Figure 1A - C , Immunofluorescence images ( inverted intensity ) of P14 - P15 IHCs from indicated genotypes , revealing differences in otoferlin fluorescence intensity and distribution . Maximum projection of confocal stacks , scale bar 10µm . D - F , Upper panels , examples for IHCs , co - labelled for otoferlin ( magenta ) and Vglut3 ( green ) and position of the line for line scans ; maximum projection of few optical sections , scale bars 5µm . Lower panels , for quantification of membrane staining , the fluorescence was normalized to the cellular fluorescence for each fluorophore , then the average of five parallel line scans through the middle of the cells for the sum of both fluorescence values ( black line ) was used to determine the position of the basal membrane . At the most basal cellular point along this line which exceeds the threshold value of 2 ( yellow diamond ) , the otoferlin - Vglut3 fluorescence difference ( blue line ) gave the value for relative otoferlinplasma membrane levels ( orange diamond ) . Insets : enlargements of basal regions . G , Otoferlin protein levels were reduced in OtofI515T / I515T IHCs ( indicates numbers represent numbers of cells ) and OtofPga / Pga IHCs compared to wild - type ( Otof + / + ) controls ( mean ± SEM ) . H , ratio of apical / basal protein levels ( above / below nuclear midline depicted as green line in A ) indicates an apical shift of otoferlin in OtofPga / Pga IHCs . I , Relative levels of membrane - bound otoferlin at the basal pole of IHCs . K , Absolute amount of otoferlin at the basal IHCplasma membrane , gained by multiplication of relative plasma membrane levels ( I ) x total cellular otoferlin protein levels ( G ) . Kruskal - Wallis test ; * * * indicates p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C, Immunofluorescence images (inverted intensity) of P14-P15 IHCs from indicated genotypes, revealing differences in otoferlin fluorescence intensity and distribution. Maximum projection of confocal stacks, scale bar 10µm.D-F, Upper panels, examples for IHCs, co-labelled for otoferlin (magenta) and Vglut3 (green) and position of the line for line scans; maximum projection of few optical sections, scale bars 5µm. Lower panels, for quantification of membrane staining, the fluorescence was normalized to the cellular fluorescence for each fluorophore, then the average of five parallel line scans through the middle of the cells for the sum of both fluorescence values (black line) was used to determine the position of the basal membrane. At the most basal cellular point along this line which exceeds the threshold value of 2 (yellow diamond), the otoferlin-Vglut3 fluorescence difference (blue line) gave the value for relative otoferlinplasma membrane levels (orange diamond). Insets: enlargements of basal regions.G, Otoferlin protein levels were reduced in OtofI515T/I515T IHCs (indicates numbers represent numbers of cells) and OtofPga/Pga IHCs compared to wild-type (Otof+/+) controls (mean±SEM).H, ratio of apical/basal protein levels (above/below nuclear midline depicted as green line in A) indicates an apical shift of otoferlin in OtofPga/Pga IHCs.I, Relative levels of membrane-bound otoferlin at the basal pole of IHCs.K, Absolute amount of otoferlin at the basal IHCplasma membrane, gained by multiplication of relative plasma membrane levels (I) x total cellular otoferlin protein levels (G). Kruskal-Wallis test; *** indicates p<0.001; **, p<0.01; *, p<0.05.


Figure 2A , ABR thresholds in OtofI515T / I515T ( red , n = 5 ) and Otof - / I515T ( blue , n = 7 ) mice were elevated compared to Otof + / + mice ( black , n = 5 ) at an age of 3 - 4 weeks . The grey dotted line indicates the maximum loudspeaker output of 90dB ; thresholds exceeding this value were set to 100dB for calculation of the mean ± SEM . At 12kHz , only the threshold increase for Otof - / I515T vs Otof + / + is significant ( Kruskal - Wallis test with Dunn ' s multiple comparisons test between all 3 groups ) . B , Grand averages of ABR waveforms ± SEM in response to 80dB click stimulation of the mice analyzed in A : The small wave preceding ABR wave I probably represents the summating potential ( SP , hair cell receptor potential ) , which is intact in Otof mutants . ABR wave I is reduced in amplitude while subsequent peaks are better preserved in OtofI515T / I515T mice ( Fig EV1 ) . C , Mean ABR wave I amplitude ± SEM for different stimulus intensities ( all differences between genotypes are significant ; 2 - way ANOVA with Tukey ' s multiple comparison test ) . D , At 8 weeks ( circles ) and 25 weeks ( open squares ) , OtofI515T / I515T mice showed highly elevated ABR thresholds compared to Otof + / + mice ( n = 7 - 8 each ; p < 0 . 001 at 12kHz , Mann - Whitney - U - test ) . Grey dotted line as in A . E , Grand averages of ABR waveforms ± SEM in response to 80dB Click stimulation in mice aged 8 weeks . OtofI515T / I515T ( n = 8 ) have drastically reduced ABR amplitudes compared to Otof + / + mice ( n = 7 ) . Otof - / - mice have no ABR ( green , n = 9 ) . F , Mean ABR wave I amplitude ± SEM for different stimulus intensities for 8 week and 25 week old OtofI515T / I515T and Otof + / + mice ( p < 0 . 001 , 2 - way ANOVA ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, ABR thresholds in OtofI515T/I515T (red, n=5) and Otof-/I515T (blue, n=7) mice were elevated compared to Otof+/+ mice (black, n=5) at an age of 3-4 weeks. The grey dotted line indicates the maximum loudspeaker output of 90dB; thresholds exceeding this value were set to 100dB for calculation of the mean ± SEM. At 12kHz, only the threshold increase for Otof-/I515T vs Otof+/+ is significant (Kruskal-Wallis test with Dunn's multiple comparisons test between all 3 groups).B, Grand averages of ABR waveforms ± SEM in response to 80dB click stimulation of the mice analyzed in A: The small wave preceding ABR wave I probably represents the summating potential (SP, hair cell receptor potential), which is intact in Otof mutants. ABR wave I is reduced in amplitude while subsequent peaks are better preserved in OtofI515T/I515T mice (Fig EV1).C, Mean ABR wave I amplitude ± SEM for different stimulus intensities (all differences between genotypes are significant; 2-way ANOVA with Tukey's multiple comparison test).D, At 8 weeks (circles) and 25 weeks (open squares), OtofI515T/I515T mice showed highly elevated ABR thresholds compared to Otof+/+ mice (n=7-8 each; p<0.001 at 12kHz, Mann-Whitney-U-test). Grey dotted line as in A.E, Grand averages of ABR waveforms ± SEM in response to 80dB Click stimulation in mice aged 8 weeks. OtofI515T/I515T (n=8) have drastically reduced ABR amplitudes compared to Otof+/+ mice (n=7). Otof-/- mice have no ABR (green, n=9).F, Mean ABR wave I amplitude ± SEM for different stimulus intensities for 8 week and 25 week old OtofI515T/I515T and Otof+/+ mice (p<0.001, 2-way ANOVA).


Figure 3A , B , No difference in voltage dependent Ca2 + - currents ( A ) and fractional activation of ICa - channels ( B ) between OtofI515T / I515T IHCs ( n = 16 ) and IHCs of Otof + / + littermates ( n = 13 ) . C , Exocytosis was recorded by measures of changes in membrane capacitance ( ΔCm , lower panel ) in response to depolarization ( left , 20ms ; right , 100ms ) to the voltage where maximum Ca2 + - currents were elicited ( upper panel ) , typically - 14mV . D , Upper panel , while for stimuli up to 20ms exocytosis from OtofI515T / I515T ( n = 13 ) and Otof + / + ( n = 11 ) IHCs was similar , sustained exocytosis , representing most likely the release of replenished vesicles , was significantly reduced in OtofI515T / I515T IHCs ( t - test ) . Lower panel , Ca2 + - current integrals were of similar size . E , Flash photolysis of caged Ca2 + elicited a smaller exocytic response in OtofI515T / I515T IHCs . F , The kinetics of the fast component from E was comparable between OtofI515T / I515T IHCs and Otof + / + littermates ( b ) . Open circles represent previously published data on IHCs of hearing wild - type mice ( a ; Beutner et al , 2001 ; Pangrsic et al , 2010 ) .

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 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 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

Figure 3A,B, No difference in voltage dependent Ca2+-currents (A) and fractional activation of ICa-channels (B) between OtofI515T/I515T IHCs (n=16) and IHCs of Otof+/+ littermates (n=13).C, Exocytosis was recorded by measures of changes in membrane capacitance (ΔCm, lower panel) in response to depolarization (left, 20ms; right, 100ms) to the voltage where maximum Ca2+-currents were elicited (upper panel), typically -14mV.D, Upper panel, while for stimuli up to 20ms exocytosis from OtofI515T/I515T (n=13) and Otof+/+ (n=11) IHCs was similar, sustained exocytosis, representing most likely the release of replenished vesicles, was significantly reduced in OtofI515T/I515T IHCs (t-test). Lower panel, Ca2+-current integrals were of similar size.E, Flash photolysis of caged Ca2+ elicited a smaller exocytic response in OtofI515T/I515T IHCs.F, The kinetics of the fast component from E was comparable between OtofI515T/I515T IHCs and Otof+/+ littermates (b). Open circles represent previously published data on IHCs of hearing wild-type mice (a; Beutner et al, 2001; Pangrsic et al, 2010).


