Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes . The three panels in each figure are as follows ( a - e , left ) FM4 - 64 ; ( center ) GFP - SKL ; ( right ) superimposed FM4 - 64 and GFP - SKL signals . ( a , f ) stage 0 ; ( b , g ) early stage 1 ; ( c , h ) late stage 1 ; ( d , i ) stage 2 ; ( e ) stage 3 . ( d , arrowhead ) During the capture of these fluorescence images , this peroxisome - containing vesicle had just pinched off from the outer vacuolar membrane . Therefore , the FM4 - 64 - stained vesicle membrane and GFP - SKL could not be superimposed in this picture .

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Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes. The three panels in each figure are as follows (a-e, left) FM4-64; (center) GFP-SKL; (right) superimposed FM4-64 and GFP-SKL signals. (a, f) stage 0; (b, g) early stage 1; (c, h) late stage 1; (d, i) stage 2; (e) stage 3. (d, arrowhead) During the capture of these fluorescence images, this peroxisome-containing vesicle had just pinched off from the outer vacuolar membrane. Therefore, the FM4-64-stained vesicle membrane and GFP-SKL could not be superimposed in this picture.


Kinetics of morphological changes in vacuoles and peroxisomalGFP - SKL during microautophagic degradation of P . pastoris in glucose adaptation . At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point . Cells were counted in three randomly selected fields , and standard deviations are shown as vertical bars .

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Kinetics of morphological changes in vacuoles and peroxisomalGFP-SKL during microautophagic degradation of P. pastoris in glucose adaptation. At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point. Cells were counted in three randomly selected fields, and standard deviations are shown as vertical bars.


Macroautophagic degradation of peroxisomes after transfer of methanol - grown P . pastoris cells to ethanol medium . ( a - e ) Electron micrographs of intermediates seen during macroautophagy . ( a ) After 1 h of ethanol adaptation , peroxisomes ( P ) were often surrounded by autophagosomal membranes , higher magnifications of which are in d and e . ( b ) After 1 h of ethanol adaptation , the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane . ( c ) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole . Magnified pictures of samples obtained at the ( d ) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes , and at the ( e ) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes . ( f ) Kinetics of morphological changes in vacuoles and peroxisomal GFP - SKL . Other conditions are described in Materials and Methods , and are the same as in Fig . 4 . ( g ) Fluorescence images taken after 2 h of ethanol adaptation of wild - type P . pastoris cells .

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Macroautophagic degradation of peroxisomes after transfer of methanol-grown P. pastoris cells to ethanol medium. (a-e) Electron micrographs of intermediates seen during macroautophagy. (a) After 1 h of ethanol adaptation, peroxisomes (P) were often surrounded by autophagosomal membranes, higher magnifications of which are in d and e. (b) After 1 h of ethanol adaptation, the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane. (c) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole. Magnified pictures of samples obtained at the (d) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes, and at the (e) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes. (f) Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL. Other conditions are described in Materials and Methods, and are the same as in Fig. 4. (g) Fluorescence images taken after 2 h of ethanol adaptation of wild-type P. pastoris cells.


Effect of inhibitors on transport of peroxisomal GFP - SKL to , and degradation in , vacuoles during ( a ) glucose adaptation and ( b ) ethanol adaptation . Wortmannin and PMSF were dissolved in DMSO , and added to the glucose medium . ( Filled circle ) Control ( 2 % DMSO ) ; ( open circle ) 1 mM wortmannin ( with 2 % DMSO ) ; ( filled square ) 3 mM PMSF ( with 2 % DMSO ) ; ( filled triangle ) 10 mg / ml cycloheximide .

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Effect of inhibitors on transport of peroxisomal GFP-SKL to, and degradation in, vacuoles during (a) glucose adaptation and (b) ethanol adaptation. Wortmannin and PMSF were dissolved in DMSO, and added to the glucose medium. (Filled circle) Control (2% DMSO); (open circle) 1 mM wortmannin (with 2% DMSO); (filled square) 3 mM PMSF (with 2% DMSO); (filled triangle) 10 mg/ml cycloheximide.


Microautophagic degradation of peroxisomes is not observed in a vacuolar protease - negative strain ( STK7 ) of P . pastoris . Kinetics of morphological changes in vacuoles and peroxisomal GFP - SKL during ( a ) glucose adaptation and ( b ) ethanol adaptation . ( c ) Fluorescence images taken after 2 h of glucose adaptation . Arrows show GFP - SKL in vacuolar matrix . Some cells showed diffusion of GFP - SKL into the vacuolar matrix .

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Microautophagic degradation of peroxisomes is not observed in a vacuolar protease-negative strain (STK7) of P. pastoris. Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL during (a) glucose adaptation and (b) ethanol adaptation. (c) Fluorescence images taken after 2 h of glucose adaptation. Arrows show GFP-SKL in vacuolar matrix. Some cells showed diffusion of GFP-SKL into the vacuolar matrix.


( a ) Visualization of alcohol oxidase activity in colonies after ( a ) glucose adaptation for 8 h and ethanol adaptation for 24 h in wild - type ( strain PPY12 ) and pag mutant cells ( strains STK1 - STK6 , Materials and Methods ) of P . pastoris . ( b ) Western - blot analysis of samples taken from cultures shifted from methanol to glucose for the indicated time . Antibodies used were AOX , alcohol oxidase ; F1 , mitochondrial F1β subunit ; G6PDH , glucose - 6 - phosphate dehydrogenase . Growth curve of the wild - type strain PPY12 and the pag4 mutant ( strain STK4 ) after shift from methanol to glucose .

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(a) Visualization of alcohol oxidase activity in colonies after (a) glucose adaptation for 8 h and ethanol adaptation for 24 h in wild-type (strain PPY12) and pag mutant cells (strains STK1-STK6, Materials and Methods) of P. pastoris. (b) Western-blot analysis of samples taken from cultures shifted from methanol to glucose for the indicated time. Antibodies used were AOX, alcohol oxidase; F1, mitochondrial F1β subunit; G6PDH, glucose-6-phosphate dehydrogenase. Growth curve of the wild-type strain PPY12 and the pag4 mutant (strain STK4) after shift from methanol to glucose.


Fluorescence images of ( a ) pag2 ( strain STK2 ) and ( b ) pag3 ( strain STK3 ) mutants of P . pastoris after a 3 h shift to glucose . ( b ) Arrows show deeply invaginated structures of vacuolar membranes surrounding peroxisomes in the pag3 mutant .

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Fluorescence images of (a) pag2 (strain STK2) and (b) pag3 (strain STK3) mutants of P. pastoris after a 3 h shift to glucose. (b) Arrows show deeply invaginated structures of vacuolar membranes surrounding peroxisomes in the pag3 mutant.


Fluorescence images of the pag4 ( strain STK4 ) mutant of P . pastoris after ( a ) 0 , ( b ) 3 , or ( c ) 6 h of glucose adaptation . The vacuolar membrane proliferated extensively and surrounded peroxisomes during glucose adaptation .

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Fluorescence images of the pag4 (strain STK4) mutant of P. pastoris after (a) 0, (b) 3, or (c) 6 h of glucose adaptation. The vacuolar membrane proliferated extensively and surrounded peroxisomes during glucose adaptation.


