EMBO/soda-nel huggingface dataset:
Activation of endogenous retroviruses during brain development causes an inflammatory response (2021)
Figure 2

Figure 2. CRISPR-Cas9 deletion of Trim28 in adult neurons in vivo.

(a) A schematic of the workflow targeting Trim28 in the mouse forebrain using AAV vectors expressing the gRNA and a nuclear RFP reporter. 8 weeks later the injected animals were analysed either by immunohistochemical analysis or nuclei isolation by FACS prior to DNA/RNA sequencing.

(b) Estimation of gene editing at the Trim28 loci using NGS-sequencing of amplicons from DNA isolated from 50.000 RFP+ nuclei per animal. One animal per group was analyzed. Black bars indicate % of the detected indels that disrupted the frameshift.

(c-d) Gene editing of the Trim28-loci resulted in a robust loss of Trim28 protein, as evaluated by IHC where the expression of Trim28 in RFP+ cells were quantified and are displayed as mean ± SEM. Approximately 600 RFP+ cells per animal and group was evaluated. Scale bar 30 μm.

(e) RNA-seq analysis of the expression of TE-families using TEtranscripts.

(f) RNA-seq analysis of the Trim28-KO and control samples, visualising full length MMERVK10C elements (left panel) and ChIP-seq analysis of H3K9me3 in adult forebrain neurons (right panel). The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram.
Activation of endogenous retroviruses during brain development causes an inflammatory response (2021)
Figure 3

Figure 3. Deletion of Trim28 during brain development results in aberrant TE expression in the adult brain.

(a) A schematic of the breeding scheme resulting in highly efficient conditional deletion of Trim28 during cortical development and analysing the adult tissue 3 months later by IHC and RNA-seq.

(b) IHC for Trim28 in the adult cortex revealed that the protein was lost in cells exposed to Cre-activity during brain development (GFP+ cells). Scale bars: low magnification 75 μm, high magnification 20μm.

(c) RNA-seq analysis of the expression of TE-families using TEtranscripts

(d) Significantly upregulated TE families upon the Trim28-KO, in which the families with the highest fold change are listed.

(e) RNA-seq analysis of full length MMERVK10Cs in the adult tissue. The location of the full length MMERVK10Cs are indicated as a thick black line under each histogram.
Activation of endogenous retroviruses during brain development causes an inflammatory response (2021)
Figure 4

Figure 4. Single nuclei RNA-seq of cortical tissue from Emx1-Cre/Trim28-KO animals and their littermates.

(a) A schematic of the workflow for the single nuclei RNA-seq of cortical tissue from Emx1-Cre/Trim28-KO animals and their littermates.

(b) UMAP showing the unbiased clustering analysis with seven different cell clusters.

(c-d) UMAP and pie-charts showing the distribution of Trim28-KO and control cells over the seven different clusters. There were no major differences in proportions of the different cell clusters between Emx1-Cre/Trim28-KO animals and controls.

(e-h) A selection of significant cell type specific changes in gene expression between Emx1-Cre/Trim28-KO animals and controls as revealed by single-nuclei RNA-seq. The black dots represent the mean value value (Wilcoxon rank sum test, (p-adj value < 0.01), n=2. For a full list, see Table EV2.
Activation of endogenous retroviruses during brain development causes an inflammatory response (2021)
Figure 5

Figure 5. Cell type specific analysis of ERV activation in Emx1-Cre/Trim28-KO animals.

(a) The workflow used to analyse ERV expression in single nuclei RNA-seq data.

(b) Mean plots show the changes of TE subfamily expression in each cell cluster upon the Emx1-Cre/Trim28-KO. A differential expression analysis performed with DESeq2 (described in material and methods) showed upregulated TEs in cell types in which Trim28 was deleted (excitatory neurons, astrocytes, oligodendrocytes and oligodendrocyte precursors), (indicated with red dots: p-adj < 0.01, log2FC > 3). The specific upregulated elements and their fold changes are listed in bar graphs under each mean plot. In cell types in which Trim28 was not deleted, TE expression remained unaffected (inhibitory neurons and microglia).
Activation of endogenous retroviruses during brain development causes an inflammatory response (2021)
Figure 6

Figure 6. Activation of ERVs during brain development results in the presence of ERV proteins in adult brain tissue and signs of an inflammatory response.

(a) IHC analysis for the microglia marker Iba1 in Emx1-Cre/Trim28-KO animals and controls. Scale bars: Low magnification 75 μm, high magnification 10 μm.

