E Lysates from 293T transiently expressing p62 - 3Myc GFP - CbpCT4 variants were immunoprecipitated using GST - GFP - Nanobody . Bound 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 transiently expressing GFP - CbpCT4 p62 - 3Myc variants were immunoprecipitated with GST - GFP - Nanobody . Bound were analyzed by immunoblotting using 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 infected with indicated S . pneumoniae strains for 2 h were fixed stained with DAPI an anti - Atg14 antibody , percentages of perinuclear - localizing Atg14 containing 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 in ( H ) are shown . Scale bars , 10 µm .

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


D GST - pulldown assays using indicated GST - fusion or GST lysates from 293T expressing GFP - Atg14 CCD or GFP . Bound 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 expressing GFP - fusion or GFP were subjected to SDS - PAGE 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 of HeLa transiently expressing GFP - fusion . Scale bars , 10 µm .

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


H , I GST - pulldown assays using indicated GST - fused or GST lysates from 293T expressing GFP - Atg14 CCD or GFP . Bound 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 , E14 . 5 n = 70 , 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 . IsolectinB4 ( IsoB4 ; ) , ERG ( red ) 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 normalized to wildtype ( WT ) mean ; one dot is two fields / mouse ; error bars : SD ; unpaired T , * p < 0 . 05 . n = 18 - 20 .

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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 , yellow in B ) . One dot is two fields / mouse ; error bars : SD ; unpaired T , * 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 gain in adult . Error bars : SD ; 2 - way ANOVA p = 0 . 0009 ; Sidak ' s multiple comparison , * * 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 . IsolectinB4 ( IsoB4 ) in black ; outer 20 % of vascularized 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 P6 developing retina . G . Radial outgrowth ( between optic nerve vascular front ) .

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


Vascularized . I . Vessel in outer 20 % of retinal vasculature ; pink in F ( vessel normalized to vascularized ) . 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.


. P , postnatal day ; w , . 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 normalized to WT mean of litter .

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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 . IsolectinB4 ( IsoB4 ) in black ; outer 20 % of vascularized in pink . Scale bar , 250 µm . P6 insets ; sprout . 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 . between optic nerve vascular front . P , postnatal day ; error bars : SD ; 2 - way ANOVA p < 0 . 0001 ; Bonferroni ' s multiple comparison , * 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 . 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 retinal front . of vessel in outer 20 % of vasculature ( pink in D ) ( vessel normalized to vascularized ) . P , postnatal day ; error bars : SD ; 2 - way ANOVA p = 0 . 0054 ; Bonferroni ' s multiple comparison , * 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 ; ) , ERG ( red ) , EdU ( green ) double - positive ( yellow ) . Lower panel shows ERG / EdU merged alone with EdU signal masked by ERG signal . Scale bar , 50 µm ; error bars : SD ; unpaired T , * 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 . Sprout from tip to base in P6 retinas ; error bars : SD ; unpaired T , * p < 0 . 05 ; n = 3 - 5 retinas , one retina / mouse .

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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 ) collagen IV ( red ) in P4 wildtype ( WT ) 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 , * * 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 pY1173 . A . Anti - VEGFR2 immunoprecipitates ( IP ) from lung lysates from wildtype ( WT ) Vegfr2Y1212F / Y1212F FVB mice , tail - vein injected with PBS or VEGF for 1 minute . Each lane ( A ) represent one 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.


as of phosphoprotein level normalized to total VEGFR2 . each dot ( B ) represent one 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 enriched in immobilized pY1212 peptide precipitate . 10 ( red oval ) were selected for further . PSM : peptide - spectrum matches . : average of spectra for 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 PI3Kp85 . HDMEC total lysate incubated with indicated immobilized peptides followed by immunoblotting for GRB2 PI3Kp85 .

