EMBO/soda-nel huggingface dataset:
The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection. (2020)
Figure 4

Figure 4. The IRE1α/XBP1s pathway modulates cytokine responses in infected BMDCs and cross-presentation of soluble secreted parasite antigens.

(A) Dotplots showing the percentage of CD11c+/MHCIIhi cells in Pru Tomato SAG1‐OVA parasite infected XBP1fl/fl, XBP1ΔDC, XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC BMDCs (upper panels) and of Tomato-positive cells (percentage of infected BMDCs: lower panels).

(B) Mean percentage ± S.E.M of infection is indicated (n=6 mice pooled from 2 independent experiments ). Tukey's ANOVA one-way test, ns: p>0,05.

(C) Mean percentage of parasitophorous vacuoles containing 1, 2, 4 or 8 parasites/vacuole (p/vac) in XBP1fl/flIRE1fl/fl, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs. Tukey's ANOVA one-way test, ns: p> 0,05. Mean values ± S.E.M. n=3 independent experiments (biological replicates).

(D) IL-6, IL-12 and IL-23 secreted cytokine levels from Pru parasite infected XBP1fl/fl, XBP1ΔDC, XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC BMDCs measured by ELISA. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; ***: p<0.001; ****: p<0.0001; mean ± S.E.M (n=6 mice pooled from 2 independent experiments).

(E) mRNA relative expression level of Il-6, Il-12 and Il-23 from XBP1fl/flIRE1fl/fl, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru parasites for 16 h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).

(F) H-2Kb‐SKL8 MHC class I presentation by XBP1fl/flIRE1fl/fl treated or not with 4µ8C, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru Tomato SAG1‐OVA at the indicated ratios, assessed with the B3Z T cell hybridoma. Unpaired Student T-test, *: p<0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments).

(G) Percentage of Pru Tomato SAG1-OVA infected BMDCs at the indicated ratios of parasites:BMDC. Bar graphs depict mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments). Tukey's ANOVA one-way test, ns (p>0,05) when p values are not identicated.

(H) Exogenous MHCI presentation of synthetic SKL8 peptide pulsed on XBP1fl/flIRE1fl/fl treated or not with 4µ8C, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs assessed with the B3Z T cell hybridoma. Bar graphs depict mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments). Unpaired Student T-test, *: p<0.05, ns (p>0,05) when p values are not identicated.

(I) Graph depicts the expression (indicated as the Mean Fluorescence Intensity) of H-2Kb by Pru Tomato SAG1‐OVA infected XBP1fl/fl IRE1fl/fl treated or not with 4μ8C, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs. Bar graphs depict mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments). Tukey's ANOVA one-way test, ns: p>0.05.
The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection. (2020)
Figure 5

Figure 5: The IRE1α/XBP1s pathway is activated in splenic cDC1 of mice infected with T. gondii.

(A, B) Expression of VenusFP by splenic cDC1 (CD26+ CD11c+ MHCII+ XCR1+ CD172a-, upper panels) and cDC2 (CD26+ CD11c+ MHCII+ XCR1- CD172a+, lower panel) from WT (A) and ERAI (B) mice non-infected (NI) or infected with Pru (6 days post-infection (dpi)) reporting on IRE1α pathway activation.

(C) Graph depicts the Mean Fluorescence Intensity measured for each condition in ERAI mice (6 dpi). Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; ***: p<0.001; ****: p<0.0001; mean ± S.E.M (n=5 mice per genotype and condition, one representative experiment from 2 independent experiments is shown).

(D) Graph depicts the expression (indicated as the Mean Fluorescence Intensity) of VenusFP by splenic monocytes, neutrophils, B and T cells from Pru infected or non-infected (NI) ERAI mice (6 dpi). Bar graphs depict mean ± S.E.M (n=5 mice per genotype and condition, one representative experiment from 2 independent experiments is shown). Unpaired Student T-test, ns: p>0,05.

(E, F) RT-qPCR analysis of the indicated UPR genes among total RNA prepared from splenic cDC1 (E) and cDC2 (F) sorted by flow cytometry 6 dpi from wild-type mice, non-infected (NI) or infected with Pru parasites. Results were normalized to the housekeeping gene Gapdh and are expressed as relative mRNA level compared to the NI condition. Bar graphs depict mean ± S.E.M. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).
The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection. (2020)
Figure 6

Figure 6. The IRE1α pathway protects mice from acute infection and promotes T cell responses.

(A) Survival curves of XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru parasites. Log-rank (Mantel-Cox) test, p=0,0004; n=10 mice per genotype.

