B Quantification of the band intensities following GST - pulldown assays with 293T cell lysates expressing the autophagy - related proteins and recombinant GST - CbpFR6 shown in Fig EV2D .

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B Quantification of the band intensities following GST-pulldown assays with 293T cell lysates expressing the autophagy-related proteins and recombinant GST-CbpFR6 shown in Fig EV2D.


C Lysates from 293T cells transiently expressing 3Myc - Atg14 or Atg5 - 3Myc , and GFP - CbpCT4 or GFP were immunoprecipitated with GFP - Trap , and the bound proteins were analyzed by immunoblotting .

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C Lysates from 293T cells transiently expressing 3Myc-Atg14 or Atg5-3Myc, and GFP-CbpCT4 or GFP were immunoprecipitated with GFP-Trap, and the bound proteins were analyzed by immunoblotting.


D Lysates from 293T cells transiently expressing GFP - CbpCT4 or GFP , and 6Myc - Atg14 were immunoprecipitated with an anti - Myc antibody and Protein G PLUS - Agarose . The bound proteins were analyzed by immunoblotting .

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D Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP, and 6Myc-Atg14 were immunoprecipitated with an anti-Myc antibody and Protein G PLUS-Agarose. The bound proteins were analyzed by immunoblotting.


E Recombinant GST - CbpFR6 or GST , and 3Myc - Atg14 were used for GST - pulldown assays . The bound proteins were analyzed by immunoblotting using an anti - Myc antibody .

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E Recombinant GST-CbpFR6 or GST, and 3Myc-Atg14 were used for GST-pulldown assays. The bound proteins were analyzed by immunoblotting using an anti-Myc antibody.


F MEFs infected with R6 ΔcbpF expressing CbpFR6 - FLAG for 2 h were subjected to IP and assayed using anti - HA agarose beads . The bound proteins were analyzed by immunoblotting using an anti - FLAG antibody .

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F MEFs infected with R6 ΔcbpF expressing CbpFR6-FLAG for 2 h were subjected to IP and assayed using anti-HA agarose beads. The bound proteins were analyzed by immunoblotting using an anti-FLAG antibody.


A Lysates from MEFs stably expressing HA - Atg14 infected with S . pneumoniae TIGR4 WT or ΔcbpC for 1 , 2 , or 3 h in the presence or absence of cycloheximide were subjected to SDS - PAGE and analyzed by immunoblotting with the indicated antibodies .

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A Lysates from MEFs stably expressing HA-Atg14 infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of cycloheximide were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.


B MEFs / HA - Atg14 cells were infected with S . pneumoniae TIGR4 WT or ΔcbpC for 1 , 2 , or 3 h in the presence or absence of Bafilomycin A1 ( BafA1 ) . The lysates were subjected to SDS - PAGE and analyzed by immunoblotting with the indicated antibodies .

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B MEFs/HA-Atg14 cells were infected with S. pneumoniae TIGR4 WT or ΔcbpC for 1, 2, or 3 h in the presence or absence of Bafilomycin A1 (BafA1). The lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies.


( C A549 cells were infected with S . pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and an anti - Atg14 Representative epifluorescence images are shown . Scale bars , 10 µm . The percentages of perinuclear - localizing Atg14 containing cells were quantified .

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(C A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and an anti-Atg14 Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of perinuclear-localizing Atg14 containing cells were quantified.


A549 cells were infected with S . pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and - GM130 antibody . Representative epifluorescence images are shown . Scale bars , 10 µm . The percentages of intact Golgi apparatus - containing cells were quantified .

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A549 cells were infected with S. pneumoniae R6 WT or ΔcbpC for 2 h and fixed and stained with DAPI and -GM130 antibody. Representative epifluorescence images are shown. Scale bars, 10 µm. The percentages of intact Golgi apparatus-containing cells were quantified.


G S . pneumoniae ( Sp ) invasion - permissive A549 cells ( marked with GFP ) and Sp invasion - non - permissive ( pIgR knocked down ) A549 cells were co - cultured at a 1 : 3 ratio . Atg14 - disappearance experiments were conducted , and representative epifluorescence images are shown . Scale bars , 10 µm . The lines show each cell shape and the arrows show invasion - permissive A549 cells marked with GFP . H The percentages of perinuclear - localizing Atg14 containing cells in ( G ) were quantified .

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G S. pneumoniae (Sp) invasion-permissive A549 cells (marked with GFP) and Sp invasion-non-permissive (pIgR knocked down) A549 cells were co-cultured at a 1:3 ratio. Atg14-disappearance experiments were conducted, and representative epifluorescence images are shown. Scale bars, 10 µm. The lines show each cell shape and the arrows show invasion-permissive A549 cells marked with GFP. H The percentages of perinuclear-localizing Atg14 containing cells in (G) were quantified.


I Lysates from 293T cells transiently expressing GFP - CbpC or GFP , and HA - Atg14 were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against HA or GFP .

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I Lysates from 293T cells transiently expressing GFP-CbpC or GFP, and HA-Atg14 were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against HA or GFP.


J Lysates from 293T cells transiently expressing GFP - CbpCT4 or GFP were immunoprecipitated using GST - GFP - Nanobody . Additionally , beads were mixed with lysates from 293T cells transiently expressing p62 - 3Myc and HA - Atg14 , and bound proteins were analyzed by immunoblotting .

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J Lysates from 293T cells transiently expressing GFP-CbpCT4 or GFP were immunoprecipitated using GST-GFP-Nanobody. Additionally, beads were mixed with lysates from 293T cells transiently expressing p62-3Myc and HA-Atg14, and bound proteins were analyzed by immunoblotting.


K A549 cells treated with the indicated siRNAs were infected with S . pneumoniae R6 WT or ΔcbpF for the indicated durations . The cells were fixed and stained with DAPI and an anti - Atg14 antibody , and percentages of perinuclear - localizing Atg14 containing cells were quantified .

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K A549 cells treated with the indicated siRNAs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated durations. The cells were fixed and stained with DAPI and an anti-Atg14 antibody, and percentages of perinuclear-localizing Atg14 containing cells were quantified.


L Lysates from MEFs stably expressing GFP - CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS - PAGE and immunoblotted using antibodies against LC3 or p62 .

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L Lysates from MEFs stably expressing GFP-CbpCT4 or GFP in the presence or absence of rapamycin or chloroquine were subjected to SDS-PAGE and immunoblotted using antibodies against LC3 or p62.


M Quantification of NanoBRET signals in 293A cells transiently expressing HaloTag - Stx17 and Nanoluc - Atg14 in the presence or absence of GFP - CbpC or GFP .

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M Quantification of NanoBRET signals in 293A cells transiently expressing HaloTag-Stx17 and Nanoluc-Atg14 in the presence or absence of GFP-CbpC or GFP.


B GST - pulldown assays performed using GST - CbpFR6 or GST and lysates from 293T cells expressing GFP - Atg14 derivatives or GFP . The bound proteins were analyzed by immunoblotting using an anti - GFP antibody .

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B GST-pulldown assays performed using GST-CbpFR6 or GST and lysates from 293T cells expressing GFP-Atg14 derivatives or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody.


D , E GST - pulldown assays using GST - CbpFR6 derivatives and lysates from 293T cells expressing GFP - Atg14 CCD or GFP . The bound proteins were analyzed by immunoblotting using an anti - GFP antibody . Each GST - CbpFR6 derivative - bound bead was confirmed by Coomassie brilliant blue ( CBB ) staining .

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D, E GST-pulldown assays using GST-CbpFR6 derivatives and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 derivative-bound bead was confirmed by Coomassie brilliant blue (CBB) staining.


H GST - pulldown assays performed using GST - CbpFR6 mutants or GST and lysates from 293T cells expressing GFP - Atg14 CCD or GFP . The bound proteins were analyzed by immunoblotting using an anti - GFP antibody . Each GST - CbpFR6 mutant - bound bead was confirmed by CBB staining .

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H GST-pulldown assays performed using GST-CbpFR6 mutants or GST and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. The bound proteins were analyzed by immunoblotting using an anti-GFP antibody. Each GST-CbpFR6 mutant-bound bead was confirmed by CBB staining.


