EMBO/soda-nel huggingface dataset:
Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-OctB2R cascade (2020)
Figure 5

Figure 5 Stx provides a buffering mechanism for Octβ2R expression in the presence of OA.

(A) Quantitative RT-PCR of stx in control and in mutant of the Tβh gene in the OA synthesis pathway. (mean±SEM: control for Tβh, 0.977±0.05125, N=3; Tβhnm18, 1.383±0.0325, N=3). (B) Quantitative RT-PCR of stx after OA treatment in w1118 control and Octβ1R, Octβ2R mutants, Octβ3R mutants. (From left to right mean±SEM: 1.000±0.0288, 0.781±0.0176, 1.477±0.043，1.403±0.0578, 1.157±0.0617, 0.881±0.0689, 1.149±0.0398, 0.953±0.0543, 1.017±0.0600, 1.090±0.0378, N=3). (C) Quantitative RT-PCR of Octβ2R after OA treatment in wildtype control and stx mutants. (From left to right: mean±SEM: 0.897±0.0721, N=4; 0.512±0.1132, N=3; 0.475±0.0320, N=4; 0.557±0.0712, N=3; 1.01±0.0085, N=2; 0.795±0.0225, N=2; 0.770±0.0705, N=2; 0.763±0.0630, N=2). (D) Model of stx function. (E) Ellipsoid body knock down of Octβ2R results in no significant change of stx. Data represent mean±SEM: 1.012±0.006, 0.635±0.027, 1.323±0.136; 0.839±0.055, 1.201±0.072, 0.790±0.052. N=3. (F) Ellipsoid body knock down of Octβ2R results in sleep rebound increase. In response to OA, ellipsoid knock down of Octβ2R rescues control phenotype. Data represent mean±SEM: 13.80±2.439, n=31; 5.465±2.036, n=25; 19.55±2.275, n=27; 12.47±2.684, n=24; 30.03±3.428, n=25; 43.67±4.616, n=25. (G-H) Sleep profile of PcXT109 mutant with or without OA treatment. In both control and PcXT109 mutant flies, the treatment of OA results in sleep decrease, n=16 (G). Quantifications of the decreasing amount showed that PcXT109 mutant fly loss significantly more sleep that control flies (From left to right mean±SEM: 13.90±1.397; 21.25±1.493, N=4) (H).

Data information: Bar graphs are presented as mean ± SEM. Statistical differences were measured using unpaired Student's t-test, n.s. indicates no significant difference, *P < 0.05, **p <0.01, and ***P < 0.001. n indicates the number of tested flies; N indicates the number of biological repetitions (For A-C and E, each repeat with a sample size of 30 individual fly heads). All the p-values are listed in supplementary table (Table EV3).
Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-OctB2R cascade (2020)
Figure 6

Figure 6. Phenotype identification of TβH mutant by video-based methods.

Sleep curve for Tβh mutant and control flies in video-based method. (B) Total sleep for Tβh mutant and control flies in video-based method (From left to right mean±SEM: 688.2±32.71, n=48; 123.2±11.68, n=46). (C) Number of Grooming behaviors in 10min during ZT3.5-4.5 (From left to right mean±SEM: 9.571±2.644, N=7; 4.429±1.343, N=7; 7.429±2.170, N=7; 24.29±3.300, N=7).

Data information: Bar graphs are presented as mean ± SEM. (B) Statistical differences were measured using non-parametric test with two-tailed Mann-Whitney test, ***P < 0.001. (C) Statistical differences were measured using unpaired Student's t-test, n.s. indicates no significant difference, **p <0.01. n indicates the number of tested flies; N indicates the number of biological repetitions. All the p-values are listed in supplementary table (Table EV3).

Expanded view figures legends
A Single-Cell Transcriptome Atlas of the Adult Human Retina (2019)
Figure 2

Figure 2: Identification of MALAT1-hi and MALAT1-lo subpopulations of rod photoreceptors.

A) Feature expression heatmap of a panel of known marker genes for rod photoreceptors across the identified 16 retinal cell clusters. Brown colour indicates ≥100 nTrans (number of transcripts).

B) Representation of the three donor retina samples in the six rod photoreceptor clusters.

C) Violin plot showing high or low expression levels of MALAT1 in rod photoreceptor clusters.

D) Distribution of rod photoreceptor populations with high MALAT1 expression (MALAT1-hi) or low MALAT1 expression (MALAT1-lo) in three donor retina samples.

