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

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

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 / .

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 O

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 .

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

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 .

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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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

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

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 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 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 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

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-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 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 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 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

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

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 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 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

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 .

O O O O 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. 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 .

O O O O O 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 B-CONTROLLED_VAR 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 . 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 .

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

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

O O O O O O O O O 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

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 .

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

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 .

O O O O B-MEASURED_VAR 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 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 .

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

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 .

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

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 .

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

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 .

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

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 - ) .

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

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 .

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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

(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 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

(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 .

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

(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 .

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 B-MEASURED_VAR O

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

(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 .

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

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

(B) 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 ) .

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

(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 ) .

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

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 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

(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 O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

(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 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 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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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

(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 )

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

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

(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 + / - .

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

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 .

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

(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 .

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

(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 .

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

(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 .

(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 .

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

(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 .

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

(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 .

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

(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 ) .

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 B-MEASURED_VAR O O O O O O O O

(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 .

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

(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.


( B ) 293 were transfected for 48 h with indicated siRNAs BAG3 expression plasmid ( BAG3 ‐ N1 ) or vector control ( N1 ) followed by same as in ( A ) .

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

(B) 293 cells were transfected for 48 h with the indicated siRNAs and BAG3 expression plasmid (BAG3‐N1) or vector control (N1) followed by the same analysis as in (A).


( C ) Diagram shows autophagic flux of 293 with differently modulated BAG1 BAG3 as described in ( A ) ( B ) . Autophagic flux was determined by strength of LC3 ‐ II accumulation in a 2 ‐ h treatment period with Pep . A / E64 . Therefore , normalised LC3 ‐ II in absence of inhibitors were subtracted from corresponding obtained in presence of Pep . A / E64 . Values are expressed as mean ± s . e . m . * P0 . 05 versus control ‐ transfected , n = 3 .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-MEASURED_VAR 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 O O 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) Diagram shows the autophagic flux of 293 cells with differently modulated BAG1 and BAG3 levels as described in (A) and (B). Autophagic flux was determined by the strength of LC3‐II accumulation in a 2‐h treatment period with Pep.A/E64. Therefore, normalised LC3‐II levels in the absence of inhibitors were subtracted from corresponding levels obtained in the presence of Pep.A/E64. Values are expressed as mean±s.e.m. *P0.05 versus control‐transfected cells, n=3.


( D ) I90 were transfected with GFP fused to LC3 ( pGFP . LC3 ) co ‐ transfected either with BAG3 ‐ N1 or N1 . After transfection for 48 h , were treated as in ( A ) of indicated were detected by immunoblot .

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

(D) I90 cells were transfected with GFP fused to LC3 (pGFP.LC3) and co‐transfected either with BAG3‐N1 or N1. After transfection for 48 h, cells were treated as in (A) and levels of indicated proteins were detected by immunoblot analysis.


( E ) I90 , transfected as in ( D ) for 24 h , were microscopically analysed for GFP ‐ LC3 . Representative pictures are shown . Bar : 20 μm . Values expressed in diagram are mean ± s . e . m . from three independent experiments ( 50 were counted per experiment ) . * P0 . 05 versus N1 , n = 3 .

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

(E) I90 cells, transfected as in (D) for 24 h, were microscopically analysed for GFP‐LC3 fluorescence. Representative pictures are shown. Bar: 20 μm. Values expressed in diagram are mean±s.e.m. from three independent experiments (50 cells were counted per experiment). *P0.05 versus N1, n=3.


( A ) I90 were transfected with BAG3 ( BAG3 ‐ N1 ) or vector control . After transfection for 48 h , of indicated were detected by western ‐ blot .

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

(A) I90 cells were transfected with BAG3 (BAG3‐N1) or vector control. After transfection for 48 h, levels of indicated proteins were detected by western‐blot analysis.


( B ) Transcript of SQSTM1 in I90 transfected as in ( A ) were compared by real ‐ time PCR . Depicted is fold ‐ increase of SQSTM1 mRNA in BAG3 ‐ N1 ‐ transfected relative to control ( N1 ) ‐ transfected . Values are expressed as mean ± s . e . m . * * P0 . 01 versus N1 , n = 3 .

