( J ) 3D X - ray tomography reconstruction of a 16 - channel oCI implanted in a gerbil cochlea via the round window . Cables and μLEDs are marked in blue , the most apical µLED is indicated by the black arrowhead ; the basilar membrane is marked in green . µLEDs have a size of 60 × 60 µm .

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

(J) 3D X-ray tomography reconstruction of a 16-channel oCI implanted in a gerbil cochlea via the round window. Cables and μLEDs are marked in blue, the most apical µLED is indicated by the black arrowhead; the basilar membrane is marked in green. µLEDs have a size of 60×60 µm.


( A - D ) Exemplar STCs in response to SGN illumination with a single μLED ( A ) , a block of four neighboring μLEDs ( B ) and all μLEDs of an oCI inserted via the round window in a CatCh - transduced gerbil ( C ) and with all µLEDs in a wildtype gerbil ( WT ; D ) . μLEDs were spaced by 250 μm .

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

(A-D) Exemplar STCs in response to SGN illumination with a single μLED (A), a block of four neighboring μLEDs (B) and all μLEDs of an oCI inserted via the round window in a CatCh-transduced gerbil (C) and with all µLEDs in a wildtype gerbil (WT; D). μLEDs were spaced by 250 μm.


( E ) Acoustic auditory brainstem responses ( average of 1000 stimuli at a stimulation rate of 10 Hz ) upon click stimulation from 20 - 100 dB SPL before ( left ) and after deafening ( right ; star : threshold ) . ( F ) Optically evoked auditory brainstem responses before ( left ) and after deafening ( right ) , elicited by ~ 35 mW pulses of 1 ms duration , delivered by a laser - coupled fiber ( 1000 stimuli presented at 10 Hz ) , recorded from the same animal . Optogenetic activation of the auditory system was still possible after deafening . Differences in amplitude and waveform before and after deafening are likely due to different positioning of recording electrodes and the optical fiber .

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

(E) Acoustic auditory brainstem responses (average of 1000 stimuli at a stimulation rate of 10 Hz) upon click stimulation from 20-100 dB SPL before (left) and after deafening (right; star: threshold). (F) Optically evoked auditory brainstem responses before (left) and after deafening (right), elicited by ~35 mW pulses of 1 ms duration, delivered by a laser-coupled fiber (1000 stimuli presented at 10 Hz), recorded from the same animal. Optogenetic activation of the auditory system was still possible after deafening. Differences in amplitude and waveform before and after deafening are likely due to different positioning of recording electrodes and the optical fiber.


( E ) Acoustic auditory brainstem responses ( average of 1000 stimuli at a stimulation rate of 10 Hz ) upon click stimulation from 20 - 100 dB SPL before ( left ) and after deafening ( right ; star : threshold ) .

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

(E) Acoustic auditory brainstem responses (average of 1000 stimuli at a stimulation rate of 10 Hz) upon click stimulation from 20-100 dB SPL before (left) and after deafening (right; star: threshold).


( G - H ) Exemplar STCs in response to SGN illumination with all µLEDs in a CatCh - transduced , kanamycin - deafened gerbil before ( G ) and after ( H ) sacrifice .

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

(G-H) Exemplar STCs in response to SGN illumination with all µLEDs in a CatCh-transduced, kanamycin-deafened gerbil before (G) and after (H) sacrifice.


( I ) Maximum strength of ICC responses ( mean ± standard deviation ) evoked by oCI stimulation with 1 ( indiv . ; n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( block ; n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , and 16 ( all ; n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) active µLEDs , and with a laser - coupled optical fiber ( ~ 35 mW ; n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 73 . 25 , 4 degrees of freedom , p = 5 . 19 * 10 - 45 ) and post - hoc multiple comparison test ; indiv . vs . block : p = 9 . 9 * 10 - 9 ; block vs . all : p = 0 . 0096 ; all vs . fiber : p = 0 . 0016 ; all CatCh vs . all WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . oCI stimulation with all µLEDs has also been performed in WT animals ( n = 7 / 7 oCIs , N = 2 / 2 hearing / deaf gerbils ) and in sacrificed CatCh - transduced animals ( post - mortem ; n = 5 / 1 oCIs in N = 5 / 1 hearing / deaf gerbils ) . Brown dots indicate data recorded from deafened animals . ( J ) Number of active electrodes ( d ´ > 1 ; mean ± standard deviation ) upon oCI stimulation with 1 ( n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , and 16 µLEDs ( n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) , and with optical fiber stimulation ( n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 30 . 18 , 4 degrees of freedom , p = 1 . 42 * 10 - 21 ) and post - hoc multiple comparison test ; indiv . vs . block : p = 6 . 8 * 10 - 5 ; block vs . all : p = 0 . 006 ; all vs . fiber : p = 0 . 25 ; all CatCh vs . all WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . Responses to oCI stimulation with all µLEDs were also observed in hearing WT animals ( n = 3 oCIs in 2 gerbils ) . No active electrodes were found in response to 4 / 7 oCI stimulations in 2 / 2 hearing / deaf wildtype gerbils ) . ( K ) Thresholds ( d ´ = 1 , measured at the best electrode ( BE ) ) for ICC activation evoked with 16 active µLEDs on an oCI ( n = 24 / 9 in N = 9 / 3 hearing / deaf gerbils ) and with a laser - coupled optical fiber ( n = 9 / 3 in N = 9 / 3 hearing / deaf gerbils ; p = 0 . 012 , Wilcoxon rank sum test ) . Central horizontal line indicates the median , lower , upper horizontal lines indicate the minimum and maximum values , and box edges indicate the 25th and 75th percentile , respectively .

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

(I) Maximum strength of ICC responses (mean ± standard deviation) evoked by oCI stimulation with 1 (indiv.; n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (block; n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 (all; n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils) active µLEDs, and with a laser-coupled optical fiber (~35 mW; n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 73.25, 4 degrees of freedom, p = 5.19*10-45) and post-hoc multiple comparison test; indiv. vs. block: p = 9.9*10-9; block vs. all: p = 0.0096; all vs. fiber: p = 0.0016; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). oCI stimulation with all µLEDs has also been performed in WT animals (n = 7/7 oCIs, N = 2/2 hearing/deaf gerbils) and in sacrificed CatCh-transduced animals (post-mortem; n = 5/1 oCIs in N = 5/1 hearing/deaf gerbils). Brown dots indicate data recorded from deafened animals. (J) Number of active electrodes (d´ > 1; mean ± standard deviation) upon oCI stimulation with 1 (n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 µLEDs (n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils), and with optical fiber stimulation (n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 30.18, 4 degrees of freedom, p = 1.42*10-21) and post-hoc multiple comparison test; indiv. vs. block: p = 6.8*10-5; block vs. all: p = 0.006; all vs. fiber: p = 0.25; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). Responses to oCI stimulation with all µLEDs were also observed in hearing WT animals (n = 3 oCIs in 2 gerbils). No active electrodes were found in response to 4/7 oCI stimulations in 2/2 hearing/deaf wildtype gerbils). (K) Thresholds (d´ = 1, measured at the best electrode (BE)) for ICC activation evoked with 16 active µLEDs on an oCI (n = 24/9 in N = 9/3 hearing/deaf gerbils) and with a laser-coupled optical fiber (n = 9/3 in N = 9/3 hearing/deaf gerbils; p = 0.012, Wilcoxon rank sum test). Central horizontal line indicates the median, lower, upper horizontal lines indicate the minimum and maximum values, and box edges indicate the 25th and 75th percentile, respectively.


( I ) Maximum strength of ICC responses ( mean ± standard deviation ) evoked by oCI stimulation with 1 ( indiv . ; n = 129 / 77 µLEDs in N = 9 / 3 hearing / deaf gerbils ) , 4 ( block ; n = 62 / 27 blocks in N = 9 / 3 hearing / deaf gerbils ) , and 16 ( all ; n = 24 / 9 oCIs in N = 9 / 3 hearing / deaf gerbils ) active µLEDs , and with a laser - coupled optical fiber ( ~ 35 mW ; n = 9 / 3 fiber stimulations in N = 9 / 3 hearing / deaf gerbils ) in CatCh - transduced gerbils ( One - way ANOVA ( F - value : 73 . 25 , 4 degrees of freedom , p = 5 . 19 * 10 - 45 ) and post - hoc multiple comparison test ; indiv . vs . block : p = 9 . 9 * 10 - 9 ; block vs . all : p = 0 . 0096 ; all vs . fiber : p = 0 . 0016 ; all CatCh vs . all WT : p = 9 . 9 * 10 - 9 ; * * : p < 0 . 01 , * * * : p < 0 . 001 , n . s . = non - significant ) . oCI stimulation with all µLEDs has also been performed in WT animals ( n = 7 / 7 oCIs , N = 2 / 2 hearing / deaf gerbils ) and in sacrificed CatCh - transduced animals ( post - mortem ; n = 5 / 1 oCIs in N = 5 / 1 hearing / deaf gerbils ) . Brown dots indicate data recorded from deafened animals .

