A Analysis of ASCO enrichment by ChIRP using two sets of independent biotinylated probes ODD and EVEN compared to negative control with probes designed against the LacZ RNA . The fold enrichment was calculated between ODD or EVEN samples against LacZ . These samples were used for protein precipitation and Mass Spectrometry analyses ( ChIRP - MS ) , from which PRP8a was identified as a potential ASCO partner .

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A Analysis of ASCO enrichment by ChIRP using two sets of independent biotinylated probes ODD and EVEN compared to negative control with probes designed against the LacZ RNA. The fold enrichment was calculated between ODD or EVEN samples against LacZ. These samples were used for protein precipitation and Mass Spectrometry analyses (ChIRP-MS), from which PRP8a was identified as a potential ASCO partner.


B Validation of PRP8a - ASCO interaction by PRP8a - RIP . U5 RNA was used as a positive control . Data information : In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT - qPCR

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B Validation of PRP8a-ASCO interaction by PRP8a-RIP. U5 RNA was used as a positive control. Data information: In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT-qPCR


B Validation of PRP8a - ASCO interaction by PRP8a - RIP . U5 RNA was used as a positive control . Data information : In , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT -

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C Immunoblot analysis was performed during PRP8a immunoprecipitation ( IP ) . The same volume of input , unbound fraction was loaded as well as 20 % of the eluted IP fraction . α - PRP8a and α - IgG : IP performed with anti - PRP8a or control rabbit IgG antibody , respectively .

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C Immunoblot analysis was performed during PRP8a immunoprecipitation (IP). The same volume of input, unbound fraction was loaded as well as 20 % of the eluted IP fraction. α-PRP8a and α-IgG : IP performed with anti-PRP8a or control rabbit IgG antibody, respectively.


: In D , the results are expressed as a percentage of the Input for PRP8a RIP followed by RT - qPCR , and IgG RIP was used as a negative control .

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: In D, the results are expressed as a percentage of the Input for PRP8a RIP followed by RT-qPCR, and IgG RIP was used as a negative control.


A ASCO transcript levels in WT and prp8 - 7 mutants .

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A ASCO transcript levels in WT and prp8-7 mutants.


prp8 - 7 leaky mutant displays similar AS events as observed in RNAi - ASCO . Quantification of SR34 isoforms splicing index by RT - qPCR .

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prp8-7 leaky mutant displays similar AS events as observed in RNAi-ASCO. Quantification of SR34 isoforms splicing index by RT-qPCR.


C prp8 - 7 leaky mutant displays similar AS events as observed in RNAi - ASCO . Quantification of ESP isoforms splicing index by RT - qPCR .

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C prp8-7 leaky mutant displays similar AS events as observed in RNAi-ASCO. Quantification of ESP isoforms splicing index by RT-qPCR.


D ASCO transcript levels in smd1b mutant . In A and D RNAs were extracted from WT , prp8 - 7 and smd1b 14 - day - old plants .

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D ASCO transcript levels in smd1b mutant. In A and D RNAs were extracted from WT, prp8-7 and smd1b 14-day-old plants.


E SmD1b can bind ASCO in vivo . U6 RNA was used as a positive control and a housekeeping gene ( HKG2 , AT4G26410 ) RNA as a negative control .

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E SmD1b can bind ASCO in vivo. U6 RNA was used as a positive control and a housekeeping gene (HKG2, AT4G26410) RNA as a negative control.


F SmD1b recognizes in vivo the RNAs of 4 genes regulated by ASCO . In E and F the results were expressed as % INPUT in SmD1b - GFP RIP and IgG RIP used as a negative control .

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F SmD1b recognizes in vivo the RNAs of 4 genes regulated by ASCO. In E and F the results were expressed as % INPUT in SmD1b-GFP RIP and IgG RIP used as a negative control.


smd1b mutant displays similar AS events as observed in RNAi - ASCO . Quantification of SR34 isoforms splicing index by RT - qPCR .

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smd1b mutant displays similar AS events as observed in RNAi-ASCO. Quantification of SR34 isoforms splicing index by RT-qPCR.


H smd1b mutant displays similar AS events as observed in RNAi - ASCO . Quantification of ESP isoforms splicing index by RT - qPCR .

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H smd1b mutant displays similar AS events as observed in RNAi-ASCO. Quantification of ESP isoforms splicing index by RT-qPCR.


( B ) Phenotypes of affected individuals . Camptodactyly , absent flexion crease , and limited forearm supination were observed .

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(B) Phenotypes of affected individuals. Camptodactyly, absent flexion crease, and limited forearm supination were observed.


( C - E ) T1 weighted MRI scan on upper limbs of subject IV : 7 . ( C ) The pronator quadratus absence of affected side was indicated by a red arrow . ( D ) No difference was found in palmar muscles . ( E ) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow . ( F - H ) T1 weighted MRI scan on upper limbs of subject IV : 8 . ( F ) Increased epimysial fat was indicated by red arrow . ( G ) Completely loss of thenar eminences of both hands were indicated by red arrows . ( H ) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows .

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(C-E) T1 weighted MRI scan on upper limbs of subject IV:7. (C) The pronator quadratus absence of affected side was indicated by a red arrow. (D) No difference was found in palmar muscles. (E) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow. (F-H) T1 weighted MRI scan on upper limbs of subject IV:8. (F) Increased epimysial fat was indicated by red arrow. (G) Completely loss of thenar eminences of both hands were indicated by red arrows. (H) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows.