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


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

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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 phosphoAkt immunostaining ( green ) of iECs from FVB wildtype ( WT ) Vegfr2Y1212F / Y1212F lungs . Hoechst 33342 ( blue ) shows nuclei . Arrows indicate accumulation of phosphoERK1 / 2 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.


accumulation expressed as phoshoERK1 / 2 - positive normalized to total phosphoERK1 / 2 by field . Each dot represents 1 field . Min , minutes of stimulation . 2 - way ANOVA p = 0 . 0248 ; Sidak ' s multiple comparison , * 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.


accumulation expressed as fold - increase of phosphoAkt in Vegfr2Y1212F / Y1212F over PBS - treated wildtype ( WT ) iECs , normalized to number of nuclei . Each dot represents 1 field . Min , minutes of stimulation . Unpaired T , * 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 total Akt on lung lysates from wiltype ( WT ) Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one mouse . 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 , * p < 0 . 05 . n = 3 experiments . 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 , * p < 0 . 05 . n = 3 experiments .

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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 ) Vegfr2Y1212F / Y1212F FVB mice tail - vein injected with PBS or VEGFA followed by circulation for time points indicated . Each lane represents one mouse . of Myc / Actin expressed as Fold change to PBS . Min , minutes of stimulation . Error bars : SD ; 2 - way ANOVA p = 0 . 0058 ; unpaired T , * 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 expression . C . Myc Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype ( WT ) mutant ( VEGFR2 Y1212F ) C57Bl / 6 FVB mice . D . of Myc / Actin expressed as Arbitrary unit . Each lane ( C ) each dot ( D ) represent one mouse . 2 - way ANOVA p < 0 . 0001 ; unpaired T , * * p < 0 . 01 . n = 5 - 7 .

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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 ) mutant ( VEGFR2 Y1212F ) C57Bl / 6 FVB mice . Each dot represents one mouse . 2 - way ANOVA p = 0 . 0041 ; Sidak ' s multiple comparison , * 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 FVB iECs . CD31 ( ) , ERG ( red ) , EdU ( green ) double - positive ( yellow ) in C57Bl / 6 ( F ) 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 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 , * p < 0 . 05 ; * * p < 0 . 01 . C57Bl / 6 n = 3 - 6 mice , FVB n = 3 - 5mice .

B-EXP_ASSAY O O O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-CELL_TYPE O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O

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 of E11 . 5 explants from C57Bl / 6 taken from wildtype ( WT ) Vegfr2Y1212F / Y1212F with ( MycOE / Cre + ) without ( MycOE / Cre - ) endothelial - specific overexpression of Myc . Endothelial marker ( CD31 ) immunostaining highlights vasculature in . Embryonic tissue blacked out to highlight sprouting .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O

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 . as fold - increase of vessel sprouting compared to WT MycOE / Cre - . Scale bar , 250 µm . Error bars : SD ; unpaired T , * * p < 0 . 01 , * * * * p < 0 . 0001 . n = 4 - 11 explants , one explant / .

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

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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 K - Ras , Angiogenesis mTORC1 gene sets in ( WT ) compared to VEGFR2 Y1212F isolated ECs . K - Ras 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 . color in shows gene expression value , expressed as a z - score across . Red color represents relative higher expression blue color represents relative lower expression value . z - score is calculated by ( x - x ' ) / s where x is gene expression , x ' is mean of gene expression across samples s is standard deviation of gene expression across samples .

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

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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 wildtype , from left to right on control plates without genotoxic agent 10 days after germination . B . wildtype , single double mutants of cycb1 were grown on control plates without genotoxic agent . Root lengths were measured 10 days after germination . C . 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 . rightmost is wee1 mutant that shows high sensitivity to HU . D . wildtype , single double mutants of cycb1 were grown on plates supplemented with 1 mM HU . Root lengths were measured 10 days after germination . E . 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 . rightmost is ku70 mutant that shows high sensitivity to BLM . F . wildtype , single 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-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-TISSUE 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 . 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 were transferred to new control plates three days after germination . Root lengths mutants were measured three days after transfer net root growth of three days is shown in graphs . I . cycb1 single mutants germinated on control plates were transferred to plates supplemented with 15 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer net root growth of three days is shown in graphs . J . cycb1 single mutants germinated on control plates were transferred to plates supplemented with 30 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer net root growth of three days is shown in graphs .

