B , WT ( SEY6210 ) , apg9Δ ( JKY007 ) , apg9Δ transformed with pAPG9 ( 414 ) centromeric plasmid were grown in SMD transferred to SD ( − N ) as described in Materials Methods . Aliquots were removed at indicated times spread onto YPD plates in triplicate . Numbers of viable colonies were determined after two to three days . APG9 gene complements starvation - sensitive phenotype of apg9Δ mutant .

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B, WT (SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with pAPG9(414) centromeric plasmid were grown in SMD and transferred to SD(−N) as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after two to three days. The APG9 gene complements the starvation-sensitive phenotype of the apg9Δ mutant.


Apg9p is an unglycosylated , integral . A , Wild - type ( WT ; SEY6210 ) , apg9Δ ( JKY007 ) , apg9Δ transformed with single copy ( CEN ) or multicopy ( 2μ ) plasmids encoding APG9 were grown to log phase in SMD . extracts were prepared analyzed by immunoblot using antiserum to Apg9p . Apg9p is detected as an ∼ 125 - kD .

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Apg9p is an unglycosylated, integral membrane protein. A, Wild-type (WT; SEY6210), apg9Δ (JKY007), and the apg9Δ strain transformed with single copy (CEN) or multicopy (2μ) plasmids encoding APG9 were grown to log phase in SMD. Protein extracts were prepared and analyzed by immunoblot using antiserum to Apg9p. Apg9p is detected as an ∼125-kD protein.


. B , Hydrophobicity of Apg9p on Kyte - Doolittle algorithm ( Kyte Doolittle 1982 ) predicts six to eight transmembrane domains .

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. B, Hydrophobicity analysis of Apg9p based on the Kyte-Doolittle algorithm (Kyte and Doolittle 1982) predicts six to eight transmembrane domains.


C , of wild - type YW5 - 1B were grown in YPD , converted to spheroplasts , osmotically lysed as described in Materials Methods . After a preclearing step to remove debris , lysates were incubated with or without 1 % Triton X - 100 on ice for 30 min , as indicated . Centrifugation at 10 , 000 g for 1 h separated lysate into supernatant ( S ) pellet ( P ) fractions , which were analyzed by immunoblots with antiserum to Apg9p . Apg9p is found in low - speed pellet in absence of Triton X - 100 . Detergent extraction completely solubilizes Apg9p causes it to appear in supernatant

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C, Cells of wild-type strain YW5-1B were grown in YPD, converted to spheroplasts, and osmotically lysed as described in Materials and Methods. After a preclearing step to remove cell debris, the lysates were incubated with or without 1% Triton X-100 on ice for 30 min, as indicated. Centrifugation at 10,000 g for 1 h separated the lysate into supernatant (S) and pellet (P) fractions, which were analyzed by immunoblots with antiserum to Apg9p. Apg9p is found in the low-speed pellet in the absence of Triton X-100. Detergent extraction completely solubilizes Apg9p and causes it to appear in the supernatant fraction


D , CTD1 harboring a multicopy 3 × HA APG9 plasmid was grown in YPD converted to spheroplasts as described in Materials Methods . A crude lysate was prepared treated with or without endo H as indicated overnight at 37 ° C analyzed by immunoblots with anti - HA antibodies or anti - CPY serum . Endo H treatment removes N - linked glycosyl residues of CPY , causing a shift in its electrophoretic mobility . In contrast , Apg9p is not glycosylated remains unchanged in presence or absence of endo H treatment .

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D, Strain CTD1 harboring a multicopy 3×HA APG9 plasmid was grown in YPD and converted to spheroplasts as described in Materials and Methods. A crude lysate was prepared and treated with or without endo H as indicated overnight at 37°C and analyzed by immunoblots with anti-HA antibodies or anti-CPY serum. Endo H treatment removes the N-linked glycosyl residues of CPY, causing a shift in its electrophoretic mobility. In contrast, Apg9p is not glycosylated and remains unchanged in the presence or absence of endo H treatment.


Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients . A B , STY1 ( pep4Δ ) was grown in YPD to log - phase , osmotically lysed . Lysates were precleared , loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , centrifuged for 2 . 5 h at 174 , 000 g as described in Materials Methods . Fractions were collected from top ( fraction number 1 ) to bottom ( fraction number 16 ) . Fractions were subjected to SDS - PAGE immunoblot with antiserum against Apg9p : in A , Kex2p ( trans Golgi network ) , Pho8p ( vacuole ) , Pma1p ( plasma ) ; in B , Sec12p ( ER ) Pep12p ( endosome ) . graphs in panels A B are from same gradient , but are presented separately for clarity .

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Apg9p does not comigrate with typical endomembrane markers in sucrose density gradients. A and B, Strain STY1 (pep4Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 2.5 h at 174,000 g as described in Materials and Methods. Fractions were collected from the top (fraction number 1) to the bottom (fraction number 16). Fractions were subjected to SDS-PAGE and immunoblot with antiserum against Apg9p and: in A, Kex2p (trans Golgi network), Pho8p (vacuole), and Pma1p (plasma membrane); and in B, Sec12p (ER) and Pep12p (endosome). The graphs in panels A and B are from the same gradient, but are presented separately for clarity.


Apg9p does not comigrate with prAPI in a that accumulates Cvt / autophagic vesicles . TK415 ( ypt7Δ ) was grown in YPD to log - phase , osmotically lysed . Lysates were precleared , centrifuged at 100 , 000 g for 1 h , pellet fraction resuspended centrifuged a second time . resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18 - 55 % ( wt / wt ) , centrifuged for 16 h at 174 , 000 g as described in Materials Methods . 16 fractions were collected starting from top subjected to SDS - PAGE immunoblot with antisera against Apg9p API . graph represents an average of two experiments .

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Apg9p does not comigrate with prAPI in a strain that accumulates Cvt/autophagic vesicles. Strain TK415 (ypt7Δ) was grown in YPD to log-phase, and osmotically lysed. Lysates were precleared, centrifuged at 100,000 g for 1 h, and the pellet fraction resuspended and centrifuged a second time. The resulting pellet fraction was subsequently loaded on a sucrose density gradient ranging from 18-55% (wt/wt), and centrifuged for 16 h at 174,000 g as described in Materials and Methods. 16 fractions were collected starting from the top and subjected to SDS-PAGE and immunoblot with antisera against Apg9p and API. The graph represents an average of two experiments.


Apg9p localizes to large perivacuolar punctate structures . Immunofluorescence microscopy of strains CTD1 ( apg9Δ ) , KSL12C ( vps4Δ ) , SKD6 - 1D ( apg6Δ ) transformed with multicopy plasmid 3 × HA APG9 . were grown in YPD to log phase ( vegetative ) , incubated further in SD ( − N ) medium with 1 mM PMSF for 3 h ( starvation ) . were fixed with formaldehyde examined by immunofluorescence microscopy as described in Materials Methods . Anti - HA FITC - conjugated antibodies mark Apg9p ( left ) , vacuole can be visualized by Nomarski optics ( middle ) . An overlay is shown in right panels .

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Apg9p localizes to large perivacuolar punctate structures. Immunofluorescence microscopy of strains CTD1 (apg9Δ), KSL12C (vps4Δ), and SKD6-1D (apg6Δ) transformed with the multicopy plasmid 3×HA APG9. Cells were grown in YPD to log phase (vegetative), and incubated further in SD(−N) medium with 1 mM PMSF for 3 h (starvation). The cells were fixed with formaldehyde and examined by immunofluorescence microscopy as described in Materials and Methods. Anti-HA and FITC-conjugated antibodies mark Apg9p (left), and the vacuole can be visualized by Nomarski optics (middle). An overlay is shown in the right panels.


In vivo examination of Apg9pGFP fusion . Wild - type ( SEY6210 ) expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase labeled with FM 4 - 64 to stain vacuoles as described in Materials Methods . labeled were viewed directly with a Leica DM IRB confocal microscope . Large , punctate Apg9pGFP structures appear juxtaposed to FM 4 - 64 - labeled vacuole membranes . In addition , smaller punctate structures appear throughout cytoplasm .

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In vivo examination of the Apg9pGFP fusion protein. Wild-type (SEY6210) cells expressing Apg9pGFP from a multicopy plasmid were grown in SMD to midlog phase and labeled with FM 4-64 to stain the vacuoles as described in Materials and Methods. The labeled cells were viewed directly with a Leica DM IRB confocal microscope. Large, punctate Apg9pGFP structures appear juxtaposed to the FM 4-64-labeled vacuole membranes. In addition, smaller punctate structures appear throughout the cytoplasm.


Ultrastructural localization of Apg9p for immunoelectron microscopy . STY1 ( pep4Δ ) expressing 3 × HA Apg9p was grown in YPD to log phase transferred to SD ( − N ) medium for 2 h . were fixed stained with anti - HA antibody 5 - nm colloidal gold - conjugated goat anti - mouse IgG as described in Materials Methods . A , Section showing autophagic bodies in vacuole lumen . B , Section showing a autophagosome . Apg9p is seen in patches near vacuole , but not on autophagic bodies or autophagosomes . AB , autophagic ; AP , autophagosome ; N , nucleus ; V , vacuole ; VM , vacuolar . Arrows point to areas of concentrated Apg9p .

