( E - F ) Western blot analysis ( E ) of VE - cadherin , Vimentin , Twist , Snail , and OTUD1 and densitometric quantification ( F ) of VE - cadherin , Vimentin , Twist and Snail in aortic tissues of the mice . β - actin was used as the loading control ( n = 5 biological replicates ) .

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

(E-F) Western blot analysis (E) of VE-cadherin, Vimentin, Twist, Snail, and OTUD1 and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues of the mice. β-actin was used as the loading control (n=5 biological replicates).


( G - H ) mRNA levels of Cdh5 , Vim , Twist1 , Snai1 ( G ) , Tgfb1 and Col1a1 ( H ) in aortic tissues of the mice were examined by real - time qPCR assay . The values were normalized to Rn18s ( n = 5 biological replicates ) .

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

(G-H) mRNA levels of Cdh5, Vim, Twist1, Snai1 (G), Tgfb1 and Col1a1 (H) in aortic tissues of the mice were examined by real-time qPCR assay. The values were normalized to Rn18s (n=5 biological replicates).


( B ) HUVECs transfected with si - OTUD1 or NC were challenged with 1 μM Ang II for 24 h . Western blot analysis of VE - cadherin , Vimentin , α - SMA , Twist and Snail were performed . GAPDH was used as the loading control .

O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O

(B) HUVECs transfected with si-OTUD1 or NC were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.


( E ) HUVECs transfected with Flag - OTUD1 or EV were challenged with 1 μM Ang II for 24 h . Western blot analysis of VE - cadherin , Vimentin , α - SMA , Twist and Snail were performed . GAPDH was used as the loading control .

O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O

(E) HUVECs transfected with Flag-OTUD1 or EV were challenged with 1 μM Ang II for 24 h. Western blot analysis of VE-cadherin, Vimentin, α-SMA, Twist and Snail were performed. GAPDH was used as the loading control.


( C ) Co - immunoprecipitation of OTUD1 and SMAD2 or SMAD3 in HUVECs transfected with Flag - OTUD1 . Exogenous Flag - OTUD1 was immunoprecipitated by anti - Flag antibody . IgG , immunoglobulin G .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O

(C) Co-immunoprecipitation of OTUD1 and SMAD2 or SMAD3 in HUVECs transfected with Flag-OTUD1. Exogenous Flag-OTUD1 was immunoprecipitated by anti-Flag antibody. IgG, immunoglobulin G.


( G ) Immunoprecipitation of SMAD3 in 293T cells co - transfected with GFP - SMAD3 , HA - Ub and Flag - OTUD1 and then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody . ( H ) Immunoprecipitation of SMAD3 in 293T cells co - transfected with GFP - SMAD3 , HA - Ub , and Flag - OTUD1 or Flag - OTUD1 ( C320S ) , and then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody . ( I ) Immunoprecipitation of SMAD3 in 293T cells co - transfected with GFP - SMAD3 , Flag - OTUD1 , and HA - Ub , HA - Ub - K48 ( K48 only ) , or HA - Ub - K63 ( K63 only ) , respectively , and then challenged with 10 μM MG132 for 6 h . Ubiquitinated SMAD3 was detected by immunoblotting using anti - HA antibody .

O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 B-SMALL_MOLECULE O O O O B-EXP_ASSAY B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O O

(G) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (H) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and Flag-OTUD1 or Flag-OTUD1(C320S), and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody. (I) Immunoprecipitation of SMAD3 in 293T cells co-transfected with GFP-SMAD3, Flag-OTUD1, and HA-Ub, HA-Ub-K48 (K48 only), or HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Ubiquitinated SMAD3 was detected by immunoblotting using anti-HA antibody.


( A - B ) 293T cells were transfected with various amount of Flag - OTUD1 . Western blot analysis ( A ) and densitometric quantification ( B ) of SMAD3 . GAPDH was used as the loading control ( n = 3 biological replicates ) .

O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

(A-B) 293T cells were transfected with various amount of Flag-OTUD1. Western blot analysis (A) and densitometric quantification (B) of SMAD3. GAPDH was used as the loading control (n=3 biological replicates).


( E - F ) Western blot analysis ( E ) and densitometric quantification ( F ) of SMAD3 in aortic tissues of the mice from four groups . β - actin was used as the loading control ( n = 5 biological replicates ) .

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

(E-F) Western blot analysis (E) and densitometric quantification (F) of SMAD3 in aortic tissues of the mice from four groups. β-actin was used as the loading control (n=5 biological replicates).


( G ) Co - immunoprecipitation of SMAD3 and SMAD4 in 293T cells co - transfected with GFP - SMAD3 , HA - Ub , and HA - Ub - K63 ( K63 only ) , respectively , and then challenged with 10 μM MG132 for 6 h . Exogenous SMAD3 was immunoprecipitated by anti - GFP antibody .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O

(G) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3, HA-Ub, and HA-Ub-K63 (K63 only), respectively, and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody.


( H ) Co - immunoprecipitation of SMAD3 and SMAD4 in 293T cells co - transfected with GFP - SMAD3 and Flag - OTUD1 and then challenged with 10 μM MG132 for 6 h . Exogenous SMAD3 was immunoprecipitated by anti - GFP antibody . IgG , immunoglobulin G .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O

(H) Co-immunoprecipitation of SMAD3 and SMAD4 in 293T cells co-transfected with GFP-SMAD3 and Flag-OTUD1 and then challenged with 10 μM MG132 for 6 h. Exogenous SMAD3 was immunoprecipitated by anti-GFP antibody. IgG, immunoglobulin G.


