+ / + ( c42 , c44 ) + / m ( c27 , c43 ) which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . General survival ( calcein - AM ) / ( EthD - 1 ) . ( B ) caspase - 3 + apoptotic . ( C ) senescence ( β - gal ) . ( D ) damage estimated by γH2AX . with > 4 γH2AX foci in nucleus were counted . ( E ) Mitochondrial ROS ( MitoSox ) . ( F ) Mitochondrial potential ( MitoID ) .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. General cell survival (calcein-AM)/death (EthD-1). (B) Cleaved caspase-3+ apoptotic cell death. (C) Cellular senescence (β-gal staining). (D) DNA damage estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted. (E) Mitochondrial ROS (MitoSox). (F) Mitochondrial membrane potential (MitoID).


+ / + ( c42 , c44 ) + / m ( c27 , c43 ) which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . Seahorse . Terminal differentiation of neural ( NSCs ) was induced oxygen consumption rate ( OCR ) extracellular acidification rate ( ECAR ) were analyzed on NSCs at differentiation day 4 . p < 0 . 001 * , n = 7 ( WT ) , 10 ( mutant ) , t - .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Seahorse test. Terminal differentiation of neural stem cells (NSCs) was induced and the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed on the NSCs at differentiation day 4. p<0.001*, n=7 (WT), 10 (mutant), t-test.


+ / + ( c42 , c44 ) + / m ( c27 , c43 ) which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . Western blot analyses for ER stress marker .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Western blot analyses for ER stress marker proteins.


+ / + ( c42 , c44 ) + / m ( c27 , c43 ) which showed similar midbrain - type dopamine ( mDA ) neuron differentiation ( Fig . 3 ) were compared at terminal differentiation day 15 . Resistance of mDA neurons against mitochondrial toxins . Differentiated mDA neurons were exposed to toxins indicated for 1 h , viable TH + mDA neurons were counted . Data are expressed as % TH + relative to untreated . Three cultures were analyzed for each . N = 3 independent experiments , * Significance at p < 0 . 05 . Scale bars , 100 µm .

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The +/+ (c42, c44) and +/m (c27, c43) cells which showed similar midbrain-type dopamine (mDA) neuron differentiation (Fig. 3) were compared at terminal differentiation day 15. Resistance of mDA neurons against mitochondrial toxins. Differentiated mDA neurons were exposed to the toxins indicated for 1 h, and viable TH+ mDA neurons were counted. Data are expressed as % TH+ cells relative to untreated. Three cultures were analyzed for each cell line. N=3 independent experiments, *Significance at p<0.05. Scale bars, 100 µm.


Representative in vitro phase - contrast ( A ) , TH / DAPI or TH ( insets ) ( B ) , TH / SYNAPSIN - stained ( C ) in cultures terminally differentiated from mutant ( c13 , c19 ) corrected hiPSCs ( c22 , c59 ) .

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Representative in vitro phase-contrast (A), TH/DAPI or TH (insets) (B), and TH/SYNAPSIN-stained (C) images in the cultures terminally differentiated from mutant (c13, c19) and corrected hiPSCs (c22, c59).


DA neuron differentiation of DA neurons were assessed by DA neuronal yields


DA neuron morphological of DA neurons were assessed by TH + fiber

DA neuron morphological of DA neurons were assessed by TH+ fiber lengths


DA neuron , synaptical of DA neurons were assessed by synaptic puncta densities


DA neuron differentiation functional maturation of DA neurons were assessed by presynaptic DA neuron releases DA were measured in media conditioned in differentiated cultures for 24 h ( D15 ) evoked by KCl - mediated depolarization for 30 min .

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DA neuron differentiation functional maturation of DA neurons were assessed by presynaptic DA neuron releases DA levels were measured in the media conditioned in the differentiated cultures for 24 h (D15) and evoked by KCl-mediated depolarization for 30 min.


breaks estimated by γH2AX . with > 4 γH2AX foci in nucleus were counted .

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DNA breaks estimated by γH2AX staining. Cells with >4 γH2AX foci in the nucleus were counted.


SA - β - gal was performed for senescence .

SA-β-gal staining was performed for cellular senescence analysis.


Mitochondrial ROS ( MitoSox ) were analyzed after terminal differentiation .

