Figure 4A - D . Representative maximum projections of confocal sections of ( A - B ) P14 and ( C - D ) P20 Wrb + / + : CreA and Wrbfl / fl : CreA apical turn organs of Corti following immunolabeling against otoferlin ( green ) and Vglut3 ( magenta ) processed and imaged under identical conditions . A ' - D ' show individual otoferlin stainings with an intensity - coded lookup table . A ' ' - D ' ' present single xz projections at a central point through a representative IHC to illustrate otoferlin subcellular distribution . Scale bars in A - D ' represent 10 μm , in A ' ' - D ' ' 2 µm . Note the dramatic reduction in otoferlin fluorescence and the alteration in its distribution as well as a change in cell shape and position of nuclei ( for detailed analysis of cell shape and nuclei position , please refer to Figure EV5 ) . E - G . Semiquantitative analysis of otoferlin immunofluorescence in the apical parts of IHCs from Wrbfl / fl : CreA mice using averaged regions of interests in cell ' s maximal projections ( outlined with a dotted line in G for a single representative IHC ) normalized to the maximum values observed in the respective control IHCs . Averaged supra nuclear coronal line profiles as illustrated with a dashed line in G are shown for ( F ) P14 - 16 and ( F ' ) P18 - 21 IHCs . * * * p < 0 . 001 versus Wrb + / + : CreAH - J . Semiquantitative analysis of otoferlin immunofluorescence in the basal parts of IHCs from Wrbfl / fl : CreA mice using averaged regions of interests in cell ' s maximal projections ( outlined with a dotted line in J for a single representative IHC ) normalized to the maximum values observed in the respective control IHCs . Averaged supranuclear coronal line profiles as illustrated with a dashed line in G are shown for ( I ) P14 - 16 and ( I ' ) P18 - 21 IHCs . * * * p < 0 . 001 versus Wrb + / + :

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

Figure 4A-D. Representative maximum projections of confocal sections of (A-B) P14 and (C-D) P20 Wrb+/+:CreA and Wrbfl/fl:CreA apical turn organs of Corti following immunolabeling against otoferlin (green) and Vglut3 (magenta) processed and imaged under identical conditions. A'-D' show individual otoferlin stainings with an intensity-coded lookup table. A''-D'' present single xz projections at a central point through a representative IHC to illustrate otoferlin subcellular distribution. Scale bars in A-D' represent 10 μm, in A''-D'' 2 µm. Note the dramatic reduction in otoferlin fluorescence and the alteration in its distribution as well as a change in cell shape and position of nuclei (for detailed analysis of cell shape and nuclei position, please refer to Figure EV5).E-G. Semiquantitative analysis of otoferlin immunofluorescence in the apical parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell's maximal projections (outlined with a dotted line in G for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supra nuclear coronal line profiles as illustrated with a dashed line in G are shown for (F) P14-16 and (F') P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreAH-J. Semiquantitative analysis of otoferlin immunofluorescence in the basal parts of IHCs from Wrbfl/fl:CreA mice using averaged regions of interests in cell's maximal projections (outlined with a dotted line in J for a single representative IHC) normalized to the maximum values observed in the respective control IHCs. Averaged supranuclear coronal line profiles as illustrated with a dashed line in G are shown for (I) P14-16 and (I') P18-21 IHCs. ***p<0.001 versus Wrb+/+:CreA


Figure 5A . Representative electron micrograph of a Wrb + / + : CreA IHC ribbon synapse . Scale bar : 120 nm . B - B ' . In Wrbfl / fl : CreA ( depicted are two consecutive ultrathin sections ) IHCs , accumulations of large partially amorphous vesicles were observed close to ribbon synapses ( red arrows ) , but also further away in the cytoplasm ( cyan arrows ) . Moreover , cisternal structures resembling ( r ) ER are found unusually close to the ribbon ( orange arrow in B ' ) . Scale bar : 120 nm . E . Quantification in random ultrathin sections ( Wrb + / + : CreA n = 28 , from two animals ; Wrbfl / fl : CreA n = 44 , from three animals ) of the numbers of membrane - proximal " MP " vesicles ( in close proximity to membrane and ribbon ) ; vesicles within a 100 nm range along the active zone membrane ; vesicles at the lower " proximal " and the upper " distal " half of the ribbon as well as vesicle > 70 nm ( " large vesicles " ) in a radius of 800 nm around the ribbon . The vesicle number was significantly reduced in Wrbfl / fl : CreA IHCs at both ribbon parts but not at the presynaptic membrane . F . Average vesicle diameters of the membrane - proximal vesicles and the vesicles at both halves of the ribbon . The mean vesicle diameter increases significantly at both parts of the ribbon in Wrbfl / fl : CreA IHCs . Data are represented as mean ± SEM . Student ' s two - sample t - test * * * p < 0 .

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

Figure 5A. Representative electron micrograph of a Wrb+/+:CreA IHC ribbon synapse. Scale bar: 120 nm.B-B'. In Wrbfl/fl:CreA (depicted are two consecutive ultrathin sections) IHCs, accumulations of large partially amorphous vesicles were observed close to ribbon synapses (red arrows), but also further away in the cytoplasm (cyan arrows). Moreover, cisternal structures resembling (r)ER are found unusually close to the ribbon (orange arrow in B'). Scale bar: 120 nm.E. Quantification in random ultrathin sections (Wrb+/+:CreA n = 28, from two animals; Wrbfl/fl:CreA n = 44, from three animals) of the numbers of membrane-proximal "MP" vesicles (in close proximity to membrane and ribbon); vesicles within a 100 nm range along the active zone membrane; vesicles at the lower "proximal" and the upper "distal" half of the ribbon as well as vesicle >70 nm ("large vesicles") in a radius of 800 nm around the ribbon. The vesicle number was significantly reduced in Wrbfl/fl:CreA IHCs at both ribbon parts but not at the presynaptic membrane.F. Average vesicle diameters of the membrane-proximal vesicles and the vesicles at both halves of the ribbon. The mean vesicle diameter increases significantly at both parts of the ribbon in Wrbfl/fl:CreA IHCs. Data are represented as mean ± SEM. Student's two-sample t-test ***p<0.001


Figure 6A . Ca2 + current - voltage relationship of P14 - P17 IHCs of Wrbfl / fl : CreA ( n = 17 IHCs ) and Wrb + / + : CreA ( n = 15 IHCs ) mice in 2 mM extracellular [ Ca2 + ] showed no change in amplitude or voltage - dependence of the Ca2 + current . Data are represented as mean ± SEM . B . Representative Ca2 + current ( top ) and Cm changes ( bottom ) in Wrbfl / fl : CreA and Wrb + / + : CreA IHCs in response to a 20 - ms step depolarizations to - 14 mV ( the potential eliciting the maximum Ca2 + current ) . C . Representative Ca2 + current ( top ) and Cm changes ( bottom ; finite impulse response filtered ) in Wrbfl / fl : CreA and Wrb + / + : CreA IHCs in response to a 100 ms step depolarizations to - 14 mV . D . Exocytic ΔCm ( top ) and corresponding Ca2 + current integrals , QCa ( bottom ) for various depolarization durations . Data are grand averages of the cells ' means . Exocytic ΔCm was reduced in the knockout from 20 ms onwards , indicating reduced sustained exocytosis . Data from OtofPga / Pga animals were replotted for direct comparison from ( Pangrsic et al , 2010 ) . E . ΔCm / QCa2 + ratio indicates a lower efficacy of Ca2 + influx in driving exocytosis in Wrbfl / fl : CreA IHCs , for stimuli of 20 ms or longer . Data represents means ± SEM ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 .

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

Figure 6A. Ca2+ current-voltage relationship of P14-P17 IHCs of Wrbfl/fl:CreA (n = 17 IHCs) and Wrb+/+:CreA (n = 15 IHCs) mice in 2 mM extracellular [Ca2+] showed no change in amplitude or voltage-dependence of the Ca2+ current. Data are represented as mean ± SEM.B. Representative Ca2+ current (top) and Cm changes (bottom) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 20-ms step depolarizations to -14 mV (the potential eliciting the maximum Ca2+ current).C. Representative Ca2+ current (top) and Cm changes (bottom; finite impulse response filtered) in Wrbfl/fl:CreA and Wrb+/+:CreA IHCs in response to a 100 ms step depolarizations to -14 mV.D. Exocytic ΔCm (top) and corresponding Ca2+ current integrals, QCa (bottom) for various depolarization durations. Data are grand averages of the cells' means. Exocytic ΔCm was reduced in the knockout from 20 ms onwards, indicating reduced sustained exocytosis. Data from OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).E. ΔCm/QCa2+ ratio indicates a lower efficacy of Ca2+ influx in driving exocytosis in Wrbfl/fl:CreA IHCs, for stimuli of 20 ms or longer. Data represents means ± SEM; *p<0.05; **p<0.01; ***p<0.001


