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

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

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

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

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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 infected with VN Wt ΔF2 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 full - gasdermin D . Equal loading was confirmed by beta actin . One representative blot of five independent repeats is shown . ( B ) of band for gasdermin D full - isoform . mean ± standard deviation five independent experiments is shown . Statistical was performed by paired two ‑ Student ' s t . , asterisk ( * ) marks a significant difference when compared to 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 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

(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 infected with VN Wt ΔF2 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 gasdermin D N - terminal C - terminal isoforms by immunoblot in supernatants ; one representative experiment of three independent repeats is shown . ( D ) of band for N - terminal gasdermin D isoform band . Three independent experiments are shown . Statistical was performed by paired two ‑ Student ' s t . , * p < 0 . 05 . ( E ) of band for C - terminal gasdermin D isoform band . Three independent experiments are shown . Statistical was performed by paired two ‑ Student ' s t . , * 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 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

(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 infected with VN Wt ΔF2 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 active caspase - 1 were detected by immunoblot in lysates ( , upper panels ) supernatants ( sup , lower panels ) from same experiment . One representative experiment of three independent repeats is shown . ( G ) of band for active caspase - 1 band . Five independent experiments are shown . Statistical was performed by paired two ‑ Student ' s t . * p < 0 . 05 . ( H ) of total pro - caspase - 1 were quantified on pellets respective supernatants . Three independent experiments are shown . Statistical was performed by paired two ‑ Student ' s t .

O O 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

(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 of whole lysate ( input ) Flag - specific immunoprecipitation ( IP ) from 293T co - transfected with expression plasmids encoding V5 - NLRP3 Flag ZsGreen - PB1 - F2 from indicated viral strains for 24 h Representative blot of three independent repeats is shown . ( B ) of NLRP3 IP band was normalized to respective NLRP3 input band ( n = 3 independent biological replicates , normalized to 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

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

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(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 were co - transfected with expression plasmids encoding for indicated V5 - tagged domains of NLRP3 for 24h . Whole lysate ( left blot ) 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 .

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

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(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 were co - transfected with expression plasmids encoding for Flag - NLRP3 , PB1 - F2 or NEK7 or combinations thereof as indicated . Whole lysate 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 : of IP bands ' relative to their input . mean ± standard deviation of three independent experiments is shown . Statistical was performed by paired two ‑ Student ' s t ( * 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

(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 were co - transfected with expression plasmids encoding for Flag - NLRP3 - LRR , PB1 - F2 or NEK7 or combinations thereof as indicated . Whole lysate 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 ) of IP bands ' relative to their input . mean ± standard deviation of three independent experiments is shown . Statistical was performed by paired two ‑ Student ' s t ( * p < 0 . 05 ) .

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(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 E : 293T were co - transfected with expression plasmids encoding for Flag - NLRP3 - LRR , PB1 - F2 or V5 - NLRP3 - Pyrin or combinations thereof as indicated . Whole lysate 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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expressing B2R constructs with C - terminal SmBiT βarr1 with N - terminal LgBiT were treated with indicated concentrations of bradykinin , ligand - induced change in luminescent signal was measured . - response curves were plotted using GraphPad Prism , pEC50 ( top ) Emax ( bottom ) were calculated . Data represent mean ± 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 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

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 B2RΔI374 but a decrease for B2RL370T B2RΔG368 + L370T mutants . HEK - 293 expressing indicated B2R mutants in presence 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 - of data ( mean ± ) from three independent experiments ( five for B2RΔG368 B2RΔI374 ) is presented as bar graphs in lower panels . Data are normalized with respect to maximal signal under control condition ( treated as 100 % ) , analyzed using Two - way - ANOVA with Bonferroni multiple comparisons ( * 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 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

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

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

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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 expressing NanoBiT - Gq indicated B2R constructs were treated different concentrations of bradykinin , ligand - induced change in luminescent signal was measured . - response curves were plotted using GraphPad Prism to calculate pEC50 ( top ) Emax ( bottom ) values included in graph . Data represent mean ± of three independent experiments with each performed in duplicate , normalized with respect to 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 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

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 expressing 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 signal intensities were quantified using densitometry presented as bar graphs . Data were normalized with respect to signal at 100nM agonist in control ( treated as 100 % ) represent mean ± of five independent experiments . Data were analyzed using Two - way - ANOVA with Bonferroni multiple comparisons ( * * * 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 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

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 expressing 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 signal intensities were quantified using densitometry presented as bar graphs . Data were normalized with respect to signal at 100nM agonist in control ( treated as 100 % ) represent mean ± of five ( six for AT1aRΔL337 ) independent experiments . Data were analyzed using Two - way - ANOVA with Bonferroni multiple comparisons ( * * * 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 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

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 K48 - polyubiquitin chains of Parkin . WT or MITOL KO HeLa 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 were fractionated into , mitochondrial ER fractions , then subjected to an IP - IB with indicated antibodies . K48 - polyubiquitin chains were immunoblotted with K48 - specific ubiquitin antibody .

