Figure 4A . Binding of Annexin V Alexa Fluor 488 to control ( Scr ) and MDGI - silenced ( shMDGI1 ) BT12 cells was measured by flow cytometric analyses five days after silencing . The analysis was performed by gating the cells into two populations : P1 ( small , granular ) and P2 ( normal - sized cells ) ( 30 000 analysed events / cell line , n = 3 ) . B . Geometric mean fluorescence ( FL - 1 ) relative to the controls ( Scr ) that was set as 1 in P1 and P2 in MDGI - silenced ( shMDGI1 ) cells . Data are represented as mean ± SD . * P < 0 . 05 , two - tailed , nonparametric Mann - Whitney ' s U - test . C , D Representative micrographs of non - treated control BT12 cells ( C ) and cells treated with 2 mM LLOMe for 4 h ( D ) before staining with the anti - galectin - 1 antibodies ( LGALS1 ) ( green ) . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm . E , F Representative micrographs of LGALS1 - stained ( green ) BT12 control ( E ) and MDGI - silenced cells ( F ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm . G , H Representative micrographs of LGALS1 - stained ( green ) BT13 control ( G ) and MDGI - silenced cells ( H ) six days after silencing . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm . I . Percentage of LGALS1 - positive puncta staining in the control ( Scr ) and MDGI - silenced ( sh1 ) cells . In total 0 . 4 - 1 . 2x104 LGALS1 - stained cells were analysed from 50 mm2 coverslip areas ( n = 6 ) . Data are represented as mean ± SD . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test . J . Quantification of the percentage of dead cells six days after MDGI silencing and LGALS1 staining ( n = 100 ) in control ( Scr ) and MDGI - silenced ( sh1 ) BT12 and BT13 cells . Data are represented as mean ± SD . * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test . K . Cytoplasmic cathepsin B activity in the control ( Scr ) and MDGI - silenced ( sh1 ) BT12 and BT13 cells . Data are represented as mean ± SD . * * P < 0 . 01 ; * * P < 0 . 001 , two - tailed , nonparametric Mann - Whitney ' s U - test . L . Number of live cells in MDGI - silenced BT12 and BT13 cells incubated without ( sh1 ) or with ( sh1 + K777 ) the pan - cathepsin inhibitor ( K777 ) after 1 , 3 and 5 days of incubation . Data shows the percentage of live cells compared to the control ( Scr ) cells set as 100 % . Data are represented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 , two - tailed , nonparametric Mann - Whitney ' s U - test .

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

Figure 4A. Binding of Annexin V Alexa Fluor 488 to control (Scr) and MDGI-silenced (shMDGI1) BT12 cells was measured by flow cytometric analyses five days after silencing. The analysis was performed by gating the cells into two populations: P1 (small, granular) and P2 (normal-sized cells) (30 000 analysed events/cell line, n = 3). B. Geometric mean fluorescence (FL-1) relative to the controls (Scr) that was set as 1 in P1 and P2 in MDGI-silenced (shMDGI1) cells. Data are represented as mean ± SD. *P < 0.05, two-tailed, nonparametric Mann-Whitney's U-test.C, D Representative micrographs of non-treated control BT12 cells (C) and cells treated with 2 mM LLOMe for 4 h (D) before staining with the anti-galectin-1 antibodies (LGALS1) (green). Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.E, F Representative micrographs of LGALS1-stained (green) BT12 control (E) and MDGI-silenced cells (F) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.G, H Representative micrographs of LGALS1-stained (green) BT13 control (G) and MDGI-silenced cells (H) six days after silencing. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.I. Percentage of LGALS1-positive puncta staining in the control (Scr) and MDGI-silenced (sh1) cells. In total 0.4-1.2x104 LGALS1-stained cells were analysed from 50 mm2 coverslip areas (n = 6). Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.J. Quantification of the percentage of dead cells six days after MDGI silencing and LGALS1 staining (n = 100) in control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.K. Cytoplasmic cathepsin B activity in the control (Scr) and MDGI-silenced (sh1) BT12 and BT13 cells. Data are represented as mean ± SD. **P < 0.01; **P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.L. Number of live cells in MDGI-silenced BT12 and BT13 cells incubated without (sh1) or with (sh1+K777) the pan-cathepsin inhibitor (K777) after 1, 3 and 5 days of incubation. Data shows the percentage of live cells compared to the control (Scr) cells set as 100 %. Data are represented as mean ± SD. *P < 0.05; **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.


Figure 5A . Proportions ( mol % ) of saturated ( Sat ) , monounsaturated ( Mono ) , diunsaturated ( Di ; total of 2 double bonds in the acyl chains ) , and polyunsaturated ( Poly ; total of 3 - 7 double bonds in the acyl chains ) lipid species from the two main phospholipid classes ( phosphatidylcholine PC and phosphatidylethanolamine PE ) of the lysosomal fractions of the BT12 and BT13 control ( Scr ) and MDGI - silenced ( shMDGI ) cells . A representative graph of two independent experiments is shown . B . Fold change of the main PC and PE species in the lysosomal membranes of MDGI - silenced ( shMDGI ) and control ( Scr ) cells ( Log2 , by mol % data ) . Abbreviations : [ Lipid class ] [ total number of acyl chain carbons ] : [ total number of double bonds in the chains ] . A representative heatmap of two independent experiments is shown . C . Profiles of ceramide ( Cer ) species ( mol % ) in the lysosomal membranes of MDGI - silenced ( shMDGI ) and control ( Scr ) BT12 and BT13 cells . Abbreviations : Cer [ number of acyl chain carbons ] : [ number of double bonds in the acyl chain ] ; all principal species containing a sphingosine chain . A representative graph of two independent experiments is shown . D . Ratio of the 24 : 1 to 24 : 0 ( mol % . mol % ) Cer species in the lysosomal membranes of MDGI - silenced ( shMDGI ) and control ( Scr ) BT12 and BT13 cells . A representative graph of two independent experiments is shown .

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

Figure 5A. Proportions (mol %) of saturated (Sat), monounsaturated (Mono), diunsaturated (Di; total of 2 double bonds in the acyl chains), and polyunsaturated (Poly; total of 3-7 double bonds in the acyl chains) lipid species from the two main phospholipid classes (phosphatidylcholine PC and phosphatidylethanolamine PE) of the lysosomal fractions of the BT12 and BT13 control (Scr) and MDGI-silenced (shMDGI) cells. A representative graph of two independent experiments is shown.B. Fold change of the main PC and PE species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) cells (Log2, by mol % data). Abbreviations: [Lipid class] [total number of acyl chain carbons]:[total number of double bonds in the chains]. A representative heatmap of two independent experiments is shown.C. Profiles of ceramide (Cer) species (mol %) in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. Abbreviations: Cer [number of acyl chain carbons]:[number of double bonds in the acyl chain]; all principal species containing a sphingosine chain. A representative graph of two independent experiments is shown.D. Ratio of the 24:1 to 24:0 (mol %.mol %) Cer species in the lysosomal membranes of MDGI-silenced (shMDGI) and control (Scr) BT12 and BT13 cells. A representative graph of two independent experiments is shown.


Figure 6A . Measurement of the BT12 , BT13 , and ZH305 glioblastoma cell viability using the MTT - assay at the indicated clemastine concentrations and time points ( n = 12 ) . A dashed line marks the 50 % cell viability . B . Measurement of the viability of normal human endothelial cells ( HuAR2T ) , normal human astrocytes ( NHA ) , and embryonic kidney ( HEK293T ) cells at the indicated clemastine concentrations and time points ( n = 12 ) . A dashed line marks the 50 % cell viability . C . Representative micrographs of BT12 , BT13 , and ZH305 glioblastoma cells treated with 1 µg / ml of clemastine for 24 h and stained with anti - galectin - 1 ( LGALS1 ) antibody ( green ) . Non - treated cells served as control . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 20 µm . D . Representative micrographs of normal human endothelial cells ( HuAR2T ) , normal human astrocytes ( NHA ) , and kidney cells ( HEK293T ) treated with 1 µg / ml clemastine for 24 h and stained with anti - LGALS1 antibody ( green ) . Non - treated cells served as control . Nuclei were visualised by using DAPI ( blue ) . Scale bar : 10 µm .

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Figure 6A. Measurement of the BT12, BT13, and ZH305 glioblastoma cell viability using the MTT-assay at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.B. Measurement of the viability of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and embryonic kidney (HEK293T) cells at the indicated clemastine concentrations and time points (n = 12). A dashed line marks the 50 % cell viability.C. Representative micrographs of BT12, BT13, and ZH305 glioblastoma cells treated with 1 µg/ml of clemastine for 24 h and stained with anti-galectin-1 (LGALS1) antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 20 µm.D. Representative micrographs of normal human endothelial cells (HuAR2T), normal human astrocytes (NHA), and kidney cells (HEK293T) treated with 1 µg/ml clemastine for 24 h and stained with anti-LGALS1 antibody (green). Non-treated cells served as control. Nuclei were visualised by using DAPI (blue). Scale bar: 10 µm.


