sections of ankle joints .

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G Effects of DL-all-rac-α-tocopherol in an acute gouty arthritis model. MSU crystals (0.5 mg) suspended in 20 μL endotoxin-free PBS or PBS control were injected intra-articularly into the tibia-tarsal joint (ankle) of C57BL/6 WT mice with or without DL-all-rac-α-tocopherol. Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin-stained sections of ankle joints.


H Effects of DL - all - rac - α - tocopherol in an MSU - induced peritonitis model . Twelve hours after DL - all - rac - α - tocopherol pretreatment , 100 μL MSU solution ( 10 mg / mL ) was intraperitoneally injected for 4 h . Peritoneal IL - 1β production was then measured by ELISA , and CD11b + Ly6G + neutrophil recruitment was measured by FACS .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-DISEASE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-EXP_ASSAY O

H Effects of DL-all-rac-α-tocopherol in an MSU-induced peritonitis model. Twelve hours after DL-all-rac-α-tocopherol pretreatment, 100 μL MSU solution (10 mg/mL) was intraperitoneally injected for 4 h. Peritoneal IL-1β production was then measured by ELISA, and CD11b+ Ly6G+ neutrophil recruitment was measured by FACS.


B . Western blot analysis of purified autophagic vesicles . Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms , respectively , and are represented with total lysate ( TL ) . Depicted are representative blots of 14 independent approaches .

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

B. Western blot analysis of purified autophagic vesicles. Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms, respectively, and are represented with total lysate (TL). Depicted are representative blots of 14 independent approaches.


A . Differential interference contrast microscopy images of purified autophagic vesicles at high ( I ) or low ( II ) dilution . Images are representative of 3 independent approaches . B . Negative stain electron microscopy images of isolated vesicles . C . Size evaluation of isolated vesicles . The diameters of approx . 60 individual vesicles were determined using EM images .

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

A. Differential interference contrast microscopy images of purified autophagic vesicles at high (I) or low (II) dilution. Images are representative of 3 independent approaches. B. Negative stain electron microscopy images of isolated vesicles. C. Size evaluation of isolated vesicles. The diameters of approx. 60 individual vesicles were determined using EM images.


D . Western blot analysis of isolated autophagic vesicles upon proteinase K digestion . Mechanically opened vesicles served as positive control . For negative control , isolates were incubated with BSA instead of proteinase K . Depicted are two different blots that are representative for 5 independent

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

D. Western blot analysis of isolated autophagic vesicles upon proteinase K digestion. Mechanically opened vesicles served as positive control. For negative control, isolates were incubated with BSA instead of proteinase K. Depicted are two different blots that are representative for 5 independent experiments


E . Phospholipids identified in autophagic vesicles isolated upon different conditions . Relative amounts were calculated based on total levels of detected phospholipids . Abbreviations are depicted in A .

O O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR 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

E. Phospholipids identified in autophagic vesicles isolated upon different conditions. Relative amounts were calculated based on total levels of detected phospholipids. Abbreviations are depicted in A.


A . Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy . Log2 - transformed fold changes . For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles , no fold changes could be calculated and they are indicated as not determinable ( nd ) . B . Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy . Log2 - transformed fold changes . For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles , no fold changes could be calculated and they are indicated as not determinable ( nd ) .

O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR 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 B-SUBCELLULAR I-SUBCELLULAR 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 B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE 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

A. Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd). B. Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd).


( A ) Classical , ( B ) Rolled , and ( C ) Rotated subpopulations of the 80S * HCVIRES complex . Left column shows an overview of the final cryo - EM reconstructions with the HCVIRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCVIRES ( pink ) , tRNA ( green ) .

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

(A) Classical, (B) Rolled, and (C) Rotated subpopulations of the 80S*HCVIRES complex. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCVIRES (pink), tRNA (green).


( D ) Subpopulations of the 80S * HCVIRES complex containing P - site tRNA . The 80S is in the classical configuration and the head tilt is not present . Left column shows an overview of the final cryo - EM reconstructions with the HCVIRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCVIRES ( pink ) , tRNA ( green ) .

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

(D) Subpopulations of the 80S*HCVIRES complex containing P-site tRNA. The 80S is in the classical configuration and the head tilt is not present. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink),40S subunit (yellow),60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts, respectively.28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray),HCVIRES (pink), tRNA (green).


( E ) The cryo - EM map was filtered according to resolution . 40S * HCV IRES . Left column shows an overview of the final cryo - EM reconstructions with the HCV IRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCV IRES ( pink ) , tRNA ( green ) .

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

(E) The cryo-EM map was filtered according to resolution. 40S*HCV IRES. Left column shows an overview of the final cryo-EM reconstructions with the HCV IRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCV IRES (pink), tRNA (green).


( A ) Comparison of 40S subunit positions in Rolled , Rotated , Classical / no head tilt ( orange ) and Classical 80S * HCVIRES complex ( yellow ) in common 60S alignment . The cryo - EM map of the Classical 80S * HCVIRES was filtered to 6 Å . Arrows indicate the direction of movement during transition between two different states . The distance changes in the 40S subunit positions resulting from the rigid body transformation are color - coded in Å units . ( B ) Comparison of the HCVIRES in Classical 80S * HCVIRES complex ( gray ) and upon P - tRNA binding ( Classical , no head tilt ; purple ) in a common 40S body alignment . The distance of the movement of domain II was indicated with an arrow and Å

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

(A) Comparison of 40S subunit positions in Rolled, Rotated, Classical/no head tilt (orange) and Classical 80S*HCVIRES complex (yellow) in common 60S alignment. The cryo-EM map of the Classical 80S*HCVIRES was filtered to 6 Å. Arrows indicate the direction of movement during transition between two different states. The distance changes in the 40S subunit positions resulting from the rigid body transformation are color-coded in Å units.(B) Comparison of the HCVIRES in Classical 80S*HCVIRES complex (gray) and upon P-tRNA binding (Classical, no head tilt; purple) in a common 40S body alignment. The distance of the movement of domain II was indicated with an arrow and Å units


( A ) Adopted secondary structure of the IRES representing the tertiary fold . ( B ) Atomic model of the HCV IRES RNA . IRES domains are presented as coloured ribbons , overlaid with experimental IRES density ( gray , semi - transparent ) . ( C ) . Molecular model of the HCV IRES ( pink ) with ribosomal interaction sites ( coloured proteins and helices ) .

