Zeta potentials of bare MNPs , NH2 - MNPs and pcMNPs in water . ( D ) The magnetic response property of bare MNPs , NH2 - MNPs and MBs in 30 s .

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Zeta potentials of bare MNPs, NH2-MNPs and pcMNPs in water. (D) The magnetic response property of bare MNPs, NH2-MNPs and MBs in 30 s.


RNA binding affinity of pcMNPs analysed by native PAGE ;

RNA binding affinity of pcMNPs analysed by native PAGE;


Dispersion stability of pcMNPs in a high - salt solution ( 50 mM NaCl ) .

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Dispersion stability of pcMNPs in a high-salt solution (50 mM NaCl).


RT - PCR assays towards viral RNA extracted by different pcMNPs - based methods . RT - PCR assays amplifying ( A ) the ORF1ab region and ( B ) the N gene in pseudoviral RNA extracted by a manual protocol .

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RT-PCR assays towards viral RNA extracted by different pcMNPs-based methods.RT-PCR assays amplifying (A) the ORF1ab region and (B) the N gene in pseudoviral RNA extracted by a manual protocol.


RT - PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol , and ( B ) RT - PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol .

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RT-PCR assays amplifying the ORF1ab region in pseudoviral RNA extracted by automated protocol, and (B) RT-PCR assays amplifying the N gene in pseudoviral RNA extracted by an automated protocol.


RT - PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT - PCR protocol , and ( B ) corresponding calibration curve .

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RT-PCR assays amplifying the N gene in pseudoviral RNA following the Conventional RT-PCR protocol, and (B) corresponding calibration curve.


RT - PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT - PCR protocol , and ( D ) corresponding calibration curve . The red line is the linear regression fit ( R2 = 0 . 998 ) .

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RT-PCR assays amplifying the N gene in pseudoviral RNA following the Direct RT-PCR protocol, and (D) corresponding calibration curve. The red line is the linear regression fit (R2 = 0.998).


a , LAMP1 + lysosome size and quantity in NRK cells ( dotted outline ) starved for hours ( h ) . Error bars show s . e . m . ( n = 3 ) .

a, LAMP1+ lysosome size and quantity in NRK cells (dotted outline) starved for hours (h). Error bars show s.e.m. (n = 3).


b , Starved NRK cells expressing LAMP1 - YFP show tubules ( arrows ) . Box is expanded on the right .

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b, Starved NRK cells expressing LAMP1-YFP show tubules (arrows). Box is expanded on the right.


c , Immuno - TEM ( gold marks LAMP1 ) of starved NRK cells . Continuity ( arrow ) illustrated between autolysosome ( red star ) and tubule .

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c, Immuno-TEM (gold marks LAMP1) of starved NRK cells. Continuity (arrow) illustrated between autolysosome (red star) and tubule.


d , LAMP1 - YFP expressing NRK - LC3 cells as in a . Arrows show reformation tubules . e , Three - dimensional reconstruction of a cell from d .

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d, LAMP1-YFP expressing NRK-LC3 cells as in a. Arrows show reformation tubules. e, Three-dimensional reconstruction of a cell from d.


f , Time - lapse images ( seconds ) of starved LAMP1 - YFP - expressing NRK cells .

f, Time-lapse images (seconds) of starved LAMP1-YFP-expressing NRK cells.


g , Time - lapse images ( minutes ) of starved LAMP1 - PAGFP or LAMP1 - RFP - expressing NRK cells after photo - activation . Scale bars , 5 µm .

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g, Time-lapse images (minutes) of starved LAMP1-PAGFP or LAMP1-RFP-expressing NRK cells after photo-activation. Scale bars, 5 µm.


a - c , Starved LAMP1 - YFP NRK cells stained with Lysotracker or DQ - BSA . c , Right , TEM of purified lysosomes . d , Magnification ( 4 × ) of images shown in c . Arrows indicate reformation tubules and arrowhead indicates autolysosome .

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a-c, Starved LAMP1-YFP NRK cells stained with Lysotracker or DQ-BSA. c, Right, TEM of purified lysosomes. d, Magnification (4×) of images shown in c. Arrows indicate reformation tubules and arrowhead indicates autolysosome.


e , f , Immunofluorescence ( e ) and immuno - EM ( f ) using LAMP1 antibody of starved LAMP1 - YFPNRKcells shown in c .

e, f, Immunofluorescence (e) and immuno-EM (f) using LAMP1 antibody of starved LAMP1-YFPNRKcells shown in c.


g , Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D . Middle panel shows 8 - h fractions 7 and 9 analysed by TEM . Fraction 10 is the gradient top .

g, Density gradient fractions of NRK cells starved for times shown and blotted for LAMP2 or Cathepsin D. Middle panel shows 8-h fractions 7 and 9 analysed by TEM. Fraction 10  is the  gradient top.


h , Starved LAMP1 - PAGFP NRK cells were photo - activated ( 4 h ) and imaged at 12 h with Lysotracker ( upper ) or DQ - BSA ( lower ) . Boxes show enlargements . R ( r ) = Pearson ' s coefficient . Scale bars , 5 µm .

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h, Starved LAMP1-PAGFP NRK cells were photo-activated (4 h) and imaged at 12 h with Lysotracker (upper) or DQ-BSA (lower). Boxes show enlargements. R(r) = Pearson's coefficient. Scale bars, 5 µm.


a , TEM ( bottom ; scale bar , 5 µm ) and fluorescence ( top ; scale bar , 10 µm ) of NRK - LC3 cells starved for times shown with enlargements ( boxes ) .

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a, TEM (bottom; scale bar, 5 µm) and fluorescence (top; scale bar, 10 µm) of NRK-LC3 cells starved for times shown with enlargements (boxes).


b , d , Immunoblots of cells shown in a with indicated antibodies ( d , after transfection with non - specific ( NS ) or atg5 RNAi ) . c , Autophagy ( black lines show independent experiments ) and phospho - S6K percentage ( densitometry of data shown in b , red line ) .

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b, d, Immunoblots of cells shown in a with indicated antibodies (d, after transfection with non-specific (NS) or atg5 RNAi). c, Autophagy (black lines  show  independent experiments) and phospho-S6K percentage (densitometry of data shown in b, red line).


e - g , LAMP1 - YFP NRK - LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h ( total , 6 h ) or 6 h ( total , 8 h ) analysed by blotting ( e ) as in b or microscopy ( f ; scale bar , 5 µm ) with quantification ( g ) . Error bars , s . e . m . ; n = 3 .

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e-g, LAMP1-YFP NRK-LC3 cells starved for 2 h and then treated with 100 nM rapamycin for another 4 h (total, 6 h) or 6 h (total, 8 h) analysed by blotting (e) as in b or microscopy (f; scale bar, 5 µm) with quantification (g). Error bars, s.e.m.; n = 3.


a , LAMP1 / LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation .

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a, LAMP1/LC3NRK cells were starved for 4 h and treated with GTPγS for 6 h of further starvation.


b , Rab7Q67L - GFP - transfected LC3 - CFP / Lamp1 - RFP NRK cells starved for 10 h .

b, Rab7Q67L-GFP-transfected LC3-CFP/Lamp1-RFP NRK cells starved for 10 h.


c , Rab7 - CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation .

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c, Rab7-CFP NRK cells starved for 2 h and treated with 100 nM rapamycin for 8 h of further starvation.


d , LAMP1 - YFP NRK - LC3 cells starved for times shown with 1 µg ml - 1 leupeptin and blotted with indicated antibodies . e , Cells in d imaged . .

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d, LAMP1-YFP NRK-LC3 cells starved for times shown with 1 µg ml-1 leupeptin and blotted with indicated antibodies. e, Cells in d imaged..


f , Fibroblast cells from patients with Scheie syndrome ( GM01256 ) or healthy controls starved and blotted with indicated antibodies . g , Cells in f imaged . Lysosome size classified by Image - ProPlus ( IPP ) software .

f, Fibroblast cells from patients with Scheie syndrome (GM01256) or healthy controls starved and blotted with indicated antibodies. g, Cells in f imaged. Lysosome size classified by Image-ProPlus (IPP) software.


A . LRP6 level is reduced during serum starvation . HEK293 cells were incubated in serum - free medium for the indicated hours . Cells were lysed and the cell lysates were analyzed by immunoblotting .

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A. LRP6 level is reduced during serum starvation. HEK293 cells were incubated in serum-free medium for the indicated hours. Cells were lysed and the cell lysates were analyzed by immunoblotting.


B . Serum starvation reduced the amount of LRP6 . HEK293 cells were incubated in serum - free medium for 3 h . Then cells were additionally incubated for 1 h after addition of 10 % serum . Cells were lysed and the cell lysates were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / β - actin and p - YAP / YAP of three independent immunoblots was quantified ( biological replicates , middle and right panel , respectively ) . N . S . : non - significant , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars indicate standard deviation of biological triplicate measurements . Student ' s t - test was used for statistical analysis .

