Left - Scheme for identifying sites that lose 5hmC their association with POU5F1 binding . Right - of 5hmC profile during AA + 2i treatment of WT pre - iPSC POU5F1 binding in pre - iPSCs or mESCs at loci identified in left panel .

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Left - Scheme for identifying sites that lose 5hmC and their association with POU5F1 binding. Right - Heatmap of 5hmC profile during AA+2i treatment of WT pre-iPSC and POU5F1 binding in pre-iPSCs or mESCs at the loci identified in the left panel.


Representative 5hmC profiles during AA + 2i conversion of WT Kdm3b - KO pre - iPSC with POU5F1 binding in pre - iPSCs mESCs . Loci were assigned to nearest gene .

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Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC with POU5F1 binding in pre-iPSCs and mESCs. Loci were assigned to the nearest gene.


ChIP - PCR for POU5F1 during AA + 2i reprogramming of WT Kdm3b - KO pre - iPSCs . Values are fold change of enrichment between WT Kdm3b KO ( WT / Kdm3bKO ) .

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ChIP-PCR for POU5F1 during AA+2i reprogramming of WT and Kdm3b-KO pre-iPSCs. Values are fold change of enrichment between WT and Kdm3b KO (WT/Kdm3bKO).


tSNE of Tet1 , Tet2 , Nanog , Cdh1 expression in single sequencing of MEF undergoing OSKM reprogramming from Tran et al 2019b . MEF , Reprogramming ( REPROG ) , mESCs are highlighted in red dotted lines . Specific reprogramming timepoints are highlighted within reprogramming cluster in arrows . Color corresponds to log10 of transcript for each .

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tSNE of Tet1, Tet2, Nanog, and Cdh1 expression in single cell RNA sequencing of MEF undergoing OSKM reprogramming from Tran et al 2019b. MEF, Reprogramming (REPROG), and mESCs cells are highlighted in red dotted lines. Specific reprogramming timepoints are highlighted within the reprogramming cluster in arrows. Color corresponds to log10 of transcript counts for each individual cell.


Bar graphs indicating number of expressing Tet1 , Tet2 , Cdh1 or Nanog from reprograming population ( Day 3 , 6 , 9 12 ) . Number of per reprogramming population is highlighted within legend .

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Bar graphs indicating number of cells expressing Tet1, Tet2, Cdh1 or Nanog from the reprograming population (Day 3, 6, 9 and 12). Number of cells per reprogramming population is highlighted within legend.


Scheme for deriving Tet1 Tet2 double knockout pre - iPSC lines . Tet1KO / KO Tet2fl / fl or Tet1WT / WT Tet2fl / fl MEFs were treated with retroviruses containing OSKM . Tet1KO / KO Tet2fl / fl or Tet1WT / WT Tet2fl / fl pre - iPSC clones were isolated treated with either Adenoviral expressed Cre recombinase or Empty vector to generate a population of pre - iPSCs with Tet1KO / KO Tet2fl / fl , Tet1KO / KO Tet2KO / KO , Tet1WT / WT Tet2fl / fl , Tet1WT / WT , Tet2KO / KO . Scale bar , 50 μm .

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Scheme for deriving Tet1 and Tet2 double knockout pre-iPSC lines. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl MEFs were treated with retroviruses containing OSKM. Tet1KO/KO Tet2fl/fl or Tet1WT/WT Tet2fl/fl pre-iPSC clones were isolated and treated with either Adenoviral expressed Cre recombinase or Empty vector to generate a population of pre-iPSCs with Tet1KO/KO Tet2fl/fl, Tet1KO/KO Tet2KO/KO, Tet1WT/WT Tet2fl/fl, and Tet1WT/WT, and Tet2KO/KO. Scale bar, 50 μm.


of Nanog positive by intracellular flow cytometry in each genotype as indicated of Tet1WT / WT Tet2fl / fl , Tet1WT / WT Tet2KO / KO , Tet1KO / KO Tet2fl / fl , Tet1KO / KO Tet2KO / KO after 10 days of AA + 2i treatment .

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Quantification of percent Nanog positive cells by intracellular flow cytometry in each genotype as indicated of Tet1WT/WT Tet2fl/fl, Tet1WT/WT Tet2KO/KO, Tet1KO/KO Tet2fl/fl, and Tet1KO/KO Tet2KO/KO after 10 days of AA+2i treatment.


RT - PCR of SPPL2c expression in different murine tissues . Total from indicated tissues was either transcribed into cDNA prior to PCR amplification ( + RT ) or , as negative control , used directly as template ( - RT ) . A fragment of actin ORF was amplified as control .

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RT-PCR analysis of SPPL2c expression in different murine tissues. Total RNA from the indicated tissues was either transcribed into cDNA prior to PCR amplification (+RT) or, as negative control, used directly as template (-RT). A fragment of the actin ORF was amplified as control.


Western Blot of total lysates from testis isolated from wild type or SPPL2c - / - using antibodies against C - ( D ) or N - terminal ( E ) epitopes of SPPL2c . Lysates from HeLa transiently expressing SPPL2c isoforms were analysed in parallel .

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Western Blot analysis of total lysates from testis isolated from wild type or SPPL2c-/- using antibodies against C- (D) or N-terminal (E) epitopes of SPPL2c. Lysates from HeLa cells transiently expressing the SPPL2c isoforms were analysed in parallel.


SPPL2c was visualised by indirect immunofluorescence in HeLa transiently expressing C - terminally Myc - tagged murine SPPL2c isoform A . For detection of SPPL2c , either anti - Myc or SPPL2c antiserum ( C - terminal epitope ) were employed as indicated together with anti - KDEL , anti - GM130 or anti - ERGIC53 as indicated in order to label ER , cis Golgi apparatus or ER - Golgi intermediate compartment ( ERGIC ) , respectively .

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SPPL2c was visualised by indirect immunofluorescence in HeLa cells transiently expressing C-terminally Myc-tagged murine SPPL2c isoform A. For detection of SPPL2c, either anti-Myc or the SPPL2c antiserum (C-terminal epitope) were employed as indicated together with anti-KDEL, anti-GM130 or anti-ERGIC53 as indicated in order to label the ER, the cis Golgi apparatus or the ER-Golgi intermediate compartment (ERGIC), respectively.


Lysates from HEK293 transiently transfected with murine SPPL2c - myc ( isoform A ) were treated with glycosidases PNGase F or Endo H as indicated prior to Western blot with anti - SPPL2c ( C - term ) . Deglycosylation of endogenously expressed LIMP2 was analysed as control .

Lysates from HEK293 cells transiently transfected with murine SPPL2c-myc (isoform A) were treated with the glycosidases PNGase F or Endo H as indicated prior to Western blot analysis with anti-SPPL2c (C-term). Deglycosylation of endogenously expressed LIMP2 was analysed as control.


N - glycosylation of endogenous SPPL2c is similar to overexpressed . Total lysates from testis of wild type or SPPL2c - / - mice were

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N-glycosylation of endogenous SPPL2c is similar to the overexpressed protein. Total lysates from testis of wild type or SPPL2c-/- mice were treated


Subcellular fractionation of postnuclear supernatant from murine testis using a self - forming Percoll density gradient . Fractions were collected from bottom to top subjected to Western blot for SPPL2c . disulfide isomerase ( PDI ) , Cathepsin D CD44 were detected as organelle marker .

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Subcellular fractionation of postnuclear supernatant from murine testis using a self-forming Percoll density gradient. Fractions were collected from bottom to top and subjected to Western blot analysis for SPPL2c. Protein disulfide isomerase (PDI), Cathepsin D and CD44 were detected as organelle marker proteins.


