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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 .

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

H-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 .

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

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 .

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

A-C 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 .

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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.


A Growth curves show proliferation reduction in ATRX depleted U87 cells . Data are expressed as means ± standard deviation ( s . d . ) , N = 2 . 500 cells / well were plated .

O O O O O O O O O O O O O O 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 Growth curves show proliferation reduction in ATRX depleted U87 cells. Data are expressed as means ± standard deviation (s.d.), N = 2. 500 cells/well were plated.


B β - gal staining revealed the increased senescent cell population in ATRX - deleted U87 cells . Upper panel , micrographs of control and ATRX - deleted U87 cells processed for ATRX immunofluorescence ( IF ) and β - gal senescence assay . Scale bar , 200 µm . D , DAPI . Lower panel , percentage of senescent cells . Data are expressed as means ± s . d . , N = 3 , unpaired 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

B β-gal staining revealed the increased senescent cell population in ATRX-deleted U87 cells. Upper panel, micrographs of control and ATRX-deleted U87 cells processed for ATRX immunofluorescence (IF) and β-gal senescence assay. Scale bar, 200 µm. D, DAPI. Lower panel, percentage of senescent cells. Data are expressed as means ± s.d., N = 3, unpaired t test.


C , D PML / TelG immuno - FISH ( C ) shows increased APB formation in ATRX - deleted U87 cells ( D ) . Scale bar , 10 µm . ( D ) Percentages of APB positive cells are expressed as means ± s . d . , N = 3 , unpaired 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

C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87 cells (D). Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.


E C - circle assays revealed enhanced C - circles formation in ATRX - deleted U87 cells . Reactions lacking DNA or ɸ29 serve as negative controls . 30 ng of indicated template DNA was used in each reaction .

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

E C-circle assays revealed enhanced C-circles formation in ATRX-deleted U87 cells. Reactions lacking DNA or ɸ29 serve as negative controls. 30 ng of indicated template DNA was used in each reaction.


G 53BP1 / TelG immuno - FISH shows increased TIF formation in ATRX - deleted U87 cells ( G ) . Arrows denote telomeres positive for 53BP1 signals . Scale bar , 10 µm . ( G ) Percentages of cells containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired 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

G 53BP1/TelG immuno-FISH shows increased TIF formation in ATRX-deleted U87 cells (G). Arrows denote telomeres positive for 53BP1 signals. Scale bar, 10 µm. (G) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


H Growth curves revealed increase of cell proliferation in ATRX - deleted U87 cells ( A - 1 and A - 2 ) after hTERT transduction . Data are expressed as means ± s . d . , N = 2 .

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

H Growth curves revealed increase of cell proliferation in ATRX-deleted U87 cells (A-1 and A-2) after hTERT transduction. Data are expressed as means ± s.d., N = 2.


I Western blots show depletion of ATRX protein expression in hTERT - transduced and ATRX - deleted A - 1 and A - 2 U87 cells .

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

I Western blots show depletion of ATRX protein expression in hTERT-transduced and ATRX-deleted A-1 and A-2 U87 cells.


A Growth curves revealed that deletion of ATRX had minor effect on proliferation of TERT - overexpressed U87 - T cells , but further removal of the exogenous floxed TERT by Ad - Cre induced rapid cell cycle arrest . Data are expressed as means ± s . d . , N = 3 .

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A Growth curves revealed that deletion of ATRX had minor effect on proliferation of TERT-overexpressed U87-T cells, but further removal of the exogenous floxed TERT by Ad-Cre induced rapid cell cycle arrest. Data are expressed as means ± s.d., N = 3.


B Telomere restriction fragment ( TRF ) analysis shows increased telomeric length heterogeneities in ATRX - deleted U87 - T and the further enhancement following Ad - Cre treatment . Genomic DNAs ( EtBr staining , left panel ) prepared from control and ATRX - deleted U87 - T cells at day 9 post control or Ad - Cre infection ( middle panel ) were assayed by hybridization with 32P - labelled ( TTAGGG ) 4 probe , followed by re - hybridization with an oligonucleotide probe specific for centromere region ( right panel ) . Genomic DNA from U2OS cells was used as an ALT positive control .

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B Telomere restriction fragment (TRF) analysis shows increased telomeric length heterogeneities in ATRX-deleted U87-T and the further enhancement following Ad-Cre treatment. Genomic DNAs (EtBr staining, left panel) prepared from control and ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (middle panel) were assayed by hybridization with 32P-labelled (TTAGGG)4 probe, followed by re-hybridization with an oligonucleotide probe specific for centromere region (right panel). Genomic DNA from U2OS cells was used as an ALT positive control.


C , D PML / TelG immuno - FISH ( C ) shows increased APB formation in ATRX - deleted U87 - T cells ( D ) at day 9 post control or Ad - Cre infection . Images are maximum - intensity projections of ~ 10 stacks captured with a 63x lens with 2x zoom . Scale bar , 10 µm . ( D ) Percentages of APB positive cells are expressed as means ± s . d . , N = 3 , unpaired t test .

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C, D PML/TelG immuno-FISH (C) shows increased APB formation in ATRX-deleted U87-T cells (D) at day 9 post control or Ad-Cre infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (D) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.


E 2D gel analysis revealed increased T - circle formation in ATRX - deleted U87 - T cells without or with Ad - Cre infection . Shown are gels stained with EtBr and blots hybridized with a TelG probe . Indicated are linear ( lin ) and open ( oc ) T - circle forms of telomeric DNA .

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E 2D gel analysis revealed increased T-circle formation in ATRX-deleted U87-T cells without or with Ad-Cre infection. Shown are gels stained with EtBr and blots hybridized with a TelG probe. Indicated are linear (lin) and open (oc) T-circle forms of telomeric DNA.


F C - circle assays shows increased C - circle formation in ATRX - deleted U87 - T cells at day 9 post control or Ad - Cre infection .

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F C-circle assays shows increased C-circle formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection.


G , H 53BP1 / TelG immuno - FISH ( G ) shows increased TIF formation in ATRX - deleted U87 - T cells at day 9 post control or Ad - Cre infection ( H ) . Scale bar , 10 µm . ( H ) Percentages of cells containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t test .

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G, H 53BP1/TelG immuno-FISH (G) shows increased TIF formation in ATRX-deleted U87-T cells at day 9 post control or Ad-Cre infection (H). Scale bar, 10 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


B Western blots of extracts from U87 cells infected with Lenti - CRISPR ( puro ) encoding individual ATRX - targeted sgRNAs . The extracts were isolated after puromycin selection , and assayed for ATRX depletion levels using antibody against ATRX or ACTIN control . The asterisks ( * ) denote the two sgATRXs ( sgA # 10 and sgA # 19 ) used in following experiments .

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B Western blots of extracts from U87 cells infected with Lenti-CRISPR(puro) encoding individual ATRX-targeted sgRNAs. The extracts were isolated after puromycin selection, and assayed for ATRX depletion levels using antibody against ATRX or ACTIN control. The asterisks (*) denote the two sgATRXs (sgA#10 and sgA#19) used in following experiments.


C ATRX immunofluorescence staining of U87 cells infected with lentiviral control sgRosa , sgA # 10 , or sgA # 19 . D , DAPI . Scale bar , 50 µm .

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C ATRX immunofluorescence staining of U87 cells infected with lentiviral control sgRosa, sgA#10, or sgA#19. D, DAPI. Scale bar, 50 µm.


