Figure 1A . Spindle in a fixed HeLa cell expressing PRC1 - GFP ( green ) and mRFP - CENP - B ( magenta ) oriented horizontally with respect to the imaging plane , as shown in the scheme . Images of different z - slices ( central plane of the spindle z = 0 , two images below , z = - 4 µm and z = - 2 µm , and above , z = + 3 µm and z = + 5 µm ) , maximum projection of a z - stack ( max z ) , and 3D projections ( 3D ) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown . Additional z - slices of this spindle are shown in Fig EV1A . B . Spindle in a fixed HeLa cell expressing PRC1 - GFP ( green ) and mRFP - CENP - B ( magenta ) oriented vertically with respect to the imaging plane , as shown in the scheme . Legend as in A . Additional z - slices of this spindle are shown in Fig EV1B . C . Correlation between the number of PRC1 - labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal ( black ) and vertical ( blue ) spindles of fixed HeLa cells in metaphase . Data points represent individual spindles , lines show linear fits . D . Pie charts showing the fraction of PRC1 - labeled bundles associated with kinetochore pairs ( blue ) , PRC1 - labled bundles not associated with kinetochores ( green ) and kinetochores not associated with PRC1 bundles ( magenta ) in horizontal ( top row ) and vertical spindles ( bottom row ) from cells with up to 70 chromosomes ( left column ) and more than 70 chromosomes ( right column ) . Horizontal spindles contained a total of 1786 kinetochore - PRC1 pairs ( n = 29 cells ) and vertical spindles 1133 kinetochore - PRC1 pairs ( n = 16 cells ) . E , F . Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1 - labeled bundles . G . Number of kinetochore pairs coupled with PRC1 - labeled bundles per unit area of the cross section of the central part of horizontal ( black ) and vertical ( blue ) spindles as a function of the number of kinetochore pairs coupled with PRC1 - labeled bundles in the spindle .

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

Figure 1A. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented horizontally with respect to the imaging plane, as shown in the scheme. Images of different z-slices (central plane of the spindle z=0, two images below, z=-4 µm and z=-2 µm, and above, z=+3 µm and z=+5 µm), maximum projection of a z-stack (max z), and 3D projections (3D) with the 3D coordinate system represented as a cuboidal box that indicates different spindle orientations are shown. Additional z-slices of this spindle are shown in Fig EV1A.B. Spindle in a fixed HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane, as shown in the scheme. Legend as in A. Additional z-slices of this spindle are shown in Fig EV1B.C. Correlation between the number of PRC1-labeled bundles and the number of pairs of sister kinetochores counted throughout horizontal (black) and vertical (blue) spindles of fixed HeLa cells in metaphase. Data points represent individual spindles, lines show linear fits.D. Pie charts showing the fraction of PRC1-labeled bundles associated with kinetochore pairs (blue), PRC1-labled bundles not associated with kinetochores (green) and kinetochores not associated with PRC1 bundles (magenta) in horizontal (top row) and vertical spindles (bottom row) from cells with up to 70 chromosomes (left column) and more than 70 chromosomes (right column). Horizontal spindles contained a total of 1786 kinetochore-PRC1 pairs (n=29 cells) and vertical spindles 1133 kinetochore-PRC1 pairs (n=16 cells).E, F. Spindle length and spindle width as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles.G. Number of kinetochore pairs coupled with PRC1-labeled bundles per unit area of the cross section of the central part of horizontal (black) and vertical (blue) spindles as a function of the number of kinetochore pairs coupled with PRC1-labeled bundles in the spindle.


Figure 2A . Central plane of the spindle in a live HeLa cell expressing PRC1 - GFP ( green ) and mRFP - CENP - B ( magenta ) oriented vertically with respect to the imaging plane . B . Examples of trajectories , with respect to the spindle ' s center of the mass , of individual PRC1 - GFP ( green ) and corresponding mRFP - CENP - B ( magenta ) signals from the spindle in A that moved together for at least 200 s . Dots represent starting points of trajectories , t = 0 s . Trajectories finish at t = 200 s . Grey circle represents the center of mass of the spindle . C . Pie chart showing the fraction of PRC1 - labeled fibers and sister kinetochores that moved together for at least 60 s ( blue , n = 226 pairs in 5 cells ) . A small fraction of PRC1 - labeled fibers ( green , n = 32 in 5 cells ) and kinetochores ( magenta , n = 16 in 5 cells ) did not move together . D . Correlation coefficients between trajectories ( with respect to the spindle ' s center of the mass ) of a kinetochore pair and the trajectories of the PRC1 - labeled fiber coupled to the kinetochore pair ( green ) , the nearest neighbor PRC1 - labeled fiber ( blue ) , the next nearest neighbor ( black ) and a randomly chosen PRC1 - labeled fiber ( grey ) ; n , the number of trajectories from 3 cells . E . Examples of scenarios observed in dynamic interplay of PRC1 - labeled bundles ( green ) and kinetochores ( magenta ) from the spindle in A . Row 1 : PRC1 - labeled bundle and kinetochore move together until they separate at 130 s . Row 2 : kinetochore is free of any PRC1 - labeled bundle until it appears at 130 s . Row 3 : kinetochore and PRC1 - labeled bundle move together . Row 4 : kinetochore moves with PRC1 - labeled bundle which merges with neighboring bundle at 169 s . Row 5 : two kinetochores move together with a single PRC1 - labeled bundle .

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

Figure 2A. Central plane of the spindle in a live HeLa cell expressing PRC1-GFP (green) and mRFP-CENP-B (magenta) oriented vertically with respect to the imaging plane.B. Examples of trajectories, with respect to the spindle's center of the mass, of individual PRC1-GFP (green) and corresponding mRFP-CENP-B (magenta) signals from the spindle in A that moved together for at least 200 s. Dots represent starting points of trajectories, t=0 s. Trajectories finish at t=200 s. Grey circle represents the center of mass of the spindle.C. Pie chart showing the fraction of PRC1-labeled fibers and sister kinetochores that moved together for at least 60 s (blue, n=226 pairs in 5 cells). A small fraction of PRC1-labeled fibers (green, n=32 in 5 cells) and kinetochores (magenta, n=16 in 5 cells) did not move together.D. Correlation coefficients between trajectories (with respect to the spindle's center of the mass) of a kinetochore pair and the trajectories of the PRC1-labeled fiber coupled to the kinetochore pair (green), the nearest neighbor PRC1-labeled fiber (blue), the next nearest neighbor (black) and a randomly chosen PRC1-labeled fiber (grey); n, the number of trajectories from 3 cells.E. Examples of scenarios observed in dynamic interplay of PRC1-labeled bundles (green) and kinetochores (magenta) from the spindle in A. Row 1: PRC1-labeled bundle and kinetochore move together until they separate at 130 s. Row 2: kinetochore is free of any PRC1-labeled bundle until it appears at 130 s. Row 3: kinetochore and PRC1-labeled bundle move together. Row 4: kinetochore moves with PRC1-labeled bundle which merges with neighboring bundle at 169 s. Row 5: two kinetochores move together with a single PRC1-labeled bundle.


Figure 3A . Top row : MG132 arrested HeLa cells stably expressing tubulin - GFP ( green ) and immunostained for endogenous PRC1 ( AlexaFluor555 shown in magenta ) in 5 individual z - images . Enlargements of the boxed region ( top : merge , middle : GFP , bottom : AlexaFluor555 ) below the first image show the bridging fiber determined as the line connecting two ends of the sister k - fibers in green channel and localization of endogenous PRC1 ( magenta ) . Corresponding maximum projections are shown to the right ( max z , left : merge , middle : GFP , right : AlexaFluor555 ) . B . Quantification of tubulin - GFP bridging fibers ( green bar ) immunostained for endogenous PRC1 ( magenta bar ) . C . Images show pole - to - pole tracking ( white curve ) of tubulin signal ( top , green ) , which was used to measure the endogenous PRC1 signal along the same contour ( middle , magenta ) , and the corresponding scheme ( bottom ) from a HeLa cell expressing tubulin - GFP and immunostained for PRC1 . Images of the same spindle without ( left ) and with the tracked contour ( right ) are shown . The graph shows normalized pole - to - pole intensity profiles ( each intensity profile was scaled so that one pole is at x = 0 and the other at x = 1 ) of endogenous PRC1 ( grey lines ) acquired in tubulin - GFP HeLa cells immunostained for PRC1 . Black line shows the mean value . D . Images show pole - to - pole tracking ( white curve ) of PRC1 - GFP signal ( top , merge ; middle , GFP ; bottom , scheme ) from a HeLa cell expressing PRC1 - GFP and mRFP - CENP - B . Images of the same spindle without ( left ) and with the tracked contour ( right ) are shown . The graph shows normalized pole - to - pole intensity profiles ( legend as in C ) of PRC1 - GFP acquired in HeLa cells expressing PRC1 - GFP and mRFP - CENP - B . E . Example of length measurement of the immunostained PRC1 signal ( LPRC1 ) in HeLa cell expressing tubulin - GFP and immunostained for PRC1 . F . Comparison of the length LPRC1 between PRC1 - GFP signal ( green , corresponds to the PRC1 fused to GFP from a PRC1 - GFP cell line ) and PRC1 - AlexaFluor555 ( magenta , corresponds to the endogenous immunostained PRC1 ) ( p = 0 . 10 ) . G . Cross section signal intensity of the immunostained endogenous PRC1 ( magenta line ) from a HeLa cell expressing tubulin - GFP and immunostained for PRC1 . Area under the peak is defined as Icross , measured at the position of the blue line as in scheme . Horizontal lines mark the background signal ( black line ) , vertical lines delimit the area ( grey ) where the signal was measured .