(b-c) The morphology of microglia in cortex and striatum (control region) of Emx1-Cre/Trim28-KO animals and controls were quantified by high content screening of Iba1 immunoreactivity, revealing differences in process length, area and number of branch points specifically in cortex, error bars mean ± SEM (unpaired t-test. p-values: 1.8x10-4, 9.6x10-4 and 5.2x10-6, respectively). A large number of photos from three control and two KO animals were analysed, see material and methods for details.

(d) Western blot revealed an increased expression of CD68 in the Emx1-Cre/Trim28-KO animals (n=5 per group), unpaired t-test, **p=0.0036, error bars mean ± SEM. The star indicates an unspecific band.

(e) An increased expression of the ERV derived protein IAP-Gag was detected in the cortex of Emx1-Cre/Trim28-KO animals (n=5 per group), unpaired t-test, ***p=0.0008, error bars mean ± SEM.

(f) Immunohistochemistry for IAP-gag visualized the presence of ERV proteins in the cortex of Emx1-Cre/Trim28-KO animals. The distribution of IAP-gag was heterogenous among Trim28-KO neurons, where it was either accumulated in a low (#) or high (°) number of aggregate-like structures in the cytoplasm, or as weak homogenous staining throughout the cytoplasm (#) or not present at all (*). Scale bars: low magnification 75μm, high magnification 5 μm.

Figure legends Extended view
Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes (2021)
Figure 1

Figure 1. Rb∆K4 knock-in mice develop normally but exhibit telomere attrition

A. Schematic structure of pRb, location of the 14 CDK sites and the four Ser/Thr-to-Ala substitutions (denoted by *) in Rb∆K4 knock-in mice in exon 23. Clustering of phosphorylation sites is as per Sanidas et al. (Sanidas et al., 2019), based on protein interaction profiles of monophosphorylated pRb species. N, N-terminus; A-B, pocket domain; L, spacer/linker; C, C-terminus which includes exon 23.

B. Western blot analysis of Rb∆K4 thymocytes with an antibody that recognizes both hyper-phosphorylated (ppRb) and hypo-phosphorylated pRb species.

C. Representative flow cytometric profiles of thymocytes from 6-week old Rb∆K4 and control mice for CD3-, CD4- and CD8-positive cell populations. Bottom, average of CD3-, CD4- and CD8 single- and double-positive cells in Rb∆K4 vs. control thymocytes showing no significant (n.s.) difference by student's t-test. Bars represent mean ±SD (standard deviation); n=6 biological replicates).

D. Short telomeres in Rb∆K4 mice. Images of splenocyte metaphase spreads hybridized with the telomere repeat probe (pink). Note shorter telomeres in Rb∆K4 splenocytes (arrows). Bottom, quantitative analysis of 10 splenocyte metaphases from one pair of Rb∆K4 and control littermate; all 5 pairs are shown in Appendix Fig. S2 (P<0.001 by two-tailed Mann-Whitney-Wilcoxon test).
Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes (2021)
Figure 2

Figure 2. Rb∆K7 knock-in mice exhibit near-normal embryogenesis and neonatal development

A. Schematic structure of pRb, location of 14 CDK sites and the seven Ser/Thr-to-Ala substitutions (denoted by *) in Rb∆K7 mice. Phosphorylation of upstream sites is indicated: arrow, phosphorylated; blocking line, not phosphorylated in Rb∆K7 thymocytes.

B. BrdU immunohistochemistry analysis of sagittal sections of E13.5 Rb∆K7 and control littermate embryos. Arrows point to lateral ventricle and liver showing similar intensity of staining/cell proliferation.

C. Left, representative flow cytometric analysis for CD3, CD4 and CD8, showing near normal T cell development in 10-day-old Rb∆K7 mice. Right, average of CD3, CD4 and CD8 single- and double-positive cell populations in Rb∆K7 vs control thymocytes with no significant difference (n.s.) by student's t-test. Results are presented as mean ±SD, n=4 biological replicates).

D. Left, images showing apparent normal division of retinal progenitors in Rb∆K7 mice. Mice were labeled with BrdU for 2 hrs; retinal sections from indicated genotypes were stained with DAPI (blue), Ki67 (green), PH3 (red) or BrdU (red) to mark nuclei, all dividing cells, mitotic or S-phase cells, respectively. Right, quantification of staining showing no significant difference among the genotypes. Bars denote mean ±SD; n=3 biological replicates). Scale bar: 20 μm.