Figure 7A . All SGNs of Wrbfl / fl : CreA mice had spontaneous firing rates ( SR ) below 20 Hz ( p > 0 . 05 , Kolmogorow - Smirnov test ) . B . Representative tuning curves from Wrbfl / fl : CreA and Wrb + / + : CreA SGNs indicating preserved active cochlear amplification despite disruption of Wrb in IHCs . C . Normal thresholds at the characteristic frequency ( CF ) in Wrbfl / fl : CreA SGNs . D - D ' ' . Mean poststimulus time histograms ( PSTH ) ± SEM of Wrbfl / fl : CreA and Wrb + / + : CreA SGNs in response to 50 ms tone bursts presented at CF , 30 dB above threshold at stimulus rates of 2 Hz ( D ) , 5 Hz ( D ' ) , or 10 Hz ( D ' ' ) . While the general response pattern was preserved , spike rates were drastically reduced in Wrbfl / fl : CreA SGNs , especially at higher stimulus rates . Data from auditory neurons ( SGN and cochlear nucleus ) of OtofPga / Pga animals were replotted for direct comparison from ( Pangrsic et al , 2010 ) . E . Spike rates in response to sound onset ( maximum rate in PSTH with 0 . 5 ms binwidth ) and adapted rates ( averaged between 35 - 45 ms after stimulus onset ) were significantly lower in Wrbfl / fl : CreA SGNs ( stimulus rate 5 Hz ; p < 0 . 001 ) . F . In line with the reduction in spike rates , first spike latency ( FSL ) following stimulus onset was greatly increased in Wrbfl / fl : CreA SGNs compared to Wrb + / + : CreA ( stimulus rate 5 Hz ; p < 0 . 001 ) . G . Illustration of the stimulus paradigm for forward masking experiments : a 100 ms masker tone presented at CF , 30 dB above threshold was followed by a silent interval of variable duration and a 15 ms probe tone ( CF , 30 dB above threshold ) . Bottom : in Wrbfl / fl : CreA SGNs , the response to the first 5 ms of the masker probe tone tone ( shown as a fraction of the response to the first 5ms of the masker ) was strongly reduced , and the half time of recovery ( from normalized curves ) was increased from 39 . 2 ± 12 . 2 ms in Wrb + / + : CreA to 215 . 7 ± 51 . 2 ms in Wrbfl / fl : CreA SGNs ( p < 0 . 001 , Mann - Whitney U test ) . Data represent means ± SEM .

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

Figure 7A. All SGNs of Wrbfl/fl:CreA mice had spontaneous firing rates (SR) below 20 Hz (p>0.05, Kolmogorow-Smirnov test).B. Representative tuning curves from Wrbfl/fl:CreA and Wrb+/+:CreA SGNs indicating preserved active cochlear amplification despite disruption of Wrb in IHCs.C. Normal thresholds at the characteristic frequency (CF) in Wrbfl/fl:CreA SGNs.D-D''. Mean poststimulus time histograms (PSTH) ± SEM of Wrbfl/fl:CreA and Wrb+/+:CreA SGNs in response to 50 ms tone bursts presented at CF, 30 dB above threshold at stimulus rates of 2 Hz (D), 5 Hz (D'), or 10 Hz (D''). While the general response pattern was preserved, spike rates were drastically reduced in Wrbfl/fl:CreA SGNs, especially at higher stimulus rates. Data from auditory neurons (SGN and cochlear nucleus) of OtofPga/Pga animals were replotted for direct comparison from (Pangrsic et al, 2010).E. Spike rates in response to sound onset (maximum rate in PSTH with 0.5 ms binwidth) and adapted rates (averaged between 35-45 ms after stimulus onset) were significantly lower in Wrbfl/fl:CreA SGNs (stimulus rate 5 Hz; p<0.001).F. In line with the reduction in spike rates, first spike latency (FSL) following stimulus onset was greatly increased in Wrbfl/fl:CreA SGNs compared to Wrb+/+:CreA (stimulus rate 5 Hz; p<0.001).G. Illustration of the stimulus paradigm for forward masking experiments: a 100 ms masker tone presented at CF, 30 dB above threshold was followed by a silent interval of variable duration and a 15 ms probe tone (CF, 30 dB above threshold). Bottom: in Wrbfl/fl:CreA SGNs, the response to the first 5 ms of the masker probe tone tone (shown as a fraction of the response to the first 5ms of the masker) was strongly reduced, and the half time of recovery (from normalized curves) was increased from 39.2 ± 12.2 ms in Wrb+/+:CreA to 215.7 ± 51.2 ms in Wrbfl/fl:CreA SGNs (p<0.001, Mann-Whitney U test). Data represent means ± SEM.


Figure 8C . Apparent decrease in insertion efficiency for the TMD mutant version . While the WT otoferlin glycosylation amounted to 3 . 84 ± 0 . 53 , the TMD mutant had only 2 . 13 ± 0 . 65 ( n = 3 , p < 0 . 05 ) . Data are represented as mean ± SEM .

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

Figure 8C. Apparent decrease in insertion efficiency for the TMD mutant version. While the WT otoferlin glycosylation amounted to 3.84 ± 0.53, the TMD mutant had only 2.13 ± 0.65 (n=3, p<0.05). Data are represented as mean ± SEM.


Figure 9A . Visual reinforcement audiometry performed in the free field : a mild hearing loss . B . Distortion product otoacoustic emissions ( DPOAEs ) were detected in each ear , whereasC . Auditory brainstem responses ( ABR ) were lacking , together providing the signature of auditory synaptopathy or neuropathy . D - E . Cochlear microphonic potentials ( CM ) , summating potential ( SP ) and compound action potential ( CAP ) recorded through transtympanic electrocochleography ( ECochG ) in response to clicks at decreasing stimulus intensities are superimposed on the corresponding potentials recorded from one normally - hearing control ( range as measured at 120 dB 4 . 31 - 28 . 02 µV in 20 normally - hearing children ) . Means and standard errors of peak latency and amplitude are also reported for each potential category . The ECochG waveform resulting from CM cancellation in the control begins with an abrupt negative deflection , the SP , followed by a negative peak , the neural CAP . In the child with the OTOF TMD mutation , the ECochG responses begin with a rapid negative deflection that peaks at the same latency as the SP in the control and has comparable amplitude . This is followed by a low - amplitude negative potential that peaks at the same CAP in controls but shows a markedly prolonged duration . In all graphs time " 0 " refers to CM onset . R : right , L : left . F . Means and standard errors of peak latency and amplitude are reported for each potential category .

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

Figure 9A. Visual reinforcement audiometry performed in the free field: a mild hearing loss.B. Distortion product otoacoustic emissions (DPOAEs) were detected in each ear, whereasC. Auditory brainstem responses (ABR) were lacking, together providing the signature of auditory synaptopathy or neuropathy.D-E. Cochlear microphonic potentials (CM), summating potential (SP) and compound action potential (CAP) recorded through transtympanic electrocochleography (ECochG) in response to clicks at decreasing stimulus intensities are superimposed on the corresponding potentials recorded from one normally-hearing control (range as measured at 120 dB 4.31-28.02 µV in 20 normally-hearing children). Means and standard errors of peak latency and amplitude are also reported for each potential category. The ECochG waveform resulting from CM cancellation in the control begins with an abrupt negative deflection, the SP, followed by a negative peak, the neural CAP. In the child with the OTOF TMD mutation, the ECochG responses begin with a rapid negative deflection that peaks at the same latency as the SP in the control and has comparable amplitude. This is followed by a low-amplitude negative potential that peaks at the same CAP in controls but shows a markedly prolonged duration. In all graphs time "0" refers to CM onset. R: right, L: left.F. Means and standard errors of peak latency and amplitude are reported for each potential category.


Figure 1A Immunostaining of a murine knee joint at 18 . 5 dpc with an anti GCP - 2 or IgG control antibody . B Immunohistochemistry for GCP - 2 ( red ) in the knee of a human 5 - month old fetus . C , D Immunostaining for GCP - 2 in an adult ( 12 week old ) mouse knee ( C ) or in adult human articular cartilage ( D ) .

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

Figure 1A Immunostaining of a murine knee joint at 18.5 dpc with an anti GCP-2 or IgG control antibody.B Immunohistochemistry for GCP-2 (red) in the knee of a human 5-month old fetus.C, D Immunostaining for GCP-2 in an adult (12 week old) mouse knee (C) or in adult human articular cartilage (D).


Figure 2GAG content as assessed by Alcian blue staining with densitometry ( A ) A HAC micromasses treated with a GCP - 2 neutralizing antibody ( n = 4 ) or IgG control ( n = 4 ) ; p values were determined by the Mann - Whitney U test . GAG content as assessed by Alcian blue staining with spectrophotometric B C28 / I2 micromasses treated with GCP - 2 siRNA ( n = 12 ) or scrambled control siRNA ( Scr ; n = 14 ) ; p values were determined by unpaired two - tailed Student ' s t test . GAG content as assessed by Alcian blue staining spectrophotometric C C3H10T½ micromasses treated for 3 days with recombinant GCP - 2 100 ng / ml ( n = 4 ) or vehicle ( n = 4 ) or BMP - 2 ( n = 4 ) ; p values were determined by ANOVA followed by Tukey HSD post hoc test . D qPCR for ACAN ( Aggrecan ) expression in C3H10T½ micromasses treated for 3 days with recombinant GCP - 2 ( 100 ng / ml ) ( n = 3 ) or vehicle ( n = 4 ) ; ACAN - Aggrecan ; E spectrophotometric quantification of GAG release ( dimethylmethylene blue assay ) in supernatant of human cartilage explants treated with recombinant GCP - 2 ( n = 5 ) or vehicle ( n = 5 ) and incubated for 24hF Toluidine blue staining of ectopic cartilage explants collected 2 weeks after subcutaneous co - implantation of HACs in a 10 : 1 ratio with growth - arrested COS7 expressing either GFP ( n = 3 ) or GCP - 2 ( n = 3 ) and quantification - on the right - hand side - of the metachromatically stained area as % of total area ; scale bar = 200µm ; G Collagen 2 ( Col2 ) immunostaining ( left ) and quantification ( right ) as % of total area ; scale bar = 100µm ; n = 3 per group ; p values were determined by unpaired two - tailed Student ' s t test .

