. D . Pilot study for dominant - negative inhibition ( DNI ) of Δ159 and Δ159 - 175 . DNI was assessed by co - transfecting 22L - ScN2a cells with ( 3F4 ) MoPrP and Δ159 or Δ159 - 175 , respectively , and testing for PK - resistant ( 3F4 ) MoPrP . Left panel shows representative immunoblot ( mAb 3F4 ) and right panel the statistical analysis of quantified PK - res levels of a triplicate experiment . Empty pcDNA3 . 1 plasmid was used as control for co - transfection in lane 1 . The error bars indicate standard deviation . * , p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Pilot study for dominant-negative inhibition (DNI) of Δ159 and Δ159-175. DNI was assessed by co-transfecting 22L-ScN2a cells with (3F4)MoPrP and Δ159 or Δ159-175, respectively, and testing for PK-resistant (3F4)MoPrP. Left panel shows representative immunoblot (mAb 3F4) and right panel the statistical analysis of quantified PK-res levels of a triplicate experiment. Empty pcDNA3.1 plasmid was used as control for co-transfection in lane 1. The error bars indicate standard deviation. *, p<0.05.


A . N - linked glycans of ΔPrPs are not complex - type . Immunoblot comparing EndoH - and non - digested lysates of ΔPrP159 and Δ159 - 175 transfected cells . Left panel was probed with mAb 3F4 mAb , detecting transiently expressed ΔPrPs . On the right panel , the membrane was re - probed with mAb 4H11 , detecting total PrP . Arrowheads depict deglycosylated ΔPrPs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. N-linked glycans of ΔPrPs are not complex-type. Immunoblot comparing EndoH- and non-digested lysates of ΔPrP159 and Δ159-175 transfected cells. Left panel was probed with mAb 3F4 mAb, detecting transiently expressed ΔPrPs. On the right panel, the membrane was re-probed with mAb 4H11, detecting total PrP. Arrowheads depict deglycosylated ΔPrPs.


B . The additional band of Δ159 - 167 represents a third N - glycan sensitive to EndoH . Upon EndoH digestion , all bands converge into a non - glycoform band ( lane 3 and 4 ) . In Δ159 - 167 ( 169 ) the third glycosylation site is not present anymore .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The additional band of Δ159-167 represents a third N-glycan sensitive to EndoH. Upon EndoH digestion, all bands converge into a non-glycoform band (lane 3 and 4). In Δ159-167(169) the third glycosylation site is not present anymore.


C . ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface . Confocal microscopy images of N2a cells transiently transfected with ( 3F4 ) MoPrP , Δ159 or Δ159 - 175 , either permeabilized ( right ) or non - permeabilized ( left ) with 0 . 2 % TX100 after fixation . Cells were immuno - labeled with mAb 3F4 . ( 3F4 ) MoPrP - expressing cells show bright immunopositivity on the cell surface , whereas ΔPrPs are not observed on the cell surface . Also intracellular staining differs strongly between ( 3F4 ) MoPrP and ΔPrP expressing cells Scale bars , 25 µm .

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

C. ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface. Confocal microscopy images of N2a cells transiently transfected with (3F4)MoPrP, Δ159 or Δ159-175, either permeabilized (right) or non-permeabilized (left) with 0.2% TX100 after fixation. Cells were immuno-labeled with mAb 3F4. (3F4)MoPrP-expressing cells show bright immunopositivity on the cell surface, whereas ΔPrPs are not observed on the cell surface. Also intracellular staining differs strongly between (3F4)MoPrP and ΔPrP expressing cells Scale bars, 25 µm.


A . ΔPrPs are insoluble in detergent solution . Immunoblots showing partition of Δ159 and Δ159 - 175 in supernatant ( Sup ) and pellet fraction after ultracentrifugation of lysates supplemented with 4 % sarcosyl . Upper panel is mAb 3F4 , lower panel is membrane re - probed with mAb 4H11 detecting also endogenous wild - type PrP besides ΔPrPs . The majority of ΔPrPs is found in the insoluble fraction in both infected and non - infected cells ( upper panel , lanes 3 , 4 , 7 and 8 ) . Wild - type PrP almost completely partitions into the soluble fraction ( Sup ) in non - infected N2a cells with present conditions . PrP in pellet fractions of non - infected N2a cells is from ΔPrPs as can be seen from the banding pattern ( Lane 7 and 8 , square bracket ) . PrP in lanes 3 and 4 ( lower panel ) is PrP27 - 30 .

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A. ΔPrPs are insoluble in detergent solution. Immunoblots showing partition of Δ159 and Δ159-175 in supernatant (Sup) and pellet fraction after ultracentrifugation of lysates supplemented with 4% sarcosyl. Upper panel is mAb 3F4, lower panel is membrane re-probed with mAb 4H11 detecting also endogenous wild-type PrP besides ΔPrPs. The majority of ΔPrPs is found in the insoluble fraction in both infected and non-infected cells (upper panel, lanes 3, 4, 7 and 8). Wild-type PrP almost completely partitions into the soluble fraction (Sup) in non-infected N2a cells with present conditions. PrP in pellet fractions of non-infected N2a cells is from ΔPrPs as can be seen from the banding pattern (Lane 7 and 8, square bracket). PrP in lanes 3 and 4 (lower panel) is PrP27-30.


B . ΔPrPs are moderately resistant to chymotrypsin digestion . Immunoblots showing chymotrypsin - resistant fragments of Δ159 and Δ159 - 175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin ( Chy ) . Note that full - length diglycoform of ΔPrP still remains at 8 µg / ml Chy ( lanes 5 and 10 , upper panel , closed arrowhead ) , whereas that of endogenous wild - type PrP is completely digested ( lanes 5 and 10 , lower panel , open arrowhead ) and only small amounts of truncated fragments remained ( curly bracket ) . Other smaller fragments in lane 5 and 10 ( square - brackets ) represent ΔPrPs .

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B. ΔPrPs are moderately resistant to chymotrypsin digestion. Immunoblots showing chymotrypsin-resistant fragments of Δ159 and Δ159-175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin (Chy). Note that full-length diglycoform of ΔPrP still remains at 8 µg/ml Chy (lanes 5 and 10, upper panel, closed arrowhead), whereas that of endogenous wild-type PrP is completely digested (lanes 5 and 10, lower panel, open arrowhead) and only small amounts of truncated fragments remained (curly bracket). Other smaller fragments in lane 5 and 10 (square-brackets) represent ΔPrPs.


A . ΔPrPs are substantially soluble in detergent without GdnHCl denaturation . Immunoblots probed with mAbs 3F4 ( left ) or 4H11 ( right , membrane re - probed ) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159 - 175 . Det denotes detergent phase , Aq aqueous phase .

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

A. ΔPrPs are substantially soluble in detergent without GdnHCl denaturation. Immunoblots probed with mAbs 3F4 (left) or 4H11 (right, membrane re-probed) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159-175. Det denotes detergent phase, Aq aqueous phase.


B . ΔPrPs are highly hydrophobic after denaturation with GdnHCl . Representative immunoblot of TX114 - treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159 - 175 almost exclusively in detergent phase ( lanes 1 - 4 ) . PIPLC treatment ( + , even lanes ) was done after denaturation with GdnHCl or not ( − , uneven lanes ) . PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions .

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

B. ΔPrPs are highly hydrophobic after denaturation with GdnHCl. Representative immunoblot of TX114-treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159-175 almost exclusively in detergent phase (lanes 1-4). PIPLC treatment (+, even lanes) was done after denaturation with GdnHCl or not (−, uneven lanes). PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions.


C . Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion . After further diluting out GdnHCl by phase - separation and removal of the aqueous phase containing GdnHCl , PIPLC was able to digest ΔPrPs . Left two panels ( upper mAb 3F4 , lower 4H11 ) are shorter expositions , right panels longer expositions to show also difference in PrP levels in the detergent phase . Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion ( e . g . upper - left panel , lanes 1 , 3 vs . 2 , 4 ) . Migration of ΔPrPs in the aqueous phase of PIPLC - digested samples was slightly slower than that of PIPLC - undigested samples ( − ) ( upper - right panel , lane 9 vs . 10 ) .

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

C. Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion. After further diluting out GdnHCl by phase-separation and removal of the aqueous phase containing GdnHCl, PIPLC was able to digest ΔPrPs. Left two panels (upper mAb 3F4, lower 4H11) are shorter expositions, right panels longer expositions to show also difference in PrP levels in the detergent phase. Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion (e.g. upper-left panel, lanes 1, 3 vs. 2, 4). Migration of ΔPrPs in the aqueous phase of PIPLC-digested samples was slightly slower than that of PIPLC-undigested samples (−) (upper-right panel, lane 9 vs. 10).


