Deletion of VE - cadherin in lymphatic endothelial of newborn pups was induced by two optimal doses of 4 - OHT at days P2 P4 . For 6 later ear punches were subjected to wholemount immunostaining of dermal vasculature . A - H ( A , B , E , F ) Overview MIPs of ear skin wholemount preparations immunostained for indicated antigens . VEC , VE - cadherin ; PRX , PROX1 . ( C , D , G , H ) Magnified views of areas in red boxes in ( A , E ) . Areas delimited by stippled yellow boxes in ( C ) ( G ) are enlarged in insets ( yellow solid boxes ) . Yellow arrows indicate widened lumen , yellow arrowheads denote oak leaf - shaped LECs with discontinuous junctions . Scale bars in ( A E ) correspond to 100 μm in ( C G ) to 50 µm . data are representative for 6 ( Cdh5lox / lox ) 5 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed from 2 litters

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

Deletion of VE-cadherin in lymphatic endothelial cells of newborn pups was induced by two optimal doses of 4-OHT at days P2 and P4. For analysis 6 weeks later ear punches were subjected to wholemount immunostaining of the dermal vasculature. A-H (A, B, E, F) Overview MIPs of ear skin wholemount preparations immunostained for the indicated antigens. VEC, VE-cadherin; PRX, PROX1. (C, D, G, H) Magnified views of the areas in the red boxes in (A, E). Areas delimited by the stippled yellow boxes in (C) and (G) are enlarged in the insets (yellow solid boxes). Yellow arrows indicate widened lumen, yellow arrowheads denote oak leaf-shaped LECs with discontinuous junctions. Scale bars in (A and E) correspond to 100 μm and in (C and G) to 50 µm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters


A - L VE - cadherin deletion was initiated either in newborn pups ( A , B , E , F ) by two applications of 4 - OHT at days P2 P4 or in mice at 11 ( C , G ) 42 ( D , H ) of ( three applications of Tamoxifen at 2 day intervals via oral gavage ) . Ear skins were prepared 6 ( A , B , E , F ) or 5 ( C , D , G , H ) after induction immunostained for indicated . Shown are MIPs of confocal tile scans ( approx . 750 µm x 750 µm ) covering 40 µm in depth . arrows in ( F ) highlight distorted partially fragmented lymphatic vessels arrowheads denote aberrantly pointed vessels ( F ) . ( I - L ) Lymphatic valves in dermis of ear were maintained for 5 despite deletion of VE - cadherin at 11 of ( yellow arrows in K , L ) . PEC1 , PECAM1 ; PRX1 , PROX1 ; VR3 , VEGFR - 3 . Scale bars correspond to 100 μm . data represent wholemount stainings from 6 ( A , E ) , 5 ( B , F ) , 6 ( C , G , K , L ) , 3 ( D , H ) 6 ( I , J ) analyzed

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A-L VE-cadherin deletion was initiated either in newborn pups (A, B, E, F) by two applications of 4-OHT at days P2 and P4 or in mice at 11 weeks (C, G) and 42 weeks (D, H) of age (three applications of Tamoxifen at 2 day intervals via oral gavage). Ear skins were prepared 6 weeks (A, B, E, F) or 5 weeks (C, D, G, H) after induction and immunostained for the indicated proteins. Shown are MIPs of confocal tile scans (approx. 750 µm x 750 µm) covering 40 µm in depth. White arrows in (F) highlight distorted and partially fragmented lymphatic vessels and white arrowheads denote aberrantly pointed vessels (F). (I-L) Lymphatic valves in the dermis of the ear were maintained for 5 weeks despite deletion of VE-cadherin at 11 weeks of age (yellow arrows in K, L). PEC1, PECAM1; PRX1, PROX1;VR3, VEGFR-3. Scale bars correspond to 100 μm. The data represent wholemount stainings from 6 (A, E), 5 (B, F), 6 (C, G, K, L), 3 (D, H) and 6 (I, J) analyzed animals


Fig . 4 : VE - cadherin is indispensable for maintenance of mesenteric lymphatic vessels at ages . A - F Deletion of VE - cadherin was induced in mice at various ( 4 ( A ) , neonate ( B ) , 6 month ( C , D , E ) or 1 year ( F ) ) by either two applications of 4 - OHT at days P2 P4 ( neonates ) or three applications of Tamoxifen at 2 day intervals via oral gavage ( other ages ) . mesentery was prepared as indicated 4 , 6 , 8 or 11 later wholemount preparations were immunostained for specified in color ( left ) . ( B ) Inset in top panel shows a magnification of outlined by dashed ( for further magnification see expanded view Fig . 1A B ) . Arrows indicate previous position of lymphatic valves ; arrowheads indicate PECAM1 + capillaries supplying intestinal fat tissue lining vessels ( magnified in expanded view Fig . 1C ) ; yellow arrowheads denote island of LECs that display intracellular VEGFR - 3 . ( C ) Arrows denote LECs that have converted from growth as tubes to sheets ; arrowheads , thinning of lymphatic vessels ; yellow arrowheads , aberrant sprouting of LECs . outlined by a box is depicted magnified in ( D ) provides details on LECs growing as a sheet of . ( E ) Formation of ectopic sprouts from valve of degenerating lymphatic vessels . ( F ) Fully deteriorated lymphatic vessel with emanating sprouts that initiate sheet - like growth . PEC1 , PECAM1 ; PRX1 , PROX1 ; VR3 , VEGFR - 3 . Scale bars = 100 μm . data represent n = 5 ( A ) , n = 5 ( B ) , n = 3 ( C - E ) n = 6 ( F ) .

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Fig. 4: VE-cadherin is indispensable for the maintenance of mesenteric lymphatic vessels at all ages. A-F Deletion of VE-cadherin was induced in mice at various age (4 weeks (A), neonate (B), 6 month (C, D, E) or 1 year (F)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages). The mesentery was prepared as indicated 4, 6, 8 or 11 weeks later and the wholemount preparations were immunostained for the proteins specified in color (left). (B) Inset in top panel shows a magnification of the area outlined by the white dashed line (for further magnification see expanded view Fig. 1A and B). Arrows indicate the previous position of lymphatic valves; white arrowheads indicate PECAM1+ capillaries supplying the intestinal fat tissue lining the vessels (magnified in expanded view Fig. 1C); yellow arrowheads denote island of LECs that display intracellular VEGFR-3 staining. (C) Arrows denote LECs that have converted from growth as tubes to sheets; white arrowheads, thinning of lymphatic vessels; yellow arrowheads, aberrant sprouting of LECs. The area outlined by a white box is depicted magnified in (D) and provides details on LECs growing as a sheet of cells. (E) Formation of ectopic sprouts from the valve area of degenerating lymphatic vessels. (F) Fully deteriorated lymphatic vessel with emanating sprouts that initiate sheet-like growth. PEC1, PECAM1; PRX1, PROX1; VR3, VEGFR-3. Scale bars = 100 μm. The data represent n=5 (A), n=5 (B), n=3 (C-E) and n=6 (F) animals.


Fig . 5 : Deletion of VE - cadherin results in fragmentation distension of intestinal lacteals . A - E Deletion of VE - cadherin was induced in mice at various ages ( neonatal ( C ) , 16 ( D ) or 1 year ( E ) ) by either two applications of 4 - OHT at days P2 P4 ( neonates ) or three applications of Tamoxifen at 2 day intervals via oral gavage ( other ages ) . Control mice of same were subjected to an identical regimen ( A , B ) . Intestinal wholemount preparations of jejunum ileum were generated 5 - 8 after Tamoxifen induction immunostained for indicated , scale bars = 100 μm . data represent wholemount stainings from 3 ( D ) , 5 ( B , E ) 6 ( A , C ) analyzed for each genotype .

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Fig. 5: Deletion of VE-cadherin results in fragmentation and distension of the intestinal lacteals. A-E Deletion of VE-cadherin was induced in mice at various ages (neonatal (C), 16 weeks (D) or 1 year (E)) by either two applications of 4-OHT at days P2 and P4 (neonates) or three applications of Tamoxifen at 2 day intervals via oral gavage (all other ages).Control mice of the same age were subjected to an identical regimen (A, B). Intestinal wholemount preparations of the jejunum and ileum were generated 5 - 8 weeks after Tamoxifen induction and immunostained for the indicated proteins, scale bars = 100 μm. The data represent wholemount stainings from 3 (D), 5 (B,E) and 6 (A, C) animals analyzed for each genotype.


