Figure 7 ( C ) CH12F3 - 2A cells were transfected with WT Phf5a or its mutants ( Phf5aR - MF ) along with siRNA against Phf5a . An anti - Flag antibody was used to immunoprecipitate and immunodetect Phf5aR - MF . An anti - Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a . The antibodies used to detect the co - associated proteins are indicated next to the respective blot .

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Figure 7(C) CH12F3-2A cells were transfected with WT Phf5a or its mutants (Phf5aR-MF) along with siRNA against Phf5a. An anti-Flag antibody was used to immunoprecipitate and immunodetect Phf5aR-MF. An anti-Phf5a immunoblot confirmed the knockdown efficiency of the endogenous Phf5a. The antibodies used to detect the co-associated proteins are indicated next to the respective blot.


Figure 8 ( B ) Western blot showing expression of endogenous and exogenously introduced Phf5a . ( C ) H2A . Z ChIP assay was performed using CH12F3 - 2A cells transfected with indicated siRNA and the Phf5a expression constructs . The data are compiled from 2 experiments and presented relative to siControl treated sample , which was set as 100 . The error bars represent the mean ± sd .

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Figure 8(B) Western blot showing expression of endogenous and exogenously introduced Phf5a.(C) H2A.Z ChIP assay was performed using CH12F3-2A cells transfected with indicated siRNA and the Phf5a expression constructs. The data are compiled from 2 experiments and presented relative to siControl treated sample, which was set as 100. The error bars represent the mean ± sd.


Figure 1C . Western blot ( WB ) analysis of splicing factors for Vector and 2FLAG - eIF4E U2OS cell lines . Myc , Mcl1 , and cyclin D1 ( CCND1 ) served as positive controls , and β - actin as a loading control . Both 2FLAG - eIF4E ( 2Flag - eIF4E ) and endogenous eIF4E ( endog - eIF4E ) are shown . Each β - actin blot corresponds to the above western blots . Experiments were carried out at least three independent times , and one representative experiment is shown . D . WB analysis of splicing factors as a function of eIF4E reduction using CRISPR - eIF4E and CRISPR - Ctrl U2OS cell lines . Myc , Mcl1 and CCND1 served as positive controls , and β - actin a loading control . Each β - actin blot corresponds to the western blots immediately above . Experiments were carried out at least three independent times , and one representative experiment is shown . H . Total RNA levels monitored by RT - qPCR corresponding to mRNA export assays shown in G . Data were normalized to Vector control to calculate fold change . The mean and standard deviation , , as well as p - values ( using Student t - test ) , were derived from three biological experiments , each carried out in technical triplicate . Data information : Student t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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Figure 1C. Western blot (WB) analysis of splicing factors for Vector and 2FLAG-eIF4E U2OS cell lines. Myc, Mcl1, and cyclin D1 (CCND1) served as positive controls, and β-actin as a loading control. Both 2FLAG-eIF4E (2Flag-eIF4E) and endogenous eIF4E (endog-eIF4E) are shown. Each β-actin blot corresponds to the above western blots. Experiments were carried out at least three independent times, and one representative experiment is shown.D. WB analysis of splicing factors as a function of eIF4E reduction using CRISPR-eIF4E and CRISPR-Ctrl U2OS cell lines. Myc, Mcl1 and CCND1 served as positive controls, and β-actin a loading control. Each β-actin blot corresponds to the western blots immediately above. Experiments were carried out at least three independent times, and one representative experiment is shown.H. Total RNA levels monitored by RT-qPCR corresponding to mRNA export assays shown in G. Data were normalized to Vector control to calculate fold change. The mean and standard deviation, , as well as p-values (using Student t-test), were derived from three biological experiments, each carried out in technical triplicate. Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001


Figure 2A . WB analysis of the impacts of S53A 2FLAG - eIF4E ( 2Flag - S53A ) relative to WT 2FLAG - eIF4E ( 2Flag - eIF4E ) on SF production . CCND1 served as positive controls , and β - actin or HSP90 as loading controls . Both 2FLAG - eIF4E ( 2Flag - eIF4E ) and endogenous eIF4E ( endog - eIF4E ) are shown . Each β - actin or HSP90 blot corresponds to the above western blots . S53A eIF4E protein levels are indistinguishable from eIF4E . Experiments were carried out using three separate clones for eIF4E , S53A and Vector controls , and one representative experiment is shown . ( Right ) Western blots were quantified by FIJI , intensities normalized to Vector control and plotted using PRISM . The mean , standard deviation , and p - values are shown . Student t - test : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . B . Polysomal loading analysis was measured to assess the relevance of eIF4E ' s capacity to increase translation efficiency on SF protein production . Top panel shows the polysomal profile ( at 254 nm ) demonstrating that 2FLAG - eIF4E overexpression did not alter the profile relative to Vector controls , consistent with previous studies Polysomes were isolated by size exclusion chromatography ( SEC ) and thus the heaviest polysomes elute first and monosomes last . ( Middle and Bottom panels ) RNAs were monitored on heavy , light or monosome polysomes using RT - qPCR and presented as a fraction for the given RNA . Known translation targets of eIF4E , MYC and VEGF was shifted to higher polysomes in eIF4E overexpressing cells as well as SF - encoding RNAs with altered polysomes ( middle ) or those that were unchanged by eIF4E including the negative control ACTB are in the bottom panel Experiments were carried out in biological duplicates using different clones . Means are shown and p - values calculated using ANOVA ( PRISM ) . Data information : ANOVA test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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Figure 2A. WB analysis of the impacts of S53A 2FLAG-eIF4E (2Flag-S53A) relative to WT 2FLAG-eIF4E (2Flag-eIF4E) on SF production. CCND1 served as positive controls, and β-actin or HSP90 as loading controls. Both 2FLAG-eIF4E (2Flag-eIF4E) and endogenous eIF4E (endog-eIF4E) are shown. Each β-actin or HSP90 blot corresponds to the above western blots. S53A eIF4E protein levels are indistinguishable from eIF4E. Experiments were carried out using three separate clones for eIF4E, S53A and Vector controls, and one representative experiment is shown. (Right) Western blots were quantified by FIJI, intensities normalized to Vector control and plotted using PRISM. The mean, standard deviation, and p-values are shown. Student t-test: * p<0.05, **p<0.01, ***p<0.001.B. Polysomal loading analysis was measured to assess the relevance of eIF4E's capacity to increase translation efficiency on SF protein production. Top panel shows the polysomal profile (at 254 nm) demonstrating that 2FLAG-eIF4E overexpression did not alter the profile relative to Vector controls, consistent with previous studies Polysomes were isolated by size exclusion chromatography (SEC) and thus the heaviest polysomes elute first and monosomes last. (Middle and Bottom panels) RNAs were monitored on heavy, light or monosome polysomes using RT-qPCR and presented as a fraction for the given RNA. Known translation targets of eIF4E, MYC and VEGF was shifted to higher polysomes in eIF4E overexpressing cells as well as SF-encoding RNAs with altered polysomes (middle) or those that were unchanged by eIF4E including the negative control ACTB are in the bottom panel Experiments were carried out in biological duplicates using different clones. Means are shown and p-values calculated using ANOVA (PRISM) . Data information: ANOVA test, * p<0.05, **p<0.01, ***p<0.001


Figure 3B . Western blot analysis of untreated ( Ctrl ) and Ribavirin treated ( Rib ) Vector and eIF4E NOMO - 1 cell lines . Myc , and Mcl1 served as positive controls , and β - actin as a loading control . Each β - actin blot corresponds to the above WBs . Experiments were carried out at least three independent times , and one representative experiment is shown . Ribavirin dose was 10uM which is clinically achievableC . WB analysis of Splicing Factors levels in primary AML samples with high ( AML - H ) or normal ( AML - N ) eIF4E levels , as well as bone marrow mononuclear cells from healthy volunteers ( Norm for normal ) . Numbers refer to different individuals . β - actin was used as a loading control . ( * = degraded sample ) . E . Total levels of UsnRNA in NOMO - 1 Vector and eIF4E cells monitored by RT - qPCR . Data were normalized to Vector control and shown as a fold change . The mean and standard deviation , as well as p - values ( using Student t - test ) were derived from three biological replicates each carried out in triplicate for all panels .

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Figure 3B. Western blot analysis of untreated (Ctrl) and Ribavirin treated (Rib) Vector and eIF4E NOMO-1 cell lines. Myc, and Mcl1 served as positive controls, and β-actin as a loading control. Each β-actin blot corresponds to the above WBs. Experiments were carried out at least three independent times, and one representative experiment is shown. Ribavirin dose was 10uM which is clinically achievableC. WB analysis of Splicing Factors levels in primary AML samples with high (AML-H) or normal (AML-N) eIF4E levels, as well as bone marrow mononuclear cells from healthy volunteers (Norm for normal). Numbers refer to different individuals. β-actin was used as a loading control. (* = degraded sample).E. Total levels of UsnRNA in NOMO-1 Vector and eIF4E cells monitored by RT-qPCR. Data were normalized to Vector control and shown as a fold change. The mean and standard deviation, as well as p-values (using Student t-test) were derived from three biological replicates each carried out in triplicate for all panels.


