E LC3 - II formation in HeLa cells transfected with Flag - tagged TFEB or TFEB mutants after TFEB RNAi . The cells were treated with or without amino acid starvation .

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

E LC3-II formation in HeLa cells transfected with Flag-tagged TFEB or TFEB mutants after TFEB RNAi. The cells were treated with or without amino acid starvation.


F GFP - p62 levels in HEK293 cells stably expressing GFP - p62 . The cells were treated with or without addition of CQ .

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

F GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were treated with or without addition of CQ.


Representative images of mCherry - Atg8a ( red ) ( G ) in Drosophila larval fat body in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as the control and DAPI staining ( blue ) was used to indicate the cell nucleus ( Scale bars , 10 µm ) .

O O O O O B-MEASURED_VAR 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

Representative images of mCherry-Atg8a (red) (G) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).


Representative images of LysoTracker ( red ) staining ( H ) in Drosophila larval fat body in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as the control and DAPI staining ( blue ) was used to indicate the cell nucleus ( Scale bars , 10 µm ) .

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

Representative images of LysoTracker (red) staining (H) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).


Quantification of mCherry - ATG8a puncta ( I ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , Student ' s t - test ) .

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

Quantification of mCherry-ATG8a puncta (I) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


Quantification of LysoTracker puncta ( J ) ( Graph represents data from three independent experiments with ≥ 30 cells per condition ; mean ± SEM ; * * * p < 0 . 001 , 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

Quantification of LysoTracker puncta (J) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding . The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR - Gal4 driver . Drosophila ( GMR - Gal4 / + ) was used as the control .

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

The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control.


C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR - Gal4 driver . The Drosophila were fed with or without CQ . Drosophila ( GMR - Gal4 / + ) was used as the control .

O O O O O O O O O O O O O O O O O O O O O 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 light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR-Gal4 driver. The Drosophila were fed with or without CQ. Drosophila (GMR-Gal4/+) was used as the control.


D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ ( 30 flies per condition were randomly selected for analyses ; * p < 0 . 05 , * * * p < 0 . 001 , ꭓ2 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

D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ (30 flies per condition were randomly selected for analyses; *p < 0.05, ***p < 0.001, ꭓ2 test).


D . Quantity of Ki67 + cells in tumour and representative histology images analysed by Immunohistochemistry ( IHC ) that described in ( A ) were shown . Ki67 expression was analysed in average number of four high power field ( HPF ) . Data information : All data are mean ± s . e . m . NS , not significant . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

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 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 O O

D. Quantity of Ki67+ cells in tumour and representative histology images analysed by Immunohistochemistry (IHC) that described in (A) were shown. Ki67 expression was analysed in average number of four high power field (HPF). Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


F . Relative mRNA expression of IL - 22 , IL - 17a , IFN - γ , IL - 1β , IL - 6 , IL - 10 , Muc1 of colon tumour described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

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

F. Relative mRNA expression of IL-22, IL-17a, IFN-γ, IL-1β, IL-6, IL-10, Muc1 of colon tumour described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


G . Representative immunoblot images ( left ) and quantification analysis of P - Stat3 , Bcl - xl , P - IκBα protein expression ( right ) in the tumour described in ( A ) were shown . Data information : All data are mean ± s . e . m . NS , not significant . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O O 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. Representative immunoblot images (left) and quantification analysis of P-Stat3, Bcl-xl, P-IκBα protein expression (right) in the tumour described in (A) were shown. Data information: All data are mean ± s.e.m. NS, not significant. *P < 0.05, **P < 0.01, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


representative histological images ( B ) of wild - type control mice or BTNL2 - KO mice treated with 2 % DSS for 8 days were shown ( n = 15 ) .

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

representative histological images (B) of wild-type control mice or BTNL2-KO mice treated with 2% DSS for 8 days were shown (n=15).


E . Relative mRNA expression of IL - 22 , RegⅢγ , RegⅢβ , Muc1 , IL - 17a , IFN - γ , IL - 1β , IL - 6 and IL - 10 of colonic tissues described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 15 . Data are representative of three biological replicates .

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

E. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β, IL-6 and IL-10 of colonic tissues described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant. **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=15. Data are representative of three biological replicates.


representative H & E images ( H ) of wild - type control mice or BTNL2 - KO mice treated with Fc or mIL - 22 - Fc ( ip . 5 μg / mouse ) at day 0 , 2 , 4 , 6 during 2 % DSS treatment were shown ( n = 12 ) . Data information Data are representative of three biological replicates H ) .

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

representative H&E images (H) of wild-type control mice or BTNL2-KO mice treated with Fc or mIL-22-Fc (ip. 5 μg/mouse) at day 0, 2, 4, 6 during 2% DSS treatment were shown (n=12). Data information Data are representative of three biological replicates H).


B . IL - 22 and IL - 17a expression in lamina propria lymphocytes ( LPLs ) after cultured in plate - coated Fc , mBTNL2 - Fc ( 30 μg / mL ) with or without IL - 23 ( 10 ng / mL ) for 20 h was analyzed by ELISA . C . IL - 22 production from cells of LPLs cultured in the presence of plate - coated Fc , mBTNL2 - Fc with anti - control Ab or anti - BTNL2 mAb for 20 h was analyzed by ELISA . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 6 Pooled data from three biological replicates were

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

B. IL-22 and IL-17a expression in lamina propria lymphocytes (LPLs) after cultured in plate-coated Fc, mBTNL2-Fc (30 μg/mL) with or without IL-23 (10 ng/mL) for 20 h was analyzed by ELISA. C. IL-22 production from cells of LPLs cultured in the presence of plate-coated Fc, mBTNL2-Fc with anti-control Ab or anti-BTNL2 mAb for 20 h was analyzed by ELISA. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Pooled data from three biological replicates were shown


D . CD4 + T cells , γδ T cells , ILC3s , CD8 + T cells sorted by FACS were cultured in the plate - coated Fc , mBTNL2 - Fc with or without IL - 23 ( 10 ng / mL ) for 20 h , and IL - 22 production was analyzed by ELISA . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 6 Pooled data from three biological replicates were

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

D. CD4+ T cells, γδ T cells, ILC3s, CD8+ T cells sorted by FACS were cultured in the plate-coated Fc, mBTNL2-Fc with or without IL-23 (10 ng/mL) for 20 h, and IL-22 production was analyzed by ELISA. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Pooled data from three biological replicates were shown


F . IL - 22 production was analyzed by Flow cytometry in LPLs of wild - type mice treated with Fc or mBTNL2 - Fc ( ip . 50 μg / mouse ) on day 0 , 2 , 4 , 6 during 2 % DSS treatment . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one mouse , n = 12 for the Fc group and n = 14 for the mBTL2 - Fc group ( F ) . Pooled data from three biological replicates were shown F .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. IL-22 production was analyzed by Flow cytometry in LPLs of wild-type mice treated with Fc or mBTNL2-Fc (ip. 50 μg/mouse) on day 0, 2, 4, 6 during 2%DSS treatment. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one mouse, n=12 for the Fc group and n=14 for the mBTL2-Fc group (F). Pooled data from three biological replicates were shown F.


