L - N Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV - nc - GFP ( L ) and LV - CHL1 shRNA - GFP ( M ) into both the sensorimotor cortex and lesion sites . High - magnification image ( N ) of the area in ( M ) ( asterisk ) . The arrows indicate the lesion sites ; the asterisk indicates the lesion epicenter . O Quantification of intensity index of BDA - labeled axons at certain distances from the lesion border in ( L and M ) . * p < 0 . 05 and * * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 9 mice in ( L ) , and n = 10 mice in ( M ) .

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L-N Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (L) and LV-CHL1 shRNA-GFP (M) into both the sensorimotor cortex and lesion sites. High-magnification image (N) of the area in (M) (asterisk). The arrows indicate the lesion sites; the asterisk indicates the lesion epicenter.O Quantification of intensity index of BDA-labeled axons at certain distances from the lesion border in (L and M). *p < 0.05 and **p < 0.01; two-way ANOVA followed by Fisher's LSD. n= 9 mice in (L), and n= 10 mice in (M).


A Co - immunostaining of PTPσ ( blue ) , NB - 3 ( green ) , and BDA ( red ) at spinal lesion sites of NB - 3 + / + ( A1 ) and NB - 3 − / − ( A6 ) mice . ( A2 - A5 and A7 - A10 ) High - magnification images of boxed areas in ( A1 and A6 ) showing PTPσ expression at the terminations of BDA - labeled corticospinal axons , respectively . Images in ( A2 and A7 ) are presented as single - channel fluorescence images in ( A3 - A5 and A8 - A10 ) , respectively . B Quantification of fluorescence intensity of PTPσ at BDA - labeled axonal terminations of NB - 3 + / + and NB - 3 − / − mice after SCI . * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test . n = 12 mice per group .

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

A Co-immunostaining of PTPσ (blue), NB-3 (green), and BDA (red) at spinal lesion sites of NB-3+/+ (A1) and NB-3−/− (A6) mice. (A2-A5 and A7-A10) High-magnification images of boxed areas in (A1 and A6) showing PTPσ expression at the terminations of BDA-labeled corticospinal axons, respectively. Images in (A2 and A7) are presented as single-channel fluorescence images in (A3-A5 and A8-A10), respectively.B Quantification of fluorescence intensity of PTPσ at BDA-labeled axonal terminations of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05; one-way ANOVA followed by Bonferroni post-test. n = 12 mice per group.


C Co - immunostaining of PTPσ and NB - 3 in the FG - labeled corticospinal neurons ( arrowheads ) of NB - 3 + / + and NB - 3 − / − mice 14 days post - injury ( dpi ) . Insets , only the signals from PTPσ and FG in NB - 3 + / + corticospinal neurons ( red arrowheads ) were visualized . D Quantification of fluorescence intensity of PTPσ in ( C ) . * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test . The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified .

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

C Co-immunostaining of PTPσ and NB-3 in the FG-labeled corticospinal neurons (arrowheads) of NB-3+/+ and NB-3−/− mice 14 days post-injury (dpi). Insets, only the signals from PTPσ and FG in NB-3+/+ corticospinal neurons (red arrowheads) were visualized.D Quantification of fluorescence intensity of PTPσ in (C). *p < 0.05; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.


E Co - immunoprecipitation of NB - 3 and PTPσ from sensorimotor cortex lysates of NB - 3 + / + mice after SCI . F Quantification of immunoprecipitated PTPσ by NB - 3 in ( E ) . * * p < 0 . 01 ; one sample t - test . Data were analyzed from 3 independent experiments each including 15 mice per group .

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E Co-immunoprecipitation of NB-3 and PTPσ from sensorimotor cortex lysates of NB-3+/+mice after SCI.F Quantification of immunoprecipitated PTPσ by NB-3 in (E). **p < 0.01; one sample t-test. Data were analyzed from 3 independent experiments each including 15 mice per group.


G PTPσ expression in NB - 3 + / + and NB - 3 − / − pyramidal neurons when co - culturing with NB - 3 - expressing astrocytes ( dashed lines and asterisks ) or astrocytes that didn ' t express NB - 3 ( dashed lines ) . Upper inset , high - magnification view of the soma ( white arrowhead ) . Lower inset , high - magnification view of the axon tip ( green arrowhead ) . H Quantification of fluorescence intensity of PTPσ in ( G ) . n . s . , not significant , and * p < 0 . 05 ; one - way ANOVA followed by Bonferroni post - test .

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

G PTPσ expression in NB-3+/+ and NB-3−/− pyramidal neurons when co-culturing with NB-3-expressing astrocytes (dashed lines and asterisks) or astrocytes that didn't express NB-3 (dashed lines). Upper inset, high-magnification view of the soma (white arrowhead). Lower inset, high-magnification view of the axon tip (green arrowhead).H Quantification of fluorescence intensity of PTPσ in (G). n.s., not significant, and *p < 0.05; one-way ANOVA followed by Bonferroni post-test.


I Expression of PTPσ , p - Akt , p - mTOR , and p - S6 ( S240 / 244 ) in pyramidal neurons that expressed PTPσ shRNA - GFP . A control plasmid expressing GFP was transfected as the negative control ( nc ) . J Quantification of fluorescence intensity of PTPσ , p - Akt , p - mTOR , and p - S6 ( S240 / 244 ) in ( I ) . * p < 0 . 05 ; one sample t - test . The intensities of more than 300 pyramidal neurons from 4 independent experiments in each group in ( H ) and ( J ) were quantified , respectively .

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I Expression of PTPσ, p-Akt, p-mTOR, and p-S6 (S240/244) in pyramidal neurons that expressed PTPσ shRNA-GFP. A control plasmid expressing GFP was transfected as the negative control (nc).J Quantification of fluorescence intensity of PTPσ, p-Akt, p-mTOR, and p-S6 (S240/244) in (I). *p < 0.05; one sample t-test. The intensities of more than 300 pyramidal neurons from 4 independent experiments in each group in (H) and (J) were quantified, respectively.


K - M Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV - nc - GFP ( K ) and LV - PTPσ shRNA - GFP ( L ) into the sensorimotor cortex . High - magnification image ( M ) of the area in ( L ) ( asterisk ) . The arrows indicate the lesion sites ; the asterisk indicates the lesion epicenter . N Quantification of the intensity index of BDA - labeled axons at certain distances from the lesion border in ( K and L ) . * p < 0 . 01 ; two - way ANOVA followed by Fisher ' s LSD . n = 8 mice in ( K ) , and n = 10 mice in ( L ) .

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K-M Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (K) and LV-PTPσ shRNA-GFP (L) into the sensorimotor cortex. High-magnification image (M) of the area in (L) (asterisk). The arrows indicate the lesion sites; the asterisk indicates the lesion epicenter.N Quantification of the intensity index of BDA-labeled axons at certain distances from the lesion border in (K and L). *p < 0.01; two-way ANOVA followed by Fisher's LSD. n = 8 mice in (K), and n = 10 mice in (L).


( B ) Flow cytometry analysis of WT to Il21r - / - splenic SWHEL B cells over time . ( C ) WT to Il21r - / - SWHEL B cell ratio within CTV division peaks . Data information : Data in ( B ) is representative of 8 - 9 biological replicates ( n = 8 - 9 ) pooled from two independent experiments with statistical analysis by one - way ANOVA with Tukey ' s post - test . * * p ≤ 0 . 01 ; * * * * p ≤ 0 . 0001 . Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) and are representative for two independent experiments .

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(B) Flow cytometry analysis of WT to Il21r-/- splenic SWHEL B cells over time. (C) WT to Il21r-/- SWHEL B cell ratio within CTV division peaks. Data information: Data in (B) is representative of 8-9 biological replicates (n = 8-9) pooled from two independent experiments with statistical analysis by one-way ANOVA with Tukey's post-test. ** p ≤ 0.01; **** p ≤ 0.0001. Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.


( E ) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks . Data information : Data show concatenated data from 4 - 5 biological replicates ( n = 4 - 5 ) and are representative for two independent experiments .

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(E) Rate of apoptosis measured by detecting active caspase 3 by flow cytometry across CTV division peaks. Data information: Data show concatenated data from 4-5 biological replicates (n = 4-5) and are representative for two independent experiments.


( B ) Flow cytometry analysis showing WT to Il21r - / - SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

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(B) Flow cytometry analysis showing WT to Il21r-/- SWHEL B cell ratio of cells that have been in S phase and thus incorporated BrdU. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.


( D ) Ratio of the proportion of BrdU positive WT and Il21r - / - SWHEL B cells with 2N DNA content 4 , 10 and 12 hours post BrdU pulse on day 3 . 5 post immunization . Data information : Data are representative of 5 - 7 biological replicates ( n = 5 - 7 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

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(D) Ratio of the proportion of BrdU positive WT and Il21r-/- SWHEL B cells with 2N DNA content 4, 10 and 12 hours post BrdU pulse on day 3.5 post immunization. Data information: Data are representative of 5-7 biological replicates (n = 5-7) from two independent experiments. Statistical analysis by one-sample t test. *p ≤ 0.05.


