Coomassie gel of recombinant ATG16L172 - 307 wild type and mutants . * indicates bacterial

Coomassie gel of recombinant ATG16L172-307 wild type and mutants. * indicates bacterial protein


Liposome co - sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes . Supernatant ( S ) and pellet ( P ) were analysed by immunoblotting against T7 tag to detect ATG16L1 . Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments ( right panel ) .

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

Liposome co-sedimentation assay using recombinant ATG16L1 protein and sonicated liposomes. Supernatant (S) and pellet (P) were analysed by immunoblotting against T7 tag to detect ATG16L1. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel).


Liposome co - sedimentation assay , as in ( C ) , using ATG16L172 - 307LE and sonicated liposomes that contain or lack PI3P . Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments ( right panel ) with error bars depicting SEM values .

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

Liposome co-sedimentation assay, as in (C), using ATG16L172-307LE and sonicated liposomes that contain or lack PI3P. Quantification of percentage protein in the pellet fraction was calculated from 3 independent experiments (right panel) with error bars depicting SEM values.


ATG16L1 - / - cells were reconstituted with ATG16L1WT - or ATG16L1LE - expression constructs and autophagy assessed during amino acid starvation ( AA starve ) followed by immunoblotting or immunofluorescence analyses . Immunofluorescence analyses of amino acid starved cells ( 2 hr ) using antibodies against WIPI2 ( red ) or ATG16L1 ( green ) . Scale bar : 9 μm . Cells as in ( A ) with the addition of 3 ' MA ( and pretreatment with the drug for 30 min ) . Scale bar : 9 μm .

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

ATG16L1-/- cells were reconstituted with ATG16L1WT- or ATG16L1LE-expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses. Immunofluorescence analyses of amino acid starved cells (2 hr) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm. Cells as in (A) with the addition of 3'MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm.


Quantification of percentage of cells positive for ATG16L1 puncta


Cells were left untreated , amino acid starved for 2 hr , or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr . Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1 . Scale bar : 10 μm .

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Cells were left untreated, amino acid starved for 2 hr, or amino acid starved for 2 hr followed by refeeding with full growth media for 1 hr. Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1. Scale bar: 10 μm.


Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr . Cell lysates were analysed using the indicated antibodies .

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

Immunoblot analyses of amino acid starved cells in the presence of BafA1 for 2 hr. Cell lysates were analysed using the indicated antibodies.


WIPI2 - / - cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr . BafA1 treatment was included in all conditions for 2 hr prior to lysis . Cell lysates were analysed by immunoblotting using the indicated antibodies . Quantification of LC3 - II / LC3 - I ratio of three independent experiments is shown on the right panel .

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

WIPI2-/- cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 hr. BafA1 treatment was included in all conditions for 2 hr prior to lysis. Cell lysates were analysed by immunoblotting using the indicated antibodies. Quantification of LC3-II/LC3-I ratio of three independent experiments is shown on the right panel.


Ferroptosis assay in ATG16L1 - / - cells stably expressing ATG16L1WT , ATG16L1LD or ATG16L1LE . Cells were cultured in amino acid free media ( AA starve ) in the presence of 10 % FBS or 10 % dialysed FBS ( diFBS ) for 24 hr . Quantification of percentage of PI - positive cells from at least three independent experiments is shown .

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

Ferroptosis assay in ATG16L1-/- cells stably expressing ATG16L1WT, ATG16L1LD or ATG16L1LE. Cells were cultured in amino acid free media (AA starve) in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 hr. Quantification of percentage of PI-positive cells from at least three independent experiments is shown.


B . Western blot analysis of purified autophagic vesicles . Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms , respectively , and are represented with total lysate ( TL ) . Depicted are representative blots of 14 independent approaches .

O O O O O O O O O O O O O O O O O O O O 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. Western blot analysis of purified autophagic vesicles. Isolations were performed using antibodies directed against LC3B or all GABARAP isoforms, respectively, and are represented with total lysate (TL). Depicted are representative blots of 14 independent approaches.


A . Differential interference contrast microscopy images of purified autophagic vesicles at high ( I ) or low ( II ) dilution . Images are representative of 3 independent approaches . B . Negative stain electron microscopy images of isolated vesicles . C . Size evaluation of isolated vesicles . The diameters of approx . 60 individual vesicles were determined using EM images .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Differential interference contrast microscopy images of purified autophagic vesicles at high (I) or low (II) dilution. Images are representative of 3 independent approaches. B. Negative stain electron microscopy images of isolated vesicles. C. Size evaluation of isolated vesicles. The diameters of approx. 60 individual vesicles were determined using EM images.


D . Western blot analysis of isolated autophagic vesicles upon proteinase K digestion . Mechanically opened vesicles served as positive control . For negative control , isolates were incubated with BSA instead of proteinase K . Depicted are two different blots that are representative for 5 independent

O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot analysis of isolated autophagic vesicles upon proteinase K digestion. Mechanically opened vesicles served as positive control. For negative control, isolates were incubated with BSA instead of proteinase K. Depicted are two different blots that are representative for 5 independent experiments


E . Phospholipids identified in autophagic vesicles isolated upon different conditions . Relative amounts were calculated based on total levels of detected phospholipids . Abbreviations are depicted in A .

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

E. Phospholipids identified in autophagic vesicles isolated upon different conditions. Relative amounts were calculated based on total levels of detected phospholipids. Abbreviations are depicted in A.


A . Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy . Log2 - transformed fold changes . For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles , no fold changes could be calculated and they are indicated as not determinable ( nd ) . B . Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy . Log2 - transformed fold changes . For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles , no fold changes could be calculated and they are indicated as not determinable ( nd ) .

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

A. Volcano plot showing the differential appearance of proteins in autophagic vesicles of EBSS treated cells in comparison to vesicles isolated under basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon EBSS treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd). B. Volcano plot showing the differential appearance of proteins in autophagic vesicles of MG132 treated cells in comparison to vesicles isolated upon basal autophagy. Log2-transformed fold changes. For proteins that were excluded from autophagic vesicles upon MG132 treatment or which exclusively appeared within these vesicles, no fold changes could be calculated and they are indicated as not determinable (nd).


Growth suppression in cancer cells after 72h of treatment with APR - 246 + / - MRP1 inhibitor MK - 571 as shown by the WST - 1 assay ( n ≥ 3 , n shown in Table S1 ) . Cross in heatmap indicates no data available .

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Growth suppression in cancer cells after 72h of treatment with APR-246 +/- MRP1 inhibitor MK-571 as shown by the WST-1 assay (n ≥ 3, n shown in Table S1). Cross in heatmap indicates no data available.


Growth suppression in human dermal fibroblasts ( HDF ) by WST - 1 assay after 72h of with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Growth suppression in human dermal fibroblasts (HDF) by WST-1 assay after 72h of with APR-246 +/- MK-571 (n = 3).


Mean IC50 values ( µM ) for APR - 246 + / - 20 μM MK - 571 of 21 cell lines . * p < 0 . 0001 , Wilcoxon test . Each dot represents one cell line . Mean IC50 values and n shown in Table S1 . ZIP synergy scores of most synergistic area sub - grouped according to TP53 gene status for 21 cell lines . Each dot represents one cell line . Scores and n are shown in Table S1 .

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

Mean IC50 values (µM) for APR-246 +/- 20 μM MK-571 of 21 cell lines. *p < 0.0001, Wilcoxon test. Each dot represents one cell line. Mean IC50 values and n shown in Table S1. ZIP synergy scores of most synergistic area sub-grouped according to TP53 gene status for 21 cell lines. Each dot represents one cell line. Scores and n are shown in Table S1.


Sub - G1 DNA content of OVCAR - 3 cells ( R248Q ) after APR - 246 + / - MK - 571 treatment as assessed by propidium iodide staining at 48h ( n = 3 ) .

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

Sub-G1 DNA content of OVCAR-3 cells (R248Q) after APR-246 +/- MK-571 treatment as assessed by propidium iodide staining at 48h (n = 3).


