C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal proliferation . ( C ) Suspension of single MB ‐ SCs isolated from Ptch1 + / − mice . MBs were cultured in medium to allow formation of neurospheres . neurospheres were dissociated treated with increasing concentrations of GlaB or DMSO only . After 7 days of treatment , number of secondary neurospheres derived from a known number of single was counted . self ‐ renewal MB ‐ SCs ' capability is expressed as of neurosphere ‐ forming ( left ) . Representative bright field of neurospheres after GlaB treatment are also shown ( right ) .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (C) Suspension of single MB‐SCs isolated from Ptch1+/−mice. MBs were cultured in stem cell medium to allow the formation of primary neurospheres. Primary neurospheres were dissociated and treated with increasing concentrations of GlaB or DMSO only. After 7 days of treatment, the number of secondary neurospheres derived from a known number of single cells was counted. The self‐renewal MB‐SCs' capability is expressed as percentage of neurosphere‐forming cells (left). Representative bright field images of tumor neurospheres after GlaB treatment are also shown (right).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . qRT - PCR show Hh , proliferation stemness target mRNA expression . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin HPRT ) .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. qRT-PCR analysis show Hh, proliferation and stemness target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT).


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal proliferation . ( D , E ) MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated for 48 h with GlaB ( 5 μM ) or DMSO only . Western blot show Hh , proliferation stemness target expression .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (D, E) MB‐SCs isolated from Ptch1+/−miceMBs were treated for 48 h with GlaB (5 μM) or DMSO only. Western blot analysis show Hh, proliferation and stemness target protein expression levels.


C - F GlaB inhibits MB ‐ SCs ' self ‐ renewal proliferation . ( F ) BrdU in MB ‐ SCs treated with GlaB ( 5 μM ) for 24 or 48 h plated on polylysinated chamber slides . Inhibition of proliferation was measured as of BrdU incorporation in comparison with DMSO ‐ treated sample .

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C-F GlaB inhibits MB‐SCs' self‐renewal and proliferation. (F) BrdU assay in MB‐SCs treated with GlaB (5 μM) for 24 or 48 h and plated on polylysinated chamber slides. Inhibition of cell proliferation was measured as percentage of BrdU incorporation in comparison with DMSO‐treated sample.


G TUNEL in MB ‐ SCs . MB ‐ SCs isolated from Ptch1 + / − miceMBs were treated with GlaB ( 5 μM ) compared to DMSO ‐ treated sample . Bottom panel shows a Western blot of caspase ‐ 3 in GlaB ‐ treated versus control .

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G TUNEL assay in MB‐SCs. MB‐SCs isolated from Ptch1+/−miceMBs were treated with GlaB (5 μM) and compared to DMSO‐treated sample. Bottom panel shows a Western blot of caspase‐3 in GlaB‐treated versus control cells.


A Change of volume during GlaB or vehicle treatment period ( 18 days ) .

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A Change of tumor volume during GlaB or vehicle treatment period (18 days).


B Representative flank allografts average volumes . Error bars indicate SD . * P 0 . 05 .

B Representative flank allografts average volumes. Error bars indicate SD. *P 0.05.


C Representative Masson ' s trichromestaining of tumors ; immunohistochemistry of Gli1 , Ki67 TUNEL stainings . Scale bars represent 35 μm for Masson ' s trichromestaining , 25 μm for Gli1 TUNEL , 10 μm for Ki67 stainings .

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C Representative Masson's trichromestaining of tumors; immunohistochemistry of Gli1, Ki67 and TUNEL stainings. Scale bars represent 35 μm for Masson's trichromestaining, 25 μm for Gli1 and TUNEL, and 10 μm for Ki67 stainings.


D of Ki67 TUNEL stainings from immunohistochemistry shown in ( C ) . Error bars indicate SD . * P 0 . 05 .

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D Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (C). Error bars indicate SD. *P 0.05.


E qRT - PCR show Hh proliferation target mRNA expression . For qRT - PCR , results were normalized to endogenous control ( β2 ‐ microglobulin HPRT ) . Data show mean ± SD of ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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E qRT-PCR analysis show Hh and proliferation target mRNA expression levels. For qRT-PCR, results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


F Western blot show Hh proliferation target expression . Data show mean ± SD of ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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F Western blot analysis show Hh and proliferation target protein expression levels. Data show the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


A Representative ( low high magnification ) of HE ( upper panel ) Ki67immunohistochemistry ( lower panel ) of a human Daoy MB ‐ derived orthotopic in NOD / SCIDmicecerebella after i . p . injections of GlaB . Scale bars represent 500 or 200 μm for HE , 150 μm or 50 μm for Ki67 . B Representative average volumes at end of treatment . Error bars indicate SD . * P 0 . 05 . C of Ki67 from immunohistochemistry shown in ( A ) . Error bars indicate SD . * P 0 . 05 .

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A Representative images (low and high magnification) of HE (upper panel) and Ki67immunohistochemistry staining (lower panel) of a human Daoy MB cell‐derived orthotopic tumor in NOD/SCIDmicecerebella after i.p. injections of GlaB. Scale bars represent 500 or 200 μm for HE staining, and 150 μm or 50 μm for Ki67 staining.B Representative tumor average volumes at the end of treatment. Error bars indicate SD. *P 0.05.C Quantification of Ki67 staining from immunohistochemistry shown in (A). Error bars indicate SD. *P 0.05.


A , B ASZ001 BCC were treated with GlaB ( 5 μM ) or DMSO only . After indicated times , a trypan blue was performed to determine ( A ) growth rate ( B ) of . Data show mean ± SD of three independent experiments . * P 0 . 05 versus DMSO .

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A, B ASZ001 BCC cells were treated with GlaB (5 μM) or DMSO only. After the indicated times, a trypan blue count was performed to determine (A) the growth rate and (B) the percentage of cell death. Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.


C Gli1 mRNA expression were determined by qRT - PCR after treatment of ASZ001 BCC with GlaB or DMSO only for indicated times . Results were normalized to endogenous control ( β2 ‐ microglobulin HPRT ) . Data show mean ± SD of three independent experiments . * P 0 . 05 versus DMSO .

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C Gli1 mRNA expression levels were determined by qRT-PCR after treatment of ASZ001 BCC cells with GlaB or DMSO only for the indicated times. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Data show the mean ± SD of three independent experiments. *P 0.05 versus DMSO.


D - H GlaB inhibits Gli1 ‐ dependent BCC growth in ASZ001 BCC allografts in vivo . ( D ) Change of volume during GlaB or vehicle treatment period ( 18 days ) .

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (D) Change of tumor volume during GlaB or vehicle treatment period (18 days).


D - H GlaB inhibits Gli1 ‐ dependent BCC growth in ASZ001 BCC allografts in vivo . ( E ) Representative flank allografts average volumes ( upper panel ) .

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (E) Representative flank allografts average volumes (upper panel).


D - H GlaB inhibits Gli1 ‐ dependent BCC growth in ASZ001 BCC allografts in vivo . ( F ) HE immunohistochemistry of Gli1 , Ki67 TUNEL of allograft samples . Scale bars represent 50 μm for HE , 25 μm for Gli1 , 25 μm for Ki67 30 μm for TUNEL stainings . ( G ) of Ki67 TUNEL stainings from immunohistochemistry shown in ( F )

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (F) HE and immunohistochemistry staining of Gli1, Ki67 and TUNEL of allograft tumor samples. Scale bars represent 50 μm for HE, 25 μm for Gli1, 25 μm for Ki67 and 30 μm for TUNEL stainings. (G) Quantification of Ki67 and TUNEL stainings from immunohistochemistry shown in (F)


D - H GlaB inhibits Gli1 ‐ dependent BCC growth in ASZ001 BCC allografts in vivo . ( H ) qRT - PCR of Hh proliferation target mRNA expression . Results were normalized to endogenous control ( β2 ‐ microglobulin HPRT ) . Shown is mean ± SD of ( n = 6 ) for each treatment . * P 0 . 05 versus Ctr .

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D-H GlaB inhibits Gli1‐dependent BCC tumor growth in ASZ001 BCC allografts in vivo. (H) qRT-PCR of Hh and proliferation target mRNA expression levels. Results were normalized to endogenous control (β2‐microglobulin and HPRT). Shown is the mean ± SD of tumor (n = 6) for each treatment. *P 0.05 versus Ctr.


Adult mouse back skin sections immunostained for LYVE1 ( red ) , αSMA ( green ) counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . LV , lymphatic vessels ; apm , arrector pili muscle .

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Adult mouse back skin sections immunostained for LYVE1 (red), αSMA (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. LV, lymphatic vessels; apm, arrector pili muscle.


Back skin sections of P55 mouse skin immunostained for LYVE1 ( red ) , Tenascin - C ( green ) counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . TNC , Tenascin - C .