I Lysates of 293T cells transiently expressing GFP - CbpCT4 mutants or GFP were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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I Lysates of 293T cells transiently expressing GFP-CbpCT4 mutants or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


J Confocal microscopy images of HeLa cells transiently expressing the indicated GFP - CbpCT4 mutants . Scale bars , 10 µm .

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J Confocal microscopy images of HeLa cells transiently expressing the indicated GFP-CbpCT4 mutants. Scale bars, 10 µm.


B Lysates from 293T cells transiently expressing p62 - 3Myc and GFP - CbpCT4 variants were immunoprecipitated using a GST - GFP - Nanobody fusion protein . Bound proteins were analyzed by immunoblotting .

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B Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using a GST-GFP-Nanobody fusion protein. Bound proteins were analyzed by immunoblotting.


E Lysates from 293T cells transiently expressing p62 - 3Myc and GFP - CbpCT4 variants were immunoprecipitated using the GST - GFP - Nanobody protein . Bound proteins were analyzed by immunoblotting .

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E Lysates from 293T cells transiently expressing p62-3Myc and GFP-CbpCT4 variants were immunoprecipitated using the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting.


G Lysates of 293T cells transiently expressing GFP - CbpCT4 and p62 - 3Myc variants were immunoprecipitated with the GST - GFP - Nanobody protein . Bound proteins were analyzed by immunoblotting using the indicated antibodies .

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G Lysates of 293T cells transiently expressing GFP-CbpCT4 and p62-3Myc variants were immunoprecipitated with the GST-GFP-Nanobody protein. Bound proteins were analyzed by immunoblotting using the indicated antibodies.


H A549 cells infected with the indicated S . pneumoniae strains for 2 h were fixed and stained with DAPI and an anti - Atg14 antibody , and the percentages of perinuclear - localizing Atg14 containing cells were quantified .

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H A549 cells infected with the indicated S. pneumoniae strains for 2 h were fixed and stained with DAPI and an anti-Atg14 antibody, and the percentages of perinuclear-localizing Atg14 containing cells were quantified.


I Representative epifluorescence images in ( H ) are shown . Scale bars , 10 µm .

I Representative epifluorescence images in (H) are shown. Scale bars, 10 µm.


D GST - pulldown assays using the indicated GST - fusion proteins or GST and lysates from 293T cells expressing GFP - Atg14 CCD or GFP . Bound proteins were analyzed by immunoblotting using an anti - GFP antibody .

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D GST-pulldown assays using the indicated GST-fusion proteins or GST and lysates from 293T cells expressing GFP-Atg14 CCD or GFP. Bound proteins were analyzed by immunoblotting using an anti-GFP antibody.


E Lysates from 293T cells expressing GFP - fusion proteins or GFP were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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E Lysates from 293T cells expressing GFP-fusion proteins or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


F Confocal images of HeLa cells transiently expressing GFP - fusion proteins . Scale bars , 10 µm .

F Confocal images of HeLa cells transiently expressing GFP-fusion proteins. Scale bars, 10 µm.


H , I GST - pulldown assays using the indicated GST - fused proteins or GST and lysates from 293T expressing GFP - Atg14 CCD or GFP . Bound proteins were analyzed by immunoblotting using an anti - GFP antibody .

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H, I GST-pulldown assays using the indicated GST-fused proteins or GST and lysates from 293T expressing GFP-Atg14 CCD or GFP. Bound proteins were analyzed by immunoblotting using an anti-GFP antibody.


Partial embryonic lethality after E11 . 5 . Chi - square , * * * * p < 0 . 0001 . E11 . 5 n = 114 embryos , E14 . 5 n = 70 embryos , Adult n = 314 born pups .

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Partial embryonic lethality after E11.5. Chi-square, **** p<0.0001. E11.5 n=114 embryos, E14.5 n=70 embryos, Adult n=314 born pups.


Hindbrain vessel morphology . IsolectinB4 ( IsoB4 ; white ) , ERG ( red ) and EdU incorporation ( green ) . EdU signal was masked with ERG signal . Scale bar , 50 µm .

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Hindbrain vessel morphology. IsolectinB4 (IsoB4; white), ERG (red) and EdU incorporation (green). EdU signal was masked with ERG signal. Scale bar, 50 µm.


Hindbrain vessel density . IsolectinB4 vessel area normalized to wildtype ( WT ) mean ; one dot is two fields / mouse ; error bars : SD ; unpaired T test , * p < 0 . 05 . n = 18 - 20 .

Hindbrain vessel density. IsolectinB4 vessel area normalized to wildtype (WT) mean; one dot is two fields/mouse; error bars: SD; unpaired T test, * p <0.05. n=18-20.


Hindbrain EC proliferation . EdU incorporation in ERG - positive ECs ( double - positive cells , yellow in B ) . One dot is two fields / mouse ; error bars : SD ; unpaired T test , * p < 0 . 05 . n = 7 - 12 .

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Hindbrain EC proliferation. EdU incorporation in ERG-positive ECs (double-positive cells, yellow in B). One dot is two fields/mouse; error bars: SD; unpaired T test, *p < 0.05. n=7-12.


Reduced weight gain in the adult . Error bars : SD ; 2 - way ANOVA p = 0 . 0009 ; Sidak ' s multiple comparison test , * * p < 0 . 01 . n = 13 - 20 mice .

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Reduced weight gain in the adult. Error bars: SD; 2-way ANOVA p=0.0009; Sidak's multiple comparison test, **p <0.01. n=13-20 mice.


P6 retina vessel morphology . IsolectinB4 ( IsoB4 ) in black ; the outer 20 % of the vascularized area in pink . Scale bar , 250 µm .

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P6 retina vessel morphology. IsolectinB4 (IsoB4) in black; the outer 20% of the vascularized area in pink. Scale bar, 250 µm.


Vessel parameters in the P6 developing retina . G . Radial outgrowth ( distance between optic nerve and vascular front ) .

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Vessel parameters in the P6 developing retina. G. Radial outgrowth (distance between optic nerve and vascular front).


Vascularized area . I . Vessel area in the outer 20 % of the retinal vasculature ; pink in F ( vessel area normalized to vascularized area ) . Each dot represents one retina / mouse ; n = 3 - 4 .

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Vascularized area. I. Vessel area in the outer 20% of the retinal vasculature; pink in F (vessel area normalized to vascularized area). Each dot represents one retina/mouse; n=3-4.


EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs . Each dot represents one retina / mouse ; error bars : SD . n = 8 - 9 .

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EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents one retina/mouse; error bars: SD. n=8-9.


Body weight . P , postnatal day ; w , weeks . Error bars : SD . n = 5 - 12 mice .

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Body weight. P, postnatal day; w, weeks. Error bars: SD. n=5-12 mice.


Hindbrain vessel parameters . Vessel density ; isolectinB4 vessel area normalized to WT mean of the litter .

Hindbrain vessel parameters. Vessel density; isolectinB4 vessel area normalized to WT mean of the litter.


Hindbrain vessel parameters . EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs .

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Hindbrain vessel parameters. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs.


P4 - P7 retina vessel morphology . IsolectinB4 ( IsoB4 ) in black ; outer 20 % of the vascularized area in pink . Scale bar , 250 µm . P6 insets ; sprout morphology . Scale bar , 50 µm .

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P4-P7 retina vessel morphology. IsolectinB4 (IsoB4) in black; outer 20% of the vascularized area in pink. Scale bar, 250 µm. P6 insets; sprout morphology. Scale bar, 50 µm.


Radial outgrowth . Distance between optic nerve and vascular front . P , postnatal day ; error bars : SD ; 2 - way ANOVA p < 0 . 0001 ; Bonferroni ' s multiple comparison test , * p < 0 . 05 . P4 n = 3 - 5 retinas , one retina / mouse , P5 n = 4 - 7 , P6 n = 9 - 9 , P7 n = 4 - 5 . Retina vascularized area . P , postnatal day ; error bars : SD ; 2 - way ANOVA p = 0 . 0013 . P4 n = 7 - 9 retinas , one retina / mouse , P5 n = 4 - 9 , P6 n = 9 - 9 , P7 n = 4 - 5 .