OVCAR - 3 cell confluency by IncuCyte ® during 72h of treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

OVCAR-3 cell confluency by IncuCyte® during 72h of treatment with APR-246 +/- MK-571 (n = 3).


Growth suppression in HCT116 WT ( n = 4 ) and R248W cells ( n = 3 ) after 72h treatment with APR - 246 + / - MRP1 inhibitor Reversan , as shown by WST - 1 .

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

Growth suppression in HCT116 WT (n = 4) and R248W cells (n = 3) after 72h treatment with APR-246 +/- MRP1 inhibitor Reversan, as shown by WST-1.


Western blot analysis of MRP1 ( Cell Signaling . ) , p53 ( DO - 1 ) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53 .

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

Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53.


Growth suppression ( WST - 1 assay ) of HCT116 WT and R248W cells transfected with MRP1 siRNA ( n ≥ 5 and n ≥ 2 respectively ) after 48h APR - 246 treatment . Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs . Data and n of individual siRNAs are shown in Fig S1J , data is also part of Fig 7B .

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

Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (n ≥ 5 and n ≥ 2 respectively) after 48h APR-246 treatment. Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs. Data and n of individual siRNAs are shown in Fig S1J, data is also part of Fig 7B.


Western blot analysis of MRP1 ( Cell Signaling . ) , p53 ( DO - 1 ) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector ( EV ) and MRP1 expression vector .

Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector (EV) and MRP1 expression vector.


Growth suppression ( WST - 1 assay ) of HCT116 WT and R248W cells transfected with empty vector ( EV ) and MRP1 vector after 72h APR - 246 treatment ( n = 4 ) . Data is also part of Fig S7E .

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

Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with empty vector (EV) and MRP1 vector after 72h APR-246 treatment (n = 4). Data is also part of Fig S7E.


Growth of Eso26 xenografts ( R248W mutant TP53 ) in mice treated with vehicle , APR - 246 ( 50mg / kg ) , MK - 571 ( 50mg / kg ) or APR - 246 and MK - 571 for 16 days . p values determined by one - way ANOVA with Tukey ' s correction at day 18 , n is indicated in the figure . Kaplan - Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle , APR - 246 ( 50mg / kg ) , MK - 571 ( 50mg / kg ) or APR - 246 and MK - 571 for 16 days . Mice that were culled at ethical endpoints prior to reaching a tumor volume of > 1400mm3 are marked as censored observations . p values were determined by Log - rank ( Mantell - Cox ) test between different treatment groups . n is indicated in the figure .

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

Growth of Eso26 xenografts (R248W mutant TP53) in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. p values determined by one-way ANOVA with Tukey's correction at day 18, n is indicated in the figure.   Kaplan-Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. Mice that were culled at ethical endpoints prior to reaching a tumor volume of >1400mm3 are marked as censored observations. p values were determined by Log-rank (Mantell-Cox) test between different treatment groups. n is indicated in the figure.


Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint ( 5 days ) or post treatment / late timepoint ( > 22 days ) after treatment initiation with APR - 246 ( 50mg / kg ) + / - MK - 571 ( 50mg / kg ) . Dots indicate individual tumors .

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Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint (5 days) or post treatment / late timepoint (>22 days) after treatment initiation with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). Dots indicate individual tumors.


Quantification of cleaved caspase 3 immunohistochemistry staining of Eso26 xenograft tumors in same tumors as shown in Fig . 2C . Dots indicate individual tumors .

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

Quantification of cleaved caspase 3 immunohistochemistry staining of Eso26 xenograft tumors in same tumors as shown in Fig. 2C. Dots indicate individual tumors.


Representative images of hematoxylin / eosin ( H & E ) staining and immunostaining of Ki67 , cleaved caspase 3 and p53 , post treatment / late timepoint ( > 22 days after treatment initiation ) with APR - 246 ( 50mg / kg ) + / - MK - 571 ( 50mg / kg ) .

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

Representative images of hematoxylin/eosin (H&E) staining and immunostaining of Ki67, cleaved caspase 3 and p53, post treatment / late timepoint (>22 days after treatment initiation) with APR-246 (50mg/kg) +/- MK-571 (50mg/kg).


Growth suppression determined by the ATP - based CTG assay in colorectal cancer patient - derived organoids ( colo - PDO ) after treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Growth suppression determined by the ATP-based CTG assay in colorectal cancer patient-derived organoids (colo-PDO) after treatment with APR-246 +/- MK-571 (n = 3).


IC50 values ( µM ) APR - 246 + / - 20 μM MK - 571 in indicated PDOs . * p = 0 . 04 , paired t test ( n = 3 except colo - PDO2 and eso - PDO3 where n = 2 ) .

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

IC50 values (µM) APR-246 +/- 20 μM MK-571 in indicated PDOs. *p = 0.04, paired t test (n = 3 except colo-PDO2 and eso-PDO3 where n = 2).


Organoid area ( in black , left y - axis ) and PI intensity ( in red , right y - axis ) of colo - PDO1 and colo - PDO2 as determined by image analysis 72h after treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Organoid area (in black, left y-axis) and PI intensity (in red, right y-axis) of colo-PDO1 and colo-PDO2 as determined by image analysis 72h after treatment with APR-246 +/- MK-571 (n = 3).


14C accumulation ( cpm / mg x ml - 1 ) in 11 cell lines after 24h 14C - APR - 246 + / - MK - 571 treatment ( n ≥ 3 ) . * p = 0 . 0006 , paired t - test . 14C - accumulation and n of individual cell lines as shown in Table S3 .

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

14C accumulation (cpm/mg x ml-1) in 11 cell lines after 24h 14C -APR-246 +/- MK-571 treatment (n ≥ 3). *p = 0.0006, paired t-test. 14C-accumulation and n of individual cell lines as shown in Table S3.


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 ( WT and R248W ) cells after 24h + treatment with 14C - APR - 246 + / - MK - 571 or Reversan ( n ≥ 3 , each dots in figure ) .

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

14C accumulation (cpm/mg x ml-1) in HCT116 (WT and R248W) cells after 24h +treatment with 14C -APR-246 +/- MK-571 or Reversan (n ≥ 3, each dots in figure).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT and R248W cells ( n ≥ 3 and ≥ 2 ) transfected with MRP1 siRNAs after 24h of 14C - APR - 246 treatment . Indicated values represent four individual MRP1 siRNAs and two individual negative control ( Ctrl ) siRNAs , where each dot is an individual siRNA . Mean of 14C - accumulation after treatment and n with individual siRNA sequences is shown in Table S4 . * p = 0 . 04 and for R248W p = 0 . 06 , Wilcoxon matched pairs signed rank test .

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

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells (n ≥ 3 and ≥ 2) transfected with MRP1 siRNAs after 24h of 14C-APR-246 treatment. Indicated values represent four individual MRP1 siRNAs and two individual negative control (Ctrl) siRNAs, where each dot is an individual siRNA. Mean of 14C-accumulation after treatment and n with individual siRNA sequences is shown in Table S4. *p = 0.04 and for R248W p = 0.06, Wilcoxon matched pairs signed rank test.


APR - 246 content in OVCAR - 3 cells ( TP53 R248Q ) 24h after treatment with APR - 246 + / - MK - 571 , ( n = 3 ) . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 .

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

APR-246 content in OVCAR-3 cells (TP53 R248Q) 24h after treatment with APR-246 +/- MK-571, (n = 3). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Glutathione - conjugated MQ ( GS - MQ ) in OVCAR - 3 cells after 24h treatment with APR - 246 + / - MK - 571 ( n = 2 ) . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 .

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

Glutathione-conjugated MQ (GS-MQ) in OVCAR-3 cells after 24h treatment with APR-246 +/- MK-571 (n = 2). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Amount of GS - MQ at indicated timepoints after incubation of GS - MQ + / - NAC ( n = 2 ) . See Fig . EV4 for chemical reaction . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 .

