( A ) S2 expressing RDH / CH2064 - GFP were treated with synthesis inhibitor cycloheximide ( CHX ) for indicated time ( h : hours ) . level of RDH / CH2064 - GFP was determined by western blotting using anti - GFP antibody . Tubulin was used as loading control . ( B ) of relative RDH / CH2064 - GFP in ( A ) . Error bars represent ± .

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(A) S2 cells expressing RDH/CH2064-GFP were treated with the protein synthesis inhibitor cycloheximide (CHX) for the indicated time (h: hours). The level of RDH/CH2064-GFP was determined by western blotting using anti-GFP antibody. Tubulin was used as loading control. (B) Quantification of the relative RDH/CH2064-GFP protein levels in (A). Error bars represent ± SEM.


( C ) S2 expressing Bmm - GFP were treated with synthesis inhibitor cycloheximide ( CHX ) for indicated time ( h : hours ) . level of Bmm - GFP was determined by western blotting using anti - GFP antibody . Tubulin was used as loading control . ( D ) of relative Bmm - GFP in ( C ) . Error bars represent ± .

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(C) S2 cells expressing Bmm-GFP were treated with the protein synthesis inhibitor cycloheximide (CHX) for the indicated time (h: hours). The level of Bmm-GFP was determined by western blotting using anti-GFP antibody. Tubulin was used as loading control. (D) Quantification of the relative Bmm-GFP protein levels in (C). Error bars represent ± SEM.


( F ) Oil red of 3rd instar larval oenocytes in different genetic backgrounds under fed or starvation conditions .

(F) Oil red staining of 3rd instar larval oenocytes in different genetic backgrounds under fed or starvation conditions.


B , C , Representative of pPLT3 : : erCFP ( cyan ) expressing PI - stained ( red ) Arabidopsis roots in Col or wox5 background , respectively . D , Mean intensities of pPLT3 : : erCFP roots summarized in box scatter plots . mean of CFP signal in Col roots was to set to 100 % . D , Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . data was statistically analyzed by one - way ANOVA Holm - Sidak post - hoc multiple comparisons . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype results from two technical replicates . B , C , Scale bars represent 10 µm . PI = propidium iodide CFP = cyan .

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B, C, Representative images of pPLT3::erCFP (cyan) expressing and PI-stained (red) Arabidopsis roots in Col or wox5 background, respectively. D, Mean fluorescence intensities of the pPLT3::erCFP roots summarized in box and scatter plots. The mean fluorescence intensity of the CFP signal in Col roots was to set to 100 %. D, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. B, C, Scale bars represent 10 µm. PI = propidium iodide CFP = cyan fluorescent protein.


E , F , Representative of pPLT3 : : PLT3 - YFP ( yellow ) expressing FM4 - 64 - stained ( red ) Arabidopsis roots in Col or wox5 mutant background , respectively . g , Mean intensities of pPLT3 : : PLT3 - YFP expressing roots summarized in box scatter plots . mean of YFP signal in Col roots was to set to 100 % . G , Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . data was statistically analyzed by one - way ANOVA Holm - Sidak post - hoc multiple comparisons . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype results from two technical replicates . , E , F , Scale bars represent 10 µm . YFP = yellow

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E, F, Representative images of pPLT3::PLT3-YFP (yellow) expressing and FM4-64-stained (red) Arabidopsis roots in Col or wox5 mutant background, respectively. g, Mean fluorescence intensities of the pPLT3::PLT3-YFP expressing roots summarized in box and scatter plots. The mean fluorescence intensity of the YFP signal in Col roots was to set to 100%. G, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. , E, F, Scale bars represent 10 µm. YFP = yellow fluorescent protein