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

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 ER in later phase of mitophagy . EGFP - MITOL knock in HeLa were transfected with HA - Parkin treated with DMSO or CCCP ( 10 µM ) as indicated times . were fixed , permeabilized subjected to immunofluorescence with indicated antibodies . Colocalization was quantified by Manders ' s coefficient . Means ± of more than 10 obtained from three independent experiments . For statistical , a one - way ANOVA with Tukey ' s multiple comparisons 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 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

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 mitochondria , is not newly synthesized . HeLa stably expressing HA - Parkin were co - transfected with vectors for MITOL - KikGR FLAG - FKBP38 . After ultraviolet ( 365 nm ) exposure to whole plate , were incubated with or without CCCP ( 10 µM ) for indicated time , monitored for red green KikGR . Scale bar , 10 μm . Higher magnification of boxed regions are shown in 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. 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 ER fraction in absence of MITOL . WT or MITOL KO HeLa stably expressing HA - Parkin were treated with DMSO or CCCP ( 10 µM ) for 48 h . Lysates of were fractionated into , mitochondrial ER fractions , then subjected to an IB with indicated antibodies . Phospho - Ser65 - Parkin is detected by using specific antibody . Right panels are of IB about Phospho - Ser65 - Parkin of ER fraction . data represent mean ± SD for three independent experiments . For statistical , a one - way ANOVA with Tukey post - 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 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

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 ER in absence of MITOL . HeLa were transfected with vectors for KikGR - Parkin . After ultraviolet ( 365 nm ) exposure to whole plate , were incubated with or without CCCP ( 10 µM ) for indicated time , monitored for red green KikGR . Scale bar , 10 μm . Higher magnification of boxed regions are shown in 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. 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 stably expressing HA - Parkin were transfected with mitochondrial targeting FLAG - FKBP38 ( FKBP38ActA ) ER targeting FLAG - FKBP38 ( FKBP38IYFFT ) , treated with DMSO or CCCP ( 10 µM ) for 24 h . Lysates of were subjected to an IB with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies . Another 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 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

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

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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 in mitophagy . WT or MITOL KO HeLa stably expressing HA - Parkin were transfected with FLAG - Bcl2 treated with DMSO or CCCP ( 10 µM ) for indicated times . were immunostained with indicated antibody . Scale bar , 1 μm .

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

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 miR - 505 correlated with overall survival in ovarian cancer . validation cohort ( n = 150 samples ) is shown . See Figure EV1 for training cohort .

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

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(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 T308 in response to miR - 155 suppression of T308 phosphorylation in response to miR - 181b .

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

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(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 PEO4 48 hours post transfection with indicated oligos . were counterstained with phalloidin DAPI .

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(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 with 10 ng / mL TGFβ1 significantly sensitized to AKT inhibition with AKT Inhibitor X . Each point represents mean of 3 experiments ± SD * denotes p - value < 0 . 05 by Student ' s T - .

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

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

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(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 burden by ( B ) nodule number ( C ) in an orthotopic xenograft model using SKOV3 . Box - - whisker plot from n = 8 . p - value from Student ' s T - . LD , low - dose ( 200 μg / kg ) ; HD , high - dose ( 400 μg / kg ) .

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

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(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 USP1 in indicated lines .

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


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

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


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

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(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 Tuj1 ( TUBB3 ) MAP2 in ES2 48 hours post transfection . were counterstained with DAPI phalloidin .

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(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 were enriched for TargetScan predicted targets . p - value from hypergeometric distribution .

(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 lines : HOSE , HBEC30 ) .

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(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 burden ascites volume in an orthotopic xenograft mouse model . Box - - whisker plot from n = 8 . p - value from Student ' s T - .

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(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 on E - cadherinplasma accumulation 48 hours after transfection . were counterstained with phalloidin DAPI .

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

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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).


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

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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).


Characterization of ER - Containing Autophagosomes ( ERAs ) during UPR ( A ) of representative DTT - treated wild - type that contain ERAs . Nuclei cytoplasm are indicated as N C , respectively . ( B ) Enlargement of representative of ERAs from different . bottom right image is likely to show a section through a cup - shaped ERA . Note that there are no connections between stacked cisternae envelope . ( C ) High magnification of ERA double envelope . ( D ) Some ERAs are found attached to or are in close proximity to ER tubules / sheets ( indicated by arrow ) . Note that section in ( A ) includes two such junctions . ( E ) High - pressure freezing / freeze substitution image of an ERA linked to an ER tubule / sheet . osmium / lead used in this technique visualizes ribosomes demonstrates that outer ERA envelope , but not stacked internal cisternae , are tightly studded with ribosomes , indicating that they originate from ER membranes . ( F ) High - pressure freezing / freeze substitution image of an ER - ERA junction using an improved protocol to visualize membranes . ( G ) Using same technique as in ( F ) , we visualized internal content of an ERA . Note that both portions of internal membranes of sequestering double envelope contain bound ribosomes , hence are likely derived from ER .

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Characterization of ER-Containing Autophagosomes (ERAs) during the UPR(A) Images of representative DTT-treated wild-type cells that contain ERAs. Nuclei and cytoplasm are indicated as N and C, respectively.(B) Enlargement of representative images of ERAs from different cells. The bottom right image is likely to show a section through a cup-shaped ERA. Note that there are no connections between the stacked cisternae and the envelope.(C) High magnification of the ERA double membrane envelope.(D) Some ERAs are found attached to or are in close proximity to ER tubules/sheets (indicated by the arrow). Note that the section in (A) includes two such junctions.(E) High-pressure freezing/freeze substitution image of an ERA linked to an ER tubule/sheet. The osmium/lead staining used in this technique visualizes ribosomes and demonstrates that the outer ERA envelope membrane, but not the stacked internal cisternae, are tightly studded with ribosomes, indicating that they originate from ER membranes.(F) High-pressure freezing/freeze substitution image of an ER-ERA junction using an improved protocol to visualize membranes.(G) Using the same technique as in (F), we visualized the internal membrane content of an ERA. Note that both portions of the internal membranes and of the sequestering double membrane envelope contain bound ribosomes, and hence are likely derived from the ER.


