Figure 2 ( B ) The N - lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA . Recombinant PKA at different concentrations ( 0 , 7 , 34 , 168 , or 840 ng ) was incubated with recombinant ( E . coli ) wild - type PI4KB ( 20 μg ) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry . Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity ( total area ) of the phosphorylated vs non - phosphorylated peptide ( 486 - 506 ) . ( C ) PI4KB phosphorylation by PKA alters the affinity for c10orf76 . The kinase activity of different variants of PI4KB ( 15 nM ) was measured in the presence of varying amounts of c10orf76 ( 23 nM - 1 . 5μM ) with 100 % PI lipid substrate ( 0 . 5 mg / L ) and 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates ( n = 3 ) . ( D ) PI4KB has the same kinase activity when Ser496 is phosphorylated or mutated to alanine . Kinase assay of PI4KB non - phosphorylated , phos - Ser496 or S496A ( 15 nM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . The data was normalized to the kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) . ( E ) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76 . His - pulldown assays of 6xHis - c10orf76 ( 3 μM ) with wild - type or RL494EA PI4KB ( 1 - 2 μM ) . ( F ) RL494EA PI4KB activity is not inhibited by c10orf76 . Kinase assays of either wild type or mutant RL494EA PI4KB ( 40 nM ) were carried out with varying concentrations of c10orf76 ( 3 . 9 nM - 2μM ) with 100 % PI lipid vesicles ( 0 . 5 mg / L ) and 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) . ( G ) Wild - type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity . Kinase assays of either wild - type and mutant PI4KB ( 10 nM ) were carried out with 100 % PI lipid vesicles ( 0 . 5 mg / L ) , 100 μM ATP , and PI4KB ( 300 nM ) on Golgi - mimic vesicles ( 0 . 5 mg / mL ) with 10 μM ATP . The data was normalized to the kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) . ( H ) FLH409AAA - c10orf76 mutant shows decreased affinity for PI4KB . His - pulldown assays of 6xHis - c10orf76 ( 1 μM ) with wild - type PI4KB ( 1 μM ) . Samples washed a total of 4 times . ( I ) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced . Kinase assay of PI4KB ( 40 nM ) and a concentration curve of c10orf76 ( 3 . 9 nM - 2μM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . The data was normalized to the kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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 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 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 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 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 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 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 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

Figure 2(B) The N-lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA. Recombinant PKA at different concentrations (0, 7, 34, 168, or 840 ng) was incubated with recombinant (E. coli) wild-type PI4KB (20 μg) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry. Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity (total area) of the phosphorylated vs non-phosphorylated peptide (486-506).(C) PI4KB phosphorylation by PKA alters the affinity for c10orf76. The kinase activity of different variants of PI4KB (15 nM) was measured in the presence of varying amounts of c10orf76 (23 nM-1.5μM) with 100% PI lipid substrate (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates (n=3).(D) PI4KB has the same kinase activity when Ser496 is phosphorylated or mutated to alanine. Kinase assay of PI4KB non-phosphorylated, phos-Ser496 or S496A (15 nM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).(E) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76. His-pulldown assays of 6xHis-c10orf76 (3 μM) with wild-type or RL494EA PI4KB (1-2 μM).(F) RL494EA PI4KB activity is not inhibited by c10orf76. Kinase assays of either wild type or mutant RL494EA PI4KB (40 nM) were carried out with varying concentrations of c10orf76 (3.9 nM-2μM) with 100% PI lipid vesicles (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).(G) Wild-type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity. Kinase assays of either wild-type and mutant PI4KB (10 nM) were carried out with 100% PI lipid vesicles (0.5 mg/L), 100 μM ATP, and PI4KB (300 nM) on Golgi-mimic vesicles (0.5 mg/mL) with 10 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).(H) FLH409AAA-c10orf76 mutant shows decreased affinity for PI4KB. His-pulldown assays of 6xHis-c10orf76 (1 μM) with wild-type PI4KB (1 μM). Samples washed a total of 4 times.(I) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced. Kinase assay of PI4KB (40 nM) and a concentration curve of c10orf76 (3.9 nM-2μM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


Figure 3 ( A ) Transfections of HEK293 cells revealed that both wild - type GFP - PI4KB and RL494EA GFP - PI4KB localize to the Golgi . ( B ) WT c10orf76 also localized to the Golgi , however , the PI4KB binding deficient mutant of c10orf76 ( FLH409AAA ) predominantly localized to the cytosol . ( D ) Mitochondria recruitment experiment with wild - type PI4KB and c10orf76 . Left : AKAP1 - FRB - CFP is localized to the mitochondria before ( top ) and 5 minutes after rapamycin ( 100 nM ) treatment ( bottom ) . Middle : mRFP - FKBP12 - PI4KB at high expression level is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 at a high expression level is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) . ( F ) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76 . Left : AKAP1 - FRB - CFP is localized to the mitochondria before ( top ) and 5 minutes after ( bottom ) rapamycin treatment . Middle : mRFP - FKBP12 - PI4KB ( RL494EA ) is located in the cytosol before rapamycin ( top ) and translocates to the mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 is located in the cytosol before ( top ) and after ( bottom ) rapamycin induction .

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 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 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

Figure 3(A) Transfections of HEK293 cells revealed that both wild-type GFP-PI4KB and RL494EA GFP-PI4KB localize to the Golgi.(B) WT c10orf76 also localized to the Golgi, however, the PI4KB binding deficient mutant of c10orf76 (FLH409AAA) predominantly localized to the cytosol.(D) Mitochondria recruitment experiment with wild-type PI4KB and c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after rapamycin (100 nM) treatment (bottom). Middle: mRFP-FKBP12-PI4KB at high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 at a high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom).(F) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after (bottom) rapamycin treatment. Middle: mRFP-FKBP12-PI4KB(RL494EA) is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 is located in the cytosol before (top) and after (bottom) rapamycin induction.


Figure 4 ( A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm . B ) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers ( B ) . ( C ) Western blot of GBF1 , PI4KB and B - Actin levels in both wild - type and c10orf76 KO HAP1 cells . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm . ( D ) Markers of Arf1 activation - the coatomer proteins COPIα / γ and βCOP act as a readout for GTP - bound Arf1 , while the native coatomer was detected with the CM1 antibody . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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 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 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

Figure 4(A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.B) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers (B). (C) Western blot of GBF1, PI4KB and B-Actin levels in both wild-type and c10orf76 KO HAP1 cells. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.(D) Markers of Arf1 activation - the coatomer proteins COPIα/γ and βCOP act as a readout for GTP-bound Arf1, while the native coatomer was detected with the CM1 antibody. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


Figure 5 ( A ) Viral infection assays determining viral titers by end - point titration at 0 hours and 8 hours in HAP1 wild - type or c10orf76 knockout cells . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild - type titres at 8 hours using a one - way ANOVA . * * , P < 0 . 01 ; N . S . , P > 0 . 05 . ( B ) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild - type and PI4KB knockout cells upon transfection of wild - type PI4KB , the complex - disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Values were statistically evaluated compared to the GalT control using a one - way ANOVA . * * , P < 0 . 01 ; * , P < 0 . 05 ; N . S . , P > 0 . 05 .

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

Figure 5(A) Viral infection assays determining viral titers by end-point titration at 0 hours and 8 hours in HAP1 wild-type or c10orf76 knockout cells. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild-type titres at 8 hours using a one-way ANOVA. **, P<0.01; N.S., P>0.05.(B) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild-type and PI4KB knockout cells upon transfection of wild-type PI4KB, the complex-disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Values were statistically evaluated compared to the GalT control using a one-way ANOVA. **, P<0.01; *, P<0.05; N.S., P>0.05.


Figure 1 ( B ) Western blot of RNAi efficiency and specificity for oligos used in ( C - G ) . HeLa Kyoto cells were incubated for 72 h with control oligos or RNAi - oligos depleting CUL4A , CUL4B or DDB1 . Specific downregulation of the indicated proteins was analyzed by immunoblotting . GAPDH controls for equal loading . ( C ) Quantification of western blot bands and the ratio between neddylated vs unneddylated CUL4 with the paralog downregulated is calculated . Graphs displays mean with SEM of N = 3 biological replicates . * * P = 0 . 001 . ( E ) Flow cytometry analysis of Hela cells treated with the indicated RNAi for 72h and immune - stained for γH2A . X , DNA staining with propidium iodide . One of three biological replicates is represented . ( G ) Live - cell imaging of mitosis in RNAi depleted HeLa Kyoto cells . Brightfield images were taken every 3 min , and mitotic duration was quantified by measuring the time from cell rounding to telophase . Mean with SEM is depicted from N = 3 biological experiments analyzing a total of 30 single cells . nsP = 0 . 9995 ; * * P = 0 . 0040 ; * * P = 0 . 0044 . Red symbols mark incomplete mitotic events during the movie . White arrows mark cells undergoing mitosis . Scale bar : 10 μm .