Figure 7A . Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells ) , and daily treated with saline vehicle ( Ve ) or 50 mg / kg clemastine ( Cle , 100 mg / kg single dose administered at day 1 ) for 12 days starting at day 15 after tumour implantation . BT12 ( parental ( wt , Ve n = 9 , Cle n = 12 ) and control shRNA infected ( Ve n = 5 , Cle n = 5 ) , ZH305 ( Ve n = 10 , Cle n = 10 ) , and BT13 ( Ve n = 10 , Cle n = 9 ) . Human glioma cells were visualised with an anti - human vimentin ( hVim , red ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 1 mm . B , C Quantification of the number of the invading single BT12 wt glioblastoma cells ( B ) and distance of the invaded cells from the primary tumour ( C ) of vehicle ( black bars , Ve ) and clemastine - treated ( white bars , Cle ) animals ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . D . Quantification of the number of secondary tumours ( diameter > 300 µm ) in the murine brain for the indicated treatments and tumour models . BT12 ( parental ( wt , Ve n = 9 , Cle n = 12 ) and control shRNA infected ( Ve n = 5 , Cle n = 5 ) , and BT13 ( Ve n = 10 , Cle n = 9 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . E . Quantification of the number of angiotropic BT12 cells that have co - opted existing blood vessels in the murine brain for the indicated treatments ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . Quantification of the number of TUNEL positive cells in the murine brain for the indicated treatments ( Ve n = 9 , Cle n = 12 ) . Data are represented as mean ± SEM . * * * * P < 0 . 0001 , two - tailed , nonparametric Mann - Whitney ' s U - test . G . Representative micrographs of consecutive brain sections ( separating distance : 9 µm ) showing xenografts treated with vehicle or clemastine and labelled with the anti - human vimentin ( red in left panels ) and TUNEL ( red in right panels ) . Nuclei were visualised by using DAPI ( white ) . Scale bar : 50 µm . H . Kaplan - Meier survival curve of the BT12 xenografted animals treated with vehicle ( green line , n = 15 ) and clemastine ( black line , n = 12 ) . Survival of the animals has been measured and pooled from two independent experiments . P = 0 . 044 , Mantel - Cox Log - rank test .

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

Figure 7A. Representative whole coronal section micrographs of murine brain intracranially implanted with various glioblastoma cells), and daily treated with saline vehicle (Ve) or 50 mg/kg clemastine (Cle, 100 mg/kg single dose administered at day 1) for 12 days starting at day 15 after tumour implantation. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), ZH305 (Ve n = 10, Cle n = 10), and BT13 (Ve n = 10, Cle n = 9). Human glioma cells were visualised with an anti-human vimentin (hVim, red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.B, C Quantification of the number of the invading single BT12 wt glioblastoma cells (B) and distance of the invaded cells from the primary tumour (C) of vehicle (black bars, Ve) and clemastine-treated (white bars, Cle) animals (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.D. Quantification of the number of secondary tumours (diameter >300 µm) in the murine brain for the indicated treatments and tumour models. BT12 (parental (wt, Ve n = 9, Cle n = 12) and control shRNA infected (Ve n = 5, Cle n = 5), and BT13 (Ve n = 10, Cle n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.E. Quantification of the number of angiotropic BT12 cells that have co-opted existing blood vessels in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.Quantification of the number of TUNEL positive cells in the murine brain for the indicated treatments (Ve n = 9, Cle n = 12). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.G. Representative micrographs of consecutive brain sections (separating distance: 9 µm) showing xenografts treated with vehicle or clemastine and labelled with the anti-human vimentin (red in left panels) and TUNEL (red in right panels). Nuclei were visualised by using DAPI (white). Scale bar: 50 µm.H. Kaplan-Meier survival curve of the BT12 xenografted animals treated with vehicle (green line, n = 15) and clemastine (black line, n = 12). Survival of the animals has been measured and pooled from two independent experiments. P = 0.044, Mantel-Cox Log-rank test.


Figure 1MIP of cerebral vessels of 3dpf Tg ( kdrl : HRAS - mCherry ) s916 embryo ( grey LUT ; inverted ) . Higher magnification panel showing two kugeln ( arrowheads ) arising from the middle mesencephalic central artery ( MMCtA ) . Diameter of kugeln at 3dpf ( mean ± s . e . m . 10 . 13 ± 0 . 49 ; n = 93 kugeln from 32 3dpf embryos ; 3 experimental repeats ) . Number of kugeln per embryo in 3 , 4 and 5dpf animals was not statistically significantly different ( mean ± s . e . m . 3dpf 5 . 47 ± 1 . 09 , 4dpf 7 . 47 ± 1 . 46 , 5dpf 7 . 09 ± 1 . 55 ; p = 0 . 8571 ; 3dpf : 175 kugeln from 32 embryos ; 4dpf : 239 kugeln from 32 embryos , 5dpf : 227 kugeln from 32 embryos ; 4 experimental repeats ; Kruskal - Wallis test ) . Kugeln ( arrowheads ) could be observed in 28dpf animals . MIP of cerebral vessels of 4dpf Tg ( fli1aep : CAAX - eGFP ) embryo ( grey LUT ; inverted ) . Higher magnification panel shows a kugel ( arrowhead ) protruding from the posterior mesencephalic central artery ( PMCtA ) . Single Z - plane micrograph of cerebral vessels of a 3dpf Tg ( gata1 : dsRed ) , injected with a Tol2 - fli1a : myr - Cherry construct showing two kugeln ( arrowheads ) in the higher magnification panel protruding from the MMCtA . Locations and proportion of kugeln on cerebral vessels ( colour coded by vessel ; total n = 107 kugeln from 34 4dpf embryos ; 3 experimental repeats ) . AMCtA - anterior mesencephalic central artery , BCA - basal communicating artery , CaDI - caudal division of internal carotid artery , CMV - communicating vessel , CtA - central arteries , MMCtA - middle mesencephalic central artery , MtA - metencephalic artery , PCS - posterior communicating segment , PMBC - posterior midbrain channel , PMCtA - posterior mesencephalic central artery , PrA - prosencephalic artery .

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

Figure 1MIP of cerebral vessels of 3dpf Tg(kdrl:HRAS-mCherry)s916 embryo (grey LUT; inverted). Higher magnification panel showing two kugeln (arrowheads) arising from the middle mesencephalic central artery (MMCtA).Diameter of kugeln at 3dpf (mean ± s.e.m. 10.13 ± 0.49; n=93 kugeln from 32 3dpf embryos; 3 experimental repeats).Number of kugeln per embryo in 3, 4 and 5dpf animals was not statistically significantly different (mean ± s.e.m. 3dpf 5.47 ± 1.09, 4dpf 7.47 ± 1.46, 5dpf 7.09 ± 1.55; p = 0.8571; 3dpf: 175 kugeln from 32 embryos; 4dpf: 239 kugeln from 32 embryos, 5dpf: 227 kugeln from 32 embryos; 4 experimental repeats; Kruskal-Wallis test).Kugeln (arrowheads) could be observed in 28dpf animals.MIP of cerebral vessels of 4dpf Tg(fli1aep:CAAX-eGFP) embryo (grey LUT; inverted). Higher magnification panel shows a kugel (arrowhead) protruding from the posterior mesencephalic central artery (PMCtA).Single Z-plane micrograph of cerebral vessels of a 3dpf Tg(gata1:dsRed), injected with a Tol2-fli1a:myr-Cherry construct showing two kugeln (arrowheads) in the higher magnification panel protruding from the MMCtA.Locations and proportion of kugeln on cerebral vessels (colour coded by vessel; total n=107 kugeln from 34 4dpf embryos; 3 experimental repeats). AMCtA - anterior mesencephalic central artery, BCA - basal communicating artery, CaDI - caudal division of internal carotid artery, CMV - communicating vessel, CtA - central arteries, MMCtA - middle mesencephalic central artery, MtA - metencephalic artery, PCS - posterior communicating segment, PMBC - posterior midbrain channel, PMCtA - posterior mesencephalic central artery, PrA - prosencephalic artery.


Figure 2MIPs of a time - series light sheet acquisition shows three different kugeln ( kugel 1 - black arrowhead ; kugel 2 - grey arrowhead ; kugel 3 - unfilled arrowhead ) protruding and retracting from parent vessels ( 2min intervals ; inverted LUT ) . Kugeln persisted for variable durations before regression into the parent vessel ( 43 kugeln from 9 4dpf embyros ; 4 experimental repeats ) . MIPs taken 20min apart showing examples of different kugel behaviour including shape changes ( asterisk ) , expansion ( circle ) , retraction ( triangle ) , or little change ( pentagon ) . MIPs taken 1h apart show that kugeln may be observed at one time - point ( red arrowhead , 55 : 20 : 00 ) but not 1h later in the same animal ( 56 : 20 : 00 ) and to develop on other vessels 1h later ( 57 : 20 : 00 ) which again have regressed by 58 : 20 : 00 . Kymographs generated by line - scanning across the diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes , while no such oscillations were observed in adjacent similarly sized cerebral vessels ( all images grey LUT ; inverted ) .

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

Figure 2MIPs of a time-series light sheet acquisition shows three different kugeln (kugel 1 - black arrowhead; kugel 2 - grey arrowhead; kugel 3 - unfilled arrowhead) protruding and retracting from parent vessels (2min intervals; inverted LUT).Kugeln persisted for variable durations before regression into the parent vessel (43 kugeln from 9 4dpf embyros; 4 experimental repeats).MIPs taken 20min apart showing examples of different kugel behaviour including shape changes (asterisk), expansion (circle), retraction (triangle), or little change (pentagon).MIPs taken 1h apart show that kugeln may be observed at one time-point (red arrowhead, 55:20:00) but not 1h later in the same animal (56:20:00) and to develop on other vessels 1h later (57:20:00) which again have regressed by 58:20:00.Kymographs generated by line-scanning across the diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes, while no such oscillations were observed in adjacent similarly sized cerebral vessels (all images grey LUT; inverted).


