EMBO/SourceData huggingface dataset:
Figure 4B. Analysis of FACS data of sorted macrophages shows the relative percentage of CD45+CD11b+F4/80+ cells in each genotype and the absolute number of sorted cells used for RNA sequencing. Representative flow cytometry gating strategy for sorting is shown in Fig EV3B. N=5-9. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test.C. Differential expression of genes was analyzed using the edgeR software package. IPA was performed with filtered lists of up- and downregulated genes that exhibited an FDR-value of <0.1 in different genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt). The number of differentially expressed genes used for analysis is indicated. Data are presented as pseudo-heatmap with magnitudes of Activation Z-scores color-coded as indicated in the legend; all non-grey comparisons are -log10(p-value)>2.5 unless otherwise indicated.D. Gene Set Enrichment Analysis (GSEA) was performed to compare transcriptomes of genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt, [all]/Act vs [all]/wt) to 3 published datasets of Tumor-Associated Macrophages (TAM) (Franklin et al. 2014; Ojalvo et al, 2009; Galletti et al, 2016). Gene sets are composed of top genes upregulated (TAM UP) or downregulated (TAM DOWN) in TAM vs normal tissue macrophages. Positive Normalized Enrichment Score (NES) corresponds to enrichment of gene set in activin-upregulated genes; negative NES corresponds to enrichment in activin-downregulated genes. Data are presented as pseudo-heatmap with magnitudes of NES color-coded as indicated in the legend; all non-grey comparisons are FDR<0.10.E. Differential GSEA was performed on combined genotype comparison [all]/Act vs [all]/wt and two datasets of human AK vs normal skin (GSE2503; Nindl et al, 2006) and GSE63107 to compare these transcriptomes to gene sets derived from top upregulated genes in these datasets (top); and gene sets derived from top upregulated genes in datasets of differential human macrophage activation (IFN-γ, TNF-α, LPS, IL-4 vs non-treated) and selected human myeloid/lymphoid cell type-specific gene expression profiles (macrophage vs monocyte, dendritic cell, and T-cell) (Xue et al, 2014; bottom). Activin target genes enriched in the AK transcriptomes are shown (right). Data are presented as pseudo-heatmaps with magnitudes of Normalized Enrichment Scores (NES) and Log2(Fold Change) color-coded as indicated in the respective legends; all non-grey comparisons are FDR<0.05 for NES and p<0.10 for Fold Change.G. Heatmap of 28 differentially expressed genes (FDR≤0.05, |log2Ratio|≥1) overlapping in all 3 comparisons (wt/Act vs wt/wt mice, HPV8/Act vs HPV8/wt and HPV8/Act vs wt/wt). Genes with a published pro-tumorigenic function are highlighted in red.
Figure 5A,B,F. Expression of Cx3cr1 (A), Spp1 (B) relative to Rps29 was analyzed by qRT-PCR in peritoneal macrophages, resident or inflammatory, treated in vitro with 10 ng/ml of activin A (Act) or untreated (Ctrl, arbitrarily set to 1). Results are combined from 2-5 independent experiments performed with cells pooled from at least 5 wt mice.C,D. Western blot analysis of total ear protein lysates of 12-14 week-old mice for osteopontin (OPN) and GAPDH (C). Each lane represents an individual mouse (n=3). (D) Band intensities were quantified and normalized to GAPDH. The expression level in one of the wt mice was arbitrarily set to 1.E. Ear skin sections from 10 week-old mice stained with anti-CD68 (red) or anti-osteopontin antibody (green); counterstained with Hoechst (blue). Osteopontin-positive macrophages in wt/Act and HPV8/Act mice are indicated with arrows. Representative of N=2 wt/wt and N=3 HPV8/wt, wt/Act and HPV8/Act mice. Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.A,B,F. Expression of Arg1 (F) relative to Rps29 was analyzed by qRT-PCR in peritoneal macrophages, resident or inflammatory, treated in vitro with 10 ng/ml of activin A (Act) or untreated (Ctrl, arbitrarily set to 1). Results are combined from 2-5 independent experiments performed with cells pooled from at least 5 wt mice.G. In situ gelatin zymography (top panel) and methylene blue staining (bottom panel) of the ear skin from 10 week-old mice. Representative images of 2-3 mice per genotype are shown. Scale bar = 500μm.H,I. Gelatin zymography of ear skin lysates from 10 week-old mice. Inverted image of a representative gel with 2 (wt/wt) or 3 (HPV8/wt, wt/Act, HPV8/Act) mice per genotype is shown in (H), quantification of the results obtained in 2 independent experiments is shown in (I). N=5 wt/wt, N=6 HPV8/wt, wt/Act and HPV8/Act mice.J. Representative pictures of ear skin from 10 week-old mice stained for MECA32 (red), LYVE1 (green); counterstained with Hoechst (blue). Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.K,L. Quantification of MECA32+ area (K) and average distance between MECA32+ vessels and basement membrane (BM) (L).