Bar graphs show time - course analyses of U87 ( E ) transduced with lentiviral LRG vector encoding sgRosa control or ATRX - targeted sgA # 10 or sgA # 19 . The graph plots the percentage of GFP + cells over time following transduction with indicated sgRNAs . Data are expressed as means ± s . d . , * denotes ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . N = 3 , unpaired t test .

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Bar graphs show time-course analyses of U87 (E) transduced with lentiviral LRG vector encoding sgRosa control or ATRX-targeted sgA#10 or sgA#19. The graph plots the percentage of GFP+ cells over time following transduction with indicated sgRNAs. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.


Bar graphs show time - course analyses of U2OS cells ( F ) transduced with lentiviral LRG vector encoding sgRosa control or ATRX - targeted sgA # 10 or sgA # 19 . The graph plots the percentage of GFP + cells over time following transduction with indicated sgRNAs . Data are expressed as means ± s . d . , * denotes ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . N = 3 , unpaired t test .

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Bar graphs show time-course analyses of U2OS cells (F) transduced with lentiviral LRG vector encoding sgRosa control or ATRX-targeted sgA#10 or sgA#19. The graph plots the percentage of GFP+ cells over time following transduction with indicated sgRNAs. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.


G , H 53BP1 / TRF2 co - immunofluorescence ( G ) revealed strongly increased TIF formation in lentiviral sgA # 10 or sgA # 19 transduced U87 cells assayed at day 45 but not day 15 post infections ( H ) . Scale bar , 15 µm . ( H ) Percentages of cells containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t test .

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G, H 53BP1/TRF2 co-immunofluorescence (G) revealed strongly increased TIF formation in lentiviral sgA#10 or sgA#19 transduced U87 cells assayed at day 45 but not day 15 post infections (H). Scale bar, 15 µm. (H) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


I , J 53BP1 / TelG immuno - FISH ( I ) revealed strongly enhanced TIF formation in lentiviral sgA # 10 or sgA # 19 transduced IMR90 cells assayed at day 35 but not day 10 post infections ( J ) . Scale bar , 10 µm . ( J ) Percentages of cells containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t test .

I, J 53BP1/TelG immuno-FISH (I) revealed strongly enhanced TIF formation in lentiviral sgA#10 or sgA#19 transduced IMR90 cells assayed at day 35 but not day 10 post infections (J). Scale bar, 10 µm. (J) Percentages of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


K Bar graphs show time - course analyses of IMR90 cells transduced with lentiviral sgRosa , sgA # 10 or sgA # 19 . Data are expressed as means ± s . d . , * denotes ns ( not significant ) ; * * , p < 0 . 01 ; * * * , p < 0 . 001 . N = 3 , unpaired t test .

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K Bar graphs show time-course analyses of IMR90 cells transduced with lentiviral sgRosa, sgA#10 or sgA#19. Data are expressed as means ± s.d., * denotes ns (not significant); **, p < 0.01; ***, p < 0.001. N = 3, unpaired t test.


A , B γH2AX / TelG immuno - FISH ( A ) revealed increased TIF formation in IMR90 cells transduced with DAXX - targeted lentiviral sgRNAs ( sgD # 1 and # 2 ) ( B ) . Scale bar , 10 µm . Arrows denote telomeres that are positive for γH2AX signals . ( B ) Percentage of cells containing > 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t test .

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A, B γH2AX/TelG immuno-FISH (A) revealed increased TIF formation in IMR90 cells transduced with DAXX-targeted lentiviral sgRNAs (sgD#1 and #2) (B). Scale bar, 10 µm. Arrows denote telomeres that are positive for γH2AX signals. (B) Percentage of cells containing > 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


C Western blots show depleted DAXX protein expression in Ad - Flp - infected cd - DAXX cells .

C Western blots show depleted DAXX protein expression in Ad-Flp-infected cd-DAXX cells.


D , E PML / TelG immune - FISH ( D ) show enhanced APB formation in Ad - Flp infected cd - DAXX cells ( cd - D # 1 and cd - D # 2 ) ( E ) . The cells were analyzed at day 18 post Ad - Flp infection . Images are maximum - intensity projections of ~ 10 stacks captured with a 63x lens with 2x zoom . Scale bar , 10 µm . ( E ) Percentages of APB + cells are expressed as means ± s . d . , N = 3 , unpaired t test .

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D, E PML/TelG immune-FISH (D) show enhanced APB formation in Ad-Flp infected cd-DAXX cells (cd-D#1 and cd-D#2) (E). The cells were analyzed at day 18 post Ad-Flp infection. Images are maximum-intensity projections of ~10 stacks captured with a 63x lens with 2x zoom. Scale bar, 10 µm. (E) Percentages of APB+ cells are expressed as means ± s.d., N = 3, unpaired t test.


Depletion of DAXX induced delayed onset of growth arrest ( F ) ( F ) 2 , 000 cells were plated at day 4 post Ad - Flp infection . Data are expressed as means ± s . d . , N = 3 .

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Depletion of DAXX induced delayed onset of growth arrest (F) (F) 2,000 cells were plated at day 4 post Ad-Flp infection. Data are expressed as means ± s.d., N = 3.


Depletion of DAXX induced C - circle formation ( G ) .

Depletion of DAXX induced C-circle formation (G).


IMR90 cells at day 25 post infection of lentiviral control or sgA # 10 were synchronized by serum starvation and switched to 15 % serum - containing medium and assayed by 53BP1 / TelG immuno - FISH ( A ) at indicated time . Arrows denote telomeres that are positive for 53BP1 signals . Scale bar , 15 µm . ( B ) Percentage of cells containing ; 4 TIFs are expressed as means ± s . d . , N = 3 , unpaired t test .

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IMR90 cells at day 25 post infection of lentiviral control or sgA#10 were synchronized by serum starvation and switched to 15% serum-containing medium and assayed by 53BP1/TelG immuno-FISH (A) at indicated time. Arrows denote telomeres that are positive for 53BP1 signals. Scale bar, 15 µm. (B) Percentage of cells containing ; 4 TIFs are expressed as means ± s.d., N = 3, unpaired t test.


C , D RPA / TRF2 co - immunofluorescence ( C ) revealed strongly increased telomeric replication stress in U87 cells at day 30 but not day 10 post lentiviral sgA # 10 infection ( D ) . Lentiviral control and sgA # 10 infected U87 cells at indicated time post - infection were released following thymidine ( Thy ) synchronization and processed for RPA and TRF2 co - staining . Scale bar , 10 µm . ( D ) Percentages of cells with > 4 RPA / TRF2 co - localized foci are expressed as means ± s . d . , N = 3 , unpaired t test .

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C, D RPA/TRF2 co-immunofluorescence (C) revealed strongly increased telomeric replication stress in U87 cells at day 30 but not day 10 post lentiviral sgA#10 infection (D). Lentiviral control and sgA#10 infected U87 cells at indicated time post-infection were released following thymidine (Thy) synchronization and processed for RPA and TRF2 co-staining. Scale bar, 10 µm. (D) Percentages of cells with > 4 RPA/TRF2 co-localized foci are expressed as means ± s.d., N = 3, unpaired t test.


A , B ATAC - seq analyses revealed progressive increased telomeric DNA accessibilities in sgA # 10 transduced IMR90 cells . ( A ) The graph plots show the cumulative percentage of total reads that contained at least indicated number of TTAGGG ( or CCCTAA ) tandem repeats . ( B ) The percentage of total reads that contains > 3 tandem telomeric repeats are expressed as means ± s . d . , N = 2 , unpaired t test .

