( B ) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125 . Mean and standard deviation are presented ( N = 3 ) . Student ' s t ‐ test was used to analyse statistical significance , * P0 . 001 and * * P0 . 0001 .

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(B) LC3 was monitored by immunofluorescence in cells treated with EBSS for 3 h in the presence or absence of 10 μM JNK inhibitor SP600125. Mean and standard deviation are presented (N=3). Student's t‐test was used to analyse statistical significance, *P0.001 and **P0.0001.


( C ) BI ‐ 1 WT and KO cells were treated with EBSS for indicated time points , in the presence or absence of 10 μM of the JNK inhibitor SP600125 . Levels of phosphorylation of JNK ( pJNK ) and LC3 ‐ II were determined by western blot . The levels of total JNK and Hsp90 are shown as control ( N = 4 ) . Image was assembled from cropped lanes of the same western blot analysis of the same gel .

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(C) BI‐1 WT and KO cells were treated with EBSS for indicated time points, in the presence or absence of 10 μM of the JNK inhibitor SP600125. Levels of phosphorylation of JNK (pJNK) and LC3‐II were determined by western blot. The levels of total JNK and Hsp90 are shown as control (N=4). Image was assembled from cropped lanes of the same western blot analysis of the same gel.


( D ) BI ‐ 1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125 , and the electrophoretic shift associated with BCL ‐ 2 phosphorylation was monitored by western blot .

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(D) BI‐1 WT and KO cells were treated with EBSS for indicated time points in the presence or absence of 10 μM SP600125, and the electrophoretic shift associated with BCL‐2 phosphorylation was monitored by western blot.


( E ) BI ‐ 1 WT and KO cells were co ‐ transfected with expression vectors for Beclin ‐ 1 - MYC , BI ‐ 1 - HA , and BCL ‐ XL - FLAG . After 24 h , cells were treated with EBSS for 2 h or left untreated . The association of MYC ‐ tagged expressed Beclin ‐ 1 and BCL ‐ XL - FLAG was assessed by immunoprecipitation of Beclin ‐ 1 followed by western blot analysis .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O

(E) BI‐1 WT and KO cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. After 24 h, cells were treated with EBSS for 2 h or left untreated. The association of MYC‐tagged expressed Beclin‐1 and BCL‐XL-FLAG was assessed by immunoprecipitation of Beclin‐1 followed by western blot analysis.


( F ) 293T cells were co ‐ transfected with expression vectors for Beclin ‐ 1 - MYC , BI ‐ 1 - HA , and BCL ‐ XL - FLAG . Cell extracts were prepared in CHAPS buffer and Beclin ‐ 1 - MYC immunoprecipitated , and the possible co ‐ precipitation of BI ‐ 1 - HA , and BCL ‐ XL - FLAG was assessed by western blot analysis ( N = 3 ) . Asterisks indicate BI ‐ 1 oligomers .

O O O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

(F) 293T cells were co‐transfected with expression vectors for Beclin‐1-MYC, BI‐1-HA, and BCL‐XL-FLAG. Cell extracts were prepared in CHAPS buffer and Beclin‐1-MYC immunoprecipitated, and the possible co‐precipitation of BI‐1-HA, and BCL‐XL-FLAG was assessed by western blot analysis (N=3). Asterisks indicate BI‐1 oligomers.


( G ) ( G ) 293T cells were co ‐ transfected as described in ( F ) and BI ‐ 1 - HA was immunoprecipitated , and the possible co ‐ precipitation of Beclin ‐ 1 - MYC , and BCL ‐ XL - FLAG determined by western blot analysis ( N = 3 ) . Figure source data can be found with the Supplementary Information .

O O O O O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(G) (G) 293T cells were co‐transfected as described in (F) and BI‐1-HA was immunoprecipitated, and the possible co‐precipitation of Beclin‐1-MYC, and BCL‐XL-FLAG determined by western blot analysis (N=3). Figure source data can be found with the Supplementary Information.


( A ) BI ‐ 1 KO MEFs were stably transduced with lentiviral vectors expressing an shRNA against beclin ‐ 1 ( shBeclin ‐ 1 ) or ire1α ( shIRE1α ) mRNA or luciferase ( shLuc ) as control . The levels of LC3 , Beclin ‐ 1 , IRE1α and Hsp90 were monitored by western blot .

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(A) BI‐1 KO MEFs were stably transduced with lentiviral vectors expressing an shRNA against beclin‐1 (shBeclin‐1) or ire1α (shIRE1α) mRNA or luciferase (shLuc) as control. The levels of LC3, Beclin‐1, IRE1α and Hsp90 were monitored by western blot.


( B ) BI ‐ 1 KO MEFs were stably transduced with lentiviral vectors as described in A . Cells were exposed to EBSS for 3 h , and then LC3 levels were analysed by western blot . Image was assembled from cropped lanes of the same western blot analysis .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O

(B) BI‐1 KO MEFs were stably transduced with lentiviral vectors as described in A. Cells were exposed to EBSS for 3 h, and then LC3 levels were analysed by western blot. Image was assembled from cropped lanes of the same western blot analysis.


( C ) Endogenous LC3 distribution was visualized using immunofluorescence and confocal microscopy in BI ‐ 1 KO / shLuc and BI ‐ 1 KO / shBeclin ‐ 1 cells . Quantification represents the visualization of at least 180 cells . Student ' s t ‐ test was used to analyse statistical significance . Mean and standard deviation are presented , * P0 . 001 , NS : non ‐ significant .

O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O 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) Endogenous LC3 distribution was visualized using immunofluorescence and confocal microscopy in BI‐1 KO/shLuc and BI‐1 KO/shBeclin‐1 cells. Quantification represents the visualization of at least 180 cells. Student's t‐test was used to analyse statistical significance. Mean and standard deviation are presented, *P0.001, NS: non‐significant.


( D ) LC3 was visualized and quantified in BI ‐ 1 KO / shLuc and BI ‐ 1 KO / shIRE1α cells described in ( B ) by immunofluorescence and confocal microscopy analysis .

O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O

(D) LC3 was visualized and quantified in BI‐1 KO/shLuc and BI‐1 KO/shIRE1α cells described in (B) by immunofluorescence and confocal microscopy analysis.


( E ) BI ‐ 1 KO cells were transiently transfected with a TRAF2 dominant ‐ negative ( TRAF2 ‐ DN ) construct or empty vector ( mock ) , and after 48 h cells were stimulated with EBSS and the levels of LC3 ‐ II and Hsp90 were monitored by western blot . Right panel : quantification of relative LC3 ‐ II levels normalized with Hsp90 and non ‐ treated cells . Mean and standard deviation are presented .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD 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-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O

(E) BI‐1 KO cells were transiently transfected with a TRAF2 dominant‐negative (TRAF2‐DN) construct or empty vector (mock), and after 48 h cells were stimulated with EBSS and the levels of LC3‐II and Hsp90 were monitored by western blot. Right panel: quantification of relative LC3‐II levels normalized with Hsp90 and non‐treated cells. Mean and standard deviation are presented.


( F ) 293T cells were co ‐ transfected with expression vectors for HA ‐ tagged IRE1α ( IRE1 - HA ) , TRAF2 - FLAG , and MYC ‐ tagged BI ‐ 1 ( BI1 - MYC ) . After 48 h of transfection , HA ‐ tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot . Right panel : the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI ‐ 1 was quantified and normalized with the expression levels observed in the inputs . For comparison , the co ‐ IP signal observed in the absence of BI ‐ 1 was normalized as 100 % co ‐ IP in each independent experiment ( N = 3 ) . Mean and standard deviation are presented , * P0 . 05 . Figure source data can be found with the Supplementary Information .

