( 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

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(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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(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.


( C ) Expression of GALNT isoforms in primary keratinocytes and HaCaT cell line . The scatter plot depicts individual RPKM values of 2 biological replicates .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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 .

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(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.


GalNAc - 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 .

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GalNAc-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

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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 .

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(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 .

(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 .

(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 ) .

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(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 ) .

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(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 .

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(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.


( 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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 ) 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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

(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.


( 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 ) .

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(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 .

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(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 .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) 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.


( 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 .

(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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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

(c,d) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e-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.


( 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 .

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

(a-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 .

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

(d-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 .

(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 .

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

(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 .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

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

(a-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 ) .

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

(g-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

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

(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 .

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

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

(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.


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

(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.


Subcellular 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 ' ) .

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

Subcellular 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 .

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(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 .

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(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.


( A ) RCC1 . 24 ‐ NeoR and ‐ NucNeoR cells were incubated for 48 h with IFN ‐ γ and in addition with 7 . 5 mM 3 ‐ MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h . Subsequently , the cells were fixed with 0 . 5 % paraformaldehyde and tested with 20 ‐ 4 / A4 cells in a GM ‐ CSF release assay . Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing .

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(A) RCC1.24‐NeoR and ‐NucNeoR cells were incubated for 48 h with IFN‐γ and in addition with 7.5 mM 3‐MA or 200 µM leupeptin or 100 µM chloroquine for the last 20 h. Subsequently, the cells were fixed with 0.5% paraformaldehyde and tested with 20‐4/A4 cells in a GM‐CSF release assay. Both NeoR and NucNeoR were presented by a pathway that involves autophagy and lysosomal processing.


( B ) LCL1 . 11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise . Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors , indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing .

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(B) LCL1.11 cells transfected with NeoR or NucNeoR expression plasmids were tested likewise. Presentation of endogenous NeoR and NucNeoR was blocked by all inhibitors, indicating that NeoR and NucNeoR presentation is dependent on autophagy and lysosomal processing.


( C ) To exclude toxic side effects of the inhibitors , LCL1 . 11 cells were incubated for 20 h with 200 ng / mL recombinant NeoR protein in the presence or absence of the inhibitors . Afterwards , residual protein and inhibitors were removed by washing and the cells were co ‐ cultured with the NeoR ‐ specific CD4 + T cell clone 20 ‐ 4 / A4 . The presentation of exogenous NeoR on MHC class II was not affected by 3 ‐ MA , but was impaired when lysosomal processing was blocked with chloroquine or leupeptin .

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(C) To exclude toxic side effects of the inhibitors, LCL1.11 cells were incubated for 20 h with 200 ng/mL recombinant NeoR protein in the presence or absence of the inhibitors. Afterwards, residual protein and inhibitors were removed by washing and the cells were co‐cultured with the NeoR‐specific CD4+ T cell clone 20‐4/A4. The presentation of exogenous NeoR on MHC class II was not affected by 3‐MA, but was impaired when lysosomal processing was blocked with chloroquine or leupeptin.


( D ) LCL1 . 11 cells were transfected with siRNA specific for Atg12 or GFP , and Atg12 mRNA levels were determined 24 h later by qPCR . The relative amount of Atg12 mRNA was reduced approximately fourfold .

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(D) LCL1.11 cells were transfected with siRNA specific for Atg12 or GFP, and Atg12 mRNA levels were determined 24 h later by qPCR. The relative amount of Atg12 mRNA was reduced approximately fourfold.


( E ) At 24 h after siRNA transfection , NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later . In cells transfected with Atg12 siRNA , but not in mock ‐ transfected cells or cells transfected with GFP siRNA , antigen presentation of both NeoR and NucNeoR was greatly reduced .

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(E) At 24 h after siRNA transfection, NeoR and NucNeoR were expressed in these cells and antigen presentation was assessed 36 h later. In cells transfected with Atg12 siRNA, but not in mock‐transfected cells or cells transfected with GFP siRNA, antigen presentation of both NeoR and NucNeoR was greatly reduced.


( A ) RCC1 . 24 cells were transfected with the NES ‐ GFP ‐ NeoR ‐ GFP ‐ NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence . As compared to untreated cells ( a ) , cells treated with 8 nM LMB for 20 h ( b ) showed a strong accumulation of the protein in the nucleus . ( a ' ) and ( b ' ) are the corresponding daylight photographs of the transfected cells .

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(A) RCC1.24 cells were transfected with the NES‐GFP‐NeoR‐GFP‐NLS expression construct and subcellular localization of the fusion protein was monitored by UV fluorescence. As compared to untreated cells (a), cells treated with 8 nM LMB for 20 h (b) showed a strong accumulation of the protein in the nucleus. (a') and (b') are the corresponding daylight photographs of the transfected cells.


( B ) NeoR ‐ and NucNeoR ‐ transfected LCL1 . 11 cells were either left untreated or incubated for 20 h with 8 nM LMB . Subsequently , the cells were fixed with 0 . 5 % paraformaldehyde and probed with the NeoR ‐ specific T cells . LMB treatment had no significant effect on the presentation of NeoR or NucNeoR on MHC class II , indicating that CRM1 ‐ dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II .

