Figure 5The optimal growth rate , i . e . the one that maximizes fitness over a cycle of famine and feast , depends on the ratio of feast and famine periods . For long periods of feast interrupted by short periods of famine , E . coli the optimal strategy is to grow fast . For long periods of starvation intermitted by short periods of growth , the optimal strategy is long survival and slow growth . For the scenario of panel A and B , $ \ frac { T _ { - } } { T _ { + } } = 48 $ , the optimum is μ = 0 . 86 h − 1 and γ = 0 . 5 d − 1 , see dashed line .

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

Figure 5The optimal growth rate, i.e. the one that maximizes fitness over a cycle of famine and feast, depends on the ratio of feast and famine periods. For long periods of feast interrupted by short periods of famine, E. coli the optimal strategy is to grow fast. For long periods of starvation intermitted by short periods of growth, the optimal strategy is long survival and slow growth. For the scenario of panel A and B, $\frac{T_{-}}{T_{+}} = 48$, the optimum is μ = 0.86 h − 1 and γ = 0.5 d − 1, see dashed line.


Figure 6Adaptation at the end of growth . When the nutrient concentration decreases , the nutrient uptake rate slows down with a Michaelis - Menten type kinetics ( top ) . Depending on the uptake affinity , bacteria will experience either a sharp or smooth decrease of growth rate ( bottom left , bright orange : 10 µM , dark orange : 1 mM ) . During this slow down , the proteome adapts ( bottom center ) , with lower affinities ( dark orange ) showing a slight improvement of proteome adaptation , which results in a slightly lower death rate ( bottom right ) . Adaptation in complex media . A medium supplemented with multiple nutrients ( one - red , two - green , ten - blue ) leads to a step - wise decrease of growth rate during nutrient depletion ( bottom left ) . Due to the extended periods of slow growth after exhaustion of primary nutrients , bacteria can adapt their proteome substantially ( bottom center ) , and decrease their death rate ( bottom right ) .

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 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

Figure 6Adaptation at the end of growth. When the nutrient concentration decreases, the nutrient uptake rate slows down with a Michaelis-Menten type kinetics (top). Depending on the uptake affinity, bacteria will experience either a sharp or smooth decrease of growth rate (bottom left, bright orange: 10 µM, dark orange: 1 mM). During this slow down, the proteome adapts (bottom center), with lower affinities (dark orange) showing a slight improvement of proteome adaptation, which results in a slightly lower death rate (bottom right).Adaptation in complex media. A medium supplemented with multiple nutrients (one - red, two - green, ten - blue) leads to a step-wise decrease of growth rate during nutrient depletion (bottom left). Due to the extended periods of slow growth after exhaustion of primary nutrients, bacteria can adapt their proteome substantially (bottom center), and decrease their death rate (bottom right).


Figure 1Live cell microscopy of U - 2 OS - TetO cells expressing rTetR - GFP ( ctrl ) , or rTetR - GFP - Kin14VIb ( Kin14VIb ) ( B ) Representative stills showing the most frequently observed metaphase localization and daughter nuclei distribution of the duplicated TetO locus ( white arrowheads ) . Chromatin was visualized through expression of H2B - mCherry , GFP depicts the TetO locus . Due to the presence of a predicted NES sequence in Kin14VIb , the TetO locus only becomes visible at NEB in Kin14VIb - expressing cells . Time = h : min , scale bar = 5 µm . Schemes illustrating the metaphase localization of the TetO locus and its subsequent distribution over daughter nuclei are shown on the right .

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Figure 1Live cell microscopy of U-2 OS-TetO cells expressing rTetR-GFP (ctrl), or rTetR-GFP-Kin14VIb (Kin14VIb) (B) Representative stills showing the most frequently observed metaphase localization and daughter nuclei distribution of the duplicated TetO locus (white arrowheads). Chromatin was visualized through expression of H2B-mCherry, GFP depicts the TetO locus. Due to the presence of a predicted NES sequence in Kin14VIb, the TetO locus only becomes visible at NEB in Kin14VIb-expressing cells. Time = h:min, scale bar = 5 µm. Schemes illustrating the metaphase localization of the TetO locus and its subsequent distribution over daughter nuclei are shown on the right.


Figure 2 ( C ) Quantification of the fraction of ctrl or Kin14VIb - expressing cells with the TetO locus observed outside the metaphase plate ( TetO out ) . ( D ) Deconstruction of ( C ) . Fraction of cells with the indicated orientation of the duplicated TetO locus and TetO chromosome . Data information : Bars represent the mean of 2 biological replicates , while dark - and light - colored dots represent the values of each experiment . ( C , D ) : N ≥ 29 cells per condition , per experiment , * * * * P < 0 . 0001 ; Fisher ' s exact test . E . Quantification of the number of cells with Mad1 - positive KTs nearby the TetO locus . In case the KTs of the TetO chromosome were aligned , we scored whether KTs in the vicinity of the TetO locus were Mad1 + . In case the KTs of the TetO chromosome could be distinguished in rTetR - GFP - Kin14VIb - expressing cells ( with either orthogonal or parallel oriented KTs ) , we scored if at least one of the KTs was Mad1 + . Data information : Bars represent the mean of 2 biological replicates , while dark - and light - colored dots represent the values of each experiment . ( E ) : N ≥ 27 cells per condition , per experiment , ( ns ) not significant ; Fisher ' s exact test .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(C) Quantification of the fraction of ctrl or Kin14VIb-expressing cells with the TetO locus observed outside the metaphase plate (TetO out). (D) Deconstruction of (C). Fraction of cells with the indicated orientation of the duplicated TetO locus and TetO chromosome. Data information: Bars represent the mean of 2 biological replicates, while dark- and light-colored dots represent the values of each experiment. (C, D): N ≥ 29 cells per condition, per experiment, ****P<0.0001; Fisher's exact test.E. Quantification of the number of cells with Mad1-positive KTs nearby the TetO locus. In case the KTs of the TetO chromosome were aligned, we scored whether KTs in the vicinity of the TetO locus were Mad1+. In case the KTs of the TetO chromosome could be distinguished in rTetR-GFP-Kin14VIb-expressing cells (with either orthogonal or parallel oriented KTs), we scored if at least one of the KTs was Mad1+. Data information: Bars represent the mean of 2 biological replicates, while dark- and light-colored dots represent the values of each experiment. (E): N ≥27 cells per condition, per experiment, (ns) not significant; Fisher's exact test.


Figure 3A - D ( A , C ) Frequencies of the different types of segregation errors observed during anaphase ( A ) and late anaphase / telophase ( C ) . Schemes illustrating the observed types of anaphase errors are shown on the right of ( A ) . Cells within the main TetO locus distribution categories in control ( 1 - 1 ) , and Kin14VIb ( 2 - 0 ) conditions were scored ( see also Fig EV1A ) . The majority of Kin14VIb - expressing anaphases display at least one stretched arm ( CENP - C in line with TetO locus at the pole ) or a bridge in line with the TetO locus at the pole ( " stretched arm or bridge in line " ) , some of them with additional bridges ( + ) . ( B , D ) IF images for CENP - C , GFP , H3S10ph of U - 2 OS TetO cells in anaphase ( B ) and late anaphase or telophase ( D ) , expressing either rTetR - GFP or rTetR - GFP - Kin14VIb . Images ( maximum intensity projections ) represent the most frequently observed anaphase and telophase errors in cells with either a 1 - 1 ( majority of events in ctrl ) , or 2 - 0 ( majority of events in Kin14VIb ) TetO locus distribution . Scale bar = 5 µm . Yellow arrowheads indicate TetO locus , red arrowheads CENP - C . Data information : ( A , C ) : Means ± S . E . M . of 3 biological replicates are shown . Dots represent the values of each experiment . N = 41 - 89 cells per condition , per experiment * * * * P < 0 . 0001 , ( ns ) not significant ; Fisher ' s exact test . ( B , D ) : for optimal visibility of the TetO foci , the brightness and contrast of all channels were linearly adjusted for individual cells .

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

Figure 3A-D (A, C) Frequencies of the different types of segregation errors observed during anaphase (A) and late anaphase/telophase (C). Schemes illustrating the observed types of anaphase errors are shown on the right of (A). Cells within the main TetO locus distribution categories in control (1-1), and Kin14VIb (2-0) conditions were scored (see also Fig EV1A). The majority of Kin14VIb-expressing anaphases display at least one stretched arm (CENP-C in line with TetO locus at the pole) or a bridge in line with the TetO locus at the pole ("stretched arm or bridge in line"), some of them with additional bridges (+). (B, D) IF images for CENP-C, GFP, H3S10ph of U-2 OS TetO cells in anaphase (B) and late anaphase or telophase (D), expressing either rTetR-GFP or rTetR-GFP-Kin14VIb. Images (maximum intensity projections) represent the most frequently observed anaphase and telophase errors in cells with either a 1-1 (majority of events in ctrl), or 2-0 (majority of events in Kin14VIb) TetO locus distribution. Scale bar = 5 µm. Yellow arrowheads indicate TetO locus, red arrowheads CENP-C. Data information: (A, C): Means ± S.E.M. of 3 biological replicates are shown. Dots represent the values of each experiment. N= 41-89 cells per condition, per experiment ****P<0.0001, (ns) not significant; Fisher's exact test. (B, D): for optimal visibility of the TetO foci, the brightness and contrast of all channels were linearly adjusted for individual cells.


