( G ) Nitric oxide ( NO ) levels in the culture media of cells relative to the IFNγ / TNFα control 24h after treatment Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

(G) Nitric oxide (NO) levels in the culture media of cells relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( A ) Immunofluorescence staining for myoglobin and myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . Quantification represents the average myotube width Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

(A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


( B ) Western blotting for iNOS and tubulin protein levels 24h after treatment . Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ / TNFα control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting for iNOS and tubulin protein levels 24h after treatment. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


( C ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) and total S6 . Quantification represents the pS6K / S6K and pS6 / S6 ratios relative to the NT control . Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

(C) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


( A ) Representative images of overall hind limb musculature . Scale bar represents 1cm

(A) Representative images of overall hind limb musculature. Scale bar represents 1cm


( B ) Western blotting for phospho - Ser79 - ACC ( pACC ) and total ACC

(B) Western blotting for phospho-Ser79-ACC (pACC) and total ACC


( C ) Cross - sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort ( n = 4 ) . Cross - sections were stained by H & E . Scale bars represent 100µm Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

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

(C) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls


( D - E ) RT - qPCR analysis of Atrogin - 1 / MAFbx ( D mRNA expression from the tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

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

(D-E) RT-qPCR analysis of Atrogin-1/MAFbx (D mRNA expression from the tibialis anterior of three mice per cohort (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls


( D - E ) RT - qPCR analysis o MuRF1 ( E ) mRNA expression from the tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

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

(D-E) RT-qPCR analysis o MuRF1 (E) mRNA expression from the tibialis anterior of three mice per cohort (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls


( A ) Cross - sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort ( n = 4 ) . Cross - sections were stained by H & E . Scale bars represent 100µm Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

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

(A) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


( B - C ) RT - qPCR analysis of Atrogin - 1 / MAFbx ( B mRNA expression from the tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

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

(B-C) RT-qPCR analysis of Atrogin-1/MAFbx (B mRNA expression from the tibialis anterior of four mice per cohort (n = 4). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


( B - C ) RT - qPCR analysis o MuRF1 ( C ) mRNA expression from the tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

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

(B-C) RT-qPCR analysis o MuRF1 (C) mRNA expression from the tibialis anterior of four mice per cohort (n = 4). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


Genes with higher dominant PAS usage ( DPU ) ( A ) or with lower APA Shannon index ( B ) express at higher levels ( liver shown as example ) . The number of genes in each group is indicated on top . Mann - Whitney U test significance levels are indicated for neighboring groups : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . The boxplots show the Q1 to Q3 quartile values ( the box limits ) , the median ( the horizontal green lines ) and values within the 1 . 5 * IQR ( the whiskers ) .

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

Genes with higher dominant PAS usage (DPU) (A) or with lower APA Shannon index (B) express at higher levels (liver shown as example). The number of genes in each group is indicated on top. Mann-Whitney U test significance levels are indicated for neighboring groups: *P < 0.05; **P < 0.01; ***P < 0.001. The boxplots show the Q1 to Q3 quartile values (the box limits), the median (the horizontal green lines) and values within the 1.5 * IQR (the whiskers).


Spearman correlations between gene expression level and dominant PAS usage ( DPU ) in each tissue for all genes ( blue ) , genes with DPU equal to or above 90 % and genes with DPU less than 90 % . Genes with DPU less than 90 % exhibit weaker Spearman correlations between gene expression level and dominant PAS usage in each tissue . An empty square indicates that the P value of Spearman ' s rho is above 0 . 01 . ( ESC : embryonic stem cells , CER : cerebellum , COR : cerebral cortex , HEA : heart , KID : kidney , LIV : liver , LUN : lung , MUS : muscle , SPL : spleen . )

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

Spearman correlations between gene expression level and dominant PAS usage (DPU) in each tissue for all genes (blue), genes with DPU equal to or above 90% and genes with DPU less than 90%. Genes with DPU less than 90% exhibit weaker Spearman correlations between gene expression level and dominant PAS usage in each tissue. An empty square indicates that the P value of Spearman's rho is above 0.01. (ESC: embryonic stem cells, CER: cerebellum, COR: cerebral cortex, HEA: heart, KID: kidney, LIV: liver, LUN: lung, MUS: muscle, SPL: spleen.)