Figure 6A. Kinetics of tumor incidence in HPV8/Act mice treated for 5 weeks with 50mg/kg anti-CSF1R antibody (AFS98) or with control rat IgG. N=10 mice per treatment group.B. Representative pictures of anti-CD68- or CD206-stained cryosections of ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R antibody or control rat IgG. Scale bar = 100μm.C. Quantification of the CD68+ or CD206+ area relative to total dermis area. N=10 mice per treatment group.D. Representative pictures of anti-BrdU (green) and anti-K14 (red) stained cryosections of ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R antibody or control rat IgG. Scale bar: 50μm.E. Quantification of BrdU+ keratinocytes per mm of basement membrane. N=8 anti-CSF1R-injected mice; N=4 rat IgG-injected mice.F. Expression of Mmp12 relative to Rps29 was analyzed by qRT-PCR in total ear skin from mice treated with anti-CSF1R antibody or with rat IgG. Expression in one of the IgG-injected mice was set to 1. N=10 per group.G. Representative pictures of the ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R or control rat IgG, stained for CD31 (red), LYVE1 (green), and counterstained with Hoechst (blue). Boxed areas are shown at higher magnification in the top-right corner. Scale bar: 100μm.H,I. Quantification of CD31+LYVE1- area (H) and number of CD31+LYVE1-vessels (I).
Figure 1Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046) worms on partial lawn of P. aeruginosa at 25°C.   Kaplan Meier survival curve of N2, odr-3(n2150) and odr-3(n2046)   worms on full lawn of P. aeruginosa at 25°C.
Figure 2Chemotactic response of N2 worms to varying dilutions of 1-undecene. n ≥ 3 assays. Error bars indicate SEM.(i) Schematic of setup used to record the movement of 5 worms each under control and 1-undecene odor exposure. (ii) Movement trajectories of N2 worms on E. coli OP50 lawn exposed to 1-undecene odor for 30 min and 60 min, each color represents the trajectory of single worm. Scale bar = 10 mm.
Figure 3Average calcium responses of AWB::GCaMP3 worms exposed to 1-undecene at indicated concentrations. Each worm was recorded for 180 s. Worms were under stimulus between 11 s-130 s, window shown in grey, and stimulus withdrawal beyond 130 s. Shaded regions around the curves represent SEM. n = 3. Two dashed box represents 10 s time window after stimulus addition and stimulus removal respectively.
Figure 4Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, odr-3(n2150) and odr-3(n2046) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01 as determined by two-tailed unpaired t-test. Error bars indicate SEM. Real time PCR analysis of irg-1, irg-2 and irg-3 genes in N2, lim-4(ky403) and lim-4(yz12) worms exposed to 1-undecene odor upon respective naive worms. n = 3. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 as determined by two-tailed unpaired t-test. Error bars indicate SEM.Kaplan Meier survival curve on the full lawn of P. aeruginosa for N2 (naive) worms and N2 worms pre exposed to 1-undecene odor. Survival assay was performed at 20°C.
Figure 1(A) Death rate versus pre-starvation growth rate on a semilog-plot. CARLOS conditions are defined in legend. CARLOS conditions that perturb nutrient quality (C, A, L and S) lead to an exponential increase of death rate. Conditions that stress the proteome (R and O) show an orthogonal exponential increase with slower growth. Each data point is a single experiment. Chemostat (C) and parts of (L) are from Ref. (Biselli et al., 2020). See Dataset EV1 for data.(C) Z-values are merged into a single 'Survival score' for each protein that measures correlation of abundance with survival across all experiments. A detailed description of the construction of the score is given in Box 1. Several GO-processes are significantly enriched (FDR levels of the Benjamini-Hochberg step-up procedure) towards higher scores. ∆Survival score is calculated as the difference in median Survival score between a process and the background of all proteins.
