B Effect of canonical noncanonical H2A ROF on histone octamer assembly . Left : Gel filtration chromatography profiles of octamers assembled by histones including various H2A family members or mutants . Highlighted in blue shadow are chromatography fractions subject to SDS - PAGE . Right : SDS - PAGE of histone octamer integrity by visualizing histone stoichiometry . stars indicate lnkH2B - H2A containing different H2A family members or mutants present in chromatography fractions . triangles indicate chromatography fractions containing majority of lnkH2B - H2A or their mutants .

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B Effect of the canonical and noncanonical H2A ROF on histone octamer assembly. Left: Gel filtration chromatography profiles of octamers assembled by core histones including various H2A family members or mutants. Highlighted in blue shadow are chromatography fractions subject to SDS-PAGE analysis. Right: SDS-PAGE analysis of histone octamer integrity by visualizing histone stoichiometry. The stars indicate lnkH2B-H2A containing different H2A family members or mutants present in chromatography fractions. The triangles indicate the chromatography fractions containing the majority of the lnkH2B-H2A or their mutants.


D , E Effects of canonical noncanonical H2A ROF on nucleosome resistance to MNase digestion . Displayed are Native - PAGE results ( D ) graph of MNase digestion efficiency calculated from quantitation of digested against total ( E ) . experiments are repeated twice . Data are mean ± SD , n = 2 .

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D,E Effects of the canonical and noncanonical H2A ROF on nucleosome resistance to MNase digestion. Displayed are Native-PAGE results (D) and graph of MNase digestion efficiency calculated from quantitation of the digested DNA against total DNA (E). All experiments are repeated twice. Data are mean ± SD, n = 2.


D SDS - PAGE of SWR1 purified from S . cerevisiae .

D SDS-PAGE analysis of SWR1 complex purified from S. cerevisiae.


E Effect of H2A . Z . 2 . 2 ROF on facilitating histone replacement . Incorporations of yeast H2A . Z - H2B dimers containing either wild type H2A . Z or Z . 2 . 2 - like H2A . Z were analyzed by SWR1 - catalyzed histone replacement . H2A . Z Z . 2 . 2 - like H2A . Z incorporated into nucleosome were resolved by 6 % Native - PAGE detected by SYBR Green western blotting .

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E Effect of H2A.Z.2.2 ROF on facilitating histone replacement. Incorporations of yeast H2A.Z-H2B dimers containing either the wild type H2A.Z or Z.2.2-like H2A.Z were analyzed by SWR1-catalyzed histone replacement assay. H2A.Z and Z.2.2-like H2A.Z incorporated into nucleosome were resolved by 6% Native-PAGE and detected by SYBR Green staining and western blotting.


A . expression PV incorporation of spike were assessed by Western blotting using an anti - S2 mAb . Lentiviral capsid components were detected using anti - p24 / 55 .

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A. Cellular expression and PV incorporation of spike were assessed by Western blotting using an anti-S2 mAb. Lentiviral capsid components were detected using anti-p24/55.


F . Western blot detection of ACE2 ( rabbit anti - ACE2 ) in lysates of stated . ACE2 was detected in HEK 293T samples after loading 3X more lysate increasing signal exposure time ( labelled in boldface ) .

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F. Western blot detection of ACE2 (rabbit anti-ACE2) in cell lysates of the stated cells. ACE2 was detected in HEK 293T samples after loading 3X more lysate and increasing signal exposure time (labelled in boldface).


J . Over - expression confirmed by Western blotting , actin CD81 were used as loading controls for ACE2 TMPRSS2 respectively .

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J. Over-expression confirmed by Western blotting, actin and CD81 were used as loading controls for ACE2 and TMPRSS2 respectively.


L . expression PV incorporation of stated spike were assessed by Western blotting . displayed samples are taken from same image have been cropped to remove irrelevant lanes / bands .

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L. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting. The displayed samples are taken from the same image and have been cropped to remove irrelevant lanes/bands.


