Figure 1 ( 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 . ( 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 . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.(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).(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).


Figure 2 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.(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.


Figure 3 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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).(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.


Figure 4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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).


Figure 5 ( 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 . ( 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 ) . 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 . 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.(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).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.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).


Figure 6 ( 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 . ( 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 .

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Figure 6(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.(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.


Figure 1 ( B ) Lineage tracing of the anagen matrix from β - actin - CreER : Confetti , 7 days after induction with either a high dose of tamoxifen ( TM ) ( top ) or low dose ( bottom ) for single cell clonal lineages . ( C ) Images of skin sections at various stages of the hair cycle , immunostained for Gata6 ( red ) , CD34 or K14 ( green ) and DNA ( blue ) . ( D ) images ( D ' ) for Gata6 inducible knockout ( iKO ) and wild type ( WT ) mice over time , showing the loss of Gata6 ( red ) from the hair follicle . Scale bars : 30 m .

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Figure 1(B) Lineage tracing of the anagen matrix from β-actin-CreER:Confetti, 7 days after induction with either a high dose of tamoxifen (TM) (top) or low dose (bottom) for single cell clonal lineages.(C) Images of skin sections at various stages of the hair cycle, immunostained for Gata6 (red), CD34 or K14 (green) and DNA (blue).(D) images (D') for Gata6 inducible knockout (iKO) and wild type (WT) mice over time, showing the loss of Gata6 (red) from the hair follicle. Scale bars: 30 m.


Figure 2 ( B ) Representative images of hematoxylin staining of skin sections from the telogen induced mice sacrificed at 2 , 4 , and 10 days ( d ) post TM induction . ( C ) Images of hematoxylin staining of skin section from the anagen induced mice . Quantification shown in Figure EV2D . Scale bars : 30 m .

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Figure 2(B) Representative images of hematoxylin staining of skin sections from the telogen induced mice sacrificed at 2, 4, and 10 days (d) post TM induction. (C) Images of hematoxylin staining of skin section from the anagen induced mice. Quantification shown in Figure EV2D. Scale bars: 30 m.


Figure 3 ( B - G ) Gata6 WT and iKO skin after 3 days after TM induction and 12 hours after BrdU labeling or 5 days after TM induction and 2 days after BrdU labeling stained with markers as indicated in corresponding color .

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Figure 3(B-G) Gata6 WT and iKO skin after 3 days after TM induction and 12 hours after BrdU labeling or 5 days after TM induction and 2 days after BrdU labeling stained with markers as indicated in corresponding color.


Figure 4 ( A ) Hair follicles exhibit immunostaining signal for DNA damage marker H2A . X ( red ) and apoptosis marker Caspase3 ( green ) as early as one day after Gata6 iKO induction . Top panel shows a section of UV irradiated epidermis as a positive control for DNA damage . Note that Caspase 3 is not expressed in the H2A . X cells of the epidermis but is found in some iKO hair follicle matrix cells . Right panels show single color images as indicated at top ( B ) Quantification ( average SD ) of the percentage of apoptotic cells per matrix as determined by counting of Caspase3 + cells ( n = 50 follicles from 3 mice per group ) ( C ) Quantification ( average SD ) of the percentage of DNA damaged cells per matrix as determined by counting of H2A . X + Caspase3 - cells ( n = 50 follicles from 3 mice per group ) . ( D ) Regional distribution of apoptosis was analyzed by dividing the matrix into four compartments as shown in the legend ( left ) and counting the fraction of Caspase3 + cells per each matrix compartments ( right ; n = 50 follicles from 3 mice per group ) . ( E ) Proliferation in the matrix compartments shown in ( D ) was assessed by BrdU labeling for 1 hour , followed by staining , and quantification ( F ) ( interior cells labeled with solid bars , exterior cells labeled with striped bars ) . Unpaired t - test : * P - value < 0 . 05 . n = 20 follicles from 2 mice per group .

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Figure 4(A) Hair follicles exhibit immunostaining signal for DNA damage marker H2A.X (red) and apoptosis marker Caspase3 (green) as early as one day after Gata6 iKO induction. Top panel shows a section of UV irradiated epidermis as a positive control for DNA damage. Note that Caspase 3 is not expressed in the H2A.X cells of the epidermis but is found in some iKO hair follicle matrix cells. Right panels show single color images as indicated at top (B) Quantification (average SD) of the percentage of apoptotic cells per matrix as determined by counting of Caspase3+ cells (n=50 follicles from 3 mice per group) (C) Quantification (average SD) of the percentage of DNA damaged cells per matrix as determined by counting of H2A.X+Caspase3- cells (n=50 follicles from 3 mice per group).(D) Regional distribution of apoptosis was analyzed by dividing the matrix into four compartments as shown in the legend (left) and counting the fraction of Caspase3+ cells per each matrix compartments (right; n=50 follicles from 3 mice per group).(E) Proliferation in the matrix compartments shown in (D) was assessed by BrdU labeling for 1 hour, followed by staining, and quantification (F) (interior cells labeled with solid bars, exterior cells labeled with striped bars). Unpaired t-test: *P-value < 0.05. n=20 follicles from 2 mice per group.