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

(B) NeoR‐ and NucNeoR‐transfected LCL1.11 cells were either left untreated or incubated for 20 h with 8 nM LMB. Subsequently, the cells were fixed with 0.5% paraformaldehyde and probed with the NeoR‐specific T cells. LMB treatment had no significant effect on the presentation of NeoR or NucNeoR on MHC class II, indicating that CRM1‐dependent nuclear export is not involved in the presentation of this nuclear antigen on MHC class II.


A . qRT - PCR analysis of RNA samples from normal human skin ( NS ) and AK for activin βA ( INHBA ) , normalized to hypoxanthine phosphoribosyltransferase ( HPRT ) . Expression levels in one of the normal skin samples were set to 1 . Scatter plots are shown . NS : N = 4 ; AK : N = 21 . ns = non - significant ( Mann - Whitney test ) .

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A. qRT-PCR analysis of RNA samples from normal human skin (NS) and AK for activin βA (INHBA), normalized to hypoxanthine phosphoribosyltransferase (HPRT). Expression levels in one of the normal skin samples were set to 1. Scatter plots are shown. NS: N=4; AK: N=21. ns = non-significant (Mann-Whitney test).


B . qRT - PCR analysis of RNA samples from normal back skin of wild - type mice ( N = 4 , n = 4 ) , non - tumorigenic back skin from HPV8 / wt mice ( N = 7 , n = 9 ) and papillomas from HPV8 / wt transgenic mice ( N = 8 , n = 11 ) for Inhba , normalized to glyceraldehyde 3 - phosphate dehydrogenase ( Gapdh ) . Expression levels in one of the wild - type back skin samples were set to 1 . Statistical significance was determined using 1 - way ANOVA and Bonferroni ` s Multiple Comparisons Test of log - transformed data . N , number of mice ; n , number of biopsies .

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B. qRT-PCR analysis of RNA samples from normal back skin of wild-type mice (N=4, n=4), non-tumorigenic back skin from HPV8/wt mice (N=7, n=9) and papillomas from HPV8/wt transgenic mice (N=8, n=11) for Inhba, normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh). Expression levels in one of the wild-type back skin samples were set to 1. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test of log-transformed data. N, number of mice; n, number of biopsies.


C - E . Sections from a tumor collected from a 64 week - old HPV8 / wt mouse ( C , D ) or from non - tumorigenic skin of 5 week - old HPV8 / wt mice ( E ) stained with an anti - activin A antibody and counterstained with hematoxylin . E : Epidermis , D : Dermis , HF : Hair follicle . ( D ) Shows a higher magnification of the boxed area in ( C ) . Scale bars : 200μm ( C ) and 100μm ( D , E ) .

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C-E. Sections from a tumor collected from a 64 week-old HPV8/wt mouse (C,D) or from non-tumorigenic skin of 5 week-old HPV8/wt mice (E) stained with an anti-activin A antibody and counterstained with hematoxylin. E: Epidermis, D: Dermis, HF: Hair follicle. (D) Shows a higher magnification of the boxed area in (C). Scale bars: 200μm (C) and 100μm (D,E).


F . Kinetics of tumor incidence in HPV8 / wt and HPV8 / Act mice ; N = 20 mice per genotype . Statistical significance was determined using Log - rank ( Mantel - Cox ) test .

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F. Kinetics of tumor incidence in HPV8/wt and HPV8/Act mice; N=20 mice per genotype. Statistical significance was determined using Log-rank (Mantel-Cox) test.


G . Age of the mice when the first tumors appeared on certain body sites . Each dot represents one tumor .

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G. Age of the mice when the first tumors appeared on certain body sites. Each dot represents one tumor.


H . Representative macroscopic pictures of ear tumors ( left panel ) and H & amp ; amp ; E - stained sections of ear acanthopapillomas ( upper and middle panel ) or an ear acanthopapilloma with trichoepitheliomatous differentiation ( lower panel ) . Boxed areas are shown at higher magnification in subsequent pictures to the right in each horizontal picture panel . E : epidermis ; D : dermis ; C : cartilage . Scale bars : 200μm ( left panel ) , 100μm ( middle panel ) or 50μm ( right panel ) .

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H. Representative macroscopic pictures of ear tumors (left panel) and H&amp;E-stained sections of ear acanthopapillomas (upper and middle panel) or an ear acanthopapilloma with trichoepitheliomatous differentiation (lower panel). Boxed areas are shown at higher magnification in subsequent pictures to the right in each horizontal picture panel. E: epidermis; D: dermis; C: cartilage. Scale bars: 200μm (left panel), 100μm (middle panel) or 50μm (right panel).


A , B . Representative pictures of ear sections from 10 week - old mice stained for δTcR ( green ) ; counterstained with Hoechst ( blue ) ( A ) and quantification of δTcR + cells in the ear epidermis ( B ) . Dotted lines indicate the epidermal - dermal border . Scale bar : 20μm . N = 6 wt / wt mice , N = 7 HPV8 / wt mice , N = 6 wt / Act mice and ( N = 8 ) HPV8 / Act mice .

