Figure 3B 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 ( BD 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 , H Confocal images showing GFP + / GFAP + / SOX2 + astrocytes of distinct DG layers co - expressing the proliferation marker MCM2 Data information : scale bars = 10 µm ( HK 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 ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B 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 (BD 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,H Confocal images showing GFP+/GFAP+/SOX2+ astrocytes of distinct DG layers co-expressing the proliferation marker MCM2 Data information: scale bars = 10 µm (HK 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)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.


Figure 4B 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 runnersE 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B 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 runnersE 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.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


Figure 5B 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 miceE 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B 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 miceE 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.


Figure 2A . 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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.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.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.


Figure 3B . 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 . 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 .

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Figure 3B. 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.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.


Figure 4B . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. 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.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.


Figure 1A . 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 .

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Figure 1A. 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.


Figure 2B . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. 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.


Figure 3A . 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 . 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 ) .

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Figure 3A. 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.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).


Figure 4B . 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 ) .

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Figure 4B. 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).


Figure 5A . 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 .

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Figure 5A. 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.


Figure 6A . 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 . 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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Figure 6A. 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.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.


Figure 7A . 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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Figure 7A. 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).


Figure 8A . 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 . 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 .

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Figure 8A. 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.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.


Figure 9A . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9A. 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.


Figure 1 ( 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 ) . ( 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 ) . ( 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 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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).(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).(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).


Figure 2 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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).(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).


Figure 3 ( 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 . ( 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 ) . ( L ) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1 - PAKI cells on day 16 of dorsal ( left , p = 0 . 0007 ( V1 - PAKI ) , p = 0 . 0002 ( + Buf ) ) and ventral ( right , p = 0 . 0083 ( V1 - PAKI ) , p = 0 . 0276 ( + Buf ) ) 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 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus V1 - PAKI group ; ( one - way ANOVA ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.(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).(L) Enrichment of RNA polymerase Ⅱ on SOX1 promoter region after Bufexamac treatment in V1-PAKI cells on day 16 of dorsal (left, p=0.0007 (V1-PAKI), p=0.0002 (+Buf)) and ventral (right, p=0.0083 (V1-PAKI), p=0.0276 (+Buf)) 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 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus V1-PAKI group ; (one-way ANOVA).


Figure 4 ( B and C ) RIP analysis for endogenous interaction between HDAC10 and SOX1 - OT V1 on day 16 of dorsal neural differentiation ( B ) , p = 0 . 0085 and ventral neural differentiation ( C ) , p = 0 . 0152 . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus IgG group ( unpaired two - tailed Student ' s t - test ) . ( D and E ) Enrichment of HDAC10 on SOX1 promoter region in control and V1 - PAKI cells on day 16 of dorsal neural differentiation ( D ) , p = 0 . 0052 and ventral neural differentiation ( E ) , p = 0 . 0001 . 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 ( unpaired two - tailed Student ' s t - test ) . ( G and H ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 , SOX1 - OT V1 - 5P or SOX1 - OT V1 - 3P overexpression 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 ( ptV1 + dox ) , p = 0 . 0001 ( pt5P + dox ) . Scale bar , 100 μm . ( I and J ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 , SOX1 - OT V1 - 5P or SOX1 - OT V1 - 3P overexpression in V1 - PAKI cells on day 16 of ventral neural differentiation ( I ) , and quantiﬁcation of SOX1 + cells ( J ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptV1 + dox ) , p = 0 . 0001 ( pt5P + dox ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * p < 0 . 05 , * * * p < 0 . 001 versus Ctrl group ; # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 versus - dox group ( one - way ANOVA ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B and C) RIP analysis for endogenous interaction between HDAC10 and SOX1-OT V1 on day 16 of dorsal neural differentiation (B), p=0.0085 and ventral neural differentiation (C), p=0.0152. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, **p<0.01, ***p<0.001 versus IgG group (unpaired two-tailed Student's t-test).(D and E) Enrichment of HDAC10 on SOX1 promoter region in control and V1-PAKI cells on day 16 of dorsal neural differentiation (D), p=0.0052 and ventral neural differentiation (E), p=0.0001. 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 (unpaired two-tailed Student's t-test).(G and H) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression 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 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of SOX1 (red) after SOX1-OT V1, SOX1-OT V1-5P or SOX1-OT V1-3P overexpression in V1-PAKI cells on day 16 of ventral neural differentiation (I), and quantiﬁcation of SOX1+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptV1+dox), p=0.0001 (pt5P+dox). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). *p<0.05, ***p<0.001 versus Ctrl group ; #p<0.05, ##p<0.01, ###p<0.001 versus -dox group (one-way ANOVA).