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 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 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 O O O O O

Figure 1(B) Western blot of RNAi efficiency and specificity for oligos used in (C-G). HeLa Kyoto cells were incubated for 72 h with control oligos or RNAi-oligos depleting CUL4A, CUL4B or DDB1. Specific downregulation of the indicated proteins was analyzed by immunoblotting. GAPDH controls for equal loading. (C) Quantification of western blot bands and the ratio between neddylated vs unneddylated CUL4 with the paralog downregulated is calculated. Graphs displays mean with SEM of N=3 biological replicates. **P = 0.001.(E) Flow cytometry analysis of Hela cells treated with the indicated RNAi for 72h and immune-stained for γH2A.X, DNA staining with propidium iodide. One of three biological replicates is represented.(G) Live-cell imaging of mitosis in RNAi depleted HeLa Kyoto cells. Brightfield images were taken every 3 min, and mitotic duration was quantified by measuring the time from cell rounding to telophase. Mean with SEM is depicted from N=3 biological experiments analyzing a total of 30 single cells. nsP= 0.9995; **P=0.0040; **P= 0.0044. Red symbols mark incomplete mitotic events during the movie. White arrows mark cells undergoing mitosis. Scale bar: 10 μm.


Figure 2 ( A ) CUL4A and CUL4B were analyzed by immunoblotting of extracts prepared from HeLa Kyoto cells arrested in S - phase ( S = thymidine ) , G2 ( R0 - 3306 ) , or mitosis ( M = nocodazole or taxol ) . One of three biological replicates is shown . H3 pS10 ( phosphorylated histone H3 at serine 10 ) was used as a mitotic marker . Same samples but separate blot for H3pS10 staining due to size differences . ( C ) Mitotic population of unperturbed cells was harvested by shake - off . Three subsequent harvests are depicted ( R1 - R3 ) . The interphase population are cells that remained attached to the culture dish . ( G ) In vitro kinase assays with purified CDK1 - cyclinB and Sf9 - purified CUL4B and mutant proteins in the presence of [ γ - 32P ] ATP . The autoradiogram ( top panel ) shows CUL4B phosphorylation , while coomassie staining ( lower panel ) controls equal loading of CUL4B and mutants ( N = 3 technical replicates for WT , P50L ; NP1 ; N = 2 for NP1 - A49T and NP1 - A49T - P50L ) . Note that the A49T mutation in the NP1 mutant restores phosphorylation of the T49 residue , but only in the presence of the adjacent proline at position 50 .

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

Figure 2(A) CUL4A and CUL4B were analyzed by immunoblotting of extracts prepared from HeLa Kyoto cells arrested in S-phase (S= thymidine), G2 (R0-3306), or mitosis (M= nocodazole or taxol). One of three biological replicates is shown. H3 pS10 (phosphorylated histone H3 at serine 10) was used as a mitotic marker. Same samples but separate blot for H3pS10 staining due to size differences.(C) Mitotic population of unperturbed cells was harvested by shake-off. Three subsequent harvests are depicted (R1-R3). The interphase population are cells that remained attached to the culture dish.(G) In vitro kinase assays with purified CDK1-cyclinB and Sf9-purified CUL4B and mutant proteins in the presence of [γ-32P]ATP. The autoradiogram (top panel) shows CUL4B phosphorylation, while coomassie staining (lower panel) controls equal loading of CUL4B and mutants (N=3 technical replicates for WT, P50L; NP1; N=2 for NP1-A49T and NP1-A49T-P50L). Note that the A49T mutation in the NP1 mutant restores phosphorylation of the T49 residue, but only in the presence of the adjacent proline at position 50.


Figure 3 ( A ) Western blot analysis to monitor CUL4A or CUL4B levels in extracts prepared from HeLaFLP control cells or HeLaFLP cells deleted for CUL4B by CRISPR / Cas9 ( ∆ CUL4BFLP ) but stably expressing no CUL4B ( - ) , CUL4B or the indicated NP1 or P50L mutants from the doxycycline - inducible promoter . GAPDH was included to control equal loading . One of three biological replicates is shown . ( D ) The expression levels of endogenous HA - tagged WT and the indicated CUL4B mutants were compared by immunoblotting with HA . II antibody . GAPDH controls for equal loading ( F ) In vitro E3 ligase activity assays using the indicated reconstituted CRL4BCRBN complexes and the neo - substrate CKlα . At time 0 , lenalidomide was added to start the reaction and CKlα ubiquitination was followed at the times indicated ( in minutes ) by immunoblotting with CKlα antibody . N ≥ 3 technical replicates for CUL4B , CUL4A , P50L , K895R and L864fs * 13 ; N = 1 for NP1 . Note that the in vitro E3 ligase activity of CRL4B and the P50L patient mutation is comparable .

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Figure 3(A) Western blot analysis to monitor CUL4A or CUL4B levels in extracts prepared from HeLaFLP control cells or HeLaFLP cells deleted for CUL4B by CRISPR/Cas9 (∆CUL4BFLP) but stably expressing no CUL4B (-), CUL4B or the indicated NP1 or P50L mutants from the doxycycline-inducible promoter. GAPDH was included to control equal loading. One of three biological replicates is shown.(D) The expression levels of endogenous HA-tagged WT and the indicated CUL4B mutants were compared by immunoblotting with HA.II antibody. GAPDH controls for equal loading(F) In vitro E3 ligase activity assays using the indicated reconstituted CRL4BCRBN complexes and the neo-substrate CKlα. At time 0, lenalidomide was added to start the reaction and CKlα ubiquitination was followed at the times indicated (in minutes) by immunoblotting with CKlα antibody. N≥3 technical replicates for CUL4B, CUL4A, P50L, K895R and L864fs*13; N=1 for NP1. Note that the in vitro E3 ligase activity of CRL4B and the P50L patient mutation is comparable.


Figure 4 ( C ) To control RNAi efficiency and specificity , HeLa Kyoto cells were incubated for 72 h with control oligos ( control ) or RNAi - oligos depleting CUL4B , LIS1 or WDR1 . Specific downregulation of the indicated proteins was analyzed by immunoblotting . GAPDH controls for equal loading . One of three biological replicates is shown . ( E ) HA - immunoprecipitation ( IP ) of extracts ( input ) prepared from unedited ( - ) or HA - CUL4B endogenously expressing HeLa Kyoto cell lines arrested in S - phase ( thymidine ) or mitosis ( nocodazole ) were probed with antibodies against LIS1 , WDR1 and DDB1 . The antibody recognizing phospho - H3 ( S19 ) controls mitotic arrest and specificity of the immunoprecipitation . ( F , G ) HeLa Kyoto endogenously expressing HA - CUL4B or the indicated NP1 or P50L mutants were arrested in mitosis with nocodazole . Extracts ( input ) and HA - immunoprecipitants were analyzed for the presence of HA - CUL4B for control and bound DDB1 and LIS1 ( F ) or DDB1 and WDR1 ( G ) , respectively . One of 3 biological replicates is shown .

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 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

Figure 4(C) To control RNAi efficiency and specificity, HeLa Kyoto cells were incubated for 72 h with control oligos (control) or RNAi-oligos depleting CUL4B, LIS1 or WDR1. Specific downregulation of the indicated proteins was analyzed by immunoblotting. GAPDH controls for equal loading. One of three biological replicates is shown.(E) HA-immunoprecipitation (IP) of extracts (input) prepared from unedited (-) or HA-CUL4B endogenously expressing HeLa Kyoto cell lines arrested in S-phase (thymidine) or mitosis (nocodazole) were probed with antibodies against LIS1, WDR1 and DDB1. The antibody recognizing phospho-H3(S19) controls mitotic arrest and specificity of the immunoprecipitation.(F, G) HeLa Kyoto endogenously expressing HA-CUL4B or the indicated NP1 or P50L mutants were arrested in mitosis with nocodazole. Extracts (input) and HA-immunoprecipitants were analyzed for the presence of HA-CUL4B for control and bound DDB1 and LIS1 (F) or DDB1 and WDR1 (G), respectively. One of 3 biological replicates is shown.


Figure 5 ( A ) Coomassie - stained SDS - PAGE of in vitro pull - down assays of FLAG - tagged LIS1 and WDR1 , and His - DDB1 , co - expressed in baculoviral Sf9 cells , F is FLAG and H is His . One of 3 biological replicates is shown . Note that DDB1 specifically interacts with LIS1 and WDR1 . ( C , D ) Western blot of in vitro pulldown assays of Strep - tagged CUL4B and FLAG - tagged LIS1 ( C ) or WDR1 ( D ) co - expressed in Sf9 cells in the presence ( + ) or absence ( - ) of DDB1 . One of 3 biological replicates is shown . Note that LIS1 and WDR1 specifically bind CUL4B , but this interaction is enhanced by the presence of DDB1 . ( G ) FP assay investigating binding of CRL4B and CRL4A to Alexa - labeled WDR1Fluorescence polarization assay comparing the binding of CRL4B and λ - PP - treated CRL4B to Alexa - labeled WDR1 . The mean with SD of N = 3 technical replicates is depicted . ( L ) Quantification of in vitro binding assays ( Strep - pull down ) of Strep - LIS1 with purified CRL4B , or the NP1 phospho - mutant ( CRL4BNP1 ) ( N = 2 technical replicates ) , or λ - PP treated CRL4B ( CRL4Bλ - PP ) ( N = 1 ) .