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

Figure 3A. Top row: MG132 arrested HeLa cells stably expressing tubulin-GFP (green) and immunostained for endogenous PRC1 (AlexaFluor555 shown in magenta) in 5 individual z-images. Enlargements of the boxed region (top: merge, middle: GFP, bottom: AlexaFluor555) below the first image show the bridging fiber determined as the line connecting two ends of the sister k-fibers in green channel and localization of endogenous PRC1 (magenta). Corresponding maximum projections are shown to the right (max z, left: merge, middle: GFP, right: AlexaFluor555).B. Quantification of tubulin-GFP bridging fibers (green bar) immunostained for endogenous PRC1 (magenta bar).C. Images show pole-to-pole tracking (white curve) of tubulin signal (top, green), which was used to measure the endogenous PRC1 signal along the same contour (middle, magenta), and the corresponding scheme (bottom) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (each intensity profile was scaled so that one pole is at x=0 and the other at x=1) of endogenous PRC1 (grey lines) acquired in tubulin-GFP HeLa cells immunostained for PRC1. Black line shows the mean value.D. Images show pole-to-pole tracking (white curve) of PRC1-GFP signal (top, merge; middle, GFP; bottom, scheme) from a HeLa cell expressing PRC1-GFP and mRFP-CENP-B. Images of the same spindle without (left) and with the tracked contour (right) are shown. The graph shows normalized pole-to-pole intensity profiles (legend as in C) of PRC1-GFP acquired in HeLa cells expressing PRC1-GFP and mRFP-CENP-B.E. Example of length measurement of the immunostained PRC1 signal (LPRC1) in HeLa cell expressing tubulin-GFP and immunostained for PRC1.F. Comparison of the length LPRC1 between PRC1-GFP signal (green, corresponds to the PRC1 fused to GFP from a PRC1-GFP cell line) and PRC1-AlexaFluor555 (magenta, corresponds to the endogenous immunostained PRC1) (p=0.10).G. Cross section signal intensity of the immunostained endogenous PRC1 (magenta line) from a HeLa cell expressing tubulin-GFP and immunostained for PRC1. Area under the peak is defined as Icross, measured at the position of the blue line as in scheme. Horizontal lines mark the background signal (black line), vertical lines delimit the area (grey) where the signal was measured.


Figure 4A . Images of HeLa cells stably expressing tubulin - GFP ( green ) and transiently mRFP - CENP - B ( magenta ) treated with control siRNA ( left images ) and siRNA targeting PRC1 ( right images ) . Enlargements of the boxed region , shown to the right of the image of the whole spindle , are focused on kinetochores and bridging fiber ( top : merge ; middle : GFP , bottom : scheme ) . B . Western blot showing PRC1 protein from unlabeled cells , tubulin - GFP HeLa cell line and PRC1 - GFP HeLa cell line . Detailed measurements are in Expanded View 4 . C . Mean ratio of signal intensities of the bridging fiber ( Ib , measured at the position of a blue line as in scheme ) and sum of the bridging and k - fiber ( Ibk , measured at the position of the orange line as in scheme ) , measured in control cells ( grey bar ) and PRC1 siRNA treated cells ( black bar ) ( p = 0 . 0003 ) . D . Mean interkinetochore distance ( dk , see scheme ) measured in control cells ( grey bar ) and PRC1 siRNA treated cells ( black bar ) ( p = 0 . 0001 ) . E . Images of individual spindles of HeLa cells expressing tubulin - GFP and immunostained for PRC1 obtained from control cells ( top ) and PRC1 siRNA treated cells ( bottom ) . All cells were imaged with the same imaging parameters . Only AlexaFluor555 channel is shown . F . Mean signal intensity I of the immunostained PRC1 in control cells ( grey bar ) and PRC1 siRNA treated cells ( black bar ) in HeLa cells expressing tubulin - GFP and immunostained for PRC1 ( p = 0 . 0001 ) . G . Images of HeLa cells stably expressing PRC1 - GFP treated with control ( left image ) and siRNA targeting PRC1 ( right image ) immunostained for PRC1 ( magenta ) . Graph showing the length of the immunostained PRC1 signal , LPRC1 ( p = 0 . 5167 ) , signal intensity , I ( p = 0 . 0003 ) and Icross ( p = 0 . 0001 ) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1 - GFP and immunostained for PRC1 . H . Images of HeLa cells stably expressing PRC1 - GFP ( green ) treated with control ( left image ) and siRNA targeting PRC1 ( right image ) immunostained for PRC1 . Graph showing the length of the PRC1 - GFP signal , LPRC1 ( p = 0 . 3407 ) , signal intensity , I ( p = 0 . 0001 ) , and Icross ( p = 0 . 0001 ) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1 - GFP and immunostained for PRC1 .

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

Figure 4A. Images of HeLa cells stably expressing tubulin-GFP (green) and transiently mRFP-CENP-B (magenta) treated with control siRNA (left images) and siRNA targeting PRC1 (right images). Enlargements of the boxed region, shown to the right of the image of the whole spindle, are focused on kinetochores and bridging fiber (top: merge; middle: GFP, bottom: scheme).B. Western blot showing PRC1 protein from unlabeled cells, tubulin-GFP HeLa cell line and PRC1-GFP HeLa cell line. Detailed measurements are in Expanded View 4.C. Mean ratio of signal intensities of the bridging fiber (Ib, measured at the position of a blue line as in scheme) and sum of the bridging and k-fiber (Ibk, measured at the position of the orange line as in scheme), measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0003).D. Mean interkinetochore distance (dk, see scheme) measured in control cells (grey bar) and PRC1 siRNA treated cells (black bar) (p=0.0001).E. Images of individual spindles of HeLa cells expressing tubulin-GFP and immunostained for PRC1 obtained from control cells (top) and PRC1 siRNA treated cells (bottom). All cells were imaged with the same imaging parameters. Only AlexaFluor555 channel is shown.F. Mean signal intensity I of the immunostained PRC1 in control cells (grey bar) and PRC1 siRNA treated cells (black bar) in HeLa cells expressing tubulin-GFP and immunostained for PRC1 (p=0.0001).G. Images of HeLa cells stably expressing PRC1-GFP treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1 (magenta). Graph showing the length of the immunostained PRC1 signal, LPRC1 (p=0.5167), signal intensity, I (p=0.0003) and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.H. Images of HeLa cells stably expressing PRC1-GFP (green) treated with control (left image) and siRNA targeting PRC1 (right image) immunostained for PRC1. Graph showing the length of the PRC1-GFP signal, LPRC1 (p=0.3407), signal intensity, I (p=0.0001), and Icross (p=0.0001) in siRNA targeting PRC1 in comparison to control HeLa cells expressing PRC1-GFP and immunostained for PRC1.


Figure 2 ( A ) Proportion change of Chinese SL isolates from different phylogenetic clades . The Chinese isolates were classified into two groups based on their sampling time ( 1993 - 2010 vs 2011 - 2020 ) . The percentage of isolates from each clade in each period was compared .

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

Figure 2(A) Proportion change of Chinese SL isolates from different phylogenetic clades. The Chinese isolates were classified into two groups based on their sampling time (1993-2010 vs 2011-2020). The percentage of isolates from each clade in each period was compared.