( I ) The MET variants by Sanger sequencing was indicated by red arrow .

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(I) The MET variants by Sanger sequencing was indicated by red arrow.


( J ) 293T cells were transfected with FLAG - tagged MET / METMut / Vector plasmids , and 48 h post transfection , cells were treated with 10 ng / mL recombinant human HGF for 1 h . Then MET / METMut protein purification and tyrosine kinase assay were conducted . Western Blot pictures representative of n = 3 experiments . METMut means p . Y1234C mutant MET . EGFR means epidermal growth factor receptor and serves as a positive control . NC means negative control .

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(J) 293T cells were transfected with FLAG-tagged MET/METMut/Vector plasmids, and 48 h post transfection, cells were treated with 10 ng/mL recombinant human HGF for 1 h. Then MET/METMut protein purification and tyrosine kinase assay were conducted. Western Blot pictures representative of n = 3 experiments. METMut means p.Y1234C mutant MET. EGFR means epidermal growth factor receptor and serves as a positive control. NC means negative control.


( A ) Gross appearance of wild - type and heterozygous newborns . ( B ) Graph showing weight of P0 mice . Sample size : WT ( n = 4 ) and Hetero ( n = 5 ) . * P < 0 . 05 , by one - way ANOVA and follow by Dunnett ' s post hoc test .

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(A) Gross appearance of wild-type and heterozygous newborns. (B) Graph showing weight of P0 mice. Sample size: WT (n = 4) and Hetero (n = 5). *P < 0.05, by one-way ANOVA and follow by Dunnett's post hoc test.


Sections of spine from wild types and heterozygotes at P0 , were conducted with HE staining and immunofluorescence staining using anti - myosin heavy chain antibody . Scale bars , 100 μm .

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Sections of spine from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.


Sections of forelimbs , hindlimbs , hand , and foot from wild types and heterozygotes at P0 , were conducted with HE staining and immunofluorescence staining using anti - myosin heavy chain antibody . Scale bars , 100 μm .

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Sections of forelimbs, hindlimbs, hand, and foot from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.


( H ) The mean number of myofibers in the muscles of the spine , forelimb , hindlimb , hand and foot was qualified . NS means no statistic significance . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed independent student ' s t test . In ( C - H ) , n = 3 .

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(H) The mean number of myofibers in the muscles of the spine, forelimb, hindlimb, hand and foot was qualified. NS means no statistic significance. *P < 0.05, **P < 0.01, by two-tailed independent student's t test. In (C-H), n = 3.


( I ) Transmission electron microscope analysis of gastrocnemius from wild - type and heterozygous newborns . Black arrows denote mitochodria . Scale bars , 2 μm . WT means wild types , Hetero means heterozygotes .

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(I) Transmission electron microscope analysis of gastrocnemius from wild-type and heterozygous newborns. Black arrows denote mitochodria. Scale bars, 2 μm. WT means wild types, Hetero means heterozygotes.


( A ) In situ hybridization of E10 . 5 embryos using Pax3 probe . Pax3 expression ( brown signal ) was observed in limb bud and demomyotome ( DM ) , respectively . Cells labeled with Pax3 were indicated by red arrow . Scale bars , 200 μm .

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(A) In situ hybridization of E10.5 embryos using Pax3 probe. Pax3 expression (brown signal) was observed in limb bud and demomyotome (DM), respectively. Cells labeled with Pax3 were indicated by red arrow. Scale bars, 200 μm.


( B ) Gross appearance of E14 . 5 embryos of indicated genotype . Scale bars , 2 . 5 mm .

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(B) Gross appearance of E14.5 embryos of indicated genotype. Scale bars, 2.5 mm.


( C ) HE staining of forelimb , hindlimb , and paraspinal muscle of E14 . 5 embryos . Scale bars , 100 μm .

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(C) HE staining of forelimb, hindlimb, and paraspinal muscle of E14.5 embryos. Scale bars, 100 μm.


( D ) Gross appearance of E16 . 5 embryos of indicated genotype . Scale bars , 2 . 5 mm .

(D) Gross appearance of E16.5 embryos of indicated genotype. Scale bars, 2.5 mm.


( E ) HE staining of forelimb , hindlimb , and paraspinal muscle of E16 . 5 embryos . n = 3 , scale bars , 200 μm .

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(E) HE staining of forelimb, hindlimb, and paraspinal muscle of E16.5 embryos. n = 3, scale bars, 200 μm.


( F ) Anti - Ki67 antibody was used to label proliferative myoblasts ( red fluorescence ) with DAPI labeled nucleuses ( blue fluorescence ) in paraspinal muscle . Scale bar , 25 μm . ( G ) Bar graph showing statistical analysis of positive rate of Ki67 - labeled nuclei , n = 3 with more than 150 cells analyzed per n , * * P < 0 . 01 , by chi - square test ( χ2 test ) . WT means wild types , Hetero means heterozygotes , and Homo means homozygotes .

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(F) Anti-Ki67 antibody was used to label proliferative myoblasts (red fluorescence) with DAPI labeled nucleuses (blue fluorescence) in paraspinal muscle. Scale bar, 25 μm. (G) Bar graph showing statistical analysis of positive rate of Ki67-labeled nuclei, n = 3 with more than 150 cells analyzed per n, **P < 0.01, by chi-square test (χ2 test). WT means wild types, Hetero means heterozygotes, and Homo means homozygotes.