(B) Number of parasite genomes per spleen and PEC from XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC infected intraperitoneally with 500 Pru parasites 6 days post-infection. Unpaired Student T-test **: p<0.01; mean ± S.E.M (n=2 independent experiments combined).

(C) mRNA relative expression of Il-6, Il-12, Ifn-g and Tnf-a by splenocytes from XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC mice infected or not (NI) with 500 Pru parasites for 6 days. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ****: p<0.0001; mean ± S.E.M (n=2 independent experiments combined).

(D, E) Quantification of the number of total (left histogram) and activated (right histogram) (IFNγ-positive) CD4+ T cells (D) and CD8+ T cells (E) in the spleen of control mice (XBP1fl/flIRE1fl/fl) and XBP1ΔDC IRE1truncDC infected for 6 days. Two-way ANOVA Tukey's multiple compare, ns: p> 0.05; *: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; mean ± S.E.M (n=2 independent experiments combined). (F) Frequency and number of cDC2 (left) and cDC1 (right) in the spleen of control mice and XBP1ΔDC IRE1truncDC infected for 6 days. Two-way ANOVA Tukey's multiple compare, ns: p> 0.05; ****: p <0.0001; mean ± S.E.M (n=2 independent experiments combined).
Sororin actively maintains sister chromatid cohesion (2016)
Figure 1

Figure 1. ESCO1 and ESCO2 are required for stable association of cohesin with chromatin

(A) Western blot of SMC3-LAP expressing HeLa cells treated with siRNA as indicated and synchronized in G2-phase. Efficiency of RNA interference and SMC3 acetylation levels were analyzed from total extracts and after chromatin fractionation (asterisks indicate unspecific signals that were depleted from antibody dilutions used in later experiments, presumably by repeated usage of the antibody samples). Note that ESCO1 levels were increased on chromatin after depletion of ESCO2, raising the possibility that a decrease in chromatin bound ESCO2 might be compensated for by ESCO1 recruitment to chromatin.

(B) Still images of an inverse fluorescence recovery after photobleaching (iFRAP) experiment. Fluorescence intensities are false-colored as indicated. Size bar, 5 μm.

(C) Graph depicting the normalized intensity after photobleaching from two biological replicates to quantify chromatin dissociation kinetics. Error bars denote standard error of the mean (s.e.m.), n = 10 cells per condition.

(D) Quantification of the relative abundance of SMC3-LAP in the stable chromatin binding mode.
Sororin actively maintains sister chromatid cohesion (2016)
Figure 2

Figure 2. SMC3 acetylation site mutants generate stable cohesin-chromatin interactions without ESCO1 and ESCO2

(A) Quantification of iFRAP experiments using HeLa cells expressing SMC3-LAP in the wild-type form (wt) or with lysines 105-106 mutated to glutamines (QQ) or arginines (RR) and synchronized in G1-phase. Error bars denote s.e.m., n > 25 cells per condition.

(B) Quantification of chromatin residence time of SMC3-LAP alleles in G1-phase.

(C) Quantification of iFRAP experiments using cells synchronized in G2-phase. Error bars denote s.e.m., n > 26 cells per condition.

(D) Quantification of stably bound SMC3-LAP alleles in G2-phase.

(E) Western blot of mouse fibroblasts expressing SMC3-LAP in the presence or absence of endogenous Smc3 after gene deletion. Extracts were prepared after subculturing two times. Cells without SMC3-LAP do not proliferate after deletion of Smc3.

(F) Western blot showing HeLa cell extracts after treatment with siRNA as indicated and synchronized in G2-phase. Note that ESCO1 immunoblot signals were also reduced in sororin depleted cells. We currently do not know if this is an effect of sororin depletion, an off-target effect or an artefact of unequal sample loading.

(G) iFRAP quantification of stably chromatin bound SMC3-LAP after siRNA treatment using cells synchronized in G2-phase. Error bars denote s.e.m., n > 11 cells per condition.
Sororin actively maintains sister chromatid cohesion (2016)
Figure 3

Figure 3. Sororin cannot stabilize cohesin–chromatin interactions before DNA replication

(A) FACS profile of cells transfected with nondegradable KEN-box mutant sororin-FLAG (sorKBM-FLAG) after synchronization in G1-phase and propidium iodide staining.

(B) Immunofluorescence microscopy experiment to determine transfection efficiency.

(C) Western blot of fractionated lysates to detect sororinKBM-FLAG.

(D) Quantification of SMC3-LAP residence time on chromatin after transfecting cells with sorKBM-FLAG in G1-phase. Error bars denote s.e.m.; n > 15 cells per condition.

(E–F) Analysis of cell cycle distribution and transfection efficiency using cells synchronized in G2-phase.