A . Degradation of ΔPrPs by lysosomal and proteasomal systems . ( Left ) N2a cells transiently transfected with Δ159 or Δ159 - 175 were treated for 7 hours with inhibitors either for lysosomal degradation ( bafilomycin A1 , Baf , 120 nM ) , autophagy ( 3 - methyladenine , 3MA , 10 mM ) , or the proteasome ( MG132 , 10 µM ) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159 - 175 ( mAb 3F4 ) . Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs , respectively . An increment in diglycoforms is observed in Baf - and 3MA - treated cells , whereas in MG132 - treated cells also the non - glycoforms are increased . ( Right ) A graph showing quantification of all PrP bands by densitometric analysis . Data from 3 independent ( one in duplicate ) experiments for Δ159 and 2 independent ( one in duplicate ) for Δ159 - 175 were statistically analyzed for mean and standard deviation ( error bars ) .

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

A. Degradation of ΔPrPs by lysosomal and proteasomal systems. (Left) N2a cells transiently transfected with Δ159 or Δ159-175 were treated for 7 hours with inhibitors either for lysosomal degradation (bafilomycin A1, Baf, 120 nM), autophagy (3-methyladenine, 3MA, 10 mM), or the proteasome (MG132, 10 µM) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159-175 (mAb 3F4). Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs, respectively. An increment in diglycoforms is observed in Baf- and 3MA-treated cells, whereas in MG132-treated cells also the non-glycoforms are increased. (Right) A graph showing quantification of all PrP bands by densitometric analysis. Data from 3 independent (one in duplicate) experiments for Δ159 and 2 independent (one in duplicate) for Δ159-175 were statistically analyzed for mean and standard deviation (error bars).


B . ΔPrPs can be localized in LAMP1 - immunopositive vesicles . Immunofluorescence analysis shows distribution of ΔPrP ( mAb 3F4 ) and LAMP1 in cells with or without 6M GdnHCl antigen - retrieval treatment . N2a cells transfected with ΔPrP159 were fixed , permeabilized and incubated without ( upper panel ) or with 6M GdnHCl ( lower panel ) . With GndHCl treatment ΔPrP is diffusely distributed ( arrowheads ) and co - localizes poorly with LAMP1 . After GdnHCl treatment ( lower panel ) , bright 3F4 - immunopositive puncta ( arrows ) are observed which partly co - localize with LAMP1 - positive structures ( merge ) . Scale bars , 10 µm .

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

B. ΔPrPs can be localized in LAMP1-immunopositive vesicles. Immunofluorescence analysis shows distribution of ΔPrP (mAb 3F4) and LAMP1 in cells with or without 6M GdnHCl antigen-retrieval treatment. N2a cells transfected with ΔPrP159 were fixed, permeabilized and incubated without (upper panel) or with 6M GdnHCl (lower panel). With GndHCl treatment ΔPrP is diffusely distributed (arrowheads) and co-localizes poorly with LAMP1. After GdnHCl treatment (lower panel), bright 3F4-immunopositive puncta (arrows) are observed which partly co-localize with LAMP1-positive structures (merge). Scale bars, 10 µm.


A . DNI efficiencies of ΔPrPs are inversely correlated to deletion size . Representative immunoblot showing PK - resistant PrPSc moiety of ( 3F4 ) MoPrP co - expressed with indicated ΔPrPs in 22L - ScN2a cells . Quantitative analysis is shown below as scheme . Relative PrPSc levels were calculated as proportion of PK - resistant ( 3F4 ) MoPrP co - transfected with each ΔPrP to that of co - transfected with empty vector control in the same experiment . Data from 4 ( for Δ159 - 165 , Δ159 - 167 and Δ159 - 167 ( 169 ) only 3 ) independent experiments were statistically analyzed for mean and standard deviation ( error bars ) .

A. DNI efficiencies of ΔPrPs are inversely correlated to deletion size. Representative immunoblot showing PK-resistant PrPSc moiety of (3F4)MoPrP co-expressed with indicated ΔPrPs in 22L-ScN2a cells. Quantitative analysis is shown below as scheme. Relative PrPSc levels were calculated as proportion of PK-resistant (3F4)MoPrP co-transfected with each ΔPrP to that of co-transfected with empty vector control in the same experiment. Data from 4 (for Δ159-165, Δ159-167 and Δ159-167(169) only 3) independent experiments were statistically analyzed for mean and standard deviation (error bars).


B . DNI of ΔPrP with a deletion in the C - terminal part of H1 ∼ H2 . Δ171 - 175 ( see Fig . 7A ) was transiently expressed in N2a cells ( left panel ) and DNI efficiency evaluated in 22L - ScN2a cells ( right panel ) ( mAb 3F4 ) . Despite the much shorter deletion size , Δ171 - 175 exerted only inefficient DNI , similar to Δ159 - 175 ( right panel ) .

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B. DNI of ΔPrP with a deletion in the C-terminal part of H1∼H2. Δ171-175 (see Fig. 7A) was transiently expressed in N2a cells (left panel) and DNI efficiency evaluated in 22L-ScN2a cells (right panel) (mAb 3F4). Despite the much shorter deletion size, Δ171-175 exerted only inefficient DNI, similar to Δ159-175 (right panel).


. B . Δ31 - 160 variants are expressed at comparable levels and have EndoH - sensitive and - resistant N - glycans . Immunoblot comparing EndoH - digested and non - digested samples from N2a cells transiently transfected with Δ31 - 160 variants , probed with anti - PrP mAb C16 - S raised against the C - terminal portion of H3 . Non - Tf , samples prepared from N2a cells without transfection . Arrowhead denotes deglycosylated Δ159 ( lane 11 ) , bracket deglycosylated fragments , square bracket EndoH - resistant fragments . Asterisks indicate non - specific bands . Δ31 - 160 variants are not completely deglycosylated by EndoH , resulting in EndoH - resistant fragments ( square bracket ) which are observed also in reducing conditions ( right panel ; + / − dithiothreitol ( DTT ) treatment ) . Note that endogenous wild - type PrPc was not detected under used conditions .

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

. B. Δ31-160 variants are expressed at comparable levels and have EndoH-sensitive and -resistant N-glycans. Immunoblot comparing EndoH-digested and non-digested samples from N2a cells transiently transfected with Δ31-160 variants, probed with anti-PrP mAb C16-S raised against the C-terminal portion of H3. Non-Tf, samples prepared from N2a cells without transfection. Arrowhead denotes deglycosylated Δ159 (lane 11), bracket deglycosylated fragments, square bracket EndoH-resistant fragments. Asterisks indicate non-specific bands. Δ31-160 variants are not completely deglycosylated by EndoH, resulting in EndoH-resistant fragments (square bracket) which are observed also in reducing conditions (right panel; +/− dithiothreitol (DTT) treatment). Note that endogenous wild-type PrPc was not detected under used conditions.


C . DNI of Δ31 - 160 variants is similarly inversely related to size of deletion and dependent on intact C - terminal H1 - H2 portion . Immunoblot probed with mAb 3F4 showing PK - resistant ( 3F4 ) MoPrP co - transfected with indicated constructs into 22L - ScN2a cells ( left panel ) . Δ31 - 160 was slightly more effective than Δ159 . Right panel shows quantification of results as obtained from a triplicate experiment , using densitometry on ImageJ . Bars illustrate mean ± standard deviation . * , p < 0 . 05 .

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C. DNI of Δ31-160 variants is similarly inversely related to size of deletion and dependent on intact C-terminal H1-H2 portion. Immunoblot probed with mAb 3F4 showing PK-resistant (3F4)MoPrP co-transfected with indicated constructs into 22L-ScN2a cells (left panel). Δ31-160 was slightly more effective than Δ159. Right panel shows quantification of results as obtained from a triplicate experiment, using densitometry on ImageJ. Bars illustrate mean ± standard deviation. *, p<0.05.


D . Δ31 - 160 forms spontaneous aggregates with moderate chymotrypsin ( Chy ) resistance . Immunoblots probed with mAbs C16 - S ( upper panel ) or 3F4 ( lower panel ) for comparing Chy - resistant fragments of Δ31 - 160 with that of Δ159 . Lysates from N2a cells expressing Δ159 or Δ31 - 160 were digested with indicated concentrations of Chy for 30 minutes and then digested with PNGaseF . A sample from non - transfected N2a cells is shown in the lane 5 . Asterisks indicate non - specific C16 - S bands . Arrowhead denotes deglycoform of Δ159 , bracket deglycoform of Δ31 - 160 .

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D. Δ31-160 forms spontaneous aggregates with moderate chymotrypsin (Chy) resistance. Immunoblots probed with mAbs C16-S (upper panel) or 3F4 (lower panel) for comparing Chy-resistant fragments of Δ31-160 with that of Δ159. Lysates from N2a cells expressing Δ159 or Δ31-160 were digested with indicated concentrations of Chy for 30 minutes and then digested with PNGaseF. A sample from non-transfected N2a cells is shown in the lane 5. Asterisks indicate non-specific C16-S bands. Arrowhead denotes deglycoform of Δ159, bracket deglycoform of Δ31-160.