Figure 3 ( A ) Bacterial resistance to desiccation . Representative isolates that are phylogenetically distinct within Clade - 1 ( n = 2 ) , Clade - 2 ( n = 1 ) , Clade - 4 ( n = 2 ) and Clade - 5 ( n = 6 ) were used for desiccation assay Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . The P values for ( A ) were calculated using unpaired t - test ( two - tailed ) ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) . ( B ) Bacterial survivability under oxidative stress . Representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) and C5 ( n = 6 ) were treated with hydrogen peroxide . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . The P values for ( B ) , were calculated using unpaired t - test ( two - tailed ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) . ( C ) Biofilm formation ability . The biofilm of the representative isolates from C1 ( n = 2 ) , C2 ( n = 1 ) , C4 ( n = 2 ) and C5 ( n = 6 ) were formed at 22 ° C for 48 h . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . The P values for ( C ) were calculated using unpaired t - test ( two - tailed ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) . ( D ) Comparison of survival rates of chick embryo after challenging SL C5 and C1 isolates . Comparison was on 6 day - post - infection ( dpi ) . Ten embryos were used for each group . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( D ) were calculated using Log - rank ( Mantel - Cox , chi - square ) test ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) . ( E ) Survival rates of zebrafish embryo after challenging SL C5 and C1 isolates . The comparison was on 14 dpi . Ten zebrafish embryos were used for each group . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . P values for ( E ) were calculated using Log - rank ( Mantel - Cox , chi - square ) test ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) . ( F ) Bacterial loads in distinct organs of mice after challenging SL C5 ( L42 ) and C1 ( L1 ) isolates . The bacterial counts in the liver , spleen , and cecum of mice were measured on 3 and 5 dpi . Ten mice were used per group . Data information : All the experiments were triplicated with biological replicates . Data are presented as mean ± SD . The P values for ( F ) were calculated using unpaired t - test ( two - tailed ) , ( * : P < 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 001 ) .

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

Figure 3(A) Bacterial resistance to desiccation. Representative isolates that are phylogenetically distinct within Clade-1 (n=2), Clade-2 (n=1), Clade-4 (n=2) and Clade-5 (n=6) were used for desiccation assay Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (A) were calculated using unpaired t-test (two-tailed) (*:P<0.05, **:P<0.01, ***:P<0.001).(B) Bacterial survivability under oxidative stress. Representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were treated with hydrogen peroxide. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (B), were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).(C) Biofilm formation ability. The biofilm of the representative isolates from C1 (n=2), C2 (n=1), C4 (n=2) and C5 (n=6) were formed at 22°C for 48 h. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (C) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).(D) Comparison of survival rates of chick embryo after challenging SL C5 and C1 isolates. Comparison was on 6 day-post-infection (dpi). Ten embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (D) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).(E) Survival rates of zebrafish embryo after challenging SL C5 and C1 isolates. The comparison was on 14 dpi. Ten zebrafish embryos were used for each group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. P values for (E) were calculated using Log-rank (Mantel-Cox, chi-square) test (*:P<0.05, **:P<0.01, ***:P<0.001).(F) Bacterial loads in distinct organs of mice after challenging SL C5 (L42) and C1 (L1) isolates. The bacterial counts in the liver, spleen, and cecum of mice were measured on 3 and 5 dpi. Ten mice were used per group. Data information: All the experiments were triplicated with biological replicates. Data are presented as mean ± SD. The P values for (F) were calculated using unpaired t-test (two-tailed), (*:P<0.05, **:P<0.01, ***:P<0.001).


Figure 1Protein levels of E2F7 and E2F8 during cell cycle progression . HeLa cells were synchronized by a double thymidine block and released into fresh medium . Cells were harvested at the indicated time points and an asynchronous ( AS ) condition was used as control . Protein levels were measured by immunoblotting and cell cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . The asterisk indicates the E2F7 - specific band . Decreased stability of E2F7 and E2F8 in nocodazole - arrested cells . HeLa cells were either treated with DMSO or nocodazole ( 50 ng / ml ) for 16 hours . Cells were harvested and lysed for immunoblotting . Protein expression of cyclin B1 was used as a marker for G2 or M , and γ - tubulin was used as loading control . Selective Cullin - RING inhibitor MLN4924 rescued the degradation of E2F7 / 8 under nocodazole - arrested condition . HeLa cells were treated with DMSO , nocodazole or nocodazole plus MLN4924 ( 0 . 1 μM ) for 16 hours . Cells were harvested and lysed for immunoblotting . cyclin B1 expression was used as a marker for G2 or M cell cycle progression , and γ - tubulin was used as loading control . Increased half - life of E2F7 / 8 by MLN4924 treatment . HeLa cells were either treated with cycloheximide ( CHX , 50 ug / ml ) with or without MLN4924 ( 0 . 1 μM ) . Protein levels of E2F7 and E2F8 were determined by immunoblotting ( left panel ) . Asterisk indicates the E2F7 - specific band . cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression , γ - tubulin was used as loading control . Quantifications ( right panels ) were performed based on two independent experiments . Bar and error bars represent mean ± SEM .

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Figure 1Protein levels of E2F7 and E2F8 during cell cycle progression. HeLa cells were synchronized by a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points and an asynchronous (AS) condition was used as control. Protein levels were measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). The asterisk indicates the E2F7-specific band.Decreased stability of E2F7 and E2F8 in nocodazole-arrested cells. HeLa cells were either treated with DMSO or nocodazole (50 ng/ml) for 16 hours. Cells were harvested and lysed for immunoblotting. Protein expression of cyclin B1 was used as a marker for G2 or M, and γ-tubulin was used as loading control.Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control.Increased half-life of E2F7/8 by MLN4924 treatment. HeLa cells were either treated with cycloheximide (CHX, 50 ug/ml) with or without MLN4924 (0.1 μM). Protein levels of E2F7 and E2F8 were determined by immunoblotting (left panel). Asterisk indicates the E2F7-specific band. cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression, γ-tubulin was used as loading control. Quantifications (right panels) were performed based on two independent experiments. Bar and error bars represent mean ± SEM.


Figure 2C - terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F . The residues on each motif were mutated to alanines ( R to A , I / L to A ) with site - direct mutagenesis PCR . HEK293 cells were transfected with either EGFP - tagged empty vector ( EGFP ) , wild - types E2F7 / 8 ( WT ) or alanine mutants . Nocodazole ( 50 ng / ml ) was added 32 hours after transfection , and MG132 ( 1 μg / ml ) was added 5 hours before harvesting at 48 hours post transfection . Cells were harvested and lysed for immunoprecipitation using anti - EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP . Schematic view showing the truncated mutants and ML / AA mutant of cyclin F ( Left ) . HEK cells were transfected with indicated constructs and co - immunoprecipitation was performed using Flag resin ( Right ) .

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Figure 2C-terminus motifs at parallel positions on E2F7 and E2F8 are essential for binding to cyclin F. The residues on each motif were mutated to alanines (R to A, I/L to A) with site-direct mutagenesis PCR. HEK293 cells were transfected with either EGFP-tagged empty vector (EGFP), wild-types E2F7/8 (WT) or alanine mutants. Nocodazole (50 ng/ml) was added 32 hours after transfection, and MG132 (1 μg/ml) was added 5 hours before harvesting at 48 hours post transfection. Cells were harvested and lysed for immunoprecipitation using anti-EGFP resin followed by immunoblotting with antibodies against cyclin F and EGFP.Schematic view showing the truncated mutants and ML/AA mutant of cyclin F (Left). HEK cells were transfected with indicated constructs and co-immunoprecipitation was performed using Flag resin (Right).


Figure 3Wild - type or mutant versions of EGFP - tagged E2F7 / 8 were co - transfected with either empty vector or Flag - tagged cyclin F in HEK293 cells . Nocodazole was added to cells 8h before harvest . 48 hours after transfection , cells were collected and lysed for immunoblotting . Knockdown of cyclin F stabilized E2F7 and E2F8 . HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA . Cells were harvested at 48 hours post transfection . Protein levels of E2F7 / 8 were analyzed by immunoblotting . Asterisk indicates the specific band of E2F7 detection . Cyclin F targets atypical E2Fs during G2 / M . HeLa cells were transfected with either scrambled siRNA ( scr ) or cyclin F siRNA ( sicyclin F ) for 24 hours . Then cells were synchronized by double thymidine block and released into fresh medium after the second block . Cells were harvested at the indicated timepoints after the release . Protein expression was measured by immunoblotting and cell cycle progression was determined by flow - cytometry ( shown in Fig EV 1A ) . Asterisk indicates the specific detection of endogenous E2F7 . Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo . HEK293 cells were transfected with HA - E2F7 / 8 , with or without Flag - cyclin F , and with HA - tagged ubiquitin . 5 hours before harvest , cells were treated with MG132 . 48 hours after transfection , HEK cells were harvested and lysed for immunoprecipitation pull - down assay with anti - HA resin followed by immunoblotting .