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A, B ATAC-seq analyses revealed progressive increased telomeric DNA accessibilities in sgA#10 transduced IMR90 cells. (A) The graph plots show the cumulative percentage of total reads that contained at least indicated number of TTAGGG (or CCCTAA) tandem repeats. (B) The percentage of total reads that contains > 3 tandem telomeric repeats are expressed as means ± s.d., N = 2, unpaired t test.


C , D ChIP assays ( C ) show reduced nucleosome densities in sgA # 10 transduced IMR90 cells ( D ) . Input and ChIP DNAs processed against indicated antibodies were assayed by dot - blotting and hybridized with 32P - labeled probes specific for TelG . ( D ) The relative enrichment was calculated after normalization of ChIP DNA signals to the respective input DNA signals . Data are expressed as means ± s . d . , N = 3 , paired t test .

C, D ChIP assays (C) show reduced nucleosome densities in sgA#10 transduced IMR90 cells (D). Input and ChIP DNAs processed against indicated antibodies were assayed by dot-blotting and hybridized with 32P-labeled probes specific for TelG. (D) The relative enrichment was calculated after normalization of ChIP DNA signals to the respective input DNA signals. Data are expressed as means ± s.d., N = 3, paired t test.


A Growth curves show the immortalization processes of lentiviral Rosa control ( sgRosa # 1 or # 2 ) or sgATRX ( sgA # 10 or # 19 ) transduced IMR90 cells . TERT transduced IMR90 culture was included as a positive control for immortalization .

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A Growth curves show the immortalization processes of lentiviral Rosa control (sgRosa#1 or #2) or sgATRX (sgA#10 or #19) transduced IMR90 cells. TERT transduced IMR90 culture was included as a positive control for immortalization.


B Quantitative RT - PCR of endogenous TERT gene expression revealed that sgATRX but not sgRosa transduced IMR90 cells adopted ALT during immortalization . Data are expressed as means ± s . d . , N = 3 , unpaired t test .

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B Quantitative RT-PCR of endogenous TERT gene expression revealed that sgATRX but not sgRosa transduced IMR90 cells adopted ALT during immortalization. Data are expressed as means ± s.d., N = 3, unpaired t test.


C Western blots show depletion of ATRX protein expression in the immortalized ALT IMR90 cells .

C Western blots show depletion of ATRX protein expression in the immortalized ALT IMR90 cells.


D C - circle analysis shows increased C - circle formation in the immortalized ALT cells . U2OS was used as a positive control , and reactions lacking DNA or ɸ29 serve as negative controls .

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D C-circle analysis shows increased C-circle formation in the immortalized ALT cells. U2OS was used as a positive control, and reactions lacking DNA or ɸ29 serve as negative controls.


E Telomere restriction fragment ( TRF ) analysis show enhanced telomeric length heterogeneities in the immortalized ALT cells .

E Telomere restriction fragment (TRF) analysis show enhanced telomeric length heterogeneities in the immortalized ALT cells.


F , G PML / TRF2 co - immunofluorescence ( F ) shows increased APB formation in the immortalized ALT cells ( G ) . Scale bar , 10 µm . ( G ) Percentages of APB positive cells are expressed as means ± s . d . , N = 3 , unpaired t test .

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F, G PML/TRF2 co-immunofluorescence (F) shows increased APB formation in the immortalized ALT cells (G). Scale bar, 10 µm. (G) Percentages of APB positive cells are expressed as means ± s.d., N = 3, unpaired t test.


Chl - HyPer2 response to chloroplast hydrogen peroxide elicited by BSMV infection in N . benthamiana epidermal cells . Ratiometric images ( F488 / 405 nm ) of fluorescence excitation at 488 and 405 nm show the oxidized state of chloroplast - targeted HyPer2 , respectively . The false - blue color indicates the chloroplasts . Scale bars , 30 μm . Data information : In ( A ) , data are representative of at least three independent experiments .

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Chl-HyPer2 response to chloroplast hydrogen peroxide elicited by BSMV infection in N. benthamiana epidermal cells. Ratiometric images (F488/405 nm) of fluorescence excitation at 488 and 405 nm show the oxidized state of chloroplast-targeted HyPer2, respectively. The false-blue color indicates the chloroplasts. Scale bars, 30 μm. Data information: In (A), data are representative of at least three independent experiments.


Quantification of the BSMV - dependent changes in Chl - HyPer2 fluorescence at 0 to 6 dpi . The percentage of ROS - positive chloroplasts ( white ) among all chloroplasts ( blue and white ) per visual field . Data information : In ( B ) , error bars indicate mean ± SEM ( n = 5 ) .

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Quantification of the BSMV-dependent changes in Chl-HyPer2 fluorescence at 0 to 6 dpi. The percentage of ROS-positive chloroplasts (white) among all chloroplasts (blue and white) per visual field. Data information: In (B), error bars indicate mean ± SEM (n = 5).


Western blot analysis of 2 - Cys Prx redox status in empty vector ( EV ) and BSMV inoculated leaves under non - reducing conditions at 3 dpi . Western blots were analyzed with anti - 2 - Cys Prx or anti - CP antibodies . The Rubisco large subunit ( RbcL ) served as a loading control . Data information : , representative data are shown , at least three independent replicates were performed with similar results .

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Western blot analysis of 2-Cys Prx redox status in empty vector (EV) and BSMV inoculated leaves under non-reducing conditions at 3 dpi. Western blots were analyzed with anti-2-Cys Prx or anti-CP antibodies. The Rubisco large subunit (RbcL) served as a loading control. Data information: , representative data are shown, at least three independent replicates were performed with similar results.


Relative mRNA levels of the sAPX , 2 - Cys Prx , GPXL , and PrxQ genes in response to BSMV infections at 1 , 3 and 5 dpi . The empty vector ( EV ) was used as a negative control , NbEF1α was used as an internal control . Data information : In ( D ) , error bars indicate mean ± SEM from three independent experiments ; * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 from Student ' s t test .

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Relative mRNA levels of the sAPX, 2-Cys Prx, GPXL, and PrxQ genes in response to BSMV infections at 1, 3 and 5 dpi. The empty vector (EV) was used as a negative control, NbEF1α was used as an internal control. Data information: In (D), error bars indicate mean ± SEM from three independent experiments; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001 from Student's t test.


BSMV accumulation in N . benthamiana leaves transiently expressing chloroplast ROS scavenging proteins . Agrobacterium harboring plasmids expressing various proteins are indicated above the panels . BSMV was agroinfiltrated into the same leaves at 1 . 5 dpi . Three days later , Western blot analyses were conducted with anti - TGB1 or anti - Flag antibodies . RbcL served as the loading control . 2 - CP , 2 - Cys Prx . Data information : representative data are shown , at least three independent replicates were performed with similar results .

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BSMV accumulation in N. benthamiana leaves transiently expressing chloroplast ROS scavenging proteins. Agrobacterium harboring plasmids expressing various proteins are indicated above the panels. BSMV was agroinfiltrated into the same leaves at 1.5 dpi. Three days later, Western blot analyses were conducted with anti-TGB1 or anti-Flag antibodies. RbcL served as the loading control. 2-CP, 2-Cys Prx. Data information: representative data are shown, at least three independent replicates were performed with similar results.


