( E ) Scr ( stably expressing scrambled shRNA ) and Mfn2 knockdown ( KD ) ( stably expressing shRNA directed against Mfn2 ) 3T3 ‐ L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h . Total and cleaved caspase 3 were detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(E) Scr (stably expressing scrambled shRNA) and Mfn2 knockdown (KD) (stably expressing shRNA directed against Mfn2) 3T3‐L1 fibroblasts were incubated in the presence or absence of 1 μM Tg for 24 h. Total and cleaved caspase 3 were detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( F ) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h . Lactate dehydrogenase ( LDH ) release was analyzed by flow cytometry to assess necrotic cell death . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(F) WT and Mfn2 KO cells were treated with 1 μM Tg for 24 h. Lactate dehydrogenase (LDH) release was analyzed by flow cytometry to assess necrotic cell death. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( G ) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z ‐ VAD ‐ fmk and stained for annexin V / PI . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT + Tg group .

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

(G) WT and Mfn2 KO cells were incubated for 24 h with or without 1 μM Tg in the presence or absence of z‐VAD‐fmk and stained for annexin V/PI. Data are mean±s.e.m. (n=4). *P0.05 versus WT+Tg group.


( H ) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h ( 3 h of pre ‐ incubation with CHX ) . Florescence microscopy images show that CHX prevents cytoplasmic vacuolization . Scale bar : 10 μm .

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

(H) Mfn2 KO cells transfected with the pEGFP plasmid were incubated with 1 μM Tg alone or in combination with 2 μM CHX for 24 h (3 h of pre‐incubation with CHX). Florescence microscopy images show that CHX prevents cytoplasmic vacuolization. Scale bar: 10 μm.


( I ) WT ( black circles ) and Mfn2 KO cells ( white circles ) were incubated with 1 μM Tg for varying times , and ALIX protein was detected by western blot . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . Source data for this figure is available on the online supplementary information page .

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

(I) WT (black circles) and Mfn2 KO cells (white circles) were incubated with 1 μM Tg for varying times, and ALIX protein was detected by western blot. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


( A , upper panel ) WT ( black circles ) or Mfn2 KO cells ( white circles ) were treated with 1 μM Tg for a range of times . ( A , lower ) Densitometric quantification . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group ; # P0 . 05 versus WT non ‐ Tg ‐ treated group .

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

(A, upper panel) WT (black circles) or Mfn2 KO cells (white circles) were treated with 1 μM Tg for a range of times. (A, lower) Densitometric quantification. Data are mean±s.e.m. (n=3). *P0.05 versus WT group; #P0.05 versus WT non‐Tg‐treated group.


( B , upper panel ) WT or Mfn2 KO cells were treated with Tg for 0 , 6 or 12 h , in the presence or absence of Bafilomycin ( Baf , 100 nM ) . LC3b ‐ I and LC3b ‐ II expression was measured by western blot . ( B , lower panel ) Densitometric quantification of LC3b ‐ II levels ( relative to tubulin ) . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT + Baf + Tg group .

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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

(B, upper panel) WT or Mfn2 KO cells were treated with Tg for 0, 6 or 12 h, in the presence or absence of Bafilomycin (Baf, 100 nM). LC3b‐I and LC3b‐II expression was measured by western blot. (B, lower panel) Densitometric quantification of LC3b‐II levels (relative to tubulin). Data are mean±s.e.m. (n=3). *P0.05 versus WT+Baf+Tg group.


( C ) WT or Mfn2 KO cells stably expressing mCherry ‐ GFP ‐ LC3b were treated with 1 μM Tg , 100 ng / ml Brefeldin ( Bref ) , or 0 . 5 μg / ml tunicamycin ( Tm ) for 24 h and examined by confocal microscopy . Scale bar : 10 μm .

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

(C) WT or Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b were treated with 1 μM Tg, 100 ng/ml Brefeldin (Bref), or 0.5 μg/ml tunicamycin (Tm) for 24 h and examined by confocal microscopy. Scale bar: 10 μm.


( D ) Quantification of mCherry + / GFP ‐ ( mCherry ‐ LC3 ) red puncta per cell ( a measure of acidic autophagosomes ) is shown . Data are mean ± s . e . m . ( n = 3 ; 100 cells were analyzed per experiment and group ) . * P0 . 05 versus WT group .

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

(D) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus WT group.


( E ) Acidic compartments of WT or Mfn2 KO cells were stained with Lysotracker Green and analyzed by flow cytometry . Data are given as mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(E) Acidic compartments of WT or Mfn2 KO cells were stained with Lysotracker Green and analyzed by flow cytometry. Data are given as mean±s.e.m. (n=3). *P0.05 versus WT group.


( F ) LAMP1 expression was immunodetected in WT and Mfn2 KO cells in basal conditions .

(F) LAMP1 expression was immunodetected in WT and Mfn2 KO cells in basal conditions.


( G ) Expression of Beclin ‐ 1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . Source data for this figure is available on the online supplementary information page .

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

(G) Expression of Beclin‐1 or LC3b transcripts in WT or Mfn2 KO cells treated with or without 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


( B ) Immunodetection of p ‐ PERK , PERK , GADD34 , p ‐ eIF2α , eIF2α , ATF4 , CHOP , and XBP ‐ 1s in WT and Mfn2 KO cells treated with 1 μM Tg for the times indicated .

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

(B) Immunodetection of p‐PERK,PERK, GADD34, p‐eIF2α, eIF2α, ATF4, CHOP, and XBP‐1s in WT and Mfn2 KO cells treated with 1 μM Tg for the times indicated.


( C ) Densitometric quantifications . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(C) Densitometric quantifications. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( D ) Immunodetection of p ‐ eIF2α , eIF2α , and ATF4 in WT and Mfn2 KO cells treated with 0 . 5 μg / ml Tunicamycin ( Tm ) for the times indicated .

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

(D) Immunodetection of p‐eIF2α, eIF2α, and ATF4 in WT and Mfn2 KO cells treated with 0.5 μg/ml Tunicamycin (Tm) for the times indicated.


( E ) Transcriptional activity driven by ATF6 . WT and Mfn2 KO cells were co ‐ transfected with the 5xATF6 ‐ GL3 and TK ‐ Renilla plasmids and treated with 1 μM Tg for 24 h after transfection . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT group . Source data for this figure is available on the online supplementary information page .

O O O O O O O O O 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

(E) Transcriptional activity driven by ATF6. WT and Mfn2 KO cells were co‐transfected with the 5xATF6‐GL3 and TK‐Renilla plasmids and treated with 1 μM Tg for 24 h after transfection. Data are mean±s.e.m. (n=4). *P0.05 versus WT group.Source data for this figure is available on the online supplementary information page.


( A ) Immunodetection of PERK in WT and Mfn2 KO cells stably expressing a scrambled shRNA ( Scr ) or a shRNA directed against PERK ( PERK KD ) .

O O O O O O O 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) Immunodetection of PERK in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed against PERK (PERK KD).


( B ) Caspase activity was detected by measurement of DEVD - AFC substrate processing in WT or Mfn2 KO cells subjected to PERK silencing and treated with 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group .

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

(B) Caspase activity was detected by measurement of DEVD-AFC substrate processing in WT or Mfn2 KO cells subjected to PERK silencing and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.


( C , D ) Total and cleaved caspase 3 levels were detected by western blot . Densitometric quantification is shown in D . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group .

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

(C, D) Total and cleaved caspase 3 levels were detected by western blot. Densitometric quantification is shown in D. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.


( E , F ) Immunodetection of LC3b ‐ I and ‐ II in WT and Mfn2 KO cells subjected to PERK silencing and treated as indicated with Tg or Baf ( 1 μM Tg ; 100 nM Baf ) for 6 h . Densitometric quantification is shown in F . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT ; # P0 . 05 versus Scr group . Source data for this figure is available on the online supplementary information page .

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

(E, F) Immunodetection of LC3b‐I and ‐II in WT and Mfn2 KO cells subjected to PERK silencing and treated as indicated with Tg or Baf (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). *P0.05 versus WT; #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.


( A ) Immunodetection of XBP ‐ 1s in WT and Mfn2 KO cells stably expressing a scrambled shRNA ( Scr ) or a shRNA directed to XBP ‐ 1 ( XBP ‐ 1 KD ) , and treated with Tg for 6 h .

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

(A) Immunodetection of XBP‐1s in WT and Mfn2 KO cells stably expressing a scrambled shRNA (Scr) or a shRNA directed to XBP‐1 (XBP‐1 KD), and treated with Tg for 6 h.


( B ) Caspase activity was detected by measurement of DEVD ‐ AFC substrate processing . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group . # P0 . 05 versus Scr group .

(B) Caspase activity was detected by measurement of DEVD‐AFC substrate processing. Data are mean±s.e.m. (n=3). *P0.05 versus WT group. #P0.05 versus Scr group.


( C , D ) Total and cleaved caspase 3 levels were detected by western blot . WT and Mfn2 KO cells subjected or not to XBP ‐ 1 silencing ( Scr , and KD ) and treated with 1 μM Tg for 24 h . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT group .

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

(C, D) Total and cleaved caspase 3 levels were detected by western blot. WT and Mfn2 KO cells subjected or not to XBP‐1 silencing (Scr, and KD) and treated with 1 μM Tg for 24 h. Data are mean±s.e.m. (n=3). *P0.05 versus WT group.


( E , F ) Immunodetection of LC3b ‐ I and ‐ II in Scr and XBP ‐ 1 KD WT or Mfn2 KO cells treated as indicated ( 1 μM Tg ; 100 nM Baf ) for 6 h . Densitometric quantification is shown in F . Data are mean ± s . e . m . ( n = 3 ) . # P0 . 05 versus Scr group .

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(E, F) Immunodetection of LC3b‐I and ‐II in Scr and XBP‐1 KD WT or Mfn2 KO cells treated as indicated (1 μM Tg; 100 nM Baf) for 6 h. Densitometric quantification is shown in F. Data are mean±s.e.m. (n=3). #P0.05 versus Scr group.