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A,B. Representative pictures of ear sections from 10 week-old mice stained for δTcR (green); counterstained with Hoechst (blue) (A) and quantification of δTcR+ cells in the ear epidermis (B). Dotted lines indicate the epidermal-dermal border. Scale bar: 20μm. N=6 wt/wt mice, N=7 HPV8/wt mice, N=6 wt/Act mice and (N=8) HPV8/Act mice.


C . Quantification of βTcR + cells in ear skin cryosections from 10 week - old wt / wt ( N = 5 ) ; HPV8 / wt ( N = 5 ) ; wt / Act ( N = 8 ) or HPV8 / Act ( N = 8 ) mice .

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C. Quantification of βTcR+ cells in ear skin cryosections from 10 week-old wt/wt (N=5); HPV8/wt (N=5); wt/Act (N=8) or HPV8/Act (N=8) mice.


D - F . Percentage of viable CD4 + βTcR + ( D ) , CD8 + βTcR + ( E ) or CD4 + CD25 + ( F ) cells among all epidermal cells analyzed by flow cytometry . Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown . Representative flow cytometry scatter plots are shown in Appendix Fig S1A - C .

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D-F. Percentage of viable CD4+ βTcR+ (D), CD8+ βTcR+ (E) or CD4+CD25+ (F) cells among all epidermal cells analyzed by flow cytometry. Results from 5 experiments with ears pooled from at least 3 mice per genotype are shown. Representative flow cytometry scatter plots are shown in Appendix Fig S1A-C.


G . Representative pictures of ear cryosections from 10 week - old mice stained with antibodies against Foxp3 ( red ) and counterstained with Hoechst ( blue ) . Insert shows higher magnification of the Foxp3 - positive cells in wt / Act skin . Dotted lines indicate the epidermal - dermal border . Scale bar : 50μm .

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G. Representative pictures of ear cryosections from 10 week-old mice stained with antibodies against Foxp3 (red) and counterstained with Hoechst (blue). Insert shows higher magnification of the Foxp3-positive cells in wt/Act skin. Dotted lines indicate the epidermal-dermal border. Scale bar: 50μm.


H . Kinetics of tumor incidence in HPV8 / Act / wt ( N = 29 ) , HPV8 / Act / CD4KO ( N = 10 ) , HPV8 / wt / wt ( N = 23 ) and HPV8 / wt / CD4KO ( N = 22 ) mice per genotype . Statistical significance was determined using the Log - rank ( Mantel - Cox ) test ; ns = non - significant .

H. Kinetics of tumor incidence in HPV8/Act/wt (N=29), HPV8/Act/CD4KO (N=10), HPV8/wt/wt (N=23) and HPV8/wt/CD4KO (N=22) mice per genotype. Statistical significance was determined using the Log-rank (Mantel-Cox) test; ns = non-significant.


A , B . Representative pictures of ear skin cryosections from 10 - 12 week - old mice stained with anti - CD206 antibodies ( green ) ; counterstained with Hoechst ( blue ) ( A ) , and quantification of CD206 + area as a percentage of total dermal area ( B ) in wt / wt ( N = 3 ) , HPV8 / wt ( N = 7 ) , wt / Act ( N = 2 ) or HPV8 / Act ( N = 8 ) mice . The basement membrane is indicated with a white dotted line .

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A,B. Representative pictures of ear skin cryosections from 10-12 week-old mice stained with anti-CD206 antibodies (green); counterstained with Hoechst (blue) (A), and quantification of CD206+ area as a percentage of total dermal area (B) in wt/wt (N=3), HPV8/wt (N=7), wt/Act (N=2) or HPV8/Act (N=8) mice. The basement membrane is indicated with a white dotted line.


C . Abundance of MerTK + CD64 + macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12 - 16 week - old mice . N = 5 - 6 . Representative flow cytometry scatter plots are shown in Appendix Fig S1D .

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C. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of normal or precancerous ear skin from 12-16 week-old mice. N = 5-6. Representative flow cytometry scatter plots are shown in Appendix Fig S1D.


D . Quantification of CD206 + area as a percentage of total dermal area in wt ( N = 7 ) and Act ( N = 6 ) mice in back skin treated 8x with TPA ( Antsiferova et al , 2011 ) .

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D. Quantification of CD206+ area as a percentage of total dermal area in wt (N=7) and Act (N=6) mice in back skin treated 8x with TPA (Antsiferova et al, 2011).


E . Abundance of MerTK + CD64 + macrophages analyzed by flow cytometry of pre - tumorigenic ear skin from 12 week - old mice expressing the CCR2 - CFP - DTR transgene ( wt / CCR2 ) or double - transgenic animals ( Act / CCR2 ) , untreated or treated with diphtheria toxin ( + DT ) 3 times every 48h and analyzed 24h after the last injection . Percentage of CD45 + CD11b + MerTK + CD64 + macrophages among live cells is shown . N = 3 control wt / CCR2 and Act / CCR2 mice ; N = 4 DT - injected wt / CCR2 and Act / CCR2 mice . Representative flow cytometry scatter plots are shown in Appendix Fig S1E .

