A , B . Representative pictures of ear skin cryosections from 10 - 12 week - old mice stained with anti - CD206 antibodies ( green ) ; counterstained with Hoechst ( blue ) ( A ) , of CD206 + as a of total dermal ( B ) in wt / wt ( N = 3 ) , HPV8 / wt ( N = 7 ) , wt / Act ( N = 2 ) or HPV8 / Act ( N = 8 ) mice . basement is indicated with a dotted .

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A,B. Representative pictures of ear skin cryosections from 10-12 week-old mice stained with anti-CD206 antibodies (green); counterstained with Hoechst (blue) (A), and quantification of CD206+ area as a percentage of total dermal area (B) in wt/wt (N=3), HPV8/wt (N=7), wt/Act (N=2) or HPV8/Act (N=8) mice. The basement membrane is indicated with a white dotted line.


C . Abundance of MerTK + CD64 + macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12 - 16 week - old mice . N = 5 - 6 . Representative flow cytometry scatter plots are shown in Appendix Fig S1D .

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C. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12-16 week-old mice. N = 5-6. Representative flow cytometry scatter plots are shown in Appendix Fig S1D.


D . of CD206 + as a of total dermal in wt ( N = 7 ) Act ( N = 6 ) mice in back skin treated 8x with TPA ( Antsiferova et al , 2011 ) .

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D. Quantification of CD206+ area as a percentage of total dermal area in wt (N=7) and Act (N=6) mice in back skin treated 8x with TPA (Antsiferova et al, 2011).


E . Abundance of MerTK + CD64 + macrophages analyzed by flow cytometry of pre - tumorigenic ear skin from 12 week - old mice expressing CCR2 - CFP - DTR transgene ( wt / CCR2 ) or double - transgenic ( Act / CCR2 ) , untreated or treated with diphtheria toxin ( + DT ) 3 times every 48h analyzed 24h after last injection . of CD45 + CD11b + MerTK + CD64 + macrophages among live is shown . N = 3 control wt / CCR2 Act / CCR2 mice ; N = 4 DT - injected wt / CCR2 Act / CCR2 mice . Representative flow cytometry scatter plots are shown in Appendix Fig S1E .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O

E. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of pre-tumorigenic ear skin from 12 week-old mice expressing the CCR2-CFP-DTR transgene (wt/CCR2) or double-transgenic animals (Act/CCR2), untreated or treated with diphtheria toxin (+DT) 3 times every 48h and analyzed 24h after the last injection. Percentage of CD45+CD11b+MerTK+CD64+ macrophages among live cells is shown. N=3 control wt/CCR2 and Act/CCR2 mice; N=4 DT-injected wt/CCR2 and Act/CCR2 mice. Representative flow cytometry scatter plots are shown in Appendix Fig S1E.


F , G . Inflammatory thioglycollate - elicited peritoneal macrophages ( F ) or mouse peripheral blood monocytes ( G ) were isolated from wt mice allowed to migrate for 24h in a Transwell . Activin A 0 . 1ng / ml ( A0 . 1 ) , 1ng / ml ( A1 ) , or 10ng / ml ( A10 ) was added to lower well . Alternatively , 10ng / ml was added to lower well insert ( A10 / A10 ) . Random migration to medium ( ctrl ) was set to 1 for each mouse ; fold over control is shown . Log - transformed data were used for statistical . Results in ( F ) are representative of 4 independent experiments performed with 8 mice in total ( results obtained in one experiment performed with 3 mice are shown ) . Results in ( G ) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice .

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F, G. Inflammatory thioglycollate-elicited peritoneal macrophages (F) or mouse peripheral blood monocytes (G) were isolated from wt mice and allowed to migrate for 24h in a Transwell assay. Activin A 0.1ng/ml (A0.1), 1ng/ml (A1), or 10ng/ml (A10) was added to the lower well. Alternatively, 10ng/ml was added to the lower well and the insert (A10/A10). Random migration to medium (ctrl) was set to 1 for each mouse; fold over control is shown. Log-transformed data were used for statistical analysis. Results in (F) are representative of 4 independent experiments performed with 8 mice in total (results obtained in one experiment performed with 3 mice are shown). Results in (G) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice.


B . of FACS data of sorted macrophages shows relative of CD45 + CD11b + F4 / 80 + in each genotype absolute number of sorted used for sequencing . Representative flow cytometry gating strategy for sorting is shown in Fig EV3B . N = 5 - 9 . Statistical significance was determined using 1 - way ANOVA Bonferroni ` s Multiple Comparisons .

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B. 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 edgeR software package . IPA was performed with filtered lists of up - 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 ) . number of differentially expressed genes used for is indicated . Data are presented as pseudo - with magnitudes of Activation Z - scores color - coded as indicated in legend ; non - grey comparisons are - log10 ( p - value ) > 2 . 5 unless otherwise indicated .

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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 ( GSEA ) was performed to compare transcriptomes of genotype comparisons ( wt / Act vs wt / wt ; HPV8 / Act vs HPV8 / wt ; HPV8 / Act vs wt / wt , [ ] / Act vs [ ] / wt ) to 3 published datasets of - 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 - with magnitudes of NES color - coded as indicated in legend ; non - grey comparisons are FDR < 0 . 10 .

