EMBO/soda-nel huggingface dataset:
The Autophagy Receptor TAX1BP1 (T6BP) improves antigen presentation by MHC-II molecules. (2022)
Figure 4

Figure 4. T6BP silencing leads to perinuclear relocalization of the MIIC.

(A) MHC-II and T6BP expressions were assessed using confocal microscopy. HeLa-CIITA cells were transfected with control and T6BP-silencing siRNAs. 48h post-treatment, MHC-II and T6BP were detected using L243 and anti-T6BP antibodies, respectively, and revealed with species specific secondary antibodies. Nuclei were stained using DAPI.

(B) Quantitative analysis using in-house ImageJ script displaying distance of each MHC-II+ vesicles to the nucleus and number of vesicles per cell. At least 20,000 vesicles from 160 cells corresponding to five biological replicates were analyzed. Right panel, quantification in the siCTRL cells of the colocalization between MHC-II+ and T6BP+ dots using Pearson's coefficient where the dotted lines (at 0.5) indicate the limit under which no significant co-localization is measured (number of cells = 54).

(C) As in A, T6BP and LAMP-1 expressions were analyzed.

(D) The localization of LAMP-1+ vesicles and the number of vesicles per cell were quantified as in B. At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed.

(E) As in A, adding Lysotracker staining.

(F) As in B, quantitative analysis of Lysotracker+ vesicles: localization to the nucleus and number of vesicles per cell. Colocalization of Lysotracker+ vesicles with MHC-II+ puncta was analyzed using JACoP plugin (scales start at 0.5 above which the % of co-localization is considered significant), number of vesicles > 2000 from at least three biological replicates were analyzed corresponding to 20 cells.

(G) As in E, CD63 and MHC-II expressions were assessed.

(H) Quantitative analysis as represented in F. At least 2000 vesicles from 20 cells corresponding to two biological replicates were analyzed.

Data information: In graphs representing the number of vesicles per cells, each dot displayed corresponds to a single cell. Within the violin plots, continuous and dotted lines correspond to medians and quartiles, respectively. Scale bars: 2μm. CTRL: control. Mann-Whitney's test; * p<0.05; ** p<0.002; *** p<0.0003; **** p<0.0001; # p>0.05.
The Autophagy Receptor TAX1BP1 (T6BP) improves antigen presentation by MHC-II molecules. (2022)
Figure 5

Figure 5. T6BP silencing leads to exacerbated CD74 (Ii) degradation.

(A) CD74 expression was assessed using Western Blot. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48h post treatment, the Iip33 CD74 isoform, a degradation product (Iip16), T6BP and actin were detected using indicated antibodies. Left panel, a representative Western Blot experiment is shown. Right panel, expressions levels five biological replicates were quantified using ImageJ and are presented as mean of ratios (+/-SD) of CD74 to actin used as control housekeeping gene expression.

(B) As in (A) assessing HLA-DR and actin expression levels. Top panel, a representative Western Blot experiment is shown. Bottom panel, in three biological replicates, HLA-DR expression was quantified using ImageJ and is presented as a mean of ratios to actin (+/-SD).

(C) As in (B) assessing HLA-DM and actin expression levels. Bottom panel, HLA-DM expression was quantified in three biological replicates and presented as mean of ratios to actin (+/-SD).

(D) CD74 expression is partially recovered by blocking lysosomal acidification. As in (A), following siRNA transfection of Hela-CIITA cells, CD74 expression was assessed. The last 16h prior to harvesting, cells were treated with chloroquine (CQ) or epoxomicin (Epo). Left panel a representative experiment is shown, membranes were blotted using anti-T6BP, -CD74 and -actin antibodies. The different degradation fragments of CD74 are indicated (Iip33, Iip22 and Iip16). Right panel, expression levels of Iip33 (top) and Iip16 (bottom) were quantified in three biological replicates using ImageJ and are presented as mean of ratios of each fragment to actin (+/-SD).