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Figure 5(A) Coomassie-stained SDS-PAGE of in vitro pull-down assays of FLAG-tagged LIS1 and WDR1, and His-DDB1, co-expressed in baculoviral Sf9 cells, F is FLAG and H is His. One of 3 biological replicates is shown. Note that DDB1 specifically interacts with LIS1 and WDR1.(C, D) Western blot of in vitro pulldown assays of Strep-tagged CUL4B and FLAG-tagged LIS1 (C) or WDR1 (D) co-expressed in Sf9 cells in the presence (+) or absence (-) of DDB1. One of 3 biological replicates is shown. Note that LIS1 and WDR1 specifically bind CUL4B, but this interaction is enhanced by the presence of DDB1.(G) FP assay investigating binding of CRL4B and CRL4A to Alexa-labeled WDR1Fluorescence polarization assay comparing the binding of CRL4B and λ-PP-treated CRL4B to Alexa-labeled WDR1. The mean with SD of N=3 technical replicates is depicted. (L) Quantification of in vitro binding assays (Strep-pull down) of Strep-LIS1 with purified CRL4B, or the NP1 phospho-mutant (CRL4BNP1) (N=2 technical replicates), or λ-PP treated CRL4B (CRL4Bλ-PP) (N=1).


Figure 6 ( C ) Extracts ( input ) prepared from S - phase ( S ; thymidine ) or mitosis ( M ; nocodazole ) - arrested HeLa Kyoto cells ( - ) and endogenously expressing HA - tagged WT CUL4B , or the NP1 or P50L mutants were immunoprecipitated with HA . II - antibodies ( IP ) . Bound ACTR3 or DDB1 was detected using specific antibodies . One of three biological replicates is shown . Note that in contrast to DDB1 , ACTR3 binds CUL4B in a phosphorylation - dependent manner . ( D ) HA - immunoprecipitants ( IP ) of extracts ( input ) prepared from unedited ( - ) controls or endogenously expressing HA - CUL4B HeLa Kyoto cells arrested in mitosis with nocodazole were probed with antibodies against DDB1 , DCTN1 ( dynactin ) and actin . One of two biological replicates is depicted . ( F ) Live cell imaging of HeLa cells expressing H2B - mcherry and lifeAct - GFP , treated for 72 h with control oligos ( siControl ) or RNAi - oligos depleting CUL4B , LIS1 or WDR1 . Representative cells are shown in time intervals of 6 min during metaphase with identical contrast settings . The unanchored spindle in CUL4B - and LIS1 deleted cells is marked with a white arrow . Scale bar : 10 μm .

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 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 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

Figure 6(C) Extracts (input) prepared from S-phase (S; thymidine) or mitosis (M; nocodazole)-arrested HeLa Kyoto cells (-) and endogenously expressing HA-tagged WT CUL4B, or the NP1 or P50L mutants were immunoprecipitated with HA.II-antibodies (IP). Bound ACTR3 or DDB1 was detected using specific antibodies. One of three biological replicates is shown. Note that in contrast to DDB1, ACTR3 binds CUL4B in a phosphorylation-dependent manner.(D) HA-immunoprecipitants (IP) of extracts (input) prepared from unedited (-) controls or endogenously expressing HA-CUL4B HeLa Kyoto cells arrested in mitosis with nocodazole were probed with antibodies against DDB1, DCTN1 (dynactin) and actin. One of two biological replicates is depicted.(F) Live cell imaging of HeLa cells expressing H2B-mcherry and lifeAct-GFP, treated for 72 h with control oligos (siControl) or RNAi-oligos depleting CUL4B, LIS1 or WDR1. Representative cells are shown in time intervals of 6 min during metaphase with identical contrast settings. The unanchored spindle in CUL4B- and LIS1 deleted cells is marked with a white arrow. Scale bar: 10 μm.


Figure 7hESCs were differentiated to generate human forebrain specific regionalised organoids . Sections were stained as indicated with antibodies against SOX2 or CTIP2 to visualize neural stem cell and neuronal cell populations , respectively . ( A ) Example images of day 35 organoids . The stained sections are also shown as merged images at different maginifications , with scale bars of 200 μm and 50 μm , respectively . ( B ) The number of cortical units per mm2 total area was quantified in unedited and ΔCUL4B brain organoids at day 35 . Ventricles were counted and the number divided by the total organoid area on 20 sections from one biological replicate .

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Figure 7hESCs were differentiated to generate human forebrain specific regionalised organoids. Sections were stained as indicated with antibodies against SOX2 or CTIP2 to visualize neural stem cell and neuronal cell populations, respectively. (A) Example images of day 35 organoids. The stained sections are also shown as merged images at different maginifications, with scale bars of 200 μm and 50 μm, respectively. (B) The number of cortical units per mm2 total area was quantified in unedited and ΔCUL4B brain organoids at day 35. Ventricles were counted and the number divided by the total organoid area on 20 sections from one biological replicate.


Figure 1C57BL / 6J mice were intraarticularly infected with MRSA ( 4 × 106 CFU ; n = 3 - 4 per group ) and left untreated for 24 hours . H . The amount of infiltrating immune cells in synovial fluid was quantified and detected using MRSA expressive of green fluorescent protein ( GFP ) within immune cells ( Scale bar : 100 μm ) . MRSA bioburden in synovial fluid was quantified in colony - forming units ( CFU ) . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) . C57BL / 6J mice were intraarticularly infected with MRSA ( 4 × 106 CFU ; n = 3 - 4 per group ) and left untreated for 24 hours . K . Paraffin - embedded tissues were sectioned and used to measure inflammation score and synovial hyperplasia . GLUT1 and MCT4 expression was detected , and the percentage of positively staining cells were determined ( Scale bar : 1 , 000 μm ) . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) . M . Human cartilage and synovial tissue were infected with MRSA ( 4 × 106 CFU ) for 24 hours . The co - expression and - localization levels of GLUT1 and MCT4 in human cartilage tissue was measured and confirmed by line profile analysis ( Scale bar : 100 μm ) . Expression of GLUT1 , MCT4 , IL - 1β , and MMP3 was measured in human synovial tissue ( Scale bar : 1 , 000 μm ) .

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Figure 1C57BL/6J mice were intraarticularly infected with MRSA (4 × 106 CFU; n = 3-4 per group) and left untreated for 24 hours. H. The amount of infiltrating immune cells in synovial fluid was quantified and detected using MRSA expressive of green fluorescent protein (GFP) within immune cells (Scale bar: 100 μm). MRSA bioburden in synovial fluid was quantified in colony-forming units (CFU). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p<0.05 or **p <0.01; N.D. = not detected).C57BL/6J mice were intraarticularly infected with MRSA (4 × 106 CFU; n = 3-4 per group) and left untreated for 24 hours. K. Paraffin-embedded tissues were sectioned and used to measure inflammation score and synovial hyperplasia. GLUT1 and MCT4 expression was detected, and the percentage of positively staining cells were determined (Scale bar: 1,000 μm). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p<0.05 or **p <0.01; N.D. = not detected).M. Human cartilage and synovial tissue were infected with MRSA (4 × 106 CFU) for 24 hours. The co-expression and -localization levels of GLUT1 and MCT4 in human cartilage tissue was measured and confirmed by line profile analysis (Scale bar: 100 μm). Expression of GLUT1, MCT4, IL-1β, and MMP3 was measured in human synovial tissue (Scale bar: 1,000 μm).


Figure 2B . Physiological changes were identified and representative images were generated . C . Blood was collected and complete blood counts ( CBC ) were measured . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ; N . S . = not significant ) . D and E . Infiltrating immune cell counts within synovial fluid and MRSA synovial fluid bioburden were quantified . MRSA bioburden in synovial tissue was quantified . F . Paraffin - embedded tissues were sectioned and measured at 7 and 14 days with respect to inflammation score , synovial hyperplasia , synovial cellularity , OARSI score , proteoglycan depletion , osteophyte formation , bone erosion , and bone formation ( Scale bar : 1 , 000 μm ) . G and H . Lactate level in synovial fluid was measured at 14 days and expression of GLUT1 , MCT4 , cleaved - IL - 1β , and NLRP3 in synovial tissue was measured with GAPDH as a loading control . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ; N . S . = not significant ) .