Figure 4A . Representative images of U - 2 OS TetO anaphases / telophases expressing rTetR - GFP or rTetR - GFP - Kin14VIb with FISH - marked centromeres of chromosome 1 ( chr1 - CEN ) . White and yellow arrowheads : chr1 - CEN segregated into the main masses of segregating chromosomes ; red arrowheads : chr1 - CEN FISH signal fragmented or splitted in a bridge ( bridging ) ; chr1 - CEN lagging between the two main masses of segregating chromosomes ( lagging ) ; chr1 - CEN late arrival ( late - equal : one CEN focus in but near the edge of a separated chromosome mass and connected to a bridge ) . For clarity , the maximum intensity projection of a subset of z stacks ( 6 - 12 / 64 ) is shown . Scale bar = 5 µm . B . Frequency of the different types of errors observed in A . The mean ( bars ) and individual values ( dots ) of 2 independent experiments are shown . Data information : In ( A ) , for optimal visibility of the chr1 - CEN FISH foci , the brightness and contrast were linearly adjusted for individual cells . ( B ) : n ≥ 45 cells per condition , per experiment . Bars represent the mean of 2 biological replicates , while dark - and light - colored dots represent the values of each experiment . * P < 0 . 05 , ( ns ) not significant . Fisher ' s exact test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of U-2 OS TetO anaphases/telophases expressing rTetR-GFP or rTetR-GFP-Kin14VIb with FISH-marked centromeres of chromosome 1 (chr1-CEN). White and yellow arrowheads: chr1-CEN segregated into the main masses of segregating chromosomes; red arrowheads: chr1-CEN FISH signal fragmented or splitted in a bridge (bridging); chr1-CEN lagging between the two main masses of segregating chromosomes (lagging); chr1-CEN late arrival (late-equal: one CEN focus in but near the edge of a separated chromosome mass and connected to a bridge). For clarity, the maximum intensity projection of a subset of z stacks (6-12/64) is shown. Scale bar = 5 µm. B. Frequency of the different types of errors observed in A. The mean (bars) and individual values (dots) of 2 independent experiments are shown. Data information: In (A), for optimal visibility of the chr1-CEN FISH foci, the brightness and contrast were linearly adjusted for individual cells. (B): n ≥ 45 cells per condition, per experiment. Bars represent the mean of 2 biological replicates, while dark- and light-colored dots represent the values of each experiment. * P<0.05, (ns) not significant. Fisher's exact test.


Figure 5C . IF for Cas9 and CENP - C of metaphases of RPE1 - dCas9 - Kin14VIb cells transduced with Chr1 - telo sgRNA in the presence or absence of 500 nM rapalog . The Chr1 - telo loci ( Cas9 foci , white arrowheads ) are aligned on the metaphase plate in control condition ( - rapalog ) , while the loci are facing either a single or opposite poles after rapalog addition to induce Kin14VIb binding to dCas9 . Scale bar = 5 µm . For clarity , the maximum intensity projection of a subset of z stacks ( 18 - 50 / 100 ) is shown . Data information : ( C for optimal visibility of the Chr1 - telo foci , the brightness and contrast of all channels were linearly adjusted for individual cells . D , Live cell microscopy of asynchronously growing RPE1 - dCas9 - Kin14VIb cells transduced with Chr1 - telo sgRNA in the presence or absence of 500 nM rapalog to induce kinesin binding to the subtelomeric locus . ( D ) Stills showing the most frequently observed metaphase localization and daughter cell distribution ( white arrowheads ) of the duplicated Chr1 - telo loci . SiR - DNA was used to visualize the DNA , GFP depicts the Chr1 - telo loci . Time ( h : min ) . Scale bar = 5 µm . Note that in the upper panel , the cell was already in mitosis when imaging started ( t = 0 : 00 ) . Data information : ( D , Cells were derived from three independent imaging experiments . N = 23 cells ( - rapalog ) , n = 41 cells ( + rapalog ) . Sp = same pole , op = opposite pole , ss = same sisters . D ) : for optimal visibility of the Chr1 - telo foci , the brightness and contrast of all channels were linearly adjusted for individual cells .

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 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

Figure 5C. IF for Cas9 and CENP-C of metaphases of RPE1-dCas9-Kin14VIb cells transduced with Chr1-telo sgRNA in the presence or absence of 500 nM rapalog. The Chr1-telo loci (Cas9 foci, white arrowheads) are aligned on the metaphase plate in control condition (- rapalog), while the loci are facing either a single or opposite poles after rapalog addition to induce Kin14VIb binding to dCas9. Scale bar = 5 µm. For clarity, the maximum intensity projection of a subset of z stacks (18-50/100) is shown. Data information: (C for optimal visibility of the Chr1-telo foci, the brightness and contrast of all channels were linearly adjusted for individual cells.D, Live cell microscopy of asynchronously growing RPE1-dCas9-Kin14VIb cells transduced with Chr1-telo sgRNA in the presence or absence of 500 nM rapalog to induce kinesin binding to the subtelomeric locus. (D) Stills showing the most frequently observed metaphase localization and daughter cell distribution (white arrowheads) of the duplicated Chr1-telo loci. SiR-DNA was used to visualize the DNA, GFP depicts the Chr1-telo loci. Time (h:min). Scale bar = 5 µm. Note that in the upper panel, the cell was already in mitosis when imaging started (t =0:00). Data information: (D, Cells were derived from three independent imaging experiments. N = 23 cells (- rapalog), n = 41 cells (+ rapalog). Sp = same pole, op = opposite pole, ss = same sisters. D): for optimal visibility of the Chr1-telo foci, the brightness and contrast of all channels were linearly adjusted for individual cells.


Figure 6B , Live cell microscopy of asynchronously growing RPE1 - dCas9 - Kin14VIb cells transduced with Chr9 - cen sgRNA and plus / minus rapalog to induce kinesin binding to the pericentromeric locus ( Movies S4 - S8 ) . ( B ) Stills of the most frequently observed metaphase orientations and daughter cell distributions of the duplicated Chr9 - cen loci ( white arrowheads ) are shown . SiR - DNA was used to visualize the DNA , GFP depicts the Chr9 - cen loci . Time ( h : min ) . Scale bar = 5 µm . Data information : Cells were derived from two independent imaging experiments . . In ( B ) , for optimal visibility of the Chr9 - cen foci , the brightness and contrast of all channels were linearly adjusted for individual cells . See Movies EV4 - S8 for equally adjusted corresponding examples . D . Time between nuclear envelop break down ( NEB ) and anaphase onset in the presence ( Kin14VIb bound to dCas9 ) or absence ( Kin14VIb expressed , but not bound to dCas9 ) of rapalog . Data information : Cells were derived from two independent imaging experiments . . ( D ) : Mean and S . D . are shown .

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

Figure 6B, Live cell microscopy of asynchronously growing RPE1-dCas9-Kin14VIb cells transduced with Chr9-cen sgRNA and plus/minus rapalog to induce kinesin binding to the pericentromeric locus (Movies S4-S8). (B) Stills of the most frequently observed metaphase orientations and daughter cell distributions of the duplicated Chr9-cen loci (white arrowheads) are shown. SiR-DNA was used to visualize the DNA, GFP depicts the Chr9-cen loci. Time (h:min). Scale bar = 5 µm. Data information: Cells were derived from two independent imaging experiments.. In (B), for optimal visibility of the Chr9-cen foci, the brightness and contrast of all channels were linearly adjusted for individual cells. See Movies EV4- S8 for equally adjusted corresponding examples.D. Time between nuclear envelop break down (NEB) and anaphase onset in the presence (Kin14VIb bound to dCas9) or absence (Kin14VIb expressed, but not bound to dCas9) of rapalog. Data information: Cells were derived from two independent imaging experiments.. (D): Mean and S.D. are shown.


Figure 7A . Representative IF images of RPE1 - dCas9 - Kin14VIb cells , showing the localization of the Chr9 - cen loci in metaphase in the absence or presence or rapalog . Magnifications of the white boxed regions ( each region showing one Chr9 - cen locus ) are shown in the corners ( scale bars = 2 µm ) . The fraction of cells with Chr9 mis - aligned and CENP - C near the locus ( Cas9 ) is indicated below the image . For clarity , the maximum intensity projection of a subset of z stacks in which the Chr9 - cen loci are in focus ( 2 - 10 / 100 ) is shown . B . Frequency of the observed metaphase localizations of the Chr9 - cen loci as shown in ( A ) . Data information : In all cases , bars represent the mean of 2 independent experiments , while dark - and light - colored dots represent the values of each experiment . ( B ) : * * * * P < 0 . 0001 , Fisher ' s exact test . N ≥ 31 cells per condition , per experiment . ( A , For optimal visibility of the Chr9 - cen foci the brightness and contrast of all channels were linearly adjusted for individual cells . C . Representative IF images of RPE1 - dCas9 - Kin14VIb cells , showing the segregation and distribution of Chr9 - cen loci in anaphase ( white arrowheads ) . Red arrowheads indicate H3S10ph - positive chromatin bridges . For clarity , the maximum intensity projection of a subset of z stacks in which the chr9 - cen loci are in focus ( 30 - 62 / 100 ) is shown . Scale bar = 2 µm . Data information : C ) : For optimal visibility of the Chr9 - cen foci H3S10ph - positive chromatin bridges , the brightness and contrast of all channels were linearly adjusted for individual cells . E . Frequency of cells with a 2 - 2 distribution of the Chr9 - telo loci . Data information : In all cases , bars represent the mean of 2 independent experiments , while dark - and light - colored dots represent the values of each experiment . : * * * * P < 0 . 0001 , Fisher ' s exact test . N ≥ 29 cells per condition , per experiment . F . Frequency of the observed segregation / distribution errors of the Chr9 - cen loci as shown in ( C ) . Data information : In all cases , bars represent the mean of 2 independent experiments , while dark - and light - colored dots represent the values of each experiment . F ) : * * * * P < 0 . 0001 , Fisher ' s exact test . N ≥ 29 cells per condition , per experiment .