A - F , Representative FM4 - 64 - stained Arabidopsis roots ( grey ) expressing pWOX5 : : mVenus - NLS ( green ) in Col , plt2 , plt3 plt2 , plt3 double mutant background in longitudinal ( A - D ) , or transversal ( E - F ) optical sections . E ' , F ' , of representative in ( E ) ( F ) in Imaris to detect expressing nuclei . E ' ' , F ' ' , Overlay of 10 roots ( biological replicates ) showing of detected ( high in red , low in blue ) in Col plt2 , plt3 double mutant roots . G , Number of nuclei ( biological replicates ) expressing pWOX5 : : mVenus - NLS in Col plt2 , plt3 double mutant roots summarized in box scatter plots . H , of WOX5 expression in µm2 in Col plt2 , plt3 double mutant roots summarized in box scatter plots . G , H Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , □ = mean value , = minimum / maximum . ( G , H ) Kruskal - Wallis ANOVA with subsequent Dunns ( G ) or one - way ANOVA post - hoc Holm - Sidak multiple comparisons was used to for statistical significance ( H ) . Asterisks indicate statistically significant differences ( α = 0 . 01 ) . Number of analysed roots ( n ) ( biological replicates ) is indicated for each genotype results from three technical replicates per genotype . Scale bars represent 10 µm ; NLS = localisation signal .

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A-F, Representative FM4-64-stained Arabidopsis roots (grey) expressing pWOX5::mVenus-NLS (green) in Col, plt2, plt3 and plt2, plt3 double mutant background in longitudinal (A-D), or transversal (E-F) optical sections. E', F', Analysis of representative images in (E) and (F) in Imaris to detect and count individual expressing nuclei. E'', F'', Overlay of 10 roots (biological replicates) showing the area of detected fluorescence (high levels in red, low levels in blue) in Col and plt2, plt3 double mutant roots. G, Number of nuclei (biological replicates) expressing pWOX5::mVenus-NLS in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. H, Area of WOX5 expression in µm2 in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. G, H Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. (G, H) Kruskal-Wallis ANOVA analysis with subsequent Dunns test (G) or one-way ANOVA and post-hoc Holm-Sidak multiple comparisons test was used to test for statistical significance (H). Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from three technical replicates per genotype. Scale bars represent 10 µm; NLS = nuclear localisation signal.


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

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A, 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.


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

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The 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.


A - H , Lifetime Imaging ( FLIM ) results of transiently expressing N . benthamiana leaf epidermal . A - H , Representative FLIM of WOX5 - mVenus ( donor only ) plus indicated acceptors after a pixel - wise mono - exponential fit of mVenus signal . lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of two high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm .

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A-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 , lifetimes in ns are summarized in combined jitter box plots . dashed represents lifetime mean value of WOX5 - mV co - expressed with free mCh as negative control . one - way ANOVA Holm - Sidak post - hoc multiple comparisons was used to for statistical significance . Samples with identical letters do not show significant differences ( α = 0 . 01 ) . Number of nuclei analyzed ( n ) ( biological replicates ) is indicated results from 2 to 9 technical replicates . Box = 25 - 75 % of percentile , whisker = 1 . 5 interquartile range , − = median , ▪ = mean value .

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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 of WOX5 - mVenus ( donor only ) after a pixel - wise mono - exponential fit of mVenus signal . lifetime of WOX5 - mVenus in ns is color - coded . Low lifetimes ( blue ) due to FRET indicate strong interaction of two high lifetimes ( red ) indicate weaker or no interaction . Scale bars represent 5 µm .

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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 , nuclei showing bodies during co - expression of WOX5 - mV PLT3 - mCh were analysed for WOX5 - mV lifetime in bodies or nucleoplasm separately ( n = 7 ( biological replicates ) , from four technical replicates . mCh = mCherry . mV = mVenus .

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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.


( a ) was analysed after activation of pathways in presence absence of enforced Atg5 expression ( 18 h cultures ) . Values are means ± s . e . m . of three independent experiments .

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(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 ) regulated by Atg5 is apoptosis . data presented are from 5 h cultures . Caspase - 3 - like activity analysed in HeLa ( graph ) . of Jurkat is also shown . numbers in lower right corner indicate of , which was determined in parallel experiments using flow

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(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 cytometry


c ) Staurosporine induced redistribution of phosphatidylserine was enhanced in Atg5 overexpressing Jurkat blocked by z - VAD - fmk . Numbers indicate results of quantitative ( ) of each dot blot .

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c) 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 by silencing of Atg5 . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) . effects of Atg5 siRNA on Atg5 GAPDH were analysed by immunoblotting . asterisks indicate P 0 . 05 . n . s . , no significant difference .

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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.


a ) Spontaneous neutrophil apoptosis . Apoptosis was associated with appearance of a 24K anti - Atg5 - reactive . 33K Atg5 disappeared during apoptosis in a time - dependent manner . HeLa overexpressing Atg5 served as positive control .

