MEFi deficient ( − / − ) or not ( + / + ) for TP53INP1 were seeded in media containing or not 10 mM NAC . Forty - eight hours later , MEFi were treated or not ( NT ) with 100 μM hydrogen peroxide ( H2O2 ) during 1 h in serum free media . Cells were left to recover for 4 h in normal media containing or not NAC ( 10 mM ) , GW - 9662 ( PPARγ inhibitor , 10 μM ) or PNU - 74654 ( Wnt / β - catenin pathway inhibitor , 50 μM ) before being harvested . Cells were observed by TEM . Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure . Scale bar represents 0 . 5 μm . Results are expressed as the mean ± SEM and are representative of three independent experiments . * P ( − / − versus + / + ; NT ) = 0 . 00086 ; # P ( H2O2 versus NT ; + / + ) = 0 . 013 .

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

MEFi deficient (−/−) or not (+/+) for TP53INP1 were seeded in media containing or not 10 mM NAC. Forty-eight hours later, MEFi were treated or not (NT) with 100 μM hydrogen peroxide (H2O2) during 1 h in serum free media. Cells were left to recover for 4 h in normal media containing or not NAC (10 mM), GW-9662 (PPARγ inhibitor, 10 μM) or PNU-74654 (Wnt/β-catenin pathway inhibitor, 50 μM) before being harvested. Cells were observed by TEM.Number of LD normalized by cytoplasmic surface area is shown in both histograms at the bottom of the figure. Scale bar represents 0.5 μm. Results are expressed as the mean ± SEM and are representative of three independent experiments. *P (−/− versus +/+; NT) = 0.00086; #P (H2O2 versus NT; +/+) = 0.013.


Cells were also observed by fluorescence microscopy after Bodipy493 / 503 staining of LD ( green ) and DAPI staining of nucleus ( blue ) . L = lipid droplets . Scale bar represents 100 μm . Results are representative of three independent experiments .

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

Cells were also observed by fluorescence microscopy after Bodipy493/503 staining of LD (green) and DAPI staining of nucleus (blue). L = lipid droplets. Scale bar represents 100 μm. Results are representative of three independent experiments.


TCLs from MEFi were analyzed by immunoblotting for β - catenin , PPARγ , ATGL , MGLL and β - tubulin . Results are representative of three independent experiments .

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

TCLs from MEFi were analyzed by immunoblotting for β-catenin, PPARγ, ATGL, MGLL and β-tubulin. Results are representative of three independent experiments.


( A ) Third instar larval skeletal musculature stained with Phalloidin ( F - actin ) . In this and subsequent figures , we assayed the ventral longitudinal muscles ( highlighted in green ) .

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

(A) Third instar larval skeletal musculature stained with Phalloidin (F-actin). In this and subsequent figures, we assayed the ventral longitudinal muscles (highlighted in green).


( B - D ) GFP - Atg8 , overexpressed using the Dmef2 - Gal4 driver , labels autophagosomes . ( B ) , starved on low - nutrientfood for 6 h ( C ) , or starved on low - nutrientfood + 2 . 5 mg / ml CQ for 6 h ( D ) . GFP - Atg8 - labeled vesicles appeared only in the starved animals ( C - D ) , localizing around the nucleus and between myofibers . ( D ) CQ treatment caused accumulation of bloated GFP - Atg8 - labeled vesicles .

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

(B-D) GFP-Atg8, overexpressed using the Dmef2-Gal4 driver, labels autophagosomes. (B), starved on low-nutrientfood for 6 h (C), or starved on low-nutrientfood +2.5 mg/ml CQ for 6 h (D). GFP-Atg8-labeled vesicles appeared only in the starved animals (C-D), localizing around the nucleus and between myofibers. (D) CQ treatment caused accumulation of bloated GFP-Atg8-labeled vesicles.


( E - G ) Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - HRP - Lamp1 animals were assayed for Lamp1 and Atg8 localization ( anti - HRP , red ; GFP , green ; DAPI , blue ) . . ( E ) High - nutrient food suppressed formation of both GFP - Atg8 and HRP - Lamp1 - labeled vesicles . ( F ) Colocalization of GFP - Atg8 and HRP - Lamp in animals starved on low - nutrient food . The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp . ( G ) Addition of CQ to the starvation diet resulted in accumulation of both GFP - Atg8 and HRP - Lamp - labeled vesicles , but they failed to colocalize .

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

(E-G) Dmef2-Gal4, UAS-GFP-Atg8/UAS-HRP-Lamp1 animals were assayed for Lamp1 and Atg8 localization (anti-HRP, red; GFP, green; DAPI, blue). . (E) High-nutrient food suppressed formation of both GFP-Atg8 and HRP-Lamp1-labeled vesicles. (F) Colocalization of GFP-Atg8 and HRP-Lamp in animals starved on low-nutrient food. The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp. (G) Addition of CQ to the starvation diet resulted in accumulation of both GFP-Atg8 and HRP-Lamp-labeled vesicles, but they failed to colocalize.


( H ) Quantification of the number of GFP - Atg8 , HRP - Lamp1 , or GFP - Atg8 + HRP - Lamp1 vesicles in starved or starved + CQ muscles .

O O O 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 B-SUBCELLULAR O O O O O B-SMALL_MOLECULE B-TISSUE O

(H) Quantification of the number of GFP-Atg8, HRP-Lamp1, or GFP-Atg8+HRP-Lamp1 vesicles in starved or starved +CQ muscles.


( I - M ) The core Atg genes are required for starvation - induced autophagy in both wild - type and CQ - treated skeletal muscles . Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1 larvae were starved on low - nutrient food for 6 h ( I ) or starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h ( J ) . Note that Atg1 knockdown completely abolished the formation of GFP - Atg8 - labeled autophagosomes ( compare I - J to C - D ) . ( K - L ) Dmef2 - Gal4 , UAS - GFP - Atg8 , Atg1Δ3d larvae failed to form GFP - Atg8 vesicles when starved or starved and treated with CQ . ( M ) Quantification of autophagy changes due to Atg gene knockdown in Dmef2 - Gal4 , UAS - GFP - Atg8 larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . Each of the 10 UAS - Atg RNAi transgenes tested caused a highly significant decrease ( p < . 01 ) in the total area of GFP - Atg8vesicles . SEM is indicated , with n = 5 ventral longitudinal muscles from individual animals .

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

(I-M) The core Atg genes are required for starvation-induced autophagy in both wild-type and CQ-treated skeletal muscles. Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1 larvae were starved on low-nutrient food for 6 h (I) or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (J). Note that Atg1 knockdown completely abolished the formation of GFP-Atg8-labeled autophagosomes (compare I-J to C-D). (K-L) Dmef2-Gal4, UAS-GFP-Atg8, Atg1Δ3d larvae failed to form GFP-Atg8 vesicles when starved or starved and treated with CQ. (M) Quantification of autophagy changes due to Atg gene knockdown in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. Each of the 10 UAS-Atg RNAi transgenes tested caused a highly significant decrease (p<.01) in the total area of GFP-Atg8vesicles. SEM is indicated, with n = 5 ventral longitudinal muscles from individual animals.


. ( N - O ) EM of muscles from Dmef2 - Gal4 , UAS - whitei larvae . Animals starved on low - nutrient food + 2 . 5 mg / ml CQ ( O ) accumulated vesicles in the intermyofibril spaces ( red asterisk ) , disrupting the integrity of the sarcomere compared to non - CQ - treated control muscles ( N ) .

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

. (N-O) EM of muscles from Dmef2-Gal4, UAS-whitei larvae. Animals starved on low-nutrient food +2.5 mg/ml CQ (O) accumulated vesicles in the intermyofibril spaces (red asterisk), disrupting the integrity of the sarcomere compared to non-CQ-treated control muscles (N).


( P ) CQ treatment increased the larval crawling time of Dmef2 - Gal4 , UAS - whitei larvae in starved animals , and weakly in fed animals . ( Q ) CQ treatment increased the larval righting time of Dmef2 - Gal4 , UAS - whitei larvae in starved but not fed animals . For both locomotor assays , SEM is indicated for n = 10 larvae ( * p < . 05 , * * p < . 01 ) .

O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY 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

(P) CQ treatment increased the larval crawling time of Dmef2-Gal4, UAS-whitei larvae in starved animals, and weakly in fed animals. (Q) CQ treatment increased the larval righting time of Dmef2-Gal4, UAS-whitei larvae in starved but not fed animals. For both locomotor assays, SEM is indicated for n = 10 larvae (*p<.05, **p<.01).