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B. Serum starvation reduced the amount of LRP6. HEK293 cells were incubated in serum-free medium for 3 h. Then cells were additionally incubated for 1 h after addition of 10% serum. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin and p-YAP/YAP of three independent immunoblots was quantified (biological replicates, middle and right panel, respectively). N.S.: non-significant, **P < 0.01 and ***P < 0.001. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.


C . Loss of LRP6 in serum starvation was seen in different cell lines . Cell lines were incubated in serum - free medium for 2 h . Cells were lysed , and the cell lysates were analyzed by immunoblotting . S . E . , short exposure ; L . E . , long exposure .

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C. Loss of LRP6 in serum starvation was seen in different cell lines. Cell lines were incubated in serum-free medium for 2 h. Cells were lysed, and the cell lysates were analyzed by immunoblotting. S.E., short exposure; L.E., long exposure.


D . Glucose starvation decreased LRP6 . HEK293 cells were incubated in glucose - free medium for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting .

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D. Glucose starvation decreased LRP6. HEK293 cells were incubated in glucose-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.


E . The decrease of LRP6 by serum starvation was mediated by the lysosomal degradation pathway . HEK293 cells were incubated in serum - free medium for 4 h with DMSO or Bafilomycin A1 ( 100 nM ) . Cells were lysed and the cell lysates were analyzed by immunoblotting .

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E. The decrease of LRP6 by serum starvation was mediated by the lysosomal degradation pathway. HEK293 cells were incubated in serum-free medium for 4 h with DMSO or Bafilomycin A1 (100 nM). Cells were lysed and the cell lysates were analyzed by immunoblotting.


Loss of LRP6 by serum starvation was mediated in a clathrin HEK293 cells were transfected with clathrin siRNA and incubated in serum - free medium for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting .

Loss of LRP6 by serum starvation was mediated in a clathrin HEK293 cells were transfected with clathrin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.


G . Loss of LRP6 by serum starvation was mediated in a caveolin - 1 - dependent manner . HEK293 cells were transfected with caveolin siRNA and incubated in serum - free medium for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting .

G. Loss of LRP6 by serum starvation was mediated in a caveolin-1-dependent manner. HEK293 cells were transfected with caveolin siRNA and incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.


H . Lipid components of serum were essential for maintaining LRP6 . HEK293 cells were incubated in serum - free medium with A2058 BSA ( Sigma ) or Fraction V BSA for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / β - actin of three independent immunoblots was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . N . S . : non - significant and * P < 0 . 05 . Student ' s t - test was used for statistical analysis .

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H. Lipid components of serum were essential for maintaining LRP6. HEK293 cells were incubated in serum-free medium with A2058 BSA (Sigma) or Fraction V BSA for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. N.S.: non-significant and *P < 0.05. Student's t-test was used for statistical analysis.


A . The levels of LRP6 and phosphorylated YAP showed inverse correlation under serum starvation . HEK293 cells were incubated in serum - free medium for the indicated times . Cells were lysed and the cell lysates were analyzed by immunoblotting .

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A. The levels of LRP6 and phosphorylated YAP showed inverse correlation under serum starvation. HEK293 cells were incubated in serum-free medium for the indicated times. Cells were lysed and the cell lysates were analyzed by immunoblotting.


B . Knockdown of LRP6 induced the phosphorylation of YAP even under nutrient - rich condition . HEK293T cells were transfected with GFP siRNA as a control and LRP6 siRNA under nutrient - rich conditions . The ratio of p - YAP / total - YAP of three independent immunoblot bands was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . * P < 0 . 05 . Student ' s t - test was used for statistical analysis .

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B. Knockdown of LRP6 induced the phosphorylation of YAP even under nutrient-rich condition. HEK293T cells were transfected with GFP siRNA as a control and LRP6 siRNA under nutrient-rich conditions. The ratio of p-YAP/total-YAP of three independent immunoblot bands was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis.


C . Overexpression of LRP6 blocked the increase of YAP phosphorylation under serum starvation conditions . HEK293T cells were transfected with empty vector ( - ) or LRP6 - EGFP as indicated in the figure . One day after transfection , cells were incubated in serum - free medium for 4 h . Cells were lysed and the cell lysates were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / β - actin and p - YAP / YAP of three independent immunoblots was quantified ( middle and right panel , respectively ) . Error bars indicate standard deviation of biological triplicate measurements . * P < 0 . 05 and * * * P < 0 . 001 . Student ' s t - test was used for statistical analysis .

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C. Overexpression of LRP6 blocked the increase of YAP phosphorylation under serum starvation conditions. HEK293T cells were transfected with empty vector (-) or LRP6-EGFP as indicated in the figure. One day after transfection, cells were incubated in serum-free medium for 4 h. Cells were lysed and the cell lysates were analyzed by immunoblotting (left panel). The ratio of LRP6/β-actin and p-YAP/YAP of three independent immunoblots was quantified (middle and right panel, respectively). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05 and ***P < 0.001. Student's t-test was used for statistical analysis.


D . Knockdown of LRP6 reduced YAP reporter activity . HEK293T cells were transfected with GFP siRNA as a control , LRP6 siRNA , pRL - TK and 8XGTIIC luciferase reporter constructs . One day after transfection , luciferase activity was measured . Error bars indicate standard deviation of biological triplicate measurements . * * * P < 0 . 005 . Student ' s t - test was used for statistical analysis .

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D. Knockdown of LRP6 reduced YAP reporter activity. HEK293T cells were transfected with GFP siRNA as a control, LRP6 siRNA, pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. ***P < 0.005. Student's t-test was used for statistical analysis.


E . Knockdown of LRP6 reduced the expression of YAP target genes . Quantitative real - time PCR assay measuring the expression of CTGF , ANKRD1 , INHBA , and LRP6 mRNA in GFP and LRP6 siRNA - transfected HEK293T cells was performed . Quantification of CTGF , ANKRD1 , INHBA , and LRP6 mRNA was normalized against the level of β - actin . Error bars indicate standard deviation of technical triplicate measurements . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Student ' s t - test was used for statistical analysis .

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E. Knockdown of LRP6 reduced the expression of YAP target genes. Quantitative real-time PCR assay measuring the expression of CTGF, ANKRD1, INHBA, and LRP6 mRNA in GFP and LRP6 siRNA-transfected HEK293T cells was performed. Quantification of CTGF, ANKRD1, INHBA, and LRP6 mRNA was normalized against the level of β-actin. Error bars indicate standard deviation of technical triplicate measurements. *P < 0.05, **P < 0.01 and ***P < 0.001. Student's t-test was used for statistical analysis.


F . Knockdown of LRP6 reduced the interaction between YAP and TEAD . HEK293T cells were transfected with EGFP - YAP , Myc - TEAD4 and siRNA for LRP6 . Cells were lysed and the cell lysates were immunoprecipitated with anti - Myc antibody and analyzed by immunoblotting . WCL , whole - cell lysates .

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F. Knockdown of LRP6 reduced the interaction between YAP and TEAD. HEK293T cells were transfected with EGFP-YAP, Myc-TEAD4 and siRNA for LRP6. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


G . Overexpression of LRP6 rescued the reduced YAP reporter activity under serum starvation conditions . HEK293T cells were transfected with an empty vector ( - ) or VSVG - LRP6 , and with pRL - TK and 8XGTIIC luciferase reporter constructs . One day after transfection , cells were incubated in serum - free medium for 4 h and luciferase activity was measured . Error bars indicate standard deviation of biological triplicate measurements . * * * P < 0 . 001 . Student ' s t - test was used for statistical analysis .

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G. Overexpression of LRP6 rescued the reduced YAP reporter activity under serum starvation conditions. HEK293T cells were transfected with an empty vector (-) or VSVG-LRP6, and with pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were incubated in serum-free medium for 4 h and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. ***P < 0.001. Student's t-test was used for statistical analysis.


H . Knockdown of LRP6 induced cytoplasmic localization of YAP . HEK293A cells were transfected with siRNA for GFP or LRP6 and immunofluorescence analysis was performed . Figure shows representative image of multiple areas . DAPI was used for nucleus staining . Scale bars : 10 μm . Quantification of nuclear YAP is shown in the right panel . Quantification was performed by counting cells that have nuclear - localized YAP per image ( n = 8 ) . Error bars indicate standard deviation . * * * P < 0 . 001 . Student ' s t - test was used for statistical analysis .

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H. Knockdown of LRP6 induced cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP or LRP6 and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear-localized YAP per image (n=8). Error bars indicate standard deviation. ***P < 0.001. Student's t-test was used for statistical analysis.