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

Amount of GS-MQ at indicated timepoints after incubation of GS-MQ +/- NAC (n = 2). See Fig. EV4 for chemical reaction. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Amount of GS - MQ by LC - MS ( green line , left axis ) and NAC - MQ ( purple line , right axis ) over time after incubation of GS - MQ with NAC ( n = 2 ) . Values on y - axes are not comparable because GS - MQ and NAC - MQ have different response signals on MS . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± SEM . See also Appendix Figure S4 .

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

Amount of GS-MQ by LC-MS (green line, left axis) and NAC-MQ (purple line, right axis) over time after incubation of GS-MQ with NAC (n = 2). Values on y-axes are not comparable because GS-MQ and NAC-MQ have different response signals on MS. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.


Mean IC50 ( µM ) values of APR - 246 in 17 different cell lines ( n ≥ 3 ) as shown by WST - 1 . * p = 0 . 03 , Mann Whitney test . Mean IC50 ( µM ) values for APR - 246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1 . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Mean IC50 (µM) values of APR-246 in 17 different cell lines (n ≥ 3) as shown by WST-1. *p = 0.03, Mann Whitney test. Mean IC50 (µM) values for APR-246 treatment and n for individual cell lines are also shown in Fig 1C and listed in Table S1. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Box - and - whisker plot of Pearson correlations between PRIMA - 1 area - under - the - curve ( AUC ) and the levels of 225 different metabolites from the DepMap portal . Central band indicates median , boxes indicate 25th and 75th percentile , and whiskers show 2 . 5th and 97 . 5th percentile outlier metabolites . High GSH and GSSG ( in red ) correlate with low PRIMA - 1 sensitivity . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Box-and-whisker plot of Pearson correlations between PRIMA-1 area-under-the-curve (AUC) and the levels of 225 different metabolites from the DepMap portal. Central band indicates median, boxes indicate 25th and 75th percentile, and whiskers show 2.5th and 97.5th percentile outlier metabolites. High GSH and GSSG (in red) correlate with low PRIMA-1 sensitivity. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG ) as shown by glutathione reductase ( GR ) re - cycling assay in H1299 R175H ( tet - off ) ( + / - doxycycline ) and H1299 - / - cells 24h after seeding ( n = 3 ) . Indicated values are fold change compared to mutant TP53 cells . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Total intracellular glutathione (GSH+GSSG) as shown by glutathione reductase (GR) re-cycling assay in H1299 R175H (tet-off) (+/- doxycycline) and H1299 -/- cells 24h after seeding (n = 3). Indicated values are fold change compared to mutant TP53 cells. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG ) as shown by GR re - cycling assay in untreated HCT116 ( R248W , WT and - / - ) cells 48h after seeding ( n = 3 , 5 or 2 respectively ) . Indicated values are fold change to mutant TP53 cells . Part of this data is shown in Fig . EV4 . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Total intracellular glutathione (GSH+GSSG) as shown by GR re-cycling assay in untreated HCT116 (R248W, WT and -/-) cells 48h after seeding (n = 3, 5 or 2 respectively). Indicated values are fold change to mutant TP53 cells. Part of this data is shown in Fig. EV4. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells ( n = 2 ) Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Thiol tracker staining by flow cytometry 48h after seeding of the indicated untreated cells (n = 2) Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Correlation between 14C accumulation ( cpm / mg x ml - 1 ) after treatment with 25 µM 14C - APR - 246 and IC50 ( µM ) values for APR - 246 in 11 cell lines ( n ≥ 3 ) ( r = - 0 . 66 , p = 0 . 03 , Pearson correlation ) . Each dot represents one cell line , 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3 . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Correlation between 14C accumulation (cpm/mg x ml-1) after treatment with 25 µM 14C-APR-246 and IC50 (µM) values for APR-246 in 11 cell lines (n ≥ 3) (r = -0.66, p = 0.03, Pearson correlation). Each dot represents one cell line, 14C accumulation data are shown in Fig 3B and values and n for individual cell lines are listed in Table S1 and S3. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


ABCC1 mRNA Expression , RSEM ( Batch normalized from Illumina HiSeq _ RNASeqV2 ) in lung cancer ( luad & lusc studies , * p < 0 . 0001 ) and colon adenocarcinoma ( coad study , * p = 0 . 005 ) grouped into having no alterations or putative driver missense mutations in TP53 . Statistical analysis by Mann Whitney test , n is indicated in the figure . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

ABCC1 mRNA Expression, RSEM (Batch normalized from Illumina HiSeq_RNASeqV2) in lung cancer (luad & lusc studies, *p<0.0001) and colon adenocarcinoma (coad study, *p=0.005) grouped into having no alterations or putative driver missense mutations in TP53. Statistical analysis by Mann Whitney test, n is indicated in the figure. Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


14C accumulation ( cpm / mg x ml - 1 ) in H1299 R175H cells ( + / - doxycycline ) and H1299 - / - cells after 24h treatment with 14C - APR - 246 ( n = 2 ) . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

14C accumulation (cpm/mg x ml-1) in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells after 24h treatment with 14C-APR-246 (n = 2). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Growth suppression in H1299 R175H cells ( + / - doxycycline ) and H1299 - / - cells treated for 72h with APR - 246 as shown by WST - 1 assay ( n = 3 ) . Data information : TP53 status is indicated for each cell line . Data are represented as mean ± SEM . See also Figure EV5 and Appendix Figure S5 .

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

Growth suppression in H1299 R175H cells (+/- doxycycline) and H1299 -/- cells treated for 72h with APR-246 as shown by WST-1 assay (n = 3). Data information: TP53 status is indicated for each cell line. Data are represented as mean ± SEM. See also Figure EV5 and Appendix Figure S5.


Total intracellular glutathione ( GSH + GSSG per 106 cells ) , as determined by GR re - cycling assay after 24h MK - 571 treatment in six different cell lines ( n ≥ 3 , except HCT116 WT n = 2 ) . * p = 0 . 014 , Paired t test . Each dot represents one cell line , see Fig . EV6A for individual cell lines and n .

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

Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24h MK-571 treatment in six different cell lines (n ≥ 3, except HCT116 WT n = 2). *p = 0.014, Paired t test. Each dot represents one cell line, see Fig. EV6A for individual cell lines and n.


Intracellular oxidized glutathione ( GSSG per 106 cells ) as shown by LC - MS in OVCAR - 3 ( R248Q TP53 ) cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Intracellular reduced glutathione ( GSH per 106 cells ) as shown by LC - MS in OVCAR - 3 cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Western blot analysis of MRP1 ( Cell Signaling ) , xCT and GAPDH of OVCAR - 3 cells treated with APR - 246 + / - MK - 571 for 24h .

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

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24h.


Western blot analysis of MRP1 ( Cell Signaling ) , xCT and GAPDH of HCT116 WT and R248W cells treated with APR - 246 + / - MK - 571 for 24h .

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

Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h.


Intracellular cystine ( CySS per 106 cells ) as shown by LC - MS in OVCAR - 3 cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

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

Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


Intracellular cysteine ( Cys per 106 cells ) as shown by LC - MS in OVCAR - 3 cells at 24h treatment with APR - 246 + / - MK - 571 ( n = 3 ) .

Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT and R248W mutant TP53 cells + - transfected with MRP1 or xCT siRNA following 24h of 14C - APR - 246 treatment + / - MK - 571 . Indicated values are mean values from four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) and two control siRNAs ( n ≥ 3 ) . Mean 14C - accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C - APR - 246 treatment is also shown in Fig 3D .

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

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W mutant TP53 cells +-transfected with MRP1 or xCT siRNA following 24h of 14C -APR-246 treatment +/- MK-571 . Indicated values are mean values from four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n = 1-2) and two control siRNAs (n ≥ 3). Mean 14C -accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C -APR-246 treatment is also shown in Fig 3D.


Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR - 246 treatment as shown by WST - 1 assay . Indicated values are mean values of four MRP1 siRNAs ( n ≥ 3 ) , three xCT siRNAs ( n = 1 - 2 ) and two control siRNAs ( n ≥ 3 ) . Data for control and MRP1 siRNA after APR - 246 single treatment are also included in Fig 1I . Values and n for individual siRNA shown in Fig . S7C .