E. Left, top, western blot analysis of Rb∆K7 thymocytes with an antibody that recognizes both hyper-phosphorylated (ppRb) and hypo-phosphorylated pRb species. Right, phospho-specific immunoblots showing absence of phosphorylation at upstream Thr350 and Ser601 or the substituted Ser773Ala site in Rb∆K7 thymocytes. Right, immuno-precipitation (IP)-Western blot analysis demonstrating phosphorylation of pRb∆K7 on Ser243/Thr246 (top) and Ser605 (bottom). n.s., non-specific band.

F. Phospho-specific immunoblots showing absence of phosphorylation at upstream Thr350, and reduced but not complete loss of phosphorylation at Ser601 (arrow) in Rb∆K4 thymocytes.
Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes (2021)
Figure 3

Figure 3. Rb∆K7 MEFs exhibit enhanced resistance to CDK2 inhibitors and spontaneous senescence in vitro

A. Effects of selected kinase inhibitors with differential effects on Rb∆K7 vs. control MEFs from initial kinome screens. Average proliferation rate of untreated MEFs is shown on the right (white column). Arrows point to pan-CDK- and CDK2/7/9-inhibitors. Bars represent mean ±SD, n=3 independent MEFs.

B. Increased resistance of Rb∆K7 vs. control MEFs to CDK2 inhibition (P=0.008 by two-tailed unpaired student's t-test). Effect of a pan CDK inhibitor was near significance (P=0.07). Bars represent mean ±SD, n=3 independent MEFs.

C. Left, representative flow cytometric analysis with 7-AAD and AnnexinV, showing a significant decrease in apoptosis in Rb∆K7 MEFs relative to control. Right, significant differences in average percentage of live (7-AAD-:AnnexinV-) vs. apoptotic (7-AAD+:AnnexinV+) cells. Bars represent mean ±SD; P value calculated by two-tailed unpaired student's t-test; n=3 independent MEFs.

D. Increased G1 phase in Rb∆K7 MEFs. Mean ±SD of propidium Iodide flow cytometry analysis showing a significant increase in G1 and decrease in S phase in synchronized Rb∆K7 MEFs relative to control (n=3 independent MEFs).

E. Representative bright field images of multi-passage MEFs showing immortalized control cells but large senescing Rb∆K7 cells (arrows, original magnification: 200x; n=4-5).

F. Representative images showing increased senescence in Rb∆K7 MEFs (arrows) determined by senescence-associated β-galactosidase assay (SA-βGAL; original magnification: 200x; P=0.0395 by two-tailed unpaired student's t-test, n=4 technical replicates (see text).
Hypophosphorylated pRb knock-in mice exhibit hallmarks of aging and vitamin C-preventable diabetes (2021)
Figure 4

Figure 4. Rb∆K7 adult mice display signs of premature aging and diabetes

A. Reduced weight (in grams) of adult Rb∆K7 (n=11) vs control littermate (n=17) males. Results are presented as mean ±SD; P values calculated by two-tailed unpaired student's t-test.

B-D. Infertility in Rb∆K7 males: (B) number of litters from breeding of homozygous Rb∆K7 (n=4) or control wildtype (n=5) males with control wild-type females. Bars represent mean ±SD; P values calculated by two-tailed unpaired student's t-test. (C) Representative images of testes from two Rb∆K7 mice compared with a control littermate. (D) Sections through seminiferous tubules. Arrows point to mature spermatids in control testes that are lacking in Rb∆K7 adult testes. Scale bar, 20 μμ.

E. Kyphosis in Rb∆K7 mice. Top, images of eight-month old Rb∆K7 mouse with kyphosis (right) and control wildtype littermate (left). Bottom, radiographs of these mice under anesthesia showing abnormal cervical curvature (arrows) in the Rb∆K7 knock-in mouse.

F-I. Delayed hair re-growth in eight-month old Rb∆K7 mice two weeks post hair plucking (F). (G) Abnormal skin histology in Rb∆K7 mice relative to control. FCL, subcutaneous fat cell layer, T, telogen; A, anagen. (H-I) Representative immunostaining for keratin 14 and PCNA showing reduced expression in Rb∆K7 hair follicles vs controls. Scale bar, 50 μm.