I . Overexpression of LRP6 induced nuclear localization of YAP under starvation conditions . HEK293A cells were transfected with LRP6 - EGFP and 1 d after transfected cells were incubated with or without serum - containing medium for 4 h , and immunofluorescence analysis was performed . Figure shows representative image of multiple areas . DAPI was used for nucleus staining . Scale bars : 10μm . Quantification of nuclear YAP is shown in the right panel . Quantification was performed by counting cells that have nuclear localized YAP per image ( n = 6 ) . Error bars indicate standard deviation . * P < 0 . 05 . Student ' s t - test was used for statistical analysis .

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I. Overexpression of LRP6 induced nuclear localization of YAP under starvation conditions. HEK293A cells were transfected with LRP6-EGFP and 1 d after transfected cells were incubated with or without serum-containing medium for 4 h, and immunofluorescence analysis was performed. Figure shows representative image of multiple areas. DAPI was used for nucleus staining. Scale bars: 10μm. Quantification of nuclear YAP is shown in the right panel. Quantification was performed by counting cells that have nuclear localized YAP per image (n=6). Error bars indicate standard deviation. *P < 0.05. Student's t-test was used for statistical analysis.


A . Knockdown of LRP6 activated LATS1 . HEK293T cells were transfected with siRNA for GFP and LRP6 . After transfection , cells were lysed and the cell lysates were analyzed by immunoblotting .

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A. Knockdown of LRP6 activated LATS1. HEK293T cells were transfected with siRNA for GFP and LRP6. After transfection, cells were lysed and the cell lysates were analyzed by immunoblotting.


B . Overexpression of LRP6 reduced the amount of the active form of LATS1 that was induced by serum starvation . HEK293T cells were transfected with empty vector ( - ) or VSVG - LRP6 . 1 d after transfection , cells were incubated in serum - free medium for 4 h . After incubation , cells were lysed , and the cell lysates were analyzed by immunoblotting .

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B. Overexpression of LRP6 reduced the amount of the active form of LATS1 that was induced by serum starvation. HEK293T cells were transfected with empty vector (-) or VSVG-LRP6. 1 d after transfection, cells were incubated in serum-free medium for 4 h. After incubation, cells were lysed, and the cell lysates were analyzed by immunoblotting.


C . Merlin and LATS1 / 2 were necessary for the LRP6 - knockdown - mediated YAP phosphorylation . HEK293T cells were transfected with siRNA for GFP , LRP6 , Merlin , and LATS1 / 2 . Cells were lysed and the cell lysates were analyzed by immunoblotting .

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C. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated YAP phosphorylation. HEK293T cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.


D . Merlin and LATS1 / 2 were necessary for the LRP6 - knockdown - mediated decrease of YAP reporter activity . HEK293T cells were seeded and transfected with siRNA for GFP , LRP6 , Merlin and LATS1 / 2 . After 1 d , these cells were transfected again with pRL - TK and 8XGTIIC luciferase reporter constructs . One day after transfection , cells were lysed and luciferase activity was measured . Error bars indicate standard deviation of biological triplicate measurements . * * P < 0 . 01 and N . S : Non - significant . Student ' s t - test was used for statistical analysis .

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

D. Merlin and LATS1/2 were necessary for the LRP6-knockdown-mediated decrease of YAP reporter activity. HEK293T cells were seeded and transfected with siRNA for GFP, LRP6, Merlin and LATS1/2. After 1 d, these cells were transfected again with pRL-TK and 8XGTIIC luciferase reporter constructs. One day after transfection, cells were lysed and luciferase activity was measured. Error bars indicate standard deviation of biological triplicate measurements. **P< 0.01 and N.S: Non-significant. Student's t-test was used for statistical analysis.


E . Merlin and LATS1 / 2 were necessary for the LRP6 knockdown - mediated - cytoplasmic localization of YAP . HEK293A cells were transfected with siRNA for GFP , LRP6 , Merlin , and LATS1 / 2 , and immunofluorescence analysis was performed . The figure shows representative images of multiple areas . DAPI was used for nucleus staining . Scale bars : 10 μm . Quantification of cells that have nuclear YAP is shown in Fig EV3B .

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

E. Merlin and LATS1/2 were necessary for the LRP6 knockdown-mediated-cytoplasmic localization of YAP. HEK293A cells were transfected with siRNA for GFP, LRP6, Merlin, and LATS1/2, and immunofluorescence analysis was performed. The figure shows representative images of multiple areas. DAPI was used for nucleus staining. Scale bars: 10 μm. Quantification of cells that have nuclear YAP is shown in Fig EV3B.


F . YAP phosphorylation by loss of LRP6 was dependent on the interaction between Merlin and LATS1 / 2 . HEK293T cells were transfected with siRNA for GFP , LRP6 , and Merlin , and after 1 d were transfected again with HA - Merlin and HA - Merlin ∆ FERM . Cells were lysed and the cell lysates were analyzed by immunoblotting . The arrow indicates endogenous Merlin and HA - Merlin , and the arrowhead indicates HA - Merlin ∆ FERM .

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

F. YAP phosphorylation by loss of LRP6 was dependent on the interaction between Merlin and LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and Merlin, and after 1 d were transfected again with HA-Merlin and HA-Merlin∆FERM. Cells were lysed and the cell lysates were analyzed by immunoblotting. The arrow indicates endogenous Merlin and HA-Merlin, and the arrowhead indicates HA-Merlin∆FERM.


G . MST1 / 2 were necessary for the LRP6 - knockdown - mediated activation of LATS1 / 2 . HEK293T cells were transfected with siRNA for GFP , LRP6 and MST1 / 2 . Cells were lysed and the cell lysates were analyzed by immunoblotting .

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

G. MST1/2 were necessary for the LRP6-knockdown-mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6 and MST1/2. Cells were lysed and the cell lysates were analyzed by immunoblotting.


H . MAP4K4 / 6 / 7 were necessary for the LRP6 knockdown mediated activation of LATS1 / 2 . HEK293T cells were transfected with siRNA for GFP , LRP6 , and MAP4K4 / 6 / 7 . Cells were lysed and the cell lysates were analyzed by immunoblotting .

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

H. MAP4K4/6/7 were necessary for the LRP6 knockdown mediated activation of LATS1/2. HEK293T cells were transfected with siRNA for GFP, LRP6, and MAP4K4/6/7. Cells were lysed and the cell lysates were analyzed by immunoblotting.


A . The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition . HEK293T cells were transfected with Flag - Merlin and LRP6 - EGFP . After transfection , cells were incubated in serum - free medium for overnight with Bafilomycin A1 ( 20 nM ) . The cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting with the indicated antibodies . WCL , whole - cell lysates .

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

A. The interaction between overexpressed Merlin and LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with Flag-Merlin and LRP6-EGFP. After transfection, cells were incubated in serum-free medium for overnight with Bafilomycin A1 (20 nM). The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.


The interaction between endogenous Merlin and LRP6 was reduced under serum starvation . HEK293T cells were incubated in serum - free medium for 4 h with Bafilomycin A1 ( 100 nM ) . The cell lysates were immunoprecipitated with anti - LRP6 antibody and analyzed by immunoblotting with the indicated antibodies . WCL , whole - cell lysates .

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

The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-LRP6 antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.


The interaction between endogenous Merlin and LRP6 was reduced under serum starvation . HEK293T cells were incubated in serum - free medium for 4 h with Bafilomycin A1 ( 100 nM ) . The cell lysates were immunoprecipitated with anti - Merlin antibody and analyzed by immunoblotting with the indicated antibodies . WCL , whole - cell lysates .

The interaction between endogenous Merlin and LRP6 was reduced under serum starvation. HEK293T cells were incubated in serum-free medium for 4 h with Bafilomycin A1 (100 nM). The cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting with the indicated antibodies. WCL, whole-cell lysates.


D . The interaction between overexpressed Merlin and LATS1 was increased under serum starvation . HEK293T cells were transfected with Myc - LATS1 and EGFP - Merlin . After transfection , cells were incubated in serum - free medium for overnight and then were lysed . The cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates .

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

D. The interaction between overexpressed Merlin and LATS1 was increased under serum starvation. HEK293T cells were transfected with Myc-LATS1 and EGFP-Merlin. After transfection, cells were incubated in serum-free medium for overnight and then were lysed. The cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


E . Knockdown of LRP6 enhanced the interaction between overexpressed Merlin and LATS1 . HEK293T cells were transfected with Myc - LATS1 , EGFP - Merlin and LRP6 siRNA . Cells were lysed and the cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates .

E. Knockdown of LRP6 enhanced the interaction between overexpressed Merlin and LATS1. HEK293T cells were transfected with Myc-LATS1, EGFP-Merlin and LRP6 siRNA. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


F . Knockdown of LRP6 enhances the interaction between endogenous Merlin and LATS1 . HEK293T cells were transfected with siRNA for GFP or LRP6 . After transfection , cells were lysed and the cell lysates were immunoprecipitated with anti - Merlin antibody and analyzed by immunoblotting . WCL , whole - cell lysates .

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

F. Knockdown of LRP6 enhances the interaction between endogenous Merlin and LATS1. HEK293T cells were transfected with siRNA for GFP or LRP6. After transfection, cells were lysed and the cell lysates were immunoprecipitated with anti-Merlin antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


A . Endogenous LRP6 and OGT interact with each other . HEK293T cells were lysed and the cell lysates were immunoprecipitated with anti - LRP6 antibody and blotted with OGT . WCL , whole - cell lysates .

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

A. Endogenous LRP6 and OGT interact with each other. HEK293T cells were lysed and the cell lysates were immunoprecipitated with anti-LRP6 antibody and blotted with OGT. WCL, whole-cell lysates.


B . LRP6 is O - GlcNAcylated . LRP6 - EGFP - transfected HEK293T cells were treated in Thiamet G ( 30 μM ) or OSMI - 1 ( 50 μM ) for 6 h with Bafiolmycin A1 ( 100nM ) . After treatment , cells were lysed and the cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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. LRP6 is O-GlcNAcylated. LRP6-EGFP-transfected HEK293T cells were treated in Thiamet G (30 μM) or OSMI-1 (50 μM) for 6 h with Bafiolmycin A1 (100nM). After treatment, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


C . LRP6 is O - GlcNAcylated . Cell lysates from HEK293T cells were incubated with sWGA - beads in the absence or presence of free GlcNAc ( 25 mM ) for 3 h . After incubation , sWGA - beads were precipitated and analyzed by immunoblotting . WCL , whole - cell lysates .

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

C. LRP6 is O-GlcNAcylated. Cell lysates from HEK293T cells were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.


D . Blocking of O - GlcNAcylation reduced the amount of LRP6 . HEK293 cells were treated with OSMI - 1 ( 50 μM ) and Bafilomycin A1 ( 100 nM ) for 6 h . Cells were lysed and the cell lysates were analyzed by immunoblotting .

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

D. Blocking of O-GlcNAcylation reduced the amount of LRP6. HEK293 cells were treated with OSMI-1 (50 μM) and Bafilomycin A1 (100 nM) for 6 h. Cells were lysed and the cell lysates were analyzed by immunoblotting.


E . The O - GlcNAcylation on LRP6 was reduced under serum starvation condition . HEK293T cells were transfected with LRP6 - EGFP . Cells were incubated in serum - free medium with Bafilomycin A1 ( 100 nM ) for 4 h . After incubation , cells were lysed and the cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates . The ratio of O - GlcNAc / EGFP from three independent immunoblots was quantified ( biological replicates , bottom panel ) . * P < 0 . 05 . Error bars indicate standard deviation of biological triplicate measurements . Student ' s t - test was used for statistical analysis .

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

E. The O-GlcNAcylation on LRP6 was reduced under serum starvation condition. HEK293T cells were transfected with LRP6-EGFP. Cells were incubated in serum-free medium with Bafilomycin A1 (100 nM) for 4 h. After incubation, cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates. The ratio of O-GlcNAc/EGFP from three independent immunoblots was quantified (biological replicates, bottom panel). *P < 0.05. Error bars indicate standard deviation of biological triplicate measurements. Student's t-test was used for statistical analysis.


F . Increase of O - GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin . LRP6 - EGFP and Flag - Merlin transfected HEK293T cells were incubated in serum - free medium with Bafilomycin A1 ( 20 nM ) and Thiamet G ( 30μM ) overnight . Cells were lysed and the cell lysates were immunoprecipitated with anti - EGFP antibody and analyzed by immunoblotting . WCL , whole - cell lysates .

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

F. Increase of O-GlcNAcylation enhanced the interaction between ectopically expressed LRP6 and Merlin. LRP6-EGFP and Flag-Merlin transfected HEK293T cells were incubated in serum-free medium with Bafilomycin A1 (20 nM) and Thiamet G (30μM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-EGFP antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


G . Enhancement of global O - GlcNAcylation blocked the serum starvation - mediated increase of interaction between overexpressed LATS1 and Merlin . HEK293T cells were transfected with Myc - LATS1 and Flag - Merlin . After transfection , cells were incubated in serum - free medium with Thiamet G ( 30μM ) and Bafilomycin A1 ( 20nM ) overnight . Cells were lysed and the cell lysates were immunoprecipitated with anti - Myc antibody and analyzed by immunoblotting . WCL , whole - cell lysates .

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

G. Enhancement of global O-GlcNAcylation blocked the serum starvation-mediated increase of interaction between overexpressed LATS1 and Merlin. HEK293T cells were transfected with Myc-LATS1 and Flag-Merlin. After transfection, cells were incubated in serum-free medium with Thiamet G (30μM) and Bafilomycin A1 (20nM) overnight. Cells were lysed and the cell lysates were immunoprecipitated with anti-Myc antibody and analyzed by immunoblotting. WCL, whole-cell lysates.


A . Nutrient starvation reduced the level of LRP6 in mouse liver . Mice were starved for 36 h and liver tissues were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / Vinculin of three independent immunoblots was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . * * P < 0 . 01 . Student ' s t - test was used for statistical analysis .

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

A. Nutrient starvation reduced the level of LRP6 in mouse liver. Mice were starved for 36 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. **P < 0.01. Student's t-test was used for statistical analysis.


B . Refeeding of nutrients rescued LRP6 in mouse liver . Mice were starved and then refed for 6 h and liver tissues were analyzed by immunoblotting ( left panel ) . The ratio of LRP6 / Vinculin of three independent immunoblots was quantified ( biological replicates , right panel ) . Error bars indicate standard deviation of biological triplicate measurements . * P < 0 . 05 . Student ' s t - test was used for statistical analysis .

B. Refeeding of nutrients rescued LRP6 in mouse liver. Mice were starved and then refed for 6 h and liver tissues were analyzed by immunoblotting (left panel). The ratio of LRP6/Vinculin of three independent immunoblots was quantified (biological replicates, right panel). Error bars indicate standard deviation of biological triplicate measurements. *P < 0.05. Student's t-test was used for statistical analysis.


C . The level of LRP6 was not reduced during nutrient starvation in mouse brain . Mice were starved and brain tissues were analyzed by immunoblotting .

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

C. The level of LRP6 was not reduced during nutrient starvation in mouse brain. Mice were starved and brain tissues were analyzed by immunoblotting.


D . LRP6 was O - GlcNAcylated in mouse liver . Liver tissues were incubated with sWGA - beads in the absence or presence of free GlcNAc ( 25 mM ) for 3 h . After incubation , sWGA - beads were precipitated and analyzed by immunoblotting . WCL , whole - cell lysates .

D. LRP6 was O-GlcNAcylated in mouse liver. Liver tissues were incubated with sWGA-beads in the absence or presence of free GlcNAc (25 mM) for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.


E . O - GlcNAcylation of LRP6 was reduced during starvation in mouse liver . LRP6 in liver tissue lysates from both fed and starved mice was equilibrated by loading twice as much lysate from the livers of starved mice on the gel . Liver tissues were incubated in sWGA - beads for 3 h . After incubation , sWGA - beads were precipitated and analyzed by immunoblotting . WCL , whole - cell lysates .

E. O-GlcNAcylation of LRP6 was reduced during starvation in mouse liver. LRP6 in liver tissue lysates from both fed and starved mice was equilibrated by loading twice as much lysate from the livers of starved mice on the gel. Liver tissues were incubated in sWGA-beads for 3 h. After incubation, sWGA-beads were precipitated and analyzed by immunoblotting. WCL, whole-cell lysates.


Western blotting analysis of Gadd45a protein expression in mouse intestine crypt lysates from WT and G3Terc - / - mice ( A ) . Quantification of Gadd45a protein expression , β - actin protein level was used as the loading control ( n = 7 mice per genotype ) , two independent experiments were carried out ( B ) .

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

Western blotting analysis of Gadd45a protein expression in mouse intestine crypt lysates from WT and G3Terc-/- mice (A). Quantification of Gadd45a protein expression, β-actin protein level was used as the loading control (n=7 mice per genotype), two independent experiments were carried out (B).


Quantification of Gadd45a - ChIP values as percentage of total telomeric DNA and ALU elements in WT and G3Terc - / - MEF cells ( n = 3 for each genotype ) .

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

Quantification of Gadd45a-ChIP values as percentage of total telomeric DNA and ALU elements in WT and G3Terc-/- MEF cells (n=3 for each genotype).


Dot - bot assay following Gadd45a - ChIP in WT and G3Terc - / - MEF cells .

