Immunofluorescent analysis of TDP - 43 in control , mutant TDP - 43 and mutant TDP - 43 + HDAC6 inhibitor after 12 h treatment . Presented immunofluorescent images of control ( C2 ) and mutant TDP - 43 ( P2 ) . Scale bar : 25µm . B Profile intensity plot of control , mutant TDP - 43 and mutant TDP - 43 + HDAC6 inhibitor . C Quantification of nucleo - cytoplasmic ratio ( N / C ) fluorescent intensity of TDP - 43 in motor neurons , each dot represents one analyzed cell , unpaired Mann - Whitney - test .

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

Immunofluorescent analysis of TDP-43 in control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor after 12 h treatment. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). Scale bar: 25µm. B Profile intensity plot of control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor. C Quantification of nucleo-cytoplasmic ratio (N/C) fluorescent intensity of TDP-43 in motor neurons, each dot represents one analyzed cell, unpaired Mann-Whitney-test.


Quantification of relative nuclear mCherry TDP - 43 on total fraction , ratio paired - t - test .

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Quantification of relative nuclear mCherry TDP-43 on total fraction, ratio paired-t-test.


Quantification of percentage of moving mitochondria ,

Quantification of percentage of moving mitochondria,


P . falciparum symptomatic ( n = 30 ) and asymptomatic ( n = 40 ) infected individuals , as well as light - microscopy and PCR parasite - negative healthy immune controls ( n = 31 ) were recruited for the study . A - F . Clinical parameters determined in the study include age ( A ) , gender ( B ) , parasitemia ( C ) , hemoglobin ( g / dL blood ) ( D ) , hematocrit ( E ) , and platelet count ( F ) . Boxes represent the 25th to 75th percentiles , whiskers show the range ( minimum to maximum ) , and lines represent the median . Significance was determined by the Kruskal - Wallis test with Dunn ' s multiple comparisons ( A , D - F ) , the Chi - square test ( B ) , and the Mann - Whitney test ( C ) using 30 ( symptomatic ) , 40 ( asymptomatic ) and 31 ( healthy immune controls ) biological replicates . * * * * p < 0 . 0001 .

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

P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. A-F. Clinical parameters determined in the study include age (A), gender (B), parasitemia (C), hemoglobin (g/dL blood) (D), hematocrit (E), and platelet count (F). Boxes represent the 25th to 75th percentiles, whiskers show the range (minimum to maximum), and lines represent the median. Significance was determined by the Kruskal-Wallis test with Dunn's multiple comparisons (A, D-F), the Chi-square test (B), and the Mann-Whitney test (C) using 30 (symptomatic), 40 (asymptomatic) and 31 (healthy immune controls) biological replicates. ****p<0.0001.


P . falciparum symptomatic ( n = 30 ) and asymptomatic ( n = 40 ) infected individuals , as well as light - microscopy and PCR parasite - negative healthy immune controls ( n = 31 ) were recruited for the study . G . Antibody titers specific for P . falciparum parasite lysate . Bars represent the mean ± SD of using 29 ( symptomatic ) , 35 ( asymptomatic ) and 26 ( healthy immune controls ) biological replicates . The dotted line depicts the average antibody background levels of malaria - naive healthy Melbourne controls . H . Mean antibody titers as determined by ELISA to the P . falciparum recombinant proteins EBA - 175 , EBA - 140 , PfRh2 , PfRh4 , PfRh5 , and PfRipr .

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P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. G. Antibody titers specific for P. falciparum parasite lysate. Bars represent the mean ± SD of using 29 (symptomatic), 35 (asymptomatic) and 26 (healthy immune controls) biological replicates. The dotted line depicts the average antibody background levels of malaria-naive healthy Melbourne controls. H. Mean antibody titers as determined by ELISA to the P. falciparum recombinant proteins EBA-175, EBA-140, PfRh2, PfRh4, PfRh5, and PfRipr.


PBMCs from P . falciparum symptomatic ( n = 16 ) and asymptomatic ( n = 24 ) infected individuals as well as healthy immune controls ( n = 24 ) were stained with a panel of metal - labelled antibodies and analysed by CyTOF . tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub - populations within gated : A . TH1 - like memory CD4 + T cells ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 + ) B . TH2 - like memory CD4 + T cells ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 - ) C . Circulating memory TFH cells ( CD19 - CD3 + CD4 + CD45RA - CXCR5 + ) D . Classical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 + ) E . Atypical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 - CD21 - ) F . Activated MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 - ) The tSNE plots in the top panel display cell density and represent the pooled data for each group , while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot . Heatmaps show the median marker expression for each FlowSOM cluster .

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

PBMCs from P. falciparum symptomatic (n=16) and asymptomatic (n=24) infected individuals as well as healthy immune controls (n=24) were stained with a panel of metal-labelled antibodies and analysed by CyTOF. tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub-populations within gated: A. TH1-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3+) B. TH2-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3-) C. Circulating memory TFH cells (CD19-CD3+CD4+CD45RA-CXCR5+) D. Classical MBCs (CD3-CD19+CD20+CD10-CD27+CD21+) E. Atypical MBCs (CD3-CD19+CD20+CD10-CD27-CD21-) F. Activated MBCs (CD3-CD19+CD20+CD10-CD27+CD21-) The tSNE plots in the top panel display cell density and represent the pooled data for each group, while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot. Heatmaps show the median marker expression for each FlowSOM cluster.


Unsupervised identification of differentially abundant high - dimensional cell populations between clinical groups was performed using CITRUS ( FDR < 5 % ) . between P . falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls . A - E . tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers , relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3 + CCR6 - memory CD4 + T cells ( A ) , CXCR3 - CCR6 - memory CD4 + T cells ( B ) , circulating memory TFH cells ( C ) , classical MBCs ( D ) , and atypical MBCs ( E ) . The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays . The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay , whiskers showing the range ( minimum to maximum ) , with lines representing the median of 16 ( symptomatic ) , 24 ( asymptomatic ) and 24 ( healthy immune controls ) biological replicates , while the pink histograms illustrate marker expression in identified differentially abundant populations , relative to background expression , shown in lilac .

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

Unsupervised identification of differentially abundant high-dimensional cell populations between clinical groups was performed using CITRUS (FDR<5%). between P. falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls. A-E. tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers, relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3+CCR6- memory CD4+ T cells (A), CXCR3-CCR6- memory CD4+ T cells (B), circulating memory TFH cells (C), classical MBCs (D), and atypical MBCs (E). The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays. The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay, whiskers showing the range (minimum to maximum), with lines representing the median of 16 (symptomatic), 24 (asymptomatic) and 24 (healthy immune controls) biological replicates, while the pink histograms illustrate marker expression in identified differentially abundant populations, relative to background expression, shown in lilac.


PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for RNA - seq analysis . A . Estimated proportions of PBMC sub - populations determined from cell - type deconvolution from study participant transcriptional profiles .

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PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. A. Estimated proportions of PBMC sub-populations determined from cell-type deconvolution from study participant transcriptional profiles.


PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for RNA - seq analysis . D . Blood transcriptional module ( BTM ) analysis showing significant BTMs differentially enriched for pairwise comparisons ( FDR < 15 % ) .

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PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. D. Blood transcriptional module (BTM) analysis showing significant BTMs differentially enriched for pairwise comparisons (FDR<15%).


Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . A . Unsupervised hierarchical clustering heat map of the 922 differentially expressed genes ( DEGs ) between symptomatic P . falciparum malaria and healthy immune controls using the complete method and Euclidian measure of distance . B . Mean - difference plot displaying genes differentially expressed between symptomatic P . falciparum malaria and healthy immune controls . Each gene is plotted as a single point determined by log - fold - change and average transcript abundance . Red genes are overrepresented , and blue genes are underrepresented in symptomatic malaria .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. A. Unsupervised hierarchical clustering heat map of the 922 differentially expressed genes (DEGs) between symptomatic P. falciparum malaria and healthy immune controls using the complete method and Euclidian measure of distance. B. Mean-difference plot displaying genes differentially expressed between symptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in symptomatic malaria.


Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . F . Upstream regulator analysis of the 922 DEGs between symptomatic P . falciparum malaria and healthy immune controls . The red lines represent a significant activation z - score of ± 2 .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. F. Upstream regulator analysis of the 922 DEGs between symptomatic P. falciparum malaria and healthy immune controls. The red lines represent a significant activation z-score of ±2.


Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . Hierarchical clustering heatmap ( G ) displaying DEGs in symptomatic P . falciparum malaria and healthy immune controls predicted to be controlled by IFN - γ and IL - 13 upstream regulators .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. Hierarchical clustering heatmap (G) displaying DEGs in symptomatic P. falciparum malaria and healthy immune controls predicted to be controlled by IFN-γ and IL-13 upstream regulators.


Gene expression profiles of PBMCs from P . falciparum symptomatic and asymptomatic infected participants were compared . C . Heat map of the 418 DEGs in P . falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls . E . Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2 - fold - change and - Log10 ( P - value ) differentially expressed between P . falciparum symptomatic and asymptomatic infected individuals . Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. C. Heat map of the 418 DEGs in P. falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls. E. Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2-fold-change and -Log10(P-value) differentially expressed between P. falciparum symptomatic and asymptomatic infected individuals. Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria.


Gene expression profiles of PBMCs from P . falciparum symptomatic and asymptomatic infected participants were compared . F . Mean RPKMs + SEM of selected genes in P . falciparum malaria symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) individuals . Dotted red lines depict transcriptional levels of healthy immune controls , Mann - Whitney test of biological replicates , * p < 0 . 05 .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. F. Mean RPKMs + SEM of selected genes in P. falciparum malaria symptomatic (n=6) and asymptomatic (n=5) individuals. Dotted red lines depict transcriptional levels of healthy immune controls, Mann-Whitney test of biological replicates,*p<0.05.


Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals and healthy immune controls were compared . A . Hierarchical clustering heat map of the 171 differentially expressed genes ( DEGs ) in P . falciparum asymptomatic and healthy immune control participants . C . Mean - difference plot displaying DEGs between asyptomatic P . falciparum malaria and healthy immune controls . Each gene is plotted as a single point determined by log - fold - change and average transcript abundance . Red genes are overrepresented , and blue genes are underrepresented in asymptomatic malaria .

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Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. A. Hierarchical clustering heat map of the 171 differentially expressed genes (DEGs) in P. falciparum asymptomatic and healthy immune control participants. C. Mean-difference plot displaying DEGs between asyptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in asymptomatic malaria.


Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals and healthy immune controls were compared . E . Mean RPKMs + SEM of selected genes in P . falciparum malaria asymptomatic individuals ( n = 5 ) and healthy immune controls ( n = 6 ) . Dotted red lines depict transcriptional levels of P . falciparum - infected symptomatic individuals , Mann - Whitney test of biological replicates , * p < 0 . 05 .

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Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. E. Mean RPKMs + SEM of selected genes in P. falciparum malaria asymptomatic individuals (n=5) and healthy immune controls (n=6). Dotted red lines depict transcriptional levels of P. falciparum-infected symptomatic individuals, Mann-Whitney test of biological replicates, *p<0.05.


A . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM + activated MBCs . B . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T - bet + CD4 + T cells .

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A. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM+ activated MBCs. B. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T-bet+ CD4+ T cells.


B - G . PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 6 ) infected individuals as well as healthy immune controls ( n = 6 ) were stained with a panel of antibodies and analyzed by flow cytometry . tSNE plots ( B ) display cell density of the pooled data for each group , ( C ) shows a projection of the FlowSOM clusters on a tSNE plot , and ( D ) is a heatmap depicting the celluar phenotype of clusters of CTLA - 4 + CD3 + CD19 - cells . Bar plots representing mean relative abundance of biological replicates + SEM of relevant clusters among clinical groups ( E ) , Mann - Whitney test , * p < 0 . 05 . The panel on the left shows the average CTLA - 4 mean fluorescent intensity ( MFI ) or 6 biological replicates in cluster 4 + SEM , Mann - Whitney test , * * p < 0 . 01 , while representative histograms of CTLA - 4 expression in cluster 4 are shown on the right ( F ) . Marker intensity within clusters 1 and 6 ( G ) .

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B-G. PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=6) infected individuals as well as healthy immune controls (n=6) were stained with a panel of antibodies and analyzed by flow cytometry. tSNE plots (B) display cell density of the pooled data for each group, (C) shows a projection of the FlowSOM clusters on a tSNE plot, and (D) is a heatmap depicting the celluar phenotype of clusters of CTLA-4+CD3+CD19- cells. Bar plots representing mean relative abundance of biological replicates + SEM of relevant clusters among clinical groups (E), Mann-Whitney test, *p<0.05. The panel on the left shows the average CTLA-4 mean fluorescent intensity (MFI) or 6 biological replicates in cluster 4 + SEM, Mann-Whitney test, **p<0.01, while representative histograms of CTLA-4 expression in cluster 4 are shown on the right (F). Marker intensity within clusters 1 and 6 (G).


Human Aβ exhibits a biphasic profile in CSF of APP transgenic miceA , B Aβ40 and Aβ42 concentrations in CSF of male APP23 mice ( heterozygous ; 3 ( n = 14 ) , 6 ( n = 12 ) , 8 ( n = 10 ) , 12 ( n = 11 ) , 19 ( n = 9 ) , and 25 ( n = 6 ) months of age ) . CSF Aβ40 ( F ( 1 , 56 ) = 22 . 351 , P < 0 . 001 ) as well as CSFAβ42 ( F ( 1 , 56 ) = 38 . 597 , P < 0 . 001 ) followed a significant quadratic trend .

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Human Aβ exhibits a biphasic profile in CSF of APP transgenic miceA, B Aβ40 and Aβ42 concentrations in CSF of male APP23 mice (heterozygous; 3 (n = 14), 6 (n = 12), 8 (n = 10), 12 (n = 11), 19 (n = 9), and 25 (n = 6) months of age). CSF Aβ40 (F(1, 56) = 22.351, P < 0.001) as well as CSFAβ42 (F(1, 56) = 38.597, P < 0.001) followed a significant quadratic trend.


C Aβ42 / 40 ratio in CSF of APP23 mice showed a delayed decrease with age ( F ( 1 , 56 ) = 53 . 894 , P < 0 . 001 ) .

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C Aβ42/40 ratio in CSF of APP23 mice showed a delayed decrease with age (F(1, 56) = 53.894, P < 0.001).


D , E Aβ40 and Aβ42 concentrations in CSF of male and female APP24 mice ( homozygous ; 2 ( n = 13 ) , 3 - 4 ( n = 16 ) , 7 - 8 ( n = 15 ) , 18 - 19 ( n = 14 ) , 24 ( n = 16 ) , and 30 ( n = 18 ) months of age ) . CSF Aβ40 followed a significant quadratic trend ( F ( 1 , 86 ) = 6 . 678 , P = 0 . 011 ) and CSF Aβ42 best fitted a cubic trend ( F ( 1 , 86 ) = 30 . 599 , P < 0 . 001 ) .

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D, E Aβ40 and Aβ42 concentrations in CSF of male and female APP24 mice (homozygous; 2 (n = 13), 3-4 (n = 16), 7-8 (n = 15), 18-19 (n = 14), 24 (n = 16), and 30 (n = 18) months of age). CSF Aβ40 followed a significant quadratic trend (F(1, 86) = 6.678, P = 0.011) and CSF Aβ42 best fitted a cubic trend (F(1, 86) = 30.599, P < 0.001).


F Aβ42 / 40 ratio in CSF of APP24 mice showed a delayed decrease with age ( F ( 1 , 86 ) = 64 . 936 , P < 0 . 001 ) .

F Aβ42/40 ratio in CSF of APP24 mice showed a delayed decrease with age (F(1, 86) = 64.936, P < 0.001).


