EMBO/soda-nel huggingface dataset:
Transfer of extracellular vesicle-microRNA controls germinal center reaction and antibody production (2020)
Figure 2

Figure 2. mmu-miR-20a-5p, mmu-miR-25-3p, and mmu-miR-155-3p are transferred during IS and correlate with BIM and PTEN downmodulation in B lymphocytes

CD4+ T and DICER-KO B cells were co-cultured for 24 hours with or without OVA, and flow-cytometry sorted B cells were analyzed by small RNA next generation sequencing (NGS).

A. Heat map showing microRNAs significantly upregulated in B cells after co-culture with T cells in the presence of OVA. Data are from 3 independent experiments (samples 1-3), and only microRNAs with an adjusted P-value < 0.02 are shown.

B. Fold increase in the B cell content of upregulated microRNAs in the presence of OVA; *P<0.05, **P<0.01, ***P<0.001.

C. Heat map showing NGS analysis of microRNAs differentially expressed in activated WT C57BL/6 CD4+ T cells and their secreted exosomes. Data are from 3 independent experiments (samples a-c), and only microRNAs with an adjusted P-value < 0.02 are shown.

D. Mean tags per million (TPMs) for the differentially expressed microRNAs under study in exosomes from activated CD4+ T cells. Bars represent the mean ± SEM.

E and F. Quantitative real-time PCR of upregulated microRNAs after co-culture of OT-II derived CD4+ T cells with DICER-KO B cells activated either with LPS+IL-4 (E) or with CD40+IgM (F). Bars represent the mean ± SEM of at least three independent experiments and data were obtained by the 2-∆∆Ct method using Biogazelle software. Results are expressed as a proportion of the microRNA expression in non-OVA containing co-cultures, with normalization to the small nucleolar RNAs RNU1A1 and RNU5G.

G and H. Quantitative real-time PCR showing the expression of down-modulated mRNA targets upon IS formation between OT-II CD4+ T cells and DICER-KO B cells pre-activated with either LPS+IL-4 (G) or CD40+IgM (H). Results are the means ± SEM of at least four independent experiments and are expressed as a proportion of mRNA expression in the non-OVA condition, with normalization to GAPDH.

I-J. Fluorescently tagged mimics of the microRNAs mmu-miR-20a-5p, mmu-miR-25-3p and mmu-miR-155-3p were nucleofected into activated B lymphocytes from C57/BL6 mice. Quantitative real-time PCR showing expression of nucleofected microRNA mimics in FACS-sorted FAM+ B cells with normalization to RNU1A1 and RNU5G (I); and Bcl2l11 and Pten mRNA targets in mimic-nucleofected B cells with normalization to GAPDH (J). A representative experiment is shown of 4 performed and bar charts summarize the mean ± SEM. Significance was assessed by paired Student t test comparing the plus and minus OVA conditions; *P<0.05, **P<0.01, ***P<0.001.

.

K. Flow cytometry analysis of mimic-nucleofected B lymphocytes. Dot-plots show B220+ IgG1+ B cells 48h after mimic nucleofection from at least 4 independent experiments and bar charts show mean values ± SEM of 4 independent experiments.

Data information: Significance was assessed by the Student t test comparing the OVA and NO OVA conditions; *P<0.05, **P<0.01, ***P<0.001.
Transfer of extracellular vesicle-microRNA controls germinal center reaction and antibody production (2020)
Figure 3

Figure 3. EV transfer is required for full B cell activation and antibody production in vivo

A. Quantitative real-time PCR (RNU1A1 and RNU5G- normalized) showing blockade of microRNA transfer from CD4+ T cells pre-treated with siRNA targeting the exosome secretion mediator Rab27. Bar charts represent the mean values ± SEM of at least 4 independent experiments.

B. Quantitative real-time PCR showing mRNA target down-modulation after IS formation between DICER-KO B cells and CD4+ T cells nucleofected with either siRab27 or scrambled control siRNA, with normalization to GAPDH. Bar charts represent the mean values ± SEM of at least 4 independent experiments.

C. Representative dot-plots, showing IgG1 expression of gated B220+ B cells after IS formation as in (G); the bar chart shows the mean fluorescence Intensity ± SEM and quantification of 3 independent experiments is shown.

