EMBO/soda-nel huggingface dataset:
Stearate-derived very long-chain fatty acids are indispensable to tumor growth (2022)
Figure 6

Figure 6. ACC1/2-DKO cells sensitize to oxidative stress and mitochondria-dependent apoptosis.

MDA-MB-231 Wt cells or ACC-DKO cells were pretreated for 24 h and then cultured in FBS medium or DFBS medium with the indicated treatment for 5 days. Treatments were as follows: z-VAD-FMK (z-VAD, 10 μM), necrostain-1 (Nec-1, 2 μM), Disulfiram (0.4 μM), ferrostain-1 (Ferr-1, 10 μM), Trolox (100 μM), N-Acetyl-L-cysteine (NAC, 3 mM), and deferoxamine mesylate (DFOM, 10 μM). Data are presented relative to the values obtained for the control cells cultured in FBS medium. Dotted line shows the survival level of cells in DFBS medium without treatment (control).

Relative cellular levels of ROS in MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium for 5 days with or without exogenous VLCFA supplement.

The inhibitory effects of H2O2 and PMS on MDA-MB-231 ACC-DKO or Wt cells cultured in FBS medium for 24 h with indicated concentrations.

The protein levels of PARP1 and caspase-3 in MDA-MB-231/ACC-DKO and Wt cultured in FBS or DFBS medium.

Annexin-V & Dead cell (7-AAD) flow cytometry analysis of MDA-MB-231/ACC-DKO and Wt cultured in DFBS medium. The right panel shows the quantification of annexin V-positive cells in the left panels.

The relative cell survival of MDA-MB-231/ACC-DKO or Wt with control vector or Bcl-2 or Bcl-xL overexpression cultured in FBS medium or DFBS medium for 5 days. The left panels show the cell survival, and the right panels verify the overexpression of Bcl-2 and Bcl-xL.

The transmembrane potential in mitochondria of MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium was detected by FACS analysis. The bar graphs show the percentage of JC-1 monomer.

MDA-MB-231/ACC-DKO and Wt cells were stained for Mito Tracker to visualize mitochondria. Scale bar, 10 μm. Mitochondrial morphology was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type.

Data information: In (A, B, C, E, F and G), data are from triplicate experiments, and all experimental data were verified in at least two independent experiments. Error bars represent mean ± SD. *P < 0.05, **P < 0.01 (t-test). In (H), asterisk shows comparison with wild type group.
Unanchored tri-NEDD8 inhibits PARP-1 to protect from oxidative stress-induced cell death (2019)
Figure 2

Figure 2 NEDP1 knockout results in the accumulation of poly-NEDD8

A (Left) Western blot analysis of large scale HALO-NEDP1 pulldowns from HEK 293 NEDP1 KO lysates. (Right) Diagram of mass spectrometry sample preparation.

B Table of the number of spectral counts from proteins with identified diGly- modified peptides, following pulldown by HALO-NEDP1 CA or the HALO-NEDP1 DAGC reduced binder control. NEDD8 is the protein most highly modified by NEDD8.

C Scatterplot of proteins identified by mass spectrometry analysis identifies NEDD8 and components of the NEDD8 conjugation pathway as being the most abundant proteins in HEK 293 NEDP1 KO lysates following NEDP1-CA pulldown. iBAQ analysis of proteins identified in A) are plotted as the log2 value of the enrichment ratio (mass spectrometry intensity of the HALO-NEDP1 CA pulldown over HALO-NEDP1 DAGC pulldown) versus the log10 value of the iBAQ intensity from the HALO-NEDP1 CA pulldown. Blue markers indicate known components of the NEDD8 pathway and red markers indicate proteins that have been identified as substrates of PARP-1 in the database of ADP-ribosylated proteins, ADPriboDB (Vivelo et al., 2017).

D Venn diagram of the proteins enriched by at least 6-fold following HALO-NEDP1 CA pulldown compared with proteins identified as PARP-1 substrates in the database of ADP-ribosylated proteins, ADPriboDB (Vivelo et al., 2017).
Unanchored tri-NEDD8 inhibits PARP-1 to protect from oxidative stress-induced cell death (2019)
Figure 4

Figure 4 NEDP1 knockout inhibits PARP-1 hyperactivation

A PARP-1 activity is reduced in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells after treatment with 600 μM H2O2 for the indicated amount of time. PAR polymer generation is induced in WT cells after H2O2 treatment but induction is reduced in NEDP1 KO cells.