O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) 293T cells were co‐transfected with expression vectors for HA‐tagged IRE1α (IRE1-HA), TRAF2-FLAG, and MYC‐tagged BI‐1 (BI1-MYC). After 48 h of transfection, HA‐tagged proteins were immunoprecipitated and the possible interaction with TRAF2 was analysed by western blot. Right panel: the percentage of TRAF2 dissociation from IRE1α by the presence or absence of BI‐1 was quantified and normalized with the expression levels observed in the inputs. For comparison, the co‐IP signal observed in the absence of BI‐1 was normalized as 100% co‐IP in each independent experiment (N=3). Mean and standard deviation are presented, *P0.05. Figure source data can be found with the Supplementary Information.


( A ) The expression of dBI ‐ 1 was knocked down in Drosophila melanogaster . Then , relative expression levels of dBI ‐ 1 mRNA were monitored by semiquantitative PCR . Actin levels were monitored as control .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O

(A) The expression of dBI‐1 was knocked down in Drosophila melanogaster. Then, relative expression levels of dBI‐1 mRNA were monitored by semiquantitative PCR. Actin levels were monitored as control.


( B ) ( B ) LC3 levels were monitored in control ( Da ‐ Gal4 > huLC3 : GFP ) or dBI ‐ 1 RNAi larvae ( Da ‐ Gal4 > huLC3 : GFP , Dcr2 , dBI ‐ 1i ) under basal or fasting conditions . Then , huLC3 - GFP levels were analysed by western blot . In addition , dBI ‐ 1i larvae were treated with 100 μM SP600125 ( added in the growing media ) .

O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O

(B) (B) LC3 levels were monitored in control (Da‐Gal4>huLC3:GFP) or dBI‐1 RNAi larvae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) under basal or fasting conditions. Then, huLC3-GFP levels were analysed by western blot. In addition, dBI‐1i larvae were treated with 100 μM SP600125 (added in the growing media).


( C ) Left panel : the presence of LC3 ‐ positive vesicles ( white arrowheads ) was monitored by confocal microscopy in control ( Da ‐ Gal4 > huLC3 > GFP ) and dBI ‐ 1 knockdown pupae ( Da ‐ Gal4 > huLC3 : GFP , Dcr2 , dBI ‐ 1i ) after 6 h of puparium formation . The organization of actin cytoskeleton was monitored by staining with phalloidin ( Ph , red ) . Nucleus was stained with Topro ( blue ) . Scale bar : left 20 μm and right 11 μm . Right panel : overexpression of dBI ‐ 1 ( dBI ‐ 1EY03662 ) delays salivary gland degradation . Overexpression of dBI ‐ 1 was confirmed by semiquantitative RT - PCR . Actin levels were monitored as loading control . Right panel : wild ‐ type control and dBI ‐ 1EY03662 pupae were analysed at 14 h after puparium formation . Superficial and internal confocal planes of the cells are presented . Scale bar : 40 μm .

O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O 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) Left panel: the presence of LC3‐positive vesicles (white arrowheads) was monitored by confocal microscopy in control (Da‐Gal4>huLC3>GFP) and dBI‐1 knockdown pupae (Da‐Gal4>huLC3:GFP, Dcr2, dBI‐1i) after 6 h of puparium formation. The organization of actin cytoskeleton was monitored by staining with phalloidin (Ph, red). Nucleus was stained with Topro (blue). Scale bar: left 20 μm and right 11 μm. Right panel: overexpression of dBI‐1 (dBI‐1EY03662) delays salivary gland degradation. Overexpression of dBI‐1 was confirmed by semiquantitative RT-PCR. Actin levels were monitored as loading control. Right panel: wild‐type control and dBI‐1EY03662 pupae were analysed at 14 h after puparium formation. Superficial and internal confocal planes of the cells are presented. Scale bar: 40 μm.


( D ) Wild ‐ type control and dBI ‐ 1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker ( red ) and Hoechst ( blue ) . Then , lysosomal content in the fat body was visualized by epifluorescence microscopy . The percentage of cells presenting lysotracker ‐ positive stain is indicated . Scale bar : 50 μm .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O B-TISSUE I-TISSUE O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O B-TISSUE I-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

(D) Wild‐type control and dBI‐1EY03662 larvae were cultured in fasting conditions for different periods of time and fat body stained with lysotracker (red) and Hoechst (blue). Then, lysosomal content in the fat body was visualized by epifluorescence microscopy. The percentage of cells presenting lysotracker‐positive stain is indicated. Scale bar: 50 μm.


( E ) Control or dBI ‐ 1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days . In all , 100 individuals were monitored in each condition . Data represent mean and standard error ( N = 3 ) . Two ‐ way ANOVA was used to analyse statistical significance between groups .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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) Control or dBI‐1 knockdown adult flies were exposed to nutrient starvation and then animal viability was monitored over time for several days. In all, 100 individuals were monitored in each condition. Data represent mean and standard error (N=3). Two‐way ANOVA was used to analyse statistical significance between groups.


( F ) Second instar dBI ‐ 1 RNAi or control larvae were grown in food supplemented with 25 μg / ml Tm dissolved in DMSO or 0 . 5 % DMSO as control . The number of individual reaching the adult fly stage was evaluated . Mean and standard error are presented ( N = 3 ) , * * P0 . 01 .

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(F) Second instar dBI‐1 RNAi or control larvae were grown in food supplemented with 25 μg/ml Tm dissolved in DMSO or 0.5% DMSO as control. The number of individual reaching the adult fly stage was evaluated. Mean and standard error are presented (N=3), **P0.01.


( A ) The levels of LC3 , Atg5 / Atg12 complex , Beclin ‐ 1 , Bcl ‐ 2 , Bcl ‐ XL , IRE1α and Hsp90 were monitored in liver protein extracts of bi ‐ 1 + / + and bi ‐ 1 − / − at 6 ‐ month ‐ old mice . Each well represents independent mice .

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(A) The levels of LC3, Atg5/Atg12 complex, Beclin‐1, Bcl‐2, Bcl‐XL, IRE1α and Hsp90 were monitored in liver protein extracts of bi‐1+/+ and bi‐1−/− at 6‐month‐old mice. Each well represents independent mice.


( B ) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver .

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(B) A similar analysis was performed for indicated markers in kidney protein extracts as described in liver.


( C ) bi ‐ 1 + / + and bi ‐ 1 − / − mice were injected with 50 ng / ml of tunicamycin by intraperitoneal injection ( N = 3 ) and then LC3 levels were monitored in liver protein extracts by western blot analysis . Right panel : quantification of relative LC3 ‐ II levels , * * P0 . 005 .

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(C) bi‐1+/+ and bi‐1−/− mice were injected with 50 ng/ml of tunicamycin by intraperitoneal injection (N=3) and then LC3 levels were monitored in liver protein extracts by western blot analysis. Right panel: quantification of relative LC3‐II levels, **P0.005.


A , B Regression plot showing the association between baseline AV45 PET ( x - axis ) and annual AV45 PET change ( y - axis ) across the entire sample ( N = 300 ) . The vertical dashed line represents the Aβ - positivity threshold of AV45 PET SUVR > 0 . 079 ( A ) . Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels ( B ) .

