EMBO/sd-character-level-ner huggingface dataset:
Figure 6A, HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in (Fig 5B). Here are shown the cell cycle profiles after 16 hrs of serum deprivation (top panels), or after an additional 24 hrs of serum re-addition in absence (middle panels), or presence of 10 µM MPA (bottom panels). The results are representative of at least three independent experiments. B, HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs. The levels of γ-H2AX were analyzed on Western blots. GAPDH was used as a loading control. Statistical analysis of the immunostaining of γ-H2AX (C) in HCT116 cells treated Total nuclear intensities were obtained on >6000 (γ-H2AX) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units. Statistical analysis of the immunostaining of 53BP1 (D) in HCT116 cells treated Total nuclear intensities were obtained on > 1000 (53BP1) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units. E, Representative images cells labeled with γ-H2AX (green), 53BP1 (red) and DAPI (blue). Scale bars correspond to 10 μm F, HCT116 cells treated were analyzed by the comet assay. The percentage of tail DNA/total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates. ****p<0.0001 by Kruskal-Wallis test
TGFβ-induced stimulation of mitochondrial bioenergetics is required for increased proline biosynthesis.Figure 4Mitochondrial basal respiration (a) and FCCP-induced maximal respiration (b) of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or mock for 2h, 4h, 8h, 24h or 48h, based on Seahorse OCR measurements. The relative values were calculated by normalizing the respective values of TGFβ-treated cells to those of mock-treated cells for each time point. Left: western blot of NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX. MitoTPNOX or mitoLbNOX expression is detected by probing for their FLAG-tag. Right: NIH-3T3 cells expressing empty vector, mitoTPNOX or mitoLbNOX were treated with TGFβ or mock for 48h and proline abundance was measured by GC-MS. Values are normalized to mock-treated empty vector expressing cells. NIH-3T3 cells were treated with rotenone, antimycin, myxothiazol or vehicle control for 1h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells. NIH-3T3 cells were treated with cobalt chloride (CoCl2) or vehicle control for 6h, and proline abundance was measured by GC-MS. Values are normalized to control-treated cells. NIH-3T3 cells were treated with TGFβ or mock for 48h, and FCCP or vehicle control was added for the last 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells. NIH-3T3 cells were treated with TGFβ or mock for 48h, and oligomycin or vehicle control was added for the last 1h or 6h of the treatment. Proline abundance was measured by GC-MS. Values are normalized to mock- and control-treated cells.
Figure 2A Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34a-/- mice during normal and aberrant alveolarization (scale bar, 50 µm). B Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization. C Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization. D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34bc-/- mice during normal and aberrant alveolarization (scale bar, 50 µm). E Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization. F Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization. G Localization of miR-34a expression by β-galactosidase activity staining in the developing lungs of P14 miR-34a::lacZ+/+ mice that were undergoing normal or aberrant alveolarization (scale bar, 50 µm; larger and some additional images are presented in Appendix Fig S2).
The effect of various mutants on the MS- and C-complexes of H.Figure 5A-G) Slices through electron cryotomograms (left panels), central slices of subtomogram averages (middle panels), and schematic representations (right panels) of the MS- and C-complexes (white arrows on the slices) of different mutants in H. pylori fliP*. Light and dark green arrows in (A and D) point to the ~53-nm and ~67-nm cytoplasmic rings. Scale bars for cryotomogram slices are 50 nm, and 20 nm for STAs. Empty panels indicate that there were not enough examples to produce a STA.
Genome-wide proteomic response to HU-induced replication stress.Figure 1(A-B) Volcano plots showing the ratio of chromatin binding in HU-treated/untreated for factors that exhibit a significantly different enrichment on chromatin in sml1Δ cells. (A) Colored dots are factors with significantly different chromatin binding scores HU-treated/untreated (adj. p<0.1 and fold-change<0.5 (blue) or >0.5 (red)). (B) Factors involved in transcription with reduced occupancy upon HU treatment are labeled (left), as well as factors involved in replication stress response with increased occupancy (right). (C-D) Phosphopeptide abundances (log2 ratio WT [HU/untreated]). Colored dots are factors with significantly different phosphopeptide scores WT [HU/untreated] <4 (blue) or >4 (red) and p value<0.05 (D) Amongst the phosphopeptides induced on HU, factors involved in transcription are highlighted (green dots, n=87).