Figure 5 ( A and B ) Immunostaining assay of ASCL1 ( green ) in control , V1 - PAKI and SOX1KO cells on day 25 of dorsal neural differentiation ( A ) , and quantiﬁcation of ASCL1 + cells ( B ) , p = 0 . 0021 ( V1 - PAKI ) , p = 0 . 0024 ( SOX1KO ) . Scale bar , 100 μm . ( C and D ) Immunostaining assay of ASCL1 ( green ) in control , V1 - PAKI and SOX1KO cells on day 25 of ventral neural differentiation ( C ) , and quantiﬁcation of ASCL1 + cells ( D ) , p = 0 . 0003 ( V1 - PAKI ) , p = 0 . 0004 ( SOX1KO ) . Scale bar , 100 μm . Data information : Data are presented as mean ± SEM from three independent experiments ( n = 3 ) . * * p < 0 . 01 , * * * p < 0 . 001 versus Ctrl group ( unpaired two - tailed Student ' s t - test ) . ( E and F ) Immunostaining assay of ASCL1 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 25 of dorsal neural differentiation ( E ) , and quantiﬁcation of ASCL1 + cells ( F ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptSOX1 + dox ) . Scale bar , 100 μm . ( G and H ) Immunostaining assay of ASCL1 ( green ) after SOX1 overexpression in V1 - PAKI cells on day 25 of ventral neural differentiation ( G ) , and quantiﬁcation of ASCL1 + cells ( H ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptSOX1 + dox ) . Scale bar , 100 μm . ( I and J ) Immunostaining assay of MAP2 ( green ) after ASCL1 overexpression in V1 - PAKI cells on day 35 of dorsal neural differentiation ( I ) , and quantiﬁcation of MAP2 + cells ( J ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptASCL1 + dox ) . Scale bar , 100 μm . ( K and L ) Immunostaining assay of GABA ( green ) after ASCL1 overexpression in V1 - PAKI cells on day 35 of ventral neural differentiation ( K ) , and quantiﬁcation of GABA + cells ( L ) , p = 0 . 0001 ( V1 - PAKI ) , p = 0 . 0001 ( ptASCL1 + 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A and B) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of dorsal neural differentiation (A), and quantiﬁcation of ASCL1+ cells (B), p=0.0021 (V1-PAKI), p=0.0024 (SOX1KO). Scale bar, 100 μm. (C and D) Immunostaining assay of ASCL1 (green) in control, V1-PAKI and SOX1KO cells on day 25 of ventral neural differentiation (C), and quantiﬁcation of ASCL1+ cells (D), p=0.0003 (V1-PAKI), p=0.0004 (SOX1KO). Scale bar, 100 μm. Data information: Data are presented as mean ±SEM from three independent experiments (n=3). **p<0.01, ***p<0.001 versus Ctrl group (unpaired two-tailed Student's t-test).(E and F) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of dorsal neural differentiation (E), and quantiﬁcation of ASCL1+ cells (F), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (G and H) Immunostaining assay of ASCL1 (green) after SOX1 overexpression in V1-PAKI cells on day 25 of ventral neural differentiation (G), and quantiﬁcation of ASCL1+ cells (H), p=0.0001 (V1-PAKI), p=0.0001 (ptSOX1+dox). Scale bar, 100 μm. (I and J) Immunostaining assay of MAP2 (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of dorsal neural differentiation (I), and quantiﬁcation of MAP2+ cells (J), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+dox). Scale bar, 100 μm. (K and L) Immunostaining assay of GABA (green) after ASCL1 overexpression in V1-PAKI cells on day 35 of ventral neural differentiation (K), and quantiﬁcation of GABA+ cells (L), p=0.0001 (V1-PAKI), p=0.0001 (ptASCL1+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).