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 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 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

Figure 7A. Representative IF images of RPE1-dCas9-Kin14VIb cells, showing the localization of the Chr9-cen loci in metaphase in the absence or presence or rapalog. Magnifications of the white boxed regions (each region showing one Chr9-cen locus) are shown in the corners (scale bars = 2 µm). The fraction of cells with Chr9 mis-aligned and CENP-C near the locus (Cas9) is indicated below the image. For clarity, the maximum intensity projection of a subset of z stacks in which the Chr9-cen loci are in focus (2-10/100) is shown. B. Frequency of the observed metaphase localizations of the Chr9-cen loci as shown in (A). Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. (B): **** P<0.0001, Fisher's exact test. N ≥ 31 cells per condition, per experiment. (A, For optimal visibility of the Chr9-cen foci the brightness and contrast of all channels were linearly adjusted for individual cells.C. Representative IF images of RPE1-dCas9-Kin14VIb cells, showing the segregation and distribution of Chr9-cen loci in anaphase (white arrowheads). Red arrowheads indicate H3S10ph-positive chromatin bridges. For clarity, the maximum intensity projection of a subset of z stacks in which the chr9-cen loci are in focus (30-62/100) is shown. Scale bar = 2 µm. Data information: C): For optimal visibility of the Chr9-cen foci H3S10ph-positive chromatin bridges, the brightness and contrast of all channels were linearly adjusted for individual cells.E. Frequency of cells with a 2-2 distribution of the Chr9-telo loci. Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. : **** P<0.0001, Fisher's exact test. N ≥ 29 cells per condition, per experiment.F. Frequency of the observed segregation/distribution errors of the Chr9-cen loci as shown in (C). Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. F): **** P<0.0001, Fisher's exact test. N ≥ 29 cells per condition, per experiment.


Figure 8C . Average percentage of the bases of the p or q arm that are ( classified as ) 0 , 1 , 3 or 4 - somy . Percentages are depicted as stacked bar plots and are determined for all copy numbers other than 2 - somy ( euploid ) .

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Figure 8C. Average percentage of the bases of the p or q arm that are (classified as) 0, 1, 3 or 4-somy. Percentages are depicted as stacked bar plots and are determined for all copy numbers other than 2-somy (euploid).


figf1 ( a - d ) Degradation of long - lived proteins in MEFs from wild - type ( 18Q - htt ) and mutant huntingtin knock - in ( 111Q - htt ) mice . ( a , b ) Rates of protein degradation after serum removal ( a ) ( a - d ) Degradation of long - lived proteins in MEFs from wild - type ( 18Q - htt ) and mutant huntingtin knock - in ( 111Q - htt ) mice . ( a , b ) Rates of protein degradation after treatment with rapamycin ( rapam ) or thapsigargin ( thapsig ) ( b ) . ( c ) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl . ( d ) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3 - methyladenine ( 3 - MA ) . ( e ) Degradation of long - lived proteins in striatal cells from wild - type ( 7Q - htt ) and mutant huntingtin knock - in mice ( 111Q - htt ) in response to different autophagic stimuli . ( f , g ) Rates of protein degradation in wild - type and HD MEFs ( f ) and striatal cells ( g ) in the absence ( control ) or presence ( Atg7 − ) of Atg7 RNA interference . ( h ) Contribution of macroautophagy ( macroaut ) to the degradation of long - lived proteins in lymphoblasts from unaffected controls ( UC ) and people with HD . Mean + s . d . of values from two to four different individuals and three or four different experiments . * P 0 . 05 versus untreated , # P 0 . 05 versus control .

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

figf1(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after serum removal (a)(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after treatment with rapamycin (rapam) or thapsigargin (thapsig) (b).(c) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl.(d) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3-methyladenine (3-MA).(e) Degradation of long-lived proteins in striatal cells from wild-type (7Q-htt) and mutant huntingtin knock-in mice (111Q-htt) in response to different autophagic stimuli.(f,g) Rates of protein degradation in wild-type and HD MEFs (f) and striatal cells (g) in the absence (control) or presence (Atg7−) of Atg7 RNA interference.(h) Contribution of macroautophagy (macroaut) to the degradation of long-lived proteins in lymphoblasts from unaffected controls (UC) and people with HD. Mean + s.d. of values from two to four different individuals and three or four different experiments. *P 0.05 versus untreated, #P 0.05 versus control.


figf2 ( a ) Top : LC3 immunostaining of 18Q - htt and 111Q - htt MEFs maintained in the presence ( + ) or absence ( - ) of serum and inhibitors of lysosomal proteolysis ( inhib + ) . Bottom : mean number of LC3 + vesicles per cell in cells maintained in the presence ( left ) or absence ( right ) of serum . n = 4 . ( b ) Top : LC3 immunoblot of the same cells after serum ( S ) removal or thapsigargin ( TG ) treatment . PI , protease inhibitors . Bottom : LC3 - II levels and LC3 - II flux ( calculated as a multiple of LC3 - II value in absence of PI ; lateral numbers ) . n = 4 . ( c ) LC3 - II values and LC3 - II flux in neuronal cultures from wild - type ( WT ) or HD94 mice ( HD ) grown on wild - type rat astrocyte monolayers , analyzed as in b . n = 4 . ( d ) LC3 staining of lymphoblasts , from three unaffected controls ( UC ) and people with HD , maintained in the presence or absence of serum . Accession numbers are indicated above panels . Right : mean number of LC3 + vesicles . Extended study in Supplementary Figure 8b , c . ( e ) Immunoblots for LC3 - II in UC and HD lymphoblasts treated or not treated with protease inhibitors . Right : LC3 - II values and LC3 - II flux . Extended study in Supplementary Figure 8a . Mean + s . d . ; # P 0 . 05 . Full - length blots in Supplementary Figure 20 .

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

figf2(a) Top: LC3 immunostaining of 18Q-htt and 111Q-htt MEFs maintained in the presence (+) or absence (-) of serum and inhibitors of lysosomal proteolysis (inhib+). Bottom: mean number of LC3+ vesicles per cell in cells maintained in the presence (left) or absence (right) of serum. n = 4.(b) Top: LC3 immunoblot of the same cells after serum (S) removal or thapsigargin (TG) treatment. PI, protease inhibitors. Bottom: LC3-II levels and LC3-II flux (calculated as a multiple of LC3-II value in absence of PI; lateral numbers). n = 4.(c) LC3-II values and LC3-II flux in neuronal cultures from wild-type (WT) or HD94 mice (HD) grown on wild-type rat astrocyte monolayers, analyzed as in b. n = 4.(d) LC3 staining of lymphoblasts, from three unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Accession numbers are indicated above panels. Right: mean number of LC3+ vesicles. Extended study in Supplementary Figure 8b,c.(e) Immunoblots for LC3-II in UC and HD lymphoblasts treated or not treated with protease inhibitors. Right: LC3-II values and LC3-II flux. Extended study in Supplementary Figure 8a. Mean + s.d.; #P 0.05. Full-length blots in Supplementary Figure 20.


figf3 ( a ) Electron microscopy of striatal neurons from control and HD94 mice grown over a wild - type ratastrocyte monolayer . Higher magnification fields show the double membrane and clear content of the cytosolic vesicles . Top right : number of vacuoles per cell profile ( 13 - 17 cell profiles in triplicate experiments ; * P 0 . 05 ) . Arrows , enlarged electron - clear vesicles . ( b ) Electron micrographs of striatal cells from mutant huntingtin knock - in ( 111Q - htt ) and wild - type ( 7Q - htt ) mice . ( c ) Electron micrographs of lymphoblasts from unaffected controls ( UC ) and people with HD , maintained in the presence or absence of serum . Arrows , enlarged electron - clear vesicles . ( d ) Immunogold for LC3 in striatal cells from mutant huntingtin knock - in mice ( 111Q - htt ) , striatal neurons from HD94 mice and lymphoblasts ( lymph ) from people with HD . Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11 .