( C ) Rate of de novo cell cycle entry 10 hours post in vivo IL - 21 ( 2 µg ) or saline pulse . The ratio of WT to Il21r - / - cells not in S / G2 at the time of pulse ( BrdU - cells ) and containing > 2N DNA content 10 hours after pulse is shown . Data information : Data are representative of 10 - 12 biological replicates ( n = 10 - 12 ) from two independent experiments . Statistical analysis by one - sample t test . * p ≤ 0 . 05 .

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(C) Rate of de novo cell cycle entry 10 hours post in vivo IL-21 (2 µg) or saline pulse. The ratio of WT to Il21r-/- cells not in S/G2 at the time of pulse (BrdU- cells) and containing >2N DNA content 10 hours after pulse is shown. Data information: Data are representative of 10-12 biological replicates (n = 10-12) from two independent experiments. Statistical analysis by one-sample t test. * p ≤ 0.05.


Phosphoflow analysis of naïve WT or Il21r - / - B cells following in vitro culture for 3 hours with or without IL - 21 ( 20 ng / mL ) and / or in the presence of BCR cross - linking ( biotinylated anti - Igκ + anti - Igλ and avidin - mediated cross - linking ) or agonistic anti - CD40 . ( A ) Exemplary p - AKT ( S473 ) staining and ( B ) quantification of p - AKT median fluorescence intensity ( MedFI ) . ( C ) Exemplary p - S6 ( Ser235 / 236 ) staining and ( D ) quantification of frequency of p - S6 positive cells Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical analysis by one - way ANOVA with Tukey ' s post - test ( D ) , t test ( B , E ) * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

Phosphoflow analysis of naïve WT or Il21r-/- B cells following in vitro culture for 3 hours with or without IL-21 (20 ng/mL) and/or in the presence of BCR cross-linking (biotinylated anti-Igκ + anti-Igλ and avidin-mediated cross-linking) or agonistic anti-CD40. (A) Exemplary p-AKT (S473) staining and (B) quantification of p-AKT median fluorescence intensity (MedFI). (C) Exemplary p-S6 (Ser235/236) staining and (D) quantification of frequency of p-S6 positive cells Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by one-way ANOVA with Tukey's post-test (D), t test (B, E) * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( F , In vitro cell culture of CTV labeled SWHEL B cells with or without IL - 21 ( 20 ng / mL ) and anti - CD40 for three days and analysis by flow cytometry . ( F ) Exemplary CTV division peaks .

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(F, In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (F) Exemplary CTV division peaks.


G ) In vitro cell culture of CTV labeled SWHEL B cells with or without IL - 21 ( 20 ng / mL ) and anti - CD40 for three days and analysis by flow cytometry . ( G ) Quantification of the proportion SWHEL B that had divided and thus diluted CTV . Data information : Data are representative of 4 biological replicates ( n = 4 ) . Statistical analysis by two - way ANOVA with correction for multiple comparison using Šidák method ( G ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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G) In vitro cell culture of CTV labeled SWHEL B cells with or without IL-21 (20 ng/mL) and anti-CD40 for three days and analysis by flow cytometry. (G) Quantification of the proportion SWHEL B that had divided and thus diluted CTV. Data information: Data are representative of 4 biological replicates (n = 4). Statistical analysis by two-way ANOVA with correction for multiple comparison using Šidák method (G). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.


( B , C ) Analysis of splenic OTII Tfh cells by flow cytometry . ( B ) Tfh cell count and ( C ) Proportion of Il21Gfp / + OTII Tfh cells transcribing the Il21 locus and thus expressing GFP . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . Statistical analysis by one - way ANOVA with Tukey ' s post - test ( B , C ) . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(B, C) Analysis of splenic OTII Tfh cells by flow cytometry. (B) Tfh cell count and (C) Proportion of Il21Gfp/+ OTII Tfh cells transcribing the Il21 locus and thus expressing GFP. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. Statistical analysis by one-way ANOVA with Tukey's post-test (B, C). * p ≤ 0.05; ** p ≤ 0.01.


Analysis of splenic WT and Il21r - / - SWHEL B cells by flow cytometry . ( D ) CTV profile of and ( E ) absolute cell count in pooled CTV division peaks ( 0 - 4 , 5 - 8 , 9 - 11 + ) . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . D , show concatenated data representative of 3 - 5 biological replicates . Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm - Šídák method . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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Analysis of splenic WT and Il21r-/- SWHEL B cells by flow cytometry. (D) CTV profile of and (E) absolute cell count in pooled CTV division peaks (0-4, 5-8, 9-11+). Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. D, show concatenated data representative of 3-5 biological replicates. Data in E were analyzed after log transformation using Multiple paired t tests and p values corrected for multiple comparisons using Holm-Šídák method. * p ≤ 0.05; ** p ≤ 0.01.


( F ) Cell surface expression ( median fluorescence intensity , MedFI ) of CD38 , IgD and FAS across individual CTV division peaks . ( G ) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression . Data information : Data are representative of 5 - 6 biological replicates ( n = 5 - 6 ) form two independent experiments . F and G show concatenated data representative of 3 - 5 biological replicates .

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(F) Cell surface expression (median fluorescence intensity, MedFI) of CD38, IgD and FAS across individual CTV division peaks. (G) Representative flow cytometry plot of plasma cell differentiation indicated by CD138 expression. Data information: Data are representative of 5-6 biological replicates (n = 5-6) form two independent experiments. F and G show concatenated data representative of 3-5 biological replicates.


( A - E ) Analysis of splenic SWHEL B cells and PC by flow cytometry . ( A ) Proportion of SWHEL B cells with a FAS + GL7 + GC phenotype , ( B ) SWHEL GC B cell count and ( C ) representation of IL - 21R deficient cells among total SWHEL GC B cells . ( D ) CD98 + CD138 + _ SWHEL PC count and ( E ) representation of IL - 21R deficient cells among total SWHEL PC . Data information : Data are representative of 6 - 10 biological replicates ( n = 6 - 10 ) from two independent experiments . Data in B , D were analyzed after log transformation with with statistical analysis by Multiple paired t tests ( A , B , D ) and p - values corrected for multiple comparisons using Holm - Šídák method or One - way ANOVA with Tukey ' s post - test ( C ) or t test ( E ) .

O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Analysis of splenic SWHEL B cells and PC by flow cytometry. (A) Proportion of SWHEL B cells with a FAS+GL7+ GC phenotype, (B) SWHEL GC B cell count and (C) representation of IL-21R deficient cells among total SWHEL GC B cells. (D) CD98+ CD138+_SWHEL PC count and (E) representation of IL-21R deficient cells among total SWHEL PC. Data information: Data are representative of 6-10 biological replicates (n = 6-10) from two independent experiments. Data in B, D were analyzed after log transformation with with statistical analysis by Multiple paired t tests (A, B, D) and p-values corrected for multiple comparisons using Holm-Šídák method or One-way ANOVA with Tukey's post-test (C) or t test (E).


Coomassie stained SDS - PAGE of McjD - C547 reconstituted in liposomes . The amount reconstituted was evaluated against detergent purified

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Coomassie stained SDS-PAGE of McjD-C547 reconstituted in liposomes. The amount reconstituted was evaluated against detergent purified protein


Chromatogram of McjD C547 ( black ) labelled with Alexa 555 ( blue ) and Alexa 647 ( red ) maleimide fluorophore using a Superdex 200 size - exclusion column . The fraction containing the highest degree of labeling is marked by a

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Chromatogram of McjD C547 (black) labelled with Alexa 555 (blue) and Alexa 647 (red) maleimide fluorophore using a Superdex 200 size-exclusion column. The fraction containing the highest degree of labeling is marked by a square


The ATPase activity of all labelled variants C547 , Y64C , L67C was determined in liposomes and compared to wild - type , wt . All labelled McjD variants show basal - and ligand - induced ATPase activities in liposomes comparable to that of wild type protein , indicating that labelling does not interfere with their activity . Error bars were calculated from two replicates from two independent reconstitutions ( mean ± standard deviation , n = 2 ) . The ruler character of FRET using the fluorophore pair Alexa555 / Cy5 was supported by data of dsDNA in Figure EV1B , where different base - pair distances between both dyes correlate directly with the apparent FRET

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The ATPase activity of all labelled variants C547, Y64C, L67C was determined in liposomes and compared to wild-type, wt. All labelled McjD variants show basal- and ligand-induced ATPase activities in liposomes comparable to that of wild type protein, indicating that labelling does not interfere with their activity. Error bars were calculated from two replicates from two independent reconstitutions (mean ± standard deviation, n=2). The ruler character of FRET using the fluorophore pair Alexa555/Cy5 was supported by data of dsDNA in Figure EV1B, where different base-pair distances between both dyes correlate directly with the apparent FRET efficiency