BSMV accumulation in 2 - Cys Prx or GPXL - silenced N . benthamiana plants using anti - TGB1 antibody . Empty vector TRV : 00 was used as negative control . Data information : representative data are shown , at least three independent replicates were performed with similar results .

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BSMV accumulation in 2-Cys Prx or GPXL-silenced N. benthamiana plants using anti-TGB1 antibody. Empty vector TRV: 00 was used as negative control. Data information: representative data are shown, at least three independent replicates were performed with similar results.


Y2H analyses of interactions between γb and NbNTRC . Various Y2H combinations are indicated on the images . Self - interactions of γb served as a positive control , and combinations containing either empty AD or BD constructs served as negative controls .

Y2H analyses of interactions between γb and NbNTRC. Various Y2H combinations are indicated on the images. Self-interactions of γb served as a positive control, and combinations containing either empty AD or BD constructs served as negative controls.


BiFC analyses of γb and NbNTRC interactions during BSMV infections . Various BiFC combinations are indicated on the left . YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false - green color and chloroplasts were visualized by chloroplast autofluorescence as a false - red color . Scale bars , 20 μm .

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BiFC analyses of γb and NbNTRC interactions during BSMV infections. Various BiFC combinations are indicated on the left. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color and chloroplasts were visualized by chloroplast autofluorescence as a false-red color. Scale bars, 20 μm.


Co - IP analyses of interactions between γb and NbNTRC in N . benthamiana . Leaf tissues were agroinfiltrated with various combinations indicated above the panels were harvested at 3 dpi . Total proteins were immunoprecipitated with anti - Flag beads . Total and immunoprecipitated ( IP ) proteins were analyzed by Western blotting with anti - GFP or anti - Flag antibodies . Data information : representative data are shown , and three replicates had similar results .

Co-IP analyses of interactions between γb and NbNTRC in N. benthamiana. Leaf tissues were agroinfiltrated with various combinations indicated above the panels were harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Total and immunoprecipitated (IP) proteins were analyzed by Western blotting with anti-GFP or anti-Flag antibodies. Data information: representative data are shown, and three replicates had similar results.


GST pull - down assays to verify interactions between γb and NbNTRC in vitro . GST - tagged NTRC or GFP were incubated with γb - His or GFP - His . Input and pull - down products were analyzed by Western blot analyses with anti - GST or anti - His antibodies . GFP - His and GST - GFP served as negative controls . Data information : representative data are shown , and three replicates had similar results .

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GST pull-down assays to verify interactions between γb and NbNTRC in vitro. GST-tagged NTRC or GFP were incubated with γb-His or GFP-His. Input and pull-down products were analyzed by Western blot analyses with anti-GST or anti-His antibodies. GFP-His and GST-GFP served as negative controls. Data information: representative data are shown, and three replicates had similar results.


Co - localization of γb and NbNTRC during BSMV infection . NbNTRC - mCherry was co - expressed with BSMVγb - GFP ( Jiang et al . , 2020 ) in N . benthamiana , and confocal analyses were conducted at 3 dpi , and chloroplast autofluorescence is displayed as a false - blue color . Scale bars , 20 μm .

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Co-localization of γb and NbNTRC during BSMV infection. NbNTRC-mCherry was co-expressed with BSMVγb-GFP (Jiang et al., 2020) in N. benthamiana, and confocal analyses were conducted at 3 dpi, and chloroplast autofluorescence is displayed as a false-blue color. Scale bars, 20 μm.


Y2H assay to analyze interactions between NbNTRC and γb mutants . Various Y2H combinations are indicated on the images . Interactions of γb and NbNTRC were confirmed earlier and served as a positive control , combinations containing an empty BD construct served as a negative control .

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Y2H assay to analyze interactions between NbNTRC and γb mutants. Various Y2H combinations are indicated on the images. Interactions of γb and NbNTRC were confirmed earlier and served as a positive control, combinations containing an empty BD construct served as a negative control.


BiFC analysis of interactions between NbNTRC and γb mutants during BSMV infection of N . benthamiana . Various BiFC combinations are indicated at the top . YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false - green color and chloroplasts were visualized by chloroplast autofluorescence as a false - red color . RbcL - fused YFPc served as a negative control . Scale bars , 20 μm .

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BiFC analysis of interactions between NbNTRC and γb mutants during BSMV infection of N. benthamiana. Various BiFC combinations are indicated at the top. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color and chloroplasts were visualized by chloroplast autofluorescence as a false-red color. RbcL-fused YFPc served as a negative control. Scale bars, 20 μm.


Co - IP analysis of interactions between NbNTRC and γb mutants . NbNTRC - 3xFlag proteins together with γb - GFP , γbH85A - GFP or γbH85C - GFP were co - expressed in N . benthamiana leaves and harvested at 3 dpi . Total proteins were IP with anti - Flag beads . Total and IP products were detected in Western blots with anti - GFP or anti - Flag antibodies . Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information : , representative data were shown , and three independent experiments showed a similar result .

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Co-IP analysis of interactions between NbNTRC and γb mutants. NbNTRC-3xFlag proteins together with γb-GFP, γbH85A-GFP or γbH85C-GFP were co-expressed in N. benthamiana leaves and harvested at 3 dpi. Total proteins were IP with anti-Flag beads. Total and IP products were detected in Western blots with anti-GFP or anti-Flag antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information: , representative data were shown, and three independent experiments showed a similar result.


GST pull - down assays to evaluate in vitro interactions between NbNTRC and γb mutants . Purified γb - His , γbH85A - His , γbH85C - His , or GFP - His proteins were incubated with GST - tagged NbNTRC . Input and pull - down products were analyzed by Western blotting with anti - GST or anti - His antibodies . GFP - His served as a negative control . Arrowhead indicates the specific band of the target protein . Data information : representative data were shown , and three independent experiments showed a similar result .

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GST pull-down assays to evaluate in vitro interactions between NbNTRC and γb mutants. Purified γb-His, γbH85A-His, γbH85C-His, or GFP-His proteins were incubated with GST-tagged NbNTRC. Input and pull-down products were analyzed by Western blotting with anti-GST or anti-His antibodies. GFP-His served as a negative control. Arrowhead indicates the specific band of the target protein. Data information: representative data were shown, and three independent experiments showed a similar result.


Analysis of VSR activities of γb mutants . Infiltrated N . benthamiana leaves co - expressing sGFP plus γbH85A - 3xFlag , γbH85C - 3xFlag or the pGD empty vector ( EV ) were observed at 5 dpi under long - wave UV illumination for GFP fluorescence . The γb - 3xFlag protein served as a positive control . The white dashed circles indicate the infiltrated zone . Western blot analysis of the GFP protein in co - infiltrated leaves at 5 dpi with anti - GFP antibody . RbcL was used as loading control . Data information : representative data were shown , and three independent experiments showed a similar result .

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Analysis of VSR activities of γb mutants. Infiltrated N. benthamiana leaves co-expressing sGFP plus γbH85A-3xFlag, γbH85C-3xFlag or the pGD empty vector (EV) were observed at 5 dpi under long-wave UV illumination for GFP fluorescence. The γb-3xFlag protein served as a positive control. The white dashed circles indicate the infiltrated zone.   Western blot analysis of the GFP protein in co-infiltrated leaves at 5 dpi with anti-GFP antibody. RbcL was used as loading control.  Data information:  representative data were shown, and three independent experiments showed a similar result.


