Growth of co S ( 2n - 1 ) in the absence of choline is lost when GPT2 or DGA1 is overexpressed , and restored by SorA . Plasmids pHEYg - 1 - DGA1 , pHEYg - 1 - GPT2 and the empty vector control ( pEV ) were transformed into the strains indicated . After preculture in SD C + , ten - fold serial dilutions were spotted on SD C + / - with or without 0 . 1 μg / mL SorA and incubated at 30 oC for 3 d .

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

Growth of co S(2n-1) in the absence of choline is lost when GPT2 or DGA1 is overexpressed, and restored by SorA. Plasmids pHEYg-1-DGA1, pHEYg-1-GPT2 and the empty vector control (pEV) were transformed into the strains indicated. After preculture in SD C+, ten-fold serial dilutions were spotted on SD C+/- with or without 0.1 μg/mL SorA and incubated at 30 oC for 3 d.


Intensity encoded GP - images of the indicated C - Laurdan - stained yeast strains cultured with or without choline , accompanied by the corresponding intensity weighted GP - histogram fitted to a single Gaussian function ( light blue line ) , and transmission image . White arrows point to plasma membranes exhibiting higher lipid packing than internal , organellar membranes ; blue horizontal arrows indicate highly packed lipid material in co S ( 2n - 1 ) . To deplete the cho2opi3 parent of PC , cells were transferred to SD C - at OD600 0 . 1 , and cultured into mid - log phase . White scale bars correspond to 10 µm ; red scale bars in transmission images correspond to 5 µm .

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 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

Intensity encoded GP-images of the indicated C-Laurdan-stained yeast strains cultured with or without choline, accompanied by the corresponding intensity weighted GP-histogram fitted to a single Gaussian function (light blue line), and transmission image. White arrows point to plasma membranes exhibiting higher lipid packing than internal, organellar membranes; blue horizontal arrows indicate highly packed lipid material in co S(2n-1). To deplete the cho2opi3 parent of PC, cells were transferred to SD C- at OD600 0.1, and cultured into mid-log phase. White scale bars correspond to 10 µm; red scale bars in transmission images correspond to 5 µm.


C Time - lapse photographs of typical patterns obtained for bacteria alone and bacteria with phage , with corresponding model simulations . Scale bar represents 1 cm .

O O O O 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 Time-lapse photographs of typical patterns obtained for bacteria alone and bacteria with phage, with corresponding model simulations. Scale bar represents 1 cm.


F Visualization of infected region . E . coli FM15 was inoculated at the center of a semi - solid agar plate and the reporter phage M13 - sfGFP was inoculated 1 - cm away from the center . Fluorescence images were captured after overnight incubation using FITC channel and an exposure time of 200 ms . Scale bar represents 1 cm . G Relative fluorescence intensity at the center and edge positions of the infection zone as shown in ( F ) . The relative intensity was obtained by dividing the detected values with the maximum value of green fluorescence intensity on the plate . Data represent mean values ± s . d . for three values of the center and six values of the edge from three biological replicates . Two - tailed t - test was used to compare two groups . * * * , p = 0 . 0005 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Visualization of infected region. E. coli FM15 was inoculated at the center of a semi-solid agar plate and the reporter phage M13-sfGFP was inoculated 1-cm away from the center. Fluorescence images were captured after overnight incubation using FITC channel and an exposure time of 200 ms. Scale bar represents 1 cm. G Relative fluorescence intensity at the center and edge positions of the infection zone as shown in (F). The relative intensity was obtained by dividing the detected values with the maximum value of green fluorescence intensity on the plate. Data represent mean values ± s.d. for three values of the center and six values of the edge from three biological replicates. Two-tailed t-test was used to compare two groups. ***, p = 0.0005.


B Microscopic images of E . coli FM15 cells carrying plasmids with gfp gene downstream of the psp promoter with or without wild - type M13 phage infection . Scale bar : 1 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

B Microscopic images of E. coli FM15 cells carrying plasmids with gfp gene downstream of the psp promoter with or without wild-type M13 phage infection. Scale bar: 1 μm.


D Photographs of a quarter of the semi - solid agar plate of a SPACE experiment using high - copy accessory plasmid to improve within - cell activity of T7 RNAP on a synthetic promoter 1D8 . An E . coli FM15 strain carrying no MP was used as control . Scale bar : 1 cm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Photographs of a quarter of the semi-solid agar plate of a SPACE experiment using high-copy accessory plasmid to improve within-cell activity of T7 RNAP on a synthetic promoter 1D8. An E. coli FM15 strain carrying no MP was used as control. Scale bar: 1 cm.


E Activity in cells of T7 RNAP and its mutants from the SPACE experiment in ( D ) . Two clones of the ancestor and five clones of the mutants obtained from the evolution towards 1D8 recognition were measured by in vivo transcriptional assay Two - tailed t - test was used to compare the two groups . * * * , p = 0 . 0001 . Data represent mean values ± s . d . .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Activity in cells of T7 RNAP and its mutants from the SPACE experiment in (D). Two clones of the ancestor and five clones of the mutants obtained from the evolution towards 1D8 recognition were measured by in vivo transcriptional assay Two-tailed t-test was used to compare the two groups. ***, p=0.0001. Data represent mean values ± s.d..


B Microscopic images of E . coli FM15 cells with or without phage infection . Scale bar represents 1 μm . C The experimental result shows raw photographs of a representative two - phage competition 20 h after initial inoculation with 105 : 1 mixture of M13w : M13s at 1 cm away from the center . The simulation result is the outcome of competition between two phages PW and PS with relative production rates of 40 and 100 , respectively . Scale bar represents 1 cm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Microscopic images of E. coli FM15 cells with or without phage infection. Scale bar represents 1 μm. C The experimental result shows raw photographs of a representative two-phage competition 20 h after initial inoculation with 105:1 mixture of M13w: M13s at 1 cm away from the center. The simulation result is the outcome of competition between two phages PW and PS with relative production rates of 40 and 100, respectively. Scale bar represents 1 cm.


D Phage titer ratio of M13s to M13w along the central line and the edge of the fan - shaped infection zone in the experimental result in ( C ) . Samples were collected from different positions as shown in the schematic and phage titer was quantified by qPCR Data represent mean values ± s . d . for three independent assays . Two - tailed t - test was used to compare two groups . * , p = 0 . 028 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Phage titer ratio of M13s to M13w along the central line and the edge of the fan-shaped infection zone in the experimental result in (C). Samples were collected from different positions as shown in the schematic and phage titer was quantified by qPCR Data represent mean values ± s.d. for three independent assays. Two-tailed t-test was used to compare two groups. *, p = 0.028.


C Fold change in the relative expression activity of the evolved RNAP mutants as compared with the wild - type T7 RNAP , for the 10 selected promoter sequences corresponding to the images in ( B ) . Results were obtained by in vitro transcriptional assay Data represent mean values ± s . d . for three biologically independent assays .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fold change in the relative expression activity of the evolved RNAP mutants as compared with the wild-type T7 RNAP, for the 10 selected promoter sequences corresponding to the images in (B). Results were obtained by in vitro transcriptional assay Data represent mean values ± s.d. for three biologically independent assays.


