EMBO/soda-nel huggingface dataset:
BRD4-PRC2 Represses Transcription of T-helper 2-Specific Negative Regulators during T-cell Differentiation (2023)
Figure 4

Fig. 4. Brd4-BD2 binds acetylated-EED to repress Foxp3 and Fbxw7 in Th2 cells.

(A) Immunoprecipitation of Flag-tagged Brd4, followed by Western blotting of HA to detect HA-Ezh2, HA-EED and HA-Suz12 in HEK293T cells transfected with Flag-Brd4 and/or HA-Ezh2, HA-EED and HA-Suz12 plasmids.

(B) HEK293T cells are transfected with Flag-Brd4 and HA-EED, HA-Ezh2, and Suz12 plasmids. Lysates are immunoprecipitated with Flag-tagged Brd4, treated in-vitro with JQ1 (5μM), MS402 (5μM), and ABBV-744 (5μM), followed by Western blotting of HA.

(C) Th2 cells were differentiated for 6 days. Lysates are immunoprecipitated with Brd4, treated in-vitro with ABBV-744 (5μM), followed by Western blotting of EED, Ezh2 and Suz12.

(D) HEK293T cells are transfected with Flag-Brd4 and HA-EED plasmids. Lysates are immunoprecipitated with HA, followed by Western blotting of HA and pan-acetylation (pan-Ac). * denotes specific bands.

(E) HEK293T cells are transfected with Flag-Brd4 and HA-EED plasmids. Lysates are immunoprecipitated with Flag-tagged Brd4, treated in-vitro with 10μM acetylated EED peptides (K19, K211, K250, K261) and non-acetylated EED-K19 peptide, followed by Western blotting of HA to detect HA-EED.

(F) HEK293T cells are transfected with Flag-Brd4, HA-EED and HA-EED(K19R) plasmids. Lysates are immunoprecipitated with Flag followed by Western blotting of HA, and vice versa.

(G) Dual-luciferase reporter assay of HEK293T cells transfected with Fbxw7-promoter, Flag-Brd4, HA-EED and HA-EED(K19R) plasmids.

Data information: All Western blotting data are representative of three independent experiments. All data represent mean±SD and are representative of three independent experiments. Data are analyzed by Paired t test. *p<0.05.
BRD4-PRC2 Represses Transcription of T-helper 2-Specific Negative Regulators during T-cell Differentiation (2023)
Figure 5

Fig. 5. Brd4 through its BD2 domain regulates transcription of Th2-key genes and -negative regulators.

(A) Flow cytometric (left) and statistical analysis (right) of IL-4 and IL-17A in mouse Th2 and Th17 cells treated with BRD4 BD2-selective inhibitor ABBV-744 (1μM) and BD1-selective inhibitor MS611 (2μM), respectively.

(B) qPCR analysis of Il4, Il5, Gata3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM. (C) Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1.

(D) Western blotting of Gata3 of mouse Th2 cells treated with ABBV-744 (500nM) and MG132 (20μM).

(E) qPCR analysis of Foxp3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM.

(F) qPCR analysis of Fbxw7 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM.

(G) Schematic diagram illustrating BRD4 functions in control of transcriptional activation and repression of key genes in chromatin that functionally cooperate with each other to regulate Th2 cell program. Histone lysine acetylation and methylation are depicted with color-coded flags in green or red, respectively.

Data information: Mouse naïve CD4+ T cells were cultured in Th2 polarization condition and treated with or without inhibitors on Day 0 and were differentiated for 6 days before analysis, unless otherwise specified. All data represent mean±SD and are representative of three independent experiments. Data are analyzed by Paired t test. *p<0.05; **p<0.01; and ***p< 0.001.
Metabolic effects of bezafibrate in mitochondrial disease (2020)
Figure 2

Figure 2. Metabolic effects of bezafibrate in patients with the m.3243A>G MTTL1 mutation.

W0, W6 and W12 refer to the week of study.

