( A - C ) SPT - QD of pHluorin tagged AMPA ( GluA1 and GluA2 ) and NMDA ( GluN1 and GluN2B ) receptor subunits using GFP - antibody pre - coupled to QD655 . Primary neurons were exposed or not to Fib - Tau ( 0 . 36nM , 60 min ) . Cumulative frequency of diffusion coefficient of all trajectories ( extracted from MSD plot , inset , A ) show a slow - down in diffusion of GluA2 - subunit but not GluA1 , GluN1 and GluN2B subunits ( A ) . To compute statistical differences , the diffusion coefficient of all trajectories per movie ( from 3 independent experiments ) were averaged ( B ) . To measure statistical differences in MSD , the explored area ( area under curve during 600 - 900ms of MSD plot ) per movie ( from 3 independent experiments ) were averaged ( C ) . Decreased diffusion coefficient and area explored for GluA2 - AMPA was observed indicating increased confinement concomitant with increased clustering ( Fig 5C ) following exposure to Fib - Tau . Mann - Whitney - test , n is number of movies ( left to right ) : 14 , 15 , 14 , 13 , 15 , 15 , 15 , and 15 . * * p < 0 . 01 ; ns = not significant .

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(A-C) SPT-QD of pHluorin tagged AMPA (GluA1 and GluA2) and NMDA (GluN1 and GluN2B) receptor subunits using GFP-antibody pre-coupled to QD655. Primary neurons were exposed or not to Fib-Tau (0.36nM, 60 min). Cumulative frequency of diffusion coefficient of all trajectories (extracted from MSD plot, inset, A) show a slow-down in diffusion of GluA2-subunit but not GluA1, GluN1 and GluN2B subunits (A). To compute statistical differences, the diffusion coefficient of all trajectories per movie (from 3 independent experiments) were averaged (B). To measure statistical differences in MSD, the explored area (area under curve during 600-900ms of MSD plot) per movie (from 3 independent experiments) were averaged (C). Decreased diffusion coefficient and area explored for GluA2-AMPA was observed indicating increased confinement concomitant with increased clustering (Fig 5C) following exposure to Fib-Tau. Mann-Whitney-test, n is number of movies (left to right): 14, 15, 14, 13, 15, 15, 15, and 15. **p<0.01; ns= not significant.


( A ) Single Particle Tracking using PALM ( SPT - PALM ) was performed on α3 - NKA - Dendra expressing primary neurons . A representative image shows an epifluorescence image and trajectories ( strands of different colors ) of minimum 5 points in length . Scale bars : 1 µm in panel ( A )

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(A) Single Particle Tracking using PALM (SPT-PALM) was performed on α3-NKA-Dendra expressing primary neurons. A representative image shows an epifluorescence image and trajectories (strands of different colors) of minimum 5 points in length. Scale bars: 1 µm in panel (A)


( B , C ) Extraction of diffusion coefficient using mean squared displacement ( MSD ) approach using minimum 5 - point trajectory length . Spine and shaft were visually identified . An upward shift of MSD curve , indicating reduced confinement was observed following exposure to Fib - Tau ( 0 . 36nM , 60 min ) ( B ) . As expected , an increased diffusion coefficient ( i . e . increased mobility ) ( C ) of α3 - NKA is observed in dendritic shaft following exposure to Fib - Tau . Mann - Whitney - test , n = 21 cells , obtained from 4 independent experiments ; Each dot represents average diffusion coefficient of all trajectories value per cell . Data information : * p < 0 . 05 ; ns = not significant .

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(B, C) Extraction of diffusion coefficient using mean squared displacement (MSD) approach using minimum 5-point trajectory length. Spine and shaft were visually identified. An upward shift of MSD curve, indicating reduced confinement was observed following exposure to Fib-Tau (0.36nM, 60 min) (B). As expected, an increased diffusion coefficient (i.e. increased mobility) (C) of α3-NKA is observed in dendritic shaft following exposure to Fib-Tau. Mann-Whitney-test, n=21 cells, obtained from 4 independent experiments; Each dot represents average diffusion coefficient of all trajectories value per cell. Data information: *p<0.05; ns= not significant.


Extraction of the number of diffusive states of α3 - NKA and the transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model The occupancy time of α3 - NKA in bound - state also decreased whereas that in free - state increased upon neuron exposure to Fib - Tau ( E ) . Box - plot represents : median , interquartile range and entire distribution , Mann - Whitney - test on averaged value per movie from 4 experiments ( n = 21 cells ) . Data information : * p < 0 . 05 ; ns = not significant .

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Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model The occupancy time of α3-NKA in bound-state also decreased whereas that in free-state increased upon neuron exposure to Fib-Tau (E). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


Extraction of the number of diffusive states of α3 - NKA and the transition rates between these diffusive states using thousands of short ( minimum 2 - point ) trajectories using Hidden Markov Model Dwell - time of α3 - NKA is also reduced in bound state indicating destabilization of native α3 - NKA - complexes following interaction with Fib - Tau ( F ) . Box - plot represents : median , interquartile range and entire distribution , Mann - Whitney - test on averaged value per movie from 4 experiments ( n = 21 cells ) . Data information : * p < 0 . 05 ; ns = not significant .

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Extraction of the number of diffusive states of α3-NKA and the transition rates between these diffusive states using thousands of short (minimum 2-point) trajectories using Hidden Markov Model Dwell-time of α3-NKA is also reduced in bound state indicating destabilization of native α3-NKA-complexes following interaction with Fib-Tau (F). Box-plot represents: median, interquartile range and entire distribution, Mann-Whitney-test on averaged value per movie from 4 experiments (n=21 cells). Data information: *p<0.05; ns= not significant.


( G - H ) Cell - surface biotinylation on primary neurons exposed or not to Fib - Tau ( 1 . 8nM , 60 min ) . The amount of α - NKA at cell surface decreased upon exposure of neurons to Fib - Tau ( G ) . Quantification was performed by normalizing the western blot band intensity with input . Paired - t - test , 3 independent experiments . Data information : * p < 0 . 05 ; ns = not significant .

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(G-H) Cell-surface biotinylation on primary neurons exposed or not to Fib-Tau (1.8nM, 60 min). The amount of α-NKA at cell surface decreased upon exposure of neurons to Fib-Tau (G). Quantification was performed by normalizing the western blot band intensity with input. Paired-t-test, 3 independent experiments. Data information: *p<0.05; ns= not significant.


( I ) Paraformaldehyde fixation α3 - NKA - pHluorin ( green , top row ) reveals endocytic spots otherwise not visible in live neurons ( green , bottom row ) . Many Fib - Tau - ATTO647 ( red ) clusters overlapped ( arrow ) with α3 - NKA - pHluorin spots ( exposure , 0 . 36nM , 60 min ) . Scale bars

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(I) Paraformaldehyde fixation α3-NKA-pHluorin (green, top row) reveals endocytic spots otherwise not visible in live neurons (green, bottom row). Many Fib-Tau-ATTO647 (red) clusters overlapped (arrow) with α3-NKA-pHluorin spots (exposure, 0.36nM, 60 min). Scale bars 2µm


( J ) The majority of the overlapping Fib - Tau - ATTO647 clusters ( red ) and α3 - NKA - pHluorin spots ( green ) localized on top of Clathrin - mRFP ( blue ) puncta . Scale bars :

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(J) The majority of the overlapping Fib-Tau-ATTO647 clusters (red) and α3-NKA-pHluorin spots (green) localized on top of Clathrin-mRFP (blue) puncta. Scale bars: 2µm


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn ( shown here in green ) or unlabeled α - Syn A concentration - dependent increase in the binding and clustering of α - Syn fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α -

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

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn (shown here in green) or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of α-Syn fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing Aβ ( given in monomer - equivalent ) of Aβ ( B ) fibrils [ in total 5 - experiments were performed with either ATTO647 labeled Aβ ( shown here in green ) or unlabeled Aβ fibrils ] . concentration - dependent increase in the binding and clustering of Aβ fibrils is seen . Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α - Syn , but not Aβ

