EMBO/sd-character-level-ner huggingface dataset:
Figure 2AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. Tumour-bearing Ly5.1+ mice were monitored for a) tumour growth over 21 days AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. After 21 days TILs were processed for flow cytometry and donor T cell numbers (Ly5.1-Ly5.2+) determined. AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. After 9 days the proportion of Ly5.2+IFNγ+TNF+ versus Ly5.2+GrzB+ TILs was determined. AT-3-OVA mammary tumour cells (1x106) were injected into the fourth inguinal mammary fat pads of female Ly5.1+ mice. Seven days after tumour injection FACS-purified 2x106 naïve CD8+CD44loCD62Lhi lymph node T cells from Ly5.2+;OT-1;Ptpn2fl/fl versus Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred into tumour-bearing Ly5.1+ mice. d) for survival over 86 days. Gene expression in tumours from mice treated with Ly5.2+;OT-1;Ptpn2fl/fl T cells 21 days post-adoptive transfer versus those re-emerging in mice treated with Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl T cells. B16.F10-OVA melanoma cells (1x105) were engrafted onto the abraded skin in the flanks of Ly5.1+ mice. 24 h after tumour cell engraftment naïve CD8+CD62LhiCD44lo lymph node T cells from Ly5.2+ OT-1;Ptpn2fl/fl versus Ly5.2+ OT-1;Lck-Cre;Ptpn2fl/fl mice were adoptively transferred and tumour incidence monitored. Epidermal lymphocytes from tumour-free mice were stained for CD69hiCD103hi and donor-derived (Ly5.2+vα2+) tissue resident memory T cells (TRMs) determined by flow cytometry. Tumour sizes in B16.F10-OVA melanoma bearing mice were determined between day 21 and 23. TILs were assessed for Ly5.2+;OT-1;Ptpn2fl/fl and Ly5.2+;OT-1;Lck-Cre;Ptpn2fl/fl donor T cell numbers by flow cytometry.
Scheme of API transport pathways to the vacuole.Figure 7Scheme of API transport pathways to the vacuole. As described in the text, proAPI forms a complex in the cytosol. This Cvt complex is then targeted to the vacuole by two pathways depending upon the nutrient conditions. In growing conditions, a double membrane vesicle of ∼140 to 160 nm forms around the Cvt complex and excludes cytosol. In contrast, when cells are starved for nitrogen, the Cvt complex is taken up into larger autophagosomes (300-900 nm) together with cytosolic components. Vacuolar delivery is achieved by fusion of the outer membrane of the Cvt vesicle or autophagosome with the vacuolar membrane. Finally, the resulting Cvt bodies or autophagic bodies are broken down by vacuolar hydrolases to release proAPI into the vacuole lumen. The precursor form of the protein is then processed into the mature hydrolase.
Sir4 H‑BRCT interacts with Tom1 and Cbf1 in a phospho-dependent manner A) Scheme of experimental design for IP-MS.Figure 5Scheme of experimental design for IP-MS. Cells lacking endogenous sir4 and expressing the Myc-tagged H‑BRCT domain of Sir4, either wt or carrying mutations (RKR) were subjected to anti-Myc immunoprecipitation followed by LC-MS/MS analyses. Volcano plot showing proteins that interact with wt Sir4 H‑BRCT but not with the RKR mutant (upper right quadrant). MST analysis of the binding interactions between Sir4 H‑BRCT and Cy5-labeled phospho (filled circles) and non-phospho peptides (empty circles) from Tom1 and Cbf1. KD represents the equilibrium dissociation constant. The KD for interactions with the non-phospho peptides could not be determined (n.d.). ΔFnorm [0/00] represents the change in fluorescence during thermophoresis normalized to the initial fluorescence. Data are represented as the mean ± SEM from at least 3 independent measurements. Extracts from asynchronous cultures of strains Sir4-Myc Tom1-HA (GA-10645), Sir4-RKR-Myc Tom1-HA (GA-10646), Tom1-HA (GA-10647) and Sir4-Myc (GA-10199) were subjected to anti-Myc IP and western blotting with indicated antibodies in the presence and absence of lambda phosphatase.
