L MEFs were co ‐ transfected with FUNDC1 ‐ Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA . 24 h post ‐ transfection , cells were treated with hypoxic ( 1 % O2 ) conditions for 12 h or FCCP ( 20 μM ) for 6 h before fixed by 4 % paraformaldehyde and stained with indicated antibodies . Scale bar , 10 μm .

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

L MEFs were co‐transfected with FUNDC1‐Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA. 24 h post‐transfection, cells were treated with hypoxic (1% O2) conditions for 12 h or FCCP (20 μM) for 6 h before fixed by 4% paraformaldehyde and stained with indicated antibodies. Scale bar, 10 μm.


AULK1 + / + and ULK1 − / − cells were cultured under hypoxic ( 1 % O2 ) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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

AULK1+/+ and ULK1−/− cells were cultured under hypoxic (1% O2) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


B ULK1 + / + and ULK1 − / − cells were treated with FCCP ( 20 μM ) for the indicated times in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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

B ULK1+/+ and ULK1−/− cells were treated with FCCP (20 μM) for the indicated times in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


C ULK1 + / + and ULK1 − / − cells were transfected with FUNDC1 − Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

C ULK1+/+ and ULK1−/− cells were transfected with FUNDC1−Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


D ULK1 − / − cells were transfected with HA ‐ ULK1 or HA ‐ ULK1 ( K46N ) , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells lysates were immunoblotted .

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

D ULK1−/− cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N), then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells lysates were immunoblotted.


EULK1 − / − cells were transfected with HA − ULK1 or HA − ULK1 and then treated with FCCP ( 20 μM ) for 24 h . Cells were harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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

EULK1−/− cells were transfected with HA−ULK1 or HA−ULK1 and then treated with FCCP (20 μM) for 24 h. Cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


F FUNDC1 ‐ knockdown MEFs were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17A ) for the indicated times . Cell lysates were used for immunoblotting .

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

F FUNDC1‐knockdown MEFs were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17A) for the indicated times. Cell lysates were used for immunoblotting.


G ULK1 + / + cells were transfected with FUNDC1 ‐ Myc , and ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) . 24 h after transfection , cells were fixed with 4 % paraformaldehyde and stained with anti ‐ Myc ( mouse ) and anti ‐ LC3 ( rabbit ) primary antibodies for immunofluorescence microscopy . Scale bar , 10 μm .

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

G ULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D). 24 h after transfection, cells were fixed with 4% paraformaldehyde and stained with anti‐Myc (mouse) and anti‐LC3 (rabbit) primary antibodies for immunofluorescence microscopy. Scale bar, 10 μm.


H ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in the absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . Cell lysates were immunoblotted .

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

H ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were immunoblotted.


I ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in the absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . Cell lysates were detected with the citrate synthase activity kit .

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

I ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were detected with the citrate synthase activity kit.


J HeLa cells were transfected with FUNDC1 ‐ Myc , FUNDC1 - Myc ( S17A ) , or FUNDC1 - Myc ( S17D ) . 24 h after transfection , cell lysates were immunoprecipitated with anti ‐ LC3 antibody .

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

J HeLa cells were transfected with FUNDC1‐Myc, FUNDC1-Myc (S17A), or FUNDC1-Myc (S17D). 24 h after transfection, cell lysates were immunoprecipitated with anti‐LC3 antibody.


A ULK1 + / + or ULK1 − / − cells were cultured under hypoxic ( 1 % O2 ) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − cells were transfected with HA ‐ ULK1 , and ULK1 − / − FUNDC1 ‐ KD cells were transfected with HA ‐ ULK1 and FUNDC1 ‐ Myc , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells were harvested and lysed for immunoblotting assays .

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

A ULK1+/+ or ULK1−/− cells were cultured under hypoxic (1% O2) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1, and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc, then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.


B ULK1 + / + or ULK1 − / − cells were treated with or without FCCP ( 20 μM ) in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − cells were transfected with HA ‐ ULK1 and ULK1 − / − FUNDC1 ‐ KD cells were transfected with HA ‐ ULK1 and FUNDC1 ‐ Myc and then treated with FCCP ( 20 μM ) for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells were harvested and lysed for immunoblotting assays .

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

B ULK1+/+ or ULK1−/− cells were treated with or without FCCP (20 μM) in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1 and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc and then treated with FCCP (20 μM) for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.


C ULK1 + / + , ULK1 − / − FUNDC1 ‐ KD , or ULK1 − / − FUNDC1 ‐ KD cells rescued by introduction of HA ‐ ULK1 and FUNDC1 ‐ Myc were cultured under hypoxic ( 1 % O2 ) conditions for 24 h or with FCCP ( 20 μM ) for 24 h . Samples were analyzed by electron microscopy . Scale bar , 2 μm . The yellow asterisks mark mitochondria . The red arrows indicate double ‐ membraned autophagic structures . The yellow arrow denotes autolysosomes .

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

C ULK1+/+, ULK1−/− FUNDC1‐KD, or ULK1−/− FUNDC1‐KD cells rescued by introduction of HA‐ULK1 and FUNDC1‐Myc were cultured under hypoxic (1% O2) conditions for 24 h or with FCCP (20 μM) for 24 h. Samples were analyzed by electron microscopy. Scale bar, 2 μm. The yellow asterisks mark mitochondria. The red arrows indicate double‐membraned autophagic structures. The yellow arrow denotes autolysosomes.


D - E The mitochondrial coverage and mitochondrial − containing AVs shown in ( C ) were quantified . Recon , reconstitution .

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

D-E The mitochondrial coverage and mitochondrial−containing AVs shown in (C) were quantified. Recon, reconstitution.


A Representative immunofluorescence microscopy staining of GFP of small intestinal tissue of LGR5 ‐ GFP knock ‐ in ( LGR5 ‐ GFPki ) mice . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . B Quantification of GFP staining intensity of intestinal stem and progenitor cells ( ISPCs ) at indicated positions in basal crypts of LGR5 ‐ GFPkimice ( n = 80 crypts from four mice ) . Each dot presents one cell . Red lines : mean values ± SEM . Unpaired two ‐ tailed Student ' s t ‐ test .

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

A Representative immunofluorescence microscopy staining of GFP of small intestinal tissue of LGR5‐GFP knock‐in (LGR5‐GFPki) mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.B Quantification of GFP staining intensity of intestinal stem and progenitor cells (ISPCs) at indicated positions in basal crypts of LGR5‐GFPkimice (n = 80 crypts from four mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.


C Representative immunofluorescence microscopy staining of Msi1 of small intestinal tissue of young mTerc + / + mice . Dashed line outlines a crypt . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . D Quantification of Msi1 staining intensity of ISPCs at indicated positions in basal crypts of young mTerc + / + mice ( n = 150 crypts from three mice ) . Each dot presents one cell . Red lines : mean values ± SEM . Unpaired two ‐ tailed Student ' s t ‐ test .

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 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

C Representative immunofluorescence microscopy staining of Msi1 of small intestinal tissue of young mTerc+/+mice. Dashed line outlines a crypt. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.D Quantification of Msi1 staining intensity of ISPCs at indicated positions in basal crypts of young mTerc+/+mice (n = 150 crypts from three mice). Each dot presents one cell. Red lines: mean values ± SEM. Unpaired two‐tailed Student's t‐test.


E Representative FACS plots showing gating of LGR5hi and LGR5lo populations within the LGR5 + ( GFP ‐ positive ) cell gating .

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

E Representative FACS plots showing gating of LGR5hi and LGR5lo populations within the LGR5+ (GFP‐positive) cell gating.


F Representative FACS plots showing gating of LGR5hi ‐ high , LGR5hi ‐ low , LGR5lo ‐ high , and LGR5lo ‐ low populations within the LGR5hi gating .

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

F Representative FACS plots showing gating of LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations within the LGR5hi gating.