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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 [ ] / Act vs [ ] / wt two datasets of human AK vs normal skin ( GSE2503 ; Nindl et al , 2006 ) GSE63107 to compare these transcriptomes to gene sets derived from top upregulated genes in these datasets ( top ) ; gene sets derived from top upregulated genes in datasets of differential human macrophage activation ( IFN - γ , TNF - α , LPS , IL - 4 vs non - treated ) selected human myeloid / lymphoid type - specific gene expression profiles ( macrophage vs monocyte , dendritic , T - ) ( Xue et al , 2014 ; bottom ) . Activin target genes enriched in AK transcriptomes are shown ( right ) . Data are presented as pseudo - heatmaps with magnitudes of Normalized Enrichment Scores ( NES ) Log2 ( Fold Change ) color - coded as indicated in respective legends ; non - grey comparisons are FDR < 0 . 05 for NES p < 0 . 10 for Fold Change .

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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 . of 28 differentially expressed genes ( FDR ≤ 0 . 05 , | log2Ratio | ≥ 1 ) overlapping in 3 comparisons ( wt / Act vs wt / wt mice , HPV8 / Act vs HPV8 / wt HPV8 / Act vs wt / wt ) . Genes with a published pro - tumorigenic function are highlighted in red .

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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.


A , 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 pooled from at least 5 wt mice .

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A,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 of total ear lysates of 12 - 14 week - old mice for osteopontin ( OPN ) GAPDH ( C ) . Each lane represents an mouse ( n = 3 ) . ( D ) Band intensities were quantified normalized to GAPDH . expression level in one of wt mice was arbitrarily set to 1 .

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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 HPV8 / Act mice are indicated with arrows . Representative of N = 2 wt / wt N = 3 HPV8 / wt , wt / Act HPV8 / Act mice . Boxed areas in top panel are shown at higher magnification in bottom panel . Scale bar top : 100μm , bottom : 20μm .

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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 pooled from at least 5 wt mice .

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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 ) methylene blue ( bottom panel ) of ear skin from 10 week - old mice . Representative of 2 - 3 mice per genotype are shown . Scale bar = 500μm .

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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 ) , of results obtained in 2 independent experiments is shown in ( I ) . N = 5 wt / wt , N = 6 HPV8 / wt , wt / Act HPV8 / Act mice .

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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 . of MECA32 + ( K ) average between MECA32 + vessels basement ( BM ) ( L ) .

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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).


A . Kinetics of incidence in HPV8 / Act mice treated for 5 with 50mg / kg anti - CSF1R antibody ( AFS98 ) or with control rat IgG . N = 10 mice per treatment group .

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A. 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 with anti - CSF1R antibody or control rat IgG . Scale bar = 100μm . C . of CD68 + or CD206 + relative to total dermis . N = 10 mice per treatment group .

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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 ) anti - K14 ( red ) stained cryosections of ear skin from 10 week - old mice treated for 5 with anti - CSF1R antibody or control rat IgG . Scale bar : 50μm . E . of BrdU + keratinocytes per mm of basement . N = 8 anti - CSF1R - injected mice ; N = 4 rat IgG - injected mice .

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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 IgG - injected mice was set to 1 . N = 10 per group .

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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 ear skin from 10 week - old mice treated for 5 with anti - CSF1R or control rat IgG , stained for CD31 ( red ) , LYVE1 ( green ) , counterstained with Hoechst ( blue ) . Boxed areas are shown at higher magnification in top - right corner . Scale bar : 100μm . H , I . of CD31 + LYVE1 - ( H ) number of CD31 + LYVE1 - vessels ( I ) .

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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).


Kaplan Meier survival curve of N2 , odr - 3 ( n2150 ) odr - 3 ( n2046 ) worms on partial lawn of P . aeruginosa at 25 ° C . Kaplan Meier survival curve of N2 , odr - 3 ( n2150 ) odr - 3 ( n2046 ) worms on full lawn of P . aeruginosa at 25 ° C .

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Kaplan 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.


Chemotactic response of N2 worms to varying dilutions of 1 - undecene . n ≥ 3 assays . Error bars indicate .

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Chemotactic response of N2 worms to varying dilutions of 1-undecene. n ≥ 3 assays. Error bars indicate SEM.


( i ) Schematic of setup used to record movement of 5 worms each under control 1 - undecene odor exposure . ( ii ) Movement trajectories of N2 worms on E . coli OP50 lawn exposed to 1 - undecene odor for 30 min 60 min , each color represents trajectory of single worm . Scale bar = 10 mm .

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(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.


Average 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 , stimulus withdrawal beyond 130 s . Shaded regions around curves represent . n = 3 . Two dashed box represents 10 s time window after stimulus addition stimulus removal respectively .

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Average 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.


Real time PCR of irg - 1 , irg - 2 irg - 3 genes in N2 , odr - 3 ( n2150 ) 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 - unpaired t - . Error bars indicate . Real time PCR of irg - 1 , irg - 2 irg - 3 genes in N2 , lim - 4 ( ky403 ) 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 - unpaired t - . Error bars indicate .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Real 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 full lawn of P . aeruginosa for N2 ( naive ) worms N2 worms pre exposed to 1 - undecene odor . Survival was performed at 20 ° C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


