F . Deconvolved inverted fluorescence micrographs of Heh1 - VN Snf7 - VC BiFC signal in a chm7Δ strain with either an empty plasmid ( " - " ; pRS416 ) or one expressing CHM7 ( pRS416 - HA - CHM7 ) . Cell borders outlined . Scale bar is 5 μm . G . Plot of the percentage of cells with BiFC signal from F . Error bars are SD from the mean of each replicate . p values from un - paired student ' s T - test where ns is p > 0 . 05 . * * * , p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Deconvolved inverted fluorescence micrographs of Heh1-VN Snf7-VC BiFC signal in a chm7Δ strain with either an empty plasmid ("-"; pRS416) or one expressing CHM7 (pRS416-HA-CHM7). Cell borders outlined. Scale bar is 5 μm.G. Plot of the percentage of cells with BiFC signal from F. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. ***, p ≤ 0.001.


A . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in the indicated strains . Scale bar is 5 μm . The percentage of cells ± SD with Chm7 - GFP foci is indicated below each panel .

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

A. Deconvolved inverted fluorescence micrographs of Chm7-GFP in the indicated strains. Scale bar is 5 μm. The percentage of cells ± SD with Chm7-GFP foci is indicated below each panel.


B . Deconvolved fluorescence images of SINC - containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7 - GFP and Nup170 - mCherry ( green , red and merged images are shown ) . Scale bar is 1 μm .

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

B. Deconvolved fluorescence images of SINC-containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7-GFP and Nup170-mCherry (green, red and merged images are shown). Scale bar is 1 μm.


C . Plot of the proportion of nup clusters with Chm7 - GFP enrichment in the indicated genetic backgrounds . Error bars are SD of the mean from 3 independent replicates .

O O O O O O O O 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. Plot of the proportion of nup clusters with Chm7-GFP enrichment in the indicated genetic backgrounds. Error bars are SD of the mean from 3 independent replicates.


D . Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7 - GFP and clustered Nup170 - mCherry in a nup133Δ strain . Scale bar is 1 μm .

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

D. Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7-GFP and clustered Nup170-mCherry in a nup133Δ strain. Scale bar is 1 μm.


E . Plot of the total fluorescence / integrated density ( in arbitrary units ( A . U . ) ) of Chm7 - GFP within ( and outside of ) the SINC . Error bars represent SD deviation of the mean from > 50 SINC or non - SINC accumulations pooled from 3 independent replicates . p values from un - paired student ' s T - tests with Welch ' s correction . * * * * is 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 O O O O O O O O O O O O O O O O

E. Plot of the total fluorescence/integrated density (in arbitrary units (A.U.)) of Chm7-GFP within (and outside of) the SINC. Error bars represent SD deviation of the mean from > 50 SINC or non-SINC accumulations pooled from 3 independent replicates. p values from un-paired student's T-tests with Welch's correction. **** is p ≤ 0.0001.


F . Correlation of the total fluorescence intensity / integrated density of SINC - enriched Chm7 - GFP and Nup170 - mCherry within individual SINCs . Linear regression calculated from 150 SINCs pooled from 3 independent replicates ; r is the linear correlation ( Pearson ' s ) coefficient .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Correlation of the total fluorescence intensity/integrated density of SINC-enriched Chm7-GFP and Nup170-mCherry within individual SINCs. Linear regression calculated from 150 SINCs pooled from 3 independent replicates; r is the linear correlation (Pearson's) coefficient.


A . 10 - fold serial dilutions of the indicated yeast strains were grown on YPD at 23 ° C for 3 days before imaging .

O O 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.10-fold serial dilutions of the indicated yeast strains were grown on YPD at 23°C for 3 days before imaging.


C . Deconvolved fluorescence micrographs of Sna3 - mCherry in the indicated genetic backgrounds . Scale bar is 5 μm .

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

C. Deconvolved fluorescence micrographs of Sna3-mCherry in the indicated genetic backgrounds. Scale bar is 5 μm.


D . Deconvolved fluorescence micrographs ( maximum intensity projections of a z - series of images ) of GFP - Nup49 in the indicated yeast strains ( arrows indicate SINCs ) . E . Plot of proportion of cells in the indicated strains with SINCs . Error bars are the SD from the mean from 3 independent replicates quantifying > 300 cells for each strain . p values from un - paired student ' s T - tests where ns is p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Deconvolved fluorescence micrographs (maximum intensity projections of a z-series of images) of GFP-Nup49 in the indicated yeast strains (arrows indicate SINCs).E. Plot of proportion of cells in the indicated strains with SINCs. Error bars are the SD from the mean from 3 independent replicates quantifying >300 cells for each strain. p values from un-paired student's T-tests where ns is p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.


A . Deconvolved inverted fluorescent micrographs of the indicated strains expressing Chm7 - GFP ( percentages ± SD of the proportion of cells with Chm7 - GFP foci is at bottom of each panel , see Figure EV5B ) . Scale bar is 5 μm .

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

A. Deconvolved inverted fluorescent micrographs of the indicated strains expressing Chm7-GFP (percentages ± SD of the proportion of cells with Chm7-GFP foci is at bottom of each panel, see Figure EV5B). Scale bar is 5 μm.


B . Plot of the percentage of cells with the indicated number of Chm7 - GFP foci . Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain . p values from 2 - way ANOVA where ns represents p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , 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 O O O O O O O O O O

B. Plot of the percentage of cells with the indicated number of Chm7-GFP foci. Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns represents p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.


C . Deconvolved inverted fluorescencemicrographs of strains expressing Chm7 - GFP after 5 h at 23 ° C ( top ) or 37 ° C ( bottom ) . Scale bar is 5 μm . Percentages ± SD of the proportion of cells with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) . D . Plots of percentage of cells from C with the indicated number of Chm7 - GFP foci at 23 ° C ( left ) and 37 ° C ( right ) . Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain . p values from 2 - way ANOVA where ns is p > 0 . 05 ; * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Deconvolved inverted fluorescencemicrographs of strains expressing Chm7-GFP after 5 h at 23°C (top) or 37°C (bottom). Scale bar is 5 μm. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).D. Plots of percentage of cells from C with the indicated number of Chm7-GFP foci at 23°C (left) and 37°C (right). Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns is p > 0.05; *, p ≤ 0.05; ***, p ≤ 0.001.


A . Plot of the percentage of nup116Δ cells incubated for 3 h at 37 ° C with nuclear rim - like accumulations of Chm7 - GFP . Error bars are SD from the mean from 3 independent replicates of > 100 cells . p values from unpaired student ' s T - test where * * * is p ≤ 0 . 001 . B . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in nup116Δ cells at either 23 ° C or grown for 3 h at 37 ° C . Percentages ± SD of the proportion of cells with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Plot of the percentage of nup116Δ cells incubated for 3 h at 37°C with nuclear rim-like accumulations of Chm7-GFP. Error bars are SD from the mean from 3 independent replicates of >100 cells. p values from unpaired student's T-test where *** is p ≤ 0.001.B. Deconvolved inverted fluorescence micrographs of Chm7-GFP in nup116Δ cells at either 23°C or grown for 3 h at 37°C. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).


C . Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37 ° C . N denotes nuclear interior . Scale bars are 100 nm .

O O O O O O O O O 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. Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37°C. N denotes nuclear interior. Scale bars are 100 nm.


D . 10 - fold serial dilutions of indicated strains grown in medium permissive ( - URA ) or restrictive ( 5 - FOA ) of the retention of a URA3 / NUP116 plasmid . Images acquired after incubation for 3 days ( left ) or 6 days ( right ) at 23 ° C .

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

D. 10-fold serial dilutions of indicated strains grown in medium permissive (-URA) or restrictive (5-FOA) of the retention of a URA3/NUP116 plasmid. Images acquired after incubation for 3 days (left) or 6 days (right) at 23°C.


A . Deconvolved fluorescence micrographs of NLS - GFP in the indicated yeast strains at either 30 ° C or after 2 h at 37 ° C . Scale bar is 5 μm . B . Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS - GFP from cells represented in A . Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain . p values from un - paired student ' s T - test where ns is p > 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 . Only chm7Δ and chm7Δapq12Δ cells have N : C ratios below 1 . 2 ( dotted red line ) . C . Re - plotting of data from B showing the percentage of cells with a N : C NLS - GFP fluorescence ratio < 1 . 2 . Error bars are the SD from the mean from 3 independent replicates of 150 cells / strain . p values from student ' s T - test where * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Deconvolved fluorescence micrographs of NLS-GFP in the indicated yeast strains at either 30°C or after 2 h at 37°C. Scale bar is 5 μm.B. Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS-GFP from cells represented in A. Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain. p values from un-paired student's T-test where ns is p > 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. Only chm7Δ and chm7Δapq12Δ cells have N:C ratios below 1.2 (dotted red line).C. Re-plotting of data from B showing the percentage of cells with a N:C NLS-GFP fluorescence ratio <1.2. Error bars are the SD from the mean from 3 independent replicates of 150 cells/strain. p values from student's T-test where *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.


C . Karyograms of trisomic ES cells shows that these cell lines have extra copies of chromosomes 6 , 8 , 11 , or 15 . The Ts6 and Ts11 ES cells harbored isochromosomes of the two identical trisomic chromosomes . Ts , trisomy .

O O O O O O O O O O O O O O O O O O O O O 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. Karyograms of trisomic ES cells shows that these cell lines have extra copies of chromosomes 6, 8, 11, or 15. The Ts6 and Ts11 ES cells harbored isochromosomes of the two identical trisomic chromosomes. Ts, trisomy.