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

(A) Adopted secondary structure of the IRES representing the tertiary fold.(B) Atomic model of the HCV IRES RNA. IRES domains are presented as coloured ribbons, overlaid with experimental IRES density (gray, semi-transparent).(C). Molecular model of the HCV IRES (pink) with ribosomal interaction sites (coloured proteins and helices).


( A ) The secondary structure of the HCVIRES domain III ( hIII1 , hIII2 , IIIe and IIId ) and helix 26 ( h26 ) from the 18S rRNA . Colors of HCVIRES Domains are the same as in Fig 3 . Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line . ( B ) The interaction core between IIId , IIIe , hIII1 of HCVIRES ( colored as in ( A ) ) and Expansion segment 7 ( ES7 ) of h26 ( dark gray ) . ( C ) The rearrangement of h26 ( red ) upon binding of HCVIRES . The 80S * HCVIRES complex ( yellow ) is compared to the elongating POST state ( gray ; PDB 5Aj0 ) ( Behrmann et al , 2015 ) . The loop closing the helix is highlighted in red and dark gray .

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-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM 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 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 B-ORGANISM O O B-SUBCELLULAR 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

(A) The secondary structure of the HCVIRES domain III (hIII1, hIII2, IIIe and IIId) and helix 26 (h26) from the 18S rRNA. Colors of HCVIRES Domains are the same as in Fig 3. Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line.(B) The interaction core between IIId, IIIe, hIII1 of HCVIRES (colored as in (A)) and Expansion segment 7 (ES7) of h26 (dark gray).(C) The rearrangement of h26 (red) upon binding of HCVIRES. The 80S*HCVIRES complex (yellow) is compared to the elongating POST state (gray; PDB 5Aj0) (Behrmann et al, 2015). The loop closing the helix is highlighted in red and dark gray.


( E ) Close up on the interactions between GGG266 - 268 of IIId ( cyan ) and CCC1116 - 1118 of ES7 ( gray ) . ( F ) Close up on the interactions between G295 , A296 and U297 of IIIe ( green ) , A136 and A288 of hIII1 ( yellow ) and U1114 , U1115 of ES7 ( gray ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Close up on the interactions between GGG266-268 of IIId (cyan) and CCC1116-1118 of ES7 (gray).(F) Close up on the interactions between G295, A296 and U297 of IIIe (green), A136 and A288 of hIII1 (yellow) and U1114, U1115 of ES7 (gray).


( A ) Comparison of the 40S subunit from Classical 80S * HCVIRES complex ( yellow ) and canonical POST state ( gray ; EMD 2875 ) ( Behrmann et al , 2015 ) in common 40S body alignment . The arrow indicates the direction of the head tilt movement . Ribosomal proteins of the 40S head ( uS7 , eS25 and eS28 ) that interact with the HCVIRES domains II and IV ( pink ) on the 40S head are illuminated with blue , green , and purple , respectively . The positions of the proteins in the canonical POST state are illuminated in lighter color . The numbers indicate interaction site of HCVIRES and the 40S subunit . ( B ) Close up view on the interaction site of HCVIRES and the 40S head region . Numbers correspond to red labels in ( A ) . The rearrangement of the uS7 loop ( blue ) compared to the Classical 80S * HCVIRES complex ( gray ) is indicated . The arrow indicates the direction of the rearrangement of the uS7 loop . ( B - 1 and B - 4 ) inter subunit view from 40S subunit . ( B - 2 and - 3 ) inter subunit view from 60S subunit .

O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR 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 B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-SUBCELLULAR O O O O O O O O O O O O O B-ORGANISM 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 B-SUBCELLULAR 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 B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O

(A) Comparison of the 40S subunit from Classical 80S*HCVIRES complex (yellow) and canonical POST state (gray; EMD 2875) (Behrmann et al, 2015) in common 40S body alignment. The arrow indicates the direction of the head tilt movement. Ribosomal proteins of the 40S head (uS7, eS25 and eS28) that interact with the HCVIRES domains II and IV (pink) on the 40S head are illuminated with blue, green, and purple, respectively. The positions of the proteins in the canonical POST state are illuminated in lighter color. The numbers indicate interaction site of HCVIRES and the 40S subunit.(B) Close up view on the interaction site of HCVIRES and the 40S head region. Numbers correspond to red labels in (A). The rearrangement of the uS7 loop (blue) compared to the Classical 80S*HCVIRES complex (gray) is indicated. The arrow indicates the direction of the rearrangement of the uS7 loop. (B-1 and B-4) inter subunit view from 40S subunit. (B-2 and -3) inter subunit view from 60S subunit.


( A , B ) Comparative solvent side views of the small subunit with the bacterial SD - chamber on the 30S from the 70S POST state ( PDB 2HGR ) ( Yusupova et al , 2006 ) and the corresponding region of the eukaryotic 40S in the classical 80S * HCV IRES complex . Analogous proteins and rRNA helices are highlighted in the same colours , respectively . ( A ) Crystal structure of the 30S subunit with mRNA . The SD - helix is established between the mRNA ( orange ) and the 3 ' end of the 16S rRNA ( blue ) . ( B ) Atomic model of the 40S subunit with the HCV IRES model ( pink ) . The mRNA - part and S2 of the IRES are shown in orange and cyan , the 3 ' - end of the 18S rRNA is shown in blue .