( A ) Sectioned third instar OreR larva stained with Periodic acid - Schiff ( PAS ) . The muscles , but not the fat body , are stained purple , indicating high levels of glycogen ( m , muscle ; bw , body wall ; fb , fat body ) .

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

(A) Sectioned third instar OreR larva stained with Periodic acid-Schiff (PAS). The muscles, but not the fat body, are stained purple, indicating high levels of glycogen (m, muscle; bw, body wall; fb, fat body).


( B ) Glycogen was also detected in muscle from Dmef2 - Gal4 , UAS - GFP - Atg8 larvae , immunostained with an antiglycogen monoclonal antibody .

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

(B) Glycogen was also detected in muscle from Dmef2-Gal4, UAS-GFP-Atg8 larvae, immunostained with an antiglycogen monoclonal antibody.


( C ) GFP - Atg8 vesicles colocalized with glycogen in Dmef2 - Gal4 , UAS - GFP - Atg8 larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h ( GFP , green ; antiglycogen , red ) .

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

(C) GFP-Atg8 vesicles colocalized with glycogen in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h (GFP, green; antiglycogen, red).


D ) HRP - Lamp1 vesicles show less colocalization with glycogen in UAS - HRP - Lamp1 ; Dmef2 - Gal4 larvae starved and treated with CQ .

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

D) HRP-Lamp1 vesicles show less colocalization with glycogen in UAS-HRP-Lamp1;Dmef2-Gal4 larvae starved and treated with CQ.


( E ) Quantification of GFP - Atg8 or HRP - Lamp1 vesicles with glycogen .

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

(E) Quantification of GFP-Atg8 or HRP-Lamp1 vesicles with glycogen.


( F - G ) EM from Dmef2 - Gal4 , UAS - whitei larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . ( F ) Double - and single - membrane vesicles containing glycogen granules accumulated between myofibers ( s , sarcomere ; m , mitochondrion ; AVs , autophagic vesicles ) . ( G ) Higher magnification view of region outlined in ( E ) . ( H ) CQ treatment is not required for glycogen autophagy as seen in an EM from a Dmef2 - Gal4 , UAS - whitei larva starved on low - nutrient food for 6 h . Arrow points to double membrane .

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

(F-G) EM from Dmef2-Gal4, UAS-whitei larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Double- and single-membrane vesicles containing glycogen granules accumulated between myofibers (s, sarcomere; m, mitochondrion; AVs, autophagic vesicles). (G) Higher magnification view of region outlined in (E). (H) CQ treatment is not required for glycogen autophagy as seen in an EM from a Dmef2-Gal4, UAS-whitei larva starved on low-nutrient food for 6 h. Arrow points to double membrane.


( A - D ) Time course of autophagy induction in Dmef2 - Gal4 , UAS - GFP - Atg8 muscles , accompanied by quantification of GFP - Atg8 and glycogen colocalization . Animals were fed for 18 h in high - nutrient food + 2 . 5 mg / ml CQ , then starved on low - nutrient food + 2 . 5 mg / ml CQ for 0 - 8 h ( antiglycogen , red ; GFP , green ; DAPI , blue ) . ( A ) At time point 0 , following 18 h in high - nutrient food + CQ , the muscles contained large amounts of glycogen with no apparent autophagy . ( B ) At 3 h of starvation , glycogen stores were still high , and GFP - Atg8 - labeled vesicles began to appear . ( C - D ) At 6 and 8 h of starvation , the majority of GFP - Atg8 - labeled vesicles colocalized with glycogen .

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

(A-D) Time course of autophagy induction in Dmef2-Gal4, UAS-GFP-Atg8 muscles, accompanied by quantification of GFP-Atg8 and glycogen colocalization. Animals were fed for 18 h in high-nutrient food +2.5 mg/ml CQ, then starved on low-nutrient food +2.5 mg/ml CQ for 0-8 h (antiglycogen, red; GFP, green; DAPI, blue). (A) At time point 0, following 18 h in high-nutrient food +CQ, the muscles contained large amounts of glycogen with no apparent autophagy. (B) At 3 h of starvation, glycogen stores were still high, and GFP-Atg8-labeled vesicles began to appear. (C-D) At 6 and 8 h of starvation, the majority of GFP-Atg8-labeled vesicles colocalized with glycogen.


( E ) Time course of glycogen levels in Dmef2 - Gal4 carcasses ( muscle + body wall ) . Animals were fed for 24 h in high - nutrient food , then starved on low - nutrient food + / − 2 . 5 mg / ml CQ for 0 - 24 h . Starvation caused reduction of glycogen levels in both untreated and CQ - treated larvae over time . However , after 6 h of starvation , CQ treatment significantly increased glycogen levels compared to controls . SEM is indicated for n = 5 - 8 samples ( * p < . 05 , * * p < . 01 ) .

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

(E) Time course of glycogen levels in Dmef2-Gal4 carcasses (muscle+body wall). Animals were fed for 24 h in high-nutrient food, then starved on low-nutrient food +/− 2.5 mg/ml CQ for 0-24 h. Starvation caused reduction of glycogen levels in both untreated and CQ-treated larvae over time. However, after 6 h of starvation, CQ treatment significantly increased glycogen levels compared to controls. SEM is indicated for n = 5-8 samples (*p<.05, **p<.01).


( F - G ) activation of the Tor pathway blocked autophagy in the muscles from larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . ( F ) Autophagy levels were high in control Dmef2 - Gal4 / UAS - whitei larvae . Muscles from ( G ) UAS - Rheb / + ; Dmef2 - Gal4 / + , ( H ) Dmef2 - Gal4 / UAS - Tsc1i , and ( I ) Dmef2 - Gal4 / UAS - gigi all failed to induce autophagy .

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

(F-G) activation of the Tor pathway blocked autophagy in the muscles from larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Autophagy levels were high in control Dmef2-Gal4/UAS-whitei larvae. Muscles from (G) UAS-Rheb/+; Dmef2-Gal4/+, (H) Dmef2-Gal4/UAS-Tsc1i, and (I) Dmef2-Gal4/UAS-gigi all failed to induce autophagy.


( A - B ) Glycogen phosphorylase is not required for glycogen autophagy ( antiglycogen , red ; GFP , green ; DAPI , blue ) . ( A ) UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h exhibited high levels of colocalization between GFP - Atg8 and glycogen . ( B ) Higher magnification of region outlined in ( A )

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

(A-B) Glycogen phosphorylase is not required for glycogen autophagy (antiglycogen, red; GFP, green; DAPI, blue). (A) UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h exhibited high levels of colocalization between GFP-Atg8 and glycogen.(B) Higher magnification of region outlined in (A)


C - F ) Dmef2 - Gal4 , UAS - GFP - Atg8 larvae with GlyP and / or Atg1 knockdown were fed on high - nutrient food for 18 h before being starved on low - nutrient food ( antiglycogen , red ; GFP , green ; DAPI , blue ) . ( C ) UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + larval muscle contained high levels of glycogen prior to starvation , indicating no defect in glycogen synthesis . ( D ) Following 24 h starvation UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + muscles contained no glycogen detectable by antibody staining . ( E ) Following 24 h of starvation Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1i muscles contained no glycogen . ( F ) Double - mutant larvae UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1i larval muscles contained high levels of glycogen after 24 h of starvation , indicating an inability to break down glycogen .

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

C-F) Dmef2-Gal4, UAS-GFP-Atg8 larvae with GlyP and/or Atg1 knockdown were fed on high-nutrient food for 18 h before being starved on low-nutrient food (antiglycogen, red; GFP, green; DAPI, blue). (C) UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ larval muscle contained high levels of glycogen prior to starvation, indicating no defect in glycogen synthesis. (D) Following 24 h starvation UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ muscles contained no glycogen detectable by antibody staining. (E) Following 24 h of starvation Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1i muscles contained no glycogen. (F) Double-mutant larvae UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1i larval muscles contained high levels of glycogen after 24 h of starvation, indicating an inability to break down glycogen.


( G ) Time course of glycogen levels in Dmef2 - Gal4 carcasses ( muscle + body wall ) with expression of UAS - RNAi transgenes targeting white , GlyP , Atg1 , or GlyP + Atg1 . Simultaneous knockdown of GlyP and Atg1 , but not either gene alone , significantly reduced glycogen degradation compared to the white control after 24 h of starvation , consistent with immunostaining ( C - F ) . Between 6 and 12 h of starvation , individual knockdown of GlyP or Atg1 caused a significant increase in glycogen levels , indicating a reduced rate of glycogen degradation . SEM is indicated for n = 5 - 8 samples . The p values were calculated relative to white RNAi control at each time point ( * p < . 05 , * * p < . 01 ) .