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Confocal single molecule analysis with ALEX of labeled McjD in detergent under different conditions as indicated


Apparent ligand binding affinity values of McjD for AMPPNP ( top ) , ATP ( middle ) , and ADP - vanadate ( bottom ) . Data points were obtained from the areas of fits from the E * histograms considering the ratio ligand - free / ( ligand - free + ligand - bound ) at the indicated substrate concentrations . Extraction of Kd values from a fit to the data is described in the Material and Methods section . Data for detergent - solubilized McjD for additional biochemical conditions is shown in Figure

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Apparent ligand binding affinity values of McjD for AMPPNP (top), ATP (middle), and ADP-vanadate (bottom). Data points were obtained from the areas of fits from the E* histograms considering the ratio ligand-free/(ligand-free+ligand-bound) at the indicated substrate concentrations. Extraction of Kd values from a fit to the data is described in the Material and Methods section. Data for detergent-solubilized McjD for additional biochemical conditions is shown in Figure EV2


Representative fluorescence time traces ( blue , donor signal ; red , acceptor signal ; black , FRET signal ; yellow , fit ) of McjD labeled with Alexa555 and Alexa647 - maleimide with indicated concentrations of corresponding substrate : ( B ) McjD L67C with 10 mM ATP and 200 µM MccJ25 , McjD Y64C with 10 mM ATP and 200 µM MccJ25 , and McjD C547 with 10 mM ATP . The respective right panels are the projections of accumulated time traces in the absence and presence of substrate for each variant . We note that differences between the setup dependent apparent FRET values are not influencing our interpretations since only relative changes of FRET efficiency are interpreted . Experimental support for proper reconstitution is provided in Figure EV3A showing longer bursts and distinct TEV cleavage for liposomes compared to

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Representative fluorescence time traces (blue, donor signal; red, acceptor signal; black, FRET signal; yellow, fit) of McjD labeled with Alexa555 and Alexa647-maleimide with indicated concentrations of corresponding substrate: (B) McjD L67C with 10 mM ATP and 200 µM MccJ25, McjD Y64C with 10 mM ATP and 200 µM MccJ25, and McjD C547 with 10 mM ATP. The respective right panels are the projections of accumulated time traces in the absence and presence of substrate for each variant. We note that differences between the setup dependent apparent FRET values are not influencing our interpretations since only relative changes of FRET efficiency are interpreted. Experimental support for proper reconstitution is provided in Figure EV3A showing longer bursts and distinct TEV cleavage for liposomes compared to detergent


Representative FRET time traces ( black , FRET signal ; yellow , fit ) of McjD NBD C547 labeled with Alexa 555 and Alexa 647 - maleimide under apo conditions at 10 ms time resolution . Approximately ~ 85 % of all apo - McjD traces show no significant fluctuations beyond shot - noise . Apo - McjD is predominantly in the low FRET and thus open state with E * values < 0 . 3 Within the complete data set of apo - McjD ( N = 80 traces ) comprising donor and acceptor , ~ 15 % , show infrequent fluctuations to a higher FRET efficiency state with a lifetime of ( C ) 82 ± 25 ms where E * values > 0 . 5 are observed during short dwells . The data set represents a total recording time of 4 . 6 mins with a temporal resolution of 10 ms . To exclude unwanted influence on McjD due to surface - immobilization , experimental verification is provided that ATP - induced NBD switching occurs similarly with detergent solubilized McjD on the surface in Figure

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Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide under apo conditions at 10 ms time resolution. Approximately ~85% of all apo-McjD traces show no significant fluctuations beyond shot-noise. Apo-McjD is predominantly in the low FRET and thus open state with E* values <0.3 Within the complete data set of apo-McjD (N = 80 traces) comprising donor and acceptor, ~15%, show infrequent fluctuations to a higher FRET efficiency state with a lifetime of (C) 82 ± 25 ms where E* values > 0.5 are observed during short dwells. The data set represents a total recording time of 4.6 mins with a temporal resolution of 10 ms. To exclude unwanted influence on McjD due to surface-immobilization, experimental verification is provided that ATP-induced NBD switching occurs similarly with detergent solubilized McjD on the surface in Figure EV4


Representative FRET time traces ( black , FRET signal ; yellow , fit ) of McjD NBD C547 labeled with Alexa 555 and Alexa 647 - maleimide in apo conditions at 10 ms time resolution . This panel shows stable ATP - loaded ( left ) and ATP - free ( right ) McjD Representative FRET time traces that show switching between ATP free McjD ( E * ~ 0 . 3 ) and ATP - loaded ( high FRET > 0 . 5 ) at ~ 4s s ( left ) and ~ 1 . 4 s ( right ) . The traces show photobleaching events of the acceptor after ~ 10 s ( left ) and ~ 3 . 3 s ( right ) Representative FRET time traces that show switching between ATP - loaded ( high FRET > 0 . 5 ) and ATP free McjD ( E * ~ 0 . 3 ) at 2 s ( left ) and 5 s ( right ) . The traces show photobleaching events after ca . 40 s ( left , donor - bleaching ) and 70 s ( right , acceptor - bleaching )

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Representative FRET time traces (black, FRET signal; yellow, fit) of McjD NBD C547 labeled with Alexa 555 and Alexa 647-maleimide in apo conditions at 10 ms time resolution. This panel shows stable ATP-loaded (left) and ATP-free (right) McjD Representative FRET time traces that show switching between ATP free McjD (E* ~ 0.3) and ATP-loaded (high FRET >0.5) at ~4s s (left) and ~1.4 s (right). The traces show photobleaching events of the acceptor after ~10 s (left) and ~3.3 s (right) Representative FRET time traces that show switching between ATP-loaded (high FRET >0.5) and ATP free McjD (E* ~ 0.3) at 2 s (left) and 5 s (right). The traces show photobleaching events after ca. 40 s (left, donor-bleaching) and 70 s (right, acceptor-bleaching)


B 18F - FDG PET - MRI at the time of the diagnosis and in the interim staging after one cycle of FOLFIRINOX .

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B 18F-FDG PET-MRI at the time of the diagnosis and in the interim staging after one cycle of FOLFIRINOX.


C H & E staining of FNB and the resection specimen . Scale bars represent 50 µm .

C H&E staining of FNB and the resection specimen. Scale bars represent 50 µm.


D Phase - contrast images of organoids ( upper panel ) and respective 2D cultures ( isolated from the biopsy before FOLFIRINOX ( ID188 ) or after the neoadjuvant therapy from the resected cancer ( ID211 ) . Scale bar represents 200µm .

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D Phase-contrast images of organoids (upper panel) and respective 2D cultures (isolated from the biopsy before FOLFIRINOX (ID188) or after the neoadjuvant therapy from the resected cancer (ID211). Scale bar represents 200µm.


E IF staining of the actin filaments ( red ) and nuclei ( blue ) of organoids in the left panel and H & E staining of embedded and sectioned organoids in the right panel ( ID188 p30 , ID211 p30 ) .

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E IF staining of the actin filaments (red) and nuclei (blue) of organoids in the left panel and H&E staining of embedded and sectioned organoids in the right panel (ID188 p30, ID211 p30).


F Normalized expression ( log2 scale ) for gene pairs evaluated during Purity independent subtyping of tumors ( PurIST ) . Normalized expression ( log2 scale ) , gene 1 = basal - like , gene 2 = classical .

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F Normalized expression (log2 scale) for gene pairs evaluated during Purity independent subtyping of tumors (PurIST). Normalized expression (log2 scale), gene 1 = basal-like, gene 2 = classical.


G Gene Set Enrichment Analysis ( GSEA ) of pre - ( ID188 , p7 ) and post - treatment ( ID211 , p6 ) PDO lines . Normalized enrichment score ( NES ) and the adjusted p - value is depicted .

G Gene Set Enrichment Analysis (GSEA) of pre-(ID188, p7) and post-treatment (ID211, p6) PDO lines. Normalized enrichment score (NES) and the adjusted p-value is depicted.


H Whole exome sequencing - based copy number profiles for ID188 ( p7 ) and ID211 ( p6 ) organoids .

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H Whole exome sequencing-based copy number profiles for ID188 (p7) and ID211 (p6) organoids.


I Allele frequencies of miss - and nonsense SNVs and Indels which are shared between both organoid lines ( purple ) , private to ID188 ( red ) or private to ID211 ( blue ) .

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I Allele frequencies of miss- and nonsense SNVs and Indels which are shared between both organoid lines (purple), private to ID188 (red) or private to ID211 (blue).


A Dose response curves of ID188 ( p27 - 29 ) and ID211 ( p26 - 28 ) organoids treated with FOLFIRINOX over three days . ATP was measured with CellTiter - Glo assays . Shown is the mean ± SD of three independent experiments .