D. Experimental design. Rag1KO-mice were adoptively transferred with WT B lymphocytes and either Rab27-KO T cells (top) or WT T cells. Thereafter mice were inoculated with sheep red blood cells, and spleens and serum were harvested after 7 days.

E. Representative plot (left) and quantification (right) of the reconstitution of the B cell and T cell compartments after adoptive transfer. The graph shows the mean percentage of CD19 and CD4 positive cells from n ≥ 5 reconstituted mice ± SEM.

F. Representative flow cytometry plot (left) and quantification (right) of GL7 and CD95/Fas expression in B cells. The graph shows the mean percentage of GL7+Fas+ cells from n ≥ 5 reconstituted mice ± SEM.

G. Representative dot-plot analysis showing CXCR4 and CD86 expression on B cells for Dark zone (DZ) and Light zone (LZ) analysis (gated on GC GL7+ Fas+ B cells). The graph shows the mean percentage of DZ (CXCR4+CD86-) and LZ (CXCR4-CD86+) populations from n ≥ 5 reconstituted mice ± SEM.

H-I. ELISA quantification of IgM (H) and IgG (I) in serum from immunized adoptively transferred mice. The graph shows the mean serum concentration of IgM and IgG from n ≥ 5 reconstituted mice, respectively ± SEM.

J. Proposed model of exosomal microRNA transfer during cognate B and T cell interactions.

Data information: Significance was assessed by unpaired Student t test comparing the OVA and NO OVA conditions; *P<0.05..

Expanded View Figure legends
Ssu72 phosphatase is a conserved telomere replication terminator (2019)
Figure 1

Figure 1. Genetic screen identifies Ssu72 as telomerase regulator.

A) We identified previously unknown telomere regulators in fission yeast using the haploid S. pombe whole-genome gene deletion library including ssu72+ (SPAC3G9.04).

B) Telomere length of wt, ssu72Δ and ssu72-C13S (point mutation at the phosphatase active site) strains were measured by Southern blot in ApaI digested genomic DNA using a telomeric probe.

C) Ssu72 recruitment to telomeres is cell cycle regulated. Ssu72 was myc-tagged at the 3' end and ChIP analysis was carried out in cell cycle synchronized populations of cdc25ts strains. Septa formation was used proxy for S-phase. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).

D) Telomere length of ssu72Δ is dependent on telomerase. Diploid strains with the appropriate genotypes were sporulated and trt1∆ ssu72∆ double mutants were streaked for multiple passages (triangle indicates increased number of generations). E) Telomerase is recruited to telomeres in the absence of Ssu72. ChIP analysis for Trt1-myc in wt and ssu72∆ was performed as described in material and methods using a non-tagged strain as a control. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).

F) Telomerase activator Ccq1 is hyperphosphorylated in ssu72∆ cells. rap1∆ cells were used as positive control for Ccq1 phosphorylation status. Western blots were performed using Ccq1-flag tagged strains.
Ssu72 phosphatase is a conserved telomere replication terminator (2019)
Figure 2

Figure 2. Ssu72 is required for telomeric C-strand stability.

A) ssu72Δ telomeres present longer G-rich overhangs than wt and rif1∆. In-gel hybridization in native and denaturing conditions was labelled with a radiolabelled C-rich telomere probe and quantified for ssDNA at the telomeres. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).

B) RPA (Rad11-GFP) is enriched at ssu72∆ telomeres. Colocalization of Rad11-GFP with Taz1-mCherry, used as a telomere marker was performed in wt and ssu72∆ cells; n =3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). More than 1000 cells were analyzed for each genotype

C) ssu72+ controls telomere length independently of rif1+. Epistasis analysis of telomere length of ssu72∆ and ssu72-C13S (catalytically inactive mutant) with rif1∆ was performed by Southern blotting of ApaI digested DNA using a telomeric probe. D) ssu72+ and stn1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and stn1-75 performed by Southern blotting of ApaI digested DNA using a telomeric probe. Two independently generated ssu72∆ stn1-75 double mutants are shown.
Ssu72 phosphatase is a conserved telomere replication terminator (2019)
Figure 3

Figure 3. Ssu72 controls Stn1 telomere recruitment and phosphorylation.