N . expression PV incorporation of stated spike were assessed by Western blotting , note change in sample order relative to panel M .

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

N. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting, note the change in sample order relative to panel M.


E . expression PV incorporation of stated spike were assessed by Western blotting .

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

E. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting.


J . Expression PV incorporation of stated spike were assessed by Western blotting .

J. Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting.


D . Expression PV incorporation of stated spike were assessed by Western blotting .

D. Cellular Expression and PV incorporation of the stated spike proteins were assessed by Western blotting.


( B ) Relative TAG in different genetic backgrounds were measured by glyceride kit . For , TAG were normalized to . Error bars represent ± . * * * : P < 0 . 001 ; NS : non - significant .

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(B) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. Error bars represent ± SEM. ***: P < 0.001; NS: non-significant.


( G ) of lipid droplet in ( C F ) . ND : normal diet ; HFD : high - fat diet . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison . Each point represents data from one fat , at least 30 were analyzed for each fat . Error bars represent ± . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

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(G) Quantification of the lipid droplet size in (C and F). ND: normal diet; HFD: high-fat diet. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were analyzed for each fat body. Error bars represent ±SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


( B 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Scale bars represent 25 µm . ( B ) RDH / CG2064 RNAi suppresses enlarged lipid droplet phenotype in CSN2 RNAi under DGAT1 overexpression ( ppl > DGAT1 ) or high - fat diet conditions . ( C ) of lipid droplet in ( B ) . HFD : high - fat diet . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison . Each point represents data from one fat , at least 30 were examined in each fat . Error bars represent ± . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

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(B 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (B) RDH/CG2064 RNAi suppresses the enlarged lipid droplet phenotype in CSN2 RNAi under DGAT1 overexpression (ppl>DGAT1) or high-fat diet conditions. (C) Quantification of the lipid droplet size in (B). HFD: high-fat diet. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


, F 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Scale bars represent 25 µm . ( F ) RDH / CG2064 overexpression increases lipid droplet in ppl > DGAT1 larvae . ( G ) of lipid droplet in ( F ) . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison . Each point represents data from one fat , at least 30 were examined in each fat . Error bars represent ± . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : non - significant .

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, F 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (F) RDH/CG2064 overexpression increases lipid droplet size in ppl>DGAT1 larvae. (G) Quantification of the lipid droplet size in (F). data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Scale bars represent 25 µm . ( H ) Relative TAG in different genetic backgrounds were measured by glyceride kit . For , TAG were normalized to . ( I ) RDH / CG2064 CG2065 deletion does not affect lipid droplet under normal or high - fat diet conditions . data were analyzed by one - way ANOVA with a post hoc Turkey ' s multiple - comparison . Each point represents data from one fat , at least 30 were examined in each fat . Error bars represent ± . * * * : P < 0 . 001 ; * * : P < 0 . 01 ; NS : 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 O O O B-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O

3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. (H) Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. (I) RDH/CG2064 and CG2065 deletion does not affect lipid droplet size under normal or high-fat diet conditions. data were analyzed by one-way ANOVA with a post hoc Turkey's multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***: P < 0.001; **: P < 0.01; NS: non-significant.


( B ) 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Overexpressing wild - type or site - mutated RDH / CG2064 increases lipid droplet in larvae with DGAT1 overexpression ( ppl > DGAT1 ) . Scale bar represents 25 µm .

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(B) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Overexpressing wild-type or site-mutated RDH/CG2064 increases the lipid droplet size in larvae with DGAT1 overexpression (ppl>DGAT1). Scale bar represents 25 µm.


( E ) Immunofluorescence imaging of RDH / CG2064 - GFP , detected with anti - GFP antibody , ER marker RFP - KDEL in 3rd instar larval fat . RDH / CG2064 co - localizes with RFP - KDEL . Scale bar represents 10 µm .

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(E) Immunofluorescence imaging of RDH/CG2064-GFP, detected with anti-GFP antibody, and the ER protein marker RFP-KDEL in 3rd instar larval fat body. RDH/CG2064 co-localizes with RFP-KDEL. Scale bar represents 10 µm.