(A) Serum FGF-21 and GDF-15 levels before, during and after treatment (two-sided Wilcoxon signed-rank test, P-value = 0.031 in all significant cases) (see Fig. EV2 for details where P1 - P6 refer to the individual patients).

(B) Volcano plot showing differences in metabolite levels between 0 and 6&12 weeks of treatment. Metabolites in histidine, alanine, aspartate and glutamine metabolism, and the TCA cycle pathways are annotated at 10% False Discovery Rate (see Dataset EV1 for the whole list of differentially regulated metabolites. Although some other metabolites showed a more pronounced difference, these did not fit into a recognised pathway). Metabolite labels are colour-coded according to the identified KEGG pathways (dark red = "Alanine, aspartate and glutamate metabolism", dark green = "Histidine metabolism" and dark blue = "Citrate cycle (TCA cycle)", with transition colour-code if the metabolite belongs to more than one KEGG pathways).

(C) Scatterplot depicting P-values from the metabolites Pathway Enrichment Analysis (y-axis) and impact values from Pathway Topology Analysis (x-axis) (see Dataset EV2 for details). KEGG pathways at 10% adjusted Holm-Bonferroni P-value are highlighted.

(D) Volcano plot of acyl-carnitine metabolites between 0 and 6&12 weeks of treatment with highlighted differential metabolites at 10% FDR.

(E) Respiratory chain complex and citrate synthase activity in skeletal muscle before and after 12 weeks of treatment. No significant differences were detected before and after 12 weeks of treatment by using the two-sided Wilcoxon signed-rank test with an empirical level of significance.

Data Information: In (A) and (E) horizontal dotted lines denote the mean values in healthy age-matched controls. In (E) solid horizontal lines and bars represent the mean ± SD of the technical replicates at each time point. Horizontal lines on (A) and (E) refer to statistical comparisons of the mean values, where ns = non-significant, *P-value ≤ 0.05.
Metabolic effects of bezafibrate in mitochondrial disease (2020)
Figure 3

Figure 3. Mitochondrial biogenesis and clinical effects of bezafibrate in patients with the m.3243A>G MTTL1 mutation.

P1 - P6 refer to individual patients.

(A) Representative examples of the quadruple immunofluorescence quantification of skeletal muscle fibres before and after treatment. The box in the top right-hand corner indicates the normal range, using two antibodies to NDUFB8 and COX I.

(B) Quantitative quadruple immunofluorescence of skeletal muscle fibres in the six patients showing a decrease (two-sided Wilcoxon signed-rank test with an empirical level of significance) in the proportion of complex IV deficient skeletal muscle fibres before and after 12 weeks of treatment (empirical P-value = 0.048).

(C) Hierarchical cluster analysis of skeletal muscle RNA-seq transcripts before and after treatment showing no clear separation between untreated and treated patients.

(D) Volcano plot depicting differentially expressed RNA-seq genes at 10% FDR after 12 weeks of treatment. Significantly different mammalian mitochondrial genes (MitoCarta 2.0) are annotated.

(E) Reactome Pathway Analysis of the differentially expressed genes at 10% FDR after 12 weeks of treatment (see Expanded View document for details).

(F) Percentage level m.3243A>G in blood, urinary sediment and skeletal muscle. No differences were detected by using the two-sided Wilcoxon signed-rank test with an empirical level of significance. W = week of the study.

(G) Cardiac parameters before and after treatment (two-sided Wilcoxon signed-rank test with an empirical level of significance). ES = end-systolic volume (ml) (empirical P-value = 0.047) and index (ml/m2) (empirical P-value = 0.046), measured by MRI. Horizontal lines on (B), (F) and (G) refer to statistical comparisons of the mean values, where ns = non-significant, *P-value ≤ 0.05. For normal reference ranges see (Hollingsworth et al., 2012)

Tables

Table 1. Clinical characteristics of the six patients with the m.3243A>G MTTL1 mutation and mitochondrial myopathy.