Figure 2(B) Knock-out of several genes with different functionalities in the cell envelope increase death rates. Growth in glycerol minimal medium, starvation induced by glycerol depletion.(C) 8 µg/ml Polymyxin B and 4 µg/ml Colistin, added daily to cultures previously grown on glucose minimal medium (reference condition) with the first dose at 24h. Antibiotic concentration was 10x minimum inhibition concentration during growth (MIC). Each data point is the average of two biological repeats. Error bars are not shown because the variation is smaller than symbol size. Viabilities indicated at <101 mean no colonies were detected from 100 µl culture.(D) Death rates for different Mg2+ concentrations. Reference condition (black circle in Fig. 1A) has 0.41 mM Mg2+. DNP+ cultures were supplemented with 1 mM 2,4-dinitrophenol during starvation in reference medium. Each data point is from a single experiment. All cultures were grown in glucose minimal medium. P-values (Student's t-test, two-tailed) are indicated in comparison to the reference condition.
Figure 3Death rate plotted versus growth rate on a semi-log scale for experiments in 'osmo-balanced' medium. Colors indicate carbon limitation (C, 'blue', acetate and glycerol), anabolic limitation (A, 'green', 3µM IPTG), ribosome limitation (R, 'yellow', 4µM Chloramphenicol), rich medium (L, 'red', LB), LacZ overexpression (O, 'grey', 10ng/ml cTc), stationary phase (S, 'white') and the glucose reference condition ('black'), as defined in Fig. 1A. The data shows weak anti-correlation with growth rate; solid black line is an exponential fit to the data (R2 = 0.34). Dashed lines are the exponential fits to the CARLOS data in Fig. 1A.
Figure 1Proteins and (C) peptides identified for platelets, erythrocytes, platelet-rich and platelet-free plasma.Selection of the most suitable quality marker proteins for platelet contamination (blue dots) and (E) erythrocyte contamination (red dots) based on their abundance, the platelet/erythrocyte to plasma ratio and the coefficient of variation. Proteins that were only detected in platelets or erythrocytes, but not in plasma are aligned on the right side of the graph.
Figure 2Protein intensities were Z-scored across the dilution series for the 29 quality markers of the erythrocyte panel and (C) for the 29 markers of the platelet panel as a function of their spike-in proportion to plasma. Whiskers indicate 10-90 percentiles and horizontal lines denote the mean. (D) Correlation of erythrocyte count to the 'contamination index' for the erythrocyte marker panel. (E) Correlation of platelet count to contamination index for the platelet marker panel.
Figure 3Volcano plot comparing 72 plasma vs. 72 serum proteomes. Proteins highlighted in yellow were chosen according to their p-value as markers for coagulation. Only the plasma enriched proteins (compared to serum) were used in the calculation of the coagulation contamination index. Ratio of the summed intensities of all plasma or serum proteins to the sum of the plasma-enriched panel proteins are plotted for all samples. Whiskers indicate the 10-90 percentile.
Figure 4Volcano plot of the proteome comparison of time point 1 vs 4. Proteins of the platelet panel are highlighted in blue and two additional significantly regulated proteins in red.Global correlation map on the left with an inset of the platelet cluster on the right. The two significant outliers of the volcano plot in (B) are marked in red. Platelet panel proteins are highlighted in blue in the inset. Red patches in the global correlation map indicate positive and blue patches negative correlations.
Figure 2A. ReDDM activation in Dronc heterozygous (+/-) intestine 7 days after temperature shift at 29 Co; esg expression (green) labels the intestinal progenitor cells, Histone-RFP (red) is a semi-permanent marker retained in differentiated cells and Pdm-1 (grey) labels differentiated ECs. The red arrows indicates the enlarged areas depicted in the insets in the entire figure. DAPI labels the DNA In the entire Figure (blue). All of the experiments in the entire figure have been made under experimental conditions that protect the epithelial integrity (intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food).B. ReDDM activation in a Drosophila intestine in which all of the progenitor cells are fully mutant for Dronc (see full genotype in methods); note the increased cell size of esg-expressing cells (GFP+, green), the co-expression of Histone-RFP (red) and the EC marker Pdm-1 (gray), and the coexpression of Pdm-1 and GFP in enlarged cells (compare panels and detailed insets from A with B). The image is a representative example of a Drosophila adult gut 7 days after temperature shift at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-CherryC. Relative number of esg-expressing cells normalised to DAPI; notice that the relative percentage of esg-labelled cells is significantly higher in Dronc fully mutant conditions (-/-) at 3 days (***; P = 0.0007) and 7d (****; p <0.0001) post temperature shift at 29 Co (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t-test, +/- n=32, -/- n=25). D. Average cell size of esg-expressing cells (μm²); note the increased cell size of Dronc fully mutant progenitor cells (-/-) at 3d (**; P = 0.0014) and 7d (***, P = 0.0007) post temperature shift at 29 Co (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed parametric t-test, 3d +/- n = 19, 3d -/- n = 28, 7d +/- n = 29, 7d -/- n = 28). E. Relative number of esg-negative cells expressing Histone-RFP normalised to DAPI; notice that the number of Histone-RFP cells without esg expression is significantly higher in Dronc fully mutant (-/-) conditions at 7d (*; P = 0.0046) (Quantifications were made using N biological replicates ≥ 2; Mann-Whitney test, +/- n = 24, -/- n = 20.
