Figure 3Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream . The green trace indicates the fluorescence of fluorescein included in the barrier stream . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream . Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0 mM bicarbonate into the stimulus stream containing 25 mM bicarbonate . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . Paired plot of rotation frequencies of individual sperm at 0 and 25 mM bicarbonate . Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca2 + . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . Paired plot of rotation frequencies in the presence and absence of extracellular Ca2 + . Fraction of motile sperm ( mean ± SD ) in a sperm population incubated in the presence ( black ) and absence ( at t = 0 ) of extracellular Ca2 + ( red ) ( n ≥ 5 ) . Distribution of rotation frequencies in populations of freely swimming sperm in the presence ( black , n = 335 ) and absence ( red , n = 224 ) of extracellular Ca2 + ( red ) .

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Figure 3Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream. The green trace indicates the fluorescence of fluorescein included in the barrier stream. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream.Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0 mM bicarbonate into the stimulus stream containing 25 mM bicarbonate. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm at 0 and 25 mM bicarbonate.Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca2+. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies in the presence and absence of extracellular Ca2+.Fraction of motile sperm (mean ± SD) in a sperm population incubated in the presence (black) and absence (at t = 0) of extracellular Ca2+ (red) (n ≥ 5).Distribution of rotation frequencies in populations of freely swimming sperm in the presence (black, n = 335) and absence (red, n = 224) of extracellular Ca2+ (red).


Figure 5Left : Bright - field image series of a freely swimming wild - type sperm cell at t = 0 , 151 , and 303 ms . Scale bar represents 10 µm . Right : Rotation frequency ( mean ± SD ) of 24 freely swimming wild - type sperm . Left : Bright - field image series of a freely swimming Catsper1 - / - sperm cell at t = 0 , 151 , and 294 ms . Scale bar represents 10 µm . Right : Rotation frequency ( mean ± SD ) of 24 freely swimming Catsper1 - / - sperm .

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Figure 5Left: Bright-field image series of a freely swimming wild-type sperm cell at t = 0, 151, and 303 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming wild-type sperm. Left: Bright-field image series of a freely swimming Catsper1-/- sperm cell at t = 0, 151, and 294 ms. Scale bar represents 10 µm. Right: Rotation frequency (mean ± SD) of 24 freely swimming Catsper1-/- sperm.


Figure 1 ( B - C ) Coimmunoprecipitation of Ams1 ( B ) and Ape4 ( C ) with Nbr1 . Endogenously TAP - tagged Nbr1 was immunoprecipitated ( IP ) using IgG Sepharose beads . Cell lysates and immunoprecipitates were examined by immunoblotting ( IB ) . ( D - E ) Subcellular localization of Ams1 ( D ) and Ape4 ( E ) . Wild - type ( WT ) , nbr1Δ , sst4Δ , and atg1Δ cells were grown to mid - log phase in EMM medium and examined by live cell imaging . In ( D ) , Ams1 was endogenously tagged at its C - terminus with mCherry . In ( E ) , Ape4 was endogenously tagged at its C - terminus with GFP and in the same cells GFP - binding - protein ( GBP ) fused 3xUb was expressed under the P41nmt1 promoter . Cpy1 is a vacuole lumen marker . Scale bar , 3 μm .

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Figure 1(B-C) Coimmunoprecipitation of Ams1 (B) and Ape4 (C) with Nbr1. Endogenously TAP-tagged Nbr1 was immunoprecipitated (IP) using IgG Sepharose beads. Cell lysates and immunoprecipitates were examined by immunoblotting (IB).(D-E) Subcellular localization of Ams1 (D) and Ape4 (E). Wild-type (WT), nbr1Δ, sst4Δ, and atg1Δ cells were grown to mid-log phase in EMM medium and examined by live cell imaging. In (D), Ams1 was endogenously tagged at its C-terminus with mCherry. In (E), Ape4 was endogenously tagged at its C-terminus with GFP and in the same cells GFP-binding-protein (GBP) fused 3xUb was expressed under the P41nmt1 promoter. Cpy1 is a vacuole lumen marker. Scale bar, 3 μm.


Figure 2Localization of Ams1 in WT , lap2Δ , ape2Δ , and ape4Δ cells . Scale bar , 3 μm . Localization of Ape4 in WT , lap2Δ , ape2Δ , and ams1Δ cells . Scale bar , 3 μm . Ams1 , Lap2 , and Ape2 did not co - immunoprecipitate with Ape4 . Endogenously TAP - tagged Ape4 was immunoprecipitated . Ams1 was endogenously tagged with an mCherry tag .

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Figure 2Localization of Ams1 in WT, lap2Δ, ape2Δ, and ape4Δ cells. Scale bar, 3 μm.   Localization of Ape4 in WT, lap2Δ, ape2Δ, and ams1Δ cells. Scale bar, 3 μm.   Ams1, Lap2, and Ape2 did not co-immunoprecipitate with Ape4. Endogenously TAP-tagged Ape4 was immunoprecipitated. Ams1 was endogenously tagged with an mCherry tag.


Figure 3The ZZ1 - L1 fragment of Nbr1 co - immunoprecipitated with Ams1 . The ZZ1 - L1 fragment of Nbr1 co - immunoprecipitated with Ape4 . The ZZ1 region but not the ZZ2 - ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ams1 . Scale bar , 3 μm . The ZZ1 region but not the ZZ2 - ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ape4 . Scale bar , 3 μm . The ZZ2 - ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Ape2 . Scale bar , 3 μm . The ZZ2 - ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Lap2 . Scale bar , 3 μm .

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Figure 3The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ams1.   The ZZ1-L1 fragment of Nbr1 co-immunoprecipitated with Ape4.   The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ams1. Scale bar, 3 μm.   The ZZ1 region but not the ZZ2-ZZ3 region in Nbr1 is necessary for vacuolar targeting of Ape4. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Ape2. Scale bar, 3 μm.   The ZZ2-ZZ3 region but not the ZZ1 region in Nbr1 is necessary for vacuolar targeting of Lap2. Scale bar, 3 μm.


Figure 6Surface plasmon resonance ( SPR ) analysis of the Nbr1 - Ams1 interaction . GST - tagged ZZ1 - L1 fragment of Nbr1 purified from E . coli was captured on a CM5 sensor chip using the GST Capture Kit . Ams1 purified from S . pombe ( two - fold serial dilutions from 1000 nM to 62 . 5 nM ) was flowed through the chip surface . SPR analysis of the Nbr1 - Ape4 interaction . GST - tagged ZZ1 - L1 fragment of Nbr1 purified from E . coli was captured on a CM5 sensor chip using the GST Capture Kit . Ape4 purified from S . pombe ( two - fold serial dilutions from 1000 nM to 62 . 5 nM ) was flowed through the chip surface .

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Figure 6Surface plasmon resonance (SPR) analysis of the Nbr1-Ams1 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ams1 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.SPR analysis of the Nbr1-Ape4 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ape4 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.


Figure 7The effects of individually mutating the first two residues of Ams1 on its Nbr1 - binding ability . Nbr1 - Ams1 interactions were examined by the Pil1 co - tethering assay . Scale bar , 3 μm . The effects of individually mutating the third residue and other residues in the U - shaped loop of Ape4 on its Nbr1 - binding ability . Nbr1 - Ape4 interactions were examined by the Pil1 co - tethering assay . Scale bar , 3 μm .

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Figure 7The effects of individually mutating the first two residues of Ams1 on its Nbr1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.The effects of individually mutating the third residue and other residues in the U-shaped loop of Ape4 on its Nbr1-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.