Effects of NbNTRC overexpression on BSMV protein accumulation . N . benthamiana leaves were first infiltrated with Agrobacterium containing various contructs indicated above the panel . After 1 . 5 dpi , BSMV was inoculated onto the leaves by agroinfiltration . Three days later , total protein was extracted for Western blotting with anti - TGB1 and anti - Flag antibodies . The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows RbcL was a loading control . Data information : representative data are shown and three eplicates similar results .

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Effects of NbNTRC overexpression on BSMV protein accumulation. N. benthamiana leaves were first infiltrated with Agrobacterium containing various contructs indicated above the panel. After 1.5 dpi, BSMV was inoculated onto the leaves by agroinfiltration. Three days later, total protein was extracted for Western blotting with anti-TGB1 and anti-Flag antibodies. The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows RbcL was a loading control. Data information: representative data are shown and three eplicates similar results.


Effects of NbNTRC overexpression on BSMV replication . Movement - deficient BSMV ( RNAα + RNAγ ) was infiltrated into leaves that had been agroinfiltrated previously with various constructs indicated above the panel . NbNTRC and viral γb proteins were detected by Western blotting with anti - Flag and anti - γb antibodies . Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows ( see Appendix Fig S4 ) . RT - qPCR analyses of viral RNA accumulation in 4B . NbPP2A was used as an internal control . Data information : representative data are shown and three eplicates similar results . In ( C ) , error bars indicate mean ± SEM from three independent experiments .

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Effects of NbNTRC overexpression on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ) was infiltrated into leaves that had been agroinfiltrated previously with various constructs indicated above the panel. NbNTRC and viral γb proteins were detected by Western blotting with anti-Flag and anti-γb antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows (see Appendix Fig S4).   RT-qPCR analyses of viral RNA accumulation in 4B. NbPP2A was used as an internal control.   Data information: representative data are shown and three eplicates similar results. In (C), error bars indicate mean ± SEM from three independent experiments.


The symptoms of infection after BSMV inoculation of a different N . benthamiana plant at 8 dpi . Magnified images of leaves ( in white boxes ) are shown in the lower panels . Scale bars , 2 cm . Statistical analysis of symptom appearance in different N . benthamiana plants after inoculation with BSMV . Western blot analysis of the virus accumulation in Figure 4D with anti - TGB1 antibody . RbcL served as loading control . Data information : , representative data are shown and three eplicates similar results . In ( E ) , error bars indicate mean ± SD from three independent experiments .

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The symptoms of infection after BSMV inoculation of a different N. benthamiana plant at 8 dpi. Magnified images of leaves (in white boxes) are shown in the lower panels. Scale bars, 2 cm.   Statistical analysis of symptom appearance in different N. benthamiana plants after inoculation with BSMV.   Western blot analysis of the virus accumulation in Figure 4D with anti-TGB1 antibody. RbcL served as loading control.   Data information: , representative data are shown and three eplicates similar results. In (E), error bars indicate mean ± SD from three independent experiments.


Left panel : systemic symptoms of N . benthamiana plants inoculated with BSMV and BSMV γb mutants . Photographs were taken at 7 dpi . Right panel : schematic representation of inoculated ( ino . ) , older systemic leaves ( o - SL ) , and younger systemic leaves ( y - SL ) in N . benthamiana plants . Scale bars , 2 cm .

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Left panel: systemic symptoms of N. benthamiana plants inoculated with BSMV and BSMV γb mutants. Photographs were taken at 7 dpi. Right panel: schematic representation of inoculated (ino.), older systemic leaves (o-SL), and younger systemic leaves (y-SL) in N. benthamiana plants. Scale bars, 2 cm.


Western blot analyses of TGB1 and CP accumulation in BSMV and BSMV γb mutant inoculated N . benthamiana leaves . Anti - TGB1 and anti - CP were used for protein detection . Data information : , representative data are shown , and three replicates had similar results . RbcL served as loading control .

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Western blot analyses of TGB1 and CP accumulation in BSMV and BSMV γb mutant inoculated N. benthamiana leaves. Anti-TGB1 and anti-CP were used for protein detection. Data information: , representative data are shown, and three replicates had similar results. RbcL served as loading control.


Western blot detection of BSMV with anti - CP antibodies in upper N . benthamiana leaves at 7 days ( o - SL ) or 28 days ( y - SL ) after infiltration . o - SL , older systemic leaves ; y - SL , younger systemic leaves . Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control .

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Western blot detection of BSMV with anti-CP antibodies in upper N. benthamiana leaves at 7 days (o-SL) or 28 days (y-SL) after infiltration. o-SL, older systemic leaves; y-SL, younger systemic leaves. Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Effects of NbNTRC overexpression on infection of BSMV and BSMV γb mutants . A . tumefaciens harboring the indicated plasmids were infiltrated into N . benthamiana leaves . Note : Agrobacterium containing NbNTRC constructs were diluted 5 - fold compared to normal use ( OD600 = 0 . 3 ) . BSMV and the γb mutants were then agroinfiltrated into the same leaves at 1 . 5 dpi . Three days later , Western blots were conducted with anti - TGB1 and anti - Flag antibodies . Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control .

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Effects of NbNTRC overexpression on infection of BSMV and BSMV γb mutants. A. tumefaciens harboring the indicated plasmids were infiltrated into N. benthamiana leaves. Note: Agrobacterium containing NbNTRC constructs were diluted 5-fold compared to normal use (OD600 = 0.3). BSMV and the γb mutants were then agroinfiltrated into the same leaves at 1.5 dpi. Three days later, Western blots were conducted with anti-TGB1 and anti-Flag antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Left panel : Western blot detection of BSMV CP accumulation in N . benthamiana leaves inoculated with BSMV and γb movement deficient mutants . Leaf samples were collected from different regions of the same leaf ( right panel ) Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control .

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Left panel: Western blot detection of BSMV CP accumulation in N. benthamiana leaves inoculated with BSMV and γb movement deficient mutants. Leaf samples were collected from different regions of the same leaf (right panel) Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Effects of γb and γb mutants on 2 - Cys Prx in N . benthamiana . Different combinantions of the pGD empty vector ( EV ) or γb and γb mutants are indicated above the panel . NbNTRC and γb proteins were detected by Western blotting with anti - Flag and anti - γb antibodies under reducing conditions , Western blot analyses of 2 - Cys Prx abundance with anti - 2 - Cys Prx antibody under non - reducing conditions . The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows , respectively . Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control .

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Effects of γb and γb mutants on 2-Cys Prx in N. benthamiana. Different combinantions of the pGD empty vector (EV) or γb and γb mutants are indicated above the panel. NbNTRC and γb proteins were detected by Western blotting with anti-Flag and anti-γb antibodies under reducing conditions, Western blot analyses of 2-Cys Prx abundance with anti-2-Cys Prx antibody under non-reducing conditions. The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows, respectively. Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


BiFC assays to identify interactions between NbNTRC and 2 - Cys Prx in N . benthamiana during BSMV infection . NbNTRC - YFPc and 2 - Cys Prx - YFPn were co - expressed with BSMV and γb mutant derivatives as indicated on the left . The EV was used as negative control . Scale bars , 30 μm . Quantificaiton of the BiFC intensities shown in Figure 6A with ImageJ software . The NbNTRC - 2 - Cys Prx interaction was normalized to that of the EV control , which was set to 1 . 0 . Data information : Representative data were shown in and three replicates had similar results . In B , error bars indicate the mean ± SEM , and a and b indicate statistically significant differences among different groups ( n = 5 , ANOVA , p - value < 0 . 05 ) .