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a) 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 . Apoptosis was associated with appearance of a 24K anti - Atg5 - reactive a reduction of 33K Atg5 in a time - dependent manner . 24K anti - Atg5 - reactive was also visible in Jurkat after treatment with actinomycin D ( Act . D ) or cycloheximide ( CHX ; 5 h treatment in both cases ) . GAPDH expression indicates equal loading in each panel . immmunoblots are representative of at least three independent experiments .

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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.


e ) Recombinant Atg5 was digested with calpain products sequenced by Edman degradation . Calpain Atg5 at Thr 193 . Comparison of Atg5 sequences of different species demonstrates presence of same calpain cleavage site in mice . alignment was perfromed with Clustal W using data obtained from GenBank . position of six - - acid deletion causing calpain resistance is indicated in red .

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e) 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 , sample was fractionated by preparative reversed - phase HPLC analysed by mass spectrometry . exact relative molecular masses for full - truncated Atg5 are indicated .

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(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 - - acid deletion surrounding cleavage site ( 35S - Atg5Δ191 - 196 ) led to a loss of calpain cleavage fragment .

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(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 Atg5 in spontaneous Fas receptor - mediated apoptosis of neutrophils . In contrast , Atg6 did not show evidence for cleavage its remained unchanged in this system . Results of 7 h cultures are shown .

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(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 in apoptotic HeLa ( 5 h staurosporine treatment ) lacking small subunit of calpain , which was silenced by using specific siRNA .

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(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 lacking endogeneous Atg5 ( due to gene silencing , see Fig . 1c ) to staurosporine - induced . Values are means ± s . e . m . of three independent experiments ( 18 h cultures ) .

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(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).


( a ) HeLa were transiently transfected with 24K 33K Atg5 for 24 h subsequently analysed by Hoechst . Values are means ± s . e . m . of three independent experiments . Bax transfection was used as a positive control . ( b ) Representative after Hoechst demonstrating fragmentation of HeLa that were transfected with 24K Atg5 using lentiviral gene transfer for 24 h . Quantitative of these experiments is shown in a . Control transfected were loaded with Mitotracker orange subsequently processed for cytochrome c immunostaining . Enforced expression of 24K Atg5 resulted in cytochrome c release into cytosol apoptosis ( condensation , shrinkage ; approximately 80 % of ) .

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(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 were transfected with 24K 33K Atg5 using lentiviral gene transfer . was observed as a consequence of enforced expression of truncated Atg5 within two days . Morphological ( Diff - Quik ) is also shown revealed that type of was apoptosis ( condensation , shrinkage ) .

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(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 that either lacked Bcl - 2 or expressed high of Bcl - 2 received truncated Atg5 or GFP by lentiviral gene transfer . In absence of Bcl - 2 , induction of apoptosis as assessed by fragmentation was observed due to enforced expression of truncated Atg5 within two days . Apoptosis was largely blocked in expressing high of Bcl - 2 . GFP transfection did not affect viability of in this system .

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(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 independent of Bcl - 2 expression . Blots were stripped reprobed with anti - GAPDH antibody .

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(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 of acidic vesicular organelles ( VAOs , red ) followed by flow cytometry in genetically modified HeLa . That 24K Atg5 did not induce autophagy was confirmed by transmission electron microscopy ( data not shown ) .

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(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 ) after 24K Atg5 gene transfer in HeLa exhibiting different autophagic activities . Values are means ± s . e . m . of three independent experiments ( lentiviral gene transfer for 24 h ) . asterisks indicate P 0 . 05 . n . s . , no significant difference . scale bars represent 10 μM .

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(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.


( a ) Confocal microscopy of HeLa were treated with anti - CD95 antibody for 5 h or left untreated . were stained with anti - Atg5 anti - VDAC antibodies . In untreated , there was only little evidence for colocalization of Atg5 VDAC . In anti - CD95 antibody treated some of Atg5 colocalized with VDAC . of colocalization were prepared using Imaris software . Numerical was performed on 10 in each group correlation coefficient calculated . error bars represent s . e . m .

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(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 both full - truncated Atg5 were present in cytosol . After 8 h incubation , some 24K Atg5 but not full - Atg5 was found in mitochondrial fraction . Mitochondrial association of truncated Atg5 coincided with release of cytochrome c into cytosol . same data were observed in staurosporine - induced apoptosis of Jurkat ( data not shown ) . C , cytosol ; M , mitochondria .