Figure 5 ( A ) Colony formation of WT and TM induced Gata6 iKO keratinocytes stained with hematoxylin . Primary keratinocytes were isolated from induced iKO newborn mice , plated on feeder cells , and stained with hematoxylin at day 7 after plating ( top ) . Phase contrast images of cells are shown below . Scale bars : 30 m . ( B ) Quantification of colony formation from ( A ) , average SD from n = 3 mice in each genotype . ( C ) Western blot of WT and iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction , blotted with actin and Gata6 antibodies , show a decrease in Gata6 protein levels after 6 hours . ( D ) Phase contrast images of WT and Gata6 iKO keratinocytes 3 days after induction with TM ( n > 3 per genotype ) . ( E - H ) Images and quantification of DNA damage in cultured keratinocytes based on staining for DNA damage marker H2A . X ( E and G ) and comet assay ( F and H ) . Arrowheads in ( F ) indicate DNA damaged cell nuclei with comet shape . Quantification of H2A . X ( G ) represents average SD from at least 300 cells per condition from 3 experiments using different cell lines . * Unpaired t - test P - values : 6h = 0 . 01 and 24h = 0 . 004 . ( H ) Quantification of comet assay represents average SD ( n = 3 ) . ( I - J ) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2 - M arrest . ( I - J ) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2 - M arrest . ( J ) Quantification of FACS cell cycle analysis represent average SD ( n = 3 per genotype ) . * Unpaired t - test P - values : G1 = 0 . 03 , S = 5x10 - 4 , G2 - M = 7x10 - 4 . ( K ) Quantification of the frequency of H2A . X + staining in keratinocytes within S phase ( EdU + ) or another cell cycle stage ( EdU - ) following a 20 minute EdU pulse labeling ( average SD , n = 5 ) . * Unpaired t - test P - values WT = 0 . 1 , iKO = 0 . 05 . Mann - Whitney U test P - values : WT = 0 . 09 , iKO = 0 . 03 . ( L - M ) Cultured keratinocytes were made quiescent by gradual serum depletion . ( L ) Quantification of EdU + cells in proliferative and serum depleted quiescent cells ( average SD , n = 4 ) . ( M ) Quantification of H2A . X + keratinocytes during proliferative and quiescent conditions ( average SD , n = 4 ) . * Unpaired t - test P - value = 0 . 04 . Mann - Whitney U test P - value = 0 . 06 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Colony formation of WT and TM induced Gata6 iKO keratinocytes stained with hematoxylin. Primary keratinocytes were isolated from induced iKO newborn mice, plated on feeder cells, and stained with hematoxylin at day 7 after plating (top). Phase contrast images of cells are shown below. Scale bars: 30 m. (B) Quantification of colony formation from (A), average SD from n=3 mice in each genotype.(C) Western blot of WT and iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction, blotted with actin and Gata6 antibodies, show a decrease in Gata6 protein levels after 6 hours.(D) Phase contrast images of WT and Gata6 iKO keratinocytes 3 days after induction with TM (n > 3 per genotype).(E-H) Images and quantification of DNA damage in cultured keratinocytes based on staining for DNA damage marker H2A.X (E and G) and comet assay (F and H). Arrowheads in (F) indicate DNA damaged cell nuclei with comet shape. Quantification of H2A.X (G) represents average SD from at least 300 cells per condition from 3 experiments using different cell lines . *Unpaired t-test P-values: 6h=0.01 and 24h=0.004. (H) Quantification of comet assay represents average SD (n=3).(I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest.(I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest. (J) Quantification of FACS cell cycle analysis represent average SD (n=3 per genotype). *Unpaired t-test P-values: G1 = 0.03, S = 5x10-4, G2-M = 7x10-4.(K) Quantification of the frequency of H2A.X+ staining in keratinocytes within S phase (EdU+) or another cell cycle stage (EdU-) following a 20 minute EdU pulse labeling (average SD, n=5). *Unpaired t-test P-values WT=0.1, iKO= 0.05. Mann-Whitney U test P-values: WT=0.09, iKO=0.03.(L-M) Cultured keratinocytes were made quiescent by gradual serum depletion. (L) Quantification of EdU+ cells in proliferative and serum depleted quiescent cells (average SD, n=4). (M) Quantification of H2A.X+ keratinocytes during proliferative and quiescent conditions (average SD, n=4). *Unpaired t-test P-value = 0.04. Mann-Whitney U test P-value=0.06.


Figure 6 ( A ) Heatmap of differentially expressed genes ( adjusted P - value < 0 . 05 and fold change > 1 . 5 ) from RNA - seq ( n = 3 different WT and iKO cell lines ) . ( B ) qRT - PCR validation of candidate genes selected from RNA - seq analysis using RNA from cultured cells ( average SD , n = 4 ) . * Unpaired t - test P - values : Edaradd = 0 . 03 , Apcdd1 = 0 . 005 , Gls2 = 7x10 - 5 , Ppargc1b = 3x10 - 4 , Rab17 = 2x10 - 5 , Foxn1 = 0 . 03 . Mann - Whitney U test P - values : Edaradd = 0 . 06 , Apcdd1 = 0 . 03 , Gls2 = 0 . 03 , Ppargc1b = 0 . 03 , Rab17 = 0 . 03 , Foxn1 = 0 . 03 . ( C ) Table summarizing selection of candidate genes from ( B ) . Genes were selected based on known function or skin phenotype related to Gata6 iKO phenotype . ( D ) Staining of ROS by DHE . LY83583 treated cells served as positive control with elevated ROS . ( E ) Quantification of fold change in ROS at 6 and 24 hours after TM compared to WT , based on fluorescence intensity of DHE ( average SD , n = 2 ) . ( F ) Analysis of transcription factor binding site overrepresentation ( oPOSSUM ) in differentially expressed genes from RNA - seq . Points represent transcription factors plotted as Z . score of the frequency of transcription factor binding site occurrences in the promoters of downregulated genes compared to the background rate of occurrences in all gene promoters and Fisher P - value of the proportion of downregulated gene promoters containing each transcription factor binding site compared to the frequency in all gene promoters . ( H ) qRT - PCR of selected genes found to be overlapping between Gata6 iKO and Eda transgenic analyzed in Gata6 WT and iKO keratinocytes ( average SD , n = 4 ) . * Unpaired t - test P - values : Mcm3 = 0 . 001 , Mcm6 = 0 . 02 , Mcm10 = 0 . 04 , Exo1 = 0 . 02 , Pold1 = 0 . 02 . Mann - Whitney U test P - values : Mcm3 = 0 . 03 , Mcm6 = 0 . 03 , Mcm10 = 0 . 03 , Exo1 = 0 . 03 , Pold1 = 0 . 06 .

O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Heatmap of differentially expressed genes (adjusted P-value < 0.05 and fold change > 1.5) from RNA-seq (n=3 different WT and iKO cell lines).(B) qRT-PCR validation of candidate genes selected from RNA-seq analysis using RNA from cultured cells (average SD, n=4). *Unpaired t-test P-values: Edaradd=0.03, Apcdd1=0.005, Gls2=7x10-5, Ppargc1b=3x10-4, Rab17=2x10-5, Foxn1=0.03. Mann-Whitney U test P-values: Edaradd=0.06, Apcdd1=0.03, Gls2=0.03, Ppargc1b=0.03, Rab17=0.03, Foxn1=0.03. (C) Table summarizing selection of candidate genes from (B). Genes were selected based on known function or skin phenotype related to Gata6 iKO phenotype.(D) Staining of ROS by DHE. LY83583 treated cells served as positive control with elevated ROS. (E) Quantification of fold change in ROS at 6 and 24 hours after TM compared to WT, based on fluorescence intensity of DHE (average SD, n=2).(F) Analysis of transcription factor binding site overrepresentation (oPOSSUM) in differentially expressed genes from RNA-seq. Points represent transcription factors plotted as Z.score of the frequency of transcription factor binding site occurrences in the promoters of downregulated genes compared to the background rate of occurrences in all gene promoters and Fisher P-value of the proportion of downregulated gene promoters containing each transcription factor binding site compared to the frequency in all gene promoters.(H) qRT-PCR of selected genes found to be overlapping between Gata6 iKO and Eda transgenic analyzed in Gata6 WT and iKO keratinocytes (average SD, n=4). *Unpaired t-test P-values: Mcm3=0.001, Mcm6=0.02, Mcm10=0.04, Exo1=0.02, Pold1=0.02. Mann-Whitney U test P-values: Mcm3=0.03, Mcm6=0.03, Mcm10=0.03, Exo1=0.03, Pold1=0.06.