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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 . 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 were transferred to new control plates three days after germination . Root lengths were measured three days after transfer net root growth of three days is shown in graphs . M . cycb1 mutants germinated on control plates were transferred to plates supplemented with 15 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer net root growth of three days is shown in graphs . N . cycb1 mutants germinated on control plates were transferred to plates supplemented with 30 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer net root growth of three days is shown in graphs .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O 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-SMALL_MOLECULE O O O O O B-TISSUE O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O B-TISSUE 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 , cdkb1 ; 1 cdkb1 ; 2 cycb1 ; 1 cycb1 ; 3 double mutant seedlings in full spectrum view of TriTek Comet Score software . Shown are comets of incubated with 50µM cisplatin for one hour then transferred to medium without cisplatin for 30 min ( recovery ) incubated without cisplatin for one hour ( control ) , respectively . B . Box plot of of tail of wild - type , cdkb1 ; 1 cdkb1 ; 2 cycb1 ; 1 cycb1 ; 3 under cisplatin treatment . Plots are on analyses of 200 per sample from random microscopic fields of three independent biological replications . of fragments in comet tail was calculated by TriTek Comet Score software . box represents interquartile range , across box indicates median values 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 - .

O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mutant after two hours of treatment with 50µM cisplatin . D . Counted number of γ - H2AX foci per detected after two hours of treatment with 50µM cisplatin in wild - type mutant . For each sample γ - H2AX foci of 100 were counted grouped into six categories : with no , 1 - 2 , 3 - 5 , 6 - 10 , 11 - 20 more than 20 foci per .

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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 show blue spots on leaves after three days of incubation on 30 μM cisplatin . Arrows indicate representative blue sectors . C . cycb1 ; 1 show blue spots on leaves after three days of incubation on 30 μM cisplatin . Arrows indicate representative blue sectors .

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

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

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

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

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

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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 wildtype indicated mutant after two hours of treatment with 50µM cisplatin .

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

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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 of purified CDK complexes phosphorylating RAD51 . RAD51 histone H1 kinase assays were performed with [ γ - 32P ] ATP as a phosphate donor . were subjected to SDS - PAGE after kinase reaction stained with coomassie brilliant blue demonstrating equal loading of substrates ( lower panels ) . Phosphorylated were detected by autoradiograph ( upper panels ) . reactions were normalized by using equal amounts of CDKs assuring equal of active CDK - cyclin complexes , blots indicate relative amounts of CDKs in reaction ( bottom panel ) . Abbreviation : p - RAD51 p - Histone H1 for [ 32P ] - phosphorylated MBP - RAD51 - His6 recombinant human histone H1 , respectively , resulting from kinase assays with radio - labeled ATP . Asterisks indicate varying amounts of cyclins that can be in 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 harboring CYCB1 ; 1 promoter a GFP fused to N - terminal part of CYCB1 ; 1 including destruction box grown on control plates imaged five days after germination . D . Transgenic Arabidopsis harboring CYCB1 ; 1 promoter a GFP fused to N - terminal part of CYCB1 ; 1 including destruction box germinated on control plates were transferred four days after germination to plates supplemented with 50 μM cisplatin 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 harboring CYCB1 ; 2 promoter a GFP fused to N - terminal part of CYCB1 ; 2 including destruction box grown on control plates imaged five days after germination . E . Transgenic Arabidopsis harboring CYCB1 ; 2 promoter a GFP fused to N - terminal part of CYCB1 ; 2 including destruction box germinated on control plates were transferred four days after germination to plates supplemented with 50 μM cisplatin 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 harboring CDKB1 ; 1 promoter fused to GUS grown on control plates , were stained imaged five days after germination . F . Transgenic Arabidopsis harboring CDKB1 ; 1 promoter fused to GUS germinated on control plates were transferred four days after germination on plates supplemented with 50 μM cisplatin were stained 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 . immunoprecipitation ( ChIP ) of wild - type PROSOG1 : SOG1 - Myc lines anti - MYC antibody . 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 wild - type . Red arrowheads indicate 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 - ) .