( C Upper part , schematic illustration of the experimental procedure . Western blot analysis of S6 Ribosomal Protein and phospho - S6 Ribosomal Protein ( Ser235 / 236 ) from whole - liver protein extracts of female and male at P8 ( C ) treated mice . Mice were sampled after 4 days of Et - OH or rapamycin treatment .

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(C Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P8 (C) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.


D ) Upper part , schematic illustration of the experimental procedure . Western blot analysis of S6 Ribosomal Protein and phospho - S6 Ribosomal Protein ( Ser235 / 236 ) from whole - liver protein extracts of female and male at P34 ( D ) treated mice . Mice were sampled after 4 days of Et - OH or rapamycin treatment .

D) Upper part, schematic illustration of the experimental procedure. Western blot analysis of S6 Ribosomal Protein and phospho-S6 Ribosomal Protein (Ser235/236) from whole-liver protein extracts of female and male at P34 (D) treated mice. Mice were sampled after 4 days of Et-OH or rapamycin treatment.


Keratinocytes were isolated from the skin of a 4 - year - old patient with severe - generalised RDEB linked to homozygous insertion - deletion in COL7A1 ( Hilal et al , 1993 ) . Cultured RDEB cells ( blue line ) were serially passaged for more than 4 months , displaying a growth potential similar to non - diseased control cells ( YF29 ) isolated from the foreskin of a newborn ( black line ) . To calculate the percentage of growing colonies , 100 to 1 , 000 cells were plated into indicator dishes at each passage . Cells were grown for 12 days , fixed and stained with rhodamine B . Colonies were scored as growing or aborted ( Barrandon Green , 1987 ) .

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Keratinocytes were isolated from the skin of a 4-year-old patient with severe-generalised RDEB linked to homozygous insertion-deletion in COL7A1 (Hilal et al, 1993). Cultured RDEB cells (blue line) were serially passaged for more than 4 months, displaying a growth potential similar to non-diseased control cells (YF29) isolated from the foreskin of a newborn (black line). To calculate the percentage of growing colonies, 100 to 1,000 cells were plated into indicator dishes at each passage. Cells were grown for 12 days, fixed and stained with rhodamine B. Colonies were scored as growing or aborted (Barrandon Green, 1987).


Clonal analysis demonstrated the presence of stem cells ( holoclones ) in a passage VII RDEB culture ( 95 % of growing colonies ) .

Clonal analysis demonstrated the presence of stem cells (holoclones) in a passage VII RDEB culture (95% of growing colonies).


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . COLVII detection in clones by immunostaining . COLVII expression ( green ) was detectable in some clones ( 6 , 17 , 22 , 58 and 61 ) and not in others ( 3 , 24 and 54 ) ; nuclei were stained with Hoechst 33342 ( blue ) . Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3 - J2 feeder cells . Scale bar : 50 μm .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation.COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm.


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . Quantitative RT - PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes . All clones shown in ( A ) were transduced but expressed different levels of COL7A1 transcripts . Clones 6 , 17 , 22 , 54 , 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors ( YF29 and OR - CA , control 1 and 2 , respectively ) . The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1 .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Quantitative RT-PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1.


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . Determination of proviral rearrangements in transduced clones . A Southern blot was performed using genomic DNA of RDEB cells , clones and the infected mass culture from which the clones were isolated . Genomic DNA was digested with EcoRV and SpeI that cut at the 3 ′ and 5 ′ end of the provirus ( Supplementary Fig S2 ) and hybridised with a 907 - bp COL7A1 probe radiolabelled with 32P isotope . The upper band corresponded to the endogenous signal . The retroviral producer line Flp293A - E1aColVII1 was used as a control for the digested 9 . 6 - kb provirus ( proviral signal ) . Smaller bands corresponded to rearranged proviruses marked with an asterisk .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk.


Single cells were isolated from a mass culture ( passage V ) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA . Clonal types were determined ( Barrandon Green , 1987 ) and listed in Supplementary Table S1 . Growing clones were expanded for further characterisation . Identification of stem cells producing COLVII . Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant , while clone 54 and surprisingly clone 22 did not ( see A ) . RDEB cells were used as a negative control and healthy donor cells as a positive control . The secreted matrix metalloproteinase 2 ( MMP2 ) was used as a loading control .

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Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green,1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.


Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII . Non - COLVII - producing holoclone 54 ( black lines ) and COLVII - producing holoclone 6 ( red lines ) were serially transferred once a week until exhaustion ( Rochat et al , 1994 ) . Theoretical number of epidermal cells available for characterisation and CEA production from corrected ( clone 6 ) and uncorrected stem cells ( clone 54 ) calculated from the day of cloning . The colony - forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling , the generation number and the total progeny of isolated stem cells . Both corrected and uncorrected epidermal stem cells show high growth potential in vitro .

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Serial cultivation of transduced and untransduced holoclones demonstrated that the growth potential of the stem cells was not affected by the production of COLVII. Non-COLVII-producing holoclone 54 (black lines) and COLVII-producing holoclone 6 (red lines) were serially transferred once a week until exhaustion (Rochat et al, 1994).Theoretical number of epidermal cells available for characterisation and CEA production from corrected (clone 6) and uncorrected stem cells (clone 54) calculated from the day of cloning. The colony-forming efficiency and the percentage of growing colonies for each passage were used to calculate the population doubling, the generation number and the total progeny of isolated stem cells. Both corrected and uncorrected epidermal stem cells show high growth potential in vitro.