Mitochondrial ROS (MitoSox) were analyzed after terminal differentiation.


mitochondrial potential ( MitoID ) were analyzed after terminal differentiation .

mitochondrial membrane potential (MitoID) were analyzed after terminal differentiation.


Accumulation of α - synuclein oligomers analyzed by western blotting . mutant ( c13 ) corrected ( c22 ) neural ( NSCs ) were transduced with α - synuclein - expressing lentiviruses . At 6 days after differentiation , were extracted fractionated to Triton X - 100 ( 1 % ) - soluble insoluble fractions . Shown is representative WB image of three independent experiments . α - Synuclein oligomers are indicated with a bracket , while monomer bands are marked with an arrowhead .

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Accumulation of α-synuclein oligomers analyzed by western blotting. The mutant (c13) and corrected (c22) neural stem cells (NSCs) were transduced with α-synuclein-expressing lentiviruses. At 6 days after differentiation, cells were extracted and fractionated to Triton X-100 (1%)-soluble and insoluble fractions. Shown is the representative WB image of three independent experiments. α-Synuclein oligomers are indicated with a bracket, while monomer bands are marked with an arrowhead.


Amphetamine - induced rotation scores . net rotation values of transplanted with differentiated mutant ( n = 7 ) corrected ( n = 6 ) pt - hiPSCs are depicted . Significant differences from mutant - transplanted at each post - transplantation time point are at * p < 0 . 05 or * * p < 0 . 001 . engraftments measured by integrated intensities in grafted with DiR - labeled pt corrected hiPSCs . * p < 0 . 05 .

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Amphetamine-induced rotation scores. The ipsilateral net rotation values of individual animals transplanted with differentiated mutant (n=7) and corrected (n=6) pt-hiPSCs are depicted. Significant differences from the mutant cell-transplanted at each post-transplantation time point are at *p<0.05 or **p<0.001. Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR-labeled pt and corrected hiPSCs. *p<0.05.


Overviews of hNCAM + / TH + grafts at 8 post - transplantation . Insets , TH + DA neuron in grafts shown with high magnification .

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Overviews of hNCAM+/TH+ cell grafts at 8 weeks post-transplantation. Insets, TH+ DA neuron morphology in grafts shown with high magnification.


Co - expression of mDA neuron - specific FOXA2 , LMX1A , NURR1 , DAT in TH + DA generated by corrected pt - hiPSC transplantation .

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Co-expression of the mDA neuron-specific FOXA2, LMX1A, NURR1, DAT in TH+ DA cells generated by the corrected pt-hiPSC transplantation.


engraftments measured by integrated intensities in grafted with DiR - labeled pt corrected hiPSCs . * p < 0 . 05 . Total number of TH + in graft ( TH + numbers / mm3 ) per .

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Cell engraftments measured by the integrated fluorescent intensities in the animals grafted with DiR-labeled pt and corrected hiPSCs. *p<0.05. Total number of TH+ cells in the graft (TH+ numbers/mm3) per animal.


LIN28A stability assays . HEK293 were transfected with pFLAG - WT - LIN28A pFLAG - LIN28A R192G . samples were prepared during 24 h of cycloheximide ( 50 μg / ml ) treatment . Representative western blot data for LIN28A level changes normalized to β - actin ( upper ) or with normalization on initial ( 0 h ) LIN28A ( lower ) . Band intensities were quantified using ImageJ , normalized values are depicted ( lower graph ) ( n = 5 ) .

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LIN28A protein stability assays. HEK293 cells were transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G. Protein samples were prepared during 24 h of cycloheximide (50 μg/ml) treatment. Representative western blot data for LIN28A protein level changes normalized to β-actin protein levels (upper) or with normalization based on initial (0 h) LIN28A protein levels (lower). Band intensities were quantified using ImageJ, and normalized values are depicted (lower graph) (n=5).


proteasome inhibitor MG132 treatment increases LIN28A ( R192G ) level . After serum starvation for 3 hours , HEK293T expressing LIN28A ( R912G ) were treated with or without MG132 ( 0 . 1 - 10 uM ) for 30 minutes , then followed by WB analyses .

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The proteasome inhibitor MG132 treatment increases LIN28A(R192G) protein level. After serum starvation for 3 hours, HEK293T cells expressing LIN28A(R912G) were treated with or without MG132 (0.1-10 uM) for 30 minutes, and then followed by WB analyses.


