EMBO/soda-nel huggingface dataset:
The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT-liver communication (2020)
Figure 5

Figure 5. PLTP lipid transfer activity is required for the metabolic effects of PLTP on glucose homeostasis

A Schematic illustration of mutant PLTP (PLTPM159E). The point mutation of PLTP coding sequence by substitution of methionine with glutamate at 159 amino acid position.

B Relative expression of Pltp in liver of mice that received adenovirus GFP (control) or PLTP. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. **P<0.01 relative to GFP by ANOVA one-way followed by Tukey's test. N.S., not significant.

C Plasma PLTP concentration in mice that received adenovirus GFP or PLTP as determined by quantitative mass spectrometry. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. ***P<0.001 relative to GFP by ANOVA one-way followed by Tukey's test. N.S., not significant.

D Plasma PLTP activity in mice that received adenovirus GFP or PLTP. n=4 for GFP, n=6 for PLTP and n=6 for PLTPM159E. ***P<0.001 PLTP relative to GFP and PLTPM159E; *P<0.05 PLTPM159E relative to GFP by ANOVA one-way followed by Tukey's test.

E Changes in body weight of mice that received AAV control or PLTP during 10 weeks of HFD. n=8 for GFP, n=10 for PLTP and n=10 for PLTPM159E. *P<0.05, **P<0.01 relative to PLTPM159E by two-way ANOVA repeated measures followed by Bonferroni's test.

F Relative Ucp1 expression in BAT of mice that received AAV GFP (control) or PLTP at 10 weeks of HFD. n=4 for GFP, n=5 for PLTP and n=4 for PLTPM159E. Tbp was used as an internal control. *P<0.05, **P<0.01 by ANOVA one-way followed by Tukey's test.

G Glucose tolerance test of mice that received AAV GFP or PLTP at 10 weeks of HFD. n=5 for GFP and n=6 for both PLTP and PLTPM159E. *P<0.05, **P<0.01, ***P<0.001 PLTP relative to GFP by two-way ANOVA repeated measures followed by Bonferroni's test. AUC of glucose, shown in the right graph, was analyzed by one-way ANOVA followed by Tukey's test. N.S., not significant.

H Plasma total cholesterol of mice that received AAV control or PLTP at 10 weeks of HFD. n=7 for all groups. *P=0.05 by ANOVA one-way followed by Fisher's LSD test. N.S., not significant.

I Plasma bile acids in (H). n=7 for all groups. ***P<0.001 PLTP relative to GFP and PLTPM159E by ANOVA one-way followed by Tukey's test.

J Plasma phospholipid in (H). n=7 for all groups. ***P<0.001 PLTP and PLTPM159E relative to GFP; *P<0.05 PLTP relative to PLTPM159E by ANOVA one-way followed by Fisher's LSD test.

Data information: All the data were represented as mean ± s.e.m.
Disentangling molecular mechanisms regulating sensitization of interferon alpha signal transduction (2020)
Figure 3

Figure 3, Model calibration with IFNα-induced signal transduction in Huh7.5 upon prestimulation and stimulation with IFNα

Growth factor-depleted Huh7.5 cells were prestimulated with 2.8 pM, 28 pM, 1400 pM IFNα or left untreated and were stimulated with 1400 pM IFNα 24 hours later. IFNα-induced signaling was measured by time-resolved quantitative immunoblotting and detected with chemiluminescence using a CCD-based camera. Data was normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration.

Model calibration with time-resolved IFNα-induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence intervals estimated from biological replicates (N=3 to N=23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively.

Model calibration with time-resolved IFNα-induced feedback transcripts upon prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα, assessed by qRT-PCR. mRNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT and TBP. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=3 to N=14) using a combined scaling and error model. Model trajectories are represented by lines.

