Figure 1 ( B ) MPL - MIL HCM assay in HeLaWT and HeLaHexaKO expressing HT - LC3B , starved in EBSS for 90min ± 100 nM BafA1 . Cells were sequentially incubated with HT ligands MIL and MPL , and HCM quantification carried out . Plasma membrane permeabilization was carried out with 4 nM Agilent XF Plasma Membrane Permeabilizer / PMP . PMP was used during the MIL staining and prior to fixation in 4 % paraformaldehyde . MPL staining followed the fixation . ( B i ) MPL - accessible membrane - bound HT - LC3B ( remaining available to MPL after MIL saturation ) . ( B ii ) MIL - accessible membrane - bound HT - LC3B ; ( B iii ) MIL / MPL puncta ratio . Circles , control samples ( full medium ) ; rectangles ( EBSS ) ; triangles ( EBSS + BafA1 ) ; green , MIL ; red , MPL ; gray , MIL / MPL ratios . Images and statistics , HCM images ( masks : white , primary objects / cells ; red , MPL profiles ; green , MIL profiles ) , one of 60 fields / well , 1 , 000 primary objects ( cells ) per well ; 6 wells per plate ( sampling error ) , triplicate plates ( independent biological replicates ) . Statistical significance was determined by one - way ANOVA followed by Tukey ' s multiple comparison test . All values are mean ± SD , n = 3 biologically independent replicates . ( C - E ) Selected electron microscopy ( EM ) images of HeLaWT and HeLaHexaKO EBSS - induced for autophagy for 90 min and their quantification ( graph in F ) . AP , autophagosomes ( phagophores or double membrane autophagosomes with content of similar electron - density to surrounding cytosol ) ; Amph , amphisomes ; AL , autolysosomes ; MVB , multivesicular bodies ; LELy , late endosomes or lysosomes . Statistics , unpaired t - test , two groups : HeLaWT and HeLaHexaKO ( 93 images each ; sample mean , SE ) . Blue single asterisk , cytoplasmic cargo inside amphisome or autolysosome ; double asterisk , phagophore structures ; arrows , phagophores or autophagosomes . ( F ) Super - resolution ( dSTORM ) of HeLaHexaKO HT - LC3B stained with MIL ( note staining of the interiors of the globular structures ) .

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

Figure 1(B) MPL-MIL HCM assay in HeLaWT and HeLaHexaKO expressing HT-LC3B, starved in EBSS for 90min ± 100 nM BafA1. Cells were sequentially incubated with HT ligands MIL and MPL, and HCM quantification carried out. Plasma membrane permeabilization was carried out with 4 nM Agilent XF Plasma Membrane Permeabilizer/PMP. PMP was used during the MIL staining and prior to fixation in 4% paraformaldehyde. MPL staining followed the fixation. (B i) MPL-accessible membrane-bound HT-LC3B (remaining available to MPL after MIL saturation). (B ii) MIL-accessible membrane-bound HT-LC3B; (B iii) MIL/MPL puncta ratio. Circles, control samples (full medium); rectangles (EBSS); triangles (EBSS+BafA1); green, MIL; red, MPL; gray, MIL/MPL ratios. Images and statistics, HCM images (masks: white, primary objects/cells; red, MPL profiles; green, MIL profiles), one of 60 fields/well, 1,000 primary objects (cells) per well; 6 wells per plate (sampling error), triplicate plates (independent biological replicates). Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD, n = 3 biologically independent replicates.(C-E) Selected electron microscopy (EM) images of HeLaWT and HeLaHexaKO EBSS-induced for autophagy for 90 min and their quantification (graph in F). AP, autophagosomes (phagophores or double membrane autophagosomes with content of similar electron-density to surrounding cytosol); Amph, amphisomes; AL, autolysosomes; MVB, multivesicular bodies; LELy, late endosomes or lysosomes. Statistics, unpaired t-test, two groups: HeLaWT and HeLaHexaKO (93 images each; sample mean, SE). Blue single asterisk, cytoplasmic cargo inside amphisome or autolysosome; double asterisk, phagophore structures; arrows, phagophores or autophagosomes. (F) Super-resolution (dSTORM) of HeLaHexaKO HT-LC3B stained with MIL (note staining of the interiors of the globular structures).


Figure 2 ( B ) Representative images of SolVit assay with HeLaWT and HeLaHexaKO as donors , and HeLaHexaKO HT - LC3B as acceptor . SolVit HCM images ( example ) . MPL + ( red ) , sealed LC3B + membranes MIL + ( green ) , unsealed LCB + membranes . ( C ) Quantification of MPL + and MIL + profiles ( 60 fields / well acquired ) : ( C i ) MPL + ( red ) puncta per field . ( C ii ) MIL + ( green ) puncta per field . ( C iii ) MIL + / MPL + profile ratios ( gray ) . Data information : Statistical significance was determined by one - way ANOVA followed by Tukey ' s multiple comparison test . All values are mean ± SD , n = 3 biologically independent replicates , each HCM experiment : 1 , 000 valid primary objects / cells per well ( max fields per well , 60 ) , 5 wells / sample .

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

Figure 2(B) Representative images of SolVit assay with HeLaWT and HeLaHexaKO as donors, and HeLaHexaKO HT-LC3B as acceptor. SolVit HCM images (example). MPL+ (red), sealed LC3B+ membranes MIL+ (green), unsealed LCB+ membranes. (C) Quantification of MPL+ and MIL+ profiles (60 fields/well acquired): (C i) MPL+ (red) puncta per field. (C ii) MIL+ (green) puncta per field. (C iii) MIL+/MPL+ profile ratios (gray). Data information: Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD, n = 3 biologically independent replicates, each HCM experiment: 1,000 valid primary objects/cells per well (max fields per well, 60), 5 wells/sample.


Figure 3 ( B ) Cells were starved in EBSS for 90 min ± 100 nM BafA1 , sequentially incubated with MIL and MPL . MPL ( B i ; red ) , marker of sealed membranes ; MIL ( B ii ; green ) , marker of unsealed membranes ; profiles quantified by HCM . ( B iii ) MIL + / MPL + puncta ratio ( gray ) , ( C ) HelaWT , HeLaHexaKO , HeLaLC3TKO , HeLaGABATKO stably expressing HT - LC3B were starved with EBSS for 90min or 6h . Cell were incubated with ± HaloTag ligand fluorescently labeled with TMR . In - gel fluoresce ( TMR ) and Western blots ( anti - HaloTag antibody ) of freed HaloTagTMR were imaged and band intensity quantified . ( D ) Quantification of in - gel fluorescence band intensity and Western blot with anti - Halo antibody .

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 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 3(B) Cells were starved in EBSS for 90 min ±100 nM BafA1, sequentially incubated with MIL and MPL. MPL (B i; red), marker of sealed membranes; MIL (B ii; green), marker of unsealed membranes; profiles quantified by HCM. (B iii) MIL+/MPL+ puncta ratio (gray),(C) HelaWT, HeLaHexaKO, HeLaLC3TKO, HeLaGABATKO stably expressing HT-LC3B were starved with EBSS for 90min or 6h. Cell were incubated with ± HaloTag ligand fluorescently labeled with TMR. In-gel fluoresce (TMR) and Western blots (anti-HaloTag antibody) of freed HaloTagTMR were imaged and band intensity quantified. (D) Quantification of in-gel fluorescence band intensity and Western blot with anti-Halo antibody.


Figure 4 ( C ) SolVit complementation / rescue analysis ( quantification ) of acceptor HeLaLC3TKO - HT - LC3B PNS with donor PNS from HeLaWT , HeLaLC3TKO or HelaGABATKO cells ± ATP , 1 h , 37 ˚ C . ( D ) SolVit complementation / rescue analysis ( quantification ) of acceptor HeLaGABATKO - HT - LC3B PNS with donor PNS from HeLaWT , HeLaLC3TKO and HelaGABATKO cells ± ATP , 1 h , 37 ˚ C .

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

Figure 4(C) SolVit complementation/rescue analysis (quantification) of acceptor HeLaLC3TKO-HT-LC3B PNS with donor PNS from HeLaWT, HeLaLC3TKO or HelaGABATKO cells ±ATP, 1 h, 37˚C. (D) SolVit complementation/rescue analysis (quantification) of acceptor HeLaGABATKO-HT-LC3B PNS with donor PNS from HeLaWT, HeLaLC3TKO and HelaGABATKO cells ±ATP, 1 h, 37˚C.


