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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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(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 O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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

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

(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 .

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

(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 .

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

(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 ) .

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

(H) 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 ) .

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

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 .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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

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 .

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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.


Knockdown of USP18 results in sustained signaling . USP18 knockdown efficiency determined by quantitative immunoblotting of cytoplasmic lysates of Huh7 . 5 transfected with USP18 siRNA relative to parental Huh7 . 5 cells and Huh7 . 5 cells transfected with non - targeting siRNA . Cells were stimulated with 1400 pM IFNα for 24 hours . Error bars represent 1σ confidence intervals estimated from biological replicates ( N = 6 to N = 10 ) ( left ) . Model fit and experimental data of Huh7 . 5 cells transfected with control siRNA or USP18 siRNA are shown . Cells were transfected with USP18 siRNA or control siRNA 1 day prior to growth factor - depletion . Next , cells were prestimulated with 1400 pM IFNα ( yellow background ) and stimulated with 1400 pM IFNα at 24 hours or untreated ( white background ) . IFNα - induced phosphorylation of STAT1 and STAT2 and induction of USP18 and tSTAT1 , comprising both phosphorylated and unphosphorylated STAT1 protein , are displayed . Experimental data were obtained by quantitative immunoblotting using chemiluminescence and CCD camera device ( ImageQuant ) . For model purposes data in control siRNA and untransfected Huh7 . 5 are combined to one condition . Data from multiple time courses scaled together is displayed as filled circles with errors representing 1σ confidence interval estimated from biological replicates ( N = 2 to N = 10 ) using a combined scaling and error model . Lines represent model trajectories .

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Knockdown of USP18 results in sustained signaling. USP18 knockdown efficiency determined by quantitative immunoblotting of cytoplasmic lysates of Huh7.5 transfected with USP18 siRNA relative to parental Huh7.5 cells and Huh7.5 cells transfected with non-targeting siRNA. Cells were stimulated with 1400 pM IFNα for 24 hours. Error bars represent 1σ confidence intervals estimated from biological replicates (N=6 to N=10) (left). Model fit and experimental data of Huh7.5 cells transfected with control siRNA or USP18 siRNA are shown. Cells were transfected with USP18 siRNA or control siRNA 1 day prior to growth factor-depletion. Next, cells were prestimulated with 1400 pM IFNα (yellow background) and stimulated with 1400 pM IFNα at 24 hours or untreated (white background). IFNα-induced phosphorylation of STAT1 and STAT2 and induction of USP18 and tSTAT1, comprising both phosphorylated and unphosphorylated STAT1 protein, are displayed. Experimental data were obtained by quantitative immunoblotting using chemiluminescence and CCD camera device (ImageQuant). For model purposes data in control siRNA and untransfected Huh7.5 are combined to one condition. Data from multiple time courses scaled together is displayed as filled circles with errors representing 1σ confidence interval estimated from biological replicates (N=2 to N=10) using a combined scaling and error model. Lines represent model trajectories.


Overexpression of Huh7 . 5 is not sufficient to explain negative memory . Induced expression of USP18 after treatment of Huh7 . 5 - TetON - USP18 and Huh7 . 5 - TetON - control cells with doxycycline for 24 hours in comparison with parental Huh7 . 5 cells treated with 1400 pM IFNα for 24 hours . Analysis was performed by quantitative immunoblotting . Error bars represent 1σ confidence intervals estimated from biological replicates ( N = 3 to N = 5 ) ( left ) . Model fits and experimental data of Huh7 . 5 - TetON - USP18 treated with doxycycline for 24 hours and stimulated with 1400 pM IFNα or parental Huh7 . 5 cells prestimulated with 0 or 1400 pM IFNα and stimulated with 1400 pM IFNα after 24 hours are shown . The dynamics of IFNα - induced phosphorylation of STAT1 and STAT2 and induction of USP18 are depicted . Experimental data was obtained by quantitative immunoblotting using chemiluminescence and CCD camera device ( ImageQuant ) . For modeling purposes data from Huh7 . 5 - TetON empty vector control and untransduced Huh7 . 5 are combined to one condition . Data is displayed as filled circles with errors representing 1σ confidence - interval estimated from biological replicates ( N = 3 to N = 4 ) using a combined scaling and error model . Line represents model trajectories .