a) mRNA expression levels of autophagic marker genes in hearts of wild-type (WT) and α-MHC-miR-212/132 transgenic (TG) mice (n=9–10).f5(a) mRNA expression levels of autophagic marker genes in hearts of wild-type (WT) and α-MHC-miR-212/132 transgenic (TG) mice (n=9-10). (b,c) Ratio of LC3II to LC3I (b) and p62 protein levels (c) in WT, miR-212/132 null (KO) and α-MHC miR-212/132 TG mice. M: Size marker (n=4-12). (d,e) Representative images (d) and quantification (e) of LC3:mCherry puncta (in red) in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=30). Nuclei are stained in blue with Hoechst33342. (f,g) Representative electron microscopy images (f) and quantification (g) of autophagic vacuoles in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=20). (h,i) Representative FACS plots (h) and quantification (i) of percent increase of autophagic flux in control and miR-212/132-overexpressing TG H9c2 cells under normal and serum/glucose-deprivation conditions (n=3-4 experiments). All values represent mean±s.e.m. *P0.05; **P0.01; ***P0.005. Scale bars, 50 μm in d and 2 μm in f. FC, fold change.
Figure 7A, Schematic of GEMM of Kras-induced adenocarcinoma. Established lung tumors in LSL-KrasG12D mice were treated with vehicle, AZD4547 (10 mg/kg/day), BI6727 (5 mg/kg/day), and HCQ (30 mg/kg/day) as indicated. B, Lung morphology and H&E staining of lung tissue sections after 3-week treatment. C, Tumor burden, average tumor size and tumor number (determined by QuPath software) after the treatment. *P<0.05, **P<0.01 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. Data are presented as mean ± SD (error bar) of three or four replicates (n=3 or 4). D, The tumor size in different treatment groups, calculated by number of tumors at the indicated size range divided by total tumor number of the treatment group. Data are the mean of four replicates (n=4). E, Tumor grade analysis (% of nodules/lung) after the treatment. The anaylsis was based on DuPage et al, (2009). Data are the mean of three replicates (n=3). F, Box plots showing the percentage of Ki67-positive cells in different treatment groups. Data are based on IHC staining of lung tissue section of LSL-KrasG12D mice. *P<0.05 and P＞0.05 (ns) by two-way ANOVA with Tukey's multiple comparisons test. The boxplots span from the first to third quartile, the whiskers extend to 1.5× the interquartile range (IQR). The horzontal line within the boxes represents the median of each group of 10 replicates (n=10). G, Working model depicting synthetic lethal interaction of FGFR1/PLK1 inhibitors and autophagy as a protective mechanism in KRAS-mutant cancer cells.
RNA degradation via autophagy in S.Figure 7Wild‐type and atg1Δ of S. pombe were grown in SD to mid‐log phase and transferred to SD(‐N) at time 0. At the indicated time points, the cells were collected; the metabolites in these cells were extracted and analyzed by LC/MS as described in . The results are presented as normalized intensities on the basis of peak height of each metabolite in wild‐type cells. All data are means of triplicates. The error bars represent the standard deviation.
Growth-death dependence.Figure 1Exponential growth of E. coli K-12, measured using optical density (OD600), in lysogenic broth (LB, white circles) or minimal medium supplemented with different carbon sources, listed in the legend on the bottom right in a greyscale. All the cultures are grown in batch mode, see Methods. Bacterial viability in colony forming units (CFU) per ml of cultures from panel A, grown until OD600 0.5, washed and re-suspended in carbon-free minimal medium. Death rates (slopes of the exponential fits) are listed in Table EV1. Initial density for fast growth (lighter shades) is lower than for slow growth (darker shades), due larger cell sizes that lead to lower cell densities per OD600. Death rates of panel B plotted against growth rates of panel A shown as circles in a grey-scale. Squares show data obtained using eight other carbon substrates, also listed in Table EV1. Color circles show data obtained by varying growth rate in a chemostat. The black line shows a linear weighted least square fit to log-transformed data, that takes uncertainty in data points into account (see Methods). Data shown as mean ± (standard deviation) SD. Two or three replicates per condition.