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Ultrastructural localization of Apg9p for immunoelectron microscopy. Strain STY1 (pep4Δ) expressing 3×HA Apg9p was grown in YPD to log phase and transferred to SD(−N) medium for 2 h. The cells were fixed and stained with anti-HA antibody and 5-nm colloidal gold-conjugated goat anti-mouse IgG as described in Materials and Methods. A, Section showing autophagic bodies in the vacuole lumen. B, Section showing a cytosolic autophagosome. Apg9p is seen in patches near the vacuole, but not on autophagic bodies or autophagosomes. AB, autophagic body; AP, autophagosome; N, nucleus; V, vacuole; VM, vacuolar membrane. Arrows point to areas of concentrated Apg9p.


Vesicle accumulation kinetic of prAPI import in conditional Apg9p mutant . A , Morphological of apg9Δ indicates an import defect at an early step in pathway . Wild - type ( YW5 - 1B ) apg9Δ ( CTD1 ) were grown in YPD ( vegetative ) or transferred to SD ( − N ) ( starvation ) for 3 h in presence of PMSF ( starvation ) examined by DIC ( Nomarski ) microscopy ( Zeiss Axioplan ) . PMSF inhibits degradation of autophagic bodies . Under starvation conditions , wild - type accumulate autophagic bodies when vesicle breakdown is blocked . However , under same conditions , apg9Δ do not accumulate autophagic bodies , indicating that Apg9p is required for a step before vesicle fusion release of autophagic into vacuolar lumen . apg9Δ transformed with APG9 plasmid shows same result as wild - type ( data not shown ) . B , apg9ts is tightly blocked for prAPI import at nonpermissive . Wild - type ( SEY6210 ) apg9Δ ( JKY007 ) transformed with apg9ts centromeric plasmid were incubated at 24 38 ° C for 5 min , - labeled for 10 min , then subjected to nonradioactive chase reactions for indicated times . Samples at each time point were immunoprecipitated with antiserum to API resolved by SDS - PAGE as described in Materials Methods . API - immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager results are presented in graph . mature API was determined by dividing mature API value over sum of precursor mature API values for each time point .

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Vesicle accumulation test and kinetic analysis of prAPI import in the temperature conditional Apg9p mutant. A, Morphological analysis of apg9Δ indicates an import defect at an early step in the pathway. Wild-type (YW5-1B) and apg9Δ (CTD1) cells were grown in YPD (vegetative) or transferred to SD(−N) (starvation) for 3 h in the presence of PMSF (starvation) and examined by DIC (Nomarski) microscopy (Zeiss Axioplan). PMSF inhibits the degradation of autophagic bodies. Under starvation conditions, wild-type cells accumulate autophagic bodies when vesicle breakdown is blocked. However, under the same conditions, apg9Δ cells do not accumulate autophagic bodies, indicating that Apg9p is required for a step before vesicle fusion and release of the autophagic body into the vacuolar lumen. The apg9Δ strain transformed with the APG9 plasmid shows the same result as wild-type (data not shown). B, The apg9ts strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (SEY6210) and apg9Δ (JKY007) cells transformed with the apg9ts centromeric plasmid were incubated at 24 and 38°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API and resolved by SDS-PAGE as described in Materials and Methods. API-immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager and the results are presented in the graph. The percent mature API was determined by dividing the mature API value over the sum of the precursor and mature API values for each time point.


of apg9ts indicates Apg9p is directly required for vesicle formation step . A , nature of prAPI binding pelleting in apg9ts . Spheroplasts of apg9ts were labeled for 10 min chased for 30 min at 38 ° C . spheroplasts were osmotically lysed in a buffer containing no salt ( 20 mM Pipes , pH 6 . 8 ) or a physiological of salt ( 100 mM KOAc , 50 mM KCl , 5 mM MgCl2 , 20 mM Pipes , pH 6 . 8 ) pelleted at 5 , 000 g . samples were immunoprecipitated with antiserum to API marker PGK . apg9ts retains salt dependent binding pelleting of prAPI . B , prAPI in apg9ts accumulates in both a - associated state a large pelletable . Spheroplasts of apg9ts ypt7Δ were labeled for 10 min chased for 30 min at 38 ° C . labeled spheroplasts were then osmotically lysed separated into supernatant ( S ) pellet ( P ) fractions by centrifugation at 5 , 000 g for 5 min . An aliquot was removed for total lysate control ( T ) . pellet fraction ( P ) was resuspended in 15 % Ficoll - 400 in gradient buffer ( 20 mM Pipes , 5 mM MgCl2 , complete EDTA - free protease inhibitor cocktail ) in presence or absence of Triton X - 100 overlaid with 13 2 % Ficoll - 400 in gradient buffer . step gradients were centrifuged at 13 , 000 g for 10 min . - containing float ( F ) , nonfloat ( NF ) , pellet ( P2 ) fractions were immunoprecipitated with antiserum to API as described in Materials Methods . position of prAPI is indicated . C , prAPI in apg9ts is protease accessible . Spheroplasts isolated from apg9ts ypt7Δ were - labeled for 10 min chased for 30 min at 38 ° C . labeled spheroplasts were then osmotically lysed separated into low - speed supernatant ( S ) pellet ( P ) fractions after a 5 , 000 g centrifugation step . pellet fractions were subjected to protease treatment in absence or presence of 0 . 2 % Triton X - 100 as described in Materials Methods . resulting samples were immunoprecipitated with antiserum to API PGK . immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager . protease - protected prAPI was determined by dividing value for prAPI in protease - treated sample by value for total prAPI before protease treatment . PGK was determined by dividing supernatant ( S ) or pellet ( P ) value over sum of S P values .

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Analysis of the apg9ts strain indicates Apg9p is directly required for the vesicle formation step. A, The nature of prAPI binding and pelleting in the apg9ts strain. Spheroplasts of apg9ts were labeled for 10 min and chased for 30 min at 38°C. The spheroplasts were osmotically lysed in a buffer containing no salt (20 mM Pipes, pH 6.8) or a physiological concentration of salt (100 mM KOAc, 50 mM KCl, 5 mM MgCl2, 20 mM Pipes, pH 6.8) and pelleted at 5,000 g. All samples were immunoprecipitated with antiserum to API and the cytosolic marker PGK. apg9ts retains the salt dependent binding and pelleting of prAPI. B, prAPI in the apg9ts strain accumulates in both a membrane-associated state and a large pelletable complex. Spheroplasts of apg9ts and ypt7Δ were labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 5,000 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 in gradient buffer (20 mM Pipes, 5 mM MgCl2, complete EDTA-free protease inhibitor cocktail) in the presence or absence of Triton X-100 and overlaid with 13 and 2% Ficoll-400 in gradient buffer. The step gradients were centrifuged at 13,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods. The position of prAPI is indicated. C, prAPI in the apg9ts strain is protease accessible. Spheroplasts isolated from apg9ts and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 38°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 5,000 g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. The resulting samples were immunoprecipitated with antiserum to API and PGK. The immunoprecipitated bands were quantified by a Molecular Dynamics STORM PhosphorImager. The percent protease-protected prAPI was determined by dividing the value for prAPI in the protease-treated sample by the value for total prAPI before protease treatment. The percent PGK was determined by dividing the supernatant (S) or pellet (P) value over the sum of the S and P values.


( b ) Volcano plots of up ( red ) down ( blue ) regulated genes ( compared to healthy control samples ) in S . Typhi S . Paratyphi A positive individuals ( Nepal Oxford ) . Black numbers indicate up - down - regulated genes compared to healthy controls .

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(b) Volcano plots of up (red) and down (blue) regulated genes (compared to healthy control samples) in S. Typhi and S. Paratyphi A positive individuals (Nepal and Oxford). Black numbers indicate the up- and down-regulated genes compared to healthy controls.


( d - e ) Scatter plots of BTMs enriched ( p > 0 . 05 ) in blood - culture positive samples in Nepal ( y - axis ) versus Oxford ( x - axis ) for typhoid fever ( d ) paratyphoid fever ( e ) . For further details on BTMs refer to reference ( Chaussabel et al . , 2014 ) .

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(d-e) Scatter plots of BTMs enriched (p>0.05) in blood-culture positive samples in Nepal (y-axis) versus Oxford (x-axis) for typhoid fever (d) and paratyphoid fever (e). For further details on BTMs refer to reference (Chaussabel et al., 2014).


( a ) Single - sample GSEA Normalised Enrichment Scores ( NES ) of IFN DC BTMs of individuals with blood - culture confirmed enteric fever in Nepal Oxford .

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(a) Single-sample GSEA Normalised Enrichment Scores (NES) of IFN and DC BTMs of individuals with blood-culture confirmed enteric fever in Nepal and Oxford.


( b ) of 500 most variably expressed genes in samples of Nepali cohort . Bar graph on top of shows of each at time of sampling . Three samples labelled as ' 03NP - CONT ' are samples that grew contaminants were thus excluded from entire .

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(b) Heatmap of the 500 most variably expressed genes in samples of the Nepali cohort. Bar graph on top of the heatmap shows temperature of each individual at the time of sampling. Three samples labelled as '03NP-CONT' are samples that grew bacterial contaminants and were thus excluded from the entire analysis.


( d ) Combined expression score for samples on 5 - gene signature for samples in discovery cohort ( top ) validation cohort ( bottom ) . Ox . CTRL : Oxford controls ( D0 ) ; CTRL : Nepali control samples . PTB : pulmonary TB ; DENV : Dengue samples ; bsPf : blood - stage P . falciparum ; SPT : S . Paratyphi A ; ST : S . Typhi . ST SPT samples are derived from challenge models as well as from Nepal .