A) Ssu72 is required for telomere recruitment of Stn1 in late S phase. ChIP analysis of stn1-myc in wt and ssu72∆ cells was performed in synchronized cdc25ts cells. n ≥ 3; *p ≤0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM).

B) 2D-gel analysis of NsiI telomeric fragments of wt and ssu72∆ strains. Smart ladder from Eurogentec was used for DNA size measurement.

C) Serine 74 substitution to a phosphomimetic aspartate amino acid (stn1-S74D) is sufficient to confer ssu72∆ telomere defects. Telomere length epistasis analysis of ssu72∆ and stn1-S74D mutants were performed by Southern blotting of ApaI digested genomic DNA using a telomeric probe.

D) Sequence alignment of Stn1 highlighting serine 74 identified in fission yeast as a phosphorylated residue.

E) Similar to ssu72∆ mutants, stn1-S74D is defective in telomere recruitment. ChIP analysis of stn1-myc and stn1-S74D-myc using a non-tagged strain as a control. n = 3; *p ≤0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).
Ssu72 phosphatase is a conserved telomere replication terminator (2019)
Figure 4

Figure 4. Ssu72 is required for polymerase α activation.

A) ssu72+ and pol1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and pol1-13 mutants was performed by Southern blotting of ApaI digested DNA using a telomeric probe.

B) Stn1-Pol1 interaction requires Ssu72 phosphatase. Immunoprecipitation experiments of Pol1-Flag with Stn1-Myc was preformed both in wt and ssu72∆ mutants.

C) Immunoprecipitation of Ten1-Flag with Stn1-Myc in either wt or ssu72∆ was performed as control for Stn1-Ten1 complex integrity.

D) Overexpression of polymerase alpha partially rescues telomere length defect of ssu72Δ mutants. Multi-copy vector with polymerase α under thiamine promoter were expressed both in wt or ssu72Δ cells.

E) Model for Ssu72 regulation of telomere replication in fission yeast. See text for details.
Ssu72 phosphatase is a conserved telomere replication terminator (2019)
Figure 5

Figure 5. Down-regulation of human SSU72 results in telomere elongation and fragility.

A) Telomere elongation of SSU72 down-regulated cells is telomerase dependent. Telomere Southern blot was carried out with HT1080 cells infected for 4 weeks with lentiviral particles carrying two independent shRNAs against SSU72 (CDS and UTR regions) and Luciferase (Luc) as control for shRNA. Knockdown efficiencies were determined by RT-qPCR using specific primers against hSSU72. Densitometry analysis is shown on Appendix Fig S5.

B) Telomere elongation of SSU72 down-regulated cells is epistatic with STN1 downregulation. TRF analysis of HT1080 cells infected with the appropriate retroviral or lentiviral particles carrying either GFP shRNA, SSU72 shRNA (against UTR), STN1 shRNA or double SSU72 shRNA/STN1 shRNA. Cells were grown for 4 weeks and DNA was isolated to carry out Southern Blot analysis. Densitometry analysis is shown on Appendix Fig S7.

C) hSSU72 down-regulation results in multi-telomeric signals (MTS) that are dependent on DNA replication. Visualization of mitotic spreads of HT1080 hSSU72 shRNA cells treated with aphidicolin and colcemid. Telomere FISH was carried out using a PNA-telomere probe. MTS are represented by arrows. Quantification of MTS per metaphase: n=4; **p ≤0.01 ****p ≤0.0001 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).
Ssu72 phosphatase is a conserved telomere replication terminator (2019)
Figure 6

Figure 6. SSU72 is required for STN1 recruitment to human telomeres.

A) hSSU72 downregulation results in telomere DNA damage foci (TIF). Cells with indicated shRNAs were fixed and IF-FISH analysis was performed using a 53BP1 antibody and a PNA-telomere probe. (Right) Quantification of cells with more than 5 telomeric 53BP1 foci: n=3; **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).

B) hSSU72 is required for efficient loading of hSTN1 at human telomeres. ChIP was preformed using anti-FLAG antibodies and Southern blotting was performed using a human telomeric probe. Quantification of 3 independent ChIP experiments *p ≤0.05 **p ≤0.01 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM).