(E and F). Analysis of CD74 proteolysis. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48h post treatment, cells were pulsed for 30 min with 35S-Met/Cys, washed and chased for 1, 2 and 4h. HLA-DR/CD74 complexes were first immunoprecipitated using Tü36 antibody (E) and then reimmunoprecipitated with VICY1 antibody (F). Samples were boiled and analyzed using SDS-PAGE. The bands corresponding to CD74 isoforms (Iip41 and Iip33) and the cleavage products (Iip16) are indicated. (E right panel) Iip16 expression was quantified using ImageJ and presented as a percentage of Iip16 normalized to the highest quantity of Iip16 detected after 1h of chase in the control condition. Results are representative of two biological replicates. Ii: invariant chain (CD74); CTRL: control.

Data information: (A-C)Wilcoxon's test; * p<0.05; # p>0.05. (D) One-way Anova statistical test combined with Bonferronis multiple comparison test was applied. Dotted lines indicate statistically significant differences between conditions.
Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition (2016)
Figure 1

Figure 1. hnRNPA3 modulates repeat RNA and poly-GA expression.

(A, B) Knockdown of hnRNPA3 increases poly-GA expression while expressions of EGFP protein levels are not altered upon knockdown of hnRNPs. The control (“x0”) vector lacks the G4C2 repeats but still contains the 5’ flanking region and 3x TAG. ANOVA, Dunnett. (C, D) Overexpression of hnRNPA3 suppresses poly-GA expression. n=2 experiments performed in duplicates. ANOVA, Tukey. (E) Increased repeat RNA upon hnRNPA3 knockdown. EGFP RNA levels are only slightly increased as compared to levels of the repeat RNA. n=2 experiments performed in duplicates. ANOVA, Dunnett. (F, G) Knockdown of hnRNPA3 increases RNA foci. n=3 replicates; two tailed paired t-test, Scale bar=20 μm. All graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.
Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition (2016)
Figure 2

Figure 2. RNA binding and nuclear transport are required for hnRNPA3 mediated repression of poly-GA production

(A-C) Rescue of repression of poly-GA and repeat RNA by wild type (wt) hnRNPA3 and hnRNPA2 but not by the RNA binding mutant hnRNPA3DxD. (B) n=3 experiments performed in duplicates; (C) n= 2 experiments performed in duplicates. ANOVA, Tukey. (D-F) Rescue of repression of poly-GA and repeat RNA by hnRNPA3WT but not the M9-NLS deletion mutant. (E) n=3 experiments performed in duplicates; (F) n=4 experiments performed in duplicates. ANOVA, Tukey. All Graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001 (See also Figure EV1).
Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition (2016)
Figure 3

Figure 3. DPR expression and deposition.

(A) poly-GA aggregates co-localize with p62-positive deposits. (B) Redistribution of nuclear TDP-43 to the cytoplasm in a poly-GA/poly-GR double-positive cell. Note that nuclei of poly-GA/poly-GR double positive cells are frequently disrupted. (C) Knockdown of hnRNPA3 increases repeat RNA expression. n=2 experiments performed in duplicates. ANOVA, Dunnett. (D) Filter trap assay probed with anti-FLAG, anti-myc or anti-HA antibodies reveal abundant DPR production. (E) Relative expression level of poly-GA, poly-GP and poly-GR normalized to GR. n=3 experiments, ANOVA, Tukey. (F) Expression of all the three DPRs is increased upon knockdown of hnRNPA3. Signals from knockdown with siRNA#8 were normalized to 1. n=3 experiments, ANOVA, Dunnett. (G) Co-expression of two/three different DPRs in single cells is increased upon hnRNPA3 knockdown. n=3 replicates, ANOVA, Dunnett. All graphs are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Scale bars=20μm. (see also Figure EV2 and EV3).
Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition (2016)
Figure 4

Figure 4. Knockdown of hnRNPA3 induces neuronal DPR accumulation.

