( B ) Following study , mice were sacrificed their organs were obtained for histopathological assessment . Hematoxylin Eosin was carried out on tissue samples .

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(B) Following the study, mice were sacrificed and their organs were obtained for histopathological assessment. Hematoxylin and Eosin staining was carried out on the tissue samples.


, Scaled reduced cost for uptake rates of at different growth rates . Colour represents scaled reduced cost value . A higher value indicates a greater impact of a given increase in acid uptake on growth rate .

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

, Scaled reduced cost analysis for uptake rates of amino acids at different growth rates. Colour represents the scaled reduced cost value. A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate.


, fold changes of CcpA mutant over wild type . Log2 fold changes were calculated from LFQ values p values were calculated on 2 - sided two sample t - with a FDR threshold of 0 . 05 S 0 equal to 1 ( Tusher et al , 2001 ) . outside significance lines ( FDR = 0 . 05 , S 0 = 1 ) are significantly changed in expression . Example sets of are highlighted are labeled with their gene names .

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

, Protein fold changes of the CcpA mutant over wild type. Log2 fold changes were calculated from LFQ values p values were calculated based on 2-sided two sample t-tests with a FDR threshold of 0.05 and S0 equal to 1 (Tusher et al, 2001). Proteins outside the significance lines (FDR = 0.05, S0 = 1) are significantly changed in expression. Example sets of proteins are highlighted and are labeled with their gene names.


( C ) Change in expression of 36 genes defining host - targeted anti - viral signature . log2 fold change at 24 hours post SARS - CoV - 2 infection from A549 are shown .

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

(C) Change in the expression levels of 36 genes defining the host-targeted anti-viral signature. log2 fold change at 24 hours post SARS-CoV-2 infection from A549 cells are shown.


( D ) Change in expression of 17 genes defining anti - cytokine signature ; log2 fold change at 24 hours post high SARS - CoV - 2 infection from A549 - ACE2 are shown .

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

(D) Change in the expression levels of 17 genes defining anti-cytokine signature; log2 fold change at 24 hours post high SARS-CoV-2 infection from A549-ACE2 cells are shown.


( A ) Representative of Mock , SARS - CoV - 2 infected ( Ctrl ) , Salmeterol - treated wells analyzed with Multiwavelength Scoring application in MetaXpress . Grayscales of were adjusted to enable direct comparison of relative of among treatments : Segmentation show how were segmented identified as spike positive . Purple , nuclei ; cyan , spike . Scale bar , 100 μm .

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

(A) Representative fluorescence images of Mock, SARS-CoV-2 infected (Ctrl), and Salmeterol-treated wells analyzed with the Multiwavelength Cell Scoring application in MetaXpress. Grayscales of the images were adjusted to enable direct comparison of the relative levels of fluorescence among the treatments: Segmentation images show how cells were segmented and identified as spike positive. Purple, nuclei; cyan, spike. Scale bar, 100 μm.


( B ) Violin plots of Vero - E6 labeled for Spike . Multi - wavelength Scoring algorithm in MetaXpress was used to determine integrated signal in as a measure of amount of Spike within each . plots show population distribution of integrated signal for of treatments . Boxes in plot show interquartile range ( IQR ) with top bottom edges marking 75th 25th percentiles , respectively . horizontal in box is median value , whiskers are defined to be 1 . 5 IQR . ordinate is a log scale . effect of treatment is assessed quantitatively by changes in median signal level , qualitatively by observing changes in modes . dashed is 3 standard deviations above mean signal in Mock samples is used as a cut - off to quantify number of that are positive or negative for Spike signal . statistics table below plots shows number of counted in each treatment group , median of population .

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

(B) Violin plots of Vero-E6 cells labeled for Spike protein. The Multi-wavelength Cell Scoring algorithm in MetaXpress was used to determine the integrated fluorescent signal in individual cells as a measure of the amount of Spike protein within each cell. The plots show the population distribution of the integrated signal for all of the treatments. The Boxes in the plot show the interquartile range (IQR) with the top and bottom edges marking the 75th and 25th percentiles, respectively. The horizontal line in the box is the median value, and the whiskers are defined to be 1.5 IQR. The ordinate is a log scale. The effect of the treatment is assessed quantitatively by changes in the median signal level, and qualitatively by observing changes in the modes. The dashed line is 3 standard deviations above the mean signal in the Mock samples and is used as a cut-off to quantify the number of cells that are positive or negative for the Spike signal. The statistics table below the plots shows the number of cells counted in each treatment group, and the median of the population.


HEK293 acceptor transfected with or without ACE2 TMPRSS2 were seeded in 384 well plates , pretreated with 7 - point gradients of compounds for 1 - 2 h , co - cultured for 4 hours with HEK293 donor expressing SARS - CoV - 2 spike GFP , or donor expressing GFP only ( no spike ) . of GFP - positive objects were acquired on a confocal high - content imager analyzed for syncytia formation integrated GFP ( total GFP ) as a measure of cytotoxicity , using a CNT algorithm as described in Materials Methods . Data are aggregate of 8 independent biological repeats ; where errors are shown they represent SD from matching concentrations in at least three experiments .

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

HEK293 acceptor cells transfected with or without ACE2 and TMPRSS2 were seeded in 384 well plates, pretreated with 7-point gradients of test compounds for 1-2 h, and co-cultured for 4 hours with HEK293 donor cells expressing SARS-CoV-2 spike and GFP, or donor cells expressing GFP only (no spike). Images of GFP-positive objects were acquired on a confocal high-content imager and analyzed for syncytia formation and integrated GFP area (total GFP) as a measure of cytotoxicity, using a CNT algorithm as described in the Materials and Methods. Data are the aggregate of 8 independent biological repeats; where errors are shown they represent SD from matching concentrations in at least three experiments.


V . cholerae wild - type ΔrpoE strains carrying indicated plasmids were grown to early stationary phase ( OD600 of 1 . 5 ) induced with L - arabinose ( 0 . 2 % final conc . ) . Expression of MicV VrrA was monitored on Northern blots . 5S rRNA served as loading control .

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V. cholerae wild-type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase (OD600 of 1.5) and induced with L-arabinose (0.2% final conc.). Expression of MicV and VrrA was monitored on Northern blots. 5S rRNA served as loading control.


V . cholerae wild - type , ΔvchM ΔvchM ΔrpoE strains harboring PmicV : : mKate2 plasmids were grown in M9 medium . Samples were collected during various growth phases analyzed for .

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V. cholerae wild-type, ΔvchM and ΔvchM ΔrpoE strains harboring PmicV::mKate2 plasmids were grown in M9 medium. Samples were collected during various growth phases and analyzed for fluorescence.


V . cholerae wild - type ΔrpoE strains carrying PmicV : : mKate2 plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) treated with ethanol ( 3 . 5 % final conc . ) , or water . was determined 180 min after ethanol treatment mKate2 of mock - treated samples were set to 1 .

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V. cholerae wild-type and ΔrpoE strains carrying PmicV::mKate2 plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.


V . cholerae wild - type ΔrpoE strains carrying PvrrA : : mKate2 ( E ) plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) treated with ethanol ( 3 . 5 % final conc . ) , or water . was determined 180 min after ethanol treatment mKate2 of mock - treated samples were set to 1 .

B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O

V. cholerae wild-type and ΔrpoE strains carrying PvrrA::mKate2 (E) plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.


V . cholerae wild - type , ΔmicV , ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0 . 2 treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates CFU / ml were determined .

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V. cholerae wild-type, ΔmicV, ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


V . cholerae ΔvrrA ΔmicV strains carrying pBAD - micV , pBAD - vrrA or an empty vector control ( pCtr ) were cultivated to early stationary phase ( OD600 of 1 . 5 ) in LB . were treated with L - arabinose ( 0 . 2 % final conc . ) samples were collected at indicated time points after induction . Northern blot was performed to determine VrrA , MicV ompT . 5S rRNA served as loading control . For comparison , samples of a wild - type carrying pCtr were collected during various growth phases , which indicated ~ 18 - fold ~ 7 - fold higher of VrrA MicV expressed from pBAD plasmids , respectively ( see Source data for quantifications ) .

