EMBO/soda-nel huggingface dataset:
The androgen receptor depends on ligand-binding domain dimerization for transcriptional activation. (2021)
Figure 7

Figure 7. Comparison of WT AR and AR W752R interactomes.

(A) Double hybrid assay in COS cells transfected with WT LBD, W752R LBD or empty vector (negative control) in the presence of the LxxLL fragment of SRC1. Average is shown and shaded areas represent SEM (biological replicates, n = 4, Tukey's multiple comparisons test, **P < 0.01, ***P < 0.001).

(B) Confocal fluorescence microscopy images of AR-WT-BirA* and AR W752R-BirA*expressing HEK293 cells treated in the presence of 50 µM biotin with 100 nM DHT or vehicle (ethanol) and with or without 0.03 µg/ml TET as indicated. AR-BirA*s were detected with anti-AR (red) and biotinylated proteins with fluorescently labeled streptavidin (green). Nuclei were visualized using DAPI. Scale bars = 20 µm.

(C) Heatmap showing the MS spectral counts of high confidence interactors (FDR < 0.05 after SAINT analysis) identified with WT AR-BirA* and AR W752R-BirA* upon DHT or vehicle exposure. Values for three biological replicates from DHT-and vehicle-exposed samples are shown. GRPEL1 and DBT were the only high confidence interactors of AR W752R-BirA*. On the right, FDRs and spectral count averages are shown for the DHT-treated samples. Spectral counts have been normalized to those of AR in each sample.

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Brain Cross-Protection against SARS-CoV-2 Variants by a Lentiviral Vaccine in New Transgenic Mice (2021)
Figure 1

Figure 1. Large permissiveness of the lungs and brain of B6.K18-hACE2IP-THV transgenic mice to SARS-CoV-2 replication.

(A) Representative genotyping results from 15 N1 B6.K18-hACE2IP-THV mice as performed by qPCR to determine their hACE2 gene copy number per genome. Dots represent individual mice.

(B) Phenotyping of the same mice, presented in the same order, inoculated i.n. with 0.3 × 105 TCID50 of SARS-CoV-2 at the age of 5-7 wks and viral RNA content was determination in the indicated organs at 3 dpi by conventional E-specific qRT-PCR. Red lines indicate the qRT-PCR limits of detection.

(C, D) Quantification (C) and images (D) of hACE2 protein expression level by Western blot from the lungs of the same mice as in panels A and B, presented in the same order.

(E, F) Kinetics of SARS-CoV-2 replication in the lungs and brain of B6.K18-hACE2IP-THV mice as followed by measuring viral RNA contents by conventional E-specific qRT-PCR (E) or by PFU counting (F).

(G) Percentage of initial weight measured from the mice of panels E and F.
Brain Cross-Protection against SARS-CoV-2 Variants by a Lentiviral Vaccine in New Transgenic Mice (2021)
Figure 3

Figure 3. Comparison of SARS-CoV-2 replication and infection-derived inflammation in B6.K18-hACE2IP-THV and B6.K18-hACE22prlmn/Jax transgenic mice.

(A) Comparative permissiveness of various organs from B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax transgenic mice (n = 5-6/group) to SARS-CoV-2 replication, as determined at 3 dpi by conventional E-specific or sub-genomic Esg-specific qRT-PCR. Red lines indicate the qRT-PCR limits of detection.

(B) Comparative quantitation of hACE-2 mRNA in the lungs and brain of B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax transgenic mice (n = 5-6/group).

(C) Heatmaps represent log2 fold change in cytokine and chemokine mRNA expression in the lungs or brain of B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax mice at 3 dpi (n = 5-6/group). Data were normalized versus untreated controls.

Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01, ns = not significant).
Brain Cross-Protection against SARS-CoV-2 Variants by a Lentiviral Vaccine in New Transgenic Mice (2021)
Figure 4

Figure 4. Vaccination with LV::S protects both lungs and central nervous system from SARS-CoV-2 infection in B6.K18-hACE2IP-THV transgenic mice.

