D . Co - immunoprecipitation analyses of Elongator complexes in patient - derived fibroblast cell . Whole cell lysates ( left ) and eluates after immune - precipitation ( right ) are shown for control fibroblasts ( ctrl ) and cultured patient fibroblasts ( Elp4Y91C / L296I ( patient 1 ) ; Elp6L118W / L118W ( patient 2 ) , Elp6L118W / L118W ( patient 3 ) . Used antibodies for immobilization ( ELP1 ) and detection ( ELP1 , ELP3 , ELP4 , ELP5 and α - tubulin ) are indicated . n = 4 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 O O O O O O O O O O O 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. Co-immunoprecipitation analyses of Elongator complexes in patient-derived fibroblast cell. Whole cell lysates (left) and eluates after immune-precipitation (right) are shown for control fibroblasts (ctrl) and cultured patient fibroblasts (Elp4Y91C/L296I (patient 1); Elp6L118W/L118W (patient 2), Elp6L118W/L118W (patient 3). Used antibodies for immobilization (ELP1) and detection (ELP1, ELP3, ELP4 , ELP5 and α-tubulin) are indicated. n = 4 independent experiments.


A . High - performance liquid chromatography ( HPLC ) coupled to mass spectrometry ( MS ) used to quantify the Elongator - dependent tRNA modification 5 - carbamoylmethyluridine ( ncm5U ) in human fibroblasts . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . B . HPLC - MS quantification of Elongator - dependent tRNA modification 5 - methoxy - carbonylmethyluridine ( mcm5U ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . C . HPLC - MS quantification of Elongator - dependent tRNA modification 5 - methoxycarbonylmethyl - 2 - thiouridine ( mcm5s2U ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . Data information : Statistical analysis for data one - way ANOVA ( α = 0 . 05 ) with a Dunnett ' s post - hoc test . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; ns - not significant ) . Data represent mean ± SEM .

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

A. High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) used to quantify the Elongator-dependent tRNA modification 5-carbamoylmethyluridine (ncm5U) in human fibroblasts. Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. B. HPLC-MS quantification of Elongator-dependent tRNA modification 5-methoxy-carbonylmethyluridine (mcm5U). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. C. HPLC-MS quantification of Elongator-dependent tRNA modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.


D . HPLC - MS quantification of Elongator - independent tRNA modification 1 - methyladenosine ( m1A ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . E . HPLC - MS quantification of Elongator - independent tRNA modification 7 - methylguanosine ( m7G ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . Data information : Statistical analysis for data one - way ANOVA ( α = 0 . 05 ) with a Dunnett ' s post - hoc test . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; ns - not significant ) . Data represent mean ± SEM .

O O O O O O O O O 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 O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HPLC-MS quantification of Elongator-independent tRNA modification 1-methyladenosine (m1A). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. E. HPLC-MS quantification of Elongator-independent tRNA modification 7-methylguanosine (m7G). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.


F . Differential expression ( DE ) of peptides in fibroblasts obtained from the individuals with ELP4 and ELP6 variants relative to control ( n = 3 technical repeats per genotype ) . Down - ( red ) and upregulated ( blue ) peptides are depicted on volcano plots .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Differential expression (DE) of peptides in fibroblasts obtained from the individuals with ELP4 and ELP6 variants relative to control (n = 3 technical repeats per genotype). Down- (red) and upregulated (blue) peptides are depicted on volcano plots.


A . Representative images of Golgi - Cox - stained brain sections in the hippocampal area of adult ( 2 - months - old ) wild - type and mutant mice . Rectangles represent CA1 region containing pyramidal neurons selected for neuron reconstruction and subsequent analyses . Scale bar : 100 μm . Abbreviations : DG - dentate gyrus , CA1 - cornu ammonis 1 , CA2 - cornu ammonis

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of Golgi-Cox-stained brain sections in the hippocampal area of adult (2-months-old) wild-type and mutant mice. Rectangles represent CA1 region containing pyramidal neurons selected for neuron reconstruction and subsequent analyses. Scale bar: 100 μm. Abbreviations: DG - dentate gyrus, CA1 - cornu ammonis 1, CA2 - cornu ammonis 2


C . Quantification of total dendritic length from dendritic tree reconstructions shown in B . n = 15 ; 5 neurons per animal and 3 animals per genotype . D . Sholl analysis of the complexity of basal dendritic arbors . n = 15 ; 5 neurons per animal and 3 animals per genotype . Data information : Statistical analysis : unpaired two - tailed t - test ( α = 0 . 05 ) with Welch ' s correction . Holm - Sidak correction was applied to adjust for multiple comparisons ( D ) . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; ns - not significant ) . Data represent mean ± SEM ( with error bars in D removed when smaller than the corresponding data point ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of total dendritic length from dendritic tree reconstructions shown in B. n = 15; 5 neurons per animal and 3 animals per genotype. D. Sholl analysis of the complexity of basal dendritic arbors. n = 15; 5 neurons per animal and 3 animals per genotype. Data information: Statistical analysis: unpaired two-tailed t-test (α = 0.05) with Welch's correction. Holm-Sidak correction was applied to adjust for multiple comparisons (D). Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns - not significant). Data represent mean ± SEM (with error bars in D removed when smaller than the corresponding data point).


E . Representative images of dendritic spines on basal dendrites of the cortical neurons . Quantification of spine density per 10 μm on secondary dendrites is shown . n = 15 ; 5 neurons per animal and 3 animals per genotype . Scale bar : 10 μm . Data information : Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; ns - not significant ) . Data represent mean ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of dendritic spines on basal dendrites of the cortical neurons. Quantification of spine density per 10 μm on secondary dendrites is shown. n = 15; 5 neurons per animal and 3 animals per genotype. Scale bar: 10 μm. Data information: Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns - not significant). Data represent mean ± SEM


( A - D ) Relative growth of ( top ) or western blot analysis of YAP in ( bottom ) the indicated prostate cancer cell lines expressing control , YAP - WT , or YAP - 5SA constructs through lentiviral infection . Of note , LNCaP and C4 - 2 express full - length AR while 22RV1 and R1 - D567 express AR splicing variants V7 and V567ES , respectively . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Relative growth of (top) or western blot analysis of YAP in (bottom) the indicated prostate cancer cell lines expressing control, YAP-WT, or YAP-5SA constructs through lentiviral infection. Of note, LNCaP and C4-2 express full-length AR while 22RV1 and R1-D567 express AR splicing variants V7 and V567ES, respectively. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( M ) Western blot analysis of LATS1 , LATS2 , YAP , phosphorylated YAP at S109 ( pS109 ) and S127 ( pS127 ) in C4 - 2 cells treated with two independent LATS1 / 2 siRNAs .

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

(M) Western blot analysis of LATS1, LATS2, YAP, phosphorylated YAP at S109 (pS109) and S127 (pS127) in C4-2 cells treated with two independent LATS1/2 siRNAs.


( Q , Western blot analysis of LATS1 , LATS2 , and YAP in ( Q ) C4 - 2 cells treated with the indicated siRNA .

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

(Q, Western blot analysis of LATS1, LATS2, and YAP in (Q) C4-2 cells treated with the indicated siRNA.


( E ) Relative mRNA levels of AR and the indicated AR target genes ( left ) and western blot analysis of AR ( right ) in C4 - 2 cells treated with increasing concentrations of XMU - MP - 1 . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * 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 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 O O O O O O O O O O O O O O O O O O

(E) Relative mRNA levels of AR and the indicated AR target genes (left) and western blot analysis of AR (right) in C4-2 cells treated with increasing concentrations of XMU-MP-1. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( G ) Relative mRNA levels of the indicated AR or YAP target genes in C4 - 2 cells treated with control ( siNC ) or YAP siRNA ( siYAP _ 1 or siYAP _ 2 ) for 24 hours and with or without 2 μM XMU - MP - 1 for 12 hours . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * 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 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 O O O O O O O O O O O O O O O O O O O O O

(G) Relative mRNA levels of the indicated AR or YAP target genes in C4-2 cells treated with control (siNC) or YAP siRNA (siYAP_1 or siYAP_2) for 24 hours and with or without 2 μM XMU-MP-1 for 12 hours. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


Relative mRNA levels of the indicated AR or YAP target genes ( J , in C4 - 2 ( J , cells expressing control ( NC ) , YAP - WT or YAP - 5SA constructs through lentiviral infection . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative mRNA levels of the indicated AR or YAP target genes (J, in C4-2 (J, cells expressing control (NC), YAP-WT or YAP-5SA constructs through lentiviral infection. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( A ) Western blot analysis of AR ( left ) and quantification of AR ( right ) in C4 - 2 cells treated CHX for the indicated time .