O O O O O O B-SMALL_MOLECULE O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Time course of glycogen levels in Dmef2-Gal4 carcasses (muscle+body wall) with expression of UAS-RNAi transgenes targeting white, GlyP, Atg1, or GlyP+Atg1. Simultaneous knockdown of GlyP and Atg1, but not either gene alone, significantly reduced glycogen degradation compared to the white control after 24 h of starvation, consistent with immunostaining (C-F). Between 6 and 12 h of starvation, individual knockdown of GlyP or Atg1 caused a significant increase in glycogen levels, indicating a reduced rate of glycogen degradation. SEM is indicated for n = 5-8 samples. The p values were calculated relative to white RNAi control at each time point (*p<.05, **p<.01).


( A - D ) Glycogen synthase ( GlyS ) is required for glycogen synthesis in D . melanogaster muscles . PAS staining for glycogen was absent in Dmef2 - Gal4 / UAS - GlySi muscles ( B ) compared to control Dmef2 - Gal4 / UAS - whitei muscles ( A ) . Antiglycogen immunostaining for glycogen was absent in Dmef2 - Gal4 / UAS - GlySi muscles ( D ) compared to Dmef2 - Gal4 / UAS - whitei control muscles ( C ) .

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

(A-D) Glycogen synthase (GlyS) is required for glycogen synthesis in D. melanogaster muscles. PAS staining for glycogen was absent in Dmef2-Gal4/UAS-GlySi muscles (B) compared to control Dmef2-Gal4/UAS-whitei muscles (A). Antiglycogen immunostaining for glycogen was absent in Dmef2-Gal4/UAS-GlySi muscles (D) compared to Dmef2-Gal4/UAS-whitei control muscles (C).


( C ) . ( E - K ) GlyS is required for the formation of large CQ - induced autophagosomes . Vesicles are much smaller in Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - GlySi larval muscle starved 6 h in low - nutrient food + 2 . 5 mg / ml CQ ( G ) than in control Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - whitei larval muscle ( E ) . ( F , H ) The difference in autophagosome size is clearly evident at high magnification .

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

(C). (E-K) GlyS is required for the formation of large CQ-induced autophagosomes. Vesicles are much smaller in Dmef2-Gal4, UAS-GFP-Atg8/UAS-GlySi larval muscle starved 6 h in low-nutrient food +2.5 mg/ml CQ (G) than in control Dmef2-Gal4, UAS-GFP-Atg8/UAS-whitei larval muscle (E). (F, H) The difference in autophagosome size is clearly evident at high magnification.


( I - K ) Quantification of autophagy changes due to GlyS gene knockdown in Dmef2 - Gal4 , UAS - GFP - Atg8 larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . SEM is indicated , with n = 5 ( I ) or n = 10 ( J - K ) ventral longitudinal muscles from individual animals ( * p < . 05 , * * p < . 01 ) . ( I ) Each of the four UAS - GlyS RNAi transgenes tested caused a significant decrease in the total area of GFP - Atg8vesicles in the muscle compared to the UAS - whitei control . ( J ) Vesicle number was unchanged by GlyS knockdown . ( K ) UAS - GlyS RNAi caused a highly significant decrease in the mean vesicle size ( area ) compared to the control .

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

(I-K) Quantification of autophagy changes due to GlyS gene knockdown in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. SEM is indicated, with n = 5 (I) or n = 10 (J-K) ventral longitudinal muscles from individual animals (*p<.05, **p<.01). (I) Each of the four UAS-GlyS RNAi transgenes tested caused a significant decrease in the total area of GFP-Atg8vesicles in the muscle compared to the UAS-whitei control. (J) Vesicle number was unchanged by GlyS knockdown. (K) UAS-GlyS RNAi caused a highly significant decrease in the mean vesicle size (area) compared to the control.


( L - M ) GlyS gene expression , monitored using a MiMIC transposon insertion ( MI01490 ) , showed expression in the larval muscle ( L ) but not the fat body ( M ) ( green , GFP ; blue , DAPI ) .

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

(L-M) GlyS gene expression, monitored using a MiMIC transposon insertion (MI01490), showed expression in the larval muscle (L) but not the fat body (M) (green, GFP; blue, DAPI).


N - O ) Larvae were starved on low - nutrient food for 6 h prior to dissection of the fat bodies . Autophagy in Cg - Gal4 / + ; UAS - GFP - Atg8 / UAS - GlySi ( O ) was not substantially different from autophagy in Cg - Gal4 / + ; UAS - GFP - Atg8 / UAS - whitei control fat bodies ( GFP , green ; DAPI , blue ) .

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

N-O) Larvae were starved on low-nutrient food for 6 h prior to dissection of the fat bodies. Autophagy in Cg-Gal4/+; UAS-GFP-Atg8/UAS-GlySi (O) was not substantially different from autophagy in Cg-Gal4/+; UAS-GFP-Atg8/UAS-whitei control fat bodies (GFP, green; DAPI, blue).


. ( P ) EM of muscle from Dmef2 - Gal4 / UAS - GlySi animal starved on low - nutrient food + CQ . Note that the intermyofibril spaces ( red asterisk ) and sarcomere structure are not distorted .

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

. (P) EM of muscle from Dmef2-Gal4/UAS-GlySi animal starved on low-nutrient food +CQ. Note that the intermyofibril spaces (red asterisk) and sarcomere structure are not distorted.


Q ) GlyS or Atg1 knockdown significantly improved the crawling time of larvae treated with CQ and starved for 6 h . SEM is indicated for n = 10 larvae . The p values were calculated relative to white RNAi control larvae ( * p < . 05 , * * p < . 01 ) .

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

Q) GlyS or Atg1 knockdown significantly improved the crawling time of larvae treated with CQ and starved for 6 h. SEM is indicated for n = 10 larvae. The p values were calculated relative to white RNAi control larvae (*p<.05, **p<.01).


( A ) Protein sequence alignment of the C - terminal region of D . melanogaster , human , and yeast Glycogen synthases . Identical residues are blue ; all other residues are red . Conserved in all three species , R593 , W609 , and S651 are underlined .

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

(A) Protein sequence alignment of the C-terminal region of D. melanogaster, human, and yeast Glycogen synthases. Identical residues are blue; all other residues are red. Conserved in all three species, R593, W609, and S651 are underlined.


( B ) Western blot / Co - immunoprecipitation ( co - IP ) showing that Flag - Atg8 binds to a Venus - GlyS or Venus - GlyS ( S651A ) protein complex in response to starvation . Flag - Atg8 is unable to co - IP with either Venus - GlyS ( R593A ) or Venus - GlyS ( W609A ) . Venus - GlyS and Venus - GlyS mutants were co - IP ' d from muscle lysate from Dmef2 - Gal4 / UAS - Flag - Atg8 or UAS - Venus - GlyS ( WT or mutant ) / + ; Dmef2 - Gal4 / UAS - Flag - Atg8third instar larvae . These were fed on high - nutrientfood for 18 h , and then transferred to fresh high - nutrientfood or low - nutrientfood for 6 h .

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

(B) Western blot/Co-immunoprecipitation (co-IP) showing that Flag-Atg8 binds to a Venus-GlyS or Venus-GlyS (S651A) protein complex in response to starvation. Flag-Atg8 is unable to co-IP with either Venus-GlyS (R593A) or Venus-GlyS (W609A). Venus-GlyS and Venus-GlyS mutants were co-IP'd from muscle lysate from Dmef2-Gal4/UAS-Flag-Atg8 or UAS-Venus-GlyS(WT or mutant)/+;Dmef2-Gal4/UAS-Flag-Atg8third instar larvae. These were fed on high-nutrientfood for 18 h, and then transferred to fresh high-nutrientfood or low-nutrientfood for 6 h.


( C ) Venus - GlyS was localized predominantly to the Flag - labeled autophagosomes , with weak staining in the rest of the cytoplasm . ( D ) Venus - GlyS ( R593A ) was found throughout the cytoplasm and did not colocalize with autophagosomes . ( E ) Venus - GlyS ( S651A ) was localized to the autophagosomes . ( F ) Venus - GlyS ( W609A ) did not colocalize with the Flag - labeled autophagosomes .

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

(C) Venus-GlyS was localized predominantly to the Flag-labeled autophagosomes, with weak staining in the rest of the cytoplasm. (D) Venus-GlyS (R593A) was found throughout the cytoplasm and did not colocalize with autophagosomes. (E) Venus-GlyS (S651A) was localized to the autophagosomes. (F) Venus-GlyS (W609A) did not colocalize with the Flag-labeled autophagosomes.


