EMBO/soda-nel huggingface dataset:
CNOT6L Couples Selective Maternal Transcript Degradation to Meiotic Cell Cycle Progression in Oocyte (2018)
Figure 4

Figure 4. RNA sequencing analyses of oocytes and zygotes derived from WT, Cnot6l-/- and Btg4-/- female mice.

A: Relative mRNA copy number dynamics in WT and Cnot6l-/- samples at the indicated stages. Error bars indicate values of the two biological replicates.

B: Box plot showing gene expression levels of WT, Cnot6l-/- and Btg4-/- oocytes at the MII stage. Genes were divided into 10 bins according to their expression levels in the WT MII oocytes. The box indicates upper and lower quantiles, the thick line in the box indicate the median, and the whiskers represent 2.5th and 97.5th percentiles. ***P < 0.001 by two-tailed Student's t test. n.s.: non-significant.

C: qRT-PCR results showing the relative expression levels of Cnot6l in oocytes collected from WT and Cnot6l-/- mice (Cnot6l-/-/WT). Error bars, s.e.m. ***P < 0.001 by two-tailed Student's t test. n = 3 biological replicates.

D: Scatter plot comparing transcripts between WT and Cnot6l-/- oocytes (at GV, MI and MII stages) and zygotes derived from these oocytes. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectively.

E: Heatmap showing transcripts that were degraded during meiotic maturation (GV/Zygote > 10) in WT oocytes. The definition of cluster I-III is described in the text.

F: Relative mRNA copy number dynamics of the three gene clusters in WT, Cnot6l-/- and Btg4-/- samples.

G: Venn diagrams showing the overlap of transcripts that were significantly degraded during GV-to-MII transition in WT, Cnot6l-/- and Btg4-/- oocytes (GV/MII > 10).

H: Venn diagrams showing the overlap of transcripts that were stabilized during GV-to-MII transition in Cnot6l-/- and Btg4-/- oocytes (GV/MII > 10 in WT, but <10 in KO).

I: Venn diagrams showing the overlap of transcripts that were destabilized in GV-MII transition (FPKM > 1; fold change (GV/MII) > 10) of WT oocytes and the genes upregulated in this process after Cnot6l knockout (FPKM > 1; fold change (Cnot6l−/−/WT at MII) > 10).
CNOT6L Couples Selective Maternal Transcript Degradation to Meiotic Cell Cycle Progression in Oocyte (2018)
Figure 6

Figure 6. Meiotic resumption-triggered deadenylation of maternal transcripts was blocked in Cnot6l-/- oocytes.

A, B: Western blot results showing the levels of CPEB1 in WT and Cnot6l-/- oocytes at the indicated time points after meiotic resumption. Total proteins from 50 (A) or 100 (B) oocytes are loaded in each lane. DDB1 (A) or α-tubulin (B) is blotted as a loading control. Experiments were performed three times with reproducible results; a representative result is shown.

C, D: Immunofluorescence (C) and quantification (D) of L-homopropargylglycine (HPG) showing the overall translation levels of MI and MII oocytes collected from WT and Cnot6l-/- mice. Scale bar, 20 μm. Error bars, standard deviations (n = 6 oocytes for each genotype). **P < 0.01, ***P < 0.001.

E: Relative mRNA copy numbers of polysome-bound transcripts in WT and Cnot6l-/- oocytes at indicated stages. Error bars indicate values of the replicates.

F: Scatter plot comparing polysome-bound transcripts between WT and Cnot6l-/- oocytes at GV, MI and MII stages, respectively. Transcripts decreased or increased more than five folds in Cnot6l-/- oocyte samples were high-lighted with blue or red, respectively.

G: Pie chart in left depicts total transcripts significantly degraded during GV-MI transitions in WT oocytes. Pie chart in right shows the polysome-association of transcripts that were deemed to be degraded during GV-MI transitions in WT oocytes, but were stabilized after Cnot6l knockout.