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A Dose response curves of ID188 (p27-29) and ID211 (p26-28) organoids treated with FOLFIRINOX over three days. ATP was measured with CellTiter-Glo assays. Shown is the mean ± SD of three independent experiments.


C Plot of the delta AUC in ID188 ( p5 + 27 ) versus ID211 ( p9 + 11 ) cells over 415 drugs . Dashed lines demark the threshold of delta AUC > 0 . 3 or < - 0 . 3 . D Heatmap of the screening hits as defined in C . AUC is color coded .

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C Plot of the delta AUC in ID188 (p5+27) versus ID211 (p9+11) cells over 415 drugs. Dashed lines demark the threshold of delta AUC >0.3 or <-0.3. D Heatmap of the screening hits as defined in C. AUC is color coded.


E - J Dose response curves of ID188 and ID211 organoids ( ID188 p27 - 29 , ID211 p26 - 28 ) or 2D lines ( ID188 p5 + 29 , ID211 p9 + 13 ) treated with ( E and F ) : Cobimetinib or ( H and I ) : Biminetinib . ( G ) Cobimetinib and ( J ) Biminetinib : GI50 values . Shown is the mean ± SD of three independent experiments .

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E - J Dose response curves of ID188 and ID211 organoids (ID188 p27-29, ID211 p26-28) or 2D lines (ID188 p5+29, ID211 p9+13) treated with (E and F): Cobimetinib or (H and I): Biminetinib. (G) Cobimetinib and (J) Biminetinib: GI50 values. Shown is the mean ± SD of three independent experiments.


K Illustration of a cohort of patients ( n = 15 ) with available primary 2D PDAC cell lines . This cohort includes the ID188 ( pre - FFX ( FOLFIRINOX ) , red ) and ID211 ( post - FFX , blue ) lines . These lines were screened for sensitivity towards three MEKi . L 15 primary human PDAC 2D cell lines were screened for MEKi ( as indicated ) sensitivity . The determined AUC was variance scaled and the z - scores are depicted . The ID188 and ID211 identity is color coded .

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K Illustration of a cohort of patients (n=15) with available primary 2D PDAC cell lines. This cohort includes the ID188 (pre-FFX (FOLFIRINOX), red) and ID211 (post-FFX, blue) lines. These lines were screened for sensitivity towards three MEKi. L 15 primary human PDAC 2D cell lines were screened for MEKi (as indicated) sensitivity. The determined AUC was variance scaled and the z-scores are depicted. The ID188 and ID211 identity is color coded.


Treatment of HeLa cells with DFP ( 1mM for 24 h ) reduces the conjugation of SUMO - 2 / 3 ( n = 5 , biological replicates ; * * , p < 0 . 01 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) does not change global SUMO - 1 - ylation ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - test ) .

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Treatment of HeLa cells with DFP (1mM for 24 h) reduces the conjugation of SUMO-2/3 (n=5, biological replicates; **, p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change global SUMO-1-ylation (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).


Treatment of HeLa cells with DFP ( 1mM for 24 h ) leads to increased levels of SENP3 ( n = 8 , biological replicates ; * * p < 0 . 01 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) does not change levels of SENP5 ( n = 5 , biological replicates ; p > 0 . 05 ; N . S . , not statistically significant ; paired t - test ) .

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Treatment of HeLa cells with DFP (1mM for 24 h) leads to increased levels of SENP3 (n=8, biological replicates; **p<0.01; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) does not change levels of SENP5 (n=5, biological replicates; p>0.05; N.S., not statistically significant; paired t-test).


DFP treatment increases SENP3 stability . DFP ( 1mM for 24 h ) - treated HeLa cells were treated with cycloheximide ( CHX ; 50μg / ml ) for the indicated durations in a chase experiment . Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated .

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DFP treatment increases SENP3 stability. DFP (1mM for 24 h)-treated HeLa cells were treated with cycloheximide (CHX; 50μg/ml) for the indicated durations in a chase experiment. Lysate samples were separated on different lanes in the same gel and immunoblotted as indicated.


Treatment of HeLa cells with DFP ( 1mM for 24 h ) decreases levels of CHIP ( n = 8 , biological replicates ; * * * p < 0 . 001 ; paired t - test ) . Treatment of HeLa cells with DFP ( 1mM for 24 h ) leads to decreased levels of CHIP mRNA ( n = 3 , biological replicates ; * p < 0 . 05 ; unpaired t - test ) .

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Treatment of HeLa cells with DFP (1mM for 24 h) decreases levels of CHIP (n=8, biological replicates; ***p<0.001; paired t-test).   Treatment of HeLa cells with DFP (1mM for 24 h) leads to decreased levels of CHIP mRNA (n=3, biological replicates; *p<0.05; unpaired t-test).


Treatment of DFP ( 1mM for 24 h ) - treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP . Histogram shows relative levels of cellular CHIP upon four different conditions ( n = 3 , biological replicates ; * * * * p < 0 . 0001 ; * * * p < 0 . 001 ; repeated measures analysis of variance followed by Tukey ' s multiple comparisons test ) .

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Treatment of DFP (1mM for 24 h)-treated HeLa cells with 50μM MG132 for the final 6 h does not recover reduced levels of CHIP induced by DFP. Histogram shows relative levels of cellular CHIP upon four different conditions (n=3, biological replicates; ****p<0.0001; *** p<0.001; repeated measures analysis of variance followed by Tukey's multiple comparisons test).


CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells . Nsi or CHIPi ( Nsi , non - specific siRNA ; CHIPi , CHIP siRNA ; concentration , 20 nM ) was transfected into HeLa cells for 48 h . Lysate samples were blotted as indicated . CHIP knockdown prevents the DFP - induced increase in SENP3 levels . Nsi , SENP3i or CHIPi ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; CHIPi , CHIP siRNA ; siRNA concentration , 20nM ) was transfected into HeLa cells for 48 h . Two days post - transfection the cells were treated with DFP for a further 24 h .

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CHIP knockdown increases levels of SENP3 but not SENP5 in HeLa cells. Nsi or CHIPi (Nsi, non-specific siRNA; CHIPi, CHIP siRNA; concentration, 20 nM) was transfected into HeLa cells for 48 h. Lysate samples were blotted as indicated.   CHIP knockdown prevents the DFP-induced increase in SENP3 levels. Nsi, SENP3i or CHIPi (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; CHIPi, CHIP siRNA; siRNA concentration, 20nM) was transfected into HeLa cells for 48 h. Two days post-transfection the cells were treated with DFP for a further 24 h.


SENP3 knockdown abolishes the DFP - induced increase in LC3 - II in HeLa cells . Nsi or SENP3i ( I ) was transfected into HeLa cells . Two days post - transfection the cells were treated with DFP for a further 24 h . Whole cell lysate samples were blotted as indicated ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ; siRNA concentration , 20nM ; DFP , 1mM for 24 h ; values normalized to the control value ; n = 3 , biological replicates ; * * * , p < 0 . 001 ; N . S . , not significant , p > 0 . 05 ; paired t - test ) .

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SENP3 knockdown abolishes the DFP-induced increase in LC3-II in HeLa cells. Nsi or SENP3i (I) was transfected into HeLa cells. Two days post-transfection the cells were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; siRNA concentration, 20nM; DFP, 1mM for 24 h; values normalized to the control value; n=3, biological replicates; ***, p<0.001; N.S., not significant, p>0.05; paired t-test).


Nsi or SENP3i ( II ) was transfected into HeLa cells with stable expression of mito - Keima grown on 6 - well plates . Two days post - transfection the cells were treated with DFP ( 1mM ; 24 h ) . Cells transfected with Nsi and treated with CQ ( 50μM ; 24 h ) in the absence or presence of DFP ( 1mM ; 24 h ) were included as a negative control for mitophagy induction . The images show overlaid fluorescence and transmitted light images of unfixed , live cells obtained using an EVOS - fl inverted LED fluorescence microscope ( Red : mitophagic puncta ; Scale bar , 100 μm ) . Histogram shows the percentages of mitophagic cells under 6 different conditions ( n = 3 , biological replicates ; * * * * , p < 0 . 0001 ; Ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test ) .

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Nsi or SENP3i (II) was transfected into HeLa cells with stable expression of mito-Keima grown on 6-well plates. Two days post-transfection the cells were treated with DFP (1mM; 24 h). Cells transfected with Nsi and treated with CQ (50μM; 24 h) in the absence or presence of DFP (1mM; 24 h) were included as a negative control for mitophagy induction. The images show overlaid fluorescence and transmitted light images of unfixed, live cells obtained using an EVOS-fl inverted LED fluorescence microscope (Red: mitophagic puncta; Scale bar, 100 μm). Histogram shows the percentages of mitophagic cells under 6 different conditions (n=3, biological replicates; ****, p<0.0001; Ordinary one-way ANOVA followed by Tukey's multiple comparisons test).