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Figure 2B. Physiological changes were identified and representative images were generated.C. Blood was collected and complete blood counts (CBC) were measured. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected; N.S. = not significant).D and E. Infiltrating immune cell counts within synovial fluid and MRSA synovial fluid bioburden were quantified. MRSA bioburden in synovial tissue was quantified. F. Paraffin-embedded tissues were sectioned and measured at 7 and 14 days with respect to inflammation score, synovial hyperplasia, synovial cellularity, OARSI score, proteoglycan depletion, osteophyte formation, bone erosion, and bone formation (Scale bar: 1,000 μm). G and H. Lactate level in synovial fluid was measured at 14 days and expression of GLUT1, MCT4, cleaved-IL-1β, and NLRP3 in synovial tissue was measured with GAPDH as a loading control. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected; N.S. = not significant).


Figure 3B . Infiltrating immune cell counts within synovial fluid were quantified . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) . C . Serum and synovial fluid lactate levels were measured and the relative luminescence intensity is shown . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) . D . Expression of GLUT1 and MMP3 in synovial tissue was measured using GAPDH as a loading control . E . Physiological changes were identified , and representative images were generated . F . Blood was collected , and complete blood counts ( CBC ) were measured . G . Paraffin - embedded tissues were sectioned and measured with respect to inflammation score , synovial hyperplasia , OARSI score , proteoglycan depletion , osteophyte formation , bone erosion , and bone formation ( Scale bar : 1 , 000 μm ) . The expression of GLUT1 was detected and the percentage of positively staining cells was determined . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 ; N . D . = not detected ) .

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Figure 3B. Infiltrating immune cell counts within synovial fluid were quantified. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).C. Serum and synovial fluid lactate levels were measured and the relative luminescence intensity is shown. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).D. Expression of GLUT1 and MMP3 in synovial tissue was measured using GAPDH as a loading control.E. Physiological changes were identified, and representative images were generated.F. Blood was collected, and complete blood counts (CBC) were measured. G. Paraffin-embedded tissues were sectioned and measured with respect to inflammation score, synovial hyperplasia, OARSI score, proteoglycan depletion, osteophyte formation, bone erosion, and bone formation (Scale bar: 1,000 μm). The expression of GLUT1 was detected and the percentage of positively staining cells was determined. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p<0.05 or **p<0.01; N.D. = not detected).


Figure 5C57BL / 6J mice were intraarticularly infected with MRSA ( 4 × 106 CFU ; n = 4 - 5 per group ) for 24 hours . Synovial fluid cells were collected and ( 5 × 105 ) seeded on antibiotic - free DMEM medium with or without DMF ( 300 µg / ml ) for 6 and 24 hours . B . MRSA expressive of green fluorescent protein ( GFP ) was detected and measured ( Scale bar : 100 μm ) . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 or * * * p < 0 . 001 or * * * * p < 0 . 0001 ; N . D . = not detected ; N . S . = not significant ) . G . Inhibition zones in disk fusion assays using vancomycin ( 1 , 000 µg ) and DMF ( 75 to 300 µg ) were measured and presented in a histogram . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 or * * * p < 0 . 001 or * * * * p < 0 . 0001 ; N . D . = not detected ; N . S . = not significant ) . J . RAW264 . 7 cells were stained with CellTracker and infected with MRSA ( 4 × 106 CFU ) for 2 hours . The cells were then washed to remove extra - cellular MRSA . Medium containing DMF ( 700 µg / ml ) , vancomycin ( 100 µg / ml ) , and DMF ( 700 µg / ml ) with vancomycin ( 100 µg / ml ) was added and GFP intensity was measured at 1 - hour intervals for a total of 12 hours . Representative images of differential fluorescence with CellTracker and GFP at 4 - hour intervals for a total of 12 hours . Intracellular and extracellular MRSA were semi - quantitatively measured at 12 hours . Data information : Error bars show means ± SD with individual data points . Two - tailed unpaired t - test analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 or * * * p < 0 . 001 or * * * * p < 0 . 0001 ; N . D . = not detected ; N . S . = not significant ) .

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Figure 5C57BL/6J mice were intraarticularly infected with MRSA (4 × 106 CFU; n = 4-5 per group) for 24 hours. Synovial fluid cells were collected and (5 × 105) seeded on antibiotic-free DMEM medium with or without DMF (300 µg/ml) for 6 and 24 hours. B. MRSA expressive of green fluorescent protein (GFP) was detected and measured (Scale bar: 100 μm). Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).G. Inhibition zones in disk fusion assays using vancomycin (1,000 µg) and DMF (75 to 300 µg) were measured and presented in a histogram. Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).J. RAW264.7 cells were stained with CellTracker and infected with MRSA (4 × 106 CFU) for 2 hours. The cells were then washed to remove extra-cellular MRSA. Medium containing DMF (700 µg/ml), vancomycin (100 µg/ml), and DMF (700 µg/ml) with vancomycin (100 µg/ml) was added and GFP intensity was measured at 1-hour intervals for a total of 12 hours. Representative images of differential fluorescence with CellTracker and GFP at 4-hour intervals for a total of 12 hours. Intracellular and extracellular MRSA were semi-quantitatively measured at 12 hours. Data information: Error bars show means ± SD with individual data points. Two-tailed unpaired t-test analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected; N.S. = not significant).


Figure 6C57BL / 6J mice were subcutaneously treated with vancomycin ( 30 mg / kg ) and rifampin ( 20 mg / kg ) for 3 days following MRSA ( 8 × 106 CFU ) infection , followed by systemic ( 14 mg / kg ) or local ( 70 μg / joint ) DMF treatment at 3 days interval for a total of 3 times ( n = 6 per group ) . D . Paraffin - embedded tissues were sectioned and measured with respect to inflammation score , synovial hyperplasia , synovial cellularity , proteoglycan depletion , osteophyte formation , bone erosion , and bone formation ( Scale bar : 2 , 000 or 500 μm ) . Bone resorption by osteoclasts was measured by tartrate - resistant acid phosphatase ( TRAP ) staining ( Scale bar : 2 , 000 μm ) . Lameness score was also measured . E . Illustrated summary of DMF ' s effect on MRSA - induced septic arthritis . Data information : In vivo experiments were repeated in at least two independent experiments . Error bars show means ± SD with individual data points . One - way ANOVA with Tukey ' s post hoc analysis was conducted to determine statistical significance ( * p < 0 . 05 or * * p < 0 . 01 or * * * p < 0 . 001 or * * * * p < 0 . 0001 ; N . D . = not detected ) .

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Figure 6C57BL/6J mice were subcutaneously treated with vancomycin (30 mg/kg) and rifampin (20 mg/kg) for 3 days following MRSA (8 × 106 CFU) infection, followed by systemic (14 mg/kg) or local (70 μg/joint) DMF treatment at 3 days interval for a total of 3 times (n = 6 per group). D. Paraffin-embedded tissues were sectioned and measured with respect to inflammation score, synovial hyperplasia, synovial cellularity, proteoglycan depletion, osteophyte formation, bone erosion, and bone formation (Scale bar: 2,000 or 500 μm). Bone resorption by osteoclasts was measured by tartrate-resistant acid phosphatase (TRAP) staining (Scale bar: 2,000 μm). Lameness score was also measured. E. Illustrated summary of DMF's effect on MRSA-induced septic arthritis. Data information: In vivo experiments were repeated in at least two independent experiments. Error bars show means ± SD with individual data points. One-way ANOVA with Tukey's post hoc analysis was conducted to determine statistical significance (*p < 0.05 or ** p < 0.01 or *** p < 0.001 or **** p < 0.0001; N.D. = not detected).


Figure 1A Representative image of tumor - bearing mice and tumors after subcutaneous injection of Hepa1 - 6 hepatoma cells into WT and Tet2 - / - ( KO ) mice . B Mean tumor volumes in WT and Tet2 - / - mice ( n = 6 ) . C Tumor weights 18 days after tumor cell injection into WT and KO mice ( n = 6 ) . D Kaplan - Meier survival curves of tumor - bearing and tumor - free WT and KO mice ( n = 15 ) . E Representative images of lungs with metastasis from WT and KO mice injected with Hepa1 - 6 cells via tail vein , as well as quantification of the number of lung metastasis ( n = 4 ) . F Representative images of tumors from WT and KO mice subcutaneously injected with Py8119 breast cancer cells , as well as quantification of tumor weights ( n = 5 ) .

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Figure 1A Representative image of tumor-bearing mice and tumors after subcutaneous injection of Hepa1-6 hepatoma cells into WT and Tet2-/- (KO) mice.B Mean tumor volumes in WT and Tet2-/- mice (n=6).C Tumor weights 18 days after tumor cell injection into WT and KO mice (n=6).D Kaplan-Meier survival curves of tumor-bearing and tumor-free WT and KO mice (n=15).E Representative images of lungs with metastasis from WT and KO mice injected with Hepa1-6 cells via tail vein, as well as quantification of the number of lung metastasis (n=4).F Representative images of tumors from WT and KO mice subcutaneously injected with Py8119 breast cancer cells, as well as quantification of tumor weights (n=5).


