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 .

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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 cells 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 ) 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 .

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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 ) , 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 .

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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 . Quantification of MECA32 + area ( K ) and average distance between MECA32 + vessels and basement membrane ( 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 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 .

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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 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 .

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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 ) 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 .

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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 the 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 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 ) .

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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 ) 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 .

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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 SEM .

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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 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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(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 .

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(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 the cell envelope increase death rates . Growth in glycerol minimal medium , starvation induced by glycerol depletion .

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(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 .

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(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 .

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(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.


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 .

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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.


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

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Proteins 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 .

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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.


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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 )

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. 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

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

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 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 :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: +/- :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 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 :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 .

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

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 .

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

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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 / +

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 / +

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

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 , 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 -

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

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 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 -

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 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 .

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

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 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 .

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

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 .

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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 . 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 .

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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 . 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 .

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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 . 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 . 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 .

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 .

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


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 .

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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 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 .

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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 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 .

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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


B 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 .

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B 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.


B 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 .

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B 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.


A 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 .

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A 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.


A 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 .

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A 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.


( b ) Total RNA sequencing of cenRNAs in yeasts . Left ; read counts per million tracks for CEN16 in wildtype ( black , top ) and DAD1E50D humanized ( red , bottom ) yeasts . Right ; heatmap of cenRNAs transcripts per million counts across all chromosomes in wildtype and DAD1E50D humanized strains . Centromere VI is marked with an asterisk to indicate that the plasmid encoding the histone plasmid also encodes a CEN6 sequence , which reads likely

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(b) Total RNA sequencing of cenRNAs in yeasts. Left; read counts per million tracks for CEN16 in wildtype (black, top) and DAD1E50D humanized (red, bottom) yeasts. Right; heatmap of cenRNAs transcripts per million counts across all chromosomes in wildtype and DAD1E50D humanized strains. Centromere VI is marked with an asterisk to indicate that the plasmid encoding the histone plasmid also encodes a CEN6 sequence, which reads likely emanant


( c ) RT - PCRs of the indicated RNAs . Lane 1 is genomic DNA , lane 2 is wildtype shuffle strain ( yDT67 ) , lane 3 is BY4742 , lane 4 is DAD1E50D humanized , lane 5 is scc4D65Y humanized , lanes 6 - 9 are the same as 2 - 5 , but without reverse transcriptase added , lane 10 is no DNA .

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(c) RT-PCRs of the indicated RNAs. Lane 1 is genomic DNA, lane 2 is wildtype shuffle strain (yDT67), lane 3 is BY4742, lane 4 is DAD1E50D humanized, lane 5 is scc4D65Y humanized, lanes 6-9 are the same as 2-5, but without reverse transcriptase added, lane 10 is no DNA.


( d ) Example images from strains with fluorescent tags on the microtubule organizing center ( Spc110 - GFP ) and the kinetochore ( Nuf2 - RFP ) . Images were taken from cultures in mid - log phase of growth .

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(d) Example images from strains with fluorescent tags on the microtubule organizing center (Spc110-GFP) and the kinetochore (Nuf2-RFP). Images were taken from cultures in mid-log phase of growth.


( e ) DASH mutants increase histone - humanization in the scc4D65Y background . Humanization rates of the scc4D65Y mutant with and without DAD1E50D or DAM1N80Y . Significance of the mean difference in 5 - FOAR frequency was determined with the Mann - Whitney test . Each dot represents one biological replicate ( n = 8 ) , central band represents the median , box extends from the 25th to 75th percentiles , and whiskers represent the minimum and maximum .

(e) DASH mutants increase histone-humanization in the scc4D65Y background. Humanization rates of the scc4D65Y mutant with and without DAD1E50D or DAM1N80Y. Significance of the mean difference in 5-FOAR frequency was determined with the Mann-Whitney test. Each dot represents one biological replicate (n = 8), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.


( f ) Ploidy analysis by flow cytometry in the scc4D65Y humanized mutants from panel d . Flow cytometry data from humanized lineages yHs5 and yHs7 are shown for aneuploid and euploid controls , respectively .

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(f) Ploidy analysis by flow cytometry in the scc4D65Y humanized mutants from panel d. Flow cytometry data from humanized lineages yHs5 and yHs7 are shown for aneuploid and euploid controls, respectively.