G . IL - 22 , IL - 17a , RORC and HIF - 1α expression in splenocytes after cultured in plate - coated Fc or mBTNL2 - Fc ( 30 μg / mL ) for 20 h was analyzed by real - time PCR . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 6 Data are representative of three biological

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. IL-22, IL-17a, RORC and HIF-1α expression in splenocytes after cultured in plate-coated Fc or mBTNL2-Fc (30 μg/mL) for 20 h was analyzed by real-time PCR. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=6 Data are representative of three biological replicates


E . Relative mRNA expression of IL - 22 , RegⅢγ , RegⅢβ , Muc1 , IL - 17a , IFN - γ , IL - 1β and IL - 6 from colonic tissues described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 15 Pooled data from three biological

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

E. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β and IL-6 from colonic tissues described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=15 Pooled data from three biological replicates


J . Relative mRNA expression of IL - 22 , RegⅢγ , RegⅢβ , Muc1 , IL - 17a , IFN - γ , IL - 1β and IL - 6 of colonic tissue described in ( F ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test Each dot represents one repetition , n = 12 Pooled data from three biological

J. Relative mRNA expression of IL-22, RegⅢγ, RegⅢβ, Muc1, IL-17a, IFN-γ, IL-1β and IL-6 of colonic tissue described in (F) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test Each dot represents one repetition, n=12 Pooled data from three biological replicates


Representative colonic tumour burden ( B ) that described in ( A ) were shown ( n = 12 ) . Data information : Data are representative of three biological replicates (

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

Representative colonic tumour burden (B) that described in (A) were shown (n=12). Data information: Data are representative of three biological replicates (B


F . Relative mRNA expression of IL - 22 , IL - 17a , IFN - γ , IL - 1β , IL - 6 , IL - 10 and Muc1 of colonic tumour described in ( A ) was shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test for F , Each dot represents one mouse , n = 12 . Pooled data from three biological

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

F. Relative mRNA expression of IL-22, IL-17a, IFN-γ, IL-1β, IL-6, IL-10 and Muc1 of colonic tumour described in (A) was shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for F, Each dot represents one mouse, n=12. Pooled data from three biological replicates


G . Representative immunoblot images ( left ) and quantification analysis of P - Stat3 , Bcl - xl , P - IκBα protein ( right ) expression of tumour described in ( A ) were shown . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test for G , Each dot represents one mouse , n = 12 . Data are representative of three biological replicates left panel of G ) . Pooled data from three biological replicates are shown in right panel of

O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunoblot images (left) and quantification analysis of P-Stat3, Bcl-xl, P-IκBα protein (right) expression of tumour described in (A) were shown. Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for G, Each dot represents one mouse, n=12. Data are representative of three biological replicates left panel of G). Pooled data from three biological replicates are shown in right panel of G


H , Quantity of Ki67 + cells in tumours and representative histology images that described in ( A ) were shown . Ki67 expression was analysed in average number of four high power field ( HPF ) . Data information : All data are mean ± s . e . m . NS , not significant . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 based on two - tailed Student ' s t test for H ) . Each dot represents one mouse , n = 12 . Pooled data from three biological replicates are shown in H .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Quantity of Ki67+ cells in tumours and representative histology images that described in (A) were shown. Ki67 expression was analysed in average number of four high power field (HPF). Data information: All data are mean ± s.e.m. NS, not significant., *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 based on two-tailed Student's t test for H). Each dot represents one mouse, n=12. Pooled data from three biological replicates are shown in H.


A . Survival of patients with colorectal cancer from Kaplan - Meier database was analyzed Left panel of A were representative of 150 technical replicates , and right panel of A were representative of 499 technical replicates .

O O O O O O O O O O O O O O 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. Survival of patients with colorectal cancer from Kaplan-Meier database was analyzed Left panel of A were representative of 150 technical replicates, and right panel of A were representative of 499 technical replicates.


representative histology images in human colorectal cancer specimens ( 1 # - 5 # ) and human colon polyp specimens ( 6 # - 7 # ) were shown . Number of cells expressed BTNL2 or IL - 22 was analysed in high power field ( HPF ) , arrows indicate cells expressed BTNL2 or IL - 22 ( B ) .

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

representative histology images in human colorectal cancer specimens (1#-5#) and human colon polyp specimens (6#-7#) were shown. Number of cells expressed BTNL2 or IL-22 was analysed in high power field (HPF), arrows indicate cells expressed BTNL2 or IL-22 (B).


D - E . Expression of BTNL2 in normal colon tissue or DSS + AOM induced tumour tissue were measured by immunoblot ( D ) and real - time PCR ( E ) ( n = 5 ) . Data information : All data are mean ± s . e . m . NS , not significant . , * * P < 0 . 01 based on two - tailed Student ' s t test for ( E ) . Each dot represents one mouse , n = 5 .

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

D-E. Expression of BTNL2 in normal colon tissue or DSS+AOM induced tumour tissue were measured by immunoblot (D) and real-time PCR (E) (n=5). Data information: All data are mean ± s.e.m. NS, not significant., **P < 0.01 based on two-tailed Student's t test for (E). Each dot represents one mouse, n=5.


( A ) Overexpression of RNF26 promoted polyubiquitination of MITA . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with a control or RNF26 expression plasmid ( 0 . 5 , 1 , 2 µg ) . Twenty - four hours after transfection , cells were subjected to immunoprecipitation ( IP ) under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - HA ( upper panel ) , anti - Flag ( middle panel ) and anti - RNF26 ( bottom panel ) . Ub , ubiquitin .

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 B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

(A) Overexpression of RNF26 promoted polyubiquitination of MITA. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with a control or RNF26 expression plasmid (0.5, 1, 2 µg). Twenty-four hours after transfection, cells were subjected to immunoprecipitation (IP) under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-HA (upper panel), anti-Flag (middle panel) and anti-RNF26 (bottom panel). Ub, ubiquitin.


( B ) RNF26 - mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26 . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and HA - Ub ( 1 µg ) together with RNF26 or its mutants ( 1 µg each ) . IP under denatured conditions and ubiquitination assay was performed as described in ( A ) .

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

(B) RNF26-mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with RNF26 or its mutants (1 µg each). IP under denatured conditions and ubiquitination assay was performed as described in (A).


( C ) UbcH5 mediated polyubiquitination of MITA by RNF26 . The RNF26 and MITA proteins were obtained by in vitro transcription and translation , then incubated with biotin - Ub , E1 and the indicated E2s . Polyubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) .

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

(C) UbcH5 mediated polyubiquitination of MITA by RNF26. The RNF26 and MITA proteins were obtained by in vitro transcription and translation, then incubated with biotin-Ub, E1 and the indicated E2s. Polyubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels).


( D ) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro . MITA , RNF26 and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and MITA were incubated with RNF26 or its mutants , followed by ubiquitination and immunoblot analysis as described in ( C ) . All experiments were repeated for at least three times with similar results .

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

(D) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro. MITA, RNF26 and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and MITA were incubated with RNF26 or its mutants, followed by ubiquitination and immunoblot analysis as described in (C). All experiments were repeated for at least three times with similar results.


( A ) RNF26 interacted with MITA . The 293 cells ( 5 × 106 ) were transfected with the indicated plasmids ( 5 µg each ) . Twenty - four hours later , cells were lysed and cell lysates were immunoprecipitated with anti - Flag or control IgG . The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti - HA or anti - Flag .

O O O B-MEASURED_VAR 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

(A) RNF26 interacted with MITA. The 293 cells (5×106) were transfected with the indicated plasmids (5 µg each). Twenty-four hours later, cells were lysed and cell lysates were immunoprecipitated with anti-Flag or control IgG. The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti-HA or anti-Flag.