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DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing Aβ (given in monomer-equivalent) of Aβ (B) fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ (shown here in green) or unlabeled Aβ fibrils]. concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing concentrations ( given in monomer - equivalent ) of α - Syn fibrils [ in total 5 - experiments were performed with either ATTO647 labeled α - Syn or unlabeled α - Syn A concentration - dependent increase in the binding and clustering of both α - Syn fibrils is seen . One - Way ANOVA with Dunnett ' s post - hoc test was performed to compare the different condition to the reference condition ( 100nM ) . The number of images analyzed is 27 , 25 , 25 , 26 and 28 in ( C ) , from left to right . The data originate from 3 to 4 independent experiments . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of α-Syn fibrils [in total 5-experiments were performed with either ATTO647 labeled α-Syn or unlabeled α-Syn A concentration-dependent increase in the binding and clustering of both α-Syn fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 27, 25, 25, 26 and 28 in (C) , from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


DIV21 neurons were co - exposed to fixed concentration of Fib - Tau ( 0 . 36nM , 60min , ATTO550 labelled , red ) and increasing concentrations ( given in monomer - equivalent ) of Aβ fibrils [ in total 5 - experiments were performed with either ATTO647 labeled Aβ or unlabeled Aβ fibrils A concentration - dependent increase in the binding and clustering of Aβ fibrils is seen . One - Way ANOVA with Dunnett ' s post - hoc test was performed to compare the different condition to the reference condition ( 100nM ) . The number of images analyzed is 18 , 50 , 50 , 50 , 25 in ( D ) , from left to right . The data originate from 3 to 4 independent experiments . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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

DIV21 neurons were co-exposed to fixed concentration of Fib-Tau (0.36nM, 60min, ATTO550 labelled, red) and increasing concentrations (given in monomer-equivalent) of Aβ fibrils [in total 5-experiments were performed with either ATTO647 labeled Aβ or unlabeled Aβ fibrils A concentration-dependent increase in the binding and clustering of Aβ fibrils is seen. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the different condition to the reference condition (100nM).The number of images analyzed is 18, 50, 50, 50, 25 in (D), from left to right. The data originate from 3 to 4 independent experiments. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


Notably , Fib - Tau clustering dramatically increased with increasing concentrations of α - Syn , but not Aβ fibrils The quantification in ( E ) includes all 5 - experiments ( n = number of images analyzed , left to right : 58 , 54 , 50 , 62 , 63 , 65 , 43 , 35 , 60 , 60 , 60 , 35 ; One - Way ANOVA with Dunnette ' s post - hoc test was performed to compare the size of Fib - Tau clusters ) . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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

Notably, Fib-Tau clustering dramatically increased with increasing concentrations of α-Syn, but not Aβ fibrils The quantification in (E) includes all 5-experiments (n = number of images analyzed, left to right: 58, 54, 50, 62, 63, 65, 43, 35, 60, 60, 60, 35; One-Way ANOVA with Dunnette's post-hoc test was performed to compare the size of Fib-Tau clusters). Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


A perfect co - localization between Fib - Tau and α - Syn clusters is also observed . The proportion of Fib - Tau clusters co - localizing with α - Syn fibrils increased with an increasing concentration of α - Syn fibrils reaching 60 - 70 % overlap A weaker , concentration independent , overlap ( 10 - 20 % ) was observed with Aβ fibrils The quantification in ( F ) is derived from 3 to 4 independent experiments . The number of images analyzed is 27 , 25 , 25 , 26 , 28 for α - Syn and 18 , 50 , 50 , 50 , 25 for Aβ from left to right . One - Way ANOVA with Dunnett ' s post - hoc test was performed to compare the co - localization differences from 100nM concentration . Box - plot represents : median , interquartile range and 10 - 90 % distribution . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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A perfect co-localization between Fib-Tau and α-Syn clusters is also observed. The proportion of Fib-Tau clusters co-localizing with α-Syn fibrils increased with an increasing concentration of α-Syn fibrils reaching 60-70% overlap A weaker, concentration independent, overlap (10-20%) was observed with Aβ fibrils The quantification in (F) is derived from 3 to 4 independent experiments. The number of images analyzed is 27, 25, 25, 26, 28 for α-Syn and 18, 50, 50, 50, 25 for Aβ from left to right. One-Way ANOVA with Dunnett's post-hoc test was performed to compare the co-localization differences from 100nM concentration. Box-plot represents: median, interquartile range and 10-90% distribution. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


( G - I ) Primary neurons were co - exposed to Fib - Tau ( 0 . 36nM , ATTO550 labelled ) and Fib - α - Syn ( 50nM , monomer - equivalent , unlabeled ) for 60min and immunolabelled for homer ( G ) or α3 - NKA ( H ) . Quantification of Fib - Tau clusters co - localized with homer or α3 - NKA ( I ) . Note an increased co - localization of Fib - Tau with homer in presence of Fib - α - Syn . Data information : Box - plot represents : median , interquartile range and 10 - 90 % distribution , Mann - Whitney - test is performed , n = 40 images from 4 - experiments , Scale bars , 5µm . * p < 0 . 05 , * * * p < 0 . 001 ; ns = not significant .

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(G-I) Primary neurons were co-exposed to Fib-Tau (0.36nM, ATTO550 labelled) and Fib-α-Syn (50nM, monomer-equivalent, unlabeled) for 60min and immunolabelled for homer (G) or α3-NKA (H). Quantification of Fib-Tau clusters co-localized with homer or α3-NKA (I). Note an increased co-localization of Fib-Tau with homer in presence of Fib-α-Syn. Data information: Box-plot represents: median, interquartile range and 10-90% distribution, Mann-Whitney-test is performed, n=40 images from 4-experiments, Scale bars, 5µm. *p<0.05, ***p<0.001; ns= not significant.


( B ) The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . Key residues involved in stabilizing the templating base ( Y505 and R517 ) are shown in black .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(B) The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). Key residues involved in stabilizing the templating base (Y505 and R517) are shown in black.


( C ) ( Left panel ) 8 - oxo - dG ( green ) preferentially adopts the pro - mutagenic syn - conformation . A simulated annealing Fo - Fc omit density map is shown for 8 - oxo - dG ( contoured at 3σ ) . ( Right panel ) 8 - oxo - dG ( anti ) ( black , modeled using 2PFO ) was overlayed with the Fo - Fc omit density map . In the anti - conformation , the C8 - carbonyl would directly clash with the 5 ′ - phosphate ( red - dotted line ) .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) (Left panel) 8-oxo-dG (green) preferentially adopts the pro-mutagenic syn-conformation. A simulated annealing Fo-Fc omit density map is shown for 8-oxo-dG (contoured at 3σ). (Right panel) 8-oxo-dG(anti) (black, modeled using 2PFO)was overlayed with the Fo-Fc omit density map. In the anti-conformation, the C8-carbonyl would directly clash with the 5′-phosphate (red-dotted line).


( D ) 8 - oxo - dG ( syn ) ( green ) is stabilized by multiple interactions , including Tyr505 and Arg517 ( grey ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(D) 8-oxo-dG(syn) (green) is stabilized by multiple interactions, including Tyr505 and Arg517 (grey).


( B ) Overview of the 8 - oxo - dG : dATP insertion complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . The primer terminal ddC and the incoming ddCTP are shown in cyan and magenta , respectively . Key residues that form the nascent base pair binding pocket ( N513 and R517 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The catalytic metal A and the nucleotide binding metal B are shown in red and neon , respectively .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming ddCTP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.


( C ) 8 - oxo - dG ( green ) establishes a Watson - Crick pair with ddCTP ( magenta ) . Binding of the 8 - oxo - dG in the anti - conformation is only possible because the C8 - carbonyl is far enough away not to clash with the 5 ′ - phosphate . Asn513 and Arg517 ( purple ) interact with the minor groove of the nascent base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 8-oxo-dG (green) establishes a Watson-Crick pair with ddCTP (magenta). Binding of the 8-oxo-dG in the anti-conformation is only possible because the C8-carbonyl is far enough away not to clash with the 5′-phosphate. Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , the primer terminus in cyan and the incoming dATP in magenta . A water molecule ( red ) and Mg2 + ( neon ) are occupying the metal A and metal B sites , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O

(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and metal B sites, respectively.