Figure 7 ( B ) qRT - PCR confirmation of the change in Edaradd mRNA expression in - vivo from mouse whole skin ( average SD , 1d n = 3 , 2d n = 4 ) . * Unpaired t - test P - values : 1d = 0 . 02 , 2d = 0 . 05 . Mann - Whitney U test P - values : 1d = 0 . 06 , 2d = 0 . 03 . ( C ) ChIP - qPCR for Gata6 at three conserved binding sites within the Edaradd promoter . GAPDH served as a negative control . Binding site 1 , 2 , and 3 are located 50 bp , 1 kb , and 1 . 5 kb upstream of the transcription start site , respectively ( average SD , n = 4 ) . * Unpaired t - test P - values GAPDH = 0 . 4 , 1 = 0 . 002 , 2 = 0 . 8 , 3 = 0 . 2 . Mann - Whitney U test P - values : GAPDH = 0 . 3 , 1 = 0 . 06 , 2 = 0 . 9 , 3 = 0 . 2 . ( D ) Western blot from whole skin lysate . Note decrease of Edaradd and activated pNF - B in the iKO . ( E - F ) Staining for Edaradd and quantification show decrease in Edaradd protein levels in the matrix of Gata6 iKO . * Unpaired t - test P - value = 3 . 5x10 - 14 . n = 100 follicles from 3 mice per genotype . ( G - H ) Staining for pNF - B and quantification show decrease of active NF - B in the Gata6 iKO matrix . * Unpaired t - test P - value = 2 . 2x10 - 16 . n = 50 follicles from 3 mice . ( I ) Rescue of Gata6 iKO keratinocyte growth by transfection with pGata6 or pEdaradd . Cell plates of keratinocytes are stained with Rhodanile Blue ( n = 3 cell lines each ) . ( J ) Quantification of H2A . X in cells stably transfected with pMock , pGata6 , or pEdaradd plasmids , followed by induction with TM for 6 hours ( average SD , n = 4 with > 500 cells counted per condition ) . * Unpaired t - test P - values = 2x10 - 4 ( iKO pGata6 vs pMock ) , 2x10 - 4 ( iKO pEdaradd vs pMock ) . ( K ) qRT - PCR analysis of Mcm10 expression in pMock , pGata6 , and pEdaradd WT and iKO expressing cells ( average SD , n = 4 ) . * Unpaired t - test P - values : 8x10 - 4 ( iKO pGata6 vs pMock ) , 0 . 03 ( iKO pEdaradd vs pMock ) . Mann - Whitney U test P - values : 0 . 03 ( iKO pGata6 vs pMock ) , 0 . 03 ( iKO pEdaradd vs pMock ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B) qRT-PCR confirmation of the change in Edaradd mRNA expression in-vivo from mouse whole skin (average SD, 1d n=3, 2d n=4). *Unpaired t-test P-values: 1d = 0.02, 2d = 0.05. Mann-Whitney U test P-values: 1d=0.06, 2d=0.03.(C) ChIP-qPCR for Gata6 at three conserved binding sites within the Edaradd promoter. GAPDH served as a negative control. Binding site 1, 2, and 3 are located 50 bp, 1 kb, and 1.5 kb upstream of the transcription start site, respectively (average SD, n=4). *Unpaired t-test P-values GAPDH=0.4, 1=0.002, 2=0.8, 3=0.2. Mann-Whitney U test P-values: GAPDH=0.3, 1=0.06, 2=0.9, 3=0.2.(D) Western blot from whole skin lysate. Note decrease of Edaradd and activated pNF-B in the iKO.(E-F) Staining for Edaradd and quantification show decrease in Edaradd protein levels in the matrix of Gata6 iKO. *Unpaired t-test P-value = 3.5x10-14. n=100 follicles from 3 mice per genotype.(G-H) Staining for pNF-B and quantification show decrease of active NF-B in the Gata6 iKO matrix. *Unpaired t-test P-value = 2.2x10-16. n=50 follicles from 3 mice.(I) Rescue of Gata6 iKO keratinocyte growth by transfection with pGata6 or pEdaradd. Cell plates of keratinocytes are stained with Rhodanile Blue (n=3 cell lines each).(J) Quantification of H2A.X in cells stably transfected with pMock, pGata6, or pEdaradd plasmids, followed by induction with TM for 6 hours (average SD, n=4 with > 500 cells counted per condition). *Unpaired t-test P-values = 2x10-4 (iKO pGata6 vs pMock), 2x10-4 (iKO pEdaradd vs pMock).(K) qRT-PCR analysis of Mcm10 expression in pMock, pGata6, and pEdaradd WT and iKO expressing cells (average SD, n=4). *Unpaired t-test P-values: 8x10-4 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock). Mann-Whitney U test P-values: 0.03 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock).