Microautophagy of peroxisomes in a pex1 - ts mutant ( strain SJH242 ) of P . pastoris expressing GFP - SKL from the constitutive glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) promoter at the restrictive temperature for peroxisome assembly ( 34 ° C ) . ( a ) Kinetics of peroxisome degradation at 34 ° C . Strain SJH242 was induced on methanol at 23 ° C , and glucose adaptation was performed at 34 ° C . ( b ) Changes in vacuole morphology were seen even in the absence of normal peroxisomes . At 0 h , SJH242 cells were grown at 34 ° C when GFP - SKL was diffuse in the cytosol and many vacuoles showed spherical morphologies . After 2 h , ∼ 20 - 30 % vacuoles showed complicated membrane structures representative of stage 2 cells .

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Microautophagy of peroxisomes in a pex1-ts mutant (strain SJH242) of P. pastoris expressing GFP-SKL from the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter at the restrictive temperature for peroxisome assembly (34°C). (a) Kinetics of peroxisome degradation at 34°C. Strain SJH242 was induced on methanol at 23°C, and glucose adaptation was performed at 34°C. (b) Changes in vacuole morphology were seen even in the absence of normal peroxisomes. At 0 h, SJH242 cells were grown at 34°C when GFP-SKL was diffuse in the cytosol and many vacuoles showed spherical morphologies. After 2 h, ∼20-30% vacuoles showed complicated membrane structures representative of stage 2 cells.


D . Plot of initial MT polymerization velocities at 37 ° C in the presence of increasing concentrations of WT or NN . Tubulin concentration = 5 μM . Each point represents an average of 3 independent measurements of the rate of turbidity increase measured at 500 nm wavelength ( OD500 / min ) . Fitted are sigmoidal dose - response curves : Y = YB + ( ( YT - YB ) / ( 1 + 10Xi - X ) ) , where YB and YT are Y values ( OD500 / min ) at the bottom and top plateaus , respectively , and Xi is the X value at the inflection point . Representative MT samples from 2 : 5 μM WT : tubulin or NN : tubulin incubations are shown in E and F , respectively . MT architecture distributions for WT and NN are shown with pie graphs .

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D. Plot of initial MT polymerization velocities at 37 °C in the presence of increasing concentrations of WT or NN. Tubulin concentration = 5 μM. Each point represents an average of 3 independent measurements of the rate of turbidity increase measured at 500 nm wavelength (OD500/min). Fitted are sigmoidal dose-response curves: Y = YB + ((YT - YB)/(1 + 10Xi - X)), where YB and YT are Y values (OD500/min) at the bottom and top plateaus, respectively, and Xi is the X value at the inflection point. Representative MT samples from 2:5 μM WT:tubulin or NN:tubulin incubations are shown in E and F, respectively. MT architecture distributions for WT and NN are shown with pie graphs.


E , F . Representative low and high magnification images of MTs nucleated for 10 min at 37 ° C from 5 μM tubulin , in the presence of : 2 μM wild - type DCX ( WT ) ( E ) or 2 μM NDC - NDC ( NN ) ( F ) .

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E, F. Representative low and high magnification images of MTs nucleated for 10 min at 37 °C from 5 μM tubulin, in the presence of: 2 μM wild-type DCX (WT) (E) or 2 μM NDC-NDC (NN) (F).


H . Representative kymographs with arrowheads indicating MT catastrophes .

H. Representative kymographs with arrowheads indicating MT catastrophes.


A . Representative low - pass filtered cryo - EM micrographs of MTs decorated with wild - type DCX ( WT ) or NDC - NDC construct ( NN ) after 30 sec or 1 h incubation at 37 ° C . Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration . The final ratio of WT or NN to tubulin was 50 : 5 μM , giving dense protein background . WT co - polymerized exclusively 13 - PF MTs , while NN both 13 - and 14 - PF MTs . Rapid ( 30 sec ) MT polymerization in excess of WT produces MTs in an intermediate GTP / GDP . Pi state , whereas long - lived WT - MTs and NN - MTs are all in the GDP state .

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A. Representative low-pass filtered cryo-EM micrographs of MTs decorated with wild-type DCX (WT) or NDC-NDC construct (NN) after 30 sec or 1 h incubation at 37 °C. Additional DCX proteins were added in excess after MT deposition on EM grids to maximise MT decoration. The final ratio of WT or NN to tubulin was 50:5 μM, giving dense protein background. WT co-polymerized exclusively 13-PF MTs, while NN both 13- and 14-PF MTs. Rapid (30 sec) MT polymerization in excess of WT produces MTs in an intermediate GTP/GDP.Pi state, whereas long-lived WT-MTs and NN-MTs are all in the GDP state.


C - F . Immunofluorescence images of esg - GFP and Delta ( Dl ) staining with the midgut section from the R4 region of 14 - day Drosophila ( C ) , 26 - day Drosophila ( D ) , 40 - day Drosophila ( E ) , and 40 - day Drosophila in response to 0 . 5 mM ALA administration , which started at day 26 after eclosion ( F ) . esg - GFP ( green ) identifies ISCs and their differentiating cells . Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue .

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C-F. Immunofluorescence images of esg-GFP and Delta (Dl) staining with the midgut section from the R4 region of 14-day Drosophila (C), 26-day Drosophila (D), 40-day Drosophila (E), and 40-day Drosophila in response to 0.5 mM ALA administration, which started at day 26 after eclosion (F). esg-GFP (green) identifies ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue.


J . Western blotting results of midguts indicate a decrease of LAS in the midguts of aged Drosophila . Loading controls , β - tubulin . K . Quantification of LAS band intensity as seen in experiments ( J ) . Error bars indicate the SD of three independent experiments . L Data information : Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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J. Western blotting results of midguts indicate a decrease of LAS in the midguts of aged Drosophila. Loading controls, β-tubulin. K. Quantification of LAS band intensity as seen in experiments (J). Error bars indicate the SD of three independent experiments. L Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


C . Quantification of the content of ALA in midguts of flies with indicated genotypes using LC - ESI - MS / MS . Error bars indicate the SD of three independent experiments . Data information : Error bars represent SDs . Student ' s t - tests , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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C. Quantification of the content of ALA in midguts of flies with indicated genotypes using LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments. Data information: Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


D - F . Immunofluorescence images of the midgut section from the R4 region in Drosophila carrying esgts - GAL4 - driven UAS - lacZ ( D , control ) , Las RNAi ( E ) , or Las RNAi in response to ALA administration ( F ) . esg - GFP ( green ) indicates ISCs and their differentiating cells . Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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D-F. Immunofluorescence images of the midgut section from the R4 region in Drosophila carrying esgts-GAL4-driven UAS-lacZ (D, control), Las RNAi (E), or Las RNAi in response to ALA administration (F). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


I - J . Immunofluorescence images of control ( FRT40A , I ) and Las RNAi ( J ) MARCM clones ( green , outlined by white dotted lines ) 10 days after clone induction ( ACI ) . Pdm1 staining ( red ) was used to visualize ECs . K . Quantification of the ratio of Pdm1 + cells per clone of MARCM clones with indicated genotypes . n is indicated . Each dot corresponds to one clone . L . Quantification of MARCM clone size of experiments in ( I - J ) . n is indicated . Each dot corresponds to one clone . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μm ( D Error bars represent SDs . Student ' s t - tests , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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I-J. Immunofluorescence images of control (FRT40A, I) and Las RNAi (J) MARCM clones (green, outlined by white dotted lines) 10 days after clone induction (ACI). Pdm1 staining (red) was used to visualize ECs. K. Quantification of the ratio of Pdm1+ cells per clone of MARCM clones with indicated genotypes. n is indicated. Each dot corresponds to one clone. L. Quantification of MARCM clone size of experiments in (I-J). n is indicated. Each dot corresponds to one clone. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm (D Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


A . Representative images and quantification of Drosophila midguts treated with the pH indicator Bromophenol Blue . Moreover , quantification of the three categories of Drosophila of indicated conditions . The three categories can be divided into : homeostasis , a well - defined acidic ( yellow colored ) copper cell region ( CCR ) is flanked by basic ( blue colored ) anterior midgut ( AM ) and posterior midgut ( PM ) . " Perturbed A " , the acidic region is lost and the whole gut is basic ; " Perturbed B " , the strongly acidic region is lost , and the remainder of the gut also becomes less basic . The numbers of quantified guts from left to right are 90 , 90 , and 90 . Error bars show the SD of three independent experiments .

