Figure 1A . Schematic depiction of the Caenorhabditis elegans phasmid cilia ( left ) . Two sensory cilia are aligned into the phasmid channel ( the dashed box and enlarged in the middle ) . Each cilium contains a ciliary base , a middle segment ( m . s . ) , and a distal segment ( d . s . ) . Right shows an inverted fluorescence Hessian - SIM image of GFP - tagged dynein - 2 heavy chain CHE - 3 in cilia , in which individual puncta were resolved . Scale bar : 1 μm . B . Frame series of an anterograde moving IFT - particle from a 7xGFP : : CHE - 3 worm . Localization precisions are shown under each frame . Time in millisecond ( ms ) ; Scale bar , 100 nm . C . Localization precisions ( mean ± SD ) of tracks from multi - GFP - labeled worms . The number of GFP copies was estimated as the following : the dynein - 2 heavy chain of CHE - 3 forms a dimer , 14 = 2 x 7 ; IFT - particle subunit CHE - 2 is also a dimer , 14 = 2 x 7 ; IFT - particle subunit DYF - 11 can be a tetramer , 12 = 4 x 3 ; the genetic cross combined 7xGFP : : CHE - 3 with DYF - 11 : : 3XGFP , 26 = 2 x 7 + 4 x 3 . D . The representative diagram shows the displacement of the cargo within the middle ciliary segment . The y - axis grid lines are spaced 16 nm . Protein CHE - 3 tagged with 7x GFP was used for tracking experiments . Trace was analyzed using the SIC fitting algorithmE . Histogram of 161 displacements detected in 15 tracks extracted from 12 WT worms . The plus and minus displacements are defined as the upward " jumping " or downward " jumping " Protein markers used for tracking experiments are CHE - 3 tagged with 7x GFP , CHE - 2 tagged with 7x GFP , or CHE - 3 tagged with 7x GFP plus DYF - 11 tagged with 3x GFP .

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 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 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 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 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

Figure 1A. Schematic depiction of the Caenorhabditis elegans phasmid cilia (left). Two sensory cilia are aligned into the phasmid channel (the dashed box and enlarged in the middle). Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.). Right shows an inverted fluorescence Hessian-SIM image of GFP-tagged dynein-2 heavy chain CHE-3 in cilia, in which individual puncta were resolved. Scale bar: 1 μm.B. Frame series of an anterograde moving IFT-particle from a 7xGFP::CHE-3 worm. Localization precisions are shown under each frame. Time in millisecond (ms); Scale bar, 100 nm.C. Localization precisions (mean ± SD) of tracks from multi-GFP-labeled worms. The number of GFP copies was estimated as the following: the dynein-2 heavy chain of CHE-3 forms a dimer, 14 = 2 x 7; IFT-particle subunit CHE-2 is also a dimer, 14 = 2 x 7; IFT-particle subunit DYF-11 can be a tetramer, 12 = 4 x 3; the genetic cross combined 7xGFP::CHE-3 with DYF-11::3XGFP, 26 = 2 x 7 + 4 x 3.D. The representative diagram shows the displacement of the cargo within the middle ciliary segment. The y-axis grid lines are spaced 16 nm. Protein CHE-3 tagged with 7x GFP was used for tracking experiments. Trace was analyzed using the SIC fitting algorithmE. Histogram of 161 displacements detected in 15 tracks extracted from 12 WT worms. The plus and minus displacements are defined as the upward "jumping" or downward "jumping" Protein markers used for tracking experiments are CHE-3 tagged with 7x GFP, CHE-2 tagged with 7x GFP, or CHE-3 tagged with 7x GFP plus DYF-11 tagged with 3x GFP.


Figure 2A . Frame series shows the off - axis movement of an IFT - particle ( # 1 ) . A neighboring particle ( # 2 ) serves as a fiduciary marker . B . Representative two - dimensional IFT - tracks from WT , kinesin - II ( klp - 11 ) , and osm - 3 null animals . Red and violet circles indicate the start and end of the tracks , respectively . Proteins used for tracking experiments are CHE - 3 tagged with 7x GFP , or DYF - 11 tagged with 3x GFP . C . Time - averaged mean - square displacement ( MSD ) versus lag time ( tlag ) of 10 - 15 tracks from WT and IFT - kinesin mutant animals . Proteins used for tracking experiments are CHE - 3 tagged with 7x GFP , CHE - 3 tagged with 7x GFP plus DYF - 11 tagged with 3x GFP , CHE - 2 tagged with 7x GFP , or DYF - 11 tagged with 3x GFP . E . Average deviation angles in 10 - 15 tracks from WT and mutant animals . Comparisons were performed with the WT . F . Representative tracks of the recombinant C . elegans kinesin - II and OSM - 3 - kinesin moving along suspended MTs in the optical trap assay . G . Representative TEM images of the middle ciliary segments from WT and IFT - kinesin mutant animals . Red arrowheads indicate ectopic singlet microtubules . Blue arrowheads indicate ectopic doublet microtubules . The schematics below depict the phenotype of each image .

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 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 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 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 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 2A. Frame series shows the off-axis movement of an IFT-particle (#1). A neighboring particle (#2) serves as a fiduciary marker.B. Representative two-dimensional IFT-tracks from WT, kinesin-II (klp-11), and osm-3 null animals. Red and violet circles indicate the start and end of the tracks, respectively. Proteins used for tracking experiments are CHE-3 tagged with 7x GFP, or DYF-11 tagged with 3x GFP.C. Time-averaged mean-square displacement (MSD) versus lag time (tlag) of 10-15 tracks from WT and IFT-kinesin mutant animals. Proteins used for tracking experiments are CHE-3 tagged with 7x GFP, CHE-3 tagged with 7x GFP plus DYF-11 tagged with 3x GFP, CHE-2 tagged with 7x GFP, or DYF-11 tagged with 3x GFP.E. Average deviation angles in 10-15 tracks from WT and mutant animals. Comparisons were performed with the WT.F. Representative tracks of the recombinant C. elegans kinesin-II and OSM-3-kinesin moving along suspended MTs in the optical trap assay.G. Representative TEM images of the middle ciliary segments from WT and IFT-kinesin mutant animals. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image.


Figure 3B . Representative two - dimensional IFT tracks from WT and OSM - 3 ( 1 or 3xGS ) animals . Red and violet circles indicate the start and end of the tracks , respectively . C . Time - averaged mean - square displacement ( MSD ) versus lag time ( tlag ) of 10 - 15 tracks from WT and mutant worms .

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Figure 3B. Representative two-dimensional IFT tracks from WT and OSM-3(1 or 3xGS) animals. Red and violet circles indicate the start and end of the tracks, respectively.C. Time-averaged mean-square displacement (MSD) versus lag time (tlag) of 10-15 tracks from WT and mutant worms.


Figure 4A . Histogram of anterograde IFT speeds of 3xGFP : : CHE - 3 at the middle ( top ) and distal ( bottom ) ciliary segments ( top ) . Comparisons were performed between the WT and osm - 3 mutants . * * * B . Schematic depiction of the C . elegans amphid and phasmid cilia . Each cilium contains a ciliary base , a middle segment ( m . s . ) , and a distal segment ( d . s . ) . C . Amphid ( top ) and phasmid ( bottom ) cilia in the WT and osm - 3 GS knock - in animals . Cilia are marked with 7xGFP : : CHE - 3 . Arrowheads indicate the ciliary base . Arrows indicate the junctions between the middle and distal segments . D . Cilium length ( mean ± SD ) in WT and osm - 3 mutant animals . Comparisons were performed between the WT and osm - 3 mutants . E . Quantifications of dye - filling defects from WT and osm - 3 mutant animals . F . Representative TEM images of the middle ciliary segments from osm - 3 ( 1xGS ) and osm - 3 ( 3xGS ) worms . Red arrowheads indicate ectopic singlet microtubules . Blue arrowheads indicate ectopic doublet microtubules . The schematics below depict the phenotype of each image .

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

Figure 4A. Histogram of anterograde IFT speeds of 3xGFP::CHE-3 at the middle (top) and distal (bottom) ciliary segments (top). Comparisons were performed between the WT and osm-3 mutants. ***B. Schematic depiction of the C. elegans amphid and phasmid cilia. Each cilium contains a ciliary base, a middle segment (m.s.), and a distal segment (d.s.). C. Amphid (top) and phasmid (bottom) cilia in the WT and osm-3 GS knock-in animals. Cilia are marked with 7xGFP::CHE-3. Arrowheads indicate the ciliary base. Arrows indicate the junctions between the middle and distal segments.D. Cilium length (mean ± SD) in WT and osm-3 mutant animals. Comparisons were performed between the WT and osm-3 mutants.E. Quantifications of dye-filling defects from WT and osm-3 mutant animals.F. Representative TEM images of the middle ciliary segments from osm-3(1xGS) and osm-3(3xGS) worms. Red arrowheads indicate ectopic singlet microtubules. Blue arrowheads indicate ectopic doublet microtubules. The schematics below depict the phenotype of each image.