Figure 1Confocal microscopy reveals typical " class B " pattern of βarr1 recruitment for V2R and B2R as reflected by first the localization at the plasma membrane and subsequently , internalization in endosomal vesicles upon agonist - stimulation . HEK - 293 cells expressing V2R / B2R and βarr1 - mCherry were stimulated with agonist ( AVP ; 100nM and Bradykinin ; 100nM ) and the localization of βarr1 was visualized using confocal microscopy . Representative images from three independent experiments are shown here , and the scale bar is 10μm . Visual scoring of images from three independent experiments revealed agonist - induced βarr1 recruitment ( i . e . membrane and endosomal localization ) in approximately 77 % of the cells for V2R ( 221 cells ) and 75 % of the cells for B2R ( 662 cells ) . Agonist - induced phosphorylation of ERK1 / 2 in HEK - 293 cells expressing either V2R or B2R in the absence ( control ; CTL ) and presence of βarr1 knock - down ( βarr1 - KD ) are measured using Western blotting . Densitometry - based quantification of data ( mean ± SEM ) from four independent experiments is presented as bar - graphs in the right panels , normalized with respect to maximal signal under control condition ( treated as 100 % ) , and analyzed using Two - way - ANOVA with Bonferroni multiple comparisons test ( * * * p < 0 . 001 , * * p < 0 . 01 ) .

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Figure 1Confocal microscopy reveals typical "class B" pattern of βarr1 recruitment for V2R and B2R as reflected by first the localization at the plasma membrane and subsequently, internalization in endosomal vesicles upon agonist-stimulation. HEK-293 cells expressing V2R/B2R and βarr1-mCherry were stimulated with agonist (AVP; 100nM and Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring of images from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 77% of the cells for V2R (221 cells) and 75% of the cells for B2R (662 cells).Agonist-induced phosphorylation of ERK1/2 in HEK-293 cells expressing either V2R or B2R in the absence (control; CTL) and presence of βarr1 knock-down (βarr1-KD) are measured using Western blotting. Densitometry-based quantification of data (mean ±SEM) from four independent experiments is presented as bar-graphs in the right panels, normalized with respect to maximal signal under control condition (treated as 100%), and analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, **p <0.01).


Figure 2Confocal microscopy reveals robust recruitment of βarr1 to B2R mutant constructs upon agonist - stimulation . HEK - 293 cells expressing either B2RWT or B2R mutants along with βarr1 - mCherry were stimulated with agonist ( Bradykinin ; 100nM ) and the localization of βarr1 was visualized using confocal microscopy . Representative images from three independent experiments are shown here , and the scale bar is 10μm . Visual scoring from three independent experiments revealed agonist - induced βarr1 recruitment ( i . e . membrane and endosomal localization ) in approximately 75 % of the cells for B2RWT ( 662 cells ) , 74 % of the cells for B2RΔG368 ( 169 cells ) , 75 % of the cells for B2RL370T ( 130 cells ) , 85 % of the cells for B2RΔG368 + L370T ( 132 cells ) and 77 % of the cells for B2RΔI374 ( 116 cells ) . HEK - 293 cells expressing B2R constructs with C - terminal SmBiT and βarr1 with N - terminal LgBiT were treated with indicated concentrations of bradykinin , and ligand - induced change in luminescent signal was measured . Concentration - response curves were plotted using GraphPad Prism , and pEC50 ( top ) and Emax ( bottom ) were calculated . Data represent mean ± SEM of five independent experiments ( three for B2RΔI374 ) , each performed in duplicate .

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Figure 2Confocal microscopy reveals robust recruitment of βarr1 to B2R mutant constructs upon agonist-stimulation. HEK-293 cells expressing either B2RWT or B2R mutants along with βarr1-mCherry were stimulated with agonist (Bradykinin; 100nM) and the localization of βarr1 was visualized using confocal microscopy. Representative images from three independent experiments are shown here, and the scale bar is 10μm. Visual scoring from three independent experiments revealed agonist-induced βarr1 recruitment (i.e. membrane and endosomal localization) in approximately 75% of the cells for B2RWT (662 cells), 74% of the cells for B2RΔG368 (169 cells), 75% of the cells for B2RL370T (130 cells), 85% of the cells for B2RΔG368+L370T (132 cells) and 77% of the cells for B2RΔI374 (116 cells).HEK-293 cells expressing B2R constructs with C-terminal SmBiT and βarr1 with N-terminal LgBiT were treated with indicated concentrations of bradykinin, and ligand-induced change in luminescent signal was measured. Concentration-response curves were plotted using GraphPad Prism, and pEC50 (top) and Emax (bottom) were calculated. Data represent mean ± SEM of five independent experiments (three for B2RΔI374), each performed in duplicate.


Figure 3Knock - 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 ) .

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Figure 3Knock-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).


Figure 4HEK - 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 .

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Figure 4HEK-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.


Figure 5HEK - 293 cells expressing the indicated receptor constructs ( pcDNA vector control for mock - transfection ) together with βarr1 - SmBiT and LgBiT - Ib30 were stimulated with the indicated concentrations of respective agonists for 10min . Subsequently , the luminescence signal generated upon the interaction of βarr1 - SmBiT and LgBiT - Ib30 was measured , normalized with basal signal ( i . e . no agonist stimulation treated as 1 ) , and plotted using GraphPad Prism . pEC50 ( top ) and Emax ( bottom ) values are mentioned in the respective graphs and data represent mean ± SEM of four independent experiments , each carried out in duplicate . HEK - 293 cells expressing the NanoBiT - Gq protein and indicated B2R constructs were treated different concentrations of bradykinin , and ligand - induced change in luminescent signal was measured . Concentration - response curves were plotted using GraphPad Prism to calculate pEC50 ( top ) and Emax ( bottom ) values included in the graph . Data represent mean ± SEM of three independent experiments with each performed in duplicate , and normalized with respect to the luminescence signal under basal ( no - agonist treatment ) condition ( treated as 1 ) .

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Figure 5HEK-293 cells expressing the indicated receptor constructs (pcDNA vector control for mock-transfection) together with βarr1-SmBiT and LgBiT-Ib30 were stimulated with the indicated concentrations of respective agonists for 10min. Subsequently, the luminescence signal generated upon the interaction of βarr1-SmBiT and LgBiT-Ib30 was measured, normalized with basal signal (i.e. no agonist stimulation treated as 1), and plotted using GraphPad Prism. pEC50 (top) and Emax (bottom) values are mentioned in the respective graphs and data represent mean±SEM of four independent experiments, each carried out in duplicate.HEK-293 cells expressing the NanoBiT-Gq protein and indicated B2R constructs were treated different concentrations of bradykinin, and ligand-induced change in luminescent signal was measured. Concentration-response curves were plotted using GraphPad Prism to calculate pEC50 (top) and Emax(bottom) values included in the graph. Data represent mean±SEM of three independent experiments with each performed in duplicate, and normalized with respect to the luminescence signal under basal (no-agonist treatment) condition (treated as 1).


Figure 7HEK - 293 cells expressing the indicated AT1aR constructs , in absence ( CTL ; control ) or presence of βarr1 knock - down ( βarr1 - KD ) were stimulated with indicated concentrations of angiotensin II ( AngII ) for 10 min . Subsequently , ERK1 / 2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs . Data were normalized with respect to the signal at 100nM agonist concentration in control cells ( treated as 100 % ) and represent mean ± SEM of five independent experiments . Data were analyzed using Two - way - ANOVA with Bonferroni multiple comparisons test ( * * * p < 0 . 001 , p < 0 . 05 ) . HEK - 293 cells expressing the indicated AT1aR constructs , in absence ( CTL ; control ) or presence of βarr1 knock - down ( βarr1 - KD ) were stimulated with indicated concentrations of angiotensin II ( AngII ) for 10 min . Subsequently , ERK1 / 2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs . Data were normalized with respect to the signal at 100nM agonist concentration in control cells ( treated as 100 % ) and represent mean ± SEM of five ( six for AT1aRΔL337 ) independent experiments . Data were analyzed using Two - way - ANOVA with Bonferroni multiple comparisons test ( * * * p < 0 . 001 , p < 0 . 05 ) .