HEK293 were transiently transfected with murine N - terminally HA - tagged heme oxygenase 1 ( HA - mHO - 1 ) alone or in combination with active or inactive ( D / A ) murine SPP or SPPL2c . Substrate were determined by Western blot with anti - HA . For detection of SPP , an antiserum recognizing both endogenous human ( h ) overexpressed murine ( m ) SPP was employed . Murine SPPL2c was visualised using antiserum generated against C - terminus of . Actin was detected to control for equal loading .

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HEK293 cells were transiently transfected with murine N-terminally HA-tagged heme oxygenase 1 (HA-mHO-1) alone or in combination with active or inactive (D/A) murine SPP or SPPL2c. Substrate levels were determined by Western blot analysis with anti-HA. For detection of SPP, an antiserum recognizing both endogenous human (h) and overexpressed murine (m) SPP was employed. Murine SPPL2c was visualised using the antiserum generated against the C-terminus of the protein. Actin was detected to control for equal protein loading.


Immunofluorescence of HeLa transiently expressing murine HO - 1 ( HA - mHO - 1 ) alone or together with active or inactive ( D / A ) SPPL2c - myc . Substrate proteases were visualised using HA Myc epitopes , respectively .

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Immunofluorescence analysis of HeLa cells transiently expressing murine HO-1 (HA-mHO-1) alone or together with active or inactive (D/A) SPPL2c-myc. Substrate and proteases were visualised using the HA and Myc epitopes, respectively.


TA Ube2J1 is not proteolysed by co - expressed SPP or SPPL2c in HEK293 . Western blot was

The TA protein Ube2J1 is not proteolysed by co-expressed SPP or SPPL2c in HEK293 cells. Western blot analysis was performed


Differential cleavage of CYB5a , RAMP4 - 2 RAMP4 by SPPL2c SPP . Substrates proteases were co - expressed in HEK293 followed by Western blot as described above . Densitometric from at least n = 3 independent experiments was performed . Substrate were normalised compared to without protease - overexpression .

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Differential cleavage of CYB5a, RAMP4-2 and RAMP4 by SPPL2c and SPP. Substrates and proteases were co-expressed in HEK293 cells followed by Western blot analysis as described above. Densitometric quantification from at least n=3 independent experiments was performed. Substrate levels were normalised and compared to cells without protease-overexpression.


HEK293 expressing HA - RAMP4 - 2 alone or in combination with SPPL2c or SPP were treated with 100 µM ( Z - LL ) 2 - ketone ( ZLL ) , 5 µM inhibitor X ( InX ) , 5 µM DAPT or DMSO as control . RAMP4 - 2 band was quantified from blots of three independent experiments normalised to just over - expressing substrate .

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HEK293 cells expressing HA-RAMP4-2 alone or in combination with SPPL2c or SPP were treated with 100 µM (Z-LL)2-ketone (ZLL), 5 µM inhibitor X (InX), 5 µM DAPT or DMSO as control. RAMP4-2 band intensity was quantified from blots of three independent experiments and normalised to cells just over-expressing the substrate.


Microsomes isolated from testis of wild type SPPL2c - / - mice were solubilised in 1 % digitonin were separated by blue native PAGE . After transfer to PVDF , SPPL2c SPP were detected using polyclonal antisera introduced above .

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Microsomes isolated from testis of wild type and SPPL2c-/- mice were solubilised in 1% digitonin and proteins were separated by blue native PAGE. After transfer to PVDF membrane, SPPL2c and SPP were detected using the polyclonal antisera introduced above.


Histochemical visualization of β - galactosidase ( β - gal ) reporter activity revealed SPPL2c expression within seminiferous tubules . Cryo - sections from PFA - fixed wild type SPPL2c - / - testis were stained with X - Gal as β - gal substrate .

Histochemical visualization of β-galactosidase (β-gal) reporter activity revealed SPPL2c expression within seminiferous tubules. Cryo-sections from PFA-fixed wild type and SPPL2c-/- testis were stained with X-Gal as β-gal substrate.


SPPL2c were analysed in germ populations from testis of wild type mice sorted for their content which was determined by Hoechst 33342 as depicted in Fig EV4D . While for 4C , 2C 1C fractions 10 µg of were loaded , for testis control samples ( = total lysates ) 20 µg of were subjected to electrophoresis . * , non - specific band .

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SPPL2c protein levels were analysed in germ cell populations from testis of wild type mice sorted for their DNA content which was determined by Hoechst 33342 staining as depicted in Fig EV4D. While for 4C, 2C and 1C fractions 10 µg of protein were loaded, for the testis control samples (=total lysates) 20 µg of protein were subjected to electrophoresis. *, non-specific band.


Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin - fixed wild type SPPL2c - / - testis . SPPL2c antiserum directed against a C - terminal epitope of protease diaminobenzidine as peroxidase substrate were employed . Nuclei were stained with hematoxylin . Scale bar , 100 µm ( upper panel ) or 50 µm ( lower panel ) .

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Immunohistochemical detection of SPPL2c was performed on cryosections of Bouin-fixed wild type and SPPL2c-/- testis. The SPPL2c antiserum directed against a C-terminal epitope of the protease and diaminobenzidine as peroxidase substrate were employed. Nuclei were stained with hematoxylin. Scale bar, 100 µm (upper panel) or 50 µm (lower panel).


SPPL2c were compared in total lysates prepared from testis of wild type mice sacrificed at of 4 , 6 , 8 or 12 by Western blotting . A testis lysate from an adult SPPL2c - / - mouse was used as negative control .

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SPPL2c levels were compared in total lysates prepared from testis of wild type mice sacrificed at the age of 4, 6, 8 or 12 weeks by Western blotting. A testis lysate from an adult SPPL2c-/- mouse was used as negative control.


of both testicles of wild type SPPL2c - / - mice ( n = 12 per genotype ) was determined normalised to .

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The weight of both testicles of wild type and SPPL2c-/- mice (n=12 per genotype) was determined and normalised to the body weight.


Paraffin sections from Bouin - fixed wild type SPPL2c - / - testis were stained with hematoxylin & eosin ( H & E ) . Scale bars , 100 µm .

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Paraffin sections from Bouin-fixed wild type and SPPL2c-/- testis were stained with hematoxylin & eosin (H&E). Scale bars, 100 µm.


In H & E stained paraffin - sections from Bouin - fixed wild type SPPL2c - / - testis , mean number of elongated spermatids per seminiferous tubule was determined . were counted in 20 tubuli in sections from n = 6 mice per genotype .

In H&E stained paraffin-sections from Bouin-fixed wild type and SPPL2c-/- testis, the mean number of elongated spermatids per seminiferous tubule was determined. Cells were counted in 20 tubuli in sections from n=6 mice per genotype.


SPPL2c is not present in mature spermatozoa . Western blot of total lysates of wild type ( + / + ) SPPL2c - / - testis , epididymis spermatozoa recovered from cauda epididymidis / vas deferens for SPPL2c .

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SPPL2c is not present in mature spermatozoa. Western blot analysis of total lysates of wild type (+/+) and SPPL2c-/- testis, epididymis and spermatozoa recovered from the cauda epididymidis/vas deferens for SPPL2c.


Mature sperm were isolated from epididymis of wild type or SPPL2c - deficient mice analysed by phase - contrast microscopy . Representative pictures of morphologically normal spermatozoa are depicted . Scale bars , 10 µm .

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Mature sperm were isolated from the epididymis of wild type or SPPL2c-deficient mice and analysed by phase-contrast microscopy. Representative pictures of morphologically normal spermatozoa are depicted. Scale bars, 10 µm.


Reduced motility of SPPL2c - / - sperm . Spermatozoa were recovered from cauda epididymidis of wild type SPPL2c - / - mice ( n = 3 ) subjected to computer - assisted sperm . Average path velocity ( VAP ) , straight velocity ( VSL ) curvilinear velocity ( VCL ) were determined in micrometer per second .

