IntelliCage was used to evaluate the memory flexibility of adult 11B mice . Four separate sessions were carried out : ( i ) 5 WT males together with 5 11B males ; ( ii ) 5 other WT males together with 5 other 11B males ; ( iii ) 5 WT females together with 7 11B females ; ( iv ) 4 other WT females together with 8 other 11B females . Data are the mean of the total 10 males ( WT ) , 10 males ( 11B ) , 9 females ( WT ) and 15 females ( 11B ) adult littermate mice . Data points were obtained at consecutive days , error bars indicate SD . ( A , B ) Quantification of the discrimination error rate in the first 100 visits during the drinking phase of the acquisition stage of adult wildtype ( WT ) and 11B mice ( male , A ; female , B ) in the IntelliCage . Overall place learning performance of WT and 11B mice was analysed using two - way ANOVA followed by Fisher ' s LSD test ; all data points during the acquisition phase were included . For males ( A ) , WT vs . 11B , P > 0 . 05 ; data over time , * * * * P < 0 . 0001 ; interaction , P > 0 . 05 . For females ( B ) , WT vs . 11B , P > 0 . 05 ; data over time , * * * * P < 0 . 0001 ; interaction , P > 0 . 05 . Note that ( i ) the two male sessions and the two female sessions yielded essentially the same results , and ( ii ) not shown in panels the two separate male sessions yielded essentially the same results , and the two separate female sessions yielded essentially the same results .

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

IntelliCage was used to evaluate the memory flexibility of adult 11B mice. Four separate sessions were carried out: (i) 5 WT males together with 5 11B males; (ii) 5 other WT males together with 5 other 11B males; (iii) 5 WT females together with 7 11B females; (iv) 4 other WT females together with 8 other 11B females. Data are the mean of the total 10 males (WT), 10 males (11B), 9 females (WT) and 15 females (11B) adult littermate mice. Data points were obtained at consecutive days, error bars indicate SD. (A, B) Quantification of the discrimination error rate in the first 100 visits during the drinking phase of the acquisition stage of adult wildtype (WT) and 11B mice (male, A; female, B) in the IntelliCage. Overall place learning performance of WT and 11B mice was analysed using two-way ANOVA followed by Fisher's LSD test; all data points during the acquisition phase were included. For males (A), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. For females (B), WT vs. 11B, P > 0.05; data over time, **** P < 0.0001; interaction, P > 0.05. Note that (i) the two male sessions and the two female sessions yielded essentially the same results, and (ii) not shown in panels the two separate male sessions yielded essentially the same results, and the two separate female sessions yielded essentially the same results.


Quantification of NANOG - positive iPSC colonies obtained after 14 days of OSKM induction in MEFs treated with either non - target or Kdm3b siRNA in FBS media . Values represent fold change over non - target control providing relative quantitation . Quantification of NANOG - positive iPSC colonies obtained after 12 or 14 days of OSKM induction in MEFs treated with either non - targeting or Kdm3b siRNA in KSR media . Values represent fold change over non - target control providing relative quantitation .

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Quantification of NANOG-positive iPSC colonies obtained after 14 days of OSKM induction in MEFs treated with either non-target or Kdm3b siRNA in FBS media. Values represent fold change over non-target control providing relative quantitation. Quantification of NANOG-positive iPSC colonies obtained after 12 or 14 days of OSKM induction in MEFs treated with either non-targeting or Kdm3b siRNA in KSR media. Values represent fold change over non-target control providing relative quantitation.


Quantification of NANOG positive colonies after retroviral expression of OSKM ( Left ) or OSK ( Right ) in MEF treated with non - targeting or siRNA targeting Kdm3 . Nanog counts were performed on Day 16 for OSKM or Day 21 for OSK reprogramming . Values represent fold change over non - target control providing relative quantitation .

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Quantification of NANOG positive colonies after retroviral expression of OSKM (Left) or OSK (Right) in MEF treated with non-targeting or siRNA targeting Kdm3. Nanog counts were performed on Day 16 for OSKM or Day 21 for OSK reprogramming. Values represent fold change over non-target control providing relative quantitation.