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

(A, B) Comparative solvent side views of the small subunit with the bacterial SD-chamber on the 30S from the 70S POST state (PDB 2HGR) (Yusupova et al, 2006) and the corresponding region of the eukaryotic 40S in the classical 80S*HCV IRES complex. Analogous proteins and rRNA helices are highlighted in the same colours, respectively. (A) Crystal structure of the 30S subunit with mRNA. The SD-helix is established between the mRNA (orange) and the 3' end of the 16S rRNA (blue).(B) Atomic model of the 40S subunit with the HCV IRES model (pink). The mRNA-part and S2 of the IRES are shown in orange and cyan, the 3'-end of the 18S rRNA is shown in blue.


( C ) Overview and zoomed view of Classical 80S * HCVIRES . The cryo - EM map of the Classical 80S * HCVIRES was filtered according to resolution . ( D and E ) mRNA exit channel from in Classical 80S * HCVIRES and POST state ( PDB 5AJ0 ) ( Behrmann et al , 2015 ) . The two structures are aligned at the 40S body . We note eS28 as part of the 40S head is differently located because of the head tilt . The surface representation was created using the pdb models . Shown are the 18S rRNA ( yellow ) , ribosomal proteins ( gray ) , eS26 ( cyan ) eS28 ( purple ) , HCVIRES ( pink ) .

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

(C) Overview and zoomed view of Classical 80S*HCVIRES. The cryo-EM map of the Classical 80S*HCVIRES was filtered according to resolution.(D and E) mRNA exit channel from in Classical 80S*HCVIRES and POST state (PDB 5AJ0) (Behrmann et al, 2015). The two structures are aligned at the 40S body. We note eS28 as part of the 40S head is differently located because of the head tilt. The surface representation was created using the pdb models. Shown are the 18S rRNA (yellow), ribosomal proteins (gray), eS26 (cyan) eS28 (purple), HCVIRES (pink).


( A ) Comparison of HCV IRES from 80S * HCV IRES Classical ( pink ) and tRNAs from POST state ( gray ) ( PDB 5AJ0 ) ( Behrmann et al , 2015 ) in a common 40S body alignment of the cryo - EM maps . ( B ) Same as above but in a common 40S head alignment .

O O O O O B-ORGANISM O O B-SUBCELLULAR O B-ORGANISM 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 B-SUBCELLULAR O 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-SUBCELLULAR O O O

(A) Comparison of HCV IRES from 80S*HCV IRES Classical (pink) and tRNAs from POST state (gray)(PDB 5AJ0) (Behrmann et al, 2015) in a common 40S body alignment of the cryo-EM maps.(B) Same as above but in a common 40S head alignment.


( C ) Comparison of the 40S subunit and bound ligands of the classical 80S * HCVIRES complex ( orange ) and 80S * HCVIRES * Met - tRNA * eIF5B * GMPPNP complex ( POST - like state ) ( yellow ) ( EMD 2683 ) ( Yamamoto et al , 2014 ) in common 60S alignment . ( D ) The models of the 80S * HCVIRES Classical complex ( gray ) , 80S * HCVIRES * Met - tRNA * eIF5B * GMPPNP complex ( POST - like state ) ( green ) ( PDB 4UPZ ) ( Yamamoto et al , 2014 ) and 80S * HCVIRES Classical / no head tilt complex ( pink ) are rigid body fitted into the corresponding densities in a common 40S body alignment . The Helix 68 from the 28S rRNA is depicted as well ( blue ) .

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

(C) Comparison of the 40S subunit and bound ligands of the classical 80S*HCVIRES complex (orange) and 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (yellow)(EMD 2683) (Yamamoto et al, 2014) in common 60S alignment.(D) The models of the 80S*HCVIRES Classical complex (gray), 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (green)(PDB 4UPZ) (Yamamoto et al, 2014) and 80S*HCVIRES Classical/no head tilt complex (pink) are rigid body fitted into the corresponding densities in a common 40S body alignment. The Helix 68 from the 28S rRNA is depicted as well (blue).


( A and B ) Comparison of the 80S * HCV IRES Classical complex and Classical / no head tilt complex ( 40S yellow , 60S blue , IRES pink , P - tRNA green ) at intersubunit view . Inter subunit bridge 1b / c and 1a are indicated as B1b / c and B1a , respectively . The cryo - EM map of classical 80S * HCV IRES classical complex was filtered to 6 Å . ( C and D ) Close up of the mRNA tunnel . The distance between helix 29 and 44 of the 18S rRNA are indicated with an arrow and Å

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(A and B) Comparison of the 80S*HCV IRES Classical complex and Classical/no head tilt complex (40S yellow, 60S blue, IRES pink, P-tRNA green) at intersubunit view. Inter subunit bridge 1b/c and 1a are indicated as B1b/c and B1a, respectively. The cryo-EM map of classical 80S*HCV IRES classical complex was filtered to 6 Å.(C and D) Close up of the mRNA tunnel. The distance between helix 29 and 44 of the 18S rRNA are indicated with an arrow and Å units


( C ) Benign breast epithelial MCF10A and breast carcinoma BT - 474 cells were embedded in 3D collagen as single cells or as spheroids , respectively , and the growth was followed for 5 days . Light micrographs show filamentous actin ( phalloidin ) and nuclei ( Hoechst ) in representative cell colonies . Quantitative assessment of the nuclei counts per colony show the induced proliferation in MCF10A cells after ECHCD1 sgRNA knockout . At 72 hours , MCF10A mock versus ECHDC1 _ sgRNA _ 1 and ECHDC1 _ sgRNA _ 2 P < 0 . 05 ; at 96 hours mock versus ECHDC1 _ sgRNA _ 1 , ECHDC1 _ sgRNA _ 2 and ECHDC1 _ sgRNA _ 3 P < 0 . 001 ; at 120 hours mock versus ECHDC1 _ sgRNA _ 1 , ECHDC1 _ sgRNA _ 2 and ECHDC1 _ sgRNA _ 3 P < 0 . 0001 . Nuclei count relative to mock 0 hours . Error bars indicate mean ± SEM ; n ≥ 10 colonies . Statistical significance was assessed with one - way ANOVA with Tukey ' s multiple comparison test . Scale bar 50 µm .

