A . , B . Transverse sections from trachea isolated from GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) P0 mice , immunostained with antibodies against acetylated y - tubulin ( ACT , cilia marker , red ) , pericentrin ( PCNT , marker for nascent centrioles , green ) , γ tubulin ( centriole marker , green ) and E - cadherin ( marker for cell boundaries , blue ) . Serial images have been merged to show the complete trachea section ( left ) . Inserts show higher magnification images for acetylated y - tubulin and pericentrin staining in GemC1WT / WT and GemC1KO / KO trachea , respectively . Scale bars , 10 µm .

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A., B. Transverse sections from trachea isolated from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice, immunostained with antibodies against acetylated y-tubulin (ACT, cilia marker, red), pericentrin (PCNT, marker for nascent centrioles, green), γ tubulin (centriole marker, green) and E-cadherin (marker for cell boundaries, blue). Serial images have been merged to show the complete trachea section (left). Inserts show higher magnification images for acetylated y-tubulin and pericentrin staining in GemC1WT/WT and GemC1KO/KO trachea, respectively. Scale bars, 10 µm.


C . McIdas and Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) P0 mice . Arrows show representative positive cells for McIdas ( green ) and FoxJ1 ( red ) in the GemC1WT / WT mice . DNA was stained with Draq - 5 ( blue ) . Scale bars , 10 µm .

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C. McIdas and Foxj1 specific antibodies were used for immunofluorescence on trachea from GemC1WT/WT (upper) or GemC1KO/KO (lower) P0 mice. Arrows show representative positive cells for McIdas (green) and FoxJ1 (red) in the GemC1WT/WT mice. DNA was stained with Draq-5 (blue). Scale bars, 10 µm.


D . Transverse sections from P0 GemC1WT / WT ( upper ) or GemC1KO / KO ( lower ) trachea , immunostained with antibodies against Keratin 5 ( Krt5 , basal cell marker , green ) , and acetylated y - tubulin ( ACT , cilia marker , red ) . DNA was stained with Draq - 5 ( blue ) . Serial images have been merged to show the complete trachea section on the left . Higher magnification images are shown on the right . Scale bars , 10 µm .

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D. Transverse sections from P0 GemC1WT/WT (upper) or GemC1KO/KO (lower) trachea, immunostained with antibodies against Keratin 5 (Krt5, basal cell marker, green), and acetylated y-tubulin (ACT, cilia marker, red). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section on the left. Higher magnification images are shown on the right. Scale bars, 10 µm.


E . Transverse sections from P0 GemC1WT / WT ( left two images ) or GemC1KO / KO ( right two images ) trachea , immunostained with antibodies against Muc5ac ( marker for mucus - expressing cells , red ) and E - cadherin ( marker for cell boundaries , green ) . DNA was stained with Draq - 5 ( blue ) . Serial images have been merged to show the complete trachea section ( left ) . Higher magnifications from the same section are shown on the right . Scale bars , 10 µm .

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E. Transverse sections from P0 GemC1WT/WT (left two images) or GemC1KO/KO (right two images) trachea, immunostained with antibodies against Muc5ac (marker for mucus-expressing cells, red) and E-cadherin (marker for cell boundaries, green). DNA was stained with Draq-5 (blue). Serial images have been merged to show the complete trachea section (left). Higher magnifications from the same section are shown on the right. Scale bars, 10 µm.


( A ) ( B ) CPT sensitivity assayed by 10 - fold serial dilutions of different mutant combinations on YPAD plates . Four biological replicates have been performed .

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(A) (B) CPT sensitivity assayed by 10-fold serial dilutions of different mutant combinations on YPAD plates. Four biological replicates have been performed.


( C ) Colony - forming unit assays to increasing CPT concentrations of the same mutant combinations as in ( B ) . Mean values ± SD are plotted . Four biological replicates have been performed .

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(C) Colony-forming unit assays to increasing CPT concentrations of the same mutant combinations as in (B). Mean values ± SD are plotted. Four biological replicates have been performed.


( D ) UV sensitivity assayed by 10 - fold serial dilutions of different mutant combinations with rad3 - 102 allele on YPAD plates after exposure to the indicated UV - C doses . Five biological replicates have been performed .

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(D) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Five biological replicates have been performed.


( F ) BIR frequencies ( Lys + survivors among total cells ) for the WT and indicated mutant strains are plotted on a logarithmic scale . Data represent the mean ± SD from 3 to 6 independent experiments . ns , non - significant difference , * P - value = 0 . 0134 , Mann - Whitney unpaired t test .

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(F) BIR frequencies (Lys+ survivors among total cells) for the WT and indicated mutant strains are plotted on a logarithmic scale. Data represent the mean ± SD from 3 to 6 independent experiments. ns, non-significant difference, * P-value = 0.0134, Mann-Whitney unpaired t test.


( C ) UV sensitivity assayed by 10 - fold serial dilutions of different mutant combinations with rad3 - 102 allele on YPAD plates after exposure to the indicated UV - C doses . Four biological replicates have been performed .

(C) UV sensitivity assayed by 10-fold serial dilutions of different mutant combinations with rad3-102 allele on YPAD plates after exposure to the indicated UV-C doses. Four biological replicates have been performed.


( D ) Synthetic combinations of rad3 - 102 mus81 ∆ with rad51 ∆ and pol32 ∆ . Tetrads dissected on YPAD medium are shown . Triangles indicate either a severe growth defect or lethality .

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(D) Synthetic combinations of rad3-102 mus81∆ with rad51∆ and pol32∆. Tetrads dissected on YPAD medium are shown. Triangles indicate either a severe growth defect or lethality.


( A ) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type , rad52 ∆ , rad51 ∆ , mus81 ∆ and pol32 ∆ cells and further released into S phase . Cells were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 1 h , and released into S phase . Three biological replicates have been performed .

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(A) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad52∆, rad51∆, mus81∆ and pol32∆ cells and further released into S phase. Cells were synchronized in G1 with α -factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Three biological replicates have been performed.


( B ) ( C ) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type , rad3 - 102 , rad3 - 102 rad51 ∆ , rad3 - 102 mus81 and rad3 - 102 mus81 - dd cells and further released into S phase . Cells were synchronized in G1 with α - factor , untreated or irradiated with 20 J / m2 UV - C , let in G1 for 2h , and released into S phase . Three biological replicates have been performed .

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(B) (C) Analysis of DNA content by flow cytometry of G1 phase synchronized wild type, rad3-102, rad3-102 rad51∆, rad3-102 mus81 and rad3-102 mus81-dd cells and further released into S phase. Cells were synchronized in G1 with α-factor, untreated or irradiated with 20 J/m2 UV-C, let in G1 for 2h, and released into S phase. Three biological replicates have been performed.


( A ) Analysis of replicated DNA tracks length by single - molecule DNA combing in WT , rad52 ∆ , rad51 ∆ , mus81 ∆ and pol32 ∆ cells exposed to CPT . Exponentially growing cells were treated with DMSO or 50 µM CPT for 2h and then pulse - labeled with 50 µM EdU for 20min . DNA fibers were combed on silanized coverslips and EdU - labeled DNA was detected by Click chemistry . Graph depicts the distribution of EdU tracks length in kb . Box and whiskers indicate 25 - 75 and 10 - 90 percentiles , respectively . Median EdU tracks length is indicated in kb ( values from two biological replicates were pooled ) . Asterisks indicate the P - value of the Mann - Whitney unpaired t test , * * : p = 0 . 0015 . The percentage of EdU track length decrease between the DMSO and CPT conditions is indicated between parentheses for each strain . Representative images of DNA fibers are shown . Red and white : EdU , green : DNA .

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(A) Analysis of replicated DNA tracks length by single-molecule DNA combing in WT, rad52∆, rad51∆, mus81∆ and pol32∆ cells exposed to CPT. Exponentially growing cells were treated with DMSO or 50 µM CPT for 2h and then pulse-labeled with 50 µM EdU for 20min. DNA fibers were combed on silanized coverslips and EdU-labeled DNA was detected by Click chemistry. Graph depicts the distribution of EdU tracks length in kb. Box and whiskers indicate 25-75 and 10-90 percentiles, respectively. Median EdU tracks length is indicated in kb (values from two biological replicates were pooled). Asterisks indicate the P-value of the Mann-Whitney unpaired t test, **: p=0.0015. The percentage of EdU track length decrease between the DMSO and CPT conditions is indicated between parentheses for each strain. Representative images of DNA fibers are shown. Red and white: EdU, green: DNA.


( C ) Analysis of replication intermediates by 2D gel electrophoresis . Replication intermediates were monitored at early origin ARS305 and region C in WT , rad3 - 102 , mus81 ∆ and rad3 - 102 mus81 ∆ cells . Cells were synchronized in G1 with α - factor and collected at the indicated time points after release into S phase . A scheme of the studied chromosomal region is shown ( drawn to scale ) . Relevant probes are indicated by gray bars , and coordinates of ARS and restriction sites are indicated in bp . Two biological replicates have been performed .