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Figure 3Wild-type or mutant versions of EGFP-tagged E2F7/8 were co-transfected with either empty vector or Flag-tagged cyclin F in HEK293 cells. Nocodazole was added to cells 8h before harvest. 48 hours after transfection, cells were collected and lysed for immunoblotting.Knockdown of cyclin F stabilized E2F7 and E2F8. HeLa and RPE cells were transfected with either scramble siRNA or pool cyclin F siRNA. Cells were harvested at 48 hours post transfection. Protein levels of E2F7/8 were analyzed by immunoblotting. Asterisk indicates the specific band of E2F7 detection.Cyclin F targets atypical E2Fs during G2/M. HeLa cells were transfected with either scrambled siRNA (scr) or cyclin F siRNA (sicyclin F) for 24 hours. Then cells were synchronized by double thymidine block and released into fresh medium after the second block. Cells were harvested at the indicated timepoints after the release. Protein expression was measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). Asterisk indicates the specific detection of endogenous E2F7.Cyclin F contributes to the ubiquitylation of E2F7 and E2F8 in vivo. HEK293 cells were transfected with HA-E2F7/8, with or without Flag-cyclin F, and with HA-tagged ubiquitin. 5 hours before harvest, cells were treated with MG132. 48 hours after transfection, HEK cells were harvested and lysed for immunoprecipitation pull-down assay with anti-HA resin followed by immunoblotting.


Figure 4Schematic view of the experimental setting for the HU - synchronized live cell imaging . 48 hours before imaging , RPE - FUCCI cells were transfected with siRNA against scramble or cyclin F . 16 hours before imaging , cells were synchronized at the G1 / S border by HU ( 2 mM ) treatment . Representative images from different channels are shown , and white arrows in Differential Interference Contrast DIC channel indicate the traced cell . Scale bar : 10µm . Quantification of the number of Ctrl ( left panel ) and 7 / 8KO ( right panel ) RPE - FUCCI cells with scr or sicyclin F that completed mitosis after HU release . For each condition , 100 cells were monitored by live cell imaging . Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours . Log - rank tests were performed to analyze the statistical significance . Schematic view of the experimental setting for live imaging of asynchronous cells . 48 hours before imaging , RPE - FUCCI cells were transfected with siRNA against scramble or cyclin F . At the start of the imaging , G1 cells ( red : mKO2 - Cdt1 > mAG1 - Geminin , 50 cells per condition ) were enrolled and subsequently monitored though their entire cell cycle until mitosis . The black dot represents mitosis . Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8 . Number of Ctrl ( left panel ) or 7 / 8KO ( right panel ) RPE - FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown . For each condition , 50 cells at G1 were monitored by live cell imaging . Each cell was followed until it successfully progressed through S and G2 phase , finished mitosis and divided into two daughter cells , for a maximum of 24 hours . Log - rank tests were performed to analyze the statistical significance . Loss of cyclin F delays the progression from G1 / S transition to mitosis . Histogram shows the time from G1 / S ( mAG1 - Geminin intensity increases to higher than 10 % of the maximum value in three consecutive imaging frames ) to completed mitosis . Only cells that finished mitosis were enrolled in this quantification . Student t - test was used to test the statistical significance , asterisks indicates the P value < 0 . 01 . Bar and error bars represent mean ± SEM . Depletion of cyclin F stalls the cell cycle at late S / G2 . After 24 hours of live cell imaging , the cell cycle progression from panel E was quantified . Histogram shows the percentage of cells at each stage ( at 24h ) over the whole population ( 50 cells per condition ) .

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

Figure 4Schematic view of the experimental setting for the HU-synchronized live cell imaging. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. 16 hours before imaging, cells were synchronized at the G1/S border by HU (2 mM) treatment. Representative images from different channels are shown, and white arrows in Differential Interference Contrast DIC channel indicate the traced cell. Scale bar: 10µm.Quantification of the number of Ctrl (left panel) and 7/8KO (right panel) RPE-FUCCI cells with scr or sicyclin F that completed mitosis after HU release. For each condition, 100 cells were monitored by live cell imaging. Each cell was followed until it successfully finished mitosis and divided into two daughter cells for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.Schematic view of the experimental setting for live imaging of asynchronous cells. 48 hours before imaging, RPE-FUCCI cells were transfected with siRNA against scramble or cyclin F. At the start of the imaging, G1 cells (red: mKO2-Cdt1 > mAG1-Geminin, 50 cells per condition) were enrolled and subsequently monitored though their entire cell cycle until mitosis. The black dot represents mitosis.Knockdown of cyclin F causes a delay in mitotic entry that is dependent on E2F7 and E2F8. Number of Ctrl (left panel) or 7/8KO (right panel) RPE-FUCCI cells with scr or cyclin F RNAi that finished mitosis during live cell imaging is shown. For each condition, 50 cells at G1 were monitored by live cell imaging. Each cell was followed until it successfully progressed through S and G2 phase, finished mitosis and divided into two daughter cells, for a maximum of 24 hours. Log-rank tests were performed to analyze the statistical significance.Loss of cyclin F delays the progression from G1/S transition to mitosis. Histogram shows the time from G1/S (mAG1-Geminin intensity increases to higher than 10% of the maximum value in three consecutive imaging frames) to completed mitosis. Only cells that finished mitosis were enrolled in this quantification. Student t-test was used to test the statistical significance, asterisks indicates the P value <0.01. Bar and error bars represent mean ± SEM.Depletion of cyclin F stalls the cell cycle at late S/G2. After 24 hours of live cell imaging, the cell cycle progression from panel E was quantified. Histogram shows the percentage of cells at each stage (at 24h) over the whole population (50 cells per condition).


Figure 5Disruption of cyclin F binding site increased stability of E2F7 . E2F7WT and E2F7R894A constructs were integrated in to HeLa / TO system . Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium , and cells were collected every 3 hours for immunoblotting . qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels . Bar and error bars represent mean ± SEM . Over - expression of E2F7R894A delays cell cycle progression through G2 - M phase . HeLa / TO cells expressing either wild - type or mutant version of E2F7 were arrested with 16h of HU , and then cells were released into fresh medium with or without doxycycline . Live cell imaging was performed to trace the G2 - M progression of HeLa / TO cells .

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Figure 5Disruption of cyclin F binding site increased stability of E2F7. E2F7WT and E2F7R894A constructs were integrated in to HeLa/TO system. Cells were arrested with HU for 16 hours before releasing into doxycycline containing medium, and cells were collected every 3 hours for immunoblotting.qPCR showing that mRNA level of E2F7WT and E2F7R894A were at comparable levels. Bar and error bars represent mean ± SEM.Over-expression of E2F7R894A delays cell cycle progression through G2-M phase. HeLa/TO cells expressing either wild-type or mutant version of E2F7 were arrested with 16h of HU, and then cells were released into fresh medium with or without doxycycline. Live cell imaging was performed to trace the G2-M progression of HeLa/TO cells.


Figure 6Heatmap showing differentially expressed genes after cyclin F knockdown , and rescued by additional E2F7 / 8 depletion . Highlighted genes are all involved in DNA repair . Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions . qPCR showing the RNA expression of atypical E2F - target genes that are involved in DNA replication or DNA repair . HeLa cells were transfected for 48 hours with siRNAs as indicated . Cells were incubated with nocodazole for 16 hours before harvesting . Data represent averages ± SEM ( n = 3 ) ; * P < 0 . 05 or * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant . Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions . HeLa cells were treated with nocodazole for 16 hours prior to harvesting .

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Figure 6Heatmap showing differentially expressed genes after cyclin F knockdown, and rescued by additional E2F7/8 depletion. Highlighted genes are all involved in DNA repair. Cells were arrested with nocodazole for 16 hours prior to harvesting to minimize bias from potential differences in cell cycle progression between the different conditions.qPCR showing the RNA expression of atypical E2F- target genes that are involved in DNA replication or DNA repair. HeLa cells were transfected for 48 hours with siRNAs as indicated. Cells were incubated with nocodazole for 16 hours before harvesting. Data represent averages ± SEM (n=3); *P<0.05 or **P<0.01 (Student's t-test). n.s.: not significant.Immunoblotting showing the protein levels of Chk1 and Rad51 in the indicated siRNA conditions. HeLa cells were treated with nocodazole for 16 hours prior to harvesting.