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Figure 7HEK-293 cells expressing the indicated AT1aR constructs, in absence (CTL; control) or presence of βarr1 knock-down (βarr1-KD) were stimulated with indicated concentrations of angiotensin II (AngII) for 10 min. Subsequently, ERK1/2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs. Data were normalized with respect to the signal at 100nM agonist concentration in control cells (treated as 100%) and represent mean±SEM of five independent experiments. Data were analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, p<0.05).HEK-293 cells expressing the indicated AT1aR constructs, in absence (CTL; control) or presence of βarr1 knock-down (βarr1-KD) were stimulated with indicated concentrations of angiotensin II (AngII) for 10 min. Subsequently, ERK1/2 phosphorylation was measured by Western blotting and signal intensities were quantified using densitometry and presented as bar graphs. Data were normalized with respect to the signal at 100nM agonist concentration in control cells (treated as 100%) and represent mean±SEM of five (six for AT1aRΔL337) independent experiments. Data were analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, p<0.05).


Figure 1A . Sorbitol stimulates YAP phosphorylation . HEK293A cells were cultured in the presence or absence of serum , and treated with 0 . 2 M sorbitol for the indicated time points . Phos - tag gel was used to assess total YAP phosphorylation based on mobility shift . Note that YAP and TAZ are recognized by the same antibody . l . e . denotes long exposure of YAP and TAZ . B . Osmotic stress induces LATS phosphorylation . HEK293A cells were treated with 0 . 2 M sorbitol for 30 or 60 minutes in the presence or absence of serum . LATS phosphorylation at the hydrophobic motif ( HM ) was detected with the phospho - specific pLATS antibody . C . Osmotic stress increases LATS kinase activity . HEK293A cells were treated with 0 . 2 M sorbitol for 30 minutes . Lats1 was immunoprecipitated and an in vitro kinase assay was performed using recombinant GST - YAP as a substrate . Phosphorylation of YAP was determined by immunoblotting with phospho - YAP ( S127 ) antibody . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) . D . Osmotic stress does not lead to YAP cytoplasmic localization . HEK293A cells were treated with 0 . 2 M sorbitol for 1 hr in the presence of serum . YAP subcellular localization was determined by immunofluorescence staining ( red ) . DAPI ( blue ) was used to stain for DNA . Scale bar : 20 μm . Quantification of more nuclear ( N > C ) or more cytosolic ( N < C ) YAP signal is determined with randomly chosen fields , each with approximately 100 cells . E . Osmotic stress does not reduce YAP target gene expression . HEK293A cells were treated with 0 . 2 M sorbitol for 4 hr . mRNA levels of CTGF and CYR61 were measured by quantitative RT - PCR and normalized to GAPDH control . Data are presented as mean ± SEM . n . s means p > 0 . 05 ( two tailed student ' s t - test , n = 3 ) . F . Osmotic stress does not disrupt the interaction between YAP and TEAD1 . HEK293A cells were treated with 0 . 2 M sorbitol for 1 hr . Endogenous YAP was immunoprecipitated with YAP antibody , and TEAD1 and YAP were detected by Western blot . Cells were serum starved for 1 hr as indicated ( - serum ) . G . Osmotic stress does not increase the interaction between YAP and 14 - 3 - 3 . HEK293A cells were transiently transfected with Flag - YAP and Myc - 14 - 3 - 3 , then treated with 0 . 2 M sorbitol for the indicated time points . Myc - 14 - 3 - 3 was immunoprecipitated and the co - precipitated Flag - YAP was detected .

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Figure 1A. Sorbitol stimulates YAP phosphorylation. HEK293A cells were cultured in the presence or absence of serum, and treated with 0.2 M sorbitol for the indicated time points. Phos-tag gel was used to assess total YAP phosphorylation based on mobility shift. Note that YAP and TAZ are recognized by the same antibody. l.e. denotes long exposure of YAP and TAZ.B. Osmotic stress induces LATS phosphorylation. HEK293A cells were treated with 0.2 M sorbitol for 30 or 60 minutes in the presence or absence of serum. LATS phosphorylation at the hydrophobic motif (HM) was detected with the phospho-specific pLATS antibody.C. Osmotic stress increases LATS kinase activity. HEK293A cells were treated with 0.2 M sorbitol for 30 minutes. Lats1 was immunoprecipitated and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP was determined by immunoblotting with phospho-YAP (S127) antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).D. Osmotic stress does not lead to YAP cytoplasmic localization. HEK293A cells were treated with 0.2 M sorbitol for 1 hr in the presence of serum. YAP subcellular localization was determined by immunofluorescence staining (red). DAPI (blue) was used to stain for DNA. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields, each with approximately 100 cells.E. Osmotic stress does not reduce YAP target gene expression. HEK293A cells were treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. n.s means p > 0.05 (two tailed student's t-test, n = 3).F. Osmotic stress does not disrupt the interaction between YAP and TEAD1. HEK293A cells were treated with 0.2 M sorbitol for 1 hr. Endogenous YAP was immunoprecipitated with YAP antibody, and TEAD1 and YAP were detected by Western blot. Cells were serum starved for 1 hr as indicated (- serum).G. Osmotic stress does not increase the interaction between YAP and 14-3-3. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the co-precipitated Flag-YAP was detected.


Figure 2A . Osmotic stress induces YAP nuclear translocation . HEK293A cells were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for 1 hr . YAP / TAZ ( red , upper panels ) or YAP ( red , lower panels ) were stained with two specific antibodies . DAPI ( blue ) was used to visualize cell nuclei . Scale bar : 20 μm . Quantification of more nuclear ( N > C ) or more cytosolic ( N < C ) YAP signal is determined with randomly chosen fields . B . Osmotic stress induces TAZ nuclear translocation . YAP knockout ( KO ) HEK293A cells were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for 1 hr . TAZ localization was determined by immunofluorescence staining with the YAP / TAZ antibody ( red ) . Scale bar : 20 μm . C . Osmotic stress induces a transient YAP nuclear translocation . HEK293A cells were serum starved for 1 hr followed by 0 . 2 M sorbitol treatment for the indicated time points . D . Osmotic stress induces YAP target gene expression . Wild type ( WT ) or YAP / TAZ double knockout ( Y / T KO ) HEK293A cells were serum starved for 1 hr and treated with 0 . 2 M sorbitol for 4 hr . mRNA levels of CTGF and CYR61 were measured by quantitative RT - PCR and normalized to GAPDH control . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) . E . Osmotic stress decreases YAP and 14 - 3 - 3 interaction . HEK293A cells were transiently transfected with Flag - YAP and Myc - 14 - 3 - 3 , and were serum starved for 16 hr . Cells were then treated with 0 . 2 M sorbitol for the indicated time points . Myc - 14 - 3 - 3 was immunoprecipitated and the associated Flag - YAP was detected by Western . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