( B ) Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 ( Casparian strip integrity factor 2 ) ligand treatment . Lignin and cellulosic ( unmodified ) cell walls are stained with Basic Fuchsin and Calcofluor White , shown in magenta and white respectively . Schematics are indicating the position of optical sections in a 3D illustration . For each condition , at least 10 roots were tested and showed similar results in two independent experiments . White arrows indicate sites of excess lignification on the cortex facing ( outer ) side . Scale Bar = 5 µm . Data information : " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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

(B) Lignin accumulation patterns at endodermal surface or median positions with or without the 100 nM CIF2 (Casparian strip integrity factor 2) ligand treatment. Lignin and cellulosic (unmodified) cell walls are stained with Basic Fuchsin and Calcofluor White, shown in magenta and white respectively. Schematics are indicating the position of optical sections in a 3D illustration. For each condition, at least 10 roots were tested and showed similar results in two independent experiments. White arrows indicate sites of excess lignification on the cortex facing (outer) side. Scale Bar = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( C , D ) Localization of SGN1 - Citrine and lignin deposition patterns in pCASP1 : : SGN1 - Citrine lines in wild - type ( Col ) and different mutant backgrounds ( sgn1 , sgn3 , cif1 cif2 ) ( C ) . myrpalm - SGN1 - Citrine localization and lignin deposition patterns in pCASP1 : : myrpalm - SGN1 - Citrine lines ( D ) . Lignin ( Basic Fuchsin ) and cell walls ( Calcoflour White ) are shown in magenta and white respectively . For this experiment , two or three independent lines were tested . From each transgenic line , 2 positions from 12 roots were observed and representative pictures are shown in the figure . Schematics are indicating the position of optical sections in a 3D illustration . White arrows in ( D ) highlight excess lignification on the pericycle - facing ( inner ) side . Scale bars = 5 µm . Data information : " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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

(C,D) Localization of SGN1-Citrine and lignin deposition patterns in pCASP1::SGN1-Citrine lines in wild-type (Col) and different mutant backgrounds (sgn1, sgn3, cif1 cif2) (C). myrpalm-SGN1-Citrine localization and lignin deposition patterns in pCASP1::myrpalm-SGN1-Citrine lines (D). Lignin (Basic Fuchsin) and cell walls (Calcoflour White) are shown in magenta and white respectively. For this experiment, two or three independent lines were tested. From each transgenic line, 2 positions from 12 roots were observed and representative pictures are shown in the figure. Schematics are indicating the position of optical sections in a 3D illustration. White arrows in (D) highlight excess lignification on the pericycle-facing (inner) side. Scale bars = 5 µm. Data information: "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( A ) Quantification of defects in CSD formation as number of holes per 100 µm in the CASP1 - GFP domain at around 10 cells after onset of CASP1 - GFP expression in 5 - day - old seedlings . In the box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper and lower whiskers extend to maximum or minimum values no further than 1 . 5 times IQR ( interquartile range , the distance between the first and third quartiles ) . One - way ANOVA was performed followed by Tukey ' s test . Different letters show significant statistical differences . ( p < 0 . 05 , One - way ANOVA and Tukey ' s test , 12 roots in total were observed for each condition in two independent assays ) .

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

(A) Quantification of defects in CSD formation as number of holes per 100 µm in the CASP1-GFP domain at around 10 cells after onset of CASP1-GFP expression in 5-day-old seedlings. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to maximum or minimum values no further than 1.5 times IQR (interquartile range, the distance between the first and third quartiles). One-way ANOVA was performed followed by Tukey's test. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test, 12 roots in total were observed for each condition in two independent assays).


( B ) Propidium iodide ( PI ) penetration assay in the presence or the absence of CIF2 . CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells . In the box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper and lower whiskers extend to the maximum or minimum values no further than 1 . 5 times IQR . Different letters show significant statistical differences . ( p < 0 . 05 , One - way ANOVA and Tukey ' s test . During two independent experiments , 10 roots in total were tested for each condition ) .

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

(B) Propidium iodide (PI) penetration assay in the presence or the absence of CIF2. CS barrier function was scored as exclusion of PI signal from the inner side of endodermal cells. In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters show significant statistical differences. (p<0.05, One-way ANOVA and Tukey's test. During two independent experiments, 10 roots in total were tested for each condition).


( C ) [ γ - 32P ] ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1 . Autoradiograph is shown on top . Coomassie stained - gel below illustrates presence and equal loading of recombinant proteins . Note that a kinase - dead SGN1 variant was used to avoid auto - phosphorylation activity of SGN1 . Also note that trigger factor represents a very big tag protein , accounting for the high migration of TF - SGN1 . Representative result of three independent experiments is shown .

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

(C) [γ-32P]ATP radioactive in vitro kinase assay of SGN3 kinase domain against SGN1. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Note that a kinase-dead SGN1 variant was used to avoid auto-phosphorylation activity of SGN1. Also note that trigger factor represents a very big tag protein, accounting for the high migration of TF-SGN1. Representative result of three independent experiments is shown.


( A ) Localization of Citrine - RBOHD ( left ) or Citrine - RBOHF ( right ) in endodermal ( en ) cells . Both proteins were expressed under the control of pCASP1 , an endodermis - specific promoter . Representative pictures are shown , 2 positions from 10 roots for each transgenic line were inspected . Scale bar = 5 µm .

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

(A) Localization of Citrine-RBOHD (left) or Citrine-RBOHF (right) in endodermal (en) cells. Both proteins were expressed under the control of pCASP1, an endodermis-specific promoter. Representative pictures are shown, 2 positions from 10 roots for each transgenic line were inspected. Scale bar = 5 µm.


( B ) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2 - hour 100 nM CIF2 peptide treatment . Arrowheads indicate excess lignification . Pictures are shown as overviews ( maximum projection ) or median sections . Lignin and cell walls are shown with magenta ( stained with Basic Fuchsin ) and gray ( stained with Calcofluor White ) respectively . Representative pictures are shown , 12 roots ( overview ) and 2 positions in 12 roots ( median section ) were inspected . Scale bars = 20 µm ( lignin overviews ) , 5 µm ( median sections ) . " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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

(B) Lignin accumulation in WT and rbohD and rbohF single mutants and a double mutant with or without 2-hour 100 nM CIF2 peptide treatment. Arrowheads indicate excess lignification. Pictures are shown as overviews (maximum projection) or median sections. Lignin and cell walls are shown with magenta (stained with Basic Fuchsin) and gray (stained with Calcofluor White) respectively. Representative pictures are shown, 12 roots (overview) and 2 positions in 12 roots (median section) were inspected. Scale bars = 20 µm (lignin overviews), 5 µm (median sections). "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( A ) Co - visualization of pCASP1 : : SGN1 - mCherry and pCASP1 : : SGN3 - GFP . Note that their respective localization at the PM has only restricted overlap at the cortical side of the CS domain ( Arrow ) . Representative picture is shown , 3 - 6 roots in 4 different transgenic lines were inspected . Scale bar = 10 µm . " inner " designates the stele - facing endodermal surface , " outer " , the cortex - facing surface .

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

(A) Co-visualization of pCASP1::SGN1-mCherry and pCASP1::SGN3-GFP. Note that their respective localization at the PM has only restricted overlap at the cortical side of the CS domain (Arrow). Representative picture is shown, 3-6 roots in 4 different transgenic lines were inspected. Scale bar = 10 µm. "inner" designates the stele-facing endodermal surface, "outer", the cortex-facing surface.


( B , C ) Overview of endodermal cells after with or without 1 µM CIF2 treatment for 24 hours . Red arrowheads are indicating ROS production sites and boxes in dotted lines are corresponding to the regions in ( sky blue and creme boxes in C and black boxes in D ) . Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment . Scale bars = 500 nm .

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

(B,C) Overview of endodermal cells after with or without 1 µM CIF2 treatment for 24 hours. Red arrowheads are indicating ROS production sites and boxes in dotted lines are corresponding to the regions in (sky blue and creme boxes in C and black boxes in D). Similar patterns were obtained in 39 or 41 cells from 5 roots with or without the peptide treatment. Scale bars = 500 nm.


( D ) In situ H2O2 detection at Casparian strips in WT , sgn3 , sgn1 , rbohF , rbohD and rbohDF with or without 24 - hour treatment of 1 µM CIF2 . Brackets and arrowheads indicate Casparian strips ( seen as uniformly whitish cell wall areas ) and H2O2 production sites ( black area ) respectively . Scale bar = 1000 nm .