D Expression activity of 10 RNAP mutants on T7 promoter and 10 promoter variants measured by in vivo transcriptional assay . Mutants are arranged in the same order as their original target promoters listed on the left side of the heatmap . Data are normalized so that the activity of wild - type RNAP on the T7 promoter is 100 ; the mean for three biologically independent replicates is

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Expression activity of 10 RNAP mutants on T7 promoter and 10 promoter variants measured by in vivo transcriptional assay. Mutants are arranged in the same order as their original target promoters listed on the left side of the heatmap. Data are normalized so that the activity of wild-type RNAP on the T7 promoter is 100; the mean for three biologically independent replicates is shown


A . Representative pseudo colour images of sypHy fluorescence in WT and BsnGT hippocampal neurons at rest , upon stimulation with 40 and 900 APs at 20Hz ( in the presence of bafilomycin A1 ) and upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation . B . Average traces of the normalised fluorescence change ( ΔF / F0 ) of sypHy in WT ( black ) and BsnGT ( red ) neurons as described in A . Intensities were normalized to the peak of NH4Cl response . C . Quantifications of mean RRP fraction in WT and BsnGT . D . Frequency distribution histograms of the response amplitudes of 1050 individual synaptic puncta ( from 6 independent experiments ) to stimulation with 40 AP . The distribution is shifted to lower values in BsnGT . E . Quantifications of mean TRP fraction in WT and BsnGT . F . Frequency distribution histograms of synaptic response amplitudes in BsnGT and WT neurons stimulated with 900 AP . Note the shift in distribution between genotypes . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . In the frequency distribution histograms ( D and F ) black lines depict superimposed Gaussian fits for each group . The sample size n ( in parentheses ) corresponds to the number of analysed imaging experiments ( B , C , E ) In D and F , the data of 1050 synapses per genotype were processed . Data is obtained from 6 ( A - F ) independent culture preparations . Significance was assessed with Student ' s t - test ; * * * p < 0 . 001 . Scale bar is 2 µm in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 pseudo colour images of sypHy fluorescence in WT and BsnGT hippocampal neurons at rest, upon stimulation with 40 and 900 APs at 20Hz (in the presence of bafilomycin A1) and upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation. B. Average traces of the normalised fluorescence change (ΔF/F0) of sypHy in WT (black) and BsnGT (red) neurons as described in A. Intensities were normalized to the peak of NH4Cl response. C. Quantifications of mean RRP fraction in WT and BsnGT. D. Frequency distribution histograms of the response amplitudes of 1050 individual synaptic puncta (from 6 independent experiments) to stimulation with 40 AP. The distribution is shifted to lower values in BsnGT. E. Quantifications of mean TRP fraction in WT and BsnGT. F. Frequency distribution histograms of synaptic response amplitudes in BsnGT and WT neurons stimulated with 900 AP. Note the shift in distribution between genotypes. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. In the frequency distribution histograms (D and F) black lines depict superimposed Gaussian fits for each group. The sample size n (in parentheses) corresponds to the number of analysed imaging experiments (B, C, E) In D and F, the data of 1050 synapses per genotype were processed. Data is obtained from 6 (A-F) independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 2 µm in A


G , Representative images of Syt1Ab uptake ( magenta ) driven by endogenous network activity in hippocampal neurons ( 18 DIV ) from WT and BsnGT mice . VGLUT1 ( green ) marks excitatory presynapses ( upper panels ) and VGAT the inhibitory ones ( lower panels ) . H . Quantification of normalized IF of Syt1Ab uptake in excitatory ( red boxes ) and inhibitory ( pink boxes ) synapses on experiments in G . I . Quantification of fraction of active ( Syt1Ab - labelled ) excitatory and inhibitory synapses on experiments illustrated in G . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The dashed lines in H depict IF in WT that were used for normalization . The sample size n ( in parentheses ) corresponds to the number of analysed images ( G , Data is obtained from independent culture preparations . Significance was assessed with Student ' s t - test ; * * * p < 0 . 001 . Scale bar is 5 µm in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Representative images of Syt1Ab uptake (magenta) driven by endogenous network activity in hippocampal neurons (18 DIV) from WT and BsnGT mice. VGLUT1 (green) marks excitatory presynapses (upper panels) and VGAT the inhibitory ones (lower panels). H. Quantification of normalized IF of Syt1Ab uptake in excitatory (red boxes) and inhibitory (pink boxes) synapses on experiments in G. I. Quantification of fraction of active (Syt1Ab-labelled) excitatory and inhibitory synapses on experiments illustrated in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The dashed lines in H depict IF in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed images (G, Data is obtained from independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 5 µm in G


J . Representative images of Syt1Ab uptake ( magenta ) upon depolarization with 50 mM KCl . Identical cultures and staining were applied as in G . K . Quantification of normalized IF of Syt1Ab uptake on experiments from J . L . Quantification of fraction of active excitatory and inhibitory synapses in J . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The dashed lines in K depict IF in WT that were used for normalization . The sample size n ( in parentheses ) corresponds to the number of analysed images J ) . Data is obtained from independent culture preparations . Significance was assessed with Student ' s t - test ; * * * p < 0 . 001 . Scale bar is 5 µm in J .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of Syt1Ab uptake (magenta) upon depolarization with 50 mM KCl. Identical cultures and staining were applied as in G. K. Quantification of normalized IF of Syt1Ab uptake on experiments from J. L. Quantification of fraction of active excitatory and inhibitory synapses in J. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The dashed lines in K depict IF in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed images J). Data is obtained from independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 5 µm in J.


A , B . Representative pseudo colour images ( A ) and average traces ( B ) of sypHy fluorescence plotted for WT and BsnGT neurons treated with a CDK5 inhibitor roscovitine ( rosc , 100µM , gray and pink trace ) or vehicle ( black and red trace ) for 30 minutes before stimulation with 40 and 900 APs in the presence of bafilomycin A . C , D . Plots show mean values of RRP ( C ) and TRP ( D ) fraction for both genotypes before and after treatment . E . Roscovitine treatment has a significantly higher effect on RRP and TRP in BsnGT neurons compared to WT . Data information : n corresponding to the number of imaging experiments done on 4 independent cell preparations is given in brackets for each analysis . In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . Significance was assessed by two - way ANOVA with Tukey ´ s multiple comparison test ( C , D ) and by Student ' s t - test ( E ) * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 2µm .

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

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons treated with a CDK5 inhibitor roscovitine (rosc, 100µM, gray and pink trace) or vehicle (black and red trace) for 30 minutes before stimulation with 40 and 900 APs in the presence of bafilomycin A. C, D. Plots show mean values of RRP (C) and TRP (D) fraction for both genotypes before and after treatment. E. Roscovitine treatment has a significantly higher effect on RRP and TRP in BsnGT neurons compared to WT. Data information: n corresponding to the number of imaging experiments done on 4 independent cell preparations is given in brackets for each analysis. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. Significance was assessed by two-way ANOVA with Tukey´s multiple comparison test (C, D) and by Student's t-test (E) *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2µm.


A . Representative immunoblot with antibody against CDK5 - dependent pSer551 of Syn1 ( pSer551Syn1 ) and total Syn1 and total protein stain used as loading control for quantification of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT and BsnGT mice . B , C . Quantification of pSer551Syn1 / Syn1 ratio ( in B ) and total Syn1 ( in C ) abundance on blots from A . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size n ( in brackets ) corresponds to the number of independently processed animals in B and C For statistics , student ' s t - test in B and C were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunoblot with antibody against CDK5-dependent pSer551 of Syn1 (pSer551Syn1) and total Syn1 and total protein stain used as loading control for quantification of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT and BsnGT mice. B, C. Quantification of pSer551Syn1 / Syn1 ratio (in B) and total Syn1 (in C) abundance on blots from A. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independently processed animals in B and C For statistics, student's t-test in B and C were used, *p≤0.05, **p < 0.01, ***p< 0.001.