Figure 6 ( A and B ) Immunostaining assay of SOX1 ( red ) in control , V1 - PAKI and SOX1KO cells on day 40 of dorsal ( A ) and ventral ( B ) organoids differentiation . Scale bar , 100 μm . ( C ) Immunostaining assay of MAP2 ( green ) in control , V1 - PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation . Scale bar , 100 μm . ( D ) Immunostaining assay of GABA ( green ) in control , V1 - PAKI and SOX1KO cells on day 40 of ventral organoids differentiation . Scale bar , 100 μm . ( E and F ) Immunostaining assay of SOX1 ( red ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of dorsal ( E ) and ventral ( F ) organoids differentiation . Scale bar , 100 μm . ( G ) Immunostaining assay of MAP2 ( green ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of dorsal organoids differentiation . Scale bar , 100 μm . ( H ) Immunostaining assay of GABA ( green ) after SOX1 - OT V1 overexpression in V1 - PAKI cells on day 40 of ventral organoids differentiation . Scale bar , 100 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A and B) Immunostaining assay of SOX1 (red) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal (A) and ventral (B) organoids differentiation. Scale bar, 100 μm. (C) Immunostaining assay of MAP2 (green) in control, V1-PAKI and SOX1KO cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (D) Immunostaining assay of GABA (green) in control, V1-PAKI and SOX1KO cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.(E and F) Immunostaining assay of SOX1 (red) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal (E) and ventral (F) organoids differentiation. Scale bar, 100 μm. (G) Immunostaining assay of MAP2 (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of dorsal organoids differentiation. Scale bar, 100 μm. (H) Immunostaining assay of GABA (green) after SOX1-OT V1 overexpression in V1-PAKI cells on day 40 of ventral organoids differentiation. Scale bar, 100 μm.


Figure 1Flow cytometric evaluation of CXCR4 surface expression using an anti - CXCR4 - PE antibody . Left : positive patient ; right : negative patient ; representative data are shown . The gating strategy is depicted in Supplementary Fig S1 . Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype . Median fluorescence intensity of surface CXCR4 expression relative to isotype control ( n = 7 - 10 patients ) . Horizontal bars indicate mean of all individual patient values ; asterisks indicate statistical significance ( plasma cells vs B cells : P = 0 . 0082 ; plasma cells vs T cells : P = 0 . 0091 ; plasma cells vs CD34 + cells : P = 0 . 011 ; plasma cells vs monocytes : P = 0 . 0423 ; Student ' s t - test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype ) . MM patients judged positive for CXCR4 expression were selected for this analysis . Representative MM bone marrow staining of a patient positive for MM CXCR4 expression : hematoxylin and eosin ( H & amp ; amp ; E ) staining . Immunohistochemistry for CD138 , CXCR4 , light chain kappa , and light chain lambda .

O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Flow cytometric evaluation of CXCR4 surface expression using an anti-CXCR4-PE antibody. Left: positive patient; right: negative patient; representative data are shown. The gating strategy is depicted in Supplementary Fig S1.Representative histograms revealing the magnitude of CXCR4 expression in myeloma cells as compared to the indicated bone marrow cell subtype.Median fluorescence intensity of surface CXCR4 expression relative to isotype control (n = 7-10 patients). Horizontal bars indicate mean of all individual patient values; asterisks indicate statistical significance (plasma cells vs B cells: P = 0.0082; plasma cells vs T cells: P = 0.0091; plasma cells vs CD34+ cells: P = 0.011; plasma cells vs monocytes: P = 0.0423; Student's t-test to compare mean plasmacell relative expression with mean relative expression of each further indicated cell subtype). MM patients judged positive for CXCR4 expression were selected for this analysis.Representative MM bone marrow staining of a patient positive for MM CXCR4 expression: hematoxylin and eosin (H&amp;E) staining. Immunohistochemistry for CD138, CXCR4, light chain kappa, and light chain lambda.