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

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

O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O

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 legA9 mutant with MOI of 0 . 5 . CFUs were scored at 1 , 24 , 48 , 72 h . ( B ) BMDMs were infected with JR32 or 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 ‐ t ‐ ) .

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

O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-EXP_ASSAY B-ORGANISM I-ORGANISM I-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O

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

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


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

O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O

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

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 , rescues colocalization with LC3 fusion with lysosomes . BMDMs were untreated or pretreated with rapamycin ( Rap ) for 1 h kept throughout infection . BMDMs were infected with L . pneumophila JR32 or leg A9 mutant for ( A ) 1 or ( B ) 24 h with an MOI of 0 . 5 . CFUs were scored at these time points .

O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-CELL_TYPE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O

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

O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C, D) 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 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 are representative of two independent experiments . Asterisks indicate significant differences ( * p < 0 . 05 ; two − t − .

O O O B-CELL_TYPE O O O B-GENEPROD O O O B-ORGANISM O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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) 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 absence of Atg 5 in 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 from 6 h infected BMDMs with JR32 or legA9 mutant . Polyubiquitin is stained red . 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 ) colocalization of L . pneumophila with polyubiquitin . Hundred were scored from each coverslips . were captured with 60 × objective magnified 3 × , scale bar = 10 μm . Data are presented as means + SD of n = 3 are representative of two independent experiments . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 01 ; two ‐ t ‐ ) .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 from 6 h infected BMDMs with JR32 or legA9 mutant . P62 is stained red with p62 antibody . 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 ) colocalization of L . pneumophila with p62 . Hundred were scored from each coverslips . were captured with 60 × objective magnified 3 × , scale bar = 10 μm .

O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD 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) 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 legA9 mutant for 6 h level of p62 was quantified in lysates by ELISA ( performed in triplicate 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 F ) or 24 h ( E G ) .

B-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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 showing downregulation of p62 at 24 48 h postnucleofection . ( A - B D - H ) Data are shown as mean + SD of n = 3 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 − t − ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) 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 , 72 h .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

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

O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O O O O O

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

O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 legA9 mutant at an MOI of 0 . 5 , 1 or 5 for 24 h . Apoptosis was quantified by Photometric enzyme immunoassay of ( apoptosis ) histone − associated − fragments ( ELISA ) .

O O O B-CELL_TYPE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O 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-EXP_ASSAY O O

(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 legA9 mutant at an MOI of 0 . 5 or 5 for 24 h . fold change in LDH release was measured from overall population of macrophages . ( A - B ) Data are shown as mean ± SD of n = 3 are representative of three independent experiments .

O O O B-CELL_TYPE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE 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 ( 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 , supernatants were collected for a final time point at day 7 ( for Vero E6 - TMPRSS2 ) or day 8 ( for Vero E6 ) . A , B ) Viral titer was determined by immune - focus forming ( focus - forming units [ FFU ] / mL ) , day 0 values are calculated on known titer of inoculum . were determined by RT - PCR from ( C , D ) or viral supernatants ( E , F ) for each time point using indicated primer - probe sets . Note that CT is plotted inversely on Y axes , so that lower concentrations are shown lower on axes . data points represent four technical replicates from two independent experiments are plotted as mean with error bars plotted as + / - . Limit of detection for infectious titer is plotted as minimum y value .

O O O O O B-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O 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-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ( RT ) or 37ºC for indicated time periods . N = 1 for A , B . For C D datapoints are from 3 different viral stocks titered in duplicate plotted as mean with error bars plotted as + / - . 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 .

O O O O O O O O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


( A _ B ) Vero E6 - TMPRSS2 ( A ) or Vero E6 ( B ) were inoculated with a total of 126 clinical NP swab samples representing a range of viral , both total E ( Clinical Sample E CT detected by WHO - E primer / probe set ) subgenomic E ( CT detected by Mills - SgE primer set developed in this study ) . Culture supernatants were harvested when displayed CPE or at day 7 if no CPE was observed earlier , clarified , presence ( blue square ) or absence ( yellow circle ) of SARS - CoV - 2 determined by immuno - focus .