E . Representative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained for pSer551Syn1 and total Syn1 . F . Quantification of IF intensity of pSer551Syn1 in synapses from WT , BsnGT , and WT cells treated with roscovitine . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size n ( in brackets ) corresponds to the number of independent images ( E , quantified from two ( F ) independent cultures per condition . For statistics , one - way ANOVA with Sidak ´ s multiple comparisons test in F were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 5 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 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

E. Representative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained for pSer551Syn1 and total Syn1. F. Quantification of IF intensity of pSer551Syn1 in synapses from WT, BsnGT, and WT cells treated with roscovitine. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images (E, quantified from two (F) independent cultures per condition. For statistics, one-way ANOVA with Sidak´s multiple comparisons test in F were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in


G . Representative images of 21 DIV hippocampal WT and BsnGT neurons at baseline activity , upon network activity silencing ( TTX , 1 µM , 72 h ) or in conditions of increased activity ( BIC , 30 µM , 48 h ) H . Quantification of experiment in G . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size n ( in brackets ) corresponds to the number of independent images , G ) quantified from four ( H ) independent cultures per condition . For statistics two - way ANOVA with Tukey ´ s multiple comparisons test in H were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 5 µm in G .

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 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 O O

G. Representative images of 21 DIV hippocampal WT and BsnGT neurons at baseline activity, upon network activity silencing (TTX, 1 µM, 72 h) or in conditions of increased activity (BIC, 30 µM, 48 h) H. Quantification of experiment in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images , G) quantified from four (H) independent cultures per condition. For statistics two-way ANOVA with Tukey´s multiple comparisons test in H were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in G.


A , B . Representative pseudo colour images ( A ) and average traces ( B ) of sypHy fluorescence plotted from WT and BsnGT hippocampal neurons treated with calcineurin inhibitor FK506 . Data information : Scale bar is 2 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 B-CONTROLLED_VAR O O O O O O O O O O

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted from WT and BsnGT hippocampal neurons treated with calcineurin inhibitor FK506. Data information: Scale bar is 2 µm.


C , D . Quantification of the RRP and TRP fractions in WT and BsnGT neurons from experiment in A , B . Data information : In all plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The sample size is given in brackets for each quantification and reflect the number of analysed imaging experiments done on 3 independently prepared cultures per genotype Statistical significance was assessed using two - way ANOVA with Tukey ´ s multiple comparison test for C and D significance is depicted as follows : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D. Quantification of the RRP and TRP fractions in WT and BsnGT neurons from experiment in A, B. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of analysed imaging experiments done on 3 independently prepared cultures per genotype Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test for C and D significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.


E . Representative Western blot on P2 fractions prepared from hippocampal tissue of WT and BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 and total SNAP25 . F . Quantification of blot in E shows lower pThr138SNAP25 to total SNAP25 IF ratio in BsnGT compared to WT . Data information : In all plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The sample size is given in brackets for each quantification and reflect the number of animals per genotype used for WB in F Statistical significance was assessed using Student ' s t - test in F , significance is depicted as follows : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative Western blot on P2 fractions prepared from hippocampal tissue of WT and BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 and total SNAP25. F. Quantification of blot in E shows lower pThr138SNAP25 to total SNAP25 IF ratio in BsnGT compared to WT. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of animals per genotype used for WB in F Statistical significance was assessed using Student's t-test in F, significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.


A , B . Representative pseudo colour images ( A ) and average traces ( B ) of sypHy fluorescence plotted for WT and BsnGT neurons without treatment ( black and red ) and upon treatment with adenylyl cyclase activator forskolin ( gray and pink ) or PKA inhibitor H89 ( light grey and orange ) . Data information : The scale bar is 2 µm in

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A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons without treatment (black and red) and upon treatment with adenylyl cyclase activator forskolin (gray and pink) or PKA inhibitor H89 (light grey and orange). Data information: The scale bar is 2 µm in A


C , D . Quantification of the RRP and TRP fraction from experiment in A and B . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in ( C and D ) , The statistical significance was assessed in C , D using two - way ANOVA with Tukey ´ s multiple comparison test * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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C, D. Quantification of the RRP and TRP fraction from experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in (C and D), The statistical significance was assessed in C, D using two-way ANOVA with Tukey´s multiple comparison test *p≤0.05, **p < 0.01, ***p< 0.001.


E . Representative immunoblot with antibody against pSer9Syn1 and total Syn1 and total protein stain of hippocampal P2 fraction from WT and BsnGT mice prepared in the presence of KN 93 to isolate PKA - dependent regulation of pSer9Syn1 . F , G . Quantification of pSer9Syn1 / Syn1 ratio ( in F ) and Syn1 ( in G ) on blots from E . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments samples prepared from individual animals in F and G , The statistical significance was assessed in F and G using Student ' s t - test as is depicted in graphs as * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative immunoblot with antibody against pSer9Syn1 and total Syn1 and total protein stain of hippocampal P2 fraction from WT and BsnGT mice prepared in the presence of KN 93 to isolate PKA-dependent regulation of pSer9Syn1. F, G. Quantification of pSer9Syn1/Syn1 ratio (in F) and Syn1 (in G) on blots from E. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments samples prepared from individual animals in F and G, The statistical significance was assessed in F and G using Student's t-test as is depicted in graphs as *p≤0.05, **p < 0.01, ***p< 0.001.


H . Representative images of hippocampal neurons from WT and BsnGT mice labelled with antibodies against pSer9Syn1 and total Syn1 . Neurons were pre - treated ( 4 µM , 1hr ) with KN93 to isolate PKA - dependent phosphorylation and then treated in addition with forskolin or H89 before fixation . I . Quantification of staining in H . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size is given in brackets and corresponds to the independent visual fields obtained from 2 independent culture preparations in I . The statistical significance was assessed in I using two - way ANOVA with Tukey ´ s multiple comparison test as * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . The scale bar is 5 µm in 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Representative images of hippocampal neurons from WT and BsnGT mice labelled with antibodies against pSer9Syn1 and total Syn1. Neurons were pre-treated (4 µM, 1hr) with KN93 to isolate PKA-dependent phosphorylation and then treated in addition with forskolin or H89 before fixation. I. Quantification of staining in H. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the independent visual fields obtained from 2 independent culture preparations in I. The statistical significance was assessed in I using two-way ANOVA with Tukey´s multiple comparison test as *p≤0.05, **p < 0.01, ***p< 0.001. The scale bar is 5 µm in H.


A , B . Representative pseudo colour images ( A ) and average traces ( B ) of sypHy fluorescence plotted for untreated WT and BsnGT neurons ( black and red ) and cultures treated with of 6 - Bnz - cAMPS ( 50 µM , 1 hour ; gray and pink ) and rolipram ( 1 µM , 30 min , light grey and orange ) . Data information : Scale bar is 2µm .

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

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for untreated WT and BsnGT neurons (black and red) and cultures treated with of 6-Bnz-cAMPS (50 µM, 1 hour; gray and pink) and rolipram (1 µM, 30 min, light grey and orange). Data information: Scale bar is 2µm.


C , D . Quantifications of RRP and TRP fractions in experiment in A and B . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The sample size is given in brackets and corresponds to the number of imaging experiments performed on three independently prepared cultures Statistical significance was assessed using two - way ANOVA with Tukey ´ s multiple comparison test in C , D : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D. Quantifications of RRP and TRP fractions in experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the number of imaging experiments performed on three independently prepared cultures Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test in C, D : *p≤0.05, **p < 0.01, ***p< 0.001.


H . Quantification of pSer145PDE4B to the total PDE4B ratio from experiment in I . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The sample size is given in brackets and corresponds to the individual animals used for sample preparation Statistical significance was assessed using Student ' s t - test in H and is given as : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of pSer145PDE4B to the total PDE4B ratio from experiment in I. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the individual animals used for sample preparation Statistical significance was assessed using Student's t-test in H and is given as: *p≤0.05, **p < 0.01, ***p< 0.001.


I . Representative immunoblot with antibody against CDK5 - dependent pSer145PDE4B and total PDE4B on hippocampal tissue from WT and BsnGT animals .