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

(I) Maximum strength of ICC responses (mean ± standard deviation) evoked by oCI stimulation with 1 (indiv.; n = 129/77 µLEDs in N = 9/3 hearing/deaf gerbils), 4 (block; n = 62/27 blocks in N = 9/3 hearing/deaf gerbils), and 16 (all; n = 24/9 oCIs in N = 9/3 hearing/deaf gerbils) active µLEDs, and with a laser-coupled optical fiber (~35 mW; n = 9/3 fiber stimulations in N = 9/3 hearing/deaf gerbils) in CatCh-transduced gerbils (One-way ANOVA (F-value: 73.25, 4 degrees of freedom, p = 5.19*10-45) and post-hoc multiple comparison test; indiv. vs. block: p = 9.9*10-9; block vs. all: p = 0.0096; all vs. fiber: p = 0.0016; all CatCh vs. all WT: p = 9.9*10-9;**: p < 0.01, ***: p < 0.001, n.s. = non-significant). oCI stimulation with all µLEDs has also been performed in WT animals (n = 7/7 oCIs, N = 2/2 hearing/deaf gerbils) and in sacrificed CatCh-transduced animals (post-mortem; n = 5/1 oCIs in N = 5/1 hearing/deaf gerbils). Brown dots indicate data recorded from deafened animals.


( A ) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via the round window , from apical to basal direction ( A - D ) . White markers define the best electrode ( BE ) , and measurement of the spread of excitation ( SoE ) is indicated in the left panel . In STCs where more than one peak was detected , the dorsal - and ventral - most electrodes have been considered as the boundaries of the STC in order to avoid underestimation of the SoE .

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

(A) Exemplar STCs in response to SGN stimulation with four blocks of four µLEDs inserted via the round window, from apical to basal direction (A-D). White markers define the best electrode (BE), and measurement of the spread of excitation (SoE) is indicated in the left panel. In STCs where more than one peak was detected, the dorsal- and ventral-most electrodes have been considered as the boundaries of the STC in order to avoid underestimation of the SoE.


( C ) Spectral spread of excitation upon SGN stimulation with µLED - based oCIs ( blocks of 4 µLEDs , solid lines ; mean ± standard error of the mean ) , acoustic ( black dashed line ) , laser - coupled optical fibers ( blue dashed line ) , as well as mono - and bipolar electrical stimulation with a clinical - style eCI ( dashed and dotted red lines , respectively ) . Average data from stimulation via optical fibers and eCIs are replotted from Dieter et al ( Dieter et al , 2019 ) .

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

(C) Spectral spread of excitation upon SGN stimulation with µLED-based oCIs (blocks of 4 µLEDs, solid lines; mean ± standard error of the mean), acoustic (black dashed line), laser-coupled optical fibers (blue dashed line), as well as mono- and bipolar electrical stimulation with a clinical-style eCI (dashed and dotted red lines, respectively). Average data from stimulation via optical fibers and eCIs are replotted from Dieter et al (Dieter et al, 2019).


a , Silver staining of LC3 complexes purified from U2OS cells expressing ZZ - Flag - LC3 in normal medium or Earle ' s balanced salt solution ( EBSS ) for 2 h . Asterisks mark centriolar satellite proteins .

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

a, Silver staining of LC3 complexes purified from U2OS cells expressing ZZ-Flag-LC3 in normal medium or Earle's balanced salt solution (EBSS) for 2 h. Asterisks mark centriolar satellite proteins.


b - d , Co - immunoprecipitation ( IP ) of OFD1 with LC3 , LC3 with PCM1 , or OFD1 with PCM1 in HEK293T cells . HA , haemagglutinin .

O O O O O O O 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-d, Co-immunoprecipitation (IP) of OFD1 with LC3, LC3 with PCM1, or OFD1 with PCM1 in HEK293T cells. HA, haemagglutinin.


e , Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown ( KD ) HEK293T cells .

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

e, Western blotting analysis of PCM1 and OFD1 protein levels in control or PCM1 knockdown (KD) HEK293T cells.


f , Co - immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells . IP efficiency is calculated as the ratio of immunoprecipitated OFD1 / LC3 .

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

f, Co-immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T cells. IP efficiency is calculated as the ratio of immunoprecipitated OFD1/LC3.


g , h , Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation ( SS ) . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments .

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

g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 hserum starvation (SS). Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.


g , h , Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation ( SS ) , 50 nM bafilomycin A1 ( Baf ) , 20 µM chloroquine ( CQ ) or 1 µM MG132 . Quantified OFD1 level was normalized with alpha - tubulin . Similar results were observed in three independent experiments .

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

g, h, Western blotting analysis of protein levels of indicated proteins in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation (SS), 50 nM bafilomycin A1 (Baf), 20 µM chloroquine (CQ) or 1 µM MG132. Quantified OFD1 level was normalized with alpha-tubulin. Similar results were observed in three independent experiments.


a , Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin ( Ac - tubulin ) , or centriole marker γ - tubulin in hTERT - RPE1 cells in normal medium ( Un ) or subjected to 24 hserum starvation ( SS ) .

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

a, Representative confocal images of endogenous OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole marker γ-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to 24 hserum starvation (SS).


b , Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5 + / + and Atg5 − / − MEFs subjected to 24 hserum starvation . a , b , Data shown represent mean ± s . d . percentage of cells with centriolar satelliteOFD1 for 100 cells per well in triplicate samples . * * * P 0 . 001 , two - tailed unpaired student ' s t - test . Similar results were observed in three independent experiments .

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

b, Representative confocal images of endogenous OFD1 and acetylated tubulin in Atg5+/+ and Atg5−/− MEFs subjected to 24 hserum starvation. a, b, Data shown represent mean ± s.d. percentage of cells with centriolar satelliteOFD1 for 100 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments.


a , b , Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation . Data shown represent mean ± s . d . percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well , respectively , in triplicate samples . * * * P 0 . 001 , two - tailed unpaired student ' s t - test . Similar results were observed in three independent experiments . Un , untreated ; SS , serum - starved .

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

a, b, Representative confocal images of cilium marker acetylated tubulin from MEFs with indicated genotype subjected to 24 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia or length of the cilia for 500 cells or 100 cells per well, respectively, in triplicate samples. ***P  0.001, two-tailed unpaired student's t-test. Similar results were observed in three independent experiments. Un, untreated; SS, serum-starved.


a , Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 ( C - 19 ) cells , quantified OFD1 level was normalized with alpha - tubulin .

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

a, Western blotting analysis of OFD1 protein levels in control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was normalized with alpha-tubulin.


b , Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation . Data shown represent mean ± s . d . percentage of cells with primary cilia for 500 cells per well in triplicate samples . * * * P 0 . 001 , two - tailed unpaired student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Quantification of percentage of cells with primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation. Data shown represent mean ± s.d. percentage of cells with primary cilia for 500 cells per well in triplicate samples. ***P 0.001, two-tailed unpaired student's t-test.


c , Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation .

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

c, Quantification of length of primary cilia in C19 cells subject to 48 h or 72 h serum starvation.


d , e , Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation . Data shown represent 100 cells per well in triplicate samples .

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

d, e, Representative confocal images of primary cilia with variable length formed in MCF7 or C19 cells subject to 72 h serum starvation. Data shown represent 100 cells per well in triplicate samples.


d , e , Representative confocal images of primary cilia with variable length formed in C19 cells subject to 72 hserum starvation . Data shown represent 100 cells per well in triplicate samples .

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

d, e, Representative confocal images of primary cilia with variable length formed in C19 cells subject to 72 hserum starvation. Data shown represent 100 cells per well in triplicate samples.


f , Scanning electron microscope analysis of primary cilia ( marked by arrows ) formed in C - 19 cells subject to 72 h serum starvation and cycling Atg5 + / + MEFs in normal medium for 24 h . Similar results were observed in three independent experiments .

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

f, Scanning electron microscope analysis of primary cilia (marked by arrows) formed in C-19 cells subject to 72 h serum starvation and cycling Atg5+/+ MEFs in normal medium for 24 h. Similar results were observed in three independent experiments.


E ) Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF PLD into hnRNPA2 LCRK droplets . Removing all charged residues except for arginine from hnRNPA2 LC ( hnRNPA2 LCCD , R ) allows partitioning of hnRNPF PLD into droplets , indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning . hnRNPA2 LCRK does not phase separate much as hnRNPA2 LC at these conditions , see Figure S1J for quantification of phase separation of variants . Conditions : 20 µM proteins , 20 mM MES pH 5 . 5 50 mM NaCl , 150 mM urea . Scale bar : 10 µm .

