Volcano plot showing DEGs in unstimulated COVID - 19 patient - derived macrophages ( SC - conv ) compared to macrophages from SARS - CoV - 2 naïve individuals ( SC - naïve ) . Negative log10 padj values are plotted against the log2 fold - change . Selected genes of interest are labeled .

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Volcano plot showing DEGs in unstimulated COVID-19 patient-derived macrophages (SC-conv) compared to macrophages from SARS-CoV-2 naïve individuals (SC-naïve). Negative log10 padj values are plotted against the log2 fold-change. Selected genes of interest are labeled.


IL - 1β concentration ( pg / ml ) in supernatants of SC - naïve individuals ( n = 7 ) which were pre - treated for 24h with FCCP ( 1µg / ml ) and H2O2 ( 10µg / ml ) . Macrophages were then incubated with LPS or S - Protein ( 4h ) and nigericin ( 2h ) as indicated in the figure . Control cells were left unstimulated or stimulated with LPS or S - protein without pre - treatment with FCSP or H2O2 ( n = 6 ) . For statistical analysis , Brown - Forsythe and Welsh ANOVA test was used .

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IL-1β concentration (pg/ml) in supernatants of SC-naïve individuals (n = 7) which were pre-treated for 24h with FCCP (1µg/ml) and H2O2 (10µg/ml). Macrophages were then incubated with LPS or S-Protein (4h) and nigericin (2h) as indicated in the figure. Control cells were left unstimulated or stimulated with LPS or S-protein without pre-treatment with FCSP or H2O2 (n = 6). For statistical analysis, Brown-Forsythe and Welsh ANOVA test was used.


Representative gene loci ( TLR2 ( top ) and S100A7A / 8 / 9 / 12 ( bottom ) H3K27ac peaks ) comparing SC - naïve ( blue ) and SC - conv ( red ) monocytes . Red peaks reach higher values than blue peaks ( representative examples of four samples for each condition ) .

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Representative gene loci (TLR2 (top) and S100A7A/8/9/12 (bottom) H3K27ac peaks) comparing SC-naïve (blue) and SC-conv (red) monocytes. Red peaks reach higher values than blue peaks (representative examples of four samples for each condition).


Quantification of IL - 1β concentrations ( pg / ml ) in the supernatant of primary macrophage cultures from patients with active / untreated tuberculosis ( n = 6 ; red / black bars ) or healthy / SARS - CoV - 2 naïve individuals ( n = 3 ; blue bars ) or the same patients after 6 months of anti - tuberculous treatment ; n = 6 ; white / red bars ) stimulated with LPS or S - protein ( 0 . 1 µg / ml ) for 4h and subsequent incubation with nigericin for 2h . For statistical analysis , two - way ANOVA with tukey post hoc test was used .

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

Quantification of IL-1β concentrations (pg/ml) in the supernatant of primary macrophage cultures from patients with active/untreated tuberculosis (n = 6; red/black bars) or healthy/SARS-CoV-2 naïve individuals (n = 3; blue bars) or the same patients after 6 months of anti-tuberculous treatment; n = 6; white/red bars) stimulated with LPS or S-protein (0.1 µg/ml) for 4h and subsequent incubation with nigericin for 2h. For statistical analysis, two-way ANOVA with tukey post hoc test was used.


G The entry efficiency of SARS - CoV - 2 and RshSTT182 pseudoviruses in HeLa - hACE2 cells . Green fluorescence indicates the entry of the pseudoviruses into the Hela - hACE2 cells . Unaltered HeLa cells were used as a negative control . H Statistics for transduction of pseudoviruses . Data represents the results of five replicates in one representative assay . All data are presented as mean ± SD . Pseudovirus infection assays were performed at least twice .

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G The entry efficiency of SARS-CoV-2 and RshSTT182 pseudoviruses in HeLa-hACE2 cells. Green fluorescence indicates the entry of the pseudoviruses into the Hela-hACE2 cells. Unaltered HeLa cells were used as a negative control. H Statistics for transduction of pseudoviruses. Data represents the results of five replicates in one representative assay. All data are presented as mean ± SD. Pseudovirus infection assays were performed at least twice.


A Flow cytometry characterization of binding between bat - ACE2 orthologs and the SARS - CoV - 2 or RshSTT182 / 200 RBDs . BHK cells expressing eGFP - fused ACE2s were stained with the indicated His - tagged proteins . The SARS - CoV - 2 NTD was used as a negative control .

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A Flow cytometry characterization of binding between bat-ACE2 orthologs and the SARS-CoV-2 or RshSTT182/200 RBDs. BHK cells expressing eGFP-fused ACE2s were stained with the indicated His-tagged proteins. The SARS-CoV-2 NTD was used as a negative control.


A Flow cytometry characterization of binding between ACE2s and the RshSTT182 / 200 or SARS - CoV - 2 RBDs . Cells stained with the RshSTT182 / 200 RBD , the SARS - CoV - 2 RBD and the SARS - CoV - 2 NTD proteins are shown in orange , marine and red , respectively . The SARS - CoV - 2 NTD was used as a negative control .

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A Flow cytometry characterization of binding between ACE2s and the RshSTT182/200 or SARS-CoV-2 RBDs. Cells stained with the RshSTT182/200 RBD, the SARS-CoV-2 RBD and the SARS-CoV-2 NTD proteins are shown in orange, marine and red, respectively. The SARS-CoV-2 NTD was used as a negative control.


D The entry efficiency of SARS - CoV - 2 and RshSTT182 pseudoviruses into HeLa - hACE2 cells . Green fluorescence indicates the entry of the pseudoviruses into the Hela - hACE2 cells . Unaltered HeLa cells were used as a negative control . E Statistics for transduction of pseudoviruses . Data represents the results of five replicates in one representative assay . All data are presented as mean ± SD . Pseudovirus infection assays were performed at least twice .

D The entry efficiency of SARS-CoV-2 and RshSTT182 pseudoviruses into HeLa-hACE2 cells. Green fluorescence indicates the entry of the pseudoviruses into the Hela-hACE2 cells. Unaltered HeLa cells were used as a negative control. E Statistics for transduction of pseudoviruses. Data represents the results of five replicates in one representative assay. All data are presented as mean ± SD. Pseudovirus infection assays were performed at least twice.


B The neutralization curve of serum samples in COVID - 19 convalescents and vaccinees against RshSTT182 and SARS - CoV - 2 are shown . Bar represents the SD of two replicates .

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B The neutralization curve of serum samples in COVID-19 convalescents and vaccinees against RshSTT182 and SARS-CoV-2 are shown. Bar represents the SD of two replicates.


C . Binding assay using MBP fused Apc1 - loop500 fragments and B56γ . Apc1 - loop500 WT or its derivatives ( ∆ 11 or 3A ) were incubated with the 35S - labelled Flag - B56γ in interphase extract ( Inter ) or anaphase extract ( Ana ) supplemented with CycB ∆ 167 at 23 ˚ C for 1 hr . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or coomassie brilliant blue ( CBB ) staining . The bar graph is quantification of bound B56γ . The intensities of MBP control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

C. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. Apc1-loop500 WT or its derivatives (∆11 or 3A) were incubated with the 35S-labelled Flag-B56γ in interphase extract (Inter) or anaphase extract (Ana) supplemented with CycB∆167 at 23˚C for 1 hr. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or coomassie brilliant blue (CBB) staining. The bar graph is quantification of bound B56γ. The intensities of MBP control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


D . Cdk - dependent in vitro kinase assay of Apc1 - loop500 . MBP fused WT or 3A Apc1 - loop500 fragment was incubated with Cdk2 - cyclin A in the presence of [ γ - 32P ] - ATP at 23 ˚ C for 10 min , separated by SDS - PAGE and detected by autoradiography .

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D. Cdk-dependent in vitro kinase assay of Apc1-loop500. MBP fused WT or 3A Apc1-loop500 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 23˚C for 10 min, separated by SDS-PAGE and detected by autoradiography.