Rat hippocampal neurons at DIV3 were transduced with lentivirus coexpressing either hnRNPA3 targeting shRNA (shA3) or a control shRNA (shCtrl) and tagRFP. Three days after transduction (DIV3+3), neurons were transfected with (G4C2)80HIGH(+0) and analyzed at (DIV3+7). (A) Neurons were fixed, immunostained and imaged by confocal microscopy. Double immunofluorescence for poly-GA aggregates (green) and tagRFP (red). Nuclei were labeled with DAPI. Scale bar represent 20 μm. (B) Efficient knockdown of endogenous rat hnRNPA3 with its targeting shRNA. (C) Poly-GA aggregates were detected in a filter trap assay using an anti-Flag antibody. (D) The amounts of poly-GA aggregates were quantified and are presented as the fold change of signals from neurons treated with the control shRNA or the repeat construct. Means ± SD of three independent experiments are shown. * p<0.05 by a Student’s t-test. n= 3 replicates.
Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition (2016)
Figure 5

Figure 5. Reduction of hnRNPA3 leads to enhanced formation of nuclear RNA foci in fibroblasts derived from patients with C9orf72 repeat extensions

(A) Fibroblasts from three individual patients with confirmed C9orf72 repeat expansions were investigated. (B) Knockdown of hnRNPA3 with two independent siRNA results in selective depletion of hnRNPA3 in fibroblasts from 3 C9orf72 repeat carriers. Actin was used as a loading control. (C) Knockdown of hnRNPA3 increases RNA foci in cells derived from C9orf72 carriers. No G4C2 repeat RNA foci are detected in fibroblast without C9orf72 repeat expansions (WT). Nucleoli were stained with anti nucleolin antibodies (green) and nuclei were stained with DAPI (blue). (D) Quantification of the relative frequency of RNA foci positive cell (RNA foci positivity) (fold change). n=3 experiments for each case. (E) Quantification of the number of RNA foci per RNA foci-positive cell (fold change). n=3 experiments for each case. (D, E) The average foci number of non-treated (N/T) fibroblast was normalized to 1 in each case. Bars indicate mean. Individual points indicate mean values obtained from a single experiment (49-214 cells were counted in single experiment). Error bars indicate 95%CI, ANOVA, Tukey-Kramer HSD, *p<0.05, **p<0.01, ***p<0.001. (F) Representative presentation of increased RNA foci in C9orf72 carriers upon lentiviral-mediated hnRNPA3 knockdown. (G) Quantification of the number of RNA foci (fold change). 3 cases were analyzed. Single points indicate an average obtained from 29-30 cells per case. Color code labels values obtained in fibroblasts derived from the three individual patients. Two-tailed t-test. Mean ± SEM. Scale bar= 10 μm.
Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition (2016)
Figure 6

Figure 6. Nuclear hnRNPA3 reduction correlates with poly-GA accumulation in patients with C9orf72 repeat expansions

(A) Double immunofluorescence staining with anti-GA (green) and anti-hnRNPA3 (red) antibodies of the granular layer of the dentate gyrus of a control case and three C9orf72 mutation cases. In C9 mutation cases with low nuclear hnRNPA3 expression (#8 & #1) more poly-GA aggregates were observed as in cases with high nuclear hnRNPA3 (#7). Inserts show examples of co-localization of poly-GA and hnRNPA3 aggregates. (B) Granular layer neurons of a C9orf72 mutation case with (white arrows) or without (orange arrows) nuclear clearance of hnRNPA3. (C) poly-GA aggregates are more frequent in C9orf72 mutation cases with lower nuclear hnRNPA3 levels than in those cases with higher nuclear hnRNPA3 levels (divided by median of nuclear hnRNPA3 intensities in 34 C9orf72 mutation cases into two subgroups). Bar graph indicates mean value. Error bars indicate 95% CI. Single points indicate mean from three micrographs per case. Note that the difference in GA positivity between both groups remains significant (p = 0.0086) after removal of the highest outlier in the low nuclear A3 group. Two-tailed t-test. Scale bar = 10 μm.
Endophilin A2 regulates B cell endocytosis and is required for germinal center and humoral responses (2021)
Figure 3

Figure 3. Endophilin A2 is required for peripheral B cell development and GC responses.