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

Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR-246 treatment as shown by WST-1 assay. Indicated values are mean values of four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n =1-2) and two control siRNAs (n ≥ 3). Data for control and MRP1 siRNA after APR-246 single treatment are also included in Fig 1I. Values and n for individual siRNA shown in Fig. S7C.


Total intracellular glutathione ( GSH + GSSG per 106 cells ) in HCT116 WT cells as shown by GR re - cycling assay 48h post transfection of siRNA . Indicated values are means from four MRP1 siRNAs ( n = 3 ) , two xCT siRNA ( n = 2 - 3 ) and two control siRNA ( n = 2 - 3 ) .

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

Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT cells as shown by GR re-cycling assay 48h post transfection of siRNA. Indicated values are means from four MRP1 siRNAs (n = 3), two xCT siRNA (n = 2-3) and two control siRNA (n = 2-3).


Total intracellular glutathione ( GSH + GSSG per 106 cells ) in HCT116 WT and R248W cells measured by GR re - cycling assay after 24h of MK - 571 or sulfasalazine ( SSZ ) treatment ( n ≥ 3 , except SSZ where n = 2 , n indicated by dots ) .

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

Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT and R248W cells measured by GR re-cycling assay after 24h of MK-571 or sulfasalazine (SSZ) treatment (n ≥ 3, except SSZ where n = 2, n indicated by dots).


14C accumulation ( cpm / mg x ml - 1 ) in HCT116 WT and R248W cells at 24h treatment with 14C - APR - 246 , combined with MK - 571 or sulfasalazine ( SSZ ) , ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots ) . Data for 14C - APR - 246 + / - MK - 571 is also shown in Fig 3C and S3D .

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

14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells at 24h treatment with 14C -APR-246, combined with MK-571 or sulfasalazine (SSZ), (n ≥ 4, except SSZ where n = 2, n indicated by dots). Data for 14C -APR-246 +/- MK-571 is also shown in Fig 3C and S3D.


Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) , or untreated ( n ≥ 4 , except SSZ where n = 2 , n indicated by dots )

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

Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ), or untreated (n ≥ 4, except SSZ where n = 2, n indicated by dots)


OVCAR - 3 ( R248Q TP53 ) confluence by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalzine ( SSZ ) ( n = 3 ) . Part of the data are shown in Fig 1G . Growth suppression in OVCAR - 3 cells as determined by IncuCyte ® during 72h treatment with APR - 246 + / - MK - 571 or sulfasalazine ( SSZ ) depicted as heatmaps ( n = 3 ) .

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

OVCAR-3 (R248Q TP53) confluence by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalzine (SSZ) (n = 3). Part of the data are shown in Fig 1G. Growth suppression in OVCAR-3 cells as determined by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ) depicted as heatmaps (n = 3).


Male , 12wk wildtype ( WT ; C57Bl / 6J ) or obese / diabetic monogenic ( db / db ; BKS . Cg - m + / + Lepr DB / J ; N = 4 / group , A - E ) , New Zealand Black ( NZB ) and polygenic obese / prediabetic New Zealand Obese ( NZO ; n = 4 / group ; F - J ) , as well as young ( i . e . 3 mo ) and aged ( i . e . 22 mo ; n = 5 / group ; K - O ) , mice were fed ad libitum ( fed ) or fasted for 24h ( fasted ) . Serum non - esterified fatty acids ( A , F , K ) , triglycerides ( B , G , L ) and ketone bodies ( C , H , M ) were measured . In addition , serumacylcarinitine profiling was conducted and medium - chain ( D , I , N ) and long - chain ( E , J , O ) acylcarnitine concentrations are shown .

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

Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic monogenic (db/db; BKS.Cg-m+/+ Lepr DB/J; N = 4/group, A-E), New Zealand Black (NZB) and polygenic obese/prediabetic New Zealand Obese (NZO; n=4/group; F-J), as well as young (i.e. 3 mo) and aged (i.e.22 mo; n = 5/group; K-O), mice were fed ad libitum (fed) or fasted for 24h (fasted). Serum non-esterified fatty acids (A, F, K), triglycerides (B, G, L) and ketone bodies (C, H, M) were measured. In addition, serumacylcarinitine profiling was conducted and medium-chain (D, I, N) and long-chain (E, J, O) acylcarnitine concentrations are shown.


In another cohort of mice , ex vivo long - chain fatty acid ( LCFA ) metabolism , including uptake ( P ) , oxidation ( Q ) and non - oxidative LCFA disposal ( NOFAD ; R ) , in precision cut liver slices from fed and fasted WT and db / db mice ( n = 3 / group ; 4 slices per mouse ) , were determined . In addition , in slices from fasted mice , glucose output was determined in the presence of incubation with NEFA ( BSA - NEFA ) or vehicle ( BSA ) ( S ) .

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

In another cohort of mice, ex vivo long-chain fatty acid (LCFA) metabolism, including uptake (P), oxidation (Q) and non-oxidative LCFA disposal (NOFAD; R), in precision cut liver slices from fed and fasted WT and db/db mice (n = 3/ group; 4 slices per mouse), were determined. In addition, in slices from fasted mice, glucose output was determined in the presence of incubation with NEFA (BSA-NEFA) or vehicle (BSA) (S).


Liver mRNA expression of Growth Arrest and DNA Damage inducible 45 alpha ( Gadd45a ; A , D , G ) , beta ( Gadd45b ; B , E , H ) and gamma ( Gadd45g ; C , F , I ) was measured in fed and fasted obese / diabetic monogenic ( db / db ( n = 4 / group ) ; A , - C ) , obese / pre - diabetic polygenic New Zealand Obese ( NZO ( n = 4 / group ; D - F ) , and aged C57Bl / 6J ( 22mo ; n = 5 / group ; G - I ) as well as corresponding lean , young wildtype ( WT ) mice ( n = 4 / group ) . Matched controls for the NZO and aged mice were New Zealand Black ( NZB ) and 12wk old C57Bl / 6J ( 12wk ) mice , respectively .

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

Liver mRNA expression of Growth Arrest and DNA Damage inducible 45 alpha (Gadd45a; A, D, G), beta (Gadd45b; B, E, H) and gamma (Gadd45g; C, F, I) was measured in fed and fasted obese/diabetic monogenic (db/db (n=4/group); A,-C), obese/pre-diabetic polygenic New Zealand Obese (NZO (n=4/group; D-F), and aged C57Bl/6J (22mo; n = 5/group; G-I) as well as corresponding lean, young wildtype (WT) mice (n=4/group). Matched controls for the NZO and aged mice were New Zealand Black (NZB) and 12wk old C57Bl/6J (12wk) mice, respectively.


Liver GADD45β as well as the housekeeping protein Valosin containing protein ( VCP ) protein expression was measured from liver samples ( J - L ) . Data are mean ± SEM . Effect of genotype , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Effect of nutritional state : # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 . The statistical test used and respective p - value outputs can be found in Appendix Table S1 .

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

Liver GADD45β as well as the housekeeping protein Valosin containing protein (VCP) protein expression was measured from liver samples (J-L). Data are mean ± SEM. Effect of genotype, * p < 0.05, ** p < 0.01, *** p < 0.001. Effect of nutritional state: # p < 0.05, ## p < 0.01, ### p < 0.001. The statistical test used and respective p-value outputs can be found in Appendix Table S1.


Male , GADD45β + / + ( WT ; n = 6 ) or GADD45β - / - mice ( KO ; n = 4 ) were fed ad libitum ( fed ) or fasted for 24h ( fasted ) , and subsequently refed for 24h . O2 consumption rate ( A ) , CO2 production rate ( B ) and respiratory exchange ratio ( C ) were measured by indirect calorimetry .

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

Male, GADD45β+/+ (WT; n=6) or GADD45β-/- mice (KO; n=4) were fed ad libitum (fed) or fasted for 24h (fasted), and subsequently refed for 24h. O2 consumption rate (A), CO2 production rate (B) and respiratory exchange ratio (C) were measured by indirect calorimetry.