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

Figure 2GAG content as assessed by Alcian blue staining with densitometry (A) A HAC micromasses treated with a GCP-2 neutralizing antibody (n=4) or IgG control (n=4); p values were determined by the Mann-Whitney U test.GAG content as assessed by Alcian blue staining with spectrophotometric B C28/I2 micromasses treated with GCP-2 siRNA (n=12) or scrambled control siRNA (Scr; n=14); p values were determined by unpaired two-tailed Student's t test.GAG content as assessed by Alcian blue staining spectrophotometric C C3H10T½ micromasses treated for 3 days with recombinant GCP-2 100 ng/ml (n=4) or vehicle (n=4) or BMP-2 (n=4); p values were determined by ANOVA followed by Tukey HSD post hoc test.D qPCR for ACAN (Aggrecan) expression in C3H10T½ micromasses treated for 3 days with recombinant GCP-2 (100 ng/ml) (n=3) or vehicle (n=4); ACAN - Aggrecan;E spectrophotometric quantification of GAG release (dimethylmethylene blue assay) in supernatant of human cartilage explants treated with recombinant GCP-2 (n=5) or vehicle (n=5) and incubated for 24hF Toluidine blue staining of ectopic cartilage explants collected 2 weeks after subcutaneous co-implantation of HACs in a 10:1 ratio with growth-arrested COS7 expressing either GFP (n=3) or GCP-2 (n=3) and quantification - on the right-hand side - of the metachromatically stained area as % of total area; scale bar =200µm;G Collagen 2 (Col2) immunostaining (left) and quantification (right) as % of total area; scale bar = 100µm; n = 3 per group; p values were determined by unpaired two-tailed Student's t test.


Figure 3A , B qPCR for ( A ) Runx2 and ( B ) Col1A1 expression in HAC micromasses treated for 24 hours with recombinant GCP - 2 ( 100 ng / ml ) or vehicle ( n = 4 ) ; Runx2 = Runt - related transcription factor 2 , Col1A1 = collagen type 1 alpha 1 ; n = 4C Alkaline phosphatase staining ( red ) in HAC monolayer after 3 week treatment with T3 hormone ( 100 ng / ml ) , T3 hormone + GCP - 2 ( 100ng / ml ) , or control medium ; scale bar 100µm . D quantification of alkaline phosphatase stained area ( n = 3 ) ; p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means . E qPCR for Col10A1 ( n = 3 ) of HAC 3 weeks after treatment with vehicle , T3 ( 100 ng / ml ) or T3 + GCP - 2 ( 100 ng / ml ) ; n = 3 ; F , H C3H10T½ monolayers were treated with osteogenic medium ( OM ) or OM + GCP - 2 ( 100 ng / ml ) : ( F ) number of cells per well positive for alkaline phosphatase staining ( n = 3 in OM and n = 4 in OM + GCP - 2 ) , ( G ) number of alkaline phosphatase positive nodules per well ( n = 7 ) , and ( H ) spectrophotometric quantification of alizarin red staining at 570nm ( n = 6 )

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

Figure 3A, B qPCR for (A) Runx2 and (B) Col1A1 expression in HAC micromasses treated for 24 hours with recombinant GCP-2 (100 ng/ml) or vehicle (n=4); Runx2 = Runt-related transcription factor 2, Col1A1 = collagen type 1 alpha 1; n=4C Alkaline phosphatase staining (red) in HAC monolayer after 3 week treatment with T3 hormone (100 ng/ml), T3 hormone + GCP-2 (100ng/ml), or control medium; scale bar 100µm.D quantification of alkaline phosphatase stained area (n=3); p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means.E qPCR for Col10A1 (n=3) of HAC 3 weeks after treatment with vehicle, T3 (100 ng/ml) or T3+GCP-2 (100 ng/ml); n=3;F, H C3H10T½ monolayers were treated with osteogenic medium (OM) or OM + GCP-2 (100 ng/ml): (F) number of cells per well positive for alkaline phosphatase staining (n=3 in OM and n=4 in OM+GCP-2), (G) number of alkaline phosphatase positive nodules per well (n=7), and (H) spectrophotometric quantification of alizarin red staining at 570nm (n=6)


Figure 4GAG binding capacity of wildtype GCP - 2 , and mutants ( K101E single mutant , K105E single mutant , GCP - 2 - D = K101E _ K105E double mutant and GCP - 2 - T = triple mutant K100E _ K101E _ K105E ) was assessed by ( B ) a heparin microtiter plate binding assay measuring the interaction of biotinylated - heparin ( b - heparin ) with immobilized proteins ( C ) heparin affinity chromatography equilibrated and run in PBS , pH 7 . 4 . In ( C ) the proteins are eluted with a salt gradient from 0 - 2 M NaCl monitored by the conductivity ( Cond ) , where 20 and 90 mS / cm correspond to 193 and 1360 mM NaCl , respectively ; GCP - 2 - T , GCP - 2 - D , K101E , K105E and WT proteins elute at 530 mM , 617 mM , 815 mM , 850 mM and 1024 mM NaCl , respectively . D , E Maximum migratory activity of WT GCP - 2 ( 50nM ) was compared to GCP - 2 mutants ( 50nM ) in ( D ) chemotaxis and ( E ) trans - endothelial migration assays in CXCR2 - expressing 300 - 19 pre B cells . WT = wildtype GCP - 2 ; K101E = GCP - 2 _ K101E single mutant ; K105E = GCP - 2 _ K105E single mutant ; D = GCP - 2 _ K101E _ K105E double mutant ; T = GCP - 2 _ K100E _ K101E _ K105E triple mutant ; n = 4 ; p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means . C28 / I2 micromasses stimulated with WT GCP - 2 , GCP - 2 triple mutant ( GCP - 2 - T ) or untreated and assessed for ( F ) AKT phosphorylation levels by western blotting ; V = vehicle treated samples , GCP - 2 = WT GCP - 2 treated samples , GCP - 2 - T = triple mutant GCP - 2 treated samples , pAKT = phosphorylated AKT , tAKT = total AKT . On the right , quantification of the density of the bands using ImageJ . Five independent experiments were analyzed . The data were scaled and p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means . C28 / I2 micromasses stimulated with WT GCP - 2 , GCP - 2 triple mutant ( GCP - 2 - T ) or untreated and assessed G spectrophotometric quantification of proteoglycan content by Alcian blue staining . p values were determined by Kruskal - Wallis test with Dunn ' s post - hoc test ; ( n = 8 ) . C28 / I2 micromasses stimulated with WT GCP - 2 , GCP - 2 triple mutant ( GCP - 2 - T ) or untreated and assessed H ACAN expression levels by qPCR ; ACAN = Aggrecan ; p values were determined by ANOVA with Tukey ' s HSD post - hoc test ; n = 4 . I - J Real time quantitative PCR of COL2A1 ( I ) and COL10A1 ( J ) mRNA normalized to β actin . p values were determined by fitting a generalised linear model followed by comparison of the estimated marginal means ; n = 4 control , 5 GCP - 2 and GCP - 2 - T for COL2A1 and n = 6 per group for COL10A1 . K Number of neutrophils counted within the intercondylar space on sections from mice killed 4 days after an intra - articular injection of adenovirus encoding GFP ( n = 3 ) , GCP - 2 ( n = 3 ) and GCP - 2 - T ( n = 3 ) ; p values were determined by fitting a generalised linear model ( family = poisson ) followed by pairwise comparison of the estimated marginal means .

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

Figure 4GAG binding capacity of wildtype GCP-2, and mutants (K101E single mutant, K105E single mutant, GCP-2-D = K101E_K105E double mutant and GCP-2-T = triple mutant K100E_ K101E_K105E) was assessed by (B) a heparin microtiter plate binding assay measuring the interaction of biotinylated-heparin (b-heparin) with immobilized proteins(C) heparin affinity chromatography equilibrated and run in PBS, pH 7.4. In (C) the proteins are eluted with a salt gradient from 0-2 M NaCl monitored by the conductivity (Cond), where 20 and 90 mS/cm correspond to 193 and 1360 mM NaCl, respectively; GCP-2-T, GCP-2-D, K101E, K105E and WT proteins elute at 530 mM, 617 mM, 815 mM, 850 mM and 1024 mM NaCl, respectively.D, E Maximum migratory activity of WT GCP-2 (50nM) was compared to GCP-2 mutants (50nM) in (D) chemotaxis and (E) trans-endothelial migration assays in CXCR2-expressing 300-19 pre B cells. WT = wildtype GCP-2; K101E = GCP-2_K101E single mutant; K105E = GCP-2_K105E single mutant; D = GCP-2_K101E_K105E double mutant; T = GCP-2_K100E_K101E_K105E triple mutant; n=4; p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means.C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed for (F) AKT phosphorylation levels by western blotting; V = vehicle treated samples, GCP-2 = WT GCP-2 treated samples, GCP-2-T = triple mutant GCP-2 treated samples, pAKT = phosphorylated AKT, tAKT = total AKT. On the right, quantification of the density of the bands using ImageJ. Five independent experiments were analyzed. The data were scaled and p values were determined by fitting a generalised linear model followed by pairwise comparison of the estimated marginal means.C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed G spectrophotometric quantification of proteoglycan content by Alcian blue staining. p values were determined by Kruskal-Wallis test with Dunn's post-hoc test; (n=8).C28/I2 micromasses stimulated with WT GCP-2, GCP-2 triple mutant (GCP-2-T) or untreated and assessed H ACAN expression levels by qPCR; ACAN = Aggrecan; p values were determined by ANOVA with Tukey's HSD post-hoc test; n=4.I-J Real time quantitative PCR of COL2A1 (I) and COL10A1 (J) mRNA normalized to β actin. p values were determined by fitting a generalised linear model followed by comparison of the estimated marginal means; n=4 control, 5 GCP-2 and GCP-2-T for COL2A1 and n=6 per group for COL10A1.K Number of neutrophils counted within the intercondylar space on sections from mice killed 4 days after an intra-articular injection of adenovirus encoding GFP (n= 3), GCP-2 (n=3) and GCP-2-T (n=3); p values were determined by fitting a generalised linear model (family=poisson) followed by pairwise comparison of the estimated marginal means.