Figure 2A , B Immunohistochemistry analysis of CD8a , CD4 ( A ) , PCNA , and Foxp3 ( B ) in tumors from WT and Tet2 - / - ( KO ) mice subcutaneously injected with Hepa1 - 6 hepatoma cells . Corresponding bar charts display positive staining , quantified using Image Pro Plus 6 . 0 software ( n = 3 ) . Pictures were captured at 400X magnification . Scale bar = 50 μm . D Tumor growth curves in WT and KO mice treated with PBS or with anti - PD - 1 Ab ( n = 3 ) . E Representatives images of day - 19 tumors from WT and KO from panel D . F Tumor weights from WT and KO mice at day 19 . G Flow cytometry analysis of splenic CD8 + T cells in WT and KO mice with or without anti - PD - 1 treatment after tumor challenge . H - K GSEA analysis showing the significant down - regulation of genes in GO biological processes : chemokine signaling pathway ( H ) , T cell receptor signaling pathway ( I ) , innate immune response ( J ) and cytokine receptor interaction ( K ) . The x - axis represents that the mouse genes are ranked from the most up - regulated ( left end ) to the most down - regulated ( right end ) in comparison of syngeneic tumor from Hepa1 - 6 tumor - bearing KO to WT mice ( n = 4 ) . The y - axis is an enrichment score ( ES ) , which goes up ( > 0 ) and down ( < 0 ) representing the enrichment of up - regulated genes and down - regulated genes , respectively . NES is the normalized enrichment score . p - value was calculated by permutation .

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 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 B-PANEL_START 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A, B Immunohistochemistry analysis of CD8a, CD4 (A), PCNA, and Foxp3 (B) in tumors from WT and Tet2-/- (KO) mice subcutaneously injected with Hepa1-6 hepatoma cells. Corresponding bar charts display positive staining, quantified using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.D Tumor growth curves in WT and KO mice treated with PBS or with anti-PD-1 Ab (n =3).E Representatives images of day-19 tumors from WT and KO from panel D.F Tumor weights from WT and KO mice at day 19.G Flow cytometry analysis of splenic CD8+ T cells in WT and KO mice with or without anti-PD-1 treatment after tumor challenge.H-K GSEA analysis showing the significant down-regulation of genes in GO biological processes: chemokine signaling pathway (H), T cell receptor signaling pathway (I), innate immune response (J) and cytokine receptor interaction (K). The x-axis represents that the mouse genes are ranked from the most up-regulated (left end) to the most down-regulated (right end) in comparison of syngeneic tumor from Hepa1-6 tumor-bearing KO to WT mice (n=4). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of up-regulated genes and down-regulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.


Figure 3A Percentages of MDSCs ( CD11b + Gr1 + ) gated from the CD45 + cell population in peripheral blood and spleen [ n = 11 , tumor - free ( - ) WT and Tet2 - / - （ KO ） mice ; n = 12 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 10 , tumor - free ( - ) WT and KO mice ; n = 13 , tumor - bearing ( + ) WT and KO mice , spleen ] . B Percentages of G - MDSCs ( CD11b + Ly6G + ) gated from the CD45 + cell population in peripheral blood and spleen [ n = 14 , tumor - free ( - ) WT and KO mice ; n = 15 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 15 , tumor - free ( - ) WT and KO mice ; n = 15 , tumor - bearing ( + ) WT and KO mice , spleen ] . C Percentages of M - MDSCs ( CD11b + Ly6C + ) gated from the CD45 + cell population in peripheral blood and spleen [ n = 5 , tumor - free ( - ) WT and KO mice ; n = 6 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 6 , tumor - free ( - ) WT and KO mice ; n = 6 , tumor - bearing ( + ) WT and KO mice , spleen ] . D Representative images of Ly6g and Ly6c immunohistochemistry in Hepa1 - 6 cell tumors from WT and KO mice . Corresponding bar charts display quantification of positive staining using Image Pro Plus 6 . 0 software ( n = 3 ) . Pictures were captured at 400X magnification . Scale bar = 50 μm . E Percentages of CD8 + T cells gated from the CD45 + cell population in peripheral blood and spleen [ n = 11 , tumor - free ( - ) WT and KO mice ; n = 5 , tumor - bearing ( + ) WT and Tet2 - / - KO mice , peripheral blood ; n = 16 , tumor - free ( - ) WT and KO mice ; n = 12 , tumor - bearing ( + ) WT and KO mice , spleen ] . F Percentages of CD4 + T cells gated from the CD45 + cell population in peripheral blood and spleen [ n = 8 , tumor - free ( - ) WT and KO mice ; n = 5 , tumor - bearing ( + ) WT and KO mice , peripheral blood ; n = 16 , tumor - free ( - ) WT and KO mice ; n = 11 , tumor - bearing ( + ) WT and KO mice , spleen ] .

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Figure 3A Percentages of MDSCs (CD11b+Gr1+) gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and Tet2-/-（KO） mice; n=12, tumor-bearing (+) WT and KO mice, peripheral blood; n=10, tumor-free (-) WT and KO mice; n=13, tumor-bearing (+) WT and KO mice, spleen]. B Percentages of G-MDSCs (CD11b+Ly6G+) gated from the CD45+ cell population in peripheral blood and spleen [n=14, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, peripheral blood; n=15, tumor-free (-) WT and KO mice; n=15, tumor-bearing (+) WT and KO mice, spleen]. C Percentages of M-MDSCs (CD11b+Ly6C+) gated from the CD45+ cell population in peripheral blood and spleen [n=5, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, peripheral blood; n=6, tumor-free (-) WT and KO mice; n=6, tumor-bearing (+) WT and KO mice, spleen]. D Representative images of Ly6g and Ly6c immunohistochemistry in Hepa1-6 cell tumors from WT and KO mice. Corresponding bar charts display quantification of positive staining using Image Pro Plus 6.0 software (n=3). Pictures were captured at 400X magnification. Scale bar = 50 μm.E Percentages of CD8+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=11, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and Tet2-/- KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=12, tumor-bearing (+) WT and KO mice, spleen]. F Percentages of CD4+ T cells gated from the CD45+ cell population in peripheral blood and spleen [n=8, tumor-free (-) WT and KO mice; n=5, tumor-bearing (+) WT and KO mice, peripheral blood; n=16, tumor-free (-) WT and KO mice; n=11, tumor-bearing (+) WT and KO mice, spleen].


Figure 4A Sorting efficiency of G - MDSCs ( CD11b + Ly6G + ) from spleens of tumor - bearing Tet2 - / - （ KO ） and WT mice . B GSEA analysis showing the significant upregulation of genes in GO biological processes : DNA replication ( left ) , cell cycle G1 - S phase transition ( middle ) , and ribosome biogenesis ( right ) . The x - axis represents that the mouse genes are ranked from the most upregulated ( left end ) to the most downregulated ( right end ) in comparison of tumor - bearing KO to WT mice from G - MDSCs of spleen ( n = 2 ) . The y - axis is an enrichment score ( ES ) , which goes up ( > 0 ) and down ( < 0 ) representing the enrichment of upregulated genes and downregulated genes , respectively . NES is the normalized enrichment score . p - value was calculated by permutation . C G - MDSCs purified from spleen of tumor - bearing WT or Tet2 - / - mice were co - cultured with CD8 + T cells at the indicated cellular ratios for T cell activation assays , quantified by CFSE - labeling and dye dilution for 3 days in vitro . Representative histograms show CFSE signals in the in vitro T cell activation assays . D Quantification of CD8 + T cell proliferation in panel C . Each point represents a co - culture experiment ( n = 3 ) . ns , not significant . Graphs show mean ± SEM and statistical analysis by Student ' s t - test .

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Figure 4A Sorting efficiency of G-MDSCs (CD11b+Ly6G+) from spleens of tumor-bearing Tet2-/- （KO） and WT mice.B GSEA analysis showing the significant upregulation of genes in GO biological processes: DNA replication (left), cell cycle G1-S phase transition (middle), and ribosome biogenesis (right). The x-axis represents that the mouse genes are ranked from the most upregulated (left end) to the most downregulated (right end) in comparison of tumor-bearing KO to WT mice from G-MDSCs of spleen (n=2). The y-axis is an enrichment score (ES), which goes up (>0) and down (<0) representing the enrichment of upregulated genes and downregulated genes, respectively. NES is the normalized enrichment score. p-value was calculated by permutation.C G-MDSCs purified from spleen of tumor-bearing WT or Tet2-/- mice were co-cultured with CD8+ T cells at the indicated cellular ratios for T cell activation assays, quantified by CFSE-labeling and dye dilution for 3 days in vitro. Representative histograms show CFSE signals in the in vitro T cell activation assays. D Quantification of CD8+ T cell proliferation in panel C. Each point represents a co-culture experiment (n=3). ns, not significant. Graphs show mean ± SEM and statistical analysis by Student's t-test.


