EMBO/soda-nel huggingface dataset:
Spatial control of nucleoporin condensation by Fragile X-related proteins (2020)
Figure 1

Figure 1 - FXR1 protein localizes to the NE and interacts with NUPs.

A Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (shown a mean value, *P < 0.05, **P < 0.01; N = 3).

B Lysates of HeLa cells stably expressing GFP alone or 3xGFP-Nup85 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (SE, short exposure, LE, long exposure) (shown a mean value, *P < 0.05; N = 3).

C Immunoprecipitation from HEK293T cell lysates using FXR1 antibody or IgG analysed by Western blot. The arrow points to the heavy chain of IgG (IgG HC) (shown a mean value, *P < 0.05; N = 3).

D HeLa cells were treated with indicated siRNAs, synchronized by double thymidine block, and released for 12 hours and analysed by immunofluorescence microscopy for the lamin B receptor (LBR) to label the NE, and FXR1.

E HeLa cells stably expressing GFP-FXR1 were analysed by immunofluorescence microscopy for GFP and mAb414, which labels FG-Nups. The magnified framed regions are shown in the corresponding numbered panels. The arrowheads indicate NE and cytoplasmic localization of GFP-FXR1.

F HeLa cells stably expressing GFP-Nup107 were synchronized by double thymidine block and released for 12 hours, permeabilized with Triton/SDS or digitonin for antibodies to access the nuclear and cytoplasmic or cytoplamsic side of the nucleus, respectively, and analysed by immunofluorescence microscopy.

Data information: Scale bars are 5 μm. Statistical significance was assessed by unpaired two-tailed Student's T-test.
Spatial control of nucleoporin condensation by Fragile X-related proteins (2020)
Figure 2

Figure 2 - FXR1 inhibits aberrant assembly of cytoplasmic Nups.

A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and release for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.

B HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and then analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules was quantified, 1000 cells were analysed for each graph (mean ±SD, **P < 0.01; ***P < 0.001, N = 3). The corresponding representative pictures are shown in Figure EV1 and the corresponding Western blot analysis is shown in Figure 3B.

C-F HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Nups present in different NPC subcomplexes are depicted in the color code corresponding to the NPC scheme shown on the right. Additional or complementary representative images and channels of cells depicted in (C) are shown in Appendix Figures S1 and S2B-D. Nuclear intensity of FG-Nups labelled by mAb414 (D), RanBP2 (E) and GFP-Nup107 (F) was quantified. 1800 cells were analysed for each graph (mean ±SD, *P < 0.05; **P < 0.01; N = 3).

G, H Asynchronously proliferating U2OS cells were treated with the indicated siRNAs and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic nucleoporin granules (G) was quantified and nuclear intensity of FG-Nups labelled by mAb414 (H) was quantified. 1600 cells were analysed in (G) and 2100 cells were analysed in (H) (mean ±SD, *P < 0.05; *** P<0.001; N = 3).

Data information: Scale bars are 5 μm. Statistical significance was assessed by unpaired two-tailed Student's T-test.
Spatial control of nucleoporin condensation by Fragile X-related proteins (2020)
Figure 3

Figure 3 - The FXR1 regulates nuclear morphology during G1 cell cycle phase.

A HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels. Arrowheads point to nuclear blebs observed in FXR1-deficient cells.

B, C HeLa cells stably expressing GFP, GFP-FXR1 wild type (WT) and GFP-FXR1 mutated in the sequence recognized by FXR1 siRNA-1 (GFP-FXR1-MUT-siRNA1) were treated with the indicated siRNAs, synchronized by double thymidine block, released for 24 hours and analysed by Western blot (B) and immunofluorescence microscopy (C). The percentage of cells with irregular nuclei was quantified, 1000 cells were analysed (mean ±SD, **P < 0.01, ***P < 0.001; N = 3). The corresponding representative pictures are shown in Figure EV1.

