EMBO/soda-nel huggingface dataset:
Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication (2020)
Figure 1

Figure 1. Timeless and Tipin are required to maintain processive replication past G4 structures in vivo.

A. The BU-1 locus as a model system to record G4-dependent replication stalling. The leading strand of a replication fork entering the locus from 3' end stochastically stalls at the +3.5 G4, leading to the formation of a region of ssDNA, with interruption of parental histone recycling and of histone modifications necessary to maintain normal expression of the locus (Schiavone et al., 2014).

B. Instability of BU-1 expression in timeless cells. FACS plots of wild type and timeless (clone 1) DT40 cells stained with anti-Bu-1 conjugated with phycoerythrin. Each line represents the Bu-1 expression profile of an individual clonal population. Unstained controls are shown in blue.

C. Fluctuation analysis for Bu-1 loss in wild-type DT40 cells and two independent timeless clones generated by CRISPR-Cas9 targeting (clones 1 and 2; Appendix Fig. S1), timeless (clone 1) complemented by expression of human Timeless cDNA and a timeless mutant on a background in which the endogenous +3.5 G4 has been deleted (ΔG4) (Schiavone et al., 2014).

D. Fluctuation analysis for Bu-1 loss in DT40 wild-type and tipin cells.

Data information: In c. and d., each symbol represents the percentage of cells in an individual clone expanded for 2-3 weeks that have lost Bu-1high expression. At least two independent fluctuation analyses were performed, with 24-36 individual clones each cell line per repeat. Bars and whiskers represent median and interquartile range, respectively. **** p < 0.0001; one-way ANOVA.
Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication (2020)
Figure 3

Figure 3. The Timeless-Tipin complex shows a preference for binding G4 DNA.

Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences.

A. ssG4: G4 flanked by single-stranded DNA; ssHP: hairpin flanked by single-stranded DNA; ss: single-stranded DNA (Appendix Table S2 for sequence details).

B. Binding affinity of Timeless-Tipin for a range of G4 DNA sequences (see Appendix Table S2 for sequence details and references). Single-stranded (ss20: 5′-6FAM-ATAAGAGTGGTTAGAGTGTA) and double-stranded (ds20: ss20 annealed to complementary sequence) DNA were also tested as controls.

Data information: Each data point is the mean of at least 3 independent experiments and the error bars indicate one SD.
Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication (2020)
Figure 4

Figure 4. The C-terminus of Timeless is required for processive G4 replication. Fluctuation analysis for the generation of Bu-1 loss variants. Top to bottom: wild type, timeless (clone 1), a timeless mutant (timeless ∆C) generated by CRISPR-Cas9 targeting exon 16 which truncates the protein removing the CTD containing both the DBD and the PARP binding domains. Then, complementation of timeless#1 with human Timeless (hTim), hTim∆816-1208 (lacking both the DBD and PBD), hTim∆816-965 (lacking the DBD) and hTim[1:1000], lacking the PBD. At least two independent fluctuation analyses were performed with 24-36 individual clones each cell line per repeat. Bars and whiskers represent median and interquartile range, respectively. * p < 0.05 and **** p < 0.0001; one-way ANOVA for comparison with the wild-type cells.
Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication (2020)
Figure 5

Figure 5. DDX11 is required for processive replication in collaboration with Timeless.

A. Enhanced recruitment of DDX11 to chromatin associated PCNA following exposure to 4 µM PDS for 24 hours. PCNA was precipitated from cross-linked chromatin and the immunoprecipitate blotted for FLAG-DDX11.

B. Fluctuation analysis for Bu-1a loss in wild-type DT40 cells, two independent ddx11 clones generated by CRISPR-Cas9 targeting (clones 1 and 2) and one ddx11 clone generated by conventional homologous recombination gene targeting (clone 3). Each symbol represents the percentage of cells in an individual clone expanded for 2-3 weeks that have lost Bu-1ahigh expression.