Figure 5B Representative flow cytometry plots of splenic G - MDSCs measured in tumor - free untreated Tet2 - / - ( KO ) and WT mice and after anti - Ly6g or PBS injections into tumor - bearing mice . Flow cytometric analysis of T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - Ly6g treatment after tumor challenge . C Tumor growth curves in WT and KO mice treated with either PBS or anti - mouse Ly6g ( n = 5 ) . D Day - 18 tumor weights at the endpoint of ( C ) [ n = 11 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 12 , anti - Ly6g antibody ( + ) injected tumor - bearing WT and KO mice ] . E Percentages of CD8 + T cells from the spleens of tumor - bearing mice with or without anti - mouse Ly6g treatment [ n = 17 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 6 , anti - Ly6g ( + ) antibody injected tumor - bearing WT and KO mice ] . F Percentages of CD4 + T cells from the spleens of tumor - bearing mice with or without anti - mouse Ly6g treatment [ n = 16 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 6 , anti - Ly6g ( + ) antibody injected tumor - bearing WT and KO mice Flow cytometric analysis of T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - Ly6g treatment after tumor challenge .

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Figure 5B Representative flow cytometry plots of splenic G-MDSCs measured in tumor-free untreated Tet2-/- (KO) and WT mice and after anti-Ly6g or PBS injections into tumor-bearing mice. Flow cytometric analysis of T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-Ly6g treatment after tumor challenge.C Tumor growth curves in WT and KO mice treated with either PBS or anti-mouse Ly6g (n=5).D Day-18 tumor weights at the endpoint of (C) [n=11, PBS (-) injected tumor-bearing WT and KO mice; n=12, anti-Ly6g antibody (+) injected tumor-bearing WT and KO mice].E Percentages of CD8+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=17, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice]. F Percentages of CD4+ T cells from the spleens of tumor-bearing mice with or without anti-mouse Ly6g treatment [n=16, PBS (-) injected tumor-bearing WT and KO mice; n=6, anti-Ly6g (+) antibody injected tumor-bearing WT and KO mice Flow cytometric analysis of T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-Ly6g treatment after tumor challenge.


Figure 6A Gene set enrichment analysis of the IL - 6 / JAK / STAT signaling pathway in G - MDSCs from tumor - bearing Tet2 - / - ( KO ) versus WT mice ( n = 2 ) . p - value was calculated by permutation . B ELISA analysis of IL - 6 , IFN - γ , and IL - 2 levels in blood from WT and KO mice upon tumor challenge ( n = 4 , tumor - bearing WT mice ; n = 3 , tumor - bearing KO mice ) . C Tumor growth curves in tumor - bearing WT and KO mice treated with PBS or anti - mouse IL - 6 antibody ( n = 5 ) . D Day - 18 tumor weights at the endpoint of ( C ) [ n = 10 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 7 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice ] . E , F Percentages of CD11b + Ly6G + G - MDSCs gated from total cells in ( E ) spleen and ( F ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 antibody [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 4 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 4 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 4 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . G , H Percentages of CD8 + T cells gated from total cells in ( G ) spleen and ( H ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . I , J Percentages of CD4 + T cells gated from total cells in ( I ) spleen and ( J ) peripheral blood from tumor - bearing mice treated with PBS or anti - mouse IL - 6 [ n = 5 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , spleen ; n = 4 , PBS ( - ) injected tumor - bearing WT and KO mice ; n = 5 , anti - IL - 6 antibody ( + ) injected tumor - bearing WT and KO mice , peripheral blood ] . Flow cytometric analysis of G - MDSCs and T cells in Tet2 - / - mice and littermate control mice ( WT ) with or without anti - IL - 6 treatment after tumor challenge .

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Figure 6A Gene set enrichment analysis of the IL-6/JAK/STAT signaling pathway in G-MDSCs from tumor-bearing Tet2-/- (KO) versus WT mice (n=2). p-value was calculated by permutation.B ELISA analysis of IL-6, IFN-γ, and IL-2 levels in blood from WT and KO mice upon tumor challenge (n=4, tumor-bearing WT mice; n=3, tumor-bearing KO mice).C Tumor growth curves in tumor-bearing WT and KO mice treated with PBS or anti-mouse IL-6 antibody (n=5).D Day-18 tumor weights at the endpoint of (C) [n=10, PBS (-) injected tumor-bearing WT and KO mice; n=7, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice].E, F Percentages of CD11b+ Ly6G+ G-MDSCs gated from total cells in (E) spleen and (F) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 antibody [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=4, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. G, H Percentages of CD8+ T cells gated from total cells in (G) spleen and (H) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. I, J Percentages of CD4+ T cells gated from total cells in (I) spleen and (J) peripheral blood from tumor-bearing mice treated with PBS or anti-mouse IL-6 [n=5, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, spleen; n=4, PBS (-) injected tumor-bearing WT and KO mice; n=5, anti-IL-6 antibody (+) injected tumor-bearing WT and KO mice, peripheral blood]. Flow cytometric analysis of G-MDSCs and T cells in Tet2-/- mice and littermate control mice (WT) with or without anti-IL-6 treatment after tumor challenge.


Figure 1 ( A , B ) Flag ‐ SENP3 or HA ‐ PELP1 was expressed in HeLa cells as indicated . Proteins were captured on Flag ‐ or HA ‐ beads and immunoblotted using antibodies directed against endogenous PELP1 or SENP3 as indicated . ( C ) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of SENP3 by western blotting with an anti ‐ SENP3 antibody . ( D - F ) Flag ‐ or HA ‐ tagged proteins were expressed in HeLa cells , captured on Flag ‐ or HA ‐ beads and subjected to immunoblotting as indicated . In all experiments , the inputs represent 2 . 5 % of the total cell lysate .

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Figure 1(A, B) Flag‐SENP3 or HA‐PELP1 was expressed in HeLa cells as indicated. Proteins were captured on Flag‐ or HA‐beads and immunoblotted using antibodies directed against endogenous PELP1 or SENP3 as indicated.(C) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of SENP3 by western blotting with an anti‐SENP3 antibody.(D-F) Flag‐ or HA‐tagged proteins were expressed in HeLa cells, captured on Flag‐ or HA‐beads and subjected to immunoblotting as indicated. In all experiments, the inputs represent 2.5% of the total cell lysate.


Figure 2 ( A , B ) Flag ‐ PELP1 and HA ‐ WDR18 were expressed in HeLa cells as indicated . HA ‐ WDR18 was captured on HA ‐ beads and the bound material was probed by immunoblotting for the presence of ectopically expressed Flag ‐ tagged PELP1 or endogenous PELP1 as indicated . ( C ) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of endogenous WDR18 by western blotting with an anti ‐ WDR18 antibody . ( D ) Flag ‐ TEX10 was expressed with HA ‐ PELP1 in HeLa cells , captured on Flag ‐ beads and proteins were immunoblotted as indicated . ( E ) HA ‐ TEX10 was expressed , captured on HA ‐ beads and associated proteins were probed for the presence of endogenous WDR18 using immunoblotting . The inputs represent 2 . 5 % of the total cell lysate .

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Figure 2(A, B) Flag‐PELP1 and HA‐WDR18 were expressed in HeLa cells as indicated. HA‐WDR18 was captured on HA‐beads and the bound material was probed by immunoblotting for the presence of ectopically expressed Flag‐tagged PELP1 or endogenous PELP1 as indicated.(C) Endogenous PELP1 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and immunocomplexes were probed for the presence of endogenous WDR18 by western blotting with an anti‐WDR18 antibody.(D) Flag‐TEX10 was expressed with HA‐PELP1 in HeLa cells, captured on Flag‐beads and proteins were immunoblotted as indicated.(E) HA‐TEX10 was expressed, captured on HA‐beads and associated proteins were probed for the presence of endogenous WDR18 using immunoblotting. The inputs represent 2.5% of the total cell lysate.


Figure 3 ( A - C ) Immunofluorescence staining of endogenous PELP1 , SENP3 , UBF1 and NPM1 in HeLa cells . Nuclei were visualized using DAPI staining .

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Figure 3(A-C) Immunofluorescence staining of endogenous PELP1, SENP3, UBF1 and NPM1 in HeLa cells. Nuclei were visualized using DAPI staining.