D-J HeLa cells stably expressing the chromatin marker histone H2B labelled with mCherry were treated with indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Time from prophase till anaphase (D), from prophase till metaphase (E), from metaphase till anaphase (F) and from anaphase till chromatin decondensation (G) was quantified. The selected frames of the movies are depicted and time is shown in minutes (H). Arrowheads point to nuclear blebs appearing during nuclear expansion of FXR1-deficient cells. Percentage of daughter cells with irregular nuclei was quantified in (I) and time from anaphase till nuclear blebs was quantified in (J). 66 cells were analysed (mean ±SD, ***P < 0.001; N = 3).

Data information: Scale bars are 5 μm. Statistical significance was assessed by unpaired two-tailed Student's T-test.
Spatial control of nucleoporin condensation by Fragile X-related proteins (2020)
Figure 4

Figure 4 - FXR1 regulates cytoplasmic Nups during early interphase.

A-D HeLa cells stably expressing GFP-Nup107 were treated with indicated siRNAs, synchronized by double thymidine block, released and analysed by live video spinning disk confocal microscopy (A). The selected frames of the movies are depicted and time is shown in minutes. The onset of anaphase is indicated. The magnified framed regions with time indicated in minutes are shown in (B). White arrowheads point to the cytoplasmic GFP-NUP107 granules appearing during nuclear expansion of control and FXR1-deficient cells, yellow arrowheads point to the fussion events of GFP-NUP107 granules with NE in control cells. The percentage of cells with cytoplasmic GFP-Nup107 granules was quantified in (C). Time from anaphase till GFP-Nup107 cytoplasmic granule formation was quantified in (D). 57 cells were analysed (mean ±SD, *P < 0.05; ns = non-significant; N = 3).

Data information: Scale bars are 5 μm (A) and 1 μm (B). Statistical significance was assessed by unpaired two-tailed Student's T-test.
Spatial control of nucleoporin condensation by Fragile X-related proteins (2020)
Figure 6

Figure 6 - FXR1 works together with the dynein-BICD2 complex to inhibit cytoplasmic Nup granules formation.

A HeLa cells stably expressing GFP alone or GFP-FXR1 were immunoprecipitated using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; N = 3).

B, C HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. Images in (B) correspond to the numbered magnified framed regions indicated in the pictures shown in

Appendix Figure S6E. Arrowheads indicate blebbed regions of nuclei. The percentage of interphasic cells with Nup granules was quantified (C), 900 cells were analysed (mean ±SD, ***P < 0.001; N = 3). The corresponding Western blot analysis is shown in Appendix Figure S6D.

D Lysates of HeLa cells stably expressing GFP alone or GFP-FXR1 were subjected to immunoprecipitation using GFP-Trap beads (GFP-IP), analysed by Western blot and quantified (mean, *P < 0.05; **P < 0.01; N = 3).

E, F HeLa cells were treated with the indicated siRNAs, synchronized by double thymidine block and released for 12 hours and analysed by immunofluorescence microscopy for FXR1 and mAb414. The magnified framed regions are shown in the corresponding numbered panels in (E). The percentage of cells with cytoplasmic Nup granules was quantified in (F), 2700 cells were analysed (mean ±SD, ***P < 0.001; N = 3).

Data information: Scale bars are 5 μm. Statistical significance was assessed by unpaired two-tailed Student's T-test (A, D) and one-way ANOVA test with Dunnett's correction (C, F).
Spatial control of nucleoporin condensation by Fragile X-related proteins (2020)
Figure 7

Figure 7 - FXR1 regulates cytoplasmic Nups by dynein-based microtubule-dependent transport.

A, B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as indicated and analysed by immunofluorescence microscopy. The percentage of cells with cytoplasmic GFP-Nup133 granules was quantified in (B), 5200 cells were analysed (mean ±SD, **P < 0.01; ***P < 0.001; N = 3).

C-E HeLa cells stably expressing GFP-Nup107 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours, treated with nocodazole to induce granule formation and washed-out as in (A) and analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted and time is shown in minutes. The magnified framed regions are depicted in the lower rows. White arrowheads point to individual GFP-Nup107-positive granules. Yellow arrowheads point to the granules undergoing fusion events. The percentage of cells with fusion/fission events of GFP-Nup107 granules was quantified in (D), 815 cells were analysed (mean ±SD, **P < 0.01; N = 3). The number of fusion/fission events per cell was quantified in (E), 815 cells were analysed (mean ±SD; N = 3).