B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O

V. cholerae ΔvrrA ΔmicV strains carrying pBAD-micV, pBAD-vrrA or an empty vector control (pCtr) were cultivated to early stationary phase (OD600 of 1.5) in LB. Cells were treated with L-arabinose (0.2% final conc.) and RNA samples were collected at the indicated time points after induction. Northern blot analysis was performed to determine VrrA, MicV and ompT levels. 5S rRNA served as loading control. For comparison, RNA samples of a wild-type strain carrying pCtr were collected during various growth phases, which indicated ~18-fold and ~7-fold higher levels of VrrA and MicV expressed from the pBAD plasmids, respectively (see Source data for quantifications).


V . cholerae ΔvrrA ΔmicV strains carrying indicated reporter plasmids ( x - axis ) either an empty vector control ( pCtr ) , pMicV , or pVrrA plasmid , were cultivated in M9 GFP was measured . of control strains was set to 1 . target genes were classified according to ( B ) : regulated by both

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V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to (B): regulated by both sRNAs


V . cholerae ΔvrrA ΔmicV strains carrying indicated reporter plasmids ( x - axis ) either an empty vector control ( pCtr ) , pMicV , or pVrrA plasmid , were cultivated in M9 GFP was measured . of control strains was set to 1 . target genes were classified according to regulated only by

B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by MicV


V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by VrrA


V . cholerae ΔvrrA ΔmicV strains carrying ompT : : gfp or ompT M1 * : : gfp fusions ( D ) , ushA : : gfp or ushA M1 * : : gfp fusions ( E ) , or lpp : : gfp or lpp M2 * : : gfp fusions ( F ) an empty vector control ( pCtr ) , micV expression plasmids ( pMicV , pMicV M1 ) or vrrA expression plasmids ( pVrrA , pVrrA M2 ) were grown in M9 GFP was measured . M1 M2 denote mutations indicated in ( A , B , C ) . of control strains was set to 1 .

B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

V. cholerae ΔvrrA ΔmicV strains carrying the ompT::gfp or ompT M1*::gfp fusions (D), ushA::gfp or ushA M1*::gfp fusions (E), or lpp::gfp or lpp M2*::gfp fusions (F) and an empty vector control (pCtr), the micV expression plasmids (pMicV, pMicV M1) or the vrrA expression plasmids (pVrrA, pVrrA M2) were grown in M9 and GFP fluorescence was measured. M1 and M2 denote the mutations indicated in (A, B, C). Fluorescence of the control strains was set to 1.


V . cholerae ΔrpoE , ΔrpoE ΔvrrA , ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD - rpoE or an empty vector control ( pCtr ) were grown to OD600 of 1 . 5 L - arabinose ( 0 . 2 % final conc . ) was added . samples were collected at indicated time points monitored for ompT ( G ) , ushA ( H ) , or lpp ( I ) using qRT - PCR .

B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

V. cholerae ΔrpoE, ΔrpoE ΔvrrA, ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD-rpoE or an empty vector control (pCtr) were grown to OD600 of 1.5 and L-arabinose (0.2% final conc.) was added. RNA samples were collected at the indicated time points and monitored for ompT (G), ushA (H), or lpp (I) levels using qRT-PCR.


V . cholerae ΔvrrA ΔmicV strains carrying ompT : : 3XFLAG gene pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to an OD600 of 2 . 0 . samples were collected analyzed for MicV , VrrA RybB expression on Northern blots . OmpT : : 3xFLAG production was tested on Western blots . RNAPα 5S rRNA served as loading controls for Western Northern blots , respectively .

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

V. cholerae ΔvrrA ΔmicV strains carrying the ompT::3XFLAG gene and pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to an OD600 of 2.0. RNA and protein samples were collected and analyzed for MicV, VrrA and RybB expression on Northern blots. OmpT::3xFLAG production was tested on Western blots. RNAPα and 5S rRNA served as loading controls for Western and Northern blots, respectively.


E . coli wild - type strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were grown in LB to an OD600 of 2 . 0 . samples were collected investigated on Northern blots SDS - PAGE , respectively . For comparison , we included E . coli insertional mutant strains ompA : : kanR ompC : : kanR for specific assignment of OmpA OmpC bands .

B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O B-SMALL_MOLECULE B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD O O

E. coli wild-type strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and investigated on Northern blots and SDS-PAGE, respectively. For comparison, we included the E. coli insertional mutant strains ompA::kanR and ompC::kanR for specific assignment of OmpA and OmpC bands.


V . cholerae wild - type ΔrpoE strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to OD600 of 0 . 2 treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates CFU / ml were determined .

B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

V. cholerae wild-type and ΔrpoE strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


V . cholerae wild - type ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or sRNA library after consecutive selection experiments ( Sel1 , Sel2 , Sel3 ) were grown in LB to OD600 of 0 . 2 . were treated with ethanol ( 3 . 5 % final conc . ) for 6 hours . Serial dilutions were prepared spotted onto agar plates . R1 R2 indicate two independent biological replicates .

B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library after consecutive selection experiments (Sel1, Sel2, Sel3) were grown in LB to OD600 of 0.2. Cells were treated with ethanol (3.5% final conc.) for 6 hours. Serial dilutions were prepared and spotted onto agar plates. R1 and R2 indicate two independent biological replicates.


V . cholerae wild - type ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or sRNA library before ( input ) or after consecutive ethanol selection experiments ( Sel1 , Sel2 , Sel3 ) were cultivated in LB to OD600 of 2 . 0 . fractions were analyzed by SDS - PAGE . indicated bands were analyzed by mass spectrometry .

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

V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library before (input) or after consecutive ethanol selection experiments (Sel1, Sel2, Sel3) were cultivated in LB to OD600 of 2.0. Membrane fractions were analyzed by SDS-PAGE. The indicated bands were analyzed by mass spectrometry.


V . cholerae ΔvrrA ΔmicV expressing ompA : : 3xFLAG gene carrying an empty vector control ( pCtr ) , or plasmids producing 15 most highly enriched sRNA variants ( sRNA variants 1 - 15 ) were grown in LB to an OD600 of 2 . 0 . samples were collected tested for sRNA OmpA : : 3xFLAG expression on Northern Western blots , respectively ( with RNAPα 5S rRNA as loading controls ) .

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

V. cholerae ΔvrrA ΔmicV cells expressing the ompA::3xFLAG gene and carrying an empty vector control (pCtr), or plasmids producing the 15 most highly enriched sRNA variants (sRNA variants 1-15) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and tested for sRNA and OmpA::3xFLAG expression on Northern and Western blots, respectively (with RNAPα and 5S rRNA as loading controls).


V . cholerae ΔvrrA ΔmicV strains carrying ompA : : gfp fusion an empty vector control ( pCtr ) or enriched sRNA expression plasmids were grown in M9 GFP was measured . of control strains was set to 1 .

B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

V. cholerae ΔvrrA ΔmicV strains carrying the ompA::gfp fusion and an empty vector control (pCtr) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1.


V . cholerae wild - type , ΔrpoE , ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0 . 2 treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates CFU / ml were determined .

V. cholerae wild-type, ΔrpoE, ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


( A ) Immunoblot showing activation of PI3K / AKT pathway ( by assessing of p - AKT Ser473 ) of ECs 24h after induction of Pik3caH1047R expression .

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(A) Immunoblot showing the activation of PI3K/AKT pathway (by assessing the levels of p-AKT Ser473) of ECs 24h after induction of Pik3caH1047R expression.


( B ) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression . Differentially expressed genes ( DEGs ) ( p adj < 0 . 05 ) in Pik3caH1047R over Pik3caWT ECs at 24h ( grey , unchanged ; orange , downregulated ; purple , upregulated ) . n = 4 biological replicates per genotype .

O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE 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) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression. Differentially expressed genes (DEGs) (p adj < 0.05) in Pik3caH1047R over Pik3caWT ECs at 24h (grey, unchanged; orange, downregulated; purple, upregulated). n=4 biological replicates per genotype.


( D ) Immunoblot showing activation of PI3K / AKT pathway of Pik3caWT Pik3caH1047R ECs after 2h starvation ( - ) 30 min stimulation with 20 % bovine serum ( FBS ) ( + ) .

O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD B-CELL_TYPE O O O O O O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O O

(D) Immunoblot showing the activation of PI3K/AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation (-) and 30 min stimulation with 20% fetal bovine serum (FBS) (+).