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Back skin sections of P55 mouse skin immunostained for LYVE1 (red), Tenascin-C (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. TNC, Tenascin-C.


Adult mouse back skin sections from different postnatal ( P ) days immunostained for LYVE1 ( red ) , Lhx2 ( green ) counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . epi , epidermis ; der , dermis .

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Adult mouse back skin sections from different postnatal (P) days immunostained for LYVE1 (red), Lhx2 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.


Histogram of of LV to HFSC positive for Lhx2 . ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Histogram of the quantification of the LV distance to HFSC positive for Lhx2. ). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Back skin sections of P49 mouse skin immunostained for LYVE1 ( red ) , CD34 ( green ) counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 100 μm . epi , epidermis ; der , dermis . arrowheads denote proximity of lymphatic capillaries to HFSC .

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Back skin sections of P49 mouse skin immunostained for LYVE1 (red), CD34 (green) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis. White arrowheads denote the proximity of lymphatic capillaries to HFSC.


Histogram of of LV to HFSC positive for CD34 . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . C , Catagen ; Te , early Telogen ; Tm , mid Telogen ; Tl , late Telogen ; A , Anagen .

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Histogram of the quantification of the LV distance to HFSC positive for CD34. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. C, Catagen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen; A, Anagen.


Back skin sections of K15CrePR + / T ; Wls ∆ / ∆ Control K15CrePR + / + ; Wlsflox / flox mice treated with mifepristone from P7 during 12 ( P90 ) , immunostained for LYVE1 ( red ) , counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 100 μm . epi , epidermis ; der , dermis .

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Back skin sections of K15CrePR+/T; Wls∆/∆ and Control K15CrePR+/+; Wlsflox/flox mice treated with mifepristone from P7 during 12 weeks (P90), immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.


Histogram of of LV angle relative to epidermis . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Histogram of the quantification of the LV angle relative to the epidermis. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Distribution of LV HF at mouse stage E15 . 5 ( A ) , E16 . 5 ( B ) E17 . 5 ( C ) . Maximum projection of whole mount immunofluorescence analyses using LYVE1 ( red ) as lymphatic endothelial marker counterstained with DAPI ( blue ) . Red arrowheads denote HF placodes . n = 3 - 4 . Scale bar , 50 μm .

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Distribution of LV and HF at the mouse embryo stage E15.5 (A), E16.5 (B) and E17.5 (C). Maximum projection images of whole mount immunofluorescence analyses using LYVE1 (red) as lymphatic endothelial marker and counterstained with DAPI (blue). Red arrowheads denote HF placodes. n= 3 - 4 embryos. Scale bar, 50 μm.


LV associated with HF further organize associating HF triads , which connect with other HF triads across skin . P70 , 70 d old mice . n = 3 - 4 mice . arrowheads denote capillaries stemming from HF triads . Scale bar , 100 μm .

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LV associated with individual HF further organize associating HF triads, which connect with other HF triads across the skin. P70, 70 d old mice. n= 3 - 4 mice. White arrowheads denote capillaries stemming from HF triads. Scale bar, 100 μm.


3D reconstructions of whole skin mounts showing a triad of HF connected by LV . n = 3 - 4 mice . Scale bar , 50 μm . arrowhead denotes an extending lymphatic capillary from a HF triad .

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3D reconstructions of whole skin mounts showing a triad of HF connected by LV. n= 3 - 4 mice. Scale bar, 50 μm. White arrowhead denotes an extending lymphatic capillary from a HF triad.


of intravital microscopy analyses showing aligned HF rows in back skin interconnected by LV in Prox1CreERT2 ; Rosa - LSL - eYPF mice . Dotted boxes denote HF triads ; arrowheads denote capillaries stemming from HF triads interconnecting to other HF triads in adjacent rows . n = 3 - 4 mice . Scale bar , 50 μm .

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Images of intravital microscopy analyses showing aligned HF rows in the back skin interconnected by LV in the Prox1CreERT2; Rosa-LSL-eYPF mice. Dotted boxes denote HF triads; White arrowheads denote capillaries stemming from HF triads interconnecting to other HF triads in adjacent rows. n= 3 - 4 mice. Scale bar, 50 μm.


Adult back skin sections from different postnatal ( P ) days immunostained for LYVE1 ( red ) counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 10 μm . arrowheads denote LV protrusion fenestrated areas . LV , lymphatic vessels ; HF , hair follicle .

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Adult back skin sections from different postnatal (P) days immunostained for LYVE1 (red) and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 10 μm. White arrowheads denote LV membrane protrusion and fenestrated areas. LV, lymphatic vessels; HF, hair follicle.


Histogram of caliber ( μm ) of LV at different postnatal days . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . A , Anagen ; C , Catagen ; T , Telogen ; Te , early Telogen ; Tm , mid Telogen ; Tl , late Telogen .

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Histogram of the caliber (μm) of LV at different postnatal days. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. A, Anagen; C, Catagen; T, Telogen; Te, early Telogen; Tm, mid Telogen; Tl, late Telogen.


H & E of back skin sections from mice treated intradermally with Control or Clodronate liposomes . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 200 μm . epi , epidermis ; der , dermis .

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H&E staining of back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. epi, epidermis; der, dermis.


LYVE1 immunofluorescence ( red ) counterstained with DAPI ( blue ) ( D ) in back skin sections from mice treated intradermally with Control or Clodronate liposomes . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 100 μm . epi , epidermis ; der , dermis .

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LYVE1 immunofluorescence (red) counterstained with DAPI (blue) (D) in back skin sections from mice treated intradermally with Control or Clodronate liposomes. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 100 μm. epi, epidermis; der, dermis.


Histogram of caliber ( μm ) of LV ( E ) in back skin sections from mice treated intradermally with Control or Clodronate liposomes .

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Histogram of the caliber (μm) of LV (E) in back skin sections from mice treated intradermally with Control or Clodronate liposomes.


Back skin sections of Controls K14ΔNβ - cateninER + / T mice immunostained for LYVE1 ( red ) , counterstained with DAPI ( blue ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 50 μm . epi , epidermis ; der , dermis .

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Back skin sections of Controls and K14ΔNβ-cateninER+/T mice immunostained for LYVE1 (red), and counterstained with DAPI (blue). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 50 μm. epi, epidermis; der, dermis.


Histogram of LV caliber in skin sections from Controls K14ΔNβ - cateninER + / T mice . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Histogram of the LV caliber in skin sections from Controls and K14ΔNβ-cateninER+/T mice. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Histogram of of HF in Telogen Anagen present in skin of control mice or mice treated with Cilostazol . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Histogram of the percentage of HF in Telogen and Anagen present in the skin of control mice or mice treated with Cilostazol. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Gene set enrichment plots ( GSEA ) of LV using public annotations showing enrichment of selected gene signatures involved in different pathways in late Telogen ( P69 , Tl ) compared with mid Telogen ( P55 , Tm ) . NES : normalized enrichment score . false discovery rate ( FDR ; q - value ) is indicated .

Gene set enrichment plots (GSEA) of LV using public annotations showing the enrichment of selected gene signatures involved in different pathways in late Telogen (P69, Tl) compared with mid Telogen (P55, Tm). NES: normalized enrichment score. The false discovery rate (FDR; q-value) is indicated.


Heat map diagram of DEGs in LV during late Telogen compared to mid Telogen , relative to biological processes , analyzed using Enrichr platform ( Chen et al . , 2013 ; Kuleshov et al . , 2016 ) . Data shown represent log2 ( FPKM + 1 ) .

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Heat map diagram of DEGs in LV during late Telogen compared to mid Telogen, relative to biological processes, analyzed using the Enrichr platform (Chen et al., 2013; Kuleshov et al., 2016). Data shown represent the log2 (FPKM+1).


Histograms of ISH analyses by RNAscope to quantify level of expression of ITGA5 ( D ) , DCN ( E ) , PKD1 ( F ) PLXND1 ( G ) , assessed by number of mRNA spots present in LYVE1 + in vicinity of HF in P55 P70 mouse back skin . n = average of 30 LYVE1 + / sample , n = 3 mice .

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Histograms of RNA ISH analyses by RNAscope to quantify the level of expression of ITGA5 (D), DCN (E), PKD1 (F) and PLXND1 (G), assessed by the number of mRNA spots present in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= average of 30 LYVE1+ cells / sample, n= 3 mice.