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E. Abundance of MerTK+CD64+ macrophages analyzed by flow cytometry of pre-tumorigenic ear skin from 12 week-old mice expressing the CCR2-CFP-DTR transgene (wt/CCR2) or double-transgenic animals (Act/CCR2), untreated or treated with diphtheria toxin (+DT) 3 times every 48h and analyzed 24h after the last injection. Percentage of CD45+CD11b+MerTK+CD64+ macrophages among live cells is shown. N=3 control wt/CCR2 and Act/CCR2 mice; N=4 DT-injected wt/CCR2 and Act/CCR2 mice. Representative flow cytometry scatter plots are shown in Appendix Fig S1E.


F , G . Inflammatory thioglycollate - elicited peritoneal macrophages ( F ) or mouse peripheral blood monocytes ( G ) were isolated from wt mice and allowed to migrate for 24h in a Transwell assay . Activin A 0 . 1ng / ml ( A0 . 1 ) , 1ng / ml ( A1 ) , or 10ng / ml ( A10 ) was added to the lower well . Alternatively , 10ng / ml was added to the lower well and the insert ( A10 / A10 ) . Random migration to medium ( ctrl ) was set to 1 for each mouse ; fold over control is shown . Log - transformed data were used for statistical analysis . Results in ( F ) are representative of 4 independent experiments performed with 8 mice in total ( results obtained in one experiment performed with 3 mice are shown ) . Results in ( G ) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice .

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F, G. Inflammatory thioglycollate-elicited peritoneal macrophages (F) or mouse peripheral blood monocytes (G) were isolated from wt mice and allowed to migrate for 24h in a Transwell assay. Activin A 0.1ng/ml (A0.1), 1ng/ml (A1), or 10ng/ml (A10) was added to the lower well. Alternatively, 10ng/ml was added to the lower well and the insert (A10/A10). Random migration to medium (ctrl) was set to 1 for each mouse; fold over control is shown. Log-transformed data were used for statistical analysis. Results in (F) are representative of 4 independent experiments performed with 8 mice in total (results obtained in one experiment performed with 3 mice are shown). Results in (G) are combined from 2 independent experiments performed with monocytes isolated from pooled blood of at least 13 wt mice.


B . Analysis of FACS data of sorted macrophages shows the relative percentage of CD45 + CD11b + F4 / 80 + cells in each genotype and the absolute number of sorted cells used for RNA sequencing . Representative flow cytometry gating strategy for sorting is shown in Fig EV3B . N = 5 - 9 . Statistical significance was determined using 1 - way ANOVA and Bonferroni ` s Multiple Comparisons Test .

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B. Analysis of FACS data of sorted macrophages shows the relative percentage of CD45+CD11b+F4/80+ cells in each genotype and the absolute number of sorted cells used for RNA sequencing. Representative flow cytometry gating strategy for sorting is shown in Fig EV3B. N=5-9. Statistical significance was determined using 1-way ANOVA and Bonferroni`s Multiple Comparisons Test.


C . Differential expression of genes was analyzed using the edgeR software package . IPA was performed with filtered lists of up - and downregulated genes that exhibited an FDR - value of < 0 . 1 in different genotype comparisons ( wt / Act vs wt / wt ; HPV8 / Act vs HPV8 / wt ; HPV8 / Act vs wt / wt ) . The number of differentially expressed genes used for analysis is indicated . Data are presented as pseudo - heatmap with magnitudes of Activation Z - scores color - coded as indicated in the legend ; all non - grey comparisons are - log10 ( p - value ) > 2 . 5 unless otherwise indicated .

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C. Differential expression of genes was analyzed using the edgeR software package. IPA was performed with filtered lists of up- and downregulated genes that exhibited an FDR-value of <0.1 in different genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt). The number of differentially expressed genes used for analysis is indicated. Data are presented as pseudo-heatmap with magnitudes of Activation Z-scores color-coded as indicated in the legend; all non-grey comparisons are -log10(p-value)>2.5 unless otherwise indicated.


D . Gene Set Enrichment Analysis ( GSEA ) was performed to compare transcriptomes of genotype comparisons ( wt / Act vs wt / wt ; HPV8 / Act vs HPV8 / wt ; HPV8 / Act vs wt / wt , [ all ] / Act vs [ all ] / wt ) to 3 published datasets of Tumor - Associated Macrophages ( TAM ) ( Franklin et al . 2014 ; Ojalvo et al , 2009 ; Galletti et al , 2016 ) . Gene sets are composed of top genes upregulated ( TAM UP ) or downregulated ( TAM DOWN ) in TAM vs normal tissue macrophages . Positive Normalized Enrichment Score ( NES ) corresponds to enrichment of gene set in activin - upregulated genes ; negative NES corresponds to enrichment in activin - downregulated genes . Data are presented as pseudo - heatmap with magnitudes of NES color - coded as indicated in the legend ; all non - grey comparisons are FDR < 0 . 10 .

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

D. Gene Set Enrichment Analysis (GSEA) was performed to compare transcriptomes of genotype comparisons (wt/Act vs wt/wt; HPV8/Act vs HPV8/wt; HPV8/Act vs wt/wt, [all]/Act vs [all]/wt) to 3 published datasets of Tumor-Associated Macrophages (TAM) (Franklin et al. 2014; Ojalvo et al, 2009; Galletti et al, 2016). Gene sets are composed of top genes upregulated (TAM UP) or downregulated (TAM DOWN) in TAM vs normal tissue macrophages. Positive Normalized Enrichment Score (NES) corresponds to enrichment of gene set in activin-upregulated genes; negative NES corresponds to enrichment in activin-downregulated genes. Data are presented as pseudo-heatmap with magnitudes of NES color-coded as indicated in the legend; all non-grey comparisons are FDR<0.10.