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BiFC assays to identify interactions between NbNTRC and 2-Cys Prx in N. benthamiana during BSMV infection. NbNTRC-YFPc and 2-Cys Prx-YFPn were co-expressed with BSMV and γb mutant derivatives as indicated on the left. The EV was used as negative control. Scale bars, 30 μm.   Quantificaiton of the BiFC intensities shown in Figure 6A with ImageJ software. The NbNTRC-2-Cys Prx interaction was normalized to that of the EV control, which was set to 1.0.   Data information: Representative data were shown in and three replicates had similar results. In B, error bars indicate the mean ± SEM, and a and b indicate statistically significant differences among different groups (n = 5, ANOVA, p-value < 0.05).


Split - LUC assays to determine NbNTRC - 2 - Cys Prx interactions in N . benthamiana during BSMV infection . NbNTRC - cLUC and 2 - Cys Prx - nLUC were co - expressed in N . benthamiana leaves infected with the indicated BSMV derivatives , Bioluminescence was observed at 3 dpi . The white dashed circles indicate the infiltrated regions . Quantification of the luciferase signals shown in Figure 6C as assessed by ImageJ software . Data information : Representative data were shown , and three replicates had similar results . In ( D ) , error bars indicate mean ± SEM ( n = 6 ) . c indicate statistically significant differences among different groups ( ANOVA , p - value < 0 . 05 ) .

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Split-LUC assays to determine NbNTRC-2-Cys Prx interactions in N. benthamiana during BSMV infection. NbNTRC-cLUC and 2-Cys Prx-nLUC were co-expressed in N. benthamiana leaves infected with the indicated BSMV derivatives, Bioluminescence was observed at 3 dpi. The white dashed circles indicate the infiltrated regions.   Quantification of the luciferase signals shown in Figure 6C as assessed by ImageJ software.   Data information: Representative data were shown , and three replicates had similar results. In (D), error bars indicate mean ± SEM (n = 6). c indicate statistically significant differences among different groups (ANOVA, p-value < 0.05).


Competitive GST pull - down assays showing the effects of γb on interactions between NbNTRC and 2 - Cys Prx . GST - NbNTRC and 2 - Cys Prx - His were incubated with increasing amounts ( 5 , 10 , 20 , and 40 μg ) of γb and its mutant derivatives . Glutathione - Sepharose beads were washed and Western blot analysis was performed using anti - GST and anti - His antibodies . Data information : representative data are shown , and three independent experiments had similar results .

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Competitive GST pull-down assays showing the effects of γb on interactions between NbNTRC and 2-Cys Prx. GST-NbNTRC and 2-Cys Prx-His were incubated with increasing amounts (5, 10, 20, and 40 μg) of γb and its mutant derivatives. Glutathione-Sepharose beads were washed and Western blot analysis was performed using anti-GST and anti-His antibodies. Data information: representative data are shown, and three independent experiments had similar results.


Analysis of NbNTRC - 2 - Cys Prx interactions in the presence of the γb protein . NbNTRC - 3xFlag and 2 - Cys Prx - GFP were co - expressed in N . benthamiana leaves . Total proteins were extracted at 4 dpi and immunoprecipitated with anti - Flag beads , co - incubated with increasing amounts ( 20 , 40 , and 60 μg ) of γb - His and γb - His mutant derivatives . Input and IP proteins were analyzed by Western blotting with anti - His , anti - GFP or anti - Flag antibodies . The uncleaved 2 - Cys Prx with cTP and cleaved 2 - Cys Prx without cTP are marked with black and gray arrows , respectively . Data information : representative data are shown , and three independent experiments had similar results .

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Analysis of NbNTRC-2-Cys Prx interactions in the presence of the γb protein. NbNTRC-3xFlag and 2-Cys Prx-GFP were co-expressed in N. benthamiana leaves. Total proteins were extracted at 4 dpi and immunoprecipitated with anti-Flag beads, co-incubated with increasing amounts (20, 40, and 60 μg) of γb-His and γb-His mutant derivatives. Input and IP proteins were analyzed by Western blotting with anti-His, anti-GFP or anti-Flag antibodies. The uncleaved 2-Cys Prx with cTP and cleaved 2-Cys Prx without cTP are marked with black and gray arrows, respectively. Data information: representative data are shown, and three independent experiments had similar results.


Chl - HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in N . benthamiana epidermal cells . The pCB301 empty vector ( EV ) was used as a negative control . Scale bars , 30 μm . The false - blue color indicates the chloroplasts . Quantification of LRSV - dependent changes in Chl - HyPer2 fluorescence at 3 dpi . Data information : , data are representative of at least three independent experiments . error bars represent mean ± SEM ( n = 5 ) ; * * p - value < 0 . 01 from Student ' s t test .

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Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in N. benthamiana epidermal cells. The pCB301 empty vector (EV) was used as a negative control. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.   Quantification of LRSV-dependent changes in Chl-HyPer2 fluorescence at 3 dpi.   Data information: , data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.


Western blot analyses of 2 - Cys Prx abundance in EV and LRSV inoculated leaves under non - reducing conditions at 3 dpi . RbcL served as a loading control . Data information : representative data are shown , and three replicates had similar results .

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Western blot analyses of 2-Cys Prx abundance in EV and LRSV inoculated leaves under non-reducing conditions at 3 dpi. RbcL served as a loading control. Data information: representative data are shown, and three replicates had similar results.


Western blot analyses to detect LRSV γb in NbNTRC - OE and NbNTRC - KO N . benthamiana plants challenged with LRSV at 8 dpi . RbcL was used as the loading control . Data information : representative data are shown , and three replicates had similar results .

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Western blot analyses to detect LRSV γb in NbNTRC-OE and NbNTRC-KO N. benthamiana plants challenged with LRSV at 8 dpi. RbcL was used as the loading control. Data information: representative data are shown, and three replicates had similar results.


Chl - HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N . benthamiana epidermal cells . Scale bars , 30 μm . The false - blue color indicates the chloroplasts . Quantification of the TYMV - dependent changes in Chl - HyPer2 fluorescence at 5 dpi . Data information : data are representative of at least three independent experiments . error bars represent mean ± SEM ( n = 5 ) ; * * p - value < 0 . 01 from Student ' s t test .

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Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N. benthamiana epidermal cells. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.   Quantification of the TYMV-dependent changes in Chl-HyPer2 fluorescence at 5 dpi.   Data information: data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.


Western blot analysis of 2 - Cys Prx abundance in EV or TYMV inoculated leaves under non - reducing conditions at 5 dpi . RbcL was used as the loading control . Data information : representative data are shown , and three replicates had similar results .

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Western blot analysis of 2-Cys Prx abundance in EV or TYMV inoculated leaves under non-reducing conditions at 5 dpi. RbcL was used as the loading control. Data information: representative data are shown, and three replicates had similar results.


RT - qPCR analysis of TYMV RNA accumulation at 8 dpi . NbEF1α served as a internal control . Data information : representative data are shown , and three replicates had similar results .

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RT-qPCR analysis of TYMV RNA accumulation at 8 dpi. NbEF1α served as a internal control. Data information: representative data are shown, and three replicates had similar results.


