EMBO/SourceData huggingface dataset:
Fig. 3. Kinetics of SARS-CoV-2 and SARS-CoV RNA synthesis in infected Vero E6 cells.(a) Hybridization analysis of viral mRNAs isolated from SARS-CoV-2- and SARS-CoV -infected Vero E6 cells, separated in an agarose gel and probed with a radiolabelled oligonucleotide recognizing the genome and subgenomic mRNAs of both viruses. Subsequently, the gel was re-hybridized to a probe specific for 18S ribosomal RNA, which was used as a loading control. (b) Analysis of the relative abundance of each of the SARS-CoV-2 and SARS-CoV transcripts. Phosphorimager quantification was performed for the bands of the samples isolated at 12, 14 and 24 h p.i., which yielded essentially identical relative abundances. The table shows the average of these three measurements. SARS-CoV-2 mRNA sizes were calculated on the basis of the position of the leader and body transcription-regulatory sequences (ACGAAC) in the viral genome (Sawicki and Sawicki 1995, Xu, Hu et al. 2003).
Fig. 4. Cross-reactivity of antisera raised against SARS-CoV structural and non-structural proteins.Selected antisera previously raised against SARS-CoV nsps and structural proteins cross-react with corresponding SARS-CoV-2 proteins. SARS-CoV-2-infected Vero E6 cells (m.o.i. of 0.3) were fixed at 12 or 24 h p.i. For immunofluorescence microscopy, cells were (double)labelled with (a) a rabbit antiserum recognising nsp4 and a mouse mAb recognising dsRNA; (b) anti-nsp4 rabbit serum and a mouse mAb directed against the N protein; (c-e) rabbit antisera recognising against nsp3, nsp13 and the M protein, respectively. Nuclear DNA was stained with Hoechst 33258. Bar, 20 µm.
Fig. 6. Assay to screen for compounds that inhibit SARS-CoV-2 replication.Inhibition of SARS-CoV-2 replication (coloured bars) was tested in Vero E6 cells by developing a CPE-reduction assay and evaluating several previously identified inhibitors of SARS-CoV, which was included for comparison (grey bars). For each compound a two-fold serial dilution series in the low-micromolar range was tested; (a) Remdesivir, (b) chloroquine, (c) Alisporivir and (d) pegylated interferon alpha-2. Cell viability was assayed using the CellTiter 96® Aqueous One Solution cell proliferation assay (MTS assay). Compound toxicity (solid line) was evaluated in parallel using mock-infected, compound-treated cells. The graphs show the results of 3 independent experiments, each performed using quadruplicate samples (mean ± SD are shown).
Figure 1A. Maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA (red) combined with immunostaining for L1 ORF1p (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. White arrow heads point to cytoplasmic foci where L1 RNA and ORF1p co-localize. Scatter plot shows the number of co-localized L1 Tf-ORF1 foci in the cytoplasm per cell (n=275 WT, 304 Dicer_KO1, 311 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 RNP foci. Bar graphs are mean values of percentage of cells with large L1 Tf-ORF1 foci co-localizing in the cytoplasm (n=3 technical replicates).B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (red), MOV10 (green) and nuclei stained with DAPI (blue). The grey square marks position of inset in the zoomed image. White arrow heads point to cytoplasmic foci where L1 ORF1p and MOV10 co-localize. Scatter plot shows the number of co-localized ORF1-MOV10 foci in the cytoplasm per cell (n= 293 WT, 295 Dicer_KO1, 295 Dicer_KO2 cells). Red arrow heads point to relatively larger sized L1 ORF1-MOV10 foci. Bar graphs are mean values of percentage of cells with large ORF1-MOV10 foci co-localizing in the cytoplasm (n=3 technical replicates).
Figure 2A. WT and Dicer_KO mESCs were treated with 0.5mM Sodium Arsenite (NaAsO2) for 20 minutes or left untreated prior to fixation with formaldehyde. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for G3BP1 (red) and MOV10 (green) with nuclei stained with DAPI (blue).B. Representative western blots showing low AGO2 protein levels in Dicer_KO as compared to WT mESCs (right side). LAMINB1 served as loading control. On the left, maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA FISH (red) combined with immunostaining for a resident protein of P-bodies, DDX6 (green) and nuclei stained with DAPI (blue). The grey square marks position of the inset. Yellow arrow heads point to cytoplasmic foci where L1 RNA and DDX6 protein co-localize.