( A ) 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 S ) lead to an exponential increase of rate . Conditions that stress proteome ( R O ) show an orthogonal exponential increase with slower growth . Each data point is a single experiment . Chemostat ( C ) parts of ( L ) are from Ref . ( Biselli et al . , 2020 ) . See Dataset EV1 for data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 that measures correlation of abundance with survival across experiments . A detailed description of construction of score is given in Box 1 . Several GO - processes are significantly enriched ( FDR of Benjamini - Hochberg step - up procedure ) towards higher scores . ∆ Survival score is calculated as difference in median Survival score between a process background of .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( B ) Knock - out of several genes with different functionalities in envelope increase rates . Growth in glycerol minimal medium , starvation induced by glycerol depletion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 4 µg / ml Colistin , added daily to cultures previously grown on glucose minimal medium ( reference condition ) with first dose at 24h . Antibiotic was 10x minimum inhibition during growth ( MIC ) . Each data point is average of two biological repeats . Error bars are not shown because variation is smaller than symbol . Viabilities indicated at < 101 mean no colonies were detected from 100 µl culture .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) 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 . cultures were grown in glucose minimal medium . P - values ( Student ' s t - , two - ) are indicated in comparison to reference condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


rate plotted versus growth rate on a semi - log scale for experiments in ' osmo - balanced ' medium . Colors indicate carbon limitation ( C , ' blue ' , acetate 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 , ' ' ) glucose reference condition ( ' black ' ) , as defined in Fig . 1A . data shows weak anti - correlation with growth rate ; solid black is an exponential fit to data ( R2 = 0 . 34 ) . Dashed lines are exponential fits to CARLOS data in Fig . 1A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Death 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.


( C ) peptides identified for platelets , erythrocytes , platelet - rich platelet - free plasma .

Proteins and (C) peptides identified for platelets, erythrocytes, platelet-rich and platelet-free plasma.


Selection of most suitable quality marker for platelet contamination ( blue dots ) ( E ) erythrocyte contamination ( red dots ) on their abundance , platelet / erythrocyte to plasma coefficient of variation . that were only detected in platelets or erythrocytes , but not in plasma are aligned on right side of graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


intensities were Z - scored across dilution series for 29 quality markers of erythrocyte panel ( C ) for 29 markers of platelet panel as a function of their spike - in proportion to plasma . Whiskers indicate 10 - 90 percentiles horizontal lines denote mean . ( D ) Correlation of erythrocyte to ' contamination index ' for erythrocyte marker panel . ( E ) Correlation of platelet to contamination index for platelet marker panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Protein 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.


Volcano plot comparing 72 plasma vs . 72 serum proteomes . highlighted in yellow were chosen according to their p - value as markers for coagulation . Only plasma enriched ( compared to serum ) were used in calculation of coagulation contamination index . of summed intensities of plasma or serum to sum of plasma - enriched panel are plotted for samples . Whiskers indicate 10 - 90 percentile .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Volcano 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.


Volcano plot of proteome comparison of time point 1 vs 4 . of platelet panel are highlighted in blue two additional significantly regulated in red .

O O O O O O O O O O O O O O O O O O O O O O O O O O

Volcano 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 left with an inset of platelet cluster on right . two significant outliers of volcano plot in ( B ) are marked in red . Platelet panel are highlighted in blue in inset . Red patches in global correlation map indicate positive blue patches negative correlations .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


. A representative example of a 7 days ( 7d ) old adult posterior midgut at low ( A ) high magnification ( B , Region 4 - Region 5 ) showing initiator caspase reporter DBS - S - QF ( Red , immunostaining anti - HA ) . Flies were reared under experimental regime that protects intestinal epithelial integrity ( intestine reared in Oxford Medium flies transferred every two days into vials with fresh food , see Figure EV1 ) at 25 Co . image on left was acquired at low magnification with 10x objective , while right panel was acquired using 40x . left right in examples correspond to independent intestines of same genotype treatment . Genotype : y1 w1118 UAS - mCD8 : : GFP . L QUAS - mtdTomato - 3xHA Act - DBS - S - QF ( BL83131 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

. A representative example of a 7 days (7d) old adult female posterior midgut at low (A) and high magnification (B, Region 4-Region 5) showing the initiator caspase reporter DBS-S-QF (Red, immunostaining anti-HA). Flies were reared under experimental regime that protects the intestinal epithelial integrity (intestine reared in Oxford Medium and flies transferred every two days into vials with fresh food, see Figure EV1) at 25 Co. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. The left and right images in all examples correspond to independent intestines of the same genotype and treatment. Genotype: y1 w1118 UAS-mCD8::GFP.L QUAS-mtdTomato-3xHA Act-DBS-S-QF (BL83131)


B . A representative example of a 7 d intestine reared at 29 Co in experimental conditions that protect epithelial integrity labelled with ReDDM - lineage tool . esg ( green signal ) labels intestinal progenitor , whilst Histone - RFP ( red signal ) acts as a semi - permanent marker that allows visualization of differentiated . Note extensive overlap between two markers absence of differentiated only showing Histone - RFP labelling , as an indication of negligible epithelial turnover . image on left was acquired at low magnification with 10x objective , while right panel was acquired using 40x . dotted outlines gut using DAPI ( not shown ) as a reference . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 C . of intestinal subpopulations labelled with ReDDM system at high magnification ( GFP Histone - RFP ) different time points post ReDDM activation ( 3 - 7 - days ) in experimental conditions that protect epithelial integrity ; note that none of populations in gut ( GFP ( n . s . no significant , P = 0 . 5267 ) or Histone - RFP ( n . s . , P = 0 . 2752 ) significantly increase in number overtime ( Quantifications were made using N ≥ 2 biological replicates , total number of guts analysed at 3 days n = 34 at 7 days n = 45 ; Mann - Whitney , C ) . Error bars represent Standard Error of Mean . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