Immunoblot analyses of TDG and TDG - SUMO in whole cell extracts of mESCs and MEFs . TDG and TDG - SUMO conjugates before ( 2i , 0 h ) and during differentiation of TDG wildtype ( TDGwt ) and TDG deficient ( TDGnull ) mESC and MEFs ( 5 µM all - trans retinoic acid ( RA , 24 h , 48 h ) . Percentages of TDG - SUMO relative to total TDG signal are indicated .

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Immunoblot analyses of TDG and TDG-SUMO in whole cell extracts of mESCs and MEFs. TDG and TDG-SUMO conjugates before (2i, 0 h) and during differentiation of TDG wildtype (TDGwt) and TDG deficient (TDGnull) mESC and MEFs (5 µM all-trans retinoic acid (RA, 24 h, 48 h). Percentages of TDG-SUMO relative to total TDG signal are indicated.


Immunoblot analyses of TDG , TDG - SUMO in whole cell extracts of mESCs TDG and TDG - SUMO before and after H2O2 treatment ( 10 mM , 15 min ) .

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Immunoblot analyses of TDG, TDG-SUMO in whole cell extracts of mESCs TDG and TDG-SUMO before and after H2O2 treatment (10 mM, 15 min).


Immunoblot analyses of TDG and TDG - SUMO in whole cell extracts of mESCs Chromatin fractionation from mESCs expressing wildtype ( ESC - TDGwt ) or SUMOylation - deficient TDG ( ESC - TDGsnm ) . Sol , chromatin unbound proteins ; Chr1 , chromatin associated proteins ; Chr2 , chromatin bound fraction ; Histone H2B , stably chromatin bound marker .

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Immunoblot analyses of TDG and TDG-SUMO in whole cell extracts of mESCs Chromatin fractionation from mESCs expressing wildtype (ESC-TDGwt) or SUMOylation-deficient TDG (ESC-TDGsnm). Sol, chromatin unbound proteins; Chr1, chromatin associated proteins; Chr2, chromatin bound fraction; Histone H2B, stably chromatin bound marker.


Immunoblot analyses of XRCC1 and , XRCC1 - SUMO , in whole cell extracts of mESCs and MEFs . XRCC1 and XRCC1 - SUMO conjugates before ( 2i , 0 h ) and during RA - induced differentiation ( 24 h , 48 h ) in mESCs and MEFs . Percentages of XRCC1 - SUMO relative to total XRCC1 signal are indicated .

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Immunoblot analyses of XRCC1 and , XRCC1-SUMO, in whole cell extracts of mESCs and MEFs. XRCC1 and XRCC1-SUMO conjugates before (2i, 0 h) and during RA-induced differentiation (24 h, 48 h) in mESCs and MEFs. Percentages of XRCC1-SUMO relative to total XRCC1 signal are indicated.


Immunoblot analyses of XRCC1 and XRCC1 - SUMO , in whole cell extracts of mESCs XRCC1 SUMOylation in native whole cells extracts before and after incubation with purified SUMO protease ( SENP2364 - 489 ) .

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Immunoblot analyses of XRCC1 and XRCC1-SUMO, in whole cell extracts of mESCs XRCC1 SUMOylation in native whole cells extracts before and after incubation with purified SUMO protease (SENP2364-489).


Immunoblot analyses of , XRCC1 and XRCC1 - SUMO in whole cell extracts of mESCs XRCC1 SUMOylation before and after exposure to H2O2 ( 5 mM , 15 min ) .

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Immunoblot analyses of , XRCC1 and XRCC1-SUMO in whole cell extracts of mESCs XRCC1 SUMOylation before and after exposure to H2O2 (5 mM, 15 min).


Immunoblot analyses of TET1 isoforms in whole cell extracts of mESCs and MEFs . TET1 and TET1s isoform protein levels in mESCs during RA induced differentiation and in MEFs .

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Immunoblot analyses of TET1 isoforms in whole cell extracts of mESCs and MEFs. TET1 and TET1s isoform protein levels in mESCs during RA induced differentiation and in MEFs.


Fluorescence microscopy of GFP - TDGwt GFP - TDGpip in mESCs . Scale bar : 100µM . Quantitation of fluorescence signals of GFP - TDGwt and GFP - TDGpip in mESCs . median with interquartile range . > 470 cells were analysed of each GFPTDGwt and GFPTDGpip ESCs .

Fluorescence microscopy of GFP-TDGwt GFP-TDGpip in mESCs. Scale bar: 100µM. Quantitation of fluorescence signals of GFP-TDGwt and GFP-TDGpip in mESCs. median with interquartile range. >470 cells were analysed of each GFPTDGwt and GFPTDGpip ESCs.


Immunoblot analysis of XRCC1 and XRCC1 - SUMO in GFP - TDGwt and GFP - TDGpip mESCs .

Immunoblot analysis of XRCC1 and XRCC1-SUMO in GFP-TDGwt and GFP-TDGpip mESCs.


" Far Western " blot analyses of BER protein interactions . Recombinant purified human TDG ( 10 µg ) , POLß ( 1 µg ) , APE1 ( 1 µg ) , and LIGIII ( 1 µg ) probed with XRCC1 and XRCC1 - SUMO ( 20 µg ) and immune - stained for XRCC1 .

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"Far Western" blot analyses of BER protein interactions. Recombinant purified human TDG (10 µg), POLß (1 µg), APE1 (1 µg), and LIGIII (1 µg) probed with XRCC1 and XRCC1-SUMO (20 µg) and immune-stained for XRCC1.


Immunoblot analysis of interactions of XRCC1 and XRCC1 - SUMO with endogenous TDG . Purified 6hisXRCC1 or in vitro modified and purified 6hisXRCC1 - SUMO was coupled to Ni - NTA agarose and incubated with mESC extracts . Input ( I ) , last wash ( LW ) and elution ( E ) fractions .

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Immunoblot analysis of interactions of XRCC1 and XRCC1-SUMO with endogenous TDG. Purified 6hisXRCC1 or in vitro modified and purified 6hisXRCC1-SUMO was coupled to Ni-NTA agarose and incubated with mESC extracts. Input (I), last wash (LW) and elution (E) fractions.


0 . 5 µM TDGwt , were incubated with biotinylated 5caC substrate ( G • 5caC DNA , 1 µM ) and homoduplex competitor DNA ( G • C DNA , 10 µM ) and equimolar amounts of XRCC1 , POLβ and LIGIII proteins .

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0.5 µM TDGwt, were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) and equimolar amounts of XRCC1, POLβ and LIGIII proteins.


Schematic of biotin - 5caC DNA substrate pulldown experiment . 0 . 5 µM TDGwt or TDGcat ( TDG ( N140A ) ) incubated with biotinylated 5caC substrate ( G • 5caC DNA , 1 µM ) and homoduplex competitor DNA ( G • C DNA , 10 µM ) as indicated and equimolar amounts of XRCC1 - SUMO ( > 50 % SUMOylated ) , POLβ and LIGIII .

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Schematic of biotin-5caC DNA substrate pulldown experiment. 0.5 µM TDGwt or TDGcat (TDG(N140A)) incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1-SUMO (>50% SUMOylated), POLβ and LIGIII.


1 µM biotinylated 5caC substrate ( G • 5caC DNA ) and 10 µM homoduplex competitor DNA ( G • C DNA ) were incubated with XRCC1 - SUMO ( 50 % SUMOylated ) and BER proteins as indicated .

