EMBO/soda-nel huggingface dataset:
Defective ribosomal products (DRiPs) challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin (2019)
Figure 3

Figure 3: Upon heat shock or proteasome inhibition, DRiPs, polyUb proteins and 20S proteasomes accumulate in PML-NBs (see also Figure EV3 and Appendix Figure S2 and Movies EV1-3, EV6 and EV7).

(A) DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h; where indicated cycloheximide was also added (CHX; 50 μg/ml). Scale bars: 5 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and heat shock (HS) at 42˚C for 2 h. PML-NBs were visualized using a PML specific antibody, followed by incubation with alexa fluor 647. For convenience the alexa fluor 647 signal is shown in green. Scale bars: 5 µm.

(B) PolyUb proteins (FK1) labelling in HeLa cells left untreated or treated with HS at 42˚C for 2 h, alone or combined with CHX (50 μg/ml). Scale bars: 5 µm.

(C) STED super-resolution microscopy showing colocalization of endogenous polyUb proteins (FK1) and PML-NBs in PML-GFP HeLa cells treated with HS at 42˚C for 2 h. Scale bars: 5 µm.

(D) Quantitation of cells shown in A-C. Number of cells counted/condition: 712 - 1343 in three independent experiments; statistical significance via One-way ANOVA; p < 10-5, ± s.e.m.

(E) DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h; where indicated, cycloheximide was also added (CHX; 50 μg/ml). Asterisks (*) indicate nucleoli. Scale bars: 10 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and MG132 (10 µM) for 4 hrs. Scale bars: 5 µm.

(F) Quantitation of cells shown in E. Number of cells counted/condition: 298 - 1713 in three independent experiments; statistical significance via One-way ANOVA; p ≤ 0.001, ± s.e.m.

(G) Intranuclear distribution of 20S proteasomes in HeLa cells left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Asterisks (*) indicate nucleoli. Scale bars: 10 µm.
Defective ribosomal products (DRiPs) challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin (2019)
Figure 4

Figure 4: Compartmentalization of DRiPs and polyUb proteins is catalyzed by the self-assembly of PML into nuclear bodies.

(A) HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, total proteins were extracted and expression levels of PML were analyzed by immunoblotting. TUBA4A was used as loading control.

(B) HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, cells were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Left panel: automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 334 - 364; p < 10-10. Right panel: automated quantitation of the number of DRiP foci/nucleus. Cell number analyzed: 334 - 364; p < 10-10. Automated DRiP segmentation is based on azide signal.

(C) DRiPs and polyUb (FK1) protein distribution in cells treated as described in (A). Automated DRiP segmentation is based on azide signal. Quantitation of the number of DRiP foci enriched for polyUb (>2) is shown; n = 590 - 2114; statistical significance via 2‐tailed Student's t‐test; p < 10-10. Scale bars: 10 µm.

(D) Immunoblotting on protein extracts from HeLa cells lipofected with non-targeting siRNA control or a specific siRNA against Ubc9. A dilution curve of the control sample is shown. TUBA4A was used as loading control.

(E) Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal.

(F) Cells lipofected for 72 h with non-targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 151 - 225; p < 10-10 (see panel G).

(G) Representative pictures showing that Ubc9-depleted cells cannot efficiently induce the self-assembly of PML-NBs upon treatment with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Scale bars: 10 µm.
Defective ribosomal products (DRiPs) challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin (2019)
Figure 5

Figure 5: PolyUb proteins are cleared from PML-NBs by chaperones (see also and Appendix Figure S3 and EV4 and Movies EV1-5).

(A) PolyUb protein (FK1) labelling in PML-GFP HeLa Kyoto cells that were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Cells were either immediately fixed or let to recover for 5 h in drug-free medium (Control), with VER (40 µM) or Eeyarestatin I (5 µM). Scale bars: 10 µm.

(B) Quantitation of the % of cells with polyUb (FK1) nuclear foci shown in (A). Number of cells counted/condition: 310 - 1258 in three independent experiments; statistical significance via One-way ANOVA; p = 0.0003 or 10-6, ± s.e.m.