A , A targeted MAPPIT screen identifies several DUBs as putative RAS interactors . A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait . pSEL ( + 2L ) - HRAS G12V was expressed in HEK293T cells together with the indicated prey . BRAF served as a positive control . Each measurement was done in triplicate . The results are expressed as a mean of normalized luciferase activity ( leptin - treated cells vs leptin - untreated cells ) . The overall mean value + 2 s . d . served as a threshold .

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

A, A targeted MAPPIT screen identifies several DUBs as putative RAS interactors. A MAPPIT array containing DUB prey library was screened with HRAS G12V as bait. pSEL(+2L)-HRAS G12V was expressed in HEK293T cells together with the indicated prey. BRAF served as a positive control. Each measurement was done in triplicate. The results are expressed as a mean of normalized luciferase activity (leptin-treated cells vs leptin-untreated cells). The overall mean value + 2 s.d. served as a threshold.


B , MAPPIT assay confirms the interaction between OTUB1 and RAS proteins . pSEL ( + 2L ) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey . Empty vector and two random baits , MAL and eDHFR , were used as negative controls . REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits . The results are expressed as a mean of normalized luciferase activity ± s . e . m ( leptin - treated cells vs leptin - untreated cells ) , n = 3 .

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

B, MAPPIT assay confirms the interaction between OTUB1 and RAS proteins. pSEL(+2L) vectors coding RAS proteins were expressed in HEK293T cells together with the indicated prey. Empty vector and two random baits, MAL and eDHFR, were used as negative controls. REM2 and EFHA1 preys that bind to the bait receptor itself was used to evaluate expression of the RAS baits. The results are expressed as a mean of normalized luciferase activity ± s.e.m (leptin-treated cells vs leptin-untreated cells), n=3.


C , NRAS Q61K mutant co - immunoprecipitates with OTUB1 . At 48 hours post - transfection with Flag - tagged NRAS Q61K and HA - tagged OTUB1 expression constructs , HA - tagged OTUB1 was immunoprecipitated with anti - HA - agarose followed by immunoblotting using anti - Flag or anti - HA antibodies .

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

C, NRAS Q61K mutant co-immunoprecipitates with OTUB1. At 48 hours post-transfection with Flag-tagged NRAS Q61K and HA-tagged OTUB1 expression constructs, HA-tagged OTUB1 was immunoprecipitated with anti-HA-agarose followed by immunoblotting using anti-Flag or anti-HA antibodies.


D , OTUB1 interacts with wt NRAS and active NRAS mutant . Flag - tagged NRAS proteins were immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing HA - tagged OTUB1 or empty vector ( V ) .

O O O O O O O O O O O O O O O O O O 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, OTUB1 interacts with wt NRAS and active NRAS mutant. Flag-tagged NRAS proteins were immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V).


E , GTP binding does not affect the complex formation between NRAS and OTUB1 . Recombinant Flag - tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA - tagged OTUB1 in the excess of GTP - γ - S or GDP , followed by immunoblotting with the indicated antibodies .

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

E, GTP binding does not affect the complex formation between NRAS and OTUB1. Recombinant Flag-tagged NRAS was incubated with lysates derived from HEK293T cells expressing HA-tagged OTUB1 in the excess of GTP-γ-S or GDP, followed by immunoblotting with the indicated antibodies.


F , OTUB1 interacts with wt KRAS . Flag - tagged KRAS was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing HA - tagged OTUB1 or empty vector ( V ) . C , D , E , F , Immunoprecipitates , IP . Whole cell lysate , WCL .

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

F, OTUB1 interacts with wt KRAS. Flag-tagged KRAS was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing HA-tagged OTUB1 or empty vector (V). C, D, E, F, Immunoprecipitates, IP. Whole cell lysate, WCL.


G , OTUB1 co - localizes with KRAS at the plasmamembrane . At 24 hours after co - transfection with GFP - tagged KRAS and HA - tagged OTUB1 , HeLa cells were immunostained with anti - HA antibody . The outlined areas are shown at higher magnification at the top of each image . Scale bar , 10 micron .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, OTUB1 co-localizes with KRAS at the plasmamembrane. At 24 hours after co-transfection with GFP-tagged KRAS and HA-tagged OTUB1, HeLa cells were immunostained with anti-HA antibody. The outlined areas are shown at higher magnification at the top of each image. Scale bar, 10 micron.


A , Suppression of OTUB1 expression increases NRAS mono - and di - ubiquitination . 6xHis - tagged ubiquitin and Flag - NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1 . Ubiquitinated NRAS was purified by Co2 + metal affinity chromatography and detected by anti - Flag antibody .

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A, Suppression of OTUB1 expression increases NRAS mono- and di-ubiquitination. 6xHis-tagged ubiquitin and Flag-NRAS were introduced into HEK293T cells expressing shGFP or shRNAs against OTUB1. Ubiquitinated NRAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.


B , C , D , Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination . 6xHis - tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA - OTUB1 , the catalytically dead mutant HA - OTUB1 C91S , or empty vector ( V ) . Ubiquitinated RAS was purified by Co2 + metal affinity chromatography and detected by anti - Flag antibody .

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B, C, D, Catalytic activity of OTUB1 is not required to inhibit RAS ubiquitination. 6xHis-tagged ubiquitin and RAS expression constructs were introduced into HEK293T cells expressing wt HA-OTUB1, the catalytically dead mutant HA-OTUB1 C91S, or empty vector (V). Ubiquitinated RAS was purified by Co2+ metal affinity chromatography and detected by anti-Flag antibody.


E , OTUB1 induces membrane RAS re - localization . Confocal imaging of HeLa cells expressing the indicated constructs . For each sample , z - stacks obtained by scanning the sample from the apical to the basal layer . Step - size , 2 micron .

O O O O O O O O O O O O O O O O O O O O O O O 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, OTUB1 induces membrane RAS re-localization. Confocal imaging of HeLa cells expressing the indicated constructs. For each sample, z-stacks obtained by scanning the sample from the apical to the basal layer. Step-size, 2 micron.


F , RAS cellular distribution expressed as percentage of cells with specific RAS localization . For quantification of RAS localization , cells were randomly imaged using IN Cell Analyzer . RAS localization ( > 200 cells ) was scored as intracellular and diffused ( IN ) , mostly at the plasmamembrane ( PM ) , or both intracellular and plasmamembrane ( PM / IN ) . p - value = 0 . 0005 as determined by Chi2 - test , n = 3 . Representative images of HeLa cells expressing GFP - tagged KRAS are shown in Appendix Figure S1 . Pull - down , PD . Whole cell lysate , WCL . Not modified , n . m .

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F, RAS cellular distribution expressed as percentage of cells with specific RAS localization. For quantification of RAS localization, cells were randomly imaged using IN Cell Analyzer. RAS localization (>200 cells) was scored as intracellular and diffused (IN), mostly at the plasmamembrane (PM), or both intracellular and plasmamembrane (PM/IN). p-value=0.0005 as determined by Chi2-test, n=3. Representative images of HeLa cells expressing GFP-tagged KRAS are shown in Appendix Figure S1. Pull-down, PD. Whole cell lysate, WCL. Not modified, n.m.


A , B , OTUB1 overexpression promotes serum - induced activation of endogenous wt RAS ( A ) or wt NRAS ( B ) . GTP - bound RAS was pulled down from HEK293T cells expressing HA - tagged OTUB1 or empty vector ( V ) using recombinant RAF1 RBD conjugated to agarose beads . Input was controlled by immunoblotting using anti - panRAS or anti - Flag

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A, B, OTUB1 overexpression promotes serum-induced activation of endogenous wt RAS (A) or wt NRAS (B). GTP-bound RAS was pulled down from HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) using recombinant RAF1 RBD conjugated to agarose beads. Input was controlled by immunoblotting using anti-panRAS or anti-Flag antibodies


C , D OTUB1 enhances serum - induced ERK1 / 2 phosphorylation in endogenous wt RAS ( C ) or wt KRAS ( D ) . Levels of phosphorylated ERK1 / 2 and total ERK1 / 2 in HEK293T cells expressing HA - tagged OTUB1 or empty vector ( V ) were analyzed by Meso Scale assay . The results are expressed as a mean of pERK1 / 2 levels relative to total ERK1 / 2 ± s . e . m . n = 2 .

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C, D OTUB1 enhances serum-induced ERK1/2 phosphorylation in endogenous wt RAS (C) or wt KRAS (D). Levels of phosphorylated ERK1/2 and total ERK1/2 in HEK293T cells expressing HA-tagged OTUB1 or empty vector (V) were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m. n=2.