Data information: Scale bars are 5 μm. Statistical significance was assessed by unpaired one-way ANOVA test with Sidak's (B) or Dunnet's (D, E) correction.
Spatial control of nucleoporin condensation by Fragile X-related proteins (2020)
Figure 8

Figure 8 - Nup localization defects can be linked to Fragile X syndrome.

A-D Human FXS-patient derived fibroblasts (FXS-1, FXS-2) and control human fibroblasts were synchronized in early G1 by Monastrol release and analysed by Western blot (A) and immunofluorescence microscopy (B, D). The percentage of cells with cytoplasmic Nup granules was quantified in (C), 283 cells were analysed (mean ±SD, ***P < 0.001; N = 3). Examples of Nup localization defects are shown in (B) and examples of nuclear architecture defects are shown in (D).

E-G Human induced pluripotent stem cells (iPSCs) derived from a FXS patient (FXS-iPSC) and the isogenic rescue cells (C1_2-iPSC) were analysed by immunofluorescence microscopy (E, G). The percentage of cells with cytoplasmic Nup133 granules was quantified in (F), 5500 cells were analysed (mean ±SD, **P < 0.01; N = 3). Examples of co-localization events of re-expressed FMRP and Nup133 are shown in (G).

H, I Mouse Embryonic Fibroblasts (MEFs) derived from the Fmr1 knock-out (KO) mice and wild type controls were synchronized in early G1 by Monastrol release and analysed by immunofluorescence microscopy (H). The percentage of cells with cytoplasmic nucleoporin granules was quantified in (I), 2400 cells were analysed (mean ±SD, *P < 0.05; N = 3).

Data information: Scale bars are 5 μm. Statistical significance was assessed by one-way ANOVA test with Sidak's correction (C), paired two-tailed Student's T-test (F) and unpaired two-tailed Student's T-test (I).
Spatial control of nucleoporin condensation by Fragile X-related proteins (2020)
Figure 9

Figure 9 - FXR1 regulates G1 cell cycle progression.

A HeLa cells were transfected with the import/export reporter plasmid XRGG-GFP, treated with the indicated siRNAs and synchronized in early G1 phase by Monastrol release. Dexamethasone was added for 3 hours to induce XRGG-GFP nuclear import. Following wash-out the nuclear export of XRGG-GFP was analysed by live video spinning disk confocal microscopy. The selected frames of the movies are depicted in Figure EV5C. The percentage of cytoplasmic XRGG-GFP over time was quantified in Figure EV5D and quantifications of individual cells from the 20 and 30 minutes time points are depicted in (A), 199 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3).

B HeLa cells stably expressing GFP-Nup133 were treated with the indicated siRNAs, synchronized by double thymidine block, released for 12 hours and analysed by immunofluorescence microscopy. The magnified framed regions are shown in the corresponding numbered panels.

C, D Asynchronously proliferating HeLa cells were treated with indicated siRNAs and analysed by immunofluorescence microscopy (C). The percentage of p-Rb positive cells was quantified in (D), 2800 cells were analysed (mean ±SD, **P < 0.01; N = 3).

E, F Asynchronously proliferating HeLa cells were treated with indicated siRNAs, incubated with EdU during 30min and analysed by immunofluorescence microscopy. The percentage of p-Rb and/or EdU positive cells was quantified in (E), 2100 cells were analysed (mean ±SD, *P < 0.05; **P < 0.01; N = 3), and the percentage of p-Rb and/or cyclin B positive cells was quantified in (F), 3300 cells were analysed (mean ±SD, *P < 0.05; N = 3).