C. Fluctuation analysis for Bu-1a loss variant generation in wild-type cells, ddx11 (clone 1) cells, ddx11 (clone 1) complemented by expression of chicken DDX11 WT cDNA, ddx11 (clone 1) complemented by expression of helicase-dead form of chicken DDX11 (K87A) cDNA, and a ddx11 clone generated in cells in which the endogenous +3.5 G4 has been deleted (ΔG4).

D. Fluctuation analysis for Bu-1 loss in two independent timeless/ddx11 double-mutant clones (#1 and #2), ddx11 expressing DDX11KAK (see Appendix Fig. S3), and fancj and fancj/ddx11 double-mutants. Fluctuation analyses for wild type, timeless #1 (Fig. 4) and ddx11 #1 (Fig. 5A) are shown for comparison.

Data information: In all cases, at least two independent fluctuation analyses were performed, with 24-36 individual clones each cell line per repeat. Bars and whiskers represent median and interquartile range, respectively. **** p < 0.0001; one-way ANOVA for comparison with wild-type cells.
Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication (2020)
Figure 6

Figure 6. Timeless and DDX11-deficient cells have impaired growth and increased H2AX phosphorylation in the presence of a G4 ligand.

A. Growth curves for DT40 wild type, ddx11, timeless and ddx11/timeless cells, with and without 4 μM pyridostatin (PDS). Cells were seeded at 5 x104 cells/ml on day 0 and the viable cells were counted each 24 h for 4 days. Bars represent SD of two independent experiments performed in duplicate. Doubling times (DMSO): WT 13 hours, timeless 18 hours, ddx11 16 hours, ddx11/timeless 24 hours. Doubling times (PDS): WT 13.6 hours, timeless 27 hours, ddx11 25.7 hours, ddx11/timeless 47.5 hours.

B. DDR signalling detected by phosphorylation of histone H2AX (γ-H2AX) by flow cytometry in untreated cells or cells exposed to 4 μM PDS for 3 days. Pale histogram, untreated; dark histogram, treated; black dotted line, positive control cells treated with 0.1 µM cisplatin, also for 3 days.

C. Quantification of γ-H2AX in DT40 wild type, ddx11, timeless and ddx11/timeless cells treated with 4 μM PDS for 3 days. The central band represents the median, the box the 25th - 75th centile and whiskers the minimum to maximum range of three independent experiments performed in duplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 unpaired, 2-tailed t-test for each pairwise comparison +/- PDS. See also Appendix Fig 4 for immunofluorescence images of the γ-H2AX signal.
Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication (2020)
Figure 7

Figure 7. Loss of DDX11 and Timeless leads to genome wide expression dysregulation of genes with G4s around the transcription start site (TSS).

A. Correlation of magnitude and direction of change of genes dysregulated (relative to wild type) in timeless vs. ddx11 DT40 cells. rs (Spearman rho) is shown for each correlation.

B. Genes dysregulated in timeless, ddx11 and fancj mutants have a higher density of G4s around their TSS. Cumulative fraction of the genes dysregulated in timeless (red), ddx11 (blue) and fancj (green) containing n (x-axis) G4 motifs within 1.5kb of the TSS compared with all genes (black). p values calculated with the Kolmogorov-Smirnov test.

C. Metagene analysis showing G4 frequency around the TSS of genes dysregulated (up or down) in timeless (left panel), ddx11 (centre panel) and fancj (right panel) compared with all genes (black line). G4 frequency is calculated separately for coding (above the x-axis) and template strands (below the x-axis).

D. A model for recognition and processing of replisome associated G4s by Timeless / DDX11. Current evidence suggests that Timeless is a constitutive component of the replication fork. We suggest that the C terminus of Timeless may help detect G4s in the vicinity of the replisome by a combination of direct recognition through the DNA binding domain and indirectly through the PARP-binding domain. It is not currently possible to distinguish whether this mechanism would operate ahead or behind the fork itself (Lerner and Sale, 2019), although recent structural evidence placing Tof1, the yeast homologue of Timeless, ahead of the fork (Baretić et al., 2020) would appear to make the first possibility more probable. However, for failure to resolve G4s ahead of the fork to result in uncoupling would require the CMG helicase to traverse the structure, as has been suggested for interstrand crosslinks (Sparks et al., 2019).