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I. Representative immunoblot with antibody against CDK5-dependent pSer145PDE4B and total PDE4B on hippocampal tissue from WT and BsnGT animals.


Relative quantification of mRNA for Ppcs , Ppcdc , and Coasy by genotype from each of the three study regions . n = 11 , 4 , 4 for WT and n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively )

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Relative quantification of mRNA for Ppcs, Ppcdc, and Coasy by genotype from each of the three study regions. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively)


Western blot and quantification for Coasy from GP by genotype and treatment status ( pPanSH = 4 ' - phosphopantetheine ) . Ponceau S was used as a loading control . n = 8 , 4 for WT and n = 8 , 6 for KO ( vehicle and pPanSH groups , respectively ) .

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

Western blot and quantification for Coasy from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as a loading control. n=8, 4 for WT and n=8, 6 for KO (vehicle and pPanSH groups, respectively).


Relative mRNA expression of Tfrc , Ireb2 , and Hamp by genotype and brain region . n = 11 , 4 , 4 for WT and n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively )

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Relative mRNA expression of Tfrc, Ireb2, and Hamp by genotype and brain region. n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively)


Total iron quantity in cytosol and mitochondria from GP using ICP - MS . The data distribution is presented using a box plot , including minimum , first quartile , median , third quartile , and maximum . The magenta - colored line represent mean . n = 6 for both

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Total iron quantity in cytosol and mitochondria from GP using ICP-MS. The data distribution is presented using a box plot, including minimum, first quartile, median, third quartile, and maximum. The magenta-colored line represent mean. n=6 for both genotypes


Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype . Quantification of aconitase activity in two tissues ( brain and liver ) from animals of each genotype by band density . n = 4 for WT , n = 5 for KO ( D - E ) Activity of pyruvate

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Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenase


Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype . Quantification of aconitase activity in two tissues ( brain and liver ) from animals of each genotype by band density . n = 4 for WT , n = 5 for KO ( D - E ) Activity of pyruvate dehydrogenase ( D ) and complex I ( E ) in GP by genotype and treatment status ( veh = vehicle ; pPanSH = 4 ' - phosphopantetheine ) . n = 5 for both genotypes and treatment groups .

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

Assay of mitochondrial versus cytosolic aconitase activity in brain compared by genotype. Quantification of aconitase activity in two tissues (brain and liver) from animals of each genotype by band density. n=4 for WT, n=5 for KO (D-E) Activity of pyruvate dehydrogenase (D) and complex I (E) in GP by genotype and treatment status (veh = vehicle; pPanSH = 4'-phosphopantetheine). n=5 for both genotypes and treatment groups.


Western blot and quantification of TfR1 and PDHE1α1 from GP by genotype and treatment status ( pPanSH = 4 ' - phosphopantetheine ) . Ponceau S was used as total protein loading control and VDAC for the mitochondrial protein loading control .

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Western blot and quantification of TfR1 and PDHE1α1 from GP by genotype and treatment status (pPanSH = 4'-phosphopantetheine). Ponceau S was used as total protein loading control and VDAC for the mitochondrial protein loading control.


Relative mRNA and protein expression of the dopamine receptor Drd1 compared by genotype , brain region and treatment status ( pPanSH = 4 ' - phosphopantetheine ) . n = 11 , 4 , 4 for WT and n = 8 , 4 , 4 for KO ( GP , SN , Cerebellum , respectively ) QRT - PCR . n = 5 for Western blot for all

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

Relative mRNA and protein expression of the dopamine receptor Drd1 compared by genotype, brain region and treatment status (pPanSH = 4'-phosphopantetheine). n=11,4,4 for WT and n=8,4,4 for KO (GP, SN, Cerebellum, respectively) QRT-PCR. n=5 for Western blot for all groups


Relative mRNA expression of Drd2 and GABA receptor Gabra3 expression by genotype and brain region . n = same as 3A .

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Relative mRNA expression of Drd2 and GABA receptor Gabra3 expression by genotype and brain region. n= same as 3A.


Western blot quantification from whole brain of tyrosine hydroxylase compared by genotype . n = 5 for WT and n = 3 for KO .

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Western blot quantification from whole brain of tyrosine hydroxylase compared by genotype. n=5 for WT and n=3 for KO.


Relative quantification of COASY mRNA in human PKAN primary cell lines . n = 5 for both genotypes and cell

Relative quantification of COASY mRNA in human PKAN primary cell lines. n= 5 for both genotypes and cell types


Relative quantification of COASY / Coasy in fresh , blood - derived lymphocytes from human and mouse compared by genotype . n = 51 , 35 for human ( control , PKAN , respectively ) and n = 5 for both mouse genotypes .

Relative quantification of COASY/Coasy in fresh, blood-derived lymphocytes from human and mouse compared by genotype. n=51,35 for human (control, PKAN, respectively) and n=5 for both mouse genotypes.


Extracellular flux analysis in human fibroblasts showing differences in OCR ( oxygen consumption rate ) and ECAR ( extracellular acidification rate ) by genotype . Oligom ; Oligomycin , FCCP ; Carbonyl cyanide - ptrifluoromethoxyphenylhydrazone , 2 - DG ; 2 - deoxy - glucose . Quantification of baseline OCR and ECAR is shown , as well . n = 3 per genotype with four 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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-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

Extracellular flux analysis in human fibroblasts showing differences in OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) by genotype. Oligom; Oligomycin, FCCP; Carbonyl cyanide-ptrifluoromethoxyphenylhydrazone, 2-DG; 2-deoxy-glucose. Quantification of baseline OCR and ECAR is shown, as well. n=3 per genotype with four technical replicates.


Complex I activity assay using fibroblast lysates . Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n = 4 for both genotypes and treatment groups .

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Complex I activity assay using fibroblast lysates. Cells were either treated with vehicle or 50μM pPanSH for three days and total protein extract was used for the Complex I Dipstick assay n=4 for both genotypes and treatment groups.


Relative mRNA expression of mitochondrially - encoded genes important for oxidative phosphorylation from complex I ( MT - ND1 ) , complex III ( MT - CYB ) , cytochrome C ( MT - CO1 ) , and ATP synthase ( MT - ATP6 ) in human mutant fibroblasts versus control cells . n = 3 for both genotypes .

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Relative mRNA expression of mitochondrially-encoded genes important for oxidative phosphorylation from complex I (MT-ND1), complex III (MT-CYB), cytochrome C (MT-CO1), and ATP synthase (MT-ATP6) in human mutant fibroblasts versus control cells. n=3 for both genotypes.


Calculation of active mitochondria using the JC - 1 assay by genotype in control and PKAN lymphoblasts .

Calculation of active mitochondria using the JC-1 assay by genotype in control and PKAN lymphoblasts.


Relative expression of Pank1α , Pank1β , total Pank1 and Pank3 in mouse brain regions . n = 8 , 8 , 4 for both genotypes ( GP , SN , Cerebellum , respectively ) .

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Relative expression of Pank1α, Pank1β, total Pank1 and Pank3 in mouse brain regions. n=8,8,4 for both genotypes (GP, SN, Cerebellum, respectively).


Relative expression of Pank1α and Coasy in GP from WT and KO mice treated for 14 days with bezafibrate ( 0 . 8 mg / g body weight ) or gemfibrozil ( 1 . 2 mg / g body weight ) . n = 8 for both genotypes treated with vehicle , n = 5 for both KO groups treated with either bezafibrate or gemfibrozil .

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 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O

Relative expression of Pank1α and Coasy in GP from WT and KO mice treated for 14 days with bezafibrate (0.8 mg/g body weight) or gemfibrozil (1.2 mg/g body weight). n=8 for both genotypes treated with vehicle, n=5 for both KO groups treated with either bezafibrate or gemfibrozil.


Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds : 4 ' - phosphopantetheine ( pPanSH ) , pantetheine , vitamin B5 , and CoA . n = 8 ( WT - veh ) , 5 ( KO - veh ) , 5 ( KO - pPanSH ) , 4 ( KO - all other intermediates ) . Correction of Coasy , Tfrc , Ireb2 , and Drd1 expression in GP by 4 ' - phosphopantetheine over a range of doses . n = 8 ( WT - veh ) , 5 ( KO - veh ) , 5 ( KO - 0 . 82 ) , 4 ( WT - 5 ) , 5 ( KO - 5 ) , 5 ( KO - 8 . 2 ) , 5 ( KO - 10 ) . 5 ( WT - 20 ) , 5 ( KO - 20 )

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

Relative quantification of Coasy mRNA from GP compared by treatment status using four compounds: 4'-phosphopantetheine (pPanSH), pantetheine, vitamin B5, and CoA. n=8 (WT-veh), 5 (KO-veh), 5 (KO-pPanSH), 4 (KO-all other intermediates). Correction of Coasy, Tfrc, Ireb2, and Drd1 expression in GP by 4'-phosphopantetheine over a range of doses. n=8 (WT-veh), 5 (KO-veh), 5 (KO-0.82), 4 (WT-5), 5 (KO-5), 5 (KO-8.2), 5 (KO-10). 5 (WT-20), 5 (KO-20)


Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH . n = 3 for both genotypes .

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Relative quantification of COASY mRNA from human fibroblasts treated with pPanSH. n=3 for both genotypes.


ECAR measured in cultured human fibroblasts by genotype and treatment status . Cells were treated for three days with 4 ' - phosphopantetheine ( pPanSH ) or vehicle ( veh ) . n = 3 for both genotypes .

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

ECAR measured in cultured human fibroblasts by genotype and treatment status. Cells were treated for three days with 4'-phosphopantetheine (pPanSH) or vehicle (veh). n=3 for both genotypes.


Relative quantification of Coasy , Tfrc and Drd1 mRNA from GP by number of days post - cessation of treatment with 5μg of 4 ' - phosphopantetheine per g of body weight . n = 11 ( WT - veh - D0 ) , 5 ( KO - veh - D0 ) , 4 ( WT - pPanSH - D0 ) , 5 ( KO - pPanSH - D0 ) , 5 ( KO - pPanSH - D1 ) , 5 ( KO - pPanSH - D2 ) , 5 ( KO - pPanSH - D3 ) , 5 ( WT - pPanSH - D7 ) , 4 ( KO - pPanSH - D7 ) ,

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

Relative quantification of Coasy, Tfrc and Drd1 mRNA from GP by number of days post-cessation of treatment with 5μg of 4'-phosphopantetheine per g of body weight. n= 11 (WT-veh-D0), 5 (KO-veh-D0), 4 (WT-pPanSH-D0), 5 (KO-pPanSH-D0), 5 (KO-pPanSH-D1), 5 (KO-pPanSH-D2), 5 (KO-pPanSH-D3), 5 (WT-pPanSH-D7), 4 (KO-pPanSH-D7),


Biological replicates of transcriptional profiles ( Tx ) measured in separately prepared cell free lysate batches of E . coli .

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Biological replicates of transcriptional profiles (Tx) measured in separately prepared cell free lysate batches of E. coli.


Correlation between transcriptional profiles ( Tx ) for regulatory sequence libraries from in vitro and in vivo measurements in E . coli . The color scale indicates the density of data points in a given area of the plot .

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Correlation between transcriptional profiles (Tx) for regulatory sequence libraries from in vitro and in vivo measurements in E. coli. The color scale indicates the density of data points in a given area of the plot.


Correlation between primary TSS calls ( in bp from ATG start codon ) of regulatory sequences from in vitro and in vivo measurements in E . coli .

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Correlation between primary TSS calls (in bp from ATG start codon) of regulatory sequences from in vitro and in vivo measurements in E. coli.


Optimization of transcriptional output using different concentrations of Mg - glutamate and K - glutamate in 10 bacterial cell - free expression systems with Broccoli as a reporter ( solid lines , optimal buffer composition shown in red ) . No DNA template controls are shown as grey dashed lines . 12 . 5 nM of DNA template was used in all systems , except L . lactis ( 50 nM used ) .

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

Optimization of transcriptional output using different concentrations of Mg-glutamate and K-glutamate in 10 bacterial cell-free expression systems with Broccoli as a reporter (solid lines, optimal buffer composition shown in red). No DNA template controls are shown as grey dashed lines. 12.5 nM of DNA template was used in all systems, except L. lactis (50 nM used).


Transcriptional activities ( Tx ) of 421 regulatory sequences that were active in all 10 bacterial cell - free expression systems .

Transcriptional activities (Tx) of 421 regulatory sequences that were active in all 10 bacterial cell-free expression systems.


Pairwise comparison of transcriptional profiles of 421 universally - active regulatory sequences between bacterial species . Example scatter plots with relatively high and low Pearson correlation in the heatmap ( marked i and ii ) are shown on left .

Pairwise comparison of transcriptional profiles of 421 universally-active regulatory sequences between bacterial species. Example scatter plots with relatively high and low Pearson correlation in the heatmap (marked i and ii) are shown on left.


Correlation between evolutionary divergences ( 16S rRNA percent identity ) and pairwise Pearson correlation of transcriptional profiles . Dashed line represents linear regression .

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

Correlation between evolutionary divergences (16S rRNA percent identity) and pairwise Pearson correlation of transcriptional profiles. Dashed line represents linear regression.


Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for ( d ) E . coli + B .

Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (d) E. coli + B. subtilis


Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for ( e ) E . coli + C . glutamicum hybrids with different mixing ratios .

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

Pairwise correlation of RS7003 transcriptional profiles between hybrid lysate and single constituent species lysates for (e) E. coli + C. glutamicum hybrids with different mixing ratios.


A : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . n = 6 for young and old mice , respectively . B : WT1 immunohistochemistry ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . n = 6 for young and old mice , respectively . C : TUNEL assay ( upper panels ) and quantification of TUNEL - positive tubular cells ( upper panel ) in glomeruli from young and aged mice . Panels are representative images of 6 young and old mice , respectively . Scale bar = 20 μm . D : Senescence - associated β - galactosidase staining ( upper panels ) and quantification of β - galactosidase - positive glomeruli ( lower panel ) in kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . Panels are representative images of 4 young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively. B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 6 for young and old mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive tubular cells (upper panel) in glomeruli from young and aged mice. Panels are representative images of 6 young and old mice, respectively. Scale bar = 20 μm. D: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. Panels are representative images of 4 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


E : p21 / griffonia simplicifolia / nephrin coimmunostaining in kidneys from young and aged mice ( left panels ) and quantification ( right panel ) of p21 - positive endothelial cells per glomeruli . Original magnification X630 . Scale bar = 10 μm . Panels are representative images of 6 young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys from young and aged mice (left panels) and quantification (right panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


F : 53BP1 / CD34 co - immunostaining in kidneys from young and aged mice ( left panels ) and quantification ( right panels ) of 53BP1 foci per glomerular endothelial cell . Original magnification X630 . Scale bar = 10 μm . Panels are representative images of 6 young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: 53BP1/CD34 co-immunostaining in kidneys from young and aged mice (left panels) and quantification (right panels) of 53BP1 foci per glomerular endothelial cell. Original magnification X630. Scale bar = 10 μm. Panels are representative images of 6 young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


I : PAI - 1 immunohistochemistry ( left panels ) and quantification of PAI - 1 - positive glomeruli ( right panel ) in kidneys from young and aged mice . Original magnification X400 . Scale bar = 20 μm . n = 5 for young and old mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : young versus old mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from young and aged mice. Original magnification X400. Scale bar = 20 μm. n = 5 for young and old mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: young versus old mice.


A : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 4 and n = 5 for RT + and RT - , respectively . B : WT1 immunohistochemistry ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 5 for RT + and RT - mice , respectively . C : Senescence - associated β - galactosidase staining ( upper panels ) and quantification of β - galactosidase - positive glomeruli ( lower panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . Scale bar = 20 μm . n = 5 for RT + and RT - mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 5 for RT+ and RT-, respectively. B: WT1 immunohistochemistry (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively. C: Senescence-associated β-galactosidase staining (upper panels) and quantification of β-galactosidase-positive glomeruli (lower panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. Scale bar = 20 μm. n = 5 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


E : p21 / griffonia simplicifolia / nephrin coimmunostaining ( left panels ) and quantification of endothelial p21 - positive cells ( right panel ) in glomeruli of kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X630 . Scale bar = 10 μm . n = 4 for RT + and RT - mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: p21/griffonia simplicifolia/nephrin coimmunostaining (left panels) and quantification of endothelial p21-positive cells (right panel) in glomeruli of kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


F : 53BP1 / CD34 coimmunostaining ( left panels ) and quantification of glomerular endothelial cell 53BP1 foci ( right panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X630 . Scale bar = 10 μm . n = 4 for RT + and RT - mice , respectively . Arrows show 53BP1 foci . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice .

F: 53BP1/CD34 coimmunostaining (left panels) and quantification of glomerular endothelial cell 53BP1 foci (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X630. Scale bar = 10 μm. n = 4 for RT+ and RT- mice, respectively. Arrows show 53BP1 foci. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


H : PAI - 1 immunohistochemistry ( left panels ) and quantification of PAI - 1 - positive glomeruli ( right panel ) in kidneys from irradiated ( RT + ) and non - irradiated ( RT - ) control mice 12 months after irradiation . Original magnification X400 . n = 4 for RT + and RT - mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : irradiated versus non - irradiated mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: PAI-1 immunohistochemistry (left panels) and quantification of PAI-1-positive glomeruli (right panel) in kidneys from irradiated (RT+) and non-irradiated (RT-) control mice 12 months after irradiation. Original magnification X400. n = 4 for RT+ and RT- mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: irradiated versus non-irradiated mice.


A : WT1 ( left upper panels ) and p21 ( left middle and lower panels ) immunostaining in 4 - , 12 - , 18 - and 24 - month - old mice . Quantification of WT1 - positive cells ( right upper panels ) , and glomerular and tubular p21 - postive cells ( right lower panels ) . Original magnification X400 . Scale bar = 20 μm . n = 4 for each group . Arrows show positive p21 nuclei , either in glomeruli or in tubules . B : WT1 ( left upper panels ) and p21 ( left middle and lower panels ) immunostaining in 4 - and 12 - month - old irradiated ( RT + ) and non - irradiated ( RT - ) mice . Quantification of WT1 - positive cells ( right upper panels ) , and glomerular and tubular p21 - postive cells ( right lower panels ) . Original magnification X400 . Scale bar = 20 μm . n = 4 for each group . Arrows show positive p21 nuclei , either in glomeruli or in tubules . Data information : Data are means ± SEM . Statistical analysis : ANOVA followed by the Tukey - Kramer 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4-, 12-, 18- and 24-month-old mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. B: WT1 (left upper panels) and p21 (left middle and lower panels) immunostaining in 4- and 12-month-old irradiated (RT+) and non-irradiated (RT-) mice. Quantification of WT1-positive cells (right upper panels), and glomerular and tubular p21-postive cells (right lower panels). Original magnification X400. Scale bar = 20 μm. n = 4 for each group. Arrows show positive p21 nuclei, either in glomeruli or in tubules. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test.


A : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . B : WT1 immunostaining ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . C : PAI - 1 immunostaining ( upper panels ) and quantification of PAI - 1 - positive glomeruli ( lower panel ) in 28 - month - old p16 INK - ATTAC mice treated with either vehicle ( AP - ) or AP20187 ( AP + ) . Original magnification X400 . Scale bar = 20 μm . n = 5 . Data information : Data are means ± SEM . Statistical analysis : t - student test : AP + versus AP - mice .

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

A: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. B: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. C: PAI-1 immunostaining (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in 28-month-old p16 INK-ATTAC mice treated with either vehicle (AP-) or AP20187 (AP+). Original magnification X400. Scale bar = 20 μm. n = 5. Data information: Data are means ± SEM. Statistical analysis: t-student test: AP+ versus AP- mice.


A : PAI - 1 immunohistochemistry ( upper panels ) and quantification of PAI - 1 - positive glomeruli ( lower panel ) in kidneys of PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 4 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . B : Morphology ( PAS staining , upper panels ) and quantification of glomerular lesions ( lower panel ) of kidneys from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 12 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . C : TUNEL assay ( upper panels ) and quantification of TUNEL - positive glomeruli ( lower panel ) in kidneys from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . D : WT1 immunostaining ( upper panels ) and quantification of WT1 - positive glomerular cells ( lower panel ) in kidneys from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X400 . Scale bar = 20 μm . n = 4 and n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . Data information : Data are means ± SEM . Statistical analysis : t - student test : PAI - 1 ∆ endo versus PAI - 1flox mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: PAI-1 immunohistochemistry (upper panels) and quantification of PAI-1-positive glomeruli (lower panel) in kidneys of PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 for PAI-1flox and PAI-1∆endo mice, respectively. B: Morphology (PAS staining, upper panels) and quantification of glomerular lesions (lower panel) of kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 12 for PAI-1flox and PAI-1∆endo mice, respectively. C: TUNEL assay (upper panels) and quantification of TUNEL-positive glomeruli (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. D: WT1 immunostaining (upper panels) and quantification of WT1-positive glomerular cells (lower panel) in kidneys from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X400. Scale bar = 20 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.


G : p21 / griffonia simplicifolia / nephrin coimmunostaining in kidneys ( upper panels ) from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age and quantification ( lower panel ) of p21 - positive endothelial cells per glomeruli . Original magnification X630 . Scale bar = 10 μm . n = 4 and n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . H : 53BP1 / CD34 coimmunostaining ( upper panels ) and quantification of glomerular 53BP1 foci in glomerular endothelial cells from PAI - 1flox and PAI - 1 ∆ endo mice at 22 months of age . Original magnification X630 . Scale bar = 10 μm . n = 4 and n = 6 for PAI - 1flox and PAI - 1 ∆ endo mice , respectively . Arrows show 53BP1 positive foci . Data information : Data are means ± SEM . Statistical analysis : t - student test : PAI - 1 ∆ endo versus PAI - 1flox mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: p21/griffonia simplicifolia/nephrin coimmunostaining in kidneys (upper panels) from PAI-1flox and PAI-1∆endo mice at 22 months of age and quantification (lower panel) of p21-positive endothelial cells per glomeruli. Original magnification X630. Scale bar = 10 μm. n = 4 and n = 6 for PAI-1flox and PAI-1∆endo mice, respectively. H: 53BP1/CD34 coimmunostaining (upper panels) and quantification of glomerular 53BP1 foci in glomerular endothelial cells from PAI-1flox and PAI-1∆endo mice at 22 months of age. Original magnification X630. Scale bar = 10 μm. n=4 and n=6 for PAI-1flox and PAI-1∆endo mice, respectively. Arrows show 53BP1 positive foci. Data information: Data are means ± SEM. Statistical analysis: t-student test: PAI-1∆endo versus PAI-1flox mice.