O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-TISSUE O O O O O O

A, B Regression plot showing the association between baseline AV45 PET (x-axis) and annual AV45 PET change (y-axis) across the entire sample (N=300). The vertical dashed line represents the Aβ-positivity threshold of AV45 PET SUVR > 0.079 (A). Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels (B).


Boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) , illustrating the differences in GE180 PET ( A ) compared between wild type C57BL / 6 mice ( n = 43 ) and APPNL - G - G ( n = 15 ) .

O 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-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O

Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in GE180 PET (A) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).


B Boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) , illustrating the differences in the rate of change in Florbetaben PET ( B ) compared between wild type C57BL / 6 mice ( n = 43 ) and APPNL - G - G ( n = 15 ) .

O O O O O O O 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-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O

B Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in the rate of change in Florbetaben PET (B) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).


C The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline , i . e . at 5 months of age ( C ) . The shaded area corresponds to the 95 % CI of the regression line .

O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O 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 The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline, i.e. at 5 months of age (C). The shaded area corresponds to the 95% CI of the regression line.


A - C Coronal ( top row ) and axial ( bottom row ) slices of average 18 - F GE - 180 TSPO ( microglia ) tracer and 18F - florbetaben amyloid - PET split up by group ( A - C ) : C57BL / 6 ( A ) , APPNL - G - F mice subgroup with a low ( < median ) GE - 180 PET ( B ) , and the APPNL - G - F mice subgroup with a high ( > median ) GE - 180 PET ( C ) . APPNL - G - F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months ( B ) compared to those at high baseline level of GE - 180 PET ( C ) .

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A-C Coronal (top row) and axial (bottom row) slices of average 18-F GE-180 TSPO (microglia) tracer and 18F-florbetaben amyloid-PET split up by group (A - C): C57BL/6 (A), APPNL-G-F mice subgroup with a low (< median) GE-180 PET (B), and the APPNL-G-F mice subgroup with a high (> median) GE-180 PET (C). APPNL-G-F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months (B) compared to those at high baseline level of GE-180 PET (C).


Effects of sTREM2 on AV1451 - PET - assessed tau pathology stratified by Braak - stages shown as boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) .

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Effects of sTREM2 on AV1451-PET-assessed tau pathology stratified by Braak-stages shown as boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range).


( A ) Vero cell was inoculated with BALF sample , and the CPEs were observed at 4 - dpi .

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(A) Vero cell was inoculated with BALF sample, and the CPEs were observed at 4-dpi.


( B ) RNAs were extracted from the cell culture supernatant , and detected using a commercial kit targeting the ORF 1ab ( red ) and N ( blue ) genes of SARS - CoV - 2 .

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(B) RNAs were extracted from the cell culture supernatant, and detected using a commercial kit targeting the ORF 1ab (red) and N (blue) genes of SARS-CoV-2.


Testing the convalescent plasma IgG antibody ( C ) using the purified SARS - CoV - 2 particles . The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer , respectively . The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory . The PBS control was serum - free .

O O O B-TISSUE B-SUBCELLULAR O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-TISSUE O O O O O O O B-ORGANISM O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O O O O O O O O O O O

Testing the convalescent plasma IgG antibody (C) using the purified SARS-CoV-2 particles. The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer, respectively. The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory. The PBS control was serum-free.


Testing the SARA - CoV - 2 - RBD specific human monoclonal antibodies ( D ) using the purified SARS - CoV - 2 particles . The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer , respectively . The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory . The PBS control was serum - free .

O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-TISSUE O O O O O O O B-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O

Testing the SARA-CoV-2-RBD specific human monoclonal antibodies (D) using the purified SARS-CoV-2 particles. The control plasmas 1 and 2 were obtained from a patient recovered from influenza A virus infection and a healthy volunteer, respectively. The control monoclonal antibody is a human monoclonal antibody specific to influenza A virus generated by our laboratory. The PBS control was serum-free.


( E ) Viruses were detected by IFA using the patient ' s plasma , and plasma from a healthy control was used as negative control . Scale bar , 100 μm .

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(E) Viruses were detected by IFA using the patient's plasma, and plasma from a healthy control was used as negative control. Scale bar, 100 μm.


( A ) . Image of negative stained SARS - CoV - 2 . Nail - like spikes can be clearly seen .

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(A). Image of negative stained SARS-CoV-2. Nail-like spikes can be clearly seen.


Enlarged view of virion boxed in ( A ) . ( C ) Zoom - in view of a spike boxed in ( B ) . The shape is depicted by red dot line . Length , the diameter of stem and spike ' s head are 23nm , 4nm and 7nm , respectively .

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Enlarged view of virion boxed in (A). (C) Zoom-in view of a spike boxed in (B). The shape is depicted by red dot line. Length, the diameter of stem and spike's head are 23nm, 4nm and 7nm, respectively.


( A ) and ( B ) . Cryo - EM images of SARS - CoV - 2 . ( C ) . Zoom - in view of the virion showed in ( A ) . Envelope and nucleocapsid are indicated by green and blue respectively , remarkable spikes are indicated by red triangles . ( D ) . Zoom - in views of the two virions showed in ( B ) , remarkable spikes are indicated by red triangles . ( E ) . Zoom - in view of the spike indicated by yellow triangle in ( C ) . The shape is depicted by yellow dot lines . ( F ) . Zoom - in view of the spike indicated by yellow triangle in ( D ) . The shape is depicted by yellow dot lines .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O 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-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O

(A) and (B). Cryo-EM images of SARS-CoV-2. (C). Zoom-in view of the virion showed in (A). Envelope and nucleocapsid are indicated by green and blue respectively, remarkable spikes are indicated by red triangles. (D). Zoom-in views of the two virions showed in (B), remarkable spikes are indicated by red triangles. (E). Zoom-in view of the spike indicated by yellow triangle in (C). The shape is depicted by yellow dot lines. (F). Zoom-in view of the spike indicated by yellow triangle in (D). The shape is depicted by yellow dot lines.


( A ) Levels of Piwil1 ( Miwi ) , Piwil2 ( Mili ) and Piwil4 ( Miwi2 ) transcripts in RNA seq . data from undifferentiated aNPCs ( Days of differentiation - DIF - 0 ) and differentiating neuroblasts ( DIF 4 - 14 ) ; insets in left and right panels show the same data with smaller scales in the ordinate axes . Data information : data are expressed as mean ± SEM , n = 3 biological replicates .

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(A) Levels of Piwil1 (Miwi), Piwil2 (Mili) and Piwil4 (Miwi2) transcripts in RNA seq. data from undifferentiated aNPCs (Days of differentiation -DIF- 0) and differentiating neuroblasts (DIF 4-14); insets in left and right panels show the same data with smaller scales in the ordinate axes. Data information: data are expressed as mean ± SEM, n = 3 biological replicates.