Deletion of VE - cadherin was induced in adult mice ( 13 ) EdU incorporation into PROX1 - positive nuclei was determined 6 after Tamoxifen administration . Shown are mesenteric wholemount stainings prepared from control ( A , B ; n = 3 ) or VE - cadherin - deleted mice ( C , D ; n = 3 ) . ( B ) ( D ) show a magnification of outlined by dashed in ( A ) ( C ) . Antigens shown in addition to EdU incorporation are depicted in colour on left side of first panel . PEC , PECAM1 ; PRX1 , PROX1 . Scale bars = 100 µm Enumeration of EdU - positive LECs . measurements were obtained from three nine confocal stacks per biological group . PROX1 - positive nuclei were counted using particle tool of Fiji . Co - localization of PROX1 EdU was determined manually in each confocal plane , using counting tool of Fiji . data represent mean ± SD . * * * * p ≤ 0 . 0001 . Two - unpaired Student ' s t

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Deletion of VE-cadherin was induced in adult mice (13 weeks) and EdU incorporation into PROX1-positive cell nuclei was determined 6 weeks after Tamoxifen administration. Shown are mesenteric wholemount stainings prepared from control (A, B; n=3) or VE-cadherin-deleted mice (C, D; n=3). (B) and (D) show a magnification of the area outlined by the white dashed line in (A) and (C). Antigens shown in addition to the EdU incorporation are depicted in colour on the left side of the first panel. PEC, PECAM1; PRX1, PROX1. Scale bars = 100 µm Enumeration of EdU-positive LECs. The measurements were obtained from three animals and nine confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji. Co-localization of PROX1 and EdU was determined manually in each confocal plane, using the cell counting tool of Fiji. The data represent mean ± SD. **** p ≤ 0.0001. Two-tailed unpaired Student's t test


Aberrantly enhanced VEGFR - 3 phosphorylation in VE - cadherin - deficient mesenteric LECs of six old mice that had received 4 - OHT injections at P2 P4 . Shown are wholemount immunostainings for antigens shown on left in color . pVR3 , pVEGFR - 3 , PRX1 , PROX1 ; PEC1 , PECAM1 . ( G ) ( I ) show a magnification of outlined by dashed box in ( F ) ( H ) . Scale bars = 50 μm . data are representative for 6 ( Cdh5lox / lox ) 5 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed from 2

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Aberrantly enhanced VEGFR-3 phosphorylation in VE-cadherin-deficient mesenteric LECs of six weeks old mice that had received 4-OHT injections at P2 and P4. Shown are wholemount immunostainings for the antigens shown on the left in color. pVR3, pVEGFR-3, PRX1, PROX1; PEC1, PECAM1. (G) and (I) show a magnification of the area outlined by the white dashed box in (F) and (H). Scale bars = 50 μm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters


Dermal , mesenteric intestinal tissue pieces of 15 week old control ( Cdh5lox / lox ) mice were dissociated total was extracted immediately . For reverse transcription 2 . 0 µg of total was used for each tissue type . Expression of lymphangiogenic factors VEGF - C VEGF - D was determined by quantitative real - time ( qRT ) - PCR using specific TaqMan probes . Samples were measured in triplicate diagrams are representative for 3 independent experiments . Relative expression ( ΔΔCt ) of target genes was normalized to control transcript

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Dermal, mesenteric and intestinal tissue pieces of 15 week old control (Cdh5lox/lox) mice were dissociated and total RNA was extracted immediately. For reverse transcription 2.0 µg of total RNA was used for each tissue type. Expression of lymphangiogenic factors VEGF-C and VEGF-D was determined by quantitative real-time (qRT)-PCR using specific TaqMan probes. Samples were measured in triplicate and diagrams are representative for 3 independent experiments. Relative expression (ΔΔCt) of target genes was normalized to the control transcript UBC


Loss of VEGFR - 3 surface expression on mesenteric LECs growing in sheets was associated with concomitant upregulation of VEGFR - 2 , scale bars = 50 μm . data are representative for 4 analyzed from 2 litters . VR2 , VEGFR - 2 ; VR3 , VEGFR - 3 , PRX1 ,

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Loss of VEGFR-3 surface expression on mesenteric LECs growing in cell sheets was associated with concomitant upregulation of VEGFR-2, scale bars = 50 μm. The data are representative for 4 analyzed animals from 2 litters.VR2, VEGFR-2; VR3, VEGFR-3, PRX1, PROX1


Expression of transcriptional co - activators YAP TAZ was analyzed in mesenteric wholemount preparations 8 after induction of VE - Cadherin deletion at 1 year of by three doses of Tamoxifen . Respective antigens depicted are indicated on top of panels in colour . highly related molecules YAP TAZ were both recognized by antibody used are therefore collectively abbreviated Y / T . Scale bars = 100

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Expression of the transcriptional co-activators YAP and TAZ was analyzed in mesenteric wholemount preparations 8 weeks after induction of VE-Cadherin deletion at 1 year of age by three doses of Tamoxifen. Respective antigens depicted are indicated on the top of the panels in colour. The highly related molecules YAP and TAZ were both recognized by the antibody used and are therefore collectively abbreviated Y/T. Scale bars = 100 μm


Fig . 7 : Loss of VE - cadherin from lymphatic endothelial results in increased circumferential actin a reduced number of focal adhesions . A , B dermal LECs were isolated as described in appendix Fig . S8 A . PdLECs were used for up to four passages control VE - cadherin - deleted pdLECs had been cultured for same number of passages . were seeded at a density of 2 . 5 x 104 on gelatine - coated 15mm glass coverslips co - stained after 3 days of culture for actin cytoskeleton ( Phalloidin ) either Vinculin ( A ) or FAK ( B ) for visualization of focal adhesions . PROX1 in ( A ) identifies LECs , nuclei in ( B ) were counterstained with Hoechst . Scale bars correspond to 50 µm . ( C , D ) Bar diagrams show number of focal adhesions / , which were enumerated using Image J from Vinculin FAK stainings shown in ( A B ) . Data are mean values + S . E . M . derived from three lines 9 viewfields of 256 µm x 256 µm per biological group . * * * p ≤ 0 . 001 . Two - unpaired Student ' s t .

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Fig. 7: Loss of VE-cadherin from lymphatic endothelial cells results in increased circumferential actin and a reduced number of focal adhesions. A, B Primary dermal LECs were isolated as described in appendix Fig. S8 A. PdLECs were used for up to four passages and control and VE-cadherin-deleted pdLECs had been cultured for the same number of passages. Cells were seeded at a density of 2.5 x 104 cells on gelatine-coated 15mm glass coverslips and co-stained after 3 days of culture for the actin cytoskeleton (Phalloidin) and either Vinculin (A) or FAK (B) for the visualization of focal adhesions. PROX1 staining in (A) identifies LECs, nuclei in (B) were counterstained with Hoechst. Scale bars correspond to 50 µm. (C, D) Bar diagrams show the number of focal adhesions/cell, which were enumerated using Image J from the Vinculin and FAK stainings shown in (A and B). Data are mean values + S.E.M. derived from three cell lines and 9 viewfields of 256 µm x 256 µm per biological group. *** p ≤ 0.001. Two-tailed unpaired Student's t test.


Fig . 8 : Enhanced collective migration in VE - cadherin - deficient lymphatic endothelial . ( A ) Collective migration of VE - cadherin - deficient control LECs was assessed in wound scratch assays . LECs were grown up to four passages seeded at a density of 5x104 on fibronectin - coated 8 well µ - chamber slides ( growth 0 . 22 cm2 ) ; scale bar 500 µm ( B ) of zone denuded from is depicted in curve diagram showing mean values ± SD of three experiments conducted in quadruplets . * p ≤ 0 . 01 . Two - unpaired Student ' s t . Expanded View Figures - Legends

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Fig. 8: Enhanced collective cell migration in VE-cadherin-deficient lymphatic endothelial cells. (A) Collective cell migration of VE-cadherin-deficient and control LECs was assessed in wound scratch assays. LECs were grown up to four passages and seeded at a density of 5x104 cells on fibronectin-coated 8 well µ-chamber slides (growth area 0.22 cm2); scale bar 500 µm (B) The area of the zone denuded from cells is depicted in the curve diagram showing mean values ± SD of three individual experiments conducted in quadruplets. * p ≤ 0.01. Two-tailed unpaired Student's t test. Expanded View Figures - Legends


A . Endogenous Cyclin B1 - YFP localisation during mitosis live in RPE1 ( still from Movie EV1 ) .

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A. Endogenous Cyclin B1-YFP localisation during mitosis live in RPE1 cells (still from Movie EV1).


Immunofluorescence ( B ) of relative Cyclin B1 MAD1 at unattached attached kinetochores in arrested in STLC .

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Immunofluorescence images (B) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC.


C . Immunofluorescence quantifications ( C ) of relative Cyclin B1 MAD1 at unattached attached kinetochores in arrested in STLC . Each dot represents a kinetochore , data is from 40 kinetochore pairs ( 13 , max 5 kinetochore pairs / ) .

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C. Immunofluorescence quantifications (C) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC. Each dot represents a kinetochore, data is from 40 kinetochore pairs (13 cells, max 5 kinetochore pairs/cell).


D , E . of relative kinetochore intensities of Cyclin B1 MAD1 in nocodazole - arrested ( noco ) treated with MPS1 inhibitors , AZ - 3146 ( 5μM ) or Reversine ( 500nM ) , either before ( D ) or after ( E ) mitotic entry .

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D,E. Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole-arrested cells (noco) treated with the MPS1 inhibitors, AZ-3146 (5μM) or Reversine (500nM), either before (D) or after (E) mitotic entry.


F . Immunofluorescence of LacI - MAD1 Cyclin B1 in U2OS containing a LacO arrays on chromosome

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F. Immunofluorescence images of LacI-MAD1 and Cyclin B1 in U2OS cells containing a LacO arrays on chromosome 1


G . Live imaging of Cyclin B1 - mCherry ( CycB1 - mCh ) in LacO - U2OS transfected with LacI - MAD1 - FL ( full : aa1 - 718 ) or various LacI - MAD1 truncations ( acid numbers indicated ) .