Figure 2Real - time PCR analysis of cxcr4 transcript expression levels in HeLa ( negative control ) and in MM cell lines MM . 1S and OPM - 2 . Shown is the mean relative expression ± SEM ( n = 3 independent experiments ) . Values are normalized to the expression of ubiquitin ( Ub ) . The asterisk indicates statistically significant differences ( HeLa vs MM . 1S : P < 0 . 001 ; HeLa vs OPM - 2 : P < 0 . 0001 ; Student ' s t - test ) . CXCR4 protein expression assessed by immunoblotting ( one representative blot out of 3 is shown ) . Binding of [ 68Ga ] Pentixafor to MM . 1S and OPM - 2 cells after the indicated incubation periods ( n = 3 per cell line and time point ) . Shown is the mean ± SEM . The difference between the OPM - 2 groups is statistically significant ; * P < 0 . 0017 ( one - way ANOVA ) . Mean tumor - to - background ratio ( TBR ) for [ 18F ] FDG ( left ) and for [ 68Ga ] Pentixafor ( right ) in MM . 1S and OPM - 2 xenograft - bearing NOD SCID mice . Shown is the mean ± SEM , n = 8 tumors ( 4 mice ) ; * P = 0 . 0111 for [ 18F ] FDG and * P = 0 . 0113 for [ 68Ga ] Pentixafor ( Student ' s t - test ) . One - way ANOVA revealed significant differences between the groups ; P < 0 . 0001 ( not graphically shown ) . Representative [ 68Ga ] Pentixafor PET images of three mice bearing MM . 1S ( right shoulder ) and OPM - 2 ( left shoulder ) tumors . Flow cytometric quantification of CXCR4 cell surface expression on resected MM . 1S and OPM - 2 tumors . Data are the mean ± SEM , n = 4 . * P = 0 . 0286 ; Mann - Whitney U - test . Correlation of [ 68Ga ] Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry . n = 8 tumors were analyzed . Mice ( n = 4 ) bearing OPM - 2 and MM . 1S xenografts were coinjected with AMD3100 ( right image , one representative mouse ) or not pretreated ( left image ) before undergoing [ 68Ga ] PentixaforPET . The white arrows point to the bladder . Quantification is shown in Supplementary Fig S3A . Source data are available online for this figure .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Real-time PCR analysis of cxcr4 transcript expression levels in HeLa (negative control) and in MM cell lines MM.1S and OPM-2. Shown is the mean relative expression ± SEM (n = 3 independent experiments). Values are normalized to the expression of ubiquitin (Ub). The asterisk indicates statistically significant differences (HeLa vs MM.1S: P < 0.001; HeLa vs OPM-2: P < 0.0001; Student's t-test).CXCR4 protein expression assessed by immunoblotting (one representative blot out of 3 is shown).Binding of [68Ga]Pentixafor to MM.1S and OPM-2 cells after the indicated incubation periods (n = 3 per cell line and time point). Shown is the mean ± SEM. The difference between the OPM-2 groups is statistically significant; *P < 0.0017 (one-way ANOVA).Mean tumor-to-background ratio (TBR) for [18F]FDG (left) and for [68Ga]Pentixafor (right) in MM.1S and OPM-2 xenograft-bearing NOD SCID mice. Shown is the mean ± SEM, n = 8 tumors (4 mice); *P = 0.0111 for [18F]FDG and *P = 0.0113 for [68Ga]Pentixafor (Student's t-test). One-way ANOVA revealed significant differences between the groups; P < 0.0001 (not graphically shown).Representative [68Ga]Pentixafor PET images of three mice bearing MM.1S (right shoulder) and OPM-2 (left shoulder) tumors.Flow cytometric quantification of CXCR4 cell surface expression on resected MM.1S and OPM-2 tumors. Data are the mean ± SEM, n = 4. *P = 0.0286; Mann-Whitney U-test.Correlation of [68Ga]Pentixafor PET mean TBR and CXCR4 cell surface expression assessed by flow cytometry. n = 8 tumors were analyzed.Mice (n = 4) bearing OPM-2 and MM.1S xenografts were coinjected with AMD3100 (right image, one representative mouse) or not pretreated (left image) before undergoing [68Ga]PentixaforPET. The white arrows point to the bladder. Quantification is shown in Supplementary Fig S3A.Source data are available online for this figure.


Figure 3 [ 68Ga ] PentixaforPET / CT and [ 18F ] FDGPET / CTA - D Maximum intensity projections ( MIP ) of [ 68Ga ] Pentixafor ( A ) and [ 18F ] FDGPET / CT ( B ) of a 68 - year - old male with histologically proven multiple myeloma indicating the better lesion - to - background contrast for [ 68Ga ] Pentixafor in the corresponding myeloma manifestations . Trans - axial views of the upper thorax ( C ) and the pelvis ( D ) underline the higher uptake values of the bone manifestations ( yellow arrows ) of [ 68Ga ] Pentixafor compared to [ 18F ] FDG .