O O O O O B-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-TISSUE O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(A_B) Vero E6-TMPRSS2 (A) or Vero E6 (B) cells were inoculated with a total of 126 clinical NP swab samples representing a range of viral RNA levels, both total E RNA (Clinical Sample E CT detected by WHO-E primer/probe set) and subgenomic E RNA (CT detected by the Mills-SgE primer set developed in this study). Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of SARS-CoV-2 determined by immuno-focus assay.


( A - B ) Supernatants from panel of infected ( A ) Vero E6 - TMPRSS2 ( B ) Vero E6 were harvested when CPE was observed viral titer ( FFU / mL measured by focus forming ) was determined for final time point of each sample that successfully isolated . Samples are colored according to day CPE was observed sample was harvested . Total E CT of original clinical sample used for viral isolation is plotted on X axis . Data shown is from subset of samples that successfully isolated in each . Limit of detection for infectious titer is 400 FFU / mL .

O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B) Supernatants from the panel of infected (A) Vero E6-TMPRSS2 and (B) Vero E6 cells were harvested when CPE was observed and the viral titer (FFU/mL measured by focus forming assay) was determined for the final time point of each sample that successfully isolated. Samples are colored according to the day CPE was observed and the sample was harvested. Total E RNA CT of the original clinical sample used for viral isolation is plotted on the X axis. Data shown is from the subset of samples that successfully isolated in each cell line. Limit of detection for infectious titer is 400 FFU/mL.


( A ) amount of subgenomic E in panel of clinical samples used to infect Vero E6 - TMPRSS2 was measured using a previously - published primer - probe set ( Wölfel - sgE )

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(A) The amount of subgenomic E RNA in the panel of clinical samples used to infect Vero E6-TMPRSS2 cells was measured using a previously-published primer-probe set (Wölfel-sgE)


( B ) For a subset of 85 NP swab samples for which sufficient material remained , SARS - CoV - 2 negative - strand E were determined by strand - specific RT - PCR with tagged primers . Data information : Culture supernatants were harvested when displayed CPE or at day 7 if no CPE was observed earlier , clarified , presence ( blue square ) or absence ( yellow circle ) of infectious SARS - CoV - 2 determined by immuno - focus .

O O O O O O O O B-TISSUE O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(B) For a subset of 85 NP swab samples for which sufficient material remained, SARS-CoV-2 negative-strand E RNA levels were determined by strand-specific RT-PCR with tagged primers. Data information: Culture supernatants were harvested when cells displayed CPE or at day 7 if no CPE was observed earlier, clarified, and presence (blue square) or absence (yellow circle) of infectious SARS-CoV-2 determined by immuno-focus assay.


of housekeeping genes GAPDH 18S rRNA , negative - strand E ( negE ) , sgE relative to total of E were measured in infected , clarified supernatants concentrated through a sucrose cushion by ultracentrifugation . Data points represent four technical replicates from two independent experiments ( Supernatant ) or three replicates ( Concentrated ) are normalized to total of E for each sample , plotted as mean with error bars plotted as + / - .

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Levels of the cellular housekeeping genes GAPDH and 18S rRNA, negative-strand E (negE), and sgE relative to total levels of E RNA were measured in infected cells, clarified supernatants and virus concentrated through a sucrose cushion by ultracentrifugation. Data points represent four technical replicates from two independent experiments (Cell and Supernatant) or three replicates (Concentrated Virus) and are normalized to the total levels of E for each sample, plotted as mean with error bars plotted as +/- SEM.


( A ) Immunoblot of BAG1 , BAG3 , Hsc / Hsp70 Hsp90 in young old I90 . For detection of BAG , an antibody directed against conserved BAG domain was used .

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(A) Immunoblot analysis of BAG1, BAG3, Hsc/Hsp70 and Hsp90 in young and old I90 cells. For detection of BAG proteins, an antibody directed against the conserved BAG domain was used.


( B ) Real ‐ time PCR of human BAG family members Hsc70 Hsp90 in young old I90 . Depicted is expression ( log2 ) of target genes in old relative to young . * P0 . 05 * * P0 . 01 versus young , n = 3 .