Visualization of an ER Marker after UPR Induction ( A ) treated with UPR - inducing drug DTT ( + DTT ) or with no drug were visualized using a fusion between translocon component Sec61 red - " cherry . " Top panels show untreated , bottom panels show representative UPR - induced . ( B ) Representative showing UPR - induced that contain ERAs ( indicated by arrows ) .

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Fluorescence Visualization of an ER Marker after UPR Induction(A) Cells treated with the UPR-inducing drug DTT (+DTT) or with no drug were visualized using a fusion protein between the translocon component Sec61 and the red-fluorescent protein "cherry." Top panels show untreated cells, and bottom panels show representative UPR-induced cells.(B) Representative images showing UPR-induced cells that contain ERAs (indicated by arrows).


Immunogold Labeling of ERAs with an Antibody Directed against an ER Marker ( A ) Representative section of a immunolabeled against a myc - tagged Sec63 , an integral ER . As a antibody , we used a rabbit polyclonal anti - myc , as a secondary , we used 15 - nm gold particles - conjugated anti - rabbit antibody . Nucleus , envelope , ER , ERA are indicated as N , NE , ER , ERA , respectively . ( B ) High magnification of an electron micrograph of a section of ER . showed that there are 5 ± 2 gold particles per linear micrometer of ER . ( C ) High magnification of ERAs . To predict how many gold particles one should expect in a particular ERA , we first calculated averaged amount of ER ( expressed as in linear micrometers ) present in an ERA ( similar to ones shown in Figure 3B ) , normalized value for its . These calculations determined that there are 20 . 8 ± 3 . 3 μm of ER per μm2 inside ERAs . These values allowed us to predict how many gold particles would be expected over a section of an ERA if it were packed with ER membranes . Two representative ERAs are shown . ERA shown in middle picture should hold 2 . 4 μm of ER inside , therefore , should have 12 gold particles . We counted 12 gold particles . ERA on right could contain 2 . 7 μm of ER should contain14 gold particles ; we counted 16 gold particles . image on right shows a representative view of a nucleoplasmic region . ( D ) of gold - labeling density per . To assess signal - to - noise of our immunogold - labeling procedure , we assessed background labeling by counting number of gold particles over an areas of nucleoplasm ( N ) over ERAs , normalized to respective areas .

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Immunogold Labeling of ERAs with an Antibody Directed against an ER Membrane Marker(A) Representative section of a cell immunolabeled against a myc-tagged Sec63, an integral ER membrane protein. As a primary antibody, we used a rabbit polyclonal anti-myc and, as a secondary, we used 15-nm gold particles-conjugated anti-rabbit antibody. Nucleus, nuclear envelope, ER, and ERA are indicated as N, NE, ER, and ERA, respectively.(B) High magnification of an electron micrograph of a section of ER. Quantification showed that there are 5 ± 2 gold particles per linear micrometer of ER.(C) High magnification of ERAs. To predict how many gold particles one should expect in a particular ERA, we first calculated and averaged the amount of ER (expressed as length in linear micrometers) present in an ERA (similar to the ones shown in Figure 3B), and normalized the value for its area. These calculations determined that there are 20.8 ± 3.3 μm of ER per μm2 inside the ERAs. These values allowed us to predict how many gold particles would be expected over a section of an ERA if it were packed with ER membranes. Two representative ERAs are shown. The ERA shown in the middle picture should hold 2.4 μm of ER inside and, therefore, should have 12 gold particles. We counted 12 gold particles. The ERA on the right could contain 2.7 μm of ER and should contain14 gold particles; we counted 16 gold particles. The image on the right shows a representative view of a nucleoplasmic region.(D) Quantification of gold-labeling density per area. To assess the signal-to-noise ratio of our immunogold-labeling procedure, we assessed background labeling by counting the number of gold particles over an areas of nucleoplasm (N) and over ERAs, and normalized the counts to the respective areas.


( A ) Wild - type transformed with a plasmid containing GFP - Atg8 were grown for 4 h in synthetic media with no drug , with UPR - inducing conditions ( + DTT + TM ) , or under nitrogen starvation conditions ( N starv ) , then harvested for preparation . extracts were analyzed by Western blotting using antibodies against GFP ( top panel ) or Hac1 ( bottom panel ) . Total was measured by BCA . Same of was loaded in each lane , transfer efficiency was checked by Ponceau . identities of different bands are indicated .

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(A) Wild-type cells transformed with a plasmid containing GFP-Atg8 were grown for 4 h in synthetic media with no drug, with UPR-inducing conditions (+DTT and +TM), or under nitrogen starvation conditions (N starv), and then harvested for protein preparation. Protein extracts were analyzed by Western blotting using antibodies against GFP (top panel) or Hac1 (bottom panel). Total protein concentration was measured by BCA protein assay. Same concentration of protein was loaded in each lane, and transfer efficiency was checked by Ponceau staining. The identities of the different bands are indicated.


( B ) Wild - type expressing GFP - Atg8 grown under conditions described above were visualized by microscopy .

(B) Wild-type cells expressing GFP-Atg8 grown under the conditions described above were visualized by fluorescence microscopy.


( C ) GFP - Atg8 was detected in extracts from untreated hac1Δ or expressing HAC1i ( + DOC ) by Western blotting using antibodies against GFP .