Figure 5B Weekly pain measurement by incapacitance , shown as the percentage of bodyweight loaded on the operated leg in GFP ( n = 15 ) vs GCP - 2 ( n = 14 ) and GFP vs GCP - 2 - T ( n = 15 ) treated mice . Circles show the mean , and error bars show 95 % confidence intervals . C Incapacitance at week 10 ( final time - point ) ; red line indicates 50 % loading ( no pain ) ; p values were determined by unpaired , two - tailed one sample Student ' s t test testing the hypothesis that mice loaded 50 % body weight on the operated limb ( n = 13 in the GFP group and n = 14 in the GCP - 2 and GCP - 2 - T groups ) . D The area under the curve ( AUC ) of incapacitance calculated starting from week 6 ; p values were determined by ANOVA with Tukey ' s HSD post - hoc test ( n = 13 in the GFP group and 14 in the GCP - 2 and GCP - 2 - T group ) . E , F Osteoarthritis severity assessed using ( E ) OARSI scoring system 10 weeks after MLI ; GFP ( n = 7 ) , GCP - 2 ( n = 8 ) and GCP - 2 - T ( n = 7 ) ; p values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means . F representative image ( Safranin O staining ) for each treatment ; arrows indicate the tidemark ; G Quantification of osteophytes area ; GFP ( n = 7 ) , GCP - 2 ( n = 7 ) and GCP - 2 - T ( n = 8 ) ; H Representative images with osteophyte highlighted I Osteophyte maturity score ( n = 7 ) ; p values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means .

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

Figure 5B Weekly pain measurement by incapacitance, shown as the percentage of bodyweight loaded on the operated leg in GFP (n= 15) vs GCP-2 (n=14) and GFP vs GCP-2-T (n=15) treated mice. Circles show the mean, and error bars show 95% confidence intervals.C Incapacitance at week 10 (final time-point); red line indicates 50% loading (no pain); p values were determined by unpaired, two-tailed one sample Student's t test testing the hypothesis that mice loaded 50% body weight on the operated limb (n=13 in the GFP group and n=14 in the GCP-2 and GCP-2-T groups). D The area under the curve (AUC) of incapacitance calculated starting from week 6; p values were determined by ANOVA with Tukey's HSD post-hoc test (n=13 in the GFP group and 14 in the GCP-2 and GCP-2-T group).E, F Osteoarthritis severity assessed using (E) OARSI scoring system 10 weeks after MLI; GFP (n= 7), GCP-2 (n=8) and GCP-2-T (n=7); p values were determined by fitting a generalized linear model followed by comparison of the estimated marginal means. F representative image (Safranin O staining) for each treatment; arrows indicate the tidemark;G Quantification of osteophytes area; GFP (n= 7), GCP-2 (n=7) and GCP-2-T (n=8); H Representative images with osteophyte highlighted I Osteophyte maturity score (n=7); p values were determined by fitting a generalized linear model followed by pairwise comparison of the estimated marginal means.


figf1 ( a ) Ba / F3 cells expressing myrPI ( 3 ) K - ER , myrPKB - ER , FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT for 4 h ( myrPI ( 3 ) K - ER , myrPKB - ER ) or 8 h ( FOXO3 ( A3 ) - ER and FOXO4 ( A3 ) - ER ) and microarray analyses were performed . Shown are the fold changes relative to control cells for glutamine synthetase ( GS ) , Mxi1 and Pink1 . Data are represented as mean values of one experiment performed in quadruplicate . ( b ) Ba / F3 cells expressing either myrPI ( 3 ) K - ER or myrPKB - ER were cytokine starved overnight and stimulated with 4 - OHT . Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR . Data are represented as mean ± s . e . m . normalized for B2M ( n = 4 ) . * P0 . 05 , * * P0 . 01 . ( c ) Ba / F3 cells expressing either myrPI ( 3 ) K - ER or myrPKB - ER were cytokine starved overnight and stimulated with 4 - OHT . Cell lysates were analysed for protein levels of phospho - FOXO3 ( T32 ) , glutamine synthetase , p27 and actin . Shown are representative blots ( n = 4 ) . ( d ) Ba / F3 cells expressing either FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT in the presence of mIL - 3 . Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR . Data are represented as mean ± s . e . m . values normalized for B2M ( n = 3 ) . * * P0 . 01 . ( e ) Ba / F3 cells expressing either FOXO3 ( A3 ) - ER or FOXO4 ( A3 ) - ER were stimulated with 4 - OHT in the presence of mIL - 3 . Cell lysates were analysed for protein levels of glutamine synthetase , p27 and actin . Shown are representative blots ( n = 3 ) . ( f ) Wild - type Ba / F3 cells were cytokine starved overnight and stimulated with mIL - 3 . Cell lysates were analysed for protein levels of phospho - FOXO3 ( T32 ) , glutamine synthetase , p27 and actin . Shown are representative blots ( n = 4 ) . ( g ) Wild - type Ba / F3 cells were incubated with LY294002 in the presence of mIL - 3 . Cell lysates were analysed for protein levels of glutamine synthetase , p27 and actin . Shown are representative blots ( n = 3 ) . ( h ) FOXO1 , 3 , 4 − / − MEFs and wild - type MEFs were incubated with LY294002 ( 10 μM ) for the indicated times . Cell RNA was isolated and relative mRNA levels of glutamine synthetase were analysed using quantitative PCR . Data are represented as mean values normalized for B2M ( n = 2 ) . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf1(a) Ba/F3 cells expressing myrPI(3)K-ER, myrPKB-ER, FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT for 4 h (myrPI(3)K-ER, myrPKB-ER) or 8 h (FOXO3(A3)-ER and FOXO4(A3)-ER) and microarray analyses were performed. Shown are the fold changes relative to control cells for glutamine synthetase (GS), Mxi1 and Pink1. Data are represented as mean values of one experiment performed in quadruplicate.(b) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ± s.e.m. normalized for B2M (n=4). *P0.05, **P0.01.(c) Ba/F3 cells expressing either myrPI(3)K-ER or myrPKB-ER were cytokine starved overnight and stimulated with 4-OHT. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).(d) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Relative mRNA levels of glutamine synthetase were analysed using quantitative rtPCR. Data are represented as mean ±  s.e.m. values normalized for B2M (n=3). **P0.01.(e) Ba/F3 cells expressing either FOXO3(A3)-ER or FOXO4(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).(f) Wild-type Ba/F3 cells were cytokine starved overnight and stimulated with mIL-3. Cell lysates were analysed for protein levels of phospho-FOXO3 (T32), glutamine synthetase, p27 and actin. Shown are representative blots (n=4).(g) Wild-type Ba/F3 cells were incubated with LY294002 in the presence of mIL-3. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=3).(h) FOXO1,3,4−/− MEFs and wild-type MEFs were incubated with LY294002 (10 μM) for the indicated times. Cell RNA was isolated and relative mRNA levels of glutamine synthetase were analysed using quantitative PCR. Data are represented as mean values normalized for B2M (n=2). Uncropped images of blots are shown in Supplementary Fig. S7.


figf2 ( a ) Ba / F3 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT and actinomycin D ( 1 μg ml − 1 ) or dimethylsulphoxide ( DMSO ) as a control in the presence of mIL - 3 for 16 h . Cell lysates were analysed for protein levels of glutamine synthetase ( GS ) , p27 and actin . Shown are representative blots ( n = 3 ) . ( b ) Glutamine synthetase reporter plasmids carrying mutations in putative FOXO - binding sites were transfected in HEK293 cells together with Renilla and FOXO3 ( A3 ) as indicated . Luciferase activity was measured 40 h after transfection . Data are depicted as relative luciferase units ( RLU ) compared with the control . Shown are mean ± s . e . m . values ( n = 3 ) ( a ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - ULK2 and stimulated with 4 - OHT and MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . Cells were stained for LC3 and analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm . ( d ) DLD1 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT . Cell lysates were analysed for protein levels of glutamine synthetase , p27 and actin . Shown are representative blots ( n = 4 ) . ( e ) The osteosarcoma cell line U2OS expressing FOXO ( A3 ) - ER was stimulated with 4 - OHT , cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin . ( f ) MSCs expressing FOXO ( A3 ) - ER were stimulated with 4 - OHT , cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin . Shown are representative blots ( n = 3 ) . ( g ) Wild - type , daf - 2 and daf - 16 mutant worms were synchronized to L1 by hypochlorite treatment and were placed on nematode growth medium agar plates . After five days worms were lysed in imidazole and analysed for glutamine synthetase activity . ( h ) Wild - type N2 worms or daf - 2 mutants were synchronized to L1 by hypochlorite treatment and placed on nematode growth medium plates with or without bacteria expressing DAF - 16 double - stranded RNA . After five days worms were lysed in imidazole and analysed for glutamine synthetase activity . ( g , h ) Shown are the means of five independent plates for each condition . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf2(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and actinomycin D (1 μg ml−1) or dimethylsulphoxide (DMSO) as a control in the presence of mIL-3 for 16 h. Cell lysates were analysed for protein levels of glutamine synthetase (GS), p27 and actin. Shown are representative blots (n=3).(b) Glutamine synthetase reporter plasmids carrying mutations in putative FOXO-binding sites were transfected in HEK293 cells together with Renilla and FOXO3(A3) as indicated. Luciferase activity was measured 40 h after transfection. Data are depicted as relative luciferase units (RLU) compared with the control. Shown are mean ± s.e.m. values (n=3)(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm.(d) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT. Cell lysates were analysed for protein levels of glutamine synthetase, p27 and actin. Shown are representative blots (n=4).(e) The osteosarcoma cell line U2OS expressing FOXO(A3)-ER was stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin.(f) MSCs expressing FOXO(A3)-ER were stimulated with 4-OHT, cells were lysed and equal amounts of proteins were analysed for levels of glutamine synthetase and actin. Shown are representative blots (n=3).(g) Wild-type, daf-2 and daf-16 mutant worms were synchronized to L1 by hypochlorite treatment and were placed on nematode growth medium agar plates. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity.(h) Wild-type N2 worms or daf-2 mutants were synchronized to L1 by hypochlorite treatment and placed on nematode growth medium plates with or without bacteria expressing DAF-16 double-stranded RNA. After five days worms were lysed in imidazole and analysed for glutamine synthetase activity. (g,h) Shown are the means of five independent plates for each condition. Uncropped images of blots are shown in Supplementary Fig. S7.