Model calibration with the amount of molecules per cell of STAT1, STAT2, IRF9 and USP18 determined 24 hours after prestimulation with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Calibrator proteins were spiked into 10 µg of total protein lysate and subjected to quantitative immunoblotting. Immunoblot detection was performed by chemiluminescence using a CCD-camera-based device. Averaged values (N=4) are displayed with standard deviations. Green squares indicate amounts estimated by the mathematical model.

Model calibration of IFNα-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 prestimulated with 0, 2.8 pM, 28 pM or 1400 pM IFNα. Nuclear lysates were subjected to quantitative immunoblotting. Experimental data is represented by filled circles with errors representing 1σ confidence-interval estimated from biological replicates (N=4 to N=22) using a combined scaling and error model. Model trajectories are represented by lines.
Disentangling molecular mechanisms regulating sensitization of interferon alpha signal transduction (2020)
Figure 6

Figure 6, Cell type-specific basal protein amounts in relation to pathway desensitization in cell lines and primary human hepatocytes

The number of molecules per cell for STAT1, STAT2, IRF9 and USP18 were simulated by the mathematical model for untreated Huh7.5 and HepG2-hNTCP cells as well as for cells stimulated with 1.2 pM and 608 pM Roferon for 24 hours.

The number of molecules per cell for STAT1, STAT2, IRF9 and USP18 were determined experimentally in primary human hepatocytes from six different patients. Cells were harvested without pretreatment and different amounts of protein calibrators were spiked in total cell lysates and lysates were subjected to immunoblotting. Detection was performed with antibodies specific to STAT1, STAT2, IRF9 or USP18 using chemiluminescense on a CCD camera-based device (ImageQuant). Average of at least N=3 are displayed with standard deviations. Data was used for calibration of a primary human hepatocyte-specific mathematical model and estimated values are shown with green squares.

Experimental data and model fits of IFNα- and Roferon-induced phosphorylation of cytoplasmic or cellular STAT1 and STAT2 in growth factor-depleted primary human hepatocytes prestimulated with 0, 2.8 or 1400 pM IFNα (patients 1-3) or 0, 1.2 or 608 pM Roferon (patients 4-6). Primary human hepatocytes from the same patients 1-6 as in (B) were used. Experimental data is represented by filled circles (N=1 per patient). Experimental errors were estimated from the signal variance of the hepatocytes prestimulated with 1400 pM IFNα. Lines indicate model fits.

Absolute and relative antiviral response is calculated by the mathematical model for each cell line and patient-derived primary human hepatocytes prestimulated with 0, 1.2 or 608 pM Roferon by simulating the amount of occupied GAS and ISRE binding sites upon stimulation with 608 pM Roferon. For the absolute antiviral response, the baseline-subtracted area under curve (ΔAUC) from 24 to 28 hours is shown. For the relative antiviral response, the ΔAUC from 24 to 28 hours is divided by the corresponding ΔAUC in cells without pre-treatment.
Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons (2018)
Figure 1

Figure 1 - Expression and localization of Nrg3 in vivo.

A) Double fluorescence in situ hybridization using Nrg3 (green) and Gad1 (red) specific probes; DAPI was used as a counter stain. Nrg3 is present in Gad1-positive (filled arrowheads) and -negative neurons. (A') and (A'') show higher magnifications of boxed areas indicated in (A). Nrg3-negative neurons are indicated by open arrowheads; note that many but not all neurons express Nrg3.

B) Comparison of mRNA levels of Nrg3, Nrg1β type III (Nrg1β−III), Nrg2β, Nrg1β type II (Nrg1β−II), Nrg2α and Nrg1β type I (Nrg1β−I) in the cortex (Cx), hippocampus (HC) and striatum (Str) of juvenile mice (P30-60) by quantitative RT-PCR. Data represent means ± SD of 4 biological replicates. 2-way-ANOVA with Bonferroni's multiple comparisons test was performed to assess statistical significance (****P<0.0001, ***P<0.001, ns=not significant).