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Figure 2A. Osmotic stress induces YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. YAP/TAZ (red, upper panels) or YAP (red, lower panels) were stained with two specific antibodies. DAPI (blue) was used to visualize cell nuclei. Scale bar: 20 μm. Quantification of more nuclear (N > C) or more cytosolic (N < C) YAP signal is determined with randomly chosen fields.B. Osmotic stress induces TAZ nuclear translocation. YAP knockout (KO) HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for 1 hr. TAZ localization was determined by immunofluorescence staining with the YAP/TAZ antibody (red). Scale bar: 20 μm.C. Osmotic stress induces a transient YAP nuclear translocation. HEK293A cells were serum starved for 1 hr followed by 0.2 M sorbitol treatment for the indicated time points.D. Osmotic stress induces YAP target gene expression. Wild type (WT) or YAP/TAZ double knockout (Y/T KO) HEK293A cells were serum starved for 1 hr and treated with 0.2 M sorbitol for 4 hr. mRNA levels of CTGF and CYR61 were measured by quantitative RT-PCR and normalized to GAPDH control. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).E. Osmotic stress decreases YAP and 14-3-3 interaction. HEK293A cells were transiently transfected with Flag-YAP and Myc-14-3-3, and were serum starved for 16 hr. Cells were then treated with 0.2 M sorbitol for the indicated time points. Myc-14-3-3 was immunoprecipitated and the associated Flag-YAP was detected by Western. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


Figure 3A . Inhibition of p38 or JNK does not block osmotic stress - induced YAP nuclear localization . HEK293A cells were pretreated with 2 μM p38 inhibitor ( SB203580 ) or 20 μM JNK inhibitor ( SP600125 ) before treatment with 0 . 2 M sorbitol for 1 hr in the absence of serum . Quantification of YAP / TAZ subcellular staining is shown . Scale bar : 20 μm . A . Inhibition of p38 or JNK does not block osmotic stress - induced YAP nuclear localization . HEK293A cells were pretreated with 2 μM p38 inhibitor ( SB203580 ) or 20 μM JNK inhibitor ( SP600125 ) before treatment with 0 . 2 M sorbitol for 1 hr in the absence of serum . Cell lysates from identically treated samples were examined for phosphorylation of p38 substrate MK - 2 and JNK substrate c - Jun . B . NLK knockout blocks osmotic stress - induced YAP nuclear localization . HEK293A cells were transiently transfected with CRISPR / Cas9 to knockout NLK . Wild type ( WT ) cells and the NLK KO cell pool were treated with 0 . 2 M sorbitol for 1 hr in the absence of serum . YAP / TAZ subcellular localization was determined by immunofluorescence staining . Scale bar : 20 μm . C . NLK induces YAP nuclear translocation . HEK293A cells were co - transfected with Flag - YAP together with vector control , Myc - NLK - WT ( wild type NLK ) , or NLK - KN ( kinase negative mutant ) . Cells were serum starved for 6 hr , and YAP localization and NLK expression were determined by Flag ( green ) and Myc ( red ) antibodies , respectively . Scale bar : 20 μm . D . NLK induces YAP phosphorylation . HA - YAP was co - transfected with NLK - WT or NLK - KN in HEK293A cells . The phos - tag gel showed NLK - WT but not NLK - KN caused a significant mobility shift of YAP . E . NLK phosphorylates YAP in vitro . NLK - WT and NLK - KN were immunoprecipitated from HEK293A cells and an in vitro kinase assay was performed using recombinant GST - YAP as a substrate in the presence of ATP - γ - S . Total phosphorylation of YAP was detected by immunoblotting with thiophosphate ester antibody , which identifies the alkylated thiophosphorylation on YAP .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Quantification of YAP/TAZ subcellular staining is shown. Scale bar: 20 μm.A. Inhibition of p38 or JNK does not block osmotic stress-induced YAP nuclear localization. HEK293A cells were pretreated with 2 μM p38 inhibitor (SB203580) or 20 μM JNK inhibitor (SP600125) before treatment with 0.2 M sorbitol for 1 hr in the absence of serum. Cell lysates from identically treated samples were examined for phosphorylation of p38 substrate MK-2 and JNK substrate c-Jun.B. NLK knockout blocks osmotic stress-induced YAP nuclear localization. HEK293A cells were transiently transfected with CRISPR/Cas9 to knockout NLK. Wild type (WT) cells and the NLK KO cell pool were treated with 0.2 M sorbitol for 1 hr in the absence of serum. YAP/TAZ subcellular localization was determined by immunofluorescence staining. Scale bar: 20 μm.C. NLK induces YAP nuclear translocation. HEK293A cells were co-transfected with Flag-YAP together with vector control, Myc-NLK-WT (wild type NLK), or NLK-KN (kinase negative mutant). Cells were serum starved for 6 hr, and YAP localization and NLK expression were determined by Flag (green) and Myc (red) antibodies, respectively. Scale bar: 20 μm.D. NLK induces YAP phosphorylation. HA-YAP was co-transfected with NLK-WT or NLK-KN in HEK293A cells. The phos-tag gel showed NLK-WT but not NLK-KN caused a significant mobility shift of YAP.E. NLK phosphorylates YAP in vitro. NLK-WT and NLK-KN were immunoprecipitated from HEK293A cells and an in vitro kinase assay was performed using recombinant GST-YAP as a substrate in the presence of ATP-γ-S. Total phosphorylation of YAP was detected by immunoblotting with thiophosphate ester antibody, which identifies the alkylated thiophosphorylation on YAP.


Figure 4A . NLK induces YAP Ser 128 phosphorylation . Flag - YAP WT or S128A mutant was co - transfected with or without Myc - NLK . Phosphorylation was determined by Western blot using YAP S128 phosphospecific antibody . B . NLK phosphorylates YAP Ser 128 in vitro . NLK - WT or NLK - KN was transfected into HEK293A cells and immunoprecipitated . An in vitro NLK kinase assay was performed using recombinant GST - YAP as a substrate . Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho - YAP ( S128 ) antibody . C . Osmotic stress induces YAP phosphorylation at Ser 128 . In the left panel , HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated . In the right panel , Flag - YAP was transfected into HEK293A cells . Flag - YAP was immunoprecipitated , YAP Ser 128 phosphorylation was detected by a phosphospecific antibody . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) . D . NLK deficiency reduces YAP S128 phosphorylation . Two CRISPR / CAS9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A . WT cells and two pools of NLK CRISPR / CAS9 transfected cells were treated with 0 . 2 M sorbitol for the indicated time points . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 4 ) . E . The S128D phosphomimetic mutant abolishes YAP interaction with 14 - 3 - 3 . Flag - YAP WT and mutant constructs were co - transfected with Myc - 14 - 3 - 3 into HEK293A cells . Cells were serum starved for 16 hrs . 14 - 3 - 3 was immunoprecipitated with Myc antibody and the associated YAP was detected with Flag antibody . F . YAP Ser 128 phosphorylation is required for disruption of YAP - 14 - 3 - 3 interaction by osmotic stress . Flag - YAP WT or Flag - YAP S128A constructs were co - transfected with Myc - 14 - 3 - 3 into HEK293A cells . Cells were serum starved for 16 hrs , and treated with 0 . 2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 hr . Refreshing medium served as a control to disrupt YAP and 14 - 3 - 3 binding . 14 - 3 - 3 was immunoprecipitated with Myc antibody , and the associated YAP was detected with Flag antibody . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. NLK induces YAP Ser 128 phosphorylation. Flag-YAP WT or S128A mutant was co-transfected with or without Myc-NLK. Phosphorylation was determined by Western blot using YAP S128 phosphospecific antibody.B. NLK phosphorylates YAP Ser 128 in vitro. NLK-WT or NLK-KN was transfected into HEK293A cells and immunoprecipitated. An in vitro NLK kinase assay was performed using recombinant GST-YAP as a substrate. Phosphorylation of YAP Ser 128 was determined by immunoblotting with phospho-YAP (S128) antibody.C. Osmotic stress induces YAP phosphorylation at Ser 128. In the left panel, HEK293A cells were treated with sorbitol and endogenous YAP was immunoprecipitated. In the right panel, Flag-YAP was transfected into HEK293A cells. Flag-YAP was immunoprecipitated, YAP Ser 128 phosphorylation was detected by a phosphospecific antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).D. NLK deficiency reduces YAP S128 phosphorylation. Two CRISPR/CAS9 gRNA plasmids targeting different sites of NLK were transfected into HEK293A. WT cells and two pools of NLK CRISPR/CAS9 transfected cells were treated with 0.2 M sorbitol for the indicated time points. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 4).E. The S128D phosphomimetic mutant abolishes YAP interaction with 14-3-3. Flag-YAP WT and mutant constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs. 14-3-3 was immunoprecipitated with Myc antibody and the associated YAP was detected with Flag antibody.F. YAP Ser 128 phosphorylation is required for disruption of YAP-14-3-3 interaction by osmotic stress. Flag-YAP WT or Flag-YAP S128A constructs were co-transfected with Myc-14-3-3 into HEK293A cells. Cells were serum starved for 16 hrs, and treated with 0.2 M sorbitol for the indicated time points or refreshed with serum containing medium for 1 hr. Refreshing medium served as a control to disrupt YAP and 14-3-3 binding.14-3-3 was immunoprecipitated with Myc antibody, and the associated YAP was detected with Flag antibody. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


