Figure 3A , Schematic representation of a longitudinal section of an Arabidopsis RM . QC cells are marked in red , CSCs are marked in dark blue , CCs in light blue . Combined mPSPI ( grey ) and EdU ( red ) staining for 24 hours ( SCN staining ) to analyse the CSC ( A - I ) and QC division phenotype ( J - R ) within the same roots are shown . B - H , Representative images of the SCN staining in Col , and the indicated single , double , and triple mutant roots . QC positions are marked by yellow arrowhead . I , Analyses of the SCN staining for CSC phenotypes . Frequencies of roots showing 0 , 1 , 2 , or 3 CSC layers are plotted as bar graphs . J , Schematic representation of a transversal section of an Arabidopsis RM . QC cells are marked in red , CEI initials are marked in turquoise . K - Q , Representative images of transversal sections with QC cells outlined in yellow . R , Analyses of the SCN staining for QC division phenotypes . Frequencies of roots showing 0 , 1 , 2 , 3 or ≥ 4 dividing QC cells are plotted as bar graphs . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from 2 - 5 technical replicates per genotype . QC = quiescent center , CSC = columella stem cell , CEI = cortex endodermis initial , SCN = stem cell niche , mPSPI = modified pseudo - Schiff propidium iodide , EdU = 5 - ethynyl - 2 ' - deoxyuridine , scale bars represent 5 µm .

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

Figure 3A, Schematic representation of a longitudinal section of an Arabidopsis RM. QC cells are marked in red, CSCs are marked in dark blue, CCs in light blue. Combined mPSPI (grey) and EdU (red) staining for 24 hours (SCN staining) to analyse the CSC (A-I) and QC division phenotype (J-R) within the same roots are shown. B-H, Representative images of the SCN staining in Col, and the indicated single, double, and triple mutant roots. QC positions are marked by yellow arrowhead. I, Analyses of the SCN staining for CSC phenotypes. Frequencies of roots showing 0, 1, 2, or 3 CSC layers are plotted as bar graphs. J, Schematic representation of a transversal section of an Arabidopsis RM. QC cells are marked in red, CEI initials are marked in turquoise. K-Q, Representative images of transversal sections with QC cells outlined in yellow. R, Analyses of the SCN staining for QC division phenotypes. Frequencies of roots showing 0, 1, 2, 3 or ≥4 dividing QC cells are plotted as bar graphs. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype. QC = quiescent center, CSC = columella stem cell, CEI = cortex endodermis initial, SCN = stem cell niche, mPSPI = modified pseudo-Schiff propidium iodide, EdU = 5-ethynyl-2'-deoxyuridine, scale bars represent 5 µm.


Figure 4The combined results of the SCN staining 3 are shown as 2D plots to visualise the correlation of the CSC layer and QC division phenotypes . Number of CSC layers are shown on the y axis and the QC division phenotype is shown on the x axis . The darker the colour , the more roots show the respective phenotype ( see colour gradient top right indicating the frequencies in percent ) . Col wild - type roots show one layer of CSCs and no EdU stained cells ( no QC division ) after 24 h EdU staining . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype and results from 2 - 5 technical replicates per genotype .

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

Figure 4The combined results of the SCN staining 3 are shown as 2D plots to visualise the correlation of the CSC layer and QC division phenotypes. Number of CSC layers are shown on the y axis and the QC division phenotype is shown on the x axis. The darker the colour, the more roots show the respective phenotype (see colour gradient top right indicating the frequencies in percent). Col wild-type roots show one layer of CSCs and no EdU stained cells (no QC division) after 24 h EdU staining. Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from 2-5 technical replicates per genotype.


Figure 7A - H , Fluorescence Lifetime Imaging ( FLIM ) results of transiently expressing N . benthamiana leaf epidermal cells . A - H , Representative FLIM images of WOX5 - mVenus ( donor only ) plus the indicated acceptors after a pixel - wise mono - exponential fit of the mVenus fluorescence signal . The fluorescence lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of the two proteins and high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm . I , Fluorescence lifetimes in ns are summarized in combined jitter and box plots . The dashed line represents the fluorescence lifetime mean value of the WOX5 - mV co - expressed with free mCh as negative control . The one - way ANOVA and Holm - Sidak post - hoc multiple comparisons test was used to test for statistical significance . Samples with identical letters do not show significant differences ( α = 0 . 01 ) . Number of nuclei analyzed ( n ) ( biological replicates ) is indicated and results from 2 to 9 technical replicates . Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , ▪ = mean value . J , Representative FLIM images of WOX5 - mVenus ( donor only ) after a pixel - wise mono - exponential fit of the mVenus fluorescence signal . The fluorescence lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of the two proteins and high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm . K , Individual nuclei showing nuclear bodies during co - expression of WOX5 - mV and PLT3 - mCh were analysed for WOX5 - mV lifetime in the nuclear bodies or nucleoplasm separately ( n = 7 ( biological replicates ) , from four technical replicates . mCh = mCherry . mV = mVenus .

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 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 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 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

Figure 7A-H, Fluorescence Lifetime Imaging (FLIM) results of transiently expressing N. benthamiana leaf epidermal cells. A-H, Representative FLIM images of WOX5-mVenus (donor only) plus the indicated acceptors after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm.I, Fluorescence lifetimes in ns are summarized in combined jitter and box plots. The dashed line represents the fluorescence lifetime mean value of the WOX5-mV co-expressed with free mCh as negative control. The one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test was used to test for statistical significance. Samples with identical letters do not show significant differences (α = 0.01). Number of nuclei analyzed (n) (biological replicates) is indicated and results from 2 to 9 technical replicates. Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, ▪ = mean value.J, Representative FLIM images of WOX5-mVenus (donor only) after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm.K, Individual nuclei showing nuclear bodies during co-expression of WOX5-mV and PLT3-mCh were analysed for WOX5-mV lifetime in the nuclear bodies or nucleoplasm separately (n = 7 (biological replicates), from four technical replicates. mCh = mCherry. mV = mVenus.


figf1 ( a ) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression ( 18 h cultures ) . Values are means ± s . e . m . of three independent experiments . ( b ) The cell death regulated by Atg5 is apoptosis . All data presented are from 5 h cultures . Caspase - 3 - like activity analysed in HeLa cells ( graph ) . Morphology of Jurkat cells is also shown . The numbers in the lower right corner indicate the percentage of cell death , which was determined in parallel experiments using flow cytometryc ) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z - VAD - fmk . Numbers indicate the results of the quantitative analysis ( percentage ) of each dot blot . d ) Reduction of drug - induced cell death by silencing of Atg5 . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) . The effects of the Atg5 siRNA on Atg5 and GAPDH levels were analysed by immunoblotting . The asterisks indicate P 0 . 05 . n . s . , no significant difference .

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 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 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) Cell death was analysed after the activation of death pathways in the presence and absence of enforced Atg5 expression (18 h cultures). Values are means ± s.e.m. of three independent experiments.(b) The cell death regulated by Atg5 is apoptosis. All data presented are from 5 h cultures. Caspase-3-like activity analysed in HeLa cells (graph). Morphology of Jurkat cells is also shown. The numbers in the lower right corner indicate the percentage of cell death, which was determined in parallel experiments using flow cytometryc) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat cells and blocked by z-VAD-fmk. Numbers indicate the results of the quantitative analysis (percentage) of each dot blot.d) Reduction of drug-induced cell death by silencing of Atg5. Values are means ± s.e.m. of three independent experiments (18 h cultures). The effects of the Atg5 siRNA on Atg5 and GAPDH levels were analysed by immunoblotting. The asterisks indicate P 0.05. n.s., no significant difference.


figf2a ) Spontaneous neutrophil apoptosis . Apoptosis was associated with the appearance of a 24K anti - Atg5 - reactive protein . 33K Atg5 disappeared during apoptosis in a time - dependent manner . HeLa cells overexpressing Atg5 served as positive control . b ) Anti - CD95 - induced apoptosis of Jurkat cells . Apoptosis was associated with the appearance of a 24K anti - Atg5 - reactive protein and a reduction of 33K Atg5 in a time - dependent manner . The 24K anti - Atg5 - reactive protein was also visible in Jurkat cells after treatment with actinomycin D ( Act . D ) or cycloheximide ( CHX ; 5 h treatment in both cases ) . GAPDH expression indicates equal loading in each panel . All immmunoblots are representative of at least three independent experiments .