Figure 1 ( A ) OCR of wt and gas - 1 mutant nematodes ( normalized to nematode size as given by the COPAS Biosort ( Time of Flight - TOF ) ) . One representative curve shown ( mean ± SEM ; n = 3 ) * * * * p value < 0 . 0001 ; Kruskal - Wallis Test , Dunn ' s post hoc correction . Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( B ) age - 1 and age - 1 ; gas - 1 mutants animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves . Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( C ) daf - 16 and daf - 16 ; age - 1 ; gas - 1 mutant animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves . ( D ) Volcano plot of differentially phosphorylated proteins in gas - 1 ( fc21 ) mutants compared to wt . Cutoffs : 20 % FDR and | log2 ( foldchange ) | > 0 . 345 ( in red ) . ( F ) Lifespan analysis of mev - 1 ( kn1 ) nematodes compared to wt , age - 1 ( hx546 ) and age - 1 ( hx546 ) ; mev - 1 ( kn1 ) animals . ( G ) ( G ) ATP measurements in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to wt ) , n = 4 . Error bars : Mean ± SEM , * p value < 0 . 05 , * * * p value = 0 . 001 , Kruskal - Wallis Test , Dunn ' s post hoc correction . ( H ) OCR of gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to TOF ) . Left panel : One representative curve ( Mean ± SEM ) . Right panel : Basal respiration in wt , gas - 1 and age - 1 ; gas - 1 animals , pooled from 3 biological replicates , * * * * p value < 0 . 0001 , ns = non - significant , Kruskal - Wallis Test , Dunn ' s post hoc correction . ( I ) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms . Scale bar : 2 μm . ( J ) Left panel : Percentage of FRAP . Each point : mean recovery of ~ 30 bleached regions ( 2 regions per animal , 15 animals per condition ) ± SEM . Right panel : values of the maximum ( total ) recovery per strain ± SEM , * * * p value < 0 . 001 , * * * * p value < 0 . 0001 , one - way ANOVA , Tukey ' s post - hoc correction . ( K ) Representative lifespan of age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and fzo - 1 RNAi bacteria . Average median lifespans ± SEM from all replicates indicated beneath representative curves . For all panels : wt = wild type , a = age - 1 , g = gas - 1 , a ; g = age - 1 ; gas - 1 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) OCR of wt and gas-1 mutant nematodes (normalized to nematode size as given by the COPAS Biosort (Time of Flight -TOF)). One representative curve shown (mean ± SEM; n=3) ****p value< 0.0001; Kruskal-Wallis Test, Dunn's post hoc correction.Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (B) age-1 and age-1; gas-1 mutants animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (C) daf-16 and daf-16; age-1; gas-1 mutant animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.(D) Volcano plot of differentially phosphorylated proteins in gas-1(fc21) mutants compared to wt. Cutoffs: 20% FDR and |log2(foldchange)| >0.345 (in red).(F) Lifespan analysis of mev-1(kn1) nematodes compared to wt, age-1(hx546) and age-1(hx546); mev-1(kn1) animals. (G)(G) ATP measurements in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (normalized to wt), n=4. Error bars: Mean ± SEM, *p value <0.05, ***p value =0.001, Kruskal-Wallis Test, Dunn's post hoc correction.(H) OCR of gas-1 and age-1; gas-1 mutant nematodes (normalized to TOF). Left panel: One representative curve (Mean ± SEM). Right panel: Basal respiration in wt, gas-1 and age-1; gas-1 animals, pooled from 3 biological replicates, ****p value <0.0001, ns= non-significant, Kruskal-Wallis Test, Dunn's post hoc correction.(I) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms. Scale bar: 2 μm. (J) Left panel: Percentage of FRAP. Each point: mean recovery of ~30 bleached regions (2 regions per animal, 15 animals per condition) ± SEM. Right panel: values of the maximum (total) recovery per strain ± SEM, ***p value <0.001, **** p value <0.0001, one-way ANOVA, Tukey's post-hoc correction.(K) Representative lifespan of age-1; gas-1 nematodes fed with empty vector (EV) and fzo-1 RNAi bacteria. Average median lifespans ± SEM from all replicates indicated beneath representative curves. For all panels: wt= wild type, a= age-1, g= gas-1, a; g= age-1; gas-1.


Figure 2 ( A ) Left panel : Venn Diagram of the number of genes significantly dysregulated in age - 1 ; gas - 1 versus wt ( green ) and gas - 1 versus wt ( red ) . Right panel : Scatter - plots of the expression of genes significantly downregulated ( left ) or upregulated ( right ) in gas - 1 and age - 1 ; gas - 1 mutants compared to wt . In black : expression levels of genes that are significantly up - or down - regulated in gas - 1 mutants compared to wt . In red : expression levels of the same genes in age - 1 ; gas - 1 mutants compared to wt . ( C ) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative curves ( D ) Volcano plot of TMT / iTRAQ - based quantitative proteomic analysis . In red , proteins significantly dysregulated in age - 1 ; gas - 1 compared to gas - 1 ( FDR < 20 % ; | log2 ( fold change ) | > 0 . 47 ) . Dashed lines indicate cutoffs . Proteins involved in carbohydrate ( green ) and lipid ( blue ) metabolism are labeled . ( H ) Left panel : Glucose quantification in wt , gas - 1 and age - 1 ; gas - 1 nematode lysates with NMR , n = 7 , ns = non - significant , Mann Whitney U test . Right panel : Quantification of total carbohydrates in the indicated strains with phenol - sulfuric assay . Error bars : mean ± SEM , 2 biological replicates ( 5 technical replicates each ) , normalized to wt and number of animals used . * * p value < 0 . 01 , ns = non - significant , Mann - Whitney U test .

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Figure 2(A) Left panel: Venn Diagram of the number of genes significantly dysregulated in age-1; gas-1 versus wt (green) and gas-1 versus wt (red). Right panel: Scatter-plots of the expression of genes significantly downregulated (left) or upregulated (right) in gas-1 and age-1; gas-1 mutants compared to wt. In black: expression levels of genes that are significantly up- or down-regulated in gas-1 mutants compared to wt. In red: expression levels of the same genes in age-1; gas-1 mutants compared to wt.(C) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative curves(D) Volcano plot of TMT/iTRAQ-based quantitative proteomic analysis. In red, proteins significantly dysregulated in age-1; gas-1 compared to gas-1 (FDR< 20%; |log2 (fold change)| >0.47). Dashed lines indicate cutoffs. Proteins involved in carbohydrate (green) and lipid (blue) metabolism are labeled.(H) Left panel: Glucose quantification in wt, gas-1 and age-1; gas-1 nematode lysates with NMR, n=7, ns= non-significant, Mann Whitney U test. Right panel: Quantification of total carbohydrates in the indicated strains with phenol-sulfuric assay. Error bars: mean ± SEM, 2 biological replicates (5 technical replicates each), normalized to wt and number of animals used. **p value < 0.01, ns= non-significant, Mann-Whitney U test.


Figure 3 ( C ) Immunoblot analysis of phospho - AAK - 2 in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( actin loading control ) . Numbers correspond to band densitometries across 3 biological replicates . ( D ) Representative lifespan of age - 1 ; gas - 1 double mutant and age - 1 ; aak - 2 ; gas - 1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves . ( E ) Lifespan assay of gas - 1 mutant animals compared to gas - 1 ; AAK - 2 overexpressing ( O / E ) nematodes and their wt counterparts . Inset : immunoblot analysis of phospho - AAK - 2 in gas - 1 ( g ) and gas - 1 ; AAK - 2 O / E ( g ; a O / E ) animals ( actin loading control ) , showing the overexpressed protein ( lower band ) and the endogenous one ( upper band ) . ( F ) Left panel : Immunoblot analysis of phospho - KIN - 1 substrates in young adult nematodes . Numbers correspond to band densitometries across 2 biological replicates . Right panel : immunoblot analysis of phospho - KIN - 1 substrates in age - 1 ; gas - 1 animals grown on bacteria carrying an empty vector ( EV ) or expressing kin - 1 RNAi . Two different kin - 1 RNAi clones ( g1 , b5 ) were used in this experiment . Numbers correspond to band densitometries of the presented immunoblot . Dashed rectangles : band corresponding to downregulated KIN - 1 phospho - substrate . ( G ) Representative lifespan assay of wt and age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and kin - 1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves . ATP measurements in : ( H ) age - 1 ( hx546 ) ; gas - 1 ( fc21 ) ( a ; g ) and age - 1 ( hx546 ) ; aak - 2 ( ok524 ) ; gas - 1 ( fc21 ) ( a ; aa ; g ) mutant nematodes ; ATP measurements in : ( I ) age - 1 ; gas - 1 ( EV ) ( a ; g ( EV ) ) and age - 1 ; gas - 1 ; kin - 1 ( RNAi ) ( a ; g ; kin - 1 ( RNAi ) ) . Error bars : Mean ± SEM , n = 4 , * p value < 0 . 05 , Mann Whitney U test . ( J ) Left panel : curves represent FRAP in age - 1 ; gas - 1 animals grown on RNAi from larval L1 ( or from egg for fzo - 1 RNAi ) to L4 stage . Each point : mean recovery of bleached regions ( 2 - 5 regions per animal , 10 - 20 animals per condition ) ± SEM . Right panel : Maximum ( total ) recovery per condition are shown , ± SEM . * p value < 0 . 05 , * * p value < 0 . 01 , * * * * p value < 0 . 0001 , Kruskal - Wallis test , Dunn ' s post - hoc correction .