. E , F , Overexpression of wt OTUB1 ( E ) or the catalytically dead mutant OTUB1 C91S mutant ( F ) promotes serum - induced MAPK activation in cell expressing wt NRAS . Whole cell lysates were analyzed by immunoblotting using the indicated antibodies .

. E, F, Overexpression of wt OTUB1 (E) or the catalytically dead mutant OTUB1 C91S mutant (F) promotes serum-induced MAPK activation in cell expressing wt NRAS. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies.


G , H , Overexpression of OTUB1 has no effect on serum - induced MAPK activation in cell expressing mutant NRAS Q61K or KRAS G12V . Whole cell lysates were analyzed by immunoblotting using antibodies as indicated . A , B , C , D , E , F , G , H Serum - starved HEK293T cells expressing the indicated constructs were stimulated with 10 % serum for the indicated time periods .

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G, H, Overexpression of OTUB1 has no effect on serum-induced MAPK activation in cell expressing mutant NRAS Q61K or KRAS G12V. Whole cell lysates were analyzed by immunoblotting using antibodies as indicated. A,B,C,D,E,F,G,H Serum-starved HEK293T cells expressing the indicated constructs were stimulated with 10% serum for the indicated time periods.


I , Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial cells ( HEK TEST ) , expressing empty vector ( V ) , myristoylated AKT1 ( myr - AKT ) , MEK1 D218 , D222 mutant ( MEKDD ) .

I, Immunoblot of OTUB1 overexpression in immortalized human embryonic kidney epithelial cells (HEK TEST), expressing empty vector (V), myristoylated AKT1 (myr-AKT), MEK1 D218,D222 mutant (MEKDD).


J , K , OTUB1 cooperates with active AKT1 to promote anchorage - independent growth . Representative images of soft - agar colonies formed by HEK TEST cells expressing the indicated constructs . The number of soft agar colonies formed by cells expressing OTUB1 compared to cells expressing an empty vector . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 2 .

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J, K, OTUB1 cooperates with active AKT1 to promote anchorage-independent growth. Representative images of soft-agar colonies formed by HEK TEST cells expressing the indicated constructs. The number of soft agar colonies formed by cells expressing OTUB1 compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=2.


B , C , OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13 . 1 copy number alteration . Pearson correlation of OTUB1 copy number ( log2 ratio ) with OTUB1 mRNA levels ( RNAseq normalized read counts , log2 transformed ) was analyzed .

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B,C, OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13.1 copy number alteration. Pearson correlation of OTUB1 copy number (log2 ratio) with OTUB1 mRNA levels (RNAseq normalized read counts, log2 transformed) was analyzed.


D , E , F , OTUB1 expression in TCGA lung adenocarcinoma ( LUAD ) and lung squamous cell carcinoma ( SCC ) patients . Patients were stratified according to their OTUB1 mRNA levels and / or their KRAS status as described in Materials and Methods . Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis . p - values were determined by two - sided t - Test . Total number of patients , n .

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D,E,F, OTUB1 expression in TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (SCC) patients. Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis. p-values were determined by two-sided t-Test. Total number of patients, n.


G , Gain of 11q13 . 1 locus is an early event in lung adenocarcinoma development . TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages ( T1 - T4 ) . Total numbers of patients , n . Statistical comparison of the sample distributions were compared using Chi - square test .

G, Gain of 11q13.1 locus is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


H , OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development . TCGA lung adenocarcinoma patients were stratified by tumor stages ( T1 - T4 ) and OTUB1 expression levels as described in Materials and Methods . Total numbers of patients , n . Statistical comparison of the sample distributions were compared using Chi - square test .

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H, OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients were stratified by tumor stages (T1-T4) and OTUB1 expression levels as described in Materials and Methods. Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


I , KRAS mutation is a late event in lung adenocarcinoma progression . TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages ( T1 - T4 ) . Total numbers of patients , n . Statistical comparison of the sample distributions were compared using Chi - square test .

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I, KRAS mutation is a late event in lung adenocarcinoma progression. TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


, Suppression of OTUB1 expression in NSCLC cell lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1 .

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, Suppression of OTUB1 expression in NSCLC cell lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1.


B , OTUB1 suppression decreases serum - induced ERK1 / 2 phosphorylation . Serum - starved A549 cells expressing shRNAs against OTUB1 or GFP were stimulated with 10 % serum for the indicated time periods . Levels of phosphorylated ERK1 / 2 and total ERK1 / 2 were analyzed by Meso Scale assay . The results are expressed as a mean of pERK1 / 2 levels relative to total ERK1 / 2 ± s . e . m ; p - values were determined by two - sided t - Test , n = 2 .

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B, OTUB1 suppression decreases serum-induced ERK1/2 phosphorylation. Serum-starved A549 cells expressing shRNAs against OTUB1 or GFP were stimulated with 10% serum for the indicated time periods. Levels of phosphorylated ERK1/2 and total ERK1/2 were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m; p-values were determined by two-sided t-Test, n=2.


C , D , OTUB1 suppression impairs the anchorage - independent growth of Western blot cancer cell lines . Representative images of soft - agar colonies formed by Western blot cells expressing shRNAs against OTUB1 or GFP . The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 .

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C, D, OTUB1 suppression impairs the anchorage-independent growth of Western blot cancer cell lines. Representative images of soft-agar colonies formed by Western blot cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.


C , D , OTUB1 suppression impairs the anchorage - independent growth of lung cancer cell lines . Representative images of soft - agar colonies formed by lung cells expressing shRNAs against OTUB1 or GFP . The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 .

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C, D, OTUB1 suppression impairs the anchorage-independent growth of lung cancer cell lines. Representative images of soft-agar colonies formed by lung cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.


E , Xenograft tumor growth of A549 cells expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice . p - value was determined by 2 - way ANOVA .

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E, Xenograft tumor growth of A549 cells expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice. p-value was determined by 2-way ANOVA.


A , OTUB1 overexpression promotes serum - induced MAPK activation in wt KRAS H1993 lung adenocarcinoma cells .

A, OTUB1 overexpression promotes serum-induced MAPK activation in wt KRAS H1993 lung adenocarcinoma cells.


B , OTUB1 overexpression does not affect serum - induced MAPK activation in mutant KRAS A549 lung adenocarcinoma cells . A , B , Serum - starved lung cells expressing Flag - OTUB1 or an empty vector ( V ) were stimulated with 10 % serum for the indicated time periods . Whole cell lysates were analyzed by immunoblotting using antibodies against pERK1 / 2 , ERK1 / 2 , Flag and vinculin .

B, OTUB1 overexpression does not affect serum-induced MAPK activation in mutant KRAS A549 lung adenocarcinoma cells. A,B, Serum-starved lung cells expressing Flag-OTUB1 or an empty vector (V) were stimulated with 10% serum for the indicated time periods. Whole cell lysates were analyzed by immunoblotting using antibodies against pERK1/2, ERK1/2, Flag and vinculin.


C , Representative immunoblots of Flag - tagged OTUB1 overexpression in lung adenocarcinoma cell

C, Representative immunoblots of Flag-tagged OTUB1 overexpression in lung adenocarcinoma cell lines


D , E , OTUB1 overexpression affects anchorage - independent growth of lung adenocarcinoma cell lines . Representative images of soft - agar colonies formed by the indicated cells expressing Flag - OTUB1 or empty vector ( V ) . The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 .

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D, E, OTUB1 overexpression affects anchorage-independent growth of lung adenocarcinoma cell lines. Representative images of soft-agar colonies formed by the indicated cells expressing Flag-OTUB1 or empty vector (V). The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.


F , G , Xenograft tumor growth of A549 , H1993 or H1437 cells expressing Flag - OTUB1 or an empty vector ( V ) subcutaneously injected into nude mice . p - values were determined by 2 - way ANOVA .

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F,G, Xenograft tumor growth of A549, H1993 or H1437 cells expressing Flag-OTUB1 or an empty vector (V) subcutaneously injected into nude mice. p-values were determined by 2-way ANOVA.


A , Representative examples of OTUB1 immunostaining in lung adenocarcinomas . Samples were scored as negative / low , medium or high staining as described in Materials and Methods . Scale bar , 100 micron .

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A, Representative examples of OTUB1 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Materials and Methods. Scale bar, 100 micron.


B , OTUB1 expression in lung non - small cell carcinomas with different KRAS mutation status . OTUB1 expression was assessed in TMA samples by IHC .

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B, OTUB1 expression in lung non-small cell carcinomas with different KRAS mutation status. OTUB1 expression was assessed in TMA samples by IHC.