(A) Timeline of prime-boost LV::S vaccination and SARS-CoV-2 challenge in B6.K18-hACE2IP-THV mice (n = 6/group). The LVs used in this experiment were integrative.

(B) Serum neutralization capacity of anti-SCoV-2 Abs in LV::S-vaccinated mice compared to sham mice (n = 6/group).

(C) Viral RNA content as determined in various organs at 3 dpi (n = 6/group) by use of conventional E-specific or sub-genomic Esg-specific qRT-PCR. Red lines indicate the qRT-PCR detection limits.

(D) Percentages of NK cells or neutrophils in the lungs of LV::S- or sham-vaccinated and SARS-CoV-2-challenged B6.K18-hACE2IP-THV transgenic mice at 3 dpi (n = 6/group). Percentages were calculated versus total lung live CD45+ cells.

(E) Relative log2 fold change in cytokine and chemokine mRNA expression in the brain of LV::S- or sham-immunized and SARS-CoV-2-challenged B6.K18-hACE2IP-THV transgenic mice at 3 dpi (n = 6/group). Means ± SD are shown. Data were normalized versus untreated controls.

Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).
Brain Cross-Protection against SARS-CoV-2 Variants by a Lentiviral Vaccine in New Transgenic Mice (2021)
Figure 5

Figure 5. Cellular and humoral immunity in LV::S-vaccinated B6.K18-hACE2IP-THV mice. B6.K18-hACE2IP-THV mice were primed (i.m.) at wk 0 and boosted (i.n.) at wk 5 (n = 5) with non-integrative LV::S. Control mice were injected with an empty LV (sham).

(A) Representative IFN-γ response by lung CD8+ T cells of as studied at wk 7 after in vitro stimulation with the indicated SCoV-2-derived peptides.

(B, C) Cytometric strategy to detect lung CD8+ T central memory (Tcm, CD44+CD62L+CD69-), T effector memory (Tem, CD44+CD62L-CD69-) and T resident memory (Trm, CD44+CD62L-CD69+CD103+) and (C) percentages of these subsets among CD8+ T-cells in LV::S-vaccinated (n = 9) or sham (n = 5) mice.

(D) Cytometric strategy to detect SCoV-2-specific CD8+ T cells by use of the H-2Db-SCoV-2:538-546 dextramer in the lungs of LV::S or sham-vaccinated mice. Inside CD8+ dextramer+ T-cell subset, Tcm, Tem and Trm have been distinguished.

(E) Percentages of dextramer+ cells were calculated versus CD8+ T cells in both mouse groups and those of Tcm, Tem and Trm were calculated versus dextramer+ CD44+ cells in LV::S-vaccinated mice (n = 4/group). N/A= not applicable.

(F, G) Anti-SCoV-2 IgG or IgA titers (F) and neutralizing activity (EC50) (G) in the sera or lung homogenates at 3 dpi. Samples from individual mice (n = 4/group) were studied.

Data information: Statistical significance of the difference between the two groups was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01).
Brain Cross-Protection against SARS-CoV-2 Variants by a Lentiviral Vaccine in New Transgenic Mice (2021)
Figure 7

Figure 7. Features of olfactive bulbs or brains in the protected LV::S- or unprotected sham-vaccinated B6.K18-hACE2IP-THV mice. Mice are those detailed in the Figure 6.

(A) Example of CD3-positive cells in an olfactory bulb from an LV::S i.m.-i.n. vaccinated and protected mice and representative results from this group versus sham-vaccinated and unprotected mice at 3 dpi (n = 7-9/group). Scale bar: 50 μm. Statistical significance was evaluated by Mann-Whitney test (**= p <0.01).

(B-D) Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups. (C, D) Innate immune cells in the olfactory bulbs (C) or brain (D). The CD11b+ Ly6C+ Ly6G+ population in the olfactory bulbs are neutrophils and the CD11b+ Ly6C+ Ly6G- cells of the brain are inflammatory monocytes.