Histograms represent mean for Jup ( H ) Emilin1 ( I ) in LYVE1 + in vicinity of HF in P55 P70 mouse back skin . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Histograms represent the mean fluorescence intensity for Jup (H) and Emilin1 (I) in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


H & E ( A ) histogram of HF ( B ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice injected with vehicles ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with CSA vehicle ( CSA ) ; Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI treated with tamoxifen , CSA intradermal Diphtheria Toxin ( CSA + DT ) starting from early Telogen ( P49 ) analyzed at end of treatments ( P58 ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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H&E staining (A) and histogram of the HF length (B) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal Diphtheria Toxin (CSA+DT) starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


Ki67 immunostaining ( C ) histogram of number of Ki67 + per HF ( D ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice injected with vehicles ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with CSA vehicle ; Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI treated with tamoxifen , CSA intradermal DT , starting from early Telogen ( P49 ) analyzed at end of treatments ( P58 ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Ki67 immunostaining (C) and histogram of the number of Ki67+ cells per HF (D) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle; and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


LYVE1 immunostaining ( E ) histogram of LV caliber ( F ) analyzed in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice injected with vehicles ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with CSA vehicle ( CSA ) ; Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with tamoxifen , CSA intradermal DT , starting from early Telogen ( P49 ) analyzed at end of treatments ( P58 ) . Scale bar , 10 μm . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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LYVE1 immunostaining (E) and histogram of the LV caliber (F) analyzed in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice injected with vehicles (Control), Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with CSA and vehicle (CSA); and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen, CSA and intradermal DT, starting from early Telogen (P49) and analyzed at the end of the treatments (P58). Scale bar, 10 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


H & E ( G ) histogram of HF ( H ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with tamoxifen intradermal DT , starting from Anagen ( P30 ) analyzed at end of treatments ( P37 ) . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice . Scale bar , 200 μm . Data represent mean value ± .

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H&E staining (G) and histogram of the HF length (H) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). n= 3 - 4 skin samples per mouse, n= 3 - 4 mice. Scale bar, 200 μm. Data represent the mean value ± SEM.


caspase 3 immunostaining ( I ) histogram of number of caspase 3 + per HF ( J ) in adult back skin sections from Prox1CreERT2 + / + ; LSL - ROSA26 - iDTRKI / KI mice ( Control ) , Prox1CreERT2 + / T ; LSL - ROSA26 - iDTRKI / KI mice treated with tamoxifen intradermal DT , starting from Anagen ( P30 ) analyzed at end of treatments ( P37 ) . Scale bar , 50 μm . n = 3 - 4 skin samples per mouse , n = 3 - 4 mice .

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Cleaved caspase 3 immunostaining (I) and histogram of the number of cleaved caspase 3+ cells per HF (J) in adult back skin sections from Prox1CreERT2+/+; LSL-ROSA26-iDTRKI/KI mice (Control), and Prox1CreERT2+/T; LSL-ROSA26-iDTRKI/KI mice treated with tamoxifen and intradermal DT, starting from Anagen (P30) and analyzed at the end of the treatments (P37). Scale bar, 50 μm. n= 3 - 4 skin samples per mouse, n= 3 - 4 mice.


A , SW620 HCT116 were treated with indicated concentrations of SB505124 ( SB ) or LGK974 ( LGK ) for 16 h . Total extracts were analyzed by WB . was analyzed by densitometry of three different experiments represented B , SW620 HCT116 expressing a shCtl or shRor2 vectors were analyzed by WB . of WBs from three biological replicates are presented in right panel . Values correspond to fold variation in shRor2 versus shCtl ( mean ± SD ) .

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A, SW620 and HCT116 cells were treated with the indicated concentrations of SB505124 (SB) or LGK974 (LGK) for 16 h. Total cell extracts were analyzed by WB. Protein quantification was analyzed by densitometry of three different experiments and represented B, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs from three biological replicates are presented in the right panel. Values correspond to fold variation in shRor2 versus shCtl cells (mean ± SD).


C , was isolated from shCtl or shRor2 SW620 HCT116 expression of indicated genes was assessed by qRT - PCR . Values correspond to fold variation in shRor2 versus shCtl . Results are mean ± SD of at least three biological replicates performed in triplicate .

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C, RNA was isolated from shCtl or shRor2 SW620 and HCT116 cells and the expression of the indicated genes was assessed by qRT-PCR. Values correspond to fold variation in shRor2 versus shCtl cells. Results are the mean ± SD of at least three biological replicates performed in triplicate.


D , SW620 HCT116 expressing a shCtl or shRor2 vectors were analyzed by WB . of WBs are presented in Appendix Fig S3D as in B .

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D, SW620 and HCT116 cells expressing a shCtl or shRor2 vectors were analyzed by WB. Quantification of the WBs are presented in Appendix Fig S3D as in B.


E , HCT116 or SW620 were treated with conditioned medium from control or Wnt5a - producing for 16 h expression of indicated genes assessed by qRT - PCR ( E ) ( mean ± SD of three biological replicates performed in triplicate in E ) .

E, HCT116 or SW620 cells were treated with conditioned medium from control or Wnt5a-producing cells for 16 h and expression of the indicated genes assessed by qRT-PCR (E) (mean ± SD of three biological replicates performed in triplicate in E).


G , WB of HT29 M6 SW480 overexpressing Snail1 . is presented in Appendix Fig S5I .

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G, WB analysis of HT29 M6 and SW480 overexpressing Snail1. Quantification is presented in Appendix Fig S5I.


I , SW620 HCT116 were transfected with control or Snail1 siRNA analyzed by qRT - PCR ( I , mean ± SD of three biological replicates performed in triplicate ) .

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I, SW620 and HCT116 cells were transfected with control or Snail1 siRNA and analyzed by qRT-PCR (I, mean ± SD of three biological replicates performed in triplicate).


A , Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 HCT116 . is presented in right panel ( mean ± SD or three biological replicates ) .

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A, Smad2 phosphorylation was determined in extracts from shCtl or shRor2 SW620 and HCT116 cells. The quantification is presented in the right panel (mean ± SD or three biological replicates).


D , MSC were treated with conditioned media from shCtl or shRor2 SW620 , D ) for indicated times . TGFβ receptor inhibitor SB505124 ( SB , 5 μM ) was added when indicated in D . were lysed analyzed by WB . of from D is represented at lower panel ( mean ± SD of three biological replicates )

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D, MSC were treated with the conditioned media from shCtl or shRor2 SW620 , D) for the indicated times. TGFβ receptor inhibitor SB505124 (SB, 5 μM) was added when indicated in D. Cells were lysed and proteins analyzed by WB. Quantification of the protein levels from D is represented at the lower panel (mean ± SD of three biological replicates)


E , GFP - labelled MSC were seeded in Matrigel - coated transwells with SW620 HCT116 , supplemented with SB when indicated . After 48 h non - invading were removed from upper surface of present at lower surface were fixed . GFP - labelled were counted in five different fields per filter by ImageJ software represented ( bottom ) . A representative picture of SW620 in each condition is shown ( top ) . bar corresponds to 500 μm . figure shows mean ± SD of three experiments .

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E, GFP-labelled MSC were seeded in Matrigel-coated transwells with SW620 and HCT116 cells, and supplemented with SB when indicated. After 48 h non-invading cells were removed from the upper surface of the membrane and cells present at the lower surface were fixed. GFP-labelled cells were counted in five different fields per filter by ImageJ software and represented (bottom). A representative picture of SW620 cells in each condition is shown (top). The bar corresponds to 500 μm. The figure shows the mean ± SD of three experiments.


C , Representative of effects of Ror2 depletion on HCT116 soft - agar colony formation ( 21 days ) ( left ) . Representative of SW620 are shown in Appendix Fig S7B . Results show mean ± SD of number of colonies in triplicate samples of three independent experiments ( right ) .

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C, Representative micrographs of the effects of Ror2 depletion on HCT116 cells soft-agar colony formation (21 days) (left). Representative micrographs of SW620 cells are shown in Appendix Fig S7B. Results show the mean ± SD of the number of colonies in triplicate samples of three independent experiments (right).


were seeded for - sphere formation in organoid complete medium . In E , SW620 were supplemented with LGK ( 10 μM ) , starting day of seeding . After four days , wells were stained ( top ) number of colonies counted represented ( bottom ) ( mean ± SD of three biological replicates ) . A more detailed micrograph of colonies is shown in Appendix Fig S7D .

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Cells were seeded for tumor-sphere formation in tumor organoid complete medium. In E, SW620 cells were supplemented with LGK (10 μM), starting the day of seeding. After four days, the wells were stained (top) and the number of colonies counted and represented (bottom) (mean ± SD of three biological replicates). A more detailed micrograph of the colonies is shown in Appendix Fig S7D.


F , was isolated from SW620 control , stably - depleted of Ror2 , treated with LGK974 for 16 h or transfected with siSnail1 expression of gene markers LGR5 , ASCL2 , SMOC2 differentiation marker KRT20 was assessed by qRT - PCR . figure shows mean ± SD of at least three biological replicates ( performed in triplicate ) represented with respect to corresponding control ( dashed ) .

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F, RNA was isolated from SW620 control, stably-depleted of Ror2, treated with LGK974 for 16 h or transfected with siSnail1 and the expression of the stem cell gene markers LGR5, ASCL2, SMOC2 and the differentiation marker KRT20 was assessed by qRT-PCR. The figure shows the mean ± SD of at least three biological replicates (performed in triplicate) represented with respect to the corresponding control (dashed line).