( G ) Mfn2 KO cells stably expressing mCherry ‐ GFP ‐ LC3b and subjected or not to XBP ‐ 1 silencing ( Scr or KD ) were treated with 1 μM Tg , 100 ng / ml Bref , or 0 . 5 μg / ml Tm for 24 h and examined by confocal microscopy . Scale bar : 10 μm .

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

(G) Mfn2 KO cells stably expressing mCherry‐GFP‐LC3b and subjected or not to XBP‐1 silencing (Scr or KD) were treated with 1 μM Tg, 100 ng/ml Bref, or 0.5 μg/ml Tm for 24 h and examined by confocal microscopy. Scale bar: 10 μm.


( H ) Quantification of mCherry + / GFP ‐ ( mCherry ‐ LC3 ) red puncta per cell ( a measure of acidic autophagosomes ) is shown . Data are mean ± s . e . m . ( n = 3 ; 100 cells were analyzed per experiment and group ) . # P0 . 05 versus Scr group . Source data for this figure is available on the online supplementary information page .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of mCherry+/GFP‐ (mCherry‐LC3) red puncta per cell (a measure of acidic autophagosomes) is shown. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). #P0.05 versus Scr group.Source data for this figure is available on the online supplementary information page.


( A ) Immunodetection of p ‐ PERK and p ‐ eIF2α in Mfn2 KO MEFs , Mfn2 knockdown 3T3 ‐ L1 fibroblasts , and Mfn2 knockdown C2C12 myoblasts .

(A) Immunodetection of p‐PERK and p‐eIF2α in Mfn2 KO MEFs, Mfn2 knockdown 3T3‐L1 fibroblasts, and Mfn2 knockdown C2C12 myoblasts.


( B ) Densitometric quantification of p ‐ PERK and p ‐ eIF2α in MEFs . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus WT cells .

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

(B) Densitometric quantification of p‐PERK and p‐eIF2α in MEFs. Data are mean±s.e.m. (n=3). *P0.05 versus WT cells.


( C , D ) Immunodetection of p ‐ eIF2α , eIF2α , CHOP , XBP ‐ 1s , LC3b , and p62 in Mfn2 ‐ deficient liver ( C ) or skeletal muscle ( D ) from tissue ‐ specific KO mice .

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

(C, D) Immunodetection of p‐eIF2α, eIF2α, CHOP, XBP‐1s, LC3b, and p62 in Mfn2‐deficient liver (C) or skeletal muscle (D) from tissue‐specific KO mice.


( E ) Co ‐ immunoprecipitation of Mfn2 and PERK in WT or Mfn2 KO cells stably expressing PERK ‐ myc or an empty vector ( pBABE ‐ myc ; negative 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) Co‐immunoprecipitation of Mfn2 and PERK in WT or Mfn2 KO cells stably expressing PERK‐myc or an empty vector (pBABE‐myc; negative control).


( F ) Co ‐ immunoprecipitation of endogenous Mfn2 and PERK in WT MEF lysates . PERK was immunoprecipitated with a C ‐ terminal antibody and co ‐ immunoprecipitation of Mfn2 was detected by western blot . Source data for this figure is available on the online supplementary information page .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co‐immunoprecipitation of endogenous Mfn2 and PERK in WT MEF lysates. PERK was immunoprecipitated with a C‐terminal antibody and co‐immunoprecipitation of Mfn2 was detected by western blot.Source data for this figure is available on the online supplementary information page.


( A ) Representative confocal images of mitochondrial morphology in scrambled ( Scr ) and PERK KD Mfn2 KO cells stained with MitoTracker Green .

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

(A) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD Mfn2 KO cells stained with MitoTracker Green.


( B ) Mitochondrial morphology quantification . Data are mean ± s . e . m . ( n = 3 ; 100 cells were analyzed per experiment and group ) . * P0 . 05 versus Scr group .

(B) Mitochondrial morphology quantification. Data are mean±s.e.m. (n=3; 100 cells were analyzed per experiment and group). *P0.05 versus Scr group.


( C , D ) Representative confocal images of mitochondrial morphology in scrambled ( Scr ) and PERK KD WT cells ( C ) or scrambled ( Scr ) and PERK KD Mfn1 KO cells ( D ) stained with MitoTracker Green .

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

(C, D) Representative confocal images of mitochondrial morphology in scrambled (Scr) and PERK KD WT cells (C) or scrambled (Scr) and PERK KD Mfn1 KO cells (D) stained with MitoTracker Green.


( E ) Representative confocal images of mitochondrial morphology in Mfn2 KO cells treated with 1 μM TUDCA or 10 mM 4 ‐ phenyl butyric acid for 6 h and stained with MitoTracker Green .

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(E) Representative confocal images of mitochondrial morphology in Mfn2 KO cells treated with 1 μM TUDCA or 10 mM 4‐phenyl butyric acid for 6 h and stained with MitoTracker Green.


( F ) Flow ‐ cytometry quantification of ROS levels in Scr and PERK KD WT and Mfn2 KO

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(F) Flow‐cytometry quantification of ROS levels in Scr and PERK KD WT and Mfn2 KO cells


( G ) or Scr and PERK KD WT and Mfn1 cells using DHR1 , 2 , 3 . Data are mean ± s . e . m . ( n = 4 ) . * P0 . 05 versus WT group , # P0 . 05 versus Scr group . Scale bars : 10 μm .

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

(G) or Scr and PERK KD WT and Mfn1 cells using DHR1,2,3. Data are mean±s.e.m. (n=4). *P0.05 versus WT group, #P0.05 versus Scr group. Scale bars: 10 μm.


( A ) Mitochondrial calcium overload in Scr and PERK KD Mfn2 KO cells . Cells were loaded with Rhod ‐ 2 and then treated with 2 . 5 mM CaCl2 ( left ) . Calcium uptake was monitored by confocal microscopy . Representative confocal images of mitochondrial morphology in WT and Mfn2 KO cells stained with Rhod ‐ 2 and incubated with 2 . 5 mM CaCl2 for 5 min ( right ) . Scale bars : 10 μm .

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

(A) Mitochondrial calcium overload in Scr and PERK KD Mfn2 KO cells. Cells were loaded with Rhod‐2 and then treated with 2.5 mM CaCl2 (left). Calcium uptake was monitored by confocal microscopy. Representative confocal images of mitochondrial morphology in WT and Mfn2 KO cells stained with Rhod‐2 and incubated with 2.5 mM CaCl2 for 5 min (right). Scale bars: 10 μm.


( B - E ) Mitochondrial oxygen consumption ( OCR ) was measured in Scr and PERK KD Mfn2 KO cells ( B ) , Scr and PERK KD WT cells ( C ) , Scr and PERK KD Mfn1 KO cells ( D ) , and WT cells stably expressing PERK ‐ myc ( E ) . Data are mean of three independent experiments . The following parameters were measured : oxygen consumption under routine conditions ( DMEM with 5 . 5 mM glucose ) , maximal respiratory capacity reached after uncoupling with FCCP , and respiratory leak , measured after inhibition of ATP synthase with oligomycin . Data are mean ± s . e . m . * P0 . 05 versus Scr group or versus the empty plasmid group .

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

(B-E) Mitochondrial oxygen consumption (OCR) was measured in Scr and PERK KD Mfn2 KO cells (B), Scr and PERK KD WT cells (C), Scr and PERK KD Mfn1 KO cells (D), and WT cells stably expressing PERK‐myc (E). Data are mean of three independent experiments. The following parameters were measured: oxygen consumption under routine conditions (DMEM with 5.5 mM glucose), maximal respiratory capacity reached after uncoupling with FCCP, and respiratory leak, measured after inhibition of ATP synthase with oligomycin. Data are mean±s.e.m. *P0.05 versus Scr group or versus the empty plasmid group.


( F , G ) Representative confocal images of mitochondrial morphology in WT cells stably expressing PERK ‐ myc stained with MitoTracker Green . Scale bars : 10 μm . Quantitative analysis of mitochondrial morphology is shown in G . Data are mean ± s . e . m . ( n = 3 ) . * P0 . 05 versus the empty plasmid group .

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

(F, G) Representative confocal images of mitochondrial morphology in WT cells stably expressing PERK‐myc stained with MitoTracker Green. Scale bars: 10 μm. Quantitative analysis of mitochondrial morphology is shown in G. Data are mean±s.e.m. (n=3). *P0.05 versus the empty plasmid group.


A . Ncl and Pus7 mRNA levels ( normalized to TBP ) at different time points after LPS stimulation in c - mycwt / wt and c - myc ∆ / ∆ cells . Data are presented as mean ± SD ; n = 3 .

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A. Ncl and Pus7 mRNA levels (normalized to TBP) at different time points after LPS stimulation in c-mycwt/wt and c-myc∆/∆ cells. Data are presented as mean ± SD; n = 3.


B . To quantify RNA synthesis , we exposed our cultures to a brief pulse of ethyl - uridine ( EU ) and measured its incorporation into RNA by light microscopy . The scatter plots show the nuclear Area as detected in the 2D plane ( x axis , in pixels ) versus the EU signal ( y axis , as arbitrary units , AU ) for each single cell identified by DAPI staining in c - mycwt / wt and c - myc ∆ / ∆ populations at the indicated time points after LPS treatment . One representative experiment out of 5 is shown .

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B. To quantify RNA synthesis, we exposed our cultures to a brief pulse of ethyl-uridine (EU) and measured its incorporation into RNA by light microscopy. The scatter plots show the nuclear Area as detected in the 2D plane (x axis, in pixels) versus the EU signal (y axis, as arbitrary units, AU) for each single cell identified by DAPI staining in c-mycwt/wt and c-myc∆/∆ populations at the indicated time points after LPS treatment. One representative experiment out of 5 is shown.


C . Variation of each expressed mRNA at 4h of LPS treatment ( log2FC , relative to time 0 ) for c - mycwt / wt ( x - axis ) and c - myc ∆ / ∆ cells ( y - axis ) , as measured by RNA - seq . Regulatory groups 1 - 6 and Grey Zone ( GZ ) are as defined in the text .