Immunodeficient SCID mice were transplanted with untransduced cultured RDEB keratinocytes ( left ) or the COLVII - secreting holoclone ( clone 6 ) ( right ) . Punch biopsies were obtained at various times post - transplantation ( PT ) , stained with haematoxylin / eosine ( H / E ) , for human leucocyte antigen - 1 ( HLA - 1 ) ( green ) and human COLVII ( red ) . RDEB keratinocytes generated a HLA - 1 - positive epidermis that adhered poorly to the dermo - epidermal junction ( DEJ ) ( arrow indicates a blister ) and absence of COLVII ( dotted line delimits the dermis ) , whereas the corrected keratinocytes produced an epidermis that adhered to the dermis and deposited COLVII ( red ) at the DEJ . Note the presence of KI67 - positive keratinocytes ( red ) in the basal and suprabasal layers in the corrected epidermis at 385 days post - transplantation , indicating that transplanted cells had self - renewed for more than a year . Scale bar : 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Immunodeficient SCID mice were transplanted with untransduced cultured RDEB keratinocytes (left) or the COLVII-secreting holoclone (clone 6) (right). Punch biopsies were obtained at various times post-transplantation (PT), stained with haematoxylin/eosine (H/E), for human leucocyte antigen-1 (HLA-1) (green) and human COLVII (red). RDEB keratinocytes generated a HLA-1-positive epidermis that adhered poorly to the dermo-epidermal junction (DEJ) (arrow indicates a blister) and absence of COLVII (dotted line delimits the dermis), whereas the corrected keratinocytes produced an epidermis that adhered to the dermis and deposited COLVII (red) at the DEJ. Note the presence of KI67-positive keratinocytes (red) in the basal and suprabasal layers in the corrected epidermis at 385 days post-transplantation, indicating that transplanted cells had self-renewed for more than a year. Scale bar: 50 μm.


Transmission electron microscopy ( TEM ) demonstrated the presence of anchoring fibrils ( arrows ) at the DEJ of the corrected epidermis ( middle right panel ) and in a normal human skin biopsy ( left panel ) . Note the absence of anchoring fibrils in the RDEB epidermis ( middle left panel ) . Detection of human COLVII by immunogold staining in the DEJ of corrected epidermis ( right panel ) . Scale bar : 250 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 O 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

Transmission electron microscopy (TEM) demonstrated the presence of anchoring fibrils (arrows) at the DEJ of the corrected epidermis (middle right panel) and in a normal human skin biopsy (left panel). Note the absence of anchoring fibrils in the RDEB epidermis (middle left panel). Detection of human COLVII by immunogold staining in the DEJ of corrected epidermis (right panel). Scale bar: 250 nm.


Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage XII ( clone 6 ) . Five specific signals ( red ) were detected on five different chromosomes . As expected , a COL7A1 probe ( red ) hybridised to the two endogenous COL7A1 alleles located on the chromosomes 3 as identified by a specific centromeric probe ( Cen 3 ) and to three other chromosomes identified as chromosomes 2 , 11 and 22 by means of specific centromeric probes ( Cen 2 , Cen 11 and Cen 22 , respectively ) . The latter corresponded to proviruses . Determination of proviral integration sites in corrected stem cell . Genomic DNA from clone 6 was submitted to whole - genome sequencing together with PCR and subsequent Sanger sequencing to identify the integration sites to the base - pair level . Mate pairs that span from the viral sequence to a human chromosome were extracted and used to estimate integration regions . The reads were mapped to the hg19 reference sequence . Three integration sites were uncovered : one in chromosome 2 , one in chromosome 11 and one in chromosome 22 , and targeted genes were identified .

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

Visualisation of COL7A1 sequence by FISH analysis in corrected stem cells at passage XII (clone 6). Five specific signals (red) were detected on five different chromosomes. As expected, a COL7A1 probe (red) hybridised to the two endogenous COL7A1 alleles located on the chromosomes 3 as identified by a specific centromeric probe (Cen 3) and to three other chromosomes identified as chromosomes 2, 11 and 22 by means of specific centromeric probes (Cen 2, Cen 11 and Cen 22, respectively). The latter corresponded to proviruses.Determination of proviral integration sites in corrected stem cell. Genomic DNA from clone 6 was submitted to whole-genome sequencing together with PCR and subsequent Sanger sequencing to identify the integration sites to the base-pair level. Mate pairs that span from the viral sequence to a human chromosome were extracted and used to estimate integration regions. The reads were mapped to the hg19 reference sequence. Three integration sites were uncovered: one in chromosome 2, one in chromosome 11 and one in chromosome 22, and targeted genes were identified.


Analysis of the level of expression of targeted genes in corrected stem cells by quantitative RT - PCR . DARS was not significantly changed in clone 6 compared to the cells before transduction . Primers used annealed downstream of the proviral integration site . Error bars represent the standard deviation of three replicates . Identification of sequence abnormalities in the two alleles of the DARS - targeted gene in clone 6 by NGS . Reads were mapped to the hg19 reference sequence . Small insertion - deletions and SNP calling were performed and compared to GWAS databases . The mapping highlighted two indels and one SNP in the DARS genomic sequence . The indels were not associated with disease and the SNP was synonymous ( CCU - CCG both codons correspond to proline ) .