Dot-bot assay following Gadd45a-ChIP in WT and G3Terc-/- MEF cells.


Kaplan - Meier survival curves of the mice with indicated genotypes ( WT , n = 44 ; Gadd45a - / - , n = 48 ; G3Terc - / - , n = 37 ; G3 - dKO , n = 41 ) . P value comparing the survival of G3Terc - / - with G3 - dKO mice is calculated with the log - rank test .

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

Kaplan-Meier survival curves of the mice with indicated genotypes (WT, n=44; Gadd45a-/-, n=48; G3Terc-/-, n=37; G3-dKO, n=41). P value comparing the survival of G3Terc-/- with G3-dKO mice is calculated with the log-rank test.


The average body weight of mice with indicated genotypes at indicated time points ( G3 - dKO Female ( n = 19 ) vs G3Terc - / - Female ( n = 17 ) , P = 0 . 004 ; G3 - dKO Male ( n = 14 ) vs G3Terc - / - Male ( n = 14 ) , P = 0 . 0038 ) . P values are calculated with log - rank test .

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

The average body weight of mice with indicated genotypes at indicated time points (G3-dKO Female (n=19) vs G3Terc-/- Female (n=17), P=0.004; G3-dKO Male (n=14) vs G3Terc-/- Male (n=14), P=0.0038). P values are calculated with log-rank test.


Whole mount images of colon samples from 7 - month old mice with indicated genotypes ( G ) . Quantification on density of colon crypts ( H ) ( n = 5 mice per group ) .

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

Whole mount images of colon samples from 7-month old mice with indicated genotypes (G). Quantification on density of colon crypts (H) (n=5 mice per group).


H & E - stained small intestine sections from 7 - month old mice with indicated genotypes ( I ) . Quantification of the number of small intestine basal crypts ( J ) ( n = 5 - 6 mice per group ) .

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

H&E-stained small intestine sections from 7-month old mice with indicated genotypes (I). Quantification of the number of small intestine basal crypts (J) (n=5-6 mice per group).


Olfm4 - positive intestinal stem cells ( indicated by arrows ) in small intestine sections ( A ) . Quantification of intestinal stem cell ( Olfm4 + ) number per 100 μm ( n = 4 - 5 mice per group ) of small intestine sections ( B ) .

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

Olfm4-positive intestinal stem cells (indicated by arrows) in small intestine sections (A). Quantification of intestinal stem cell (Olfm4+) number per 100 μm (n=4-5 mice per group) of small intestine sections (B).


Representative images of small intestine crypt culture ( day 2 ) . Arrows mark the surviving cysts ( C ) . Quantification of numbers of cysts per culture well ( D ) ( n = 4 - 5 mice / genotype ) .

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

Representative images of small intestine crypt culture (day 2). Arrows mark the surviving cysts (C). Quantification of numbers of cysts per culture well (D)(n=4-5 mice/genotype).


In vitro crypt culture ( day 8 ) from small intestine crypts isolated from mice with indicated genotypes . Arrows mark the crypt - like structure ( E ) . The number of organoids per culture wells ( n = 4 - 5 mice per group ) . Two hundred crypts are seeded per well ( F ) .

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

In vitro crypt culture (day 8) from small intestine crypts isolated from mice with indicated genotypes. Arrows mark the crypt-like structure (E). The number of organoids per culture wells (n=4-5 mice per group). Two hundred crypts are seeded per well (F).


Representative images of TUNEL staining in small intestine sections . Arrows indicate TUNEL positive cells ( G ) . Quantification of TUNEL positive cells per crypt ( n = 5 mice per group ) ( H ) .

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

Representative images of TUNEL staining in small intestine sections. Arrows indicate TUNEL positive cells (G). Quantification of TUNEL positive cells per crypt (n=5 mice per group) (H).


Representative images of phosphorylated histone 3 ( pH3 ) antibody staining in mouse small intestine . Arrows indicate pH3 positive cells ( I ) . Quantification of pH3 positive crypt cells per 100 μm of small intestines ( n = 4 - 6 mice per group ) ( J ) .

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

Representative images of phosphorylated histone 3 (pH3) antibody staining in mouse small intestine. Arrows indicate pH3 positive cells (I). Quantification of pH3 positive crypt cells per 100 μm of small intestines (n=4-6 mice per group) (J).


Representative images of immunostaining for p53 on small intestine sections . Arrows indicate p53 - positive cells ( A ) . Quantification of the percentage of p53 positive cells per crypt ( n = 5 - 6 mice per group ) ( B ) .

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

Representative images of immunostaining for p53 on small intestine sections. Arrows indicate p53-positive cells (A). Quantification of the percentage of p53 positive cells per crypt (n=5-6 mice per group) (B).


Representative images of γH2AX antibody staining on small intestine paraffin sections from WT , Gadd45a - / - , G3Terc - / - , and G3 - dKO mice ( 7 months old ) . Arrows indicate γH2AX positive nuclei ( C ) . Quantification of the percentage of γH2AX positive cells per small intestine crypt ( n = 4 - 6 mice per group ) ( D ) .

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

Representative images of γH2AX antibody staining on small intestine paraffin sections from WT, Gadd45a-/-, G3Terc-/-, and G3-dKO mice (7 months old). Arrows indicate γH2AX positive nuclei (C). Quantification of the percentage of γH2AX positive cells per small intestine crypt (n=4-6 mice per group) (D).


Representative images of small intestine crypt sections stained with antibody against pATM ( Ser - 1981 ) ( E ) . Quantification of frequencies of pATM ( Ser - 1981 ) positive crypt cells ( n = 4 - 6 of 7 - month old mice per genotype ) ( F ) .

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

Representative images of small intestine crypt sections stained with antibody against pATM (Ser-1981)(E). Quantification of frequencies of pATM (Ser-1981) positive crypt cells (n=4-6 of 7-month old mice per genotype) (F).


Representative image of the anaphase bridge ( indicated with arrows ) in the basal crypt of G3Terc - / - mice ( G ) . The percentage of cells with anaphase bridges in pH3 positive basal crypt cell population ( n = 4 - 6 mice per genotype ) ( H ) .

Representative image of the anaphase bridge (indicated with arrows) in the basal crypt of G3Terc-/- mice (G). The percentage of cells with anaphase bridges in pH3 positive basal crypt cell population (n=4-6 mice per genotype) (H).


Heat map indicating the relative expression levels of DDR and cell cycle - related gene transcripts in MEFs from indicated genotypes ( Data are extracted from RNAseq datasets ) .

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Heat map indicating the relative expression levels of DDR and cell cycle-related gene transcripts in MEFs from indicated genotypes (Data are extracted from RNAseq datasets).


CpG island methylation status of subtelomeric regions on chromosome 1q . gDNAs from MEF with indicated genotypes were PCR amplified and analysed by bisulfite sequencing . Yellow and blue bars denote the frequencies of methylated and unmethylated CpG islands , respectively , at each position ( A ) . Quantification of subtelomeric CpG island methylation ( B ) .

CpG island methylation status of subtelomeric regions on chromosome 1q. gDNAs from MEF with indicated genotypes were PCR amplified and analysed by bisulfite sequencing. Yellow and blue bars denote the frequencies of methylated and unmethylated CpG islands, respectively, at each position (A). Quantification of subtelomeric CpG island methylation (B).


Telomeric quantification and dot - bot assay of tri - methyl H3K9 ( H3K9me3 ) - ChIP in indicated

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Telomeric quantification and dot-bot assay of tri-methyl H3K9 (H3K9me3)-ChIP in indicated genotypes


Telomeric quantification and dot - bot assay of acetylated H3K9 ( H3K9ac ) - ChIP in indicated

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Telomeric quantification and dot-bot assay of acetylated H3K9 (H3K9ac)-ChIP in indicated genotypes


Telomeric quantification and dot - bot assay of HP1a - ChIP in indicated

Telomeric quantification and dot-bot assay of HP1a-ChIP in indicated genotypes


Representative images of small intestine organoids from WT and G3Terc - / - mice incubated with or without APE1 inhibitor ( CRT0044876 , 10μM ) treatment . Arrows indicate the crypt - like structure in organoid cultures ( A ) . Quantification of organoid number ( per well ) and size ( in 104 μm2 ) ( n = 3 - 4 mice per genotype / treatment ) ( B - C ) .

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Representative images of small intestine organoids from WT and G3Terc-/- mice incubated with or without APE1 inhibitor (CRT0044876, 10μM) treatment. Arrows indicate the crypt-like structure in organoid cultures (A). Quantification of organoid number (per well) and size (in 104 μm2) (n=3-4 mice per genotype/treatment) (B-C).