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(C) Analysis of replication intermediates by 2D gel electrophoresis. Replication intermediates were monitored at early origin ARS305 and region C in WT, rad3-102, mus81∆ and rad3-102 mus81∆ cells. Cells were synchronized in G1 with α-factor and collected at the indicated time points after release into S phase. A scheme of the studied chromosomal region is shown (drawn to scale). Relevant probes are indicated by gray bars, and coordinates of ARS and restriction sites are indicated in bp. Two biological replicates have been performed.


( A ) Analysis of nuclear Rad52 - GFP foci formation . An illustrative image of the experimental setup is shown . DIC , differential interference contrast ; yellow , Rad52 - GFP ; red , mCHERRY - Pus1 ( nuclear compartment marker ) . Scale bar = 5 µm .

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(A) Analysis of nuclear Rad52-GFP foci formation. An illustrative image of the experimental setup is shown. DIC, differential interference contrast; yellow, Rad52-GFP; red, mCHERRY-Pus1 (nuclear compartment marker). Scale bar = 5 µm.


( B ) ( C ) Kinetic analysis of Rad52 foci formation . Wild - type and mus81 ∆ cells containing Rad52 - YFP and mCHERRY - Pus1 were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 30 min , and released into S phase . Cells were collected at the indicated time points and visualized by fluorescence microscopy . Mean ± SEM of cells with Rad52 foci are shown for each time point . Flow cytometry profiles corresponding the experimental setup are shown . Four biological replicates have been performed .

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(B) (C) Kinetic analysis of Rad52 foci formation. Wild-type and mus81∆ cells containing Rad52-YFP and mCHERRY-Pus1 were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 30 min, and released into S phase. Cells were collected at the indicated time points and visualized by fluorescence microscopy. Mean ± SEM of cells with Rad52 foci are shown for each time point. Flow cytometry profiles corresponding the experimental setup are shown. Four biological replicates have been performed.


( D ) Mms4 - Flag10 phosphorylation analyzed by immunoblot in wild type cells exposed to CPT . Wild - type cells were synchronized in G1 with α - factor , treated with DMSO or 50 µM CPT , let in G1 for 1 h , and released into S phase . Cells were collected at the indicated time points and Mms4 was immunodetected with anti - Flag antibodies . Clb2 immunodetection serves as a marker for G2 phase entry . FACS profiles corresponding the experimental setup are also shown . Two biological replicates have been performed .

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(D) Mms4-Flag10 phosphorylation analyzed by immunoblot in wild type cells exposed to CPT. Wild-type cells were synchronized in G1 with α-factor, treated with DMSO or 50 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points and Mms4 was immunodetected with anti-Flag antibodies. Clb2 immunodetection serves as a marker for G2 phase entry. FACS profiles corresponding the experimental setup are also shown. Two biological replicates have been performed.


( E ) CPT sensitivity assayed by 10 - fold serial dilutions of S - MUS81 and G2 - MUS81 alleles compared to the wild type , mus81 , mms4 ∆ and cdc5 - 2 mutants . Three biological replicates have been performed .

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(E) CPT sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles compared to the wild type, mus81, mms4∆ and cdc5-2 mutants. Three biological replicates have been performed.


( F ) UV sensitivity assayed by 10 - fold serial dilutions of S - MUS81 and G2 - MUS81 alleles in combination with rad3 - 102 on YPAD plates after exposure to the indicated UV - C doses . Three biological replicates have been performed .

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(F) UV sensitivity assayed by 10-fold serial dilutions of S-MUS81 and G2-MUS81 alleles in combination with rad3-102 on YPAD plates after exposure to the indicated UV-C doses. Three biological replicates have been performed.


( A ) ( B ) Pulsed - field gel electrophoresis ( PFGE ) analysis of wild type , mus81 ∆ , cdc5 - 2 and G2 - MUS81 cells in response to CPT . Cells were synchronized in G1 with α - factor , treated with DMSO or 100 µM CPT , let in G1 for 1 h , and released into S phase . Cells were collected at the indicated time points . DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE . Upper panel : agarose gel stained with ethidium bromide ; lower panel : Southern blot using a chromosome IV specific probe . JMs , joint molecules accumulated in the gel wells . The mean value of JMs relative to the total amount of DNA is indicated for each time point . Two to three biological replicates have been performed .

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(A) (B) Pulsed-field gel electrophoresis (PFGE) analysis of wild type, mus81∆, cdc5-2 and G2-MUS81 cells in response to CPT. Cells were synchronized in G1 with α-factor, treated with DMSO or 100 µM CPT, let in G1 for 1 h, and released into S phase. Cells were collected at the indicated time points. DNA contents was analyzed by flow cytometry and the DNA extracted in agarose plugs was analyzed by PFGE. Upper panel: agarose gel stained with ethidium bromide; lower panel: Southern blot using a chromosome IV specific probe. JMs, joint molecules accumulated in the gel wells. The mean value of JMs relative to the total amount of DNA is indicated for each time point. Two to three biological replicates have been performed.


( C ) ( D ) PFGE analysis of rad52 ∆ and rad52 ∆ mus81 ∆ cells in response to CPT performed as in ( A ) ( B ) . The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the + CPT 140 min time points in rad52 ∆ and rad52 ∆ mus81 ∆ cells are shown . Two biological replicates have been performed .

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(C) (D) PFGE analysis of rad52∆ and rad52∆ mus81∆ cells in response to CPT performed as in (A) (B). The gel has been stained with Ethidium bromide and densitometry profiles corresponding to the +CPT 140 min time points in rad52∆ and rad52∆ mus81∆ cells are shown. Two biological replicates have been performed.


( A ) Akt is deubiquitinated during fasting . Left : Soluble fractions of TA muscles from fed and fasted mice were analyzed by SDS - PAGE and immunoblot using Akt antibody . Right : densitometric measurement of presented blots ( n = 3 ) . * p < 0 . 05 vs . fed by one - tailed t - test . Data are represented as mean ± SEM .

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(A) Akt is deubiquitinated during fasting. Left: Soluble fractions of TA muscles from fed and fasted mice were analyzed by SDS-PAGE and immunoblot using Akt antibody. Right: densitometric measurement of presented blots (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.


( B ) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted ( 2d ) mice . Percipitated were analyzed by immunoblotting using an anti - ubiquitin conjugates . Mouse IgG was used as a control for non - specific binding .

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(B) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted (2d) mice. Percipitated were analyzed by immunoblotting using an anti-ubiquitin conjugates. Mouse IgG was used as a control for non-specific binding.


) D ( Ubiquitinated Akt is a substrate for USP1 in vitro . Top : Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice , and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi - well plate . The dividing line indicates the removal of an intervening lane for presentation purposes . Bottom : Densitometric measurements of presented blots . Data is presented as the ratio between ubiquitinated Akt to total Akt in each well .

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)D( Ubiquitinated Akt is a substrate for USP1 in vitro. Top: Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice, and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi-well plate. The dividing line indicates the removal of an intervening lane for presentation purposes. Bottom: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each well.


( E ) shRNA - mediated knockdown of USP1 in Hela cells . Soluble extracts were analyzed by immunoblotting .

(E) shRNA-mediated knockdown of USP1 in Hela cells. Soluble extracts were analyzed by immunoblotting.


( F ) USP1 deubiquitinates Akt during fasting in vivo . Left : Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS - PAGE and immunoblotting using anti - Akt . The actin blot serves as a loading control . Right : densitometric measurements of presented blots ( n = 3 ) . Data is presented as ratio of ubiquitinated Akt to total Akt normalized to actin . * p < 0 . 05 vs . fed by one - tailed t - test . Data are represented as mean ± SEM .

(F) USP1 deubiquitinates Akt during fasting in vivo. Left: Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using anti-Akt. The actin blot serves as a loading control. Right: densitometric measurements of presented blots (n=3). Data is presented as ratio of ubiquitinated Akt to total Akt normalized to actin. * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.


( G ) USP1 removes K63 - linked polyubiquitin chains on Akt in vivo . Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1 ( C90S ) or control plasmids . Mouse IgG was used as a control for non - specific binding . Protein precipitates were subjected to immunoblotting with an antibody against K63 - linked polyubiquitin conjugates .

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(G) USP1 removes K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1(C90S) or control plasmids. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.


( H ) USP1 removes polyubiquitin chains linked to K8 on Akt . Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1 ( C90S ) , HA - Akt ( K8R ) or control from fasted mice , and protein precipitates were analyzed by immunoblotting with anti - Akt antibody . Mouse IgG was used as a control for non - specific binding . Right : Densitometric measurements of presented blots . Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane ( n = 2 ) .

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(H) USP1 removes polyubiquitin chains linked to K8 on Akt. Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1(C90S) , HA-Akt(K8R) or control from fasted mice, and protein precipitates were analyzed by immunoblotting with anti-Akt antibody. Mouse IgG was used as a control for non-specific binding. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).