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A. Representative images and quantification of Drosophila midguts treated with the pH indicator Bromophenol Blue. Moreover, quantification of the three categories of Drosophila of indicated conditions. The three categories can be divided into: homeostasis, a well-defined acidic (yellow colored) copper cell region (CCR) is flanked by basic (blue colored) anterior midgut (AM) and posterior midgut (PM). "Perturbed A", the acidic region is lost and the whole gut is basic; "Perturbed B", the strongly acidic region is lost, and the remainder of the gut also becomes less basic. The numbers of quantified guts from left to right are 90, 90, and 90. Error bars show the SD of three independent experiments.


C . Excretion of Drosophila treated with Bromophenol Blue . Images of deposits and quantification of deposit numbers are shown . Excretions are quantified in 30 fields for each group of 12 Drosophila . Tests were repeated as three independent experiments . Data information : The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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C. Excretion of Drosophila treated with Bromophenol Blue. Images of deposits and quantification of deposit numbers are shown. Excretions are quantified in 30 fields for each group of 12 Drosophila. Tests were repeated as three independent experiments. Data information: The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


G . Survival ( percentage ) of female W1118 Drosophila with and without supplementation of ALA as indicated . The numbers of quantified flies : 100 ( W1118 + 0 . 5 mM ALA ) and 100 ( W1118 + 0 mM ALA ) . Three independent experiments were conducted . Data information : Error bars represent SDs . p - values for lifespan curves were calculated by the log - rank test . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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G. Survival (percentage) of female W1118 Drosophila with and without supplementation of ALA as indicated. The numbers of quantified flies: 100 (W1118 + 0.5 mM ALA) and 100 (W1118 + 0 mM ALA). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan curves were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


I . Survival ( percentage ) of female Canton - S Drosophila with and without supplementation of ALA as indicated . The numbers of quantified flies : 100 ( Canton - S + 0 mM ALA ) , 100 ( Canton - S + 0 . 5 mM ALA ) . Three independent experiments were conducted . J . Survival ( percentage ) of female Canton - S Drosophila with and without supplementation of ALA at 26 - day as indicated . The numbers of quantified flies : 100 ( Canton - S + 0 mM ALA ) and 100 ( Canton - S + 0 . 5 mM ALA ) . Three independent experiments were conducted . Data information : Error bars represent SDs . p - values for lifespan curves ( were calculated by the log - rank test . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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

I. Survival (percentage) of female Canton-S Drosophila with and without supplementation of ALA as indicated. The numbers of quantified flies: 100 (Canton-S + 0 mM ALA), 100 (Canton-S + 0.5 mM ALA). Three independent experiments were conducted. J. Survival (percentage) of female Canton-S Drosophila with and without supplementation of ALA at 26-day as indicated. The numbers of quantified flies: 100 (Canton-S + 0 mM ALA) and 100 (Canton-S + 0.5 mM ALA). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan curves ( were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


A - F . Immunofluorescence images of midgut sections from the R4 region in Drosophila carrying esgts - GAL4 - driven expression of CAT cDNA ( A ) , CAT cDNA with AD4 administration ( B ) , CAT cDNA with ALA administration ( C ) , Keap1 RNAi ( D ) , Keap1 RNAi with AD4 administration ( E ) , or Keap1 RNAi with ALA administration ( F ) . esg - GFP ( green ) represents ISCs and their differentiating cells . Dl staining ( red ) was used to visualize ISCs . G . Quantification of the number of esg - GFP + cells , Dl + cells of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA , CAT cDNA , CAT cDNA with AD4 administration , CAT cDNA with ALA administration , Keap1 RNAi , Keap1 RNAi with AD4 administration , or Keap1 RNAi with ALA administration . n is indicated . The numbers of quantified guts from left to right are 18 , 19 , 20 , 18 , 18 , 19 , 19 , 18 , 19 , 20 , 18 , 18 , 19 , and 19 . H . Quantification of the number of pH3 + cells and luciferase activity of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA , CAT cDNA , CAT cDNA with AD4 administration , CAT cDNA with ALA administration , Keap1 RNAi , Keap1 RNAi with AD4 administration , or Keap1 RNAi with ALA administration . The numbers of quantified guts from left to right are 18 , 19 , 20 , 18 , 18 , 19 , and 19 . I - Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. Immunofluorescence images of midgut sections from the R4 region in Drosophila carrying esgts-GAL4-driven expression of CAT cDNA (A), CAT cDNA with AD4 administration (B), CAT cDNA with ALA administration (C), Keap1 RNAi (D), Keap1 RNAi with AD4 administration (E), or Keap1 RNAi with ALA administration (F). esg-GFP (green) represents ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. G. Quantification of the number of esg-GFP+ cells, Dl+ cells of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA, CAT cDNA, CAT cDNA with AD4 administration, CAT cDNA with ALA administration, Keap1 RNAi, Keap1 RNAi with AD4 administration, or Keap1 RNAi with ALA administration. n is indicated. The numbers of quantified guts from left to right are 18, 19, 20, 18, 18, 19, 19, 18, 19, 20, 18, 18, 19, and 19. H. Quantification of the number of pH3+ cells and luciferase activity of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA, CAT cDNA, CAT cDNA with AD4 administration, CAT cDNA with ALA administration, Keap1 RNAi, Keap1 RNAi with AD4 administration, or Keap1 RNAi with ALA administration. The numbers of quantified guts from left to right are 18, 19, 20, 18, 18, 19, and 19. I- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


I - L . Representative images of the midgut section from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( I ) , Las RNAi ( J ) , Las RNAi and CAT cDNA ( K ) , or Las RNAi and Keap1 RNAi ( L ) . esg - GFP ( green ) indicates ISCs and their differentiating cells . Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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

I-L. Representative images of the midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (I), Las RNAi (J), Las RNAi and CAT cDNA (K), or Las RNAi and Keap1 RNAi (L). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


A . Volcano plots of differentially expressed genes in pair - wise comparison of 40 - day Drosophila treated without ALA administration to 40 - day Drosophila treated with 0 . 5 mM ALA administration . Blue symbols indicate significantly downregulated mass bins ( Log2 FC < - 0 . 6 and p < 0 . 05 ) , red symbols indicate significantly upregulated mass bins ( Log2 FC > 0 . 6 and p < 0 . 05 ) , and gray symbols indicate mass bins that were not significantly changed .

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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Volcano plots of differentially expressed genes in pair-wise comparison of 40-day Drosophila treated without ALA administration to 40-day Drosophila treated with 0.5 mM ALA administration. Blue symbols indicate significantly downregulated mass bins (Log2 FC < -0.6 and p < 0.05), red symbols indicate significantly upregulated mass bins (Log2 FC > 0.6 and p < 0.05), and gray symbols indicate mass bins that were not significantly changed.