B. A representative example of a 7 d intestine reared at 29 Co in experimental conditions that protect the epithelial integrity and labelled with ReDDM cell-lineage tool. esg (green cytoplasmic signal) labels intestinal progenitor cells, whilst Histone-RFP (red nuclear signal) acts as a semi-permanent marker that allows the visualization of differentiated cells. Note the extensive overlap between the two markers and the absence of differentiated cells only showing the Histone-RFP labelling, as an indication of negligible epithelial turnover. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. White dotted line outlines the gut using DAPI staining (not shown) as a reference. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019 C. Quantification of intestinal cell subpopulations labelled with ReDDM system at high magnification (GFP and Histone-RFP) and different time points post ReDDM activation (3- and 7-days) in experimental conditions that protect the epithelial integrity; note that none of the cell populations in the gut (GFP (n.s. no significant , P = 0.5267) or Histone-RFP (n.s. , P =0.2752) significantly increase in number overtime (Quantifications were made using N ≥ 2 biological replicates, total number of guts analysed at 3 days n =34 and at 7 days n=45; Mann-Whitney, C). Error bars represent Standard Error of the Mean. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019)


D . A representative example of an adult posterior midgut at low ( A ) high magnification ( B , Region 4 - Region 5 ) showing initiator caspase reporter DBS - S - QF ( Red , immunostaining anti - HA ) after 16 hours of paraquat treatment in Oxford food at 25 Co ; note expansion of labelling with DBS - S - QF to large intestinal ( ECs ) ( compare D with A ) . image on left was acquired at low magnification with 10x objective , while right panel was acquired using 40x . dotted outlines gut using DAPI as a reference . Genotype : y1 w1118 UAS - mCD8 : : GFP . L QUAS - mtdTomato - 3xHA Act - DBS - S - QF ( BL83131 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. A representative example of an adult female posterior midgut at low (A) and high magnification (B, Region 4-Region 5) showing the initiator caspase reporter DBS-S-QF (Red, immunostaining anti-HA) after 16 hours of paraquat treatment in Oxford food at 25 Co; note the expansion of the labelling with DBS-S-QF to large intestinal cells (ECs) (compare D with A). The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. White dotted line outlines the gut using DAPI staining as a reference. Genotype: y1 w1118 UAS-mCD8::GFP.L QUAS-mtdTomato-3xHA Act-DBS-S-QF (BL83131)


E . ReDDM lineage - tracing in an adult intestine reared in Oxford Medium paraquat ( 20 mM ) during 16 hours at 29 Co ; note abundance of Histone - RFP without GFP signal , as an indication of epithelial damage subsequent differentiation of progenitor . image on left was acquired at low magnification with 10x objective , while right panel was acquired using 40x . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 ) F . of ReDDM labelling at high magnification after paraquat treatment ; note statistically significant increase ( * * ; P = 0 . 0099 ) of Histone - RFP expressing without GFP signal ( Quantifications were made using N ≥ 2 biological replicates ; Unpaired two - t , 3d n = 9 , 7d n = 7 ) . Error bars represent Standard Error of Mean . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP ( Irene Miguel Aliaga ) / Cyo ; UAS - Histone - RFP ( BL7019 ) TubG80ts ( BL7019 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O

E. ReDDM lineage-tracing in an adult intestine reared in Oxford Medium and paraquat (20 mM) during 16 hours at 29 Co; note the abundance of Histone-RFP cells without GFP signal, as an indication of epithelial damage and subsequent differentiation of progenitor cells. The image on the left was acquired at low magnification with the 10x objective, while the right panel was acquired using the 40x. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019) F. Quantification of ReDDM labelling at high magnification after paraquat treatment; note the statistically significant increase (**; P = 0.0099) of Histone-RFP expressing cells without GFP signal (Quantifications were made using N ≥ 2 biological replicates; Unpaired two-tailed t test, 3d n = 9, 7d n =7). Error bars represent Standard Error of the Mean. Genotype: w1118; esg-Gal4 UAS-CD8-GFP (Irene Miguel Aliaga)/ Cyo; UAS-Histone-RFP (BL7019) TubG80ts (BL7019)


H . Representative example of an intestine 7d old expressing two copies of effector caspase inhibitor P35 under regulation of esg - gal4 ( green immunostaining with antibody against P35 ) at 29 Co . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - P35 ( BL5072 ) ; UAS - P35 ( BL5073 ) / + I . ReDDM lineage - tracing system in a Drosophila intestine expressing two copies of effector caspase inhibitor P35 under regulation of esg - Gal4 experimental conditions that protect epithelial integrity at 29 Co . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - P35 ( BL5072 ) ; UAS - Histone - RFP TubG80ts / UAS - P35 ( BL5073 ) J . ReDDM corresponding to intestines described in ( I ) ; no significant increase in either esg ( n . s . , P = 0 . 1352 ) or Histone - RFP ( n . s . , P = 0 . 9801 ) number is observed ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - t , 3d n = 12 , 7d n = 11 ) . Error bars represent Standard Error of Mean . DAPI ( blue ) labels nuclei in panels A , D H . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - P35 ( BL5072 ) ; UAS - Histone - RFP TubG80ts / UAS - P35 ( BL5073 )

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O

H. Representative example of an intestine 7d old expressing two copies of the effector caspase inhibitor P35 under the regulation of esg-gal4 (green immunostaining with antibody against P35) at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-P35 (BL5072); UAS-P35 (BL5073)/+ I. ReDDM lineage-tracing system in a Drosophila intestine expressing two copies of the effector caspase inhibitor P35 under the regulation of esg-Gal4 and experimental conditions that protect the epithelial integrity at 29 Co. Genotype: w1118; esg-Gal4 UAS-CD8-GFP/ UAS-P35 (BL5072); UAS-Histone-RFP TubG80ts / UAS-P35 (BL5073) J. ReDDM quantification corresponding to the intestines described in (I); no significant increase in either esg (n.s. , P = 0.1352) or Histone-RFP (n.s. , P = 0.9801) cell number is observed (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, 3d n = 12, 7d n = 11). Error bars represent Standard Error of the Mean. DAPI (blue) labels the nuclei in panels A, D and H. Genotype: w1118; esg-Gal4 UAS-CD8-GFP/ UAS-P35 (BL5072); UAS-Histone-RFP TubG80ts / UAS-P35 (BL5073)