Figure 3Double transgenic visualizing endothelial membrane ( red ) and endothelial nuclei ( green ) . Endothelial nuclei ( arrowhead ) were observed close to but never within the kugel ( unfilled arrowhead ) . Double transgenic showing endothelial membrane ( red ) and endothelial F - actin ( green ) . F - actin was found to localize at the neck of the kugelnDouble transgenic showing endothelial membrane ( red ) and endothelial cytoplasm ( green ) . The cytoplasmic reporter was visible in the parent vessel but not in the kugel ( unfilled arrowhead ) . Triple transgenic showing endothelial membrane ( red ) , neurons ( green ) and erythrocytes ( red ) . Surrounding neurons were excluded from the volume of the kugel ( unfilled arrowhead ) and erythrocytes were not observed inside kugeln ( arrowhead ) . Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo ( 100nM 1 hour ; * * p = 0 . 0041 ; control n = 21 embryos 8 . 05 ± 1 . 76 ( mean ± s . e . m . ) ; Latrunculin n = 21 embryos 17 . 19 ± 2 . 93 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) . Latrunculin B treatment statistically significantly reduced kugel diameter ( * p = 0 . 0164 ; control n = 169 kugeln from 21 embryos 7 . 56 ± 2 . 25 ( mean ± s . e . m . ) ; Latrunculin n = 361 kugeln from 21 embryos 5 . 91 ± 1 . 78 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - test ) . Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo ( 25 µM 1h ; * * * * p < 0 . 0001 ; control n = 22 embryos 3 . 77 ± 0 . 56 ( mean ± s . e . m . ) , Blebbistatin n = 24 embryos 1 . 08 ± 0 . 22 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) . Blebbistatin treatment had no effect on kugel diameter ( p = 0 . 3731 ; control n = 83 kugeln from 22 embryos 6 . 27 ± 0 . 72 ( mean ± s . e . m . ) , Blebbistatin n = 26 kugeln from 24 embryos 7 . 02 ± 0 . 89 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

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

Figure 3Double transgenic visualizing endothelial membrane (red) and endothelial nuclei (green). Endothelial nuclei (arrowhead) were observed close to but never within the kugel (unfilled arrowhead).Double transgenic showing endothelial membrane (red) and endothelial F-actin (green). F-actin was found to localize at the neck of the kugelnDouble transgenic showing endothelial membrane (red) and endothelial cytoplasm (green). The cytoplasmic reporter was visible in the parent vessel but not in the kugel (unfilled arrowhead).Triple transgenic showing endothelial membrane (red), neurons (green) and erythrocytes (red). Surrounding neurons were excluded from the volume of the kugel (unfilled arrowhead) and erythrocytes were not observed inside kugeln (arrowhead).Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo (100nM 1 hour; **p = 0.0041; control n=21 embryos 8.05 ± 1.76 (mean ± s.e.m.); Latrunculin n=21 embryos 17.19 ± 2.93 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).Latrunculin B treatment statistically significantly reduced kugel diameter (*p = 0.0164; control n=169 kugeln from 21 embryos 7.56 ± 2.25 (mean ± s.e.m.); Latrunculin n=361 kugeln from 21 embryos 5.91 ± 1.78 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo (25 µM 1h; ****p <0.0001; control n=22 embryos 3.77 ± 0.56 (mean ± s.e.m.), Blebbistatin n=24 embryos 1.08 ± 0.22 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).Blebbistatin treatment had no effect on kugel diameter (p = 0.3731; control n=83 kugeln from 22 embryos 6.27 ± 0.72 (mean ± s.e.m.), Blebbistatin n=26 kugeln from 24 embryos 7.02 ± 0.89 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Figure 4MIP of the cerebral vessels of a 4dpf embryo before exsanguination ( grey LUT ; inverted ) . MIP of the same embryo after exsanguination , which did not alter kugel size . Time - lapse imaging of an embryo with transiently halted cardiac contraction ( using Tricaine ) . Despite absent blood flow kugeln still changed shape ( grey arrowhead ) , retained shape ( white arrowhead ) or protruded and retracted ( black arrowhead ; time post cessation of flow is indicated on micrographs ; 3dpf ; grey inverted LUT ) . Time - lapse of an embryo with transiently halted cardiac contraction as in ( C ) showed that kugel diameter still oscillates ( time post cessation of flow ) . Dextran microangiography filled perfused vessels with dextran ( arrowhead ) , while dextran was not observed to enter kugeln ( unfilled arrowhead ) . Inhibition of cardiac contraction by tnnt2a morpholino ( MO ) knockdown statistically significantly reduced kugel number per embryo ( * * * * p < 0 . 0001 ; control n = 20 embryos 5 . 10 ± 1 . 47 ( mean ± s . e . m . ) , tnnt2a MO = 18 embryos 0 . 06 ± 0 . 06 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

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

Figure 4MIP of the cerebral vessels of a 4dpf embryo before exsanguination (grey LUT; inverted). MIP of the same embryo after exsanguination, which did not alter kugel size.Time-lapse imaging of an embryo with transiently halted cardiac contraction (using Tricaine). Despite absent blood flow kugeln still changed shape (grey arrowhead), retained shape (white arrowhead) or protruded and retracted (black arrowhead; time post cessation of flow is indicated on micrographs; 3dpf; grey inverted LUT). Time-lapse of an embryo with transiently halted cardiac contraction as in (C) showed that kugel diameter still oscillates (time post cessation of flow).Dextran microangiography filled perfused vessels with dextran (arrowhead), while dextran was not observed to enter kugeln (unfilled arrowhead).Inhibition of cardiac contraction by tnnt2a morpholino (MO) knockdown statistically significantly reduced kugel number per embryo (****p<0.0001; control n=20 embryos 5.10 ± 1.47 (mean ± s.e.m.), tnnt2a MO=18 embryos 0.06 ± 0.06 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).


Figure 5Kugeln were studied in the double - transgenic Tg ( fli1a : LifeAct - mClover ) sh467 , Tg ( kdrl : HRAS - mCherry ) s916 . BLECs were mClover positive and mCherry - negative ( BLECs - black arrowheads ; kugeln - white arrowheads ) . No direct physical interaction between kugeln ( BLECs - black arrowheads ; kugeln - white arrowheads ) and BLECs was observed ( n = 21 4dpf embryos ; 3 experimental repeats ) . Depth - coded MIPs showed that BLECs and kugeln are present on different anatomical planes ( depths ; purple ventral ( v ) , white dorsal ( d ) ; BLECs - black arrowheads ; kugeln - white arrowheads ) . Ccbe1 morpholino ( MO ) injection led to a loss of lymphatics ( white arrowhead ) . Kugel number was not statistically significantly altered by ccbe1 morpholino ( MO ) knockdown ( p = 0 . 3496 ; control MO n = 22 embryos 0 . 95 ± 0 . 28 ( mean ± s . e . m . ) , ccbe1 MO n = 23 embryos 0 . 57 ± 0 . 14 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) . Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown ( p = 0 . 8783 ; control MO n = 21 kugeln from 22 embryos 7 . 75 ± 1 . 29 ( mean ± s . e . m . ) , ccbe1 MO n = 13 kugeln from 23 embryos 8 . 93 ± 2 . 35 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) . Injection of IgG - conjugated Alexa 647 into the tectum showed no uptake ( blue ; unfilled arrowhead ) by kugeln ( white arrowhead ; 140 kugeln from 17 4dpf embryos ; 2 experimental repeats ) .

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

Figure 5Kugeln were studied in the double-transgenic Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916. BLECs were mClover positive and mCherry-negative (BLECs - black arrowheads; kugeln - white arrowheads).No direct physical interaction between kugeln (BLECs - black arrowheads; kugeln - white arrowheads) and BLECs was observed (n=21 4dpf embryos; 3 experimental repeats). Depth-coded MIPs showed that BLECs and kugeln are present on different anatomical planes (depths; purple ventral (v), white dorsal (d); BLECs - black arrowheads; kugeln - white arrowheads).Ccbe1 morpholino (MO) injection led to a loss of lymphatics (white arrowhead).Kugel number was not statistically significantly altered by ccbe1 morpholino (MO) knockdown (p = 0.3496; control MO n=22 embryos 0.95 ± 0.28 (mean ± s.e.m.), ccbe1 MO n=23 embryos 0.57 ± 0.14 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).Kugel diameter was not statistically significantly altered by ccbe1 MO knockdown (p = 0.8783; control MO n=21 kugeln from 22 embryos 7.75 ± 1.29 (mean ± s.e.m.), ccbe1 MO n=13 kugeln from 23 embryos 8.93 ± 2.35 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).Injection of IgG-conjugated Alexa 647 into the tectum showed no uptake (blue; unfilled arrowhead) by kugeln (white arrowhead; 140 kugeln from 17 4dpf embryos; 2 experimental repeats).


Figure 6The possible interaction of macrophages with kugeln was studied in the transgenic Tg ( kdrl : HRAS - mCherry ) s916 , Tg ( fms : GAL4 . VP16 ) i186 , Tg ( UAS - E1b : nfsB . mCherry ) il149 , visualizing macrophages and the vasculature simultaneously . Coloured depth - coding indicates the different anatomical depths of kugeln and macrophages ( ventral purple , dorsal white ) . Maximum intensity projections of z - stacks acquired every 10 minutes during a time - lapse showed no interaction of macrophages ( unfilled arrowhead ) with kugeln ( filled arrowhead ; n = 21 kugeln in 8 3dpf embryos ) . Macrophages are all white , indicating they are on a different z - plane to the vessels seen .