( E , F ) ( E ) cycle by flow cytometry of Pik3caWT Pik3caH1047R ECs after cultured either without serum ( - ) or 20 % serum ( + ) for 24h . ( F ) of proliferative ECs ( in S G2M phases ) from Fig 1E . Error bars are s . e . m . n ≥ 3 biological replicates per genotype . Statistical was performed by nonparametric Mann - Whitney . * * p < 0 . 01 * * * * p < 0 . 0001 were considered statistically significant .

O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD B-CELL_TYPE O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E, F) (E) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum (-) or 20% serum (+) for 24h. (F) Percentage of proliferative ECs (in S and G2M phases) from Fig 1E. Error bars are s.e.m. n ≥ 3 biological replicates per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 and ****p < 0.0001 were considered statistically significant.


( B ) Representative of control EC - Pik3caH1047R / WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels . Scale bars : 150 μm .

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(B) Representative images of control and EC-Pik3caH1047R/WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels. Scale bars: 150 μm.


( D ) of IB4 - positive areas per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype .

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(D) Quantification of IB4-positive areas per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype.


( E ) Representative of EC - mTmG mouse retinas isolated at indicated time points stained with IB4 GFP . Scale bars : 150 μm . ( F ) of GFP / IB4 per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical was performed by nonparametric Mann - Whitney . * p < 0 . 05 was considered statistically significant .

O O O O O B-CELL_TYPE O O B-ORGANISM B-TISSUE O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative images of EC-mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP. Scale bars: 150 μm. (F) Quantification of GFP/IB4 ratio per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


( A , B ) Representative of EC - Pik3caH1047R / WT ( A ) EC - mTmG ( B ) P6 retinas from mice treated with decreasing doses of 4 - OHT on P1 . Retinas were stained for blood vessels ( IB4 ) GFP as indicated . Scale bars : 150 μm .

O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O B-TISSUE O B-ORGANISM O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE O B-EXP_ASSAY O B-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O O O O O O O

(A, B) Representative images of EC-Pik3caH1047R/WT (A) and EC-mTmG (B) P6 retinas from mice treated with decreasing doses of 4-OHT on P1. Retinas were stained for blood vessels (IB4) and GFP as indicated. Scale bars: 150 μm.


( C ) of GFP / IB4 of EC - mTmG retinas . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical was performed by nonparametric Mann - Whitney . * p < 0 . 05 was considered statistically significant .

O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of GFP/IB4 ratio of EC-mTmG retinas. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


( D ) of IB4 - positive per retina in Pik3caH1047R / WT retinas . Data presented as a of control for each 4 - OHT dose . Error bars are s . e . m . n = 4 retinas per genotype . Data information : Statistical was performed by nonparametric Mann - Whitney . * p < 0 . 05 was considered statistically significant .

O O O O B-GENEPROD O O O B-TISSUE O B-GENEPROD O O B-TISSUE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of IB4-positive area per retina in Pik3caH1047R/WT retinas. Data presented as a percentage of the control for each 4-OHT dose. Error bars are s.e.m. n = 4 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


( A ) Representative of EC - Pik3caH1047R / WT P6 retinas isolated from mice treated with 0 . 125 mg / kg of 4 - OHT on P1 . Retinas were immunostained for blood vessels ( IB4 ) . Red asterisks show arteries arterioles yellow arrowheads veins . Scale bars : 150 μm ( left panel ) 30 μm ( right , high magnification panels ) . ( B ) Representative of P6 retinas from control EC - Pik3caH1047R / WT mice treated with 0 . 125 mg / kg 4 - OHT on P1 , following immunostaining for p - S6 ( S235 / 236 ) blood vessels ( IB4 ) . Scale bars : 150 μm ( left panels ) 30 μm ( right panels , high magnification ) . ( C ) Representative control EC - Pik3caH1047R / WT P6 retinas immunostained for blood vessels ( IB4 ) , EC nuclei ( Erg ) EdU . Scale bars : 150 μm ( left panels ) 30 μm ( right panels , high magnification ) . ( D - G ) of ( D ) p - S6 ( S235 / 236 ) ( presented as a fold change of vehicle - treated control ) , ( E ) EC proliferation by EdU , ( F ) EC number by Erg positive ( G ) retinal vascularity by IB4 - positive in control EC - Pik3caH1047R / WT retinas . Error bars are s . e . m . n > 5 retinas per genotype . Statistical was performed by nonparametric Mann - Whitney . * * p < 0 . 01 was considered statistically significant .

O O O O O B-CELL_TYPE O B-GENEPROD O O O B-TISSUE O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-TISSUE O B-EXP_ASSAY O B-TISSUE I-TISSUE O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O B-TISSUE O O 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-TISSUE O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O B-TISSUE B-EXP_ASSAY O B-TISSUE I-TISSUE O B-GENEPROD O O B-CELL_TYPE B-SUBCELLULAR O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O B-GENEPROD O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of EC-Pik3caH1047R/WT P6 retinas isolated from mice treated with 0.125 mg/kg of 4-OHT on P1. Retinas were immunostained for blood vessels (IB4). Red asterisks show arteries and arterioles and yellow arrowheads veins. Scale bars: 150 μm (left panel) and 30 μm (right, high magnification panels). (B) Representative images of P6 retinas from control and EC-Pik3caH1047R/WT mice treated with 0.125 mg/kg 4-OHT on P1, following immunostaining for p-S6 (S235/236) and blood vessels (IB4). Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (C) Representative control and EC-Pik3caH1047R/WT P6 retinas immunostained for blood vessels (IB4), EC nuclei (Erg) and EdU. Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (D-G) Quantification of (D) p-S6 (S235/236) intensity (presented as a fold change of vehicle-treated control), (E) EC proliferation by EdU staining, (F) EC number by Erg positive cells and (G) retinal vascularity by IB4-positive area in control and EC-Pik3caH1047R/WT retinas. Error bars are s.e.m. n > 5 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 was considered statistically significant.


( B , C ) Representative of P6 retinas isolated from control EC - Pik3caH1047R / WT mouse . Blood vessels were stained with IB4 . Lower panels showing high magnification of representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panel ) 30 μm ( lower panels ) . ( D - G ) of ( D ) retinal vascularity by IB4 , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU , ( G ) pS6 ( S235 / 236 ) ( presented as a fold change of vehicle - treated control ) . Bars represent mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical was performed by nonparametric Mann - Whitney . * p < 0 . 05 * * p < 0 . 01 were considered statistically significant .

O O O O O O O O B-TISSUE O O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O B-TISSUE I-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O B-CELL_TYPE B-SUBCELLULAR O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD B-EXP_ASSAY O O O O B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O 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, C) Representative images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panel) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


( B , C ) Representative confocal of P6 retinas isolated from control EC - Pik3caH1047R / WT mouse . Blood vessels were stained with IB4 . Lower panels showing high magnification of representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) 30 μm ( lower panels ) . ( D - G ) of ( D ) retinal vascularity by IB4 , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU , ( G ) pS6 ( S235 / 236 ) ( presented as a fold change of vehicle - treated control ) . Bars represent mean ± s . e . m . n ≥ 3 retinas per genotype . Statistical was performed by nonparametric Mann - Whitney . * p < 0 . 05 * * p < 0 . 01 were considered statistically significant .

O O O O O O B-EXP_ASSAY O O B-TISSUE O O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O B-TISSUE I-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O B-CELL_TYPE B-SUBCELLULAR O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD B-EXP_ASSAY O O O O B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O 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, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 3 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


( B , C ) Representative confocal of P6 retinas isolated from control EC - Pik3caH1047R / WT mouse . Blood vessels were stained with IB4 . Lower panels showing high magnification of representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) 30 μm ( lower panels ) . ( D - G ) of ( D ) retinal vascularity by IB4 , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU , ( G ) pS6 ( S235 / 236 ) ( presented as a fold change of vehicle - treated control ) . Bars represent mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical was performed by nonparametric Mann - Whitney . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 were considered statistically significant .

O O O O O O B-EXP_ASSAY O O B-TISSUE O O O B-CELL_TYPE O B-GENEPROD O O B-ORGANISM O B-TISSUE I-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-TISSUE I-TISSUE O B-GENEPROD O O B-CELL_TYPE B-SUBCELLULAR O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_TYPE O O B-GENEPROD B-EXP_ASSAY O O O O B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O 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, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.


