Figure 3 ( 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 . ( 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 .

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

Figure 3(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.(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.


Figure 4 ( 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 . ( 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 . ( 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 .

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

Figure 4(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.(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.(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.


Figure 5 ( 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 ) . ( 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 .

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

Figure 5(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).(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.


Figure 1Relative 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 . 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 . 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 .

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

Figure 1Relative 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.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.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.


Figure 2Expression 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 ) . 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 ) . 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 .

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

Figure 2Expression 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).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).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.


Figure 3HUVECs 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 . 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 . 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 blotHUVECs 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 .

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

Figure 3HUVECs 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.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.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 blotHUVECs 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.


Figure 4Transcriptome - 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 . 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 . Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation . Scale bars , 2000 μm .

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

Figure 4Transcriptome-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.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.Montage of representative images and quantification of 2D capillary network formation assays on HUVECs subjected to the above experimental manipulation. Scale bars, 2000 μm.


Figure 5Sanger 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 . 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 . 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 .

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

Figure 5Sanger 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.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.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.


Figure 1Box - 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 thresholdProtein - 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 assayProtein - 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 assayCo - 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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Figure 1Box-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 thresholdProtein-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 assayProtein-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 assayCo-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.


Figure 2Heat 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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Figure 2Heat 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.


Figure 3Time 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 resultsDiurnal 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 shadingWestern 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 controlBES1 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 controlBES1 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 antibodiesIn 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 .

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Figure 3Time 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 resultsDiurnal 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 shadingWestern 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 controlBES1 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 controlBES1 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 antibodiesIn 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.


Figure 4Col - 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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Figure 4Col-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.


Figure 5Col - 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 resultsActivation 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 resultsIncreased 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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Figure 5Col-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 resultsActivation 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 resultsIncreased 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 1Figure 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 1Figure 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.


Figure 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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Figure 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.


Figure 3 • 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 . • 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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Figure 3• 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.•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.


Figure 4 • 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4• 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.


Figure 1A . 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 . 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 . 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 .

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

Figure 1A. 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.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.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.


Figure 2A . 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 . 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 µmD . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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.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 µmD. 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.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.


Figure 3A , 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 . 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 .

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Figure 3A, 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.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.


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

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

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


Figure 6A . Growth curve of blood stage parasites in C57BL / 6 mice infected by the bite of 10 mosquitoes ( left ) or 10 . 000 sporozoites intra venously ( right ) at two different times post mosquito infection as indicated . Shown is the mean ± SEM . Mice infected with Concavin ( - ) parasites by bite represent 2 independent biological replicates with 3 mice each . Wild type bite back and Concavin ( - ) intra venous injections represent one biological replicate using 3 or 4 mice respectively . C . Liver cell invasion assay of concavin - gfp and concavin ( - ) parasites . Parasites are positively stained for CSP in case they remain extracellular . Both , normal and deformed parasites were detected intracellularly . Graph shows quantification of CSP positive ( red , hence extracellular ) and negative ( grey , hence intracellular ) sporozoites . Small graph shows the percentage of deformed and normally shaped intracellular concavin ( - ) sporozoites . Data points represent the 4 individual biological replicates with n indicating the numbers of sporozoites observed . Shown is the mean ± SEM . P - values are calculated using the Mann Whitney test . Scale bar : 5 µm . D . Liver - stage development of concavin ( - ) parasites compared to wildtype , both expressing cytoplasmic GFP ( white ) . Parasite size was measured 24h and 48h post infection . Data points represent individual parasites from 4 independent biological replicates . Shown is the mean ± SEM . P - values calculated using the Mann Whitney test . Scale bar : 5 µm . E . Sporozoite ejection of immobilized concavin ( - ) infected mosquitoes on glass slides and quantification of ejected sporozoites from 20 mosquitoes . * indicates individual sporozoites in the ejected saliva . Scale bar : 10 µm . F , G . Only normally shaped concavin ( - ) sporozoites released by salivary glands move on helical paths ( arrows ) through polyacrylamide gels that mimic the skin ( F ) . Quantification of two individual experiments Scale bar 10 µm . ( G ) : only normal shaped sporozoites were able to migrate through the gel .