( B ) Western blot ( inset ) and quantification ( bar graph ) of Mili protein abundance in DIF0 aNPCs and differentiating neuroblasts upon viral - induced neurogenesis ( DIF4 - 14 ) . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni

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(B) Western blot (inset) and quantification (bar graph) of Mili protein abundance in DIF0 aNPCs and differentiating neuroblasts upon viral-induced neurogenesis (DIF4-14). Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test


( C , D ) Western blot ( insets ) and quantification ( bar graphs ) of Miwi and Mili protein abundance in lysates from postnatal mouse testis , hippocampus and undifferentiated aNPCs cultures . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) Western blot (insets) and quantification (bar graphs) of Miwi and Mili protein abundance in lysates from postnatal mouse testis, hippocampus and undifferentiated aNPCs cultures. Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test


( E ) Western blot ( inset ) and quantification ( bar graph ) of Mili protein abundance in lysates from cultured mouse hippocampal neurons and undifferentiated aNPCs . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by two - tailed Student ' s t - test ( in E ) .

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(E) Western blot (inset) and quantification (bar graph) of Mili protein abundance in lysates from cultured mouse hippocampal neurons and undifferentiated aNPCs. Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by two-tailed Student's t-test (in E).


( F ) Representative immunofluorescence micrograph of Mili ( white ) and Td + NSCs ( red ) in hippocampal subgranular zone ( SGZ ) ; arrows indicate Td + Mili + double - positive cells . Data information : In F : GCL , granular cell layer ; H , Hilus . The scale bars represent 10 µm .

O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O B-TISSUE O O O O O O O O

(F) Representative immunofluorescence micrograph of Mili (white) and Td+ NSCs (red) in hippocampal subgranular zone (SGZ); arrows indicate Td+ Mili + double-positive cells. Data information: In F: GCL, granular cell layer; H, Hilus. The scale bars represent 10 µm.


( G ) Scheme of the experiment ( top ) and Mili mRNA expression in sorted Td + and Td - cells after in vivo transduction with split - Cre viruses in the hippocampus ( bottom ) . Data information : data are expressed as mean ± SEM , n = 3 biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 as assessed by one - way ANOVA with Bonferroni test G )

O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Scheme of the experiment (top) and Mili mRNA expression in sorted Td+ and Td- cells after in vivo transduction with split-Cre viruses in the hippocampus (bottom). Data information: data are expressed as mean ± SEM, n = 3 biological replicates. *p < 0.05, **p < 0.01, **** p < 0.0001 as assessed by one-way ANOVA with Bonferroni test G)


( E ) Mili mRNA expression ( left bar graph ) ; western blot ( inset ) and quantification ( right bar graph ) of Mili protein abundance in aNPCs upon viral transduction of scrambled shRNA ( Control ) or shRNA targeting Mili ( Mili KD ) . Data information : data are expressed as mean ± SEM unless differently indicated , n = 3 biological replicates ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test .

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(E) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in aNPCs upon viral transduction of scrambled shRNA (Control) or shRNA targeting Mili (Mili KD). Data information: data are expressed as mean ± SEM unless differently indicated, n = 3 biological replicates ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test.


Western blot ( G ) of the endogenous piCS after co - immunoprecipitation ( IP ) with endogenous Mili ( IP Mili ) , or control IgG ( IP IgG ) in lysates of DIF4 aNPCs .

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Western blot (G) of the endogenous piCS after co-immunoprecipitation (IP) with endogenous Mili (IP Mili), or control IgG (IP IgG) in lysates of DIF4 aNPCs.


( C ) Mili mRNA expression ( left bar graph ) ; western blot ( inset ) and quantification ( right bar graph ) of Mili protein abundance in lysates from the DG of mouse hippocampi 48 hours after the injection of scrambled ( Control ) or GapmeR1 against Mili ( Mili KD ) . Data information : data are expressed as mean ± SEM , n = 3 ( C , biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test .

O O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O B-ORGANISM B-TISSUE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mili mRNA expression (left bar graph); western blot (inset) and quantification (right bar graph) of Mili protein abundance in lysates from the DG of mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 against Mili (Mili KD). Data information: data are expressed as mean ± SEM, n = 3 (C, biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test.


( E ) Representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP at 30 dpi of scrambled ( Control ) and GapmeR1 against Mili ( Mili KD ) ; Right panels : Fold change in GFAP fluorescence intensity level ( upper graph ) in a hippocampal region of interest ( ROI ) of 500µm2 in brain slices upon Mili KD compared to Control ; Gfap mRNA levels ( lower graph ) in the DG from mouse hippocampi 48 hours after the injection of scrambled ( Control ) or GapmeR1 ( Mili KD ) . Data information : data are expressed as mean ± SEM , n = 3 E mRNA and 5 ( E biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . SGZ , subgranular zone . The scale bars represent 100 µm ( E ,

O O O O B-EXP_ASSAY O O O B-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-TISSUE O B-ORGANISM B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O

(E) Representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP at 30 dpi of scrambled (Control) and GapmeR1 against Mili (Mili KD); Right panels: Fold change in GFAP fluorescence intensity level (upper graph) in a hippocampal region of interest (ROI) of 500µm2 in brain slices upon Mili KD compared to Control; Gfap mRNA levels (lower graph) in the DG from mouse hippocampi 48 hours after the injection of scrambled (Control) or GapmeR1 (Mili KD). Data information: data are expressed as mean ± SEM, n = 3 E mRNA and 5 (E biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. SGZ, subgranular zone. The scale bars represent 100 µm (E,


( F ) ( Left ) representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP ( green ) , BrdU ( red ) , NeuN ( white ) and nuclear DNA ( blue ) at 30 dpi of scrambled ( Control ) or GapmeR1 against Mili ( Mili KD ) ; ( right graphs ) percentages of NeuN + BrdU + ( white arrowheads in the images ) , or GFAP + BrdU + ( yellow arrowheads in the images ) double - positive cells over total BrdU + cells . Data information : data are expressed as mean ± SEM , n = F ) biological replicates . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . GCL , granular cell layer , SGZ , subgranular zone . The scale bars represent 100 µm F ) .

O O O O O O O B-EXP_ASSAY O O O B-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O

(F) (Left) representative immunofluorescence micrograph of postnatal hippocampal sections immunostained for GFAP (green), BrdU (red), NeuN (white) and nuclear DNA (blue) at 30 dpi of scrambled (Control) or GapmeR1 against Mili (Mili KD); (right graphs) percentages of NeuN+BrdU+ (white arrowheads in the images), or GFAP+BrdU+ (yellow arrowheads in the images) double-positive cells over total BrdU+ cells. Data information: data are expressed as mean ± SEM, n = F) biological replicates. ∗p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. GCL, granular cell layer, SGZ, subgranular zone. The scale bars represent 100 µm F).


( B ) Representative bright - field microscopy images ( left ) and quantification ( right ) of ß - galactosidase + aNPCs as percent of total cells upon Mili KD , or control 48 h after induction of spontaneous differentiation . Data information : data are expressed as mean ± SEM , n = 5 ( B biological replicates p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . The scale bars represent 50 µm ( B ,

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative bright-field microscopy images (left) and quantification (right) of ß-galactosidase+ aNPCs as percent of total cells upon Mili KD, or control 48 h after induction of spontaneous differentiation. Data information: data are expressed as mean ± SEM, n = 5 (B biological replicates p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm (B,


( C ) Representative fluorescence microscopy images ( left ) and quantification ( right ) of control or Mili KD neuroblasts 48h after spontaneous differentiation , immunostained with anti - BrdU ( white ) and Ki67 ( purple ) antibodies . ( Right ) Percentage of BrdU + and Ki67 - cells over BrdU + cells . Data information : data are expressed as mean ± SEM , n = C ) biological replicates p < 0 . 05 , ∗ ∗ p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , as assessed by two - tailed Student ' s t - test . The scale bars represent 50 µm C )

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative fluorescence microscopy images (left) and quantification (right) of control or Mili KD neuroblasts 48h after spontaneous differentiation, immunostained with anti-BrdU (white) and Ki67 (purple) antibodies. (Right) Percentage of BrdU+ and Ki67- cells over BrdU+ cells. Data information: data are expressed as mean ± SEM, n = C) biological replicates p < 0.05, ∗∗p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed by two-tailed Student's t-test. The scale bars represent 50 µm C)


F , Chow fed hIGFREO had similar body mass to WT , however , hIGFREO did not gain as much weight as WT after 8 weeks of HFD ( n = 6 - 10 mice per group ) .