A . ReDDM activation in Dronc heterozygous ( + / - ) intestine 7 days after shift at 29 Co ; esg expression ( green ) labels intestinal progenitor , Histone - RFP ( red ) is a semi - permanent marker retained in differentiated Pdm - 1 ( grey ) labels differentiated ECs . red arrows indicates enlarged areas depicted in insets in entire figure . DAPI labels In entire Figure ( blue ) . of experiments in entire figure have been made under experimental conditions that protect epithelial integrity ( intestine reared in Oxford Medium flies transferred every two days into vials with fresh food ) .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. 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 of progenitor are fully mutant for Dronc ( see full genotype in methods ) ; note increased of esg - expressing ( GFP + , green ) , co - expression of Histone - RFP ( red ) EC marker Pdm - 1 ( gray ) , coexpression of Pdm - 1 GFP in enlarged ( compare panels detailed insets from A with B ) . image is a representative example of a Drosophila adult gut 7 days after 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 -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

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-Cherry


C . Relative number of esg - expressing normalised to DAPI ; notice that relative of esg - labelled is significantly higher in Dronc fully mutant conditions ( - / - ) at 3 days ( * * * ; P = 0 . 0007 ) 7d ( * * * * ; p < 0 . 0001 ) post shift at 29 Co ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - t - , + / - n = 32 , - / - n = 25 ) .

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C. 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 of esg - expressing ( μm² ) ; note increased of Dronc fully mutant progenitor ( - / - ) at 3d ( * * ; P = 0 . 0014 ) 7d ( * * * , P = 0 . 0007 ) post shift at 29 Co ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - parametric t - , 3d + / - n = 19 , 3d - / - n = 28 , 7d + / - n = 29 , 7d - / - n = 28 ) .

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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 expressing Histone - RFP normalised to DAPI ; notice that number of Histone - RFP 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 , + / - n = 24 , - / - n = 20 .

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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.


. A 7d intestine Dronc heterozygous ( + / - ) intestine in which intestinal express GFP under regulation of Dl - Gal4 . of experiments described in figure were performed in Oxford medium following an experimental regime that protects epithelial integrity ( flies transferred every two days into vials with fresh food ) . DAPI ( blue ) labels in entire Figure . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / + ( recombinant chromosome made for this study ; parental delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP was obtained from Maria Dominguez ) B . A 7d intestine in which Intestinal have become Dronc homozygous mutant ( - / - ) upon expressing UAS - Flp under regulation of Dl - Gal4 . Dl - expressing are labelled with UASGFP ( green ) ; there are no morphological differences between A B . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry C . Relative number of GFP + Delta - expressing normalised to DAPI ; there is no significant increase in intestinal number between heterozygous homozygous Dronc mutant conditions ( ns ; P = 0 . 9231 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - t , + / - n = 22 , - / - n = 13 ) . Error bars represent Standard Deviation of Mean . Genotypes : + / - : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry - / - : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry D . Average Delta ( μm² ) ; notice that does not change between heterozygous homozygous Dronc mutant ISCs ( ns ; P = 0 . 9694 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - t , + / - n = 42 , - / - n = 21 ) . Error bars represent Standard Deviation of mean . Genotypes :

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. A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Dl-Gal4. All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity (flies transferred every two days into vials with fresh food). DAPI (blue) labels the DNA in the entire Figure. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO/+ (recombinant chromosome made for this study; the parental line delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP was obtained from Maria Dominguez) B. A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Dl-Gal4. Dl-expressing cells are labelled with UASGFP (green); there are no morphological differences between A and B. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Relative number of GFP+ Delta-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (ns; P = 0.9231) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 22, -/- n = 13). Error bars represent Standard Deviation of the Mean. Genotypes: +/- :w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry -/- : w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry D. Average Delta cell size (μm²); notice that the cell size does not change between heterozygous and homozygous Dronc mutant ISCs (ns; P = 0.9694) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 42, -/- n = 21). Error bars represent Standard Deviation of the mean. Genotypes:


. A 7d intestine Dronc heterozygous ( + / - ) intestine in which intestinal express GFP under regulation of Dl - Gal4 . of experiments described in figure were performed in Oxford medium following an experimental regime that protects epithelial integrity ( flies transferred every two days into vials with fresh food ) . DAPI ( blue ) labels in entire Figure . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / + ( recombinant chromosome made for this study ; parental delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP was obtained from Maria Dominguez ) B . A 7d intestine in which Intestinal have become Dronc homozygous mutant ( - / - ) upon expressing UAS - Flp under regulation of Dl - Gal4 . Dl - expressing are labelled with UASGFP ( green ) ; there are no morphological differences between A B . Genotype : w1118 ; ; delta - Gal4 UAS - GFP TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry C . Relative number of GFP + Delta - expressing normalised to DAPI ; there is no significant increase in intestinal number between heterozygous homozygous Dronc mutant conditions ( ns ; P = 0 . 9231 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - t , + / - n = 22 , - / - n = 13 ) . Error bars represent Standard Deviation of Mean . Genotypes :