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Reduced motility of SPPL2c-/- sperm cells. Spermatozoa were recovered from the cauda epididymidis of wild type and SPPL2c-/- mice (n=3) and subjected to computer-assisted sperm analysis. Average path velocity (VAP), straight line velocity (VSL) and curvilinear velocity (VCL) were determined in micrometer per second.


Mean litter sizes for different breeding schemes . data depicted are on n = 12 ( + / + X + / + ) , n = 57 ( + / - X + / - ) , n = 11 ( - / - X - / - ) , n = 10 ( - / - X + / + ) n = 11 ( + / + X - / - ) different breedings , respectively .

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Mean litter sizes for different breeding schemes. The data depicted are based on n=12 (+/+X+/+), n=57 (+/-X+/-), n=11 (-/-X-/-), n=10 (-/-X+/+) and n=11 (+/+X-/-) different breedings, respectively.


Carbonate - washed total fractions from wild type SPPL2c - / - testis ( n = 5 ) were subjected to a label - free quantitative proteome . For each quantified , a log2 between SPPL2c - deficient wild type samples was calculated . Negative values indicate a depletion positive values an enrichment in SPPL2c - / - samples . In depicted volcano plot negative log10 of p value ( y axis ) is plotted versus log2 of SPPL2c - / - versus wild type samples ( x axis ) . above significance level of p < 0 . 01 ( unpaired Student ' s t ) are coloured in red . hyperbolic curve indicates threshold for a permutation - FDR correction for multiple hypotheses with p = 0 . 05 s0 = 0 . 1 .

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Carbonate-washed total membrane fractions from wild type and SPPL2c-/- testis (n=5) were subjected to a label-free quantitative proteome analysis. For each quantified protein, a log2 intensity ratio between SPPL2c-deficient and wild type samples was calculated. Negative values indicate a depletion and positive values an enrichment in SPPL2c-/- samples. In the depicted volcano plot the negative log10 of the p value (y axis) is plotted versus the log2 intensity ratio of SPPL2c-/- versus wild type samples (x axis). All proteins above the significance level of p<0.01 (unpaired Student's t test) are coloured in red. The hyperbolic curve indicates the threshold for a permutation-based FDR correction for multiple hypotheses with p = 0.05 and s0 = 0.1.


HeLa were transiently transfected with N - terminally HA - tagged PLN ( HA - PLN ) inactive ( D / A ) SPPL2c - myc expressed were visualised by indirect immunofluorescence via their HA Myc epitopes .

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HeLa cells were transiently transfected with N-terminally HA-tagged PLN (HA-PLN) and inactive (D/A) SPPL2c-myc The expressed proteins were visualised by indirect immunofluorescence via their HA and Myc epitopes.


HEK293 were transiently transfected with HA - PLN alone or in combination with active or inactive ( D / A ) murine SPPL2c Isoform A ( D ) or Isoform B ( E ) . Substrate processing was analysed by Western blotting with anti - HA . SPPL2c expression was confirmed with antiserum against C - terminal epitope . Actin was detected to confirm equal loading .

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HEK293 cells were transiently transfected with HA-PLN alone or in combination with active or inactive (D/A) murine SPPL2c Isoform A (D) or Isoform B (E). Substrate processing was analysed by Western blotting with anti-HA. SPPL2c expression was confirmed with the antiserum against the C-terminal epitope. Actin was detected to confirm equal protein loading.


Subcellular localisation of PLN mutants was analysed by indirect immunofluorescence using anti - HA in combination with anti - disulphide isomerase A6 ( PDIA6 ) to stain ER . 37 - 41A 47 - 51 PLN mutants were not expressed in relevant amounts .

Subcellular localisation of the PLN mutants was analysed by indirect immunofluorescence using anti-HA in combination with anti-protein disulphide isomerase A6 (PDIA6) to stain the ER. The 37-41A and 47-51 PLN mutants were not expressed in relevant amounts.


HEK293 were transiently transfected with wild type or mutated HA - PLN alone or together with SPPL2c isoform A .

HEK293 cells were transiently transfected with wild type or mutated HA-PLN alone or together with SPPL2c isoform A.


of n = 5 experiments as shown in ( H ) . For each PLN variant , residual PLN in SPPL2c co - expressing is depicted as % of PLN in absence of protease .

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Quantification of n=5 experiments as shown in (H). For each PLN variant, the residual PLN in SPPL2c co-expressing cells is depicted as % of PLN in absence of the protease.


Accumulation of endogenous PLN in testis of SPPL2c - / - mice . PLN was immuno - precipitated ( IP ) from equal amounts of total lysates of wild type or SPPL2c - / - testis with mouse monoclonal antibody . Western blot of IPs was performed with rabbit monoclonal PLN antibody . SPPL2c actin were detected from total lysates directly .

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Accumulation of endogenous PLN in the testis of SPPL2c-/- mice. PLN was immuno-precipitated (IP) from equal protein amounts of total lysates of wild type or SPPL2c-/- testis with the mouse monoclonal antibody. Western blot analysis of the IPs was performed with the rabbit monoclonal PLN antibody. SPPL2c and actin were detected from total lysates directly.


of normalised PLN / Actin ratios from n = 3 biological replicates per genotype .

Quantification of normalised PLN/Actin ratios from n=3 biological replicates per genotype.


Expression of PLN in murine testis from wild type ( wt ) SPPL2c - / - ( 2c - / - ) was quantified by qPCR .

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Expression of PLN in murine testis from wild type (wt) SPPL2c-/- (2c-/-) was quantified by qPCR.


Immunohistochemical detection of PLN in paraformaldehyde ( PFA ) - fixed testis from wild type SPPL2c - / - mice . Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control diaminobenzidine as peroxidase substrate .

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Immunohistochemical detection of PLN in paraformaldehyde (PFA)-fixed testis from wild type and SPPL2c-/- mice. Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control and diaminobenzidine as peroxidase substrate.


Testis suspensions were stained with 2 . 5 µM DRAQ5 to determine amount . 1C - co = elongated spermatids , 1C = other haploid including round spermatids , 2C = spermatogonia , secondary spermatocytes , Sertoli , other somatic , 4C = spermatocytes , G2 spermatogonia .

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Testis suspensions were stained with 2.5 µM DRAQ5 to determine cellular DNA amount. 1C-co = elongated spermatids, 1C = other haploid cells including round spermatids, 2C = spermatogonia, secondary spermatocytes, Sertoli cells, other somatic cells, 4C = primary spermatocytes, G2 spermatogonia.


Intracellular Ca2 + concentrations were analysed in testis suspensions from either wild type or SPPL2c - deficient mice using Ca2 + - sensitive probe Fluo4 - AM . germ populations were identified Bars indicate Median Fluo4 - AM ± SD of 8 samples from 3 mice per genotype normalised to those of wild type samples .

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Intracellular Ca2+ concentrations were analysed in testis suspensions from either wild type or SPPL2c-deficient mice using the Ca2+-sensitive probe Fluo4-AM. Individual germ cell populations were identified Bars indicate Median Fluo4-AM fluorescence ± SD of 8 individual samples from 3 mice per genotype normalised to those of wild type samples.


Median forward scatter ( FSC ) was controlled in 1C - co population from datasets Median FSC ± SD of 8 samples from 3 mice per genotype .

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Median forward scatter (FSC) was controlled in the 1C-co population from the datasets Median FSC ± SD of 8 individual samples from 3 mice per genotype.


Expression of SPPL2c in human testis . ( H ) Western blot of lysates from HEK293 expressing human ( h ) SPPL2c , human murine testis using antisera against a C - terminal epitope of murine SPPL2c or human SPPL2c as indicated .

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Expression of SPPL2c in human testis. (H) Western blot analysis of lysates from HEK293 cells expressing human (h) SPPL2c, human and murine testis using antisera against a C-terminal epitope of murine SPPL2c or human SPPL2c as indicated.