Fig.Figure 4A and B Influenza virus specific activation of the murine FcγRI, FcγRII, FcγRIII and FcγRIV by antibodies in pooled pre-challenge sera (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11 mice per group). MDCKII cells were infected with Cal/7/9 (A) or PR8 (B) before mouse serum and FcγR-expressing reporter cells were added. Influenza specific Fc-FcγR interaction induces the production of IL-2 in the FcγR-expressing reporter cell, which was measured by anti-IL2 ELISA (n = 3, technical duplicates). Mean ± SE. C HA-specific activation of FcγR by AAV-HA, AAV-cHA or WIV pooled pre-challenge sera (n = 18 mice per group). MDCKII cells were transfected with wildtype HA (pAAV-HA) or a stalk-only HA (pAAV-mHL1) before mouse serum and FcγR-I-expressing reporter cells were added (n = 2, technical duplicates). Mean ± SE.
Features of FRH critical for helicase and clock function.Figure 6(A) Key residues for FRH function shown on the large-cell structure: K208 and S324 compose the ATP binding site, E294 lies between the ADP and RNA pockets and participates in ATP hydrolysis; P871 resides in the elbow region of the arch domain. K811, R806, R712 and K766 contribute to a positively charged surface on the KOW domain. R806H disrupts the clock. Q131, G132 and V142 in the N-terminus provide interactions to the helicase RecA domains (FRH domains are colored according to Fig1, ADP and RNA are in orange bonds). (B) Same as (A) after rotation about a horizontal axis. (C) Close-up of the ADP-binding pocket and N-terminal interaction for the large-cell structure (cyan ribbons) and the small-cell structure (blue ribbons). (D) Electrostatic potential surface of FRH (blue positive, red negative) shown with the same orientation as in (B). (E) Close-up of the KOW domain showing the large number of basic (aqua sticks) and acidic side chains (yellow sticks) on the surface that also includes the mutational sites R806, R712, K811 and K766 (grey sticks)
Chemical genomics screen across four S.Figure 1Core genome phylogeny of part of the Saccharomyces Genome Resequencing Project (SGRP) yeast isolates; coloured dots indicate the four strains whose KOs library was screened in this study. Schematic of the chemical genomics screen; each strain's KO library was robotically plated on 1536 solid agar plates and each KO colony size was used as a proxy for fitness in each condition. Reproducibility of the S-scores using the two batches used in the screening. Reproducibility of the S-scores using genes having multiple independent colonies plated in the screening. Clustered heatmap of the whole chemical genomics screen; each subsection belongs to an individual strain's KO library. Grey cells indicate missing values.
Figure 2(A) Schematic depiction of BET1 indicating the position and conservation of the variants identified in this study. TMD = transmembrane domain. (B) Sashimi plots comparing muscle RNA sequencing reads in P1 (red) and two control muscle samples (blue and green) at the exons 3-5 of BET1. (C) Schematics of normal BET1 exon 3-4 splicing (top) and the various aberrant splice products associated with the paternally inherited p.(Asp68His) variant in P1. Splice variant one utilizes normal splicing generating a full-length BET1 protein with the Asp68His substitution. Splice variant two skips exon 4 to non-coding transcript (NR_133908) exon 5, resulting in a shift of the reading frame and a premature termination codon after 18 amino acids. Splice variant three utilizes a cryptic splice acceptor within exon 4, deleting the first 34 nucleotides of exon 4, and producing again a frameshift and a premature termination. The maternally inherited frameshift variant c.134delC introduces a premature stop codon in exon 2 and is predicted to induce nonsense mediated decay (NMD).
Figure 6A, Growth curve of a KRAS-mutant lung cancer PDX (BE564T) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, ***P<0.001 and P＞0.05 (ns) by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (5 mice/group); error bar: SD B, C, Relative tumor volume (B) and weights (C) of PDX (BE564T) tumors after the treatment. *P<0.05 and ***P<0.001 by unpaired two-tailed t-test. (C) Data are the mean of tumor weights of each group (5 mice/group); error bar: SD D, E, H&E and IHC analysis (Ki67 and Caspase-3) of PDX (BE 564T) tumors after the treatment (D). Quantification of the IHC data for the positivity of Ki-67 and Caspase-3 are shown in (E). *P<0.05 and **P<0.01 by two-way ANOVA with Tukey's multiple comparisons test. Data are shown as mean ± SD (error bar) of three independent experiments (n=3). F-K, Growth curves (F, I) of KRAS-mutant lung cancer PDXs (PF139, PF563) treated with vehicle, HCQ (30 mg/kg/day), AZD4547 (10 mg/kg/day) and BI6727 (5 mg/kg/day), alone or in combination. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey's multiple comparisons test. Data are the mean of tumor volume of each group (4 mice/group); error bar: SD Relative tumor volume (G, J) and weights (H, K) of the PDX tumors after the treatment. *P<0.05, **P<0.01, ***P<0.001 and P＞0.05 (ns) by unpaired two-tailed t-test. Data are the mean ± SD (error bar) of tumor weights of each group (4 mice/group).