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

(A) Western blot analysis of AR (left) and quantification of AR (right) in C4-2 cells treated CHX for the indicated time.


Western blot analysis of AR and YAP ( I , in C4 - 2 cells stably expressing Tet - O - YAP - 5SA and treated with 0 . 2 μg / ml doxycycline ( Dox ) for the indicated periods of time . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of AR and YAP (I, in C4-2 cells stably expressing Tet-O-YAP-5SA and treated with 0.2 μg/ml doxycycline (Dox) for the indicated periods of time. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


Western blot analysis of AR and YAP L ) in C4 - 2 cells stably expressing Tet - O - YAP - 5SAS94A and treated with 0 . 2 μg / ml doxycycline ( Dox ) for the indicated periods of time .

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

Western blot analysis of AR and YAP L) in C4-2 cells stably expressing Tet-O-YAP-5SAS94A and treated with 0.2 μg/ml doxycycline (Dox) for the indicated periods of time.


relative mRNA levels of TEAD1 and TEAD4 and the indicated AR target genes ( B ) C4 - 2 cells treated with two independent TEAD1 / 3 / 4 siRNA or control siRNA . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

relative mRNA levels of TEAD1 and TEAD4 and the indicated AR target genes (B) C4-2 cells treated with two independent TEAD1/3/4 siRNA or control siRNA. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( D ) Relative mRNA levels of the indicated AR target genes ( left ) and western blot analysis of TEAD ( right ) in C4 - 2 cells expression the control or TEAD4 constructs and treated with vehicle or XMU - MP - 1 . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * 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 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 O O O O O O O O O O O O O O O O O O

(D) Relative mRNA levels of the indicated AR target genes (left) and western blot analysis of TEAD (right) in C4-2 cells expression the control or TEAD4 constructs and treated with vehicle or XMU-MP-1. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( H - J ) HEK - 293T cells transfected with the indicated constructs . Cell lysates were immunoprecipitated with the indicate antibodies . Immunoprecipitates and cell lysates ( input ) were analyzed by western blot with the indicated antibodies . ( K ) In vitro binding assay using GST or GST - TEAD4 purified from bacteria and Flag - AR immunopurified from HEK293 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 O O 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-J) HEK-293T cells transfected with the indicated constructs. Cell lysates were immunoprecipitated with the indicate antibodies. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies. (K) In vitro binding assay using GST or GST-TEAD4 purified from bacteria and Flag-AR immunopurified from HEK293 cells.


HEK - 293T cells transfected with the indicated AR , TEAD constructs . Cell lysates were immunoprecipitated with the indicate antibodies . Immunoprecipitates and cell lysates ( input ) were analyzed by western blot with the indicated antibodies .

HEK-293T cells transfected with the indicated AR, TEAD constructs. Cell lysates were immunoprecipitated with the indicate antibodies. Immunoprecipitates and cell lysates (input) were analyzed by western blot with the indicated antibodies.


( O - P ) ChIP - qPCR ( O ) and RT - PCR analyses show that exogenous expression of wild type ( TEAD4 - WT ) but not the DNA binding deficient form ( TEAD4m ) of TEAD4 rescued AR binding to the promoter / enhancer regions of KLK2 and KLK3 ( O ) as well as the expression of these genes ( P ) in C4 - 2 cells treated with TEAD1 / 3 / 4 siRNA . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(O-P) ChIP-qPCR (O) and RT-PCR analyses show that exogenous expression of wild type (TEAD4-WT) but not the DNA binding deficient form (TEAD4m) of TEAD4 rescued AR binding to the promoter/enhancer regions of KLK2 and KLK3 (O) as well as the expression of these genes (P) in C4-2 cells treated with TEAD1/3/4 siRNA. Data information: Results are representatives of three independent experiments. Data are ± SD. *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( D ) YAP - 5SA but not YAP - 5SAS94A inhibited AR / TEAD interaction . C4 - 2 cells stably expressing Tet - O - GFP , Tet - O - YAP - 5SA or Tet - O - YAP - 5SAS94A were treated with 0 . 2 μg / ml doxycycline ( Dox ) for 10 hours , followed by CoIP and western blot analyses with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) YAP-5SA but not YAP-5SAS94A inhibited AR/TEAD interaction. C4-2 cells stably expressing Tet-O-GFP, Tet-O-YAP-5SA or Tet-O-YAP-5SAS94A were treated with 0.2 μg/ml doxycycline (Dox) for 10 hours, followed by CoIP and western blot analyses with the indicated antibodies.


( G - H ) ChIP - qPCR analysis of AR ( G ) and TEAD ( H ) binding to the promoter / enhancer regions of KLK2 , KLK3 and CTGF in Tet - O - YAP - 5SA expressing C4 - 2 cells treated with 0 . 2 μg / ml doxycycline ( Dox ) for the indicated time . Data information : Results are representatives of three independent experiments . Data ± SD . ns : not significant , * * P < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-H) ChIP-qPCR analysis of AR (G) and TEAD (H) binding to the promoter/enhancer regions of KLK2, KLK3 and CTGF in Tet-O-YAP-5SA expressing C4-2 cells treated with 0.2 μg/ml doxycycline (Dox) for the indicated time. Data information: Results are representatives of three independent experiments. Data ± SD. ns: not significant, **P<0.01, ***P<0.001 (Student's t-test).


Western blot analysis of YAP and TEAD ( left ) and mRNA levels of the indicated genes ( right ) in C4 - 2 cells treated with siYAP / TAZ for 18 ( A ) , 36 ( B ) , hours . Data information : Results are representatives of three independent experiments . Data ± SD . ns : not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blot analysis of YAP and TEAD (left) and mRNA levels of the indicated genes (right) in C4-2 cells treated with siYAP/TAZ for 18 (A), 36 (B), hours. Data information: Results are representatives of three independent experiments. Data ± SD. ns: not significant, *P<0.05, **P<0.01, ***P<0.001 (Student's t-test).


( A - F ) YAP activation inhibited AR positive prostate cancer growth in xenografts bearing C4 - 2 or 22RV1 tumors that stably express Tet - O - YAP - 5SA . Male NOD scid gamma ( NSG ) mice bearing C4 - 2 tumor cells stably expressing a Tet - O - EGFP construct were used as control . Male NOD scid gamma ( NSG ) mice bearing the indicated tumors were injected i . p . with PBS containing Dox ( 20mg / kg ) or PBS daily for the indicated time . Tumor growth curve ( A , D ) , photograph of tumor samples ( B , E ) , and quantification of tumor weight ( C , F ) at the end of treatment were shown . XMU - MP - 1 inhibited AR positive prostate cancer growth in xenografts bearing C4 - 2 ( G - I ) , tumors . Male NOD scid gamma ( NSG ) mice bearing the indicated tumors were treated daily with XUM - MP - 1 at the indicated concentrations or with vehicle for the indicated periods of time . Tumor growth curve ( G , J , images of tumor samples ( H , K , and quantification of tumor weight ( I at the end of treatment were shown . Data information : Data are ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . n = 7 mice for each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) YAP activation inhibited AR positive prostate cancer growth in xenografts bearing C4-2 or 22RV1 tumors that stably express Tet-O-YAP-5SA. Male NOD scid gamma (NSG) mice bearing C4-2 tumor cells stably expressing a Tet-O-EGFP construct were used as control. Male NOD scid gamma (NSG) mice bearing the indicated tumors were injected i.p. with PBS containing Dox (20mg/kg) or PBS daily for the indicated time. Tumor growth curve (A, D), photograph of tumor samples (B, E), and quantification of tumor weight (C, F) at the end of treatment were shown. XMU-MP-1 inhibited AR positive prostate cancer growth in xenografts bearing C4-2 (G-I), tumors. Male NOD scid gamma (NSG) mice bearing the indicated tumors were treated daily with XUM-MP-1 at the indicated concentrations or with vehicle for the indicated periods of time. Tumor growth curve (G, J, images of tumor samples (H, K, and quantification of tumor weight (I at the end of treatment were shown. Data information: Data are ± SD. **P<0.01, ***P<0.001 (Student's t-test). n=7 mice for each group.