figf3 ( a ) Ba / F3 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT in the presence of mIL - 3 . Cell lysates were analysed for glutamine synthetase ( GS ) activity in an enzyme assay ( left ) . In addition , the expression of glutamine synthetase , p27 and actin was determined using western blot ( right ) . Shown are the mean ± s . e . m . ( n = 3 ) and representative blots of these experiments . * * P0 . 01 . ( b ) Ba / F3 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT for 16 h in the presence of mIL - 3 together with MSO , as indicated . Cell lysates were analysed for glutamine synthetase activity in an enzyme assay ( left ) . Furthermore the expression levels of glutamine synthetase , p27 and actin were determined using western blot ( right ) . Shown are the mean ± s . e . m . ( n = 3 ) and representative blots of these experiments . * P0 . 05 , * * P0 . 01 . ( c ) Ba / F3 cells expressing myrPI ( 3 ) K - ER were cytokine - starved overnight . The next day , cells were washed in PBS and put in a medium without serum , with or without 4 - OHT . At the times indicated , medium samples were taken and analysed for amino acid levels by high - performance liquid chromatography . Cells were lysed and equal amounts of protein were analysed by western blotting for levels of phospho - PKB ( S473 ) , phospho - FOXO3 ( T32 ) and actin ( inset ) . Shown are the mean of relative amino acid levels , compared with t = 0 ( n = 2 ) , and representative blots of these experiments . ( d ) Ba / F3 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT in serum - free medium containing mIL - 3 . The medium was analysed for amino acid levels by high - performance liquid chromatography . Cells were lysed and analysed for protein levels of glutamine synthetase , p27 and actin ( inset ) . Shown are the mean ± s . e . m . of relative amino acid levels , compared with t = 0 ( n = 4 ) , and representative blots of these experiments . * * P0 . 01 . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

figf3(a) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in the presence of mIL-3. Cell lysates were analysed for glutamine synthetase (GS) activity in an enzyme assay (left). In addition, the expression of glutamine synthetase, p27 and actin was determined using western blot (right). Shown are the mean ± s.e.m. (n=3) and representative blots of these experiments. **P0.01.(b) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT for 16 h in the presence of mIL-3 together with MSO, as indicated. Cell lysates were analysed for glutamine synthetase activity in an enzyme assay (left). Furthermore the expression levels of glutamine synthetase, p27 and actin were determined using western blot (right). Shown are the mean ±  s.e.m. (n=3) and representative blots of these experiments. *P0.05, **P0.01.(c) Ba/F3 cells expressing myrPI(3)K-ER were cytokine-starved overnight. The next day, cells were washed in PBS and put in a medium without serum, with or without 4-OHT. At the times indicated, medium samples were taken and analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and equal amounts of protein were analysed by western blotting for levels of phospho-PKB (S473), phospho-FOXO3 (T32) and actin (inset). Shown are the mean of relative amino acid levels, compared with t=0 (n=2), and representative blots of these experiments.(d) Ba/F3 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT in serum-free medium containing mIL-3. The medium was analysed for amino acid levels by high-performance liquid chromatography. Cells were lysed and analysed for protein levels of glutamine synthetase, p27 and actin (inset). Shown are the mean ±  s.e.m. of relative amino acid levels, compared with t=0 (n=4), and representative blots of these experiments. **P0.01. Uncropped images of blots are shown in Supplementary Fig. S7.


figf4 ( a ) DLD1 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT with or without MSO . After 24 h , the cells were starved of serum , amino acids and glucose in D - PBS containing the indicated inhibitors and stimulated with amino acids for 10 min . Cell lysates were analysed for protein levels of phospho - S6K ( Thr 389 ) and actin . Shown are representative blots of three independent experiments . ( b , c ) DLD1 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT and MSO ( 0 . 25 M ) for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy . ( b ) Shown are representative pictures ( n = 4 ) . Scale bars , 100 μm . ( c ) Quantification of co - localization between mTOR and LAMP2 as seen in b . Depicted are the mean ± s . e . m . of the percentage of co - localized pixels from four experiments . * P0 . 05 , * * P0 . 01 . ( d ) DLD1 cells expressing FOXO3 ( A3 ) - ER were treated with or without 4 - OHT , MSO and BafA1 ( 200 nM ) . After 24 h , cell lysates were analysed for protein levels of LC3 and tubulin . Shown are representative blots ( n = 4 ) . ( e ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transfected with glutamine synthetase ( GS ) siRNA or a non - targeting siRNA and stimulated with 4 - OHT in glutamine - free DMEM containing 0 . 1 % FBS . After 24 h , cell lysates were analysed for protein levels of glutamine synthetase , LC3 and actin . Shown are representative blots ( n = 2 ) ( f ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transfected with glutamine synthetasesiRNA or non - template ( NT ) control siRNA and stimulated with 4 - OHT for 16 h . Cells were lysed and equal amounts of proteins were analysed for levels of p62 or glutamine synthetase . Shown are representative blots ( n = 2 ) . ( g ) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h . Cell lysates were analysed for protein levels of LC3 and actin . Shown are representative blots ( n = 4 ) . Uncropped images of blots are shown in Supplementary Figs S7 and S8 .

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

figf4(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT with or without MSO. After 24 h, the cells were starved of serum, amino acids and glucose in D-PBS containing the indicated inhibitors and stimulated with amino acids for 10 min. Cell lysates were analysed for protein levels of phospho-S6K (Thr 389) and actin. Shown are representative blots of three independent experiments.(b,c) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO (0.25 M) for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy. (b) Shown are representative pictures (n=4). Scale bars, 100 μm. (c) Quantification of co-localization between mTOR and LAMP2 as seen in b. Depicted are the mean ±  s.e.m. of the percentage of co-localized pixels from four experiments. *P0.05, **P0.01.(d) DLD1 cells expressing FOXO3(A3)-ER were treated with or without 4-OHT, MSO and BafA1 (200 nM). After 24 h, cell lysates were analysed for protein levels of LC3 and tubulin. Shown are representative blots (n=4).(e) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetase (GS) siRNA or a non-targeting siRNA and stimulated with 4-OHT in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of glutamine synthetase, LC3 and actin. Shown are representative blots (n=2)(f) DLD1 cells expressing FOXO3(A3)-ER were transfected with glutamine synthetasesiRNA or non-template (NT) control siRNA and stimulated with 4-OHT for 16 h. Cells were lysed and equal amounts of proteins were analysed for levels of p62 or glutamine synthetase. Shown are representative blots (n=2).(g) DLD1 cells were stimulated with LY294002 or PKB inhibitor VIII for 24 h. Cell lysates were analysed for protein levels of LC3 and actin. Shown are representative blots (n=4). Uncropped images of blots are shown in Supplementary Figs S7 and S8.


figf5 ( a ) DLD1 cells expressing FOXO3 ( A3 ) - ER were stimulated with 4 - OHT and MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . Cells were stained for LC3 and ER and analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm . ( b ) Quantification of LC3 - positive spots as seen in a using ImageJ software . Depicted are mean ± s . e . m . of the number of LC3 - positive spots divided by the number of DAPI - positive cells ( n = 4 ) . * P0 . 05 . ( c ) MSCs expressing FOXO3 ( A3 ) - ER were treated with or without 4 - OHT and BafA1 ( 100 nM ) . At the indicated time points , cell lysates were analysed for protein levels of glutamine synthetase ( GS ) , LC3 , pS6 ( Ser 235 / 236 ) and actin . Shown are representative blots ( n = 2 ) . ( d ) IL - 3 was withdrawn from Ba / F3 cells in the presence or absence of BafA1 ( 100 nM ) as indicated . Cell lysates were analysed for protein levels of glutamine synthetase , LC3 and hsp90 . Shown are representative blots ( n = 2 ) . ( e ) DLD1 cells were stimulated with l - glutamine ( l - Gln ) in glutamine - free DMEM containing 0 . 1 % FBS . After 24 h , cell lysates were analysed for protein levels of LC3 , p62 , p - S6K ( Thr 389 ) and actin . Shown are representative blots ( n = 3 ) . ( f ) DLD1 cells were starved and stimulated with glutamine ( 5 and 10 mM ) . Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy , and the co - localization between mTOR and LAMP2 was quantified by ImageJ . Depicted are the means of the percentage of co - localized pixels ( n = 2 ) . ( g ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - WIPI - 1 and subsequently stimulated with 4 - OHT in the presence or absence of MSO in glutamine - free DMEM containing 0 . 1 % FCS or medium without amino acids for 24 h . GFP - WIPI - 1 puncta formation analysis was performed by confocal microscopy . Depicted are the percentages of cells positive for GFP - WIPI - 1 puncta . Shown are the mean ± s . e . m . ( n = 4 ) . * P0 . 05 and * * P0 . 01 . Uncropped images of blots are shown in Supplementary Fig . S8 .