Figure 4A , Spontaneous rates of SGNs from OtofI515T / I515T ( red , n = 35 ) and Otof + / + littermates ( black , n = 39 ) were not significantly different ( p = 0 . 83 , Kolmogorov - Smirnov test ) . B - D , Averaged poststimulus time histograms ± SEM from OtofI515T / I515T mice ( n = 25 - 32 ) and Otof + / + littermate SGNs ( n = 13 - 27 ) to stimulation with 50ms tone bursts at the characteristic frequency ( CF ) of each fiber , 30dB above threshold at indicated stimulus rates . E , Quantification of onset ( largest 0 . 5ms bin , Mann - Whitney U test ) and adapted responses ( averaged 35 - 45ms from response onset , t - test ) from data in B - D . F , The jitter of the first sound - evoked spike was significantly increased ( p < 0 . 001 , Mann - Whitney U test ) but retained its inverse correlation with spike rates . G , Spike rate increases with rising stimulus intensity were significantly shallower in OtofI515T / I515T SGNs , both for repetitive stimulation with 50ms tone bursts ( left , p = 0 . 014 , t - test ) and for continuous stimulation with amplitude modulated tones ( right , p < 0 . 001 , Mann - Whitney U test ) . H , Phase locking to amplitude modulated tones ( assessed as the maximal synchronization index ) was typically less precise in OtofI515T / I515T SGNs than in Otof + / + SGNs ( p = 0 . 09 , Mann - Whitney U test ) . I , 100ms masker tone and 15ms probe tones ( both at CF , 30dB above threshold ) were separated by silent intervals of variable duration . Inter - masker intervals were 500ms for Otof + / + , and 1000ms for OtofI515T / I515T . The ratio of probe and masker onset responses revealed enhanced RRP depletion after stimulation in OtofI515T / I515T ( pink ; mean ± SEM red ) compared to Otof + / + ( grey ; mean ± SEM black ; for 4ms interval : p = 0 . 001 , t - test ) and a slowed time course of recovery ( x : half time of recovery , taken from normalized recovery functions ; p < 0 . 001 , Mann - Whitney U test ) . K , Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps . 5 OtofI515T / I515T mice ( pink , mean ± SEM red ) avoided drinking less efficiently than 2 Otof + / + mice ( grey , mean ± SEM black ) for shorter gap durations . See also Figs S2 / S3 .

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 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 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 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 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

Figure 4A, Spontaneous rates of SGNs from OtofI515T/I515T (red, n=35) and Otof+/+ littermates (black, n=39) were not significantly different (p=0.83, Kolmogorov-Smirnov test).B-D, Averaged poststimulus time histograms ±SEM from OtofI515T/I515T mice (n=25-32) and Otof+/+ littermate SGNs (n=13-27) to stimulation with 50ms tone bursts at the characteristic frequency (CF) of each fiber, 30dB above threshold at indicated stimulus rates.E, Quantification of onset (largest 0.5ms bin, Mann-Whitney U test) and adapted responses (averaged 35-45ms from response onset, t-test) from data in B-D.F, The jitter of the first sound-evoked spike was significantly increased (p<0.001, Mann-Whitney U test) but retained its inverse correlation with spike rates.G, Spike rate increases with rising stimulus intensity were significantly shallower in OtofI515T/I515T SGNs, both for repetitive stimulation with 50ms tone bursts (left, p=0.014, t-test) and for continuous stimulation with amplitude modulated tones (right, p<0.001, Mann-Whitney U test).H, Phase locking to amplitude modulated tones (assessed as the maximal synchronization index) was typically less precise in OtofI515T/I515T SGNs than in Otof+/+SGNs (p=0.09, Mann-Whitney U test).I, 100ms masker tone and 15ms probe tones (both at CF, 30dB above threshold) were separated by silent intervals of variable duration. Inter-masker intervals were 500ms for Otof+/+, and 1000ms for OtofI515T/I515T. The ratio of probe and masker onset responses revealed enhanced RRP depletion after stimulation in OtofI515T/I515T (pink; mean ±SEM red) compared to Otof+/+ (grey; mean ±SEM black; for 4ms interval: p=0.001, t-test) and a slowed time course of recovery (x: half time of recovery, taken from normalized recovery functions; p<0.001, Mann-Whitney U test).K, Mice learned to drink water when continuous noise was present but avoided drinking when the noise was interrupted by silent gaps. 5 OtofI515T/I515T mice (pink, mean ±SEM red) avoided drinking less efficiently than 2 Otof+/+ mice (grey, mean ±SEM black) for shorter gap durations. See also Figs S2/S3.


Figure 5A , Exemplary ABR traces ( Click 100dB ) from one OtofI515T / I515T mouse recorded at indicated bullatemperatures during local heating . Note that ABRs never disappeared completely , but wave I amplitude changed reversibly with temperature . B , C , ABR wave I amplitudes in OtofI515T / I515T ( A , Click 100dB ) and Otof + / + mice ( B , Click 80dB ) decreased with increasing temperature in the bulla . Each color represents a different experiment , dashed lines are line fits . Open symbols indicate the beginning of the experiments ; subsequent recordings are connected by lines . The four indicated data points in B correspond to ABRs in A . D , Explanted organ of Corti from an Otof + / + mouse at P4 after two days in vitro ( DIV2 ) at 37 ° C immunostained against otoferlin ( magenta ) and Vglut3 ( green ) . Note the intense immunostaining of otoferlin at the plasma membrane . Scale bar 10 µm . E - J , Explanted organs of Corti from Otof - / - mice at P4 were transfected by GeneGun with otoferlin cDNA and eGFP and immunostained at DIV2 . E , Example cell transfected with wild - type mouseotoferlin cDNA . F , G , Representative cells transfected with mouseotoferlin including the 20 amino acids of the RXR motif ( see Fig EV4 ) . Cells in G were incubated at 38 . 5 ° C for 30 min prior to fixation . The RXR - otoferlin transfected cells show a membrane localization of otoferlin surrounding the Vglut3 immunofluorescence / eGFP fluorescence at the basal pole of the cells ( arrows ) , similar as in controls ( A , B ) . H , J , Both the human RXR motif and the Ile515Thr mutation were introduced in mouse cDNA , and cells were incubated at 37 ° C ( H ) or for 30min at 38 . 5 ° C ( J ) before fixation . Here , green fluorescence surrounds the otoferlin immunofluorescence ( arrows ) , suggesting loss of otoferlin from the plasma membrane . K , Quantification of the relative plasma membrane levels of otoferlin ( as in Fig 1 ) revealed normal plasma membrane abundance when the human RXR motif was present , but a strong reduction for otoferlin with RXR and Ile515Thr ( individual cells ; mean ± SEM ; 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Exemplary ABR traces (Click 100dB) from one OtofI515T/I515T mouse recorded at indicated bullatemperatures during local heating. Note that ABRs never disappeared completely, but wave I amplitude changed reversibly with temperature.B,C, ABR wave I amplitudes in OtofI515T/I515T (A, Click 100dB) and Otof+/+ mice (B, Click 80dB) decreased with increasing temperature in the bulla. Each color represents a different experiment, dashed lines are line fits. Open symbols indicate the beginning of the experiments; subsequent recordings are connected by lines. The four indicated data points in B correspond to ABRs in A.D, Explanted organ of Corti from an Otof+/+ mouse at P4 after two days in vitro (DIV2) at 37°C immunostained against otoferlin (magenta) and Vglut3 (green). Note the intense immunostaining of otoferlin at the plasma membrane. Scale bar 10 µm.E-J, Explanted organs of Corti from Otof-/- mice at P4 were transfected by GeneGun with otoferlin cDNA and eGFP and immunostained at DIV2.E, Example cell transfected with wild-type mouseotoferlin cDNA.F,G, Representative cells transfected with mouseotoferlin including the 20 amino acids of the RXR motif (see Fig EV4). Cells in G were incubated at 38.5°C for 30 min prior to fixation. The RXR-otoferlin transfected cells show a membrane localization of otoferlin surrounding the Vglut3 immunofluorescence/eGFP fluorescence at the basal pole of the cells (arrows), similar as in controls (A,B).H,J, Both the human RXR motif and the Ile515Thr mutation were introduced in mouse cDNA, and cells were incubated at 37°C (H) or for 30min at 38.5°C (J) before fixation. Here, green fluorescence surrounds the otoferlin immunofluorescence (arrows), suggesting loss of otoferlin from the plasma membrane.K, Quantification of the relative plasma membrane levels of otoferlin (as in Fig 1) revealed normal plasma membrane abundance when the human RXR motif was present, but a strong reduction for otoferlin with RXR and Ile515Thr (individual cells; mean ±SEM; t-test).


Figure 6A - E , Capacitance increments recorded from Otof + / + ( black ) and OtofI515T / I515T ( red ) IHCs . Individual cells recorded at indicated temperatures ( light circles ) , mean ± SEM for perforated patch clamp experiments ( t - test ) . F , G , Summary of the capacitance changes and Ca2 + - current integrals for Otof + / + ( F ) and OtofI515T / I515T IHCs ( G ) at the different temperatures illustrates the drastic increase in exocytosis for physiological temperature . * Significant differences compared to RT measurements are indicated with colors of the respective temperature , and between PT and high temperature in violet ( t - test ) . H - J , Otoferlin immunofluorescence in Otof + / + IHCs ( upper panel ) and OtofI515T / I515T IHCs ( middle and lower panels ) of explanted organs of Corti at P7 - P8 after incubation at indicated temperatures for 24h ; maximum projections of z - stacks , inverted images , scale bar 10µm . The same imaging settings have been applied in all experiments , and the same lookup table was applied for the upper and middle panels . Images of lower panels are enhanced compared to middle panels ( lookup table covering full data range of only this genotype ) to visualize otoferlin distribution . H - J , Otoferlin immunofluorescence in Otof + / + IHCs ( upper panel ) and OtofI515T / I515T IHCs ( middle and lower panels ) of explanted organs of Corti at P7 - P8 after incubation at indicated temperatures for 24h ; maximum projections of z - stacks , inverted images , scale bar 10µm . The same imaging settings have been applied in all experiments , and the same lookup table was applied for the upper and middle panels . Images of lower panels are enhanced compared to middle panels ( lookup table covering full data range of only this genotype ) to visualize otoferlin distribution . K , Quantification of otoferlin immunofluorescence in Otof + / + IHCs ( black bars , n = 98 - 137 cells , 4 - 5 experiments ) and OtofI515T / I515T IHCs ( red bars , n = 90 - 97 cells ; Kruskal - Wallis test as in L - N ) , indicate reduced protein levels with increasing temperature . L , Apical / basal otoferlin protein distribution , revealing a significant apical shift of otoferlin and Vglut3 for OtofI515T / I515T IHCs ( red symbols ) at 38 . 5 ° C compared to Otof + / + ( grey / black symbols ) . M , Relative levels of membrane bound otoferlin were lowered with increasing temperature . N , Absolute otoferlin membrane immunostaining strongly decreased with temperature .