Lysates of 3rd instar larval fat samples of different genetic backgrounds were immunoprecipitated with anti - Flag antibody then probed by anti - GFP or anti - Flag antibody in a western blot . Plin2 , but not Plin1 or Bmm , is specifically coimmunoprecipitated with RDH / CG2064 ( A ) .

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Lysates of 3rd instar larval fat body samples of different genetic backgrounds were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin2, but not Plin1 or Bmm, is specifically coimmunoprecipitated with RDH/CG2064 (A).


( E ) Lysates of Drosophila S2 expressing full - Plin1 or Plin1 with a C - terminal deletion ( Plin1ΔC ) were immunoprecipitated with anti - Flag antibody then probed by anti - GFP or anti - Flag antibody in a western blot . Plin1ΔC coimmunoprecipitated with RDH / CG2064 .

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(E) Lysates of Drosophila S2 cells expressing full-length Plin1 or Plin1 with a C-terminal deletion (Plin1ΔC) were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin1ΔC coimmunoprecipitated with RDH/CG2064.


( F ) 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . Under high - fat conditions , RDH / CG2064 overexpression increases lipid droplet , Plin2 RNAi suppresses enlargement induced by RDH / CG2064 overexpression . Scale bar represents 25 µm .

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(F) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Under high-fat conditions, RDH/CG2064 overexpression increases the lipid droplet size, and Plin2 RNAi suppresses the enlargement induced by RDH/CG2064 overexpression. Scale bar represents 25 µm.


( A , B of Plin2 - GFP in 3rd instar larval fat of different genotypes . Scale bar represents 25 µm . ( A ) CSN2 RNAi or RDH / CG2064 overexpression reduces peripheral localization of Plin2 - GFP increases localization of Plin2 - GFP on small peri - lipid droplets ( boxed ) . DAPI ( blue ) stains nuclei . arrows indicate peripheral lipid droplet . Red arrows indicate medial lipid droplets . ( B ) peripheral localization of Plin2 - GFP is greatly reduced in fat from larvae with CSN2 RNAi or RDH / CG2064 overexpression . were taken by focusing on periphery of fat .

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

(A, B Images of Plin2-GFP in 3rd instar larval fat body cells of different genotypes. Scale bar represents 25 µm. (A) CSN2 RNAi or RDH/CG2064 overexpression reduces the peripheral localization of Plin2-GFP and increases the localization of Plin2-GFP on small peri-nuclear lipid droplets (boxed). DAPI (blue) stains nuclei. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. (B) The peripheral localization of Plin2-GFP is greatly reduced in fat body cells from larvae with CSN2 RNAi or RDH/CG2064 overexpression. Images were taken by focusing on the periphery of fat cells.


C , of Plin2 - GFP in 3rd instar larval fat of different genotypes . Scale bar represents 25 µm . ( C ) In fat from ppl > DGAT1 larvae with CSN2 RNAi or RDH / CG2064 overexpression , localization of Plin2 - GFP is greatly reduced at periphery is dramatically increased on large medial lipid droplets . arrows indicate peripheral lipid droplet . Red arrows indicate medial lipid droplets . ( D ) plots of in ( C ) . GFP along across a lipid droplet was measured by ImageJ . In ppl > DGAT1 larvae with CSN2 RNAi or RDH / CG2064 overexpression , Plin2 - GFP localization on large medial lipid droplets is represented by two peaks . More than 30 lipid droplets were measured for each genotype . plot shown is a typical image curve for indicated genotype .

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

C, Images of Plin2-GFP in 3rd instar larval fat body cells of different genotypes. Scale bar represents 25 µm. (C) In fat body cells from ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, the localization of Plin2-GFP is greatly reduced at the periphery and is dramatically increased on large medial lipid droplets. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. (D) Fluorescence intensity line plots of the images in (C). GFP intensity along the line across a lipid droplet was measured by ImageJ. In ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, Plin2-GFP localization on large medial lipid droplets is represented by two peaks. More than 30 lipid droplets were measured for each genotype. The plot shown is a typical image curve for the indicated genotype.


