( I , J ) Left are representative traces showing D - CPP - sensitive currents recorded from cultured hippocampal neurons ( I ) or slices ( J ) incubated in 0Mg or Pic , respectively , with or without Cyclo . Black lines : application of D - CPP . Right are statistics of D - CPP - sensitive current density from 18 - 26 ( I ) or 16 - 18 slices ( 15 rats ) ( J ) . Unless stated , 10 µM cyclopamine was used . Data were mean + . * p < 0 . 05 , * * < 0 . 01 , * * * p < 0 . 001 vs . Ctrl ; # p < 0 . 05 , # # < 0 . 01 , # # # p < 0 . 001 vs . 0Mg or Pic ; & amp ; amp ; p < 0 . 05 vs . 0Mg + IgG . C - E used one - way ANOVA . H - J used student ' s t .

O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

(I,J) Left are representative traces showing the D-CPP-sensitive currents recorded from cultured hippocampal neurons (I) or slices (J) incubated in 0Mg or Pic, respectively, with or without Cyclo. Black lines: application of D-CPP. Right are statistics of D-CPP-sensitive current density from 18-26 cells (I) or 16-18 slices (15 rats) (J). Unless stated, 10 µM cyclopamine was used. Data were mean + SEM. *p<0.05,**<0.01,***p<0.001 vs. Ctrl; #p<0.05,##<0.01,###p<0.001 vs. 0Mg or Pic; &amp;p<0.05 vs. 0Mg + IgG. C-E used one-way ANOVA. H-J used student's t test.


( A ) Glutamate assayed by HPLC in medium of neurons incubated with indicated agents . n = 4 - 6 .

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(A) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents. n=4-6.


( B ) paired - of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle ( Ctrl ) or Shh ( 13 - 14 slices , 6 rats ) . Insets : representative traces recorded in response to paired stimuli with different intervals .

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(B) The paired-pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle (Ctrl) or Shh (13-14 slices, 6 rats). Insets: representative traces recorded in response to paired pulse stimuli with different intervals.


Statistics of 3H - glutamate uptake by neurons treated with indicated agents ( C , n = 10 )

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Statistics of 3H-glutamate uptake by neurons treated with indicated agents (C, n=10)


Statistics of 3H - glutamate uptake by neurons transfected with two lentivirus - RNAi against EAAC1 ( R2 R3 ) or nonsense RNAi ( Non ) in response to vehicle ( Ctrl ) or Shh ( D , n = 3 - 6 ) . No difference between R2 or R3 in Ctrl those in Shh . Inset of ( D ) : representative western - blots for EAAC1 .

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Statistics of 3H-glutamate uptake by neurons transfected with two lentivirus-based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n=3-6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative western-blots for EAAC1.


Aspartate ( Asp ) - evoked currents at - 70 mV from hippocampal neurons transfected with RNAi or Non ( E , n = 17 - 23 ) or treated with indicated agents ( F , n = 16 - 18 ) .

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Aspartate (Asp)-evoked currents at -70 mV from hippocampal neurons transfected with RNAi or Non (E, n=17-23) or treated with indicated agents (F, n=16-18).


( G ) Upper , representative western - blots of EAAC1 Smo from HEK293 transfected with empty vectors ( Ctrl ) , constitutively active form of Smoothened ( SmoA1 ) , EAAC1 , or EAAC1 plus SmoA1 . Lower , 3H - glutamate uptake by transfected with indicated vectors . n = 9 .

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(G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9.


( H , I ) Shh effects on 3H - glutamate uptake ( H , n = 6 ) or Asp - evoked currents ( I , n = 17 - 23 ) of neurons with or without pertussis toxin ( PTX ) pretreatment . For E , F , I , uppers : representative traces . Shh : 500 ng / mL . Data were mean ± . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Ctrl or Non ( Ctrl ) with student ' s t .

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(H,I) Shh effects on 3H-glutamate uptake (H, n=6) or Asp-evoked currents (I, n=17-23) of neurons with or without pertussis toxin (PTX) pretreatment. For E,F,I, the uppers: representative traces. Shh: 500 ng/mL. Data were mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl or Non (Ctrl) with student's t test.


Effects of Cyclo ( A - C , n = 14 ) on progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) number of stimulations required to reach equivalent seizure ( C , F , I , L ) . In A - F , Ctrl are vehicle - treated mice .

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Effects of Cyclo (A-C, n=14) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.