Figure 1 ( B ) Growth inhibition upon overexpression of HOS3 - NLS driven by the GAL1 promoter is suppressed by overexpression of the KAT Esa1 . 10 - fold serial dilutions of the indicated strains transformed with an empty vector or with the indicated plasmids , were spotted onto SC - Glu and SC - Gal medium ( to activate the GAL1 promoter ) and incubated at 25 ° C for 3 days . Note that HOS3 - NLS ( under the control of the native HOS3 promoter ) does not affect cell growth . ( C ) esa1 - ts and gcn5Δ esa1 - ts mutants have bud emergence defects . ( Top ) Cells of the indicated strains were arrested in G1 by treatment with ɑ - factor for 2 . 5 h at 25 ºC , shifted to 37 ºC for 1 h and released from the G1 arrest at 37 ºC . Cells were fixed at the indicated times and the presence of buds was assessed by microscopy . Mean and SEM are derived from n = number of independent experiments . At least 200 cells were scored for each strain and time point . ( Bottom ) Bright field images of the indicated strains 60 min after the ɑ - factor washout . Arrowheads point to cell buds . Scale bar , 4 µm . ( D ) Inactivation of ESA1 and GCN5 delays DNA replication . Cells of the indicated genotypes were synchronised as in panel C , and DNA content was evaluated by flow cytometry . Numbers indicate time in minutes after the release . This experiment was repeated three times with similar results ; one experiment is shown .

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 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

Figure 1(B) Growth inhibition upon overexpression of HOS3-NLS driven by the GAL1 promoter is suppressed by overexpression of the KAT Esa1. 10-fold serial dilutions of the indicated strains transformed with an empty vector or with the indicated plasmids, were spotted onto SC-Glu and SC-Gal medium (to activate the GAL1 promoter) and incubated at 25 °C for 3 days. Note that HOS3-NLS (under the control of the native HOS3 promoter) does not affect cell growth.(C) esa1-ts and gcn5Δ esa1-ts mutants have bud emergence defects. (Top) Cells of the indicated strains were arrested in G1 by treatment with ɑ-factor for 2.5 h at 25 ºC, shifted to 37 ºC for 1 h and released from the G1 arrest at 37 ºC. Cells were fixed at the indicated times and the presence of buds was assessed by microscopy. Mean and SEM are derived from n = number of independent experiments. At least 200 cells were scored for each strain and time point. (Bottom) Bright field images of the indicated strains 60 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.(D) Inactivation of ESA1 and GCN5 delays DNA replication. Cells of the indicated genotypes were synchronised as in panel C, and DNA content was evaluated by flow cytometry. Numbers indicate time in minutes after the release. This experiment was repeated three times with similar results; one experiment is shown.


Figure 2 ( A ) Overexpression of Esa1 and Gcn5 KATs leads to increased acetylation levels of the nuclear basket nucleoporin Nup60 . ( Top ) Nup60 - GFP was immunoprecipitated from extracts of the indicated strains , and its acetylation state probed with anti - AcLys antibodies . ( Bottom ) Total extracts probed with anti - HA antibodies to verify KAT overexpression . ( B ) nup60 - KN partially rescues the budding defect of esa1 - ts cells . ( Left ) Budding of cells of the indicated strains were determined as in Figure 1C . At least 200 cells were scored for each strain and time point . Data from 3 independent experiments is represented as mean and SEM ( esa1 - ts , esa1 - ts nup60 - KN ) . ( Right ) Bright field images of the indicated strains 40 min after the ɑ - factor washout . Arrowheads point to cell buds . Scale bar , 4 µm . ( C ) mRNA levels of CLN2 , CDC21 , SVS1 , and RNR1 were determined for cells of the indicated strains after G1 arrest and release at restrictive temperature , with samples collected at indicated times . Data from 3 independent experiments is represented as mean and SEM . ( D ) nup60 - KN mutation partially rescues the delay in synthesis of the G1 / S cyclin Cln2 in esa1 - ts cells . Cells of the indicated strains were processed as in ( B ) and the amount of Cln2 - HA protein at the indicated times was assessed by western blot . % of budded cells is indicated below for each corresponding strain and time point . Note the slow , inefficient budding in esa1 - ts mutants . In WT , the reduction of Cln2 at 60 min and its increase at 90 min reflects the start of a second cycle , which is absent in esa1 - ts cells . ( E ) nup60 - KN partially rescues the Whi5 export defect of esa1 - ts daughter cells . Composite of bright field and Whi5 - mCherry ( left ) and quantification of Whi5 nuclear export ( right ) in mother ( M ) and daughter ( D ) cells of the indicated strains . Whi5 export ( arrows ) is delayed in esa1 - ts mothers and daughters compared to WT ( p < 000 . 1 , Log - rank Mantel - Cox test ) ; esa1 - ts nup60 - KN advances Whi5 export relative to esa1 - ts in daughters ( p = 0 . 0105 ) , but not in mothers ( p > 0 . 05 ) . 3 z - confocal slices spaced 0 . 5 µm were acquired every 3 min ; maximum projections of selected timepoints are shown . Time is indicated in minutes ; t = 0 marks Whi5 nuclear import . Scale bar , 5 µm . n = number of cells , pooled from two independent experiments with similar results .

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

Figure 2(A) Overexpression of Esa1 and Gcn5 KATs leads to increased acetylation levels of the nuclear basket nucleoporin Nup60. (Top) Nup60-GFP was immunoprecipitated from extracts of the indicated strains, and its acetylation state probed with anti-AcLys antibodies. (Bottom) Total extracts probed with anti-HA antibodies to verify KAT overexpression.(B) nup60-KN partially rescues the budding defect of esa1-ts cells. (Left) Budding of cells of the indicated strains were determined as in Figure 1C. At least 200 cells were scored for each strain and time point. Data from 3 independent experiments is represented as mean and SEM (esa1-ts, esa1-ts nup60-KN). (Right) Bright field images of the indicated strains 40 min after the ɑ-factor washout. Arrowheads point to cell buds. Scale bar, 4 µm.(C) mRNA levels of CLN2, CDC21, SVS1, and RNR1 were determined for cells of the indicated strains after G1 arrest and release at restrictive temperature, with samples collected at indicated times. Data from 3 independent experiments is represented as mean and SEM.(D) nup60-KN mutation partially rescues the delay in synthesis of the G1/S cyclin Cln2 in esa1-ts cells. Cells of the indicated strains were processed as in (B) and the amount of Cln2-HA protein at the indicated times was assessed by western blot. % of budded cells is indicated below for each corresponding strain and time point. Note the slow, inefficient budding in esa1-ts mutants. In WT, the reduction of Cln2 at 60 min and its increase at 90 min reflects the start of a second cycle, which is absent in esa1-ts cells.(E) nup60-KN partially rescues the Whi5 export defect of esa1-ts daughter cells. Composite of bright field and Whi5-mCherry (left) and quantification of Whi5 nuclear export (right) in mother (M) and daughter (D) cells of the indicated strains. Whi5 export (arrows) is delayed in esa1-ts mothers and daughters compared to WT (p < 000.1, Log-rank Mantel-Cox test); esa1-ts nup60-KN advances Whi5 export relative to esa1-ts in daughters (p=0.0105), but not in mothers (p > 0.05). 3 z-confocal slices spaced 0.5 µm were acquired every 3 min; maximum projections of selected timepoints are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. n = number of cells, pooled from two independent experiments with similar results.


Figure 3 ( A ) Composite of bright field and Whi5 - tdTomato ( BF / Whi5 ) and CLN2 - PP7 mRNA labelled with PCP - NLS - GFP ( CLN2 mRNA ) in mother ( M ) and daughter ( D ) cells of the indicated strains . Arrows indicate nuclear foci . Brighter GFP nucleoplasmic areas may correspond to the nucleolus . Insets show enhanced - contrast images to visualise cytoplasmic mRNA particles ( arrowheads ) . Numbers indicate minutes relative to Whi5 nuclear import . Maximum projections of whole - cell Z - stacks are shown for Whi5 and CLN2 mRNA except in the inset and in selected esa1 - ts images , where single Z - slices are shown for clarity . Scale bar : 4 µm ( inset : 1 µm ) . ( B ) Fraction of cells of the indicated strains with nuclear CLN2 - PP7 mRNA foci , aligned relative to Whi5 import . Symbols represent individual values ; lines were generated by smoothing of the nearest 3 neighbouring values ( 0th order polynomial ) . ( C ) Fraction of cells of the indicated strains with nuclear CLN2 - PP7 mRNA foci , aligned relative to Whi5 export . ( D ) Mean fluorescence intensity of nuclear CLN2 mRNA foci at the time of Whi5 export , normalised relative to the nuclear background . Data information boxes include 50 % of data points , lines represent the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; * * , p ≤ 0 . 01 ; n . s . , p > 0 . 05 , ordinary one - way ANOVA with Tukey multiple comparisons test . Adjusted p - values in ( D ) : WT vs . esa1 - ts p < 0 . 0001 , WT vs . esa1 - ts nup60 - KN p < 0 . 0001 , esa1 - ts vs . esa1 - ts nup60 - KN p = 0 . 9845 Foci were scored in individual Z - slices spanning the entire cell volume . n = number of cells , pooled from two independent experiments with similar results . ( E ) Number of cytoplasmic CLN2 - PP7 mRNA foci in early G1 ( 5 minutes after Whi5 import ) and at the G1 / S transition ( 5 min after Whi5 export ) . Data information : boxes include 50 % of data points , lines represent the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; * * , p ≤ 0 . 01 ; n . s . , p > 0 . 05 , ordinary one - way ANOVA with Tukey multiple comparisons test . Adjusted p - values in ( E ) : WT vs . esa1 - ts p < 0 . 0001 , esa1 - ts vs . esa1 - ts nup60 - KN p = 0 . 002 . Foci were scored in individual Z - slices spanning the entire cell volume . n = number of cells , pooled from two independent experiments with similar results . ( F ) The position of nuclear CLN2 - PP7 foci ( marked with arrowheads in the examples ) was scored relatively to the nuclear periphery ( visualised with PCP - NLS - GFP ) in the indicated strains . The fraction of non - perinuclear CLN2 - PP7 foci is significantly increased in esa1 - ts cells compared to the WT and rescued by nup60 - KN mutation ( two - sided Fisher ' s exact test p = 0 . 0209 and p = 0 . 0227 ) . Scale bar , 1 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 O O O O O 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 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 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 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