Expanded View Figure Legends
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 1

Figure 1. Tau protein levels in brain tissues of hTau-WT and hTau-A152T mice and controls.

Tau levels in the cortex (A, C–E) and hippocampus (B, F–H) of 4–10-month-old mice were determined by western blot analysis. (A, B) Representative western blots of cortical (A) and hippocampal (B) homogenates from mice of the indicated genotypes show levels of hTau (HT7) and total tau (Tau5). Actin served as a loading control. (C–H) Quantitation of western blot signals for full-length tau detected with the mTau/hTau cross-reactive antibodies Tau5 (C, F) or EP2456Y (D, G) or the hTau-specific antibody Tau12 (E, H). n = 3–8 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (E, H) or by Welch’s one-way ANOVA with post-hoc two-tailed Welch’s t test (C, D, F, G). P values were Holm-adjusted for multiple comparisons. Values are means ± SEM.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 2

Figure 2. Levels of tau, tau fragments and tau mRNA in brain tissues of hTau-WT and hTau-A152T mice and controls.

Tau expression in the cortex (A, C, E) and hippocampus (B, D, F) of 4–10-month-old mice was determined by quantitative RT-PCR (A, B) and western blot analysis (C–F). (A, B) hTau mRNA levels in tissue homogenates were determined by quantitative RT-PCR. (C, D) Western blots from Figure 1A and B were enhanced to better reveal tau fragments of ~40 kDa. (E, F) Quantitation of western blot signals for tau fragments of ~40 kDa detected with Tau5 in tissue homogenates. n = 3–4 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 by two-tailed unpaired t test (A, B) or by Welch’s one-way ANOVA with post-hoc two-tailed Welch’s t test (E, F). P values were Holm-adjusted. Values are means ± SEM.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 3

Figure 3. hTau distribution, phosphorylation patterns, and astrogliosis in hTau-WT and hTau-A152T mice.

Coronal brain sections from 2–4-month-old mice of the indicated genotypes were immunostained with different tau antibodies. (A–I) Sections of hemibrain (A–C), whole hippocampus (D–F), and CA1 region (G–I) immunostained for hTau (HT7). (J–O) Hippocampal sections immunostained with phosphorylation-dependent tau antibodies: PHF1 (p396, 404) (J–L), AT8 (pSer 199, 202, and pThr 205) (M–O). (P–R) Representative photomicrographs of GFAP immunostaining of the CA1 region (P, Q) and quantitation of GFAP immunoreactivity (R). Scale bars: 1 mm (A–C), 300 μm (D–F and J–O), 100 μm (G–I), 25 μm (P, Q). n = 4 mice per genotype. *P < 0.05 by one-way ANOVA with post-hoc Tukey test. Values are means ± SEM.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 4

Figure 4. Reversibility of abnormal tau accumulation and astrogliosis in hTau-A152T mice.

Six-month-old hTau-A152T(L1) mice and NTG controls were or were not treated with DOX for 2 months and analyzed at 8 months of age by immunohistochemistry. (A–I) Untreated hTau-A152T(L1) mice had increased neuronal labeling with the hTau antibody HT7 (A), the phosphorylation-dependent tau antibody PHF1 (D), and the conformation-dependent tau antibody MC1 (G). HT7 (A) and PHF1 (D) staining patterns were similar. MC1 (G) specifically stained hippocampal mossy fibers. In hTau-A152T(L1) mice, DOX reduced hippocampal staining with HT7, PHF1, and MC1 (B, E, H) to the levels in DOX-treated NTG controls (C, F, I). (J–M) DOX also normalized CA1 levels of GFAP immunoreactivity (IR) in hTau-A152T(L1) mice, as shown by representative photomicrographs (J–L) and quantitation of GFAP immunoreactivity (M). Scale bars: 300 µm (A–I), 25 µm (J–L). n = 3–4 mice per genotype. *P < 0.05 by two-tailed Welch’s t test without Holm adjustment. Values are means ± SEM. a.u., arbitrary units.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 5

Figure 5. Impaired nesting behavior of hTau-A152T mice.