A - C . Immunofluorescence images of esg - GFP and Lysotracker staining with the midgut section from the R4 region in 14 - day WT flies ( A ) , 40 - day WT flies ( B ) , 40 - day WT flies with ALA administration ( C ) . esg - GFP ( green ; outlined by dotted lines ) , lysotracker ( red ) . esg - GFP + cells are outlined by red dotted lines in the images of lysotracker staining channel . D . Quantification of the dot number of Lysotracker in esg positive cells from experiments ( A - C ) . n is indicated . The numbers of quantified guts from left to right are 9 , 11 , and 8 . E . Quantification of the dot size of Lysotracker in esg positive cells from experiments ( A - C ) . n is as indicated . F - Data information : DAPI stained nuclei are shown in blue . Scale bars represent 5 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C. Immunofluorescence images of esg-GFP and Lysotracker staining with the midgut section from the R4 region in 14-day WT flies (A), 40-day WT flies (B), 40-day WT flies with ALA administration (C). esg-GFP (green; outlined by dotted lines), lysotracker (red). esg-GFP+ cells are outlined by red dotted lines in the images of lysotracker staining channel. D. Quantification of the dot number of Lysotracker in esg positive cells from experiments (A-C). n is indicated. The numbers of quantified guts from left to right are 9, 11, and 8. E. Quantification of the dot size of Lysotracker in esg positive cells from experiments (A-C). n is as indicated. F- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 5 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


F - H . Expression of esg - GAL4 - driven UAS - GFP - mCherry - Atg8a in 14 - day Drosophila ( F ) , 40 - day Drosophila ( G ) , 40 - day Drosophila with ALA administration started at the middle age ( 26 days ) ( H ) . GFP ( green ) and mCherry ( red ) . The red arrows indicate the autophagosomes . I . Quantification of the dot number of mCherry in esg positive cells from experiments ( F - H ) . n is indicated . The numbers of quantified guts from left to right are 11 , 10 , and 12 . J . Quantification of the dot size of mCherry in esg positive cells from experiments ( F - H ) . n is as indicated . K - Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H. Expression of esg-GAL4-driven UAS-GFP-mCherry-Atg8a in 14-day Drosophila (F), 40-day Drosophila (G), 40-day Drosophila with ALA administration started at the middle age (26 days) (H). GFP (green) and mCherry (red). The red arrows indicate the autophagosomes. I. Quantification of the dot number of mCherry in esg positive cells from experiments (F-H). n is indicated. The numbers of quantified guts from left to right are 11, 10, and 12. J. Quantification of the dot size of mCherry in esg positive cells from experiments (F-H). n is as indicated. K- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


K - L . Immunofluorescence images of esg - GFP and Lysotracker staining with the midgut section from the R4 region in 14 - day flies carrying esgts - GAL4 - driven UAS - lacZ ( K ) , and 14 - day flies carrying esgts - GAL4 - driven Las RNAi ( L ) . esg - GFP ( green ; outlined by dotted lines ) , lysotracker ( red ) . esg - GFP + cells are outlined by dotted lines . M . Quantification of the dot number of Lysotracker in esg positive cells from experiments ( K - L ) . n is indicated . The numbers of quantified guts from left to right are 10 , and 9 . N . Quantification of the dot size of Lysotracker in esg positive cells from experiments ( K - L ) . n is as indicated . O - Data information : DAPI stained nuclei are shown in blue . Scale bars represent 5 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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

K-L. Immunofluorescence images of esg-GFP and Lysotracker staining with the midgut section from the R4 region in 14-day flies carrying esgts-GAL4-driven UAS-lacZ (K), and 14-day flies carrying esgts-GAL4-driven Las RNAi (L). esg-GFP (green; outlined by dotted lines), lysotracker (red). esg-GFP+ cells are outlined by dotted lines. M. Quantification of the dot number of Lysotracker in esg positive cells from experiments (K-L). n is indicated. The numbers of quantified guts from left to right are 10, and 9. N. Quantification of the dot size of Lysotracker in esg positive cells from experiments (K-L). n is as indicated. O- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 5 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


O - P . Expression of esg - GAL4 - driven UAS - GFP - mCherry - Atg8a in 14 - day Drosophila carrying esgts - GAL4 - driven UAS - lacZ ( flies were cultured at 18 ° C and transferred to 29 ° C after flies eclosion ) ( O ) , and 14 - day Drosophila with Las depleted in ISCs and EBs ( flies were cultured at 18 ° C and transferred to 29 ° C after flies eclosion ) ( P ) . GFP ( green ) and mCherry ( red ) . The red arrows indicate the autophagosomes . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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

O-P. Expression of esg-GAL4-driven UAS-GFP-mCherry-Atg8a in 14-day Drosophila carrying esgts-GAL4-driven UAS-lacZ (flies were cultured at 18 °C and transferred to 29 °C after flies eclosion) (O), and 14-day Drosophila with Las depleted in ISCs and EBs (flies were cultured at 18 °C and transferred to 29 °C after flies eclosion) (P). GFP (green) and mCherry (red). The red arrows indicate the autophagosomes. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


A - C . Immunofluorescence images of midgut section from the R4 region in 40 - day flies ( A ) , 40 - day flies with spermidine administration started at 26th day after fly eclosion ( B ) , 40 - day flies with spermidine and ALA administration started at 26th day after fly eclosion ( C ) . esg - GFP ( green ) indicates ISCs and their differentiating cells . Dl ( red ) staining was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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

A-C. Immunofluorescence images of midgut section from the R4 region in 40-day flies (A), 40-day flies with spermidine administration started at 26th day after fly eclosion (B), 40-day flies with spermidine and ALA administration started at 26th day after fly eclosion (C). esg-GFP (green) indicates ISCs and their differentiating cells. Dl (red) staining was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


F - I . Representative images of the midgut section from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( F , control ) , Las RNAi ( G ) , Las RNAi and Atg5 cDNA ( H ) , or Las RNAi with spermidine administration ( I ) . GFP ( green ) and Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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

F-I. Representative images of the midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (F, control), Las RNAi (G), Las RNAi and Atg5 cDNA (H), or Las RNAi with spermidine administration (I). GFP (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


N . Survival ( percentage ) of female Drosophila with indicated genotypes . The numbers of quantified Drosophila : 100 ( UAS - lacZ ) , 100 ( Las RNAi ) , 100 ( Las RNAi + UAS - Atg5 ) , and 100 ( Las RNAi + UAS - Atg8a ) . Three independent experiments were conducted . Data information : Error bars represent SDs . p - values for lifespan curves ( N ) were calculated by the log - rank test . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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

N. Survival (percentage) of female Drosophila with indicated genotypes. The numbers of quantified Drosophila: 100 (UAS-lacZ), 100 (Las RNAi), 100 (Las RNAi + UAS-Atg5), and 100 (Las RNAi + UAS-Atg8a). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan curves (N) were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


A - E . Expressions of Rab5 - GFP reporter in ISCs of 14 - day WT Drosophila ( A ) , 40 - day WT Drosophila ( B ) , 40 - day Drosophila with ALA administration ( C ) , 14 - day Drosophila carrying esgts - GAL4 > UAS - lacZ ( D ) , and 14 - day Drosophila carrying esgts - GAL4 > Las RNAi ( E ) . GFP ( green ) . F . Quantification of fluorescence intensity of RAB5 - GFP in experiments ( A - E ) . Each dot corresponds to one cell . n is indicated . The numbers of quantified guts from left to right are 13 , 11 , 12 , 10 and 13 . G Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - tests , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E. Expressions of Rab5-GFP reporter in ISCs of 14-day WT Drosophila (A), 40-day WT Drosophila (B), 40-day Drosophila with ALA administration (C), 14-day Drosophila carrying esgts-GAL4>UAS-lacZ (D), and 14-day Drosophila carrying esgts-GAL4>Las RNAi (E). GFP (green). F. Quantification of fluorescence intensity of RAB5-GFP in experiments (A-E). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 13, 11, 12, 10 and 13. G Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