J. Hyperglycemia in Rb∆K7 but not Rb∆K4 mice. Blood glucose in 10-12-month old fasting Rb∆K7 (left, n=11) or 18-month old fasting Rb∆K4 (right, n=15) mice versus control littermates (n=6 and n=8, respectively). Mean ±SD; P values by two-tailed unpaired student's t-test.
Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin (2020)
Fig. 4

Analysis of nCoV-2019 receptor usage.Determination of virus infectivity in HeLa cells with or without the expression of ACE2. h, human; b, Rhinolophus sinicus bat; c, civet; s, swine (pig); m, mouse. ACE2 protein (green), viral protein (red) and nuclei (blue) was shown. Scale bar=10 um.
IFN-stimulated Metabolite Transporter ENT3 Facilitates Viral Genome Release (2022)
Figure 1

Figure 1. LPS-activated pathways upregulate the expression of ENT3 in BMDMs.

(A) BMDMs were treated with E. coli (MOI=100) for 30 minutes and further cultured for various length of time as indicated. The dynamic of ENT3 expression was determined by measuring Slc29a3 transcripts via RT-qPCR.

(B) Phagocytosis inhibitor, Cytochalasin D (CytoD), pretreated BMDMs were incubated with E. coli (MOI=100) and subjected for Slc29a3 expression analysis via RT-qPCR 6 hours post-infection.

(C) The bacterial effector molecules generated from different treatments. Live, heat-killed (HK), gentamicin-killed (GK) E. coli, or metabolite-containing supernatant (Meta), were prepared.

(D) Different bacterial components were applied as stimulants to BMDMs. Slc29a3 expression was analyzed after 30 minutes of stimulation and a total of 6 hours incubation.

(E) BMDMs were treated with different doses of LPS for 30 minutes, harvested 6 hours post-stimulation, and subjected for RT-qPCR analysis.

Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.
IFN-stimulated Metabolite Transporter ENT3 Facilitates Viral Genome Release (2022)
Figure 2

Figure 2. LPS induces ENT3 expression through the TLR4-TRIF axis.

(A) The Schematic diagram of TLR4 signaling pathways.

(B) BMDMs from WT or MyD88-/- mice were treated with 10 ng/ml of LPS or live E. coli (MOI=100) and subjected for Slc29a3 expression analysis.

(C) WT or (D) MyD88-/- BMDMs were stimulated with 10 ng/ml LPS or live E. coli (MOI=100) in the presence of 10 µM TBK inhibitor, MRT 67307, and analyzed for Slc29a3 expression.

(E) The 10 ng/ml LPS-treated BMDMs were examined for Ifnb1 expression after 30 minutes of stimulation and a total of 6 hours incubation.

(F) BMDMs were treated with 100 U/ml of IFN-β for various length of time and subjected for Slc29a3 expression.

Data information: The results shown are combined from three biological replicates (n=3), unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.
IFN-stimulated Metabolite Transporter ENT3 Facilitates Viral Genome Release (2022)
Figure 3

Figure 3. Type I IFN upregulates ENT3 transcripts through an IFNAR1-STAT1 axis.

(A) The brief schematic diagram of type I IFN downstream signaling cascades.

(B-E) BMDMs from WT, IFNAR1-/- (B), or STAT1-/- (C) mice were treated with 10 ng/ml LPS, live E. coli (MOI=100), or 100 U/ml IFN-β, harvested and examined for Slc29a3 induction. BMDMs from WT, IFNAR1-/- (D) or STAT1-/- (E) mice were treated with 100 U/ml IFN-β, 50 µg/ml poly(I:C), or EMCV (MOI=10), harvested and examined for Slc29a3 induction.

(F-H) The expression dynamic of Slc29a3 was profiled in BMDMs treated with two different doses of IFN-α (F), IFN-β (G), IFN-γ (H) for various length of time as indicated.

Data information: The expression level was calculated relative to Rpl19 and normalized to untreated group as 1. The results shown in B to E are from three biological replicates (n=3) and in F to H from two biological replicates (n=2). Unpaired two-tailed Student's t-test was used for statistical analysis, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.
IFN-stimulated Metabolite Transporter ENT3 Facilitates Viral Genome Release (2022)
Figure 4

Figure 4. Slc29a3 has a STAT1 binding site at its promoter region.

(A) The induction fold change (treated/untreated) of Slc29a3 and selected ISG transcripts were curated from INTERFEROME database. Shown is data extracted from 250 IU/ml IFN-β treated BMDMs.

(B) Potential STAT1 interacting sites on Slc29a3 were identified using the ChIP-Atlas database. The red bars marked the STAT1 binding sites while the height of the bars represented the frequency of STAT1 binding on the specified region.