(MIDD: mitochondrially inherited diabetes and deafness, NMDAS = Newcastle Mitochondrial Disease Adult Scale).

Symptom

duration (years)

Expanded View Figure Legends
UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy (2022)
Figure 1

Figure 1. UBXD8 dually localizes to mitochondria and the ER.

Schematic of the domain structure and membrane topology of UBXD8, UBXD2, and UBXD6. UBA: ubiquitin-associated; HP: hairpin; UAS: ubiquitin associating; UBX: Ubiquitin regulatory X; CC: coiled-coil.

Immunofluorescence analysis of exogenously-expressed FLAG-UBXD8, FLAG-UBXD2, and FLAG-UBXD6 in U2OS cells. Scale bar, 5 μm.

Schematic, western blot, and immunofluorescence analysis of the 3×FLAG-UBXD8 knockin U2OS cell line. Scale bar, 5 μm.

Immunofluorescence analysis of endogenous UBXD8 in U2OS cells. Scale bar, 5 μm.

Immunofluorescence analysis of endogenous UBXD8 in WT and ∆UBXD8 HeLa cells. Scale bar, 5 μm.

Data information: In (B, C, D, E), mitochondria were visualized by anti-Tom20 immunostaining; ER was labeled by ER-DsRed or by anti-PDI immunostaining.
UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy (2022)
Figure 2

Figure 2. UBXD8 associates with mitochondrial and ER ubiquitin E3 ligases and recruits VCP to mitochondria and the ER.

Blue native gel analysis of UBXD8 in mitochondrial fractions purified from WT and ∆UBXD8 HeLa cells.

Immunoprecipitation analysis of UBXD8 interaction with mitochondrial (MARCH5 and MUL1) and ER (RNF185) ubiquitin E3 ligases. HeLa cells were infected with lentivirus to stably express HA-tagged ubiquitin E3 ligases. Whole cell lysates were prepared for anti-HA immunoprecipitation.

Immunoprecipitation analysis of UBXD8 interaction with MARCH5 and RNF185. Whole cell lysates from 3×FLAG-UBXD8 knockin HEK293T cells were prepared for anti-FLAG immunoprecipitation. MARCH5 and RNF185: short exposure for whole cell lysate blots, long exposure for FLAG-IP blots.

Fractionation analysis of UBXD8's effect on VCP recruitment to mitochondria and the ER. Whole cell lysate, mitochondria, and ER fractions were purified (see methods for detail) from WT, ∆UBXD8, and ∆UBXD8 HeLa cells rescued with UBXD8 or the UBX* mutant. UBX*-UBXD8 carries four point mutations (K367A, F407A, P408G, R409A) in the UBX domain to disrupt the UBXD8-VCP interaction. For each sample, 5 μg proteins were loaded for western blot analysis.

Quantitative analysis of VCP levels in the mitochondria and ER fractions from the indicated HeLa cells. Data are shown as mean ± SE from three biological repeats. Statistics: one-way ANOVA; **P < 0.01, ***P < 0.001.
UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy (2022)
Figure 3

Figure 3. Both UBXD8 and UBXD2 (ER VCP adaptor) participate in mitochondrial protein degradation.

A, B. Western blot (A) and quantitative (B) analyses of MiD49 and Mcl1 degradation in WT and ∆MARCH5 HeLa cells. Cycloheximide (CHX: 200 μg/ml; protein synthesis inhibitor); MG132: 20 μM, proteasome inhibitor.

C, D. Western blot (C) and quantitative (D) analyses of Mcl1 degradation in the indicated HeLa cells.

E, F. Western blot (E) and quantitative (F) analyses of MiD49 degradation in the indicated HeLa cells.