H: Pie chart in left depicts total transcripts significantly degraded during GV-MII transitions in WT oocytes. Pie chart in right shows the polysome-association of transcripts that were deemed to be degraded during GV-MII transitions in WT oocytes, but were stabilized after Cnot6l knockout.

I: Quantitative RT-PCR results showing the relative levels of indicated transcripts in association with polysomes in WT and Cnot6l-/- oocytes at GV, MI and MII stages. Error bars, standard deviations (n = 3 biological repeats).
CNOT6L Couples Selective Maternal Transcript Degradation to Meiotic Cell Cycle Progression in Oocyte (2018)
Figure 7

Figure 7. Role of (A+U)-rich element (ARE)-binding protein ZFP36L2 in CNOT6L-mediated maternal mRNA decay.

A: Presence of putative AREs (AUUUA) in transcripts of WT oocyte. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (MII/GV) in WT oocytes.

B: Presence of putative AREs in oocyte transcripts stabilized by Cnot6l-/- knockout. Transcripts containing no ARE or transcripts containing 1 or more AREs were subdivided into frequency groups according to the log2 fold change (Cnot6l-/-/WT) at MII stage.

C: Co-IP results showing interaction of ZFP36L2 with CNOT6L and CNOT7. HeLa cells were co-transfected with plasmids expressing HA-ZFP36L2 and FLAG-CNOT6L/7 for 48 h before immunoprecipitation. Experiments were performed three times with reproducible results; a representative result is shown.

D: RNA immunoprecipitation results showing interaction of CNOT6L with indicated transcripts, with or without the presence of ZFP36L2. HeLa cells were co-transfected with plasmids expressing FLAG-ZFP36L2 and HA-CNOT6L for 48 h before immunoprecipitation using an anti-HA antibody. mRNAs recovered from the immunoprecipitates were subjected to qRT-PCR. Error bars, standard deviations (n = 3 biological repeats). ***P < 0.001 by two-tailed Student's t test.
CNOT6L Couples Selective Maternal Transcript Degradation to Meiotic Cell Cycle Progression in Oocyte (2018)
Figure 8

Figure 8. CNOT6L is a downstream effector of ERK1/2 in regulating oocyte meiotic cell cycle progression. A, B: Illustration (A) and fluorescence microscopy results (B) showing the translation activities of the Cnot6l 3&quot;-UTR (WT and CPE mutated) in GV- (maintained by 2 μM milrinone) or MII- (released from milrinone) arrested oocytes. GFP signal indicated translational activation of Cnot6l 3&quot;-UTR. An in vitro transcribed and polyadenylated mCherry mRNA was co-microinjected as a positive control. Scale bar, 100 μm. C: Relative intensity of GFP signal in (B) after normalization by mCherry signal in the same oocyte. Error bars, s.e.m. *P < 0.05; ***P < 0.001 by two-tailed Student&quot;s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicated (n). D, E: Illustration (D) and fluorescence microscopy results (E) showing the expression of GFP-fused Cnot6l 3&quot;-UTR in GV and MII oocytes with or without U0126 treatment (20 μM). Scale bar, 100 μm. F: Relative intensity of GFP signal in (E) after normalization by mCherry signal in the same oocyte. Error bars, s.e.m. ***P < 0.001 by two-tailed Student&quot;s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicated (n). G: Rates of PB2 emission and normal spindle assembly in oocytes cultured with or without U0126. Fully grown GV oocytes were microinjected with mRNAs encoding CNOT6L, CNOT7 and/or BTG4 and are released from milrinone at 12 h after microinjection. Then the oocytes were further culture for 24 h with or without adding U0126 to the medium. Error bars, s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student&quot;s t test. n.s.: non-significant. The numbers of analyzed oocytes were indicated (n). H: A diagram showing role of CCR4-NOT deadenylase complex in targeting maternal mRNA decay. During the onset of oocyte meiotic resumption, MAPK cascade and CPEB1 triggers translational activation of maternal transcripts including those encoding BTG4, CNOT7, CNOT6L, and ZFP36L2 (Belloc & Mendez, 2008, Ma et al., 2015, Sha et al., 2017, Yu et al., 2016b). CNOT6L and other CCR4-NOT components are important downstream effectors of ERK1 and ERK2 in the regulation of spindle assembly and meiotic cell cycle progression in oocytes. During oocyte maturation, RNA-binding protein ZFP36L2 associates with CNOT6L and functions as a CCR4-NOT adaptor in triggering the degradation of ARE-containing transcripts. At a later stage of MZT, an alternative adaptor BTG4 binds to translation initiation factor eIF4E and thereby recruits the CCR4-NOT complex to the actively translated mRNAs. The stepwise recruitment of different adaptors by different catalytic subunits mediates stage-specific degradation of maternal mRNAs by the CCR4-NOT deadenylase. The question mark means that the direct involvement of CNOT6L in BTG4-mediated MZT process remains inconclusive because Cnot6l null oocytes had severe meiosis defects before MZT. Extensive View Figure legends:
A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation (2022)
Figure 1