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(C) Benign breast epithelial MCF10A and breast carcinoma BT-474 cells were embedded in 3D collagen as single cells or as spheroids, respectively, and the growth was followed for 5 days. Light micrographs show filamentous actin (phalloidin) and nuclei (Hoechst) in representative cell colonies. Quantitative assessment of the nuclei counts per colony show the induced proliferation in MCF10A cells after ECHCD1 sgRNA knockout. At 72 hours, MCF10A mock versus ECHDC1_sgRNA_1 and ECHDC1_sgRNA_2 P < 0.05; at 96 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.001; at 120 hours mock versus ECHDC1_sgRNA_1, ECHDC1_sgRNA_2 and ECHDC1_sgRNA_3 P < 0.0001. Nuclei count relative to mock 0 hours. Error bars indicate mean ± SEM; n ≥ 10 colonies. Statistical significance was assessed with one-way ANOVA with Tukey's multiple comparison test. Scale bar 50 µm.


( E ) Measured metabolite levels of intermediates in propanoate metabolism in select breast cancer cell lines with or without the ECHDC1 rCCS status ( n = 7 in both groups ) . Boxes represent the interquartile range , whiskers represent the range of the values and solid line within the box correspond to the median value . Outlier points indicates values not included between the whiskers . Statistical significance was assessed with Wilcoxon test .

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(E) Measured metabolite levels of intermediates in propanoate metabolism in select breast cancer cell lines with or without the ECHDC1 rCCS status (n=7 in both groups). Boxes represent the interquartile range, whiskers represent the range of the values and solid line within the box correspond to the median value. Outlier points indicates values not included between the whiskers. Statistical significance was assessed with Wilcoxon test.


( H ) Survival analysis based on mRNA expression levels of DDX27 in patients with endometrial cancer in the TCGA dataset . Expression levels were divided into 2 classes , high ( n = 203 ) and low ( n = 322 ) , based on mean expression level of DDX27 ( logFPKM = 18 . 27 ) . Patients in the high class showed lower survival probability than those in the low class ( P = . 4 . 2 × 10 - 4 ; log - rank test ) .

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(H) Survival analysis based on mRNA expression levels of DDX27 in patients with endometrial cancer in the TCGA dataset. Expression levels were divided into 2 classes, high (n=203) and low (n=322), based on mean expression level of DDX27 (logFPKM=18.27). Patients in the high class showed lower survival probability than those in the low class (P=.4.2×10-4; log-rank test).


( I ) mRNA expression levels of DDX27 in PTEN mutated ( Mut , n = 302 ) and PTEN wild type ( WT , n = 224 ) endometrial patient tumors in the TCGA dataset . Triangles correspond to the median value . P - value was calculated with Wilcoxon test .

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(I) mRNA expression levels of DDX27 in PTEN mutated (Mut, n=302) and PTEN wild type (WT, n=224) endometrial patient tumors in the TCGA dataset. Triangles correspond to the median value. P-value was calculated with Wilcoxon test.


A FBN2 mRNA levels in multiple human tissues . Human tissue cDNAs were used as templates for PCR of FBN2 , followed by agarose gel analyses . GAPDH levels served as loading controls ( biological replicates , n = 2 ) .

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A FBN2 mRNA levels in multiple human tissues. Human tissue cDNAs were used as templates for PCR of FBN2, followed by agarose gel analyses. GAPDH levels served as loading controls (biological replicates, n=2).


B FBN2 mRNA levels in mouse tissues based on quantitative RT - PCR . Data were normalized based on levels in adipose tissues .

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B FBN2 mRNA levels in mouse tissues based on quantitative RT-PCR. Data were normalized based on levels in adipose tissues.


C Comparison of FBN2 transcript levels between human and mouse placentas . Common primers for FBN2 and GAPDH in both species were used for RT - PCR analyses ( biological replicates , n = 3 ) .

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C Comparison of FBN2 transcript levels between human and mouse placentas. Common primers for FBN2 and GAPDH in both species were used for RT-PCR analyses (biological replicates, n=3) .


D Immuno - histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy . During early pregnancy , placensin immunostaining was found in cytotrophoblasts ( c ) , whereas syncytiotrophoblasts ( s ) showed weak staining . At 38 weeks of pregnancy , strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts ( arrows ) . Staining using E - cadherin antibodies confirmed the identity of cytotrophoblasts ( Inset showing higher magnification E - cadherin ( E - Cad ) staining ) . Lower panels depict sections stained with nonimmune IgG as negative controls . Scale bars , 100 μm .

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D Immuno-histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy. During early pregnancy, placensin immunostaining was found in cytotrophoblasts (c), whereas syncytiotrophoblasts (s) showed weak staining. At 38 weeks of pregnancy, strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts (arrows). Staining using E-cadherin antibodies confirmed the identity of cytotrophoblasts (Inset showing higher magnification E-cadherin (E-Cad) staining). Lower panels depict sections stained with nonimmune IgG as negative controls. Scale bars, 100 μm.


E Immunoblotting of placensin in human placental villi ( term pregnancy ) . Samples from three individual patients were processed with or without N - glycosidase pre - treatment to reveal untreated and deglycosylated placensin .

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E Immunoblotting of placensin in human placental villi (term pregnancy). Samples from three individual patients were processed with or without N-glycosidase pre-treatment to reveal untreated and deglycosylated placensin.


F For prokaryotic cell expression , a cDNA fragment corresponding to residues 2779 to 2912 of the human FBN2 coding region was appended with GST and 6 - histidine tags and sub - cloned into the PGEX - 6P - 1 vector for expression in E . coli . Left panel : Coomassie blue staining of proteins in the SDS - PAGE gel before and after digestion with the PreScission protease . 1 : Flow through ; 2 : Washes ; 3 : Before PreScission treatment ; 4 : After PreScission treatment . Right panel : Immunoblotting using placensin antibodies .