G , H Aβ40 and Aβ42 in the CSF of female APP51 mice ( heterozygous ; 3 ( n = 6 ) , 15 ( n = 8 ) , and 24 ( n = 8 ) months of age ; 22 mice in total ) . CSF Aβ40 ( F ( 1 , 19 ) = 37 . 349 , P < 0 . 001 ) as well as CSFAβ42 ( F ( 1 , 19 ) = 107 . 670 , P < 0 . 001 ) followed a significant quadratic trend .

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G, H Aβ40 and Aβ42 in the CSF of female APP51 mice (heterozygous; 3 (n = 6), 15 (n = 8), and 24 (n = 8) months of age; 22 mice in total). CSF Aβ40 (F(1, 19) = 37.349, P < 0.001) as well as CSFAβ42 (F(1, 19) = 107.670, P < 0.001) followed a significant quadratic trend.


I Aβ42 / 40 ratio in CSF of APP51 mice showed a delayed decrease with age ( F ( 1 , 19 ) = 26 . 367 , P < 0 . 001 ) .

I Aβ42/40 ratio in CSF of APP51 mice showed a delayed decrease with age (F(1, 19) = 26.367, P < 0.001).


HumanAβ in CSF and brain of APP transgenic miceA APP23CSFAβ40 and Aβ42 in the same animals as shown in Fig1 . CSFAβ42 and Aβ40 are expressed as percentages of levels measured in the youngest age group .

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HumanAβ in CSF and brain of APP transgenic miceA APP23CSFAβ40 and Aβ42 in the same animals as shown in Fig1. CSFAβ42 and Aβ40 are expressed as percentages of levels measured in the youngest age group.


B , C Aβ40 and Aβ42 ( pmol / g wet brain ) in the FA - soluble brain extract from the same APP23mice showed a robust increase with age ; ANOVA revealed a significant cubic trend ( F ( 1 , 56 ) = 221 . 114 , P < 0 . 001 and F ( 1 , 56 ) = 370 . 947 , P < 0 . 001 , respectively ) .

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B, C Aβ40 and Aβ42 (pmol/g wet brain) in the FA-soluble brain extract from the same APP23mice showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 56) = 221.114, P < 0.001 and F(1, 56) = 370.947, P < 0.001, respectively).


D APP24 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentage of the youngest age group .

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D APP24 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentage of the youngest age group.


E , F Aβ40 and Aβ42 ( pmol / g wet brain ) in the brain from the same APP24mice also showed a robust increase with age ; ANOVA revealed a significant cubic trend ( F ( 1 , 86 ) = 202 . 173 , P < 0 . 001 and F ( 1 , 86 ) = 139 . 941 , P < 0 . 001 , respectively ) .

E, F Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP24mice also showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 86) = 202.173, P < 0.001 and F(1, 86) = 139.941, P < 0.001, respectively).


G APP51 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentages of levels in the youngest age group .

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G APP51 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentages of levels in the youngest age group.


H , I Aβ40 and Aβ42 ( pmol / g wet brain ) in the brain from the same APP51 mice showed a robust increase with age ; ANOVA revealed a significant quadratic trend ( F ( 1 , 19 ) = 12 . 960 , P = 0 . 002 and F ( 1 , 19 ) = 19 . 366 , P < 0 . 001 , respectively ) .

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H, I Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP51 mice showed a robust increase with age; ANOVA revealed a significant quadratic trend (F(1, 19) = 12.960, P = 0.002 and F(1, 19) = 19.366, P < 0.001, respectively).


Aβplaque pathology in the brains of APP23miceAβimmunostaining ( CN3 antibody , dark blue ) in 25 - μm sagittal brain sections shows only sparse Aβ deposits primarily in the frontal cortex of 6 - to 8 - month - old APP23mice . At 12 months and thereafter , there is a progressive increase in plaque number and size and a progressive involvement of different brain regions . Insets highlight the plaque characteristics at the different ages . Scale bar , 100 μm . The mean number of Aβ plaques per section per hemibrain increased with age in 3 - to 12 - month - old mice . Only four mice were analyzed in the 3 - month - old age group , as APP23mice do not develop plaques at this age ( Sturchler - Pierrat et al , 1997 ) . The 6 - , 8 - , and 12 - month - old age groups included 12 , 10 , and 11 mice , respectively ( these are the same mice that were used for CSF and brainAβ measurements ) . Note that the Aβ plaques became too numerous and often could no longer be individually distinguished in the age groups > 12 months of age . Data are represented as group means ± SEM .

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Aβplaque pathology in the brains of APP23miceAβimmunostaining (CN3 antibody, dark blue) in 25-μm sagittal brain sections shows only sparse Aβ deposits primarily in the frontal cortex of 6- to 8-month-old APP23mice. At 12 months and thereafter, there is a progressive increase in plaque number and size and a progressive involvement of different brain regions. Insets highlight the plaque characteristics at the different ages. Scale bar, 100 μm.The mean number of Aβ plaques per section per hemibrain increased with age in 3- to 12-month-old mice. Only four mice were analyzed in the 3-month-old age group, as APP23mice do not develop plaques at this age (Sturchler-Pierrat et al, 1997). The 6-, 8-, and 12-month-old age groups included 12, 10, and 11 mice, respectively (these are the same mice that were used for CSF and brainAβ measurements). Note that the Aβ plaques became too numerous and often could no longer be individually distinguished in the age groups > 12 months of age. Data are represented as group means ± SEM.


Brain sAPPβ shows an age - related increase in APP23 , APP24 , and APP51 micesAPPβ was measured in Triton X - 100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group . Swedish sAPPβ showed an age - dependent increase in APP23 mice following a linear trend ( F ( 1 , 83 ) = 52 . 914 , P < 0 . 001 ) ; APP23 from two independent batches were included in this analysis ( see Materials and Methods and Supplementary Fig S2 for details ) . Swedish sAPPβ showed an age - dependent increase in APP24mice following a linear trend ( F ( 1 , 84 ) = 11 . 264 , P = 0 . 001 ) . Human wild - type sAPPβ showed an age - dependent increase in APP51 following a quadratic trend ( F ( 1 , 18 ) = 68 . 980 , P < 0 . 001 ) . Data information : Post hoc Dunnett ' s test group comparisons were always conducted between the youngest group and all other groups . All data are represented as group means ± SEM ; * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 . For absolute values , see Supplementary Fig S2 .

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Brain sAPPβ shows an age-related increase in APP23, APP24, and APP51 micesAPPβ was measured in Triton X-100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group.Swedish sAPPβ showed an age-dependent increase in APP23 mice following a linear trend (F(1, 83) = 52.914, P < 0.001); APP23 from two independent batches were included in this analysis (see Materials and Methods and Supplementary Fig S2 for details).Swedish sAPPβ showed an age-dependent increase in APP24mice following a linear trend (F(1, 84) = 11.264, P = 0.001).Human wild-type sAPPβ showed an age-dependent increase in APP51 following a quadratic trend (F(1, 18) = 68.980, P < 0.001).Data information: Post hoc Dunnett's test group comparisons were always conducted between the youngest group and all other groups. All data are represented as group means ± SEM; *P < 0.05; **P < 0.01; and ***P < 0.001. For absolute values, see Supplementary Fig S2.


( B ) Quantification of embryos that expressing the ZGA reporter ( MERVL : : tdTomato ) after injection with a siRNA - repressor or mRNA - inducer . N , the total number of embryos analyzed for each condition . Error bars , mean ± S . D . ; n = 3 biological replicates per group .

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(B) Quantification of embryos that expressing the ZGA reporter (MERVL::tdTomato) after injection with a siRNA-repressor or mRNA-inducer. N, the total number of embryos analyzed for each condition. Error bars, mean ± S.D.; n = 3 biological replicates per group.


( C ) Representative fluorescence image of SCNT embryos derived by different methods . The SCNT embryos produced by transfer of MERVL : : tdTomato cumulus cells ( B6D2F1 background ) into WT enucleated oocytes . n = 3 biological replicates per group . Scale bar , 100 μm .