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F, Chow fed hIGFREO had similar body mass to WT, however, hIGFREO did not gain as much weight as WT after 8 weeks of HFD (n=6-10 mice per group).


G , Representative images of difference in fat and water distribution shown by magnetic resonance ( MR ) imaging in hIGFREO and WT . Scale bar = 1cm .

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G, Representative images of difference in fat and water distribution shown by magnetic resonance (MR) imaging in hIGFREO and WT. Scale bar = 1cm.


hIGFREO glucose intolerance measured by glucose tolerance test ( n = 5 - 7 mice per group ) .

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hIGFREO glucose intolerance measured by glucose tolerance test (n=5-7 mice per group).


G , H , Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve ( n = 10 - 12 mice per group ) .

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G, H, Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve (n=10-12 mice per group).


hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates ( WT ) after HFD . ( n = 4 per genotype ) .

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hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates (WT) after HFD. (n=4 per genotype).


D , Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT ( n = 6 per genotype ) .

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D, Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT (n= 6 per genotype).


Faith ' s phylogenetic diversity ( PD ) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF - 1R endothelial overexpressing mice ( hIGFREO ) mice and wildtype littermates ( WT ) mice after high fat diet feeding ( n = 4 - 5 mice per group ) . Chao - 1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT ( n = 4 - 5 mice per group ) .

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Faith's phylogenetic diversity (PD) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF-1R endothelial overexpressing mice (hIGFREO) mice and wildtype littermates (WT) mice after high fat diet feeding (n=4-5 mice per group). Chao-1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT (n=4-5 mice per group).


B , Bi , human IGF - 1R endothelial overexpressing mice ( hIGFREO ) had comparable weight gain as wildtype littermates ( WT ) after 8 weeks of HFD + antibiotics ( ABs ) when compared to WT . Bii , both hIGFREO and WT gained significant weight compared to chow fed mice ( n = 7 = 9 mice per group ) .

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B, Bi, human IGF-1R endothelial overexpressing mice (hIGFREO) had comparable weight gain as wildtype littermates (WT) after 8 weeks of HFD + antibiotics (ABs) when compared to WT. Bii, both hIGFREO and WT gained significant weight compared to chow fed mice (n=7=9 mice per group).


D , E , There was no difference in hIGFREO and WT glucose tolerance ( as measured by glucose tolerance test and area under the curve ( AUC ) ) ( n = 7 - 9 mice per group ) .

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D, E, There was no difference in hIGFREO and WT glucose tolerance (as measured by glucose tolerance test and area under the curve (AUC)) (n=7-9 mice per group).


Characteristic micrographs for both data sets , respectively ( left and right ) .

Characteristic micrographs for both data sets, respectively (left and right).


g - h ) Representative β - catenin ChIP - qPCR of indicated target genes in wt and TCF1 / LEF1 long isoform double KO ( DLKO ) clones ( g ) and Kaiso KO clones ( h ) in RPMI8402 cells . Enrichment of each ChIP is normalized to its respective negative IgG control , indicated by a red - dashed line . DLKO1 , DLKO2 , KKO1 and KKO2 designate independent single - cell derived clones . Graph represents the mean and sd of 3 technical replicates .

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g-h) Representative β-catenin ChIP-qPCR of indicated target genes in wt and TCF1/LEF1 long isoform double KO (DLKO) clones (g) and Kaiso KO clones (h) in RPMI8402 cells. Enrichment of each ChIP is normalized to its respective negative IgG control, indicated by a red-dashed line. DLKO1, DLKO2, KKO1 and KKO2 designate independent single-cell derived clones. Graph represents the mean and sd of 3 technical replicates.


e ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell line ( right panel ) after knockdown of β - catenin ( left panel ) compared to control . Graph represents the mean and sd of 6 independent experiments . f ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell line after treatment with the β - catenin inhibitor ICG - 001 ( 10µM , overnight ) . Graph represents the mean and sd of 3 independent experiments . g ) Relative mRNA expression of β - catenin target genes in the RPMI8402 cell lines ( right panel ) after knockdown of ZBTB33 / Kaiso ( left panel ) compared to control . Graph represents the mean and sd of 3 independent experiments . Data information : Statistical significance was determined by two - sided Student ' s t test .

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e) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line (right panel) after knockdown of β-catenin (left panel) compared to control. Graph represents the mean and sd of 6 independent experiments. f) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell line after treatment with the β-catenin inhibitor ICG-001 (10µM, overnight). Graph represents the mean and sd of 3 independent experiments. g) Relative mRNA expression of β-catenin target genes in the RPMI8402 cell lines (right panel) after knockdown of ZBTB33/Kaiso (left panel) compared to control. Graph represents the mean and sd of 3 independent experiments. Data information: Statistical significance was determined by two-sided Student's t test.


c ) Kaplan - Meier curves representing disease free ( DSF , top panel ) and overall survival ( OS , bottom panel ) probability for groups of patients P1 ( N = 7 ) , P2 ( N = 5 ) , P3 ( N = 4 ) , P4 ( N = 9 ) and P5 ( N = 15 ) obtained in a ) . Time is represented in years . Statistical significance among groups was determined by log - rank test .

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c) Kaplan-Meier curves representing disease free (DSF, top panel) and overall survival (OS, bottom panel) probability for groups of patients P1 (N = 7), P2 (N = 5), P3 (N = 4), P4 (N = 9) and P5 (N = 15) obtained in a). Time is represented in years. Statistical significance among groups was determined by log-rank test.


a ) Effect of Vincristine ( VCR ) treatment on cell viability of RPMI8402 cells transduced with sh β - catenin or sh - control . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * p ≤ 0 . 001 of IC50 comparison with respect to sh control Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for a ( at day 0 )

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a) Effect of Vincristine (VCR) treatment on cell viability of RPMI8402 cells transduced with sh β-catenin or sh-control. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. *** p ≤ 0.001 of IC50 comparison with respect to sh control Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for a (at day 0)


Effect of VCR ( b ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 comparison with respect VCR - treated cells . Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for , b , ( at day 0 )

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Effect of VCR (b) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 comparison with respect VCR-treated cells. Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for , b, (at day 0)


Methotrexate ( MTX ) ( c left ) , L - Asparaginase ( L - ASP ) ( c central ) or Cytarabine ( Ara - C ) ( c right ) treatment in the presence or absence of increasing doses of the β - catenin inhibitor ICG - 001 . Dose - response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments . * * * * p ≤ 0 . 0001 , * * * p ≤ 0 . 001 , * * p ≤ 0 . 01 , * p ≤ 0 . 05 of IC50 Data information : For all applicable figure panels , data represent mean ± SD of three independent experiments . Statistical significance was determined by two - sided Student ' s t test for c , ( at day 0 )

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Methotrexate (MTX) (c left), L-Asparaginase (L-ASP) (c central) or Cytarabine (Ara-C) (c right) treatment in the presence or absence of increasing doses of the β-catenin inhibitor ICG-001. Dose-response curves represent the logistic fitting and individual points correspond to mean ± SD of three independent experiments. **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05 of IC50 Data information: For all applicable figure panels, data represent mean ± SD of three independent experiments. Statistical significance was determined by two-sided Student's t test for c, (at day 0)


c ) Percentage of RPMI8402 cells PB , BM , spleen and liver at sacrifice . RPMI8402 cells identified as human CD45 + HLA - ABC + CD5 + by flow cytometry and gated on DAPI negative cells . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by two - sided Student ' s t test .