( A - B ) Representative images ( A ) of H & E staining of aortic sections ( scale bar = 25 μm ) and quantification of arterial wall thickness ( B ) ; n = 5 biological replicates . E , endothelium ; M , media ; A , adventitia . ( C - D ) Representative images ( C ) of Masson ' s Trichrome staining of aortic sections ( scale bar = 25 μm ) and quantification of collagen deposition ( D ) ; n = 5 biological replicates .

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

(A-B) Representative images (A) of H&E staining of aortic sections (scale bar=25 μm) and quantification of arterial wall thickness (B); n=5 biological replicates. E, endothelium; M, media; A, adventitia. (C-D) Representative images (C) of Masson's Trichrome staining of aortic sections (scale bar=25 μm) and quantification of collagen deposition (D); n=5 biological replicates.


( E - F ) Western blot analysis ( E ) and densitometric quantification ( F ) of VE - cadherin , Vimentin , Twist and Snail in aortic tissues . β - actin was used as the loading control ( n = 5 biological replicates ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O

(E-F) Western blot analysis (E) and densitometric quantification (F) of VE-cadherin, Vimentin, Twist and Snail in aortic tissues. β-actin was used as the loading control (n=5 biological replicates).


( C ) Immunocytochemistry showing post - synaptic marker PSD - 95 ( red ) , pre - synaptic marker Synapsin - I ( green ) , dendritic marker Map2 ( blue ) and co - localization of PSD - 95 and Synapsin - I from dissociated Nedd4 - 2 WT or Nedd4 - 2 cKO hippocampal neurons at DIV 16 . Representative secondary dendrites ( left ) and quantification of colocalized synaptic puncta number and area ( right ) ( n = 36 for Nedd4 - 2 WT and Nedd4 - 2 cKO neurons ) are shown . Scale bar : 5 µm .

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

(C) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from dissociated Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons at DIV 16. Representative secondary dendrites (left) and quantification of colocalized synaptic puncta number and area (right) (n = 36 for Nedd4-2 WT and Nedd4-2 cKO neurons) are shown. Scale bar: 5 µm.


( F ) A train of high frequency stimulation ( HFS ; 100 Hz for 1s ) induced early - phase LTP ( E - LTP ) at Schaffer collateral synapses in Nedd4 - 2 WT ( n = 7 slices from 6 mice ) and Nedd4 - 2 cKO ( n = 6 slices from 5 mice ) mice . Representative fEPSP traces were recorded before and 60 min after LTP induction ( Scale bars : 0 . 5 mV , 5 ms ) . Summary bar graphs showing the fEPSP slopes measured 50 - 60 min after a HFS at Schaffer collateral synapses was on the right . ( G ) Late - phase LTP ( L - LTP ) induced by four trains of HFS in Nedd4 - 2 WT slices ( n = 7 slices from 7 mice ) and Nedd4 - 2 cKO slices ( n = 9 slices from 8 mice ) . Representative fEPSP traces were recorded before and 180 min after LTP induction ( Scale bars : 0 . 5 mV , 5 ms ) . Summary bar graphs showing the fEPSP slopes measured 160 - 180 min after L - LTP induction at Schaffer collateral synapses was on the right .

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

(F) A train of high frequency stimulation (HFS; 100 Hz for 1s) induced early-phase LTP (E-LTP) at Schaffer collateral synapses in Nedd4-2 WT (n = 7 slices from 6 mice) and Nedd4-2 cKO (n = 6 slices from 5 mice) mice. Representative fEPSP traces were recorded before and 60 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 50-60 min after a HFS at Schaffer collateral synapses was on the right. (G) Late-phase LTP (L-LTP) induced by four trains of HFS in Nedd4-2 WT slices (n = 7 slices from 7 mice) and Nedd4-2 cKO slices (n = 9 slices from 8 mice). Representative fEPSP traces were recorded before and 180 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 160-180 min after L-LTP induction at Schaffer collateral synapses was on the right.


( B ) Barnes maze test from Nedd4 - 2 WT ( n = 8 ) or Nedd4 - 2 cKO ( n = 8 ) mice ( 6 - 8 weeks old ) . Schematic representation of Barnes maze ( top left ) . Mice were trained for 4 days with three training trials on each day to locate the escape box ( black circle ) . Learning curve during 4 training days was shown by the escape latency and the number of errors before escaping ( top right ) . Schematic representation of Barnes maze on probe trials ( bottom left ) . Quadrant occupancy on probe trial at day5 and at day12 ( bottom right ) were assessed by the time spent in the target area ( green quadrant ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Barnes maze test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of Barnes maze (top left). Mice were trained for 4 days with three training trials on each day to locate the escape box (black circle). Learning curve during 4 training days was shown by the escape latency and the number of errors before escaping (top right). Schematic representation of Barnes maze on probe trials (bottom left). Quadrant occupancy on probe trial at day5 and at day12 (bottom right) were assessed by the time spent in the target area (green quadrant).