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

Figure 6A. Growth curve of blood stage parasites in C57BL/6 mice infected by the bite of 10 mosquitoes (left) or 10.000 sporozoites intra venously (right) at two different times post mosquito infection as indicated. Shown is the mean ± SEM. Mice infected with Concavin(-) parasites by bite represent 2 independent biological replicates with 3 mice each. Wild type bite back and Concavin(-) intra venous injections represent one biological replicate using 3 or 4 mice respectively.C. Liver cell invasion assay of concavin-gfp and concavin(-) parasites. Parasites are positively stained for CSP in case they remain extracellular. Both, normal and deformed parasites were detected intracellularly. Graph shows quantification of CSP positive (red, hence extracellular) and negative (grey, hence intracellular) sporozoites. Small graph shows the percentage of deformed and normally shaped intracellular concavin(-) sporozoites. Data points represent the 4 individual biological replicates with n indicating the numbers of sporozoites observed. Shown is the mean ± SEM. P-values are calculated using the Mann Whitney test. Scale bar: 5 µm.D. Liver-stage development of concavin(-) parasites compared to wildtype, both expressing cytoplasmic GFP (white). Parasite size was measured 24h and 48h post infection. Data points represent individual parasites from 4 independent biological replicates. Shown is the mean ± SEM. P-values calculated using the Mann Whitney test. Scale bar: 5 µm.E. Sporozoite ejection of immobilized concavin(-) infected mosquitoes on glass slides and quantification of ejected sporozoites from 20 mosquitoes. * indicates individual sporozoites in the ejected saliva. Scale bar: 10 µm.F, G. Only normally shaped concavin(-) sporozoites released by salivary glands move on helical paths (arrows) through polyacrylamide gels that mimic the skin (F). Quantification of two individual experiments Scale bar 10 µm. (G): only normal shaped sporozoites were able to migrate through the gel.


Figure 7A . Percentage of mosquitoes depositing WT or concavin ( - ) sporozoites in the skin of a mouse during a bite ( left ) and sporozoites deposited during a mosquito bite ( right ) . P - values are calculated using the Fishers exact test ( left ) and the Mann Whitney test ( right ) . Bite transmission experiments were repeated over 3 independent mosquito infections for the KO and 2 independent mosquito infections for the WT . For each mosquito infection 2 - 3 mice were imaged , for a total of 3 - 5 ears . B . Maximum fluorescence intensity projections encoded by color for time from movies showing migrating sporozoites after mosquito - bite transmission . Graphs and camembert diagrams below show fraction of motile and immotile sporozoites after 10 , 20 and 30 minutes of recording , numbers analysed as well as sporozoite speed . Pooled data from 4 - 5 WT or 5 - 8 concavin ( - ) bite sites per time point . Scale bar : 50 µm . P - values are calculated using the Mann Whitney test . C . Motile WT or concavin ( - ) sporozoites entering blood vessels during the first hour following micro - injection . Number above bars shows numbers of sporozoites observed . Pooled data from four WT or five concavin ( - ) experiments . P - values are calculated using the Fishers exact test . D , E ( D ) Deformation and ( E ) disintegration of concavin ( - ) sporozoites migrating in the skin . Individual images corresponding to the frames shown on the right are indicated in distinct colours in the maximum projection ( left ) of 13 - min movies . Arrowheads indicate constrictions of the parasites . Graphs below time - lapse show that deformation and disintegration are preceded by a decrease in speed . Color of dots correspond to the time - points displayed in the time - lapse images . Scale bars : 10 µm . F . Percentage of deformation and disintegration events observed in 54 WT and 57 concavin ( - ) sporozoites that were tracked for at least 10 min . Pooled data from 5 WT or 10 concavin ( - ) bite sites .

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

Figure 7A. Percentage of mosquitoes depositing WT or concavin(-) sporozoites in the skin of a mouse during a bite (left) and sporozoites deposited during a mosquito bite (right). P-values are calculated using the Fishers exact test (left) and the Mann Whitney test (right). Bite transmission experiments were repeated over 3 independent mosquito infections for the KO and 2 independent mosquito infections for the WT. For each mosquito infection 2-3 mice were imaged, for a total of 3-5 ears.B. Maximum fluorescence intensity projections encoded by color for time from movies showing migrating sporozoites after mosquito-bite transmission. Graphs and camembert diagrams below show fraction of motile and immotile sporozoites after 10, 20 and 30 minutes of recording, numbers analysed as well as sporozoite speed. Pooled data from 4-5 WT or 5-8 concavin (-) bite sites per time point. Scale bar: 50 µm. P-values are calculated using the Mann Whitney test.C. Motile WT or concavin(-) sporozoites entering blood vessels during the first hour following micro-injection. Number above bars shows numbers of sporozoites observed. Pooled data from four WT or five concavin(-) experiments. P-values are calculated using the Fishers exact test.D, E (D) Deformation and (E) disintegration of concavin(-) sporozoites migrating in the skin. Individual images corresponding to the frames shown on the right are indicated in distinct colours in the maximum projection (left) of 13-min movies. Arrowheads indicate constrictions of the parasites. Graphs below time-lapse show that deformation and disintegration are preceded by a decrease in speed. Color of dots correspond to the time-points displayed in the time-lapse images. Scale bars: 10 µm.F. Percentage of deformation and disintegration events observed in 54 WT and 57 concavin(-) sporozoites that were tracked for at least 10 min. Pooled data from 5 WT or 10 concavin(-) bite sites.