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c) Percentage of RPMI8402 cells PB, BM, spleen and liver at sacrifice. RPMI8402 cells identified as human CD45+HLA-ABC+CD5+ by flow cytometry and gated on DAPI negative cells. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.


g ) H & E staining of representative images per each mouse treated as indicated . h ) Percentage of liver area covered by cellular colonies per 10X micrograph . 4 different images were quantified per animal and mean values per animal are represented by dots . Control : animals left untreated ( n = 2 ) ; Chemo + DMSO : animals receiving chemotherapy and vehicle ( n = 4 ) ; Chemo + ICG - 001 : animals receiving chemotherapy and the β - catenin inhibitor ICG - 001 ( n = 4 ) Data information : For all applicable figure panels , data from Chemo + DMSO and Chemo + ICG - 001 groups are mean ± SD and dots represent individual values per animal . Control group data is only represented by individual values per animal . Statistical significance between Chemo + DMSO and Chemo + ICG - 001 groups was determined by two - sided Student ' s t test .

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g) H&E staining of representative images per each mouse treated as indicated. h) Percentage of liver area covered by cellular colonies per 10X micrograph. 4 different images were quantified per animal and mean values per animal are represented by dots. Control: animals left untreated (n = 2); Chemo + DMSO: animals receiving chemotherapy and vehicle (n = 4); Chemo + ICG-001: animals receiving chemotherapy and the β-catenin inhibitor ICG-001 (n = 4) Data information: For all applicable figure panels, data from Chemo + DMSO and Chemo + ICG-001 groups are mean ± SD and dots represent individual values per animal. Control group data is only represented by individual values per animal. Statistical significance between Chemo + DMSO and Chemo + ICG-001 groups was determined by two-sided Student's t test.


Representative micrographs of a time - lapse microscopy of K and KM non - regressed tumour cells ( H2B - GFP green ) . Top : Mitotic cell with a chromatin bridge ( yellow arrow ) . Bottom : Mitotic cell with misalignment and cytokinesis failure resulting in binucleation ( yellow arrow ) .

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Representative micrographs of a time-lapse microscopy of K and KM non-regressed tumour cells (H2B-GFP green). Top: Mitotic cell with a chromatin bridge (yellow arrow). Bottom: Mitotic cell with misalignment and cytokinesis failure resulting in binucleation (yellow arrow).


Percentage of mitotic errors in K and KM primary tumours and in K and KM non - regressed tumours . Percentage of cells in K and KM primary tumours and in K and KM non - regressed tumours with the indicated mitotic errors . Scale bar 20 µm .

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Percentage of mitotic errors in K and KM primary tumours and in K and KM non-regressed tumours. Percentage of cells in K and KM primary tumours and in K and KM non-regressed tumours with the indicated mitotic errors. Scale bar 20 µm.


Somatic copy number alterations in each individual Kras and Kras / Mad2 non - regressed tumours . 7 K non - regressed and 16 KM non - regressed tumours were sequenced . Focal deletion ( DEL , green ) , Focal amplification ( AMP , light green ) , whole chromosome gain ( WCG , dark grey ) and loss ( WCL , light grey ) , partial chromosome gain ( PCG , dark blue ) and loss ( PCL , light blue ) and gross chromosomal rearrangement ( GCR , red ) .

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Somatic copy number alterations in each individual Kras and Kras/Mad2 non-regressed tumours. 7 K non-regressed and 16 KM non-regressed tumours were sequenced. Focal deletion (DEL, green), Focal amplification (AMP, light green), whole chromosome gain (WCG, dark grey) and loss (WCL, light grey), partial chromosome gain (PCG, dark blue) and loss (PCL, light blue) and gross chromosomal rearrangement (GCR, red).


Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene . Colour of the block corresponds to the segment mean ( the degree to which a genome segment is lost ( blue ) or gained ( red ) .

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Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene. Colour of the block corresponds to the segment mean (the degree to which a genome segment is lost (blue) or gained (red).


Immunostaining of phospho - cMet in non - regressed tumours carrying or not the amplification in chromosome 6 . Yellow numbers indicate the total number of cMet positive cells / total number of cells counted . Scale bar 100 µm .

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Immunostaining of phospho-cMet in non-regressed tumours carrying or not the amplification in chromosome 6. Yellow numbers indicate the total number of cMet positive cells/total number of cells counted. Scale bar 100 µm.


Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification . 0 indicates when doxycycline was removed . Each colour represents one tumour . Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth .

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Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification. 0 indicates when doxycycline was removed. Each colour represents one tumour. Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth.


Genome - wide log2 - ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non - regressed tumour showing no amplification in the primary tumour ( upper panels ) and a small amplification in the non - regressed tumours ( bottom panels , yellow arrow ) .

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Genome-wide log2-ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non-regressed tumour showing no amplification in the primary tumour (upper panels) and a small amplification in the non-regressed tumours (bottom panels, yellow arrow).


Immunostaining of phospho - cMet in 3 biopsied primary tumours ( PT ) and their corresponding non - regressed tumours ( KM5 , KM7 and KM8 , which are also shown in Fig 2C ) . Scale bar 100 µm .

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Immunostaining of phospho-cMet in 3 biopsied primary tumours (PT) and their corresponding non-regressed tumours (KM5, KM7 and KM8, which are also shown in Fig 2C). Scale bar 100 µm.


Representative two - dimensional scatter plots constructed with overlaid dPCR data of the reference ( VIC ) and cMet ( FAM ) from one tumour without cMet amplification and one with cMet amplified . Dots represent results of independent PCR reactions in the wells of a digital PCR chip . Reactions in the bottom left corner ( yellow ) are negative for both targets , while the ones in the top right corner ( green ) are double positives . Reactions in the top left ( blue ) and bottom right ( red ) corners are positive for cMet and the reference targets , respectively .

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Representative two-dimensional scatter plots constructed with overlaid dPCR data of the reference (VIC) and cMet (FAM) from one tumour without cMet amplification and one with cMet amplified. Dots represent results of independent PCR reactions in the wells of a digital PCR chip. Reactions in the bottom left corner (yellow) are negative for both targets, while the ones in the top right corner (green) are double positives. Reactions in the top left (blue) and bottom right (red) corners are positive for cMet and the reference targets, respectively.


Representative photographs of FISH staining with a probe for Met ( red signal ) and a probe for a reference gene EML4 ( green signals ) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots ( white arrows ) . The lower panel shows an example of a tumour with cMet amplification ( several red dots ) and 2 green dots ( yellow arrow ) . Scale bar 10 µm .