( A ) Inhibition of USP1 increases PI3K - Akt - FoxO signaling during fasting . Soluble fractions of normal and atrophying muscles expressing shLacz or USP1 ( C90S ) were analyzed by SDS - PAGE and immunoblot . The black line indicate the removal of intervening lanes for presentation purposes . Right : densitometric measurements of presented pAkt ( T308 ) , pAkt ( S473 ) , and Akt blots . Data is presented as ratio of phosphorylated Akt to total Akt ( n = 3 ) . * p < 0 . 05 vs . fed by one - tailed t - test . Data are represented as mean ± SEM .

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(A) Inhibition of USP1 increases PI3K-Akt-FoxO signaling during fasting. Soluble fractions of normal and atrophying muscles expressing shLacz or USP1(C90S) were analyzed by SDS-PAGE and immunoblot. The black line indicate the removal of intervening lanes for presentation purposes. Right: densitometric measurements of presented pAkt (T308), pAkt (S473), and Akt blots. Data is presented as ratio of phosphorylated Akt to total Akt (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.


( B ) Downregulation of USP1 reduces MuRF1 and Atrogin1 expression during fasting . Quantitative RT - PCR of mRNA preparations from atrophying and control muscles expressing shLacz or shUSP1 ( 80 % transfection efficiency ) using primers for MuRF1 and Atrogin1 . Data are plotted as the mean fold change relative to fed control . n = 4 . * , P < 0 . 05 vs . shLacz in fed . # , P < 0 . 05 vs . shLacz in fasting by one - tailed t - test . Data are represented as mean ± SEM .

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(B) Downregulation of USP1 reduces MuRF1 and Atrogin1 expression during fasting. Quantitative RT-PCR of mRNA preparations from atrophying and control muscles expressing shLacz or shUSP1 (80% transfection efficiency) using primers for MuRF1 and Atrogin1. Data are plotted as the mean fold change relative to fed control. n = 4. *, P < 0.05 vs. shLacz in fed. #, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.


( C ( Downregulation of USP1 markedly reduces muscle fiber atrophy . Cross - sectional areas of 500 fibers transfected with shUSP1 ( that express GFP , green bars ) vs . 500 nontransfected fibers ( black bars ) in the same muscle . n = 5 mice . A representative image is shown ; Laminin staining is in red ; Bar , 50μm .

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(C( Downregulation of USP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shUSP1 (that express GFP, green bars) vs. 500 nontransfected fibers (black bars) in the same muscle. n = 5 mice. A representative image is shown; Laminin staining is in red; Bar, 50μm.


( D ) Downregulation of USP1attenuates the loss of muscle mass during fasting . shUSP1 was delivered to more than 60 % of muscle fibers . Mean weights of electroporated muscles are plotted as the percent weight loss . n = 6 . # , P < 0 . 05 vs . shLacz in fed ; * , P < 0 . 05 vs . shLacz in fasting by one - tailed t - test . Data are represented as mean ± SEM .

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(D) Downregulation of USP1attenuates the loss of muscle mass during fasting. shUSP1 was delivered to more than 60% of muscle fibers. Mean weights of electroporated muscles are plotted as the percent weight loss. n = 6. #, P < 0.05 vs. shLacz in fed; *, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.


( E ) USP1 inhibition significantly increases glucose tolerance in mice during fasting . Left : mice injected i . p . with specific USP1 Inhibitor ( 12ug / gr body weight ) or saline during fasting ( 2d ) were subjected to glucose tolerance test . Blood glucose levels were measured at the indicated time points following glucose injection ( 1 mg / gr body weight ) . Data is depicted in a graph as mg / dL glucose ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 vs . mice injected with saline by one - tailed t - test . Data are represented as mean ± SEM . Right : soluble fractions of muscles from injected mice were analyzed by SDS - PAGE and immunoblot .

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

(E) USP1 inhibition significantly increases glucose tolerance in mice during fasting. Left: mice injected i.p. with specific USP1 Inhibitor (12ug/gr body weight) or saline during fasting (2d) were subjected to glucose tolerance test. Blood glucose levels were measured at the indicated time points following glucose injection (1 mg/gr body weight). Data is depicted in a graph as mg/dL glucose (n=3). * p<0.05, ** p<0.005, *** p<0.0005 vs. mice injected with saline by one-tailed t-test. Data are represented as mean ±SEM. Right: soluble fractions of muscles from injected mice were analyzed by SDS-PAGE and immunoblot.


( A ) During fasting , downregulation of PHLPP1 enhances Akt phosphorylation at S473 . Soluble fractions of muscles transfected with shLacz or shPHLPP1 from fasted mice ( 2d ) were analyzed by immunoblotting . Right : densitometric measurements of presented blots . Data is presented as ratio of shPHLPP1 to shLacz ( n = 3 for shLacz and n = 4 for shPHLPP1 ) . * p < 0 . 05 vs . shLacz in fasting by one - tailed t - test . Data are represented as mean ± SEM .

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(A) During fasting, downregulation of PHLPP1 enhances Akt phosphorylation at S473. Soluble fractions of muscles transfected with shLacz or shPHLPP1 from fasted mice (2d) were analyzed by immunoblotting. Right: densitometric measurements of presented blots. Data is presented as ratio of shPHLPP1 to shLacz (n=3 for shLacz and n=4 for shPHLPP1). * p<0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.


( B ) PHLPP1 downregulation increases rates of protein synthesis during fasting . Mice were injected with puromycin , and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody . Analysis of right ( R ) and left ( L ) limbs for each mouse is shown .

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(B) PHLPP1 downregulation increases rates of protein synthesis during fasting. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Analysis of right (R) and left (L) limbs for each mouse is shown.


( C ) Downregulation of PHLPP1 markedly reduces muscle fiber atrophy . Cross - sectional areas of 500 fibers transfected with shPHLPP1 ( that express GFP , green bars ) vs . 500 nontransfected fibers ( black bars ) in the same muscle . n = 4 mice . A representative image is shown ; Laminin staining is in red ; Bar , 50μm .

(C) Downregulation of PHLPP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shPHLPP1 (that express GFP, green bars) vs. 500 nontransfected fibers (black bars) in the same muscle. n = 4 mice. A representative image is shown; Laminin staining is in red; Bar, 50μm.


( D ) USP1 / UAF1 interact with PHLPP1 in normal muscle . USP1 was immunoprecipitated from the soluble fraction of normal muscles . Mouse IgG was used as a control for non - specific binding . Precipitates were analyzed by immunoblotting .

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(D) USP1/UAF1 interact with PHLPP1 in normal muscle. USP1 was immunoprecipitated from the soluble fraction of normal muscles. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting.


( C ( In fasting , Akt sediments to glycerol gradient fractions containing USP1 / UAF1 , PHLPP1 , and TSC1 / TSC2 . Muscle homogenates from fed or fasted mice were separated on 10 % - 40 % glycerol gradient , and alternate fractions were analyzed by SDS - PAGE and immunoblot . Red rectangle marks fractions containing USP1 / UAF1 , PHLPP1 , TSC1 and TSC2 in fed conditions .

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(C( In fasting, Akt sediments to glycerol gradient fractions containing USP1/UAF1, PHLPP1, and TSC1/TSC2. Muscle homogenates from fed or fasted mice were separated on 10%-40% glycerol gradient, and alternate fractions were analyzed by SDS-PAGE and immunoblot. Red rectangle marks fractions containing USP1/UAF1, PHLPP1, TSC1 and TSC2 in fed conditions.


( D ) During fasting , Akt is recruited to USP1 - PHLPP1 - TSC1 complex . TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or USP1 ( C90S ) from fed or fasted mice . Mouse IgG was used as a control for non - specific binding . Precipitates were analyzed by immunoblotting . Black lines indicate the removal of intervening lanes for presentation purposes .

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(D) During fasting, Akt is recruited to USP1-PHLPP1-TSC1 complex. TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or USP1(C90S) from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Black lines indicate the removal of intervening lanes for presentation purposes.


( E ) During fasting , inhibition of USP1 reduces rates of protein synthesis . Mice were injected with puromycin , and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody . Right : Densitometric measurement of the presented blot . n = 3 . * , P < 0 . 05 vs . shLacz by one - tailed t - test . Data are represented as mean ± SEM .

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(E) During fasting, inhibition of USP1 reduces rates of protein synthesis. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.


( F ) Inhibition of USP1 results in TSC1 accumulation . Soluble fraction of muscles expressing shLacz or USP1 ( C90S ) from fasted mice were analyzed by SDS - PAGE and immunoblotting . Right : Densitometric measurement of the presented blot . n = 3 . * , P < 0 . 05 vs . shLacz . Data is presented as protein content ( AU ) in USP1 ( C90S ) vs . shLacz by one - tailed t - test . Data are represented as mean ± SEM .

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(F) Inhibition of USP1 results in TSC1 accumulation. Soluble fraction of muscles expressing shLacz or USP1(C90S) from fasted mice were analyzed by SDS-PAGE and immunoblotting. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz. Data is presented as protein content (AU) in USP1(C90S) vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.