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figf2a) Spontaneous neutrophil apoptosis. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein. 33K Atg5 disappeared during apoptosis in a time-dependent manner. HeLa cells overexpressing Atg5 served as positive control.b) Anti-CD95-induced apoptosis of Jurkat cells. Apoptosis was associated with the appearance of a 24K anti-Atg5-reactive protein and a reduction of 33K Atg5 in a time-dependent manner. The 24K anti-Atg5-reactive protein was also visible in Jurkat cells after treatment with actinomycin D (Act. D) or cycloheximide (CHX; 5 h treatment in both cases). GAPDH expression indicates equal loading in each panel. All immmunoblots are representative of at least three independent experiments.


figf3e ) Recombinant Atg5 was digested with calpain and the products sequenced by Edman degradation . Calpain cleaved Atg5 at Thr 193 . Comparison of Atg5 sequences of different species demonstrates the presence of the same calpain cleavage site in mice . The sequence alignment was perfromed with Clustal W using sequence data obtained from GenBank . The position of the six - amino - acid deletion causing calpain resistance is indicated in red . ( d ) Following calpain - mediated cleavage of recombinant Atg5 in vitro , the sample was fractionated by preparative reversed - phase HPLC and analysed by mass spectrometry . The exact relative molecular masses for full - length and truncated Atg5 are indicated . ( f ) A six - amino - acid deletion surrounding the cleavage site ( 35S - Atg5Δ191 - 196 ) led to a loss of the calpain cleavage fragment . ( g ) Calpain inhibition by ALLN blocked both cleavage of Bax and Atg5 in spontaneous and Fas receptor - mediated apoptosis of neutrophils . In contrast , Atg6 did not show evidence for cleavage and its levels remained unchanged in this system . Results of 7 h cultures are shown . ( h ) Atg5 is not cleaved in apoptotic HeLa cells ( 5 h staurosporine treatment ) lacking the small subunit of calpain , which was silenced by using specific siRNA . ( i ) Calpain resistant Atg5Δ191 - 196 was unable to sensitize HeLa cells lacking endogeneous Atg5 ( due to gene silencing , see Fig . 1c ) to staurosporine - induced cell death . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) .

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figf3e) Recombinant Atg5 was digested with calpain and the products sequenced by Edman degradation. Calpain cleaved Atg5 at Thr 193. Comparison of Atg5 sequences of different species demonstrates the presence of the same calpain cleavage site in mice. The sequence alignment was perfromed with Clustal W using sequence data obtained from GenBank. The position of the six-amino-acid deletion causing calpain resistance is indicated in red.(d) Following calpain-mediated cleavage of recombinant Atg5 in vitro, the sample was fractionated by preparative reversed-phase HPLC and analysed by mass spectrometry. The exact relative molecular masses for full-length and truncated Atg5 are indicated.(f) A six-amino-acid deletion surrounding the cleavage site (35S-Atg5Δ191-196) led to a loss of the calpain cleavage fragment.(g) Calpain inhibition by ALLN blocked both cleavage of Bax and Atg5 in spontaneous and Fas receptor-mediated apoptosis of neutrophils. In contrast, Atg6 did not show evidence for cleavage and its levels remained unchanged in this system. Results of 7 h cultures are shown.(h) Atg5 is not cleaved in apoptotic HeLa cells (5 h staurosporine treatment) lacking the small subunit of calpain, which was silenced by using specific siRNA.(i) Calpain resistant Atg5Δ191-196 was unable to sensitize HeLa cells lacking endogeneous Atg5 (due to gene silencing, see Fig. 1c) to staurosporine-induced cell death. Values are means ± s.e.m. of three independent experiments (18 h cultures).


figf4 ( a ) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining . Values are means ± s . e . m . of three independent experiments . Bax transfection was used as a positive control . ( b ) Representative images after Hoechst staining demonstrating nuclear fragmentation of HeLa cells that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h . Quantitative analysis of these experiments is shown in a . Control and transfected cells were loaded with Mitotracker orange and subsequently processed for cytochrome c immunostaining . Enforced expression of 24K Atg5 resulted in cytochrome c release into the cytosol and apoptosis ( nuclear condensation , cell shrinkage ; approximately 80 % of the cells ) . ( c ) Jurkat cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer . Cell death was observed as a consequence of enforced expression of truncated Atg5 within two days . Morphological analysis ( Diff - Quik ) is also shown and revealed that the type of death was apoptosis ( nuclear condensation , cell shrinkage ) . ( d ) CEM cells that either lacked Bcl - 2 or expressed high levels of Bcl - 2 received truncated Atg5 or GFP by lentiviral gene transfer . In the absence of Bcl - 2 , induction of apoptosis as assessed by DNA fragmentation was observed due to enforced expression of truncated Atg5 within two days . Apoptosis was largely blocked in cells expressing high levels of Bcl - 2 . GFP transfection did not affect viability of the cells in this system . ( e ) Staurosporine - induced Atg5 cleavage ( 5 h treatment ) in CEM cells independent of Bcl - 2 expression . Blots were stripped and reprobed with anti - GAPDH antibody . ( f ) Detection of autophagic activity by staining of acidic vesicular organelles ( VAOs , red ) followed by flow cytometry analysis in genetically modified HeLa cells . That 24K Atg5 did not induce autophagy was confirmed by transmission electron microscopy ( data not shown ) . ( g ) Cell death after 24K Atg5 gene transfer in HeLa cells exhibiting different autophagic activities . Values are means ± s . e . m . of three independent experiments ( lentiviral gene transfer for 24 h ) . The asterisks indicate P 0 . 05 . n . s . , no significant difference . The scale bars represent 10 μM .

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 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 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 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 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 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

figf4(a) HeLa cells were transiently transfected with 24K and 33K Atg5 for 24 h and subsequently analysed by Hoechst staining. Values are means ± s.e.m. of three independent experiments. Bax transfection was used as a positive control. (b) Representative images after Hoechst staining demonstrating nuclear fragmentation of HeLa cells that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h. Quantitative analysis of these experiments is shown in a. Control and transfected cells were loaded with Mitotracker orange and subsequently processed for cytochrome c immunostaining. Enforced expression of 24K Atg5 resulted in cytochrome c release into the cytosol and apoptosis (nuclear condensation, cell shrinkage; approximately 80% of the cells).(c) Jurkat cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer. Cell death was observed as a consequence of enforced expression of truncated Atg5 within two days. Morphological analysis (Diff-Quik) is also shown and revealed that the type of death was apoptosis (nuclear condensation, cell shrinkage).(d) CEM cells that either lacked Bcl-2 or expressed high levels of Bcl-2 received truncated Atg5 or GFP by lentiviral gene transfer. In the absence of Bcl-2, induction of apoptosis as assessed by DNA fragmentation was observed due to enforced expression of truncated Atg5 within two days. Apoptosis was largely blocked in cells expressing high levels of Bcl-2. GFP transfection did not affect viability of the cells in this system.(e) Staurosporine-induced Atg5 cleavage (5 h treatment) in CEM cells independent of Bcl-2 expression. Blots were stripped and reprobed with anti-GAPDH antibody.(f) Detection of autophagic activity by staining of acidic vesicular organelles (VAOs, red) followed by flow cytometry analysis in genetically modified HeLa cells. That 24K Atg5 did not induce autophagy was confirmed by transmission electron microscopy (data not shown).(g) Cell death after 24K Atg5 gene transfer in HeLa cells exhibiting different autophagic activities. Values are means ± s.e.m. of three independent experiments (lentiviral gene transfer for 24 h). The asterisks indicate P 0.05. n.s., no significant difference. The scale bars represent 10 μM.


figf5 ( a ) Confocal microscopy of HeLa cells were treated with anti - CD95 antibody for 5 h or left untreated . Cells were stained with anti - Atg5 and anti - VDAC antibodies . In untreated cells , there was only little evidence for colocalization of Atg5 and VDAC . In anti - CD95 antibody treated cells some of the Atg5 colocalized with VDAC . The images of colocalization were prepared using Imaris software . Numerical analysis was performed on 10 cells in each group and the correlation coefficient calculated . The error bars represent the s . e . m . ( b ) Subcellular fractionation of blood neutrophils that were left untreated to induce spontaneous apoptosis . After 4 h incubation , cleavage of Atg5 was evident and both full - length and truncated Atg5 were present in the cytosol . After 8 h incubation , some 24K Atg5 but not full - length Atg5 was found in the mitochondrial fraction . Mitochondrial association of truncated Atg5 coincided with the release of cytochrome c into the cytosol . The same data were observed in staurosporine - induced apoptosis of Jurkat cells ( data not shown ) . C , cytosol ; M , mitochondria . ( c ) Cytochrome c release assay . Truncated and full - length recombinant Atg5 were added to isolated mitochondria and incubated at 37 ° C for 1 h . Supernatants and mitochondrial pellets were then analysed for cytochrome c by immunoblotting . Truncated Bid ( tBid ) was used as a positive control for mitochondrial permeabilization . Truncated Atg5 released cytochrome c in a concentration - dependent manner , indicating mitochondrial permeabilization . Full - length Atg5 had only little effect in this system . ( d ) HeLa cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer for 24 h and analysed for caspase - 3 cleavage . GAPDH expression demonstrates equal loading . ( e ) Jurkat cells were treated with staurosporine for 5 h or left untreated . Staurosporine induced Atg5 cleavage and both full - length and truncated Atg5 were present in total lysates . Truncated but not full - length Atg5 was detected in Bcl - xL immunoprecipitates obtained from apoptotic Jurkat cells . No evidence for physical interaction between Atg5 and Bax was obtained . The lower panels demonstrate that both Bax and Bcl - xL were enriched following immunoprecipitation . The asterisk indicates P 0 . 05 . The scale bars represent 10 μM .