HDX-MS analysis of active and inactive BakΔTM.Figure 5(a) The HDX values after 120 min of BakΔTM bound to ΔH5 nanodiscs are subtracted from the values with soluble BakΔTM and plotted against the amino acid sequence. α-helical secondary structure is indicated at the top. (b) The HDX data displayed in (a) are color-coded on the Bak∆TM structure. Higher fractional uptake in the unbound state is colored in red, while less uptake is colored in blue. (c The HDX of inactive BakΔTM subtracted from the HDX of BidBH3-activated BakΔTM, both bound to nanodiscs, is color coded on the BakΔTM structure , d) The HDX of inactive BakΔTM subtracted from the HDX of BidBH3-activated BakΔTM, both bound to nanodiscs, is plotted against the amino acid sequence in (d). In this case, the HDX rates after 10 sec were used for figure preparation. The position of the α-helical secondary structure elements (α1-α8) is indicated at the top.
Svb activator induces stem cell proliferationA esgts midguts expressing GFP alone (control), or expressing OvoB, and carrying a Dl-lacZ transgene that marks ISCs.Figure 3esgts midguts expressing GFP alone (control), or expressing OvoB, and carrying a Dl-lacZ transgene that marks ISCs. Samples were stained for GFP (green) and ß-Gal (red); the graph shows quantification of ß-Gal-positive cells (ISCs). Quantification of number of mitotic PH3-positive cells/midgut in esgts guts expressing GFP alone (control), or expressing OvoB and svb-RNAi; Y axis is drawn as log(10). esgts midguts expressing GFP alone (control) or expressing SvbACT. Samples were stained for GFP (green), and ß-catenin (purple), DE-Cadherin (white) or Scribble (yellow). Top and bottom pictures show separate channels for a same region. esgts midguts expressing GFP alone (control), or expressing miR8, OvoB, and mir8 + OvoB. Samples were stained for GFP (green) and ß-catenin (purple). The graph shows quantification of GFP-positive cells for each genotype. The Y axis is drawn using a log(10) scale. esgts midguts expressing GFP alone (control), or expressing Notch Intra Cellular Domain (NICD), and NICD + OvoB. In closeups (right), DAPI is shown in purple for improved contrast; the arrow highlights a cell with intermediate phenotype.
Inverse expression of the transcription factors c-Myb and c-Maf/MafB in fetal monocytes and pMΦ.Figure 5Schematic of the samples for RNA-seq analysis. pMΦ from E10.5 YS (pMΦ-Ys), E14.5 liver (pMΦ-Li), and E17.5 lung (pMΦ-Lu) as well as fetal Mo form E14.5 liver (Fetal Mo-Li) and E17.5 lung (Fetal Mo-Lu) were sorted from WT embryos, respectively. pMΦ-Ys, pMΦ-Li and Fetal Mo-Li were i.n. transferred to neonatal Csf2ra-/- mice. Six weeks later, pMΦ-Ys-, pMΦ-Li- and Fetal Mo-Li-derived mature AM (mAM-pMΦ-Ys, mAM-pMΦ-Li, mAM-Fetal Mo-Li) were sorted from the BAL of recipients, respectively. AM from 6-week old WT were sorted as control. Principal component analysis (B) and clustering matrix (C) of the transcriptomes of precursors and mature AM described above in A. Heatmap of AM signature-up genes (D), AM signature-down genes (E), and AM-specific transcription factors (F) in the precursors and mature AM described above in A. the data are from one RNA-seq experiment (1 - 2 biological replicates per group). qPCR (n = 3 biological replicates) analysis of Myb, Maf, Mafb, and Pparg expression in different fetal precursors from WT embryos and mature AM from WT adult mice. Gene Ontology (GO) term enrichment maps for Cellular Component (CC) of pMΦ and fetal Mo from E14.5 fetal liver. The maps show the top enriched gene networks with the FDR q value < 0.1. The line thickness connecting two gene set nodes corresponds to the number of genes overlapping between the two sets. Red nodes indicate gene sets enriched within genes strongly downregulated in fetal Mo. Blue nodes indicate gene sets enriched within genes strongly upregulated in fetal Mo compared to pMΦ.