( G ) mTOR inhibition with rapamycin results in reduced USP1 protein content and enhanced Akt phosphorylation at T308 . However , a simultaneous downregulation of USP1 is required to promote TSC1 accumulation . Soluble fraction of muscles expressing shLacz or gUSP1 ( in vivo CRISPR ) from fed and fasted mice injected i . p . with rapamycin ( 6mg / kg body weight ) or saline were analyzed by SDS - PAGE and immunoblot .

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(G) mTOR inhibition with rapamycin results in reduced USP1 protein content and enhanced Akt phosphorylation at T308. However, a simultaneous downregulation of USP1 is required to promote TSC1 accumulation. Soluble fraction of muscles expressing shLacz or gUSP1 (in vivo CRISPR) from fed and fasted mice injected i.p. with rapamycin (6mg/kg body weight) or saline were analyzed by SDS-PAGE and immunoblot.


( A ) During fasting , inhibition of Dab2 - Akt association reduces the interaction of Akt with USP1 - PHLPP1 - TSC1 complex . Left : TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2 - DN from fed or fasted mice . Mouse IgG was used as a control for non - specific binding . Precipitates were analyzed by immunoblotting . Right : densitometric measurement of PHLPP1 and TSC1 blots , and data is presented as PHLPP1 / TSC1 ratio .

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(A) During fasting, inhibition of Dab2-Akt association reduces the interaction of Akt with USP1-PHLPP1-TSC1 complex. Left: TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Right: densitometric measurement of PHLPP1 and TSC1 blots, and data is presented as PHLPP1/TSC1 ratio.


( B ) Dab2 promotes Akt deubiquitination in vivo . Left : Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2 - DN from fed and fasted mice using a specific antibody . Mouse IgG was used as a control for non - specific binding . Protein precipitates were analyzed by Western blot analysis using Akt antibody . Right : Densitometric measurements of presented blots . Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane ( n = 2 ) .

(B) Dab2 promotes Akt deubiquitination in vivo. Left: Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were analyzed by Western blot analysis using Akt antibody. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).


( C ( Dab2 promotes cleavage of K63 - linked polyubiquitin chains on Akt in vivo . Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2 - DN from fed and fasted mice using a specific antibody . Mouse IgG was used as a control for non - specific binding . Protein precipitates were subjected to immunoblotting with an antibody against K63 - linked polyubiquitin conjugates .

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(C( Dab2 promotes cleavage of K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.


( D ) Inhibition of Dab2 - Akt association enhances Akt phosphorylation in fasting . Soluble fraction of TA muscles transfected with shLacz or Dab2 - DN from fed and fasted mice were analyzed by SDS - PAGE and immunoblotting using the indicated antibodies . Right : densitometric measurement of presented blots . Data are mean ± SEM , n = 3 . * p < 0 . 05 . vs . shLacz in fed conditions , by one - tailed t - test .

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(D) Inhibition of Dab2-Akt association enhances Akt phosphorylation in fasting. Soluble fraction of TA muscles transfected with shLacz or Dab2-DN from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Right: densitometric measurement of presented blots. Data are mean ±SEM, n=3. * p<0.05. vs. shLacz in fed conditions, by one-tailed t-test.


Relative expression levels of the HIF1A - AS2 transcript in the indicated cell lines under normoxia condition were monitored by qRT - PCR analysis and represented as 39 minus Ct values .

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Relative expression levels of the HIF1A-AS2 transcript in the indicated cell lines under normoxia condition were monitored by qRT-PCR analysis and represented as 39 minus Ct values.


Sanger sequencing validation of RNA editing events on HIF1A - AS2 . The editing frequencies of the indicated sites were quantitated based on three independent experiments and shown in Table EV 1 . Asterisks mark the sites undergoing statistically significant increases in RNA editing .

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Sanger sequencing validation of RNA editing events on HIF1A-AS2. The editing frequencies of the indicated sites were quantitated based on three independent experiments and shown in Table EV 1. Asterisks mark the sites undergoing statistically significant increases in RNA editing.


RNA - IP of ADAR1 in HUVECs , followed by real - time PCR analysis of the HIF - 1A and HIF1A - AS2 RNAs . The relative enrichment of transcript association was normalized to the IgG . GAPDH was used as a non - binding control .

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RNA-IP of ADAR1 in HUVECs, followed by real-time PCR analysis of the HIF-1A and HIF1A-AS2 RNAs. The relative enrichment of transcript association was normalized to the IgG. GAPDH was used as a non-binding control.


Cells were cultured under normal or hypoxic condition . Relative levels of the HIF1A - AS transcripts in total vs . poly ( A ) + RNA fractions ( F ) were determined by qRT - PCR .

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Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in total vs. poly(A)+ RNA fractions (F) were determined by qRT-PCR.


Cells were cultured under normal or hypoxic condition . Relative levels of the HIF1A - AS transcripts in subcellular fractions ( G ) , were determined by qRT - PCR .

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Cells were cultured under normal or hypoxic condition. Relative levels of the HIF1A-AS transcripts in subcellular fractions (G), were determined by qRT-PCR.


FISH analysis of the spatial distribution of HIF - 1A mRNA ( red ) and HIF1A - AS2 transcripts ( white ) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs . Nuclei were counterstained by DAPI . Scale bars , 10 μm .

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FISH analysis of the spatial distribution of HIF-1A mRNA (red) and HIF1A-AS2 transcripts (white) in HUVECs subjected to mock or CoCl2 treatment for 5 hrs. Nuclei were counterstained by DAPI. Scale bars, 10 μm.


Expression of HIF1A - AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides ( ODN ) - based knockdown . Cells were then treated with CoCl2 for 5 hrs to mimic hypoxic condition , and subjected to gene expression analyses : The RNA abundance of HIF - 1A and HIF1A - AS2 was analyzed by real - time PCR and normalized by 18s rRNA ( A ) .

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Expression of HIF1A-AS2 was suppressed in HUVECs by oligo phosphodiester nucleotides (ODN)-based knockdown. Cells were then treated with CoCl2 for 5 hrs to mimic hypoxic condition, and subjected to gene expression analyses: The RNA abundance of HIF-1A and HIF1A-AS2 was analyzed by real-time PCR and normalized by 18s rRNA (A).


HIF - 1α protein level was monitored by western blot , with β - actin as the internal control ( B ) .

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HIF-1α protein level was monitored by western blot, with β-actin as the internal control (B).


Nuclear run - on assays were performed to assess the extent of nascent transcription of target genes under mock vs . CoCl2 treatment ( C )

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Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes under mock vs. CoCl2 treatment (C)


Nuclear run - on assays were performed to assess the extent of nascent transcription of target genes in the HIF1A - AS2 knockdown cells ( D ) .

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Nuclear run-on assays were performed to assess the extent of nascent transcription of target genes in the HIF1A-AS2 knockdown cells (D).


HIF - 1A mRNA expression was detected in a region - specific manner ( 5 ' vs . 3 ' ) by primers corresponding to exons 1 - 2 ( E1 - 2 ) and exon 14 - 15 ( E14 - 15 ) . To further monitor transcription activity , ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells under CoCl2 treatment ( E ) , The precipitated DNA fragments were quantitatively analyzed by real - time PCR using primers for the HIF1A - AS2 and HIF - 1A promoters , and normalized to the values of IgG . HIF1A - AS2 corresponds to the transcribed region .

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HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells under CoCl2 treatment (E), The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region.


HIF - 1A mRNA expression was detected in a region - specific manner ( 5 ' vs . 3 ' ) by primers corresponding to exons 1 - 2 ( E1 - 2 ) and exon 14 - 15 ( E14 - 15 ) . To further monitor transcription activity , ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells in HIF1A - AS2 - knockdown vs . control cells ( F ) . The precipitated DNA fragments were quantitatively analyzed by real - time PCR using primers for the HIF1A - AS2 and HIF - 1A promoters , and normalized to the values of IgG . HIF1A - AS2 corresponds to the transcribed region .

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HIF-1A mRNA expression was detected in a region-specific manner (5' vs. 3') by primers corresponding to exons 1-2 (E1-2) and exon 14-15 (E14-15). To further monitor transcription activity, ChIP was performed to characterize RNA polymerase II binding on chromatin isolated from MCF7 cells in HIF1A-AS2-knockdown vs. control cells (F). The precipitated DNA fragments were quantitatively analyzed by real-time PCR using primers for the HIF1A-AS2 and HIF-1A promoters, and normalized to the values of IgG. HIF1A-AS2 corresponds to the transcribed region.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . The expression levels of ADAR1 , HIF - 1A and HIF1A - AS2 transcripts were analyzed by qRT - PCR .

HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. The expression levels of ADAR1, HIF-1A and HIF1A-AS2 transcripts were analyzed by qRT-PCR.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . Nuclear run - on assay was done to measure the newly synthesized HIF - 1A and HIF1A - AS2 RNAs in ADAR1 - knockdown cells , followed by real - time PCR analysis of the indicated transcripts .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Nuclear run-on assay was done to measure the newly synthesized HIF-1A and HIF1A-AS2 RNAs in ADAR1-knockdown cells, followed by real-time PCR analysis of the indicated transcripts.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . ChIP analysis of RNA polII binding on chromatin region of the HIF1A - AS2 and HIF - 1A promoters . The extent of occupancy was compared between control and ADAR1 - knockdown cells . The precipitated DNA fragments were quantitatively analyzed by real - time PCR and normalized by the values of IgG .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. ChIP analysis of RNA polII binding on chromatin region of the HIF1A-AS2 and HIF-1A promoters. The extent of occupancy was compared between control and ADAR1-knockdown cells. The precipitated DNA fragments were quantitatively analyzed by real-time PCR and normalized by the values of IgG.


Chromatin binding of ADAR1 to HIF1A - AS2 and HIF - 1A promoters under nomoxia ( 20 % O2 ) vs . hypoxia ( 1 % O2 , 5 hrs ) conditions , as assessed by ChIP .

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Chromatin binding of ADAR1 to HIF1A-AS2 and HIF-1A promoters under nomoxia (20% O2) vs. hypoxia (1% O2, 5 hrs) conditions, as assessed by ChIP.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . The 3 ' - end extension of HIF1A - AS2 RNA transcription in ADAR1 knockdown cells ( GFP , control ) was examined by PCR with primers corresponding to the depicted regions of HIF1A - AS2 locus . GAPDH serves as an internal control . Sequence locations are shown in kbp and relative to the transcription start site .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. The 3'-end extension of HIF1A-AS2 RNA transcription in ADAR1 knockdown cells (GFP, control) was examined by PCR with primers corresponding to the depicted regions of HIF1A-AS2 locus. GAPDH serves as an internal control. Sequence locations are shown in kbp and relative to the transcription start site.


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . Expression levels of HIF - 1α protein ( F ) by western

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Expression levels of HIF-1α protein (F) by western blot


HUVECs were transfected with ADAR1 - targeting siRNAs and subjected to hypoxia treatment for 5 hrs . Expression levels of HIF - 1α downstream genes ( G ) by real - time PCR , as indicated . qRT - PCR results were normalized by the expression of 18s rRNA .

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HUVECs were transfected with ADAR1-targeting siRNAs and subjected to hypoxia treatment for 5 hrs. Expression levels of HIF-1α downstream genes (G) by real-time PCR, as indicated. qRT-PCR results were normalized by the expression of 18s rRNA.


Transcriptome - wide changes in the cells subjected to CoCl2 treatment , HIF1A - AS2 or ADAR1 knockdown were profiled by RNA - Sequencing . For genes that undergo up - regulation upon CoCl2 treatment ( n = 505 ) , overall expression profiles of the different cell groups are represented by the heatmap . The expression values ( represented by normalized read counts ) were displayed in shades of red or blue ( linear scale ) relative to the means of all corresponding values within individual experimental groups . Clusters of genes ( based on unsupervised hierarchical clustering ) are indicated and denoted by their experimental types / conditions .

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Transcriptome-wide changes in the cells subjected to CoCl2 treatment, HIF1A-AS2 or ADAR1 knockdown were profiled by RNA-Sequencing. For genes that undergo up-regulation upon CoCl2 treatment (n = 505), overall expression profiles of the different cell groups are represented by the heatmap. The expression values (represented by normalized read counts) were displayed in shades of red or blue (linear scale) relative to the means of all corresponding values within individual experimental groups. Clusters of genes (based on unsupervised hierarchical clustering) are indicated and denoted by their experimental types/conditions.


GSEA comparing the gene expression profile by gene up - regulated upon CoCl2 treatment and those down - regulated in ADAR1 knockdown . The curves correspond to the observed enrichment score profiles in the CoCl2 gene set to the ADAR1 knockdown gene sets . The bar ‐ code plot below enrichment score plot denote the rank of each member in the CoCl2 up - regulated gene list .

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GSEA comparing the gene expression profile by gene up-regulated upon CoCl2 treatment and those down-regulated in ADAR1 knockdown. The curves correspond to the observed enrichment score profiles in the CoCl2 gene set to the ADAR1 knockdown gene sets. The bar‐code plot below enrichment score plot denote the rank of each member in the CoCl2 up-regulated gene list.


HUVECs were transfected with the indicated combinations of siRNA ( GFP as control versus ADAR1 ) and ODN ( GFP as control versus HIF1A - AS2 ) , followed by CoCl2 treatment for 5 hrs . The expression levels of ADAR1 and HIF - 1α protein ( E ) were analyzed respectively by immunoblotting .

HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. The expression levels of ADAR1 and HIF-1α protein (E) were analyzed respectively by immunoblotting.


HUVECs were transfected with the indicated combinations of siRNA ( GFP as control versus ADAR1 ) and ODN ( GFP as control versus HIF1A - AS2 ) , followed by CoCl2 treatment for 5 hrs . For cells in ( E ) , RNA expression of heat shock protein - encoding genes ( HSPA1A , HSPA1B , and HSPA6 ) with reported roles in angiogenesis were profiled by qRT - PCR .

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HUVECs were transfected with the indicated combinations of siRNA (GFP as control versus ADAR1) and ODN (GFP as control versus HIF1A-AS2), followed by CoCl2 treatment for 5 hrs. For cells in (E), RNA expression of heat shock protein-encoding genes (HSPA1A, HSPA1B, and HSPA6) with reported roles in angiogenesis were profiled by qRT-PCR.


Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation . Scale bars , 2000 μm .

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Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation. Scale bars, 2000 μm.


Sanger sequencing validation of RNA editing of the LIMD1 ( A ) and VHL ( B ) transcripts .

Sanger sequencing validation of RNA editing of the LIMD1 (A) and VHL (B) transcripts.


Downregulation of ADAR1 levels in MCF7 cells was done using specific siRNAs . Cells were then subjected to hypoxia treatment for 5 hrs . The extent of knockdown and gene alterations was monitored by western blotting using antibodies targeting LIMD1 , VHL , PHD1 , PHD2 , ADAR1 and HIF - 1α , as indicated β - actin was used as loading control .

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Downregulation of ADAR1 levels in MCF7 cells was done using specific siRNAs. Cells were then subjected to hypoxia treatment for 5 hrs. The extent of knockdown and gene alterations was monitored by western blotting using antibodies targeting LIMD1, VHL, PHD1, PHD2, ADAR1 and HIF-1α, as indicated β-actin was used as loading control.


To characterize subcellular distribution of target transcripts , real - time PCR analysis was done on the fractionated MCF7 cells with the indicated experimental treatments .

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To characterize subcellular distribution of target transcripts, real-time PCR analysis was done on the fractionated MCF7 cells with the indicated experimental treatments.


Translation efficiency was determined by quantifying transcript association with the polyribosome fraction , using ADAR1 - depleted MCF7 cells with hypoxia treatment . The abundance of LIMD1 in polysomes was analyzed by real - time PCR and normalized by the eEF1α levels .

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Translation efficiency was determined by quantifying transcript association with the polyribosome fraction, using ADAR1-depleted MCF7 cells with hypoxia treatment. The abundance of LIMD1 in polysomes was analyzed by real-time PCR and normalized by the eEF1α levels.


To inspect HIF - 1α protein abundance and modification with ubiquitin , anti - HIF - 1α , anti - ubiquitylation ( Ub ) , and anti - VHL immunoprecipitation experiments were performed , coupled with immunoblotting detection of ubiquitylation and HIF - 1α , as indicated . MCF7 cells transfected with ADAR1 siRNA and treated with hypoxia were used . Protein signals were quantified using the ImageJ software . The fold changes of protein levels were determined on the basis of immunoreactive signals normalized by the input ( In ) , with the control ( siGFP ) group being represented as 1 .

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To inspect HIF-1α protein abundance and modification with ubiquitin, anti- HIF-1α, anti-ubiquitylation (Ub), and anti-VHL immunoprecipitation experiments were performed, coupled with immunoblotting detection of ubiquitylation and HIF-1α, as indicated. MCF7 cells transfected with ADAR1 siRNA and treated with hypoxia were used. Protein signals were quantified using the ImageJ software. The fold changes of protein levels were determined on the basis of immunoreactive signals normalized by the input (In), with the control (siGFP) group being represented as 1.