Figure 5A . NLK does not induce nuclear translocation of the S128A mutant YAP . HEK293A cells were co - transfected with Myc - NLK and Flag - YAP WT or S128A mutant . Cells were serum starved for 6 hr , and YAP localization and NLK expression were determined by Flag ( green ) and Myc ( red ) antibodies , respectively . Scale bar : 20 μm . B . YAP Ser 128 phosphorylation is required for osmotic stress - induced YAP nuclear localization . HEK293A YAP KO cells reconstituted with Flag - YAP WT , YAP S128D , or YAP S128A mutant were treated with 0 . 2 M sorbitol for 1 hr . YAP subcellular localization was determined by Flag immunofluorescence staining ( green ) . Scale bar : 20 μm .

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Figure 5A. NLK does not induce nuclear translocation of the S128A mutant YAP. HEK293A cells were co-transfected with Myc-NLK and Flag-YAP WT or S128A mutant. Cells were serum starved for 6 hr, and YAP localization and NLK expression were determined by Flag (green) and Myc (red) antibodies, respectively. Scale bar: 20 μm.B. YAP Ser 128 phosphorylation is required for osmotic stress-induced YAP nuclear localization. HEK293A YAP KO cells reconstituted with Flag-YAP WT, YAP S128D, or YAP S128A mutant were treated with 0.2 M sorbitol for 1 hr. YAP subcellular localization was determined by Flag immunofluorescence staining (green). Scale bar: 20 μm.


Figure 6A . YAP S128D reconstituted cells show growth advantage under hyperosmotic conditions . YAP / TAZ dKO HEK293A cells with stable expression of YAP WT , S128A , and S128D were cultured in the absence ( left panel ) or presence of 0 . 2M sorbitol ( right panel ) for the indicated amount of time . Cell numbers were counted and normalized to day 0 . Data are presented as mean ± SEM , n = 3 . B . YAP S128D reconstituted cells have lower cell death in a hyperosmotic environment . Cell cycle analyses of YAP / TAZ dKO HEK293A cells with stable expression of YAP WT , S128A , and S128D after 0 . 2 M sorbitol treatment were determined using flow cytometry . Propidium iodine ( PI ) was used for DNA staining ( Fig EV5 ) . Quantification of sub - G1 cells is shown . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) . C . YAP S128D reconstituted cells show reduced apoptosis under hyperosmotic conditions . Annexin V analyses of YAP / TAZ dKO HEK293A cells with stable expression of YAP WT , S128A , and S128D after 0 . 2 M sorbitol treatment were determined using flow cytometry . PE - Annexin V and 7 - AAD stained for phospholipid phosphatidylserine ( PS ) and DNA , respectively . Data are presented as mean ± SEM . * p < 0 . 05 ( two tailed student ' s t - test , n = 3 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. YAP S128D reconstituted cells show growth advantage under hyperosmotic conditions. YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D were cultured in the absence (left panel) or presence of 0.2M sorbitol (right panel) for the indicated amount of time. Cell numbers were counted and normalized to day 0. Data are presented as mean ± SEM, n = 3.B. YAP S128D reconstituted cells have lower cell death in a hyperosmotic environment. Cell cycle analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. Propidium iodine (PI) was used for DNA staining (Fig EV5). Quantification of sub-G1 cells is shown. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).C. YAP S128D reconstituted cells show reduced apoptosis under hyperosmotic conditions. Annexin V analyses of YAP/TAZ dKO HEK293A cells with stable expression of YAP WT, S128A, and S128D after 0.2 M sorbitol treatment were determined using flow cytometry. PE-Annexin V and 7-AAD stained for phospholipid phosphatidylserine (PS) and DNA, respectively. Data are presented as mean ± SEM. *p < 0.05 (two tailed student's t-test, n = 3).


Figure 1TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress . ( A ) ROS levels in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) left untreated , after 6 h of nutrient starvation or 24 h of metabolic stress . ROS levels were measured by flow cytometry after DCF treatment . The results are expressed as the mean DCF fluorescence ( and standard deviation ) , from three independent experiments . TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress . ( B ) Basal , nutrient starvation - induced ( 5 h ) or metabolic stress - induced ( 18 h ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , measured by flow cytometry after DCF treatment . The results are expressed as the mean intensity of cell fluorescence ( and standard deviation ) . * represents significant difference from control conditions ( P < 0 . 05 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress. (A) ROS levels in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) left untreated, after 6 h of nutrient starvation or 24 h of metabolic stress. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation), from three independent experiments.TIGAR regulates intracellular ROS levels in response to nutrient starvation or metabolic stress.(B) Basal, nutrient starvation-induced (5 h) or metabolic stress-induced (18 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05).


Figure 2TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( A ) ( Left panel ) confocal microscopy images of the fluorescence in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clone TIGAR # 7 ) or control cells ( clone Cont # 1 ) and infected with an adenovirus expressing GFP - LC3 for 16 h . Cells were then left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . ( Right panel ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta from three independent experiments . The mean values with standard deviation are presented . TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( B ) ( Left panel ) Confocal microscopy images of the fluorescence in U2OS cells stably expressing GFP - LC3 and transfected with scrambled or TIGAR siRNAs . After 48 h transfection , cells were then left untreated , exposed to nutrient starvation for 5 h or to metabolic stress for 18 h . ( Right panel ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta from three independent experiments . The mean values with standard deviation are presented . TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( C ) ( Left panel ) Western blot showing the expression levels of endogenous LC3 - I , LC3 - II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs , and 48 h later exposed to nutrient starvation for 0 , 2 . 5 and 6 h . ( Middle panel ) Western blot showing the expression levels of endogenous LC3 - I , LC3 - II and TIGAR in U2OS stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) and left untreated . ( Right panel ) Western blot showing the expression levels of p62 , COX - IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated . Actin expression was examined as a loading control . TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress . ( D ) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0 , 1 , 3 , 5 and 8 h ; * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (A) (Left panel) confocal microscopy images of the fluorescence in U2OS cells stably over-expressing Flag-tagged-TIGAR (clone TIGAR#7) or control cells (clone Cont#1) and infected with an adenovirus expressing GFP-LC3 for 16 h. Cells were then left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(B) (Left panel) Confocal microscopy images of the fluorescence in U2OS cells stably expressing GFP-LC3 and transfected with scrambled or TIGAR siRNAs. After 48 h transfection, cells were then left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h. (Right panel) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta from three independent experiments. The mean values with standard deviation are presented.TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress. (C) (Left panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs, and 48 h later exposed to nutrient starvation for 0, 2.5 and 6 h. (Middle panel) Western blot showing the expression levels of endogenous LC3-I, LC3-II and TIGAR in U2OS stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) and left untreated. (Right panel) Western blot showing the expression levels of p62, COX-IV and TIGAR in U2OS cells transfected with scrambled or TIGAR siRNAs and left untreated. Actin expression was examined as a loading control.TIGAR expression modulates autophagy in response to nutrient starvation or metabolic stress.(D) Western blot showing the expression levels of endogenous TIGAR in U2OS cells after exposure to nutrient starvation or metabolic stress for 0, 1, 3, 5 and 8 h; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