G - I mRNA expression of indicated genes relative to GAPDH in LGR5hi ‐ high , LGR5hi ‐ low , LGR5lo ‐ high , and LGR5lo ‐ low populations ( n = 3 mice ) . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ 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

G-I mRNA expression of indicated genes relative to GAPDH in LGR5hi‐high, LGR5hi‐low, LGR5lo‐high, and LGR5lo‐low populations (n = 3 mice). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A mRNA expression of indicated components of the canonical Wnt / β ‐ catenin pathway in freshly isolated small intestinal crypts of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 5 mice per group ) .

A mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway in freshly isolated small intestinal crypts of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 5 mice per group).


B Axin2 mRNA expression in LGR5 + cells of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and mTerc + / + mice ( n = 3 mice per group ) .

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

B Axin2 mRNA expression in LGR5+ cells of 12‐ to 16‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).


C mRNA expression of indicated components of the canonical Wnt / β ‐ catenin pathway and of the p53 target gene p21 in cultured crypts of 2 ‐ month ‐ old G3 mTerc − / − mice and mTerc + / + mice ( n = 3 mice per group ) .

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

C mRNA expression of indicated components of the canonical Wnt/β‐catenin pathway and of the p53 target gene p21 in cultured crypts of 2‐month‐old G3 mTerc−/−mice and mTerc+/+mice (n = 3 mice per group).


A - J Flow cytometry analysis of freshly isolated crypt cells from the small intestine of young ( 2 - 3 month ) and old ( 12 - 16 month ) LGR5 ‐ GFPki , mTerc + / + mice and LGR5 ‐ GFPki , G3 mTerc − / − mice ( n = 4 mice per group ) . ( A , B , D , E , H , I ) Representative FACS plots depicting the analysis of LGR5 + cells . Note the reduction in LGR5 + cells in G3 mTerc − / − mice with the remaining cells showing almost exclusively weak expression of GFP ( LGR5lo ) and that within the fraction of LGR5hi cells , the cells with high LGR5 ‐ reporter activity ( LGR5hi ‐ high ) are preferentially depleted in response to IR . ( C , F ) Quantification of ( C ) the number of LGR5 + cells and ( F ) the number of LGR5hi and LGR5lo cells . ( G ) Quantification of the percentage of LGR5hi cells and LGR5lo cells within the fraction of LGR5 + cells . ( J ) Quantification of the survival rate of LGR5hi ‐ high cells and LGR5hi ‐ low cells within the fraction of LGR5hi cells comparing old G3 mTerc − / − mice to mTerc + / + mice .

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

A-J Flow cytometry analysis of freshly isolated crypt cells from the small intestine of young (2-3 month) and old (12-16 month) LGR5‐GFPki, mTerc+/+mice and LGR5‐GFPki, G3 mTerc−/−mice (n = 4 mice per group). (A, B, D, E, H, I) Representative FACS plots depicting the analysis of LGR5+ cells. Note the reduction in LGR5+ cells in G3 mTerc−/−mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo) and that within the fraction of LGR5hi cells, the cells with high LGR5‐reporter activity (LGR5hi‐high) are preferentially depleted in response to IR. (C, F) Quantification of (C) the number of LGR5+ cells and (F) the number of LGR5hi and LGR5lo cells. (G) Quantification of the percentage of LGR5hi cells and LGR5lo cells within the fraction of LGR5+ cells. (J) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing old G3 mTerc−/−mice to mTerc+/+mice.


K - O Immunofluorescence microscopy staining of GFP in basal crypts of 9 ‐ month ‐ old LGR5 ‐ GFPki , G3 mTerc − / − mice and age ‐ matched LGR5 ‐ GFPki , mTerc + / + mice ( n = 2 mice per group ) . ( K , L ) Representative pictures are given . Dashed lines outline the crypts . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . ( M ) Quantification of absolute number of GFP + ISPCs at indicated positions at the crypt base per 50 crypts . ( N ) The histogram depicts the depletion rate ( % ) of LGR5 + cells in LGR5 ‐ GFPki , G3 mTerc − / − mice compared to age ‐ matched LGR5 ‐ GFPki , mTerc + / + mice at indicated positions in the basal crypts . ( O ) Quantification of GFP staining intensity of ISPCs at indicated positions in positively stained basal crypts . Note that the staining intensity of position 4 cells is equal in mTerc + / + mice and G3 mTerc − / − mice and is significantly lower than position 1 and 2 cells of mTerc + / + mice .

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

K-O Immunofluorescence microscopy staining of GFP in basal crypts of 9‐month‐old LGR5‐GFPki, G3 mTerc−/−mice and age‐matched LGR5‐GFPki, mTerc+/+mice (n = 2 mice per group). (K, L) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (M) Quantification of absolute number of GFP+ISPCs at indicated positions at the crypt base per 50 crypts. (N) The histogram depicts the depletion rate (%) of LGR5+ cells in LGR5‐GFPki, G3 mTerc−/−mice compared to age‐matched LGR5‐GFPki, mTerc+/+mice at indicated positions in the basal crypts. (O) Quantification of GFP staining intensity of ISPCs at indicated positions in positively stained basal crypts. Note that the staining intensity of position 4 cells is equal in mTerc+/+mice and G3 mTerc−/−mice and is significantly lower than position 1 and 2 cells of mTerc+/+mice.


A - I Staining of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( A , B ) H & amp ; amp ; E staining , ( C , F , I ) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of ( C ) spindle ‐ shaped cells in between and adjacent to the Paneth cells . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ 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 O O O O O O O O O O O O O O O O O O O O O O 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-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of (A, B) H&amp;E staining, (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (C) spindle‐shaped cells in between and adjacent to the Paneth cells. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - I Staining of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( D , E ) PCNA staining . Arrowheads and numbers indicate the position of ISPCs in the basal crypts . Scale bars : 20 μm . ( C , F , I ) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of cells staining positive for ( F ) PCNA . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/−mice and age‐matched mTerc+/+mice (n = 4-5 mice per group). Representative pictures of (D, E) PCNA staining. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of cells staining positive for (F) PCNA . Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - I Staining of small intestinal tissue sections of 12 ‐ to 16 ‐ month ‐ old G3 mTerc − / − mice and age ‐ matched mTerc + / + mice ( n = 4 - 5 mice per group ) . Representative pictures of ( ( G , H ) Msi1 staining are given . Arrowheads and numbers indicate the position of ISPCs in the basal crypts . Scale bars : 20 μm . ( C , F , I ) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of ( I ) Msi1 . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

A-I Staining of small intestinal tissue sections of 12‐ to 16‐month‐old G3 mTerc−/− mice and age‐matched mTerc+/+ mice (n = 4-5 mice per group). Representative pictures of ((G, H) Msi1 staining are given. Arrowheads and numbers indicate the position of ISPCs in the basal crypts. Scale bars: 20 μm. (C, F, I) Quantification of the absolute numbers of ISPCs at the indicated positions in the crypt base per 50 crypts as determined by counting of (I) Msi1. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


J , K Representative pictures of Olfm4 in situ hybridization on small intestinal sections of 9 ‐ month ‐ old G3 mTerc − / − mice ( K ) and age ‐ matched mTerc + / + ( J ) mice . Arrowheads point to positive cells . Dashed lines outline the crypts . Note that the remaining ISPCs in G3 mTerc − / − mice were mostly located in position 4 above the Paneth cells . Scale bar : 20 μm .

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 O O O

J, K Representative pictures of Olfm4 in situ hybridization on small intestinal sections of 9‐month‐old G3 mTerc−/− mice (K) and age‐matched mTerc+/+ (J) mice. Arrowheads point to positive cells. Dashed lines outline the crypts. Note that the remaining ISPCs in G3 mTerc−/− mice were mostly located in position 4 above the Paneth cells. Scale bar: 20 μm.