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(d) Combined expression score for samples based on the 5-gene signature for samples in the discovery cohort (top) and validation cohort (bottom). Ox.CTRL: Oxford controls (D0); CTRL: Nepali control samples. PTB: pulmonary TB; DENV: Dengue samples; bsPf: blood-stage P. falciparum; SPT: S. Paratyphi A; ST: S. Typhi. ST and SPT samples are derived from the challenge models as well as from Nepal.


( b ) Dot plot of prediction probability of being class EF versus expression score calculated on bases of 5 - gene signature ( on gene array data ) .

(b) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5-gene signature (based on gene array data).


( c ) qPCR gene expression scores of 5 - gene signature ( ∆ ∆ CT over PPIA ) for CTRLs , 03NP - sEF , 03NP - SPT 03NP - ST samples from Nepal . Yellow diamonds in 03NP - sEF category represent 9 classified as EF on random forest algorithm .

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(c) qPCR gene expression scores of the 5-gene signature (∆∆CT over PPIA) for CTRLs, 03NP-sEF, 03NP-SPT and 03NP-ST samples from Nepal. Yellow diamonds in the 03NP-sEF category represent the 9 patients classified as EF based on the random forest algorithm.


( d ) qPCR expression values ( ∆ ∆ Ct over PPIA ) of 5 - gene signature in control samples ( Oxford Nepal ) , S . Paratyphi A ( 03NP - SPT ) or S . Typhi ( 03NP - ST ) in Nepal , samples at day 7 after challenge of participants who stayed well following challenge with S . Typhi ( nD7 ) , or typhoid diagnosis after challenge ( TD ) in Vi - TCV

(d) qPCR expression values (∆∆Ct over PPIA) of the 5-gene signature in control samples (Oxford and Nepal), S. Paratyphi A (03NP-SPT) or S. Typhi (03NP-ST) in Nepal, samples at day 7 after challenge of participants who stayed well following challenge with S. Typhi (nD7), or typhoid diagnosis after challenge (TD) in the Vi-TCV study


( e ) Combined qPCR expression score of 5 - gene signature . Black arrows indicate outlier samples .

(e) Combined qPCR expression score of the 5-gene signature. Black arrows indicate outlier samples.


( f ) CRP for samples of which data was available ( CRP was only measured in Oxford CHIM ) . D0 = pre - challenge baseline Vi - TCV study ; nD7 = day 7 samples of participants who stayed well following challenge ( Vi - TCV study ) ; SPT = S . Paratyphi A ( 03NP ) ; ST = S . Typhi ( 03NP ) ; TD = Typhoid diagnosis ( Vi - TCV study ) . ( g ) Spearman ' s rank correlation of 5 - gene combined expression score ( left ) ( only nD7 TD samples from Oxford CHIM - Vi - TCV SPT ST cases from Nepal at presentation to hospital were included ) ( right ) CRP ( CRP was only available for Oxford CHIM - Vi - TCV samples we excluded D0 baseline measures ) .

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(f) Temperature and CRP for samples of which data was available (CRP was only measured in the Oxford CHIM). D0 = pre-challenge baseline Vi-TCV study; nD7 = day 7 samples of participants who stayed well following challenge (Vi-TCV study); SPT = S. Paratyphi A (03NP); ST = S. Typhi (03NP); TD = Typhoid diagnosis (Vi-TCV study). (g) Spearman's rank correlation of the 5-gene combined expression score and (left) temperature (only nD7 and TD samples from the Oxford CHIM - Vi-TCV and SPT and ST cases from Nepal at presentation to hospital were included) and (right) CRP (CRP was only available for Oxford CHIM - Vi-TCV samples and we excluded D0 baseline measures).


A . NLK but not kinase - negative NLK ( Flag - NLK - KM ; K155M ) caused mobility shift of EGFP - YAP . EGFP - YAP was transfected with empty vector , Flag - NLK - WT , or Flag - NLK - KM into HEK293T . Mobility shift was shown by immunoblotting .

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A. NLK but not kinase-negative NLK (Flag-NLK-KM; K155M) caused mobility shift of EGFP-YAP. EGFP-YAP was transfected with empty vector, Flag-NLK-WT, or Flag-NLK-KM into HEK293T cells. Mobility shift was shown by immunoblotting.


B . NLK phosphorylates YAP in vitro . Flag - NLK - WT , or Flag - NLK - KM was immunoprecipitated with anti - Flag antibody from lysates of HEK293T transfected with each plasmid subjected to in vitro kinase with bacterially purified GST - YAP in presence of [ 32P - ATP ] . Phosphorylation of GST - YAP autophosphorylation of NLK were examined by autoradiography ( top panel ) , Coomassie Blue showed similar of GST - YAP Flag - NLK ( bottom panel ) .

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B. NLK phosphorylates YAP in vitro. Flag-NLK-WT, or Flag-NLK-KM was immunoprecipitated with anti-Flag antibody from cell lysates of HEK293T cells transfected with each plasmid and subjected to in vitro kinase assay with bacterially purified GST-YAP in the presence of [32P-ATP]. Phosphorylation of GST-YAP and autophosphorylation of NLK were examined by autoradiography (top panel), and Coomassie Blue staining showed similar levels of GST-YAP and Flag-NLK (bottom panel).


C . NLK interacts with YAP at endogenous level . Immunoprecipitates from HEK293T lysates with indicated antibody were blotted with anti - YAP or anti - NLK antibody .

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C. NLK interacts with YAP at the endogenous level. Immunoprecipitates from HEK293T cell lysates with indicated antibody were blotted with anti-YAP or anti-NLK antibody.


B . NLK - WT but not NLK - KM reduces Ser127 phosphorylation of endogenous YAP . lysates from HEK293 transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in figure ( left panel ) . of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± . * * P < 0 . 01 . Student t - was used for statistical .

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B. NLK-WT but not NLK-KM reduces Ser127 phosphorylation of endogenous YAP. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. **P < 0.01. Student t-test was used for statistical analysis.


C . Deficiency of NLK leads to increased YAP phosphorylation at Ser127 . Lysates from wild type two independent NLK knockout HEK293 were immunoblotted with antibodies indicated in figure ( left panel ) . of p - YAP ( S127 ) or p - YAP ( S397 ) / total - YAP of three independent western blots was quantified ( right panel ) . Data are presented as mean ± . * P < 0 . 05 * * P < 0 . 01 . Student t - was used for statistical .

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C. Deficiency of NLK leads to increased YAP phosphorylation at Ser127. Lysates from wild type and two independent NLK knockout HEK293 cells were immunoblotted with antibodies indicated in the figure (left panel). The ratio of p-YAP(S127) or p-YAP(S397)/total-YAP of three independent western blots was quantified (right panel). Data are presented as mean ± sem. *P < 0.05 and **P < 0.01. Student t-test was used for statistical analysis.


D . Ectopic expression of NLK attenuates YAP phosphorylation at Ser127 while increases phosphorylation at Ser128 . lysates from HEK293 transfected with empty vector ( - ) , Flag - NLK - WT , or Flag - NLK - KM were immunoblotted with antibodies shown in figure .

D. Ectopic expression of NLK attenuates YAP phosphorylation at Ser127 while increases phosphorylation at Ser128. Cell lysates from HEK293 cells transfected with empty vector (-), Flag-NLK-WT, or Flag-NLK-KM were immunoblotted with antibodies shown in the figure.


E . Substitution of Ser127 with alanine enhances phosphorylation at Ser128 , vice versa . lysates from HEK293T transfected with empty vector ( - ) , EGFP - YAP , EGFP - YAP - S127A , or EGFP - YAP - S128A were immunoblotted with antibodies shown in figure .

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E. Substitution of Ser127 with alanine enhances phosphorylation at Ser128, and vice versa. Cell lysates from HEK293T cells transfected with empty vector (-), EGFP-YAP, EGFP-YAP-S127A, or EGFP-YAP-S128A were immunoblotted with antibodies shown in the figure.


A . Ectopic expression of NLK attenuates interaction between YAP 14 - 3 - 3 . Lysates from HEK293T transfected with plasmids indicated in figure were immunoprecipitated with anti - HA antibody immunoblotted with anti - GFP antibody ( top two panels ) . Whole lysates ( WCL ) were immunoblotted with antibodies shown in figure ( bottom three panels ) .

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A. Ectopic expression of NLK attenuates interaction between YAP and 14-3-3. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-HA antibody and immunoblotted with anti-GFP antibody (top two panels). Whole cell lysates (WCL) were immunoblotted with antibodies shown in the figure (bottom three panels).


B . Deficiency of NLK leads to increased interaction between YAP 14 - 3 - 3 . Lysates from NLK knockout HEK293 were immunoprecipitated with anti - YAP antibody immunoblotted with anti - 14 - 3 - 3 antibody ( top two panels ) . WCL were immunoblotted with antibodies indicated in figure ( bottom three panels ) .

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B. Deficiency of NLK leads to increased interaction between YAP and 14-3-3. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-YAP antibody and immunoblotted with anti-14-3-3 antibody (top two panels). WCL were immunoblotted with antibodies indicated in the figure (bottom three panels).


C . Phosphorylation status of Ser127 or Ser128 determines interaction between 14 - 3 - 3 YAP . Lysates from HEK293T transfected with plasmids indicated in figure were immunoprecipitated with anti - Flag antibody immunoblotted with anti - HA antibodies ( top two panels ) . Lysates were immunoblotted with anti - HA or anti - Flag antibodies ( bottom two panels ) . " n . s . " arrow indicates a non - specific band also visible in non - transfected lane .