Figure 3TIGAR expression modulates autophagy independently of p53 . ( A ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta . U2OS cells stably expressing GFP - LC3 were transfected with scrambled or TIGAR siRNAs , and 48 h after transfection , cells were left untreated ( t0 ) or treated with Bafilomycin A1 ( 100 nM ) for 1 or 2 h ( t1 and t2 ) . The percentage of cells with GFP - LC3 puncta was calculated at the indicated time points . Data are shown as the mean and standard deviation from three independent experiments . ( B ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta . U2OS cells stably expressing GFP - LC3 were cotransfected with scrambled or TIGAR siRNAs , and scrambled or p53 siRNA . After 48 h transfection , cells were left untreated or exposed for 5 h to nutrient starvation . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments . ( C ) Western blot showing the expression levels of endogenous p53 and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs , and scrambled or p53 siRNA , and 48 h later exposed to nutrient starvation for 5 h . Actin expression was examined as a loading control . ( D ) Quantitation of the percentage of GFP - LC3 - positive cells displaying GFP puncta . U2OS cells stably expressing GFP - LC3 were cotransfected with scrambled or TIGAR siRNAs , and scrambled or DRAM siRNA1 / 2 . After 48 h transfection , cells were left untreated or exposed for 5 h to nutrient starvation . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments ; * represents significant difference from starved control conditions ( P < 0 . 05 ) ; @ represents significant difference from untreated control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3TIGAR expression modulates autophagy independently of p53. (A) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were transfected with scrambled or TIGAR siRNAs, and 48 h after transfection, cells were left untreated (t0) or treated with Bafilomycin A1 (100 nM) for 1 or 2 h (t1 and t2). The percentage of cells with GFP-LC3 puncta was calculated at the indicated time points. Data are shown as the mean and standard deviation from three independent experiments.(B) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(C) Western blot showing the expression levels of endogenous p53 and TIGAR in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled or p53 siRNA, and 48 h later exposed to nutrient starvation for 5 h. Actin expression was examined as a loading control.(D) Quantitation of the percentage of GFP-LC3-positive cells displaying GFP puncta. U2OS cells stably expressing GFP-LC3 were cotransfected with scrambled or TIGAR siRNAs, and scrambled or DRAM siRNA1/2. After 48 h transfection, cells were left untreated or exposed for 5 h to nutrient starvation. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments; * represents significant difference from starved control conditions (P<0.05); @ represents significant difference from untreated control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


Figure 4 ( A ) U2OS cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments . ( B ) ( Left panel ) ROS levels in U2OS cells left untreated or treated with 0 . 5 or 1 mM of H2O2 for 24 h . ROS levels were measured by flow cytometry after DCF treatment . The results are expressed as the mean DCF fluorescence ( and standard deviation ) from three independent experiments . ( Right panel ) Quantitation of the percentage of GFP - LC3 puncta positive cells for cells treated as described above . Data are shown as the mean and standard deviation from three independent experiments . ( C ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably expressing GFP - LC3 were transfected with vector pCHER1A expressing the mCherry gene as control , or expression plasmids for Flag - tagged - TIGAR or HA - tagged - FBPase - 2 . After 48 h transfection , cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments . ( D ) Quantitation of the percentage of GFP - LC3 puncta positive cells . Cells were left untreated , exposed to nutrient starvation for 6 h with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) , AO2 ( glutathione ethyl ester ( 4 mM ) ) or AO3 ( ethyl pyruvate ( 4 mM ) ) for 24 h . U2OS cells stably expressing GFP - LC3 were transfected with vector pCHER1A expressing the mCherry gene as control , or expression plasmid for Flag - tagged - TIGAR . After 48 h transfection , cells were treated . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation from three independent experiments . ( E ) ( Left panel ) Quantitation of the percentage of GFP - LC3 puncta positive cells . Cells were left untreated , exposed to nutrient starvation for 5 h or to metabolic stress for 18 h , with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h . U2OS cells stably over - expressing GFP - LC3 in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 . ( Right panel ) Basal or nutrient starvation - induced ( 5 h ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 ( NAC ( 2 mM ) and L - ascorbic acid ( 2 mM ) ) for 24 h , measured by flow cytometry after DCF treatment . The results are expressed as the mean intensity of cell fluorescence ( and standard deviation ) . * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) U2OS cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(B) (Left panel) ROS levels in U2OS cells left untreated or treated with 0.5 or 1 mM of H2O2 for 24 h. ROS levels were measured by flow cytometry after DCF treatment. The results are expressed as the mean DCF fluorescence (and standard deviation) from three independent experiments. (Right panel) Quantitation of the percentage of GFP-LC3 puncta positive cells for cells treated as described above. Data are shown as the mean and standard deviation from three independent experiments.(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmids for Flag-tagged-TIGAR or HA-tagged-FBPase-2. After 48 h transfection, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(D) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 6 h with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)), AO2 (glutathione ethyl ester (4 mM)) or AO3 (ethyl pyruvate (4 mM)) for 24 h. U2OS cells stably expressing GFP-LC3 were transfected with vector pCHER1A expressing the mCherry gene as control, or expression plasmid for Flag-tagged-TIGAR. After 48 h transfection, cells were treated. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation from three independent experiments.(E) (Left panel) Quantitation of the percentage of GFP-LC3 puncta positive cells. Cells were left untreated, exposed to nutrient starvation for 5 h or to metabolic stress for 18 h, with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2. (Right panel) Basal or nutrient starvation-induced (5 h) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2 with or without treatment with AO1 (NAC (2 mM) and L-ascorbic acid (2 mM)) for 24 h, measured by flow cytometry after DCF treatment. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


Figure 5 ( A ) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA . ( B ) RT - PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA . ( C ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably over - expressing GFP - LC3 in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , and either scrambled , ATG5 siRNA or ATG10 siRNA . After 48 h , cells were left untreated or exposed to nutrient starvation for 5 h . ( D ) ROS levels in U2OS cells in the presence of either scrambled , TIGAR siRNA1 or TIGAR siRNA2 , and scrambled , ATG5 siRNA or ATG10 siRNA , measured by flow cytometry after DCF treatment . After 48 h , cells were left untreated or exposed to nutrient starvation for 5 h . The results are expressed as the mean intensity of cell fluorescence ( and standard deviation ) . * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Western blot showing knockdown of ATG5 protein expression by ATG5 siRNA.(B) RT-PCR showing knockdown of ATG10 mRNA expression by ATG10 siRNA.(C) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing GFP-LC3 in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and either scrambled, ATG5 siRNA or ATG10 siRNA. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h.(D) ROS levels in U2OS cells in the presence of either scrambled, TIGAR siRNA1 or TIGAR siRNA2, and scrambled, ATG5 siRNA or ATG10 siRNA, measured by flow cytometry after DCF treatment. After 48 h, cells were left untreated or exposed to nutrient starvation for 5 h. The results are expressed as the mean intensity of cell fluorescence (and standard deviation). * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