Immunoblot of H3K9me1 / 2 histone demethylase in WT or Kdm3b CRISPR targeted pre - iPSC clones . These pre - iPSCs carry a Nanog - GFP reporter . Parent line ( WT ) , and two Kdm3b - KO clones using gRNA targeting either exon 1 ( KO # 1 ) or exon 2 ( KO # 2 ) are shown . RNA Polymerase II levels are the loading control .

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Immunoblot of H3K9me1/2 histone demethylase in WT or Kdm3b CRISPR targeted pre-iPSC clones. These pre-iPSCs carry a Nanog-GFP reporter. Parent line (WT), and two Kdm3b-KO clones using gRNA targeting either exon 1 (KO#1) or exon 2 (KO#2) are shown. RNA Polymerase II levels are the loading control.


Quantification of Nanog - GFP positive cells after 10 days of AA + 2i treatment of WT or Kdm3b - deleted pre - iPSCs .

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Quantification of Nanog-GFP positive cells after 10 days of AA+2i treatment of WT or Kdm3b-deleted pre-iPSCs.


Immunoblot of H3K9 methylation levels upon addition of H3K9 methyltransferase inhibitor UNC0638 in WT or Kdm3b - KO pre - iPSCs .

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Immunoblot of H3K9 methylation levels upon addition of H3K9 methyltransferase inhibitor UNC0638 in WT or Kdm3b-KO pre-iPSCs.


Quantification of percent Nanog - GFP positive cells after 10 days of AA + 2i or AA + 2i with 5µM UNC0638 in WT or Kdm3b deleted pre - iPSCs .

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Quantification of percent Nanog-GFP positive cells after 10 days of AA+2i or AA+2i with 5µM UNC0638 in WT or Kdm3b deleted pre-iPSCs.


Pearson correlation of transcripts per million ( TPM ) of WT and Kdm3b - KO pre - iPSC upon DMSO or AA + 2i treatment for 2 or 10 days and mESC , reprogramming populations from Chronis et . al ( 2017 ) , ( labelled as CC ) .

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Pearson correlation of transcripts per million (TPM) of WT and Kdm3b-KO pre-iPSC upon DMSO or AA+2i treatment for 2 or 10 days and mESC, reprogramming populations from Chronis et. al (2017), (labelled as CC).


k - means clustering of the union of DEG ( FDR > 0 . 99 ) between day 2 and DMSO in WT and Kdm3b - KO pre - iPSCs . Values represent a log2 - fold change of day 2 over DMSO . Key pluripotency genes are shown on the right . k - means clustering of the union of DEG ( FDR > 0 . 99 ) between day 10 and DMSO in WT and Kdm3b - KO pre - iPSCs . Values represent a log2 - fold change of day 10 over DMSO . Key pluripotency genes or epigenetic regulators are shown on the right .

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k-means clustering of the union of DEG (FDR > 0.99) between day 2 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 2 over DMSO. Key pluripotency genes are shown on the right. k-means clustering of the union of DEG (FDR > 0.99) between day 10 and DMSO in WT and Kdm3b-KO pre-iPSCs. Values represent a log2-fold change of day 10 over DMSO. Key pluripotency genes or epigenetic regulators are shown on the right.


Expression of Tet enzymes and Tdg during AA + 2i treatment in WT or Kdm3b - KO pre - iPSCs . Error bars represent the standard deviation of two biological replicates from the RNA - Seq sample .

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Expression of Tet enzymes and Tdg during AA+2i treatment in WT or Kdm3b-KO pre-iPSCs. Error bars represent the standard deviation of two biological replicates from the RNA-Seq sample.


Quantification of percent Nanog - GFP cells of WT , Kdm3b - KO and Kdm3b - KO pre - iPSC clones expressing Tet1 catalytic domain ( Tet1CD ) , Nanog , or both .

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Quantification of percent Nanog-GFP cells of WT, Kdm3b-KO and Kdm3b-KO pre-iPSC clones expressing Tet1 catalytic domain (Tet1CD), Nanog, or both.


Representative 5hmC profiles during AA + 2i conversion of WT pre - iPSCs . Illustration of the 5hmC pattern is shown in the bottom panel .