( B ) Western blots of F - actin to G - actin ratio with or without cLTP induction from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured neurons . Schematic representation of the experimental design for cLTP inductions ( top left ) . Representative western blots ( bottom left ) and quantification of F - actin to G - actin from Nedd4 - 2 WT or Nedd4 - 2 cKO ( right ) cultures after cLTP induction and recovery in normal aCSF for 10 or 30 min before lysis ( n = 11 and 8 for Nedd4 - 2 WT and Nedd4 - 2 cKO cultures , respectively ) . For quantification , data from cLTP induction were normalized to data from control ( CTL ) condition .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-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-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-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) Western blots of F-actin to G-actin ratio with or without cLTP induction from Nedd4-2 WT or Nedd4-2 cKO cultured neurons. Schematic representation of the experimental design for cLTP inductions (top left). Representative western blots (bottom left) and quantification of F-actin to G-actin from Nedd4-2 WT or Nedd4-2 cKO (right) cultures after cLTP induction and recovery in normal aCSF for 10 or 30 min before lysis (n = 11 and 8 for Nedd4-2 WT and Nedd4-2 cKO cultures, respectively). For quantification, data from cLTP induction were normalized to data from control (CTL) condition.


( A ) Western blots of Nedd4 - 2 , total cofilin , phospho ( p ) - cofilin and GAPDH from Nedd4 - 2 WT or Nedd4 - 2 cKO forebrains at postnatal day 14 . Representative western blots and quantification of total cofilin relative to GAPDH and p - cofilin relative to total cofilin ( n = 6 for Nedd4 - 2 WT and Nedd4 - 2 cKO brains ) were shown .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O

(A) Western blots of Nedd4-2, total cofilin, phospho (p)-cofilin and GAPDH from Nedd4-2 WT or Nedd4-2 cKO forebrains at postnatal day 14. Representative western blots and quantification of total cofilin relative to GAPDH and p-cofilin relative to total cofilin (n = 6 for Nedd4-2 WT and Nedd4-2 cKO brains) were shown.


( B ) Immunocytochemistry of cofilin , p - cofilin and dendritic marker MAP2 from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons at DIV 16 . Representative dendrites of Nedd4 - 2 WT and Nedd4 - 2 cKO neurons and quantification of total cofilin relative to MAP2 and p - cofilin relative to total cofilin are shown ( n = 25 and 26 for Nedd4 - 2 WT and Nedd4 - 2 cKO neurons , respectively ) . Scale bar : 5 µm .

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

(B) Immunocytochemistry of cofilin, p-cofilin and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16. Representative dendrites of Nedd4-2 WT and Nedd4-2 cKO neurons and quantification of total cofilin relative to MAP2 and p-cofilin relative to total cofilin are shown (n = 25 and 26 for Nedd4-2 WT and Nedd4-2 cKO neurons, respectively). Scale bar: 5 µm.


( C ) Immunocytochemistry showing cofilin , p - cofilin , and dendritic marker MAP2 from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16 . Representative dendrites and quantification from Nedd4 - 2 WT or Nedd4 - 2 cKO cultures are shown ( n = 36 - 39 neurons ) . Scale bar : 5 µm . For quantification , data from cLTP induction were normalized to data from control ( CTL ) condition .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_TYPE O O 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) Immunocytochemistry showing cofilin, p-cofilin, and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16. Representative dendrites and quantification from Nedd4-2 WT or Nedd4-2 cKO cultures are shown (n = 36-39 neurons). Scale bar: 5 µm. For quantification, data from cLTP induction were normalized to data from control (CTL) condition.


( A ) Immunocytochemistry showing post - synaptic marker PSD - 95 ( red ) , pre - synaptic marker Synapsin - I ( green ) , dendritic marker Map2 ( blue ) and co - localization of PSD - 95 and Synapsin - I from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons at DIV 16 treated with TAT , TAT - S3 , or TAT - pS3 peptide for 12 hrs ( 10 μM ) . Representative secondary dendrites of Nedd4 - 2 WT ( top left ) and Nedd4 - 2 cKO ( bottom left ) neurons are shown . Quantification of colocalized synaptic puncta number and synaptic puncta area for Nedd4 - 2 WT ( top right ) and Nedd4 - 2 cKO ( bottom right ) neurons is on the right ( n = 32 - 36 neurons ) . Scale bar : 5 µm .

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

(A) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16 treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM). Representative secondary dendrites of Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) neurons are shown. Quantification of colocalized synaptic puncta number and synaptic puncta area for Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons is on the right (n = 32-36 neurons). Scale bar: 5 µm.


( B ) Patch - clamp recordings from Nedd4 - 2 WT or Nedd4 - 2 cKO hippocampal neurons treated with TAT , TAT - S3 , or TAT - pS3 peptide for 12 hrs ( 10 μM ) at DIV 14 - 17 . Representative mEPSC traces from Nedd4 - 2 WT ( top left ) and Nedd4 - 2 cKO ( bottom left ) are shown . Quantification of mEPSC amplitude and frequency from Nedd4 - 2 WT ( top right ) and Nedd4 - 2 cKO ( bottom right ) neurons are shown ( n = 22 - 24 neurons ) .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O

(B) Patch-clamp recordings from Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM) at DIV 14-17. Representative mEPSC traces from Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) are shown. Quantification of mEPSC amplitude and frequency from Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons are shown (n = 22-24 neurons).


( C ) Barnes maze test after intraperitoneally injecting TAT ( n = 8 ) or TAT - pS3 ( n = 8 ) peptide in Nedd4 - 2 WT mice ( top ) , or TAT ( n = 9 ) or TAT - pS3 ( n = 8 ) peptides in Nedd4 - 2 cKO mice ( bottom ) . Learning curve during 4 training days of Barnes maze task showing the escape latency , number of errors before escaping , and the quadrant occupancy on probe trial at day 5 and at day 12 were assessed .