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F For prokaryotic cell expression, a cDNA fragment corresponding to residues 2779 to 2912 of the human FBN2 coding region was appended with GST and 6-histidine tags and sub-cloned into the PGEX-6P-1 vector for expression in E. coli. Left panel: Coomassie blue staining of proteins in the SDS-PAGE gel before and after digestion with the PreScission protease. 1: Flow through; 2: Washes; 3: Before PreScission treatment; 4: After PreScission treatment. Right panel: Immunoblotting using placensin antibodies.


G Immunoblotting of recombinant placensin generated in E . coli . and CHO cells . For CHO cell expression , placensin cDNA sequence was subcloned into an adenoviral vector pAV [ Exp ] - CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later . Recombinant placensin proteins secreted from CHO cells ( b ) showed higher molecular weight than those from bacteria ( a ) . Following treatment with N - glycosidase to remove N - linked carbohydrate side chains , prokaryotic - and eukaryotic cell - derived placensin proteins showed similar sizes .

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G Immunoblotting of recombinant placensin generated in E. coli. and CHO cells. For CHO cell expression, placensin cDNA sequence was subcloned into an adenoviral vector pAV[Exp]-CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later. Recombinant placensin proteins secreted from CHO cells (b) showed higher molecular weight than those from bacteria (a). Following treatment with N-glycosidase to remove N-linked carbohydrate side chains, prokaryotic- and eukaryotic cell-derived placensin proteins showed similar sizes.


A Prokaryotic cell - derived placensin stimulated cAMP production ( at 10 min . ) , and PKA activity ( at 30 min . ) by primary mouse hepatocytes and hepatocyte - derived AML12 cells ( biological replicates , n = 5 ) .

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A Prokaryotic cell-derived placensin stimulated cAMP production (at 10 min.), and PKA activity (at 30 min.) by primary mouse hepatocytes and hepatocyte-derived AML12 cells (biological replicates, n=5).


B Placensin stimulation of glucose secretion ( at 5h ) and key gluconeogenesis gene transcripts ( at 1h ) in both cell types ( biological replicates , n = 4 ) . Transcript levels for PEPCK and G6Pase were determined using quantitative RT - PCR . Treatment with glucagon ( 10 nM ) served as positive controls .

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B Placensin stimulation of glucose secretion (at 5h) and key gluconeogenesis gene transcripts (at 1h) in both cell types (biological replicates, n=4). Transcript levels for PEPCK and G6Pase were determined using quantitative RT-PCR. Treatment with glucagon (10 nM) served as positive controls.


C Liver from adult female mice was perfused in situ with placensin ( 1 . 0 ug / pulse ) or PBS before measurement of hepatic glucose output from inferior vena cava ( biological replicates , n = 3 ) .

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C Liver from adult female mice was perfused in situ with placensin (1.0 ug/pulse) or PBS before measurement of hepatic glucose output from inferior vena cava (biological replicates, n=3).


D Adult female mice were injected intraperitoneally with placensin ( 30 ug / injection ) with blood sampling at different time points for glucose levels ( biological replicates , n = 5 ) .

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D Adult female mice were injected intraperitoneally with placensin (30 ug/injection) with blood sampling at different time points for glucose levels (biological replicates, n=5).


A Left panel : Immunoblotting of placensin content in HTR - 8 / SVneo , but not BeWo , JAR , and JEG3 , cells . Right panel : Time - dependent secretion of placensin by HTR - 8 / SVneo , but not JAR , cells monitored using an ELISA ( biological replicates , n = 4 - 5 ) .

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A Left panel: Immunoblotting of placensin content in HTR-8/SVneo, but not BeWo, JAR, and JEG3, cells. Right panel: Time-dependent secretion of placensin by HTR-8/SVneo, but not JAR, cells monitored using an ELISA (biological replicates, n=4-5).


B Knockdown of FBN2 in HTR - 8 / SVneo cells led to decreases in FBN2 transcripts and placensin secretion . Cells were treated with FBN2 siRNA ( SiFBN2 ; 20 nM ) or control siRNA ( SiC ; 20 nM ) for 6h . Following media change , cells and conditioned media were collected at 48h after incubation . Placensin transcripts and proteins were determined by RT - PCR and ELISA ( biological replicates , n = 8 ) , respectively . Lower panel : immunoblotting of secreted placensin by HTR - 8 / Svneo .

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B Knockdown of FBN2 in HTR-8/SVneo cells led to decreases in FBN2 transcripts and placensin secretion. Cells were treated with FBN2 siRNA (SiFBN2; 20 nM) or control siRNA (SiC; 20 nM) for 6h. Following media change, cells and conditioned media were collected at 48h after incubation. Placensin transcripts and proteins were determined by RT-PCR and ELISA (biological replicates, n=8), respectively. Lower panel: immunoblotting of secreted placensin by HTR-8/Svneo.


C Placensin stimulation of cAMP production and PKA activities by HTR - 8 / SVneo cells . Cells were treated with placensin for 10 min . before cAMP measurement ( biological replicates , n = 5 ) . For PKA activities , cells were treated with placensin ( 50 ng / ml ) for 30 min . Treatment with forskolin ( 10 uM ) served as positive controls .

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C Placensin stimulation of cAMP production and PKA activities by HTR-8/SVneo cells. Cells were treated with placensin for 10 min. before cAMP measurement (biological replicates, n=5). For PKA activities, cells were treated with placensin (50 ng/ml) for 30 min. Treatment with forskolin (10 uM) served as positive controls.


D Placensin stimulation of transcript levels for MMP9 but not MMP2 ( biological replicates , n = 12 ) . Cells were treated with placensin for 24h before RT - PCR analyses of transcript levels .