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

figf3(a) Electron microscopy of striatal neurons from control and HD94 mice grown over a wild-type ratastrocyte monolayer. Higher magnification fields show the double membrane and clear content of the cytosolic vesicles. Top right: number of vacuoles per cell profile (13-17 cell profiles in triplicate experiments; *P 0.05). Arrows, enlarged electron-clear vesicles.(b) Electron micrographs of striatal cells from mutant huntingtin knock-in (111Q-htt) and wild-type (7Q-htt) mice.(c) Electron micrographs of lymphoblasts from unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Arrows, enlarged electron-clear vesicles.(d) Immunogold for LC3 in striatal cells from mutant huntingtin knock-in mice (111Q-htt), striatal neurons from HD94 mice and lymphoblasts (lymph) from people with HD. Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11.


figf4 ( a ) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q - htt and 111Q - htt mice . Insets : higher magnification of single vesicles . Right : percentage of vesicles with electron - clear ( light ) , vesiculated ( multivesicular ) or electron - dense ( dark ) contents . Mean + s . e . m . of three different isolations ( > ∼ 1 , 000 autophagic vacuoles ) . ( b ) Bidimensional electrophoresis and Sypro Ruby staining of these fractions . Left , intact vacuoles ; right , vacuole contents . Horizontal dimension , isoelectric point ( pH ) ; vertical , molecular mass ( kDa ) . * P 0 . 05 .

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figf4(a) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q-htt and 111Q-htt mice. Insets: higher magnification of single vesicles. Right: percentage of vesicles with electron-clear (light), vesiculated (multivesicular) or electron-dense (dark) contents. Mean + s.e.m. of three different isolations (> ∼1,000 autophagic vacuoles).(b) Bidimensional electrophoresis and Sypro Ruby staining of these fractions. Left, intact vacuoles; right, vacuole contents. Horizontal dimension, isoelectric point (pH); vertical, molecular mass (kDa). *P 0.05.


figf5 ( a ) Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes ( APHL ) isolated from livers of 18Q - htt ( 18Q ) and 111Q - htt ( 111Q ) mice . Cyt , cytochrome ; Dyn IC , dynein intermediate chain ; ADRP , adipose differentiation - related protein ; Polyubq , polyubiquitin . Bottom : amount of each protein in 111Q - htt sample , as a multiple of its amount in 18Q - htt sample . Mean + s . d . ; n = 4 . ( b ) Immunoblots for htt and LC3 in homogenates ( Homog ) , cytosol and fractions enriched in APHs and enriched in autophagolysosomes ( APHL ) isolated from livers of wild - type ( 18Q - htt ) and mutant huntingtin knock - in mice ( 111Q - htt ) . Representative one of four experiments with duplicated samples . * P 0 . 05 . Full - length blots in Supplementary Figure 20 .

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 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

figf5(a) Immunoblot for the indicated proteins in fractions enriched in APHs and autophagolysosomes (APHL) isolated from livers of 18Q-htt (18Q) and 111Q-htt (111Q) mice. Cyt, cytochrome; Dyn IC, dynein intermediate chain; ADRP, adipose differentiation-related protein; Polyubq, polyubiquitin. Bottom: amount of each protein in 111Q-htt sample, as a multiple of its amount in 18Q-htt sample. Mean + s.d.; n = 4.(b) Immunoblots for htt and LC3 in homogenates (Homog), cytosol and fractions enriched in APHs and enriched in autophagolysosomes (APHL) isolated from livers of wild-type (18Q-htt) and mutant huntingtin knock-in mice (111Q-htt). Representative one of four experiments with duplicated samples. *P 0.05. Full-length blots in Supplementary Figure 20.


figf6 ( a ) Immunoblot for htt or p62 from total APHs ( T ) and their corresponding membranes ( Mbr ) and matrices ( Mtx ) isolated from livers of wild - type htt ( 18Q ) and 111Q - htt mutant huntingtin knock - in mice ( 111Q ) . Left , representative immunoblots . Right , distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots . AV , autophagic vacuoles . Mean + s . d . * P 0 . 05 compared to wild - type values . ( b ) Filter retardation analysis of the same fractions as in a , blotted for htt . Negative signal indicated absence of aggregates retained in the filter . ( c ) Immunoblot for ubiquitin in homogenates ( Homog ) , cytosol and the autophagic fractions described in a , using 6 % ( top ) and 16 % ( bottom ) gels . Dotted line separates stacking and running parts of gel . ( d ) Immunoblot for htt , LC3 and p62 , of brain homogenates ( Homog ) and fractions enriched in APHs from the same mouse groups as in a . ( e ) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co - immunoprecipitation for htt under low stringency . Input for 111Q - htt autophagic vacuoles was one - third of that for 18Q - htt to avoid antibody saturation . Levels of htt ( top ) and p62 ( bottom ) in the input , immunoprecipitate ( IP ) and flow through ( FT ) are shown . Full - length blots in Supplementary Figure 21 .

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

figf6(a) Immunoblot for htt or p62 from total APHs (T) and their corresponding membranes (Mbr) and matrices (Mtx) isolated from livers of wild-type htt (18Q) and 111Q-htt mutant huntingtin knock-in mice (111Q). Left, representative immunoblots. Right, distribution of htt and p62 between Mbr and Mtx calculated by densitometric quantification in six such immunoblots. AV, autophagic vacuoles. Mean + s.d. *P 0.05 compared to wild-type values.(b) Filter retardation analysis of the same fractions as in a, blotted for htt. Negative signal indicated absence of aggregates retained in the filter.(c) Immunoblot for ubiquitin in homogenates (Homog), cytosol and the autophagic fractions described in a, using 6% (top) and 16% (bottom) gels. Dotted line separates stacking and running parts of gel.(d) Immunoblot for htt, LC3 and p62, of brain homogenates (Homog) and fractions enriched in APHs from the same mouse groups as in a.(e) Immunoblot for htt and p62 of membranes from autophagic vacuoles shown in d subjected to co-immunoprecipitation for htt under low stringency. Input for 111Q-htt autophagic vacuoles was one-third of that for 18Q-htt to avoid antibody saturation. Levels of htt (top) and p62 (bottom) in the input, immunoprecipitate (IP) and flow through (FT) are shown. Full-length blots in Supplementary Figure 21.


figf7 ( a ) Neutral lipids in MEFs from 18Q - htt and 111Q - htt mice stained with BODIPY 493 / 503 . ( b ) Electron micrographs of livers from 18Q - htt and 111Q - htt mice . Right : number of lipid droplets ( LD ) per cell profile ( prof . ) , mean area of LDs and percentage of cellular area occupied by LDs . Mean + s . d . ; n = 3 . ( c - e ) Neutral lipids in striatal cells from 7Q - htt and 111Q - httmice ( c ) , grown on a monolayer of their own astrocytes ( c - e ) Neutral lipids in primary striatal neurons from 18Q - htt and 111Q - httmice grown on a monolayer of their own astrocytes ( d ) ( c - e ) Neutral lipids in lymphoblasts from a control ( UC ) and HD - affected human ( e ) stained with BODIPY 493 / 503 . MAP2 staining highlights neurons . Extended study in Supplementary Figure 15b . ( f ) Electron micrographs of a lymphoblast from an individual with HD ( with 78 and 15 polyglutamine repeats in the two alleles ) . Green arrows , LDs . ( g ) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects . Numbers of polyglutamine repeats are shown below . Mean + s . d . of > 100 cell profiles . Dotted red line , mean value of the respective samples . * P 0 . 05 . ( h ) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals ( top ) and two individuals with HD ( bottom ) . Nuclei are highlighted with hematoxylin . Lipid droplets are visible as red puncta . Subject identifiers are shown in each panel . ( i ) The percentage of total cellular area occupied by lipid droplets ( left ) and the average number of lipid droplets per cell ( right ) was calculated for each of the samples by quantification of eight or nine different fields . * P 0 . 001 ; analysis of variance , P 0 . 01 for each person with HD versus each control individual by Tukey post hoc test . Subject identifiers are as in h .

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

figf7(a) Neutral lipids in MEFs from 18Q-htt and 111Q-htt mice stained with BODIPY 493/503.(b) Electron micrographs of livers from 18Q-htt and 111Q-htt mice. Right: number of lipid droplets (LD) per cell profile (prof.), mean area of LDs and percentage of cellular area occupied by LDs. Mean + s.d.; n = 3.(c-e) Neutral lipids in striatal cells from 7Q-htt and 111Q-httmice (c), grown on a monolayer of their own astrocytes(c-e) Neutral lipids in primary striatal neurons from 18Q-htt and 111Q-httmice grown on a monolayer of their own astrocytes (d)(c-e) Neutral lipids in lymphoblasts from a control (UC) and HD-affected human (e) stained with BODIPY 493/503. MAP2 staining highlights neurons. Extended study in Supplementary Figure 15b.(f) Electron micrographs of a lymphoblast from an individual with HD (with 78 and 15 polyglutamine repeats in the two alleles). Green arrows, LDs.(g) Fraction of cellular cytosol occupied by LDs quantified in lymphoblasts from three UC and four HD subjects. Numbers of polyglutamine repeats are shown below. Mean + s.d. of >100 cell profiles. Dotted red line, mean value of the respective samples. *P 0.05.(h) Oil Red O staining of striatal tissue from brain of two different unaffected control individuals (top) and two individuals with HD (bottom). Nuclei are highlighted with hematoxylin. Lipid droplets are visible as red puncta. Subject identifiers are shown in each panel.(i) The percentage of total cellular area occupied by lipid droplets (left) and the average number of lipid droplets per cell (right) was calculated for each of the samples by quantification of eight or nine different fields. *P 0.001; analysis of variance, P 0.01 for each person with HD versus each control individual by Tukey post hoc test. Subject identifiers are as in h.