E) Fluorescent in situ hybridization analysis of human peripheral retina showing heterogeneous levels of MALAT1 expression in the rod photoreceptors located in the outer nuclear layer (ONL). INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm.
A Single-Cell Transcriptome Atlas of the Adult Human Retina (2019)
Figure 3

Figure 3: MALAT1 subpopulations of rod photoreceptors is not due to donor variation. Canonical correlation analysis was used to effectively correct donor-specific variations in rod photoreceptors.

A, B) (A) t-SNE visualization of human retinal cells colored by annotation of 13 transcriptionally distinct clusters (CCA0-CCA12) and (B) their distribution in 3 donor retina samples.

C) Feature expression heatmap showing expression patterns of 7 rod photoreceptor markers across 12 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥50 nTrans (number of transcripts).

D) t-SNE plots showing expression of MALAT1.

E) Expression pattern of MALAT1 in the rod photoreceptor showing MALAT1-hi (CCA1, CCA10) and MALAT1-lo (CCA0) subpopulations. x-axis depicts normalized transcript levels.

F) t-SNE plots showing expression of major ribosomal genes.

G) Heatmap of differentially expressed genes between the two MALAT1-hi clusters CCA1 and CCA10. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered in CCA1 and CCA10.
A Single-Cell Transcriptome Atlas of the Adult Human Retina (2019)
Figure 4

Figure 4: Loss of MALAT1 expression in rod photoreceptors with longer post-mortem time. Fluorescent in situ hybridization analysis of the same donor human peripheral retina at different time points post-mortem (7 and 13 hours, Retina 7), showing decreases in MALAT1-hi rod subpopulations in the outer nuclear layer (ONL) at later time point. INL: inner nuclear layer; OPL: outer plexiform layer. Scale bar = 20µm. White arrows indicated MALAT1-hi rod photoreceptors.
A Single-Cell Transcriptome Atlas of the Adult Human Retina (2019)
Figure 5

Figure 5: Assessment of cone photoreceptor and glial cell types in human retina .

A) Feature expression heatmap showing the expression of 11 known cone photoreceptor markers across 16 retinal cell clusters. Brown colour indicates ≥10 nTrans (number of transcripts).

B) The proportion of cone photoreceptor subtypes identified in population C10, based on expression of OPN1LW / OPN1MW (L/M cones) and OPN1SW (S cones).

C) Scatter plots showing expression of OPN1LW/OPN1MW or OPN1SW in individual cone photoreceptors for population C10. The colour depicts expression level for OPN1LW/OPN1MW in individual cells.

D) Heatmap of top 20 differentially expressed genes between L/M cones and S cones. The colour depicts normalised gene expression (z-score capped at 2.5).

E) Expression pattern of glial markers in Muller glia (C9) and retinal astrocytes (C16). x-axis depicts normalized transcript levels.
Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. (2013)
Figure 1

Expression of ΔPrPs and dominant-negative inhibition (DNI) of Δ159 and Δ159–175 in persistently prion-infected cells.

A. Schematic illustration of prion protein secondary structure elements comprising two β-strands (B1 and B2) and three α-helices (H1–3) and post-translational modifications (two N-linked glycans and glycosylphosphatidylinositol anchor) along with position and extent of deletion of ΔPrPs constructs. Deletions in the H1∼H2 portion have a common N-terminal end (residue 159) and gradually extend into the C-terminal direction. All constructs have a 3F4 epitope tag (methionines at residue 108 and 111). Underlined residues denote B1. B. Comparison of ΔPrP expression levels. Representative immunoblot is shown for detection of (3F4)MoPrP and ΔPrPs in transiently transfected N2a cells using mAb 3F4. ΔPrPs are similar in expression level glycosylation appearance, except for Δ159–167 which has an extra fragment larger than the diglycoform (lane 7, arrow). C. ΔPrPs are not converted into PK-resistant PrP in prion-infected cells. 22L-ScN2a cells were transiently transfected with (3F4)MoPrP, Δ159 or Δ159–175, and cell lysates digested with PK or not. Immunoblot was done using mAb 3F4. D. Pilot study for dominant-negative inhibition (DNI) of Δ159 and Δ159–175. DNI was assessed by co-transfecting 22L-ScN2a cells with (3F4)MoPrP and Δ159 or Δ159–175, respectively, and testing for PK-resistant (3F4)MoPrP. Left panel shows representative immunoblot (mAb 3F4) and right panel the statistical analysis of quantified PK-res levels of a triplicate experiment. Empty pcDNA3.1 plasmid was used as control for co-transfection in lane 1. The error bars indicate standard deviation. *, p<0.05.
Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. (2013)
Figure 2

ΔPrPs are non-complex glycosylated and retained intracellularly.