In a distinct cohort , male GADD45β + / + ( WT ; n = 5 ) or GADD45β - / - mice ( KO ; n = 8 ) were fed ad libitum ( fed ) or fasted for 24h ( fasted ) . Serumnon - esterified fatty acids ( D ) and ketone bodies ( E ) as well as serum ( F ) and liver ( G ) triglycerides ( TG ) were measured .

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

In a distinct cohort, male GADD45β+/+ (WT; n=5) or GADD45β-/- mice (KO; n= 8) were fed ad libitum (fed) or fasted for 24h (fasted). Serumnon-esterified fatty acids (D) and ketone bodies (E) as well as serum (F) and liver (G) triglycerides (TG) were measured.


Serum TG ( H ) , NEFA ( I ) , and blood glucose ( BG , J ) concentrations during an oral lipid tolerance test in overnight fasted GADD45β + / + ( WT ; n = 5 ) or GADD45β - / - ( KO ; n = 5 ) mice .

O B-MEASURED_VAR O O O O B-MEASURED_VAR 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

Serum TG (H), NEFA (I), and blood glucose (BG, J) concentrations during an oral lipid tolerance test in overnight fasted GADD45β+/+ (WT; n = 5) or GADD45β-/- (KO; n=5) mice.


Bloodglucose excursion during and intraperitoneal insulin tolerance test ( K ) as well as fasting bloodglucose ( L ) , seruminsulin ( M ) , and HOMA - IR ( N ) in GADD45β + / + ( WT ; n = 6 ) or GADD45β - / - mice ( KO , n = 9 ) chronically fed a normal - ( NFD ) or high ( HFD ) fat diet . Data are mean ± SEM .

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

Bloodglucose excursion during and intraperitoneal insulin tolerance test (K) as well as fasting bloodglucose (L), seruminsulin (M), and HOMA-IR (N) in GADD45β+/+ (WT; n = 6) or GADD45β-/- mice (KO, n = 9) chronically fed a normal- (NFD) or high (HFD) fat diet. Data are mean ± SEM.


Male , GADD45β + / + ( WT ) or GADD45β - / - mice ( KO ) were fed ad libitum ( fed ) or fasted for 24h ( fasted ) and ex vivo liver slice long - chain fatty acid ( LCFA ) metabolism was measured including LCFA uptake ( A ) , oxidation ( B ) and non - oxidative LCFA disposal ( NOFAD ) was calculated ( C ) .

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

Male, GADD45β+/+ (WT) or GADD45β-/- mice (KO) were fed ad libitum (fed) or fasted for 24h (fasted) and ex vivo liver slice long-chain fatty acid (LCFA) metabolism was measured including LCFA uptake (A), oxidation (B) and non-oxidative LCFA disposal (NOFAD) was calculated (C).


In addition , glucose production was measured ( D ) in the presence ( BSA - NEFA ) or absence ( BSA ) of extracellular fatty acids ( n = 4 / group with 4 liver slices per mouse liver ) .

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

In addition, glucose production was measured (D) in the presence (BSA-NEFA) or absence (BSA) of extracellular fatty acids (n=4/group with 4 liver slices per mouse liver).


Gadd45b mRNA expression was measured from fractionated parenchymal hepatocytes ( Hepas ) as well as non - parenchymal cells ( NP ) from C57Bl / 6J mice fed ad libitum ( fed ) or fasted for 24h ( fasted ) ; n = 3 / group ( E ) .

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

Gadd45b mRNA expression was measured from fractionated parenchymal hepatocytes (Hepas) as well as non-parenchymal cells (NP) from C57Bl/6J mice fed ad libitum (fed) or fasted for 24h (fasted); n=3/group (E).


Male , C57Bl / 6J mice with ( AAV - G45b miR ) or without ( AAV - NC miR ) liver - hepatocyte - restricted GADD45β silencing were fed or fasted for 24h ( n = 6 / group ) and serum levels of non - esterified fatty acids ( NEFA ; F ) as well as liver triglyceride ( TG ) concentration ( G ) were measured .

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

Male, C57Bl/6J mice with (AAV-G45b miR) or without (AAV-NC miR) liver-hepatocyte-restricted GADD45β silencing were fed or fasted for 24h (n = 6/group) and serum levels of non-esterified fatty acids (NEFA; F) as well as liver triglyceride (TG) concentration (G) were measured.


Male , GADD45β + / + ( WT ; n = 16 ) or GADD45β - / - mice ( KO ; n = 15 ) fasted for 24h ( fasted ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 7 - 8 / group ) . LiverTG concentration ( H ) was measured .

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

Male, GADD45β+/+ (WT; n=16) or GADD45β-/- mice (KO; n=15) fasted for 24h (fasted) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 7-8/group). LiverTG concentration (H) was measured.


Male , C57Bl / 6J mice with ( AAV - G45b miR ; n = 15 ) or without ( AAV - NC miR , n = 13 ) liver - hepatocyte - restricted GADD45β silencing were chronically fed a normal ( NFD ) or high ( HFD ) fat diet ( n = 6 - 8 / group ) . Fasting blood glucose ( I ) and serum insulin ( J ) were measured from which HOMA - IR was calculated ( K ) .

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

Male, C57Bl/6J mice with (AAV-G45b miR; n= 15) or without (AAV-NC miR, n = 13) liver-hepatocyte-restricted GADD45β silencing were chronically fed a normal (NFD) or high (HFD) fat diet (n = 6-8/group). Fasting blood glucose (I) and serum insulin (J) were measured from which HOMA-IR was calculated (K).


Male , 12wk wildtype ( WT ; C57Bl / 6J ) or obese / diabetic ( db / db ; BKS . Cg - m + / + Lepr DB / J ) with ( AD - G45b OE ) or without ( AD - NC ) prior liver - restricted GADD45β over - expression were fasted and blood glucose ( A ) and serum insulin ( B ) were measured from which HOMA - IR was calculated ( C ) ( n = 4 - 6 / group ) . Data are mean ± SEM . Effect of genotype / viral manipulation , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Effect of nutritional state : # 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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Male, 12wk wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) prior liver-restricted GADD45β over-expression were fasted and blood glucose ( A) and serum insulin (B) were measured from which HOMA-IR was calculated (C) (n = 4-6/group). Data are mean ± SEM. Effect of genotype/viral manipulation, * p < 0.05, ** p < 0.01, *** p < 0.001. Effect of nutritional state: # p < 0.05, ## p < 0.01, ### p < 0.001.


Liver GADD45B mRNA expression from men with ( T2D ) or without ( NGT ) type 2 diabetes ( n = 14 - 23 / group ) .

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Liver GADD45B mRNA expression from men with (T2D) or without (NGT) type 2 diabetes (n = 14-23/group).


Scatter plots fasting plasma triglycerides ( E ) and glucose ( F ) in correlation with liver GADD45B mRNA expression ( n = 37 ) . Inserts show r2 values and p values from Spearman ' s correlation test . The statistical test used and respective p - value outputs can be found in Appendix Table S1 .

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

Scatter plots fasting plasma triglycerides (E) and glucose (F) in correlation with liver GADD45B mRNA expression (n = 37). Inserts show r2 values and p values from Spearman's correlation test. The statistical test used and respective p-value outputs can be found in Appendix Table S1.


Male , GADD45β + / + ( WT ; n = 16 ) or GADD45β - / - mice ( KO ; n = 15 ) fasted for 24h ( fasted ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 7 - 8 / group ) . Liver mRNA expression of Gadd45b ( A ) as well as fatty acid metabolic genes ( B ) encompassing transport ( Slc28a2 , Slc27a5 , Cd36 ) , intracellular binding ( Fabp1 , Dbi ) and metabolism ( Acly , Dgat1 , Atgl , Hsl ) .