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 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 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

Analysis of the level of expression of targeted genes in corrected stem cells by quantitative RT-PCR. DARS was not significantly changed in clone 6 compared to the cells before transduction. Primers used annealed downstream of the proviral integration site. Error bars represent the standard deviation of three replicates.Identification of sequence abnormalities in the two alleles of the DARS-targeted gene in clone 6 by NGS. Reads were mapped to the hg19 reference sequence. Small insertion-deletions and SNP calling were performed and compared to GWAS databases. The mapping highlighted two indels and one SNP in the DARS genomic sequence. The indels were not associated with disease and the SNP was synonymous (CCU-CCG both codons correspond to proline).


Upper panel : expression of proteins associated with the acquisition of immortalisation process involved in senescence , in evasion of growth suppression and in apoptosis ( Hanahan Weinberg , 2011 ) . The level of P53 , P21 , RAS and P16 was similar in clone 6 , untransduced cells and mass culture from four independent infections , significantly different from the squamous cell carcinoma cell line SCC - 13 . Lower panel : the phosphorylation state of the PRB restriction point was maintained in clone 6 and untransduced recessive dystrophic epidermolysis bullosa ( RDEB ) cells , whereas PRB was heavily phosphorylated in transformed cells ( SCC - 13 ) . Appropriate loading controls were used for each cellular extract ( GAPDH for cytoplasmic extracts , histone H3 for nuclear extracts and tubulin for whole - cell extracts . H3 for histone H3 , ppRB for hyperphosphorylated pRB and pRB for hypophosphorylated PRB ) .

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

Upper panel: expression of proteins associated with the acquisition of immortalisation process involved in senescence, in evasion of growth suppression and in apoptosis (Hanahan Weinberg, 2011). The level of P53, P21, RAS and P16 was similar in clone 6, untransduced cells and mass culture from four independent infections, significantly different from the squamous cell carcinoma cell line SCC-13. Lower panel: the phosphorylation state of the PRB restriction point was maintained in clone 6 and untransduced recessive dystrophic epidermolysis bullosa (RDEB) cells, whereas PRB was heavily phosphorylated in transformed cells (SCC-13). Appropriate loading controls were used for each cellular extract (GAPDH for cytoplasmic extracts, histone H3 for nuclear extracts and tubulin for whole-cell extracts. H3 for histone H3, ppRB for hyperphosphorylated pRB and pRB for hypophosphorylated PRB).


Transduced clone 6 had a diploid karyotype at passage XVI ( see also Fig5A ) .

Transduced clone 6 had a diploid karyotype at passage XVI (see also Fig5A).


Transduced clones were not tumorigenic . The tumorigenic potential of the clones was tested by tumour formation in nude mice . Transduced RDEB holoclones ( blue ) [ clone 6 ( square , n = 4 ) passage X , clone 22 ( circle , n = 2 ) passage XI , clone 54 ( cross , n = 4 ) passage X ] were not tumorigenic when injected subcutaneously in athymic mice as were untransduced RDEB keratinocytes ( passage VII ) . SCC - 13 , a squamous cell carcinoma cell line ( black ) ( lozenge , n = 2 ) , was used as a positive control .

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

Transduced clones were not tumorigenic. The tumorigenic potential of the clones was tested by tumour formation in nude mice. Transduced RDEB holoclones (blue) [clone 6 (square, n = 4) passage X, clone 22 (circle, n = 2) passage XI, clone 54 (cross, n = 4) passage X] were not tumorigenic when injected subcutaneously in athymic mice as were untransduced RDEB keratinocytes (passage VII). SCC-13, a squamous cell carcinoma cell line (black) (lozenge, n = 2), was used as a positive control.


To test whether the transduced RDEB keratinocytes had disseminated after the generation of an epidermis onto SCID mice ( see Fig4C ) , the internal organs of the recipient mice ( 3 mice for clone 6 and 2 mice for clone 22 ) were harvested 385 days post - transplantation and analysed for COL7A1 proviral sequences . No COL7A1 sequence was detected . A mouse transplanted with a holoclone obtained from a healthy donor ( YF29 ) was used as an internal control . PCR - positive controls were genomic DNA from transduced cells ( holoclone 6 ) and cDNAs isolated from healthy keratinocytes . B - actin was run as a loading control .

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

To test whether the transduced RDEB keratinocytes had disseminated after the generation of an epidermis onto SCID mice (see Fig4C), the internal organs of the recipient mice (3 mice for clone 6 and 2 mice for clone 22) were harvested 385 days post-transplantation and analysed for COL7A1 proviral sequences. No COL7A1 sequence was detected. A mouse transplanted with a holoclone obtained from a healthy donor (YF29) was used as an internal control. PCR-positive controls were genomic DNA from transduced cells (holoclone 6) and cDNAs isolated from healthy keratinocytes. B-actin was run as a loading control.


A . Volcano plot showing the fold change ( y - axis ) versus adjusted ( adj . ) p - value ( x - axis ) of the beta cell transcriptomes between chow and HFD - fed mice ( 16 weeks ) . Genes highlighted in red or green are based on the thresholds of Log2 fold change > 1 and adj . P value < 0 . 01 ( two - tailed paired student ' s t - test ) . Genes with > 10 fold upregulated expression are labeled with their name . Pbk gene expression is indicated by the arrow . RNAseq data was available from https : / / doi . org / 10 . 1371 / journal . pone . 0213299 . t004 .