O O O B-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 B-GENEPROD I-GENEPROD I-GENEPROD 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 B-GENEPROD I-GENEPROD I-GENEPROD 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 O O O O O O O

(C) Barnes maze test after intraperitoneally injecting TAT (n = 8) or TAT-pS3 (n = 8) peptide in Nedd4-2 WT mice (top), or TAT (n = 9) or TAT-pS3 (n = 8) peptides in Nedd4-2 cKO mice (bottom). Learning curve during 4 training days of Barnes maze task showing the escape latency, number of errors before escaping, and the quadrant occupancy on probe trial at day 5 and at day 12 were assessed.


A . SDS - PAGE analysis of purified sACES1211 showed a single band of ~ 170 kDa and thus indicates extensive N - glycosylation ( protein - only molecular weight is 138 kDa ) .

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

A. SDS-PAGE analysis of purified sACES1211 showed a single band of ~170 kDa and thus indicates extensive N-glycosylation (protein-only molecular weight is 138 kDa).


B . SEC - HPLC analysis of the respective samples of purified sACES1211 , truncated N - domain , or truncated C - domain showed a single symmetric peak for each protein with calibrated molecular weights of 244 kDa , 147 kDa , and 75 kDa , respectively . The sACES1211 sample did not show any peaks corresponding to single - domain or homodimer molecular weights .

O O B-EXP_ASSAY O B-EXP_ASSAY 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 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 O O O O O O O O O O O

B. SEC-HPLC analysis of the respective samples of purified sACES1211, truncated N-domain, or truncated C-domain showed a single symmetric peak for each protein with calibrated molecular weights of 244 kDa, 147 kDa, and 75 kDa, respectively. The sACES1211 sample did not show any peaks corresponding to single-domain or homodimer molecular weights.


C . Native PAGE analysis showed a major diffuse band of ~ 260 kDa and a minor band of ~ 500 kDa for purified sACES1211 , representing monomeric and dimeric forms of the protein . Commercial gel filtration standards and bovine serum albumin ( BSA ) were used to estimate the molecular weight . Purified truncated N - domain was used as an additional molecular weight marker for sACES1211 and showed a single diffuse band of ~ 160 kDa .

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 B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-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 B-GENEPROD O O O O O O O O O O O

C. Native PAGE analysis showed a major diffuse band of ~260 kDa and a minor band of ~500 kDa for purified sACES1211, representing monomeric and dimeric forms of the protein. Commercial gel filtration standards and bovine serum albumin (BSA) were used to estimate the molecular weight. Purified truncated N-domain was used as an additional molecular weight marker for sACES1211 and showed a single diffuse band of ~160 kDa.


( A , B ) B - ALL progression measured as ( A ) absolute number or ( B ) percentage of OFP + cells in peripheral blood of transplanted mice ( mean ± SD , each dot represents an individual mouse , CTRL = 6 mice , IFNγ = 10 mice ; * * * * p ≤ 0 . 001 , Two - Way ANOVA ; mice in blue and yellow ( B ) were also analyzed by single - cell RNA

O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O B-GENEPROD O O B-ORGANISM 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 O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY

(A, B) B-ALL progression measured as (A) absolute number or (B) percentage of OFP+ cells in peripheral blood of transplanted mice (mean ± SD, each dot represents an individual mouse, CTRL = 6 mice, IFNγ = 10 mice; **** p ≤ 0.001, Two-Way ANOVA; mice in blue and yellow (B) were also analyzed by single-cell RNA sequencing


( F , G ) Percentage of CD4 + and CD8 + cells within OFP - BM cells ( F ) and maturation state ( G ) of CD8 + lymphocytes ( CD62L - CD44 - double negative , CD62L - CD44 + effector memory , CD62L + CD44 - naive and CD62L + CD44 + central memory ) in the BM immune infiltrate at 12 - and 17 - days after B - ALL ( mean ± SD , each dot represents an individual mouse ; * * p = 0 . 005 , * * * * p ≤ 0 . 001 , Two - Way ANOVA ) .

O O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O 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

(F, G) Percentage of CD4+ and CD8+ cells within OFP- BM cells (F) and maturation state (G) of CD8+ lymphocytes (CD62L-CD44- double negative, CD62L-CD44+ effector memory, CD62L+CD44- naive and CD62L+CD44+ central memory) in the BM immune infiltrate at 12- and 17-days after B-ALL (mean ± SD, each dot represents an individual mouse; ** p = 0.005, **** p ≤ 0.001, Two-Way ANOVA).


( A ) Engraftment of CD45 . 2 donor cells and lineage composition following transplantation of wild - type ( WT ) or IFNγ receptor 1 knock out ( KO ) HSPCs transduced with either the control Tie2 . NGFR ( CTRL ) or Tie2 . IFNγ ( IFNγ ) LV ( mean ± SD , each dot represents an individual mouse , WT CTRL = 7 mice , WT IFNγ = 7 mice , KO CTRL = 7 mice , KO IFNγ = 7 mice ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O B-GENEPROD O O B-ORGANISM O O O O O B-ORGANISM O O B-GENEPROD O O B-ORGANISM O O

(A) Engraftment of CD45.2 donor cells and lineage composition following transplantation of wild-type (WT) or IFNγ receptor 1 knock out (KO) HSPCs transduced with either the control Tie2.NGFR (CTRL) or Tie2.IFNγ (IFNγ) LV (mean ± SD, each dot represents an individual mouse, WT CTRL = 7 mice, WT IFNγ = 7 mice, KO CTRL = 7 mice, KO IFNγ = 7 mice).