figf8 ( a ) Top : immunofluorescence for the mitochondrial marker COX IV in 18Q - htt and 111Q - htt MEFs maintained in the presence or absence of serum . Bottom : number of mitochondria per cell . Mean + s . d . of 10 - 20 cells in three different experiments . ( b ) Electron micrographs of striatal neurons from wild - type ( Ctr ) and HD94 mice grown over a wild - type ratastrocyte monolayer . Arrows , abnormally short ( black ) or abnormally long ( green ) mitochondria . Right : number of mitochondria per cell profile . Mean + s . d . of ten cells per group in triplicates . ( c ) Left : electron micrographs of lymphoblasts from controls ( UC ) or people with HD . Arrows as in b . Right : percentage of cellular area occupied by mitochondria in different individuals . Line indicates population mean . ( d , e ) Striatal cells from 7Q - htt and 111Q - htt mice ( d ) stained with Mitotracker and Mito - ROS . Right , merged images . Percentage of colocalization is indicated at the bottom in dMEFs from 18Q - htt and 111Q - httmice ( e ) stained with Mitotracker and Mito - ROS . Right , merged images . Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e . CCCP was added to control cells in e as a positive control for depolarization . * P 0 . 05 . ( f , g ) Striatal cells from 7Q - htt and 111Q - htt mice untreated or treated with vinblastine were co - stained for LC3 and Mitotracker ( f ) . Arrows , colocalization events . Extended study in Supplementary Figure 16 . ( f , g ) Striatal cells from 7Q - htt and 111Q - httmice untreated or treated with vinblastine were co - stained for LC3 and BODIPY 493 / 503 ( g ) . Arrows , colocalization events . Extended study in Supplementary Figure 16 .

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

figf8(a) Top: immunofluorescence for the mitochondrial marker COX IV in 18Q-htt and 111Q-htt MEFs maintained in the presence or absence of serum. Bottom: number of mitochondria per cell. Mean + s.d. of 10-20 cells in three different experiments.(b) Electron micrographs of striatal neurons from wild-type (Ctr) and HD94 mice grown over a wild-type ratastrocyte monolayer. Arrows, abnormally short (black) or abnormally long (green) mitochondria. Right: number of mitochondria per cell profile. Mean + s.d. of ten cells per group in triplicates.(c) Left: electron micrographs of lymphoblasts from controls (UC) or people with HD. Arrows as in b. Right: percentage of cellular area occupied by mitochondria in different individuals. Line indicates population mean.(d,e) Striatal cells from 7Q-htt and 111Q-htt mice (d) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in dMEFs from 18Q-htt and 111Q-httmice (e) stained with Mitotracker and Mito-ROS. Right, merged images. Percentage of colocalization is indicated at the bottom in d and is displayed in the graph at the bottom in e. CCCP was added to control cells in e as a positive control for depolarization. *P 0.05.(f,g) Striatal cells from 7Q-htt and 111Q-htt mice untreated or treated with vinblastine were co-stained for LC3 and Mitotracker (f). Arrows, colocalization events. Extended study in Supplementary Figure 16.(f,g) Striatal cells from 7Q-htt and 111Q-httmice untreated or treated with vinblastine were co-stained for LC3 and BODIPY 493/503 (g). Arrows, colocalization events. Extended study in Supplementary Figure 16.


Figure 1Cellular locations of native and virally mediated Kcnq1 expression in the cochleaA Diagram showing the major landmarks of the cochlear section to facilitate comparison of data shown in ( B - F ) . B - F Immunolabeling results of Kcnq1 ( green ) in cochlear cryosections are shown for uninjected WT ( B ) , untreated Kcnq1 − / − ( C ) , injected WT ( D ) , and Kcnq1 − / − mice given injections into the ST ( E ) or SM ( F ) . Cell nuclei were outlined by counterstaining with either DAPI ( B - D , F ) or Qnuclear deep red ( E ) . Scale bars represent approximately 100 μm . Meaning of white arrows are given in the text .

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Figure 1Cellular locations of native and virally mediated Kcnq1 expression in the cochleaA Diagram showing the major landmarks of the cochlear section to facilitate comparison of data shown in (B-F).B-F Immunolabeling results of Kcnq1 (green) in cochlear cryosections are shown for uninjected WT (B), untreated Kcnq1−/− (C), injected WT (D), and Kcnq1−/− mice given injections into the ST (E) or SM (F). Cell nuclei were outlined by counterstaining with either DAPI (B-D, F) or Qnuclear deep red (E). Scale bars represent approximately 100 μm. Meaning of white arrows are given in the text.


Figure 2Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cellsKcnq1 and cellnuclei were , respectively , labeled green and purple . The SV consists of three layers of cells ; marginal cells are the first layer of cells on the side of the endolymphatic space . Cryosection through the SV of WT mice . The large arrow points to the native Kcnq1 ( labeled green ) in WT mice . Smaller arrows show nuclei of the marginal cells . Cryosection through the SV of a Kcnq1 − / − mouse injected with AAV expressing Kcnq1 . Labeled in green ( bigger arrow ) is the AAV1 - expressed Kcnq1 , found only in the apical membrane of the marginal cells . Smaller arrows show nuclei of the marginal cells . Arrowheads show Kcnq1immunolabeling in fibrocytes outside the SV . Cryosection through the lateral wall of Kcnq1 − / − mice showing intracellular distribution of Kcnq1immunolabeling ( green ) in fibrocytes . Cryosection through the spiral ganglia of cochlea of treated Kcnq1 − / − mice showing immunolabeling ( green ) in the cells of spiral ganglia ( arrows ) . Data information : Purple staining in all panels is counterstaining with DAPI showing the locations of cellnuclei . Scale bars represent appropriately 50 μm .

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Figure 2Polarized intracellular trafficking of native and virally expressed Kcnq1 was found specifically in the marginal cellsKcnq1 and cellnuclei were, respectively, labeled green and purple. The SV consists of three layers of cells; marginal cells are the first layer of cells on the side of the endolymphatic space.Cryosection through the SV of WT mice. The large arrow points to the native Kcnq1 (labeled green) in WT mice. Smaller arrows show nuclei of the marginal cells.Cryosection through the SV of a Kcnq1−/−mouse injected with AAV expressing Kcnq1. Labeled in green (bigger arrow) is the AAV1-expressed Kcnq1, found only in the apical membrane of the marginal cells. Smaller arrows show nuclei of the marginal cells. Arrowheads show Kcnq1immunolabeling in fibrocytes outside the SV.Cryosection through the lateral wall of Kcnq1−/−mice showing intracellular distribution of Kcnq1immunolabeling (green) in fibrocytes.Cryosection through the spiral ganglia of cochlea of treated Kcnq1−/−mice showing immunolabeling (green) in the cells of spiral ganglia (arrows).Data information: Purple staining in all panels is counterstaining with DAPI showing the locations of cellnuclei. Scale bars represent appropriately 50 μm.


Figure 3Quantification of Kcnq1 - positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled ( red ) by phalloidin conjugated with rhodamine . A Immunolabeling results ( Kcnq1 labeled in green ) in WT mice . B , C Immunolabeling results ( Kcnq1 labeled in green ) of treated Kcnq1 − / − mice , middle ( B ) and apical ( C ) turns , respectively . D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT ( gray bars , left ) , untreated Kcnq1 − / − ( middle ) , and treated Kcnq1 − / − mice ( black bars , right ) . Data are given as mean ± SD ( n = 6 ) . E , F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine . Results from WT ( E ) and untreated Kcnq1 − / − mice ( F ) are compared . Data information : Scale bars represent approximately 50 μm .

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Figure 3Quantification of Kcnq1-positive marginal cells and the effect of treatment on the cellular organization of the marginal cells in the SVThe membranes of marginal cells are labeled (red) by phalloidin conjugated with rhodamine.A Immunolabeling results (Kcnq1 labeled in green) in WT mice.B, C Immunolabeling results (Kcnq1 labeled in green) of treated Kcnq1−/−mice, middle (B) and apical (C) turns, respectively.D The percentage of marginal cells having positive Kcnq1immunolabeling signal above a visually detectable level is shown for WT (gray bars, left), untreated Kcnq1−/− (middle), and treated Kcnq1−/−mice (black bars, right). Data are given as mean ± SD (n = 6).E, F The organization of marginal cells in the SV is outlined by labeling with phalloidin conjugated with rhodamine. Results from WT (E) and untreated Kcnq1−/−mice (F) are compared.Data information: Scale bars represent approximately 50 μm.


Figure 4Comparison of cochlear morphologyA - C Cochlear sections obtained from WT ( A ) , nontreated Kcnq1 − / − ( B ) , and treated Kcnq1 − / − mice ( C ) were compared . Major landmarks of the cochlear sections are labeled and pointed by arrows . Scale bars represent approximately 50 μm .