Figure 4A. Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes), Prospero (red nuclei of EEs) and Dronc-TurboID (green shows Streptavidin labelled proteins by Dronc-TurboID); note the preferential enrichment of Streptavidin staining is preferentially enriched in the intestinal progenitor cells (white arrows indicate an intestinal progenitor cell upregulating Dronc).Representative image of a Drosophila posterior midgut showing the expression of Armadillo (red membranes) and Actin-Dronc-GFP-Myc (green, anti-GFP); note the preferential enrichment of Dronc expression in the small groups of cells formed by intestinal progenitor cells (arrow). High magnification of the picture shown in B. The white arrow indicates a presumptive ISC showing low levels of Dronc compared to the flanking GFP+ EBs. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc. Transversal Z-section of the cells shown in C. The arrow indicates a presumptive small ISC expressing high levels of Arm and low levels of Dronc-GFP-Myc.E. The enteroblast marker Su(H) (red, immunostaining against Beta-galactosidase) strongly co-localises with high levels of Dronc expression (green, immunostaining against GFP); white arrow indicates the enlarged area depicted in the insets.F. There is extensive overlap between the expression of the EB marker Su(H) (green, immunostaining against Beta-galactosidase; white arrow) and the apical caspase reporter DBS-S-QF (red, immunostaining against HA), but it is also present in progenitor cells without Su(H) expression (yellow arrowheads). DAPI (Blue) labels cell nuclei in all the panels.
Figure 3a. Lipid droplet time course. Representative photomicrographs showing lipid droplet abundance in ARPE19 cells treated with DFP starting at 7 h and maximal at 24 h. Neutral lipids were revealed by BODIPY 493/503. Nuclei are counterstained with Hoescht33342. Inset shows detail of LDs in a cell treated with DFP for 24 h. Arrows highlight LD accumulation. Scale bars =5 μm.b. Mitophagy time course. Representative photomicrographs showing time course of DFP treatment in mitophagy reporter cells (human ARPE-19 cells with stable expression of mito-QC). Mitochondrial networks are visible in yellow and mitolysosomes as red-only puncta signifying mitophagy. Nuclei are counterstained with Hoescht33342. Mitophagy appears maximal after 24 h of DFP treatment, with little difference between control and 7 h conditions. Inset shows details of mitolysosomes within the mitochondrial network of a highly mitophagic cell following DFP treatment. Arrows highlight mitolysosomes. Scale bar = 5 μm.c. Lipid droplets accumulate at mitochondria when cells are treated with DFP. Representative photomicrographs showing lipid droplet and mitochondria in ARPE19 cells treated with DFP at 7 h and at 24 h. Neutral lipids were revealed by BODIPY 493/503 and mitochondria by Mitotracker. Nuclei are counterstained with Hoescht33342. Inset shows detail of LDs in close proximity to mitochondria in cells treated with DFP for 24 h. Scale bar = 5 μm.d. Quantification of lipid droplet area per cell over time. Lipid droplet area was quantified per cell area using a pipeline generated in CellProfiler. n=3 with at least 50 cells quantified per biological replicate for each condition. Data represent means ±SEM.e. Quantification of mitophagy over time. Mitophagy index per cell is measured as a ratio of the number of mitolysosomes per mitochondrial content. n=3 with at least 50 cells quantified per biological replicate for each condition. Data represent means ±SEM.f. Quantification of lipid droplet and mitochondria proximity over time. n=2 with at least 50 cells quantified per biological replicate for each condition. Ratios between Control and 24h DFP; P<0.05.
Figure 4d. DFP-induced Lipid droplet accumulation is dependent on DGAT1. Representative photomicrographs of ARPE19 cells treated with DFP (1mM) for 7 h followed by treatment with LD inhibitors (5 μM) as indicated. Cells were fixed after 24 h of DFP treatment. Neutral lipid droplets were stained by BODIPY493/503 and imaged by confocal microscopy after 24 h of DFP treatment ('i' denotes the presence of inhibitor e.g. DGAT1i = DGAT1-inhibition). Scale bar = 5 μm.e. Quantitation of lipid droplets area per cell from experiments in 2d. Data are represented as mean ± SEM (n=3 with at least 80 cells analysed per condition in each biological replicate).