Figure 6 ( A ) Quantitation of the percentage of GFP - LC3 puncta positive cells . U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) were left untreated or exposed to nutrient starvation for 6 h , with or without treatment with Z - VAD - FMK for 24 h . The percentage of cells with GFP - LC3 puncta was calculated , and data are shown as the mean and standard deviation of the mean from three independent experiments . ( B ) Apoptosis in U2OS cells stably over - expressing Flag - tagged - TIGAR ( clones TIGAR # 5 and TIGAR # 7 ) or control cells ( clones Cont # 1 and Cont # 3 ) , as measured by cells with a sub - G1 DNA content . Cells were transfected with either scrambled , TIGAR siRNA1 or TIGAR siRNA2 . Cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . ( C ) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs , and scrambled , ATG5 siRNA or ATG10 siRNA , as measured by cells with a sub - G1 DNA content . After 48 h , cells were left untreated , exposed to nutrient starvation for 6 h or to metabolic stress for 24 h . Data are shown as the mean and standard deviation from three independent experiments . In each case , the increase in apoptosis after knockdown of ATG5 or ATG10 , compared with the matched control , was statistically significant ; * represents significant difference from control conditions ( P < 0 . 05 ) ; # represents a lack of significant difference from control conditions ( P > 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Quantitation of the percentage of GFP-LC3 puncta positive cells. U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3) were left untreated or exposed to nutrient starvation for 6 h, with or without treatment with Z-VAD-FMK for 24 h. The percentage of cells with GFP-LC3 puncta was calculated, and data are shown as the mean and standard deviation of the mean from three independent experiments.(B) Apoptosis in U2OS cells stably over-expressing Flag-tagged-TIGAR (clones TIGAR#5 and TIGAR#7) or control cells (clones Cont#1 and Cont#3), as measured by cells with a sub-G1 DNA content. Cells were transfected with either scrambled, TIGAR siRNA1 or TIGAR siRNA2. Cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h.(C) Apoptosis in U2OS cells cotransfected with scrambled or TIGAR siRNAs, and scrambled, ATG5 siRNA or ATG10 siRNA, as measured by cells with a sub-G1 DNA content. After 48 h, cells were left untreated, exposed to nutrient starvation for 6 h or to metabolic stress for 24 h. Data are shown as the mean and standard deviation from three independent experiments. In each case, the increase in apoptosis after knockdown of ATG5 or ATG10, compared with the matched control, was statistically significant; * represents significant difference from control conditions (P<0.05); # represents a lack of significant difference from control conditions (P>0.05).


Figure 1The central axial sections of the reconstructions . Note the C - terminal domain of gp104 and other low - density features that do not visualize well in the isosurface representation ( panels ( A - C ) ) . Letters G , H and I indicate the position of the cross - sections shown in the homonymously labeled panels below . A 69 Å - thick slice of the reconstruction of the extended sheath cryoEM map orthogonal to the sixfold axis . The proximal and distal parts of one of the three fibers that run at 82 ° to the sixfold axis are indicated with a semitransparent cyan and magenta lines , respectively . Sections of the spontaneously and urea contracted sheath structures that cut through the baseplate / proximal part of the fiber plane , whose position in the reconstructions is indicated with a red line in panels ( E and F ) . The putative direction of the distal part of the fiber that is completely disordered in the reconstructions is indicated with a solar cross symbol .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1The central axial sections of the reconstructions. Note the C-terminal domain of gp104 and other low-density features that do not visualize well in the isosurface representation (panels (A-C)). Letters G, H and I indicate the position of the cross-sections shown in the homonymously labeled panels below. A 69 Å-thick slice of the reconstruction of the extended sheath cryoEM map orthogonal to the sixfold axis. The proximal and distal parts of one of the three fibers that run at 82° to the sixfold axis are indicated with a semitransparent cyan and magenta lines, respectively. Sections of the spontaneously and urea contracted sheath structures that cut through the baseplate/proximal part of the fiber plane, whose position in the reconstructions is indicated with a red line in panels (E and F). The putative direction of the distal part of the fiber that is completely disordered in the reconstructions is indicated with a solar cross symbol.


Figure 7Percentage of phage particles in the extended , intermediate , and contracted states after 5 , 20 and 80 minutes of incubation with L . monocytogenes cell walls . The numbers above the bars show the number of particles contained in the tomograms . The average length of the tail and the standard deviation for each of the three states is given next to the color legend . The pre - attachment state . Here and elsewhere in this figure , the EM image in the lower panel is a slice through the cryoET reconstruction . Note that in the presence of the Listeria cell wall the gp108 fibers are no longer in their stowed conformation but instead are fully or partially unraveled . Gp108 fibers attach to the host cell wall but the sheath is not yet contracted . RBP / TF gp106 ( and possibly gp107 ) pyramids reorient to point toward the cell surface , change their conformation , and bind to the cell wall . The baseplate - proximal part of the sheath is in a contracted state , whereas the capsid - proximal part is still in an extended state . The baseplate - proximal part of the sheath contains a segment where the tail tube is clearly visible because it no longer interacts with the sheath ( lower panel ) . The post - attachment state . The tail is fully contracted and the tail tube has pierced through the cell wall . The capsid appears full indicating that DNA injection has not yet begun .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Percentage of phage particles in the extended, intermediate, and contracted states after 5, 20 and 80 minutes of incubation with L. monocytogenes cell walls. The numbers above the bars show the number of particles contained in the tomograms. The average length of the tail and the standard deviation for each of the three states is given next to the color legend.The pre-attachment state. Here and elsewhere in this figure, the EM image in the lower panel is a slice through the cryoET reconstruction. Note that in the presence of the Listeria cell wall the gp108 fibers are no longer in their stowed conformation but instead are fully or partially unraveled. Gp108 fibers attach to the host cell wall but the sheath is not yet contracted. RBP/TF gp106 (and possibly gp107) pyramids reorient to point toward the cell surface, change their conformation, and bind to the cell wall. The baseplate-proximal part of the sheath is in a contracted state, whereas the capsid-proximal part is still in an extended state. The baseplate-proximal part of the sheath contains a segment where the tail tube is clearly visible because it no longer interacts with the sheath (lower panel). The post-attachment state. The tail is fully contracted and the tail tube has pierced through the cell wall. The capsid appears full indicating that DNA injection has not yet begun.


Figure 2B : Growth charts of 2 probands each showing typical growth patterns of self - limited DP , without compromised linear growth before puberty . Solid horizontal black lines connect green dots representing bone age to red dots at the equivalent chronological age .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B: Growth charts of 2 probands each showing typical growth patterns of self-limited DP, without compromised linear growth before puberty. Solid horizontal black lines connect green dots representing bone age to red dots at the equivalent chronological age.


Figure 3C : Biological consequences of the 2 identified N - terminal mutations . Both WT and mutant N - terminal protein fragments ( p . Arg156Leu and p . Glu161Lys ) were expressed in HEK293 cells as demonstrated by western blotting . The GFP - tagged protein products of both were not detected in the conditioned media of mammalian cells , as compared to wild type ( WT ) , and appear to be retained in the intracellular compartment ( mean ± s . e . m . ; n = 3 ) . Ponceau redstaining is shown to demonstrate equal protein loading for conditioned media . UT - untransfected negative control ; nd - not detected ; * : p = 0 . 01094 ( WT vs p . Arg156Leu ) and p = 0 . 04408 ( WT vs p . Glu161Lys ) by 2 - tailed t - test . It has to date not been possible to test cytoplasmic retention for the two C - terminal mutations due to difficulty expressing the full length or C - terminal protein fragment in mammalian cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3C: Biological consequences of the 2 identified N-terminal mutations. Both WT and mutant N-terminal protein fragments (p.Arg156Leu and p.Glu161Lys) were expressed in HEK293 cells as demonstrated by western blotting. The GFP-tagged protein products of both were not detected in the conditioned media of mammalian cells, as compared to wild type (WT), and appear to be retained in the intracellular compartment (mean ± s.e.m.; n = 3). Ponceau redstaining is shown to demonstrate equal protein loading for conditioned media. UT - untransfected negative control; nd - not detected; *: p=0.01094 (WT vs p.Arg156Leu) and p=0.04408 (WT vs p.Glu161Lys) by 2-tailed t-test. It has to date not been possible to test cytoplasmic retention for the two C-terminal mutations due to difficulty expressing the full length or C-terminal protein fragment in mammalian cells.