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

(B) Transcript levels of SQSTM1 in I90 cells transfected as in (A) were compared by real‐time PCR analysis. Depicted is the fold‐increase of SQSTM1 mRNA levels in BAG3‐N1‐transfected cells relative to control (N1)‐transfected cells. Values are expressed as mean±s.e.m. **P0.01 versus N1 cells, n=3.


( C ) I90 were transfected with a BAG3 ‐ GFP fusion plasmid for 48 h followed by indirect immunofluorescence of endogenous SQSTM1 . ( a ) Direct of BAG3 ‐ GFP ( green ) , ( b ) indirect immunofluorescence of SQSTM1 ( red ) ( c ) stainings of ( a ) ( b ) overlapped . DAPI ( blue ) was used to stain . Representative pictures are shown . Bar : 10 μm .

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 B-MEASURED_VAR 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

(C) I90 cells were transfected with a BAG3‐GFP fusion plasmid for 48 h followed by indirect immunofluorescence staining of endogenous SQSTM1. (a) Direct fluorescence of BAG3‐GFP (green), (b) indirect immunofluorescence of SQSTM1 (red) and (c) the stainings of (a) and (b) overlapped. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 10 μm.


( D ) Co ‐ IP studies testing interaction of SQSTM1 BAG3 . I90 were transfected as in ( A ) . After 24 h , SQSTM1 BAG3 were immunoprecipitated followed by of co ‐ sedimented , as indicated . As negative control purified rabbit ( rb ) mouse ( ms ) IgG was used . Left panel shows relative amounts of in lysates used for Co ‐ IP ( Input ) .

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

(D) Co‐IP studies testing the interaction of SQSTM1 and BAG3. I90 cells were transfected as in (A). After 24 h, SQSTM1 and BAG3 were immunoprecipitated followed by analysis of co‐sedimented proteins, as indicated. As negative control purified rabbit (rb) and mouse (ms) IgG was used. Left panel shows relative amounts of proteins in lysates used for Co‐IP (Input).


( A ) Proteasomal chymotrypsin cathepsin activity in lysates from young ( Y ) old ( O ) I90 was determined using specific probes , as described in Material methods section . Values are expressed are mean ± s . e . m . * P0 . 05 * * P0 . 01 versus young , n = 3 .

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

(A) Proteasomal chymotrypsin and cathepsin activity in lysates from young (Y) and old (O) I90 cells was determined using specific fluorescence probes, as described in the Material and methods section. Values are expressed are mean±s.e.m. *P0.05 and **P0.01 versus young, n=3.


( B ) Real ‐ time PCR of LC3 , WIPI1 SQSTM1 mRNA in young old I90 . Depicted is mean expression ( log2 ) ± s . e . m . of target genes in old relative to young . * * P0 . 01 * * * P0 . 001 versus young , n = 3 .

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

(B) Real‐time PCR analysis of LC3, WIPI1 and SQSTM1 mRNA levels in young and old I90 cells. Depicted is the mean expression ratio (log2) ±s.e.m. of target genes in old cells relative to young cells. **P0.01 and ***P0.001 versus young, n=3.


( C ) Immunoblot of LC3 SQSTM1 in young old I90 .

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

(C) Immunoblot analysis of LC3 and SQSTM1 in young and old I90 cells.


( D ) Young old I90 were treated for 2 h with bafilomycin A1 ( BafA1 , 2 μM ) or DMSO as control followed by immunoblot of indicated .

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

(D) Young and old I90 cells were treated for 2 h with bafilomycin A1 (BafA1, 2 μM) or DMSO as control followed by immunoblot analysis of indicated proteins.


( E ) Immunoblot of WIPI1 expression in young old I90 .

(E) Immunoblot analysis of WIPI1 expression in young and old I90 cells.


( F ) Indirect immunofluorescence of endogenous LC3 ( green ) , SQSTM1 ( red ) WIPI1 ( ) in I90 of young old . DAPI ( blue ) was used to stain . Representative pictures are shown . Bar : 20 μm . Diagrams show of with indicated characteristics counted as in Figure 3E .

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

(F) Indirect immunofluorescence staining of endogenous LC3 (green), SQSTM1 (red) and WIPI1 (white) in I90 cells of young and old age. DAPI (blue) was used to stain DNA. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells with indicated characteristics counted as in Figure 3E.