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

Figure 6The possible interaction of macrophages with kugeln was studied in the transgenic Tg(kdrl:HRAS-mCherry)s916, Tg(fms:GAL4.VP16)i186, Tg(UAS-E1b:nfsB.mCherry)il149, visualizing macrophages and the vasculature simultaneously. Coloured depth-coding indicates the different anatomical depths of kugeln and macrophages (ventral purple, dorsal white).Maximum intensity projections of z-stacks acquired every 10 minutes during a time-lapse showed no interaction of macrophages (unfilled arrowhead) with kugeln (filled arrowhead; n=21 kugeln in 8 3dpf embryos). Macrophages are all white, indicating they are on a different z-plane to the vessels seen.


Figure 7VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number ( * * * * p < 0 . 0001 ; DMSO control n = 30 embryos 8 . 53 ± 2 . 62 ( mean ± s . e . m . ) ; AV951 n = 31 embryos 21 . 10 ± 2 . 71 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; Mann - Whitney U test ) . Mean kugel diameter was not statistically significantly different after AV951 treatment ( p = 0 . 7890 ; DMSO control n = 243 kugeln from 30 embryos 9 . 94 ± 0 . 76 ( mean ± s . e . m . ) ; AV951 n = 652 kugeln from 31 embryos 9 . 73 ± 0 . 32 ( mean ± s . e . m . ) ; 4dpf ; 4 experimental repeats ; Student ' s t - test ) . DAF - FM staining , a vital dye for nitric oxide ( NO ) , showed that 57 . 56 % of kugeln were positive for nitric oxide reactivity ( 2 . 5µM incubation from 96 - 102hpf ; n = 22 4dpf embryos ; 118 of 205 kugeln filled ; 3 experimental repeats ) . Images show representative " filled " ( DAF - FM positive ) and " unfilled " ( DAF - FM negative ) kugeln . Time - lapse acquisition with DAF - FM revealed that kugeln contained NO early in their biogenesis ( grey LUT ; inverted ) . Application of LysoTracker , a vital dye that stains lysosomes or acidic compartments , showed that 17 . 08 % of kugeln contained acidic contents ( 8 . 33µM ; 96 - 101hpf ; n = 22 4dpf embryos ; 62 of 363 kugeln filled ; 3 experimental repeats ) . Images show representative " filled " ( Lysotracker positive ) and " unfilled " ( Lysotracker negative ) kugeln . The nitric oxide synthase ( NOS ) inhibitor L - NAME had no statistically significant effect on kugel number per embryo ( 0 . 5mM L - NAME 18h ; p = 0 . 4870 ; control n = 22 embryos 14 . 27 ± 3 . 35 ( mean ± s . e . m . ) , L - NAME n = 24 embryos 7 . 46 ± 1 . 61 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) . Diameter of kugeln was not affected by L - NAME ( p = 0 . 4161 ; control n = 315 kugeln from 22 embryos 8 . 77 ± 0 . 49 ( mean ± s . e . m . ) , L - NAME n = 179 kugeln from 24 8 . 66 ± 0 . 71 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) .

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

Figure 7VEGF inhibition by 2h treatment with AV951 statistically significantly increased kugel number (****p<0.0001; DMSO control n=30 embryos 8.53 ± 2.62 (mean ± s.e.m.); AV951 n=31 embryos 21.10 ± 2.71 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Mann-Whitney U test).Mean kugel diameter was not statistically significantly different after AV951 treatment (p = 0.7890; DMSO control n=243 kugeln from 30 embryos 9.94 ± 0.76 (mean ± s.e.m.); AV951 n=652 kugeln from 31 embryos 9.73 ± 0.32 (mean ± s.e.m.); 4dpf; 4 experimental repeats; Student's t-test).DAF-FM staining, a vital dye for nitric oxide (NO), showed that 57.56% of kugeln were positive for nitric oxide reactivity (2.5µM incubation from 96-102hpf; n=22 4dpf embryos; 118 of 205 kugeln filled; 3 experimental repeats). Images show representative "filled" (DAF-FM positive) and "unfilled" (DAF-FM negative) kugeln.Time-lapse acquisition with DAF-FM revealed that kugeln contained NO early in their biogenesis (grey LUT; inverted).Application of LysoTracker, a vital dye that stains lysosomes or acidic compartments, showed that 17.08% of kugeln contained acidic contents (8.33µM; 96-101hpf; n=22 4dpf embryos; 62 of 363 kugeln filled; 3 experimental repeats). Images show representative "filled" (Lysotracker positive) and "unfilled" (Lysotracker negative) kugeln.The nitric oxide synthase (NOS) inhibitor L-NAME had no statistically significant effect on kugel number per embryo (0.5mM L-NAME 18h; p = 0.4870; control n=22 embryos 14.27 ± 3.35 (mean ± s.e.m.), L-NAME n=24 embryos 7.46 ± 1.61 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).Diameter of kugeln was not affected by L-NAME (p = 0.4161; control n=315 kugeln from 22 embryos 8.77 ± 0.49 (mean ± s.e.m.), L-NAME n=179 kugeln from 24 8.66 ± 0.71 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).


Figure 8Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT ( * * * * p < 0 . 0001 ; control n = 24 embryos 9 . 38 ± 1 . 77 ( mean ± s . e . m . ) , DAPT n = 24 embryos 0 . 92 ± 0 . 28 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Mann - Whitney U test ) . Mean kugel diameter was not statistically significantly altered by DAPT treatment ( p = 0 . 0832 ; control n = 225 kugeln from 24 embryos 8 . 64 ± 0 . 54 ( mean ± s . e . m . ) ; DAPT n = 22 kugeln from 24 embryos 6 . 88 ± 0 . 86 ( mean ± s . e . m . ) ; 4dpf ; 3 experimental repeats ; Student ' s t - test ) . A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln ( white arrowheads pointing to kugeln ; n = 122 kugeln from 23 3dpf embryos ; 2 experimental repeats ) . Studying Notch signalling in the Notch reporter line Tg ( TP1glob : venusPest ) s940 , showed high Notch levels in the mid - brain , but , again , no difference in expression at the sites of kugeln ( n = 45 kugeln from 19 4dpf embryos ; 2 experimental repeats ) . Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV - 939 ( * * * p = 0 . 0003 ; control n = 22 embryos 1 . 32 ± 0 . 24 ( mean ± s . e . m . ) ; XAV - 939 n = 21 embryos 4 . 29 ± 0 . 71 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) . Kugel diameter was not statistically significantly altered by XAV - 939 ( p = 0 . 4098 ; control n = 29 kugeln from 22 embryos 8 . 78 ± 0 . 76 ( mean ± s . e . m . ) ; XAV - 939 n = 90 kugeln from 21 embryos 7 . 99 ± 0 . 56 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Student ' s t - test ) Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV ( p 0 . 0359 ; control n = 22 embryos 1 . 04 ± 0 . 34 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 21 embryos 4 . 24 ± 1 . 28 ( mean ± s . e . m . ) ; 3dpf ; 3 experimental repeats ; Mann - Whitney U test ) . The diameter of kugel was not statistically significantly altered by GSK3 inhibition ( p 0 . 5555 ; control n = 23 kugeln from 22 embryos 7 . 26 ± 0 . 89 ( mean ± s . e . m . ) ; GSK3 inhibitor n = 89 kugeln from 21 embryos 8 . 09 ± 0 .

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

Figure 8Kugel number was significantly decreased by inhibition of Notch signalling by 12h treatment with 50µM DAPT (****p<0.0001; control n=24 embryos 9.38 ± 1.77 (mean ± s.e.m.), DAPT n=24 embryos 0.92 ± 0.28 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Mann-Whitney U test).Mean kugel diameter was not statistically significantly altered by DAPT treatment (p = 0.0832; control n=225 kugeln from 24 embryos 8.64 ± 0.54 (mean ± s.e.m.); DAPT n=22 kugeln from 24 embryos 6.88 ± 0.86 (mean ± s.e.m.); 4dpf; 3 experimental repeats; Student's t-test).A transgenic dll4 reporter line showed no indication of different dll4 expression at the sites of kugeln (white arrowheads pointing to kugeln; n=122 kugeln from 23 3dpf embryos; 2 experimental repeats).Studying Notch signalling in the Notch reporter line Tg(TP1glob:venusPest)s940, showed high Notch levels in the mid-brain, but, again, no difference in expression at the sites of kugeln (n=45 kugeln from 19 4dpf embryos; 2 experimental repeats).Kugel number was statistically significantly increased by inhibition of Wnt signalling by 4h treatment with 10µm XAV-939 (***p = 0.0003; control n=22 embryos 1.32 ± 0.24 (mean ± s.e.m.); XAV-939 n=21 embryos 4.29 ± 0.71 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).Kugel diameter was not statistically significantly altered by XAV-939 (p = 0.4098; control n=29 kugeln from 22 embryos 8.78 ± 0.76 (mean ± s.e.m.); XAV-939 n=90 kugeln from 21 embryos 7.99 ± 0.56 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Student's t-test)Kugel number was statistically significantly increased by activation of Wnt signalling by 4h treatment with 10µm GSK3 inhibition XV (p 0.0359; control n=22 embryos 1.04 ± 0.34 (mean ± s.e.m.); GSK3 inhibitor n=21 embryos 4.24 ± 1.28 (mean ± s.e.m.); 3dpf; 3 experimental repeats; Mann-Whitney U test).The diameter of kugel was not statistically significantly altered by GSK3 inhibition (p 0.5555; control n=23 kugeln from 22 embryos 7.26 ± 0.89 (mean ± s.e.m.); GSK3 inhibitor n=89 kugeln from 21 embryos 8.09 ± 0.97