C : Relative mRNA expression of senescence markers ( p16 and p21 ) and SASP molecules ( PAI - 1 , IL6 , IL8 ) in GEnC at early ( p9 ) or late ( p17 ) passage . n = 5 independent experiments . Data information : Data are means ± SEM . Statistical analysis : t - student

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Relative mRNA expression of senescence markers (p16 and p21) and SASP molecules (PAI-1, IL6, IL8) in GEnC at early (p9) or late (p17) passage. n = 5 independent experiments. Data information: Data are means ± SEM. Statistical analysis: t-student test


E : Immunofluorescence of paxilin ( green ) and phalloidin ( red ) in podocytes stimulated with control medium , supernatant ( SN ) from glomerular endothelial cells from early ( p9 ) or late ( p17 ) passage and treated either with vehicle or Tiplaxtinin , a PAI - 1 inhibitor ( upper panel ) . Quantification of focal adhesions ( lower panel ) was performed by counting the number of paxilin - positive spots in 30 cells from three independent experiments . Original magnification X1000 . Scale bar = 20 μm . Data information : Data are means ± SEM . Statistical analysis : ANOVA followed by the Tukey - Kramer

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

E: Immunofluorescence of paxilin (green) and phalloidin (red) in podocytes stimulated with control medium, supernatant (SN) from glomerular endothelial cells from early (p9) or late (p17) passage and treated either with vehicle or Tiplaxtinin, a PAI-1 inhibitor (upper panel). Quantification of focal adhesions (lower panel) was performed by counting the number of paxilin-positive spots in 30 cells from three independent experiments. Original magnification X1000. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test


A : PAI - 1 staining ( left panel ) and quantification ( right panel ) in glomeruli of kidney biopsies from young ( < 40 years of age , n = 8 ) or old ( > 80 years of age , n = 10 ) kidney donors at time of transplantation ( M0 ) . Original magnification X400 . Scale bar = 20 μm . B : p16 staining ( left panel ) and quantification ( right panel ) in glomeruli of kidney biopsies from young ( < 40 years of age , n = 8 ) or old ( > 80 years of age , n = 10 ) kidney donors at time of transplantation ( M0 ) . The arrows point to the staining . Original magnification X400 . Scale bar = 20 μm . Data information : Data are means ± SEM . Statistical analysis : t - student

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: PAI-1 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). Original magnification X400. Scale bar = 20 μm. B: p16 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). The arrows point to the staining. Original magnification X400. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: t-student test


F : Quantification of the glomerulosclerosis score at time of ( M0 ) , or 12 months after ( M12 ) kidney transplantation from elderly donors in patients displaying a negative ( PAI - 1 - , n = 17 ) or positive ( PAI - 1 + , n = 13 ) glomerular PAI - 1 staining at time of transplantation ( M0 ) . Data information : Data are means ± SEM . Statistical analysis : ANOVA followed by the Tukey - Kramer

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F: Quantification of the glomerulosclerosis score at time of (M0), or 12 months after (M12) kidney transplantation from elderly donors in patients displaying a negative (PAI-1-, n = 17) or positive (PAI-1+, n = 13) glomerular PAI-1 staining at time of transplantation (M0). Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test


H : Urinary PAI - 1 measured by ELISA in a cohort of elderly patients with ( n = 48 ) or without ( n = 48 ) chronic kidney disease . Data information : Data are means ± SEM . Statistical analysis : t - student

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H: Urinary PAI-1 measured by ELISA in a cohort of elderly patients with (n = 48) or without (n = 48) chronic kidney disease. Data information: Data are means ± SEM. Statistical analysis: t-student test


( a ) Representative micro - CT pictures of hind legs of 20 - 30 week - old control ( A20WT ) and A20myel - KO littermates . Note the severe osteoporosis in A20myel - KO mice . ( b ) Trabecular parameters , calculated on micro - CT scans of hind legs of 20 - 30 week - old control ( A20WT ) and A20myel - KO littermates . Each dot represents an individual mouse ( A20WT , n = 10 ; A20myel - KO , n = 8 ) .

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(a) Representative micro-CT pictures of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Note the severe osteoporosis in A20myel-KO mice. (b) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20myel-KO littermates. Each dot represents an individual mouse (A20WT, n=10; A20myel-KO, n=8).


( a ) Levels of TNF and IL - 6 in serum of A20WT , A20OC - KO and A20myel - KO mice . Each dot represents an individual mouse ( A20WT , n = 11 ; A20OC - KO , n = 11 and A20myel - KO , n = 14 ) . Data are expressed as mean ± SEM . *

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(a) Levels of TNF and IL-6 in serum of A20WT, A20OC-KO and A20myel-KO mice. Each dot represents an individual mouse (A20WT, n=11; A20OC-KO, n=11 and A20myel-KO, n=14). Data are expressed as mean ± SEM. *


( c ) Trabecular parameters , calculated on micro - CT scans of hind legs of 20 - 30 week - old control ( A20WT ) and A20OC - KO littermates . Each dot represents an individual mouse ( A20WT , n = 6 ; A20OC - KO , n = 9 ) .

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(c) Trabecular parameters, calculated on micro-CT scans of hind legs of 20-30 week-old control (A20WT) and A20OC-KO littermates. Each dot represents an individual mouse (A20WT, n=6; A20OC-KO, n=9).


( e ) Representative pictures of TRAP - stained sections from tibia of 20 week - old A20WT and A20OC - KO mice . Red arrows indicate osteoclasts .

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(e) Representative pictures of TRAP-stained sections from tibia of 20 week-old A20WT and A20OC-KO mice. Red arrows indicate osteoclasts.


( f ) Quantification of the number of TRAP - positive cells on sections from tibia of 20 week - old A20WT and A20OC - KO mice . Each dot represents an individual mouse ( A20WT , n = 5 ; A20OC - KO , n = 6 ) .

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(f) Quantification of the number of TRAP-positive cells on sections from tibia of 20 week-old A20WT and A20OC-KO mice. Each dot represents an individual mouse (A20WT, n=5; A20OC-KO, n=6).


( d ) Measurement of NF - κB luciferase activity in lysates of HEK293T cells transiently transfected with an NF - κB reporter plasmid , an expression plasmid for β - galactosidase ( βgal ) , an expression plasmid for RANK , and with either an empty vector or a vector expressing wild - type A20 . Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc / βgal to normalize for possible differences in transfection efficiency .

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(d) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transiently transfected with an NF-κB reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and with either an empty vector or a vector expressing wild-type A20. Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency.


( e ) Western blot analysis of whole cell lysates from BMDMs cultures isolated from A20WT and A20myel - KO mice and stimulated with RANKL ( 100ng / ml ) for the indicated time periods . Actin is shown as a loading control .

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(e) Western blot analysis of whole cell lysates from BMDMs cultures isolated from A20WT and A20myel-KO mice and stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.


( f ) Wild - type and A20 deficient BMDMs were stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for RANK , A20 , TRAF6 , HOIP and Sharpin .

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(f) Wild-type and A20 deficient BMDMs were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for RANK, A20, TRAF6, HOIP and Sharpin.


( g ) BMDMs from A20WT and A20myel - KO mice were stimulated with RANKL ( 100ng / ml ) for the indicated time periods and immunoblotted to visualize p100 processing to p52 . Actin is shown as a loading control .

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(g) BMDMs from A20WT and A20myel-KO mice were stimulated with RANKL (100ng/ml) for the indicated time periods and immunoblotted to visualize p100 processing to p52. Actin is shown as a loading control.


( a ) Measurement of NF - κB luciferase activity in lysates of HEK293T cells transfected with an NF - κB luciferase reporter plasmid , an expression plasmid for β - galactosidase ( βgal ) , an expression plasmid for RANK , and plasmids encoding different A20 variants : A20WT , A20DUB ( C103A mutation ) , A20ZnF4 ( C624A / C627A ) , A20ZnF7 ( C775A / C779A ) , and A20ZnF4ZnF7 ( C624A / C627A / C775A / C779A ) . Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc / βgal to normalize for possible differences in transfection efficiency . Data are expressed as the mean of technical triplicates ± SD .