Figure 7Loss of cyclin F induced E2F7 / 8 - dependent Homologous Recombination deficiency . HeLa cells that were stably transformed with pDR - GFP were transfected with siRNA as indicated . 48h after the initial transfection , cells were harvested for Flow - cytometry . GFP positive cells were gated ( Fig EV5A ) . Relative HR efficiency were adjusted to the scramble siRNA condition . Data represent averages ± SEM ( n = 3 ) ; * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant . Loss of cyclin F induced E2F7 / 8 - dependent γ - H2AX accumulation . HeLa cells were transfected with indicated siRNA for 24 hours , and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ - H2AX . DAPI was used to stain the cell nucleus . Relative intensity of γ - H2AX was quantified by Image J software , and 150 cells were quantified for each condition . Red bars represent averages ; * * P < 0 . 01 ( Student ' s t - test ) . n . s . : not significant . Scale bar 20 μm . Loss of cyclin F increased DNA damage recovery time before cell division . RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours , and then treated with HU for 16 hours . At the beginning of the imaging , only the single cells with at least one 53BP1 foci were traced , till the time frame that no 53BP1 foci was observed . The mitotic progression of the cells was defined as the duration from damage recovery to cell division . Histogram shows the damage recovery time ( green ) and the mitotic progression ( black ) of 50 cells for each condition . Chi - square analysis was performed to test the statistical significance ( P < 0 . 01 ) . Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7 / 8 . The cumulative curves represent the add - up number of cells that overcome the DNA damage lesions , for a time frame of 24 hours . For each condition , 50 cells were quantified . Log - rank tests were performed to analyze the statistical significance .

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Figure 7Loss of cyclin F induced E2F7/8-dependent Homologous Recombination deficiency. HeLa cells that were stably transformed with pDR-GFP were transfected with siRNA as indicated.48h after the initial transfection, cells were harvested for Flow-cytometry. GFP positive cells were gated (Fig EV5A). Relative HR efficiency were adjusted to the scramble siRNA condition. Data represent averages ± SEM (n=3); **P<0.01 (Student's t-test). n.s.: not significant.Loss of cyclin F induced E2F7/8-dependent γ-H2AX accumulation. HeLa cells were transfected with indicated siRNA for 24 hours, and then treated with nocodazole for 16 hours before fixation for immunofluorescence staining of γ-H2AX. DAPI was used to stain the cell nucleus. Relative intensity of γ-H2AX was quantified by Image J software, and 150 cells were quantified for each condition. Red bars represent averages; **P<0.01 (Student's t-test). n.s.: not significant. Scale bar 20 μm.Loss of cyclin F increased DNA damage recovery time before cell division. RPE cells integrated with the 53BP1 construct were transfected with indicated siRNA for 24 hours, and then treated with HU for 16 hours. At the beginning of the imaging, only the single cells with at least one 53BP1 foci were traced, till the time frame that no 53BP1 foci was observed. The mitotic progression of the cells was defined as the duration from damage recovery to cell division. Histogram shows the damage recovery time (green) and the mitotic progression (black) of 50 cells for each condition. Chi-square analysis was performed to test the statistical significance (P<0.01).Knockdown of cyclin F caused a delay in DNA lesion recovery that is dependent on E2F7/8. The cumulative curves represent the add-up number of cells that overcome the DNA damage lesions, for a time frame of 24 hours. For each condition, 50 cells were quantified. Log-rank tests were performed to analyze the statistical significance.


Figure 1ALP assay . The cells expressing Pho8Δ60p were cultured in SD + CA medium ( open bars ) and shifted to SD ( − N ) medium for three hours ( filled bars ) . The ALP activity of the lysates was measured to estimate autophagic activity . Wild - type cells , TN124 ; Δapg8 mutant harboring vector , TK303 ; Δapg8 mutant harboring APG8 on a centromeric vector , TK301 ; Δapg8 mutant harboring 3 × HA - tagged APG8 plasmid , TK307 .

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Figure 1ALP assay. The cells expressing Pho8Δ60p were cultured in SD+CA medium (open bars) and shifted to SD(−N) medium for three hours (filled bars). The ALP activity of the lysates was measured to estimate autophagic activity. Wild-type cells, TN124; Δapg8 mutant harboring vector, TK303; Δapg8 mutant harboring APG8 on a centromeric vector, TK301; Δapg8 mutant harboring 3 × HA-tagged APG8 plasmid, TK307.


Figure 3Expression of APG8 . A , The lysates prepared from the growing cells were subjected to immunoblotting with the anti - Apg8p antibody . Lane 1 , wild - type cells ( YW5 - 1B ) ; lane 2 , Δapg8 cells harboring APG8 on a multicopy vector ( TK201 ) ; lane 3 , Δapg8 cells ( TK404 ) . B , The wild - type ( YW5 - 1B ) cells were cultured in YEPD medium until 1 - 2 × 107 cells / ml ( 0 h ) and shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , 3 , 4 . 5 , and 6 h at 30 ° C . The amount of Apg8p was estimated by immunoblotting with anti - Apg8p antibody . Each lane has 10 μg of total protein . C , YW5 - 1B cells were cultured in YEPD medium until 1 - 2 × 107 cells / ml ( 0 h ) and shifted to SD ( − N ) medium for 0 . 5 , 1 , 2 , and 6 h at 30 ° C , and mRNA was prepared from each culture as described in Materials and Methods . APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe . Each lane has 4 μg of total mRNA . D , YW5 - 1B cells were cultured in YEPD medium until 1 × 107 cells / ml at 30 ° C . Rapamycin was added to the culture at a final concentration of 0 . 2 μg / ml and the cells were further incubated in YEPD medium for two hours at 30 ° C . Western blotting was performed with the anti - Apg8p antibody . Each lane has 20 μg of total protein .

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Figure 3Expression of APG8. A, The lysates prepared from the growing cells were subjected to immunoblotting with the anti-Apg8p antibody. Lane 1, wild-type cells (YW5-1B); lane 2, Δapg8 cells harboring APG8 on a multicopy vector (TK201); lane 3, Δapg8 cells (TK404).B, The wild-type (YW5-1B) cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, 3, 4.5, and 6 h at 30°C. The amount of Apg8p was estimated by immunoblotting with anti-Apg8p antibody. Each lane has 10 μg of total protein.C, YW5-1B cells were cultured in YEPD medium until 1-2 × 107 cells/ml (0 h) and shifted to SD(−N) medium for 0.5, 1, 2, and 6 h at 30°C, and mRNA was prepared from each culture as described in Materials and Methods. APG8 mRNA and ACT1 mRNA were detected by Northern blotting with each specific probe. Each lane has 4 μg of total mRNA.D, YW5-1B cells were cultured in YEPD medium until 1 × 107 cells/ml at 30°C. Rapamycin was added to the culture at a final concentration of 0.2 μg/ml and the cells were further incubated in YEPD medium for two hours at 30°C. Western blotting was performed with the anti-Apg8p antibody. Each lane has 20 μg of total protein.


Figure 4Subcellular fractionation and solubilization of Apg8p . The wild - type cells ( YW5 - 1B ) growing logarithmically in YEPD medium or starved in SD ( − N ) medium for three hours were lysed . A , The lysates ( T ) were centrifuged at 13 , 000 g for 15 min to generate pellet ( LSP ) and supernatant ( LSS ) . LSS fraction was further centrifuged at 100 , 000 g for one hour and separated to pellet ( HSP ) and supernatant ( HSS ) . LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates . Equal volume of each sample was applied to each well , and immunoblotting was performed with anti - Apg8p antibody . B , The lysates ( T ) were sonicated and centrifuged at 100 , 000 g for one hour to separate pellet ( P ) and supernatant ( S ) . The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody , respectively . C , The sonicated lysates were centrifuged at 100 , 000 g for one hour to generate pellet fraction . The pellet fraction was treated with 2 % Triton X - 100 ( TX - 100 ) for 30 min on ice . The sample was centrifuged at 100 , 000 g for one hour and separated to supernatant ( S ) and pellet ( P ) fractions . The distribution of Apg8p was examined by immunoblotting with the anti - Apg8p antibody .

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Figure 4Subcellular fractionation and solubilization of Apg8p. The wild-type cells (YW5-1B) growing logarithmically in YEPD medium or starved in SD(−N) medium for three hours were lysed. A, The lysates (T) were centrifuged at 13,000 g for 15 min to generate pellet (LSP) and supernatant (LSS). LSS fraction was further centrifuged at 100,000 g for one hour and separated to pellet (HSP) and supernatant (HSS). LSP and HSP were resuspended in an equal volume of the lysis buffer to the original lysates. Equal volume of each sample was applied to each well, and immunoblotting was performed with anti-Apg8p antibody.B, The lysates (T) were sonicated and centrifuged at 100,000 g for one hour to separate pellet (P) and supernatant (S). The distribution of CPY and Apg8p was examined by immunoblotting with each specific antibody, respectively.C, The sonicated lysates were centrifuged at 100,000 g for one hour to generate pellet fraction. The pellet fraction was treated with 2% Triton X-100 (TX-100) for 30 min on ice. The sample was centrifuged at 100,000 g for one hour and separated to supernatant (S) and pellet (P) fractions. The distribution of Apg8p was examined by immunoblotting with the anti-Apg8p antibody.