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

figf5(a) DLD1 cells expressing FOXO3(A3)-ER were stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and ER and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm. (b) Quantification of LC3-positive spots as seen in a using ImageJ software. Depicted are mean ±  s.e.m. of the number of LC3-positive spots divided by the number of DAPI-positive cells (n=4). *P0.05.(c) MSCs expressing FOXO3(A3)-ER were treated with or without 4-OHT and BafA1 (100 nM). At the indicated time points, cell lysates were analysed for protein levels of glutamine synthetase (GS), LC3, pS6 (Ser 235/236) and actin. Shown are representative blots (n=2).(d) IL-3 was withdrawn from Ba/F3 cells in the presence or absence of BafA1 (100 nM) as indicated. Cell lysates were analysed for protein levels of glutamine synthetase, LC3 and hsp90. Shown are representative blots (n=2).(e) DLD1 cells were stimulated with l-glutamine (l-Gln) in glutamine-free DMEM containing 0.1% FBS. After 24 h, cell lysates were analysed for protein levels of LC3, p62, p-S6K (Thr 389) and actin. Shown are representative blots (n=3).(f) DLD1 cells were starved and stimulated with glutamine (5 and 10 mM). Cells were stained for mTOR and LAMP2 and analysed by confocal microscopy, and the co-localization between mTOR and LAMP2 was quantified by ImageJ. Depicted are the means of the percentage of co-localized pixels (n=2).(g) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-WIPI-1 and subsequently stimulated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS or medium without amino acids for 24 h. GFP-WIPI-1 puncta formation analysis was performed by confocal microscopy. Depicted are the percentages of cells positive for GFP-WIPI-1 puncta. Shown are the mean ±  s.e.m. (n=4). *P0.05 and **P0.01. Uncropped images of blots are shown in Supplementary Fig. S8.


figf6 ( a ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - ULK2 and stimulated with 4 - OHT and MSO for 24 h in glutamine - free DMEM containing 0 . 1 % FCS . Cells were stained for LC3 and analysed by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bars , 20 μm . ( b ) DLD1 cells expressing FOXO3 ( A3 ) - ER were transiently transfected with GFP - WIPI - 1 and subsequently treated with 4 - OHT in the presence or absence of MSO in glutamine - free DMEM containing 0 . 1 % FCS . After 24 h , cells were analysed for GFP and p62 expression by confocal microscopy . Shown are representative pictures ( n = 3 ) . Scale bar , 20 μm . ( c ) DLD1 cells expressing FOXO3 ( A3 ) - ER were treated with 4 - OHT and MSO in glutamine - free DMEM containing 0 . 1 % FCS . After 24 h cells were analysed for endogenous Atg12 puncta formation by confocal microscopy . Shown are the mean ± s . d . ( n = 3 ) . * P0 . 05 . ( d ) DLD1 cells expressing FOXO ( A3 ) - ER were transfected with glutamine synthetase ( GS ) siRNA or non - template ( NT ) control siRNA , and stimulated with 4 - OHT for 24 h with or without 3 - methyladenine ( 3 - MA ) . Apoptosis was determined by FACS analysis after labelling cells with annexin V ( AxV ) - phycoerythrin and DAPI . The graph shows relative fold versus control samples , mean ± s . e . m . ( n = 3 ) . * P0 . 05 .

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

figf6(a) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-ULK2 and stimulated with 4-OHT and MSO for 24 h in glutamine-free DMEM containing 0.1% FCS. Cells were stained for LC3 and analysed by confocal microscopy. Shown are representative pictures (n=3). Scale bars, 20 μm.(b) DLD1 cells expressing FOXO3(A3)-ER were transiently transfected with GFP-WIPI-1 and subsequently treated with 4-OHT in the presence or absence of MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h, cells were analysed for GFP and p62 expression by confocal microscopy. Shown are representative pictures (n=3). Scale bar, 20 μm.(c) DLD1 cells expressing FOXO3(A3)-ER were treated with 4-OHT and MSO in glutamine-free DMEM containing 0.1% FCS. After 24 h cells were analysed for endogenous Atg12 puncta formation by confocal microscopy. Shown are the mean ± s.d. (n=3). *P0.05.(d) DLD1 cells expressing FOXO(A3)-ER were transfected with glutamine synthetase (GS) siRNA or non-template (NT) control siRNA, and stimulated with 4-OHT for 24 h with or without 3-methyladenine (3-MA). Apoptosis was determined by FACS analysis after labelling cells with annexin V (AxV)-phycoerythrin and DAPI. The graph shows relative fold versus control samples, mean ±  s.e.m. (n=3). *P0.05.


Figure 1A - E Scatter plots showing the association of each of the p - tau biomarkers with age in the Aβ - negative ( A - ; blue ; n = 250 ) and the Aβ - positive ( A + ; red ; n = 131 ) groups . The solid lines indicate the regression line and the 95 % confidence intervals for each of the groups . For each group , the standardized regression coefficients ( β ) and the P - values were computed using a linear model adjusting for sex . Additionally , we computed the ' Age x Aβ status ' interaction term . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; N , N - terminal ; p - tau , phosphorylated tau .

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

Figure 1A-E Scatter plots showing the association of each of the p-tau biomarkers with age in the Aβ-negative (A-; blue; n = 250) and the Aβ-positive (A+; red; n = 131) groups. The solid lines indicate the regression line and the 95% confidence intervals for each of the groups. For each group, the standardized regression coefficients (β) and the P-values were computed using a linear model adjusting for sex. Additionally, we computed the 'Age x Aβ status' interaction term. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; p-tau, phosphorylated tau.


Figure 2 ( A , B , C , D , E ) Dot and box - plot comparing each of the p - tau biomarker between the Aβ - negative ( A - ; blue ; n = 250 ) and the Aβ - positive ( A + ; red ; n = 131 ) groups . Aβ positivity was defined as CSF Aβ42 / 40 ratio < 0 . 071 . ( F , G , H , I , J ) Scatter plots depicting the changes between each p - tau biomarker as a function of CSF Aβ42 / 40 . The horizontal - axes directions were inverted ; lower CSF Aβ42 / 40 ratio reflects higher Aβ pathology . For each Aβ status group , we computed the standardized regression coefficients ( β ) and the P - values , adjusted for age and sex . The solid lines indicate the regression line and the 95 % confidence intervals for each of the Aβ status groups . The dashed green lines indicate the CSF Aβ42 / 40 cutoff . Participants were also colour - coded based on the Aβ PET CL scale ( ≤ 12CL , black ; > 12CL , turquoise ; Aβ PET non - available , grey ) .

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

Figure 2(A, B, C, D, E) Dot and box-plot comparing each of the p-tau biomarker between the Aβ-negative (A-; blue; n = 250) and the Aβ-positive (A+; red; n =131) groups. Aβ positivity was defined as CSF Aβ42/40 ratio < 0.071.(F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of CSF Aβ42/40. The horizontal-axes directions were inverted; lower CSF Aβ42/40 ratio reflects higher Aβ pathology. For each Aβ status group, we computed the standardized regression coefficients (β) and the P-values, adjusted for age and sex. The solid lines indicate the regression line and the 95% confidence intervals for each of the Aβ status groups. The dashed green lines indicate the CSF Aβ42/40 cutoff. Participants were also colour-coded based on the Aβ PET CL scale (≤12CL, black; >12CL, turquoise; Aβ PET non-available, grey).


Figure 3 ( A , B , C , D , E ) Dot and box - plot comparing each of the p - tau biomarker between the Aβ negative ( A - ; blue ; n = 287 ) and the Aβ positive ( A + ; red ; n = 42 ) groups . Aβ positivity was defined with Aβ PET visual read . ( F , G , H , I , J ) Scatter plots depicting the changes between each p - tau biomarker as a function of Aβ PET Centiloids ( CL ) . The standardized regression coefficients ( β ) and the P - values were computed using a linear model adjusting for age and sex . The solid lines indicate the regression line and the 95 % confidence intervals . The dashed green lines indicate the CL12 and CL30 cutoffs . Participants were also colour - coded based on the CSF Aβ42 / 40 ratio ( A - , black ; A + , turquoise ) . ( K , L , M , N , O ) Dot and box - plots depicting comparison between each of the p - tau biomarker between Centiloid scale groups : ( i ) ≤ 12CL ( blue ; n = 278 ) , ( ii ) 12 - 30CL ( subthreshold Aβ pathology group ; grey ; n = 28 ) , ( iii ) > 30CL ( red ; n = 25 ) . The box - plots depict the median ( horizontal bar ) , interquartile range ( IQR , hinges ) and 1 . 5 x IQR ( whiskers ) .

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

Figure 3(A, B, C, D, E) Dot and box-plot comparing each of the p-tau biomarker between the Aβ negative (A-; blue; n = 287) and the Aβ positive (A+; red; n = 42) groups. Aβ positivity was defined with Aβ PET visual read.(F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of Aβ PET Centiloids (CL). The standardized regression coefficients (β) and the P-values were computed using a linear model adjusting for age and sex. The solid lines indicate the regression line and the 95% confidence intervals. The dashed green lines indicate the CL12 and CL30 cutoffs. Participants were also colour-coded based on the CSF Aβ42/40 ratio (A-, black; A+, turquoise).(K, L, M, N, O) Dot and box-plots depicting comparison between each of the p-tau biomarker between Centiloid scale groups: (i) ≤12CL (blue; n = 278), (ii) 12 - 30CL (subthreshold Aβ pathology group; grey; n = 28), (iii) >30CL (red; n = 25). The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers).