SENP3 knockdown abolishes DFP - induced mitophagic autolysosomes . Nsi or SENP3i ( I ) together with mito - pHfluorin were transfected into HeLa cells in the absence or presence of DFP ( 24 h ) , and were analyzed using the mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Magenta : SENP3 ; Scale bar , 10 μm ; n = 28 cells per condition from two individual experiments ; * * * * p < 0 . 0001 ; unpaired t - test ) .

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SENP3 knockdown abolishes DFP-induced mitophagic autolysosomes. Nsi or SENP3i (I) together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), and were analyzed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Magenta: SENP3; Scale bar, 10 μm; n=28 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test).


Fis1 knockdown abolishes DFP - induced LC3 - II . Fis1 siRNA was introduced into HeLa cells for 48 h , and the cells were treated with DFP for further 24 h ( Nsi , non - specific siRNA ; Fis1i , Fis1 siRNA ; siRNA concentration , 20nM ; DFP , 1mM ) . Whole cell lysate samples were blotted as indicated . Values are presented as mean ± SEM and are normalized to the control value ( n = 3 , biological replicates ; * * , p < 0 . 01 ; paired t - test ) .

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

Fis1 knockdown abolishes DFP-induced LC3-II. Fis1 siRNA was introduced into HeLa cells for 48 h, and the cells were treated with DFP for further 24 h (Nsi, non-specific siRNA; Fis1i, Fis1 siRNA; siRNA concentration, 20nM; DFP, 1mM). Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=3, biological replicates; **, p<0.01; paired t-test).


Fis1 knockout abolishes DFP - induced LC3 - II . Fis1 + / + or Fis1 - / - cells were treated with DFP for 24 h . Whole cell lysate samples were blotted as indicated . Values are presented as mean ± SEM and are normalized to the control value ( n = 6 , biological replicates ; * , p < 0 . 05 ; paired t - test ) .

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

Fis1 knockout abolishes DFP-induced LC3-II. Fis1+/+ or Fis1-/- cells were treated with DFP for 24 h. Whole cell lysate samples were blotted as indicated. Values are presented as mean ± SEM and are normalized to the control value (n=6, biological replicates; *, p<0.05; paired t-test).


Fis1 knockdown prevents DFP - induced mitophagic autolysosomes . Nsi or Fis1i together with mito - pHfluorin were transfected into HeLa cells in the absence or presence of DFP ( 24 h ) , which were analysed using the mito - pHfluorin construct 72 h post - transfection ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Yellow : mitochondria labelled by mCherry - SEP - A ; Blue : Fis1 ; Scale bar , 10 μm ; n = 25 cells per condition from three individual experiments ; * * * p < 0 . 001 ; unpaired - t test ) .

B-MEASURED_VAR 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 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 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

Fis1 knockdown prevents DFP-induced mitophagic autolysosomes. Nsi or Fis1i together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), which were analysed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Blue: Fis1; Scale bar, 10 μm; n=25 cells per condition from three individual experiments; *** p<0.001; unpaired-t test).


Flag - Fis1 is deSUMOylated by SENP3 . SENP3 knockdown enhances Fis1 SUMOylation . Nsi or SENP3i was transfected into HEK293 cells expressing Flag - Fis1 , His - SUMO - 2 and Ubc9 for 48 h . His - pulldown and lysate samples were detected by immunoblotting for Flag , SENP3 or β - actin . Values are presented as mean ± SEM ( n = 3 , biological replicates ; * p < 0 . 05 ; paired - t test ) .

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

Flag-Fis1 is deSUMOylated by SENP3. SENP3 knockdown enhances Fis1 SUMOylation. Nsi or SENP3i was transfected into HEK293 cells expressing Flag-Fis1, His-SUMO-2 and Ubc9 for 48 h. His-pulldown and lysate samples were detected by immunoblotting for Flag, SENP3 or β-actin. Values are presented as mean ± SEM (n=3, biological replicates; * p<0.05; paired-t test).


The SUMOylation status of Fis1 influences its mitochondrial localization . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG was expressed in HeLa cells for 48 h . Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated ( the upper panel ) . Histograms ( the right panel ) show normalized levels of Flag - Fis1 WT or Flag - Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction ( Value are presented as mean ± SEM and are normalized to the control value ; n = 7 , biological replicates ; * , p < 0 . 05 ; paired t - test ) .

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

The SUMOylation status of Fis1 influences its mitochondrial localization. Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG was expressed in HeLa cells for 48 h. Whole cell lysates and cytosolic and mitochondrial fractions were prepared and blotted as indicated (the upper panel). Histograms (the right panel) show normalized levels of Flag-Fis1 WT or Flag-Fis1 K149R mutant associated with the cytosolic or mitochondrial fraction (Value are presented as mean ± SEM and are normalized to the control value; n=7, biological replicates; *, p<0.05; paired t-test).


The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum ( ER ) . Flag - Fis1 WT , Flag - Fis1 SUMOylation deficient mutant K149R or Flag - Fis1 - SUMO - 2ΔGG along with GFP - Cb5 as an ER marker were expressed in HeLa cells for 48 h . Immunocytochemistry was then performed ( Green : ER labelled by GFP - Cb5 ; Blue : different Flag - Fis1 forms ; Scale bar , 10 μm )

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

The SUMOylation status of Fis1 may influence its association with the Endoplasmic Reticulum (ER). Flag-Fis1 WT, Flag-Fis1 SUMOylation deficient mutant K149R or Flag-Fis1-SUMO-2ΔGG along with GFP-Cb5 as an ER marker were expressed in HeLa cells for 48 h. Immunocytochemistry was then performed (Green: ER labelled by GFP-Cb5; Blue: different Flag-Fis1 forms; Scale bar, 10 μm)


Expressing a SUMOylation - deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP - induced LC3 - II induction . pcDNA3 , Flag - Fis1 WT or Flag - Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h ( Nsi , non - specific siRNA ; SENP3i , SENP3 siRNA ( I ) ; siRNA concentration , 20nM ) , and the cells were treated with DFP for a further 24 h ( DFP , 1mM ) . Lysate samples were blotted as indicated .

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

Expressing a SUMOylation-deficient Fis1 mutant reverses the effect of SENP3 knockdown on DFP-induced LC3-II induction. pcDNA3, Flag-Fis1 WT or Flag-Fis1 K149R mutant was transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA (I); siRNA concentration, 20nM), and the cells were treated with DFP for a further 24 h (DFP, 1mM). Lysate samples were blotted as indicated.


SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP - Fis1 K149R in the presence of DFP . CFP - Fis1 WT or CFP - Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h , and the cells were treated with DFP for a further 24 h ( Red : mCherry - A or puncta indicating occurrences of mitophagy marked by white arrows ; Green : SEP - A ; Cyan / blue : CFP - Fis1 ; Magenta , SENP3 ; Scale bar , 10 μm ) ; Values are presented as mean ± SEM ( n = 21 cells per condition from two individual experiments ; * * * * p < 0 . 0001 ; unpaired t - test ) . Knockdown of SENP3 was further confirmed by immunoblotting ( the lower right panel ) .

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

SENP3 depletion does not abolish mitophagic puncta detected in HeLa cells expressing SUMOylation deficient CFP-Fis1 K149R in the presence of DFP. CFP-Fis1 WT or CFP-Fis1 K149R mutant were transfected into HeLa cells in which SENP3 was depleted using siRNA for 48 h, and the cells were treated with DFP for a further 24 h (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Cyan/blue: CFP-Fis1; Magenta, SENP3; Scale bar, 10 μm); Values are presented as mean ± SEM (n=21 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test). Knockdown of SENP3 was further confirmed by immunoblotting (the lower right panel).


B . BrdU - sensitized U - 2 - OS cells stably expressing GFP - NLS - CDKL5 were pre - incubated with DMSO ( mock ) , olaparib ( 5 μM ) , talazoparib ( 50 nM ) or PDD00017273 ( 0 . 3 μM ) for 1 h prior to micro - irradiation and live - imaging at the indicated times post - irradiation . One of three independent experiments is shown . Scale bar is 10

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

B. BrdU-sensitized U-2-OS cells stably expressing GFP-NLS-CDKL5 were pre-incubated with DMSO (mock), olaparib (5 μM), talazoparib (50 nM) or PDD00017273 (0.3 μM) for 1 h prior to micro-irradiation and live-imaging at the indicated times post-irradiation. One of three independent experiments is shown. Scale bar is 10 μm


E . BrdU - sensitized parental or PARP1Δ / Δ , PARP2Δ / Δ , PARP1 / 2Δ / Δ U - 2 - OS cells transiently expressing GFP NLS - CDKL5 were subjected to 355 nm line micro - irradiation followed by time lapse imaging . One of two independent experiments is shown . Scale bar is 10

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E. BrdU-sensitized parental or PARP1Δ/Δ, PARP2Δ/Δ, PARP1/2Δ/Δ U-2-OS cells transiently expressing GFP NLS-CDKL5 were subjected to 355 nm line micro-irradiation followed by time lapse imaging. One of two independent experiments is shown. Scale bar is 10 μm


G . Cells subjected to the workflow in F . were detergent - extracted and fixed before staining with anti - GFP or fibrillarin ( nucleoli ) . Scale bar is 10 μm H . Quantification of the detergent - insoluble GFP - NLS - CDKL5 signal ( minus nucleolar signal ) . The mean ± SD from three biological experiments is shown . Statistical significance was assessed by one - way - ANOVA - test . Asterisks * * indicate P - value of < 0 . 01 ; ns - not significant .