( C ) B - ALL progression measured as absolute number of OFP + cells in the peripheral blood ( mean ± SD , each dot represents an individual mouse ; * p = 0 . 0286 at day 10 ; p = 0 . 0174 at day 12 ; * * * p = 0 . 0002 ; * * * * p ≤ 0 . 0001 ; Ordinary Two - Way ANOVA ) .

O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O 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 O O O O O O O O O O O

(C) B-ALL progression measured as absolute number of OFP+ cells in the peripheral blood (mean ± SD, each dot represents an individual mouse; * p = 0.0286 at day 10; p = 0.0174 at day 12; *** p = 0.0002; **** p ≤ 0.0001; Ordinary Two-Way ANOVA).


( E ) Percentage of CD8 + T lymphocytes within OFP - CD45 + BM cells ( mean ± SD , each dot represents an individual mouse ) .

O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O B-ORGANISM O O

(E) Percentage of CD8+ T lymphocytes within OFP- CD45+ BM cells (mean ± SD, each dot represents an individual mouse).


( B , C ) Violin plots show the distribution of IFNγ ( B ) and MHC - II gene ( C ) signatures for each experimental condition , within each custom cell - type . Wilcox Rank Sum Test for IFNγ signature , condition IFNγ d12 vs . CTR d12 : B - ALL p = 2 . 68x10 - 19 , B cells p = 0 . 5 , plasma cells p = 0 . 78 ; Wilcox Rank Sum Test for MHC - II signature , condition IFNγ d12 vs . CTRL d12 : B - ALL p = 4 . 26x10 - 46 , B cells p = 1x10 - 9 , plasma cells p = 0 . 32 .

O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-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 B-GENEPROD O O O B-GENEPROD O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O

(B, C) Violin plots show the distribution of IFNγ (B) and MHC-II gene (C) signatures for each experimental condition, within each custom cell-type. Wilcox Rank Sum Test for IFNγ signature, condition IFNγ d12 vs. CTR d12: B-ALL p= 2.68x10-19, B cells p=0.5, plasma cells p= 0.78; Wilcox Rank Sum Test for MHC-II signature, condition IFNγ d12 vs. CTRL d12: B-ALL p= 4.26x10-46, B cells p=1x10-9, plasma cells p= 0.32.


( F ) Expression of Mki67 transcripts in B - ALL cells from CTRL d12 and IFNγ d12 conditions , extracted from the scRNAseq dataset ( * * * * p ≤ 0 . 0001 , Welch ' s test ) .

O O O O O B-GENEPROD O O B-DISEASE I-DISEASE I-DISEASE O O O O O B-GENEPROD 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

(F) Expression of Mki67 transcripts in B-ALL cells from CTRL d12 and IFNγ d12 conditions, extracted from the scRNAseq dataset (**** p ≤ 0.0001, Welch's test).


( I ) Loss of IFNγ response over time in B - ALL cells from the IFNγ group , potentially facilitated by reduced expression of Ifngr1 , Ifngr2 , Jak1 , Stat1 and Irf1 ( data extracted from the scRNAseq dataset statistical analysis by Two - Way ANOVA ) .

O O O O O B-GENEPROD O O O O B-DISEASE I-DISEASE I-DISEASE O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O

(I) Loss of IFNγ response over time in B-ALL cells from the IFNγ group, potentially facilitated by reduced expression of Ifngr1, Ifngr2, Jak1, Stat1 and Irf1 (data extracted from the scRNAseq dataset statistical analysis by Two-Way ANOVA).


Combination gene therapy with IFNα , TNFα and IFNγ , expressed from the Tie2 vector platform . CTRL ( Tie2 . NGFR ) , 2 independent experiments , n = 9 mice ; IFNα , 1 experiment , n = 2 mice ; IFNα / IFNγ , 1 experiment , n = 4 mice ; IFNα / TNFα , 1 experiment , n = 3 mice ; IFNγ / TNFα , 2 independent experiments , n = 19 mice . ( C ) Percentage of MHC II + macrophages identified by F4 / 80 expression in the spleen ( mean ± SD , each dot represents an individual mouse ; * p = 0 . 0484 , * * p = 0 . 0049 , ordinary One - Way ANOVA ) . ( D ) Percentage of CD8 + T lymphocytes within CD45 positive and OFP negative splenic cells ( mean ± SD , each dot represents an individual mouse ; * p = 0 . 0356 , ordinary One - Way ANOVA ) . (

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

Combination gene therapy with IFNα, TNFα and IFNγ, expressed from the Tie2 vector platform. CTRL (Tie2.NGFR), 2 independent experiments, n = 9 mice; IFNα, 1 experiment, n = 2 mice; IFNα/IFNγ, 1 experiment, n= 4 mice; IFNα/TNFα, 1 experiment, n = 3 mice; IFNγ/TNFα, 2 independent experiments, n = 19 mice. (C) Percentage of MHC II+ macrophages identified by F4/80 expression in the spleen (mean ± SD, each dot represents an individual mouse; *p = 0.0484, **p = 0.0049, ordinary One-Way ANOVA). (D) Percentage of CD8+ T lymphocytes within CD45 positive and OFP negative splenic cells (mean ± SD, each dot represents an individual mouse; *p = 0.0356, ordinary One-Way ANOVA). (