Figure 3A. Representative western blots showing L1 ORF1p and MOV10 levels in the indicated cell lines. Immunoblot with antibody recognizing TUBULIN and coomassie stained membranes depict the loading, asterisk marks position of non-specific band in the ORF1 immunoblot (n=3 technical replicates).B. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for L1 ORF1p (green) combined with RNA FISH for L1 Tf RNA (red) and nuclei stained with DAPI (blue) (n=3 technical replicates).C. Representative images of BlastR colonies stained with crystal violet blue of indicated cell lines is shown on the left. Cells were transfected with either mutant reporter plasmid (L1N21A) or retrotransposition competent reporter (L1WT) as shown in the scheme with timeline for the experiment on the top. Bar graphs on the right depict the average number of BlastR colonies (n=3 biological replicates).
Figure 4A. Scheme and quantification of relative luminescence from luciferase assays validating post-transcriptional regulation of MOV10 by miRNAs in HEK293T cells (n=4 biological replicates).B. Scheme and quantification of relative luminescence from luciferase assays in Hek293T cells to confirm specificity of post-transcriptional regulation of MOV10 by miRNAs with either wild-type (WT) or Mutant (MUT) reporters where the MicroRNA Response Element (MRE) for the depicted miRNAs in the 3'UTR of MOV10 were mutated (n=3 biological replicates).C. Scheme and representative images for western blot analysis and corresponding quantification to assess miRNA mediated regulation on MOV10 expression in mESCs (n=3 biological replicates).D. Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis where miRNA mediated downregulation of MOV10 in Drosha_KO mESCs is accompanied by an increase in L1 retrotransposition (n=3 biological replicates).
Figure 5A. Scheme of experiment and representative images along with quantitation showing induction of L1 RNP aggregate formation upon ectopic MOV10 expression. Images are maximum intensity projections across Z stacks from indicated mESCs stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10 tagged with HA, and nuclei stained with DAPI (blue). White arrow heads point to cytoplasmic foci where L1 ORF1p and HA-MOV10 co-localize. Data collected from 289 Ctrl, 275 L1UP Cl1 and 296 L1UP Cl2 mESCs (n=3 biological replicates).B. Scheme and representative images from colony forming assays for plasmid based retrotransposition analysis in Ctrl and L1UP mESCs comparing rate of retrotransposition upon transfection with Empty Vector (EV) or ectopic expression of HA-MOV10 (n=3 biological replicates).
Figure 6A. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of full length L1 and HA-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for L1 ORF1p (red) combined with immunostaining for HA (green) to detect ectopically expressed MOV10, and nuclei stained with DAPI (blue). The bottom panel shows cells where accumulation of ORF1-MOV10 was observed in relatively large foci compared to cells where co-localization of the two proteins was mostly in relatively small foci (top panel), 68-119 transfected cells were analyzed per experiment (n=3 biological replicates).B. Scheme of experiment and representative images showing RNP aggregate formation upon ectopic expression of HA-ORF1 or HA-ORF1 in conjunction with T7-MOV10 in WT mESCs. Images are maximum intensity projections across Z stacks stained for HA (green) and T7 (red) to detect ectopically expressed ORF1p and MOV10 respectively, and nuclei stained with DAPI (blue). 100-147 transfected cells were analyzed per experiment (n=3 biological replicates).
Figure 1Heatmaps of AIB1-YAP co-activated, YAP activated, AIB1-YAP corepressed, and YAP repressed genes shown in panel (A). Red and blue indicate high and low levels, respectively. RNA-sequencing was performed in triplicate and statistics in Panel (A-B) were calculated by EdgeR with Benjamini-Hochberg multiple test correction in R.Select genes are regulated by YAP and AIB1, others are YAP regulated independently of AIB1. Endogenous gene expression changes assessed by RT-qPCR.Significant genomic co-occupancy between AIB1 and TEAD in normal breast and breast cancer cells. Overlap of AIB1 and TEAD4 ChIP-seq peaks in MCF10A and MCF7 cell lines. Select peaks from MCF10A ChIP-seq shown. ChIP-sequencing performed in triplicate.TEAD binding motifs are enriched within AIB1 occupied sites across the genome. HOMER de novo motif analysis of AIB1 ChIP-seq peaks in MCF10A and MCF7 cells.AIB1 modulates TEAD transcriptional activity in a YAP dependent manner. HEK293T cells were transfected with CTGF-promoter reporter and expression vectors of indicated proteins.AIB1 is engaged with TEAD on co-activated target promoters in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.AIB1 is engaged with TEAD on co-activated target promoters in early stage breast cancer cells. Sequential ChIP-reChIP performed with indicated antibodies in MCFDCIS cells. Levels of precipitated chromatin assessed by qPCR.