Figure 4A . Endogenous eIF4E co - immunoprecipitated with SFs in the nuclear fractions of U2OS cells . Immunoprecipitations ( IP ) were carried out using U2OS nuclear lysates and anti - eIF4E antibody ( eIF4E - IP ) or appropriate IgG control ( IgG - IP ) . IP samples along with Input ( 2 % ) and supernatants ( Sn ) after IPs were analyzed by WB using antibodies as indicated . B . eIF4E co - immunoprecipitated with SFs in the nuclear fractions of NOMO - 1 cells . IPs were carried out using eIF4E overexpressing NOMO - 1 nuclear lysates and anti - eIF4E antibody ( eIF4E - IP ) or appropriate IgG control ( IgG - IP ) . IP samples were analyzed by WB as above and carried out in three biological replicates . C . eIF4E interaction with splicing factors depends on the presence of RNAs . The eIF4E IP was divided into two and treated with buffer or RNase . After elution , 2 % of the nuclear extract and the IPs were resolved on SDS - PAGE and analyzed by western blotting with indicated antibodies . Representative gels of three biological replicates are shown ( middle panel ) and intensities measured using FIJI are shown in scatter plot ( bottom panel ) generated in PRISM with means depicted as horizontal lines and standard deviation as vertical lines . Each point represents an independent experiment . Fraction purity was assessed with the indicated antibodies ( C top panel ) . Data information : ( C , The number of independent experiments for each target is shown which varies from n = 3 - 4 replicates . Error bars representative standard deviations ( vertical line ) .

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Figure 4A. Endogenous eIF4E co-immunoprecipitated with SFs in the nuclear fractions of U2OS cells. Immunoprecipitations (IP) were carried out using U2OS nuclear lysates and anti-eIF4E antibody (eIF4E-IP) or appropriate IgG control (IgG-IP). IP samples along with Input (2%) and supernatants (Sn) after IPs were analyzed by WB using antibodies as indicated. B. eIF4E co-immunoprecipitated with SFs in the nuclear fractions of NOMO-1 cells. IPs were carried out using eIF4E overexpressing NOMO-1 nuclear lysates and anti-eIF4E antibody (eIF4E-IP) or appropriate IgG control (IgG-IP). IP samples were analyzed by WB as above and carried out in three biological replicates.C. eIF4E interaction with splicing factors depends on the presence of RNAs. The eIF4E IP was divided into two and treated with buffer or RNase. After elution, 2% of the nuclear extract and the IPs were resolved on SDS-PAGE and analyzed by western blotting with indicated antibodies. Representative gels of three biological replicates are shown (middle panel) and intensities measured using FIJI are shown in scatter plot (bottom panel) generated in PRISM with means depicted as horizontal lines and standard deviation as vertical lines. Each point represents an independent experiment. Fraction purity was assessed with the indicated antibodies (C top panel). Data information: (C, The number of independent experiments for each target is shown which varies from n=3-4 replicates. Error bars representative standard deviations (vertical line).


Figure 6C . Survival analyses for of High - eIF4E patients and Normal - eIF4E patients for which specimens were studied The rest of the cohort is shown for comparison . Log Rank test was performed and shows a significant difference in survival between the High - eIF4E group ( 10 specimens ) and the rest of the cohort ( 432 specimens ) , as well as between High - eIF4E and Normal - eIF4E groups ( 11 specimens ) ( p - value < 0 . 05 )

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Figure 6C. Survival analyses for of High-eIF4E patients and Normal-eIF4E patients for which specimens were studied The rest of the cohort is shown for comparison. Log Rank test was performed and shows a significant difference in survival between the High-eIF4E group (10 specimens) and the rest of the cohort (432 specimens), as well as between High-eIF4E and Normal-eIF4E groups (11 specimens) (p-value < 0.05)


Figure 7C . Validation of splicing targets identified by rMATs in U2OS ( upper panel ) and NOMO - 1 ( lower panel ) cell lines . RT - qPCR analysis using specific primers for each splicing event normalized to the corresponding total levels of that given transcript Data were normalized to Vector control to calculate fold change . The mean and standard deviation , as well as p - values were derived from three biological replicates . P - values were calculated between eIF4E overexpressing and Vector cells ( when the asterisk is over eIF4E ) , and eIF4E cells treated with Ribavirin vs eIF4E overexpressing untreated cells ( for the asterisk over Ribavirin ) . Data information : Student t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001D . eIF4E associates with both pre - mRNA splicing substrates and their spliced products . The enrichment of mRNAs for splicing targets in eIF4E - RIPs versus input mRNAs from the nuclear fractions of 2FLAG - eIF4E U2OS cells monitored by RT - qPCR using primers for the specific splice event ( S for " spliced " ) , pre - mRNA specific primers to the relevant intron or intron - exon boundary ( NS for " unspliced " ) , and primers specific to common regions present in all spliced and not - spliced mRNAs ( All ) . Data were normalized to input samples and presented as a fold change . The mean , standard deviation and p - values ( Student t - test ) were derived from three independent experiments ( each carried out in triplicate ) . Data information : Student t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 .

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Figure 7C. Validation of splicing targets identified by rMATs in U2OS (upper panel) and NOMO-1 (lower panel) cell lines. RT-qPCR analysis using specific primers for each splicing event normalized to the corresponding total levels of that given transcript Data were normalized to Vector control to calculate fold change. The mean and standard deviation, as well as p-values were derived from three biological replicates. P-values were calculated between eIF4E overexpressing and Vector cells (when the asterisk is over eIF4E), and eIF4E cells treated with Ribavirin vs eIF4E overexpressing untreated cells (for the asterisk over Ribavirin). Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001D. eIF4E associates with both pre-mRNA splicing substrates and their spliced products. The enrichment of mRNAs for splicing targets in eIF4E-RIPs versus input mRNAs from the nuclear fractions of 2FLAG-eIF4E U2OS cells monitored by RT-qPCR using primers for the specific splice event (S for "spliced"), pre-mRNA specific primers to the relevant intron or intron-exon boundary (NS for "unspliced"), and primers specific to common regions present in all spliced and not-spliced mRNAs (All). Data were normalized to input samples and presented as a fold change. The mean, standard deviation and p-values (Student t-test) were derived from three independent experiments (each carried out in triplicate). Data information: Student t-test, * p<0.05, **p<0.01, ***p<0.001


Figure 1 ( A - F ) Representative PAS - stained sections of the pons ( A ) and hippocampal ( C ) regions of the brain and heart ( E ) of one year old HOIL - 1 [ C458S ] and WT mice are shown . Scale bar = 100 μm . Arrow heads indicate α - amylase - resistant PAS - positive polyglucosan deposits . Graphs showing α - amylase resistant PAS scores of the pons ( B ) and hippocampal ( D ) regions of the brain and the heart ( F ) of HOIL - 1 [ C458S ] ( red ) and WT ( blue ) mice aged 0 . 5 , 1 . 0 and 1 . 5 years . The number of biological replicates analysed at each age is indicated . The word zero highlighted in blue indicates that no α - amylase resistant , PAS - positive material could be detected in the WT mice . The error bars show mean + SEM . Statistical significance between the genotypes was calculated by using two - way ANOVA and Šidák ' s multiple comparison ' s test . * * * * denotes p < 0 . 0001 .

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Figure 1(A-F) Representative PAS-stained sections of the pons (A) and hippocampal (C) regions of the brain and heart (E) of one year old HOIL-1[C458S] and WT mice are shown. Scale bar = 100 μm. Arrow heads indicate α-amylase-resistant PAS-positive polyglucosan deposits. Graphs showing α-amylase resistant PAS scores of the pons (B) and hippocampal (D) regions of the brain and the heart (F) of HOIL-1[C458S] (red) and WT (blue) mice aged 0.5, 1.0 and 1.5 years. The number of biological replicates analysed at each age is indicated. The word zero highlighted in blue indicates that no α-amylase resistant, PAS-positive material could be detected in the WT mice. The error bars show mean + SEM. Statistical significance between the genotypes was calculated by using two-way ANOVA and Šidák's multiple comparison's test. **** denotes p<0.0001.


Figure 2 ( A ) HOIL - 1 activity towards purified bovine liver glycogen was measured using Cy5 - labelled ubiquitin . Control assays lacking ATP , UBE2L3 , HOIL - 1 and glycogen are indicated , as are subsequent treatments with 50 μg / mL human salivary α ‑ amylase and 1 . 5 M hydroxylamine ( NH2OH ) . The gel has been stained using the periodic acid - Schiff ( PAS ) method to visualise glycogen ( purple - magenta ) and Coomassie Blue stain to visualise protein . ( B ) Prior to PAS and Coomassie staining , the gel shown in ( A ) was stained with Flamingo fluorescent protein stain ( shown in red ) and glycogen ubiquitylation observed by visualising the fluorescent Cy5 - ubiquitin signal ( shown in green ) . ( D ) In vitro ubiquitylation of maltoheptaose by HOIL - 1 . Assays were treated with α ‑ amylase or hydroxylamine as indicated . Reaction products were detected by Coomassie staining .