LC - MS / MS . Immunoprecipitated FLAG - LIN28A WT was loaded on an SDS gel ( left upper ) . Bold red sequences indicate peptides matching LIN28A ( left lower ) . Right , LC - MS / MS spectra of WT LIN28A .

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LC-MS/MS analysis. Immunoprecipitated FLAG-LIN28A WT was loaded on an SDS gel (left upper). Bold red sequences indicate peptides matching LIN28A (left lower). Right, LC-MS/MS spectra of WT LIN28A.


localization of WT LIN28A LIN28A ( R192G ) . HeLa transfected with pFLAG - WT - LIN28 pFLAG - LIN28A ( R192G ) were stained with FLAG antibody counterstained with DAPI .

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Cytoplasmic localization of WT LIN28A and LIN28A(R192G) proteins. HeLa cells transfected with pFLAG-WT-LIN28 and pFLAG-LIN28A(R192G) were stained with FLAG antibody and counterstained with DAPI.


LET - 7 microRNA regulatory activity of LIN28A . HeLa were transfected with plasmids expressing FLAG - tagged WT - LIN28A , LIN28AR192G , or mock pFLAG vector ( control ) , Let - 7a , b , c were quantified by RT - qPCR . level of was normalized to that of U6 snRNA . p < 0 . 05 , n = 26 from 6 independent experiments , ns = not significant , ANOVA .

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LET-7 microRNA regulatory activity of LIN28A. HeLa cells were transfected with plasmids expressing FLAG-tagged WT- LIN28A, LIN28AR192G, or mock pFLAG vector (control), and Let-7a, b, and c levels were quantified by RT-qPCR. The level of RNA was normalized to that of U6 snRNA. p<0.05, n=26 from 6 independent experiments, ns = not significant, ANOVA.


Affinity of LIN28A binding to its cognate mRNA 3 ′ UTR region . Upper : schematic for pmirGLO - LIN28A used in this , a bicistronic Firefly / Renilla luciferase ( FLuc / RLuc ) plasmid containing part of LIN28A 3 ′ UTR containing putative LIN28A binding sites ( black dots ) Wilbert et al . , 2012 ( ) . Lower : HeLa were transiently co - transfected with pmirGLO - LIN28A 3 ′ UTR along with pFLAG - WT - LIN28A , pFLAG - LIN28A R192G , or mock pFLAG control . LIN28A - mRNA binding activities were determined by relative luciferase activity of FLuc normalized to those of RLuc . * p < 0 . 05 , n = 18 from 6 independent experiments .

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Affinity of LIN28A protein binding to its cognate mRNA 3′UTR region. Upper: schematic for pmirGLO-LIN28A used in this assay, a bicistronic Firefly/Renilla luciferase (FLuc/RLuc) plasmid containing the part of the LIN28A 3′UTR containing the putative LIN28A protein binding sites (black dots)Wilbert et al., 2012(). Lower: HeLa cells were transiently co-transfected with pmirGLO-LIN28A 3′UTR along with pFLAG-WT-LIN28A, pFLAG-LIN28A R192G, or mock pFLAG control. LIN28A protein-mRNA binding activities were determined by relative luciferase activity levels of FLuc normalized to those of RLuc. *p<0.05, n=18 from 6 independent experiments.


LIN28A - RHA co - IP assays in HEK293 transfected with pFLAG - WT - LIN28A pFLAG - LIN28A R192G .

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LIN28A-RHA co-IP assays in HEK293 cells transfected with pFLAG-WT-LIN28A and pFLAG-LIN28A R192G.


GSEA showing enrichment of BCC [ 28 ] , well - differentiated EMT containing SCC [ 33 ] for YAP gene signatures obtained from mouse hepatocellular carcinoma [ 40 ] human breast cancer [ 39 ] , human oral squamous carcinoma [ 42 ] human colorectal cancer [ 41 ] . Well - diff SCC : well - differentiated SCC ; EMT SCC : EMT - containing SCC

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GSEA analysis showing enrichment of BCC [28], well-differentiated and EMT containing SCC [33] for YAP gene signatures obtained from mouse hepatocellular carcinoma [40]human breast cancer cell line [39], human oral squamous cell carcinoma [42] and human colorectal cancer [41]. Well-diff SCC: well-differentiated SCC; EMT SCC: EMT-containing SCC


( A ) Venn diagram showing overlap between four published YAP signatures [ 39 - 42 ]