(E) Brain H&E histology at 3 dpi. The top right and both bottom panels show examples, in two different mice, of leukocyte clusters (arrows) alongside the ventricular wall. No such clusters were detected in the LV::S i.m.-i.n. vaccinated mice (top left panel). Scale bar: 200 μm. The close up view (bottom right panel) highlights the thickened, disorganized ependymal lining, compared to the normal ependymal cells and cilia of an LV::S i.m.-i.n. vaccinated mouse (top left panel). Scale bar: 50 μm.
Brain Cross-Protection against SARS-CoV-2 Variants by a Lentiviral Vaccine in New Transgenic Mice (2021)
Figure 8

Figure 8. Full protective capacity of LV::S against the SARS-CoV-2 Gamma variant.

(A) Timeline of LV::S i.m.-i.n. immunization and challenge with SARS-CoV-2 Gamma in B6.K18-hACE2IP-THV mice (n = 5/group). The LVs used in this experiment were non-integrative. Olfactory bulbs, brains and lungs were collected at 3 dpi.

(B) Brain or lung viral RNA contents, determined by conventional E-specific or sub-genomic Esg-specific qRT-PCR at 3 dpi. Two mice out of the 5 sham-vaccinated mice did not have detectable viral RNA in the lungs despite high viral RNA content in the brain and hACE2 mRNA expression levels comparable to that of the other mice in the same group.

(C) Neutralizing activity (EC50) of sera from individual LV::S-vaccinated mice against pseudo-viruses harboring SCoV-2 from the ancestral strain or D614G, Alpha, Beta or Gamma variants. Red asterisk indicates significance versus ancestral, blue asterisk indicates significance versus D614G variant, while orange asterisk indicates significance versus Alpha variant. Statistical comparisons were made at the respective boosting timepoint. In homologous settings, sera from mice immunized with LV::SBeta or LV::SGamma, fully inhibited pseudoviruses bearing S from Beta or Gamma, validating the assay for all pseudo-viruses used.

(D) Cytometric analysis of CD8+ T cells in pooled olfactory bulbs of LV::S i.m.-i.n. vaccinated and protected mice versus sham-vaccinated and unprotected mice.

(E) Wild type or µMT KO mice (n = 5-9/group) were injected by LV::S or sham following the time line shown in (A), then pretreated with Ad5::hACE2 4 days before challenge with the ancestral SARS-CoV-2 strain. Lung viral RNA contents were determined at 3 dpi.

(F) T-splenocyte responses in LV::S-primed and -boosted C57BL/6 WT mice or sham controls (n = 3-5/group), evaluated by IFN-γ ELISPOT using 15-mer peptides encompassing SCoV-2 MHC-I-restricted epitopes.

Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001).

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The RNA Polymerase II subunit RPB-9 recruits the Integrator complex to terminate C. elegans piRNA transcription (2020)
Figure 1

Figure 1. rpb-9 is required for piRNA pathway integrity.

A) Schematic of the EMS mutagenesis screen carried out using the piRNA sensor and identification of a novel allele (mj261) of the rpb-9 gene.

B) rpb-9 locus, in scale (top) and comparisons of RPB-9 protein domains to homologs in other species (bottom). The introduced mutation is highlighted in red.

C) Expression patterns of rpb-9 in adult animals by RNA-sequencing.

D-G) Quantification of piRNA sensor expression. Representative DIC and fluorescence microscopy images of piRNA sensor expression in wild type (mjIs144) (D) and rpb‑9 (mj261) animals (two images with varying GFP expression are shown, scale bar = 20 µm) (E), validation of transgene expression by RT-qPCR (n=3, error bars represent SD) (F) and representative DIC and fluorescence microscopy images of piRNA sensor expression in the rpb-9 rescue (mj261; mjSi70) animals (two images with varying GFP expression are shown). Scale bar = 20 µm (G).
The RNA Polymerase II subunit RPB-9 recruits the Integrator complex to terminate C. elegans piRNA transcription (2020)
Figure 2

Figure 2. rpb-9 is required for the initiation step of the piRNA pathway.