A. N-linked glycans of ΔPrPs are not complex-type. Immunoblot comparing EndoH- and non-digested lysates of ΔPrP159 and Δ159–175 transfected cells. Left panel was probed with mAb 3F4 mAb, detecting transiently expressed ΔPrPs. On the right panel, the membrane was re-probed with mAb 4H11, detecting total PrP. Arrowheads depict deglycosylated ΔPrPs. B. The additional band of Δ159–167 represents a third N-glycan sensitive to EndoH. Upon EndoH digestion, all bands converge into a non-glycoform band (lane 3 and 4). In Δ159–167(169) the third glycosylation site is not present anymore. C. ΔPrPs are mainly distributed in intracellular compartments rather than on the cell surface. Confocal microscopy images of N2a cells transiently transfected with (3F4)MoPrP, Δ159 or Δ159–175, either permeabilized (right) or non-permeabilized (left) with 0.2% TX100 after fixation. Cells were immuno-labeled with mAb 3F4. (3F4)MoPrP-expressing cells show bright immunopositivity on the cell surface, whereas ΔPrPs are not observed on the cell surface. Also intracellular staining differs strongly between (3F4)MoPrP and ΔPrP expressing cells Scale bars, 25 µm.
Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. (2013)
Figure 3

ΔPrPs spontaneously form aggregates that are detergent-insoluble and relatively chymotrypsin-resistant.

A. ΔPrPs are insoluble in detergent solution. Immunoblots showing partition of Δ159 and Δ159–175 in supernatant (Sup) and pellet fraction after ultracentrifugation of lysates supplemented with 4% sarcosyl. Upper panel is mAb 3F4, lower panel is membrane re-probed with mAb 4H11 detecting also endogenous wild-type PrP besides ΔPrPs. The majority of ΔPrPs is found in the insoluble fraction in both infected and non–infected cells (upper panel, lanes 3, 4, 7 and 8). Wild-type PrP almost completely partitions into the soluble fraction (Sup) in non-infected N2a cells with present conditions. PrP in pellet fractions of non-infected N2a cells is from ΔPrPs as can be seen from the banding pattern (Lane 7 and 8, square bracket). PrP in lanes 3 and 4 (lower panel) is PrP27-30. B. ΔPrPs are moderately resistant to chymotrypsin digestion. Immunoblots showing chymotrypsin-resistant fragments of Δ159 and Δ159–175 from transiently transfected N2a cell lysates digested with indicated concentrations of chymotrypsin (Chy). Note that full-length diglycoform of ΔPrP still remains at 8 µg/ml Chy (lanes 5 and 10, upper panel, closed arrowhead), whereas that of endogenous wild-type PrP is completely digested (lanes 5 and 10, lower panel, open arrowhead) and only small amounts of truncated fragments remained (curly bracket). Other smaller fragments in lane 5 and 10 (square-brackets) represent ΔPrPs.
Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. (2013)
Figure 4

ΔPrPs have a GPI-anchor attached.

A. ΔPrPs are substantially soluble in detergent without GdnHCl denaturation. Immunoblots probed with mAbs 3F4 (left) or 4H11 (right, membrane re-probed) showing ΔPrPs and endogenous PrP in detergent and aqueous phases of TX114 lysates prepared from N2a cells transfected with Δ159 or Δ159–175. Det denotes detergent phase, Aq aqueous phase. B. ΔPrPs are highly hydrophobic after denaturation with GdnHCl. Representative immunoblot of TX114-treated lysates after denaturation with 3 M GdnHCl and developed with mAb 3F4 showing Δ159 and Δ159–175 almost exclusively in detergent phase (lanes 1–4). PIPLC treatment (+, even lanes) was done after denaturation with GdnHCl or not (−, uneven lanes). PIPLC treatment did not significantly influence ΔPrP partitioning under these conditions. C. Hydrophobicity of ΔPrPs combined with sensitivity to PIPLC digestion. After further diluting out GdnHCl by phase-separation and removal of the aqueous phase containing GdnHCl, PIPLC was able to digest ΔPrPs. Left two panels (upper mAb 3F4, lower 4H11) are shorter expositions, right panels longer expositions to show also difference in PrP levels in the detergent phase. Under these conditions a substantial reduction of ΔPrPs in the detergent phase was achieved by PIPLC digestion (e.g. upper-left panel, lanes 1, 3 vs. 2, 4). Migration of ΔPrPs in the aqueous phase of PIPLC-digested samples was slightly slower than that of PIPLC-undigested samples (−) (upper-right panel, lane 9 vs. 10).
Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. (2013)
Figure 5

ΔPrPs are degraded by both lysosomal and proteasomal degradation systems.