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Overexpression of Huh7.5 is not sufficient to explain negative memory. Induced expression of USP18 after treatment of Huh7.5-TetON-USP18 and Huh7.5-TetON-control cells with doxycycline for 24 hours in comparison with parental Huh7.5 cells treated with 1400 pM IFNα for 24 hours. Analysis was performed by quantitative immunoblotting. Error bars represent 1σ confidence intervals estimated from biological replicates (N=3 to N=5) (left). Model fits and experimental data of Huh7.5-TetON-USP18 treated with doxycycline for 24 hours and stimulated with 1400 pM IFNα or parental Huh7.5 cells prestimulated with 0 or 1400 pM IFNα and stimulated with 1400 pM IFNα after 24 hours are shown. The dynamics of IFNα-induced phosphorylation of STAT1 and STAT2 and induction of USP18 are depicted. Experimental data was obtained by quantitative immunoblotting using chemiluminescence and CCD camera device (ImageQuant). For modeling purposes data from Huh7.5-TetON empty vector control and untransduced Huh7.5 are combined to one condition. Data is displayed as filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3 to N=4) using a combined scaling and error model. Line represents model trajectories.


The number of molecules per cell for STAT1 , STAT2 , IRF9 and USP18 were determined experimentally in primary human hepatocytes from six different patients . Cells were harvested without pretreatment and different amounts of protein calibrators were spiked in total cell lysates and lysates were subjected to immunoblotting . Detection was performed with antibodies specific to STAT1 , STAT2 , IRF9 or USP18 using chemiluminescense on a CCD camera - based device ( ImageQuant ) . Average of at least N = 3 are displayed with standard deviations . Data was used for calibration of a primary human hepatocyte - specific mathematical model and estimated values are shown with green squares .

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The number of molecules per cell for STAT1, STAT2, IRF9 and USP18 were determined experimentally in primary human hepatocytes from six different patients. Cells were harvested without pretreatment and different amounts of protein calibrators were spiked in total cell lysates and lysates were subjected to immunoblotting. Detection was performed with antibodies specific to STAT1, STAT2, IRF9 or USP18 using chemiluminescense on a CCD camera-based device (ImageQuant). Average of at least N=3 are displayed with standard deviations. Data was used for calibration of a primary human hepatocyte-specific mathematical model and estimated values are shown with green squares.


Experimental data and model fits of IFNα - and Roferon - induced phosphorylation of cytoplasmic or cellular STAT1 and STAT2 in growth factor - depleted primary human hepatocytes prestimulated with 0 , 2 . 8 or 1400 pM IFNα ( patients 1 - 3 ) or 0 , 1 . 2 or 608 pM Roferon ( patients 4 - 6 ) . Primary human hepatocytes from the same patients 1 - 6 as in ( B ) were used . Experimental data is represented by filled circles ( N = 1 per patient ) . Experimental errors were estimated from the signal variance of the hepatocytes prestimulated with 1400 pM IFNα . Lines indicate model fits .

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Experimental data and model fits of IFNα- and Roferon-induced phosphorylation of cytoplasmic or cellular STAT1 and STAT2 in growth factor-depleted primary human hepatocytes prestimulated with 0, 2.8 or 1400 pM IFNα (patients 1-3) or 0, 1.2 or 608 pM Roferon (patients 4-6). Primary human hepatocytes from the same patients 1-6 as in (B) were used. Experimental data is represented by filled circles (N=1 per patient). Experimental errors were estimated from the signal variance of the hepatocytes prestimulated with 1400 pM IFNα. Lines indicate model fits.