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C. Variation of each expressed mRNA at 4h of LPS treatment (log2FC, relative to time 0) for c-mycwt/wt (x-axis) and c-myc∆/∆ cells (y-axis), as measured by RNA-seq. Regulatory groups 1-6 and Grey Zone (GZ) are as defined in the text.


D . Numbers of genes classified in the different regulatory categories at the indicated time points after LPS stimulation .

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D. Numbers of genes classified in the different regulatory categories at the indicated time points after LPS stimulation.


E . Variations ( log2FC ) in mature and precursor RNAs ( as measured by RNA - seq ) and in the modeled rates ( RNA synthesis , processing and degradation ) for Myc dependent and independent genes . The grey scale represents the starting level for each parameter in unstimulated cells .

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E. Variations (log2FC) in mature and precursor RNAs (as measured by RNA-seq) and in the modeled rates (RNA synthesis, processing and degradation) for Myc dependent and independent genes. The grey scale represents the starting level for each parameter in unstimulated cells.


A . Overlap between Myc ChIP - seq peaks . For each time - point indicated at the bottom , the percentage of peaks overlapping over ≥ 1 bp with any other time - point ( Reference sample ) is reported . Peaks showed consistent distributions along the time - course , almost all those called in untreated cells being included in LPS - stimulated

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A. Overlap between Myc ChIP-seq peaks. For each time-point indicated at the bottom, the percentage of peaks overlapping over ≥1 bp with any other time-point (Reference sample) is reported. Peaks showed consistent distributions along the time-course, almost all those called in untreated cells being included in LPS-stimulated samples


B , C . Distribution of Myc at promoters ( B ) and distal sited ( C ) in LPS - treated B - cells ( 0 , 2 , 4 8h ) and in B - cells in vivo , in either control c - mycwt / wt ( C ) , pre - tumoral Eµ - myc mice ( P ) or tumors ( T ) 4 [ ] . Each row represents a promoter - associated Myc peak within a 6 kb genomic interval ( centered on the midpoint of the peak ) . The heatmap includes every annotated promoter in chromosome 1 ( as representative of the whole genome ) that was called as Myc - associated by ChIP - seq in at least one sample . For the same intervals , we also show the distribution of H3K4me1 , H3K4me3 , and H3K27ac in the in vivo control sample , and gene annotations ( exons in red , introns in pink ; + sense , - antisense strand ) .

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B, C. Distribution of Myc at promoters (B) and distal sited (C) in LPS-treated B-cells (0, 2, 4 8h) and in B-cells in vivo, in either control c-mycwt/wt (C), pre-tumoral Eµ-myc mice (P) or tumors (T) 4[]. Each row represents a promoter-associated Myc peak within a 6 kb genomic interval (centered on the midpoint of the peak). The heatmap includes every annotated promoter in chromosome 1 (as representative of the whole genome) that was called as Myc-associated by ChIP-seq in at least one sample. For the same intervals, we also show the distribution of H3K4me1, H3K4me3, and H3K27ac in the in vivo control sample, and gene annotations (exons in red, introns in pink; + sense, - antisense strand).


D , E . Numbers of Myc peaks at promoters ( - 2kb , + 1kb ) ( D ) and distal sites ( E ) at different time points after LPS stimulation . The shadings mark the subsets of peaks associated with H3K4me3 ( D ) or H3K4me1 ( E ) , with or without H3K27ac .

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D, E. Numbers of Myc peaks at promoters (-2kb, +1kb) (D) and distal sites (E) at different time points after LPS stimulation. The shadings mark the subsets of peaks associated with H3K4me3 (D) or H3K4me1 (E), with or without H3K27ac.


F . Percentage of active promoters ( positive for H3K4me3 and H3K27ac ) or distal sites ( H3K4me1 and H3K27ac ) that overlap with at least a Myc peak at the different time points after LPS stimulation ( 0 , 2 , 4 , 8h ) or in vivo ( C , P , T1 , T2 , T3 ) .

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F. Percentage of active promoters (positive for H3K4me3 and H3K27ac) or distal sites (H3K4me1 and H3K27ac) that overlap with at least a Myc peak at the different time points after LPS stimulation (0, 2, 4, 8h) or in vivo (C, P, T1, T2, T3).


G . Quantitative heatmap showing Myc signal intensities ( read counts normalized by library size ) at each bound region in chromosome 1 , ranked based on the signal in the control sample ( 0h LPS ) .

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G. Quantitative heatmap showing Myc signal intensities (read counts normalized by library size) at each bound region in chromosome 1, ranked based on the signal in the control sample (0h LPS).


H . Percentage of Myc peaks at promoters ( top ) or distal sites ( bottom ) that contain either canonical ( CACGTG ) or variant E - boxes ( CACGCG , CATGCG , CACGAG , CATGTG ) 83637 [ , , ] in a 200 bp window centered on the peak summit .

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H. Percentage of Myc peaks at promoters (top) or distal sites (bottom) that contain either canonical (CACGTG) or variant E-boxes (CACGCG, CATGCG, CACGAG, CATGTG) 83637[,,] in a 200 bp window centered on the peak summit.


I . Myc enrichment by ChIP - seq at different time points after LPS treatment for peaks located either in promoters or distal sites and containing the indicated DNA motifs . In the boxplot , the central tendency marks the median , the box ranges mark the upper and lower quartiles , and the whiskers extend to the upper and lower extreme datapoints which are no more than 1 . 5 times the interquartile range from the box . The Myc ChIP - seq time - course was performed on a unique pool of cells , obtained from > 10 mice .

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I. Myc enrichment by ChIP-seq at different time points after LPS treatment for peaks located either in promoters or distal sites and containing the indicated DNA motifs. In the boxplot, the central tendency marks the median, the box ranges mark the upper and lower quartiles, and the whiskers extend to the upper and lower extreme datapoints which are no more than 1.5 times the interquartile range from the box. The Myc ChIP-seq time-course was performed on a unique pool of cells, obtained from > 10 mice.


A . Scatter plots correlating the variations in Myc share at promoters ( averaged between the 2 , 4 and 8h time points ) and in RNA synthesis ( at 8h ) , relative to untreated cells , for Myc - dependent and independent genes . The Spearman correlation between the two parameters is reported .

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A. Scatter plots correlating the variations in Myc share at promoters (averaged between the 2, 4 and 8h time points) and in RNA synthesis (at 8h), relative to untreated cells, for Myc-dependent and independent genes. The Spearman correlation between the two parameters is reported.


B . The ability of the Myc share in predicting the transcriptional outcome can be represented in terms of a Receiver operating characteristic ( ROC ) curve , which represents the Sensitivity ( true positive rate ) and Specificity ( true negative rate ) of a predictor ( i . e . Myc share ) in discriminating between two classes ( i . e . up - and down - regulated genes ) , using different thresholds . The largest the area under the curve ( AUC ) , the largest is the predictive power , in a scale from 0 . 5 ( random classification : grey line ) to 1 ( perfect predictor ) . For each system , the dot corresponds to the variation of Myc at which promoters begin increasing their share of Myc binding .

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B. The ability of the Myc share in predicting the transcriptional outcome can be represented in terms of a Receiver operating characteristic (ROC) curve, which represents the Sensitivity (true positive rate) and Specificity (true negative rate) of a predictor (i.e. Myc share) in discriminating between two classes (i.e. up- and down-regulated genes), using different thresholds. The largest the area under the curve (AUC), the largest is the predictive power, in a scale from 0.5 (random classification: grey line) to 1 (perfect predictor). For each system, the dot corresponds to the variation of Myc at which promoters begin increasing their share of Myc binding.


C . Fold - change in Myc levels at each bound promoter ( at 4h LPS , relative to time zero ) as a function of the initial binding intensity at time zero ( expressed as log2 of the coverage in a 200 bp window around the summit of the peak ) . Peaks containing a canonical E - box are highlighted in black .

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C. Fold-change in Myc levels at each bound promoter (at 4h LPS, relative to time zero) as a function of the initial binding intensity at time zero (expressed as log2 of the coverage in a 200 bp window around the summit of the peak). Peaks containing a canonical E-box are highlighted in black.


D . Percentage of Myc - bound promoters within the indicated transcriptionally regulatory categories that contains a canonical or variant E - box . The overall chi - square is 207 . 7 with a p - value < 0 . 00001 . The contribution to the chi - square of each category is reported above the corresponding bar in the plot .

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D. Percentage of Myc-bound promoters within the indicated transcriptionally regulatory categories that contains a canonical or variant E-box. The overall chi-square is 207.7 with a p-value <0.00001. The contribution to the chi-square of each category is reported above the corresponding bar in the plot.


B , C . Boxplots reporting LPS - induced changes ( Log2FC ) for each of the four RNAPII kinetic rates in c - mycwt / wt ( wt ) and c - myc ∆ / ∆ cells for Myc - dependent ( B ) and Myc - independent ( C ) induced and repressed genes . p - values obtained with two - sample Wilcoxon tests for the comparison between c - mycwt / wt and c - myc ∆ / ∆ cells are reported . The RNAPII ChIP - seq time - course was performed on two biological replicates ( each on pools of cells from > 10 mice ) , and reads were merged after sequencing .

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B, C. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for Myc-dependent (B) and Myc-independent (C) induced and repressed genes. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported. The RNAPII ChIP-seq time-course was performed on two biological replicates (each on pools of cells from > 10 mice), and reads were merged after sequencing.


D , E . Heatmaps showing the variations in RNAPII kinetic rates ( Log2FC ) on Myc - dependent ( D ) and Myc - independent ( E ) genes during the LPS time - course . The grey scale represents the starting level for each parameter in unstimulated cells . The first column on the left reports the changes in Myc share for the same genes . Genes were grouped using hierarchical clustering .

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D, E. Heatmaps showing the variations in RNAPII kinetic rates (Log2FC) on Myc-dependent (D) and Myc-independent (E) genes during the LPS time-course. The grey scale represents the starting level for each parameter in unstimulated cells. The first column on the left reports the changes in Myc share for the same genes. Genes were grouped using hierarchical clustering.