Figure 1: - In vitro reconstitution of active UFM1 E3 ligase.

Comparison of size exclusion chromatography profiles of UFL1 expressed alone (Red) and co-expressed UFL1/UFBP1 complex (Black) run under identical buffer conditions on a SuperdexTM 200 Increase 10/300 GL column. Molecular weight standards are shown in grey. Fractions corresponding to each peak were collected and analysed on 4-12% denaturing SDS-PAGE followed by Coomassie staining and is shown below.

Mass photometry analysis of co-purified UFL1/UFBP1 complex. The theoretical and experimental molecular weights are indicated above.

Immunoblot comparing UFL1 autoUFMylation (Top) and UFM1 chain synthesis (Middle) in the presence of UFL1 expressed alone and in complex with UFBP1. (Bottom) Assays to monitor UFMylation of substrates using purified TRIP4. Reaction products were separated on a 4-12% SDS-PAGE gel under reducing conditions and analysed by immunoblotting using indicated antibodies.

In vitro reconstitution of 60S ribosome UFMylation. Purified 60S ribosome was incubated with either UFL1 on its own or UFL1/UFBP1 complex together with UBA5, UFC1 and UFM1 for indicated time points at 30°C.

Immunoblot showing formation of free UFM1 chains in the presence (Lane 3,4) and absence of UFL1/UFBP1 (Lane 1,2). (Bottom) Graphical representation of the linkage composition of di-UFM1 chains obtained from LC-MS/MS analysis.

Immunoblot analysing polyUFMylation (top) and UFL1 autoUFMylation (bottom) in the presence of indicated Lys to Arg (K-R) and Lys-less(K0) mutants of UFM1.

Immunoblot showing in vitro UFMylation assay to check for formation of di-UFM1 chains in the presence of WT, Lys-less (K0) and single lys mutants (K only) of UFM1.

Data information: Data shown in C-G are representative of at least 3 independent experiments
A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation (2022)
Figure 2

Figure 2: - UFL1/UFBP1 complex activates UFC1 for aminolysis

Schematic showing the workflow for substrate independent single turnover lysine discharge assays.

Coomassie stained gels monitoring discharge of UFM1 from UFC1 in the presence of indicated free amino acids. UBE2D3 is used as a positive control for lysine and cysteine discharge.

Multiple sequence alignment of 1-410 a.a. region of UFL1 from various organisms to highlight the degree of conservation of cysteine residues in this region.

Immunoblot showing in vitro UFMylation assays in the presence of Cys to Ala (indicated as C-A) mutants of UFL1 to check for UFBP1 UFMylation (top), UFL1 autoUFMylation (middle) and substrate UFMylation (bottom).