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Figure 2(A) HOIL-1 activity towards purified bovine liver glycogen was measured using Cy5-labelled ubiquitin. Control assays lacking ATP, UBE2L3, HOIL-1 and glycogen are indicated, as are subsequent treatments with 50 μg/mL human salivary α‑amylase and 1.5 M hydroxylamine (NH2OH). The gel has been stained using the periodic acid-Schiff (PAS) method to visualise glycogen (purple-magenta) and Coomassie Blue stain to visualise protein.(B) Prior to PAS and Coomassie staining, the gel shown in (A) was stained with Flamingo fluorescent protein stain (shown in red) and glycogen ubiquitylation observed by visualising the fluorescent Cy5-ubiquitin signal (shown in green).(D) In vitro ubiquitylation of maltoheptaose by HOIL-1. Assays were treated with α‑amylase or hydroxylamine as indicated. Reaction products were detected by Coomassie staining.


Figure 3 ( F ) HOIL - 1 activity against the indicated maltosaccharides ( 2 mM ) was assayed and visualised by staining with Coomassie protein stain . Where indicated , assays were treated with 1 . 5 M hydroxylamine . Met1 - linked ubiquitin tetramer was also included in these assays . ( H ) In vitro ubiquitylation of the indicated cyclodextrins ( 2 mM ) by HOIL - 1 , visualised by Coomassie staining . Met1 - linked ubiquitin tetramer was also included in these assays .

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Figure 3(F) HOIL-1 activity against the indicated maltosaccharides (2 mM) was assayed and visualised by staining with Coomassie protein stain. Where indicated, assays were treated with 1.5 M hydroxylamine. Met1-linked ubiquitin tetramer was also included in these assays.(H) In vitro ubiquitylation of the indicated cyclodextrins (2 mM) by HOIL-1, visualised by Coomassie staining. Met1-linked ubiquitin tetramer was also included in these assays.


Figure 4 ( A ) 1 μg of each of the indicated recombinant proteins was incubated with amylose resin for 60 min , the beads were pelleted by centrifugation , and proteins in the pellet ( P ) and supernatant ( S ) were compared to the protein input ( I ) by means of western blotting with the appropriate antibodies . ( B ) Bovine liver glycogen and potato starch - derived amylopectin were incubated for 60 min with the indicated recombinant proteins , centrifuged for 90 min at 100 , 000 × g , and protein in the resulting supernatants ( S ) and pellets ( P ) analysed by western blotting with the appropriate antibodies . Protein without polysaccharide was used as a negative control ( Buffer ) . ( C ) Amylose binding assays of full - length ( 1 - 1072 ) and truncated HOIP protein , visualised by immunoblotting . Shown on the right are schematic representations of the truncated HOIP constructs used . Images not drawn to scale . PUB , peptide N - glycanase and UBA - and UBX - containing protein domain ; ZF , zinc finger domain ; NZF , Npl4 zinc finger domain ; UBA , ubiquitin - associated domain ; RBR , RING - in - between - RING domain ; LDD , linear ubiquitin chain determining domain . ( D ) Amylose binding assays of full - length ( 1 - 387 ) and truncated Sharpin mutants along with schematic representations of the Sharpin constructs used . Images not shown to scale . PH , pleckstrin homology domain ; LTM , LUBAC - tethering motif ; UBL , ubiquitin - like domain .

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Figure 4(A) 1 μg of each of the indicated recombinant proteins was incubated with amylose resin for 60 min, the beads were pelleted by centrifugation, and proteins in the pellet (P) and supernatant (S) were compared to the protein input (I) by means of western blotting with the appropriate antibodies.(B) Bovine liver glycogen and potato starch-derived amylopectin were incubated for 60 min with the indicated recombinant proteins, centrifuged for 90 min at 100,000 × g, and protein in the resulting supernatants (S) and pellets (P) analysed by western blotting with the appropriate antibodies. Protein without polysaccharide was used as a negative control (Buffer).(C) Amylose binding assays of full-length (1-1072) and truncated HOIP protein, visualised by immunoblotting. Shown on the right are schematic representations of the truncated HOIP constructs used. Images not drawn to scale. PUB, peptide N-glycanase and UBA- and UBX-containing protein domain; ZF, zinc finger domain; NZF, Npl4 zinc finger domain; UBA, ubiquitin-associated domain; RBR, RING-in-between-RING domain; LDD, linear ubiquitin chain determining domain.(D) Amylose binding assays of full-length (1-387) and truncated Sharpin mutants along with schematic representations of the Sharpin constructs used. Images not shown to scale. PH, pleckstrin homology domain; LTM, LUBAC-tethering motif; UBL, ubiquitin-like domain.


Figure 5 ( A ) Maltoheptaose ubiquitylation by HOIL - 1 was assayed at 30 ° C in the presence of Met1 - or Lys63 - linked ubiquitin dimers for the indicated times and visualised by Coomassie staining . ( B ) Maltoheptaose ubiquitylation by HOIL - 1 was assayed at 30 ° C in the presence of Met1 - or Lys63 - linked ubiquitin tetramers for the indicated times and visualised by Coomassie staining . ( C ) HOIL - 1 [ T203A / F204A / R210A ] was assayed at 30 ° C in the absence or presence of the indicated ubiquitin dimers and tetramers . These mutations have been reported to impair the binding of ubiquitin to the NZF domain and their approximate location in HOIL - 1 is indicated ( schematic not to scale ) . ( D ) Assay of HOIL - 1 [ Δ194 - 222 ] lacking the NZF core domain at 30 ° C in the absence or presence of the indicated ubiquitin oligomers . ( E ) Allosteric activation of HOIL - 1 [ 233 - 510 ] ( lacking the LTM , UBL and NZF domains at the N ‑ terminus ) by ubiquitin oligomers .

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Figure 5(A) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin dimers for the indicated times and visualised by Coomassie staining.(B) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin tetramers for the indicated times and visualised by Coomassie staining.(C) HOIL-1[T203A/F204A/R210A] was assayed at 30°C in the absence or presence of the indicated ubiquitin dimers and tetramers. These mutations have been reported to impair the binding of ubiquitin to the NZF domain and their approximate location in HOIL-1 is indicated (schematic not to scale).(D) Assay of HOIL-1[Δ194-222] lacking the NZF core domain at 30°C in the absence or presence of the indicated ubiquitin oligomers.(E) Allosteric activation of HOIL-1[233-510] (lacking the LTM, UBL and NZF domains at the N‑terminus) by ubiquitin oligomers.


Figure 6 ( B ) Time - course assay performed at 30 ° C featuring wild - type HOIL - 1 in the absence or presence of the indicated ubiquitin oligomers , visualised by Coomassie staining . ( C ) HOIL - 1 [ I358R ] , assayed as in ( B ) . ( D ) HOIL - 1 [ F381A / E383A ] , assayed as in ( B ) . ( E ) The truncated HOIL - 1 [ 233 - 510 ; I358R ] mutant was assayed as in ( B ) . ( F ) The truncated HOIL - 1 [ 233 - 510 ; F381A / E383A ] mutant was assayed as in ( B ) .

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Figure 6(B) Time-course assay performed at 30°C featuring wild-type HOIL-1 in the absence or presence of the indicated ubiquitin oligomers, visualised by Coomassie staining.(C) HOIL-1[I358R], assayed as in (B). (D) HOIL-1[F381A/E383A], assayed as in (B).(E) The truncated HOIL-1[233-510; I358R] mutant was assayed as in (B). (F) The truncated HOIL-1[233-510; F381A/E383A] mutant was assayed as in (B).


Figure 7 ( A ) Requirements for en bloc transfer of linear ubiquitin dimer to maltoheptaose were investigated by leaving out the reaction components indicated . Reaction products were detected by staining with Coomassie . ( B ) Requirements for en bloc transfer of Met1 - linked ubiquitin tetramer to maltoheptaose were investigated by leaving out the reaction components indicated . ( C ) Time - course assay performed at 30 ° C in the presence of Met1 - linked ubiquitin oligomers in which the two most C - terminal residues were either glycine ( normal ubiquitin ) or alanine to prevent conjugation to substrates ( GG / AA ) . ( D ) Maltoheptaose ubiquitylation was assayed for 60 s at 30 ° C in the presence of the indicated ubiquitin tetramers .

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

Figure 7(A) Requirements for en bloc transfer of linear ubiquitin dimer to maltoheptaose were investigated by leaving out the reaction components indicated. Reaction products were detected by staining with Coomassie.(B) Requirements for en bloc transfer of Met1-linked ubiquitin tetramer to maltoheptaose were investigated by leaving out the reaction components indicated.(C) Time-course assay performed at 30°C in the presence of Met1-linked ubiquitin oligomers in which the two most C-terminal residues were either glycine (normal ubiquitin) or alanine to prevent conjugation to substrates (GG/AA).(D) Maltoheptaose ubiquitylation was assayed for 60 s at 30°C in the presence of the indicated ubiquitin tetramers.


Figure 1MPK4 ‐ HA is detected in PAT1 ‐ Myc immunoprecipitates from double transgenic Arabidopsis plants . Immunoblots of input and anti ‐ Myc IPs probed with anti ‐ HA and anti ‐ Myc antibodies . Left panel , input ; right panel , anti ‐ Myc IP . Yeast pat1Δ mutants are unable to grow at 37 ° C while wild ‐ type ( B4742 ) strain grows at 30 ° C ( left panel ) and 37 ° C ( right ) . Growth at 37 ° C is restored in pat1Δ expressing AtPAT1 . Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures . Co ‐ IP between PAT1 ‐ HA and LSM1 ‐ GFP . Proteins were transiently co ‐ expressed in N . benthamiana and tissue harvested 3 days post ‐ infiltration . Immunoblots of GFP IPs ( top panel ) and inputs ( 20 μg each , bottom panel ) . Immunoblots were cut in half and probed with anti ‐ HA antibodies and anti ‐ GFP antibodies . Agarose gel electrophoresis of 5 ′ RACE PCR to detect capped transcripts of EIF4A1 , UGT87A2 and EXPL1 in 14 ‐ day ‐ old Col ‐ 0 and pat1 ‐ 1 .