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(C) GFP-Atg8 was detected in extracts from untreated hac1Δ cells or cells expressing HAC1i (+DOC) by Western blotting using antibodies against GFP.


( D ) Western blot using antibodies against GFP of extracts from hac1Δ , ire1Δ , or vps4Δ pep4Δ expressing GFP - Atg8 . Mutant were grown under regular conditions , UPR - inducing conditions ( + DTT ) , or nitrogen starvation conditions ( N starv ) .

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(D) Western blot using antibodies against GFP of extracts from hac1Δ, ire1Δ, or vps4Δ pep4Δ cells expressing GFP-Atg8. Mutant cells were grown under regular conditions, UPR-inducing conditions (+DTT), or nitrogen starvation conditions (N starv).


Localization of GFP - Atg8 during UPR InductionSome of DTT - treated shown in Figure 4B expressing GFP - Atg8 Sec61 - cherry ( as an ER marker ) were visualized using microscopy . GFP - Atg8 localizes in close proximity to ERAs detected by ER marker .

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Localization of GFP-Atg8 during UPR InductionSome of the DTT-treated cells shown in Figure 4B expressing GFP-Atg8 and Sec61-cherry (as an ER marker) were visualized using fluorescence microscopy. GFP-Atg8 localizes in close proximity to the ERAs detected by the ER marker.


Atg8 Other ATG Genes Are Necessary during UPR InductionSerial dilutions for wild - type , hac1Δ , atg1Δ , atg8Δ , atg9Δ , atg16Δ , atg20Δ deletion vps4Δ pep4Δ double deletion were grown on rich - media plates with no drug ( YPD ) or with different concentrations of tunicamycin ( TM ; 0 . 2 or 1 . 0 μg / ml ) . atg19Δ gave an identical result to other autophagy genes shown here ( unpublished data ) .

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Atg8 and Other ATG Genes Are Necessary during UPR InductionSerial dilutions for wild-type, hac1Δ, atg1Δ, atg8Δ, atg9Δ, atg16Δ, and atg20Δ deletion cells and vps4Δ pep4Δ double deletion cells were grown on rich-media plates with no drug (YPD) or with different concentrations of tunicamycin (TM; 0.2 or 1.0 μg/ml). atg19Δ gave an identical result to the other autophagy genes shown here (unpublished data).


A Left : Overlay image of a confocal recording of an apoptotic U2OS surrounded by non - apoptotic labeled with Bax ( green ) Tom22 ( red ) . Bax translocates to mitochondria in apoptotic . Right : display of two color channels as shown in overlay image . asterisk denotes apoptotic . Scale bars : 10 µm .

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A Left: Overlay image of a confocal recording of an apoptotic U2OS cell surrounded by non-apoptotic cells labeled with Bax (green) and Tom22 (red). Bax translocates to the mitochondria in apoptotic cells. Right: Individual display of the two color channels as shown in the overlay image. The asterisk denotes the apoptotic cell. Scale bars: 10 µm.


B , C Dual color image of an apoptotic U2OS decorated with antibodies against Bax ( green ) Tom20 ( red ) . Bax signal was recorded in STED mode Tom20 signal in diffraction - limited confocal mode . ( B ) ( C ) display same data set . small are magnifications of areas indicated by rectangles . In ( B ) color table was adjusted to brightest Bax signal in image resulting in a clear display of clusters . In ( C ) color table was adjusted such that dimmer Bax - rings are visible clusters are saturated . arrows point to Bax - rings . Scale bars : 1 µm .

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B, C Dual color image of an apoptotic U2OS cell decorated with antibodies against Bax (green) and Tom20 (red). The Bax signal was recorded in the STED mode and the Tom20 signal in the diffraction-limited confocal mode. (B) and (C) display the same data set. The small images are magnifications of the areas indicated by the rectangles. In (B) the color table was adjusted to the brightest Bax signal in the image resulting in a clear display of the clusters. In (C) the color table was adjusted such that the dimmer Bax-rings are visible and the clusters are saturated. The arrows point to Bax-rings. Scale bars: 1 µm.


D Optical sections of an apoptotic mitochondrion labelled for Bax ( green ) Tom22 ( red ) . Shown are three optical sections a maximum projection of entire image stack . Scale bar : 0 . 5µm .

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D Optical sections of an apoptotic mitochondrion labelled for Bax (green) and Tom22 (red). Shown are three optical sections and a maximum intensity projection of the entire image stack. Scale bar: 0.5µm.


E Bax - rings on apoptotic mitochondria of various mammalian types . Shown are apoptotic mitochondria labelled for Bax ( green ) Tom22 ( red ) in HT1080 , SH - SY5Y , HeLa CV - 1 , as indicated . Scale bars : 0 . 5 µm .

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E Bax-rings on apoptotic mitochondria of various mammalian cell types. Shown are apoptotic mitochondria labelled for Bax (green) and Tom22 (red) in HT1080, SH-SY5Y, HeLa and CV-1 cells, as indicated. Scale bars: 0.5 µm.


F of diameter of Bax - rings in apoptotic U2OS . Only long ring - axis was measured . Bax - rings smaller than 200 nm exist , but were not considered .

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F Quantification of the diameter of Bax-rings in apoptotic U2OS cells. Only the long ring-axis was measured. Bax-rings smaller than 200 nm exist, but were not considered.


Apoptotic mitochondria were decorated with antibodies against Bax ( green ) Tom22 ( red ) . Bax signal was recorded in STED mode . Shown are color channels as well as an overlay ( from top to bottom ) of several apoptotic mitochondria . We used Lagrange interpolation to double number of pixels . Note that throughout this manuscript except for contrast stretching no image manipulation was applied . Scale bar : 0 . 5 µm .