Single-turnover lysine discharge assays in the presence and absence of full length UFL1/UFBP1 complex. The reaction was stopped by addition of non-reducing SDS loading buffer (1x final) and analysed for discharge on a 4-12% SDS PAGE gel under non-reducing conditions. Top gel - LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained

Data information: Data in B, D, E are representative of at least 3 independent experiments
A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation (2022)
Figure 3

Figure 3: - Biochemical characterization of UFL1/UFBP1 complex

Cartoon representation of predicted full length human UFL1/UFBP1 complex using AlphaFfold. UFL1 and UFBP1 are shown in blue and teal colors respectively. The dimeric interface of UFL1 and UFBP1 is highlighted in the dotted box. WH: Winged helix; pWH: Partial Winged helix; NTR: N-terminal region; CTR: C-terminal region.

View of the dimeric interface of UFL1/UFBP1 complex highlighting the formation of a full composite WH domain by two partial winged helix domains of UFL1 (blue) and UFBP1 (teal).

Close up view of the interface of UFL1/UFBP1 complex highlighting the interactions at the pWH-pWH' domain interface centred on UFL1 Leu45 (L45) and UFBP1 Ile283 (I283).

Immunoblot from immunoprecipitation (IP) assays to validate the predicted model of UFL1/UFBP1 interaction in cells. UFL1WT-3xFLAG and UFL1L45R-3xFLAG were transiently overexpressed along with UFBP1-SBP in HEK293T-UFL1 KO cells and subjected to separate pulldowns using anti-FLAG antibody or streptavidin. Immunoprecipitated material was run on a 4-12% SDS PAGE gel and analysed by immunoblotting using indicated antibodies.

Schematic representation of UFL1/UFBP1 constructs with different domain boundaries designed to identify the minimal catalytic region of UFL1/UFBP1 complex required for aminolysis.

Quantitative representation of single turnover Lysine discharge assays to identify the minimal boundaries of UFL1 (left) and UFBP1(right) required for activation of UFC1. (n=3, mean±SD). A representative gel image used for the quantitative analysis is shown in Fig EV4A-B.

Immunoblot showing in vitro UFMylation assay to monitor formation of free di-UFM1 chains in the presence of UFL1/UFBP1 complexes bearing different domain boundaries.

Immunoblot of in vitro assay monitoring UFMylation of 60S Ribosome in the presence of different UFL1/UFBP1 truncations as depicted in (E).

Data information: Data in F-H are representative of at least 3 independent experiments
A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation (2022)
Figure 4

Figure 4: - Role of CDK5RAP3 in UFMylation

CDK5RAP3 forms a stable complex with UFL1/UFBP1 in vitro. 30 µg of UFL1/UFBP1 was mixed with 15 µg of CDK5RAP3 and loaded on a SuperdexTM 200 Increase 3.2/300 column analytical gel filtration column. (Bottom) Fractions were collected and analysed on 4-12% SDS PAGE under reducing conditions and visualized by Coomassie staining.

In vitro UFMylation assay in the presence of increasing concentrations of CDK5RAP3 to monitor the E3 ligase activity of UFL1/UFBP1 complex. The reaction products were run on a 4-12% SDS-PAGE gel under reducing conditions and immunoblotting was performed using indicated antibodies.

Single turnover lysine discharge assays to check for activation of UFC1 by UFL1/UFBP1 in the presence and absence of CDK5RAP3. The reaction products were run on a 4-12% SDS PAGE and visualized by Oriole staining.

Quantification of discharge of UFM1 from UFC1 in the presence and absence of CDK5RAP3 as seen in Fig 4D, n=3 biological replicates, mean ±SD.

Substrate UFMylation assays using purified 60S Ribosomes in the presence of increasing concentration of CDK5RAP3. Reaction was performed for 10 min, stopped by addition of SDS Loading dye and analysed on an SDS-PAGE gel under reducing conditions followed by immunoblotting with indicated antibodies.

Immunoblot analysis monitoring UFMylation of Histone H4 in the presence of increasing concentrations of CDK5RAP3 (1 µM, 2 µM, 3 µM). UFL1/UFBP1 concentrations - 1 µM.
A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation (2022)
Figure 5

Figure 5: - Role of TAK motif in UFC1's activity

Comparison of catalytic sites of UBE2D3 (PDB ID:5EGG, shown in green) and UFC1 (PDB ID:2Z6O, shown in pink) shown as cartoons. The residues of the conserved HPN and TAK motif of UBE2D3 and UFC1 respectively are shown in ball and stick representation.