E . Differential GSEA was performed on combined genotype comparison [ all ] / Act vs [ all ] / wt and two datasets of human AK vs normal skin ( GSE2503 ; Nindl et al , 2006 ) and GSE63107 to compare these transcriptomes to gene sets derived from top upregulated genes in these datasets ( top ) ; and gene sets derived from top upregulated genes in datasets of differential human macrophage activation ( IFN - γ , TNF - α , LPS , IL - 4 vs non - treated ) and selected human myeloid / lymphoid cell type - specific gene expression profiles ( macrophage vs monocyte , dendritic cell , and T - cell ) ( Xue et al , 2014 ; bottom ) . Activin target genes enriched in the AK transcriptomes are shown ( right ) . Data are presented as pseudo - heatmaps with magnitudes of Normalized Enrichment Scores ( NES ) and Log2 ( Fold Change ) color - coded as indicated in the respective legends ; all non - grey comparisons are FDR < 0 . 05 for NES and p < 0 . 10 for Fold Change .

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

E. Differential GSEA was performed on combined genotype comparison [all]/Act vs [all]/wt and two datasets of human AK vs normal skin (GSE2503; Nindl et al, 2006) and GSE63107 to compare these transcriptomes to gene sets derived from top upregulated genes in these datasets (top); and gene sets derived from top upregulated genes in datasets of differential human macrophage activation (IFN-γ, TNF-α, LPS, IL-4 vs non-treated) and selected human myeloid/lymphoid cell type-specific gene expression profiles (macrophage vs monocyte, dendritic cell, and T-cell) (Xue et al, 2014; bottom). Activin target genes enriched in the AK transcriptomes are shown (right). Data are presented as pseudo-heatmaps with magnitudes of Normalized Enrichment Scores (NES) and Log2(Fold Change) color-coded as indicated in the respective legends; all non-grey comparisons are FDR<0.05 for NES and p<0.10 for Fold Change.


G . Heatmap of 28 differentially expressed genes ( FDR ≤ 0 . 05 , | log2Ratio | ≥ 1 ) overlapping in all 3 comparisons ( wt / Act vs wt / wt mice , HPV8 / Act vs HPV8 / wt and HPV8 / Act vs wt / wt ) . Genes with a published pro - tumorigenic function are highlighted in red .

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G. Heatmap of 28 differentially expressed genes (FDR≤0.05, |log2Ratio|≥1) overlapping in all 3 comparisons (wt/Act vs wt/wt mice, HPV8/Act vs HPV8/wt and HPV8/Act vs wt/wt). Genes with a published pro-tumorigenic function are highlighted in red.


A , B , F . Expression of Cx3cr1 ( A ) , Spp1 ( B ) relative to Rps29 was analyzed by qRT - PCR in peritoneal macrophages , resident or inflammatory , treated in vitro with 10 ng / ml of activin A ( Act ) or untreated ( Ctrl , arbitrarily set to 1 ) . Results are combined from 2 - 5 independent experiments performed with cells pooled from at least 5 wt mice .

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A,B,F. Expression of Cx3cr1 (A), Spp1 (B) relative to Rps29 was analyzed by qRT-PCR in peritoneal macrophages, resident or inflammatory, treated in vitro with 10 ng/ml of activin A (Act) or untreated (Ctrl, arbitrarily set to 1). Results are combined from 2-5 independent experiments performed with cells pooled from at least 5 wt mice.


C , D . Western blot analysis of total ear protein lysates of 12 - 14 week - old mice for osteopontin ( OPN ) and GAPDH ( C ) . Each lane represents an individual mouse ( n = 3 ) . ( D ) Band intensities were quantified and normalized to GAPDH . The expression level in one of the wt mice was arbitrarily set to 1 .

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C,D. Western blot analysis of total ear protein lysates of 12-14 week-old mice for osteopontin (OPN) and GAPDH (C). Each lane represents an individual mouse (n=3). (D) Band intensities were quantified and normalized to GAPDH. The expression level in one of the wt mice was arbitrarily set to 1.


E . Ear skin sections from 10 week - old mice stained with anti - CD68 ( red ) or anti - osteopontin antibody ( green ) ; counterstained with Hoechst ( blue ) . Osteopontin - positive macrophages in wt / Act and HPV8 / Act mice are indicated with arrows . Representative of N = 2 wt / wt and N = 3 HPV8 / wt , wt / Act and HPV8 / Act mice . Boxed areas in top panel are shown at higher magnification in bottom panel . Scale bar top : 100μm , bottom : 20μm .

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E. Ear skin sections from 10 week-old mice stained with anti-CD68 (red) or anti-osteopontin antibody (green); counterstained with Hoechst (blue). Osteopontin-positive macrophages in wt/Act and HPV8/Act mice are indicated with arrows. Representative of N=2 wt/wt and N=3 HPV8/wt, wt/Act and HPV8/Act mice. Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.