Plasma levels of IFNα were measured in a cohort of 36 patients with chronic HBV .

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Plasma levels of IFNα were measured in a cohort of 36 patients with chronic HBV.


( C ) Western blot showing that Hel2 ( 1 - 315 ) is defective in RQC but not in NGD . The arrest products derived from the R ( CGN ) 12 reporter in ltn1 ∆ cells expressing truncated Hel2 mutant protein were detected with an anti - GFP antibody .

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(C) Western blot showing that Hel2(1-315) is defective in RQC but not in NGD. The arrest products derived from the R(CGN)12 reporter in ltn1∆ cells expressing truncated Hel2 mutant protein were detected with an anti-GFP antibody.


( D ) Northern blot showing the 5 ' NGD - IM derived from the R ( CGN ) 12 reporter in ski2 ∆ cells expressing the indicated Hel2 mutant proteins . 5 ' NGD - IMs were detected with a DIG - labelled GFP probe .

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(D) Northern blot showing the 5' NGD-IM derived from the R(CGN)12 reporter in ski2∆ cells expressing the indicated Hel2 mutant proteins. 5' NGD-IMs were detected with a DIG-labelled GFP probe.


( E ) Primer extension mapping of 5 ' ends of 3 ' NGD - intermediates in Hel2 - WT or Hel2 ( 1 - 315 ) mutant cells at nucleotide resolution . . The primer extension samples were analysed using 5 % TBE - Urea - PAGE and detected by fluorescence . Non - specific reverse transcription ( ReTr ) products are indicated by asterisks .

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(E) Primer extension mapping of 5' ends of 3' NGD-intermediates in Hel2-WT or Hel2(1-315) mutant cells at nucleotide resolution. . The primer extension samples were analysed using 5% TBE-Urea-PAGE and detected by fluorescence. Non-specific reverse transcription (ReTr) products are indicated by asterisks.


( A ) Northern blot analysis demonstrating that K63 - linked ubiquitination was required for an endonucleolytic cleavage by R ( CGN ) 12 . The ski2 ∆ UB - WT and ski2 ∆ ub - K63R cells were transformed with the R ( CGN ) 12 reporter , and total RNA samples were separated by 2 % agarose / MOPS gel . 5 ' NGD - IMs in the cells were detected as in Fig 1D .

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(A) Northern blot analysis demonstrating that K63-linked ubiquitination was required for an endonucleolytic cleavage by R(CGN)12. The ski2∆UB-WT and ski2∆ub-K63R cells were transformed with the R(CGN)12 reporter, and total RNA samples were separated by 2% agarose/MOPS gel. 5'NGD-IMs in the cells were detected as in Fig 1D.


( B ) Western blot showing that K63 - linked ubiquitination was required for RQC . Protein samples from UB - WT , UB - WT ltn1 ∆ , ub - K63R , ub - K63R ltn1 ∆ cells expressing the GFP - R ( CGN ) 12 - HIS3 reporter were subjected to Western blot analysis using an anti - GFP antibody to detect the arrest products . Note the accumulation of RQC - specific CAT - tails in lane 2 .

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(B) Western blot showing that K63-linked ubiquitination was required for RQC. Protein samples from UB-WT, UB-WT ltn1∆, ub-K63R, ub-K63R ltn1∆ cells expressing the GFP-R(CGN)12-HIS3 reporter were subjected to Western blot analysis using an anti-GFP antibody to detect the arrest products. Note the accumulation of RQC-specific CAT-tails in lane 2.


( C ) Western blot analysis demonstrating polyubiquitination of uS10 : Affinity - tagged Hel2 - Flag - TEV - ProteinA ( FTP ) was co - purified with ribosomes harbouring HA - tagged wild type uS10 ( uS10 - 3HA ribosomes ) or uS10 mutated in its ubiquitination sites ( K6 / 8R ) . Western blots of whole protein extracts were performed using an anti - HA antibody to detect polyubiquitinated uS10 - 3HA .