( B ) Overview of the 8 - oxo - dG : dATP insertion complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) containing a templating 8 - oxo - dG ( green ) . The primer terminal ddC and the incoming dATP are shown in cyan and magenta , respectively . Key residues that form the nascent base pair binding pocket ( N513 and R517 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The catalytic metal A and the nucleotide binding metal B are shown in red and neon , respectively .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Overview of the 8-oxo-dG:dATP insertion complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) containing a templating 8-oxo-dG (green). The primer terminal ddC and the incoming dATP are shown in cyan and magenta, respectively. Key residues that form the nascent base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The catalytic metal A and the nucleotide binding metal B are shown in red and neon, respectively.


( C ) 8 - oxo - dG ( green ) establishes a Hoogsteen base pair with dATP ( magenta ) . Asn513 and Arg517 ( purple ) interact with the minor groove of the nascent base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 8-oxo-dG (green) establishes a Hoogsteen base pair with dATP (magenta). Asn513 and Arg517 (purple) interact with the minor groove of the nascent base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , the primer terminus in cyan and the incoming dATP in magenta . A water molecule ( red ) and Mg2 + ( neon ) are occupying the metal A and B sites , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O

(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, the primer terminus in cyan and the incoming dATP in magenta. A water molecule (red) and Mg2+ (neon) are occupying the metal A and B sites, respectively.


( A and B ) Overlay of the nascent base pair in the insertion structures with an undamaged reference structure ( 2PFO , black ) . Binding of 8 - oxo - dG ( green ) in both the anti - ( A ) or syn - ( B ) conformation involves a repositioning of the 5 ′ - phosphate ( red arrow ) . Furthermore , the Arg514 side chain ( purple ) that normally stacks with the templating base and interacts with the 5 ′ - phosphate ( blue - dotted line ) , also repositions ( black arrow ) . In both structures , Arg514 interacts with the C6 - carbonyl of the templating 8 - oxo - dG ( anti ) ( A ) or the C6 - amino group of the incoming dATP ( B ) . The incoming nucleotide is shown in magenta .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

(A and B) Overlay of the nascent base pair in the insertion structures with an undamaged reference structure (2PFO, black). Binding of 8-oxo-dG (green) in both the anti- (A) or syn-(B) conformation involves a repositioning of the 5′-phosphate (red arrow). Furthermore, the Arg514 side chain (purple) that normally stacks with the templating base and interacts with the 5′-phosphate (blue-dotted line), also repositions (black arrow). In both structures, Arg514 interacts with the C6-carbonyl of the templating 8-oxo-dG(anti) (A) or the C6-amino group of the incoming dATP (B). The incoming nucleotide is shown in magenta.


( B ) Overview of the 8 - oxo - dG : dC extension complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) and an incoming dUMPNPP ( magenta ) . The primer terminal dC and 8 - oxo - dG are shown in cyan and magenta , respectively . Key residues that form the primer terminal base pair binding pocket ( N513 , R517 and Glu529 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The nucleotide binding metal ( B ) is shown in neon .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dC and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513, R517 and Glu529) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.


( C ) 8 - oxo - dG ( orange ) establishes a Watson - Crick base pair with dC ( cyan ) at the primer terminus . Tyr505 , Arg517 , and Glu529 ( purple ) interact with the minor groove of the base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 8-oxo-dG (orange) establishes a Watson-Crick base pair with dC (cyan) at the primer terminus. Tyr505, Arg517, and Glu529 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , incoming dUMPNPP in magenta , and primer terminal dC in cyan . Expectedly , Mg2 + ( neon ) is occupying the metal B site . However , the metal A is absent in the structure , resulting in a non - catalytic position of the 3 ′ - OH . This is consistent with other structures containing an incoming non - hydrolyzable analog .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dC in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.


( B ) Overview of the 8 - oxo - dG : dC extension complex . The 39 - kDa catalytic domain ( 8 - kDa subdomain not shown for clarity ) is colored by subdomain : fingers ( salmon ) , palm ( yellow ) and thumb ( purple ) . The polymerase is in complex with a 1 - nt gapped 16 - mer oligo ( grey ) and an incoming dUMPNPP ( magenta ) . The primer terminal dA and 8 - oxo - dG are shown in cyan and magenta , respectively . Key residues that form the primer terminal base pair binding pocket ( N513 and R517 ) and are involved in catalysis ( D427 , D429 and D490 ) are shown in black . The nucleotide binding metal ( B ) is shown in neon .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overview of the 8-oxo-dG:dC extension complex. The 39-kDa catalytic domain (8-kDa subdomain not shown for clarity) is colored by subdomain: fingers (salmon), palm (yellow) and thumb (purple). The polymerase is in complex with a 1-nt gapped 16-mer oligo (grey) and an incoming dUMPNPP (magenta). The primer terminal dA and 8-oxo-dG are shown in cyan and magenta, respectively. Key residues that form the primer terminal base pair binding pocket (N513 and R517) and are involved in catalysis (D427, D429 and D490) are shown in black. The nucleotide binding metal (B) is shown in neon.


( C ) 8 - oxo - dG ( orange ) establishes a Hoogsteen base pair with dA ( cyan ) at the primer terminus . Tyr505 and Arg517 ( purple ) interact with the minor groove of the base pair . The base pair geometry ( C1 ′ distance and λ angles ) is indicated at the bottom of the figure . A simulated annealing Fo - Fc omit density map for the pair is also shown ( contoured at 3σ ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 8-oxo-dG (orange) establishes a Hoogsteen base pair with dA (cyan) at the primer terminus. Tyr505 and Arg517 (purple) interact with the minor groove of the base pair. The base pair geometry (C1′ distance and λ angles) is indicated at the bottom of the figure. A simulated annealing Fo-Fc omit density map for the pair is also shown (contoured at 3σ).


( D ) Overlay of the primer terminus and active site with an undamaged reference structure ( 2PFO , black ) . The catalytic aspartic acids are shown in yellow , incoming dUMPNPP in magenta , and primer terminal dA in cyan . Expectedly , Mg2 + ( neon ) is occupying the metal B site . However , the metal A is absent in the structure , resulting in a non - catalytic position of the 3 ′ - OH . This is consistent with other structures containing an incoming non - hydrolyzable analog .

(D) Overlay of the primer terminus and active site with an undamaged reference structure (2PFO, black). The catalytic aspartic acids are shown in yellow, incoming dUMPNPP in magenta, and primer terminal dA in cyan. Expectedly, Mg2+ (neon) is occupying the metal B site. However, the metal A is absent in the structure, resulting in a non-catalytic position of the 3′-OH. This is consistent with other structures containing an incoming non-hydrolyzable analog.


( A ) Glu529 ( purple ) forms a hydrogen bond with the C2 - amino group of the primer terminal templating 8 - oxo - dG ( orange ) in the anti - conformation . ( B ) This interaction is abolished when 8 - oxo - dG ( orange ) adopts the syn - conformation . An overlay with the 8 - oxo - dG ( anti ) - containing structure reveals that Glu529 ( black ) would likely clash ( red - dotted line ) with the C8 - carbonyl of 8 - oxo - dG ( syn ) . Repositioning of Glu529 ( purple ) places the side chain 4 . 5 Å away from the C8 - carbonyl . ( C ) Comparison of catalytic efficiencies for extension past an 8 - oxo - dG ( anti ) : dC , 8 - oxo - dG ( syn ) : dA and dG : dC base pairs at the primer terminus using WT and E529A Pol λ .