C , OTUB1 up - regulation is an early event in lung adenocarcinoma development . Lung adenocarcinoma patients expressing different levels of OTUB1 were stratified by tumor stages ( T1 - T3 ) . Total number of patients , n .

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C, OTUB1 up-regulation is an early event in lung adenocarcinoma development. Lung adenocarcinoma patients expressing different levels of OTUB1 were stratified by tumor stages (T1-T3). Total number of patients, n.


D , Representative examples of phosphorylated - ERK1 / 2 immunostaining in lung adenocarcinomas . Samples were scored as negative / low , medium or high staining as described in Material and Methods . Scale bar , 100 micron .

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D, Representative examples of phosphorylated-ERK1/2 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Material and Methods. Scale bar, 100 micron.


E , OTUB1 levels correlates with increased levels of phosphorylated - ERK1 / 2 in wt KRAS lung adenocarcinomas . Association between OTUB1 and pERK1 / 2 expression levels was assessed in the same tumor samples by IHC .

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E, OTUB1 levels correlates with increased levels of phosphorylated-ERK1/2 in wt KRAS lung adenocarcinomas. Association between OTUB1 and pERK1/2 expression levels was assessed in the same tumor samples by IHC.


F , Ki67 expression in lung adenocarcinomas expressing different levels of OTUB1 . Ki67 expression was assessed by IHC . The percentage of Ki67 - positive cells was counted , and the samples were scored as negative / low , < 10 % ; medium , between 10 and 40 % ; high , > 40 % . See Figure EV4 for representative images of Ki67 staining .

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F, Ki67 expression in lung adenocarcinomas expressing different levels of OTUB1. Ki67 expression was assessed by IHC. The percentage of Ki67-positive cells was counted, and the samples were scored as negative/low, <10%; medium, between 10 and 40%; high, > 40%. See Figure EV4 for representative images of Ki67 staining.


G , Wt KRAS lung adenocarcinoma TCGA patients with medium OTUB1 expression have poorer overall survival .

G, Wt KRAS lung adenocarcinoma TCGA patients with medium OTUB1 expression have poorer overall survival.


H , OTUB1 expression levels are not associated with survival rate of KRAS - mutant lung adenocarcinoma TCGA patients . G , H , Patients were stratified according to their OTUB1 mRNA levels and / or their KRAS status as described in Materials and Methods . Overall survival of lung adenocarcinoma patients expressing different levels of OTUB1 mRNA as measured by Kaplan - Meier curves . p - values were determined by Log Rank Test .

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H, OTUB1 expression levels are not associated with survival rate of KRAS-mutant lung adenocarcinoma TCGA patients. G,H, Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Overall survival of lung adenocarcinoma patients expressing different levels of OTUB1 mRNA as measured by Kaplan-Meier curves. p-values were determined by Log Rank Test.


( A ) Identified acetylated histone peptides plotted against their log2 SILAC ratio ( 1h after 16 J / m2 UV / mock ) , ranked by SILAC

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(A) Identified acetylated histone peptides plotted against their log2 SILAC ratio (1h after 16 J/m2 UV/mock), ranked by SILAC ratio


( C ) Western blot of acid extracted histones and WCE from HeLa cells , treated with HATi ( CTK7A , 100 μM and CPTH2 , 50 μM ) or HDACi ( TSA , 1 μM ) for 4h or mock treated as

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(C) Western blot of acid extracted histones and WCE from HeLa cells, treated with HATi (CTK7A, 100 μM and CPTH2, 50 μM) or HDACi (TSA, 1 μM) for 4h or mock treated as indicated


( D ) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV - irradiation ( 16 J / m2 ) . Blots were stained with α - acetyl - lysine ( top panels , high and low exposure ) , α - histone H4 ( middle panel ) and α - tubulin ( bottom panel )

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(D) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV-irradiation (16 J/m2). Blots were stained with α-acetyl-lysine (top panels, high and low exposure), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( F ) Representative western blots of HeLa WCE from different time points after UV - C irradiation ( 16J / m2 ) using the indicated histone modification specific antibodies . Histone H4 and tubulin were used as loading

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(F) Representative western blots of HeLa WCE from different time points after UV-C irradiation (16J/m2) using the indicated histone modification specific antibodies. Histone H4 and tubulin were used as loading controls


( A ) Representative western blots of histone acetylation levels of HeLa cells lysed at indicated time points after UV irradiation ( 16 J / m2 , left panel ) , α - amanitin ( second panel , 100 µg / ml ) , THZ1 ( third panel , 1 μM ) or flavopiridol ( right panel , 1 μM ) treatment . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) and α - tubulin ( bottom panel )

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(A) Representative western blots of histone acetylation levels of HeLa cells lysed at indicated time points after UV irradiation (16 J/m2, left panel), α-amanitin (second panel, 100 µg/ml), THZ1 (third panel, 1 μM) or flavopiridol (right panel, 1 μM) treatment. Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( C ) Representative western blots of histone acetylation levels of HeLa cells pre - treated with transcription inhibitors ( mock ( left ) , THZ1 ( middle , 1 μM ) or flavopiridol ( right , 1 μM ) an hour before UV irradiation and lysed at the indicated time points after UV ( 16J / m2 ) . Blots were stained with the indicated

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(C) Representative western blots of histone acetylation levels of HeLa cells pre-treated with transcription inhibitors (mock (left), THZ1 (middle, 1 μM) or flavopiridol (right, 1 μM) an hour before UV irradiation and lysed at the indicated time points after UV (16J/m2). Blots were stained with the indicated antibodies


( A ) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation ( 16 J / m2 ) , HU / AraC treatment ( 100 mM / 10 μM ) or MMC treatment ( 10 µg / ml ) . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) and α - tubulin ( bottom panel )

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(A) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation (16 J/m2), HU/AraC treatment (100 mM/10 μM) or MMC treatment (10 µg/ml). Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)


( C ) Representative Western blots , stained with the indicated antibodies of HeLa cells pre - treated o / n with an S - phase inhibitor ( PHA 767491 hydrochloride , 10 μM ) and lysed at indicated time points after UV ( 16 J / m2 )

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(C) Representative Western blots, stained with the indicated antibodies of HeLa cells pre-treated o/n with an S-phase inhibitor (PHA 767491 hydrochloride, 10 μM) and lysed at indicated time points after UV (16 J/m2)


C Top : Schematic illustration of the miR - 17 - 92 cluster with colours marking miRNAs belonging to the same miRNA family . Bottom : TaqMan analysis of the mature miR - 17 - 92 miRNAs in Srsf3 knockout ( KO ) and control ( Ctrl ) iPSCs ( * P < 0 . 05 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 3 , biological replicates ) .

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C Top: Schematic illustration of the miR-17-92 cluster with colours marking miRNAs belonging to the same miRNA family. Bottom: TaqMan analysis of the mature miR-17-92 miRNAs in Srsf3 knockout (KO) and control (Ctrl) iPSCs (*P<0.05, ***P<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=3, biological replicates).


B TaqMan analysis of the six miR - 17 - 92 miRNAs in HEK293T cells overexpressing the WT - miR - 17 - 92 , 17 / 20a - ∆ CNNC or total - ∆ CNNC construct ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One - way ANOVA , mean as ± SEM , n = 4 , biological replicates ) .

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B TaqMan analysis of the six miR-17-92 miRNAs in HEK293T cells overexpressing the WT-miR-17-92, 17/20a-∆CNNC or total-∆CNNC construct (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, One-way ANOVA, mean as ± SEM, n=4, biological replicates).


C Western blot analysis of HA - tagged SRSF3 - FL or SRSF3 - ∆ RS expression in LIM1215 cells ( detection with a HA - antibody ) .

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C Western blot analysis of HA-tagged SRSF3-FL or SRSF3-∆RS expression in LIM1215 cells (detection with a HA-antibody).


D TaqMan analysis of the six miR - 17 - 92 miRNAs in LIM1215 cells overexpressing empty pCGN vector , SRSF3 - FL or SRSF3 - ∆ RS constructs ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One - way ANOVA , data as mean ± SEM , n = 3 , biological replicates ) .

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D TaqMan analysis of the six miR-17-92 miRNAs in LIM1215 cells overexpressing empty pCGN vector, SRSF3-FL or SRSF3-∆RS constructs (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, One-way ANOVA, data as mean ± SEM, n=3, biological replicates).


B Visualization and comparison of WT miR - 17 - 92 SHAPE reactivities in control and SRSF3 - KD cells . First panel : Mean control ( blue ) and SRSF3 - KD ( red ) SHAPE reactivities , calculated over 11nt sliding windows . Second panel : ∆ SHAPE reactivity differences between control and SRSF3 - KD . Regions with higher reactivity in control are marked with blue shading and regions with higher reactivity in SRSF3 - KD with red shading . Third panel : The SRSF3 - KD to control reactivity ratios . The black boxes mark the CNNC sites .