( B ) Representative sequencing chromatograms of PIK3CA TEK mutant VM - derived ECs . Arrows show detected point mutations .

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

(B) Representative sequencing chromatograms of PIK3CA and TEK mutant VM-derived ECs. Arrows show the detected point mutations.


( C ) Representative confocal of PIK3CA TEK - derived ECs immunostained for VE - cadherin ( EC - specific junctional ) ERG ( EC - specific transcription factor ) . nuclei were visualised with DAPI . Scale bars : 30 μm .

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

(C) Representative confocal images of PIK3CA and TEK patient-derived ECs immunostained for VE-cadherin (EC-specific junctional protein) and ERG (EC-specific transcription factor). Cell nuclei were visualised with DAPI. Scale bars: 30 μm.


( D ) Immunoblot showing activation of PI3K / AKT / mTORC1 pathway ( by assessing of p - AKT p - S6 ) among different PIK3CA TEK - derived ECs . HUVEC were used as wild - type control .

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

(D) Immunoblot showing the activation of PI3K/AKT/mTORC1 pathway (by assessing the levels of p-AKT and p-S6) among different PIK3CA and TEK patient-derived ECs. Primary HUVEC were used as wild-type control.


( E - F ) Immunoblot showing impact of miransertib at increasing doses on PI3K / AKT / mTORC1 pathway ( by assessing p - AKT p - S6 ) in ( E ) PIK3CA ( F ) TEK mutant - derived ECs .

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(E-F) Immunoblot showing the impact of miransertib at increasing doses on PI3K/AKT/mTORC1 pathway (by assessing p-AKT and p-S6 levels) in (E) PIK3CA and (F) TEK mutant patient-derived ECs.


( G ) Wild - type ( HUVECs ) , PIK3CA TEK mutant EC viability upon treatment with miransertib for 72h at different doses assessed by MTS . Fitting curves 95 % CI IC50 values for wild - type ( HUVECs ) , PIK3CA TEK ECs are shown . For PIK3CA TEK - mutant ECs n = 3 biological replicates ( ) ; for wild - type ECs ( HUVECs ) n = 4 technical replicates PIK3CA - mutant vs . TEK - mutant p = 0 . 501 ; PIK3CA - mutant vs . HUVECs p = 0 . 0815 ; TEK - mutant vs . HUVECs : p = 0 . 4085 . Statistical was performed by comparison of best - fit values using extra sum - of - squares F .

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

(G) Wild-type (HUVECs), PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay. Fitting curves and 95% CI IC50 values for wild-type (HUVECs), PIK3CA and TEK ECs are shown. For PIK3CA and TEK-mutant ECs n = 3 biological replicates (patients); for wild-type ECs (HUVECs) n = 4 technical replicates PIK3CA-mutant vs. TEK-mutant p = 0.501; PIK3CA-mutant vs. HUVECs p = 0.0815; TEK-mutant vs. HUVECs: p = 0.4085. Statistical analysis was performed by comparison of best-fit values using the extra sum-of-squares F test.


Relative iNKT yield in various tissues from TN mouse lines compared to C57BL / 6 mouse lines . Tissues were isolated from indicated C57BL / 6 or iNKT TN mouse lines stained with anti - CD3 CD1d ( PBS57 ) tetramer .

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Relative iNKT cell yield in various tissues from TN mouse lines compared to C57BL/6 mouse lines. Tissues were isolated from indicated C57BL/6 or iNKT TN mouse lines and stained with anti-CD3 and CD1d (PBS57) tetramer.


Spleen , mLN , sdLN , liver , adipose , lung lymphocytes from Jα18 - / - or Vα14 mice were stimulated in vitro with RAW - CD1d 1μg α - GalCer . An additional sample of Vα14 lymphocytes from each organ was plated with RAW - CD1d but no α - GalCer . Supernatants were collected after 24 hours cytokine determined by cytokine bead array . Error bars are SD of mean values from three different mice per group . Results shown are representative of two independent experiments where n = 3 biological replicates .

B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE B-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD B-ORGANISM O O O O O B-CELL_LINE O B-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD B-CELL_TYPE O O O O O O B-CELL_LINE O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

Spleen, mLN, sdLN, liver, adipose, and lung lymphocytes from Jα18-/- or Vα14 mice were stimulated in vitro with RAW-CD1d cells and 1μg α-GalCer. An additional sample of Vα14 lymphocytes from each organ was plated with RAW-CD1d cells but no α-GalCer. Supernatants were collected after 24 hours and cytokine concentration determined by cytokine bead array. Error bars are SD of mean values from three different mice per group. Results shown are representative of two independent experiments where n=3 biological replicates.


Lymphocytes from indicated tissues of C57BL / 6 Vα14 mice were stained with anti - CD3 CD1d ( PBS57 ) tetramer , before they were fixed , permeabilized , stained with antibodies to T - bet , RORγt , PLZF . Results shown are gated on CD3 + CD1d - tetramer + .

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

Lymphocytes from the indicated tissues of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer, before they were fixed, permeabilized, and stained with antibodies to T-bet, RORγt, and PLZF. Results shown are gated on CD3+CD1d-tetramer+ cells.


of CD3 + CD1d - tetramer + iNKT in each organ that stained positively for PLZF , T - bet , RORγt are shown . * * p < 0 . 01 , Mann - Whitney . Error bars are SD .

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

The percentage of CD3+ CD1d-tetramer+ iNKT cells in each organ that stained positively for PLZF, T-bet, and RORγt are shown. **p<0.01, Mann-Whitney test. Error bars are SD.


Lymphocytes from Peyer ' s patches of C57BL / 6 Vα14 mice were stained with anti - CD3 CD1d ( PBS57 ) tetramer . of lymphocytes that were CD3 + CD1d - tetramer + iNKT among Peyer ' s patches of C57BL / 6 , Vα14 , Jα18 - / - mice are shown . Mann - Whitney . Error bars are SD . C57BL / 6 n = 22 ; Vα14 n = 16 ; Jα18 - / - n = 8 .

B-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O B-GENEPROD B-ORGANISM O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O

Lymphocytes from the Peyer's patches of C57BL/6 and Vα14 mice were stained with anti-CD3 and CD1d (PBS57) tetramer. Percentage of lymphocytes that were CD3+CD1d-tetramer+ iNKT cells among the Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=22; Vα14 n=16; Jα18-/- n=8.


( C - E ) Mice were analyzed for total fecal IgA by ELISA . Mann - Whitney . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 .

O O O O O B-ORGANISM O O O O B-TISSUE B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O

(C-E)Mice were analyzed for total fecal IgA by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.


( C - E ) Mice were analyzed for total fecal IgG1 ( D ) or total serum IgG1 ( E ) by ELISA . Mann - Whitney . Error bars are SD . C57BL / 6 n = 14 ; Vα14 n = 11 ; Jα18 - / - n = 13 .

O O O O O B-ORGANISM O O O O B-TISSUE B-GENEPROD O O O O O B-TISSUE B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O

(C-E)Mice were analyzed for total fecal IgG1 (D) or total serum IgG1 (E) by ELISA. Mann-Whitney test. Error bars are SD. C57BL/6 n=14; Vα14 n=11; Jα18-/- n=13.


Percentages of total B that were IgG1 + in spleen , mLN , Peyer ' s patches of C57BL / 6 , Vα14 , Jα18 - / - mice are shown . Mann - Whitney . Error bars are SD . C57BL / 6 n = 15 ; Vα14 n = 15 ; Jα18 - / - n =

B-EXP_ASSAY O O B-CELL_TYPE O O B-GENEPROD O O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O

Percentages of total B cells that were IgG1+ in the spleen, mLN, and Peyer's patches of C57BL/6, Vα14, and Jα18-/- mice are shown. Mann-Whitney test. Error bars are SD. C57BL/6 n=15; Vα14 n=15; Jα18-/- n=5


CD1d ( PBS57 ) tetramer + CD3 + were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4 + CD3 + CD1d tetramer - from PP ( PP CD4 ) . RNAseq was performed . of FPKM values for indicated Tfh genes across each RNAseq sample .

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CD1d (PBS57) tetramer+ CD3+ cells were sorted from spleens or PP of 3 different Vα14 TN mice along with CD4+CD3+CD1d tetramer- cells from PP (PP CD4). RNAseq was performed. Heatmap of FPKM values for the indicated Tfh genes across each RNAseq sample.