G. Immunofluorescence analysis of MiD49-FLAG localization in the indicated HeLa cells. Scale bar, 5 μm.

Data information: Data are shown as mean ± SE from three biological repeats (B, D, F). Statistics: two-tailed unpaired Student's t-test (B, D, F); *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant.
UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy (2022)
Figure 4

Figure 4. UBXD8 degrades mitochondrial and ER substrates in cis and in trans.

Schematic of mito-UBXD8 and ER-UBXD8.

Fractionation analysis of the subcellular localization of mito-UBXD8 and ER-UBXD8. Whole cell lysate, mitochondria, and ER fractions were purified from the indicated HeLa cells. Exogenously-expressed mito-UBXD8 has similar expression level with endogenous UBXD8 in WT cells; exogenously-expressed WT UBXD8 and ER-UBXD8 have similar expression levels.

Immunofluorescence analysis of mito-UBXD8 and ER-UBXD8 localization in U2OS cells. Scale bar, 5 μm.

D, E. Western blot (D) and quantitative (E) analyses of Mcl1 degradation in the indicated HeLa cells. ∆UBXD8 HeLa cells were rescued with vector, WT-, mito-, and ER-UBXD8. Data are shown as mean ± SE from three biological repeats (E). Statistics: two-tailed unpaired Student's t-test (E); *P < 0.05, **P < 0.01, ***P < 0.001.

Cartoon illustration of the substrate degradation by mitochondrial and ER adaptor-VCP complexes.
UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy (2022)
Figure 5

Figure 5. UBXD8 knockout sensitizes cells to mitochondrial stresses and apoptotic insults.

A-C, Growth tests of the indicated HeLa cells in response to oligomycin (A), chloramphenicol (B), and ethidium bromide (EB) (C) treatment. Oligo: 10 μM; Chloramphenicol: 300 μg/ml; EB: 50 ng/ml.

Cell survival analysis of the indicated HeLa cells treated with Actinomycin D (ActD, 1 μM) or Doxorubicin (Doxo, 10 μM). Representative images are shown. Cell viability was measured by Cell Titer-Glo. Scale bar, 50 μm.

Western blot analysis of apoptosis activation (caspase cleavage and PARP cleavage) in the indicated HeLa cells treated similarly as in (D).

Cell survival analysis of WT and ∆UBXD8 MCF7 cells treated with ActD (1 μM) or Doxo (10 μM). Cell viability was measured by Cell Titer-Glo.

Cell survival analysis of the indicated HeLa cells. 7 × 105 cells in one well of 6-well plate were treated with ActD (200 nM) for 4 hours or Doxo (50 nM) for 2 hours and subsequently cultured in normal medium for 21 days. Surviving colonies were stained by 0.1% (W/V) methylene blue. Representative images and quantifications of the surviving colony numbers are shown.

Data information: Data are shown as mean ± SE from three biological repeats (A, B, C, D, F, G). Statistics: two-tailed unpaired Student's t-test (A, B, C, D, F); one-way ANOVA (G); *P < 0.05, **P < 0.01, ***P < 0.001.
UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy (2022)
Figure 6

Figure 6. UBXD8 mediates the degradation of multiple BH3-only proteins and restrains apoptosis via Noxa degradation.

Western blot analysis of BH3-domain containing proteins in the indicated HeLa cells treated with Doxo (10 μM). Three proteins with enhanced level in ∆UBXD8 cells (Noxa, Bik, and Bnip3) are highlighted in red.

Western blot and quantitative analysis of Noxa degradation in the indicated HeLa cells.

Western blot and quantitative analysis of Bik and Bnip3 degradation in the indicated HeLa cells.

D-G. Western blot (D, F) and cell viability (E, G) analysis of the indicated HeLa cells. Cell viability was measured by Cell Titer-Glo. Dox: 10 μM.

Data information: Data are shown as mean ± SE from three biological repeats (B, C, E, G). Statistics: two-tailed unpaired Student's t-test (B, C, E, G); *P < 0.05, **P < 0.001, ***P < 0.001.
DDX3X and Specific Initiation Factors Modulate FMR1 Repeat-Associated Non-AUG Initiated translation (2019)
Figure 2

Figure 2. Knockdown of Belle Mitigates Repeat-Associated Toxicity by Inhibiting RAN Translation in Drosophila.