Figure 3(A) Composite of bright field and Whi5-tdTomato (BF/Whi5) and CLN2-PP7 mRNA labelled with PCP-NLS-GFP (CLN2 mRNA) in mother (M) and daughter (D) cells of the indicated strains. Arrows indicate nuclear foci. Brighter GFP nucleoplasmic areas may correspond to the nucleolus. Insets show enhanced-contrast images to visualise cytoplasmic mRNA particles (arrowheads). Numbers indicate minutes relative to Whi5 nuclear import. Maximum projections of whole-cell Z-stacks are shown for Whi5 and CLN2 mRNA except in the inset and in selected esa1-ts images, where single Z-slices are shown for clarity. Scale bar: 4 µm (inset: 1 µm).(B) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 import. Symbols represent individual values; lines were generated by smoothing of the nearest 3 neighbouring values (0th order polynomial). (C) Fraction of cells of the indicated strains with nuclear CLN2-PP7 mRNA foci, aligned relative to Whi5 export.(D) Mean fluorescence intensity of nuclear CLN2 mRNA foci at the time of Whi5 export, normalised relative to the nuclear background. Data information boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (D): WT vs. esa1-ts p < 0.0001, WT vs. esa1-ts nup60-KN p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.9845 Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.(E) Number of cytoplasmic CLN2-PP7 mRNA foci in early G1 (5 minutes after Whi5 import) and at the G1/S transition (5 min after Whi5 export). Data information: boxes include 50% of data points, lines represent the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; **, p ≤ 0.01; n.s., p > 0.05, ordinary one-way ANOVA with Tukey multiple comparisons test. Adjusted p-values in (E): WT vs. esa1-ts p < 0.0001, esa1-ts vs. esa1-ts nup60-KN p = 0.002. Foci were scored in individual Z-slices spanning the entire cell volume. n = number of cells, pooled from two independent experiments with similar results.(F) The position of nuclear CLN2-PP7 foci (marked with arrowheads in the examples) was scored relatively to the nuclear periphery (visualised with PCP-NLS-GFP) in the indicated strains. The fraction of non-perinuclear CLN2-PP7 foci is significantly increased in esa1-ts cells compared to the WT and rescued by nup60-KN mutation (two-sided Fisher's exact test p = 0.0209 and p = 0.0227). Scale bar, 1 µm.


Figure 4 ( B ) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3 - NLS overexpression . 10 - fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC - Glu and SC - Gal medium and incubated at 25 ° C for 3 days . ( C ) A high - copy plasmid containing SAC3 rescues the lethality of Hos3 - NLS overexpression . Strains carrying a high copy plasmid containing the SAC3 ORF ( nucleotides 467 - 3905 ) , or the empty vector , were grown ( D ) Overexpression of Hos3 - NLS promotes nuclear accumulation of mRNA . ( Left ) Cultures of the indicated strains were treated with galactose overnight to induce HOS3 - NLS expression , cells were fixed , and FISH was performed using Cy3 - Oligo ( dT ) . ( Right ) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions . Data information : , arrows point to polyadenylated RNA in the nucleus , which was visualised by DAPI staining . Data from 3 independent biological replicates ( 7h30 ) is represented as mean and s . d . , and data from 2 independent biological replicates ( 0 and 3h30 ) as mean and range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . At least 200 cells were scored for each time - point and condition . Scale bar , 4 µm . ( E ) Inactivation of Esa1 impairs export of poly ( A ) RNA . ( Left ) Cultures of the indicated strains were incubated at 37 ºC . ( Right ) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions Data information : , arrows point to polyadenylated RNA in the nucleus , which was visualised by DAPI staining . Data from 3 independent biological replicates ( 7h30 ) is represented as mean and s . d . , and data from 2 independent biological replicates ( 0 and 3h30 ) as mean and range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . At least 200 cells were scored for each time - point and condition . Scale bar , 4 µm . ( F ) nup60 - KN mutation partially rescues the mRNA export defects of esa1 - ts . Cells of the indicated strains were processed Data information : arrows point to polyadenylated RNA in the nucleus , which was visualised by DAPI staining . Data from 3 independent biological replicates ( 7h30 ) is represented as mean and s . d . , and data from 2 independent biological replicates ( 0 and 3h30 ) as mean and range . * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . At least 200 cells were scored for each time - point and condition . Scale bar , 4 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 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 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 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 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

Figure 4(B) Overexpression of MEX67 and MTR2 rescues the lethality of Hos3-NLS overexpression. 10-fold serial dilutions of the indicated strains transformed with the indicated plasmids spotted onto SC-Glu and SC-Gal medium and incubated at 25 °C for 3 days.(C) A high-copy plasmid containing SAC3 rescues the lethality of Hos3-NLS overexpression. Strains carrying a high copy plasmid containing the SAC3 ORF (nucleotides 467-3905), or the empty vector, were grown(D) Overexpression of Hos3-NLS promotes nuclear accumulation of mRNA. (Left) Cultures of the indicated strains were treated with galactose overnight to induce HOS3-NLS expression, cells were fixed, and FISH was performed using Cy3-Oligo(dT). (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions. Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.(E) Inactivation of Esa1 impairs export of poly(A) RNA. (Left) Cultures of the indicated strains were incubated at 37 ºC. (Right) The fraction of cells with nuclear mRNA accumulation was determined for the indicated strains and conditions Data information: , arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.(F) nup60-KN mutation partially rescues the mRNA export defects of esa1-ts. Cells of the indicated strains were processed Data information: arrows point to polyadenylated RNA in the nucleus, which was visualised by DAPI staining. Data from 3 independent biological replicates (7h30) is represented as mean and s.d., and data from 2 independent biological replicates (0 and 3h30) as mean and range. *, p ≤ 0.05; ***, p ≤ 0.001; ns, p > 0.05, two-tailed unpaired t-test. At least 200 cells were scored for each time-point and condition. Scale bar, 4 µm.


Figure 5 ( A ) Depletion of Hos3 or expression of acetyl - mimic Nup60 ( nup60 - KN ) increases the nuclear localisation of Sac3 in G1 . Cells of the indicated strains were imaged by time - lapse microscopy and the fluorescence levels of the indicated proteins were determined in G1 ( after cytokinesis ) . The NPC component Nup49 was used as a control for Nuclear Pore Complex protein levels . Fluorescence intensity was measured in sum projections of whole - cell Z - stacks , by segmentation of either the nuclear area in the mCherry channel , or of the whole cell in the brightfield channel . The ratio of Sac3 / Nup49 intensities was then normalised relative to the mean intensity of wild - type mothers . Data information arrowheads point to daughter cells boxes include 50 % of data points , the line represents the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . Scale bar , 2 µm . n = number of cells , pooled from three independent experiments with similar results . ( B ) Inactivation of Esa1 decreases Sac3 nuclear levels . Wild - type ( WT ) and esa1 - ts cells were arrested in mitosis by treatment with nocodazole at 25 ºC , shifted to 37 ºC , released from the mitosis block in fresh medium at 37 ºC , and imaged by time - lapse microscopy . Fluorescence levels were quantified in G1 Data information : arrowheads point to daughter cells boxes include 50 % of data points , the line represents the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . Scale bar , 2 µm . n = number of cells , pooled from three independent experiments with similar results . ( C ) Rapamycin - dependent dimerisation abolishes Sac3 mother / daughter asymmetries . NUP60 - mCherry - FKBP SAC3 - GFP - FRB cells were incubated with rapamycin ( RAPA ) to trigger FRB - FKBP heterodimerization , or with DMSO as control . Fluorescence levels were quantified in G1 cells 15 to 30 minutes after addition of the drug . Data information arrowheads point to daughter cells , boxes include 50 % of data points , the line represents the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 0001 ; ns , p > 0 . 05 , two - tailed unpaired t - test . Scale bar , 2 µm . n = number of cells , pooled from three independent experiments with similar results . ( D ) Sac3 anchoring to the nuclear basket advances Start in esa1 - ts daughter cells . Composite of bright field and Whi5 - mGFP ( top ) and quantification of Whi5 nuclear export timing ( bottom ) in wild - type ( WT ) and esa1 - ts mother ( M ) and daughter ( D ) cells treated with either rapamycin ( RAPA ) or DMSO and expressing Nup60 - FRB and Sac3 - mCherry - FKBP . Sac3 anchoring to Nup60 does not alter Whi5 export timing in WT mother or daughter cells ( DMSO vs RAPA , p > 0 . 05 , Log - rank Mantel - Cox test ) , but it advances Whi5 export in esa1 - ts daughters ( p = 0 . 0001 ) . Whi5 export efficiency was slightly improved also in mother cells ( p = 0 . 0374 ) . 8 z - confocal slices spaced 0 . 4 µm were acquired every 3 min ; maximum projections are shown . Time is indicated in minutes ; t = 0 marks Whi5 nuclear import . Scale bar , 5 µm . Data information : arrowheads point to Whi5 export .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Depletion of Hos3 or expression of acetyl-mimic Nup60 (nup60-KN) increases the nuclear localisation of Sac3 in G1. Cells of the indicated strains were imaged by time-lapse microscopy and the fluorescence levels of the indicated proteins were determined in G1 (after cytokinesis). The NPC component Nup49 was used as a control for Nuclear Pore Complex protein levels. Fluorescence intensity was measured in sum projections of whole-cell Z-stacks, by segmentation of either the nuclear area in the mCherry channel, or of the whole cell in the brightfield channel. The ratio of Sac3/Nup49 intensities was then normalised relative to the mean intensity of wild-type mothers. Data information arrowheads point to daughter cells boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.(B) Inactivation of Esa1 decreases Sac3 nuclear levels. Wild-type (WT) and esa1-ts cells were arrested in mitosis by treatment with nocodazole at 25 ºC, shifted to 37 ºC, released from the mitosis block in fresh medium at 37 ºC, and imaged by time-lapse microscopy. Fluorescence levels were quantified in G1 Data information: arrowheads point to daughter cells boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.(C) Rapamycin-dependent dimerisation abolishes Sac3 mother/daughter asymmetries. NUP60-mCherry-FKBP SAC3-GFP-FRB cells were incubated with rapamycin (RAPA) to trigger FRB-FKBP heterodimerization, or with DMSO as control. Fluorescence levels were quantified in G1 cells 15 to 30 minutes after addition of the drug. Data information arrowheads point to daughter cells, boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.0001; ns, p > 0.05, two-tailed unpaired t-test. Scale bar, 2 µm. n = number of cells, pooled from three independent experiments with similar results.(D) Sac3 anchoring to the nuclear basket advances Start in esa1-ts daughter cells. Composite of bright field and Whi5-mGFP (top) and quantification of Whi5 nuclear export timing (bottom) in wild-type (WT) and esa1-ts mother (M) and daughter (D) cells treated with either rapamycin (RAPA) or DMSO and expressing Nup60-FRB and Sac3-mCherry-FKBP. Sac3 anchoring to Nup60 does not alter Whi5 export timing in WT mother or daughter cells (DMSO vs RAPA, p > 0.05, Log-rank Mantel-Cox test), but it advances Whi5 export in esa1-ts daughters (p = 0.0001). Whi5 export efficiency was slightly improved also in mother cells (p = 0.0374). 8 z-confocal slices spaced 0.4 µm were acquired every 3 min; maximum projections are shown. Time is indicated in minutes; t=0 marks Whi5 nuclear import. Scale bar, 5 µm. Data information: arrowheads point to Whi5 export.