Spleen , mLN , PP lymphocytes from Vα14 C57BL / 6 mice were stimulated with PMA ionomycin . Lymphocytes were stained with anti - CD3 , CD1d ( PBS57 ) tetramer , anti - IL - 4 , anti - IFNγ . Percentages of iNKT non - iNKT T within population of CD3 + IL - 4 + CD3 + IFNγ + , n = 5 mice for C57BL / 6 group n = 4 mice for Vα14 group . Error bars are SD .

B-TISSUE O B-TISSUE O B-TISSUE B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-CELL_TYPE O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O

Spleen, mLN, and PP lymphocytes from Vα14 and C57BL/6 mice were stimulated with PMA and ionomycin. Lymphocytes were stained with anti-CD3, CD1d (PBS57) tetramer, anti-IL-4, and anti-IFNγ. Percentages of iNKT cells and non-iNKT T cells within the population of CD3+IL-4+ cells and CD3+IFNγ+ cells, n = 5 mice for C57BL/6 group and n = 4 mice for Vα14 group. Error bars are SD.


Vα14 TN mice were administered α - GalCer either 2μg intravenously or 5μg by oral gavage . Mice were given Brefeldin A intraperitoneally after 30 min , tissues were harvested 3 hours later . from spleen , mesenteric lymph node , Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , stained with antibodies to IL - 4 , IFNγ , IL - 17 . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT .

B-GENEPROD O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O B-ORGANISM O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-CELL_TYPE O

Vα14 TN mice were administered α-GalCer either 2μg intravenously or 5μg by oral gavage. Mice were given Brefeldin A intraperitoneally after 30 min, and tissues were harvested 3 hours later. Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with antibodies to IL-4, IFNγ, and IL-17. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells.


of data from C , n = 2 mice per group . Mann - Whitney . Error bars are .

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Quantification of data from C, n=2 mice per group. Mann-Whitney test. Error bars are SEM.


Pooled spleen LN from a Vα14 TN mouse were cocultured with wild type B with or without 1μg α - GalCer with or without agnostic anti - CD40 . IgM was measured by ELISA of culture supernatants 4 days later .

O B-TISSUE I-TISSUE O O B-GENEPROD O B-ORGANISM O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O

Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with wild type B cells with or without 1μg α-GalCer and with or without agnostic anti-CD40. IgM was measured by ELISA of culture supernatants 4 days later.


Pooled spleen LN from a Vα14 TN mouse were cocultured with anti - CD40 activated wild type B with or without 1μg α - GalCer with blocking antibody to CD1d ( 1μg / mL , clone 1B1 ) or isotype control as indicated . IgM , IgG1 , IgA were measured by ELISA of 4 day culture supernatants .

O B-TISSUE I-TISSUE O O B-GENEPROD O B-ORGANISM O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O

Pooled spleen and LN cells from a Vα14 TN mouse were cocultured with anti-CD40 activated wild type B cells with or without 1μg α-GalCer and with blocking antibody to CD1d (1μg/mL, clone 1B1) or isotype control as indicated. IgM, IgG1, and IgA were measured by ELISA of 4 day culture supernatants.


Spleen , mLN or PP from Vα14 TN iNKT mice PP from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B . 1μg α - GalCer was added to cultures to specifically activate iNKT . IL - 4 were measured by ELISA of culture supernatants .

B-TISSUE O B-TISSUE O B-TISSUE O B-GENEPROD O B-CELL_TYPE B-ORGANISM B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O

Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. IL-4 were measured by ELISA of culture supernatants.


Spleen , mLN or PP from Vα14 TN iNKT mice PP from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B . 1μg α - GalCer was added to cultures to specifically activate iNKT . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 ( D - E ) were measured by ELISA of culture supernatants .

B-TISSUE O B-TISSUE O B-TISSUE O B-GENEPROD O B-CELL_TYPE B-ORGANISM B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O

Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1 (D-E) were measured by ELISA of culture supernatants.


Spleen , mLN or PP from Vα14 TN iNKT mice PP from an IL - 4 - / - mouse 1μg α - GalCer was added to cultures to specifically activate iNKT . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + IgA + class switched B were enumerated by flow cytometry after 4 days of

B-TISSUE O B-TISSUE O B-TISSUE O B-GENEPROD O B-CELL_TYPE B-ORGANISM B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ and IgA+ class switched B cells were enumerated by flow cytometry after 4 days of coculture


Spleen , mLN or PP from Vα14 TN iNKT mice PP from an IL - 4 - / - mouse were cocultured with anti - CD40 activated wild type or CD1d - / - B . 1μg α - GalCer was added to cultures to specifically activate iNKT . Blocking antibodies to IL - 4 or isotype were added as indicated . IgG1 + class switched B were enumerated by flow cytometry after 4 days of

B-TISSUE O B-TISSUE O B-TISSUE O B-GENEPROD O B-CELL_TYPE B-ORGANISM B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

Spleen, mLN or PP cells from Vα14 TN iNKT mice and PP cells from an IL-4-/- mouse were cocultured with anti-CD40 activated wild type or CD1d-/- B cells. 1μg α-GalCer was added to the cultures to specifically activate iNKT cells. Blocking antibodies to IL-4 or isotype were added as indicated. IgG1+ class switched B cells were enumerated by flow cytometry after 4 days of coculture


OVA - specific antibody titers from feces were determined by ELISA from mice in ( A ) . Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after final boost ) . Error bars are SD .

B-GENEPROD O O O O O B-TISSUE O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

OVA-specific antibody titers from feces were determined by ELISA from the mice in (A). Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.


OVA - specific antibody titers from feces blood were determined by ELISA from mice in Shown are optical density values for samples collected on day 52 post - vaccination ( 7 days after final boost ) . Error bars are SD .

B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

OVA-specific antibody titers from feces and blood were determined by ELISA from the mice in Shown are optical density values for samples collected on day 52 post-vaccination (7 days after the final boost). Error bars are SD.


from spleen , mesenteric lymph node , Peyer ' s patches of Vα14 TN mice were permeabilized , fixed , stained with indicated antibodies . Plots shown are gated on CD1d - ( PBS57 ) - tetramer + CD3 + iNKT . of data from E , n = 3 mice per group . Mann - Whitney . Error bars are . * p < 0 . 05 .

O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O B-ORGANISM O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

Cells from spleen, mesenteric lymph node, and Peyer's patches of Vα14 TN mice were permeabilized, fixed, and stained with the indicated antibodies. Plots shown are gated on CD1d-(PBS57)-tetramer+ CD3+ iNKT cells. Quantification of data from E, n=3 mice per group. Mann-Whitney test. Error bars are SEM. *p<0.05.


Differential expression comparing transcriptomes of spleen iNKT versus PP - iNKT .

O B-EXP_ASSAY O O O B-TISSUE B-CELL_TYPE O B-TISSUE O B-CELL_TYPE O

Differential expression analysis comparing transcriptomes of spleen iNKT cells versus PP-iNKT cells.


of FPKM values from genes uniquely upregulated in PP - iNKT as compared to both spleen iNKT PP CD4 . Red indicates higher expression blue indicates lower expression .

O O O O O O O O B-TISSUE O B-CELL_TYPE O O O O B-TISSUE B-CELL_TYPE B-TISSUE B-GENEPROD O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O

Heatmap of FPKM values from genes uniquely upregulated in PP-iNKT cells as compared to both spleen iNKT and PP CD4 cells. Red indicates higher expression and blue indicates lower expression.


Spleen PP from Vα14 TN mice were stained with indicated antibodies , analyzed by flow cytometry gated on CD1d ( PBS57 ) tetramer + CD3 + .

B-TISSUE I-TISSUE O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O O

Spleen and PP cells from Vα14 TN mice were stained with the indicated antibodies, analyzed by flow cytometry and gated on CD1d (PBS57) tetramer+CD3+ cells.


of data from D , n = 11 mice per group . Mann - Whitney . Error bars are .

Quantification of data from D, n=11 mice per group. Mann-Whitney test. Error bars are SEM.


( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic . SL2 were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h was induced with 10 μM ecdysone for 24 h . ( a ) viability was assayed by trypan blue . Data are mean from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - ) . ( b ) viability using Muse ( Millipore , USA ) . Data are mean from three independent experiments , with error bars representing s . d . * P0 . 05 ( Student ' s t - ) .