E . Cdk can promote Apc1 - loop500 and B56 interaction . MBP fused WT or 3A Apc1 - loop500 fragment was incubated in the presence or absence of Cdk2 - cyclin A at 30 ˚ C for 60 min . MBP - fused peptides ( - / + kinase ) were isolated and incubated with purified Flag - B56γ at 23 ˚ C for 30 min . The bound proteins were recovered by amylose beads and analysed by SDS - PAGE and immunoblotting with indicated antibodies . F . Quantification of E . Bound B56γ to WT Apc1 - loop500 control ( - Cdk ) was arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

E. Cdk can promote Apc1-loop500 and B56 interaction. MBP fused WT or 3A Apc1-loop500 fragment was incubated in the presence or absence of Cdk2-cyclin A at 30˚C for 60 min. MBP-fused peptides (-/+ kinase) were isolated and incubated with purified Flag-B56γ at 23˚C for 30 min. The bound proteins were recovered by amylose beads and analysed by SDS-PAGE and immunoblotting with indicated antibodies. F. Quantification of E. Bound B56γ to WT Apc1-loop500 control (-Cdk) was arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


A . Cdc20 binding assay in Xenopus egg extracts . The purified recombinant wild - type ( WT ) or B56 binding site mutant APC / C ( 1 - L557A / V560A ) was incubated with APC / C - depleted ( ∆ APC ) interphase extract ( Inter ) or ∆ APC anaphase extract supplemented with CycB ∆ 167 ( Ana ) at 23 ˚ C for indicated times . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . pApc1 ( pS314 / pS318 ) is a phospho - site specific antibody that binds only when both S314 and S318 are phosphorylated . B . Quantification of A . The intensities of WT control in interphase were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant wild-type (WT) or B56 binding site mutant APC/C (1-L557A/V560A) was incubated with APC/C-depleted (∆APC) interphase extract (Inter) or ∆APC anaphase extract supplemented with CycB∆167 (Ana) at 23˚C for indicated times. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. pApc1 (pS314/pS318) is a phospho-site specific antibody that binds only when both S314 and S318 are phosphorylated. B. Quantification of A. The intensities of WT control in interphase were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


C . B56 binding site mutant APC / C ( 1 - L557A / V560A ) is less active in ubiquitylation assay than WT APC / C . The purified recombinant WT APC / C or B56 binding motif mutant APC / C ( 1 - L557A / V560A ) was incubated with ∆ APC anaphase extract . The recovered APC / C - Cdc20 complex was subjected to ubiquitylation assay using 35S - labelled cyclin B as a substrate . Samples were taken at indicated time points and analysed by SDS - PAGE and autoradiography . D . Quantification of C . Error bars , s . e . m . from three independent experiments .

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

C. B56 binding site mutant APC/C (1-L557A/V560A) is less active in ubiquitylation assay than WT APC/C. The purified recombinant WT APC/C or B56 binding motif mutant APC/C (1-L557A/V560A) was incubated with ∆APC anaphase extract. The recovered APC/C-Cdc20 complex was subjected to ubiquitylation assay using 35S-labelled cyclin B as a substrate. Samples were taken at indicated time points and analysed by SDS-PAGE and autoradiography. D. Quantification of C. Error bars, s.e.m. from three independent experiments.


A . ( left panel ) Cdc20 binding to APC / C loop domain mutants in interphase extract . The purified recombinant WT or Apc1 mutant APC / Cs ( 1 - ∆ L300 , 1 - ∆ L300 / ∆ 11 or 1 - ∆ L300 / L557A / V560A ) were incubated with ∆ APC interphase extract at 23 ˚ C for 1 hr . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . ( right panel ) Quantification of bound Cdc20 . The intensities of 1 - ∆ L300 control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments . B . ( left panel ) Cdc20 binding to APC / C loop domain mutants in anaphase extracts . Same as A , but ∆ APC anaphase extract was used and incubated at 23 ˚ C for 50 min in anaphase extract . ( right panel ) Quantification of bound Cdc20 . The intensities of 1 - ∆ L300 control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. (left panel) Cdc20 binding to APC/C loop domain mutants in interphase extract. The purified recombinant WT or Apc1 mutant APC/Cs (1-∆L300, 1-∆L300/∆11 or 1-∆L300/L557A/V560A) were incubated with ∆APC interphase extract at 23˚C for 1 hr. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments. B. (left panel) Cdc20 binding to APC/C loop domain mutants in anaphase extracts. Same as A, but ∆APC anaphase extract was used and incubated at 23˚C for 50 min in anaphase extract. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


A . ( left panel ) Cdk - dependent in vitro kinase assay of Apc1 - loop300 . 3xFlag - tagged WT or 7T Apc1 - loop300 fragment was incubated with Cdk2 - cyclin A in the presence of [ γ - 32P ] - ATP at 30 ˚ C for 30 min , and separated by SDS - PAGE and detected by autoradiography . ( right panel ) 32P - phosphorylated WT or 7T Apc1 - loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS - PAGE followed by autoradiography . B . Quantification of removal of radioactivity in A . Error bars , s . e . m . from three independent experiments .

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

A. (left panel) Cdk-dependent in vitro kinase assay of Apc1-loop300. 3xFlag-tagged WT or 7T Apc1-loop300 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 30˚C for 30 min, and separated by SDS-PAGE and detected by autoradiography. (right panel) 32P-phosphorylated WT or 7T Apc1-loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS-PAGE followed by autoradiography. B. Quantification of removal of radioactivity in A. Error bars, s.e.m. from three independent experiments.


C . Cdc20 - 5A efficiently binds to the B56 binding site mutant APC / C . The purified recombinant WT APC / C or Apc1 - loop500 mutant APC / C ( 1 - L557A / V560A ) was incubated with WT Cdc20 or non - phosphorylatable Cdc20 mutant ( 5A ) in ∆ APC ∆ Cdc20 anaphase extract at 23 ˚ C for 55 min . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . D . Quantification of C . The intensities of WT APC / C control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

C. Cdc20-5A efficiently binds to the B56 binding site mutant APC/C. The purified recombinant WT APC/C or Apc1-loop500 mutant APC/C (1-L557A/V560A) was incubated with WT Cdc20 or non-phosphorylatable Cdc20 mutant (5A) in ∆APC∆Cdc20 anaphase extract at 23˚C for 55 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. D. Quantification of C. The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


E . The APC / C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A - B56γ at 23 ˚ C . Samples were taken at the indicated time points and analysed by SDS - PAGE and immunoblotting with antibodies , including phospho - site specific antibodies for pCdc20 ( pT79 ) and pApc1 ( pS314 / pS318 ) .

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E. The APC/C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A-B56γ at 23˚C. Samples were taken at the indicated time points and analysed by SDS-PAGE and immunoblotting with antibodies, including phospho-site specific antibodies for pCdc20 (pT79) and pApc1 (pS314/pS318).


Cell count of Ronin fl / del and Ronin del / del ESC . Data represent mean ± SD , n = three independent experiments .

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Cell count of Ronin fl/del and Ronin del/del ESC. Data represent mean ± SD, n = three independent experiments.


Annexin V cell death assay of Ronin fl / del and Ronin del / del cultured for 6 days . Two - tailed unpaired Student ' s t test , data represent mean ± SD , n = three independent experiments .

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

Annexin V cell death assay of Ronin fl/del and Ronin del/del cultured for 6 days. Two-tailed unpaired Student's t test, data represent mean ± SD, n = three independent experiments.


Ronin fl / del and Ronin del / del ESC stained for Ronin ( left panel ) or Oct4 ( right panel ) . The nuclei are counterstained with DAPI .

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

Ronin fl/del and Ronin del/del ESC stained for Ronin (left panel) or Oct4 (right panel). The nuclei are counterstained with DAPI.


Haematoxylin / eosin staining of paraffin sections of Ronin fl / del and ecto - Ronin teratomas .

B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O B-GENEPROD B-DISEASE O

Haematoxylin/eosin staining of paraffin sections of Ronin fl/del and ecto-Ronin teratomas.


In vitro differentiation of Ronin fl / del , ecto - Ronin ( grown in the presence of Dox ) and Ronin del / del ESC .

O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE O

In vitro differentiation of Ronin fl/del, ecto-Ronin (grown in the presence of Dox) and Ronin del/del ESC.


Volcano plot of gene expression in Ronin del / del day 4 vs . Ronin fl / del ( left panel ) and Ronin del / del day 8 vs . Ronin fl / del ESC ( right panel ) with annotated naïve and primed pluripotency markers .

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

Volcano plot of gene expression in Ronin del/del day 4 vs. Ronin fl/del (left panel) and Ronin del/del day 8 vs. Ronin fl/del ESC (right panel) with annotated naïve and primed pluripotency markers.


Volcano plot of gene expression in Ronin del / del day 8 vs . Ronin del / del day 4 ESC .

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

Volcano plot of gene expression in Ronin del/del day 8 vs. Ronin del/del day 4 ESC.


Analysis of nascent protein synthesis using OP - puro assay . Ronin fl / del cells treated with protein synthesis inhibitor ( cycloheximide ) are used as negative control . The nuclei are counterstained with DAPI . Quantification of the OP - puro signal , normalised to DAPI . Ordinary one - way Anova data represent mean ± SD ; n = three independent experiments for each genotype .

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

Analysis of nascent protein synthesis using OP-puro assay. Ronin fl/del cells treated with protein synthesis inhibitor (cycloheximide) are used as negative control. The nuclei are counterstained with DAPI.   Quantification of the OP-puro signal, normalised to DAPI. Ordinary one-way Anova data represent mean ± SD; n = three independent experiments for each genotype.