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Figure 3(C) Immunoblot analysis of phospho-AAK-2 in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (actin loading control). Numbers correspond to band densitometries across 3 biological replicates.(D) Representative lifespan of age-1; gas-1 double mutant and age-1; aak-2; gas-1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves.(E) Lifespan assay of gas-1 mutant animals compared to gas-1; AAK-2 overexpressing (O/E) nematodes and their wt counterparts. Inset: immunoblot analysis of phospho-AAK-2 in gas-1 (g) and gas-1; AAK-2 O/E (g; a O/E) animals (actin loading control), showing the overexpressed protein (lower band) and the endogenous one (upper band).(F) Left panel: Immunoblot analysis of phospho-KIN-1 substrates in young adult nematodes. Numbers correspond to band densitometries across 2 biological replicates. Right panel: immunoblot analysis of phospho-KIN-1 substrates in age-1; gas-1 animals grown on bacteria carrying an empty vector (EV) or expressing kin-1 RNAi. Two different kin-1 RNAi clones (g1, b5) were used in this experiment. Numbers correspond to band densitometries of the presented immunoblot. Dashed rectangles: band corresponding to downregulated KIN-1 phospho-substrate.(G) Representative lifespan assay of wt and age-1; gas-1 nematodes fed with empty vector (EV) and kin-1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves.ATP measurements in: (H) age-1(hx546); gas-1(fc21) (a; g) and age-1(hx546); aak-2(ok524); gas-1(fc21) (a; aa; g) mutant nematodes;ATP measurements in: (I) age-1; gas-1 (EV) (a; g (EV)) and age-1; gas-1; kin-1 (RNAi) (a; g; kin-1(RNAi)). Error bars: Mean ± SEM, n=4, *p value <0.05, Mann Whitney U test.(J) Left panel: curves represent FRAP in age-1; gas-1 animals grown on RNAi from larval L1 (or from egg for fzo-1 RNAi) to L4 stage. Each point: mean recovery of bleached regions (2-5 regions per animal, 10-20 animals per condition) ± SEM. Right panel: Maximum (total) recovery per condition are shown, ± SEM. *p value< 0.05, **p value< 0.01, ****p value< 0.0001, Kruskal-Wallis test, Dunn's post-hoc correction.


Figure 4A ) Volcano plot of lipidomic profiling in gas - 1 versus age - 1 ; gas - 1 mutants . Red stars : significantly altered lipids in age - 1 ; gas - 1 versus gas - 1 ( 5 % FDR ; n = 5 ) . B ) NMR - 1D targeted metabolic profiling in age - 1 ; gas - 1 mutants ( normalized to gas - 1 ) . Dashed lines indicate fold change cutoff ( | log2 ( fold change ) | < 1 . 23 ) . Red bars : not significant , Light blue bars : FDR < 10 % , | log2 ( fold change ) | < 1 . 23 , Dark blue bars : FDR < 10 % , | log2 ( fold change ) | > 1 . 23 . Error bar : mean + SEM , n = 7 . D ) Left panel : qRT - PCR of xdh - 1 ( normalized to wt ) . Error bars : Mean ± SEM , n = 6 . * * * * p value < 0 . 0001 , * p value < 0 . 05 , ns = non - significant , Kruskal - Wallis with Dunn ' s post hoc test . Right : xanthine oxidase activity levels normalized to animal number . Error bars : mean ± SEM , n = 2 , ns = non - significant , Mann Whitney U test . F ) Immunoblot analysis of phospho - Akt ( S473 ) , pan Akt , phospho - PRAS40 ( T246 ) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0 . 2 % DMSO ( - ) or AMP ( 2 μM ) , ENPF ( 2 μM ) and PTX ( 36 μM ) ( + ) for 24 h ( actin loading control ) . ( G ) Immunoblot analysis of phospho - PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated ( actin loading control ) . For all immunoblot panels : numbers correspond to band densitometries across 2 - 3 biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Volcano plot of lipidomic profiling in gas-1 versus age-1; gas-1 mutants. Red stars: significantly altered lipids in age-1; gas-1 versus gas-1 (5% FDR; n=5).B) NMR-1D targeted metabolic profiling in age-1; gas-1 mutants (normalized to gas-1). Dashed lines indicate fold change cutoff (|log2(fold change)| <1.23). Red bars: not significant, Light blue bars: FDR<10%, |log2(fold change)| <1.23, Dark blue bars: FDR <10%, |log2(fold change)| >1.23. Error bar: mean + SEM, n=7.D) Left panel: qRT-PCR of xdh-1 (normalized to wt). Error bars: Mean ± SEM, n=6. ****p value< 0.0001, *p value< 0.05, ns= non-significant, Kruskal-Wallis with Dunn's post hoc test. Right: xanthine oxidase activity levels normalized to animal number. Error bars: mean ± SEM, n=2, ns= non-significant, Mann Whitney U test.F) Immunoblot analysis of phospho-Akt (S473), pan Akt, phospho-PRAS40 (T246) and total PRAS40 in WT and NDUFA9 KO HAP1 cells treated with 0.2% DMSO (-) or AMP (2 μM), ENPF (2 μM) and PTX (36 μM) (+) for 24 h (actin loading control).(G) Immunoblot analysis of phospho-PKA substrates in WT and NDUFA9 KO HAP1 cells treated as indicated (actin loading control). For all immunoblot panels: numbers correspond to band densitometries across 2-3 biological replicates.