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Radial outgrowth. Distance between optic nerve and vascular front. P, postnatal day; error bars: SD; 2-way ANOVA p<0.0001; Bonferroni's multiple comparison test, *p<0.05. P4 n=3-5 retinas, one retina/mouse, P5 n=4-7, P6 n=9-9, P7 n=4-5. Retina vascularized area. P, postnatal day; error bars: SD; 2-way ANOVA p=0.0013. P4 n=7-9 retinas, one retina/mouse, P5 n=4-9, P6 n=9-9, P7 n=4-5.


Retina vessel density in the retinal front . Quantification of the vessel area in the outer 20 % of the vasculature ( pink in D ) ( vessel area normalized to vascularized area ) . P , postnatal day ; error bars : SD ; 2 - way ANOVA p = 0 . 0054 ; Bonferroni ' s multiple comparison test , * p < 0 . 05 , * * p < 0 . 01 . P4 n = 3 - 5 retinas , one retina / mouse , P5 n = 4 - 6 , P6 n = 3 - 5 , P7 n = 4 - 5 .

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Retina vessel density in the retinal front. Quantification of the vessel area in the outer 20% of the vasculature (pink in D) (vessel area normalized to vascularized area). P, postnatal day; error bars: SD; 2-way ANOVA p=0.0054; Bonferroni's multiple comparison test, *p<0.05, ** p <0.01. P4 n=3-5 retinas, one retina/mouse, P5 n=4-6, P6 n=3-5, P7 n=4-5.


Proliferation of retinal ECs . H . EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs , at P6 .

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Proliferation of retinal ECs. H. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs, at P6.


IsolectinB4 ( IsoB4 ; white ) , ERG ( red ) , EdU ( green ) and double - positive cells ( yellow ) . Lower panel shows ERG / EdU merged staining alone with EdU signal masked by ERG signal . Scale bar , 50 µm ; error bars : SD ; unpaired T test , * p < 0 . 05 . n = 5 - 8 retinas , one retina / mouse .

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IsolectinB4 (IsoB4; white), ERG (red), EdU (green) and double-positive cells (yellow). Lower panel shows ERG/EdU merged staining alone with EdU signal masked by ERG signal. Scale bar, 50 µm; error bars: SD; unpaired T test, *p < 0.05. n=5-8 retinas, one retina/mouse.


Retina sprout length . Sprout length from tip to base in P6 retinas ; error bars : SD ; unpaired T test , * p < 0 . 05 ; n = 3 - 5 retinas , one retina / mouse .

Retina sprout length. Sprout length from tip to base in P6 retinas; error bars: SD; unpaired T test, *p<0.05; n=3-5 retinas, one retina/mouse.


Vessel stability . IsolectinB4 ( IsoB4 , green ) and collagen IV ( red ) in P4 wildtype ( WT ) and Vegfr2Y1212F / Y1212F retinas . Arrows ; collagen IV - positive empty sleeves . A ; artery . Scale bar , 50 µm .

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Vessel stability. IsolectinB4 (IsoB4, green) and collagen IV (red) in P4 wildtype (WT) and Vegfr2Y1212F /Y1212F retinas. Arrows; collagen IV-positive empty sleeves. A; artery. Scale bar, 50 µm.


Vessel stability . Collagen IV empty sleeves around main arteries in P4 - P7 retinas , three fields / retina / mouse . Error bars : SD ; 2 - way ANOVA p = 0 . 0580 ; Bonferroni ' s multiple comparison test , * * p < 0 . 01 . P4 n = 7 - 9 , P5 n = 6 - 12 , P6 n = 3 - 5 , P7 n = 4 .

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Vessel stability. Collagen IV empty sleeves around main arteries in P4-P7 retinas, three fields/retina/mouse. Error bars: SD; 2-way ANOVA p=0.0580; Bonferroni's multiple comparison test, ** p <0.01. P4 n=7-9, P5 n=6-12, P6 n=3-5, P7 n=4.


Immunoblot showing VEGFR2 phosphorylation on pY1212 , pY949 and pY1173 . A . Anti - VEGFR2 immunoprecipitates ( IP ) from lung lysates from wildtype ( WT ) and Vegfr2Y1212F / Y1212F FVB mice , tail - vein injected with PBS or VEGF for 1 minute . Each lane ( A ) represent one individual mouse .

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Immunoblot showing VEGFR2 phosphorylation on pY1212, pY949 and pY1173. A. Anti-VEGFR2 immunoprecipitates (IP) from lung lysates from wildtype (WT) and Vegfr2Y1212F /Y1212F FVB mice, tail-vein injected with PBS or VEGF for 1 minute. Each lane (A) represent one individual mouse.


Quantification as percentage of phosphoprotein level normalized to total VEGFR2 . each dot ( B ) represent one individual mouse .

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Quantification as percentage of phosphoprotein level normalized to total VEGFR2. each dot (B) represent one individual mouse.


Summary plot of 51 proteins enriched in the immobilized pY1212 peptide precipitate . 10 proteins ( red oval ) were selected for further analysis . PSM : peptide - spectrum matches . Area : average area of spectra for the three most abundant peptides in arbitrary units .

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Summary plot of 51 proteins enriched in the immobilized pY1212 peptide precipitate. 10 proteins (red oval) were selected for further analysis. PSM: peptide-spectrum matches. Area: average area of spectra for the three most abundant peptides in arbitrary units.


Blot for peptide - enriched GRB2 and PI3Kp85 . HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 and PI3Kp85 .

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

Blot for peptide-enriched GRB2 and PI3Kp85. HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 and PI3Kp85.


VEGFR2 / GRB2 and VEGFR2 / PI3Kp85 complexes in isolated ( i ) ECs . E . PLA for VEGFR2 / GRB2 and VEGFR2 / PI3Kp85 complexes ( black puncta ) in iECs from FVB wildtype ( WT ) and Vegfr2Y1212F / Y1212F lungs . Hoechst 33342 show nuclei ( grey ) . Scale bar , 20 µm . F , G . Fold - increase over PBS - treated sample ; each dot represents the mean of 6 fields . 2 - way ANOVA p = 0 . 0206 ( F ) , p = 0 . 0368 ( G ) ; Sidak ' s multiple comparison test , * p < 0 . 05 . n = 3 experiments .

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

VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes in isolated (i)ECs. E. PLA for VEGFR2/GRB2 and VEGFR2/PI3Kp85 complexes (black puncta) in iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 show nuclei (grey). Scale bar, 20 µm. F, G. Fold-increase over PBS-treated sample; each dot represents the mean of 6 fields. 2-way ANOVA p=0.0206 (F), p=0.0368 (G); Sidak's multiple comparison test, * p <0.05. n=3 experiments.


phosphoERK1 / 2 and phosphoAkt immunostaining ( green ) of iECs from FVB wildtype ( WT ) and Vegfr2Y1212F / Y1212F lungs . Hoechst 33342 ( blue ) shows nuclei . Arrows indicate nuclear accumulation of phosphoERK1 / 2 and phosphoAkt , respectively . Scale bar , 20 µm .

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phosphoERK1/2 and phosphoAkt immunostaining (green) of iECs from FVB wildtype (WT) and Vegfr2Y1212F/Y1212F lungs. Hoechst 33342 (blue) shows nuclei. Arrows indicate nuclear accumulation of phosphoERK1/2 and phosphoAkt, respectively. Scale bar, 20 µm.


Nuclear accumulation expressed as phoshoERK1 / 2 - positive nuclear area normalized to the total phosphoERK1 / 2 area by field . Each dot represents 1 field . Min , minutes of stimulation . 2 - way ANOVA p = 0 . 0248 ; Sidak ' s multiple comparison test , * p < 0 . 05 , * * p < 0 . 01 . n = 3 experiments .

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Nuclear accumulation expressed as phoshoERK1/2-positive nuclear area normalized to the total phosphoERK1/2 area by field. Each dot represents 1 field. Min, minutes of stimulation. 2-way ANOVA p=0.0248; Sidak's multiple comparison test, * p <0.05, ** p <0.01. n=3 experiments.


Nuclear accumulation expressed as fold - increase of nuclear phosphoAkt area in Vegfr2Y1212F / Y1212F over PBS - treated wildtype ( WT ) iECs , normalized to the number of nuclei . Each dot represents 1 field . Min , minutes of stimulation . Unpaired T test , * p < 0 . 05 . n = 3 experiments .