A - I Three ‐ month ‐ old mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR ( n = 5 mice per group ) . ( A - F ) Representative pictures of PCNA staining . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . Note the depletion of PCNA + CBC cells in position 1 and 2 in between the Paneth cells at 3 - 48 h after IR and the regeneration of these cells at 4 - 6 days after IR . ( G ) Quantification of PCNA + ISPCs at the indicated positions in basal crypts at the indicated time points after IR . Mean values ± SEM are given .

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

A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (A-F) Representative pictures of PCNA staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. Note the depletion of PCNA+ CBC cells in position 1 and 2 in between the Paneth cells at 3-48 h after IR and the regeneration of these cells at 4-6 days after IR. (G) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts at the indicated time points after IR. Mean values ± SEM are given.


A - I Three ‐ month ‐ old mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR ( n = 5 mice per group ) . ( H , I ) Representative pictures of Olfm4 in situ hybridization . Arrowheads point to positive cells . Dashed lines outline the crypts in irradiated samples . Scale bar : 20 μm . Note the selective survival of ISPCs above the Paneth cells at 24 h after IR .

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

A-I Three‐month‐old mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR (n = 5 mice per group). (H, I) Representative pictures of Olfm4 in situ hybridization. Arrowheads point to positive cells. Dashed lines outline the crypts in irradiated samples. Scale bar: 20 μm. Note the selective survival of ISPCs above the Paneth cells at 24 h after IR.


J - X Three ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR . ( J - N ) Representative pictures of GFP staining at indicated time points after irradiation . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm .

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J-X Three‐month‐old LGR5‐GFPki, mTerc+/+ mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (J-N) Representative pictures of GFP staining at indicated time points after irradiation. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm.


J - X Three ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were exposed to 12 Gy γ ‐ irradiation . Small intestinal tissue was collected at indicated time points after IR . ( O - X ) Flow cytometry analysis of freshly isolated basal crypt cells at the indicated time points after IR ( n = 4 - 10 mice per group ) . ( O ) The histogram depicts the number of LGR5 + cells in freshly isolated basal crypts at the indicated time point after IR . ( P , Q ) Flow cytometry analysis was used to determine the percentage of ( P ) LGR5hi cells and ( Q ) LGR5lo cells within the fraction of LGR5 + cells . ( R ‐ W ) Representative FACS plots of small intestinal crypt cells of non ‐ irradiated mice , 24 h after irradiation . Note the reduction in LGR5 + cells in irradiated mice with the remaining cells showing almost exclusively weak expression of GFP ( LGR5lo ) . ( X ) Quantification of the survival rate of LGR5hi ‐ high cells and LGR5hi ‐ low cells within the fraction of LGR5hi cells comparing irradiated mice to non ‐ irradiated mice . Note the preferential depletion of LGR5hi ‐ high cells within LGR5hi cell population upon IR . Mean values ± SEM are given . NIR , non ‐ irradiated ; IR , irradiated .

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

J-X Three‐month‐old LGR5‐GFPki, mTerc+/+mice were exposed to 12 Gy γ‐irradiation. Small intestinal tissue was collected at indicated time points after IR. (O-X) Flow cytometry analysis of freshly isolated basal crypt cells at the indicated time points after IR (n = 4-10 mice per group). (O) The histogram depicts the number of LGR5+ cells in freshly isolated basal crypts at the indicated time point after IR. (P, Q) Flow cytometry analysis was used to determine the percentage of (P) LGR5hi cells and (Q) LGR5lo cells within the fraction of LGR5+ cells. (R‐W) Representative FACS plots of small intestinal crypt cells of non‐irradiated mice, 24 h after irradiation. Note the reduction in LGR5+ cells in irradiated mice with the remaining cells showing almost exclusively weak expression of GFP (LGR5lo). (X) Quantification of the survival rate of LGR5hi‐high cells and LGR5hi‐low cells within the fraction of LGR5hi cells comparing irradiated mice to non‐irradiated mice. Note the preferential depletion of LGR5hi‐high cells within LGR5hi cell population upon IR. Mean values ± SEM are given. NIR, non‐irradiated; IR, irradiated.


B , C LGR5hi and LGR5lo cells were freshly isolated from 3 ‐ month ‐ old LGR5 ‐ GFPki mice 6 h after 12 Gy γ ‐ irradiation ( IR ) or from non ‐ irradiated ( NIR ) mice ( n = 3 mice per group ) . ( B ) qPCR analysis of relative mRNA expression of apoptosis ‐ related genes compared to GAPDH . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ 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 O O O O O O

B, C LGR5hi and LGR5lo cells were freshly isolated from 3‐month‐old LGR5‐GFPki mice 6 h after 12 Gy γ‐irradiation (IR) or from non‐irradiated (NIR) mice (n = 3 mice per group). (B) qPCR analysis of relative mRNA expression of apoptosis‐related genes compared to GAPDH. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


B , C LGR5hi and LGR5lo cells were freshly isolated from 3 ‐ month ‐ old LGR5 ‐ GFPki mice 6 h after 12 Gy γ ‐ irradiation ( IR ) or from non ‐ irradiated ( NIR ) mice ( n = 3 mice per group ) . ( C ) Representative Western blot analysis of cell lysates for the expression of phospho ‐ p53 and cleaved caspase ‐ 3 .

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B, C LGR5hi and LGR5lo cells were freshly isolated from 3‐month‐old LGR5‐GFPki mice 6 h after 12 Gy γ‐irradiation (IR) or from non‐irradiated (NIR) mice (n = 3 mice per group). (C) Representative Western blot analysis of cell lysates for the expression of phospho‐p53 and cleaved caspase‐3.


D , E Three ‐ month ‐ old mTerc + / + mice were γ ‐ irradiated with 12 Gy . Small intestinal tissue was collected at 3 h after IR ( n = 3 mice per group ) . ( D ) Representative picture of TUNEL staining . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . ( E ) Percentage of TUNEL ‐ positive ISPCs at indicated positions in basal crypts . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ 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 O O O O O O O O O O O O O O O O O O O O

D, E Three‐month‐old mTerc+/+ mice were γ‐irradiated with 12 Gy. Small intestinal tissue was collected at 3 h after IR (n = 3 mice per group). (D) Representative picture of TUNEL staining. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (E) Percentage of TUNEL‐positive ISPCs at indicated positions in basal crypts. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


F - P Two ‐ month ‐ old LGR5 ‐ GFPki , p53 + / + mice and LGR5 ‐ GFPki , p53 − / − mice were exposed to 12 Gy γ ‐ irradiation ( n = 4 mice per group ) . Basal crypts were isolated at 24 h after IR . ( F - H ) Flow cytometry analysis of the survival rate of LGR5 + cells ( F ) , LGR5hi and LGR5lo cells ( G ) , and LGR5hi ‐ high and LGR5hi ‐ low cells ( H ) of irradiated mice ( 24 h after IR ) compared to non ‐ irradiated mice ( NIR ) . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test . ( I - P ) Representative FACS plots of LGR5 + cells in freshly isolated basal crypts from irradiated and non ‐ irradiated mice of indicated genotypes . Note the stronger rescue of survival rate of LGR5hi ‐ high than the LGR5hi ‐ low cells upon p53 deletion . NIR , non ‐ irradiated ; IR , irradiated .

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

F-P Two‐month‐old LGR5‐GFPki, p53+/+ mice and LGR5‐GFPki, p53−/− mice were exposed to 12 Gy γ‐irradiation (n = 4 mice per group). Basal crypts were isolated at 24 h after IR. (F-H) Flow cytometry analysis of the survival rate of LGR5+ cells (F), LGR5hi and LGR5lo cells (G), and LGR5hi‐high and LGR5hi‐low cells (H) of irradiated mice (24 h after IR) compared to non‐irradiated mice (NIR). Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test. (I-P) Representative FACS plots of LGR5+ cells in freshly isolated basal crypts from irradiated and non‐irradiated mice of indicated genotypes. Note the stronger rescue of survival rate of LGR5hi‐high than the LGR5hi‐low cells upon p53 deletion. NIR, non‐irradiated; IR, irradiated.