A. Degradation of ΔPrPs by lysosomal and proteasomal systems. (Left) N2a cells transiently transfected with Δ159 or Δ159–175 were treated for 7 hours with inhibitors either for lysosomal degradation (bafilomycin A1, Baf, 120 nM), autophagy (3-methyladenine, 3MA, 10 mM), or the proteasome (MG132, 10 µM) and cell lysates analyzed in immunoblot for expression of Δ159 and Δ159–175 (mAb 3F4). Square bracket and bracket denote diglycoforms and nonglycoforms of ΔPrPs, respectively. An increment in diglycoforms is observed in Baf- and 3MA-treated cells, whereas in MG132-treated cells also the non-glycoforms are increased. (Right) A graph showing quantification of all PrP bands by densitometric analysis. Data from 3 independent (one in duplicate) experiments for Δ159 and 2 independent (one in duplicate) for Δ159–175 were statistically analyzed for mean and standard deviation (error bars). B. ΔPrPs can be localized in LAMP1-immunopositive vesicles. Immunofluorescence analysis shows distribution of ΔPrP (mAb 3F4) and LAMP1 in cells with or without 6M GdnHCl antigen-retrieval treatment. N2a cells transfected with ΔPrP159 were fixed, permeabilized and incubated without (upper panel) or with 6M GdnHCl (lower panel). With GndHCl treatment ΔPrP is diffusely distributed (arrowheads) and co-localizes poorly with LAMP1. After GdnHCl treatment (lower panel), bright 3F4-immunopositive puncta (arrows) are observed which partly co-localize with LAMP1-positive structures (merge). Scale bars, 10 µm.
Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. (2013)
Figure 6

Inverse correlation of DNI and size of deletion and importance of positioning of deletion.

A. DNI efficiencies of ΔPrPs are inversely correlated to deletion size. Representative immunoblot showing PK-resistant PrPSc moiety of (3F4)MoPrP co-expressed with indicated ΔPrPs in 22L-ScN2a cells. Quantitative analysis is shown below as scheme. Relative PrPSc levels were calculated as proportion of PK-resistant (3F4)MoPrP co-transfected with each ΔPrP to that of co-transfected with empty vector control in the same experiment. Data from 4 (for Δ159–165, Δ159–167 and Δ159–167(169) only 3) independent experiments were statistically analyzed for mean and standard deviation (error bars). B. DNI of ΔPrP with a deletion in the C-terminal part of H1∼H2. Δ171–175 (see Fig. 7A) was transiently expressed in N2a cells (left panel) and DNI efficiency evaluated in 22L-ScN2a cells (right panel) (mAb 3F4). Despite the much shorter deletion size, Δ171–175 exerted only inefficient DNI, similar to Δ159–175 (right panel).
Critical significance of the region between Helix 1 and 2 for efficient dominant-negative inhibition by conversion-incompetent prion protein. (2013)
Figure 7

ΔPrPs lacking in addition the N-terminal region still exert efficient DNI.