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Nuclear accumulation expressed as fold-increase of nuclear phosphoAkt area in Vegfr2Y1212F/Y1212F over PBS-treated wildtype (WT) iECs, normalized to the number of nuclei. Each dot represents 1 field. Min, minutes of stimulation. Unpaired T test, *p < 0.05. n=3 experiments.


Immunoblot for phosphoERK1 / 2 , total ERK1 / 2 , phosphoAkt and total Akt on lung lysates from wiltype ( WT ) and Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one individual mouse . Quantification of phosphoERK1 / 2 / total ERK1 / 2 expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0025 ; unpaired T test , * p < 0 . 05 . n = 3 experiments . Quantification of phosphoAkt / total Akt expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0003 ; unpaired T test , * p < 0 . 05 . n = 3 experiments .

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

Immunoblot for phosphoERK1/2, total ERK1/2, phosphoAkt and total Akt on lung lysates from wiltype (WT) and Vegfr2Y1212F /Y1212F FVB mice tail-vein injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse. Quantification of phosphoERK1/2 /total ERK1/2 expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0025; unpaired T test, *p < 0.05. n=3 experiments. Quantification of phosphoAkt/total Akt expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0003; unpaired T test, *p < 0.05. n=3 experiments.


Immunoblot for Myc on lung lysates from wiltype ( WT ) and Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one individual mouse . Quantification of Myc / Actin expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0058 ; unpaired T test , * p < 0 . 05 , * * p < 0 . 01 . n = 3 experiments .

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Immunoblot for Myc on lung lysates from wiltype (WT) and Vegfr2Y1212F /Y1212F FVB mice tail-vein injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse. Quantification of Myc/Actin expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0058; unpaired T test, *p < 0.05, ** p <0.01. n=3 experiments.


Myc protein expression . C . Myc and Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype ( WT ) and mutant ( VEGFR2 Y1212F ) C57Bl / 6 and FVB mice . D . Quantification of Myc / Actin expressed as Arbitrary unit . Each lane ( C ) and each dot ( D ) represent one individual mouse . 2 - way ANOVA p < 0 . 0001 ; unpaired T test , * * p < 0 . 01 . n = 5 - 7 .

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

Myc protein expression. C. Myc and Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. D. Quantification of Myc/Actin expressed as Arbitrary unit. Each lane (C) and each dot (D) represent one individual mouse. 2-way ANOVA p <0.0001; unpaired T test, ** p <0.01. n=5-7.


Baseline transcriptional level of Myc from lung isolated ECs from untreated wildtype ( WT ) and mutant ( VEGFR2 Y1212F ) C57Bl / 6 and FVB mice . Each dot represents one individual mouse . 2 - way ANOVA p = 0 . 0041 ; Sidak ' s multiple comparison test , * p < 0 . 05 . n = 4 - 5 .

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Baseline transcriptional level of Myc from lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. Each dot represents one individual mouse. 2-way ANOVA p=0.0041; Sidak's multiple comparison test, * p <0.05. n=4-5.


Proliferation of C57Bl / 6 and FVB iECs . CD31 ( white ) , ERG ( red ) , EdU ( green ) and double - positive cells ( yellow ) in C57Bl / 6 ( F ) and FVB ( G ) iECs . EdU signal was masked with ERG signal .

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Proliferation of C57Bl/6 and FVB iECs. CD31 (white), ERG (red), EdU (green) and double-positive cells (yellow) in C57Bl/6 (F) and FVB (G) iECs. EdU signal was masked with ERG signal.


Proliferation of C57Bl / 6 and FVB iECs . EdU incorporation in ERG - positive ECs normalized to total ERG - positive ECs . Each dot represents 2 fields / well , one well / mouse . Scale bar , 100 µm . Error bars : SD ; 2 - way ANOVA p = 0 . 0843 ( H ) , p = 0 . 0474 ( I ) ; unpaired T test , * p < 0 . 05 ; * * p < 0 . 01 . C57Bl / 6 n = 3 - 6 mice , FVB n = 3 - 5mice .

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Proliferation of C57Bl/6 and FVB iECs. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents 2 fields/well, one well/mouse. Scale bar, 100 µm. Error bars: SD; 2-way ANOVA p =0.0843 (H), p=0.0474 (I); unpaired T test, *p < 0.05; **p < 0.01. C57Bl/6 n=3-6 mice, FVB n=3-5mice.


Rescue of angiogenic sprouting by Myc overexpression in E11 . 5 C57Bl / 6 Vegfr2Y1212F / Y1212F explant tissue . Representative images of E11 . 5 explants from C57Bl / 6 embryos taken from wildtype ( WT ) and Vegfr2Y1212F / Y1212F littermates with ( MycOE / Cre + ) and without ( MycOE / Cre - ) endothelial - specific overexpression of Myc . Endothelial cell marker ( CD31 ) immunostaining highlights the vasculature in white . Embryonic tissue blacked out to highlight sprouting area .

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Rescue of angiogenic sprouting by Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. Representative images of E11.5 explants from C57Bl/6 embryos taken from wildtype (WT) and Vegfr2Y1212F /Y1212F littermates with (MycOE/Cre +) and without (MycOE/Cre-) endothelial-specific overexpression of Myc. Endothelial cell marker (CD31) immunostaining highlights the vasculature in white. Embryonic tissue blacked out to highlight sprouting area.


Rescue of angiogenic sprouting by Myc overexpression in E11 . 5 C57Bl / 6 Vegfr2Y1212F / Y1212F explant tissue . Quantification as fold - increase of vessel sprouting area compared to WT MycOE / Cre - . Scale bar , 250 µm . Error bars : SD ; unpaired T test , * * p < 0 . 01 , * * * * p < 0 . 0001 . n = 4 - 11 explants , one explant / embryo .

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Rescue of angiogenic sprouting by Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. Quantification as fold-increase of vessel sprouting area compared to WT MycOE/Cre -. Scale bar, 250 µm. Error bars: SD; unpaired T test, ** p <0.01, **** p <0.0001. n=4-11 explants, one explant/embryo.


Rescue of Ec proliferation . Myc overexpression in E11 . 5 C57Bl / 6 Vegfr2Y1212F / Y1212F explant tissue . ERG ( red ) , EdU ( green ) and double - positive cells ( yellow ; arrows ) .

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Rescue of Ec proliferation. Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. ERG (red), EdU (green) and double-positive cells (yellow; arrows).


Rescue of Ec proliferation . Quantification of EdU / ERG double positive ECs normalized to total ECs . Scale bar , 50 µm . Error bars : SD ; unpaired T test , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 - 7 explants , one explant / embryo .

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Rescue of Ec proliferation. Quantification of EdU/ERG double positive ECs normalized to total ECs. Scale bar, 50 µm. Error bars: SD; unpaired T test, *** p <0.001, **** p <0.0001. n=3-7 explants, one explant/embryo.


GSEAs showing upregulation of genes belonging to the K - Ras , Angiogenesis and mTORC1 gene sets in ( WT ) compared to VEGFR2 Y1212F isolated ECs . K - Ras and Angiogenesis gene sets derived from C57Bl / 6 iECs kept in basal condition ; mTORC1 gene set derived from C57Bl / 6 iECs treated with VEGFA for 3h . See Supplementary Table 2 . Enrichment score ( ES ) , normalized ES ( NES ) are shown . P - values < 0 . 05 were regarded as significant . Heat maps displaying gene regulation in gene sets shown in A . The color in the heatmap shows the gene expression value , expressed as a z - score across animals . Red color represents relative higher expression and blue color represents relative lower expression value . The z - score is calculated by ( x - x ' ) / s where x is individual gene expression , x ' is the mean of the gene expression across samples and s is standard deviation of the gene expression across samples .