(A) CRISPR-targeted bone marrow chimera workflow, showing flow cytometry detection of donor-derived (GFP+), sgRNA-containing (mCherry+) cells.

(B) Development of B cell populations in chimeras' bone marrow and spleen from Cd4- or Sh3gl1-targeted HSCs. Percentage of mCherry+ cells at each stage is normalized to initial infection rate of transferred lineage- cells, and shown as mean + SEM. N =11-12 mice. *p=0.0286, ***p<0.0001 using two-way ANOVA.

(C) MZ development in mCherry+ B cell compartment.

(D) Representative stain of surface CD86 and CD69 on adoptively transferred HEL-specific follicular B cells 24 hours post SRBC-HEL immunization.

(E) Quantification of surface CD86 and CD69 mean fluorescence intensity (MFI). Data show mean and SEM from 6 mice across 2 experiments.

(F) Fractions of mCherry+ cells in GC, PC, IgG1+ class-switched and follicular B cells at 4-, 8- and 14-days post SRBC-HEL immunization. N = 8-10 mice; data shows mean and SEM. *p<0.05; ***p<0.001; ****p<0.0001 using 2-way ANOVA with multiple comparisons.
Endophilin A2 regulates B cell endocytosis and is required for germinal center and humoral responses (2021)
Figure 4

Figure 4. Endophilin A2 knockout mouse mimics the phenotype of CRISPR-targeted chimeras and exhibits reduced levels of affinity matured serum antibodies upon immunization.

(A) Characterization of B cell populations in bone marrow, spleen and lymph node at steady state. Data show mean and SEM from N = 5 mice. P, significance in t tests performed for follicular (FO) and MZ cell counts.

(B) Spleen size in Sh3gl1-/- and WT littermates at 12 weeks of age.

(C) Surface levels of MHCII, BAFFR, CD40 and LPAM1 in Sh3gl1-/- and WT littermates. N = 4-5 mice. P, statistical significance from unpaired t tests. Data show mean ± SEM.

(D) Quantification of B220+Fas+CD38- GC population as percentage of total splenic B cells and NP-specific cells as a percentage of the GC population 14 days after NP-CGG immunization in alum. Data show mean ± SEM from N = 6 mice, p, significance in unpaired t tests.

(E-F) Serum antibodies collected pre-immunization and 7- or 14-days post NP-CGG immunization, detected by NP7-BSA ELISA using isotype-specific secondary antibodies. Data show mean ± SEM from N = 3 experiments with statistical significance from 2-way ANOVA with multiple comparisons.

(G) Ratio of binding to NP7 and NP25, as measured by ELISA. Data show mean ± SEM; N = 4-6 mice. Statistical significance from 2-way ANOVA with multiple comparisons.

(H) Numbers of Tfh cells 14 days post-immunization, identified as a CD4+CD44+PD-1+CXCR5+ population. Mean and SEM of N = 4 mice.
Endophilin A2 regulates B cell endocytosis and is required for germinal center and humoral responses (2021)
Figure 5

Figure 5. Endophilin A2 positively regulates BCR internalization but is dispensable for antigen presentation.

(A) Soluble anti-IgM internalization in CRISPR-targeted follicular B cells. N = 11- 12 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA.

(B) Soluble anti-IgD internalization. N = 3 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA.

(C) Soluble anti-IgG internalization in GC B cells from SRBC-immunized mice. N = 4 mice. Data show mean ± SEM. P, statistical significance from two-way ANOVA.

(D) Internalization of anti-IgM from PMS quantified in fixed naïve B cells at 10 min post synapse formation. N = 172 and 97 cells analyzed in one out of 2 representative experiments. Data show mean ± SEM. P = 0.0001 in unpaired t test.

(E) Surface IgM or IgD MFI relative to WT in naïve splenic follicular B cells. N = 3 mice. Data show mean ± SEM.

(F) IgM degradation following total cell surface biotinylation, measured as loss of biotin-labelled membrane IgM (mIgM) in cell lysates over time.