Figure 5 ( A ) GST pulldown analysis of in vitro translated and radiolabeled [ 35S ] Myc - VPS37A with GST or GST tagged LC3A , LC3B , LC3C , GABARAP , GABARAP - L1 and GABARAP - L2 . 2 % of input was loaded . ( B ) Co - IP analysis of interactions between FLAG - VPS37A , GFP - LC3A and GFP - GABARAP ( GABA ) in HEK293T cells . 5 % of input was loaded . ( F ) Co - IP between GFP - LC3A with FLAG - VPS37AFL , UEVVPS37A and VPS37AdeltaUEV . ( G ) Co - IP analyses in HEK293T cells expressing FLAG - VPS37AFL , FLAG - VPS37AdeltaN ( 1 - 90 ) , VPS37AdeltaN ( 1 - 20 ) and FLAG - Nter - EGFP and co - transfected with GFP - GABA . 5 % of input was loaded . ( H ) Co - IP analyses in HEK293T cells expressing FLAG - VPS37AFL , FLAG - VPS37AdeltaN ( 1 - 90 ) , VPS37AdeltaN ( 1 - 20 ) and FLAG - Nter - EGFP and co - transfected with GFP - LC3A . 5 % of input was loaded . ( I ) Complementation of VPS37A KO with VPS37A constructs that have or lack mATG8 - binding region . Quantification of MIL + puncta in Huh7WT HT - LC3B ( circles ) and Huh7VPS37AKO HT - LC3B cells ( squares ) , transfected with empty FLAG , full length VPS37A ( FLAG - VPS37AFL ) or mutant VPS37A ( FLAG - VPS37AdeltaN ( 1 - 20 ) ) . Data information : Statistical significance was determined by one - way ANOVA followed by Tukey ' s multiple comparison test . All values are means ± SD , n = 3 biologically independent replicates , each HCM experiment : 1 , 000 valid primary objects / well , 6 wells / sample . ( J ) HCM images , examples corresponding to Fig 5I , 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 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 B-PANEL_START 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 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

Figure 5(A) GST pulldown analysis of in vitro translated and radiolabeled [35S] Myc-VPS37A with GST or GST tagged LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 and GABARAP-L2. 2% of input was loaded.(B) Co-IP analysis of interactions between FLAG-VPS37A, GFP-LC3A and GFP-GABARAP(GABA) in HEK293T cells. 5% of input was loaded.(F) Co-IP between GFP-LC3A with FLAG-VPS37AFL, UEVVPS37A and VPS37AdeltaUEV.(G) Co-IP analyses in HEK293T cells expressing FLAG-VPS37AFL, FLAG-VPS37AdeltaN (1-90), VPS37AdeltaN(1-20) and FLAG-Nter-EGFP and co-transfected with GFP-GABA. 5% of input was loaded. (H) Co-IP analyses in HEK293T cells expressing FLAG-VPS37AFL, FLAG-VPS37AdeltaN (1-90), VPS37AdeltaN(1-20) and FLAG-Nter-EGFP and co-transfected with GFP-LC3A. 5% of input was loaded.(I) Complementation of VPS37A KO with VPS37A constructs that have or lack mATG8-binding region. Quantification of MIL+ puncta in Huh7WT HT-LC3B (circles) and Huh7VPS37AKO HT-LC3B cells (squares), transfected with empty FLAG, full length VPS37A (FLAG-VPS37AFL) or mutant VPS37A (FLAG-VPS37AdeltaN(1-20)). Data information: Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are means ± SD, n = 3 biologically independent replicates, each HCM experiment: 1,000 valid primary objects/well, 6 wells/sample. (J) HCM images, examples corresponding to Fig 5I, scale bar, 10 μm.


Figure 6 ( B ) MPL - MIL HCM assay in Huh7WT and Huh7VPS37AKO stably expressing HT - LC3B . Quantification : ( B i ) membrane - permeant HT ligand ( MPL ) staining of LC3B - II sequestered within sealed membranes . ( B ii ) MIL staining of membrane - bound HT - LC3B - II accessible to the cytosol and ( B iii ) MIL / MPL puncta ratio . ( F - H ) MPL - MIL HCM assay in Huh7WT ( control or CHMP2A siRNA - treated cells ; immunoblot in F ) stably expressing HT - LC3B during CCCP - induced mitophagy in ± BafA1 ( 100 nM ) . ( G ) Quantification : ( G i ) MPL + sealed membranes ; ( G ii ) MIL + unsealed membranes ; ( G iii ) MIL / MPL puncta ratio . ( H ) HCM images corresponding to panel G .

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

Figure 6(B) MPL-MIL HCM assay in Huh7WT and Huh7VPS37AKO stably expressing HT-LC3B. Quantification: (B i) membrane-permeant HT ligand (MPL) staining of LC3B-II sequestered within sealed membranes. (B ii) MIL staining of membrane-bound HT-LC3B-II accessible to the cytosol and (B iii) MIL/MPL puncta ratio.(F-H) MPL-MIL HCM assay in Huh7WT (control or CHMP2A siRNA-treated cells; immunoblot in F) stably expressing HT-LC3B during CCCP-induced mitophagy in ± BafA1 (100 nM). (G) Quantification: (G i) MPL+ sealed membranes; (G ii) MIL+ unsealed membranes; (G iii) MIL/MPL puncta ratio. (H) HCM images corresponding to panel G.


Figure 7 ( B ) Quantification of puncta : ( B - i ) MPL + puncta ( red ) , sealed LCB + membranes ; ( B - ii ) MIL + ( green ) puncta , unsealed / ligand - accessible LCB + membranes ; ( B - iii ) MIL / MPL ratios ( gray ) . ( C ) Quantification of MIL + puncta ( unsealed / ligand - accessible LC3B + membranes ) in Huh7WT ( circles ) or Huh7VPS37AKO ( squares ) cells stably expressing HT - LC3B . Cells were transfected with empty FLAG vector , FLAG - VPS37A full length ( VPS37AFL ) , FLAG - VPS37AdeltaN ( 1 - 90 ) , or FLAG - VPS37AdeltaN ( 1 - 20 ) constructs . CCCP , 6 h treatment . ( D ) HCM images of Huh7VPS37AKO HT - LC3B with VPS37A constructs ( VPS37AFull , FLAG - VPS37AdeltaN ( 1 - 90 ) , FLAG - VPS37AdeltaN ( 1 - 20 ) and FLAG - VPS37ANter - GFP ) . FLAG - Tag VPS37A constructs , expression detected as red ; and MIL + puncta , pseudo color green . Data information : Statistical significance was determined by one - way ANOVA followed by Tukey ' s multiple comparison test . All values are mean ± SD , n = 3 biologically independent experiments , each HCM experiment : 1 , 000 valid primary objects / cells per well , 6 wells / sample , Scale bar , 10μm .

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 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 O O O O O O O O O O O O O O O O O O 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 7(B) Quantification of puncta: (B-i) MPL+ puncta (red), sealed LCB+ membranes; (B-ii) MIL+ (green) puncta, unsealed/ligand-accessible LCB+ membranes; (B-iii) MIL/MPL ratios (gray).(C) Quantification of MIL+ puncta (unsealed/ligand-accessible LC3B+ membranes) in Huh7WT (circles) or Huh7VPS37AKO (squares) cells stably expressing HT-LC3B. Cells were transfected with empty FLAG vector, FLAG-VPS37A full length (VPS37AFL), FLAG-VPS37AdeltaN(1-90), or FLAG-VPS37AdeltaN(1-20) constructs. CCCP, 6 h treatment. (D) HCM images of Huh7VPS37AKO HT-LC3B with VPS37A constructs (VPS37AFull, FLAG-VPS37AdeltaN(1-90), FLAG-VPS37AdeltaN(1-20) and FLAG-VPS37ANter-GFP). FLAG-Tag VPS37A constructs, expression detected as red; and MIL+ puncta, pseudo color green. Data information: Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. All values are mean ± SD, n = 3 biologically independent experiments, each HCM experiment: 1,000 valid primary objects/cells per well, 6 wells/sample, Scale bar, 10μm.


Figure 3Representative HDX - MS curves showing the protected regions in CD53 and CD2 . The HDX experiments were performed ( manually or with a LEAP automation system ) in triplicate . Error bars show the standard deviation . D Representative HDX - MS curves showing the protected regions in CD81 and CD19 . The HDX experiments were manually performed in triplicate . Error bars show the standard deviation .

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 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

Figure 3Representative HDX-MS curves showing the protected regions in CD53 and CD2. The HDX experiments were performed (manually or with a LEAP automation system) in triplicate. Error bars show the standard deviation.D Representative HDX-MS curves showing the protected regions in CD81 and CD19. The HDX experiments were manually performed in triplicate. Error bars show the standard deviation.


Figure 5A CD81 knockout of 293T cells . The knockout cell line was generated by CRISPR - Cas9 . The immunoblots using CD81 antibody and actin antibody were conducted on the same film . B CD81 facilitates CD19 maturation . CD81 and CD19 ( 300 ng DNA ) were transfected into the CD81 - knockout 293T cells . Anti - FLAG immunoblot was performed for CD19 with a C - FLAG tag . The deglycosylation was performed by digesting the cell lysate with PNGase F or Endo H , following the manufacturers ' instructions . The deglycosylation pattern of CD19 species ( p : precursor , m and m ' : two mature forms ) differs because the glycosylation content of these species is differentC Trypsin digestion of CD19 . The digestion was performed at 37 ° C with intact cells . The mature CD19 forms ( m and m ' ) can be digested because they are surface expressed , whereas the intracellular precursor form ( p ) is protected from the digestion . E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19 . Chol : mutations at the cholesterol - binding site of CD81 . CD81 constructs and CD19 ( 300 ng DNA ) were co - transfected into the CD81 - knockout 293T cells . Top , Anti - FLAG immunoblot was performed for CD19 with a C - FLAG tag . In presence of wildtype CD81 ( wt ) , most CD19 is in the properly glycosylated mature form ( m ) , and the overglycosylated m ' form is not observed . With the EC1 mutants ( A4 in particular ) , however , m ' emerges and m shifts to high MW , indicating an alteration in the CD19 glycosylation . The level of intracellular p form is low with the wildtype CD81 or with the mutants . Bottom , Control experiment showing similar expression level of wild - type CD81 and CD81 mutants . Anti - V5 immunoblotting was conducted for the CD81 constructs with a C - V5 tag , and anti - actin immunoblotting was conducted on the same film .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O 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 5A CD81 knockout of 293T cells. The knockout cell line was generated by CRISPR-Cas9. The immunoblots using CD81 antibody and actin antibody were conducted on the same film.B CD81 facilitates CD19 maturation. CD81 and CD19 (300 ng DNA) were transfected into the CD81-knockout 293T cells. Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. The deglycosylation was performed by digesting the cell lysate with PNGase F or Endo H, following the manufacturers' instructions. The deglycosylation pattern of CD19 species (p: precursor, m and m': two mature forms) differs because the glycosylation content of these species is differentC Trypsin digestion of CD19. The digestion was performed at 37 °C with intact cells. The mature CD19 forms (m and m') can be digested because they are surface expressed, whereas the intracellular precursor form (p) is protected from the digestion.E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19. Chol: mutations at the cholesterol-binding site of CD81. CD81 constructs and CD19 (300 ng DNA) were co-transfected into the CD81-knockout 293T cells. Top, Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. In presence of wildtype CD81 (wt), most CD19 is in the properly glycosylated mature form (m), and the overglycosylated m' form is not observed. With the EC1 mutants (A4 in particular), however, m' emerges and m shifts to high MW, indicating an alteration in the CD19 glycosylation. The level of intracellular p form is low with the wildtype CD81 or with the mutants. Bottom, Control experiment showing similar expression level of wild-type CD81 and CD81 mutants. Anti-V5 immunoblotting was conducted for the CD81 constructs with a C-V5 tag, and anti-actin immunoblotting was conducted on the same film.