A Representative photographs of fly eyes expressing (CGG)90-EGFP under a GMR-GAL4 driver, with various belle disruptions.

B Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with belle disruptions (Mann-Whitney U test with Bonferonni corrections for multiple comparisons; n=35-77/genotype).

C, D Longevity assays of (CGG)90-EGFP; Tub5-GS (Log-rank Mantel-Cox test with Bonferroni corrections for multiple comparisons; n=110-219/genotype) and (CGG)90-EGFP; ElaV-GS (n=147-299/genotype) flies with belle knockdown.

E Western blots of the FMRpolyG-EGFP RAN product in (CGG)90-EGFP; Tub5-GS flies with and without belle knockdown by two independent shRNAs.

F Quantitation of FMRpolyG-EGFP band density, normalized to β tubulin band density, from blots in E (Student's t test; n=4-5/genotype).

G Abundance of (CGG)90-EGFP mRNA normalized to RPL32 mRNA, following belle knockdown, determined by qRT-PCR (n=8/genotype).

H Western blot of AUG-driven EGFP in EGFP; Tub5-GS flies with and without belle knockdown.

I Quantitation of EGFP band density, normalized to β tubulin band density, from blot in H (n=4/genotype).

Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).
DDX3X and Specific Initiation Factors Modulate FMR1 Repeat-Associated Non-AUG Initiated translation (2019)
Figure 3

Figure 3. Knockdown of DDX3X Inhibits RAN Translation in Cultured Human Cells.

A Dose-response curves showing the effects of two independent anti-DDX3X siRNAs on the expression of AUG-NL-3xF (top) and (CGG)100 +1 NL-3xF (bottom) reporters. Plasmid-based reporters were transfected into HeLa cells 24 hours after knockdown, and reporter expression was quantified by luminescence. Nanoluciferase (NL) luminescence has been normalized to luminescence from firefly luciferase (FF), which was co-transfected, in order to control for transfection variability. Asterisks refer to comparisons between anti-DDX3X siRNAs and siRNAs against EGFP (siEGFP; two-way ANOVA with Dunnett's multiple comparisons test; n=12/condition).

B, C (CGG)n +1 and (CGG)n +2 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition).

D, E Western blots of FMRpolyG-NL-3xF and FMRpolyA-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.

Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).
DDX3X and Specific Initiation Factors Modulate FMR1 Repeat-Associated Non-AUG Initiated translation (2019)
Figure 4

Figure 4. DDX3X Facilitates Expression of RAN Products at the Level of Translation.

A Expression of in vitro transcribed AUG, +1 (CGG)100, and +2 (CGG)100 NL-3xF RNAs following DDX3X knockdown in HeLa cells, expressed as NL luminescence normalized to FF luminescence. (Student's t test with Bonferroni corrections for multiple comparisons; n=21/condition).

B Abundance of reporter mRNAs following DDX3X knockdown and plasmid-reporter transfection, determined by qRT-PCR. (n=7/condition.) This panel depicts data as means ± SEM.

C Expression of AUG-NL-3xF and +1 (CGG)100 NL-3xF in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X (two-way ANOVA with Tukey's multiple comparisons test; n=4/condition).

D Enrichment of HSPA1A and +1 (CGG)100 NL-3xF mRNA following anti-DDX3X RIP, relative to incubation with isotype control IgG. MALAT RNA, in contrast, is not enriched (Student's t test, n=3). Data from the additional replicate is presented in Appendix Figure S8A.

E Expression of +1 and +2 (CGG)100 NL-3xF plasmid reporters with and without an AUG inserted 5' to the CGG repeat, with and without DDX3X knockdown. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing either +1 or +2 (CGG)100 NL-3xF and those expressing the respective AUG-driven variant (two-way ANOVA with Tukey's multiple comparisons test; n=11-12/condition).