A) Schematic of the germline-specific pie-1::gfp::h2b::pie-1 sensor (top) and schematic of the experiment (bottom).

B) Representative fluorescence images of transgene expression in wild-type, rpb-9 (mj261), hrde-1 (tm1200) and prg1 (n4357); prg-2 (n4358) animals in the parental (P0) and inheriting (G1-G6) generations. Scale bar = 50 µm.

C) Quantification of the percentage of desilenced individuals in wild-type, rpb-9 (mj261), prg-1 (n4357); prg-2 (n4358) and hrde-1 (tm1200) populations across generations.
The RNA Polymerase II subunit RPB-9 recruits the Integrator complex to terminate C. elegans piRNA transcription (2020)
Figure 3

Figure 3. rpb-9 represses two DNA transposon families and a subset of somatic genes. A) Differential expression analysis of transposable elements in rpb-9 (mj261) mutants versus wild type (polyA-selected RNA-seq libraries). [p<=0.01, log2(fold change)>=1, DESeq2 analysis]

B) Table showing the numbers of matching piRNAs (with 0, 1, 2 and 3 mismatches respectively) for each of the upregulated transposon families.

C) Genome-wide differential gene expression analysis of rpb-9 (mj261) mutants versus wild-type (polyA-selected RNA-seq libraries). [p<=0.01, log2(fold change)>1 or <-1, DESeq2 analysis].

D) Differential expression analysis of germline-specific and soma-specific genes, as classified in (Reinke et al, 2004). "All genes" is shown for comparison. (PolyA-selected RNA-seq libraries, n=3).

E-G) Pairwise correlation plots of rpb-9 (mj261) and prg-1 (n4357) (E), hrde-1 (tm1200) (F) or gonad-dissected prg-1 (n4357) (Reed et al, 2020) (G) transcriptomes. Differentially expressed genes in rpb-9 (mj261) are shown in black, shared upregulated genes in red, shared downregulated genes in blue. The piRNA sensor transcript is highlighted in green. (Total RNA "Ribo-Zero" RNA-seq libraries).
The RNA Polymerase II subunit RPB-9 recruits the Integrator complex to terminate C. elegans piRNA transcription (2020)
Figure 4

Figure 4. rpb-9 affects RNA pol II binding to a subset of genes.

A-C) Analysis of RNA Pol II binding (RPB-1/AMA-1 ChIP-seq) at upregulated genes in rpb-9 (mj261) mutants, as defined in Figure 3C. Class I: upregulated genes with increased RNA Pol II binding (A); Class II: upregulated genes with invariant RNA Pol II binding (B); Class III: upregulated genes with reduced RNA Pol II binding (C). n=3 biological replicates are shown per each genotype. (PolyA-selected RNA-seq libraries). Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers.

D) RNA Pol II binding profile over the piRNA sensor locus.

E) RNA Pol II binding profile over the Chapaev-2 and CEMUDR1 DNA transposons, scaled to 1kb.
The RNA Polymerase II subunit RPB-9 recruits the Integrator complex to terminate C. elegans piRNA transcription (2020)
Figure 5

Figure 5. rpb-9 is required for the robust production of piRNA-pathway dependent HRDE-1-associated secondary 22G siRNAs.

A) Enrichment analysis of RPB-9-bound proteins in rpb‑9::ollas (mj604) versus wild type by IP/MS. [p<=0.05, log2(fold change)>1 or <-1, Welch t-test]. n=4 technical replicates (pooled).

B) Transgene transcript length is not affected in rpb-9 (mj261) mutants. Scheme of the gfp unspliced transcript showing the positions of RT-qPCR primers (top) and pairwise ratios (3′ to 5′) of unspliced gfp amounts along the transcript as indicated by the primer pairs (bottom). (n=3, error bars represent SD). ns= not significant (two-tailed t-test).

C) Transcriptome binning according to increasing 22G siRNA density in wild-type animals (grey, n=3). Mean normalized rpb-9 (mj261) mRNA reads (red, n=3) are overlaid with mean normalized wild-type mRNA reads (grey). (PolyA-selected RNA-seq libraries). The piRNA sensor transcript is indicated with red (rpb-9 (mj261)) and grey (wild type) dots. Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers.