D . CGH analysis of trisomic ES cell lines . The relative gene copy number is plotted using a log2 scale .

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

D. CGH analysis of trisomic ES cell lines. The relative gene copy number is plotted using a log2 scale.


E . CGH analysis of the genome stability of trisomic ES cells .

E. CGH analysis of the genome stability of trisomic ES cells.


F . CGH analysis of the genome integrity of trisomic ES cell subclones .

F. CGH analysis of the genome integrity of trisomic ES cell subclones.


A . Cumulative growth curves of trisomic and wild - type ES cells . The number of ES cells was counted at the indicated days in culture . The cell lines were tested in triplicates . Error bars , ± S . D . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cumulative growth curves of trisomic and wild-type ES cells. The number of ES cells was counted at the indicated days in culture. The cell lines were tested in triplicates. Error bars, ±S.D. * P < 0.05, ** P < 0.01.


B . Cell cycle distribution of trisomic and wild - type ES cell lines . The percentage of cells in G1 , S , and G2 / M phases was determined via flow cytometry . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cell cycle distribution of trisomic and wild-type ES cell lines. The percentage of cells in G1, S, and G2/M phases was determined via flow cytometry. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


C . The percentage of Annexin V + apoptotic cells was quantified . Error bars , ± S . D . n = 3 .

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

C. The percentage of Annexin V+ apoptotic cells was quantified. Error bars, ±S.D. n=3.


D . Trisomic and wild - type ES cells were treated with Cycloheximide or MG132 for 48 hours at the indicated doses . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

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D. Trisomic and wild-type ES cells were treated with Cycloheximide or MG132 for 48 hours at the indicated doses. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


A . The coloning - formation rates of the trisomic and wild - type ES cells . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O 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 coloning-formation rates of the trisomic and wild-type ES cells. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


B . Alkaline phosphatase ( AP ) staining of trisomic and wild - type ES cells after LIF withdrawal ( LIF 0 U / ml ) for 5 days . Scale bar , 500 μm . C . Evaluation of AP positive and negative colonies after LIF withdrawal . Black bars , undifferentiated colonies ; orange bars , partially differentiated colonies ; green bars , totally differentiated colonies . Error bars , ± S . D . n = 3 . * * , P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Alkaline phosphatase (AP) staining of trisomic and wild-type ES cells after LIF withdrawal (LIF 0 U/ml) for 5 days. Scale bar, 500 μm.C. Evaluation of AP positive and negative colonies after LIF withdrawal. Black bars, undifferentiated colonies; orange bars, partially differentiated colonies; green bars, totally differentiated colonies. Error bars, ±S.D. n=3. **, P < 0.01.


D . Boxplots of EB diameter at the indicated days of EB formation . Each boxplot was plotted for more than 20 EBs . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O 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. Boxplots of EB diameter at the indicated days of EB formation. Each boxplot was plotted for more than 20 EBs. * P < 0.05, ** P < 0.01.


E . Time course analysis of marker genes expression during EB formation by RT - qPCR . Error bars , ± S . D . n = 3 .

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E. Time course analysis of marker genes expression during EB formation by RT-qPCR. Error bars, ±S.D. n=3.


F . Quantification of Oct4 , Nanog and Rex1 expression in trisomic and wild - type ES cell derived EBs at day 8 of differentiation . Error bars , ± S . D . n = 3 . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of Oct4, Nanog and Rex1 expression in trisomic and wild-type ES cell derived EBs at day 8 of differentiation. Error bars, ±S.D. n=3. ** P < 0.01.


A . Teratomagrowth curves of trisomic and wild - type ES cells after subcutaneous injection into SCIDmice . 1 × 106 cells were injected per site ( 6 sites / per group ) . Error bars , ± S . D . * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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. Teratomagrowth curves of trisomic and wild-type ES cells after subcutaneous injection into SCIDmice. 1×106 cells were injected per site (6 sites/per group). Error bars, ±S.D. * P < 0.05.


B . Four - week teratomas derived from wild - type and trisomic ( Ts8 or Ts11 ) ES cell lines . 5 × 105 cells were injected per site ( 8 sites / per group ) .

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B. Four-week teratomas derived from wild-type and trisomic (Ts8 or Ts11) ES cell lines. 5×105 cells were injected per site (8 sites/per group).


C . Representative images of undifferentiated regions in Ts8 ES cell derived teratomas ( middle , top and bottom ) and the primitive neural rosettes in the teratomas derived from Ts15 ES cells ( right , top and bottom ) . The images of mature neuroectoderm from wild - type ES cell formed teratomas ( left , top and bottom ) were shown . The bottom panels were the enlarged views of the white dotted boxes of the top panels . Hematoxylin and eosin staining . Scale bars , 50 μm .

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

C. Representative images of undifferentiated regions in Ts8 ES cell derived teratomas (middle, top and bottom) and the primitive neural rosettes in the teratomas derived from Ts15 ES cells (right, top and bottom). The images of mature neuroectoderm from wild-type ES cell formed teratomas (left, top and bottom) were shown. The bottom panels were the enlarged views of the white dotted boxes of the top panels. Hematoxylin and eosin staining. Scale bars, 50 μm.


D . Immunohistochemistry analysis of Oct4 expression in wild - type , Homo - 11 , Ts6 , Ts8 , Ts11 , or Ts15 ES cellteratomas produced via the injection of 5 × 105 ES cells . Scale bar , 100 μm .

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

D. Immunohistochemistry analysis of Oct4 expression in wild-type, Homo-11, Ts6, Ts8, Ts11, or Ts15 ES cellteratomas produced via the injection of 5×105 ES cells. Scale bar, 100 μm.


E . AP staining of the primary cultured teratomacells derived from trisomic and wild - type ES cells . Ts6 , Ts8 , or Ts11 ESC teratomacells grew ES - like colonies that were stained positive for AP . Scale bar , 200 μm .

E. AP staining of the primary cultured teratomacells derived from trisomic and wild-type ES cells. Ts6, Ts8, or Ts11 ESC teratomacells grew ES-like colonies that were stained positive for AP. Scale bar, 200 μm.


F . The expression of marker genes in cultured teratoma cells ( p3 ) . Error bars , ± S . D . n = 3 . * * P < 0 . 01 .

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F. The expression of marker genes in cultured teratoma cells (p3). Error bars, ±S.D. n=3. ** P < 0.01.


B . Karyograms of Di8 , Di11 and Di15 ES cell lines . Di8 was the corrected ES cell line from Ts8 , showing diploid karyotype and Y chromosome loss . Di11 and Di15 were from Ts11 and Ts15 ES cells respectively .

B. Karyograms of Di8, Di11 and Di15 ES cell lines. Di8 was the corrected ES cell line from Ts8, showing diploid karyotype and Y chromosome loss. Di11 and Di15 were from Ts11 and Ts15 ES cells respectively.


C . AP staining of trisomic and the corrected diploid ES cells after LIF withdrawal ( LIF 0 U / ml ) for 5 days . Scale bar , 500 μm .

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C. AP staining of trisomic and the corrected diploid ES cells after LIF withdrawal (LIF 0 U/ml) for 5 days. Scale bar, 500 μm.


D . Teratomagrowth curves of trisomic and the corrected diploid ES cells . The injected cell number was 5 × 105 / site ( 8 sites / per group ) . Error bars , ± S . D . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O 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. Teratomagrowth curves of trisomic and the corrected diploid ES cells. The injected cell number was 5×105/site (8 sites/per group). Error bars, ±S.D. ** P < 0.01.


E . Scatterplot of four - week teratoma weight . The teratomas were formed by the corrected diploid ES cells and their paternal trisomic ES cells . * P < 0 . 05 .

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E. Scatterplot of four-week teratoma weight. The teratomas were formed by the corrected diploid ES cells and their paternal trisomic ES cells. * P < 0.05.


F . RT - qPCR analysis of some gene expression in trisomic and wild - type ES cells . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

F. RT-qPCR analysis of some gene expression in trisomic and wild-type ES cells. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


A . Morphology of EBs grew for 8 days derived from individual or mixed ES cell lines . Hematoxylin and eosin staining . Scale bar , 100 μm .

A. Morphology of EBs grew for 8 days derived from individual or mixed ES cell lines. Hematoxylin and eosin staining. Scale bar, 100 μm.


B . Quantification of Nanog expression in trisomic , wild - type , and mixed ES cell - derived EBs cultured for 8 days . Error bars , ± S . D . n = 3 . * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O 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. Quantification of Nanog expression in trisomic, wild-type, and mixed ES cell-derived EBs cultured for 8 days. Error bars, ±S.D. n=3. ** P < 0.01.


C . The morphology of EBs at day 10 of differentiation . M15 was the normal EB formation condition and was used as control . Black arrows indicate the cystic structures .

C. The morphology of EBs at day 10 of differentiation. M15 was the normal EB formation condition and was used as control. Black arrows indicate the cystic structures.


D . Boxplots of EBs diameter at day 10 of EB formation under M15 , CM , and M15 supplemented with Bmp4 conditions . Each boxplot was plotted for more than 10 EBs . EBs cultured under CM or Bmp4 condition were compared to EBs of the same cell line cultured under M15 condition . * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Boxplots of EBs diameter at day 10 of EB formation under M15, CM, and M15 supplemented with Bmp4 conditions. Each boxplot was plotted for more than 10 EBs. EBs cultured under CM or Bmp4 condition were compared to EBs of the same cell line cultured under M15 condition. * P < 0.05, ** P < 0.01.