B , C , Representative of Fibronectin Vimentin in consecutive sections from paraffin - embedded tumors obtained when tumors were initially detected ( days 18 or 22 ) . with an anti - human antibody is also enclosed . scale bar corresponds to 10 μm ( B ) . Fibronectin Vimentin were quantified as described in Methods ( six tumors , three areas per ) represented in C ( mean ± SD ) .

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B, C, Representative images of Fibronectin and Vimentin staining in consecutive sections from paraffin-embedded tumors obtained when tumors were initially detected (days 18 or 22). Staining with an anti-human antibody is also enclosed. The scale bar corresponds to 10 μm (B). Fibronectin and Vimentin staining were quantified as described in Methods (six tumors, three areas per tumor) and represented in C (mean ± SD).


D , Representative of invading front of tumors . scale bar corresponds to 4 μM . E , of tumors showing muscle invasion .

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D, Representative images of the invading front of the tumors. The scale bar corresponds to 4 μM. E, Percentage of tumors showing muscle invasion.


F , G , In vivo growth of MTO subcutaneously injected in NOD / SCID mice ( F ) ( 5x105 / flank ; eight tumors per condition ) treated with WntC59 ( 10 mg / kg / day in drinking water ) or vehicle . of NOD / SCID mice at day 26 is presented in G . In F G , mean ± SD of eight tumors is shown .

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F, G, In vivo tumor growth of MTO cells subcutaneously injected in NOD/SCID mice (F) (5x105 cells/flank; eight tumors per condition) and treated with WntC59 (10 mg/kg/day in the drinking water) or vehicle. Tumor weight of NOD/SCID mice at day 26 is presented in G. In F and G, the mean ± SD of the eight tumors is shown.


I , J , WB ( I ) ( J ) of MTO infected with shRor2 or shCtl . of WB is also presented ( I , right ) . Results are mean ± SD of three biological replicates ( performed in triplicated in J ) .

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I, J, WB (I) and RNA analysis (J) of MTO cells infected with shRor2 or shCtl. Quantification of the WB is also presented (I, right). Results are the mean ± SD of three biological replicates (performed in triplicated in J).


N , O , C57BL6 mice were injected in spleen with MTO treated since day 1 with WntC59 or vehicle . In N , kinetics of in vivo luminescence ( mean ± SD of values in five mice ) ; in O , of mice showing in vivo luminescence .

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N, O, C57BL6 mice were injected in the spleen with MTO cells and treated since day 1 with WntC59 or vehicle. In N, kinetics of in vivo luminescence (mean ± SD of the values in five mice); in O, the percentage of mice showing in vivo luminescence.


A - C , Viability was assayed by MTT in indicated treated with LGK when specified supplemented with cisplatin for 24 h . In B , cisplatin resistance was assessed in shRor2 transfected with a Snail1 expression plasmid or corresponding empty plasmid . figure shows mean ± SD of results of three experiments .

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A-C, Viability was assayed by MTT in the indicated cells treated with LGK when specified and supplemented with cisplatin for 24 h. In B, cisplatin resistance was assessed in shRor2 cells transfected with a Snail1 expression plasmid or the corresponding empty plasmid. The figure shows the mean ± SD of the results of three experiments.


D , were treated with cisplatin for indicated times . Total extracts were lysed analyzed by WB .

D, Cells were treated with cisplatin for the indicated times. Total extracts were lysed and analyzed by WB.


I , MTO viability was determined as above ( mean ± SD of results of three - four experiments ) .

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I, MTO cell viability was determined as above (mean ± SD of the results of three-four experiments).


A representative immunoblots of lamin B1 in striatum ( 8w , 12w 20w ; N = 6 for both genotypes ; 30w ; N = 4 5 for WT R6 / 1 mice , respectively ) , hippocampus ( 8w ; N = 8 6 for WT R6 / 1 mice , respectively ; 12w ; N = 7 6 for WT R6 / 1 mice , respectively ; 20w N = 5 6 for WT R6 / 1 mice , respectively ; 30w ; N = 8 5 for WT R6 / 1 mice , respectively ) cortex ( 8w ; N = 5 4 for WT R6 / 1 mice , respectively ; 12w 20w ; N = 6 for both genotypes ; 30w ; N = 6 5 for WT R6 / 1 mice , respectively ) . Data information : Data is expressed as a of controls . Each point corresponds to value from an sample . Bars represent mean ± S . E . M . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 as compared with WT mice ( two - unpaired Student ' s t ) . Tubulin was used as loading control . Exact P values are reported in Appendix Table S3 .

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A Quantification and representative immunoblots of lamin B1 in the striatum (8w, 12w and 20w; N = 6 for both genotypes; 30w; N = 4 and 5 for WT and R6/1 mice, respectively), hippocampus (8w; N = 8 and 6 for WT and R6/1 mice, respectively; 12w; N = 7 and 6 for WT and R6/1 mice, respectively; 20w N = 5 and 6 for WT and R6/1 mice, respectively; 30w; N = 8 and 5 for WT and R6/1 mice, respectively) and cortex (8w; N = 5 and 4 for WT and R6/1 mice, respectively; 12w and 20w; N = 6 for both genotypes; 30w; N = 6 and 5 for WT and R6/1 mice, respectively). Data information: Data is expressed as a percentage of controls. Each point corresponds to the value from an individual sample. Bars represent the mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with WT mice (two-tailed unpaired Student's t test). Tubulin was used as loading control. Exact P values are reported in Appendix Table S3.


B representative immunoblots of lamin B2 in striatum ( 8w , 12w 20w ; N = 6 for both genotypes , 30w ; N = 5 for both genotypes ) , hippocampus ( 8w ; N = 8 6 for WT R6 / 1 mice , respectively ; 12w ; N = 6 7 for WT R6 / 1 mice , respectively ; 20w ; N = 5 for both genotypes ; 30w ; N = 8 5 for WT R6 / 1 mice , respectively ) cortex ( 8w 12w ; N = 6 for both genotypes ; 20w ; N = 5 for both genotypes ; 30w ; N = 8 5 for WT R6 / 1 mice , respectively ) . Data information : Data is expressed as a of controls . Each point corresponds to value from an sample . Bars represent mean ± S . E . M . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 as compared with WT mice ( two - unpaired Student ' s t ) . Tubulin was used as loading control . Exact P values are reported in Appendix Table S3 .

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B Quantification and representative immunoblots of lamin B2 in the striatum (8w, 12w and 20w; N = 6 for both genotypes, 30w; N = 5 for both genotypes), hippocampus (8w; N = 8 and 6 for WT and R6/1 mice, respectively; 12w; N = 6 and 7 for WT and R6/1 mice, respectively; 20w; N = 5 for both genotypes; 30w; N = 8 and 5 for WT and R6/1 mice, respectively) and cortex (8w and 12w; N = 6 for both genotypes; 20w; N = 5 for both genotypes; 30w; N = 8 and 5 for WT and R6/1 mice, respectively). Data information: Data is expressed as a percentage of controls. Each point corresponds to the value from an individual sample. Bars represent the mean ± S.E.M. *P < 0.05, **P < 0.01 and ***P < 0.001 as compared with WT mice (two-tailed unpaired Student's t test). Tubulin was used as loading control. Exact P values are reported in Appendix Table S3.


A Mouse brain tissue was processed for immunohistochemistry by combining anti - lamin B1 ( red ) anti - NeuN ( green ) antibodies . Representative ( maximal Z - projections ) showing distribution of lamin B1 in striatum of 30 - week - old wild - type ( WT ) R6 / 1 mice . Yellow arrowheads show co - localization between lamin B1 NeuN , arrowheads show NeuN negative lamin B1 positive nuclei . Scale bar 50 μm 25 μm for low high magnification , respectively .

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A Mouse brain tissue was processed for immunohistochemistry by combining anti-lamin B1 (red) and anti-NeuN (green) antibodies. Representative images (maximal Z-projections) showing the distribution of lamin B1 in the striatum of 30-week-old wild-type (WT) and R6/1 mice. Yellow arrowheads show co-localization between lamin B1 and NeuN, and white arrowheads show NeuN negative lamin B1 positive nuclei. Scale bar 50 μm and 25 μm for low and high magnification, respectively.


B Lamin B1 distribution in human putamen was analysed by immunohistochemistry . Antibody against lamin B1 ( red ) was combined with DAPI - Fluoromount G ( blue ) to label nuclei . Representative show distribution of lamin B1 in putamen of non - affected individuals ( CTL ) HD at different stages of disease ( VS II - IV : Vonsattel grades ) . Yellow arrowheads indicate MSNs glial , respectively . Scale bar 50 μm 20 μm for low high magnification , respectively .