Immunohistochemical detection of SPPL2c with monoclonal 1A1 antibody in paraffin - sections from Bouin ' s - fixed human testis . In addition , sections were incubated with control immunoglobulins . Arrows indicate elongated spermatids .

Immunohistochemical detection of SPPL2c with the monoclonal 1A1 antibody in paraffin-sections from Bouin's-fixed human testis. In addition, sections were incubated with control immunoglobulins. Arrows indicate elongated spermatids.


A intrinsic of NAD ( P ) H co - localizes extensively with mitochondria ( marked by Mitotracker CMXRos )

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A The intrinsic fluorescence of NAD(P)H co-localizes extensively with mitochondria (marked by Mitotracker CMXRos)


B 2P - FLIM detects fraction of NAD ( P ) H bound to enzyme partners ( a2 % )

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B 2P-FLIM detects the fraction of NAD(P)H bound to enzyme partners (a2%)


a2 % values ( starved , after insulin or R + L addition ) were recorded pixel by pixel for mouse cortical ( C neurons . Each colored in center panel graphs refers to a single field of view containing 2 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view before after stimulation , respectively . Using same raw data , relative contributions of NADH NADPH to total a2 % values were calculated ( Blacker et al , 2014 ) are shown in right panel bar graphs . NADH NAD ( P ) H contributed an average of 63 % 37 % , respectively , of total a2 % values . statistical analyses were performed using Student ' s two - unpaired t -

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a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel for mouse cortical (C neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests


D a2 % values ( starved , after insulin or R + L addition ) were recorded pixel by pixel fo human ( D ) neurons . Each colored in center panel graphs refers to a single field of view containing 2 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view before after stimulation , respectively . Using same raw data , relative contributions of NADH NADPH to total a2 % values were calculated ( Blacker et al , 2014 ) are shown in right panel bar graphs . NADH NAD ( P ) H contributed an average of 63 % 37 % , respectively , of total a2 % values . statistical analyses were performed using Student ' s two - unpaired t -

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D a2% values (starved, and after insulin or R+L addition) were recorded pixel by pixel fo human (D) neurons. Each colored line in the center panel graphs refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view before and after stimulation, respectively. Using the same raw data, the relative contributions of NADH and NADPH to the total a2% values were calculated (Blacker et al, 2014) and are shown in the right panel bar graphs. NADH and NAD(P)H contributed an average of 63% and 37%, respectively, of the total a2% values. All statistical analyses were performed using Student's two-tailed unpaired t-tests


Wild type ( WT ) mouse cortical neurons cultured for 10 days in vitro ( A were serum - starved in Hank ' s balanced salt solution for 2 hours . Next , were imaged immediately ( starved ) , after which either ( R + L ) or insulin were added . Thirty minutes later were imaged again . Finally , mTOR inhibitor , Torin1 was added , after an additional 30 minutes , were imaged for last time . Histograms represent changes in fraction of enzyme - bound NAD ( P ) H ( a2 % ) for each condition . Note that insulin significantly increased a2 % values , which was reversed by Torin1 - mediated inhibition of mTORC1 . Average data from 5 fields of view per condition are shown . Statistical analyses were performed using Student ' s two - unpaired t -

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Wild type (WT) mouse cortical neurons cultured for 10 days in vitro (A were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test


human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor ( B ) were serum - starved in Hank ' s balanced salt solution for 2 hours . Next , were imaged immediately ( starved ) , after which either ( R + L ) or insulin were added . Thirty minutes later were imaged again . Finally , mTOR inhibitor , Torin1 was added , after an additional 30 minutes , were imaged for last time . Histograms represent changes in fraction of enzyme - bound NAD ( P ) H ( a2 % ) for each condition . Note that insulin significantly increased a2 % values , which was reversed by Torin1 - mediated inhibition of mTORC1 . Average data from 5 fields of view per condition are shown . Statistical analyses were performed using Student ' s two - unpaired t -

human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells (B) were serum-starved in Hank's balanced salt solution for 2 hours. Next, the cells were imaged immediately (starved), after which either amino acids (R+L) or insulin were added. Thirty minutes later the cells were imaged again. Finally, the mTOR inhibitor, Torin1 was added, and after an additional 30 minutes, the cells were imaged for the last time. Histograms represent changes in the fraction of enzyme-bound NAD(P)H (a2%) for each condition. Note that insulin and amino acids significantly increased the a2% values, which was reversed by Torin1-mediated inhibition of mTORC1. Average data from 5 fields of view per condition are shown. Statistical analyses were performed using Student's two-tailed unpaired t-test


C Raptor - depleted , WT cortical neurons were imaged by 2P - FLIM before 30 minutes after treatment with insulin . Inhibiting mTORC1 by depleting Raptor with shRNA made mitochondria insensitive to insulin . Shown here are average data from 4 fields of view of a single replicate out of a total of 3 replicates . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - unpaired t -

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C Raptor-depleted, WT cortical neurons were imaged by 2P-FLIM before and 30 minutes after treatment with insulin. Inhibiting mTORC1 by depleting Raptor with shRNA made mitochondria insensitive to insulin. Shown here are average data from 4 fields of view of a single replicate out of a total of 3 replicates. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor were serum - starved in Hank ' s balanced salt solution ( HBSS ) for 2 hours . Next , ( R + L ) , insulin , insulin + R + L , or oxidative phosphorylation inhibitor , NaN3 , were added , or HBSS was replaced with complete medium . One hour later reactive oxygen species ( ROS ) , ATP oxygen consumption were measured . data were collected from 3 independent assays in which each experimental condition contained 6 - 8 replicates . Error bars represent ± s . e . m . Statistical analyses were performed using Student ' s two - unpaired t -

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A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitor cells were serum-starved in Hank's balanced salt solution (HBSS) for 2 hours. Next, amino acids (R+L), insulin, insulin + R+L, or the oxidative phosphorylation inhibitor, NaN3, were added, or the HBSS was replaced with complete medium. One hour later cellular reactive oxygen species (ROS), ATP levels and oxygen consumption were measured. The data were collected from 3 independent assays in which each experimental condition contained 6-8 replicates. Error bars represent ± s.e.m. Statistical analyses were performed using Student's two-tailed unpaired t-test


B MP - PAM imaging of wild type ( WT ) mouse cerebral cortex through an open - skull window 1 hour after topical application of ( R + L ) . A decrease in blood oxygenation of cortical vasculature was observed , indicating elevated oxygen extraction consumption due to upregulation of mitochondrial activity . Data were obtained from 3 mice , each of which was measured once for O2 saturation , oxygen extraction fraction ( OEF ) cerebral metabolic rate of oxygen ( CMRO2 ) . Error bars represent ± s . e .

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B MP-PAM imaging of wild type (WT) mouse cerebral cortex through an open-skull window 1 hour after topical application of amino acids (R+L). A decrease in blood oxygenation of the cortical vasculature was observed, indicating elevated oxygen extraction and consumption due to upregulation of mitochondrial activity. Data were obtained from 3 mice , each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Error bars represent ± s.e.m


C MP - PAM imaging of WT mouse cerebral cortex through an open - skull window beginning at baseline , which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin . Baseline of O2 saturation blood flow speed were recorded , after which R + L was applied to open skull window . One hour later , O2 saturation blood flow speed were recorded again . Data were obtained from 4 mice , each of which was measured once for O2 saturation , oxygen extraction fraction ( OEF ) cerebral metabolic rate of oxygen ( CMRO2 ) . Note that no significant changes in O2 saturation or OEF were observed after R + L stimulation , in contrast to what was observed in absence of rapamycin ( Fig 3B ) . Error bars represent ± s . e .