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

(A) Glu529 (purple) forms a hydrogen bond with the C2-amino group of the primer terminal templating 8-oxo-dG (orange) in the anti-conformation. (B) This interaction is abolished when 8-oxo-dG (orange) adopts the syn-conformation. An overlay with the 8-oxo-dG(anti)-containing structure reveals that Glu529 (black) would likely clash (red-dotted line) with the C8-carbonyl of 8-oxo-dG(syn). Repositioning of Glu529 (purple) places the side chain 4.5 Å away from the C8-carbonyl. (C) Comparison of catalytic efficiencies for extension past an 8-oxo-dG(anti):dC, 8-oxo-dG(syn):dA and dG:dC base pairs at the primer terminus using WT and E529A Pol λ.


( A ) Undamaged structure containing a templating dG at the primer terminus ( 2PFO ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at the primer terminus . ( B ) Undamaged structure containing a templating dA at the primer terminus ( 1XSN ) was overlayed with a structure containing an 8 - oxo - dG : dC base pair ( black ) or an 8 - oxo - dG : dA mispair ( red ) at the primer terminus .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O

(A) Undamaged structure containing a templating dG at the primer terminus (2PFO) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus. (B) Undamaged structure containing a templating dA at the primer terminus (1XSN) was overlayed with a structure containing an 8-oxo-dG:dC base pair (black) or an 8-oxo-dG:dA mispair (red) at the primer terminus.


( A ) Gross morphology of control ( Ctrl ) , Sas - 4 − / − , Sas - 4 − / − 53bp1 − / − , and Sas - 4 − / − Usp28 − / − embryos at E9 . 5 . At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text . Scale bar = 500 μm .

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

(A) Gross morphology of control (Ctrl), Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. At least five embryos were considered per genotype and all the double mutant embryos exhibited the rescue criteria mentioned in the text. Scale bar = 500 μm.


( B , C ) Immunostaining for p53 ( B ) and Cleaved - Caspase3 ( Cl - CASP3 , C ) on transverse sections of Ctrl , Sas - 4 − / − , Sas - 4 − / − 53bp1 − / − , and Sas - 4 − / − Usp28 − / − embryos at E9 . 5 . The area shown encompasses the ventral neural tube and surrounding mesenchyme . Asterisks in ( B ) denote non - specific staining of blood cells . Scale bar = 50 μm . ( D , E ) Quantifications of the p53 ( D ) and Cl - CASP3 ( E ) fluorescence signals shown in ( B ) and ( C ) , respectively . Three embryos per genotype were used for the quantifications . * * * p < 0 . 001 , * p < 0 . 05 ( two - tailed student ' s T - test , one - way ANOVA with Tukey ' s multiple comparisons tests gave similar results ) . Bars represent mean ± s . d . ( D ) Ctrl : 1 . 00 ± 0 . 06 ( n = 1770 cells ) ; Sas - 4 − / − : 6 . 84 ± 0 . 34 ( n = 275 ) , Sas - 4 − / − 53bp1 − / − : 1 . 85 ± 0 . 75 ( n = 690 ) , Sas - 4 − / − Usp28 − / − : 1 . 50 ± 0 . 48 ( n = 975 ) . ( E ) Ctrl : 1 . 00 ± 0 . 70 ( n = 1690 ) ; Sas - 4 − / − : 7 . 88 ± 2 . 62 ( n = 175 ) ; Sas - 4 − / − 53bp1 − / − : 1 . 43 ± 0 . 71 ( n = 1030 ) ; Sas - 4 − / − Usp28 − / − : 1 . 57 ± 0 . 44 ( n = 950 ) .

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

(B, C) Immunostaining for p53 (B) and Cleaved-Caspase3 (Cl-CASP3, C) on transverse sections of Ctrl, Sas-4−/−, Sas-4−/− 53bp1−/−, and Sas-4−/− Usp28−/− embryos at E9.5. The area shown encompasses the ventral neural tube and surrounding mesenchyme. Asterisks in (B) denote non-specific staining of blood cells. Scale bar = 50 μm. (D, E) Quantifications of the p53 (D) and Cl-CASP3 (E) fluorescence signals shown in (B) and (C), respectively. Three embryos per genotype were used for the quantifications. *** p < 0.001, *p<0.05 (two-tailed student's T-test, one-way ANOVA with Tukey's multiple comparisons tests gave similar results). Bars represent mean ± s.d. (D) Ctrl: 1.00 ± 0.06 (n=1770 cells); Sas-4−/−: 6.84 ± 0.34 (n=275), Sas-4−/− 53bp1−/−: 1.85 ± 0.75 (n=690), Sas-4−/− Usp28−/−: 1.50 ± 0.48 (n=975). (E) Ctrl: 1.00 ± 0.70 (n=1690); Sas-4−/−: 7.88 ± 2.62 (n=175); Sas-4−/− 53bp1−/−: 1.43 ± 0.71 (n=1030); Sas-4−/− Usp28−/−: 1.57 ± 0.44 (n=950).


( A , B ) Whole - mount immunostaining and quantification for p53 on Ctrl and Sas - 4 − / − embryos at E7 . 5 ( A ) and E6 . 5 ( B ) . Representative sagittal planes are shown and the dotted lines demarcate the epiblast . Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below , normalized to control embryos in the same batch at E7 . 5 ( 4 embryos ) and at E6 . 5 ( 9 embryos ) . * * * p < 0 . 001 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . Scale bars = 100 μm . ( A ) Ctrl : 1 . 04 ± 0 . 00 ( n = 495 cells ) ; Sas - 4 - / - : 1 . 65 ± 0 . 19 ( n = 850 ) . ( B ) Ctrl : 1 . 00 ± 0 . 18 ( n = 1665 ) ; Sas - 4 - / - : 1 . 16 ± 0 . 44 ( n = 1330 )

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

(A, B) Whole-mount immunostaining and quantification for p53 on Ctrl and Sas-4−/− embryos at E7.5 (A) and E6.5 (B). Representative sagittal planes are shown and the dotted lines demarcate the epiblast. Quantification of nuclear p53 fluorescence intensity in the epiblast is shown below, normalized to control embryos in the same batch at E7.5 (4 embryos) and at E6.5 (9 embryos). *** p < 0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bars = 100 μm. (A) Ctrl: 1.04 ± 0.00 (n=495 cells); Sas-4-/-: 1.65 ± 0.19 (n=850). (B) Ctrl: 1.00 ± 0.18 (n=1665); Sas-4-/-: 1.16 ± 0.44 (n=1330)


( C , D ) Immunostaining for 53BP1 and USP28 on sagittal sections of WT embryos at E6 . 5 ( C ) or sagittal planes of whole - mount immunostaining at E5 . 5 ( D ) . Quantification of the fluorescent signals in the epiblast ( Ep ) versus visceral endoderm ( VE ) are shown below . Four embryos per genotype were used for the quantifications . * * * * p < 0 . 0001 , * * * p < 0 . 001 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . Scale bar = 50 μm . ( C ) 53BP1 in Ep : 1 . 00 ± 0 . 10 ( n = 225 cells ) ; 53BP1 in VE : 0 . 37 ± 0 . 06 ( n = 105 ) ; USP28 in Ep : 1 . 00 ± 0 . 08 ( n = 235 ) ; USP28 in VE : 0 . 17 ± 0 . 05 ( n = 90 ) . ( D ) 53BP1 in Ep : 1 . 00 ± 0 . 24 ( n = 225 ) ; 53BP1 in VE : 0 . 68 ± 0 . 14 ( n = 115 ) ; USP28 in Ep : 1 . 00 ± 0 . 23 ( n = 280 ) ; USP28 in VE : 0 . 28 ± 0 . 00 ( n = 110 ) .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(C, D) Immunostaining for 53BP1 and USP28 on sagittal sections of WT embryos at E6.5 (C) or sagittal planes of whole-mount immunostaining at E5.5 (D). Quantification of the fluorescent signals in the epiblast (Ep) versus visceral endoderm (VE) are shown below. Four embryos per genotype were used for the quantifications. **** p < 0.0001, ***p<0.001 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 50 μm. (C) 53BP1 in Ep: 1.00 ± 0.10 (n=225 cells); 53BP1 in VE: 0.37 ± 0.06 (n=105); USP28 in Ep: 1.00 ± 0.08 (n=235); USP28 in VE: 0.17 ± 0.05 (n=90). (D) 53BP1 in Ep: 1.00 ± 0.24 (n=225); 53BP1 in VE: 0.68 ± 0.14 (n=115); USP28 in Ep: 1.00 ± 0.23 (n=280); USP28 in VE: 0.28 ± 0.00 (n=110).