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

. A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Dl-Gal4. All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity (flies transferred every two days into vials with fresh food). DAPI (blue) labels the DNA in the entire Figure. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO/+ (recombinant chromosome made for this study; the parental line delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP was obtained from Maria Dominguez) B. A 7d intestine in which the Intestinal Stem Cells have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Dl-Gal4. Dl-expressing cells are labelled with UASGFP (green); there are no morphological differences between A and B. Genotype: w1118; ;delta-Gal4 UAS-GFP TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Relative number of GFP+ Delta-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (ns; P = 0.9231) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 22, -/- n = 13). Error bars represent Standard Deviation of the Mean. Genotypes:


. A 7d intestine Dronc heterozygous ( + / - ) intestine in which intestinal express GFP under regulation of Su ( H ) - Gal4 . Genotype : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / + A 7d intestine in which Enteroblasts have become Dronc homozygous mutant ( - / - ) upon expressing UAS - Flp under regulation of Su ( H ) - Gal4 . Su ( H ) - Gal4 - expressing are labelled with UASGFP ( green ) UAS - Histone - RFP ; note increase in when compared to E absence of Histone - RFP without GFP signal . Genotype : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry Relative number of Su ( H ) - Gal4 - expressing normalised to DAPI ; there is no significant increase in intestinal number between heterozygous homozygous Dronc mutant conditions ( n . s . ; P = 0 . 5099 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney , + / - n = 30 , - / - n = 29 ) . Error bars represent Standard Deviation of mean . Genotypes : + / - : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 ; Su ( H ) - Gal4 UAS - GFP ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry H . Average Su ( H ) ( μm² ) , there is a significant increase in Su ( H ) Dronc null ( - / - ) when compared to heterozygous control condition ( * * ; P = 0 . 0064 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney , + / - n = 49 , - / - n = 33 ) . Error bars represent Standard Deviation of mean . Genotypes :

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

. A 7d intestine Dronc heterozygous (+/-) intestine in which intestinal stem cells express GFP under the regulation of Su(H)-Gal4. Genotype: w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/+ A 7d intestine in which the Enteroblasts have become Dronc homozygous mutant (-/-) upon expressing UAS-Flp under the regulation of Su(H)-Gal4. Su(H)-Gal4-expressing cells are labelled with UASGFP (green) and UAS-Histone-RFP; note the increase in cell size when compared to E and the absence of Histone-RFP cells without GFP signal. Genotype: w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/ UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry Relative number of Su(H)-Gal4-expressing cells normalised to DAPI; there is no significant increase in intestinal stem cell number between heterozygous and homozygous Dronc mutant conditions (n.s. ; P = 0.5099) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 30, -/- n = 29). Error bars represent Standard Deviation of the mean. Genotypes: +/- : w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/+ -/- : w1118; Su(H)-Gal4 UAS-GFP; TubG80ts UAS-Histone-RFP DroncKO/ UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry H. Average Su(H) cell size (μm²), there is a significant increase in Su(H) Dronc null cells (-/-) when compared to the heterozygous control condition (**; P = 0.0064) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 49, -/- n = 33). Error bars represent Standard Deviation of the mean. Genotypes:


A . Representative image of a Drosophila posterior midgut showing expression of Armadillo ( red membranes ) , Prospero ( red nuclei of EEs ) Dronc - TurboID ( green shows Streptavidin labelled by Dronc - TurboID ) ; note preferential enrichment of Streptavidin is preferentially enriched in intestinal progenitor ( arrows indicate an intestinal progenitor upregulating Dronc ) .

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A. 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 expression of Armadillo ( red membranes ) Actin - Dronc - GFP - Myc ( green , anti - GFP ) ; note preferential enrichment of Dronc expression in small groups of formed by intestinal progenitor ( arrow ) . High magnification of picture shown in B . arrow indicates a presumptive ISC showing low of Dronc compared to flanking GFP + EBs . arrow indicates a presumptive small ISC expressing high of Arm low of Dronc - GFP - Myc . Transversal Z - section of shown in C . arrow indicates a presumptive small ISC expressing high of Arm low of Dronc - GFP - Myc .

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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 . enteroblast marker Su ( H ) ( red , immunostaining against Beta - galactosidase ) strongly co - localises with high of Dronc expression ( green , immunostaining against GFP ) ; arrow indicates enlarged depicted in insets .

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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 expression of EB marker Su ( H ) ( green , immunostaining against Beta - galactosidase ; arrow ) apical caspase reporter DBS - S - QF ( red , immunostaining against HA ) , but it is also present in progenitor without Su ( H ) expression ( yellow arrowheads ) . DAPI ( Blue ) labels nuclei in panels .

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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.


A . Representative ReDDM labeling of a Drosophila Dronc heterozygous ( + / - ) intestine overexpressing an RNAi against Notch for 3 days ; note lack of fully differentiated Histone - RFP as EC ( red ) without esg expression ( green , GFP ) . DAPI labels nuclei in panels of experiments described in figure were performed in Oxford medium following an experimental regime that protects epithelial integrity . Genotype : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + B . Representative ReDDM labelling of a Drosophila Dronc mutant homozygous ( - / - ) intestine overexpressing an RNAi against Notch for 3 post shift at 29 oC ; note lack of differentiated Histone - RFP as EC ( red ) without esg expression ( green , GFP ) , as well as increase of GFP - positive ( compare A to B ) . Genotype : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry C . of number of Histone - RFP normalised to DAPI ( proxy of progenitor proliferation obtained from experiments shown in A B panels ) ; note statically significant increase in Histone - RFP positive in Dronc homozygous mutant intestines compared to controls ( * * ; P = 0 . 0080 ) ( Quantifications were made using N ≥ 2 biological replicates ; unpaired two - t , + / - n = 8 , - / - n = 15 ) . Error bars represent Standard Deviation of mean . Genotypes : + / - : w1118 UAS - Notch - RNAi ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 UAS - Notch - RNAi ( Joaquin Navascués ) ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O