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Figure 4Comparison of cochlear morphologyA-C Cochlear sections obtained from WT (A), nontreated Kcnq1−/− (B), and treated Kcnq1−/− mice (C) were compared. Major landmarks of the cochlear sections are labeled and pointed by arrows. Scale bars represent approximately 50 μm.


Figure 5Waveforms of ABRs are compared in WT , untreated Kcnq1 − / − , and treated Kcnq1 − / − mice , as labeled above data traces . Series of averaged ABR data traces were evoked from tone - burst sounds with intensities ranging from 20 to 90 dB SPL . Summary of averaged ABR thresholds at various frequencies for different groups of mice ; untreated Kcnq1 − / − ( open triangles ) , treated Kcnq1 − / − ( filled triangles ) , injected WT ( filled circles ) , and uninjected WT ( filled squares ) . Plot legends are given in the figure . The plot with open circles connected with dashed lines represents average data from the five best cases of treated Kcnq1 − / − mice . Error bars represent standard error of the mean . Click - evoked ABR thresholds of WT ( filled squares ) , treated Kcnq1 − / − mice ( filled triangles ) , untreated Kcnq1 − / − mice ( open squares ) measured 4 - 30 weeks after mice were born . Error bars represent standard error of the mean . Upward arrows indicate that click - ABR thresholds were at the maximal sound level that could be reliably measured by the system .

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Figure 5Waveforms of ABRs are compared in WT, untreated Kcnq1−/−, and treated Kcnq1−/− mice, as labeled above data traces. Series of averaged ABR data traces were evoked from tone-burst sounds with intensities ranging from 20 to 90 dB SPL.Summary of averaged ABR thresholds at various frequencies for different groups of mice; untreated Kcnq1−/− (open triangles), treated Kcnq1−/− (filled triangles), injected WT (filled circles), and uninjected WT (filled squares). Plot legends are given in the figure. The plot with open circles connected with dashed lines represents average data from the five best cases of treated Kcnq1−/− mice. Error bars represent standard error of the mean.Click-evoked ABR thresholds of WT (filled squares), treated Kcnq1−/− mice (filled triangles), untreated Kcnq1−/− mice (open squares) measured 4-30 weeks after mice were born. Error bars represent standard error of the mean. Upward arrows indicate that click-ABR thresholds were at the maximal sound level that could be reliably measured by the system.


Figure 3A subplantar injection of 5 % of formalin ( 20 µl ) was administered in the right hind paw of Balb / c mice and an equal volume of PBS was injected in left hind paw . After 1 hour of formalin injection , Balb / c mice were treated with a single dose of either PBS ( vehicle control ) or Diclofenac ( 10 mg / kg ) or different concentrations of rPPE2 ( 2 mg / kg and 3 mg / kg ) via intraperitoneal route and after three hours of treatment , mice were sacrificed and the paw sections were prepared and stained with Toluidine blue to quantify mast cell population . ( A ) Photographs of representative sections were visualized at 20X magnification ( scale bar = 50 µm ) . ( B ) Counting of mast cells was performed in Toluidine blue stained paw sections using ImageJ software and were normalized per unit area ( mm2 ) . Data information : Data shown are Mean ± SEM of 8 mice . For ( B , unpaired t - test was applied to calculate p values .

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Figure 3A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice and an equal volume of PBS was injected in left hind paw. After 1 hour of formalin injection, Balb/c mice were treated with a single dose of either PBS (vehicle control) or Diclofenac (10 mg/kg) or different concentrations of rPPE2 (2 mg/kg and 3 mg/kg) via intraperitoneal route and after three hours of treatment, mice were sacrificed and the paw sections were prepared and stained with Toluidine blue to quantify mast cell population. (A) Photographs of representative sections were visualized at 20X magnification (scale bar = 50 µm). (B) Counting of mast cells was performed in Toluidine blue stained paw sections using ImageJ software and were normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. For (B, unpaired t-test was applied to calculate p values.


Figure 5 ( B - E ) In another experiment , NIH - 3T3 cells ( B , C ) or MEFs ( D , E ) were incubated with either PBS or rPPE2 ( 3 µg / ml ) . After 45 minutes of rPPE2 treatment , cells were fixed , permeabilized , and stained with anti - PPE2 Ab . DAPI was used to stain the nucleus . Nuclear localization of rPPE2 in NIH - 3T3 ( C ) and MEFs ( E ) was measured by calculating the colocalization coefficient . Data information : Data represent Mean ± SEM of more than 50 cells of three independent experiments . For Figure C , E unpaired t - test was applied to calculate p values . ( F - G ) ( F ) NIH - 3T3 cells were transiently transfected with either pEGFP or pEGFP - PPE2 . After 24 hours of transfection , cells were fixed and observed under a confocal microscope . DAPI was used to stain the nucleus . ( G ) Nuclear localization of rPPE2 was measured by calculating colocalization coefficient . Data information : Data represent Mean ± SEM of more than 50 cells of three independent experiments . For Figure G , unpaired t - test was applied to calculate p values .

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Figure 5(B-E) In another experiment, NIH-3T3 cells (B, C) or MEFs (D, E) were incubated with either PBS or rPPE2 (3 µg/ml). After 45 minutes of rPPE2 treatment, cells were fixed, permeabilized, and stained with anti-PPE2 Ab. DAPI was used to stain the nucleus. Nuclear localization of rPPE2 in NIH-3T3 (C) and MEFs (E) was measured by calculating the colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure C, E unpaired t-test was applied to calculate p values.(F-G) (F) NIH-3T3 cells were transiently transfected with either pEGFP or pEGFP-PPE2. After 24 hours of transfection, cells were fixed and observed under a confocal microscope. DAPI was used to stain the nucleus. (G) Nuclear localization of rPPE2 was measured by calculating colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure G, unpaired t-test was applied to calculate p values.


Figure 6 ( D ) Lysates prepared from the same samples used in C were resolved on SDS - PAGE and checked for GFP expression by Western blotting using anti - GFP Ab . GAPDH was used as a loading control . ( E ) Varying concentrations of rPPE2 protein were incubated with [ γ - 32P ] - ATP oligonucleotides of 60 bp spanning SCF promoter . DNA - protein complexes were resolved on 7 % native polyacrylamide gel . In cold competition reactions , equimolar cold / unlabeled 60 bp oligonucleotides were used ( arrow represents PPE2 - oligo complex ) . The data shown are representative of 3 independent experiments .

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Figure 6(D) Lysates prepared from the same samples used in C were resolved on SDS-PAGE and checked for GFP expression by Western blotting using anti-GFP Ab. GAPDH was used as a loading control.(E) Varying concentrations of rPPE2 protein were incubated with [γ-32P]-ATP oligonucleotides of 60 bp spanning SCF promoter. DNA-protein complexes were resolved on 7% native polyacrylamide gel. In cold competition reactions, equimolar cold/unlabeled 60 bp oligonucleotides were used (arrow represents PPE2-oligo complex). The data shown are representative of 3 independent experiments.


Figure 8A subplantar injection of 5 % of formalin ( 20 µl ) was administered in the right hind paw of Balb / c mice , and an equal volume of PBS was injected in left hind paw . After induction of inflammation , Balb / c mice were treated with PBS or peptide ( 8 mg / kg ) via the intraperitoneal route . After 3 hours , paw tissues were harvested and used for cDNA synthesis . B ) ( B ) Next , Toluidine blue staining was done to check the mast cell population . Photographs of representative sections were visualized at 20X magnification ( scale bar = 100 µm ) . A subplantar injection of 5 % of formalin ( 20 µl ) was administered in the right hind paw of Balb / c mice , and an equal volume of PBS was injected in left hind paw . After induction of inflammation , Balb / c mice were treated with PBS or peptide ( 8 mg / kg ) via the intraperitoneal route . After 3 hours , paw tissues were harvested and used for cDNA synthesis . ( C ) Counting of mast cells was performed in Toluidine blue - stained paw sections using ImageJ software and was normalized per unit area ( mm2 ) . Data information : Data shown are Mean ± SEM of 8 mice . unpaired t - test was applied to calculate p values .

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Figure 8A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice, and an equal volume of PBS was injected in left hind paw. After induction of inflammation, Balb/c mice were treated with PBS or peptide (8 mg/kg) via the intraperitoneal route. After 3 hours, paw tissues were harvested and used for cDNA synthesis. B) (B) Next, Toluidine blue staining was done to check the mast cell population. Photographs of representative sections were visualized at 20X magnification (scale bar = 100 µm).A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice, and an equal volume of PBS was injected in left hind paw. After induction of inflammation, Balb/c mice were treated with PBS or peptide (8 mg/kg) via the intraperitoneal route. After 3 hours, paw tissues were harvested and used for cDNA synthesis. (C) Counting of mast cells was performed in Toluidine blue-stained paw sections using ImageJ software and was normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. unpaired t-test was applied to calculate p values.