Figure 4 ( B ) HeLa cells were transfected with siRNA duplexes targeting PELP1 , TEX10 , WDR18 , MDN1 or a control siRNA as indicated . Downregulation of the respective proteins was analysed by western blotting as indicated ( WB , three lower panels ) . At 72 h after transfection , cells were pulse labelled with 32P ‐ orthophosphate for 1 h and chased for 2 . 5 h . RNA was separated on a denaturing agarose gel and detected by ethidium bromide ( EtBr ) staining and autoradiography after drying of the gel . ( C ) The signal intensities of the 28S and 32S rRNA forms were quantified by phosphoimager analysis and the ratio of 28S : 32S rRNA was calculated . Values represent the average of four ( two for siMDN1 ) independent experiments . Error bars indicate s . d . ( D ) PELP1 is associated with ITS2 ‐ containing RNA species . Endogenous PELP1 was immunoprecipitated from HeLa cells . In total , 10 % of the immunoprecipitated material was analysed by western blotting ( lower panel ) . The remaining material was used for RNA isolation and cDNA synthesis . The cDNA was used as template for PCR amplification using primers within the ITS2 / 28S region or GAPDH as a control . Input corresponds to 10 % of the cell lysate . Vertical line in the upper panel indicates removal of irrelevant adjacent lanes . ( E ) Pre ‐ ribosomal particles were separated by sucrose gradient centrifugation of nuclear extracts . Proteins were recovered from the fractions by TCA precipitation and further analysed by SDS - PAGE and western blotting using antibodies as indicated . Vertical lines solely indicate loading onto two separate gels . ( F ) Nucleolar export of pre ‐ 60S particles is strongly compromised in cells depleted of PELP1 , TEX10 , WDR18 , MDN1 or SENP3 . HeLa cells expressing YFP ‐ RpL27 from a tetracycline ‐ inducible promoter were transfected with the indicated siRNAs . After 24 h , expression of YFP ‐ RpL27 was induced and 72 h later the localization of RpL27 was monitored . Pictures were taken using identical exposure times .

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Figure 4(B) HeLa cells were transfected with siRNA duplexes targeting PELP1, TEX10, WDR18, MDN1 or a control siRNA as indicated. Downregulation of the respective proteins was analysed by western blotting as indicated (WB, three lower panels). At 72 h after transfection, cells were pulse labelled with 32P‐orthophosphate for 1 h and chased for 2.5 h. RNA was separated on a denaturing agarose gel and detected by ethidium bromide (EtBr) staining and autoradiography after drying of the gel.(C) The signal intensities of the 28S and 32S rRNA forms were quantified by phosphoimager analysis and the ratio of 28S:32S rRNA was calculated. Values represent the average of four (two for siMDN1) independent experiments. Error bars indicate s.d.(D) PELP1 is associated with ITS2‐containing RNA species. Endogenous PELP1 was immunoprecipitated from HeLa cells. In total, 10% of the immunoprecipitated material was analysed by western blotting (lower panel). The remaining material was used for RNA isolation and cDNA synthesis. The cDNA was used as template for PCR amplification using primers within the ITS2/28S region or GAPDH as a control. Input corresponds to 10% of the cell lysate. Vertical line in the upper panel indicates removal of irrelevant adjacent lanes.(E) Pre‐ribosomal particles were separated by sucrose gradient centrifugation of nuclear extracts. Proteins were recovered from the fractions by TCA precipitation and further analysed by SDS-PAGE and western blotting using antibodies as indicated. Vertical lines solely indicate loading onto two separate gels.(F) Nucleolar export of pre‐60S particles is strongly compromised in cells depleted of PELP1, TEX10, WDR18, MDN1 or SENP3. HeLa cells expressing YFP‐RpL27 from a tetracycline‐inducible promoter were transfected with the indicated siRNAs. After 24 h, expression of YFP‐RpL27 was induced and 72 h later the localization of RpL27 was monitored. Pictures were taken using identical exposure times.


Figure 5 ( A ) HA ‐ PELP1 was expressed alone or in combination with SUMO1 or SUMO2 in the presence or absence of SV5 ‐ tagged SENP3 in HeLa cells . Expression of the respective proteins was verified by western blotting . Detection of β ‐ tubulin served as loading control . ( B ) SUMO modification of endogenous PELP1 was monitored by anti ‐ PELP1 immunoblotting in HeLa cells transfected with siRNAs as indicated . Depletion of the respective proteins was verified by western blotting . ( C ) HeLa cells were transfected with siRNAs and plasmids as indicated . His ‐ SUMO conjugates were recovered on Ni ‐ NTA beads and subjected to western blotting using anti ‐ PELP1 antibodies . ( D ) HA ‐ PELP1 or HA ‐ PELP1K826R was expressed in the presence of SUMO1 or SUMO2 in HeLa cells . Expression of the respective proteins was analysed by western blotting . Detection of β ‐ tubulin served as loading control . ( E , F ) Flag ‐ p14ARF and SUMO were expressed in HeLa cells and SUMO modification of ectopically expressed HA ‐ PELP1 or endogenous PELP1 was monitored by immunoblotting with anti ‐ HA or anti ‐ PELP1 antibodies as indicated . Arrowheads indicate SUMO - PELP1 conjugates .

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Figure 5(A) HA‐PELP1 was expressed alone or in combination with SUMO1 or SUMO2 in the presence or absence of SV5‐tagged SENP3 in HeLa cells. Expression of the respective proteins was verified by western blotting. Detection of β‐tubulin served as loading control.(B) SUMO modification of endogenous PELP1 was monitored by anti‐PELP1 immunoblotting in HeLa cells transfected with siRNAs as indicated. Depletion of the respective proteins was verified by western blotting.(C) HeLa cells were transfected with siRNAs and plasmids as indicated. His‐SUMO conjugates were recovered on Ni‐NTA beads and subjected to western blotting using anti‐PELP1 antibodies.(D) HA‐PELP1 or HA‐PELP1K826R was expressed in the presence of SUMO1 or SUMO2 in HeLa cells. Expression of the respective proteins was analysed by western blotting. Detection of β‐tubulin served as loading control.(E, F) Flag‐p14ARF and SUMO were expressed in HeLa cells and SUMO modification of ectopically expressed HA‐PELP1 or endogenous PELP1 was monitored by immunoblotting with anti‐HA or anti‐PELP1 antibodies as indicated. Arrowheads indicate SUMO-PELP1 conjugates.


Figure 6 ( A ) Flag ‐ SUMO2 was expressed in HeLa cells by transient transfection . Flag ‐ SUMO2 ‐ expressing cells were visualized by indirect immunofluorescence . The localization of endogenous PELP1 was monitored in Flag ‐ SUMO2 ‐ positive cells ( green arrowheads ) or untransfected cells ( white arrowheads ) using anti ‐ PELP1 antibody . ( B - D ) HeLa cells were transfected with siRNAs as indicated . After fixation and permeabilization , the localization of PELP1 , LAS1L , TEX10 or PES1 was monitored by indirect immunofluorescence using the corresponding antibodies . ( E ) HeLa cells were depleted of endogenous PELP1 and reconstituted with siRNA resistant constructs expressing either wild ‐ type HA ‐ tagged PELP1 or a Flag ‐ tagged PELP1 - SUMO2 fusion protein . The localization of the respective proteins was monitored by indirect immunofluorescence using either anti ‐ HA ‐ or anti ‐ Flag ‐ antibodies . ( F ) The localization of LAS1L or SENP3 was analysed in cells expressing Flag ‐ PELP1 - SUMO2 . Indirect immunofluorescence was done using anti ‐ LAS1L or anti ‐ SENP3 antibodies . Nuclei were counterstained with DAPI .

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Figure 6(A) Flag‐SUMO2 was expressed in HeLa cells by transient transfection. Flag‐SUMO2‐expressing cells were visualized by indirect immunofluorescence. The localization of endogenous PELP1 was monitored in Flag‐SUMO2‐positive cells (green arrowheads) or untransfected cells (white arrowheads) using anti‐PELP1 antibody.(B-D) HeLa cells were transfected with siRNAs as indicated. After fixation and permeabilization, the localization of PELP1, LAS1L, TEX10 or PES1 was monitored by indirect immunofluorescence using the corresponding antibodies.(E) HeLa cells were depleted of endogenous PELP1 and reconstituted with siRNA resistant constructs expressing either wild‐type HA‐tagged PELP1 or a Flag‐tagged PELP1-SUMO2 fusion protein. The localization of the respective proteins was monitored by indirect immunofluorescence using either anti‐HA‐ or anti‐Flag‐antibodies.(F) The localization of LAS1L or SENP3 was analysed in cells expressing Flag‐PELP1-SUMO2. Indirect immunofluorescence was done using anti‐LAS1L or anti‐SENP3 antibodies. Nuclei were counterstained with DAPI.