Effects of 5E1 ( D - F , n = 16 - 20 ) , on progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) number of stimulations required to reach equivalent seizure ( C , F , I , L ) . In A - F , Ctrl are vehicle - treated mice .

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Effects of 5E1 (D-F, n=16-20), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.


Effects of ablation of Smo in CaMKIIα - positive neurons ( G - I ) , on progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) number of stimulations required to reach equivalent seizure ( C , F , I , L ) . In G - I , Ctrl : Smofl / fl induced by tamoxifen ; CaMK : Smofl / flCaMKIIα - CreERT2 induced by tamoxifen , n = 14 - 20 .

O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O

Effects of ablation of Smo in CaMKIIα-positive neurons (G-I), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In G-I, Ctrl: Smofl/fl induced by tamoxifen; CaMK: Smofl/flCaMKIIα-CreERT2 induced by tamoxifen, n=14-20.


Effects of ablation of Smo in Aldh1l1 - positive astrocytes ( J - L ) on progression of kindling including seizure class ( A , D , G , J ) , evoked electrographic seizure duration ( ESD ) ( B , E , H , K ) number of stimulations required to reach equivalent seizure ( C , F , I , L ) . In J - L , Ctrl : Smo + / fl ; Aldh1l1 : Smo + / flAldh1l1 - Cre , n = 19 - 20 .

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Effects of ablation of Smo in Aldh1l1-positive astrocytes (J-L) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In J-L, Ctrl: Smo+/fl; Aldh1l1: Smo+/flAldh1l1-Cre, n=19-20.


( M ) Frequency of spontaneous seizures ( class 4 - 5 ) in mice administrated with Cyclo or vehicle ( Ctrl ) at 4th , 6th 8th week after pilocarpine SE induction . n = 22 - 23 .

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(M) Frequency of spontaneous seizures (class 4-5) in mice administrated with Cyclo or vehicle (Ctrl) at 4th, 6th and 8th week after pilocarpine SE induction. n=22-23.


( N ) of mice with class 5 seizures within 8 after pilocarpine SE induction . n = 22 - 23 . Cyclo : 10 mg / kg . 5E1 : 900 ng / mouse . Data were mean ± from at least three independent experiments . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 vs . Ctrl with student ' s t .

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(N) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction. n=22-23. Cyclo: 10 mg/kg. 5E1: 900 ng/mouse. Data were mean ± SEM from at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl with student's t test.


Morphological changes of vacuolar membranes peroxisomal GFP - SKL during microautophagic degradation of peroxisomes after glucose adaptation . Double of methanol - grown of P . pastoris at various times after switch to glucose medium . ( a ) 0 h , ( b ) 30 min , ( c ) 2 h .

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Morphological changes of vacuolar membranes and peroxisomal GFP-SKL during microautophagic degradation of peroxisomes after glucose adaptation. Double fluorescence of methanol-grown cells of P. pastoris cells at various times after the switch to glucose medium. (a) 0 h, (b) 30 min, (c) 2 h.


electron microscope showing various stages during microautophagic degradation of peroxisomes . three panels in each figure are as follows ( a - e , left ) FM4 - 64 ; ( center ) GFP - SKL ; ( right ) superimposed FM4 - 64 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 capture of these , this peroxisome - containing vesicle had just pinched off from outer vacuolar . Therefore , FM4 - 64 - stained vesicle GFP - SKL could not be superimposed in this picture .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O

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 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 . were counted in three randomly selected fields , standard deviations are shown as vertical bars .

O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O

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 to ethanol medium . ( a - e ) Electron 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 e . ( b ) After 1 h of ethanol adaptation , autophagosomal surrounding peroxisomes appears to have fused with vacuolar . ( c ) At 2 h a peroxisome in form of an autophagic is seen within vacuole . Magnified pictures of samples obtained at ( d ) 20 min time point showing endoplasmic reticulum in close proximity to membranes surrounding peroxisomes , at ( e ) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes . ( f ) Kinetics of morphological changes in vacuoles peroxisomal GFP - SKL . Other conditions are described in Materials Methods , are same as in Fig . 4 . ( g ) taken after 2 h of ethanol adaptation of wild - type P . pastoris .

O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O O O 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-SMALL_MOLECULE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O

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 , degradation in , vacuoles during ( a ) glucose adaptation ( b ) ethanol adaptation . Wortmannin PMSF were dissolved in DMSO , added to 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 .