Crypts of 2 ‐ to 3 ‐ month ‐ old LGR5ki mice were cultured and γ ‐ irradiated with 6 Gy on day 10 of culture . Sixteen hours before irradiation , cells were exposed to the indicated treatment ( n = 4 independent experiments per group ) . A - J Representative FACS plots of indicated treatments are given . NIR , non ‐ irradiated ; IR , irradiated . K - P Flow cytometry analysis of the percentage of LGR5 + , LGR5hi , and LGR5lo cells of irradiated cultures ( 24 h after IR ) compared to the percentage in non ‐ irradiated controls ( NIR ) set to 100 % . ( K - M ) Note that inhibition of Wnt signaling by DKK1 treatment or R ‐ spondin reduction ( 50 % Rspo ) partially rescued the decrease in LGR5 + cells in irradiated cultures . ( N - P ) Note that activation of Wnt signaling by 6 ‐ BIO treatment aggravated the decrease in LGR5 + cells in irradiated cultures . Mean values ± SEM are given . Unpaired two ‐ tailed Student ' s t ‐ test .

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Crypts of 2‐ to 3‐month‐old LGR5ki mice were cultured and γ‐irradiated with 6 Gy on day 10 of culture. Sixteen hours before irradiation, cells were exposed to the indicated treatment (n = 4 independent experiments per group).A-J Representative FACS plots of indicated treatments are given. NIR, non‐irradiated; IR, irradiated.K-P Flow cytometry analysis of the percentage of LGR5+, LGR5hi, and LGR5lo cells of irradiated cultures (24 h after IR) compared to the percentage in non‐irradiated controls (NIR) set to 100%. (K-M) Note that inhibition of Wnt signaling by DKK1 treatment or R‐spondin reduction (50% Rspo) partially rescued the decrease in LGR5+ cells in irradiated cultures. (N-P) Note that activation of Wnt signaling by 6‐BIO treatment aggravated the decrease in LGR5+ cells in irradiated cultures. Mean values ± SEM are given. Unpaired two‐tailed Student's t‐test.


A - G Two ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were i . v . injected with LRP6 neutralization antibody , and IgG serves as a negative control . Thirty ‐ six hours after injection , small intestinal tissue was collected for GFPimmunofluorescence microscopy staining . ( A - F ) Representative pictures are given . Dashed lines outline the crypts . Arrowheads and numbers indicate cell positions in the crypts . Scale bar : 20 μm . ( G ) Quantification of GFP staining intensity of cells at indicated positions in basal crypts ( n = 3 mice per group , fifty positively stained cells were measured per mouse ) . Note the GFP staining intensity was substantially reduced in anti ‐ LRP6 antibody ‐ injected group with intact maintenance of ISPCs at the crypt bottom as shown by DAPI staining .

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A-G Two‐month‐old LGR5‐GFPki, mTerc+/+mice were i.v. injected with LRP6 neutralization antibody, and IgG serves as a negative control. Thirty‐six hours after injection, small intestinal tissue was collected for GFPimmunofluorescence microscopy staining. (A-F) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm. (G) Quantification of GFP staining intensity of cells at indicated positions in basal crypts (n = 3 mice per group, fifty positively stained cells were measured per mouse). Note the GFP staining intensity was substantially reduced in anti‐LRP6 antibody‐injected group with intact maintenance of ISPCs at the crypt bottom as shown by DAPI staining.


H - L Two ‐ month ‐ old mTerc + / + mice were i . v . injected with anti ‐ LRP6 antibody 12 h before γ ‐ irradiation . Small intestinal tissue was collected at 24 h after IR ( n = 3 - 4 mice per group ) . IgG serves as a negative control . ( H - K ) Representative pictures of PCNA staining of small intestine of non ‐ irradiated mice ( H , I ) or 6 Gy ‐ irradiated mice ( J , K ) . Note the anti ‐ LRP6 antibody injection did not induce loss of CBC cells at position 1 and 2 in non ‐ irradiated mice . Arrowheads and numbers indicate ISPC positions in the crypts . Scale bar : 20 μm . ( L ) Quantification of PCNA + ISPCs at the indicated positions in basal crypts 24 h after IR with indicated doses or non ‐ irradiated mice .

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H-L Two‐month‐old mTerc+/+ mice were i.v. injected with anti‐LRP6 antibody 12 h before γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 3-4 mice per group). IgG serves as a negative control. (H-K) Representative pictures of PCNA staining of small intestine of non‐irradiated mice (H, I) or 6 Gy‐irradiated mice (J, K). Note the anti‐LRP6 antibody injection did not induce loss of CBC cells at position 1 and 2 in non‐irradiated mice. Arrowheads and numbers indicate ISPC positions in the crypts. Scale bar: 20 μm. (L) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts 24 h after IR with indicated doses or non‐irradiated mice.


M - R Two ‐ month ‐ old LGR5 ‐ GFPki , mTerc + / + mice were i . v . injected with LRP6 neutralization antibody 12 h before 12 Gy γ ‐ irradiation . IgG serves as a negative control . Small intestinal tissue was collected at 24 h after IR ( n = 4 mice per group ) . ( M - P ) Representative FACS plots of indicated treatments . ( Q ) Flow cytometry analysis of the number of LGR5 + ISPCs of irradiated mice compared to the number in non ‐ irradiated mice ( NIR ) set to 100 % . ( R ) Quantification of the number of LGR5 + , LGR5hi , and LGR5lo cells of mice with indicated treatment by flow cytometry . Note that the total LGR5 + cell frequency was not significantly different between anti ‐ LRP6 antibody ‐ injected and control IgG ‐ injected mice , though the LGR5hi fraction was reduced in the LRP6 antibody ‐ injected group .

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M-R Two‐month‐old LGR5‐GFPki, mTerc+/+ mice were i.v. injected with LRP6 neutralization antibody 12 h before 12 Gy γ‐irradiation. IgG serves as a negative control. Small intestinal tissue was collected at 24 h after IR (n = 4 mice per group). (M-P) Representative FACS plots of indicated treatments. (Q) Flow cytometry analysis of the number of LGR5+ ISPCs of irradiated mice compared to the number in non‐irradiated mice (NIR) set to 100%. (R) Quantification of the number of LGR5+, LGR5hi, and LGR5lo cells of mice with indicated treatment by flow cytometry. Note that the total LGR5+cell frequency was not significantly different between anti‐LRP6 antibody‐injected and control IgG‐injected mice, though the LGR5hi fraction was reduced in the LRP6 antibody‐injected group.


A - C GFPimmunofluorescence microscopy staining of small intestine from 2 ‐ month ‐ old LGR5 ‐ GFPki , APC + / + mice and LGR5 ‐ GFPki , APCmin / + mice . ( A ) Quantification of GFP staining intensity of cells at indicated positions in basal crypts ( n = 3 mice per group , 50 positively stained cells were measured per mouse ) . Note the GFP staining intensity was increased in APCmin / + mice , particularly in position 4 and 5 cells . ( B , C ) Representative pictures are given . Dashed lines outline the crypts . Arrowheads and numbers indicate cell positions in the crypts . Scale bar : 20 μm .

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A-C GFPimmunofluorescence microscopy staining of small intestine from 2‐month‐old LGR5‐GFPki, APC+/+ mice and LGR5‐GFPki, APCmin/+mice. (A) Quantification of GFP staining intensity of cells at indicated positions in basal crypts (n = 3 mice per group, 50 positively stained cells were measured per mouse). Note the GFP staining intensity was increased in APCmin/+ mice, particularly in position 4 and 5 cells. (B, C) Representative pictures are given. Dashed lines outline the crypts. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm.