( G ) peripheral lipid droplets in larval fat from different genetic backgrounds were stained by BODIPY ( green ) . CSN3 RNAi or RDH / CG2064 overexpression reduces number of peripheral lipid droplets in both wild - type ppl > DGAT1 larvae .

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(G) The peripheral lipid droplets in larval fat body cells from different genetic backgrounds were stained by BODIPY (green). CSN3 RNAi or RDH/CG2064 overexpression reduces the number of peripheral lipid droplets in both wild-type and ppl>DGAT1 larvae.


( A , Confocal of Bmm - GFP ( green ) in 3rd instar larval fat bodies of different genotypes . DAPI ( blue ) stains nuclei . Scale bar represents 25 µm . ( A ) Knocking down CSN2 or overexpressing RDH / CG2064 in with DGAT1 overexpression ( ppl > DGAT1 ) decreases level lipid droplet localization of Bmm - GFP . ( B ) GFP along across a lipid droplet in ( A ) was measured by ImageJ . lipid droplet localization of Bmm - GFP , represented by two peaks , is clearly visible in fat from ppl > DGAT1 larvae , but it is lost in fat from ppl > DGAT1 larvae with CSN2 RNAi or overexpression of RDH / CG2064 . More than 30 lipid droplets of each genotype were measured . One typical image curve is shown for each genotype .

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(A, Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (A) Knocking down CSN2 or overexpressing RDH/CG2064 in animals with DGAT1 overexpression (ppl>DGAT1) decreases the level and lipid droplet localization of Bmm-GFP. (B) The GFP intensity along the line across a lipid droplet in (A) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, is clearly visible in fat cells from ppl>DGAT1 larvae, but it is lost in fat cells from ppl>DGAT1 larvae with CSN2 RNAi or overexpression of RDH/CG2064. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.


F ) Confocal of Bmm - GFP ( green ) in 3rd instar larval fat bodies of different genotypes . DAPI ( blue ) stains nuclei . Scale bar represents 25 µm . ( F ) Overexpression of Plin2 decreases level lipid droplet localization of Bmm - GFP in fat from larvae with DGAT1 overexpression . ( G ) GFP along across a lipid droplet from ( F ) was measured by ImageJ . lipid droplet localization of Bmm - GFP , represented by two peaks , was evident in fat from ppl > DGAT1 larvae , but is lost in ppl > DGAT1 larvae with overexpression of Plin2 . More than 30 lipid droplets of each genotype were measured . One typical image curve is shown for each genotype .

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

F) Confocal images of Bmm-GFP (green) in 3rd instar larval fat bodies of different genotypes. DAPI (blue) stains nuclei. Scale bar represents 25 µm. (F) Overexpression of Plin2 decreases the level and lipid droplet localization of Bmm-GFP in fat cells from larvae with DGAT1 overexpression. (G) GFP intensity along the line across a lipid droplet from (F) was measured by ImageJ. The lipid droplet localization of Bmm-GFP, represented by two peaks, was evident in fat cells from ppl>DGAT1 larvae, but is lost in ppl>DGAT1 larvae with overexpression of Plin2. More than 30 lipid droplets of each genotype were measured. One typical image curve is shown for each genotype.


( H ) 3rd instar larval fat bodies were stained by BODIPY ( green ) for lipid droplets DAPI ( blue ) for nuclei . bmm RNAi increases lipid droplet in fat from larvae with DGAT1 overexpression . Scale bar represents 25 µm .

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(H) 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. bmm RNAi increases the lipid droplet size in fat cells from larvae with DGAT1 overexpression. Scale bar represents 25 µm.


( 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 ) .

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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 ) .

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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 .

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(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 .

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(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 .

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(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 .

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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 ) .

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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 .

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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 .

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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 .

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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 .

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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.