(C) Quantitation of the number of PML-NBs enriched for polyUb (>2); n = 611 - 2612; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.

(D) Quantitation of the % of cells with polyUb proteins (FK1) sequestered in nuclear foci. HeLa cells were treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h. Then, cells were either immediately fixed or let to recover for 3 h in drug-free medium (control), with VER (40 µM) or Eeyarestatin I (5 µM). Number of cells counted/condition: 593 - 1016 in three independent experiments; statistical significance via One-way ANOVA; p = 10-3, ± s.e.m.

(E) Quantitation of the number of PML-NBs enriched for polyUb (>2) in HeLa cells treated as described in (D) but with a recovery time of 2 h; n = 1155 - 3283; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.

(F) Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to the following treatments: untreated (Control); MG132 (10 µM) and OP-puro (25 µM) for 4 h, alone or combined with CHX (50 μg/ml). Where indicated cells were allowed to recover for 5 hrs in drug-free medium (Control) or with VER (40 µM). Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.

(G) Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to heat shock (HS) at 42˚C for 2 hrs. Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.
Defective ribosomal products (DRiPs) challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin (2019)
Figure 6

Figure 6: DRiPs and mCherry-VHL convert PML-NBs into an amyloid solid-like state that chaperones can revert (see also Figure EV4 and Movies EV1-9).

(A) PML-GFP HeLa Kyoto cells were lipofected with a cDNA encoding for mCherry-VHL. 24 h post-transfection, cells were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Where indicated (OP-puro + MG132 + CHX), translation was inhibited during stress with cycloheximide (CHX; 50 μg/ml). In the lower panel, cells were allowed to recover for 5 h in drug-free medium (Control), or with VER (40 µM). The subcellular distribution of PML-GFP and mCherry-VHL was studied by live-cell confocal imaging. Representative pictures are shown. Scale bars: 5 µm.

(B-F) Quantitation of the fluorescence intensity recovery after bleach of PML-GFP. PML-GFP HeLa Kyoto overexpressing mCherry-VHL for 24 h and treated as described in (A) were analyzed. The mean of 19-23 FRAP curves and the fitting curves are shown in black and red respectively. In gray, the SD is shown.

(G) Intranuclear distribution of 20S proteasomes, HSP70, VCP and DRiPs in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h and allowed to recover for 5 h in drug-free medium (control), with VER (40 µM), Eeyarestatin I (5 µM) or MG132 (10 µM). Scale bars: 5 µm.

(H) GFP-PSMA7 HeLa Kyoto cells were left untreated or treated as described in A. Cells were subjected to FRAP to analyze GFP-PSMA7 mobility. Quantitation of three populations of GFP-PSMA7 molecules is shown: fast moving, slow moving and immobile; statistical significance via One-way ANOVA; n = 12 - 14 ± SEM.
Defective ribosomal products (DRiPs) challenge nuclear function by impairing nuclear condensate dynamics and immobilizing ubiquitin (2019)
Figure 7

Figure 7: Sequestration of ubiquitin in solid PML-NBs impairs the recruitment of 53BP1 at fragile chromosomal sites (see also Figure EV5).

(A) Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h, followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI). Where indicated, cells were treated with MG132, VER or EeyI alone for 5 h. Scale bars: 20 µm.

(B) Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (A) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 1112 - 1537 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.

(C) HeLa cells were lipofected with cDNAs encoding for HA-Ubiquitin, Flag-Ubiquitin or Flag-VCP 24 h post-transfection, cells were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM). Cells were processed for staining of HA, Flag and endogenous 53BP1. * indicates cells that do not express HA- or Flag-Ubiquitin and show no rescue of 53BP1 foci formation after stress. Scale bars: 20 µm.

(D) Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (C) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 710 - 938 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.