D - F Two ‐ month ‐ old APC + / + mice and APCmin / + mice were exposed to 6 Gy γ ‐ irradiation . Small intestinal tissue was collected at 24 h after IR ( n = 3 mice per group ) . ( D ) Quantification of PCNA + ISPCs at the indicated positions in basal crypts 24 h after IR . IR , irradiated . ( E , F ) Representative pictures of PCNA staining are given . Arrowheads and numbers indicate cell positions in the crypts . Scale bar : 20 μm .

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D-F Two‐month‐old APC+/+ mice and APCmin/+ mice were exposed to 6 Gy γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 3 mice per group). (D) Quantification of PCNA+ ISPCs at the indicated positions in basal crypts 24 h after IR. IR, irradiated. (E, F) Representative pictures of PCNA staining are given. Arrowheads and numbers indicate cell positions in the crypts. Scale bar: 20 μm.


G - K Two ‐ month ‐ old LGR5 ‐ GFPki , APC + / + mice and LGR5 ‐ GFPki , APCmin / + mice were exposed to 6 Gy γ ‐ irradiation . Small intestinal tissue was collected at 24 h after IR ( n = 4 mice per group ) . ( G ) Flow cytometry analysis of the number of LGR5 + ISPCs of irradiated mice compared to the number in non ‐ irradiated mice ( NIR ) set to 100 % . ( H - K ) Representative FACS plots of indicated groups are given .

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G-K Two‐month‐old LGR5‐GFPki, APC+/+ mice and LGR5‐GFPki, APCmin/+ mice were exposed to 6 Gy γ‐irradiation. Small intestinal tissue was collected at 24 h after IR (n = 4 mice per group). (G) Flow cytometry analysis of the number of LGR5+ ISPCs of irradiated mice compared to the number in non‐irradiated mice (NIR) set to 100%. (H-K) Representative FACS plots of indicated groups are given.


B Dose response curves for viability of parental cells ( in red ) or selected clones ( in black ) upon treatment with NFR for 48h . The green dashed box highlight concentration in the physiologically relevant range . Curves are mean ± s . e . m . of 3 independent experiments performed in triplicate . P values were calculated using 2way ANOVA analysis of variance between parental cells and each individual clone . Bar graph represents the EC50 ( half maximal effective concentration ) of the dose responses . Data are mean ± s . e . m . of 3 independent experiments performed in triplicate . P values were calculated using 1way ANOVA analysis of variance ( P value in red ) followed by Dunnett ' s multiple comparison tests . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Dose response curves for viability of parental cells (in red) or selected clones (in black) upon treatment with NFR for 48h. The green dashed box highlight concentration in the physiologically relevant range. Curves are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 2way ANOVA analysis of variance between parental cells and each individual clone. Bar graph represents the EC50 (half maximal effective concentration) of the dose responses. Data are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 1way ANOVA analysis of variance (P value in red) followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01.


C The parental population and the NFR resistant clones were analyzed for eEF2K mRNA expression by real - time PCR relative to β - actin ( mean and s . e . m . from 3 independent batches of cells are shown ) . P values were calculated using 1way ANOVA analysis of variance followed by Dunnett ' s multiple comparison tests . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 .

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 O O O O

C The parental population and the NFR resistant clones were analyzed for eEF2K mRNA expression by real-time PCR relative to β-actin (mean and s.e.m. from 3 independent batches of cells are shown). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001.


D The parental population and representative NFR resistant clones were analyzed by WB for eFF2K total protein level . Tubulin is used as loading control . Histogram represents mean and s . e . m of relative eEF2K level quantified from WB of 4 different batches of cells collected at different dates ( see also source data ) . P values were calculated using 1way ANOVA analysis of variance followed by Dunnett ' s multiple comparison tests . * * * P value ≤ 0 . 001 .

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 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

D The parental population and representative NFR resistant clones were analyzed by WB for eFF2K total protein level. Tubulin is used as loading control. Histogram represents mean and s.e.m of relative eEF2K level quantified from WB of 4 different batches of cells collected at different dates (see also source data). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *** P value ≤ 0.001.


A Immunoblot analysis of eEF2 Thr56 phosphorylation ( P - eEF2 ) in HeLa cells treated for 6h with increasing doses of NFR , and compared with 10 μg / ml tunicamycin ( TM ) , 200 nM rapamycin ( Rapa . ) or 1h starvation ( Starv . ) . Tubulin is used as loading control .

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

A Immunoblot analysis of eEF2 Thr56 phosphorylation (P-eEF2) in HeLa cells treated for 6h with increasing doses of NFR, and compared with 10 μg/ml tunicamycin (TM), 200 nM rapamycin (Rapa.) or 1h starvation (Starv.). Tubulin is used as loading control.


B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR , 200 nM rapamycin ( Rapa . ) , 1h starvation ( Starv . ) or 1 mM of the AMPK activator AICAR , were analyzed by immunoblot with antibodies directed against total or phosphorylated eEF2 ( Thr56 ) .

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

B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, 200 nM rapamycin (Rapa.), 1h starvation (Starv.) or 1 mM of the AMPK activator AICAR, were analyzed by immunoblot with antibodies directed against total or phosphorylated eEF2 (Thr56).


C Three representative NFR - resistant HeLa clones were analyzed by Immunoblot for eEF2K expression level and eEF2 phosphorylation upon NFR and compared to parental HeLa cells . For treatments , medium was replaced for 6h with medium containing DMSO ( Mock ) , increasing doses of NFR or 10 μg / ml tunicamycin ( TM ) , or for 1h with PBS for starvation ( Starv . ) . Tubulin is used as loading control .

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

C Three representative NFR-resistant HeLa clones were analyzed by Immunoblot for eEF2K expression level and eEF2 phosphorylation upon NFR and compared to parental HeLa cells. For treatments, medium was replaced for 6h with medium containing DMSO (Mock), increasing doses of NFR or 10 μg/ml tunicamycin (TM), or for 1h with PBS for starvation (Starv.). Tubulin is used as loading control.


D Immunoblot analysis of eEF2 Thr56 phosphorylation in HeLa cells treated for 6h with increasing doses of different HIV - PIs as indicated or the NFR metabolite M8 ( hydroxy - tert - butylamide ) . Molecular structures of the different HIV - PIs used are indicated . Each panel is representative of at least 3 independent experiments .

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 B-CONTROLLED_VAR 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 O O O

D Immunoblot analysis of eEF2 Thr56 phosphorylation in HeLa cells treated for 6h with increasing doses of different HIV-PIs as indicated or the NFR metabolite M8 (hydroxy-tert-butylamide). Molecular structures of the different HIV-PIs used are indicated. Each panel is representative of at least 3 independent experiments.


B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR , Rapamycin ( Rapa ) , AICAR or starved for indicated time in PBS ( Starv . ) , were analyzed by immunoblot using indicated antibodies . Tubulin is used as loading control .

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

B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for indicated time in PBS (Starv.), were analyzed by immunoblot using indicated antibodies. Tubulin is used as loading control.


C A potent mTOR inhibition does not impair NFR - mediated eEF2 phosphorylation . WT MEFs were treated with the indicated concentrations of Rapamycin or with vehicle ( Mock ) . After 30 minutes , indicated doses of NFR were added and cells were incubated for additional 6h and analyzed for phosphorylated S6R and eEF2 . Tubulin is used as loading control .

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

C A potent mTOR inhibition does not impair NFR-mediated eEF2 phosphorylation. WT MEFs were treated with the indicated concentrations of Rapamycin or with vehicle (Mock). After 30 minutes, indicated doses of NFR were added and cells were incubated for additional 6h and analyzed for phosphorylated S6R and eEF2. Tubulin is used as loading control.


D NFR - mediated eEF2 phosphorylation is not affected in AMPKα1α2 dKO . AMPKα1α2 WT and dKO MEF treated for 6h with indicated concentration of NFR , Rapamycin ( Rapa ) , AICAR or starved for 1h in PBS ( Starv . ) , were analyzed by immuoblot with indicated antibodies . * anti - total AMPKα antibody give an unspecific band with a slightly higher molecular weight in dKO cells . Tubulin is used as loading control . Each panel is representative of at least 3 independent experiments .