( B ) RNF26 was physiologically associated with MITA . THP - 1 cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins . NC , negative control .

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 O 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) RNF26 was physiologically associated with MITA. THP-1 cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins. NC, negative control.


( C ) RNF26 is localized to the ER and mitochondria . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Twenty - four hours after transfection , the cells were stained with Mito - Tracker Red for 15 minutes or left untreated . The cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis .

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

(C) RNF26 is localized to the ER and mitochondria. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Twenty-four hours after transfection, the cells were stained with Mito-Tracker Red for 15 minutes or left untreated. The cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis.


D ) Subcellular distribution of RNF26 . The 293 cells ( 5 × 107 ) were infected with SeV for the indicated time points or left uninfected . Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins .

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

D) Subcellular distribution of RNF26. The 293 cells (5×107) were infected with SeV for the indicated time points or left uninfected. Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins.


( E ) RNF26 and MITA were colocalized to the ER . HeLa cells ( 2 × 105 ) were transfected with the indicated plasmids ( 0 . 5 µg each ) . Eighteen hours after transfection , cells were infected with SeV or HSV - 1 for 6 hours or left uninfected , followed by ER - Tracker Blue / White staining for 30 minutes . Cells were fixed with 4 % paraformaldehyde and subjected for confocal microscopy analysis . All experiments were repeated for at least three times with similar results .

O O O B-MEASURED_VAR 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 O O O O

(E) RNF26 and MITA were colocalized to the ER. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Eighteen hours after transfection, cells were infected with SeV or HSV-1 for 6 hours or left uninfected, followed by ER-Tracker Blue/White staining for 30 minutes. Cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis. All experiments were repeated for at least three times with similar results.


( A ) RNF26 mediated polyubiquitination of MITA at K150 . The 293 cells ( 5 × 106 ) were transfected with HA - Ub ( 1 µg ) and RNF26 ( 1 µg ) together with Flag - MITA or the indicated mutants ( 5 µg each ) . Twenty - four hours after transfection , cells were subjected IP under denatured conditions with anti - Flag and immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated .

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

(A) RNF26 mediated polyubiquitination of MITA at K150. The 293 cells (5×106) were transfected with HA-Ub (1 µg) and RNF26 (1 µg) together with Flag-MITA or the indicated mutants (5 µg each). Twenty-four hours after transfection, cells were subjected IP under denatured conditions with anti-Flag and immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.


B ) RNF26 targeted MITA for polyubiquitination at K150 in vitro . RNF26 , MITA and its mutants were obtained by in vitro transcription and translation . Biotin - Ub , E1 , UbcH5 and RNF26 were incubated with MITA or its mutants . The ubiquitination of MITA was examined by immunoblot analysis with HRP - streptavidin ( top panel ) . The inputs of RNF26 and MITA were analyzed by immunoblots with anti - MITA and anti - RNF26 ( bottom panels ) . All experiments were repeated for at least three times with similar results .

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

B) RNF26 targeted MITA for polyubiquitination at K150 in vitro. RNF26, MITA and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and RNF26 were incubated with MITA or its mutants. The ubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels). All experiments were repeated for at least three times with similar results.


( A and B ) RNF26 targeted MITA for K11 - linked polyubiquitination . The 293 cells ( 5 × 106 ) were transfected with Flag - MITA ( 5 µg ) and RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Twenty - four hours after transfection , cells were subjected for IP under denatured conditions with limited amount of anti - Flag ( 0 . 5 µg ) so that equal amount of Flag - MITA is pulled down . The immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - Flag ( lower panel ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated . Ub - AKR , all lysine residues of ubiquitin were mutated to arginine .

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

(A and B) RNF26 targeted MITA for K11-linked polyubiquitination. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Twenty-four hours after transfection, cells were subjected for IP under denatured conditions with limited amount of anti-Flag (0.5 µg) so that equal amount of Flag-MITA is pulled down. The immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated. Ub-AKR, all lysine residues of ubiquitin were mutated to arginine.


( C ) Effects of RNF26 - RNAi plasmids on the expression of RNF26 . In the upper panels , the 293 cells ( 1 × 106 ) were transfected with the expression plasmids for RNF26 - Flag ( 0 . 5 µg ) and HA - β - actin ( 0 . 1 µg ) together with the indicated RNAi plasmids ( 1 µg each ) . Twenty - four hours after transfection , whole cell lysates were analyzed by immunoblots with anti - Flag or anti - HA . In the lower panels , 293 cells were transduced with a control or RNF26 - RNAi by retrovirus mediated gene transfer . Cells ( 1 × 106 ) were lysed and whole cell lysates were analyzed by immunoblots with anti - RNF26 or anti - β - actin .

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 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 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-MEASURED_VAR O O O O O O O

(C) Effects of RNF26-RNAi plasmids on the expression of RNF26. In the upper panels, the 293 cells (1×106) were transfected with the expression plasmids for RNF26-Flag (0.5 µg) and HA-β-actin (0.1 µg) together with the indicated RNAi plasmids (1 µg each). Twenty-four hours after transfection, whole cell lysates were analyzed by immunoblots with anti-Flag or anti-HA. In the lower panels, 293 cells were transduced with a control or RNF26-RNAi by retrovirus mediated gene transfer. Cells (1×106) were lysed and whole cell lysates were analyzed by immunoblots with anti-RNF26 or anti-β-actin.


( D ) Immunoblot analysis of Flag - tagged ubiquitin expression in THP - 1 cells stably transfected with Flag - Ub - K11O plasmid . Whole cell lysates of THP - 1 - Flag - Ub - K11O - RNF26 - RNAi and control cells ( 1 × 106 ) were analyzed by immunoblots with antibodies against the indicated proteins . RNF26 - RNAi # 1 was used here and in the following experiments if not noted .

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 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-MEASURED_VAR O O O O O O O O O O O O O O O O

(D) Immunoblot analysis of Flag-tagged ubiquitin expression in THP-1 cells stably transfected with Flag-Ub-K11O plasmid. Whole cell lysates of THP-1-Flag-Ub-K11O-RNF26-RNAi and control cells (1×106) were analyzed by immunoblots with antibodies against the indicated proteins. RNF26-RNAi #1 was used here and in the following experiments if not noted.


( E and F ) Effects of RNF26 knockdown on virus - induced K11 - linked polyubiquitination of endogenous MITA . In ( E ) , THP - 1 - Flag - Ub - K11O - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti - MITA . The immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panels ) or anti - MITA ( lower panels ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins . In ( F ) , 293 - HA - Ub - K11O cells ( 2 × 107 ) were transfected with a control or RNF26 - RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , puromycin ( 1 µg / mL ) was added into the culture medium . The cells were selected for twenty - four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in ( E ) . All experiments were repeated for at least three times with similar results .

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

(E and F) Effects of RNF26 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. In (E), THP-1-Flag-Ub-K11O-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti-MITA. The immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panels) or anti-MITA (lower panels). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins. In (F), 293-HA-Ub-K11O cells (2×107) were transfected with a control or RNF26-RNAi plasmid (10 µg each). Twelve hours after transfection, puromycin (1 µg/mL) was added into the culture medium. The cells were selected for twenty-four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in (E). All experiments were repeated for at least three times with similar results.