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

(D) In situ H2O2 detection at Casparian strips in WT, sgn3, sgn1, rbohF, rbohD and rbohDF with or without 24-hour treatment of 1 µM CIF2. Brackets and arrowheads indicate Casparian strips (seen as uniformly whitish cell wall areas) and H2O2 production sites (black area) respectively. Scale bar = 1000 nm.


( E ) Quantification of ROS production as a number of dark pixels area ( n = 23 - 44 sites from 5 roots of each condition in one experiment . ) . In the box plot , boxes indicate ranges from first to third quartiles , bold central lines display median . Upper and lower whiskers extend to the maximum or minimum values no further than 1 . 5 times IQR . Different letters mean significant statistical differences . ( p < 0 . 01 , One - way ANOVA and Tukey ' s test . )

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

(E) Quantification of ROS production as a number of dark pixels area (n = 23 - 44 sites from 5 roots of each condition in one experiment.). In the box plot, boxes indicate ranges from first to third quartiles, bold central lines display median. Upper and lower whiskers extend to the maximum or minimum values no further than 1.5 times IQR. Different letters mean significant statistical differences. (p<0.01, One-way ANOVA and Tukey's test.)


( A ) [ γ - 32P ] ATP radioactive in vitro kinase assay of TF - SGN1 against GST - N - terminal cytoplasmic domains of RBOHD or F . Autoradiograph is shown on top . Coomassie stained - gel below illustrates presence and equal loading of recombinant proteins . Experiments were done independently three times with similar results .

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

(A) [γ-32P]ATP radioactive in vitro kinase assay of TF-SGN1 against GST-N-terminal cytoplasmic domains of RBOHD or F. Autoradiograph is shown on top. Coomassie stained-gel below illustrates presence and equal loading of recombinant proteins. Experiments were done independently three times with similar results.


( B ) HEK293Tcell based NOX activation assay . Cells were transfected with the indicated plasmid combinations . The phosphatase inhibitor Calyculin A was added directly before the start of the measurements . Each data point represents the mean of six wells analyzed in parallel , bars indicate S . D . . Experiments were repeated three

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

(B) HEK293Tcell based NOX activation assay. Cells were transfected with the indicated plasmid combinations. The phosphatase inhibitor Calyculin A was added directly before the start of the measurements. Each data point represents the mean of six wells analyzed in parallel, bars indicate S.D.. Experiments were repeated three times


( A ) CASP1 - GFP and lignin deposition in WT , sgn3 , rbohD , rbohF and rbohDF . CASP1 - GFP and lignin ( fuchsin ) are presented in green and magenta respectively . Pictures were obtained from more than 10 roots from each background with similar results . Scale bar = 10 µm .

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

(A) CASP1-GFP and lignin deposition in WT, sgn3, rbohD, rbohF and rbohDF. CASP1-GFP and lignin (fuchsin) are presented in green and magenta respectively. Pictures were obtained from more than 10 roots from each background with similar results. Scale bar = 10 µm.


( B ) Time lapse imaging of single - or cotreatment of 10 nM CIF2 with 25 µM cycloheximide ( CHX ) on CASP1 - GFP in cif1 cif2 . Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium . Scale bar = 10 µm ( See also movie EV1 ) . ( C ) Quantification of ( B ) . Relative numbers of holes in CASP1 - GFP domain after single - or co - treatment with CIF2 or CHX from the pictures in ( B ) . Bars are S . D . * indicates statistical significance from all other conditions ( p < 0 . 01 ) after one - way ANOVA and Tukey test . 6 roots in total for each condition were observed during two independent tests .

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

(B) Time lapse imaging of single- or cotreatment of 10 nM CIF2 with 25 µM cycloheximide (CHX) on CASP1-GFP in cif1 cif2. Seedlings were pretreated with or without CHX for 30 min and transferred onto each medium. Scale bar = 10 µm (See also movie EV1). (C) Quantification of (B). Relative numbers of holes in CASP1-GFP domain after single- or co-treatment with CIF2 or CHX from the pictures in (B). Bars are S.D. * indicates statistical significance from all other conditions (p<0.01) after one-way ANOVA and Tukey test. 6 roots in total for each condition were observed during two independent tests.


( D ) Fold - change of 930 genes ( p < 0 . 05 and log2 ( Fold change ) ≥ 1 or ≤ - 1 at least one time point in one genotype ) after CIF2 treatment at indicated time points in WT , cif1 , 2 and sgn3 . Degree of the fold changes are shown in color code as indicated .

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

(D) Fold-change of 930 genes (p<0.05 and log2(Fold change) ≥1 or ≤ -1 at least one time point in one genotype) after CIF2 treatment at indicated time points in WT, cif1,2 and sgn3. Degree of the fold changes are shown in color code as indicated.


( E ) Relative expression levels of PER15 to CLATHRIN control in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) . ( F ) Fold changes of PER15 in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistical significance differences ( p < 0 . 01 , ANOVA and Tukey test ) .

O O O O B-EXP_ASSAY 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 O O 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 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

(E) Relative expression levels of PER15 to CLATHRIN control in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (F)Fold changes of PER15 in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistical significance differences (p < 0.01, ANOVA and Tukey test).


( G ) Relative expression levels of PER49 to CLATHRIN control in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) . ( H ) Fold changes of PER49 in each genotype with or without 2 - hour CIF2 treatment . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) .

(G) Relative expression levels of PER49 to CLATHRIN control in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (H) Fold changes of PER49 in each genotype with or without 2-hour CIF2 treatment. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).


( I ) Relative fold changes of PER15 in pCASP1 : : SGN1 - Citrine and pCASP1 : : myrpalm - SGN1 - Citrine compared to the expression level in WT . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) . ( J ) Relative fold changes of PER49 in pCASP1 : : SGN1 - Citrine and pCASP1 : : myrpalm - SGN1 - Citrine compared to the expression level in WT . Bars are S . D . ( n = 3 ) . Different characters indicate statistically significant differences ( p < 0 . 01 , ANOVA and Tukey test ) .

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

(I)Relative fold changes of PER15 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test). (J)Relative fold changes of PER49 in pCASP1::SGN1-Citrine and pCASP1::myrpalm-SGN1-Citrine compared to the expression level in WT. Bars are S.D. (n=3). Different characters indicate statistically significant differences (p < 0.01, ANOVA and Tukey test).


A Immunostaining of HeLa cells probed for FITC ‐ labeled streptavidin and DNA and the merge streptavidin ( red ) and DNA ( blue ) . Cell surface was labeled using amine biotinylation . Scale bar , 10 μm .

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

A Immunostaining of HeLa cells probed for FITC‐labeled streptavidin and DNA and the merge streptavidin (red) and DNA (blue). Cell surface was labeled using amine biotinylation. Scale bar, 10 μm.


B Western blot analysis of cell surface preparation . The surface ‐ biotinylated cells were lysed using a detergent containing lysis buffer , incubated with and then eluted from streptavidin beads ( i . e . , cell sur . fraction ) . Cell surface fraction and the whole ‐ cell lysate were blotted against a cell surface protein , EGFR , and a cytoplasmic protein , actin .

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

B Western blot analysis of cell surface preparation. The surface‐biotinylated cells were lysed using a detergent containing lysis buffer, incubated with and then eluted from streptavidin beads (i.e., cell sur. fraction). Cell surface fraction and the whole‐cell lysate were blotted against a cell surface protein, EGFR, and a cytoplasmic protein, actin.


B Western blot confirmation of the mitosis ‐ selective cell surface localization of PCDH7 . Western blotting analysis of cell surface protein preparation from mitosis and interphase cells . The whole ‐ cell lysate and elution from streptavidin beads were blotted against TFRC , EGFR , actin , and phospho ‐ Histone H3 . TFRC protein was enriched in interphase cell surface preparation in comparison with mitosis preparation , while PCDH7 protein was enriched in mitotic cell surface preparation .

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

B Western blot confirmation of the mitosis‐selective cell surface localization of PCDH7. Western blotting analysis of cell surface protein preparation from mitosis and interphase cells. The whole‐cell lysate and elution from streptavidin beads were blotted against TFRC, EGFR, actin, and phospho‐Histone H3. TFRC protein was enriched in interphase cell surface preparation in comparison with mitosis preparation, while PCDH7 protein was enriched in mitotic cell surface preparation.


C Western blot confirmation of the mitosis ‐ selective cell surface localization of PCDH1 . Whole ‐ cell lysate samples were subjected to SDS - PAGE and blotted with the anti ‐ PCDH1 , anti ‐ β ‐ actin , and anti ‐ phospho ‐ Histone H3 antibodies , as indicated on the left . Cell surface fraction samples were blotted with the anti ‐ PCDH1 and anti ‐ EGFR antibodies .