F . Boxplots reporting LPS - induced changes ( Log2FC ) for each of the four RNAPII kinetic rates in c - mycwt / wt ( wt ) and c - myc ∆ / ∆ cells for genes of clusters 1 and 9 . p - values obtained with two - sample Wilcoxon tests for the comparison between c - mycwt / wt and c - myc ∆ / ∆ cells are reported .

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F. Boxplots reporting LPS-induced changes (Log2FC) for each of the four RNAPII kinetic rates in c-mycwt/wt (wt) and c-myc∆/∆ cells for genes of clusters 1 and 9. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.


G . Changes in Myc share in LPS treated versus untreated cells for the different clusters of panel d . p - values obtained with two - sample Wilcoxon tests for the comparison between c - mycwt / wt and c - myc ∆ / ∆ cells are reported .

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G. Changes in Myc share in LPS treated versus untreated cells for the different clusters of panel d. p-values obtained with two-sample Wilcoxon tests for the comparison between c-mycwt/wt and c-myc∆/∆ cells are reported.


A Heart rate ( HR ) of WT and Adcy9 - / - mice averaged over 5 min of non - active versus sustained activity on home cage running wheels . Student ' s t - test P values for indicated comparisons are shown . Circles represent data from individual animals . The boxplot central band is the median while whiskers represent the 10th and 90th percentiles .

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A Heart rate (HR) of WT and Adcy9-/- mice averaged over 5 min of non-active versus sustained activity on home cage running wheels. Student's t-test P values for indicated comparisons are shown. Circles represent data from individual animals. The boxplot central band is the median while whiskers represent the 10th and 90th percentiles.


B , C HR and R - R interval of WT ( black bars ) and Adcy9 - / - mice ( red bars ) before and after ISO ( 1 μg / g )

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B,C HR and R-R interval of WT (black bars) and Adcy9-/- mice (red bars) before and after ISO (1 μg/g) injection


D Representative ECG recordings of 7 mo WT and Adcy9 - / - mice , ~ 55 min post ISO - injection .

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D Representative ECG recordings of 7 mo WT and Adcy9-/- mice, ~55 min post ISO-injection.


E , F HR variability was calculated before and after ISO injection using the root mean square of successive RR interval differences ( RMSSD ; panel ( E ) ) and percentage of sequential R - R intervals differing by > 6 ms ( pNN6 ; panel ( F ) ) methods .

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E,F HR variability was calculated before and after ISO injection using the root mean square of successive RR interval differences (RMSSD; panel (E)) and percentage of sequential R-R intervals differing by >6 ms (pNN6; panel (F)) methods.


A , B PLA assay performed in neonatal cardiomyocytes expressing GFP - Flag or AC9 - Flag using antibodies against Flag and endogenous POPDC1 , POPDC2 or Gβγ . Images ( A ) ; scale bar is 20 um ; nuclear staining by DAPI shown in blue ) and quantitation ( B ) of PLA signal are shown .

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A,B PLA assay performed in neonatal cardiomyocytes expressing GFP-Flag or AC9-Flag using antibodies against Flag and endogenous POPDC1, POPDC2 or Gβγ. Images (A); scale bar is 20 um; nuclear staining by DAPI shown in blue) and quantitation (B) of PLA signal are shown.


( D ) Quantification of COS7 cells expressing BiFC constructs for AC9 , POPDC 1 - 3 . The expressed proteins tagged with VN ( top line ) and VC ( bottom line ) are shown .

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(D) Quantification of COS7 cells expressing BiFC constructs for AC9, POPDC 1-3. The expressed proteins tagged with VN (top line) and VC (bottom line) are shown.


( E ) Representative live - cell images of indicated BiFC combinations in HEK293 cells ( n > 20 cells ; scale bar : 10 μm ) . Quantification of POPDC1 - VN : AC9 - VC is shown in Fig 3 and 7 .

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(E) Representative live-cell images of indicated BiFC combinations in HEK293 cells (n>20 cells; scale bar: 10 μm). Quantification of POPDC1-VN:AC9-VC is shown in Fig 3 and 7.


A HEK293 cells expressing Flag - AC9 in the presence or absence of Myc - tagged POPDC1 or - 2 were subjected to co - immunoprecipitation ( Co - IP ) with anti - MYC and assayed for AC activity with 300 nM Gαs - GTPγS . Kruskal - Wallis One Way ANOVA analysis on Ranks was performed ( n = 6 experiments , P = 0 . 003 between all groups ) with multiple comparisons to AC9 control by Dunn ' s method and Mann - Whitney Rank Sum Test ( * * P = 0 . 002 ) .

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A HEK293 cells expressing Flag-AC9 in the presence or absence of Myc-tagged POPDC1 or -2 were subjected to co-immunoprecipitation (Co-IP) with anti-MYC and assayed for AC activity with 300 nM Gαs-GTPγS. Kruskal-Wallis One Way ANOVA analysis on Ranks was performed (n=6 experiments, P=0.003 between all groups) with multiple comparisons to AC9 control by Dunn's method and Mann-Whitney Rank Sum Test (**P=0.002).


B A portion of the lysates and Co - IP from ( A ) were subjected to western blot ( WB ) analysis . Sf9 cells expressing Flag - AC9 served as a positive WB control . Molecular weight markers are denoted as M . Quantitation of Flag - AC9 WB by One Way ANOVA , n = 3 - 4 experiments , with comparisons by Tukey test , * * P = 0 . 003 , * * * P = 0 . 001 .

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B A portion of the lysates and Co-IP from (A) were subjected to western blot (WB) analysis. Sf9 cells expressing Flag-AC9 served as a positive WB control. Molecular weight markers are denoted as M. Quantitation of Flag-AC9 WB by One Way ANOVA, n=3-4 experiments, with comparisons by Tukey test, **P=0.003, ***P=0.001.


( C ) HEK293 cells expressing Flag - AC9 + / - POPDC1 - Myc or POPDC2 - Myc were subjected to Co - IP with anti - FLAG and subjected to WB analysis with anti - MYC and anti - FLAG . Quantitation of anti - MYC WB by One Way ANOVA , n = 4 experiments , with comparisons by Tukey test , * * P = 0 . 003 , * * * P < 0 . 001 ) .

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(C) HEK293 cells expressing Flag-AC9 +/- POPDC1-Myc or POPDC2-Myc were subjected to Co-IP with anti-FLAG and subjected to WB analysis with anti-MYC and anti-FLAG. Quantitation of anti-MYC WB by One Way ANOVA, n=4 experiments, with comparisons by Tukey test, **P=0.003, ***P<0.001).


( D ) HEK293 cells expressing POPDC1 - Myc + / - GFP - tagged AC9 and control TM proteins EGFR , or LAMP1 were subjected to Co - IP with anti - MYC . Western blotting of lysates and Co - IPs for GFP ( top ) and MYC ( bottom ) are shown ( n = 3 experiments ) .

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(D) HEK293 cells expressing POPDC1-Myc +/- GFP-tagged AC9 and control TM proteins EGFR, or LAMP1 were subjected to Co-IP with anti-MYC. Western blotting of lysates and Co-IPs for GFP (top) and MYC (bottom) are shown (n=3 experiments).


( F ) BiFC of AC9 and indicated POPDC1 truncations in COS - 7 cells . Kruskal - Wallis One Way ANOVA analysis was performed ( n = 4 experiments ) with multiple comparisons by Tukey test , * * * P < 0 . 001 compared to VN control , * * P = 0 . 008 .

(F) BiFC of AC9 and indicated POPDC1 truncations in COS-7 cells. Kruskal-Wallis One Way ANOVA analysis was performed (n=4 experiments) with multiple comparisons by Tukey test, ***P<0.001 compared to VN control, **P=0.008.


( G ) Co - IP with anti - MYC in COS - 7 cells expressing Flag - AC9 and indicated Myc - tagged POPDC1 truncations . Western blotting with anti - AC9 and anti - MYC ( POPDC1 ) of Co - IP and lysates is shown . Kruskal - Wallis One Way ANOVA analysis was performed ( n = 3 - 5 experiments , P = 0 . 006 ) , with multiple comparisons by Dunn ' s method ( * P = 0 . 021 , * * P = 0 . 01 ) .

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(G) Co-IP with anti-MYC in COS-7 cells expressing Flag-AC9 and indicated Myc-tagged POPDC1 truncations. Western blotting with anti-AC9 and anti-MYC (POPDC1) of Co-IP and lysates is shown. Kruskal-Wallis One Way ANOVA analysis was performed (n=3-5 experiments, P=0.006), with multiple comparisons by Dunn's method (*P=0.021, **P=0.01).


A Co - localization of CFP - TREK - 1 with the BiFC signal from AC9 - VN : AC9 - VC homodimer ( top ) and POPDC1 - VN : AC9 - VC complex ( bottom ) in HEK293 cells .

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A Co-localization of CFP-TREK-1 with the BiFC signal from AC9-VN:AC9-VC homodimer (top) and POPDC1-VN:AC9-VC complex (bottom) in HEK293 cells.


B The lifetime distribution of Cerulean - tagged proteins + / - the indicated YFP - tagged proteins expressed in HEK293 cells . Mann - Whitney Rank Sum Test was performed ( * * * P < 0 . 001 ; n = cell number indicated on each bar ) .

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B The lifetime distribution of Cerulean-tagged proteins +/- the indicated YFP-tagged proteins expressed in HEK293 cells. Mann-Whitney Rank Sum Test was performed (***P<0.001; n=cell number indicated on each bar).


C Co - IP of Myc - tagged POPDC1 ( anti - MYC ) pulled down both Flag - AC9 and YFP - tagged TREK - 1 ( n = 3 experiments ) . One - way ANOVA was performed with multiple comparisons by Holm - Sidak method ( # P = 0 . 007 compared to control ) .

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C Co-IP of Myc-tagged POPDC1 (anti-MYC) pulled down both Flag-AC9 and YFP-tagged TREK-1 (n=3 experiments). One-way ANOVA was performed with multiple comparisons by Holm-Sidak method (#P=0.007 compared to control).