A ) Effects of RNF26 knockdown on virus - induced polyubiquitination of endogenous MITA . The THP - 1 - RNF26 - RNAi or control cells ( 2 × 107 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - Ub ( K48 ) ( upper panel ) , anti - Ub ( K63 ) ( middle panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins .

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

A) Effects of RNF26 knockdown on virus-induced polyubiquitination of endogenous MITA. The THP-1-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Ub(K48) (upper panel), anti-Ub(K63) (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.


( B ) Effects of RNF5 knockdown on virus - induced K11 - linked polyubiquitination of endogenous MITA . The 293 - HA - Ub - K11O cells ( 2 × 107 ) were transfected with the indicated RNAi plasmid ( 10 µg each ) . Twelve hours after transfection , the cells were selected with puromycin ( 1 µg / mL ) for twenty - four hours and infected with SeV for the indicated time points or left uninfected . Cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies .

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

(B) Effects of RNF5 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. The 293-HA-Ub-K11O cells (2×107) were transfected with the indicated RNAi plasmid (10 µg each). Twelve hours after transfection, the cells were selected with puromycin (1 µg/mL) for twenty-four hours and infected with SeV for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies.


( C ) RNF26 and RNF5 competed with each other on MITA polyubiquitination . The 293 cells ( 5 × 106 ) were transfected with MITA ( 5 µg ) , Flag - Ub - K48O and HA - Ub - K11O ( 1 µg each ) together with indicated amount of RNF26 and RNF5 . Twenty - four hours after transfection , cell lysates were subjected to IP under denatured conditions with anti - MITA and the immunoprecipitates were analyzed by immunoblots with anti - Flag ( upper panel ) , anti - HA ( middle panel ) or anti - MITA ( lower panel ) . The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins .

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

(C) RNF26 and RNF5 competed with each other on MITA polyubiquitination. The 293 cells (5×106) were transfected with MITA (5 µg), Flag-Ub-K48O and HA-Ub-K11O (1 µg each) together with indicated amount of RNF26 and RNF5. Twenty-four hours after transfection, cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panel), anti-HA (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.


( D ) Effects of RNF26 knockdown on virus - triggered MITA degradation . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected . Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins ( upper immunoblots ) . The relative protein levels of MITA in reference to β - actin were analyzed by Quantity One program and data shown are mean ± S . D . of three independent experiments ( lower histographs ) .

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

(D) Effects of RNF26 knockdown on virus-triggered MITA degradation. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins (upper immunoblots). The relative protein levels of MITA in reference to β-actin were analyzed by Quantity One program and data shown are mean ± S.D. of three independent experiments (lower histographs).


( A ) Effects of RNF26 knockdown on SeV - triggered activation of the IFN - β promoter . The 293 cells ( 2 × 105 ) were transfected with the IFN - β promoter reporter ( 0 . 1 µg ) and the indicated RNAi plasmid ( 0 . 5 µg each ) . Thirty hours after transfection , cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

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

(A) Effects of RNF26 knockdown on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and the indicated RNAi plasmid (0.5 µg each). Thirty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( B and C ) Effects of RNF26 knockdown on virus - triggered induction of IFN - β in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( B )

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

(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B)


( B and C ) Effects of RNF26 knockdown on virus - triggered induction of IFN - β in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( B ) or ELISA analysis (

O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-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 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 and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C


( D ) Effects of RNF26 knockdown on virus - triggered induction of IFNB1 gene in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with VSV , EMCV or ECTV for the indicated time points or left uninfected before quantitative real - time PCR analysis was performed as in ( B ) .

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 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

(D) Effects of RNF26 knockdown on virus-triggered induction of IFNB1 gene in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with VSV, EMCV or ECTV for the indicated time points or left uninfected before quantitative real-time PCR analysis was performed as in (B).


( E and F ) Effects of RNF26 knockdown on virus - triggered induction of TNFα in THP - 1 cells . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected followed by quantitative real - time PCR ( E ) and ELISA analysis ( F ) .

O 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 O O O O O O O O O O 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 and F) Effects of RNF26 knockdown on virus-triggered induction of TNFα in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (E) and ELISA analysis (F).


( G ) Effects of RNF26 knockdown on virus - triggered phosphorylation of TBK1 , IRF3 and IκBα . The THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were infected with SeV or HSV - 1 for the indicated time points or left uninfected , whole cell lysates were analyzed by immunoblots with anti - p - TBK1 , anti - TBK1 , anti - p - IRF3 , anti - IRF3 , anti - p - IκBα , anti - IκBα , anti - RNF26 or anti - β - actin as indicated . All experiments were repeated for at least three times with similar results . The bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR 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 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 B-MEASURED_VAR O O O B-MEASURED_VAR 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 O O O O O O O O O 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) Effects of RNF26 knockdown on virus-triggered phosphorylation of TBK1, IRF3 and IκBα. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected, whole cell lysates were analyzed by immunoblots with anti-p-TBK1, anti-TBK1, anti-p-IRF3, anti-IRF3, anti-p-IκBα, anti-IκBα, anti-RNF26 or anti-β-actin as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


A ) Effects of RNF26 knockdown on IRF3 level . In the left panel , whole cell lysates of THP - 1 - RNF26 - RNAi or control cells ( 1 × 106 ) were analyzed by immunoblots with the indicated antibodies . In the right panel , cells ( 1 × 106 ) were subjected to quantitative real - time PCR analysis .

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

A) Effects of RNF26 knockdown on IRF3 level. In the left panel, whole cell lysates of THP-1-RNF26-RNAi or control cells (1×106) were analyzed by immunoblots with the indicated antibodies. In the right panel, cells (1×106) were subjected to quantitative real-time PCR analysis.


B ) Effects of RNF26 on IRF3 stability . The 293 cells ( 1 × 106 ) were transfected with HA - IRF3 ( 0 . 5 µg ) and HA - β - actin ( 0 . 1 µg ) together with RNF26 or its mutant ( 0 . 3 µg each ) . Twenty - four hours later , whole cell lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

O O O O B-MEASURED_VAR 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 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 B-MEASURED_VAR O

B) Effects of RNF26 on IRF3 stability. The 293 cells (1×106) were transfected with HA-IRF3 (0.5 µg) and HA-β-actin (0.1 µg) together with RNF26 or its mutant (0.3 µg each). Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( C ) Effects of RNF26 and its mutant on SeV - triggered activation of the IFN - β promoter . The 293 cells ( 2 × 105 ) were transfected with the IFN - β promoter reporter ( 0 . 1 µg ) and RNF26 or its mutant ( 0 . 1 µg each ) . Twenty hours after transfection , cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed .

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

(C) Effects of RNF26 and its mutant on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and RNF26 or its mutant (0.1 µg each). Twenty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.


( D ) Effects of RNF26 on IRF3 ubiquitination . The 293 cells ( 5 × 106 ) were transfected with Flag - IRF3 ( 5 µg ) and RNF26 ( 1 µg ) together with HA - Ub or its mutants ( 1 µg each ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment . The cell lysates were subjected to IP under denatured conditions with anti - Flag and the immunoprecipitates were analyzed by immunoblots with anti - HA ( upper panels ) or anti - Flag ( lower panels ) . The whole cell lysates were analyzed by immunoblots with anti - Flag or anti - RNF26 as indicated .