XMU - MP - 1 inhibited AR positive prostate cancer growth in xenografts bearing 22RV1 tumors . Male NOD scid gamma ( NSG ) mice bearing the indicated tumors were treated daily with XUM - MP - 1 or with vehicle for the indicated periods of time . Tumor growth curve J , Data information : Data are ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - test ) . n = 7 mice for each group .

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

XMU-MP-1 inhibited AR positive prostate cancer growth in xenografts bearing 22RV1 tumors. Male NOD scid gamma (NSG) mice bearing the indicated tumors were treated daily with XUM-MP-1 or with vehicle for the indicated periods of time. Tumor growth curve J, Data information: Data are ± SD. **P<0.01, ***P<0.001 (Student's t-test). n=7 mice for each group.


THP - 1 derived macrophages were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . Viral protein expression for PB1 - F2 ( A ) was detected by immunoblotting at 24 h post - infection ( hpi ) Equal loading was confirmed by detection of beta actin .

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

THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Viral protein expression for PB1-F2 (A) was detected by immunoblotting at 24 h post-infection (hpi) Equal loading was confirmed by detection of beta actin.


B THP - 1 derived macrophages were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . Viral protein expression for NP ( B ) was detected by immunoblotting at indicated time points , respectively . Equal loading was confirmed by detection of beta actin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Viral protein expression for NP (B) was detected by immunoblotting at indicated time points, respectively. Equal loading was confirmed by detection of beta actin.


( C ) THP - 1 derived macrophages were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . Levels of viral M1 mRNA expression were detected by RT q ‑ PCR at 16 hpi . The mean ± standard deviation of three independent biological samples is shown . Statistical analysis was performed by students t - test .

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

(C) THP-1 derived macrophages were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Levels of viral M1 mRNA expression were detected by RT q‑PCR at 16 hpi. The mean ± standard deviation of three independent biological samples is shown. Statistical analysis was performed by students t-test.


( D ) THP - 1 derived macrophages were infected with VN and PR8 Wt and ΔF2 strains for 24 h at MOI 10 . Levels of IL - 1β were quantified by ELISA from the supernatants . The mean ± standard deviation of seven independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are indicated .

(D) THP-1 derived macrophages were infected with VN and PR8 Wt and ΔF2 strains for 24 h at MOI 10. Levels of IL-1β were quantified by ELISA from the supernatants. The mean ± standard deviation of seven independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


( E ) THP - 1 control and knockout cells for NLRP3 were mock treated or infected with the VN Wt and ΔF2 strains for 24 h at MOI 10 and IL - 1β levels were assessed by ELISA from the supernatants . The mean ± standard deviation of five independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are 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 O O O O O O O O O O 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) THP-1 control and knockout cells for NLRP3 were mock treated or infected with the VN Wt and ΔF2 strains for 24 h at MOI 10 and IL-1β levels were assessed by ELISA from the supernatants. The mean ± standard deviation of five independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


( F ) Immunoblot detection for NLRP3 on control and NLRP3 knockout THP - 1 cells . Equal loading was confirmed by specific blotting against beta actin . Representative blot of three independent repeats is shown .

O O O O O O O O O O O O O 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) Immunoblot detection for NLRP3 on control and NLRP3 knockout THP-1 cells. Equal loading was confirmed by specific blotting against beta actin. Representative blot of three independent repeats is shown.


( G ) Human primary macrophages differentiated from PBMCs from two healthy donors were infected with H5N1 Wt and ΔF2 for 5 h at MOI 10 . Levels of IL - 1β were measured by ELISA in technical replicates . The mean of two independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are 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 O O O O 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) Human primary macrophages differentiated from PBMCs from two healthy donors were infected with H5N1 Wt and ΔF2 for 5 h at MOI 10. Levels of IL-1β were measured by ELISA in technical replicates. The mean of two independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


( H ) THP - 1 derived macrophages were infected with a human H3N2 IAV strain ( Wyo ) Wt and ΔF2 for 24 h at MOI 4 . Levels of IL - 1β were measured by ELISA from the supernatants . The mean ± standard deviation of four independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are 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 O O O O O O O O O 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) THP-1 derived macrophages were infected with a human H3N2 IAV strain (Wyo) Wt and ΔF2 for 24 h at MOI 4. Levels of IL-1β were measured by ELISA from the supernatants. The mean ± standard deviation of four independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


( I ) Detection of viral matrix protein M1 by immunoblotting in cells infected with the H3N2 Wt and ΔF2 strains .

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

(I) Detection of viral matrix protein M1 by immunoblotting in cells infected with the H3N2 Wt and ΔF2 strains.


( J ) Human primary macrophages isolated and differentiated from PBMCs from three healthy donors were infected with H3N2 Wt and ΔF2 for 24 h at MOI 4 . Levels of IL - 1β were measured by ELISA . The mean ± standard deviation of three independent experiments is shown . Statistical analysis was performed by one - way ANOVA , p - values are indicated .

(J) Human primary macrophages isolated and differentiated from PBMCs from three healthy donors were infected with H3N2 Wt and ΔF2 for 24 h at MOI 4. Levels of IL-1β were measured by ELISA. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by one-way ANOVA, p-values are indicated.


Primary human alveolar macrophages from BALF of five pediatric patients ( patients are color coded ) were infected with the human H3N2 strain ( Wyo ) Wt and ΔF2 for 16 h at MOI 1 . ( K ) Levels of IL - 1β were measured by ELISA from the supernatants . The mean ± standard deviation is shown .

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

Primary human alveolar macrophages from BALF of five pediatric patients (patients are color coded) were infected with the human H3N2 strain (Wyo) Wt and ΔF2 for 16 h at MOI 1. (K) Levels of IL-1β were measured by ELISA from the supernatants. The mean ± standard deviation is shown.


Primary human alveolar macrophages from BALF of five pediatric patients ( patients are color coded ) were infected with the human H3N2 strain ( Wyo ) Wt and ΔF2 for 16 h at MOI 1 . Levels of viral M1 mRNA ( L ) , Mx1 mRNA ( M ) and IP10 mRNA ( N ) expression were detected by RT q ‑ PCR . The mean ± standard deviation for cells from four patients are shown . Individiual patients are color coded .

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

Primary human alveolar macrophages from BALF of five pediatric patients (patients are color coded) were infected with the human H3N2 strain (Wyo) Wt and ΔF2 for 16 h at MOI 1. Levels of viral M1 mRNA (L), Mx1 mRNA (M) and IP10 mRNA (N) expression were detected by RT q‑PCR. The mean ± standard deviation for cells from four patients are shown. Individiual patients are color coded.


( A - D ) THP - 1 cells were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . Flow cytometry results for annexin V single positive ( A and C ) and annexin V and zombie dye double positive ( B and D ) cells . Percentage of positive cells are depicted at 12 hpi or 24hpi .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. Flow cytometry results for annexin V single positive (A and C) and annexin V and zombie dye double positive (B and D) cells. Percentage of positive cells are depicted at 12 hpi or 24hpi.


( E and F ) THP - 1 cells were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . LDH release measured from supernatants of mock treated or infected cells at 12 hpi ( E ) or 24hpi ( F ) .

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

(E and F) THP-1 cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi (E) or 24hpi (F).


G THP - 1 Caspase 1 - / - cells were infected with the wildtype ( Wt ) and ΔF2 H5N1 ( VN ) virus at MOI 10 . LDH release measured from supernatants of mock treated or infected cells at 12 hpi or 24hpi . n = 3 for 12hpi , n = 5 for 24hpi . Medians are indicated , statistical analysis was performed by one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 THP-1 Caspase 1 -/- cells were infected with the wildtype (Wt) and ΔF2 H5N1 (VN) virus at MOI 10. LDH release measured from supernatants of mock treated or infected cells at 12 hpi or 24hpi. n=3 for 12hpi, n=5 for 24hpi. Medians are indicated, statistical analysis was performed by one-way ANOVA.