G A hypothetical model how Fragile X-related proteins spatialy regulate nucleoporin condensation. FXR proteins (blue) interact with cytoplasmic soluble Nups (red circles) and dynein (green) and facilitate their localization to the NE during early G1. This function of FXR proteins inhibits formation of aberrant cytoplasmic Nup assemblies, the Cytoplasmic nucleoporin Granules (CNGs), contributing to the equilibrium of NE-NPCs and driving the G1-specific protein export and maintenance of nuclear shape and cell cycle progression. Silencing of FXR proteins, for instance in FXS patients, leads to the accumulation of CNGs, nuclear atypia, protein export defects, and defects in cell cycle progression, which may contribute to the pathology of FXS.

Data information: Scale bars are 5 μm. Statistical significance was assessed by Kruskal-Wallis test with Dunn's correction (A), unpaired two-tailed Student's T-test (D) and One-way ANOVA test with Sidak's correction (E, F).

Expanded View Figure Legends
MiR-182-3p targets TRF2 and impairs tumor growth of triple negative breast cancer (2022)
Figure 1

Figure 1. miR-182-3p reduces TRF2 expression in different cancer cell lines.

A. Schematic representation of luciferase screening approach. Upper panel shows the four target predictions software used for in silico analysis. Bottom panel indicates the main steps performed in the high-throughput screening.

B. Upper panel, sequence interaction of miR-182-3p with the target site of the wild type 3'UTR of TRF2 in human. Bottom panel, generation of mutant 3'UTR of TRF2 luciferase construct containing the deletion of target site for miR-182-3p.

C-E. Luciferase reporter assay in HeLa cells using the synthetic miR-Control or miR-182-3p in combination with the wild type (C) or the mutant 3'UTR of TRF2 construct (D) or the wild type 3'UTR of TRF1 (E).

F,G. Western blotting for TRF2 expression in Telomerase Positive (HeLa, HCT116, MDA-MB-231, MDA-MB-436) and ALT positive (U2-OS, Saos-2) cells transiently transfected with miR-Control or miR-182-3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control.

H. U2-OS cells transiently transfected with the miR-Control, miR-182-3p or miR-182-3p inhibitor were assayed by quantitative immunofluorescence for TRF2 three days post-transfection. Left panel, representative images. Scale bar: 10µm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N=number of analysed nuclei. Red bar indicates mean value.

I. U2-OS cells transfected as described in (H) were assayed by immuno-fluorescence combined with telomeric FISH. Left panel, representative images of co-localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10µm. Right panel, co-localizations were analysed using imageJ software. N=number of analysed nuclei.

Data information: For C-G and I, data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by Student's t-test; for (H), P values are determined by Mann-Whitney test.
MiR-182-3p targets TRF2 and impairs tumor growth of triple negative breast cancer (2022)
Figure 2

Figure 2. miR-182-3p induces telomeric and pericentromeric DNA damage by TRF2 abrogation.

A. Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA-MB-231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analysed.

B. Representative images and enlargements of co-localizations (white arrowheads) relative to the experiment described in A. Scale bar: 10µm.

C. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA-MB-231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX positive cells with ≥1 PIFs per nucleus were analysed.

D. Representative images of co-localizations (white arrowheads) relative to the experiment described in C. Scale bar: 10µm.

E. Quantification of TIFs in MDA-MB-231 cells over-expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified.

F. Representative images and enlargements relative to the experiment described in E. White arrowheads indicate co-localizations events. Scale bar: 10µm.

G. Quantification of PIFs in MDA-MB-231 cells over-expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX positive cells with ≥1 PIFs per nucleus were analysed.

H. Representative images relative to the experiment described in G. White arrowheads indicate co-localizations events. Scale bar: 10µm.

Data information: For A,C,E,G data are shown as mean ± SD. Three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. At least 60 nuclei were analysed for each experimental condition. All the experiments were performed three days post-transfection with the indicated miRNAs or siRNAs.
MiR-182-3p targets TRF2 and impairs tumor growth of triple negative breast cancer (2022)
Figure 3

Figure 3. Ectopic expression of miR-182-3p reduces cell growth by apoptosis activation

A,B. MDA-MB-436 and MDA-MB-231 cells underwent two rounds of transfection with miR-Control, miR-182-3p or miR-182-3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24h up to a maximum of 3 days. The percentage of cell confluence was analysed.