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

(D) Effects of RNF26 on IRF3 ubiquitination. The 293 cells (5×106) were transfected with Flag-IRF3 (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Eighteen hours after transfection, 3-MA (500 ng/mL) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment. The cell lysates were subjected to IP under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panels) or anti-Flag (lower panels). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.


( E ) Effects of inhibitors on RNF26 - mediated destabilization of IRF3 . The 293 cells ( 1 × 106 ) were transfected with the indicated plasmids as described in ( B ) . Eighteen hours after transfection , 3 - MA ( 500 ng / mL ) , NH4Cl ( 25 mM ) or MG132 ( 100 µM ) was added into culture medium for four hours . Whole cell lysates were analyzed by immunoblots with anti - HA or anti - RNF26 .

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 B-MEASURED_VAR O

(E) Effects of inhibitors on RNF26-mediated destabilization of IRF3. The 293 cells (1×106) were transfected with the indicated plasmids as described in (B). Eighteen hours after transfection, 3-MA (500 ng/mL), NH4Cl (25 mM) or MG132 (100 µM) was added into culture medium for four hours. Whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.


( F ) Effects of ATG12 knockdown on RNF26 - mediated destabilization of IRF3 . The 293 - ATG12 - RNAi or control cells ( 1 × 106 ) were transfected with the indicated plasmids . Twenty - four hours later , whole cell lysates were analyzed by immunoblots with anti - HA , anti - RNF26 or anti - ATG12 as indicated . All experiments were repeated for at least three times with similar results . The bar graphs show mean ± S . D . ( n = 3 ) of a representative experiment performed in triplicate .

O O O O O B-CONTROLLED_VAR 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 O 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 O O O 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) Effects of ATG12 knockdown on RNF26-mediated destabilization of IRF3. The 293-ATG12-RNAi or control cells (1×106) were transfected with the indicated plasmids. Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA, anti-RNF26 or anti-ATG12 as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.


( B ) Direct sequencing of PARN in a control , P1 and her parents .

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

(B) Direct sequencing of PARN in a control, P1 and her parents.


( D ) Direct sequencing of PARN in a control , P2 and her parents .

(D) Direct sequencing of PARN in a control, P2 and her parents.


( E ) Aberrant splicing products detected in P2 and her mother but not in P2 ' s father nor in a healthy control . GAPDH RT - PCR was performed as control .

O O O O O O O O O O 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) Aberrant splicing products detected in P2 and her mother but not in P2's father nor in a healthy control. GAPDH RT-PCR was performed as control.


( F ) Detection of the exon6 / 7 deletion by PCR with specific primers ( 1F / 2R ) .

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

(F) Detection of the exon6/7 deletion by PCR with specific primers (1F/2R).


( G ) PARN detection in cell lysates from P1 and P2 and a healthy control . Actin was used as loading control .

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

(G) PARN detection in cell lysates from P1 and P2 and a healthy control. Actin was used as loading control.


( H ) In vitro deadenylation activity using protein extracts from control and P1 and P2 SV40T - transformed fibroblasts . Mixing protein extracts from control together with P1 or P2 eliminated the deadenylation defect whereas pairwise mixing of P1 and P2 failed to complement the deadenylation defect .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro deadenylation activity using protein extracts from control and P1 and P2 SV40T-transformed fibroblasts. Mixing protein extracts from control together with P1 or P2 eliminated the deadenylation defect whereas pairwise mixing of P1 and P2 failed to complement the deadenylation defect.


( A ) Mean telomere length ( kb ) of whole blood cells from patients and their parents estimated with the TRF method .

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) Mean telomere length (kb) of whole blood cells from patients and their parents estimated with the TRF method.


( B ) Representative picture of telomeric signals used for Q - FISH analysis on metaphase spreads from SV40T - transfomed fibroblasts and Muntjac cells used to normalize the signals . ( C ) Individual and mean values of Q - FISH analyses for control and patients . Mean fluorescence intensity of Muntjac cells was set to 1 . Error bars indicate s . d . . ( D ) Graphical representation of ( C ) showing the average of fluorescence ratios ( relative to Muntjac ) . Error bars indicate s . e . m . . The non - parametric Kruskal - Wallis test was applied to compare relative fluorescence values between Ctl and P1 , and Ctl and P2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative picture of telomeric signals used for Q-FISH analysis on metaphase spreads from SV40T-transfomed fibroblasts and Muntjac cells used to normalize the signals. (C) Individual and mean values of Q-FISH analyses for control and patients. Mean fluorescence intensity of Muntjac cells was set to 1. Error bars indicate s.d.. (D) Graphical representation of (C) showing the average of fluorescence ratios (relative to Muntjac). Error bars indicate s.e.m.. The non-parametric Kruskal-Wallis test was applied to compare relative fluorescence values between Ctl and P1, and Ctl and P2.


( E ) Representative pictures of nuclei from two controls and patient ' s primary fibroblasts showing 53BP1 foci ( green ) and telomeres ( red , detected by Telo - FISH ) . Yellow arrows indicate telomere dysfunction - induced foci ( TIF ) . The scale bar corresponds to 5µm . ( F ) Quantification of ( E ) in primary fibroblasts from two healthy controls ( passages > 4 ) and from patient 1 ( passages < 4 ) . Control 1 : n = 224 ; Control 2 : n = 263 ; Patient 1 : n = 231 . Averages are shown and error bars indicate s . e . m . . Unpaired Student ' s t tests were applied when indicated .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative pictures of nuclei from two controls and patient's primary fibroblasts showing 53BP1 foci (green) and telomeres (red, detected by Telo-FISH). Yellow arrows indicate telomere dysfunction-induced foci (TIF). The scale bar corresponds to 5µm. (F) Quantification of (E) in primary fibroblasts from two healthy controls (passages > 4) and from patient 1 (passages < 4). Control 1: n = 224; Control 2: n = 263; Patient 1: n = 231. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied when indicated.


( G ) Representative pictures of SA - β - galactosidase staining in control ( passage 15 ) and P1 ' s primary fibroblasts ( passage 6 ) . Results are expressed as the percentage of SA - β - galactosidase - positive cells ( averages , lower panel ) . Control : n = 436 ; P1 : n = 436 . A test to compare two population proportions was applied .

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

(G) Representative pictures of SA-β-galactosidase staining in control (passage 15) and P1's primary fibroblasts (passage 6). Results are expressed as the percentage of SA-β-galactosidase-positive cells (averages, lower panel). Control: n = 436; P1: n = 436. A test to compare two population proportions was applied.


( H ) Representative pictures of chromosomes with normal or aberrant telomeres detected by Telo - FISH . The scale bar corresponds to 1µm . ( I ) Quantification of terminal deletions and sister telomere losses ( J ) from three independent experiments ( counted chromatids : Ctl1 : n = 2224 ; Ctl2 : n = 5696 ; P1 : n = 4764 ; P2 : n = 7908 ) . Averages are shown and χ2 - tests were applied to compare Ctl1 / 2 with either P1 or P2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative pictures of chromosomes with normal or aberrant telomeres detected by Telo-FISH. The scale bar corresponds to 1µm. (I) Quantification of terminal deletions and sister telomere losses (J) from three independent experiments (counted chromatids: Ctl1: n=2224; Ctl2: n= 5696; P1: n=4764; P2: n=7908). Averages are shown and χ2-tests were applied to compare Ctl1/2 with either P1 or P2.