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C MP-PAM imaging of WT mouse cerebral cortex through an open-skull window beginning at baseline, which corresponded to 30 minutes after suppression of mTORC1 activity by a single topical application of 1 µM rapamycin. Baseline levels of O2 saturation and blood flow speed were recorded, after which R+L was applied to the open skull window. One hour later, O2 saturation and blood flow speed were recorded again. Data were obtained from 4 mice, each of which was measured once for O2 saturation, oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2). Note that no significant changes in O2 saturation or OEF were observed after R+L stimulation, in contrast to what was observed in the absence of rapamycin (Fig 3B). Error bars represent ± s.e.m


A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitors ( Control ) , or otherwise identical expressing Flag - Raptor fused to either plasma targeting signal of H - Ras or lysosomal targeting signal of Rheb were serum - starved in Hank ' s balanced salt solution for 2 hours , then imaged by 2P - FLIM before 30 minutes after addition of insulin . Each colored refers to a single field of view containing 5 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view . Statistical analyses were performed using Student ' s two - unpaired t - ( ns : not significant ) . Expression of Flag - Raptor fusion was confirmed by western blotting ( right panel ) . Note that NiMA was supported when mTORC1 was targeted to lysosomes ( Flag - Raptor Rheb15 ) , but not to plasma ( Flag - Raptor H - Ras25 ) . Western blots are representative of 3 independent

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A Human neurons differentiated in culture for 30 days from ReNcell VM neuronal progenitors (Control), or otherwise identical cells expressing Flag-Raptor fused to either the plasma membrane targeting signal of H-Ras or the lysosomal targeting signal of Rheb were serum-starved in Hank's balanced salt solution for 2 hours, and then imaged by 2P-FLIM before and 30 minutes after addition of insulin. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test (ns: not significant). Expression of the Flag-Raptor fusion proteins was confirmed by western blotting (right panel). Note that NiMA was supported when mTORC1 was targeted to lysosomes (Flag-Raptor Rheb15), but not to the plasma membrane (Flag-Raptor H-Ras25). Western blots are representative of 3 independent assays


B Mouse cortical neurons starved in Hank ' s balanced salt solution for 2 hours were treated with AβOs , insulin or ( R + L ) for 30 minutes , then were fixed double labeled with anti - mTOR , either anti - LAMP1 or Mitotracker CMXRos . Error bars represent ± s . e . m . Data are representative of 3 independent assays

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B Mouse cortical neurons starved in Hank's balanced salt solution for 2 hours were treated with AβOs, insulin or amino acids (R+L) for 30 minutes, and then were fixed and double labeled with anti-mTOR, and either anti-LAMP1 or Mitotracker CMXRos. Error bars represent ± s.e.m. Data are representative of 3 independent assays


C Reducing Bcl - xL expression in WT mouse cortical neurons does not affect NiMA . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - unpaired t -

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C Reducing Bcl-xL expression in WT mouse cortical neurons does not affect NiMA. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


A replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3 - hour of thymidine analog , EdU . were labeled additionally with mitochondrial marker , Mitotracker CMXRos , with an antibody to neuron marker ,

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A DNA replication in mitochondrial replisomes was detected in mouse cortical neurons after a 3-hour pulse of the thymidine analog, EdU. Cells were labeled additionally with the mitochondrial marker, Mitotracker CMXRos, and with an antibody to the neuron marker, MAP2


B of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit mtDNA replication by ~ 60 % , an effect that was blocked by AβOs . Error bars represent ± s . e . m . Statistical analyses were performed using Student " s two - unpaired t - are representative of 3 independent

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B Quantification of EdU uptake into mouse cortical neuron replisomes showed that nutrients inhibit mtDNA replication by ~60%, an effect that was blocked by AβOs. Error bars represent ± s.e.m. Statistical analyses were performed using Student"s two-tailed unpaired t-test and are representative of 3 independent a


C WT mouse cortical neurons expressing either Flag - Raptor H - Ras25 or Flag - Raptor Rheb15 were serum - starved in AβO - containing Hank " s balanced salt solution for 2 hours . Next , were - labeled for 3 hours with EdU . Note that forcing mTORC1 to plasma ( Flag - Raptor H - Ras25 ) yielded an ~ 80 % increase in EdU incorporation into mtDNA in presence of AβOs regardless of whether or not neurons had been stimulated with insulin + R + L . In contrast , EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes ( Flag - Raptor - Rheb15 ) . Error bars represent ±

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C WT mouse cortical neurons expressing either Flag-Raptor H-Ras25 or Flag-Raptor Rheb15 were serum-starved in AβO-containing Hank"s balanced salt solution for 2 hours. Next, the cells were pulse-labeled for 3 hours with EdU. Note that forcing mTORC1 to the plasma membrane (Flag-Raptor H-Ras25) yielded an ~80% increase in EdU incorporation into mtDNA in the presence of AβOs regardless of whether or not the neurons had been stimulated with insulin+R+L. In contrast, EdU uptake into mitochondrial replisomes was not stimulated by AβOs in neurons whose mTORC1 was forced to lysosomes (Flag-Raptor-Rheb15). Error bars represent ±


A Unmodified ReNcell VM human neuronal progenitor were co - plated with comparable Ren - GFP or Ren - APPSL into 3 - dimensional Matrigel matrices , differentiated into neurons for 60 days . GFP is produced by both Ren - GFP Ren - APPSL , latter also produce pathogenic human APP with Swedish London mutations , secrete Aβ that becomes entrapped in Matrige Following neuronal differentiation , 2P - FLIM was used to monitor NiMA in ReNcell VM neurons , which were discriminated from Ren - GFP Ren - APPSL neurons because they lack GFP . Note that NiMA was observed in ReNcell VM neurons when they were co - cultured with Ren - GFP neurons , but not when they were co - cultured with Ren - APPSL neurons . Each colored refers to a single field of view containing 5 - 15 , each pair of solid dotted lines of same color refers to same field of view . Statistical analyses were performed using Student ' s two - unpaired t -

A Unmodified ReNcell VM human neuronal progenitor cells were co-plated with comparable Ren-GFP or Ren-APPSL cells into 3-dimensional Matrigel matrices, and differentiated into neurons for 60 days. Cytosolic GFP is produced by both Ren-GFP and Ren-APPSL cells, and the latter also produce pathogenic human APP with the Swedish and London mutations, and secrete Aβ that becomes entrapped in the Matrige Following neuronal differentiation, 2P-FLIM was used to monitor NiMA in the ReNcell VM neurons, which were discriminated from Ren-GFP and Ren-APPSL neurons because they lack GFP. Note that NiMA was observed in ReNcell VM neurons when they were co-cultured with Ren-GFP neurons, but not when they were co-cultured with Ren-APPSL neurons. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


B ReNcell VM were differentiated into neurons for 30 days in 2D cultures . Subsequent AβO exposure for 16 hours blocked NiMA , but expression of Flag - Raptor - Rheb15 , which forces mTORC1 to lysosomes , prevented NiMA inhibition by AβOs . Each colored refers to a single field of view containing 5 - 15 , each identically colored pair of solid dotted lines refers to same field of view . Flag - Raptor - Rheb15 expression was confirmed by western blot ting . Statistical analyses were performed using Student ' s two - unpaired t - . Western blots are representative of 3 independent

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B ReNcell VM cells were differentiated into neurons for 30 days in 2D cultures. Subsequent AβO exposure for 16 hours blocked NiMA, but expression of Flag-Raptor-Rheb15, which forces mTORC1 to lysosomes, prevented NiMA inhibition by AβOs. Each colored line refers to a single field of view containing 5-15 cells, and each identically colored pair of solid and dotted lines refers to the same field of view. Flag-Raptor -Rheb15 expression was confirmed by western blotting. Statistical analyses were performed using Student's two-tailed unpaired t-test. Western blots are representative of 3 independent assays