Identification of patients with a two-exon deletion UQCRH and generation of a mouse model with the equivalent deletion of Uqcrh.Figure 1A Pedigree of the family carrying a two-exon deletion of UQCRH. Affected individuals (black-filled squares) present with episodes of vomiting, metabolic acidosis, high lactate and hypoglycaemia. B Position of the identified UQCRH deletion in the two affected patients. Empty bars, untranslated region (UTR); Filled bars, translated sequence; thin blue lines, intron; red line underline the two deleted exons. C Scheme of the genomic targeting strategy by a Cre recombinase resulting in the deletion of two flanking loxP exons in the Uqcrh gene of the mouse model. SA indicates splice acceptor site; pA, poly-A. D Alignment of amino acids of human UQCRH, human pseudo-gene UQCRHL and mouse Uqcrh. In red the deleted amino acids in human and mouse. The empty box indicates variation in amino acid residues.
Figure 3(A) Chemical structure of MMV007839. The chemical name of MMV007839 is 2-hydroxy-7-methoxy-2-(perfluoroethyl) chroman-4-one and its chemical formula is C12H9F5O4. (B) Cryo-EM density map of pentameric PfFNT viewed from the parasite's cytoplasm. Densities of the five bound MMV007839 within the pentamer are colored red. The five protomers of pentameric PfFNT are colored yellow, slate, orange, purple and gray. (C) Ribbon diagram of the 2.78-Å resolution structure of pentameric PfFNT-MMV007839 viewed from the parasite's cytoplasm. The five protomers of pentameric PfFNT are colored yellow, slate, orange, purple and gray. (D) Ribbon diagram of pentameric PfFNT-MMV007839 viewed from the extracellular side of the parasite. The five protomers of pentameric PfFNT are colored yellow, slate, orange, purple and gray. (E) Ribbon diagram of pentameric PfFNT-MMV007839 viewed from the parasite's membrane plane. The five protomers of pentameric PfFNT are colored yellow, slate, orange, purple and gray. Densities of the five bound MMV007839 are in red meshes. (F) The MMV007839 of PfFNT. The bound MMV007839 is colored green. Density of the bound MMV007839 is in red mesh. Residues involved in forming the inhibitor binding site are colored yellow.
Figure 3A. Knockdown of lnc-AP, as well as the endogenous pep-AP in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. si-NC was the abbreviation of commercial normal control of siRNA. B. CCK-8 assay of inhibition ratio by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA. C. IF detection of H2AFX for DNA damage by L-OHP in two groups of CRC cells with down- or up-regulation of lnc-AP, and nuclei were stained with DAPI (n = 3, biological replicates, mean±SEM, Dunnett-t test). D. Prediction of potential transcription factors for the source gene of lnc-AP by Match. E. PCR confirmation of siRNA of the predicted transcription factors in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. si-NC was the abbreviation of commercial normal control of siRNA. F. PCR analysis of effect of the predicted transcription factors on lnc-AP in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. si-NC was the abbreviation of commercial normal control of siRNA. G. Verification of the interaction between CEBPA and the upstream region of AP002387 via ChIP assay followed by PCR (n = 3, biological replicates, mean±SEM, t test). Data information: * for P < 0.05; ** for P < 0.01 H. PCR analysis of lnc-AP attenuation and restoration in HCT116 and SW480 cells (n = 3, biological replicates, mean±SEM, Dunnett-t test). Data information: * for P < 0.05; ** for P < 0.01; NS for no significance. Control referred to the corresponding plasmid vector and si-NC was the abbreviation of commercial normal control of siRNA. I. WB verification of pep-AP attenuation and restoration in HCT116 cells. J. CCK-8 assay of inhibition ratio by L-OHP in HCT116 and SW480 cells with lnc-AP/pep-AP attenuation and restoration (n = 3, biological replicates, mean±SEM). Data information: * si-NC was the abbreviation of commercial normal control of siRNA.