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(a) Measurement of NF-κB luciferase activity in lysates of HEK293T cells transfected with an NF-κB luciferase reporter plasmid, an expression plasmid for β-galactosidase (βgal), an expression plasmid for RANK, and plasmids encoding different A20 variants: A20WT, A20DUB (C103A mutation), A20ZnF4 (C624A/C627A), A20ZnF7 (C775A/C779A), and A20ZnF4ZnF7 (C624A/C627A/C775A/C779A). Cell lysates were analysed for luciferase and βgal activity and values are plotted as Luc/βgal to normalize for possible differences in transfection efficiency. Data are expressed as the mean of technical triplicates ± SD.


( c - d ) Western blot analysis of whole cell lysates from BMDMs differentiated from A20WT and A20ZnF4ZnF7 mice , stimulated with RANKL ( 100ng / ml ) for the indicated time periods . Actin is shown as a loading control .

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(c-d) Western blot analysis of whole cell lysates from BMDMs differentiated from A20WT and A20ZnF4ZnF7 mice, stimulated with RANKL (100ng/ml) for the indicated time periods. Actin is shown as a loading control.


( e ) BMDMs isolated from control ( A20WT ) , A20ZnF7 , A20ZnF4ZnF7 and A20DUB mice were stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for A20 . Actin was used as a loading control .

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(e) BMDMs isolated from control (A20WT), A20ZnF7, A20ZnF4ZnF7 and A20DUB mice were stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.


( f ) Wild - type BMDMs were pretreated for 1h with HOIPIN - 8 ( 50µM ) or left untreated and stimulated with GST - RANKL ( 1 μg / ml ) for the indicated time periods . The RANK signaling complex was immunoprecipitated using glutathione - sepharose beads and immunoblotted for A20 . Actin was used as a loading control .

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(f) Wild-type BMDMs were pretreated for 1h with HOIPIN-8 (50µM) or left untreated and stimulated with GST-RANKL (1 μg/ml) for the indicated time periods. The RANK signaling complex was immunoprecipitated using glutathione-sepharose beads and immunoblotted for A20. Actin was used as a loading control.


( B ) Proteasome inhibitor treatment ( 10 μM MG132 for 1 to 8 h ) promotes accumulation of a ~ 110KD fragment ( asterisk ) in HEK293A cells .

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(B) Proteasome inhibitor treatment (10 μM MG132 for 1 to 8 h) promotes accumulation of a ~110KD fragment (asterisk) in HEK293A cells.


( C ) The ~ 110KD fragment ( asterisk ) is related to AMOT . Wild - type ( WT ) and AMOT knockout ( KO ) HEK293A cells were treated with 10 μM proteasome inhibitor bortezomib ( BTZ ) for 6 h and subjected to Western blotting analysis using the indicated antibodies .

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(C) The ~110KD fragment (asterisk) is related to AMOT. Wild-type (WT) and AMOT knockout (KO) HEK293A cells were treated with 10 μM proteasome inhibitor bortezomib (BTZ) for 6 h and subjected to Western blotting analysis using the indicated antibodies.


( F and G ) Serum and LPA treatments induce AMOT degradation and promote accumulation of a ~ 110 kDa protein ( asterisk ) in HEK293A cells . Cells were serum - starved overnight and then treated with 10 % FBS or 1 μM LPA for the indicated time . The phosphorylation levels of YAP proteins were detected by Phos - tag gel and pYAP immunoblotting . Protein quantification is shown below . Data represent mean ± SEM from three independent experiments . L . E . indicates long exposure .

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(F and G) Serum and LPA treatments induce AMOT degradation and promote accumulation of a ~110 kDa protein (asterisk) in HEK293A cells. Cells were serum-starved overnight and then treated with 10% FBS or 1 μM LPA for the indicated time. The phosphorylation levels of YAP proteins were detected by Phos-tag gel and pYAP immunoblotting. Protein quantification is shown below. Data represent mean ± SEM from three independent experiments. L.E. indicates long exposure.


C ) Nelfinavir ( NFV ) and Lopinavir ( LPV ) block AMOT cleavage . HEK293A cells were pre - treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h . Cell lysates were then subjected to Western blotting .

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C) Nelfinavir (NFV) and Lopinavir (LPV) block AMOT cleavage. HEK293A cells were pre-treated with protease inhibitors overnight followed by 10 μM BTZ treatment for 6 h. Cell lysates were then subjected to Western blotting.


( F ) Deletion of DDI2 blocks AMOT cleavage . Wild - type ( WT ) and DDI2 KO cells were serum - starved overnight and then treated with 1 μM LPA for 2 h .

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(F) Deletion of DDI2 blocks AMOT cleavage. Wild-type (WT) and DDI2 KO cells were serum-starved overnight and then treated with 1 μM LPA for 2 h.


( G ) Both DDI1 and DDI2 mediate AMOT cleavage . Ectopically expressed FLAG - tagged DDI1 or DDI2 rescues AMOT cleavage in DDI2 KO HEK293A cells .

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(G) Both DDI1 and DDI2 mediate AMOT cleavage. Ectopically expressed FLAG-tagged DDI1 or DDI2 rescues AMOT cleavage in DDI2 KO HEK293A cells.


K ) The aspartic catalytic - site mutant DDI2 exhibits a dominant negative effect through inactivation of wild - type DDI2 in HEK293A cells . Wild - type HEK293A cells were transfected with the indicated plasmids and treated with 1 μM LPA ( K ) before harvest .

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K) The aspartic catalytic-site mutant DDI2 exhibits a dominant negative effect through inactivation of wild-type DDI2 in HEK293A cells. Wild-type HEK293A cells were transfected with the indicated plasmids and treated with 1 μM LPA (K) before harvest.


( A ) Ubiquitination is required for AMOT cleavage . HEK293A cells were treated with 100 nM E1 inhibitor MLN7243 overnight followed by 10 μM BTZ for 6 h . Cell lysates were then subjected to Western blotting .

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(A) Ubiquitination is required for AMOT cleavage. HEK293A cells were treated with 100 nM E1 inhibitor MLN7243 overnight followed by 10 μM BTZ for 6 h. Cell lysates were then subjected to Western blotting.


( B and C ) The TNKS1 / 2 - RNF146 axis is crucial for AMOT cleavage . Wild - type ( WT ) , TNKS1 / 2 double KO ( dKO ) and RNF146 KO HEK293A cells were treated with the proteasome inhibitor MG132 for 6 h before harvest . Cell lysates were subjected to Western blotting analysis using the indicated antibodies .

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(B and C) The TNKS1/2-RNF146 axis is crucial for AMOT cleavage. Wild-type (WT), TNKS1/2 double KO (dKO) and RNF146 KO HEK293A cells were treated with the proteasome inhibitor MG132 for 6 h before harvest. Cell lysates were subjected to Western blotting analysis using the indicated antibodies.


( H and I ) AMOT cleavage states in different cell lines with NF2 - wild - type or NF2 - deficiency .

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(H and I) AMOT cleavage states in different cell lines with NF2-wild-type or NF2-deficiency.


( K ) Serum , LPA , and S1P stimulate PARylation and ubiquitination of AMOT . Serum - starved cells were treated with 10 % FBS , 1 μM LPA , or 1 μM S1P for 4 h and subjected to an immunoprecipitation ( IP ) assay .

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(K) Serum, LPA, and S1P stimulate PARylation and ubiquitination of AMOT. Serum-starved cells were treated with 10% FBS, 1 μM LPA, or 1 μM S1P for 4 h and subjected to an immunoprecipitation (IP) assay.