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

(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (a) The cell viability was assayed by trypan blue staining. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test). (b) Cell count and viability assay using Muse (Millipore, USA). Data are mean from three independent experiments, with error bars representing s.d. *P0.05 (Student's t-test).


( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic . SL2 were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h was induced with 10 µM ecdysone for 24 h . ( c ) Caspase activity was measured from total lysate on DEVD - AMC , represented as fold increase relative to control RNAi . Data are mean from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - ) .

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

(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 µM ecdysone for 24 h. (c) Caspase activity was measured from total cell lysate on DEVD-AMC, represented as fold increase relative to control RNAi. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).


( a - d ) RNAi - mediated knockdown of dUTX inhibits ecdysone - induced apoptotic . SL2 were treated with two independent dsRNAs to dUTX or a control gene ( GFP ) for 48 h was induced with 10 μM ecdysone for 24 h . ( d ) level of knockdown of dUTX was quantificated by qPCR normalized against ribosomal rp49 . Relative dUTX expression following knockdown using two independent dsRNA are shown ( n = 3 ) with error bars representing s . d . * P0 . 05 ( Student ' s t - ) .

O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 μM ecdysone for 24 h. (d) The level of knockdown of dUTX was quantificated by qPCR and normalized against ribosomal protein rp49. Relative dUTX expression levels following knockdown using the two independent dsRNA are shown (n=3) with error bars representing s.d. *P0.05 (Student's t-test).


( a , b ) dUTX directly interacts with ecdysone receptor ( EcR / Usp ) . ( a ) GST GST - dUTX - NR containing hormone domain were used in pull - down experiments with 35S - labelled EcR Usp in presence of ethanol control ( − ) or 10 μM ecdysone ( + ) . GST input are shown in Coomassie Brilliant blue - stained gel , GST is labelled with arrow head dUTX - NR with an asterisk . EcR Usp inputs are shown labelled with arrows .

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

(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (a) GST and GST-dUTX-NR containing the nuclear hormone domain were used in pull-down experiments with 35S-labelled EcR and Usp in the presence of ethanol control (−) or 10 μM ecdysone (+). The GST input proteins are shown in Coomassie Brilliant blue-stained gel, GST is labelled with arrow head and dUTX-NR with an asterisk. The EcR and Usp inputs are shown labelled with arrows.


( a , b ) dUTX directly interacts with ecdysone receptor ( EcR / Usp ) . ( b ) Immunoprecipitation ( IP ) of EcR dUTX in SL2 . were transfected with HA - dUTX lysates were immunoprecipitated using anti - HA or a control IgG isotype antibody . immunoprecipitates were analysed by western blotting using anti - EcR anti - HA antibodies .

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

(a,b) dUTX directly interacts with ecdysone receptor (EcR/Usp). (b) Immunoprecipitation (IP) of EcR and dUTX in SL2 cells. Cells were transfected with HA-dUTX and the cell lysates were immunoprecipitated using anti-HA or a control IgG isotype antibody. The immunoprecipitates were analysed by western blotting using the anti-EcR and anti-HA antibodies.


( c ) Colocalization of dUTX EcR on salivary polytene chromosomes . Immunostaining of salivary polytene chromosome prepared from third instar larvae expressing FLAG - dUTX with dUTX ( green ) EcR ( red ) , with Hoechst - stained ( blue ) . Arrows indicate examples of colocalized bands . No signal was detected when immunostaining with secondary antibody only . Scale bar represent 5 μm .

O O O O O B-GENEPROD I-GENEPROD O B-TISSUE O O O B-EXP_ASSAY O B-TISSUE B-SUBCELLULAR I-SUBCELLULAR O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

(c) Colocalization of dUTX and EcR on salivary gland polytene chromosomes. Immunostaining of salivary glands polytene chromosome prepared from third instar larvae expressing FLAG-dUTX with dUTX (green) and EcR (red), with Hoechst-stained DNA (blue). Arrows indicate examples of colocalized bands. No signal was detected when immunostaining with secondary antibody only. Scale bar represent 5 μm.


( a ) Histology of paraffin sections at 14 h 24 h RPF shows intact salivary present in dUTX1 compared with control at 24 h RPF . Ovals indicate position of salivary fragments . Scale bar represent 50 μm . of salivary phenotypic data was at 24 h RPF .

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(a) Histology analysis of paraffin sections at 14 h and 24 h RPF shows intact salivary glands present in dUTX1 compared with control at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data was at 24 h RPF.


( b , c ) Caspase activity are reduced in dUTX1 salivary at 14 h RPF . ( b ) caspase - 3 antibody ( green ) Lamin antibody ( red ) are shown in control dUTX1 salivary at 14 h RPF . Scale bar represents 50 μm . ( c ) Caspase activity was measured from 14 h RPF control dUTX1 salivary using DEVD - AMC as a substrate . Data are mean from three independent experiments each using at least 20 salivary from control dUTX1 for preparing extracts . error bars represent s . e . m . * P - value0 . 05 ( Student ' s t - ) .

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(b,c) Caspase staining and activity are reduced in dUTX1 salivary glands at 14 h RPF. (b) Cleaved caspase-3 antibody (green) and Lamin antibody (red) staining are shown in control and dUTX1 salivary glands at 14 h RPF. Scale bar represents 50 μm. (c) Caspase activity was measured from 14 h RPF control and dUTX1 salivary glands using DEVD-AMC as a substrate. Data are mean from three independent experiments each using at least 20 salivary glands from control and dUTX1 for preparing cell extracts. The error bars represent s.e.m. *P-value0.05 (Student's t-test).


( d ) Autophagy examined using mCherry - Atg8a ( red ) nuclei stained with Hoechst ( blue ) . Scale bar represents 100 μm . quantitation of mCherry - Atg8a as mean pixel per ( control n = 242 , dUTX1 n = 224 ) , with error bars representing s . e . m . * P - value0 . 05 ( Student ' s t - ) .

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(d) Autophagy puncta examined using mCherry-Atg8a (red) and nuclei stained with Hoechst (blue). Scale bar represents 100 μm. The quantitation of the mCherry-Atg8a puncta as the mean fluorescent pixel intensity per cell (control n=242, dUTX1 n=224), with error bars representing s.e.m. *P-value0.05 (Student's t-test).


( a ) of , dronc , drice rpr transcripts were measured from third instar larvae ( L3 ) , 6 h 14 h RPF salivary from control ( blue ) dUTX1 ( red ) by qPCR expressed relative to internal control gene rp49 .

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(a) Levels of dark, dronc, drice and rpr transcripts were measured from third instar larvae (L3), 6 h and 14 h RPF salivary glands from control (blue) and dUTX1 (red) by qPCR and expressed relative to the internal control gene rp49.


( b ) transcript of Atg genes relative to internal control gene rp49 in salivary of control ( blue ) dUTX1 ( red ) were analysed by qPCR at L3 , 6 h 14 h RPF . Data in panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - ) .

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(b) The transcript levels of Atg genes relative to the internal control gene rp49 in the salivary glands of control (blue) and dUTX1 (red) were analysed by qPCR at L3, 6 h and 14 h RPF. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).


( a , b ) occupancy of dUTX on promoter regions of apoptosis Atg genes was detected by immunoprecipitation ( ChIP ) . SL2 overexpressing HA - dUTX were - immunoprecipitated with anti - HA anti - GFP ( IgG control ) antibodies following ecdysone treatment . qPCR was used to assess binding to promoters of , dronc , drice rpr ( a )

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(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of dark, dronc, drice and rpr (a)


( a , b ) occupancy of dUTX on promoter regions of apoptosis Atg genes was detected by immunoprecipitation ( ChIP ) . SL2 overexpressing HA - dUTX were - immunoprecipitated with anti - HA anti - GFP ( IgG control ) antibodies following ecdysone treatment . qPCR was used to assess binding to promoters of Atg1 , Atg2 , Atg3 , Atg4 , Atg5 , Atg6 , Atg7 , Atg8a , Atg9 , Atg12 Atg18 ( b ) , expressed relative to internal control gene Gapdh . Results are shown as fold enrichment of % input compared with IgG control .