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 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 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

figf5(a) Confocal microscopy of HeLa cells were treated with anti-CD95 antibody for 5 h or left untreated. Cells were stained with anti-Atg5 and anti-VDAC antibodies. In untreated cells, there was only little evidence for colocalization of Atg5 and VDAC. In anti-CD95 antibody treated cells some of the Atg5 colocalized with VDAC. The images of colocalization were prepared using Imaris software. Numerical analysis was performed on 10 cells in each group and the correlation coefficient calculated. The error bars represent the s.e.m.(b) Subcellular fractionation of blood neutrophils that were left untreated to induce spontaneous apoptosis. After 4 h incubation, cleavage of Atg5 was evident and both full-length and truncated Atg5 were present in the cytosol. After 8 h incubation, some 24K Atg5 but not full-length Atg5 was found in the mitochondrial fraction. Mitochondrial association of truncated Atg5 coincided with the release of cytochrome c into the cytosol. The same data were observed in staurosporine-induced apoptosis of Jurkat cells (data not shown). C, cytosol; M, mitochondria.(c) Cytochrome c release assay. Truncated and full-length recombinant Atg5 were added to isolated mitochondria and incubated at 37 °C for 1 h. Supernatants and mitochondrial pellets were then analysed for cytochrome c by immunoblotting. Truncated Bid (tBid) was used as a positive control for mitochondrial permeabilization. Truncated Atg5 released cytochrome c in a concentration-dependent manner, indicating mitochondrial permeabilization. Full-length Atg5 had only little effect in this system.(d) HeLa cells were transfected with 24K and 33K Atg5 using lentiviral gene transfer for 24 h and analysed for caspase-3 cleavage. GAPDH expression demonstrates equal loading.(e) Jurkat cells were treated with staurosporine for 5 h or left untreated. Staurosporine induced Atg5 cleavage and both full-length and truncated Atg5 were present in total lysates. Truncated but not full-length Atg5 was detected in Bcl-xL immunoprecipitates obtained from apoptotic Jurkat cells. No evidence for physical interaction between Atg5 and Bax was obtained. The lower panels demonstrate that both Bax and Bcl-xL were enriched following immunoprecipitation. The asterisk indicates P 0.05. The scale bars represent 10 μM.


Figure 1A slice through a filtered tomogram showing SR vesicles in contact with a putative T - tubule . Individual RyR1 molecules are indicated by the red circles . The dense protein coat on the surface of the SR vesicles correspond to the SERCA pump . Areas circled in yellow indicate accumulation of density inside the SR . Scale bar : 20 nm . A slice through the structure of RyR1 in contact with putative TT - membrane . A middle slice through the in situ structure of RyR1 at 12 . 6 - Å resolution . The observed additional transmembrane density is indicated by the red arrows . Scale bars in B , C : 10 nm . Slice through the structures of apoRyR1 with the putative TT - membrane ( left ) and the standalone apoRyR1 ( right , with the flipped handendness and reduced resolution for comparison ) 22 Å away from the middle slice ( Y = 92 in the volume coordinates ) . Scale bar : 10 nm . The location of the CaM / S100A1 binding site is marked with red circles in slices through maps of the in situ structure ( left ) and the single particle structure ( EMDB : 8393 ) in the same orientation as the in situ structure and filtered to 15 Å ( right ) . Scale bar : 10 nm .

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Figure 1A slice through a filtered tomogram showing SR vesicles in contact with a putative T-tubule. Individual RyR1 molecules are indicated by the red circles. The dense protein coat on the surface of the SR vesicles correspond to the SERCA pump. Areas circled in yellow indicate accumulation of density inside the SR. Scale bar: 20 nm. A slice through the structure of RyR1 in contact with putative TT-membrane. A middle slice through the in situ structure of RyR1 at 12.6-Å resolution. The observed additional transmembrane density is indicated by the red arrows. Scale bars in B, C: 10 nm.Slice through the structures of apoRyR1 with the putative TT-membrane (left) and the standalone apoRyR1 (right, with the flipped handendness and reduced resolution for comparison) 22 Å away from the middle slice (Y = 92 in the volume coordinates). Scale bar: 10 nm.The location of the CaM/S100A1 binding site is marked with red circles in slices through maps of the in situ structure (left) and the single particle structure (EMDB: 8393) in the same orientation as the in situ structure and filtered to 15 Å (right). Scale bar: 10 nm.


Figure 2A slice through the average structure of the apo - state at a position 22 Å away from the middle slice shown in Fig . 1C . The intra - SR extension is boxed on the density in red . Representative SR vesicles with different distributions of inner - SR density : segregated , evenly distributed , empty , and empty with filaments . RyR1 particles are circled in red , areas circled in yellow indicate accumulation of density inside the SR . The fractions of vesicles that fall into each density category for each RyR1 sample ( EDTA : n = 64 ; 1 mM Ca2 + : n = 32 ; 5 mM Ca2 + : n = 21 , technical replicates ) . Blue corresponds to the fraction of SR lumen vesicles showing evenly distributed density , orange to segregated density , and grey to empty vesicles . Structures of RyR1 determined in presence of increasing Ca2 + concentration ( 1 mM and 5 mM ) , all with intra - SR extensions . Scale bars : 10 nm in ( A ) and ( E ) , 20 nm in ( C ) .

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Figure 2A slice through the average structure of the apo-state at a position 22 Å away from the middle slice shown in Fig. 1C. The intra-SR extension is boxed on the density in red.Representative SR vesicles with different distributions of inner-SR density: segregated, evenly distributed, empty, and empty with filaments. RyR1 particles are circled in red, areas circled in yellow indicate accumulation of density inside the SR.The fractions of vesicles that fall into each density category for each RyR1 sample (EDTA: n=64; 1 mM Ca2+: n = 32; 5 mM Ca2+: n=21, technical replicates). Blue corresponds to the fraction of SR lumen vesicles showing evenly distributed density, orange to segregated density, and grey to empty vesicles.Structures of RyR1 determined in presence of increasing Ca2+ concentration (1 mM and 5 mM), all with intra-SR extensions. Scale bars: 10 nm in (A) and (E), 20 nm in (C).


Figure 3B is domain representation on the grayscale image . Please note the location of two transmembrane densities : yellow - shown in Figure 1C , magenta - shown in Figure 3B . Slices through the results of classification of the particles in the global average into 4 classes at resolutions 15 . 4 , 18 , 18 , and 27 Å , respectively , with the percentages of the total particles in each class shown beneath . Red arrows show domain movements compared to class 1 . The first five modes of correlated movements . Direction of motion is indicated by the arrows . Informativity of the respective modes measured as variance of the corresponding mode divided by variance of the first mode . Scale bars : 10 nm .

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Figure 3B is domain representation on the grayscale image. Please note the location of two transmembrane densities: yellow - shown in Figure 1C, magenta - shown in Figure 3B.Slices through the results of classification of the particles in the global average into 4 classes at resolutions 15.4, 18, 18, and 27 Å, respectively, with the percentages of the total particles in each class shown beneath. Red arrows show domain movements compared to class 1.The first five modes of correlated movements. Direction of motion is indicated by the arrows. Informativity of the respective modes measured as variance of the corresponding mode divided by variance of the first mode. Scale bars: 10 nm.


Figure 4Structure of ryRyR1 at a resolution of 17 . 5 Å . Two views are through the middle slice ( top ) of the reconstruction and at the level of sarcoplasmic extentions 22 Å below ( bottom panels ) . The movement of the outer domains is associated with a visible change in membrane curvature as compared to the apo - structure . This movement is highlighted by red arrows in the right panels . Scale bar : 10 nm .

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Figure 4Structure of ryRyR1 at a resolution of 17.5 Å. Two views are through the middle slice (top) of the reconstruction and at the level of sarcoplasmic extentions 22 Å below (bottom panels). The movement of the outer domains is associated with a visible change in membrane curvature as compared to the apo-structure. This movement is highlighted by red arrows in the right panels. Scale bar: 10 nm.


Figure 2A . Total lipids were extracted from PfCDPK7 - KO or 3D7 parasites and separated by HPTLC . After quantification by GC - MS content relative to total fatty acids was determined . Relative abundance of individual phospholipids was quantified . Disruption of PfCDPK7 caused a significant decrease in PC ( mean ± SEM , n = 3 ; biological replicates , * * * p < 0 . 001 , t - test ) . E . 3D7 or PfCDPK7 - KO parasites were cultured in the presence or absence of 200 μM choline in culture medium . After 3 days , parasitemia was determined by counting parasites on thin blood smears ( mean ± SEM , n = 6 ; biological replicates , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA ) .

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Figure 2A. Total lipids were extracted from PfCDPK7-KO or 3D7 parasites and separated by HPTLC. After quantification by GC-MS content relative to total fatty acids was determined. Relative abundance of individual phospholipids was quantified. Disruption of PfCDPK7 caused a significant decrease in PC (mean± SEM, n=3; biological replicates, ***p<0.001, t-test).E. 3D7 or PfCDPK7-KO parasites were cultured in the presence or absence of 200 μM choline in culture medium. After 3 days, parasitemia was determined by counting parasites on thin blood smears (mean± SEM, n=6; biological replicates, *p< 0.05, ***p<0.001, ANOVA).