C) Schematic display of the structure of Nrg3 before (left) and after cleavage by Bace1 (right). The following domains of Nrg3 are indicated: black, N-terminal intracellular domain and transmembrane domain; blue, extracellular domain; red EGF domain; black, stalk region with Bace1 cleavage site indicated by an arrow and C-terminal transmembrane domain; green, intracellular domain.

D) Detection of Nrg3 by western blot analysis in fractions from brain lysates: S1, crude lysate; S2, cytosol and light membranes; S2', cytosol; LM, light membranes; P2', crude synaptosomes; S3', synaptosomal cytoplasm; SV, synaptic vesicles; P3, synaptosomal membranes; SPM, synaptic plasma membranes.

E, F) Immunohistochemical analysis in the hippocampus of wildtype (E,E',E'') and ErbB4 mutant mice (F) at 2 month of age using Nrg3-, ErbB4- and parvalbumin (Parv)-specific antibodies. The white box in (E) is displayed magnified in (E',E''). In control mice, Nrg3 is enriched on dendrites of ErbB4+ PV interneurons, compared to the surrounding neuropil (E',E''). In ErbB4 mutants, the Nrg3 enrichment on PV interneurons is not apparent (F).

G) Nrg3, ErbB4 and GluA4 were identified by immunohistology and co-localized in synaptic puncta on dendrites of interneurons in the stratum radiatum of the hippocampal CA1. Note that (G) displays Nrg3/ErbB4, (G') Nrg3/GluA4, and (G'') ErbB4/GluA4 signals of the same triple-stained image; false colors were assigned for better signal visualization.

H) Quantification of GluA4 puncta present on ErbB4+ dendrites in the CA1 stratum radiatum of adult wildtype (wt) and Nrg3-/- mice (age P90-120). Data are presented as box plots with Tukey's whiskers and outliers; means are indicated by a Plus symbols. n=65 (wt) and n=62 (Nrg3-/-) from 5 animals each. Unpaired t-test (2-tailed) with Welch's correction was performed to assess statistical significance (****P<0.0001).

Data information: Scale bars: 500 µm (A,E), 50 µm (F), 5 µm (G'').
Senataxin homolog Sen1 is required for efficient termination of RNA polymerase III transcription. (2019)
Figure 1

Figure 1. Sen1 associates predominantly with RNAP3-transcribed genes. (ABC) A-C Snapshots of ChIP-seq signals of Sen1 (this study), RNAP3 (this study) and RNAP2 (data from (Larochelle et al, 2018)) across (A) a 25 kb region of chromosome 3 containing several tRNA and 5S rRNA genes, (B) the U6 snRNA snu6 and (C) the srp7 loci.

(D) Comparison of the distribution of ChIP-seq reads across the indicated categories of genes for Sen1 (this study) and Seb1 (data from (Lemay et al, 2016)).

(E) Genome-wide pairwise Pearson correlation coefficient matrix at a resolution of 10 bp.

(F) ChIP-qPCR analysis of Flag-tagged Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates).

(G) ChIP-qPCR analysis of the Flag-tagged RNAP3 subunit Rpc37 and Flag-tagged Sen1 across the SPCTRNAARG.10 tRNA locus, whose TATA box was either mutated (TATA-) or not (TATA+). The mutations introduced to disrupt the putative TATA boxes are indicated in red above the graphs. The enrichment values were normalized to SPCTRNATHR.10 (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).
Senataxin homolog Sen1 is required for efficient termination of RNA polymerase III transcription. (2019)
Figure 2

Figure 2: Sen1 is required for normal RNAP3 transcription.

(A) (left) ChIP-qPCR analysis of Rpc37 in the presence or absence of Sen1 at the indicated loci in a population of cycling cells (mean ± standard deviation from 4 biological replicates). (right) Western blot analysis of Rpc37 protein levels in the presence or absence of Sen1. Tubulin was used as a loading control.