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(C) Western blot analysis demonstrating polyubiquitination of uS10: Affinity-tagged Hel2-Flag-TEV-ProteinA (FTP) was co-purified with ribosomes harbouring HA-tagged wild type uS10 (uS10-3HA ribosomes) or uS10 mutated in its ubiquitination sites (K6/8R). Western blots of whole protein extracts were performed using an anti-HA antibody to detect polyubiquitinated uS10-3HA.


( D ) Western blot analysis showing that uS10 - polyubiquitination in Hel2 - bound ribosomes was mainly K63 - linked : Affinity tagged Hel2 ( Hel2 - ProteinA - TEV - His6 ; PTH - Hel2 ) was co - purified with ribosomes from a strain expressing wild type ubiquitin ( UB - WT ) or K63R mutant ubiquitin ( ub - K63R ) and HA - tagged uS10 ( uS10 - 3HA ) . Western blot analysis of whole protein extracts was performed using anti - HA antibody .

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(D) Western blot analysis showing that uS10-polyubiquitination in Hel2-bound ribosomes was mainly K63-linked: Affinity tagged Hel2(Hel2-ProteinA-TEV-His6; PTH-Hel2) was co-purified with ribosomes from a strain expressing wild type ubiquitin (UB-WT) or K63R mutant ubiquitin (ub-K63R) and HA-tagged uS10 (uS10-3HA). Western blot analysis of whole protein extracts was performed using anti-HA antibody.


( E ) Western blot of in vitro polyubiquitination assays of uS10 . Reactions were performed with the indicated components including His - tagged ubiquitin and several ubiquitin mutants . Polyubiquitinated HA - tagged uS10 ( uS10 - 3HA ) was detected by Western blot analysis using an anti - HA antibody .

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(E) Western blot of in vitro polyubiquitination assays of uS10. Reactions were performed with the indicated components including His-tagged ubiquitin and several ubiquitin mutants. Polyubiquitinated HA-tagged uS10 (uS10-3HA) was detected by Western blot analysis using an anti-HA antibody.


( F - H ) Northern blot analysis of NGD - cleavage sites in the absence of uS10 ubiquitination or RQT complex components : The full - length GFP - R ( CGN ) 12 - FLAG - HIS3 mRNA and 5 ' NGD - intermediates ( 5 ' NGD - IM ) or 3 ' NGD - intermediates ( 3 ' NGD - IM ) were detected in the indicated mutant cells by Northern blotting with DIG - labelled probes . 5 ' NGD - intermediates were detected by DIG - labelled GFP probe and 3 ' NGD - intermediates were detected by the DIG - labelled HIS3 probe . SCR1 was used as loading control . FL = full - length . Note the upstream shift of NGD cleavage sites in lanes F4 , G4 , H6 , and H8 .

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(F-H) Northern blot analysis of NGD-cleavage sites in the absence of uS10 ubiquitination or RQT complex components: The full-length GFP-R(CGN)12-FLAG-HIS3 mRNA and 5' NGD-intermediates (5' NGD-IM) or 3' NGD-intermediates (3' NGD-IM) were detected in the indicated mutant cells by Northern blotting with DIG-labelled probes. 5' NGD-intermediates were detected by DIG-labelled GFP probe and 3' NGD-intermediates were detected by the DIG-labelled HIS3 probe. SCR1 was used as loading control. FL = full-length. Note the upstream shift of NGD cleavage sites in lanes F4, G4, H6, and H8.


( I ) Mapping of 5 ' ends of 3 ' NGD - intermediates as demonstrated in Fig 1E . Non - specific reverse transcription ( ReTr ) products are indicated by asterisks . Note that both uS10 ubiquitination and Slh1 / Rqt2 were required for an endonucleolytic cleavage within the road - blocked ribosome ( X1 - X4 ) .