D Co - IP of TREK - 1 ( anti - GFP ) pulls down Flag - AC9 and endogenous POPDC1 . AKAP79 is not pulled down in complex ( n = 3 experiments ) .

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D Co-IP of TREK-1 (anti-GFP) pulls down Flag-AC9 and endogenous POPDC1. AKAP79 is not pulled down in complex (n=3 experiments).


A IP - AC assay with IgG versus anti - TREK - 1 in heart homogenates from WT and Adcy9 - / - mice ; AC activity stimulated with 300 nM Gαs - GTPγS . IP - AC with anti - AKAP150 is shown as a positive control . Two - way ANOVA ( overall P < 0 . 001 , n = 3 mice per genotype ) with multiple comparisons by Holm - Sidak test ; # P < 0 . 001 for indicated comparison or to respective IgG controls . IgG versus anti - TREK - 1 in Adcy9 - / - was analyzed by Student ' s t - test ( * * P = 0 . 009 , n = 3 ) . A portion of the co - IP was subjected to western blotting with anti - TREK - 1 and anti - AKAP150 .

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A IP-AC assay with IgG versus anti-TREK-1 in heart homogenates from WT and Adcy9-/- mice; AC activity stimulated with 300 nM Gαs-GTPγS. IP-AC with anti-AKAP150 is shown as a positive control. Two-way ANOVA (overall P<0.001, n=3 mice per genotype) with multiple comparisons by Holm-Sidak test; #P<0.001 for indicated comparison or to respective IgG controls. IgG versus anti-TREK-1 in Adcy9-/- was analyzed by Student's t-test (**P=0.009, n=3). A portion of the co-IP was subjected to western blotting with anti-TREK-1 and anti-AKAP150.


B IP - AC assay with IgG versus anti - TREK in WT and Popdc1 - / - mouse heart homogenates ; AC activity stimulated as in ( A ) . Statistics as in ( A ) , n = 4 mice per genotype , # P < 0 . 001 . Paired t - test within Popdc1 - / - , P = 0 . 123 .

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B IP-AC assay with IgG versus anti-TREK in WT and Popdc1-/- mouse heart homogenates; AC activity stimulated as in (A). Statistics as in (A), n=4 mice per genotype, #P<0.001. Paired t-test within Popdc1-/-, P=0.123.


C Total AC activity in heart homogenates from WT or Popdc1 - / - ( P1 - / - ) mice stimulated with 300 nM Gαs - GTPγS or 100 μM calcium and 300 nM calmodulin .

C Total AC activity in heart homogenates from WT or Popdc1-/- (P1-/-) mice stimulated with 300 nM Gαs-GTPγS or 100 μM calcium and 300 nM calmodulin.


D IP - AC assay with anti - MYC from cell lysates expressing AC1 or AC8 in the presence or absence of Myc - tagged POPDC1 . AC activity stimulated with 100 μM calcium and 300 nM calmodulin . Student ' s t - test ( n = 3 experiments , * * P = 0 . 0011 and * P = 0 . 012 ) .

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D IP-AC assay with anti-MYC from cell lysates expressing AC1 or AC8 in the presence or absence of Myc-tagged POPDC1. AC activity stimulated with 100 μM calcium and 300 nM calmodulin. Student's t-test (n=3 experiments, **P=0.0011 and *P=0.012).


E IP - AC assay with IgG versus anti - TREK - 1 in WT and Popdc1 - / - mouse heart homogenates ; AC activity stimulated as in ( D ) . Student ' s t - test ( * P = 0 . 03 ; * * P = 0 . 005 , n = 4 mice per genotype ) .

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E IP-AC assay with IgG versus anti-TREK-1 in WT and Popdc1-/- mouse heart homogenates; AC activity stimulated as in (D). Student's t-test (*P=0.03; **P=0.005, n=4 mice per genotype).


A Treatment of HEK293 cells for 10 min in the presence of 10 µM ISO reduced the amount of Flag - AC9 and POPDC1 - Myc pulled down by TREK - 1 co - IP .

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A Treatment of HEK293 cells for 10 min in the presence of 10 µM ISO reduced the amount of Flag-AC9 and POPDC1-Myc pulled down by TREK-1 co-IP.


B , C Quantification ( IP / input ) of Flag - AC9 ( B ) and POPDC1 - Myc ( C ) between ISO treatment groups by two - tailed paired Student ' s t - test ( * P = 0 . 028 , * * P = 0 . 007 ; n = 3 biological replicates ) .

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B, C Quantification (IP/input) of Flag-AC9 (B) and POPDC1-Myc (C) between ISO treatment groups by two-tailed paired Student's t-test (*P=0.028, **P=0.007; n=3 biological replicates).


, B BiFC signal between the indicated VN - and VC - tagged proteins ( POPDC1 , P1 ) . B Percent decrease of BiFC signal in ( A ) with ISO treatment ( 10 μM , 10 min at 37 ° C ) . Paired t - test for each condition ( vehicle versus ISO ) using raw data prior to calculation of percent of vehicle control ( n = 6 ; * P < 0 . 05 ; * * P < 0 .

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, B BiFC signal between the indicated VN- and VC-tagged proteins (POPDC1, P1). B Percent decrease of BiFC signal in (A) with ISO treatment (10 μM, 10 min at 37°C). Paired t-test for each condition (vehicle versus ISO) using raw data prior to calculation of percent of vehicle control (n=6; *P<0.05; **P<0.01


C ISO dose - response curves for BiFC interactions between POPDC1 : AC9 , TREK - 1 : AC9 , and TREK - 1 with a catalytically inactive AC9 ( TREK - 1 : AC9D ) .

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C ISO dose-response curves for BiFC interactions between POPDC1:AC9, TREK-1:AC9, and TREK-1 with a catalytically inactive AC9 (TREK-1:AC9D).


D TREK - 1 : AC9 BiFC signal is absent in COS - 7 cells lacking detectable POPDC1 . TREK - 1 : TREK - 1 BiFC is a positive control . One - way ANOVA with multiple comparisons by Holm Sidak method ( n = 3 experiments ; * * * P < 0 . 001 ) .

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D TREK-1:AC9 BiFC signal is absent in COS-7 cells lacking detectable POPDC1. TREK-1:TREK-1 BiFC is a positive control. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3 experiments; ***P<0.001).


E Overexpression of POPDC1 truncation of Popeye domain ( POPDC1 - Δ172 ) , but not WT POPDC1 , abolishes ISO reduction of TREK - 1 : AC9 BiFC signal . One - way ANOVA with multiple comparisons by Holm Sidak method ( n = 3 - 4 experiments ; * * * P < 0 . 001 compared to control ) .

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E Overexpression of POPDC1 truncation of Popeye domain (POPDC1-Δ172), but not WT POPDC1, abolishes ISO reduction of TREK-1:AC9 BiFC signal. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4 experiments; ***P<0.001 compared to control).


A Representative two - electrode voltage clamp measurements in Xenopus oocytes injected with TREK - 1 alone ( orange ) or TREK - 1 co - injected with cRNAs encoding the indicated proteins . Voltage was ramped from - 120 to + 40 mV . B Relative TREK - 1 current amplitudes at + 40 mV normalized to TREK - 1 injected alone . The number of oocytes is given within the bar graphs ( n = 37 - 97 ) . 3 - 5 batches of oocytes were used for each condition .

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A Representative two-electrode voltage clamp measurements in Xenopus oocytes injected with TREK-1 alone (orange) or TREK-1 co-injected with cRNAs encoding the indicated proteins. Voltage was ramped from -120 to +40 mV. B Relative TREK-1 current amplitudes at +40 mV normalized to TREK-1 injected alone. The number of oocytes is given within the bar graphs (n=37-97). 3-5 batches of oocytes were used for each condition.


C Relative currents normalized to TREK - 1 plus POPDC1 in the absence and presence of theophylline . Data are plotted as mean ± SEM .

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C Relative currents normalized to TREK-1 plus POPDC1 in the absence and presence of theophylline. Data are plotted as mean ± SEM.


D , E Oocytes were treated with 0 . 5 mM theophylline prior to voltage - clamp measurements as in ( A , B ) .

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D, E Oocytes were treated with 0.5 mM theophylline prior to voltage-clamp measurements as in (A, B).


Immunoblot ( IB ) analyses of the levels of β - catenin and RAS in DLD1 , WT - HCT116 , MT - HCT116 , HepG2 , and SW48 cells transfected with Myc - axin for 36

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Immunoblot (IB) analyses of the levels of β-catenin and RAS in DLD1, WT-HCT116, MT-HCT116, HepG2, and SW48 cells transfected with Myc-axin for 36 hr


Effects of β - catenin knock - down on polyubiquitination - dependent degradation of RAS . IB analyses of MT - HCT116 cells transfected with control or two independent β - catenin siRNAs for 48

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Effects of β-catenin knock-down on polyubiquitination-dependent degradation of RAS. IB analyses of MT-HCT116 cells transfected with control or two independent β-catenin siRNAs for 48 hr


Polyubiquitination assays of MT - HCT116 cells cotransfected with two independent β - catenin siRNAs and HA - ubiquitin ( Ub ) for 72 hr in the presence of the proteasomal inhibitor MG132 , and WCLs were immunoprecipitated with an anti - pan - RAS

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Polyubiquitination assays of MT-HCT116 cells cotransfected with two independent β-catenin siRNAs and HA-ubiquitin (Ub) for 72 hr in the presence of the proteasomal inhibitor MG132, and WCLs were immunoprecipitated with an anti-pan-RAS antibody


IB analyses of HEK293 cells transfected with WT - or S33Y - Flag - β - catenin with or without HA - GSK3β for 36

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IB analyses of HEK293 cells transfected with WT- or S33Y-Flag-β-catenin with or without HA-GSK3β for 36 hr


IB analyses of HEK293 cells transfected with β - catenin siRNA or control siRNA for 48 h and then treated with L - cell - conditioned medium ( L - CM ) or Wnt3a - CM for 8

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IB analyses of HEK293 cells transfected with β-catenin siRNA or control siRNA for 48 h and then treated with L-cell-conditioned medium (L-CM) or Wnt3a-CM for 8 hr