(E) Colony formation assay. The number of colonies surviving 10 days after plating is shown. Control cells or cells treated with MG132 (10 µM) and puromycin (5 µg/ml), followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI) are shown. Where indicated, cells were treated with VER or EeyI alone for 5 h. Number of colonies counted/condition: up to 526 in three independent experiments; statistical significance via One-way ANOVA; p < 0.03, ± s.e.m.

Figure legend FOR EV FIGURES
MicroRNA-181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2 (2021)
Figure 1

Figure 1. Peripheral γδ T cells from metastatic cancer patients display impaired effector functions and increased expression of miR-181a.

(A) γδ T cell concentration in the peripheral blood from healthy donors and indicated metastatic cancer patients.

(B) RT-PCR analysis of the expression of NKG2D in γδ T cells isolated from indicated samples.

(C) RT-PCR analysis of the expression of IFN-γ and TNF-α in γδ T cells isolated from the peripheral blood of healthy controls and metastatic cancer patients.

(D) RT-PCR analysis of the expression of miR-125b-5p and miR-99a-5p in γδ T cells isolated from indicated samples.

(E) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from indicated samples.

Data information: (A-E) Error bars represent the mean ± SEM. CTRL F=Control Females (n=25-27 independent biological samples); BC=Breast Cancer (n=79-83 independent biological samples); CTRL M=Control Males (n=19-20 independent biological samples); PC=Prostate Cancer (n=29-36 independent biological samples). Statistical analysis was performed using the unpaired Student's t-test with Welch's correction. ns=not significant. *P < 0.05, **P < 0.01 and ****P < 0.0001. All experiments were performed with two technical replicates.
MicroRNA-181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2 (2021)
Figure 2

Figure 2. miR-181a is downregulated upon type 1 effector γδ T cell differentiation.

(A) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymic biopsies, cultured for 11 days with IL-7 or IL-2 (n=12-15 independent biological samples).

(B) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells freshly isolated from thymic biopsies (Thymus) and PBLs (n=6-14 independent biological samples).

(C-D) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) in γδ T cells isolated from thymus (C) or from PBLs (D) and cultured with the indicated cytokines, respectively for 11 days (C) or for 4-6 days (D). When indicated, γδ PBLs were also co-cultured with an anti-TGF-β blocking antibody. Data are normalized to the value obtained in IL-7 cultures (n=4-10 independent biological samples).

(E-F) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) versus FACS analysis of the expression of TNF-α (left panel), IFN-γ (middle panel) and NKGD2 (right panel) in γδ T cells isolated from thymus (E) and PBLs (F). Samples were either freshly isolated or cultured for 11 days (γδ thymocytes) or for 6 days (γδ PBLs) with the indicated cytokines (n=5 independent biological samples).

Data information: (A-D) Error bars represent the mean ± SEM. (A, C-D) Statistical analysis was performed using the paired Student's t-test. (B) Statistical analysis was performed using the Mann Whitney U test. (E-F) The Pearson's correlation coefficient (r) was used to measure the strength of association between two variables. ns=not significant. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. All experiments were performed with two technical replicates.
MicroRNA-181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2 (2021)
Figure 3

Figure 3. miR-181a overexpression impairs γδ T cell functional differentiation.

(A) Retroviral transduction workflow for γδ thymocytes.

(B) Gating strategy for identification of the GFP+ versus GFP- cells.

(C) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) (normalized to the values obtained with the empty virus) in transduced (GFP+) and untransduced (GFP-) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=4 independent biological samples).

(D) FACS analysis of the expression of Annexin V in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).

(E) RT-PCR analysis of the expression of Bcl2 and Bcl-xL (normalized to the values obtained with the empty virus) in transduced (GFP+) γδ thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=5 independent biological samples).

(F) FACS-derived expression of indicated surface and intracellular markers in miR-181a versus empty transduced (GFP+) and untransduced (GFP-) γδ T thymocytes, cultured with IL-7 plus IL-2 for 11 days (n=7-11 independent biological samples).