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

Figure 6A-E, Capacitance increments recorded from Otof+/+ (black) and OtofI515T/I515T (red) IHCs. Individual cells recorded at indicated temperatures (light circles), mean ±SEM for perforated patch clamp experiments (t-test).F,G, Summary of the capacitance changes and Ca2+-current integrals for Otof+/+ (F) and OtofI515T/I515T IHCs (G) at the different temperatures illustrates the drastic increase in exocytosis for physiological temperature. *Significant differences compared to RT measurements are indicated with colors of the respective temperature, and between PT and high temperature in violet (t-test).H-J, Otoferlin immunofluorescence in Otof+/+ IHCs (upper panel) and OtofI515T/I515T IHCs (middle and lower panels) of explanted organs of Corti at P7-P8 after incubation at indicated temperatures for 24h; maximum projections of z-stacks, inverted images, scale bar 10µm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution.H-J, Otoferlin immunofluorescence in Otof+/+ IHCs (upper panel) and OtofI515T/I515T IHCs (middle and lower panels) of explanted organs of Corti at P7-P8 after incubation at indicated temperatures for 24h; maximum projections of z-stacks, inverted images, scale bar 10µm. The same imaging settings have been applied in all experiments, and the same lookup table was applied for the upper and middle panels. Images of lower panels are enhanced compared to middle panels (lookup table covering full data range of only this genotype) to visualize otoferlin distribution.K, Quantification of otoferlin immunofluorescence in Otof+/+ IHCs (black bars, n=98-137 cells, 4-5 experiments) and OtofI515T/I515T IHCs (red bars, n=90-97 cells; Kruskal-Wallis test as in L-N), indicate reduced protein levels with increasing temperature.L, Apical/basal otoferlin protein distribution, revealing a significant apical shift of otoferlin and Vglut3 for OtofI515T/I515T IHCs (red symbols) at 38.5°C compared to Otof+/+ (grey/black symbols).M, Relative levels of membrane bound otoferlin were lowered with increasing temperature.N, Absolute otoferlin membrane immunostaining strongly decreased with temperature.


Figure 7A - C , Pre - embedding immunogold labelling for EM visualizes otoferlin localization in random ultrathin sections through the basal part of IHCs in Otof + / + ( A ) and OtofI515T / I515T ( B ) but not in Otof - / - ( C ) mice . IHCs are highlighted in beige . Scale bar 100nm . D - G , Magnified synaptic ribbons ( R ) and postsynaptic afferent boutons of SGNs ( Aff ) , displaying otoferlin immunogold labelling at active zone membranes ( pink arrowheads ) but not around the synaptic ribbon , using saponin permeabilization ( D - F ) , or Triton - X - 100 treatment ( G ) . H , Vglut3 immunogold labelling adjacent to the ribbon as expected for synaptic vesicle . Scale bar 200nm . I , J , Some otoferlin labelled structures bear clathrin coats ( red arrowheads ) , suggesting that they originate from bulk endocytosis . Scale bar 100nm . K , Otoferlin labelled vesicles were on average larger in OtofI515T / I515T IHCs ( OtofI515T / I515T , n = 86 vesicles , 18 images ; Otof + / I + , n = 82 vesicles , 6 images ; Wilcoxon - rank - sum - test , as in L - M ) . L , Quantification of immunogold clusters at the plasma membrane revealed a strong trend towards reduced levels of otoferlin in OtofI515T / I515T ( 6 images each ) . M , Distal from the plasma membrane , immunogold labels were reduced in OtofI515T / I515T IHCs . N , Representative electron micrographs of IHCribbon synapses in OtofI515T / I515Tand Otof + / ( conventional embedding without immunogold ) after pre - incubation for 10min at the indicated temperature followed by high K + stimulation or 0 Ca2 + inhibition for 1min 45sec . Scale bars 100nm . P - Q , Synaptic vesicles with > 45 nm diameter appeared at 39 ° C in random sections ; fraction of large membrane - proximal ( P ; Kruskal - Wallis test ) and ribbon - associated ( Q ; one way ANOVA followed by Tukey ' s test ) synaptic vesicles ( see also Appendix Table S1 ) .

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 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 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 O O O O O O O O O O O O O O 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-C, Pre-embedding immunogold labelling for EM visualizes otoferlin localization in random ultrathin sections through the basal part of IHCs in Otof+/+ (A) and OtofI515T/I515T (B) but not in Otof-/- (C) mice. IHCs are highlighted in beige. Scale bar 100nm.D-G, Magnified synaptic ribbons (R) and postsynaptic afferent boutons of SGNs (Aff), displaying otoferlin immunogold labelling at active zone membranes (pink arrowheads) but not around the synaptic ribbon, using saponin permeabilization (D-F), or Triton-X-100 treatment (G).H, Vglut3 immunogold labelling adjacent to the ribbon as expected for synaptic vesicle. Scale bar 200nm.I,J, Some otoferlin labelled structures bear clathrin coats (red arrowheads), suggesting that they originate from bulk endocytosis. Scale bar 100nm.K, Otoferlin labelled vesicles were on average larger in OtofI515T/I515T IHCs (OtofI515T/I515T, n=86 vesicles, 18 images; Otof+/I+, n=82 vesicles, 6 images; Wilcoxon-rank-sum-test, as in L-M).L, Quantification of immunogold clusters at the plasma membrane revealed a strong trend towards reduced levels of otoferlin in OtofI515T/I515T (6 images each).M, Distal from the plasma membrane, immunogold labels were reduced in OtofI515T/I515T IHCs.N, Representative electron micrographs of IHCribbon synapses in OtofI515T/I515Tand Otof+/ (conventional embedding without immunogold) after pre-incubation for 10min at the indicated temperature followed by high K+ stimulation or 0 Ca2+ inhibition for 1min 45sec. Scale bars 100nm.P-Q, Synaptic vesicles with >45 nm diameter appeared at 39°C in random sections; fraction of large membrane-proximal (P; Kruskal-Wallis test) and ribbon-associated (Q; one way ANOVA followed by Tukey's test) synaptic vesicles (see also Appendix Table S1).


Figure 8A - D , Exemplary virtual sections through EM tomograms for stimulatory condition at 36 ° C and 39oC . Scale bar 100nm . F - I , 3D reconstruction of the ribbon - associated pool of vesicles ( front view ) . Active zone membrane is depicted in blue , presynaptic density in pink , and ribbons in red , scale bars 100nm . F - I , 3D reconstruction of the membrane - proximal pool ( top view ) and J - M , the ribbon - associated pool of vesicles ( front view ) . Active zone membrane is depicted in blue , presynaptic density in pink , and ribbons in red , scale bars 100nm . N - Q , Cumulative probability distribution of the diameter of round vesicles as well as the longest axis of flattened vesicles in the membrane - proximal pool ( N - O ) and ribbon - associated pool ( P - Q ) . N - P , one way ANOVA followed by Tukey ' s test ; Q , Kruskal - Wallis test . Round vesicles tested in both the pools appeared larger in OtofI515T / I515T IHC synapses but the long axis of flattened vesicles were comparable for both the genotypes ( see also Table EV1 ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8A-D, Exemplary virtual sections through EM tomograms for stimulatory condition at 36°C and 39oC. Scale bar 100nm.F-I, 3D reconstruction of the ribbon-associated pool of vesicles (front view). Active zone membrane is depicted in blue, presynaptic density in pink, and ribbons in red, scale bars 100nm.F-I, 3D reconstruction of the membrane-proximal pool (top view) and J-M, the ribbon-associated pool of vesicles (front view). Active zone membrane is depicted in blue, presynaptic density in pink, and ribbons in red, scale bars 100nm.N-Q, Cumulative probability distribution of the diameter of round vesicles as well as the longest axis of flattened vesicles in the membrane-proximal pool (N-O) and ribbon-associated pool (P-Q). N-P, one way ANOVA followed by Tukey's test; Q, Kruskal-Wallis test. Round vesicles tested in both the pools appeared larger in OtofI515T/I515T IHC synapses but the long axis of flattened vesicles were comparable for both the genotypes (see also Table EV1).


f1 ( a ) H3R expression in cultured ratcortical neurons at DIV7 after OGD / R injury ( 2 h OGD and 24 h reperfusion ) shown by western blot ( n = 6 per condition ; * * P0 . 01 versus control with Student ' s t - test ) . Full - size blots are shown in Supplementary Fig . 9 . ( b ) When the H3R antagonist thioperamide ( THIO , 10 − 6 mol l − 1 at reperfusion ) and the agonist immepip ( IMME , 10 − 6 mol l − 1 at reperfusion ) were administered , viability was tested in cultured neurons by 3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 2 , 5 - diphenyltetrazolium bromide ( MTT ) assay after OGD / R ( n = 7 per condition ; * * * P0 . 001 with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) . ( c ) 2 , 3 , 5 - triphenyltetrazolium chloride - stained brain sections showing the infarct area in WT and H3R − / − mice receiving saline , the H3R antagonists A331440 , clobenpropit ( CLOB ) and thioperamide , ( THIO ; 5 mg kg − 1 , i . p . , immediately at reperfusion and 6 h later ) , and the H3R agonist immepip ( IMME , 1 μg , i . c . v . , at reperfusion ) after I / R ( 1 h MCAO followed by 24 h reperfusion ; scale bar , 5 mm ) . The bar graphs show infarct volume ( d ) and neurological score ( e ) ( n = 6 - 8 mice per condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 001 versus WT saline group with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m .