Effects of KYA1797K or XAV939 on degradation of β - catenin and RAS in isogenic HCT116 cells harboring WT - or MT - CTNNB1 . IB analyses of β - catenin and RAS levels in WT - or MT - HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24

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Effects of KYA1797K or XAV939 on degradation of β-catenin and RAS in isogenic HCT116 cells harboring WT- or MT-CTNNB1. IB analyses of β-catenin and RAS levels in WT- or MT-HCT116 cells treated with 5 or 20 µM of KYA1797K or XAV939 for 24 hr


ICC analyses of WT - or MT - HCT116 cells treated with 20 µM of KYA1797K for 24 hr . Scale bars , 10

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ICC analyses of WT- or MT-HCT116 cells treated with 20 µM of KYA1797K for 24 hr. Scale bars, 10 µm


For polyubiquitination assays , WT - or MT - HCT116 cells were transfected with FLAG - Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132 . WCLs were immunoprecipitated with β - catenin or pan - RAS antibody and subjected to IB analyses to detect each

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For polyubiquitination assays, WT- or MT-HCT116 cells were transfected with FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K for 24 hr in the presence of MG132. WCLs were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analyses to detect each protein


The 3 - ( 4 , 5 - dimethylthiazol - 2yl ) - 2 - 5 - diphenyltetrazolium bromide ( MTT ) assays were performed for measurements of growth of WT - or MT - HCT116 cells treated with the indicated concentrations of KYA1797K . Data are presented as the mean ± SD ( n = 3 )

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The 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) assays were performed for measurements of growth of WT- or MT-HCT116 cells treated with the indicated concentrations of KYA1797K. Data are presented as the mean ± SD (n = 3)


Analyses of the interaction between β - catenin and RAS . WCLs of DLD1 cells were immunoprecipitated with β - catenin or pan - RAS antibody and subjected to IB

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Analyses of the interaction between β-catenin and RAS. WCLs of DLD1 cells were immunoprecipitated with β-catenin or pan-RAS antibody and subjected to IB analyses


IB analyses of indicated proteins in the samples immunoprecipitated with anti - β - catenin antibody with WCLs of HEK293 cells treated with Wnt3a -

IB analyses of indicated proteins in the samples immunoprecipitated with anti-β-catenin antibody with WCLs of HEK293 cells treated with Wnt3a-CM


Left panel : MT - HCT116 cell extracts were fractionated as cytosolic and nuclear portions and subjected to IB analyses , respectively . Right panel : The cytosolic and nuclear fractions were immunoprecipitated with anti - β - catenin antibody and subjected to IB

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Left panel: MT-HCT116 cell extracts were fractionated as cytosolic and nuclear portions and subjected to IB analyses, respectively. Right panel: The cytosolic and nuclear fractions were immunoprecipitated with anti-β-catenin antibody and subjected to IB analyses


β - Catenin - RAS interaction was visualized by in situ proximity ligation assay in HEK293 cells treated with L - CM or Wnt3a - CM . Scale bars , 20 µm . The proximity ligation signal was quantified in 10 randomly distributed microscopic images that contained > 100

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β-Catenin-RAS interaction was visualized by in situ proximity ligation assay in HEK293 cells treated with L-CM or Wnt3a-CM. Scale bars, 20 µm. The proximity ligation signal was quantified in 10 randomly distributed microscopic images that contained > 100 cells


In vitro pulldown analyses to identify β - catenin binding region of RAS . Upper panel : Schematic of the HRAS full - length ( FL ) or two deletion mutants . Lower panel : Purified GST - HRAS - FL , - ΔHVR , or - Δ131 - 165 proteins were pulled - down with purified His - β - catenin - FL protein and subjected to IB

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In vitro pulldown analyses to identify β-catenin binding region of RAS. Upper panel: Schematic of the HRAS full-length (FL) or two deletion mutants. Lower panel: Purified GST-HRAS-FL, -ΔHVR, or -Δ131-165 proteins were pulled-down with purified His-β-catenin-FL protein and subjected to IB analyses


IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG - β - catenin and Myc - HRAS - FL , - ΔHVR , or - Δ131 - 165 for 32

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IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-FL, -ΔHVR, or -Δ131-165 for 32 hr


IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG - β - catenin and Myc - HRAS - WT , - 2A , or - 2E for 32

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IB analyses of IP with FLAG antibody in WCLs of HEK293 cells cotransfected with FLAG-β-catenin and Myc-HRAS-WT, -2A, or -2E for 32 hr


Upper panel : Schematic of the β - catenin - FL or two deletion mutants . Lower panel : Purified His - β - catenin - FL , ΔN86 , or ΔC110 proteins were pull - down with purified GST - HRAS - FL protein and subjected to IB

Upper panel: Schematic of the β-catenin-FL or two deletion mutants. Lower panel: Purified His-β-catenin-FL, ΔN86, or ΔC110 proteins were pull-down with purified GST-HRAS-FL protein and subjected to IB analyses


IB analyses of IP with Myc antibody with WCLs of HEK293 cells cotransfected with Myc - HRAS and GFP - β - catenin - FL , - ΔN86 , or - ΔC110 for 32

IB analyses of IP with Myc antibody with WCLs of HEK293 cells cotransfected with Myc-HRAS and GFP-β-catenin-FL, -ΔN86, or -ΔC110 for 32 hr


Fluorescence spectroscopy analyses of the interaction between β - catenin and KRAS using purified recombinant proteins , intact β - catenin and KRAS . Molar ratios of β - catenin and KRAS are presented . A . U . , absorbance

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Fluorescence spectroscopy analyses of the interaction between β-catenin and KRAS using purified recombinant proteins, intact β-catenin and KRAS. Molar ratios of β-catenin and KRAS are presented. A.U., absorbance units


Nuclear magnetic resonance ( NMR ) titration for 15N - labeled KRAS ( full - length , 1 - 188 ) with β - catenin ( C - terminal fragment , 661 - 781 ) . The titration points are indicated in red ( 1 : 0 ) , green ( 1 : 5 ) , and blue ( 1 : 10 ) . A gradual perturbation in chemical shift is indicated by the

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Nuclear magnetic resonance (NMR) titration for 15N-labeled KRAS (full-length, 1-188) with β-catenin (C-terminal fragment, 661-781). The titration points are indicated in red (1:0), green (1:5), and blue (1:10). A gradual perturbation in chemical shift is indicated by the arrow


Effects of KYA1797K on the interaction and phosphorylation of β - catenin and pan - RAS . WT - or MT - HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132 . WCLs were immunoprecipitated with pan - RAS antibody and subjected to IB

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Effects of KYA1797K on the interaction and phosphorylation of β-catenin and pan-RAS. WT- or MT-HCT116 cells were treated with 20 µM of KYA1797K in the presence of MG132. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses


Effects of Wnt3a - CM treatment on the formation of β - catenin destruction complex and phosphorylation of β - catenin and RAS in HEK293 cells treated with L - CM or Wnt3a - CM in the presence of MG132 for 8 hr . WCLs were immunoprecipitated with β - catenin or pan - RAS antibodies and subjected to IB

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Effects of Wnt3a-CM treatment on the formation of β-catenin destruction complex and phosphorylation of β-catenin and RAS in HEK293 cells treated with L-CM or Wnt3a-CM in the presence of MG132 for 8 hr. WCLs were immunoprecipitated with β-catenin or pan-RAS antibodies and subjected to IB analyses


IB analyses were performed to detect β - catenin and pan - RAS in DLD1 cells cotransfected with FLAG - β - catenin - WT or - MT and Myc - KRAS - WT or - 2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA - GSK3β for 24 hr ,

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IB analyses were performed to detect β-catenin and pan-RAS in DLD1 cells cotransfected with FLAG-β-catenin-WT or -MT and Myc-KRAS-WT or -2A for 24 hr and then treated or transfected with 20 µM of KYA1797K or HA-GSK3β for 24 hr, respectively


IB analyses to determine the interaction between β - catenin and BBR peptide by in vitro pull - down assays using purified His - β - catenin and GST - BBR

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IB analyses to determine the interaction between β-catenin and BBR peptide by in vitro pull-down assays using purified His-β-catenin and GST-BBR proteins


IB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG - β - catenin and GFP - BBR or - Mock in the presence of proteasome inhibitor , ALLN , for 24

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IB analyses of IP with FLAG antibody and WCLs from HEK293 cells cotransfected with FLAG-β-catenin and GFP-BBR or -Mock in the presence of proteasome inhibitor, ALLN, for 24 hr


Effects of BBR overexpression on KYA1797K - induced RAS degradation . I analyses to detect levels of RAS in MT - HCT116 cells transfected with GFP - Mock or - BBR for 24 hr and then treated with 20 µM of KYA1797K for 24

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Effects of BBR overexpression on KYA1797K-induced RAS degradation. I analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hr


Effects of BBR overexpression on KYA1797K - induced RAS degradation ICC ( D ; Scale bars , 10 µm ) analyses to detect levels of RAS in MT - HCT116 cells transfected with GFP - Mock or - BBR for 24 hr and then treated with 20 µM of KYA1797K for 24

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Effects of BBR overexpression on KYA1797K-induced RAS degradation ICC (D; Scale bars, 10 µm) analyses to detect levels of RAS in MT-HCT116 cells transfected with GFP-Mock or -BBR for 24 hr and then treated with 20 µM of KYA1797K for 24 hr


MT - HCT116 cells were cotransfected with GFP - Mock or - BBR and FLAG - Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr , and WCLs were immunoprecipitated with pan - RAS

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MT-HCT116 cells were cotransfected with GFP-Mock or -BBR and FLAG-Ub for 24 hr and then treated with 20 µM of KYA1797K in the presence of MG132 for 8 hr, and WCLs were immunoprecipitated with pan-RAS antibody


IB analyses of IP with β - catenin antibody in MT - HCT116 cells transfected with GFP - BBR - WT , - 2E , or GFP - Mock in the presence of ALLN for 24