(A)Venn diagram showing the overlap between four published YAP signatures [39-42]


( B ) Venn diagram showing overlap between YAP signatures obtained in Oral SCC Breast ( YAP Signature 1 ) CRC HCC ( YAP Signature 2 ) . (

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(B) Venn diagram showing the overlap between the YAP signatures obtained in Oral SCC and Breast (YAP Signature 1) and CRC and HCC (YAP Signature 2) .(C


. ( C ) Venn diagram showing overlap between YAP Signature 1 genes upregulated by more than 1 . 5 fold in BCC compared to IFE [

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.(C) Venn diagram showing the overlap between YAP Signature 1 and the genes upregulated by more than 1.5 fold in BCC compared to IFE[28


( D ) Venn diagram showing overlap between YAP Signature 1 genes upregulated by more than 1 . 5 fold in well - differentiated SCC versus IFE in EMT containing SCC versus HF [ 33 ]

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(D) Venn diagram showing the overlap between YAP Signature 1 and the genes upregulated by more than 1.5 fold in well-differentiated SCC versus IFE and in EMT containing SCC versus HF[33]


( E ) Venn diagram showing genes commonly upregulated in YAP signature 1 in BCCs , well - differentiated EMT - containing

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(E) Venn diagram showing the genes commonly upregulated in YAP signature 1 and in BCCs, well-differentiated and EMT-containing SCCs


( F ) Venn diagram showing overlap between YAP signature 2 genes upregulated by more than 1 . 5 fold in BCC compared to IFE [ 28 ]

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(F) Venn diagram showing the overlap between YAP signature 2 and the genes upregulated by more than 1.5 fold in BCC compared to IFE [28]


( G ) Venn diagram showing overlap between YAP signature 2 genes upregulated by more than 1 . 5 fold in well - differentiated SCC versus IFE in EMT containing SCC versus HF [

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(G) Venn diagram showing the overlap between YAP signature 2 and the genes upregulated by more than 1.5 fold in well-differentiated SCC versus IFE and in EMT containing SCC versus HF [33


( H ) Venn diagram showing genes commonly upregulated YAP Signature 2 in BCCs , well - differentiated EMT - containing SCCs . HCC : hepatocellular carcinoma ; CRC : colorectal cancer ; Well - diff SCC : well - differentiated SCC ; EMT SCC : EMT containing SCC

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(H) Venn diagram showing the genes commonly upregulated the YAP Signature 2 and in BCCs, well-differentiated and EMT-containing SCCs. HCC: hepatocellular carcinoma; CRC: colorectal cancer; Well-diff SCC: well-differentiated SCC; EMT SCC: EMT containing SCC


A . Anterior lateral of two with ELP6 variants showing dysmorphic features at 20 ( 2 ) 19 ( 3 ) years of .

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A. Anterior and lateral photographs of two patients with ELP6 variants showing dysmorphic features at 20 (Patient 2) and 19 (Patient 3) years of age.


B . Mid - sagittal ( I ) , coronal ( II ) transversal ( III ) MRI scans of 1 with newly identified ELP4 variants 2 3 with ELP6 variants . Atrophy of cerebellum with prominence of cerebellar folia is marked by circles . Arrows indicate thin corpus callosum , red arrowheads point to enlarged extra - axial CSF spaces black arrowheads to cerebral sulci in keeping with cerebral cerebellar volume loss .

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B. Mid-sagittal (I), coronal (II) and transversal (III) MRI scans of Patient 1 with newly identified ELP4 variants and Patient 2 and Patient 3 with ELP6 variants. Atrophy of the cerebellum with prominence of the cerebellar folia is marked by circles. Arrows indicate thin corpus callosum, red arrowheads point to enlarged extra-axial CSF spaces and black arrowheads to cerebral sulci in keeping with cerebral and cerebellar volume loss.


C . EEG of 3 . Left panel : 1 - 2 Hz left fronto - temporal spike multi - spike slow wave discharges ( Fp1 - F7 T3 - T5 ) spread to homologous same hemisphere opposite hemisphere , predominantly seen on bipolar montage EEG . Right panel : Spike - multi - spike slow wave activities together with fast rhythms originating from bilateral fronto - temporal - frontocentral regions indicate electrographic seizure activity . Described changes are marked by circles .