A. Representative ReDDM labeling of a Drosophila Dronc heterozygous (+/-) intestine overexpressing an RNAi against Notch for 3 days; note the lack of fully differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP). DAPI staining labels cell nuclei in panels All of the experiments described in the figure were performed in Oxford medium following an experimental regime that protects the epithelial integrity. Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + B. Representative ReDDM labelling of a Drosophila Dronc mutant homozygous (-/-) intestine overexpressing an RNAi against Notch for 3 post temperature shift at 29 oC; note the lack of differentiated Histone-RFP cells as EC (red) without esg expression (green, GFP), as well as the increase of GFP-positive cells (compare A to B). Genotype: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry C. Quantification of the number of Histone-RFP cells normalised to DAPI (proxy of progenitor cell proliferation obtained from the experiments shown in A and B panels); note the statically significant increase in Histone-RFP positive cells in Dronc homozygous mutant intestines compared to controls (**; P =0.0080) (Quantifications were made using N ≥ 2 biological replicates; unpaired two-tailed t test, +/- n = 8, -/- n = 15). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118 UAS-Notch-RNAi; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118 UAS-Notch-RNAi (Joaquin Navascués); esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


D . Drosophila Dronc heterozygous intestine overexpressing Notch intracellular domain for 7 days post shift at 29 oC ; intestinal esg - positive progenitor ( green ( GFP ) red ( Histone - RFP ) . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ( Joaquin Navascués ) ; TubG80ts UAS - Histone - RFP DroncKO / + E . Drosophila Dronc homozygous intestine overexpressing Notch intracellular domain for 7d post shift at 29 oC ; notice that Dronc deficiency accelerates elimination of intestinal progenitor induced by Notch overactivation ( compare D E ) . arrows indicate position of insets 500μm from posterior region . Note complete loss of esg labelled in this region . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA - Cherry F . Relative number of esg - positive to DAPI in either heterozygous or homozygous Dronc mutant esg overexpressing Notch - Intra ; note significant reduction of esg - expressing ( * * * * ; P < 0 . 0001 ) ( Quantifications were made using N ≥ 2 biological replicates ; Mann - Whitney , + / - n = 17 , - / - n = 17 ) . Error bars represent Standard Deviation of mean . Genotypes : + / - : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / + E. Drosophila Dronc homozygous intestine overexpressing the Notch intracellular domain for 7d post temperature shift at 29 oC; notice that the Dronc deficiency accelerates the elimination of intestinal progenitor cells induced by Notch overactivation (compare D and E). The white arrows indicate the position of insets 500μm from the posterior region. Note the complete loss of esg labelled cells in this region. Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry F. Relative number of esg-positive cells to DAPI in either heterozygous or homozygous Dronc mutant esg cells overexpressing Notch-Intra; note the significant reduction of esg-expressing cells (****; P < 0.0001) (Quantifications were made using N ≥ 2 biological replicates; Mann-Whitney test, +/- n = 17, -/- n = 17). Error bars represent Standard Deviation of the mean. Genotypes: +/-: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118; esg-Gal4 UAS-CD8 -GFP / UAS-Notchintra; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


D . Drosophila Dronc heterozygous intestine overexpressing Notch intracellular domain for 7 days post shift at 29 oC ; intestinal esg - positive progenitor ( green ( GFP ) red ( Histone - RFP ) . Genotype : w1118 ; esg - Gal4 UAS - CD8 - GFP / UAS - Notchintra ( Joaquin Navascués ) ; TubG80ts UAS - Histone - RFP DroncKO / +

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D. Drosophila Dronc heterozygous intestine overexpressing the Notch intracellular domain for 7 days post temperature shift at 29 oC; intestinal esg-positive progenitor cells (green (GFP) and red (Histone-RFP). Genotype: w1118; esg-Gal4 UAS-CD8-GFP / UAS-Notchintra (Joaquin Navascués); TubG80ts UAS-Histone-RFP DroncKO / +


G . Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC . esg expression in green ( GFP ) labels intestinal progenitors , whilst Su ( H ) - lacZ ( red ) distinguishes a subpopulation of EBs within esg - expressing . Genotype : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / +

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G. Representative image of a Drosophila Dronc heterozygous intestines 7 days after transfer to 29 oC. esg expression in green (GFP) labels all intestinal progenitors cells, whilst Su(H)-lacZ (red) distinguishes a subpopulation of EBs within the esg-expressing cells. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / +


H . Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC . Note , transcription of Su ( H ) in large GFP ( - ) , suggestive of an aberrant upregulation of Notch - signalling within fully differentiated ECs . Genotype : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

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H. Representative image of a Drosophila Dronc KO intestine 7 days after transfer to 29 oC. Note, the transcription of Su(H) in large cells GFP (-), suggestive of an aberrant upregulation of Notch-signalling within fully differentiated ECs. Genotype: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


I . Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat . Note absence of large Su ( H ) positive , GFP ( - ) compared to ( H ) . arrows indicate enlarged depicted in insets . Genotypes : + / - : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / + - / - : w1118 , Su ( H ) GBE - LacZ ; esg - Gal4 UAS - CD8 - GFP / + ; TubG80ts UAS - Histone - RFP DroncKO / UAS - Flippase ( BL8209 ) FRT Dronc - GFP - APEX FRT suntag - HA -