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Apoptotic mitochondria were decorated with antibodies against Bax (green) and Tom22 (red). The Bax signal was recorded in the STED mode. Shown are the individual color channels as well as an overlay (from top to bottom) of several apoptotic mitochondria. We used Lagrange interpolation to double the number of pixels. Note that throughout this manuscript except for contrast stretching no image manipulation was applied. Scale bar: 0.5 µm.


A , B Three - color of mitochondria of unchallenged ( A ) apoptotic ( B ) U2OS . From left to right : Tom22 , Bax ( recorded in STED - mode ) , cytochrome c an overlay of three channels . arrows denote Bax - rings . Scale bars : 2 µm .

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A, B Three-color images of mitochondria of unchallenged (A) and apoptotic (B) U2OS cells. From left to right: Tom22, Bax (recorded in the STED-mode), cytochrome c and an overlay of all three channels. The arrows denote Bax-rings. Scale bars: 2 µm.


A In apoptotic Drp1 knockdown U2OS , cytochrome c release is delayed . Confocal overview image of an apoptotic Drp1 knockdown labelled with antibodies against Tom20 ( red ) , cytochrome c ( blue ) Bax ( green ) . Large image : Overlay of three channels . Small : Magnifications of indicated by rectangle in large image . Shown are channels , as indicated . arrows point to mitochondria that had not released cytochrome c ; asterisks denote mitochondria that had released cytochrome c . Scale bars : 10 µm ( overview ) 2 µm ( magnifications ) .

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A In apoptotic Drp1 knockdown U2OS cells, cytochrome c release is delayed. Confocal overview image of an apoptotic Drp1 knockdown cell labelled with antibodies against Tom20 (red), cytochrome c (blue) and Bax (green). Large image: Overlay of all three channels. Small images: Magnifications of the area indicated by the rectangle in the large image. Shown are the individual channels, as indicated. The arrows point to mitochondria that had not released cytochrome c; the asterisks denote mitochondria that had released cytochrome c. Scale bars: 10 µm (overview) and 2 µm (magnifications).


B In Drp1 knockdown Bax - ring formation cytochrome c release is not correlated . Three - color of apoptotic mitochondria of Drp1 knockdown . From left to right : Tom22 , Bax ( recorded in STED - mode ) , cytochrome c an overlay of three channels . arrows point to Bax - rings . Scale bar : 1 µm .

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B In Drp1 knockdown cells Bax-ring formation and cytochrome c release is not correlated. Three-color images of apoptotic mitochondria of Drp1 knockdown cells. From left to right: Tom22, Bax (recorded in the STED-mode), cytochrome c and an overlay of all three channels. The arrows point to Bax-rings. Scale bar: 1 µm.


Drp1 knockdown were treated with DMSO ( control ) or with actinomycin D for 14 h to induce apoptosis were decorated with antisera against Bax , cytochrome c , Mic27 or Mic60 ( see Appendix Figure S4 ) . Three - color were taken , whereby Mic27 or Mic60 were recorded in STED mode . Relying on Bax cytochrome c signals , we discriminated between healthy apoptotic mitochondria before after cytochrome c release . distributions of Mic27 or Mic60 were analyzed by determining normalized variance of , which is a sensitive measure for distribution of labelled . numbers within columns represent numbers of analyzed error bars represent standard error of mean .

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Drp1 knockdown cells were treated with DMSO (control) or with actinomycin D for 14 h to induce apoptosis and were decorated with antisera against Bax, cytochrome c, and Mic27 or Mic60 (see Appendix Figure S4). Three-color images were taken, whereby Mic27 or Mic60 were recorded in the STED mode. Relying on the Bax and the cytochrome c signals, we discriminated between healthy and apoptotic mitochondria before and after cytochrome c release. The distributions of Mic27 or Mic60 were analyzed by determining the normalized variance of the fluorescence intensity, which is a sensitive measure for the distribution of the labelled protein. The numbers within the columns represent the numbers of images analyzed and the error bars represent the standard error of the mean.


Shown are seven optical sections through a mitochondrion of an apoptotic U2OS . Top middle row : Tom22 Bax ( recorded in STED ) channel , respectively . Bottom row : overlay ( Tom22 : red ; Bax : green ) . Note absence of Tom22 signal in enclosed by Bax - ring . Displayed are raw data . Scale bar : 0 . 5 µm .

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Shown are seven optical sections through a mitochondrion of an apoptotic U2OS cell. Top and middle row: Tom22 and Bax (recorded in STED) channel, respectively. Bottom row: overlay (Tom22: red; Bax: green). Note the absence of Tom22 signal in the area enclosed by the Bax-ring. Displayed are raw data. Scale bar: 0.5 µm.


principle : RNAi was performed with luciferase - expressing that were challenged with or without CTLs bi - specific antibody . Before readout , supernatant was removed remaining intact were lysed to measure residual - - associated luciferase . To identify immune - checkpoint regulators , difference between normalized luciferase measurements for conditions with CTLs without CTLs was calculated . siRNAs enhancing CTL cytotoxicity would only reduce normalized luciferase under conditions with CTLs ; hence , difference between luciferase measurements will be > 0 .