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G. Live imaging of Cyclin B1-mCherry (CycB1-mCh) in LacO-U2OS cells transfected with LacI-MAD1-FL (full length: aa1-718) or various LacI-MAD1 truncations (amino acid numbers indicated).


H , I . Immunofluorescence ( H ) quantifications ( I ) of Cyclin B1 MAD1 kinetochore in control ( MAD1 - WT ) or MAD1β HeLa ( two independent clones : C13 or C24 ) treated with nocodazole .

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H,I. Immunofluorescence images (H) and quantifications (I) of Cyclin B1 and MAD1 kinetochore levels in control (MAD1-WT) or MAD1β HeLa cells (two independent clones: C13 or C24) treated with nocodazole.


J , K . Immunofluorescence ( J ) ( K ) of Cyclin B1 MAD1 kinetochore localisation in doxycycline - inducible MAD1α β knockouts treated with or without dox for 10 days then arrested in nocodazole . were selected that had full MAD1 knockout in doxycycline treatment ( this constituted approximately 30 % of ) .

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J,K. Immunofluorescence images (J) and quantification (K) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline-inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole. Cells were selected that had full MAD1 knockout in the doxycycline treatment (this constituted approximately 30% of cells).


L . Relative kinetochore volumes occupied by Cyclin B1 MAD1 ( relative to CenpC ) in nocodazole - arrested MAD1α MAD1β ( calculated from experiments shown in ( H , I ) .

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L. Relative kinetochore volumes occupied by Cyclin B1 and MAD1 (relative to CenpC) in nocodazole-arrested MAD1α and MAD1β cells (calculated from experiments shown in (H,I).


A . Elution profiles SDS - PAGE for SEC runs on indicated column of Cyclin B1 : CDK1 ( blue profile ) , MBP - MAD1 : MAD2 ( red ) , their combination ( green ) . Note that same Cyclin B1 : CDK1 elution profile SDS - PAGE is also displayed as reference in panels D F to improve clarity . For same reason , MBP - MAD1 : MAD2 elution profile SDS - PAGE is also displayed in panels 2A - C . B . Elution profiles SDS - PAGE for SEC runs on indicated column of CDK1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , their combination ( green ) . C . Elution profiles SDS - PAGE for SEC runs on indicated column of Cyclin B1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , their combination ( green ) . D . Elution profiles SDS - PAGE for SEC runs of Cyclin B1 : CDK1 ( blue ) , MBP - MAD1 ∆ 93 : MAD2 ( green ) , their combination ( red ) .

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A. Elution profiles and SDS-PAGE for SEC runs on the indicated column of the Cyclin B1:CDK1 complex (blue profile), MBP-MAD1:MAD2 (red), and their combination (green). Note that the same Cyclin B1:CDK1 elution profile and SDS-PAGE is also displayed as reference in panels D and F to improve clarity. For the same reason, the MBP-MAD1:MAD2 elution profile and SDS-PAGE is also displayed in panels 2A-C. B. Elution profiles and SDS-PAGE for SEC runs on the indicated column of CDK1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). C. Elution profiles and SDS-PAGE for SEC runs on the indicated column of Cyclin B1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). D. Elution profiles and SDS-PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD1∆93:MAD2 (green), and their combination (red).


F . Elution profiles SDS - PAGE for SEC runs of Cyclin B1 : CDK1 ( blue ) , MBP - MAD13EK : MAD2 ( E52K , E53K , E56K mutations ; red ) , their combination ( green ) . In A - D , potential binding partners were combined at a of 5 µM .

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F. Elution profiles and SDS-PAGE for SEC runs of Cyclin B1:CDK1 (blue), MBP-MAD13EK:MAD2 (E52K, E53K, and E56K mutations; red), and their combination (green). In A-D, individual potential binding partners were combined at a concentration of 5 µM.


A . Western blot of indicated vsv - MAD1 - WT or 3EK HeLa clones treated with or without doxycycline for 10 days .

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A. Western blot analysis of indicated vsv-MAD1-WT or 3EK HeLa clones treated with or without doxycycline for 10 days.


B . Immunofluorescence showing MAD1 ZW10 kinetochore in nocodazole - arrested MAD1 - WT - C13 3EK - C14 just after envelope breakdown ( early prometaphase ) or later in mitosis when is condensed ( late prometaphase ) . Note , that early late prometaphase was defined on level of condensation .

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B. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested MAD1-WT-C13 AND 3EK-C14 just after nuclear envelope breakdown (early prometaphase) or later in mitosis when the chromatin is condensed (late prometaphase). Note, that early and late prometaphase was defined based on the level of chromatin condensation.


C , D . Relative kinetochore volumes occupied by MAD1 ( C ) ZW10 ( D ) ( relative to CenpC ) in MAD1 - WT - 3EK in early late prometaphase .

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C,D. Relative kinetochore volumes occupied by MAD1 (C) and ZW10 (D) (relative to CenpC) in MAD1-WT and -3EK cells in early and late prometaphase.


E . of MAD1 kinetochore from indicated MAD1 - WT 3EK clones treated as in ( B ) .

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E. Quantification of MAD1 kinetochore intensity from indicated MAD1-WT and 3EK clones treated as in (B).


A - C . Immunofluorescence ( A ) quantifications ( B , C ) of relative MAD1 MAD2 kinetochore in indicated lines arrested in nocodazole treated with / without AZ - 3146 for 30 min to inhibit MPS1 . In kinetochore graphs , each dot represents a , horizontal lines indicate median vertical bars show 95 % confidence interval . Note , when these vertical bars do not overlap difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . kinetochore graphs display data that is relative to WT C11 controls in nocodazole , which are normalised to 1 . mean level of normalised controls is indicated by dotted lines . 30 from 3 experiments . Scale bars = 5µM . D . Duration of mitotic arrest in indicated lines arrested in nocodazole then treated with 1 . 25 µM AZ - 3146 . Graph shows cumulative mean ( ± ) of 3 experiments , 50 per condition per experiment .

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A-C. Immunofluorescence images (A) and quantifications (B,C) of relative MAD1 and MAD2 kinetochore levels in indicated cells lines arrested in nocodazole and treated with/without AZ-3146 for 30 min to inhibit MPS1. In all kinetochore intensity graphs, each dot represents a cell, horizontal lines indicate the median and vertical bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). All kinetochore intensity graphs display data that is relative to WT C11 controls in nocodazole, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines. 30 cells from 3 experiments. Scale bars = 5µM. D. Duration of mitotic arrest in indicated cell lines arrested in nocodazole and then treated with 1.25 µM AZ-3146. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


A . Immunofluorescence of MAD1 MAD1 - pT716 kinetochore in nocodazole - arrested RPE1 treated with or without AZ - 3146 . Scale bars = 5µM .

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A. Immunofluorescence images of MAD1 and MAD1-pT716 kinetochore levels in nocodazole-arrested RPE1 cells treated with or without AZ-3146. Scale bars = 5µM.


B . Electron of rotary - shadowed MAD193 - 718 : MAD2 ( top row ) , MBP - MAD1 : MAD2 ( middle row ) , MBP - MAD1 : MAD2 in with GST - CDK1 : Cyclin B1 ( bottom row ) . Scale bar = 50 nm .

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B. Electron micrographs of rotary-shadowed MAD193-718:MAD2 (top row), MBP-MAD1:MAD2 (middle row), and MBP-MAD1:MAD2 in complex with GST-CDK1:Cyclin B1 (bottom row). Scale bar = 50 nm.


C . Quantifications ( top ) corresponding immunofluorescence ( underneath ) of relative kinetochore MAD1 MAD1 - pT716 in nocodazole - arrested of MAD1 - WT - C14 MAD1 - 3EK - C12 treated with different doses of AZ - 3146 for 30 min . MG132 was included at time of AZ - 3146 addition to prevent mitotic exit . Each dot represents a , horizontal lines indicate median error bars show 95 % confidence interval . Note , when these vertical bars do not overlap difference is considered statistically significant at a level of at least p < 0 . 05 ( see methods ) . 30 from 3 experiments . Both kinetochore graphs display data that is relative to WT - C14 untreated controls , which are normalised to 1 . mean level of normalised controls is indicated by dotted lines .

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C. Quantifications (top) and corresponding immunofluorescence images (underneath) of relative kinetochore MAD1 and MAD1-pT716 levels in nocodazole-arrested of MAD1-WT-C14 and MAD1-3EK-C12 cells treated with different doses of AZ-3146 for 30 min. MG132 was included at the time of AZ-3146 addition to prevent mitotic exit. Each dot represents a cell, horizontal lines indicate the median and error bars show the 95% confidence interval. Note, when these vertical bars do not overlap the difference is considered statistically significant at a level of at least p<0.05 (see methods). 30 cells from 3 experiments. Both kinetochore intensity graphs display data that is relative to the WT-C14 untreated controls, which are normalised to 1. The mean level of the normalised controls is indicated by the dotted lines.


D . Duration of mitotic arrest in MAD1 - WT C14 or MAD1 - 3EK C12 arrested in nocodazole then treated with indicated concentrations of AZ - 3146 . Note , 1 . 25 µM AZ - 3146 data is displayed in Figure 4D , but also included here to allow comparison with other drug doses . Graph shows cumulative mean ( ± ) of 3 experiments , 50 per condition per experiment .