( G ) Old young I90 were treated for 1 h with DMSO as control ( C ) , lactacystin ( L , 2 μM ) or NH4Cl ( 20 mM ) plus leupeptin ( Leu , 5 μM ) ( N , NH4Cl / Leu ) . Western ‐ blot analyses were performed for detection of indicated . In diagram ( right panel ) , of polyUb ‐ SQSTM1 are depicted after normalisation to corresponding Tubulin . Values are expressed as mean ± s . e . m . * P0 . 05 versus old control , # P0 . 05 versus young control , n = 3 .

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

(G) Old and young I90 cells were treated for 1 h with DMSO as control (C), lactacystin (L, 2 μM) or NH4Cl (20 mM) plus leupeptin (Leu, 5 μM) (N, NH4Cl/Leu). Western‐blot analyses were performed for detection of indicated proteins. In the diagram (right panel), levels of polyUb‐proteins and SQSTM1 are depicted after normalisation to corresponding Tubulin levels. Values are expressed as mean±s.e.m. *P0.05 versus old control, #P0.05 versus young control, n=3.


( H ) I90 of old ( upper panel ) young ( lower panel ) were transfected with atg7 or nonsense ( nons ) siRNA . After transfection for 4 days , were treated with NH4Cl / Leu or DMSO for 1 h followed by fractioning of lysates in TritonX ‐ 100 ( TX ‐ 100 ) soluble insoluble material . Equal amounts of both fractions were directed to immunoblot for of indicated . Gapdh Histone H3 were used as loading controls of soluble insoluble fractions , respectively .

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

(H) I90 cells of old (upper panel) and young (lower panel) age were transfected with atg7 or nonsense (nons) siRNA. After transfection for 4 days , the cells were treated with NH4Cl/Leu or DMSO for 1 h followed by fractioning of cell lysates in TritonX‐100 (TX‐100) soluble and insoluble material. Equal protein amounts of both fractions were directed to immunoblot analysis for analysis of indicated proteins. Gapdh and Histone H3 were used as loading controls of soluble and insoluble fractions, respectively.


( I ) Transmission electron microscopic of young ( a ) old ( b ) I90 . Magnifications of marked areas in ( a ) ( b ) are shown in ( c ) ( d ) , respectively . An arrow indicates an autolysosome , arrow heads indicate autophagosomes . M , mitochondrion . N , nucleus .

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

(I) Transmission electron microscopic analysis of young (a) and old (b) I90 cells. Magnifications of marked areas in (a) and (b) are shown in (c) and (d), respectively. An arrow indicates an autolysosome, and arrow heads indicate autophagosomes. M, mitochondrion. N, nucleus.


( A ) Immunoblot of indicated of young ( Y ) old ( O ) I90 upon fractionation as described in Figure 5H .

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) Immunoblot analysis of indicated proteins of young (Y) and old (O) I90 cells upon fractionation as described in Figure 5H.


( B , C ) After transfection for 96 h of old I90 with bag3 or nonsense ( nons ) siRNA , BAG1 BAG3 mRNA were analysed by immunoblot ( B ) real ‐ time PCR ( C ) , respectively . Transcript in bag3 siRNA are depicted as mean log2 expression ± s . e . m . relative to nons siRNA . * P0 . 05 * * * P0 . 001 versus nons , n = 3 .

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

(B, C) After transfection for 96 h of old I90 cells with bag3 or nonsense (nons) siRNA, BAG1 and BAG3 protein and mRNA levels were analysed by immunoblot (B) and real‐time PCR (C) analysis, respectively. Transcript levels in bag3 siRNA cells are depicted as the mean log2 expression ratio ±s.e.m. relative to nons siRNA cells. *P0.05 and ***P0.001 versus nons, n=3.


( D ) Indirect immunofluorescence of endogenous LC3 ( green ) WIPI1 ( ) in old I90 transfected with nons ( a , b ) bag3 siRNA ( c , d ) for 96 h . DAPI ( blue ) was used as a marker . Representative pictures are shown . Bar : 20 μm . Diagrams show of counted as in Figure 3E .

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

(D) Indirect immunofluorescence analysis of endogenous LC3 (green) and WIPI1 (white) in old I90 cells transfected with nons (a, b) and bag3 siRNA (c, d) for 96 h. DAPI (blue) was used as a nuclear marker. Representative pictures are shown. Bar: 20 μm. Diagrams show percentage of cells counted as in Figure 3E.