Representative images of γH2AX antibody staining on dissociated organoid cells from WT and G3Terc - / - organoid cultures with or without APE1 inhibitor ( CRT0044876 , 10μM ) ( DNAs are stained with DAPI ) ( D ) . Quantification of γH2AX positive cells ( n = 3 - 4 mice per group ) ( E ) .

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Representative images of γH2AX antibody staining on dissociated organoid cells from WT and G3Terc-/- organoid cultures with or without APE1 inhibitor (CRT0044876, 10μM) (DNAs are stained with DAPI) (D). Quantification of γH2AX positive cells (n=3-4 mice per group) (E).


Telomeric DNA quantification and dot - blot assay of H3K9me3 -

Telomeric DNA quantification and dot-blot assay of H3K9me3-ChIP


Telomeric DNA quantification and dot - blot assay of H3K9ac -

Telomeric DNA quantification and dot-blot assay of H3K9ac-ChIP


Telomeric DNA quantification and dot - blot assay of HP1a -

Telomeric DNA quantification and dot-blot assay of HP1a-ChIP


Representative images of Olfm4 staining in small intestine sections from mice treated with or without APE1 inhibitor ( CRT0044876 , CRT ) . Arrows indicate Olfm4 positive intestine stem cells ( I ) . Quantification of Olfm4 positive intestine stem cells ( n = 4 mice per group ) ( J ) .

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Representative images of Olfm4 staining in small intestine sections from mice treated with or without APE1 inhibitor (CRT0044876, CRT). Arrows indicate Olfm4 positive intestine stem cells (I). Quantification of Olfm4 positive intestine stem cells (n=4 mice per group) (J).


The knockdown efficiency of shRNA - GADD45A in human WI38 fibroblasts .

The knockdown efficiency of shRNA-GADD45A in human WI38 fibroblasts.


The proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A .

The proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A.


Representative images of β - gal staining in human fibroblast cultures with control and shRNA - GADD45A treatments at passage 54 ( C ) . Quantification of the percentage of β - gal positive cells in human fibroblasts with control and shRNA - GADD45A treatments ( duplicates per group ) ( D ) .

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Representative images of β-gal staining in human fibroblast cultures with control and shRNA-GADD45A treatments at passage 54 (C). Quantification of the percentage of β-gal positive cells in human fibroblasts with control and shRNA-GADD45A treatments (duplicates per group) (D).


Representative images of GADD45A and γH2AX staining in colon sections from young ( 35 - 50 y / o ) and old ( 75 - 85 y / o ) people ( E ) . Quantification of the percentage of GADD45A positive cells in young and old human colon tissues ( n = 8 per age group ) ( F ) . Quantification of the percentage of γH2AX positive cells in young and old human colon tissues ( n = 6 per age group ) ( G ) .

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Representative images of GADD45A and γH2AX staining in colon sections from young (35-50 y/o) and old (75-85 y/o) people (E). Quantification of the percentage of GADD45A positive cells in young and old human colon tissues (n=8 per age group) (F). Quantification of the percentage of γH2AX positive cells in young and old human colon tissues (n=6 per age group) (G).


Body weights of WT and Rcan1 - KO mice fed normal chow diet ( NC ) for 20 weeks starting at 8 - 10 weeks of age ( male , n = 15 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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Body weights of WT and Rcan1-KO mice fed normal chow diet (NC) for 20 weeks starting at 8-10 weeks of age (male, n=15 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


Body weights of WT and Rcan1 - KO mice fed a high - fat diet ( HFD ) for 20 weeks starting at 8 - 10 weeks of age ( male , n = 15 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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Body weights of WT and Rcan1-KO mice fed a high-fat diet (HFD) for 20 weeks starting at 8-10 weeks of age (male, n=15 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


C Food consumption per gram of body weight gained ( g / g ∆ BW ) over the first 15 weeks on NC or HFD , starting at 8 weeks of age ( male , n = 9 each ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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C Food consumption per gram of body weight gained (g/g ∆BW) over the first 15 weeks on NC or HFD, starting at 8 weeks of age (male, n=9 each). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


Cholesterol remaining in the feces of WT and Rcan1 - KO mice fed a HFD , measured as mMol per mg of feces ( males , n = 4 over 3 days ) . Cholesterol content of NC and the HFD chow ( input ) were measured for comparison . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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

Cholesterol remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). Cholesterol content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


triglyceride remaining in the feces of WT and Rcan1 - KO mice fed a HFD , measured as mMol per mg of feces ( males , n = 4 over 3 days ) . triglyceride content of NC and the HFD chow ( input ) were measured for comparison . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

triglyceride remaining in the feces of WT and Rcan1-KO mice fed a HFD, measured as mMol per mg of feces (males, n=4 over 3 days). triglyceride content of NC and the HFD chow (input) were measured for comparison. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


MRI measures of total body weight , percentage lean mass , and percent fat mass prior to and after 6 weeks on the HFD ( males , n = 5 ) . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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MRI measures of total body weight, percentage lean mass, and percent fat mass prior to and after 6 weeks on the HFD (males, n=5). Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


MRI image of WT and KO males following 8 weeks on a HFD . Yellow delineates visceral WAT . Pink delineates subcutaneous WAT . Quantification graphed on the right . Data information : values shown are mean ± SEM * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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MRI image of WT and KO males following 8 weeks on a HFD. Yellow delineates visceral WAT. Pink delineates subcutaneous WAT. Quantification graphed on the right. Data information: values shown are mean ±SEM * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


Representative H & E staining of gWAT from WT and KO males after 25 weeks on NC or HFD . Scale bar = 100 μm . K Quantification of cross - sectional area ( CSA ) of adipocytes in K ( n = 3 animals , 3 images / each , 75 - 100 adipocytes ) . Scale bar = 100 μm . Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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Representative H&E staining of gWAT from WT and KO males after 25 weeks on NC or HFD. Scale bar = 100 μm. K Quantification of cross-sectional area (CSA) of adipocytes in K (n=3 animals, 3 images/each, 75-100 adipocytes). Scale bar = 100 μm. Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


L Transcript levels for inflammatory markers Monocyte chemoattractant protein - 1 ( Mcp - 1 ) , macrophage antigen F4 / 80 ( F480 ) , tumor necrosis factor - alpha ( Tnfα ) , and tumor necrosis factor - beta ( Tnfβ ) in gWAT of WT and KO after 25 weeks on NC or HFD ( males , n = 4 - 5 ) . Transcript levels were normalized to 18S Data information : values shown are mean ± SD * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons , except for A and B , which used multiple t - tests ) .

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L Transcript levels for inflammatory markers Monocyte chemoattractant protein-1 (Mcp-1), macrophage antigen F4/80 (F480), tumor necrosis factor-alpha (Tnfα), and tumor necrosis factor-beta (Tnfβ) in gWAT of WT and KO after 25 weeks on NC or HFD (males, n=4-5). Transcript levels were normalized to 18S Data information: values shown are mean ±SD * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (two-way ANOVA with multiple comparisons, except for A and B, which used multiple t-tests).


A , B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD , with quantification of relative stain intensity ( n = 3 , 3 images each ) . Scale bar = 100 μm . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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A, B Oil Red O staining of livers from WT and KO males after 25 weeks on NC or HFD, with quantification of relative stain intensity (n=3, 3 images each). Scale bar = 100 μm. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


C Transcript levels for genes involved in lipid metabolism and gluconeogenesis in livers of WT and KO mice after 25 weeks on NC or HFD . Stearoyl - Coenzyme A desaturase ( Scd1 ) , fatty acid transporter ( CD36 ) , glycerol - 3 - phosphate acyltransferase ( Gpat ) , carnitine palmitoyltransferase I ( Cpt1 ) , peroxisome proliferator - activated gamma ( PPARγ ) , pyruvate dehydrogenase acetyl - transferase ( Pdk4 ) , PPARγ coactivator 1 alpha ( PGC1α ) , diacylglycerol O - acyltransferase ( Dgat ) , and facilitated glucose transporter ( Glut4 ) . ( males , n = 4 - 5 ) . Transcript levels were normalized to 18S . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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C Transcript levels for genes involved in lipid metabolism and gluconeogenesis in livers of WT and KO mice after 25 weeks on NC or HFD. Stearoyl-Coenzyme A desaturase (Scd1), fatty acid transporter (CD36), glycerol-3-phosphate acyltransferase (Gpat), carnitine palmitoyltransferase I (Cpt1), peroxisome proliferator-activated gamma (PPARγ), pyruvate dehydrogenase acetyl-transferase (Pdk4), PPARγ coactivator 1 alpha (PGC1α), diacylglycerol O-acyltransferase (Dgat), and facilitated glucose transporter (Glut4). (males, n=4-5). Transcript levels were normalized to 18S. Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


D - F Serum levels of free fatty acids ( FAA ) , insulin , and leptin in WT and KO after 25 weeks on NC or HFD ( males , n = 8 - 10 ) . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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D-F Serum levels of free fatty acids (FAA), insulin, and leptin in WT and KO after 25 weeks on NC or HFD (males, n=8-10). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Glucose tolerance test ( GTT ) after 25 weeks on NC or HFD . GTT was performed following an overnight fast Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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Glucose tolerance test (GTT) after 25 weeks on NC or HFD. GTT was performed following an overnight fast Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Insulin tolerance test ( ITT ) after 25 weeks on NC or HFD . , ITT following a three hour fast ( males , n = 6 ) . Data information : values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 ; * * * = p < 0 . 001 ; * * * * = p < 0 . 0001 ( two - way ANOVA with multiple comparisons ) .