IB analyses of IP with β-catenin antibody in MT-HCT116 cells transfected with GFP-BBR-WT, -2E, or GFP-Mock in the presence of ALLN for 24 hr


IB analyses of MT - HCT116 cells transfected with GFP - Mock , - BBR - WT , or - BBR - 2E for 24 hr and then treated with 20 µM of KYA1797K for 24

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IB analyses of MT-HCT116 cells transfected with GFP-Mock, -BBR-WT, or -BBR-2E for 24 hr and then treated with 20 µM of KYA1797K for 24 hr


MT - HCT116 cells were transfected with GFP - Mock , - BBR - WT , or - BBR - 2E together with FLAG - Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells . WCLs were immunoprecipitated with pan - RAS antibody and subjected to IB

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MT-HCT116 cells were transfected with GFP-Mock, -BBR-WT, or -BBR-2E together with FLAG-Ub with or without 20 µM of KYA1797K for 24 hr and MG132 was treated for 8 hr before harvesting cells. WCLs were immunoprecipitated with pan-RAS antibody and subjected to IB analyses


Effects of KYA1797K and PTD - BBR on RAS degradation IB assay of MT - HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr ,

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Effects of KYA1797K and PTD-BBR on RAS degradation IB assay of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively


Effects of KYA1797K and PTD - BBR o cell growth MTT assays of MT - HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr , respectively . Data are presented as the mean ± SD ( n = 3 )

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Effects of KYA1797K and PTD-BBR o cell growth MTT assays of MT-HCT116 or SW48 cells treated with indicated conditions for 24 and 96 hr, respectively. Data are presented as the mean ± SD (n = 3)


Effects of overexpression of GFP - BBR - WT or - 2E mutant on KYA1797K - induced growth and transformation of CRC cells . MT - HCT116 or SW48 cells were transfected with GFP - BBR - WT , - 2E , or GFP - Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell

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Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions MTT assays were performed for measurements of cell growth


Effects of overexpression of GFP - BBR - WT or - 2E mutant on KYA1797K - induced growth and transformation of CRC cells . MT - HCT116 or SW48 cells were transfected with GFP - BBR - WT , - 2E , or GFP - Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular

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Effects of overexpression of GFP-BBR-WT or -2E mutant on KYA1797K-induced growth and transformation of CRC cells. MT-HCT116 or SW48 cells were transfected with GFP-BBR-WT, -2E, or GFP-Mock for 24 hr and then treated with KYA1797K via indicated conditions foci formation assay were performed to detect cellular transformation


Effects of cotreatments with KYA1797K and PTD - BBR on xenografted tumor growth . MT - HCT116 cells were subcutaneously injected in nude mice with subsequent i . p . injection of vehicle , 20 mg / kg of KYA1797K , or a cotreatment of PTD - BBR ( 25 mg / kg ) and KYA1797K ( 20 mg / kg ) for 20 d . Tumor volumes ( upper panel ) were measured every 4 days , and tumor weights ( lower panel ) were measured at the times of sacrifice . Data are presented as the mean ± SD ( four mice per group )

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Effects of cotreatments with KYA1797K and PTD-BBR on xenografted tumor growth. MT-HCT116 cells were subcutaneously injected in nude mice with subsequent i.p. injection of vehicle, 20 mg/kg of KYA1797K, or a cotreatment of PTD-BBR (25 mg/kg) and KYA1797K (20 mg/kg) for 20 d. Tumor volumes (upper panel) were measured every 4 days, and tumor weights (lower panel) were measured at the times of sacrifice. Data are presented as the mean ± SD (four mice per group)


WCLs prepared from tumor tissues were immunoprecipitated with anti - β - catenin antibody . WCLs or co - IP samples were subjected to IB

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WCLs prepared from tumor tissues were immunoprecipitated with anti-β-catenin antibody. WCLs or co-IP samples were subjected to IB analyses


IHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI . Scale bar , 20

IHC analyses of tissue sections incubated with the indicated antibodies and then counterstained with DAPI. Scale bar, 20 µm


( A ) smFISH targeting Bmal1 , Cry1 and Nr1d1 in wild - type NIH 3T3 cells at 17h , 29h and 25h after synchronisation with Dex , respectively . Nuclei are stained with DAPI ( blue ) . Each fluorescent dot ( white ) corresponds to a single transcript . Segmented cell boundaries are delineated in grey . The blue and white channels represent maximum z - projections . Scale bar : 20μm .

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(A) smFISH targeting Bmal1, Cry1 and Nr1d1 in wild-type NIH 3T3 cells at 17h, 29h and 25h after synchronisation with Dex, respectively. Nuclei are stained with DAPI (blue). Each fluorescent dot (white) corresponds to a single transcript. Segmented cell boundaries are delineated in grey. The blue and white channels represent maximum z-projections. Scale bar: 20μm.


( B ) Number of Bmal1 , Cry1 , and Nr1d1 transcripts per cell as a function of time after treatment with Dex . These data combine all smFISH hybridizations from the 4h sampled Nr1d1 / Cry1 and Bmal1 / Cry1 experiment . Each dot shows the average over one replicate from an independent slide ( Methods ) . The solid lines represent fits using a two - harmonic cosinor model ( Equation ( 1 ) , Methods ) for each gene individually .

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(B) Number of Bmal1, Cry1, and Nr1d1 transcripts per cell as a function of time after treatment with Dex. These data combine all smFISH hybridizations from the 4h sampled Nr1d1/Cry1 and Bmal1/Cry1 experiment. Each dot shows the average over one replicate from an independent slide (Methods). The solid lines represent fits using a two-harmonic cosinor model (Equation (1), Methods) for each gene individually.


( D ) Distributions of Nr1d1 , Cry1 and Bmal1 transcripts at 21h , 29h and 37h after synchronisation with Dex . μ represents the mean of the distribution , CV represent the coefficient of variation ( standard deviation / mean ) , and N is the number of cells at the given time point . Total number of cells analysed for the Nr1d1 / Cry1 pair : 21h - 449 ; 25h - 414 ; 29h - 521 ; 33h - 463 ; 37h - 477 ; 41h - 429 . Total number of cells analysed for the Bmal1 / Cry1 pair : 17h - 465 ; 21h - 490 ; 25h - 504 ; 29h - 436 ; 33h - 407 ; 37h - 454 ; 41h - 404 .

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(D) Distributions of Nr1d1, Cry1 and Bmal1 transcripts at 21h, 29h and 37h after synchronisation with Dex. μ represents the mean of the distribution, CV represent the coefficient of variation (standard deviation / mean), and N is the number of cells at the given time point. Total number of cells analysed for the Nr1d1/Cry1 pair: 21h - 449; 25h - 414; 29h - 521; 33h - 463; 37h - 477; 41h - 429. Total number of cells analysed for the Bmal1/Cry1 pair: 17h - 465; 21h - 490; 25h - 504; 29h - 436; 33h - 407; 37h - 454; 41h - 404.


( A ) Log conditional mean mRNA count for a given area as a function of the log area . Each cell is sorted by area by placing it in one of 20 bins that range from the 5th to the 95th percentile . The conditional mean given the area E [ m | A ] is then the mean mRNA count of each bin . The area is normalised such that the average area is equal to one . Solid lines represent a least - squares linear fit to the data .

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(A) Log conditional mean mRNA count for a given area as a function of the log area. Each cell is sorted by area by placing it in one of 20 bins that range from the 5th to the 95th percentile. The conditional mean given the area E[m|A] is then the mean mRNA count of each bin. The area is normalised such that the average area is equal to one. Solid lines represent a least-squares linear fit to the data.


( B ) Top image : Dual - channel smFISH targeting Bmal1 and Cry1 simultaneously , taken at 17h after Dex synchronisation . Blue , nuclei stained with DAPI ; green dots , Bmal1 transcripts ; red , Cry1 transcripts . Bottom image : Dual - channel smFISH targeting Nr1d1 and Cry1 simultaneously , taken at 25h after Dex synchronisation . Blue , nuclei stained with DAPI ; green dots , Nr1d1 transcripts ; red , Cry1 transcripts . Scale bar : 20μm .

(B) Top image: Dual-channel smFISH targeting Bmal1 and Cry1 simultaneously, taken at 17h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Bmal1 transcripts; red, Cry1 transcripts. Bottom image: Dual-channel smFISH targeting Nr1d1 and Cry1 simultaneously, taken at 25h after Dex synchronisation. Blue, nuclei stained with DAPI; green dots, Nr1d1 transcripts; red, Cry1 transcripts. Scale bar: 20μm.


( C ) Bivariate distributions of mRNA counts per cell for dual - channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1 . Each dot corresponds to a single cell , and the contours represent KDE estimates of the density . Time represents the number of hours after Dex synchronisation , and R represents the Pearson correlation coefficient .

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(C) Bivariate distributions of mRNA counts per cell for dual-channel smFISH targeting either Bmal1 and Cry1 or Nr1d1 and Cry1. Each dot corresponds to a single cell, and the contours represent KDE estimates of the density. Time represents the number of hours after Dex synchronisation, and R represents the Pearson correlation coefficient.


( C ) Posterior parameter estimates . Burst frequency is plotted as a function of time , where the solid line represents the posterior mean and the shaded area represents the 90 % confidence interval . Posterior probability densities are shown for the average burst size , β ( which controls the dependence between burst size and cell area ) and ρ ( representing the correlation in burst size between genes ( in log space ) ) , where β is inferred from model M2 and the average burst size and ρ are calculated from model M4 .

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(C) Posterior parameter estimates. Burst frequency is plotted as a function of time, where the solid line represents the posterior mean and the shaded area represents the 90% confidence interval. Posterior probability densities are shown for the average burst size, β (which controls the dependence between burst size and cell area) and ρ (representing the correlation in burst size between genes (in log space)), where β is inferred from model M2 and the average burst size and ρ are calculated from model M4.


( D ) Comparison of the probability density of the data ( kernel density estimates ) with the model ( using model M4 ) . To estimate the probability from the model , the data set was simulated 15 times using the posterior mean parameter values combined with the measured cell areas .