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B Lamin B1 distribution in human putamen was analysed by immunohistochemistry. Antibody against lamin B1 (red) was combined with DAPI-Fluoromount G (blue) to label nuclei. Representative images show the distribution of lamin B1 in the putamen of non-affected individuals (CTL) and HD patients at different stages of the disease (VS II-IV: Vonsattel grades). Yellow and white arrowheads indicate MSNs and glial cells, respectively. Scale bar 50 μm and 20 μm for low and high magnification, respectively.


C distribution of lamin B1 in putamen of HD was analysed by immunohistochemistry . Anti - lamin B1 antibody ( red ) was combined with anti - GFAP antibody ( green ) nuclei were labelled with DAPI - Fluoromount G ( blue ) . Representative show distribution of lamin B1 at Vonsattel grade III . Yellow arrowheads indicate MSNs arrowheads indicate GFAP - positive . Scale bar 10 μm .

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C The distribution of lamin B1 in the putamen of HD patients was analysed by immunohistochemistry. Anti-lamin B1 antibody (red) was combined with anti-GFAP antibody (green) and nuclei were labelled with DAPI-Fluoromount G (blue). Representative images show the distribution of lamin B1 at Vonsattel grade III. Yellow arrowheads indicate MSNs and white arrowheads indicate GFAP-positive cells. Scale bar 10 μm.


A Representative showing distribution of lamin B1 . On left , showing maximal Z - projection . Scale bar 150 μm . Small correspond to CA1 DG magnified showing independent DAPI , GFAP Lamin B1 channels , merge , of representative confocal Z - stack . Scale bar 50 μm .

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A Representative images showing the distribution of lamin B1. On the left, images showing maximal Z-projection. Scale bar 150 μm. Small images correspond to CA1 and DG magnified images showing independent DAPI, GFAP and Lamin B1 channels, and merge, of representative confocal Z-stack images. Scale bar 50 μm.


B Left , representative showing distribution of lamin B1 in nuclei of CA - 1 hippocampal neurons from WT R6 / 1 mice along different Z axis planes ( 1 - 6 ) . On right , a 3D reconstruction of Z - stack confocal generated using ImageJ . Scale bar 20 μm .

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B Left, representative images showing the distribution of lamin B1 in the nuclei of CA-1 hippocampal neurons from WT and R6/1 mice along different Z axis planes (1-6). On the right, a 3D reconstruction of the Z-stack confocal images generated using ImageJ. Scale bar 20 μm.


A Graphs show of different parameters in mice striatal MSNs nuclei ( Ctip2 + / NeuN + ) . N = 4 for both genotypes . An average of 5 , 000 nuclei was analysed for each sample . Representative are shown . Scale bar 7 μm .

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A Graphs show the quantification of different parameters in mice striatal MSNs nuclei (Ctip2+/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm.


B Graphs show of different parameters in mice striatal interneurons ( Ctip2 - / NeuN + ) . N = 4 for both genotypes . An average of 5 , 000 nuclei was analysed for each sample . Representative are shown . Scale bar 7 μm .

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B Graphs show the quantification of different parameters in mice striatal interneurons (Ctip2-/NeuN+). N = 4 for both genotypes. An average of 5,000 nuclei was analysed for each sample. Representative images are shown. Scale bar 7 μm.


C Graphs show of lamin B1 circularity in 30 - week - old R6 / 1 mouse striatal neurons nuclei with ( mHtt + ) or without ( mHtt - ) inclusions . N = 6 . EM48 antibody was used to label mHtt inclusions . Representative are shown . Scale bar 7 μm .

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C Graphs show the quantification of lamin B1 intensity and circularity in 30-week-old R6/1 mouse striatal neurons nuclei with (mHtt +) or without (mHtt -) nuclear inclusions. N = 6. EM48 antibody was used to label mHtt inclusions. Representative images are shown. Scale bar 7 μm.


D Graphs showing lamin B1 circularity in MSNs nuclei from putamen of HD at different stages of disease ( VS : Vonsattel grade ) corresponding controls ( CTL : non - affected individuals ) . N = 6 for CTL , N = 3 for VS I - II N = 4 for VS III - IV . An average of 50 nuclei was examined for each sample . Representative ( maximal Z - projections ) are shown . Scale bar 7μm .

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D Graphs showing lamin B1 intensity and circularity in MSNs nuclei from the putamen of HD patients at different stages of the disease (VS: Vonsattel grade) and corresponding controls (CTL: non-affected individuals). N = 6 for CTL, N = 3 for VS I-II and N = 4 for VS III-IV. An average of 50 nuclei was examined for each sample. Representative images (maximal Z-projections) are shown. Scale bar 7μm.


E Graphs show of different parameters in hippocampal CA1 ( Ctip2 + / Prox1 - ) DG ( Ctip2 + / Prox1 + ) neurons nuclei . N = 4 for each genotype . Representative are shown . Scale bar 7 μm .

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E Graphs show the quantification of different parameters in hippocampal CA1 ( Ctip2+/Prox1-) and DG (Ctip2+/Prox1+) neurons nuclei. N = 4 for each genotype. Representative images are shown. Scale bar 7 μm.


A Graphs show of lamin B1 circularity . N = 3 . An average of 12 nuclei was examined in each culture . Data are expressed as a of controls . Bars represent mean ± S . E . M . * P < 0 . 05 * * P < 0 . 01 as compared to mApple - C1 control neurons ( two - unpaired Student ' s t ) . Exact P values are reported in Appendix Table S3 . B Representative showing striatal neurons transfected with mApple - C1 or with mApple - LB1 , both in red . Neuronal nuclei were stained with DAPI - Fluoromount G ( blue ) . Lamin B1 is shown in . Scale bar 10 μm .

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

A Graphs show the quantification of lamin B1 intensity and circularity. N = 3. An average of 12 nuclei was examined in each culture. Data are expressed as a percentage of controls. Bars represent the mean ± S.E.M. *P < 0.05 and **P < 0.01 as compared to mApple-C1 control neurons (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3. B Representative images showing primary striatal neurons transfected with mApple-C1 or with mApple-LB1, both in red. Neuronal nuclei were stained with DAPI-Fluoromount G (blue). Lamin B1 is shown in white. Scale bar 10 μm.


A Graphs show of lamin B1 circularity . N = 3 . An average of 12 nuclei was examined in each culture . Data are expressed as a of controls . Bars represent mean ± S . E . M . * P < 0 . 05 * * P < 0 . 01 as compared to mApple - C1 control neurons ( two - unpaired Student ' s t ) . Exact P values are reported in Appendix Table S3 .

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

A Graphs show the quantification of lamin B1 intensity and circularity. N = 3. An average of 12 nuclei was examined in each culture. Data are expressed as a percentage of controls. Bars represent the mean ± S.E.M. *P < 0.05 and **P < 0.01 as compared to mApple-C1 control neurons (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.


B - D permeability in 30 - week - old wild - type ( WT ) R6 / 1 mice striatal MSNs , CA1 DG neurons nuclei , respectively . ( B C ) N = 4 for each condition ; ( D ) N = 5 for each condition . An average of 25 nuclei were analysed for each sample

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B-D Nuclear permeability in 30-week-old wild-type (WT) and R6/1 mice striatal MSNs, CA1 and DG neurons nuclei, respectively.(B and C) N = 4 for each condition; (D) N = 5 for each condition. An average of 25 nuclei were analysed for each sample


B - D permeability in 30 - week - old wild - type ( WT ) R6 / 1 mice striatal MSNs , CA1 DG neurons nuclei , respectively . ( B C ) N = 4 for each condition ; ( D ) N = 5 for each condition . An average of 25 nuclei were analysed for each

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A UCSC genome browser capture of lamin B1 ChIP - seq signal ( log ( LB1 / Input ) ) LADs discovered in wild - type ( WT LAD ) R6 / 1 ( R6 / 1 LADs ) mice in combination with NPC lamin B1 DamID ( top ) . Black arrows highlight differences in identified LADs between WT R6 / 1 mice ChIP - seq data . Boxplots of LADs ( log10 ( LADs + 1 ) ) of LADs score ( log10 ( LADs score + 1 ) ) obtained by EDD ( bottom ) from WT ( N = 3 ) R6 / 1 ( N = 3 ) lamin B1 ChIP - seq data . Hipp , hippocampus . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median .

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A UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) and LADs discovered in wild-type (WT LAD) and R6/1 (R6/1 LADs) mice in combination with NPC lamin B1 DamID (top). Black arrows highlight differences in identified LADs between WT and R6/1 mice ChIP-seq data. Boxplots of LADs size (log10(LADs size + 1)) and of LADs score (log10(LADs score + 1)) obtained by EDD (bottom) from WT (N=3) and R6/1 (N=3) lamin B1 ChIP-seq data. Hipp, hippocampus. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.