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(C) Representative fluorescence image of SCNT embryos derived by different methods. The SCNT embryos produced by transfer of MERVL::tdTomato cumulus cells (B6D2F1 background) into WT enucleated oocytes. n = 3 biological replicates per group. Scale bar, 100 μm.


( D ) Stacked bar plots show the fraction of embryos at blastocyst stages after injection with different mRNA , as indicated . Error bars , mean ± S . D . ; n = 3 biological replicates per group ; N , total number of embryos analyzed for each condition ; * * P < 0 . 01 , * * * P < 0 . 001 as compared with control group , by two - tailed Student ' s t - test .

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(D) Stacked bar plots show the fraction of embryos at blastocyst stages after injection with different mRNA, as indicated. Error bars, mean ± S.D.; n = 3 biological replicates per group; N, total number of embryos analyzed for each condition; **P < 0.01, ***P < 0.001 as compared with control group, by two-tailed Student's
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( E ) Representative live - cell images of dynamic ZGA - reporter expression during SCNT embryo development . The time after initial observation is shown at the bottom of the image . n = 3 biological replicates per group . Scale bar , 50 μm .

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(E) Representative live-cell images of dynamic ZGA-reporter expression during SCNT embryo development. The time after initial observation is shown at the bottom of the image. n = 3 biological replicates per group. Scale bar, 50 μm.


( G ) Preimplantation development of SCNT embryos . The percentage of embryos reaching each indicated stage is shown . The D - SCNT embryos produced by transfer of dox - Dux cumulus cell ( B6D2F1 background ) into WT enucleated oocytes ( B6D2F1 background ) . Error bars , mean ± S . D . ; n = 3 biological replicates per group . Scale bar , 100 μm .

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(G) Preimplantation development of SCNT embryos. The percentage of embryos reaching each indicated stage is shown. The D-SCNT embryos produced by transfer of dox-Dux cumulus cell (B6D2F1 background) into WT enucleated oocytes (B6D2F1 background). Error bars, mean ± S.D.; n = 3 biological replicates per group. Scale bar, 100 μm.


( J ) Image of full - term cloned mice derived by canonical SCNT and D - SCNT method ( left ) . The birth rate of SCNT embryos derived by different methods is indicated ( right ) . N , the total number of cloned pups obtained from each condition .

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(J) Image of full-term cloned mice derived by canonical SCNT and D-SCNT method (left). The birth rate of SCNT embryos derived by different methods is indicated (right). N, the total number of cloned pups obtained from each condition.


( B ) Durations of dox addition at different time points during chemical induction ( upper ) and assessment of AP + colonies ( bottom ) . Error bars , mean ± S . D . ; n = 3 biological replicates per condition ; * * P < 0 . 01 , * * * P < 0 . 001 as compared with condition - 1 , by two - tailed Student ' s t - test . n . s . , not significant .

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(B) Durations of dox addition at different time points during chemical induction (upper) and assessment of AP+ colonies (bottom). Error bars, mean ± S.D.; n = 3 biological replicates per condition; **P < 0.01, ***P < 0.001 as compared with condition-1, by two-tailed Student's
 t-test. n.s., not significant.


( C ) Morphological changes at distinct time points during D - CiPS induction . Scale bar , 100 μm .

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(C) Morphological changes at distinct time points during D-CiPS induction. Scale bar, 100 μm.


( D ) Representative images of the number of AP + positive colonies . n = 5 biological replicates per condition .

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(D) Representative images of the number of AP+ positive colonies. n = 5 biological replicates per condition.


( E ) RT - qPCR analysis of select ZGA - related genes in dox - untreated D - CiPSCs and canonical CiPSCs at passage 1 . The value in CiPSCs was set as 1 , and data shown are mean expression values relative to GAPDH . Error bars , mean ± S . E . M . ; n = 3 biological replicates per group ; * * * P < 0 . 001 by two - tailed Student ' s t - test .

(E) RT-qPCR analysis of select ZGA-related genes in dox-untreated D-CiPSCs and canonical CiPSCs at passage 1. The value in CiPSCs was set as 1, and data shown are mean expression values relative to GAPDH. Error bars, mean ± S.E.M.; n = 3 biological replicates per group; ***P < 0.001 by two-tailed Student's t-test.


( F ) Representative immunostaining images of D - CiPSCs expressing pluripotency markers . n = 5 biological replicates . Scale bar , 50 μm .

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(F) Representative immunostaining images of D-CiPSCs expressing pluripotency markers. n = 5 biological replicates. Scale bar, 50 μm.


( G ) Histology by H & E staining of teratoma tissues derived by D - CiPS cells . n = 3 biological replicates . Scale bar , 200 μm .

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(G) Histology by H&E staining of teratoma tissues derived by D-CiPS cells. n = 3 biological replicates. Scale bar, 200 μm.


( I ) Karyotype analysis of D - CiPSCs and canonical CiPSCs . The majority of D - CiPSCs and CiPSCs cells possessed a normal karyotype . Scale bar , 10 μm .

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(I) Karyotype analysis of D-CiPSCs and canonical CiPSCs. The majority of D-CiPSCs and CiPSCs cells possessed a normal karyotype. Scale bar, 10 μm.


( J ) Bisulfite sequencing analysis of demethylation of Oct4 and Nanog promoters in D - CiPSCs or canonical CiPSCs . Filled and empty squares represent methylated and unmethylated CpGs , respectively .

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(J) Bisulfite sequencing analysis of demethylation of Oct4 and Nanog promoters in D-CiPSCs or canonical CiPSCs. Filled and empty squares represent methylated and unmethylated CpGs, respectively.


Ookinete formation in vitro . After 12 h of culture , the ookinetes were Giemsa - stained and analyzed for ookinete morphology . The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes .

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Ookinete formation in vitro. After 12 h of culture, the ookinetes were Giemsa-stained and analyzed for ookinete morphology. The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes.


Oocyst counts in the mosquitoes at day 7 post blood - feeding . x / y on the top is the number of mosquitoes containing oocyst / the number of mosquitoes dissected ; the percentage number is the mosquito infection prevalence .

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Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence.


Co - staining of P28 and GAP45 ( IMC protein ) in female gametes ( P28 + only ) and zygotes ( P28 + / GAP45 + ) of the 17XNL , Δap2 - o3 and Δcdpk4 strains . Percentages of female gametes and zygotes were indicated respectively . n is the number of cells counted . Scale bars = 5 μm . Note : the signal of P28 and GAP45 is reduced similarly in all Δap2 - o3 female gametes and zygotes compared to that of 17XNL .

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Co-staining of P28 and GAP45 (IMC protein) in female gametes (P28+ only) and zygotes (P28+/GAP45+) of the 17XNL, Δap2-o3 and Δcdpk4 strains. Percentages of female gametes and zygotes were indicated respectively. n is the number of cells counted. Scale bars = 5 μm. Note: the signal of P28 and GAP45 is reduced similarly in all Δap2-o3 female gametes and zygotes compared to that of 17XNL.


Day 7 midgut oocyst counts from mosquitoes infected with parasites , including 17XNL , Δap2 - o3 , Δmap2 , or Δnek4 strain alone as well as mixtures of Δmap2 / Δnek4 , Δap2 - o3 / Δmap2 , and Δap2 - o3 / Δnek4 . Δnek4 and Δmap2 are female and male gamete - defect parasites , respectively . n is the number of mosquitoes dissected , Mann - Whitney test applied , two experiments repeated .

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Day 7 midgut oocyst counts from mosquitoes infected with parasites, including 17XNL, Δap2-o3, Δmap2, or Δnek4 strain alone as well as mixtures of Δmap2/Δnek4, Δap2-o3/Δmap2, and Δap2-o3/Δnek4. Δnek4 and Δmap2 are female and male gamete-defect parasites, respectively. n is the number of mosquitoes dissected, Mann-Whitney test applied, two experiments repeated.


Transcript expression heatmap by RNA - seq of 26 genes involving DNA replication and DNA repair between DFsc7 ( WT ) and DFsc7 ; Δap2 - o3 ( KO ) strains .