(B) (left) Total RNA from the indicated strains were separated on a 2,8% agarose gel. (right) Quantification of overall tRNA levels (mean ± standard deviation from 3 biological replicates).

(C) Strand-specific RT-qPCR was used to quantify the indicated RNAP3 transcripts. Transcript levels were normalized to act1 (mean ± standard deviation from 3 biological replicates).
Senataxin homolog Sen1 is required for efficient termination of RNA polymerase III transcription. (2019)
Figure 3

Figure 3: Sen1 regulates RNAP3 recruitment in an R-loop independent manner.

Cells were grown in minimal medium during 18 hours to induce the strong expression of RnhA.

(A) R-ChIP using a catalytically-inactive RNase H1 (Rnh1-D129N) was used to quantify R-loop formation at RNAP3-transcribed genes (mean ± standard deviation from 4 biological replicates).

(B) (left) ChIP-qPCR of the 13myc-tagged RNAP3 subunit Rpc25 in the indicated strains at the indicated loci (mean ± standard deviation of 4 biological replicates). (right) Western blot analysis of Myc-tagged Rpc25 and Flag-tagged RnhA protein levels in the indicated strains. Tubulin was used as a loading control.
Senataxin homolog Sen1 is required for efficient termination of RNA polymerase III transcription. (2019)
Figure 4

Figure 4: Sen1 is required for efficient RNAP3 transcription termination.

(A-C) Snapshots of ChIP-seq signals of the RNAP3 subunits Rpc1 and Rpc2 in the presence or absence of Sen1 across a representative (A) tRNA gene, (B) 5S rRNA gene and (C) srp7. Boxed regions highlight the increased density of reads in the downstream region of genes in the absence of Sen1.

(D) Average ChIP-seq profile of Rpc1 and Rpc2 across all isolated tRNA and 5S rRNA genes in the presence and absence of Sen1.

(E) Scanning of Rpc37-3flag occupancy at three different tDNA loci in the absence of Sen1 by ChIP-qPCR (mean ± standard deviation from 6 biological replicates).
Senataxin homolog Sen1 is required for efficient termination of RNA polymerase III transcription. (2019)
Figure 5

Figure 5: Lack of Sen1 produces extended read-through tRNA transcripts.

(A) Cells of the indicated genotypes that carried or not the DRT5T dimeric tRNA construct (schematized on top) were grown either in the presence of the optimum concentration of adenine (left) or in the presence of a limiting concentration of adenine (right). Two independent clones of the same genotype (#1 and #2) were used. See text for details. 3F refers to the 3 Flag epitope tag at the C-terminus of Rpc37.

(B) Tetrad dissection was used to show that the double mutant sen1∆ rpc37-V189D is dead.

(C) Strand-specific RT-qPCR was used to quantify the levels of read-through transcripts (see Methods). The mean ± standard deviation from 4 biological replicates is represented here. P-values were obtained using the Wilcoxon-Mann Whitney statistical test.

(D) Northern blot analysis of the tRNA SPATRNAPRO.02 using an intron-specific probe (TCTAAACTCAGCATACAAGTGGGG). U5 snRNA was used as a loading control.

(E) Sequence of the ~350 nt-long read-through transcript at SPATRNAPRO.02. Residues in blue represent the sequence of the mature tRNA. Residues in red represent potential terminator sequences. Red arrows show the 3' end nucleotide of the read-through transcripts. The numbers indicate the number of times the sequenced transcripts terminated at the indicated position.
Senataxin homolog Sen1 is required for efficient termination of RNA polymerase III transcription. (2019)
Figure 6

Figure 6. A strong terminator sequence compensates for lack of Sen1.

(A) Sequence of the engineered strong terminator (arg10-23T) at the SPCTRNAARG.10 gene.

(B) ChIP-qPCR analysis of Rpc37 around SPCTRNAARG.10 gene in the strong terminator mutant (mean ± standard deviation from 4 biological replicates).