Figure 3A - B . 3D7 ( A ) or parasites overexpressing EK - GFP ( B ) were used to prepare protein lysates followed by immunoprecipitation using anti - PfCDPK7 or anti - GFP antibody , respectively . Subsequently , protein lysate or indicated IP were subjected to Western blotting using anti - PMT ( A ) , anti - GFP ( B ) . PMT was found to co - IP with PfCDPK7 ( A , lane 2 ) and EK - GFP with PfCDPK7 ( B , lane 3 ) . C . 3D7 and PfCDPK7 - KO parasites at schizont stage ( ~ 40 h ) were used for Western blotting using anti - PMT antisera . Actin served as a loading control . Right panel : Densitometry was performed for bands corresponding to PMT in Western blots and the measurements were normalized to Actin . Fold change in PMT was determined with respect to 3D7 ( mean ± SEM , n = 4 ; biological replicates , * * p < 0 . 01 , t - test ) .

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Figure 3A-B. 3D7 (A) or parasites overexpressing EK-GFP (B) were used to prepare protein lysates followed by immunoprecipitation using anti-PfCDPK7 or anti-GFP antibody, respectively. Subsequently, protein lysate or indicated IP were subjected to Western blotting using anti-PMT (A), anti-GFP (B). PMT was found to co-IP with PfCDPK7 (A, lane 2) and EK-GFP with PfCDPK7 (B, lane 3).C. 3D7 and PfCDPK7-KO parasites at schizont stage (~40 h) were used for Western blotting using anti-PMT antisera. Actin served as a loading control. Right panel: Densitometry was performed for bands corresponding to PMT in Western blots and the measurements were normalized to Actin. Fold change in PMT was determined with respect to 3D7 (mean± SEM, n=4; biological replicates, **p<0.01, t-test).


Figure 4A . Parasites overexpressing EK - GFP or its S37A mutant were synchronized and parasitemia was determined at the end of first ( 48 h . p . i ) and second cycle ( 96 h . p . i ) . Fold change in parasitemia of EK _ S37A parasites with respect to WT EK overexpressing parasites was compared , which was significantly reduced ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , ANOVA ) . C . IFA was performed on parasites expressing WT EK - GFP or its S37A mutant 6 - 12h post - invasion to detect ( i ) RAP1 , which is transferred to PV post invasion , and ( ii ) MSP1 which is located at the PVM . Parasite expressing WT EK - GFP exhibited typical round ring like shape . In contrast , several parasites expressing the S37A mutant exhibited irregular morphology with much smaller vacuolar space and in some parasites RAP1 was found outside the PV ( Scale bar - 1 μm ) . E . Parasites EK - GFP or its S37A mutant were synchronized and parasites were allowed to mature to schizonts / segmentors . The graph shows average number of merozoites per schizont from three biological replicates . Each replicate represents an average of merozoites from 30 - 40 schizonts . ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , t - test ) .

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Figure 4A. Parasites overexpressing EK-GFP or its S37A mutant were synchronized and parasitemia was determined at the end of first (48 h.p.i) and second cycle (96 h.p.i). Fold change in parasitemia of EK_S37A parasites with respect to WT EK overexpressing parasites was compared, which was significantly reduced (mean± SEM, n=3; biological replicates, *p<0.05, ANOVA).C. IFA was performed on parasites expressing WT EK-GFP or its S37A mutant 6-12h post-invasion to detect (i) RAP1, which is transferred to PV post invasion, and (ii) MSP1 which is located at the PVM. Parasite expressing WT EK-GFP exhibited typical round ring like shape. In contrast, several parasites expressing the S37A mutant exhibited irregular morphology with much smaller vacuolar space and in some parasites RAP1 was found outside the PV (Scale bar-1 μm).E. Parasites EK-GFP or its S37A mutant were synchronized and parasites were allowed to mature to schizonts/segmentors. The graph shows average number of merozoites per schizont from three biological replicates. Each replicate represents an average of merozoites from 30-40 schizonts. (mean± SEM, n=3; biological replicates, *p<0.05, t-test).


Figure 5A . 3D7 parasites were treated with DMSO or 50nM MMV048 for 24h . Subsequently , parasites were fixed and IFA was performed to detect ER marker BiP and PfCDPK7 . While PfCDPK7 was present in puncta proximal to ER ( left panel ) , inhibitor treatment caused a loss in this vesicular localization and resulted in a more diffuse distribution of the enzyme ( right panel ) ( Scale bar - 1 μm ) . C . 3D7 parasites were treated with DMSO or 50nM MMV048 for 20 - 24h . Subsequently , IFA was performed ~ 24h post - invasion to detect RAP1 and MSP1 ( left panel ) Right panel , Quantitation of parasites exhibiting abnormal staining for RAP1 outside the PV was observed ( mean ± SEM , n = 3 ; biological replicates , * * p < 0 . 01 , t - test ; Scale bar - 1 μm ) .

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Figure 5A. 3D7 parasites were treated with DMSO or 50nM MMV048 for 24h. Subsequently, parasites were fixed and IFA was performed to detect ER marker BiP and PfCDPK7. While PfCDPK7 was present in puncta proximal to ER (left panel), inhibitor treatment caused a loss in this vesicular localization and resulted in a more diffuse distribution of the enzyme (right panel) (Scale bar-1 μm).C. 3D7 parasites were treated with DMSO or 50nM MMV048 for 20-24h. Subsequently, IFA was performed ~24h post-invasion to detect RAP1 and MSP1 (left panel) Right panel, Quantitation of parasites exhibiting abnormal staining for RAP1 outside the PV was observed (mean± SEM, n=3; biological replicates, **p<0.01, t-test; Scale bar-1 μm).


Figure 6A - B . 3D7 parasites were synchronized and treated with 10nM UCT943 or 50nM MMV048 for 10 - 12h . Subsequently , parasites were incubated with 14C - Cho ( A ) or 14C - Eth ( B ) and were harvested after 12h . Total lipids were extracted and separated by TLC . The radiolabeled lipids were detected by phosphorimaging and quantitated by densitometry ( upper panel ) . Bottom Panel ( A and B ) , fold change in PC and PE formation in inhibitor treated parasites with respect to DMSO treatment was determined ( mean ± SEM , n = 4 ; biological replicates , * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ; ns , not significant ) . C - D . UCT943 and MMV048 treated 3D7 ( C ) or EK - GFP overexpressing parasites ( D ) were subjected to Western blotting using anti - PMT ( C ) and anti - GFP ( D ) antibodies ( left panels ) . Right Panels : densitometry was performed for bands corresponding to PMT ( C ) or EK - GFP ( D ) , which were normalized with respect to Actin . Fold change in PMT or EK was determined with respect to DMSO treated parasites ( Mean ± SEM , n = 4 ; biological replicates , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA ) .

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Figure 6A-B. 3D7 parasites were synchronized and treated with 10nM UCT943 or 50nM MMV048 for 10-12h. Subsequently, parasites were incubated with 14C-Cho (A) or 14C-Eth (B) and were harvested after 12h. Total lipids were extracted and separated by TLC. The radiolabeled lipids were detected by phosphorimaging and quantitated by densitometry (upper panel). Bottom Panel (A and B), fold change in PC and PE formation in inhibitor treated parasites with respect to DMSO treatment was determined (mean±SEM, n=4; biological replicates, **p<0.01, ***p<0.001, ANOVA; ns, not significant).C-D. UCT943 and MMV048 treated 3D7 (C) or EK-GFP overexpressing parasites (D) were subjected to Western blotting using anti-PMT (C) and anti-GFP (D) antibodies (left panels). Right Panels: densitometry was performed for bands corresponding to PMT (C) or EK-GFP (D), which were normalized with respect to Actin. Fold change in PMT or EK was determined with respect to DMSO treated parasites (Mean± SEM, n=4; biological replicates, *p<0.05, ***p<0.001, ANOVA).


Figure 7B . Ring - infected erythrocytes were treated with MMV048 for 24h and then incubated with FM4 - 64 and DAPI to assess lipid uptake . Subsequently , FM4 - 64 associated fluorescence was detected by fluorescence microscopy ( upper panel ) or quantitated by FACS ( lower panel ) and % change in FM4 - 64 uptake in inhibitor - treated parasites was determined . ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , t - test ; Scale bar - 1 μm ) . D . Trophozoite stage infected erythrocytes were incubated with FM4 - 64 and DAPI to assess lipid uptake by 3D7 or PfCDPK7 - KO parasites . Subsequently , FM4 - 64 associated fluorescence was visualized by live microscopy ( top panel ) and also analyzed by FACS and % change in FM4 - 64 uptake in KO parasites was determined ( bottom panel ) ( mean ± SEM , n = 3 ; biological replicates , * p < 0 . 05 , t - test ; Scale bar - 1 μm ) .

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Figure 7B. Ring-infected erythrocytes were treated with MMV048 for 24h and then incubated with FM4-64 and DAPI to assess lipid uptake. Subsequently, FM4-64 associated fluorescence was detected by fluorescence microscopy (upper panel) or quantitated by FACS (lower panel) and % change in FM4-64 uptake in inhibitor-treated parasites was determined. (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm).D. Trophozoite stage infected erythrocytes were incubated with FM4-64 and DAPI to assess lipid uptake by 3D7 or PfCDPK7-KO parasites. Subsequently, FM4-64 associated fluorescence was visualized by live microscopy (top panel) and also analyzed by FACS and % change in FM4-64 uptake in KO parasites was determined (bottom panel) (mean± SEM, n=3; biological replicates, *p<0.05, t-test; Scale bar-1 μm).