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Representative 5hmC profiles during AA+2i conversion of WT pre-iPSCs. Illustration of the 5hmC pattern is shown in the bottom panel.


Heatmap of k - means clustered 5hmC patterns with histone modifications in pre - iPSCs and mESCs from Chronis et al ( 2017 ) .

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Heatmap of k-means clustered 5hmC patterns with histone modifications in pre-iPSCs and mESCs from Chronis et al (2017).


Representative profiles of regions that undergo demethylation through a 5hmC intermediate . Top - 5hmC profile during AA + 2i treatment . Middle - Percent methylation via bisulfite sequencing in MEFs and iPSCs , data from Rais et al 2013 . Bottom - Percent 5hmC via Tet - assisted bisulfite sequencing of MEFs expressing OSKM for 0 or 5 days , data from Hu et al 2014 .

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Representative profiles of regions that undergo demethylation through a 5hmC intermediate. Top - 5hmC profile during AA+2i treatment. Middle - Percent methylation via bisulfite sequencing in MEFs and iPSCs, data from Rais et al 2013. Bottom - Percent 5hmC via Tet-assisted bisulfite sequencing of MEFs expressing OSKM for 0 or 5 days, data from Hu et al 2014.


Left - Quantification of percent Nanog - GFP positive colonies . Right - Total Nanog - GFP positive colonies obtained after ten days of AA + 2i treatment in Tdg - depleted pre - iPSC ( Tdg ) or control ( Empty ) .

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Left - Quantification of percent Nanog-GFP positive colonies. Right - Total Nanog-GFP positive colonies obtained after ten days of AA+2i treatment in Tdg-depleted pre-iPSC (Tdg) or control (Empty).


Quantification of percent Nanog - GFP positive cells of WT or Kdm3b - KO pre - IPSC clones expressing Nanog , Tet1CD , or Nanog + Tet1CD after 15 days of AA + 2i or AA + 2i with 4 or 5µM of UNC0638 .

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Quantification of percent Nanog-GFP positive cells of WT or Kdm3b-KO pre-IPSC clones expressing Nanog, Tet1CD, or Nanog+Tet1CD after 15 days of AA+2i or AA+2i with 4 or 5µM of UNC0638.


Left - Heatmap of K - mean clustered 5hmC patterns in WT pre - iPSC with 5hmC profile of Kdm3b - KO pre - iPSC treated with AA + 2i for 0 , 2 or 10 days . Middle - Metaplot of 5hmC signal on day 10 between WT ( orange ) and Kdm3b - KO ( black ) for each cluster . Y - axis scale is in normalized coverage data ( 1x106 / Total Count ) . Right - Illustration of 5hmC pattern in WT and Kdm3b - KO during AA + 2i treatment .

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Left - Heatmap of K-mean clustered 5hmC patterns in WT pre-iPSC with 5hmC profile of Kdm3b-KO pre-iPSC treated with AA+2i for 0, 2 or 10 days. Middle - Metaplot of 5hmC signal on day 10 between WT (orange) and Kdm3b-KO (black) for each cluster. Y-axis scale is in normalized coverage data (1x106/Total Count). Right - Illustration of 5hmC pattern in WT and Kdm3b-KO during AA+2i treatment.


Representative 5hmC profiles during AA + 2i conversion of WT and Kdm3b - KO pre - iPSC .

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Representative 5hmC profiles during AA+2i conversion of WT and Kdm3b-KO pre-iPSC.


Scheme for identifying genes with both misregulated 5hmC and expression . Heatmap of two - fold misregulated 5hmC and two - fold differential expression between WT and Kdm3b - KO after 10 days of AA + 2i treatment ( 607 genes ) . Peaks are sorted by 5hmC signal in WT . RNA seq values represent a log2 - fold change of day 10 over DMSO for either WT or Kdm3b KO pre - iPSCs .

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Scheme for identifying genes with both misregulated 5hmC and expression. Heatmap of two-fold misregulated 5hmC and two-fold differential expression between WT and Kdm3b-KO after 10 days of AA+2i treatment (607 genes). Peaks are sorted by 5hmC signal in WT. RNA seq values represent a log2-fold change of day 10 over DMSO for either WT or Kdm3b KO pre-iPSCs.