(C) Strand specific RT-qPCR was used to quantify read-through transcripts at SPCTRNAARG.10 (arg10) and SPCTRNASER.09 (ser09) in the strong terminator mutant (mean ± standard deviation from 4 biological replicates; p-value obtained using the Wilcoxon-Mann Whitney statistical test).

EXPANDED VIEW FIGURE LEGENDS
tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription (2016)
Figure 1

Figure 1. Rpp30 mutations induce premature arrest of oogenesis in Drosophila.

A. Ovaries from control heterozygous Rpp3018.2 flies (left), homozygous Rpp3018.2 or transheterozygous Rpp3018.2/Rpp30PE flies (middle) and homozygous Rpp3018.2 carrying a copy of ubiRpp30GFP transgene (right). Scale bar, 100µm. B. Rpp30 is a subunit of RNase P (red), a ribozyme involved in pre-tRNA processing by cleaving the 5’ tail. The RNase Z (blue) cuts the 3’ tail of the pre-tRNA, and CCA motif is added, forming mature tRNA ready to bind an amino acid (aa). C. Gene region corresponding to Rpp30 (RNAse P protein 30, adapted from Flybase). The P-element insertion (Rpp30PE, dark grey) and the point mutation Leucine to Proline (Rpp3018.2, arrow) are shown. D. Ovaries were dissected, lysed and protein extracts analyzed by Western Blot using anti-Rpp30, anti- GFP or anti-tubulin antibodies. Orange arrows indicate overexpressed and endogenous Rpp30 signals. Genotypes are numbered as 1,2,3. E. Rpp30-dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds: Left. Homozygous Rpp3018.2 ovariole expressing ubiquitous ubiRpp30GFP (st, stage). Right. Transheterozygous Rpp3018.2/Rpp30PE ovarioles overexpressing germline specific nanosGal4,UASRpp30GFP. Note follicular somatic cells with no Rpp30GFP expression (arrowhead). Magnifications show nuclear Rpp30 localization, Rpp30 perinuclear foci (arrows), and Orb (a specific oocyte marker). Scale bar, 10µm. F. flp/FRT clones mutant for Rpp3018.2specifically in the germline (star) and/or the follicular cells (arrows) are detected by the absence of GFP. Magnifications show mutant oocytes with Orb mislocalization next to the karyosome (dotted circle) instead of being localized to the posterior, as seen in stage 3 (st.3) wild-type clones. Scale bar, 10µm.
tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription (2016)
Figure 2

Figure 2. Rpp30 is required for pre-tRNA processing.

Whole fly RNA extracts were used to study tRNA processing by Northern Blot. Top box : the three different probes used are depicted : 5’ and 3’ probes (red and green, respectively) can detect full pre-tRNAs and tRNAs intermediates, but will not detect mature tRNAs. An Internal Probe (IP, blue) corresponding to the anti-codon region was used to detect all non-mature and mature tRNAs. The genotypes used in this experiment are numbered at the bottom (1-6). Northern Blot panels: tRNA-his 5’ probe (top), tRNA-his 3’ probe (middle) and tRNA-his IP (bottom). U6 probe was used as loading control.
tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription (2016)
Figure 3

Figure 3. Rpp30 mutation leads to the activation of several checkpoint proteins and to

replication stress.