Combination of IFNγ gene therapy with immuno - oncology drugs . CTRL ( Tie2 . NGFR ) + antibody isotype , n = 6 mice ; CTRL + αCTLA4 antibody , n = 5 mice ; CTRL + αLAG3 antibody , n = 5 mice ; CTRL + 1 - Methyltryptophan ( 1 - MT ) , n = 5 mice ; IFNγ ( Tie2 . IFNγ ) + antibody isotype , n = 7 mice ; IFNγ + αCTLA4 antibody , n = 7 mice ; IFNγ + αLAG3 antibody , n = 7 mice ; IFNγ + 1 - MT , n = 5 mice . ( F ) Percentage of MHC II + macrophages identified by F4 / 80 expression in the spleen ( mean ± SD , each dot represents an individual mouse ; * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 as compared to CTRL isotype , ordinary One - Way ANOVA ) . ( G ) Percentage of CD8 + T lymphocytes within OFP negative BM cells ( mean ± SD , each dot represents an individual mouse ) . S

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

Combination of IFNγ gene therapy with immuno-oncology drugs. CTRL (Tie2.NGFR) + antibody isotype, n = 6 mice; CTRL + αCTLA4 antibody, n= 5 mice; CTRL + αLAG3 antibody, n = 5 mice; CTRL + 1-Methyltryptophan (1-MT), n = 5 mice; IFNγ (Tie2.IFNγ) + antibody isotype, n = 7 mice; IFNγ + αCTLA4 antibody, n = 7 mice; IFNγ + αLAG3 antibody, n = 7 mice; IFNγ +1-MT, n = 5 mice. (F) Percentage of MHC II+ macrophages identified by F4/80 expression in the spleen (mean ± SD, each dot represents an individual mouse; **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001 as compared to CTRL isotype, ordinary One-Way ANOVA). (G) Percentage of CD8+ T lymphocytes within OFP negative BM cells (mean ± SD, each dot represents an individual mouse). S


( A ) Representative traces showing SQR - driven oxygen consumption in 3x106 control ( C ) and five patientsfibroblasts ( P1 - 5 ) in the absence ( solid traces ) or presence ( dashed traces ) of CoQ10 in the culture medium . See Materials and Methods for the role and concentration of the compounds used . ( B ) Relative rates of SQR - driven oxygen consumption are normalized to CIV respiration rates .

O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O 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 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 B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O

(A) Representative traces showing SQR-driven oxygen consumption in 3x106 control (C) and five patientsfibroblasts (P1-5) in the absence (solid traces) or presence (dashed traces) of CoQ10 in the culture medium. See Materials and Methods for the role and concentration of the compounds used. (B) Relative rates of SQR-driven oxygen consumption are normalized to CIV respiration rates.


( A ) Representative western blot showing the level of SQR , TST , ETHE1 and SUOX proteins in three control ( C1 - 3 ) and four patients fibroblasts ( P1 - 4 ) . ( B - E ) Relative levels of proteins normalized to Vinculin .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-GENEPROD O

(A) Representative western blot showing the level of SQR, TST, ETHE1 and SUOX proteins in three control (C1-3) and four patients fibroblasts (P1-4). (B-E) Relative levels of proteins normalized to Vinculin.


( A ) Representative western blots showing the level of SQR , TST , ETHE1 and SUOX proteins in two controls ( C1 , C2 ) and two patients fibroblasts ( P3 , P4 ) . ( B - E ) Relative levels of proteins normalized to Vinculin .

(A) Representative western blots showing the level of SQR, TST, ETHE1 and SUOX proteins in two controls (C1, C2) and two patients fibroblasts (P3, P4). (B-E) Relative levels of proteins normalized to Vinculin.


( A ) Representative western blots showing SQR , TST , ETHE1 and SUOX of two control fibroblasts treated with DMSO ( - ) or with DMSO + 4 - NB ( + ) . ( B ) Quantification of proteins levels in fibroblasts treated with 4 - NB relative to control .

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

(A) Representative western blots showing SQR, TST, ETHE1 and SUOX of two control fibroblasts treated with DMSO (-) or with DMSO+ 4-NB (+). (B) Quantification of proteins levels in fibroblasts treated with 4-NB relative to control.


( C ) Representative western blots showing ADCK3 , SQR , TST , ETHE1 and SUOX in two control ( EV ) and six ADCK3 - depleted clones ( ADCK3 RNAi ) . ( D ) Quantification of proteins levels in ADCK3 - depleted clones ( ADCK3 RNAi ) relative to controls ( EV ) .

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

(C) Representative western blots showing ADCK3, SQR, TST, ETHE1 and SUOX in two control (EV) and six ADCK3-depleted clones (ADCK3 RNAi). (D) Quantification of proteins levels in ADCK3-depleted clones (ADCK3 RNAi) relative to controls (EV).