Figure 6A Migration of naïve Cd53 - / - cultured primary pre - B cells to 100 ng / mL CXCL12 compared to WT littermate controls ( n = 5 mice per group ) over three independent experiments . Bars and error bars represent mean ± standard error of the mean . * * p < 0 . 01 by two - way ANOVA . B Migration of Cd53 - / - pre - B cells transduced with empty , control , or mutant constructs in response to CXCL12 gradient , 3 days after transduction ( n = 2 - 5 mice per group ) over two independent experiments . Bars and error bars represent mean ± standard error of the mean . * * p < 0 . 01 and * * * p < 0 . 001 by two - way ANOVA .

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 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

Figure 6A Migration of naïve Cd53-/- cultured primary pre-B cells to 100 ng/mL CXCL12 compared to WT littermate controls (n = 5 mice per group) over three independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 by two-way ANOVA.B Migration of Cd53-/- pre-B cells transduced with empty, control, or mutant constructs in response to CXCL12 gradient, 3 days after transduction (n = 2-5 mice per group) over two independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 and ***p < 0.001 by two-way ANOVA.


Figure 1E - F , Candidate gene regions with neurogenesis - associated de novo methylation ( E ) and demethylation ( F ) . Depicted are mean methylation levels per group at individual CpGs within the specified genomic regions . Stars indicate significantly differentially methylated CpGs with q < 0 . 05 ( logistic regression with multiple testing correction using SLIM method ) and methylation difference greater than 20 % .

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Figure 1E-F, Candidate gene regions with neurogenesis-associated de novo methylation (E) and demethylation (F). Depicted are mean methylation levels per group at individual CpGs within the specified genomic regions. Stars indicate significantly differentially methylated CpGs with q < 0.05 (logistic regression with multiple testing correction using SLIM method) and methylation difference greater than 20 %.


Figure 2D , Dnmt3a / b - KO did not affect NPC proliferation . Depicted are data points for every sample with genotype means ± standard errors of the mean ( SEM ) . E , Reduced percentage of dead cells ( Annexin V / propidium iodide ( PI ) double - positive ) in KO cultures at 46 h after start of differentiation . Depicted are data points for every sample with genotype means ± SEM . F - G , NPCs from WT and KO mice differentiated into astrocytes ( GFAP - positive ) and neurons ( Tubb3 - positive ) . Increased numbers of both neurons and astrocytes were generated from KO NPCs . Depicted are data points for every sample with genotype means ± SEM . Scale bar : 100 µm . H , High magnification fluorescent image of differentiated neurons . Scale bar : 20 µm . I , Sholl analysis of Tubb3 - labeled WT and KO neurons ( n = 29 neurons , WT ; n = 30 neurons , KO ) . Reported p - value corresponds to genotype effect from two - way ANOVA . Asterisks highlight data points with p < 0 . 05 after multiple testing adjustment ( Holm method ) of repeated t - tests . Depicted are means ± SEM . J , High magnification fluorescent image of differentiated astrocytes . Scale bar : 20 µm . K , Astrocytes from KO cultures covered a larger area than WT astrocytes . Data points represent means of individual cultures ( n > 30 astrocytes per culture ) . Depicted are data points per sample with genotype means ± SEM .

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Figure 2D, Dnmt3a/b-KO did not affect NPC proliferation. Depicted are data points for every sample with genotype means ± standard errors of the mean (SEM). E, Reduced percentage of dead cells (Annexin V/propidium iodide (PI) double-positive) in KO cultures at 46 h after start of differentiation. Depicted are data points for every sample with genotype means ± SEM. F-G, NPCs from WT and KO mice differentiated into astrocytes (GFAP-positive) and neurons (Tubb3-positive). Increased numbers of both neurons and astrocytes were generated from KO NPCs. Depicted are data points for every sample with genotype means ± SEM. Scale bar: 100 µm.H, High magnification fluorescent image of differentiated neurons. Scale bar: 20 µm. I, Sholl analysis of Tubb3-labeled WT and KO neurons (n = 29 neurons, WT; n = 30 neurons, KO). Reported p-value corresponds to genotype effect from two-way ANOVA. Asterisks highlight data points with p < 0.05 after multiple testing adjustment (Holm method) of repeated t-tests. Depicted are means ± SEM. J, High magnification fluorescent image of differentiated astrocytes. Scale bar: 20 µm. K, Astrocytes from KO cultures covered a larger area than WT astrocytes. Data points represent means of individual cultures (n > 30 astrocytes per culture). Depicted are data points per sample with genotype means ± SEM.


Figure 3H , Dnmt3a / b - dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons . Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS ( left ) and expression fold changes in KO versus WT neurons as determined by qRT - PCR ( right ; n = 4 cultures per genotype ; p - values from Mann - Whitney test ; depicted are data points for every sample with genotype means ± SEM ) .

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Figure 3H, Dnmt3a/b-dependent loss of DNA methylation at neuronal gene candidates was associated with reduced transcription in neurons. Depicted are mean CpG methylation levels at specified genomic regions as determined by RRBS (left) and expression fold changes in KO versus WT neurons as determined by qRT-PCR (right; n = 4 cultures per genotype; p-values from Mann-Whitney test; depicted are data points for every sample with genotype means ± SEM).


Figure 4A , No difference in numbers of Ki67 - positive , proliferating cells was observed between the subgranular zone ( SGZ ) of Dnmt3a / b - WT and Dnmt3a / b - KO mice one week after tamoxifen administration . Depicted are data points for every animal with genotype means ± SEM . B , Bright field image of Ki67 - positive cells stained using diaminobenzidine method . Scale bar : 100 µm . C , KO of Dnmt3a / b did not affect numbers of new - born neurons in the dentate gyrus . Mice were administered with tamoxifen and analyzed 5 weeks after the first injection . BrdU was injected 3 . 5 weeks before analysis . Depicted are data points for every animal with genotype means ± SEM . D , Fluorescence image of BrdU / NeuN - positive new - born neurons . Scale bar : 25 µm .

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Figure 4A, No difference in numbers of Ki67-positive, proliferating cells was observed between the subgranular zone (SGZ) of Dnmt3a/b-WT and Dnmt3a/b-KO mice one week after tamoxifen administration. Depicted are data points for every animal with genotype means ± SEM. B, Bright field image of Ki67-positive cells stained using diaminobenzidine method. Scale bar: 100 µm. C, KO of Dnmt3a/b did not affect numbers of new-born neurons in the dentate gyrus. Mice were administered with tamoxifen and analyzed 5 weeks after the first injection. BrdU was injected 3.5 weeks before analysis. Depicted are data points for every animal with genotype means ± SEM. D, Fluorescence image of BrdU/NeuN-positive new-born neurons. Scale bar: 25 µm.


Figure 5B , Representative fluorescence image of mature and immature neurons . Scale bar : 50 µm . C , KO mice exhibit reduced total numbers of Calb2 - positive cells relative to Dcx - positive cells in the dentate gyrus . F , Representative fluorescence image of the immunostaining used for detection of mature neurons . Scale bar : 100 µm . G , Sholl analysis of Gfp - positive cells revealed reduced dendritic outgrowth of adult - born neurons in KO mice ( n = 20 neurons , WT ; n = 23 neurons , KO ) . Depicted p - value reports genotype effect from two - way ANOVA ( distance to soma : p < 0 . 001 ) . Asterisks highlight data points with p < 0 . 05 after multiple testing correction ( Holm method ) of repeated t - tests . Data points are means ± SEM . Experimental scheme as depicted in Fig . 4E . I , Adult - born neurons of KO mice exhibit reduced numbers of dendritic spines in the outer molecular layer ( n = 32 neurons from 3 mice , WT ; n = 46 neurons from 4 mice , KO ) . Depicted are individual data points for every neuron with genotype means ± SEM ( p - value from unpaired t - test ) . J , Mean spine size per animal was reduced in KO mice ( n = 50 spines per mouse ) . K , Mean percentage of mushroom spines ( diameter of spine head ≥ 0 . 45 µm ) per animal was lower in KO mice compared to WT mice . L , Representative fluorescence image of dendritic spines in WT and KO mice . Scale bar : 5 µm .