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Figure 3[68Ga]PentixaforPET/CT and [18F]FDGPET/CTA-D Maximum intensity projections (MIP) of [68Ga]Pentixafor (A) and [18F]FDGPET/CT (B) of a 68-year-old male with histologically proven multiple myeloma indicating the better lesion-to-background contrast for [68Ga]Pentixafor in the corresponding myeloma manifestations. Trans-axial views of the upper thorax (C) and the pelvis (D) underline the higher uptake values of the bone manifestations (yellow arrows) of [68Ga]Pentixafor compared to [18F]FDG.


Figure 4Visual comparison of [ 18F ] FDG - and [ 68Ga ] Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer ( total : n = 14 ) . Number of patients ( total n = 14 ) for whom imaging with [ 18F ] FDG PET ( FDG , n = 2 ) or [ 68Ga ] Pentixafor PET ( Pentixafor , n = 7 ) was superior , with comparable positivity ( comparable , n = 3 ) , and with dual imaging providing complementary visual information ( complementary , n = 2 ) .

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Figure 4Visual comparison of [18F]FDG- and [68Ga]Pentixafor PET scansNumber of patients with visual positivity for the indicated PET tracer (total: n = 14).Number of patients (total n = 14) for whom imaging with [18F]FDG PET (FDG, n = 2) or [68Ga]Pentixafor PET (Pentixafor, n = 7) was superior, with comparable positivity (comparable, n = 3), and with dual imaging providing complementary visual information (complementary, n = 2).


Figure 5 [ 68Ga ] PentixaforPET / MRimagesA - C Coronal views of [ 68Ga ] Pentixafor ( A ) , T2 STIR weighted MRI ( B ) and CTbone window ( C ) of a maleuptake with histologically proven multiple myeloma . The increased [ 68Ga ] Pentixaforbone correlates with the hyperintense T2 STIR signal ; however , the myeloma manifestations are underestimated in the corresponding bone window CT .

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Figure 5[68Ga]PentixaforPET/MRimagesA-C Coronal views of [68Ga]Pentixafor (A), T2 STIR weighted MRI (B) and CTbone window (C) of a maleuptake with histologically proven multiple myeloma. The increased [68Ga]Pentixaforbone correlates with the hyperintense T2 STIR signal; however, the myeloma manifestations are underestimated in the corresponding bone window CT.


Figure 1 ( A ) The activities ( mUnits / g of protein ) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and ∆ 4 - CYB cells and normalized by the percentage of citrate synthase ( CS ) activity . Results are expressed as mean ± SD ( n = 4 - 6 biological replicates ) . Unpaired Student ' s t - test * * p = 0 . 0100 ; * * * p = 0 . 0002 ; * * * * p < 0 . 0001 . ( B ) Complex I in gel activity assays ( IGA ) after Blue - native gel electrophoresis ( BNGE ) of WT and ∆ 4 - CYB samples solubilized either with 1 . 6 mg DDM / mg protein or 4 mg digitonin / mg protein . The gels were incubated in the reaction mixture for 1 . 5 hours ( lighter signals in DDM gels ) or were left to continue the reaction for 24 hours to obtain darker signals ( DDM and Digitonin gels ) . ( C ) BNGE , Western blot and immunodetection , with anti - NDUFS1 ( cI ) , anti - ATP5A ( cV ) and anti - SDHB ( cII ) antibodies , of samples from the WT cybrids and from ∆ 4 - CYB clones E ( # 17 . 3E ) and B ( # 17 . 3B ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) The activities (mUnits/g of protein) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and ∆4-CYB cells and normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=4-6 biological replicates). Unpaired Student's t-test **p=0.0100; ***p=0.0002; ****p<0.0001.(B) Complex I in gel activity assays (IGA) after Blue-native gel electrophoresis (BNGE) of WT and ∆4-CYB samples solubilized either with 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 hours (lighter signals in DDM gels) or were left to continue the reaction for 24 hours to obtain darker signals (DDM and Digitonin gels).(C) BNGE, Western blot and immunodetection, with anti-NDUFS1 (cI), anti-ATP5A (cV) and anti-SDHB (cII) antibodies, of samples from the WT cybrids and from ∆4-CYB clones E (#17.3E) and B (#17.3B).