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

E) Substitution of all arginines in hnRNPA2 LC with lysine prevents the partitioning of hnRNPF PLD into hnRNPA2 LCRK droplets. Removing all charged residues except for arginine from hnRNPA2 LC (hnRNPA2 LCCD,R) allows partitioning of hnRNPF PLD into droplets, indicating arginine in hnRNPA2 LC is required and necessary for hnRNPF partitioning. hnRNPA2 LCRK does not phase separate much as hnRNPA2 LC at these conditions, see Figure S1J for quantification of phase separation of variants. Conditions: 20 µM proteins, 20 mM MES pH 5.5 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.


A ) AlexaFluor488 - tagged ( green ) hnRNPA2 LC undergoes LLPS , while AlexaFluor555 - tagged ( red ) TOG D1 does not . However , TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1 : 1 ratio . Conditions : 20 µM indicated protein ( ~ 1 % fluorescently tagged ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 10 µm . hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD and TOG D1 samples were made concurrently . B ) Similar to hnRNPF PLD , AlexaFluor555 - tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488 - tagged FUS LC droplets with both proteins at 300 µM . Conditions : 300 µM proteins ( ~ 1 % fluorescently tagged ) , 20 mM MES pH 5 . 5 150 mM NaCl , 150 mM urea . Scale bar : 10 µm .

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

A) AlexaFluor488-tagged (green) hnRNPA2 LC undergoes LLPS, while AlexaFluor555-tagged (red) TOG D1 does not. However, TOG D1 partitions into hnRNPA2 LC droplets when mixed at a 1:1 ratio. Conditions: 20 µM indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. hnRNPA2 LC control duplicated from Figure 1A as hnRNPF PLD and TOG D1 samples were made concurrently. B) Similar to hnRNPF PLD, AlexaFluor555-tagged TOG D1 does not undergo LLPS at 300 µM or partition into AlexaFluor488-tagged FUS LC droplets with both proteins at 300 µM. Conditions: 300 µM proteins (~1% fluorescently tagged), 20 mM MES pH 5.5 150 mM NaCl, 150 mM urea. Scale bar: 10 µm.


E ) AlexaFluor555 - tagged hnRNPF PLD or FL and AlexaFluor405 - tagged TOG D1 partition simultaneously into AlexaFluor488 - tagged hnRNPA2 LC droplets . Conditions : 20 µM of each indicated protein ( ~ 1 % fluorescently tagged ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 20 µm .

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

E) AlexaFluor555-tagged hnRNPF PLD or FL and AlexaFluor405-tagged TOG D1 partition simultaneously into AlexaFluor488-tagged hnRNPA2 LC droplets. Conditions: 20 µM of each indicated protein (~1% fluorescently tagged), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 20 µm.


C ) Fluorescence micrographs showing that although TOG D1 and hnRNPF PLD partition into hnRNPA2 LC droplets ( rows 1 and 3 ) , they are unable to partition into tyrosine phosphorylated hnRNPA2 LC droplets ( rows 2 and 4 ) . Conditions : 20 µM proteins ( ~ 1 % fluorescently labeled ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 10 µm . D ) Phosphomimic variants containing 5 or 12 serine to glutamate substitutions ( hnRNPA2 LC5E and hnRNPA2 LC12E , respectively ) both allow partitioning of TOG D1 and hnRNPF PLD , even though hnRNPA2 LC12E barely phase separates at these conditions , indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 and hnRNPF PLD . See Appendix Figure S7B for quantification of LLPS of hnRNPA2 LC5E and hnRNPA2 LC12E . Conditions : 20 µM proteins ( ~ 1 % fluorescently labeled ) , 20 mM MES pH 5 . 5 , 50 mM NaCl , 150 mM urea . Scale bar : 10 µm .

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

C) Fluorescence micrographs showing that although TOG D1 and hnRNPF PLD partition into hnRNPA2 LC droplets (rows 1 and 3), they are unable to partition into tyrosine phosphorylated hnRNPA2 LC droplets (rows 2 and 4). Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm. D) Phosphomimic variants containing 5 or 12 serine to glutamate substitutions (hnRNPA2 LC5E and hnRNPA2 LC12E, respectively) both allow partitioning of TOG D1 and hnRNPF PLD, even though hnRNPA2 LC12E barely phase separates at these conditions, indicating that increased negative charge is insufficient to prevent partitioning of TOG D1 and hnRNPF PLD. See Appendix Figure S7B for quantification of LLPS of hnRNPA2 LC5E and hnRNPA2 LC12E. Conditions: 20 µM proteins (~1% fluorescently labeled), 20 mM MES pH 5.5, 50 mM NaCl, 150 mM urea. Scale bar: 10 µm.


A ) After cleavage of a C - terminal maltose binding protein solubility tag , hnRNPA2 FL WT undergoes LLPS and disease mutants D290V and P298L aggregate . In contrast , phosphorylated hnRNPA2 FL WT and P298L do not undergo LLPS or aggregation in the time frame tested . Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous aggregates in the time frame tested . Conditions : 20 µM proteins , 20 mM Tris pH 7 . 4 , 50 mM NaCl . Scale bar : 10

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

A) After cleavage of a C-terminal maltose binding protein solubility tag, hnRNPA2 FL WT undergoes LLPS and disease mutants D290V and P298L aggregate. In contrast, phosphorylated hnRNPA2 FL WT and P298L do not undergo LLPS or aggregation in the time frame tested. Phosphorylated hnRNPA2 FL D290V can form some structures resembling liquid droplets but does not form amorphous aggregates in the time frame tested. Conditions: 20 µM proteins, 20 mM Tris pH 7.4, 50 mM NaCl. Scale bar: 10 µm


D ) After 22 hours of paraquat induced oxidative stress , both HRPA - 1mScarlet and HRPA - 1HsLCWTmScarlet assemble more than when unstressed ( ANOVA p < 0 . 0001 ) . No significant differences were found within the no stress ( p = 0 . 8 ) stress groups ( p = 0 . 45 ) by ANOVA , indicating that there is no difference in assembly between HRPA - 1mScarlet and HRPA - 1HsLCWTmScarlet . N = 12 animals per genotype .

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

D) After 22 hours of paraquat induced oxidative stress, both HRPA-1mScarlet and HRPA-1HsLCWTmScarlet assemble more than when unstressed (ANOVA p<0.0001). No significant differences were found within the no stress (p=0.8) stress groups (p=0.45) by ANOVA, indicating that there is no difference in assembly between HRPA-1mScarlet and HRPA-1HsLCWTmScarlet. N = 12 animals per genotype.


F ) There is no significant difference in number of spots in HRPA - 1HsLCWTmScarlet lines with or without Fyn , although there seems to be a trend of reduced spots in HRPA - 1HsLCD290VmScarlet lines in animals also expressing Fyn * ; this difference is not statistically significant ( ANOVA ) . N = 12 animals per genotype . G ) After exposure to 22 hours paraquat induced oxidative stress there is no significant difference between HRPA - 1HsLCWTmScarlet lines with or without Fyn , although there seems to be a trend of reduced spots in HRPA - 1HsLCD290VmScarlet lines in animals also expressing Fyn * ; this difference is not statistically significant ( ANOVA ) . N = 12 animals per genotype .

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

F) There is no significant difference in number of spots in HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype. G) After exposure to 22 hours paraquat induced oxidative stress there is no significant difference between HRPA-1HsLCWTmScarlet lines with or without Fyn, although there seems to be a trend of reduced spots in HRPA-1HsLCD290VmScarlet lines in animals also expressing Fyn*; this difference is not statistically significant (ANOVA). N = 12 animals per genotype.


D ) Loss of C . elegans TDP - 43 ortholog , tdp - 1 , rescues glutamatergic tail / phasmid neurodegeneration caused by hrpa - 1HsLCD290V after 22 hours paraquat induced oxidative stress , but not hrpa - 1 ( ∆ ) . N = 4 - 12 animals / genotype / trial , significance from two tailed t - test ; tdp - 1 ∆ = tdp - 1 ( tgx58 ) . E ) Loss of C . elegans TDP - 43 ortholog , tdp - 1 , rescues glutamatergic tail / phasmid neurodegeneration caused by hrpa - 1HsLCD290V after 22 hours paraquat induced oxidative stress , but not hrpa - 1 ( ∆ ) . N = 4 - 12 animals / genotype / trial , significance from two tailed t - test ; tdp - 1 ∆ = tdp - 1 ( ok803 ) .

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

D) Loss of C. elegans TDP-43 ortholog, tdp-1, rescues glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not hrpa-1(∆). N=4-12 animals/genotype/trial, significance from two tailed t-test; tdp-1∆=tdp-1(tgx58). E) Loss of C. elegans TDP-43 ortholog, tdp-1, rescues glutamatergic tail/phasmid neurodegeneration caused by hrpa-1HsLCD290V after 22 hours paraquat induced oxidative stress, but not hrpa-1(∆). N=4-12 animals/genotype/trial, significance from two tailed t-test; tdp-1∆=tdp-1(ok803).