Figure 4A - J : Igsf10 expression was not observed at E10 . 5 ( A , sagittal section ) but readily detectable at E12 . 5 , in the NM ( B and C , frontal sections ) and E14 . 5 , along the migratory path of GnRH neurons ( D , frontal section ) . Igsf10 positive cells were negative for Isolectin b4 ( marker of vasculature ) at all developmental stages ( E , at E14 . 5 ) . Incubation with the sense probe resulted in no signal at all stages ( E14 . 5 shown in F ) . Peripherin - positive cells olfactory axons were extending within the Igsf10 - positive NM ( G and H ) . Cxcl12 mRNA expression is shown in I ( E14 . 5 , frontal section ) . GnRH neurons are located in the MPOA by E17 . 5 ( J , frontal section ) in an Igsf10 negative area . K - N : In human 11pcw brains , IGSF10 expression pattern was similar to that observed in mouse , with GnRH neurons interspersed in an IGSF10 - positive NM ( K - N , frontal sections , see also schematic in P ) . IGSF10 positive cells were also detected in the olfactory bulb ( OB , panel O ) . Sense probe resulted in no specific signal ( panel N ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-J: Igsf10 expression was not observed at E10.5 (A, sagittal section) but readily detectable at E12.5, in the NM (B and C, frontal sections) and E14.5, along the migratory path of GnRH neurons (D, frontal section). Igsf10 positive cells were negative for Isolectin b4 (marker of vasculature) at all developmental stages (E, at E14.5). Incubation with the sense probe resulted in no signal at all stages (E14.5 shown in F). Peripherin-positive cells olfactory axons were extending within the Igsf10-positive NM (G and H). Cxcl12 mRNA expression is shown in I (E14.5, frontal section). GnRH neurons are located in the MPOA by E17.5 (J, frontal section) in an Igsf10 negative area.K-N: In human 11pcw brains, IGSF10 expression pattern was similar to that observed in mouse, with GnRH neurons interspersed in an IGSF10-positive NM (K-N, frontal sections, see also schematic in P). IGSF10 positive cells were also detected in the olfactory bulb (OB, panel O). Sense probe resulted in no specific signal (panel N).


Figure 5A : Levels of Igsf10 expression in native NIH3T3 cells , and cells stably expressing a scrambled ( sh - scr ) or Igsf10 ( sh - Igsf10 ) shRNA . B and C : migration of GN11 cells from aggregates into NIH3T3 sh - scr ( B ) or sh - Igsf10 ( C ) . D : Analysis of the migration index ( see Methods ) . E : RT - PCR analysis in zebrafish of total embryos and larvae showing the expression of Igsf10 mRNA at different time points . Expected band size : 905 bp . F : Representative time - lapse analysis of mispair control ( Control Sp - MO and Control ATG - MO ) and Igsf10 morpholinos ( Igsf10 Sp - MO and Igsf10 ATG - MO ) injected Tg ( gnrh3 : EGFP ) embryos and larvae , at different time - points . Images show lateral view of live larvaehead ( 48 and 72 hpf ) and dorsal view ( 4 and 5 dpf ) of live larvaehead ; anterior is left . White arrowheads indicate normal GnRH3 neuron clusters in the olfactory area of control embryos . Brackets show the extension of the projections towards the hypothalamus . White arrows indicate examples of abnormal GnRH3 neurons scattered in the olfactory area of morphants accompanied by lack of projections toward the hypothalamus , indicated with Δ ( hpf - hours post fertilisation ; dpf - days post fertilisation . ) Scale bar , 100 um ( panels at 48 and 72hpf in F ) , 150 um ( panels at 4 and 5dpf in F ) . Panel G : Effect of Igsf10 Splice - site morpholino injection observed at 48hrs . Quantification of the percentage of embryos showing an abnormal GnRH3 - neuron phenotype was significantly higher in Igsf10 Splice - site - MO ( Sp - MO ) injected embryos compared to relative controls . * : p = 0 . 02422 , * * : p = 0 . 00101 , * * * : p = 0 . 00020 , * * * * : p = 0 . 00001 .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A: Levels of Igsf10 expression in native NIH3T3 cells, and cells stably expressing a scrambled (sh-scr) or Igsf10 (sh-Igsf10) shRNA.B and C: migration of GN11 cells from aggregates into NIH3T3 sh-scr (B) or sh-Igsf10 (C).D: Analysis of the migration index (see Methods).E: RT-PCR analysis in zebrafish of total embryos and larvae showing the expression of Igsf10 mRNA at different time points. Expected band size: 905 bp.F: Representative time-lapse analysis of mispair control (Control Sp-MO and Control ATG-MO) and Igsf10 morpholinos (Igsf10 Sp-MO and Igsf10 ATG-MO) injected Tg(gnrh3:EGFP) embryos and larvae, at different time-points. Images show lateral view of live larvaehead (48 and 72 hpf) and dorsal view (4 and 5 dpf) of live larvaehead; anterior is left. White arrowheads indicate normal GnRH3 neuron clusters in the olfactory area of control embryos. Brackets show the extension of the projections towards the hypothalamus. White arrows indicate examples of abnormal GnRH3 neurons scattered in the olfactory area of morphants accompanied by lack of projections toward the hypothalamus, indicated with Δ (hpf - hours post fertilisation; dpf - days post fertilisation.) Scale bar, 100 um (panels at 48 and 72hpf in F), 150 um (panels at 4 and 5dpf in F). Panel G: Effect of Igsf10 Splice-site morpholino injection observed at 48hrs. Quantification of the percentage of embryos showing an abnormal GnRH3-neuron phenotype was significantly higher in Igsf10 Splice-site-MO (Sp-MO) injected embryos compared to relative controls. *: p=0.02422, **: p=0.00101, ***: p=0.00020, ****: p=0.00001.


Figure 1D . The relative abundance of valine ( Val ) and leucine - isoleucine ( Leu - Ile ) . N = 18 ; Boxplot , central band stands for median , boxes stand for 50 % of the data , whiskers stand for min or max of the data E . The relative abundance of 3 - hydroxybutyrate ( 3 - HOB ) and acetylcarnitine ( AC ) . N = 18 ; Boxplot , central band stands for median , boxes stand for 50 % of the data , whiskers stand for min or max of the data Data information : Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction . F . The concentration of FGF21 in serum . Control , N = 18 ; POI , N = 20 ; Boxplot , central band stands for median , boxes stand for 50 % of the data , whiskers stand for min or max of the data G . The concentration of GDF15 in serum . Control , N = 14 ; POI , N = 9 ; Boxplot , central band stands for median , boxes stand for 50 % of the data , whiskers stand for min or max of the data . Data information : Error bars stand for SEM of biological repeats . The p value was calculated by two - tailed t - test with 2 - way ANOVA correction .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1D. The relative abundance of valine (Val) and leucine-isoleucine (Leu-Ile). N=18; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data E. The relative abundance of 3-hydroxybutyrate (3-HOB) and acetylcarnitine (AC). N=18; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.F. The concentration of FGF21 in serum. Control, N=18; POI, N=20; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data G. The concentration of GDF15 in serum. Control, N=14; POI, N=9; Boxplot, central band stands for median, boxes stand for 50% of the data, whiskers stand for min or max of the data. Data information: Error bars stand for SEM of biological repeats. The p value was calculated by two-tailed t-test with 2-way ANOVA correction.