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1 µM biotinylated 5caC substrate (G•5caC DNA) and 10 µM homoduplex competitor DNA (G•C DNA) were incubated with XRCC1-SUMO (50% SUMOylated) and BER proteins as indicated.


0 . 5 µM human TDGwt protein were incubated with 1 µM biotinylated 5caC substrate ( G • 5caC DNA ) , 0 . 5 µM SUMO and 10 µM homoduplex competitor DNA ( G • C DNA ) as indicated . Shown is an immunoblot analysis of TDG and free SUMO .

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0.5 µM human TDGwt protein were incubated with 1 µM biotinylated 5caC substrate (G•5caC DNA), 0.5 µM SUMO and 10 µM homoduplex competitor DNA (G•C DNA) as indicated. Shown is an immunoblot analysis of TDG and free SUMO.


" Far Western " blot analyses of TDGwt and TDGE310Q with 6hisXRCC1 and 6hisXRCC1 - SUMO . TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes and probed with XRCC1 or XRCC1 - SUMO ( probe : XRCC1 or XRCC1SUMO ) where indicated . TDG and XRCC1 probes were detected by immunoblotting with anti TDG and anti XRCC1 antibodies as indicated .

"Far Western" blot analyses of TDGwt and TDGE310Q with 6hisXRCC1 and 6hisXRCC1-SUMO. TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes and probed with XRCC1 or XRCC1-SUMO (probe:XRCC1 or XRCC1SUMO) where indicated. TDG and XRCC1 probes were detected by immunoblotting with anti TDG and anti XRCC1 antibodies as indicated.


Schematic of biotin - 5caC DNA substrate pulldown experiment . Immunoblot analysis of purified TDGwt or TDG SUMO - binding mutant ( TDGE310Q ) , 6hisXRCC1 / XRCC1 - SUMO and 6his BER proteins bound to Streptavidin ( St ) Biotin - 5caC DNA substrate . 0 . 5 µM TDGwt or TDG SUMO - binding mutant ( TDGE310Q ) were incubated with biotinylated 5caC substrate ( G • 5caC DNA , 1 µM ) and homoduplex competitor DNA ( G • C DNA , 10 µM ) as indicated and equimolar amounts of XRCC1 , POLβ , APE1 and LIGIII proteins . Grey ovals , BERosome .

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Schematic of biotin-5caC DNA substrate pulldown experiment. Immunoblot analysis of purified TDGwt or TDG SUMO-binding mutant (TDGE310Q), 6hisXRCC1/XRCC1-SUMO and 6his BER proteins bound to Streptavidin (St) Biotin-5caC DNA substrate. 0.5 µM TDGwt or TDG SUMO-binding mutant (TDGE310Q) were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1, POLβ, APE1 and LIGIII proteins. Grey ovals, BERosome.


Reconstitution SUMO conjugation / de - conjugation reaction with purified SAE1 / 2 , UBC9 , SUMO protease SENP2364 - 489 ) , free SUMO in equimolar amounts and 20 - fold molar excess over TDG . TDG SUMOylation was performed in the presence or absence of AP - site containing synthetic DNA double - strands as ( as indicated ( G • AP ) . TDG and TDG - SUMO were detected by immunoblotting . SUMOylation of DNA ( G • AP ) bound TDG is strongly reduced but a 20 - fold molar excess of free SUMO compensates the inhibitory effect of DNA .

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Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA.


Reconstitution SUMO conjugation / de - conjugation reaction with purified SAE1 / 2 , UBC9 , SUMO protease SENP2364 - 489 ) , free SUMO in equimolar amounts and 20 - fold molar excess over TDG . TDG SUMOylation was performed in the presence or absence of AP - site containing synthetic DNA double - strands as ( as indicated ( G • AP ) . TDG and TDG - SUMO were detected by immunoblotting . SUMOylation of DNA ( G • AP ) bound TDG is strongly reduced but a 20 - fold molar excess of free SUMO compensates the inhibitory effect of DNA . SUMOylation assay as in ( A ) with SUMO provided as XRCC1 - SUMO instead of free SUMO .

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Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMOylation assay as in (A) with SUMO provided as XRCC1-SUMO instead of free SUMO.


Reconstitution SUMO conjugation / de - conjugation reaction with purified SAE1 / 2 , UBC9 , SUMO protease SENP2364 - 489 ) , free SUMO in equimolar amounts and 20 - fold molar excess over TDG . TDG SUMOylation was performed in the presence or absence of AP - site containing synthetic DNA double - strands as ( as indicated ( G • AP ) . TDG and TDG - SUMO were detected by immunoblotting . SUMOylation of DNA ( G • AP ) bound TDG is strongly reduced but a 20 - fold molar excess of free SUMO compensates the inhibitory effect of DNA . SUMO transfer as depicted in ( B ) with additional APE1 , POLß , LIGIII , and TDG .

Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMO transfer as depicted in (B) with additional APE1, POLß, LIGIII, and TDG.


Immunoblot analysis of XRCC1 , TDG , TDG - SUMO in TDGwt ( wt ) , TDGsnm ( snm ) and XRCC1null mESCs ( null ) .

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Immunoblot analysis of XRCC1, TDG, TDG-SUMO in TDGwt (wt), TDGsnm (snm) and XRCC1null mESCs (null).


Immunoblot analysis of TDG and TDG - SUMO in TDGwt ( wt ) and XRCC1null mESCs ( null ) exposed to H2O2 as indicated .

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Immunoblot analysis of TDG and TDG-SUMO in TDGwt (wt) and XRCC1null mESCs (null) exposed to H2O2 as indicated.


Top : Schematic of the SUMO - mediated TDG - BERosome ( grey oval ) formation and DNA processing . Bottom : Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double - strands in presence of dynamic SUMO - conjugation / de - conjugation . Left panels lanes 3 - 14 : BER reconstitution with XRCC1 or XRCC1 - SUMO as indicated . Right panels lanes 15 - 21 : BER reconstitution with free SUMO or XRCC1 - SUMO as SUMO donor as indicated . ds59merDNA substrates : lane 1 - 2 CG / CG HpaII methylation sensitive restriction site , cleavable by HpaII ; lane 3 - 6 , 8 - 14 , 15 - 17 , 21 HpaII methylation sensitive restriction site restriction site with a caC ( 5caCG / CG ) , not cleavable by HpaII ; lane 7 and 20 repaired Product ( CC / CG ) that is cleavable by HpaII after excision / repair of caC / CG to CG / CG by BER . Percentage of total signal of fully repaired product is indicated . ss59merDNA , single stranded DNA .

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Top: Schematic of the SUMO-mediated TDG-BERosome (grey oval) formation and DNA processing. Bottom: Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double-strands in presence of dynamic SUMO-conjugation/de-conjugation. Left panels lanes 3-14: BER reconstitution with XRCC1 or XRCC1-SUMO as indicated. Right panels lanes 15-21: BER reconstitution with free SUMO or XRCC1-SUMO as SUMO donor as indicated. ds59merDNA substrates: lane 1-2 CG/CG HpaII methylation sensitive restriction site, cleavable by HpaII; lane 3-6, 8-14, 15-17, 21 HpaII methylation sensitive restriction site restriction site with a caC (5caCG/CG), not cleavable by HpaII; lane 7 and 20 repaired Product (CC/CG) that is cleavable by HpaII after excision/repair of caC/CG to CG/CG by BER. Percentage of total signal of fully repaired product is indicated. ss59merDNA, single stranded DNA.