Fractions of orthologous genes in different groups exhibiting different subcellular localizations of mRNA isoforms in rat neurons . Same : isoforms enriched in same cellular location . Diff : isoforms enriched in different cellular locations , i . e . one isoform in neuropil and another one in soma . ( Fisher exact test : * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) .

Fractions of orthologous genes in different groups exhibiting different subcellular localizations of mRNA isoforms in rat neurons. Same: isoforms enriched in same cellular location. Diff: isoforms enriched in different cellular locations, i.e. one isoform in neuropil and another one in soma. (Fisher exact test: *P < 0.05; **P < 0.01; ***P < 0.001).


2D clustering of Pearson correlation coefficients between all pairwise sample combinations of C57BL / 6J ( BL6 ) or SPRET / EiJ ( SPR ) allelic PAS usages from 5 , 264 genes ( see Methods ) . 2D clustering of Pearson correlation coefficients between all pairwise combinations of BL6 or SPR allelic gene expression levels from 5 , 264 genes . All clustering was performed hierarchically using Pearson correlations between samples .

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

2D clustering of Pearson correlation coefficients between all pairwise sample combinations of C57BL/6J (BL6) or SPRET/EiJ (SPR) allelic PAS usages from 5,264 genes (see Methods). 2D clustering of Pearson correlation coefficients between all pairwise combinations of BL6 or SPR allelic gene expression levels from 5,264 genes. All clustering was performed hierarchically using Pearson correlations between samples.


Representative Ca2 + signals in sperm from a patient with deafness - infertility syndrome lacking functional CatSper channels ( CATSPER2 - / - ) ( red ) and a healthy donor ( black ) , evoked by progesterone , PGE1 , NH4Cl , or ionomycin . NH4Cl increases the intracellular pH . Bar graph : Amplitudes ( n = 4 ; mean ± SD ) of Ca2 + signals in CATSPER2 - / -

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

Representative Ca2+ signals in sperm from a patient with deafness-infertility syndrome lacking functional CatSper channels (CATSPER2-/-) (red) and a healthy donor (black), evoked by progesterone, PGE1, NH4Cl, or ionomycin. NH4Cl increases the intracellular pH. Bar graph: Amplitudes (n = 4; mean ± SD) of Ca2+ signals in CATSPER2-/- sperm


Representative monovalent CatSper currents in CATSPER2 - / - sperm ( blue , green , orange , purple , brown ) and in sperm from a healthy donor ( black ) and corresponding current - voltage relationship ( right ) . The membrane voltage was stepped from - 100 mV to + 100 mV in increments of 10 mV from a holding potential of - 80 mV . Outward and inward current amplitudes ( mean ± SD ) at + 100 mV and - 100 mV , respectively , in CATSPER2 - / - sperm ( color code : panel B ) and sperm from healthy donors ( black ) .

Representative monovalent CatSper currents in CATSPER2-/- sperm (blue, green, orange, purple, brown) and in sperm from a healthy donor (black) and corresponding current-voltage relationship (right). The membrane voltage was stepped from -100 mV to +100 mV in increments of 10 mV from a holding potential of -80 mV. Outward and inward current amplitudes (mean ± SD) at +100 mV and -100 mV, respectively, in CATSPER2-/- sperm (color code: panel B) and sperm from healthy donors (black).


Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2 - / - sperm form DIS patients using antibodies directed against CatSper 3 ( D ) or CatSper 4 ( E ) ; DNA was labelled with DAPI ( blue ) . Scale bars represent 10 µm .

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

Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2-/- sperm form DIS patients using antibodies directed against CatSper 3 (D) or CatSper 4 (E); DNA was labelled with DAPI (blue). Scale bars represent 10 µm.


3D - STORM image in xy - projection of sperm from a healthy donor labelled with the anti - CatSper 3 antibody ( left ) . Axial projection of the boxed region ( right ) . Scale bars represent 5 µm in xy - projections and 200 nm in axial projections .