Figure 2IPs from RNase A-treated S-phase HeLa cell lysate using the SLBP antibody or IgG (A) Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots IPs from RNase A-treated S-phase HeLa cell lysate using the ALYREF antibody or IgG (B). Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots (C) Pull-downs of in vitro translated 35S labelled SLBP and luciferase (Cntl) using MBP or MBP-ALYREF in the presence of RNase A. The proteins pulled down were visualized by coomassie staining (left) or phosphoimager (right). 3% of input was loaded. (D) GST-SLBP, GST-UAP56 and GST were used for pull-down of purified MBP-ALYREF or MBP in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE, followed by coomassie staining. 37.5% of input proteins were loaded. (E) (Top) Domain schematic representation of SLBP. (Bottom) Flag IPs from RNase A-treated HeLa cell lysate individually expressing indicated Flag tagged proteins, followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot (E) (Top) Domain schematic representation of SLBP. followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot except that Flag IPs were carried out from HA-SLBP stable expression cells transfected with plasmids expressing ALYREF fragments. (G) Western blotting to examine the KD efficiency of SLBP. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to western analysis (H) Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant. Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to RT-qPCRs (I). Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant. (J) Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot. (K) Graphic displays that SLBP functions in recruiting ALYREF to histone mRNAs.
Figure 4(A) LN229 cells transduced with a pool of four siRNAs against AMOTL1 (+) or a scrambled siRNA (-) were treated with DMSO or thapsigargin (TG) and subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane was quantified by Image J and is shown under the blot. (B) LN229 cells stably transduced with 6x-Histidine-tagged Ubiquitin (His-Ubi) were then stably transduced with indicated shRNAs against AMOTL1 or a scrambled shRNA control (sh-Co.). These cells were detached, lysed and subjected to nickel-charged affinity purification followed by western blotting for endogenous Merlin. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot. (C) A diagram illustrating that AMOTL1 mediates the NEDD4L-Merlin interaction. (D) Merlin-depleted LN229 cells stably expressing Flag-HA-tagged Merlin were transduced with scrambled shRNA (sh-Co.) or two distinct shRNAs targeting AMOTL1, treated with DMSO or thapsigargin (TG) and subjected to Flag immunoprecipitation followed by western blotting. (E) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. PY: PPEY motif; LY: LPTY motif. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot. (F) Merlin-depleted LN229 cells stably transduced with Flag-tagged wild-type Merlin or the indicated mutants were treated with DMSO or thapsigargin (TG). The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot. (G) HEK293T cells were transfected with the indicated constructs. The cells were lysed and subjected to immunoprecipitation with a Flag antibody. The lysate and immunoprecipitated products were subjected to western blotting. The ratio of mono-ubiquitinated to native Merlin in each lane of the lysate blot was quantified by Image J and is shown under the blot.