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D. Duration of mitotic arrest in MAD1-WT C14 or MAD1-3EK C12 cells arrested in nocodazole and then treated with indicated concentrations of AZ-3146. Note, the 1.25 µM AZ-3146 data is displayed in Figure 4D, but also included here to allow comparison with other drug doses. Graph shows cumulative mean (±SEM) of 3 experiments, 50 cells per condition per experiment.


shows dynamic expression of genes in SF3A / 3B ( E ) SR family ( F ) at each indicated differentiation stage . Both heatmaps were row normalized . heatmaps were scaled with Z - Score using log2 ( FPKM ) expression of indicated components . n = 3 technical replicates .

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The heatmap shows the dynamic expression of genes in the SF3A/3B complex (E) and SR family (F) at each indicated differentiation stage. Both heatmaps were row normalized. The heatmaps were scaled with Z-Score using the log2(FPKM) expression of indicated components. n=3 technical replicates.


mRNA expression of splicing regulator SRSF2 splicing factor PCBP2 during hematopoietic differentiation were measured by RT - qPCR . ACTB gene was used as a control . Results given are mean ± standard deviation ( SD ) . P - values were determined by an unpaired two - Student ' s t - . n ≥ 3 biological replicates .

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The mRNA expression of splicing regulator SRSF2 and splicing factor PCBP2 during hematopoietic differentiation were measured by RT-qPCR. The ACTB gene was used as a control. Results given are mean ± standard deviation (SD). P-values were determined by an unpaired two-tailed Student's t-test. n ≥ 3 biological replicates.


experimental schematic ( upper panel ) . APLNR + on day 2 were FACS - purified treated with various dosages of PLB from day 2 . 5 to 5 during inducing hematopoietic differentiation . On day 5 , generation of CD31 + CD34 + EPCs CD31 + CD34 + CD73 - HEPs were assessed . representative FACS plots showed frequency of APLNR + , CD31 + CD34 + EPCs CD31 + CD34 + CD73 - HEPs without or with PLB treatment . bar graphs show of CD31 + CD34 + EPCs CD31 + CD34 + CD73 - HEPs without or with PLB treatment .

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The experimental schematic (upper panel). APLNR+ cells on day 2 were FACS-purified and treated with various dosages of PLB from day 2.5 to 5 during inducing hematopoietic differentiation. On day 5, the generation of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs were assessed. The representative FACS plots showed the frequency of APLNR+ cells, CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment. The bar graphs show the percentage of CD31+CD34+ EPCs and CD31+CD34+CD73- HEPs without or with PLB treatment.


bar graph represents of CD31 + CD34 + from ( H ) . P - values were calculated by one - way ANOVA followed by Tukey ' s .

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The bar graph represents the percentage of CD31+CD34+ cells from (H). P-values were calculated by one-way ANOVA followed by Tukey's test.


bar graph representing of CD31 + CD34 + from ( K ) . P - values were determined by an unpaired two - Student ' s t - .

The bar graph representing the percentage of CD31+CD34+ cells from (K). P-values were determined by an unpaired two-tailed Student's t-test.


representative FACS plots show frequency of CD31 + CD34 + EPCs in GFP + population at day 5 with 1 . 25 nM PLB supplementation alone or in combination with NUMB _ S ectopic expression from day 2 . 5 . bar graph denotes of CD31 + CD34 + in GFP + as described in ( F ) . P - values were determined by an unpaired two - Student ' s t - .

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The representative FACS plots show the frequency of CD31+CD34+ EPCs in the GFP+ population at day 5 with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5. The bar graph denotes the percentage of CD31+CD34+ in GFP+ cells as described in (F). P-values were determined by an unpaired two-tailed Student's t-test.


On day 5 of hematopoietic differentiation , inclusion or exclusion of NUMB exon 9 were detected by RT - PCR with 1 . 25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2 . 5 . bar graph showing signaling of each specific band from RT - PCR electropherogram .

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On day 5 of hematopoietic differentiation, the inclusion or exclusion of NUMB exon 9 were detected by RT-PCR with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5. The bar graph showing the signaling intensity of each specific band from the RT-PCR electropherogram.


top panel depicts location of specific primers targeting NUMB _ S NUMB _ L isoform . bottom bar graph showing expression of NUMB _ S NUMB _ L in on day 5 of differentiation with 1 . 25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2 . 5 with RT - qPCR .

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The top panel depicts the location of specific primers targeting NUMB_S and NUMB_L isoform. The bottom bar graph showing the expression of NUMB_S and NUMB_L in cells on day 5 of differentiation with 1.25 nM PLB supplementation or SRSF2 exogenous expression alone or their combinational treatment from day 2.5 with RT-qPCR.


plotting expression of NUMB _ S key components in NOTCH signaling in day 2 - APLNR + , day 5 - DMSO treated day 5 - PLB treated ( 2 . 5 nM ) , respectively . NOTCH components were divided into groups of upregulated , downregulated , unchanged by comparison between DMSO PLB treated . was scaled with Z - Score using log2 ( FPKM ) expression of indicated genes . n = 2 technical

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The heatmap plotting the expression of NUMB_S and the key components in NOTCH signaling in day 2-APLNR+ cells, day 5-DMSO treated and day 5-PLB treated cells (2.5 nM), respectively. The NOTCH components were divided into groups of upregulated, downregulated, and unchanged by comparison between DMSO and PLB treated cells. The heatmap was scaled with Z-Score using the log2(FPKM) expression of indicated genes. n=2 technical replicates


representative FACS plots indicating generation of CD31 + CD34 + EPCs on day 5 of differentiation with or without HES1 overexpression . DOX was treated from day 2 . 5 to 5 to induce HES1 overexpression . Statistical of of CD31 + CD34 + EPCs of ( H ) .

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The representative FACS plots indicating the generation of CD31+CD34+ EPCs on day 5 of differentiation with or without HES1 overexpression. DOX was treated from day 2.5 to 5 to induce HES1 overexpression. Statistical analysis of the percentage of CD31+CD34+ EPCs of (H).


B In vivo transduction profile of luciferase reporter vectors displaying variants of enriched library capsid motifs or unmodified wild type AAV2 control capsid . Four after i . v . injection of 5x1010 genomic particles / mouse containing a CMV - luciferase reporter gene , luminescence was measured in brain control organs . Data are shown as bars ( mean ) with plotted data points ( n = 3 / group , 8 - 12 ) .

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B In vivo transduction profile of luciferase reporter vectors displaying variants of the enriched library capsid sequence motifs or unmodified wild type AAV2 control capsid. Four weeks after i.v. injection of 5x1010 genomic particles/mouse containing a CMV-luciferase reporter gene, luminescence was measured in the brain and control organs. Data are shown as bars (mean) with plotted individual data points (n = 3 animals/group, age 8-12 weeks).


A Luciferase reporter gene vectors displaying brain - targeted peptide NRGTEWD ( " BR1 " ) , unmodified wild type AAV2 capsid , or previously reported brain - targeting peptide DSPAHPS ( PPS ) . Vectors were intravenously injected into mice ( 5x1010 genomic particles / mouse ) . Panels show representative examples of n = 5 , 8 - 12 per group . were imaged in dorsal ( left panel ) , ventral ( second from left panel ) , lateral ( second from right right panel ) position , 14 days after vector injection . B Close - up imaging of AAV - BR1 - treated mice . Mice were imaged in dorsal lateral position in a different color scheme allowing detailed visualization of transduced brain in living , even as late as at day 264 after vector injection . C Virtual sections : sagittal , coronal , transaxial ( left panels ) three - dimensional reconstruction ( right panel ) of luminescence of a mouse injected with BR1 vector ( as in A ) . were obtained by measuring different wavelengths of emitted ( Living Image software ) , confirming brain as exclusive source of luminescence .

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A Luciferase reporter gene vectors displaying the brain-targeted peptide NRGTEWD ("BR1"), unmodified wild type AAV2 capsid, or the previously reported brain-targeting peptide DSPAHPS (PPS). Vectors were intravenously injected into mice (5x1010 genomic particles/mouse). Panels show representative examples of n = 5 animals, age 8-12 weeks per group. Animals were imaged in dorsal (left panel), ventral (second from left panel), and lateral (second from right and right panel) position, 14 days after vector injection.B Close-up imaging of AAV-BR1-treated mice. Mice were imaged in dorsal and lateral position in a different color scheme allowing detailed visualization of the transduced brain in living animals, even as late as at day 264 after vector injection.C Virtual sections : sagittal, coronal, and transaxial (left panels) and three-dimensional reconstruction (right panel) of the luminescence images of a mouse injected with BR1 vector (as in A). Images were obtained by measuring different wavelengths of the emitted light (Living Image software), confirming the brain as exclusive source of luminescence.


AAV - BR1 vector harboring luciferase gene under control of CAG promoter was administered intravenously ( 5x1010 genomic particles / mouse , 8 ) . Long - term transgene expression was analyzed by in vivo bioluminescence imaging at 16 time points during a 665 - day period ( n = 2 ) . Original of analyzed ( above ) of luminescence in brain as region of interest = ROI ( bottom ) .