G - K . Expressions of Rab7 - GFP reporter in ISCs of 14 - day WT Drosophila ( G ) , 40 - day WT Drosophila ( H ) , 40 - day Drosophila with ALA administration ( I ) , 14 - day Drosophila carrying esgts - GAL4 > UAS - lacZ ( J ) , and 14 - day Drosophila carrying esgts - GAL4 > Las RNAi ( K ) . GFP ( green ) . L . Quantification of fluorescence intensity of RAB7 - GFP in experiments ( G - K ) . Each dot corresponds to one cell . n is indicated . The numbers of quantified guts from left to right are 11 , 10 , 11 , 13 and 12 . M Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - tests , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

G-K. Expressions of Rab7-GFP reporter in ISCs of 14-day WT Drosophila (G), 40-day WT Drosophila (H), 40-day Drosophila with ALA administration (I), 14-day Drosophila carrying esgts-GAL4>UAS-lacZ (J), and 14-day Drosophila carrying esgts-GAL4>Las RNAi (K). GFP (green). L. Quantification of fluorescence intensity of RAB7-GFP in experiments (G-K). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 10, 11, 13 and 12. M Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


M - Q . Immunofluorescence images of midgut section from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( M , control ) , constitutively active form of RAB5 ( N ) , constitutively active form of RAB5 with ALA administration ( O ) , constitutively active form of RAB7 ( P ) , or constitutively active form of RAB7 with ALA administration ( Q ) . GFP ( green ) and Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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

M-Q. Immunofluorescence images of midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (M, control), constitutively active form of RAB5 (N), constitutively active form of RAB5 with ALA administration (O), constitutively active form of RAB7 (P), or constitutively active form of RAB7 with ALA administration (Q). GFP (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


A - D . Representative images of midgut sections from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( A , control ) , Las RNAi ( B ) , Las RNAi and RAB5 - CA ( C ) , or Las RNAi and RAB7 - CA ( D ) . GFP ( green ) , Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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

A-D. Representative images of midgut sections from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (A, control), Las RNAi (B), Las RNAi and RAB5-CA (C), or Las RNAi and RAB7-CA (D). GFP (green), Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


C . Survival ( percentage ) of female Drosophila of indicated genotypes . The numbers of quantified Drosophila : 100 ( UAS - lacZ ) , 100 ( Las RNAi ) , 100 ( Las RNAi + UAS - Rab5 - CA ) and 100 ( Las RNAi + UAS - Rab7 - CA ) . Three independent experiments were conducted Data information : Error bars represent SDs . p - values for lifespan curves ( C ) were calculated by the log - rank test . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

O O O O 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival (percentage) of female Drosophila of indicated genotypes. The numbers of quantified Drosophila: 100 (UAS-lacZ), 100 (Las RNAi), 100 (Las RNAi + UAS-Rab5-CA) and 100 (Las RNAi + UAS-Rab7-CA). Three independent experiments were conducted Data information: Error bars represent SDs. p-values for lifespan curves (C) were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


F - I . Immunofluorescence images of dpERK staining of midgut sections of the R4 region of 40 - day Drosophila ( F ) , 40 - day Drosophila with ALA administration ( G ) , 14 - day Drosophila carrying esgts - GAL4 - driven UAS - lacZ ( H ) , and Drosophila carrying esgts - GAL4 - driven Las RNAi ( I ) . dpERK ( red ) . J . Quantification of the fluorescence intensity of dpERK in experiments ( F - I ) . Each dot corresponds to one cell . n is indicated . The numbers of quantified guts from left to right are 11 , 12 , 12 , and 13 . K Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μm Error bars represent SDs . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I. Immunofluorescence images of dpERK staining of midgut sections of the R4 region of 40-day Drosophila (F), 40-day Drosophila with ALA administration (G), 14-day Drosophila carrying esgts-GAL4-driven UAS-lacZ (H), and Drosophila carrying esgts-GAL4-driven Las RNAi (I). dpERK (red). J. Quantification of the fluorescence intensity of dpERK in experiments (F-I). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 12, 12, and 13. K Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm Error bars represent SDs. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


K - M . Representative images of midgut sections from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( K , control ) , Las RNAi ( L ) , or Las RNAi and dominant negative form of EGFR ( EGFR - DN , M ) . GFP ( green ) and Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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

K-M. Representative images of midgut sections from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (K, control), Las RNAi (L), or Las RNAi and dominant negative form of EGFR (EGFR-DN, M). GFP (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


D . Representative time - lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP - TMD ( ER ) ( green ) and SiR - lysosome ( orange ) . Zoom insets highlight regions of interest containing a static ( upper panels ) or mobile endosome ( lower panels ) as denoted by the white arrows . First row shows ER ( green ) , the second row shows MDA generated ER movement ( magenta ) , third row shows a merge of the two channels and the fourth row shows a merge of ER ( green ) and LE / Lys ( orange ) . Scale bars : full size 10µm , zoom inset 3µm . Images relate to movies EV2 and 3 . E . Kymograph of the mobile vesicle in panel ( D ) . F . MDA quantification of ( D ) . Mobility of static and mobile endosomes was quantified and normalized to the static endosomes . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 20 cells per condition from 3 independent experiments ) . All images acquired for 90 frames at 4Hz . Significance two - tailed student t - test , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (ER) (green) and SiR-lysosome (orange). Zoom insets highlight regions of interest containing a static (upper panels) or mobile endosome (lower panels) as denoted by the white arrows. First row shows ER (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Scale bars: full size 10µm, zoom inset 3µm. Images relate to movies EV2 and 3. E. Kymograph of the mobile vesicle in panel (D). F. MDA quantification of (D). Mobility of static and mobile endosomes was quantified and normalized to the static endosomes. Graph represents mean ± s.d. of analyses at multiple locations within the same cell(n=20 cells per condition from 3 independent experiments). All images acquired for 90 frames at 4Hz. Significance two-tailed student t-test, **** P<0.0001.


C . Representative time - lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP - TMD ( green ) marked ER and SiR - lysosome - stained LE / Lys ( orange ) . Zoom insets highlight regions of interest containing a mobile endosome as denoted by the white arrow . First row represents ER ( GFP - TMD ) ( green ) , the second row shows MDA generated ER movement ( magenta ) , third row shows a merge of the two preceding channels and the fourth row shows a merge of ER ( green ) and LE / Lys ( orange ) . Samples were treated with either DMSO or 2µm paclitaxel . Scale bar : 30µm and zoom insets 3µm . All images acquired for 90 frames at 1Hz . Images related to movie EV7 and 8 D . Kymograph of panel ( C ) . E . MDA quantification of panel ( C ) normalized to control cells , showing the average ER movement surrounding LE / Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 20 cells per condition from 3 independent experiments ) . Significance two - tailed student t - test * * * * P < 0 . 0001 ; ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) marked ER and SiR-lysosome-stained LE/Lys (orange) . Zoom insets highlight regions of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either DMSO or 2µm paclitaxel. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz.Images related to movie EV7 and 8 D. Kymograph of panel (C). E. MDA quantification of panel (C) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n = 20 cells per condition from 3 independent experiments). Significance two-tailed student t-test **** P<0.0001; ns = not significant.