C Confocal images of rNSC - derived astrocytes expressing GFP ( green ) and astrocyte - specific markers ( ACSBG1 , GFAP , S100β ; magenta ) at different timepoints ( P7 , P10 , P14 ) ; lower pictures depict single - channel images the astrocyte markers ( grey ) used above . Data information : ; scale bars ( C = 10 µm ,

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C Confocal images of rNSC-derived astrocytes expressing GFP (green) and astrocyte-specific markers (ACSBG1, GFAP, S100β; magenta) at different timepoints (P7, P10, P14); lower pictures depict single-channel images the astrocyte markers (grey) used above. Data information: ; scale bars (C = 10 µm,


D Representative pictures of recombined astrocytes ( GFP + / ACSBG1 + ; arrowheads ) during DG development ( P3 , P7 , P10 , P14 ) . E , Quantification of total numbers of recombined astrocytes ( GFP + / ACSBG1 + ) per area at postnatal timepoints in the total DG ( E ) Data information : All data are represented as mean ± SEM ; number of experimental animals throughout figure : n = 5 ( P3 ) and n = 6 ( P7 , P10 , P14 ) , ( E indicated by red dots dotted lines mark GZ borders ; scale bars ( D = 50 µm .

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D Representative pictures of recombined astrocytes (GFP+/ACSBG1+; arrowheads) during DG development (P3, P7, P10, P14). E, Quantification of total numbers of recombined astrocytes (GFP+/ACSBG1+) per area at postnatal timepoints in the total DG (E) Data information: All data are represented as mean ± SEM; number of experimental animals throughout figure: n = 5 (P3) and n = 6 (P7, P10, P14), (E indicated by red dots dotted lines mark GZ borders; scale bars (D = 50 µm.


, F Quantification of total numbers of recombined astrocytes ( GFP + / ACSBG1 + ) per area at postnatal timepoints in the distinct DG compartments ( F ) , i . e . hilus , GZ , and ML indicated by different colours .

, F Quantification of total numbers of recombined astrocytes (GFP+/ACSBG1+) per area at postnatal timepoints in the distinct DG compartments (F), i.e. hilus, GZ, and ML indicated by different colours.


G Representative images of proliferating astrocytes ( ACSBG1 + , cyan ; arrowheads ) in combination with cell cycle markers KI67 or MCM2 ( magenta ; P3 , P7 : KI67 ; P10 , P14 : MCM2 ) . Data information : ; dotted lines mark GZ borders ; scale bars G ) = 50 µm .

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G Representative images of proliferating astrocytes (ACSBG1+, cyan; arrowheads) in combination with cell cycle markers KI67 or MCM2 (magenta; P3, P7: KI67; P10, P14: MCM2). Data information: ; dotted lines mark GZ borders; scale bars G) = 50 µm.


H Confocal image of proliferating astrocytes at P10 ( ACSBG1 + , cyan ; MCM2 + , magenta ) that did not express NESTIN ( white ) . I , J Quantification of total proliferating astrocytes ( ACSBG1 + / KI67 + for P3 / P7 ; ACSBG1 + / MCM2 + for P10 / P14 ) per area at distinct timepoints per total DG ( I ) or hilus , GZ and ML ( J ) . Data information : All data are represented as mean ± SEM ; number of experimental animals throughout figure : n = 5 ( P3 ) and n = 6 ( P7 , P10 , P14 ) , I ) indicated by red dots ; scale bars , H = 10 µm ,

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H Confocal image of proliferating astrocytes at P10 (ACSBG1+, cyan; MCM2+, magenta) that did not express NESTIN (white). I, J Quantification of total proliferating astrocytes (ACSBG1+/KI67+ for P3/P7; ACSBG1+/MCM2+ for P10/P14) per area at distinct timepoints per total DG (I) or hilus, GZ and ML (J). Data information: All data are represented as mean ± SEM; number of experimental animals throughout figure: n = 5 (P3) and n = 6 (P7, P10, P14), I) indicated by red dots; scale bars ,H = 10 µm,


K Confocal images of proliferating rNSC - derived astrocytes ( GFP + / ACSBG1 + / MCM2 + ; arrowheads ) at P10 . Data information : scale bars K ) = 10

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K Confocal images of proliferating rNSC-derived astrocytes (GFP+/ACSBG1+/MCM2+; arrowheads) at P10. Data information: scale bars K) = 10 µm


B Representative pictures of P28 animals that received BrdU at P3 , P7 , P14 or P21 . For cell type identification of generated progeny , immunostainings against NESTIN ( white ) , GFAP ( green ) , and BrdU ( magenta ) were performed . rNSCs were identified as NESTIN + / GFAP + / BrdU + with a radial process ( as indicated with arrows ) , neurons as NESTIN - / GFAP - / BrdU + cells , and astrocytes as NESTIN - / GFAP + / BrdU + cells in the hilus , GZ , and ML ( arrowheads ) . Data information : dotted lines label GZ borders ; scale bars = 50 µm .

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B Representative pictures of P28 animals that received BrdU at P3, P7, P14 or P21. For cell type identification of generated progeny, immunostainings against NESTIN (white), GFAP (green), and BrdU (magenta) were performed. rNSCs were identified as NESTIN+/GFAP+/BrdU+ with a radial process (as indicated with arrows), neurons as NESTIN-/GFAP-/BrdU+ cells, and astrocytes as NESTIN-/GFAP+/BrdU+ cells in the hilus, GZ, and ML (arrowheads). Data information: dotted lines label GZ borders; scale bars = 50 µm.


C Quantification of total numbers of generated cell types ( rNSCs , neurons , astrocytes ) per area at distinct timepoints as indicated by different colours . Data information : Values represent mean ± SEM ; number of experimental animals : ( C n = 5 ( P3 , P21 ) , n = 4 ( P7 , P14 ) ;

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C Quantification of total numbers of generated cell types (rNSCs, neurons, astrocytes) per area at distinct timepoints as indicated by different colours. Data information: Values represent mean ± SEM; number of experimental animals: (C n = 5 (P3, P21), n = 4 (P7, P14);


D Quantification of total numbers of newly generated astrocytes in the hilus , GZ , and ML at P3 , P7 , P14 , P21 . Data information : Values represent mean ± SEM ; number of experimental animals , D ) n = 5 ( P3 , P21 ) , n = 4 ( P7 , P14 )

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D Quantification of total numbers of newly generated astrocytes in the hilus, GZ, and ML at P3, P7, P14, P21. Data information: Values represent mean ± SEM; number of experimental animals ,D) n = 5 (P3, P21), n = 4 (P7, P14)


G Representative pictures of immunostainings against cell type - specific proteins ( all depicted in white ) . Recombined cells were identified by GFP , rNSCs by their radial process labelled with NESTIN ( P3 , P7 ) or GFAP ( P10 , P14 ) , astrocytes by ACSBG1 , immature neurons expressed DCX , and proliferating cells the cell cycle markers KI67 ( P3 , P7 ) or MCM2 ( P10 , P14 ) . H Quantification of the percentages of GFP + recombined cell types out of all recombined cells at distinct timepoints . Intermediate precursor cells ( IPCs ) identified by TBR2 expression were added to DCX + neuroblasts . I Quantification of the proportions of proliferating cell types ( rNSCs , IPCs / neuroblasts , astrocytes ) out of all proliferating cells at P3 , P7 , P10 , P14 . Data information : Values represent mean ± SEM ; number of experimental animals : ( H , I ) n = 5 ( P3 ) , = 6 ( P7 , P10 , P14 ) ; dotted lines label GZ borders ; scale bars = 50 µm .

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G Representative pictures of immunostainings against cell type-specific proteins (all depicted in white). Recombined cells were identified by GFP, rNSCs by their radial process labelled with NESTIN (P3, P7) or GFAP (P10, P14), astrocytes by ACSBG1, immature neurons expressed DCX, and proliferating cells the cell cycle markers KI67 (P3, P7) or MCM2 (P10, P14). H Quantification of the percentages of GFP+ recombined cell types out of all recombined cells at distinct timepoints. Intermediate precursor cells (IPCs) identified by TBR2 expression were added to DCX+ neuroblasts. I Quantification of the proportions of proliferating cell types (rNSCs, IPCs/neuroblasts, astrocytes) out of all proliferating cells at P3, P7, P10, P14. Data information: Values represent mean ± SEM; number of experimental animals: (H,I) n = 5 (P3), = 6 (P7, P10, P14); dotted lines label GZ borders; scale bars = 50 µm.