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Transcript expression heatmap by RNA-seq of 26 genes involving DNA replication and DNA repair between DFsc7 (WT) and DFsc7;Δap2-o3 (KO) strains.


Representative fluorescence microscopy images of mCherry and DPOD2 : : GFP expression in female and male gametocytes of the DTS1 ( ccp2 : : mCherry ; dpod2 : : gfp ) and DTS1 ; Δap2 - o3 strains . mCherry is specifically expressed in female gametocytes . Flow cytometry detection of mCherry and DPOD2 : : GFP expression in female and male gametocytes of the DTS1 and DTS1 ; Δap2 - o3 strains . The female and male gametocyte populations are circled by solid and dashed line respectively . E , F Similar analysis ( as in C , D ) in DTS2 ( ccp2 : : mCherry ; dpod1 : : gfp ) and DTS2 ; Δap2 - o3 strains . G , H Similar analysis ( as in C , D ) in DTS3 ( ccp2 : : mCherry ; rpa1 : : gfp ) and DTS3 ; Δap2 - o3 strains . I , J Similar analysis ( as in C , D ) in DTS4 ( ccp2 : : mCherry ; mcm7 : : gfp ) and DTS4 ; Δap2 - o3 strains .

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Representative fluorescence microscopy images of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 (ccp2::mCherry;dpod2::gfp) and DTS1;Δap2-o3 strains. mCherry is specifically expressed in female gametocytes. Flow cytometry detection of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 and DTS1;Δap2-o3 strains. The female and male gametocyte populations are circled by solid and dashed line respectively. E,F Similar analysis (as in C, D) in DTS2 (ccp2::mCherry;dpod1::gfp) and DTS2;Δap2-o3 strains. G,H Similar analysis (as in C, D) in DTS3 (ccp2::mCherry;rpa1::gfp) and DTS3;Δap2-o3 strains. I,J Similar analysis (as in C, D) in DTS4 (ccp2::mCherry;mcm7::gfp) and DTS4;Δap2-o3 strains.


EMSA using the recombinant GST - fused AP2 domain of AP2 - O3 and a synthesized DNA probe containing three repeats of predicted motif . GST was used as a negative control . An arrowhead indicates the shifted band .

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EMSA using the recombinant GST-fused AP2 domain of AP2-O3 and a synthesized DNA probe containing three repeats of predicted motif. GST was used as a negative control. An arrowhead indicates the shifted band.


ChIP - qPCR validation of the binding between AP2 - O3 and the upstream region of four male genes The regions detected via ChIP - qPCR are indicated by pink line ( in the peak ) and purple line ( out of the peak ) . mean ± SEM from three independent experiments .

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ChIP-qPCR validation of the binding between AP2-O3 and the upstream region of four male genes The regions detected via ChIP-qPCR are indicated by pink line (in the peak) and purple line (out of the peak). mean±SEM from three independent experiments.


qRT - PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID .

qRT-PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID.


Western blot of GAP45 protein expression in non - activated ( NAG ) and activated gametocyte ( AG ) of 17XNL and Δap2 - o3 strains . BiP as loading control .

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Western blot of GAP45 protein expression in non-activated (NAG) and activated gametocyte (AG) of 17XNL and Δap2-o3 strains. BiP as loading control.


IFA of AP2 - O3 protein expression in activated gametocytes of Pccp2 strains .

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IFA of AP2-O3 protein expression in activated gametocytes of Pccp2 strains.


( a ) Brains of the ' rescued ' mice ( Becn1 − / − ; Becn1EGFP / + ) and of control Becn1 + / + littermates were lysed and subjected to immune isolation using an anti - GFP antibody . Proteins bound to anti - GFP antibody were isolated , detected and identified by SDS - PAGE , Coomassie brilliant blue staining and mass spectrometry . Seven bands pulled down by anti - GFP antibody specifically from ' rescued ' mice lysate are numbered ( this is a copy of image from our previous report with modification ) .

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(a) Brains of the 'rescued' mice (Becn1−/−; Becn1EGFP/+) and of control Becn1+/+ littermates were lysed and subjected to immune isolation using an anti-GFP antibody. Proteins bound to anti-GFP antibody were isolated, detected and identified by SDS-PAGE, Coomassie brilliant blue staining and mass spectrometry. Seven bands pulled down by anti-GFP antibody specifically from 'rescued' mice lysate are numbered (this is a copy of image from our previous report with modification).


( b ) Mouse brain lysates were subjected to co - immunoprecipitation ( co - IP ) followed by WB analysis using the indicated antibodies .

(b) Mouse brain lysates were subjected to co-immunoprecipitation (co-IP) followed by WB analysis using the indicated antibodies.


( c ) Mouse brain lysates were subjected to co - IP by NRBF2 antibody and followed by WB analysis using the indicated antibodies .

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(c) Mouse brain lysates were subjected to co-IP by NRBF2 antibody and followed by WB analysis using the indicated antibodies.


( d ) Gel filtration analysis . The supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10 / 30 column and each fraction was immunoblotted with the indicated antibodies . Relative amounts of each fraction determined by Odyssey software are plotted .

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(d) Gel filtration analysis. The supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10/30 column and each fraction was immunoblotted with the indicated antibodies. Relative amounts of each fraction determined by Odyssey software are plotted.


( e ) The deficiency of Atg14L impairs the protein interaction between Beclin 1 and NRBF2 . NIH3T3 cells were transfected with Atg14L or NRBF2 RNAi for 72 h , respectively , and then were lysed and subjected to IP using Beclin 1 antibody . The immunoprecipitants were immunoblotted using the indicated antibodies .

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(e) The deficiency of Atg14L impairs the protein interaction between Beclin 1 and NRBF2. NIH3T3 cells were transfected with Atg14L or NRBF2 RNAi for 72 h, respectively, and then were lysed and subjected to IP using Beclin 1 antibody. The immunoprecipitants were immunoblotted using the indicated antibodies.


( f ) Direct binding between Atg14L and NRBF2 . Flag - tagged Atg14L purified from HEK293T cells overexpressing Flag - Atg14L was incubated with GST or NRBF2 - GST purified from Escherichia coli . The GST pull - down assay was performed to examine the direct binding between Flag - Atg14L and NRBF2 - GST .

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(f) Direct binding between Atg14L and NRBF2. Flag-tagged Atg14L purified from HEK293T cells overexpressing Flag-Atg14L was incubated with GST or NRBF2-GST purified from Escherichia coli. The GST pull-down assay was performed to examine the direct binding between Flag-Atg14L and NRBF2-GST.


( b ) NRBF2 KO MEFs were transfected with CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP for 48 h . Atg14L - linked Vps34 was pulled down by an anti - Atg14L antibody and subjected to KA . IP products and inputs were immunoblotted using the antibodies indicated . ( c ) Quantification of IPed Vps34 kinase activity ( PI ( 3 ) P ) , Vps34 and Vps15 protein versus IPed Atg14L protein . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , multiple t - test are followed by Bonferroni correction , n = 4 ( otherwise indicated , n means number of independent experiments ; NS , not significant ) ) .

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

(b) NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 48 h. Atg14L-linked Vps34 was pulled down by an anti-Atg14L antibody and subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (c) Quantification of IPed Vps34 kinase activity (PI(3)P), Vps34 and Vps15 protein versus IPed Atg14L protein. (Data are shown as mean±s.e.m., P values are indicated on the figures, multiple t-test are followed by Bonferroni correction, n=4 (otherwise indicated, n means number of independent experiments; NS, not significant)).


( d ) Overexpression of NRBF2 enhances Atg14L - linked Vps34kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and Flag - Atg14L in the presence of CFP or NRBF2 - CFP . Atg14L - linked Vps34 is pulled down by an anti - Flag antibody and subjected to KA ( upper panel ) . IP products and inputs were immunoblotted using the antibodies indicated ( upper panel ) . ( e ) Quantification of Vps34kinase activity ( lower panel ) shows the significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on the figures , one sample t - test versus hypothetical mean 1 , n = 5 ) .