(G) Quantification of stably chromatin-bound SMC3-LAP from cells expressing nondegradable sororin in G2-phase. Cells were treated with siRNA as indicated.

(H) Western blot showing fractionated and nuclease digested chromatin from cells synchronized in G1 and G2-phase after immunoprecipitation with FLAG antibodies.
Sororin actively maintains sister chromatid cohesion (2016)
Figure 4

Figure 4. The genome-wide association of sororin with cohesin occurs exclusively on replicated DNA

(A) Examples of sororin (dotted arrows) and SMC3 ChIP-seq and BrdU DIP-seq data from early S-phase as compared to G2-phase using the Integrated Genome Browser (IGB; Nicol et al, 2009).

(B) Alignment of sororin, BrdU and BrdU-negative control sequencing bins in early S-phase to human chromosome 18 (GRCh37/hg19). Bars correspond to called peaks, curves to read enrichments after smoothing (k=25 kbp).

(C) Quantification of the co-occupancy of BrdU incorporation and sororin localization in early S-phase and sororin in G2-phase depicted as p-value distributions calculated with IntervalStats (Chikina & Troyanskaya, 2012) and compared to randomized controls. Sororin peaks were identified as overlapping peaks of 4 samples in early S-phase and 2 samples in G2-phase, using only the common peaks.

(D) Quantification of the co-occupancy of SMC3 in early S-phase and in G2-phase with BrdU incorporation in early S-phase.
Sororin actively maintains sister chromatid cohesion (2016)
Figure 5

Figure 5. The CDCA5 gene encoding sororin is essential for development, cell proliferation and proper cohesion

(A) Schematic representation of wild-type (wt), targeted (flx) and deleted (Δ) Cdca5 alleles (left) and Southern blot of mouse genomic DNA (right). Red triangles, loxP sites.

(B) Observed and expected genotype distribution among offspring of heterozygous Cdca5 Δ/+ mice (newborn mice, n = 136; E9.5, n = 54).

(C) Gene deletion and G0 phase synchronization protocol for mouse embryonic fibroblasts isolated from Cdca5 flx/flx mice expressing ERT2-Cre.

(D) Western blot of cell extracts to analyze depletion of sororin protein from fibroblasts after tamoxifen-induced Cre expression. Extracts were prepared either one or two days after release from the arrest as indicated. Chr, fractionated chromatin; sol, soluble proteins.

(E) Proliferation curve of cells with the indicated genotype after tamoxifen treatment.

(F) Observed cell cycle distribution of fibroblasts treated with tamoxifen under starvation and subcultured in rich medium.

(G) Quantification of mitotic cells with misaligned chromosomes after Cdca5 deletion and proliferation for 36 to 60 hours. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition.

(H) Phenotypic classification of nuclear morphology after Cdca5 deletion and proliferation for 5 to 7 days. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition.

(I) Western blot showing protein depletion efficiency. Cre expression was induced with tamoxifen for 24 hours in proliferating Smc3 and Cdca5 conditional knockout immortalized fibroblasts.

(J) Analysis of chromosome spreads after Smc3 or Cdca5 deletion and nocodazole treatment. Representative images from every category are shown; size bar, 10 µm; error bars denote s.e.m.; n > 500 per genotype.
Sororin actively maintains sister chromatid cohesion (2016)
Figure 6

Figure 6. A sororin degron allele supports viability and sister chromatid cohesion in mouse fibroblasts

(A) Schematic illustration of the LAP-AID-tag integrated into the bacterial artificial chromosome containing the mouse Cdca5 locus.

(B) Immunofluorescence staining of cells expressing sor-LAP-AID and the F-box helper protein after treatment with auxin. Antibodies against GFP mark the fusion protein and Aurora B antibody stains cells in G2/M-phase. Scale bar, 10 µm.

(C) Quantification of immunofluorescence after treating cells with auxin. Error bars denote s.e.m.; a.u., arbitrary units.

(D) Time course quantification of GFP immunofluorescence signal decrease after auxin addition.

(E) Analysis of chromosome spreads after auxin treatment.

(F) Western blot showing cell extracts after treatment with auxin.
Sororin actively maintains sister chromatid cohesion (2016)
Figure 7

Figure 7. Sororin turns over rapidly on chromatin

(A) Cell cycle distribution of immortalized Cdca5 Δ/Δ sor-LAP-AID fibroblasts after 24-hour incubation with aphidicolin and release.

(B) Western blot showing cell extracts after incubation with aphidicolin and release.

(C) Still images of a fluorescence recovery after photobleaching experiment. Size bar, 5 μm. Bleach area radius, 2 µm.