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

A.Volcano plot showing the fold change (y-axis) versus adjusted (adj.) p-value (x-axis) of the beta cell transcriptomes between chow and HFD-fed mice (16 weeks). Genes highlighted in red or green are based on the thresholds of Log2 fold change > 1 and adj. P value < 0.01 (two-tailed paired student's t-test). Genes with >10 fold upregulated expression are labeled with their name. Pbk gene expression is indicated by the arrow. RNAseq data was available from https://doi.org/10.1371/journal.pone.0213299.t004.


L . Pbk KD in INS - 1 cells suppresses cell growth . Western blot data showed the decreased Pbk expression level in INS - 1 cells with Pbk - targeted shRNA transduction . Cell growth curve was from three independent experimets ( n = 3 ) . * * * P < 0 . 0001 ( Vector : shRNA - # 1 ) , * P = 0 . 0181 ( Vector : shRNA - # 5 ) , ( Two way ANOVA ) . M . The role of ectopic expression of Pbk - WT and Pbk - Mut on INS - 1 cell growth . Western blot data showed the overexpression of V5 - flaged wild type and mutant Pbk in INS - 1 cells . Cell growth curve was from three independent experimets ( n = 3 ) . * * P = 0 . 0069 ( Two way ANOVA ) . ns , not statistically significant difference .

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

L. Pbk KD in INS-1 cells suppresses cell growth. Western blot data showed the decreased Pbk expression level in INS-1 cells with Pbk-targeted shRNA transduction. Cell growth curve was from three independent experimets (n=3). ***P < 0.0001 (Vector:shRNA-#1), *P = 0.0181 (Vector:shRNA-#5) , (Two way ANOVA). M. The role of ectopic expression of Pbk-WT and Pbk-Mut on INS-1 cell growth. Western blot data showed the overexpression of V5-flaged wild type and mutant Pbk in INS-1 cells. Cell growth curve was from three independent experimets (n=3). **P = 0.0069 (Two way ANOVA). ns, not statistically significant difference.


F . Islet number in each pancreas section on 11 - week - old male PbkKI / KI and PbkWT / WT mice ( n = 4 for each group ) . * P = 0 . 0443 ( two - tailed unpaired student ' s t - test ) . G . Beta cell mass comparison between 11 - week - old male PbkKI / KI and PbkWT / WT mice ( n = 4 for each group ) . * * P = 0 . 0015 ( two - tailed unpaired student ' s t - test ) .

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F. Islet number in each pancreas section on 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). *P = 0.0443 (two-tailed unpaired student's t-test). G. Beta cell mass comparison between 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). **P = 0.0015 (two-tailed unpaired student's t-test).


K , L . Representative images of double immuno - staining for insulin ( green ) and BrdU ( red ) of pancreas from PbkWT / WT ( K ) or PbkKI / KI ( L ) mice with 5 weeks HFD feeding . Islet area was circled with white dashed line . Nuclei were labeled by DAPI ( blue ) . Scale bar : 50 μm .

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K, L. Representative images of double immuno-staining for insulin (green) and BrdU (red) of pancreas from PbkWT/WT (K) or PbkKI/KI (L) mice with 5 weeks HFD feeding. Islet area was circled with white dashed line. Nuclei were labeled by DAPI (blue). Scale bar: 50 μm.


E , F . JunD KD in INS - 1 cells using shRNA upregulated Pbk protein levels ( E ) and mRNA levels ( F ) . qPCR data was from three independent experimets ( n = 3 ) . * * * P = 0 . 0001 ( shRNA - # 1 ) , * * * P = 0 . 0007 ( shRNA - # 2 ) ( two - tailed unpaired student ' s t - test ) . G , H . JunD KD in PIME cells using shRNA increased Pbk protein levels ( G ) and mRNA levels ( H ) . qPCR data was from three independent experimets ( n = 3 ) . * P = 0 . 0236 ( shRNA - # 1 ) , * P = 0 . 0175 ( shRNA - # 2 ) ( two - tailed unpaired student ' s t - test ) .

O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, F. JunD KD in INS-1 cells using shRNA upregulated Pbk protein levels (E) and mRNA levels (F). qPCR data was from three independent experimets (n=3).***P = 0.0001 (shRNA-#1), ***P = 0.0007 (shRNA-#2) (two-tailed unpaired student's t-test). G, H. JunD KD in PIME cells using shRNA increased Pbk protein levels (G) and mRNA levels (H). qPCR data was from three independent experimets (n=3).*P = 0.0236 (shRNA-#1), *P = 0.0175 (shRNA-#2) (two-tailed unpaired student's t-test).


N . JunD overexpression in PIME cells suppressed Pbk protein levels ( M , Lane 2 vs Lane 1 ) , but did not affect Pbk expression in menin - null PIME cells ( M , Lane 4 vs Lane 3 ) .

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N. JunD overexpression in PIME cells suppressed Pbk protein levels (M, Lane 2 vs Lane 1), but did not affect Pbk expression in menin-null PIME cells (M, Lane 4 vs Lane 3).