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

3D-STORM image in xy-projection of sperm from a healthy donor labelled with the anti-CatSper 3 antibody (left). Axial projection of the boxed region (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.


3D - STORM images in xy - projection of CATSPER2 - / - sperm ( left ) labelled with the anti - CatSper 3 ( upper panel ) and anti - CatSper 4 ( lower panel ) antibody . Axial projection of the boxed regions ( right ) . Scale bars represent 5 µm in xy - projections and 200 nm in axial projections .

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

3D-STORM images in xy-projection of CATSPER2-/- sperm (left) labelled with the anti-CatSper 3 (upper panel) and anti-CatSper 4 (lower panel) antibody. Axial projection of the boxed regions (right). Scale bars represent 5 µm in xy-projections and 200 nm in axial projections.


Dark - field microscopy of a single sperm ; shown are single frames obtained at t = 0 , 48 , 96 , and 136 ms . Scale bar = 25

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

Dark-field microscopy of a single sperm; shown are single frames obtained at t = 0, 48, 96, and 136 ms. Scale bar = 25 µm


Dark - field imaging of a sperm population ; left : single frames at t = 185 , 363 , 540 , and 718 ms . Sperm selected for analysis are highlighted ( 1 - 4 ) . Right : Temporal change in the brightness ( blinking ) of sperm heads . The blue lines correspond to the time - points of the single frames . Scale bar = 25 µm .

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

Dark-field imaging of a sperm population; left: single frames at t = 185, 363, 540, and 718 ms. Sperm selected for analysis are highlighted (1-4). Right: Temporal change in the brightness (blinking) of sperm heads. The blue lines correspond to the time-points of the single frames. Scale bar = 25 µm.


Representative distribution of rotation frequencies of freely swimming non - capacitated ( 0 mM bicarbonate ; black ; n = 218 ) and capacitated sperm ( 25 mM bicarbonate ; red ; n = 232 ) determined by dark - field imaging .

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

Representative distribution of rotation frequencies of freely swimming non-capacitated (0 mM bicarbonate; black; n = 218) and capacitated sperm (25 mM bicarbonate; red; n = 232) determined by dark-field imaging.


Rolling frequency ( mean ± SD ) of non - capacitated ( 0 mM bicarbonate , n = 1 , 455 ) and capacitated sperm ( 25 mM bicarbonate , n = 1 , 097 ) .

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

Rolling frequency (mean ± SD) of non-capacitated (0 mM bicarbonate, n = 1,455) and capacitated sperm (25 mM bicarbonate, n = 1,097).


Rolling frequency ( mean ± SD ) of freely swimming sperm in 0 ( n = 1 , 175 ) , 0 . 2 ( n = 832 ) , and 1 % ( w / v ) ( n = 599 ) methyl cellulose . .

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

Rolling frequency (mean ± SD) of freely swimming sperm in 0 (n = 1,175), 0.2 (n = 832), and 1 % (w/v) (n = 599) methyl cellulose. .


Bright - field images of an optically trapped sperm cell , obtained at t = 0 , 55 , 155 , and 185 ms . Scale bar represents 10 µm .

Bright-field images of an optically trapped sperm cell, obtained at t = 0, 55, 155, and 185 ms. Scale bar represents 10 µm.


Representative time - course of the rotation frequency of trapped sperm ( each sperm cell is represented by a different color ) . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis . .

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

Representative time-course of the rotation frequency of trapped sperm (each sperm cell is represented by a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis..


Distribution of rotation frequencies in trapped sperm ( n = 32 ; mean frequency ± SD 6 . 0 ± 2 . 1 Hz ) .

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

Distribution of rotation frequencies in trapped sperm (n = 32; mean frequency ± SD 6.0 ± 2.1 Hz).


Image series of a sperm cell trapped parallel to the optical axis ; images were obtained at t = 0 , 95 , 165 , and 205 ms . The red bar indicates the 360 ° rotation of the tip of the head . Scale bar represents 10 µm .