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

Male, GADD45β+/+ (WT; n=16) or GADD45β-/- mice (KO; n=15) fasted for 24h (fasted) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 7-8/group). Liver mRNA expression of Gadd45b (A) as well as fatty acid metabolic genes (B) encompassing transport (Slc28a2, Slc27a5, Cd36), intracellular binding (Fabp1, Dbi) and metabolism (Acly, Dgat1, Atgl, Hsl).


C : Representative immunoblots of FATP2 , CD36 , FABP1 and GADD45β from liver whole tissue lysate ( W ) as well as fractionated organelles / intraceuular structures including nuclei ( N ) , mitochondria ( MT ) , microsomes ( MS ) and cytoplasm ( C ) , from GADD45β + / + ( WT ) and GADD45β - / - ( KO ) mice . D : Quantified band densities of FABP1 enrichment from fractions in C ( n = 4 / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Representative immunoblots of FATP2, CD36, FABP1 and GADD45β from liver whole tissue lysate (W) as well as fractionated organelles/intraceuular structures including nuclei (N), mitochondria (MT), microsomes (MS) and cytoplasm (C), from GADD45β+/+ (WT) and GADD45β-/- (KO) mice. D: Quantified band densities of FABP1 enrichment from fractions in C (n = 4/group).


E : Liver fraction enrichment of FABP1 from male , GADD45β + / + ( WT ) or GADD45β - / - ( KO ) fasted for 24h with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 4 / group ) . Insert shows a representative FABP1 immunoblot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Liver fraction enrichment of FABP1 from male, GADD45β+/+ (WT) or GADD45β-/- (KO) fasted for 24h with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 4/group). Insert shows a representative FABP1 immunoblot.


F : Liver fraction enrichment of FABP1 from obese / diabetic male db / db mice fasted for 24h with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( n = 4 / group ) . Insert shows a representative FABP1 immunoblot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Liver fraction enrichment of FABP1 from obese/diabetic male db/db mice fasted for 24h with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (n = 4/group). Insert shows a representative FABP1 immunoblot.


G : FABP1 and GADD45B immunoblots from Flag - immunoprecipitations ( IP - FLAG ) or mock IP ( IP - HA ) from liver input samples from mice with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression . Shown is a representative immunoblot from 3 separate experiments using 3 different input samples per condition .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: FABP1 and GADD45B immunoblots from Flag-immunoprecipitations (IP-FLAG) or mock IP (IP-HA) from liver input samples from mice with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression. Shown is a representative immunoblot from 3 separate experiments using 3 different input samples per condition.


H : Liver long chain acyl - CoA ( LC - acyl - CoA ) concentrations were determined in GADD45β + / + ( WT ) or GADD45β - / - ( KO ) mice ( H ; n = 6 / group ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( I ; n = 5 / group ) .

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

H: Liver long chain acyl-CoA (LC-acyl-CoA) concentrations were determined in GADD45β+/+ (WT) or GADD45β-/- (KO) mice (H; n=6/ group) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (I; n =5/group).


Liver LC - acyl - CoA concentrations were determined in wildtype ( WT ; C57Bl / 6J ) or obese / diabetic ( db / db ; BKS . Cg - m + / + Lepr DB / J ) with ( AD - G45b OE ) or without ( AD - NC ) liver - restricted GADD45β over - expression ( J ; n = 4 / group ) .

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

Liver LC-acyl-CoA concentrations were determined in wildtype (WT; C57Bl/6J) or obese/diabetic (db/db; BKS.Cg-m+/+ Lepr DB/J) with (AD-G45b OE) or without (AD-NC) liver-restricted GADD45β over-expression (J; n=4/group).


Concentration of total cell - free DNA ( cfDNA ) in 13 CSF samples . Samples in which SCNAs were detected are shown in dark blue , and tended to have higher levels of total

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

Concentration of total cell-free DNA (cfDNA) in 13 CSF samples. Samples in which SCNAs were detected are shown in dark blue, and tended to have higher levels of total cfDNA


Relative copy number estimation in 20 genes of interest , and 18 large genomic regions , determined by sWGS of CSF from 13 glioma patients . Genes are ordered by genomic position , and their chromosomal locations are indicated in parentheses . Amplifications are shown in dark blue , deletions in orange , and copy number neutral regions are in grey . The top green bar indicates the tumor largest length dimension ( < 42 mm vs > 42 mm ) , and the top violet bar indicates the glioma subtype . SCNAs were more frequently detected in CSF from patients with large glioblastoma

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 copy number estimation in 20 genes of interest, and 18 large genomic regions, determined by sWGS of CSF from 13 glioma patients. Genes are ordered by genomic position, and their chromosomal locations are indicated in parentheses. Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in grey. The top green bar indicates the tumor largest length dimension (<42 mm vs >42 mm), and the top violet bar indicates the glioma subtype. SCNAs were more frequently detected in CSF from patients with large glioblastoma tumors


SCNAs determined by sWGS in 4 tumor subparts ( T1 - T4 ) and the CSF sample , collected from patient G1 . Amplifications are shown in dark blue , deletions in orange , and copy number neutral regions are in dark grey . sWGS from plasma and urine samples collected at the same time as the CSF sample showed no

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

SCNAs determined by sWGS in 4 tumor subparts (T1 - T4) and the CSF sample, collected from patient G1. Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in dark grey. sWGS from plasma and urine samples collected at the same time as the CSF sample showed no SCNAs


Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 ( 4 tumor subparts and 1 CSF sample ) . Amplifications are shown in dark blue , deletions in orange , and copy number neutral regions are in light

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

Heat map summarizing the SCNAs detected by sWGS of 28 genes of interest in tumor biopsies and CSF from patient G1 (4 tumor subparts and 1 CSF sample). Amplifications are shown in dark blue, deletions in orange, and copy number neutral regions are in light grey


Heat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples . Shared detection in tissue and CSF is indicated in green , detection of the alteration only in tissue in orange , and non - detection in blue . The top bars indicate the cfDNA concentration ( copies / mL ; in a range of purples ) , the size of the tumors ( in a range of browns ) , the type of glioma ( in a range of blues ) , and whether the tumor was in direct contact with the CSF or not ( based on MRI , green or red , respectively ) . Samples are ranked from the left to right by decreasing concentration of cfDNA ( copies / mL )

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

Heat map summarizing detection of EGFR and PTEN alterations in tumor tissue and in CSF samples. Shared detection in tissue and CSF is indicated in green, detection of the alteration only in tissue in orange, and non-detection in blue. The top bars indicate the cfDNA concentration (copies/mL; in a range of purples), the size of the tumors (in a range of browns), the type of glioma (in a range of blues), and whether the tumor was in direct contact with the CSF or not (based on MRI, green or red, respectively). Samples are ranked from the left to right by decreasing concentration of cfDNA (copies/mL)


A ) Fragment size distribution , determined by sWGS , in CSF ( blue line ) , plasma ( red dashed line ) and urine ( black dashed line ) from patient G1 . The 3 samples were collected simultaneously before initiation of treatment . B ) Fragment size distribution of cfDNA , determined by sWGS , in CSF from 13 glioma patients . The samples contained a high fraction of reads from DNA fragments with lengths corresponding to ~ 145 bp and ~ 167 bp . The fragment size profile of cfDNA from the plasma sample from patient G1 is shown in red . All samples were collected simultaneously before initiation of treatment C ) Cumulative frequency analysis of the average density of fragment size in CSF ( blue ) and plasma ( red ) . The vertical dashed line represents 167

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A) Fragment size distribution, determined by sWGS, in CSF (blue line), plasma (red dashed line) and urine (black dashed line) from patient G1. The 3 samples were collected simultaneously before initiation of treatment. B) Fragment size distribution of cfDNA, determined by sWGS, in CSF from 13 glioma patients. The samples contained a high fraction of reads from DNA fragments with lengths corresponding to ~145 bp and ~167 bp. The fragment size profile of cfDNA from the plasma sample from patient G1 is shown in red. All samples were collected simultaneously before initiation of treatment C) Cumulative frequency analysis of the average density of fragment size in CSF (blue) and plasma (red). The vertical dashed line represents 167 bp