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Assay principle: RNAi was performed with luciferase-expressing cells that were challenged with or without CTLs and bi-specific antibody. Before readout, cell supernatant was removed and the remaining intact cells were lysed to measure the residual-cell-associated luciferase. To identify immune-checkpoint regulators, the difference between normalized luciferase measurements for conditions with CTLs and without CTLs was calculated. siRNAs enhancing CTL cytotoxicity would only reduce normalized luciferase levels under conditions with CTLs; hence, the difference between luciferase measurements will be > 0.


Luc - CTL performed at different T to MCF7 with PBMC - derived CD8 + T anti - EpCAM x CD3 bi - specific antibody ( ○ ) . Anti - CD19 × CD3 bi - specific Ab ( ▪ ) was used as a specificity control since CD19 is a B - lymphocyte - specific antigen therefore this bsAb fails to cross - link to T . Lower luciferase indicates higher lysis . Error bars denote ± .

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Luc-CTL assay performed at different T cell to MCF7 cell ratio with PBMC-derived CD8+ T cells and anti-EpCAM x CD3 bi-specific antibody (○). Anti-CD19 × CD3 bi-specific Ab (▪) was used as a specificity control since CD19 is a B-lymphocyte-specific antigen and therefore this bsAb fails to cross-link tumor to T cells. Lower luciferase intensity indicates higher lysis. Error bars denote ± SEM.


Luc - CTL was performed with MCF7 transfected with control or PD - L1 - specific siRNAs cocultured with or without CTLs bsAb . For each condition , luciferase activity of PD - L1 - siRNA - treated was normalized to that of control siRNA ; n = 8 . Error bars denote ± .

Luc-CTL assay was performed with MCF7 cells transfected with control or PD-L1-specific siRNAs and cocultured with or without CTLs and bsAb. For each condition, the luciferase activity of PD-L1-siRNA-treated cells was normalized to that of the control siRNA; n = 8. Error bars denote ± SEM.


Comparison between Luc - CTL ( ▪ ) classical chromium ( Cr ) - release ( ○ ) with MCF7 as target survivin - specific T as effector at varying effector to target ( E : T ) ratios . Error bars denote ± .

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Comparison between the Luc-CTL assay (▪) and the classical chromium (Cr)-release assay (○) with MCF7 as target cells and survivin-specific T cells as effector cells at varying effector to target (E:T) ratios. Error bars denote ± SEM.


B Correlation between replicates for screen 2 for both viability toxicity sets , as determined by Pearson rank correlation .

B Correlation between replicates for screen 2 for both viability and toxicity sets, as determined by the Pearson rank correlation test.


C - E Graphical summary of gene function related to modification of T - mediated lysis cellviability for screens 1 , 2 , 3 , respectively . Positive score = reduced cancer cellviability ; negative score = increased viability . X - axis : influence on cellviability without addition of T . Y - axis : influence on cellviability with addition of T . Appropriate immune - suppressive ( PD - L1 , CEACAM - 6 , GAL - 3 ) lethality ( UBC , PLK - 1 ) controls , along with few positive ( GRM4 , GRK5 ) negative ( CCR9 , IL8 ) candidate immune - modulatory hits are highlighted herein .

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C-E Graphical summary of gene function related to modification of T cell-mediated tumor lysis and cellviability for screens 1, 2, and 3, respectively. Positive score = reduced cancer cellviability; negative score = increased viability. X-axis: influence on cellviability without addition of T cells. Y-axis: influence on cellviability with addition of T cells. Appropriate immune-suppressive (PD-L1, CEACAM-6, GAL-3) and lethality (UBC, PLK-1) controls, along with few positive (GRM4, GRK5) and negative (CCR9, IL8) candidate immune-modulatory hits are highlighted herein.


A MCF7 were transfected with described siRNA sequences harvested after 72 h for mRNA estimation using RT - PCR ( upper ) immunoblot ( lower ) , respectively . GAPDH beta - actin were used as controls for normalization , respectively .

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A MCF7 cells were transfected with the described siRNA sequences and harvested after 72 h for mRNA and protein estimation using RT-PCR (upper) and immunoblot (lower) analysis, respectively. GAPDH and beta-actin were used as controls for RNA and protein normalization, respectively.


B Luc - CTL cytotoxicity with PBMC - derived CTLs bi - specific Ab as effector population MCF7 as target , which were transfected with ( s1 - s4 ) or pooled CCR9 siRNA sequences . PD - L1 non - specific control siRNAs were used as positive negative controls , respectively , for CTL - mediated cytotoxicity .

B Luc-CTL cytotoxicity assay with PBMC-derived CTLs and bi-specific Ab as effector population and MCF7 as target cells, which were transfected with individual (s1-s4) or pooled CCR9 siRNA sequences. PD-L1 and non-specific control siRNAs were used as positive and negative controls, respectively, for CTL-mediated cytotoxicity.


C , D Cr - release showing % specific lysis of MCF7 by survivin - specific T at different ratios upon CCR9 knockdown ( C ) or overexpression ( D ) . MCF7 were transfected with either CCR9 siRNA s1 ( Δ ) , pooled siRNA sequences ( ○ ) , positive control PD - L1 ( □ ) , non - specific control siRNA ( ▪ ) ( C ) or with pCMV6 - AC - His control vector ( ▪ ) pCMV6 - AC - His - CCR9 expression construct ( ○ ) ( D ) 72 h prior to .

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C, D Cr-release assay showing % specific lysis of MCF7 cells by survivin-specific T cells at different ratios upon CCR9 knockdown (C) or overexpression (D). MCF7 cells were transfected with either CCR9 siRNA s1 (Δ), pooled siRNA sequences (○), positive control PD-L1 (□), and non-specific control siRNA (▪) (C) or with pCMV6-AC-His control vector (▪) and pCMV6-AC-His-CCR9 expression construct (○) (D) 72 h prior to the assay.