C UCSC genome browser capture of lamin B1 ChIP - seq signal ( log ( LB1 / Input ) ) LADs discovered in wild - type ( WT LADs ) R6 / 1 ( R6 / 1 LADs ) mice ; common ( green ) , wild - type ( WT ) specific ( WT spec . LADs , red ) R6 / 1 specific ( R6 / 1 spec . LADs , blue ) LADs identified by EDD ( top , WT ( N = 3 ) , R6 / 1 ( N = 3 ) ) . Black arrows highlight WT specific LADs not identified in R6 / 1 mice . Boxplot of average ( log10 ( LADs + 1 ) ) for common , WT specific R6 / 1 specific LADs ( right ) . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median .

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C UCSC genome browser capture of lamin B1 ChIP-seq signal (log(LB1/Input)) LADs discovered in wild-type (WT LADs) and R6/1 (R6/1 LADs) mice; and common (green), wild-type (WT) specific (WT spec. LADs, red) and R6/1 specific (R6/1 spec. LADs, blue) LADs identified by EDD (top, WT (N=3), R6/1 (N=3)). Black arrows highlight WT specific LADs not identified in R6/1 mice. Boxplot of average size (log10(LADs size + 1)) for common, WT specific and R6/1 specific LADs (right). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.


D Lamin B1 enrichment in common , wild - type ( WT , N = 3 ) specific ( spec ) R6 / 1 ( N = 3 ) specific LADs ( log10 ( LB1 / Input reads + 1 ) in hippocampus ( Hipp ; left ) . Bar graphs of significant ( Benjamini ' s adjusted P - value < 0 . 05 ) Biological Processes terms from DAVID for genes within common WT specific LADs ( right ) . Gene - term enrichment was estimated by DAVID using a modified Fisher ' s exact Benjamini multiple correction . Bars represents - log10 ( Benjamini ' s adjusted P - value ) . Exact P values are reported in Appendix Table S3 . E Representaive immunoblot showing lamin B1 in nucleoplasm ( Nucleop ) ( Chrom ) in hippocampus of 30 - week - old wild - type ( WT , N = 7 ) R6 / 1 ( N = 7 ) mice . TBP , TATA binding .

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D Lamin B1 enrichment in common, wild-type (WT, N=3) specific (spec) and R6/1 (N=3) specific LADs (log10(LB1/Input reads +1) in hippocampus (Hipp; left). Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes within common and WT specific LADs (right). Gene-term enrichment was estimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3. E Representaive immunoblot showing lamin B1 levels in the nucleoplasm (Nucleop) and chromatin (Chrom) in the hippocampus of 30-week-old wild-type (WT, N = 7) and R6/1 (N = 7) mice. TBP, TATA binding protein.


D Lamin B1 enrichment in common , wild - type ( WT , N = 3 ) specific ( spec ) R6 / 1 ( N = 3 ) specific LADs ( log10 ( LB1 / Input reads + 1 ) in hippocampus ( Hipp ; left ) . Bar graphs of significant ( Benjamini ' s adjusted P - value < 0 . 05 ) Biological Processes terms from DAVID for genes within common WT specific LADs ( right ) . Gene - term enrichment was estimated by DAVID using a modified Fisher ' s exact Benjamini multiple correction . Bars represents - log10 ( Benjamini ' s adjusted P - value ) . Exact P values are reported in Appendix Table S3 .

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D Lamin B1 enrichment in common, wild-type (WT, N=3) specific (spec) and R6/1 (N=3) specific LADs (log10(LB1/Input reads +1) in hippocampus (Hipp; left). Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes within common and WT specific LADs (right). Gene-term enrichment was estimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3.


B UCSC genome browser capture of wild - type ( WT , N = 3 ) R6 / 1 ( N = 3 ) mice hippocampus ATAC - seq data , hippocampal H3K9ac unchanged , closed in R6 / 1 open in R6 / 1 accessible detected regions ( left ) in Tuba1α , Gabra2α Dlx2 gene locus . Arrows in blue ( closed in R6 / 1 ) red ( open in R6 / 1 ) indicate differential accessible regions . Bar graphs of significant ( Benjamini ' s adjusted P - value < 0 . 05 ) Biological Processes terms from DAVID for genes associated with decreased ( right top ) or increased ( right bottom ) accessibility regions . Gene - term enrichment westimated by DAVID using a modified Fisher ' s exact Benjamini multiple correction . Bars represents - log10 ( Benjamini ' s adjusted P - value ) . Exact P values are reported in Appendix Table S3 .

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B UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, hippocampal H3K9ac and unchanged, closed in R6/1 and open in R6/1 accessible detected regions (left) in Tuba1α, Gabra2α and Dlx2 gene locus. Arrows in blue (closed in R6/1) and red (open in R6/1) indicate differential accessible regions. Bar graphs of significant (Benjamini's adjusted P-value < 0.05) Biological Processes terms from DAVID for genes associated with decreased (right top) or increased (right bottom) chromatin accessibility regions. Gene-term enrichment westimated by DAVID using a modified Fisher's exact test and Benjamini multiple correction test. Bars represents the -log10 (Benjamini's adjusted P-value). Exact P values are reported in Appendix Table S3.


C Boxplots showing average gene expression ( log10 ( FPKMs + 1 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 ) in R6 / 1 ( N = 9 ) versus WT ( N = 9 ) mice ( left ) . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median . showing expression profile ( log10 ( FPKMs + 1 ) ) of genes up - or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 mice ( mid ) . Genes are ranked by degree of expression . Numbers in colour scale show correspondence between gene expression values colours . Boxplots showing average TSS accessibility ( log10 ( ATAC reads + 1 ) , N = 3 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 versus WT mice hippocampus ( right ) . Exact P values are reported in Appendix Table S3 . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median . D Average gene expression ( closed or open regions FPKMs / unchanged regions FPKMs ) for genes associated with differential accessible regions in R6 / 1 ( N = 9 ) versus WT ( N = 9 ) mice ( adjusted P - value < 0 . 05 , N = 3 ) . Each point corresponds to value from an sample . Data is shown as mean ± S . E . M . * P < 0 . 05 as compared with WT mice ( two - unpaired Student ' s t ) . Exact P values are reported in Appendix Table S3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Boxplots showing average gene expression (log10(FPKMs +1) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2) in R6/1 (N=9) versus WT (N=9) mice (left). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. Heatmap showing expression profile (log10 (FPKMs +1)) of genes up- or down-regulated (adjusted P-value<0.001, |FC|>2, N=9) in R6/1 mice (mid). Genes are ranked by the degree of expression. Numbers in colour scale show the correspondence between gene expression values and colours. Boxplots showing average TSS chromatin accessibility (log10(ATAC reads +1), N=3) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2, N=9) in R6/1 versus WT mice hippocampus (right). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. D Average gene expression (closed or open regions FPKMs/ unchanged regions FPKMs) for genes associated with differential accessible regions in R6/1 (N=9) versus WT (N=9) mice (adjusted P-value < 0.05, N=3). Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. *P < 0.05 as compared with WT mice (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.


C Boxplots showing average gene expression ( log10 ( FPKMs + 1 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 ) in R6 / 1 ( N = 9 ) versus WT ( N = 9 ) mice ( left ) . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median . showing expression profile ( log10 ( FPKMs + 1 ) ) of genes up - or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 mice ( mid ) . Genes are ranked by degree of expression . Numbers in colour scale show correspondence between gene expression values colours . Boxplots showing average TSS accessibility ( log10 ( ATAC reads + 1 ) , N = 3 ) for genes up or down - regulated ( adjusted P - value < 0 . 001 , | FC | > 2 , N = 9 ) in R6 / 1 versus WT mice hippocampus ( right ) . Exact P values are reported in Appendix Table S3 . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Boxplots showing average gene expression (log10(FPKMs +1) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2) in R6/1 (N=9) versus WT (N=9) mice (left). The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. Heatmap showing expression profile (log10 (FPKMs +1)) of genes up- or down-regulated (adjusted P-value<0.001, |FC|>2, N=9) in R6/1 mice (mid). Genes are ranked by the degree of expression. Numbers in colour scale show the correspondence between gene expression values and colours. Boxplots showing average TSS chromatin accessibility (log10(ATAC reads +1), N=3) for genes up or down-regulated (adjusted P-value < 0.001, |FC|>2, N=9) in R6/1 versus WT mice hippocampus (right). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.