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Figure 5B, Representative fluorescence image of mature and immature neurons. Scale bar: 50 µm. C, KO mice exhibit reduced total numbers of Calb2-positive cells relative to Dcx-positive cells in the dentate gyrus. F, Representative fluorescence image of the immunostaining used for detection of mature neurons. Scale bar: 100 µm. G, Sholl analysis of Gfp-positive cells revealed reduced dendritic outgrowth of adult-born neurons in KO mice (n = 20 neurons, WT; n = 23 neurons, KO). Depicted p-value reports genotype effect from two-way ANOVA (distance to soma: p < 0.001). Asterisks highlight data points with p < 0.05 after multiple testing correction (Holm method) of repeated t-tests. Data points are means ± SEM. Experimental scheme as depicted in Fig. 4E. I, Adult-born neurons of KO mice exhibit reduced numbers of dendritic spines in the outer molecular layer (n = 32 neurons from 3 mice, WT; n = 46 neurons from 4 mice, KO). Depicted are individual data points for every neuron with genotype means ± SEM (p-value from unpaired t-test). J, Mean spine size per animal was reduced in KO mice (n = 50 spines per mouse). K, Mean percentage of mushroom spines (diameter of spine head ≥ 0.45 µm) per animal was lower in KO mice compared to WT mice. L, Representative fluorescence image of dendritic spines in WT and KO mice. Scale bar: 5 µm.


Figure 6A - B , Quantification of adult - born ( BrdU - positive ) cells in the dentage gyrus ( DG ) of Dnmt3a / b - KO and WT mice after five weeks or three months of environmental enrichment ( ENR ) or standard housing ( STD ) . BrdU was injected 4 . 5 weeks before analysis . Depicted p - values correspond to genotype effects from two - way ANOVA . Influences of ENR were significant with p < 0 . 01 . Depicted are data points for every animal with group means ± SEM . D , Fluorescence images of c - Fos - positive cells in the DG and CA3 of WT and KO mice . Scale bar : 200 µm . E , Numbers of c - Fos - positive cells in the DG without ( STD ) and with acute stimulation in ENR for 1 h ( n = 2 , WT STD ; n = 2 , KO STD ; n = 5 , WT ENR ; n = 5 , KO ENR ) . F , Numbers of c - Fos - positive cells in suprapyramidal and infrapyramidal blades of the DG after acute stimulation in ENR ( n = 5 mice , WT ; n = 5 mice , KO ) . G , Percentage of new - born neurons ( BrdU / NeuN - positive ) that expressed c - Fos after ENR ( n = 5 mice , WT ; n = 5 mice , KO ) . H , Representative fluorescent image of c - Fos expression in BrdU / NeuN - positive cells in the DG . Scale bar : 25 µm . I , Percentage of c - Fos - positive cells that expressed Dcx in WT and KO mice ( n = 5 mice , WT ; n = 4 mice , KO ) . J , Representative fluorescent image of c - Fos - positive cells and Dcx - positive cells in the DG . Scale bar : 100 µm . L , Fluorescent image of c - Fos expression in Parvalbumin ( Pv ) - positive interneurons in CA3 . Scale bar : 50 µm . M , A higher percentage of Pv - positive interneurons in CA3 expressed c - Fos in KO mice ( n = 4 mice , WT ; n = 4 mice , KO ) .

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Figure 6A-B, Quantification of adult-born (BrdU-positive) cells in the dentage gyrus (DG) of Dnmt3a/b-KO and WT mice after five weeks or three months of environmental enrichment (ENR) or standard housing (STD). BrdU was injected 4.5 weeks before analysis. Depicted p-values correspond to genotype effects from two-way ANOVA. Influences of ENR were significant with p < 0.01. Depicted are data points for every animal with group means ± SEM.D, Fluorescence images of c-Fos-positive cells in the DG and CA3 of WT and KO mice. Scale bar: 200 µm. E, Numbers of c-Fos-positive cells in the DG without (STD) and with acute stimulation in ENR for 1 h (n = 2, WT STD; n = 2, KO STD; n = 5, WT ENR; n = 5, KO ENR). F, Numbers of c-Fos-positive cells in suprapyramidal and infrapyramidal blades of the DG after acute stimulation in ENR (n = 5 mice, WT; n = 5 mice, KO). G, Percentage of new-born neurons (BrdU/NeuN-positive) that expressed c-Fos after ENR (n = 5 mice, WT; n = 5 mice, KO). H, Representative fluorescent image of c-Fos expression in BrdU/NeuN-positive cells in the DG. Scale bar: 25 µm. I, Percentage of c-Fos-positive cells that expressed Dcx in WT and KO mice (n = 5 mice, WT; n = 4 mice, KO). J, Representative fluorescent image of c-Fos-positive cells and Dcx-positive cells in the DG. Scale bar: 100 µm. L, Fluorescent image of c-Fos expression in Parvalbumin (Pv)-positive interneurons in CA3. Scale bar: 50 µm. M, A higher percentage of Pv-positive interneurons in CA3 expressed c-Fos in KO mice (n = 4 mice, WT; n = 4 mice, KO).


Figure 7D , Average time animals needed to reach the platform per day . Note that KO mice showed increased path length and latency on days 4 and 5 . E , Time mice spent on day 5 in the quadrant in which the goal was located during the acquisition phase .

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Figure 7D, Average time animals needed to reach the platform per day. Note that KO mice showed increased path length and latency on days 4 and 5. E, Time mice spent on day 5 in the quadrant in which the goal was located during the acquisition phase.


Figure 1 ( B ) The transcriptional levels of DmATG1 in the third instar larvae were analysed by qRT - PCR . Ribosomal protein 49 ( rp49 ) was used as an internal control ; n = 3 . Bars indicate mean ± s . d . ( C ) Images of the larvae of denoted genotypes at 3 days ( top ) and 6 days ( middle ) after egg ‐ laying ( AEL ) , and quantification of the larvae that developed into the mid - late third instar stage of each genotype ( bottom ) ; n = 3 . Bars indicate mean ± s . d . Fifteen larvae of each genotype were examined in each experiment . ( D ) Suppression of lipid vesicle aggregation in the fat body of dTOR mutants by reduced gene dosage of DmATG1 . The fat body images of the second / early third instar larvae of denoted genotypes under fully fed conditions . ( E ) Images of the larval salivary glands of denoted genotypes . Hoechst33342 ( pseudo - coloured green ) and phalloidin - TRITC ( red ) were used to visualize the nuclei and cell boundary of larval salivary gland cells , respectively . ( F ) Quantitative analysis of cell and nuclear sizes in Fig 1E ; n = 5 . Bars indicate mean ± s . d . DmATG1 , Drosophila Autophagy ‐ specific gene 1 ; dTOR , Drosophila TOR ; qRT - PCR , quantitative real ‐ time reverse transcriptase - PCR .

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Figure 1(B) The transcriptional levels of DmATG1 in the third instar larvae were analysed by qRT-PCR. Ribosomal protein 49 (rp49) was used as an internal control; n=3. Bars indicate mean±s.d.(C) Images of the larvae of denoted genotypes at 3 days (top) and 6 days (middle) after egg‐laying (AEL), and quantification of the larvae that developed into the mid-late third instar stage of each genotype (bottom); n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment.(D) Suppression of lipid vesicle aggregation in the fat body of dTOR mutants by reduced gene dosage of DmATG1. The fat body images of the second/early third instar larvae of denoted genotypes under fully fed conditions.(E) Images of the larval salivary glands of denoted genotypes. Hoechst33342 (pseudo-coloured green) and phalloidin-TRITC (red) were used to visualize the nuclei and cell boundary of larval salivary gland cells, respectively.(F) Quantitative analysis of cell and nuclear sizes in Fig 1E; n=5. Bars indicate mean±s.d. DmATG1, Drosophila Autophagy‐specific gene 1; dTOR, Drosophila TOR; qRT-PCR, quantitative real‐time reverse transcriptase-PCR.


Figure 2 ( A ) Quantification of adults that survived after eclosion for denoted genotypes ; n = 3 . Bars indicate mean ± s . d . Fifteen larvae of each genotype were examined in each experiment . ( B , C ) Activation of dS6K in DmATG1 mutants . Phosphorylation of dS6K was examined by immunoblot analyses using phosphospecific dS6K T398 antibody in the third instar larvae or pupae of denoted genotypes . Tubulinimmunoblot was used as a loading control . Rheb expressing flies ( hs > Rheb ) were used as a positive control ( C ) . hs > Rheb larvae were subjected to heat shock at 37 ° C for 1 h and then incubated at 30 ° C for 3 h before sample preparation to induce Rheb expression . For quantification , the levels of dS6K phosphorylation in each genotype were measured using Adobe Photoshop , and normalized to the tubulin levels . Results are expressed as a fold change compared with the wild ‐ type controls ( w1118 ) . DmATG1 , Drosophila Autophagy ‐ specific gene 1 ; dS6k , Drosophila S6 kinase .

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Figure 2(A) Quantification of adults that survived after eclosion for denoted genotypes; n=3. Bars indicate mean±s.d. Fifteen larvae of each genotype were examined in each experiment.(B,C) Activation of dS6K in DmATG1 mutants. Phosphorylation of dS6K was examined by immunoblot analyses using phosphospecific dS6K T398 antibody in the third instar larvae or pupae of denoted genotypes. Tubulinimmunoblot was used as a loading control. Rheb expressing flies (hs>Rheb) were used as a positive control (C). hs>Rheb larvae were subjected to heat shock at 37°C for 1 h and then incubated at 30°C for 3 h before sample preparation to induce Rheb expression. For quantification, the levels of dS6K phosphorylation in each genotype were measured using Adobe Photoshop, and normalized to the tubulin levels. Results are expressed as a fold change compared with the wild‐type controls (w1118). DmATG1, Drosophila Autophagy‐specific gene 1; dS6k, Drosophila S6 kinase.