Nesting behavior of mice at 10–14 months of age was scored 0–7 by an investigator blinded to genotype. n = 17 NTG, 12 CaMKII-tTA, 13 hTau-WT(L32), and 10 hTau-A152T(L1). Nest building was significantly impaired only in hTau-A152T(L1) mice (P < 0.01 at 2 and 6 h) and CaMKII-tTA mice (P < 0.05 at 2 h, P < 0.01 at 6 hours) by nonparametric Wilcoxon rank-sum test with gatekeeping approach and Holm adjustment. Values are means ± SEM.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 6

Figure 6. Age-dependent impairment of learning and memory of hTau-A152T mice in the MWM.

Mice from cohorts 1–3 were tested in the MWM at the indicated ages. (A–D, M, N) Learning curves of mice in cohorts 1 (A–D) and 3 (M, N). Data at day 0 are from the first trial on day 1. Data from cohort 2 are shown in EV5B–G. Compared with NTG controls, only hTau-A152T(L1) mice showed significant learning impairments at 4–6, 17–19 (cohort 1, P < 0.001) and 17–21 (cohort 3, P < 0.01) months of age by Cox proportional hazards model analysis with mixed effects and Holm adjustment for the following comparisons for each age range: NTG versus CaMKII-tTA, NTG versus TRE-hTau-A152T(L1), and NTG versus hTau-A152T(L1) for cohort 1, and NTG versus CaMKII-tTA, NTG versus hTau-WT(L32), NTG versus hTau-A152T(L1), and hTau-WT(L32) versus hTau-A152T(L1) for cohort 3. (E–L, O–V) Performance of mice from cohorts 1 (E–L), 3 (O–R) and 1–3 combined (S–V) in a probe trial (platform removed) 24 h after the last training trial. (E–H, O, P, S, T) Latency to reach original platform location. (I–L, Q, R, U, V) Number of times mice crossed the original platform location (black bars) or the average of their crossings of corresponding locations in the three other quadrants (white bars). Cohort 1: n = 12 (A–L) NTG; 13 (A, B, E, F, I, J), 11 (C, G, K), or 9 (D, H, L) TRE-hTau-A152T(L1); 11 (A, B, E, F, I, J), 10 (C, G, K), or 9 (D, H, L) CaMKII-tTA; and 14 (A, B, E, F, I, J), 13 (C, G, K), or 12 (D, H, L) hTau-A152T(L1) mice. Cohort 3: n = 17 (M–R) NTG; 12 (M, O, Q) or 11 (N, P, R) CaMKII-tTA; 13 (M, O, Q) or 12 (N, P, R) hTau-WT(L32); and 10 (M–R) hTau-A152T(L1) mice. Cohorts 1+2+3: n = 47 (S, U), or 39 (T, V) NTG; 11 (S, U) or 9 (T, V) TRE-hTau-A152T(L1); 34 (S, U) or 31 (T, V) CaMKII-tTA; 13 (S, U) or 12 (T, V) hTau-WT(L32); and 43 (S, U) or 36 (T, V) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Cox proportional hazards/mixed effects model analysis (A–D, M, N), one-way ANOVA with post-hoc Tukey (E–H, O, P) or Dunnett’s (S, T) test, or one-tailed paired t test with Holm adjustment (I–L, Q, R, U, V). Values are means ± SEM.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 7

Figure 7. Normal sociability of hTau-A152T-WT(L32) and hTau-A152T(L1) mice.