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(I) Mapping of 5' ends of 3' NGD-intermediates as demonstrated in Fig 1E. Non-specific reverse transcription (ReTr) products are indicated by asterisks. Note that both uS10 ubiquitination and Slh1/Rqt2 were required for an endonucleolytic cleavage within the road-blocked ribosome (X1-X4).


Both RQC and NGDRQC + are triggered by a ( CGA - CCG ) dicodon containing arrest sequence in vivo . ( B ) Western blot showing the arrest products derived from the GFP - X - FLAG - HIS3 reporter . Translation products were detected by western blot using an anti - GFP antibody .

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Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (B) Western blot showing the arrest products derived from the GFP-X-FLAG-HIS3 reporter. Translation products were detected by western blot using an anti-GFP antibody.


Both RQC and NGDRQC + are triggered by a ( CGA - CCG ) dicodon containing arrest sequence in vivo . ( C ) Northern blot for the 5 ' NGD - IM derived from the ( CGA - CCG ) reporter in ski2 ∆ cells . 5 ' - NGD - IMs were detected with a DIG - labelled GFP probe . SCR1 was used as a load control .

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Both RQC and NGDRQC+ are triggered by a (CGA-CCG) dicodon containing arrest sequence in vivo. (C) Northern blot for the 5' NGD-IM derived from the (CGA-CCG) reporter in ski2∆ cells. 5'-NGD-IMs were detected with a DIG-labelled GFP probe. SCR1 was used as a load control.


( D ) Western blot of test translations using the ( CGA - CCG ) dicodon stalling mRNA reporter shown in ( A ) . The mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2 ∆ uS10 - 3HA strain . Expression of the translation products ( free His - and V5 tagged truncated uL4 protein and the same protein attached to tRNA ) was visualized with an anti - V5 antibody .

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(D) Western blot of test translations using the (CGA-CCG) dicodon stalling mRNA reporter shown in (A). The mRNA reporter was added to a yeast in vitro translation extract obtained from a ski2∆uS10-3HA strain. Expression of the translation products (free His- and V5 tagged truncated uL4 protein and the same protein attached to tRNA) was visualized with an anti-V5 antibody.


( E ) Sucrose gradient fractions ( top ) and western blot analysis ( bottom ) of the ( CGA - CCG ) dicodon - stalled RNC samples . After the translation reaction , the RNCs were affinity - purified as indicated in ( C ) . The eluate was loaded on a 10 - 50 % sucrose gradient and fractionated . Each collected fraction was analysed using anti - HA antibody to detect uS10 - HA . Note that disomes are preferentially polyubiquitinated over monosomes .

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(E) Sucrose gradient fractions (top) and western blot analysis (bottom) of the (CGA-CCG) dicodon-stalled RNC samples. After the translation reaction, the RNCs were affinity-purified as indicated in (C). The eluate was loaded on a 10-50 % sucrose gradient and fractionated. Each collected fraction was analysed using anti-HA antibody to detect uS10-HA. Note that disomes are preferentially polyubiquitinated over monosomes.


( A ) Western blot analysis after overexpression of Hel2 in strains expressing HA - tagged ribosomal proteins using an anti - HA antibody . Note the increase of the polyubiquitinated eS7 as well as uS10 and uS3 .

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(A) Western blot analysis after overexpression of Hel2 in strains expressing HA-tagged ribosomal proteins using an anti-HA antibody. Note the increase of the polyubiquitinated eS7 as well as uS10 and uS3.


( C ) Western blot analysis showing the role of Hel2 and Not4 in eS7A ubiquitination : Both copies of eS7 ( eS7A and eS7B ) were deleted and transformed with a plasmid containing HA - tagged eS7A ( eS7 - WT ) or eS7A mutated in the four potential ubiquitination sites ( 4KR ) . Whole protein extracts we obtained from eS7AΔeS7BΔ cells or cells with an additional deletion in E3 ligases Hel2 and Not4 ( hel2Δ and not4Δ ) . Note that the four mutated lysine residues were responsible for Not4 - dependent monoubiquitination .