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 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 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

f1(a) H3R expression in cultured ratcortical neurons at DIV7 after OGD/R injury (2 h OGD and 24 h reperfusion) shown by western blot (n=6 per condition; **P0.01 versus control with Student's t-test). Full-size blots are shown in Supplementary Fig. 9.(b) When the H3R antagonist thioperamide (THIO, 10−6 mol l−1 at reperfusion) and the agonist immepip (IMME, 10−6 mol l−1 at reperfusion) were administered, viability was tested in cultured neurons by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after OGD/R (n=7 per condition; ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).(c) 2,3,5-triphenyltetrazolium chloride-stained brain sections showing the infarct area in WT and H3R−/− mice receiving saline, the H3R antagonists A331440, clobenpropit (CLOB) and thioperamide, (THIO; 5 mg kg−1, i.p., immediately at reperfusion and 6 h later), and the H3R agonist immepip (IMME, 1 μg, i.c.v., at reperfusion) after I/R (1 h MCAO followed by 24 h reperfusion; scale bar, 5 mm).The bar graphs show infarct volume (d) and neurological score (e) (n=6-8 mice per condition, *P0.05, **P0.01, ***P0.001 versus WT saline group with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


f2 ( a ) TUNEL staining of apoptotic cells in penumbral brain slices in vivo ( scale bar , 100 μm ) when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after tMCAO . TUNEL - positive cells are green , and all cells are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) . ( b , d ) H3R was inhibited by thioperamide , and cleaved caspase - 3 protein expression is shown by western blot in WT and H3R − / − mice after tMCAO ( b ) ( n = 6 - 7 per condition ; * * P0 . 01 , * * * P0 . 001 with analysis of variances ( ANOVAs ) followed by Bonferroni / Dunn post hoc test ) ( c ) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide ( scale bar , 50 μm ) after OGD / R . TUNEL - positive cells are green , and all cells are stained with 4 ' , 6 - diamidino - 2 - phenylindole ( blue ) . ( b , d ) H3R was inhibited by thioperamide , and cleaved caspase - 3 protein expression is shown by western blot in WT and H3R − / − mice in cultured neurons subjected to OGD / R ( d ) ( n = 8 per condition ; * P0 . 05 , * * * P0 . 001 with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m . Full - size blots are shown in Supplementary Fig . 10 .

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 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 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

f2(a) TUNEL staining of apoptotic cells in penumbral brain slices in vivo (scale bar, 100 μm) when H3R was inhibited with thioperamide (scale bar, 50 μm) after tMCAO. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).(b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice after tMCAO (b) (n=6-7 per condition; **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by Bonferroni/Dunn post hoc test)(c) TUNEL staining of apoptotic cells in cultured neurons when H3R was inhibited with thioperamide (scale bar, 50 μm) after OGD/R. TUNEL-positive cells are green, and all cells are stained with 4',6-diamidino-2-phenylindole (blue).(b,d) H3R was inhibited by thioperamide, and cleaved caspase-3 protein expression is shown by western blot in WT and H3R−/−mice in cultured neurons subjected to OGD/R (d) (n=8 per condition; *P0.05, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 10.


f3 ( a ) 2 , 3 , 5 - Triphenyltetrazolium chloride ( TTC ) - stained brain sections from WT and HDC − / − mice showing the infarct area in those receiving saline , the H3R antagonist thioperamide ( THIO ) , the H1R antagonist pyrilamine ( PYRI ; 10 mg kg − 1 , i . p . , at reperfusion ) , the H2R antagonist cimetidine ( CIME ; 10 mg kg − 1 , i . p . , at reperfusion ) and α - FMH ( 50 mg kg − 1 , i . p . , 2 h before ischemia ) after 24 h reperfusion ( n = 6 per condition ; scale bar , 5 mm ) . ( b ) Infarct volumes and ( c ) neurological deficit scores ( * P0 . 05 , * * P0 . 01 , * * * P0 . 001 with analysis of variances followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m .

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 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

f3(a) 2,3,5-Triphenyltetrazolium chloride (TTC)-stained brain sections from WT and HDC−/− mice showing the infarct area in those receiving saline, the H3R antagonist thioperamide (THIO), the H1R antagonist pyrilamine (PYRI; 10 mg kg−1, i.p., at reperfusion), the H2R antagonist cimetidine (CIME; 10 mg kg−1, i.p., at reperfusion) and α-FMH (50 mg kg−1, i.p., 2 h before ischemia) after 24 h reperfusion (n=6 per condition; scale bar, 5 mm).(b) Infarct volumes and (c) neurological deficit scores (*P0.05, **P0.01, ***P0.001 with analysis of variances followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


f4 ( a ) Akt / GSK - 3β / mTOR / P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD / R . LiCl ( 5 mmol l − 1 immediately at reperfusion ) was used to rephosphorylate GSK - 3β / mTOR / P70S6K . The digits below represent the semiquantified optidensity of the bands , the control bands were defined as 1 . 00 . ( b ) Representative western blots showing the phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signaling in H3R − / − mice after OGD / R injury . The digits below represent the semiquantified optidensity of the bands , the control bands were defined as 1 . 00 . ( c ) Representative images showing LC3 - positive autophagic vacuoles ( red ) with 4 ' , 6 - diamidino - 2 - phenylindole staining ( blue ) after administration of thioperamide and LiCl with OGD / R ( scale bar , 10 μm ) . ( d ) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD / R ( n = 7 , * * P0 . 01 , * * * P0 . 001 with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) . ( e ) Representative western blots and bar graph showing the expression of LC3 - I and LC3 - II in steady - state autophagy and autophagic flux under OGD / R injury when thioperamide ( THIO ) and immepip ( IMME ) were administered ( n = 6 ; * * P0 . 01 , * * * P0 . 001 with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m . Full - size blots are shown in Supplementary Fig . 11 .

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 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 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

f4(a) Akt/GSK-3β/mTOR/P70S6K phosphorylation assessed by western blots after thioperamide administration and OGD/R. LiCl (5 mmol l−1 immediately at reperfusion) was used to rephosphorylate GSK-3β/mTOR/P70S6K. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.(b) Representative western blots showing the phosphorylation of Akt/GSK-3β/mTOR/P70S6K signaling in H3R−/− mice after OGD/R injury. The digits below represent the semiquantified optidensity of the bands, the control bands were defined as 1.00.(c) Representative images showing LC3-positive autophagic vacuoles (red) with 4',6-diamidino-2-phenylindole staining (blue) after administration of thioperamide and LiCl with OGD/R (scale bar, 10 μm).(d) Cell viability assessed by MTT when thioperamide and LiCl were administered after OGD/R (n=7, **P0.01, ***P0.001 with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).(e) Representative western blots and bar graph showing the expression of LC3-I and LC3-II in steady-state autophagy and autophagic flux under OGD/R injury when thioperamide (THIO) and immepip (IMME) were administered (n=6; **P0.01, ***P0.001 with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 11.


f5 ( a ) Representative immunohistochemical staining of LC3 ( green ) along with NeuN staining ( red ) in WT and H3R − / − mice ( scale bar , 25 μm ) . ( b , c ) When thioperamide was administered in vivo , LC3 expression was assessed by western blot after I / R in WT mice ( b ) ( * P0 . 05 , * * * P0 . 001 ; n = 6 mice per condition , with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) , and in H3R − / − mice ( c ) ( * * * P0 . 001 versus H3R − / − sham group ; n = 5 - 6 mice per condition , with ANOVAs followed by the Bonferroni / Dunn post hoc test . ) Data are presented as mean ± s . e . m . Full - size blots are shown in Supplementary Fig . 12 .

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 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

f5(a) Representative immunohistochemical staining of LC3 (green) along with NeuN staining (red) in WT and H3R−/− mice (scale bar, 25 μm).(b,c) When thioperamide was administered in vivo, LC3 expression was assessed by western blot after I/R in WT mice (b) (*P0.05, ***P0.001; n=6 mice per condition, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test), and in H3R−/− mice (c) (***P0.001 versus H3R−/− sham group; n=5-6 mice per condition, with ANOVAs followed by the Bonferroni/Dunn post hoc test.) Data are presented as mean±s.e.m. Full-size blots are shown in Supplementary Fig. 12.


f6 ( a ) Western blots showing the inhibitory effect of 3 - MA on autophagy in I / R . Full - size blots are shown in Supplementary Fig . 13 . ( b ) TTC - stained sections showing the infarct area after administration of 3 - MA ( 100 nmol , i . c . v . , at reperfusion ) in WT and H3R − / − mice ( scale bar , 5 mm ) , and ( c ) bar graph showing infarct volume ( n = 6 - 8 mice per condition ; * P0 . 05 , * * * P0 . 001 , with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) . ( d ) TTC - stained sections ( scale bar , 5 mm ) and bar graph showing the infarct area after administration of thioperamide in Atg5 + / − mice ( n = 4 - 6 mice per condition ) . ( e ) In cultured neurons , viability was tested by MTT assay after 3 - MA ( 2 . 5 mmol l − 1 at reperfusion ) and thioperamide was administered in OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . ( f ) In cultured neurons , viability was tested by MTT after siRNA for Atg7 and OGD / R ( n = 7 per condition ; * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . ( g ) Viability was tested by MTT assay on Atg5 + / + and Atg5 − / − MEFs after thioperamide administration after OGD / R ( n = 7 per condition ; * * P0 . 01 , * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Data are presented as mean ± s . e . m .