E Cr - release showing % specific lysis of MDA - MB - 231 breast by survivin - specific T at different ratios upon CCR9 knockdown ( ○ ) in comparison to control knockdown ( ▪ ) .

E Cr-release assay showing % specific lysis of MDA-MB-231 breast tumor cell line by survivin-specific T cells at different ratios upon CCR9 knockdown (○) in comparison to the control knockdown (▪).


F , G Cr - release showing lysis of - derived melanoma ( M579 - A2 ) by - infiltrating lymphocytes ( TIL 412 ) ( F ) or lysis of PANC - 1 pancreatic adenocarcinoma by - derived pancreatic TIL 53 ( G ) at different E : T ratios upon CCR9 ( ○ ) or control ( ▪ ) knockdown .

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F, G Cr-release assay showing lysis of patient-derived melanoma cells (M579-A2) by tumor-infiltrating lymphocytes (TIL 412) (F) or lysis of PANC-1 pancreatic adenocarcinoma cells by patient-derived pancreatic TIL 53 (G) at different E:T ratios upon CCR9 (○) or control (▪) knockdown.


A , B ELISpot showing IFN - γ ( A ) granzyme B ( B ) secretion by survivin - specific T , as spot numbers , upon CCR9 knockdown ( black bars ) in MCF7 compared to control knockdown ( bars ) . T ( TC ) alone ( grey bars ) were used as control for background spot numbers .

A, B ELISpot assay showing IFN-γ (A) and granzyme B (B) secretion by survivin-specific T cells, as spot numbers, upon CCR9 knockdown (black bars) in MCF7 cells compared to the control knockdown (white bars). T cells (TC) alone (grey bars) were used as control for background spot numbers.


C Luminex showing cytokine in supernatant from coculture of survivin - specific TC either CCR9hi MCF7 ( transfected with CCR9 - specific siRNA ) or CCR9lo MCF7 ( transfected with control siRNA ) .

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C Luminex assay showing cytokine levels in the supernatant from the coculture of survivin-specific TC and either CCR9hi MCF7 (transfected with CCR9-specific siRNA) or CCR9lo MCF7 (transfected with control siRNA) cells.


D Phospho - plex showing phospho - STAT in survivin - specific TC upon encountering CCR9hi or CCR9lo MCF7 . Log2 of mean ( MFI ) of respective analytes to unstimulated TC is plotted herein .

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D Phospho-plex analysis showing the phospho-STAT levels in survivin-specific TC upon encountering CCR9hi or CCR9lo MCF7 cells. Log2 ratio of mean fluorescent intensity (MFI) of the respective analytes to the unstimulated TC is plotted herein.


E Immunoblot showing phospho - STAT1 in CCR9hi - treated , CCR9lo - treated or unstimulated TC using phospho - specific STAT1 ( pTyr701 ) antibody . Beta - actin was used as loading control .

E Immunoblot analysis showing the phospho-STAT1 levels in the CCR9hi-treated, CCR9lo-treated or unstimulated TC using the phospho-specific STAT1 (pTyr701) antibody. Beta-actin was used as the loading control.


A ELISA showing CCL25 in lysates from indicated tumorcell lines . CCR9 knockdown ( k . d . ) in MCF7 was achieved using specific shRNA ( see Materials Methods ) .

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A ELISA showing CCL25 levels in cell lysates from indicated tumorcell lines. CCR9 knockdown (k.d.) in MCF7 cells was achieved using specific shRNA (see Materials and Methods).


B Cr - release showing % specific lysis of MCF7 by survivin TC upon CCL25 ( □ ) or CCR9 ( ○ ) inhibition using specific siRNAs in comparison to control siRNA ( ▪ ) . Mean ± are depicted herein .

B Cr-release assay showing % specific lysis of MCF7 cells by survivin TC upon CCL25 (□) or CCR9 (○) inhibition using specific siRNAs in comparison to the control siRNA (▪). Mean ± SEM are depicted herein.


C MCF7 were transfected with control or CCR9 - specific siRNAs , 48 h later , supernatants ( CCR9lo or CCR9hi SSN , respectively ) were used to culture survivin TCs overnight . Supernatant - treated TCs were then used as effector against CCR9lo or CCR9hi MCF7 in Cr - release along with wild - type MCF7 . Mean ± are depicted herein .

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C MCF7 cells were transfected with control or CCR9-specific siRNAs, and 48 h later, the supernatants (CCR9lo or CCR9hi SSN, respectively) were used to culture survivin TCs overnight. Supernatant-treated TCs were then used as effector cells against CCR9lo or CCR9hi MCF7 tumor cells in the Cr-release assay along with wild-type MCF7 cells. Mean ± SEM are depicted herein.


D Cr - release showing % specific lysis of MCF7 that were pre - treated with or without pertussis toxin ( PTX ) , or knocked down for CCR9 using specific siRNA . Mean ± are depicted herein .

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D Cr-release assay showing % specific lysis of MCF7 cells that were pre-treated with or without pertussis toxin (PTX), or knocked down for CCR9 using specific siRNA. Mean ± SEM are depicted herein.