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(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 . Truncated full - recombinant Atg5 were added to isolated mitochondria incubated at 37 ° C for 1 h . Supernatants 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 - dependent manner , indicating mitochondrial permeabilization . Full - Atg5 had only little effect in this system .

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(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 were transfected with 24K 33K Atg5 using lentiviral gene transfer for 24 h analysed for caspase - 3 cleavage . GAPDH expression demonstrates equal loading .

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(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 were treated with staurosporine for 5 h or left untreated . Staurosporine induced Atg5 cleavage both full - truncated Atg5 were present in total lysates . Truncated but not full - Atg5 was detected in Bcl - xL immunoprecipitates obtained from apoptotic Jurkat . No evidence for physical interaction between Atg5 Bax was obtained . lower panels demonstrate that both Bax Bcl - xL were enriched following immunoprecipitation . asterisk indicates P 0 . 05 . scale bars represent 10 μM .

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(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.


A slice through a filtered tomogram showing SR vesicles in contact with a putative T - tubule . RyR1 molecules are indicated by red circles . dense coat on surface of SR vesicles correspond to SERCA pump . Areas circled in yellow indicate accumulation of density inside SR . Scale bar : 20 nm . A slice through structure of RyR1 in contact with putative TT - . A middle slice through in situ structure of RyR1 at 12 . 6 - Å resolution . observed additional transmembrane density is indicated by red arrows . Scale bars in B , C : 10 nm .

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A 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 structures of apoRyR1 with putative TT - ( left ) standalone apoRyR1 ( right , with flipped handendness reduced resolution for comparison ) 22 Å away from middle slice ( Y = 92 in volume coordinates ) . Scale bar : 10 nm .

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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.


location of CaM / S100A1 binding site is marked with red circles in slices through maps of in situ structure ( left ) single particle structure ( EMDB : 8393 ) in same orientation as in situ structure filtered to 15 Å ( right ) . Scale bar : 10 nm .

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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.


A slice through average structure of apo - state at a position 22 Å away from middle slice shown in Fig . 1C . intra - SR extension is boxed on density in red .

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A 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 , empty with filaments . RyR1 particles are circled in red , areas circled in yellow indicate accumulation of density inside SR .

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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.


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 fraction of SR lumen vesicles showing evenly distributed density , orange to segregated density , grey to empty vesicles .

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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 + ( 1 mM 5 mM ) , with intra - SR extensions . Scale bars : 10 nm in ( A ) ( E ) , 20 nm in ( C ) .

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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).


B is domain representation on grayscale image . Please note location of two transmembrane densities : yellow - shown in Figure 1C , magenta - shown in Figure 3B .

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B 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 results of classification of particles in global average into 4 classes at resolutions 15 . 4 , 18 , 18 , 27 Å , respectively , with percentages of total particles in each class shown beneath . Red arrows show domain movements compared to class 1 .

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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.


first five modes of correlated movements . Direction of motion is indicated by arrows . Informativity of respective modes measured as variance of corresponding mode divided by variance of first mode . Scale bars : 10 nm .

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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.


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

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Structure 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.


A . Total lipids were extracted from PfCDPK7 - KO or 3D7 separated by HPTLC . After by GC - MS content relative to total fatty was determined . Relative abundance of phospholipids was quantified . Disruption of PfCDPK7 caused a significant decrease in PC ( mean ± , n = 3 ; biological replicates , * * * p < 0 . 001 , t - ) .

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A. 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 were cultured in presence or absence of 200 μM choline in culture medium . After 3 days , parasitemia was determined by counting on thin blood smears ( mean ± , n = 6 ; biological replicates , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA ) .

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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).


A - B . 3D7 ( A ) or overexpressing EK - GFP ( B ) were used to prepare lysates followed by immunoprecipitation using anti - PfCDPK7 or anti - GFP antibody , respectively . Subsequently , 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 ) EK - GFP with PfCDPK7 ( B , lane 3 ) .

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A-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 PfCDPK7 - KO 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 measurements were normalized to Actin . Fold change in PMT was determined with respect to 3D7 ( mean ± , n = 4 ; biological replicates , * * p < 0 . 01 , t - ) .

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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).