Box - plot showing the enrichment scores ( E - scores ) of all possible 8 - mers containing the G - box ( CACGTG ) , PBE - box ( CATGTG ) , and BRRE elements ( CGTGC / TG ) . Boxes represent the 25 - 75 % quartiles and the black line the median of distribution . Bars indicate the 1 - 25 % ( above ) and 75 - 100 % ( below ) quartiles . E - scores above 0 . 4 denote that binding of the proteins towards the indicated DNA element is statistically significant . Dashed blue line indicates the 0 . 4

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Box-plot showing the enrichment scores (E-scores) of all possible 8-mers containing the G-box (CACGTG), PBE-box (CATGTG), and BRRE elements (CGTGC/TG). Boxes represent the 25-75% quartiles and the black line the median of distribution. Bars indicate the 1-25% (above) and 75-100% (below) quartiles. E-scores above 0.4 denote that binding of the proteins towards the indicated DNA element is statistically significant. Dashed blue line indicates the 0.4 threshold


Protein - binding microarrays ( PBM ) were probed with PIF4 - His , MBP - BES1 and the PIF4 - His / MBP - BES1 complex . For PIF4 - BES1 complex purification , both proteins were co - expressed in E . coli using the double expression pCOLADuet - 1 vector . An anti - MBP antibody was used for BES1 and BES1 - PIF4 signal detection , while PIF4 was detected with an anti - His antibody Electrophoretic mobility shift assays ( EMSA ) showing interaction of the PIF4 , BES1 , and PIF4 - BES1 proteins with the conserved G - box , PBE - , and BRRE - elements in the PIL1 ( At2g46970 ) , PRE5 ( At3g28857 ) , and DWF4 ( At3g50660 ) promoters . Increasing amounts of protein were used for the

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Protein-binding microarrays (PBM) were probed with PIF4-His, MBP-BES1 and the PIF4-His/MBP-BES1 complex. For PIF4-BES1 complex purification, both proteins were co-expressed in E. coli using the double expression pCOLADuet-1 vector. An anti-MBP antibody was used for BES1 and BES1-PIF4 signal detection, while PIF4 was detected with an anti-His antibody Electrophoretic mobility shift assays (EMSA) showing interaction of the PIF4, BES1, and PIF4-BES1 proteins with the conserved G-box, PBE-, and BRRE-elements in the PIL1 (At2g46970), PRE5 (At3g28857), and DWF4 (At3g50660) promoters. Increasing amounts of protein were used for the assay


Protein - binding microarrays ( PBM ) were probed with PIF4 - His , MBP - BES1 and the PIF4 - His / MBP - BES1 complex . For PIF4 - BES1 complex purification , both proteins were co - expressed in E . coli using the double expression pCOLADuet - 1 vector . An anti - MBP antibody was used for BES1 and BES1 - PIF4 signal detection , while PIF4 was detected with an anti - His antibody BES1 binds both BRRE - and G - box elements as a homodimer . A deletion of BES1 ( delN ) fused to MBP ( MBP - delN ) and the complete protein ( MBP - BES1 ) were co - expressed in E . coli . Formation of intermediate mobility bands , indicative of DNA recognition by a dimeric form of the protein was tested in EMSA assays . A signal corresponding to the dimeric full - length MBP - BES1 and MBP - delN complex was detected with both DNA probes . Increasing amounts of the proteins were used in the

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Protein-binding microarrays (PBM) were probed with PIF4-His, MBP-BES1 and the PIF4-His/MBP-BES1 complex. For PIF4-BES1 complex purification, both proteins were co-expressed in E. coli using the double expression pCOLADuet-1 vector. An anti-MBP antibody was used for BES1 and BES1-PIF4 signal detection, while PIF4 was detected with an anti-His antibody BES1 binds both BRRE- and G-box elements as a homodimer. A deletion of BES1 (delN) fused to MBP (MBP-delN) and the complete protein (MBP-BES1) were co-expressed in E.coli. Formation of intermediate mobility bands, indicative of DNA recognition by a dimeric form of the protein was tested in EMSA assays. A signal corresponding to the dimeric full-length MBP-BES1 and MBP-delN complex was detected with both DNA probes. Increasing amounts of the proteins were used in the assay


Co - expression of the PIF4 and BES1 effector constructs leads to synergistic pPIL1 activation , and reverses BES1 - dependent inhibition of the pDWF4 reporter . The pPIL1 and pDWF4 promoters including three G - boxes ( green boxes ) and two BRRE - elements ( orange boxes ) were fused to the firefly luciferase reporter gene ( LUC ) and co - transfected with 35S : : PIF4 , 35S : : BES1 and 35S : : bes1 - D effector constructs into Nicotiana benthamiana leaves . Leaf discs were collected 48 hours after infiltration and luciferase activity was measured in a microplate luminometer .

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Co-expression of the PIF4 and BES1 effector constructs leads to synergistic pPIL1 activation, and reverses BES1-dependent inhibition of the pDWF4 reporter. The pPIL1 and pDWF4 promoters including three G-boxes (green boxes) and two BRRE- elements (orange boxes) were fused to the firefly luciferase reporter gene (LUC) and co-transfected with 35S::PIF4, 35S::BES1 and 35S::bes1-D effector constructs into Nicotiana benthamiana leaves. Leaf discs were collected 48 hours after infiltration and luciferase activity was measured in a microplate luminometer.


Heat map and fold - change graph representation of the genes identified in A . Expression data were obtained from Genevestigator and heatmap and clustering analyses were performed using the MeV software . PIF + BES - UP indicates genes up - regulated by PIF and BES and identified as targets of both the PIF and BES factors . BES - DOWN are BES repressed targets , while BES - UP correspond to BES induced targets . The PIF - UP cluster includes PIF induced genes directly bound by the PIF factors .

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Heat map and fold-change graph representation of the genes identified in A. Expression data were obtained from Genevestigator and heatmap and clustering analyses were performed using the MeV software. PIF+BES-UP indicates genes up-regulated by PIF and BES and identified as targets of both the PIF and BES factors. BES-DOWN are BES repressed targets, while BES-UP correspond to BES induced targets. The PIF-UP cluster includes PIF induced genes directly bound by the PIF factors.


Time course analysis of BES1 protein accumulation levels . pBES1 : : BES1 - GFP seedlings were grown under short days for 6 days and samples were harvested each 2 hours for a 24 h interval . Total protein extracts were used for western blot analysis with an anti - GFP antibody to detect the BES1 - GFP protein . Hybridization with anti - RPT5 antibody is included as a loading control . Dark and light periods are represented as black and white bars , respectively . The experiment was repeated twice with similar

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Time course analysis of BES1 protein accumulation levels. pBES1::BES1-GFP seedlings were grown under short days for 6 days and samples were harvested each 2 hours for a 24 h interval. Total protein extracts were used for western blot analysis with an anti-GFP antibody to detect the BES1-GFP protein. Hybridization with anti-RPT5 antibody is included as a loading control. Dark and light periods are represented as black and white bars, respectively. The experiment was repeated twice with similar results


Diurnal oscillation of BES1 and BZR1 transcripts . Col - 0 seedlings were grown for 6 days under short days and samples were harvested every 3 hours . BES1 and BZR1 expression levels were determined by qRT - PCR analyses and normalized to PP2A . Relative levels are the mean of three technical replicates and error bars represent standard deviation . The experiment was performed three times with similar results . Dark periods are indicated by

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Diurnal oscillation of BES1 and BZR1 transcripts. Col-0 seedlings were grown for 6 days under short days and samples were harvested every 3 hours. BES1 and BZR1 expression levels were determined by qRT-PCR analyses and normalized to PP2A. Relative levels are the mean of three technical replicates and error bars represent standard deviation. The experiment was performed three times with similar results. Dark periods are indicated by shading


Western blot analyses of 35S : : BES - GFP seedlings grown for six days under short days . Samples were collected every two hours , starting two hours before lights on ( ZT - 2 ) to ZT6 . Western blots were hybridized with an anti - GFP antibody to detect the BES1 - GFP protein , and anti - RPT5 as a loading

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Western blot analyses of 35S::BES-GFP seedlings grown for six days under short days. Samples were collected every two hours, starting two hours before lights on (ZT-2) to ZT6. Western blots were hybridized with an anti-GFP antibody to detect the BES1-GFP protein, and anti-RPT5 as a loading control


BES1 but not BZR1 is stabilized in the light . 35S : : BES1 - GFP and 35S : : BZR1 - GFP seedlings were grown for 6 days in continuous white light ( WL ) , red light ( RL ) , and continuous dark ( DARK ) , and the BES1 and BZR1 proteins analyzed by western blot using an anti - GFP antibody . Hybridization with an anti - RPT5 antibody was used as a loading

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BES1 but not BZR1 is stabilized in the light. 35S::BES1-GFP and 35S::BZR1-GFP seedlings were grown for 6 days in continuous white light (WL), red light (RL), and continuous dark (DARK), and the BES1 and BZR1 proteins analyzed by western blot using an anti-GFP antibody. Hybridization with an anti-RPT5 antibody was used as a loading control


BES1 accumulation is independent of brassinosteroid levels . Six day - old 35S : : BES - GFP seedlings were treated overnight with 100 nM epi - brassinolide ( BL ) or 0 . 5 µM brassinazole ( BRZ ) . Material was collected at the indicated time points and analyzed by western blot hybridization with anti - GFP and - RPT5

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BES1 accumulation is independent of brassinosteroid levels. Six day-old 35S::BES-GFP seedlings were treated overnight with 100 nM epi-brassinolide (BL) or 0.5 µM brassinazole (BRZ). Material was collected at the indicated time points and analyzed by western blot hybridization with anti-GFP and -RPT5 antibodies


In vivo analysis of PIF4 - BES1 complex formation . Six day - old pPIF4 : : PIF4 - HA seedlings grown under short day conditions were collected at ZT0 , ZT1 and ZT8 . PIF4 - HA was immunoprecipitated with an anti - HA antibody and the pulled - down BES1 protein immunodetected with an anti - BES antibody . * Indicates a non - specific band by the primary antibody used to immunoprecipitation .