( A ) THP - 1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6 . 7 μM of nigericin ( nig ) for 45 min were analyzed by immunoblotting . Detection of the full - length gasdermin D . Equal loading was confirmed by beta actin . One representative blot of five independent repeats is shown . ( B ) Quantification of band intensity for the gasdermin D full - length isoform . The mean ± standard deviation five independent experiments is shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . , The asterisk ( * ) marks a significant difference when compared to the mock group * * p < 0 . 01 .

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

(A) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of the full-length gasdermin D. Equal loading was confirmed by beta actin. One representative blot of five independent repeats is shown. (B) Quantification of band intensity for the gasdermin D full-length isoform. The mean ± standard deviation five independent experiments is shown. Statistical analysis was performed by the paired two‑tailed Student's t test., The asterisk (*) marks a significant difference when compared to the mock group ** p < 0.01.


( C ) THP - 1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6 . 7 μM of nigericin ( nig ) for 45 min were analyzed by immunoblotting . Detection of cleaved gasdermin D N - terminal and C - terminal isoforms by immunoblot in supernatants ; one representative experiment of three independent repeats is shown . ( D ) Quantification of band intensity for N - terminal gasdermin D isoform band . Three independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . , * p < 0 . 05 . ( E ) Quantification of band intensity for C - terminal gasdermin D isoform band . Three independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Detection of cleaved gasdermin D N-terminal and C-terminal isoforms by immunoblot in supernatants; one representative experiment of three independent repeats is shown. (D) Quantification of band intensity for N-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05. (E) Quantification of band intensity for C-terminal gasdermin D isoform band. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test., * p < 0.05.


( F ) THP - 1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6 . 7 μM of nigericin ( nig ) for 45 min were analyzed by immunoblotting . Pro - caspase - 1 and active caspase - 1 were detected by immunoblot in cell lysates ( cell , upper panels ) and supernatants ( sup , lower panels ) from the same experiment . One representative experiment of three independent repeats is shown . ( G ) Quantification of band intensity for active caspase - 1 band . Five independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test . * p < 0 . 05 . ( H ) Levels of total pro - caspase - 1 were quantified on pellets and respective supernatants . Three independent experiments are shown . Statistical analysis was performed by the paired two ‑ tailed Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) THP-1 cells infected with VN Wt and ΔF2 virus at a MOI 10 for 24 h or treated with 6.7 μM of nigericin (nig) for 45 min were analyzed by immunoblotting. Pro-caspase-1 and active caspase-1 were detected by immunoblot in cell lysates (cell, upper panels) and supernatants (sup, lower panels) from the same experiment. One representative experiment of three independent repeats is shown. (G) Quantification of band intensity for active caspase-1 band. Five independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test. * p < 0.05. (H) Levels of total pro-caspase-1 were quantified on pellets and respective supernatants. Three independent experiments are shown. Statistical analysis was performed by the paired two‑tailed Student's t test.


( A ) Western blot analysis of whole cell lysate ( input ) and Flag - specific immunoprecipitation ( IP ) from 293T cells co - transfected with expression plasmids encoding V5 - NLRP3 and Flag ZsGreen - PB1 - F2 from indicated viral strains for 24 h Representative blot of three independent repeats is shown . ( B ) Quantification of the NLRP3 IP band was normalized to the respective NLRP3 input band ( n = 3 independent biological replicates , normalized to the respective input ) . Mean values + - SD are 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of whole cell lysate (input) and Flag-specific immunoprecipitation (IP) from 293T cells co-transfected with expression plasmids encoding V5-NLRP3 and Flag ZsGreen-PB1-F2 from indicated viral strains for 24 h Representative blot of three independent repeats is shown. (B) Quantification of the NLRP3 IP band was normalized to the respective NLRP3 input band (n=3 independent biological replicates, normalized to the respective input). Mean values +- SD are indicated.


( E ) 293T cells co transfected with expression plasmids encoding different truncation mutants for Flag Zsgreen - PB1 - F2 from VN ( H5N1 ) and V5 - NLRP3 were processed as described in ( A ) . Representative blot of three independent repeats is shown .

O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells co transfected with expression plasmids encoding different truncation mutants for Flag Zsgreen-PB1-F2 from VN (H5N1) and V5-NLRP3 were processed as described in (A). Representative blot of three independent repeats is shown.


( B ) 293T cells were co - transfected with expression plasmids encoding for the indicated V5 - tagged domains of NLRP3 for 24h . Whole cell lysate ( left blot ) and flag specific IP ( right blot ) were analyzed by immunoblotting . Equal loading was confirmed by blotting against tubulin . Representative blots of two independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with expression plasmids encoding for the indicated V5-tagged domains of NLRP3 for 24h. Whole cell lysate (left blot) and flag specific IP (right blot) were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of two independent experiments are shown.


( C ) 293T cells were co ‑ transfected with expression plasmids encoding Flag - NLRP3 , V5 ‑ PB1 - F2 and V5 ‑ ASC for 24h . Whole cell lysate and flag specific IP were analyzed by immunoblotting for indicated antigens . Equal loading of whole cell lysate was assured by blotting against beta actin . Representative blot of two independent repeats is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co‑transfected with expression plasmids encoding Flag-NLRP3, V5‑PB1-F2 and V5‑ASC for 24h. Whole cell lysate and flag specific IP were analyzed by immunoblotting for indicated antigens. Equal loading of whole cell lysate was assured by blotting against beta actin. Representative blot of two independent repeats is shown.


( D ) 293T cells were co - transfected with expression plasmids encoding for Flag - NLRP3 , PB1 - F2 or NEK7 or combinations thereof as indicated . Whole cell lysate and Flag IP were analyzed by immunoblotting . Equal loading was confirmed by blotting against beta actin . Representative blots of three independent experiments are shown . Right panel : Quantification of IP bands ' intensity relative to their input . The mean ± standard deviation of three independent experiments is shown . Statistical analysis was performed by paired two ‑ tailed Student ' s t test ( * p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3, PB1-F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and Flag IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown. Right panel: Quantification of IP bands' intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‑tailed Student's t test (*p< 0.05).


( E ) As in D : 293T cells were co - transfected with expression plasmids encoding for Flag - NLRP3 - LRR , PB1 - F2 or NEK7 or combinations thereof as indicated . Whole cell lysate and NLRP3 - LRR IP were analyzed by immunoblotting . Equal loading was confirmed by blotting against beta actin . Representative blots of three independent experiments are shown . ( F ) Quantification of IP bands ' intensity relative to their input . The mean ± standard deviation of three independent experiments is shown . Statistical analysis was performed by paired two ‑ tailed Student ' s t test ( * p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) As in D: 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3-LRR, PB1-F2 or NEK7 or combinations thereof as indicated. Whole cell lysate and NLRP3-LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against beta actin. Representative blots of three independent experiments are shown. (F) Quantification of IP bands' intensity relative to their input. The mean ± standard deviation of three independent experiments is shown. Statistical analysis was performed by paired two‑tailed Student's t test (*p< 0.05).


G ) As in D and E : 293T cells were co - transfected with expression plasmids encoding for Flag - NLRP3 - LRR , PB1 - F2 or V5 - NLRP3 - Pyrin or combinations thereof as indicated . Whole cell lysate and NLRP3 - LRR IP were analyzed by immunoblotting . Equal loading was confirmed by blotting against tubulin . Representative blots of three independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) As in D and E: 293T cells were co-transfected with expression plasmids encoding for Flag-NLRP3-LRR, PB1-F2 or V5-NLRP3-Pyrin or combinations thereof as indicated. Whole cell lysate and NLRP3-LRR IP were analyzed by immunoblotting. Equal loading was confirmed by blotting against tubulin. Representative blots of three independent experiments are shown.