( E ) Same as in Figure 5D but old transfected as in ( A ) were used . Values expressed in diagram are mean ± s . e . m . * P0 . 05 versus nons , n = 3 .

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

(E) Same analysis as in Figure 5D but old cells transfected as in (A) were used. Values expressed in the diagram are mean±s.e.m. *P0.05 versus nons, n=3.


( F ) Same as in Figure 5G but old I90 transfected as in ( A ) were used . C , control ; L , lactacystin , N , NH4Cl / Leu . Values are expressed as mean ± s . e . m . * P0 . 05 versus nons control , or as indicated , n = 3 .

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

(F) Same analysis as in Figure 5G but old I90 cells transfected as in (A) were used. C, control; L, lactacystin, N, NH4Cl/Leu. Values are expressed as mean±s.e.m. *P0.05 versus nons control, or as indicated, n=3.


( G , H ) Same as in ( F ) but young I90 48 h after transfection with a BAG3 expression plasmid ( BAG3 ‐ N1 ) or vector control ( N1 ) together with nons or sqstm1 siRNA ( G ) nons or atg7 siRNA ( H ) , as indicated , were used . * , # , ∼ + , P0 . 05 versus C N1 , C BAG3 ‐ N1 , C BAG3 ‐ N1 sqstm1 or atg7 C N1 sqstm1 or atg7 , n = 3 .

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

(G, H) Same analysis as in (F) but young I90 cells 48 h after transfection with a BAG3 expression plasmid (BAG3‐N1) or vector control (N1) together with nons or sqstm1 siRNA (G) and nons or atg7 siRNA (H), as indicated, were used. *, #, ∼ and +, P0.05 versus C N1, C BAG3‐N1, C BAG3‐N1 sqstm1 or atg7 and C N1 sqstm1 or atg7, n=3.


( A ) extracts from cerebellum ( CER ) of young ( Y , 3 months ) old ( O , 24 months ) mice were analysed for BAG1 BAG3 expression by immunoblot .

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

(A) Protein extracts from cerebellum (CER) of young (Y, 3 months) and old (O, 24 months) mice were analysed for BAG1 and BAG3 expression by immunoblot analysis.


( B ) Real ‐ time PCR of indicated mRNA in cerebellum of young old mice . Depicted is log2 expression of target genes in old mice relative to young mice . Values are expressed as mean ± s . e . m . * P0 . 05 * * P0 . 01 versus young , n = 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Real‐time PCR analysis of indicated mRNA levels in cerebellum of young and old mice. Depicted is the log2 expression ratio of target genes in old mice relative to young mice. Values are expressed as mean±s.e.m. *P0.05 and **P0.01 versus young, n=3.


( C ) Immunoblot as in ( A ) but in cortex ( CTX ) , hippocampus ( HIP ) mid ‐ brain ( MB ) .

O O O 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) Immunoblot analysis as in (A) but in cortex (CTX), hippocampus (HIP) and mid‐brain (MB).


( D ) Western ‐ blot of indicated in different brain regions of young old mice .

(D) Western‐blot analysis of indicated proteins in different brain regions of young and old mice.


( E ) Total cathepsin specific cathepsin B activity in brain extracts from young old mice was determined using probes Z ‐ FR ‐ AMC Z ‐ RR ‐ AMC , respectively , as described in Material methods section . Values are expressed as mean ± s . e . m . * P0 . 05 versus young , n = 3 .

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

(E) Total cathepsin and specific cathepsin B activity in brain extracts from young and old mice was determined using fluorescence probes Z‐FR‐AMC and Z‐RR‐AMC, respectively, as described in the Material and methods section. Values are expressed as mean±s.e.m. *P0.05 versus young, n=3.


( F ) Expression of indicated in hippocampal astrocytic neuronal cultures from young ( 2 months ) old ( 24 months ) rats . Detection of GLT1 NeuN served as astrocyte neuron markers , respectively . Note NeuN signals in astrocytic cultures . We attributed these signals to presence of resting nuclei of dead neurons , which we observed under microscope .

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

(F) Expression analysis of indicated proteins in primary hippocampal astrocytic and neuronal cell cultures from young (2 months) and old (24 months) rats. Detection of GLT1 and NeuN served as astrocyte and neuron markers, respectively. Note the NeuN signals in astrocytic cultures. We attributed these signals to the presence of resting nuclei of dead neurons, which we observed under the microscope.