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B Visualization and comparison of WT miR-17-92 SHAPE reactivities in control and SRSF3-KD cells. First panel: Mean control (blue) and SRSF3-KD (red) SHAPE reactivities, calculated over 11nt sliding windows. Second panel: ∆SHAPE reactivity differences between control and SRSF3-KD. Regions with higher reactivity in control are marked with blue shading and regions with higher reactivity in SRSF3-KD with red shading. Third panel: The SRSF3-KD to control reactivity ratios. The black boxes mark the CNNC sites.


G Visualization and comparison of total - ∆ CNNC miR - 17 - 92 SHAPE reactivities in control and SRSF3 - KD cells . First panel : Mean control ( grey ) and SRSF3 - KD ( orange ) SHAPE reactivities , calculated over 11nt sliding windows . Second panel : ∆ SHAPE reactivity differences between control and SRSF3 - KD . Regions with higher reactivity in control are marked with grey shading and regions with higher reactivity in SRSF3 - KD with orange shading . Third panel : The SRSF3 - KD to control reactivity ratios . The red boxes mark the mutated CNNC sites .

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G Visualization and comparison of total-∆CNNC miR-17-92 SHAPE reactivities in control and SRSF3-KD cells. First panel: Mean control (grey) and SRSF3-KD (orange) SHAPE reactivities, calculated over 11nt sliding windows. Second panel: ∆SHAPE reactivity differences between control and SRSF3-KD. Regions with higher reactivity in control are marked with grey shading and regions with higher reactivity in SRSF3-KD with orange shading. Third panel: The SRSF3-KD to control reactivity ratios. The red boxes mark the mutated CNNC sites.


B RT - qPCR quantification of Srsf3 and Cdkn1a ( p21 ) mRNA levels in Srsf3 - knockout ( KO ) and control ( Ctrl ) iPSCs ( * p < 0 . 05 , * * * < 0 . 001 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 3 , biological replicates ) .

B RT-qPCR quantification of Srsf3 and Cdkn1a (p21) mRNA levels in Srsf3-knockout (KO) and control (Ctrl) iPSCs (*p<0.05, ***<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=3, biological replicates).


C Quantification of Cdkn1a mRNA expression by RT - qPCR during reprogramming in Srsf3 - KO and control cells ( * * * p < 0 . 001 , * * * * p < 0 . 0001 , Two - way ANOVA , data as mean ± SEM , n = 3 , biological replicates ) .

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C Quantification of Cdkn1a mRNA expression by RT-qPCR during reprogramming in Srsf3-KO and control cells (***p<0.001, ****p<0.0001, Two-way ANOVA, data as mean ± SEM, n=3, biological replicates).


D RT - qPCR quantification of CDKN1A ( p21 ) levels in HEK293T cells overexpressing empty vector , WT - miR - 17 - 92 , 17 / 20a - ∆ CNNC and total - CNNC ∆ expression constructs ( * p < 0 . 05 , * * p < 0 . 01 , One - way ANOVA , data as mean ± SEM , n = 3 , biological replicates ) .

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D RT-qPCR quantification of CDKN1A (p21) levels in HEK293T cells overexpressing empty vector, WT-miR-17-92, 17/20a-∆CNNC and total-CNNC∆ expression constructs (*p<0.05, **p<0.01, One-way ANOVA, data as mean ± SEM, n=3, biological replicates).


E RT - qPCR quantification of CDKN1A ( p21 ) levels in LIM1215 cells over expressing empty vector , SRSF3 - FL and SRSF3 - ∆ RS constructs ( * * p < 0 . 01 , One - way ANOVA , data as mean ± SEM , n = 3 , biological replicates ) . F RT - qPCR quantification of CDKN1A ( p21 ) levels in LIM1215 cells overexpressing scrambled control , miR - 17 , miR - 20a or miR - 17 + miR - 20a mimics ( * * p < 0 . 01 , One - way ANOVA , data as mean ± SEM , n = 3 , biological replicates ) .

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E RT-qPCR quantification of CDKN1A (p21) levels in LIM1215 cells over expressing empty vector, SRSF3-FL and SRSF3-∆RS constructs (**p<0.01, One-way ANOVA, data as mean ± SEM, n=3, biological replicates). F RT-qPCR quantification of CDKN1A (p21) levels in LIM1215 cells overexpressing scrambled control, miR-17, miR-20a or miR-17+miR-20a mimics (**p<0.01, One-way ANOVA, data as mean ± SEM, n=3, biological replicates).


A Western blot analysis of SRSF3 and CDKN1A expression in SRSF3 - T2A - GFP overexpressing ( SRSF3 ) and control ( GFP ) LIM1215 cells . GAPDH served as a loading control .

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A Western blot analysis of SRSF3 and CDKN1A expression in SRSF3-T2A-GFP overexpressing (SRSF3) and control (GFP) LIM1215 cells. GAPDH served as a loading control.


B TaqMan analysis of miR - 17 - 92 miRNAs in SRSF3 - T2A - GFP overexpressing ( SRSF3 - GFP ) and GFP - only control ( GFP ) LIM1215 cells ( * p < 0 . 05 , * * * * p < 0 . 001 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 4 , biological replicates ) .

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B TaqMan analysis of miR-17-92 miRNAs in SRSF3-T2A-GFP overexpressing (SRSF3-GFP) and GFP-only control (GFP) LIM1215 cells (*p<0.05, ****p<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=4, biological replicates).


C RT - qPCR quantification of CDKN1A ( p21 ) mRNA levels in SRSF3 - T2A - GFP overexpressing ( SRSF3 - GFP ) and control ( GFP ) LIM1215 cells ( * * p < 0 . 01 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 3 , biological replicates ) .

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C RT-qPCR quantification of CDKN1A (p21) mRNA levels in SRSF3-T2A-GFP overexpressing (SRSF3-GFP) and control (GFP) LIM1215 cells (**p<0.01, two-tailed unpaired Student's t-test, data as mean ± SEM, n=3, biological replicates).


D Left : Schematic of the Firefly luciferase reporter constructs Right : Luciferase activity in SRSF3 overexpressing ( SRSF3 - GFP ) and control ( GFP ) LIM1215 cells transfected with the reporter constructs ( * * p < 0 . 01 , * * * p < 0 . 001 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 4 , biological replicates ) .

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D Left: Schematic of the Firefly luciferase reporter constructs Right: Luciferase activity in SRSF3 overexpressing (SRSF3-GFP) and control (GFP) LIM1215 cells transfected with the reporter constructs (**p<0.01, ***p<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=4, biological replicates).


F Quantification of fraction of cells in G1 , S and G2 phases in SRSF3 overexpressing cells ( SRSF3 - GFP ) relative to control ( GFP ) LIM1215 cells ( * p < 0 . 05 , * * p < 0 . 01 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 4 , biological replicates ) .

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F Quantification of fraction of cells in G1, S and G2 phases in SRSF3 overexpressing cells (SRSF3-GFP) relative to control (GFP) LIM1215 cells (*p<0.05, **p<0.01, two-tailed unpaired Student's t-test, data as mean ± SEM, n=4, biological replicates).


A , B RT - qPCR quantification of SRSF3 ( A ) and CDKN1A ( B ) mRNA levels in colorectal tumours and their paired normal samples ( * p < 0 . 05 , * * p < 0 . 01 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 25 , biological replicates ) . N = normal , T = tumour .

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A, B RT-qPCR quantification of SRSF3 (A) and CDKN1A (B) mRNA levels in colorectal tumours and their paired normal samples (*p<0.05, **p<0.01, two-tailed unpaired Student's t-test, data as mean ± SEM, n=25, biological replicates). N=normal, T=tumour.


D Association between SRSF3 expression and tumour ( T ) stage . T1 ( n = 3 ) , T2 ( n = 5 ) , T3 ( n = 11 ) and T4 ( n = 3 ) tumours , SRSF3 fold change as the dependent variable . Linear regression analysis ( central band = median , boxes = 25th and 75th percentile and whiskers = min and max , n = biological replicates ) . E Association between SRSF3 expression and tumour differentiation status . Moderately ( Mod . ) differentiated tumours ( n = 19 ) and poorly differentiated tumours ( n = 5 ) , SRSF3 fold change as the dependent variable . Linear regression analysis ( central band = median , boxes = 25th and 75th percentile and whiskers = min and max ; n = biological replicates ) .

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D Association between SRSF3 expression and tumour (T) stage. T1 (n=3), T2 (n=5), T3 (n=11) and T4 (n=3) tumours, SRSF3 fold change as the dependent variable. Linear regression analysis (central band=median, boxes =25th and 75th percentile and whiskers=min and max, n=biological replicates). E Association between SRSF3 expression and tumour differentiation status. Moderately (Mod.) differentiated tumours (n=19) and poorly differentiated tumours (n=5), SRSF3 fold change as the dependent variable. Linear regression analysis (central band=median, boxes =25th and 75th percentile and whiskers=min and max; n=biological replicates).