Transmission electron microscopy analysis of mitochondrial structure in Ronin fl / del and Ronin del / del ESC . Mitochondria analysed , n > 150 for each condition .

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

Transmission electron microscopy analysis of mitochondrial structure in Ronin fl/del and Ronin del/del ESC. Mitochondria analysed, n > 150 for each condition.


Quantification of the OCR analysis . Kruskal - Wallis test , data represent mean ± SD , n = 16 technical replicates per genotype in three independent experiments .

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

Quantification of the OCR analysis. Kruskal-Wallis test, data represent mean ± SD, n = 16 technical replicates per genotype in three independent experiments.


Blastocyst ( E4 . 5 ) and diapause embryos stained for Ronin , Sox2 and Sox17 . Egg cylinder stage ( E5 . 5 ) embryo stained for Ronin and Sox17 . Nuclei are counterstained with DAPI .

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

Blastocyst (E4.5) and diapause embryos stained for Ronin, Sox2 and Sox17. Egg cylinder stage (E5.5) embryo stained for Ronin and Sox17. Nuclei are counterstained with DAPI.


Transmission electron microscopy analysis of mitochondrial morphology in the epiblast of E4 . 5 , diapause and E5 . 5 embryo . Mitochondria analysed , E4 . 5 ( n mitochondria = 55 ) , diapause ( n mitochondria = 62 ) , E5 . 5 ( n mitochondria = 65 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O B-TISSUE O O O O O O O O O O B-TISSUE O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O

Transmission electron microscopy analysis of mitochondrial morphology in the epiblast of E4.5, diapause and E5.5 embryo. Mitochondria analysed, E4.5 (n mitochondria = 55), diapause (n mitochondria = 62), E5.5 (n mitochondria = 65).


E4 . 5 chimeric embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in E4 . 5 blastocysts , Mann - Whitney test , data represent mean ± SD ; Ronin fl / del n embryos = 26 , Ronin del / del n embryos = 27 . Diapause embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in diapause embryos , unpaired Student ' s t test , data represent mean ± SD , Ronin fl / del n embryos = 4 , Ronin del / del n embryos = 12 . E5 . 5 chimeric embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in E5 . 5 embryos , unpaired Student ' s t test , data represent mean ± SD , Ronin fl / del n embryos = 21 , Ronin del / del n embryos = 15 . Reactivated E5 . 5 chimeric embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in reactivated E5 . 5 embryos , Mann - Whitney test , data represent mean ± SD , Ronin fl / del n embryos = 17 , Ronin del / del n embryos = 12 .

O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O B-GENEPROD O O O O O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O B-GENEPROD O O O O B-TISSUE O O O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O B-GENEPROD O O O O B-TISSUE O O O O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O B-GENEPROD O O O O B-TISSUE O O O

E4.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E4.5 blastocysts, Mann-Whitney test, data represent mean ± SD; Ronin fl/del n embryos = 26, Ronin del/del n embryos = 27.   Diapause embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in diapause embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 4, Ronin del/del n embryos = 12.   E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E5.5 embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 21, Ronin del/del n embryos = 15.   Reactivated E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in reactivated E5.5 embryos, Mann-Whitney test, data represent mean ± SD, Ronin fl/del n embryos = 17, Ronin del/del n embryos = 12.


a ) Auto - ubiquitination assays in which the E3 acts as both an E3 and as proxy substrate were performed with GST - HHARIRBR , T7 - Triad1ΔAri , GST - ParkinRBR , and Flag - BRCA1 / BARD1 and either UbcH5WT or UbcH5L104Q as the E2 . Products were visualized by western blotting against indicated tags on E3s . Times given are post - ATP addition .

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

a) Auto-ubiquitination assays in which the E3 acts as both an E3 and as proxy substrate were performed with GST-HHARIRBR, T7-Triad1ΔAri, GST-ParkinRBR, and Flag-BRCA1/BARD1 and either UbcH5WT or UbcH5L104Q as the E2. Products were visualized by western blotting against indicated tags on E3s. Times given are post-ATP addition.


b ) Overlay of ( 1H , 15N ) - HSQC - TROSY spectra of 15N - UbcH5 - O - 15N - Ub in the absence ( black ) and presence ( red ) of 0 . 5mol equiv . HHARI RING1 . A subset of UbcH5 peaks , but not Ub peaks , shift and broaden upon HHARI RING1 binding .

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

b) Overlay of (1H,15N)-HSQC-TROSY spectra of 15N-UbcH5-O-15N-Ub in the absence (black) and presence (red) of 0.5mol equiv. HHARI RING1. A subset of UbcH5 peaks, but not Ub peaks, shift and broaden upon HHARI RING1 binding.


a ) Histogram of CSPs between free 15N - UbcH7 and 15N - UbcH7 - O - Ub identifies UbcH7 residues affected by conjugation to Ub . Active site ( Ser86 ) is indicated with a star and cross - over helix residues ( 101 - 113 ) are marked with a gray cylinder . INSET . CSPs greater than 1 stdv ( > 0 . 115 ppm ) are highlighted in yellow on a surface representation of UbcH7 ( PDB 1fbv ) . The view on the left shows the surface that contains the UbcH7 cross - over helix .

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

a) Histogram of CSPs between free 15N-UbcH7 and 15N-UbcH7-O-Ub identifies UbcH7 residues affected by conjugation to Ub. Active site (Ser86) is indicated with a star and cross-over helix residues (101-113) are marked with a gray cylinder. INSET. CSPs greater than 1 stdv (> 0.115 ppm) are highlighted in yellow on a surface representation of UbcH7 (PDB 1fbv). The view on the left shows the surface that contains the UbcH7 cross-over helix.


b ) Region of 1H - 15N - HSQC TROSY spectra containing resonances of the cross - over helix residues S107 and Q106 are overlaid : unconjugated 15N - UbcH7 ( blue ) , 15N - UbcH7 - O - Ub ( black ) , and 15N - UbcH7 - O - UbI44A ( red ) . Peaks representing S107 and Q106 in the context of UbcH7 ~ Ub - I44A ( red ) are closer to peaks observed in free UbcH7 ( blue ) than UbcH7 ~ Ub - WT ( black ) indicating that I44A of Ub disrupts interactions with the cross - over helix of UbcH7 .

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

b) Region of 1H-15N-HSQC TROSY spectra containing resonances of the cross-over helix residues S107 and Q106 are overlaid: unconjugated 15N-UbcH7 (blue), 15N-UbcH7-O-Ub (black), and 15N-UbcH7-O-UbI44A (red). Peaks representing S107 and Q106 in the context of UbcH7~Ub-I44A (red) are closer to peaks observed in free UbcH7 (blue) than UbcH7~Ub-WT (black) indicating that I44A of Ub disrupts interactions with the cross-over helix of UbcH7.


a ) Region of 1H - 15N - HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid : free 15N - Ub ( blue ) , UbcH7 - O - 15N - Ub ( black ) , and HHARI RING1 - bound UbcH7 - O - 15N - Ub ( red ) . Black arrow highlights perturbation upon conjugation to UbcH7 ( black arrow ) and red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7 .

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

a) Region of 1H-15N-HSQC TROSY spectra that contains Gln49 Ub resonance are overlaid: free 15N-Ub (blue), UbcH7-O-15N-Ub (black), and HHARI RING1-bound UbcH7-O-15N-Ub (red). Black arrow highlights perturbation upon conjugation to UbcH7 (black arrow) and red arrow highlights perturbation of conjugated Ub upon HHARI RING1 binding to UbcH7.


b ) Region of 1H - 13C - HSQC spectra of 13C - UbcH7 ( blue ) , 13C - UbcH7 - O - Ub ( black ) , and HHARI RING1 - bound 13C - UbcH7 - O - Ub ( red ) that includes the methyl ( 13CH3 ) resonance of the surface - exposed UbcH7 cross - over helix residue , Ala 110 ( black arrow ) is shown . The 13CH3 peak of Ala110 either broadens dramatically or shifts to an unknown position in the spectrum of 13C - UbcH7 - O - Ub ( black ) and reappears at its position in free UbcH7 in the presence of HHARI RING1 , consistent with disruption of closed UbcH7 ~ Ub conformations . Pairwise overlays and larger spectra are provided in Appendix Fig S3 .