Figure 2A ) Solid phase transfection of nontargeting ( scrambled ) or PLK1 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 24 , 48 and 72 hours and imaged after DNA staining with Hoechst . Green arrowheads indicate examples of prometaphase arrested cells , and the red arrowheads indicate examples of dead cells due to Plk1 downregulation . Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments . Scale bar , 20 μm . B ) Solid phase transfection of nontargeting ( scrambled ) or CCNA2 targeting gRNAs into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours and imaged after DNA staining with Hoechst . C ) Quantification of nuclear size measurements from Fig 2B . Data derived from 3 independent experiments . P value ( scrambled vs CCNA2 ) < 2e - 16 , Kolmogorov - Smirnov test . D ) Solid phase transfection of nontargeting ( scrambled ) , GOLGA2 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Golga2 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . E ) Quantification of experiments in Fig 2D . Data derived from 3 independent experiments . P value ( scrambled vs GOLGA2 ) < 2e - 16 , Mann - Whitney U test . F ) Solid phase transfection of nontargeting ( scrambled ) , MKI67 targeting gRNA complexes into Cas9 expressing RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Ki67 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . G ) Quantification of experiments in Fig 2F . Data derived from 3 independent experiments . P value ( scrambled vs MKI67 ) < 2e - 16 , Mann - Whitney U test . H ) Comparison of phenotypic penetrance with liquid and solid phase transfection . RPE - 1 , HEK293T , NCI - H358 and NCI - N87 cells were transfected with scrambled or POLR2A targeting gRNAs in in two different cell numbers ( 2250 or 20000 cells / well ) . 5 days post transfection cell viability were measured by CellTiter - Glo . Boxplots represent values from at least 3 independent experiments containing 3 technical replicates . I ) Cell viability measurements after solid phase transfection targeting POLR2A in a panel of cell lines . Cas9 expressing cell lines were transfected with scrambled and POLR2A targeting gRNA and cell viability was assessed after 5 days . The raw values are background subtracted and normalized to the mock controls . Results are from at least 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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Figure 2A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 24, 48 and 72 hours and imaged after DNA staining with Hoechst. Green arrowheads indicate examples of prometaphase arrested cells, and the red arrowheads indicate examples of dead cells due to Plk1 downregulation. Phenotypic penetrance is indicated at the top of each panel with standard deviation derived from 2 independent experiments. Scale bar, 20 μm.B) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting gRNAs into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. C) Quantification of nuclear size measurements from Fig 2B. Data derived from 3 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test.D) Solid phase transfection of nontargeting (scrambled), GOLGA2 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Golga2 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. E) Quantification of experiments in Fig 2D. Data derived from 3 independent experiments. P value (scrambled vs GOLGA2) < 2e-16, Mann-Whitney U test. F) Solid phase transfection of nontargeting (scrambled), MKI67 targeting gRNA complexes into Cas9 expressing RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. G) Quantification of experiments in Fig 2F. Data derived from 3 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test. H) Comparison of phenotypic penetrance with liquid and solid phase transfection. RPE-1, HEK293T, NCI-H358 and NCI-N87 cells were transfected with scrambled or POLR2A targeting gRNAs in in two different cell numbers (2250 or 20000 cells/well). 5 days post transfection cell viability were measured by CellTiter-Glo. Boxplots represent values from at least 3 independent experiments containing 3 technical replicates.I) Cell viability measurements after solid phase transfection targeting POLR2A in a panel of cell lines. Cas9 expressing cell lines were transfected with scrambled and POLR2A targeting gRNA and cell viability was assessed after 5 days. The raw values are background subtracted and normalized to the mock controls. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


Figure 3A ) Solid phase transfection of nontargeting ( scrambled ) or CCNA2 targeting RNP complexes into WT RPE - 1TP53 - / - cells . Cells were fixed after 72 hours and imaged after DNA staining with Hoechst . Scale bar , 50μm . B ) Quantification of experiments in Fig 3A . Data derived from 2 independent experiments . P value ( scrambled vs CCNA2 ) < 2e - 16 , Kolmogorov - Smirnov test . C ) Solid phase transfection of nontargeting ( scrambled ) , MKI67 targeting RNP complexes into WT RPE - 1TP53 - / - cells . Cells were fixed after 72 hours , stained with Ki67 antibody ( red ) , and Hoechst ( blue ) to mark DNA and imaged . Scale bar , 50μm . D ) Quantification of experiments in Fig 3C . Data derived from 2 independent experiments . P value ( scrambled vs MKI67 ) < 2e - 16 , Mann - Whitney U test . E ) Four cell lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA . 5 days post transfection , cell viability in each well was measured . Results are from at least 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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Figure 3A) Solid phase transfection of nontargeting (scrambled) or CCNA2 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours and imaged after DNA staining with Hoechst. Scale bar, 50μm. B) Quantification of experiments in Fig 3A. Data derived from 2 independent experiments. P value (scrambled vs CCNA2) < 2e-16, Kolmogorov-Smirnov test. C) Solid phase transfection of nontargeting (scrambled), MKI67 targeting RNP complexes into WT RPE-1TP53 -/- cells. Cells were fixed after 72 hours, stained with Ki67 antibody (red), and Hoechst (blue) to mark DNA and imaged. Scale bar, 50μm. D) Quantification of experiments in Fig 3C. Data derived from 2 independent experiments. P value (scrambled vs MKI67) < 2e-16, Mann-Whitney U test. E) Four cell lines were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


Figure 4A ) Viability of NCI - N87 and NCI - H358 cells upon transfection of 45 different gRNAs by solid phase transfection . Cas9 expressing NCI - N87 and NCI - H358 cell were seeded on pre - coated plates . 5 days post transfection , cell viability in each well were measured by CellTiter - Glo . Values were background subtracted , normalized to scrambled controls and plotted against each other . Blue dots represent scrambled controls , whereas the yellow dots represent POLR2A gRNAs . Green dots represent the genes that affect the viability in a cell line dependent manner . Results are representative of 3 independent experiments . B , C ) Logarithm of the odds ( LOD ) scores of cancer associated gene KOs in NCI - N87 and NCI - H358 cells derived from screens with gRNAs ( B ) and RNPs ( C ) . Bottom panels show distribution of positive ( POLR2A ) and negative ( scrambled ) controls for each experiment . LOD score of 3 is highlighted with a dashed line , indicating statistical significance .

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Figure 4A) Viability of NCI-N87 and NCI-H358 cells upon transfection of 45 different gRNAs by solid phase transfection. Cas9 expressing NCI-N87 and NCI-H358 cell were seeded on pre-coated plates. 5 days post transfection, cell viability in each well were measured by CellTiter-Glo. Values were background subtracted, normalized to scrambled controls and plotted against each other. Blue dots represent scrambled controls, whereas the yellow dots represent POLR2A gRNAs. Green dots represent the genes that affect the viability in a cell line dependent manner. Results are representative of 3 independent experiments.B, C) Logarithm of the odds (LOD) scores of cancer associated gene KOs in NCI-N87 and NCI-H358 cells derived from screens with gRNAs (B) and RNPs (C). Bottom panels show distribution of positive (POLR2A) and negative (scrambled) controls for each experiment. LOD score of 3 is highlighted with a dashed line, indicating statistical significance.


Figure 5A ) Solid phase transfection of nontargeting ( scrambled ) or PLK1 targeting RNP complexes into human primary lung fibroblasts ( HLF - 1 ) , primary lung adenocarcinoma cells ( LAD - 1 ) or RPE - 1TP53 - / - cells . 72 hours post transfection cells were stained with Hoechst and imaged . Boxplots represent values from 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs Plk1 ) < 0 . 05 . In the right panels , representative images for each cell line is shown after RNP transfection targeting PLK1 . Arrowheads indicate the cells that are arrested in prometaphase due to Plk1 downregulation . Scale bars indicate 20μm . B ) Two human primary lung fibroblasts ( HLF ) and three primary tumor cell lines derived from patients suffering from either lung squamous carcinoma ( LSC ) or lung adenocarcinoma ( LAD ) were transfected with RNP complexes with scrambled or POLR2A targeting gRNA . 5 days post transfection , cell viability in each well was measured . Results are from at least 3 independent experiments containing 3 technical replicates . For all cell lines , P values ( scrambled vs POLR2A ) < 0 . 005 .

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Figure 5A) Solid phase transfection of nontargeting (scrambled) or PLK1 targeting RNP complexes into human primary lung fibroblasts (HLF-1), primary lung adenocarcinoma cells (LAD-1) or RPE-1TP53 -/- cells. 72 hours post transfection cells were stained with Hoechst and imaged. Boxplots represent values from 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs Plk1) < 0.05. In the right panels, representative images for each cell line is shown after RNP transfection targeting PLK1. Arrowheads indicate the cells that are arrested in prometaphase due to Plk1 downregulation. Scale bars indicate 20μm.B) Two human primary lung fibroblasts (HLF) and three primary tumor cell lines derived from patients suffering from either lung squamous carcinoma (LSC) or lung adenocarcinoma (LAD) were transfected with RNP complexes with scrambled or POLR2A targeting gRNA. 5 days post transfection, cell viability in each well was measured. Results are from at least 3 independent experiments containing 3 technical replicates. For all cell lines, P values (scrambled vs POLR2A) < 0.005.