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 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

G. Cells subjected to the workflow in F. were detergent-extracted and fixed before staining with anti-GFP or fibrillarin (nucleoli). Scale bar is 10 μm H. Quantification of the detergent-insoluble GFP-NLS-CDKL5 signal (minus nucleolar signal). The mean ± SD from three biological experiments is shown. Statistical significance was assessed by one-way-ANOVA-test. Asterisks ** indicate P-value of <0.01; ns - not significant.


B . BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS , the GFP - NLS - CDKL5 deletion mutants shown in A , or full length ( FL ) GFP - NLS - CDKL5 were subjected to line micro - irradiation ( 355 nm ) and time lapse imaging . Three independent experiments were performed , and one representative experiment is shown . Scale bar is 10

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B. BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS, the GFP-NLS-CDKL5 deletion mutants shown in A, or full length (FL) GFP-NLS-CDKL5 were subjected to line micro-irradiation (355 nm) and time lapse imaging. Three independent experiments were performed, and one representative experiment is shown. Scale bar is 10 μm


D . Same as in B except that the GFP - NLS tagged CDKL5 fragments indicated were examined . Scale bar is 10

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D. Same as in B except that the GFP-NLS tagged CDKL5 fragments indicated were examined. Scale bar is 10 μm


F . Recombinant fragments of CDKL5 fused to GST ( 1 . 2 , 2 . 5 , 5 , 10 µg ) , or GST , were dot - blotted on nitrocellulose membrane and then incubated with synthetic PAR . PAR binding was detected by far western blotting . APLF was used as positive control . One of three independent experiments is

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F. Recombinant fragments of CDKL5 fused to GST (1.2, 2.5, 5, 10 µg), or GST, were dot-blotted on nitrocellulose membrane and then incubated with synthetic PAR. PAR binding was detected by far western blotting. APLF was used as positive control. One of three independent experiments is shown


( G - H ) . U - 2 - OS ( Flp - In T - Rex ) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min . Extracts were subjected to immunoprecipitation with antibodies against CDKL5 ( G ) or PAR ( H ) ( or non - specific IgG as control ) . Precipitates , and input lysates , were analyzed by western blotting using the indicated antibodies . One of two independent experiments is

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

(G-H) .U-2-OS (Flp-In T-Rex) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min. Extracts were subjected to immunoprecipitation with antibodies against CDKL5 (G) or PAR (H) (or non-specific IgG as control). Precipitates, and input lysates, were analyzed by western blotting using the indicated antibodies. One of two independent experiments is shown


A . ( Top ) BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS - CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro - irradiation ( 405 nm ) . ( Bottom ) Quantitation of spot intensities . Data represent the mean ± SEM of two independent experiments ; > 50 micro - irradiated cells per point . Scale bar is 10

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A. (Top) BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro-irradiation (405 nm). (Bottom) Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated cells per point. Scale bar is 10 μm


BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS - CDKL5 , mCherry - XRCC1 or mCherry - FAN1 were pre - incubated with indicated transcription inhibitors prior to line micro - irradiation ( 355 nm ) and time lapse imaging . One of three independent experiments is shown . Scale bar is 10

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 B-MEASURED_VAR 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

BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5, mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation (355 nm) and time lapse imaging. One of three independent experiments is shown. Scale bar is 10 μm


E . Same as B - D . except that BrdU - sensitized cells stably expressing GFP - NLS - CDKL5 were also pre - incubated with olaparib as control . Cells were subjected to line micro - irradiation , fixed and then subjected to indirect immunofluorescence using antibodies against GFP , PAR and γH2AX . Scale bar is 10

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E. Same as B-D. except that BrdU-sensitized cells stably expressing GFP-NLS-CDKL5 were also pre-incubated with olaparib as control. Cells were subjected to line micro-irradiation, fixed and then subjected to indirect immunofluorescence using antibodies against GFP, PAR and γH2AX. Scale bar is 10 μm


F . Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging . Scale bar is 10

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F. Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging. Scale bar is 10 μm


B . Boxplots showing VSN - normalised intensity of phospho - peptides corresponding to EP400 pSer729 , ELOA pSer311and TTDN1 pSer40 from the experiment in A . The central band of the boxplot indicates the median value , while the hinges represent the first and third quartile ( bottom and top of boxplot , respectively ) . The whiskers extend to the largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times the interquartile range from their respective hinge . In all cases the data was derived from 3 biological replicates .

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

B. Boxplots showing VSN-normalised intensity of phospho-peptides corresponding to EP400 pSer729, ELOA pSer311and TTDN1 pSer40 from the experiment in A. The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. In all cases the data was derived from 3 biological replicates.


C . Boxplots of the VSN - adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG - EP400 , FLAG - ELOA and FLAG - TTDN1 from the experiment in A . The central band of the boxplot indicates the median value , while the hinges represent the first and third quartile ( bottom and top of boxplot , respectively ) . The whiskers extend to the largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times the interquartile range from their respective hinge . Datapoints further removed than the whiskers are plotted individually . The experiment was conducted using three biological replicates within each respective group , each TMT channel represents a single biological replicate .

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

C. Boxplots of the VSN-adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG-EP400, FLAG-ELOA and FLAG-TTDN1 from the experiment in A.The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. Datapoints further removed than the whiskers are plotted individually. The experiment was conducted using three biological replicates within each respective group, each TMT channel represents a single biological replicate.


A . HEK293 cells were co - transfected with CDKL5 ( wild type " WT " or kinase - dead " KD " K42R mutant ) fused to an NLS , and FLAG - ELOA ( wild type " WT " or a S311A mutant " SA " ) . Anti - FLAG precipitates or cell extracts were probed with the antibodies indicated . One of three independent experiments is shown . B . Same as A . showing a range of pathogenic ( red ) and benign ( blue ) CDKL5 variants .

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 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 B-MEASURED_VAR O O

A. HEK293 cells were co-transfected with CDKL5 (wild type "WT" or kinase-dead "KD" K42R mutant) fused to an NLS, and FLAG-ELOA (wild type "WT" or a S311A mutant "SA"). Anti-FLAG precipitates or cell extracts were probed with the antibodies indicated. One of three independent experiments is shown. B. Same as A. showing a range of pathogenic (red) and benign (blue) CDKL5 variants.


( C - E ) . Wild type ( WT ) , CDKL5 - disrupted ( CDKL5Δ / Δ ) or siRNA - transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks . Quantification of ELOA - pSer311 signal at the laser tracks is shown . Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated ( n ) . For simplicity , only intensities greater than zero are shown . Statistical significance was assessed by one - way - ANOVA - test or unpaired t test with Welch ' s correction . Asterisks * * * * indicate P - values of < 0 . 0001 . Scale bar is 10

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(C-E). Wild type (WT), CDKL5-disrupted (CDKL5Δ/Δ) or siRNA-transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks. Quantification of ELOA-pSer311 signal at the laser tracks is shown. Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated (n). For simplicity, only intensities greater than zero are shown. Statistical significance was assessed by one-way-ANOVA-test or unpaired t test with Welch's correction. Asterisks **** indicate P-values of <0.0001. Scale bar is 10 μm


D . ( Left ) Quantification of transcription in U - 2 - OS 263 IFII reporter cells from experiment in B . > 150 cells were analysed per condition per experiment . The mean ± SD from four independent experiments is shown . ( Right ) Quantitative RT - PCR analysis of YFP - MS2 mRNA in U - 2 - OS 263 reporter cells . Data represent mean ± SD in different biological replicates as indicated ( n ) . Statistical significance for all the data was assessed by two - way - ANOVA - test , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; ns - not significant .

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D. (Left) Quantification of transcription in U-2-OS 263 IFII reporter cells from experiment in B. >150 cells were analysed per condition per experiment. The mean ± SD from four independent experiments is shown. (Right) Quantitative RT-PCR analysis of YFP-MS2 mRNA in U-2-OS 263 reporter cells. Data represent mean ± SD in different biological replicates as indicated (n). Statistical significance for all the data was assessed by two-way-ANOVA-test, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant.