Data information: (C-F) Error bars represent the mean ± SEM. Statistical analysis was performed using the paired Student's t-test. ns=not significant. *P < 0.05 and **P < 0.01. All experiments were performed with two technical replicates.
MicroRNA-181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2 (2021)
Figure 4

Figure 4. miR-181a targets Map3k2 and Notch2 mRNAs.

(A) Putative binding sites of miR-181a(-5p and -2-3p) in the 3′ UTR region of Map3k2 and Notch2. Potential miR-181a(-5p and -2-3p) gene targets were selected based on predictions obtained in referenced mirDIP v4.1database. The predicted binding regions that were amplified for the luciferase assay are highlighted in grey.

(B) RT-PCR analysis of the expression of the indicated genes associated with Mapk, Notch, Irf4 or Stat1 signaling in sorted GFP+ γδ thymocytes transduced with empty or miR-181a viral vector. Data are normalized to the value obtained with the empty virus. Samples were cultured with IL-7 plus IL-2 for 11 days (n=3-5 independent biological samples).

(C) Dual luciferase reporter assay performed to verify binding between miR-181a and Map3k2, Notch2, Irf4 or Stat1 mRNA. HEK293 T cells were co-transfected with a pmirGLO Dual-Luciferase miRNA Target Expression Vector containing either the WT or mutated (Mut) 3′ UTR target sites plus miR-181a. A negative construct (without miR-181a binding sites) was included. Firefly/Renilla ratios were normalized to those obtained for the empty vector (n=4 independent biological samples).

(D) RT-PCR analysis of the expression of miR-181a(-5p and -2-3p) versus MAP3K2 (left panel) or NOTCH2 (right panel) expression in γδ T cells isolated from thymic biopsies. Samples were cultured for 11 days with the indicated cytokines (n=6 independent biological samples).

(E-G) RT-PCR analysis in circulating γδ T cells of Map3k2 or Notch2 expression (E), Nkg2d or Tnfa expression (F), and NKG2D or TNF-α protein expression (G) after siRNA-mediated knockdown of Map3k2 or Notch2. In each experiment, a control condition was also used where a nontargeting negative control siRNA was transfected. γδ were cultured with IL-7 for 4 days (n=6 independent biological samples).

Data information: (B, C, E-G) Error bars represent the mean ± SEM. Statistical analysis was performed using the paired Student's t-test. (D) The Pearson's correlation coefficient (r) was used to measure the strength of association between two variables. ns=not significant. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. All experiments were performed with two technical replicates.
Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis (2023)
Figure 1

Figure 1 Ch25h expression in the central nervous system during EAE

(A) RNAscope fluorescence in-situ hybridization of Ch25h transcripts (red) in the spinal cord of non-immunized mice (NI) (left panels) and mice 17 days after EAE immunization (peak disease) (2 right panels) shown in endothelial cells (Isolectin B4 (IsoB4) green top panels) and activated macrophages/microglia (Ionized calcium biding adaptor molecule 1 (IBA1) green lower panels). Nuclei are shown in blue. Scale bars, 100 μm; insets, 20 μm.

(B) Quantitative analysis of Ch25h mRNA expression in the spinal cord of NI and EAE mice comparing expression in macrophages/microglia (MG) and endothelial cells (EC), n = 6 biological replicates/group.

(C) Construct of Ch25h-eGFPfl/fl mice.

(D) Flow cytometry analysis of Ch25h-eGFP reporter expression in CNS endothelial cells (CNS ECs: Live cells CD45-Ter119-CD13-CD31+). Wild-type mice were used as negative controls for eGFP signal. CNS ECs from Ch25h-eGFP (Ch25hfl/fl) mice and Ch25h-eGFP fl/fl-Ve-CadherinCreERT2 (Ch25hECKO) mice where the Cre recombinase is expressed in the endothelial cells are compared. Non-immunized mice (NI) are compared with mice at the peak of EAE 16 days after immunization.

(E) Percentage of eGFP+ CNS ECs in the same condition as in (D). Symbols indicate individual mice and bars indicate mean ± SD. Ch25hfl/fl NI: n = 4 biological replicates, Ch25hfl/fl EAE: n = 5 biological replicates, Ch25hECKO NI: n = 4 biological replicates, Ch25hECKO EAE: n = 4 biological replicates. Representative results of 1 experiment.