F . Representative images of n = 3 of COS7 cells stably expressing GFP - TMD ( green ) to mark the ER and SiR - lysosome stained LE / Lys ( orange ) merged . Zoom insets highlight region of interest containing a mobile endosome as denoted by the white arrow . First row represents ER ( GFP - TMD ) ( green ) , the second row shows MDA generated ER movement ( magenta ) , third row shows a merge of the two preceding channels and the fourth row shows a merge of ER ( green ) and LE / Lys ( orange ) . Samples were treated with either siControl or siKTN1 . Scale bar : 30µm and zoom insets 3µm . All images acquired for 90 frames at 1Hz . Images related to movie EV9 and 10 G . Kymograph of panel ( F ) . H . MDA quantification of panel ( F ) normalized to control cells , showing the average ER movement surrounding LE / Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions , respectively . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 20 cells per condition from 3 independent experiments ) . Significance two - tailed student t - test . * * * * P < 0 . 0001 ; ns = not significant

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of n = 3 of COS7 cells stably expressing GFP-TMD (green) to mark the ER and SiR-lysosome stained LE/Lys (orange) merged. Zoom insets highlight region of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either siControl or siKTN1. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz. Images related to movie EV9 and 10 G. Kymograph of panel (F). H. MDA quantification of panel (F) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions, respectively. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=20 cells per condition from 3 independent experiments). Significance two-tailed student t-test. **** P<0.0001; ns = not significant


A . Representative time - lapse spinning disk confocal images of n = 3 live COS7 cells stably expressing mCherry - KDEL ( green ) and transiently expressing GFP - RAB7 ( orange ) . Zoom insets shows a region of interest containing a mobile RAB7 - positive endosome as denoted by the white arrows . First row represents the ER ( mCherry - KDEL ) ( green ) , the second row shows MDA - generated ER movement ( magenta ) , third row shows a merge of the two preceding channels and the fourth row shows a merge of ER ( green ) and GFP - RAB7 ( orange ) . Scale bar : 10µm , zoom insets 3µm . Images related to movie EV11 . Images were acquired for 90 frames at 1Hz .

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

A. Representative time-lapse spinning disk confocal images of n = 3 live COS7 cells stably expressing mCherry-KDEL (green) and transiently expressing GFP-RAB7 (orange). Zoom insets shows a region of interest containing a mobile RAB7-positive endosome as denoted by the white arrows. First row represents the ER (mCherry-KDEL) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and GFP-RAB7 (orange). Scale bar: 10µm, zoom insets 3µm. Images related to movie EV11. Images were acquired for 90 frames at 1Hz.


B . Kymograph of panel ( A ) . C . MDA quantification of panel ( A ) and figures EV2A - G normalized to 1 showing average ER movement surrounding static endosomes and mobile endosomes . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 15 cells per condition from 3 independent experiments ) . All images acquired for 90 frames at 1Hz . Significance two - tailed student t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Kymograph of panel (A). C. MDA quantification of panel (A) and figures EV2A-G normalized to 1 showing average ER movement surrounding static endosomes and mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. *P<0.05, ** P<0.01, **** P<0.0001 ns = not significant.


E . Representative time - lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP - TMD ( green ) and in lower panels transiently expressing mScarlet - RILP ( magenta ) and SiR - lysosome - stained LE / Lys ( orange ) . Zoom insets shows region of interest containing ER ( GFP - TMD ) . First row represents ER ( GFP - TMD ) ( green ) , the second row shows MDA - generated ER movement ( magenta ) , third row shows a merge of the two preceding channels . Scale bar : 10µm , zoom insets 3µm . Images related to movie EV19 and movie EV20 . Images were acquired for 90 frames at 1Hz . F . MDA quantification of panel ( E ) and figure EV3A normalized to 1 showing total average ER movement . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 15 cells per condition from 3 independent experiments ) . All images acquired 90 frames at 1Hz . Significance two - tailed student t - test . * * * * P < 0 . 0001 ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) and in lower panels transiently expressing mScarlet-RILP (magenta) and SiR-lysosome-stained LE/Lys (orange). Zoom insets shows region of interest containing ER (GFP-TMD). First row represents ER (GFP-TMD) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels. Scale bar: 10µm, zoom insets 3µm. Images related to movie EV19 and movie EV20. Images were acquired for 90 frames at 1Hz. F. MDA quantification of panel (E) and figure EV3A normalized to 1 showing total average ER movement. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired 90 frames at 1Hz. Significance two-tailed student t-test. **** P<0.0001 ns = not significant.


B . Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD - GFP ( green ) and SiR - lysosome - stained endosomes / lysosomes ( orange ) . COS7 cells in the middle panels overexpress mCherry - RILP ( magenta ) . The COS7 cells in the lower panels overexpress mCherry - TMEM55B ( magenta ) . Zoom insets show region of interest containing the ER ( TMD - GFP ) ( green ) and junction analysis of the skeletonized ER where the number of ER junctions representing the quantified average . Scale bars : 15µm . zoom insets : 3µm . C . Quantification of the number of ER junctions per µm2 from panel ( B ) as resulted from ER junction analysis . Graph represents mean ± s . d . of analyses at multiple locations within one cell ( n = 15 from 3 independent experiments ) . Significance two - tailed student t - test . * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange). COS7 cells in the middle panels overexpress mCherry-RILP (magenta). The COS7 cells in the lower panels overexpress mCherry-TMEM55B (magenta). Zoom insets show region of interest containing the ER (TMD-GFP) (green) and junction analysis of the skeletonized ER where the number of ER junctions representing the quantified average. Scale bars: 15µm. zoom insets: 3µm. C. Quantification of the number of ER junctions per µm2 from panel (B) as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell (n=15 from 3 independent experiments). Significance two-tailed student t-test. ***P<0.001.


A . Representative images of time - lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP - TMD ( green ) and SiR - lysosome - stained LE / Lys ( orange ) treated with either siControl or co - treated with siVAPA , siVAPB and siMOSPD2 . Zoom insets show regions of interest containing mobile LE / Lys as denoted by the white arrow . First row represents ER ( TMD - GFP ) ( green ) , the second row shows MDA generated ER movement ( magenta ) , third row shows a merge of the two preceding channels and the fourth row shows a merge of ER ( green ) and SiR - lysosome stained LE / Lys ( orange ) . Scale bar : 5µm , zoom insets 3µm . Images related to movie EV40 and 47 . Images were acquired for 90 frames at 1Hz .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (green) and SiR-lysosome-stained LE/Lys (orange) treated with either siControl or co-treated with siVAPA, siVAPB and siMOSPD2. Zoom insets show regions of interest containing mobile LE/Lys as denoted by the white arrow. First row represents ER (TMD-GFP) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and SiR-lysosome stained LE/Lys (orange). Scale bar: 5µm, zoom insets 3µm. Images related to movie EV40 and 47. Images were acquired for 90 frames at 1Hz.


B . Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD - GFP ( green ) and SiR - lysosome - stained endosomes / lysosomes ( orange ) treated with siControl or with siVAPA , siVAPB and siMOSPD2 . Zoom insets show region of interest containing ER ( TMD - GFP ) ( green ) and junction analysis of skeletonized ER where number of ER junctions representing the quantified average . Scale bars : 10µm . zoom insets : 3µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange) treated with siControl or with siVAPA, siVAPB and siMOSPD2. Zoom insets show region of interest containing ER (TMD-GFP) (green) and junction analysis of skeletonized ER where number of ER junctions representing the quantified average. Scale bars: 10µm. zoom insets: 3µm.