Figure 1 ( c ) Substrate independent in vitro SUMOylation assay with SUMO2WT , SUMO2K11Ac or SUMO2K11Q was performed and analyzed by immunoblotting with the indicated antibodies . ( d ) Substrate independent in vitro SUMOylation assay using either SUMO2WT or SUMO2K11Ac in the presence of increasing amounts of ZNF451 - N . Reaction products were analyzed by immunoblotting with the indicated antibodies . Note that - when compared to ( c ) assays were done at reduced concentrations of E1 and E2 ( e ) In vitro SUMOylation assay using a fixed amount of SUMO2WT ( 220 ng ) with increasing concentrations of SUMO2K11Ac . Ratios ranged from 3 : 1 ( unmodified / K11Ac ) to 1 : 2 ( unmodified / K11Ac ) ( f ) In vitro SUMOylation assay , using SUMO2WT or SUMO2K11Ac , with p53 as a substrate . p53 was generated by in vitro translation / transcription in a rabbit reticulocyte lysate system . SUMO chain formation on p53 was analyzed by immunoblotting with α - p53 antibodies .

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Figure 1(c) Substrate independent in vitro SUMOylation assay with SUMO2WT, SUMO2K11Ac or SUMO2K11Q was performed and analyzed by immunoblotting with the indicated antibodies.(d) Substrate independent in vitro SUMOylation assay using either SUMO2WT or SUMO2K11Ac in the presence of increasing amounts of ZNF451-N. Reaction products were analyzed by immunoblotting with the indicated antibodies. Note that - when compared to (c) assays were done at reduced concentrations of E1 and E2(e) In vitro SUMOylation assay using a fixed amount of SUMO2WT (220 ng) with increasing concentrations of SUMO2K11Ac. Ratios ranged from 3:1 (unmodified/K11Ac) to 1:2 (unmodified/K11Ac)(f) In vitro SUMOylation assay, using SUMO2WT or SUMO2K11Ac, with p53 as a substrate. p53 was generated by in vitro translation/transcription in a rabbit reticulocyte lysate system. SUMO chain formation on p53 was analyzed by immunoblotting with α-p53 antibodies.


Figure 2 ( a ) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac ( 2 . 65 μM each ) with recombinant SIRT1 in the presence or absence of NAD + . ( b ) In vitro deacetylation of recombinant SUMO2WT , SUMO2K11Ac , SUMO2K33Ac and SUMO2K35Ac . Immunoblotting was performed with antibodies as indicated . ( c ) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 ( 0 . 74 µM , 1 . 5 μM , 3 µM ) , followed by immunoblotting with α - SUMO2K11Ac . ( d ) Combination of in vitro deacetylation and substrate independent in vitro SUMOylation with SUMO2WT and SUMO2K11Ac in the presence or absence of SIRT1 ( 1 . 5 μM ) . ( e ) In vitro deacetylation of SUMO2WT or SUMO2K11Ac with or without addition of SIRT1 , followed by substrate dependent in vitro SUMOylation of p53 . ( f ) Enrichment of acetylated proteins after SIRT1 inhibition in HeLa cells . Western blot analysis of SUMO2K11Ac after treatment of cells with the SIRT1 inhibitior NAM ( 10 mM ) for 5h and subsequent enrichment for acetylated proteins via acetyl - lysine immunoprecipitation . As a control cells were either treated with control siRNA or siSUMO2 / 3 . The samples were analyzed by immunoblotting with α - SUMO2 / 3 and α - SUMO2K11Ac antibodies . The α - SUMO2 / 3Ac and α - SUMO2K11Ac reactive species migrating at ~ 200 kDa are marked with an asterisk .