Figure 1Representative images of colon tumors were shown. Scale bars, 10 mm. Tumor number, tumor size and tumor load in each mouse was measured. Histological analysis of colon tumors was shown by hematoxylin and eosin (HE) staining. Tumors were microscopically analyzed and classified as low or high grade. Histological score was assessed by a pathologist. Scale bars, 25 µm. Tumor tissues were stained for Ki67 and p-STAT3. The percentage of Ki67-positive and p-STAT3-positive cells was quantified. Scale bars, 25 µm. Proportion of CD11b+F4/80+ and RORγt+/CD45+lin- cells was analyzed in colonic LP cells and mLNs among tumor-bearing WT and Dectin-3-/- mice by flow cytometry. Relative expression of Il-6, Il-22, Cxcl1 and Il-17a in LP cells from tumor-bearing WT and Dectin-3-/- mice were detected using qPCR.
Figure 7Violin plots of the log scale normalized expression of LIF receptor subunits, IL6ST (A), LIFR (B), LIF (C), IL6R (D) in all cell types in the human choroid Note the wide distribution of IL6ST (gp130), the common subunit to all receptors of the IL-6 family (A). In contrast, LIFR is strongly expressed in ECs and to a lesser extent in fibroblasts and pericytes (B). LIF is expressed in fibroblasts, macrophages/monocytes and pericytes and minimally in ECs (C). The specific IL-6 receptor (IL6R) is highly expressed in macrophage/monocytes (D), while LIFR has little expression in these cells (B). Violin plots of the log scale normalized expression of LIF receptor subunits KDR (E) in all cell types in the human choroid KDR shows, as expected, high expression selectively in EC (E). (F) Cell clusters in uniform manifold approximation and projection (UMAP) space.
Figure 6(A) A431 WT cells with seipin tagged at its chromosomal locus with sfGFP and stably expressing ER marker plasmid (BFP-KDEL) were delipidated and treated with OA for 20 h, fixed and stained with LipidTox Red. Single confocal plane of an airyscan z-stack. (B) WT and SKO cells were treated with OA for 20 h, fixed and stained with LD540. Maximum projections of deconvolved confocal z-stacks. Dashed lines indicate cell boundaries. (C) Analysis of LD areas of cells treated as in (A) and imaged with widefield microscopy. Bars: % of LDs/field of view in indicated size category, mean ± SEM, n=25-30 fields of view (100-120 ROIs of not clustered LD areas from 100-120 cells), 2 experiments, p**< 0.005 (unpaired T-test). See also Appendix Figure S5D. (D) TEM micrographs of WT and SKO cells treated with OA for 20 h. (E) WT and SKO cells were treated as in (B), LDs were visualized with LD540 and cells imaged live with widefield microscopy. Exemplary trajectories of peripheral LDs tracked for 40 s. (F) Analysis of cells treated and imaged as in (E). Bars: 1 - [Pearson's colocalization coefficient between subsequent frames/cell], ± SEM, n=187-221 cells, 2 experiments, **p<0.005 (Mann Whitney test). (G) WT and SKO cells were treated with OA for 20 h, fixed and stained with anti-ACSL3 antibodies and LD540. Maximum intensity projections of deconvolved confocal z-stacks. Insets show small and large LDs. (H) Analysis of (G). Bars: % of small (diameter <0.5 µm) or large (diameter >0.5 µm) LDs/cell in the indicated category, mean ± SEM, n=10 ROIs from 10 cells/treatment (305-581 LDs), **p<0.005 (unpaired T-test). (I) WT and SKO cells stably expressing HPos-Cherry were incubated with OA for 20 h, and LDs were labelled with LipidTox Green prior to imaging. The HPos-Cherry signal was bleached and signal recovery to LDs was monitored. (J) Analysis of (I). Data is normalized to both pre-bleach and post-bleach intensities, ± SEM, n=51-61 bleached LD areas/genotype, 3 experiments.