(A–C) One cohort of mice was assessed with the social approach test at young (A) and old (B) age and another cohort of mice at middle age (C). Mice were placed individually into the empty center chamber of a three-chamber apparatus. One side-chamber contained an empty cup, and the other an identical cup with an unfamiliar live mouse in it. Close contact with each cup was monitored for 10 min. n = 12 (A, B) or 17 (C) NTG; 13 (A) or 10 (B) TRE-hTau-A152T(L1); 12 (A), 9 (B), or 12 (C) CamKII-tTA; 13 (C) hTau-WT(L32); and 14 (A), 12 (B), or 10 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-tailed paired t test with Holm adjustment. Values are means ± SEM.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 8

Figure 8. Age dependent neuronal cell loss in hTau-A152T(L1) mice.

(A–F) Coronal brain sections obtained from mice at 4–6 (A–C) or 20–23 (D–F) months of age were immunostained for NeuN. The number of NeuN-positive cells with neuronal morphology was determined in dentate gyrus (A, D), CA3 (B, E), and CA1 (C, F). n = 11 (A–C) or 18 (D–F) NTG; 11 (A–C) or 5 (D–F) CamKII-tTA; 7 (A–C) or 9 (D–F) hTau-WT(L32); and 14 (A–C) or 10 (D–F) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Tukey test. Values are means ± SEM.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 9

Figure 9. Increased synaptic transmission in hTau-WT and hTau-A152T mice.

fEPSPs in CA3 stratum lucidum were induced by stimulation of mossy fibers in acute hippocampal slices. (A) Representative fEPSP traces from slices of 4–8-month-old mice. (B, C) Input/output curves reflecting the change in the fEPSP slope as a function of the fiber volley amplitude from slices obtained at 4–8 (B) or 20 (C) months of age. (D, E) Short-term plasticity assessed by measuring the PPR (second pulse/first pulse) with 50-msec interpulse interval at 4–8 (D) or 20 (E) months of age. (F) Mossy fiber LTP at 20 months of age. n (mice/slices) = 5–9/8–21 (NTG), 2–5/5–12 (CaMKII-tTA), 3–6/8–14 (TRE-hTau), 3–6/8–14 (hTau-WT), and 5–6/8–14 (hTau-A152T). *P < 0.05, **P < 0.01, ***P < 0.001 versus NTG by linear regression analysis with Holm adjustment (B, C) or by one-way ANOVA and Dunnett’s test (D, E). No significant difference was detected by two-way ANOVA in (F). Values are means ± SEM.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 10

Figure 10. hTau-A152T enhances network hypersynchrony.

(A–C) Subdural EEG recordings from freely behaving mice at 4–9 months of age before (A top trace, B) and after (A bottom trace, C) injection of PTZ at a dose (30 mg/kg) that did not produce convulsions. (A) Representative EEG traces from an hTau-A152T(L1) mouse. Arrows indicate epileptic discharges. (B, C) Quantitation of spikes per hour at baseline (B) and of spikes per 20-min intervals after PTZ injection (C). n = 31 (B) or 23 (C) NTG; 13 (B) or 11 (C) CaMKII-tTA; 12 (B) or 10 (C) hTau-WT(L32); and 22 (B) or 21 (C) hTau-A152T(L1) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by Welch’s one-way ANOVA and two-tailed Welch’s t test with Holm adjustment (B) or by two-way repeated-measures ANOVA and Tukey test (C). Values are means ± SEM.
Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice (2016)
Figure 11

Figure 11. Poor survival of mice coexpressing hAPP/A and hTau-A152T.