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

Image series of a sperm cell trapped parallel to the optical axis; images were obtained at t = 0, 95, 165, and 205 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm.


Representative distribution of rotation frequencies of freely swimming CatSper - deficient sperm incubated under non - capacitating ( 0 mM bicarbonate ; black ; n = 73 ) and capacitating conditions ( 25 mM bicarbonate ; red ; n = 272 ) .

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

Representative distribution of rotation frequencies of freely swimming CatSper-deficient sperm incubated under non-capacitating (0 mM bicarbonate; black; n = 73) and capacitating conditions (25 mM bicarbonate; red; n = 272).


Rolling frequency ( mean ± SD ) of freely swimming CATSPER2 - / - sperm incubated under non - capacitating ( 0 mM bicarbonate , n = 1009 ) and capacitating ( 25 mM bicarbonate , n = 946 ) conditions .

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

Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm incubated under non-capacitating (0 mM bicarbonate, n = 1009) and capacitating (25 mM bicarbonate, n = 946) conditions.


Rolling frequency ( mean ± SD ) of freely swimming CATSPER2 - / - sperm in 0 ( n = 457 ) , 0 . 2 ( n = 389 ) , and 1 % ( w / v ) ( n = 187 ) methyl cellulose .

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

Rolling frequency (mean ± SD) of freely swimming CATSPER2-/- sperm in 0 (n = 457), 0.2 (n = 389), and 1 % (w/v) (n = 187) methyl cellulose.


Image series of a CATSPER2 - / - sperm cell optically trapped perpendicular to the optical axis ; images were obtained at t = 0 , 75 , 150 , and 225 ms . Scale bar represents 10 µm . Image series of a CATSPER2 - / - sperm cell optically trapped parallel to the optical axis ; images were obtained at t = 0 , 60 , 195 , and 320 ms . The red bar indicates the 360 ° rotation of the tip of the head . Scale bar represents 10 µm . Representative time courses of the rotation frequencies of optically trapped CATSPER2 - / - sperm ( each sperm is represented in a different color ) . Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis .

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

Image series of a CATSPER2-/- sperm cell optically trapped perpendicular to the optical axis; images were obtained at t = 0, 75, 150, and 225 ms. Scale bar represents 10 µm. Image series of a CATSPER2-/- sperm cell optically trapped parallel to the optical axis; images were obtained at t = 0, 60, 195, and 320 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 µm. Representative time courses of the rotation frequencies of optically trapped CATSPER2-/- sperm (each sperm is represented in a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis.


Rotation 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 .

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

Rotation 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 .

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

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 + .

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

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 ) .

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

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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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).


Left : 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 .

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

Left: 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.


( 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 ) .

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

(B-C) 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 .

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

(D-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.


Localization 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 .

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

Localization 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 .

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

Ams1, Lap2, and Ape2 did not co-immunoprecipitate with Ape4. Endogenously TAP-tagged Ape4 was immunoprecipitated. Ams1 was endogenously tagged with an mCherry tag.


The 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 .

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.


Surface 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 .

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

Surface 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 .

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

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.


The 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 .

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

The 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 .

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.


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


A . 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 .

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

A. 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 .

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

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 ) .

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

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 .

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

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 ) .

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

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 ) .

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

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 .

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

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.


A . 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 .

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

A. 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 .

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

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 .

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

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 ) .

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

D. 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 ) .

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

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).


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 . Quantification of YAP / TAZ subcellular staining is shown . Scale bar : 20 μm .

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

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. 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 .

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

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 .

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

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 .

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

C. 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 .

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

D. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. 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.


A . 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 .

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

A. 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 ) .

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

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 ) .

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

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 .

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

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).


A . 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 .

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

A. 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 .

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

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.


A . 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 .

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

A. 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 ) .

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

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

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).


TIGAR 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 .

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

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

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).


TIGAR 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 .

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

TIGAR 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 .

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

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 .

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

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 ) .

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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).


TIGAR 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 .

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

TIGAR 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 .

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

(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 .

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

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

(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).


( 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 .

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(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 .

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

(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 .

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

(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 .

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

(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.