( A ) Validation of PARN KO in HT1080 cells by Western blot . GAPDH is used as loading control .

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

(A) Validation of PARN KO in HT1080 cells by Western blot. GAPDH is used as loading control.


( B ) In vitro deadenylation activity assay using protein extracts from HT1080 ( WT ) and HT1080PARN KO ( PARN KO ) cells .

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) In vitro deadenylation activity assay using protein extracts from HT1080 (WT) and HT1080PARN KO (PARN KO) cells.


( C ) TRAP assay using successive dilutions ( 500 ng , 250 ng , 125 ng and 62 . 5 ng ) of cell extracts from WT or PARN KO cells . An internal control ( IC ) for PCR was used .

O O O O O 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

(C) TRAP assay using successive dilutions (500 ng, 250 ng, 125 ng and 62.5 ng) of cell extracts from WT or PARN KO cells. An internal control (IC) for PCR was used.


( D ) qRT - PCR analysis of hTR expression in WT and PARN KO cells . Expression levels were normalized first to ACTB and then to WT . Three independent RNA extractions were performed for each cell line . Error bars indicate s . e . m . .

O O O O 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 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) qRT-PCR analysis of hTR expression in WT and PARN KO cells. Expression levels were normalized first to ACTB and then to WT. Three independent RNA extractions were performed for each cell line. Error bars indicate s.e.m..


( E ) Representative pictures of FISH against hTR in WT and PARN KO cells . Arrows indicate hTR foci . Scale bar 5 μm .

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 O O O O

(E) Representative pictures of FISH against hTR in WT and PARN KO cells. Arrows indicate hTR foci. Scale bar 5 μm.


( F ) PARN detection by Western blot in the indicated conditions . Expression was induced by incubating cells with 10 ng / ml doxycycline for 72 h . Actin is used as loading control . PARN / actin ratios , normalized to doxycycline - treated WT / PARN cells , are shown below .

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

(F) PARN detection by Western blot in the indicated conditions. Expression was induced by incubating cells with 10 ng/ml doxycycline for 72 h. Actin is used as loading control. PARN/actin ratios, normalized to doxycycline-treated WT/PARN cells, are shown below.


( G ) In vitro deadenylation activity assay using protein extracts from doxycycline - treated WT / empty , WT / PARN , KO / empty and KO / PARN cells .

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

(G) In vitro deadenylation activity assay using protein extracts from doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells.


( H ) qRT - PCR analysis of the indicated gene transcripts in doxycycline - treated WT / empty , WT / PARN , KO / empty and KO / PARN cells . Expression levels were normalized first to ACTB and then to WT / empty . Three independent doxycycline inductions were performed for each cell line . Averages are shown and error bars indicate s . e . m . . Unpaired Student ' s t tests were applied .

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 O O O O O O O O O O O

(H) qRT-PCR analysis of the indicated gene transcripts in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to WT/empty. Three independent doxycycline inductions were performed for each cell line. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied.


( I ) Representative western blot analysis of PARN , TRF2 and p53 . Actin was used as loading control . Mean TRF2 / Actin levels from two independent experiments are indicated below .

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

(I) Representative western blot analysis of PARN, TRF2 and p53. Actin was used as loading control. Mean TRF2/Actin levels from two independent experiments are indicated below.


( J ) Same as ( H ) for p53 and p21 transcripts . Averages are shown and error bars indicate s . e . m . . Unpaired Student ' s t tests were applied .

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

(J) Same as (H) for p53 and p21 transcripts. Averages are shown and error bars indicate s.e.m.. Unpaired Student's t tests were applied.


( K ) qRT - PCR analysis of TERRA from the indicated chromosome ends in doxycycline - treated KO / empty and KO / PARN cells . Expression levels were normalized first to ACTB and then to KO / empty . Three independent doxycycline inductions were performed for each cell line . Error bars indicate s . e . m . .

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

(K) qRT-PCR analysis of TERRA from the indicated chromosome ends in doxycycline-treated KO/empty and KO/PARN cells. Expression levels were normalized first to ACTB and then to KO/empty. Three independent doxycycline inductions were performed for each cell line. Error bars indicate s.e.m..


( A ) Analysis of telomeric aberrations by Telo - FISH in doxycycline - treated WT / empty , WT / PARN , KO / empty and KO / PARN cells . Two independent experiments were performed . Counted chromatids : WT / empty : n = 1824 ; WT / PARN : n = 1744 ; KO / empty : n = 1452 ; KO / PARN : n = 1920 . Averages are shown and χ2 - tests were applied to compare KO / empty with either WT / empty or KO / PARN .

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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O

(A) Analysis of telomeric aberrations by Telo-FISH in doxycycline-treated WT/empty, WT/PARN, KO/empty and KO/PARN cells. Two independent experiments were performed. Counted chromatids: WT/empty: n=1824; WT/PARN: n= 1744; KO/empty: n=1452; KO/PARN: n=1920. Averages are shown and χ2-tests were applied to compare KO/empty with either WT/empty or KO/PARN.


( B ) TRF analysis of telomere length ( kb ) in KO / empty and KO / PARN cells treated with 10 ng / ml Dox for 72h . A control Western blot with PARN and Actin is shown below for the corresponding samples .

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

(B) TRF analysis of telomere length (kb) in KO/empty and KO/PARN cells treated with 10 ng/ml Dox for 72h. A control Western blot with PARN and Actin is shown below for the corresponding samples.


( A ) HT1080 ( WT ) and HT1080PARN KO ( KO ) cells stably transfected with GFP plasmid ( empty ) were transfected with siRNAs against p53 for 72h before RNA extraction and qRT - PCR . siRNAs against luciferase ( siLuci ) were used as control . p53 expression levels were normalized first to ACTB and then to WT / empty / siLuci cells . Experiment was performed in triplicate . Error bars indicate s . e . m .

O O O 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 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

(A) HT1080 (WT) and HT1080PARN KO (KO) cells stably transfected with GFP plasmid (empty) were transfected with siRNAs against p53 for 72h before RNA extraction and qRT-PCR. siRNAs against luciferase (siLuci) were used as control. p53 expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m.


( B ) Cells were treated as in ( A ) and transcript levels of the indicated genes were measured by qRT - PCR . cDNA expression levels were normalized first to ACTB and then to WT / empty / siLuci cells . Experiment was performed in triplicate . Error bars indicate s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells were treated as in (A) and transcript levels of the indicated genes were measured by qRT-PCR. cDNA expression levels were normalized first to ACTB and then to WT/empty/siLuci cells. Experiment was performed in triplicate. Error bars indicate s.e.m.


( C ) qRT - PCR analyses of the indicated genes were performed on RNA extracted from P1 ' s primary or SV40T - transformed fibroblasts . cDNA expression levels were normalized to primary fibroblasts .

O O O O O O O O O O O O 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) qRT-PCR analyses of the indicated genes were performed on RNA extracted from P1's primary or SV40T-transformed fibroblasts. cDNA expression levels were normalized to primary fibroblasts.


( C ) Northern blot analysis of pre - rRNAs from control and patient B - LCLs . ( D ) Log2 values of 18S - E / 21S and 18S - EFL / 21S for P1 and P2 were normalized to the values of the control and presented in a graphical format , as Log averages + standard deviation for two independent 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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) Northern blot analysis of pre-rRNAs from control and patient B-LCLs. (D) Log2 values of 18S-E/21S and 18S-EFL/21S for P1 and P2 were normalized to the values of the control and presented in a graphical format, as Log averages + standard deviation for two independent experiments.