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C. Phosphorylation status of Ser127 or Ser128 determines interaction between 14-3-3 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA antibodies (top two panels). Lysates were immunoblotted with anti-HA or anti-Flag antibodies (bottom two panels). "n.s." arrow indicates a non-specific band also visible in the non-transfected lane.


D . Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP . Immunofluorescence was performed in HeLa transfected with EGFP - YAP , EGFP - YAP - S127A , EGFP - YAP - S128A , or EGFP - YAP - S128D at high density . Figure shows a representative of multiple areas . Nuclei were stained with Hoechst 33342 . Scale bars : 20 μm .

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D. Phosphorylation status of Ser127 or Ser128 determines subcellular localization of YAP. Immunofluorescence analysis was performed in HeLa cells transfected with EGFP-YAP, EGFP-YAP-S127A, EGFP-YAP-S128A, or EGFP-YAP-S128D at high cell density. Figure shows a representative of multiple areas. Nuclei were stained with Hoechst 33342. Scale bars: 20 μm.


E . NLK knockout leads to increased localization of endogenous YAP . Immunoblot shows significant reduction of NLK level in NLK knockout HEK293 pools ( top panel ) .

E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. Immunoblot analysis shows significant reduction of NLK level in NLK knockout HEK293 cell pools (top panel).


E . NLK knockout leads to increased localization of endogenous YAP . To determine subcellular localization of YAP indirect immunofluorescence was performed in NLK knockout HEK293 pools ( bottom panel ) . Figure shows a representative of multiple areas . Nuclei were stained with DAPI . Scale bars : 20 μm .

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E. NLK knockout leads to increased cytoplasmic localization of endogenous YAP. To determine subcellular localization of YAP indirect immunofluorescence analysis was performed in NLK knockout HEK293 cell pools (bottom panel). Figure shows a representative of multiple areas. Nuclei were stained with DAPI. Scale bars: 20 μm.


A . Localization of endogenous YAP NLK in low ( LD ) or high density ( HD ) MCF10A ( upper panel ) or NIH3T3 ( bottom panel ) was examined by indirect immunofluorescence . Nuclei were stained with DAPI . Scale bars : 20 μm .

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A. Localization of endogenous YAP and NLK in low (LD) or high density (HD) MCF10A (upper panel) or NIH3T3 cells (bottom panel) was examined by indirect immunofluorescence. Nuclei were stained with DAPI. Scale bars: 20 μm.


B . Level of NLK decreases in a density - dependent manner . Lysates from low ( LD ) high density ( HD ) of MCF10A , HEK293T , NIH3T3 were immunoblotted with antibodies indicated in figure .

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B. Level of NLK decreases in a cell density-dependent manner. Lysates from low (LD) and high density (HD) of MCF10A, HEK293T, and NIH3T3 cells were immunoblotted with antibodies indicated in the figure.


C . Level of endogenous NLK mRNA is unaffected , whereas expression of YAP target genes was reduced , at high density . Quantitative real - time PCR analyses for expression of AMOTL2 , CTGF , CYR61 , NLK in low ( LD ) high density ( HD ) NIH3T3 were performed . of AMOTL2 , CTGF , CYR61 , NLK mRNA was normalized with level of Gapdh . Data represent average values from a representative of multiple experiments performed in triplicate . Error bars indicate standard deviations of triplicate measurements . Data are presented as mean ± . * P < 0 . 05 * * * P < 0 . 005 . Student t - was used for statistical .

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C. Level of endogenous NLK mRNA is unaffected, whereas the expression of YAP target genes was reduced, at high cell density. Quantitative real-time PCR analyses for expression of AMOTL2, CTGF, CYR61, and NLK in low (LD) and high density (HD) NIH3T3 cells were performed. Quantification of AMOTL2, CTGF, CYR61, and NLK mRNA was normalized with the level of Gapdh. Data represent average values from a representative of multiple experiments performed in triplicate. Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05 and ***P < 0.005. Student t-test was used for statistical analysis.


A . Knockout of NLK reduces interaction between endogenous YAP TEAD . Lysates from NLK knockout HEK293 were immunoprecipitated with anti - TEAD1 antibody immunoblotted with anti - TEAD1 antibody or anti - YAP antibody ( top panel ) . Whole lysates ( WCL ) were immunoblotted with antibodies indicated in figure .

A. Knockout of NLK reduces the interaction between endogenous YAP and TEAD. Lysates from NLK knockout HEK293 cells were immunoprecipitated with anti-TEAD1 antibody and immunoblotted with anti-TEAD1 antibody or anti-YAP antibody (top panel). Whole cell lysates (WCL) were immunoblotted with antibodies indicated in the figure.


B . Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 YAP . Lysates from HEK293T transfected with plasmids indicated in figure were immunoprecipitated with anti - Flag antibody immunoblotted with indicated antibodies in figure .

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B. Phosphorylation status of Ser127 or Ser128 determines interaction between TEAD1 and YAP. Lysates from HEK293T cells transfected with plasmids indicated in the figure were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies in the figure.


C . Downregulation of nemo with RNAi achieved in posterior compartment of wing imaginal discs ( co - expression of GFP serves to mark posterior compartment ) using hh - Gal4 driver results in decreased expression of Yorkie target gene DIAP1 . Two different RNAi constructs produce similar effect . Panels below show Z - stacks of magnified areas around A / P border ( left ) . of DIAP1 achieved upon posterior - downregulation of nemo , measured as normalized posterior - to - anterior compared to similar expression of GFP . Data are present as mean ± , n = 8 discs . * P < 0 . 05 . Student t - was used for statistical ( right ) . Scale bars : 50 µm .

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C. Downregulation of nemo with RNAi achieved in the posterior compartment of wing imaginal discs (co-expression of GFP serves to mark the posterior compartment) using the hh-Gal4 driver results in decreased expression of the Yorkie target gene DIAP1. Two different RNAi constructs produce similar effect. Panels below show Z-stacks of magnified areas around the A/P border (left). Quantification of DIAP1 levels achieved upon posterior-downregulation of nemo, measured as normalized posterior-to-anterior levels and compared to similar expression of GFP. Data are present as mean ± sem, n=8 discs. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.


D . Yorkie is epistatic to Nemo : co - overexpression of constitutively active mutant yki [ S168A ] together with nmo - RNAi produces typical yki [ S168A ] : severe overgrowth of affected with concomitant increase in yki target gene expression ( DIAP1 ) . Due to lethality of hh - Gal4 - driven co - overexpression , another posterior driver en - Gal4 was used instead ; anti - Hh ( green ) was used to mark posterior compartment ( left panel ) . posterior DIAP1 in UAS - RNAi - nemo ; en - Gal4 UAS - yki [ S168A ] ; en - Gal4 , presented as in ( C ) ; n = 3 - 6 discs . Posterior DIAP1 in UAS - RNAi - nemo ; en - Gal4 are also significantly different from control UAS - GFP ; en - Gal4 discs ( right panel ) . Data are presented as mean ± . * * * P < 0 . 005 . Student t - was used for statistical ( right ) . Scale bars : 50 µm .

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D. Yorkie is epistatic to Nemo: co-overexpression of the constitutively active mutant yki[S168A] together with nmo-RNAi produces typical yki[S168A] phenotypes: severe overgrowth of the affected area with concomitant increase in yki target gene expression (DIAP1). Due to lethality of the hh-Gal4-driven co-overexpression, another posterior driver en-Gal4 was used instead; anti-Hh staining (green) was used to mark the posterior compartment (left panel). Quantification posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 and UAS-yki[S168A]; en-Gal4, presented as in (C); n= 3-6 discs. Posterior DIAP1 levels in UAS-RNAi-nemo; en-Gal4 are also significantly different from control UAS-GFP; en-Gal4 discs (right panel). Data are presented as mean ± sem. ***P < 0.005. Student t-test was used for statistical analysis (right). Scale bars: 50 µm.


E . Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout . HEK293 wild type or HEK293 NLK knockout were transfected with indicated plasmids ( " EV " stands for " empty vector " ) , wounded areas after 0 24 hr were measured by using TScratch program ( left panel ) . Graphs show of unfilled areas at 24 hr compared to 0 hr , average values from a representative of multiple experiments performed in triplicate ( right - top panel ) . Lysates were immunoblotted with antibodies indicated in figure ( right - bottom panel ) . Error bars indicate standard deviations of triplicate measurements . Data are presented as mean ± . * P < 0 . 05 . Student t - was used for statistical ( right ) . Scale bars : 200 μm .

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E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ("EV" stands for "empty vector"), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel). Lysates were immunoblotted with antibodies indicated in the figure (right-bottom panel). Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± sem. *P < 0.05. Student t-test was used for statistical analysis (right). Scale bars: 200 μm.


E . Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout . HEK293 wild type or HEK293 NLK knockout were transfected with indicated plasmids ( " EV " stands for " empty vector " ) , wounded areas after 0 24 hr were measured by using TScratch program ( left panel ) . Graphs show of unfilled areas at 24 hr compared to 0 hr , average values from a representative of multiple experiments performed in triplicate ( right - top panel ) .

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E. Mutant forms of YAP which have different phosphorylation status of Ser127 or S128 differentially rescue retarded wound healing caused by NLK knockout. HEK293 wild type or HEK293 NLK knockout cells were transfected with indicated plasmids ("EV" stands for "empty vector"), and the wounded areas after 0 and 24 hr were measured by using the TScratch program (left panel). Graphs show percentage of unfilled areas at 24 hr compared to 0 hr, and average values from a representative of multiple experiments performed in triplicate (right-top panel).


Immunoblot of ADAR1 p110 , ADAR1 p150 ADAR2 expression in various organs isolated from wild - type ( WT ) mice . expression of GAPDH is shown as a reference .