D) Distribution of piRNA targets (as defined in (Bagijn et al, 2012) and of HDRE-1- and CSR-1-dependent 22G siRNAs across bins as defined in (C)).

E) TE binning according to increasing 22G siRNA density in wild-type animals (grey, n=3). Mean normalized rpb-9 (mj261) mRNA reads (red, n=3) are overlaid with mean normalized wild-type mRNA reads (grey). (PolyA-selected RNA-seq libraries). The CEMUDR1 and Chapaev-2 transcripts are indicated with red (rpb-9 (mj261)) and grey (wild type) box plots. Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers.

F) Distribution of Class I, Class II and Class II genes (as defined in Figure 4A-C) across bins as defined in (C)).

G) Distribution of normalized 22G siRNA reads mapping over the piRNA sensor.

H) Distribution of normalized reads mapping over the CEMUDR1 and Chapaev-2 DNA transposons.
The RNA Polymerase II subunit RPB-9 recruits the Integrator complex to terminate C. elegans piRNA transcription (2020)
Figure 6

Figure 6. rpb-9 is required for the generation of mature piRNAs.

A) Expression levels of mature piRNAs in wild-type, rpb-9 (mj261), rpb-9 rescue (mj261; mjSi70) and prg-1 (n4357) animals (n=3, hyper-geometric test). Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers.

B) Mature piRNA expression along chromosome IV coordinates (motif-dependent piRNA clusters I and II) in wild-type and rpb-9 (mj261) animals.

C) Northern blot quantification of mature piRNA 21UR-1 levels in wild-type, prg-1 (n4357), mut-7 (mj253) and rpb-9 (mj261) animals.

D) Quantification of the levels of the mature piRNAs 21UR-4997 and 21UR-8099 by small RNA sequencing (raw reads normalized to library size, n=3). Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers.
The RNA Polymerase II subunit RPB-9 recruits the Integrator complex to terminate C. elegans piRNA transcription (2020)
Figure 7

Figure 7. rpb-9 is required for transcription termination at piRNA loci.

A) Median precursor length shift of nascent (left) and nucleoplasmic (right) piRNA precursors in rpb-9 (mj261) mutants and rescue (mj261; mjSi70) animals compared to wild-type (n=2). Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers.

B) Distribution of nascent piRNA precursor lengths in rpb-9 (mj261) and rescue (mj261; mjSi70) animals compared to wild type.

C) Peak positions of nascent piRNA precursor length distributions in 2000 subsamples of 5000 precursor sequences sampled without replacement. Central bands represent the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers.

D) Distributions of nucleoplasmic piRNA precursor lengths in rpb-9 (mj261) and rescue (mj261; mjSi70) animals compared to wild type.

E) Distribution of 5′ ends of short capped RNA reads mapping to motif-dependent piRNA loci. Total counts of read 5′ ends mapping within a 50bp window of motif-dependent piRNA loci, aggregated by position relative to 21U-RNA 5′ U sites. A comparable enrichment of reads initiating 2 nt upstream of 21U-RNA 5′ U sites is observed across replicates and genotypes.

F) rpb-9 recruits Integrator at piRNA loci. Representative single-plan immunofluorescence images of Integrator (ints-6::GFP) and PRDE-1 (prde-1::mCherry) localization in wild-type and rpb-9 (mj261) animals. Scale bar = 5 µm.

G) Co-localization analysis of PRDE-1::mCHERRY and INTS-6::GFP foci in wild-type and rpb-9 (mj261) animals. Central bands represent the median, notches represent the confidence interval around the median, boxes represent the 25th and 75th percentiles and whiskers represent the lowest and highest values, excluding outliers. n (nuclei/genotype) > 16. (Welch's t-test, p = 1.7e-17).