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(B) Real‐time PCR analysis of human BAG family members and Hsc70 and Hsp90 in young and old I90 cells. Depicted is the expression ratio (log2) of target genes in old cells relative to young cells. *P0.05 and **P0.01 versus young, n=3.


( C ) Co ‐ IP of Hsc / Hsp70 interaction with BAG1 BAG3 in young ( Y ) old ( O ) I90 . Upper panel shows relative amounts of in lysates used for Co ‐ IP ( Input ) . Middle panel shows of BAG1 BAG3 found in Hsc / Hsp70 immunoprecipitates . Lower panel shows of Hsc / Hsp70 co ‐ sedimented upon immunoprecipitation of BAG3 .

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(C) Co‐IP analysis of Hsc/Hsp70 interaction with BAG1 and BAG3 in young (Y) and old (O) I90 cells. Upper panel shows relative amounts of proteins in cell lysates used for Co‐IP (Input). Middle panel shows levels of BAG1 and BAG3 found in Hsc/Hsp70 immunoprecipitates. Lower panel shows levels of Hsc/Hsp70 co‐sedimented upon immunoprecipitation of BAG3.


( D , E ) 293 were treated with indicated agents for 8 h followed by immunoblot of indicated .

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(D, E) 293 cells were treated with the indicated agents for 8 h followed by immunoblot analysis of indicated proteins.


( A ) 293 were transfected with bag1 , bag3 or nonsense ( nons ) siRNAs , as indicated . After 48 h , were transfected with d2GFP expression plasmid together with half amounts of indicated siRNAs . After additional 24 h , of indicated were detected by immunoblot .

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(A) 293 cells were transfected with bag1, bag3 or nonsense (nons) siRNAs, as indicated. After 48 h, cells were transfected with d2GFP expression plasmid together with half the amounts of the indicated siRNAs. After additional 24 h, levels of indicated proteins were detected by immunoblot analysis.


( B ) 293 were transfected with indicated siRNAs for 48 h followed by real ‐ time PCR of BAG1 BAG3 mRNA . Depicted is mean relative expression ( log2 ) ± s . e . m . * P0 . 05 * * * P0 . 001 versus nons , n = 3 .

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(B) 293 cells were transfected with indicated siRNAs for 48 h followed by real‐time PCR analysis of BAG1 and BAG3 mRNA levels. Depicted is the mean relative expression ratio (log2) ±s.e.m. *P0.05 and ***P0.001 versus nons, n=3.


( C ) BAG1 knockdown was performed in 293 for 48 h . Thereafter , were transfected with GFP or GFP ‐ UPS reporter genes as indicated . After additional 24 h , of GFP ‐ positive were detected by western ‐ blot using a GFP antibody .

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(C) BAG1 knockdown was performed in 293 cells for 48 h. Thereafter, cells were transfected with GFP or GFP‐based UPS reporter genes as indicated. After additional 24 h, levels of GFP‐positive proteins were detected by western‐blot analysis using a GFP antibody.


( D , E ) Immunoblot of indicated from d2HEK treated with different concentrations of MG132 for 5 h ( D ) or transfected with indicated amounts of BAG1S plasmid for 24 h ( E ) .

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(D, E) Immunoblot analysis of indicated proteins from d2HEK cells treated with different concentrations of MG132 for 5 h (D) or transfected with indicated amounts of BAG1S plasmid for 24 h (E).


( A ) 293 were transfected with nonsense ( nons ) , bag1 or bag3 siRNAs for 48 h then treated for 2 h with lysosomal inhibitors pepstatinA E64 ( both 10 μg / ml ; Pep . A / E64 ) or DMSO as control , followed by immunoblot of indicated .

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(A) 293 cells were transfected with nonsense (nons), bag1 or bag3 siRNAs for 48 h and then treated for 2 h with the lysosomal inhibitors pepstatinA and E64 (both 10 μg/ml; Pep.A/E64) or DMSO as control, followed by immunoblot analysis of the indicated proteins.