B , C . Quantitative PCR with reverse transcription ( qRT - PCR ) analysis of SLCO5a1 ( B ) and RYR2 expression levels ( C ) ( left panels ) U - 2 - OS HA - ER - I - PpoI cells depleted of CDKL5 transiently transfected with FLAG - tagged CDKL5 wild type ( WT ) or a K42R - mutated kinase dead ( KD ) mutant , or empty vector , at the times indicated after inducing I - PpoI . The mean ± SD from two qPCR replicates of two independent experiments is shown . Statistical significance for all the data was assessed by two - way - ANOVA - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; ns - not significant . I - PpoI - mediated cutting efficiency in the relevant gene is shown in the right - hand

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B, C. Quantitative PCR with reverse transcription (qRT-PCR) analysis of SLCO5a1 (B) and RYR2 expression levels (C) (left panels) U-2-OS HA-ER-I-PpoI cells depleted of CDKL5 transiently transfected with FLAG-tagged CDKL5 wild type (WT) or a K42R-mutated kinase dead (KD) mutant, or empty vector, at the times indicated after inducing I-PpoI. The mean ± SD from two qPCR replicates of two independent experiments is shown. Statistical significance for all the data was assessed by two-way-ANOVA-test *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant. I-PpoI-mediated cutting efficiency in the relevant gene is shown in the right-hand panel


A . Heatmap summary of Aft1 induction experiment showing all genes that change with | log2 ( fold - change ) | > 1 . 5 .

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A. Heatmap summary of Aft1 induction experiment showing all genes that change with |log2(fold-change)| > 1.5.


( D ) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp ( without ATG ) to the sequence from 5 ' cap of pri - miR - 31 to ATG site or which mutated to ATT of the sORF . Cells were selected by G418 for 2 days . Analysis of the GFP expression by confocal microscopy or flow cytometry , scale bar 100 μm .

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(D) NIH 3T3 cells transfected by equal amounts of plasmid fused with gfp (without ATG) to the sequence from 5' cap of pri-miR-31 to ATG site or which mutated to ATT of the sORF. Cells were selected by G418 for 2 days. Analysis of the GFP expression by confocal microscopy or flow cytometry, scale bar 100 μm.


( E ) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP - N1 - miPEP31 or pEGFP - N1 , the red arrow indicate predicted size of miPEP31 - GFP band .

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(E) Immunoblot analysis of GFP and miPEP31 in NIH 3T3 cells transfected by pEGFP-N1-miPEP31 or pEGFP-N1, the red arrow indicate predicted size of miPEP31-GFP band.


( F ) Flow - fish to detect the localization of pri - miR - 31 . The location of pri - miR - 31 was analyzed by confocal microscopy . Scale bar 5 μm .

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(F) Flow-fish to detect the localization of pri-miR-31. The location of pri-miR-31 was analyzed by confocal microscopy. Scale bar 5 μm.


( G ) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31 . Values are expressed as fold changes relative to controls and normalized to GAPDH , the red arrow indicate predicted size of miPEP31 .

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(G) Immunoblot analysis of multiple mouse tissues with a specific antibody against miPEP31. Values are expressed as fold changes relative to controls and normalized to GAPDH, the red arrow indicate predicted size of miPEP31.


( H ) Immunoblot analysis of spleen , lymph node and thymus from WT or miR - 31 - / - mice with miPEP31 antibody , the red arrow indicate predicted size of miPEP31 .

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(H) Immunoblot analysis of spleen, lymph node and thymus from WT or miR-31-/- mice with miPEP31 antibody, the red arrow indicate predicted size of miPEP31.


( A ) CD4 + T cells were activated with anti - CD3 / 28 and treated with different concentration of FAM - miPEP31 for 24 hours , the fluorescein was detected by Flow Cytometry .

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(A) CD4+ T cells were activated with anti-CD3/28 and treated with different concentration of FAM-miPEP31 for 24 hours, the fluorescein was detected by Flow Cytometry.


( B ) Confocal Microscopy was used to analyze the fluorescence of FAM - miPEP31 in anti - CD3 / 28 activated CD4 + T cells treated with 10 μM FAM - miPEP31 . Scale bar 5 μm .

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(B) Confocal Microscopy was used to analyze the fluorescence of FAM-miPEP31 in anti-CD3/28 activated CD4+ T cells treated with 10 μM FAM-miPEP31. Scale bar 5 μm.


Anti - CD3 / 28 activated CD4 + T cells were cultured with 10 μM miPEP31 for 24 hours . ( C ) Western Blotting analysis of Cytochrome C and Histone H2A in total lysates and nuclear lysates .

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Anti-CD3/28 activated CD4+ T cells were cultured with 10 μM miPEP31 for 24 hours. (C) Western Blotting analysis of Cytochrome C and Histone H2A in total lysates and nuclear lysates.


Anti - CD3 / 28 activated CD4 + T cells were cultured with 10 μM miPEP31 for 24 hours . ( D ) ELISA to detect the concentration of miPEP31 in protein of total cell and nuclear extracts in CD4 + T and Treg cells treated with or without 10 μM miPEP31 . Data in ( D ) are presented as mean ± SEM of four biological replicates . ns , not significant ( CD4 + T versus Treg ) , unpaired two - tailed Student ' s t - test . .

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Anti-CD3/28 activated CD4+ T cells were cultured with 10 μM miPEP31 for 24 hours. (D) ELISA to detect the concentration of miPEP31 in protein of total cell and nuclear extracts in CD4+ T and Treg cells treated with or without 10 μM miPEP31. Data in (D) are presented as mean ± SEM of four biological replicates. ns, not significant (CD4+ T versus Treg), unpaired two-tailed Student's t-test..


( E ) Naïve CD4 + T cells were isolated from WT C57BL / 6 mice , and activated by anti - CD3 / 28 for 48 hours . Subsequently , FAM - labeled miPEP31 was added . Imaging Flow Cytometry analysis of FAM labeled miPEP31 in CD4 + T cells . Scale bar 7 μm .

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(E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, FAM-labeled miPEP31 was added. Imaging Flow Cytometry analysis of FAM labeled miPEP31 in CD4+ T cells. Scale bar 7 μm.


( F , G ) C57BL / 6 mice were injected intravenously with 100 μg FAM - labeled miPEP31 . Splenocytes were isolated and stained for CD4 and DAPI . Imaging Flow Cytometry analysis of miPEP31 in CD4 + T cells ( F ) and non - CD4 splenocytes ( G ) , scale bar 10 μm .

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(F, G) C57BL/6 mice were injected intravenously with 100 μg FAM-labeled miPEP31. Splenocytes were isolated and stained for CD4 and DAPI. Imaging Flow Cytometry analysis of miPEP31 in CD4+ T cells (F) and non-CD4 splenocytes (G) , scale bar 10 μm.


( D , E ) Naïve CD4 + T cells were isolated from WT C57BL / 6 mice , and activated by anti - CD3 / 28 for 48 hours . Subsequently , 10 μM FAM - labeled miPEP31 was added for 20 min at 4 ℃ or 37 ℃ . Confocal Microscopy analysis of the fluorescence . Scale bar 10 μm . The localization of miPEP31 with DAPI was shown in ( E ) . Data are representative nuclear co - localization of miPEP31 in four representative images from four biological experiments . ( mean ± SEM ) . * * * * p < 0 . 0001 , unpaired two - tailed Student ' s t - test .

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(D, E) Naïve CD4+ T cells were isolated from WT C57BL/6 mice, and activated by anti-CD3/28 for 48 hours. Subsequently, 10 μM FAM-labeled miPEP31 was added for 20 min at 4℃ or 37℃. Confocal Microscopy analysis of the fluorescence. Scale bar 10 μm. The localization of miPEP31 with DAPI was shown in (E). Data are representative nuclear co-localization of miPEP31 in four representative images from four biological experiments. (mean ± SEM). ****p < 0.0001, unpaired two-tailed Student's t-test.


scPEP or miPEP31 was injected intravenously every 3 days starting from day 8 post immunization . ( D , E ) H & E and Fast blue staining of paraffin sections of spinal cords derived from scPEP or miPEP31 treated mice on day 15 after immunization . Histopathology scores of inflammation and demyelination were shown in ( E ) . Scale bars , 70 μm . Data in ( C , E ) are presented as mean ± SEM of three independent experiments with nine to ten mice per group in each . * * * *

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scPEP or miPEP31 was injected intravenously every 3 days starting from day 8 post immunization. (D, E) H&E and Fast blue staining of paraffin sections of spinal cords derived from scPEP or miPEP31 treated mice on day 15 after immunization. Histopathology scores of inflammation and demyelination were shown in (E). Scale bars, 70 μm. Data in (C, E) are presented as mean ± SEM of three independent experiments with nine to ten mice per group in each. ****


IL - 1β concentrations in supernatants of macrophages , unstimulated ( control ) ( B ) or stimulated with SP / N ( C ) or LPS / N ( D ) .