Mean fragment size density in patients with detected SCNAs in the CSF ( yellow ) , and those with no detected SCNAs ( dark blue )

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Mean fragment size density in patients with detected SCNAs in the CSF (yellow), and those with no detected SCNAs (dark blue)


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Log2 ratio of the difference in cfDNA fragment sizes between CSF samples with detected SCNAs and those samples in which no SCNAs were detected


The amplitude of the 10 bp periodic peaks in fragment size is a signature for non - tumor DNA in the CSF . The amplitude of the 10 bp periodic peaks in fragment length distribution ( in the range between 75 and 150 bp , see Methods ) when compared to the mean copy number alteration calculated from sWGS revealed a significant negative correlation ( Pearson - 0 . 85 ; p = 0 . 0002 )

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The amplitude of the 10 bp periodic peaks in fragment size is a signature for non-tumor DNA in the CSF. The amplitude of the 10 bp periodic peaks in fragment length distribution (in the range between 75 and 150 bp, see Methods) when compared to the mean copy number alteration calculated from sWGS revealed a significant negative correlation (Pearson -0.85; p=0.0002)


A The distribution of the GFP - tagged Hxt2 changes during the cell division . In small and medium budded cells ( white arrowheads ) , Hxt2 is mainly present in the membrane of the mother cell . In large budded cells , Hxt2 is distributed over the entire plasma membrane ( yellow arrowheads ) .

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A The distribution of the GFP-tagged Hxt2 changes during the cell division. In small and medium budded cells (white arrowheads), Hxt2 is mainly present in the membrane of the mother cell. In large budded cells, Hxt2 is distributed over the entire plasma membrane (yellow arrowheads).


B HXT2 mRNA localization correlate with that of the protein . FISH with an HXT2 - specific probe . Early in the cell cycle the mRNA is predominantly found in the mother cell ( white arrowheads ) while it is equally distributed after meta - to - anaphase transition ( MAT ) ( yellow arrowheads ) . C Quantification of ( B ) . The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated . Values > 1 indicate a stronger signal in the mother , < 1 a stronger signal in the bud and around 1 equal distribution . Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed . Boxes represent the interquartile range from the 25th to the 75th percentile with the median . Whiskers represent the 10th and 90th percentile , respectively .

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B HXT2 mRNA localization correlate with that of the protein. FISH with an HXT2-specific probe. Early in the cell cycle the mRNA is predominantly found in the mother cell (white arrowheads) while it is equally distributed after meta-to-anaphase transition (MAT) (yellow arrowheads). C Quantification of (B). The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.


D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton . Cells were either treated for 15 min with 30 mg / ml latrunculin A or with 30 mg / ml benomyl . White arrowheads point to small - budded and yellow arrowheads to large budded cells . E Quantification of ( D ) of three in dependent experiments with at least 50 cells each . Values > 1 indicate a stronger signal in the mother , < 1 a stronger signal in the bud and around 1 equal distribution . Boxes represent the interquartile range from the 25th to the 75th percentile with the median . Whiskers represent the 10th and 90th percentile , respectively .

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D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton. Cells were either treated for 15 min with 30 mg/ml latrunculin A or with 30 mg/ml benomyl. White arrowheads point to small-budded and yellow arrowheads to large budded cells. E Quantification of (D) of three in dependent experiments with at least 50 cells each. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.


A , Inhibiting translation by either applying Cycloheximide ( CHX ) or Verrucarin A ( VerruA ) leads to the retention of HXT2 mRNA in the mother cell after MAT . Chloro : chloroform ; solvent control for VerruA ; NT : not treated ; control for CHX .

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A, Inhibiting translation by either applying Cycloheximide (CHX) or Verrucarin A (VerruA) leads to the retention of HXT2 mRNA in the mother cell after MAT. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.


FISH of CHX or VerruA - treated cells . Chloro : chloroform ; solvent control for VerruA ; NT : not treated ; control for CHX .

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FISH of CHX or VerruA-treated cells. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.


C CHX treatment leads to increased transcript stability . Quantitative PCR of HXT2 mRNA in CHX - treated cells in comparison to prt1 - 1 mutant cells . The data represents 3 independent experiments and the standard deviation is given .

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C CHX treatment leads to increased transcript stability. Quantitative PCR of HXT2 mRNA in CHX-treated cells in comparison to prt1-1 mutant cells. The data represents 3 independent experiments and the standard deviation is given.


D Translation of HXT2 mRNA is more active late in the cell cycle . The HXT2 mRNA level bound to either di - / trisomes and polysomes ( ≥ 4 ribosomes ) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1 / S or M - phase followed by qPCR . At G1 / S similar levels of the HXT2 mRNA were found in the different ribosome fractions , whereas in M - phase more mRNA was bound to polysomes , suggesting a more active translation in M - phase .

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D Translation of HXT2 mRNA is more active late in the cell cycle. The HXT2 mRNA level bound to either di-/trisomes and polysomes (≥ 4 ribosomes) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1/S or M-phase followed by qPCR. At G1/S similar levels of the HXT2 mRNA were found in the different ribosome fractions, whereas in M-phase more mRNA was bound to polysomes, suggesting a more active translation in M-phase.


E The localization of HXT2 mRNA is independent of the Signal Recognition Particle ( SRP ) . Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA .

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E The localization of HXT2 mRNA is independent of the Signal Recognition Particle (SRP). Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA.


A Deletion of SCP160 , ASC1 , or BFR1 increases HXT2 mRNA signal in the bud . FISH experiment with deletions in various RNA binding proteins .

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A Deletion of SCP160, ASC1, or BFR1 increases HXT2 mRNA signal in the bud. FISH experiment with deletions in various RNA binding proteins.


B The effect of ∆ scp160 on HXT2 mRNA localization is independent of its increased ploidy . Scp160 truncations that lack either the last two ( ∆ C2 ) or the last four ( ∆ C4 ) KH domains or a Tet - off SCP160 construct , in which expression is blocked by the addition of doxycycline ( Doxy )

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B The effect of ∆scp160 on HXT2 mRNA localization is independent of its increased ploidy. Scp160 truncations that lack either the last two (∆C2) or the last four (∆C4) KH domains or a Tet-off SCP160 construct, in which expression is blocked by the addition of doxycycline (Doxy)


D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud . Treatment of cells with forskolin ( + Forsko ) or using a ∆ bcy1 strain , causes HXT2 mRNA to be bud - enriched .

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D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud. Treatment of cells with forskolin (+Forsko) or using a ∆bcy1 strain, causes HXT2 mRNA to be bud-enriched.


E HXT2 mRNA enrichment upon refeeding is Ras2 - dependent .

E HXT2 mRNA enrichment upon refeeding is Ras2-dependent.


A Table of the glucose affinity of the four most important hexose transporters . B FISH of HXT1 , 3 and 4 under glucose rich conditions . White arrowheads depict cells before , yellow arrowheads cells after MAT . C Quantification of ( B ) .

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A Table of the glucose affinity of the four most important hexose transporters. B FISH of HXT1, 3 and 4 under glucose rich conditions. White arrowheads depict cells before, yellow arrowheads cells after MAT. C Quantification of (B).


D Hyperactive PKA promotes enrichment of all HXTs after MAT .

D Hyperactive PKA promotes enrichment of all HXTs after MAT.


E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation .

E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation.


F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding . Daughters and mothers were separated through elutriation . qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions .

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F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding. Daughters and mothers were separated through elutriation. qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions.


G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2 - GFP transporter levels at the plasma membrane of the daughter cells . Daughter cells were enriched through elutriation . Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment . Western blot analysis confirms the higher amount of Hxt2 - GFP transporter protein present in daughter cells under glucose - shift

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G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2-GFP transporter levels at the plasma membrane of the daughter cells. Daughter cells were enriched through elutriation. Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment. Western blot analysis confirms the higher amount of Hxt2-GFP transporter protein present in daughter cells under glucose-shift conditions


A RNA degradation is not essential for asymmetric HXT2 mRNA localization . FISH of wild - type and ∆ xrn1 cells .