Figure 3 ( A , B ) HEK 293T cells transfected with denoted plasmids were ( A ) deprived of nutrients for 90 min , and then replenished with DMEM for 30 min or ( B ) stimulated with 50 ng / ml epidermal growth factor ( EGF ) for 30 min . The S6K immune complexes were analysed by immunoblot using appropriate antibodies ( top three panels ) . Flag ( ATG1α ) blots were completed from the same cell lysates ( bottom panel ) . ( C ) HEK 293T cells transfected with control siRNA , ATG1α siRNA or ATG1β siRNA were deprived of nutrients for 90 min , and then the phosphorylation and protein levels of endogenous S6K ( top two panels ) and S6 ( bottom two panels ) were determined . ( D ) HEK 293T cells transfected with control siRNA or ATG1α siRNA were deprived of nutrients for 90 min , and then replenished with DMEM for 30 min . The phosphorylation and protein levels of endogenous S6K were examined . For quantification , the levels of S6K Thr 389 phosphorylation were measured using Adobe Photoshop , and normalized to the S6K protein levels . Results are expressed as a fold change compared with the first indicated sample . ( E ) HEK293T cells were transfected with control siRNA or ATG1α siRNA with pEGFP ‐ C1 ( a transfection marker , green ) , and nutrient ‐ starved for 90 min , fixed and stained with phosphospecific S6 antibody ( red ) . ATG1 , Autophagy ‐ specific gene 1 ; HA , haemagglutinin ; KI , ATG1α Lys 46 Ile ( kinase ‐ dead ) mutant ; siRNA , short interfering RNA ; S6K , S6 kinase ; WT , wild type .

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Figure 3(A,B) HEK 293T cells transfected with denoted plasmids were (A) deprived of nutrients for 90 min, and then replenished with DMEM for 30 min or (B) stimulated with 50 ng/ml epidermal growth factor (EGF) for 30 min. The S6K immune complexes were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel).(C) HEK 293T cells transfected with control siRNA, ATG1α siRNA or ATG1β siRNA were deprived of nutrients for 90 min, and then the phosphorylation and protein levels of endogenous S6K (top two panels) and S6 (bottom two panels) were determined.(D) HEK 293T cells transfected with control siRNA or ATG1α siRNA were deprived of nutrients for 90 min, and then replenished with DMEM for 30 min. The phosphorylation and protein levels of endogenous S6K were examined. For quantification, the levels of S6K Thr 389 phosphorylation were measured using Adobe Photoshop, and normalized to the S6K protein levels. Results are expressed as a fold change compared with the first indicated sample.(E) HEK293T cells were transfected with control siRNA or ATG1α siRNA with pEGFP‐C1 (a transfection marker, green), and nutrient‐starved for 90 min, fixed and stained with phosphospecific S6 antibody (red). ATG1, Autophagy‐specific gene 1; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase‐dead) mutant; siRNA, short interfering RNA; S6K, S6 kinase; WT, wild type.


Figure 4 ( A , B ) HEK 293T cells were transfected with HA - Akt ( A ) or Myc - RSK ( B ) , and / or Flag - ATG1α WT or KI plasmids . The immunoprecipitated Akt and RSK proteins were analysed by immunoblot using appropriate antibodies ( top three panels ) . Flag ( ATG1α ) blots were completed from the same cell lysates ( bottom panel ) . ( C ) HEK 293T cells were transfected with HA - S6K Thr 389 Glu mutant and / or Flag - ATG1α WT or KI . The immunoprecipitated S6K mutants were analysed by immunoblot using anti ‐ phosphospecific S6K Thr 229 ( top panel ) and HA ( S6K ) antibodies ( middle panel ) . Flag ( ATG1α ) blot was completed from the same cell lysates ( bottom panel ) . ATG1 , Autophagy ‐ specific gene 1 ; EGF , epidermal growth factor ; HA , haemagglutinin ; KI , ATG1α Lys 46 Ile ( kinase ‐ dead ) mutant ; RSK , ribosomal S6 kinase ; WT , wild type .

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Figure 4(A,B) HEK 293T cells were transfected with HA-Akt (A) or Myc-RSK (B), and/or Flag-ATG1α WT or KI plasmids. The immunoprecipitated Akt and RSK proteins were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel).(C) HEK 293T cells were transfected with HA-S6K Thr 389 Glu mutant and/or Flag-ATG1α WT or KI. The immunoprecipitated S6K mutants were analysed by immunoblot using anti‐phosphospecific S6K Thr 229 (top panel) and HA (S6K) antibodies (middle panel). Flag (ATG1α) blot was completed from the same cell lysates (bottom panel). ATG1, Autophagy‐specific gene 1; EGF, epidermal growth factor; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase‐dead) mutant; RSK, ribosomal S6 kinase; WT, wild type.


Figure 1 ( B ) Met - Phe formation monitored upon rapidly mixing initiation complexes ( 80S IC ) with ternary complexes eEF1A - GTP - [ 14C ] Phe - tRNAPhe in the absence ( orange , 0 . 13 ± 0 . 02 s - 1 ) or presence ( cyan , 0 . 15 ± 0 . 02 s - 1 ) of eEF3 ( 2 µM ) in a quench - flow apparatus , and the extent of peptide formation was analyzed by HPLC and radioactivity counting . Data are normalized to Met - Phe formation in the absence of eEF3 with the maximum value in the dataset set to 1 . Data are presented as mean ± SEM of n = 3 biological replicates . ( C ) Met - Phe - Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe - tRNAPhe ( 80S 2C ) with ternary complexes eEF1A - GTP - [ 14C ] Val - tRNAVal in the presence of eEF2 and eEF3 ( green , 0 . 15 ± 0 . 01 s - 1 ) , eEF2 ( red ) or eEF3 ( blue ) . Data presented as mean ± SEM of n = 3 biological replicates . ( D ) Met - Phe - Val formation monitored with 80S 2C incubated with eEF2 and eEF3 ( green , 0 . 4 ± 0 . 14 s - 1 ) , eEF2 ( red , 1 . 0 ± 0 . 5 s - 1 ) or eEF3 ( blue ) , or in the absence of eEF2 and eEF3 ( brown ) for 15 min before adding with ternary complexes eEF1A - GTP - [ 14C ] Val - tRNAVal and monitoring the kinetics of Val incorporation . Data presented as mean ± SEM of n = 3 . Data information : Datasets in ( C ) and ( D ) are normalized to Met - Phe - Val formation in presence of eEF2 and eEF3 with the maximum value set to 1 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Met-Phe formation monitored upon rapidly mixing initiation complexes (80S IC) with ternary complexes eEF1A-GTP-[14C]Phe-tRNAPhe in the absence (orange, 0.13±0.02 s-1) or presence (cyan, 0.15±0.02 s-1) of eEF3 (2 µM) in a quench-flow apparatus, and the extent of peptide formation was analyzed by HPLC and radioactivity counting. Data are normalized to Met-Phe formation in the absence of eEF3 with the maximum value in the dataset set to 1. Data are presented as mean ± SEM of n = 3 biological replicates. (C) Met-Phe-Val formation monitored upon rapidly mixing 80S complexes carrying MetPhe-tRNAPhe (80S 2C) with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green, 0.15±0.01 s-1), eEF2 (red) or eEF3 (blue). Data presented as mean ± SEM of n = 3 biological replicates. (D) Met-Phe-Val formation monitored with 80S 2C incubated with eEF2 and eEF3 (green, 0.4±0.14 s-1), eEF2 (red, 1.0±0.5 s-1) or eEF3 (blue), or in the absence of eEF2 and eEF3 (brown) for 15 min before adding with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal and monitoring the kinetics of Val incorporation. Data presented as mean ± SEM of n = 3. Data information: Datasets in (C) and (D) are normalized to Met-Phe-Val formation in presence of eEF2 and eEF3 with the maximum value set to 1.


Figure 2 ( B ) mRNA - tRNA translocation monitored in a time - resolved Pmn assay upon mixing 80S 2C with Pmn in the absence ( brown triangles ) or presence of eEF2 and eEF3 ( green , 0 . 13 ± 0 . 01 min - 1 ) , eEF2 ( red , 0 . 030 ± 0 . 005 min - 1 ) or eEF3 ( blue , 0 . 10 ± 0 . 01 min - 1 ) . Data are normalized to Met - Phe - Pmn formation in presence of eEF2 with the maximum value set to 1 . Data presented as mean ± SEM of n = 3 . ( C ) Dissociation of deacylated tRNA from the E site by the fluorescence change of tRNAfMet ( Flu ) upon rapidly mixing of 80S 2C with buffer ( grey ) , or with ternary complexes eEF1A - GTP - [ 14C ] Val - tRNAVal in the presence of eEF2 and eEF3 ( green ) , eEF2 ( red ) , eEF3 ( blue ) , or eEF2 and eEF3 without the ternary complex ( magenta ) in a stopped - flow apparatus . Each trace is an average of 5 - 7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction . ( D ) tRNAfMet ( Flu ) co - eluting with 80S 2C incubated without factors ( orange ) , with eEF2 ( red ) , with eEF2 and eEF3 ( magenta ) , or with eEF2 , eEF3 and TC - Val ( green ) for 15 min before loading on a BioSuite 450 size - exclusion column to separate ribosome - bound and - unbound tRNA . Data are normalized to tRNAfMet ( Flu ) bound to 80S 2C in the absence of eEF2 / eEF3 with the maximum value set to 100 % . Data presented as mean ± SEM of n = 3 . ( E ) Dissociation of tRNAfMet ( Flu ) from 80S 2C deacylated tRNA incubated with eEF2 and eEF3 ( green ) or eEF2 ( red ) for 15 min before adding with ternary complexes . Each trace is an average of 5 - 7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction .

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Figure 2(B) mRNA-tRNA translocation monitored in a time-resolved Pmn assay upon mixing 80S 2C with Pmn in the absence (brown triangles) or presence of eEF2 and eEF3 (green, 0.13±0.01 min-1), eEF2 (red, 0.030±0.005 min-1) or eEF3 (blue, 0.10±0.01 min-1). Data are normalized to Met-Phe-Pmn formation in presence of eEF2 with the maximum value set to 1. Data presented as mean ± SEM of n = 3.(C) Dissociation of deacylated tRNA from the E site by the fluorescence change of tRNAfMet(Flu) upon rapidly mixing of 80S 2C with buffer (grey), or with ternary complexes eEF1A-GTP-[14C]Val-tRNAVal in the presence of eEF2 and eEF3 (green), eEF2 (red), eEF3 (blue), or eEF2 and eEF3 without the ternary complex (magenta) in a stopped-flow apparatus. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.(D) tRNAfMet(Flu) co-eluting with 80S 2C incubated without factors (orange), with eEF2 (red), with eEF2 and eEF3 (magenta), or with eEF2, eEF3 and TC-Val (green) for 15 min before loading on a BioSuite 450 size-exclusion column to separate ribosome-bound and -unbound tRNA. Data are normalized to tRNAfMet(Flu) bound to 80S 2C in the absence of eEF2/eEF3 with the maximum value set to 100%. Data presented as mean ± SEM of n = 3.(E) Dissociation of tRNAfMet(Flu) from 80S 2C deacylated tRNA incubated with eEF2 and eEF3 (green) or eEF2 (red) for 15 min before adding with ternary complexes. Each trace is an average of 5-7 individual time courses and normalized at 1 for the fluorescence at the start of the reaction.


Figure 3 ( F ) Codon - specific ribosome occupancies for 21 nt RPFs from WT and eEF3d cells ( two biological replicates ) . CGA and AGG codons are shown in red and purple , respectively . Variance for each dataset is indicated . ( G ) Scatter plot of codon - specific occupancies for 28 nt RPFs comparing eEF3d to WT cells from libraries prepared with CHX + ANS . Trend line ( blue ) and its slope are shown . Codons that encode proline and glycine are colored in purple and green , respectively .

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Figure 3(F) Codon-specific ribosome occupancies for 21 nt RPFs from WT and eEF3d cells (two biological replicates). CGA and AGG codons are shown in red and purple, respectively. Variance for each dataset is indicated.(G) Scatter plot of codon-specific occupancies for 28 nt RPFs comparing eEF3d to WT cells from libraries prepared with CHX+ANS. Trend line (blue) and its slope are shown. Codons that encode proline and glycine are colored in purple and green, respectively.


Figure 116 days after tumor inoculation , B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis . D , Percentage ( left ) and absolute numbers ( right ) of CD8 + T cells in tumors ( n = 4 ) . Each data point represents one mouse . Data are presented as means ± s . d . and analyzed by two - tailed unpaired Student ' s t - test . 16 days after tumor inoculation , B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis . E , Representative plots ( left ) and the percentage ( right ) of IFNγ producing CD8 + T cells ( n = 5 ) . Each data point represents one mouse . Data are presented as means ± s . d . and analyzed by two - tailed unpaired Student ' s t - test . 16 days after tumor inoculation , B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis . G , Percentage ( left ) and absolute numbers ( right ) of Treg cells ( n = 4 ) . Each data point represents one mouse . Data are presented as means ± s . d . and analyzed by two - tailed unpaired Student ' s t - test . 16 days after tumor inoculation , B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis . I , The proportion of Treg cells expressing the indicated surface marker ( n = 5 ) . Each data point represents one mouse . Data are presented as means ± s . d . and analyzed by two - tailed unpaired Student ' s t - test .

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Figure 116 days after tumor inoculation, B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis. D, Percentage (left) and absolute numbers (right) of CD8+ T cells in tumors (n=4). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.16 days after tumor inoculation, B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis. E, Representative plots (left) and the percentage (right) of IFNγ producing CD8+ T cells (n=5). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.16 days after tumor inoculation, B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis. G, Percentage (left) and absolute numbers (right) of Treg cells (n=4). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.16 days after tumor inoculation, B16F10 tumors from WT and Ythdf2 cKO mice were digested for flow cytometry analysis. I, The proportion of Treg cells expressing the indicated surface marker (n=5). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.


Figure 2splenocytes from 1 - year - old WT and Ythdf2 cKO mice were analyzed by flow cytometry ( n = 4 ) . C , Representative plots ( left ) and the percentage ( right ) of naïve ( CD62LhiCD44lo ) , central memory ( cMem , CD62LhiCD44hi ) , and effector memory ( eMem , CD62LloCD44hi ) T cells . Data are presented as means ± s . d . and were analyzed by two - tailed unpaired Student ' s t - test . splenocytes from 1 - year - old WT and Ythdf2 cKO mice were analyzed by flow cytometry ( n = 4 ) . D , Representative plot ( left ) of FOXP3 expression level and the FOXP3 expression MFI ( right ) in splenic Treg cells . Data are presented as means ± s . d . and were analyzed by two - tailed unpaired Student ' s t - test . splenocytes from 1 - year - old WT and Ythdf2 cKO mice were analyzed by flow cytometry ( n = 4 ) . E , Representative plots ( left ) and percentage ( right ) of FOXP3 expressing Treg cells in CD4 T cells . Data are presented as means ± s . d . and were analyzed by two - tailed unpaired Student ' s t - test . splenocytes from 1 - year - old WT and Ythdf2 cKO mice were analyzed by flow cytometry ( n = 4 ) . F , Representative plot ( left ) of FOXP3 Treg cells PD - 1 expression and the MFI value ( right ) in WT and Ythdf2 cKO Treg cells . Data are presented as means ± s . d . and were analyzed by two - tailed unpaired Student ' s t - test .

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Figure 2splenocytes from 1-year-old WT and Ythdf2 cKO mice were analyzed by flow cytometry (n=4). C, Representative plots (left) and the percentage (right) of naïve (CD62LhiCD44lo), central memory (cMem, CD62LhiCD44hi), and effector memory (eMem, CD62LloCD44hi) T cells. Data are presented as means ± s.d. and were analyzed by two-tailed unpaired Student's t-test.splenocytes from 1-year-old WT and Ythdf2 cKO mice were analyzed by flow cytometry (n=4). D, Representative plot (left) of FOXP3 expression level and the FOXP3 expression MFI (right) in splenic Treg cells. Data are presented as means ± s.d. and were analyzed by two-tailed unpaired Student's t-test.splenocytes from 1-year-old WT and Ythdf2 cKO mice were analyzed by flow cytometry (n=4). E, Representative plots (left) and percentage (right) of FOXP3 expressing Treg cells in CD4 T cells. Data are presented as means ± s.d. and were analyzed by two-tailed unpaired Student's t-test.splenocytes from 1-year-old WT and Ythdf2 cKO mice were analyzed by flow cytometry (n=4). F, Representative plot (left) of FOXP3 Treg cells PD-1 expression and the MFI value (right) in WT and Ythdf2 cKO Treg cells. Data are presented as means ± s.d. and were analyzed by two-tailed unpaired Student's t-test.


Figure 3B , Functional assay with WT ( n = 5 ) or Ythdf2 cKO Treg ( n = 5 ) cells isolated from B16F10 tumors on day 16 . B , Percentage of IFNγ producing CD8 + T cells after coculture . Each data point represents one mouse . Data are presented as means ± s . d . and analyzed by two - tailed unpaired Student ' s t - test . E , F , Ratio of Annexin V + Treg cells in the B16F10 tumor ( E , n = 4 ) or spleen ( F , n = 3 ) . Each data point represents one mouse . Data are presented as means ± s . d . and analyzed by two - tailed unpaired Student ' s t - test . G , Representative plots ( left ) and ratio ( right ) of GFP + / GFP - Treg cells in B16F10 tumors from Foxp3CreGFP / + Ythdf2fl / + or Foxp3CreGFP / + Ythdf2fl / fl mice .

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Figure 3B, Functional assay with WT (n=5) or Ythdf2 cKO Treg (n=5) cells isolated from B16F10 tumors on day 16. B, Percentage of IFNγ producing CD8+ T cells after coculture. Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.E, F, Ratio of Annexin V+ Treg cells in the B16F10 tumor (E, n=4) or spleen (F, n=3). Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.G, Representative plots (left) and ratio (right) of GFP+/GFP- Treg cells in B16F10 tumors from Foxp3CreGFP/+Ythdf2fl/+ or Foxp3CreGFP/+Ythdf2fl/fl mice.


Figure 4E , IGV visualization showing m6A and YTHDF2 binding at Nlrc3 , Nfkbie , and Traf3 loci in CD4 + T cells ( GSE188853 ) .

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Figure 4E, IGV visualization showing m6A and YTHDF2 binding at Nlrc3, Nfkbie, and Traf3 loci in CD4+ T cells (GSE188853).


Figure 5B16F10 tumors were developed in mice receiving anti - TNFα or control IgG treatments and harvested on day 15 after tumor injection ( n = 5 , WT + anti - TNFα n = 4 ) . C , Ratio of tumor - infiltrating Treg cells in CD4 + T cells . B16F10 tumors were developed in mice receiving anti - TNFα or control IgG treatments and harvested on day 15 after tumor injection ( n = 5 , WT + anti - TNFα n = 4 ) . D , Percentage of apoptotic Treg cells in the tumor . In C and D , data are presented as means ± s . d . and analyzed by two - tailed unpaired Student ' s t - test . E , Relative mRNA levels of indicated genes in WT or Ythdf2 cKO Treg cells before or after 24 hours TNFα stimulation . Each data point represents one mouse . Data are presented as means ± s . d . and analyzed by two - tailed unpaired Student ' s t - test . F , WT or Ythdf2 cKO Treg cells from the spleen were stimulated with 10 ng / mL TNFα in vitro . 24 hours post TNFα stimulation , cells were treated with Actinomycin D . Decay of Nlrc3 , Nfkbie , and Traf3 was monitored by RT - qPCR ( n = 3 , biological replicates ) . Data are presented as means ± s . d . and analyzed two - way ANOVA . H , Functional assay with WT ( n = 4 ) or Ythdf2 cKO Treg ( n = 5 ) cells isolated from B16F10 tumors on day 16 . H , Percentage of IFNγ producing CD8 T cells after coculture . Each data point represents one mouse . Data are presented as means ± s . d . and analyzed by two - tailed unpaired Student ' s t - test .