H) Positions of unique 5′ monophosphate small RNA reads mapping at piRNA promoters after removal of reads >15 nt initiating at annotated piRNA 5′ U sites. The average signal is normalized to sequences per million of mapped reads.
The BCL-2 family member BID plays a role during embryonic development in addition to its BH3-only protein function by acting in parallel to BAX, BAK and BOK (2022)
Figure 3

Figure 3. Bok-/-Bak-/-Bax-/-Bid-/- quadruple knockout (QKO) foetuses displayed brain and craniofacial anomalies at E18.5

A-C) Heads of E18.5 foetuses were serially sectioned in coronal orientation and stained with H&E. Representative images of rostro-occipital level-matched sections from age-matched wild-type (n = 2), Bok-/-Bak-/-Bax-/- triple knockout (TKO; n = 4) and QKO foetuses (n = 4) depicting the forebrain, nasal cavity, and tongue are shown. BP, bony palate; Cx, cerebral cortex; ES, epidural space; In, incisor; LV; lateral ventricle; Ma, mandible; MC, mouth cavity; NC, nasal cavity; NS, nasal septum; St, striatum; SVZ, subventricular zone; To, tongue; WP, whisker follicles. Scale bar = 850 µm (A), 1200 µm (B, C).

D) Morphometric assessment of indicated tissue areas. Data of individual animals are displayed as circles with means and standard error of the mean. Data were analysed by unpaired, two-tailed Student's t-test. Related data normalised to mean bodyweight are displayed in Figure EV1.
The BCL-2 family member BID plays a role during embryonic development in addition to its BH3-only protein function by acting in parallel to BAX, BAK and BOK (2022)
Figure 4

Figure 4. Anomalies in the eyes of Bok-/-Bak-/-Bax-/-Bid-/- quadruple knockout (QKO) foetuses at E18.5 of development

A-C) Representative H&E-stained coronal serial sections of the eye from E18.5 wild-type (n = 2), Bok-/-Bak-/-Bax-/- triple knockout (n = 3), and Bok-/-Bak-/-Bax-/-Bid-/- quadruple knockout (n = 3) foetuses.

D) Densitometry of hyaloid cavity tissue section as an assessment of blood vessel endothelial cells present. Data of individual animals are displayed as circles with means and standard error of the mean and were analysed by unpaired, two-tailed Student's t-test.

HC, hyaloid cavity (future vitreous body); HV, hyaloid vessels; Le, lens; NC nasal cavity; NR, neural retinal; PR, pigmented retina; UM, upper molar.

Scale bar = 1470 µm.
The BCL-2 family member BID plays a role during embryonic development in addition to its BH3-only protein function by acting in parallel to BAX, BAK and BOK (2022)
Figure 9

Figure 9. BID can trigger partial cytochrome c release from isolated BAX/BAK/BOK-deficient mitochondria when applied at high levels.

(A) Estimation of BID protein levels in TKO MEFs. DKO MEFs re-expressing human BAK, TKO MEFs, and two human leukaemia cell lines RS4;11 and MV4;11, were lysed and analysed by Western blot for BID levels, using a rat monoclonal antibody raised against human BID (anti-hBID clone 2D1) that also recognises mouse BID. Also present are samples of the human FL-BID protein added at the equivalent concentrations used in (B) and (C). On the right gel, duplicate samples of DKO and TKO cells and permeabilised DKO and TKO cells (from B and C) showing that endogenous mouse BID is cytosolic in these cells. Data shown are representative of 2 independent experiments.

(B, C) Partial cytochrome c release by FL-BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID. DKO MEFs re-expressing human BAK or TKO MEFs were permeabilised, and then the cells incubated with the indicated concentrations of Bim BH3 peptide, cBID or FL-BID, alone or in the indicated combinations, at 30oC for 30 min. Cells were then co-stained for cytochrome c and TOM20 and analysed by flow cytometry. Left hand panels show median fluorescence data from replicate experiments (squares denote experiment 1, circles denote experiment 2). Right hand panels show histograms of cytochrome c staining and TOM20 staining from experiment 2 (treatments are as listed in the left-hand panels).

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