Figure 7 ( A ) LAS1L was generated by in vitro transcription / translation and incubated with recombinant E1 and E2 enzymes and SUMO1 or SUMO2 , respectively , in the presence of ATP . In the control reaction ( lane 1 ) SUMO was not added . ( B ) HeLa cells were transfected with siRNAs and plasmids as indicated . Expression of the respective proteins was verified by western blotting . Detection of β ‐ tubulin served as loading control . ( C ) HeLa cells were transfected with siRNAs and plasmids as indicated . His ‐ SUMO2 conjugates were captured on magnetic Ni ‐ NTA beads and subjected to western blotting using anti ‐ LAS1L antibodies . Vertical lines indicate removal of irrelevant neighbouring lanes from the initial gel ( see Supplementary Figure 14 ) .

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Figure 7(A) LAS1L was generated by in vitro transcription/translation and incubated with recombinant E1 and E2 enzymes and SUMO1 or SUMO2, respectively, in the presence of ATP. In the control reaction (lane 1) SUMO was not added.(B) HeLa cells were transfected with siRNAs and plasmids as indicated. Expression of the respective proteins was verified by western blotting. Detection of β‐tubulin served as loading control.(C) HeLa cells were transfected with siRNAs and plasmids as indicated. His‐SUMO2 conjugates were captured on magnetic Ni‐NTA beads and subjected to western blotting using anti‐LAS1L antibodies. Vertical lines indicate removal of irrelevant neighbouring lanes from the initial gel (see Supplementary Figure 14).


Figure 1C ) A representative selection of 2D classes of UBR5 . UBR - UBR box ,

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Figure 1C) A representative selection of 2D classes of UBR5. UBR - UBR box,


Figure 3B ) Sucrose gradient sedimentation profiles of GFPAKIRIN2 in the presence or absence of UBR5 . Gels were imaged using fluorescent imaging ( 488nm ) to visualise the recombinant GFP tag , and stain - free imaging to visualise all proteins . Gels of assays are a representative of at least three experimental replicates . Reactions in B were performed at 37 ° C . The final concentration of E3 enzyme in B : 0 . 3µMC ) Ubiquitination of GFPAKIRIN2 . D ) Ubiquitination of GFPUbAKIRIN2 . E ) Ubiquitination activity of wild - type and mutant UBR5ΔUBA on GFPUbAKIRIN2 substrate . Gels were imaged using fluorescent imaging ( 488nm ) to visualise the recombinant GFP tag , and stain - free imaging to visualise all proteins . Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels . Gels of assays are a representative of at least three experimental replicates . Reactions C were performed at 37 ° C . Reactions in D performed at 25 ° C . The final concentration of E3 enzyme in C , D : 0 . 3µMF ) Sucrose gradient sedimentation profiles of GFPUbAKIRIN2 in the absence ( upper panel ) or in the presence of UBR5wt ( middle panel ) , and UBR5ΔUBA ( lower panel ) . Gels were imaged using fluorescent imaging ( 488nm ) to visualise the recombinant GFP tag , and stain - free imaging to visualise all proteins . Gels of assays are a representative of at least three experimental replicates . G ) Ubiquitination activity of wild - type and oligomerisation - defective mutants on GFPUbAKIRIN2 . Gels were imaged using fluorescent imaging ( 488nm ) to visualise the recombinant GFP tag , and stain - free imaging to visualise all proteins . Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels . Gels of assays are a representative of at least three experimental replicates . Reactions in G were performed at 25 ° C . The final concentration of E3 enzyme , in G : 0 . 2µM .

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Figure 3B) Sucrose gradient sedimentation profiles of GFPAKIRIN2 in the presence or absence of UBR5. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels of assays are a representative of at least three experimental replicates. Reactions in B were performed at 37°C. The final concentration of E3 enzyme in B : 0.3µMC) Ubiquitination of GFPAKIRIN2. D) Ubiquitination of GFPUbAKIRIN2. E) Ubiquitination activity of wild-type and mutant UBR5ΔUBA on GFPUbAKIRIN2 substrate. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels. Gels of assays are a representative of at least three experimental replicates. Reactions C were performed at 37°C. Reactions in D performed at 25°C. The final concentration of E3 enzyme in C, D: 0.3µMF) Sucrose gradient sedimentation profiles of GFPUbAKIRIN2 in the absence (upper panel) or in the presence of UBR5wt (middle panel), and UBR5ΔUBA (lower panel). Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels of assays are a representative of at least three experimental replicates.G) Ubiquitination activity of wild-type and oligomerisation-defective mutants on GFPUbAKIRIN2. Gels were imaged using fluorescent imaging (488nm) to visualise the recombinant GFP tag, and stain-free imaging to visualise all proteins. Gels were subsequently stained using Coomassie staining to visualise ubiquitin levels. Gels of assays are a representative of at least three experimental replicates. Reactions in G were performed at 25°C. The final concentration of E3 enzyme , in G: 0.2µM.


Figure 4C ) A selection of 2D classes from Fig 1C . Arrows point to the position of the HECT domain . Scale bar : 100Å . Reactions in C were performed at 25 ° CF ) Ubiquitination of GFPUbAKIRIN2 by UBR5 and UBR5ΔPL and G ) isolated UBR5HECT . Gels were imaged as described in Figure 3 . Gels of assays are a representative of at least two experimental replicates . All reactions were performed using a final concentration of 0 . 3µM E3 .

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Figure 4C) A selection of 2D classes from Fig 1C. Arrows point to the position of the HECT domain. Scale bar: 100Å. Reactions in C were performed at 25°CF) Ubiquitination of GFPUbAKIRIN2 by UBR5 and UBR5ΔPL and G) isolated UBR5HECT. Gels were imaged as described in Figure 3. Gels of assays are a representative of at least two experimental replicates. All reactions were performed using a final concentration of 0.3µM E3.


Figure 1 ( A ) Multiple sequence alignment of Atg8 homologues . Amino ‐ acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal _ W program . Underlined residues represent mutated amino acids in Atg8 . ( B ) Alignment of Atg8 predicted three ‐ dimensional structure with its mammalian homologues . SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation . Orange represents the predicted Atg8 structure , and red , blue and green represent GATE ‐ 16 , GABARAP and LC3 , respectively . ( C ) Three ‐ dimensional model of Atg8 . The predicted structure contains a ubiquitin fold decorated by two further amino ‐ terminal helices ( amino acids Tyr 49 , Leu 50 , Phe 77 and Phe 79 are marked in green ) .

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Figure 1(A) Multiple sequence alignment of Atg8 homologues. Amino‐acid sequences of Atg8 and its mammalian homologues are aligned using the Clustal_W program. Underlined residues represent mutated amino acids in Atg8.(B) Alignment of Atg8 predicted three‐dimensional structure with its mammalian homologues. SWISS MODEL was used to predict Atg8 structure and PyMol was used for presentation. Orange represents the predicted Atg8 structure, and red, blue and green represent GATE‐16, GABARAP and LC3, respectively.(C) Three‐dimensional model of Atg8. The predicted structure contains a ubiquitin fold decorated by two further amino‐terminal helices (amino acids Tyr 49, Leu 50, Phe 77 and Phe 79 are marked in green).


Figure 2 ( A ) Cells were grown to mid ‐ exponential phase and then incubated in the presence of 3 . 5 % galactose to induce expression of GFP - Atg8 . Aminopeptidase I ( API ) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti ‐ Atg8 ( right bottom panel ) , anti ‐ GFP ( left bottom panel ) or anti ‐ API antibodies ( upper panel ) . ( B ) Cells were pulse - labelled with [ 35S ] methionine and chased on non - radioactive starvation medium . Aliquots were taken at the indicated times and acid - soluble small peptides generated by proteolysis were determined . ( C ) Cells were grown in control or in starvation medium and visualized by light microscopy . Arrows point to the pre ‐ autophagosomal structure ( PAS ) , and arrowheads point to GFP within the vacuoles . ( D ) Cell extracts were subjected to SDS - polyacrylamide gel electrophoresis followed by immunoblot analysis using anti ‐ GFP antibodies . GFP , green fluorescent protein ; mAPI , mature aminopeptidase I ; pAPI , premature aminopeptidase I ; WT , wild type .

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Figure 2(A) Cells were grown to mid‐exponential phase and then incubated in the presence of 3.5% galactose to induce expression of GFP-Atg8. Aminopeptidase I (API) maturation was tested in cells incubated in control or starvation medium by western blot analysis using anti‐Atg8 (right bottom panel), anti‐GFP (left bottom panel) or anti‐API antibodies (upper panel).(B) Cells were pulse-labelled with [35S]methionine and chased on non-radioactive starvation medium. Aliquots were taken at the indicated times and acid-soluble small peptides generated by proteolysis were determined.(C) Cells were grown in control or in starvation medium and visualized by light microscopy. Arrows point to the pre‐autophagosomal structure (PAS), and arrowheads point to GFP within the vacuoles.(D) Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis using anti‐GFP antibodies. GFP, green fluorescent protein; mAPI, mature aminopeptidase I; pAPI, premature aminopeptidase I; WT, wild type.