(G) Western densitometry quantifying IgM degradation assay. Proportion of biotin detected is normalized to the initial WT condition (left) or to the initial amount for each genotype (right). Data show mean ± SEM from three independent tests. P, statistical significance from two-way ANOVA.

(H) MHCII antigen presentation in SWHEL B cells 24 hours post adoptive transfer and immunization with Ea-HEL-SRBC conjugate as detected by anti-Y-Ae surface stain.

(I) Relative anti-Y-Ae MFI in CRISPR-targeted B cells normalized to HEL-only immunized mice. N = 10 immunized mice across 3 experiments. Data show mean ± SEM.

(J) In vitro antigen presentation in CRISPR-targeted B cells measured by anti-Y-Ae. N = 8 independent cultures in 2 experiments. Data show mean ± SEM.

(K) Proliferation (CFSE dilution) of OTII CD4 cells in co-cultures with anti-Igκ-OVA pulsed B cells; and quantification from 6-8 mice. Data show mean ± SEM.

(L) Relative anti-Y-Ae MFI in GC from immunised wildtype and Sh3gl1-/- mice. N = 11 from 2 experiments. Data show mean ± SEM.
Endophilin A2 regulates B cell endocytosis and is required for germinal center and humoral responses (2021)
Figure 6

Figure 6. Endophilin A2 is required for B cell iron-dependent oxidative phosphorylation and cell expansion.

(A) CRISPR-targeted follicular B cell proliferation after 3 days of CD40L stimulation. mCherry+ populations were quantified within individual DDAO peaks. N = 3 mice. P, statistical significance calculated by two-way ANOVA reached p<0.0001 for all proliferation peaks.

(B) CD80 mRNA levels in B cells following 24 hours of CD40L culture, relative to untreated B cells. Data from RNAseq of N = 3 mice.

(C-D) Isolated naïve B cells from CRISPR-targeted chimeras after 3-day culture in stated cytokines. (C) mCherry percentage in targeted primary cell cultures. N = 15 mice. *p<0.05, **p<0.01, ****p<0.0001 using two-way ANOVA. (D) AnnexinV surface stain in targeted cells. N = 3-4 mice.

(E) mCherry+ cell percentage in CRISPR-targeted Ramos cells over time. N = 6 independent experiments.

(F) Cell counts at passage 3-6 of CRISPR-targeted puromycin-selected Ramos cells. N = 4 independent infections. P, statistical significance from unpaired t test.

(G) Oxygen consumption rate (OCR) in CRISPR-targeted Ramos cells. N = 2 independent infections. P, two-way ANOVA of basal and maximum OCR.

(H) OCR in B cells isolated from Sh3gl1-/- and WT littermates. N = 9 mice. P, two-way ANOVA of basal and maximum OCR.

(I) OCR in T cells isolated from Sh3gl1-/- and WT littermates. N = 4 mice.

(J-M) Cell cultures with full RPMI media supplemented with 50 μg/ml FeSO4 or 5 μM hemin chloride. (J) SH3GL1-targeted Ramos cells. N = 4 independent infections. P, statistical significance from two-way ANOVA from both treatments versus RPMI. (K) Control sgRNA-targeted Ramos cells. (L) Sh3gl1-/- isolated B cells, maintained in CD40L culture. N = 3 mice. P, significance calculated by two-way ANOVA.

(M) B cells isolated from WT littermates, maintained in CD40L culture.

(N) OCR in Sh3gl1-/- or WT B cells after overnight culture in CD40L, supplemented with 5 μM hemin chloride. N = 6 mice. P, two-way ANOVA with matched samples for basal and maximum OCR

(O) TfR internalization assay in Ramos cells using biotinylated anti-CD71. 4 independent CRISPR infections in 2 experiments. P, significance calculated using two-way ANOVA and indicated in corresponding color.

(P,Q) Mouse transferrin internalization in B cells from SRBC-immunized WT and Sh3gl1-/- littermates. N = 7 mice per genotype in 2 experiments. P, statistical significance analyzed using two-way ANOVA. (P) Naïve B cells. (Q) GC B cells.

Data information: All data show means ± SEM.