Figure 1 ( B ) A fragment of genomic DNA ( 2 . 2 kb ) was amplified by PCR and in vitro sgRNA - guided Cas9 - mediated cleavage assay was performed with each of the sgRNAs . sgRNA - targeting sites are indicated by arrows , genomic DNA size is indicated by asterisk .

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Figure 1(B) A fragment of genomic DNA (2.2 kb) was amplified by PCR and in vitro sgRNA-guided Cas9-mediated cleavage assay was performed with each of the sgRNAs. sgRNA-targeting sites are indicated by arrows, genomic DNA size is indicated by asterisk.


Figure 4 ( C ) Immunohistochemistry of day 8 spleen sections for host mouse receiving 5x105 isolated CD45 . 2 B cells from CLK09 ( left ) and CLK19 ( right ) KI mouse . Green , B220 ; Blue , CD3 ; White , CD45 . 2 ; Red , GL7 . White arrows indicate the CD45 . 2 B cells .

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Figure 4(C) Immunohistochemistry of day 8 spleen sections for host mouse receiving 5x105 isolated CD45.2 B cells from CLK09 (left) and CLK19 (right) KI mouse. Green, B220; Blue, CD3; White, CD45.2; Red, GL7.White arrows indicate the CD45.2 B cells.


Figure 6 ( A ) CD45 . 2 frequency among GCs induced by eOD - GT8 60 mer at different time points . Adoptively transferred mice with 1 in 104 of precursor frequency were immunized with eOD - GT8 60mer . On days 8 , 15 and 36 , the splenic B cells were isolated to measure the CD45 . 2 cell frequency in GC B cells by FACS using the markers mentioned above by FACS . Gated plots represent the CD45 . 2 frequency among GC B cells . ( B ) Quantification of CD45 . 2 frequency among GCs at three time points . X - axis represents adoptively transferred mice groups with different precursor frequency ( 1 in 104 , 2 in 105 , 3 in 106 ) at three time point , y axis represents CD45 . 2 frequency among GC . Each circle represents one mouse . For CLK21 and CLK09 panel , n = 5 mice in each group . For CLK19 panel , n = 3 mice in each group . Bars indicate mean ± SD from mice in each group . ( C ) IgG titers detected by ELISA . eOD - GT8 or eOD - GT8 KO soluble proteins were used to coat ELISA plates . Sera from adoptively transferred mice at different time points were used to detect by ELISA . Bars indicate geometric mean and geometric SD from mice in each group . For CLK21 and CLK09 panel , n = 5 mice in each group . For CLK19 panel , n = 3 mice in each group . X - axis represents precursor frequency , and Y - axis represents the change of area under curve ( AUC coated eOD - GT8 - AUC coated eOD - GT8 KO ) . Each circle represents one mouse .

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Figure 6(A) CD45.2 frequency among GCs induced by eOD-GT8 60 mer at different time points. Adoptively transferred mice with 1 in 104 of precursor frequency were immunized with eOD-GT8 60mer. On days 8, 15 and 36, the splenic B cells were isolated to measure the CD45.2 cell frequency in GC B cells by FACS using the markers mentioned above by FACS. Gated plots represent the CD45.2 frequency among GC B cells. (B) Quantification of CD45.2 frequency among GCs at three time points. X-axis represents adoptively transferred mice groups with different precursor frequency (1 in 104, 2 in 105, 3 in 106) at three time point, y axis represents CD45.2 frequency among GC. Each circle represents one mouse. For CLK21 and CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. Bars indicate mean ± SD from mice in each group.(C) IgG titers detected by ELISA. eOD-GT8 or eOD-GT8 KO soluble proteins were used to coat ELISA plates. Sera from adoptively transferred mice at different time points were used to detect by ELISA. Bars indicate geometric mean and geometric SD from mice in each group. For CLK21 and CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. X-axis represents precursor frequency, and Y-axis represents the change of area under curve (AUC coated eOD-GT8 -AUC coated eOD-GT8 KO). Each circle represents one mouse.


Figure 1Phenotypes of 4 - d - old Col , two cbf1 mutants ( cbf1 - 1 and cbf1 - 2 ) and two CBF1 - OE lines ( CBF1 - myc and CBF1 - flag ) grown at 22 ° C in darkness ( D ) or continuous W , R , FR , and B light . Scale bar = 1 mm . Hypocotyl lengths of Col , two cbf1 mutants and two CBF1 - OE lines grown at 22 ° C in D or continuous W , R , FR , and B light . Error bars represent SD from 20 seedlings . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( two - tailed t - test ) for the indicated genotype compared with Col . NS , not significant .

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

Figure 1Phenotypes of 4-d-old Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown at 22°C in darkness (D) or continuous W, R, FR, and B light. Scale bar = 1 mm.   Hypocotyl lengths of Col, two cbf1 mutants and two CBF1-OE lines grown at 22°C in D or continuous W, R, FR, and B light. Error bars represent SD from 20 seedlings. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col. NS, not significant.


Figure 2qRT - PCR analysis showing the relative expression of CBF1 in 4 - d - old wild - type ( Col ) seedlings grown at 22 ° C in darkness ( D ) or continuous W , R , FR , and B light . Error bars represent SD of three technical replicates . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . GUS staining of 4 - d - old homozygous CBF1p : GUS transgenic seedlings grown at 22 ° C in D or continuous W , R , FR , and B light . Scale bar = 1 mm . qRT - PCR data showing that CBF1 expression is induced by different light regimes . Wild - type ( Col ) seedlings were first grown at 22 ° C in D for 4 d , and then transferred to continuous W , FR , R , or B light for the indicated times ranging from 1 h to 24 h . Error bars represent SD of three technical replicates . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . Immunoblots showing CBF1 protein levels in nuclear fractions from 4 - d - old wild - type ( Col ) seedlings grown at 22 ° C in D or continuous W , R , FR , and B light . Anti - H3 was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of CBF1 normalized to those of loading control , respectively . The ratio of the first clear band was set to 100 for each blot . Immunoblots showing CBF1 - myc protein levels in 4 - d - old CBF1 - myc seedlings grown at 22 ° C in D or continuous W , R , FR , and B light . anti - HSP was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of CBF1 - myc normalized to those of loading control , respectively . The ratio of the first clear band was set to 100 for each blot . F , G Immunoblots showing the levels of CBF1 - myc proteins after exposure to white light . CBF1 - myc seedlings were first grown at 22 ° C for 4 d in D , and then transferred to continuous W light for the indicated time periods within 1 h ( F ) or ranging from 1 h to 24 h ( G ) . anti - HSP was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1 - myc normalized to those of loading control , respectively . The ratio of the first clear band was set to 100 for each blot . Immunoblots showing the levels of CBF1 - myc proteins after exposure to FR ( H ) , R ( I ) or B ( J ) light . CBF1 - myc seedlings were first grown at 22 ° C for 4 d in D , and then transferred to continuous FR , R or B light for the indicated time periods ranging from 1 h to 24 h . anti - HSP was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1 - myc normalized to those of loading control , respectively . The ratio of the first clear band was set to 100 for each blot .

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 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 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 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 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

Figure 2qRT-PCR analysis showing the relative expression of CBF1 in 4-d-old wild-type (Col) seedlings grown at 22°C in darkness (D) or continuous W, R, FR, and B light. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).GUS staining of 4-d-old homozygous CBF1p:GUS transgenic seedlings grown at 22°C in D or continuous W, R, FR, and B light. Scale bar = 1 mm.qRT-PCR data showing that CBF1 expression is induced by different light regimes. Wild-type (Col) seedlings were first grown at 22°C in D for 4 d, and then transferred to continuous W, FR, R, or B light for the indicated times ranging from 1 h to 24 h. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).Immunoblots showing CBF1 protein levels in nuclear fractions from 4-d-old wild-type (Col) seedlings grown at 22°C in D or continuous W, R, FR, and B light. Anti-H3 was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.Immunoblots showing CBF1-myc protein levels in 4-d-old CBF1-myc seedlings grown at 22°C in D or continuous W, R, FR, and B light. anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.F, G Immunoblots showing the levels of CBF1-myc proteins after exposure to white light. CBF1-myc seedlings were first grown at 22°C for 4 d in D, and then transferred to continuous W light for the indicated time periods within 1 h (F) or ranging from 1 h to 24 h (G). anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.Immunoblots showing the levels of CBF1-myc proteins after exposure to FR (H), R (I) or B (J) light. CBF1-myc seedlings were first grown at 22°C for 4 d in D, and then transferred to continuous FR, R or B light for the indicated time periods ranging from 1 h to 24 h. anti-HSP was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of CBF1 or CBF1-myc normalized to those of loading control, respectively. The ratio of the first clear band was set to 100 for each blot.