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Figure 1MPK4‐HA is detected in PAT1‐Myc immunoprecipitates from double transgenic Arabidopsis plants. Immunoblots of input and anti‐Myc IPs probed with anti‐HA and anti‐Myc antibodies. Left panel, input; right panel, anti‐Myc IP.Yeast pat1Δ mutants are unable to grow at 37°C while wild‐type (B4742) strain grows at 30°C (left panel) and 37°C (right). Growth at 37°C is restored in pat1Δ expressing AtPAT1. Serial dilutions of each yeast strain were plated on YPAD agar plates and grown at the indicated temperatures.Co‐IP between PAT1‐HA and LSM1‐GFP. Proteins were transiently co‐expressed in N. benthamiana and tissue harvested 3 days post‐infiltration. Immunoblots of GFP IPs (top panel) and inputs (20 μg each, bottom panel). Immunoblots were cut in half and probed with anti‐HA antibodies and anti‐GFP antibodies.Agarose gel electrophoresis of 5′ RACE PCR to detect capped transcripts of EIF4A1, UGT87A2 and EXPL1 in 14‐day‐old Col‐0 and pat1‐1.


Figure 2MPK4 and MPK6 were immunoprecipitated from extracts of Col ‐ 0 seedlings treated with 200 nM flg22 for 10 min . For the negative control ( − ) , extracts were incubated with agarose beads without antibodies . IPs were incubated with His6 ‐ PAT1 , His6 ‐ PAT1 S208A or MBP for 60 min at 37 ° C before boiling and SDS ‐ PAGE . Autoradiogram ( top panel ) , Coomassie ‐ stained gel for loading control ( bottom ) .

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Figure 2MPK4 and MPK6 were immunoprecipitated from extracts of Col‐0 seedlings treated with 200 nM flg22 for 10 min. For the negative control (−), extracts were incubated with agarose beads without antibodies. IPs were incubated with His6‐PAT1, His6‐PAT1 S208A or MBP for 60 min at 37°C before boiling and SDS‐PAGE. Autoradiogram (top panel), Coomassie‐stained gel for loading control (bottom).


Figure 3pat1 mutants have a serrated leaf , semi ‐ dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions , genotypes as indicated . Col ‐ 0 and eds1 ‐ 2 plants and pat1 ‐ 1 and pat1 ‐ 1 / eds1 ‐ 2 , respectively , were placed in the middle of the same pots before the pictures were taken .

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Figure 3pat1 mutants have a serrated leaf, semi‐dwarf phenotypePlants photographed at 4 weeks of growth in short day conditions, genotypes as indicated. Col‐0 and eds1‐2 plants and pat1‐1 and pat1‐1/eds1‐2, respectively, were placed in the middle of the same pots before the pictures were taken.


Figure 4PR gene expression is elevated in mpk4 ‐ 2 and pat1 ‐ 1 mutants . Four ‐ week ‐ old plants were used for RNA extraction , followed by qRT ‐ PCR . Fold ‐ change in PR1 ( gray bars ) and PR2 ( black ) expression is relative to Col ‐ 0 , normalized to UBQ10 . Standard error of the mean is indicated by errors bars ( n = 3 ) . Statistical significance between the mean values was determined by ANOVA followed by Fisher ' s LSD test , P ‐ values are only shown for mean values significantly different from Col ‐ 0 . pat1 is more resistant to colonization by P . syringae pv . tomato DC3000 . Bacteria were syringe ‐ infiltrated and samples taken 3 days post ‐ infiltration . Data are shown as log10 ‐ transformed colony ‐ forming units / cm2 leaf tissue ( cfu / cm2 ) . Standard error of the mean is indicated by errors bars ( n = 4 ) . Statistical significance between the mean values was determined by ANOVA followed by Fisher ′ s LSD test .

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Figure 4PR gene expression is elevated in mpk4‐2 and pat1‐1 mutants. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test, P‐values are only shown for mean values significantly different from Col‐0.pat1 is more resistant to colonization by P. syringae pv. tomato DC3000. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are shown as log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher′s LSD test.


Figure 5Confocalmicroscopy with root elongation zones . Five ‐ day ‐ old Col ‐ 0 / PAT1 ‐ GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min ( second panel ) followed by treatment with 100 μg / ml cycloheximide for 60 min ( bottom panel ) . The scale bar corresponds to 10 μm . Confocalmicroscopy with 5 ‐ day ‐ old VCS ‐ GFP seedlings treated as in ( A ) . The scale bar corresponds to 10 μm . PAT1 protein is induced by PAMP treatment . Immunoblot detection of equal amounts of protein from Col ‐ 0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water ( 180 min after infiltration ) . Immunoblots were probed with anti ‐ PAT1 antibodies . Negative control pat1 ‐ 1 was loaded for comparison . Coomassie brilliant blue protein loading control is indicated by CBB . Anti ‐ GFP immunoblotting of proteins extracted from Col ‐ 0 / PAT1 ‐ GFP seedlings treated over a time course with flg22 ( top panel ) and Coomassie brilliant blue ( CBB ) ‐ stained PVDF loading control is shown ( bottom panel ) . No PAT1 ‐ GFP was detected in negative control Col ‐ 0 .

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

Figure 5Confocalmicroscopy with root elongation zones. Five‐day‐old Col‐0/PAT1‐GFP seedlings were treated with 1 μM flg22 on glass slides for 20 min (second panel) followed by treatment with 100 μg/ml cycloheximide for 60 min (bottom panel). The scale bar corresponds to 10 μm.Confocalmicroscopy with 5‐day‐old VCS‐GFP seedlings treated as in (A). The scale bar corresponds to 10 μm.PAT1 protein is induced by PAMP treatment. Immunoblot detection of equal amounts of protein from Col‐0 seedlings at times in minutes as indicated following vacuum infiltration with 1 μM flg22 or water (180 min after infiltration). Immunoblots were probed with anti‐PAT1 antibodies. Negative control pat1‐1 was loaded for comparison. Coomassie brilliant blue protein loading control is indicated by CBB.Anti‐GFP immunoblotting of proteins extracted from Col‐0/PAT1‐GFP seedlings treated over a time course with flg22 (top panel) and Coomassie brilliant blue (CBB)‐stained PVDF loading control is shown (bottom panel). No PAT1‐GFP was detected in negative control Col‐0.


Figure 6Elevated PR gene expression in pat1 mutants is suppressed by summ2 ‐ 8 . Four ‐ week ‐ old plants were used for RNA extraction , followed by qRT ‐ PCR . Fold ‐ change in PR1 ( gray bars ) and PR2 ( black bars ) expression is relative to Col ‐ 0 , normalized to UBQ10 . Standard error of the mean is indicated by errors bars ( n = 3 ) . pat1 resistance to P . syringae pv . tomato DC3000 is suppressed by summ2 ‐ 8 . Bacteria were syringe ‐ infiltrated and samples taken 3 days post ‐ infiltration . Data are log10 ‐ transformed colony ‐ forming units / cm2 leaf tissue ( cfu / cm2 ) . Standard error of the mean is indicated by errors bars ( n = 4 ) . 14 ‐ day ‐ old seedlings grown on ms plates were used for RNA extraction . Next , 1 μg of RNA from each sample was treated with XRN1 ( NEB ) or mock treated before RT ‐ qPCR . Data were normalized to ACT2 , and transcript levels were compared between XRN1 and mock ‐ treated RNA for each genotype . Standard error of the mean is indicated by error bars ( n = 4 ) . Statistical significance between the mean values was determined by ANOVA followed by Fisher ' s LSD test . Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR , which include Ser208 , in Col ‐ 0 and mkk1 / 1 ‐ summ2 before and after flg22 treatment . The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment . The ratios were obtained using quantitative iTRAQ mass spectrometry .

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

Figure 6Elevated PR gene expression in pat1 mutants is suppressed by summ2‐8. Four‐week‐old plants were used for RNA extraction, followed by qRT‐PCR. Fold‐change in PR1 (gray bars) and PR2 (black bars) expression is relative to Col‐0, normalized to UBQ10. Standard error of the mean is indicated by errors bars (n = 3).pat1 resistance to P. syringae pv. tomato DC3000 is suppressed by summ2‐8. Bacteria were syringe‐infiltrated and samples taken 3 days post‐infiltration. Data are log10‐transformed colony‐forming units/cm2 leaf tissue (cfu/cm2). Standard error of the mean is indicated by errors bars (n = 4).14‐day‐old seedlings grown on ms plates were used for RNA extraction. Next, 1 μg of RNA from each sample was treated with XRN1 (NEB) or mock treated before RT‐qPCR. Data were normalized to ACT2, and transcript levels were compared between XRN1 and mock‐treated RNA for each genotype. Standard error of the mean is indicated by error bars (n = 4). Statistical significance between the mean values was determined by ANOVA followed by Fisher's LSD test.Phosphorylation of the PAT1 peptide SSFVSYPPPGSISPDQR, which include Ser208, in Col‐0 and mkk1/1‐summ2 before and after flg22 treatment. The phosphorylation stoichiometry is illustrated relative to Col PAT1 without flg22 treatment. The ratios were obtained using quantitative iTRAQ mass spectrometry.