A Boxplots of average expression ( log10 ( FPKMs + 1 ) ) for genes sublist 1 - 5 ( lowest to highest expression ) for wild - type ( WT , N = 3 ) mice ( left ) . Pie charts of gene distribution among generated sublists ( 1 - 5 ) for genes ( middle ) genes in LADs ( right ) in WT mice . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median . B Average gene expression ( FPKMs ) of genes found in common wild - type ( WT , N = 3 ) specific LADs for WT ( N = 9 ) R6 / 1 ( N = 9 ) mice . Each point corresponds to value from an sample . Data is shown as mean ± S . E . M . * P < 0 . 05 as compared to WT mice ( two - unpaired Student ' s t ) . Exact P values are reported in Appendix Table S3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Boxplots of average expression (log10(FPKMs +1)) for genes sublist 1-5 (lowest to highest expression) for wild-type (WT, N=3) mice (left). Pie charts of gene distribution among generated sublists (1-5) for all genes (middle) and genes in LADs (right) in WT mice. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. B Average gene expression (FPKMs) of genes found in common and wild-type (WT, N=3) specific LADs for WT (N=9) and R6/1 (N=9) mice. Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. * P < 0.05 as compared to WT mice (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.


A Boxplots of average expression ( log10 ( FPKMs + 1 ) ) for genes sublist 1 - 5 ( lowest to highest expression ) for wild - type ( WT , N = 3 ) mice ( left ) . Pie charts of gene distribution among generated sublists ( 1 - 5 ) for genes ( middle ) genes in LADs ( right ) in WT mice . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Boxplots of average expression (log10(FPKMs +1)) for genes sublist 1-5 (lowest to highest expression) for wild-type (WT, N=3) mice (left). Pie charts of gene distribution among generated sublists (1-5) for all genes (middle) and genes in LADs (right) in WT mice. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.


C Average accessibility ( normalized read ) of genes found in common wild - type ( WT , N = 3 ) specific LADs for WT ( N = 3 ) R6 / 1 ( N = 3 ) mice . Each point corresponds to value from an sample . Data is shown as mean ± S . E . M . Wilcoxon Mann Whitney was used for statistical . Exact P values are reported in Appendix Table S3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Average chromatin accessibility (normalized read counts) of genes found in common and wild-type (WT, N=3) specific LADs for WT (N=3) and R6/1 (N=3) mice. Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. Wilcoxon Mann Whitney test was used for statistical analysis. Exact P values are reported in Appendix Table S3.


D Venn diagram showing total number of genes found in common wild - type ( WT ) specific LADs regions ( left ) . Bar graph showing number of up - down - regulated genes found in common WT specific LADs regions filtering only according to adjusted P - value ( adjusted P - value < 0 . 001 ) or additionally with fold change ( adjusted P - value < 0 . 001 , | FC | > 2 ) obtained from deseq2 differential expression ( see methods ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Venn diagram showing the total number of genes found in common and wild-type (WT) specific LADs regions (left). Bar graph showing the number of up- and down-regulated genes found in common and WT specific LADs regions filtering only according to adjusted P-value (adjusted P-value <0.001) or additionally with fold change (adjusted P-value <0.001, |FC| > 2) obtained from deseq2 differential expression analysis (see methods).


E UCSC genome browser capture of wild - type ( WT , N = 3 ) R6 / 1 ( N = 3 ) mice hippocampus ATAC - seq data , lamin B1 ChIP - seq ( log ( Lb1 ChIP / Input ) ) for WT ( N = 3 ) R6 / 1 ( N = 3 ) mice hippocampus , hippocampal H3K9ac , CTCF H3K9me3 for WT mice hippocampus in Fos locus ( left ) . Enhancer regions ( E1 - E5 ) are indicated with arrows . Boxplot of lamin B1 enrichment ( log10 ( Lb1 ChIP / Input ) ) for regions with unchanged , decreased ( closed in R6 / 1 ) increased ( opened in R6 / 1 ) accessibility in R6 / 1 ( N = 3 ) mice hippocampus . * P < 0 . 05 as compared to WT ( N = 3 ) mice . ( Wilcoxon Mann Whitney ) . Exact P values are reported in Appendix Table S3 . bottom top of boxes are first third quartiles , within represents median . whiskers denote interval within 1 . 5 times interquartile range ( IQR ) from median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, lamin B1 ChIP-seq (log(Lb1 ChIP/Input)) for WT (N=3) and R6/1 (N=3) mice hippocampus, hippocampal H3K9ac, CTCF and H3K9me3 for WT mice hippocampus in Fos locus (left). Enhancer regions (E1-E5) are indicated with arrows. Boxplot of lamin B1 enrichment (log10(Lb1 ChIP/Input)) for regions with unchanged, decreased (closed in R6/1) and increased (opened in R6/1) chromatin accessibility in R6/1 (N=3) mice hippocampus. * P < 0.05 as compared to WT (N=3) mice. (Wilcoxon Mann Whitney test). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.


B , C Graphs show of time exploring each object with respect to total exploration time in ( B ) NOLT ( C ) NORT , 5 after treatment ( Veh , vehicle ; BA , betulinic acid ; WT , wild - type ) . * * * * P < 0 . 0001 compared to corresponding old location / object . WT veh N = 13 ; R6 / 1 veh N = 10 ; R6 / 1 + BA N = 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

B,C Graphs show the percentage of time exploring each object with respect to the total exploration time in the (B) NOLT and (C) NORT, 5 weeks after treatment (Veh, vehicle; BA, betulinic acid; WT, wild-type). ****P < 0.0001 compared to the corresponding old location/object. WT veh N = 13; R6/1 veh N = 10; R6/1+BA N = 10.


D Accelerating rotarod was assessed after 7 of treatment . * * P < 0 . 01 * * * P < 0 . 001 compared to vehicle - treated wild - type ( WT ) mice ; # P < 0 . 05 compared to vehicle - treated R6 / 1 mice . WT veh N = 13 ; R6 / 1 veh N = 9 ; R6 / 1 + BA N = 9 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Accelerating rotarod was assessed after 7 weeks of treatment. **P < 0.01 and ***P < 0.001 compared to vehicle-treated wild-type (WT) mice; #P < 0.05 compared to vehicle-treated R6/1 mice. WT veh N = 13; R6/1 veh N = 9; R6/1+BA N = 9.


E Lamin B1 were analysed by Western blot in striatum ( WT veh N = 10 ; R6 / 1 veh N = 6 ; R6 / 1 + BA N = 7 ) , hippocampus ( WT veh N = 12 ; R6 / 1 veh N = 8 ; R6 / 1 + BA N = 7 ) cortex ( WT veh N = 11 ; R6 / 1 veh N = 6 ; R6 / 1 + BA N = 7 ) after 12 of treatment . * P < 0 . 05 , * * P < 0 . 05 compared to vehicle - treated wild - type ( WT ) mice # P < 0 . 05 compared to vehicle - treated R6 / 1 mice . Representative immunoblots of lamin B1 α - tubulin ( as loading control ) for each treatment group are shown .

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

E Lamin B1 levels were analysed by Western blot in the striatum (WT veh N = 10; R6/1 veh N = 6; R6/1+BA N = 7), hippocampus (WT veh N = 12; R6/1 veh N = 8; R6/1+BA N = 7) and cortex (WT veh N = 11; R6/1 veh N = 6; R6/1+BA N = 7) after 12 weeks of treatment. *P < 0.05, **P < 0.05 compared to vehicle-treated wild-type (WT) mice and #P < 0.05 compared to vehicle-treated R6/1 mice. Representative immunoblots of lamin B1 and α-tubulin (as loading control) for each treatment group are shown.


F Lamin B1 were analysed by FANSI in hippocampal CA1 neurons nuclei from wild - type ( WT ) R6 / 1 mice after 12 of treatment . * P < 0 . 05 compared to vehicle - treated WT mice . * P < 0 . 05 as compared to vehicle - treated WT mice . Lamin B1 : WT veh N = 6 ; R6 / 1 veh N = 5 ; R6 / 1 + BA N = 4 ; Lamin B1 circularity : WT veh N = 6 ; R6 / 1 veh N = 5 ; R6 / 1 + BA N = 5 .

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

F Lamin B1 intensity and nuclear morphology were analysed by FANSI in hippocampal CA1 neurons nuclei from wild-type (WT) and R6/1 mice after 12 weeks of treatment. *P < 0.05 compared to vehicle-treated WT mice. *P < 0.05 as compared to vehicle-treated WT mice. Lamin B1 intensity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 4; Lamin B1 circularity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 5.


G permeability was analysed by FRAP in hippocampal CA1 neurons nuclei after 12 of treatment . * p < 0 . 05 compared to vehicle - treated wild - type ( WT ) mice . WT veh N = 4 ; R6 / 1 veh N = 5 ; R6 / 1 + BA N = 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Nuclear permeability was analysed by FRAP in hippocampal CA1 neurons nuclei after 12 weeks of treatment. *p < 0.05 compared to vehicle-treated wild-type (WT) mice. WT veh N = 4; R6/1 veh N = 5; R6/1+BA N = 6.


( A ) Purification of Halo - tagged cohesin tetramer . Human cohesin tetramer tagged by Halo was purified from insect Alexa488 dye was conjugated to Halo - tag ( cohesinHalo488 ) .

O O O O O O O O O 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) Purification of the Halo-tagged cohesin tetramer. Human cohesin tetramer tagged by Halo was purified from insect cells and Alexa488 dye was conjugated to Halo-tag (cohesinHalo488).