G ) Expression of Fyn * in sensory glutamatergic neurons ( osm - 10 promoter , unc - 54 3 ' UTR ) reduces glutamatergic tail / phasmid neuron degeneration in hrpa - 1HsLCD290V animals after 22 hours paraquat induced oxidative stress . N = 9 - 12 animals / genotype / trial , significance from two tailed t - test . H ) Expression of an empty Fyn control ( Fynempty ) in sensory glutamatergic neurons ( osm - 10 promoter , unc - 54 3 ' UTR ) does not alter glutamatergic phasmid neuron degeneration in hrpa - 1HsLCD290V animals after 22 hours paraquat induced oxidative stress . N = 9 - 12 animals / genotype / trial .

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

G) Expression of Fyn* in sensory glutamatergic neurons (osm-10 promoter, unc-54 3'UTR) reduces glutamatergic tail/phasmid neuron degeneration in hrpa-1HsLCD290V animals after 22 hours paraquat induced oxidative stress. N=9-12 animals/genotype/trial, significance from two tailed t-test. H) Expression of an empty Fyn control (Fynempty) in sensory glutamatergic neurons (osm-10 promoter, unc-54 3'UTR) does not alter glutamatergic phasmid neuron degeneration in hrpa-1HsLCD290V animals after 22 hours paraquat induced oxidative stress. N=9-12 animals/genotype/trial.


( F ) 5 - day - old Col - 0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh weight . Shown is the mean of relative fresh weight compared to the MS medium control - / + SD , n = 30 - 32 biological replicates from 2 pooled experiments ( one - way ANOVA , Tukey post - hoc test , a - b p < 0 . 001 ) .

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(F) 5-day-old Col-0Cas9 WT and CRISPR glv seedlings were treated with 100 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=30-32 biological replicates from 2 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b p<0.001).


( G ) Ethylene production was measured in Col - 0Cas9 WT and CRISPR glv 3 . 5 h after elicitation with 500 nM flg22 or co - treatment with 1 µM GLV2 . Ethylene production was normalized to the average of WT responses to flg22 set as 1 . Shown is the mean of n = 11 - 12 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc test , a - b p < 0 . 01 ) .

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

(G) Ethylene production was measured in Col-0Cas9 WT and CRISPR glv 3.5 h after elicitation with 500 nM flg22 or co-treatment with 1 µM GLV2. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=11-12 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.01).


( H ) Mean cfu of Pto DC3000 COR - 4 days after spray inoculation of Col - 0Cas9 WT and CRISPR glv ; n = 12 biological replicates from 3 pooled experiments - / + SD ( Students T - Test , * p < 0 . 05 ) .

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

(H) Mean cfu of Pto DC3000 COR- 4 days after spray inoculation of Col-0Cas9 WT and CRISPR glv; n=12 biological replicates from 3 pooled experiments-/+ SD (Students T-Test, *p<0.05).


( B ) ROS burst in WT after elicitation with 10 nM flg22 , 1 μM GLV2 , co - treatment or pre - treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22 . Shown is the mean of total ROS production ( Total RLU ) over 40 minutes normalized to the flg22 response set as 100 % - / + SD , n = 96 - 120 biological replicates from 15 pooled experiments ( one - way ANOVA , Tukey post - hoc test , a - b / c / d , p < 0 . 001 ; b - c , p < 0 . 05 ; b - d , p < 0 .

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

(B) ROS burst in WT after elicitation with 10 nM flg22, 1 μM GLV2, co-treatment or pre-treatment with 1 μM GLV2 for 5 h prior to elicitation with flg22. Shown is the mean of total ROS production (Total RLU) over 40 minutes normalized to the flg22 response set as 100% -/+ SD, n=96-120 biological replicates from 15 pooled experiments (one-way ANOVA, Tukey post-hoc test, a-b/c/d, p<0.001; b-c, p<0.05; b-d, p<0.001


( D ) PR1 expression was measured in 10 - day - old WT seedlings treated for 24 h with 100 nM flg22 , 1 μM GLV2 or co - treatment . Expression was normalized to the UBQ house - keeping gene . Shown is the mean of n = 4 . Symbol colors represent different biological

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

(D) PR1 expression was measured in 10-day-old WT seedlings treated for 24 h with 100 nM flg22, 1 μM GLV2 or co-treatment. Expression was normalized to the UBQ house-keeping gene. Shown is the mean of n=4. Symbol colors represent different biological replicates


( B ) Ethylene production was measured in the indicated genotypes 3 . 5 h after elicitation with mock or 500 nM flg22 . Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1 . Shown is the mean of n = 18 biological replicates from 3 pooled experiments - / + SD ( one - way ANOVA , Tukey post - hoc test , a - b p < 0 . 001 ; a - c , p < 0 . 001 ; b - c , p < 0 . 001 ) .

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

(B) Ethylene production was measured in the indicated genotypes 3.5 h after elicitation with mock or 500 nM flg22. Ethylene accumulation was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=18 biological replicates from 3 pooled experiments -/+ SD (one-way ANOVA, Tukey post-hoc test, a-b p<0.001; a-c, p<0.001; b-c, p<0.001).


( E ) Ethylene production was measured in WT and rgi5x and rgi5x pRGI3 : : RGI3 # 2 and # 3 3 . 5 h after elicitation with 500 nM flg22 . Ethylene production was normalized to the average of WT responses to flg22 set as 1 . Shown is the mean of n = 8 biological replicates from 2 pooled experiments - / + SD ( one - way ANOVA , Dunnett post - hoc test , a - b p < 0 . 05 ) .

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

(E) Ethylene production was measured in WT and rgi5x and rgi5x pRGI3::RGI3 #2 and #3 3.5 h after elicitation with 500 nM flg22. Ethylene production was normalized to the average of WT responses to flg22 set as 1. Shown is the mean of n=8 biological replicates from 2 pooled experiments -/+ SD (one-way ANOVA, Dunnett post-hoc test, a-b p<0.05).


( F ) 5 - day - old seedlings of the indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh weight . Shown is the mean of relative fresh weight compared to the MS medium control - / + SD , n = 36 biological replicates from 3 pooled experiments ( Students T - test , * * * p < 0 . 001 ) .

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

(F) 5-day-old seedlings of the indicated genotypes were treated with 100 nM elf18 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=36 biological replicates from 3 pooled experiments (Students T-test, ***p<0.001).


( C ) 5 - day - old seedlings of WT , rgi5x and rgi5x pRGI3 : : RGI3 # 2 and # 3 were treated with 10 nM flg22 or co - treated with 1 μM GLV2 for 7 days before measuring fresh weight . Shown is the mean of relative fresh weight compared to the MS medium control - / + SD , n = 32 biological replicates pooled from 4 experiments ( Students T - test , * * p < 0 . 01 ) .

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

(C) 5-day-old seedlings of WT, rgi5x and rgi5x pRGI3::RGI3 #2 and #3 were treated with 10 nM flg22 or co-treated with 1 μM GLV2 for 7 days before measuring fresh weight. Shown is the mean of relative fresh weight compared to the MS medium control -/+ SD, n=32 biological replicates pooled from 4 experiments (Students T-test, **p<0.01).


( D ) Quantitative analysis of synthetic GLV2 or GLV2 - S peptide binding to RGI3ECD using microscale thermophoresis . Shown is the mean - / + SD , n = 3 technical replicates .

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

(D) Quantitative analysis of synthetic GLV2 or GLV2-S peptide binding to RGI3ECD using microscale thermophoresis. Shown is the mean -/+ SD, n=3 technical replicates.


( E ) Co - immunoprecipitation experiment upon co - expression of BAK1 - HA and RGI3 - GFP in N . benthamiana . IP was performed using GFP - TRAP agarose beads . Western blots for protein detection were probed with α - GFP or α - HA antibodies . Similar results were obtained in 3 independent experiments .

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

(E) Co-immunoprecipitation experiment upon co-expression of BAK1-HA and RGI3-GFP in N. benthamiana. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Similar results were obtained in 3 independent experiments.


( C ) Co - immunoprecipitation experiment upon co - expression of FLS2 - HA , BAK1 - HA and RGI3 - GFP in N . benthamiana . Protein extraction was performed 30 min after mock , 1 μM flg22 or 1 μM GLV2 treatment . IP was performed using GFP - TRAP agarose beads . Western blots for protein detection were probed with α - GFP or α - HA antibodies . Data information : Similar results were obtained in 3 independent experiments .

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

(C) Co-immunoprecipitation experiment upon co-expression of FLS2-HA, BAK1-HA and RGI3-GFP in N. benthamiana. Protein extraction was performed 30 min after mock, 1 μM flg22 or 1 μM GLV2 treatment. IP was performed using GFP-TRAP agarose beads. Western blots for protein detection were probed with α-GFP or α-HA antibodies. Data information: Similar results were obtained in 3 independent experiments.


( B ) Quantitative real - time PCR of FLS2 transcripts from 12 - day - old seedlings treated with mock or 1 μM GLV2 for the indicated time , n = 3 biological replicates . UBQ was used as a house keeping gene . Students T - test revealed no statistical difference for each of the different timepoints . Data information : Similar results were obtained in 3 independent experiments .