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

b) Region of 1H-13C-HSQC spectra of 13C-UbcH7 (blue), 13C-UbcH7-O-Ub (black), and HHARI RING1-bound 13C-UbcH7-O-Ub (red) that includes the methyl (13CH3) resonance of the surface-exposed UbcH7 cross-over helix residue, Ala 110 (black arrow) is shown. The 13CH3 peak of Ala110 either broadens dramatically or shifts to an unknown position in the spectrum of 13C-UbcH7-O-Ub (black) and reappears at its position in free UbcH7 in the presence of HHARI RING1, consistent with disruption of closed UbcH7~Ub conformations. Pairwise overlays and larger spectra are provided in Appendix Fig S3.


c ) Regions of 1H - 15N - HSQC - TROSY spectra of UbcH7 - O - 15N - Ub in the absence ( black ) and presence ( red ) of a RING1 domain from the RBR E3s HHARI ( left ) or RNF144 ( middle ) or a canonical RING domain of BRCA1 / BARD1 ( right ) . The perturbations on Ub due to binding of HHARI RING1 and RNF144 RING1 are remarkably similar , while binding of the canonical RING domain of BRCA1 / BARD1 has no observable effect on the Ub spectrum . Gray boxes mark area expanded in a )

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

c) Regions of 1H-15N-HSQC-TROSY spectra of UbcH7-O-15N-Ub in the absence (black) and presence (red) of a RING1 domain from the RBR E3s HHARI (left) or RNF144 (middle) or a canonical RING domain of BRCA1/BARD1 (right). The perturbations on Ub due to binding of HHARI RING1 and RNF144 RING1 are remarkably similar, while binding of the canonical RING domain of BRCA1/BARD1 has no observable effect on the Ub spectrum. Gray boxes mark area expanded in a)


b ) LEFT . Auto - ubiquitination assays were performed with wild - type HHARIRBR or an active site - dead mutant ( C357A - HHARIRBR ) . Products were visualized by western blotting against GST . The zero time point was taken immediately prior to ATP addition , all other times are post - ATP addition . RIGHT . Identical assays as shown on left were performed with the UBR - hybrid . The active site - dead mutant ( C357A ) retains substantial auto - ubiquitination activity signifying that UbcH5 ~ Ub is able to transfer Ub directly onto the GST - UBR - hybrid construct , bypassing the RING2 active site Cys .

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

b) LEFT. Auto-ubiquitination assays were performed with wild-type HHARIRBR or an active site-dead mutant (C357A- HHARIRBR). Products were visualized by western blotting against GST. The zero time point was taken immediately prior to ATP addition, all other times are post-ATP addition. RIGHT. Identical assays as shown on left were performed with the UBR-hybrid. The active site-dead mutant (C357A) retains substantial auto-ubiquitination activity signifying that UbcH5~Ub is able to transfer Ub directly onto the GST-UBR-hybrid construct, bypassing the RING2 active site Cys.


a ) LEFT . E3 auto - ubiquitination assays were performed using various Ub mutants ( I44A , R42A , Q49A , Q49E , and V70A ) and the RBR E3s HHARIRBR ( left ) and ParkinRBR ( right ) . Products were visualized by western blotting against HA - Ub . Samples were analyzed 30 minutes after ATP addition .

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

a) LEFT. E3 auto-ubiquitination assays were performed using various Ub mutants (I44A, R42A, Q49A, Q49E, and V70A) and the RBR E3s HHARIRBR (left) and ParkinRBR (right). Products were visualized by western blotting against HA-Ub. Samples were analyzed 30 minutes after ATP addition.


c ) UbcH7 ~ Ub conjugates were preformed with either with WT - Ub or I44A - Ub . After the addition of apyrase to quench the charging reaction , UbcH7 ~ UbWT ( left ) or UbcH7 ~ UbI44A ( right ) was incubated with ParkinRBR . The disappearance of each UbcH7 ~ Ub species and appearance of auto - ubiquitinated E3 was visualized under non - reducing conditions by western blotting for HA - Ub . Time was recorded post addition of ParkinRBR . E2 ~ Ub conjugated with I44A - Ub does not disappear over the time course of the reaction .

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

c) UbcH7~Ub conjugates were preformed with either with WT-Ub or I44A-Ub. After the addition of apyrase to quench the charging reaction, UbcH7~UbWT (left) or UbcH7~UbI44A (right) was incubated with ParkinRBR. The disappearance of each UbcH7~Ub species and appearance of auto-ubiquitinated E3 was visualized under non-reducing conditions by western blotting for HA-Ub. Time was recorded post addition of ParkinRBR. E2~Ub conjugated with I44A-Ub does not disappear over the time course of the reaction.


d ) UbcH7 conjugated with WT - Ub , I44A - Ub , or V70A Ub as indicated was incubated with either H887A - HOIPRBR - LDD ( left ) or H359A - HHARIRBR ( right ) mutants that allow trapping of the E3 ~ Ub thioester with WT - Ub ( Stieglitz et al , 2013 ; [ 29 ] ) . While a HOIP ~ Ub thioester species is observed when UbcH7 was charged with WT - Ub , no detectable transfer occurs with the I44A - Ub conjugate ( left ) . Similarly , V70A - Ub shows reduced formation of the HHARI ~ Ub thioester ( right ) . In addition to blotting for HA - Ub , the blot on the right was also blotted for GST - HHARI .

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

d) UbcH7 conjugated with WT-Ub, I44A-Ub, or V70A Ub as indicated was incubated with either H887A-HOIPRBR-LDD (left) or H359A-HHARIRBR (right) mutants that allow trapping of the E3~Ub thioester with WT-Ub (Stieglitz et al, 2013;[29]). While a HOIP~Ub thioester species is observed when UbcH7 was charged with WT-Ub, no detectable transfer occurs with the I44A-Ub conjugate (left). Similarly, V70A-Ub shows reduced formation of the HHARI~Ub thioester (right). In addition to blotting for HA-Ub, the blot on the right was also blotted for GST-HHARI.


a ) Top panel . Regions of 1H - 15N - HSQC TROSY spectra of 15N - HHARI RING2 in the absence ( black ) and presence of excess Ub ( red ) . Middle panel . Identical spectral region , but in the presence of Ub - V70A ( red ) . Bottom panel . Identical spectral region , but spectra are from a truncated HHARI RING2 construct that lacks N - terminal residues 325 - 335 ( HHARI RING2 - ΔL ) .

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a) Top panel. Regions of 1H-15N-HSQC TROSY spectra of 15N-HHARI RING2 in the absence (black) and presence of excess Ub (red). Middle panel. Identical spectral region, but in the presence of Ub-V70A (red). Bottom panel. Identical spectral region, but spectra are from a truncated HHARI RING2 construct that lacks N-terminal residues 325-335 (HHARI RING2-ΔL).


d ) Mutations in the Ub binding surface of RING2 decrease activity in E3 auto - ubiquitination assays . Time points ( 10min ) are visualized by western blotting for the GST - tag on HHARI . Relative activity of HHARI WT and mutant forms is clearest when the intensity of the unmodified HHARI band is compared .

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d) Mutations in the Ub binding surface of RING2 decrease activity in E3 auto-ubiquitination assays. Time points (10min) are visualized by western blotting for the GST-tag on HHARI. Relative activity of HHARI WT and mutant forms is clearest when the intensity of the unmodified HHARI band is compared.


e ) 1H - 15N - HSQC TROSY spectra of 15N - Ub demonstrate binding by WT HHARI RING2 , but not by mutant HHARI RING2 constructs that exhibit decreased auto - ubiquitination activity . Overlay of 15N - Ub spectra in the absence ( black ) and presence of WT - HHARI RING2 ( red ) , W336A - HHARI RING2 ( blue ) , T341N - HHARI RING2 ( green ) , or E352A - HHARI RING2 ( purple ) . HHARI mutations that exhibited reduced binding to Ub also show decreased ubiquitination activity in panel d ) .

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e) 1H-15N-HSQC TROSY spectra of 15N-Ub demonstrate binding by WT HHARI RING2, but not by mutant HHARI RING2 constructs that exhibit decreased auto-ubiquitination activity. Overlay of 15N-Ub spectra in the absence (black) and presence of WT-HHARI RING2 (red), W336A-HHARI RING2 (blue), T341N-HHARI RING2 (green), or E352A-HHARI RING2 (purple). HHARI mutations that exhibited reduced binding to Ub also show decreased ubiquitination activity in panel d).


( a ) UVRAG interaction with C - Vps . 293T cells were co - transfected with Flag - UVRAG and HA - Vps16 , HA - Vps11 , HA - Vps18 , HA - Vps33 or HA - Vps39 . Whole - cell lysates ( WCLs ) were used for immunoprecipitation ( IP ) with an anti - Flag antibody , followed by immunoblotting ( IB ) with an anti - HA antibody . 1 % WCL was used as the input .