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AAV-BR1 vector harboring the luciferase gene under control of the CAG promoter was administered intravenously (5x1010 genomic particles/mouse, age 8 weeks). Long-term transgene expression was analyzed by in vivo bioluminescence imaging at 16 time points during a 665-day period (n = 2 animals). Original images of analyzed animals (above) and quantification of luminescence in the brain as the region of interest = ROI (bottom).


Luciferase activity vector copy numbers were determined in tissue lysates 14 days after vector administration ( 5x1010 genomic particles / mouse , 8 - 12 ) . A Vector - mediated luminescence . Transgene expression was measured in AAV - BR1 harboring luciferase gene under control of CAG promoter control vectors ( AAV - PPS wild type rAAV2 ) in brain off - target control organs ( left panel ) . Comparison of luminescence in brain mediated by AAV - BR1 control vectors ( right panel ) , * * * * p < 0 . 0001 ( BR1 : brain vs . ) , * * * p = 0 . 0002 ( PPS : heart vs . brain / muscle ) , * * * p = 0 . 0001 ( PPS : heart vs . lung / liver / spleen / kidney ) , * p = 0 . 0101 ( WT : heart vs . lung ) , * p = 0 . 0120 ( WT : heart vs . spleen ) , * p = 0 . 0148 ( WT : heart vs . kidney ) , * p = 0 . 0113 ( WT : heart vs . muscle ) , * * p = 0 . 0011 ( Brain : BR1 vs . PPS ) , * * * p = 0 . 0009 ( Brain : BR1 vs . WT ) .

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Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration (5x1010 genomic particles/mouse, age 8-12 weeks).A Vector-mediated luminescence. Transgene expression was measured in AAV-BR1 harboring the luciferase gene under control of the CAG promoter and control vectors (AAV-PPS and wild type rAAV2) in brain and off-target control organs (left panel). Comparison of luminescence in the brain mediated by AAV-BR1 and control vectors (right panel), ****p < 0.0001 (BR1: brain vs. all), ***p = 0.0002 (PPS: heart vs. brain/ muscle), ***p = 0.0001 (PPS: heart vs. lung/ liver/ spleen/ kidney), *p = 0.0101 (WT: heart vs. lung), *p = 0.0120 (WT: heart vs. spleen), *p = 0.0148 (WT: heart vs. kidney), *p = 0.0113 (WT: heart vs. muscle), **p = 0.0011 (Brain: BR1 vs. PPS), ***p = 0.0009 (Brain: BR1 vs. WT).


Luciferase activity vector copy numbers were determined in tissue lysates 14 days after vector administration ( 5x1010 genomic particles / mouse , 8 - 12 ) . B Biodistribution of AAV - BR1 control vectors ( AAV - PPS wild type rAAV2 ) in brain off - target control organs , excluding spleen ( left panel ) . Genome copy numbers of AAV - BR1 control vectors ( AAV - PPS wild type rAAV2 ) in brain ( right panel ) , * * * * p < 0 . 0001 ( BR1 : brain vs . liver / kidney / muscle ) , * * * p = 0 . 0006 ( BR1 : brain vs . heart ) , * * * p = 0 . 0003 ( BR1 : brain vs . lung ) , * * p = 0 . 0025 ( PPS : kidney vs . muscle ) , * p = 0 . 0378 ( PPS : kidney vs . lung ) , * p = 0 . 0114 ( PPS : kidney vs . liver ) , * * * * * p < 0 . 0001 ( WT : liver vs . ) , * * p = 0 . 0028 ( Brain : BR1 vs . PPS / WT ) .

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Luciferase activity and vector copy numbers were determined in tissue lysates 14 days after vector administration (5x1010 genomic particles/mouse, age 8-12 weeks).B Biodistribution of AAV-BR1 and control vectors (AAV-PPS and wild type rAAV2) in brain and off-target control organs, excluding spleen (left panel). Genome copy numbers of AAV-BR1 and control vectors (AAV-PPS and wild type rAAV2) in the brain (right panel), ****p < 0.0001 (BR1: brain vs. liver/ kidney/ muscle), ***p = 0.0006 (BR1: brain vs. heart), ***p = 0.0003 (BR1: brain vs. lung), **p = 0.0025 (PPS: kidney vs. muscle), *p = 0.0378 (PPS: kidney vs. lung), *p = 0.0114 (PPS: kidney vs. liver), *****p < 0.0001 (WT: liver vs. all), **p = 0.0028 (Brain: BR1 vs. PPS/ WT).


C57BL / 6 mice ( 8 ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of CAG promoter . show representative examples of n = 6 mice . A Representative from cerebellum , olfactory bulb , striatum , cerebral cortex , spinal cord , 14 days after vector injection . BR1 - eGFP - transduced ( green ) were positive for endothelial marker CD31 ( red ) . Scale bars represent 250 µm .

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C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.A Representative images from cerebellum, olfactory bulb, striatum, cerebral cortex, and the spinal cord, 14 days after vector injection. BR1-eGFP-transduced cells (green) were positive for the endothelial marker CD31 (red). Scale bars represent 250 µm.


C57BL / 6mice ( 8 ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of CAG promoter . show representative examples of n = 6 mice . B Higher magnification confocal microscopy . endothelial marker CD31 ( red ) colocalizes with vector - mediated eGFP expression ( green ) . Scale bars represent 50 µm ( upper panel ) or 10 µm ( lower panel ) .

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C57BL/6mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.B Higher magnification confocal microscopy images. The endothelial marker CD31 (red) colocalizes with vector-mediated eGFP expression (green). Scale bars represent 50 µm (upper panel) or 10 µm (lower panel).


C57BL / 6 mice ( 8 ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of CAG promoter . show representative examples of n = 6 mice . C CD13 of pericytes . vector - mediated eGFP expression pattern ( green ) does not colocalize with CD13 ( red ) . Scale bars represent 250 µm ( upper panel ) or 10 µm ( lower panel ) .

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C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.C CD13 staining of pericytes. The vector-mediated eGFP expression pattern (green) does not colocalize with CD13 (red). Scale bars represent 250 µm (upper panel) or 10 µm (lower panel).


C57BL / 6 mice ( 8 ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of CAG promoter . show representative examples of n = 6 mice . D Aquaporin 4 of astrocyte end - feet . vector - mediated eGFP expression ( green ) does not colocalize with aquaporin 4 ( red ) . Scale bars represent 100 µm ( upper panel ) or 10 µm ( lower panel ) .

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C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.D Aquaporin 4 staining of astrocyte end-feet. The vector-mediated eGFP expression (green) does not colocalize with aquaporin 4 (red). Scale bars represent 100 µm (upper panel) or 10 µm (lower panel).


C57BL / 6 mice ( 8 ) were injected with AAV - BR1 harboring an eGFP reporter gene under control of CAG promoter . show representative examples of n = 6 mice . E Expression of eGFP in brain endothelial prepared from C57BL / 6 mice , 14 days after injection with AAV - BR1 - eGFP . Scale bars represent 500 µm ( upper panel ) or 100 µm ( lower panel ) .

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C57BL/6 mice (age 8 weeks) were injected with AAV-BR1 harboring an eGFP reporter gene under control of the CAG promoter. Images show representative examples of n = 6 mice.E Expression of eGFP in primary brain endothelial cells prepared from C57BL/6 mice, 14 days after injection with AAV-BR1-eGFP. Scale bars represent 500 µm (upper panel) or 100 µm (lower panel).


Two after vector injection ( 1 . 8 x1011 genomic particles / ) , into 16 - old mice , Cre - mediated gene recombination driven by CAG promoter was observed mainly in brain endothelial ( lower panel , red ) . No recombination was observed in control without BR1 - iCre - injection ( upper panel ) . CD31 ( green ) was used as a marker for brain endothelial . Panels show representative examples of n = 3 . Scale bars represent 250 µm .

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Two weeks after vector injection (1.8 x1011 genomic particles/ animal), into 16-weeks old mice, Cre-mediated gene recombination driven by the CAG promoter was observed mainly in brain endothelial cells (lower panel, red). No recombination was observed in control animals without BR1-iCre-virus injection (upper panel). CD31 (green) was used as a marker for brain endothelial cells. Panels show representative examples of n = 3 animals. Scale bars represent 250 µm.


BR1 - mediated expression of NEMO or eGFP was driven by CAG promoter . A Immunostaining of cerebral microvessels . Treatment with AAV - BR1 - NEMO normalized string vessels ( arrows , highlighted in square inset ) in Nemo - / + mice as compared to Nemo - / + mice treated with AAV - BR1 - eGFP control vector at postnatal day 8 ( P8 ) . upper left panel shows in untreated wild type control mice . String vessels were identified as capillaries that have lost CD31 - positive ( green ) endothelial but stain for basement collagen IV ( red ) . Scale bars represent 200 µm . lower right panel summarizes quantitative of string vessels in Nemo - / + control mice ( NemoFl or wild type ) at P0 ( n = 3 / group ) at P8 ( n = 6 / group , * p = 0 . 0125 ) . String vessels in cerebral cortex were quantified as of total vessel lengths .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.A Immunostaining of cerebral microvessels. Treatment with AAV-BR1-NEMO normalized string vessels (white arrows, highlighted in white square inset) in Nemo-/+ mice as compared to Nemo-/+ mice treated with the AAV-BR1-eGFP control vector at postnatal day 8 (P8). The upper left panel shows the staining in untreated wild type control mice. String vessels were identified as capillaries that have lost CD31-positive (green) endothelial cells but stain for the basement membrane protein collagen IV (red). Scale bars represent 200 µm. The lower right panel summarizes quantitative analysis of string vessels in Nemo-/+ and control mice (NemoFl or wild type) at P0 (n=3 animals/ group) and at P8 (n=6 animals/ group, *p = 0.0125). String vessels in the cerebral cortex were quantified as percentage of total vessel lengths.