Distribution of peptides containing sulfhydrated cysteine residues relative to their H / L ratios as determined by the BTA analysis of cell extracts isolated from C1 and P1 patient cells ( indicated in A ) and C2 and P4 fibroblasts ( B ) . Values of H / L ratios are plotted against the number of identified peptides . The gray line marks the H / L ratio > 2 , consisting of cysteine - containing peptides in proteins from patient cell lines that exhibited higher reactivity with H2S compared with the respective control . Indicated on the X axes are representative sensitive cysteine peptides , which are common between the ones found in the RedoxDB database , and by the BTA assay .

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Distribution of peptides containing sulfhydrated cysteine residues relative to their H/L ratios as determined by the BTA analysis of cell extracts isolated from C1 and P1 patient cells (indicated in A) and C2 and P4 fibroblasts (B). Values of H/L ratios are plotted against the number of identified peptides. The gray line marks the H/L ratio >2, consisting of cysteine-containing peptides in proteins from patient cell lines that exhibited higher reactivity with H2S compared with the respective control. Indicated on the X axes are representative sensitive cysteine peptides, which are common between the ones found in the RedoxDB database, and by the BTA assay.


( A ) Representative western blots showing the levels of SQR , TST , ETHE1 and SUOX protein in the kidney of three wild - type ( WT ) and three mutant ( Mut ) mice . ( B - E ) Relative levels of proteins normalized to Vinculin .

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(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein in the kidney of three wild-type (WT) and three mutant (Mut) mice. (B-E) Relative levels of proteins normalized to Vinculin.


( A ) Representative western blots showing the levels of SQR , TST , ETHE1 and SUOX protein from the brain of 5 wild - type ( WT ) and five mutant ( Mut ) mice ( B - E ) Relative levels of proteins normalized to Vinculin .

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(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein from the brain of 5 wild-type (WT) and five mutant (Mut) mice (B-E) Relative levels of proteins normalized to Vinculin.


( A ) Total GSH amount normalized to protein from kidney and ( B ) brain of wild - type ( WT ) and mutant ( Mut ) mice . Error bars represent SDs of at least 3 animals per group .

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(A) Total GSH amount normalized to protein from kidney and (B) brain of wild-type (WT) and mutant (Mut) mice. Error bars represent SDs of at least 3 animals per group.


( C ) Thiosulfate levels in urine and ( D ) plasma normalized to protein amount . Error bars represent SDs of 9 animals per group ( urine ) and 6 animals per group ( plasma ) .

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(C) Thiosulfate levels in urine and (D) plasma normalized to protein amount. Error bars represent SDs of 9 animals per group (urine) and 6 animals per group (plasma).


( E ) H2S amount normalized to tissue from kidney and ( F ) brain . Error bars represent SDs of 7 / 8 animals per group .

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(E) H2S amount normalized to tissue from kidney and (F) brain. Error bars represent SDs of 7/8 animals per group.


( G ) Acylcarnitine C4 , C5 and C6 levels from plasma , normalized to volume . Error bars represent SDs of 5 wild - type and 3 mutants .

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(G) Acylcarnitine C4, C5 and C6 levels from plasma, normalized to volume. Error bars represent SDs of 5 wild-type and 3 mutants.


T2 - weighed brain MRIs of patient A ( D1 - 3 ) at 28 months of age and patient B at ( D4 - 6 ) at 16 months of age , showing global brain atrophy as well as periventricular and deep white matter hyperintensities .

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T2-weighed brain MRIs of patient A (D1-3) at 28 months of age and patient B at (D4-6) at 16 months of age, showing global brain atrophy as well as periventricular and deep white matter hyperintensities.


Quantifications of HOPS / CORVET subunits in fibroblast lysates ( top ) and representative immunoblots ( bottom ) , analyzed as in ( C ) . ( E ) VPS33A , ( F ) VPS11 , ( G ) VPS18 .

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Quantifications of HOPS/CORVET subunits in fibroblast lysates (top) and representative immunoblots (bottom), analyzed as in (C). (E) VPS33A, (F) VPS11, (G) VPS18.


Immunoblots ( left ) and quantifications normalized to control ( right ) of VPS16 , VPS33A and VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control ( empty vector ) .

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Immunoblots (left) and quantifications normalized to control (right) of VPS16, VPS33A and VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control (empty vector).


Analysis of the early endosome marker EEA1 in fibroblasts transduced with control or VPS16 - expressing lentivirus by confocal microscopy . ( A ) Representative confocal micrographs . Scale bar , 20 μm . ( B ) Quantification of EEA1 - stained puncta number and intensities ( ca 80 cells analyzed for each of n = 3 biological replicates ) .

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Analysis of the early endosome marker EEA1 in fibroblasts transduced with control or VPS16-expressing lentivirus by confocal microscopy. (A) Representative confocal micrographs. Scale bar, 20 μm. (B) Quantification of EEA1-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).


Analysis of intracellular Transferrin trafficking . Fibroblasts fed with fluorescently labeled Transferrin were co - stained with LysoTracker ( G ) or an antibody against LAMP2 ( H ) . Representative images ( left ) of fibroblasts transduced with control virus , with magnified areas demonstrating co - localized puncta ( arrowheads ) . Scale bars 10 μm or 2 μm ( inserts ) . Quantifications ( right ) of the co - localization between Transferrin and LysoTracker or LAMP2 , respectively , expressed as Pearson correlation coefficients . Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles ( n = 10 optical sections from 3 independent experiments ) .