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

f6(a) Western blots showing the inhibitory effect of 3-MA on autophagy in I/R. Full-size blots are shown in Supplementary Fig. 13.(b) TTC-stained sections showing the infarct area after administration of 3-MA (100 nmol, i.c.v., at reperfusion) in WT and H3R−/− mice (scale bar, 5 mm), and (c) bar graph showing infarct volume (n=6-8 mice per condition; *P0.05, ***P0.001, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).(d) TTC-stained sections (scale bar, 5 mm) and bar graph showing the infarct area after administration of thioperamide in Atg5+/−mice (n=4-6 mice per condition).(e) In cultured neurons, viability was tested by MTT assay after 3-MA (2.5 mmol l−1 at reperfusion) and thioperamide was administered in OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).(f) In cultured neurons, viability was tested by MTT after siRNA for Atg7 and OGD/R (n=7 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).(g) Viability was tested by MTT assay on Atg5+/+and Atg5−/− MEFs after thioperamide administration after OGD/R (n=7 per condition; **P0.01, ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test). Data are presented as mean±s.e.m.


f7 ( a ) Representative co - immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD / R . Full - size blots are shown in Supplementary Fig . 14 . ( b ) Cell viability was tested by MTT assay after administration of Tat - H3RCT414 - 436 ( 10 − 6 mol l − 1 at reperfusion ) under OGD / R ( data are presented as mean ± s . e . m . ; n = 7 per condition ; * P0 . 05 , * * P0 . 01 , with analysis of variances ( ANOVAs ) followed by the Bonferroni / Dunn post hoc test ) . ( c ) Representative western blots showing the effect of Tat - H3RCT414 - 436 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling and LC3 expression in OGD / R . Full - size blots are shown in Supplementary Fig . 14 . ( d ) Representative images of the effect of Tat - H3RCT414 - 436 on LC3 - positive autophagic puncta ( red ) in 4 ' , 6 - diamidino - 2 - phenylindole - stained ( blue ) neurons after OGD / R ( scale bar , 10 μm ) . ( e ) Western blots showing the effect of siRNA for CLIC4 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling and LC3 expression in OGD / R . Full - size blots are shown in Supplementary Fig . 14 . ( f ) Representative western blots and bar graph showing the expression of LC3 - I and LC3 - II on steady - state autophagy and autophagic flux under OGD / R when CLIC4 was knocked down ( data are presented as mean ± s . e . m . ; n = 6 ; * P0 . 05 , * * P0 . 01 ; , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Full - size blots are shown in Supplementary Fig . 14 . ( g ) Cell viability was assessed by MTT assay after siRNA for CLIC4 under OGD / R ( data are presented as mean ± s . e . m . ; n = 8 per condition ; * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc 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 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 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 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 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

f7(a) Representative co-immunoprecipitation results showing the effect of thioperamide on the interaction of H3R with CLIC4 during OGD/R. Full-size blots are shown in Supplementary Fig. 14.(b) Cell viability was tested by MTT assay after administration of Tat-H3RCT414-436 (10−6 mol l−1 at reperfusion) under OGD/R (data are presented as mean±s.e.m.; n=7 per condition; *P0.05, **P0.01, with analysis of variances (ANOVAs) followed by the Bonferroni/Dunn post hoc test).(c) Representative western blots showing the effect of Tat-H3RCT414-436 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.(d) Representative images of the effect of Tat-H3RCT414-436 on LC3-positive autophagic puncta (red) in 4',6-diamidino-2-phenylindole-stained (blue) neurons after OGD/R (scale bar,10 μm).(e) Western blots showing the effect of siRNA for CLIC4 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.(f) Representative western blots and bar graph showing the expression of LC3-I and LC3-II on steady-state autophagy and autophagic flux under OGD/R when CLIC4 was knocked down (data are presented as mean±s.e.m.; n=6; *P0.05, **P0.01; , with ANOVAs followed by the Bonferroni/Dunn post hoc test). Full-size blots are shown in Supplementary Fig. 14.(g) Cell viability was assessed by MTT assay after siRNA for CLIC4 under OGD/R (data are presented as mean±s.e.m.; n=8 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


Figure 1 ( C ) Graph showing log10 CFU / gland at 4 days post - inoculation with the indicated strains ( n ≥ 5 ) . Each circle represents the values obtained in individual animals , whereas mean ± s . d . is represented with lines inside each group . No statistical differences were found between infection groups using a one - sided ANOVA followed by the post - hoc Fisher ' s PLSD test .

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Figure 1(C) Graph showing log10 CFU/gland at 4 days post-inoculation with the indicated strains (n≥5). Each circle represents the values obtained in individual animals, whereas mean ± s.d. is represented with lines inside each group. No statistical differences were found between infection groups using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test.


Figure 2Plots showing the RT - qPCR analysis conducted to quantify relative expression of the indicated interleukins coding genes in the mammary glands . Results are expressed as the mean ± s . d ( n = 10 for PBS , n = 5 for WT and n = 4 for CV2 ) of the 2 - ΔΔCt relative expression values of the indicated interleukins ; the value from each individual animal was calculated from three technical replicates . The values obtained in the PBS group were used as control for normalization of gene expression ( = 1 ) . Statistical analysis was performed using a one - sided ANOVA followed by the post - hoc Fisher ' s PLSD test : ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 005 ) .

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

Figure 2Plots showing the RT-qPCR analysis conducted to quantify relative expression of the indicated interleukins coding genes in the mammary glands. Results are expressed as the mean ± s.d (n=10 for PBS, n=5 for WT and n=4 for CV2) of the 2-ΔΔCt relative expression values of the indicated interleukins; the value from each individual animal was calculated from three technical replicates. The values obtained in the PBS group were used as control for normalization of gene expression (=1). Statistical analysis was performed using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test: (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005).


Figure 3 ( B ) Comparison of the turbidimetric assay results obtained with mpn142 and mpn142Opt secretion signals . Culture supernatants ( n = 1 ) were collected at 0 h , 5 h or 24 h post - inoculation as indicated . WT strain and medium ( Hayflick ) were added as controls .

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Figure 3(B) Comparison of the turbidimetric assay results obtained with mpn142 and mpn142Opt secretion signals. Culture supernatants (n=1) were collected at 0 h, 5 h or 24 h post-inoculation as indicated. WT strain and medium (Hayflick) were added as controls.


Figure 4 ( A , B ) Mature S . aureus biofilms were allowed to develop for 24 h in polystyrene plates and then treated for the indicated time intervals with cell suspensions , or culture supernatants of the CV2 or the CV2 - DispB strains ( A ) or the WT or the WT - DispB strain ( B ) . Biofilm presence was assessed by crystal violet staining and included a negative staining control ( i . e . , crystal violet without biofilm ) . The results are expressed as mean ± s . d . of OD 595 nm absorbance values obtained from three different biological replicates ( n = 3 ) and statistically compared by one - sided ANOVA followed by the post - hoc Fisher ' s PLSD test : * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 ; * * * * P ≤ 0 .

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

Figure 4(A, B) Mature S. aureus biofilms were allowed to develop for 24 h in polystyrene plates and then treated for the indicated time intervals with cell suspensions, or culture supernatants of the CV2 or the CV2-DispB strains (A) or the WT or the WT-DispB strain (B). Biofilm presence was assessed by crystal violet staining and included a negative staining control (i.e., crystal violet without biofilm). The results are expressed as mean ± s.d. of OD 595 nm absorbance values obtained from three different biological replicates (n=3) and statistically compared by one-sided ANOVA followed by the post-hoc Fisher's PLSD test: *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005; ****P ≤ 0.00005


Figure 5 ( C , D ) Plots showing results obtained from the in vitro or ex vivo dispersion assays with the indicated strains and from the staining control . Each circle represents the OD 595 nm values obtained for individual catheters ( n ≥ 4 ) , whereas mean ± s . d . is represented with lines inside each group . Results from one - sided ANOVA followed by Fisher ' s PLSD test are shown for treatments statistically different from those based on strains not secreting dispersin B ( i . e . , the WT or CV2 strains ) . * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 .

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

Figure 5(C, D) Plots showing results obtained from the in vitro or ex vivo dispersion assays with the indicated strains and from the staining control. Each circle represents the OD 595 nm values obtained for individual catheters (n≥4), whereas mean ± s.d. is represented with lines inside each group. Results from one-sided ANOVA followed by Fisher's PLSD test are shown for treatments statistically different from those based on strains not secreting dispersin B (i.e., the WT or CV2 strains). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.


Figure 6 ( C , D ) Plots showing the SUV60 variation ( % ) between day 1 and day 4 of the different treatments . Each circle represents the SUV60 variation obtained for individual animals ( n ≥ 4 ) , whereas mean ± s . d . is represented with lines inside each group . Data below the dotted lines indicate that the SUV 60 values decreased at D4 post - treatment . Results from one - sided ANOVA followed by Fisher ' s PLSD test are shown . * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 .