Figure 7Proteins were transiently co ‐ expressed ( PAT1 ‐ GFP + MPK4 ‐ HA or SUMM2 ‐ HA ) in N . benthamiana and tissue harvested 2 days post ‐ infiltration . Anti ‐ HA immunoblots of inputs ( 20 μg each ) are shown in the bottom panel . Immunoblots of GFP IPs were probed with anti ‐ HA antibodies , then stripped and probed with anti ‐ GFP antibodies . Molecular weights are shown in kDa on the right . Confocalimages of yellow fluorescent protein complementation . PAT1 ‐ Nc was transiently co ‐ expressed with either SUMM2 ‐ Nn or At4g12010 in N . benthamiana . Confocalmicroscopy on leaf disks was conducted 2 days post ‐ infiltration . Merged pictures show overlay of brightfield , chlorophyll and eYFP . The scale bar corresponds to 25 μm .

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Figure 7Proteins were transiently co‐expressed (PAT1‐GFP + MPK4‐HA or SUMM2‐HA) in N. benthamiana and tissue harvested 2 days post‐infiltration. Anti‐HA immunoblots of inputs (20 μg each) are shown in the bottom panel. Immunoblots of GFP IPs were probed with anti‐HA antibodies, then stripped and probed with anti‐GFP antibodies. Molecular weights are shown in kDa on the right.Confocalimages of yellow fluorescent protein complementation. PAT1‐Nc was transiently co‐expressed with either SUMM2‐Nn or At4g12010 in N. benthamiana. Confocalmicroscopy on leaf disks was conducted 2 days post‐infiltration. Merged pictures show overlay of brightfield, chlorophyll and eYFP. The scale bar corresponds to 25 μm.


Figure 1A Analysis of MNase - digested chromatin DNA fragments from wild - type ( WT , sizes indicated in black ) and nap1Δ ( N , sizes indicated in red ) cells by agarose gel electrophoresis . Diagram illustrating reduction of array length in nap1Δ cells . B Frequency distribution of nucleosomal DNA size ( WT in grey fill with nap1Δ in blue traces ) from H3 ChIP - seq . C Calculation of nucleosome fuzziness as the standard deviation of nucleosome ( or tag ) positions . See also Appendix Fig S1 .

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Figure 1A Analysis of MNase-digested chromatin DNA fragments from wild-type (WT, sizes indicated in black) and nap1Δ (N, sizes indicated in red) cells by agarose gel electrophoresis. Diagram illustrating reduction of array length in nap1Δ cells.B Frequency distribution of nucleosomal DNA size (WT in grey fill with nap1Δ in blue traces) from H3 ChIP-seq.C Calculation of nucleosome fuzziness as the standard deviation of nucleosome (or tag) positions. See also Appendix Fig S1.


Figure 2B Ribbon view of the asymmetric crystallographic unit containing a ( yNap1c2 - H2AΔ14 - H2BΔ28 ) 6 assembly . For clarity only layer 1 of the complex is shown . Layer 1 contains three protomers A - C that are related by two perpendicular non - crystallographic dyads ( ) . C Side - view of the complex . The layer 2 omitted from panel B is shown in grey . The yNap1 dimer is shown in green and yellow , H2A in red and H2B in blue . See also Fig EV1 . D View of the yNap1 - H2A - H2B protomer complex with labeling of secondary structure elements .

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Figure 2B Ribbon view of the asymmetric crystallographic unit containing a (yNap1c2-H2AΔ14-H2BΔ28)6 assembly. For clarity only layer 1 of the complex is shown. Layer 1 contains three protomers A-C that are related by two perpendicular non-crystallographic dyads ().C Side-view of the complex. The layer 2 omitted from panel B is shown in grey. The yNap1 dimer is shown in green and yellow, H2A in red and H2B in blue. See also Fig EV1.D View of the yNap1-H2A-H2B protomer complex with labeling of secondary structure elements.


Figure 3A Overview of the yNap1c2 - H2AΔ14 - H2BΔ28 complex . B Residues R17 , R20 , R29 , R32 , R35 and K36 of H2A make contacts with the acidic HBR1 in yNap1 comprising residues D201 , D205 and E310 . C Residues K75 , R77 and R81 of H2A make polar contacts with the HBR2 comprising residues E332 , D333 and E339 in α8 of yNap1 . D In vitro GST - pulldown assay showing the role of residues in the HBR interfaces in H2A - H2B binding . The mutant labeled HBR1 ( lane 6 ) contains the triple mutation D201R , D205R , E310R and HBR2 ( lane 12 ) the triple mutations E332 , D333 , E336 . HBR1 + 2 ( lane 13 ) contains all six mutations . The mutant labeled HBR1 + 2A ( lane 14 ) has Alanine replacements in the six HBR residues . Top panel : Bound . Bottom panel : Input . E Disruption of the Nap1 - histone interaction by H2A mutation . Bottom panel : Inputs of the coexpressed proteins ( soluble extract ) and top panel the material bound to the glutathione affinity column were analyzed by SDS - PAGE and Coomassie staining . F Controls showing input ( bottom ) and bound ( top ) fractions . See also Fig EV2 .

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Figure 3A Overview of the yNap1c2-H2AΔ14-H2BΔ28 complex.B Residues R17, R20, R29, R32, R35 and K36 of H2A make contacts with the acidic HBR1 in yNap1 comprising residues D201, D205 and E310.C Residues K75, R77 and R81 of H2A make polar contacts with the HBR2 comprising residues E332, D333 and E339 in α8 of yNap1.D In vitro GST-pulldown assay showing the role of residues in the HBR interfaces in H2A-H2B binding. The mutant labeled HBR1 (lane 6) contains the triple mutation D201R, D205R, E310R and HBR2 (lane 12) the triple mutations E332, D333, E336. HBR1+2 (lane 13) contains all six mutations. The mutant labeled HBR1+2A (lane 14) has Alanine replacements in the six HBR residues. Top panel: Bound. Bottom panel: Input.E Disruption of the Nap1-histone interaction by H2A mutation. Bottom panel: Inputs of the coexpressed proteins (soluble extract) and top panel the material bound to the glutathione affinity column were analyzed by SDS-PAGE and Coomassie staining.F Controls showing input (bottom) and bound (top) fractions. See also Fig EV2.


Figure 5A EM analysis of the yNap1c2 - H2AΔ14 - H2BΔ28 complex . Representative rawimage of negatively - stained particles . B Representative particles ( 1st row ) , class averages coming from the projection matching procedure ( 2nd row ) and comparison of the class averages with projections from a final model generated after four projection matching cycles ( 3rd row ) . C From left to right : Crystal structure of the ( yNap1c2 - H2A - H2B ) 6 complex in two different orientations ( brown ) ; 60 Å - filtered structure of the complex ( magenta ) ; 3D reconstruction of the complex from negatively stained particles ( green ) and fit of the crystal structure into the EM map . D - I Mass spectra of yNap1 variants in the absence and presence of H2A - H2B . yNap1 in complex in the absence ( D ) or presence of H2A - H2B ( E ) . yNap1c in the absence ( F ) or bound to H2A - H2B ( G ) . The oligomerization mutant yNap1 _ IF1 alone ( H ) or in complex with H2A _ IF2Δ14 - H2BΔ28 ( I ) . yNap1 is shown in green circles and H2A - H2B as red and blue asterisks .

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Figure 5A EM analysis of the yNap1c2-H2AΔ14-H2BΔ28 complex. Representative rawimage of negatively-stained particles.B Representative particles (1st row), class averages coming from the projection matching procedure (2nd row) and comparison of the class averages with projections from a final model generated after four projection matching cycles (3rd row).C From left to right: Crystal structure of the (yNap1c2-H2A-H2B)6 complex in two different orientations (brown); 60 Å-filtered structure of the complex (magenta); 3D reconstruction of the complex from negatively stained particles (green) and fit of the crystal structure into the EM map.D-I Mass spectra of yNap1 variants in the absence and presence of H2A-H2B. yNap1 in complex in the absence (D) or presence of H2A-H2B (E). yNap1c in the absence (F) or bound to H2A-H2B (G). The oligomerization mutant yNap1_IF1 alone (H) or in complex with H2A_IF2Δ14-H2BΔ28 (I). yNap1 is shown in green circles and H2A-H2B as red and blue asterisks.