O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O

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 ( STK7 ) of P . pastoris . Kinetics of morphological changes in vacuoles peroxisomal GFP - SKL during ( a ) glucose adaptation ( b ) ethanol adaptation . ( c ) taken after 2 h of glucose adaptation . Arrows show GFP - SKL in vacuolar matrix . Some showed diffusion of GFP - SKL into vacuolar matrix .

O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O

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

O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O B-ORGANISM O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

(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.


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


of pag4 ( STK4 ) mutant of P . pastoris after ( a ) 0 , ( b ) 3 , or ( c ) 6 h of glucose adaptation . vacuolar proliferated extensively 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 ( SJH242 ) of P . pastoris expressing GFP - SKL from constitutive glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) promoter at restrictive for peroxisome assembly ( 34 ° C ) . ( a ) Kinetics of peroxisome degradation at 34 ° C . SJH242 was induced on methanol at 23 ° C , glucose adaptation was performed at 34 ° C . ( b ) Changes in vacuole were seen even in absence of normal peroxisomes . At 0 h , SJH242 were grown at 34 ° C when GFP - SKL was diffuse in cytosol many vacuoles showed spherical morphologies . After 2 h , ∼ 20 - 30 % vacuoles showed complicated structures representative of stage 2 .

O O B-SUBCELLULAR O O B-GENEPROD O O O O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O

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 presence of increasing concentrations of WT or NN . Tubulin = 5 μM . Each point represents an average of 3 independent measurements of 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 YT are Y values ( OD500 / min ) at bottom top plateaus , respectively , Xi is X value at inflection point . Representative MT samples from 2 : 5 μM WT : tubulin or NN : tubulin incubations are shown in E F , respectively . MT architecture distributions for WT NN are shown with pie graphs .

O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

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 high magnification of MTs nucleated for 10 min at 37 ° C from 5 μM tubulin , in 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 .

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H. Representative kymographs with arrowheads indicating MT catastrophes.


A . Representative low - pass filtered cryo - EM 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 were added in excess after MT deposition on EM grids to maximise MT decoration . final of WT or NN to tubulin was 50 : 5 μM , giving dense background . WT co - polymerized exclusively 13 - PF MTs , while NN both 13 - 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 NN - MTs are in GDP state .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-GENEPROD 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-GENEPROD O O O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O

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

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR O O O O O

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 midguts of aged Drosophila . Loading controls , β - tubulin . K . of LAS band as seen in experiments ( J ) . Error bars indicate SD of three independent experiments . L Data information : Error bars represent SDs . Student ' s t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , 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 . of content of ALA in midguts of flies with indicated genotypes using LC - ESI - MS / MS . Error bars indicate SD of three independent experiments . Data information : Error bars represent SDs . Student ' s t - , * * * * p < 0 . 0001 , 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 of midgut section from 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 their differentiating . Dl ( 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 of control ( FRT40A , I ) Las RNAi ( J ) MARCM clones ( green , outlined by dotted lines ) 10 days after clone induction ( ACI ) . Pdm1 ( red ) was used to visualize ECs . K . of of Pdm1 + per clone of MARCM clones with indicated genotypes . n is indicated . Each dot corresponds to one clone . L . of MARCM clone 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 - , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

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

O O O O B-ORGANISM B-TISSUE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O O O O O O 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-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O

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 . of deposits of deposit numbers are shown . Excretions are quantified in 30 fields for each group of 12 Drosophila . were repeated as three independent experiments . Data information : statistical used in other panels were student ' s t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , 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 ( ) of W1118 Drosophila with without supplementation of ALA as indicated . numbers of quantified flies : 100 ( W1118 + 0 . 5 mM ALA ) 100 ( W1118 + 0 mM ALA ) . Three independent experiments were conducted . Data information : Error bars represent SDs . p - values for lifespan curves were calculated by log - rank . statistical used in other panels were student ' s t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