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Figure 2(a) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac (2.65 μM each) with recombinant SIRT1 in the presence or absence of NAD+.(b) In vitro deacetylation of recombinant SUMO2WT, SUMO2K11Ac, SUMO2K33Ac and SUMO2K35Ac. Immunoblotting was performed with antibodies as indicated.(c) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 (0.74 µM, 1.5 μM, 3 µM), followed by immunoblotting with α-SUMO2K11Ac.(d) Combination of in vitro deacetylation and substrate independent in vitro SUMOylation with SUMO2WT and SUMO2K11Ac in the presence or absence of SIRT1 (1.5 μM).(e) In vitro deacetylation of SUMO2WT or SUMO2K11Ac with or without addition of SIRT1, followed by substrate dependent in vitro SUMOylation of p53.(f) Enrichment of acetylated proteins after SIRT1 inhibition in HeLa cells. Western blot analysis of SUMO2K11Ac after treatment of cells with the SIRT1 inhibitior NAM (10 mM) for 5h and subsequent enrichment for acetylated proteins via acetyl-lysine immunoprecipitation. As a control cells were either treated with control siRNA or siSUMO2/3. The samples were analyzed by immunoblotting with α-SUMO2/3 and α-SUMO2K11Ac antibodies. The α-SUMO2/3Ac and α-SUMO2K11Ac reactive species migrating at ~200 kDa are marked with an asterisk.


Figure 3 ( b ) Western blot analysis with α - RGS - His and α - HA was performed to monitor poly - SUMOylation of HA - PML . The remaining samples were measured by LC - MS / MS and final data analysis was done with MaxQuant . A K11 SUMOylated SUMO2 peptide was identified and is depicted with an Andromeda score of 49 . 3 . Intensities for this SUMO2 peptide for the control ( gray ) and arsenic ( purple ) treated samples are blotted as a bar graph . The diagram shows a representative dataset derived from two replicates . The corresponding peptide is displayed below the graph . The SUMOylation site is marked with SUMO2 ( green ) and peptide fragment ions are indicated by y - ( red ) and b - ( blue ) . ( c ) The degradation of the PML reactive band ( ~ 120 kDa ) was determined by immunoblotting . HeLa cells were either mock transfected or transfected with siRNA directed against SUMO1 / 2 / 3 . The expression of RGS - His - tagged SUMO2WT or SUMO2K11Q was induced by DOX for 12 h . Prior to lysis , cells were treated with 1 μM As2O3 for 6 h . Western blot analysis for wild - type SUMO2 and SUMO2K11Q were blotted with α - RGS - His . Immunoblotting with α - Tubulin served as a loading control . Immunoblots for knockdown controls ( SUMO1 and SUMO2 / 3 ) can be

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

Figure 3(b) Western blot analysis with α-RGS-His and α-HA was performed to monitor poly-SUMOylation of HA-PML. The remaining samples were measured by LC-MS/MS and final data analysis was done with MaxQuant. A K11 SUMOylated SUMO2 peptide was identified and is depicted with an Andromeda score of 49.3. Intensities for this SUMO2 peptide for the control (gray) and arsenic (purple) treated samples are blotted as a bar graph. The diagram shows a representative dataset derived from two replicates. The corresponding peptide is displayed below the graph. The SUMOylation site is marked with SUMO2 (green) and peptide fragment ions are indicated by y- (red) and b- (blue).(c) The degradation of the PML reactive band (~ 120 kDa) was determined by immunoblotting. HeLa cells were either mock transfected or transfected with siRNA directed against SUMO1/2/3. The expression of RGS-His-tagged SUMO2WT or SUMO2K11Q was induced by DOX for 12 h. Prior to lysis, cells were treated with 1 μM As2O3 for 6 h. Western blot analysis for wild-type SUMO2 and SUMO2K11Q were blotted with α-RGS-His. Immunoblotting with α-Tubulin served as a loading control. Immunoblots for knockdown controls (SUMO1 and SUMO2/3) can be found


Figure 4 ( b ) Immunoblotting of samples with α - RGS - His ( SUMO variants ) and α - PCNA as loading control . ( c ) Bar graph , comparing the identified SUMO - SUMO2 linkages of control and heat shock samples for SUMO2WT ( gray ) and SUMO2K11Q ( purple ) . The relative abundance ( y - axis ) of the indicated SUMO - SUMO2 linkage sites ( x - axis ) are shown . The measured intensity for K11 in the SUMO2WT under control conditions was set to 100 % and the relative abundance for the individual residues was calculated in relation to this value . One of two independent replicates is shown .