Figure 1Double fluorescent whole mount in situ hybridization ( WM - ISH ) to detect her6 ( green ) and her9 ( magenta ) ; coronal view ( left panel ) and transversal section ( right panel ) , scale bar 20μm ; 30 - 32hpf ; annotations denote anterior ( A ) , posterior ( P ) , otic vesicle ( ov ) , dorsal ( D ) and ventral ( V ) . Representative time series example of Her6 : : Venus expression during development , in the midbrain and hindbrain . Confocal images represented as 2D Maximum projection ; longitudinal view ; scale bar 50μm ; otic vesicle ( ov ) ; also included in Movie EV1 . r1 : rhombomere 1 , r2 : rhombomere 2 , r3 : rhombomere 3 , r4 : rhombomere 4 , r5 : rhombomere 5 , r6 : rhombomere 6 . Intensity mean of Her6 : : Venus per rhombomere area over development grouped by expression level , related to the r1 - r6 regions in panel ( c ) : ( d ) r1 and r2 ; ( e ) r3 and r4 ; ( f ) r5 and r6 . Transversal view of r6 in Her6 : : Venus embryos over time ; Her6 : : Venus protein expression domains : a ventral domain ( arrows ) and a more dorsal lateral domain ( arrowheads ) ; the caax - mRFP was used as membrane marker ( magenta ) ; scale bars 20μm ; images at 30 - 40hpf are maximum projection of 4 z - stacks from Movie EV2 . Quantification of Her6 : : Venus ( + ) cell number ( green ) compared to total cell number ( black ) over development . Proportional changes in Her6 : : Venus ( + ) cell numbers during development ; bars indicate median and interquartile range of counts collected from 3 different z - stacks per embryo and one embryo per condition ; one - way ANOVA with Bonferroni multiple comparison test with significance p < 0 . 01Nuclear abundance and nuclear concentration of Her6 : : Venus protein in homozygous embryos at different stages during development measured by fluorescence correlation spectroscopy ( FCS ) ; bars indicate median and interquartile range of ( 19hpf : 6 embryos , 50 cells ) , ( 29hpf : 6 embryos , 86 cells ) , ( 33hpf : 5 embryos , 76 cells ) , ( 35hpf : 6 embryos , 59 cells ) and ( 48hpf : 6 embryos , 89 cells ) ; Kruskal - Wallis with Dunn ' s multiple comparison test , significance p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * .

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

Figure 1Double fluorescent whole mount in situ hybridization (WM-ISH) to detect her6 (green) and her9 (magenta); coronal view (left panel) and transversal section (right panel), scale bar 20μm; 30-32hpf; annotations denote anterior (A), posterior (P), otic vesicle (ov), dorsal (D) and ventral (V).Representative time series example of Her6::Venus expression during development, in the midbrain and hindbrain. Confocal images represented as 2D Maximum projection; longitudinal view; scale bar 50μm; otic vesicle (ov); also included in Movie EV1. r1: rhombomere 1, r2: rhombomere 2, r3: rhombomere 3, r4: rhombomere 4, r5: rhombomere 5, r6: rhombomere 6.Intensity mean of Her6::Venus per rhombomere area over development grouped by expression level, related to the r1-r6 regions in panel (c) : (d) r1 and r2; (e) r3 and r4; (f) r5 and r6.Transversal view of r6 in Her6::Venus embryos over time; Her6::Venus protein expression domains: a ventral domain (arrows) and a more dorsal lateral domain (arrowheads); the caax-mRFP was used as membrane marker (magenta); scale bars 20μm; images at 30-40hpf are maximum projection of 4 z-stacks from Movie EV2.Quantification of Her6::Venus(+) cell number (green) compared to total cell number (black) over development.Proportional changes in Her6::Venus(+) cell numbers during development; bars indicate median and interquartile range of counts collected from 3 different z-stacks per embryo and one embryo per condition; one-way ANOVA with Bonferroni multiple comparison test with significance p<0.01Nuclear abundance and nuclear concentration of Her6::Venus protein in homozygous embryos at different stages during development measured by fluorescence correlation spectroscopy (FCS); bars indicate median and interquartile range of (19hpf: 6 embryos, 50 cells), (29hpf: 6 embryos, 86 cells), (33hpf: 5 embryos, 76 cells), (35hpf: 6 embryos, 59 cells) and (48hpf: 6 embryos, 89 cells); Kruskal-Wallis with Dunn's multiple comparison test, significance p<0.05*, p<0.001***, p<0.0001****.


Figure 23D confocal representative images of a single neural progenitor cell ( red arrow ) tracked over time , data starting from 34hpf ; scale bar 10μm , images from Movie EV3 . Single cell time series of Her6 : : Venus , mKeima - H2B and Her6 : : Venus signal normalized by mKeima - H2B corresponding to the cell in ( c ) .

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Figure 23D confocal representative images of a single neural progenitor cell (red arrow) tracked over time, data starting from 34hpf; scale bar 10μm, images from Movie EV3.Single cell time series of Her6::Venus, mKeima-H2B and Her6::Venus signal normalized by mKeima-H2B corresponding to the cell in (c).


Figure 3Chromogenic WM - ISH of miR - 9 using miR - 9 LNA 5 ' - Dig observed at different stages during development ; longitudinal view , anterior to the left . Transverse section of double fluorescent WM - ISH for her6 ( green ) and mir - 9 - 4 ( magenta ) imaged in fixed embryo at 31hpf ; scale bar 30μm . Confocal imaging of CTRL and MBSm embryo showing Her6 : : Venus expression in the hindbrain , rhombomeres 3 to 6 ( r3 - r6 ) over the course of development ; longitudinal view , scale 30μm , otic vesicle ( ov ) . Images are representing 2D maximum projection . Her6 : : Venus expression ( red arrows ) in hindbrain , rhombomere 6 of live CTRL and MBSm embryos at 52hpf ; edge of ventricular zone shown in yellow ; transversal view ; scale 30μm . Her6 : : Venus protein abundance in CTRL versus MBSm homozygous embryos observed in hindbrain , r6 between 34hpf and 69hpf ; bars indicate median and interquartile range of ( 34hpf CTRL : 36 cells , 1 embryo ) , ( 34hpf MBSm : 35 cells , 1 embryo ) , ( 48 ± 2hpf CTRL : 172 cells , 7 embryos ) , ( 48 ± 2hpf MBSm : 124 cells , 5 embryos ) , ( 56 ± 2hpf CTRL : 69 cells , 4 embryos ) , ( 56 ± 2hpf MBSm : 31 cells , 2 embryos ) , ( 69hpf CTRL : 12 cells , 1 embryo ) , ( 69hpf MBSm : 6 cells , 2 embryos ) ; Kruskal - Wallis with Dunn ' s multiple comparison test , significance : p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * . Comparative profiles of Her6 protein abundance over development in CTRL and MBSm embryos quantified as median of single cell abundance data in ( f ) .

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

Figure 3Chromogenic WM-ISH of miR-9 using miR-9 LNA 5'-Dig observed at different stages during development; longitudinal view, anterior to the left.Transverse section of double fluorescent WM-ISH for her6 (green) and mir-9-4 (magenta) imaged in fixed embryo at 31hpf; scale bar 30μm.Confocal imaging of CTRL and MBSm embryo showing Her6::Venus expression in the hindbrain, rhombomeres 3 to 6 (r3-r6) over the course of development; longitudinal view, scale 30μm, otic vesicle (ov). Images are representing 2D maximum projection.Her6::Venus expression (red arrows) in hindbrain, rhombomere 6 of live CTRL and MBSm embryos at 52hpf; edge of ventricular zone shown in yellow; transversal view; scale 30μm. Her6::Venus protein abundance in CTRL versus MBSm homozygous embryos observed in hindbrain, r6 between 34hpf and 69hpf; bars indicate median and interquartile range of (34hpf CTRL: 36 cells, 1 embryo), (34hpf MBSm: 35 cells, 1 embryo), (48±2hpf CTRL: 172 cells, 7 embryos), (48±2hpf MBSm: 124 cells, 5 embryos), (56±2hpf CTRL: 69 cells, 4 embryos), (56±2hpf MBSm: 31 cells, 2 embryos), (69hpf CTRL: 12 cells, 1 embryo), (69hpf MBSm: 6 cells, 2 embryos); Kruskal-Wallis with Dunn's multiple comparison test, significance: p<0.05*, p<0.001***, p<0.0001****. Comparative profiles of Her6 protein abundance over development in CTRL and MBSm embryos quantified as median of single cell abundance data in (f).


Figure 4Representative examples of single cell oscillators observed in CTRL ( a ) and MBSm ( b ) embryos imaged from 34hpf onwards ; time series represent Her6 : : Venus expression relative to mKeima - H2B ( top panel ) and detrended relative signal ( bottom panel ) ; parameters reported for log - likelihood ratio ( LLR ) and period correspond to KOUosc . Pairwise analysis comparing proportion of oscillators in CTRL versus MBSm embryos indicated as dots ; paired t test with two tail significance p < 0 . 01 * * . Pairwise analysis comparing periods observed in CTRL versus MBSm oscillatory cells ; dots indicate median per experiment per condition ; paired t test with two tail not significant . Representative examples of detrended Her6 : : Venus relative to mKeima - H2B in non - oscillatory single cells observed in CTRL ( f ) and MBSm ( g ) embryos imaged from 34hpf onwards .