( E ) Northern blot analysis of pre - rRNAs from HT1080PARN WT and HT1080PARN KO cells , transduced with an empty vector . ( F ) Ectopic expression of PARN was induced in HT1080PARN KO cells and compared to cells transduced with an empty vector . Western blot relative to actin assessed PARN levels . ( G ) Quantitative analyses of 18S - E / 21S and 18S - E FL / 21S ratios were as described in ( D ) for HT1080PARN KO cells relative to HT1080PARN WT ( left panel ; see ( E ) ) , and for HT1080PARN KO cells rescued by ectopic expression of PARN relative to HT1080PARN KO cells transduced with an empty expression vector ( right panel ; see ( F ) ) , with Log averages + standard deviation for four independent 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 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-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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

(E) Northern blot analysis of pre-rRNAs from HT1080PARN WT and HT1080PARN KO cells, transduced with an empty vector. (F) Ectopic expression of PARN was induced in HT1080PARN KO cells and compared to cells transduced with an empty vector. Western blot relative to actin assessed PARN levels. (G) Quantitative analyses of 18S-E/21S and 18S-E FL/21S ratios were as described in (D) for HT1080PARN KO cells relative to HT1080PARN WT (left panel; see (E)), and for HT1080PARN KO cells rescued by ectopic expression of PARN relative to HT1080PARN KO cells transduced with an empty expression vector (right panel; see (F)), with Log averages + standard deviation for four independent experiments.


( A ) Western blot analysis of Parn expression in control and Parn + / - MEFs .

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

(A) Western blot analysis of Parn expression in control and Parn+/- MEFs.


( B ) Northern blot analysis of total RNAs from WT and Parn + / - MEFs .

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

(B) Northern blot analysis of total RNAs from WT and Parn+/- MEFs.


( D ) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn + / - intercrosses . n indicates the number of animal analyzed . Statistical significance ( P = 1 . 5417E - 08 ) of the differences between the observed and expected genotype distributions was assessed by χ2 - test . ( E ) Frequencies of expected and observed E11 . 5 embryos of the indicated genotype obtained from Parn + / - intercrosses . n indicates the number of animal analysed . Statistical significance ( P = 0 . 02535 ) of the differences between the observed and expected genotype distributions was assessed by χ2 - test .

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 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

(D) Frequencies of expected and observed newborns of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analyzed. Statistical significance (P = 1.5417E-08) of the differences between the observed and expected genotype distributions was assessed by χ2-test. (E) Frequencies of expected and observed E11.5 embryos of the indicated genotype obtained from Parn+/- intercrosses. n indicates the number of animal analysed. Statistical significance (P = 0.02535) of the differences between the observed and expected genotype distributions was assessed by χ2-test.


( F ) , ( G ) p53 knock - out does not rescue Parn - / - . No Parn - / - p53 - / - mouse was born from various combinations of Parn + / - and either p53 + / - or p53 - / - crosses ( 49 and 56 pups , respectively ) . Statistical significance ( P = 0 . 000886 ) of the differences between the observed and expected genotype distributions was assessed by χ2 - test .

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

(F), (G) p53 knock-out does not rescue Parn-/-. No Parn-/- p53-/- mouse was born from various combinations of Parn+/- and either p53+/- or p53-/- crosses (49 and 56 pups, respectively). Statistical significance (P = 0.000886) of the differences between the observed and expected genotype distributions was assessed by χ2-test.


C . Binding assay using MBP fused Apc1 - loop500 fragments and B56γ . Apc1 - loop500 WT or its derivatives ( ∆ 11 or 3A ) were incubated with the 35S - labelled Flag - B56γ in interphase extract ( Inter ) or anaphase extract ( Ana ) supplemented with CycB ∆ 167 at 23 ˚ C for 1 hr . The bound proteins were recovered by amylose beads , separated by SDS - PAGE and detected by autoradiography or coomassie brilliant blue ( CBB ) staining . The bar graph is quantification of bound B56γ . The intensities of MBP control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

C. Binding assay using MBP fused Apc1-loop500 fragments and B56γ. Apc1-loop500 WT or its derivatives (∆11 or 3A) were incubated with the 35S-labelled Flag-B56γ in interphase extract (Inter) or anaphase extract (Ana) supplemented with CycB∆167 at 23˚C for 1 hr. The bound proteins were recovered by amylose beads, separated by SDS-PAGE and detected by autoradiography or coomassie brilliant blue (CBB) staining. The bar graph is quantification of bound B56γ. The intensities of MBP control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


D . Cdk - dependent in vitro kinase assay of Apc1 - loop500 . MBP fused WT or 3A Apc1 - loop500 fragment was incubated with Cdk2 - cyclin A in the presence of [ γ - 32P ] - ATP at 23 ˚ C for 10 min , separated by SDS - PAGE and detected by autoradiography .

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

D. Cdk-dependent in vitro kinase assay of Apc1-loop500. MBP fused WT or 3A Apc1-loop500 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 23˚C for 10 min, separated by SDS-PAGE and detected by autoradiography.


E . Cdk can promote Apc1 - loop500 and B56 interaction . MBP fused WT or 3A Apc1 - loop500 fragment was incubated in the presence or absence of Cdk2 - cyclin A at 30 ˚ C for 60 min . MBP - fused peptides ( - / + kinase ) were isolated and incubated with purified Flag - B56γ at 23 ˚ C for 30 min . The bound proteins were recovered by amylose beads and analysed by SDS - PAGE and immunoblotting with indicated antibodies . F . Quantification of E . Bound B56γ to WT Apc1 - loop500 control ( - Cdk ) was arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

E. Cdk can promote Apc1-loop500 and B56 interaction. MBP fused WT or 3A Apc1-loop500 fragment was incubated in the presence or absence of Cdk2-cyclin A at 30˚C for 60 min. MBP-fused peptides (-/+ kinase) were isolated and incubated with purified Flag-B56γ at 23˚C for 30 min. The bound proteins were recovered by amylose beads and analysed by SDS-PAGE and immunoblotting with indicated antibodies. F. Quantification of E. Bound B56γ to WT Apc1-loop500 control (-Cdk) was arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


A . Cdc20 binding assay in Xenopus egg extracts . The purified recombinant wild - type ( WT ) or B56 binding site mutant APC / C ( 1 - L557A / V560A ) was incubated with APC / C - depleted ( ∆ APC ) interphase extract ( Inter ) or ∆ APC anaphase extract supplemented with CycB ∆ 167 ( Ana ) at 23 ˚ C for indicated times . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . pApc1 ( pS314 / pS318 ) is a phospho - site specific antibody that binds only when both S314 and S318 are phosphorylated . B . Quantification of A . The intensities of WT control in interphase were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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 B-CONTROLLED_VAR 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 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

A. Cdc20 binding assay in Xenopus egg extracts. The purified recombinant wild-type (WT) or B56 binding site mutant APC/C (1-L557A/V560A) was incubated with APC/C-depleted (∆APC) interphase extract (Inter) or ∆APC anaphase extract supplemented with CycB∆167 (Ana) at 23˚C for indicated times. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. pApc1 (pS314/pS318) is a phospho-site specific antibody that binds only when both S314 and S318 are phosphorylated. B. Quantification of A. The intensities of WT control in interphase were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