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(a,b) The occupancy of dUTX on the promoter regions of apoptosis and Atg genes was detected by chromatin immunoprecipitation (ChIP). SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. qPCR was used to assess the binding to the promoters of Atg1, Atg2, Atg3, Atg4, Atg5, Atg6, Atg7, Atg8a, Atg9, Atg12 and Atg18 (b), expressed relative to the internal control gene Gapdh. Results are shown as the fold enrichment of % input compared with IgG control.


( c ) abundance of H3K27me3 on promoters of apoptosis autophagy genes detected by ChIP . SL2 knocked down for dUTX were - immunoprecipitated with anti - H3K27me3 anti - GFP ( IgG control ) antibodies following ecdysone treatment . Results are shown as fold enrichment of % input compared with RNAi control .

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(c) The abundance of H3K27me3 on the promoters of apoptosis and autophagy genes detected by ChIP. SL2 cells knocked down for dUTX were chromatin-immunoprecipitated with anti-H3K27me3 and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control.


( d ) occupancy of EcR on promoter regions of , dronc Atg1 by ChIP expressed relative to internal control gene rp49 . was immunoprecipitated with anti - EcR - B1 anti - GFP ( IgG control ) antibodies from SL2 following ecdysone treatment . Results are shown as fold enrichment of % input compared with IgG control . Data in panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Student ' s t - ) .

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(d) The occupancy of EcR on the promoter regions of dark, dronc and Atg1 by ChIP expressed relative to the internal control gene rp49. Chromatin was immunoprecipitated with anti-EcR-B1 and anti-GFP (IgG control) antibodies from SL2 cell following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).


( a ) of BR - C E93 transcripts were measured in salivary isolated from third instar larvae ( L3 ) , 6 h 14 h RPF control ( blue ) dUTX1 ( red ) using qPCR expressed relative to internal control gene rp49 .

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(a) Levels of BR-C and E93 transcripts were measured in salivary glands isolated from third instar larvae (L3), 6 h and 14 h RPF control (blue line) and dUTX1 (red line) using qPCR and expressed relative to the internal control gene rp49.


( b ) occupancy of dUTX on promoter regions of BR - C E93 genes detected by ChIP . SL2 overexpressing HA - dUTX were - immunoprecipitated with anti - HA anti - GFP ( IgG control ) antibodies following ecdysone treatment . Results are shown as fold enrichment of % input compared with IgG control . In right panel abundance of H3K27me3 on promoters of BR - C E93 genes as detected by ChIP is shown . SL2 knockdown for dUTX ( dUTX RNAi ) were - immunoprecipitated with anti - H3K27me3 rabbit IgG ( control ) antibodies following ecdysone treatment . Results are shown as fold enrichment of % input compared with RNAi control . Data in panels are expressed as means from three independent experiments , with error bars representing s . e . m . * P0 . 05 ( Students t - ) .

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(b) The occupancy of dUTX on the promoter regions of BR-C and E93 genes detected by ChIP. SL2 cells overexpressing HA-dUTX were chromatin-immunoprecipitated with anti-HA and anti-GFP (IgG control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with IgG control. In the right hand panel abundance of H3K27me3 on the promoters of BR-C and E93 genes as detected by ChIP is shown. SL2 cells knockdown for dUTX (dUTX RNAi) were chromatin-immunoprecipitated with anti-H3K27me3 and rabbit IgG (control) antibodies following ecdysone treatment. Results are shown as the fold enrichment of % input compared with RNAi control. Data in all panels are expressed as means from three independent experiments, with error bars representing s.e.m. *P0.05 (Students t-test).


( a ) Mosaic clones of dUTX1 ( hsFLP ; ubi - GFP FRT40A / dUTX1 FRT40A ) from third instar larval salivary with wild - type marked by - localized GFP ( green ) . dUTX1 clones have high of H3K27me3 ( red ) compared with neighbouring control marked by GFP . Scale bar represents 50 μm .

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(a) Mosaic clones of dUTX1 (hsFLP; ubi-GFP FRT40A/dUTX1 FRT40A) from third instar larval salivary glands with wild-type cells marked by nuclear-localized GFP (green). dUTX1 clones have high levels of H3K27me3 (red) compared with the neighbouring control cells marked by GFP. Scale bar represents 50 μm.


( b ) Western blot of level of H3K27me3 in control dUTX1 larval salivary . Alpha - tubulin H3 serve as loading controls .

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(b) Western blot analysis of the level of H3K27me3 in control and dUTX1 larval salivary glands. Alpha-tubulin and H3 serve as loading controls.


( c ) Expression of dUTX wild - type ( dUTXWT ) dUTX catalytic mutant ( dUTXJmj ) in salivary ( using Sgs3 - GAL4 driver ) of dUTX1 . Histology of paraffin sections at 24 h RPF shows intact salivary present in dUTX1 in dUTX1 ; dUTXJmj * compared with control dUTX1 ; dUTXWT at 24 h RPF . Ovals indicate position of salivary fragments . Scale bar represent 50 μm . of salivary phenotypic data at 24 h RPF is shown on right .

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(c) Expression of dUTX wild-type (dUTXWT) and dUTX catalytic mutant (dUTXJmj) in the salivary glands (using Sgs3-GAL4 driver) of dUTX1. Histology analysis of paraffin sections at 24 h RPF shows intact salivary glands present in dUTX1 and in dUTX1; dUTXJmj* compared with control and dUTX1; dUTXWT at 24 h RPF. Ovals indicate the position of salivary glands and fragments. Scale bar represent 50 μm. Quantification of the salivary gland phenotypic data at 24 h RPF is shown on the right.


( B ) Scatterplots represent SILAC / SUMO - IP after EGF treatment from three biological replicates . Each dot represents a that is either present in same between untreated EGF treated ( blue dots ) or is significantly more present in one of samples ( red yellow dots ) .

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(B) Scatterplots represent SILAC/SUMO-IP quantification after EGF treatment from three biological replicates. Each dot represents a protein that is either present in the same ratio between untreated and EGF treated cells (dark blue dots) or is significantly more present in one of the samples (red and yellow dots).


( C ) Left panel : Bar graph depicting mass spectrometry results of 11 with altered SUMOylation ( significant hits with P < 0 . 0001 ) , as well as three non - changing SUMO2 , RanGAP1 TRIM28 . Right panel : highlighted in bold in left panel were validated by SUMO - IP / immunoblotting from serum starved HeLa without or with 10 min EGF stimulation .

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(C) Left panel: Bar graph depicting mass spectrometry results of 11 proteins with altered SUMOylation (significant hits with P<0.0001), as well as the three non-changing proteins SUMO2, RanGAP1 and TRIM28. Right panel: Proteins highlighted in bold in the left panel were validated by SUMO-IP/immunoblotting from serum starved HeLa cells without or with 10 min EGF stimulation.


( D ) Time course experiment : serum starved HeLa were treated with 100 nM EGF , samples were harvested at indicated times subjected to SUMO2 IP followed by immunoblotting with indicated antibodies . IRF2BP1 , IRF2BP2 TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment .

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(D) Time course experiment: serum starved HeLa cells were treated with 100 nM EGF, samples were harvested at indicated times and subjected to SUMO2 IP followed by immunoblotting with the indicated antibodies. IRF2BP1, IRF2BP2 and TRIM24 are rapidly but transiently deSUMOylated upon EGF treatment.


( B ) Identification of endogenous SUMO - sites in IRF2BP1 ( left panel ) IRF2BP2 ( right panel ) . HeLa were transfected with HA - tagged wildtype ( WT ) or mutant ( KR ) , endogenous SUMO - IPs were performed HA - signal was analyzed by immunoblotting . Mutation of C - terminal SUMO - site abolished SUMOylation of IRF2BP1 IRF2BP2 in HeLa .

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(B) Identification of the endogenous SUMO-sites in IRF2BP1 (left panel) and IRF2BP2 (right panel). HeLa cells were transfected with HA-tagged wildtype (WT) or mutant (KR) proteins, endogenous SUMO-IPs were performed and the HA-signal was analyzed by immunoblotting. Mutation of the C-terminal SUMO-site abolished SUMOylation of IRF2BP1 and IRF2BP2 in HeLa cells.


( E ) Stable HeLa expressing pIRES - empty vector , IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non - targeting ( nt ) siRNA . Exogenous siRNA - resistant IRF2BP1 was expressed at low similar to endogenous IRF2BP1 . * refers to an unspecific band .