B Confocal image of an adult DG populated by distinct cells identified by cell type - specific protein expression and morphology . All cells newly - generated within the 12 days incorporated BrdU ( magenta ) ; rNSCs were identified by expression of SOX2 ( cyan ) , localization of their cell bodies to the SGZ , and a GFAP + radial process ( green ) ; IPCs were located within the SGZ and express SOX2 only ( cyan ) ; neuroblasts in the SGZ and GZ were labelled by DCX ( white ; arrows ) ; astrocytes in the hilus , GZ , and ML ( arrowheads ) expressed SOX2 ( cyan ) . C Quantification of the percentages of generated cell types out of all generated cells ( rNSCs , IPCs + neuroblasts , astrocytes ) in the DG . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : ( C n = 5 ; dotted lines border SGZ and GZ ; scale bars = 50 µm (

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B Confocal image of an adult DG populated by distinct cells identified by cell type-specific protein expression and morphology. All cells newly-generated within the 12 days incorporated BrdU (magenta); rNSCs were identified by expression of SOX2 (cyan), localization of their cell bodies to the SGZ, and a GFAP+ radial process (green); IPCs were located within the SGZ and express SOX2 only (cyan); neuroblasts in the SGZ and GZ were labelled by DCX (white; arrows); astrocytes in the hilus, GZ, and ML (arrowheads) expressed SOX2 (cyan). C Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs + neuroblasts, astrocytes) in the DG. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C n = 5 ; dotted lines border SGZ and GZ; scale bars = 50 µm (B


D Representative picture of astrocytes located to distinct DG layers ( hilus GZ , ML ) . Co - expression of BrdU / SOX2 / GFAP revealed that all DG compartments harboured astrocytes which were newly generated during adulthood ( arrowheads ) . E Quantification of the proportions of generated astrocytes per DG layer out of all generated DG astrocytes . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : E ) n = 5 , dotted lines border GZ ; scale bars = 50 µm D ,

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D Representative picture of astrocytes located to distinct DG layers (hilus GZ, ML). Co-expression of BrdU/SOX2/GFAP revealed that all DG compartments harboured astrocytes which were newly generated during adulthood (arrowheads). E Quantification of the proportions of generated astrocytes per DG layer out of all generated DG astrocytes. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): E) n = 5, dotted lines border GZ; scale bars = 50 µm D,


F Representative image of proliferating astrocytes ( SOX2 + / MCM2 + ; cyan and magenta , respectively ; arrowheads ) of all DG layers . G Quantification of the percentages of proliferating astrocytes in distinct DG layers over all proliferating DG astrocytes . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : ( G ) n = 4 , dotted lines border GZ ; scale bars = 50 µm F ,

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F Representative image of proliferating astrocytes (SOX2+/MCM2+; cyan and magenta, respectively; arrowheads) of all DG layers. G Quantification of the percentages of proliferating astrocytes in distinct DG layers over all proliferating DG astrocytes. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (G) n = 4, dotted lines border GZ; scale bars = 50 µm F,


H Confocal images showing GFP + / GFAP + / SOX2 + astrocytes of distinct DG layers co - expressing the proliferation marker MCM2 Data information : scale bars = 10 µm (

H Confocal images showing GFP+/GFAP+/SOX2+ astrocytes of distinct DG layers co-expressing the proliferation marker MCM2 Data information: scale bars = 10 µm (H


K Representative image of recombined cells ( GFP + , green ) of different cellular identity : rNSCs [ SOX2 + ( cyan ) with a NESTIN + radial process ( white ) ] , IPCs ( SOX2 + nuclei in the SGZ ; cyan ) , DCX + neuroblasts in the SGZ and GZ ( magenta ) , and astrocytes ( SOX2 + cells in the hilus , GZ , and ML ; cyan ) . L Quantification of the proportions of recombined cell types over all recombined DG cells ( rNSCs , IPCs + neuroblasts , astrocytes ) . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : ( L n = 3 ; SGZ and GZ ; scale bars = 50 µm K ,

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K Representative image of recombined cells (GFP+, green) of different cellular identity: rNSCs [SOX2+ (cyan) with a NESTIN+ radial process (white)], IPCs (SOX2+ nuclei in the SGZ; cyan), DCX+ neuroblasts in the SGZ and GZ (magenta), and astrocytes (SOX2+ cells in the hilus, GZ, and ML; cyan). L Quantification of the proportions of recombined cell types over all recombined DG cells (rNSCs, IPCs + neuroblasts, astrocytes). Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (L n = 3; SGZ and GZ; scale bars = 50 µm K,


M Quantification of the percentages of rNSC - derived astrocytes in distinct DG layers over all recombined DG astrocytes . N Confocal images of proliferating rNSC - derived astrocytes ( GFP + / SOX2 + / MCM2 + ; arrowheads ) in hilus and GZ . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : M ) n = 3 scale bars = 10 µm N )

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M Quantification of the percentages of rNSC-derived astrocytes in distinct DG layers over all recombined DG astrocytes. N Confocal images of proliferating rNSC-derived astrocytes (GFP+/SOX2+/MCM2+; arrowheads) in hilus and GZ. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): M) n = 3 scale bars = 10 µm N)


Q Representative examples of field views captured within the hippocampal fissure ( indicated by the abundant large vessels ) , the GZ with its densely packed granule neurons and the hilus containing much larger neuronal somata ; expression of GCaMP7 virus ( green ) , labelling of astrocytes by hGFAP - RFP ( red ) . Data information : scale bars = 50 µm Q ,

Q Representative examples of field views captured within the hippocampal fissure (indicated by the abundant large vessels), the GZ with its densely packed granule neurons and the hilus containing much larger neuronal somata; expression of GCaMP7 virus (green), labelling of astrocytes by hGFAP-RFP (red). Data information: scale bars = 50 µm Q,


R Examples of locally dividing astrocytes ( arrows ) derived from two experimental animals . Arrowheads depict landmarks that served for identification . Data information : scale bars = 50 µm R ) . dpi = days post injection .

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R Examples of locally dividing astrocytes (arrows) derived from two experimental animals. Arrowheads depict landmarks that served for identification. Data information: scale bars = 50 µm R). dpi = days post injection.


B Representative images of BrdU - incorporating newly - generated cells in control and running hGFAPeGFP animals . BrdU + cells ( magenta ) were assigned to distinct cell types : rNSCs [ identified by their SOX2 + ( cyan ) cell bodies residing in the SGZ with a GFAP + radial process ( green ) ] ; IPCs / neuroblasts [ SOX2 + ( cyan ) nuclei located to the SGZ , DCX + cells ( white ) in the SGZ and GZ , respectively ] , and astrocytes [ SOX2 + cells ( cyan ) in the hilus , GZ , and ML ] . Arrows mark newly generated neuronally committed cells ( BrdU + / DCX + ) , while arrowheads indicate BrdU + astrocytes ( SOX2 + ) . Data information : dotted lines border SGZ and GZ ; scale bars = 50 µm .

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B Representative images of BrdU-incorporating newly-generated cells in control and running hGFAPeGFP animals. BrdU+ cells (magenta) were assigned to distinct cell types: rNSCs [identified by their SOX2+ (cyan) cell bodies residing in the SGZ with a GFAP+ radial process (green)]; IPCs/neuroblasts [SOX2+ (cyan) nuclei located to the SGZ, DCX+ cells (white) in the SGZ and GZ, respectively], and astrocytes [SOX2+ cells (cyan) in the hilus, GZ, and ML]. Arrows mark newly generated neuronally committed cells (BrdU+/DCX+), while arrowheads indicate BrdU+ astrocytes (SOX2+). Data information: dotted lines border SGZ and GZ; scale bars = 50 µm.


C Quantification of total numbers of generated cell types ( BrdU + ) per area in the DGs of control and runner animals . rNSCs : * * * * p < 0 . 0001 ; IPCs / neuroblasts : * * * p = 0 . 0007 ; astrocytes : * * p = 0 . 0032 ; unpaired t - test . D Quantification of the percentages of generated cell types out of all generated cells ( rNSCs , IPCs + neuroblasts , astrocytes ) in the DGs of controls and runners ; not significant , unpaired t - test . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : ( C , D ) n = 5 for controls , n = 6 for

C Quantification of total numbers of generated cell types (BrdU+) per area in the DGs of control and runner animals. rNSCs: **** p < 0.0001; IPCs/neuroblasts: *** p = 0.0007; astrocytes: ** p = 0.0032; unpaired t-test. D Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs + neuroblasts, astrocytes) in the DGs of controls and runners; not significant, unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C,D) n = 5 for controls, n = 6 for runners


E Representative images of proliferating ( MCM2 + ; magenta ) cells of adult control and running hGFAPeGFP animals ; rNSCs , IPCs ( arrows ) and astrocytes ( arrowheads ) were identified by criteria described in ( B ) . F Quantification of total numbers of proliferating cell types ( MCM2 + ) per area in the DG of controls and runners . rNSCs : * * p = 0 . 0052 ; IPCs : * * * p = 0 . 0008 ; astrocytes : * p = 0 . 0210 ; unpaired t - test . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : ; ( F ) n = 4 for controls , n = 5 for runners dotted lines border SGZ and GZ ; scale bars = 50 µm .

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E Representative images of proliferating (MCM2+; magenta) cells of adult control and running hGFAPeGFP animals; rNSCs, IPCs (arrows) and astrocytes (arrowheads) were identified by criteria described in (B). F Quantification of total numbers of proliferating cell types (MCM2+) per area in the DG of controls and runners. rNSCs: ** p = 0.0052; IPCs: *** p = 0.0008; astrocytes: * p = 0.0210; unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): ; (F) n = 4 for controls, n = 5 for runners dotted lines border SGZ and GZ; scale bars = 50 µm.


H Representative images of recombined ( GFP + ; green ) cells of distinct cellular identities : rNSCs [ SOX2 + ( cyan ) with a NESTIN + radial process ( white ) ] , IPCs ( SOX2 + nuclei in the SGZ ; cyan ) , DCX + neuroblasts in the SGZ and GZ ( magenta ) , and astrocytes ( SOX2 + cells in the hilus , GZ , and ML ; cyan ) . Data information : dotted lines border SGZ and GZ ; scale bars = 50 µm .

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H Representative images of recombined (GFP+; green) cells of distinct cellular identities: rNSCs [SOX2+ (cyan) with a NESTIN+ radial process (white)], IPCs (SOX2+ nuclei in the SGZ; cyan), DCX+ neuroblasts in the SGZ and GZ (magenta), and astrocytes (SOX2+ cells in the hilus, GZ, and ML; cyan). Data information: dotted lines border SGZ and GZ; scale bars = 50 µm.