A , B , F . Expression of Arg1 ( F ) relative to Rps29 was analyzed by qRT - PCR in peritoneal macrophages , resident or inflammatory , treated in vitro with 10 ng / ml of activin A ( Act ) or untreated ( Ctrl , arbitrarily set to 1 ) . Results are combined from 2 - 5 independent experiments performed with cells pooled from at least 5 wt mice .

A,B,F. Expression of Arg1 (F) relative to Rps29 was analyzed by qRT-PCR in peritoneal macrophages, resident or inflammatory, treated in vitro with 10 ng/ml of activin A (Act) or untreated (Ctrl, arbitrarily set to 1). Results are combined from 2-5 independent experiments performed with cells pooled from at least 5 wt mice.


G . In situ gelatin zymography ( top panel ) and methylene blue staining ( bottom panel ) of the ear skin from 10 week - old mice . Representative images of 2 - 3 mice per genotype are shown . Scale bar = 500μm .

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G. In situ gelatin zymography (top panel) and methylene blue staining (bottom panel) of the ear skin from 10 week-old mice. Representative images of 2-3 mice per genotype are shown. Scale bar = 500μm.


H , I . Gelatin zymography of ear skin lysates from 10 week - old mice . Inverted image of a representative gel with 2 ( wt / wt ) or 3 ( HPV8 / wt , wt / Act , HPV8 / Act ) mice per genotype is shown in ( H ) , quantification of the results obtained in 2 independent experiments is shown in ( I ) . N = 5 wt / wt , N = 6 HPV8 / wt , wt / Act and HPV8 / Act mice .

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H,I. Gelatin zymography of ear skin lysates from 10 week-old mice. Inverted image of a representative gel with 2 (wt/wt) or 3 (HPV8/wt, wt/Act, HPV8/Act) mice per genotype is shown in (H), quantification of the results obtained in 2 independent experiments is shown in (I). N=5 wt/wt, N=6 HPV8/wt, wt/Act and HPV8/Act mice.


J . Representative pictures of ear skin from 10 week - old mice stained for MECA32 ( red ) , LYVE1 ( green ) ; counterstained with Hoechst ( blue ) . Boxed areas in top panel are shown at higher magnification in bottom panel . Scale bar top : 100μm , bottom : 20μm . K , L . Quantification of MECA32 + area ( K ) and average distance between MECA32 + vessels and basement membrane ( BM ) ( L ) .

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J. Representative pictures of ear skin from 10 week-old mice stained for MECA32 (red), LYVE1 (green); counterstained with Hoechst (blue). Boxed areas in top panel are shown at higher magnification in bottom panel. Scale bar top: 100μm, bottom: 20μm.K,L. Quantification of MECA32+ area (K) and average distance between MECA32+ vessels and basement membrane (BM) (L).


A . Kinetics of tumor incidence in HPV8 / Act mice treated for 5 weeks with 50mg / kg anti - CSF1R antibody ( AFS98 ) or with control rat IgG . N = 10 mice per treatment group .

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A. Kinetics of tumor incidence in HPV8/Act mice treated for 5 weeks with 50mg/kg anti-CSF1R antibody (AFS98) or with control rat IgG. N=10 mice per treatment group.


B . Representative pictures of anti - CD68 - or CD206 - stained cryosections of ear skin from 10 week - old mice treated for 5 weeks with anti - CSF1R antibody or control rat IgG . Scale bar = 100μm . C . Quantification of the CD68 + or CD206 + area relative to total dermis area . N = 10 mice per treatment group .

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B. Representative pictures of anti-CD68- or CD206-stained cryosections of ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R antibody or control rat IgG. Scale bar = 100μm.C. Quantification of the CD68+ or CD206+ area relative to total dermis area. N=10 mice per treatment group.


D . Representative pictures of anti - BrdU ( green ) and anti - K14 ( red ) stained cryosections of ear skin from 10 week - old mice treated for 5 weeks with anti - CSF1R antibody or control rat IgG . Scale bar : 50μm . E . Quantification of BrdU + keratinocytes per mm of basement membrane . N = 8 anti - CSF1R - injected mice ; N = 4 rat IgG - injected mice .

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D. Representative pictures of anti-BrdU (green) and anti-K14 (red) stained cryosections of ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R antibody or control rat IgG. Scale bar: 50μm.E. Quantification of BrdU+ keratinocytes per mm of basement membrane. N=8 anti-CSF1R-injected mice; N=4 rat IgG-injected mice.


F . Expression of Mmp12 relative to Rps29 was analyzed by qRT - PCR in total ear skin from mice treated with anti - CSF1R antibody or with rat IgG . Expression in one of the IgG - injected mice was set to 1 . N = 10 per group .

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

F. Expression of Mmp12 relative to Rps29 was analyzed by qRT-PCR in total ear skin from mice treated with anti-CSF1R antibody or with rat IgG. Expression in one of the IgG-injected mice was set to 1. N=10 per group.


G . Representative pictures of the ear skin from 10 week - old mice treated for 5 weeks with anti - CSF1R or control rat IgG , stained for CD31 ( red ) , LYVE1 ( green ) , and counterstained with Hoechst ( blue ) . Boxed areas are shown at higher magnification in the top - right corner . Scale bar : 100μm . H , I . Quantification of CD31 + LYVE1 - area ( H ) and number of CD31 + LYVE1 - vessels ( I ) .