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

D NFR-mediated eEF2 phosphorylation is not affected in AMPKα1α2 dKO. AMPKα1α2 WT and dKO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for 1h in PBS (Starv.), were analyzed by immuoblot with indicated antibodies. * anti-total AMPKα antibody give an unspecific band with a slightly higher molecular weight in dKO cells. Tubulin is used as loading control. Each panel is representative of at least 3 independent experiments.


A - B EEF2K contributes to the decreased translation observed with NFR without impacting translation in presence of TM . A . Quantification of newly synthesized proteins at 0 , 2 , 4 and 6h after 20 μM NFR ( left panels ) or 10 μg / ml TM ( right panels ) treatment in MEF eEF2K + / + and eEF2K - / - . Treated cells were labeled for 15 minutes with 35S - methionine and visualized by SDS PAGE and subsequent autoradiography . Autoradiographies from four different experiments ( see also source data ) were quantified and results show percentage of translation compared to untreated cells . The mean and s . e . m of four independent metabolic labeling experiments are shown . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 obtained using 2way ANOVA analysis of variance ( in red ) followed by Bonferroni posttest ( in black ) . B . eEF2K + / + , eEF2K - / - , eIF2αWT and eIF2αS51A MEFs were treated for 6h with indicated doses of NFR , 200 nM rapamycin , or 10 μg / ml TM , or for 30 min with 10 μg / ml CHX . 35S - methionine incorporation was measured by liquid scintillation counting . Data are shown as the percentage of translation compared to untreated cells . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 obtained using 2way ANOVA analysis of variance followed by Bonferroni posttest .

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

A-B EEF2K contributes to the decreased translation observed with NFR without impacting translation in presence of TM. A. Quantification of newly synthesized proteins at 0, 2, 4 and 6h after 20 μM NFR (left panels) or 10 μg/ml TM (right panels) treatment in MEF eEF2K +/+ and eEF2K-/-. Treated cells were labeled for 15 minutes with 35S-methionine and visualized by SDS PAGE and subsequent autoradiography. Autoradiographies from four different experiments (see also source data) were quantified and results show percentage of translation compared to untreated cells. The mean and s.e.m of four independent metabolic labeling experiments are shown. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001 obtained using 2way ANOVA analysis of variance (in red) followed by Bonferroni posttest (in black). B. eEF2K+/+, eEF2K-/-, eIF2αWT and eIF2αS51A MEFs were treated for 6h with indicated doses of NFR, 200 nM rapamycin, or 10 μg/ml TM, or for 30 min with 10 μg/ml CHX. 35S-methionine incorporation was measured by liquid scintillation counting. Data are shown as the percentage of translation compared to untreated cells. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001 obtained using 2way ANOVA analysis of variance followed by Bonferroni posttest.


C Representative polysome profiles of eEF2K WT and KO MEF treated 6h with NFR or TM . Area under curve for Sub - polysomes ( S ) and Polysomes ( P ) used to calculate the P / S ratio were indicated ( see also raw data provided ) . Bar graph represents ratio normalized to untreated cells . OD254 nm is optical density at 254 nm . Data showed mean ± s . e . m . of P / S ratio calculated from 3 independent experiments . P values were calculated using 1way ANOVA analysis of variance followed by Bonferroni posttest ; * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 .

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

C Representative polysome profiles of eEF2K WT and KO MEF treated 6h with NFR or TM. Area under curve for Sub-polysomes (S) and Polysomes (P) used to calculate the P/S ratio were indicated (see also raw data provided). Bar graph represents ratio normalized to untreated cells. OD254 nm is optical density at 254 nm. Data showed mean ±s.e.m. of P/S ratio calculated from 3 independent experiments. P values were calculated using 1way ANOVA analysis of variance followed by Bonferroni posttest; *P value ≤ 0.05, ** P value ≤ 0.01.


D The ribosome half - transit time in MEFs eEF2K + / + and eEF2K - / - was determined as described in Materials and Methods . Incorporation of 35S - Methionine into total protein within the PMS and PRS was obtained by linear regression analysis . Presented graphs are representative of two ( CHX 1 μg / ml for 30 min and Starvation 30 min in PBS in eEF2K + / + cells ) to four ( NFR 20μM for 6h in eEF2K + / + and eEF2K - / - cells ) independent experiments . Indicated values represent the x displacement measurement ( in time ) between the PMS line at 300sec and the PRS line ( see also raw data provided ) . Histogram represents mean and s . e . m . of the ribosome half transit time from four independent experiments . P values were calculated using two tails unpaired Student ' s T - tests ; * P value ≤ 0 . 05 .

O O O O O O O O O B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ribosome half-transit time in MEFs eEF2K+/+ and eEF2K-/- was determined as described in Materials and Methods. Incorporation of 35S-Methionine into total protein within the PMS and PRS was obtained by linear regression analysis. Presented graphs are representative of two (CHX 1 μg/ml for 30 min and Starvation 30 min in PBS in eEF2K+/+ cells) to four (NFR 20μM for 6h in eEF2K+/+ and eEF2K-/- cells) independent experiments. Indicated values represent the x displacement measurement (in time) between the PMS line at 300sec and the PRS line (see also raw data provided). Histogram represents mean and s.e.m. of the ribosome half transit time from four independent experiments. P values were calculated using two tails unpaired Student's T-tests; *P value ≤ 0.05.


A MEF eEF2K WT , KO or KO reconstituted with human eEF2K ( Flag - eEF2K ) were analyzed for cell growth at indicated times . Fold change ( means ± s . e . m of triplicate ) of the cell number just before treatment are shown . Histogram shows percentage of growth inhibition after 72h of 10 μM NFR . P values are 1way ANOVA with Bonferroni posttest calculated from 3 independent experiments . * * * P value ≤ 0 . 001 .

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

A MEF eEF2K WT, KO or KO reconstituted with human eEF2K (Flag-eEF2K) were analyzed for cell growth at indicated times. Fold change (means ± s.e.m of triplicate) of the cell number just before treatment are shown. Histogram shows percentage of growth inhibition after 72h of 10 μM NFR. P values are 1way ANOVA with Bonferroni posttest calculated from 3 independent experiments. *** P value ≤ 0.001.


B - F Dose response curves for cell viability after 48h NFR measured using MTS assay . Curves and bar graph for EC50 are mean ± s . e . m . of 3 independent experiments performed in triplicate . For curves , p values are 2way ANOVA analysis of variance . For bar graph , p values are two tails unpaired Student ' s T - tests ( B , E ) , or 1way ANOVA with Bonferroni ( C ) or Dunnett ' s multiple comparison posttests ( D and F ) . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 . ( B , C ) eEF2K - / - MEFs show a decrease susceptibly to NFR compared to eEF2K + / + controls ( B ) whereas eEF2K reconstitution restores NFR sensitivity ( C ) .

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

B-F Dose response curves for cell viability after 48h NFR measured using MTS assay. Curves and bar graph for EC50 are mean ± s.e.m. of 3 independent experiments performed in triplicate. For curves, p values are 2way ANOVA analysis of variance. For bar graph, p values are two tails unpaired Student's T-tests (B, E), or 1way ANOVA with Bonferroni (C) or Dunnett's multiple comparison posttests (D and F). *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. (B,C) eEF2K -/- MEFs show a decrease susceptibly to NFR compared to eEF2K +/+ controls (B) whereas eEF2K reconstitution restores NFR sensitivity (C).


B - F Dose response curves for cell viability after 48h NFR measured using MTS assay . Curves and bar graph for EC50 are mean ± s . e . m . of 3 independent experiments performed in triplicate . For curves , p values are 2way ANOVA analysis of variance . For bar graph , p values are two tails unpaired Student ' s T - tests ( B , E ) , or 1way ANOVA with Bonferroni ( C ) or Dunnett ' s multiple comparison posttests ( D and F ) . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 . ( D - F ) , HeLa ( D ) , A549 ( E ) and MCF7 ( F ) clones with CRISPR - Cas9 generated eEF2K deficiency ( CrEEF2K ) show a decrease susceptibly to NFR compare to control cells ( CrLuci ) .