Figure 6 ( A ) Time Lapse microscopy of WT and nup60 - KN cells expressing GAL1pr : sfGFP and Nup60 - mCherry at the indicated times of galactose induction . Scale bar , 4 µm . ( B ) Depletion of Hos3 , and expression of acetyl - mimic Nup60 ( nup60 - KN ) enhances GAL1 expression . WT , hos3Δ , nup60 - KN and nup60 - KN hos3Δ cells were shifted to galactose and imaged by time - lapse microscopy to monitor GAL1pr : sfGFP expression during 7 hours . Nuclear fluorescence was scored by segmentation of the nuclear area in the mCherry channel and mean fluorescence of nuclear GFP and Nup60 - mCherry was quantified from sum projections of whole - cell Z - stacks at the indicated times . At least 200 cells were scored for each strain and time point . Shaded areas indicate the SEM . ( C ) mRNA levels of GAL1 were determined for wild type ( WT ) and nup60 - KN cells at the indicated times after galactose addition . One of two independent experiments with similar results is shown ( mean and SEM from 3 technical replicates ) . ( D ) The GFP intensity of mother / daughter pairs for cells in B , at 5 - minute intervals after galactose addition ( left ) and 425 min after galactose addition ( right ) . Boxes include 50 % of data points , the line represents the median and whiskers extend to maximum and minimum values . * * * * , p ≤ 0 . 001 ; ns , p > 0 . 05 , two - tailed paired t - test . n = number of cells . One of two independent experiments with similar results is shown . ( E ) Expression of GAL1pr : sfGFP induced with galactose ( left ) or ß - estradiol ( right ) , in the presence of the ß - estradiol - dependent transactivator Gal4 - ER - VP16 . Smooth lines show GFP fluorescence intensity ( left x axis ) ; lines with circles show the difference in GFP intensity between the indicated strains ( right x axis ) . The difference between wild type and hos3 ∆ , and between wild type and nup60 - KN , increases continuously over time in response to galactose , but not to ß - estradiol .

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Figure 6(A) Time Lapse microscopy of WT and nup60-KN cells expressing GAL1pr:sfGFP and Nup60-mCherry at the indicated times of galactose induction. Scale bar, 4 µm.(B) Depletion of Hos3, and expression of acetyl-mimic Nup60 (nup60-KN) enhances GAL1 expression. WT, hos3Δ, nup60-KN and nup60-KN hos3Δ cells were shifted to galactose and imaged by time-lapse microscopy to monitor GAL1pr:sfGFP expression during 7 hours. Nuclear fluorescence was scored by segmentation of the nuclear area in the mCherry channel and mean fluorescence of nuclear GFP and Nup60-mCherry was quantified from sum projections of whole-cell Z-stacks at the indicated times. At least 200 cells were scored for each strain and time point. Shaded areas indicate the SEM.(C) mRNA levels of GAL1 were determined for wild type (WT) and nup60-KN cells at the indicated times after galactose addition. One of two independent experiments with similar results is shown (mean and SEM from 3 technical replicates).(D) The GFP intensity of mother/daughter pairs for cells in B, at 5-minute intervals after galactose addition (left) and 425 min after galactose addition (right). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. ****, p ≤ 0.001; ns, p > 0.05, two-tailed paired t-test. n = number of cells. One of two independent experiments with similar results is shown.(E) Expression of GAL1pr:sfGFP induced with galactose (left) or ß-estradiol (right), in the presence of the ß-estradiol-dependent transactivator Gal4-ER-VP16. Smooth lines show GFP fluorescence intensity (left x axis); lines with circles show the difference in GFP intensity between the indicated strains (right x axis). The difference between wild type and hos3∆, and between wild type and nup60-KN, increases continuously over time in response to galactose, but not to ß-estradiol.


Figure 7 ( A ) Cells of the indicated strains were incubated with galactose in the presence of either rapamycin ( RAPA ) to induce FRB - FKBP heterodimerization , or DMSO as control . GAL1pr : sfGFP expression was monitored over time ( B ) ( Top ) Representative images of the indicated cells in rapamycin or DMSO , 250 min after galactose addition . Scale bar 4 µm . ( Bottom ) Mother / daughter pairs were quantified as in Figure 6D at 250 min after galactose addition ( pink shaded area in A ) . Boxes include 50 % of data points , the line represents the median and whiskers extend to maximum and minimum values . * * , p ≤ 0 . 01 ; * , p ≤ 0 . 05 ; ns , p > 0 . 05 , two - tailed paired t - test for M - D comparisons , unpaired for comparisons between strains . n = number of cells . One of two independent experiments with similar results is shown .

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Figure 7(A) Cells of the indicated strains were incubated with galactose in the presence of either rapamycin (RAPA) to induce FRB-FKBP heterodimerization, or DMSO as control. GAL1pr:sfGFP expression was monitored over time(B) (Top) Representative images of the indicated cells in rapamycin or DMSO, 250 min after galactose addition. Scale bar 4 µm. (Bottom) Mother/daughter pairs were quantified as in Figure 6D at 250 min after galactose addition (pink shaded area in A). Boxes include 50% of data points, the line represents the median and whiskers extend to maximum and minimum values. **, p ≤ 0.01; *, p ≤ 0.05; ns, p > 0.05, two-tailed paired t-test for M-D comparisons, unpaired for comparisons between strains. n = number of cells. One of two independent experiments with similar results is shown.


Figure 1 ( C ) Expression of GALNT isoforms in primary keratinocytes and HaCaT cell line . The scatter plot depicts individual RPKM values of 2 biological replicates . ( D ) Expression of GalNAc - T1 , - T2 , and - T3 in human skin ( upper panel ) and HaCaT keratinocyte organoids ( lower panel ) . Frozen human skin or HaCaT keratinocyte organotypic skin models were stained using antibodies for the GalNAc - T isoforms . Scale bar - 25 μm . ( E ) Phenotypic characterization of organotypic models made with HaCaT WT or GALNT1 / 2 / 3 KO keratinocytes . IHC of tissue sections stained for differentiation marker keratin 10 ( upper panel ) or proliferation marker Ki67 ( lower panel ) . Scale bar - 50 μm . Red arrows - flattened cells ; red asterisks - K10 - negative region in suprabasal / granular layers ; purple asterisks - pyknotic nuclei ; green asterisks - increase in Ki67 positive cells . ( F ) Quantification of epidermal thickness of skin organotypic models . Epidermal thickness was measured in 5 distinct images ( 4 positions / image ) of 4 clones of GALNT isoform KO or WT ( 4 different tissues ) and is presented as averages + SD . Due to high ZFN KO phenotypic inter - clonal variation and to exclude off target effects , 5 clones of ZFN and 3 clones of CRISPR KO ( each targeted by a different gRNA ) were used for GALNT3 KO . ANOVA followed by Dunnet ' s multiple comparison test was used to compare mean epidermal thicknesses of different KOs to WT . * p ≤ 0 . 05 ; * * * * p ≤ 0 . 0001 . ( G ) Quantification of keratin 10 - negative area in skin organotypic tissue sections . Keratin 10 - negative area was measured in 5 distinct images of 4 clones of GALNT isoform KO or WT ( 4 different tissues ) and is presented as averages + SD . Due to high ZFN KO phenotypic inter - clonal variation and to exclude off target effects , 5 clones of ZFN and 3 clones of CRISPR KO ( each targeted by a different gRNA ) were used for GALNT3 KO . ANOVA followed by Dunnet ' s multiple comparison test was used to compare mean areas of different KOs to WT . * * * * p ≤ 0 . 0001 . ( H ) RNA Seq expression data of select differentiation markers , expressed in spinous and granular layers of the epidermis ( cartoon ) . Expression data is presented as log2 ( average KO / average WT ) . Significantly changed transcripts , identified in R analysis , are marked with asterisks .