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Figure 5B16F10 tumors were developed in mice receiving anti-TNFα or control IgG treatments and harvested on day 15 after tumor injection (n=5, WT + anti-TNFα n=4). C, Ratio of tumor-infiltrating Treg cells in CD4+ T cells.B16F10 tumors were developed in mice receiving anti-TNFα or control IgG treatments and harvested on day 15 after tumor injection (n=5, WT + anti-TNFα n=4). D, Percentage of apoptotic Treg cells in the tumor. In C and D, data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.E, Relative mRNA levels of indicated genes in WT or Ythdf2 cKO Treg cells before or after 24 hours TNFα stimulation. Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.F, WT or Ythdf2 cKO Treg cells from the spleen were stimulated with 10 ng/mL TNFα in vitro. 24 hours post TNFα stimulation, cells were treated with Actinomycin D. Decay of Nlrc3, Nfkbie, and Traf3 was monitored by RT-qPCR (n=3, biological replicates). Data are presented as means ± s.d. and analyzed two-way ANOVA.H, Functional assay with WT (n=4) or Ythdf2 cKO Treg (n=5) cells isolated from B16F10 tumors on day 16. H, Percentage of IFNγ producing CD8 T cells after coculture. Each data point represents one mouse. Data are presented as means ± s.d. and analyzed by two-tailed unpaired Student's t-test.


Figure 1 ( A ) H1299 cells were transfected with Myc - β - catenin , CK1α and MDMX mutants . Phosphorylation of β - catenin S45 was determined by western blot . Co - expression of MDMX blocked the increase of pS45 level . MDMXSG ( W200S / W201G ) and MDMX - 1 - 300 were defective for CK1α binding . ( B ) GST - β - catenin - 1 - 200 was incubated with purified FLAG - CK1α , FLAG - MDMX and ATP at indicated ratios . Phosphorylation of β - catenin S45 was determined by western blot . MDMXS289A contains mutation of CK1α phosphorylation site . ( C ) Kinetic characterization of CK1α inhibition by MDMX . Reaction velocity as a function of β - catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α . Data were fitted to the Michaelis - Menten equation . The resulting parameter values are shown in the table . ( D ) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α . Data were fitted to the Michaelis - Menten equation .

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Figure 1(A) H1299 cells were transfected with Myc-β-catenin, CK1α and MDMX mutants. Phosphorylation of β-catenin S45 was determined by western blot. Co-expression of MDMX blocked the increase of pS45 level. MDMXSG (W200S/W201G) and MDMX-1-300 were defective for CK1α binding.(B) GST-β-catenin-1-200 was incubated with purified FLAG-CK1α, FLAG-MDMX and ATP at indicated ratios. Phosphorylation of β-catenin S45 was determined by western blot. MDMXS289A contains mutation of CK1α phosphorylation site.(C) Kinetic characterization of CK1α inhibition by MDMX. Reaction velocity as a function of β-catenin peptide substrate and increasing MDMX concentrations at 100 µM ATP and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation. The resulting parameter values are shown in the table.(D) Reaction velocity as a function of ATP and increasing MDMX concentrations at 800 µM peptide substrate and 2 nM CK1α. Data were fitted to the Michaelis-Menten equation.


Figure 2 ( A , B ) H1299 cells and human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX and analyzed by western blot . ( C ) H1299 cells were transiently transfected with CK1α plasmid and analyzed for β - catenin phosphorylation by western blot . ( D ) Cytoplasmic and nuclear fractions of H1299 cells stably expressing MDMX were analyzed for indicated markers . ( E ) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β - catenin S45 kinase activity in a titration assay . ( F ) Extracts of H1299 cells separately transfected with MDMX or CK1α were mixed in the presence of CK1α inhibitors . In vitro formation of MDMX - CK1α complex was detected by MDMX IP followed by CK1α western blot .

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Figure 2(A, B) H1299 cells and human foreskin fibroblasts HFF were stably infected with lentivirus expressing MDMX and analyzed by western blot.(C) H1299 cells were transiently transfected with CK1α plasmid and analyzed for β-catenin phosphorylation by western blot.(D) Cytoplasmic and nuclear fractions of H1299 cells stably expressing MDMX were analyzed for indicated markers.(E) Extract of H1299 expressing MDMX at indicated amounts were analyzed for β-catenin S45 kinase activity in a titration assay.(F) Extracts of H1299 cells separately transfected with MDMX or CK1α were mixed in the presence of CK1α inhibitors. In vitro formation of MDMX-CK1α complex was detected by MDMX IP followed by CK1α western blot.


Figure 3 ( A , B , C , D ) Cells were treated with MDMX siRNA for 48 hrs and analyzed for β - catenin pS45 level by western blot . ( E ) H1299 and 293T cells were treated with indicated siRNA for 48 hrs . The activity of β - catenin S45 kinase in cell extracts was determined using an ELISA assay . The results are average of 3 experiments ( mean + / - SD ) . * p < 0 . 05 ( Student ' s t test ) .

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Figure 3(A, B, C, D) Cells were treated with MDMX siRNA for 48 hrs and analyzed for β-catenin pS45 level by western blot.(E) H1299 and 293T cells were treated with indicated siRNA for 48 hrs. The activity of β-catenin S45 kinase in cell extracts was determined using an ELISA assay. The results are average of 3 experiments (mean +/- SD). *p<0.05 (Student's t test).


Figure 4 ( B GST - β - catenin - 1 - 200 was incubated with ATP , CK1α and GST - MDMX mutants at indicated ratios . The level of pS45 was determined by western blot . C , D ) GST - β - catenin - 1 - 200 was incubated with ATP , CK1α and GST - MDMX mutants at indicated ratios . The level of pS45 was determined by western blot .

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Figure 4(B GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.C, D) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX mutants at indicated ratios. The level of pS45 was determined by western blot.


Figure 5 ( B ) Purified FLAG - CK1α was incubated with ATP , GST - MDMX , GST - β - catenin - 1 - 200 , pDI peptide ( LTFEHYWAQLTS , 5 µM ) , and pDI3A peptide ( LTAEHYAAQATS , 5 µM ) . β - catenin pS45 level was determined by western blot . ( C ) H1299 cells were cotransfected with MDMX , CK1α and p53 mutants . MDMX - CK1α binding was determined by IP - western blot . ( D ) Endogenous p53 in A549 ( wt ) and Panc1 ( R273H ) cells were depleted by CRISPR / Cas9 knockout . β - catenin pS45 level was determined by western blot . ( E ) The β - catenin S45 kinase activity in A549 and Panc1 cells was

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Figure 5(B) Purified FLAG-CK1α was incubated with ATP, GST-MDMX, GST-β-catenin-1-200, pDI peptide (LTFEHYWAQLTS, 5 µM), and pDI3A peptide (LTAEHYAAQATS, 5 µM). β-catenin pS45 level was determined by western blot.(C) H1299 cells were cotransfected with MDMX, CK1α and p53 mutants. MDMX-CK1α binding was determined by IP-western blot.(D) Endogenous p53 in A549 (wt) and Panc1 (R273H) cells were depleted by CRISPR/Cas9 knockout. β-catenin pS45 level was determined by western blot.(E) The β-catenin S45 kinase activity in A549 and Panc1 cells was determined


Figure 6 ( A ) H1299 cells were transfected with MDMX and CK1α mutants . MDMX - CK1α binding was determined by IP - western blot . ( B ) GST - β - catenin - 1 - 200 was incubated with ATP , CK1α and GST - MDMX . The level of pS45 was determined by western blot . ( C ) GST - β - catenin - 1 - 200 was incubated with identical amounts of purified FLAG - CK1α mutants and ATP . The level of pS45 was determined by western blot . ( D ) . ELISA plates coated with GST - β - catenin - 1 - 200 was incubated with purified FLAG - CK1α mutants , ATP , and MDMX . The level of pS45 was determined by ELISA assay . The results are average of 3 experiments ( mean + / - SD ) .

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Figure 6(A) H1299 cells were transfected with MDMX and CK1α mutants. MDMX-CK1α binding was determined by IP-western blot.(B) GST-β-catenin-1-200 was incubated with ATP, CK1α and GST-MDMX. The level of pS45 was determined by western blot.(C) GST-β-catenin-1-200 was incubated with identical amounts of purified FLAG-CK1α mutants and ATP. The level of pS45 was determined by western blot.(D). ELISA plates coated with GST-β-catenin-1-200 was incubated with purified FLAG-CK1α mutants, ATP, and MDMX. The level of pS45 was determined by ELISA assay. The results are average of 3 experiments (mean +/- SD).