( a ) Growth assays of wild type , ipl1 - 2 mutants , and ipl1 - 2 kinetochore double mutants at permissive ( 24ºC ) and non - permissive temperature ( 32ºC ) . Ten - fold serial dilutions of ~ 1 . 0 OD600 yeast cultures were spotted onto YPD solid media and grown at the indicated temperatures for four and three days , respectively .

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(a) Growth assays of wild type, ipl1-2 mutants, and ipl1-2 kinetochore double mutants at permissive (24ºC) and non-permissive temperature (32ºC). Ten-fold serial dilutions of ~1.0 OD600 yeast cultures were spotted onto YPD solid media and grown at the indicated temperatures for four and three days, respectively.


( b ) Flow cytometry analysis of wild type , ipl1 - 2 mutants , and ipl1 - 2 kinetochore double mutants at the permissive ( 24 ˚ C ) and non - permissive temperature ( 30ºC ) . Yeast cultures were grown to logarithmic phase at 24 ˚ C and divided and grown at 24 ˚ C and 30 ˚ C for 6 hours before cells were collected and processed for DNA content analysis using Sytox Green stain .

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(b) Flow cytometry analysis of wild type, ipl1-2 mutants, and ipl1-2 kinetochore double mutants at the permissive (24˚C) and non-permissive temperature (30ºC). Yeast cultures were grown to logarithmic phase at 24˚C and divided and grown at 24˚C and 30˚C for 6 hours before cells were collected and processed for DNA content analysis using Sytox Green stain.


( c ) Growth assays of wild type , mad3 ∆ mutants , and mad3 ∆ DAD1E50D double mutants on YPD with and without 30 µg / ml benomyl . Ten - fold serial dilutions of ~ 1 . 0 OD600 yeast cultures were spotted onto the indicated solid media and grown at 30 ˚ C for three days .

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(c) Growth assays of wild type, mad3∆ mutants, and mad3∆ DAD1E50D double mutants on YPD with and without 30 µg/ml benomyl. Ten-fold serial dilutions of ~1.0 OD600 yeast cultures were spotted onto the indicated solid media and grown at 30˚C for three days.


( b ) Histone - humanization assay for various dam1N80X mutants . 5 - FOA is used to counter - select the yeast histone plasmid , forcing growth with the human histone plasmid . Yeast were serially diluted from a starting culture of 1 . 0 OD600 .

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(b) Histone-humanization assay for various dam1N80X mutants. 5-FOA is used to counter-select the yeast histone plasmid, forcing growth with the human histone plasmid. Yeast were serially diluted from a starting culture of 1.0 OD600.


( f ) Rupture force assay from experiments at three concentrations of DASH complex incubated with the beads . Briefly , 3 . 5 pM of beads were incubated with the indicated concentrations of DASH complex ( representing a ratio of DASH complex to beads of 714 : 1 , 1428 : 1 , and 2860 : 1 ) . These DASH - decorated beads were then washed before suspending in a reaction mix containing free tubulin and 5 nM free DASH complex . Two way ANOVA tests of the mean difference in rupture force are shown with multiple test corrections ( false discovery rate ) . Each dot represents an individual rupture force measurement ( n ≥ 21 ; exact number shown below each box between parentheses ) , central band represents the median , box extends from the 25th to 75th percentiles , and whiskers represent the minimum and maximum .

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(f) Rupture force assay from experiments at three concentrations of DASH complex incubated with the beads. Briefly, 3.5 pM of beads were incubated with the indicated concentrations of DASH complex (representing a ratio of DASH complex to beads of 714:1, 1428:1, and 2860:1). These DASH-decorated beads were then washed before suspending in a reaction mix containing free tubulin and 5 nM free DASH complex. Two way ANOVA tests of the mean difference in rupture force are shown with multiple test corrections (false discovery rate). Each dot represents an individual rupture force measurement (n ≥ 21; exact number shown below each box between parentheses), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.


( g ) Survival curves for each DASH complex are shown at the indicated concentrations . Log - rank tests were performed to test whether the survival curves were significantly different from one another . Error bars represent the standard error and are displayed as the shaded area .

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(g) Survival curves for each DASH complex are shown at the indicated concentrations. Log-rank tests were performed to test whether the survival curves were significantly different from one another. Error bars represent the standard error and are displayed as the shaded area.