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

B-F Dose response curves for cell viability after 48h NFR measured using MTS assay. Curves and bar graph for EC50 are mean ± s.e.m. of 3 independent experiments performed in triplicate. For curves, p values are 2way ANOVA analysis of variance. For bar graph, p values are two tails unpaired Student's T-tests (B, E), or 1way ANOVA with Bonferroni (C) or Dunnett's multiple comparison posttests (D and F). *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. (D-F), HeLa (D), A549 (E) and MCF7 (F) clones with CRISPR-Cas9 generated eEF2K deficiency (CrEEF2K) show a decrease susceptibly to NFR compare to control cells (CrLuci).


G NFR - mediated toxicity assessed using AnnexinV / PI staining and FACS analysis after 24h treatment . Histogram shows the percentage of dead cells ( AV + / PI + ) . Data are mean ± s . e . m . of 3 independent experiments . P values are 2way ANOVA with Bonferroni posttest . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 NFR-mediated toxicity assessed using AnnexinV/PI staining and FACS analysis after 24h treatment. Histogram shows the percentage of dead cells (AV+/PI+). Data are mean ± s.e.m. of 3 independent experiments. P values are 2way ANOVA with Bonferroni posttest. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001.


A Tumor volumes of eEF2K - / - and eEF2K + / + RasV12 engraft implanted subcutaneously in AGR129mice . Treatment with NFR or vehicle was initiated 6 days post - implantation . Data are mean of tumor volume ± s . e . m . ( n = 8 per group ) . P values were calculated using 2way ANOVA followed by Bonferroni posttest comparing WT tumor with vehicle or NFR treatment and KO tumors with vehicle or NFR treatment ; * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 . Experiment is representative of 2 performed in same conditions .

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

A Tumor volumes of eEF2K -/- and eEF2K +/+ RasV12 engraft implanted subcutaneously in AGR129mice. Treatment with NFR or vehicle was initiated 6 days post-implantation. Data are mean of tumor volume ± s.e.m. (n = 8 per group). P values were calculated using 2way ANOVA followed by Bonferroni posttest comparing WT tumor with vehicle or NFR treatment and KO tumors with vehicle or NFR treatment; *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. Experiment is representative of 2 performed in same conditions.


B Immunoblots for total and phosphorylated eEF2 of tumor engraftment from ( A ) . ( NS , Non - Specific signal . ) C Bar graph shows relative phosphorylation intensities of eEF2 compared with total eEF2 determined by densitometry analysis . Mean ± s . e . m . are shown ( n = 4 ) . P values were calculated using 2way ANOVA followed by Bonferroni posttest ; * * P value ≤ 0 . 01 .

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B Immunoblots for total and phosphorylated eEF2 of tumor engraftment from (A). (NS, Non-Specific signal.)C Bar graph shows relative phosphorylation intensities of eEF2 compared with total eEF2 determined by densitometry analysis. Mean ± s.e.m. are shown (n=4). P values were calculated using 2way ANOVA followed by Bonferroni posttest ; **P value ≤ 0.01.


( A ) Immunoblot analysis . Primary hepatocytes prepared from wild - type mice were infected with adenovirus expressing GFP or NBR1 under the CAG promoter for 48 hrs . Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - test .

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 O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with adenovirus expressing GFP or NBR1 under the CAG promoter for 48 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


( B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in the hepatocytes expressing GFP . The experiments were performed three times . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - 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

(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


( C ) Immunofluorescence microscopy . Wild type hepatocytes were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' s t - test .

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 B-MEASURED_VAR 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 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

(C) Immunofluorescence microscopy. Wild type hepatocytes were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.


( D ) FRAP assay . Wild type hepatocytes cultured in glass - bottom plates were infected with GFP - NBR1 adenovirus for 48 hrs . The signal recovery after photobleaching was measured , quantified from at least three independent biological replicates , and is represented as means ± s . e . Bar : 1 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FRAP assay. Wild type hepatocytes cultured in glass-bottom plates were infected with GFP-NBR1 adenovirus for 48 hrs. The signal recovery after photobleaching was measured, quantified from at least three independent biological replicates, and is represented as means ± s.e. Bar: 1 µm.


( A ) Immunoblot analysis . Primary hepatocytes prepared from wild - type mice were infected with adenovirus expressing GFP - Nrf2 and GFP or NBR1 for 48 hrs . Total cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * * P < 0 . 01 as determined by Welch ' s t - 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 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 O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with adenovirus expressing GFP-Nrf2 and GFP or NBR1 for 48 hrs. Total cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. **P < 0.01 as determined by Welch's t-test.


( B ) Immunoblot analysis . Primary hepatocytes prepared from p62f / f and p62f / f ; Alb - Cre mice were infected with adenovirus against GFP or NBR1 for 48 hrs . Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin ( cytosolic proteins ) or Lamin B ( nuclear proteins ) . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - test .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis. Primary hepatocytes prepared from p62f/f and p62f/f; Alb-Cre mice were infected with adenovirus against GFP or NBR1 for 48 hrs. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin (cytosolic proteins) or Lamin B (nuclear proteins). Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


( C ) Immunofluorescence microscopy . Primary hepatocytes from wild type mice were infected with adenovirus expressing GFP - Nrf2 and LacZ or NBR1 for 48 hrs . More than 100 cells were assayed for the nuclear signal of Nrf2 . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' s t - test . Bars : 10 µm .

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 B-CONTROLLED_VAR 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

(C) Immunofluorescence microscopy. Primary hepatocytes from wild type mice were infected with adenovirus expressing GFP-Nrf2 and LacZ or NBR1 for 48 hrs. More than 100 cells were assayed for the nuclear signal of Nrf2. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test. Bars: 10 µm.


( D ) Total RNAs were prepared from primary hepatocytes described in ( B ) . Values were normalized against the amount of mRNA in the p62f / f hepatocytes expressing GFP . The experiments were performed at least three times . Data are are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - 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 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 O O O O O O O O O O O O O O

(D) Total RNAs were prepared from primary hepatocytes described in (B). Values were normalized against the amount of mRNA in the p62f/f hepatocytes expressing GFP. The experiments were performed at least three times. Data are are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


( A ) Immunofluorescence microscopy . Primary hepatocytes prepared from p62f / f mice were infected with or without adenovirus Cre recombinase for 48 hrs and then additionally infected with adenovirus LacZ or NBR1 . 24 hrs after the infection , the hepatocytes were immunostained with anti - Keap1 , anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 10 µm .

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

(A) Immunofluorescence microscopy. Primary hepatocytes prepared from p62f/f mice were infected with or without adenovirus Cre recombinase for 48 hrs and then additionally infected with adenovirus LacZ or NBR1. 24 hrs after the infection, the hepatocytes were immunostained with anti-Keap1, anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 10 µm.


( B ) Immunoprecipitation analysis . Crude extracts were prepared from primary hepatocytes as described in ( A ) and then immunoprecipitated with anti - FLAG ( as a negative control ) or anti - NBR1 antibody . The resulting samples were analyzed by immunoblot analysis . Data shown are representative of three separate experiments .

O O O O O O O O O O O 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

(B) Immunoprecipitation analysis. Crude extracts were prepared from primary hepatocytes as described in (A) and then immunoprecipitated with anti-FLAG (as a negative control) or anti-NBR1 antibody. The resulting samples were analyzed by immunoblot analysis. Data shown are representative of three separate experiments.