Expanded View figure legends
Coordinated control of adiposity and growth by anti-anabolic kinase ERK7 (2020)
Figure 1

Figure 1: ERK7 inhibits lipid storage in the fat body

A. Schematic presentation showing the generation of ERK71 mutants by CRISPR/Cas9 targeting.

B. ERK71 mutants have elevated triacylglycerol (TAG) levels (N=4 replicates of ≥10 larvae/ replicate for each genotype).

C. Fat body specific depletion of ERK7 by RNAi (BDSC 56939) leads to increased TAG levels (N=4 replicates of 10 larvae/replicate for each genotype).

D. Representative immunofluorescent images of lipid droplet (LipidTOX) and nuclear (DAPI) staining in control and ERK71 mutant fat bodies of third instar larvae. Scale bar: 50 µm.

E. ERK71 mutant fat body cells contain more lipid droplets than control cells (N=30 cells for each genotype).

F. After fed [13C]glucose, the ERK71 mutant fat bodies display elevated [13C]TAG/PE and unlabeled TAG/PE molar ratios (measured by mass spectrometry-based lipidomics, N=3 replicates of 15 fat bodies/replicate, TAG=triacylglycerol, PE=phosphatidylethanolamine)

G. ERK7 overexpression in the fat body results in reduced TAG levels, while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the TAG storage (N=4 replicates of ≥10 larvae/replicate for each genotype).

H- J ERK7 expressing, GFP-marked, fat body clone (H, marked by yellow dotted line; scale bar: 50 µm) contains less (I) and smaller (J) lipid droplets than control cells (marked by white dotted line), visualized by LipidTOX staining (N=11 for control and 4 for ERK7 overexpression).

K. qRT-PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation. Expression of RP49 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).

Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). * p<0.05, ** p<0.01, *** p<0.001 (Student's t test).
Coordinated control of adiposity and growth by anti-anabolic kinase ERK7 (2020)
Figure 2

Figure 2: ERK7 overexpression inhibits lipid biosynthesis

A. Fatty acid composition of total lipids in CG>control and CG>ERK7 larvae, measured by gas chromatography (N=3 replicates of 15 larvae/replicate for each genotype).

B. Schematic presentation of biosynthesis of fatty acids and their likely chain shortening by peroxisomal partial β-oxidation.

C. Amount of glucose-derived [13C]-labeled carbons incorporated into the TAG pool of control and CG>ERK7 fat bodies (N=3 replicates of 15 fat bodies/replicate for each genotype and diet).

D. Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels (N=3 replicates of 15 larvae/replicate for each genotype).

E. Circulating glucose levels are decreased in ERK71 mutants (N=4 replicates of 15 larvae/replicate for each genotype).

Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). * p<0.05, ** p<0.01, *** p<0.001 (Student's t test).
Coordinated control of adiposity and growth by anti-anabolic kinase ERK7 (2020)
Figure 3

Figure 3: ERK7 regulates lipid metabolism gene expression

A. Four-way plot (t-statistics) presenting the ERK7-dependent fasting-responsive genes (adj.p.val < 0.05). Dark blue indicates significant in both comparisons; dark turquoise, significant in one comparison; light turquoise, not significant.

B. Venn diagrams of the fasting-downregulated/ERK7-dependent transcriptome (adj.p.val < 0.05) and fasting upregulated/ERK7-dependent transcriptome (adj.p.val < 0.05).

C. Bar plots of selected ERK7-regulated pathways with overrepresentation of significantly changing genes (adj.p.val <0.05, LFC <-0.5 or LFC >0.5). The bar plot shows enrichment (-log 10 adj.p.val, generated by piano package). Blue color denotes downregulated in mutants and red denotes upregulated in mutants. G - Gene Ontology (GO), K - KEGG Pathway, R - Reactome Pathway Database.

D. Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism. Color key displays scaled log2 gene expression.

E, F. Expression analysis of Seipin, Agpat3, ATPCL, fabp, Lip4 and CG34448 by qRT-PCR. RP49 was used for normalization (N=3 replicates of ≥10 larvae/replicate for each genotype).

Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). * p<0.05, ** p<0.01, *** p<0.001 (Student's t test).
Coordinated control of adiposity and growth by anti-anabolic kinase ERK7 (2020)
Figure 4

Figure 4: Fat body ERK7 inhibits cell autonomous and systemic growth

A. Weight of control and ERK71 mutant larvae at 72h after egg deposition (AED) (N=4 replicates of 12 larvae/replicate for each genotype).

B. Pupariation kinetics of control and ERK71 mutants (N=4 replicates of 30 larvae/replicate for each genotype). p<0.0001, analyzed by Log-rank test.

C. ERK71 mutants fail to suppress growth upon 24 h of starvation (N≥4 replicates of 15 larvae/replicate for each genotype).

D. ERK71 mutant larvae (N=4 replicates of 24 larvae/replicate for each genotype, p<0.01 analyzed by Log-rank test) are sensitive to nutrient deprivation.

E, F. Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to a moderately accelerated pupariation rate (E, p<0.001 analyzed by Log-rank test, N=8 replicates of 30 larvae/replicate for each genotype) and an increase in larval weight at 72h AED (F, N=5 replicates of 15 larvae/replicate for each genotype).

G. A representative image showing that fat body-specific overexpression of ERK7 by the CG-GAL4 driver (CG>ERK7) impairs larval growth. Scale bar: 0.5 mm.

H. Ectopic expression of ERK7 leads to delayed pupariation (p<0.0001, analyzed by Log-rank test), while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence pupariation rate (N=4 replicates of 30 larvae/replicate for each genotype).

I. In contrast to ERK7 wt, kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the pupal volume (N=4 replicates of 10 pupae/replicate for each genotype).

Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). ** p<0.01, *** p<0.001, ns - not significant (Student's t test).
Coordinated control of adiposity and growth by anti-anabolic kinase ERK7 (2020)
Figure 5

Figure 5: ERK7 regulates nucleolar size and ribosome biogenesis

A. Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus. Scale bar: 30 µm.

B, C. ERK71 mutant fat bodies display increased nuclear area (B, N>160 cells, obtained from 5 independent fat bodies) and increased nucleolar/nuclear area ratio (C, N>80 cells for nucleolus/nucleus ratio, obtained from 5 independent fat bodies), when compared to control fat bodies.

D-F. ERK7 expressing fat body clones (D, marked by GFP, scale bar: 20 µm) display reduced nuclear area (E) and reduced nucleolar/nuclear area ratio (F), when compared to control cells. Nuclei were visualized by DAPI staining, nucleoli were visualized by anti-Fibrillarin antibodies (N=10).

G, H. Expression analysis of Pol I (G) and Pol III (H) target RNAs by qRT-PCR. Expression of CDK7 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).

Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). * p<0.05, ** p<0.01, *** p<0.001, ns - not significant (Student's t test).
Coordinated control of adiposity and growth by anti-anabolic kinase ERK7 (2020)
Figure 6

Figure 6: PWP1 acts downstream of ERK7 to control growth and lipid storage

A. ERK7 overexpression leads to phosphorylation of PWP1, as shown by appearance of an extra band on Phos-tag Western blot (marked with red arrow). Insulin-inducible bands (dark blue) remain unresponsive to ERK7.

B. Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae. Scale bar: 20 µm.

C. PWP1 localization displays increased nucleolar/nuclear ratio in ERK71 mutant fat bodies compared to control (N=20 cells from 5 independent fat bodies per genotype).

D. Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae. ERK7 overexpression (GFP marked clones) alters the subcellular localization of PWP1. Scale bar: 30 µm.

E. PWP1 localization displays reduced nuclear/cytoplasmic ratio in ERK7 overexpressing clones, compared to control cells (N=10 cells from at least 8 different fat bodies per genotype).

F. Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels (N=4 replicates of ≥10 larvae/replicate for each genotype).

G. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body (CG-GAL4) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm.

H, I. Fat body specific knockdown of PWP1 suppresses increased lipid droplet number (H; N>30 cells per genotype) and increased nuclear area (I; N>45 cells per genotype) caused by ERK7 knockdown (BDSC 56939).

Data information: N stands for the number of biological replicates. Error bars display standard deviation (SD). ** p<0.01, *** p<0.001 (Student's t test).