BR1 - mediated expression of NEMO or eGFP was driven by CAG promoter . B of active caspase 3 - positive endothelial at P8 ( n = 5 / group , * p = 0 . 0201 ) .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.B Quantification of active caspase 3-positive endothelial cells at P8 (n= 5 animals/ group, *p = 0.0201).


BR1 - mediated expression of NEMO or eGFP was driven by CAG promoter . C Albumin in brain tissue as indicator for BBB leakage . In Nemo - / + mice treated with AAV - BR1 - NEMO , less albumin was found in brain tissue , indicating less BBB leakage . Representative Western blot with albumin from P8 Nemo - / + mice treated with AAV - BR1 - NEMO or AAV - BR1 - eGFP control vector , respectively , as well as untreated wild type ( WT ) control mice . Right panel : quantitative of Western blots ( n = 4 / group , * p = 0 . 0283 )

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.C Albumin in brain tissue as indicator for BBB leakage. In Nemo-/+ mice treated with AAV-BR1-NEMO, less albumin was found in brain tissue, indicating less BBB leakage. Representative Western blot with albumin from P8 Nemo-/+ mice treated with AAV-BR1-NEMO or AAV-BR1-eGFP control vector, respectively, as well as untreated wild type (WT) control mice. Right panel: quantitative analysis of the Western blots (n = 4 animals/ group, *p = 0.0283)


BR1 - mediated expression of NEMO or eGFP was driven by CAG promoter . D of vector - treated mice . WT control or Nemo - / + mice at P8 treated with AAV - BR1 - NEMO or AAV - BR1 - eGFP ( n = 18 / group , * p = 0 . 0038 ) .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter.D Body weight of vector-treated mice. WT control or Nemo-/+ mice at P8 treated with AAV-BR1-NEMO or AAV-BR1-eGFP (n= 18 animals/ group, *p = 0.0038).


BR1 - mediated expression of NEMO or eGFP was driven by CAG promoter . were at of 8 - 12 . A Representative immunostainings of cerebral microvessels . String vessels ( arrows , highlighted in square inset ) were significantly reduced in NemobeKO mice treated with AAV - BR1 - NEMO compared to NemobeKO mice treated with AAV - BR1 - eGFP control vector . NemoFl mice served as control . Scale bars represent 200 µm . B of string vessel lengths in cerebral cortex as of total vessel lengths . NemobeKO NemoFl mice were treated with AAV - BR1 - NEMO or AAV - BR1 - eGFP control vector ( n = 9 NemoFl + BR1 - eGFP , 10 NemobeKO + BR1 - eGFP , 9 NemoFl + BR1 - NEMO , 13 NemobeKO + BR1 - NEMO ) , * * * * p < 0 . 0001 . C Total vessel measured as total CD31 - positive vessels . Vessels were restored in NemobeKO mice treated with AAV - BR1 - NEMO compared to AAV - BR1 - eGFP injected mice . NemoFl mice served as a control ( n = 9 NemoFl + BR1 - eGFP , 10 NemobeKO + BR1 - eGFP , 9 NemoFl + BR1 - NEMO , 13 NemobeKO + BR1 - NEMO ) , * * * p = 0 . 0007 , * * p = 0 . 0038 ( NemoKO : eGFP vs . NemoFl : NEMO ) , * * p = 0 . 0014 ( NemoKO : NEMO vs . NemoKO : eGFP ) .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.A Representative immunostainings of cerebral microvessels. String vessels (white arrows, highlighted in white square inset) were significantly reduced in NemobeKO mice treated with AAV-BR1-NEMO compared to NemobeKO mice treated with AAV-BR1-eGFP control vector. NemoFl mice served as control animals. Scale bars represent 200 µm.B Quantification of string vessel lengths in the cerebral cortex as percentage of total vessel lengths. NemobeKO and NemoFl mice were treated with AAV-BR1-NEMO or AAV-BR1-eGFP control vector (n=9 NemoFl animals + BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ****p < 0.0001.C Total vessel length measured as total CD31-positive vessels. Vessels were restored in NemobeKO mice treated with AAV-BR1-NEMO compared to the AAV-BR1-eGFP injected mice. NemoFl mice served as a control (n= 9 NemoFl animals +BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ***p = 0.0007, **p = 0.0038 (NemoKO:eGFP vs. NemoFl:NEMO ), **p = 0.0014 (NemoKO:NEMO vs. NemoKO:eGFP).


BR1 - mediated expression of NEMO or eGFP was driven by CAG promoter . were at of 8 - 12 . D IgG albuminWestern blots of brain lysates . Less leakage in BBB was detected in NemobeKO mice treated with AAV - BR1 - NEMO than in NemobeKO mice injected with AAV - BR1 - eGFP ( right panel indicates quantified gel under various treatment conditions ( n = 5 per group ) . NemoFl mice served as controls , * * * * p < 0 . 0001 .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.D IgG and albuminWestern blots of brain lysates. Less leakage in the BBB was detected in NemobeKO mice treated with AAV-BR1-NEMO than in NemobeKO mice injected with AAV-BR1-eGFP (the right panel indicates the quantified gel intensity under the various treatment conditions (n= 5 animals per group). NemoFl mice served as controls, ****p < 0.0001.


BR1 - mediated expression of NEMO or eGFP was driven by CAG promoter . were at of 8 - 12 . E Quantitative immunoglobulin of coronal brain sections of NemoFl NemobeKO mice . Ig extravasation was significantly reduced in AAV - BR1 - NEMO - treated mice ( n = 9 NemoFl + BR1 - eGFP , 10 NemobeKO + BR1 - eGFP , 9 NemoFl + BR1 - NEMO , 13 NemobeKO + BR1 - NEMO ) , * * * * p < 0 . 0001 .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.E Quantitative immunoglobulin staining of coronal brain sections of NemoFl and NemobeKO mice. Ig extravasation was significantly reduced in AAV-BR1-NEMO-treated mice (n= 9 NemoFl animals + BR1-eGFP, 10 NemobeKO animals + BR1-eGFP, 9 NemoFl animals + BR1-NEMO, and 13 NemobeKO animals + BR1-NEMO), ****p < 0.0001.


BR1 - mediated expression of NEMO or eGFP was driven by CAG promoter . were at of 8 - 12 . F Effect of AAV - BR1 vector on BBB permeability . No vector ( left ) or empty AAV - BR1 vector ( right ) was injected i . v . to wild type mice BBB permeability was assessed by extravasation of tracer sodium fluorescein ( n = 7 per group ) . No significant difference was detected ( n . s . ) .

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BR1-mediated expression of NEMO or eGFP was driven by the CAG promoter. All animals were at the age of 8-12 weeks.F Effect of AAV-BR1 vector on BBB permeability. No vector (left) or empty AAV-BR1 vector (right) was injected i.v. to wild type mice and BBB permeability was assessed by extravasation of the fluorescent tracer sodium fluorescein (n=7 animals per group). No significant difference was detected (n.s.).


A LC3 punctum formation in WT GCN5 KO HeLa ( Scale bars , 10 µm ) .

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A LC3 punctum formation in WT and GCN5 KO HeLa cells (Scale bars, 10 µm).


Immunoblot showing LC3 - II formation in WT or GCN5 KO HeLa in presence or absence of lysosome inhibitor Baf .

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Immunoblot showing LC3-II formation in WT or GCN5 KO HeLa cells in the presence or absence of the lysosome inhibitor Baf.


Immunoblot showing LC3 - II formation MB - 3 - treated HeLa in presence or absence of lysosome inhibitor Baf .

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Immunoblot showing LC3-II formation MB-3-treated HeLa cells in the presence or absence of the lysosome inhibitor Baf.


D Formation of LC3 in GCN5 KO HeLa overexpressing Myc - tagged GCN5 or GCN5 - E575Q ( Scale bars , 10 µm ) .

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D Formation of LC3 puncta in GCN5 KO HeLa cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).


E of LC3 in were treated with or without Baf ( Graph represents data from three independent experiments with ≥ 30 per condition ; mean ± ; * * * p < 0 . 001 , Student ' s t - ) .

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E Quantification of LC3 puncta in cells The cells were treated with or without Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


Formation of LC3 in HeLa overexpressing GFP - GCN5 with or without Baf

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Formation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatment


Formation of LC3 in HeLa overexpressing GFP - GCN5 with or without Baf treatment ( Graph represents data from three independent experiments with ≥ 30 per condition ; mean ± ; * p < 0 . 05 , * * p < 0 . 01 , Student ' s t - ; Scale bar , 10 µm ) .

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Formation of LC3 puncta in HeLa cells overexpressing GFP-GCN5 with or without Baf treatment (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bar, 10 µm).


H LC3 - II formation in GFP - GCN5 - overexpressing HeLa .

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H LC3-II formation in GFP-GCN5-overexpressing HeLa cells.