C . MDA quantification of ER of panels ( A , B ) and fig . EV4B - G normalized to control cells showing average ER movement surrounding mobile endosomes . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 30 cells per condition from 3 independent experiments ) . All images acquired for 90 frames at 1Hz . Significance two - tailed student t - test . * * P < 0 . 01 , * * * * P < 0 . 0001 , ns = not significant D . MDA quantification of SiR - lysosome - stained LE / Lys of ( A , B ) and fig . EV4B - G normalized to 1 showing total average endosomal movement . Graph represents mean ± s . d . of n = 15 cells per condition from 3 independent experiments . Significance two tailed student t - test . ns = not significant . E . Quantification of number of ER junctions per µm2 from ( B ) and fig . H - M as resulted from ER junction analysis . Graph represents mean ± s . d . of analyses at multiple locations within one cell of n = 15 from 3 independent experiments . Significance two - tailed student t - test . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MDA quantification of ER of panels (A, B) and fig. EV4B-G normalized to control cells showing average ER movement surrounding mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=30 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. **P<0.01, ****P< 0.0001, ns=not significant D. MDA quantification of SiR-lysosome-stained LE/Lys of (A, B) and fig. EV4B - G normalized to 1 showing total average endosomal movement. Graph represents mean ±s.d. of n = 15 cells per condition from 3 independent experiments. Significance two tailed student t-test. ns= not significant. E. Quantification of number of ER junctions per µm2 from (B) and fig. H - M as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell of n = 15 from 3 independent experiments. Significance two-tailed student t-test. *P<0.05, **P<0.01 ***P<0.001, ****P< 0.0001, ns=not significant.


ASPC - 1 , MiaPaCa - 2 , Colo - 357 , Panc - 1 , Panc - 89 and PancTu - 1 cells were grown in the absence or presence of the indicated concentrations of omeprazole , 5 - fluorouracile , pantoprazole or gemcitabine , respectively , for 4 days to determine the IC50 values . The IC50 ies and the sigmoidicities are gradually differing between the cell lines . In lower concentrations a slight growth - stimulatory effect ( hormesis ) was observed .

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

ASPC-1, MiaPaCa-2, Colo-357, Panc-1, Panc-89 and PancTu-1 cells were grown in the absence or presence of the indicated concentrations of omeprazole, 5-fluorouracile, pantoprazole or gemcitabine, respectively, for 4 days to determine the IC50 values. The IC50 ies and the sigmoidicities are gradually differing between the cell lines. In lower concentrations a slight growth-stimulatory effect (hormesis) was observed.


The cell lines ASPC - 1 , MiaPaCa - 2 , Colo357 , Panc - 1 , Panc89 and PancTu1 were untreated or treated with 5 - FU alone or in combination with the indicated concentrations of OMP for 4 days . At lower doses of 5 - FU alone ( black lines , 0 µg / ml OMP ) a growth - stimulatory effect ( hormesis ) was observed in the cell lines ASPC - 1 , Panc - 1 and PancTu - 1 . The data points show the means of 8 measurements . The curves are fitted in these cell lines using the Brain - Cousens model ( see sub - section 4 . 11 ) . In MiaPaCa - 2 and Panc - 89 cells no hormesis occured , these curves were fitted using a three parameter logistic model . In Colo357 cells the curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations ( data points shown ) . The red , green and blue lines indicate the dose - effect curves of 5 - FU when various concentrations of OMP were added ( 10 , 20 and 40 µg / ml , respectively ) . In ASPC - 1 and Panc - 1 cells the hormesis of 5 - FU was reversed and in PancTu - 1 cell it was mitigated by OMP depending on the concentration of 5 - FU . In MiaPaCa - 2 cells we found an additive interaction of 5 - FU with OMP . In Panc - 89 cells the interaction was antagonistic .

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

The cell lines ASPC-1, MiaPaCa-2 , Colo357, Panc-1, Panc89 and PancTu1 were untreated or treated with 5-FU alone or in combination with the indicated concentrations of OMP for 4 days. At lower doses of 5-FU alone (black lines, 0 µg/ml OMP) a growth-stimulatory effect (hormesis) was observed in the cell lines ASPC-1, Panc-1 and PancTu-1. The data points show the means of 8 measurements. The curves are fitted in these cell lines using the Brain-Cousens model (see sub-section 4.11). In MiaPaCa-2 and Panc-89 cells no hormesis occured, these curves were fitted using a three parameter logistic model. In Colo357 cells the curves could not be fitted by any pharmacodynamic model due to large standard errors at these lower concentrations (data points shown). The red, green and blue lines indicate the dose-effect curves of 5-FU when various concentrations of OMP were added (10, 20 and 40 µg/ml, respectively). In ASPC-1 and Panc-1 cells the hormesis of 5-FU was reversed and in PancTu-1 cell it was mitigated by OMP depending on the concentration of 5-FU. In MiaPaCa-2 cells we found an additive interaction of 5-FU with OMP. In Panc-89 cells the interaction was antagonistic.


Acridine Orange was added to living untreated ASPC - 1 and MiaPaCa - 2 cells and cells treated with 5 - FU , OMP or the combination of both for 30 minutes or 24 hours . Microscopical life images were taken at 525 nm ( green ) and 650 nm ( red ) to detect changes in the lysosomal pH value ( three images per plate , three plates per group ) . The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann - Whitney - U - test . Significant differences compared to control are marked by * . In ASPC - 1 cells , after 30 min of treatment , intralysosomal acidity increased upon treatment with OMP ( p : 0 . 0051 ) and 5 - FU + OMP ( p < 0 . 0001 ) . After 24 hours , the acidity is increased upon all treatment regimens ( 5 - FU - p : 0 . 0002 ; OMP - p < 0 . 00001 ; OMP + 5 - FU - p : 0 . 037 ) . In MiaPaCa - 2 cells the acidity is elevated after 30 min upon 5 - FU ( p : 0 . 005 ) and decreased after treatment with OMP ( p : 0 . 037 ) , 5 - FU ( p : 0 . 00026 ) and 5 - FU + OMP ( p : 0 . 011 ) after 24 hours .

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

Acridine Orange was added to living untreated ASPC-1 and MiaPaCa-2 cells and cells treated with 5-FU , OMP or the combination of both for 30 minutes or 24 hours. Microscopical life images were taken at 525 nm (green) and 650 nm (red) to detect changes in the lysosomal pH value (three images per plate, three plates per group). The red to green fluorescence ratio of the lysosomes of treated cells were compared to the control groups by the Mann-Whitney-U-test. Significant differences compared to control are marked by *. In ASPC-1 cells, after 30 min of treatment, intralysosomal acidity increased upon treatment with OMP (p:0.0051) and 5-FU+OMP (p<0.0001). After 24 hours, the acidity is increased upon all treatment regimens (5-FU - p:0.0002; OMP - p<0.00001; OMP+5-FU - p:0.037). In MiaPaCa-2 cells the acidity is elevated after 30 min upon 5-FU (p:0.005) and decreased after treatment with OMP (p:0.037), 5-FU (p:0.00026) and 5-FU+OMP (p:0.011) after 24 hours.


( A ) ASPC - 1 cell without treatment ( 800 fold ) . ( B ) ASPC - 1 cell undergoing apoptosis upon 160 µg / ml OMP after 24 hours ( 800 fold ) . Vacuolisation of the cytoplasma and condensation of the nucleus are visible . ( C ) Phagophores and autophagosomes in a segment of an ASPC - 1 cell treated with omeprazole 80 µg / ml for 24 hours ( 2800fold enlargement ) . The phagophores are characterised by a cup - like shape ( white arrows ) . Autophagosomes are closed particles , the number of which is increased in treated cells ( black arrows ) . ( D ) Early phagophores and autophagosomes are also found in MiaPaCa - 2 cells treated with OMP 80 µg / ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex . In contrast to ASPC - 1 cells , early signs of apoptosis such as vacuolization , are also present .