Figure 1A , Seedlings of WT , abs3 - 1D , FD11 - 55 , and arf2 - 20 before and after 6 d C - deprivation . B , Seedlings of WT , abs3 - 1D , FD18 - 70 , and pif5 - 10 before and after 6 d C - deprivation . C and D , RT - qPCR analysis of ABS3 expression levels in 7 - d - old seedlings of the same genotypes as shown in A ( C ) and B ( D ) . Fold changes were calculated with respect to the expression level in the WT . two - tailed t test , * * * * , p < 0 . 0001 , n . s . , not significant . data are means ± standard deviation ( s . d . ) of 3 biological replicates . E and F , Chlorophyll and total cellular protein content reduction in genotypes shown in A ( E ) and B ( F ) after 6 d C - deprivation . data are means ± standard deviation ( s . d . ) of 3 biological replicates . G and H , RT - qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A ( G ) and B ( H ) before and after 4 d C - deprivation . Fold changes were calculated with respect to the expression level in the WT before C - deprivation . data are means ± standard deviation ( s . d . ) of 3 biological replicates .

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Figure 1A, Seedlings of WT, abs3-1D, FD11-55, and arf2-20 before and after 6 d C-deprivation. B, Seedlings of WT, abs3-1D, FD18-70, and pif5-10 before and after 6 d C-deprivation.C and D, RT-qPCR analysis of ABS3 expression levels in 7-d-old seedlings of the same genotypes as shown in A (C) and B (D). Fold changes were calculated with respect to the expression level in the WT. two-tailed t test, ****, p<0.0001, n.s., not significant. data are means ± standard deviation (s.d.) of 3 biological replicates.E and F, Chlorophyll and total cellular protein content reduction in genotypes shown in A (E) and B (F) after 6 d C-deprivation. data are means ± standard deviation (s.d.) of 3 biological replicates.G and H, RT-qPCR analysis of ORE1 and SGR1 expression levels in genotypes shown in A (G) and B (H) before and after 4 d C-deprivation. Fold changes were calculated with respect to the expression level in the WT before C-deprivation. data are means ± standard deviation (s.d.) of 3 biological replicates.


Figure 2C , Heatmap of log2FC values of 2 , 587 ABS3 - regulated genes ( abs3 - 1D vs . WT ) under C - deprivation in indicated comparisons . F , Heatmap of log2FC values of 1 , 326 ARF2 , PIF5 , and C - deprivation co - regulated genes in indicated comparisons . C - deprivation : abs3 - 1D 4 d C - deprivation vs . abs3 - 1D 0 d C - deprivation ; ARF2 : abs3 - 1D vs . arf2 - 20 abs3 - 1D after 4 d C - deprivation ; PIF5 : abs3 - 1D vs . abs3 - 1D pif5 - 10 after 4 d C - deprivation . Enlarged were expression changes of senescence genes identified in DAVID GO

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Figure 2C, Heatmap of log2FC values of 2,587 ABS3-regulated genes (abs3-1D vs. WT) under C-deprivation in indicated comparisons.F, Heatmap of log2FC values of 1,326 ARF2, PIF5, and C-deprivation co-regulated genes in indicated comparisons. C-deprivation: abs3-1D 4 d C-deprivation vs. abs3-1D 0 d C-deprivation; ARF2: abs3-1D vs. arf2-20 abs3-1D after 4 d C-deprivation; PIF5: abs3-1D vs. abs3-1D pif5-10 after 4 d C-deprivation. Enlarged were expression changes of senescence genes identified in DAVID GO analysis


Figure 3A , Interaction between ARF2 and PIF5 in the yeast two - hybrid assay . Growth of yeast cells co - transformed with indicated BD and AD vectors was monitored on quadruple dropout medium ( QDO , SD - Ade / - His / - Leu / - Trp ) and QDO medium containing X - α - gal . B , Interaction between ARF2 and PIF5 in protoplast BiFC assays . YN - ARF2 and PIF5 - YC were coexpressed with the nuclear marker NLS - mCherry in Arabidopsis mesophyll protoplasts . Coexpression of YN - ARF2 and YC alone or YN alone with PIF5 - YC served as negative controls . Bars , 10 μm . C , Direct interaction between GST - ARF2 and MBP - PIF5 - His in in vitro GST pull - down assays . D , Co - immunoprecipitation of ARF2 - HA with PIF5 - GFP in vivo . ARF2 - HA was coexpressed with PIF5 - GFP or GFP alone in protoplasts . Proteins co - immunoprecipitated by GFP - Trap beads were immunoblotted with anti - GFP and anti - HA antibodies .

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Figure 3A, Interaction between ARF2 and PIF5 in the yeast two-hybrid assay. Growth of yeast cells co-transformed with indicated BD and AD vectors was monitored on quadruple dropout medium (QDO, SD-Ade/-His/-Leu/-Trp) and QDO medium containing X-α-gal.B, Interaction between ARF2 and PIF5 in protoplast BiFC assays. YN-ARF2 and PIF5-YC were coexpressed with the nuclear marker NLS-mCherry in Arabidopsis mesophyll protoplasts. Coexpression of YN-ARF2 and YC alone or YN alone with PIF5-YC served as negative controls. Bars, 10 μm.C, Direct interaction between GST-ARF2 and MBP-PIF5-His in in vitro GST pull-down assays.D, Co-immunoprecipitation of ARF2-HA with PIF5-GFP in vivo. ARF2-HA was coexpressed with PIF5-GFP or GFP alone in protoplasts. Proteins co-immunoprecipitated by GFP-Trap beads were immunoblotted with anti-GFP and anti-HA antibodies.


Figure 4A , Seedlings of WT , pARF2 : ARF2 - GFP OEs in the WT background , pif4 - 2 pif5 - 10 , and pARF2 : ARF2 - GFP OEs in the pif4 - 2 pif5 - 10 background before and after 6 d C - deprivation . B , RT - qPCR analysis of ARF2 expression levels in 7 - d - old seedlings of the same genotypes as shown in A . Fold changes were calculated with respect to the expression level in the WT . data are means ± s . d . of 3 biological replicates . C , Chlorophyll and total cellular protein content reduction in genotypes shown in A after 6 d C - deprivation . Color legends in C are the same as in B . data are means ± s . d . of 3 biological replicates . D , Seedlings of WT , pPIF5 : PIF5 - GFP OEs in the WT background , arf2 - 20 , and pPIF5 : PIF5 - GFP OEs in the arf2 - 20 background before and after 6 d C - deprivation . E , RT - qPCR analysis of PIF5 expression levels in 7 - d - old seedlings of the same genotypes as shown in D . Fold changes were calculated with respect to the expression level in the WT . data are means ± s . d . of 3 biological replicates . F , Chlorophyll and total cellular protein content reduction in genotypes shown in D after 6 d C - deprivation . Color legends in F are the same as in E . data are means ± s . d . of 3 biological replicates . G , Seedlings of WT , arf2 - 20 , pif5 - 10 , and arf2 - 20 pif5 - 10 before and after 12 d C - deprivation . H , Chlorophyll and total cellular protein content reduction in genotypes shown in G after 12 d C - deprivation . data are means ± s . d . of 3 biological replicates . I , Seedlings of WT , abs3 - 1D , arf2 - 20 abs3 - 1D , pif5 - 10 abs3 - 1D , and arf2 - 20 pif5 - 10 abs3 - 1D before and after 8 d C - deprivation . J , Chlorophyll and total cellular protein content reduction in genotypes shown in I after 8 d C - deprivation . data are means ± s . d . of 3 biological replicates .

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Figure 4A, Seedlings of WT, pARF2:ARF2-GFP OEs in the WT background, pif4-2 pif5-10, and pARF2:ARF2-GFP OEs in the pif4-2 pif5-10 background before and after 6 d C-deprivation.B, RT-qPCR analysis of ARF2 expression levels in 7-d-old seedlings of the same genotypes as shown in A. Fold changes were calculated with respect to the expression level in the WT. data are means ± s.d. of 3 biological replicates.C, Chlorophyll and total cellular protein content reduction in genotypes shown in A after 6 d C-deprivation. Color legends in C are the same as in B. data are means ± s.d. of 3 biological replicates.D, Seedlings of WT, pPIF5:PIF5-GFP OEs in the WT background, arf2-20, and pPIF5:PIF5-GFP OEs in the arf2-20 background before and after 6 d C-deprivation.E, RT-qPCR analysis of PIF5 expression levels in 7-d-old seedlings of the same genotypes as shown in D. Fold changes were calculated with respect to the expression level in the WT. data are means ± s.d. of 3 biological replicates.F, Chlorophyll and total cellular protein content reduction in genotypes shown in D after 6 d C-deprivation. Color legends in F are the same as in E. data are means ± s.d. of 3 biological replicates.G, Seedlings of WT, arf2-20, pif5-10, and arf2-20 pif5-10 before and after 12 d C-deprivation.H, Chlorophyll and total cellular protein content reduction in genotypes shown in G after 12 d C-deprivation. data are means ± s.d. of 3 biological replicates.I, Seedlings of WT, abs3-1D, arf2-20 abs3-1D, pif5-10 abs3-1D, and arf2-20 pif5-10 abs3-1D before and after 8 d C-deprivation.J, Chlorophyll and total cellular protein content reduction in genotypes shown in I after 8 d C-deprivation. data are means ± s.d. of 3 biological replicates.