(A–D) Numbers of offspring from crosses of hAPP-J20 mice with hTau-A152T(L1) (A, B) or hTau-WT(L32) (C, D) mice that were (A, C) or were not (B, D) treated with DOX. (E) Numbers of offspring from crosses of lower expresser hAPP-J9 mice with hTau-A152T(L1) mice that were not treated with DOX. n = 141 (A), 112 (B), 94 (C), 83 (D), and 282 (E) mice per cohort. Yields from two crosses indicated significant deviations from Mendelian inheritance: hAPP-J20 x TRE-hTau-A152T(L1) x CaMKII-tTA without DOX (Holm-adjusted P = 0.003, unadjusted P = 0.0008) and hAPP-J9 x TRE-hTau-WT(L32) x CaMKII-tTA without DOX (adjusted and unadjusted P < 0.0001) by chi-square goodness-of-fit test. (F) Subdural EEG recordings from untreated 3–10-month-old mice were analyzed to compare their number of spikes at baseline. n = 8 NTG, 6 hAPP-J9, 6 hTau-A152T(L1), and 8 hAPP-J9/hTau-A152T(L1). *P < 0.05, **P < 0.01 by logistic regression analysis for three-way interaction of the three transgenes with Holm adjustment (A–E) or by two-tailed Welch’s t test with Holm adjustment (F).
Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3 (2020)
Figure 1

Figure 1: Loss of SEZ6 selectively changes the levels of GluK2 and GluK3 at the neuronal surface.

A) Workflow of the surface analysis performed in SEZ6KO and WT neurons. SEZ6 flox/flox neurons were infected with CRE (SEZ6KO) and GFP (WT) virus at DIV2 and metabolic labeling was started at DIV5. Surface analysis was performed at DIV7.

B) Protein classification of the total proteins (3209, left panel) and of the glycoproteins (571, right panel) detected in 3 out of 3 experiments. More than 90% of the glycoproteins detected are classified as membrane proteins, as expected for the surface labeling.

C) Changes of the 3209 proteins detected on the cell surface of SEZ6KO neurons compared to WT. The average log2 fold change of SEZ6KO neurons over WT (log2 SEZ6KO/WT) is plotted against the negative log10 of each protein's p-value according to a one-sample t test. Blue labeled dots are considered as hits (including GluK2 and GluK3) and are proteins with a p-value less than 0.05 (neg. log10(0.05)=1.3; horizontal dotted line) and a fold-change to less than -0.5 or more than 0.5 (vertical dotted lines). GluK2 and GluK3 are labeled in pink, GluA1 and GluA2 are indicated with green lines and dots and GluN1 and GluN2B are marked with red lines and dots.
Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3 (2020)
Figure 2

Figure 2: Validation of GluK2/3 reduction on the surface of SEZ6KO neurons by immunoblot. A) SEZ6KO and WT neurons were biotinylated with Sulfo-NHS-Biotin and surface proteins were enriched by streptavidin bead pull-down. The GluK2/3 antibody cannot discriminate the subunits 2 and 3 (Lerma & Marques, 2013), therefore the band is commonly indicated with the labeling GluK2/3. GluK2/3, GluA2 and GluN2B surface levels were quantified, normalized to SEZ6L2 surface levels (negative control) in the same sample (GluK2/3 / SEZ6L2) and divided by the WT levels, with the ratio for WT being set to 1.0 (plot shows mean ± S.E.M., at least 10 replicates in 4 independent biological experiments, Mann-Whitney ****p-value<0.005).

B) mRNA levels of GluA1, GluA2, GluK2, GluK3 and GluN2B were quantified in SEZ6KO neurons, normalized to GAPDH and β-actin mRNA in the same sample and compared to the mRNA levels in WT neurons. No difference in GluK2/3 mRNA was detected in SEZ6KO neurons compared to WT (plot shows mean ± SD, 6 replicates in 2 independent biological experiments, multiple t-test was used).

C) Editing of GluK2 mRNA was tested in SEZ6KO and WT neurons. The editing value was calculated as (intensity of 376 [edited] / intensity of [376 (edited) + 269 (unedited)]) × 100 and normalized to the band at 76 bp. No difference was detected in SEZ6KO neurons compared to WT (plot shows mean ± S.E.M., 6 replicates in 2 independent biological experiments, Mann-Whitney test was used).