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A RNA degradation is not essential for asymmetric HXT2 mRNA localization. FISH of wild-type and ∆xrn1 cells.


B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud . Cells were treated upon glucose addition with either 120 mg / ml benomyl for 15 min , 30 mg / ml latrunculin A for 30 min or 200 µM CK - 666 for 30 min . HXT2 mRNA was detect by FISH .

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B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud. Cells were treated upon glucose addition with either 120 mg/ml benomyl for 15 min, 30 mg/ml latrunculin A for 30 min or 200 µM CK-666 for 30 min. HXT2 mRNA was detect by FISH.


C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function . HXT2 mRNA FISH with ∆ bim1 and ∆ kar9 strains . Arrows point to a nucleus with HXT2 signal . D Quantification of the daughter cell enrichment from data displayed in ( C ) . E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in ( C ) . Percentage of cells with asymmetric HXT2 mRNA staining is displayed .

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C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function. HXT2 mRNA FISH with ∆bim1 and ∆kar9 strains. Arrows point to a nucleus with HXT2 signal. D Quantification of the daughter cell enrichment from data displayed in (C). E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in (C). Percentage of cells with asymmetric HXT2 mRNA staining is displayed.


A Deletion of NUP2 and MLP1 / 2 reduced HXT2 mRNA enrichment in the bud . FISH under glucose shift conditions with strains in which different NPC components were deleted . B Quantification of ( A ) .

A Deletion of NUP2 and MLP1/2 reduced HXT2 mRNA enrichment in the bud. FISH under glucose shift conditions with strains in which different NPC components were deleted. B Quantification of (A).


C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift . FISH - IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84 - HA . White arrows point to HXT2 mRNA signals on the nuclear envelope .

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C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift. FISH-IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84-HA. White arrows point to HXT2 mRNA signals on the nuclear envelope.


D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift . Stills of time lapse movies . HXT2 mRNA was visualized using the MS2 - GFP system . Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus . Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus . Cyan : Nup84 - mCherry ; white arrowhead : HXT2 mRNA - MS2 + MCP - GFP .

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D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift. Stills of time lapse movies. HXT2 mRNA was visualized using the MS2-GFP system. Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus. Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus. Cyan: Nup84-mCherry; white arrowhead: HXT2 mRNA-MS2 + MCP-GFP.


HXT2 only strains grow faster than the other HXT only strain . Growth was assessed 2 - 3 days after incubation at 30 ° C .

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HXT2 only strains grow faster than the other HXT only strain. Growth was assessed 2-3 days after incubation at 30°C.


D Quantification of competition assay . qPCR with primers against either HXT1 , 2 , 3 or 4 was performed . HXT gene abundance was normalized against actin .

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D Quantification of competition assay. qPCR with primers against either HXT1, 2, 3 or 4 was performed. HXT gene abundance was normalized against actin.


E Strains that lack HXT2 grow slower when coming from quiescence compared to WT .

E Strains that lack HXT2 grow slower when coming from quiescence compared to WT.


F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96 % of cells independently of the cell cycle . Arrows point to examples of HXT2 - U1A mRNA in the bud early and late in the cell cycle .

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F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96% of cells independently of the cell cycle. Arrows point to examples of HXT2-U1A mRNA in the bud early and late in the cell cycle.


G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2 .

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G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2.


H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers . Cells were re - fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points .

H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers. Cells were re-fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points.


( A ) Michaelis - Menten analysis of the reduction of 2 - hydroxyacetophenone ( 2 - HAP ) . The initial velocities ( V0 / [ Etot ] ) are given for the individual subunits ( i . e . , normalized to subunit not oligomer concentration ) . Enzyme assays were conducted at 30 ˚ C in 100 mM sodium phosphate ( pH 6 . 0 ) using 500 µM NADPH and 100 nM SCR or 1 µM SCR - his6 . Enzyme activity at each substrate concentration was assayed at least three and usually four times . The data points show the average values for these technical replicates , while the error bars show the standard deviations .

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(A) Michaelis-Menten analysis of the reduction of 2-hydroxyacetophenone (2-HAP). The initial velocities (V0/[Etot]) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH and 100 nM SCR or 1 µM SCR-his6. Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.


Comparison of kcat values obtained from Michaelis - Menten analyses of the reduction of four different substrates . The turnover rates ( kcat ) are given for the individual subunits ( i . e . , normalized to subunit not oligomer concentration ) . Enzyme assays were conducted at 30 ˚ C in 100 mM sodium phosphate ( pH 6 . 0 ) using 500 µM NADPH and 100 nM SCR or 1 µM SCR - his6 . The data points show the average values for these technical replicates , while the error bars show the standard deviations .

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Comparison of kcat values obtained from Michaelis-Menten analyses of the reduction of four different substrates. The turnover rates (kcat) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH and 100 nM SCR or 1 µM SCR-his6. The data points show the average values for these technical replicates, while the error bars show the standard deviations.


( A ) Analytical gel - filtration of purified his8 - MBP - SCR2 ( black ) , strep - SCR ( yellow ) , and his8 - MBP - SCR2 / strep - SCR on a Superdex 200 Increase column at 20 ˚ C in 150 mM NaCl , 50 mM Tris - HCl , pH 8 . 0 .

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(A) Analytical gel-filtration of purified his8-MBP-SCR2 (black), strep-SCR (yellow), and his8-MBP-SCR2/strep-SCR on a Superdex 200 Increase column at 20 ˚C in 150 mM NaCl, 50 mM Tris-HCl, pH 8.0.


( B ) Michaelis - Menten kinetic analyses of NADPH - dependent reduction of 2 - hydroxyacetophenone and methyl acetoacetate at 30 ˚ C in 100 mM sodium phosphate , pH 6 . 0 . The turnover rate ( V0 / [ Etot ] ) is given for the individual subunits Enzyme activity at each substrate concentration was assayed at least three and usually four times . The data points show the average values for these technical replicates , while the error bars show the standard deviations .

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(B) Michaelis-Menten kinetic analyses of NADPH-dependent reduction of 2-hydroxyacetophenone and methyl acetoacetate at 30 ˚C in 100 mM sodium phosphate, pH 6.0. The turnover rate (V0/[Etot]) is given for the individual subunits Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.


( D ) Western blot analysis of ADAR1 expression in E13 . 5 immortalized MEFs of the indicated genotypes + / - interferon - β ( IFNβ ) . Data from two independent cell lines per genotype .

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(D) Western blot analysis of ADAR1 expression in E13.5 immortalized MEFs of the indicated genotypes +/- interferon-β (IFNβ). Data from two independent cell lines per genotype.


( G ) Survival analysis of mice of the indicated genotypes ; number as indicated for each genotype . Statistical difference determined by Log - rank ( Mantel - Cox ) test .

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(G) Survival analysis of mice of the indicated genotypes; number as indicated for each genotype. Statistical difference determined by Log-rank (Mantel-Cox) test.


( H ) Total white blood cell counts ( WBC ) in peripheral blood ( PB ) .

(H) Total white blood cell counts (WBC) in peripheral blood (PB).


( J - L ) Peripheral blood ( J ) red blood cell ( RBC ) , ( K ) hematocrit ( HCT ) and ( L ) platelets .

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(J-L) Peripheral blood (J) red blood cell (RBC), (K) hematocrit (HCT) and (L) platelets.


( C ) Survival analysis of mice of the indicated genotypes ; number as indicated for each genotype . No statistical difference between genotypes by Log - rank ( Mantel - Cox ) test . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) .

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(C) Survival analysis of mice of the indicated genotypes; number as indicated for each genotype. No statistical difference between genotypes by Log-rank (Mantel-Cox) test. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4).


( E ) Peripheral blood leukocyte populations ( by lineage ) between genotypes at day 0 and day 28 . ; R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . All BM , spleen and thymic analysis at day 28 post tamoxifen

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Peripheral blood leukocyte populations (by lineage) between genotypes at day 0 and day 28. ; R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM, spleen and thymic analysis at day 28 post tamoxifen treatment