A Cortical mouse neurons from tau knockout ( KO ) mice were serum - starved in Hank ' s balanced salt solution for 2 hours . were then imaged for NiMA , after which insulin or were immediately added , were then imaged again 30 minutes later Data information : Each colored lin refers to a single field of view containing 2 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view . Statistical analyses were performed using Student ' s two - unpaired t -

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A Cortical mouse neurons from tau knockout (KO) mice were serum-starved in Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin or amino acids were immediately added, and the cells were then imaged again 30 minutes later Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum - starved in AβO - containing Hank ' s balanced salt solution for 2 hours . were then imaged for NiMA , after which insulin was immediately added , were then imaged again 30 minutes later . Note that NiMA occurred in absence or presence of AβOs , which inhibited NiMA in wild type human neuron Data information : Each colored lin refers to a single field of view containing 2 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view . Statistical analyses were performed using Student ' s two - unpaired t -

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B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum-starved in AβO-containing Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin was immediately added, and the cells were then imaged again 30 minutes later. Note that NiMA occurred in the absence or presence of AβOs, which inhibited NiMA in wild type human neuron Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test


Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to ( R + L Each colored refers to a single field of view containing 5 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view . Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 , a downstream target of mTORC1 - S6K pathway . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - unpaired t -

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Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids (R+L Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


Wild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored refers to a single field of view containing 5 - 15 , each pair of solid dotted lines ( same color ) refers to same field of view . Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 , a downstream target of mTORC1 - S6K pathway . Western blots are representative of 3 independent assays . Statistical analyses were performed using Student ' s two - unpaired t -

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Wild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test


( D Human fibroblasts deficient in Tsc2 ( TSC2 Δexons1 - 14 ) were imaged before 30 minutes after insulin addition . Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum - starved in Hank ' s balanced salt solution for 2 hours before stimulation with insulin or , were imaged before 30 minutes after stimulation . Each colored refers to a single field of view containing 2 - 5 , each pair of solid dotted lines of same color refers to same field of view Statistical analyses were performed using Student ' s two - unpaired t -

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(D Human fibroblasts deficient in Tsc2 (TSC2 Δexons1-14) were imaged before and 30 minutes after insulin addition. Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view Statistical analyses were performed using Student's two-tailed unpaired t-test


( E ) Re - expression of human Tsc2 rescues mitochondrial responses to insulin . Cultures were serum - starved in Hank ' s balanced salt solution for 2 hours before stimulation with insulin or , were imaged before 30 minutes after stimulation . Each colored refers to a single field of view containing 2 - 5 , each pair of solid dotted lines of same color refers to same field of view . Re - expression of Flag - WT Tsc2 was corroborated by indirect immun o ( IF ) . Statistical analyses were performed using Student ' s two - unpaired t -

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(E) Re-expression of human Tsc2 rescues mitochondrial responses to insulin. Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Re-expression of the Flag-WT Tsc2 was corroborated by indirect immunofluorescence (IF). Statistical analyses were performed using Student's two-tailed unpaired t-test


C Sequences of synthetic PrP alleles differing in octarepeat region of mousePrP . Conserved histidines in octarepeats 2 - 5 are coloured blue missense mutations are shown in red . Overproduction of C2 fragment is indicated by + + signs to right of panel .

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C Sequences of synthetic PrP alleles differing in the octarepeat region of mousePrP. Conserved histidines in octarepeats 2-5 are coloured blue and missense mutations are shown in red. Overproduction of C2 fragment is indicated by ++ signs to the right of the panel.


A Lysates from RK13 transfected with WT , S1 , S3 S3 . F88W PrP were probed with antibody Sha31 , 9A2 12B2 after digestion with PNGaseF .

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A Lysates from RK13 cells transfected with WT, S1, S3 and S3.F88W PrP were probed with antibody Sha31, 9A2 and 12B2 after digestion with PNGaseF.


B , C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 lysates . A schematic of different lengths of C2 caused by single mutations in OR is shown in lower portion of ( C ) .

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B, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).


D Lysates of N2a , HEK , SMB - PS SH - SY5Y not transfected ( − ) transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF analysed by Western blot using Sha31 .

D Lysates of N2a, HEK, SMB-PS and SH-SY5Y cells not transfected (−) and transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF and analysed by Western blot using Sha31.


E of lysates from RK13 transfected with plasmids incorporating Phe substitutions into hydrophobic domain of WT PrP , S1 PrP S3 PrP . Lysates were PNGaseF - digested before Western blot using Sha31 antibody . F1 : A114F , F2 : G118F , F3 : G122F , F4 :

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Analysis of lysates from RK13 cells transfected with plasmids incorporating Phe substitutions into the hydrophobic domain of WT PrP, S1 PrP and S3 PrP. Lysates were PNGaseF-digested before Western blot using the Sha31 antibody. F1: A114F, F2: G118F, F3: G122F, F4: G126F


A Brain homogenates of TgPrP ( WT ) , TgPrP ( S1 ) - 17 TgPrP ( S3 . F88W ) - 35 mice were analysed by Western blot before after PNGaseF digestion using PrP antibodies Sha31 , 12B2 9A2 . Prnp0 / 0 , WT brain homogenates were used as controls .

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A Brain homogenates of TgPrP(WT), TgPrP(S1)-17 and TgPrP(S3.F88W)-35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31, 12B2 and 9A2. Prnp0/0, and WT brain homogenates were used as controls.


B of C2 to full - PrP in TgPrP ( S3 . F88W ) - 35 WT mice ( left panel , n = 7 , P = 1 . 46E - 06 ) in RK13 expressing WT S3 PrP ( right panel , n = 5 , P = 4 . 01E - 05 ) is shown . Values are presented as mean ± . Unpaired , two - t - , * * * * P < 0 . 0001 .

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B The ratio of C2 to full-length PrP in TgPrP(S3.F88W)-35 and WT mice (left panel, n = 7, P = 1.46E-06) and in RK13 cells expressing WT and S3 PrP (right panel, n = 5, P = 4.01E-05) is shown. Values are presented as mean ± SEM. Unpaired, two-tailed t-test, ****P < 0.0001.


C Different tissue homogenates from a TgPrP ( S3 . F88W ) - 35 mouse were PNGaseF - digested analysed for PrP using antibody 1A6 . FL , full - PrP ; ns , non - specific signal detected in immunoblotting procedure .

C Different tissue homogenates from a TgPrP(S3.F88W)-35 mouse were PNGaseF-digested and analysed for PrP using the antibody 1A6. FL, full-length PrP; ns, non-specific signal detected in the immunoblotting procedure.


A Sections of sciatic nerve from TgPrP ( S3 . F88W ) - 14 mice ( 610 days old ) TgPrP ( S3 . F88W ) - 35 mice ( 614 , 570 614 days old , respectively ) were analysed using toluidine blue ( upper row ) , with third panels for each showing a hypermyelinated phenotype . Scale bar in left panel represents 50 μm . lower rows show EM analyses of mice of indicated genotypes . Scale bar is 10 μm for panels 1 - 4 , with fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3 . F88W PrP ( scale bar , 5 μm ) .

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A Sections of sciatic nerve from TgPrP(S3.F88W)-14 female mice (all 610 days old) and TgPrP(S3.F88W)-35 female mice (614, 570 and 614 days old, respectively) were analysed using toluidine blue staining (upper row), with the third panels for each line showing a hypermyelinated phenotype. Scale bar in left panel represents 50 μm. The lower rows show EM analyses of mice of the indicated genotypes. Scale bar is 10 μm for panels 1-4, with the fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3.F88W PrP (scale bar, 5 μm).


B Western blot of sciatic nerve extracts ( 10 μg ) from mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain ( see Fig3 ) . asterisk indicates a greater than full - fragment present in TgPrP ( S3 . F88W ) mice , possibly corresponding to incomplete removal of N - terminal signal peptide .