Figure 5Immunofluorescent staining of Δapg8 cells expressing 3 × HA - Apg8p . A , The Δapg8 cells harboring 3 × HA - tagged APG8 plasmid , TK114 cells , were grown until logarithmic phase . The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts . The spheroplasts were permeabilized by 0 . 5 % Triton X - 100 , and incubated with the anti - HA antibody , 16B12 , followed with the FITC - conjugated anti - mouse IgG . Left , Fluorescence image of 3 × HA - Apg8p ; right , Nomarski image of the cells . Arrows show punctate signals that are a little larger than tiny dot signals . Bars , 10 μm . B , Immunofluorescent staining of YW5 - 1B cells before ( 0 h ) and after ( 3 h ) shift to starvation ( negative control ) . Left , Fluorescence image ; right , Nomarski image . Bars , 10 μm . C , The cells were shifted to starvation for 0 , 0 . 5 , and 3 h . Upper panels , Fluorescence images of 3 × HA - Apg8p ; lower panels , Nomarski images of the cell . Bars , 5 μm .

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Figure 5Immunofluorescent staining of Δapg8 cells expressing 3 × HA-Apg8p. A, The Δapg8 cells harboring 3 × HA-tagged APG8 plasmid, TK114 cells, were grown until logarithmic phase. The cells were fixed by formaldehyde and were treated with Zymolyase 100T to generate spheroplasts. The spheroplasts were permeabilized by 0.5% Triton X-100, and incubated with the anti-HA antibody, 16B12, followed with the FITC-conjugated anti-mouse IgG. Left, Fluorescence image of 3 × HA-Apg8p; right, Nomarski image of the cells. Arrows show punctate signals that are a little larger than tiny dot signals. Bars, 10 μm. B, Immunofluorescent staining of YW5-1B cells before (0 h) and after (3 h) shift to starvation (negative control). Left, Fluorescence image; right, Nomarski image. Bars, 10 μm. C, The cells were shifted to starvation for 0, 0.5, and 3 h. Upper panels, Fluorescence images of 3 × HA-Apg8p; lower panels, Nomarski images of the cell. Bars, 5 μm.


Figure 6Change of Apg8p localization in Δpep4 cells after shift to starvation . The Δapg8Δpep4 cells harboring 3 × HA - tagged APG8 plasmid , TK116 cells , were shifted to starvation for 0 , 0 . 5 , 1 , 3 , and 6 h . Immunofluorescence microscopy was performed as described in Fig . 5 . A - E , FITC staining of 3 × HA - Apg8p . F - J , Nomarski images of the cells . Arrows show an autophagic body . Bars , 5 μm .

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Figure 6Change of Apg8p localization in Δpep4 cells after shift to starvation. The Δapg8Δpep4 cells harboring 3 × HA-tagged APG8 plasmid, TK116 cells, were shifted to starvation for 0, 0.5, 1, 3, and 6 h. Immunofluorescence microscopy was performed as described in Fig. 5. A-E, FITC staining of 3 × HA-Apg8p. F-J, Nomarski images of the cells. Arrows show an autophagic body. Bars, 5 μm.


Figure 7Immunostaining image of a cell containing autophagic bodies in the vacuole . The Δapg8Δpep4 cells expressing 3 × HA - Apg8p , TK116 cells , were shifted to SD ( − N ) medium for two hours . Cells were immunolabeled with anti - N mAb , 16B12 , followed by 10 - nm gold - conjugated goat anti - mouse IgG . AB , vacuole ; N , nucleus ; V , vacuole .

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Figure 7Immunostaining image of a cell containing autophagic bodies in the vacuole. The Δapg8Δpep4 cells expressing 3 × HA-Apg8p, TK116 cells, were shifted to SD(−N) medium for two hours. Cells were immunolabeled with anti-N mAb, 16B12, followed by 10-nm gold-conjugated goat anti-mouse IgG. AB, vacuole; N, nucleus; V, vacuole.


Figure 8Immuno - EM of autophagosomes . A and B , Mature autophagosome . C and D , Premature autophagosome . Arrows show expanded regions of the intramembrane space of the premature autophagosomes . TK116 cells were incubated in SD ( − N ) medium for one hour ( D ) , two hours ( B and C ) , or three hours ( A ) . The localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by the incubation with 5 - nm gold - ( A and D ) or 10 - nm gold - ( B and C ) conjugated goat anti - mouse IgG . AP , autophagosome ; N , nucleus ; V , vacuole .

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Figure 8Immuno-EM of autophagosomes. A and B, Mature autophagosome. C and D, Premature autophagosome. Arrows show expanded regions of the intramembrane space of the premature autophagosomes. TK116 cells were incubated in SD(−N) medium for one hour (D), two hours (B and C), or three hours (A). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A and D) or 10-nm gold- (B and C) conjugated goat anti-mouse IgG. AP, autophagosome; N, nucleus; V, vacuole.


Figure 9Possible intermediate structures of autophagosome . A , A membrane sac under construction . Arrows show gold particles . B and C , Isolation membranes . B , Isolation membrane is detected along the arrows . C , The small arrow shows a semicircular isolation membrane and a large arrow marks its open area . D , Apg8p - residing structures gathered in the area close to the vacuole . TK116 cells were incubated in SD ( − N ) medium for one hour ( A and C ) , or two hours ( B and D ) . The localization of 3 × HA - Apg8p was detected with anti - HA antibody , followed by the incubation with 5 - nm gold - ( A ) or 10 - nm gold - ( B - D ) conjugated goat anti - mouse IgG . V , vacuole .

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Figure 9Possible intermediate structures of autophagosome. A, A membrane sac under construction. Arrows show gold particles. B and C, Isolation membranes. B, Isolation membrane is detected along the arrows. C, The small arrow shows a semicircular isolation membrane and a large arrow marks its open area. D, Apg8p-residing structures gathered in the area close to the vacuole. TK116 cells were incubated in SD(−N) medium for one hour (A and C), or two hours (B and D). The localization of 3 × HA-Apg8p was detected with anti-HA antibody, followed by the incubation with 5-nm gold- (A) or 10-nm gold- (B-D) conjugated goat anti-mouse IgG. V, vacuole.


Figure 10Fine morphology of Δypt7 and Δapg8 cells and proteinase - K accession to proAPI in these mutants . A , EM image of Δypt7 cell starved in SD ( − N ) medium for four hours . Autophagosomes ( AP , arrows ) were accumulated . B , EM image of the starved Δapg8 cell . C - E , The representatives of the membrane structures detected in the starved Δapg8 cells . C , Autophagosome - like structure indistinguishable from autophagosome . D and E , Aberrant multivesicular structures . N , nucleus ; V , vacuole . F , Proteinase K - sensitivity of proAPI . Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD ( − N ) medium for 4 . 5 h . The lysates were treated with 100 μg / ml proteinase K in the presence or absence of Triton X - 100 . Asterisk shows mature API and digestion product of proAPI by proteinase K .

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Figure 10Fine morphology of Δypt7 and Δapg8 cells and proteinase-K accession to proAPI in these mutants. A, EM image of Δypt7 cell starved in SD(−N) medium for four hours. Autophagosomes (AP, arrows) were accumulated. B, EM image of the starved Δapg8 cell. C-E, The representatives of the membrane structures detected in the starved Δapg8 cells. C, Autophagosome-like structure indistinguishable from autophagosome. D and E, Aberrant multivesicular structures. N, nucleus; V, vacuole. F, Proteinase K-sensitivity of proAPI. Cell lysates were prepared from Δypt7 and Δapg8 cells starved in SD(−N) medium for 4.5 h. The lysates were treated with 100 μg/ml proteinase K in the presence or absence of Triton X-100. Asterisk shows mature API and digestion product of proAPI by proteinase K.


Figure 1B The Na + and K + contents and Na + / K + ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d . C Linear regression of root Na + / K + ratio under salt stress ( 150 mM NaCl for 1 d ) and fruit weight in 195 accessions from three groups . R2 , coefficient of determination . n is the accession number of the three groups . D The Na + / K + ratios in roots of TS - 21 , TS - 422 , TS - 577 , and TS - 670 after treatment with 150 mM NaCl for the indicated time ( n = 3 biological repeats ) . E , F Salt - resistance assay of two PIM accessions ( TS - 21 and TS - 422 ) and two BIG ( TS - 577 and TS - 670 ) . 20 - day - old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks ( E ) . The survival rates were obtained from at least 12 plants in three repeated experiments ( F ) .