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(d) Overexpression of NRBF2 enhances Atg14L-linked Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and Flag-Atg14L in the presence of CFP or NRBF2-CFP. Atg14L-linked Vps34 is pulled down by an anti-Flag antibody and subjected to KA (upper panel). IP products and inputs were immunoblotted using the antibodies indicated (upper panel). (e) Quantification of Vps34kinase activity (lower panel) shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


( a ) Starvation - induced long - lived protein degradation is impaired after NRBF2 knockdown . Compared with control ( con ) siRNA , NRBF2 siRNA decreases long - lived protein degradation ( proteolysis rate ) in NIH3T3 cells under starvation ( ST ) condition . This difference is diminished when the starved cells are co - treated with wortmannin ( WM , 100 nM ) . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , one - way analysis of variance with Dunnett as post hoc test , n = 4 ; NS , not significant ) .

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(a) Starvation-induced long-lived protein degradation is impaired after NRBF2 knockdown. Compared with control (con) siRNA, NRBF2 siRNA decreases long-lived protein degradation (proteolysis rate) in NIH3T3 cells under starvation (ST) condition. This difference is diminished when the starved cells are co-treated with wortmannin (WM, 100 nM). (Data are shown as mean±s.e.m., P values are indicated on the figures, one-way analysis of variance with Dunnett as post hoc test, n=4; NS, not significant).


( b ) Rapamycin ( Rap ) - induced autophagy is impaired in the NRBF2 KO cells . The MEFs were treated with ( dimethyl sulfoxide ) DMSO or 50 μg ml − 1 of Rap for 4 h then the cell lysates were immunoprobed with anti - LC3 and anti - p62 antibodies . ( c ) LC3 - II and p62 levels were quantified and normalized with β - actin . Quantification results show that Rap - induced LC3 lipidation ( upper ) and p62 degradation ( lower ) were markedly impaired in the NRBF2 KO MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , one sample t - test ( WT con versus KO con ) or unpaired t - test ( WT Rap versus KO Rap ) ) . p62 blots quantification , n = 6 ; LC3 blots quantification , n = 3 ) .

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

(b) Rapamycin (Rap)-induced autophagy is impaired in the NRBF2 KO cells. The MEFs were treated with (dimethyl sulfoxide) DMSO or 50 μg ml−1 of Rap for 4 h then the cell lysates were immunoprobed with anti-LC3 and anti-p62 antibodies. (c) LC3-II and p62 levels were quantified and normalized with β-actin. Quantification results show that Rap-induced LC3 lipidation (upper) and p62 degradation (lower) were markedly impaired in the NRBF2 KO MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figures, one sample t-test (WT con versus KO con) or unpaired t-test (WT Rap versus KO Rap)). p62 blots quantification, n=6; LC3 blots quantification, n=3).


( d , e ) The MEFs were subjected to Rap ( 50 μg ml − 1 ) or starvation ( HBSS ) treatment then stained with anti - LC3 ( d ) or anti - WIPI2 ( e ) antibody . ( f ) The LC3 and WIPI2 puncta per cell were counted and quantified . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , independent samples Kruskal - Wallis test followed by pairwise comparisons , n = 30 randomly selected cells , a representative result from three independent repeats ) .

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(d,e) The MEFs were subjected to Rap (50 μg ml−1) or starvation (HBSS) treatment then stained with anti-LC3 (d) or anti-WIPI2 (e) antibody. (f) The LC3 and WIPI2 puncta per cell were counted and quantified. (Data are shown as mean±s.e.m., P values are indicated on the figures, independent samples Kruskal-Wallis test followed by pairwise comparisons, n=30 randomly selected cells, a representative result from three independent repeats).


( g ) NRBF2 - CFP stable cells were starved in HBSS for 4 h and then subjected to WIPI2 staining . Under starved condition , NRBF2 - CFP forms puncta structures that co - localized with WIPI2 . This is a representative image from three independent experiments .

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(g) NRBF2-CFP stable cells were starved in HBSS for 4 h and then subjected to WIPI2 staining. Under starved condition, NRBF2-CFP forms puncta structures that co-localized with WIPI2. This is a representative image from three independent experiments.


( h ) The NRBF2 KO MEFs were transfected with CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP for 24 h and then followed by co - immunostaining with GFP and WIPI2 antibodies . ( i ) The WIPI2 puncta number per cell in GFP - positive cells were counted and quantified . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , independent samples Kruskal - Wallis test followed by pairwise comparisons , n = 30 randomly selected cells , a representative result from two independent repeats ) . Scale bars , 20 μm , or length indicated on the figure . CM , culture medium .

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(h) The NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 24 h and then followed by co-immunostaining with GFP and WIPI2 antibodies. (i) The WIPI2 puncta number per cell in GFP-positive cells were counted and quantified. (Data are shown as mean±s.e.m., P values are indicated on the figures, independent samples Kruskal-Wallis test followed by pairwise comparisons, n=30 randomly selected cells, a representative result from two independent repeats). Scale bars, 20 μm, or length indicated on the figure. CM, culture medium.


( a ) NRBF2 KO MEFs display impaired autophagosome acidification . WT and NRBF2 KO MEFs are transiently transfected with mCherry - GFP - LC3B . WT MEFs expressing mCherry - GFP - LC3B contain many red - only puncta along with yellow ( presence of both red and green ) puncta . The number of both red - only puncta and yellow puncta markedly increases after rapamycin ( Rap ; 50 μg ml − 1 , 4 h ) or starvation ( HBSS , 4 h ) treatment , suggesting the increased autophagolysosomes and nascent autophagosomes . In contrast , NRBF2 KO MEFs expressing mCherry - GFP - LC3B contain quite fewer red - only puncta and yellow puncta , even after Rap or starvation treatment . ( b ) The quantification results show that both the numbers of red - only puncta and percentage of red - only puncta ( verses total puncta ) in KO MEFs were markedly reduced . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , Mann - Whitney U - test , n = 20 randomly selected cells , a representative result from three independent repeats ) , indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells . Scale bars , 20 μm or 10 μm ( amplified regions ) .

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

(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry-GFP-LC3B. WT MEFs expressing mCherry-GFP-LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta markedly increases after rapamycin (Rap; 50 μg ml−1, 4 h) or starvation (HBSS, 4 h) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry-GFP-LC3B contain quite fewer red-only puncta and yellow puncta, even after Rap or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were markedly reduced. (Data are shown as mean±s.e.m., P values are indicated on the figures, Mann-Whitney U-test, n=20 randomly selected cells, a representative result from three independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bars, 20 μm or 10 μm (amplified regions).


( c ) Autophagolysome formation is impaired in NRBF2 KO MEFs . WT and NRBF2 KO MEFs were transiently transfected with RFP - LC3 then stained with LAMP2 antibody . The co - localization between RFP - LC3 and LAMP2 are illustrated by line profile . RFP - LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs ( as indicated by white arrows ) , suggesting the impairment of autophagolysosome formation in KO cells . Scale bars , 20 μm or 10 μm ( amplified regions ) . This is a representative image from three independent experiments .

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(c) Autophagolysome formation is impaired in NRBF2 KO MEFs. WT and NRBF2 KO MEFs were transiently transfected with RFP-LC3 then stained with LAMP2 antibody. The co-localization between RFP-LC3 and LAMP2 are illustrated by line profile. RFP-LC3 puncta recruit much stronger LAMP2 signal in WT MEFs than in NRBF2 KO MEFs (as indicated by white arrows), suggesting the impairment of autophagolysosome formation in KO cells. Scale bars, 20 μm or 10 μm (amplified regions). This is a representative image from three independent experiments.


( d ) Overexpression of NRBF2 enhances UVRAG - linked Vps34 kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and FLAG - UVRAG , in the presence of CFP or NRBF2 - CFP . UVRAG - linked Vps34 was pulled down by an anti - FLAG antibody and subjected to KA . IP products and inputs are immunoblotted using the antibodies indicated . ( e ) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on the figures , one sample t - test versus hypothetical mean 1 , n = 5 ) .