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(D) Comparison of the probability density of the data (kernel density estimates) with the model (using model M4). To estimate the probability from the model, the data set was simulated 15 times using the posterior mean parameter values combined with the measured cell areas.


D Heatmaps showing pairwise comparison of MPRA activity for all constructs ( mRNA / DNA ) between donors - left panel : resting CD4 T cells ; right panel : stimulated CD4 T cells .

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D Heatmaps showing pairwise comparison of MPRA activity for all constructs (mRNA/DNA) between donors - left panel: resting CD4 T cells; right panel: stimulated CD4 T cells.


B Activity of each allele at 10 positive control SNPs and 1 negative control SNP in stimulated T cells . GATA1 , NF - κB , and RUNX1 constructs were designed to include a binding site for the indicated transcription factors ( + ) or with that site disrupted ( - ) . Fixed effects meta - analysis P value is shown : * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 . Box and whisker plots represent median and IQR ( box ) and min - to - max ( whiskers ) .

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B Activity of each allele at 10 positive control SNPs and 1 negative control SNP in stimulated T cells. GATA1, NF-κB, and RUNX1 constructs were designed to include a binding site for the indicated transcription factors (+) or with that site disrupted (-). Fixed effects meta-analysis P value is shown: *<0.05; **<0.01, ***<0.001, ****<0.0001. Box and whisker plots represent median and IQR (box) and min-to-max (whiskers).


D Expression - modulating effect of each SNP [ log2 ( OR ) ] as measured in MPRA and validation experiments . OR were calculated using the median activity of allelic constructs and are presented with respect to the risk allele ( luciferase : n = 5 , MPRA : n = 12 ) .

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D Expression-modulating effect of each SNP [log2(OR)] as measured in MPRA and validation experiments. OR were calculated using the median activity of allelic constructs and are presented with respect to the risk allele (luciferase: n=5, MPRA: n=12).


A Pie chart depicting fine - mapping results ( Huang et al , 2017 ) ( posterior probabilities ) for an IBD - associated locus on 21q21 ( left panel ) . MPRA results in resting ( centre panel ) and stimulated CD4 T cells ( right panel ) showing that the putative causal variant has significant expression - modulating effect .

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A Pie chart depicting fine-mapping results (Huang et al, 2017) (posterior probabilities) for an IBD-associated locus on 21q21 (left panel). MPRA results in resting (centre panel) and stimulated CD4 T cells (right panel) showing that the putative causal variant has significant expression-modulating effect.


B Pie chart depicting Bayesian fine - mapping results for an AS - associated locus on 2p15 ( left panel ) . MPRA results in stimulated T cells ( right panel ) showing that rs6759298 has a significant expression - modulating effect ( the strongest of any variant at this locus ) while the other candidate SNPs have negligible effects .

B Pie chart depicting Bayesian fine-mapping results for an AS-associated locus on 2p15 (left panel). MPRA results in stimulated T cells (right panel) showing that rs6759298 has a significant expression-modulating effect (the strongest of any variant at this locus) while the other candidate SNPs have negligible effects.


C GWAS results ( Liu et al , 2015 ) at a Crohn ' s disease and multiple sclerosis - associated locus on 6p23 ( left panel ) . MPRA for candidate SNPs in stimulated T cells ( right panel ) identifying a SNP ( rs34421390 ) with by far the greatest expression - modulating effect at this locus ( blue , risk allele reduces expression ; red , risk allele increases expression ) . Dotted horizontal line represents significance threshold ( corrected for multiple - testing ) .

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C GWAS results (Liu et al, 2015) at a Crohn's disease and multiple sclerosis-associated locus on 6p23 (left panel). MPRA for candidate SNPs in stimulated T cells (right panel) identifying a SNP (rs34421390) with by far the greatest expression-modulating effect at this locus (blue, risk allele reduces expression; red, risk allele increases expression). Dotted horizontal line represents significance threshold (corrected for multiple-testing).


D GWAS results at a Type 1 Diabetes - associated locus on 14q32 ( Onengut - Gumuscu et al , 2015 ; left panel ) . MPRA for the candidate SNPs in stimulated T cells ( right panel ) . The construct with the largest and most significant effect contains the risk alleles for 2 SNPs ( rs1988588 and rs3902659 ) , each of which has a smaller effect when tested individually ( position indicated by vertical dotted line ) . Dotted horizontal line represents significance threshold ( corrected for multiple - testing ) .

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D GWAS results at a Type 1 Diabetes-associated locus on 14q32 (Onengut-Gumuscu et al, 2015; left panel). MPRA for the candidate SNPs in stimulated T cells (right panel). The construct with the largest and most significant effect contains the risk alleles for 2 SNPs (rs1988588 and rs3902659), each of which has a smaller effect when tested individually (position indicated by vertical dotted line). Dotted horizontal line represents significance threshold (corrected for multiple-testing).


A IBD GWAS results ( Liu et al , 2015 ) at a multi - disease - associated locus on chromosome 6q23 .

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A IBD GWAS results (Liu et al, 2015) at a multi-disease-associated locus on chromosome 6q23.


C A single variant ( rs6927172 ) has the largest and most significant expression - modulating activity in resting ( left panel ) and stimulated CD4 T cells ( right panel ) with the risk allele reducing transcription . Plots represent median and IQR ( box ) and min - to - max ( whiskers ) . FDR - corrected meta - analysis P value shown .

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C A single variant (rs6927172) has the largest and most significant expression-modulating activity in resting (left panel) and stimulated CD4 T cells (right panel) with the risk allele reducing transcription. Plots represent median and IQR (box) and min-to-max (whiskers). FDR-corrected meta-analysis P value shown.


E Allele - specific NF - κB binding in CD4 T cells from rs6927172 heterozygotes , demonstrating reduced NF - κB binding to the risk allele following stimulation ( n = 8 ; one - sample t - test , two - tailed ) .

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E Allele-specific NF-κB binding in CD4 T cells from rs6927172 heterozygotes, demonstrating reduced NF-κB binding to the risk allele following stimulation (n=8; one-sample t-test, two-tailed).


F Allele - specific expression of enhancer RNA in heterozygous CD4 T cells . DNA used for technical control ( n = 6 ; paired t - test ; two - tailed ) .

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F Allele-specific expression of enhancer RNA in heterozygous CD4 T cells. DNA used for technical control (n=6; paired t-test; two-tailed).


G Genome - wide H3K27ac ChIP - seq in stimulated CD4 T cells from major - and minor - allele homozygotes at rs6927172 ( n = 6 ) . Upper panels show input - normalised H3K27ac signals plotted against enhancer rank . Super - enhancers are defined above the inflection point of the curve . Lower panels show H3K27ac reads from a major ( left ) and a minor ( risk ) allele homozygote ( right ) in a 9kb window around rs6927172 .

G Genome-wide H3K27ac ChIP-seq in stimulated CD4 T cells from major- and minor-allele homozygotes at rs6927172 (n=6). Upper panels show input-normalised H3K27ac signals plotted against enhancer rank. Super-enhancers are defined above the inflection point of the curve. Lower panels show H3K27ac reads from a major (left) and a minor (risk) allele homozygote (right) in a 9kb window around rs6927172.


I Expression of genes on 6q23 in CD4 T cells from 131 patients with active IBD , stratified by rs6927172 genotype ( qPCR ; one - way ANOVA ) . Error bars represent SD . Expression of IL20RA and IL22RAR2 not detected .

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I Expression of genes on 6q23 in CD4 T cells from 131 patients with active IBD, stratified by rs6927172 genotype (qPCR; one-way ANOVA). Error bars represent SD. Expression of IL20RA and IL22RAR2 not detected.


B Primary CD4 T cell editing efficiency for indicated combinations of 5 ' and 3 ' gRNAs ( n = 6 for DB , DH , FH , and n = 2 for FB ) . Distribution of indels assessed using ICE .

B Primary CD4 T cell editing efficiency for indicated combinations of 5' and 3' gRNAs (n=6 for DB, DH, FH, and n=2 for FB). Distribution of indels assessed using ICE.


D Expression of genes on 6q23 in EU - containing mRNA ( EU added at time of stimulation ) showing that deletion of the NF - κB binding site specifically reduces transcription of TNFAIP3 , but not other genes at this locus ( n = 6 ; one sample t - test ) . Representative data from the DH gRNA combination .

D Expression of genes on 6q23 in EU-containing mRNA (EU added at time of stimulation) showing that deletion of the NF-κB binding site specifically reduces transcription of TNFAIP3, but not other genes at this locus (n=6; one sample t-test). Representative data from the DH gRNA combination.


E Expression of CD69 , an activation marker , following CRISPR editing with indicated gRNA combinations or the non - targeting ( negative ) control ( NTC ) - data shown for ATTO - 550 positive ( RNP - containing ) and negative cells ( n = 6 ; paired t - test , one - tailed ) . Inset flow cytometry plot depicting representative gating of ATTO - 550 positive and negative cells .

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E Expression of CD69, an activation marker, following CRISPR editing with indicated gRNA combinations or the non-targeting (negative) control (NTC) - data shown for ATTO-550 positive (RNP-containing) and negative cells (n=6; paired t-test, one-tailed). Inset flow cytometry plot depicting representative gating of ATTO-550 positive and negative cells.


F Correlation between editing efficiency ( total indel rate ) and levels of phosphorylated IκBα in CD4 T cells ( normalised to the mean fluorescence intensity in NTC ; n = 9 , linear regression ) .

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F Correlation between editing efficiency (total indel rate) and levels of phosphorylated IκBα in CD4 T cells (normalised to the mean fluorescence intensity in NTC; n=9, linear regression).


G Secretion of effector cytokines following deletion of the NF - κB binding site - Th1 ( left panel , IFNγ ) , Th17 ( centre panel , IL - 17A ) and Th2 subsets ( right panel , IL - 4 ) ( n = 6 , paired t - test , one - tailed ) .

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G Secretion of effector cytokines following deletion of the NF-κB binding site - Th1 (left panel, IFNγ), Th17 (centre panel, IL-17A) and Th2 subsets (right panel, IL-4) (n=6, paired t-test, one-tailed).