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(C) Western blot analysis showing the role of Hel2 and Not4 in eS7A ubiquitination: Both copies of eS7 (eS7A and eS7B) were deleted and transformed with a plasmid containing HA-tagged eS7A (eS7-WT) or eS7A mutated in the four potential ubiquitination sites (4KR). Whole protein extracts we obtained from eS7AΔeS7BΔ cells or cells with an additional deletion in E3 ligases Hel2 and Not4 (hel2Δ and not4Δ). Note that the four mutated lysine residues were responsible for Not4-dependent monoubiquitination.


( D ) Western blot showing that Not4 was required for Hel2 - mediated polyubiquitination of eS7 in Hel2 - bound ribosomal complexes : Cells expressing HA - tagged eS7A or the eS7 - 4KR mutant , as well as PTH - tagged Hel2 in eS7AΔeS7BΔ and eS7AΔeS7BΔnot4Δ background were probed for eS7 - ubiquitination . Either cell lysates or affinity - purified Hel2 - ribosome complexes were used . The levels of the ubiquitinated eS7A in the lysates and in the affinity - purified samples with PTH - Hel2 were determined by Western blot analysis using an anti - HA antibody to detect eS7 .

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(D) Western blot showing that Not4 was required for Hel2-mediated polyubiquitination of eS7 in Hel2-bound ribosomal complexes: Cells expressing HA-tagged eS7A or the eS7-4KR mutant, as well as PTH-tagged Hel2 in eS7AΔeS7BΔ and eS7AΔeS7BΔnot4Δ background were probed for eS7-ubiquitination. Either cell lysates or affinity-purified Hel2-ribosome complexes were used. The levels of the ubiquitinated eS7A in the lysates and in the affinity-purified samples with PTH-Hel2 were determined by Western blot analysis using an anti-HA antibody to detect eS7.


( E ) Western blotting of in vitro ubiquitination assays of eS7A . The reactions were performed with the indicated components including His - tagged ubiquitin . Ribosomes were purified from Hel2Δ cells expressing HA - tagged eS7A . Polyubiquitinated HA - tagged eS7A ( eS7A - 3HA ) was detected by Western blot analysis using an anti - HA antibody . We observed Hel2 - mediated polyubiquitination of the monoubiquitinated eS7A .

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(E) Western blotting of in vitro ubiquitination assays of eS7A. The reactions were performed with the indicated components including His-tagged ubiquitin. Ribosomes were purified from Hel2Δ cells expressing HA-tagged eS7A. Polyubiquitinated HA-tagged eS7A (eS7A-3HA) was detected by Western blot analysis using an anti-HA antibody. We observed Hel2-mediated polyubiquitination of the monoubiquitinated eS7A.


( F ) Western blot analysis of eS7A in vitro ubiquitination assays showing that Hel2 - mediated polyubiquitination of eS7 was mainly K63 - linked and required Not4 - dependent monoubiquitination : These assays were performed similarly as described in ( E ) except yeast strains were lacking Not4 ( not4Δ ) . Reactions were performed with the indicated components including His - tagged ubiquitin and several ubiquitin mutants and eS7A - ubiquitination was monitored using an anti - HA antibody .

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(F) Western blot analysis of eS7A in vitro ubiquitination assays showing that Hel2-mediated polyubiquitination of eS7 was mainly K63-linked and required Not4-dependent monoubiquitination: These assays were performed similarly as described in (E) except yeast strains were lacking Not4 (not4Δ). Reactions were performed with the indicated components including His-tagged ubiquitin and several ubiquitin mutants and eS7A-ubiquitination was monitored using an anti-HA antibody.