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 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 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 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 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 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

Figure 1(C) Expression of GALNT isoforms in primary keratinocytes and HaCaT cell line. The scatter plot depicts individual RPKM values of 2 biological replicates.(D) Expression of GalNAc-T1, -T2, and -T3 in human skin (upper panel) and HaCaT keratinocyte organoids (lower panel). Frozen human skin or HaCaT keratinocyte organotypic skin models were stained using antibodies for the GalNAc-T isoforms. Scale bar - 25 μm.(E) Phenotypic characterization of organotypic models made with HaCaT WT or GALNT1/2/3 KO keratinocytes. IHC of tissue sections stained for differentiation marker keratin 10 (upper panel) or proliferation marker Ki67 (lower panel). Scale bar - 50 μm. Red arrows - flattened cells; red asterisks - K10-negative region in suprabasal/granular layers; purple asterisks - pyknotic nuclei; green asterisks - increase in Ki67 positive cells.(F) Quantification of epidermal thickness of skin organotypic models. Epidermal thickness was measured in 5 distinct images (4 positions/image) of 4 clones of GALNT isoform KO or WT (4 different tissues) and is presented as averages +SD. Due to high ZFN KO phenotypic inter-clonal variation and to exclude off target effects, 5 clones of ZFN and 3 clones of CRISPR KO (each targeted by a different gRNA) were used for GALNT3 KO. ANOVA followed by Dunnet's multiple comparison test was used to compare mean epidermal thicknesses of different KOs to WT. * p ≤ 0.05; **** p ≤ 0.0001.(G) Quantification of keratin 10-negative area in skin organotypic tissue sections. Keratin 10-negative area was measured in 5 distinct images of 4 clones of GALNT isoform KO or WT (4 different tissues) and is presented as averages +SD. Due to high ZFN KO phenotypic inter-clonal variation and to exclude off target effects, 5 clones of ZFN and 3 clones of CRISPR KO (each targeted by a different gRNA) were used for GALNT3 KO. ANOVA followed by Dunnet's multiple comparison test was used to compare mean areas of different KOs to WT. **** p ≤ 0.0001.(H) RNA Seq expression data of select differentiation markers, expressed in spinous and granular layers of the epidermis (cartoon). Expression data is presented as log2(average KO/average WT). Significantly changed transcripts, identified in R analysis, are marked with asterisks.


Figure 3 ( A ) Numbers of quantified O - glycopeptides identified with ETD fragmentation with either dimethyl labeling or TMT labeling approach in COSMC KO or WT background , respectively . ( B ) Numbers of differentially glycosylated peptides in the two approaches , using select filters on quantification values . ( C ) Numbers of unambiguous unique differential O - glycosites identified with the two different approaches . The stacked bar graph differentiates between glycosites identified on peptides with single and multiple HexNAc / HexHexNAc modifications . ( E ) Analysis of differential glycosites identified with the TMT labeling approach using the Qluqore Omics Explorer software . The heatmap represents O - glycosites most significantly contributing to sample variation , using ANOVA with a q value cutoff of 0 . 1 , where each compared sample consists of three individual KO clones . Each horizontal line depicts the median quantification of glycopeptides contributing to the same glycosite . Alternative differentially glycosylated site positions are separated by semicolons on sites derived from glycopeptides with multiple HexNAc / HexHexNAc modifications .

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 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 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

Figure 3(A) Numbers of quantified O-glycopeptides identified with ETD fragmentation with either dimethyl labeling or TMT labeling approach in COSMC KO or WT background, respectively. (B) Numbers of differentially glycosylated peptides in the two approaches, using select filters on quantification values. (C) Numbers of unambiguous unique differential O-glycosites identified with the two different approaches. The stacked bar graph differentiates between glycosites identified on peptides with single and multiple HexNAc/HexHexNAc modifications.(E) Analysis of differential glycosites identified with the TMT labeling approach using the Qluqore Omics Explorer software. The heatmap represents O-glycosites most significantly contributing to sample variation, using ANOVA with a q value cutoff of 0.1, where each compared sample consists of three individual KO clones. Each horizontal line depicts the median quantification of glycopeptides contributing to the same glycosite. Alternative differentially glycosylated site positions are separated by semicolons on sites derived from glycopeptides with multiple HexNAc/HexHexNAc modifications.


Figure 4GalNAc - T1 affects the organization and function of the endomembrane system . HaCaT WT and GALNT1 KO cells were stained for intracellular trafficking markers ( scale bar - 10 μm ) . ( A ) Clathrin ; ( B ) Rab5 ; ( C ) Rab11 . GalNAc - T1 affects the organization and function of the endomembrane system . ( D ) Steady state intracellular pH measurement in in HaCaT WT and GALNT1 KO cells . Data is presented as average ± SD of 2 independent experiments . ANOVA followed by Dunnet ' s multiple comparison test ( E ) GalNAc - T2 is important for cell to matrix adhesion . HaCaT WT and GALNT2 KO cells were used in a cell to matrix adhesion assay using plates coated with different ECM components . Data is presented as average + SD of 3 independent experiments and is normalized to WT . ANOVA followed by Dunnet ' s multiple comparison test was used to compare mean relative adhesion of 2 different clones of GALNT2 KOs to WT . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 . ( F ) Western blot for GalNAc - T2 specific target and hemidesmosome component COL17A1 . ( G ) Quantification of Western blot bands for COL17A1 ( full length ) normalized to β actin . ( I ) GalNAc - T2 may affect tight junction formation and integrity . Upper panel , TEM of WT and GALNT2 KO organotypic tissue sections , where tight junctions are marked with asterisks . Lower panel , IF staining of WT and GALNT2 KO for tight junction marker occludin ( scale bar - 50 μm ) . Inserts depict 3x zoomed in partial images ( scale bar - 50 μm ) . ( J ) Expression of cyclin A ( green ) in HaCaT WT and GALNT3 KO organotypic tissue , visualized by IF ( scale bar - 20 μm ) . ( L - N ) Co - culture of HaCaT WT or GALNT3 KO keratinocytes with N / TERT1 keratinocytes induces cellular differentiation . Cells co - cultured on coverslips were stained for cleaved Notch 1 intracellular domain ( L ) , cytokeratin 10 ( M ) , or GalNAc - T3 ( N ; inserts depict 2x zoomed in partial images ( scale bar - 20 μm ) ) . Green - relevant staining , red - N / TERT1 cells ( labeled with CMTMTR ) , blue - DAPI , scale bar - 20 μm . Boundaries between HaCaT and N / TERT - 1 cells in the green panel of ( L ) are marked with a dashed line .

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 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 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 B-PANEL_START 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 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 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 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

Figure 4GalNAc-T1 affects the organization and function of the endomembrane system. HaCaT WT and GALNT1 KO cells were stained for intracellular trafficking markers (scale bar - 10 μm). (A) Clathrin; (B) Rab5; (C) Rab11.GalNAc-T1 affects the organization and function of the endomembrane system. (D) Steady state intracellular pH measurement in in HaCaT WT and GALNT1 KO cells. Data is presented as average ± SD of 2 independent experiments. ANOVA followed by Dunnet's multiple comparison test(E) GalNAc-T2 is important for cell to matrix adhesion. HaCaT WT and GALNT2 KO cells were used in a cell to matrix adhesion assay using plates coated with different ECM components. Data is presented as average + SD of 3 independent experiments and is normalized to WT. ANOVA followed by Dunnet's multiple comparison test was used to compare mean relative adhesion of 2 different clones of GALNT2 KOs to WT. * p ≤ 0.05; ** p ≤ 0.01.(F) Western blot for GalNAc-T2 specific target and hemidesmosome component COL17A1.(G) Quantification of Western blot bands for COL17A1 (full length) normalized to β actin.(I) GalNAc-T2 may affect tight junction formation and integrity. Upper panel, TEM of WT and GALNT2 KO organotypic tissue sections, where tight junctions are marked with asterisks. Lower panel, IF staining of WT and GALNT2 KO for tight junction marker occludin (scale bar - 50 μm). Inserts depict 3x zoomed in partial images (scale bar - 50 μm).(J) Expression of cyclin A (green) in HaCaT WT and GALNT3 KO organotypic tissue, visualized by IF (scale bar - 20 μm).(L-N) Co-culture of HaCaT WT or GALNT3 KO keratinocytes with N/TERT1 keratinocytes induces cellular differentiation. Cells co-cultured on coverslips were stained for cleaved Notch 1 intracellular domain (L), cytokeratin 10 (M), or GalNAc-T3 (N; inserts depict 2x zoomed in partial images (scale bar - 20 μm)). Green - relevant staining, red - N/TERT1 cells (labeled with CMTMTR), blue - DAPI, scale bar - 20 μm. Boundaries between HaCaT and N/TERT-1 cells in the green panel of (L) are marked with a dashed line.


figf1 ( a ) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine , and then analyzed by western blot using antibodies to LC3 ( top ) , p62 ( middle ) or vinculin ( bottom ) . ( b ) Immunofluorescence for p62 and DAPI in the SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28 ( n = 3 p62 for each ) . Dotted lines indicate boundaries of the SVZ and granular zone ( GZ ) . The arrows and arrowheads mark larger and smaller p62 + aggregates , respectively , in the striatum ( ST ) and SVZ . ( c ) TEM of mitochondria in SVZ tissue from Ctrl and FIP200GFAP cKO mice at P28 and P56 ( n = 2 mice for each , > 30 cells for each mouse ) . Arrows mark mitochondria . ( d ) TEM of mitochondria in cultured neurospheres from Ctrl and FIP200GFAP cKO mice ( n = 2 mice for each , > 30 cells for each mouse ) . Arrows mark mitochondria . ( e , f ) DHE and DAPI fluorescence in the dentate gyrus ( e ) and SVZ ( f ) of Ctrl and FIP200GFAP cKO mice at P28 ( n = 5 mice for each ) . Dotted lines indicate the boundaries of the GZ ( e ) and SVZ ( f ) . Arrows mark cells in the SGZ ( e ) and SVZ ( f ) , and arrowheads mark cells in surround regions ( the GZ in e and the ST in f ) . LV , lateral ventricle ; ML , molecular layer . Full - length blots are presented in Supplementary Figure 11 . ( a ) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine , and then analyzed by western blot using antibodies to LC3 ( top ) , p62 ( middle ) or vinculin ( bottom ) .