A . overexpressing EK - GFP or its S37A mutant were synchronized parasitemia was determined at end of first ( 48 h . p . i ) second cycle ( 96 h . p . i ) . Fold change in parasitemia of EK _ S37A with respect to WT EK overexpressing was compared , which was significantly reduced ( mean ± , n = 3 ; biological replicates , * p < 0 . 05 , ANOVA ) .

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A. 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 expressing WT EK - GFP or its S37A mutant 6 - 12h post - invasion to detect ( i ) RAP1 , which is transferred to PV post invasion , ( ii ) MSP1 which is located at PVM . expressing WT EK - GFP exhibited typical round ring like shape . In contrast , several expressing S37A mutant exhibited irregular with much smaller vacuolar space in some RAP1 was found outside PV ( Scale bar - 1 μm ) .

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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 . EK - GFP or its S37A mutant were synchronized were allowed to mature to schizonts / segmentors . graph shows average number of merozoites per schizont from three biological replicates . Each replicate represents an average of merozoites from 30 - 40 schizonts . ( mean ± , n = 3 ; biological replicates , * p < 0 . 05 , t - ) .

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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).


A . 3D7 were treated with DMSO or 50nM MMV048 for 24h . Subsequently , were fixed IFA was performed to detect ER marker BiP PfCDPK7 . While PfCDPK7 was present in proximal to ER ( left panel ) , inhibitor treatment caused a loss in this vesicular localization resulted in a more diffuse distribution of enzyme ( right panel ) ( Scale bar - 1 μm ) .

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A. 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 were treated with DMSO or 50nM MMV048 for 20 - 24h . Subsequently , IFA was performed ~ 24h post - invasion to detect RAP1 MSP1 ( left panel ) Right panel , Quantitation of exhibiting abnormal for RAP1 outside PV was observed ( mean ± , n = 3 ; biological replicates , * * p < 0 . 01 , t - ; Scale bar - 1 μm ) .

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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).


A - B . 3D7 were synchronized treated with 10nM UCT943 or 50nM MMV048 for 10 - 12h . Subsequently , were incubated with 14C - Cho ( A ) or 14C - Eth ( B ) were harvested after 12h . Total lipids were extracted separated by TLC . radiolabeled lipids were detected by phosphorimaging quantitated by densitometry ( upper panel ) . Bottom Panel ( A B ) , fold change in PC PE formation in inhibitor treated with respect to DMSO treatment was determined ( mean ± , n = 4 ; biological replicates , * * p < 0 . 01 , * * * p < 0 . 001 , ANOVA ; ns , not significant ) .

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A-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 MMV048 treated 3D7 ( C ) or EK - GFP overexpressing ( D ) were subjected to Western blotting using anti - PMT ( C ) 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 ( Mean ± , n = 4 ; biological replicates , * p < 0 . 05 , * * * p < 0 . 001 , ANOVA ) .

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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).


B . Ring - infected erythrocytes were treated with MMV048 for 24h then incubated with FM4 - 64 DAPI to assess lipid uptake . Subsequently , FM4 - 64 associated was detected by microscopy ( upper panel ) or quantitated by FACS ( lower panel ) % change in FM4 - 64 uptake in inhibitor - treated was determined . ( mean ± , n = 3 ; biological replicates , * p < 0 . 05 , t - ; Scale bar - 1 μm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 DAPI to assess lipid uptake by 3D7 or PfCDPK7 - KO . Subsequently , FM4 - 64 associated was visualized by live microscopy ( top panel ) also analyzed by FACS % change in FM4 - 64 uptake in KO was determined ( bottom panel ) ( mean ± , n = 3 ; biological replicates , * p < 0 . 05 , t - ; Scale bar - 1 μm ) .

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

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).


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

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(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.


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

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

(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.


( 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 36 , splenic B were isolated to measure CD45 . 2 frequency in GC B by FACS using markers mentioned above by FACS . Gated plots represent CD45 . 2 frequency among GC B . ( B ) 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 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 .

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

(A) 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 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 geometric SD from mice in each group . For CLK21 CLK09 panel , n = 5 mice in each group . For CLK19 panel , n = 3 mice in each group . X - axis represents precursor frequency , Y - axis represents change of under curve ( AUC coated eOD - GT8 - AUC coated eOD - GT8 KO ) . Each circle represents one mouse .