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Insulin tolerance test (ITT) after 25 weeks on NC or HFD. , ITT following a three hour fast (males, n=6). Data information: values shown are mean ±SD. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 (two-way ANOVA with multiple comparisons).


Oxygen consumption ( VO2 ) of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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Oxygen consumption (VO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


carbon dioxide production ( VCO2 ) of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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

carbon dioxide production (VCO2) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


respiratory exchange ratio VCO2 / VO2 ( RER ) of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

respiratory exchange ratio VCO2/VO2 (RER) of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


physical activity of 10 - 12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system ( males , n = 6 ) . Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM . Data information : values shown are average ± SD . * * * = p < 0 . 001 ( two - way ANOVA with multiple comparisons ) .

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physical activity of 10-12 week old WT and KO mice on normal chow were measured using the CLAM metabolic cage system (males, n=6). Bar graphs show the average value of metabolic measurements made within the 60 minutes flanking 6AM or 6PM. Data information: values shown are average ±SD. *** = p < 0.001 (two-way ANOVA with multiple comparisons).


Transcript levels for Rcan1 . 1 and Rcan1 . 4 during differentiation of 3T3 - L1 adipocytes , measured at 0 , 1 , 2 , 3 , 4 , 6 and 24 hours following the addition MDI as well as at 3 , 7 and 10 days as indicated ( n = 3 ) . * = p < 0 . 05 ; * * = p < 0 . 01 , student ' s t - test , unpaired , vs 0 time point . Data information : Transcript levels were normalized to 18S . All values shown are mean ±

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Transcript levels for Rcan1.1 and Rcan1.4 during differentiation of 3T3-L1 adipocytes, measured at 0, 1, 2, 3, 4, 6 and 24 hours following the addition MDI as well as at 3, 7 and 10 days as indicated (n=3). * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, vs 0 time point. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


B Western blot for RCAN1 . 1 , RCAN1 . 4 and tubulin ( TUB ) in whole cell extracts of 3T3 - L1 adipocytes transfected with a control si - RNA or ones targeting either Rcan1 . 4 or Rcan1 . 1 ( 20 μg per lane ) .

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B Western blot for RCAN1.1, RCAN1.4 and tubulin (TUB) in whole cell extracts of 3T3-L1 adipocytes transfected with a control si-RNA or ones targeting either Rcan1.4 or Rcan1.1 (20 μg per lane).


C Representative images of 3T3 - L1 adipocytes transfected with the indicated si - RNAs prior to differentiation and stained with Oil Red O on day 10 . Scale bar = 100 μm .

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C Representative images of 3T3-L1 adipocytes transfected with the indicated si-RNAs prior to differentiation and stained with Oil Red O on day 10. Scale bar = 100 μm.


D Western blot for adiponectin ( ADPOQ ) released into the media during differentiation by 3T3 - L1 adipocytes transfected with the indicated si - RNAs . Ponceau stained image provides loading control .

D Western blot for adiponectin (ADPOQ) released into the media during differentiation by 3T3-L1 adipocytes transfected with the indicated si-RNAs. Ponceau stained image provides loading control.


E Transcript levels for Pgc1 - α , in 3T3 - L1 adipocytes transfected with the indicated si - RNAs and assayed over 10 - days of differentiation ( n = 3 ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ±

E Transcript levels for Pgc1-α, in 3T3-L1 adipocytes transfected with the indicated si-RNAs and assayed over 10-days of differentiation (n=3). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


Representative bright - field images showing lipid accumulation at 10 - days differentiation of Ing - svf cells treated with the indicated siRNA . Scale bar = 50 μm .

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Representative bright-field images showing lipid accumulation at 10-days differentiation of Ing-svf cells treated with the indicated siRNA. Scale bar = 50 μm.


G Transcript levels for Adrenoceptor beta 3 ( Adrb3 ) , uncoupling protein 1 ( Ucp1 ) , PPARγ coactivator 1 alpha ( PGC1α ) , and deiodinase type II ( Dio2 ) Ing - svf cultures transfected with control siRNA ( C ) or one targeting Rcan1 ( KD ) then maintained in growth media ( G ) , or differentiated for ten - days ( D ) with and without the addition of 100 mM isoproterionol for 6 hours prior to harvesting ( D + ISO ) ( n = 2 in triplicate ) . * = p < 0 . 05 ; * * = p < 0 . 01 , two - way ANOVA with multiple comparisons . Data information : Transcript levels were normalized to 18S . All values shown are mean ±

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G Transcript levels for Adrenoceptor beta 3 (Adrb3), uncoupling protein 1 (Ucp1), PPARγ coactivator 1 alpha (PGC1α), and deiodinase type II (Dio2) Ing-svf cultures transfected with control siRNA (C) or one targeting Rcan1 (KD) then maintained in growth media (G), or differentiated for ten-days (D) with and without the addition of 100 mM isoproterionol for 6 hours prior to harvesting (D+ISO) (n=2 in triplicate). * = p < 0.05; ** = p < 0.01, two-way ANOVA with multiple comparisons. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


H , I Transcript levels for the indicated panel of genes relevant to adipocyte differentiation in Platelet - derived growth factor receptor alpha - positive ( PDGFRα + ) and lineage - negative ( Lin - ) stromal vascular cells isolated by FACS sorting from sWAT of WT ( black bars ) and KO ( red bars ) . In H , isolated cells were maintained in growth media . In I , cultures were treated with MDI then differentiated for 10 days . * = p < 0 . 05 ; * * = p < 0 . 01 , student ' s t - test , unpaired , WT vs . KO . Data information : Transcript levels were normalized to 18S . All values shown are mean ±

H, I Transcript levels for the indicated panel of genes relevant to adipocyte differentiation in Platelet-derived growth factor receptor alpha-positive (PDGFRα+) and lineage-negative (Lin-) stromal vascular cells isolated by FACS sorting from sWAT of WT (black bars) and KO (red bars). In H, isolated cells were maintained in growth media. In I, cultures were treated with MDI then differentiated for 10 days. * = p < 0.05; ** = p < 0.01, student's t-test, unpaired, WT vs. KO. Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD


A Representative H & E histological sections of sWAT from WT and KO mice probed with anti - UCP1 antibodies . Mice were either maintained at 24 ° C ( RT ) or housed at 6 ° C for 5 days . Scale bar = 200 μm .

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A Representative H&E histological sections of sWAT from WT and KO mice probed with anti-UCP1 antibodies. Mice were either maintained at 24°C (RT) or housed at 6°C for 5 days. Scale bar = 200 μm.


Transcript levels of Pgc1α and Ucp1 in sWAT of male and female WT and KO mice after 24 hours at RT or 4 ° C ( n = Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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Transcript levels of Pgc1α and Ucp1 in sWAT of male and female WT and KO mice after 24 hours at RT or 4°C (n= Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


C Transcript levels of Ucp1 , Pgc1α , Adrb3 , retinoblastoma 1 ( Rb1 ) , zinc finger protein 423 ( Zfp423 ) , and PR domain containing 16 ( Prdm16 ) were quantified in sWAT of WT and KO mice after 25 weeks on a HFD ( n = 4 - 5 ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ; unpaired student ' s t -

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C Transcript levels of Ucp1, Pgc1α, Adrb3, retinoblastoma 1 (Rb1), zinc finger protein 423 (Zfp423), and PR domain containing 16 (Prdm16) were quantified in sWAT of WT and KO mice after 25 weeks on a HFD (n=4-5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. ; unpaired student's t-test


D Transcript levels of Pgc1α - 1b and Pgc1α - 1a isoforms in sWAT of WT and KO 8 week old females maintained at 24 ° C ( RT ) or housed at 4 ° C for 24 hours ( n = 5 ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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D Transcript levels of Pgc1α-1b and Pgc1α-1a isoforms in sWAT of WT and KO 8 week old females maintained at 24°C (RT) or housed at 4°C for 24 hours (n=5). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


E . Western blot for RCAN1 in various skeletal muscle groups . Each lane was loaded with 20 μg of total protein from plantaris ( PL ) , soleus ( SOL ) , extensor digitorum longus ( EDL ) , tibialis anterior ( TA ) , red gastrocnemius ( RG ) , white gastrocnemius ( WG ) , white vastus lateralis ( WV ) , heart ventricle ( HT ) , atria , diaphragm ( Dia ) , and Tong . The lane containing brain extract was loaded with 10 μg of protein . Antibody specificity was validated by running 20 μg of protein from the heart of an Rcan1 - KO animal ( HT - KO ) . The asterisks * indicate the location of putative proteolytic fragments of RCAN1 . 1 .