Figure 6A Native PAGE gel showing the result of an affinity shift assay scanned to detect AF488 - ( left ) or AF647 - labeled DNA ( right ) . 31 nM - 4 µM of yNap1 - H2A - H2B ( lanes 6 - 13 ) were incubated with a mixture of 0 . 8 µM AF488 - labeled H3 - H4 - DNA tetrasome complexes and 0 . 8 µM AF647 - labeled DNA ( lane 5 ) . Lanes 1 - 3 show migration of tetrasomes ( tetra ) , nucleosomes ( nucl ) or free H2A - H2B obtained by salt deposition onto AF647 - labeled DNA . B Intensity of the nucleosome band ( nucl ) is plotted . The value at 4 µM of yNap1 - H2A - H2B ( lane 13 ) was set to 100 % . For non - specific DNA binding , the value in the absence of H2A - H2B ( lane 5 ) was set to 100 % intensity and disappearance of the free DNA AF647 band was plotted . Each curve is representative of three independent experiments performed on different days . Standard deviations are shown . C Analysis of MNase - digested chromatin DNA fragments from wild - type ( BY4741 ) , nap1Δ , and nap1Δ cells complemented with different yNap1 expression plasmids . Total MNase units per 50 ml of culture are indicated on the bottom of the lanes . Kb + : DNA Marker .

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Figure 6A Native PAGE gel showing the result of an affinity shift assay scanned to detect AF488- (left) or AF647-labeled DNA (right). 31 nM - 4 µM of yNap1-H2A-H2B (lanes 6-13) were incubated with a mixture of 0.8 µM AF488-labeled H3-H4-DNA tetrasome complexes and 0.8 µM AF647-labeled DNA (lane 5). Lanes 1-3 show migration of tetrasomes (tetra), nucleosomes (nucl) or free H2A-H2B obtained by salt deposition onto AF647-labeled DNA.B Intensity of the nucleosome band (nucl) is plotted. The value at 4 µM of yNap1-H2A-H2B (lane 13) was set to 100%. For non-specific DNA binding, the value in the absence of H2A-H2B (lane 5) was set to 100% intensity and disappearance of the free DNA AF647 band was plotted. Each curve is representative of three independent experiments performed on different days. Standard deviations are shown.C Analysis of MNase-digested chromatin DNA fragments from wild-type (BY4741), nap1Δ, and nap1Δ cells complemented with different yNap1 expression plasmids. Total MNase units per 50 ml of culture are indicated on the bottom of the lanes. Kb+: DNA Marker.


Figure 1A ) Western blot analysis of Mpl in healthy donors ' ( HD ) platelets shows two distinct bands representing the mature glycoprotein ( top band ) and the ER ‐ core glycosylated form ( bottom band ) . The ratio is ~ 1 . B ) Western blot analysis of HEL whole cell lysate after transfection with cDNA coding for JAK2WT or JAK2V617F , alone or cotransfected with cDNA coding for Mpl . C ) Summary of the ratios of mature : immature Mpl for at least three independent ( 2 for the JAK2V617F transfection ) blotting experiments under the protocol shown in ( B ) . Based on two − tailed Student t − test statistical analysis , there is a significant difference ( p = 0 . 0001 ) in the ratio of Mpl isoforms in cells transfected with JAK2WT by comparison with control cells . Differences were also statistically significant in HEL cells transfected with either JAK2V617F or JAK2L611V / V617F by comparison to controls ( p = 0 . 0159 and p = 0 . 0009 , respectively ) . The mature : immature ratio is also significantly higher in cells transfected with JAK2WT than in cells transfected with JAK2V617F or JAK2L611V / V617F ( p < 0 . 01 ) . D ) Western blot analysis of K562 lysates after transfection with cDNA coding for Mpl , alone or cotransfected with cDNA coding for JAK2WT or JAK2V617F . E ) Summary of data in ( A ) , ( B ) and ( D ) , showing the relative band intensity of mature and immature Mpl and normalized for levels of β ‐ actin in each lane . F ) Immunoprecipitation of the Mpl ‐ Jak2 complex in different cell lines analyzed by immunoblotting with anti ‐ Mpl and anti ‐ JAK2 antibodies .

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Figure 1A) Western blot analysis of Mpl in healthy donors' (HD) platelets shows two distinct bands representing the mature glycoprotein (top band) and the ER‐core glycosylated form (bottom band). The ratio is ~ 1.B) Western blot analysis of HEL whole cell lysate after transfection with cDNA coding for JAK2WT or JAK2V617F, alone or cotransfected with cDNA coding for Mpl.C) Summary of the ratios of mature:immature Mpl for at least three independent (2 for the JAK2V617F transfection) blotting experiments under the protocol shown in (B). Based on two−tailed Student t−test statistical analysis, there is a significant difference (p = 0.0001) in the ratio of Mpl isoforms in cells transfected with JAK2WT by comparison with control cells. Differences were also statistically significant in HEL cells transfected with either JAK2V617F or JAK2L611V/V617F by comparison to controls (p = 0.0159 and p = 0.0009, respectively). The mature:immature ratio is also significantly higher in cells transfected with JAK2WT than in cells transfected with JAK2V617F or JAK2L611V/V617F (p < 0.01).D) Western blot analysis of K562 lysates after transfection with cDNA coding for Mpl, alone or cotransfected with cDNA coding for JAK2WT or JAK2V617F.E) Summary of data in (A), (B) and (D), showing the relative band intensity of mature and immature Mpl and normalized for levels of β‐actin in each lane.F) Immunoprecipitation of the Mpl‐Jak2 complex in different cell lines analyzed by immunoblotting with anti‐Mpl and anti‐JAK2 antibodies.


Figure 2Confocal fluorescent images of HEL cells ( JAK2V617F + / + ) after immuno ‐ fluorescence labeling of endogenous Mpl ( A ) , or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 ( B ) . C ) Confocal fluorescence image of K562 cells ( JAK2WT + / + ) transiently transfected with cDNA coding for MplmOrange2 . Red arrow in this image points to plasma membrane localization . D and E ) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno ‐ fluorescent labeling of endogenous JAK2 in K562 ( D ) or HEL ( E ) cells . Profiles of fluorescence ( across individual cells and indicated by a white arrow ) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane ( black arrow heads ) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL . F and G ) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine ( F ) or JAK2V617FmCitrine ( G ) in K562 cells . Profiles of fluorescence ( white arrows ) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine , especially at the plasma membrane ( black arrow heads ) , but a very poor colocalization with JAK2V617FmCitrine ( grey arrow heads ) . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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Figure 2Confocal fluorescent images of HEL cells (JAK2V617F+/+) after immuno‐fluorescence labeling of endogenous Mpl (A), or after transient transfection with cDNA coding for a chimeric Mpl protein fused to mOrange2 (B).C) Confocal fluorescence image of K562 cells (JAK2WT+/+) transiently transfected with cDNA coding for MplmOrange2. Red arrow in this image points to plasma membrane localization.D and E) Confocal fluorescent images obtained after transient expression of MplmOrange2 and immuno‐fluorescent labeling of endogenous JAK2 in K562 (D) or HEL (E) cells. Profiles of fluorescence (across individual cells and indicated by a white arrow) are showing a good colocalization of Mpl and JAK2WT in K562 cells at the plasma membrane (black arrow heads) but no colocalization have been observed between MplmOrange2 and JAK2V617F in HEL.F and G) Confocal fluorescent images obtained after transient coexpression of MplmOrange2 and either JAK2WTmCitrine (F) or JAK2V617FmCitrine (G) in K562 cells. Profiles of fluorescence (white arrows) are showing a very good colocalization of MplmOrange2 with JAK2WTmCitrine, especially at the plasma membrane (black arrow heads), but a very poor colocalization with JAK2V617FmCitrine (grey arrow heads). Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


Figure 3A ) K562 cells labeled live for 30 min with an anti ‐ CD235a ‐ Atto655 ( glycophorin A ) antibody before treatment with the Duolink reagents . B ) K562Mpl ‐ mKO2 cells non ‐ treated with the Duolink reagents . C and D ) HEL cells primed live for 30 min with an anti ‐ CD235a ‐ Atto655 antibody before treatment with the Duolink reagents . E and F ) K562Mpl ‐ mKO2 cells submitted to the Duolink reaction . White arrow heads are pointing at example of Duolink positive reactions at the plasma membrane , while green arrow heads are pointing at positive Duolink reactions within the cytoplasm . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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Figure 3A) K562 cells labeled live for 30 min with an anti‐CD235a‐Atto655 (glycophorin A) antibody before treatment with the Duolink reagents.B) K562Mpl‐mKO2 cells non‐treated with the Duolink reagents.C and D) HEL cells primed live for 30 min with an anti‐CD235a‐Atto655 antibody before treatment with the Duolink reagents.E and F) K562Mpl‐mKO2 cells submitted to the Duolink reaction. White arrow heads are pointing at example of Duolink positive reactions at the plasma membrane, while green arrow heads are pointing at positive Duolink reactions within the cytoplasm. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


Figure 4HEL ( A ) and K562 ( B ) cells treated with non ‐ targeting ( NT ) or anti ‐ JAK2 siRNA analyzed by western blot . C ) Confocal live cell ( upper panel ) , or fixed cells ( lower left panel ) , imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti ‐ JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells . After treatment with anti ‐ JAK2 siRNA , Mpl strongly colocalizes with the ER marker , calnexin . This colocalization in treated vs non ‐ treated cells is quantified and shown in Figure C lower right panel . Nuclei are labeled with Hoechst and scale bars = 5 µm .