Figure 5 ( A ) Effect of methylxanthines on wt lifespan . Average median lifespans ± SEM from all replicates indicated beneath representative curve . ( B ) Locomotory activity of 10 - day - old gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines . Values represent arbitrary units from 3 biological replicates with 4 technical replicates each , 35 worms per technical replicate . Error bars : mean ± SEM , * * p value < 0 . 01 , * * * p value < 0 . 001 , one - way ANOVA , Dunnett ' s post - hoc correction . ( C ) Effect of methylxanthines on the survival of gas - 1 mutants . Average median lifespans ± SEM from all replicates indicated beneath representative curves . ( D ) Pharyngeal pumps / minute in young adult gas - 1 mutant nematodes treated with vehicle or methylxanthines . Error bars : mean ± SEM , n = 18 . * * * * p value < 0 . 0001 , one - way ANOVA , Dunnett ' s post - hoc correction . ( E - F ) Immunoblot analysis of ( E ) KIN - 1 phospho - substrates in young adult gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines ( actin loading control ) . ( E - F ) Immunoblot analysis of ( F ) phospho - AAK - 2 in young adult gas - 1 mutant nematodes treated with vehicle ( DMSO ) or methylxanthines ( actin loading control ) . ( G ) Left panel : FRAP in vehicle or PTX - treated gas - 1 animals . Each point : mean recovery of 14 bleached regions ( 2 regions per animal , 7 animals per condition ) ± SEM . Right panel : maximum ( total ) recovery per condition ± SEM . * * p value < 0 . 01 unpaired t test . For all immunoblot panels : numbers correspond to band densitometries across biological replicates and for all lifespan curves : the legend shows Mean median lifespan ± SEM across all biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Effect of methylxanthines on wt lifespan. Average median lifespans ± SEM from all replicates indicated beneath representative curve.(B) Locomotory activity of 10-day-old gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines. Values represent arbitrary units from 3 biological replicates with 4 technical replicates each, 35 worms per technical replicate. Error bars: mean ± SEM, **p value< 0.01, ***p value< 0.001, one-way ANOVA, Dunnett's post-hoc correction.(C) Effect of methylxanthines on the survival of gas-1 mutants. Average median lifespans ± SEM from all replicates indicated beneath representative curves.(D) Pharyngeal pumps/minute in young adult gas-1 mutant nematodes treated with vehicle or methylxanthines. Error bars: mean ± SEM, n=18. ****p value< 0.0001, one-way ANOVA, Dunnett's post-hoc correction.(E-F) Immunoblot analysis of (E) KIN-1 phospho-substrates in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).(E-F) Immunoblot analysis of (F) phospho-AAK-2 in young adult gas-1 mutant nematodes treated with vehicle (DMSO) or methylxanthines (actin loading control).(G) Left panel: FRAP in vehicle or PTX-treated gas-1 animals. Each point: mean recovery of 14 bleached regions (2 regions per animal, 7 animals per condition) ± SEM. Right panel: maximum (total) recovery per condition ± SEM. **p value< 0.01 unpaired t test. For all immunoblot panels: numbers correspond to band densitometries across biological replicates and for all lifespan curves: the legend shows Mean median lifespan ± SEM across all biological replicates.


Figure 1 ( A ) FAK + / + and FAK − / − murine SCC cells were lysed and FAK , Ret or Src immunoprecipitated and immunoblotting carried out using anti ‐ FAK , anti ‐ Ret , anti ‐ Src and anti ‐ actin . ( B ) Cells were fixed and stained with either anti ‐ Ret ( green in the upper left panels ) or anti ‐ Ret PY1062 ( green in the lower left panels ) and DAPI ( blue ) . Merged and zoomed images are shown . Solid arrows indicate localization at adhesions while broken arrows show localization in puncta . Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . Lysates were immunoblotted with anti ‐ Ret PY1062 and anti ‐ actin ( lower right panels ) . ( C ) Cells were fixed and stained with anti ‐ Ret ( green ) and anti ‐ FAK ( red ) . Merged images are also shown . Solid arrows indicate co ‐ localization between Ret and FAK at adhesions , while broken arrows indicate its absence in puncta . Scale bars , 20 μM . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection ; SCC , squamous cell carcinoma .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) FAK+/+ and FAK−/− murine SCC cells were lysed and FAK, Ret or Src immunoprecipitated and immunoblotting carried out using anti‐FAK, anti‐Ret, anti‐Src and anti‐actin.(B) Cells were fixed and stained with either anti‐Ret (green in the upper left panels) or anti‐Ret PY1062 (green in the lower left panels) and DAPI (blue). Merged and zoomed images are shown. Solid arrows indicate localization at adhesions while broken arrows show localization in puncta. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). Lysates were immunoblotted with anti‐Ret PY1062 and anti‐actin (lower right panels).(C) Cells were fixed and stained with anti‐Ret (green) and anti‐FAK (red). Merged images are also shown. Solid arrows indicate co‐localization between Ret and FAK at adhesions, while broken arrows indicate its absence in puncta. Scale bars, 20 μM. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection; SCC, squamous cell carcinoma.


Figure 2Ret localizes to Src ‐ positive autophagosomes in FAK ‐ deficient SCC cells . Cells were fixed and stained with either ( A ) anti ‐ Ret ( green ) and anti ‐ PY416 Src ( red ) or ( B ) anti ‐ PY416 Src ( red ) and anti ‐ LC3B ( green ) . Merged and zoomed images are shown . Solid arrows indicate co ‐ localization at adhesions , while broken arrows show co ‐ localization in intracellular puncta . ( C ) Cells were transfected with scrambled , Atg5 or Atg7 siRNA , trypsinized and allowed to readhere before immunoblotting with anti ‐ Atg5 , anti ‐ Atg7 and anti ‐ actin ( right panels ) . Cells were fixed and stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) and DAPI ( blue ) . Merged images are shown . Solid arrows indicate co ‐ localization between Ret and paxillin at adhesions , while broken arrows indicate its absence in puncta . ( D ) Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . ( E ) Cells , either untreated or treated with chloroquine for 24 h , were fixed and stained with anti ‐ Ret ( green ) and DAPI ( blue ) . Scale bars , 20 μM . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection ; SCC , squamous cell carcinoma ; siRNA , short interfering RNA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Ret localizes to Src‐positive autophagosomes in FAK‐deficient SCC cells. Cells were fixed and stained with either (A) anti‐Ret (green) and anti‐PY416 Src (red) or (B) anti‐PY416 Src (red) and anti‐LC3B (green). Merged and zoomed images are shown. Solid arrows indicate co‐localization at adhesions, while broken arrows show co‐localization in intracellular puncta.(C) Cells were transfected with scrambled, Atg5 or Atg7 siRNA, trypsinized and allowed to readhere before immunoblotting with anti‐Atg5, anti‐Atg7 and anti‐actin (right panels). Cells were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Merged images are shown. Solid arrows indicate co‐localization between Ret and paxillin at adhesions, while broken arrows indicate its absence in puncta.(D) Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3).(E) Cells, either untreated or treated with chloroquine for 24 h, were fixed and stained with anti‐Ret (green) and DAPI (blue). Scale bars, 20 μM. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection; SCC, squamous cell carcinoma; siRNA, short interfering RNA.