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Representative photographs of FISH staining with a probe for Met (red signal) and a probe for a reference gene EML4 (green signals) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots (white arrows). The lower panel shows an example of a tumour with cMet amplification (several red dots) and 2 green dots (yellow arrow). Scale bar 10 µm.


Relative volume of tumours grown in 12 Rag2 - / - animals after treatment with Tepotinib or vehicle control ( 3K and 3KM tumours for each condition ) . No statistical significance was found by One - way ANOVA . Mean + / - SEM .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Relative volume of tumours grown in 12 Rag2-/- animals after treatment with Tepotinib or vehicle control (3K and 3KM tumours for each condition). No statistical significance was found by One-way ANOVA. Mean +/- SEM.


Relative volume of tumours grown in Rag2 - / - animals after treatment with Tepotinib or vehicle control . One - way ANOVA , Tukey ' s multiple comparison . Ns : not significant ; * * p < 0 . 024 . Mean + / - SEM . A total of 19 Rag2 - / - mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls . For tumours with no cMet , 13 Rag2 - / - mice were treated with Tepotinib and 9 were used as control .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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-GENEPROD O O O B-ORGANISM O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O

Relative volume of tumours grown in Rag2-/- animals after treatment with Tepotinib or vehicle control. One-way ANOVA, Tukey's multiple comparison. Ns: not significant; ** p<0.024. Mean +/- SEM. A total of 19 Rag2-/- mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls. For tumours with no cMet, 13 Rag2-/- mice were treated with Tepotinib and 9 were used as control.


Immunostaining of phospho - cMet in the same tumours grown in Rag2 - / - mice after treatment with Tepotinib or vehicle control . Scale bar 200 µm .

B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O

Immunostaining of phospho-cMet in the same tumours grown in Rag2-/- mice after treatment with Tepotinib or vehicle control. Scale bar 200 µm.


Quantification of pH3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control .

O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O

Quantification of pH3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.


Quantification of caspase 3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control .

O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O

Quantification of caspase 3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.


A , B Representative size - exclusion chromatography profiles of the MICU1 - MICU2 complex and chimaera in 2 mM EGTA ( A ) and 2 mM CaCl2 ( B ) conditions .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O

A, B Representative size-exclusion chromatography profiles of the MICU1-MICU2 complex and chimaera in 2 mM EGTA (A) and 2 mM CaCl2 (B) conditions.


A The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA . B The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2 + .

O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O

A The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA. B The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2+.


C Comparison of binding of MICU1 and EMRE peptide in both conditions .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O

C Comparison of binding of MICU1 and EMRE peptide in both conditions.


D MICU1 - MICU2 heterodimer interacts with the EMRE peptide at a 1 : 1 ratio .

O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O

D MICU1-MICU2 heterodimer interacts with the EMRE peptide at a 1:1 ratio.


E Carbachol ( CCh ) - induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt - EMRE and scramble peptides . Solid lines , mean ; shaded regions , ± SEM , n = 3 .

O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-CELL_LINE O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O

E Carbachol (CCh)-induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt-EMRE and scramble peptides. Solid lines, mean; shaded regions, ± SEM, n = 3.


F Resting mitochondrial Ca2 + levels in mt - EMRE and scramble peptides treated cells , evaluated via ratiometric imaging of the mitochondrial targeted mt - riG6m . Mean ± s . d . , student t - test , * * p < 0 . 01 , n = 3 . Data information : Error bars for some points are too small to be visible .

O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Resting mitochondrial Ca2+ levels in mt-EMRE and scramble peptides treated cells, evaluated via ratiometric imaging of the mitochondrial targeted mt-riG6m. Mean ± s.d., student t-test, **p < 0.01, n = 3. Data information: Error bars for some points are too small to be visible.


G Mean mitochondrial Ca2 + peaks response to 100 μM CCh . Mean ± s . d . , student t - test , * * * p ≤ 0 . 001 , n = 3 . Data information : Error bars for some points are too small to be visible .

O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Mean mitochondrial Ca2+ peaks response to 100 μM CCh. Mean ± s.d., student t-test, ***p ≤ 0.001, n = 3. Data information: Error bars for some points are too small to be visible.


H Representative [ Ca2 + ] c clearance traces of U2OS cells treated with PBS and mt - EMRE peptide .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O B-CELL_LINE O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-GENEPROD O O

H Representative [Ca2+]c clearance traces of U2OS cells treated with PBS and mt-EMRE peptide.


I Quantification of mitochondria Ca2 + uptake rate in response to 100 μM histamine . Mean ± s . d . , student t - test , n . s . , not significant , n = 4 . Data information : Error bars for some points are too small to be visible .

O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Quantification of mitochondria Ca2+ uptake rate in response to 100 μM histamine. Mean ± s.d., student t-test, n.s., not significant, n = 4. Data information: Error bars for some points are too small to be visible.


5C or mES medium was used during reprogramming . On day 15 , Oct4GFP + colonies were counted ( A ) .

O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O

5C or mES medium was used during reprogramming. On day 15, Oct4GFP+ colonies were counted (A).


5C or mES medium was used during reprogramming . The percentages of Oct4GFP + cells were also determined with FACS ( B ) .

O O O O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O

5C or mES medium was used during reprogramming. The percentages of Oct4GFP+ cells were also determined with FACS (B).


( C ) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs .

O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-CELL_TYPE O

(C) The expression of several core pluripotency markers was determined with qPCR at different time points during reprogramming and normalized against those in MEFs.


( D - E ) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument . Increase in extracellular acidification rate ( ECAR ) after adding glucose was considered as glycolysis ability of the cells ( D ) , while the decrease in oxygen consumption rate ( OCR ) was considered as the ATP production ability of the cells ( E ) .

O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O

(D-E) Energy metabolism was analyzed on day 6 during reprogramming with the Seahorse instrument. Increase in extracellular acidification rate (ECAR) after adding glucose was considered as glycolysis ability of the cells (D), while the decrease in oxygen consumption rate (OCR) was considered as the ATP production ability of the cells (E).


( F - G ) Expression of Hif1α was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming ( F ) . The numbers of Oct4GFP + colonies were determined on day 15 ( G ) .

O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(F-G) Expression of Hif1α was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming (F). The numbers of Oct4GFP+ colonies were determined on day 15 (G).


( H - I ) Expression of Pdk1 / 2 was modulated with a retrovirus system , and oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) were used during reprogramming ( H ) . The numbers of Oct4GFP + colonies were determined on day 15 ( I ) .

O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(H-I) Expression of Pdk1/2 was modulated with a retrovirus system, and oligomycin (1 µM) and 2-DG (5 mM) were used during reprogramming (H). The numbers of Oct4GFP+ colonies were determined on day 15 (I).


( A - B ) 5C and mES medium were used during reprogramming . Representative alkaline phosphatase ( AP ) staining on day 15 was provided in ( A ) . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 ( B ) .

O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O

(A-B) 5C and mES medium were used during reprogramming. Representative alkaline phosphatase (AP) staining on day 15 was provided in (A). AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (B).


( C ) Thirty - two epigenetic factors were selected because they had higher expression in 5C - Oct4GFP - than in mES - Oct4GFP - . Their expression in ESCs , iPSCs , and pre - iPSCs in two previous reported assay ( GSE14012 and GSE10871 ) was also listed .