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

(B) Quantitative real-time PCR of FLS2 transcripts from 12-day-old seedlings treated with mock or 1 μM GLV2 for the indicated time, n=3 biological replicates. UBQ was used as a house keeping gene. Students T-test revealed no statistical difference for each of the different timepoints. Data information: Similar results were obtained in 3 independent experiments.


( C ) Protein extraction was performed from 12 - day - old seedlings of the indicated genotypes . Western blot was probed with α - FLS2 antibodies . ( D ) Protein extraction was performed from 12 - day - old seedlings after 1 hour treatment with mock or 50 μM MG132 . Western blots were probed with α - FLS2 antibodies . Data information : Similar results were obtained in 3 independent experiments .

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

(C) Protein extraction was performed from 12-day-old seedlings of the indicated genotypes. Western blot was probed with α-FLS2 antibodies. (D) Protein extraction was performed from 12-day-old seedlings after 1 hour treatment with mock or 50 μM MG132. Western blots were probed with α-FLS2 antibodies. Data information: Similar results were obtained in 3 independent experiments.


A ) H1299 cell line was transfected with increasing concentrations of MDM2 , and the RB + 5 ´ UTR exogenous ( left panel ) and endogenous ( right panel ) levels were evaluated . B ) Evaluation of the RB expression as in ( A ) but inducing DNA damage with doxorubicin 1 µM during 16 h .

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

A) H1299 cell line was transfected with increasing concentrations of MDM2, and the RB+5´UTR exogenous (left panel) and endogenous (right panel) levels were evaluated. B) Evaluation of the RB expression as in (A) but inducing DNA damage with doxorubicin 1 µM during 16 h.


C ) Evaluation of the RB levels in an MDM2 dose - dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in HEK 293 cell line . D ) Evaluation of the RB levels in a MDM2 dose - dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in C33 cell line .

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C) Evaluation of the RB levels in an MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in HEK 293 cell line. D) Evaluation of the RB levels in a MDM2 dose-dependent fashion under normal and genotoxic stress induced by doxorubicin 16 h in C33 cell line.


E ) Evaluation of the endogenous RB expression using H1299 cell line , under normal conditions , transfecting the cells with an increasing amount of the phosphomimetic mutant MDM2 ( S395D ) . F ) Evaluation of the endogenous RB as in ( E ) but using C33 cell line .

E) Evaluation of the endogenous RB expression using H1299 cell line, under normal conditions, transfecting the cells with an increasing amount of the phosphomimetic mutant MDM2(S395D). F) Evaluation of the endogenous RB as in (E) but using C33 cell line.


A ) Evaluation of RB mRNA levels , normalised by GAPDH , in an MDM2 dose - dependent manner . The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress . Endogenous levels ( upper panel ) ; exogenous levels ( middle panel ) ; endogenous levels of RB but using the MDM2 ( S395D ) under normal conditions ( lower panel ) .

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A) Evaluation of RB mRNA levels, normalised by GAPDH, in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Endogenous levels (upper panel); exogenous levels (middle panel); endogenous levels of RB but using the MDM2(S395D) under normal conditions (lower panel).


B ) Kinetic of the endogenous RB expression in H1299 cell line from 0 to 48h . The cells were treated with cichlohexamide ( CHX ) was added to stop translation , under normal conditions ( upper panel ) and treated with doxorubicin ( lower panel ) . The cells were transfected with MDM2 .

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B) Kinetic of the endogenous RB expression in H1299 cell line from 0 to 48h. The cells were treated with cichlohexamide (CHX) was added to stop translation, under normal conditions (upper panel) and treated with doxorubicin (lower panel). The cells were transfected with MDM2.


C ) Kinetic of the endogenous RB expression in H1299 under genotoxic stress conditions in presence ( upper panel ) or in absence ( lower panel ) of CHX .

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C) Kinetic of the endogenous RB expression in H1299 under genotoxic stress conditions in presence (upper panel) or in absence (lower panel) of CHX.


D ) Evaluation of the levels of expression of RB treated with doxorubicin to generate stress in absence of CHX .

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D) Evaluation of the levels of expression of RB treated with doxorubicin to generate stress in absence of CHX.


Different constructs of RB were transfected in H1299 - RBKO treated with doxorubicin 16 h to induce genotoxic stress ; the RB constructs levels of expression were evaluated using HA antibody . The bar diagrams show the quantification of the western blots data . A ) Evaluation of the small pocket expression in an MDM2 dose - dependent manner . B ) Cells were transfected with the RB large pocket construct .

Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. A) Evaluation of the small pocket expression in an MDM2 dose-dependent manner. B) Cells were transfected with the RB large pocket construct.


Different constructs of RB were transfected in H1299 - RBKO treated with doxorubicin 16 h to induce genotoxic stress ; the RB constructs levels of expression were evaluated using HA antibody . The bar diagrams show the quantification of the western blots data . C ) Cells were transfected with RB full length without the 5 ´ URT ( named RB - HA ) . D ) Cells were transfected with the RB full - length plus 260 nt of its own 5 ´ UTR ( named RB + 5 ´ UTR ) .

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Different constructs of RB were transfected in H1299-RBKO treated with doxorubicin 16 h to induce genotoxic stress; the RB constructs levels of expression were evaluated using HA antibody. The bar diagrams show the quantification of the western blots data. C) Cells were transfected with RB full length without the 5´URT (named RB-HA). D) Cells were transfected with the RB full-length plus 260 nt of its own 5´ UTR (named RB+5´UTR).


E ) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP . Cells were treated with doxorubicin ( 1 µM for 16 h ) or DMSO .

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E) The binding of MDM2 to the RB mRNA in H1299 cells as estimated using RNA CoIP. Cells were treated with doxorubicin (1 µM for 16 h) or DMSO.


F ) RT - qPCR of RB and GAPDH of the MDM2 - immunoprecipitation from the polysomal fractionation ( see EV2D ) . Data are representative of three similar independent experiments .

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F) RT-qPCR of RB and GAPDH of the MDM2-immunoprecipitation from the polysomal fractionation (see EV2D). Data are representative of three similar independent experiments.


G ) PLA assay using anti - RB and anti - MDM2 antibodies , H1299 cell were treated or not with etoposide to induce stress , the endogenous proteins were visualised . Cell nuclei were visualised with DAPI blue . The red dots show the interaction between the two proteins . The scale bar corresponds to 20 μm .

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G) PLA assay using anti-RB and anti-MDM2 antibodies, H1299 cell were treated or not with etoposide to induce stress, the endogenous proteins were visualised. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the two proteins. The scale bar corresponds to 20 μm.


H ) RNA - PLA assay using rabbit anti - MDM2 and a mouse biotin - antibody . H1299 - RBKO was stable transfected with RB - HA or RB + 5 ´ UTR . Cell nuclei were visualised with DAPI blue . The red dots show the interaction between the protein and the mRNA . The scale bar corresponds to 20 μm .

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H) RNA-PLA assay using rabbit anti-MDM2 and a mouse biotin-antibody. H1299- RBKO was stable transfected with RB-HA or RB+5´UTR. Cell nuclei were visualised with DAPI blue. The red dots show the interaction between the protein and the mRNA. The scale bar corresponds to 20 μm.


A ) Cell cycle analysis ( FACS ) of C33 cell line after treatment with etoposide . The DNA contents of the cells were evaluated by propidium iodide staining , ( PE - A ; Phycoerythrin ) . The inset shows the % of the cells in each cell cycle phase .

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A) Cell cycle analysis (FACS) of C33 cell line after treatment with etoposide. The DNA contents of the cells were evaluated by propidium iodide staining, (PE-A; Phycoerythrin). The inset shows the % of the cells in each cell cycle phase.


B ) The cell cycle analysis of synchronised C33 cell line during the time .

B) The cell cycle analysis of synchronised C33 cell line during the time.


C ) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide , ( EV ; empty vector ) . D ) The same as in ( C ) but under normal cellular conditions .

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C) Evaluation of increasing concentrations of MDM2 on the cell cycle of C33 cell line after genotoxic stress induced with etoposide, (EV; empty vector). D) The same as in (C) but under normal cellular conditions.


E ) RB levels of the cells after 14 h of the release of nocodazole treatment . Etoposide was used to induce genotoxic stress .

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E) RB levels of the cells after 14 h of the release of nocodazole treatment. Etoposide was used to induce genotoxic stress.


F ) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment . Data information : Data are means ± s . d . of three independent experiments , student t test was used to calculate statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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F) Evaluation of the G1 cell cycle phase after 14 h of the release of nocodazole treatment. Data information: Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, **p <0.01).


A ) Ubiquitination of endogenous RB in C33 cells synchronised in the G1 or G2 / M cell cycle phases under stress conditions .