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Figure 4HEL (A) and K562 (B) cells treated with non‐targeting (NT) or anti‐JAK2 siRNA analyzed by western blot.C) Confocal live cell (upper panel), or fixed cells (lower left panel), imaging of K562 cells expressing MpleGFP or MplmKO2 treated with NT or anti‐JAK2 siRNA showing a difference of distribution of intracellular Mpl in the siRNA treated cells compare to the control cells. After treatment with anti‐JAK2 siRNA, Mpl strongly colocalizes with the ER marker, calnexin. This colocalization in treated vs non‐treated cells is quantified and shown in Figure C lower right panel. Nuclei are labeled with Hoechst and scale bars = 5 µm.


Figure 5Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells . A - M ) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 ( A - F ) or HEL ( G - M ) cells . Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image . Black arrows in the plots point to overlap between MplmKO2 and either ER ‐ tracker ( C , I ) , or Sec31a ( B , J ) that marks ER exit sites . MplmOrange2 did not colocalize with markers for the Golgi ( Golgin ‐ 97 ) or early endosomes ( EEA1 ) in either cell line . However , MplmKO2 colocalized greatly with autophagic ( LC3 ) ( E , L ) and lysosomal ( LAMP1 ) ( F , M ) markers . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient . Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells . A - M ) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 ( A - F ) or HEL ( G - M ) cells . Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image . Black arrows in the plots point to overlap between MplmKO2 and either ER ‐ tracker ( C , I ) , or Sec31a ( B , J ) that marks ER exit sites . MplmOrange2 did not colocalize with markers for the Golgi ( Golgin ‐ 97 ) or early endosomes ( EEA1 ) in either cell line . However , MplmKO2 colocalized greatly with autophagic ( LC3 ) ( E , L ) and lysosomal ( LAMP1 ) ( F , M ) markers . Nuclei are labeled with Hoechst and scale bars = 5 µm . R : Pearson ' s correlation coefficient .

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Figure 5Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells. A-M) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 (A-F) or HEL (G-M) cells. Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image. Black arrows in the plots point to overlap between MplmKO2 and either ER‐tracker (C,I), or Sec31a (B,J) that marks ER exit sites. MplmOrange2 did not colocalize with markers for the Golgi (Golgin‐97) or early endosomes (EEA1) in either cell line. However, MplmKO2 colocalized greatly with autophagic (LC3) (E,L) and lysosomal (LAMP1) (F,M) markers. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.Intracellular Mpl pools partially colocalize with ER markers and colocalize with autophagic and lysosomal markers in K562 and HEL cells. A-M) Confocal fluorescent images obtained after transient expression of MplmOrange2 or MplmKO2 and indicated labeling in K562 (A-F) or HEL (G-M) cells. Plots at right of each image show fluorescence intensities for each emission spectrum at points along the line drawn across each image. Black arrows in the plots point to overlap between MplmKO2 and either ER‐tracker (C,I), or Sec31a (B,J) that marks ER exit sites. MplmOrange2 did not colocalize with markers for the Golgi (Golgin‐97) or early endosomes (EEA1) in either cell line. However, MplmKO2 colocalized greatly with autophagic (LC3) (E,L) and lysosomal (LAMP1) (F,M) markers. Nuclei are labeled with Hoechst and scale bars = 5 µm. R: Pearson's correlation coefficient.


Figure 6Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 ( A ) and HEL ( B ) cells . Electron micrographs of K562 ( C ) and HEL ( D ) cells transiently expressing MplmKO2 ‐ miniSOG . Dark precipitate indicates the presence of the miniSOG ‐ tagged Mpl protein after photo ‐ precipitation of DAB . White boxes indicate original location of higher magnification images with corresponding letters . E - G ) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C , showing MplmKO2 ‐ miniSOG dark precipitate into autophagic carrier vesicles ( black arrows , Figure E ) . Osmiophilic reaction products also heavily stain mature AP in these preparations ( black arrows , Figure G ) . H ) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D , cells transiently expressing MplmKO2 - miniSOG , showing different stage of formation of APs ( black arrows numbered from stages 1 - 4 ) . Scale bars = 5 µm ( A - D ) or 100 nm ( E - H ) .

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Figure 6Confocal fluorescent images obtained after transient expression of Mpl tagged with mKO2 and miniSOG in K562 (A) and HEL (B) cells. Electron micrographs of K562 (C) and HEL (D) cells transiently expressing MplmKO2‐miniSOG. Dark precipitate indicates the presence of the miniSOG‐tagged Mpl protein after photo‐precipitation of DAB. White boxes indicate original location of higher magnification images with corresponding letters.E-G) High magnification electron micrograph of K562 cells treated in the same conditions as Figure C, showing MplmKO2‐miniSOG dark precipitate into autophagic carrier vesicles (black arrows, Figure E). Osmiophilic reaction products also heavily stain mature AP in these preparations (black arrows, Figure G).H) High magnification electron micrograph of HEL cells treated in the same conditions as Figure D, cells transiently expressing MplmKO2-miniSOG , showing different stage of formation of APs (black arrows numbered from stages 1-4). Scale bars = 5 µm (A-D) or 100 nm (E-H).


Figure 7A ) Western blot analysis and quantification of streptavidin ‐ immunoprecipitated surface ‐ biotinylated proteins , probed with an anti ‐ Mpl antibodyB and C ) Confocal images of K562 cells transiently expressing MplmOrange2 . Cells in ( B ) are untreated . Cells in ( C ) were stimulated 60 ' with Tpo , washed and chased for 15 min before imaging . D ) Quantification of plasma membrane fluorescence in resting and Tpo ‐ stimulated cells . Mean fluorescence at the membrane was estimated by averaging the total fluorescence after values equal to or below background . A two ‐ tailed Student t ‐ test shows a significant difference in the membrane fluorescence after stimulation with Tpo followed by Tpo removal . Scale bars = 5 µm . E ) Western blot analysis of protein phosphorylation in HEL , K562Mpl ‐ mKO2 and K562Mpl ‐ mOrange2 cells after Tpo stimulation ( 50 ng / mL Tpo , 10 min ) .

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Figure 7A) Western blot analysis and quantification of streptavidin‐immunoprecipitated surface‐biotinylated proteins, probed with an anti‐Mpl antibodyB and C) Confocal images of K562 cells transiently expressing MplmOrange2. Cells in (B) are untreated. Cells in (C) were stimulated 60' with Tpo, washed and chased for 15 min before imaging.D) Quantification of plasma membrane fluorescence in resting and Tpo‐stimulated cells. Mean fluorescence at the membrane was estimated by averaging the total fluorescence after values equal to or below background. A two‐tailed Student t‐test shows a significant difference in the membrane fluorescence after stimulation with Tpo followed by Tpo removal. Scale bars = 5 µm.E) Western blot analysis of protein phosphorylation in HEL, K562Mpl‐mKO2 and K562Mpl‐mOrange2 cells after Tpo stimulation (50 ng/mL Tpo, 10 min).


Figure 8A and B ) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl ‐ mKO2 cells after treatments for 60 min with Tpo , ionomycin ( I ) , PMA , Mon or A23187 . C ) Total lysate , surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl ‐ mKO2 cells treated with Mon or A23187 for 15 h . D ) Western blot analysis of HEL cells treated , or not , with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng / mL for 30 min . The quantification of the blot is shown . E ) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3 − MA ( 3 − Methyladenine ) or Rp . F ) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R ‐ V5 6 h post ‐ transfection . G ) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55 - V5 or HA - STX5 .

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Figure 8A and B) Surface biotinylation and total lysates western blot analysis of HEL or K562Mpl‐mKO2 cells after treatments for 60 min with Tpo, ionomycin (I), PMA, Mon or A23187.C) Total lysate, surface biotinylation and exosomal fraction western blot analysis of HEL and K562Mpl‐mKO2 cells treated with Mon or A23187 for 15 h.D) Western blot analysis of HEL cells treated, or not, with Bafilomycin A1 at 100 nm for 3 h and then stimulated or not with Tpo at 25 ng/mL for 30 min. The quantification of the blot is shown.E) Surface biotinylation and total lysates western blot analysis of HEL cells after treatments for 30 min with Tpo or for 3 h with 3−MA (3−Methyladenine) or Rp.F) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing ATG5 K130R‐V5 6 h post‐transfection.G) Total lysate and surface biotinylation western blot analysis of HEL cells overexpressing either GRASP55-V5 or HA-STX5.


Figure 1A , Distribution and classification of MAP4K3 / GLK - positive cells in 20 ml BALFs of all 12 individuals ( A ) , Φ denotes macrophages ; N denotes neutrophils ; T denotes T cells ; E denotes epithelial cells . 3 healthy controls ( HC , # 1 - # 3 ) , 3 mild COVID - 19 patients ( # 1 - # 3 ) , and 6 severe COVID - 19 patients ( # 1 - # 6 ) ( B ) from Cohort # 1 . Data were shown in UMAP . Single - cell gene expression of MAP4K3 ( GLK ) in individual cells was shown in color scale . C , D The percentages of MAP4K3 / GLK - positive KRT18 + epithelial ( C ) or KRT18 + TPPP3 + ciliated epithelial cells ( D ) in BALFs from Cohort # 1 . Data information : In ( C , D ) , * , P value < 0 . 05 ( ANOVA test ) ; § , P value < 0 . 05 ( Dunnett ' s test ) . E , F GLK mRNA levels in KRT18 + epithelial ( E ) or KRT18 + TPPP3 + ciliated epithelial cells ( F ) of Cohort # 1 . n ( cell number ) = 3 , 14 , 33 for HC ; n = 65 , 43 , 74 for mild COVID - 19 patients ; n = 164 , 62 , 10 , 4 , 74 , 66 for severe COVID - 19 patients ( E ) . n = 25 , 43 , 36 for HC ; n = 209 , 244 , 141 for mild COVID - 19 patients ; n = 189 , 223 , 330 , 792 , 80 , 243 for severe COVID - 19 patients ( F ) . Data information : In ( E , F ) , * , P value < 0 . 05 ; * * , P value < 0 . 01 ; * * * , P value < 0 . 001 ; * * * * , P value < 0 . 0001 ( Kruskal - Wallis test ) .