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D Placensin stimulation of transcript levels for MMP9 but not MMP2(biological replicates, n=12). Cells were treated with placensin for 24h before RT-PCR analyses of transcript levels.


E Placensin stimulation of MMP9 , but not MMP2 , activities without affecting cell proliferation . After 24h of treatment with placensin , media were collected for zymographic and densitometric analyses as well as determination of total cell numbers ( biological replicates , n = 4 ) .

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E Placensin stimulation of MMP9, but not MMP2, activities without affecting cell proliferation. After 24h of treatment with placensin, media were collected for zymographic and densitometric analyses as well as determination of total cell numbers (biological replicates, n=4).


F Placensin stimulation of HTR - 8 / SVneo cell invasion . After placensin treatment for 24h , cell invasiveness was determined using the Transwell assay ( left panel : micrographs of cells ; right panel : fold changes in number of migrated cells , biological replicates , n = 4 ) . Cells were incubated with mitomycin C to rule out effects on cell proliferation . Scale bars : 100μm . C : control .

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F Placensin stimulation of HTR-8/SVneo cell invasion. After placensin treatment for 24h, cell invasiveness was determined using the Transwell assay (left panel: micrographs of cells; right panel: fold changes in number of migrated cells, biological replicates, n=4). Cells were incubated with mitomycin C to rule out effects on cell proliferation. Scale bars: 100μm. C: control.


A Immunoblotting of serum placensin from patients at second trimester ( 1 - 3 ) , term pregnancy ( 4 - 6 ) using specific placensin antibodies .

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A Immunoblotting of serum placensin from patients at second trimester (1-3), term pregnancy (4-6) using specific placensin antibodies.


B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients . Numbers in parentheses represent number of patients .

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B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients. Numbers in parentheses represent number of patients.


IFNα dose - dependency of STAT1 and STAT2 phosphorylation in Huh7 . 5 cells . Cells were seeded 24 hours prior to the start of the experiment . Three hours before stimulation , cells were growth factor - depleted and were subsequently stimulated with the indicated concentrations of IFNα . Cytoplasmic protein lysates were collected one hour after the stimulation and phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701 , or STAT2 phosphorylated on tyrosine residue 690 . Data points are displayed as dots with 1σ confidence interval estimated from biological replicates ( N = 1 to N = 38 ) using a combined scaling and error model . Data is approximated with a sigmoidal function and signals corresponding to a low dose ( 2 . 8 pM IFNα ) , a medium dose ( 28 pM IFNα ) and a high dose ( 1400 pM IFNα ) are displayed with dashed lines .

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IFNα dose-dependency of STAT1 and STAT2 phosphorylation in Huh7.5 cells. Cells were seeded 24 hours prior to the start of the experiment. Three hours before stimulation, cells were growth factor-depleted and were subsequently stimulated with the indicated concentrations of IFNα. Cytoplasmic protein lysates were collected one hour after the stimulation and phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. Data points are displayed as dots with 1σ confidence interval estimated from biological replicates (N=1 to N=38) using a combined scaling and error model. Data is approximated with a sigmoidal function and signals corresponding to a low dose (2.8 pM IFNα), a medium dose (28 pM IFNα) and a high dose (1400 pM IFNα) are displayed with dashed lines.


Representative immunoblot of IFNα - induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7 . 5 cells prestimulated for 24 hours with 2 . 8 pM IFNα , 1400 pM IFNα or without prestimulation . Time points after presimulation are indicated . 20 µg of cytoplasmic lysates were analyzed using antibodies for the indicated targets . Phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701 , or STAT2 phosphorylated on tyrosine residue 690 . An asterisk indicates pSTAT1β . Calnexin served as loading control . Molecular weights are indicated on the left . Immunoblot detection was performed with chemiluminescence employing a CCD camera device ( ImageQuant ) . Quantification of immunoblots of IFNα - induced phosphorylation of cytoplasmic and nuclear STAT1 and STAT2 in Huh7 . 5 cells prestimulated with 2 . 8 pM IFNα , 1400 pM IFNα or without pretreatment . Time points after presimulation are displayed . Data is displayed as fold change relative to untreated cells . Errors were estimated with a combined scaling and error model , comprising 1σ confidence interval estimated from biological replicates ( N = 3 ) . Dashed lines indicate smoothing splines .

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

Representative immunoblot of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 cells prestimulated for 24 hours with 2.8 pM IFNα, 1400 pM IFNα or without prestimulation. Time points after presimulation are indicated. 20 µg of cytoplasmic lysates were analyzed using antibodies for the indicated targets. Phosphorylation of STAT1 and STAT2 was detected by immunoblot utilizing antibodies recognizing STAT1 phosphorylated on tyrosine residue 701, or STAT2 phosphorylated on tyrosine residue 690. An asterisk indicates pSTAT1β. Calnexin served as loading control. Molecular weights are indicated on the left. Immunoblot detection was performed with chemiluminescence employing a CCD camera device (ImageQuant).   Quantification of immunoblots of IFNα-induced phosphorylation of cytoplasmic and nuclear STAT1 and STAT2 in Huh7.5 cells prestimulated with 2.8 pM IFNα, 1400 pM IFNα or without pretreatment. Time points after presimulation are displayed. Data is displayed as fold change relative to untreated cells. Errors were estimated with a combined scaling and error model, comprising 1σ confidence interval estimated from biological replicates (N=3). Dashed lines indicate smoothing splines.