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

Figure 4Representative examples of single cell oscillators observed in CTRL (a) and MBSm (b) embryos imaged from 34hpf onwards; time series represent Her6::Venus expression relative to mKeima-H2B (top panel) and detrended relative signal (bottom panel); parameters reported for log-likelihood ratio (LLR) and period correspond to KOUosc .Pairwise analysis comparing proportion of oscillators in CTRL versus MBSm embryos indicated as dots; paired t test with two tail significance p<0.01**.Pairwise analysis comparing periods observed in CTRL versus MBSm oscillatory cells; dots indicate median per experiment per condition; paired t test with two tail not significant.Representative examples of detrended Her6::Venus relative to mKeima-H2B in non-oscillatory single cells observed in CTRL (f) and MBSm (g) embryos imaged from 34hpf onwards.


Figure 6Representative examples of triple fluorescent whole mount WM - ISH labelling of gfap ( green ) , elavl3 ( magenta ) and her6 ( grey ) domains of expression ( top panels ) in hindbrain rhombomere6 ( r6 ) in wild - type embryo observed at 34hpf ; merged images indicate how the her6 expression domain overlaps with the progenitor zone ( NP = gfap ( + ) / elavl3 ( - ) ) , transition zone ( T = gfap ( + ) / elavl3 ( + ) ) and neurogenic zone ( N = gfap ( - ) / elavl3 ( + ) ) ; ( bottom - right panel ) schematic representation showing the her6 domain spanning the NP , T and N zones with quantification of distances from dorsal ; transversal view ; annotations denote dorsal ( D ) and ventral ( V ) ; scale bar 30μm . Single molecule fluorescent in situ hybridization ( smFISH ) showing her6 ( green ) , elavl3 ( magenta ) and dapi nuclear staining ( blue ) obtained from hindbrain ( r6 ) sections of wild type embryo at 34hpf ; head arrows indicates examples of co - existence of transcriptional active sites for her6 and elavl3 ; scale bar 3μm . Triple fluorescent WM - ISH for gfap ( green ) and elavl3 ( magenta ) and her6 ( grey ) in the hindbrain ( r6 ) of CTRL ( top ) and MBSm ( bottom ) embryos at 52hpf ; dorsal ( D ) ; ventral ( V ) ; scale bar 30μm . Magnification of inset from merged gfap / elavl3 in ( c ) showing CTRL ( top ) and MBSm ( bottom ) embryos with corresponding NP / T / N and NP / T zones respectively ; dorsal ( D ) ; ventral ( V ) ; scale bar 30μm . Normalized intensity mean of elavl3 and gfap along the DV axis spanning the NP / T / N zones in CTRL and NP / T zones in MBSm respectively ; region of interest ( ROI ) delineates high elavl3 versus gfap in CTRL ( T and N zones ) while in MBSm only the T zone is observed , overlap of elavl3 and gfap intensity mean peaks . Normalized mean of elavl3 and gfap intensities in ROI observed in : ( f ) uninjected ( UI ) ( 5 embryos , 62 slices ) , ( g ) control ( CTRL ) ( 5 embryos , 35 slices ) and ( h ) MBSm ( 8 embryos , 62 slices ) conditions ; bars represent mean and SEM ; multiple t - test with Benjamini , Krieger and Yekuteli discovery , significance : p < 0 . 05 * , p < 0 . 001 * * * , p < 0 . 0001 * * * * . Transverse sections of double fluorescent WM - ISH for gfap ( green ) and neuroD4 ( blue ) in the hindbrain ( r6 ) of CTRL ( top ) and MBSm ( bottom ) embryos at 52hpf ; dorsal ( D ) ; ventral ( V ) ; scale bar 30μm . Chromogenic WM - ISH showing neuroD4 expression intensity in : uninjected ( UI ) 51 / 56 embryos ( 91 % ) , CTRL 50 / 54 embryos ( 93 % ) and mutant ( MBSm ) ; 36 / 65 embryos ( 55 % ) ; longitudinal view , scale bar 100μm .

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

Figure 6Representative examples of triple fluorescent whole mount WM-ISH labelling of gfap (green), elavl3 (magenta) and her6 (grey) domains of expression (top panels) in hindbrain rhombomere6 (r6) in wild-type embryo observed at 34hpf; merged images indicate how the her6 expression domain overlaps with the progenitor zone (NP=gfap(+)/elavl3(-)), transition zone (T=gfap(+)/elavl3(+)) and neurogenic zone (N=gfap(-)/elavl3(+)); (bottom-right panel) schematic representation showing the her6 domain spanning the NP, T and N zones with quantification of distances from dorsal; transversal view; annotations denote dorsal (D) and ventral (V); scale bar 30μm.Single molecule fluorescent in situ hybridization (smFISH) showing her6 (green), elavl3 (magenta) and dapi nuclear staining (blue) obtained from hindbrain (r6) sections of wild type embryo at 34hpf; head arrows indicates examples of co-existence of transcriptional active sites for her6 and elavl3; scale bar 3μm.Triple fluorescent WM-ISH for gfap (green) and elavl3 (magenta) and her6 (grey) in the hindbrain (r6) of CTRL (top) and MBSm (bottom) embryos at 52hpf; dorsal (D); ventral (V); scale bar 30μm. Magnification of inset from merged gfap/elavl3 in (c) showing CTRL (top) and MBSm (bottom) embryos with corresponding NP/T/ N and NP/T zones respectively; dorsal (D); ventral (V); scale bar 30μm. Normalized intensity mean of elavl3 and gfap along the DV axis spanning the NP/T/N zones in CTRL and NP/T zones in MBSm respectively; region of interest (ROI) delineates high elavl3 versus gfap in CTRL (T and N zones) while in MBSm only the T zone is observed, overlap of elavl3 and gfap intensity mean peaks.Normalized mean of elavl3 and gfap intensities in ROI observed in: (f) uninjected (UI) (5 embryos, 62 slices), (g) control (CTRL) (5 embryos, 35 slices) and (h) MBSm (8 embryos, 62 slices) conditions; bars represent mean and SEM; multiple t-test with Benjamini, Krieger and Yekuteli discovery, significance: p<0.05*, p<0.001***, p<0.0001****.Transverse sections of double fluorescent WM-ISH for gfap (green) and neuroD4 (blue) in the hindbrain (r6) of CTRL (top) and MBSm (bottom) embryos at 52hpf; dorsal (D); ventral (V); scale bar 30μm.Chromogenic WM-ISH showing neuroD4 expression intensity in: uninjected (UI) 51/56 embryos (91%), CTRL 50/54 embryos (93%) and mutant (MBSm); 36/65 embryos (55 %); longitudinal view, scale bar 100μm.


Figure 2 ( b ) Experimental validation of predicted synergistic SR - based combinational therapies in head and neck cancer : A table summarizing the experimentally observed synergism between primary drugs and their predicted rescuer - targeting treatments in 5 HNSC cell lines , based on drug treatment experiments . Synergism was estimated using standard Fa - CI analysis . The table displays the average combination index ( CI ; synergism CI < 1 , additivity effect CI = 1 , antagonism CI > 1 , NAN indeterminate CI ) at 50 % growth inhibition ( Fraction affected ) . Combinations that are synergistic are colored blue ( black otherwise ) for each cell lines tested . The inset shows an example of CI calculation for BYL719 and Dasatinib combination in HN12 cell lines based on the corresponding dose matrix ( number indicates % cell viability at 48h , n = 3 ) , and Fa - CI curve .

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Figure 2(b) Experimental validation of predicted synergistic SR-based combinational therapies in head and neck cancer: A table summarizing the experimentally observed synergism between primary drugs and their predicted rescuer-targeting treatments in 5 HNSC cell lines, based on drug treatment experiments. Synergism was estimated using standard Fa-CI analysis. The table displays the average combination index (CI; synergism CI < 1, additivity effect CI = 1, antagonism CI > 1, NAN indeterminate CI) at 50% growth inhibition (Fraction affected). Combinations that are synergistic are colored blue (black otherwise) for each cell lines tested. The inset shows an example of CI calculation for BYL719 and Dasatinib combination in HN12 cell lines based on the corresponding dose matrix (number indicates % cell viability at 48h, n = 3), and Fa-CI curve.


Figure 3 ( a ) Experimental testing of the predicted SR ( DU ) rescuers of DNMT1 via drug combination experiments in 18 NSCLC cell lines insensitive to Decitabine . The matrix displays drug interactions between Decitabine , a DNMT1 inhibitor , and inhibitors of its predicted rescuer genes ( X - axis ) across 18 NSCLC cell lines ( Y - axis ) that are insensitive to Decitabine . Row labels present rescuer genes and their inhibitors . Colors in the matrix show whether the interactions found are significantly synergistic ( red ) , antagonistic ( green ) or non - significant ( in gray ) . Values in the matrix show average synergism ( < 1 synergism and > 1 antagonism Thirteen predicted DU - SR rescuers ( red lines ) , two predicted DD SR rescuers ( green lines ) of DNMT1 and one random control ( JAK3i ) were tested .