Box plot of expression for genes that are two - fold misregulated 5hmC and expression between WT and Kdm3b - KO on day 10 ( 607 genes ) .

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Box plot of expression for genes that are two-fold misregulated 5hmC and expression between WT and Kdm3b-KO on day 10 (607 genes).


Heatmap of POU5F1 bound peaks in both mESC and pre - IPSC ( Cluster 1 ) and mESC alone ( Cluster 2 ) with 5hmC levels during Day 0 , 2 and 10 of AA + 2i treatment .

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Heatmap of POU5F1 bound peaks in both mESC and pre-IPSC (Cluster 1) and mESC alone (Cluster 2) with 5hmC levels during Day 0, 2 and 10 of AA+2i treatment.


Metaplots of POU5F1 binding for Cluster 1 ( Left ) and Cluster 2 ( Right ) regions in pre - iPSCs and mESCs .

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Metaplots of POU5F1 binding for Cluster 1 (Left) and Cluster 2 (Right) regions in pre-iPSCs and mESCs.


Metaplots of 5hmC Enrichment for Cluster 1 ( Left ) and Cluster 2 ( Right ) regions for Day 0 , 2 and 10 of AA + 2i treatment .

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Metaplots of 5hmC Enrichment for Cluster 1 (Left) and Cluster 2 (Right) regions for Day 0, 2 and 10 of AA+2i treatment.


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 .

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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 and Kdm3b - KO pre - iPSC with POU5F1 binding in pre - iPSCs and mESCs . Loci were assigned to the 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 and Kdm3b - KO pre - iPSCs . Values are fold change of enrichment between WT and 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 , 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 .

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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 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 .

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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 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 .

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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.


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 the overexpressed protein . 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 and subjected to Western blot analysis for SPPL2c . Protein disulfide isomerase ( PDI ) , Cathepsin D and CD44 were detected as organelle marker proteins .

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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 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 .

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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 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 .

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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.


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

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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 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 .

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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 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 .

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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 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 .

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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 and SPPL2c - / - testis were stained with X - Gal as β - gal substrate .

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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 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 .

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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 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 ) .

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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 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 .

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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.


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

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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 and 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 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 .

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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 analysis of total lysates of wild type ( + / + ) and SPPL2c - / - testis , epididymis and spermatozoa recovered from the 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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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

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


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 .

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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 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 .

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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.


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

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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 and SPPL2c - / - mice . Cryosections were stained using a rabbit monoclonal PLN antibody or normal rabbit IgG as negative control and 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 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 .

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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 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 .

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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 the 1C - co population from the datasets Median FSC ± SD of 8 individual 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 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 .

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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 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 .

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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 The intrinsic fluorescence 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 the 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 , 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 -

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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 , 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 -

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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 , 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 -

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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 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 -

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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 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

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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 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 -

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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 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 .

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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 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 .

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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 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

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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 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

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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 - tailed unpaired t - test

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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 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 ,

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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 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

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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 , 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 ±

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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 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 -

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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 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 blot ting . Statistical analyses were performed using Student ' s two - tailed unpaired t - test . 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 . 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 -

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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 . 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 -

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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 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 -

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

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 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 -

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

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 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 -

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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 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 immun o fluorescence ( IF ) . Statistical analyses were performed using Student ' s two - tailed unpaired t - test

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

(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 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 .

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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 cells transfected with WT , S1 , S3 and S3 . F88W PrP were probed with antibody Sha31 , 9A2 and 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 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 ) .

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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 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 .

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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 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 :

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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 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 .

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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 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 .

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 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

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 and analysed for PrP using the antibody 1A6 . FL , full - length PrP ; ns , non - specific signal detected in the immunoblotting procedure .

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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 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 ) .

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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 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 .

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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 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 .

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

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 and analysed by Western blot using ( A ) Sha31 antibody and ( B ) after PK and 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 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 .

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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 The pattern of PrPSc deposition ( A - D ) and GFAP immunostaining ( E - H ) is presented . I - P A higher power view of H & amp ; 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 ) .

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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 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 ) .

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

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 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 ) .

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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 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

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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