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(a) UVRAG interaction with C-Vps. 293T cells were co-transfected with Flag-UVRAG and HA-Vps16, HA-Vps11, HA-Vps18, HA-Vps33 or HA-Vps39. Whole-cell lysates (WCLs) were used for immunoprecipitation (IP) with an anti-Flag antibody, followed by immunoblotting (IB) with an anti-HA antibody. 1% WCL was used as the input.


( b ) Interaction between endogenous UVRAG and C - Vps subunits . WCLs of 293T cells were used for IP with control serum ( control ) or an anti - UVRAG antibody , followed by IB with the indicated antibodies . The right panel shows endogenous protein expression .

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(b) Interaction between endogenous UVRAG and C-Vps subunits. WCLs of 293T cells were used for IP with control serum (control) or an anti-UVRAG antibody, followed by IB with the indicated antibodies. The right panel shows endogenous protein expression.


( c ) Confocal microscopy analysis of the colocalization of endogenous UVRAG and Vps16 or Vps18 in HeLa cells . Insets highlight colocalization .

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(c) Confocal microscopy analysis of the colocalization of endogenous UVRAG and Vps16 or Vps18 in HeLa cells. Insets highlight colocalization.


( d ) Confocal microscopy analysis of the colocalization of UVRAG with C - Vps subunits in HeLa cells transfected with Flag - UVRAG , and epitope - tagged C - Vps subunits as indicated .

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(d) Confocal microscopy analysis of the colocalization of UVRAG with C-Vps subunits in HeLa cells transfected with Flag-UVRAG, and epitope-tagged C-Vps subunits as indicated.


( e ) Colocalization of UVRAG - C - Vps in early endosomes . HeLa cells were co - transfected with EGFP - Vps16 and Flag - UVRAG , stained with antibodies to LAMP1 , LAMP2 , EEA1 and Rab5 , followed by confocal microscopy . All images are representative of at least three independent experiments . Scale bars , 5 μm . The raw data for a and b are shown in Supplementary Information , Fig .

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(e) Colocalization of UVRAG-C-Vps in early endosomes. HeLa cells were co-transfected with EGFP-Vps16 and Flag-UVRAG, stained with antibodies to LAMP1, LAMP2, EEA1 and Rab5, followed by confocal microscopy. All images are representative of at least three independent experiments. Scale bars, 5 μm. The raw data for a and b are shown in Supplementary Information, Fig. S6


( a ) The N - terminal C2 domain or the C - terminal region of UVRAG interacts individually with Vps16 . 293T cells were co - transfected with HA - Vps16 , together with vector , Flag - UVRAG or Flag - UVRAG mutants . WCLs were immunoprecipitated with anti - Flag followed by immunoblotting with an anti - HA antibody .

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(a) The N-terminal C2 domain or the C-terminal region of UVRAG interacts individually with Vps16. 293T cells were co-transfected with HA-Vps16, together with vector, Flag-UVRAG or Flag-UVRAG mutants. WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody.


( b ) HeLa cells were transfected with EGFP - Vps16 together with Flag - UVRAG mutants as indicated and stained with anti - Flag ( red ) , followed by confocal microscopy . Scale bars , 5 μm .

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(b) HeLa cells were transfected with EGFP-Vps16 together with Flag-UVRAG mutants as indicated and stained with anti-Flag (red), followed by confocal microscopy. Scale bars, 5 μm.


( c ) Interactions of the UVRAG C - terminal truncated mutants with Vps16 . At 48 h post - transfection with HA - Vps16 and Flag - UVRAG C - terminal mutants , 293T WCLs were immunoprecipitated with anti - Flag , followed by immunoblotting with anti - HA .

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(c) Interactions of the UVRAG C-terminal truncated mutants with Vps16. At 48 h post-transfection with HA-Vps16 and Flag-UVRAG C-terminal mutants, 293T WCLs were immunoprecipitated with anti-Flag, followed by immunoblotting with anti-HA.


( d ) UVRAG C2 domain or C - terminal region deletion abolishes Vps16 binding . At 48 h post - transfection with HA - Vps16 together with Flag - UVRAG or its mutants , 293T WCLs were immunoprecipitated with anti - Flag followed by immunoblotting with an anti - HA antibody .

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(d) UVRAG C2 domain or C-terminal region deletion abolishes Vps16 binding. At 48 h post-transfection with HA-Vps16 together with Flag-UVRAG or its mutants, 293T WCLs were immunoprecipitated with anti-Flag followed by immunoblotting with an anti-HA antibody.


( a ) HeLa . Vec and HeLa . UVRAG cells transfected with HA - Vps16 and GFP - LC3 were treated with 2 μM rapamycin for 2 h , and processed for confocal microscopy ( left panel ; scale bars , 5 μm ) . The percentage of GFP - LC3 punctae positive for HA - Vps16 staining was quantified ( right panel ; data are mean ± s . e . m . , n = 60 , * P 0 . 01 ) .

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(a) HeLa.Vec and HeLa.UVRAG cells transfected with HA-Vps16 and GFP-LC3 were treated with 2 μM rapamycin for 2 h, and processed for confocal microscopy (left panel; scale bars, 5 μm). The percentage of GFP-LC3 punctae positive for HA-Vps16 staining was quantified (right panel; data are mean ± s.e.m., n = 60, *P 0.01).


( b ) Effects of Vps16 on UVRAG - mediated autophagosome formation . Light microscopic quantification of autophagosomes in HeLa . Vec and HeLa . UVRAG cells ( upper left panel ) or in HCT116 . Vec and HCT116 . UVRAG cells ( bottom left panel ) when transfected with GFP - LC3 together with control siRNA or Vps16 siRNA ( data are mean ± s . e . m . , n = 60 for HeLa cells ; n = 450 for HCT116 cells , * P 0 . 01 ; * * P 0 . 001 ) . Immunoblotting of Vps16 and tubulin are shown in the right panel .

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(b) Effects of Vps16 on UVRAG-mediated autophagosome formation. Light microscopic quantification of autophagosomes in HeLa.Vec and HeLa.UVRAG cells (upper left panel) or in HCT116.Vec and HCT116.UVRAG cells (bottom left panel) when transfected with GFP-LC3 together with control siRNA or Vps16 siRNA (data are mean ± s.e.m., n = 60 for HeLa cells; n = 450 for HCT116 cells, *P 0.01; **P 0.001). Immunoblotting of Vps16 and tubulin are shown in the right panel.


( a ) UVRAG enhances the colocalization efficiency of GFP - LC3 with LAMP1 . HeLa . Vec and HeLa . UVRAG cells transfected with GFP - LC3 were either untreated or treated with rapamycin ( 2 μM ) in the absence or presence of bafilomycin A1 ( 0 . 1 μM ) and stained for LAMP1 . Insets highlight the colocalization . The percentage of LAMP1 - positive autophagosomes was calculated in each setting ( data are mean ± s . e . m . , n = 200 ; 5 independent experiments , * * P 0 . 01 ; * P 0 . 05 ) .

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(a) UVRAG enhances the colocalization efficiency of GFP-LC3 with LAMP1. HeLa.Vec and HeLa.UVRAG cells transfected with GFP-LC3 were either untreated or treated with rapamycin (2 μM) in the absence or presence of bafilomycin A1 (0.1 μM) and stained for LAMP1. Insets highlight the colocalization. The percentage of LAMP1-positive autophagosomes was calculated in each setting (data are mean ± s.e.m., n = 200; 5 independent experiments, **P 0.01; *P 0.05).


( b ) UVRAG enhances the acquisition of CD63 by autophagosomes . The GFP - LC3 - transfected HeLa . Vec and HeLa . UVRAG cells were treated as described in a and stained for CD63 and the percentage of CD63 - positive autophagosomes was calculated ( data are mean ± s . e . m . , n = 200 , * * P 0 . 01 ; * P 0 . 05 ) .

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(b) UVRAG enhances the acquisition of CD63 by autophagosomes. The GFP-LC3-transfected HeLa.Vec and HeLa.UVRAG cells were treated as described in a and stained for CD63 and the percentage of CD63-positive autophagosomes was calculated (data are mean ± s.e.m., n = 200, **P 0.01; *P 0.05).


( c ) UVRAG promotes autophagic degradation of long - lived proteins . HeLa . Vec and HeLa . UVRAG cells were incubated for 16 h with L - 3H - Leu ( 1 μCi ml - 1 ) . The degradation of long - lived proteins was measured at the indicated time points in complete medium ( NC ) , EBSS alone ( Starv . ) , or EBSS + 0 . 1 μM bafilomycin A1 ( Starv . + BalfA1 ) . Results ( data are mean ± s . e . m . of triplicates ) are representative of 2 independent experiments . Scale bars , 5 μm .