Data information: ns: non-significant, *: P ≤ 0.05, **: P ≤ 0.005, *** P ≤ 0.0005, ****: P ≤ 0.00005. P values were determined by two-way ANOVA with Sidak's post hoc test.
Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis (2023)
Figure 2

Figure 2 Ch25h deletion in blood endothelial cells dampens EAE

(A) EAE disease course in Ch25hECKO and Cre negative littermates (Ch25hfl/fl). Top panel: EAE clinical score. Bars indicate mean ± SEM. Bottom panel: Survival analysis of EAE, depicting disease incidence. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph.

(B) As in (A) except that Ch25h fl/fl- Pdgfb-iCreERT2 mice that express Cre recombinase in blood endothelial cells (Ch25hBECKO) are shown. Bars indicate mean ± SEM. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph.

(C) As in (A) except that Ch25hfl/fl- Prox1-CreERT2 mice that express Cre recombinase in lymphatic endothelial cells (Ch25hLECKO) are shown. Bars indicate mean ± SEM. Representative results of 2 experiments with a minimum of n = 5 biological replicates/group as indicated on the graph.

(D, E) Histopathological quantifications (D) and representatives staining (E) of spinal cord sections of immunized Ch25hfl/fl mice (n = 7 biological replicates) and Ch25hECKO mice (n = 10 biological replicates) at day 21 post-immunization for cellular infiltration (HE). Five sections per mouse were quantified. Scale bars 200 µm (top panels), 100 µm (bottom panels).

Data information: ns = non-significant, *: P < 0.05, **: P ≤ 0.005, ***: P ≤ 0.0005, ****: P ≤ 0.00005. P values were determined by two-way ANOVA with Sidak's post hoc test (top panels A,B,C) and log-rank (mantel-cox) test (bottom panels A,B,C) or by two-tailed unpaired t-test (D).
Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis (2023)
Figure 3

Figure 3 Transcriptomic alterations in Ch25hECKO CNS endothelial cells.

(A) RT-qPCR analysis of Ch25h expression in primary mouse brain microvascular endothelial cells (pMBMECs) isolated from Ch25hfl/fl and Ch25hECKO mice. Cells were left unstimulated (NS) or stimulated with IL-1β. n = 5 biological replicates/group. The experiment was performed 3 times.

(B) Venn diagram of differentially expressed genes (DEG) between Ch25hECKO and Ch25hfl/fl pMBMECs assessed by RNA sequencing comparing non-stimulated (NS) and IL-1β stimulated cells. n = 3 biological replicates/group. The experiment was realized once.

(C) Dot plot of gene set enrichment analysis (GSEA) showing selected pathways. NES = normalized enrichment score.

(D) GSEA comparing enrichment of genes related to unsaturated fatty acid biosynthesis in Ch25hECKO vs Ch25hfl/fl IL-1β stimulated pMBMECs. NES = normalized enrichment score, FDR= false discovery rate q-value.

(E) Heatmap showing normalized expression (z-scores) of gene counts from the RNAseq analysis related to the unsaturated fatty-acid biosynthetic pathway.

(F) RT-qPCR of pMBMECs isolated from Ch25hECKO and Ch25hfl/fl mice and stimulated with IL-1β. n = 4 biological replicates/group. The experiment was realized once.

(G) RT-qPCR of CNS endothelial cells sorted from Ch25hECKO and Ch25hfl/fl mice during EAE (day 10 after immunization). Ch25hECKO n = 3 biological replicates, Ch25hfl/fl n = 4 biological replicates. The experiment was performed once.

Data information: Bars indicate mean ± SD. ns= non-significant, *: P ≤ 0.05, **: P ≤ 0.005, ****: P ≤ 0.00005. P values were determined by two-way ANOVA with Sidak's post hoc test (A) or by two-tailed unpaired t-test (F, G).