Figure 3 ( A ) FAK − / − cells stably re ‐ expressing FAK ‐ wt , FAK ‐ Y397F , FAK ‐ KD or FAK ‐ Y4F ‐ Y9F were immunoblotted using anti ‐ FAK , anti ‐ FAK Y397 , anti ‐ FAK Y861 , anti ‐ FAK Y925 , anti ‐ Src , anti ‐ Ret and anti ‐ actin . FAK and Ret were immunoprecipitated from these cells and then samples were immunoblotted with anti ‐ Src , anti ‐ FAK or anti ‐ Ret . ( B ) Cells were fixed and stained with anti ‐ Ret ( green ) and either anti ‐ FAK ( red in the upper panels ) or anti ‐ PY416 Src ( red in the lower panels ) . Merged images are shown . Solid arrows indicate Ret in adhesions , while broken arrows show Ret in puncta . Scale bars , 20 μM . ( C ) Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . FAK , focal adhesion kinase ; Ret , rearranged during transfection .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) FAK−/− cells stably re‐expressing FAK‐wt, FAK‐Y397F, FAK‐KD or FAK‐Y4F‐Y9F were immunoblotted using anti‐FAK, anti‐FAK Y397, anti‐FAK Y861, anti‐FAK Y925, anti‐Src, anti‐Ret and anti‐actin. FAK and Ret were immunoprecipitated from these cells and then samples were immunoblotted with anti‐Src, anti‐FAK or anti‐Ret.(B) Cells were fixed and stained with anti‐Ret (green) and either anti‐FAK (red in the upper panels) or anti‐PY416 Src (red in the lower panels). Merged images are shown. Solid arrows indicate Ret in adhesions, while broken arrows show Ret in puncta. Scale bars, 20 μM.(C) Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). FAK, focal adhesion kinase; Ret, rearranged during transfection.


Figure 4 ( A ) FAK + / + cells were treated with dasatinib , then lysates were immunoblotted with anti ‐ FAK , anti ‐ FAK Y397 , anti ‐ FAK Y925 , anti ‐ PY416 Src , anti ‐ Src and anti ‐ actin . FAK was immunoprecipitated and then immunoblotting performed using anti ‐ Src , anti ‐ FAK or anti ‐ Ret . ( B ) Cells were treated with dasatinib , then fixed and stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) and DAPI ( blue ) . Solid arrows indicate co ‐ localization in adhesions . Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . ( C ) FAK − / − cells treated with dasatinib were fixed and stained with anti ‐ Ret ( green ) , anti ‐ paxillin ( red ) and DAPI ( blue ) . Solid arrows indicate co ‐ localization in adhesions , while broken arrows show lack of co ‐ localization in puncta . Scale bars , 20 μM . Quantification of percentage of cells that contained Ret in intracellular puncta is shown . Data are presented as mean ± s . d . and the significance calculated using a Student ' s t ‐ test ( n = 3 ) . ( D ) Lysates from untreated and dasatinib ‐ treated FAK − / − cells were immunoblotted with anti ‐ Ret , anti ‐ PY416 Src , anti ‐ Src and anti ‐ actin . Ret was also immunoprecipitated from cells and then immunoblotted using anti ‐ Src and anti ‐ Ret . DAPI , 4 , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; FAK , focal adhesion kinase ; Ret , rearranged during transfection .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) FAK+/+ cells were treated with dasatinib, then lysates were immunoblotted with anti‐FAK, anti‐FAK Y397, anti‐FAK Y925, anti‐PY416 Src, anti‐Src and anti‐actin. FAK was immunoprecipitated and then immunoblotting performed using anti‐Src, anti‐FAK or anti‐Ret.(B) Cells were treated with dasatinib, then fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3). (C) FAK−/− cells treated with dasatinib were fixed and stained with anti‐Ret (green), anti‐paxillin (red) and DAPI (blue). Solid arrows indicate co‐localization in adhesions, while broken arrows show lack of co‐localization in puncta. Scale bars, 20 μM. Quantification of percentage of cells that contained Ret in intracellular puncta is shown. Data are presented as mean±s.d. and the significance calculated using a Student's t‐test (n=3).(D) Lysates from untreated and dasatinib‐treated FAK−/− cells were immunoblotted with anti‐Ret, anti‐PY416 Src, anti‐Src and anti‐actin. Ret was also immunoprecipitated from cells and then immunoblotted using anti‐Src and anti‐Ret. DAPI, 4,6‐diamidino‐2‐phenylindole; FAK, focal adhesion kinase; Ret, rearranged during transfection.


Figure 1 ( a ) Immunofluorescence labeling of wildtype mouse dorsal skin samples ( Dnmt1f / f , skin from P3 ) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte - specific knockout ( Dnmt1Δ / Δep ) results in the almost absence of a specific staining in the epidermis and in hair follicles . Dashed lines indicate the dermal - epidermal border and dotted lines indicate hair follicles . ( d ) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5 - mC labeling of P3 skin . Dashed lines indicate the dermal - epidermal border and dotted lines indicate hair follicles . ( h ) Hematoxylin and Eosin ( H & E ) staining , immunolabeling for KERATIN 10 ( KRT10 ) , LORICRIN ( LOR ) and KERATIN 6 ( KRT6 ) expression of dorsal skin sections ( P5 ) of control and Dnmt1Δ / Δep mice .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(a) Immunofluorescence labeling of wildtype mouse dorsal skin samples (Dnmt1f/f, skin from P3) shows expression of DNMT1 throughout epidermal layers whereas keratinocyte-specific knockout (Dnmt1Δ/Δep) results in the almost absence of a specific staining in the epidermis and in hair follicles. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.(d) Knockout of Dnmt1 results in lower levels of DNA methylation in the epidermis and in hair follicles as determined by anti 5-mC labeling of P3 skin. Dashed lines indicate the dermal-epidermal border and dotted lines indicate hair follicles.(h) Hematoxylin and Eosin (H&E) staining, immunolabeling for KERATIN 10 (KRT10), LORICRIN (LOR) and KERATIN 6 (KRT6) expression of dorsal skin sections (P5) of control and Dnmt1Δ/Δep mice.