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A) Ubiquitination of endogenous RB in C33 cells synchronised in the G1 or G2/M cell cycle phases under stress conditions.


B ) In vitro RB ubiquitination by MDM2 wild type and the phosphomimetic mutant MDM2 ( S395D ) .

B) In vitro RB ubiquitination by MDM2 wild type and the phosphomimetic mutant MDM2(S395D).


C ) In vitro RB ubiquitination by MDM2 ( S395D ) in presence or absence of RB mRNA .

C) In vitro RB ubiquitination by MDM2(S395D) in presence or absence of RB mRNA.


D ) A representative clonogenic assay . Increasing concentrations of MDM2 were transfected in H1299 - RBKO stable transfected with RB - HA or RB + 5 ´ UTR . The cells were treated with doxorubicin to induce genotoxic stress conditions . Data are means ± s . d . of three independent experiments , student t test was used to calculate statistical significance ( * p < 0 . 05 ) .

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D) A representative clonogenic assay. Increasing concentrations of MDM2 were transfected in H1299-RBKO stable transfected with RB-HA or RB+5´UTR. The cells were treated with doxorubicin to induce genotoxic stress conditions. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05).


E ) Evaluation of cyclin E mRNA levels in an MDM2 dose - dependent manner . The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress . Data are means ± s . d . of three independent experiments , student t test was used to calculate statistical significance ( * p < 0 . 05 , * * * p < 0 . 001 ) .

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E) Evaluation of cyclin E mRNA levels in an MDM2 dose-dependent manner. The H1299 cells were treated with doxorubicin during 16 h to induce genotoxic stress. Data are means ± s.d. of three independent experiments, student t test was used to calculate statistical significance (*p <0.05, ***p< 0.001).


The response of cytosolic and mitochondrial matrix - localized roGFP2 - Tsa2ΔCR ( C ) probes to boli of exogenous H2O2 at the indicated concentrations . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium .

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The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (C) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


The response of cytosolic and mitochondrial matrix - localized Grx1 - roGFP2 ( D ) probes to boli of exogenous H2O2 at the indicated concentrations . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium .

The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (D) probes to boli of exogenous H2O2 at the indicated concentrations. Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


The response of cytosolic and mitochondrial matrix - localized roGFP2 - Tsa2ΔCR ( E ) probes to 10 µM Complex antimycin A ( a complex III inhibitor ) . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium .

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The response of cytosolic and mitochondrial matrix-localized roGFP2-Tsa2ΔCR (E) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


The response of cytosolic and mitochondrial matrix - localized Grx1 - roGFP2 ( F ) probes to 10 µM Complex antimycin A ( a complex III inhibitor ) . Probes were expressed in wild - type BY4742 yeast cells grown to exponential phase in SGal ( - Leu ) medium .

The response of cytosolic and mitochondrial matrix-localized Grx1-roGFP2 (F) probes to 10 µM Complex antimycin A (a complex III inhibitor). Probes were expressed in wild-type BY4742 yeast cells grown to exponential phase in SGal (-Leu) medium.


( G ) The response of mitochondrial matrix - localized ( left panel ) and cytosolic ( right panel ) roGFP2 - Tsa2ΔCR probes expressed in BY4742 wild - type and Δtsa1Δtsa2 cells , to the addition of exogenous H2O2 at the indicated concentrations . Cells were grown to exponential phase in SGal ( - Leu ) medium . The lighter colored curves are controls , showing the probe response upon the addition of water .

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(G) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes expressed in BY4742 wild-type and Δtsa1Δtsa2 cells, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls, showing the probe response upon the addition of water.


( H ) The response of mitochondrial matrix - localized ( left panel ) and cytosolic ( right panel ) roGFP2 - Tsa2ΔCR probes , expressed in wild - type and Δtsa1Δtsa2 cells , to the addition of 10 µM antimycin A . Cells were grown to exponential phase in SGal ( - Leu ) medium . The lighter colored curves are controls showing the probe responses upon addition of 0 . 1 % ( v / v ) ethanol .

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(H) The response of mitochondrial matrix-localized (left panel) and cytosolic (right panel) roGFP2-Tsa2ΔCR probes, expressed in wild-type and Δtsa1Δtsa2 cells, to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. The lighter colored curves are controls showing the probe responses upon addition of 0.1% (v/v) ethanol.


( I ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type and Δpor1 cells , to boli at exogenous H2O2 at the indicated concentrations . Cells were grown to exponential phase in SGal ( - Leu ) medium . Lighter colored curves are controls showing the probe response upon the addition of water .

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(I) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells, to boli at exogenous H2O2 at the indicated concentrations. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of water.


( J ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type and Δpor1 cells to the addition of 10 µM antimycin A . Cells were grown to exponential phase in SGal ( - Leu ) medium . Lighter colored curves are controls showing the probe response upon the addition of 0 . 1 % ( v / v ) ethanol .

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(J) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δpor1 cells to the addition of 10 µM antimycin A. Cells were grown to exponential phase in SGal (-Leu) medium. Lighter colored curves are controls showing the probe response upon the addition of 0.1% (v/v) ethanol.


( A ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe , expressed in wild - type and Δprx1 , to the addition of exogenous H2O2 at the indicated concentrations . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Lighter colored curves are controls showing the probe response to the addition of water .

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(A) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe, expressed in wild-type and Δprx1, to the addition of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Lighter colored curves are controls showing the probe response to the addition of water.


( B , C ) The response of a cytosolic roGFP2 - Tsa2ΔCR probe , expressed in wild - type , Δtrx3 and Δprx1 cells , to the addition of 0 . 1 mM ( B ) or 1 mM ( C ) exogenous H2O2 . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase .

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(B,C) The response of a cytosolic roGFP2-Tsa2ΔCR probe, expressed in wild-type, Δtrx3 and Δprx1 cells, to the addition of 0.1 mM (B) or 1 mM (C) exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.


( D ) The response of a cytosolic roGFP2 - Tsa2ΔCR probe , in Δtrx3 cells transformed with a Trx3 plasmid and Δprx1 cells transformed with a Prx1 plasmid , to the addition of 0 . 1 mM exogenous H2O2 . Cells were grown in SGal medium lacking the appropriate amino acids for plasmid selection and harvested at early exponential phase . The light grey curve in both panels represents the wild - type control , treated with the same concentration of exogenous H2O2 .

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(D) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δtrx3 cells transformed with a Trx3 plasmid and Δprx1 cells transformed with a Prx1 plasmid, to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for plasmid selection and harvested at early exponential phase. The light grey curve in both panels represents the wild-type control, treated with the same concentration of exogenous H2O2.


( E ) The profile of mRNA expression of the yeast redox or redox - related enzymes in Δprx1 + empty vector cells , compared to Δprx1 + Prx1 - WT cells , both grown to an early exponential phase in SGal ( - Ura ) medium ( n = 3 biological replicates , with cells obtained from 3 independent cultures ) .

(E) The profile of mRNA expression of the yeast redox or redox-related enzymes in Δprx1+empty vector cells, compared to Δprx1+Prx1-WT cells, both grown to an early exponential phase in SGal (-Ura) medium (n = 3 biological replicates, with cells obtained from 3 independent cultures).


( F ) The response of a cytosolic roGFP2 - Tsa2ΔCR probe , in Δprx1 cells or Δctt1Δprx1 cells transformed with either an empty plasmid or a Prx1 - WT plasmid to the addition of 0 . 1 mM exogenous H2O2 . Cells were grown in SGal medium lacking the appropriate amino acids for selection and harvested in early exponential

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(F) The response of a cytosolic roGFP2-Tsa2ΔCR probe, in Δprx1 cells or Δctt1Δprx1 cells transformed with either an empty plasmid or a Prx1-WT plasmid to the addition of 0.1 mM exogenous H2O2. Cells were grown in SGal medium lacking the appropriate amino acids for selection and harvested in early exponential phase


( A ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in wild - type , Δtsa1Δtsa2 , Δpor1 , Δtrr2 , Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase .

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(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtsa1Δtsa2, Δpor1, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase.


( B ) Left panel : The response of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in wild - type , Δtrr2 , Δtrx3 or Δprx1 cells to a bolus of 2 . 5 mM H2O2 . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Right panel : Scheme depicting Prx1 and its peroxidatic cysteine at position 91 .

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(B) Left panel: The response of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in wild-type, Δtrr2, Δtrx3 or Δprx1 cells to a bolus of 2.5 mM H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: Scheme depicting Prx1 and its peroxidatic cysteine at position 91.


( C ) The response of a mitochondrial matrix - localized roGFP2 - Tsa2ΔCR probe to exogenous 2 . 5 mM H2O2 . The probe was expressed in wild - type cells transformed with an empty vector or in ∆ prx1 cells transformed with either an empty vector , a vector encoding wild - type Prx1 or a vector encoding Prx1 - C91A , grown in SGal ( - Leu , - Ura ) . ( D ) The response to of a mitochondrial matrix - localized Grx1 - roGFP2 sensor expressed in either wild - type cells transformed with an empty vector or in ∆ prx1 cells transformed with either an empty vector , a Prx1 - WT or a Prx1 - C91A plasmid to exogenous H2O2 at a concentration of 2 . 5 mM . Cells were grown to SGal ( - Leu , - Ura ) medium and harvested at early exponential phase .