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(E) Stable HeLa cells expressing pIRES-empty vector, IRF2BP1 WT or IRF2BP1 K579R were treated with siRNA against endogenous IRF2BP1 or non-targeting (nt) siRNA. Exogenous siRNA-resistant IRF2BP1 was expressed at low levels similar to endogenous IRF2BP1. * refers to an unspecific band.


( F ) Wt mutant IRF2BP1 localizes in nucleus . After knockdown of endogenous IRF2BP1 , stable IRF2BP1 ( WT or K579R ) lines were immunostained for IRF2BP1 . Exogenous IRF2BP1 variants show a similar localization . Scale bar = 10 µm .

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(F) Wt and mutant IRF2BP1 localizes in the nucleus. After knockdown of endogenous IRF2BP1, stable IRF2BP1 (WT or K579R) cell lines were immunostained for IRF2BP1. Exogenous IRF2BP1 variants show a similar nuclear localization. Scale bar = 10 µm.


( G ) IRF2BP1 wt mutant associate with to a similar extent . HeLa were lysed in 0 . 075 % NP40 ( Input ) . After centrifugation , nuclei were incubated vortexed with a extract ( NE ) buffer containing 170 mM NaCl . eluates were collected procedure was repeated using a NE buffer with higher salt concentrations , first 290 mM , then 420 mM . Wildtype IRF2BP1 , SUMO - deficient K579R mutant SUMOylated wildtype form ( * ) behave similarly .

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(G) IRF2BP1 wt and mutant associate with chromatin to a similar extent. HeLa cells were lysed in 0.075 % NP40 (Input). After centrifugation, the nuclei were incubated and vortexed with a nuclear extract (NE) buffer containing 170 mM NaCl. The eluates were collected and the procedure was repeated using a NE buffer with higher salt concentrations, first 290 mM, then 420 mM. Wildtype IRF2BP1, the SUMO-deficient K579R mutant and the SUMOylated wildtype form (*) all behave similarly.


( A ) Stable IRF2BP1 lines ( knocked - down for endogenous IRF2BP1 ) were used to perform a microarray experiment under serum starvation upon treatment with EGF for 1 hour . A subset of 38 EGF - dependent genes are differentially regulated in IRF2BP1 wildtype compared to IRF2BP1 K579R lines ( at least 1 . 5 - fold ) . Among them are DUSP1 ( arrow ) , ATF3 , Fos Egr2 . Each microarray was performed in triplicates , bars show log2 values of fold changes for wt ( black bars ) for KR ( grey bars ) .

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(A) Stable IRF2BP1 cell lines (knocked-down for endogenous IRF2BP1) were used to perform a microarray experiment under serum starvation and upon treatment with EGF for 1 hour. A subset of 38 EGF-dependent genes are differentially regulated in IRF2BP1 wildtype cells compared to IRF2BP1 K579R cell lines (at least 1.5-fold). Among them are DUSP1 (arrow), ATF3, Fos and Egr2. Each microarray was performed in triplicates, the bars show log2 values of the fold changes for wt cells (black bars) and for KR cells (grey bars).


( B ) IP reveals association of human IRF2BP1 with proximal DUSP1 promoter in HeLa . Gene architecture of human DUSP1 . primers at - 243 / - 67 ( " - 67 " ) , - 473 / - 224 ( " - 224 " ) - 1170 / - 961 ( " - 916 " ) relative to TSS were used for ChIP experiments . IRF2BP1 IRF2BP2 bind to promoter region of DUSP1 between nucleotides - 243 - 67 .

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(B) Chromatin IP reveals association of human IRF2BP1 with the proximal DUSP1 promoter in HeLa cells. Gene architecture of human DUSP1. The primers at -243/-67 ("-67"), -473/-224 ("-224") and -1170/-961 ("-916") relative to the TSS were used for ChIP experiments. IRF2BP1 and IRF2BP2 bind to the promoter region of DUSP1 between nucleotides -243 and -67.


( C ) ChIP / qPCR experiments reveal preferential IRF2BP1 binding to promoter region of DUSP1 between - 243 - 67 . Data show mean ( bar ) data points from 2 biological replicates .

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(C) ChIP/qPCR experiments reveal preferential IRF2BP1 binding to the promoter region of DUSP1 between -243 and -67. Data show mean (bar) and individual data points from 2 biological replicates.


( D ) IRF2BP1 wildtype K579R mutant both bind DUSP1 promoter in absence or presence of EGF . Stable lines expressing IRF2BP1 wildtype or K579R were knocked - down for endogenous IRF2BP1 , followed by IRF2BP1 ChIP DUSP1 qPCR of its promoter region - 243 - 67 . Data show means + / - from 3 biological replicates .

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(D) IRF2BP1 wildtype and the K579R mutant both bind the DUSP1 promoter in the absence or presence of EGF. Stable cell lines expressing IRF2BP1 wildtype or K579R were knocked-down for endogenous IRF2BP1, followed by IRF2BP1 ChIP and DUSP1 qPCR of its promoter region -243 and -67. Data show means +/- SEM from 3 biological replicates.


( E ) Q - PCR data after EGF treatment in IR2BP1 wildtype K579R lines after knockdown of endogenous IRF2BP1 . Data show means + / - from 3 biological replicates .

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(E) Q-PCR data after EGF treatment in IR2BP1 wildtype and K579R cell lines after knockdown of the endogenous IRF2BP1. Data show means +/- SEM from 3 biological replicates.


( F ) IRF2BP1 wildtype V578A lines after knockdown of endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q - PCR ( left panel ) , data show means + / − from 5 biological replicates ( left panel ) , IRF2BP1 . As expected , IRF2BP1 V578A is not SUMOylated ( right panel ) . * refers to an unspecific band .

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(F) IRF2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1 were analysed for DUSP1 transcription after EGF treatment by q-PCR (left panel), data show means +/− SEM from 5 biological replicates (left panel), and IRF2BP1 protein levels. As expected, IRF2BP1 V578A is not SUMOylated (right panel). * refers to an unspecific band.


( G ) Q - PCR data for immediate early genes after EGF treatment in IR2BP1 wildtype V578A lines after knockdown of endogenous IRF2BP1 . Data show means + / − from 4 biological replicates .

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(G) Q-PCR data for immediate early genes after EGF treatment in the IR2BP1 wildtype and V578A cell lines after knockdown of the endogenous IRF2BP1. Data show means +/− SEM from 4 biological replicates.


( A ) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 ATF3 expression upon knockdown of IRF2BP1 . Uba2 beta - actin serve as independent loading controls .

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(A) Immunoblotting of HeLa lysates at indicated times after EGF treatment reveals enhanced DUSP1 and ATF3 expression upon knockdown of IRF2BP1. Uba2 and beta-actin serve as independent loading controls.


( C ) Wound healing : HeLa were incubated with nt or IRF2BP1 siRNA for 2 days , grown to 90 % confluency analysed for wound closure with or without addition of 100 ng / ml EGF . Data show means of relative wound density + / - from 4 biological replicates .

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(C) Wound healing assay: HeLa cells were incubated with nt or IRF2BP1 siRNA for 2 days, grown to 90% confluency and analysed for wound closure with or without addition of 100 ng/ml EGF. Data show means of the relative wound density +/- SEM from 4 biological replicates.


( D ) Proliferation : HeLa upon knockdown of IRF2BP1 IRF2BP1 were analysed for density over 6 days in growth medium . One representative biological experiment is shown with means + / - from 5 technical replicates .

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(D) Proliferation assay: HeLa cells upon knockdown of IRF2BP1 and IRF2BP1 were analysed for cell density over 6 days in growth medium. One representative biological experiment is shown with means +/-SEM from 5 technical replicates.


( E ) IRF2BP1 , IRF2BP2 , IRF2BP1 + IRF2BP2 were knocked - down in HeLa for 72 hours gene expression data were recorded by microarray ; non - targeting siRNAs were used as a control . IRF2BP1 IRF2BP2 have distinct overlapping functions . Experiments were done in triplicates .

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(E) IRF2BP1, IRF2BP2, IRF2BP1+IRF2BP2 were knocked-down in HeLa cells for 72 hours and gene expression data were recorded by microarray analysis; non-targeting siRNAs were used as a control. IRF2BP1 and IRF2BP2 have distinct and overlapping functions. Experiments were done in triplicates.