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

Analysis of intracellular Transferrin trafficking. Fibroblasts fed with fluorescently labeled Transferrin were co-stained with LysoTracker (G) or an antibody against LAMP2 (H). Representative images (left) of fibroblasts transduced with control virus, with magnified areas demonstrating co-localized puncta (arrowheads). Scale bars 10 μm or 2 μm (inserts). Quantifications (right) of the co-localization between Transferrin and LysoTracker or LAMP2, respectively, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n=10 optical sections from 3 independent experiments).


Confocal micrographs of fibroblasts transduced with control or VPS16 - expressing lentivirus and labeled with LysoTracker . Scale bar , 20 μm . Quantification of LysoTracker - stained puncta number and intensities ( ca 80 cells analyzed for each of n = 3 biological replicates ) .

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Confocal micrographs of fibroblasts transduced with control or VPS16-expressing lentivirus and labeled with LysoTracker. Scale bar, 20 μm. Quantification of LysoTracker-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).


Representative immunoblots ( bottom ) and summary quantifications ( top ) of the levels of LAMP1 ( C ) and LAMP2 ( D ) in fibroblasts , normalized to levels of actin ( ACTB ) and expressed as % of controls ( n = 3 biological replicates ) . Analysis of lysosomal Cathepsin D ( CtsD ) processing by immunoblotting . Immunoblot to detect CtsD isoforms ( left ) and quantification ( right ) of processing , calculated as the percentage of CtsD divided by the sum of all isoforms ( n = 3 biological replicates ) .

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Representative immunoblots (bottom) and summary quantifications (top) of the levels of LAMP1 (C) and LAMP2 (D) in fibroblasts, normalized to levels of actin (ACTB) and expressed as % of controls (n=3 biological replicates). Analysis of lysosomal Cathepsin D (CtsD) processing by immunoblotting. Immunoblot to detect CtsD isoforms (left) and quantification (right) of processing, calculated as the percentage of CtsD divided by the sum of all isoforms (n=3 biological replicates).


Analysis of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . ( F ) Representative confocal micrographs ( optical sections ) of fibroblasts imaged live under basal or serum - starved conditions . Scale bar , 20 μm .

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Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. (F) Representative confocal micrographs (optical sections) of fibroblasts imaged live under basal or serum-starved conditions. Scale bar, 20 μm.


Analysis of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . Quantification of autophagosomes , defined as green + red puncta ( G ) and autophago - lysosomes , calculated as the number of red ( but not green ) puncta divided by the total number of puncta . Colored horizontal bars denote median values and whiskers 5 and 95 percentiles ( n = 50 - 113 cells from a total of 3 independent experiments ; for exact values see Appendix Table S1 ) .

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Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. Quantification of autophagosomes, defined as green + red puncta (G) and autophago-lysosomes, calculated as the number of red (but not green) puncta divided by the total number of puncta. Colored horizontal bars denote median values and whiskers 5 and 95 percentiles (n=50-113 cells from a total of 3 independent experiments; for exact values see Appendix Table S1).


Myelination , as reported using the Tg ( mbp : gfp - caax ) transgenic , is significantly reduced in vps16 crispants . The yellow box indicates the region assessed in D . Scale bar 100 μm . Bar graphs represent data as mean ± SEM . Statistical comparisons by Mann - Whitney U tests ( n = 6 ) . * * p < 0 . 01 .

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Myelination, as reported using the Tg(mbp:gfp-caax) transgenic, is significantly reduced in vps16 crispants. The yellow box indicates the region assessed in D. Scale bar 100 μm. Bar graphs represent data as mean ±SEM. Statistical comparisons by Mann-Whitney U tests (n=6). ** p < 0.01.


LysoTracker signal colocalizes with the astroglial reporter slc1a2b : citrine and does not appear to overlap with the pan - neuronal reporter elavl3 : GCaMP5G .

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LysoTracker signal colocalizes with the astroglial reporter slc1a2b:citrine and does not appear to overlap with the pan-neuronal reporter elavl3:GCaMP5G.


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 .

O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O B-TISSUE O 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 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 O O B-GENEPROD 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

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


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

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


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

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


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

O O O O B-EXP_ASSAY O O O B-TISSUE 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 B-GENEPROD O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD B-EXP_ASSAY 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

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


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

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

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


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

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

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


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

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


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

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

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

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


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

O O O O B-EXP_ASSAY O O B-TISSUE 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 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 O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O 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 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 O O O B-GENEPROD O O 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 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 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

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


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

O O O O O O O O B-TISSUE 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 B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O 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 O B-GENEPROD B-EXP_ASSAY 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 and p < 0 . 05 ) , red symbols indicate significantly upregulated mass bins ( Log2 FC > 0 . 6 and p < 0 . 05 ) , and gray symbols indicate mass bins that were not significantly changed .

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

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


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

O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE B-EXP_ASSAY O O B-TISSUE O 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 O O O B-SMALL_MOLECULE B-EXP_ASSAY 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 B-TISSUE O O O O 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 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

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


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

O O O O O O 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 O O B-GENEPROD O 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 B-GENEPROD O B-GENEPROD 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 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 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

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


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

O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE B-EXP_ASSAY O O B-TISSUE O 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 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 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 B-TISSUE O O 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 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

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


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

O O O O O O 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 O B-ORGANISM O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O 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 O B-GENEPROD O O 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 O

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


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

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

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

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

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


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

O O O O 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 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 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 B-TISSUE 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

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


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

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


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

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

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

O O B-EXP_ASSAY O O O O O B-ORGANISM O 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 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 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.