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

figf1(a) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine, and then analyzed by western blot using antibodies to LC3 (top), p62 (middle) or vinculin (bottom).(b) Immunofluorescence for p62 and DAPI in the SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28 (n = 3 p62 for each). Dotted lines indicate boundaries of the SVZ and granular zone (GZ). The arrows and arrowheads mark larger and smaller p62+ aggregates, respectively, in the striatum (ST) and SVZ.(c) TEM of mitochondria in SVZ tissue from Ctrl and FIP200GFAP cKO mice at P28 and P56 (n = 2 mice for each, >30 cells for each mouse). Arrows mark mitochondria.(d) TEM of mitochondria in cultured neurospheres from Ctrl and FIP200GFAP cKO mice (n = 2 mice for each, >30 cells for each mouse). Arrows mark mitochondria.(e,f) DHE and DAPI fluorescence in the dentate gyrus (e) and SVZ (f) of Ctrl and FIP200GFAP cKO mice at P28 (n = 5 mice for each). Dotted lines indicate the boundaries of the GZ (e) and SVZ (f). Arrows mark cells in the SGZ (e) and SVZ (f), and arrowheads mark cells in surround regions (the GZ in e and the ST in f). LV, lateral ventricle; ML, molecular layer. Full-length blots are presented in Supplementary Figure 11.(a) Lysates extracted from the SVZ of three different Ctrl and FIP200GFAP cKO mice treated with chloroquine, and then analyzed by western blot using antibodies to LC3 (top), p62 (middle) or vinculin (bottom).


figf2 ( a ) Hematoxylin and eosin staining of the dentate gyrus ( DG ) and SVZ from Ctrl and FIP200GFAP cKO mice at P28 . Lines indicate the boundaries of the DG and SVZ . Arrows mark cells in the SVZ . ( b - e ) Immunofluorescence for GFAP , nestin or SOX2 and DAPI in the DG ( b , c ) from Ctrl and FIP200GFAP cKO mice at P28 . The boxed areas in c - e are shown in more detail in insets ( c ) . Dotted lines indicate boundaries of the granular zone ( GZ ) and SGZ ( b ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( b , c ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes . ( b - e ) Immunofluorescence for GFAP , nestin and DAPI in the SVZ ( d , e ) from Ctrl and FIP200GFAP cKO mice at P28 . The boxed areas in c - e are shown in more detail in panels below ( d , e ) . Dotted lines indicate boundaries of the SVZ ( d , e ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( b , c ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes . ( b - e ) Immunofluorescence for GFAP , nestin or SOX2 and DAPI in the SVZ ( d , e ) from Ctrl and FIP200GFAP cKO mice at P28 . The boxed areas in c - e are shown in more detail in panels below ( d , e ) . Dotted lines indicate boundaries of the SVZ ( d , e ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( b , c ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes . ( f ) Immunofluorescence for PSA - NCAM in the RMS ( boxed areas ) , SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28 . Lines indicate the boundaries of the DG and SVZ . Arrows mark PSA - NCAM + cells in the RMS , SVZ and DG . ( g ) Immunofluorescence for NeuN in the olfactory bulb ( OB ) and DG of Ctrl and FIP200GFAP cKO mice at P31 . Lines indicate the layers of the OB and boundaries of the DG . Boxed areas in top panels are shown in more detail for staining of NeuN in insets . CC , corpus callosum ; E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum ; IGL , internal granular layer ; MCL , mitral cell layer ; EPL , external plexiform layer ; GL , glomerular layer .

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 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 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 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 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

figf2(a) Hematoxylin and eosin staining of the dentate gyrus (DG) and SVZ from Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the DG and SVZ. Arrows mark cells in the SVZ.(b-e) Immunofluorescence for GFAP, nestin or SOX2 and DAPI in the DG (b,c) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in insets (c). Dotted lines indicate boundaries of the granular zone (GZ) and SGZ (b). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes.(b-e) Immunofluorescence for GFAP, nestin and DAPI in the SVZ (d,e) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in panels below (d,e). Dotted lines indicate boundaries of the SVZ (d,e). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes.(b-e) Immunofluorescence for GFAP, nestin or SOX2 and DAPI in the SVZ (d,e) from Ctrl and FIP200GFAP cKO mice at P28. The boxed areas in c-e are shown in more detail in panels below (d,e). Dotted lines indicate boundaries of the SVZ (d,e). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (b,c), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes.(f) Immunofluorescence for PSA-NCAM in the RMS (boxed areas), SVZ and DG of Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the DG and SVZ. Arrows mark PSA-NCAM+ cells in the RMS, SVZ and DG.(g) Immunofluorescence for NeuN in the olfactory bulb (OB) and DG of Ctrl and FIP200GFAP cKO mice at P31. Lines indicate the layers of the OB and boundaries of the DG. Boxed areas in top panels are shown in more detail for staining of NeuN in insets. CC, corpus callosum; E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer.


figf3 ( a - e ) Primary ( 1st ) and secondary ( 2nd ) neurospheres from Ctrl and FIP200GFAP cKO mice at P0 and P28 . Representative P0 phase contrast images are shown in a . Mean ± s . e . m . of number ( b , d ) and size ( c , e ) of primary and secondary neurospheres from three independent experiments are shown ( n = 3 mice for each ) . ( f ) Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) and DAPI staining of the SVZ and dentate gyrus ( DG ) from Ctrl and FIP200GFAP cKO mice at P28 . Lines indicate the boundaries of the SVZ . Numbered boxed areas are shown in more detail as insets for staining of TUNEL and DAPI in the SVZ and DG . ( g ) Short - term BrdU incorporation in the SVZ and DG of Ctrl and FIP200GFAPcKO mice at P28 . Dotted lines indicate the boundaries of the SVZ and granular zone ( GZ ) . Boxed areas are shown in more detail in the insets . Lines indicate the boundaries of the SVZ and GZ . Arrows mark BrdU + cells . CC , corpus callosum ; LV , lateral ventricle ; ST , striatum . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 . ( h ) Long - term retention of BrdU in the SVZ and dentate gyrus of Ctrl and FIP200GFAP cKO mice at P31 . Lines indicate the boundaries of the SVZ and GZ . Arrows mark BrdU + cells . CC , corpus callosum ; LV , lateral ventricle ; ST , striatum . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 .

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 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 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

figf3(a-e) Primary (1st) and secondary (2nd) neurospheres from Ctrl and FIP200GFAP cKO mice at P0 and P28. Representative P0 phase contrast images are shown in a. Mean ± s.e.m. of number (b,d) and size (c,e) of primary and secondary neurospheres from three independent experiments are shown (n = 3 mice for each).(f) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and DAPI staining of the SVZ and dentate gyrus (DG) from Ctrl and FIP200GFAP cKO mice at P28. Lines indicate the boundaries of the SVZ. Numbered boxed areas are shown in more detail as insets for staining of TUNEL and DAPI in the SVZ and DG.(g) Short-term BrdU incorporation in the SVZ and DG of Ctrl and FIP200GFAPcKO mice at P28. Dotted lines indicate the boundaries of the SVZ and granular zone (GZ). Boxed areas are shown in more detail in the insets. Lines indicate the boundaries of the SVZ and GZ. Arrows mark BrdU+ cells. CC, corpus callosum; LV, lateral ventricle; ST, striatum. *P 0.05; **P 0.01; ***P 0.001.(h) Long-term retention of BrdU in the SVZ and dentate gyrus of Ctrl and FIP200GFAP cKO mice at P31. Lines indicate the boundaries of the SVZ and GZ. Arrows mark BrdU+ cells. CC, corpus callosum; LV, lateral ventricle; ST, striatum. *P 0.05; **P 0.01; ***P 0.001.


figf4 ( a ) Lysates extracted from neurospheres of Ctrl , FIP200GFAP cKO , Trp53GFAP cKO and 2cKO mice and analyzed by western blot using antibodies to p53 and actin . ( b ) Mean ± s . e . m . of relative mRNA level ( normalized to Ctrl mice as 1 ) of Cdkn1a , Bbc3 , Fas , Pax6 and Stat3 in neurospheres of Ctrl , FIP200GFAP cKO , 2cKO and Trp53GFAP cKO mice are shown ( n = 3 mice for each ) . ( c , d ) Mean ± s . e . m . of the number ( c ) and size ( d ) of primary and secondary neurospheres from Ctrl , FIP200GFAP cKO , 2cKO and Trp53GFAP cKO mice at P28 ( n = 3 mice for each ) . ( e - h ) Analysis of immunofluorescence or staining on frozen - sectioned neurospheres for nestin , Ki67 , TUNEL , PAX6 , phosphorylated STAT3 ( pSTAT3 ) and DAPI , as indicated . Mean ± s . e . m . of the percentage of Ki67 + ( e ) , BrdU + ( f ) and TUNEL + ( g ) cells in neurospheres from Ctrl , FIP200GFAP cKO , 2cKO and Trp53GFAP cKO mice are shown ( n = 3 mice for each , counting of > 500 cells for each mouse ) . Representative images are shown in h . * P 0 . 01 . Full - length blots are presented in Supplementary Figure 11 .