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

Figure 6(C, D) Plots showing the SUV60 variation (%) between day 1 and day 4 of the different treatments. Each circle represents the SUV60 variation obtained for individual animals (n≥4), whereas mean ± s.d. is represented with lines inside each group. Data below the dotted lines indicate that the SUV 60 values decreased at D4 post-treatment. Results from one-sided ANOVA followed by Fisher's PLSD test are shown. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.


Figure 1 ( A ) Yeast clones harbouring empty bait vector ( pYTH9 , mock ) or those encoding ScAtg8 , LC3B , Atg12 and SUMO1 were transformed with empty prey vector ( pACT2 , mock ) or those encoding fragments of ATG4A , ATG4B , NBR1 , p62 and full - length Nix . Interaction was assessed by yeast growth on the SD ‐ W / ‐ L / ‐ H medium with a β ‐ gal assay . ( B ) GST pulldown assays using cell extracts of Flag ‐ Nix ‐ expressing COS7 cells and immobilized GST or the indicated GST fusions . Coprecipitated Nix was detected with Flag antibodies . ( C ) Purified GST ‐ tagged NixΔTM was cleaved from the GST moiety by thrombin , purified and used for precipitation by GST fusion proteins . Precipitated proteins were analysed by western blotting ( WB ) with Nix antibodies . Ponceau S staining was used to visualize GST ‐ fusion proteins . ( D ) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated ( IP ) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies . β ‐ gal , β ‐ galactosidase ; BafA1 , bafilomycin ‐ A1 ; CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; GST , glutathione ‐ S ‐ transferase ; IgG , immunoglobulin G ; NixΔTM , recombinant Nix lacking a TM ; SUMO1 , small ubiquitin ‐ like modifier 1 ; TCL , total cell lysate ; TM , transmembrane domain .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O 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 1(A) Yeast clones harbouring empty bait vector (pYTH9, mock) or those encoding ScAtg8, LC3B, Atg12 and SUMO1 were transformed with empty prey vector (pACT2, mock) or those encoding fragments of ATG4A, ATG4B, NBR1, p62 and full-length Nix. Interaction was assessed by yeast growth on the SD‐W/‐L/‐H medium with a β‐gal assay.(B) GST pulldown assays using cell extracts of Flag‐Nix‐expressing COS7 cells and immobilized GST or the indicated GST fusions. Coprecipitated Nix was detected with Flag antibodies.(C) Purified GST‐tagged NixΔTM was cleaved from the GST moiety by thrombin, purified and used for precipitation by GST fusion proteins. Precipitated proteins were analysed by western blotting (WB) with Nix antibodies. Ponceau S staining was used to visualize GST‐fusion proteins.(D) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated (IP) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies. β‐gal, β‐galactosidase; BafA1, bafilomycin‐A1; CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; GST, glutathione‐S‐transferase; IgG, immunoglobulin G; NixΔTM, recombinant Nix lacking a TM; SUMO1, small ubiquitin‐like modifier 1; TCL, total cell lysate; TM, transmembrane domain.


Figure 2 ( C ) Lysates of COS7 cells transfected with Flag ‐ wt ‐ Nix or the indicated mutants were incubated with immobilized GST ‐ LC3A . The coprecipitated proteins were detected by western blotting ( WB ) with Flag antibodies . BNIP3 , Bcl2 / E1B 19kDa ‐ interacting protein 3 ‐ like protein ; Dr , Danio reio ; GST , glutathione ‐ S ‐ transferase ; Hs , Homo sapiens ; LIR , LC3 ‐ interacting region ; Mm , Mus musculus ; wt , wild type ; Xl , Xenopus laevis .

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

Figure 2(C) Lysates of COS7 cells transfected with Flag‐wt‐Nix or the indicated mutants were incubated with immobilized GST‐LC3A. The coprecipitated proteins were detected by western blotting (WB) with Flag antibodies. BNIP3, Bcl2/E1B 19kDa‐interacting protein 3‐like protein; Dr, Danio reio; GST, glutathione‐S‐transferase; Hs, Homo sapiens; LIR, LC3‐interacting region; Mm, Mus musculus; wt, wild type; Xl, Xenopus laevis.


Figure 3Nix recruits EGFP ‐ GABARAP ‐ L1 to stressed mitochondria in reconstituted Nix − / − MEFs . Nix − / − MEFs cotransfected with EGFP ‐ GABARAP ‐ L1 and wt ‐ Nix ( A ) , or Nix ‐ W35A ( B ) , were incubated with and without mitochondrial poison CCCP for 3 h . Cells were then fixed and analysed for colocalization between EGFP ‐ GABARAP ‐ L1 and Nix ( stained with Flag antibodies ) . The final panel in each row shows colocalization between Flag ‐ Nix ‐ Cy3 and GABARAP ‐ L1 using the ' colocalization ' highlighter plug ‐ in for ImageJ software ( NIH , Bethesda , MA , USA ) . ( C ) Quantification was achieved by counting the number of complete colocalizations , EGFP / Cy3 per cell for approximately 30 cells per condition . Results are expressed as a percentage of colocalization ( EGFP : Cy3 ) in control and CCCP ‐ treated cells . Error bars in ( C ) show the standard deviation ( s . d . ) of three independent experiments . Statistical analysis was performed using the Mann - Whitney t ‐ test . CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; DMSO , dimethyl sulfoxide ; EGFP , enhanced green fluorescent protein ; MEF , mouse embryonic fibroblast ; wt , wild type .

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 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

Figure 3Nix recruits EGFP‐GABARAP‐L1 to stressed mitochondria in reconstituted Nix−/− MEFs. Nix−/− MEFs cotransfected with EGFP‐GABARAP‐L1 and wt‐Nix (A), or Nix‐W35A (B), were incubated with and without mitochondrial poison CCCP for 3 h. Cells were then fixed and analysed for colocalization between EGFP‐GABARAP‐L1 and Nix (stained with Flag antibodies). The final panel in each row shows colocalization between Flag‐Nix‐Cy3 and GABARAP‐L1 using the 'colocalization' highlighter plug‐in for ImageJ software (NIH, Bethesda, MA, USA).(C) Quantification was achieved by counting the number of complete colocalizations, EGFP/Cy3 per cell for approximately 30 cells per condition. Results are expressed as a percentage of colocalization (EGFP:Cy3) in control and CCCP‐treated cells. Error bars in (C) show the standard deviation (s.d.) of three independent experiments. Statistical analysis was performed using the Mann-Whitney t‐test. CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; wt, wild type.


Figure 4 ( A ) Flow cytometry of reticulocyte ‐ enriched blood , cultured in vitro for 3 days , stained with MTR . The blood was taken from mice transplanted with Nix − / − bone marrow and reconstituted with wt ‐ Nix and Nix ‐ W35A viruses . Reticulocytes were induced by phenylhydrazine treatment . The viruses also express GFP . ( B ) Mitochondrial clearance in the GFP ‐ positive fraction of the reticulocyte ‐ enriched blood , as described in panel ( A ) . Mice were reconstituted with wt ‐ Nix , LIR ‐ W35A or empty vector viruses . Data from Nix − / − and Nix + / + mice are shown for comparison . ( C ) Anti ‐ Flag , anti ‐ GFP and anti ‐ β ‐ actin western blots of fetal liver cells transduced with Flag ‐ Nix and Flag ‐ W35A ‐ Nix viruses , normalized to total protein . GFP , green fluorescent protein ; LIR , LC3 ‐ interacting region ; MTR , MitoTracker Red ; wt , wild type .

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 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

Figure 4(A) Flow cytometry of reticulocyte‐enriched blood, cultured in vitro for 3 days, stained with MTR. The blood was taken from mice transplanted with Nix−/− bone marrow and reconstituted with wt‐Nix and Nix‐W35A viruses. Reticulocytes were induced by phenylhydrazine treatment. The viruses also express GFP.(B) Mitochondrial clearance in the GFP‐positive fraction of the reticulocyte‐enriched blood, as described in panel (A). Mice were reconstituted with wt‐Nix, LIR‐W35A or empty vector viruses. Data from Nix−/− and Nix+/+ mice are shown for comparison.(C) Anti‐Flag, anti‐GFP and anti‐β‐actin western blots of fetal liver cells transduced with Flag‐Nix and Flag‐W35A‐Nix viruses, normalized to total protein. GFP, green fluorescent protein; LIR, LC3‐interacting region; MTR, MitoTracker Red; wt, wild type.


Figure 1 ( A ) Western Blot for pUb on HeLa control and FBXO7 - / - cells after treatment with AO for indicated times . ( B ) Quantification of pUb signal relative to loading . Western Blots run in triplicates from three independent clones . Error bars depict S . D . . Data from three biological replicates . One - way ANOVA with multiple comparisons . p ( * * ) = 0 . 0011 ; p ( * * * * ) < 0 . 0001 . ( D ) Western Blot for pUb on extracts from day 12 iNeuron control and FBXO7 - / - cells after treatment with BafA or AO for indicated times .

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 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

Figure 1(A) Western Blot for pUb on HeLa control and FBXO7-/- cells after treatment with AO for indicated times.(B) Quantification of pUb signal relative to loading. Western Blots run in triplicates from three independent clones. Error bars depict S.D.. Data from three biological replicates. One-way ANOVA with multiple comparisons. p(**) = 0.0011; p(****)<0.0001.(D) Western Blot for pUb on extracts from day 12 iNeuron control and FBXO7-/- cells after treatment with BafA or AO for indicated times.