Growth rate trajectories for duplicate experiments ( n = 2 evolution experiments per substrate condition ) ( green purple ) for example case of D - lyxose . Population growth rates are plotted against cumulative divisions . Clones were isolated for whole genome sequencing at notable growth - rate plateaus as indicated by arrows . Mutations gained at each plateau are highlighted beside arrows ( mutations arising earlier along trajectory persisted in later sequenced clones ) .

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

Growth rate trajectories for duplicate experiments (n=2 evolution experiments per substrate condition) (green and purple) for the example case of D-lyxose. Population growth rates are plotted against cumulative cell divisions. Clones were isolated for whole genome sequencing at notable growth-rate plateaus as indicated by the arrows. Mutations gained at each plateau are highlighted beside the arrows (mutations arising earlier along the trajectory persisted in later sequenced clones).


YihS V314L + R315S mutant enzyme activity on D - mannose D - lyxose . LC - MS was used to analyze YihS activity at saturating substrate concentrations to compare turnover rates on each substrate . Product formation was followed over time at a constant enzyme . Turnover rates were calculated using linear regression ( n = 3 replicates for each enzyme , Dataset EV4 ) . error bars represent standard deviation ( n = 3 ) of peak .

B-MEASURED_VAR 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 O O O O O O

YihS V314L + R315S mutant enzyme activity on D-mannose and D-lyxose. LC-MS was used to analyze YihS activity at saturating substrate concentrations to compare turnover rates on each substrate. Product formation was followed over time at a constant enzyme concentration. Turnover rates were calculated using linear regression (n=3 replicates for each enzyme, Dataset EV4). The error bars represent standard deviation (n=3) of the peak area.


Turnover ratios of substrate conversion of D - lyxose / D - mannose are shown for wild type YihS mutant YihS enzymes . A < 1 indicates a higher turnover rate on D - mannose compared to D - lyxose . Error bars represent standard error ( n = 3 ) calculated from linear regression .

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

Turnover ratios of substrate conversion of D-lyxose / D-mannose are shown for the wild type YihS and mutant YihS enzymes. A ratio < 1 indicates a higher turnover rate on D-mannose compared to D-lyxose. Error bars represent standard error (n=3) calculated from the linear regression analysis.


Expression data ( RNAseq ) for significantly differentially expressed genes ( q - value < 0 . 05 , FDR - adjusted p - value , n = 2 biological replicates for each condition ) . Scatter plot shows log2 ( fold change ) of gene expression data comparing endpoint to initial populations for Exp . 1 Exp . 2 ( grey dots ) with location of gene in reference genome as x - axis . Those genes that are associated with AraC transcription units are highlighted ( red dots for Exp . 1 blue dots for Exp . 2 ) . Above plot , transcription units are labeled green if AraC activates expression ( in presence of arabinose ) or red if AraC represses expression of those genes .

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

Expression data (RNAseq) for significantly differentially expressed genes (q-value < 0.05, FDR-adjusted p-value, n=2 biological replicates for each condition). Scatter plot shows log2(fold change) of gene expression data comparing endpoint to initial populations for Exp. 1 and Exp. 2 (grey dots) with the location of the gene in the reference genome as the x-axis. Those genes that are associated with AraC transcription units are highlighted (red dots for Exp. 1 and blue dots for Exp. 2). Above the plot, the transcription units are labeled green if AraC activates expression (in the presence of arabinose) or red if AraC represses expression of those genes.


Growth rate of various weaned ( starting point of static phase ) optimized ( endpoint of static phase ) strains with or without fucK or araB genes knocked out . Strains were grown in triplicate ( n = 3 ) on M9 minimal media with D - arabinose as sole carbon source . colored bars represent calculated mean growth rate error bars represent standard deviation . p - values reported were calculated using a two - sided Welch ' s t - .

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

Growth rate analysis of various weaned (starting point of static phase) and optimized (endpoint of static phase) strains with or without fucK or araB genes knocked out. Strains were grown in triplicate (n=3) on M9 minimal media with D-arabinose as the sole carbon source. The colored bars represent the calculated mean growth rate and the error bars represent the standard deviation. The p-values reported were calculated using a two-sided Welch's t-test.