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

I. Representative image of a 7d Drosophila Dronc heterozygous intestines following a 16hr treatment with paraquat. Note the absence of large Su(H) positive, GFP (-) cells compared to (H). The white arrows indicate the enlarged area depicted in the insets. Genotypes: +/-: w1118, Su(H)GBE-LacZ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / + -/-: w1118, Su(H)GBE-LacZ ; esg-Gal4 UAS-CD8-GFP / + ; TubG80ts UAS-Histone-RFP DroncKO / UAS-Flippase (BL8209) FRT Dronc-GFP-APEX FRT suntag-HA-Cherry


a . Lipid droplet time course . Representative photomicrographs showing lipid droplet abundance in ARPE19 treated with DFP starting at 7 h maximal at 24 h . Neutral lipids were revealed by BODIPY 493 / 503 . Nuclei are counterstained with Hoescht33342 . Inset shows detail of LDs in a treated with DFP for 24 h . Arrows highlight LD accumulation . Scale bars = 5 μm .

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a. 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 ( human ARPE - 19 with stable expression of mito - QC ) . Mitochondrial networks are visible in yellow mitolysosomes as red - only signifying mitophagy . Nuclei are counterstained with Hoescht33342 . Mitophagy appears maximal after 24 h of DFP treatment , with little difference between control 7 h conditions . Inset shows details of mitolysosomes within mitochondrial network of a highly mitophagic following DFP treatment . Arrows highlight mitolysosomes . Scale bar = 5 μm .

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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 are treated with DFP . Representative photomicrographs showing lipid droplet mitochondria in ARPE19 treated with DFP at 7 h at 24 h . Neutral lipids were revealed by BODIPY 493 / 503 mitochondria by Mitotracker . Nuclei are counterstained with Hoescht33342 . Inset shows detail of LDs in close proximity to mitochondria in treated with DFP for 24 h . Scale bar = 5 μm .

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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 . of lipid droplet per over time . Lipid droplet was quantified per using a pipeline generated in CellProfiler . n = 3 with at least 50 quantified per biological replicate for each condition . Data represent means ± .

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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 . of mitophagy over time . Mitophagy index per is measured as a of number of mitolysosomes per mitochondrial content . n = 3 with at least 50 quantified per biological replicate for each condition . Data represent means ± .

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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 . of lipid droplet mitochondria proximity over time . n = 2 with at least 50 quantified per biological replicate for each condition . Ratios between Control 24h DFP ; P < 0 . 05 .

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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.


d . DFP - induced Lipid droplet accumulation is dependent on DGAT1 . Representative photomicrographs of ARPE19 treated with DFP ( 1mM ) for 7 h followed by treatment with LD inhibitors ( 5 μM ) as indicated . were fixed after 24 h of DFP treatment . Neutral lipid droplets were stained by BODIPY493 / 503 imaged by confocal microscopy after 24 h of DFP treatment ( ' i ' denotes presence of inhibitor e . g . DGAT1i = DGAT1 - inhibition ) . Scale bar = 5 μm .

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d. 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 per from experiments in 2d . Data are represented as mean ± ( n = 3 with at least 80 analysed per condition in each biological replicate ) .

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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).


a . ARPE - 19 ULK1 CRISPR KO with / without re - introduction of FLAG - ULK1 were treated with DFP for 24h as indicated . were fixed stained with BODIPY493 / 503 to visualise lipid droplets ( green ) or DAPI to visualise nuclei ( blue ) . Scale bar , 10 μm .

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a. 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 . from 5a , n = 3 independent experiments , statical by one - way ANOVA . * = P < 0 . 05 ; * * P = < 0 . 01 . Median represented by dashed , quartiles by dotted lines .

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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 lines in 5a by immunoblot of lysates .

c. Authentication of cell lines in 5a by immunoblot of protein lysates.


f . Representative confocal photomicrographs showing pattern of endogenous mitochondrial NIX in human ARPE - 19 treated with DFP ± DGAT1i / 2i . Samples were immunolabelled with antibodies to NIX ( green ) ATPB ( red ) . Scale bar = 2 μm . g . Representative confocal photomicrograpghs of Cathepsin - reactive endolysosomes in human ARPE - 19 treated with with DFP ± DGAT1i / 2i . Inset shows examples of lysosomal . Scale bar = 5 μm . h . Colocalization quantitation for experiments shown in 6g . One - way ANOVA with Bonferroni ' s post hoc . * * * * P < 0 . 0001 . Median represented by dashed , quartiles by dotted lines n = 2 biological replicates . i . Colocalization quantitation for ULK1 / WIPI2 immunolabelling in human ARPE - 19 treated with DFP ± DGAT1i / 2i . Median represented by dashed , 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 . * * * * = P < 0 . 0001 . Median represented by dashed , quartiles by dotted lines n = 2 biological replicates .

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f. 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.


c . Representative photomicrographs of larval neuronal bodies expressing matrix - QC reporter RNAi for mdy or control driven with nSyb - GAL4 . GFP is shown in green , mCherry is shown in magenta . Mitolysosomes ( mCherry - only ) are shown in greyscale . Scale bar = 5 μm .

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c. 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 of mitophagy ( mitolysosome per neuronal ) . Data are shown as violin plot with median ( dashed ) quartile range ( dotted lines ) ; n = 5 - 6 , with 23 - 40 per . One - way ANOVA with Šidák ' s post - correction for multiple samples ; * * = P < 0 .

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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