( A ) Immunostaining for the centrosome marker γ - tubulin ( TUBG ) on Ctrl and Sas - 4 − / − primary mESCs . The bottom panels are magnifications of the areas marked in the top panels . Scale bars = 20 μm ( top ) and 10 μm ( bottom ) .

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

(A) Immunostaining for the centrosome marker γ-tubulin (TUBG) on Ctrl and Sas-4−/− primary mESCs. The bottom panels are magnifications of the areas marked in the top panels. Scale bars = 20 μm (top) and 10 μm (bottom).


( B ) Immunostaining and quantification for p53 on Ctrl and Sas - 4 − / − primary mESCs in pluripotent and partially differentiated ( diff . ) conditions . The quantification of p53 fluorescence intensities was normalized to Ctrl ( Four independent experiments ) . * * p < 0 . 01 , * p < 0 . 05 ( two - tailed student ' s T - test , the comparison of the genotypes in the " Pluripotent " condition was not significant using the one - way ANOVA with Tukey ' s multiple comparisons tests ) . Bars represent mean ± s . d . Scale bar = 50 μm . Pluripotent Ctrl : 1 . 00 ± 0 . 02 ( n = 2030 cells ) ; pluripotent Sas - 4 - / - : 1 . 17 ± 0 . 10 ( n = 2170 ) ; partially diff . Ctrl : 0 . 62 ± 0 . 13 ( n = 2745 ) ; partially diff . Sas - 4 - / - : 1 . 12 ± 0 . 24 ( n = 2000 ) .

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

(B) Immunostaining and quantification for p53 on Ctrl and Sas-4−/− primary mESCs in pluripotent and partially differentiated (diff.) conditions. The quantification of p53 fluorescence intensities was normalized to Ctrl (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test, the comparison of the genotypes in the "Pluripotent" condition was not significant using the one-way ANOVA with Tukey's multiple comparisons tests). Bars represent mean ± s.d. Scale bar = 50 μm. Pluripotent Ctrl: 1.00 ± 0.02 (n=2030 cells); pluripotent Sas-4-/-: 1.17 ± 0.10 (n=2170); partially diff. Ctrl: 0.62 ± 0.13 (n=2745); partially diff. Sas-4-/-: 1.12 ± 0.24 (n=2000).


( C , D ) Three - day growth curves of WT and Sas - 4 − / − ( C ) or p53 − / − and Sas - 4 − / − p53 − / − ( D ) primary mESCs in the indicated conditions starting with 105 cells on Day 0 ( Four independent experiments ) . * * p < 0 . 01 , * p < 0 . 05 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . For details of the measurements , see Materials and Methods ( Tables 3 and 4 ) .

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

(C, D) Three-day growth curves of WT and Sas-4−/− (C) or p53−/− and Sas-4−/− p53−/− (D) primary mESCs in the indicated conditions starting with 105 cells on Day 0 (Four independent experiments). ** p < 0.01, * p < 0.05 (two-tailed student's T-test). Bars represent mean ± s.d. For details of the measurements, see Materials and Methods (Tables 3 and 4).


( A ) Sagittal planes of whole - mount immunostaining and quantification for TUBG on WT mouse embryos from E3 . 5 to E6 . 5 . The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass ( E3 . 5 ) or epiblast ( E5 . 5 and E6 . 5 ) . Bars represent mean ± s . d ( two - tailed student ' s T - test or one - way ANOVA with Tukey ' s multiple comparisons tests ) . Scale bars : E3 . 5 and E5 . 5 = 15 μm ; E6 . 5 = 30 μm and 3 μm ( insets ) . E3 . 5 : 88 ± 26 % ( n = 73 cells from 5 embryos ) ; E5 . 5 : 92 ± 10 % ( n = 270 from 3 embryos ) ; E6 . 5 : 79 ± 12 % ( n = 12 , 082 from 3 embryos ) .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Sagittal planes of whole-mount immunostaining and quantification for TUBG on WT mouse embryos from E3.5 to E6.5. The insets are magnifications of selected centrosomes from the dotted areas denoting the inner cell mass (E3.5) or epiblast (E5.5 and E6.5). Bars represent mean ± s.d (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests). Scale bars: E3.5 and E5.5 = 15 μm; E6.5 = 30 μm and 3 μm (insets). E3.5: 88 ± 26% (n=73 cells from 5 embryos); E5.5: 92 ± 10% (n=270 from 3 embryos); E6.5: 79 ± 12% (n=12,082 from 3 embryos).


( B , C ) Representative gSTED super - resolution images of immunostaining for TUBG and either CEP164 ( B ) or ODF2 ( C ) . Whole - mount staining was used for the CEP164 data as well as for the E3 . 5 blastocysts for ODF2 , whereas staining on sections was used for ODF2 at E5 . 5 and E6 . 5 . Quantification of the percentage of centrosomes ( TUBG ) with CEP164 or ODF2 are shown on the right . * * * * p < 0 . 0001 , * * * p < 0 . 001 , * p < 0 . 05 ( two - tailed student ' s T - test or one - way ANOVA with Tukey ' s multiple comparisons tests ; the ODF2 data between E5 . 5 and E6 . 5 was not significant using the latter test ) . Bars represent mean ± s . d . Scale bar = 0 . 5 μm . CEP164 at E3 . 5 : 2 ± 2 % , ( n = 92 centrosomes from 4 embryos ) ; E5 . 5 : 50 ± 2 % ( n = 249 from 3 embryos ) ; E6 . 5 : 62 ± 0 % ( n = 856 from 3 embryos ) . ODF2 at E3 . 5 : 20 ± 9 % ( n = 108 from 5 embryos ) ; E5 . 5 : 51 ± 4 % ( n = 294 from 5 embryos ) ; E6 . 5 : 60 ± 2 % ( n = 1 , 183 from 4 embryos ) .

O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) Representative gSTED super-resolution images of immunostaining for TUBG and either CEP164 (B) or ODF2 (C). Whole-mount staining was used for the CEP164 data as well as for the E3.5 blastocysts for ODF2, whereas staining on sections was used for ODF2 at E5.5 and E6.5. Quantification of the percentage of centrosomes (TUBG) with CEP164 or ODF2 are shown on the right. **** p < 0.0001, *** p < 0.001, * p < 0.05 (two-tailed student's T-test or one-way ANOVA with Tukey's multiple comparisons tests; the ODF2 data between E5.5 and E6.5 was not significant using the latter test). Bars represent mean ± s.d. Scale bar = 0.5 μm. CEP164 at E3.5: 2 ± 2%, (n=92 centrosomes from 4 embryos); E5.5: 50 ± 2% (n=249 from 3 embryos); E6.5: 62 ± 0% (n=856 from 3 embryos). ODF2 at E3.5: 20 ± 9% (n=108 from 5 embryos); E5.5: 51 ± 4% (n=294 from 5 embryos); E6.5: 60 ± 2% (n=1,183 from 4 embryos).