Figure 3In vitro pull - down of PHYA - C1 ( A ) and PHYB - C1 ( B ) with CBF1 . The His - tagged PHYA / B - N ( photosensory domain ) , PHYA / B - C1 ( PAS - related domain ) , and PHYA / B - C2 ( histidine kinase - related domain ) proteins pulled down with GST - tagged CBF1 or GST were detected by anti - His antibody . Input , 6 % of the His - tagged purified target proteins used in pull - down assays . LCI assays showing that CBF1 interacted with phyA in living plant cells . Scale bar = 1 cm . D , E Co - IP assays showing that CBF1 interacted with phyA ( D ) and phyB ( E ) in vivo . ( D ) homozygous CBF1 - myc seedlings were first grown at 22 ° C in continuous FR light for 4 d , then the total proteins were extracted and subsequently treated with 5 - min R light , or 5 - min R light followed by 5 - min FR light ( R + FR ) before immunoprecipitation . ( E ) homozygous CBF1 - myc seedlings were first grown at 22 ° C in continuous R light for 4 d , then the total proteins were extracted and subsequently treated with 5 - min FR light , or 5 - min FR light followed by 5 - min R light ( FR + R ) before immunoprecipitation . After light treatments , total proteins were incubated with anti - c - myc Affinity Gel ( Sigma - Aldrich ) . The total and precipitated proteins were examined by immunoblotting using antibodies against myc , phyA , phyB , and RPN6 , respectively . The numbers below anti - phyA and anti - phyB blots indicate the relative band intensities of co - precipitated phyA or phyB normalized to those of precipitated CBF1 - myc , respectively . The ratio of the first clear band was set to 100 for each blot .

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 B-PANEL_START 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 O O O O O O O O O O O O O O O O O O O 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 3In vitro pull-down of PHYA-C1 (A) and PHYB-C1 (B) with CBF1. The His-tagged PHYA/B-N (photosensory domain), PHYA/B-C1 (PAS-related domain), and PHYA/B-C2 (histidine kinase-related domain) proteins pulled down with GST-tagged CBF1 or GST were detected by anti-His antibody. Input, 6% of the His-tagged purified target proteins used in pull-down assays.LCI assays showing that CBF1 interacted with phyA in living plant cells. Scale bar = 1 cm.D, E Co-IP assays showing that CBF1 interacted with phyA (D) and phyB (E) in vivo. (D) homozygous CBF1-myc seedlings were first grown at 22°C in continuous FR light for 4 d, then the total proteins were extracted and subsequently treated with 5-min R light, or 5-min R light followed by 5-min FR light (R+FR) before immunoprecipitation. (E) homozygous CBF1-myc seedlings were first grown at 22°C in continuous R light for 4 d, then the total proteins were extracted and subsequently treated with 5-min FR light, or 5-min FR light followed by 5-min R light (FR+R) before immunoprecipitation. After light treatments, total proteins were incubated with anti-c-myc Affinity Gel (Sigma-Aldrich). The total and precipitated proteins were examined by immunoblotting using antibodies against myc, phyA, phyB, and RPN6, respectively. The numbers below anti-phyA and anti-phyB blots indicate the relative band intensities of co-precipitated phyA or phyB normalized to those of precipitated CBF1-myc, respectively. The ratio of the first clear band was set to 100 for each blot.


Figure 4Immunoblots showing the levels of PIF4 and PIF5 proteins at 22 ° C in 4 - d - old Col , cbf1 - 1 , cbf1 - 2 , and CBF1 - myc seedlings grown in darkness ( D ) or continuous W or R light . Anti - RPN6 was used as a sample loading control . numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of RPN6 , respectively . Phenotypes and hypocotyl lengths of 4 - d - old Col , cbf1 - 1 , pif4 pif5 , cbf1 pif4 pif5 , 35S : PIF4 and 35S : PIF4 cbf1 seedlings grown at 22 ° C in continuous W or R light . Error bars represent SD from 20 seedlings . * * P < 0 . 01 and * * * P < 0 . 001 ( two - tailed t - test ) for the indicated pairs of seedlings . Scale bar = 1 mm . NS , not significant . Immunoblots showing the PIF4 protein levels in 4 - d - old Col , 35S : PIF4 , and 35S : PIF4 cbf1 seedlings grown at 22 ° C in D or continuous W or R light . Anti - RPN6 was used as a sample loading control . numbers below the immunoblots indicate the relative band intensities of PIF4 normalized to those of RPN6 , respectively .

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 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 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

Figure 4Immunoblots showing the levels of PIF4 and PIF5 proteins at 22°C in 4-d-old Col, cbf1-1, cbf1-2, and CBF1-myc seedlings grown in darkness (D) or continuous W or R light. Anti-RPN6 was used as a sample loading control. numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of RPN6, respectively.Phenotypes and hypocotyl lengths of 4-d-old Col, cbf1-1, pif4 pif5, cbf1 pif4 pif5, 35S:PIF4 and 35S:PIF4 cbf1 seedlings grown at 22°C in continuous W or R light. Error bars represent SD from 20 seedlings. **P < 0.01 and ***P < 0.001 (two-tailed t-test) for the indicated pairs of seedlings. Scale bar = 1 mm. NS, not significant.Immunoblots showing the PIF4 protein levels in 4-d-old Col, 35S:PIF4, and 35S:PIF4 cbf1 seedlings grown at 22°C in D or continuous W or R light. Anti-RPN6 was used as a sample loading control. numbers below the immunoblots indicate the relative band intensities of PIF4 normalized to those of RPN6, respectively.


Figure 5Yeast three - hybrid assays showing that CBF1 represses the interaction of the Pfr - phyB with PIF4 in yeast cells . AD - PIF4 , phyB - BD and CBF1 were expressed in the yeast strain Y190 as indicated . The β - galactosidase activities were measured by liquid culture assays using ONPG as the substrate . Error bars represent SD of three independent yeast clones . Yeast three - hybrid assays showing that CBF1 represses the interaction of the Pfr - phyB with PIF5 ( B ) in yeast cells . AD - PIF4 , phyB - BD and CBF1 were expressed in the yeast strain Y190 as indicated . The β - galactosidase activities were measured by liquid culture assays using ONPG as the substrate . Error bars represent SD of three independent yeast clones . LCI assays showing that CBF1 inhibits phyB interaction with PIF4 ( C ) in plant cells . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . LCI assays showing that CBF1 inhibits phyB interaction with PIF5 ( D ) in plant cells . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . Semi - in vivo pull - down assays showing that CBF1 inhibits phyB interaction with PIF4 ( E ) The total proteins extracted from 4 - d - old 35S : phyB - GFP seedlings grown at 22 ° C in continuous R light served as the bait , and GST - PIF4 fusion proteins served as the prey . Equivalent amounts of phyB - GFP protein extract and GST - PIF4 fusion proteins , and increasing amounts of His - CBF1 were added as indicated before anti - GFP IP assays were performed . The pulled - down proteins were subjected to immunoblotting with antibodies against phyB , GST and His , respectively . Numbers below the anti - GST blots indicate the relative band intensities of co - precipitated GST - PIF4 normalized to those of precipitated phyB - GFP , respectively . The ratio of the first clear band was set to 100 for each blot . Semi - in vivo pull - down assays showing that CBF1 inhibits phyB interaction with PIF5 ( F ) . The total proteins extracted from 4 - d - old 35S : phyB - GFP seedlings grown at 22 ° C in continuous R light served as the bait , and GST - PIF5 fusion proteins served as the prey . Equivalent amounts of phyB - GFP protein extract and GST - PIF5 fusion proteins , and increasing amounts of His - CBF1 were added as indicated before anti - GFP IP assays were performed . The pulled - down proteins were subjected to immunoblotting with antibodies against phyB , GST and His , respectively . Numbers below the anti - GST blots indicate the relative band intensities of co - precipitated GST - PIF5 normalized to those of precipitated phyB - GFP , respectively . The ratio of the first clear band was set to 100 for each blot .

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 O O 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 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 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

Figure 5Yeast three-hybrid assays showing that CBF1 represses the interaction of the Pfr-phyB with PIF4 in yeast cells. AD-PIF4, phyB-BD and CBF1 were expressed in the yeast strain Y190 as indicated. The β-galactosidase activities were measured by liquid culture assays using ONPG as the substrate. Error bars represent SD of three independent yeast clones.Yeast three-hybrid assays showing that CBF1 represses the interaction of the Pfr-phyB with PIF5 (B) in yeast cells. AD-PIF4, phyB-BD and CBF1 were expressed in the yeast strain Y190 as indicated. The β-galactosidase activities were measured by liquid culture assays using ONPG as the substrate. Error bars represent SD of three independent yeast clones.LCI assays showing that CBF1 inhibits phyB interaction with PIF4 (C) in plant cells. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).LCI assays showing that CBF1 inhibits phyB interaction with PIF5 (D) in plant cells. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).Semi-in vivo pull-down assays showing that CBF1 inhibits phyB interaction with PIF4 (E) The total proteins extracted from 4-d-old 35S:phyB-GFP seedlings grown at 22°C in continuous R light served as the bait, and GST-PIF4 fusion proteins served as the prey. Equivalent amounts of phyB-GFP protein extract and GST-PIF4 fusion proteins, and increasing amounts of His-CBF1 were added as indicated before anti-GFP IP assays were performed. The pulled-down proteins were subjected to immunoblotting with antibodies against phyB, GST and His, respectively. Numbers below the anti-GST blots indicate the relative band intensities of co-precipitated GST-PIF4 normalized to those of precipitated phyB-GFP, respectively. The ratio of the first clear band was set to 100 for each blot.Semi-in vivo pull-down assays showing that CBF1 inhibits phyB interaction with PIF5 (F). The total proteins extracted from 4-d-old 35S:phyB-GFP seedlings grown at 22°C in continuous R light served as the bait, and GST-PIF5 fusion proteins served as the prey. Equivalent amounts of phyB-GFP protein extract and GST-PIF5 fusion proteins, and increasing amounts of His-CBF1 were added as indicated before anti-GFP IP assays were performed. The pulled-down proteins were subjected to immunoblotting with antibodies against phyB, GST and His, respectively. Numbers below the anti-GST blots indicate the relative band intensities of co-precipitated GST-PIF5 normalized to those of precipitated phyB-GFP, respectively. The ratio of the first clear band was set to 100 for each blot.