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Figure 3(a) Experimental testing of the predicted SR (DU) rescuers of DNMT1 via drug combination experiments in 18 NSCLC cell lines insensitive to Decitabine. The matrix displays drug interactions between Decitabine, a DNMT1 inhibitor, and inhibitors of its predicted rescuer genes (X-axis) across 18 NSCLC cell lines (Y-axis) that are insensitive to Decitabine. Row labels present rescuer genes and their inhibitors. Colors in the matrix show whether the interactions found are significantly synergistic (red), antagonistic (green) or non-significant (in gray). Values in the matrix show average synergism (<1 synergism and >1 antagonism Thirteen predicted DU-SR rescuers (red lines), two predicted DD SR rescuers (green lines) of DNMT1 and one random control (JAK3i) were tested.


Figure 6The transcriptomic alterations of rescuer genes post PD1 / PDL1 blockade in patient tumor biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - PD1 ( b ) Each panel displays the expression fold change of each predicted rescuer gene ( rows ) for different tumor samples ( columns ) and the P - value of over - all paired Wilcoxon test of the expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models . The transcriptomic alterations of rescuer genes post CTLA4 blockade in patient tumor biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - CTLA4 ( c ) Each panel displays the expression fold change of each predicted rescuer gene ( rows ) for different tumor samples ( columns ) and the P - value of over - all paired Wilcoxon test of the expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models . The transcriptomic alterations of rescuer genes post PD1 / PDL1 and CTLA4 blockade in patient tumor biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after PD1 + CTLA4 combination therapies ( d ) . Each panel displays the expression fold change of each predicted rescuer gene ( rows ) for different tumor samples ( columns ) and the P - value of over - all paired Wilcoxon test of the expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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Figure 6The transcriptomic alterations of rescuer genes post PD1/PDL1 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-PD1 (b) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.The transcriptomic alterations of rescuer genes post CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-CTLA4 (c) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.The transcriptomic alterations of rescuer genes post PD1/PDL1 and CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after PD1 + CTLA4 combination therapies (d). Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


Figure 1A Representative pictures of embryos at 18 . 5 days postcoitum ( E18 . 5 ) and hematoxylin and eosin staining of whole - mount embryo sections from PR - SET7loxp / Alfp - Cre ( PR - SET7ΔHepE ) mice and control littermates ( WT ) . B Hematoxylin and eosin staining of liver sections . C Immunohistochemical staining of liver sections with albumin antibody . D qPCR analysis of Albumin , Afp and HNF4 mRNA levels . Bars represent mean values of mRNA levels normalized to glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) mRNA and SEM from samples of four individual mice . * P - value < 0 . 01 . E Immunohistochemical staining of liver sections with cyclin B1 antibody . F Immunohistochemical staining of liver sections with γH2AX antibody .

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Figure 1A Representative pictures of embryos at 18.5 days postcoitum (E18.5) and hematoxylin and eosin staining of whole-mount embryo sections from PR-SET7loxp/Alfp-Cre (PR-SET7ΔHepE) mice and control littermates (WT).B Hematoxylin and eosin staining of liver sections.C Immunohistochemical staining of liver sections with albumin antibody.D qPCR analysis of Albumin,Afp and HNF4 mRNA levels. Bars represent mean values of mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and SEM from samples of four individual mice. *P-value < 0.01.E Immunohistochemical staining of liver sections with cyclin B1 antibody.F Immunohistochemical staining of liver sections with γH2AX antibody.


Figure 2A Macroscopic appearance of livers in 120 - day - old ( P120 ) wild - type ( WT ) and PR - SET7loxp / Alb - Cre ( KO ) mice . Note , small adenomatous foci in KO livers . B Representative hematoxylin and eosin staining of liver sections from P120 wild - type ( WT ) and PR - SET7loxp / Alb - Cre ( PR - SET7ΔHepA ) mice . Arrows show three areas containing morphologically different hepatocytes . Right panels : zoom - in to Area - A - ' normal zone ' , to Area - B - ' necrotic zone ' and to Area - C - ' regenerative zone ' . C Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with HNF4 antibody . D TUNEL staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) . Note that cells containing enlarged nuclei ( white arrows ) are TUNEL negative . E Immunohistological staining with γH2AX and albumin ( Alb ) antibodies .

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Figure 2A Macroscopic appearance of livers in 120-day-old (P120) wild-type (WT) and PR-SET7loxp/Alb-Cre (KO) mice. Note, small adenomatous foci in KO livers.B Representative hematoxylin and eosin staining of liver sections from P120 wild-type (WT) and PR-SET7loxp/Alb-Cre (PR-SET7ΔHepA) mice. Arrows show three areas containing morphologically different hepatocytes. Right panels: zoom-in to Area-A-'normal zone', to Area-B-'necrotic zone' and to Area-C-'regenerative zone'.C Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with HNF4 antibody.D TUNEL staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT). Note that cells containing enlarged nuclei (white arrows) are TUNEL negative.E Immunohistological staining with γH2AX and albumin (Alb) antibodies.


Figure 3A Electronmicroscopic images of cells containing enlarged nuclei in P120 PR - SET7ΔHepA livers and normal hepatocytes in control littermates ( WT ) . White arrows indicate : ( Nuc ) nuclei ; ( ER ) endoplasmic reticulum ; ( Mit ) mitochondrium ; ( PM ) plasma membrane ; ( a ) swollen mitochondria ; ( b ) swollen endoplasmic reticulum ; ( c ) disorganized endoplasmic reticulum ; ( d ) disrupted plasma membrane ; and ( e ) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes . B Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with H4K20Me1 and H4K20Me3 antibodies . Normal ( Area - A ) , Necrotic ( Area - B ) and Regenerative ( Area - C ) hepatocyte - containing areas are indicated . Arrows show the loss of H4K20Me1 signal from the large necrotic nuclei . C - F Partial ( 2 / 3rd ) hepatectomy was performed in 45 - day - old PR - SET7ΔHepA mice and control littermates ( WT ) . Two weeks later , at postnatal day 60 ( P60 ) liver sections were stained with hematoxylin and eosin ( C ) or γH2AX ( D ) or H4K20Me1 ( E ) or HNF4 and F4 / 80 ( F ) antibodies .

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Figure 3A Electronmicroscopic images of cells containing enlarged nuclei in P120 PR-SET7ΔHepA livers and normal hepatocytes in control littermates (WT). White arrows indicate: (Nuc) nuclei; (ER) endoplasmic reticulum; (Mit) mitochondrium; (PM) plasma membrane; (a) swollen mitochondria; (b) swollen endoplasmic reticulum; (c) disorganized endoplasmic reticulum; (d) disrupted plasma membrane; and (e) disorganized pieces of endoplasmic reticulum encircling mitochondria to form autophagosomes.B Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with H4K20Me1 and H4K20Me3 antibodies. Normal (Area-A), Necrotic (Area-B) and Regenerative (Area-C) hepatocyte-containing areas are indicated. Arrows show the loss of H4K20Me1 signal from the large necrotic nuclei.C-F Partial (2/3rd) hepatectomy was performed in 45-day-old PR-SET7ΔHepA mice and control littermates (WT). Two weeks later, at postnatal day 60 (P60) liver sections were stained with hematoxylin and eosin (C) or γH2AX (D) or H4K20Me1 (E) or HNF4 and F4/80 (F) antibodies.


Figure 4A , B Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with Ki67 , albumin ( Alb ) and A6 or HNF4 antibodies as indicated . Arrows depict the different areas in the sections as indicated above ( Fig . 1B ) . Asterisks ( * ) indicate Ki67 + hepatocytes . Small arrows ( > ) indicate Ki67 + non - hepatic cells . Note the lack of co - staining of cells with HNF4 and A6 antibodies . C Immunohistological staining of liver sections from P120 PR - SET7ΔHepA mice and control littermates ( WT ) with FGF7 and A6 antibodies . D Relative mRNA levels of FGF7 . Bars represent mean FGF7 mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 45 days or 120 - day - old mice . The data are presented as fold over values obtained with wild - type P45 samples . * P - value < 0 . 01 .

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Figure 4A, B Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with Ki67, albumin (Alb) and A6 or HNF4 antibodies as indicated. Arrows depict the different areas in the sections as indicated above (Fig.1B). Asterisks (*) indicate Ki67+ hepatocytes. Small arrows (>) indicate Ki67+ non-hepatic cells. Note the lack of co-staining of cells with HNF4 and A6 antibodies.C Immunohistological staining of liver sections from P120 PR-SET7ΔHepA mice and control littermates (WT) with FGF7 and A6 antibodies.D Relative mRNA levels of FGF7. Bars represent mean FGF7 mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 45 days or 120-day-old mice. The data are presented as fold over values obtained with wild-type P45 samples. *P-value < 0.01.


Figure 5A Analysis of reactive oxygen species ( ROS ) accumulation in frozen liver sections from 120 - day - old ( P120 ) and 240 - day - old ( P240 ) PR - SET7ΔHepA mice and control littermates ( WT ) . B Immunohistological staining of liver sections from P240 animals with phospho - STAT3 antibody . C Western blot analysis of liver extracts from P45 , P120 and P240 mice with STAT3 and phospho - STAT3 antibodies . D Macroscopic appearance of livers in P240 PR - SET7ΔHepA mice and control littermates ( WT ) . E Serum alanine aminotransferase ( ALT ) levels in P45 , P120 and P240 mice . Bars represent mean values of ALT levels and SEM from liver extracts of five individual mice . * P - value < 0 . 01 . F Representative hematoxylin and eosin staining of liver sections from P240 PR - SET7ΔHepA mice and control littermates ( WT ) . Two different magnifications are shown . G Relative mRNA levels of selected oncofetal genes . Bars represent mean Afp , H19 , GPC - 3 and CTGF mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 240 - day - old mice . The data are presented as fold over values obtained with wild - type samples . * P - value < 0 . 01 .