I GFP - p62 in HEK293 stably expressing GFP - p62 . were cultured with GCN5 siRNA with or without CQ .

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I GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were cultured with GCN5 siRNA with or without CQ.


J PDLIM1 IFT20 in GCN5 KO HEK293 with or without transfection of GFP - GCN5 addition of CQ .

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J PDLIM1 and IFT20 protein levels in GCN5 KO HEK293 cells with or without transfection of GFP-GCN5 and addition of CQ.


K Representative of mCherry - Atg8a ( red ) DAPI ( blue ) in Drosophila larval fat in which dGcn5 is overexpressed ( OE ) or silenced ( KD ) using pan - fat driver ( cg - GAL4 ) . Drosophila ( cg - GAL4 / + ) was used as control ( Graph represents data from three independent experiments with ≥ 30 per condition ; mean ± ; * p < 0 . 05 , * * * p < 0 . 001 , Student ' s t - ; Scale bars , 10 µm ) .

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K Representative images of mCherry-Atg8a (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD) using the pan-fat body driver (cg-GAL4). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, ***p < 0.001, Student's t-test; Scale bars, 10 µm).


A LAMP1 ( green ) DAPI ( blue ) in WT GCN5 KO HEK293 ( Scale bars , 10 µm ) .

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A LAMP1 puncta (green) and DAPI (blue) in WT and GCN5 KO HEK293 cells (Scale bars, 10 µm).


B - activated sorting of WT GCN5 KO HEK293 stained with LysoTracker . of 10 , 000 per sample was measured .

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B Fluorescence-activated cell sorting analysis of WT and GCN5 KO HEK293 cells stained with LysoTracker. Fluorescence intensity of 10,000 cells per sample was measured.


C Immunoblot showing lysosomal in three independent clones of GCN5 KO HEK293 . CTSD HC , cathepsin D heavy chain .

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C Immunoblot showing lysosomal protein levels in three independent clones of GCN5 KO HEK293 cells. CTSD HC, cathepsin D heavy chain.


D LAMP1 in GCN5 KO HEK293 overexpressing Myc - tagged GCN5 or GCN5 - E575Q ( Scale bars , 10 µm ) .

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D LAMP1 puncta in GCN5 KO HEK293 cells overexpressing Myc-tagged GCN5 or GCN5-E575Q (Scale bars, 10 µm).


E of LAMP1 in ( A ) ( D ) ( Graph represents data from three independent experiments with ≥ 30 per condition ; mean ± ; * * * p < 0 . 001 , Student ' s t - ) .

E Quantification of LAMP1 puncta in (A) and (D) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


F Hexosaminidase activity in GCN5 KO HEK293 ( mean ± ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - ) .

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F Hexosaminidase activity in GCN5 KO HEK293 cells (mean ± SEM; n = 3 independent experiments; ***p < 0.001, Student's t-test).


G Degradation of EGFR in WT GCN5 KO HEK293 in presence or absence of CQ .

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G Degradation of EGFR in WT and GCN5 KO HEK293 cells in the presence or absence of CQ.


H Representative of LysoTracker ( red ) DAPI ( blue ) in Drosophila larval fat in which dGcn5 is overexpressed ( OE ) or silenced ( KD ) . Drosophila ( cg - GAL4 / + ) was used as control ( Graph represents data from three independent experiments with ≥ 30 per condition ; mean ± ; * p < 0 . 05 , * * p < 0 . 01 , Student ' s t - ; Scale bars , 10 µm ) .

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H Representative images of LysoTracker staining (red) and DAPI (blue) in Drosophila larval fat body in which dGcn5 is overexpressed (OE) or silenced (KD). Drosophila (cg-GAL4/+) was used as the control (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; *p < 0.05, **p < 0.01, Student's t-test; Scale bars, 10 µm).


A of LC3 in WT , GCN5 KO GCN5 / TFEB DKO HeLa in presence or absence of Baf ( Graph represents data from three independent experiments with ≥ 30 per condition ; mean ± ; * * * p < 0 . 001 , Student ' s t - ) .

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A Quantification of LC3 puncta in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


B Immunoblot showing LC3 - II formation in WT , GCN5 KO GCN5 / TFEB DKO HeLa in presence or absence of Baf .

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B Immunoblot showing LC3-II formation in WT, GCN5 KO and GCN5/TFEB DKO HeLa cells in the presence or absence of Baf.


C of LAMP1 in WT , GCN5 KO GCN5 / TFEB DKO HEK293 ( Graph represents data from three independent experiments with ≥ 30 per condition ; mean ± ; * * * p < 0 . 001 , Student ' s t - ) .

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C Quantification of LAMP1 puncta in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


D Immunoblot showing LAMP1 in WT , GCN5 KO GCN5 / TFEB DKO HEK293 .

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D Immunoblot showing LAMP1 levels in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells.


E Acetylation of TFEB - Flag in stable TFEB - Flag - expressing HEK293 with overexpression of histone acetyltransferases as indicated . TFEB - Flag was immunoprecipitated with anti - Flag beads analyzed by immunoblot using anti - acetyl - lysine ( Ace - lys ) .

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E Acetylation of TFEB-Flag in stable TFEB-Flag-expressing HEK293 cells with overexpression of individual histone acetyltransferases as indicated. TFEB-Flag was immunoprecipitated with anti-Flag beads and analyzed by immunoblot using anti-acetyl-lysine (Ace-lys).


F TFEB - Flag acetylation in stable TFEB - Flag - expressing HEK293 transfected with Myc - tagged GCN5 or GCN5 - E575Q after incubation with GCN5 siRNA for 48 h .

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F TFEB-Flag acetylation in the stable TFEB-Flag-expressing HEK293 cells transfected with Myc-tagged GCN5 or GCN5-E575Q after incubation with GCN5 siRNA for 48 h.


G Acetylation of endogenous TFEB in MEF treated with GCN5 inhibitor MB - 3 or p300 / CBP inhibitor C646 . H3 , histone H3 . Ace - H3 ( K9 ) Ace - H3 ( K27 ) were used to show activity of GCN5 p300 , respectively .

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G Acetylation of endogenous TFEB in MEF cells treated with the GCN5 inhibitor MB-3 or the p300/CBP inhibitor C646. H3, histone H3. Ace-H3 (K9) and Ace-H3 (K27) were used to show the activity of GCN5 and p300, respectively.


H Co - precipitation of GCN5 with TFEB . TFEB was immunoprecipitated from MEF using anti - TFEB , precipitates were analyzed using anti - GCN5 .

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H Co-precipitation of GCN5 with TFEB. TFEB was immunoprecipitated from MEF cells using anti-TFEB, and the precipitates were analyzed using anti-GCN5.


I In vitro acetylation assays of TFEB . Purified recombinant TFEB was incubated with Myc - GCN5 or Myc - GCN5 - E575Q immunoprecipitated from HEK293 .

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I In vitro acetylation assays of TFEB. Purified recombinant TFEB was incubated with Myc-GCN5 or Myc-GCN5-E575Q immunoprecipitated from HEK293 cells.


J Acetylation of Flag - tagged TFEB or TFEB mutants expressed in HEK293 . 3KR : Lys 116 , Lys 274 , Lys 279 were replaced by Arg .

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J Acetylation of Flag-tagged TFEB or TFEB mutants expressed in HEK293 cells. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg.


A RT - qPCR of expression of TFEB target genes in WT , GCN5 KO GCN5 / TFEB DKO HEK293 . Data information : In this Figure , data are presented as mean ± ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - .

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A RT-qPCR analysis of the expression of TFEB target genes in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


B RT - qPCR of dMitf target gene expression in Drosophila larval fat with silencing of dGcn5 . Data information : In this Figure , data are presented as mean ± ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - .

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B RT-qPCR analysis of dMitf target gene expression in Drosophila larval fat body with silencing of dGcn5. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


C Expression of TFEB target genes in HEK293 transfected with or without TFEB - WT or TFEB - 3KR . Data information : In this Figure , data are presented as mean ± ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - .

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C Expression of TFEB target genes in HEK293 cells transfected with or without TFEB-WT or TFEB-3KR. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


D , E Luciferase activity measured in WT , GCN5 KO GCN5 / TFEB DKO HEK293 ( D ) , in HEK293 with TFEB - WT or TFEB - 3KR transfection ( E ) . were transfected or co - transfected with a TFEB - luciferase reporter . 3KR : Lys 116 , Lys 274 , Lys 279 were replaced by Arg . Data information : In this Figure , data are presented as mean ± ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - .

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D, E Luciferase activity measured in WT, GCN5 KO and GCN5/TFEB DKO HEK293 cells (D), and in HEK293 cells with TFEB-WT or TFEB-3KR transfection (E). The cells were transfected or co-transfected with a TFEB-luciferase reporter. 3KR: Lys 116, Lys 274, and Lys 279 were replaced by Arg. Data information: In this Figure, data are presented as mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test.


A Subcellular localization of TFEB in WT or GCN5 KO HEK293 treated with or without Torin1 ( Scale bars , 10 µm ) .

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A Subcellular localization of TFEB in WT or GCN5 KO HEK293 cells treated with or without Torin1 (Scale bars, 10 µm).