(C) STAT1 binding to Irf1 GAS region was used as a positive control validating the quality of IFN-β stimulation. BMDMs were stimulated with 100 U/ml of IFN-β for 4 hours and proceeded with qChIP.

(D-F) Primers targeting 3' flanking region 2 (D), 3' flanking region 1 (E), and promoter region (F) were used to quantify the STAT1 binding in BMDMs upon 100 U/ml IFN-β after 4 hour stimulation via qChIP assay.

Data information: The results in C and D-F were generated from two biological replicates (n=2) with four technical repeats each (n=4). One representative example is shown. Unpaired two-tailed Student's t-test was used for statistical analysis, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.
IFN-stimulated Metabolite Transporter ENT3 Facilitates Viral Genome Release (2022)
Figure 5

Figure 5. The expression of ENT3 is responsive to viral infection, yet Cx3cr1cre/+ENT3fl/fl mice are resistant to EMCV infection.

(A-C) (A) BMDMs were infected with EMCV (MOI=10) and harvested at indicated time points. (B) THP-1 derived macrophages were challenged with EV71 (MOI=5) and collected at different time points. (C) BMDMs were co-cultured with 50 µg/ml poly (I:C) with various length of time and harvested. The mRNA expression of Slc29a3 or SLC29A3 was measured by RT-qPCR. The expression level was calculated relative to Rpl19 or RPLP0 and normalized to untreated group at time 0 as 1. Results are combined from three biological replicates (n=3)

(D, E) ENT3fl/fl and Cx3cr1cre/+ENT3fl/fl mice (n=12) were challenged with EMCV (104 PFU, i.p.) and monitored for the survival (D) and disease severity (E) for two weeks. Survival results were analyzed by log-rank (Mantel-Cox) test, *p < 0.05.

(F, G) ENT3fl/fl and Cx3cr1cre/+ENT3fl/fl mice were challenged with 107 PFU EMCV and measured the viral titer in the brain at post-infection day 3 by plaque assay (n=6). Shown are representative plaque assay images (F) and the quantification (G).

(H) ENT3fl/fl and Cx3cr1cre/+ENT3fl/fl mice were challenged with 107 PFU EMCV and measured the serum IFN-β level at post-infection day 1 and 3 (day 0, n=3-4; post-infection, n=6-9).

Data information: Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, n.s. no significance. Data are shown as mean ± SEM.
IFN-stimulated Metabolite Transporter ENT3 Facilitates Viral Genome Release (2022)
Figure 6

Figure 6. EMCV fails to replicate in ENT3-/- BMDMs.

(A) WT and ENT3-/- BMDMs were infected with EMCV (MOI=10), and the culture supernatants were harvested 24 hours post-infection to measure the production of IFN-α, IFN-β, and IFN-γ by ELISA. Shown are combined results from three biological replicates (n=3).

(B) WT and ENT3-/- BMDMs were challenged with 2 µg/ml of liposome-packed poly (I:C) for the length of time indicated and harvested to evaluate the Ifnb1 expression by RT-qPCR. Shown are combined results from three biological replicates (n=3).

(C) WT and ENT3-/- BMDMs were infected with EMCV (MOI=10) for 2 hours and allowed for viral replication for the various length of time as indicated. The expression level of EMCV-2A2B was determined by RT-qPCR. Representative data from three biological replicates (n=3) are shown.

(D, E) The mitochondrial respiratory capacity (D) and glycolysis capacity (E) of WT and ENT3-/- BMDMs treated with 1000 U/ml IFN-β for 6 hours were measured by extracellular flux analysis. Shown are the representative results of OCR and ECAR kinetics from six biological replicates (n=6).

(F) GSEA of RNAseq results generated from WT and ENT3-/- BMDMs treated with 1000 U/ml IFN-β for 4 hours. Shown are the significantly enriched pathways in ENT3-/- BMDMs.

(G, H) The replication of EMCV in WT (G) and ENT3-/- BMDMs (H) were evaluated in the presence of exogenous 1 or 10 µM of ribonucleosides (A, U, C, G) 9 hours post-infection (MOI=10). The expression of EMCV-2A2B was determined by RT-qPCR. Shown are results from three to four biological replicates (n=3-4).

Data information: In (B) the Ifnb1 relative gene expression was normalized to Rpl19. In (C, G, H) the EMCV-2A2B expression level was calculated relative to Rpl19 and normalized to untreated group at time 0 as 1. Unpaired two-tailed Student's t-test was used for statistical analysis, *p < 0.05, ***p < 0.001. n.s. no significance. The combined data are shown as mean ± SEM.