Induction of interferon - stimulated genes upon prestimulation with 1400 pM IFNα ( yellow background ) and stimulation with 1400 pM IFNα ( white background ) in Huh7 . 5 cells , assessed by qRT - PCR . RNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT and TBP and were displayed as fold change . Peak of gene expression is indicated . Data points displayed as dots with 1σ confidence - interval estimated from biological replicates ( N = 4 to N = 14 ) using a combined scaling and error model . Dashed lines indicate smoothing splines . Induction of interferon - stimulated genes upon prestimulation with 2 . 8 pM IFNα ( yellow background ) and stimulation with 1400 pM IFNα ( white background ) in Huh7 . 5 cells , assessed by qRT - PCR . RNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT and TBP and were displayed as fold change . Peak of gene expression is indicated . Data points are displayed as dots with 1σ confidence interval estimated from biological replicates ( N = 4 to N = 6 ) using a combined scaling and error model . Dashed lines indicate smoothing splines .

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

Induction of interferon-stimulated genes upon prestimulation with 1400 pM IFNα (yellow background) and stimulation with 1400 pM IFNα (white background) in Huh7.5 cells, assessed by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP and were displayed as fold change. Peak of gene expression is indicated. Data points displayed as dots with 1σ confidence-interval estimated from biological replicates (N=4 to N=14) using a combined scaling and error model. Dashed lines indicate smoothing splines.   Induction of interferon-stimulated genes upon prestimulation with 2.8 pM IFNα (yellow background) and stimulation with 1400 pM IFNα (white background) in Huh7.5 cells, assessed by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP and were displayed as fold change. Peak of gene expression is indicated. Data points are displayed as dots with 1σ confidence interval estimated from biological replicates (N=4 to N=6) using a combined scaling and error model. Dashed lines indicate smoothing splines.


Growth factor - depleted Huh7 . 5 cells were prestimulated with 2 . 8 pM , 28 pM , 1400 pM IFNα or left untreated and were stimulated with 1400 pM IFNα 24 hours later . IFNα - induced signaling was measured by time - resolved quantitative immunoblotting and detected with chemiluminescence using a CCD - based camera . Data was normalized to reference proteins Calnexin or HDAC1 , scaled and subjected to model calibration . Model calibration with time - resolved IFNα - induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα . Cytoplasmic lysates were subjected to quantitative immunoblotting . Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 23 ) using a combined scaling and error model . Model trajectories are represented by lines . pSTAT1 , pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690 , respectively . tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1 , STAT2 , respectively .

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

Growth factor-depleted Huh7.5 cells were prestimulated with 2.8 pM, 28 pM, 1400 pM IFNα or left untreated and were stimulated with 1400 pM IFNα 24 hours later. IFNα-induced signaling was measured by time-resolved quantitative immunoblotting and detected with chemiluminescence using a CCD-based camera. Data was normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration.   Model calibration with time-resolved IFNα-induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively.


Model calibration with time - resolved IFNα - induced feedback transcripts upon prestimulation with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα , assessed by qRT - PCR . mRNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT and TBP . Experimental data is represented by filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 3 to N = 14 ) using a combined scaling and error model . Model trajectories are represented by lines .

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

Model calibration with time-resolved IFNα-induced feedback transcripts upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα, assessed by qRT-PCR. mRNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3 to N=14) using a combined scaling and error model. Model trajectories are represented by lines.


Model calibration with the amount of molecules per cell of STAT1 , STAT2 , IRF9 and USP18 determined 24 hours after prestimulation with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα . Calibrator proteins were spiked into 10 µg of total protein lysate and subjected to quantitative immunoblotting . Immunoblot detection was performed by chemiluminescence using a CCD - camera - based device . Averaged values ( N = 4 ) are displayed with standard deviations . Green squares indicate amounts estimated by the mathematical model .

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

Model calibration with the amount of molecules per cell of STAT1, STAT2, IRF9 and USP18 determined 24 hours after prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Calibrator proteins were spiked into 10 µg of total protein lysate and subjected to quantitative immunoblotting. Immunoblot detection was performed by chemiluminescence using a CCD-camera-based device. Averaged values (N=4) are displayed with standard deviations. Green squares indicate amounts estimated by the mathematical model.


Model calibration of IFNα - induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7 . 5 prestimulated with 0 , 2 . 8 pM , 28 pM or 1400 pM IFNα . Nuclear lysates were subjected to quantitative immunoblotting . Experimental data is represented by filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 4 to N = 22 ) using a combined scaling and error model . Model trajectories are represented by lines .

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

Model calibration of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 prestimulated with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Nuclear lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=4 to N=22) using a combined scaling and error model. Model trajectories are represented by lines.


Model predictions of IFNα - induced dynamics of occupied GAS bindings sites ( OccGASbs ) ( left panel ) , of the sum of the pSTAT1 : pSTAT2 heterodimers and the pSTAT1 : pSTAT2 : IRF9 trimers ( middle panel ) and of the pSTAT1 : pSTAT2 : IRF9 trimers ( right panel ) in untreated Huh7 . 5 cells and in Huh7 . 5 cells prestimulated for 24 hours with 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα . Lines with shading represent model predictions with 68 % confidence intervals using the prediction profile likelihood method . For experimental validation of the dynamics of OccGASbs , electrophoretic mobility shift assays ( EMSA ) were performed using nuclear protein lysates obtained from untreated Huh7 . 5 cells or Huh7 . 5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα . Lysates were incubated with radioactively - labeled oligonucleotides probes harboring the GAS - binding region of the human IRF1 promoter . Samples were resolved on a native polyacrylamide gel and radioactivity was visualized and quantified from three independent experiments ( left panel ) . For experimental validation of the dynamics of the sum of the pSTAT1 : pSTAT2 heterodimers and the pSTAT1 : pSTAT2 : IRF9 trimers , immunoprecipitations ( IP ) were performed using total cell lysates obtained from untreated Huh7 . 5 cells or Huh7 . 5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα . Lysates were subjected to immunoprecipitation with antibodies recognizing STAT2 and phosphorylated STAT1 was detected with quantitative immunoblotting ( IB ) ( middle panel ) . For experimental validation of the dynamics of the pSTAT1 : pSTAT2 : IRF9 trimers , immunoprecipitations were performed using total cell lysates obtained from untreated Huh7 . 5 cells or Huh7 . 5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα . Lysates were subjected to immunoprecipitation ( IP ) with antibodies recognizing IRF9 and phosphorylated STAT1 was detected with quantitative immunoblotting ( IB ) ( right panel ) . Antibodies and the corresponding proteins in the complexes are underlined . Experimental data is represented by filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 3 ) using a combined scaling and error model .