Figure 6Phenotypes of Col , two cbf1 mutants ( cbf1 - 1 and cbf1 - 2 ) and two CBF1 - OE lines ( CBF1 - myc and CBF1 - flag ) grown under LD conditions at 22 ° C , 17 ° C and 4 ° C , respectively . The seeds of different genotypes were germinated under LD ( 16 - h - light / 8 - h - dark photoperiod ) conditions at 22 ° C for 2 d , respectively , and then grown under LD conditions at 22 ° C , 17 ° C , or 4 ° C , for additional 6 d . Scale bar = 1 mm . Hypocotyl lengths of Col , two cbf1 mutants ( cbf1 - 1 and cbf1 - 2 ) and two CBF1 - OE lines ( CBF1 - myc and CBF1 - flag ) grown under LD conditions at 22 ° C , 17 ° C or 4 ° C . Error bars represent SD from 10 seedlings . * * * P < 0 . 001 ( two - tailed t - test ) for the indicated genotype compared with Col . NS , not significant . The ratios of hypocotyl lengths at 17 ° C versus 22 ° C for the indicated seedlings grown under LD conditions . Error bars represent SD from 10 seedlings . * * * P < 0 . 001 ( two - tailed t - test ) for the indicated genotype compared with Col . qRT - PCR assays showing the CBF1 transcript levels in Col seedlings grown under LD conditions at 22 ° C , 17 ° C and 4 ° C . Col seeds were germinated under LD conditions at 22 ° C for 2 d , and then grown under LD conditions at 22 ° C , 17 ° C or 4 ° C for additional 6 d . The samples were collected at ZT8 , and then subjected to qRT - PCR assays . Error bars represent SD of three technical replicates . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc test ( P < 0 . 05 ) . E , F Immunoblots showing the protein levels of PIF4 ( E ) and PIF5 ( F ) in Col , cbf1 - 1 , cbf1 - 2 , and CBF1 - myc seedlings grown under LD conditions at 22 ° C , 17 ° C and 4 ° C . The seeds of different genotypes were germinated under LD conditions at 22 ° C for 2 d , and then grown under LD conditions at 22 ° C , 17 ° C or 4 ° C for additional 6 d . The samples were collected at ZT20 , and then subjected to immunoblotting . Anti - RPN6 was used as a sample loading control . Numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of loading control , respectively . The ratio of the first band was set to 100 for each blot .

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

Figure 6Phenotypes of Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown under LD conditions at 22°C, 17°C and 4°C, respectively. The seeds of different genotypes were germinated under LD (16-h-light/8-h-dark photoperiod) conditions at 22°C for 2 d, respectively, and then grown under LD conditions at 22°C, 17°C, or 4°C, for additional 6 d. Scale bar = 1 mm.Hypocotyl lengths of Col, two cbf1 mutants (cbf1-1 and cbf1-2) and two CBF1-OE lines (CBF1-myc and CBF1-flag) grown under LD conditions at 22°C, 17°C or 4°C. Error bars represent SD from 10 seedlings. ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col. NS, not significant.The ratios of hypocotyl lengths at 17°C versus 22°C for the indicated seedlings grown under LD conditions. Error bars represent SD from 10 seedlings. ***P < 0.001 (two-tailed t-test) for the indicated genotype compared with Col.qRT-PCR assays showing the CBF1 transcript levels in Col seedlings grown under LD conditions at 22°C, 17°C and 4°C. Col seeds were germinated under LD conditions at 22°C for 2 d, and then grown under LD conditions at 22°C, 17°C or 4°C for additional 6 d. The samples were collected at ZT8, and then subjected to qRT-PCR assays. Error bars represent SD of three technical replicates. Different letters represent significant differences by one-way ANOVA with Duncan's post hoc test (P < 0.05).E, F Immunoblots showing the protein levels of PIF4 (E) and PIF5 (F) in Col, cbf1-1, cbf1-2, and CBF1-myc seedlings grown under LD conditions at 22°C, 17°C and 4°C. The seeds of different genotypes were germinated under LD conditions at 22°C for 2 d, and then grown under LD conditions at 22°C, 17°C or 4°C for additional 6 d. The samples were collected at ZT20, and then subjected to immunoblotting. Anti-RPN6 was used as a sample loading control. Numbers below the immunoblots indicate the relative band intensities of PIF4 or PIF5 normalized to those of loading control, respectively. The ratio of the first band was set to 100 for each blot.


Figure 1A ) Left panel : experimental strategy . Hematopoietic stem cells ( HSPC ) and progenitors were characterized and isolated using specific sets of surface markers . All cells express CD34 . Upon differentiation , progenitors start to express CD38 in contrast to HSPC ( blue ) . Progenitors engaged in the myeloid ( magenta ) and lymphoid ( green ) pathways express respectively CD33 and CD19 markers . Right panel : FACS gating strategy to isolate HSPC ( further referred as CD38 - ( blue box ) , and cells engaged in myeloid and lymphoid differentiation pathways ( referred to as CD33 + [ magenta box ] and CD19 + [ green box ] , respectively ) . B ) For each population , equatorial Z stacks of two representative nuclei are shown in the left panel ( chromatin is shown in blue ( DAPI staining ) and nuclear envelop in white ( Sun2 staining ) . Scale bar : 5 µm . The corresponding 3D reconstructions are presented in the right panel . Convex and non - convex surfaces are shown in blue and yellow , respectively . C ) Nucleus - invagination ratio is defined as the ratio of the non - convex area over the total nucleus surface area . Nuclei are larger and deformed in CD33 + ( magenta , n = 106 ) than CD38 - and CD19 + cells ( blue [ n = 120 ] and green [ n = 115 ] , respectively ; 3 independent experiments ; Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 , Mann - Whitney test ; n . s : non significant ) .

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 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

Figure 1A) Left panel: experimental strategy. Hematopoietic stem cells (HSPC) and progenitors were characterized and isolated using specific sets of surface markers. All cells express CD34. Upon differentiation, progenitors start to express CD38 in contrast to HSPC (blue). Progenitors engaged in the myeloid (magenta) and lymphoid (green) pathways express respectively CD33 and CD19 markers. Right panel: FACS gating strategy to isolate HSPC (further referred as CD38- (blue box), and cells engaged in myeloid and lymphoid differentiation pathways (referred to as CD33+ [magenta box] and CD19+ [green box], respectively).B) For each population, equatorial Z stacks of two representative nuclei are shown in the left panel (chromatin is shown in blue (DAPI staining) and nuclear envelop in white (Sun2 staining). Scale bar: 5 µm. The corresponding 3D reconstructions are presented in the right panel. Convex and non-convex surfaces are shown in blue and yellow, respectively. C) Nucleus-invagination ratio is defined as the ratio of the non-convex area over the total nucleus surface area. Nuclei are larger and deformed in CD33+ (magenta, n=106) than CD38- and CD19+ cells (blue [n=120] and green [n=115], respectively; 3 independent experiments; Bars correspond to mean ± SD; ****: p<0.0001, Mann-Whitney test; n.s: non significant).


Figure 2A ) For each population ( CD38 - and CD33 + cells ) , Z stacks of two representative cells are shown in the left panel . Microtubules are shown in green , actin in magenta , centrosome in white and chromatin in blue . In the middle panel , inverted image the corresponding actin staining is shown . In the left panel , the corresponding 3D projection of microtubules is presented as inverted image . Scale bar 5 µm . B ) Microtubules are tightly associated with the nucleus . Z stacks of representative CD38 - and CD33 + cells are shown . Microtubules are shown in green , nucleus envelope ( Sun2 immuno - staining ) in magenta and chromatin in blue . Scale bar 5 µm . C ) As schematized , centrosome - to - nucleus center distance ( d ) over nucleus center - to - nucleus convex envelope ( R ) ratio is calculated to extract relative position of the centrosome to the center . This parameter is significantly lower in myeloid progenitors ( n = 129 ) than in HSPC ( n = 200 ; 3 independent experiments ; * * * * ; p < 0 . 0001 . Mann - Whitney test ) indicating that centrosome becomes internalized within an invaginated region upon differentiation . D ) p150 glued associates with the nuclear membrane of HSPC ( CD38 - ) and myeloid progenitors ( CD33 + ) cells . Z stacks of representative cells are shown . Image contrast has been inverted . Stainings show DAPI , Sun2 and p150 glued . Scale bar 5µm . E ) The dynein adaptor BicD2 associates with the nuclear membrane of HSPC ( CD38 - ) and myeloid progenitors ( CD33 + ) cells . Z stacks of representative cells are shown . Image contrast has been inverted . Stainings show DAPI and BicD2 . Scale bar 5µm .