Immunoblot showing the in vitro UFMylation assay to compare the activity of UFC1 Wild type and mutants in the presence of UFL1/UFBP1 complex.

In vitro UFMylation assay to compare the activity of UFC1 Wild type and UFC1 T106I in the presence of UFL1/UFBP1 complex.

Quantification of lysine and cysteine discharge of UFM1 from UFC1 WT and other indicated mutants (n=3 biological replicates, mean ±SD). The first time point in cysteine discharge assays indicated as '0' represents reaction products before addition of cysteine. Following addition of 5 mM cysteine, the reaction was incubated at RT for the specified time duration. The reaction was stopped by addition of non-reducing SDS loading buffer (1x final) and analysed as described above.

A representative gel used for lysine quantification is given in Fig S1C-D

Substrate UFMylation assays using purified 60S Ribosomes in the presence of UFC1 Wild type and different mutants of UFC1.
A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation (2022)
Figure 6

Figure 6:- Role of the N-terminal helix of UFC1 in UFMylation

Multiple sequence alignment of UFC1 homologs from various organisms. A graphical representation of the secondary structure is extracted from the crystal structure of UFC1 (PDB ID:2Z6O)

Schematic representation of domain features of UFC1WT and UFC1ΔN.

In vitro UFMylation assays in the presence of Wild type UFC1 (UFC1WT) and UFC1 lacking the N-terminal helix (UFC1ΔN).

Assay to compare Lys discharge activities of UFC1WT and UFC1ΔN on its own, in the presence of UFL1/UFBP1 or in the presence of preassembled UFL1/UFBP1/CDK5RAP3 complex. Top gel: LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained (Representative of three independent experiments).

E-G. (E) Quantitative analysis of the intrinsic Lys reactivity (Lysine 25 mM) of UFC1WT and in UFC1ΔN in the absence of E3 ligase; (F) in the presence of UFL1/UFBP1; (G) in the presence of preassembled UFL1/UFBP1/CDK5RAP3. (E - G: n=3 biological replicates, mean ±SD).

H. Effect of deletion of N -term helix of UFC1 in 60S ribosome UFMylation. Purified 60S ribosomes (50 nM) were incubated with 0.5 µM UBA5, 1 µM UFM1, 100 nM UFL1/UFBP1 complex in the presence of either 1 µM UFC1WT or 1 µM UFC1ΔN at 30°C. The reaction was stopped at indicated time points and run on a 4-12% SDS PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies. (Right - shorter time duration. Left - Longer time duration).

Extended View Figure Legend
Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders. (2020)
Figure 1

Fig. 1. Hyperactive Beclin-1 protects against hyperammonemia and activation of hepatic autophagy improves the phenotype of OTC-deficient mice.

(A) Blood ammonia in 8-9-week-old C57BL/6J wild-type (WT) mice (n=8) and Becn1F121A mice with constitutive activation of autophagy (n=8) at baseline and 30 min after i.p. injection of NH4Cl (10 mmol/kg). **p<0.01 (Unpaired t-test). ns: not statistically significant difference.

(B) Urinary orotic acid of 12-week-old spf-ash mice treated with TB-1 (15 mg/kg, i.p) or vehicle at various times as indicated by the arrows (n=5 mice/group). *p<0.05 (Two-way ANOVA).

(C; D) Western blotting and densitometric quantifications of autophagy markers (LC3II: autophagosomes; p62 and NBR1: cargo receptors) in livers of spf-ash mice harvested after ten days of treatment with TB-1 or vehicle. β-actin was used as loading control. n=5 mice/group. **p<0.01, *p<0.05 (Unpaired t-test).

(E) Survival curves of spf-ash mice fed with a high protein diet (HPD) for ten days and treated with TB-1 alone or combined with scavenger drug and L-Arginine, or treated with scavenger drug and L-Arginine, or left untreated. WT control were included (n=5/group). ***p<0.001 (Log-rank Mantel-Cox test).