J , K . The effect of T5224 on the binding of JunD , menin , HDAC3 , and acetylated histone3 at the Pbk locus in PIME cells ( J ) or INS - 1 cells ( K ) . Three independent experimets ( n = 3 ) . * P = 0 . 0490 ( J , JunD ) , * P = 0 . 0479 ( J , Menin ) , * P = 0 . 0154 ( J , HDAC3 ) , * * P = 0 . 0029 ( J , Ac - H3 ) , * P = 0 . 0104 ( K , JunD ) , * * P = 0 . 0024 ( K , Menin ) , * * * P = 0 . 0002 ( K , HDAC3 ) , * * * P = 0 . 0001 ( K , Ac - H3 ) , ( two - tailed unpaired student ' s t - test ) . ns , not statistically significant difference .

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J, K. The effect of T5224 on the binding of JunD, menin, HDAC3, and acetylated histone3 at the Pbk locus in PIME cells (J) or INS-1 cells (K). Three independent experimets (n=3). *P = 0.0490 (J, JunD), *P = 0.0479 (J, Menin), *P = 0.0154 (J, HDAC3), **P = 0.0029 (J, Ac-H3), *P = 0.0104 (K, JunD), **P = 0.0024 (K, Menin), ***P = 0.0002 (K, HDAC3), ***P = 0.0001 (K, Ac-H3), (two-tailed unpaired student's t-test). ns, not statistically significant difference.


F . Mouse primary islets treated with MI - 503 for 3 days were subjected ChIP assay to detect the binding of menin , HDAC3 , JunD , as well as histone acetylation level at the Pbk locus . Three independent experimets ( n = 3 ) . * P = 0 . 0218 ( Menin ) , * P = 0 . 0145 ( HDAC3 ) , * P = 0 . 0184 ( Ac - H3 ) ( two - tailed unpaired student ' s t - test ) . ns , not statistically significant difference .

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F. Mouse primary islets treated with MI-503 for 3 days were subjected ChIP assay to detect the binding of menin, HDAC3, JunD, as well as histone acetylation level at the Pbk locus. Three independent experimets (n=3). *P = 0.0218 (Menin), *P = 0.0145 (HDAC3), *P = 0.0184 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.


G . MI - 503 increases proliferation of menin - expressing PIME cells but not menin - null PIME cells . Three independent experimets ( n = 3 ) . * * * P < 0 . 0001 ( Two - way ANOVA ) .

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G. MI-503 increases proliferation of menin-expressing PIME cells but not menin-null PIME cells. Three independent experimets (n=3). ***P < 0.0001 (Two-way ANOVA).


K - M . 5 days of MI - 503 treatment impacts the expression of Pbk at protein level ( K ) and mRNA level ( L ) , as well as ki67 mRNA levels ( M ) in human primary islets ( donor number is four ) . qPCR data was from three independent experimets ( n = 3 ) . * * P = 0 . 0036 ( L , 10 nM ) , * * P = 0 . 0024 ( L , 50 nM ) , * * P = 0 . 0073 ( L , 100 nM ) , * P = 0 . 0237 ( M , 50 nM ) ( two - tailed unpaired student ' s t - test ) . ns , not statistically significant difference .

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K-M. 5 days of MI-503 treatment impacts the expression of Pbk at protein level (K) and mRNA level (L), as well as ki67 mRNA levels (M) in human primary islets (donor number is four). qPCR data was from three independent experimets (n=3). **P = 0.0036 (L, 10 nM), **P = 0.0024 (L, 50 nM), **P = 0.0073 (L, 100 nM), *P = 0.0237 (M, 50 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference.


E - L . Representative images of insulin ( green ) and Ki67 ( red ) double staining on mouse islets with 8 wks MI treatment ( E - H ) , and insulin ( green ) or Pbk ( red ) double staining on mouse islets ( I - L ) . Nuclei were labeled by DAPI ( blue ) . Islet area was circled by white dashed line . Ki67 and Pbk staining are denoted by white arrows . Scale bars : 50 μm .

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E-L. Representative images of insulin (green) and Ki67 (red) double staining on mouse islets with 8 wks MI treatment (E-H), and insulin (green) or Pbk (red) double staining on mouse islets (I-L). Nuclei were labeled by DAPI (blue). Islet area was circled by white dashed line. Ki67 and Pbk staining are denoted by white arrows. Scale bars: 50 μm.


D , E . Double staining for insulin ( green ) and BrdU ( red ) of mice islets after 12 weeks of vehicle ( D ) or MI ( E ) administration . Islet area was circled by white dashed line . Nuclei were labeled by DAPI ( blue ) . BrdU staining is indicated by arrows . Scale bar : 50 μm . F , G . Quantification of BrdU positive beta cells ( F ) and beta cell mass ( G ) of MI - 463 treated HFD - fed mice or control mice . Five mice for each group , and at least 10 islet images per mouse were analyzed . * * P = 0 . 0059 , * P = 0 . 0103 ( two - tailed unpaired student ' s t - test ) .

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D, E. Double staining for insulin (green) and BrdU (red) of mice islets after 12 weeks of vehicle (D) or MI (E) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). BrdU staining is indicated by arrows. Scale bar:50 μm. F, G. Quantification of BrdU positive beta cells (F) and beta cell mass (G) of MI-463 treated HFD-fed mice or control mice. Five mice for each group, and at least 10 islet images per mouse were analyzed. **P = 0.0059, *P = 0.0103 (two-tailed unpaired student's t-test).


H , I . Double staining for insulin ( green ) and Pbk ( red ) of mice islets after 12 weeks of vehicle ( H ) or MI ( I ) administration . Islet area was circled by white dashed line . Nuclei were labeled by DAPI ( blue ) . Pbk staining is indicated by arrows . Scale bar : 50 μm . J . Quantification of Pbk staining - positive β - cells . Five mice for each group , and at least 10 islet images per mouse were analyzed . * P = 0 . 0120 ( two - tailed unpaired student ' s t - test ) .