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

(C) 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.


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

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

Phenotypes 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.


qRT - PCR showing relative expression of CBF1 in 4 - d - old wild - type ( Col ) seedlings grown at 22 ° C in darkness ( D ) or continuous W , R , FR , B . Error bars represent SD of three technical replicates . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc ( P < 0 . 05 ) .

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qRT-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 of 4 - d - old homozygous CBF1p : GUS transgenic seedlings grown at 22 ° C in D or continuous W , R , FR , B . Scale bar = 1 mm .

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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 regimes . Wild - type ( Col ) seedlings were first grown at 22 ° C in D for 4 d , then transferred to continuous W , FR , R , or B for 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 ( P < 0 . 05 ) .

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

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 in fractions from 4 - d - old wild - type ( Col ) seedlings grown at 22 ° C in D or continuous W , R , FR , B . Anti - H3 was used as a sample loading control . Numbers below immunoblots indicate relative band intensities of CBF1 normalized to those of loading control , respectively . of first clear band was set to 100 for each blot .

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

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 in 4 - d - old CBF1 - myc seedlings grown at 22 ° C in D or continuous W , R , FR , B . anti - HSP was used as a sample loading control . Numbers below immunoblots indicate relative band intensities of CBF1 - myc normalized to those of loading control , respectively . of first clear band was set to 100 for each blot .

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

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 of CBF1 - myc after exposure to . CBF1 - myc seedlings were first grown at 22 ° C for 4 d in D , then transferred to continuous W for 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 immunoblots indicate relative band intensities of CBF1 or CBF1 - myc normalized to those of loading control , respectively . of first clear band was set to 100 for each blot .

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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 of CBF1 - myc after exposure to FR ( H ) , R ( I ) or B ( J ) . CBF1 - myc seedlings were first grown at 22 ° C for 4 d in D , then transferred to continuous FR , R or B for indicated time periods ranging from 1 h to 24 h . anti - HSP was used as a sample loading control . Numbers below immunoblots indicate relative band intensities of CBF1 or CBF1 - myc normalized to those of loading control , respectively . of first clear band was set to 100 for each blot .

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

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.


In vitro pull - down of PHYA - C1 ( A ) PHYB - C1 ( B ) with CBF1 . His - tagged PHYA / B - N ( photosensory domain ) , PHYA / B - C1 ( PAS - related domain ) , PHYA / B - C2 ( histidine kinase - related domain ) pulled down with GST - tagged CBF1 or GST were detected by anti - His antibody . Input , 6 % of His - tagged purified target used in pull - down assays .

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In 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 . Scale bar = 1 cm .

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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 ) phyB ( E ) in vivo . ( D ) homozygous CBF1 - myc seedlings were first grown at 22 ° C in continuous FR for 4 d , then total were extracted subsequently treated with 5 - min R , or 5 - min R followed by 5 - min FR ( R + FR ) before immunoprecipitation . ( E ) homozygous CBF1 - myc seedlings were first grown at 22 ° C in continuous R for 4 d , then total were extracted subsequently treated with 5 - min FR , or 5 - min FR followed by 5 - min R ( FR + R ) before immunoprecipitation . After treatments , total were incubated with anti - c - myc Affinity Gel ( Sigma - Aldrich ) . total precipitated were examined by immunoblotting using antibodies against myc , phyA , phyB , RPN6 , respectively . numbers below anti - phyA anti - phyB blots indicate relative band intensities of co - precipitated phyA or phyB normalized to those of precipitated CBF1 - myc , respectively . of first clear band was set to 100 for each blot .

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

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.


Immunoblots showing of PIF4 PIF5 at 22 ° C in 4 - d - old Col , cbf1 - 1 , cbf1 - 2 , CBF1 - myc seedlings grown in darkness ( D ) or continuous W or R . Anti - RPN6 was used as a sample loading control . numbers below immunoblots indicate relative band intensities of PIF4 or PIF5 normalized to those of RPN6 , respectively .

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Immunoblots 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.


hypocotyl lengths of 4 - d - old Col , cbf1 - 1 , pif4 pif5 , cbf1 pif4 pif5 , 35S : PIF4 35S : PIF4 cbf1 seedlings grown at 22 ° C in continuous W or R . Error bars represent SD from 20 seedlings . * * P < 0 . 01 * * * P < 0 . 001 ( two - t - ) for indicated pairs of seedlings . Scale bar = 1 mm . NS , not significant .