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Figure 5A Analysis of reactive oxygen species (ROS) accumulation in frozen liver sections from 120-day-old (P120) and 240-day-old (P240) PR-SET7ΔHepA mice and control littermates (WT).B Immunohistological staining of liver sections from P240 animals with phospho-STAT3 antibody.C Western blot analysis of liver extracts from P45, P120 and P240 mice with STAT3 and phospho-STAT3 antibodies.D Macroscopic appearance of livers in P240 PR-SET7ΔHepA mice and control littermates (WT).E Serum alanine aminotransferase (ALT) levels in P45, P120 and P240 mice. Bars represent mean values of ALT levels and SEM from liver extracts of five individual mice. *P-value < 0.01.F Representative hematoxylin and eosin staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT). Two different magnifications are shown.G Relative mRNA levels of selected oncofetal genes. Bars represent mean Afp, H19, GPC-3 and CTGF mRNA levels normalized to GAPDH mRNA and SEM from samples of 5 individual 240-day-old mice. The data are presented as fold over values obtained with wild-type samples. *P-value < 0.01.


Figure 6A , B Immunohistological staining of liver sections from P240 PR - SET7ΔHepA mice and control littermates ( WT ) with Afp and HNF4 ( A ) or Ki - 67 and albumin ( Alb ) ( B ) antibodies . C - E Double staining of liver sections with A6 and HNF4 ( C ) or Sox9 and Alb ( D ) or CD133 and HNF4 ( E ) antibodies . Right panels show larger magnifications of stainings performed in PR - SET7ΔHepA liver sections .

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Figure 6A, B Immunohistological staining of liver sections from P240 PR-SET7ΔHepA mice and control littermates (WT) with Afp and HNF4 (A) or Ki-67 and albumin (Alb) (B) antibodies.C-E Double staining of liver sections with A6 and HNF4 (C) or Sox9 and Alb (D) or CD133 and HNF4 (E) antibodies. Right panels show larger magnifications of stainings performed in PR-SET7ΔHepA liver sections.


Figure 7A P240 PR - SET7ΔHepA liver sections were double - stained with A6 and Sox9 ( upper panel ) or CD133 and Sox9 ( lower panel ) . Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI - stained cells in 5 high - power fields ( HPF ) in liver sections of 3 different mice . B Cell duplication rate of primary hepatocytes from P240 PR - SET7ΔHepA livers in culture . C Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with primary hepatocytes from P240 PR - SET7ΔHepA livers or Hepa 1 - 6 cells . Right panel shows average tumor volumes ( n = 3 ) dissected at the indicated time points following injection . D , E Hematoxylin and eosin and immunohistological staining with A6 , HNF4 , Sox9 and CD133 antibodies of tumor sections from xenografts dissected 20 or 30 days after injection with primary hepatocytes from P240 PR - SET7ΔHepA livers .

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Figure 7A P240 PR-SET7ΔHepA liver sections were double-stained with A6 and Sox9 (upper panel) or CD133 and Sox9 (lower panel). Pie charts at the right represent average percentages of cells stained positively with the indicated markers after counting DAPI-stained cells in 5 high-power fields (HPF) in liver sections of 3 different mice.B Cell duplication rate of primary hepatocytes from P240 PR-SET7ΔHepA livers in culture.C Pictures of representative subcutaneous xenograft tumors dissected from immunodeficient mice 20 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers or Hepa 1-6 cells. Right panel shows average tumor volumes (n = 3) dissected at the indicated time points following injection.D, E Hematoxylin and eosin and immunohistological staining with A6, HNF4, Sox9 and CD133 antibodies of tumor sections from xenografts dissected 20 or 30 days after injection with primary hepatocytes from P240 PR-SET7ΔHepA livers.


Figure 1A Age , gender and BMI information of the Ashkenazi Jewish Centenarian cohort . Table showing the age , gender and BMI information in control and centenarian groups . Pie chart showing the percentage of male / female individuals in control and centenarian groups . Right panel showing the age and BMI distribution of all individuals recruited in targeted capture - seq . Data were presented as mean and SD , Students t - test , two tailed , * * * p < 0 . 001 . B , C Histograms of normalized allele counts in individuals 75 + years old compared to all age groups ( N = 125 , 748 ) across ( B ) all chromosome 19 SNPs and small insertions / deletions , and ( C ) missense SNPs and indels only . Dotted vertical lines show centSIRT6 . E Michaelis - Menten kinetic parameters as calculated by saturation curves using differential concentrations of myristoylated peptide or NAD + . n = 3 technical replicates ( TR ) ; error bars = SD .

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Figure 1A Age, gender and BMI information of the Ashkenazi Jewish Centenarian cohort. Table showing the age, gender and BMI information in control and centenarian groups. Pie chart showing the percentage of male/female individuals in control and centenarian groups. Right panel showing the age and BMI distribution of all individuals recruited in targeted capture-seq. Data were presented as mean and SD, Students t-test, two tailed, *** p<0.001.B, C Histograms of normalized allele counts in individuals 75+ years old compared to all age groups (N=125,748) across (B) all chromosome 19 SNPs and small insertions/deletions, and (C) missense SNPs and indels only. Dotted vertical lines show centSIRT6.E Michaelis-Menten kinetic parameters as calculated by saturation curves using differential concentrations of myristoylated peptide or NAD+. n=3 technical replicates (TR); error bars=SD.


Figure 2A , B Deacetylase activity on H3K9 ( A ) and H3K18 ( B ) residues shows reduced activity in centSIRT6 allele . Designer histones were incubated with purified SIRT6 and 5 mM NAD + , then resolved by SDS - PAGE and analyzed by immunoblotting with acetyl - specific histone antibodies . n = 3 TR ; error bars = SD . Students t - test , two tailed . Asterisks indicate p < 0 . 05 compared to WT , color corresponds to allele . H3K9ac , H3K18ac and H3 are blots , SIRT6 loading Coomassie stain HeLa histone preps were also probedC Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels . Relative fraction represents the portion of the peptide encompassing H3K9 - 17 compared to the summed total of all of the peptide quantitation values for the same region . n = 3 biological replicates ( BR ) ; error bars = SD . Students t - test , two tailedD Whole cell histone H3 acetylation levels in cumate - inducible SIRT6 human fibroblasts , assessed by Western blot . Cu , cumate ; PQ , paraquat . Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot , and administered accordingly to respective cell lines Cells were incubated with appropriate cumate dose for 48hrs prior to harvest , with PQ - treated cells receiving PQ 24hrs after initial cumate induction . E - H Relative abundance of H3K9ac at SIRT6 - dependent NF - κB target genes . Anti - H3K9ac antibodies were used to perform ChIP in the absence ( - Cu ) or presence of ( + Cu ) cumate - induced SIRT6 expression . Samples were normalized using 10 % input . n = 3 BR ; error bars = SD . Students t - test , two tailed . Black asterisk indicate p < 0 . 05 compared to - Cu condition . Red asterisk indicated p < 0 . 05 compared to + Cu WT . I , J Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity . Anti - H3K18ac antibodies were used to perform ChIP in the absence ( - Cu ) or presence of ( + Cu ) cumate - induced SIRT6 expression . Samples were normalized using 10 % input . n = 3 TR ; error bars = SD . Students t - test , two tailed . Black asterisk indicate p < 0 . 05 compared to - Cu condition .

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

Figure 2A, B Deacetylase activity on H3K9 (A) and H3K18 (B) residues shows reduced activity in centSIRT6 allele. Designer histones were incubated with purified SIRT6 and 5 mM NAD+, then resolved by SDS-PAGE and analyzed by immunoblotting with acetyl-specific histone antibodies. n=3 TR; error bars=SD. Students t-test, two tailed. Asterisks indicate p<0.05 compared to WT, color corresponds to allele. H3K9ac, H3K18ac and H3 are blots, SIRT6 loading Coomassie stain HeLa histone preps were also probedC Quantitative mass spec of histone H3 peptide purified from human cells expressing different SIRT6 alleles did not reveal a difference in acetylation levels. Relative fraction represents the portion of the peptide encompassing H3K9-17 compared to the summed total of all of the peptide quantitation values for the same region. n=3 biological replicates (BR); error bars=SD. Students t-test, two tailedD Whole cell histone H3 acetylation levels in cumate-inducible SIRT6 human fibroblasts, assessed by Western blot. Cu, cumate; PQ, paraquat. Cumate dosage required for equivalent SIRT6 protein abundance was determined by Western blot, and administered accordingly to respective cell lines Cells were incubated with appropriate cumate dose for 48hrs prior to harvest, with PQ-treated cells receiving PQ 24hrs after initial cumate induction.E-H Relative abundance of H3K9ac at SIRT6-dependent NF-κB target genes. Anti-H3K9ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 BR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition. Red asterisk indicated p<0.05 compared to +Cu WT.I, J Relative abundance of H3K18ac at satellite repeats associated with SIRT6 deacetylase activity. Anti-H3K18ac antibodies were used to perform ChIP in the absence (-Cu) or presence of (+Cu) cumate-induced SIRT6 expression. Samples were normalized using 10% input. n=3 TR; error bars=SD. Students t-test, two tailed. Black asterisk indicate p<0.05 compared to -Cu condition.