B PARP-1 activity is rescued by re-expression of NEDP1 in NEDP1 KO cells. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells transfected with the indicated constructs. 72 hours after transfection, cells were treated with the indicated amount of H2O2 and processed for Western blot analysis. PAR polymer generation is reduced in NEDP1 KO cells but can be rescued by the transient re-expression of NEDP1.

C PARP-1 inhibitor Olaparib protects WT cells from H2O2 treatment to the same extent as NEDP1 KO. U2OS cells were plated in 96 well plates and 24 hours later pre-treated with Olaparib (10 μM) for 1 hour before treatment with the indicated concentration of H2O2. 24 hours later, cell viability was measured using the CellTiterGlo assay. NEDP1 deletion protects cells from H2O2 treatment to the same extent as PARP-1 inhibition in WT cells. Olaparib does not provide additional protection to NEDP1 cells from H2O2 treatment. Graphs represent the mean ± S.E.M. of the percent survival compared to untreated cells. Two-Way ANOVA with Bonferroni post-hoc test: n=3, **** p < 0.0001

D AIF translocation to the nucleus is impaired in NEDP1 KO cells. WT and NEDP1 KO cells were plated on coverslips and 24 hours later were treated with H2O2 (600 μM) for the indicated amount of time and then processed for immunofluorescence analysis using α-AIF antibodies and DAPI staining. PARP-1 dependent cell death is induced in WT U2OS cells, as indicated by translocation of AIF from the mitochondria to the nucleus (Scale bar 10 μm).

E Total nuclear AIF intensity from (D) was measured with ImageJ and plotted as a vertical scatter plot with the group mean intensity indicated with a black bar. AIF has translocated to the nucleus after WT cells were exposed to H2O2 but not in NEDP1 KO cells. Kruskal-Wallis test with Dunn's multiple comparison post-hoc test: n=3, **** p < 0.0001 , n.s denotes not statistically significant.

F NEDP1 KO cells express normal levels of PARP-1 and AIF. Western blot analysis of whole cell lysates from WT and NEDP1 KO U2OS cells shows that the proteins necessary for the induction of PARP-1 dependent cell death are present at equal levels in both cell lines.
Unanchored tri-NEDD8 inhibits PARP-1 to protect from oxidative stress-induced cell death (2019)
Figure 6

Figure 6 NEDD8 trimers are acetylated to increase binding to the second zinc finger of PARP-1

A 2D gel electrophoresis of lysate from WT cells followed by Western blot analysis with α-NEDD8 antibody indicates free NEDD8 migrates near its predicted isoelectic point (pI) of 6.59

B 2D Gel electrophoresis of pulldowns from NEDP1 KO lysate with GST-Zn1+2 followed by Western blot analysis with α-NEDD8 antibody. Some of the 25-kDa NEDD8 band migrates to the same isoelectric point as unconjugated NEDD8 (denoted by white triangle), indicating the species is most likely an unanchored NEDD8 trimer. However, the majority of NEDD8 trimer migrates to multiple spots of lower pI (indicated in the brackets), which suggests that NEDD8 trimers undergo a post-translational modification that either adds a negative charge or blocks a positive charge on the NEDD8 trimer.

C Overexpression of HDAC1 or HDAC2 decreases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). Cells were also co-transfected, as indicated, with empty FLAG vector, HDAC1-FLAG, or HDAC2-FLAG. 24 hours post transfection, cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.

D HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were transfected with GFP or GFP fused to the first two zinc finger domains of PARP-1 (GFP-Zn1+2). 24 hours later, cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and immunoprecipitation was performed on the lysates with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.

E HDAC inhibition increases PARP-1-NEDD8 trimer binding. NEDP1 KO U2OS cells were left untreated or treated with the HDAC inhibitor sodium butyrate (NaB) (10 mM) for 4 hours. Cells were harvested and lysates were incubated with recombinant Zn1-GFP or Zn2-GFP (25 nM) for 1 hour followed by immunoprecipitation with GFP-Trap. Immunoprecipitated proteins were resolved by SDS-PAGE followed by Western blot analysis with the indicated antibodies.

F Overexpression of HDAC1 or HDAC2 in NEDP1 KO cells can rescue the induction of PAR polymer following H2O2 treatment. U2OS WT or NEDP1 KO cells were transfected with the indicated FLAG vectors. 48 post-transfection cells, were treated with H2O2 (600 μM) for the indicated amount of time and harvested directly in sample loading buffer. Lysates were resolved by SDS-PAGE and processed for Western blot analysis with the indicated antibodies.
Unanchored tri-NEDD8 inhibits PARP-1 to protect from oxidative stress-induced cell death (2019)
Figure 7

Figure 7 NEDP1 activity but not UBE2M activity is inhibited by oxidative stress.