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C. EEG of Patient 3. Left panel: 1-2 Hz left fronto-temporal spike and multi-spike slow wave discharges (Fp1-F7 and T3-T5) spread to the homologous same hemisphere and opposite hemisphere, predominantly seen on bipolar montage EEG. Right panel: Spike-and multi-spike slow wave activities together with fast rhythms originating from bilateral fronto-temporal-frontocentral regions indicate electrographic seizure activity. Described changes are marked by circles.


C . Pull - down experiments of assembled mElp123456 complexes carrying - derived mutations . mElp123WT subcomplexes were immobilized on Dynabeads ( strep ) via Elp3 incubated with Elp456 variants . inputs pull downs were loaded on SDS - PAGE gels visualized by Coomassie . molecular mass marker is indicated on left ( M ) .

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C. Pull-down experiments of the assembled mElp123456 complexes carrying patients-derived mutations. The mElp123WT subcomplexes were immobilized on the Dynabeads (strep) via Elp3 protein and incubated with the Elp456 variants. The inputs and pull downs were loaded on the SDS-PAGE gels and visualized by Coomassie staining. The molecular mass marker is indicated on the left (M).


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


( A - D ) Relative growth of ( top ) or western blot of YAP in ( bottom ) indicated prostate cancer lines expressing control , YAP - WT , or YAP - 5SA constructs through lentiviral infection . Of note , LNCaP C4 - 2 express full - AR while 22RV1 R1 - D567 express AR splicing variants V7 V567ES , respectively . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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

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


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

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

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


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

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

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


( E ) Relative mRNA of AR indicated AR target genes ( left ) western blot of AR ( right ) in C4 - 2 treated with increasing concentrations of XMU - MP - 1 . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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

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


( G ) Relative mRNA of indicated AR or YAP target genes in C4 - 2 treated with control ( siNC ) or YAP siRNA ( siYAP _ 1 or siYAP _ 2 ) for 24 hours with or without 2 μM XMU - MP - 1 for 12 hours . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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 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

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


Relative mRNA of indicated AR or YAP target genes ( J , in C4 - 2 ( J , expressing control ( NC ) , YAP - WT or YAP - 5SA constructs through lentiviral infection . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

O 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 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

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


( A ) Western blot of AR ( left ) of AR ( right ) in C4 - 2 treated CHX for indicated time .

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

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


Western blot of AR YAP ( I , in C4 - 2 stably expressing Tet - O - YAP - 5SA treated with 0 . 2 μg / ml doxycycline ( Dox ) for indicated periods of time . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

O O O B-MEASURED_VAR I-MEASURED_VAR 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

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


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

O O O B-MEASURED_VAR I-MEASURED_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

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


relative mRNA of TEAD1 TEAD4 indicated AR target genes ( B ) C4 - 2 treated with two independent TEAD1 / 3 / 4 siRNA or control siRNA . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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

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


( D ) Relative mRNA of indicated AR target genes ( left ) western blot of TEAD ( right ) in C4 - 2 expression control or TEAD4 constructs treated with vehicle or XMU - MP - 1 . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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

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


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

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

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


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

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

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


( O - P ) ChIP - qPCR ( O ) RT - PCR analyses show that exogenous expression of wild type ( TEAD4 - WT ) but not binding deficient form ( TEAD4m ) of TEAD4 rescued AR binding to promoter / enhancer regions of KLK2 KLK3 ( O ) as well as expression of these genes ( P ) in C4 - 2 treated with TEAD1 / 3 / 4 siRNA . Data information : Results are representatives of three independent experiments . Data are ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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


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

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

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


( G - H ) ChIP - qPCR of AR ( G ) TEAD ( H ) binding to promoter / enhancer regions of KLK2 , KLK3 CTGF in Tet - O - YAP - 5SA expressing C4 - 2 treated with 0 . 2 μg / ml doxycycline ( Dox ) for indicated time . Data information : Results are representatives of three independent experiments . Data ± SD . ns : not significant , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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

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


Western blot of YAP TEAD ( left ) mRNA of indicated genes ( right ) in C4 - 2 treated with siYAP / TAZ for 18 ( A ) , 36 ( B ) , hours . Data information : Results are representatives of three independent experiments . Data ± SD . ns : not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Student ' s t - ) .

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

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


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

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 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 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 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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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 . ( K ) of IL - 1β were measured by ELISA from supernatants . mean ± standard deviation is shown .

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


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 .

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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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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

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

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 .

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

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

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

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

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 B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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).