Figure 2 ( A ) 3D - SIM images of HeLa control , PINK1 - / - and FBXO7 - / - cell lines after AO - induced mitophagy . Cells were stained for nuclear DNA ( DAPI ) , mitochondria ( HSP60 ) and pUb . Zoom - ins of regions of interested are enlarged in the middle panel . 3D - surface renderings of insets are shown on the right . Scale bar = 5 µm or 1 µm . ( B , C ) Evaluation of 3D - SIM images from HeLa datasets . The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy - induction are plotted . Error bars depict S . D . from 8 - 14 measured cells per condition . Two - way ANOVA with multiple comparisons ; p ( * * * * ) < 0 . 0001 . ( D ) 3D - SIM images of iN day 12 control , PINK1 - / - and FBXO7 - / - cell lines after AO - induced mitophagy . Cells were stained for nuclear DNA ( DAPI ) , mitochondria ( HSP60 ) and pUb . Zoom - ins of regions of interest are enlarged in the middle panel . 3D - surface renderings of insets are shown on the right . Scale bar = 5 µm or 1 µm . ( E , F ) Evaluation of 3D - SIM images from iNeuron datasets . The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy - induction are plotted . Error bars depict S . D . from 7 - 14 measured cells per condition . One - way ANOVA with multiple comparisons , p ( * * * * ) < 0 . 0001 . ( G ) Confocal images of iNeuron d12 Control , PINK1 - / - and FBXO7 - / - cell lines after AO - induced mitophagy . Cells were stained for nuclear DNA ( Hoechst33342 ) , mitochondria ( HSP60 ) and pUb . Scale bar = 10 µm and 5 µm . ( H ) Evaluation of pUb volume after mitophagy induction . Error bars depict S . D . from three biological replicates . One - way ANOVA with multiple comparisons ; p ( * * * * ) < 0 . 0001 .

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 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 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 O O O O O O O O O O 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) 3D-SIM images of HeLa control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (DAPI), mitochondria (HSP60) and pUb. Zoom-ins of regions of interested are enlarged in the middle panel. 3D-surface renderings of insets are shown on the right. Scale bar = 5 µm or 1 µm.(B,C) Evaluation of 3D-SIM images from HeLa datasets. The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy-induction are plotted. Error bars depict S.D. from 8-14 measured cells per condition. Two-way ANOVA with multiple comparisons; p(****)<0.0001.(D) 3D-SIM images of iN day 12 control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (DAPI), mitochondria (HSP60) and pUb. Zoom-ins of regions of interest are enlarged in the middle panel. 3D-surface renderings of insets are shown on the right. Scale bar = 5 µm or 1 µm.(E,F) Evaluation of 3D-SIM images from iNeuron datasets. The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy-induction are plotted. Error bars depict S.D. from 7-14 measured cells per condition. One-way ANOVA with multiple comparisons, p(****)<0.0001.(G) Confocal images of iNeuron d12 Control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (Hoechst33342), mitochondria (HSP60) and pUb. Scale bar = 10 µm and 5 µm. (H) Evaluation of pUb volume after mitophagy induction. Error bars depict S.D. from three biological replicates. One-way ANOVA with multiple comparisons; p(****)<0.0001.


Figure 3 ( B ) Mean Acidic : Neutral mtKeima per - cell ratios measured by flow cytometry for HeLa cells expressing Parkin ndicating the number of hours treated with AO ( Antimycin A ( 5 µM ) and Oligomycin ( 10 µM ) ) or three hours with 25 nM BafilomycinA ( BafA ) . Error bars depict S . D . from biological triplicate measurements from three independent clones . Two - way ANOVA with multiple comparisons ; p ( * * * * ) < 0 . 0001 . ( C ) Bar graph showing the number of mtDNA puncta / cell with or without treatment with AO ( 6 h ) in HeLa control or FBXO7 - / - cells . Error bars depict S . D . from triplicate technical replicates from two independent clones . One - way ANOVA with multiple comparisons ; p ( * * * * ) < 0 . 0001 ( D ) Indicated hESCs expressing mtKeima were differentiated to iNeurons ( day 12 ) and were subjected to image analysis . Scale bar , 5 μm . ( E ) The number of red - shifted Keima foci per cell is plotted , where each dot represents the average foci - number per image stack , originating from 7 - 12 image stacks of iNeuron differentiations . Error bars represent S . D . . One - way ANOVA with multiple comparisons ; p ( * * * ) = 0 . 0001 , p ( * * * * ) < 0 . 0001 . ( F ) mtKeima mitophagy flux readout of indicated iNeuron genotypes . Cells were treated with AO for either 1 or 6 h and mitophagic flux measured by flow cytometry ( > 10 , 000 cells ) . Pooled data from biological replicates is shown , normalized to 6h BafA treated cells .

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

Figure 3(B) Mean Acidic:Neutral mtKeima per-cell ratios measured by flow cytometry for HeLa cells expressing Parkin ndicating the number of hours treated with AO (Antimycin A (5 µM) and Oligomycin (10 µM)) or three hours with 25 nM BafilomycinA (BafA). Error bars depict S.D. from biological triplicate measurements from three independent clones. Two-way ANOVA with multiple comparisons; p(****)<0.0001.(C) Bar graph showing the number of mtDNA puncta/cell with or without treatment with AO (6 h) in HeLa control or FBXO7-/- cells. Error bars depict S.D. from triplicate technical replicates from two independent clones. One-way ANOVA with multiple comparisons; p(****)<0.0001(D) Indicated hESCs expressing mtKeima were differentiated to iNeurons (day 12) and were subjected to image analysis. Scale bar, 5 μm. (E) The number of red-shifted Keima foci per cell is plotted, where each dot represents the average foci-number per image stack, originating from 7-12 image stacks of iNeuron differentiations. Error bars represent S.D.. One-way ANOVA with multiple comparisons; p(***)=0.0001, p(****)<0.0001.(F) mtKeima mitophagy flux readout of indicated iNeuron genotypes. Cells were treated with AO for either 1 or 6 h and mitophagic flux measured by flow cytometry (>10,000 cells). Pooled data from biological replicates is shown, normalized to 6h BafA treated cells.


Figure 4 ( A ) Workflow for analysis of total protein abundance in HeLa cells expressing Parkin with and without depolarization with AO ( 16 h ) . Cell extracts from triplicates were digested with trypsin prior to 18 - plex TMT labelling and analysis by mass spectrometry . ( F ) PINK1 levels in control cells and in two FBXO7 - / - clones were measured by TMT - proteomics in fed cells , from biological triplicates from two independent clones .

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Figure 4(A) Workflow for analysis of total protein abundance in HeLa cells expressing Parkin with and without depolarization with AO (16 h). Cell extracts from triplicates were digested with trypsin prior to 18-plex TMT labelling and analysis by mass spectrometry.(F) PINK1 levels in control cells and in two FBXO7-/- clones were measured by TMT-proteomics in fed cells, from biological triplicates from two independent clones.


Figure 5 ( B ) Log2 FC for the indicated mitochondrial protein - groups in control or FBXO7 - / - cells at day 0 , 4 or 12 during neurogenesis is shown in violin plots . ( C ) Log2 FC for the indicated cellular organelle proteins in control or FBXO7 - / - cells at day 0 , 4 or 12 during neurogenesis is shown in violin plots . ( F , G ) Mitochondrial morphology in iNeurons ( iN ) was assessed using confocal imaging after staining cells with HSP60 to detect mitochondria and DAPI to identify nuclei , in either fed cells or cells treated with AO ( 3 h ) ( left panel ) . The number of median # of mitochondria / cell , median mitochondrial length , and median mitochondrial circularity is shown . Error bars depict S . D . from triplicates from three differentiations ( 9 - 12 image stacks each ) , as shown in panel I left , center , and right , respectively . Two - way ANOVA with multiple comparisons ; p ( * ) = 0 . 0416 , p ( * * * * ) < 0 . 0001 . Scale bar = 10 µm . ( H ) Western Blot analysis on HeLa control and FBXO7 - / - whole cell lysate , probed for the mitochondrial fission adapter MiD51 .

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

Figure 5(B) Log2 FC for the indicated mitochondrial protein-groups in control or FBXO7-/- cells at day 0, 4 or 12 during neurogenesis is shown in violin plots. (C) Log2 FC for the indicated cellular organelle proteins in control or FBXO7-/- cells at day 0, 4 or 12 during neurogenesis is shown in violin plots.(F,G) Mitochondrial morphology in iNeurons (iN) was assessed using confocal imaging after staining cells with HSP60 to detect mitochondria and DAPI to identify nuclei, in either fed cells or cells treated with AO (3 h) (left panel). The number of median # of mitochondria/cell, median mitochondrial length, and median mitochondrial circularity is shown. Error bars depict S.D. from triplicates from three differentiations (9-12 image stacks each), as shown in panel I left, center, and right, respectively. Two-way ANOVA with multiple comparisons; p(*)=0.0416, p(****)<0.0001. Scale bar = 10 µm.(H) Western Blot analysis on HeLa control and FBXO7-/- whole cell lysate, probed for the mitochondrial fission adapter MiD51.