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 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 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 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

figf4(a) Lysates extracted from neurospheres of Ctrl, FIP200GFAP cKO, Trp53GFAP cKO and 2cKO mice and analyzed by western blot using antibodies to p53 and actin.(b) Mean ± s.e.m. of relative mRNA level (normalized to Ctrl mice as 1) of Cdkn1a, Bbc3, Fas, Pax6 and Stat3 in neurospheres of Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice are shown (n = 3 mice for each).(c,d) Mean ± s.e.m. of the number (c) and size (d) of primary and secondary neurospheres from Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice at P28 (n = 3 mice for each).(e-h) Analysis of immunofluorescence or staining on frozen-sectioned neurospheres for nestin, Ki67, TUNEL, PAX6, phosphorylated STAT3 (pSTAT3) and DAPI, as indicated. Mean ± s.e.m. of the percentage of Ki67+ (e), BrdU+ (f) and TUNEL+ (g) cells in neurospheres from Ctrl, FIP200GFAP cKO, 2cKO and Trp53GFAP cKO mice are shown (n = 3 mice for each, counting of >500 cells for each mouse). Representative images are shown in h. *P 0.01. Full-length blots are presented in Supplementary Figure 11.


figf5 ( a - c ) Hematoxylin and eosin staining of the dentate gyrus and dentate gyrus ( DG ) of P28mice ( a ) . Arrows mark cells in the SVZ . Mean ± s . e . m . of the SVZ cellularity ( b ) and DG area ( c ) per section are shown . Dotted lines indicate the boundaries of the SVZ and DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ( d - m ) Immunofluorescence of the DG ( d - h ) of P28mice ( d , e , i , j ) . Boxed areas are shown in more detail in insets ( e , j ) and / or panels below ( i , j ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( d , e ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes . Mean ± s . e . m . of the number of GFAP + nestin + and GFAP + SOX2 + radial glia ( f , h ) , and NSCs ( k , m ) , and GFAP + nestin − astrocytes ( g , l ) per section are shown . Dotted lines indicate the boundaries of the SVZ and DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ( d - m ) Immunofluorescence of the SVZ ( i - m ) of P28mice ( d , e , i , j ) . Boxed areas are shown in more detail in insets ( e , j ) and / or panels below ( i , j ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( d , e ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes . Mean ± s . e . m . of the number of GFAP + nestin + and GFAP + SOX2 + radial glia ( f , h ) , and NSCs ( k , m ) , and GFAP + nestin − astrocytes ( g , l ) per section are shown . Dotted lines indicate the boundaries of the SVZ and DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ( n , o ) Mean ± s . e . m . of the number of TUNEL + cells per 100 SVZ cells ( n ) or per 1 mm2 DG area ( o ) of P28 mice . ( p - r ) BrdU retention in the SVZ and DG of P31mice . BrdU + cells are marked by arrows ( p ) . Mean ± s . e . m . of the number of BrdU + cells per section are shown ( q , r ) . Dotted lines indicate the boundaries of the SVZ and DG ( a , i , j , p ) or granular zone ( GZ ; d , e , p ) . E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . NS , not significant ; * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ( s , t ) Mean ± s . e . m . of percentage of GFAP + nestin + BrdU + cells of total BrdU retained cells in the SVZ ( s ) and SGZ ( t ) of P31mouse . n = 5 mice , ≥ 4 sections per mouse , > 500 cells counted per mouse in n , > 20 BrdU + cells counted per mouse .

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 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 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 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 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

figf5(a-c) Hematoxylin and eosin staining of the dentate gyrus and dentate gyrus (DG) of P28mice (a). Arrows mark cells in the SVZ. Mean ± s.e.m. of the SVZ cellularity (b) and DG area (c) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001(d-m) Immunofluorescence of the DG (d-h) of P28mice (d,e,i,j). Boxed areas are shown in more detail in insets (e,j) and/or panels below (i,j). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,e), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (f,h), and NSCs (k,m), and GFAP+nestin− astrocytes (g,l) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001(d-m) Immunofluorescence of the SVZ (i-m) of P28mice (d,e,i,j). Boxed areas are shown in more detail in insets (e,j) and/or panels below (i,j). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,e), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (f,h), and NSCs (k,m), and GFAP+nestin− astrocytes (g,l) per section are shown. Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001(n,o) Mean ± s.e.m. of the number of TUNEL+ cells per 100 SVZ cells (n) or per 1 mm2 DG area (o) of P28 mice.(p-r) BrdU retention in the SVZ and DG of P31mice. BrdU+ cells are marked by arrows (p). Mean ± s.e.m. of the number of BrdU+ cells per section are shown (q,r). Dotted lines indicate the boundaries of the SVZ and DG (a,i,j,p) or granular zone (GZ; d,e,p). E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. NS, not significant; *P 0.05; **P 0.01; ***P 0.001(s,t) Mean ± s.e.m. of percentage of GFAP+nestin+BrdU+ cells of total BrdU retained cells in the SVZ (s) and SGZ (t) of P31mouse. n = 5 mice, ≥4 sections per mouse, >500 cells counted per mouse in n, >20 BrdU+ cells counted per mouse.


figf6 ( a - f ) Immunofluorescence for PSA - NCAM ( a - c ) or NeuN ( d - f ) in the dentate gyrus ( DG ) , SVZ , RMS and olfactory bulb ( OB ) of P28mice . Lines indicate boundaries of the DG ( a , d ) , SVZ ( a ) , and layers of the OB ( d ) . Boxed areas in a indicate the RMS . Arrows mark PSA - NCAM + cells . Mean ± s . e . m . of the number of PSA - NCAM + cells ( b , c ) or NeuN + cells ( e , f ) per section are shown . LV , lateral ventricle ; ML , molecular layer ; IGL , internal granular layer ; MCL , mitral cell layer ; EPL , external plexiform layer ; GL , glomerular layer . NS , not significant ; * P 0 . 05 . ( g - i ) Immunofluorescence of BrdU retention cells in the DG and OB of P31 mice . Boxed areas in left column of g are shown in more detail in right column . Dotted lines indicate the boundaries of the DG ( left ) and GZ ( right ) . Arrows mark NeuN + BrdU + cells and arrowheads mark NeuN − BrdU + cells in the DG . Mean ± s . e . m . of the percentage of NeuN + BrdU + cells of total BrdU retained cells in the DG ( h ) and OB ( i ) are shown . ( j , k ) Mean ± s . e . m . of percentage of GFAP + nestin − BrdU + cells among total BrdU + retained cells in the SVZ ( j ) and SGZ ( k ) . ( l , m ) DHE and DAPI fluorescence at P28 in the DG ( l ) and SVZ ( m ) from FIP200GFAP cKO mice with or without NAC treatment , 2cKO mice and Trp53GFAP cKO mice . Dotted lines indicate the boundaries of the granular zone ( GZ ) ( l ) and SVZ ( m ) . Arrows mark cells in the SGZ ( l ) and SVZ ( m ) and arrowheads mark cells in surrounding regions ( the GZ in l and the striatum ( ST ) in m ) . n = 5 mice , ≥ 4 sections per mouse , > 20 ( h , j , k ) or > 200 ( i ) BrdU + cells counted per mouse . cKO ( n ) , NAC non - responder cKO mice ; LV , lateral ventricle ; ML , molecular layer ; IGL , internal granular layer ; MCL , mitral cell layer ; EPL , external plexiform layer ; GL , glomerular layer . NS , not significant ; * P 0 . 05 .