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Immunoblot analysis of ADAR1 p110, ADAR1 p150 and ADAR2 expression in various organs isolated from wild-type (WT) mice. The expression of GAPDH is shown as a reference.


relative expression of ADAR1 p150 mRNA ( B ) total ADAR1 mRNA ( C ) in sorted DN , DP , 4SP 8SP thymocytes isolated from WT mice . relative expression of ADAR1 p150 mRNA ( B ) total ADAR1 mRNA ( C ) were normalized to level of expression in DN thymocytes ( DN values set to 1 ) . Values represent relative gene expression normalized to GAPDH mRNA are displayed as mean ± ( n = 5 ; Mann - Whitney U - , * p < 0 . 05 , * * p < 0 . 01 ) .

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The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. The relative expression of ADAR1 p150 mRNA (B) and total ADAR1 mRNA (C) were normalized to the level of expression in DN thymocytes (DN values set to 1). Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 5; Mann-Whitney U-test, *p < 0.05, **p < 0.01).


relative expression of type I interferon - stimulated genes ( ISGs ) in sorted DN , DP , 4SP 8SP thymocytes isolated from WT mice . Values represent relative gene expression normalized to GAPDH mRNA are displayed as mean ± ( n = 3 ; Mann - Whitney U - , * p < 0 . 05 ) . relative expression of ISGs was normalized against level of expression in DN thymocytes ( DN values set to 1 ) .

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The relative expression of type I interferon-stimulated genes (ISGs) in sorted DN, DP, 4SP and 8SP thymocytes isolated from WT mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The relative expression of ISGs was normalized against the level of expression in DN thymocytes (DN values set to 1).


Total number of thymocytes in wild - type ( WT ) CD4 + - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice .

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Total number of thymocytes in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice.


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - CD4 anti - CD8 antibodies analyzed by flow cytometry . Representative are shown ( B ) .

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (B).


percentages of DN , DP , 4SP , 8SP thymocytes were compared between WT Cd4cre A1fl / fl mice ( C ) .

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The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (C).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 anti - TCRβ antibodies were gated by high expression of TCRβ ( TCRβhigh ) . Representative are shown ( D ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and were gated by the high expression of TCRβ (TCRβhigh). Representative images are shown (D).


percentages of DN , DP , 4SP , 8SP thymocytes were compared between WT Cd4cre A1fl / fl mice ( E ) .

The percentages of DN, DP, 4SP, and 8SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (E).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD69 anti - MHC - I antibodies were gated on 4SP thymocytes . Representative are shown ( upper panel ) . percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , CD69 - MHC - I - ( M2 ) thymocytes were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with antibodies against CD5 analyzed by flow cytometry . Representative flow cytometric for DP 4SP thymocytes are shown ( upper panel ) . percentages of CD5 - DP 4SP thymocytes were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with antibodies against CD5 and analyzed by flow cytometry. Representative flow cytometric images for DP and 4SP thymocytes are shown (upper panel). The percentages of CD5- DP and 4SP thymocytes were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - CD4 , anti - CD8 , anti - CD25 anti - Foxp3 antibodies were gated on 4SP thymocytes . Representative are shown ( upper panel ) . percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg ) were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared between WT and Cd4cre A1fl/fl mice (lower panel).


CD4 + - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice were crossed with HY - TCR + mice to generate Cd4cre A1fl / fl HY - TCR + mice . Thymocytes isolated from ( A ) ( B ) HY - TCR + or Cd4cre A1fl / fl HY - TCR + mice were stained with anti - CD4 anti - CD8 antibodies analyzed by flow cytometry . Representative are shown ( left panels ) . absolute number of DP 8SP thymocytes were compared between HY - TCR + Cd4cre A1fl / fl HY - TCR + mice ( right panels ) .

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

CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with HY-TCR+ mice to generate Cd4cre A1fl/fl HY-TCR+ mice. Thymocytes isolated from male (A) and female (B) HY-TCR+ or Cd4cre A1fl/fl HY-TCR+ mice were stained with anti-CD4 and anti-CD8 antibodies and analyzed by flow cytometry. Representative images are shown (left panels). The absolute number of DP and 8SP thymocytes were compared between HY-TCR+ and Cd4cre A1fl/fl HY-TCR+ mice (right panels).


Thymocytes isolated from WT Cd4cre A1fl / fl mice were labeled with Proliferation Dye then stimulated with plate - bound anti - CD3 anti - CD28 antibodies for 4 days . Apoptotic ( Annexin V + ) proliferating thymocytes ( reduced of Proliferation Dye ) were determined by flow cytometry . Representative flow cytometric are shown ( left panel ) . percentages of either Annexin V + or Annexin V - proliferating thymocytes were compared between WT ( n = 5 ) Cd4cre A1fl / fl ( n = 4 ) mice ( right panel ) .

Thymocytes isolated from WT and Cd4cre A1fl/fl mice were labeled with Cell Proliferation Dye and then stimulated with plate-bound anti-CD3 and anti-CD28 antibodies for 4 days. Apoptotic (Annexin V+) and proliferating thymocytes (reduced intensity of Proliferation Dye) were determined by flow cytometry. Representative flow cytometric images are shown (left panel). The percentages of either Annexin V+ or Annexin V- proliferating thymocytes were compared between WT (n = 5) and Cd4cre A1fl/fl (n = 4) mice (right panel).


relative expression of anti - apoptotic genes in sorted 4SP thymocytes isolated from WT Cd4cre A1fl / fl mice . Values represent relative gene expression normalized to Actinβ mRNA are displayed as mean ± ( n = 3 ; Mann - Whitney U - , * p < 0 . 05 , N . S . ; not significant ) . mean value for relative expression of each anti - apoptotic gene in WT 4SP thymocytes was set as one .

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

The relative expression of anti-apoptotic genes in sorted 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to Actinβ mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05, N.S.; not significant). The mean value for the relative expression of each anti-apoptotic gene in WT 4SP thymocytes was set as one.


Immunoblot of total phosphorylated ZAP70 PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from WT Cd4cre A1fl / fl mice . expression of GAPDH is shown as a reference . Representative are shown ( left panel ) . band of p - ZAP70 p - PLCγ1 was normalized to that of total ZAP70 PLCγ1 ,

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

Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from WT and Cd4cre A1fl/fl mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively


Incidence of spontaneous colitis observed in wild - type ( WT ) CD4 + - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice ( n = 11 for each group ;

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Incidence of spontaneous colitis observed in wild-type (WT) and CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice (n = 11 for each group;


Representative of HE of colon dissected from a WT Cd4cre A1fl / fl mouse . Scale bar , 200 μm .

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Representative images of HE staining of the colon dissected from a WT and Cd4cre A1fl/fl mouse. Scale bar, 200 μm.


Lymphocytes isolated from colonic lamina propria of WT Cd4cre A1fl / fl mice were stained with anti - IFNγ , anti - IL17 or anti - Foxp3 antibodies together with anti - CD4 antibodies analyzed by flow cytometry . Representative are shown ( left panel ) . absolute number of IFNγ - , IL17 - , or Foxp3 - expressing CD4 + T were compared between WT Cd4cre A1fl / fl mice ( right panel ) .

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

Lymphocytes isolated from the colonic lamina propria of WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ, anti-IL17 or anti-Foxp3 antibodies together with anti-CD4 antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The absolute number of IFNγ-, IL17-, or Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (right panel).


Splenocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - IFNγ anti - IL17 together with anti - CD4 antibodies were gated on CD4 + . Representative flow cytometry are shown ( upper panel ) . percentages of IFNγ - IL17 - secreting CD4 + T were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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

Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Splenocytes isolated from WT Cd4cre A1fl / fl mice were stained with anti - Foxp3 together with anti - CD4 antibodies were gated on CD4 + . Representative flow cytometry are shown ( upper panel ) . percentages of Foxp3 - expressing CD4 + T were compared between WT Cd4cre A1fl / fl mice ( lower panel ) .

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

Splenocytes isolated from WT and Cd4cre A1fl/fl mice were stained with anti-Foxp3 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (upper panel). The percentages of Foxp3-expressing CD4+ T cells were compared between WT and Cd4cre A1fl/fl mice (lower panel).


Effector T ( Teff ) isolated from spleen of a WT mouse were stimulated with soluble anti - CD3 ( 5 μg / ml ) in presence of mitomycin C - treated splenocytes were cultured with regulatory T ( Treg ) isolated from WT Cd4cre A1fl / fl mice at indicated ratios . After 72 h , proliferative activity was measured . Values are displayed as mean ± ( n = 3 for each group ; Mann - Whitney U - , N . S . ; not significant ) . mean value for suppression of proliferative activity of Teff in a condition without Treg was set as 0 % .

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

Effector T (Teff) cells isolated from the spleen of a WT mouse were stimulated with soluble anti-CD3 (5 μg/ml) in the presence of mitomycin C-treated splenocytes and were cultured with regulatory T (Treg) cells isolated from WT and Cd4cre A1fl/fl mice at the indicated ratios. After 72 h, the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, N.S.; not significant). The mean value for the suppression of proliferative activity of Teff cells in a condition without Treg cells was set as 0%.


editing in 3 ′ UTR ( C ) intronic region ( D ) of each gene indicated in figures with their chromosomal location was validated by direct Sanger sequencing frequency of editing was compared among wild - type ( WT ) DP , WT 4SP thymocytes , 4SP thymocytes isolated from CD4 + - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice .