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Figure 4(b) Immunoblotting of samples with α-RGS-His (SUMO variants) and α-PCNA as loading control.(c) Bar graph, comparing the identified SUMO-SUMO2 linkages of control and heat shock samples for SUMO2WT (gray) and SUMO2K11Q (purple). The relative abundance (y-axis) of the indicated SUMO-SUMO2 linkage sites (x-axis) are shown. The measured intensity for K11 in the SUMO2WT under control conditions was set to 100% and the relative abundance for the individual residues was calculated in relation to this value. One of two independent replicates is shown.


Figure 1A . ROCK2 immunohistochemistry stained sections of normal and stages I , II and III cases of human pancreas adenocarcinoma . Scale bar = 50 µm ( left ) . Histoscores of ROCK2 staining ( right ) in normal pancreas ( n = 5 ) , and pancreas adenocarcinoma stage I ( n = 22 ) , stage II ( n = 46 ) and stage III / IV ( n = 4 ) . One - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . B . Overall survival of 107 patients without alterations versus 37 patients with ROCK1 and / or ROCK2 gene amplification , significantly increased mRNA or truncation mutation from TCGA research network . Survival p value determined by Log - Rank test . C , D . Log2 median - centered ROCK1 or ROCK2 RNA expression in normal ( n = 39 ) vs PDAC ( n = 39 ) , normal ( n = 5 ) vs pancreatic adenocarcinoma ( PAC ) ( n = 12 ) , or normal ( n = 6 ) vs pancreatic carcinoma ( PC ) ( n = 11 ) samples from indicated studies . Exact p values determined by Mann - Whitney test . E . ROCK1 and ROCK2 mRNA expression in human PDAC ( n = 146 ) from the TCGA research network . Significance ( p value ) of slope deviation from 0 determined by Deming regression . F . Representative hematoxylin and eosin ( H & amp ; amp ; E ) and ROCK2 immunohistochemistry stained sections of normal mouse pancreas , acinar - to - ductal metaplasia ( ADM ) , pancreatic intraepithelial neoplasia ( PanIN ) stages 1 - 3 , and PDAC from indicated genotype mice . Scale bar = 50 µm . G . Quantification of ROCK2 staining in epithelial cells of normal , ADM / PanIN1 ( KC ) , PanIN2 ( KPC ) , PanIN3 ( KPC ) and PDAC ( KPC ) pancreatic tissue ( n = 5 mice per group ) . One - way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test . H . Survival analysis of Pdx1 - Cre ; LSL - KRasG12D / + ; LSL - Trp53R172H / + ; LSL - ROCK2 : ER ( RKPC ) mice without ( n = 21 ) or with conditional ROCK activation with tamoxifen citrate ( n = 19 ) . Survival p value determined by Log - Rank test .

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

Figure 1A. ROCK2 immunohistochemistry stained sections of normal and stages I, II and III cases of human pancreas adenocarcinoma. Scale bar = 50 µm (left). Histoscores of ROCK2 staining (right) in normal pancreas (n=5), and pancreas adenocarcinoma stage I (n=22), stage II (n=46) and stage III/IV (n=4). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.B. Overall survival of 107 patients without alterations versus 37 patients with ROCK1 and/or ROCK2 gene amplification, significantly increased mRNA or truncation mutation from TCGA research network. Survival p value determined by Log-Rank test.C, D. Log2 median-centered ROCK1 or ROCK2 RNA expression in normal (n=39) vs PDAC (n=39), normal (n=5) vs pancreatic adenocarcinoma (PAC) (n=12), or normal (n=6) vs pancreatic carcinoma (PC) (n=11) samples from indicated studies. Exact p values determined by Mann-Whitney test.E. ROCK1 and ROCK2 mRNA expression in human PDAC (n=146) from the TCGA research network. Significance (p value) of slope deviation from 0 determined by Deming regression.F. Representative hematoxylin and eosin (H&amp;E) and ROCK2 immunohistochemistry stained sections of normal mouse pancreas, acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) stages 1-3, and PDAC from indicated genotype mice. Scale bar = 50 µm.G. Quantification of ROCK2 staining in epithelial cells of normal, ADM/PanIN1 (KC), PanIN2 (KPC), PanIN3 (KPC) and PDAC (KPC) pancreatic tissue (n=5 mice per group). One-way ANOVA with multiplicity adjusted exact p value by post hoc Tukey multiple comparison test.H. Survival analysis of Pdx1-Cre; LSL-KRasG12D/+; LSL-Trp53R172H/+; LSL-ROCK2:ER (RKPC) mice without (n=21) or with conditional ROCK activation with tamoxifen citrate (n=19). Survival p value determined by Log-Rank test.