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(c) UVRAG promotes autophagic degradation of long-lived proteins. HeLa.Vec and HeLa.UVRAG cells were incubated for 16 h with L-3H-Leu (1 μCi ml-1). The degradation of long-lived proteins was measured at the indicated time points in complete medium (NC), EBSS alone (Starv.), or EBSS + 0.1 μM bafilomycin A1 (Starv.+BalfA1). Results (data are mean ± s.e.m. of triplicates) are representative of 2 independent experiments. Scale bars, 5 μm.


( a ) Colocalization of GFP - LC3 and LAMP1 . HeLa cells expressing wild - type or mutant UVRAG were transfected with GFP - LC3 and incubated under normal conditions or treated with rapamycin ( 2 μM ) . Autophagosome fusion with LAMP + structures was analysed by confocal microscopy ( left panel ; rapamycin - treated ) and quantified ( right upper panel ; data are mean ± s . e . m . , n = 100 ) . Arrows and insets show the LAMP1 + autophagosomes . The number of GFP - LC3 - positive dots per cell was counted using a fluorescence microscope ( right bottom panel ; data are mean ± s . e . m . , n = 60 , * P 0 . 05 ; * * P 0 . 0001 ) .

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(a) Colocalization of GFP-LC3 and LAMP1. HeLa cells expressing wild-type or mutant UVRAG were transfected with GFP-LC3 and incubated under normal conditions or treated with rapamycin (2 μM). Autophagosome fusion with LAMP+ structures was analysed by confocal microscopy (left panel; rapamycin-treated) and quantified (right upper panel; data are mean ± s.e.m., n = 100). Arrows and insets show the LAMP1+ autophagosomes. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope (right bottom panel; data are mean ± s.e.m., n = 60, *P 0.05; **P 0.0001).


( b ) Vps16 or Vps18 knockdown inhibits UVRAG - mediated autophagosome maturation . HeLa . Vec ( left ) and HeLa . UVRAG ( right ) cells were transfected with GFP - LC3 together with control siRNA , Vps16 siRNA , or Vps18 siRNA , followed by confocal microscopy . Arrows in the top panel denote the autophagosomes with LAMP1 staining . Immunoblotting of Vps16 , or Vps18 and tubulin are shown in the middle panel . The percentage of LAMP1 + autophagosomes was calculated ( data are mean ± s . e . m . , n = 50 , * P 0 . 05 ) . Scale bars , 5 μm . The raw data of the immunoblots are shown in Supplementary Information , Fig . S6 .

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(b) Vps16 or Vps18 knockdown inhibits UVRAG-mediated autophagosome maturation. HeLa.Vec (left) and HeLa.UVRAG (right) cells were transfected with GFP-LC3 together with control siRNA, Vps16 siRNA, or Vps18 siRNA, followed by confocal microscopy. Arrows in the top panel denote the autophagosomes with LAMP1 staining. Immunoblotting of Vps16, or Vps18 and tubulin are shown in the middle panel. The percentage of LAMP1+ autophagosomes was calculated (data are mean ± s.e.m., n = 50, *P 0.05). Scale bars, 5 μm. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.


( a ) UVRAG expression promotes the recruitment of Rab7 GTPase to autophagosomes . HeLa . Vec and HeLa . UVRAG cells transfected with GFP - Rab7 and RFP - LC3 were maintained either under normal conditions ( NC ) or treated with rapamycin , followed by confocal microscopy . Insets highlight the Rab7 acquisition of autophagosomes . The percentage of Rab7 + - autophagosomes was quantified ( right panel ; data are mean ± s . e . m . , n = 60 , * * * P 0 . 001 ; * * P 0 . 01 ) . Scale bars , 5 μm .

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(a) UVRAG expression promotes the recruitment of Rab7 GTPase to autophagosomes. HeLa.Vec and HeLa.UVRAG cells transfected with GFP-Rab7 and RFP-LC3 were maintained either under normal conditions (NC) or treated with rapamycin, followed by confocal microscopy. Insets highlight the Rab7 acquisition of autophagosomes. The percentage of Rab7+-autophagosomes was quantified (right panel; data are mean ± s.e.m., n = 60, ***P 0.001; **P 0.01). Scale bars, 5 μm.


( b ) Rab7 GTPase activity is increased with UVRAG expression . At 48 h post - transfection with GFP - Rab7 together with vector , UVRAG or UVRAGΔN ( 1 - 147 ) vector , 293T WCLs were used for immunoprecipitation with anti - GFP , followed by the Rab7 GTPase activity assay . Left panel : Autoradiographs of the GTP hydrolysis products analysed by TLC . Right panel : Quantification of the percentage of GTP hydrolyzed by Rab7 ( data are mean n = 2 independent experiments ) .

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(b) Rab7 GTPase activity is increased with UVRAG expression. At 48 h post-transfection with GFP-Rab7 together with vector, UVRAG or UVRAGΔN(1-147) vector, 293T WCLs were used for immunoprecipitation with anti-GFP, followed by the Rab7 GTPase activity assay. Left panel: Autoradiographs of the GTP hydrolysis products analysed by TLC. Right panel: Quantification of the percentage of GTP hydrolyzed by Rab7 (data are mean n = 2 independent experiments).


( c ) Increased Rab7 - RILP interaction UVRAG is expressed . HCT116 . vector , HCT116 . UVRAG and HCT116 . UVRAGΔN ( 1 - 147 ) cells were transfected with GFP - Rab7 together with the GST - tagged Rab7 binding domain of RILP ( GST - RILPRBD , residues 243 - 318 ) . WCLs were used for GST pulldown , followed by immunoblotting with an anti - GFP antibody . The raw data of the immunoblots are shown in Supplementary Information , Fig . S6 .

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(c) Increased Rab7-RILP interaction UVRAG is expressed. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔN(1-147) cells were transfected with GFP-Rab7 together with the GST-tagged Rab7 binding domain of RILP (GST-RILPRBD, residues 243-318). WCLs were used for GST pulldown, followed by immunoblotting with an anti-GFP antibody. The raw data of the immunoblots are shown in Supplementary Information, Fig. S6.


( a ) EGF - stimulated endocytosis and post - endocytic trafficking . Uptake of Alexa Fluor 488 - EGF ( green ) by HCT116 . Vec ( left panel ) and HCT116 . UVRAG ( right panel ) cells was followed by confocal microscopy . At the indicated times , cells were fixed and stained with antibodies to LAMP1 ( red ) , and Topro - 3 ( blue , to stain the nucleus ) . Magnified images of the insets ( fourth row ) highlight trafficking of EGF towards LAMP1 - positive structures .

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(a) EGF-stimulated endocytosis and post-endocytic trafficking. Uptake of Alexa Fluor 488-EGF (green) by HCT116.Vec (left panel) and HCT116.UVRAG (right panel) cells was followed by confocal microscopy. At the indicated times, cells were fixed and stained with antibodies to LAMP1 (red), and Topro-3 (blue, to stain the nucleus). Magnified images of the insets (fourth row) highlight trafficking of EGF towards LAMP1-positive structures.


( b ) EGFR degradation . HCT116 . Vec , HCT116 . UVRAG and HCT116 . UVRAGΔN ( 1 - 147 ) cells were treated with EGF ( 200 ng ml − 1 ) at 37 ° C for the indicated times . WCLs were subjected to immunoblotting with an anti - EGFR antibody . EGFR at each time point was quantified and indicated as a percentage relative to that at time zero ( right panel ; data are mean ± s . e . m . , n = 3 ) .

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(b) EGFR degradation. HCT116.Vec, HCT116.UVRAG and HCT116.UVRAGΔN(1-147) cells were treated with EGF (200 ng ml−1) at 37 °C for the indicated times. WCLs were subjected to immunoblotting with an anti-EGFR antibody. EGFR at each time point was quantified and indicated as a percentage relative to that at time zero (right panel; data are mean ± s.e.m., n = 3).


( c ) DQ - green BSA dequenching assay . Representative confocal microscopy images of DQ - BSA proteolysis ( left panel ) and flow cytometry analysis ( right panel ) of RAW264 . 7 cells expressing an empty vector ( RAW264 . 7 . Vec ; dotted black line ) , wild - type UVRAG ( red line ) or UVRAGΔN ( 1 - 147 ) mutant ( green line ) loaded with 10 μg ml − 1 DQ green - BSA for the indicated times . Background ( grey line ) represents samples without the BSA load .

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(c) DQ-green BSA dequenching assay. Representative confocal microscopy images of DQ-BSA proteolysis (left panel) and flow cytometry analysis (right panel) of RAW264.7 cells expressing an empty vector (RAW264.7.Vec; dotted black line), wild-type UVRAG (red line) or UVRAGΔN(1-147) mutant (green line) loaded with 10 μg ml−1 DQ green-BSA for the indicated times. Background (grey line) represents samples without the BSA load.