Figure 6A, B. Western blots showing levels of EJC proteins or HNRNPA1 (control) in input, IgG IP or EIF4A3 IP following overexpression (OE) of CASC3 wild-type (WT) and EJC binding deficient (HDAA) mutant proteins in (A) HeLa Tet-off cells, and (B) 3BKO#3 HeLa Tet-off cells. Ramps above lanes indicate expression levels of the CASC3 proteins. C. Western blots showing levels of EJC/UPF proteins and HNRNPA1 in input and FLAG followed by EIF4A3 tandem IP from HCT116 cells expressing the FLAG-tagged endogenous protein indicated above each lane. Quantifications of UPF3B and CASC3 protein enrichment from two replicates are shown at the bottom. D. Meta-exon plot showing read distributions within the 100 nucleotide (nt) window from the exon 3′ end in RIPiT-Seq replicates of MAGOH:EIF4A3, UPF3B:EIF4A3, and CASC3:EIF4A3. The black vertical line indicates the -24 nt position. E. Venn diagram showing the degree of overlap between genes significantly enriched in CASC3:EIF4A3 EJC and UPF3B:EIF4A3 EJC occupancy as compared to MAGOH:EIF4A3 EJC occupancy. F. Scatter plot comparing log2-transformed fold change in occupancy of CASC3:EIF4A3 EJC as compared to MAGOH:EIF4A3 EJC (x-axis) and UPF3B:EIF4A3 EJC compared to MAGOH:EIF4A3 EJC. Each dot represents a gene where gene-level occupancy of each EJC composition was quantified at the canonical position for EJC footprints. Pearson correlation coefficient is shown on the top left. G. Box plots comparing occupancy of a specific EJC composition on mRNA (indicated on y-axis; normalized by gene expression from RNA-Seq) between genes of different NMD efficiency. Up, no, and down stands for upregulation, no obvious change or downregulation of a representative PTC+ isoform for each gene (see material and methods) that is used to reflect overall NMD efficiency for each gene. In the box plots, central band represents the median, boxes represent the interquartile range (IQR), whiskers indicate maximum (top) and minimum (bottom) values (1.5-times the highest or lowest IQR values, respectively), and circles represent the outliers. p-values are from Wilcoxon signed-rank test comparing the two indicated distributions. H. Box plots as in G comparing specific EJC composition occupancy relative to EJC core occupancy (normalized by MAGOH:EIF4A3 RIPiT-Seq) between genes of different NMD efficiency. Up, no, down, central band, boxes, and whiskers are as in G. I. Bar plots showing fold changes measured by isoform specific RT-qPCR of PTC+ and PTC- isoform from genes indicated on the bottom in WT and CASC3-KO HCT116 cells. Relative levels from each replicate are shown by white circles. Error bars indicate standard error of means.
Bone metastasis is inhibited by therapeutic induction of cell-intrinsic (HDACi) and systemic type I IFN (A) ELISA of IFN-α production by RM1 BD Irf- cells ± single agent MS275 and poly I:C treatment or 48 h pre-treatment with MS275 (pMS) prior to poly I:C stimulation (n = 6).Figure 5(A) ELISA of IFN-α production by RM1 BD Irf- cells ± single agent MS275 and poly I:C treatment or 48 h pre-treatment with MS275 (pMS) prior to poly I:C stimulation (n = 6). (B) Mean fluorescence intensity of H2-Kb staining on RM1 BD Irf- cells by FACS ± MS275, poly I:C or combination treatment (n = 3). (C) FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1) FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1) with (D) representative FACS plots shown. (E) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 BD Irf- cells ± MS275, poly I:C or combination treatment (n = 6 per group; log-rank (Mantel-Cox) test). (F) Mean total tumor burden in bone (femurs, tibias, spine and humerus) at endpoint by bioluminescent intensity score (log2 p/sec/cm2/sr) with values < 4.0x104 representing zero burden (n = 24 bones per group; 6 mice per group) and representative bioluminescent imaging of tumor burden in spine (all shown in Appendix Figure S5E).