In vivo analysis of PIF4-BES1 complex formation. Six day-old pPIF4::PIF4-HA seedlings grown under short day conditions were collected at ZT0, ZT1 and ZT8. PIF4-HA was immunoprecipitated with an anti-HA antibody and the pulled-down BES1 protein immunodetected with an anti-BES antibody. * Indicates a non-specific band by the primary antibody used to immunoprecipitation.


Col - 0 ( black ) , PIF4 - OX ( red ) and pifq ( blue ) seedlings were grown for 6 days under short days and samples harvested at the indicated time points . Total RNA was used for qRT - PCR quantification of PIF + BES - UP and the BR biosynthetic BES - DOWN transcripts , using PP2A as endogenous control . Error bars represent SD of three technical replicates . Dark periods are shown in grey . Relative expression of the XTR7 , PRE5 , IAA19 and BR6ox2 genes in PIF4 - OX lines are referred to the right y axis of the graph . The experiment was repeated three times with similar results .

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Col-0 (black), PIF4-OX (red) and pifq (blue) seedlings were grown for 6 days under short days and samples harvested at the indicated time points. Total RNA was used for qRT-PCR quantification of PIF+BES-UP and the BR biosynthetic BES-DOWN transcripts, using PP2A as endogenous control. Error bars represent SD of three technical replicates. Dark periods are shown in grey. Relative expression of the XTR7, PRE5, IAA19 and BR6ox2 genes in PIF4-OX lines are referred to the right y axis of the graph. The experiment was repeated three times with similar results.


Col - 0 and pifq seedlings were grown under short days at 22ºC and 28ºC , and samples were collected at the indicated time points . Expression of BR biosynthetic genes was analyzed by qRT - PCR , using the constitutive PP2A gene as internal control . Error bars represent SD of three technical replicates . The experiment was repeated twice with similar

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Col-0 and pifq seedlings were grown under short days at 22ºC and 28ºC, and samples were collected at the indicated time points. Expression of BR biosynthetic genes was analyzed by qRT-PCR, using the constitutive PP2A gene as internal control. Error bars represent SD of three technical replicates. The experiment was repeated twice with similar results


Activation of the XTR7 PIF + BES - UP target was studied as a positive control of the experiment . Error bars represent SD of three technical replicates . Dark periods are shown in grey . The experiment was performed three times with similar

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Activation of the XTR7 PIF+BES-UP target was studied as a positive control of the experiment. Error bars represent SD of three technical replicates. Dark periods are shown in grey. The experiment was performed three times with similar results


Increased levels of de - phosphorylated BES1 at elevated temperatures depend on PIFs activation of BR synthesis . Col - 0 and pifq seedlings were grown for 6 days under short days and 22ºC or 28ºC , on half strength MS media ( mock ) or media supplemented with 0 . 5 µM BRZ . BES1 was analyzed by western blot hybridization with an anti - BES1 antibody . RPT5 was used as a loading control .

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Increased levels of de-phosphorylated BES1 at elevated temperatures depend on PIFs activation of BR synthesis. Col-0 and pifq seedlings were grown for 6 days under short days and 22ºC or 28ºC, on half strength MS media (mock) or media supplemented with 0.5 µM BRZ. BES1 was analyzed by western blot hybridization with an anti-BES1 antibody. RPT5 was used as a loading control.


Figure 1 : Growth Charts and Photographs of Affected Subjects • A Family 1 - Subject II . 1 . • B Family 1 - Subject II . 3 . • C Family 2 - Subject II . 3 . • D Family 2 - Subject II . 4 . • E Family 2 - Subject II . 5 . • F Top Row - Representative X - rays showing narrow fibulae and elongated digits in Subject II . 1 of Family 1 . Bottom Row - Lower extremities and foot from Subject II . 5 and hand from Subject II . 4 , both from Family 2 .

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Figure 1: Growth Charts and Photographs of Affected Subjects•A Family 1-Subject II.1.•B Family 1-Subject II.3.•C Family 2-Subject II.3.•D Family 2-Subject II.4.•E Family 2-Subject II.5.•F Top Row -Representative X-rays showing narrow fibulae and elongated digits in Subject II.1 of Family 1. Bottom Row - Lower extremities and foot from Subject II.5 and hand from Subject II.4, both from Family 2.


• B Pedigree and representative Sanger sequencing trace of the mutation identified in family 1 . • C Pedigree and representative Sanger sequencing trace of the mutation identified in family 2 .

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• B Pedigree and representative Sanger sequencing trace of the mutation identified in family 1.•C Pedigree and representative Sanger sequencing trace of the mutation identified in family 2.


• A Assessment of proteolytic activity present in culture media of human embryonic kidney 293T cells transfected with cDNA encoding PAPP - A2 , PAPP - A2 ( D643fs ) ( family 1 ) , or PAPP - A2 ( A1033V ) ( family 2 ) . Radiolabeled substrates tested include IGFBP - 3 , IGFBP - 4 , and IGFBP - 5 . Cleavage was visualized by autoradiography following SDS - PAGE . Positions of intact IGFBPs ( i ) and cleavage products ( c ) are indicated .

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• A Assessment of proteolytic activity present in culture media of human embryonic kidney 293T cells transfected with cDNA encoding PAPP-A2, PAPP-A2(D643fs) (family 1), or PAPP-A2(A1033V) (family 2). Radiolabeled substrates tested include IGFBP-3, IGFBP-4, and IGFBP-5. Cleavage was visualized by autoradiography following SDS-PAGE. Positions of intact IGFBPs (i) and cleavage products (c) are indicated.


B Western blots following nonreducing or reducing SDS - PAGE of media from transfected cells , as indicated . Polyclonal antibodies raised against the N - terminal portion of PAPP - A2 were used for detection .

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B Western blots following nonreducing or reducing SDS-PAGE of media from transfected cells, as indicated. Polyclonal antibodies raised against the N-terminal portion of PAPP-A2 were used for detection.


• C Comparison of proteolytic activity similar to the experiment of panel A , except that equimolar concentrations of PAPP - A2 and PAPP - A2 ( A1033V ) were used . The variant from Family 1 , carrying a frameshift mutation N - terminal to the proteolytic domain of PAPP - A2 , did not show any detectable expression . All gels and blots are representative of three independent experiments .

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•C Comparison of proteolytic activity similar to the experiment of panel A, except that equimolar concentrations of PAPP-A2 and PAPP-A2 (A1033V) were used. The variant from Family 1, carrying a frameshift mutation N-terminal to the proteolytic domain of PAPP-A2, did not show any detectable expression. All gels and blots are representative of three independent experiments.


• A Concentrations of endogenous insulin - like growth factor ( IGF ) - I in the different fractions obtained by neutral chromatography on a Superdex 200 column . Serum samples from the four siblings of family 1 were analyzed and compared to a non - related prepubertal control , as all siblings were prepubertal . The amount of IGF - I detected in the fractions corresponding to the ternary complex ( TC ) was markedly greater in the homozygous affected subjects ( II · 1 and II · 3 ) compared to the unaffected siblings ( II . 2 and II . 4 ) and the prepubertal control . No IGF - I was detected in the fractions corresponding to the binary complex ( BC ) or free IGF - I in any of the subjects . This is most likely due to the fact that under physiological conditions 80 - 85 % of IGF - I circulates in the TC and after the separation procedure , the amount of IGF - I in the BC or free was below the level of detection of our IGF - I assay . These results clearly demonstrate that in the affected patients the amount of IGF - I circulating in the TC is markedly elevated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Concentrations of endogenous insulin-like growth factor (IGF)-I in the different fractions obtained by neutral chromatography on a Superdex 200 column. Serum samples from the four siblings of family 1 were analyzed and compared to a non-related prepubertal control, as all siblings were prepubertal. The amount of IGF-I detected in the fractions corresponding to the ternary complex (TC) was markedly greater in the homozygous affected subjects (II·1 and II·3) compared to the unaffected siblings (II.2 and II.4) and the prepubertal control. No IGF-I was detected in the fractions corresponding to the binary complex (BC) or free IGF-I in any of the subjects. This is most likely due to the fact that under physiological conditions 80-85% of IGF-I circulates in the TC and after the separation procedure, the amount of IGF-I in the BC or free was below the level of detection of our IGF-I assay. These results clearly demonstrate that in the affected patients the amount of IGF-I circulating in the TC is markedly elevated.