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

RNA editing in the 3′UTR (C) and intronic region (D) of each gene indicated in the figures with their chromosomal location was validated by direct Sanger sequencing and the frequency of editing was compared among wild-type (WT) DP, WT 4SP thymocytes, and 4SP thymocytes isolated from CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice.


relative expression of each interferon - stimulated gene ( ISG ) indicated in figure in sorted DP 4SP thymocytes isolated from WT Cd4cre A1fl / fl mice . Values represent relative gene expression normalized to GAPDH mRNA are displayed as mean ± ( n = 3 ; Mann - Whitney U - , * p < 0 . 05 ) . mean value for relative expression of each ISG in WT DP thymocytes was set as one .

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

The relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted DP and 4SP thymocytes isolated from WT and Cd4cre A1fl/fl mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in WT DP thymocytes was set as one.


Immunoblot of total phosphorylated STAT1 expression in thymocytes isolated from WT Cd4cre A1fl / fl mice ( n = 2 for each group ) . expression of GAPDH is shown as a reference .

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

Immunoblot analysis of total and phosphorylated STAT1 expression in thymocytes isolated from WT and Cd4cre A1fl/fl mice (n = 2 for each group). The expression of GAPDH is shown as a reference.


CD4 + - specific Adar1 knockout ( Cd4cre A1fl / fl ) mice were crossed with MDA5 knockout ( Ifih1 - / - ) mice to generate Cd4cre A1fl / fl Ifih1 - / - mice . Thymocytes isolated from Ifih1 - / - Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 anti - TCRβ antibodies analyzed by flow cytometry . Representative are shown ( left panel ) . of DN , DP , 4SP 8SP thymocytes were compared among Ifih1 - / - ( n = 4 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 5 ) Cd4cre A1fl / fl ( n = 7 ) mice ( right panel ) . same data shown in Fig 2C was used for Cd4cre A1fl / fl mice .

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

CD4+ cell-specific Adar1 knockout (Cd4cre A1fl/fl) mice were crossed with MDA5 knockout (Ifih1-/-) mice to generate Cd4cre A1fl/fl Ifih1-/- mice. Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8 and anti-TCRβ antibodies and analyzed by flow cytometry. Representative images are shown (left panel). The percentage of DN, DP, 4SP and 8SP thymocytes were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl (n = 7) mice (right panel). The same data shown in Fig 2C was used for Cd4cre A1fl/fl mice.


relative expression of each interferon - stimulated gene ( ISG ) indicated in figure in sorted 4SP thymocytes isolated from Ifih1 - / - Cd4cre A1fl / fl Ifih1 - / - mice . Values represent relative gene expression normalized to GAPDH mRNA are displayed as mean ± ( n = 3 ; Mann - Whitney U - , * p < 0 . 05 ) . mean value for relative expression of each ISG in Ifih1 - / - 4SP thymocytes was set as one .

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

The relative expression of each interferon-stimulated gene (ISG) indicated in the figure in sorted 4SP thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. Values represent the relative gene expression normalized to GAPDH mRNA and are displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, *p < 0.05). The mean value for the relative expression of each ISG in Ifih1-/- 4SP thymocytes was set as one.


Immunoblot of total phosphorylated ZAP70 PLCγ1 expression in anti - CD3 - stimulated thymocytes isolated from Ifih1 - / - Cd4cre A1fl / fl Ifih1 - / - mice . expression of GAPDH is shown as a reference . Representative are shown ( left panel ) . band of p - ZAP70 p - PLCγ1 was normalized to that of total ZAP70 PLCγ1 , respectively displayed as mean ± ( n = 3 ; Mann - Whitney U - , N . S . ; not significant ) ( right panel ) by setting mean value of stimulated Ifih1 - / - samples at 60 sec as 1 .

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

Immunoblot analysis of total and phosphorylated ZAP70 and PLCγ1 expression in anti-CD3-stimulated thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice. The expression of GAPDH is shown as a reference. Representative images are shown (left panel). The band intensity of p-ZAP70 and p-PLCγ1 was normalized to that of total ZAP70 and PLCγ1, respectively and displayed as the mean ± SEM (n = 3; Mann-Whitney U-test, N.S.; not significant) (right panel) by setting the mean value of stimulated Ifih1-/- samples at 60 sec as 1.


Thymocytes isolated from Ifih1 - / - Cd4cre A1fl / fl Ifih1 - / - mice were stimulated with plate - bound anti - CD3 ( 5 μg / ml ) anti - CD28 ( 1 μg / ml ) antibodies for 72 h proliferative activity was measured . Values are displayed as mean ± ( n = 3 for each group ; Mann - Whitney U - , * p < 0 . 05 ) . mean value for activity of Ifih1 - / - thymocytes in an unstimulated condition was set as one . For comparative , same data shown in Appendix Fig S1B was used for Cd4cre A1fl / fl mice .

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

Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) antibodies for 72 h and the proliferative activity was measured. Values are displayed as the mean ± SEM (n = 3 for each group; Mann-Whitney U-test, *p < 0.05). The mean value for the activity of Ifih1-/- thymocytes in an unstimulated condition was set as one. For comparative analysis, the same data shown in Appendix Fig S1B was used for Cd4cre A1fl/fl mice.


Thymocytes isolated from Ifih1 - / - Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 , anti - CD69 anti - MHC - I antibodies were gated on 4SP thymocytes . Representative are shown ( upper panel ) . percentages of CD69 + MHC - I - ( SM ) , CD69 + MHC - I + ( M1 ) , CD69 - MHC - I - ( M2 ) thymocytes were compared among Ifih1 - / - ( n = 5 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 3 ) Cd4cre A1fl / fl ( n = 3 ) mice ( lower panel ) . same data shown in Fig 2F was used for Cd4cre A1fl / fl mice .

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

Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD69 and anti-MHC-I antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD69+MHC-I- (SM), CD69+MHC-I+ (M1), and CD69-MHC-I- (M2) thymocytes were compared among Ifih1-/- (n = 5), Cd4cre A1fl/fl Ifih1-/- (n = 3) and Cd4cre A1fl/fl (n = 3) mice (lower panel). The same data shown in Fig 2F was used for Cd4cre A1fl/fl mice.


Thymocytes isolated from Ifih1 - / - Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - CD4 , anti - CD8 , anti - CD25 anti - Foxp3 antibodies were gated on 4SP thymocytes . Representative are shown ( upper panel ) . percentages of CD25 + Foxp3 + 4SP thymocytes ( thymic Treg ) were compared among Ifih1 - / - , Cd4cre A1fl / fl Ifih1 - / - Cd4cre A1fl / fl mice ( lower panel ) . same data shown in Fig 2H was used for Cd4cre A1fl / fl mice .

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

Thymocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-CD4, anti-CD8, anti-CD25 and anti-Foxp3 antibodies and were gated on 4SP thymocytes. Representative images are shown (upper panel). The percentages of CD25+Foxp3+ 4SP thymocytes (thymic Treg cells) were compared among Ifih1-/-, Cd4cre A1fl/fl Ifih1-/- and Cd4cre A1fl/fl mice (lower panel). The same data shown in Fig 2H was used for Cd4cre A1fl/fl mice.


Splenocytes isolated from Ifih1 - / - Cd4cre A1fl / fl Ifih1 - / - mice were stained with anti - IFNγ anti - IL17 together with anti - CD4 antibodies were gated on CD4 + . Representative flow cytometry are shown ( left panel ) . percentages of IFNγ - IL17 - secreting CD4 + T were compared among Ifih1 - / - ( n = 4 ) , Cd4cre A1fl / fl Ifih1 - / - ( n = 5 ) Cd4cre A1fl / fl mice ( n = 6 ) ( right panel ) . same data shown in Fig 4E was used for Cd4cre A1fl / fl mice .

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

Splenocytes isolated from Ifih1-/- and Cd4cre A1fl/fl Ifih1-/- mice were stained with anti-IFNγ and anti-IL17 together with anti-CD4 antibodies and were gated on CD4+ cells. Representative flow cytometry images are shown (left panel). The percentages of IFNγ- and IL17-secreting CD4+ T cells were compared among Ifih1-/- (n = 4), Cd4cre A1fl/fl Ifih1-/- (n = 5) and Cd4cre A1fl/fl mice (n = 6) (right panel). The same data shown in Fig 4E was used for Cd4cre A1fl/fl mice.


No incidence of spontaneous colitis observed in Ifih1 - / - ( n = 5 ) Cd4cre A1fl / fl Ifih1 - / - ( n = 11 ) mice . For side - by - side comparison with Cd4cre A1fl / fl

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

No incidence of spontaneous colitis observed in Ifih1-/- (n = 5) and Cd4cre A1fl/fl Ifih1-/- (n = 11) mice. For side-by-side comparison with Cd4cre A1fl/fl mice


Representative of HE of colon dissected from an Ifih1 - / - a Cd4cre A1fl / fl Ifih1 - / - mouse . Scale bar , 200 μm .

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Representative images of HE staining of colon dissected from an Ifih1-/- and a Cd4cre A1fl/fl Ifih1-/- mouse. Scale bar, 200 μm.