( A - C ) Northern blots probing for the 5 ' NGD - IM in mutant cells expressing the R ( CGN ) 12 reporter . ( A ) 5 ' NGD - IM detection in not4 ∆ ski2 ∆ , hel2 ∆ ski2 ∆ , slh1 ∆ ski2 ∆ , not4 ∆ slh1 ∆ ski2 ∆ . Quantification of full length and the 5 ' NGD - IM relative to the loading control ( SCR1 ) is given below . Note the size difference of 5 ' - NGD IMs resulting from NGDRQC + and NGDRQC - and that the shorter 5 ' - NGD intermediate ( representing intermediates of the NGDRQC - pathway ) was reduced in not4 ∆ slh1 ∆ ski2 ∆ mutant cells .

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(A-C) Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (A) 5' NGD-IM detection in not4∆ski2∆, hel2∆ski2∆, slh1∆ski2∆, not4∆ slh1∆ski2∆. Quantification of full length and the 5'NGD-IM relative to the loading control (SCR1) is given below. Note the size difference of 5'-NGD IMs resulting from NGDRQC+ and NGDRQC- and that the shorter 5'-NGD intermediate (representing intermediates of the NGDRQC- pathway) was reduced in not4∆slh1∆ski2∆ mutant cells.


Northern blots probing for the 5 ' NGD - IM in mutant cells expressing the R ( CGN ) 12 reporter . ( B ) Northern blot as in ( A ) except uS10 - WTski2 ∆ or uS10 - K6 / 8Rski2 ∆ mutant cells with or without a deletion of Not4 ( not4 ∆ ) were used . Note that the shorter 5 ' - NGD intermediates are also reduced in ski2 ∆ not4 ∆ uS10 - K6 / 8R cells .

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Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (B) Northern blot as in (A) except uS10-WTski2∆ or uS10-K6/8Rski2∆ mutant cells with or without a deletion of Not4 (not4∆) were used. Note that the shorter 5'-NGD intermediates are also reduced in ski2∆not4∆uS10-K6/8R cells.


Northern blots probing for the 5 ' NGD - IM in mutant cells expressing the R ( CGN ) 12 reporter . ( C ) Cells expressing HA - tagged eS7A or the eS7A - 4KR mutant in a ski2ΔeS7AΔeS7BΔ or ski2ΔeS7AΔeS7BΔslh1Δ backgrounds were probed for 5 ' - NGD intermediates by Northern blotting . Note that the four mutated lysine residues in eS7 were responsible for NGDRQC - in the R ( CGN ) 12 reporter .

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Northern blots probing for the 5' NGD-IM in mutant cells expressing the R(CGN)12 reporter. (C) Cells expressing HA-tagged eS7A or the eS7A-4KR mutant in a ski2ΔeS7AΔeS7BΔ or ski2ΔeS7AΔeS7BΔslh1Δ backgrounds were probed for 5'-NGD intermediates by Northern blotting. Note that the four mutated lysine residues in eS7 were responsible for NGDRQC- in the R(CGN)12 reporter.


( D ) Western blot analysis showing that K83 and K84 of eS7A are main target sites for Hel2 and Not4 - mediated ubiquitination : Similarly , as in Fig 6C , eS7AΔeS7BΔ mutant cells were used and wild type and single mutants of HA - tagged eS7A were expressed . Ubiquitinated eS7 - HA was monitored using an anti - HA antibody .

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(D) Western blot analysis showing that K83 and K84 of eS7A are main target sites for Hel2 and Not4-mediated ubiquitination: Similarly, as in Fig 6C, eS7AΔeS7BΔ mutant cells were used and wild type and single mutants of HA-tagged eS7A were expressed. Ubiquitinated eS7-HA was monitored using an anti-HA antibody.


( E ) Northern Blot analysis probing for the levels of 5 ' NGD - IM derived from the R ( CGN ) 12 reporter expressed in eS7AΔeS7BΔski2Δ cells and HA - tagged eS7 or eS7 mutants . We observed , that ubiquitination at K83 or K84 of eS7A is mainly responsible for NGDRQC - .