Figure 2A . H & amp ; amp ; E stained sections of cell invasion into collagen matrix after 8 days . Scale bar = 100 µm . Invasion index of KPC cells in the presence or absence of 10 µM H1152 ROCK inhibitor . Means ± SEM ( n = 9 for untreated , n = 8 for H1152 ) , p value by unpaired t test . B . Schematic representation of ROCK domains ( RBD , Rho binding domain ; PH , pleckstrin homology domain ; CR , cysteine - rich ) . Conditionally - activated human ROCK1 , human ROCK2 and GFP control fusion proteins ( EGFP , enhanced green fluorescent protein ; hbER , estrogen receptor hormone binding domain ) were expressed in KPflC mouse PDAC cells and blotted with anti - GFP antibody . C . KPflC cells expressing GFP : ER , ROCK1 : ER or ROCK2 : ER fusion proteins were treated for 24 h with EtOH vehicle or 1 µM 4HT in the presence or absence of 1 µM or 10 µM H1152 . Immunoblotting shows endogenous ROCK1 and ROCK2 , ER - fusions , and phosphorylation of MLC2 ( T18S19 ) . Total MLC ( MRCL3 / MRLC2 / MYL9 ) and glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) were blotted as loading controls . D . H & amp ; amp ; E stained sections of cell invasion into collagen matrix after 8 days . Scale bar = 100 µm . Invasion index of KPflC cells treated with 1 µM 4HT . Means ± SEM ( n = 6 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett ' s multiple comparison test . E . Cell proliferation determined by Ki67 immunofluorescence . Scale bar : 20 µm . F . Quantification of cell number at the collagen matrix surface per 0 . 046 mm2 field . Means ± SEM ( n = 30 ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett ' s multiple comparison test . G . Ki67 positive cell percentages at the surface and within the collagen matrix . Means ± SEM ( n = 30 ; n = 12 for GFP : ER / Matrix ) , one - way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett ' s multiple comparison test . H . Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle ( - ) or 4HT on uncoated plastic surfaces . Means ± SEM ( n = 3 ) . I . Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle ( - ) or 4HT on collagen1 - coated surfaces . Means ± SEM ( n = 3 ) .

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

Figure 2A. H&amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPC cells in the presence or absence of 10 µM H1152 ROCK inhibitor. Means ± SEM (n=9 for untreated, n=8 for H1152), p value by unpaired t test.B. Schematic representation of ROCK domains (RBD, Rho binding domain; PH, pleckstrin homology domain; CR, cysteine-rich). Conditionally-activated human ROCK1, human ROCK2 and GFP control fusion proteins (EGFP, enhanced green fluorescent protein; hbER, estrogen receptor hormone binding domain) were expressed in KPflC mouse PDAC cells and blotted with anti-GFP antibody.C. KPflC cells expressing GFP:ER, ROCK1:ER or ROCK2:ER fusion proteins were treated for 24 h with EtOH vehicle or 1 µM 4HT in the presence or absence of 1 µM or 10 µM H1152. Immunoblotting shows endogenous ROCK1 and ROCK2, ER-fusions, and phosphorylation of MLC2 (T18S19). Total MLC (MRCL3/MRLC2/MYL9) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were blotted as loading controls.D. H&amp;E stained sections of cell invasion into collagen matrix after 8 days. Scale bar = 100 µm. Invasion index of KPflC cells treated with 1 µM 4HT. Means ± SEM (n=6), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.E. Cell proliferation determined by Ki67 immunofluorescence. Scale bar: 20 µm.F. Quantification of cell number at the collagen matrix surface per 0.046 mm2 field. Means ± SEM (n=30), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.G. Ki67 positive cell percentages at the surface and within the collagen matrix. Means ± SEM (n=30; n=12 for GFP:ER/Matrix), one-way ANOVA with multiplicity adjusted exact p value by post hoc Dunnett's multiple comparison test.H. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on uncoated plastic surfaces. Means ± SEM (n=3).I. Viable cell relative to starting cell numbers were determined after 24 h or 48 h treatment with vehicle (-) or 4HT on collagen1-coated surfaces. Means ± SEM (n=3).