Figure 4A - C ROC analysis was performed to test the accuracy to discriminate between Aβ - positive ( A + ) from Aβ - negative ( A - ) individuals . Aβ positivity was defined as CSF Aβ42 / 40 < 0 . 071 ( A ) , Aβ PET positive visual read ( B ) or Aβ PET Centiloid ( CL ) > 12 ( C ) . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; NfL , neurofilament light ; N , N - terminal ; p - tau , phosphorylated tau ; t - tau , total tau .

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

Figure 4A-C ROC analysis was performed to test the accuracy to discriminate between Aβ-positive (A+) from Aβ-negative (A-) individuals. Aβ positivity was defined as CSF Aβ42/40 < 0.071 (A), Aβ PET positive visual read (B) or Aβ PET Centiloid (CL) > 12 (C). Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.


Figure 5A - D Dot and box - plot showing the levels of each p - tau biomarker in each of the AT groups . Aβ - positive ( A + ) was defined by a CSF Aβ42 / 40 < 0 . 071 and Tau - positive ( T + ) by an Elecsys CSF Mid - p - tau181 > 24 pg / ml . The box - plots depict the median ( horizontal bar ) , interquartile range ( IQR , hinges ) and 1 . 5 x IQR ( whiskers ) . The horizontal dashed line indicates the median of the p - tau biomarker in the A - T - group . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; N , N - terminal ; p - tau , phosphorylated tau .

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

Figure 5A-D Dot and box-plot showing the levels of each p-tau biomarker in each of the AT groups. Aβ-positive (A+) was defined by a CSF Aβ42/40 < 0.071 and Tau-positive (T+) by an Elecsys CSF Mid-p-tau181 > 24 pg/ml. The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers). The horizontal dashed line indicates the median of the p-tau biomarker in the A-T- group. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; p-tau, phosphorylated tau.


Figure 6The graphs represent the z - scores changes of each CSF biomarker as a function of CSF Aβ42 / 40 ratio ( as proxy of disease progression ) using a robust local weighted regression method . The z - scores were calculated using the mean and the SD of each CSF biomarker in the A - T - group as a reference . The solid lines depict the trajectory of each CSF biomarker . The dashed lines depict the trajectories of the plasma biomarkers . The vertical black dashed line indicates the CSF Aβ42 / 40 cutoff for A + . Note that the CSF p - tau biomarkers reach the 2 z - scores ( depicted with an horizontal dashed line ) with the following sequence : Mid - p - tau231 , N - p - tau181 , N - p - tau217 , Mid - p - tau181 and t - tau . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; NfL , Neurofilament light ; N , N - terminal ; p - tau , phosphorylated tau ; t - tau , total tau .

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

Figure 6The graphs represent the z-scores changes of each CSF biomarker as a function of CSF Aβ42/40 ratio (as proxy of disease progression) using a robust local weighted regression method. The z-scores were calculated using the mean and the SD of each CSF biomarker in the A-T- group as a reference. The solid lines depict the trajectory of each CSF biomarker. The dashed lines depict the trajectories of the plasma biomarkers. The vertical black dashed line indicates the CSF Aβ42/40 cutoff for A+. Note that the CSF p-tau biomarkers reach the 2 z-scores (depicted with an horizontal dashed line) with the following sequence: Mid-p-tau231, N-p-tau181, N-p-tau217, Mid-p-tau181 and t-tau. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, Neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.


Figure 1E . Ectopic expression of CG14044 in the wing pouch ( GFP area indicated by the dotted line ) induces caspase activation and DNA fragmentation ( TUNEL ) in the larval disc , melanization in the pupal wing ( arrow head ) and a structure defect in the adult wing . Data information : Scale bars , 100 μm in E ( cleaved caspase ) and 20 μm in E ( TUNEL ) and 500 μm in E ( pupae and adult wing ) . H . Deletion of the BH3 motif suppresses Sayonara - induced caspase activation , which was detected by the caspase sensor GC3Ai . I . Quantification of GC3Ai positive areas with Synr WT and the BH3 motif deletion mutant . Data information : Statistical significance was determined using one - way ANOVA with Dunnett ' s post hoc test . Scale bars , 100 μm

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

Figure 1E. Ectopic expression of CG14044 in the wing pouch (GFP area indicated by the dotted line) induces caspase activation and DNA fragmentation (TUNEL) in the larval disc, melanization in the pupal wing (arrow head) and a structure defect in the adult wing. Data information: Scale bars, 100 μm in E (cleaved caspase) and 20 μm in E (TUNEL) and 500 μm in E (pupae and adult wing).H. Deletion of the BH3 motif suppresses Sayonara-induced caspase activation, which was detected by the caspase sensor GC3Ai. I. Quantification of GC3Ai positive areas with Synr WT and the BH3 motif deletion mutant. Data information: Statistical significance was determined using one-way ANOVA with Dunnett's post hoc test. Scale bars, 100 μm in


Figure 2A - D . The Synr - induced wing structural defect is suppressed by knockdown of Debcl , Buffy or DCP1 . Data information : Scale bars , 500 μm in AF . Synr - induced caspase activation is suppressed by knockdown of Debcl . G . GC3Ai signals are suppressed by knockdown of Debcl or Buffy Data information : Scale bars , 50 μm in F and GH - I . Lysates from HEK293T cells that express Flag - Debcl or Flag - Buffy were incubated with GST or GST - Synr bound to glutathione - Sepharose . Flag - Debcl or Flag - Buffy is pulled down with GST - Synr but not with GST . J . Deletion of neither the coiled coil domain nor the BH3 motif suppresses binding Flag - Debcl to GST - Synr . O . Debcl can be photo - crosslinked with Synr at BPA that corresponds to Y124 and F141 . BPA at Y340 was used as a negative control . Red arrowheads indicate covalently crosslinked products . P . Buffy is also photo - crosslinked with Synr at BPA that corresponds to Y124 and F141 . Red arrowheads indicate covalently crosslinked products . R . HA - Debcl is detected after two series of immunoprecipitation . The first immunoprecipitation was performed for Flag - Buffy , followed by Flag peptide - mediated elution and secondary precipitation for Myc - Synr . HA - Debcl can be detected after this sequential immunoprecipitation .

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

Figure 2A-D. The Synr-induced wing structural defect is suppressed by knockdown of Debcl, Buffy or DCP1. Data information: Scale bars, 500 μm in AF. Synr-induced caspase activation is suppressed by knockdown of Debcl. G. GC3Ai signals are suppressed by knockdown of Debcl or Buffy Data information: Scale bars, 50 μm in F and GH-I. Lysates from HEK293T cells that express Flag-Debcl or Flag-Buffy were incubated with GST or GST-Synr bound to glutathione-Sepharose. Flag-Debcl or Flag-Buffy is pulled down with GST-Synr but not with GST.J. Deletion of neither the coiled coil domain nor the BH3 motif suppresses binding Flag-Debcl to GST-Synr.O. Debcl can be photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. BPA at Y340 was used as a negative control. Red arrowheads indicate covalently crosslinked products. P. Buffy is also photo-crosslinked with Synr at BPA that corresponds to Y124 and F141. Red arrowheads indicate covalently crosslinked products.R. HA-Debcl is detected after two series of immunoprecipitation. The first immunoprecipitation was performed for Flag-Buffy, followed by Flag peptide-mediated elution and secondary precipitation for Myc-Synr. HA-Debcl can be detected after this sequential immunoprecipitation.


Figure 3A . Ectopic expression of Synr induces autolysosome accumulation , which was detected by Lysotracker , in the wing disc . B . Ectopic expression of Synr also induces autophagosome accumulation , which was detected by mCherry - atg8a . The dotted square regions are magnified in the right pictures . Data information : Scale bars , 100 μm in A and H , 50 μm in BD - F . Knockdown of Atg2 or Atg8a suppresses the wing structural defect that is induced by Synr . Data information : Scale bars , 500 μm in D . Synr - induced caspase activation is suppressed by inhibition of autophagy ( atg2 , atg8a RNAis ) in a similar manner to apoptosis inhibition ( Debcl , Dcp - 1 RNAis ) . Data information : Scale bars , 100 μm

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

Figure 3A. Ectopic expression of Synr induces autolysosome accumulation, which was detected by Lysotracker, in the wing disc. B. Ectopic expression of Synr also induces autophagosome accumulation, which was detected by mCherry-atg8a. The dotted square regions are magnified in the right pictures. Data information: Scale bars, 100 μm in A and H, 50 μm in BD-F. Knockdown of Atg2 or Atg8a suppresses the wing structural defect that is induced by Synr. Data information: Scale bars, 500 μm in D.Synr-induced caspase activation is suppressed by inhibition of autophagy (atg2, atg8a RNAis) in a similar manner to apoptosis inhibition (Debcl, Dcp-1 RNAis). Data information: Scale bars, 100 μm in


Figure 4A . Inhibition of Synr suppresses the wing defect that is induced by p53 , a potent apoptosis inducer . B . Inhibition of Synr suppresses p53 - induced caspase activation . Data information : Statistical significance was determined using a two - tailed unpaired t - test ( B ) , Scale bar , 500 μm in AD . Inhibition of Synr in the midgut enterocytes enhances resistance to starvation . E . The synr mutant also exhibits enhanced resistance to starvation . Data information : Statistical significance was determined using a log - rank ( Mantel - Cox ) test ( D - E ) .