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

G. Representative pictures of the ear skin from 10 week-old mice treated for 5 weeks with anti-CSF1R or control rat IgG, stained for CD31 (red), LYVE1 (green), and counterstained with Hoechst (blue). Boxed areas are shown at higher magnification in the top-right corner. Scale bar: 100μm.H,I. Quantification of CD31+LYVE1- area (H) and number of CD31+LYVE1-vessels (I).


Growth curve of NIH - 3T3 cells treated with TGFβ ( 2 ng / mL for all experiments ) or vehicle alone ( mock ) for the indicated time . The cell number at the indicated days relative to the number at the start of the treatment ( d0 ) is shown . The dotted line represents the cell number at d0 .

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Growth curve of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or vehicle alone (mock) for the indicated time. The cell number at the indicated days relative to the number at the start of the treatment (d0) is shown. The dotted line represents the cell number at d0.


NIH - 3T3 cells were treated with TGFβ or mock for 48h and incubated with O - propargyl - puromycin ( OPP ) for the last 60 min . The translation rate was determined by flow cytometry for OPP incorporation into proteins . A representative plot is shown on the left , and the quantification is shown on the right . Values are relative to mock - treated cells .

NIH-3T3 cells were treated with TGFβ or mock for 48h and incubated with O-propargyl-puromycin (OPP) for the last 60 min. The translation rate was determined by flow cytometry for OPP incorporation into proteins. A representative plot is shown on the left, and the quantification is shown on the right. Values are relative to mock-treated cells.


Western blot of NIH - 3T3 cells treated with TGFβ or mock for 48h .

Western blot of NIH-3T3 cells treated with TGFβ or mock for 48h.


Western blot of ECM from NIH - 3T3 cells grown in the presence of absence of TGFβ and of cell lysates from NIH - 3T3 cells grown on a parallel plate . Note the absence of tubulin and the electromobility shift of collagens in ECM extracts .

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Western blot of ECM from NIH-3T3 cells grown in the presence of absence of TGFβ and of cell lysates from NIH-3T3 cells grown on a parallel plate. Note the absence of tubulin and the electromobility shift of collagens in ECM extracts.


ECM was produced from NIH - 3T3 cells according to ( d ) , and collagen abundance was measured by picrosirius red staining , normalized to the packed cell volume before ECM extraction , and expressed relative to mock - treated cells .

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ECM was produced from NIH-3T3 cells according to (d), and collagen abundance was measured by picrosirius red staining, normalized to the packed cell volume before ECM extraction, and expressed relative to mock-treated cells.


NIH - 3T3 cells were treated with TGFβ or mock for 48h . The medium was replaced in the last 12h of the treatment ( with the same treatments as before ) , and glucose consumption from the fresh media were measured using the YSI bioanalyzer .

NIH-3T3 cells were treated with TGFβ or mock for 48h. The medium was replaced in the last 12h of the treatment (with the same treatments as before), and glucose consumption from the fresh media were measured using the YSI bioanalyzer.


ATP production rate from glycolysis ( JATPgly ) and mitochondrial oxidative phosphorylation ( JATPox ) from NIH - 3T3 cells treated with TGFβ or mock for 48h , calculated using Seahorse data shown in ( k ) and EV Figure 1f .

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ATP production rate from glycolysis (JATPgly) and mitochondrial oxidative phosphorylation (JATPox) from NIH-3T3 cells treated with TGFβ or mock for 48h, calculated using Seahorse data shown in (k) and EV Figure 1f.


Pearson correlation of mitochondrial ATP production rate ( JATPox ) and OPP incorporation of NIH - 3T3 cells treated with TGFβ or mock for 2h , 4h , 8h , 24h and 48h , relative to mock - treated cells . Also see EV Figure 1k and l . Note that the datapoints of the 2h and 4h measurements overlap .

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Pearson correlation of mitochondrial ATP production rate (JATPox) and OPP incorporation of NIH-3T3 cells treated with TGFβ or mock for 2h, 4h, 8h, 24h and 48h, relative to mock-treated cells. Also see EV Figure 1k and l. Note that the datapoints of the 2h and 4h measurements overlap.


NIH - 3T3 cells were treated with TGFβ or mock for 48h . The medium was replaced in the last 12h of the treatment ( with the same treatments as before ) Glutamine consumption measured as in ( g ) .

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

NIH-3T3 cells were treated with TGFβ or mock for 48h. The medium was replaced in the last 12h of the treatment (with the same treatments as before) Glutamine consumption measured as in (g).


NIH - 3T3 cells were treated with TGFβ or mock for 48h , and the oxygen consumption rate ( OCR ) before and after treatment with mitochondrial inhibitors was measured using the Seahorse bioanalyzer . Oligo , Oligomycin ; Rot / Anti - A , Rotenone / Antimycin - A ; PCV , packed cell volume .

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NIH-3T3 cells were treated with TGFβ or mock for 48h, and the oxygen consumption rate (OCR) before and after treatment with mitochondrial inhibitors was measured using the Seahorse bioanalyzer. Oligo, Oligomycin; Rot/Anti-A, Rotenone/Antimycin-A; PCV, packed cell volume.