B Subcellular localization of Flag - tagged TFEB or TFEB mutants in HEK293 treated with or without Torin1 . were stained with anti - Flag antibody ( Scale bars , 10 µm ) .

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B Subcellular localization of Flag-tagged TFEB or TFEB mutants in HEK293 cells treated with or without Torin1. Cells were stained with anti-Flag antibody (Scale bars, 10 µm).


C ChIP - qPCR of TFEB binding to promoter of its target genes CLCN7 , GLA CTSD . Normal mouse IgG primers against upstream region lacking CLEAR sites ( up ) were used as negative controls ( mean ± ; n = 3 independent experiments ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - ) .

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C ChIP-qPCR analysis of TFEB binding to the promoter of its target genes CLCN7, GLA and CTSD. Normal mouse IgG and primers against the upstream region lacking CLEAR sites (up) were used as negative controls (mean ± SEM; n = 3 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, Student's t-test).


D Electrophoresis mobility shift of TFEB binding to promoter of GLA . A fragment from GLA promoter containing TFEB binding site was incubated with purified recombinant TFEB or each of TFEB mutants , was subjected to electrophoresis . Asterisk indicates fragment from GAPDH promoter which was used as a TFEB non - binding control .

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D Electrophoresis mobility shift assay of TFEB binding to the promoter of GLA. A DNA fragment from the GLA promoter containing the TFEB binding site was incubated with purified recombinant TFEB or each of the TFEB mutants, and was subjected to electrophoresis. Asterisk indicates the DNA fragment from the GAPDH promoter which was used as a TFEB non-binding DNA control.


F Co - precipitation of Flag - tagged TFEB TFEB mutants with Myc - TFEB in HEK293T . Immunoprecipitation was carried out with anti - Myc beads , precipitates were analyzed using anti - Flag .

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F Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-TFEB in HEK293T cells. Immunoprecipitation was carried out with anti-Myc beads, and the precipitates were analyzed using anti-Flag.


Co - precipitation of Flag - tagged TFEB TFEB mutants with Myc - tagged MITF ( G ) in HEK293T .

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Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged MITF (G) in HEK293T cells.


Co - precipitation of Flag - tagged TFEB TFEB mutants with Myc - tagged TFE3 ( H ) in HEK293T .

Co-precipitation of Flag-tagged TFEB and TFEB mutants with Myc-tagged TFE3 (H) in HEK293T cells.


I TFEB homodimer formation detected by glutaraldehyde ( GA ) crosslinking . Purified recombinant GST - tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde , then products were analyzed by immunoblotting using anti - TFEB .

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I TFEB homodimer formation detected by glutaraldehyde (GA) crosslinking. Purified recombinant GST-tagged TFEB or TFEB mutants was incubated with or without glutaraldehyde, then the products were analyzed by immunoblotting using anti-TFEB.


A Acetylation of TFEB - Flag , Myc - GCN5 histone H3 in HEK293 . were either starved of glucose or , or treated with Torin1 or MB - 3 .

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A Acetylation of TFEB-Flag, Myc-GCN5 and histone H3 in HEK293 cells. The cells were either starved of glucose or amino acids, or treated with Torin1 or MB-3.


B Acetylation of endogenous TFEB in HeLa .

B Acetylation of endogenous TFEB in HeLa cells.


C GCN5 activity measured by in vitro acetylation using Myc - GCN5 immunoprecipitated from HEK293T purified histone H3 as substrate . ( mean ± ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - ) .

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C GCN5 activity measured by in vitro acetylation assay using Myc-GCN5 immunoprecipitated from HEK293T cells and purified histone H3 as the substrate. (mean ± SEM; n = 3 independent experiments; ***p < 0.001, Student's t-test).


D Co - precipitation of TFEB - Flag with Myc - TFEB in HEK293

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D Co-precipitation of TFEB-Flag with Myc-TFEB in HEK293 cells


E LC3 - II formation in HeLa transfected with Flag - tagged TFEB or TFEB mutants after TFEB RNAi . were treated with or without acid starvation .

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E LC3-II formation in HeLa cells transfected with Flag-tagged TFEB or TFEB mutants after TFEB RNAi. The cells were treated with or without amino acid starvation.


F GFP - p62 in HEK293 stably expressing GFP - p62 . were treated with or without addition of CQ .

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F GFP-p62 levels in HEK293 cells stably expressing GFP-p62. The cells were treated with or without addition of CQ.


Representative of mCherry - Atg8a ( red ) ( G ) in Drosophila larval fat in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as control DAPI ( blue ) was used to indicate nucleus ( Scale bars , 10 µm ) .

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Representative images of mCherry-Atg8a (red) (G) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).


Representative of LysoTracker ( red ) ( H ) in Drosophila larval fat in which dMitf or dMitf - 2KQ ( K445Q / K450Q ) was overexpressed . Drosophila ( cg - GAL4 / + ) was used as control DAPI ( blue ) was used to indicate nucleus ( Scale bars , 10 µm ) .

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Representative images of LysoTracker (red) staining (H) in Drosophila larval fat body in which dMitf or dMitf-2KQ (K445Q/K450Q) was overexpressed. Drosophila (cg-GAL4/+) was used as the control and DAPI staining (blue) was used to indicate the cell nucleus (Scale bars, 10 µm).


of mCherry - ATG8a ( I ) ( Graph represents data from three independent experiments with ≥ 30 per condition ; mean ± ; * * * p < 0 . 001 , Student ' s t - ) .

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Quantification of mCherry-ATG8a puncta (I) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


of LysoTracker ( J ) ( Graph represents data from three independent experiments with ≥ 30 per condition ; mean ± ; * * * p < 0 . 001 , Student ' s t - ) .

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Quantification of LysoTracker puncta (J) (Graph represents data from three independent experiments with ≥ 30 cells per condition; mean ± SEM; ***p < 0.001, Student's t-test).


level of insoluble Tau in head of adult Drosophila with or without CQ feeding . indicated genes were transgenically expressed in eyes of Drosophila using GMR - Gal4 driver . Drosophila ( GMR - Gal4 / + ) was used as control .

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The level of insoluble Tau protein in the head of adult Drosophila with or without CQ feeding. The indicated genes were transgenically expressed in the eyes of the Drosophila using the GMR-Gal4 driver. Drosophila (GMR-Gal4/+) was used as the control.


C Representative of adult Drosophila eyes expressing indicated transgenes under control of GMR - Gal4 driver . Drosophila were fed with or without CQ . Drosophila ( GMR - Gal4 / + ) was used as control .

C Representative light micrographs of adult Drosophila eyes expressing the indicated transgenes under the control of the GMR-Gal4 driver. The Drosophila were fed with or without CQ. Drosophila (GMR-Gal4/+) was used as the control.


D Distribution of different eye following genetic manipulations Drosophila were fed with or without CQ ( 30 flies per condition were randomly selected for analyses ; * p < 0 . 05 , * * * p < 0 . 001 , ꭓ2 ) .

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D Distribution of the different eye phenotypes following genetic manipulations The Drosophila were fed with or without CQ (30 flies per condition were randomly selected for analyses; *p < 0.05, ***p < 0.001, ꭓ2 test).


( A ) EAE severity in 6 - to - 8 - week - old ( left panel ) ( right panel ) wild - type ( WT ) ERα - / - mice ( n = 7 - 9 mice per group ) . ( B ) EAE sensitivity to IFNβ in 6 - to - 8 - week - old ( top panel ) ( bottom panel ) WT ERα - / - mice . Vehicle ( Veh ) or IFNβ ( 47 μg / kg ) were i . p . injected . every other day from 0 to 10 dpi ( n = 8 - 12 mice 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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

(A) EAE severity in 6-to-8-week-old female (left panel) and male (right panel) wild-type (WT) and ERα-/- mice (n=7-9 mice per group). (B) EAE sensitivity to IFNβ in 6-to-8-week-old female (top panel) and male (bottom panel) WT and ERα-/- mice. Vehicle (Veh) or IFNβ (47 μg/kg) were i.p. injected. every other day from 0 to 10 dpi (n= 8-12 mice per group).


( D ) Sensitivity of EAE to IFNβ in Cyp19 - / - ovary - transferred WT mice WT ovary - transferred Cyp19 - / - mice ( n = 5 - 6 mice ) .

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(D) Sensitivity of EAE to IFNβ in Cyp19-/- ovary-transferred female WT mice and WT ovary-transferred Cyp19-/- mice (n=5-6 mice).


( E ) Estradiol in LNs ( A : axillary , B : branchial , C : cervical , I : inguinal ) in sham - operated OVX mice ( n = 5 mice per group ) .

(E) Estradiol levels in LNs (A: axillary, B: branchial, C: cervical, and I: inguinal) in sham-operated and OVX mice (n=5 mice per group).


( A ) Brightfield micrograph of ventral horns of Golgi - Cox stained lumber ( L ) 5 spinal cord section of WT ERα - / - EAE mice at 33 dpi with quantitation of stained in gray matter . dotted indicates division between gray matter , V . H . represents ventral horn ( n = 5 mice 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 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

(A) Brightfield micrograph of ventral horns of Golgi-Cox stained lumber (L)5 spinal cord section of male WT and ERα-/- EAE mice at 33 dpi with quantitation of area stained in gray matter. The white dotted line indicates the division between white and gray matter, V.H. represents the ventral horn (n=5 mice per group).