( D ) Immunostaining and quantification of the area of TUBG and PCNT signals from E5 . 5 and E6 . 5 centrosomes in epiblast cells . The separate channels for the marked centriole are shown in the insets . * * * p < 0 . 001 , * * p < 0 . 01 ( two - tailed student ' s T - test ) . Bars represent mean ± s . d . Scale bar = 2 μm . Quantification of PCNT at E5 . 5 : 0 . 22 ± 0 . 01 μm ( n = 103 centrosomes from 3 embryos ) ; E6 . 5 : 0 . 31 ± 0 . 02 μm ( n = 342 from 3 embryos ) . Quantification of TUBG at E5 . 5 : 0 . 15 ± 0 . 02 μm ( n = 108 from 4 embryos ) ; E6 . 5 : 0 . 25 ± 0 . 01 μm ( n = 311 from 3 embryos ) .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunostaining and quantification of the area of TUBG and PCNT signals from E5.5 and E6.5 centrosomes in epiblast cells. The separate channels for the marked centriole are shown in the insets. *** p < 0.001, ** p < 0.01 (two-tailed student's T-test). Bars represent mean ± s.d. Scale bar = 2 μm. Quantification of PCNT at E5.5: 0.22 ± 0.01 μm (n=103 centrosomes from 3 embryos); E6.5: 0.31 ± 0.02 μm (n=342 from 3 embryos). Quantification of TUBG at E5.5: 0.15 ± 0.02 μm (n=108 from 4 embryos); E6.5: 0.25 ± 0.01 μm (n=311 from 3 embryos).


MEFs expressing vector , TRPML - 1 , or GRP1 - DD were treated with BSA or palmitate ( 250 μM ) for 3 h and stained as in ( A ) . aspect ratio ( O ) , branch length ( P ) , and roundness ( Q ) of mitochondria in the cells in ( N ) .

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

MEFs expressing vector, TRPML-1, or GRP1-DD were treated with BSA or palmitate (250 μM) for 3 h and stained as in (A). aspect ratio (O), branch length (P), and roundness (Q) of mitochondria in the cells in (N).


TMRE ( 100 nM ) and MitoTracker green ( 200 nM ) staining in MEFs treated with BSA or palmitate ( 250 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . TMRE intensity in ( A ) on a per cell basis . MitoSOX ( 5 μM ) and MitoTracker Green ( 200 nM ) staining in MEFs treated with vehicle ( DMSO ) or C2 - ceramide ( 50 μM ) for 12 h after a 3 h pre - treatment with vehicle or SH - BC - 893 ( 893 , 5 μM ) . MitoSOX intensity for ( C ) on a per cell basis .

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

TMRE (100 nM) and MitoTracker green (200 nM) staining in MEFs treated with BSA or palmitate (250 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). TMRE intensity in (A) on a per cell basis. MitoSOX (5 μM) and MitoTracker Green (200 nM) staining in MEFs treated with vehicle (DMSO) or C2-ceramide (50 μM) for 12 h after a 3 h pre-treatment with vehicle or SH-BC-893 (893, 5 μM). MitoSOX intensity for (C) on a per cell basis.


NAD ( P ) H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in ( A ) 4 - 8 h after treatment with vehicle ( n = 5 ) or 120 mg / kg SH - BC - 893 ( n = 4 ) by gavage at ZT8 . 5 . Mice were sacrificed and imaged in pairs ( SD and HFD ) .

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

NAD(P)H autofluorescence evaluated by confocal microscopy in freshly resected livers from mice in (A) 4-8 h after treatment with vehicle (n=5) or 120 mg/kg SH-BC-893 (n=4) by gavage at ZT8.5. Mice were sacrificed and imaged in pairs (SD and HFD).


Citrate synthase staining in the arcuate nucleus of mice in ( E ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 3 .

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

Citrate synthase staining in the arcuate nucleus of mice in (E) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=3.


imaging was performed in the areas of the cortex


NAD ( P ) H autofluorescence was evaluated in epididymal fat ( eWAT ) freshly resected from the mice in ( A ) 4 h after treatment with vehicle or 120 mg / kg SH - BC - 893 by gavage at ZT8 . 5 ; n = 5 . NAD ( P ) H intensity from ( B ) calculated on a per cell basis ; 16 - 25 cells from each of the 5 mice per group were quantified ( 87 - 109 total cells per treatment group ) .

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NAD(P)H autofluorescence was evaluated in epididymal fat (eWAT) freshly resected from the mice in (A) 4 h after treatment with vehicle or 120 mg/kg SH-BC-893 by gavage at ZT8.5; n=5. NAD(P)H intensity from (B) calculated on a per cell basis; 16 - 25 cells from each of the 5 mice per group were quantified (87 - 109 total cells per treatment group).


TMRE ( 100 nM ) and MitoTracker Green staining ( 200 nM ) in eWAT resected from mice in ( D ) treated as in ( B , C ) ; n = 5 . TMRE ( F ) or MitoTracker ( G ) intensity for ( E ) calculated on a per cell basis ; 15 - 24 cells from each of the 5 mice in each group were quantified ( 99 - 104 total cells per treatment group ) .

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

TMRE (100 nM) and MitoTracker Green staining (200 nM) in eWAT resected from mice in (D) treated as in (B,C); n=5. TMRE (F) or MitoTracker (G) intensity for (E) calculated on a per cell basis; 15 - 24 cells from each of the 5 mice in each group were quantified (99 - 104 total cells per treatment group).


MitoSOX ( 5 μM ) and MitoTracker Green staining in eWAT resected from the mice in ( D ) treated as in ( B and E ) ; n = 5 . MitoSOX ( I ) or MitoTracker ( J ) intensity calculated as in ( F and G ) ; 10 - 13 cells from each of the 5 mice in each group were quantified ( 57 - 58 total cells per treatment group ) .

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

MitoSOX (5 μM) and MitoTracker Green staining in eWAT resected from the mice in (D) treated as in (B and E); n=5. MitoSOX (I) or MitoTracker (J) intensity calculated as in (F and G); 10 - 13 cells from each of the 5 mice in each group were quantified (57 - 58 total cells per treatment group).


Chop , Xbp1s , Atf6 , and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg / kg SH - BC - 893 on Monday , Wednesday , and Friday for the final 3 weeks ; n = 7 - 9 .

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

Chop, Xbp1s, Atf6, and Grp78 mRNA measured in eWAT resected from mice maintained on a HFD for 13 weeks and gavaged with vehicle or 120 mg/kg SH-BC-893 on Monday, Wednesday, and Friday for the final 3 weeks; n=7-9.


Plasma adiponectin ( A ) or leptin ( B ) in mice maintained on a SD or HFD for 8 - 9 weeks then gavaged with vehicle or 120 mg / kg SH - BC - 893 4 h prior to blood collection . Pair - fed mice were provided with the amount of food consumed by SH - BC - 893 - treated mice in 4 h but left untreated ; n = 8 - 10 .

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

Plasma adiponectin (A) or leptin (B) in mice maintained on a SD or HFD for 8-9 weeks then gavaged with vehicle or 120 mg/kg SH-BC-893 4 h prior to blood collection. Pair-fed mice were provided with the amount of food consumed by SH-BC-893-treated mice in 4 h but left untreated; n=8-10.


Average daily distance run by HFD - fed mice treated with vehicle ( n = 8 ) or SH - BC - 893 ( 120 mg / kg , n = 6 ) on Mondays , Wednesdays , and Fridays over 4 weeks when housed with a running wheel .

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Average daily distance run by HFD-fed mice treated with vehicle (n=8) or SH-BC-893 (120 mg/kg, n=6) on Mondays, Wednesdays, and Fridays over 4 weeks when housed with a running wheel.


Body weight change in the mice in ( O , P ) over the 24 h after the first treatment ( Q ) or after the 3 week course of treatment ( R ) ; n = 9 .

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Body weight change in the mice in (O,P) over the 24 h after the first treatment (Q) or after the 3 week course of treatment (R); n=9.


Representative H & E stained liver sections from the mice shown in Fig . 6F after 4 weeks of treatment with vehicle or 120 mg / kg SH - BC - 893 . Scale bar , 10 μm .

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Representative H&E stained liver sections from the mice shown in Fig. 6F after 4 weeks of treatment with vehicle or 120 mg/kg SH-BC-893. Scale bar, 10 μm.


Blood glucose levels ( F ) or area under the curve ( AUC , ( G ) ) during an oral glucose tolerance test performed in the same mice as in ( E ) .