G SRSF3 and CDKN1A expression in TCGA COAD data ( version 2016 _ 01 _ 28 ; tumour n = 459 and normal n = 41 ( biological replicates ) ; RSEM , RNA - seq by Expectation Maximation ; * * p < 0 . 01 , two - tailed unpaired Student ' s t - test , central band = median , dotted lines = 25th and 75th percentile ) .

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G SRSF3 and CDKN1A expression in TCGA COAD data (version 2016_01_28; tumour n=459 and normal n=41 (biological replicates); RSEM, RNA-seq by Expectation Maximation; **p<0.01, two-tailed unpaired Student's t-test, central band=median, dotted lines =25th and 75th percentile).


A . Anterior and lateral photographs of two patients with ELP6 variants showing dysmorphic features at 20 ( Patient 2 ) and 19 ( Patient 3 ) years of age .

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A. Anterior and lateral photographs of two patients with ELP6 variants showing dysmorphic features at 20 (Patient 2) and 19 (Patient 3) years of age.


B . Mid - sagittal ( I ) , coronal ( II ) and transversal ( III ) MRI scans of Patient 1 with newly identified ELP4 variants and Patient 2 and Patient 3 with ELP6 variants . Atrophy of the cerebellum with prominence of the cerebellar folia is marked by circles . Arrows indicate thin corpus callosum , red arrowheads point to enlarged extra - axial CSF spaces and black arrowheads to cerebral sulci in keeping with cerebral and cerebellar volume loss .

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B. Mid-sagittal (I), coronal (II) and transversal (III) MRI scans of Patient 1 with newly identified ELP4 variants and Patient 2 and Patient 3 with ELP6 variants. Atrophy of the cerebellum with prominence of the cerebellar folia is marked by circles. Arrows indicate thin corpus callosum, red arrowheads point to enlarged extra-axial CSF spaces and black arrowheads to cerebral sulci in keeping with cerebral and cerebellar volume loss.


C . EEG of Patient 3 . Left panel : 1 - 2 Hz left fronto - temporal spike and multi - spike slow wave discharges ( Fp1 - F7 and T3 - T5 ) spread to the homologous same hemisphere and opposite hemisphere , predominantly seen on bipolar montage EEG . Right panel : Spike - and multi - spike slow wave activities together with fast rhythms originating from bilateral fronto - temporal - frontocentral regions indicate electrographic seizure activity . Described changes are marked by circles .

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C. EEG of Patient 3. Left panel: 1-2 Hz left fronto-temporal spike and multi-spike slow wave discharges (Fp1-F7 and T3-T5) spread to the homologous same hemisphere and opposite hemisphere, predominantly seen on bipolar montage EEG. Right panel: Spike-and multi-spike slow wave activities together with fast rhythms originating from bilateral fronto-temporal-frontocentral regions indicate electrographic seizure activity. Described changes are marked by circles.


C . Pull - down experiments of the assembled mElp123456 complexes carrying patients - derived mutations . The mElp123WT subcomplexes were immobilized on the Dynabeads ( strep ) via Elp3 protein and incubated with the Elp456 variants . The inputs and pull downs were loaded on the SDS - PAGE gels and visualized by Coomassie staining . The molecular mass marker is indicated on the left ( M ) .

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C. Pull-down experiments of the assembled mElp123456 complexes carrying patients-derived mutations. The mElp123WT subcomplexes were immobilized on the Dynabeads (strep) via Elp3 protein and incubated with the Elp456 variants. The inputs and pull downs were loaded on the SDS-PAGE gels and visualized by Coomassie staining. The molecular mass marker is indicated on the left (M).


D . Co - immunoprecipitation analyses of Elongator complexes in patient - derived fibroblast cell . Whole cell lysates ( left ) and eluates after immune - precipitation ( right ) are shown for control fibroblasts ( ctrl ) and cultured patient fibroblasts ( Elp4Y91C / L296I ( patient 1 ) ; Elp6L118W / L118W ( patient 2 ) , Elp6L118W / L118W ( patient 3 ) . Used antibodies for immobilization ( ELP1 ) and detection ( ELP1 , ELP3 , ELP4 , ELP5 and α - tubulin ) are indicated . n = 4 independent experiments .

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D. Co-immunoprecipitation analyses of Elongator complexes in patient-derived fibroblast cell. Whole cell lysates (left) and eluates after immune-precipitation (right) are shown for control fibroblasts (ctrl) and cultured patient fibroblasts (Elp4Y91C/L296I (patient 1); Elp6L118W/L118W (patient 2), Elp6L118W/L118W (patient 3). Used antibodies for immobilization (ELP1) and detection (ELP1, ELP3, ELP4 , ELP5 and α-tubulin) are indicated. n = 4 independent experiments.


A . High - performance liquid chromatography ( HPLC ) coupled to mass spectrometry ( MS ) used to quantify the Elongator - dependent tRNA modification 5 - carbamoylmethyluridine ( ncm5U ) in human fibroblasts . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . B . HPLC - MS quantification of Elongator - dependent tRNA modification 5 - methoxy - carbonylmethyluridine ( mcm5U ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . C . HPLC - MS quantification of Elongator - dependent tRNA modification 5 - methoxycarbonylmethyl - 2 - thiouridine ( mcm5s2U ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . Data information : Statistical analysis for data one - way ANOVA ( α = 0 . 05 ) with a Dunnett ' s post - hoc test . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; ns - not significant ) . Data represent mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) used to quantify the Elongator-dependent tRNA modification 5-carbamoylmethyluridine (ncm5U) in human fibroblasts. Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. B. HPLC-MS quantification of Elongator-dependent tRNA modification 5-methoxy-carbonylmethyluridine (mcm5U). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. C. HPLC-MS quantification of Elongator-dependent tRNA modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.


D . HPLC - MS quantification of Elongator - independent tRNA modification 1 - methyladenosine ( m1A ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . E . HPLC - MS quantification of Elongator - independent tRNA modification 7 - methylguanosine ( m7G ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . Data information : Statistical analysis for data one - way ANOVA ( α = 0 . 05 ) with a Dunnett ' s post - hoc test . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; ns - not significant ) . Data represent mean ± SEM .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HPLC-MS quantification of Elongator-independent tRNA modification 1-methyladenosine (m1A). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. E. HPLC-MS quantification of Elongator-independent tRNA modification 7-methylguanosine (m7G). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.


F . Differential expression ( DE ) of peptides in fibroblasts obtained from the individuals with ELP4 and ELP6 variants relative to control ( n = 3 technical repeats per genotype ) . Down - ( red ) and upregulated ( blue ) peptides are depicted on volcano plots .

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F. Differential expression (DE) of peptides in fibroblasts obtained from the individuals with ELP4 and ELP6 variants relative to control (n = 3 technical repeats per genotype). Down- (red) and upregulated (blue) peptides are depicted on volcano plots.


A . Representative images of Golgi - Cox - stained brain sections in the hippocampal area of adult ( 2 - months - old ) wild - type and mutant mice . Rectangles represent CA1 region containing pyramidal neurons selected for neuron reconstruction and subsequent analyses . Scale bar : 100 μm . Abbreviations : DG - dentate gyrus , CA1 - cornu ammonis 1 , CA2 - cornu ammonis

A. Representative images of Golgi-Cox-stained brain sections in the hippocampal area of adult (2-months-old) wild-type and mutant mice. Rectangles represent CA1 region containing pyramidal neurons selected for neuron reconstruction and subsequent analyses. Scale bar: 100 μm. Abbreviations: DG - dentate gyrus, CA1 - cornu ammonis 1, CA2 - cornu ammonis 2


C . Quantification of total dendritic length from dendritic tree reconstructions shown in B . n = 15 ; 5 neurons per animal and 3 animals per genotype . D . Sholl analysis of the complexity of basal dendritic arbors . n = 15 ; 5 neurons per animal and 3 animals per genotype . Data information : Statistical analysis : unpaired two - tailed t - test ( α = 0 . 05 ) with Welch ' s correction . Holm - Sidak correction was applied to adjust for multiple comparisons ( D ) . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; ns - not significant ) . Data represent mean ± SEM ( with error bars in D removed when smaller than the corresponding data point ) .

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C. Quantification of total dendritic length from dendritic tree reconstructions shown in B. n = 15; 5 neurons per animal and 3 animals per genotype. D. Sholl analysis of the complexity of basal dendritic arbors. n = 15; 5 neurons per animal and 3 animals per genotype. Data information: Statistical analysis: unpaired two-tailed t-test (α = 0.05) with Welch's correction. Holm-Sidak correction was applied to adjust for multiple comparisons (D). Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns - not significant). Data represent mean ± SEM (with error bars in D removed when smaller than the corresponding data point).