E , F MCF7 transfected with control siRNA ( CCR9hi ) or CCR9 siRNA ( CCR9lo ) were cocultured with survivin TCs for 12 h . Gene microarray was performed with total extracted from purified T after coculture . Volcano plot ( E ) illustrating fold change ( FC ; log2 ) in gene expression intensities compared with P - value ( − log2 ) between CCR9hi - CCR9lo - treated TCs . Horizontal bar at y = 4 . 32 represents a statistical significance of P = 0 . 05 ( genes in gray below this did not reach significance ) . LogFC cutoff at ± 0 . 5 is represented by vertical lines . representation of top upregulated ( LogFC > 0 . 5 ) downregulated ( LogFC < − 0 . 85 ) genes ( F ) with P ≤ 0 . 05 . replicates per sample group are shown herein .

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E, F MCF7 cells transfected with control siRNA (CCR9hi) or CCR9 siRNA (CCR9lo) were cocultured with survivin TCs for 12 h. Gene microarray was performed with the total RNA extracted from purified T cells after the coculture. Volcano plot (E) illustrating fold change (FC; log2) in gene expression intensities compared with P-value (−log2) between CCR9hi- and CCR9lo-treated TCs. Horizontal bar at y = 4.32 represents a statistical significance of P = 0.05 (genes in gray below this line did not reach significance). LogFC cutoff at ± 0.5 is represented by the vertical lines. Heatmap representation of the top upregulated (LogFC > 0.5) and downregulated (LogFC < −0.85) genes (F) with P ≤ 0.05. Individual replicates per sample group are shown herein.


A Cr - release showing TIL 209 - mediated lysis of CCR9 + M579 - A2 ( transduced with control shRNA ) or CCR9 − M579 - A2 ( transduced with CCR9 - specific shRNA ) . Curves represent mean ± .

A Cr-release assay showing TIL 209-mediated lysis of CCR9+ M579-A2 (transduced with control shRNA) or CCR9− M579-A2 cells (transduced with CCR9-specific shRNA). Curves represent mean ± SEM.


C , D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9 + or CCR9 − M579 - A2tumors in TIL - treated mice ( C ) or PBS alone group ( D ) . Statistical difference was calculated using unpaired one - sided Mann - Whitney U - .

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C, D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9+ or CCR9−M579-A2tumors in TIL-treated mice (C) or the PBS alone group (D). Statistical difference was calculated using the unpaired one-sided Mann-Whitney U-test.


A ) SH - SY5Y neuroblastoma were either cultivated in RPMI + 10 % FCS or starved in HBSS with or without addition of LY294002 or rapamycin . After 16 h PINK1 mRNA expression was determined by RT - qPCR PINK1 mRNA content of kept in RPMI + 10 % FCS was set as 1 . Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression , comparable to positive control LY294002 ; n = 4 ; * expression changes compared to PINK1 expression in RPMI + 10 % FCS : LY294002 : p < 0 . 01 ; rapamycin : p < 1 × 10 − 4 ; HBSS : p < 0 . 005 ; HBSS + LY294002 : p < 5 × 10 − 6 ; HBSS + rapamycin : p < 0 . 0005 ; # expression changes compared to PINK1 expression in RPMI + 10 % FCS + LY294002 : p < 0 . 05 ; + expression changes compared to PINK1 expression in RPMI + 10 % FCS + rapamycin : p < 0 . 0005 .

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

A) SH-SY5Y neuroblastoma cells were either cultivated in RPMI+10% FCS or starved in HBSS with or without addition of LY294002 or rapamycin. After 16 h PINK1 mRNA expression was determined by RT-qPCR and the PINK1 mRNA content of cells kept in RPMI+10% FCS was set as 1. Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression, comparable to the positive control LY294002; n = 4; * expression changes compared to the PINK1 expression in RPMI+10% FCS: LY294002: p<0.01; rapamycin: p<1×10−4; HBSS: p<0.005; HBSS+LY294002: p<5×10−6; HBSS+ rapamycin: p<0.0005; # expression changes compared to the PINK1 expression in RPMI+10% FCS+LY294002: p<0.05; + expression changes compared to the PINK1 expression in RPMI+10% FCS+rapamycin: p<0.0005.


B ) SH - SY5Y were either stably transduced with a control ( nt ) shRNA or a shRNA directed against PINK1 cultivated in RPMI medium containing 5 % or 10 % FCS . Their PINK1 mRNA content was determined by RT - qPCR PINK1 mRNA content of nt kept in medium with 10 % FCS was set as 1 . Serum reduction increased PINK1 mRNA in nt in accordance with data shown in Fig . 1A , while stable PINK1 knockdown ( kd ) reduced PINK1 content under both conditions ; n = 3 ; * expression changes compared to PINK1 expression in RPMI + 10 % FCS : PINK1 kd 10 % FCS p < 0 . 0005 ; PINK1 induction by 5 % FCS : p < 0 . 05 ; # expression changes compared to PINK1 expression in RPMI + 5 % FCS : PINK1 kd 5 % FCS : p < 0 . 005 .

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

B) SH-SY5Y cells were either stably transduced with a control (nt) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5% or 10% FCS. Their PINK1 mRNA content was determined by RT-qPCR and PINK1 mRNA content of nt cells kept in medium with 10% FCS was set as 1. Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig. 1A, while stable PINK1 knockdown (kd) reduced PINK1 content under both conditions; n = 3; * expression changes compared to the PINK1 expression in RPMI+10% FCS: PINK1 kd 10% FCS p<0.0005; PINK1 induction by 5% FCS: p<0.05; # expression changes compared to the PINK1 expression in RPMI+5% FCS: PINK1 kd 5% FCS: p<0.005.