I Quantification of the total numbers of recombined cell types ( GFP + ) per area in the DG of controls and runners ; rNSC : p = 0 . 074 ; IPCs / neuroblasts : * p = 0 . 0460 ; astrocytes : p = 0 . 2314 ; unpaired t - test . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : ( I ) n =

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I Quantification of the total numbers of recombined cell types (GFP+) per area in the DG of controls and runners; rNSC: p = 0.074; IPCs/neuroblasts: * p = 0.0460; astrocytes: p = 0.2314; unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (I) n = 3


B Representative confocal images of BrdU - incorporating cells ( magenta ) assigned to distinct cell types of 8 week - , 14 month - , and 21 month - old hGFAPeGFP mice : rNSCs [ identified by their SOX2 + ( green ) cell bodies residing in the SGZ with a GFAP + radial process ( cyan ) ] ; IPCs / neuroblasts [ SOX2 + ( green ) nuclei located to the SGZ , DCX + cells ( white ) in the SGZ and GZ , respectively ] , and astrocytes [ SOX2 + cells ( green ) in the hilus , GZ , and ML ] . Data information : ; dotted lines border SGZ and GZ ; scale bars = 50 µm .

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B Representative confocal images of BrdU-incorporating cells (magenta) assigned to distinct cell types of 8 week-, 14 month-, and 21 month-old hGFAPeGFP mice: rNSCs [identified by their SOX2+ (green) cell bodies residing in the SGZ with a GFAP+ radial process (cyan)]; IPCs/neuroblasts [SOX2+ (green) nuclei located to the SGZ, DCX+ cells (white) in the SGZ and GZ, respectively], and astrocytes [SOX2+ cells (green) in the hilus, GZ, and ML]. Data information: ; dotted lines border SGZ and GZ; scale bars = 50 µm.


C Quantification of total numbers of generated cell types ( BrdU + ; rNSCs , IPCs + neuroblasts , astrocytes ) per area in the DGs of 8 week - , 14 month - , and 21 month - old hGFAPeGFP animals . rNSCs : * * * p = 0 . 00076 , * * p = 0 . 0014 ; IPCs / neuroblasts * * * * p < 0 . 0001 ; astrocytes : * * * p = 0 . 0008 ; * p = 0 . 0403 ; one - way ANOVA with Tukey ' s post hoc test . D Quantification of the percentages of generated cell types out of all generated cells ( rNSCs , IPCs + neuroblasts , astrocytes ) in the DG of 8 week - , 14 month - , and 21 month - old animals . rNSCs : not significant ; IPCs / neuroblasts * * * * p < 0 . 0001 , * * * p = 0 . 0001 ; astrocytes : * * p < 0 . 0019 , * * * * p < 0 . 0001 ; one - way ANOVA with Tukey ' s post hoc test . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : ( C , D ) n = 5 in 8 week - old mice , n = 6 in 14 month - old mice , n = 5 in 21 month - old

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

C Quantification of total numbers of generated cell types (BrdU+; rNSCs, IPCs + neuroblasts, astrocytes) per area in the DGs of 8 week-, 14 month-, and 21 month-old hGFAPeGFP animals. rNSCs: *** p = 0.00076, ** p = 0.0014; IPCs/neuroblasts **** p < 0.0001; astrocytes: *** p = 0.0008; * p = 0.0403; one-way ANOVA with Tukey's post hoc test. D Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs + neuroblasts, astrocytes) in the DG of 8 week-, 14 month-, and 21 month-old animals. rNSCs: not significant; IPCs/neuroblasts **** p < 0.0001, *** p = 0.0001; astrocytes: ** p < 0.0019, **** p < 0.0001; one-way ANOVA with Tukey's post hoc test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C,D) n = 5 in 8 week-old mice, n = 6 in 14 month-old mice, n = 5 in 21 month-old mice


E Representative images of proliferating cells ( MCM2 + ; magenta ) : rNSCs [ SOX2 + cell nuclei ( cyan ) residing in the SGZ with a NESTIN + radial process ( white ) ] ; IPCs [ SOX2 + nuclei ( cyan ) located to the SGZ ] , and astrocytes [ SOX2 + cells ( cyan ) in the hilus , GZ , and ML ; arrowheads ] . F Quantification of total numbers of proliferating ( MCM2 + ) cell types per area in the DGs of 8 week - , 14 month - , and 21 month - old animals . rNSCs : not significant ; IPCs * * * * p < 0 . 0001 ; astrocytes : * p = 0 . 0216 ; * p = 0 . 0266 ; one - way ANOVA with Tukey ' s post hoc test . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : ( F ) n = 4 in 8 week - old mice , n = 6 in 14 month - and 21 month - old mice dotted lines border SGZ and GZ ; scale bars = 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of proliferating cells (MCM2+; magenta): rNSCs [SOX2+ cell nuclei (cyan) residing in the SGZ with a NESTIN+ radial process (white)]; IPCs [SOX2+ nuclei (cyan) located to the SGZ], and astrocytes [SOX2+ cells (cyan) in the hilus, GZ, and ML; arrowheads]. F Quantification of total numbers of proliferating (MCM2+) cell types per area in the DGs of 8 week-, 14 month-, and 21 month-old animals. rNSCs: not significant; IPCs **** p < 0.0001; astrocytes: * p = 0.0216; * p = 0.0266; one-way ANOVA with Tukey's post hoc test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (F) n = 4 in 8 week-old mice, n = 6 in 14 month- and 21 month-old mice dotted lines border SGZ and GZ; scale bars = 50 µm.


G Confocal images of 8 week - and 14 month - old NestinCreERT2 ; GFP animals , which received tamoxifen for five days ( 10 times , every 12 h ) and were killed 12 days after the last tamoxifen shot . The majority of recombined cells were rNSCs [ SOX2 + cell nuclei ( cyan ) residing in the SGZ with a NESTIN + radial process ( white ) ] and IPCs / neuroblasts [ SOX2 + ( cyan ) nuclei located to the SGZ , DCX + cells ( magenta ) in the SGZ and GZ , respectively ] , and only very few astrocytes [ SOX2 + cells ( cyan ) in the hilus , GZ , and ML ] were generated by initially recombined rNSCs . H Quantification of total numbers of recombined ( GFP + ) rNSCs , IPCs / neuroblasts , astrocytes per area in the DGs of 8 week - and 14 month - old animals . rNSCs : * p = 0 . 0460 ; IPCs / neuroblasts : * * * * < 0 . 0001 ; astrocytes : not significant ; unpaired t - test . Data information : All data are represented as mean ± SEM ; number of experimental animals ( indicated by red dots ) : ; ( H ) n = 3 in 8 week - old mice , n = 5 in 14 month - old mice ; dotted lines border SGZ and GZ ; scale bars = 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Confocal images of 8 week- and 14 month-old NestinCreERT2; GFP animals, which received tamoxifen for five days (10 times, every 12 h) and were killed 12 days after the last tamoxifen shot. The majority of recombined cells were rNSCs [SOX2+ cell nuclei (cyan) residing in the SGZ with a NESTIN+ radial process (white)] and IPCs/neuroblasts [SOX2+ (cyan) nuclei located to the SGZ, DCX+ cells (magenta) in the SGZ and GZ, respectively], and only very few astrocytes [SOX2+ cells (cyan) in the hilus, GZ, and ML] were generated by initially recombined rNSCs. H Quantification of total numbers of recombined (GFP+) rNSCs, IPCs/neuroblasts, astrocytes per area in the DGs of 8 week- and 14 month-old animals. rNSCs: * p = 0.0460; IPCs/neuroblasts: **** < 0.0001; astrocytes: not significant; unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): ; (H) n = 3 in 8 week-old mice, n = 5 in 14 month-old mice ; dotted lines border SGZ and GZ; scale bars = 50 µm.


A . Ubiquitin - vinyl sulfone ( UbVS ) assays of RING2 release by addition of pUbl and pUb in trans to R0RBR parkin . Assays were performed with incubation of 2 μM wild - type or W403A R0RBR purified in a 1 : 1 complex with pUb . Crosslinking was initiated by addition of 10 μM UbVS in the presence of pUbl or pUb ∆ G76 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Ubiquitin-vinyl sulfone (UbVS) assays of RING2 release by addition of pUbl and pUb in trans to R0RBR parkin. Assays were performed with incubation of 2 μM wild-type or W403A R0RBR purified in a 1:1 complex with pUb. Crosslinking was initiated by addition of 10 μM UbVS in the presence of pUbl or pUb∆G76.


B . Inhibition of R0RBR activation by the K211N mutation .

B. Inhibition of R0RBR activation by the K211N mutation.


C . UbVS assays of RING2 release by various type of pUb .

C. UbVS assays of RING2 release by various type of pUb.


D . Autoubiquitination assays of parkin ligase activity induced by pUbl or pUb ∆ G76 in trans . 2 µM GST - R0RBR in complex with pUb was incubated with 50 nM E1 , 2 µM UbcH7 , 100 µM ubiquitin and 4 mM ATP .

O O O O O O O O O O O O O O O O O O O O O O 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. Autoubiquitination assays of parkin ligase activity induced by pUbl or pUb∆G76 in trans. 2 µM GST-R0RBR in complex with pUb was incubated with 50 nM E1, 2 µM UbcH7, 100 µM ubiquitin and 4 mM ATP.