E . Representative images of dendritic spines on basal dendrites of the cortical neurons . Quantification of spine density per 10 μm on secondary dendrites is shown . n = 15 ; 5 neurons per animal and 3 animals per genotype . Scale bar : 10 μm . Data information : Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; ns - not significant ) . Data represent mean ±

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E. Representative images of dendritic spines on basal dendrites of the cortical neurons. Quantification of spine density per 10 μm on secondary dendrites is shown. n = 15; 5 neurons per animal and 3 animals per genotype. Scale bar: 10 μm. Data information: Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns - not significant). Data represent mean ± SEM


Hydrolysis of fluorogenic substrates ( Suc - LLVY - AMC ) for the chymotrypsin - like activity of human ( B ) or mouse ( C ) 20S constitutive proteasome ( CP ) or 20S immunoproteasome ( IP ) at various PRN1126 concentrations . Data are presented as the means ± s . d . from quadruplicate assays . The highest fluorescence value was set to 100 % . The experiments were repeated twice with similar results .

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Hydrolysis of fluorogenic substrates (Suc-LLVY-AMC) for the chymotrypsin-like activity of human (B) or mouse (C) 20S constitutive proteasome (CP) or 20S immunoproteasome (IP) at various PRN1126 concentrations. Data are presented as the means ± s.d. from quadruplicate assays. The highest fluorescence value was set to 100%. The experiments were repeated twice with similar results.


Presentation of UTY246 - 254 on splenocytes after exposure to indicated concentrations of PRN1126 . Data are presented as the mean absorbance ± s . d . of three replicate cultures . The experiment has been performed twice , yielding similar results .

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

Presentation of UTY246-254 on splenocytes after exposure to indicated concentrations of PRN1126. Data are presented as the mean absorbance ± s.d. of three replicate cultures. The experiment has been performed twice, yielding similar results.


Flow cytometry analysis of H - 2Kb surface expression on splenocytes derived from C57BL / 6 mice treated with the indicated concentrations of PRN1126 overnight . Pooled data from three independent experiments ( n = 9 ) are shown as the means of median fluorescent intensity ± SEM .

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

Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 mice treated with the indicated concentrations of PRN1126 overnight. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM.


Splenocytes from C57BL / 6 mice ( C ) were exposed ( continuous treatment ) to 300 nM PRN1126 or vehicle ( DMSO ) or 300 nM ONX 0914 and stimulated with LPS overnight . IL - 6 concentrations in the supernatant were analyzed by ELISA . ( C ) IL - 6 concentrations are presented as mean and individual data points from triplicate wells . The experiment has been performed twice , yielding similar results .

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Splenocytes from C57BL/6 mice (C) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. (C) IL-6 concentrations are presented as mean and individual data points from triplicate wells. The experiment has been performed twice, yielding similar results.


Splenocytes from human PBMCs ( D ) were exposed ( continuous treatment ) to 300 nM PRN1126 or vehicle ( DMSO ) or 300 nM ONX 0914 and stimulated with LPS overnight . IL - 6 concentrations in the supernatant were analyzed by ELISA . Data are presented as single dots from three independent donors . The highest cytokine concentration was set to 100 % . * P < 0 . 05 . Unpaired student ´ s t - test .

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Splenocytes from human PBMCs (D) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. Data are presented as single dots from three independent donors. The highest cytokine concentration was set to 100%. *P < 0.05. Unpaired student´s t-test.


Colitis was induced by oral administration of 3 % DSS for 5 d . Mice were treated daily ( s . c . ) with either PRN1126 ( 40 mg / kg ) , or ONX 0914 ( 10 mg / kg ) , or vehicle . Data points represent mean ± SEM of 15 mice pooled from three independent experiments .

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

Colitis was induced by oral administration of 3 % DSS for 5 d. Mice were treated daily (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. Data points represent mean ± SEM of 15 mice pooled from three independent experiments.


Mice were immunized with MOG35 - 55 peptide and were monitored daily for clinical symptoms of EAE . Mice were treated three times a week ( s . c . ) with either PRN1126 ( 40 mg / kg ) , or ONX 0914 ( 10 mg / kg ) , or vehicle . All data were statistically compared to the vehicle treated group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Two - way ANOVA . Shown are the means of the clinical scores ± SEM ( n = 6 per group ) .

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Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated three times a week (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. All data were statistically compared to the vehicle treated group. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA. Shown are the means of the clinical scores ± SEM (n = 6 per group).


Hydrolysis of fluorogenic substrates ( Ac - PAL - AMC ) for LMP2 - activity of human ( upper panel ) or mouse ( lower panel ) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914 . Data are presented as the means of fluorescence ± s . d . from quadruplicate assays . The experiments were repeated three times with similar results .

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Hydrolysis of fluorogenic substrates (Ac-PAL-AMC) for LMP2-activity of human (upper panel) or mouse (lower panel) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results.


Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914 . Ficoll enriched lymphocytes from C57BL / 6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro . SDS - PAGE and immunoblotting against indicated proteins was performed . Shown are representative western blots out of three independent experiments with similar outcome .

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Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914. Ficoll enriched lymphocytes from C57BL/6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro. SDS-PAGE and immunoblotting against indicated proteins was performed. Shown are representative western blots out of three independent experiments with similar outcome.


Flow cytometry analysis of H - 2Kb surface expression on splenocytes derived from C57BL / 6 wild type ( left side ) or LMP7 - / - ( right side ) mice treated with vehicle ( DMSO ) , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) overnight . Unstained splenocytes were used as negative control . Pooled data from three independent experiments ( n = 9 ) are shown as the means of median fluorescent intensity ± SEM . All data were statistically compared to the DMSO treated group . * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA .

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

Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 wild type (left side) or LMP7-/- (right side) mice treated with vehicle (DMSO), ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) overnight. Unstained splenocytes were used as negative control. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM. All data were statistically compared to the DMSO treated group. **P < 0.01, ***P < 0.001. One-way ANOVA.


Splenocytes from C57BL / 6 mice ( A ) or human PBMCs ( B ) were incubated ( continuous treatment ) with DMSO , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) and stimulated with LPS overnight . The IL - 6 concentration in the supernatant was analyzed by ELISA . The highest IL - 6 concentration from each experiment was set to 100 % . ( A ) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates . ( B ) Data are presented as the mean ± SEM of 6 different healthy donors . All data were statistically compared to the DMSO treated group .

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Splenocytes from C57BL/6 mice (A) or human PBMCs (B) were incubated (continuous treatment) with DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) and stimulated with LPS overnight. The IL-6 concentration in the supernatant was analyzed by ELISA. The highest IL-6 concentration from each experiment was set to 100%. (A) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates. (B) Data are presented as the mean ± SEM of 6 different healthy donors. All data were statistically compared to the DMSO treated group.


The differentiation of CD4 + T cells isolated from the spleens of C57BL / 6 mice that were exposed ( continuous treatment ) to DMSO , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) and that were stimulated with plate bound antibodies to CD3 / CD28 in the presence of TGF - β and IL - 6 and scavenging antibodies neutralising IL - 4 and IFN - γ was measured in 3 days cultures . IL - 17 expression was detected by intracellular cytokine staining after a short restimulation with PMA / Ionomycin . IL - 17 expression in activated T cells cultured in the absence of Th17 - polarizing conditions is shown as a comparison ( no cocktail ) . Values reflect the percentage of CD4 + cells that are IL - 17A + . Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates . All data were statistically compared to the DMSO treated group .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 differentiation of CD4+ T cells isolated from the spleens of C57BL/6 mice that were exposed (continuous treatment) to DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+ LU-001i (300 nM each) and that were stimulated with plate bound antibodies to CD3/CD28 in the presence of TGF-β and IL-6 and scavenging antibodies neutralising IL-4 and IFN-γ was measured in 3 days cultures. IL-17 expression was detected by intracellular cytokine staining after a short restimulation with PMA/Ionomycin. IL-17 expression in activated T cells cultured in the absence of Th17-polarizing conditions is shown as a comparison (no cocktail). Values reflect the percentage of CD4+ cells that are IL-17A+. Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates. All data were statistically compared to the DMSO treated group.


Colitis was induced by oral administration of 3 % DSS . Mice were treated daily ( s . c . ) with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the begin of the experiment . Data points represent means ± SEM of 15 mice pooled from three independent experiments . ( A ) The body weight of individual mice was monitored daily and the percent weight loss ( y - axis ) was plotted versus time ( x - axis ) . All data were statistically compared to the vehicle treated group . *

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

Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. (A) The body weight of individual mice was monitored daily and the percent weight loss (y-axis) was plotted versus time (x-axis). All data were statistically compared to the vehicle treated group. *