A . Blood - stage growth rate of concavin ( - ) parasites in comparison to wild type . Data points represent parasites growing in individual mice , n indicates total number . P - value is calculated using the Mann Whitney test . Shown is the mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Blood-stage growth rate of concavin(-) parasites in comparison to wild type. Data points represent parasites growing in individual mice, n indicates total number. P-value is calculated using the Mann Whitney test. Shown is the mean ± SEM.


B . Gametocyte to ookinete conversion of 3 biological replicates . P - value calculated using the Mann Whitney test . Shown is the mean ± SEM . C . Average speed of moving ookinetes . Data points represent individual ookinetes , n indicates total number of cells from 3 biological replicates . P - values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison . D . Ookinete images of wild type and concavin ( - ) parasites revealing similar cell shapes . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O

B. Gametocyte to ookinete conversion of 3 biological replicates. P-value calculated using the Mann Whitney test. Shown is the mean ± SEM. C. Average speed of moving ookinetes. Data points represent individual ookinetes, n indicates total number of cells from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison. D. Ookinete images of wild type and concavin(-) parasites revealing similar cell shapes. Scale bar 5 µm.


E . Oocyst development of concavin ( - ) parasites compared to wild type . Data points represent individual midguts observed between d12 - 17 post infection from 3 independent cage feeds . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Oocyst development of concavin(-) parasites compared to wild type. Data points represent individual midguts observed between d12-17 post infection from 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by Dunns multiple comparison.


F . Mosquito infections resulted in deformed sporozoites in the salivary gland . Shown are example images of different sporozoites arranged to illustrate their rounding up over time . G . Quantification of sporozoite rounding up over time in the midgut ( MG ) the haemolymph ( HL ) and salivary gland ( SG ) at the indicated days post infection from three biological replicates . Sporozoites were classified as either normal , deformed or round as illustrated in F .

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

F. Mosquito infections resulted in deformed sporozoites in the salivary gland. Shown are example images of different sporozoites arranged to illustrate their rounding up over time. G. Quantification of sporozoite rounding up over time in the midgut (MG) the haemolymph (HL) and salivary gland (SG) at the indicated days post infection from three biological replicates. Sporozoites were classified as either normal, deformed or round as illustrated in F.


H . Average speed of salivary gland sporozoites . Data points represent individual sporozoites , n indicates total number from 3 biological replicates . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison 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

H. Average speed of salivary gland sporozoites. Data points represent individual sporozoites, n indicates total number from 3 biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


I . Selected trajectories of manually tracked sporozoites .

I. Selected trajectories of manually tracked sporozoites.


A . Oocyst development of concavin ( - ) parasites complemented with either the P . berghei gene or the P . falciparum orthologue fused to GFP . Data points represent individual midguts observed between d12 - 17 post infection of 3 independent cage feeds . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison 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

A. Oocyst development of concavin(-) parasites complemented with either the P. berghei gene or the P. falciparum orthologue fused to GFP. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


B . Average speed of salivary gland sporozoites . Data points represent individual sporozoites of 3 independent biological replicates . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison 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

B. Average speed of salivary gland sporozoites. Data points represent individual sporozoites of 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


C . Sum table of mosquito infections of wild type , concavin ( - ) and complemented lines expressing either P . berghei ( Pb ) or P . falciparum ( Pf ) concavin - GFP . Numbers determined on d17 post mosquito infection . Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections . Note the difference of normally shaped and total sporozoites for concavin ( - ) parasites . Images show infection of salivary glands . Scale bar : 100

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Sum table of mosquito infections of wild type, concavin(-) and complemented lines expressing either P. berghei (Pb) or P. falciparum (Pf) concavin-GFP. Numbers determined on d17 post mosquito infection. Infection rate contains 3 different countings of at least 20 mosquitoes from different mosquito infections. Note the difference of normally shaped and total sporozoites for concavin(-) parasites. Images show infection of salivary glands. Scale bar: 100 µm


D . Pb concavin - GFP and Pf concavin - GFP localization in blood and mosquito stages . Nuclei ( blue ) stained with Hoechst . Scale bar : 5 µm .

D. Pb concavin-GFP and Pf concavin-GFP localization in blood and mosquito stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.


E . Localization of P . berghei concavin - GFP in liver stages . Nuclei ( blue ) stained with Hoechst . Scale bar : 5 µm .

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E. Localization of P. berghei concavin-GFP in liver stages. Nuclei (blue) stained with Hoechst. Scale bar: 5 µm.


F . Immunofluorescence images of permeabilized and unpermeabilized concavin - GFP expressing salivary gland sporozoites stained with an anti - GFP antibody . Note that a GFP signal could only be detected after permeabilization , excluding concavin localization on the parasite surface as illustrated in the model . Scale bar : 5 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

F. Immunofluorescence images of permeabilized and unpermeabilized concavin-GFP expressing salivary gland sporozoites stained with an anti-GFP antibody. Note that a GFP signal could only be detected after permeabilization, excluding concavin localization on the parasite surface as illustrated in the model. Scale bar: 5 µm.


A , B . Time series of concavin - GFP ( A ) PhiL1 - GFP ( B ) sporozoites before and after bleaching and quantification of the fluorescence signal over time of 3 technical replicates . * indicates time of bleaching . Shown is the mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B. Time series of concavin-GFP (A) PhiL1-GFP (B) sporozoites before and after bleaching and quantification of the fluorescence signal over time of 3 technical replicates. * indicates time of bleaching. Shown is the mean ± SD.


C . Super resolution ( STED ) imaging of PhiL1 - GFP and CSP as well as concavin - GFP and CSP . Cells were stained with an anti - GFP antibody in combination with Atto - 594 ( green ) in addition to an anti - CSP staining in combination with Atto - 647 . Images were deconvolved using the Richardson - Lucy algorithm . The distance between the 2 signal peaks was measured using the plot profile of the respective channels in Fiji . Data points represent distance in individual sporozoites at the center of the cell from 2 independent biological replicates . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test . Scale bar : 1 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Super resolution (STED) imaging of PhiL1-GFP and CSP as well as concavin-GFP and CSP. Cells were stained with an anti-GFP antibody in combination with Atto-594 (green) in addition to an anti-CSP staining in combination with Atto-647. Images were deconvolved using the Richardson-Lucy algorithm. The distance between the 2 signal peaks was measured using the plot profile of the respective channels in Fiji. Data points represent distance in individual sporozoites at the center of the cell from 2 independent biological replicates. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test. Scale bar: 1 µm.


D . Localization of PhiL1 - GFP ( green ) in concavin ( - ) parasites . Nuclei ( blue ) stained with Hoechst . Scale bar 5 µm . E . Localization of SiR - Tubulin ( red ) in concavin ( - ) parasites . Nuclei ( blue ) stained with Hoechst . Scale bar 5 µm . F . Localization of CSP ( red ) in concavin ( - ) parasites . Nuclei ( blue ) stained with Hoechst . Scale bar 5 µm . Data information : Asterisks in D - F indicate apical part of the sporozoite .

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

D. Localization of PhiL1-GFP (green) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. E. Localization of SiR-Tubulin (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. F. Localization of CSP (red) in concavin(-) parasites. Nuclei (blue) stained with Hoechst. Scale bar 5 µm. Data information: Asterisks in D-F indicate apical part of the sporozoite.


B . Expression and localization of concavinC7A - GFP in blood stage parasites . Scale bar 5 µm .

B. Expression and localization of concavinC7A-GFP in blood stage parasites. Scale bar 5 µm.


C . Oocysts number in infected mosquitoes . Data points represent individual midguts observed between d12 - 17 post infection of 3 independent cage feeds . Shown is the mean ± SEM . P - value calculated using the Mann Whitney 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

C. Oocysts number in infected mosquitoes. Data points represent individual midguts observed between d12-17 post infection of 3 independent cage feeds. Shown is the mean ± SEM. P-value calculated using the Mann Whitney test.


D . Quantification of concavinC7A - GFP cell shapes at the indicated days from midgut ( MG ) or salivary gland ( SG ) derived sporozoites . Numbers above bars indicate investigated sporozoites .

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

D. Quantification of concavinC7A-GFP cell shapes at the indicated days from midgut (MG) or salivary gland (SG) derived sporozoites. Numbers above bars indicate investigated sporozoites.


E . Average speed of salivary gland sporozoites . Data points represent individual sporozoites from 3 independent biological replicates . Shown is the mean ± SEM . P - values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test .

E. Average speed of salivary gland sporozoites. Data points represent individual sporozoites from 3 independent biological replicates. Shown is the mean ± SEM. P-values are calculated using the Kruskal Wallis test followed by the Dunns multiple comparison test.


F . Localization of concavinC7A - GFP in normal and deformed sporozoites ( arrowheads point to deformations ) . Scale bar : 5 µm .

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F. Localization of concavinC7A-GFP in normal and deformed sporozoites (arrowheads point to deformations). Scale bar: 5 µm.