C,D. Cell number of MDA-MB-436 (C) and MDA-MB-231 (D) cells and TRF2 expression were analysed by automatic cell count and by western blotting at the end of the experiment described in A and B. Actin was used as loading control.

E. Two-dimensional scatter plots of Annexin V analysis performed in MDA-MB-436 at the end of the second cycle of transfection with miR-Control, miR-182-3p or miR-182-3p inhibitor. Red boxes indicate early and late apoptotic cells.

F. Quantification of Annexin V positive cells (%) of experiment described in E.

G. Two-dimensional scatter plots of Annexin V analysis performed in MDA-MB-231 as described in E.

H. Quantification of Annexin V positive cells (%) of experiment described in G.

I,J. MDA-MB-436 cells over-expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72h post-transfection.

Data information: For A,B data are shown as mean ± SEM. For C,D,F,H,I,J, data are shown as mean ± SD. For A-D and I, three independent experiments were performed (n=3). P values are determined by unpaired two-tailed t test. For F, H and J, two different biological replicates were performed.
MiR-182-3p targets TRF2 and impairs tumor growth of triple negative breast cancer (2022)
Figure 4

Figure 4. Ectopic expression of miR-182-3p reduces cell growth by senescence activation in normal cells

A. Western blotting for TRF2 expression in BJ cells transiently transfected with miR-Control or miR-182-3p. The graph represents the quantification of three independent experiments. Representative images are shown, actin was used as loading control. Unspecific bands are indicated with (*).

B, C. Mean of γH2AX foci per nucleus was analysed in BJ cells 72h post-transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in C.

D. Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72h post-transfection with the indicated miRNAs. Left panel: the mean number of TIFs per nucleus was analysed. Right panel: representative images and enlargements of co-localizations.

E. Cells number of BJ cells was analysed by automatic cell count at the end of the second round of transfection with miR-Control or miR-182-3p.

F. FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in E.

G. β-Galactosidase assay in BJ cells after two rounds of transfection with mimic miR-Control or miR-182-3p. Left panel: analysis of β-Galactosidase positive cells. Right panel: representative images. H-J. IL-6 (H), CXCL1 (I), IL-8 (J) factors were analysed by ELISA to evaluated the Senescence-associated secretory phenotype (SASP) in BJ cells treated as indicated in G.

Data information: For B-J, a student t test was used to calculate statistical significance. Scale bars (10µm). P values are indicated.
MiR-182-3p targets TRF2 and impairs tumor growth of triple negative breast cancer (2022)
Figure 5

Figure 5. LNPs-miR-182-3p treatment inhibits tumor growth in vivo

A,B. MDA-MB-231 (A) and MDA-MB-436 (B) tumor xenografts were treated with LNPs-empty, LNPs-miR-Control or by LNPs-miR-182-3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20µg of LNPs-miR-Control, LNPs-miR-182-3p or equivalent volume of LNPs-empty as indicated in the scheduling. The mean of tumor volumes (n=5 per group) is shown.

C,D. Tumors from mice treated in A and B were processed to measure miR-182-3p expression by TaqMan qPCR.

E. Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA-MB-231 human breast cancer xenografts. Scale bar: 50μm.

F. The histograms show: the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, Tunel or CD31 staining. The analyses were performed on 3 mice per group, the points represent the number of field analysed for each condition.

G, H. Luminescent MDA-MB-436 cells were injected into the brain and monitored by IVIS imaging system. After one week from implant, treatment with LNPs-miR-Control and LNPs-miR-182-3p was performed as indicated in A and B. Representative images from in vivo (upper panel) or ex-vivo (bottom panel) brain tumors are shown in G. Boxplots (H) show the measurement of photons for each brain tumor (n=5 per group) acquired at the indicated times.

Data information: For A, B and F, data are shown as mean ± SD. For C, D and H, the line in the middle of the box plot denote a median value, the limits of box represent the interquartile range (25th to 75th percentiles), while, the whiskers denote the minimum to maximum values. For A-D and H, P values are determined by unpaired two-tailed t test; for (F), P values are determined by Mann-Whitney test.