NIH - 3T3 cells were treated with TGFβ or mock for 48h and reduced ( GSH ) and oxidized ( GSSG ) glutathione were measured by LC - MS / MS . Shown is the GSH / GSSG ratio .

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NIH-3T3 cells were treated with TGFβ or mock for 48h and reduced (GSH) and oxidized (GSSG) glutathione were measured by LC-MS/MS. Shown is the GSH/GSSG ratio.


NIH - 3T3 cells were treated with TGFβ for 48h or mock and incubated with CM - H2DCFDA for the last 30 min . ROS were measured by flow cytometry for CM - H2DCFDA . Values are relative to mock - treated cells .

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NIH-3T3 cells were treated with TGFβ for 48h or mock and incubated with CM-H2DCFDA for the last 30 min. ROS were measured by flow cytometry for CM-H2DCFDA. Values are relative to mock-treated cells.


Comparison of the amino acid representation in all human proteins and all collagens . The x - axis denotes individual amino acids shown by single letter code .

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Comparison of the amino acid representation in all human proteins and all collagens. The x-axis denotes individual amino acids shown by single letter code.


Glycine and proline representation in protein acid hydrolysates of untreated NIH - 3T3 cells and their secreted ECM , produced as in Figure 1d . Amino acids were measured by GC - MS .

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Glycine and proline representation in protein acid hydrolysates of untreated NIH-3T3 cells and their secreted ECM, produced as in Figure 1d. Amino acids were measured by GC-MS.


Tracing of [ U - 13C ] L - glutamine ( [ U - 13C ] Gln ) into indicated metabolites . NIH - 3T3 cells were treated with TGFβ ( 2 ng / mL for all experiments ) or mock for 48h , and the medium was replaced ( including all treatments ) with DMEM lacking L - glutamine and supplemented with [ U - 13C ] Gln for the last 8h . Metabolic steady state was reached at this time point ( see kinetic labeling curves in Figure EV3b ) . Metabolites were measured by LC - MS . The left graph shows the total pool size normalized to the packed cell volume ; the right side shows the percent labeling . αKG , alpha - ketoglutarate ; AU , arbitrary units .

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Tracing of [U-13C] L-glutamine ([U-13C] Gln) into indicated metabolites. NIH-3T3 cells were treated with TGFβ (2 ng/mL for all experiments) or mock for 48h, and the medium was replaced (including all treatments) with DMEM lacking L-glutamine and supplemented with [U-13C] Gln for the last 8h. Metabolic steady state was reached at this time point (see kinetic labeling curves in Figure EV3b). Metabolites were measured by LC-MS. The left graph shows the total pool size normalized to the packed cell volume; the right side shows the percent labeling. αKG, alpha-ketoglutarate; AU, arbitrary units.


Glutamine flux into glutamate , αKG and proline in NIH - 3T3 cells treated with TGFβ or mock for 48h , relative to mock - treated cells .

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Glutamine flux into glutamate, αKG and proline in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells.


Abundance of indicated metabolites in NIH - 3T3 cells treated with TGFβ or mock for 48h , relative to mock - treated cells . Metabolites were measured by GC - MS .

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Abundance of indicated metabolites in NIH-3T3 cells treated with TGFβ or mock for 48h, relative to mock-treated cells. Metabolites were measured by GC-MS.


Representative MS2 spectra of the CO1A1 peptide in ECM generated with unlabeled ( [ 12C ] Gln , left ) and fully labeled glutamine ( [ U - 13C ] Gln , right ) . Peaks representing fully labeled ( m + 5 , m + 10 ) proline are highlighted in red . Peptides were measured by LC - MS .

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Representative MS2 spectra of the CO1A1 peptide in ECM generated with unlabeled ([12C] Gln, left) and fully labeled glutamine ([U-13C] Gln, right). Peaks representing fully labeled (m+5, m+10) proline are highlighted in red. Peptides were measured by LC-MS.


Relative abundance of the analytical peptide containing unlabeled ( m + 0 ) or one / two fully labelled prolines ( m + 5 / m + 10 ) from the CO1A1 peptide in ECM derived from NIH - 3T3 cells treated with TGFβ or mock in the presence of fully labelled L - glutamine ( [ U - 13C ] Gln ) . Peptides were measured by LC - MS . The amount of ECM analyzed was normalized to the packed cell volume of cells grown on a parallel plate under identical conditions .

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Relative abundance of the analytical peptide containing unlabeled (m+0) or one/two fully labelled prolines (m+5/m+10) from the CO1A1 peptide in ECM derived from NIH-3T3 cells treated with TGFβ or mock in the presence of fully labelled L-glutamine ([U-13C] Gln). Peptides were measured by LC-MS. The amount of ECM analyzed was normalized to the packed cell volume of cells grown on a parallel plate under identical conditions.


Western blot of NIH - 3T3 cells treated with TGFβ ( 2 ng / mL for all experiments ) or mock for 48h in the presence of 0 . 5 % FBS .

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Western blot of NIH-3T3 cells treated with TGFβ (2 ng/mL for all experiments) or mock for 48h in the presence of 0.5% FBS.