figf1 ( a ) Induction of GFP + dots in differentiated THP - 1 cells stably expressing GFP - LC3 , left untransfected or transfected for 6 h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , then exposed to control antibody ( Control Ab ) , antibody to neutralize IL - 1β ( Anti - IL - 1β ) , dimethyl sulfoxide ( DMSO ) or 3 - MA ( 5 mM ) ; results are presented as quantification of GFP - LC3 + dots ( top ) and images of individual cells ( middle ) . Scale bars , 10 μm . ( b ) Expression of AIM2 and beclin - 1 in cells transfected with control siRNA or siRNA targeting AIM2 or beclin - 1 ; numbers below lanes indicate band intensity ( ratio of cells transfected with targeting siRNA to those transfected with control siRNA ) . ( c ) Induction of GFP + dots in differentiated THP - 1 cells stably expressing GFP - LC3 and treated with various siRNAs ( below graph ) , quantified 6 h after transfection with poly ( dA : dT ) ( + ) or no transfection ( - ) . ( d ) Immunoblot analysis of the expression of LC3 - I and LC3 - II ( above ) in wild - type ( WT ) and Aim2 − / − BMDMs left untransfected or transfected for 6 h with poly ( dA : dT ) ; numbers below lanes indicate division of the ratio of induced LC3 - I to induced LC3 - II by the ratio of basal LC3 - I to basal LC3 - II ( band intensities ) . Below , immunoblot analysis of IL - 1β in supernatants after overnight culture . ( e ) Immunoblot analysis of LC3 - I and LC3 - II in or wild - type , ASC - deficient ( Pycard - / - ; called ' Asc - / - ' here ) or caspase - 1 - deficient ( Casp1 - / - ) BMDMs primed with LPS ( 20 ng / ml ) and then left untransfected or transfected with poly ( dA : dT ) or left untreated or treated with nigericin for 4 h ; numbers below lanes as in d . Actin serves as a loading control throughout . NS , not significant ; * P 0 . 05 and * * P 0 . 01 ( unpaired t - test ) . Data are from three individual

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 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 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 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

figf1(a) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3, left untransfected or transfected for 6 h with poly(dA:dT) (1.5 μg/ml), then exposed to control antibody (Control Ab), antibody to neutralize IL-1β (Anti-IL-1β), dimethyl sulfoxide (DMSO) or 3-MA (5 mM); results are presented as quantification of GFP-LC3+ dots (top) and images of individual cells (middle). Scale bars, 10 μm.(b) Expression of AIM2 and beclin-1 in cells transfected with control siRNA or siRNA targeting AIM2 or beclin-1; numbers below lanes indicate band intensity (ratio of cells transfected with targeting siRNA to those transfected with control siRNA).(c) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3 and treated with various siRNAs (below graph), quantified 6 h after transfection with poly(dA:dT) (+) or no transfection (-).(d) Immunoblot analysis of the expression of LC3-I and LC3-II (above) in wild-type (WT) and Aim2−/− BMDMs left untransfected or transfected for 6 h with poly(dA:dT); numbers below lanes indicate division of the ratio of induced LC3-I to induced LC3-II by the ratio of basal LC3-I to basal LC3-II (band intensities). Below, immunoblot analysis of IL-1β in supernatants after overnight culture.(e) Immunoblot analysis of LC3-I and LC3-II in or wild-type, ASC-deficient (Pycard-/-; called 'Asc-/-' here) or caspase-1-deficient (Casp1-/-) BMDMs primed with LPS (20 ng/ml) and then left untransfected or transfected with poly(dA:dT) or left untreated or treated with nigericin for 4 h; numbers below lanes as in d. Actin serves as a loading control throughout. NS, not significant; *P 0.05 and **P 0.01 (unpaired t-test). Data are from three individual experiments


figf2 ( a ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells transfected for 3 h with Cy3 - labeled poly ( dA : dT ) ( 1 . 5 μg / ml ) . Yellow dots ( Merge ) indicate colocalization of green ( LC - 3 ) and red ( Cy3 ) signals . Scale bars , 10 μm . ( b ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells transfected to express Cy3 - poly ( dA : dT ) , imaged every 15 min starting 1 h after transfection ( numbers in bottom left corners indicate time in min ( m ) ) . Arrow indicates coalescence of the Cy3 and GFP signals ( also in bottom right image ) . ( c ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells sham - transfected with no poly ( dA : dT ) ( Control ) or transfected with poly ( dA : dT ) , then fixed and immunostained for ASC ( red ) . Insets ( bottom right ) , 5 × enlargement of areas indicated by arrowheads at left . Scale bars , 10 μm ( top ) or 20 μm ( bottom ) . ( d ) Confocal microscopy of differentiated THP - 1 cells transfected with poly ( dA : dT ) and immunostained for LAMP - 1 and ASC ; below , z - axis projections . Scale bars , 10 μm . ( e ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells treated for 2 h with LPS ( 500 ng / ml ) and ATP ( 3 mM ) , then immunostained for ASC ( red ) : left image in each pair , three - dimensional volume renderings reconstructed from z - stack images ; right image in each pair , surface - segmentation models ( 2 × enlargement ) . Scale bars , 10 μm . ( f ) Electron microscopy of differentiated THP - 1 cells stimulated with LPS and ATP ( left ) or transfected with poly ( dA : dT ) ( right ) for 2 h and immunostained for ASC , visualized with 15 - nm gold - conjugated protein A . Red arrows indicate ASC immunostaining . Right image in each pair , 4 . 5 × enlargement of outlined area at left . Scale bars , 500 nm . Data are representative of three experiments ( a , c ) or two experiments ( b , d - f ) .

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 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 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 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

figf2(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected for 3 h with Cy3-labeled poly(dA:dT) (1.5 μg/ml). Yellow dots (Merge) indicate colocalization of green (LC-3) and red (Cy3) signals. Scale bars, 10 μm.(b) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected to express Cy3-poly(dA:dT), imaged every 15 min starting 1 h after transfection (numbers in bottom left corners indicate time in min (m)). Arrow indicates coalescence of the Cy3 and GFP signals (also in bottom right image).(c) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells sham-transfected with no poly(dA:dT) (Control) or transfected with poly(dA:dT), then fixed and immunostained for ASC (red). Insets (bottom right), 5× enlargement of areas indicated by arrowheads at left. Scale bars, 10 μm (top) or 20 μm (bottom).(d) Confocal microscopy of differentiated THP-1 cells transfected with poly(dA:dT) and immunostained for LAMP-1 and ASC; below, z-axis projections. Scale bars, 10 μm.(e) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for ASC (red): left image in each pair, three-dimensional volume renderings reconstructed from z-stack images; right image in each pair, surface-segmentation models (2× enlargement). Scale bars, 10 μm.(f) Electron microscopy of differentiated THP-1 cells stimulated with LPS and ATP (left) or transfected with poly(dA:dT) (right) for 2 h and immunostained for ASC, visualized with 15-nm gold-conjugated protein A. Red arrows indicate ASC immunostaining. Right image in each pair, 4.5× enlargement of outlined area at left. Scale bars, 500 nm. Data are representative of three experiments (a,c) or two experiments (b,d-f).


figf3 ( a ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells left untransfected ( Control ) or transfected for 2 h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , then immunostained for endogenous AIM2 ( b ) Confocal microscopy of differentiated THP - 1 cells transfected for 3 h with poly ( dA : dT ) , then immunostained for LAMP - 1 and AIM2 . ( c ) Confocal microscopy of differentiated THP - 1 cells left untreated or treated for 2 h with LPS ( 500 ng / ml ) and ATP ( 3 mM ) , then immunostained for endogenous LC3 and NLRP3 . Scale bars , 10 μm . Data are representative of at least three 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 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 O O O O O O O

figf3(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells left untransfected (Control) or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), then immunostained for endogenous AIM2(b) Confocal microscopy of differentiated THP-1 cells transfected for 3 h with poly(dA:dT), then immunostained for LAMP-1 and AIM2.(c) Confocal microscopy of differentiated THP-1 cells left untreated or treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for endogenous LC3 and NLRP3. Scale bars, 10 μm. Data are representative of at least three experiments.


figf4 ( a ) Immunoblot analysis of AIM2 , ASC and LC3 - II in lysates of differentiated THP - 1 cells left untransfected or transfected with poly ( dA : dT ) ( 1 . 5 μg / ml ) and left untreated or treated with 3 - MA , rapamycin or amino - acid starvation , then separated into an inflammasome - enriched fraction ( top ) or an autophagosome - enriched fraction ( middle ) . Bottom , immunoblot analysis of total cell lysates . ( b ) Immunoblot analysis of unprocessed and processed caspase - 1 and IL - 1β in lysates and supernatants of differentiated THP - 1 cells treated as in a . ( c ) Immunoblot analysis of LC3 - I and LC3 - II ( top ) in lysates of mouse BMDMs primed with LPS ( 20 ng / ml ) and left untreated or treated with alum , then left unexposed or exposed for 5 h to live ( Live TB ) or dead ( Dead TB ) M . tuberculosis ( numbers below lanes as in Fig . 1d ) . Below , immunoblot analysis of IL - 1β in supernatants of LPS - primed mouse BMDMs infected overnight with live M . tuberculosis in the presence or absence of 3 - MA ( 2 mM ) ; treatment with LPS and ATP serves as a positive control . Data are representative of at least two 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) Immunoblot analysis of AIM2, ASC and LC3-II in lysates of differentiated THP-1 cells left untransfected or transfected with poly(dA:dT) (1.5 μg/ml) and left untreated or treated with 3-MA, rapamycin or amino-acid starvation, then separated into an inflammasome-enriched fraction (top) or an autophagosome-enriched fraction (middle). Bottom, immunoblot analysis of total cell lysates.(b) Immunoblot analysis of unprocessed and processed caspase-1 and IL-1β in lysates and supernatants of differentiated THP-1 cells treated as in a.(c) Immunoblot analysis of LC3-I and LC3-II (top) in lysates of mouse BMDMs primed with LPS (20 ng/ml) and left untreated or treated with alum, then left unexposed or exposed for 5 h to live (Live TB) or dead (Dead TB) M. tuberculosis (numbers below lanes as in Fig. 1d). Below, immunoblot analysis of IL-1β in supernatants of LPS-primed mouse BMDMs infected overnight with live M. tuberculosis in the presence or absence of 3-MA (2 mM); treatment with LPS and ATP serves as a positive control. Data are representative of at least two experiments.