O O B-EXP_ASSAY O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. 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 ( ) of Canton - S Drosophila with without supplementation of ALA as indicated . numbers of quantified flies : 100 ( Canton - S + 0 mM ALA ) , 100 ( Canton - S + 0 . 5 mM ALA ) . Three independent experiments were conducted . J . Survival ( ) of Canton - S Drosophila with without supplementation of ALA at 26 - day as indicated . numbers of quantified flies : 100 ( Canton - S + 0 mM ALA ) 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 log - rank . statistical used in other panels were student ' s t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of midgut sections from 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 their differentiating . Dl ( red ) was used to visualize ISCs . G . of number of esg - GFP + , Dl + 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 . numbers of quantified guts from left to right are 18 , 19 , 20 , 18 , 18 , 19 , 19 , 18 , 19 , 20 , 18 , 18 , 19 , 19 . H . of number of pH3 + 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 . numbers of quantified guts from left to right are 18 , 19 , 20 , 18 , 18 , 19 , 19 . I - Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μm Error bars represent SDs . Student ' s t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

O O O O B-EXP_ASSAY O B-TISSUE O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O 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-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-TISSUE 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-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of midgut section from R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( I ) , Las RNAi ( J ) , Las RNAi CAT cDNA ( K ) , or Las RNAi Keap1 RNAi ( L ) . esg - GFP ( green ) indicates ISCs their differentiating . Dl ( 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 B-TISSUE O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR 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 p < 0 . 05 ) , red symbols indicate significantly upregulated mass bins ( Log2 FC > 0 . 6 p < 0 . 05 ) , 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 B-ORGANISM O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of esg - GFP Lysotracker with midgut section from 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 + are outlined by red dotted lines in of lysotracker channel . D . of dot number of Lysotracker in esg positive from experiments ( A - C ) . n is indicated . numbers of quantified guts from left to right are 9 , 11 , 8 . E . of dot of Lysotracker in esg positive 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 - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 middle ( 26 days ) ( H ) . GFP ( green ) mCherry ( red ) . red arrows indicate autophagosomes . I . of dot number of mCherry in esg positive from experiments ( F - H ) . n is indicated . numbers of quantified guts from left to right are 11 , 10 , 12 . J . of dot of mCherry in esg positive 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 - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of esg - GFP Lysotracker with midgut section from R4 region in 14 - day flies carrying esgts - GAL4 - driven UAS - lacZ ( K ) , 14 - day flies carrying esgts - GAL4 - driven Las RNAi ( L ) . esg - GFP ( green ; outlined by dotted lines ) , lysotracker ( red ) . esg - GFP + are outlined by dotted lines . M . of dot number of Lysotracker in esg positive from experiments ( K - L ) . n is indicated . numbers of quantified guts from left to right are 10 , 9 . N . of dot of Lysotracker in esg positive 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 - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-SMALL_MOLECULE O B-TISSUE O O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 transferred to 29 ° C after flies eclosion ) ( O ) , 14 - day Drosophila with Las depleted in ISCs EBs ( flies were cultured at 18 ° C transferred to 29 ° C after flies eclosion ) ( P ) . GFP ( green ) mCherry ( red ) . red arrows indicate autophagosomes . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of midgut section from 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 ALA administration started at 26th day after fly eclosion ( C ) . esg - GFP ( green ) indicates ISCs their differentiating . Dl ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR 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 of midgut section from R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( F , control ) , Las RNAi ( G ) , Las RNAi Atg5 cDNA ( H ) , or Las RNAi with spermidine administration ( I ) . GFP ( green ) Dl ( 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 B-TISSUE O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR 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 ( ) of Drosophila with indicated genotypes . numbers of quantified Drosophila : 100 ( UAS - lacZ ) , 100 ( Las RNAi ) , 100 ( Las RNAi + UAS - Atg5 ) , 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 log - rank . statistical used in other panels were student ' s t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) , 14 - day Drosophila carrying esgts - GAL4 > Las RNAi ( E ) . GFP ( green ) . F . of of RAB5 - GFP in experiments ( A - E ) . Each dot corresponds to one . n is indicated . numbers of quantified guts from left to right are 13 , 11 , 12 , 10 13 . G Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) , 14 - day Drosophila carrying esgts - GAL4 > Las RNAi ( K ) . GFP ( green ) . L . of of RAB7 - GFP in experiments ( G - K ) . Each dot corresponds to one . n is indicated . numbers of quantified guts from left to right are 11 , 10 , 11 , 13 12 . M Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , 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 of midgut section from 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 ) Dl ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

O O O O B-EXP_ASSAY O B-TISSUE O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR 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 of midgut sections from R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( A , control ) , Las RNAi ( B ) , Las RNAi RAB5 - CA ( C ) , or Las RNAi RAB7 - CA ( D ) . GFP ( green ) , Dl ( 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 B-TISSUE O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR 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 ( ) of Drosophila of indicated genotypes . numbers of quantified Drosophila : 100 ( UAS - lacZ ) , 100 ( Las RNAi ) , 100 ( Las RNAi + UAS - Rab5 - CA ) 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 log - rank . statistical used in other panels were student ' s t - . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , non - significant ( NS ) represents p > 0 . 05 .