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Blood glucose levels (F) or area under the curve (AUC, (G)) during an oral glucose tolerance test performed in the same mice as in (E).


Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH - BC - 893 from the mice in Fig . 5D , E or in 9 week old SD - fed mice treated with vehicle ( same mice as in Fig . 5J , K , 0 weeks on HFD ) ; n = 8 . Insulin levels in the plasma collected 4 h after treatment with vehicle or SH - BC - 893 from the same mice as in Fig . 5A , B ; n = 8 .

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

Insulin levels in plasma collected 4 h after the indicated treatment with vehicle or SH-BC-893 from the mice in Fig. 5D,E or in 9 week old SD-fed mice treated with vehicle (same mice as in Fig. 5J,K, 0 weeks on HFD); n=8. Insulin levels in the plasma collected 4 h after treatment with vehicle or SH-BC-893 from the same mice as in Fig. 5A,B; n=8.


( B ) Flow cytometry analysis of WT to Il21r - / - splenic SWHEL B cells over time . ( C ) WT to Il21r - / - SWHEL B cell ratio within CTV division peaks . Data information : Data in ( B ) is representative of 8 - 9 biological replicates ( n = 8 - 9 ) pooled from two independent experiments with statistical analysis by one - way ANOVA with Tukey ' s post - test . * * p ≤ 0 . 01 ; * * * * p ≤ 0 . 0001 . Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) and are representative for two independent experiments .

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(B) Flow cytometry analysis of WT to Il21r-/- splenic SWHEL B cells over time. (C) WT to Il21r-/- SWHEL B cell ratio within CTV division peaks. Data information: Data in (B) is representative of 8-9 biological replicates (n = 8-9) pooled from two independent experiments with statistical analysis by one-way ANOVA with Tukey's post-test. ** p ≤ 0.01; **** p ≤ 0.0001. Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.


( E ) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks . Data information : Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) and are representative for two independent experiments .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks. Data information: Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.


( B ) Flow cytometry analysis showing WT to Il21r - / - SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

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

(B) Flow cytometry analysis showing WT to Il21r-/- SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.


( D ) Ratio of the proportion of BrdU positive WT and Il21r - / - SWHEL B cells with 2N DNA content 4 , 10 and 12 hours post BrdU pulse on day 3 . 5 post immunization . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

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(D) Ratio of the proportion of BrdU positive WT and Il21r-/- SWHEL B cells with 2N DNA content 4, 10 and 12 hours post BrdU pulse on day 3.5 post immunization. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.


( C ) Rate of de novo cell cycle entry 10 hours post in vivo IL - 21 ( 2 µg ) or saline pulse . The ratio of WT to Il21r - / - cells not in S / G2 at the time of pulse ( BrdU - cells ) and containing > 2N DNA content 10 hours after pulse is shown . Data information : Data are representative of 10 - 12 biological replicates ( n = 10 - 12 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

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

(C) Rate of de novo cell cycle entry 10 hours post in vivo IL-21 (2 µg) or saline pulse. The ratio of WT to Il21r-/- cells not in S/G2 at the time of pulse (BrdU- cells) and containing >2N DNA content 10 hours after pulse is shown. Data information: Data are representative of 10-12 biological replicates (n = 10-12) from two independent experiments. Statistical analysis by one-sample t test. * p ≤ 0.05.


Phosphoflow analysis of naïve WT or Il21r - / - B cells following in vitro culture for 3 hours with or without IL - 21 ( 20 ng / mL ) and / or in the presence of BCR cross - linking ( biotinylated anti - Igκ + anti - Igλ and avidin - mediated cross - linking ) or agonistic anti - CD40 . ( A ) Exemplary p - AKT ( S473 ) staining and ( B ) quantification of p - AKT median fluorescence intensity ( MedFI ) . ( C ) Exemplary p - S6 ( Ser235 / 236 ) staining and ( D ) quantification of frequency of p - S6 positive cells Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical analysis by one - way ANOVA with Tukey ' s post - test ( D ) , t test ( B , E ) * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

Phosphoflow analysis of naïve WT or Il21r-/- B cells following in vitro culture for 3 hours with or without IL-21 (20 ng/mL) and/or in the presence of BCR cross-linking (biotinylated anti-Igκ + anti-Igλ and avidin-mediated cross-linking) or agonistic anti-CD40. (A) Exemplary p-AKT (S473) staining and (B) quantification of p-AKT median fluorescence intensity (MedFI). (C) Exemplary p-S6 (Ser235/236) staining and (D) quantification of frequency of p-S6 positive cells Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by one-way ANOVA with Tukey's post-test (D), t test (B, E) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( F , In vitro cell culture of CTV labeled SWHEL B cells with or without IL - 21 ( 20 ng / mL ) and anti - CD40 for three days and analysis by flow cytometry . ( F ) Exemplary CTV division peaks .

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(F, In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (F) Exemplary CTV division peaks.


G ) In vitro cell culture of CTV labeled SWHEL B cells with or without IL - 21 ( 20 ng / mL ) and anti - CD40 for three days and analysis by flow cytometry . ( G ) Quantification of the proportion SWHEL B that had divided and thus diluted CTV . Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical analysis by two - way ANOVA with correction for multiple comparison using Šidák method ( G ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

G) In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (G) Quantification of the proportion SWHEL B that had divided and thus diluted CTV. Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by two-way ANOVA with correction for multiple comparison using Šidák method (G). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( B , C ) Analysis of splenic OTII Tfh cells by flow cytometry . ( B ) Tfh cell count and ( C ) Proportion of Il21Gfp / + OTII Tfh cells transcribing the Il21 locus and thus expressing GFP . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . Statistical analysis by one - way ANOVA with Tukey ' s post - test ( B , C ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(B, C) Analysis of splenic OTII Tfh cells by flow cytometry. (B) Tfh cell count and (C) Proportion of Il21Gfp/+ OTII Tfh cells transcribing the Il21 locus and thus expressing GFP. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. Statistical analysis by one-way ANOVA with Tukey's post-test (B, C). * p ≤ 0.05; ** p ≤ 0.01.


Analysis of splenic WT and Il21r - / - SWHEL B cells by flow cytometry . ( D ) CTV profile of and ( E ) absolute cell count in pooled CTV division peaks ( 0 - 4 , 5 - 8 , 9 - 11 + ) . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . D , show concatenated data representative of 3 - 5 biological replicates . Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm - Šídák method . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

Analysis of splenic WT and Il21r-/- SWHEL B cells by flow cytometry. (D) CTV profile of and (E) absolute cell count in pooled CTV division peaks (0-4, 5-8, 9-11+). Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. D, show concatenated data representative of 3-5 biological replicates. Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm-Šídák method. * p ≤ 0.05; ** p ≤ 0.01.


( F ) Cell surface expression ( median fluorescence intensity , MedFI ) of CD38 , IgD and FAS across individual CTV division peaks . ( G ) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . F and G show concatenated data representative of 3 - 5 biological replicates .

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(F) Cell surface expression (median fluorescence intensity, MedFI) of CD38, IgD and FAS across individual CTV division peaks. (G) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. F and G show concatenated data representative of 3-5 biological replicates.


( A - E ) Analysis of splenic SWHEL B cells and PC by flow cytometry . ( A ) Proportion of SWHEL B cells with a FAS + GL7 + GC phenotype , ( B ) SWHEL GC B cell count and ( C ) representation of IL - 21R deficient cells among total SWHEL GC B cells . ( D ) CD98 + CD138 + _ SWHEL PC count and ( E ) representation of IL - 21R deficient cells among total SWHEL PC . Data information : Data are representative of 6 - 10 biological replicates ( n = 6 - 10 ) from two independent experiments . Data in B , D were analyzed after log transformation with with statistical analysis by Multiple paired t tests ( A , B , D ) and p - values corrected for multiple comparisons using Holm - Šídák method or One - way ANOVA with Tukey ' s post - test ( C ) or t test ( E ) .