Figure 2ANCO1 is recruited to YAP-TEAD by AIB1. Positive Proximity Ligation Assay (PLA) signal (white foci, falsely colored 594nm signal) shown between ANCO1-TEAD, ANCO1-YAP, AIB1-TEAD, and AIB1-YAP, in control cells are lost in AIB1 knockdown. Representative images of Proximity Ligation Assay (PLA) between indicated proteins in MCF10A shGFP (control) and shAIB1 cell lines using antibodies (species) as indicated; Scale bars shown indicate 25μm.YAP overexpression represses, AIB1 knockdown dysregulates, and ANCO1 knockdown amplifies genes at the 1q21.3 locus in MCF10A and MCFDCIS cells. Gene set enrichment analysis of RNA-seq data and cDNA array data generated from MCF10A cell lines as indicated.YAP represses 1q21.3 localized genes. Levels of endogenous S100 and SPPR genes in MCF10A cell lines following overexpression of YAP/S94A; levels were assessed by RT-qPCR.ANCO1 knockdown increases expression of S100 and SPPR genes in MCF10A and MCFDCIS cells; levels were assessed by RT-qPCR.YAP driven suppression of 1q21 genes is reverted upon ANCO1 knockdown. Levels of endogenous 1q21.3 genes were assessed in YAP or S94A-expressing, control or shANCO1 MCF10A cells by RT-qPCR.TEAD is engaged at annotated enhancer regions within 1q21.3. Analysis of TEAD4 ChIP-seq in MDA-MB-231(GSE66081), MCF7, and A549 Cells (GSE32465) at 1q21.3 with enhancers scored by GeneHancer. Enhancer regions of interest indicated between dotted lines.TEAD, YAP, AIB1, and ANCO1 are engaged at annotated enhancer regions within 1q21.3. Chromatin Immunoprecipitation (ChIP) performed with indicated antibodies in MCF10A cells.AIB1 is engaged with TEAD on superenhancer sites at 1q21.3 in normal cells. Sequential ChIP-reChIP performed with indicated antibodies in MCF10A cells. Levels of precipitated chromatin assessed by qPCR.ANCO1 is recruited to superenhancers at 1q21.3 by AIB1 and is significantly reduced at 1q21.3 following AIB1 knockdown. ChIP performed with ANCO1 antibodies in control or shAIB1 cells. Levels of precipitated chromatin assessed by qPCR.
Figure 3YAP driven aberrant sphere formation of MCF10A cell lines is ablated by AIB1 knockdown, but further enhanced by ANCO1 knockdown. Indicated cell lines were plated on Matrigel and allowed to form mammospheres for 7 days. 10x and 40x magnifications shown, Scale bar = 200μm.Quantification of aberrant spheres per field displayed in panel (A), n= number of spheres.Quantification of size (arbitrary units) of aberrant spheres per field displayed in panel (A), relative to control YAP S94A shGFP spheres. n= number of spheres.MCFDCIS xenograft tumors have increased expression of AIB1-YAP, EMT, and 1q21.3 genes during the invasive transition. Schematic of experiment shown. Gene set enrichment analysis of RNA-seq data generated from MCFDCIS xenografts at indicated time points.Invasive lesions have significantly less ANCO1 than encapsulated DCIS lesions. Representative images of H&E, p63, and ANCO1 stained MCFDCIS xenografts as DCIS and invasive lesions. Yellow circles indicate DCIS lesions and black arrows indicate invasive lesions. 20x magnification; Scale bar = 100μm. Quantification of ANCO1 positivity in DCIS and Invasive lesions shown in panel (E).
Figure 4ANCO1 levels decrease in breast cancer. ANCO1 mRNA expression from normal tissue (GTEX) and Tumor (TCGA) for breast samples.Representative IHC images of ANCO1 staining in normal human breast and DCIS lesions. DCIS lesions can be stratified to either ANCO1 negative (center panel), ANCO1 positive (right panel). Scale bar = 50μm.High ANCO1 levels are a good prognostic indicator in Basal Breast Cancer in patients. KM plot showing progression free survival in patients with stratified ANCO1 expression. n= 360 patients.Patient breast tumor samples with reduced ANCO1 levels have high expression of 1q21.3 genes, without altered AIB1-YAP co-activated gene expression. Dot plots showing TCGA Pan-Cancer data accessed via Xenabroswer. Patients were stratified to high or low expression of ANCO1 by median expression.
Figure 1B: Log2 gene expression fold changes of significantly induced genes (FDR = 1%) across different treatment scenarios. Gene induction of immediate, delayed and late responding genes is sustained upon constant activation (ON scenario) and transient upon two-hour pulse activation (ON/OFF scenario). Genes not significantly induced upon parallel CYHX treatment were considered secondary response genes. Genes were ranked by their model-derived response time.