A. Rpp3018.2 homozygous early oogenesis arrest is partially rescued by p53 mutation. Scale bar,

100μm. B. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is rescued by chk2 mutation (Rpp3018.2 , mnkp6 and Rpp3018.2/ Rpp30PE, mnkp6). Scale bar, 100μm. Star: example of one rescued stage 9 egg chamber. Arrow: example of non-rescued egg chambers. C. Germline clones mutant for Rpp3018.2 were immunostained for PCNA (red). DAPI is in blue. Magnifications: PCNA signal in a control or mutant nurse cell. Scale bar, 10μm. D. Germline clones mutant for Rpp3018.2 were immunostained for Brf (pol-III) (red). DAPI is in blue. A Z-projection of Brf staining is shown. The arrow points Brf aggregates in a mutant chamber. Scale bar, 10μm. E. Early oogenesis arrest found in Rpp3018.2 homozygous ovaries is partially rescued by claspin mutation (Rpp3018.2; claspinEP/TM3). Scale bar, 100μm. F. Quantification of rescued ovaries harboring at least one stage 9 egg chamber in genotypes numbered from 1 to 11 after one day or several days on yeast. Grey bars (1-4) are control flies. n= number of ovaries. G. Top panel. Early oogenesis arrest found in Rpp30182./PE transheterozygous ovaries is rescued by sqd-S-HA overexpression. Scale bars: 100µm. Low panel. Rpp3018.2/PE ; sqd-S-HA/sqd-S-HA ovaries were dissected and stained for Orb (green), HA (squid, red) and DAPI (blue). Scale bar, 10µm.
tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription (2016)
Figure 5

Figure 5. Rpp30 mutation leads to a collapse of piRNAs.

A. Small RNAs extracted from ovaries of different genotypes were sequenced. Size histograms of normalized RNA populations matching Transposable Elements (TE) are shown. Red: sense. Blue: antisense. endo-siRNA (21nt) and piRNAs (23-28 nt) are indicated. Rpp30 mutations in two different genetic backgrounds (Rpp3018.2 and Rpp3018.2/Rpp30PE) specifically alters piRNA populations, compared to heterozygous control ovaries. white virgin ovaries were used as developmental control. Rpp3018.2 carrying the ubiRpp30GFP transgene (genetic background control) rescues general piRNA populations. aubHN/aubQC42 was used as positive control. B. Specific normalized unique mapper piRNA populations (23-28nt) from the major piRNA clusters are shown for the indicated genotypes. The scale of reads is indicated on the left. C. The ping-pong signal calculated on multi-mappers piRNA populations is shown for the different genotypes. D. Nuage specific markers (Aub, Ago3 and Mael) are shown in stage 3 wild-type and germline mutant clones for Rpp3018.2. Scale bar, 10µm. E-H. Double mutant ovaries for Rpp30 and chk2 (Rpp3018.2, mnkp6) rescued the specific and general piRNA populations, nuage morphology and the characteristic ping-pong signal. Scale bar, 10µm.
tRNA processing defects induce replication stress and Chk2-dependent disruption of piRNA transcription (2016)
Figure 6

Figure 6. Rpp30 mutation leads to transposable elements overexpression.

A. Ovaries of different genotypes were dissected for RNA extraction. RT-qPCR was performed using transposable elements specific oligonucleotides. The boxplots show several pooled transposon fold changes obtained for different genotypes after normalization to control heterozygous Rpp3018.2/CyO. Homozygous and transheterozygous Rpp30 flies (yellow and orange, respectively) show transposon overexpression, whereas Rpp3018.2/Rpp30PE ovaries carrying the ubiRpp30GFP transgene are similar to control. aubHN/QC42 ovaries (grey) were used as positive control. Arrows point to some highly expressed transposons. B. Scatter plot showing the expression of canonical transposable elements from RNA sequencing data obtained from white virgins versus Rpp3018.2 ovaries. Arrows point to P-element (P-ele) and I-element (I-ele). Transposons are indicated as red or black dots (significant or not significant fold change, respectively). Genes are indicated as yellow or grey dots (significant or not significant fold change, respectively). C. Significant transposons fold changes (p<0.05) were pooled and shown in a boxplot (grey). Note that the median value (black bar) is above 0. Arrow indicates the P-element (P-ele). Fold changes of individual transposons are shown in a barplot. Red bars, upregulated transposons. Blue bars, downregulated transposons. D. Testes from control heterozygous, transheterozygous Rpp3018.2/Rpp30PE or aubHN/QC42 were fixed and stained for Stellate crystal formation. Z-projections are shown.