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 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 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

figf6(a-f) Immunofluorescence for PSA-NCAM (a-c) or NeuN (d-f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28mice. Lines indicate boundaries of the DG (a,d), SVZ (a), and layers of the OB (d). Boxed areas in a indicate the RMS. Arrows mark PSA-NCAM+ cells. Mean ± s.e.m. of the number of PSA-NCAM+ cells (b,c) or NeuN+ cells (e,f) per section are shown. LV, lateral ventricle; ML, molecular layer; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. NS, not significant; *P 0.05.(g-i) Immunofluorescence of BrdU retention cells in the DG and OB of P31 mice. Boxed areas in left column of g are shown in more detail in right column. Dotted lines indicate the boundaries of the DG (left) and GZ (right). Arrows mark NeuN+BrdU+ cells and arrowheads mark NeuN−BrdU+ cells in the DG. Mean ± s.e.m. of the percentage of NeuN+BrdU+ cells of total BrdU retained cells in the DG (h) and OB (i) are shown. (j,k) Mean ± s.e.m. of percentage of GFAP+nestin−BrdU+ cells among total BrdU+ retained cells in the SVZ (j) and SGZ (k).(l,m) DHE and DAPI fluorescence at P28 in the DG (l) and SVZ (m) from FIP200GFAP cKO mice with or without NAC treatment, 2cKO mice and Trp53GFAP cKO mice. Dotted lines indicate the boundaries of the granular zone (GZ) (l) and SVZ (m). Arrows mark cells in the SGZ (l) and SVZ (m) and arrowheads mark cells in surrounding regions (the GZ in l and the striatum (ST) in m). n = 5 mice, ≥4 sections per mouse, >20 (h,j,k) or >200 (i) BrdU+ cells counted per mouse. cKO(n), NAC non-responder cKO mice; LV, lateral ventricle; ML, molecular layer; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. NS, not significant; *P 0.05.


figf7 ( a - c ) Hematoxylin and eosin staining ( a ) of the SVZ and dentate gyrus ( DG ) of P28mice treated by NAC or vehicle ( Veh ) control . Dotted lines indicate boundaries for the SVZ and DG . Arrows mark cells in the SVZ . ( a - c ) Hematoxylin and eosin staining ( a ) of the SVZ and dentate gyrus ( DG ) of P28 mice treated by NAC or vehicle ( Veh ) control . Dotted lines indicate boundaries for the SVZ and DG . Arrows mark cells in the SVZ . Mean ± s . e . m . of the SVZ cellularity ( b ) and DG area ( c ) per section are shown . ( d - m ) Immunofluorescence of the DG ( d - h ) and SVZ ( i - m ) of P28 mice treated by NAC or vehicle control . Representative images are shown in d , g ( DG ) and k , m ( SVZ ) . Dotted lines indicate boundaries of the granular zone ( GZ ) ( d , g ) and SVZ ( k , m ) . Boxed areas in g , k , m are shown in more detail in insets ( g , m ) and / or panels below ( k , m ) . Lines indicate boundaries of the GZ ( d , g ) and SVZ ( k , m ) . Arrows mark GFAP + nestin + and GFAP + SOX2 + NSCs with radial glial morphology ( d , g ) , and arrowheads mark GFAP + nestin − and GFAP + SOX2 − astrocytes . Mean ± s . e . m . of the number of GFAP + nestin + and GFAP + SOX2 + radial glia ( e , h ) , and NSCs ( i , l ) , and GFAP + nestin − astrocytes ( f , j ) per section are shown . ( n , o ) Mean ± s . e . m . of the number of TUNEL + cells per 100 SVZ cells ( n ) or per 1 mm2 DG area ( o ) of P28mice treated by NAC or vehicle control . n = 5 mice , ≥ 4 sections per mouse , > 500 cells counted per NAC in n . cKO ( n ) , NAC non - responder cKO mice ; E , ependymal cells ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum . * * P 0 . 01 .

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 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

figf7(a-c) Hematoxylin and eosin staining (a) of the SVZ and dentate gyrus (DG) of P28mice treated by NAC or vehicle (Veh) control. Dotted lines indicate boundaries for the SVZ and DG. Arrows mark cells in the SVZ.(a-c) Hematoxylin and eosin staining (a) of the SVZ and dentate gyrus (DG) of P28 mice treated by NAC or vehicle (Veh) control. Dotted lines indicate boundaries for the SVZ and DG. Arrows mark cells in the SVZ. Mean ± s.e.m. of the SVZ cellularity (b) and DG area (c) per section are shown.(d-m) Immunofluorescence of the DG (d-h) and SVZ (i-m) of P28 mice treated by NAC or vehicle control. Representative images are shown in d,g (DG) and k,m (SVZ). Dotted lines indicate boundaries of the granular zone (GZ) (d,g) and SVZ (k,m). Boxed areas in g,k,m are shown in more detail in insets (g,m) and/or panels below (k,m). Lines indicate boundaries of the GZ (d,g) and SVZ (k,m). Arrows mark GFAP+nestin+ and GFAP+SOX2+ NSCs with radial glial morphology (d,g), and arrowheads mark GFAP+nestin− and GFAP+SOX2− astrocytes. Mean ± s.e.m. of the number of GFAP+nestin+ and GFAP+SOX2+ radial glia (e,h), and NSCs (i,l), and GFAP+nestin− astrocytes (f,j) per section are shown. (n,o) Mean ± s.e.m. of the number of TUNEL+ cells per 100 SVZ cells (n) or per 1 mm2 DG area (o) of P28mice treated by NAC or vehicle control. n = 5 mice, ≥4 sections per mouse, >500 cells counted per NAC in n. cKO(n), NAC non-responder cKO mice; E, ependymal cells; LV, lateral ventricle; ML, molecular layer; ST, striatum. **P 0.01.


figf8 ( a - f ) Immunofluorescence for PSA - NCAM ( a - c ) or NeuN ( d - f ) in the dentate gyrus ( DG ) , SVZ , RMS and olfactory bulb ( OB ) of P28 mice treated with NAC or vehicle ( Veh ) control . Representative images are shown for PSA - NCAM ( a ) and NeuN ( d ) . Dotted lines indicate boundaries of the DG ( a , d ) , SVZ ( a ) and layers of the OB ( d ) . Arrows mark PSA - NCAM + cells in a . Mean ± s . e . m . of the number of PSA - NCAM + cells in SVZ ( b ) and DG ( normalized to 1 mm2 area ) ( c ) , and the number of NeuN + cells in DG ( e ) and IGL of OB ( per mm2 ) ( f ) per section are shown ( n = 5 mice for each ; ≥ 4 sections for each mouse ) . cKO ( n ) , NAC non - responder cKO mice ; GZ , granular zone ; LV , lateral ventricle ; ML , molecular layer ; ST , striatum ; IGL , internal granular layer ; MCL , mitral cell layer ; EPL , external plexiform layer ; GL , glomerular layer . * * P 0 . 01 .

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

figf8(a-f) Immunofluorescence for PSA-NCAM (a-c) or NeuN (d-f) in the dentate gyrus (DG), SVZ, RMS and olfactory bulb (OB) of P28 mice treated with NAC or vehicle (Veh) control. Representative images are shown for PSA-NCAM (a) and NeuN (d). Dotted lines indicate boundaries of the DG (a,d), SVZ (a) and layers of the OB (d). Arrows mark PSA-NCAM+ cells in a. Mean ± s.e.m. of the number of PSA-NCAM+ cells in SVZ (b) and DG (normalized to 1 mm2 area) (c), and the number of NeuN+ cells in DG (e) and IGL of OB (per mm2) (f) per section are shown (n = 5 mice for each; ≥4 sections for each mouse). cKO(n), NAC non-responder cKO mice; GZ, granular zone; LV, lateral ventricle; ML, molecular layer; ST, striatum; IGL, internal granular layer; MCL, mitral cell layer; EPL, external plexiform layer; GL, glomerular layer. **P 0.01.


Figure 1Subcellular localization of NeoR and NucNeoR and T cell recognition of NeoR ‐ and NucNeoR ‐ expressing cells . RCC1 . 24 cells were transfected with the NeoR ( A ) and NucNeoR ( B ) expression constructs . After 2 days , the subcellular localization of the proteins was analyzed by immunofluorescence using the anti c ‐ myc antibody 9E10 . While NeoR was evenly distributed in nucleus and cytoplasm ( a ) , NucNeoR was confined to the cell nucleus ( b ) . Daylight photographs of the stained cells are shown in ( a ' ) and ( b ' ) . ( C ) NeoR ‐ and NucNeoR ‐ transfected RCC1 . 24 cells were treated with 100 U / mL IFN ‐ γ for 48 h to induce MHC class II expression . Subsequently , the cells were washed and co ‐ cultured with the CD4 clone 20 ‐ 4 / A4 , which recognizes a peptide derived from NeoR on HLA ‐ DP3 . GM ‐ CSF secretion by the T cells was measured 20 h later by ELISA . Both transfectants were recognized after IFN ‐ γ treatment , indicating that directing NeoR into the cell nucleus does not abrogate antigen presentation . ( D ) LCL1 . 11 cells were transfected with NeoR or NucNeoR expression plasmids and antigen presentation was assessed 48 h later with the NeoR ‐ specific T cell clone 20 ‐ 4 / A4 . Antigen ‐ transfected LCL1 . 11 cells were recognized irrespectively of NeoR localization , while untransfected cells or cells transfected with a GFP ‐ expressing control plasmid were not recognized .

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 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

Figure 1Subcellular localization of NeoR and NucNeoR and T cell recognition of NeoR‐ and NucNeoR‐expressing cells. RCC1.24 cells were transfected with the NeoR (A) and NucNeoR (B) expression constructs. After 2 days, the subcellular localization of the proteins was analyzed by immunofluorescence using the anti c‐myc antibody 9E10. While NeoR was evenly distributed in nucleus and cytoplasm (a), NucNeoR was confined to the cell nucleus (b). Daylight photographs of the stained cells are shown in (a') and (b').(C) NeoR‐ and NucNeoR‐transfected RCC1.24 cells were treated with 100 U/mL IFN‐γ for 48 h to induce MHC class II expression. Subsequently, the cells were washed and co‐cultured with the CD4 clone 20‐4/A4, which recognizes a peptide derived from NeoR on HLA‐DP3. GM‐CSF secretion by the T cells was measured 20 h later by ELISA. Both transfectants were recognized after IFN‐γ treatment, indicating that directing NeoR into the cell nucleus does not abrogate antigen presentation.(D) LCL1.11 cells were transfected with NeoR or NucNeoR expression plasmids and antigen presentation was assessed 48 h later with the NeoR‐specific T cell clone 20‐4/A4. Antigen‐transfected LCL1.11 cells were recognized irrespectively of NeoR localization, while untransfected cells or cells transfected with a GFP‐expressing control plasmid were not recognized.