E . UbVS assays of RING2 release with R0RBR purified without pUb bound .

E. UbVS assays of RING2 release with R0RBR purified without pUb bound.


F . Autoubiquitination assays of parkin ligase activity with GST - R0RBR without pUb bound . The percentage of unmodified parkin band in each lane is indicated under the gel .

O O O O O O O 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. Autoubiquitination assays of parkin ligase activity with GST-R0RBR without pUb bound. The percentage of unmodified parkin band in each lane is indicated under the gel.


B . Phosphorylation of wild - type ( WT ) parkin and chimeric Ub - R0RBR by PINK1 . Reactions were analyzed on a Phos - tag SDS - PAGE gel to assess the level of phosphorylation .

O O O O O O O O O O O O O O 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. Phosphorylation of wild-type (WT) parkin and chimeric Ub-R0RBR by PINK1. Reactions were analyzed on a Phos-tag SDS-PAGE gel to assess the level of phosphorylation.


C . Release of RING2 by Ub - R0RBR chimera . Ubiquitin - vinyl sulfone assays ( 10 μM UbVS ) were performed with 3 μM phosphorylated and non - phosphorylated full - length parkin and Ub - R0RBR .

C. Release of RING2 by Ub-R0RBR chimera. Ubiquitin-vinyl sulfone assays (10 μM UbVS) were performed with 3 μM phosphorylated and non-phosphorylated full-length parkin and Ub-R0RBR.


D . Ubiquitination activity of Ub - R0RBR chimera . Autoubiquitination assays of phosphorylated full - length parkin and Ub - R0RBR ( 3 . 3 µM with 3 µm UbcH7 and 75 µM S65A ubiquitin ) were performed to assess the impact of pUbl substitution by pUb on parkin activity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Ubiquitination activity of Ub-R0RBR chimera. Autoubiquitination assays of phosphorylated full-length parkin and Ub-R0RBR (3.3 µM with 3 µm UbcH7 and 75 µM S65A ubiquitin) were performed to assess the impact of pUbl substitution by pUb on parkin activity.


B . UbVS assays of release of RING2 by K6 - or K48 - linked ( pUb ) 4 chains . 10 μM UbVS was added to 2 μM R0RBR in the presence of increasing amount of K6 ( pUb ) 4 or K48 ( pUb ) 4 chains .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. UbVS assays of release of RING2 by K6- or K48-linked (pUb)4 chains. 10 μM UbVS was added to 2 μM R0RBR in the presence of increasing amount of K6(pUb)4 or K48(pUb)4 chains.


C . Autoubiquitination assays of parkin activation by K6 ( pUb ) 4 or K48 ( pUb ) 4 . Assays with 2 µM GST - R0RBR , 50 nM E1 , 2 µM UbcH7 and 100 µM ubiquitin were performed in the presence of increasing amount of K6 ( pUb ) 4 or K48 ( pUb ) 4 chains .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Autoubiquitination assays of parkin activation by K6(pUb)4 or K48(pUb)4. Assays with 2 µM GST-R0RBR, 50 nM E1, 2 µM UbcH7 and 100 µM ubiquitin were performed in the presence of increasing amount of K6(pUb)4 or K48(pUb)4 chains.


D . Competition between K6 ( pUb ) 4 chains and monomeric pUb . UbVs ( 10 μM ) was added to 20 nM of WT and K211N R0RBR in the presence of monomeric pUb and / or K6 ( pUb ) 4 chains . Reactions were resolved on SDS - PAGE and visualized with anti - parkin antibody by western blotting . The percentage of unmodified parkin band in each lane is indicated under the gel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Competition between K6(pUb)4 chains and monomeric pUb. UbVs (10 μM) was added to 20 nM of WT and K211N R0RBR in the presence of monomeric pUb and/or K6(pUb)4 chains. Reactions were resolved on SDS-PAGE and visualized with anti-parkin antibody by western blotting. The percentage of unmodified parkin band in each lane is indicated under the gel.


A . The localization of NSUN3 was analysed in HEK293 cells stably expressing NSUN3 - GFP ( green ) . NSUN3 - GFP and staining with a Mitotracker ( red ) are shown separately and in an overlay with DAPI to indicate nuclei . The scale bar represents 5 m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 localization of NSUN3 was analysed in HEK293 cells stably expressing NSUN3-GFP (green). NSUN3-GFP and staining with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.


B . To analyse submitochondrial localization of NSUN3 , human mitochondria were isolated and either left untreated , swollen in hypotonic buffer ( Mitoplasts ) or solubilized by sonication ( Sonic . ) before treatment with different amounts of Proteinase K ( PK ) where indicated , followed by SDS - PAGE and Western blotting using antibodies against human TIM44 , TIM23 , TOM70 or FLAG - tagged NSUN3 . Note that TIM44 localises to the matrix , while the N - terminus of TIM23 localises to the intermembrane space and TOM70 is largely exposed on the mitochondrial surface . The asterisk indicates a cross - reaction of the TOM70 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. To analyse submitochondrial localization of NSUN3, human mitochondria were isolated and either left untreated, swollen in hypotonic buffer (Mitoplasts) or solubilized by sonication (Sonic.) before treatment with different amounts of Proteinase K (PK) where indicated, followed by SDS-PAGE and Western blotting using antibodies against human TIM44, TIM23, TOM70 or FLAG-tagged NSUN3. Note that TIM44 localises to the matrix, while the N-terminus of TIM23 localises to the intermembrane space and TOM70 is largely exposed on the mitochondrial surface. The asterisk indicates a cross-reaction of the TOM70 antibody.


B . HEK293 cells expressing NSUN3 - HisPrcFLAG ( NSUN3 ) or the HisPrcFLAG - tag alone ( FLAG ) were either not crosslinked ( - ) , UV crosslinked ( UV ) or treated with 5 - azacytidine ( 5 - AzaC ) . The protein - RNA complexes were affinity purified and the bound RNA was trimmed , end - labelled with 32P phosphate and ligated to linkers . Protein - RNA complexes were separated by SDS - PAGE , transferred to nitrocellulose and exposed to an X - ray film .

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

B. HEK293 cells expressing NSUN3-HisPrcFLAG (NSUN3) or the HisPrcFLAG-tag alone (FLAG) were either not crosslinked (-), UV crosslinked (UV) or treated with 5-azacytidine (5-AzaC). The protein-RNA complexes were affinity purified and the bound RNA was trimmed, end-labelled with 32P phosphate and ligated to linkers. Protein-RNA complexes were separated by SDS-PAGE, transferred to nitrocellulose and exposed to an X-ray film.


G . 5 - AzaC crosslinking was performed and RNA associated with wildtype NSUN3 , the catalytically inactive NSUN3 mutant ( C265A ) or the FLAG tag alone was isolated as described in ( B ) . The RNA analysed by northern blot using probes against the mt - tRNAMet , mt - tRNAPro and mt - tRNAGlu . Inputs ( 0 . 1 % ) are shown on the left and eluates ( 50 % ) on the right .

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

G. 5-AzaC crosslinking was performed and RNA associated with wildtype NSUN3, the catalytically inactive NSUN3 mutant (C265A) or the FLAG tag alone was isolated as described in (B). The RNA analysed by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.


A . In vitro methylation reactions were performed using recombinant His14 - MBP - NSUN3 ( NSUN3 ) or the catalytically inactive mutant His14 - MBP - NSUN3 - C265A ( C265A ) , 3H - labelled S - adenosyl methionine as a methyl group donor and in vitro transcribed mt - tRNAMet , mt - tRNAPro and mt - tRNAGlu . The RNA was then separated on a denaturing polyacrylamide gel , stained with ethidium bromide ( EtBr ) to indicate inputs and exposed to an X - ray film to analyse methylation ( 3H - Me ) .

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

A. In vitro methylation reactions were performed using recombinant His14-MBP-NSUN3 (NSUN3) or the catalytically inactive mutant His14-MBP-NSUN3-C265A (C265A), 3H-labelled S-adenosyl methionine as a methyl group donor and in vitro transcribed mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. The RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr) to indicate inputs and exposed to an X-ray film to analyse methylation (3H-Me).


B . The distribution of Illumina sequence reads along the mt - tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV ( light grey ) or 5 - AzaC crosslinking ( dark grey ) is given as reads per million mapped reads . The position of the anticodon is indicated by a bar .

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

B. The distribution of Illumina sequence reads along the mt-tRNAMet sequence obtained from CRAC experiments with NSUN3 after UV (light grey) or 5-AzaC crosslinking (dark grey) is given as reads per million mapped reads. The position of the anticodon is indicated by a bar.


D . In vitro methylation assays were performed as described in ( A ) with His14 - MBP - NSUN3 and in vitro transcribed wildtype mt - tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in ( C ) . Two exposure times of X - ray films are shown : 16 hours ( short ) , 3 days ( long ) .

O O O 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

D. In vitro methylation assays were performed as described in (A) with His14-MBP-NSUN3 and in vitro transcribed wildtype mt-tRNAMet and cytidine mutants of the anticodon stem and loop region indicated in (C). Two exposure times of X-ray films are shown: 16 hours (short), 3 days (long).


E . In vitro methylation assay of in vitro transcribed mt - tRNAMet and chemically synthesized mt - tRNAMet containing an m5C modification at the wobble position . The experiment and analysis were performed as described in ( A ) .

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

E. In vitro methylation assay of in vitro transcribed mt-tRNAMet and chemically synthesized mt-tRNAMet containing an m5C modification at the wobble position. The experiment and analysis were performed as described in (A).