Confocal microscopy reveals typical " class B " pattern of βarr1 recruitment for V2R and B2R as reflected by first the localization at the plasma membrane and subsequently , internalization in endosomal vesicles upon agonist - stimulation . HEK - 293 cells expressing V2R / B2R and βarr1 - mCherry were stimulated with agonist ( AVP ; 100nM and Bradykinin ; 100nM ) and the localization of βarr1 was visualized using confocal microscopy . Representative images from three independent experiments are shown here , and the scale bar is 10μm . Visual scoring of images from three independent experiments revealed agonist - induced βarr1 recruitment ( i . e . membrane and endosomal localization ) in approximately 77 % of the cells for V2R ( 221 cells ) and 75 % of the cells for B2R ( 662 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal microscopy reveals typical "class B" pattern of βarr1 recruitment for V2R and B2R as reflected by first the localization at the plasma membrane and subsequently, internalization in endosomal vesicles upon agonist-stimulation. HEK-293 cells expressing V2R/B2R and βarr1-mCherry were stimulated with agonist (AVP; 100nM and Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring of images from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 77% of the cells for V2R (221 cells) and 75% of the cells for B2R (662 cells).


Agonist - induced phosphorylation of ERK1 / 2 in HEK - 293 cells expressing either V2R or B2R in the absence ( control ; CTL ) and presence of βarr1 knock - down ( βarr1 - KD ) are measured using Western blotting . Densitometry - based quantification of data ( mean ± SEM ) from four independent experiments is presented as bar - graphs in the right panels , normalized with respect to maximal signal under control condition ( treated as 100 % ) , and analyzed using Two - way - ANOVA with Bonferroni multiple comparisons test ( * * * p < 0 . 001 , * * p < 0 . 01 ) .

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

Agonist-induced phosphorylation of ERK1/2 in HEK-293 cells expressing either V2R or B2R in the absence (control; CTL) and presence of βarr1 knock-down (βarr1-KD) are measured using Western blotting. Densitometry-based quantification of data (mean ±SEM) from four independent experiments is presented as bar-graphs in the right panels, normalized with respect to maximal signal under control condition (treated as 100%), and analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, **p <0.01).


Confocal microscopy reveals robust recruitment of βarr1 to B2R mutant constructs upon agonist - stimulation . HEK - 293 cells expressing either B2RWT or B2R mutants along with βarr1 - mCherry were stimulated with agonist ( Bradykinin ; 100nM ) and the localization of βarr1 was visualized using confocal microscopy . Representative images from three independent experiments are shown here , and the scale bar is 10μm . Visual scoring from three independent experiments revealed agonist - induced βarr1 recruitment ( i . e . membrane and endosomal localization ) in approximately 75 % of the cells for B2RWT ( 662 cells ) , 74 % of the cells for B2RΔG368 ( 169 cells ) , 75 % of the cells for B2RL370T ( 130 cells ) , 85 % of the cells for B2RΔG368 + L370T ( 132 cells ) and 77 % of the cells for B2RΔI374 ( 116 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal microscopy reveals robust recruitment of βarr1 to B2R mutant constructs upon agonist-stimulation. HEK-293 cells expressing either B2RWT or B2R mutants along with βarr1-mCherry were stimulated with agonist (Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 75% of the cells for B2RWT (662 cells), 74% of the cells for B2RΔG368 (169 cells), 75% of the cells for B2RL370T (130 cells), 85% of the cells for B2RΔG368+L370T (132 cells) and 77% of the cells for B2RΔI374 (116 cells).


HEK - 293 cells expressing B2R constructs with C - terminal SmBiT and βarr1 with N - terminal LgBiT were treated with indicated concentrations of bradykinin , and ligand - induced change in luminescent signal was measured . Concentration - response curves were plotted using GraphPad Prism , and pEC50 ( top ) and Emax ( bottom ) were calculated . Data represent mean ± SEM of five independent experiments ( three for B2RΔI374 ) , each performed in duplicate .

O 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

HEK-293 cells expressing B2R constructs with C-terminal SmBiT and βarr1 with N-terminal LgBiT were treated with indicated concentrations of bradykinin, and ligand-induced change in luminescent signal was measured. Concentration-response curves were plotted using GraphPad Prism, and pEC50 (top) and Emax (bottom) were calculated. Data represent mean ± SEM of five independent experiments (three for B2RΔI374), each performed in duplicate.


Knock - down of βarr1 leads to an increase in agonist - induced ERK1 / 2 phosphorylation for B2RΔG368 and B2RΔI374 but a decrease for B2RL370T and B2RΔG368 + L370T mutants . HEK - 293 cells expressing the indicated B2R mutants in presence and absence of βarr1 knock - down were stimulated with indicated doses of bradykinin ( Brady ) for 10 min followed by detection of phosphorylated ERK1 / 2 using Western blotting . Densitometry - based quantification of data ( mean ± SEM ) from three independent experiments ( five for B2RΔG368 and B2RΔI374 ) is presented as bar graphs in the lower panels . Data are normalized with respect to maximal signal under control condition ( treated as 100 % ) , and analyzed using Two - way - ANOVA with Bonferroni multiple comparisons test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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

Knock-down of βarr1 leads to an increase in agonist-induced ERK1/2 phosphorylation for B2RΔG368 and B2RΔI374 but a decrease for B2RL370T and B2RΔG368+L370T mutants. HEK-293 cells expressing the indicated B2R mutants in presence and absence of βarr1 knock-down were stimulated with indicated doses of bradykinin (Brady) for 10 min followed by detection of phosphorylated ERK1/2 using Western blotting. Densitometry-based quantification of data (mean±SEM) from three independent experiments (five for B2RΔG368 and B2RΔI374) is presented as bar graphs in the lower panels. Data are normalized with respect to maximal signal under control condition (treated as 100%), and analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (*p <0.05; **p <0.01; ***p<0.001).


HEK - 293 cells expressing V2R , B2R , B2RΔG / L370Tor B2RL370T along with βarr1 - mCherry and YFP - tagged intrabody30 ( Ib30 - YFP ) were stimulated with their respective agonists ( AVP , 100nM for V2R ) and ( bradykinin , 1μM for B2R ) and the localization of βarr1 and Ib30 were visualized . Although βarr1 gets recruited for all four constructs , Ib30 - YFP does not colocalize with βarr1 either at the surface or upon internalization in case of B2RWT . Representative data from 3 - 5 independent experiments are shown and the scale bar is 10μm . Visual scoring from 3 - 5 independent experiments revealed agonist - induced βarr1 recruitment ( i . e . membrane and endosomal localization ) in approximately 82 % of the cells for V2R ( 691 cells ) , 10 % for B2RWT ( 254 cells ) , 63 % for B2RL370T ( 273 cells ) and 83 % for B2RΔG368 + L370T ( 158 cells ) . Pearson ' s correlation coefficients ( PCC ) were measured to assess the colocalization of βarr1 and Ib30 using JACoP plugin in ImageJ , and the mean ± sem for the basal , surface and internalized panels , respectively , are presented here . B2RWT - 0 . 32 ± 0 . 02 from 16 cells , 0 . 18 ± 0 . 01 from 32 cells , 0 . 25 ± 0 . 02 from 31 cells ; B2RΔG / L370T - 0 . 32 ± 0 . 03 from 13 cells , 0 . 92 ± 0 . 01 from 18 cells , 0 . 90 ± 0 . 01 from 16 cells ; B2RL370T - 0 . 26 ± 0 . 01 from 10 cells , 0 . 85 ± 0 . 01 from 11 cells , 0 . 87 ± 0 . 01 from 25 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HEK-293 cells expressing V2R, B2R, B2RΔG/L370Tor B2RL370T along with βarr1-mCherry and YFP-tagged intrabody30 (Ib30-YFP) were stimulated with their respective agonists (AVP, 100nM for V2R) and (bradykinin, 1μM for B2R) and the localization of βarr1 and Ib30 were visualized. Although βarr1 gets recruited for all four constructs, Ib30-YFP does not colocalize with βarr1 either at the surface or upon internalization in case of B2RWT. Representative data from 3-5 independent experiments are shown and the scale bar is 10μm. Visual scoring from 3-5 independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 82% of the cells for V2R (691 cells), 10% for B2RWT (254 cells), 63% for B2RL370T (273 cells) and 83% for B2RΔG368+L370T (158 cells). Pearson's correlation coefficients (PCC) were measured to assess the colocalization of βarr1 and Ib30 using JACoP plugin in ImageJ, and the mean±sem for the basal, surface and internalized panels, respectively, are presented here. B2RWT - 0.32±0.02 from 16 cells, 0.18±0.01 from 32 cells, 0.25±0.02 from 31 cells; B2RΔG/L370T - 0.32±0.03 from 13 cells, 0.92±0.01 from 18 cells, 0.90±0.01 from 16 cells; B2RL370T - 0.26±0.01 from 10 cells, 0.85±0.01 from 11 cells, 0.87±0.01 from 25 cells.