( B ) CohesinHalo488 loaded onto in presence of Scc2 - Scc4 was incubated in buffer with or without TEV protease . After high - salt washing , CohesinHalo488 intensities on were measured . Red bars denote median , lower , upper quartile values ( n ≥ 98 ; * p < 0 . 0001 , two - Mann - Whitney ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O 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) CohesinHalo488 loaded onto DNA in the presence of Scc2-Scc4 was incubated in the buffer with or without TEV protease. After high-salt washing, CohesinHalo488 intensities on DNA were measured. Red bars denote the median, lower, and upper quartile values (n ≥ 98; *p < 0.0001, two-tailed Mann-Whitney test).


( C ) Kymograph of a single - interacting cohesinHalo488 particle . CohesinHalo488 is loaded onto in presence of Scc2 - Scc4 , after high - salt washing , single cohesin particles were observed in presence of ATP 100 mM KCl . Scale bar , 2 μm . ( D ) Histogram of net displacement ( xfinal - xinitial ) of cohesin particles treated as in ( C ) . magenta curve represents Gaussian fit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kymograph of a single DNA-interacting cohesinHalo488 particle. CohesinHalo488 is loaded onto DNA in the presence of Scc2-Scc4 and, after high-salt washing, single cohesin particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 2 μm.(D) Histogram of the net displacement (xfinal - xinitial) of cohesin particles treated as in (C). The magenta curve represents the Gaussian fit.


( E ) CohesinHalo488 was loaded washed in high salt as in ( C ) , cohesin particles were observed in presence of ATP 100 mM or 750 mM KCl . Kymograph of - associated cohesin particles under high - salt conditions ( 750 mM KCl , right ) mean square displacement ( MSD ) versus time ( left ) are shown . Dotted lines are regression lines used to estimate diffusion coefficient ( D ) . Scale bar , 2 μm .

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

(E) CohesinHalo488 was loaded and washed in high salt as in (C), and the cohesin particles were observed in the presence of ATP and 100 mM or 750 mM KCl. Kymograph of DNA-associated cohesin particles under high-salt conditions (750 mM KCl, right) and the mean square displacement (MSD) versus time (left) are shown. Dotted lines are regression lines used to estimate the diffusion coefficient (D). Scale bar, 2 μm.


( F ) MSD versus time for - bound cohesinWT - Halo488 or cohesinKA - Halo488 particles . CohesinWT - Halo488 or cohesinKA - Halo488 was loaded onto in presence of Scc2 - 4 ATP . After high - salt washing , cohesin particles on were observed in presence of 1 mM AMP - PCP or ATP 100 mM KCl . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

(F) MSD versus time for DNA-bound cohesinWT-Halo488 or cohesinKA-Halo488 particles. CohesinWT-Halo488 or cohesinKA-Halo488 was loaded onto DNA in the presence of Scc2-4 and ATP. After high-salt washing, the cohesin particles on DNA were observed in the presence of 1 mM AMP-PCP or ATP and 100 mM KCl. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( A ) Kymograph of CohesinHalo488 in presence or absence of Wapl - Pds5 . CohesinHalo488 was loaded onto in presence of Scc2 - Scc4 washed in high - salt buffer . - bound cohesin was further incubated with or without Wapl - Pds5 , then cohesinHalo488 particles were observed in presence of ATP 100 mM KCl . Scale bar , 1 μm . ( B ) MSD versus time of cohesin particles in ( A ) are shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(A) Kymograph of CohesinHalo488 in the presence or absence of Wapl-Pds5. CohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with or without Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 1 μm.(B) MSD versus time of cohesin particles in (A) are shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( C ) Acetylated - or unacetylated - cohesinHalo488 was loaded onto in presence of Scc2 - Scc4 washed in high - salt buffer . - bound cohesin was further incubated with Wapl - Pds5 , then cohesinHalo488 particles were observed in presence of ATP 100 mM KCl . MSD versus time is shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(C) Acetylated- or unacetylated-cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( D ) Acetylated cohesinHalo488 was treated as in ( C ) , - bound cohesin was further incubated with buffer , Wapl - Pds5 , or Wapl - Pds5 plus Sororin , then cohesinHalo488 particles were observed in presence of ATP 100 mM KCl . MSD versus time is shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(D) Acetylated cohesinHalo488 was treated as in (C), the DNA-bound cohesin was further incubated with buffer, Wapl-Pds5, or Wapl-Pds5 plus Sororin, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( A ) Acetylated cohesinHalo488 was loaded onto in presence of Scc2 - Scc4 washed in high - salt buffer . - bound cohesin was further incubated with Wapl - Pds5 plus Sororin , then treated with buffer , Plk1 , or Plk1 plus BI4834 . CohesinHalo488 particles were observed in presence of ATP 100 mM KCl . MSD versus time is shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(A) Acetylated cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5 plus Sororin, and then treated with buffer, Plk1, or Plk1 plus BI4834. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( B ) Acetylated cohesinHalo488 was treated as in ( A ) , or treated with CDK1 or Aurora B rather than Plk1 . Sororin on was measured after immunofluorescence microscopy with anti - Sororin antibody ( n > 100 , * p < 0 . 0001 , two - Mann - Whitney ) .

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

(B) Acetylated cohesinHalo488 was treated as in (A), or treated with CDK1 or Aurora B rather than Plk1. Sororin intensity on DNA was measured after immunofluorescence microscopy with anti-Sororin antibody (n > 100, *p < 0.0001, two-tailed Mann-Whitney test).


( C ) Acetylated cohesinHalo488 was treated as in ( A ) but treated with Aurora B rather than Plk1 . CohesinHalo488 particles were observed in presence of ATP 100 mM KCl . MSD versus time is shown . D indicates diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

(C) Acetylated cohesinHalo488 was treated as in (A) but treated with Aurora B rather than Plk1. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


( A ) Stretched was incubated in HSS in presence or absence of geminin . After washing HSS , endogenous Smc3 ( green ) Mcm2 ( magenta ) were detected by immunofluorescence . was counterstained with SYTOX ( gray ) . Scale bar , 2 µm . ( B ) Numbers of Smc3 Mcm2 signals were counted on 10 μm shown in ( A ) . ( 22 DNAs per condition , n = 3 , mean ± s . e . m . * p < 0 . 0001 , two - Mann - Whitney ) .

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

(A) Stretched DNA was incubated in the HSS in the presence or absence of geminin. After washing the HSS, endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Scale bar, 2 µm.(B) Numbers of Smc3 and Mcm2 signals were counted on 10 μm DNA shown in (A). (22 DNAs per condition, n = 3, mean ± s.e.m. *p < 0.0001, two-tailed Mann-Whitney test).


( C ) formed in HSS in presence of geminin , cohesinHalo488 ( green ) was loaded onto with Scc2 - Scc4 . was detected by immunostaining of histone H3 ( magenta ) . was counterstained with SYTOX ( gray ) . distributions of cohesin particles histone H3 are shown as - scan plots ( bottom ) . Scale bar , 5 μm . ( D ) Cohesin particles observed in ( C ) were categorized as indicated ( > 37 particles per condition . n = 6 , mean ± s . e . m . ) . ( E ) Cohesin particle numbers for each condition . Red bars denote median , lower , upper quartile values ( n ≥ 12 , * p < 0 . 0004 , unpaired t - ) . ( F ) Colocalization of Mcm2 with Smc3 was quantified ( 50 Mcm2 particles per condition , n = 3 , mean ± s . e . m . )

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(C) Chromatin formed in the HSS in the presence of geminin, and cohesinHalo488 (green) was loaded onto the chromatin with Scc2-Scc4. Chromatin was detected by immunostaining of histone H3 (magenta). DNA was counterstained with SYTOX (gray). The distributions of cohesin particles and histone H3 are shown as line-scan plots (bottom). Scale bar, 5 μm.(D) Cohesin particles observed in (C) were categorized as indicated (> 37 particles per condition. n = 6, mean ± s.e.m.).(E) Cohesin particle numbers for each condition. Red bars denote the median, lower, and upper quartile values (n ≥ 12, *p < 0.0004, unpaired t-test).(F) Colocalization of Mcm2 with Smc3 was quantified (50 Mcm2 particles per condition, n = 3, mean ± s.e.m.)


( A ) Stretched was incubated in HSS . Endogenous Smc3 ( green ) Mcm2 ( magenta ) were detected by immunofluorescence . was counterstained with SYTOX ( gray ) . Colocalization of Smc3 Mcm2 was quantified ( bottom , n = 3 , mean ± s . e . m . ) . Scale bar , 2 μm .

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

(A) Stretched DNA was incubated in the HSS. Endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Colocalization of Smc3 and Mcm2 was quantified (bottom, n = 3, mean ± s.e.m.). Scale bar, 2 μm.