EMBO/soda-nel huggingface dataset:
Protein abundance of AKT and ERK pathway components governs cell-type-specific regulation of proliferation (2017)
Figure 5

Figure 5. Evaluation of the effects of AKT inhibitor AKT VIII and MEK inhibitor U0126 on signaling and cell cycle.

A Epo-induced signaling upon AKT or MEK inhibitor treatment. Growth-factor deprived cells were pretreated for half an hour with AKT VIII or U0126, respectively, and subsequently stimulated with 5 U/ml Epo. PDI served as loading control.

B Model calibration with integrated pAKT, ppERK and pS6 data upon inhibitor treatment and 5 U/ml Epo stimulation of mCFU-E, 32D-EpoR and BaF3-EpoR cells. Area under curve from time-resolved, quantitative immunoblotting data was calculated and is represented by filled circles. Error bars represent standard deviation estimated by an error model. Solid lines represent model trajectories.

C Analysis of cell-cycle indicator genes upon AKT VIII and U0126 treatment. Growth-factor deprived cells were pretreated for half an hour indicated doses of a single inhibitor, followed by stimulation with 5 U/ml Epo for 0 h and 3 h. The expression of cyclinD2, cyclinG2 and p27 was measured by quantitative RT-PCR and normalized to the Rpl32 gene. Genes were selected based on microarray analysis. Experimental data is shown as fold change to unstimulated cells with mean ± standard deviation, N=3. Welch Modified Two-Sample t-Test, n.s. not significant, * p<0.05, ** p<0.01, *** p<0.005.
Protein abundance of AKT and ERK pathway components governs cell-type-specific regulation of proliferation (2017)
Figure 7

Figure 7. Prediction of cell-type-specific proliferation.

A Upper panel: Model prediction of Epo-dependent proliferation in mCFU-E and BaF3EpoR cells overexpressing SHIP1 or PTEN. Solid lines represent model trajectories.

Lower panel: Experimental validation of PTEN and SHIP1 overexpression effects on Epo-dependent proliferation. Proliferation was assessed using [3H]-Thymidine incorporation 14 h (mCFU-E) or 38 h (BaF3-EpoR) after retroviral transduction with PTEN or SHIP1 construct for overexpression. Data represented as mean ± standard deviation, N=3. EC50 values are given.

B Upper panel: Model prediction of proliferation upon combined inhibitor treatment and 5 U/ml Epo stimulation. Maximum proliferation was scaled to 1.

Lower panel: Experimental validation of proliferation upon combined inhibitor treatment and 5 U/ml Epo stimulation. Proliferation was measured as cell numbers after 14 h (mCFU-E) or 38 h (BaF3-EpoR, 32D-EpoR) with Coulter Counter. Maximum was scaled to 1.
Selective termination of lncRNA transcription promotes epigenetic silencing and cell differentiation (2017)
Figure 1

Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (A) Box-plot of the enrichment of the RNAs identified by Mmi1 RNA-IPs coupled to high throughput sequencing. The log of the average enrichments obtained from two independent RNA-IPs is plotted. The enrichment is relative to the no antibody RNA-IPs conducted in parallel of Mmi1 RNA-IPs. The mRNAs and lncRNAs enriched at least two folds in both Mmi1 RNA-IPs are shown in blue and green, respectively. Crosses represent newly identified Mmi1 targets and dots known targets. (B) LncRNAs predicted to be targets of Mmi1 with a high confidence by our computational approach. (C) Surface representation of Mmi1 YTH domain. Area corresponding to conserved residues (according to the alignment shown in Fig EV 2A) and located at the surface are highlighted in green. Mutated residues in Mmi1 YTH domain are indicated in blue for the ones located within the aromatic cage, in purple for the ones surrounding the cage, and in red for the rest. (D) RNA-IPs showing the impact of Mmi1 YTH domain point mutations on Mmi1’s binding to ssm4 mRNA. The lower part shows a Western blot monitoring the protein level of WT and mutant Mmi1 proteins in the cells used for the RNA-IPs. Loading was monitored using an anti-Tub1 (tubulin) antibody. (E, F) RNA-IPs showing the YTH-dependent association of Mmi1 to the lncRNAs identified in (A) and (B). Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for (D, E and F).
Selective termination of lncRNA transcription promotes epigenetic silencing and cell differentiation (2017)
Figure 2

Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (A) Upper part, scheme of S. pombe sexual differentiation. Lower part, microscopy images showing WT and mmi1∆ cells, and mmi1∆ cells expressing Mmi1-R351E or Mmi1-R381E mutant proteins. Images were taken after 24 hours of induction of sexual differentiation by growth on SPAS medium. The percentage of cells that underwent differentiation and iodine vapor assays, conducted on the corresponding patches, are shown at the bottom of each image. (B) Mating assay showing the percentage of zygotes forming over time in the same cells as in (A). (C) Scheme of nam1-byr2 locus. Mmi1 UNAAAC binding motifs are depicted by white lines. (D) Scheme of nam1-byr2 locus in nam1-1 cells highlighting the 8 UNAAAC motifs mutated (red lines). (E) Mmi1 RNA-IPs showing the specific loss of binding of Mmi1 to nam1-1 lncRNA but not to mei4 mRNA, another target of Mmi1. (F) Mating assay showing the percentage of zygotes formed over time in WT and nam1-1 cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) (B, E and F). (G) Western blots showing the level of Flag-Byr2 protein over the first 4 hours of sexual differentiation in WT and nam1-1 cells. Tubulin (Tub1) level was used as a loading control. (H) Microscopy images of WT (h90) cells transformed with an empty plasmid (Control) and nam1-1 cells transformed with either an empty plasmid (Control) or a plasmid expressing Byr2 protein (Byr2), after 24 hours of induction of sexual differentiation. Scale bar, 10 µm.
Selective termination of lncRNA transcription promotes epigenetic silencing and cell differentiation (2017)
Figure 3

Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (A) Northern blots showing nam1 and byr2 RNA levels during the first 4 hours of sexual differentiation. Ribosomal RNAs (rRNAs) stained with ethidium bromide were used as loading controls. Black lines indicate probes used to detect nam1 and byr2 RNAs. (B) ChIPs showing the occupancy of the elongating RNAPII (RNAPII-S2P) over nam1-byr2 locus, in WT and nam1-1 cells. RNAPII-S2P was immunoprecipitated with an antibody recognizing the heptameric repeats (present in the C-terminal domain of the polymerase) when it is phosphorylated on its serine 2. Black lines, genomic regions investigated. (C) RT-qPCRs showing the accumulation of nam1 read-through transcripts (RT1-qPCR) and nam1 lncRNAs (RT2-qPCR) in cells with or without the transcription terminator Ttef inserted at the 3’ end of nam1 (scheme). Black arrow, primer used for the strand-specific reverse transcription (RT); Black line, location of the region amplified by PCR. (D) Microscopy images of, respectively, WT (h90), nam1-1 and nam1-1-Ttef cells, after induction of sexual differentiation for 24 hours. The percentage of sporulation and iodine vapor assays are shown at the bottom of the images. Scale bar, 10 µm. (E) RT-qPCRs showing the accumulation of nam1 read-through transcripts in mmi1∆ and rrp6∆ cells. Black arrow and line as in (C). (F) ChIPs monitoring the enrichment of H3K9me2 over nam1-byr2 locus and mei4 gene in WT and rrp6∆ cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).
Selective termination of lncRNA transcription promotes epigenetic silencing and cell differentiation (2017)
Figure 4

Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (A) Upper part, TNAAAC-rich sequence present in nam5, 6 and 7 lncRNAs. Lower part, schematic representation of S. pombe three centromeres showing the different pericentromeric DNA repeats susceptible to produce nam5, 6 and 7 (green arrows). (B) Northern blot showing the level of nam5/6/7 lncRNA population in mmi1-ts3, dcr1∆, clr4∆ single mutant cells and in mmi1-ts3 dcr1∆ and mmi1-ts3 clr4∆ double mutant cells, at the permissive (25ºC) and restrictive (36ºC) temperatures. (C) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts, in the same cells and conditions as in (B). Red arrow heads point to the expected PCR products while the other bands correspond to non-specific PCR products. From the scheme and the agarose gels: black arrows, primers used for the three different reverse transcriptions; black lines and red numbers, expected PCR products of the three different RT-PCRs; M, DNA ladder Markers; tub1, tubulin control. (D) RT-qPCRs showing the levels of nam5/6/7 lncRNA population in the double mutant rrp6∆ dcr1∆ cells, relative to the single mutant rrp6∆ and dcr1∆ cells. (E) RNA-IPs showing that Rrp6-Myc13 binds to nam5/6/7 lncRNAs in a Mmi1-dependent manner. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (D, E). P value was calculated using a two-tailed Student’s t-test.
Selective termination of lncRNA transcription promotes epigenetic silencing and cell differentiation (2017)
Figure 5

Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (A) ChIPs assessing the localization of Mmi1 to pericentromeric DNA in WT and clr4∆ cells. (B) ChIPs showing the localization of Mmi1 (dashed line) and H3K9me2 (black line) to the pericentromeric nam5/6/7 DNA regions during the progression of the cell cycle. Cell synchronization was achieved by using cdc25-ts cells (see experimental procedures for more details). Cell synchronization was monitored by measuring the percentage of cells with a septum (right part). (C) RT-qPCRs monitoring the levels of nam5/6/7 (left part) and dg (middle part) lncRNA populations during the cell cycle. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (A, B and C). (D) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts in G2/M and G1/S phases from synchronized cells as in (B). Red arrow heads point to the expected PCR products. The other bands are non-specific PCR products. From the scheme: black arrows, primers used for the reverse transcriptions; black lines and red numbers, regions amplified by PCR.
Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates (2020)
Figure 1

Figure 1. Enzymatic degradation of RNA causes protein aggregation.

A Diagram showing the experimental design.

B SDS-PAGE analysis of soluble (Supernatant) and insoluble proteins (Pellet) from human neurons after treatment for one hour with a mixture of RNase A and RNase T1 (A/T1), or vehicle (Ve-).

C Protein aggregation (Pellet) after incubation with different ribonucleases or DNase I in the presence of either EDTA or Mg2+. Ribonucleases used were RNase A (A), RNase T1 (T1), a mixture of RNase A and RNase T1 (A/T1), RNase 1f (1f), and RNase V1 (V1).
Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates (2020)
Figure 2

Figure 2. Enzymatic degradation of RNA induces aggregation of proteins associated with neurodegenerative diseases.

A-B Western blot analysis of soluble and aggregated proteins after RNase treatment of lysate from human neurons (A), or HEK293 cells expressing GFP-Aβ or GFP (B).

C Effect of an RNase A inhibitor (RNasin) on RNase A mediated protein aggregation in neuronal cell lysate.
Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates (2020)
Figure 3

Figure 3. RNA is required for renaturation and function of proteins aggregated by enzymatic degradation of RNA in human neuronal lysates.

A Diagram showing the renaturing assay.

B Effect of RNA/protein ratio on protein aggregation after renaturing.

C Coomassie-stained gel of soluble (Sup 1/2) and aggregated (Pel 1/2) proteins after removal of GuHCl in the presence (+) or absence (-) of total RNA. Asterix (*) denotes added RNase A.

D As in (c) but analysed by Western blot.

E ATP-hydrolysing activity of proteins renatured with total RNA or buffer (Ve-) after removal of GuHCl.

Data information: All experiments were performed with total RNA. Data in (b and e) are expressed in arbitrary units (AU) and represent the mean ± s.d of two (b) or three (e) independent experiments. **=p<0.01, *=p<0.05 by ANOVA post-hoc analysis.
Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates (2020)
Figure 4

Figure 4. Features of solubilising nucleic acids.

A Genomic attributes of RNA associated with renatured NF-H, Tau, or Aβ. Inset shows which fractions of the peaks that derive from exons or introns have their origin in coding or non-coding transcripts, respectively.

B Characterization of DNA associated with soluble, renatured Aβ or all proteins (Total), captured either by immunoprecipitation (Aβ ) or absorption to membranes (Total).

C Sequence logo of the computationally identified motif (M1) in the solubilising genomic DNA of the Total samples.
Mutant MRPS5 affects mitoribosomal accuracy and confers stress-related behavioral alterations (2018)
Figure 1

Figure 1 - Determination of translational accuracy in cell-free translation assays.

A, B Misreading (A) and read-through (B) were measured by dual luciferase gain-of-function assays using wild-type mitohybrid ribosomes (n=8 misreading, n=4 read-through), mutant A1555G mitohybrid ribosomes (n=8 misreading, n=4 read-through), E. coli wild-type ribosomes (n=9), E.coli mutant A127Y ribosomes (n=9), M. smegmatis merodiploid wild-type ribosomes (n=9), and M. smegmatis merodiploid mutant S152Y ribosomes (n=9). Mistranslation was determined by assessing the misreading at His245 (CAC codon) of the F-luc gene that was replaced by the near-cognate codon CGC or the non-cognate codon AGA, both encoding for Arg. To determine read-through the Asp357 (GAC codon) of the F-luc gene was replaced by the nonsense TGA stop codon. Results are derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity. Wild-type samples were set as 1, error bars indicate SEM; **p<0.01, ***p<0.005, ****p<0.001, ns, not significant (Student's t-test).

C Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA. Following immunoprecipitation, the MT-CO1 band was quantified by autoradiography, wild-type mitohybrid ribosomes were set as 1 (n=3), error bars indicate SEM; ****p<0.001 (Student's t-test). Products of immunoprecipitation as resolved by SDS-Page are shown exemplarily in Fig S2E.

D Ratio of 35S-Cysteine and 35S-Methionine incorporation by in-vitro translation in the presence of tobramycin using wild-type mitohybrid ribosomes, mutant A1555G mitohybrid ribosomes, and MT-CO1 mRNA; the MT-CO1 band was quantified by autoradiography.

E Misreading of wild-type mitohybrid ribosomes and mutant A1555G mitohybrid ribosomes in the presence of tobramycin. Misreading was assessed by dual luciferase assay, and results derived by calculating mutant hFluc/hRluc activity related to wild-type hFluc/hRluc activity.

F In-vitro translation of MT-CO1-TGA mRNA and MT-CO1-AGA mRNA using mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). Autoradiography of immunoprecipitated MT-CO1 proteins; top: 35S-Met labelling, bottom: 14C-Lys labelling. In-organello translated 35S-Met labelled MT-CO1 was used as a marker. * MT-CO1, ** MT-CO1 extended by read-through, MT-CO1 AGA-polyA and MT-CO1 TGA-polyA constructs used for in-vitro translation are schematized.

G Ratio of 14C-Lys and 35S-Met labelled immunoprecipitated MT-CO1 proteins. In-vitro translation using MT-CO1-TGA mRNA, MT-CO1-AGA mRNA, and mutant A1555G mitohybrid ribosomes in the presence of tobramycin (0 - 20 µM). The corresponding MT-CO1 band was quantified by autoradiography.
Mutant MRPS5 affects mitoribosomal accuracy and confers stress-related behavioral alterations (2018)
Figure 2

Figure 2 - Assessment of mitoribosomal misreading in in-organello translation.

A Autoradiography of in-organello mitochondrial translation products derived from HEK293 wild-type cells in the presence of tobramycin, incorporation of 35S-Met and 35S-Cys in MT-CO1 protein. Following in-organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in-organello translation in the presence of 1000 µM tobramycin. MT-CO1 bands were quantified and ratio of 35S-Cys / 35S-Met calculated, the 35S-Cys / 35S-Met ratio in the absence of tobramycin was set as 1 (n=3), error bars indicate SEM.

B Ratios of 35S-Cys / 35S-Met labelled MT-CO1 and MT-CO2 proteins synthesized in in-organello translation and effect of tobramycin. Following in-organello translation in the presence of tobramycin (1000 µM, 2000 µM) proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 and MT-CO2 bands were quantified and ratio of 35S-Cys / 35S-Met incorporation calculated (n=3), error bars indicate SEM; *p<0.05, ns, not significant (Student's t-test).

C Ratio of 35S-Cys and 35S-Met labelled MT-CO1 protein synthesized in in-organello translation using mitochondria from HEK293 MRPS5 wild-type cells and HEK MRPS5 mutant V336Y cells. Following in-organello translation, proteins were immunoprecipitated and analyzed by autoradiography. MT-CO1 bands quantified and ratios calculated. The ratio of the MRPS5 wild-type was set as 1 (n=8 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).

D Autoradiography of in-organello mitochondrial translation products derived from HEK293 MRPS5 wild-type cells in the presence of tobramycin (0-750 µM). Proteins were 35S-Met labeled and loaded either directly on SDS-PAGE (total, top) or following poly-lysine immunoprecipitation (bottom). * MT-CO1 and ** MT-CO1 with poly-lysine extension.

E Quantification of MT-CO1 proteins in HEK293 MRPS5 wild-type cells and HEK293 MRPS5 mutant V336Y cells. In-organello mitochondrial translation in the presence of 35S-Met and 750 µM tobramycin. Proteins were loaded either directly on SDS-PAGE (total) or following poly-lysine immunoprecipitation and analyzed by autoradiography. For total protein samples the MT-CO1 band was quantified, for poly-lysine immunoprecipitated samples the extended MT-CO1 band was quantified and the ratio of extended MT-CO1/total MT-CO1 calculated. The ratio of MRPS5 wild-type was set as 1 (MRPS5 wild-type, n=5 clones, MRPS5 V336Y, n=7 clones), error bars indicate SD; ***p<0.0001 (Student's t-test).
Mutant MRPS5 affects mitoribosomal accuracy and confers stress-related behavioral alterations (2018)
Figure 6

Figure 6 - Noise-induced hearing damage.