HEK - 293 cells expressing the indicated receptor constructs ( pcDNA vector control for mock - transfection ) together with βarr1 - SmBiT and LgBiT - Ib30 were stimulated with the indicated concentrations of respective agonists for 10min . Subsequently , the luminescence signal generated upon the interaction of βarr1 - SmBiT and LgBiT - Ib30 was measured , normalized with basal signal ( i . e . no agonist stimulation treated as 1 ) , and plotted using GraphPad Prism . pEC50 ( top ) and Emax ( bottom ) values are mentioned in the respective graphs and data represent mean ± SEM of four independent experiments , each carried out in duplicate .

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

HEK-293 cells expressing the indicated receptor constructs (pcDNA vector control for mock-transfection) together with βarr1-SmBiT and LgBiT-Ib30 were stimulated with the indicated concentrations of respective agonists for 10min. Subsequently, the luminescence signal generated upon the interaction of βarr1-SmBiT and LgBiT-Ib30 was measured, normalized with basal signal (i.e. no agonist stimulation treated as 1), and plotted using GraphPad Prism. pEC50 (top) and Emax (bottom) values are mentioned in the respective graphs and data represent mean±SEM of four independent experiments, each carried out in duplicate.


HEK - 293 cells expressing the NanoBiT - Gq protein and indicated B2R constructs were treated different concentrations of bradykinin , and ligand - induced change in luminescent signal was measured . Concentration - response curves were plotted using GraphPad Prism to calculate pEC50 ( top ) and Emax ( bottom ) values included in the graph . Data represent mean ± SEM of three independent experiments with each performed in duplicate , and normalized with respect to the luminescence signal under basal ( no - agonist treatment ) condition ( treated as 1 ) .

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

HEK-293 cells expressing the NanoBiT-Gq protein and indicated B2R constructs were treated different concentrations of bradykinin, and ligand-induced change in luminescent signal was measured. Concentration-response curves were plotted using GraphPad Prism to calculate pEC50 (top) and Emax(bottom) values included in the graph. Data represent mean±SEM of three independent experiments with each performed in duplicate, and normalized with respect to the luminescence signal under basal (no-agonist treatment) condition (treated as 1).


HEK - 293 cells expressing the indicated AT1aR constructs , in absence ( CTL ; control ) or presence of βarr1 knock - down ( βarr1 - KD ) were stimulated with indicated concentrations of angiotensin II ( AngII ) for 10 min . Subsequently , ERK1 / 2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs . Data were normalized with respect to the signal at 100nM agonist concentration in control cells ( treated as 100 % ) and represent mean ± SEM of five independent experiments . Data were analyzed using Two - way - ANOVA with Bonferroni multiple comparisons test ( * * * p < 0 . 001 , p < 0 . 05 ) .

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

HEK-293 cells expressing the indicated AT1aR constructs, in absence (CTL; control) or presence of βarr1 knock-down (βarr1-KD) were stimulated with indicated concentrations of angiotensin II (AngII) for 10 min. Subsequently, ERK1/2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs. Data were normalized with respect to the signal at 100nM agonist concentration in control cells (treated as 100%) and represent mean±SEM of five independent experiments. Data were analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, p<0.05).


HEK - 293 cells expressing the indicated AT1aR constructs , in absence ( CTL ; control ) or presence of βarr1 knock - down ( βarr1 - KD ) were stimulated with indicated concentrations of angiotensin II ( AngII ) for 10 min . Subsequently , ERK1 / 2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs . Data were normalized with respect to the signal at 100nM agonist concentration in control cells ( treated as 100 % ) and represent mean ± SEM of five ( six for AT1aRΔL337 ) independent experiments . Data were analyzed using Two - way - ANOVA with Bonferroni multiple comparisons test ( * * * p < 0 . 001 , p < 0 . 05 ) .

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

HEK-293 cells expressing the indicated AT1aR constructs, in absence (CTL; control) or presence of βarr1 knock-down (βarr1-KD) were stimulated with indicated concentrations of angiotensin II (AngII) for 10 min. Subsequently, ERK1/2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs. Data were normalized with respect to the signal at 100nM agonist concentration in control cells (treated as 100%) and represent mean±SEM of five (six for AT1aRΔL337) independent experiments. Data were analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, p<0.05).


G . Endogenous MITOL enhances the K48 - polyubiquitin chains of Parkin . WT or MITOL KO HeLa cells stably expressing HA - Parkin were treated with CCCP ( 10 µM ) for indicated time . MG132 ( 30 μM ) was added 3 h after CCCP treatment . Lysates of cells were fractionated into cytosolic , mitochondrial and ER fractions , and then subjected to an IP - IB assay with the indicated antibodies . K48 - polyubiquitin chains were immunoblotted with K48 - specific ubiquitin antibody .

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

G. Endogenous MITOL enhances the K48-polyubiquitin chains of Parkin. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for indicated time. MG132 (30 μM) was added 3 h after CCCP treatment. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IP-IB assay with the indicated antibodies. K48-polyubiquitin chains were immunoblotted with K48-specific ubiquitin antibody.


E . MITOL regulates mitophagy . MITOL WT HeLa cells , MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0 . 5µg ( + ) and 5µg ( + + ) stably expressing HA ‐ Parkin and mt ‐ Keima were treated with CCCP ( 10µM ) for 8h . Then , mKeima was measured at 488 ( pH 7 ) and 561 ( pH 4 ) nm lasers using Flow Cytometer . Percentages of mitophagy were calculated from 30 , 000 cells in each independent experiment . Data represent the mean ± SD of three independent experiments . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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

E. MITOL regulates mitophagy. MITOL WT HeLa cells, MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0.5µg (+) and 5µg (++) stably expressing HA‐Parkin and mt‐Keima were treated with CCCP (10µM) for 8h. Then, mKeima was measured at 488 (pH 7) and 561 (pH 4) nm lasers using Flow Cytometer. Percentages of mitophagy were calculated from 30,000 cells in each independent experiment. Data represent the mean ± SD of three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


C . Endogenous MITOL translocates to the ER in later phase of mitophagy . EGFP - MITOL knock in HeLa cells were transfected with HA - Parkin and treated with DMSO or CCCP ( 10 µM ) as indicated times . Cells were fixed , permeabilized and subjected to immunofluorescence analysis with the indicated antibodies . Colocalization was quantified by Manders ' s coefficient . Means ± SEM of more than 10 cells obtained from three independent experiments . For statistical analysis , a one - way ANOVA with Tukey ' s multiple comparisons test were performed , * * * * P < 0 . 0001 . Scale bar represents 1 μm .

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

C. Endogenous MITOL translocates to the ER in later phase of mitophagy. EGFP-MITOL knock in HeLa cells were transfected with HA-Parkin and treated with DMSO or CCCP (10 µM) as indicated times. Cells were fixed, permeabilized and subjected to immunofluorescence analysis with the indicated antibodies. Colocalization was quantified by Manders's coefficient. Means ± SEM of more than 10 cells obtained from three independent experiments. For statistical analysis, a one-way ANOVA with Tukey's multiple comparisons test were performed, ****P < 0.0001. Scale bar represents 1 μm.


E . ER - localized MITOL is transported from the mitochondria , and is not newly synthesized protein . HeLa cells stably expressing HA - Parkin were co - transfected with vectors for MITOL - KikGR and FLAG - FKBP38 . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel . Right panel is schematic experimental model for KikGR in this study .

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

E. ER-localized MITOL is transported from the mitochondria, and is not newly synthesized protein. HeLa cells stably expressing HA-Parkin were co-transfected with vectors for MITOL-KikGR and FLAG-FKBP38. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel. Right panel is schematic experimental model for KikGR in this study.


C . Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL . WT or MITOL KO HeLa cells stably expressing HA - Parkin were treated with DMSO or CCCP ( 10 µM ) for 48 h . Lysates of cells were fractionated into cytosolic , mitochondrial and ER fractions , and then subjected to an IB assay with the indicated antibodies . Phospho - Ser65 - Parkin is detected by using specific antibody . Right panels are the quantification of IB about Phospho - Ser65 - Parkin protein levels of the ER fraction . The data represent the mean ± SD for three independent experiments . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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

C. Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 48 h. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IB assay with the indicated antibodies. Phospho-Ser65-Parkin is detected by using specific antibody. Right panels are the quantification of IB about Phospho-Ser65-Parkin protein levels of the ER fraction. The data represent the mean ± SD for three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


D . Parkin is detected at the ER in the absence of MITOL . HeLa cells were transfected with vectors for KikGR - Parkin . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel .

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

D. Parkin is detected at the ER in the absence of MITOL. HeLa cells were transfected with vectors for KikGR-Parkin. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel.


D . MITOL KO increases degradation of ER targeting FKBP38 . HeLa cells stably expressing HA - Parkin were transfected with mitochondrial targeting FLAG - FKBP38 ( FKBP38ActA ) and ER targeting FLAG - FKBP38 ( FKBP38IYFFT ) , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies . Another cell lysates were subsequently separated on a Phos - tag gel , followed by IB with anti - Parkin antibodies ( Phos - tag ) .

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

D. MITOL KO increases degradation of ER targeting FKBP38. HeLa cells stably expressing HA-Parkin were transfected with mitochondrial targeting FLAG-FKBP38 (FKBP38ActA) and ER targeting FLAG-FKBP38 (FKBP38IYFFT), and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies. Another cell lysates were subsequently separated on a Phos-tag gel, followed by IB with anti-Parkin antibodies (Phos-tag).


E - G . MITOL partly prevents CCCP - induced cell death by reducing Parkin - mediated degradation of FKBP38 . WT or MITOL KO HeLa cells stably expressing HA - Parkin and HeLa cells were transfected with the indicated vectors , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting the percentage of dead cells using fluorescence microscopy . Data represent the mean ± SD of five independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . KR , FKBP38 K271 / 273R .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G. MITOL partly prevents CCCP-induced cell death by reducing Parkin-mediated degradation of FKBP38. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were transfected with the indicated vectors, and treated with DMSO or CCCP (10 µM) for 24 h. Cells were stained with propidium iodide (PI) for counting the percentage of dead cells using fluorescence microscopy. Data represent the mean ± SD of five independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. KR, FKBP38 K271/273R.


A . Mislocalization of Bcl - 2 is observed in MITOL KO cells in mitophagy . WT or MITOL KO HeLa cells stably expressing HA - Parkin were transfected with FLAG - Bcl2 and treated with DMSO or CCCP ( 10 µM ) for the indicated times . Cells were immunostained with indicated antibody . Scale bar , 1 μm .

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

A. Mislocalization of Bcl-2 is observed in MITOL KO cells in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin were transfected with FLAG-Bcl2 and treated with DMSO or CCCP (10 µM) for the indicated times. Cells were immunostained with indicated antibody. Scale bar, 1 μm.


B . MITOL prevents caspase - dependent cell death in mitophagy . WT or MITOL KO HeLa cells stably expressing HA - Parkin and HeLa cells were treated with DMSO or CCCP ( 10 µM ) with or without Z - VAD - FMK ( 10 μM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting fluorescence microscopy of the percentage of dead cells . Data represent the mean ± SD of three independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . C . Cell death in mitophagy caused by MITOL deficiency depends on calcium leak . WT and MITOL KO HeLa cells stably expressing HA - Parkin or HeLa cells were treated with DMSO or CCCP ( 10 µM ) with or without BAPTA - AM ( 10 μM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting fluorescence microscopy of the percentage of dead cells . Data represent the mean ± SD of three independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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

B. MITOL prevents caspase-dependent cell death in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were treated with DMSO or CCCP (10 µM) with or without Z-VAD-FMK (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. C. Cell death in mitophagy caused by MITOL deficiency depends on calcium leak. WT and MITOL KO HeLa cells stably expressing HA-Parkin or HeLa cells were treated with DMSO or CCCP (10 µM) with or without BAPTA-AM (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


( C ) Expression of miRNAs miR - 146a and miR - 505 correlated with overall survival in ovarian cancer patients . The validation cohort ( n = 150 samples ) is shown . See Figure EV1 for the training cohort .

O O O O O O O O O O O O O O O O O O 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) Expression of miRNAs miR-146a and miR-505 correlated with overall survival in ovarian cancer patients. The validation cohort (n=150 samples) is shown. See Figure EV1 for the training cohort.


( A ) The consequence of miR - 155 and miR - 181b mimics on cell viability , 5 days post transfection , relative to a negative control miRNA mimic is shown across a panel of normal and ovarian cancer cell lines as indicated . Each data point is the mean of n = 3 / cell line . In the screen , miR - 155 was found to reduce cell viability ( Z - score < - 2 ) in one cell line , and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors . miR - 181 was found to reduce viability ( Z - score < - 2 ) in 2 cell lines , and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors . N , normal cell lines ( IHH , HOSE , HBEC3 , HBEC13 , HBEC30 , and HBEC34 ) , EOC , epithelial ovarian cancer cell lines . miR - 155 data points for each cell line are shown in black while miR - 181b data points are displayed in red .

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

(A) The consequence of miR-155 and miR-181b mimics on cell viability, 5 days post transfection, relative to a negative control miRNA mimic is shown across a panel of normal and ovarian cancer cell lines as indicated. Each data point is the mean of n=3/cell line. In the screen, miR-155 was found to reduce cell viability (Z-score < -2) in one cell line, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. miR-181 was found to reduce viability (Z-score < -2) in 2 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. N, normal cell lines (IHH, HOSE, HBEC3, HBEC13, HBEC30, and HBEC34), EOC, epithelial ovarian cancer cell lines. miR-155 data points for each cell line are shown in black while miR-181b data points are displayed in red.


( B ) Immunoblots indicate suppression of serum - induced AKT phosphorylation at S473 and T308 in response to miR - 155 and suppression of T308 phosphorylation in response to miR - 181b .

O O O O O O O O O O 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) Immunoblots indicate suppression of serum-induced AKT phosphorylation at S473 and T308 in response to miR-155 and suppression of T308 phosphorylation in response to miR-181b.


( C ) miR - 155 and miR - 181b reduced cell viability in GDC0941 - sensitive breast cancer cell lines . Error bars indicate mean ± SD ( n = 3 ) .

(C) miR-155 and miR-181b reduced cell viability in GDC0941-sensitive breast cancer cell lines. Error bars indicate mean ± SD (n=3).


( D ) Differential localization of E - cadherin in PEO1 and PEO4 cells 48 hours post transfection with the indicated oligos . Cells were counterstained with phalloidin and DAPI .

O O O O O O O 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) Differential localization of E-cadherin in PEO1 and PEO4 cells 48 hours post transfection with the indicated oligos. Cells were counterstained with phalloidin and DAPI.


( E ) Stimulation of PEO4 cells with 10 ng / mL TGFβ1 significantly sensitized the cells to AKT inhibition with AKT Inhibitor X . Each point represents the mean of 3 experiments ± SD and * denotes p - value < 0 . 05 by 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 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

(E) Stimulation of PEO4 cells with 10 ng/mL TGFβ1 significantly sensitized the cells to AKT inhibition with AKT Inhibitor X. Each point represents the mean of 3 experiments ± SD and * denotes p-value < 0.05 by Student's T-test.


( F ) Cell viability upon expression of miR - 155 or miR - 181a mimics in 41 NSCLC cell lines and 5 human bronchial epithelial cell lines . Each data point is the mean of n = 3 / cell line . R2 from Pearson correlation .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Cell viability upon expression of miR-155 or miR-181a mimics in 41 NSCLC cell lines and 5 human bronchial epithelial cell lines. Each data point is the mean of n=3/cell line. R2 from Pearson correlation.