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

H, I. Double staining for insulin (green) and Pbk (red) of mice islets after 12 weeks of vehicle (H) or MI (I) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). Pbk staining is indicated by arrows. Scale bar: 50 μm. J. Quantification of Pbk staining-positive β-cells. Five mice for each group, and at least 10 islet images per mouse were analyzed. *P = 0.0120 (two-tailed unpaired student's t-test).


K . Quantification of the percentage with pbk and BrdU co - staining cells among total Pbk positive cells in HFD - fed mouse islets . The mean values of five mice from 5 sections for each mouse were presented .

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K. Quantification of the percentage with pbk and BrdU co-staining cells among total Pbk positive cells in HFD-fed mouse islets. The mean values of five mice from 5 sections for each mouse were presented.


O , P . GTT data for MI - 463 or vehicle treated PbkKI / KI or PbkWT / WT mice ( n = 3 for each group ) on 8 weeks HFD ( O ) ( * P = 0 . 0133 ( Two way ANOVA ) , ns , not statistically significant difference . ) and AUC of GTT data ( P ) ( * P = 0 . 0442 ( two - tailed unpaired student ' s t - test ) , ns , not statistically significant difference . ) .

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O, P. GTT data for MI-463 or vehicle treated PbkKI/KI or PbkWT/WT mice (n=3 for each group) on 8 weeks HFD (O) (*P = 0.0133 (Two way ANOVA), ns, not statistically significant difference.) and AUC of GTT data (P) (*P = 0.0442 (two-tailed unpaired student's t-test), ns, not statistically significant difference.).


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 .

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, 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 ,

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

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 (

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

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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 ,

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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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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+) 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 .

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

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 .

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

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 =

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

I Quantification of 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 .

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

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 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 B-MEASURED_VAR 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-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 O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times .

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

A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.


B , H1299 cells transfected with a control siRNA ( siCont . ) or an RNF20 - specific siRNA ( siRNF20 ) and a wild - type H2B ( H2BWT ) or a K120R - mutated H2B ( H2BK120R ) were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h . Representative phase - contrast images were recorded ( magnification , × 20 ) , and the surviving cells were counted . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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

B, H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


C , Lipid ROS levels were assessed by flow cytometry after C11 - BODIPY staining in cells treated Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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

C, Lipid ROS levels were assessed by flow cytometry after C11-BODIPY staining in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


D , H1299 cells treated were subjected to transmission electron microscopy . Representative images are shown .

D, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown.


E , Control H1299 cells ( transfected with siCont . or H2BWT ) and H2Bub1 - depleted H1299 cells ( transfected with siRNF20 or H2BK120R ) were treated with erastin either with or without a ferroptosis - specific inhibitor , ferrostatin - 1 ( 2 μM ) , for 24 h . Representative phase - contrast images were recorded ( magnification , × 20 ) , and the surviving cells were counted . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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

E, Control H1299 cells (transfected with siCont. or H2BWT) and H2Bub1-depleted H1299 cells (transfected with siRNF20 or H2BK120R) were treated with erastin either with or without a ferroptosis-specific inhibitor, ferrostatin-1 (2 μM), for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


A , qRT - PCR ( left ) and Western blot ( right ) analyses of H1299 cells transfected with a control siRNA ( siCont . ) or an RNF20 - specific siRNA ( siRNF20 ) and a wild - type H2B ( H2BWT ) or a K120R - mutated H2B ( H2BK120R ) for 24 h . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : 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 B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, qRT-PCR (left) and Western blot (right) analyses of H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) for 24 h. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


B , Chromatin immunoprecipitation ( ChIP ) assay was carried out with anti - H2Bub1 antibodies in H1299 cells ( left ) or 293T cells either untreated or treated with 20 μM erastin for 24 h ( right ) . The intergenic region was used as a negative control for the occupancy of H2Bub1 . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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 B-CONTROLLED_VAR 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

B, Chromatin immunoprecipitation (ChIP) assay was carried out with anti-H2Bub1 antibodies in H1299 cells (left) or 293T cells either untreated or treated with 20 μM erastin for 24 h (right). The intergenic region was used as a negative control for the occupancy of H2Bub1. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


C , Intracellular GSH levels were examined in H1299 cells treated as indicated , and bar graphs are shown . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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

C, Intracellular GSH levels were examined in H1299 cells treated as indicated, and bar graphs are shown. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


D , H1299 cells transfected as indicated were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h . Representative phase - contrast images were recorded ( magnification , × 20 ) . E , Surviving cells from the assay shown in ( D ) were counted . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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

D, H1299 cells transfected as indicated were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20). E, Surviving cells from the assay shown in (D) were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


G , Affected metal ion - binding genes were selected and subjected to cluster analysis .

G, Affected metal ion-binding genes were selected and subjected to cluster analysis.


H , Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells .

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

H, Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells.


I , Labile iron levels examined were quantified . Data information : Bars and error bars are mean ± s . d . , n = 3 independent repeats . Two - tailed unpaired Student ' s t - test was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

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

I, Labile iron levels examined were quantified. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) for 48 h .

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

Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.


Western blot analysis was performed with H1299 cells transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) for 48 h .

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

Western blot analysis was performed with H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.