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

(A-E) Analysis of splenic SWHEL B cells and PC by flow cytometry. (A) Proportion of SWHEL B cells with a FAS+GL7+ GC phenotype, (B) SWHEL GC B cell count and (C) representation of IL-21R deficient cells among total SWHEL GC B cells. (D) CD98+ CD138+_SWHEL PC count and (E) representation of IL-21R deficient cells among total SWHEL PC. Data information: Data are representative of 6-10 biological replicates (n = 6-10) from two independent experiments. Data in B, D were analyzed after log transformation with with statistical analysis by Multiple paired t tests (A, B, D) and p-values corrected for multiple comparisons using Holm-Šídák method or One-way ANOVA with Tukey's post-test (C) or t test (E).


Western blotting in the WT , Trim71 knockout ( Trim71 _ KO ) , and the Trim71 mutant ( R783H ) mESCs . The signals from the pluripotency factors Nanog and Oct4 were obtained through short ( ~ 10 seconds ) exposure .

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Western blotting in the WT, Trim71 knockout (Trim71_KO), and the Trim71 mutant (R783H) mESCs. The signals from the pluripotency factors Nanog and Oct4 were obtained through short (~10 seconds) exposure.


Colony formation assay for mESCs . The mESCs were cultured in 15 % FBS + Lif for 7 days , and the resultant colonies were fixed and stained for AP .

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Colony formation assay for mESCs. The mESCs were cultured in 15%FBS + Lif for 7 days, and the resultant colonies were fixed and stained for AP.


Exit pluripotency assay for mESCs . The mESCs were induced to exit pluripotency in medium without Lif for 2 days and then switched to 2i + Lif medium for 5 days . The resultant colonies were fixed and stained for AP .

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Exit pluripotency assay for mESCs. The mESCs were induced to exit pluripotency in medium without Lif for 2 days and then switched to 2i+Lif medium for 5 days. The resultant colonies were fixed and stained for AP.


Western blotting of pluripotency factors during EB formation . The signals from the pluripotency factors Nanog and Oct4 were obtained through long ( 5 - 10min ) exposure .

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Western blotting of pluripotency factors during EB formation. The signals from the pluripotency factors Nanog and Oct4 were obtained through long (5-10min) exposure.


Immunofluorescence ( IF ) staining showing the expression level of Rex1 in mESCs cultured in the stemness ( 2i + Lif ) and differentiating ( 15 % FBS - Lif ) conditions .

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Immunofluorescence (IF) staining showing the expression level of Rex1 in mESCs cultured in the stemness (2i + Lif) and differentiating (15% FBS - Lif) conditions.


Relative intensity of IF signals from individual cells in experiment associated with E . The results represent the means ( ± SD ) of three independent IF stainings .

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Relative intensity of IF signals from individual cells in experiment associated with E. The results represent the means (± SD) of three independent IF stainings.


Expression of neural lineage markers during mESCs neural differentiation .

Expression of neural lineage markers during mESCs neural differentiation.


IF staining of Nestin . The quantifications represent the means ( ± SD ) of three independent experiments .

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IF staining of Nestin. The quantifications represent the means (± SD) of three independent experiments.


Distribution of the WT Trim71 and the R783H Trim71 mutant binding regions in the mouse genome . In each CLIP - seq , there are two biological replicates . The binding regions present in both of the biological replicates are used for the analysis .

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Distribution of the WT Trim71 and the R783H Trim71 mutant binding regions in the mouse genome. In each CLIP-seq, there are two biological replicates. The binding regions present in both of the biological replicates are used for the analysis.


CLIP - qRT - PCR for the identified target mRNAs of the Trim71 mutant R783H . The results represent the means ( ± SD ) of three independent experiments .

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CLIP-qRT-PCR for the identified target mRNAs of the Trim71 mutant R783H. The results represent the means (±SD) of three independent experiments.


UCSC genome browser snapshot for the CLIP - seq data from Trim71 and the Trim71 mutant R783H in the Lsd1 locus . The red box indicates the binding region of the Trim71 mutant R783H . The inputs are from the size - matched input samples in the CLIP - seq analysis .

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UCSC genome browser snapshot for the CLIP-seq data from Trim71 and the Trim71 mutant R783H in the Lsd1 locus. The red box indicates the binding region of the Trim71 mutant R783H. The inputs are from the size-matched input samples in the CLIP-seq analysis.


B . A representative set of Western blots in the WT and the R783H Trim71 mESCs . C . Quantification of the target proteins in the WT and the R783H Trim71 mESCs . Data information : The results represent the means ( ± SD ) of three independent experiments . One - Way ANOVA was used to determine the significance of the difference , * p < 0 . 05 . The Western blots listed in B is the Biological Replicate

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B. A representative set of Western blots in the WT and the R783H Trim71 mESCs. C. Quantification of the target proteins in the WT and the R783H Trim71 mESCs. Data information: The results represent the means (± SD) of three independent experiments. One-Way ANOVA was used to determine the significance of the difference, *p<0.05. The Western blots listed in B is the Biological Replicate 1


Western blotting in the WT and the Trim71 ( R783H ) mESCs .

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Western blotting in the WT and the Trim71 (R783H) mESCs.


Quantification of Lsd1 protein and mRNA levels . Gapdh and 18S rRNA were used for normalization in protein and mRNA quantifications , respectively .

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Quantification of Lsd1 protein and mRNA levels. Gapdh and 18S rRNA were used for normalization in protein and mRNA quantifications, respectively.


Polysome analysis in the WT and the Trim71 ( R783H ) mESCs .

Polysome analysis in the WT and the Trim71 (R783H) mESCs.


Quantification of the indicated mRNA distribution in the RNP , 80S , and polysome fractions from the WT and the Trim71 ( R783H ) mESCs .

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Quantification of the indicated mRNA distribution in the RNP, 80S, and polysome fractions from the WT and the Trim71 (R783H) mESCs.


Western blotting in the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) .

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Western blotting in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).


Quantification of Lsd1 protein and mRNA in the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) .

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Quantification of Lsd1 protein and mRNA in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).


Polysome analysis in the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) . Quantification of the indicated mRNA distribution in the RNP , 80S , and polysome fractions from the WT mESCs expressing an empty vector , Flag - Trim71 , Flag - Trim71 ( R783H ) .

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Polysome analysis in the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H). Quantification of the indicated mRNA distribution in the RNP, 80S, and polysome fractions from the WT mESCs expressing an empty vector, Flag-Trim71, Flag-Trim71(R783H).


Deletion of the Trim71 mutant R783H binding site in Lsd1 mRNA ' s 3 ' UTR .

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Deletion of the Trim71 mutant R783H binding site in Lsd1 mRNA's 3'UTR.


CLIP - RIP followed by qRT - PCR to examine mRNAs associated with the Trim71 and the Trim71 mutant R783H in the WT , Trim71 ( R783H ) , CLIP ∆ , and Trim71 ( R783H ) / CLIP ∆ mESCs . The mRNA signals from the E14 mESCs were set as 1 for relative comparison .

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CLIP-RIP followed by qRT-PCR to examine mRNAs associated with the Trim71 and the Trim71 mutant R783H in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs. The mRNA signals from the E14 mESCs were set as 1 for relative comparison.


Western blotting in the WT , Trim71 ( R783H ) , CLIP ∆ , and Trim71 ( R783H ) / CLIP ∆ mESCs .

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Western blotting in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs.


Quantification of Lsd1 protein and mRNA in the WT , Trim71 ( R783H ) , CLIP ∆ , and Trim71 ( R783H ) / CLIP ∆ mESCs . Beta - Tubulin and 18S rRNA were used for normalization in the protein and mRNA quantification , respectively .

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Quantification of Lsd1 protein and mRNA in the WT, Trim71(R783H), CLIP∆, and Trim71(R783H)/CLIP∆ mESCs. Beta-Tubulin and 18S rRNA were used for normalization in the protein and mRNA quantification, respectively.


Exit pluripotency assay for mESCs.