Figure 7 ( A ) MEF cell lines derived from 3 p53 - / - ; MDMXSG / SG and 3 p53 - / - ; MDMXwt / wt embryos were analyzed for β - catenin pS45 level by western blot . ( B ) CK1α activity in MEF cell extracts was determined using ELISA kinase activity assay . The results are average of 3 experiments ( mean + / - SD ) . * p < 0 . 01 ( Student ' s t test ) . ( C ) Volcano plot of differential gene expression between p53 - / - ; MDMXSG / SG and p53 - / - ; MDMXwt / wt MEF lines . ( D ) Multiple Wnt signaling genes were down regulated in p53 - / - ; MDMXSG / SG MEF cell lines . ( E ) RT - PCR confirmation of Wnt pathway genes down - regulated in p53 - / - ; MDMXSG / SG MEF ( average of 3 cell lines , mean + / - SD ) and thymus from 1 month old mice ( average of 3 mice , mean + / - SD ) . * * p < 0 . 01 , * p < 0 . 05 ( Student ' s t test ) . ( F ) p53 - / - ; MDMXSG / SG and p53 - / - ; MDMXwt / wt cells were treated with conditioned medium containing Wnt3a . Axin2 mRNA level was determined by RT - PCR ( average of 3 experiments , mean + / - SD ) . * p < 0 . 05 ( Student ' s t test ) .

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Figure 7(A) MEF cell lines derived from 3 p53-/-;MDMXSG/SG and 3 p53-/-;MDMXwt/wt embryos were analyzed for β-catenin pS45 level by western blot.(B) CK1α activity in MEF cell extracts was determined using ELISA kinase activity assay. The results are average of 3 experiments (mean +/- SD). *p<0.01 (Student's t test).(C) Volcano plot of differential gene expression between p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt MEF lines.(D) Multiple Wnt signaling genes were down regulated in p53-/-;MDMXSG/SG MEF cell lines.(E) RT-PCR confirmation of Wnt pathway genes down-regulated in p53-/-;MDMXSG/SG MEF (average of 3 cell lines, mean +/- SD) and thymus from 1 month old mice (average of 3 mice, mean +/- SD). **p<0.01, *p<0.05 (Student's t test).(F) p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt cells were treated with conditioned medium containing Wnt3a. Axin2 mRNA level was determined by RT-PCR (average of 3 experiments, mean +/- SD). *p<0.05 (Student's t test).


Figure 8 ( A ) Identical starting number of cells from the MEF lines were serially passaged following identical schedules . The differences in cell number were determined after 3 and 5 passages . The results are average of 3 experiments ( mean + / - SD ) . ( B ) Identical number of cells from the MEF lines were plated at low density and cultured for 14 days . Colony formation efficiency was determined by crystal violate staining . ( C ) Survival curves of p53 - / - ; MDMXSG / SG and p53 - / - ; MDMXwt / wt mice . Thymic lymphomas were the major cause of mortality in both genotypes .

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Figure 8(A) Identical starting number of cells from the MEF lines were serially passaged following identical schedules. The differences in cell number were determined after 3 and 5 passages. The results are average of 3 experiments (mean +/- SD).(B) Identical number of cells from the MEF lines were plated at low density and cultured for 14 days. Colony formation efficiency was determined by crystal violate staining.(C) Survival curves of p53-/-;MDMXSG/SG and p53-/-;MDMXwt/wt mice. Thymic lymphomas were the major cause of mortality in both genotypes.


Figure 1D . The morphology of oocytes and embryos from female patients with MOS variants . The day of oocyte retrieval was defined as day 0 . Polar bodies were indicated by black arrows . Scale bar = 20 µm .

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Figure 1D. The morphology of oocytes and embryos from female patients with MOS variants. The day of oocyte retrieval was defined as day 0. Polar bodies were indicated by black arrows. Scale bar=20 µm.


Figure 2B . The immunofluorescence images show the pERK1 / 2 ( red ) dynamics in immature ( GV , n = 5 ) , mature ( MII , n = 10 ) , and fertilized oocytes from unidentified control patients ( n = 6 ) , as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants . pERK1 / 2 activation was inhibited after U0126 treatment ( n = 6 ) . FITC - α - tubulin ( Green ) and DAPI ( blue ) were used for co - staining . Scale bar = 10 µm . C . Western blot analysis of the MEK1 / 2 and ERK1 / 2 activation after transfection of FLAG - tagged wild - type MOS and identified MOS variants in HEK 293 cells . F . Immunofluorescence of FLAG ( green ) and pERK1 / 2 ( grey ) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs . The signal of mCherry was directly captured after fixation , with DAPI staining for DNA visualization ( blue ) . ( n = over 30 oocytes in each group ) . Scale bar = 10 µm .

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Figure 2B. The immunofluorescence images show the pERK1/2 (red) dynamics in immature (GV, n=5), mature (MII, n=10), and fertilized oocytes from unidentified control patients (n=6), as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants. pERK1/2 activation was inhibited after U0126 treatment (n=6). FITC-α-tubulin (Green) and DAPI (blue) were used for co-staining. Scale bar=10 µm.C. Western blot analysis of the MEK1/2 and ERK1/2 activation after transfection of FLAG-tagged wild-type MOS and identified MOS variants in HEK 293 cells.F. Immunofluorescence of FLAG (green) and pERK1/2 (grey) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs. The signal of mCherry was directly captured after fixation, with DAPI staining for DNA visualization (blue). (n=over 30 oocytes in each group). Scale bar=10 µm.


Figure 3A . Immunofluorescence of FLAG ( red ) and F - actin ( gray ) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild - type human MOS or MOS variants mRNAs and culture in medium with 2 . 5 µM milrinone for 24 h , followed by release to maturation ( n = 15 - 20 oocytes each group ) . Scale bar = 10 µm . C . The immunofluorescence of F - actin ( red ) and TPX2 ( green ) in WT and Erk1 / 2oo - / - oocytes , using DAPI ( blue ) co - staining ( n = 15 - 20 oocytes for each group ) . The zoomed images of F - actin are displayed in gray . Scale bar = 10 µm . D . The box plot summarizing the relative intensities of F - actin between control and ERK1 / 2oo - / - oocytes ( n = 7 - 10 oocytes for each group ) . E . Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1 / 2oo - / - mice . The MII oocyte , zygote , 2 - cell and blastocyst embryos were harvested in vivo at 14 , 24 , 48 , and 96 h post - hCG , respectively ( n = 3 mice for each timepoints in two groups ) . Scale bar = 100 µm . F , G . Bar graphs shows the blastocyst percentage ( F ) and fragment percentage ( G ) in wild - type ( n = 51 ) and maternal ERK1 / 2 deletion embryos ( n = 63 ) .

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Figure 3A. Immunofluorescence of FLAG (red) and F-actin (gray) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild-type human MOS or MOS variants mRNAs and culture in medium with 2.5 µM milrinone for 24 h, followed by release to maturation (n=15-20 oocytes each group). Scale bar= 10 µm.C. The immunofluorescence of F-actin (red) and TPX2 (green) in WT and Erk1/2oo-/- oocytes, using DAPI (blue) co-staining (n=15-20 oocytes for each group). The zoomed images of F-actin are displayed in gray. Scale bar=10 µm. D. The box plot summarizing the relative intensities of F-actin between control and ERK1/2oo-/- oocytes (n=7-10 oocytes for each group).E. Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1/2oo-/- mice. The MII oocyte, zygote, 2-cell and blastocyst embryos were harvested in vivo at 14, 24, 48, and 96 h post-hCG, respectively (n=3 mice for each timepoints in two groups). Scale bar=100 µm. F, G. Bar graphs shows the blastocyst percentage (F) and fragment percentage (G) in wild-type (n=51) and maternal ERK1/2 deletion embryos (n=63).


Figure 4H . The bar graphs show the expression levels of selected MOS - regulated decay genes ( n = 2 or 3 biological replicates ) .

Figure 4H. The bar graphs show the expression levels of selected MOS-regulated decay genes (n=2 or 3 biological replicates).


Figure 5H . Heatmap showing the expression level of selected maternal genes , mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1 / 2 inactivation .

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Figure 5H. Heatmap showing the expression level of selected maternal genes, mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1/2 inactivation.


Figure 6A . The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO ( control , Ctrl , n = 11 ) or 20 uM U0126 treatment ( n = 12 ) . B . The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO ( control , Ctrl , n = 19 ) or 20 uM U0126 ( n = 17 ) . CC . The representative images showing the JC - 1 monomers ( green ) and aggregates ( red ) of mouse oocytes after treatment with DMSO ( control , Ctrl , n = 34 ) or U0126 ( n = 40 ) in in vitro culture medium for 16 h . Scale bar = 100 µm . D . The bar graph of ratio of intensities of JC - 1 aggregates to those of monomers in ( C ) . The intensities of JC - 1 were quantified by Image J software . The oocytes number used was labeled . EE . Representative images displaying JC - 1 monomers ( green ) , aggregates ( red ) and bright filed images of GV and MII oocytes from wild - type ( WT ) and Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big and multiple polar bodies . Scale bar = 100 µm . F . Confocal images of JC - 1 monomers ( green ) and aggregates ( red ) of GV and MII oocytes from wild - type ( WT ) and Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big and multiple polar bodies , indicated by red arrows . Hoechst 33342 ( blue ) was co - stained to represent nucleus or chromosome . Scale bar = 10 µm .

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Figure 6A. The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO (control, Ctrl, n=11) or 20 uM U0126 treatment (n=12). B. The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO (control, Ctrl, n=19) or 20 uM U0126 (n=17). CC. The representative images showing the JC-1 monomers (green) and aggregates (red) of mouse oocytes after treatment with DMSO (control, Ctrl, n=34) or U0126 (n=40) in in vitro culture medium for 16 h. Scale bar=100 µm. D. The bar graph of ratio of intensities of JC-1 aggregates to those of monomers in (C). The intensities of JC-1 were quantified by Image J software. The oocytes number used was labeled. EE. Representative images displaying JC-1 monomers (green), aggregates (red) and bright filed images of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies. Scale bar=100 µm.F. Confocal images of JC-1 monomers (green) and aggregates (red) of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies, indicated by red arrows. Hoechst 33342 (blue) was co-stained to represent nucleus or chromosome. Scale bar=10 µm.