A In vitro de-neddylation by NEDP1 is sensitive to oxidation. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. NEDD8 reactions were stopped by the addition of NAE inhibitor MLN4924 (3 μM). NEDD8 reactions were incubated with GST-NEDP1 at 30°C for the indicated amount of time under reducing (50 μM DTT) or oxidizing conditions (50 μM DTT + 150 μM H2O2). Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.

B In vitro neddylation reactions with UBE2M are insensitive to H2O2. Poly-NEDD8 reactions were prepared with NAE, UBE2M, and NEDD8 as in Figure 1E. Before reactions were started, increasing concentrations of H2O2 (as indicated) were added to the NEDD8 reactions. Subsequently the reactions were incubated at 30°C for the indicated amount of time. Reactions were stopped by the addition of LDS sample loading buffer and resolved by SDS-PAGE before processing for Western blot analysis with the indicated antibodies.
Hypothalamic CDK4 regulates thermogenesis by modulating sympathetic innervation of adipose tissues (2020)
Figure 1

Figure 1 - Increased whole-body oxygen consumption and lipid utilization, and improved resistance to cold exposure in Cdk4-/- mice.

A-F Indirect calorimetry was performed using the Oxymax/CLAMS system in Cdk4+/+ (n=6) and Cdk4-/- (n=5) mice. Whole-body oxygen consumption rate (VO2) (A-B), energy expenditure (EE) (C-D) and respiratory exchange ratio (RER) (E-F) were measured at 24°C and during a cold challenge at 6°C during both the light (white rectangle) and dark (black rectangle) phases over a 2-day period.

G-I CalR was used to GLM-regression plot for each group corresponding to the association between energy balance and total body mass at 24°C (G) and the association between energy balance and total body mass at 6°C (H). The CalR interface displays each mouse as a dot, and the standard error of mean for each group in grey. The « mass effect » and « group effect » were analysed using a generalised linear model (GLM) using body weight as a covariate. The results of this analysis are shown in table (I) for Cdk4+/+ (n=6) and Cdk4-/- (n=5) mice.

J-K Basal rectal temperature was measured at 24°C (J), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (K) in Cdk4+/+ (n=10) and Cdk4-/- (n=8) mice.

Data information: All data are shown as the mean ± SEM; Student's t-test was used for statistical analysis. *P<0.05, **P<0.01, ***P<0.001.
Hypothalamic CDK4 regulates thermogenesis by modulating sympathetic innervation of adipose tissues (2020)
Figure 2

Figure 2 - Cdk4 deficiency promotes thermogenesis and mitochondrial biogenesis in iBAT.

A Gross morphology of iBAT from Cdk4+/+ and Cdk4-/- mice.

B iBAT mass normalized to body weight of Cdk4+/+ (n=11) and Cdk4-/- (n=10) mice.

C Hematoxylin-eosin staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm) from Cdk4+/+ and Cdk4-/- mice.

D-E Western blot analysis of UCP1 and CDK4 protein expression (D) and quantified protein levels of UCP1 (E) (n=4 biological replicates) in iBAT. HSP90 was used as loading control.

F Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4+/+ (n=6) and Cdk4-/- (n=6) mice as assessed by RT-PCR.

G-H Transmission electron microscopy (TEM) images of iBAT (G) (scale bar 5 μm (upper panel) and 1 μm (lower panel, LD indicates lipid droplet and M mitochondria)) and quantification of lipid droplet number (LD) and mitochondrial volume (Mito) (H) (Cdk4+/+ (n=4) and Cdk4-/- (n=4)).

I Respirometry analysis of iBAT as measured by the Oroboros system (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).

Data information: All data are shown as the mean ± SEM; Student's t-test (B, F, H, I) and Mann-Whitney U test (E) were used for statistical analyses. *P<0.05, **P<0.01.
Hypothalamic CDK4 regulates thermogenesis by modulating sympathetic innervation of adipose tissues (2020)
Figure 3

Figure 3 - BAT specific Cdk4 deletion does not affect the thermogenic characteristics of iBAT.

A-B Rectal temperature was measured at 24°C (A), and rectal temperature was monitored for 6 hours during acute cold exposure at 4°C (B) in Cdk4flox/flox (n=9) and Cdk4flox/flox Ucp1-Cre mice (n=9).

C Gross morphology of iBAT from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice

D iBAT mass compared to body weight of Cdk4flox/flox (n=8) and Cdk4flox/flox Ucp1-Cre (n=7) mice.

E Hematoxylin-eosin (H&E) staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm (upper panel) and 50 μm (lower panel)) from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice.