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

O O O O B-EXP_ASSAY O O O B-TISSUE O O O O O O O O B-ORGANISM O O O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of midgut sections from R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( K , control ) , Las RNAi ( L ) , or Las RNAi dominant negative form of EGFR ( EGFR - DN , M ) . GFP ( green ) Dl ( 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 B-TISSUE O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY B-SUBCELLULAR 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 of n = 3 of COS7 stably expressing GFP - TMD ( ER ) ( green ) SiR - lysosome ( orange ) . Zoom insets highlight regions of interest containing a static ( upper panels ) or mobile endosome ( lower panels ) as denoted by arrows . First row shows ER ( green ) , second row shows MDA generated ER movement ( magenta ) , third row shows a merge of two channels fourth row shows a merge of ER ( green ) LE / Lys ( orange ) . Scale bars : full 10µm , zoom inset 3µm . relate to movies EV2 3 . E . Kymograph of mobile vesicle in panel ( D ) . F . MDA of ( D ) . Mobility of static mobile endosomes was quantified normalized to static endosomes . Graph represents mean ± s . d . of analyses at multiple locations within same ( n = 20 per condition from 3 independent experiments ) . acquired for 90 frames at 4Hz . Significance two - student t - , * * * * P < 0 . 0001 .

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

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

O O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-SUBCELLULAR B-EXP_ASSAY B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of n = 3 live COS7 stably expressing mCherry - KDEL ( green ) transiently expressing GFP - RAB7 ( orange ) . Zoom insets shows a region of interest containing a mobile RAB7 - positive endosome as denoted by arrows . First row represents ER ( mCherry - KDEL ) ( green ) , second row shows MDA - generated ER movement ( magenta ) , third row shows a merge of two preceding channels fourth row shows a merge of ER ( green ) GFP - RAB7 ( orange ) . Scale bar : 10µm , zoom insets 3µm . related to movie EV11 . were acquired for 90 frames at 1Hz .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O 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 of panel ( A ) figures EV2A - G normalized to 1 showing average ER movement surrounding static endosomes mobile endosomes . Graph represents mean ± s . d . of analyses at multiple locations within same ( n = 15 per condition from 3 independent experiments ) . acquired for 90 frames at 1Hz . Significance two - student t - . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ns = not significant .

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

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of n = 3 of COS7 stably expressing TMD - GFP ( green ) SiR - lysosome - stained endosomes / lysosomes ( orange ) . COS7 in middle panels overexpress mCherry - RILP ( magenta ) . COS7 in lower panels overexpress mCherry - TMEM55B ( magenta ) . Zoom insets show region of interest containing ER ( TMD - GFP ) ( green ) junction of skeletonized ER where number of ER junctions representing quantified average . Scale bars : 15µm . zoom insets : 3µm . C . of number of ER junctions per µm2 from panel ( B ) as resulted from ER junction . Graph represents mean ± s . d . of analyses at multiple locations within one ( n = 15 from 3 independent experiments ) . Significance two - student t - . * * * P < 0 . 001 .

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

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY B-SUBCELLULAR O B-SUBCELLULAR O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-SUBCELLULAR B-EXP_ASSAY B-SUBCELLULAR O B-SUBCELLULAR O O O 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 of n = 3 of COS7 stably expressing TMD - GFP ( green ) SiR - lysosome - stained endosomes / lysosomes ( orange ) treated with siControl or with siVAPA , siVAPB siMOSPD2 . Zoom insets show region of interest containing ER ( TMD - GFP ) ( green ) junction of skeletonized ER where number of ER junctions representing quantified average . Scale bars : 10µm . zoom insets : 3µm .

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

O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O B-EXP_ASSAY B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PancTu - 1 were grown in absence or presence of indicated concentrations of omeprazole , 5 - fluorouracile , pantoprazole or gemcitabine , respectively , for 4 days to determine IC50 values . IC50 ies sigmoidicities are gradually differing between lines . In lower concentrations a slight growth - stimulatory effect ( hormesis ) was observed .

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.


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

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

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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-SUBCELLULAR O O 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-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE 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.