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(d) Overexpression of NRBF2 enhances UVRAG-linked Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and FLAG-UVRAG, in the presence of CFP or NRBF2-CFP. UVRAG-linked Vps34 was pulled down by an anti-FLAG antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of Vps34 kinase activity normalized with immunoprecipitated Vps34 shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


( a ) The gross anatomical views of representative 4 % paraformaldehyde - fixed livers and quantification of liver index . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test , n = 3 mice each group ) . Scale bar , 1 cm .

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(a) The gross anatomical views of representative 4% paraformaldehyde-fixed livers and quantification of liver index. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=3 mice each group). Scale bar, 1 cm.


( b ) The haematoxylin and eosin ( HE ) and CD45 staining of 10 - month - old WT and NRBF2 KO mice livers . The NRBF2 KO liver shows mild increase of ductular reaction ( yellow arrow ) and isolated necrosis ( red arrows ) . The necrotic foci were further confirmed by CD45 staining ( black arrow ) . This is a representative image from four mice per genotype . Scale bar , 50 μm . ( c ) The number of necrotic foci and ductular reaction scores were recorded under bright - field microscope . At least 10 randomly selected fields ( 10 × ) from each mouse and four mice per genotype were analysed . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures . Ductular reaction scores , unpaired Student ' s t - test ; necrotic foci , Mann - Whitney U - test , n = 4 mice each group ) . BD , bile duct ; CV , central vein ; PV , portal vein .

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(b) The haematoxylin and eosin (HE) and CD45 staining of 10-month-old WT and NRBF2 KO mice livers. The NRBF2 KO liver shows mild increase of ductular reaction (yellow arrow) and isolated necrosis (red arrows). The necrotic foci were further confirmed by CD45 staining (black arrow). This is a representative image from four mice per genotype. Scale bar, 50 μm. (c) The number of necrotic foci and ductular reaction scores were recorded under bright-field microscope. At least 10 randomly selected fields (10 × ) from each mouse and four mice per genotype were analysed. (Data are shown as mean±s.e.m., P values are indicated on the figures. Ductular reaction scores, unpaired Student's t-test; necrotic foci, Mann-Whitney U-test, n=4 mice each group). BD, bile duct; CV, central vein; PV, portal vein.


( a ) WB analysis shows accumulation of p62 in the liver of NRBF2 KO mice . Liver lysates were immunoprobed with antibodies indicated . p62 protein levels are upregulated in the KO mouse liver . ( b ) The quantification result shows that the p62 level increased more than threefold in the NRBF2 KO miceliver compared with WT miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test ; WT , n = 5 mice ; KO , n = 4 mice ) .

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(a) WB analysis shows accumulation of p62 in the liver of NRBF2 KO mice. Liver lysates were immunoprobed with antibodies indicated. p62 protein levels are upregulated in the KO mouse liver. (b) The quantification result shows that the p62 level increased more than threefold in the NRBF2 KO miceliver compared with WT miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test; WT, n=5 mice; KO, n=4 mice).


( c ) WB analysis shows accumulation of high - molecular weight ( HMW ) ubiquitin in the liver of NRBF2 KO mice . Insoluble fractions of liver lysates were immunoprobed with an anti - ubiquitin antibody , n = 4 mice per genotype .

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(c) WB analysis shows accumulation of high-molecular weight (HMW) ubiquitin in the liver of NRBF2 KO mice. Insoluble fractions of liver lysates were immunoprobed with an anti-ubiquitin antibody, n=4 mice per genotype.


( d ) Atg14L - linked Vps34kinase activity is impaired in the mice of NRBF2 KO mice . Atg14L - linked Vps34 is pulled down by an anti - Atg14L antibody and is subjected to KA . IP products and inputs were immunoblotted using the antibodies indicated . ( e ) Quantification of IPed Vps34kinase activity versus IPed Atg14L shows the significant difference between WT and NRBF2 KO miceliver . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 4 mice each group ) .

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(d) Atg14L-linked Vps34kinase activity is impaired in the mice of NRBF2 KO mice. Atg14L-linked Vps34 is pulled down by an anti-Atg14L antibody and is subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity versus IPed Atg14L shows the significant difference between WT and NRBF2 KO miceliver. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=4 mice each group).


( f ) Focal accumulation of p62 in the liver of NRBF2 KO mice . Liver frozen sections ( 15 μm in thickness ) were stained with an anti - p62 antibody . Focal accumulation of p62 is observed in the KO mice liver but not in the WT mice liver . Scale bars , 100 μm or 20 μm ( amplified regions ) . This is a representative image from four mice per genotype .

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(f) Focal accumulation of p62 in the liver of NRBF2 KO mice. Liver frozen sections (15 μm in thickness) were stained with an anti-p62 antibody. Focal accumulation of p62 is observed in the KO mice liver but not in the WT mice liver. Scale bars, 100 μm or 20 μm (amplified regions). This is a representative image from four mice per genotype.


( g ) Accumulated p62 colocalizes with NQO1 . Liver frozen sections were double stained with an anti - p62 antibody and an anti - NQO1 antibody . This is a representative image from four mice per genotype . Scale bars , 20 μm .

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(g) Accumulated p62 colocalizes with NQO1. Liver frozen sections were double stained with an anti-p62 antibody and an anti-NQO1 antibody. This is a representative image from four mice per genotype. Scale bars, 20 μm.


WT and NRBF2 KO MEFs were treated with DMSO , 1 μM thapsigargin ( TP ) or 1 μM tunicamycin ( TN ) for 24 or 48 h . ( a ) Then cells were either imaged under phase - contrast microscope or ( b ) stained with propidium iodide ( PI ) and subjected to cell death rate analysis by flow cytometry . The representative images of cell morphology after treatments and quantitative analysis of PI - positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 5 ) . Scale bars , 100 μm ; Con , control .

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

WT and NRBF2 KO MEFs were treated with DMSO, 1 μM thapsigargin(TP) or 1 μM tunicamycin (TN) for 24 or 48 h. (a) Then cells were either imaged under phase-contrast microscope or (b) stained with propidium iodide (PI) and subjected to cell death rate analysis by flow cytometry. The representative images of cell morphology after treatments and quantitative analysis of PI-positive cells rate show marked increase of cell death in the NRBF2 KO MEFs compared with WT MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=5). Scale bars, 100 μm; Con, control.


( c ) Re - introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress . NRBF2 - CFP stably expressing cells were established in the NRBF2 KO background . Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry . The cell death rate in CFP - positive ( + ) population ( NRBF2 - CFP expression ) and negative ( − ) population ( no NRBF2 - CFP expression ) were recorded and analysed , respectively . ( Data are shown as mean ± s . e . m . , P values are indicated on the figure , unpaired Student ' s t - test , n = 3 ) .

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 O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O 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) Re-introducing NRBF2 to NRBF2 KO MEFs reverses the vulnerability of cells to ER stress. NRBF2-CFP stably expressing cells were established in the NRBF2 KO background. Cells were treated with DMSO or 1 μM TP for 24 h then stained with PI and subjected to cell death rate analysis by flow cytometry. The cell death rate in CFP-positive (+) population (NRBF2-CFP expression) and negative (−) population (no NRBF2-CFP expression) were recorded and analysed, respectively. (Data are shown as mean±s.e.m., P values are indicated on the figure, unpaired Student's t-test, n=3).


( d ) ER stress inducers trigger caspase - 8 and caspase - 3 activation in NRBF2 KO MEFs . MEFs were treated with DMSO , 1 μM TP or 1 μM TN for 24 h , and then immunoprobed with indicated antibodies . Marked accumulation of cleaved caspase - 8 ( C - Cas 8 ; p43 and p18 units ) and cleaved caspase - 3 ( C - Cas 3 ) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN . This is a representative image from three independent experiments .