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IL-1β concentrations in supernatants of macrophages, unstimulated (control) (B) or stimulated with SP/N (C) or LPS/N (D).


E . SARS - CoV - 2 spike ELISA of the corresponding plasma : ( EC50 titer ) of samples collected at T0 ( n = 35 individuals ) , T1 ( n = 28 individuals ) or T2 ( n = 44 individuals ) .

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E. SARS-CoV-2 spike ELISA of the corresponding plasma: (EC50 titer) of samples collected at T0 (n = 35 individuals), T1 (n = 28 individuals) or T2 (n = 44 individuals).


F . Immunofluorescence microscopy images of ASC specks ( green ) in T2 macrophages . Nuclei were stained with DAPI ( blue ) . Scale bar indicates 20 µm . Arrow indicates ASC speck formation with magnified illustration in the right corner of the image .

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F. Immunofluorescence microscopy images of ASC specks (green) in T2 macrophages. Nuclei were stained with DAPI (blue). Scale bar indicates 20 µm. Arrow indicates ASC speck formation with magnified illustration in the right corner of the image.


G , H . ASC specks were quantified microscopically at T0 ( G ) ( n = 7 individuals ) and T2 ( H ) ( n = 8 individuals ) upon indicated stimuli .

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G, H. ASC specks were quantified microscopically at T0 (G) (n = 7 individuals) and T2 (H) (n = 8 individuals) upon indicated stimuli.


I . Cleaved gasdermin D ( lysate ) and cleaved IL - 1β ( supernatant ) detected by Western Blot in macrophages stimulated with LPS / N or SP / N at T0 and T2 .

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I. Cleaved gasdermin D (lysate) and cleaved IL-1β (supernatant) detected by Western Blot in macrophages stimulated with LPS/N or SP/N at T0 and T2.


J , K . Cell death quantification of macrophages by flow cytometry in unstimulated or SP / N stimulated cells at T0 ( n = 5 individuals ) ( J ) and T2 ( n = 5 individuals ) ( K ) using the viability dye Zombie UV . LPS / N was used as positive control .

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J, K. Cell death quantification of macrophages by flow cytometry in unstimulated or SP/N stimulated cells at T0 (n = 5 individuals) (J) and T2 (n = 5 individuals) (K) using the viability dye Zombie UV. LPS/N was used as positive control.


L . LDH release of macrophages ( n = 6 individuals ) stimulated with SP / N at T2 compared to unstimulated cells . M . IL - 1β concentrations of MCC950 treated T2 macrophage ( n = 44 individuals ) stimulation with SP / N .

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L. LDH release of macrophages (n = 6 individuals) stimulated with SP/N at T2 compared to unstimulated cells. M. IL-1β concentrations of MCC950 treated T2 macrophage (n = 44 individuals) stimulation with SP/N.


B . Volcano plot showing DEGs in the same samples . Negative log10 padj - values are plotted against the log2 fold - change . Selected genes are labeled .

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B. Volcano plot showing DEGs in the same samples. Negative log10 padj-values are plotted against the log2 fold-change. Selected genes are labeled.


C , D . CLEC4D ( C ) and CLEC4E ( D ) surface expression on patient - derived macrophages prior ( n = 5 individuals ) or post ( n = 5 individuals ) vaccination were quantified by flow cytometry . Graphs indicate the mean fluorescence intensity ( MFI ) .

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C, D. CLEC4D (C) and CLEC4E (D) surface expression on patient-derived macrophages prior (n = 5 individuals) or post (n = 5 individuals) vaccination were quantified by flow cytometry. Graphs indicate the mean fluorescence intensity (MFI).


E , F . SP or LPS as a control PAMP were incubated with affinity purified CLRs ( ratio : 10 vs . 1 for SP ) for 1h and then added to primary T2 macrophages ( n = 5 individuals ) . Subsequently , IL - 1β was quantified in the supernatants . ( E ) shows absolute values for SP ; ( F ) shows the fold change calculated with the corresponding unstimulated control for SP and LPS .

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E, F. SP or LPS as a control PAMP were incubated with affinity purified CLRs (ratio: 10 vs. 1 for SP) for 1h and then added to primary T2 macrophages (n = 5 individuals). Subsequently, IL-1β was quantified in the supernatants. (E) shows absolute values for SP; (F) shows the fold change calculated with the corresponding unstimulated control for SP and LPS.


G . Monocytes were isolated prior ( n = 5 individuals ) or after vaccination ( n = 5 individuals ) and differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin . H . Monocytes were isolated prior ( T0 ; n = 35 individuals ) or after vaccination [ 2 weeks after 1st ( T1 ; n = 28 individuals ) , 2 weeks ( T2 ; n = 38 individuals ) and 10 weeks ( T3 ; n = 33 individuals ) after 2nd vaccination ] and differentiated to macrophages . IL - 1β concentrations in supernatants of macrophages , stimulated with the SP S2 subunit SP are shown .

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G. Monocytes were isolated prior (n = 5 individuals) or after vaccination (n = 5 individuals) and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages were measured after stimulation with the SP S1 subunit SP and nigericin. H. Monocytes were isolated prior (T0; n = 35 individuals) or after vaccination [2 weeks after 1st (T1; n = 28 individuals), 2 weeks (T2; n = 38 individuals) and 10 weeks (T3; n =33 individuals) after 2nd vaccination] and differentiated to macrophages. IL-1β concentrations in supernatants of macrophages, stimulated with the SP S2 subunit SP are shown.


B . Phospho - SYK Immunofluorescence - microscopy in macrophages before and after vaccination . Nuclei stained with DAPI . Scale bars indicate 20 µm .

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B. Phospho-SYK Immunofluorescence-microscopy in macrophages before and after vaccination. Nuclei stained with DAPI. Scale bars indicate 20 µm.


C . Quantification of pSYK positive macrophages by flow cytometry before ( n = 3 individuals ) and 14 d after vaccination ( n = 3 individuals ) . Total SYK was measured as control .

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C. Quantification of pSYK positive macrophages by flow cytometry before (n = 3 individuals) and 14 d after vaccination (n = 3 individuals). Total SYK was measured as control.


D . Western blot analysis of phospho - NF - κB and NLRP3 in cell lysates of macrophages ( T0 ; T2 ) stimulated with LPS / N or SP / N .

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D. Western blot analysis of phospho-NF-κB and NLRP3 in cell lysates of macrophages (T0; T2) stimulated with LPS/N or SP/N.


E . Macrophages were incubated with IKK - β - inhibitor KINK - 1 ( n = 10 individuals ) and SYK - inhibitors entospletinib ( n = 20 individuals ) , R406 ( n = 13 individuals ) prior to SP / nigericin stimulation . IL - 1β concentrations measured with ELISA . DMSO - treated , SP / N - stimulated and untreated unstimulated macrophages were used as control ( both n = 44 individuals ) .

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E. Macrophages were incubated with IKK-β-inhibitor KINK-1 (n = 10 individuals) and SYK-inhibitors entospletinib (n = 20 individuals), R406 (n = 13 individuals) prior to SP/nigericin stimulation. IL-1β concentrations measured with ELISA. DMSO-treated, SP/N-stimulated and untreated unstimulated macrophages were used as control (both n = 44 individuals).


F . IL - 1β quantification in THP - 1 wild - type cells incubated with SYK inhibitors entospletinib ( n = 4 independent experiments ) and R406 ( n = 2 independent experiments ) or in THP - 1 SYKKO cells stimulated with SP / N ( n = 5 independent experiments ) .

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F. IL-1β quantification in THP-1 wild-type cells incubated with SYK inhibitors entospletinib (n = 4 independent experiments) and R406 (n = 2 independent experiments) or in THP-1 SYKKO cells stimulated with SP/N (n = 5 independent experiments).


G . ASC formation quantified by immunofluorescence - microscopy in SP / N stimulated primary macrophages after vaccination ( n = 5 individuals ) .

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G. ASC formation quantified by immunofluorescence-microscopy in SP/N stimulated primary macrophages after vaccination (n = 5 individuals).


H . Quantification of cell death in T2 macrophages ( n = 4 individuals ) treated with R406 prior to SP / N - stimulation .

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H. Quantification of cell death in T2 macrophages (n = 4 individuals) treated with R406 prior to SP/N-stimulation.


I - K . mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP / N - stimulated macrophages without inhibitor ( I [ exemplary results ] , J [ T0 : n = 5 individuals ; T2 : n = 5 individuals ] )

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I-K. mROS quantified by flow cytometry using MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages without inhibitor (I [exemplary results], J [T0: n = 5 individuals; T2: n = 5 individuals])


mROS quantified MitoSOX red staining at T0 and T2 in SP / N - stimulated macrophages with R406 treatment ( n = 4 individuals ) ( K ) .

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

mROS quantified MitoSOX red staining at T0 and T2 in SP/N-stimulated macrophages with R406 treatment (n = 4 individuals) (K).