A. Schematic illustration of Δ171–175, Δ31–160 and its variants. Δ31–160 variants lack the entire part between the polybasic motif (PB, italic and bold) and residue 161. SS, signal sequence. B. Δ31–160 variants are expressed at comparable levels and have EndoH-sensitive and –resistant N-glycans. Immunoblot comparing EndoH-digested and non-digested samples from N2a cells transiently transfected with Δ31–160 variants, probed with anti-PrP mAb C16-S raised against the C-terminal portion of H3. Non-Tf, samples prepared from N2a cells without transfection. Arrowhead denotes deglycosylated Δ159 (lane 11), bracket deglycosylated fragments, square bracket EndoH-resistant fragments. Asterisks indicate non-specific bands. Δ31–160 variants are not completely deglycosylated by EndoH, resulting in EndoH-resistant fragments (square bracket) which are observed also in reducing conditions (right panel; +/− dithiothreitol (DTT) treatment). Note that endogenous wild-type PrPc was not detected under used conditions. C. DNI of Δ31–160 variants is similarly inversely related to size of deletion and dependent on intact C-terminal H1–H2 portion. Immunoblot probed with mAb 3F4 showing PK-resistant (3F4)MoPrP co-transfected with indicated constructs into 22L-ScN2a cells (left panel). Δ31–160 was slightly more effective than Δ159. Right panel shows quantification of results as obtained from a triplicate experiment, using densitometry on ImageJ. Bars illustrate mean ± standard deviation. *, p<0.05. D. Δ31–160 forms spontaneous aggregates with moderate chymotrypsin (Chy) resistance. Immunoblots probed with mAbs C16-S (upper panel) or 3F4 (lower panel) for comparing Chy-resistant fragments of Δ31–160 with that of Δ159. Lysates from N2a cells expressing Δ159 or Δ31–160 were digested with indicated concentrations of Chy for 30 minutes and then digested with PNGaseF. A sample from non-transfected N2a cells is shown in the lane 5. Asterisks indicate non-specific C16-S bands. Arrowhead denotes deglycoform of Δ159, bracket deglycoform of Δ31–160.
MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms (2018)
Figure 2

Figure 2. Binding of the NAD metabolite ADP-ribose is specific to macroH2A1.1 and conserved.

A Isothermal calorimetry (ITC) assays using ADP-ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2.

B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.
MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms (2018)
Figure 3

Figure 3. MacroH2A proteins repress chromatin expansion at DNA damage sites; macroH2A1.1 being the most potent in the reduction.

A The diameter (d) of the fluorescence signaling of H2B tagged with photo-activatable GFP (PAGFP-H2B) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser.

B Chromatin expansion in absence and presence of intercalating DNA dye (- and + DNA damage) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage from 3 biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values (*, p < 0.05 using unpaired, two-tailed Student's T-test).

C Quantified chromatin expansion in cells not transfected (control) and cells transfected with each mCherry-tagged histone (as indicated). Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 compared to mCherry-H2A-expressing cells using unpaired, two-tailed Student's T-test).

D Chromatin expansion in Hela cells containing a doxocyclin (doxo.)-inducible macroH2A1.1 transgene. Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti-macroH2A1.1-immunoblotting. Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 using unpaired, two-tailed Student's T-test).
MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms (2018)
Figure 5

Figure 5. The linker domain of macroH2A2 is able to maintain heterochromatic structures.

A Western blot showing the expression of macroH2A proteins in control HepG2 cells, knock-down for macroH2A1 and macroH2A2 (DKD) and DKD with ectopic expression of the indicated YFP-tagged constructs. H3 is used as a loading control.

B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed-in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z-projections of confocal stacks.

C-D Quantification of the nuclear area and number of H3K9me3 foci per nucleus, respectively (n>50 cells, 2 biol. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).
The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours. (2015)
Figure 1

Characterisation of lung tissue expressing <sup>V600</sup><sup>E</sup>BRAF

(Top) PCR detection of the recombined BrafLox−V600E allele (Lox-V600E) in BVE mouse tissues at 8 weeks of age without tamoxifen induction. Substantial recombination is observed in the lung, while weaker recombination is also detected in the liver. No recombination was detected in hematopoietic tissues (bone marrow and spleen) even after 40 cycles of amplification (right). (Bottom) H&E staining of lung sections from wild-type (WT, 10 weeks of age), BVE (1–10 weeks of age) and Braf+/LSL−V600E mouse 8 weeks after nasal delivery of AdCre is indicated below. V600EBRAF expression induced by two different methods causes similar pathology showing papillary adenomas accompanied by stroma development. Scale bars, 100 μm.

Ki67 immunostaining of lung sections from BVE mice at 3–10 weeks of age. Scale bars, 25 μm. The right bar graph summarises %Ki67+ cells in tumours at different ages post-partum. Four to six mice were analysed for each age group as indicated, and more than 3,500 tumour cells per mouse were evaluated. The data represent mean ± SD.

Representative H&E staining and Mac2 immunohistochemistry of serial lung sections from 10-week-old BVE mice at low (upper panels) and high (lower panels) magnifications. Scale bars, 100 μm (upper panels) or 25 μm (lower panels).

Source data are available online for this figure.