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

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 PIF4 in 4 - d - old Col , 35S : PIF4 , 35S : PIF4 cbf1 seedlings grown at 22 ° C in D or continuous W or R . Anti - RPN6 was used as a sample loading control . numbers below immunoblots indicate relative band intensities of PIF4 normalized to those of RPN6 , respectively .

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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.


Yeast three - hybrid assays showing that CBF1 represses interaction of Pfr - phyB with PIF4 in yeast . AD - PIF4 , phyB - BD CBF1 were expressed in yeast Y190 as indicated . β - galactosidase activities were measured by liquid culture assays using ONPG as substrate . Error bars represent SD of three independent yeast clones .

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

Yeast 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 interaction of Pfr - phyB with PIF5 ( B ) in yeast . AD - PIF4 , phyB - BD CBF1 were expressed in yeast Y190 as indicated . β - galactosidase activities were measured by liquid culture assays using ONPG as substrate . Error bars represent SD of three independent yeast clones .

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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 . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc ( P < 0 . 05 ) .

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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 . Different letters represent significant differences by one - way ANOVA with Duncan ' s post hoc ( 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 ) total extracted from 4 - d - old 35S : phyB - GFP seedlings grown at 22 ° C in continuous R served as bait , GST - PIF4 fusion served as prey . Equivalent amounts of phyB - GFP extract GST - PIF4 fusion , increasing amounts of His - CBF1 were added as indicated before anti - GFP IP assays were performed . pulled - down were subjected to immunoblotting with antibodies against phyB , GST His , respectively . Numbers below anti - GST blots indicate relative band intensities of co - precipitated GST - PIF4 normalized to those of precipitated phyB - GFP , respectively . of first clear band was set to 100 for each blot .

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

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 ) . total extracted from 4 - d - old 35S : phyB - GFP seedlings grown at 22 ° C in continuous R served as bait , GST - PIF5 fusion served as prey . Equivalent amounts of phyB - GFP extract GST - PIF5 fusion , increasing amounts of His - CBF1 were added as indicated before anti - GFP IP assays were performed . pulled - down were subjected to immunoblotting with antibodies against phyB , GST His , respectively . Numbers below anti - GST blots indicate relative band intensities of co - precipitated GST - PIF5 normalized to those of precipitated phyB - GFP , respectively . of first clear band was set to 100 for each blot .

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

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.


of Col , two cbf1 mutants ( cbf1 - 1 cbf1 - 2 ) two CBF1 - OE lines ( CBF1 - myc CBF1 - flag ) grown under LD conditions at 22 ° C , 17 ° C 4 ° C , respectively . seeds of different genotypes were germinated under LD ( 16 - h - / 8 - h - photoperiod ) conditions at 22 ° C for 2 d , respectively , then grown under LD conditions at 22 ° C , 17 ° C , or 4 ° C , for additional 6 d . Scale bar = 1 mm .

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

Phenotypes 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 cbf1 - 2 ) two CBF1 - OE lines ( CBF1 - myc 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 - t - ) for indicated genotype compared with Col . NS , not significant .

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

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.


ratios of hypocotyl lengths at 17 ° C versus 22 ° C for indicated seedlings grown under LD conditions . Error bars represent SD from 10 seedlings . * * * P < 0 . 001 ( two - t - ) for indicated genotype compared with Col .

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

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 CBF1 transcript in Col seedlings grown under LD conditions at 22 ° C , 17 ° C 4 ° C . Col seeds were germinated under LD conditions at 22 ° C for 2 d , then grown under LD conditions at 22 ° C , 17 ° C or 4 ° C for additional 6 d . samples were collected at ZT8 , 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 ( P < 0 . 05 ) .

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

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 of PIF4 ( E ) PIF5 ( F ) in Col , cbf1 - 1 , cbf1 - 2 , CBF1 - myc seedlings grown under LD conditions at 22 ° C , 17 ° C 4 ° C . seeds of different genotypes were germinated under LD conditions at 22 ° C for 2 d , then grown under LD conditions at 22 ° C , 17 ° C or 4 ° C for additional 6 d . samples were collected at ZT20 , then subjected to immunoblotting . Anti - RPN6 was used as a sample loading control . Numbers below immunoblots indicate relative band intensities of PIF4 or PIF5 normalized to those of loading control , respectively . of first band was set to 100 for each blot .