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

Figure 4A. Inhibition of Synr suppresses the wing defect that is induced by p53, a potent apoptosis inducer. B. Inhibition of Synr suppresses p53-induced caspase activation. Data information: Statistical significance was determined using a two-tailed unpaired t-test (B), Scale bar, 500 μm in AD. Inhibition of Synr in the midgut enterocytes enhances resistance to starvation. E. The synr mutant also exhibits enhanced resistance to starvation. Data information: Statistical significance was determined using a log-rank (Mantel-Cox) test (D-E).


f1 ( b ) in N2 wild type ( black diamonds ) and ung - 1 mutants ( red squares ) , ( c ) following the depletion of EXO - 3 ( red triangles ) , APN - 1 ( blue circles ) or control RNAi ( L4440 , black diamonds ) in the N2 wild - type background , and ( d ) in msh - 2 ( ok2410 ) ( orange triangles ) , msh - 6 ( pk2504 ) ( green circles ) and mlh - 1 ( ok1917 ) ( grey squares ) mutants . ( e ) Western blottings showing expression of MSH - 6 protein in the wild type strain grown on E . coli expressing control ( N2 ) or the indicated RNAi , as well as in msh - 6 ( pk2504 ) and msh - 2 ( ok2410 ) mutants ( top panel ) , and expression of MSH - 2 : : GFP fusion protein in transgenic worms grown on E . coli expressing RNAi for MSH - 2 , MSH - 6 or empty vector control as indicated ( lower panel ) . Actin was used as the loading control . ( f ) F1 survival after depletion of MSH - 2 and MSH - 6 in the mlh - 1 ( ok1917 ) mutant . ( g , h ) F1 survival after depletion of the AP endonucleases EXO - 3 and APN - 1 in ( g ) the msh - 6 ( pk2504 ) or ( h ) msh - 2 ( ok2410 ) mutant background . Kruskal - Wallis one - way analysis on ranks ( P = 0 . 963 and P = 0 . 985 in h and g , respectively ) . Mann - Whitney rank - sum test failed to identify a difference in median survival after depleting EXO - 3 in the msh - 6 mutant ( g , P = 0 . 929 ) . ( i ) F1 survival following depletion of the indicated genes in exo - 3 ( tm4374 ) mutants . ( j ) F1 survival in msh - 6 ( pk2504 ) , exo - 3 ( tm4374 ) and exo - 3 ; msh - 6 double mutants . ( k ) F1 survival in exo - 3 ( tm4374 ) , mlh - 1 ( ok1917 ) and exo - 3 ; mlh - 1 double mutants . All survival curves ( b - d , f - k ) show the mean ± s . d . for each data point from three independent experiments .

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

f1(b) in N2 wild type (black diamonds) and ung-1 mutants (red squares), (c) following the depletion of EXO-3 (red triangles), APN-1 (blue circles) or control RNAi (L4440, black diamonds) in the N2 wild-type background, and (d) in msh-2(ok2410) (orange triangles), msh-6(pk2504) (green circles) and mlh-1(ok1917) (grey squares) mutants.(e) Western blottings showing expression of MSH-6 protein in the wild type strain grown on E. coli expressing control (N2) or the indicated RNAi, as well as in msh-6(pk2504) and msh-2(ok2410) mutants (top panel), and expression of MSH-2::GFP fusion protein in transgenic worms grown on E. coli expressing RNAi for MSH-2, MSH-6 or empty vector control as indicated (lower panel). Actin was used as the loading control.(f) F1 survival after depletion of MSH-2 and MSH-6 in the mlh-1(ok1917) mutant. (g,h) F1 survival after depletion of the AP endonucleases EXO-3 and APN-1 in (g) the msh-6(pk2504) or (h) msh-2(ok2410) mutant background. Kruskal-Wallis one-way analysis on ranks (P=0.963 and P=0.985 in h and g, respectively). Mann-Whitney rank-sum test failed to identify a difference in median survival after depleting EXO-3 in the msh-6 mutant (g, P=0.929). (i) F1 survival following depletion of the indicated genes in exo-3(tm4374) mutants. (j) F1 survival in msh-6(pk2504), exo-3(tm4374) and exo-3;msh-6 double mutants. (k) F1 survival in exo-3(tm4374), mlh-1(ok1917) and exo-3;mlh-1 double mutants. All survival curves (b-d,f-k) show the mean±s.d. for each data point from three independent experiments.


f2 ( a ) Immunofluorescence images showing anti - RPA - 1 - positive foci in response to 5 - FU after depleting the indicated genes by RNAi or empty vector control RNAi ( L4440 ; scale bars , 5 μm ) . b ) Quantification of the fraction ( % ) of 5 - FU - treated embryos that contain RPA - 1 - positive foci . ( c ) Quantification of the fraction ( % ) of 5 - FU - treated embryos that contain RPA - 1 - positive foci in N2 , mlh - 1 ( ok1917 ) and exo - 1 ( tm1842 ) mutants fed control ( L4440 ) or RNAi targeting APN - 1 ( apn - 1 ) . ( d ) Immunofluorescence showing CHK - 1 phosphorylation at Ser139 in response to 5 - FU in N2 but not in msh - 6 ( pk2504 ) mutants or in N2 after apn - 1 ( RNAi ) ( scale bars , 5 μm ) . ( e ) Quantification of the fraction ( % ) of 5 - FU - treated embryos with phospho - CHK - 1 foci . ( f ) Western blottings showing H1X . 101 levels in N2 and msh - 6 mutants , treated ( + ) or not ( − ) with 5 - FU . Actin was used as loading control . ( g ) Quantification of H1X . 101 levels relative to actin in control versus 5 - FU - treated worms . ( b , c , e , g ) Bar graphs show the mean ± s . d . from three independent experiments .

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

f2(a) Immunofluorescence images showing anti-RPA-1-positive foci in response to 5-FU after depleting the indicated genes by RNAi or empty vector control RNAi (L4440; scale bars, 5 μm).b) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci.(c) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci in N2, mlh-1(ok1917) and exo-1(tm1842) mutants fed control (L4440) or RNAi targeting APN-1 (apn-1).(d) Immunofluorescence showing CHK-1 phosphorylation at Ser139 in response to 5-FU in N2 but not in msh-6(pk2504) mutants or in N2 after apn-1(RNAi) (scale bars, 5 μm). (e) Quantification of the fraction (%) of 5-FU-treated embryos with phospho-CHK-1 foci.(f) Western blottings showing H1X.101 levels in N2 and msh-6 mutants, treated (+) or not (−) with 5-FU. Actin was used as loading control. (g) Quantification of H1X.101 levels relative to actin in control versus 5-FU-treated worms. (b,c,e,g) Bar graphs show the mean±s.d. from three independent experiments.


f3a ) Autophagy induction in response to 5 - FU measured in a GFP : : LGG - 1 reporter strain fed control RNAi ( L4440 ) or RNAi targeting the indicated genes ( scale bars , 10 μm ) . ( b ) Immunofluorescence showing anti - VPS - 34 staining in dissected embryos in the absence or presence of 5 - FU ( scale bar , 5 μm ) . ( c ) Western blot analysis of embryonic extracts with and without the addition of 5 - FU or the autophagy inhibitor 3 - MA , detecting the ~ 40 - kDa GFP : : LGG - 1 fusion protein and the cleaved ~ 25 - kDa product . ( d ) F1 survival measured after depletion of BEC - 1 ( red triangles ) and ATG - 7 ( blue squares ) as compared with worms fed control RNAi ( black diamonds ) . The survival curve shows the mean ± s . d . for each data point from three independent experiments . ( e ) Induction of autophagy ( % GFP - positive embryos ) following depletion of the indicated genes . ( f ) Immunofluorescence showing anti - VPS - 34 staining in N2 and atl - 1 ( tm853 ) embryos in the absence or presence of 5 - FU ( scale bar , 5 μm ) . ( g ) The fraction ( as % of untreated control ) of embryos with VPS - 34 - positive foci following 5 - FU treatment . ( h ) Induction of autophagy ( % GFP - positive embryos ) in control ( GFP : : LGG - 1 ) and atm - 1 ( gk186 ) mutants ( atm - 1 ; GFP : : LGG - 1 ) . ( e , g , h ) Bar graphs represent mean ± s . d . from three independent experiments .

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

f3a) Autophagy induction in response to 5-FU measured in a GFP::LGG-1 reporter strain fed control RNAi (L4440) or RNAi targeting the indicated genes (scale bars, 10 μm).(b) Immunofluorescence showing anti-VPS-34 staining in dissected embryos in the absence or presence of 5-FU (scale bar, 5 μm).(c) Western blot analysis of embryonic extracts with and without the addition of 5-FU or the autophagy inhibitor 3-MA, detecting the ~40-kDa GFP::LGG-1 fusion protein and the cleaved ~25-kDa product.(d) F1 survival measured after depletion of BEC-1 (red triangles) and ATG-7 (blue squares) as compared with worms fed control RNAi (black diamonds). The survival curve shows the mean±s.d. for each data point from three independent experiments.(e) Induction of autophagy (% GFP-positive embryos) following depletion of the indicated genes.(f) Immunofluorescence showing anti-VPS-34 staining in N2 and atl-1(tm853) embryos in the absence or presence of 5-FU (scale bar, 5 μm).(g) The fraction (as % of untreated control) of embryos with VPS-34-positive foci following 5-FU treatment.(h) Induction of autophagy (% GFP-positive embryos) in control (GFP::LGG-1) and atm-1(gk186) mutants (atm-1; GFP::LGG-1). (e,g,h) Bar graphs represent mean±s.d. from three independent experiments.