O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-CELL_TYPE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

(C) Thirty-two epigenetic factors were selected because they had higher expression in 5C-Oct4GFP- than in mES-Oct4GFP-. Their expression in ESCs, iPSCs, and pre-iPSCs in two previous reported assay (GSE14012 and GSE10871) was also listed.


( D ) The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , or Wdr5 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O

(D) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15.


During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming ( E ) .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O

During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The expression of pluripotency markers was determined with qPCR on day 6 during reprogramming (E).


During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The numbers of Oct4GFP + colonies were determined on day 15 ( F ) .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The numbers of Oct4GFP+ colonies were determined on day 15 (F).


During reprogramming with mES medium , Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 were overexpressed simultaneously with the four Yamanaka factors ( OKMS + 5F ) or Oct4 ( O + 5F ) . All factors were delivered simultaneously via a retrovirus system on day 0 and 1 . Reprogramming with only Yamanaka factors ( OKMS ) or Oct4 ( O ) served as control . The histone methylation on core pluripotency loci was determined on day 6 with ChIP - qPCR ( G ) .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O

During reprogramming with mES medium, Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 were overexpressed simultaneously with the four Yamanaka factors (OKMS+5F) or Oct4 (O+5F). All factors were delivered simultaneously via a retrovirus system on day 0 and 1. Reprogramming with only Yamanaka factors (OKMS) or Oct4 (O) served as control. The histone methylation on core pluripotency loci was determined on day 6 with ChIP-qPCR (G).


( A ) The enrichment of HIF1α , SNAI2 , TWIST1 / 2 , and ZEB1 binding sites on the promoters of Bmi1 , Ctcf , Ezh2 , Kdm2b , and Wdr5 was determined by Pscan Zambelli et al , 2009 ( ) . The resulted Z - scores were listed . " Max . M " suggested the maximum Z - score generated with SNAI2 , TWIST1 / 2 , and ZEB1 binding sites on one indicated promoter . " Average " suggested the average Z - score of all transcriptional factors tested in the Pscan software on one indicated promoter .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O 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) The enrichment of HIF1α, SNAI2, TWIST1/2, and ZEB1 binding sites on the promoters of Bmi1, Ctcf, Ezh2, Kdm2b, and Wdr5 was determined by Pscan Zambelli et al, 2009(). The resulted Z-scores were listed. "Max. M" suggested the maximum Z-score generated with SNAI2, TWIST1/2, and ZEB1 binding sites on one indicated promoter. "Average" suggested the average Z-score of all transcriptional factors tested in the Pscan software on one indicated promoter.


The expression of H1f1α , Snai2 , Twist1 / 2 , and Zeb1 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming with mES ( B ) The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs . The comparisons were performed between all groups and corresponding Flag groups .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O

The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with mES (B) The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups.


The expression of H1f1α , Snai2 , Twist1 / 2 , and Zeb1 was modulated with overexpression or sh - RNA - mediated knockdown via a retrovirus system during reprogramming with 5C medium ( C ) . The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs . The comparisons were performed between all groups and corresponding Flag groups .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O

The expression of H1f1α, Snai2, Twist1/2, and Zeb1 was modulated with overexpression or sh-RNA-mediated knockdown via a retrovirus system during reprogramming with 5C medium (C). The expression of Bmi1, Ctcf, Ezh2, Kdm2b, Wdr5 was determined on day 6 with qPCR and normalized against those in MEFs. The comparisons were performed between all groups and corresponding Flag groups.


pre - iPSCs were isolated during reprogramming with mES - Vc , mES or 5C medium . These pre - iPSCs were further cultured with a different medium ( mES - Vc and mES medium ) for seven days . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . The numbers of Oct4GFP + colonies were determined on day 7 ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

pre-iPSCs were isolated during reprogramming with mES-Vc, mES or 5C medium. These pre-iPSCs were further cultured with a different medium (mES-Vc and mES medium) for seven days. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The numbers of Oct4GFP+ colonies were determined on day 7 (E).


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . AP + Oct4GFP - and AP + Oct4GFP + colonies were counted on day 15 ( G ) .

O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. AP+Oct4GFP- and AP+Oct4GFP+ colonies were counted on day 15 (G).


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . In addition , energy metabolism was determined on day 6 with Seahorse

O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. In addition, energy metabolism was determined on day 6 with Seahorse instrument


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . Cell migration was determined by measuring the migration rate with live - cell imaging on day 6 ( J ) .

O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. Cell migration was determined by measuring the migration rate with live-cell imaging on day 6 (J).


Reprogramming was also performed with mES - Vc or mES medium . Additional factors ( Hif1α , Hif1α overexpression ; TGFβ , TGFβ1 / 2 / 3 1 ng / ml each ; 3F , Ctcf , Kdm2b , and Wdr5 overexpression ) were used simultaneously with mES medium . The expression of Bmi1 , Ctcf , Ezh2 , Kdm2b , or Wdr5 was determined on day 6 with qPCR ( K ) .

O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O

Reprogramming was also performed with mES-Vc or mES medium. Additional factors (Hif1α, Hif1α overexpression; TGFβ, TGFβ1/2/3 1 ng/ml each; 3F, Ctcf, Kdm2b, and Wdr5 overexpression) were used simultaneously with mES medium. The expression of Bmi1, Ctcf, Ezh2, Kdm2b, or Wdr5 was determined on day 6 with qPCR (K).


Expression of Hif1α was modulated via a retrovirus system or small - molecule compounds , oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) , during reprogramming . The expression of mesenchymal markers ( A )

O O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O O

Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers (A)


Expression of Hif1α was modulated via a retrovirus system or small - molecule compounds , oligomycin ( 1 µM ) and 2 - DG ( 5 mM ) , during reprogramming . The expression of mesenchymal markers cell migration ( B ) was determined with qPCR and live - cell imaging , respectively , on day 6 .

O O B-GENEPROD O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

Expression of Hif1α was modulated via a retrovirus system or small-molecule compounds, oligomycin (1 µM) and 2-DG (5 mM), during reprogramming. The expression of mesenchymal markers cell migration (B) was determined with qPCR and live-cell imaging, respectively, on day 6.


Repsox ( 1 μM ) was used on day 2 - 7 to inhibit early EMT during reprogramming with 5C medium . Except for OK + M + S group , the four Yamanaka factors were delivered simultaneously . Energy metabolism was determined on day 6 with Seahorse instrument ( E - F ) .

B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. Except for OK+M+S group, the four Yamanaka factors were delivered simultaneously. Energy metabolism was determined on day 6 with Seahorse instrument (E-F).


Repsox ( 1 μM ) was used on day 2 - 7 to inhibit early EMT during reprogramming with 5C medium . The expression of glycolysis markers was determined on day 6 with qPCR ( G ) .

B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O

Repsox (1 μM) was used on day 2-7 to inhibit early EMT during reprogramming with 5C medium. The expression of glycolysis markers was determined on day 6 with qPCR (G).


On day 15 during reprogramming , the expression of E - cadherin ( E - CAD ) and N - cadherin ( N - CAD ) , was determined with immunofluorescence together with the morphology in Oct4GFP + colonies ( A , Scale bar , 25 μM ) .

O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O

On day 15 during reprogramming, the expression of E-cadherin (E-CAD) and N-cadherin (N-CAD), was determined with immunofluorescence together with the morphology in Oct4GFP+ colonies (A, Scale bar, 25 μM).