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

Model predictions of IFNα-induced dynamics of occupied GAS bindings sites (OccGASbs) (left panel), of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers (middle panel) and of the pSTAT1:pSTAT2:IRF9 trimers (right panel) in untreated Huh7.5 cells and in Huh7.5 cells prestimulated for 24 hours with 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα. Lines with shading represent model predictions with 68% confidence intervals using the prediction profile likelihood method. For experimental validation of the dynamics of OccGASbs, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were incubated with radioactively-labeled oligonucleotides probes harboring the GAS-binding region of the human IRF1 promoter. Samples were resolved on a native polyacrylamide gel and radioactivity was visualized and quantified from three independent experiments (left panel). For experimental validation of the dynamics of the sum of the pSTAT1:pSTAT2 heterodimers and the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations (IP) were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation with antibodies recognizing STAT2 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (middle panel). For experimental validation of the dynamics of the pSTAT1:pSTAT2:IRF9 trimers, immunoprecipitations were performed using total cell lysates obtained from untreated Huh7.5 cells or Huh7.5 cells that were prestimulated for 24 hours with 280 pM IFNα and then stimulated with 1400 pM IFNα. Lysates were subjected to immunoprecipitation (IP) with antibodies recognizing IRF9 and phosphorylated STAT1 was detected with quantitative immunoblotting (IB) (right panel). Antibodies and the corresponding proteins in the complexes are underlined. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3) using a combined scaling and error model.


Model predictions of IFNα - induced dynamics of occupied GAS bindings sites ( OccGASbs ) and of occupied ISRE binding sites ( OccISREbs ) in Huh7 . 5 cells without prestimulation and in cells prestimulated for 24 hours with 28 pM and 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα . Model predictions were performed using the prediction profile likelihood method . Lines with shading represent model predictions with 68 % confidence intervals . For experimental validation , growth factor - depleted Huh7 . 5 cells were prestimulated with 0 pM , 28 pM and 280 pM IFNα . After 24 hours cells were stimulated with 1400 pM IFNα and IFNα - induced expression of target genes was measured by qRT - PCR . RNA levels were normalized to the geometric mean of reference genes GAPDH , HPRT and TBP , averaged and displayed as fold change , represented by filled circles with errors representing standard error of the mean calculated from biological replicates ( N = 3 ) . Except for gene - specific parameters ( mRNA synthesis and degradation rates , time - delay parameter and Hill coefficient ) , qRT - PCR data were used for model validation but not for model calibration .

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

Model predictions of IFNα-induced dynamics of occupied GAS bindings sites (OccGASbs) and of occupied ISRE binding sites (OccISREbs) in Huh7.5 cells without prestimulation and in cells prestimulated for 24 hours with 28 pM and 280 pM IFNα that were subsequently stimulated with 1400 pM IFNα. Model predictions were performed using the prediction profile likelihood method. Lines with shading represent model predictions with 68% confidence intervals. For experimental validation, growth factor-depleted Huh7.5 cells were prestimulated with 0 pM, 28 pM and 280 pM IFNα. After 24 hours cells were stimulated with 1400 pM IFNα and IFNα-induced expression of target genes was measured by qRT-PCR. RNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP, averaged and displayed as fold change, represented by filled circles with errors representing standard error of the mean calculated from biological replicates (N=3). Except for gene-specific parameters (mRNA synthesis and degradation rates, time-delay parameter and Hill coefficient), qRT-PCR data were used for model validation but not for model calibration.


Dose - dependent sensitization of signal transduction induced by the therapeutic interferon Roferon . Growth factor - depleted Huh7 . 5 were prestimulated with 0 , with 1 . 2 or 608 pM Roferon and stimulated with 608 pM Roferon 24 hours later . Concentrations of Roferon correspond to equipotent concentrations of IFNα . Nuclear lysates were subjected to quantitative immunoblotting and Roferon - induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescense utilizing a CCD - based camera device ( ImageQuant ) . Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 8 ) using a combined scaling and error model . Model trajectories are represented by lines . Dose - dependent sensitization of signal transduction induced by Roferon in HepG2 - hNTCP . Growth factor - depleted HepG2 - hNTCP were prestimulated with 0 , 1 . 8 or 887 pM Roferon and stimulated with 887 pM Roferon 24 hours later . Nuclear lysates were subjected to quantitative immunoblotting and Roferon - induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescence utilizing a CCD - based camera device ( ImageQuant ) . Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates ( N = 3 ) using a combined scaling and error model . Model trajectories are represented by lines .

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

Dose-dependent sensitization of signal transduction induced by the therapeutic interferon Roferon. Growth factor-depleted Huh7.5 were prestimulated with 0, with 1.2 or 608 pM Roferon and stimulated with 608 pM Roferon 24 hours later. Concentrations of Roferon correspond to equipotent concentrations of IFNα. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescense utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=8) using a combined scaling and error model. Model trajectories are represented by lines.   Dose-dependent sensitization of signal transduction induced by Roferon in HepG2-hNTCP. Growth factor-depleted HepG2-hNTCP were prestimulated with 0, 1.8 or 887 pM Roferon and stimulated with 887 pM Roferon 24 hours later. Nuclear lysates were subjected to quantitative immunoblotting and Roferon-induced phosphorylation of STAT1 and STAT2 was detected by chemiluminescence utilizing a CCD-based camera device (ImageQuant). Filled circles represent scaled data with errors representing 1σ confidence intervals estimated from biological replicates (N=3) using a combined scaling and error model. Model trajectories are represented by lines.