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B Western blot analysis of sciatic nerve protein extracts (10 μg) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain (see Fig3). The asterisk indicates a greater than full-length fragment present in TgPrP(S3.F88W) mice, possibly corresponding to incomplete removal of the N-terminal signal peptide.


C of fibre in aged ( 492 - 608 days old ) , representing of nerve occupied by fibres versus total cross - sectional of nerve . S1 S3 . F88W alleles rescue Prnp0 / 0 nerves to a similar as WT allele , but variance P - value is greater for two Tg lines expressing TgPrP ( S3 . F88W ) allele . Unpaired , two - t - , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Sample sizes exact P - values compared with Prnp0 / 0 ( n = 9 ) were as follows : TgPrP ( WT ) n = 6 , P = 3 . 63E - 05 ; Prnp0 / 0 , n = 9 ; TgPrP ( S1 ) - 17 n = 5 , P = 3 . 54E - 05 ; TgPrP ( S1 ) - 19 n = 3 , P = 6 . 35E - 06 ; TgPrP ( S3 . F88W ) - 14 n = 3 , P = 3 . 70E - 03 ; TgPrP ( S3 . F88W ) - 35 n = 7 , P = 7 . 18E - 03 .

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C Quantification of fibre morphology in aged animals (492-608 days old), representing the percentage of the nerve occupied by fibres versus the total cross-sectional area of the nerve. The S1 and S3.F88W alleles rescue Prnp0/0 nerves to a similar percentage as the WT allele, but the variance and P-value is greater for the two Tg lines expressing the TgPrP(S3.F88W) allele. Unpaired, two-tailed t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Sample sizes and exact P-values compared with Prnp0/0 (n = 9) were as follows: TgPrP(WT) n = 6, P = 3.63E-05; Prnp0/0, n = 9; TgPrP(S1)-17 n = 5, P = 3.54E-05; TgPrP(S1)-19 n = 3, P = 6.35E-06; TgPrP(S3.F88W)-14 n = 3, P = 3.70E-03; and TgPrP(S3.F88W)-35 n = 7, P = 7.18E-03.


A , B Infected brain homogenates were PK - digested analysed by Western blot using ( A ) Sha31 antibody ( B ) after PK PNGaseF digestion using Sha31 .

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A, B Infected brain homogenates were PK-digested and analysed by Western blot using (A) Sha31 antibody and (B) after PK and PNGaseF digestion using Sha31.


C Conformational stability on RML - infected brain homogenates incubated with increasing concentrations of GdnHCl . best - fit alignments of data are presented . Genotypes are noted on panels . Source data are available online for this figure .

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C Conformational stability assay on RML-infected brain homogenates incubated with increasing concentrations of GdnHCl. The best-fit alignments of the data are presented. Genotypes are noted on the individual panels.Source data are available online for this figure.


Pathology of RML - infected miceA - H pattern of PrPSc deposition ( A - D ) GFAP immunostaining ( E - H ) is presented . I - P A higher power view of H & amp ; amp ; E stained hippocampus ( I - L ) thalamus ( M - P ) to reveal vacuolation is presented . Data information : of same genotype are shown within a column , incubation times of presented with RML prion isolate are shown ( days ) . Mice expressing PrP S1 S3 alleles ( central columns ) exhibit attenuated PrPSc deposition , gliosis spongy change . Pathological features of RML isolate are not altered upon sequential passage through TgPrP ( S3 . 88W ) - 35 mice ( see schematic at bottom of diagram indicating sequential passages finally , passage back into WT mice , compare data in first fourth columns ) .

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Pathology of RML-infected miceA-H The pattern of PrPSc deposition (A-D) and GFAP immunostaining (E-H) is presented.I-P A higher power view of H&amp;E stained hippocampus (I-L) and thalamus (M-P) to reveal vacuolation is presented.Data information: Animals of the same genotype are shown within a column, and incubation times of the presented animals with the RML prion isolate are shown (days). Mice expressing PrP S1 and S3 alleles (central columns) exhibit attenuated PrPSc deposition, gliosis and spongy change. Pathological features of the RML isolate are not altered upon sequential passage through TgPrP(S3.88W)-35 mice (see schematic at bottom of the diagram indicating sequential passages and finally, passage back into WT mice, compare the data in first and fourth columns).


A Spot in scrapie using WT , TgPrP ( S1 ) - 17 TgPrP ( S3 . F88W ) - 14 brain homogenates ( 0 . 1 % or 0 . 01 % ) from mice infected with RML are shown in upper panel ( WT vs S1 P = 2 . 09E - 05 vs S3 P = 4 . 62E - 08 ) with numbers corrected for PrPC expression as per Table1 presented in lower panel ( WT vs S1 P = 1 . 04E - 13 vs S3 P = 3 . 39E - 20 ; two - t - , n = 3 ) .

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A Spot counts in the scrapie cell assay using WT, TgPrP(S1)-17 and TgPrP(S3.F88W)-14 brain homogenates (0.1% or 0.01%) from mice infected with RML are shown in the upper panel (WT vs S1 P = 2.09E-05 and vs S3 P = 4.62E-08) with numbers corrected for PrPC expression levels as per Table1 presented in the lower panel (WT vs S1 P = 1.04E-13 and vs S3 P = 3.39E-20; two-tailed t-test, n = 3).


B A comparison of incubation time vs PrPC : PrPSc for mice expressing different of WT PrP ( WT , TgPrP ( WT ) , tga20 ) , S1 PrP ( TgPrP ( S1 ) - 17 , TgPrP ( S1 ) - 19 , TgPrP ( S1 ) - 39 ) S3 PrP ( TgPrP ( S3 . F88W ) - 14 , TgPrP ( S3 . F88W ) - 35 ) .

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B A comparison of incubation time vs PrPC:PrPSc ratio for mice expressing different levels of WT PrP (WT, TgPrP(WT), tga20), S1 PrP (TgPrP(S1)-17, TgPrP(S1)-19, TgPrP(S1)-39) and S3 PrP (TgPrP(S3.F88W)-14, TgPrP(S3.F88W)-35).


B ) Co - immunoprecipitation to elucidate interaction between Suv39h1 HP1 . Whole - extracts of HEK293T expressing 3xFlag - Suv39h1 or 3xFlag - ΔN - Suv39h1 were subjected to immunoprecipitation with anti - Flag tag antibody followed by immunoblotting with indicated antibodies . IgG represents heavy or

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B) Co-immunoprecipitation analysis to elucidate the interaction between Suv39h1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Suv39h1 or 3xFlag-ΔN-Suv39h1 were subjected to immunoprecipitation with the anti-Flag tag antibody followed by immunoblotting with the indicated antibodies. IgG represents the heavy or light chain


F ) Co - immunoprecipitation to elucidate interaction between Setdb1 HP1 . Whole - extracts of HEK293T expressing 3xFlag - Setdb1 or 3xFlag - Setdb1 mutants were subjected to immunoprecipitation with anti - HP1β antibody immunocomplex was analyzed by immunoblotting with indicated antibodies .

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F) Co-immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Setdb1 or 3xFlag-Setdb1 mutants were subjected to immunoprecipitation with the anti-HP1β antibody The immunocomplex was analyzed by immunoblotting with the indicated antibodies.


G ) decay of HP1 interaction - defective Setdb1 mutant . mESC lines stably expressing 3xFlag - Setdb1 or 3xFlag - Setdb1 - 3A were treated with CHX MG132 for indicated times . Stacked bar plots show summarized amounts of Setdb1 ( right ) . Data represented as mean ± SD ( n = 4 , biological replicates ) . * * p < 0 . 01 ( Tukey ' s honestly significant difference ) .

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G) Protein decay analysis of HP1 interaction-defective Setdb1 mutant. mESC lines stably expressing 3xFlag-Setdb1 or 3xFlag-Setdb1-3A were treated with CHX and MG132 for the indicated times. Stacked bar plots show the summarized protein amounts of Setdb1 (right). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test).