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

C Western blot confirmation of the mitosis‐selective cell surface localization of PCDH1. Whole‐cell lysate samples were subjected to SDS-PAGE and blotted with the anti‐PCDH1, anti‐β‐actin, and anti‐phospho‐Histone H3 antibodies, as indicated on the left. Cell surface fraction samples were blotted with the anti‐PCDH1 and anti‐ EGFR antibodies.


A Tubulin and PCDH7immunostaining of unsynchronized HeLa cells : tubulin , green ; PCDH7 , red ; DNA , blue . PCDH7 localizes to the mitotic cell surface ( arrows ) . PCDH7 staining is barely detectable in the surrounding interphase cells . Scale bar , 5 μm . B Quantification of PCDH7 signal on the cell surface of immunostained cells in mitosis ( n = 6 , red ) and interphase ( n = 7 , blue ) . Bars show mean ± SEM .

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

A Tubulin and PCDH7immunostaining of unsynchronized HeLa cells: tubulin, green; PCDH7, red; DNA, blue. PCDH7 localizes to the mitotic cell surface (arrows). PCDH7 staining is barely detectable in the surrounding interphase cells. Scale bar, 5 μm.B Quantification of PCDH7 signal on the cell surface of immunostained cells in mitosis (n = 6, red) and interphase (n = 7, blue). Bars show mean ± SEM.


C PCDH7 ‐ EGFP ‐ transfected unsynchronized U2OS cells , stably expressing mCherry ‐ tubulin , at mitosis ( top ) and interphase ( bottom ) . PCDH7 , green ; tubulin , red ; DNA , blue . Scale bar , 10 μm .

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

C PCDH7‐EGFP‐transfected unsynchronized U2OS cells, stably expressing mCherry‐tubulin, at mitosis (top) and interphase (bottom). PCDH7, green; tubulin, red; DNA, blue. Scale bar, 10 μm.


D Immunostaining of HeLa Kyoto cells stably expressing PCDH7 ‐ LAP ‐ tagged BAC transgenes ( green ) probed for tubulin ( red ) and DNA ( blue ) . Top panel shows mitotic cell those were treated with 5 μM STC for 10 h , and bottom panel shows interphase cells . Scale bar , 10 μm .

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

D Immunostaining of HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes (green) probed for tubulin (red) and DNA (blue). Top panel shows mitotic cell those were treated with 5 μM STC for 10 h, and bottom panel shows interphase cells. Scale bar, 10 μm.


A Confocal microscopy images representing the localization of PCDH7 . U2OS cells expressing full ‐ length ( WT ) PCDH7 at mitosis ( top ) and interphase ( middle ) . Cytoplasmic deleted ( CytΔ ) PCDH7 ‐ expressing cells at mitosis ( middle ) and interphase ( bottom ) . PCDH7 , green ; DNA , blue . Scale bar , 20 μm . B Quantification of PCDH7 : : GFP ( WT and CytΔ ) protein localization on the cell surface in mitosis [ WT ( n = 11 ) , CytΔ ( n = 6 ) , red ] and interphase [ WT ( n = 25 ) , CytΔ ( n = 20 ) , blue ] . Interphase cytΔ PCDH7 localizes to the cell surface more than WT ( full length ) PCDH7 ( insets ) . Bars show mean ± SEM .

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

A Confocal microscopy images representing the localization of PCDH7. U2OS cells expressing full‐length (WT) PCDH7 at mitosis (top) and interphase (middle). Cytoplasmic deleted (CytΔ) PCDH7‐expressing cells at mitosis (middle) and interphase (bottom). PCDH7, green; DNA, blue. Scale bar, 20 μm.B Quantification of PCDH7::GFP (WT and CytΔ) protein localization on the cell surface in mitosis [WT (n = 11), CytΔ (n = 6), red] and interphase [WT (n = 25), CytΔ (n = 20), blue]. Interphase cytΔ PCDH7 localizes to the cell surface more than WT (full length) PCDH7 (insets). Bars show mean ± SEM.


C In interphase co ‐ localization between PCDH7 and ER marker KDEL . HeLa cells were transiently transfected with PCDH7 : : GFP ( green ) ( ectopic ) , fixed and stained for ER marker KDEL ( red ) ( interphase , top panel ) . Interphase ( middle panel ) and mitotic ( bottom panel ) HeLa cells were fixed and stained against ( endogenous ) PCDH7 and KDEL . Scale bar , 5 μm .

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C In interphase co‐localization between PCDH7 and ER marker KDEL. HeLa cells were transiently transfected with PCDH7::GFP (green) (ectopic), fixed and stained for ER marker KDEL (red) (interphase, top panel). Interphase (middle panel) and mitotic (bottom panel) HeLa cells were fixed and stained against (endogenous) PCDH7 and KDEL. Scale bar, 5 μm.


D PCDH7 and PCDH1 enriched on endoplasmic reticulum fraction . Whole ‐ cell lysate , cytoplasmic , endoplasmic , and mitochondrial fractions were blotted against an endoplasmic reticulum protein , KDEL , a cytoplasmic protein , actin , a mitochondrial protein Cox ‐ 2 and PCDH7 , PCDH1 .

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D PCDH7 and PCDH1 enriched on endoplasmic reticulum fraction. Whole‐cell lysate, cytoplasmic, endoplasmic, and mitochondrial fractions were blotted against an endoplasmic reticulum protein, KDEL, a cytoplasmic protein, actin, a mitochondrial protein Cox‐2 and PCDH7, PCDH1.


A PCDH7 antibody detects a single band of ˜ 110 kDa in luciferase RNAi ( luc ) , PCDH1 RNAi HeLa cell lysates , but this band is not detectable in PCDH7 and PCDH1 and PCDH7 RNAi cells . α ‐ tubulin was used as a loading control ( left ) . HeLa Kyoto cells stably expressing PCDH7 ‐ LAP ‐ tagged BAC transgenes treated with luciferase RNAi ( control ) and 150 nM ( 1 × ) and 300 nM ( 2 × ) PCDH1 esiRNA and blotted against anti ‐ GFP , anti ‐ PCDH1 , and anti ‐ actin antibodies . PCDH1 bands were reduced in PCDH1 RNAi cells , whereas mouse GFP : : PCDH1 was not affected by the esiRNA treatment ( right ) .

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

A PCDH7 antibody detects a single band of ˜110 kDa in luciferase RNAi (luc), PCDH1 RNAi HeLa cell lysates, but this band is not detectable in PCDH7 and PCDH1 and PCDH7 RNAi cells. α‐tubulin was used as a loading control (left). HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with luciferase RNAi (control) and 150 nM (1×) and 300 nM (2×) PCDH1 esiRNA and blotted against anti‐GFP, anti‐PCDH1, and anti‐actin antibodies. PCDH1 bands were reduced in PCDH1 RNAi cells, whereas mouse GFP::PCDH1 was not affected by the esiRNA treatment (right).


B Rounding pressure plots of MYH9 ( positive control , n = 7 ) , luc ( negative control , n = 12 ) , PCDH7 ( n = 11 ) , PCDH1 ( n = 12 ) , and PCDH1 and PCDH7 ( n = 11 ) RNAi cells . Bars show mean ± SEM ( left ) . Rounding pressure plots HeLa Kyoto cells stably expressing PCDH7 ‐ LAP ‐ tagged BAC transgenes treated with MYH9 ( positive control , n = 7 ) , luc ( negative control , n = 14 ) , PCDH1 ( n = 13 ) esiRNA . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; ns , P > 0 . 05 ( non ‐ significant ) .

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

B Rounding pressure plots of MYH9 (positive control, n = 7), luc (negative control, n = 12), PCDH7 (n = 11), PCDH1 (n = 12), and PCDH1 and PCDH7 (n = 11) RNAi cells. Bars show mean ± SEM (left). Rounding pressure plots HeLa Kyoto cells stably expressing PCDH7‐LAP‐tagged BAC transgenes treated with MYH9 (positive control, n = 7), luc (negative control, n = 14), PCDH1 (n = 13) esiRNA. *P 0.05; **P 0.01; ***P 0.001; ns, P > 0.05 (non‐significant).