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

GSEAs showing upregulation of genes belonging to the K-Ras, Angiogenesis and mTORC1 gene sets in (WT) compared to VEGFR2 Y1212F isolated ECs. K-Ras and Angiogenesis gene sets derived from C57Bl/6 iECs kept in basal condition; mTORC1 gene set derived from C57Bl/6 iECs treated with VEGFA for 3h. See Supplementary Table 2. Enrichment score (ES), normalized ES (NES) are shown. P-values <0.05 were regarded as significant. Heat maps displaying gene regulation in gene sets shown in A. The color in the heatmap shows the gene expression value, expressed as a z-score across animals. Red color represents relative higher expression and blue color represents relative lower expression value. The z-score is calculated by (x-x')/s where x is individual gene expression, x' is the mean of the gene expression across samples and s is standard deviation of the gene expression across samples.


Myc - regulated gene expression downstream of VEGFR2 pY1212 . Quantitative PCR analysis of a set of known Myc - regulated transcripts in C57Bl / 6 ( C , D ) and FVB ( E , F ) adult lungs at basal ( C , E ) and at 1 hour after tail - vein administration of VEGFA ( D , F ) . FASN , fatty acid synthase ; Ldha and - b , Lactate dehydrogenase A and B ; Acod1 , Aconitate Decarboxylase 1 ; Ppat , Amidophosphoribosyltransferase ; Pde4d , cAMP - specific 3 ' , 5 ' - cyclic phosphodiesterase 4D ; Prkaca , cAMP - dependent protein kinase catalytic subunit alpha . Error bars : SD ; 2 - way ANOVA between WT and Vegfr2Y1212F / Y1212F p = 0 . 0453 ( C ) , p < 0 . 0001 ( D ) , p = 0 . 0018 ( E ) , p = 0 . 09 ( F ) ; unpaired T test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . C57Bl / 6 n = 8 - 11 mice , FVB n = 9 - 14 mice .

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

Myc-regulated gene expression downstream of VEGFR2 pY1212. Quantitative PCR analysis of a set of known Myc-regulated transcripts in C57Bl/6 (C, D) and FVB (E, F) adult lungs at basal (C, E) and at 1 hour after tail-vein administration of VEGFA (D, F). FASN, fatty acid synthase; Ldha and -b, Lactate dehydrogenase A and B; Acod1, Aconitate Decarboxylase 1; Ppat, Amidophosphoribosyltransferase; Pde4d, cAMP-specific 3',5'-cyclic phosphodiesterase 4D; Prkaca, cAMP-dependent protein kinase catalytic subunit alpha. Error bars: SD; 2-way ANOVA between WT and Vegfr2Y1212F /Y1212F p=0.0453 (C), p < 0.0001 (D), p=0.0018 (E), p=0.09 (F); unpaired T test, *p < 0.05, **p < 0.01, ***p < 0.001. C57Bl/6 n=8-11 mice, FVB n=9-14 mice.


A . cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 and the wildtype , from left to right on control plates without genotoxic agent 10 days after germination . B . The wildtype , single and double mutants of cycb1 were grown on control plates without genotoxic agent . Root lengths were measured 10 days after germination . C . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 1 mM hydroxy urea ( HU ) 10 days after germination . The rightmost plant is the wee1 mutant that shows high sensitivity to HU . D . The wildtype , single and double mutants of cycb1 were grown on plates supplemented with 1 mM HU . Root lengths were measured 10 days after germination . E . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 0 , 6 μg / mL bleomycin ( BLM ) 10 days after germination . The rightmost plant is the ku70 mutant that shows high sensitivity to BLM . F . The wildtype , single and double mutants of cycb1 were grown on plates supplemented with 0 , 6 μg / mL BLM . Root lengths were measured 10 days after germination .

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

A. cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 and the wildtype, from left to right on control plates without genotoxic agent 10 days after germination.B. The wildtype, single and double mutants of cycb1 were grown on control plates without genotoxic agent. Root lengths were measured 10 days after germination.C. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 1 mM hydroxy urea (HU) 10 days after germination. The rightmost plant is the wee1 mutant that shows high sensitivity to HU.D. The wildtype, single and double mutants of cycb1 were grown on plates supplemented with 1 mM HU. Root lengths were measured 10 days after germination.E. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 0,6 μg/mL bleomycin (BLM) 10 days after germination. The rightmost plant is the ku70 mutant that shows high sensitivity to BLM.F. The wildtype, single and double mutants of cycb1 were grown on plates supplemented with 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.


G . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 15 μM cisplatin 6 days after germination , i . e . 3 days after transfer from control plates . H . cycb1 mutants were germinated on control plates and were transferred to new control plates three days after germination . Root lengths mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . I . cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . J . cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs .

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

G. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 15 μM cisplatin 6 days after germination, i.e. 3 days after transfer from control plates.H. cycb1 mutants were germinated on control plates and were transferred to new control plates three days after germination. Root lengths mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.I. cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.J. cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.


K . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 30 μM cisplatin 6 days after germination , i . e . 3 days after transfer from control plates . L . cycb1 double mutants germinated on control plates and were transferred to new control plates three days after germination . Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs . M . cycb1 mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . N . cycb1 mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs .

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

K. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 30 μM cisplatin 6 days after germination, i.e. 3 days after transfer from control plates.L. cycb1 double mutants germinated on control plates and were transferred to new control plates three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.M. cycb1 mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.N. cycb1 mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.


A . Representative examples of comets of 21 days old wild - type plants , cdkb1 ; 1 cdkb1 ; 2 and cycb1 ; 1 cycb1 ; 3 double mutant seedlings in full spectrum view of the TriTek Comet Score software . Shown are comets of plants incubated with 50µM cisplatin for one hour and then transferred to medium without cisplatin for 30 min ( recovery ) and plants incubated without cisplatin for one hour ( control ) , respectively . B . Box plot of percentage of tail DNA of wild - type cells , cdkb1 ; 1 cdkb1 ; 2 and cycb1 ; 1 cycb1 ; 3 under cisplatin treatment . Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replications . The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software . The box represents the interquartile range , the line across the box indicates the median values and whiskers represent 5 - 95 percentile values . Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99 . 99 % calculated with Student ' s t - test .

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

A. Representative examples of comets of 21 days old wild-type plants, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 double mutant seedlings in full spectrum view of the TriTek Comet Score software. Shown are comets of plants incubated with 50µM cisplatin for one hour and then transferred to medium without cisplatin for 30 min (recovery) and plants incubated without cisplatin for one hour (control), respectively.B. Box plot of percentage of tail DNA of wild-type cells, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 under cisplatin treatment. Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replications. The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software. The box represents the interquartile range, the line across the box indicates the median values and whiskers represent 5-95 percentile values. Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99.99% calculated with Student's t-test.


C . Immunostaining of γ - H2AX foci in wild - type plants and mutant cells after two hours of treatment with 50µM cisplatin . D . Counted number of γ - H2AX foci per cell detected after two hours of treatment with 50µM cisplatin in wild - type and mutant plants . For each sample the γ - H2AX foci of 100 cells were counted and grouped into six categories : cells with no , 1 - 2 , 3 - 5 , 6 - 10 , 11 - 20 and more than 20 foci per cell .

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

C. Immunostaining of γ-H2AX foci in wild-type plants and mutant cells after two hours of treatment with 50µM cisplatin.D. Counted number of γ-H2AX foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the γ-H2AX foci of 100 cells were counted and grouped into six categories: cells with no, 1-2, 3-5, 6-10, 11-20 and more than 20 foci per cell.


B . Wild - type plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin . Arrows indicate representative blue sectors . C . cycb1 ; 1 plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin . Arrows indicate representative blue sectors .

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

B. Wild-type plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.C. cycb1;1 plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.


D . Graphs show numbers of blue sectors per plant grown without drug treatment . E . Graphs show numbers of blue sectors per plant grown after incubation on 15 μM cisplatin for three days . F . Graphs show numbers of blue sectors per plant grown after incubation on 30 μM cisplatin for three days .

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

D. Graphs show numbers of blue sectors per plant grown without drug treatment.E. Graphs show numbers of blue sectors per plant grown after incubation on 15 μM cisplatin for three days.F. Graphs show numbers of blue sectors per plant grown after incubation on 30 μM cisplatin for three days.