Figure 7A-D (A) Plasma concentrations of non-esterified fatty acids (NEFA) (CTRL N=7, BAM15 N=8, P=0.0073), (B) leptin (N=8 per group, P<0.0001), (C) GDF15 (N=7 per group, P=0.035), and (D) FGF21 (N=7 per group, P=0.0026) after 3-weeks of CTRL or BAM15 treatment. E, F (E) Total (basal: P=0.015) and (F) complete (basal: P=0.0137) oxidation of 200 μM [1-14C]palmitate to CO2 from mixed gastrocnemius muscle homogenates (N=7 per group). (G) Routine (R) oxygen flux, leak (L), OXPHOS (P) and ETC (E) capacity in permeabilized mixed gastrocnemius fibers (N=6 per group). PM: pyruvate and malate, ADP: adenosine diphosphate, GS: glutamate and succinate, Tm: tetramethyl-p-phenylenediamine, PalM: palmitoylcarnitine and malate, Oct: octanoylcarnitine, and Dur: duroquinol. (H) Representative immunoblots of phosphorylated and total AMPK and ACC from iWAT, and mixed gastrocnemius muscle (I-K) Densitometric quantification (N=4 per group; iWAT p-AMPKT172: P=0.0186, iWAT p-ACCS79: P=0.0083, mixed gastrocnemius p-AMPKT172: P=0.0286). (L-N) mRNA expression of transcriptional regulators of lipogenesis and lipolysis in iWAT (N=7 per group). Srebf1 (P<0.0001), Mlxipl (P=0.0018), Scd1 (P<0.0001), FASN (P=0.0009), LIPE (P=0.0015), Pnpla2 (P<0.0001), PPARg (P<0.0001).
Figure 2A) Detail of the S1 ubiquitin binding site of SARS2 PLpro, bound to ubiquitin (left) and ISG15 (right), highlighting differential interactions of ubiquitin with the Fingers subdomain, and of ISG15 with the Thumb subdomain of PLpro. Labelled residues were mutated, see Fig EV3. B) Fluorescence polarisation assays against ubiquitin-TAMRA and ISG15-TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n=2 for each mutant, and compared to wild-type PLpro as shown in Appendix Fig S1A. Catalytic efficiencies were calculated as described in Methods. C) Gel based analysis of PLpro variant activity against Lys48-triubiquitin and proISG15. Experiments were performed in duplicate, see Fig EV3 and Source Data for uncropped gels.
Figure 5Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet-inducible expression of human p53EE. Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0.5 µg/ml Doxo. Cell death measured by flow cytometry using propidium iodide exclusion in human lung adenocarcinoma H1299 cells with tet-inducible expression of human p53EE. Cells with overexpression of human p53EE mutant display a trend of increased cell death after treatment with 0.5 µg/ml Doxo. This effect is enhanced by addition of Nutlin and blocked by the pan-caspase inhibitor QVDOph, but not the ferroptosis inhibitor Ferrostatin-1 (Ferr) as control. (A) n=3; (B) n=2. Apoptosis analysis using Annexin V staining in cells from (A). n=3. mRNA expression analysis (RTqPCR) of p53 target genes following treatment with 0.5 µg/ml Doxo ± 10 µM Nutlin. KO, tet-induced H1299-pInd cells; EE, tet-induced H1299-pInd-p53EE cells; WT, H460 cells. mRNA expression was normalized to β-actin; n=3. Proximity ligation assay for p53 with Tom20 in indicated untreated (Mock) or Doxo-treated (0.1 µg/ml, 24 hours) H1299 cells following tet-induction. Shown are representative cells with PLA signals (red) counterstained with DAPI (blue). Quantification of proximity ligation assays for p53EE with Tom20, Bcl-2, Bcl-xL and Bak in H1299 cells. Doxo treatment (0.1 µg/ml, 24 hours) as indicated. Plotted is the average number of PLA signals per cell for 10 fields of view. Mitochondrial membrane potential of control and p53EE expressing H1299 cells in the absence and presence of increasing concentrations of BID BH3 peptide. Treatment with the mitochondrial depolarizer CCCP is shown as a positive control; n=3. Apoptosis measured by flow cytometry using Annexin V in H1299 cells with tet-inducible expression of the indicated human p53 mutants; n=3. mRNA expression analysis (RTqPCR) of p53 target genes normalized to β-actin; n=3.