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(E) Northern Blot analysis probing for the levels of 5' NGD-IM derived from the R(CGN)12 reporter expressed in eS7AΔeS7BΔski2Δ cells and HA-tagged eS7 or eS7 mutants. We observed, that ubiquitination at K83 or K84 of eS7A is mainly responsible for NGDRQC-.


( F ) The levels of polyubiquitinated eS7A were increased by the overproduction of wild - type Hel2 ( FL ) but neither Hel2ΔRING nor Hel2 ( 1 - 315 ) mutants . eS7A - 3HAhel2Δ mutant cells harbouring the indicated plasmids expressing wild - type Hel2 ( FL ) , Hel2ΔRING or Hel2 ( 1 - 315 ) mutant proteins were harvested . Protein samples were analysed by Western blotting with an anti - HA ( Top panel ) or anti - FLAG antibody ( Bottom panel ) .

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(F) The levels of polyubiquitinated eS7A were increased by the overproduction of wild-type Hel2 (FL) but neither Hel2ΔRING nor Hel2(1-315) mutants. eS7A-3HAhel2Δ mutant cells harbouring the indicated plasmids expressing wild-type Hel2 (FL), Hel2ΔRING or Hel2(1-315) mutant proteins were harvested. Protein samples were analysed by Western blotting with an anti-HA (Top panel) or anti-FLAG antibody (Bottom panel).


( G ) Hel2 ( 1 - 315 ) polyubiquitinates eS7A but not uS10 . Western blotting after an in vitro ubiquitination assay using the Hel2 - ( 1 - 315 ) mutant and purified ribosomes containing HA - tagged uS10 .

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(G) Hel2(1-315) polyubiquitinates eS7A but not uS10. Western blotting after an in vitro ubiquitination assay using the Hel2-(1-315) mutant and purified ribosomes containing HA-tagged uS10.


M - O : Schematic describing the measurement of a cell ' s approach angle to the nearest point on the AER ( M ) . Merged , immunofluorescent images of WT ( top ) , slit3sq49 ( centre ) and s1pr2te273 ( below ) mesenchymal cells in sqet37Et background stained for EGFP ( green ) , Gamma tubulin ( red ) and DAPI ( blue ) . White lines run from the centre of nucleus to the nearest point on the AER , through the MTOC ( N ) . Graph depicting the approach angles to AER of leading ( Tier 1 ) , following ( Tier 2 ) and trailing ( Tier 3 ) cells of WT , slit3sq49 and s1pr2te273 embryos ( O ) . A minimum of 30 cells were measured for each tier of each genotype . * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; ANOVA with Fisher ' s post - test .

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M-O: Schematic describing the measurement of a cell's approach angle to the nearest point on the AER (M). Merged, immunofluorescent images of WT (top), slit3sq49 (centre) and s1pr2te273 (below) mesenchymal cells in sqet37Et background stained for EGFP (green), Gamma tubulin (red) and DAPI (blue). White lines run from the centre of nucleus to the nearest point on the AER, through the MTOC (N). Graph depicting the approach angles to AER of leading (Tier 1), following (Tier 2) and trailing (Tier 3) cells of WT, slit3sq49 and s1pr2te273 embryos (O). A minimum of 30 cells were measured for each tier of each genotype. *p<0.05; **p<0.005; ***p<0.001; ANOVA with Fisher's post-test.


C - E : Lateral Nomarski images of 48hpf larval fins which are WT ( C ) or slit3 + / sq49 heterozygotes ( D ) and are injected with 300µM fn1a morpholino . Blisters are observed only when there is reduced Fn1a in slit3 heterozygotes ( quantified in E ) .

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C-E: Lateral Nomarski images of 48hpf larval fins which are WT (C) or slit3+/sq49 heterozygotes (D) and are injected with 300µM fn1a morpholino. Blisters are observed only when there is reduced Fn1a in slit3 heterozygotes (quantified in E).