A . The wildtype and CDKA ; 1 - DE mutants were grown on control plates for 10 days . Root lengths were measured 10 days after germination . B . The wildtype and CDKA ; 1 - DE mutants were grown on plates containing 1 mM hydroxy urea for 10 days . The mutant wee1 was used as a positive control for hydroxy urea sensitivity . Root lengths were measured 10 days after germination . C . The wildtype and CDKA ; 1 - DE mutants were grown on plates containing 0 , 6 μg / mL bleomycin for 10 days . The mutant ku70 was used as a positive control for bleomycin sensitivity . Root lengths were measured 10 days after germination . D . The wildtype , cdkb1 ; 1 , cdkb1 ; 2 and the double mutant cdkb1 ; 1 cdkb1 ; 2 were grown on control plates for 10 days . Root lengths were measured 10 days after germination . E . The wildtype , cdkb1 ; 1 , cdkb1 ; 2 and the double mutant cdkb1 ; 1 cdkb1 ; 2 were grown on plates containing 1 mM hydroxy urea for 10 days The mutant wee1 was used as a positive control for hydroxy urea sensitivity . Root lengths were measured 10 days after germination . F . The wildtype , cdkb1 ; 1 , cdkb1 ; 2 and the double mutant cdkb1 ; 1 cdkb1 ; 2 were grown on plates containing 0 , 6 μg / mL bleomycin for 10 days . The mutant ku70 was used as a positive control for bleomycin sensitivity . Root lengths were measured 10 days after germination .

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

A. The wildtype and CDKA;1-DE mutants were grown on control plates for 10 days. Root lengths were measured 10 days after germination.B. The wildtype and CDKA;1-DE mutants were grown on plates containing 1 mM hydroxy urea for 10 days. The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.C. The wildtype and CDKA;1-DE mutants were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.D. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on control plates for 10 days. Root lengths were measured 10 days after germination.E. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 1 mM hydroxy urea for 10 days The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.F. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.


G . The wildtype , CDKA ; 1 - DE ( grey bar ) and the double mutant cdkb1 ; 1cdkb1 ; 2 ( purple bar ) were grown on control plates and were transferred to plates containing 15 μM or 30 μM cisplatin three days after germination . Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs . H . Graphs represent the ratio of the mean growth rate on 15 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype , CDKA ; 1 - DE and cdkb1 ; 1cdkb1 ; 2 . I . Graphs represent the ratio of the mean growth rate on 30 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype , CDKA ; 1 - DE and cdkb1 ; 1cdkb1 ; 2 .

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

G. The wildtype, CDKA;1-DE (grey bar) and the double mutant cdkb1;1cdkb1;2(purple bar) were grown on control plates and were transferred to plates containing 15 μM or 30 μM cisplatin three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.H. Graphs represent the ratio of the mean growth rate on 15 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2.I. Graphs represent the ratio of the mean growth rate on 30 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2.


J - N . Images show a wild - type plant , CDKA ; 1 - DE and the double mutant cdkb1 ; 1 cdkb1 ; 2 ( from left to right ) on the indicated day after germination and the indicated drug treatment .

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

J-N. Images show a wild-type plant, CDKA;1-DE and the double mutant cdkb1;1 cdkb1;2 (from left to right) on the indicated day after germination and the indicated drug treatment.


A . WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 μM or 30 μM cisplatin , respectively , three days after germination . Root lengths were measured three days after transfer and the net root growth of three days is shown in the graph . Asterisk indicates significant differences within a 5 % confidence interval ( Student ' s t - test ) . B . Image shows a wild - type plant ( left ) and rad51 mutant ( right ) grown on control plates . Images were taken 6 days after germination . C . Image shows a wild - type plant ( left ) and rad51 mutant ( right ) germinated on control plates and transferred to plates supplemented with 15 μM three days after germination . Images were taken 6 days after germination , i . e . 3 days after transfer to cisplatin .

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

A. WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 μM or 30 μM cisplatin, respectively, three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graph. Asterisk indicates significant differences within a 5% confidence interval (Student's t-test).B. Image shows a wild-type plant (left) and rad51 mutant (right) grown on control plates. Images were taken 6 days after germination.C. Image shows a wild-type plant (left) and rad51 mutant (right) germinated on control plates and transferred to plates supplemented with 15 μM three days after germination. Images were taken 6 days after germination, i.e. 3 days after transfer to cisplatin.


D . Immunostaining of RAD51 foci in the wildtype and indicated mutant cells after two hours of treatment with 50µM cisplatin .

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

D. Immunostaining of RAD51 foci in the wildtype and indicated mutant cells after two hours of treatment with 50µM cisplatin.


E . Counted number of RAD51 foci per cell detected after two hours of treatment with 50µM cisplatin in wild - type and mutant plants . For each sample the RAD51 foci of 100 cells were counted and grouped into six categories : cells with 0 , 1 , 2 , 3 , 4 or > 5 foci per cell .

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E. Counted number of RAD51 foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the RAD51 foci of 100 cells were counted and grouped into six categories: cells with 0, 1, 2, 3, 4 or >5 foci per cell.


F . In vitro kinase assay of purified CDK complexes phosphorylating RAD51 . RAD51 and histone H1 kinase assays were performed with [ γ - 32P ] ATP as a phosphate donor . Proteins were subjected to SDS - PAGE after the kinase reaction and stained with coomassie brilliant blue demonstrating the equal loading of the substrates ( lower panels ) . Phosphorylated proteins were detected by autoradiograph ( upper panels ) . The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK - cyclin complexes , protein blots indicate the relative amounts of the CDKs in the reaction ( bottom panel ) . Abbreviation : p - RAD51 and p - Histone H1 for [ 32P ] - phosphorylated MBP - RAD51 - His6 and recombinant human histone H1 , respectively , resulting from kinase assays with radio - labeled ATP . Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure . 1 : without kinase , 2 : CDKA ; 1 - CYCA2 ; 3 , 3 : CDKB1 ; 1 - CYCA2 ; 3 , 4 : CDKB1 ; 1 - CYCD2 ; 1 , 5 : CDKA ; 1 - CYCB1 ; 1 , 6 : CDKB1 ; 1 - CYCB1 ; 1 .

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F. In vitro kinase assay of purified CDK complexes phosphorylating RAD51. RAD51 and histone H1 kinase assays were performed with [γ-32P]ATP as a phosphate donor. Proteins were subjected to SDS-PAGE after the kinase reaction and stained with coomassie brilliant blue demonstrating the equal loading of the substrates (lower panels). Phosphorylated proteins were detected by autoradiograph (upper panels). The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK-cyclin complexes, protein blots indicate the relative amounts of the CDKs in the reaction (bottom panel).Abbreviation: p-RAD51 and p-Histone H1 for [32P]-phosphorylated MBP-RAD51-His6 and recombinant human histone H1, respectively, resulting from kinase assays with radio-labeled ATP. Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure. 1: without kinase, 2: CDKA;1-CYCA2;3, 3: CDKB1;1-CYCA2;3, 4: CDKB1;1-CYCD2;1, 5: CDKA;1-CYCB1;1, 6: CDKB1;1-CYCB1;1.


A . Transgenic Arabidopsis plants harboring the CYCB1 ; 1 promoter and a GFP fused to the N - terminal part of CYCB1 ; 1 including the destruction box grown on control plates and imaged five days after germination . D . Transgenic Arabidopsis plants harboring the CYCB1 ; 1 promoter and a GFP fused to the N - terminal part of CYCB1 ; 1 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application .

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A. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box grown on control plates and imaged five days after germination.D. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.


B . Transgenic Arabidopsis plants harboring the CYCB1 ; 2 promoter and a GFP fused to the N - terminal part of CYCB1 ; 2 including the destruction box grown on control plates and imaged five days after germination . E . Transgenic Arabidopsis plants harboring the CYCB1 ; 2 promoter and a GFP fused to the N - terminal part of CYCB1 ; 2 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application .

B. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box grown on control plates and imaged five days after germination.E. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.


C . Transgenic Arabidopsis plants harboring CDKB1 ; 1 promoter fused to GUS grown on control plates , were stained and imaged five days after germination . F . Transgenic Arabidopsis plants harboring CDKB1 ; 1 promoter fused to GUS germinated on control plates and were transferred four days after germination on plates supplemented with 50 μM cisplatin and were stained and imaged 24h after drug application .

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C. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS grown on control plates, were stained and imaged five days after germination.F. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS germinated on control plates and were transferred four days after germination on plates supplemented with 50 μM cisplatin and were stained and imaged 24h after drug application.