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

(A) ASPC-1 cell without treatment (800 fold). (B) ASPC-1 cell undergoing apoptosis upon 160 µg/ml OMP after 24 hours (800 fold). Vacuolisation of the cytoplasma and condensation of the nucleus are visible. (C) Phagophores and autophagosomes in a segment of an ASPC-1 cell treated with omeprazole 80 µg/ml for 24 hours (2800fold enlargement). The phagophores are characterised by a cup-like shape (white arrows). Autophagosomes are closed particles, the number of which is increased in treated cells (black arrows). (D) Early phagophores and autophagosomes are also found in MiaPaCa-2 cells treated with OMP 80 µg/ml after 24 hours in a perinuclear region containing lysosomes and the Golgi complex. In contrast to ASPC-1 cells, early signs of apoptosis such as vacuolization, are also present.


( E ) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa - 2 and ASPC - 1 cells untreated or treated with 5 - FU , OMP or the combination of both for 24 hours with standard errors . Significant differences compared to control are marked by * . In ASPC - 1 cells there were significant differences compared to the control in the OMP group ( p : 0 . 03 ) and the 5 - FU + OMP group ( p : 0 . 03 ) . In MiaPaCa - 2 cells the 5 - FU + OMP group differed significantly from control ( p < 0 . 001 ) .

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

(E) Barchart of the numbers of autophagosomes and lysosomes per cell in MiaPaCa-2 and ASPC-1 cells untreated or treated with 5-FU, OMP or the combination of both for 24 hours with standard errors. Significant differences compared to control are marked by *. In ASPC-1 cells there were significant differences compared to the control in the OMP group (p: 0.03) and the 5-FU+OMP group (p: 0.03). In MiaPaCa-2 cells the 5-FU+OMP group differed significantly from control (p<0.001).


The ASPC - 1 cell line is shown without and upon various treatment regimens ( OMP , 5 - FU or 5 - FU + OMP combination ) . The nodes of this network symbolize metabolite signals , their colours correspond to relative signal intensity ( when compared to an external standard ) as indicated in the heatmap scale below . The signal intensity is linearly related to the intracellular concentration . The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale . The lines between the boxes symbolize strongly simplified metabolic pathways . The colours of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange ( p < 0 . 05 ) , red lines indicate p < 0 . 01 . The most obvious changes is that the PC / Cho ratio is significantly lower in the OMP group compared to the control group . Upon 5 - FU , the Cho / Acetate ratio decrease is the only significant change . In the 5 - FU + OMP group , there are several significant changes , i . e . the FACH2 / CH = CH ratio is significantly higher . Moreover , the Cho / Acetate ratio changed upon 5 - FU + OMP as in the 5 - FU group , but also the citrate / GSH ratio . The cellular biochemical effects involve mainly the fatty acid and phospholipid metabolism pointing to membrane anabolism . Abbreviations : Gln - glutamine , Ala - alanine , PC - phosphatidylcholine , Cho - Choline , Lac1 + FACH2 - methyl group signal of lactate and methylene groups of the fatty acids , Lac2 - methylene group of lactate , CH = CH - protons of methin groups of unsaturated fatty acids .

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

The ASPC-1 cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colours of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. The most obvious changes is that the PC/Cho ratio is significantly lower in the OMP group compared to the control group. Upon 5-FU, the Cho/Acetate ratio decrease is the only significant change. In the 5-FU+OMP group, there are several significant changes, i.e.the FACH2/CH = CH ratio is significantly higher. Moreover, the Cho/Acetate ratio changed upon 5-FU+OMP as in the 5-FU group, but also the citrate/GSH ratio. The cellular biochemical effects involve mainly the fatty acid and phospholipid metabolism pointing to membrane anabolism. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline, Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.


The cell line is shown without and upon various treatment regimens ( OMP , 5 - FU or 5 - FU + OMP combination ) . The nodes of this network symbolize metabolite signals , their colours correspond to relative signal intensity ( when compared to an external standard ) as indicated in the heatmap scale below . The signal intensity is linearly related to the intracellular concentration . The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale . The lines between the boxes symbolize strongly simplified metabolic pathways . The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange ( p < 0 . 05 ) , red lines indicate p < 0 . 01 . Upon OMP , the PC / Cho ratios are significantly lower compared to control . Furthermore the acetate / FACH2 ratio is significantly decreased in the OMP group . The latter also showed a higher CH = CH level , the ratio to FACH2 is , however , decreased . Upon 5 - FU and 5 - FU + OMP , similar changes could be observed . Furthermore , in contrast to ASPC - 1 cells , the Ala / Gln / AMP pathway is also involved . Abbreviations : Gln - glutamine , Ala - alanine , PC - phosphatidylcholine , Cho - Choline . Lac1 + FACH2 - methyl group signal of lactate and methylene groups of the fatty acids , Lac2 - methylene group of lactate , CH = CH - protons of methin groups of unsaturated fatty acids .

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

The cell line is shown without and upon various treatment regimens (OMP, 5-FU or 5-FU+OMP combination). The nodes of this network symbolize metabolite signals, their colours correspond to relative signal intensity (when compared to an external standard) as indicated in the heatmap scale below. The signal intensity is linearly related to the intracellular concentration. The background of the nodes are left blank when the signal intensity is out of the range indicated by the heatmap scale. The lines between the boxes symbolize strongly simplified metabolic pathways. The colors of these lines indicate significant differences of the signal intensity ratios of the connected metabolites compared to the control group when orange (p<0.05), red lines indicate p<0.01. Upon OMP, the PC/Cho ratios are significantly lower compared to control. Furthermore the acetate/FACH2 ratio is significantly decreased in the OMP group. The latter also showed a higher CH = CH level, the ratio to FACH2 is, however, decreased. Upon 5-FU and 5-FU+OMP, similar changes could be observed. Furthermore, in contrast to ASPC-1 cells, the Ala/Gln/AMP pathway is also involved. Abbreviations: Gln - glutamine, Ala - alanine, PC - phosphatidylcholine, Cho - Choline. Lac1+FACH2 - methyl group signal of lactate and methylene groups of the fatty acids, Lac2 - methylene group of lactate, CH = CH - protons of methin groups of unsaturated fatty acids.


( B ) Western blot analysis of LAMP - 1 , a late endosome marker , which was nearly identically distributed over the first two fractions compared to Cathepsin - D , in the controle group , but ocurred only in the first fraction in the OMP treated group . β - COP as an indicator of the Golgi - complex , is found strongly in the lower fraction in the control group , but very weakly in the OMP treated group . Cathepsin - D , an early endosome marker , which can be found in the upper and middle fraction of the control group , but regarding the OMP group , it was only found in the first one .

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

(B) Western blot analysis of LAMP-1, a late endosome marker, which was nearly identically distributed over the first two fractions compared to Cathepsin-D, in the controle group, but ocurred only in the first fraction in the OMP treated group. β-COP as an indicator of the Golgi-complex, is found strongly in the lower fraction in the control group, but very weakly in the OMP treated group. Cathepsin - D, an early endosome marker, which can be found in the upper and middle fraction of the control group, but regarding the OMP group, it was only found in the first one.