( C ) Primary hepatocytes prepared from p62f / f ; Alb - Cre mice were co - infected with adenovirus GFP or NBR1 in combination with wild - type p62 or the mutants for 48 hrs . Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 as determined by Welch ' s t - test .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary hepatocytes prepared from p62f/f; Alb-Cre mice were co-infected with adenovirus GFP or NBR1 in combination with wild-type p62 or the mutants for 48 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test.


( D ) RT qPCR analysis . Total RNAs were prepared from hepatocytes described in ( C ) . Values were normalized against the amount of mRNA in the hepatocytes expressing GFP . The experiments were performed three times . Data are shown as means ± s . e . * P < 0 . 05 , and * * P < 0 . 01 as determined by Welch ' s t - 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

(D) RT qPCR analysis. Total RNAs were prepared from hepatocytes described in (C). Values were normalized against the amount of mRNA in the hepatocytes expressing GFP. The experiments were performed three times. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.


( A ) Immunoblot analysis . Primary hepatocytes prepared from Nrf2f / f and Nrf2f / f ; Alb - Cre mice were infected with adenovirus expressing GFP or NBR1 for 48 hrs . Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin ( cytosolic proteins ) or Lamin B ( nuclear proteins ) . Data are shown as means ± s . e . * P < 0 . 05 , and * * P < 0 . 01 as determined by Welch ' s t - test .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis. Primary hepatocytes prepared from Nrf2f/f and Nrf2f/f; Alb-Cre mice were infected with adenovirus expressing GFP or NBR1 for 48 hrs. Cytosolic and nuclear fractions were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin (cytosolic proteins) or Lamin B (nuclear proteins). Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.


( B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in the Nrf2f / f hepatocytes expressing GFP . The experiments were performed at least three times . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - 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

(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the Nrf2f/f hepatocytes expressing GFP. The experiments were performed at least three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


( C ) Immunofluorescence microscopy . Hepatocytes isolated from Nrf2f / f and Nrf2f / f ; Alb - Cre mice were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' s t - test .

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

(C) Immunofluorescence microscopy. Hepatocytes isolated from Nrf2f/f and Nrf2f/f; Alb-Cre mice were infected with LacZ or NBR1 adenovirus for 48 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.


( A ) Immunoblot analysis . Primary hepatocytes prepared from wild - type mice were infected with the indicated adenovirus for 48 hrs and subsequently treated with 100 nM Bafilomycin A1 ( BafA1 ) for 24 hrs or cultured in amino acid - deprived medium ( Stv ) for 12 hrs . Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies . Data shown are representative of three separate experiments . Bar graphs show the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - 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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis. Primary hepatocytes prepared from wild-type mice were infected with the indicated adenovirus for 48 hrs and subsequently treated with 100 nM Bafilomycin A1 (BafA1) for 24 hrs or cultured in amino acid-deprived medium (Stv) for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with the indicated antibodies. Data shown are representative of three separate experiments. Bar graphs show the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.


( B ) Fluorescence microscopy . p62 - deficient MEFs were co - transfected with mCherry - GFP - p62 in combination with wild - type NBR1 or NBR1 D50R mutant . 48 hrs after the transfection , fluorescence images were observed . Bars : 10 µm . The number of puncta positive for only red - signal in each MEFs was counted in more than 100 cells . Bar graphs are represented as means ± s . e . * * * P < 0 . 001 as determined by Welch ' s t - 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O 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) Fluorescence microscopy. p62-deficient MEFs were co-transfected with mCherry-GFP-p62 in combination with wild-type NBR1 or NBR1 D50R mutant. 48 hrs after the transfection, fluorescence images were observed. Bars: 10 µm. The number of puncta positive for only red-signal in each MEFs was counted in more than 100 cells. Bar graphs are represented as means ± s.e. ***P < 0.001 as determined by Welch's t-test.


( A ) Immunoblot analysis . Primary hepatocytes were isolated from wild - type ( wt ) , p62f / f ; Alb - Cre ( p62KO ) and Nrf2f / f ; Alb - Cre ( Nrf2KO ) mice . The hepatocytes were challenged with 10 μM sodium arsenite [ As ( III ) ] for 12 hrs . Cell lysates were prepared and subjected to immunoblot analysis with anti - NBR1 and anti - Actin antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 , and * * P < 0 . 01 as determined by Welch ' s t - test .

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

(A) Immunoblot analysis. Primary hepatocytes were isolated from wild-type (wt), p62f/f; Alb-Cre (p62KO) and Nrf2f/f; Alb-Cre (Nrf2KO) mice. The hepatocytes were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs. Cell lysates were prepared and subjected to immunoblot analysis with anti-NBR1 and anti-Actin antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, and **P < 0.01 as determined by Welch's t-test.


( B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in each non - treated hepatocytes . The experiments were performed three times . Data are shown as means ± s . e . * * P < 0 . 01 and * * * P < 0 . 001 as determined by Welch ' s t - 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

(B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in each non-treated hepatocytes. The experiments were performed three times. Data are shown as means ± s.e. **P < 0.01 and ***P < 0.001 as determined by Welch's t-test.


( C ) Immunofluorescence microscopy . Primary hepatocytes isolated from wild - type mice were challenged with 10 μM sodium arsenite [ As ( III ) ] for 12 hrs and then immunostained with anti - p62 and anti - NBR1 antibodies . Each inset is a magnified image . Bars : 20 µm . The number and size of p62 - bodies were measured in more than 100 cells . Data are shown as means ± s . e . * * * P < 0 . 001 as determined by Welch ' s t - test .

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

(C) Immunofluorescence microscopy. Primary hepatocytes isolated from wild-type mice were challenged with 10 μM sodium arsenite [As (III)] for 12 hrs and then immunostained with anti-p62 and anti-NBR1 antibodies. Each inset is a magnified image. Bars: 20 µm. The number and size of p62-bodies were measured in more than 100 cells. Data are shown as means ± s.e. ***P < 0.001 as determined by Welch's t-test.


A ) Immunoblot analysis . Primary hepatocytes were isolated from Nbr1f / f mice and then infected with adenovirus GFP or Cre for 48 hrs . Thereafter , the hepatocytes were divided into 3 groups : 1 . Cultured in regular medium ( - ) , 2 . Treated with 10 μM As ( III ) for 12 hr [ As ( III ) ] and 3 . After treated with 10 μM As ( III ) for 12 hr , cultured in regular medium for another 12 hr ( Wash ) . Cell lysates were prepared and subjected to immunoblot analysis with indicated antibodies . Data shown are representative of three separate experiments . Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin . Data are shown as means ± s . e . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 as determined by Welch ' s t - test .

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 O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis. Primary hepatocytes were isolated from Nbr1f/f mice and then infected with adenovirus GFP or Cre for 48 hrs. Thereafter, the hepatocytes were divided into 3 groups: 1. Cultured in regular medium (-), 2. Treated with 10 μM As (III) for 12 hr [As (III)] and 3. After treated with 10 μM As (III) for 12 hr, cultured in regular medium for another 12 hr (Wash). Cell lysates were prepared and subjected to immunoblot analysis with indicated antibodies. Data shown are representative of three separate experiments. Bar graphs indicate the quantitative densitometric analysis of the indicated proteins relative to actin. Data are shown as means ± s.e. *P < 0.05, **P < 0.01, and ***P < 0.001 as determined by Welch's t-test.


B ) Real - time PCR . Total RNAs were prepared from primary hepatocytes described in ( A ) . Values were normalized against the amount of mRNA in the Nbr1f / f hepatocytes expressing GFP under normal culture conditions . The experiments were performed at least three times . Data are shown as means ± s . e . * P < 0 . 05 as determined by Welch ' s t - 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 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 O O O O O O O O O O O O O O O O O

B) Real-time PCR. Total RNAs were prepared from primary hepatocytes described in (A). Values were normalized against the amount of mRNA in the Nbr1f/f hepatocytes expressing GFP under normal culture conditions. The experiments were performed at least three times. Data are shown as means ± s.e. *P < 0.05 as determined by Welch's t-test.