Figure 2 ( c ) Flow cytometric analysis of the respective immune cell populations from the epidermis of control and Dnmt1Δ / Δep mice of different postnatal age . Cells are shown as percent of viable cells . Data are mean ± SEM , two - tailed t - test . ns , not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 , n = 3 ( P3 ) , 4 ( P5 ) and 4 ( P6 + P7 ) . Data information : Scale bars , 50 µm . ( d ) Relative mRNA expression levels of immune related genes in control and Dnmt1Δ / Δep mice at P3 ( n = 3 ) , P5 ( n = 5 ) and P7 ( n ≥ 3 ) . Data are mean ± SEM . P values are calculated using one - way ANOVA with post hoc Tukey multiple comparison test , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 . Data information : Scale bars , 50 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(c) Flow cytometric analysis of the respective immune cell populations from the epidermis of control and Dnmt1Δ/Δep mice of different postnatal age. Cells are shown as percent of viable cells. Data are mean ± SEM, two-tailed t-test. ns, not significant, * p≤0.05, ** p≤0.01, **** p≤0.0001, n=3 (P3), 4 (P5) and 4 (P6 + P7). Data information: Scale bars, 50 µm.(d) Relative mRNA expression levels of immune related genes in control and Dnmt1Δ/Δep mice at P3 (n=3), P5 (n=5) and P7 (n≥3). Data are mean ± SEM. P values are calculated using one-way ANOVA with post hoc Tukey multiple comparison test, * p≤0.05, ** p≤0.01, *** p≤0.001 , **** p≤0.0001. Data information: Scale bars, 50 µm.


Figure 3 ( a ) Relative mRNA expression levels of epidermal Intracisternal A - type particle ( IAP ) during different postnatal time points ( P0 n ≥ 3 , P3 n = 3 , P5 n = 5 , P7 n ≥ 3 mice ) , which were compared using one way - ANOVA with post hoc Tukey multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 . ( c ) Relative expression of immune related genes of the epidermis from P7 of control ( n ≥ 7 ) , Dnmt1Δ / Δep , ( n ≥ 8 ) , MAVS knockout ( Mavs - / - , n = 7 ) and Dnmt1Δ / Δep Mavs - / - ( n = 6 ) mice were compared using one way - ANOVA with post hoc Holm - Sidak multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(a) Relative mRNA expression levels of epidermal Intracisternal A-type particle (IAP) during different postnatal time points (P0 n≥3, P3 n=3, P5 n=5, P7 n≥3 mice), which were compared using one way-ANOVA with post hoc Tukey multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01.(c) Relative expression of immune related genes of the epidermis from P7 of control (n≥7), Dnmt1Δ/Δep, (n≥8), MAVS knockout (Mavs-/-, n=7) and Dnmt1Δ/Δep Mavs-/- (n=6) mice were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001, **** p≤0.0001.


Figure 4Immortalized human keratinocytes ( NHEK SV - Tert3 - 5 ) were treated with 5 - aza - 2 ′ - deoxycytidine ( DAC ) or vehicle ( PBS ) . ( c , d ) PicoGreen staining of PBS - and DAC - treated keratinocytes for 48h detected micronuclei ( arrow ) , which were analyzed in a blinded manner , and compared using two - tailed t - test . Data are mean ± SEM , * p ≤ 0 . 05 ( n = 3 biological replicates ) ( d ) . Data information : Scale bars , 20 µmImmortalized human keratinocytes ( NHEK SV - Tert3 - 5 ) were treated with 5 - aza - 2 ′ - deoxycytidine ( DAC ) or vehicle ( PBS ) . ( e ) Co - labeling of DNA by PicoGreen and by cGAS in PBS and DAC treated keratinocytes . Within enlarged sections on the right arrows point at DNA / cGAS double - positive blebs and micronuclei . ( f ) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way - ANOVA with post hoc Dunnett multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ( n = 4 biological replicates ) . Data information : Scale bars , , 50

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Immortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (c, d) PicoGreen staining of PBS- and DAC-treated keratinocytes for 48h detected micronuclei (arrow), which were analyzed in a blinded manner, and compared using two-tailed t-test. Data are mean ± SEM, * p≤0.05 (n=3 biological replicates) (d). Data information: Scale bars, 20 µmImmortalized human keratinocytes (NHEK SV-Tert3-5) were treated with 5-aza-2′-deoxycytidine (DAC) or vehicle (PBS). (e) Co-labeling of DNA by PicoGreen and by cGAS in PBS and DAC treated keratinocytes. Within enlarged sections on the right arrows point at DNA/cGAS double-positive blebs and micronuclei. (f) Relative expression of CCL5 and IFT2 in keratinocytes treated with PBS or DAC for 45 h or 72 h using one way-ANOVA with post hoc Dunnett multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, ** p≤0.01, *** p≤0.001 (n=4 biological replicates). Data information: Scale bars, , 50 µm


Figure 5 ( c ) Representative images of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f / f , Dnmt1Δ / Δep , and Dnmt1Δ / Δep Cgas - / - mice . Dashed lines indicate the dermal - epidermal border . ( d ) Quantification of CD45 - positve cells . Tissue sections approximately 1 cm in length were scanned and the area of CD45 positive cells per area of nuclei was quantified using ImageJ . Sections obtained from five mice per genotype were analyzed and calculated values were compared using one way - ANOVA with post hoc Holm - Sidak multiple comparison test . Data are mean ± SEM . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . Data information : Scale bars , 100 µm ( e ) Epidermis obtained from P7 control , Dnmt1Δ / Δep , Cgas - / - and Dnmt1Δ / Δep Cgas - / - mice was analyzed for relative mRNA expression levels of immune related genes using one way - ANOVA with post hoc Holm - Sidak multiple comparison test . Data are mean ± SEM . ns not significant , * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 . At least 7 mice per genotype were used .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(c) Representative images of CD45 immunofluorescence labeling of P7 dorsal skin sections from Dnmt1f/f, Dnmt1Δ/Δep, and Dnmt1Δ/Δep Cgas-/- mice. Dashed lines indicate the dermal-epidermal border. (d) Quantification of CD45-positve cells. Tissue sections approximately 1 cm in length were scanned and the area of CD45 positive cells per area of nuclei was quantified using ImageJ. Sections obtained from five mice per genotype were analyzed and calculated values were compared using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ** p≤0.01, *** p≤0.001. Data information: Scale bars, 100 µm(e) Epidermis obtained from P7 control, Dnmt1Δ/Δep, Cgas-/- and Dnmt1Δ/Δep Cgas-/- mice was analyzed for relative mRNA expression levels of immune related genes using one way-ANOVA with post hoc Holm-Sidak multiple comparison test. Data are mean ± SEM. ns not significant, * p≤0.05, **** p≤0.0001. At least 7 mice per genotype were used.