( d ) Effect of UVRAG on endosome fusion . RAW264 . 7 . Vec and RAW264 . 7 . UVRAG cells were pulse - labelled with Oregon green 514 - avidin and dextran - 568 for 10 min and chased for 30 min . Cells were then labelled with biotin - BSA for 10 min , and processed for confocal microscopy ( left panel ) . In the absence of biotin - BSA , Oregon green514 - avidin showed very low fluorescence ( first row of confocal image ) . The percentage of dextran - 568 - labelled endosomes with visible Oregon green staining ( denoted by arrows in the left panel ) was calculated from approximately 20 different cells in 2 independent experiments ( mean , right panel ) . Scale bars , 5 μm . The raw data of the immunoblots in b are shown in the Supplementary Information , Fig . S6 .

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(d) Effect of UVRAG on endosome fusion. RAW264.7.Vec and RAW264.7.UVRAG cells were pulse-labelled with Oregon green 514-avidin and dextran-568 for 10 min and chased for 30 min. Cells were then labelled with biotin-BSA for 10 min, and processed for confocal microscopy (left panel). In the absence of biotin-BSA, Oregon green514-avidin showed very low fluorescence (first row of confocal image). The percentage of dextran-568-labelled endosomes with visible Oregon green staining (denoted by arrows in the left panel) was calculated from approximately 20 different cells in 2 independent experiments (mean, right panel). Scale bars, 5 μm. The raw data of the immunoblots in b are shown in the Supplementary Information, Fig. S6.


( B - C ) Survival ( B ) and weight loss ( C ) . N = 10 mice for each group . The P - value of survival curve by Gehan - Breslow - Wilcoxon test is shown .

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(B-C) Survival (B) and weight loss (C). N = 10 mice for each group. The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown.


( E ) Cytokine expression levels of Tnfa , Ifng , Il1a , and Il1b were measured by RT - qPCR normalized by Ppia . Data are presented as mean ± SEM , N = 4 mice per group . * , p < 0 . 05 by unpaired Student ' s t - test with two sided .

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(E) Cytokine expression levels of Tnfa, Ifng, Il1a, and Il1b were measured by RT-qPCR normalized by Ppia. Data are presented as mean ± SEM, N = 4 mice per group. *, p < 0.05 by unpaired Student's t-test with two sided.


( B - C ) Catalytically active sACE22 . v2 . 4 - IgG1 ( B ) and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 ( C ) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte ( 0 - 60 s ) and returned to buffer to measure dissociation ( 60 - 360 s ) . RBD concentrations are indicated on the right of the sensorgrams .

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(B-C) Catalytically active sACE22.v2.4-IgG1 (B) and catalytically dead sACE22.v2.4(NN)-IgG1 (C) were immobilized on BLI biosensors that were transferred to solutions of gamma RBD as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the sensorgrams.


( D - E ) Catalytically active sACE22 . v2 . 4 - IgG1 and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 were aerosolized ( 7 . 5 ml protein at 8 . 3 mg / ml in 25 minutes ) and delivered by inhalation to K18 - hACE2 transgenic mice at 12 h , 48 h , and 84 h post - inoculation with SARS - CoV - 2 gamma variant . 10 mice in each group were observed for survival ( D ) and weight loss ( E ) . The P - value of survival curve by Gehan - Breslow - Wilcoxon test is shown . Error bars for mouse weight are centered on the mean and show SEM . Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1 .

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(D-E) Catalytically active sACE22.v2.4-IgG1 and catalytically dead sACE22.v2.4(NN)-IgG1 were aerosolized (7.5 ml protein at 8.3 mg/ml in 25 minutes) and delivered by inhalation to K18-hACE2 transgenic mice at 12 h, 48 h, and 84 h post-inoculation with SARS-CoV-2 gamma variant. 10 mice in each group were observed for survival (D) and weight loss (E). The P-value of survival curve by Gehan-Breslow-Wilcoxon test is shown. Error bars for mouse weight are centered on the mean and show SEM. Catalytically active and inactive proteins were tested in the same experiment versus PBS control shown in Figure 1.


( B - C ) Survival ( B ) and weight loss ( C ) following treatment with an IgG1 isotype control ( red ) versus catalytically active ( blue ) and dead ( green ) sACE22 . v2 . 4 - IgG1 . N = 8 mice for IgG1 group and catalytic active group , N = 9 for catalytic dead group . Error bars for mouse weight are centered on the mean and show SEM . Gehan - Breslow - Wilcoxon test was applied to analyze the survival curves . P = 0 . 0099 for catalytic active group compared to catalytic dead group . P = 0 . 017 for catalytic dead group compared to IgG1 control .

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

(B-C) Survival (B) and weight loss (C) following treatment with an IgG1 isotype control (red) versus catalytically active (blue) and dead (green) sACE22.v2.4-IgG1. N = 8 mice for IgG1 group and catalytic active group, N = 9 for catalytic dead group. Error bars for mouse weight are centered on the mean and show SEM. Gehan-Breslow-Wilcoxon test was applied to analyze the survival curves. P = 0.0099 for catalytic active group compared to catalytic dead group. P = 0.017 for catalytic dead group compared to IgG1 control.


( D - E ) Catalytically active sACE22 . v2 . 4 - IgG1 ( D ) and catalytically dead sACE22 . v2 . 4 ( NN ) - IgG1 ( E ) were immobilized on BLI biosensors and transferred to solutions of RBD ( original variant ) as the soluble analyte ( 0 - 60 s ) and returned to buffer to measure dissociation ( 60 - 360 s ) . RBD concentrations are indicated on the right of the BLI sensorgrams .

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(D-E) Catalytically active sACE22.v2.4-IgG1 (D) and catalytically dead sACE22.v2.4(NN)-IgG1 (E) were immobilized on BLI biosensors and transferred to solutions of RBD (original variant) as the soluble analyte (0-60 s) and returned to buffer to measure dissociation (60-360 s). RBD concentrations are indicated on the right of the BLI sensorgrams.


( A ) Binding of monomeric sACE2 - 8his proteins to Expi293F cells expressing S of BA . 1 omicron was measured by flow cytometry .

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(A) Binding of monomeric sACE2-8his proteins to Expi293F cells expressing S of BA.1 omicron was measured by flow cytometry.


( B ) Binding of dimeric sACE22 - IgG1 proteins was compared to the binding of antibodies authorized for therapeutic use in COVID - 19 patients . Binding to Expi293F cells expressing BA . 1 omicron S was measured by flow cytometry .

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(B) Binding of dimeric sACE22-IgG1 proteins was compared to the binding of antibodies authorized for therapeutic use in COVID-19 patients. Binding to Expi293F cells expressing BA.1 omicron S was measured by flow cytometry.


( C ) Neutralization of BA . 1 omicron pseudovirus . sACE22 - IgG1 ( grey ) or sACE22 . v2 . 4 - IgG1 ( blue ) were incubated with pseudovirus for 1 h before adding to HeLa - hACE2 - 11 cells . Infection 48 h later was measured by luciferase reporter gene expression . Data are mean ± SD , N = 3 independent biological replicates .

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(C) Neutralization of BA.1 omicron pseudovirus. sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) were incubated with pseudovirus for 1 h before adding to HeLa-hACE2-11 cells. Infection 48 h later was measured by luciferase reporter gene expression. Data are mean ± SD, N = 3 independent biological replicates.


( D - E ) Authentic BA . 1 omicron virus ( isolate USA / MD - HP20874 / 2021 ) was incubated with sACE22 - IgG1 ( D ) or sACE22 . v2 . 4 - IgG1 ( E ) for 1 h and added to Calu - 3 cells . Infection 48 h later was measured by RT - qPCR for the viral N gene . 3 µM remdesivir ( black columns ) is a positive neutralization control .

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(D-E) Authentic BA.1 omicron virus (isolate USA/MD-HP20874/2021) was incubated with sACE22-IgG1 (D) or sACE22.v2.4-IgG1 (E) for 1 h and added to Calu-3 cells. Infection 48 h later was measured by RT-qPCR for the viral N gene. 3 µM remdesivir (black columns) is a positive neutralization control.


( B - C ) Survival ( B ) and weight loss ( C ) . N = 8 ( IgG1 control ) or 9 ( sACE22 . v2 . 4 - IgG1 treated ) mice . Error bars for mouse weight are centered on the mean and show SEM .

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(B-C) Survival (B) and weight loss (C). N = 8 (IgG1 control) or 9 (sACE22.v2.4-IgG1 treated) mice. Error bars for mouse weight are centered on the mean and show SEM.


( E ) Viral Spike , Nsp , and Rdrp mRNA levels in lung tissue were measured by RT - qPCR at Days 8 and 14 , normalized to Ppia transcript levels . Data are presented as mean ± SEM , N = 4 mice per group . ns , not significant ; * * , p < 0 . 01 by two - ways ANOVA , corrected by Tukey .