F Expression of NL-3xF plasmids with initiator AUG codons mutated to near-AUG codons, with and without DDX3X knockdown (two-way ANOVA with Dunnett's multiple comparisons test; n=18-24/condition). Black asterisks refer to comparisons between siEGFP-treated and siDDX3X-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter; white asterisks refer to comparisons between siDDX3X-treated cells expressing +1 (CGG)100 NL-3xF and those expressing a different reporter.

G Expression of in vitro transcribed near-AUG reporter RNAs in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X. Experiments with independent, replicate lysates are presented in Appendix Figure S7C (n=4/group).

Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).
DDX3X and Specific Initiation Factors Modulate FMR1 Repeat-Associated Non-AUG Initiated translation (2019)
Figure 5

Figure 5. EIF4B and EIF4H Modulate RAN Translation and General Translation in Drosophila and Cultured Human Cells.

A Representative photographs of GMR-GAL4; (CGG)90-EGFP fly eyes expressing manipulations of eIF4B and eIF4H. (B) Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with eIF4B/H manipulations (Mann-Whitney U test with Bonferroni corrections for multiple comparisons; n=26-55/genotype).

C, D Expression of plasmid-based AUG-NL and +1 (CGG)100 NL-3xF reporters (C), or co-transfected AUG-FF reporters (D), following knockdown of EIF4B or EIF4H. Black asterisks refer to comparisons between siEGFP- and siEIF4B/H-treated cells; pink and blue asterisks refer to comparisons between siEIF4B- (pink) or siEIF4H- (blue) treated cells expressing AUG-NL-3xF and those expressing +1 (CGG)100 (two-way ANOVA with Tukey's multiple comparisons test, n=9/condition).

E Expression of plasmid-based AUG-NL-3xF and (CGG)100 +1 NL-3xF reporters with and without over-expression of EIF4B, EIF4H, or both (two-way ANOVA with Dunnett's multiple comparisons test; n=20/condition). Asterisks refer to comparisons between cells over-expressing either EGFP or EIF4B, EIF4H, or EIF4B and EIF4H and expressing the same reporter.

Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).
DDX3X and Specific Initiation Factors Modulate FMR1 Repeat-Associated Non-AUG Initiated translation (2019)
Figure 6

Figure 6: EIF1 and EIF5 modulate RAN translation by determining AUG start-codon specificity.

A Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF1 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF1-transfected cells; green asterisks refer to comparisons between EIF1-transfected cells expressing AUG-NL-3xF and those expressing a different reporter.

B Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF5 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF5-transfected cells; pink asterisks refer to comparisons between EIF5-transfected cells expressing AUG-NL-3xF and those expressing a different reporter. For all panels, *** P≤0.001, **** P≤0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥3 replicates).
DDX3X and Specific Initiation Factors Modulate FMR1 Repeat-Associated Non-AUG Initiated translation (2019)
Figure 7

Figure 7. Knockdown of DDX3X Mitigates (CGG)100 Toxicity in Primary Rodent Neurons.

A Sample micrographs collected by automated longitudinal fluorescence microscopy, demonstrating the automated determination of cell death.

B Anti-DDX3X western blot of B35 cells transfected with either of two independent anti-DDX3X LNAs or a control LNA.

C Expression of EGFP in primary rat neurons transfected with (CGG)100 (+1) EGFP and either anti-DDX3X LNAs or a control (one-way ANOVA with Tukey's multiple comparisons test; n=2408-5689 cells/condition). All graphs depict pooled data, normalized first within the replicate.

D, E Transfection of anti-DDX3X LNA #1 (D) or #2 (E) reduced the cumulative risk of death in (CGG)100 (+1) EGFP-expressing neurons (Cox proportional hazard analysis; n=2408-3676 cells/condition).

Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).