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(C) The response of a mitochondrial matrix-localized roGFP2-Tsa2ΔCR probe to exogenous 2.5 mM H2O2. The probe was expressed in wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a vector encoding wild-type Prx1 or a vector encoding Prx1-C91A, grown in SGal (-Leu, -Ura). (D) The response to of a mitochondrial matrix-localized Grx1-roGFP2 sensor expressed in either wild-type cells transformed with an empty vector or in ∆prx1 cells transformed with either an empty vector, a Prx1-WT or a Prx1-C91A plasmid to exogenous H2O2 at a concentration of 2.5 mM. Cells were grown to SGal (-Leu, -Ura) medium and harvested at early exponential phase.


( B ) ' Acute stress - washout ' assay . Δprx1 cells containing a Prx1 - WT plasmid and expressing a mitochondrial matrix - localized Grx1 - roGFP2 probe were grown to exponential phase in SGal ( - Leu , - Ura ) medium harvested and treated with either 0 , 1 , 5 , 10 , 25 or 50 mM H2O2 for 10 mins . After removal of H2O2 and resuspension in fresh buffer the response of a mitochondrial matrix - localized Grx1 - roGFP2 probe to the addition of 1 mM H2O2 was measured . The middle panel is an enlargement of the left panel . The graph in the right panel shows the steady state Grx1 - roGFP2 oxidation following H2O2 pre - treatment as well as the maximum Grx1 - roGFP2 oxidation in response to the subsequent second H2O2 treatment .

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(B) 'Acute stress-washout' assay. Δprx1 cells containing a Prx1-WT plasmid and expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. After removal of H2O2 and resuspension in fresh buffer the response of a mitochondrial matrix-localized Grx1-roGFP2 probe to the addition of 1 mM H2O2 was measured. The middle panel is an enlargement of the left panel. The graph in the right panel shows the steady state Grx1-roGFP2 oxidation following H2O2 pre-treatment as well as the maximum Grx1-roGFP2 oxidation in response to the subsequent second H2O2 treatment.


' Acute stress - washout ' assay . Δprx1 cells expressing a mitochondrial matrix - localized Grx1 - roGFP2 probe were grown to exponential phase in SGal ( - Leu , - Ura ) medium harvested and treated with either 0 , 1 , 5 , 10 , 25 or 50 mM H2O2 for 10 mins . ( C ) The same experiment as in ( B ) performed with Δprx1 cells transformed with a Prx1 - C91A plasmid .

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'Acute stress-washout' assay. Δprx1 cells expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. (C) The same experiment as in (B) performed with Δprx1 cells transformed with a Prx1-C91A plasmid.


( D ) ∆ prx1 cells transformed with a vector encoding wild - type Prx1 , a vector encoding the matrix - targeted D - aminooxidase ( su9 - DAO ) and expressing a mitochondrial matrix - localized Grx1 - roGFP2 were grown to exponential phase in SGal ( - Leu , - Ura , - His ) and then pre - treated with 0 . 15 M D - alanine for 0 , 0 . 5 , 1 . 5 hours . Afterwards , the cells were washed and the response of Grx1 - roGFP2 to the addition of 1 mM H2O2 was measured .

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(D) ∆prx1 cells transformed with a vector encoding wild-type Prx1, a vector encoding the matrix-targeted D-aminooxidase (su9-DAO) and expressing a mitochondrial matrix-localized Grx1-roGFP2 were grown to exponential phase in SGal (-Leu, -Ura, -His) and then pre-treated with 0.15 M D-alanine for 0, 0.5, 1.5 hours. Afterwards, the cells were washed and the response of Grx1-roGFP2 to the addition of 1 mM H2O2 was measured.


( E ) Left panel : the response of mitochondrial matrix - localized roGFP2 - Prx1 and roGFP2 probes expressed in wild - type cells to exogenous H2O2 at the indicated concentrations . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Right panel : model illustrating that hyperoxidation of the Prx1 moiety in the roGFP2 - Prx1 sensor leads to a roGFP2 - like behavior .

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(E) Left panel: the response of mitochondrial matrix-localized roGFP2-Prx1 and roGFP2 probes expressed in wild-type cells to exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: model illustrating that hyperoxidation of the Prx1 moiety in the roGFP2-Prx1 sensor leads to a roGFP2-like behavior.


( H ) Redox shift assays of Prx1 . Δprx1 cells transformed with a plasmid encoding wild - type Prx1 were grown to early exponential phase in SGal ( - Ura ) medium . Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins . Exposure to H2O2 oxidizes the single cysteine of Prx1 to a state that cannot be reduced with TCEP .

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(H) Redox shift assays of Prx1. Δprx1 cells transformed with a plasmid encoding wild-type Prx1 were grown to early exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins. Exposure to H2O2 oxidizes the single cysteine of Prx1 to a state that cannot be reduced with TCEP.


( J ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 , expressed in wild - type cells grown to the addition of exogenous 1 mM H2O2 . Cells were grown in SGal ( - Leu ) and harvested at early exponential phase . Left panel , the pre - treatment efficiently inhibits the ability of Prx1 to transfer oxidizing equivalents to EGSH . Right panel , newly synthesized Prx1 is required to recover the transfer of oxidizing equivalents to EGSH in the mitochondrial matrix .

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(J) The response of a mitochondrial matrix-localized Grx1-roGFP2, expressed in wild-type cells grown to the addition of exogenous 1 mM H2O2. Cells were grown in SGal (-Leu) and harvested at early exponential phase. Left panel, the pre-treatment efficiently inhibits the ability of Prx1 to transfer oxidizing equivalents to EGSH. Right panel, newly synthesized Prx1 is required to recover the transfer of oxidizing equivalents to EGSH in the mitochondrial matrix.


( B ) The response of a mitochondrial matrix - localized roGFP2 - Prx1 - P233stop probe expressed in wild - type cells to the indicated concentrations of exogenous H2O2 . Cells were grown in SGal ( - Leu ) medium and harvested at early exponential phase . Error bars represent the standard deviation ( n = 3 biological replicates , with cells obtained from 3 independent cultures for every strain and probe combinations . For each biological replicate three technical replicates were performed ) .

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(B) The response of a mitochondrial matrix-localized roGFP2-Prx1-P233stop probe expressed in wild-type cells to the indicated concentrations of exogenous H2O2. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).


( C ) Maleimide - based gel - shift assay to assess Prx1 cysteine redox state . Wild - type cells transformed with an empty plasmid or ∆ prx1 cells with either an empty plasmid or a plasmid encoding wild - type Prx1 , the hyperoxidation - resistant Prx1 - P233stop or the inactive Prx1 - C91A variants , were grown to exponential phase in SGal ( - Ura ) medium . Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins and then directly treated with the alkylating agent mmPEG24 .

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(C) Maleimide-based gel-shift assay to assess Prx1 cysteine redox state. Wild-type cells transformed with an empty plasmid or ∆prx1 cells with either an empty plasmid or a plasmid encoding wild-type Prx1, the hyperoxidation-resistant Prx1-P233stop or the inactive Prx1-C91A variants, were grown to exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins and then directly treated with the alkylating agent mmPEG24.


( D ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 probe , expressed in Δprx1 cells containing a plasmid encoding either wild - type Prx1 , Prx1 - P233stop or Prx1C91A to the addition of 1 mM exogenous H2O2 . Cells had been pre - treated with either 10 mM H2O2 or with water as a control . For these experiments , cells were grown in SGal ( - Leu , - Ura ) medium and harvested at early exponential phase . OxD refers to the degree of sensor oxidation . Error bars represent the standard deviation ( n = 3 biological replicates , with cells obtained from 3 independent cultures for every strain and probe combinations . For each biological replicate three technical replicates were performed ) .

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(D) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe, expressed in Δprx1 cells containing a plasmid encoding either wild-type Prx1, Prx1-P233stop or Prx1C91A to the addition of 1 mM exogenous H2O2. Cells had been pre-treated with either 10 mM H2O2 or with water as a control. For these experiments, cells were grown in SGal (-Leu, -Ura) medium and harvested at early exponential phase. OxD refers to the degree of sensor oxidation. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).


( E ) Hydrogen peroxide ' acute stress ' assay . Δprx1 cells co - transformed with empty vector or with a vector encoding either wild - type Prx1 , the P233stop or the C91A variants were grown in SD ( - Ura ) and treated with the indicated concentrations of H2O2 for 30 mins . Afterwards , the cells were diluted and a fixed volume plated on YPD plates . The number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a percentage relative to the 0 mM treatment . Error bars represent standard deviation ( n = 5 - 8 biological replicates , with cells taken from independent cultures for each individual biological replicate ) .