F-G Western blot analysis of UCP1 and CDK4 protein expression (F) and quantified protein levels of UCP1(G) (n=4 biological replicates) in iBAT. HSP90 was used as a loading control.

H Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4flox/flox (n=5) and Cdk4flox/flox Ucp1-Cre (n=6) mice as assessed by RT-PCR.

Data information: All data are shown as the mean +/- SEM. Student's t-test was used for statistical analysis. ***P<0.001.
Hypothalamic CDK4 regulates thermogenesis by modulating sympathetic innervation of adipose tissues (2020)
Figure 4

Figure 4 - Activation of thermogenesis by increased sympathetic innervation of BAT in Cdk4-/- mice is blunted by β-adrenergic antagonist treatment.

A-B TH immunohistochemical staining in iBAT sections (A) (scale bar 20 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (B) (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).

C Hematoxylin-eosin staining in scWAT showing multilocular adipocytes in Cdk4-/- (scale bar 50 μm).

D Expression of thermogenic genes and brown adipocyte markers in scWAT of Cdk4+/+ (n=5) and Cdk4-/- (n=6) mice as assessed by RT-PCR.

E-F Acute cold test (4°C) after treatment with the β-adrenergic antagonist propranolol (Prop) in Cdk4+/+ (n=7) and Cdk4-/- (n=6) animals or with vehicle (NaCl) (Cdk4+/+ (n=7) and Cdk4-/- (n=6)) (E) and corresponding quantification of the area under the curve (AUC).

Data information: All data are shown as the mean ± SEM; Student's t-test was used for statistical analysis. *P<0.05, **P<0.01.
Hypothalamic CDK4 regulates thermogenesis by modulating sympathetic innervation of adipose tissues (2020)
Figure 5

Figure 5 - Cdk4 deficiency in VMH SF1 neurons leads to increased heat production during cold exposure.

A Cdk4 mRNA expression in the dissected VMH of Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre (n=6) mice

B-C Rectal temperature was measured at 24°C (B), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (C) in Cdk4flox/flox (n=15) and Cdk4flox/flox Sf1-Cre mice (n=15).

D Hematoxylin-eosin (H&E) staining of iBAT sections (scale bar 100 μm) from Cdk4flox/flox and Cdk4flox/flox Sf1-Cre mice.

E Expression of thermogenic genes and brown adipocyte markers expression in iBAT Cdk4flox/flox (n=9) and Cdk4flox/flox Sf1-Cre mice (n=9) mice as assessed by RT-PCR.

F-G TH immunohistochemical (IHC) staining in iBAT sections (F) (scale bar 40 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (G) (Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre mice (n=5)).

H-I Western blot analysis (H) and quantification of p-CREBS133 protein expression following 6 hours of cold exposure (4°C) (I) (n=3 biological replicates). HSP90 was used as a loading control.

Data information: All data are shown as the mean +/- SEM. Student's t-test (A, B, C, E, G) and Mann-Whitney U test (I) were used for statistical analyses. *P<0.05, **P<0.01, ***P<0.001.
Hypothalamic CDK4 regulates thermogenesis by modulating sympathetic innervation of adipose tissues (2020)
Figure 6

Figure 6 - Activation of thermoregulatory brain regions in Cdk4-/- mice.

A-D Confocal illustrations of frontal hypothalamic sections from Cdk4+/+ mice (A, C, N=3) and Cdk4-/- mice (B, D, N=3). Low magnification showing nuclei (Hoechst, left pannel) and c-Fos activated-cells (white, right pannel) in the mPOA (A1-2, B1-2) and the ARC, the VMH, the DMH, the LH (C1-2, D1-2). High magnifications corresponding to the frame area in A1-2, B1-2, C2, D2 and showing c-Fos-IR cells (A3-4, B3-4, C3-6, D3-6). Scale bars (A1-2, B1-2, C1-2, D1-2): 200μm, (A3-4, B3-4, C3-4, D3-4): 50μm.

E Quantification of c-Fos IR cells in brain sections of Cdk4+/+ and Cdk4-/- mice (n=15-24 slices according hypothalamic nucleus and genotype).

Data information: All data are shown as the mean ± SEM; Mann-Whitney U test was used for statistical analysis. **P<0.01.

Expanded View Figure Legends
C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency (2022)
Figure 1

Figure 1. AID C-terminal tail is required for its targeting to IgH substrates.

(A) CRISPR-guided AID∆C protein could support CSR in Aicda-/- CH12F3 cells. Left, CRISPR-guided AID is schematically illustrated with each component listed. Right, CRISPR-guided AID initiated CSR levels to IgA. Blue arrows represent multiple CRISPR recognition sites. The number in the parentheses indicates CRISPR recognition sites in each S region. Three biological replicates are plotted by dots and mean with standard deviation (SD).