Noise exposure and ABR measurements. Exposure of mice to broadband noise (2-10 kHz) of either 108 or 110 dB SPL resulted in elevations of auditory thresholds. Thresholds in Mrps5V338Y/V338Y mutant mice (black bars) were significantly higher after exposure than thresholds in littermates (open bars), (p<0.0001, two-way ANOVA for combined 108 and 110 dB data). Frequencies of 12, 24 and 32 kHz were selected for measurement to cover the mid-frequency to high-frequency regions of the cochlea. n=6 for baseline littermates; n=6 for baseline mutants; n=3 for all noise exposures. Graphs show mean and SEM.
Circadian time- and sleep-dependent modulation of cortical parvalbumin-positive inhibitory neurons (2022)
Figure 1

Figure 1. Distinct inhibitory output from PV neurons between the light and dark cycle.

A. Experimental demonstration. A1. Illustration of the mapping principle between ChR2-expressed PV neurons (green) and regular pyramidal neuron (Pyr, orange). A2. Acute slices were obtained and recorded at ZT0 or ZT12. A3. Coronal brain atlas showing the primary visual cortex (Top left panel, modified from MBL mouse brain atlas). Yellow box highlights the region of experiment. Top right panel, the reference image taken with a 10x objective from a slice prepared from a PV:Ai32 mouse. The 0.45 mm2 shaded cubic area was divided into 15 x 15 grids for independent light stimulation. The white arrowhead indicates the position of the recording electrode. Bottom panel, representative raw traces of light-evoked IPSCs (LEIPSCs) recorded from one pyramidal neuron. A4. The spatial distribution of the LEIPSCs from the example in A3. A5. The heatmap of the LEIPSCs in A4. B. The average heatmaps at ZT0 and ZT12 after soma alignment. Soma position was indicated by the blue arrow head. C. Comparison of the cumulative distribution (Kolmogorov-Smirnov test [K-S test]; P = 0.002) and the mean (Inset. ZT0: 813.3 ± 53.13 pA; ZT12: 1306 ± 93.73 pA; P < 0.0001; Mann-Whitney U test) of the max LEIPSC amplitude between ZT0 and ZT12. D. Comparison of the cumulative distribution (K-S test, P = 0.0016) and the mean (Inset. ZT0: 10.44 ± 1.44 nA; ZT12: 18.87 ± 1.44 nA; P < 0.0001; Mann-Whitney U test) of the sum LEIPSC amplitude between ZT0 and ZT12. E. The average heatmaps of LEIPSCs after pia aligned to the reference image in A3. Dark and light blue overlay traces on the side of the map depicted the marginal distribution of the sum LEIPSCs for ZT0 (Dark blue) and ZT12 (Light blue). F. Comparison of the cumulative distribution (K-S test, P = 0.0021) and the mean (Inset, ZT0: 0.067 ± 0.004 mm2; ZT12: 0.089 ± 0.0036 mm2; P = 0.0003; Mann-Whitney U test) inhibitory input size between ZT0 and ZT12.

Data information: Sample sizes used in this figure: ZT0: 48 cells from 9 mice; ZT12: 51 cells from 9 mice. Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.
Circadian time- and sleep-dependent modulation of cortical parvalbumin-positive inhibitory neurons (2022)
Figure 5

Figure 5. Spontaneous activity of PV neurons oscillates naturally during the light/dark cycle.

A1. Diagram illustrating the in vivo two-photon imaging timeline. A2. GCaMP6s (green) was expressed in V1 of the PV:Ai9 mice (red: td-Tomato). Scale: 500 µm. A3. Schematics of the calcium imaging showing a mouse that was head-fixed and free running on an air-suspended arena during the imaging section. Locomotor activity was simultaneously monitored (bottom left) along with Ca2+ imaging (Top right. Scale: 100 µm). Example spontaneous ΔF/F0 traces (bottom right). B. Top panel, Example spontaneous ΔF/F0 traces from three PV neurons repetitively imaged at ZT0 and ZT12. Bottom panels, Heatmaps showing the spontaneous ΔF/F0 time-series traces of all PV neurons imaged at ZT0 and ZT12. The color code represents the magnitude of ΔF/F0(%). C. Cumulative probability distribution of the sum ΔF/F0 of all PV neurons imaged at ZT0 and ZT12 (K-S test; P = 0.02). Inset: the mean sum ΔF/F0 (ZT0: 401.6 ± 19.55; ZT12: 559.1 ± 35.29; P = 0.0002; Mann-Whitney U test). D. Left panel, diagram depicting repetitive imaging protocol. Right panel, mean activity of PV neurons indicated by Z score imaged at four timepoints.

Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.