A Indicated tail - anchored ( TA ) were in vitro translated in rabbit reticulocyte lysate ( RRL ) using RNAs containing a stop codon . Translations were carried out in presence of 2 μM HisTrx or HisTrx - SGTA ( HT - SGTA ) , followed by incubation with HisPur Cobalt resin Bound were eluted at high imidazole , samples resolved by SDS - PAGE results visualised by phosphorimaging . Sec61β 3R carries three Arg residues within Sec61β transmembrane domain ( TMD ) which abolish its hydrophobic character was used as a negative control for SGTA binding . A schematic representation of a TA - is also shown . Open circles indicate TA - selectively bound by HisTrx - SGTA . RAMP4 - stress - associated endoplasmic reticulum 1 ; Syb2 - synaptobrevin 2 ; Synt5 - syntaxin 5 . B As for ( A ) but total translation products ( input ) were resolved by SDS - PAGE results visualised by phosphorimaging . When total products are analysed high amounts of haemoglobin in RRL distort migration of Sec61β its variant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Indicated tail-anchored (TA) proteins were in vitro translated in rabbit reticulocyte lysate (RRL) using RNAs containing a stop codon. Translations were carried out in the presence of 2 μM HisTrx or HisTrx-SGTA (HT-SGTA), followed by incubation with HisPur Cobalt resin Bound proteins were eluted at high imidazole concentration, samples resolved by SDS-PAGE and results visualised by phosphorimaging. Sec61β 3R carries three Arg residues within Sec61β transmembrane domain (TMD) which abolish its hydrophobic character and was used as a negative control for SGTA binding. A schematic representation of a TA-protein is also shown. Open circles indicate TA-proteins selectively bound by HisTrx-SGTA. RAMP4 - stress-associated endoplasmic reticulum protein 1; Syb2 - synaptobrevin 2; Synt5 - syntaxin 5. B As for (A) but total translation products (input) were resolved by SDS-PAGE and results visualised by phosphorimaging. When total products are analysed high amounts of haemoglobin in the RRL distort the migration of Sec61β and its variant.


RNAs coding for C99 variants containing a stop codon introduced either after complete coding or before predicted TMD were used . E As for ( D ) but total translation products were subjected to immunoprecipation with either anti - C99 antibody ( for full - variants ) or with anti - β - amyloid antibody ( for products lacking TMD ) . A filled square indicates inefficiently translated APP - C99 - TMD immunoprecipitated with anti - β - amyloid antibody .

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

RNAs coding for C99 variants and containing a stop codon introduced either after the complete coding sequence or before the predicted TMD were used. E As for (D) but total translation products were subjected to immunoprecipation with either anti-C99 antibody (for the full-length variants) or with anti-β-amyloid antibody (for products lacking the TMD). A filled square indicates inefficiently translated APP-C99-TMD immunoprecipitated with anti-β-amyloid antibody.


A Full - polypeptides were translated in presence of 2 μM HisTrx or HisTrx - SGTA using RNAs lacking a stop codon either stabilised at ribosome with cycloheximide ( CHX ) or released with puromycin ( puro ) . Reactions were incubated with HisPur Cobalt resin , bound eluted at high imidazole , resolved by SDS - PAGE visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX - treated samples .

A Full-length polypeptides were translated in the presence of 2 μM HisTrx or HisTrx-SGTA using RNAs lacking a stop codon and either stabilised at the ribosome with cycloheximide (CHX) or released with puromycin (puro). Reactions were incubated with HisPur Cobalt resin, bound proteins eluted at high imidazole concentration, resolved by SDS-PAGE and visualised by phosphorimaging Filled circles indicate slower migrating species of radiolabelled products selectively enriched in CHX-treated samples.


total translation reactions were treated with RNaseA immunoprecipitated with anti - C99

total translation reactions were treated with RNaseA and immunoprecipitated with anti-C99 antibody


Following in vitro translation of indicated in presence of 2 μM HisTrx - SGTA recovery on HisPur Cobalt resin eluted fractions were subjected to RNaseA treatment ( C ) - tRNA " " + tRNA " indicate tRNA - lacking tRNA - bound species , respectively .

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

Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin the eluted fractions were subjected to RNaseA treatment (C) -tRNA" and "+ tRNA" indicate tRNA-lacking and tRNA-bound protein species, respectively.


D Following in vitro translation of indicated in presence of 2 μM HisTrx - SGTA recovery on HisPur Cobalt resin precipitation with hexadecyltrimethylammonium bromide ( CTAB ) ( D ) ; - tRNA " " + tRNA " indicate tRNA - lacking tRNA - bound species , respectively .

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D Following in vitro translation of the indicated proteins in the presence of 2 μM HisTrx-SGTA and recovery on HisPur Cobalt resin precipitation with hexadecyltrimethylammonium bromide (CTAB) (D) ;-tRNA" and "+ tRNA" indicate tRNA-lacking and tRNA-bound protein species, respectively.


B Variants of PPLssKO - C99 were translated in presence of 2 μM HisTrx - SGTA using templates lacking a stop codon terminating either before ( " - TMD " ) or at indicated acid after TMD . Pull - down with HisPur Cobalt resin was carried out as described in Materials Methods , eluted material resolved by SDS - PAGE results visualised by phosphorimaging . tRNA - bound species were verified by treating PPLssKO - C99 variant terminating immediately after TMD ( TMD + 0 ) with RNaseA ( lane 9 ) .

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B Variants of PPLssKO-C99 were translated in the presence of 2 μM HisTrx-SGTA using templates lacking a stop codon and terminating either before ("-TMD") or at indicated amino acid distance after the TMD. Pull-down with HisPur Cobalt resin was carried out as described in Materials and Methods, eluted material resolved by SDS-PAGE and results visualised by phosphorimaging. tRNA-bound species were verified by treating PPLssKO-C99 variant terminating immediately after the TMD (TMD + 0) with RNaseA (lane 9).


translation reactions were carried out using RNAs coding for indicated TA - lacking a stop codon . Sec61β 3R was used as a control without a functional TMD . Filled circles indicate tRNA - bound TA - species recovered with HisTrx - SGTA .

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translation reactions were carried out using RNAs coding for the indicated TA-proteins and lacking a stop codon. Sec61β 3R was used as a control protein without a functional TMD. Filled circles indicate tRNA-bound TA-protein species recovered with HisTrx-SGTA.


D Rabbit reticulocyte lysate was left untreated ( " untreat . " ) , incubated with control immobilised antibodies ( " contr . depl . " ) or immunodepleted of Hbs1L , a factor that together with Pelota ABCE1 [ 4 ] , mediates splitting of ribosomes stalled on truncated mRNAs ( see diagram ) . These lysates were used to translate FLAG - tagged Sec61β from an mRNA lacking a stop codon in presence of 2 μM HisTrx or HisTrx - SGTA samples were processed as described for ( B ) .

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D Rabbit reticulocyte lysate was left untreated ("untreat."), incubated with control immobilised antibodies ("contr. depl.") or immunodepleted of Hbs1L, a factor that together with Pelota and ABCE1 [4], mediates splitting of ribosomes stalled on truncated mRNAs (see diagram). These lysates were used to translate FLAG-tagged Sec61β from an mRNA lacking a stop codon in the presence of 2 μM HisTrx or HisTrx-SGTA and samples were processed as described for (B).


E tRNA - bound species of FLAG - tagged Sec61β from experiment shown in panel ( D ) were quantified expressed relative to value obtained for control - depleted RRL . Shown is mean with standard error of mean for n = 4 biological replicates . Statistical significance was calculated using unpaired t with Welch ' s correction . ns - not significant .

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E tRNA-bound species of FLAG-tagged Sec61β from experiment shown in panel (D) were quantified and expressed relative to the value obtained for the control-depleted RRL. Shown is the mean with standard error of mean for n=4 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction. ns - not significant.


Sec61β wild - type ( WT ) 3R variant tagged at their N - terminus with FLAG epitope were translated in RRL from RNAs lacking a stop codon . Ribosome - nascent chain complexes ( RNCs ) were recovered using anti - FLAG affinity resin , beads were washed , bound eluted with 3xFLAG peptide RNCs isolated by pelleting through a sucrose cushion Samples were resolved by SDS - PAGE immunoblotted for endogenous rabbit SGTA , FLAG - tagged translation products , of 40S ( Rps3 ) 60S ( Rpl17 ) ribosomal subunits or analysed by phosphorimaging ( 35S ) . 3xFLAG peptide eluted material ( lanes 4 5 ) corresponds to ~ 10 % of sedimented RNCs ( lanes 7 8 ) .

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Sec61β wild-type (WT) and the 3R variant tagged at their N-terminus with the FLAG epitope were translated in RRL from RNAs lacking a stop codon. Ribosome-nascent chain complexes (RNCs) were recovered using anti-FLAG affinity resin, beads were washed, bound proteins eluted with 3xFLAG peptide and RNCs isolated by pelleting through a sucrose cushion Samples were resolved by SDS-PAGE and immunoblotted for endogenous rabbit SGTA, FLAG-tagged translation products, proteins of the 40S (Rps3) and 60S (Rpl17) ribosomal subunits or analysed by phosphorimaging (35S). The 3xFLAG peptide eluted material (lanes 4 and 5) corresponds to ~10% of the sedimented RNCs (lanes 7 and 8).


A , B PPL - C99 - TMD ( A ) PPL - C99FL ( B ) were in vitro translated in RRL from mRNAs lacking a stop codon in presence of 2 μM HisTrx or HisTrx - SGTA ε - TDBA - Lys - tRNA analogue . Reactions were irradiated with UV to induce photo - cross - linking , RNCs isolated adducts immunoprecipitated with mouse anti - SRP54 , mouse anti - SGTA or a control antibody as described in Materials Methods . Samples were resolved by SDS - PAGE results visualised by phosphorimaging . C - terminal 40 acid residues located in ribosomal exit tunnel are indicated . " x SRP54 " " x HisTrx - SGTA " indicate cross - linking adducts between translated nascent chain endogenous SRP54 or recombinant HisTrx - SGTA , respectively . Arrows indicate unmodified translation products .