(D) Graph depicting the normalized intensity after photobleaching to quantify turnover of sororin (n = 15).

(E) Quantification of the relative abundance of sororin-LAP-AID on chromatin. Error bars denote s.e.m.

(F) Quantification of the chromatin residence time of sor-LAP-AID at different times. Error bars denote s.e.m.
Single-cell RNA sequencing of motoneurons identifies regulators of synaptic wiring in Drosophila embryos (2022)
Figure 4

Figure 4: Ectopic homeodomain TF expression changes muscle target choice in a predictive manner.

(A) Left panel: Schematic drawing of a stage 17 Drosophila embryonic head, highlighting the stereotypic innervation of the anterior feeding muscles by specific MNs, MHE (green) by MN2a, which only expresses Dfd, MHD (red) by MN3, which only expresses Scr, and LR (purple) by MN2c, which only expresses Lab. Right panel: Representative confocal images of early stage 17 Drosophila embryonic heads, highlighting the expression pattern of the anterior Hox TFs Lab, Dfd and Scr, showing that Lab is expressed in the LR muscle, Dfd in the MHE muscle and Scr in the MHD muscle. The muscles are indicated by white dashed lines.

(B) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and animals mis-expressing the Hox TFs Lab, Dfd and Scr by means of the elav-GAL4 driver. A zoom on the projections (FasII staining) of MN2a and MN3 to the MHE and MHD muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions.

(C) Quantification of different phenotypes manifesting in MN2a and MN3 after Hox mis-expression in comparison to control animals: MN2a axons projecting to the MHE muscle, representing the wild-type (wt) innervation pattern of MN2a (magenta); MN3 axons projecting to the MHD muscle, representing the wild-type (wt) innervation pattern of MN3 (cyan); MN2a or MN3 axons projecting to muscles other than the MHE or MHD termed wiring defects (dark grey); abnormal synaptic morphologies at MN2a or MN3 derived axon terminals termed terminal defects (light grey). Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi. Each experiment was performed in triplicates, innervation rates were calculated from n=16 for elav>lab, n=18 for elav>Dfd and n=25 for elav>Scr. p-values between two genetic conditions were calculated by a two-sided Fisher test. # p <0 .1, * p < 0.05, **<0.01.
Single-cell RNA sequencing of motoneurons identifies regulators of synaptic wiring in Drosophila embryos (2022)
Figure 6

Figure 6: Ig genes affect synaptic wiring specificity in accordance with the motoneuronal homeodomain cluster map.

(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced neuronal DIPkappa (elav>DIPkappaRNAi) as well as in animals, which mis-express DIPgamma or dpr1 in neuronal cells (elav>DIPgamma, elav>dpr1). Single channels focusing on the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right represents a schematic drawing of the confocal images shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions.

(B) Chart highlighting the expression of the homeodomain cluster genes Dfd, Scr and mirr as well as the Ig genes DIPgamma and DIPkappa in MN2a and MN3, showing that both Ig genes are only expressed in MN2a and not in MN3.

(C) Quantification of different phenotypes manifesting in MN2a and MN3 after neuronal RNA interference or mis-expression of Ig genes (DIPgamma, DIPkappa, dpr11, dpr1) in comparison to control animals: MN2a axons projecting to the MHE muscle, representing the wild-type (wt) innervation pattern of MN2a (magenta); MN3 axons projecting to the MHD muscle, representing the wild-type (wt) innervation pattern of MN3 (cyan); MN2a or MN3 axons projecting to muscles other than the MHE or MHD termed wiring defects (dark grey); abnormal synaptic morphologies at MN2a or MN3 derived axon terminals termed terminal defects (light grey). Each experiment was performed in triplicates. Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi. p-values between two genetic conditions were calculated by a two-sided Fisher test. * p < 0.05, **<0.01, *** p < 0.005.
Single-cell RNA sequencing of motoneurons identifies regulators of synaptic wiring in Drosophila embryos (2022)
Figure 8

Figure 8: Synaptic targeting requires the homeodomain TF Dfd in neurons and muscles.

(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced Dfd expression in neurons (elav>DfdRNAi) or in muscles (Mef2>DfdRNAi) or both in both tissues (elav;Mef2>DfdRNAi). The single channels for the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and perturbation conditions.

(B) Quantitative analysis of phenotypes. Each experiment was performed in triplicates, innervation rates were calculated from n=21 for elav>DfdRNAi, n=23 for Mef2>DfdRNAi n=20 for elav;Mef2>DfdRNAi animals. Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi, p-values between two genetic conditions were calculated by a two-sided Fisher test. *** p < 0.005.