Figure 3B: pERK2 log2 fold change upon sustained activation (left) and deduced input function (right) used for model fitting. Average pERK2 log2 fold change upon 4OHT treatment equals 100% signalling amplitude.C: Measured gene expression kinetics and the resulting maximum likelihood fit of the gene expression model of all significantly induced primary response genes (FDR = 1%). Gene expression is shown as percentage of response amplitude. Mean error is calculated as the mean of absolute residuals serving as a goodness of fit measure.
Figure 4B: Signalling input conditions (left side shows deduced input function, right side shows pERK2 measurements): Sustained ERK signalling (4OHT), two-hour pulse ERK signalling (4OHT+U0126), growth factor signalling (EGF: Epidermal growth factor, FGF: Fibroblast growth factor). Deduced input functions: 100% signalling amplitude corresponds to mean induction in training condition (4OHT). Growth factor-induced input functions are linear interpolations of pERK2 log2 fold changes relative to mean induction in test condition.C: Predictions are verified with actual gene expression data. Heat maps show log2 fold changes of induced mRNAs. P: Model prediction. D: Gene expression data. E: Mean error = mean of absolute residuals.
Figure 5 Figure 5 - Immediate late genes (ILGs) have long mRNA half-lives, are transcribed immediately and have GC-rich promoters. A: Boxplot comparison of mRNA half-life estimates based on modelling of gene induction (model-derived), transcriptional shutdown (ActD-derived) and metabolic labelling (4SU-derived). Estimates from 4OHT-pretreated HEK293∆RAF1:ER cells (ON panel) are more appropriate to characterise induced genes than estimates from unstimulated cells (OFF panel). Genes not assigned to any cluster are shown in grey. C: Log2 fold changes of transcription rate in 4OHT-treated HEK293∆RAF1:ER cells derived from metabolic labelling (4SU) RNA-sequencing data document immediate transcription of IEGs and ILGs but delayed transcription of DEGs.
Figure 6 Figure 6 - Immediate late genes (ILGs) translate signal duration into response amplitude. A: Upper panel: pERK2 log2 fold changes upon different input scenarios (sustained: 4OHT; two-hour pulse: 4OHT+U0126). Lower panel: Response amplitude across temporal clusters and signal durations. Bold lines show median cluster amplitude at each time point. C: qPCR validation to test different ERK signal durations. HEK293∆RAF1:ER cells were treated with 4OHT and U0126 for different periods of time to generate signal duration scenarios of 0.5 to 8 hours (cf. Fig EV1 C). mRNAs of IEGs EGR1 and FOS relay signal duration to response duration, whereas ILGs CLU and FOSL1 decode signal duration to response amplitude (qPCR data for all 17 validated mRNAs is shown in Fig EV6 A).E: Quantification of western blots to present protein log2 fold changes of sample genes upon sustained ERK signalling (4OHT-induced) and transient ERK signalling (EGF-induced) in HEK293∆RAF1:ER cells.
Figure 2A. Ubiquitin-vinyl sulfone (UbVS) assays of RING2 release by addition of pUbl and pUb in trans to R0RBR parkin. Assays were performed with incubation of 2 μM wild-type or W403A R0RBR purified in a 1:1 complex with pUb. Crosslinking was initiated by addition of 10 μM UbVS in the presence of pUbl or pUb∆G76.B. Inhibition of R0RBR activation by the K211N mutation.C. UbVS assays of RING2 release by various type of pUb.D. Autoubiquitination assays of parkin ligase activity induced by pUbl or pUb∆G76 in trans. 2 µM GST-R0RBR in complex with pUb was incubated with 50 nM E1, 2 µM UbcH7, 100 µM ubiquitin and 4 mM ATP.E. UbVS assays of RING2 release with R0RBR purified without pUb bound.F. Autoubiquitination assays of parkin ligase activity with GST-R0RBR without pUb bound. The percentage of unmodified parkin band in each lane is indicated under the gel.
Figure 3B. Phosphorylation of wild-type (WT) parkin and chimeric Ub-R0RBR by PINK1. Reactions were analyzed on a Phos-tag SDS-PAGE gel to assess the level of phosphorylation.C. Release of RING2 by Ub-R0RBR chimera. Ubiquitin-vinyl sulfone assays (10 μM UbVS) were performed with 3 μM phosphorylated and non-phosphorylated full-length parkin and Ub-R0RBR.D. Ubiquitination activity of Ub-R0RBR chimera. Autoubiquitination assays of phosphorylated full-length parkin and Ub-R0RBR (3.3 µM with 3 µm UbcH7 and 75 µM S65A ubiquitin) were performed to assess the impact of pUbl substitution by pUb on parkin activity.