B . In vitro methylation assays using [ 3H - methyl ] - labelled S - adenosyl methionine , the in vitro transcripts of the mt - tRNAMet mutants described in ( A ) and recombinant His14 - MBP - NSUN3 . RNA was then separated on a denaturing polyacrylamide gel , stained with ethidium bromide ( EtBr ) , dried and exposed to an X - ray film to detect methylated transcripts ( 3H - Me ) .

O O O 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

B. In vitro methylation assays using [3H-methyl]-labelled S-adenosyl methionine, the in vitro transcripts of the mt-tRNAMet mutants described in (A) and recombinant His14-MBP-NSUN3. RNA was then separated on a denaturing polyacrylamide gel, stained with ethidium bromide (EtBr), dried and exposed to an X-ray film to detect methylated transcripts (3H-Me).


C . In vitro methylation assay using chemically synthesised ASL . The experiment and analysis were performed as described in ( B ) .

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. In vitro methylation assay using chemically synthesised ASL. The experiment and analysis were performed as described in (B).


A . The localization of ABH1 was analysed by immunofluorescence in HEK293 cells . ABH1 ( green ) localisation and mitochondria stained with a Mitotracker ( red ) are shown separately and in an overlay with DAPI to indicate nuclei . The scale bar represents 5 m .

O O O O O O O O O O O O O O O O O O O O O O O O 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 localization of ABH1 was analysed by immunofluorescence in HEK293 cells. ABH1 (green) localisation and mitochondria stained with a Mitotracker (red) are shown separately and in an overlay with DAPI to indicate nuclei. The scale bar represents 5 m.


B . HEK293 cells expressing ABH1 - HisPrcFLAG ( ABH1 ) or the HisPrcFLAG - tag alone ( FLAG ) were UV crosslinked ( UV ) and protein - RNA complexes were affinity purified . Co - precipitated RNA was isolated and analysis by northern blot using probes against the mt - tRNAMet , mt - tRNAPro and mt - tRNAGlu . Inputs ( 0 . 1 % ) are shown on the left and eluates ( 50 % ) on the right .

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

B. HEK293 cells expressing ABH1-HisPrcFLAG (ABH1) or the HisPrcFLAG-tag alone (FLAG) were UV crosslinked (UV) and protein-RNA complexes were affinity purified. Co-precipitated RNA was isolated and analysis by northern blot using probes against the mt-tRNAMet, mt-tRNAPro and mt-tRNAGlu. Inputs (0.1%) are shown on the left and eluates (50%) on the right.


A . In vitro transcribed mt - tRNAMet was methylated at C34 using recombinant NSUN3 and 3H - labelled S - adenosyl methionine as a methyl group donor . Radiolabelled mt - tRNAMet was re - extracted and then subjected to oxidation assays without protein ( - ) , with maltose binding protein ( MBP ) , with the dioxygenase FTO or using wildtype ( ABH1 ) or mutant ( R338A , D233A ) His14 - MBP - ABH1 . Besides ABH1 controls lacking a - ketoglutarate ( KG ) or Fe2 + ions , all samples contained a - ketoglutarate and Fe2 + ions . After oxidation , RNA was precipitated and tritium released upon oxidation of radiolabelled mt - tRNAMet was quantified in the supernatant and counts per minute ( CPM ) are shown for experiments performed in triplicate with error bars indicating ± SD ( upper panel ) . Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X - ray film to analyse the tritium retained ( 3H - Me ) .

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

A. In vitro transcribed mt-tRNAMet was methylated at C34 using recombinant NSUN3 and 3H-labelled S-adenosyl methionine as a methyl group donor. Radiolabelled mt-tRNAMet was re-extracted and then subjected to oxidation assays without protein (-), with maltose binding protein (MBP), with the dioxygenase FTO or using wildtype (ABH1) or mutant (R338A, D233A) His14-MBP-ABH1. Besides ABH1 controls lacking a-ketoglutarate (KG) or Fe2+ ions, all samples contained a-ketoglutarate and Fe2+ ions. After oxidation, RNA was precipitated and tritium released upon oxidation of radiolabelled mt-tRNAMet was quantified in the supernatant and counts per minute (CPM) are shown for experiments performed in triplicate with error bars indicating ± SD (upper panel). Pelleted RNA was separated on a denaturing polyacrylamide gel and exposed to an X-ray film to analyse the tritium retained (3H-Me).


B . Synthetic anticodon stem loop ( ASL ) was radioactively labelled and subjected to oxidation assays that were performed and analysed as described in ( A ) using no protein ( - ) , MBP or wildtype His14 - MBP - ABH1 ( ABH1 ) . Experiments were performed in triplicate with error bars indicating ± SD .

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B. Synthetic anticodon stem loop (ASL) was radioactively labelled and subjected to oxidation assays that were performed and analysed as described in (A) using no protein (-), MBP or wildtype His14-MBP-ABH1 (ABH1). Experiments were performed in triplicate with error bars indicating ± SD.


C . Anion exchange HPLC analysis was performed on synthetic m5C containing ASL ( 20nt ) before and after oxidation by ABH1 . The small shift in retention time indicates formation of f5C modified RNA . The ABH1 oxidation product was then treated with NaBH4 to generate hm5C - modified RNA . All three samples were analysed by ESI - MS and the molecular weight ( m . w . ) is indicated on the HPLC trace . Only the f5C - containing RNA was labelled efficiently with 1 - ethyl - 2 , 3 , 3 - trimethylindoleninium - 5 - sulfonate ( TMI ) .

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C. Anion exchange HPLC analysis was performed on synthetic m5C containing ASL (20nt) before and after oxidation by ABH1. The small shift in retention time indicates formation of f5C modified RNA. The ABH1 oxidation product was then treated with NaBH4 to generate hm5C-modified RNA. All three samples were analysed by ESI-MS and the molecular weight (m.w.) is indicated on the HPLC trace. Only the f5C-containing RNA was labelled efficiently with 1-ethyl-2,3,3-trimethylindoleninium-5-sulfonate (TMI).


D . The different retention times of m5C , hm5C and f5C - modified ASL RNA were confirmed by co - injection of samples shown in ( C ) . HPLC was performed as in C .

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D. The different retention times of m5C, hm5C and f5C-modified ASL RNA were confirmed by co-injection of samples shown in (C). HPLC was performed as in C.


A . MTIF2 - dependent reading of initiation codons AUG , AUA or AUU in the P site of the ribosome by unmodified ( unmod . ) or C34 - modified [ 14C ] Met - tRNAMet . Binding was determined by nitrocellulose filtration and [ 14C ] Met - tRNAMet retrieved on the membrane was quantified by scintillation counting . Binding in the absence of ribosomes ( no 70S ) or mRNA ( no mRNA ) served as controls . Data from three independent experiments are presented with error bars indicating ± SEM . The statistical significance of the results was analysed by T - test and is indicated by the asterisks in the graph .

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A. MTIF2-dependent reading of initiation codons AUG, AUA or AUU in the P site of the ribosome by unmodified (unmod.) or C34-modified [14C]Met-tRNAMet. Binding was determined by nitrocellulose filtration and [14C]Met-tRNAMet retrieved on the membrane was quantified by scintillation counting. Binding in the absence of ribosomes (no 70S) or mRNA (no mRNA) served as controls. Data from three independent experiments are presented with error bars indicating ± SEM. The statistical significance of the results was analysed by T-test and is indicated by the asterisks in the graph.


B . TUFM - dependent recognition of A site codons during elongation . Data from three independent experiments are presented with error bars indicating ± SEM and statistical analysis as in ( A ) .

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B. TUFM-dependent recognition of A site codons during elongation. Data from three independent experiments are presented with error bars indicating ± SEM and statistical analysis as in (A).


A . HeLa cells were transfected with two different siRNAs against NSUN3 ( siNSUN3 _ 1 , siNSUN3 _ 2 ) , ABH1 ( siABH1 _ 1 , siABH1 _ 2 ) or with nontarget ( siNT ) siRNA and the knockdown efficiency was analysed by quantitative PCR . The relative abundance of the NSUN3 or ABH1 mRNA was normalized to GAPDH levels . Data are presented as mean ± SD .

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A. HeLa cells were transfected with two different siRNAs against NSUN3 (siNSUN3_1, siNSUN3_2), ABH1 (siABH1_1, siABH1_2) or with nontarget (siNT) siRNA and the knockdown efficiency was analysed by quantitative PCR. The relative abundance of the NSUN3 or ABH1 mRNA was normalized to GAPDH levels. Data are presented as mean ± SD.


B . Chemically synthesised f5C modified mt - tRNAMet and total RNA from wild - type ( WT ) cells or those transfected with siRNAs as in ( A ) were treated with TMI to specifically label f5C residues . Primer extension , using a radiolabelled antisense primer , was performed under limited dNTP conditions . Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder and RNAs were detected using a phosphorimager .

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B. Chemically synthesised f5C modified mt-tRNAMet and total RNA from wild-type (WT) cells or those transfected with siRNAs as in (A) were treated with TMI to specifically label f5C residues. Primer extension, using a radiolabelled antisense primer, was performed under limited dNTP conditions. Products were separated on a denaturing polyacrylamide gel alongside a sequencing ladder and RNAs were detected using a phosphorimager.


C . Primer extension reactions were performed on total RNA from cells transfected with siRNAs as described in B . Stops corresponding to position C34 in mt - tRNAMet were quantified in three independent experiments and results are shown graphically as mean ± SD .