B ) expression of H3K9 MTs histone modification in HP1 - cTKO were monitored by immunoblotting . intensities were quantified using ImageJ software summarized on right . thin gray bold colored show data means of experimental replicates ( n ≥ 3 , biological replicates ) , respectively .

B) Protein expression levels of H3K9 MTs and histone modification levels in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and means of the experimental replicates (n ≥ 3, biological replicates), respectively.


C ) Comparison of H3K9me3 between HP1 - deficient Suv39h / Setdb1 - deficient . H3K9me were quantified using ImageJ software are summarized at bottom ; horizontal represents mean of each . More than 75 nuclei were counted for each . * * p < 0 . 01 ( Tukey ' s honestly significant difference ) .

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C) Comparison of H3K9me3 levels between HP1-deficient cells and Suv39h/Setdb1-deficient cells. H3K9me levels were quantified using ImageJ software and are summarized at the bottom; the horizontal line represents the mean intensity of each cell. More than 75 nuclei were counted for each staining. **p < 0.01 (Tukey's honestly significant difference test).


D ) Electron microscopy of HP1 - cTKO in absence of 4OHT ( left ) of treated with 4OHT for 4 days ( right ) . Boxes represent enlarged .

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D) Electron microscopy images of HP1-cTKO cells in the absence of 4OHT (left) and of cells treated with 4OHT for 4 days (right). Boxes represent enlarged images.


B ) Subcellular distribution of H3K9 MTs in HP1 - cTKO was examined by immunoblotting . α - Tubulin , Ezh2 , histone H3 were used as representative markers for each fraction . Stacked bar plots show abundance ratios of H3K9 MTs among three fractions ( right ) .

B) Subcellular distribution of H3K9 MTs in the HP1-cTKO cells was examined by immunoblotting. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction. Stacked bar plots show the abundance ratios of H3K9 MTs among the three fractions (right).


B , Elucidation of - interaction between HP1 H3K9 MTs by co - immunoprecipitation . Whole - extracts of HEK293T co - expressing HA - HP1β mutants epitope - tagged of interest were subjected to immunoprecipitation with anti - HA tag antibody . immunocomplex was analyzed by immunoblotting with indicated antibodies . intensities were quantified using ImageJ software , normalized , shown on top .

B, Elucidation of protein-protein interaction between HP1 and H3K9 MTs by co-immunoprecipitation analysis. Whole-cell extracts of HEK293T cells co-expressing HA-HP1β mutants and epitope-tagged proteins of interest were subjected to immunoprecipitation with the anti-HA tag antibody. The immunocomplex was analyzed by immunoblotting with the indicated antibodies. The intensities were quantified using ImageJ software, normalized, and shown on the top.


E ) Evaluation of HP1β mutants for maintaining H3K9me2 / 3 ensuring stability of H3K9 MTs . H3K9me2 / 3 of H3K9 MTs in HP1 - cTKO lines expressing 3xFlag - hHP1β mutants were compared before after 4OHT treatment . intensities were quantified with ImageJ software are summarized on right . Data represented as mean ± SD ( n > 3 , biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Welch ' s ) .

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E) Evaluation of HP1β mutants for maintaining H3K9me2/3 and ensuring protein stability of H3K9 MTs. H3K9me2/3 levels and protein levels of H3K9 MTs in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).


B ) expression of Jmjd1a / b in HP1 - cTKO were monitored by immunoblotting . intensities were quantified using ImageJ software summarized on right . thin gray bold colored show data mean of experimental replicates , respectively .

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B) Protein expression levels of Jmjd1a/b in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and the mean of experimental replicates, respectively.


C ) Evaluation of HP1β mutants for ensuring stability of Jmjd1a / b . of Jmjd1a / b in HP1 - cTKO lines expressing 3xFlag - hHP1β mutants were compared before after 4OHT treatment . intensities were quantified with ImageJ software are summarized on right . Data represented as mean ± SD ( n > 3 , biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Welch ' s ) .

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C) Evaluation of HP1β mutants for ensuring protein stability of Jmjd1a/b. Protein levels of Jmjd1a/b in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).


C ) Impact of Suv39h / Setdb1 / G9a depletion on HP1 - mediated stability regulation of Jmjd1a / b . Jmjd1a / b were destabilized in mESCs lacking Suv39h / Setdb1 / G9a , even though HP1 was present .

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C) Impact of Suv39h/Setdb1/G9a depletion on HP1-mediated protein stability regulation of Jmjd1a/b. Jmjd1a/b were destabilized in mESCs lacking Suv39h/Setdb1/G9a, even though HP1 was present.


D ) Functional evaluation of H3K9me2 / 3 for confining HP1 to . Subcellular localizations of HP1α , β γ in TT2 mESCs lacking Suv39h / Setdb1 / G9a were compared . α - Tubulin , Ezh2 , histone H3 were used as representative markers for each fraction .

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D) Functional evaluation of H3K9me2/3 for confining HP1 to chromatin. Subcellular localizations of HP1α, β and γ in TT2 and mESCs lacking Suv39h/Setdb1/G9a were compared. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction.


Growth suppression in cancer after 72h of treatment with APR - 246 + / - MRP1 inhibitor MK - 571 as shown by WST - 1 ( n ≥ 3 , n shown in Table S1 ) . Cross in 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 after 72h of with APR - 246 + / - MK - 571 ( n = 3 ) .

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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 lines . * p < 0 . 0001 , Wilcoxon . Each dot represents one . Mean IC50 values n shown in Table S1 . ZIP synergy scores of most synergistic sub - grouped according to TP53 gene status for 21 lines . Each dot represents one . Scores n are shown in Table S1 .

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

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 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 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 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 ) R248W ( 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 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 of MRP1 ( Signaling . ) , p53 ( DO - 1 ) GAPDH of HCT116 WT R248W 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

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 ) of HCT116 WT R248W transfected with MRP1 siRNA ( n ≥ 5 n ≥ 2 respectively ) after 48h APR - 246 treatment . Indicated values are average of four siRNAs targeting MRP1 two negative control siRNAs . Data n of 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 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

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

O O O O O O O O O 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 72h after transfection with empty vector (EV) and MRP1 expression vector.


Growth suppression ( WST - 1 ) of HCT116 WT R248W transfected with empty vector ( EV ) 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 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 MK - 571 for 16 days . p values determined by one - way ANOVA with Tukey ' s correction at day 18 , n is indicated in figure . Kaplan - Meier plot of time to reach 1400 mm3 volume in mice treated with vehicle , APR - 246 ( 50mg / kg ) , MK - 571 ( 50mg / kg ) or APR - 246 MK - 571 for 16 days . Mice that were culled at ethical endpoints prior to reaching a volume of > 1400mm3 are marked as censored observations . p values were determined by Log - rank ( Mantell - Cox ) between different treatment groups . n is indicated in 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


of Ki67 immunohistochemistry 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 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 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 O O O O

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.


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

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 of hematoxylin / eosin ( H & E ) immunostaining of Ki67 , caspase 3 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 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 ATP - CTG in colorectal cancer - 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 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 ( n = 3 except colo - PDO2 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

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 ( in black , left y - axis ) PI ( in red , right y - axis ) of colo - PDO1 colo - PDO2 as determined by image 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 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 lines after 24h 14C - APR - 246 + / - MK - 571 treatment ( n ≥ 3 ) . * p = 0 . 0006 , paired t - . 14C - accumulation n of lines as shown in Table S3 .

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

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

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 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 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

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 ( 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 ± . 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 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

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 after 24h treatment with APR - 246 + / - MK - 571 ( n = 2 ) . Data information : Indicated values are assessed by LC - MS . Data are represented as mean ± . 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 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

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 ± . 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

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

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

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

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

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 B-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

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.