G . Chromatin immunoprecipitation ( ChIP ) of wild - type plants and PROSOG1 : SOG1 - Myc lines and anti - MYC antibody . The promoter region of CYCB1 ; 1 is enriched in SOG1 - MYC after cisplatin treatment . H . Structure of genes tested by ChIP with an antibody anti - MYC in PROSOG1 : SOG1 - Myc and wild - type plants . Red arrowheads indicate the primer binding sites for PCR . A total of four regions were tested as indicated by arrowheads . Asterisks indicates significant differences within a 5 % confidence interval ( Student ' s t - test ) .

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G. Chromatin immunoprecipitation (ChIP) of wild-type plants and PROSOG1:SOG1-Myc lines and anti-MYC antibody. The promoter region of CYCB1;1 is enriched in SOG1-MYC after cisplatin treatment.H. Structure of genes tested by ChIP with an antibody anti-MYC in PROSOG1:SOG1-Myc and wild-type plants. Red arrowheads indicate the primer binding sites for PCR. A total of four regions were tested as indicated by arrowheads. Asterisks indicates significant differences within a 5% confidence interval (Student's t-test).


A . The wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 were grown on control plates and root lengths were measured 10 days after germination . B . The wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 were grown on plates containing 0 , 6 μg / mL BLM . Root lengths were measured 10 days after germination . C . Graph represents the ratio of the mean growth rate on 0 , 6 µg / mL BLM compared to control experiments on plates lacking BLM for the wildtype , ku70 , cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 .

A. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on control plates and root lengths were measured 10 days after germination.B. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on plates containing 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.C. Graph represents the ratio of the mean growth rate on 0,6 µg/mL BLM compared to control experiments on plates lacking BLM for the wildtype, ku70, cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70.


D . Image shows the wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 ( from left to right ) grown on control plates 10 days after germination . E . Images show the wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 ( from left to right ) on plates containing 0 , 6 μg / mL BLM .

D. Image shows the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) grown on control plates 10 days after germination.E. Images show the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) on plates containing 0,6 μg/mL BLM.


( A ) BMDMs were infected with L . pneumophila JR32 or the legA9 mutant with MOI of 0 . 5 . CFUs were scored at 1 , 24 , 48 , and 72 h . ( B ) BMDMs were infected with JR32 or the legA9 mutant harboring empty PL , or legA9 + legA9 pBC ‐ KS + PL . ( A , B ) Data are presented as means ± SD of at least three independent experiments . Asterisks indicate significant differences , ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; two ‐ tailed t ‐ test ) .

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(A) BMDMs were infected with L. pneumophila JR32 or the legA9 mutant with MOI of 0.5. CFUs were scored at 1, 24, 48, and 72 h. (B) BMDMs were infected with JR32 or the legA9 mutant harboring empty PL, or legA9+ legA9 pBC‐KS+ PL. (A, B) Data are presented as means ± SD of at least three independent experiments. Asterisks indicate significant differences, (*p < 0.05, **p < 0.01, ***p < 0.001; two‐tailed t‐test).


Four female C57BL / 6 mice / group received 1 × 106 of JR32 or legA9 mutant bacteria intratracheally . Lungs were homogenized and plated for CFUs counting at ( C ) 4 or ( D ) 48 h postinfection . ( C , D ) Data are shown as mean + SD of n = 4 and are representative as one of two independent experiments . Asterisks indicate significant differences ( * * p < 0 . 01 two ‐ tailed t ‐ test )

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Four female C57BL/6 mice/group received 1 × 106 of JR32 or legA9 mutant bacteria intratracheally. Lungs were homogenized and plated for CFUs counting at (C) 4 or (D) 48 h postinfection. (C, D) Data are shown as mean + SD of n = 4 and are representative as one of two independent experiments. Asterisks indicate significant differences (**p < 0.01 two‐tailed t‐test)


( A ) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria . Nuclei were stained blue with DAPI and L . pneumophila stained green with L . pneumophila antibody . Lyso − tracker red was used to stain acidified lysosomes . White arrows show L . pneumophila colocalization with lysotracker .

(A) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria. Nuclei were stained blue with DAPI and L. pneumophila stained green with L. pneumophila antibody. Lyso−tracker red was used to stain acidified lysosomes. White arrows show L. pneumophila colocalization with lysotracker.


( B ) Percent colocalization of L . pneumophila with lyso ‐ tracker .

(B) Percent colocalization of L. pneumophila with lyso‐tracker.


( C ) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria . LC3 is stained red with LC3 antibody . White arrows show L . pneumophila colocalization with LC3 .

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(C) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria. LC3 is stained red with LC3 antibody. White arrows show L. pneumophila colocalization with LC3.


( D ) Percent colocalization of L . pneumophila with LC3 . Hundred bacteria were scored from each coverslips . Images were captured with the 60 × objective and magnified 3 × , scale bar = 10 μm . ( A - D ) Data are presented as mean + SD of n = 3 and are representative of three independent experiments . Asterisks indicate significant differences ( * * p < 0 . 01 , * * * p < 0 . 001 ; two ‐ tailed t ‐ test ) .

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(D) Percent colocalization of L. pneumophila with LC3. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. (A-D) Data are presented as mean + SD of n = 3 and are representative of three independent experiments. Asterisks indicate significant differences (**p < 0.01, ***p < 0.001; two‐tailed t‐test).


Induction of autophagy in macrophages restricts legA9 mutant replication , and rescues colocalization with LC3 and fusion with lysosomes . BMDMs were untreated or pretreated with rapamycin ( Rap ) for 1 h and kept throughout the infection . BMDMs were infected with L . pneumophila JR32 or the leg A9 mutant for ( A ) 1 or ( B ) 24 h with an MOI of 0 . 5 . CFUs were scored at these time points .

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Induction of autophagy in macrophages restricts legA9 mutant replication, and rescues colocalization with LC3 and fusion with lysosomes. BMDMs were untreated or pretreated with rapamycin (Rap) for 1 h and kept throughout the infection. BMDMs were infected with L. pneumophila JR32 or the leg A9 mutant for (A) 1 or (B) 24 h with an MOI of 0.5. CFUs were scored at these time points.


( C , D ) Percent colocalization of L . pneumophila with ( C ) lyso ‐ tracker and ( D ) LC3 . Hundred bacteria were scored from each coverslips . ( A - D ) Data are presented as mean + SD of n = 3 and are representative of three independent experiments . Asterisks indicate significant differences ( * * p < 0 . 01 ; two ‐ tailed t ‐ test ) .

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(C, D) Percent colocalization of L. pneumophila with (C) lyso‐tracker and (D) LC3. Hundred bacteria were scored from each coverslips. (A-D) Data are presented as mean + SD of n = 3 and are representative of three independent experiments. Asterisks indicate significant differences (**p < 0.01; two‐tailed t‐test).


( A ) BMDMs derived from WT and Atg5 − / − mice were infected with L . pneumophila JR32 or legA9 mutant at an MOI of 0 . 5 . Data are shown as mean ± SD of n = 3 and are representative of two independent experiments . Asterisks indicate significant differences ( * p < 0 . 05 ; two − tailed t − test .

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(A) BMDMs derived from WT and Atg5−/− mice were infected with L. pneumophila JR32 or legA9 mutant at an MOI of 0.5. Data are shown as mean ± SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05; two−tailed t−test.


( B ) Western blot showing the absence of Atg 5 in the knockout . Data are representative of three independent experiments .

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(B) Western blot showing the absence of Atg 5 in the knockout. Data are representative of three independent experiments.


( A ) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria . Polyubiquitin is stained red . White arrows show L . pneumophila colocalization with polyubiquitin .

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(A) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria. Polyubiquitin is stained red. White arrows show L. pneumophila colocalization with polyubiquitin.


( B ) Percent colocalization of L . pneumophila with polyubiquitin . Hundred bacteria were scored from each coverslips . Images were captured with the 60 × objective and magnified 3 × , scale bar = 10 μm . Data are presented as means + SD of n = 3 and are representative of two independent experiments . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 01 ; two ‐ tailed t ‐ test ) .