B Flow cytometry analysis of HeLa cells treated with anti ‐ PCDH7 antibody , fixed and stained for phospho ‐ Histone H3 and DNA . Forty ‐ two percent of unsorted HeLa cella are in G2 / M phase . When cells are sorted based on their phospho ‐ Histone H3 ( middle ) expression , phospho ‐ Histone H3 + ( high expression ) cells are preferentially mitotic ( 87 . 3 % in G2 / M phase ) and 42 % of those are PCDH7 + . Similarly , PCDH7 + ( high expression ) cells are preferentially mitotic ( 82 . 1 % in G2 / M phase ) and 79 % of those are phospho ‐ Histone H3 + ( bottom ) .

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

B Flow cytometry analysis of HeLa cells treated with anti‐PCDH7 antibody, fixed and stained for phospho‐Histone H3 and DNA. Forty‐two percent of unsorted HeLa cella are in G2/M phase. When cells are sorted based on their phospho‐Histone H3 (middle) expression, phospho‐Histone H3+ (high expression) cells are preferentially mitotic (87.3% in G2/M phase) and 42% of those are PCDH7+. Similarly, PCDH7+ (high expression) cells are preferentially mitotic (82.1% in G2/M phase) and 79% of those are phospho‐Histone H3+ (bottom).


( a ) Lipid phosphatase activity of Paladin , wild - type and phosphatase - dead C / S mutant , towards PI ( 4 , 5 ) P2 and PI ( 3 , 4 , 5 ) P3 substrates . Positive control ; wild - type phosphatase and tensin homolog ( PTEN ) ; negative control ; lipid phosphatase - dead C124S PTEN . Mean ± SEM , Paired t - test , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(a) Lipid phosphatase activity of Paladin, wild-type and phosphatase-dead C/S mutant, towards PI(4,5)P2 and PI(3,4,5)P3 substrates. Positive control; wild-type phosphatase and tensin homolog (PTEN); negative control; lipid phosphatase-dead C124S PTEN. Mean±SEM, Paired t-test, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( b ) Representative confocal image of HDMEC stained for Paladin ( green ) , Vascular Endothelial Cadherin ( VEC , red ) and nuclei ( DAPI , blue ) , scale bar : 10 μm . White boxes in the upper image are showed at higher magnification below , scale bar : 3 µm .

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(b) Representative confocal image of HDMEC stained for Paladin (green), Vascular Endothelial Cadherin (VEC, red) and nuclei (DAPI, blue), scale bar: 10 μm. White boxes in the upper image are showed at higher magnification below, scale bar: 3 µm.


( c ) HDMEC stained for VEGFR2 ( green ) , Paladin ( red ) and nuclei ( DAPI , blue ) and with or without stimulation of 50 ng / ml VEGF - A for 0 , 2 or 10 min . Scale bar : 10 µm . ( d ) Quantification of VEGFR2 - Paladin co - staining before and after VEGF - A stimulation as shown in ( C ) . Mean ± SEM , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O B-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE 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 O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 O O O O O O O O O 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) HDMEC stained for VEGFR2 (green), Paladin (red) and nuclei (DAPI, blue) and with or without stimulation of 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (d) Quantification of VEGFR2-Paladin co-staining before and after VEGF-A stimulation as shown in (C). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( e ) HDMEC analysed using Proximity Ligand Assay ( PLA ) for Paladin and VEGFR2 . Green dots indicate complex formation , VE - cadherin ( red ) and nuclei ( DAPI , blue ) . Cells stimulated with 50 ng / ml VEGF - A for 0 , 2 or 10 min . Scale bar : 10 µm . ( f ) Quantification of ( e ) , the number of PLA Paldin - VEGFR2 complexes per cell at 0 , 2 and 10 minutes after VEGF - A stimulation ( top ) . Quantification of ( e ) , average distance for the PLA complexes to the nearest VE - cadherin positive junction at 0 , 2 and 10 minutes after VEGF - A stimulation ( bottom ) . Mean ± SEM , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(e) HDMEC analysed using Proximity Ligand Assay (PLA) for Paladin and VEGFR2. Green dots indicate complex formation, VE-cadherin (red) and nuclei (DAPI, blue). Cells stimulated with 50 ng/ml VEGF-A for 0, 2 or 10 min. Scale bar: 10 µm. (f) Quantification of (e), the number of PLA Paldin-VEGFR2 complexes per cell at 0, 2 and 10 minutes after VEGF-A stimulation (top). Quantification of (e), average distance for the PLA complexes to the nearest VE-cadherin positive junction at 0, 2 and 10 minutes after VEGF-A stimulation (bottom). Mean±SEM, One-way ANOVA. n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( g ) HDMEC stained for Paladin ( green ) and EEA1 ( red ) , colocalization in yellow , and nuclei ( DAPI , blue ) after VEGF - A stimulation for 0 , 2 or 10 minutes , a representative image from a single confocal plane is shown , scale bar : 10 µm . White boxes in the upper image are showed at higher magnification below , scale bar 3 µm . ( h ) Quantification of ( g ) , EEA1 / Paladin double positive particles per field of view at 0 , 2 and 10 minutes after VEGF - A stimulation . Mean ± SEM , One - way ANOVA . n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(g) HDMEC stained for Paladin (green) and EEA1 (red), colocalization in yellow, and nuclei (DAPI, blue) after VEGF-A stimulation for 0, 2 or 10 minutes, a representative image from a single confocal plane is shown, scale bar: 10 µm. White boxes in the upper image are showed at higher magnification below, scale bar 3 µm. (h) Quantification of (g), EEA1/Paladin double positive particles per field of view at 0, 2 and 10 minutes after VEGF-A stimulation. Mean±SEM, One-way ANOVA. n= 3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( a ) Cell surface VEGFR2 levels detected by cell surface biotinylation , using thiol - cleavable sulfo - NHS - SS - biotin , of HDMEC transfected with PALD1 siRNA ( # 1 and # 2 ) or non - targeting control ( " c " ) siRNA , followed by VEGF - A stimulation ( 50 ng / ml ) for indicated time periods . Total lysates ( input ) and streptavidin ( SA ) pull down , immunoblotted for VEGFR2 , Paladin , and actin . ' No biotin ctrl ' , cells not treated with sulfo - NHS - SS - biotin . ( b ) Quantification of data in ( a ) . VEGFR2 surface levels ( data pooled for the indicated time points ) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC . n = 4 for each time point , biological replicates , Mean ± SEM . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O B-GENEPROD B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY 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 O O O O O O O O O O O O O O O O O O O O O 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) Cell surface VEGFR2 levels detected by cell surface biotinylation, using thiol-cleavable sulfo-NHS-SS-biotin, of HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control ("c") siRNA, followed by VEGF-A stimulation (50 ng/ml) for indicated time periods. Total lysates (input) and streptavidin (SA) pull down, immunoblotted for VEGFR2, Paladin, and actin. 'No biotin ctrl', cells not treated with sulfo-NHS-SS-biotin. (b) Quantification of data in (a). VEGFR2 surface levels (data pooled for the indicated time points) normalized to total VEGFR2 levels and compared between control and siRNA treated HDMEC. n=4 for each time point, biological replicates, Mean±SEM. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( c ) Internalized pool of VEGFR2 after VEGF - A treatment ( 50 ng / ml ) of non - transfected HDMEC ( " NT " ) or HDMEC transfected with PALD1 siRNA ( # 1 and # 2 ) or non - targeting control siRNA ( " c " ) . Cell surface biotinylation was performed prior to VEGF - A stimulation and at indicated time points , remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down . Immunoblotting of the total lysate ( input ) and SA pull down fraction for VEGFR2 , Paladin and actin . ( d ) Quantification of data in ( c ) . Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples . Mean ± SEM , unpaired t - test for indicated time points , normalized to cntrl siRNA sample . n = 3 for each time point , biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(c) Internalized pool of VEGFR2 after VEGF-A treatment (50 ng/ml) of non-transfected HDMEC ("NT") or HDMEC transfected with PALD1 siRNA (#1 and #2) or non-targeting control siRNA ("c"). Cell surface biotinylation was performed prior to VEGF-A stimulation and at indicated time points, remaining cell surface biotin was stripped and the internalized pool of VEGFR2 was collected by SA pull down. Immunoblotting of the total lysate (input) and SA pull down fraction for VEGFR2, Paladin and actin. (d) Quantification of data in (c). Data were normalized to total VEGFR2 levels in the lysate after subtraction of signals in biotinylated and stripped samples. Mean±SEM, unpaired t-test for indicated time points, normalized to cntrl siRNA sample. n=3 for each time point, biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( e ) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown . Representative images of VEGFR2 ( green ) / EEA1 ( red ) double - positive ( yellow ) vesicles in negative control siRNA , and PALD1 KD # 2 siRNA - silenced HDMEC at 0 , 2 and 10 min of VEGF - A stimulation ( 50 ng / ml ) . DAPI in blue , scale bar : 10 µm . Inset shows only EEA1 channel , scale bar : 10 µm . ( f ) Quantification of ( e ) , number of EEA1 positive ( top ) or VEGFR2 - EEA1 double - positive vesicles ( bottom ) per field of view . Mean ± SEM , Two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(e) Analysis of EEA1 and VEGFR2 vesicles following PALD1 knockdown. Representative images of VEGFR2 (green)/EEA1 (red) double-positive (yellow) vesicles in negative control siRNA, and PALD1 KD#2 siRNA-silenced HDMEC at 0, 2 and 10 min of VEGF-A stimulation (50 ng/ml). DAPI in blue, scale bar: 10 µm. Inset shows only EEA1 channel, scale bar: 10 µm. (f) Quantification of (e), number of EEA1 positive (top) or VEGFR2-EEA1 double-positive vesicles (bottom) per field of view. Mean±SEM, Two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