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A, B PPL-C99-TMD (A) and PPL-C99FL (B) were in vitro translated in RRL from mRNAs lacking a stop codon in the presence of 2 μM HisTrx or HisTrx-SGTA and ε-TDBA-Lys-tRNA analogue. Reactions were irradiated with the UV light to induce photo-cross-linking, RNCs isolated and adducts immunoprecipitated with mouse anti-SRP54, mouse anti-SGTA or a control antibody as described in Materials and Methods. Samples were resolved by SDS-PAGE and results visualised by phosphorimaging. C-terminal 40 amino acid residues located in the ribosomal exit tunnel are indicated. "x SRP54" and "x HisTrx-SGTA" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively. Arrows indicate unmodified translation products.


C PPL - C99FL variants lacking a stop codon carrying indicated Lys to Arg substitutions were in vitro translated in presence of 2 μM HisTrx - SGTA . Reactions were processed using chicken anti - SGTA antibody for immunoprecipitation of SGTA adducts . D Cross - linking adducts between ribosome - stalled PPL - C99FL lysine mutants recombinant HisTrx - SGTA shown in panel ( C ) were quantified , any differences in translation efficiency accounted for cross - linking efficiency expressed relative to wild - type ( WT ) . Shown is mean with standard error of mean for n = 3 biological replicates . Statistical significance was calculated using unpaired t with Welch ' s correction .

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C PPL-C99FL variants lacking a stop codon and carrying the indicated Lys to Arg substitutions were in vitro translated in the presence of 2 μM HisTrx-SGTA. Reactions were processed using chicken anti-SGTA antibody for immunoprecipitation of SGTA adducts. D Cross-linking adducts between ribosome-stalled PPL-C99FL lysine mutants and recombinant HisTrx-SGTA shown in panel (C) were quantified, any differences in translation efficiency accounted for and cross-linking efficiency expressed relative to the wild-type (WT) protein. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.


A , B PPL - C99FL lacking a stop codon was in vitro translated in RRL in presence of canine pancreatic microsomes or buffer control in reactions supplemented with ε - TDBA - Lys - tRNA analogue . Reactions were carried out in presence ( A ) or absence ( B ) of 2 μM HisTrx - SGTA . Samples were subjected to UV - induced photo - cross - linking processed as described for Fig 5 . " x SRP54 " " x HisTrx - SGTA " indicate cross - linking adducts between translated nascent chain endogenous SRP54 or recombinant HisTrx - SGTA , respectively , whilst " endo . SGTA " indicates cross - linking adducts with endogenous SGTA . Cross - linking products between stalled PPL - C99FL SGTA are also indicated with arrowheads , whilst arrows indicate unmodified translation products .

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A, B PPL-C99FL lacking a stop codon was in vitro translated in RRL in the presence of canine pancreatic microsomes or buffer control in reactions supplemented with ε-TDBA-Lys-tRNA analogue. Reactions were carried out in the presence (A) or absence (B) of 2 μM HisTrx-SGTA. Samples were subjected to UV light-induced photo-cross-linking and processed as described for Fig 5. "x SRP54" and "x HisTrx-SGTA" indicate cross-linking adducts between the translated nascent chain and endogenous SRP54 or recombinant HisTrx-SGTA, respectively, whilst "endo. SGTA" indicates cross-linking adducts with endogenous SGTA. Cross-linking products between stalled PPL-C99FL and SGTA are also indicated with arrowheads, whilst arrows indicate unmodified translation products.


C , D chemical cross - linking using DSS reagent was carried out . E DSS - mediated HisTrx - SGTA cross - linking efficiency to PPL - C99FL in absence or presence of ER - derived microsomes was quantified . For each repeat , of cross - linked SGTA adduct in absence of microsomes was set as 100 % . Shown is mean with standard error of mean for n = 3 biological replicates . Statistical significance was calculated using unpaired t with Welch ' s correction .

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C, D chemical cross-linking using DSS reagent was carried out. E DSS-mediated HisTrx-SGTA cross-linking efficiency to PPL-C99FL in the absence or presence of ER-derived microsomes was quantified. For each repeat, intensity of the cross-linked SGTA adduct in the absence of microsomes was set as 100%. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction.


A Indicated precursor were translated in vitro using RRL supplemented with 20 μM HA - tagged ubiquitin 2 μM of either HisTrx or HisTrx - SGTA . RNCs were isolated , ubiquitinated nascent chains recovered using anti - HA agarose , samples resolved by SDS - PAGE analysed by phosphorimaging . Ubiquitinated precursor species were assigned on SDS - PAGE electrophoretic mobility are indicated in red . A schematic diagram of used is shown ~ 40 residues of nascent chain located in ribosomal exit tunnel are indicated . " ss " - signal , " TMD " - transmembrane domain .

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A Indicated precursor proteins were translated in vitro using RRL supplemented with 20 μM HA-tagged ubiquitin and 2 μM of either HisTrx or HisTrx-SGTA. RNCs were isolated, ubiquitinated nascent chains recovered using anti-HA agarose, samples resolved by SDS-PAGE and analysed by phosphorimaging. Ubiquitinated precursor protein species were assigned based on SDS-PAGE electrophoretic mobility and are indicated in red. A schematic diagram of the proteins used is shown and the ~40 residues of the nascent chain located in the ribosomal exit tunnel are indicated. "ss" - signal sequence, "TMD" - transmembrane domain.


B , clearly resolved ubiquitinated species of indicated ( labelled 1 to 4 ) were quantified their in HisTrx - SGTA containing samples shown relative to samples supplemented with HisTrx control . Shown is mean with standard error of mean for n = 3 biological replicates . Statistical significance was calculated using unpaired t with Welch ' s correction . ns - not significant .

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B Individual, clearly resolved ubiquitinated species of the indicated proteins (labelled 1 to 4) were quantified and their intensity in HisTrx-SGTA containing samples shown relative to samples supplemented with HisTrx control protein. Shown is the mean with standard error of mean for n=3 biological replicates. Statistical significance was calculated using unpaired t test with Welch's correction. ns - not significant.


G Verify mobility of GFP ( control ) mRNA GFP - MdCAX3 mRNA by RT - PCR in Mx leaf , , root . recombinant vectors ( F ) were transformed into A . tumefaciens GV3101 introduced into Mx leaves by vacuum infiltration .

G Verify the mobility of GFP (control) mRNA and GFP-MdCAX3 mRNA by RT-PCR in Mx leaf, stem, and root. The recombinant vectors (F) were transformed into A. tumefaciens GV3101 and introduced into Mx leaves by vacuum infiltration.


H GFP image of Mx roots under different treatment . Nuclei were labeled using DAPI . Scale Bar : 100 μm .

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H GFP fluorescence image of the Mx roots under different treatment. Nuclei were labeled using DAPI. Scale Bar: 100 μm.


H ( 488 / 440 ) image of BCECF in roots of MdCAX3 - suppressed in leaves Md / Mx . Scale bars : 100 μm .

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H Ratio (488/440) image of BCECF fluorescence in the roots of MdCAX3-suppressed in leaves Md/Mx. Scale bars: 100 μm.


I Rhizosphere acidification of MdCAX3 - suppressed in leaves Md / Mx . Bromocresol purple was used as a pH indicator for visualization . Scale bars : 1 cm .

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I Rhizosphere acidification of MdCAX3-suppressed in leaves Md/Mx. Bromocresol purple was used as a pH indicator for visualization. Scale bars: 1 cm.


F GUS in N . benthamiana leaves after transient co - expression of pro35S : : GFP with proMxCXIP1 : GUS or proMbCXIP1 : GUS , co - expression with pBI101 : GUS as a control .

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F GUS staining in N. benthamiana leaves after transient co-expression of pro35S::GFP with proMxCXIP1:GUS or proMbCXIP1:GUS, and co-expression with pBI101:GUS as a control.


G Interaction between MdCXIP1 MdCAX3 tested by yeast two - hybrid ( Y2H ) assays . A dilution series of NMY51 yeast co - expressing MdCXIP1 MdCAX3 cultured on SD / II SDIV screening media . pPBT3 - N ( empty vector ) co - expressed with MdCXIP1 was used as a negative control . X - gal was used in SD / - Trp / - Leu / - His / - Ade screening medium to further for possible interactions .

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G Interaction between MdCXIP1 and MdCAX3 tested by yeast two-hybrid (Y2H) assays. A dilution series of NMY51 yeast cells co-expressing MdCXIP1 and MdCAX3 cultured on SD/II and SDIV screening media. pPBT3-N (empty vector) co-expressed with MdCXIP1 was used as a negative control. X-gal was used in SD/-Trp/-Leu/-His/-Ade screening medium to further test for possible interactions.


I Interaction between MdCXIP1 MdCAX3 N - terminal autoinhibitory domain in an in vitro pull - down . His - MdCXIP1 , GST GST - N - MdCAX3 fusion were expressed in Escherichia coli BL21 , GST alone was used as a negative control . ' + ' indicates presence " − " indicates absence . IB , immunoblot .

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I Interaction between MdCXIP1 and the MdCAX3 N-terminal autoinhibitory domain in an in vitro pull-down assay. The His-MdCXIP1, GST and GST-N-MdCAX3 fusion proteins were expressed in the Escherichia coli BL21 strain, and the GST protein alone was used as a negative control. '+' indicates presence and "−" indicates absence. IB, immunoblot.