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The eukaryotic replisome tolerates leading-strand base damage by replicase switching (2021)
Figure 1

Figure 1. The replisome is inherently tolerant of a leading-strand Tg lesion.

(A) Schematic of the ARS306 leading-strand lesion template showing the origin of replication (Ori) and leading- (red) and lagging-strand (blue) replication products. The SmaI restriction site is shown; cleavage is used to reduce heterogeneity in product sizes for denaturing gel analysis.

(B) Schematic of the uncoupled fork and uncoupled product generated by persistent stalling of leading-strand synthesis.

(C, D) Standard replication assays on undamaged (UD), thymine glycol (Tg), and cyclobutane pyrimidine dimer (CPD) templates in the presence (C) and absence (D) of Pol η, Pol ζ, Rev1, Rad6-Rad18, Uba1, and ubiquitin (TLS machinery). Reactions contained 2.5 nM Pol δ.
The eukaryotic replisome tolerates leading-strand base damage by replicase switching (2021)
Figure 2

Figure 2. A leading-strand Tg causes uncoupling.

(A) Schematic showing the possible replication products and oligonucleotide restart products for the assay in (B). Green lines represent the restart oligonucleotides and the dashed line indicates extension of the stall product which will occur due to bypass of the Tg after oligonucleotide binding.

(B) Oligonucleotide competition assay on UD and Tg templates. S; scrambled oligonucleotide.

(C) Lane profiles of lanes 4-6 in (B) normalised to the run off product. FL; FL-lead, OR; oligonucleotide restart, S; Stall, RO; Run off, OF; Okazaki fragments.

(D) Pulse chase reaction on UD and Tg templates performed as shown. Where present, Pol δcat (10 nM) was added directly after addition of the chase.

(E) Standard replication reaction performed in the absence and presence of RFC/PCNA on the Tg template.
The eukaryotic replisome tolerates leading-strand base damage by replicase switching (2021)
Figure 3

Figure 3. Pol δ facilitates bypass of a leading-strand Tg.

(A) Outline of pulse chase reaction used in (B-E).­­

(B) Pulse chase reactions on the Tg template in the absence and presence of 10 nM Pol δ analysed on a denaturing gel.

(C) Schematic of stall and bypass products generated by SwaI and BamHI cleavage (D and E).

(D) Pulse chase reaction on the Tg template in the absence and presence of 10 nM Pol δ analysed on a urea polyacrylamide gel.

(E) Titration of Pol δ in a pulse chase reaction on the Tg template. Pol δ was added after the 0 min time point was taken.

(F) Quantification of bypass 5 min after addition of the chase for the data in (E).

(G) Titration of Pol δ or Pol ε (1, 2.5, 5, 7.5, 10 nM)­­ into a primer extension assay on a Tg template in the presence of PCNA and RFC. The primer/template sequence and location of the lesion are shown above.
The eukaryotic replisome tolerates leading-strand base damage by replicase switching (2021)
Figure 4

Figure 4. Pol δ likely performs insertion and extension during Tg bypass

(A) Schematic showing insertion and extension at the Tg site in the sequence context used in replication assays.

(B) Mapping the 3ʹ-end of the stalled nascent leading strand. Pulse chase reaction on Tg and CPD templates. Where present, Pol δ (10 nM) was added immediately after the 0 min time point. Products were cleaved with SwaI and BamHI and resolved alongside a sequencing ladder corresponding to the nascent strand. dd; dideoxy.
The eukaryotic replisome tolerates leading-strand base damage by replicase switching (2021)
Figure 5

Figure 5. Tg bypass in the absence and presence of Pol δ is error-free.

(A) Primer extension reactions on the Tg template with 5 nM Pol δ or Pol ε and either dATP, dCTP, dGTP, or dTTP.

(B) Outline of the method used to isolate and sequence replicated leading strands from reconstituted replication reactions.

(C) Analysis of nucleotide incorporation opposite Tg from replication reactions processed as shown in (B). Three individual experiments were performed, two in the presence and one in the absence of Pol δ (10 nM), giving 99, 97, and 95 successful sequencing reads respectively.