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(E) Viral Spike, Nsp, and Rdrp mRNA levels in lung tissue were measured by RT-qPCR at Days 8 and 14, normalized to Ppia transcript levels. Data are presented as mean ± SEM, N = 4 mice per group. ns, not significant; **, p < 0.01 by two-ways ANOVA, corrected by Tukey.


( A ) Using flow cytometry , binding to Expi293F cells expressing S of BA . 2 omicron was measured for monomeric sACE2 - 8his proteins . ( B ) Binding of antibodies and dimeric sACE22 - IgG1 proteins to Expi293F cells expressing BA . 2 omicron S measured by flow cytometry .

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(A) Using flow cytometry, binding to Expi293F cells expressing S of BA.2 omicron was measured for monomeric sACE2-8his proteins. (B) Binding of antibodies and dimeric sACE22-IgG1 proteins to Expi293F cells expressing BA.2 omicron S measured by flow cytometry.


( C ) BA . 2 omicron pseudovirus was incubated with sACE22 - IgG1 ( grey ) or sACE22 . v2 . 4 - IgG1 ( blue ) for 1 h and added to HeLa - hACE2 - 11 cells . Infection was measured after 48 h .

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(C) BA.2 omicron pseudovirus was incubated with sACE22-IgG1 (grey) or sACE22.v2.4-IgG1 (blue) for 1 h and added to HeLa-hACE2-11 cells. Infection was measured after 48 h.


( D - E ) Binding of monomeric sACE2 - 8his proteins ( D ) and dimeric IgG1 proteins ( E ) to Expi293F cells expressing BA . 4 / BA . 5 omicron S as measured by flow cytometry .

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(D-E) Binding of monomeric sACE2-8his proteins (D) and dimeric IgG1 proteins (E) to Expi293F cells expressing BA.4/BA.5 omicron S as measured by flow cytometry.


( A and B ) ARPE - 19 cells were transfected with siRNA duplexes to raptor , RagA + B , p18 , or nontarget . 60 h after transfection , cells were infected with adenovirus expressing either TFEB - FLAG - WT ( A ) or TFEB - FLAG - S211A ( B ) . 12 h later , cells were fixed , permeabilized with 0 . 2 % Triton X - 100 , and double stained with antibodies against FLAG ( used to detect TFEB ) and mTOR . Bars , 10 µm . ( C ) Quantification of A . ( D ) Quantification of B . Values are means ± SD of three independent experiments . * * * , P < 0 . 001 .

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(A and B) ARPE-19 cells were transfected with siRNA duplexes to raptor, RagA+B, p18, or nontarget. 60 h after transfection, cells were infected with adenovirus expressing either TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG (used to detect TFEB) and mTOR. Bars, 10 µm. (C) Quantification of A. (D) Quantification of B. Values are means ± SD of three independent experiments. ***, P < 0.001.


( A ) ARPE - 19 cells were nucleofected with the indicated Rag - expressing plasmids . 6 h after nucleofection , cells were infected with adenovirus expressing TFEB - FLAG - WT . 12 h later , cells were lysed and subjected to immunoprecipitation with the anti - FLAG antibody . The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG ( used to detect TFEB - WT ) .

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(A) ARPE-19 cells were nucleofected with the indicated Rag-expressing plasmids. 6 h after nucleofection, cells were infected with adenovirus expressing TFEB-FLAG-WT. 12 h later, cells were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG (used to detect TFEB-WT).


( B ) ARPE - 19 cells were nucleofected with the indicated Rag - and TFEB - GFP - expressing plasmids . 18 h later , cells were lysed and subjected to immunoprecipitation with the anti - HA antibody ( used to immunoprecipitate Rag proteins ) . The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP ( used to detect Rag proteins and TFEB - WT , respectively ) .

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(B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-GFP-expressing plasmids. 18 h later, cells were lysed and subjected to immunoprecipitation with the anti-HA antibody (used to immunoprecipitate Rag proteins). The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP (used to detect Rag proteins and TFEB-WT, respectively).


( C ) ARPE - 19 coexpressing TFEB and the indicated Rag - expressing plasmids were lysed and subjected to immunoprecipitation with the anti - FLAG antibody . The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG ( used to detect Rag proteins and TFEB - WT , respectively ) .

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(C) ARPE-19 coexpressing TFEB and the indicated Rag-expressing plasmids were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG (used to detect Rag proteins and TFEB-WT, respectively).


( D ) Immunoblotting analysis of coimmunoprecipitated TFEB - FLAG - S211A with Rag heterodimers . IP , immunoprecipitation .

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(D) Immunoblotting analysis of coimmunoprecipitated TFEB-FLAG-S211A with Rag heterodimers. IP, immunoprecipitation.


( A ) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin - 1 for 1 h . Cells were then lysed and subjected to immunoblotting with antibodies against TFEB ( used to detect endogenous TFEB ) and actin .

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(A) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin-1 for 1 h. Cells were then lysed and subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB) and actin.


( B ) HeLa cells were treated as indicated in A , and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation . Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB ( used to detect endogenous TFEB ) , Lamp1 , or Histone H3 .

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(B) HeLa cells were treated as indicated in A, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB), Lamp1, or Histone H3.


( C ) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin - 1 as indicated in A .

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

(C) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin-1 as indicated in A.


( D ) HeLa cells were starved in serum - and amino acid - free medium ( starvation ) for 3 h and analyzed by immunoblotting with antibodies against TFEB .

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

(D) HeLa cells were starved in serum- and amino acid-free medium (starvation) for 3 h and analyzed by immunoblotting with antibodies against TFEB.


( E ) HeLa cells were incubated in normal medium ( control ) and serum - and amino acid - free medium ( Starvation ) for 4 h or starved for 4 h followed by restimulation with amino acids ( starvation + amino acids ) for 30 min and analyzed by immunofluorescence with antibodies against TFEB ( endogenous TFEB is shown in green ) and Lamp1 ( red ) . The region within the dotted box is magnified in the insets .

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

(E) HeLa cells were incubated in normal medium (control) and serum- and amino acid-free medium (Starvation) for 4 h or starved for 4 h followed by restimulation with amino acids (starvation + amino acids) for 30 min and analyzed by immunofluorescence with antibodies against TFEB (endogenous TFEB is shown in green) and Lamp1 (red). The region within the dotted box is magnified in the insets.


( F ) HeLa cells were nucleofected with the indicated Rag - expressing plasmids . 18 h later , cells were lysed , and RagB / D heterodimers were pulled down using glutathione - Sepharose beads . Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST ( used to detect endogenous TFEB and Rag proteins , respectively ) . Bars , 10 µm .

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

(F) HeLa cells were nucleofected with the indicated Rag-expressing plasmids. 18 h later, cells were lysed, and RagB/D heterodimers were pulled down using glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST (used to detect endogenous TFEB and Rag proteins, respectively). Bars, 10 µm.


( A ) ARPE - 19 cells were infected with adenovirus expressing TFEB - FLAG - WT . 16 h later , cells were incubated with 250 nM Torin - 1 for 1 h or starved in serum - and amino acid - free medium for 3 h . Cells were then fixed , permeabilized with 0 . 2 % saponin , and double stained with antibodies against TFEB ( used to detect recombinant TFEB ) and Lamp1 .

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

(A) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-WT. 16 h later, cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 3 h. Cells were then fixed, permeabilized with 0.2% saponin, and double stained with antibodies against TFEB (used to detect recombinant TFEB) and Lamp1.


( B ) ARPE - 19 cells were infected with adenovirus expressing TFEB - FLAG - S211A . 12 h later , cells were starved in serum - and amino acid - free medium for 4 h ( starvation ) or kept in complete medium ( control ) . Cells were then fixed , permeabilized with 0 . 2 % Triton X - 100 , and stained with antibodies against FLAG ( used to detect TFEB - S211A ) .

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

(B) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-S211A. 12 h later, cells were starved in serum- and amino acid-free medium for 4 h (starvation) or kept in complete medium (control). Cells were then fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB-S211A).


( C ) HeLa cells were incubated with 250 nM Torin - 1 for 1 h or starved in serum - and amino acid - free medium for 4 h with the addition of Torin - 1 during the last hour of starvation . Cells were then washed , fixed , permeabilized with 0 . 2 % Triton X - 100 , and double stained with antibodies against TFEB ( used to detect endogenous TFEB ) and Lamp1 . Regions within the dotted boxes are magnified in the insets .

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

(C) HeLa cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 4 h with the addition of Torin-1 during the last hour of starvation. Cells were then washed, fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against TFEB (used to detect endogenous TFEB) and Lamp1. Regions within the dotted boxes are magnified in the insets.