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Figure 1B The Na+ and K+ contents and Na+/K+ ratio in roots and shoots of three groups of 369 accessions after treatment with 150 mM NaCl for 1 d.C Linear regression of root Na+/K+ ratio under salt stress (150 mM NaCl for 1 d) and fruit weight in 195 accessions from three groups. R2, coefficient of determination. n is the accession number of the three groups.D The Na+/K+ ratios in roots of TS-21, TS-422, TS-577, and TS-670 after treatment with 150 mM NaCl for the indicated time (n = 3 biological repeats).E, F Salt-resistance assay of two PIM accessions (TS-21 and TS-422) and two BIG (TS-577 and TS-670). 20-day-old seedlings grown in soil were treated with 200 mM NaCl for 3 weeks (E). The survival rates were obtained from at least 12 plants in three repeated experiments (F).


Figure 2A Manhattan plot displaying the GWAS results of Na + / K + ratio in root . The red - dashed line indicates the Bonferroni - adjusted significance threshold ( P = 1 . 0 × 10 - 7 ) . Red arrow indicates the significant SNP signal of Na + / K + ratio associated with SlHAK20 . B The nucleotide diversity ratios between PIM and CER , and between CER and BIG on chromosome 4 . The black - dashed horizontal lines indicate top 10 % threshold for entire chromosome 4 ( 1 . 82 πPIM / πCER for domestication and 4 . 27 πCER / πBIG for improvement ) . The red arrows indicate the position of SlHAK20 in the sweeps . π , nucleotide diversity . C SlHAK20 - based association mapping and pairwise LD analysis . Dots represent SNPs . Indel48 is highlighted in red . The indel - 2381 , indel - 1818 in the promoter region and five nonsynonymous variants are marked in blue . These eight variants are related to the pairwise LD diagram with dashed lines . D Haplotypes of SlHAK20 among tomato natural variations . The distribution of Na + / K + ratio in each haplotype group is exhibited by a box plot . The n indicates the number of accessions belonging to each haplotype . In the box plots , the middle line indicates the median , the box indicates the range of the percentiles of the total data using Turkey method , the whiskers indicate the interquartile range and the outer dots are outliers . Significant difference was determined by Student ' s t - test . E Allele distribution of the SlHAK20 locus at position in PIM , CER , and BIG groups . The n indicates the accession number .

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Figure 2A Manhattan plot displaying the GWAS results of Na+/K+ ratio in root. The red-dashed line indicates the Bonferroni-adjusted significance threshold (P = 1.0 × 10-7). Red arrow indicates the significant SNP signal of Na+/K+ ratio associated with SlHAK20.B The nucleotide diversity ratios between PIM and CER, and between CER and BIG on chromosome 4. The black-dashed horizontal lines indicate top 10% threshold for entire chromosome 4 (1.82 πPIM/πCER for domestication and 4.27 πCER/πBIG for improvement). The red arrows indicate the position of SlHAK20 in the sweeps. π, nucleotide diversity.C SlHAK20-based association mapping and pairwise LD analysis. Dots represent SNPs. Indel48 is highlighted in red. The indel-2381, indel-1818 in the promoter region and five nonsynonymous variants are marked in blue. These eight variants are related to the pairwise LD diagram with dashed lines.D Haplotypes of SlHAK20 among tomato natural variations. The distribution of Na+/K+ ratio in each haplotype group is exhibited by a box plot. The n indicates the number of accessions belonging to each haplotype. In the box plots, the middle line indicates the median, the box indicates the range of the percentiles of the total data using Turkey method, the whiskers indicate the interquartile range and the outer dots are outliers. Significant difference was determined by Student's t-test.E Allele distribution of the SlHAK20 locus at position in PIM, CER, and BIG groups. The n indicates the accession number.


Figure 3The expression pattern of SlHAK20 in tomato . The GUS activity was examined in seedling ( left ) and root ( right ) of 21 - days - old SlHAK20TS - 21pro : GUS transgenic tomato ( B ) . Bars , 2 cm . The expression pattern of SlHAK20 in tomato . Cross - sections of hypocotyl ( C ) of SlHAK20TS - 21pro : GUS transgenic plants . The scale bar is 400 μm in ( C ) The expression pattern of SlHAK20 in tomato . Cross - sections of root he scale bar is 200 μm in ( D ) . E Subcellular localization of SlHAK20 - YFP in tomato . The SlHAK20Hap1 - YFP ( left ) and SlHAK20Hap2 - YFP ( right ) were localized in the plasma membrane . YFP , yellow florescence protein . FM4 - 64 , a lipophilic styryl compound as a red fluorescent marker of plasma membrane . Bar , 20 μm . F SlHAK20 complements the K + uptake - defective yeast mutant assayed using a dose - response method . The K + uptake - defective yeast mutant R5421 transformed with SlHAK20Hap1 , SlHAK20Hap2 , and vector ( p416 - GPD ) were cultured in AP medium containing indicated concentrations of K + for 24 h . The data points represent three replicates . G Na + uptake analysis of yeast cells expressing SlHAK20 . Shown are the Na + concentrations in the medium over the time of culture of the ANT3 yeast cells transformed with SlHAK20Hap1 , SlHAK20Hap2 , or empty vector . The initial NaCl concentration in the medium was 60 μM . The data are shown as the means ± SD ( n = 4 ) . H Na + uptake kinetic analysis of SlHAK20 in yeast cells . The curves show concentration - dependent Na + inﬂux in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in the presence of 0 , 5 , 10 , 20 , 30 , 40 , 50 , 60 , 100 , 150 or 200 μM NaCl in the medium . The data are expressed as mean ± SD ( n = 3 ) .

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Figure 3The expression pattern of SlHAK20 in tomato. The GUS activity was examined in seedling (left) and root (right) of 21-days-old SlHAK20TS-21pro:GUS transgenic tomato (B). Bars, 2 cm.The expression pattern of SlHAK20 in tomato. Cross-sections of hypocotyl (C) of SlHAK20TS-21pro:GUS transgenic plants. The scale bar is 400 μm in (C)The expression pattern of SlHAK20 in tomato. Cross-sections of root he scale bar is 200 μm in (D).E Subcellular localization of SlHAK20-YFP in tomato. The SlHAK20Hap1-YFP (left) and SlHAK20Hap2-YFP (right) were localized in the plasma membrane. YFP, yellow florescence protein. FM4-64, a lipophilic styryl compound as a red fluorescent marker of plasma membrane. Bar, 20 μm.F SlHAK20 complements the K+ uptake-defective yeast mutant assayed using a dose-response method. The K+ uptake-defective yeast mutant R5421 transformed with SlHAK20Hap1, SlHAK20Hap2, and vector (p416-GPD) were cultured in AP medium containing indicated concentrations of K+ for 24 h. The data points represent three replicates.G Na+ uptake analysis of yeast cells expressing SlHAK20. Shown are the Na+ concentrations in the medium over the time of culture of the ANT3 yeast cells transformed with SlHAK20Hap1, SlHAK20Hap2, or empty vector. The initial NaCl concentration in the medium was 60 μM. The data are shown as the means ± SD (n = 4).H Na+ uptake kinetic analysis of SlHAK20 in yeast cells. The curves show concentration- dependent Na+ inﬂux in yeast expressing SlHAK20Hap1 or SlHAK20Hap2 in the presence of 0, 5, 10, 20, 30, 40, 50, 60, 100, 150 or 200 μM NaCl in the medium. The data are expressed as mean ± SD (n = 3).


Figure 4Na + / K + ratio contents in roots of TS - 21 wild type and slhak20 mutants . Data are shown as means ± SD ( n = 4 ) . Na + contents in roots of TS - 21 wild type and slhak20 mutants . Data are shown as means ± SD ( n = 4 ) . K + contents in roots of TS - 21 wild type and slhak20 mutants . Data are shown as means ± SD ( n = 4 ) . Na + contents in the xylem sap of TS - 21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days . Data represent means ± SD ( n = 4 ) . K + contents in the xylem sap of TS - 21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days . Data represent means ± SD ( n = 4 ) . G Shoot height of TS - 21 , slhak20 - 1 , and slhak20 - 2 . Plant height of 24 - day - old TS - 21 , slhak20 - 1 , and slhak20 - 2 were measured after growing under normal conditions for 14 days , followed by salt treatment for 14 days , and recovery for 10 days . Values are means ± SD ( n = 10 plants of each genotype ) . H Salt resistance of TS - 21 wild type and slhak20 mutants . TS - 21 , slhak20 - 1 , and slhak20 - 2 grown under normal growth conditions for 24 days , followed by a treatment with 200 mM NaCl for 14 days , and then recovered for 10 days . Control plants grown under normal growth conditions for 38 days . Scale bars , 5 cm .