A : on phase contrast microscopy for parental fibroblast lines ( upper panel ) iPSCs ( lower panel ) from control Detroit 551 , WS5A CP2A POLG ( scale bars , 50 µm ) .

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

A: Morphology on phase contrast microscopy for parental fibroblast lines (upper panel) and iPSCs (lower panel) from control Detroit 551, WS5A and CP2A POLG patients (scale bars, 50 µm).


B : Immunofluorescence of markers POU5F1 ( green ) SSEA4 ( red ) : upper panel - Detroit 551 control iPSCs , middle panel - WS5A iPSCs lower panel - CP2A iPSCs ( Scale bar , 100 µm ) . Nuclei are stained with DAPI ( blue ) .

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

B: Immunofluorescence staining of stem cell markers POU5F1 (green) and SSEA4 (red): upper panel - Detroit 551 control iPSCs, middle panel - WS5A iPSCs and lower panel - CP2A iPSCs (Scale bar, 100 µm). Nuclei are stained with DAPI (blue).


C : RT - qPCR of gene expression for LIN28A , NANOG , POU5F1 for iPSCs from control Detroit 551 , WS5A CP2A POLG ( n = 7 , technical replicates per for ESCs ; n = 4 , technical replicates per clone for control , WS5A CP2A iPSCs ) . gene expression of clones is assessed with fold change using comparative ΔΔCt method by normalizing iPSCs to ESC1 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 O O O O O O O O O O O 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: RT-qPCR quantification of gene expression for LIN28A, NANOG, and POU5F1 for all iPSCs from control Detroit 551, WS5A and CP2A POLG patients (n=7, technical replicates per line for ESCs; n=4, technical replicates per clone for control, WS5A and CP2A iPSCs). The gene expression of the individual clones is assessed with fold change using the comparative ΔΔCt method by normalizing iPSCs to ESC1.


D , E : Flow cytometric of expression level of SSEA4 ( D , n = 9 , technical replicates per for ESCs ; n = 5 , technical replicates per clone for control iPSCs ; n = 3 , technical replicates per clone for WS5A iPSCs , n = 8 , technical replicates per clone for CP2A iPSCs ) POU5F1 ( E , n = 9 , technical replicates per for ESCs ; n = 5 , technical replicates per clone for control iPSCs ; n = 3 , technical replicates per clone for WS5A iPSCs , n = 8 , technical replicates per clone for CP2A iPSCs ) for ESCs iPSCs for both ESC control lines iPSCs generated from Detroit 551 control , WS5A CP2A fibroblasts .

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

D, E: Flow cytometric quantification of expression level of SSEA4 (D, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) and POU5F1 (E, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) for ESCs and iPSCs for both ESC control lines and iPSCs generated from Detroit 551 control, WS5A and CP2A fibroblasts.


A : Representative confocal of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN ( red ) HNF4A ( green ) ( a ) ( scale bar , 100 µm ) , mesodermal derived cardiomyocytes with positive expression of TNNT2 ( red ) ( b ) ( scale bar , 100 µm ) ectodermal derived dopaminergic neurons with positive expression of TH ( green ) MAP2 ( red ) ( c ) ( scale bar , 10 µm ) . Nuclei are stained with DAPI ( blue ) .

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

A: Representative confocal images of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN (red) and HNF4A (green) (a) (scale bar, 100 µm), mesodermal derived cardiomyocytes with positive expression of TNNT2 (red) (b) (scale bar, 100 µm) and ectodermal derived dopaminergic neurons with positive expression of TH (green) and MAP2 (red) (c) (scale bar, 10 µm). Nuclei are stained with DAPI (blue).


B : Confocal of mitochondrial for iPSC lines with co - of MTG ( upper panel ) TMRE ( lower panel ) ( scale bars , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

O O O O O O O O O O O O O O O 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: Confocal images of mitochondrial morphology for iPSC lines with co-staining of MTG (upper panel) and TMRE (lower panel) (scale bars, 25 µm). Nuclei are stained with DAPI (blue).


Flow cytometric of iPSCs generated from Detroit 551 , WS5A CP2A fibroblasts for mitochondrial volume ( MTG ) ( C , n = 6 , technical replicates per clone for control CP2A ; n = 5 , technical replicates per clone for WS5A )

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

Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for mitochondrial volume (MTG) (C, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A)