( A ) Consequence of miR - 517a on EOC cell viability and 41 NSCLC cell lines ( as in 2A ) . N , normal cell lines ( IHH , HOSE , HBEC3 , HBEC13 , HBEC30 , and HBEC34 ) . miR - 517a was found to significantly reduce cell viability ( Z - score < - 2 ) in 5 cell lines in the screen , and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Consequence of miR-517a on EOC cell viability and 41 NSCLC cell lines (as in 2A). N, normal cell lines (IHH, HOSE, HBEC3, HBEC13, HBEC30, and HBEC34). miR-517a was found to significantly reduce cell viability (Z-score < -2) in 5 cell lines in the screen, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors.


( B , C ) In vivo delivery of neutral liposome incorporated miR - 517a mimic reduced tumor burden by weight ( B ) and nodule number ( C ) in an orthotopic xenograft model using SKOV3 cells . Box - and - whisker plot from n = 8 . p - value from Student ' s T - Test . LD , low - dose ( 200 μg / kg ) ; HD , high - dose ( 400 μg / kg ) .

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

(B, C) In vivo delivery of neutral liposome incorporated miR-517a mimic reduced tumor burden by weight (B) and nodule number (C) in an orthotopic xenograft model using SKOV3 cells. Box-and-whisker plot from n=8. p-value from Student's T-Test. LD, low-dose (200 μg/kg); HD, high-dose (400 μg/kg).


( D ) Correlation of the consequence of ARCN1 depletion and miR - 517a sensitivity in 12 NSCLC cell lines ( plot as in 2D ) .

O O O 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) Correlation of the consequence of ARCN1 depletion and miR-517a sensitivity in 12 NSCLC cell lines (plot as in 2D).


( E ) Immunoblots indicate miR - 517a - induced depletion of ARCN1 and USP1 in the indicated cell lines .

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

(E) Immunoblots indicate miR-517a-induced depletion of ARCN1 and USP1 in the indicated cell lines.


( F ) Consequence of the indicated siRNAs and miR - 517a on the viability of the indicated cell lines .

(F) Consequence of the indicated siRNAs and miR-517a on the viability of the indicated cell lines.


( G ) Immunoblots indicate that miR - 517a suppresses expression of ARCN1 in multiple miR - 517a sensitive cell lines .

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

(G) Immunoblots indicate that miR-517a suppresses expression of ARCN1 in multiple miR-517a sensitive cell lines.


( A ) Consequence of miR - 124 on EOC cell viability ( as in 2A ) . N , normal cell lines ( IHH and HOSE ) . In the screen , miR - 124 was found to significantly reduce cell viability ( Z - score < - 2 ) in 6 cell lines , and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Consequence of miR-124 on EOC cell viability (as in 2A). N, normal cell lines (IHH and HOSE). In the screen, miR-124 was found to significantly reduce cell viability (Z-score < -2) in 6 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors.


( B ) miR - 124 induced expression of neuronal marker proteins Tuj1 ( TUBB3 ) and MAP2 in ES2 cells 48 hours post transfection . Cells were counterstained with DAPI and phalloidin .

(B) miR-124 induced expression of neuronal marker proteins Tuj1 (TUBB3) and MAP2 in ES2 cells 48 hours post transfection. Cells were counterstained with DAPI and phalloidin.


( C ) miR - 124 responsive genes in PEO1 cells were enriched for TargetScan predicted targets . p - value from hypergeometric distribution .

O 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) miR-124 responsive genes in PEO1 cells were enriched for TargetScan predicted targets. p-value from hypergeometric distribution.


( A ) Immunoblots indicate miR - 124 - induced SIX4 depletion . siSIX4 is shown as an antibody control .

(A) Immunoblots indicate miR-124-induced SIX4 depletion. siSIX4 is shown as an antibody control.


( B ) Consequence of SIX4 depletion on EOCcell viability ( as in 2A , N , normal cell lines : HOSE , HBEC30 ) .

O O 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) Consequence of SIX4 depletion on EOCcell viability (as in 2A, N, normal cell lines: HOSE, HBEC30).


( C ) in vivo knockdown of SIX4 using neutral liposome incorporated siRNA reduced tumor burden and ascites volume in an orthotopic xenograft mouse model . Box - and - whisker plot from n = 8 . p - value from Student ' s T - Test .

(C) in vivo knockdown of SIX4 using neutral liposome incorporated siRNA reduced tumor burden and ascites volume in an orthotopic xenograft mouse model. Box-and-whisker plot from n=8. p-value from Student's T-Test.


( D ) Consequence of SIX4 depletion in PEO1 cells on E - cadherinplasma membrane accumulation 48 hours after transfection . Cells were counterstained with phalloidin and DAPI .

O O O O O 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) Consequence of SIX4 depletion in PEO1 cells on E-cadherinplasma membrane accumulation 48 hours after transfection. Cells were counterstained with phalloidin and DAPI.


( E ) Immunoblots indicate consequence of SIX4 depletion on LKB1 accumulation and AMPK pathway activation .

(E) Immunoblots indicate consequence of SIX4 depletion on LKB1 accumulation and AMPK pathway activation.


ER Proliferation under UPR - Inducing Conditions ( A ) Determination of ER abundance in control and UPR - induced cells . Representative cells are shown . The UPR was induced in wild - type cells by addition of DTT . Ultrastructure of control cells and UPR - induced cells was analyzed using ImageJ . The lower images show traces of cortical ER ( represented in magenta ) and the nuclear envelope ( NE , in blue ) . Vacuoles , nuclei , and mitochondria are indicated as V , N , and M , respectively . ( B ) Quantification of the ER proliferation during the UPR . UPR was induced and cells were collected for EM at the indicated time points . Length of the ER ( as traced in [ A ] ) was measured and divided by the area of the section . Data are plotted relative to time 0 . Measurements for each time point correspond to the mean of 25 independent cell images . ( C ) Expression of HAC1i was induced by addition of 100 μM DOC for 3 h . ER was quantified as described above in ( B ) .

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

ER Proliferation under UPR-Inducing Conditions(A) Determination of ER abundance in control and UPR-induced cells. Representative cells are shown. The UPR was induced in wild-type cells by addition of DTT. Ultrastructure of control cells and UPR-induced cells was analyzed using ImageJ. The lower images show traces of cortical ER (represented in magenta) and the nuclear envelope (NE, in blue). Vacuoles, nuclei, and mitochondria are indicated as V, N, and M, respectively.(B) Quantification of the ER proliferation during the UPR. UPR was induced and cells were collected for EM at the indicated time points. Length of the ER (as traced in [A]) was measured and divided by the area of the section. Data are plotted relative to time 0. Measurements for each time point correspond to the mean of 25 independent cell images.(C) Expression of HAC1i was induced by addition of 100 μM DOC for 3 h. ER was quantified as described above in (B).


The ER Morphologically Changes during the UPR ( A ) Control cells and UPR - induced cells were used to analyze and follow the ER within a single cell using EM . Boxes indicate the areas magnified in ( B ) . Cells shown here correspond to the full section of the images labeled " + 140 nm " in ( B ) . ( B ) Serial section of control and UPR - induced cells . Sections are separated by 70 nm on the z - axis . ER is represented in magenta and NE in blue . ( C ) Electro micrographs from control and UPR - induced cells showing that the distance between ER membranes increases during the UPR . For a better preservation of the ultrastructure , samples for this experiment were prepared using high - pressure freezing / freeze substitution techniques ( see Material and Methods ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ER Morphologically Changes during the UPR(A) Control cells and UPR-induced cells were used to analyze and follow the ER within a single cell using EM. Boxes indicate the areas magnified in (B). Cells shown here correspond to the full section of the images labeled "+140 nm" in (B).(B) Serial section of control and UPR-induced cells. Sections are separated by 70 nm on the z-axis. ER is represented in magenta and NE in blue.(C) Electro micrographs from control and UPR-induced cells showing that the distance between ER membranes increases during the UPR. For a better preservation of the ultrastructure, samples for this experiment were prepared using high-pressure freezing/freeze substitution techniques (see Material and Methods).