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

(d) ER stress inducers trigger caspase-8 and caspase-3 activation in NRBF2 KO MEFs. MEFs were treated with DMSO, 1 μM TP or 1 μM TN for 24 h, and then immunoprobed with indicated antibodies. Marked accumulation of cleaved caspase-8 (C-Cas 8; p43 and p18 units) and cleaved caspase-3 (C-Cas 3) are observed in the NRBF2 KO MEFs but not WT MEFs treated with TP or TN. This is a representative image from three independent experiments.


( e ) C - Cas 8 preferentially localizes at p62 aggregation foci . MEFs were treated with DMSO or 1 μM TP for 24 h , and then co - stained with an anti - p62 antibody and an anti - cleaved caspase - 8 antibody . The C - Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci . Scale bars , 20 μm . This is a representative image from three independent experiments .

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

(e) C-Cas 8 preferentially localizes at p62 aggregation foci. MEFs were treated with DMSO or 1 μM TP for 24 h, and then co-stained with an anti-p62 antibody and an anti-cleaved caspase-8 antibody. The C-Cas 8 fluorescence signals are triggered in NRBF2 KO MEFs treated with TP and preferentially localize at p62 aggregation foci. Scale bars, 20 μm. This is a representative image from three independent experiments.


( a ) NRBF2 is critical for the assembly of Vps34 - Vps15 and Atg14L - Beclin 1 complex . Brain lysate from WT and NRBF2 KO mice were subjected to co - IP by Vps34 antibody and Atg14L antibody . The IP products were subjected to in vitro lipid KA and WB analysis using the indicated antibodies . ( b , c ) Quantification results shows that Vps34 - Vps15 and Atg14L - Beclin 1 complex interactions as well as Atg14L - linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , unpaired Student ' s t - test , n = 4 mice in each group ; NS , not significant ) .

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

(a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid KA and WB analysis using the indicated antibodies. (b,c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are markedly impaired in NRBF2 KO mice. (Data are shown as mean±s.e.m., P values are indicated on the figures, unpaired Student's t-test, n=4 mice in each group; NS, not significant).


( d ) Overexpression of NRBF2 enhances overall Vps34kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP . Total Vps34 was pulled down by an anti - myc antibody and subjected to KA . IP products and inputs are immunoblotted using the antibodies indicated . ( e ) Quantification of IPed Vps34kinase activity , Vps15 protein normalized with IPed Vps34 shows that NRBF2 - CFP , but not CFP , dMIT - CFP or dCCD - CFP significantly enhance Vps34kinase activity and Vps34 - Vps15 interaction . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures . IPed Vps15 / IPed Vps34 , one sample t - test versus hypothetical mean 1 , Bonferroni correction , n = 3 ; IPed Vps34kinase activity / IPed Vps34 , multiple t - test followed by Bonferroni correction , n = 3 ) .

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

(d) Overexpression of NRBF2 enhances overall Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to KA. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34kinase activity and Vps34-Vps15 interaction. (Data are shown as mean±s.e.m., P values are indicated on the figures. IPed Vps15/IPed Vps34, one sample t-test versus hypothetical mean 1, Bonferroni correction, n=3; IPed Vps34kinase activity/IPed Vps34, multiple t-test followed by Bonferroni correction, n=3).


A NHF , Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation , then immunostained with LAMP1 antibody . Dashed lines in images outline cell boundaries . Scale bar , 10 μm . C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A . The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods . n = 3 per group in which 30 cells were counted per condition per experiment . Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test .

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

A NHF, Lowe 1676 and Lowe 3265 cells were grown in complete media or 5 h serum starvation, then immunostained with LAMP1 antibody. Dashed lines in images outline cell boundaries. Scale bar, 10 μm. C Quantitative analyses of lysosome perinuclear distribution in the experiments shown in A. The intracellular distribution of LAMP1 positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


B Wildtype ( Ocrl + / y ) and Ocrl - / - MEF cells were grown in complete media or 5 h serum starvation , then immunostained with indicated antibodies . Dashed lines in images outline cell boundaries . Scale bar , 10 μm .

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B Wildtype (Ocrl+/y) and Ocrl-/- MEF cells were grown in complete media or 5 h serum starvation, then immunostained with indicated antibodies. Dashed lines in images outline cell boundaries. Scale bar, 10 μm.


D , E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B . The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods . n = 3 per group in which 30 cells were counted per condition per experiment . Data information : , data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test .

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D, E Quantitative analyses of lysosome and mTOR perinuclear distribution in the experiments shown in B. The intracellular distribution of mTOR positive vesicles was quantified as described in the Methods. n = 3 per group in which 30 cells were counted per condition per experiment. Data information: , data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


I Snap images of WT ( Ocrl + / y ) , OCRL knockout ( Ocrl - / - ) and IOB MEFs transfected with lysosome - GFP . After transfection , WT , OCRL knockout and IOB MEFs were left in serum starvation for 8 h . Scale bar , 5 μm . J Quantitative analyses of tubulated lysosomes in the experiments shown in H . n = 3 per group in which 10 cells were counted per condition per experiment . D Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test .

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I Snap images of WT (Ocrl+/y), OCRL knockout (Ocrl-/-) and IOB MEFs transfected with lysosome-GFP. After transfection, WT, OCRL knockout and IOB MEFs were left in serum starvation for 8 h. Scale bar, 5 μm. J Quantitative analyses of tubulated lysosomes in the experiments shown in H. n = 3 per group in which 10 cells were counted per condition per experiment. D Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test.


A Immunoblot analysis of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K in NHF , Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF cells , three independent experiments . B Immunoblot analysis of endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K in WT ( Ocrl + / y ) and IOB MEFs grown in complete medium or 5 h serum starvation . p - p70 S6K ( Thr389 ) over total p70 S6K ratios normalized to NHF cells , three independent experiments . C Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test unless otherwise stated .

O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-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 B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K in NHF, Lowe 1676 and Lowe 3265 cells grown in complete media or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. B Immunoblot analysis of endogenous p-mTOR (Ser2448), total mTOR , p-p70 S6K (Thr389) and total p70 S6K in WT (Ocrl+/y) and IOB MEFs grown in complete medium or 5 h serum starvation. p-p70 S6K (Thr389) over total p70 S6K ratios normalized to NHF cells, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.


D NHF and Lowe 1676 cells were grown in complete media , amino - acid ( aa ) / FBS starved for 5 h , or starved and then recovered in amino - acid / FBS - containing medium , then immunoblotted using antibodies against endogenous p - mTOR ( Ser2448 ) , total mTOR , p - p70 S6K ( Thr389 ) and total p70 S6K . Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , * * * ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , n . s . , not significant , calculated by two - tailed Student ' s t - test unless otherwise stated .

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

D NHF and Lowe 1676 cells were grown in complete media, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunoblotted using antibodies against endogenous p-mTOR (Ser2448), total mTOR, p-p70 S6K (Thr389) and total p70 S6K. Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, ***≤ 0.001, ****P ≤ 0.0001, n.s., not significant, calculated by two-tailed Student's t-test unless otherwise stated.


A Representative images of NHF , Lowe 1676 and Lowe 3265 cells immunostained with anti - α - tubulin ( red ) and anti - γ - tubulin ( green ) antibodies . Scale bar , 10 μm . B Quantitative analyses of focused microtubule arrays numbers at γ - tubulin ( microtubule organizing center MTOC ) measured in the experiments shown in A . n = 3 cells per group , three independent experiments . C Data information : data are presented as mean ± SEM . * ≤ 0 . 05 , * * ≤ 0 . 01 , calculated by two - way ANOVA , multiple comparisons .

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

A Representative images of NHF, Lowe 1676 and Lowe 3265 cells immunostained with anti-α-tubulin (red) and anti-γ-tubulin (green) antibodies. Scale bar, 10 μm. B Quantitative analyses of focused microtubule arrays numbers at γ-tubulin (microtubule organizing center MTOC) measured in the experiments shown in A. n = 3 cells per group, three independent experiments. C Data information: data are presented as mean ± SEM. * ≤ 0.05, ** ≤ 0.01, calculated by two-way ANOVA, multiple comparisons.