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E. Western blot for RCAN1 in various skeletal muscle groups. Each lane was loaded with 20 μg of total protein from plantaris (PL), soleus (SOL), extensor digitorum longus (EDL), tibialis anterior (TA), red gastrocnemius (RG), white gastrocnemius (WG), white vastus lateralis (WV), heart ventricle (HT), atria, diaphragm (Dia), and Tong. The lane containing brain extract was loaded with 10 μg of protein. Antibody specificity was validated by running 20 μg of protein from the heart of an Rcan1-KO animal (HT-KO). The asterisks * indicate the location of putative proteolytic fragments of RCAN1.1.


F Transcript levels of Sln in skeletal muscle ( tibialis anterior ) of 8 - 10 week old WT and KO mice housed at RT . ( n = 4 - 5 each gender ) . G Transcript levels of Sln in skeletal muscle ( tibialis anterior ) of 8 - 10 week old male WT and KO animals housed at RT . ( n = 5 ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple comparisons F , ; unpaired student ' s t - test in G .

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F Transcript levels of Sln in skeletal muscle (tibialis anterior) of 8-10 week old WT and KO mice housed at RT. (n=4-5 each gender). G Transcript levels of Sln in skeletal muscle (tibialis anterior ) of 8-10 week old male WT and KO animals housed at RT. (n=5).Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons F, ; unpaired student's t-test in G.


H Representative western blot showing SLN protein in soleus muscle of 8 - 10 week old WT and KO animals housed at RT . GAPDH was used as a loading control .

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H Representative western blot showing SLN protein in soleus muscle of 8-10 week old WT and KO animals housed at RT. GAPDH was used as a loading control.


J Activity of the Sln - Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin ( CnA * ) and Rcan1 . Luciferase activity was normalized to beta - galactosidase activity from a co - transfected vector . ( n = 3 , assayed in duplicate ) . Data information : Transcript levels were normalized to 18S . All values shown are mean ± SD . * = p < 0 . 05 ; * * = p < 0 . 01 . ( two - way ANOVA with multiple

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J Activity of the Sln-Luc reporter in C2C12 myoblasts transiently transfected with an empty control vector or ones expressing constitutively active calcineurin (CnA*) and Rcan1. Luciferase activity was normalized to beta-galactosidase activity from a co-transfected vector. (n=3, assayed in duplicate). Data information: Transcript levels were normalized to 18S. All values shown are mean ±SD. * = p < 0.05; ** = p < 0.01. (two-way ANOVA with multiple comparisons


A Correlation of Rcan1 expression in liver and gonadal adipose tissue with HOMA - IR , using male mice from over 100 strains from the hybrid mouse diversity panel . Regression line ( red ) , r = biweight midcorrelation , p = p - value .

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A Correlation of Rcan1 expression in liver and gonadal adipose tissue with HOMA-IR, using male mice from over 100 strains from the hybrid mouse diversity panel. Regression line (red), r = biweight midcorrelation, p = p-value.


B , C Correlation of Rcan1 expression in gonadal adipose and liver with body weight , plasma insulin and plasma triglycerides , in a data set comparing backgrounds susceptible ( BTBR background ) or resistant ( C57BL / 6 background ) to diabetes when carrying the leptinob / ob ( ob ) mutation . Measures are reported as the ratio of the mean log10 intensity ( ml ratio ) . Regression line ( black ) , r = linear regression , p = p - value .

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B, C Correlation of Rcan1 expression in gonadal adipose and liver with body weight, plasma insulin and plasma triglycerides, in a data set comparing backgrounds susceptible (BTBR background) or resistant (C57BL/6 background) to diabetes when carrying the leptinob/ob (ob) mutation. Measures are reported as the ratio of the mean log10 intensity (ml ratio). Regression line (black), r = linear regression, p = p-value.


Schematic overview of the sample collection procedure for single - cell PCR ( scPCR ) . Single IHC cytoplasms from acutely dissected organs of Corti of C57BL / 6J ( Wt ) mice after hearing onset were aspirated and processed for scPCR as depicted . ( B ' ) Expression analysis of endophilins - A1 - 3 from individually isolated IHC cytoplasms using RT - PCR from a representative experimental run . Please note that for these experiments , negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in bath solution . HPRT was used as a housekeeping gene .

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Schematic overview of the sample collection procedure for single-cell PCR (scPCR). Single IHC cytoplasms from acutely dissected organs of Corti of C57BL/6J (Wt) mice after hearing onset were aspirated and processed for scPCR as depicted. (B') Expression analysis of endophilins-A1-3 from individually isolated IHC cytoplasms using RT-PCR from a representative experimental run. Please note that for these experiments, negative bath control samples from before and after the isolation procedure were an essential requirement to ensure lack of contamination from cellular debris in bath solution. HPRT was used as a housekeeping gene.


Immunoblotting of tissue lysates from p15 Wt ( C57BL / 6J ) , 1 - SKO , 1 / 2 - DKO and 1 / 3 - DKO revealed protein expression of endophilins - A1 and - A2 in the murine cochlea and ensured antibody specificity . Unfortunately , none of the commercially available endophilin - A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts , but rather appeared to ( also ) detect A1 . γ - adaptin was used as loading control . All antibody epitopes localize to the distinct C - terminal regions of the different endophilin - A family members . Hi , hippocampus ; Ce , cerebellum ; Co , pooled cochleae from a single individual of the indicated genotype ; Md , modioli ( micro - dissected and pooled from 10 Wt animals ) ; OC , organs of Corti ( micro - dissected and pooled from 10 Wt animals ) .

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Immunoblotting of tissue lysates from p15 Wt (C57BL/6J), 1-SKO, 1/2-DKO and 1/3-DKO revealed protein expression of endophilins-A1 and -A2 in the murine cochlea and ensured antibody specificity. Unfortunately, none of the commercially available endophilin-A3 antibodies we tested in these experiments gave a specific signal for A3 in cochlear extracts, but rather appeared to (also) detect A1. γ-adaptin was used as loading control. All antibody epitopes localize to the distinct C-terminal regions of the different endophilin-A family members. Hi, hippocampus; Ce, cerebellum; Co, pooled cochleae from a single individual of the indicated genotype; Md, modioli (micro-dissected and pooled from 10 Wt animals); OC, organs of Corti (micro-dissected and pooled from 10 Wt animals).


Ca2 + - current - voltage relationships evoked by incremental 15 ms step depolarizations from - 87 mV to + 58 mV revealed a ~ 25 % reduction of ICa in 1 / 3 DKO and TKO mice . ( A ' ) Due to the perinatal lethality of TKO mice , detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically - cultured organs of Corti after 7 days in vitro ( DIV ) . C57BL / 6J ( Wt ) and 1 / 3 - DKO served as controls . Please note that all recordings from cultured IHCs were performed at [ Ca2 + ] e of 10 mM to maximize IHC exocytic performance . ( A " ) Quantification and statistical analysis of individual maximum ICa amplitudes ( Imax ) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes . ( * p = 0 . 046 , one - way ANOVA with post - hoc Tukey ' s ) .

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Ca2+-current-voltage relationships evoked by incremental 15 ms step depolarizations from -87 mV to +58 mV revealed a ~25% reduction of ICa in 1/3 DKO and TKO mice. (A') Due to the perinatal lethality of TKO mice, detailed electrophysiological characterization of TKO IHCs had to be performed on organotypically-cultured organs of Corti after 7 days in vitro (DIV). C57BL/6J (Wt) and 1/3-DKO served as controls. Please note that all recordings from cultured IHCs were performed at [Ca2+]e of 10 mM to maximize IHC exocytic performance. (A") Quantification and statistical analysis of individual maximum ICa amplitudes (Imax) of the respective genotypes revealed a significant reduction in Imax in both endophilin mutant genotypes. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).


Ca2 + - current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1 / 3 - DKO cells . ( B ' ) Quantification and statistical analysis of the residual current at the end of the test pulse . ( * p = 0 . 046 , one - way ANOVA with post - hoc Tukey ' s ) .

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Ca2+-current inactivation was probed by test pulses of 100 ms to the Imax potential and revealed a significantly stronger inactivation phenotype in TKO IHCs when directly compared to Wt and 1/3-DKO cells. (B') Quantification and statistical analysis of the residual current at the end of the test pulse. (*p = 0.046, one-way ANOVA with post-hoc Tukey's).