(F) Blood ammonia levels determined after 4 days under HPD (n=5 mice/group); ***p<0.001, **p<0.01, *p<0.05 (One-way ANOVA).

Data information: Treatments in (E, F): Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M. Exact p values are reported in Appendix Table S1.
Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders. (2020)
Figure 2

Fig. 2. Enhancement of liver autophagy improves survival, increases ureagenesis, and corrects metabolic defects of ASL-deficient mice.

(A) Survival curves of AslNeo/Neo mice and age-matched wild-type (WT) controls treated with TB-1 (15 mg/kg, i.p., every 48 hours starting at day 10 of age) or vehicle. WT + Vehicle n=8; WT + TB-1 n=8; AslNeo/Neo + Vehicle n=20; AslNeo/Neo + TB-1 n=16. *p<0.05 (Log-rank Mantel-Cox test).

(B) Isotopic enrichment of 15N-labeled urea in blood, 20 min after i.p. injection of 15NH4Cl tracer (4 mmol/kg) in AslNeo/Neo mice treated with TB-1 (n=15) or vehicle (n=9). *p<0.05 (Unpaired t-test).

(C) Representative Western blotting bands of LC3, p62 and NBR1 in livers of AslNeo/Neo mice treated with TB-1 or vehicle. GAPDH and β-actin were used as loading controls.

(D) Densitometric quantifications. AslNeo/Neo + Vehicle n=5; AslNeo/Neo + TB-1 n=9. **p<0.01, *p<0.05 (Unpaired t-test).

(E) Argininosuccinate in dried blood spots (DBS) of WT and AslNeo/Neo mice injected with TB-1 or vehicle (n=4-14 mice/group). ***p<0.001, *p<0.05 (One-way ANOVA).

(F) Isotopic enrichment of 15N-labeled argininosuccinate in livers of AslNeo/Neo mice treated with TB-1 (n=11) or vehicle (n=5). *p<0.05 (Unpaired t-test).

(G) Orthogonal Projection to Latent Structure-Discriminant Analysis (OPLS-DA) score plot obtained from high-resolution 1H-NMR spectroscopy performed on livers of vehicle-treated AslNeo/Neo mice (n=6), WT controls (n=4) and AslNeo/Neo mice injected with TB-1 (n=6). A statistical model with R2=0.78 (goodness of fit), Q2=0.57 (power in prediction) and p=0.0056 was obtained. See also Appendix Figure S1.

Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference. Exact p values are reported in Appendix Table S1.
Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders. (2020)
Figure 3

Fig. 3. Enhancement of autophagy reduces hepatocellular injury and glycogen storage in ASL-deficient mice.

(A) Hematoxylin and eosin (H&E, upper panels) and periodic acid Schiff (PAS, lower panels) staining of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bars: 500 µm.

(B) Computational analysis of PAS staining (n≥4 mice/group). **p<0.01, *p<0.05 (One-way ANOVA).

(C) Quantification of hepatic glycogen in vehicle (n=6)- and TB-1-treated AslNeo/Neo mice (n=12) compared to WT (n=5) controls. **p<0.01, *p<0.05 (One-way ANOVA).

(D) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo/Neo mice treated with either with TB-1 or vehicle (n=4 mice/group). *p<0.05 (One-way ANOVA). GAPDH was used as loading control.

(E) Representative electron microscopy images of liver samples harvested from WT and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bar: 900 nm.

Data information: All values are shown as averages ± S.E.M. Exact p values are reported in Appendix Table S1.
Beclin-1-mediated activation of autophagy improves proximal and distal urea cycle disorders. (2020)
Figure 4

Fig. 4. TB-1 reduces intra-nuclear glycogen deposition in ASL-deficient mice.

(A) Representative electron microscopy images of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. False color on the images indicates glycogen within the nuclei. Scale bar: 1 μm.

(B) Quantification of nuclei containing glycogen (approx. 180 nuclei were analyzed in total per condition, n=3 mice/group).