Figure 4B. UbVS assays of release of RING2 by K6- or K48-linked (pUb)4 chains. 10 μM UbVS was added to 2 μM R0RBR in the presence of increasing amount of K6(pUb)4 or K48(pUb)4 chains.C. Autoubiquitination assays of parkin activation by K6(pUb)4 or K48(pUb)4. Assays with 2 µM GST-R0RBR, 50 nM E1, 2 µM UbcH7 and 100 µM ubiquitin were performed in the presence of increasing amount of K6(pUb)4 or K48(pUb)4 chains.D. Competition between K6(pUb)4 chains and monomeric pUb. UbVs (10 μM) was added to 20 nM of WT and K211N R0RBR in the presence of monomeric pUb and/or K6(pUb)4 chains. Reactions were resolved on SDS-PAGE and visualized with anti-parkin antibody by western blotting. The percentage of unmodified parkin band in each lane is indicated under the gel.
figf1(a-c) At 12-16 h post-transfection with GFP-LC3, NIH3T3 (a, b), MEF (b) or HCT116 (c) cells containing vector or the FLIP gene were treated with Hank's solution for 2-4 h (a) or with 2 μM rapamycin for 1-3 h (b, c). Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 10 μm (a). The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01; **P 0.05) (b, c). At 60 h post-transfection with control siRNA or cFLIP siRNA, MEF cells were transfected with GFP-LC3 (c).(d) Autophagosomes were visualized through scanning electron microscopy. Scale bar, 50 nm. The arrows indicate autophagosomes. Immunoblots (IB) of cFLIP and actin expression are shown in Supplementary Information, Fig. S1c.(e, f) NIH3T3 vector and NIH3T3-KSHV-vFLIP cells (e) or TREX-BCBL-Vector and TREX-BCBL-vFLIP cells (f) were treated with Hank's solution or 2 μM rapamycin for 2-4 h, respectively, followed by IB with anti-LC3 and anti-actin antibody.(g) At 12-16 h post-transfection with GFP-LC3, TREX-BCBL-vector and TREX-BCBL-vFLIP cells were treated with doxycycline for 24 h, followed by incubation with 2 μM rapamycin for an additional 12 h. GFP-LC3 was detected using an inverted fluorescence microscope. Scale bar, 5μm. The number of GFP-LC3-positive dots per cell was counted using a fluorescence microscope. Data are mean ± s.e.m.; n = 200-300 cells; three independent experiments; *P 0.01).(h) Autophagosomes were visualized by scanning electron microscopy. Scale bar, 50 nm.
figf2(a) BCBL cells were used for immunoprecipitation (IP) with mouse IgG or anti-Atg3, followed by immunoblotting (IB) with anti-cFLIP antibody (left panel). NIH3T3-KSHV-vFLIP cells were used for IP with mouse IgG or anti-Flag antibody, followed by IB with anti-Atg3 antibody (right panel).(b) FLIP and Atg3 interaction after autophagy induction. HCT116 cells were treated with or without 2 μM rapamycin or Hank's solution (HBSS) for 4 h before IP with anti-Atg3 antibody, followed by IB with anti-cFLIP antibody.(c) Schematic diagram of human Atg3 (top). N, amino terminus; FR, flexible region; C, carboxy terminus. At 48 h post-transfection with GST or GST-Atg3 along with Flag-vFLIP (left) or GST or GST-Atg3 along with GFP-LC3 (right), HEK293T cells were used for GST pulldown, followed by IB with the indicated antibodies.(d) Schematic diagram of KSHV vFLIP (top). The black boxes indicate the K-α2 and K-α4 peptides. At 48 h post-transfection with V5-Atg3 and either GST-vFLIP DED1 mutants (middle) or GST-vFLIP DED2 mutants (bottom), HEK293T cells were used for GST pulldown, followed by IB with anti-V5 antibody.(e) At 48 h post-transfection with GST, GST-Atg3, and either Flag-vFLIP or Flag-vFLIP mAtg3, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag antibody.(f) At 48 h post-transfection with GST-Atg3 and GFP-LC3 along with increasing amount of Flag-vFLIP, HEK293T cells were used for GST pulldown, followed by IB with anti-GFP or anti-Flag antibody.(g) NIH3T3 vector and NIH3T3 vFLIP cells were treated with 2 μM rapamycin for 4 h and used for IP with anti-Atg3 antibody, followed by IB with anti-LC3 antibody. Finally, whole-cell lysates (WCLs) were used for IB with the indicated antibodies to show expression (a-g).