C . B56 binding site mutant APC / C ( 1 - L557A / V560A ) is less active in ubiquitylation assay than WT APC / C . The purified recombinant WT APC / C or B56 binding motif mutant APC / C ( 1 - L557A / V560A ) was incubated with ∆ APC anaphase extract . The recovered APC / C - Cdc20 complex was subjected to ubiquitylation assay using 35S - labelled cyclin B as a substrate . Samples were taken at indicated time points and analysed by SDS - PAGE and autoradiography . D . Quantification of C . Error bars , s . e . m . from three independent 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 O O O O O 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. B56 binding site mutant APC/C (1-L557A/V560A) is less active in ubiquitylation assay than WT APC/C. The purified recombinant WT APC/C or B56 binding motif mutant APC/C (1-L557A/V560A) was incubated with ∆APC anaphase extract. The recovered APC/C-Cdc20 complex was subjected to ubiquitylation assay using 35S-labelled cyclin B as a substrate. Samples were taken at indicated time points and analysed by SDS-PAGE and autoradiography. D. Quantification of C. Error bars, s.e.m. from three independent experiments.


A . ( left panel ) Cdc20 binding to APC / C loop domain mutants in interphase extract . The purified recombinant WT or Apc1 mutant APC / Cs ( 1 - ∆ L300 , 1 - ∆ L300 / ∆ 11 or 1 - ∆ L300 / L557A / V560A ) were incubated with ∆ APC interphase extract at 23 ˚ C for 1 hr . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . ( right panel ) Quantification of bound Cdc20 . The intensities of 1 - ∆ L300 control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments . B . ( left panel ) Cdc20 binding to APC / C loop domain mutants in anaphase extracts . Same as A , but ∆ APC anaphase extract was used and incubated at 23 ˚ C for 50 min in anaphase extract . ( right panel ) Quantification of bound Cdc20 . The intensities of 1 - ∆ L300 control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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-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 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 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 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

A. (left panel) Cdc20 binding to APC/C loop domain mutants in interphase extract. The purified recombinant WT or Apc1 mutant APC/Cs (1-∆L300, 1-∆L300/∆11 or 1-∆L300/L557A/V560A) were incubated with ∆APC interphase extract at 23˚C for 1 hr. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments. B. (left panel) Cdc20 binding to APC/C loop domain mutants in anaphase extracts. Same as A, but ∆APC anaphase extract was used and incubated at 23˚C for 50 min in anaphase extract. (right panel) Quantification of bound Cdc20. The intensities of 1-∆L300 control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


A . ( left panel ) Cdk - dependent in vitro kinase assay of Apc1 - loop300 . 3xFlag - tagged WT or 7T Apc1 - loop300 fragment was incubated with Cdk2 - cyclin A in the presence of [ γ - 32P ] - ATP at 30 ˚ C for 30 min , and separated by SDS - PAGE and detected by autoradiography . ( right panel ) 32P - phosphorylated WT or 7T Apc1 - loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS - PAGE followed by autoradiography . B . Quantification of removal of radioactivity in A . Error bars , s . e . m . from three independent experiments .

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-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 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

A. (left panel) Cdk-dependent in vitro kinase assay of Apc1-loop300. 3xFlag-tagged WT or 7T Apc1-loop300 fragment was incubated with Cdk2-cyclin A in the presence of [γ-32P]-ATP at 30˚C for 30 min, and separated by SDS-PAGE and detected by autoradiography. (right panel) 32P-phosphorylated WT or 7T Apc1-loop300 fragment was incubated in anaphase extract and removal of radioactivity was analysed by SDS-PAGE followed by autoradiography. B. Quantification of removal of radioactivity in A. Error bars, s.e.m. from three independent experiments.


C . Cdc20 - 5A efficiently binds to the B56 binding site mutant APC / C . The purified recombinant WT APC / C or Apc1 - loop500 mutant APC / C ( 1 - L557A / V560A ) was incubated with WT Cdc20 or non - phosphorylatable Cdc20 mutant ( 5A ) in ∆ APC ∆ Cdc20 anaphase extract at 23 ˚ C for 55 min . The APC / C was recovered with Apc3 monoclonal antibody ( AF3 . 1 ) beads and the bound proteins were analysed by SDS - PAGE and immunoblotting with indicated antibodies . D . Quantification of C . The intensities of WT APC / C control were arbitrarily set to 1 . 0 . Error bars , s . e . m . from three independent experiments .

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

C. Cdc20-5A efficiently binds to the B56 binding site mutant APC/C. The purified recombinant WT APC/C or Apc1-loop500 mutant APC/C (1-L557A/V560A) was incubated with WT Cdc20 or non-phosphorylatable Cdc20 mutant (5A) in ∆APC∆Cdc20 anaphase extract at 23˚C for 55 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads and the bound proteins were analysed by SDS-PAGE and immunoblotting with indicated antibodies. D. Quantification of C. The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, s.e.m. from three independent experiments.


E . The APC / C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A - B56γ at 23 ˚ C . Samples were taken at the indicated time points and analysed by SDS - PAGE and immunoblotting with antibodies , including phospho - site specific antibodies for pCdc20 ( pT79 ) and pApc1 ( pS314 / pS318 ) .

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-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

E. The APC/C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A-B56γ at 23˚C. Samples were taken at the indicated time points and analysed by SDS-PAGE and immunoblotting with antibodies, including phospho-site specific antibodies for pCdc20 (pT79) and pApc1 (pS314/pS318).


D HCT116 , Lovo and RKO cells were transfected with control or p53 siRNAs . 48 h after transfection , total protein was then analyzed by western blot analysis . E HCT116 , Lovo and RKO cells were transfected with empty vector and p53 plasmid . 48 h after transfection , total protein was then analyzed by western blot analysis .

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

D HCT116, Lovo and RKO cells were transfected with control or p53 siRNAs. 48 h after transfection, total protein was then analyzed by western blot analysis. E HCT116, Lovo and RKO cells were transfected with empty vector and p53 plasmid. 48 h after transfection, total protein was then analyzed by western blot analysis.


F Representative immunofluorescence staining of the p53 ( green ) and METTL14 ( red ) proteins in p53 - WT HCT116 , Lovo and RKO cells and p53 - MT HT29 ( p53R273H ) , SW480 ( p53R273H / P309S ) , SW620 ( p53R273H ) and SW1116 ( p53A159D ) cells . Nuclei were stained with DAPI ( blue ) . Scale bars = 10 μm . The relative mean fluorescence density was analyzed by ImageJ .

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

F Representative immunofluorescence staining of the p53 (green) and METTL14 (red) proteins in p53-WT HCT116, Lovo and RKO cells and p53-MT HT29 (p53R273H), SW480 (p53R273H/P309S), SW620 (p53R273H) and SW1116 (p53A159D) cells. Nuclei were stained with DAPI (blue). Scale bars = 10 μm. The relative mean fluorescence density was analyzed by ImageJ.


I Western blot analysis of METTL14 levels after transfection with empty vector , wild - type or mutant p53 plasmids in HCT116 ( p53 - / - ) cells .

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 B-CONTROLLED_VAR O O O O O O

I Western blot analysis of METTL14 levels after transfection with empty vector, wild-type or mutant p53 plasmids in HCT116 (p53-/-) cells.