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

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.


A ) Left panel : experimental strategy . Hematopoietic ( HSPC ) progenitors were characterized isolated using specific sets of surface markers . express CD34 . Upon differentiation , progenitors start to express CD38 in contrast to HSPC ( blue ) . Progenitors engaged in myeloid ( magenta ) lymphoid ( green ) pathways express respectively CD33 CD19 markers . Right panel : FACS gating strategy to isolate HSPC ( further referred as CD38 - ( blue box ) , engaged in myeloid lymphoid differentiation pathways ( referred to as CD33 + [ magenta box ] CD19 + [ green box ] , respectively ) .

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

A) 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 left panel ( is shown in blue ( DAPI ) envelop in ( Sun2 ) . Scale bar : 5 µm . corresponding 3D reconstructions are presented in right panel . Convex non - convex surfaces are shown in blue yellow , respectively . C ) Nucleus - invagination is defined as of non - convex over total nucleus surface . Nuclei are larger deformed in CD33 + ( magenta , n = 106 ) than CD38 - CD19 + ( blue [ n = 120 ] green [ n = 115 ] , respectively ; 3 independent experiments ; Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 , Mann - Whitney ; n . s : non significant ) .

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

B) For 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).


A ) For each population ( CD38 - CD33 + ) , Z stacks of two representative are shown in left panel . Microtubules are shown in green , actin in magenta , centrosome in in blue . In middle panel , inverted image corresponding actin is shown . In left panel , corresponding 3D projection of microtubules is presented as inverted image . Scale bar 5 µm .

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

A) 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 nucleus . Z stacks of representative CD38 - CD33 + are shown . Microtubules are shown in green , nucleus envelope ( Sun2 immuno - ) in magenta in blue . Scale bar 5 µm .

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

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 ( d ) over nucleus center - to - nucleus convex envelope ( R ) is calculated to extract relative position of centrosome to center . This parameter is significantly lower in myeloid progenitors ( n = 129 ) than in HSPC ( n = 200 ; 3 independent experiments ; * * * * ; p < 0 . 0001 . Mann - Whitney ) indicating that centrosome becomes internalized within an invaginated region upon differentiation .

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

C) 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 of HSPC ( CD38 - ) myeloid progenitors ( CD33 + ) . Z stacks of representative are shown . Image contrast has been inverted . Stainings show DAPI , Sun2 p150 glued . Scale bar 5µm .

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

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 ) dynein adaptor BicD2 associates with of HSPC ( CD38 - ) myeloid progenitors ( CD33 + ) . Z stacks of representative are shown . Image contrast has been inverted . Stainings show DAPI BicD2 . Scale bar 5µm .

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

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.


B ) nucleus is progressively deformed over time in culture . For each time point , equatorial Z stacks of two representative are presented . Left panel : inverted image of ( DAPI ) . Right panel : microtubules are shown in green in blue . Scale bar : 5 µm . C ) Nucleus volume ( left panel ) invagination ( right panel ) increase to attain similar to those of freshly isolated CD33 + ( 24 h , n = 165 , 4 donors ; 48 h , n = 143 , 3 donors ; 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ; + Bleb , blebbistatin - treated ; + Bleb + NZ , blebbistatin nocodazole - treated ; + TX , taxol - treated ; + CilioD , ciliobrevin - D - treated ) Z stacks of 2 representative are displayed . Microtubules are shown in green , in blue , centrosome in actin in magenta . Asterisks indicate regions where microtubules are not in contact with nucleus . Scale bar 5 μm .

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

D) 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 volume ( left panel ) , invagination ( right panel ) in indicated conditions . Non - treated at 48 hours ( n = 137 , 3 donors ) 72 hours ( n = 143 , 3 donors ) ; blebbistatin - treated ( n = 96 / 3 donors ) , blebbistatin nocodazole treated ( n = 66 , 2 donors ) , taxol - treated ( n = 122 , 3 donors ) , ciliobrevin - D - treated ( n = 128 , 3 donors ) . Bars correspond to mean ± SD ; * * * * : p < 0 . 0001 . * * : p < 0 . 01 , n . s : non significant ; Mann - Whitney .

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

F) 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.