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(B) Percent colocalization of L. pneumophila with polyubiquitin. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm. Data are presented as means + SD of n = 3 and are representative of two independent experiments. Asterisks indicate significant differences (*p < 0.05, **p < 0.01, ***p < 0.01; two‐tailed t‐test).


( A ) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria . P62 is stained red with p62 antibody . White arrows show L . pneumophila colocalization with p62 .

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(A) Representative images from 6 h infected BMDMs with JR32 or legA9 mutant bacteria. P62 is stained red with p62 antibody. White arrows show L. pneumophila colocalization with p62.


( B ) Percent colocalization of L . pneumophila with p62 . Hundred bacteria were scored from each coverslips . Images were captured with the 60 × objective and magnified 3 × , scale bar = 10 μm .

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(B) Percent colocalization of L. pneumophila with p62. Hundred bacteria were scored from each coverslips. Images were captured with the 60× objective and magnified 3×, scale bar = 10 μm.


( C ) BMDMs were infected with JR32 or the legA9 mutant for 6 h and the level of p62 protein was quantified in cell lysates by ELISA ( performed in triplicate and the SD are too small to detect ) .

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(C)BMDMs were infected with JR32 or the legA9 mutant for 6 h and the level of p62 protein was quantified in cell lysates by ELISA (performed in triplicate and the SD are too small to detect).


BMDMs were nucleofected with scrambled control ( sictrl ) or siRNA against p62 ( sip 62 ) . Twenty ‐ four hours after nucleofection , murine macrophages were infected with JR32 or legA9 mutant with an MOI of 0 . 5 for 1 h ( D and F ) or 24 h ( E and G ) .

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BMDMs were nucleofected with scrambled control (sictrl) or siRNA against p62 (sip 62). Twenty‐four hours after nucleofection, murine macrophages were infected with JR32 or legA9 mutant with an MOI of 0.5 for 1 h (D and F) or 24 h (E and G).


( H ) Western blot analysis showing the downregulation of p62 at 24 and 48 h postnucleofection . ( A - B and D - H ) Data are shown as mean + SD of n = 3 and are representative of three independent experiments . ( C ) Data are representative of one experiment n = 3 . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 01 ; two − tailed t − test ) .

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(H) Western blot analysis showing the downregulation of p62 at 24 and 48 h postnucleofection. (A-B and D-H) Data are shown as mean + SD of n = 3 and are representative of three independent experiments. (C) Data are representative of one experiment n = 3. Asterisks indicate significant differences (*p < 0.05, **p < 0.01; two−tailed t−test).


( A ) Caspase − 1 KO ( casp − 1 − / − ) macrophages were infected with L . pneumophila JR32 or legA9 with an MOI of 0 . 5 for 1 , 24 , 48 , and 72 h .

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(A) Caspase−1 KO (casp−1−/−) macrophages were infected with L. pneumophila JR32 or legA9 with an MOI of 0.5 for 1, 24, 48, and 72 h.


( B ) CFUs were measured at the indicated time points . Levels of IL ‐ 1β were detected in supernatants of WT or casp ‐ 1 − / − BMDMs infected with JR32 or the legA9 mutant after 24 h . WT BMDMs were either not treated ( NT ) or infected with L . pneumophila JR32 or legA9 mutant bacteria for 2 h .

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(B) CFUs were measured at the indicated time points. Levels of IL‐1β were detected in supernatants of WT or casp‐1−/− BMDMs infected with JR32 or the legA9 mutant after 24 h. WT BMDMs were either not treated (NT) or infected with L. pneumophila JR32 or legA9 mutant bacteria for 2 h.


Activation of casp ‐ 1 or casp ‐ 7 was detected in cell extracts using ( C ) casp ‐ 1 or ( D ) casp ‐ 7 antibodies , respectively . ( A - D ) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments .

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Activation of casp‐1 or casp‐7 was detected in cell extracts using (C) casp‐1 or (D) casp‐7 antibodies, respectively. (A-D) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.


( A ) BMDMs were not infected ( NT ) or infected with L . pneumophila JR32 or the legA9 mutant at an MOI of 0 . 5 , 1 or 5 for 24 h . Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic ( apoptosis ) histone − associated − DNA fragments ( ELISA ) .

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(A) BMDMs were not infected (NT) or infected with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5, 1 or 5 for 24 h. Apoptosis was quantified by Photometric enzyme immunoassay analysis of cytoplasmic (apoptosis) histone−associated−DNA fragments (ELISA).


( B ) BMDMs were either not treated ( NT ) or treated with L . pneumophila JR32 or the legA9 mutant at an MOI of 0 . 5 or 5 for 24 h . The fold change in LDH release was measured from the overall population of macrophages . ( A - B ) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O 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) BMDMs were either not treated (NT) or treated with L. pneumophila JR32 or the legA9 mutant at an MOI of 0.5 or 5 for 24 h. The fold change in LDH release was measured from the overall population of macrophages. (A-B) Data are shown as mean ± SD of n = 3 and are representative of three independent experiments.


( A - F ) Vero E6 - TMPRSS2 ( A , C , E ) or Vero - E6 cells ( B , D , F ) were infected with SARS - CoV - 2 WA1 at an MOI of 0 . 001 . Supernatants were collected twice daily for two days , daily for an additional two days , and cells and supernatants were collected for a final time point at day 7 ( for Vero E6 - TMPRSS2 cells ) or day 8 ( for Vero E6 cells ) . A , B ) Viral titer was determined by immune - focus forming assay ( focus - forming units [ FFU ] / mL ) , day 0 values are calculated based on known titer of inoculum . RNA levels were determined by RT - PCR from cells ( C , D ) or viral supernatants ( E , F ) for each time point using the indicated primer - probe sets . Note that CT is plotted inversely on the Y axes , so that lower RNA concentrations are shown lower on the axes . All data points represent four technical replicates from two independent experiments and are plotted as mean with error bars plotted as + / - SEM . Limit of detection for infectious titer is plotted as the minimum y value .

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

(A-F) Vero E6-TMPRSS2 (A, C, E) or Vero-E6 cells (B, D, F) were infected with SARS-CoV-2 WA1 at an MOI of 0.001. Supernatants were collected twice daily for two days, daily for an additional two days, and cells and supernatants were collected for a final time point at day 7 (for Vero E6-TMPRSS2 cells) or day 8 (for Vero E6 cells). A, B) Viral titer was determined by immune-focus forming assay (focus-forming units [FFU]/mL), day 0 values are calculated based on known titer of inoculum. RNA levels were determined by RT-PCR from cells (C, D) or viral supernatants (E, F) for each time point using the indicated primer-probe sets. Note that CT is plotted inversely on the Y axes, so that lower RNA concentrations are shown lower on the axes. All data points represent four technical replicates from two independent experiments and are plotted as mean with error bars plotted as +/- SEM. Limit of detection for infectious titer is plotted as the minimum y value.


( A - D ) Pooled patient samples at high ( 5 . 7x104 copies ) , medium ( 3 . 6x102 copies ) or low ( 2x101 copies ) concentrations ( A , B ) , or stocks of SARS - CoV - 2 WA1 ( C , D ) were subjected to - 80ºC storage interrupted by ( A , C ) successive freeze - thaw cycles or ( B , D ) storage at 4ºC , room temperature ( RT ) or 37ºC for the indicated time periods . N = 1 for A , B . For C and D datapoints are from 3 different viral stocks titered in duplicate and plotted as mean with error bars plotted as + / - SEM . For C raw values are plotted while values are normalized to starting titer for D . Limit of detection for infectious titer in C is indicated by a dashed line .

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

(A-D) Pooled patient samples at high (5.7x104 copies), medium (3.6x102 copies) or low (2x101 copies) concentrations (A, B), or stocks of SARS-CoV-2 WA1 (C, D) were subjected to -80ºC storage interrupted by (A, C) successive freeze-thaw cycles or (B, D) storage at 4ºC, room temperature (RT) or 37ºC for the indicated time periods. N=1 for A, B. For C and D datapoints are from 3 different viral stocks titered in duplicate and plotted as mean with error bars plotted as +/- SEM. For C raw values are plotted while values are normalized to starting titer for D. Limit of detection for infectious titer in C is indicated by a dashed line.