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Figure 4Na+/K+ ratio contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).Na+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).K+ contents in roots of TS-21 wild type and slhak20 mutants. Data are shown as means ± SD (n = 4).Na+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).K+ contents in the xylem sap of TS-21 and slhak20 mutants after 50 mM NaCl treatment for the indicated days. Data represent means ± SD (n = 4).G Shoot height of TS-21, slhak20-1, and slhak20-2. Plant height of 24-day-old TS-21, slhak20-1, and slhak20-2 were measured after growing under normal conditions for 14 days, followed by salt treatment for 14 days, and recovery for 10 days. Values are means ± SD (n = 10 plants of each genotype).H Salt resistance of TS-21 wild type and slhak20 mutants. TS-21, slhak20-1, and slhak20-2 grown under normal growth conditions for 24 days, followed by a treatment with 200 mM NaCl for 14 days, and then recovered for 10 days. Control plants grown under normal growth conditions for 38 days. Scale bars, 5 cm.


Figure 5 Salt - tolerance assay of TS - 670 and slhak20 mutant plants . TS - 670 , slhak20 - 3 , and slhak20 - 4 grown under normal growth conditions for 23 days , and then treated with 150 mM NaCl for 5 days . Control plants grown under normal growth conditions for 28 days . Plant height and fresh weight of TS - 670 , slhak20 - 3 , and slhak20 - 4 under normal and salt stress conditions . Shoot height f 23 - day - old TS - 670 , slhak20 - 3 , and slhak20 - 4 were quantified after growing under normal conditions for 5 days , or salt treatment with 150 mM NaCl for 5 days . Values are means ± SD ( n = 10 plants of each genotype ) . Plant height and fresh weight of TS - 670 , slhak20 - 3 , and slhak20 - 4 under normal and salt stress conditions . resh weight f 23 - day - old TS - 670 , slhak20 - 3 , and slhak20 - 4 were quantified after growing under normal conditions for 5 days , or salt treatment with 150 mM NaCl for 5 days . Values are means ± SD ( n = 10 plants of each genotype ) . Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative plants ere analyzed among these genotypes . ontrol plants were grown under normal growth conditions for 28 days Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative fresh weight of shoots were analyzed among these genotypes . Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured . Values are means ± SD ( n = 12 plants of each genotype for fresh weight Phenotype of NIP and oshak4 / 17 double mutant plants under normal and salt stress conditions . 20 - day - old seedlings of NIP and oshak4 / 17 mutants were treated with 100 mM NaCl for 8 days . The pictures show representative survival rate were analyzed among these genotypes .

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Figure 5 Salt-tolerance assay of TS-670 and slhak20 mutant plants. TS-670, slhak20-3, and slhak20-4 grown under normal growth conditions for 23 days, and then treated with 150 mM NaCl for 5 days. Control plants grown under normal growth conditions for 28 days.  Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. Shoot height f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype). Plant height and fresh weight of TS-670, slhak20-3, and slhak20-4 under normal and salt stress conditions. resh weight f 23-day-old TS-670, slhak20-3, and slhak20-4 were quantified after growing under normal conditions for 5 days, or salt treatment with 150 mM NaCl for 5 days. Values are means ± SD (n = 10 plants of each genotype).  Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative plants ere analyzed among these genotypes. ontrol plants were grown under normal growth conditions for 28 days  Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative fresh weight of shoots were analyzed among these genotypes. Control plants were grown under normal growth conditions for 28 days and the fresh weights of shoots were measured. Values are means ± SD (n = 12 plants of each genotype for fresh weight  Phenotype of NIP and oshak4/17 double mutant plants under normal and salt stress conditions. 20-day-old seedlings of NIP and oshak4/17 mutants were treated with 100 mM NaCl for 8 days. The pictures show representative survival rate were analyzed among these genotypes.


Figure 6A The transcript levels of SlHAK20 in TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines grown under normal growth conditions . B The protein levels of SlHAK20Hap1 - YFP in TS - 670 and transgenic plants . ACTIN was used as loading control . Phenotypes of transgenic lines in soil under salt stress . The survival rates were obtained from at least 8 plants in four repeated experiments38 - day - old plants grown in soil as control . 20 - day - old TS - 670 and Hap1OE - 1 to - 3 were treated with 175 mM NaCl for 3 weeks and recovered for 2 weeksThe Na + in shoots and roots of TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . The 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 and Hap1OE - 3 plants grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days . The K + in shoots and roots of TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . The 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 and Hap1OE - 3 plants grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days . The Na + / K + ratio in shoots and roots of TS - 670 and three independent SlHAK20Hap1 - YFP transgenic lines during salt stress . The 21 - day - old TS - 670 , Hap1OE - 1 , Hap1OE - 2 and Hap1OE - 3 plants grown in 0 . 25 × Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days .

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Figure 6A The transcript levels of SlHAK20 in TS-670 and three independent SlHAK20Hap1-YFP transgenic lines grown under normal growth conditions.B The protein levels of SlHAK20Hap1-YFP in TS-670 and transgenic plants. ACTIN was used as loading control.Phenotypes of transgenic lines in soil under salt stress. The survival rates were obtained from at least 8 plants in four repeated experiments38-day-old plants grown in soil as control. 20-day-old TS-670 and Hap1OE-1 to -3 were treated with 175 mM NaCl for 3 weeks and recovered for 2 weeksThe Na+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.The K+ in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.The Na+/K+ ratio in shoots and roots of TS-670 and three independent SlHAK20Hap1-YFP transgenic lines during salt stress. The 21-day-old TS-670, Hap1OE-1, Hap1OE-2 and Hap1OE-3 plants grown in 0.25× Hoagland which were then treated with 50 mM NaCl for additional 0 and 7 days.


Figure 1A . Quantitative analysis of developmental expression of Lrig1 mRNA in rat hippocampus by real - time RT - PCR . The results are shown as mean SEM of n = 3 independent assays . The levels of Lrig1 mRNA were normalized using the expression of the housekeeping gene Tbp ( TATA binding protein ) . The insert shows the expression of Lrig1 in embryonic E17 . 5 rat hippocampus examined by RT - PCR . Control sample without reverse transcriptase ( - RT ) is also shown . B . Localization of Lrig1 ( red ) in coronal sections from P15 ratbrain by immunofluorescence . Anti - Lrig1ECD antibodies label dentate gyrus cells , CA1 - CA3 hippocampalneurons and pyramidal corticalneurons ( * ) . Scale bar , 400 m . C - E . Confocal image of cortical ( C ) and hippocampal ( D - E ) pyramidal neurons stained with rabbit anti - Lrig1ECD antibody . Arrows indicate Lrig1 staining in proximal segments of apical dendrites of CA1 - CA3hippocampal and pyramidal cortical neurons ( layer V ) . Scale bars , 20 m . F . Immunofluorescence staining of Lrig1 ( red ) with anti - Lrig1ECD antibody and the neuronal marker ßIII tubulin ( green ) in dissociated hippocampal cells cultured for 7 DIV . Yellow indicates neuronal expression of Lrig1 . Scale bar , 15 m . G . Immunofluorescence staining of Lrig1 ( red ) with anti - Lrig1ECD antibody and the astrocytic marker S100 ( green ) in dissociated hippocampal cells cultured for 7 DIV . Scale bar , 20 m . H . Localization of Lrig1 ( red ) with anti - Lrig1ECD antibody and the somatodendritic marker MAP - 2 ( blue ) by immunocytochemistry in dissociated rat hippocampalneurons after 12 DIV . Scale bar , 20 m .

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Figure 1A. Quantitative analysis of developmental expression of Lrig1 mRNA in rat hippocampus by real-time RT-PCR. The results are shown as mean SEM of n=3 independent assays. The levels of Lrig1 mRNA were normalized using the expression of the housekeeping gene Tbp (TATA binding protein). The insert shows the expression of Lrig1 in embryonic E17.5 rat hippocampus examined by RT-PCR. Control sample without reverse transcriptase (-RT) is also shown.B. Localization of Lrig1 (red) in coronal sections from P15 ratbrain by immunofluorescence. Anti-Lrig1ECD antibodies label dentate gyrus cells, CA1-CA3 hippocampalneurons and pyramidal corticalneurons (*). Scale bar, 400 m.C-E. Confocal image of cortical (C) and hippocampal (D-E) pyramidal neurons stained with rabbit anti-Lrig1ECD antibody. Arrows indicate Lrig1 staining in proximal segments of apical dendrites of CA1-CA3hippocampal and pyramidal cortical neurons (layer V). Scale bars, 20 m.F. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the neuronal marker ßIII tubulin (green) in dissociated hippocampal cells cultured for 7 DIV. Yellow indicates neuronal expression of Lrig1. Scale bar, 15 m.G. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the astrocytic marker S100 (green) in dissociated hippocampal cells cultured for 7 DIV. Scale bar, 20 m.H. Localization of Lrig1 (red) with anti-Lrig1ECD antibody and the somatodendritic marker MAP-2 (blue) by immunocytochemistry in dissociated rat hippocampalneurons after 12 DIV. Scale bar, 20 m.