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C. Primer extension reactions were performed on total RNA from cells transfected with siRNAs as described in B. Stops corresponding to position C34 in mt-tRNAMet were quantified in three independent experiments and results are shown graphically as mean ± SD.


D . RNA from wild - type HeLa cells and cells treated with siRNAs against NSUN3 or ABH1 ( as in A ) was either first reduced with NaBH4 or directly treated with bisulfite . After deamination and desulfonation , mt - tRNAMetRNAs were reverse transcribed , amplified , cloned and sequenced . The proportions of thymine ( grey ) generated by bisulfite - conversion or non - converted cytosine ( black ) at position 34 of mt - tRNAMet are shown . Note that for sequences from non - reduced samples , thymine can originate from unmodified or f5C - containing mt - tRNAMet , while in reduced samples it originates from unmodified cytosine .

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

D. RNA from wild-type HeLa cells and cells treated with siRNAs against NSUN3 or ABH1 (as in A) was either first reduced with NaBH4 or directly treated with bisulfite. After deamination and desulfonation, mt-tRNAMetRNAs were reverse transcribed, amplified, cloned and sequenced. The proportions of thymine (grey) generated by bisulfite-conversion or non-converted cytosine (black) at position 34 of mt-tRNAMet are shown. Note that for sequences from non-reduced samples, thymine can originate from unmodified or f5C-containing mt-tRNAMet, while in reduced samples it originates from unmodified cytosine.


A . HeLa cells were treated with non - target siRNAs ( siNT ) or those targeting NSUN3 ( siNSUN3 _ 1 or siNSUN3 _ 2 ) or ABH1 ( siABH1 _ 1 or siABH1 _ 2 ) for 72 h before labelling of mitochondrial translation products with [ 35S ] methionine . Protein samples were separated by SDS - PAGE then transferred to a membrane . Labelled proteins were detected using a phosphorimager and the levels of tubulin were determined by western blotting using an antibody against the endogenous protein for normalisation . B . Mitochondrially - translated proteins that could be clearly detected were quantified in three independent experiments and the results are shown graphically as mean ± SD .

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A. HeLa cells were treated with non-target siRNAs (siNT) or those targeting NSUN3 (siNSUN3_1 or siNSUN3_2) or ABH1 (siABH1_1 or siABH1_2) for 72 h before labelling of mitochondrial translation products with [35S]methionine. Protein samples were separated by SDS-PAGE then transferred to a membrane. Labelled proteins were detected using a phosphorimager and the levels of tubulin were determined by western blotting using an antibody against the endogenous protein for normalisation.B. Mitochondrially-translated proteins that could be clearly detected were quantified in three independent experiments and the results are shown graphically as mean ± SD.


( A ) QRT - PCR assay of PAX6 , SOX1 , NKX2 - 1 , NKX2 - 2 , TBR1 and TBR2 during dorsal ( up ) and ventral ( down ) neural differentiation . ( B ) QRT - PCR assay of SOX1 - OT V1 during dorsal ( up ) and ventral ( down ) neural differentiation Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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(A) QRT-PCR assay of PAX6, SOX1, NKX2-1, NKX2-2, TBR1 and TBR2 during dorsal (up) and ventral (down) neural differentiation. (B) QRT-PCR assay of SOX1-OT V1 during dorsal (up) and ventral (down) neural differentiation Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( F and G ) Immunostaining assay of PAX6 ( green ) and SOX1 ( red ) in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( F ) , and quantiﬁcation of SOX1 + cells ( G ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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(F and G) Immunostaining assay of PAX6 (green) and SOX1 (red) in control and V1-PAKI cells on day 16 of dorsal neural differentiation (F), and quantiﬁcation of SOX1+ cells (G), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( H and I ) Immunostaining assay of MAP2 ( green ) in control and V1 - PAKI cells on day 35 of dorsal neural differentiation ( H ) , and quantiﬁcation of MAP2 + cells ( I ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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(H and I) Immunostaining assay of MAP2 (green) in control and V1-PAKI cells on day 35 of dorsal neural differentiation (H), and quantiﬁcation of MAP2+ cells (I), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( J and K ) Immunostaining assay of PAX6 ( green ) and SOX1 ( red ) in control and V1 - PAKI cells on day 16 of ventral neural differentiation ( J ) , and quantiﬁcation of SOX1 + cells ( K ) , p = 0 . 0003 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

(J and K) Immunostaining assay of PAX6 (green) and SOX1 (red) in control and V1-PAKI cells on day 16 of ventral neural differentiation (J), and quantiﬁcation of SOX1+ cells (K), p=0.0003. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( L and M ) Immunostaining assay of GABA ( green ) in control and V1 - PAKI cells on day 35 of ventral neural differentiation ( L ) , and quantiﬁcation of GABA + cells ( M ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( unpaired two - tailed Student ' s t - test ) .

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(L and M) Immunostaining assay of GABA (green) in control and V1-PAKI cells on day 35 of ventral neural differentiation (L), and quantiﬁcation of GABA+ cells (M), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 (unpaired two-tailed Student's t-test).


( C and D ) Immunostaining assay of MAP2 ( green ) in control and SOX1KO cells on day 35 of dorsal neural differentiation ( C ) , and quantiﬁcation of MAP2 + cells ( D ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

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(C and D) Immunostaining assay of MAP2 (green) in control and SOX1KO cells on day 35 of dorsal neural differentiation (C), and quantiﬁcation of MAP2+ cells (D), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( F and G ) Immunostaining assay of GABA ( green ) in control and SOX1KO cells on day 35 of ventral neural differentiation ( F ) , and quantiﬁcation of GABA + cells ( G ) , p = 0 . 0001 . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and G) Immunostaining assay of GABA (green) in control and SOX1KO cells on day 35 of ventral neural differentiation (F), and quantiﬁcation of GABA+ cells (G), p=0.0001. Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).


( H and I ) Immunostaining assay of MAP2 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 35 of dorsal neural differentiation ( H ) , and quantiﬁcation of MAP2 + cells ( I ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( V1 - PAKI + ptSOX1 + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ; # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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(H and I) Immunostaining assay of MAP2 (green) after SOX1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (H), and quantiﬁcation of MAP2+ cells (I), p=0.0001 (V1-PAKI), p=0.0001 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).


( J and K ) Immunostaining assay of GABA ( green ) after SOX1 overexpression in V1 - PAKI cells on day 35 of ventral neural differentiation ( J ) , and quantiﬁcation of GABA + cells ( K ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0002 ( V1 - PAKI + ptSOX1 + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * * p < 0 . 001 versus Ctrl group ; # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

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 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

(J and K) Immunostaining assay of GABA (green) after SOX1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (J), and quantiﬁcation of GABA+ cells (K), p=0.0001 (V1-PAKI), p=0.0002 (V1-PAKI+ptSOX1+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). ***p<0.001 versus Ctrl group ; ###p<0.001 versus -dox group (one-way ANOVA).


( A and B ) Enrichment of H3K14Ac ( left ) and H3K27Ac ( right ) on SOX1 promoter region in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( A ) , p = 0 . 0046 ( H3K14Ac ) , p = 0 . 0223 ( H3K27Ac ) and ventral neural differentiation ( B ) , p = 0 . 0499 ( H3K14Ac ) , p = 0 . 0022 ( H3K27Ac ) . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) .

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(A and B) Enrichment of H3K14Ac (left) and H3K27Ac (right) on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (A), p=0.0046 (H3K14Ac), p=0.0223 (H3K27Ac) and ventral neural differentiation (B), p=0.0499 (H3K14Ac), p=0.0022 (H3K27Ac). Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01 versus Ctrl group (unpaired two-tailed Student's t-test).


( C Immunostaining assay of SOX1 ( red ) after 4 HDAC inhibitors ( 4i ) , including RG2833 ( 2833 , 2μM ) , CAY10683 ( CAY , 2μM ) , PCI - 34051 ( 34051 , 5μM ) and Bufexamac ( Buf , 12 . 5μM ) , treatment or remove one inhibitor from these 4 HDAC inhibitors ( 4i ) in V1 - PAKI cells on day 16 of dorsal neural differentiation Scale bar , 100 μm .

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(C Immunostaining assay of SOX1 (red) after 4 HDAC inhibitors (4i), including RG2833 (2833, 2μM), CAY10683 (CAY, 2μM), PCI-34051 (34051, 5μM) and Bufexamac (Buf, 12.5μM), treatment or remove one inhibitor from these 4 HDAC inhibitors (4i) in V1-PAKI cells on day 16 of dorsal neural differentiation Scale bar, 100 μm.


( G and H ) Immunostaining assay of SOX1 ( red ) after Bufexamac treatment in V1 - PAKI cells on day 16 of dorsal neural differentiation ( G ) , and quantiﬁcation of SOX1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( + Buf ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ; ( one - way ANOVA ) .

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 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

(G and H) Immunostaining assay of SOX1 (red) after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal neural differentiation (G), and quantiﬁcation of SOX1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).


( J and K ) Immunostaining assay of MAP2 ( green ) after Bufexamac treatment in V1 - PAKI cells on day 35 of dorsal neural differentiation ( J ) , and quantiﬁcation of MAP2 + cells ( K ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( + Buf ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ( one - way ANOVA ) .

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 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

(J and K) Immunostaining assay of MAP2 (green) after Bufexamac treatment in V1-PAKI cells on day 35 of dorsal neural differentiation (J), and quantiﬁcation of MAP2+ cells (K), p=0.0001 (V1-PAKI), p=0.0001 (+Buf). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group (one-way ANOVA).