Figure 5a. ARPE-19 ULK1 CRISPR KO cells with/without re-introduction of FLAG-ULK1 were treated with DFP for 24h as indicated. Cells were fixed and stained with BODIPY493/503 to visualise lipid droplets (green) or DAPI to visualise nuclei (blue). Scale bar, 10 μm.b. Quantification from 5a, n=3 independent experiments, statical analysis by one-way ANOVA. *=P<0.05; **P=<0.01. Median represented by dashed line, quartiles by dotted lines.c. Authentication of cell lines in 5a by immunoblot of protein lysates.
Figure 6f. Representative confocal photomicrographs showing the pattern of endogenous mitochondrial NIX in human ARPE-19 cells treated with DFP ±DGAT1i/2i. Samples were immunolabelled with primary antibodies to NIX (green) and ATPB (red). Scale bar = 2 μm. g. Representative confocal photomicrograpghs of Cathepsin-reactive endolysosomes in human ARPE-19 cells treated with with DFP ±DGAT1i/2i. Inset shows examples of lysosomal phenotypes. Scale bar = 5 μm. h. Colocalization quantitation for experiments shown in 6g. One-way ANOVA with Bonferroni's post hoc test. ****P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates. i. Colocalization quantitation for ULK1/WIPI2 immunolabelling in human ARPE-19 cells treated with DFP ±DGAT1i/2i. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates. j. Quantitation of lysosome positioning in DFP ±DGAT1i/2i. One-way ANOVA with Bonferroni's post hoc test. ****=P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates.
Figure 7c. Representative photomicrographs of larval neuronal cell bodies expressing the matrix-QC reporter and RNAi for mdy or control driven with nSyb-GAL4. GFP is shown in green, mCherry is shown in magenta. Mitolysosomes (mCherry-only puncta) are shown in greyscale. Scale bar = 5 μm.d. Quantitative analysis of mitophagy (mitolysosome per neuronal cell body). Data are shown as violin plot with median (dashed line) and quartile range (dotted lines); n = 5-6 animals, with 23-40 cells per animal. One-way ANOVA with Šidák's post-test correction for multiple samples; **=P<0.01
Figure 2B Average seizure score of KA-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h. * p < 0.05 compared to WT mice (two-way Repeated Measures ANOVA; interaction effect of time and genotype; F1,666 = 10.05, p = 0.0016). KA-treated WT mice (n=16), KA-treated Fxr2 KO mice (n=12). Data are presented as mean ± Standard Error of the Mean (SEM). D Average seizure score of pilocarpine-treated WT and Fxr2 KO mice over time from t=0 (injection time) to 2 h (two-way Repeated Measures ANOVA). Pilocarpine-treated WT mice (n=6), pilocarpine-treated Fxr2 KO mice (n=6). Data are presented as mean ± SEM.
Figure 3B PSD-95 mRNA had lower read counts in Fxr2 KO samples (n=3) compared to WT samples (n=3) (+ adjusted p < 0.1, two-tailed Student's t-test). Data are presented as mean ± SEM.
Figure 5A Representative Western blot and quantification of GluK5, PSD-95, GluN2B and mGluR5 proteins. Endogenous GluK5, PSD-95, GluN2B and mGluR5 in Fxr2 KO mice (n=9) compared to WT mice (n=11). * p < 0.05, ** p < 0.01, significant difference between WT and Fxr2 KO (two-tailed Student's t-test: GluK5: t18 = 0.87, p = 0.394; PSD-95: t18 = 2.29, p < 0.05; GluN2B: t18 = 3.09, p < 0.01; mGluR5: t18 = 3.74, p < 0.01). Quantified bands are highlighted with an asterisk or squared bracket. Data are presented as mean ± SEM.
Figure 6A Bar plot showing phospho-ERK1/2 over total ERK1/2 levels in vehicle- and KA-treated WT (closed and open black circles, vehicle-treated WT n=6; KA-treated WT n=6) and Fxr2 KO mice (closed and open magenta circles, vehicle-treated Fxr2 KO n=5; KA-treated Fxr2 KO n=6). *** p < 0.001 comparison between vehicle- and KA-treated WT (two-way ANOVA: interaction effect between genotype and treatment: F1,19 = 24.66, p < 0.001). Data are presented as mean ± SEM. B Phospho-ERK1/2 levels in vehicle- and pilocarpine-treated WT (n=4-6) and Fxr2 KO mice (n=4-6). Both genotypes responded with long-lasting seizures upon pilocarpine administration * p < 0.05 (two-way ANOVA: main effect for treatment: F1,16 = 10.98, p < 0.01). Data are presented as mean ± SEM.