(B) CRISPR-guided AID generated breaks at the S region in Aicda-/- CH12F3 cells were quantitatively detected by PEM-seq. Top, schematic illustration shows the bait SaCas9 site at Iγ3 region and CRISPR-guided AID variants targeting sites at S regions. Bottom, percentages of translocation junctions between Iγ3 and Sμ or Sα regions are plotted as mean with SD of three biological replicates in the bar plot.

(C) AID-AIDE58Q dimer proteins were expressed in AID-deficient CSR-activated B cells, AID dimer is schematically illustrated on top and CSR levels to IgG1 are shown. Three biological replicates are plotted by dots and mean with SD is shown.

(D) CSR levels to IgA in Aicda-/- CH12F3-miniS cells with indicated AID variants or CRISPR-guided AID variants. Levels of CSR to IgA are plotted and mean with SD of three to six biological replicates.

(E) Mutation profiles of miniS region in Ung-/-Msh2-/-Aicda-/- miniS cell lines complemented with indicated AID. Mutation frequency at each nucleotide along the 756-bp miniS region is plotted as a bar graph with green error bars, representing mean with standard error of the mean (SEM) of three biological replicates. Positions of AID-preferred AGCT and other RGYW motifs are marked with orange and yellow respectively under each plot.

(F) Mutation profiles of miniS region in Ung-/-Msh2-/-Aicda-/- miniS cell lines expressing CRISPR-guided AID variants. Mutation frequency at each nucleotide is plotted as mean with SEM of three biological replicates in the bar graph with green error bar. Plots are labeled as in (B). The sgRNA targeting position is indicated with blue lines at the bottom.

Data information: For panels A, B, and C, one-way ANOVA followed by Dunnett's multiple comparisons test was used for significance assessment. Unpaired two-tailed Student's t-test was performed for panel D. For all panels, ****, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05; ns, p>0.05.
C-terminal deletion-induced condensation sequesters AID from IgH targets in immunodeficiency (2022)
Figure 2

Figure 2. The condensation of AID-delC reduces its activity in CSR.

(A) Representative images showing the localization of AID (green), AIDR190X (green), and endogenous IgH loci (red) in nucleofected Aicda-/- CH12F3 cells. The IgH loci are visualized using Sμ-gRNA and indicated by white arrows. An illustration of CRISPR-Sirius IgH locus visualization is shown on top. Scale bar, 5 µm.

(B) OptoDroplet formation in CH12F3 cells. Schematic illustration of light-induced optoDroplet system is depicted on top. AID variants were tagged to the N terminus of the optoDroplet construct and blue light was employed to promote the formation of puncta three times followed by imaging each time. Representative images after the third blue light activation in Aicda-/- CH12F3 cells with the indicated AID variants are shown at the bottom. White dashed line and green line depict the shapes of nucleus and nucleolus based on Hoechst staining or GFP-FBL, respectively. Scale bar, 5 µm.

(C) Representative images showing optoDroplet formation of indicated AID variants in HEK293T cells. CRM1 inhibitor (CRM1i) was used for selective trapping of AID in nuclei. Scale bar, 5 µm.

(D) The fluorescence intensity of indicated AID variants in each HEK293T cell nucleus was measured and normalized relative to the fluorescence intensity of the whole cell. Data are shown as box plots. Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. "+" indicates the mean level. Each dot indicates one cell, and more than 20 cells represent two biological replicate samples were counted. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ****, p<0.0001; *, p<0.05; ns, p>0.05.

(E) Intensity recovery lines for optoAID∆C droplets in FRAP experiment in HEK293T cells. Bleaching occurs at t = 0 s. The background-subtracted fluorescence intensities are normalized by pre-bleach values. The mean with SD of 10 pairs of optoAIDR190X and 10 pairs of optoAIDcry is shown, respectively.

(F) Formation of puncta in primary B cells complemented with AID-FUS-GFP or AID-FUSm-GFP. FUSm contains 13 mutations on either Y or R residue. Left: Representative images showing cellular localization of AID-FUS-GFP and AID-FUSm-GFP with and without CRM1 inhibitor (CRM1i) treatment in Aicda-/- primary B cells. Scale bar, 5 µm. Right: Quantification of the number of cells with condensed puncta (3 biological replicates). Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.

(G) CSR levels to IgG1 in activated Aicda-/- primary B cells complemented with the indicated protein. Left, representative flow cytometry plots measuring CSR to IgG1. Co-expressed GFP was used to indicate the infected fractions. Right, the mean with SD of three biological replicates is shown in the bar plot. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.