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 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 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 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 2A) For each population (CD38- and CD33+ cells), Z stacks of two representative cells are shown in the left panel. Microtubules are shown in green, actin in magenta, centrosome in white and chromatin in blue. In the middle panel, inverted image the corresponding actin staining is shown. In the left panel, the corresponding 3D projection of microtubules is presented as inverted image. Scale bar 5 µm.B) Microtubules are tightly associated with the nucleus. Z stacks of representative CD38- and CD33+ cells are shown. Microtubules are shown in green, nucleus envelope (Sun2 immuno-staining) in magenta and chromatin in blue. Scale bar 5 µm.C) As schematized, centrosome-to-nucleus center distance (d) over nucleus center-to-nucleus convex envelope (R) ratio is calculated to extract relative position of the centrosome to the center. This parameter is significantly lower in myeloid progenitors (n=129) than in HSPC (n=200; 3 independent experiments; ****; p<0.0001. Mann-Whitney test) indicating that centrosome becomes internalized within an invaginated region upon differentiation.D) p150 glued associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI, Sun2 and p150 glued . Scale bar 5µm.E) The dynein adaptor BicD2 associates with the nuclear membrane of HSPC (CD38-) and myeloid progenitors (CD33+) cells. Z stacks of representative cells are shown. Image contrast has been inverted. Stainings show DAPI and BicD2. Scale bar 5µm.


Figure 3B ) The nucleus is progressively deformed over time in culture . For each time point , equatorial Z stacks of two representative cells are presented . Left panel : inverted image of chromatin ( DAPI ) . Right panel : microtubules are shown in green and chromatin in blue . Scale bar : 5 µm . C ) Nucleus volume ( left panel ) and invagination ratio ( right panel ) increase to attain levels similar to those of freshly isolated CD33 + cells ( 24 h , n = 165 , 4 donors ; 48 h , n = 143 , 3 donors ; and 72h , n = 192 , 4donors ; CD33 + , n = 227 , 5 donors ; Bars correspond to mean ± SD ; n . s : non significant ; * * * : p < 0 . 001 , * * * * : p < 0 . 0001 ; Mann Whitney test ) . D ) For each condition ( CTL , untreated cells ; + Bleb , blebbistatin - treated cells ; + Bleb + NZ , blebbistatin and nocodazole - treated cells ; + TX , taxol - treated cells ; and + CilioD , ciliobrevin - D - treated cells ) Z stacks of 2 representative cells are displayed . Microtubules are shown in green , chromatin in blue , centrosome in white and actin in magenta . Asterisks indicate regions where microtubules are not in contact with the nucleus . Scale bar 5 μm . F ) Quantifications of nuclear volume ( left panel ) , invagination ratio ( right panel ) in the indicated conditions . Non - treated cells at 48 hours ( n = 137 , 3 donors ) and 72 hours ( n = 143 , 3 donors ) ; and blebbistatin - treated cells ( n = 96 / 3 donors ) , blebbistatin and nocodazole treated cells ( n = 66 , 2 donors ) , taxol - treated cells ( n = 122 , 3 donors ) , and ciliobrevin - D - treated cells ( n = 128 , 3 donors ) . Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 . * * : p < 0 . 01 , n . s : non significant ; Mann - Whitney 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3B) The nucleus is progressively deformed over time in culture. For each time point, equatorial Z stacks of two representative cells are presented. Left panel: inverted image of chromatin (DAPI). Right panel: microtubules are shown in green and chromatin in blue. Scale bar: 5 µm. C) Nucleus volume (left panel) and invagination ratio (right panel) increase to attain levels similar to those of freshly isolated CD33+ cells (24 h, n=165, 4 donors; 48 h, n=143, 3 donors; and 72h, n=192, 4donors; CD33+, n=227, 5 donors; Bars correspond to mean ± SD; n.s: non significant; ***: p<0.001, ****: p<0.0001; Mann Whitney test). D) For each condition (CTL, untreated cells; +Bleb, blebbistatin-treated cells; +Bleb+NZ, blebbistatin and nocodazole-treated cells; +TX, taxol-treated cells; and +CilioD, ciliobrevin-D-treated cells) Z stacks of 2 representative cells are displayed. Microtubules are shown in green, chromatin in blue, centrosome in white and actin in magenta. Asterisks indicate regions where microtubules are not in contact with the nucleus. Scale bar 5 μm.F) Quantifications of nuclear volume (left panel), invagination ratio (right panel) in the indicated conditions. Non-treated cells at 48 hours (n=137, 3 donors) and 72 hours (n=143, 3 donors); and blebbistatin-treated cells (n= 96/3 donors), blebbistatin and nocodazole treated cells (n=66, 2 donors), taxol-treated cells (n=122, 3 donors), and ciliobrevin-D-treated cells (n=128, 3 donors). Bars correspond to mean ± SD; ****: p<0.0001. **: p<0.01, n.s: non significant; Mann-Whitney test.


Figure 4A ) Spatial distribution of lamin A / C in the nuclei of HSPC ( CD38 - ) , myeloid progenitors ( CD33 + ) and lymphoid progenitors ( CD19 + ) . Inverted images of equatorial Z plane of DAPI and lamin A / C are shown . Scale bar : 5 μm . B ) Line scans of lamin A / C signal along nucleus contour of representative cells are presented for each cell population . The corresponding variations in lamin A / C intensity and nucleus curvature are plotted in blue and in red , respectively . Negative curvature values correspond to local nuclear invaginations . Red asterisk underlines local nucleus invaginations . C ) Dispersion of A / C signal along the nucleus periphery is similarly low in CD38 - ( n = 17 , 3 donors ) , CD33 + ( n = 61 , 3 donors ) and CD19 + ( n = 11 , 3 donors ) indicative of an homogenous distribution of the protein along the nuclear membrane . ( Bars correspond to mean ± SD ; n . s : non significant ; Mann Whitney test ) . D ) Dispersion of lamin B signal along the nucleus periphery is similarly low in CD38 - ( n = 131 , 3 donors ) , and CD19 + ( n = 74 , 3 donors ) indicative of a homogenous distribution of the protein along the nuclear membrane . But is significantly increased in CD33 + ( n = 146 , 3 donors ) . Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 ; Mann Whitney test . E - F - G ) Spatial distribution of lamin B and local measurements of lamin B intensity and curvature of nuclear membrane in CD38 - ( E ) , CD33 + ( F ) and CD19 + ( G ) cells . Inverted images of the equatorial Z plane of DAPI ( left panel ) and lamin B ( right panel ) of a representative nucleus are shown for each population . Scale bar : 5 μm . The variations in lamin B signal intensity and curvature are plotted in blue in red , respectively . Asterisks indicate local invaginations of the nuclear membrane . In the bottom graphs , positive and negative curvatures are shown in grey and red , respectively . The absence of curvature ( curvature = 0 ) is highlighted as a dashed line . In CD33 + cells , negative curvature of the nuclear membrane are associated with low lamin B intensities .

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 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

Figure 4A) Spatial distribution of lamin A/C in the nuclei of HSPC (CD38-), myeloid progenitors (CD33+) and lymphoid progenitors (CD19+). Inverted images of equatorial Z plane of DAPI and lamin A/C are shown. Scale bar: 5 μm. B) Line scans of lamin A/C signal along nucleus contour of representative cells are presented for each cell population. The corresponding variations in lamin A/C intensity and nucleus curvature are plotted in blue and in red, respectively. Negative curvature values correspond to local nuclear invaginations. Red asterisk underlines local nucleus invaginations. C) Dispersion of A/C signal along the nucleus periphery is similarly low in CD38- (n=17, 3 donors), CD33+ (n=61, 3 donors) and CD19+ (n=11, 3 donors) indicative of an homogenous distribution of the protein along the nuclear membrane. (Bars correspond to mean ± SD; n.s: non significant; Mann Whitney test). D) Dispersion of lamin B signal along the nucleus periphery is similarly low in CD38- (n=131, 3 donors), and CD19+ (n=74, 3 donors) indicative of a homogenous distribution of the protein along the nuclear membrane. But is significantly increased in CD33+ (n=146, 3 donors). Bars correspond to mean ± SD; ****:p<0.0001; Mann Whitney test. E-F-G) Spatial distribution of lamin B and local measurements of lamin B intensity and curvature of nuclear membrane in CD38- (E), CD33+ (F) and CD19+ (G) cells. Inverted images of the equatorial Z plane of DAPI (left panel) and lamin B (right panel) of a representative nucleus are shown for each population. Scale bar: 5 μm. The variations in lamin B signal intensity and curvature are plotted in blue in red, respectively. Asterisks indicate local invaginations of the nuclear membrane. In the bottom graphs, positive and negative curvatures are shown in grey and red, respectively. The absence of curvature (curvature =0) is highlighted as a dashed line. In CD33+ cells, negative curvature of the nuclear membrane are associated with low lamin B intensities.