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(E) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with empty vector or with a vector encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SD (-Ura) and treated with the indicated concentrations of H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 5-8 biological replicates, with cells taken from independent cultures for each individual biological replicate).


( A ) Hydrogen peroxide ' acute stress ' assay . Δprx1 cells co - transformed with an empty plasmid or a plasmid encoding Prx1 , Tsa1 , PRDX3 , PRDX5 , PRDX6 , PfAOP or PfAOP - L109M were grown in SD medium lacking the appropriate amino acids for plasmid selection and pre - treated with 0 , 1 , 5 , 10 , 25 mM H2O2 for 30 mins . Afterwards , the cells were diluted and a fixed volume was plated on YPD plates . The number of viable colonies was counted after 2 days of growth at 30 ° C , here represented as a percentage relative to the 0 mM pre - treatment . Error bars represent standard deviation ( n = 3 - 8 biological replicates , with cells taken from independent cultures for each individual biological replicate ) . Significance was assessed with a Student ' s 2 - tailed , unpaired , t - test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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

(A) Hydrogen peroxide 'acute stress' assay. Δprx1 cells co-transformed with an empty plasmid or a plasmid encoding Prx1, Tsa1, PRDX3, PRDX5, PRDX6, PfAOP or PfAOP-L109M were grown in SD medium lacking the appropriate amino acids for plasmid selection and pre-treated with 0, 1, 5, 10, 25 mM H2O2 for 30 mins. Afterwards, the cells were diluted and a fixed volume was plated on YPD plates. The number of viable colonies was counted after 2 days of growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3-8 biological replicates, with cells taken from independent cultures for each individual biological replicate). Significance was assessed with a Student's 2-tailed, unpaired, t-test. *p < 0.05; **p < 0.01; ***p < 0.001.


( B ) The efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with cell death upon exposure to acute H2O2 stress . Data from viability and matrix Grx1 - roGFP2 responses during acute H2O2 stress were correlated .

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(B) The efficiency of transfer of oxidation from H2O2 to EGSH strongly correlates with cell death upon exposure to acute H2O2 stress. Data from viability and matrix Grx1-roGFP2 responses during acute H2O2 stress were correlated.


( A ) The response of a mitochondrial matrix - localized Grx1 - roGFP2 probe to 1 mM H2O2 . in BY4742 wild - type cells with an empty vector , in Δglr1 cells transformed either with an empty vector or a vector encoding wild - type Glr1 or the cytosolic form of Glr1 , where the MTS - encoding region was removed , or in Δglr1Δprx1 cells transformed either with an empty vector or a vector encoding the cytosolic form of Glr1 . Cells were grown to exponential phase in SGal ( - Leu , - Ura ) medium . Error bars represent the standard deviation ( n = 3 biological replicates , with cells obtained from 3 independent cultures for every strain and probe combinations . For each biological replicate three technical replicates were performed ) .

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(A) The response of a mitochondrial matrix-localized Grx1-roGFP2 probe to 1 mM H2O2. in BY4742 wild-type cells with an empty vector, in Δglr1 cells transformed either with an empty vector or a vector encoding wild-type Glr1 or the cytosolic form of Glr1, where the MTS-encoding region was removed, or in Δglr1Δprx1 cells transformed either with an empty vector or a vector encoding the cytosolic form of Glr1. Cells were grown to exponential phase in SGal (-Leu, -Ura) medium. Error bars represent the standard deviation (n = 3 biological replicates, with cells obtained from 3 independent cultures for every strain and probe combinations. For each biological replicate three technical replicates were performed).


( B ) Growth curve of wild - type , Δprx1 , Δglr1 and Δglr1Δprx1 cells in SD medium complemented with all amino acids ( n = 3 biological replicates ) . Significance for the difference in the time the cultures reach 50 % of their maximal OD600 was assessed with the t - test . Error bars ( as ribbon ) represent the standard deviation ( n = 4 biological replicates , with cells taken from independent cultures for each individual biological replicate ) .

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(B) Growth curve of wild-type, Δprx1, Δglr1 and Δglr1Δprx1 cells in SD medium complemented with all amino acids (n = 3 biological replicates). Significance for the difference in the time the cultures reach 50% of their maximal OD600 was assessed with the t-test. Error bars (as ribbon) represent the standard deviation (n = 4 biological replicates, with cells taken from independent cultures for each individual biological replicate).


( C ) H2O2 ' acute stress ' assay . Wild - type , Δprx1 , Δglr1 and Δglr1Δprx1 cells pre - grown in SGal medium complemented with all amino acids to early exponential phase . Cells were treated with H2O2 at the indicated concentrations for 30 mins . Subsequently cells were diluted , and a fixed volume plated on YPD plates . The number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a percentage relative to the 0 mM pre - treatment . Error bars represent standard deviation ( n = 3 biological replicates , with cells taken from independent cultures for each individual biological replicate ) . An inset with a different scale on the y - axis is presented for the 1 and 5 mM concentration to allow better interpretation .

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(C) H2O2 'acute stress' assay. Wild-type, Δprx1, Δglr1 and Δglr1Δprx1 cells pre-grown in SGal medium complemented with all amino acids to early exponential phase. Cells were treated with H2O2 at the indicated concentrations for 30 mins. Subsequently cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM pre-treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate). An inset with a different scale on the y-axis is presented for the 1 and 5 mM concentration to allow better interpretation.


( D ) H2O2 ' acute stress ' assay . Δprx1Δglr1 cells co - transformed with an empty plasmid or with a plasmid encoding either wild - type Prx1 , the P233stop or the C91A variants were grown in SGal ( - Ura ) medium to early exponential phase . Cells were treated with the indicated amounts of H2O2 for 30 mins . Subsequently , the cells were diluted , and a fixed volume plated on YPD plates . The number of viable colonies was counted after 2 days growth at 30 ° C , here represented as a percentage relative to the 0 mM treatment . Error bars represent standard deviation ( n = 3 biological replicates , with cells taken from independent cultures for each individual biological replicate ) .

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(D) H2O2 'acute stress' assay. Δprx1Δglr1 cells co-transformed with an empty plasmid or with a plasmid encoding either wild-type Prx1, the P233stop or the C91A variants were grown in SGal (-Ura) medium to early exponential phase. Cells were treated with the indicated amounts of H2O2 for 30 mins. Subsequently, the cells were diluted, and a fixed volume plated on YPD plates. The number of viable colonies was counted after 2 days growth at 30°C, here represented as a percentage relative to the 0 mM treatment. Error bars represent standard deviation (n = 3 biological replicates, with cells taken from independent cultures for each individual biological replicate).


c , Knockdown of NetB in the eye disc of GMR - RasV12 flies enhances survival . d , NetB heterozygous mutant flies survive better over Rasv12 - induced oncogenic stress . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by Dunnett ' s multiple comparisons test ( c ) and two - tailed unpaired t - test d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 .

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c, Knockdown of NetB in the eye disc of GMR-RasV12 flies enhances survival. d, NetB heterozygous mutant flies survive better over Rasv12-induced oncogenic stress. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test (c) and two-tailed unpaired t-test d, *P < 0.05, **P <0.01, ****P <0.0001.


f , qRT - PCR with mRNA from the eye disc confirms higher expression of NetB in GMR - RasV12 flies Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by two - tailed unpaired t - test * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 .

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f, qRT-PCR with mRNA from the eye disc confirms higher expression of NetB in GMR-RasV12 flies Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by two-tailed unpaired t-test *P < 0.05, **P <0.01, ****P <0.0001.


g - h , Oncogenic Ras expression in the eye disc induces NetB protein , which was detected by GFP signal using CPTI - 000748 , a protein trap of NetB . Single confocal z - section images are shown . i , Quantification of GFP signals in g and h . Data information : Data points indicate biological replicates . Data are mean ± s . e . m . and the statistical significance was determined by one - way ANOVA followed by two - tailed unpaired t - test * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 . Scale bar , 50 μm .

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g-h, Oncogenic Ras expression in the eye disc induces NetB protein, which was detected by GFP signal using CPTI-000748, a protein trap of NetB. Single confocal z-section images are shown. i, Quantification of GFP signals in g and h. Data information: Data points indicate biological replicates. Data are mean ± s.e.m. and the statistical significance was determined by one-way ANOVA followed by two-tailed unpaired t-test *P < 0.05, **P <0.01, ****P <0.0001. Scale bar, 50 μm.


a - b , The amount of NetB protein increases in the fat body GMR - RasV12 ( a ) compared to control flies ( b ) . CPTI - 000748 , a protein trap of NetB , labels endogenous NetB . Data information : Scale bar , 20 μm ( a -

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a-b, The amount of NetB protein increases in the fat body GMR-RasV12 (a) compared to control flies (b). CPTI-000748, a protein trap of NetB, labels endogenous NetB. Data information: Scale bar, 20 μm (a-b