( g ) HDMEC stained for PI ( 4 , 5 ) P2 ( cyan ) , VE - cadherin ( red ) and Paladin ( yellow in inset ) following treatment using negative control or PALD1 ( KD # 2 ) siRNA . VEGF - A stimulation for 0 , 2 or 10 minutes ( 50 ng / ml ) . DAPI in blue . Scale bar : 10 µm ( h ) Quantification of ( g ) , Total PI ( 4 , 5 ) P2 signal ( top ) or intracellular PI ( 4 , 5 ) P2 not overlapping with VE - cadherin ( bottom ) . Mean ± SEM , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O B-CELL_TYPE B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O 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 O O O O O 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) HDMEC stained for PI(4,5)P2 (cyan), VE-cadherin (red) and Paladin (yellow in inset) following treatment using negative control or PALD1 (KD#2) siRNA. VEGF-A stimulation for 0, 2 or 10 minutes (50 ng/ml). DAPI in blue. Scale bar: 10 µm (h) Quantification of (g), Total PI(4,5)P2 signal (top) or intracellular PI(4,5)P2 not overlapping with VE-cadherin (bottom). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


Signalling downstream of VEGF - A / VEGFR2 assessed in HDMEC , untreated ( NT , non - transfected ) , transfected with non - targeting siRNA ( c / cntrl ) , or with two different PALD1 - targeting siRNAs ( KD # 1 and KD # 2 ) , and treated with VEGF - A for 0 - 10 and 0 - 20 min , respectively . Immunoblotting of cell lysates for phosphorylated ( p ) VEGFR2 ( pY1175 ) , total VEGFR2 Actin served as loading control ( a PALD1 knockdown was verified by blotting for Paladin . Quantification of pVEGFR2 ( normalized to total VEGFR2 ) ( b ) Mean ± SEM , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 .

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

Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated (p) VEGFR2 (pY1175), total VEGFR2 Actin served as loading control (a PALD1 knockdown was verified by blotting for Paladin. Quantification of pVEGFR2 (normalized to total VEGFR2) (b) Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01.


Signalling downstream of VEGF - A / VEGFR2 assessed in HDMEC , untreated ( NT , non - transfected ) , transfected with non - targeting siRNA ( c / cntrl ) , or with two different PALD1 - targeting siRNAs ( KD # 1 and KD # 2 ) , and treated with VEGF - A for 0 - 10 and 0 - 20 min , respectively . Immunoblotting of cell lysates for phosphorylated Erk1 / 2 ( pT202 and pY204 ) , and total Erk . Actin served as loading control c ) . PALD1 knockdown was verified by blotting for Paladin . Quantification of pErk1 / 2 ( normalized to total Erk1 / 2 ) ( d ) . Mean ± SEM , two - way ANOVA , n = 3 biological replicates . Data information : * p < 0 . 05 , * * p < 0 . 01 .

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

Signalling downstream of VEGF-A/VEGFR2 assessed in HDMEC, untreated (NT, non-transfected), transfected with non-targeting siRNA (c/cntrl), or with two different PALD1-targeting siRNAs (KD#1 and KD#2), and treated with VEGF-A for 0-10 and 0-20 min, respectively. Immunoblotting of cell lysates for phosphorylated Erk1/2 (pT202 and pY204), and total Erk. Actin served as loading control c). PALD1 knockdown was verified by blotting for Paladin. Quantification of pErk1/2 (normalized to total Erk1/2) (d). Mean±SEM, two-way ANOVA, n=3 biological replicates. Data information: *p < 0.05, **p < 0.01.


( e - f ) Immunoblotting on total heart lysates from adult Pald1 + / + and Pald1 - / - mice , tail - vein injected with VEGF - A ( 0 . 25µg / g body weight ) or PBS for the indicated time points , for Paladin , phosphorylated and total levels of VEGFR2 , Erk1 / 2 and β2 - microglobulin ( β 2 - MG , loading control ) . ( f ) Quantification of pVEGFR pY1173 normalized to total VEGFR2 ( n = 3 ) , total VEGFR2 levels normalized to total loading control ( n = 4 ) , pT202 / pY204 Erk1 / 2 normalized to Erk1 / 2 ( n = 5 ) . Mean ± SEM , multiple t - test with Holm - Sidak correction ( total VEGFR2 ) , two - way ANOVA ( others ) . Data information : * p < 0 . 05 , * * p < 0 . 01 .

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

(e-f) Immunoblotting on total heart lysates from adult Pald1+/+ and Pald1-/- mice, tail-vein injected with VEGF-A (0.25µg/g body weight) or PBS for the indicated time points, for Paladin, phosphorylated and total levels of VEGFR2, Erk1/2 and β2-microglobulin (β 2-MG, loading control). (f) Quantification of pVEGFR pY1173 normalized to total VEGFR2 (n=3), total VEGFR2 levels normalized to total loading control (n=4), pT202/pY204 Erk1/2 normalized to Erk1/2 (n=5). Mean±SEM, multiple t-test with Holm-Sidak correction (total VEGFR2), two-way ANOVA (others). Data information: *p < 0.05, **p < 0.01.


( a , b ) Delayed vascular outgrowth and hyperdense vascular front in isolectinB4 - stained P5 retina from Pald1 - / - mouse compared with Pald1 + / + ( a ) . Orange arrow indicates radial expansion of the vascular plexus in the Pald1 + / + retina . Scale bar : 1 mm . Quantification of radial expansion ( b ) as shown in ( a ) normalized to wild type litter mates . Mean ± SEM , unpaired t - test . n = 14 litters , 18 wild type and 23 knock - out pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE O O B-GENEPROD O O O B-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, b) Delayed vascular outgrowth and hyperdense vascular front in isolectinB4-stained P5 retina from Pald1-/- mouse compared with Pald1+/+ (a). Orange arrow indicates radial expansion of the vascular plexus in the Pald1+/+ retina. Scale bar: 1 mm. Quantification of radial expansion (b) as shown in (a) normalized to wild type litter mates. Mean±SEM, unpaired t-test. n=14 litters, 18 wild type and 23 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( c , d ) Increased filopodia number in Pald1 - / - mouse retina at vascular front , visualized by isolectinB4 staining ( c ) . Scale bar : 50 μm . Quantification of filopodia per 100 µm at the vascular front ( d ) . Mean ± SEM , unpaired t - test . n = 3 litters , 7 wild type and 5 knock - out pups . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(c, d) Increased filopodia number in Pald1-/- mouse retina at vascular front, visualized by isolectinB4 staining (c). Scale bar: 50 μm. Quantification of filopodia per 100 µm at the vascular front (d). Mean±SEM, unpaired t-test. n=3 litters, 7 wild type and 5 knock-out pups. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.


( e , f ) P5 retina vascular front ( isolectinB4 , green ) and endothelial nuclei ( Erg , red ) ( e ) . Scale bar : 100 μm . Magenta stippled square area quantified in ( f ) . Vascular density was determined in the capillary bed ( left ) ( 11 wild type , 12 knock - out ) and in area around veins ( right ) ( 10 wild type , 9 knock - out , biological replicates ) . Mean ± SEM , unpaired t - test . Data information : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(e, f) P5 retina vascular front (isolectinB4, green) and endothelial nuclei (Erg, red) (e). Scale bar: 100 μm. Magenta stippled square area quantified in (f). Vascular density was determined in the capillary bed (left) (11 wild type, 12 knock-out) and in area around veins (right) (10 wild type, 9 knock-out, biological replicates). Mean±SEM, unpaired t-test. Data information: *p < 0.05, **p < 0.01, ***p < 0.001.