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Figure 1A, Distribution and classification of MAP4K3/GLK-positive cells in 20 ml BALFs of all 12 individuals (A), Φ denotes macrophages; N denotes neutrophils; T denotes T cells; E denotes epithelial cells.3 healthy controls (HC, #1 - #3), 3 mild COVID-19 patients (#1 - #3), and 6 severe COVID-19 patients (#1 - #6) (B) from Cohort #1. Data were shown in UMAP. Single-cell gene expression of MAP4K3 (GLK) in individual cells was shown in color scale.C, D The percentages of MAP4K3/GLK-positive KRT18+ epithelial (C) or KRT18+ TPPP3+ ciliated epithelial cells (D) in BALFs from Cohort #1. Data information: In (C, D), *, P value < 0.05 (ANOVA test); §, P value < 0.05 (Dunnett's test).E, F GLK mRNA levels in KRT18+ epithelial (E) or KRT18+ TPPP3+ ciliated epithelial cells (F) of Cohort #1. n (cell number) = 3, 14, 33 for HC; n = 65, 43, 74 for mild COVID-19 patients; n = 164, 62, 10, 4, 74, 66 for severe COVID-19 patients (E). n = 25, 43, 36 for HC; n = 209, 244, 141 for mild COVID-19 patients; n = 189, 223, 330, 792, 80, 243 for severe COVID-19 patients (F). Data information: In (E, F), *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001 (Kruskal-Wallis test).


Figure 2Immunoblotting analysis of the endogenous GLK and vinculin proteins from the lysates of HCC827 lung epithelial cells infected with SARS - CoV - 2 pseudovirus ( A ) Data information : Data shown are representative results of three ( A , independent experiments . Real - time PCR analysis of mouse GLK mRNA levels in HCC827 lung epithelial cells infected with SARS - CoV - 2 pseudovirus ( B ) The mRNA levels of GLK were normalized to GAPDH mRNA levels . n = 2 ( technical replicates ) per group . Data information : In ( B data are presented as means . Data shown are representative results of three B , independent experiments . Endogenous GLK levels were increased by the SARS - CoV - 2 spike protein . Immunoblotting analysis of the endogenous GLK and vinculin proteins from the lysates of HCC827 lung epithelial cells treated with the SARS - CoV - 2 spike protein ( C ) . Data information : Data shown are representative results of three C , independent experiments . Real - time PCR analysis of mouse GLK mRNA levels in HCC827 lung epithelial cells treated with the SARS - CoV - 2 spike protein ( D ) . The mRNA levels of GLK were normalized to GAPDH mRNA levels . n = 2 ( technical replicates ) per group . Data information : In D ) , data are presented as means . Data shown are representative results of three D ) independent experiments .

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Figure 2Immunoblotting analysis of the endogenous GLK and vinculin proteins from the lysates of HCC827 lung epithelial cells infected with SARS-CoV-2 pseudovirus (A) Data information: Data shown are representative results of three (A, independent experiments.Real-time PCR analysis of mouse GLK mRNA levels in HCC827 lung epithelial cells infected with SARS-CoV-2 pseudovirus (B) The mRNA levels of GLK were normalized to GAPDH mRNA levels. n = 2 (technical replicates) per group. Data information: In (B data are presented as means. Data shown are representative results of three B, independent experiments.Endogenous GLK levels were increased by the SARS-CoV-2 spike protein. Immunoblotting analysis of the endogenous GLK and vinculin proteins from the lysates of HCC827 lung epithelial cells treated with the SARS-CoV-2 spike protein (C). Data information: Data shown are representative results of three C, independent experiments.Real-time PCR analysis of mouse GLK mRNA levels in HCC827 lung epithelial cells treated with the SARS-CoV-2 spike protein (D). The mRNA levels of GLK were normalized to GAPDH mRNA levels. n = 2 (technical replicates) per group. Data information: In D), data are presented as means. Data shown are representative results of three D) independent experiments.


Figure 3A ZetaView nanoparticle tracking analysis of particle numbers and sizes of CD63 + extracellular vesicles ( EVs ) isolated from the supernatants of HCC827 lung epithelial cancer cells , which were transfected with GLK wild - type or GLK kinase - dead ( K45E ) mutant . EVs were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns . Data information : Data shown are representative results of two ( A ) independent experiments . B Immunoblotting of ACE2 and CD63 proteins in exosomes isolated from GLK wild - type - or GLK ( K45E ) kinase - dead - overexpressing HCC827 cells . Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns . Arrowhead denotes glycosylated ACE2 proteins ; asterisk denotes non - glycosylated ACE2 proteins . Data information : Data shown are representative results of three ( B , independent experiments . C Immunoblotting of ACE2 and CD63 proteins in serum exosomes isolated from the sera of 4 healthy controls ( HC ) and 7 COVID - 19 patients from NHRI Biobank for COVID - 19 patients in Taiwan ( Cohort # 3 ) . Serum collection days from the onset are shown ; treatment of oxygen therapy on the patient is also indicated . Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns . Arrowhead denotes glycosylated ACE2 proteins ; asterisk denotes non - glycosylated ACE2 proteins . D Confocal microscopy analysis of Flag - tagged ACE2 proteins ( red ) in recipient cells after incubation with exosomes for 72 h . Exosomes were isolated sequentially from the supernatants of Flag - ACE2 - overexpressing or GFP - GLK plus Flag - ACE2 - coexpressing HCC827 cells using ExoQuick kits and then ExoQuick ULTRA columns . Arrows denote Flag - tagged ACE2 proteins . Original magnification , × 630 ; scale bars , 10 μm . Data information : Data shown are representative results of three D , independent experiments . E Confocal microscopy analysis of SARS - CoV - 2 spike ( S ) protein ( green ) and RFP - labeled early endosomes ( red ) in exosome - recipient HCC827 epithelial cells . After incubation with exosomes for 72 h , recipient cells were treated with the spike protein ( S ) for another 24 h . Yellow color suggests the localization of S protein in early endosomes . Original magnification , × 630 ; scale bars , 25 μm . Data information : Data shown are representative results of three E , independent experiments . F Cell entry eﬃciencies of SARS - CoV - 2 pseudovirus into exosome - recipient HCC827 epithelial cells were measured by luciferase activity and presented as relative light units ( RLU ) at 24 h post infection . n = 3 ( biological replicates ) . Data information : In ( F ) , data are presented as means ± SEM . * * , P value < 0 . 01 ( ANOVA test ) . Data shown are representative results of three F ) independent experiments .

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

Figure 3A ZetaView nanoparticle tracking analysis of particle numbers and sizes of CD63+ extracellular vesicles (EVs) isolated from the supernatants of HCC827 lung epithelial cancer cells, which were transfected with GLK wild-type or GLK kinase-dead (K45E) mutant. EVs were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Data information: Data shown are representative results of two (A) independent experiments.B Immunoblotting of ACE2 and CD63 proteins in exosomes isolated from GLK wild-type- or GLK (K45E) kinase-dead-overexpressing HCC827 cells. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. Data information: Data shown are representative results of three (B, independent experiments.C Immunoblotting of ACE2 and CD63 proteins in serum exosomes isolated from the sera of 4 healthy controls (HC) and 7 COVID-19 patients from NHRI Biobank for COVID-19 patients in Taiwan (Cohort #3). Serum collection days from the onset are shown; treatment of oxygen therapy on the patient is also indicated. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins.D Confocal microscopy analysis of Flag-tagged ACE2 proteins (red) in recipient cells after incubation with exosomes for 72 h. Exosomes were isolated sequentially from the supernatants of Flag-ACE2-overexpressing or GFP-GLK plus Flag-ACE2-coexpressing HCC827 cells using ExoQuick kits and then ExoQuick ULTRA columns. Arrows denote Flag-tagged ACE2 proteins. Original magnification, ×630; scale bars, 10 μm. Data information: Data shown are representative results of three D, independent experiments.E Confocal microscopy analysis of SARS-CoV-2 spike (S) protein (green) and RFP-labeled early endosomes (red) in exosome-recipient HCC827 epithelial cells. After incubation with exosomes for 72 h, recipient cells were treated with the spike protein (S) for another 24 h. Yellow color suggests the localization of S protein in early endosomes. Original magnification, ×630; scale bars, 25 μm. Data information: Data shown are representative results of three E, independent experiments.F Cell entry eﬃciencies of SARS-CoV-2 pseudovirus into exosome-recipient HCC827 epithelial cells were measured by luciferase activity and presented as relative light units (RLU) at 24 h post infection. n = 3 (biological replicates). Data information: In (F), data are presented as means ± SEM. **, P value < 0.01 (ANOVA test). Data shown are representative results of three F) independent experiments.