( A ) FTCD located in mitochondria causes mitochondria aggregation during mitosis . Mitotic cells were collected by flushing from HeLa Tet - off cells in which either FTCDwt - HA - MAO or FTCD ( R382A ) - HA - MAO had been induced for 24 hrs . The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis . Cells in which FTCDwt - HA - MAO was induced for 28 hrs without synchronization were used as the asynchronous control group . Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA . Panels a - f display representative images . Scale bar = 10 μm . ( B ) The results of quantification of ( A ) . Results are shown as the mean ± SD of seven sets of independent experiments , with 100 cells counted in each group in each independent experiment . An asterisk indicates a significant difference at P < 0 . 01 compared with the others ( Bonferroni method ) .

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(A) FTCD located in mitochondria causes mitochondria aggregation during mitosis. Mitotic cells were collected by flushing from HeLa Tet-off cells in which either FTCDwt-HA-MAO or FTCD(R382A)-HA-MAO had been induced for 24 hrs. The mitotic cells were further cultured for 4 hrs to enable complete cytokinesis. Cells in which FTCDwt-HA-MAO was induced for 28 hrs without synchronization were used as the asynchronous control group. Cells were fixed and visualized with a monoclonal antibody to mitochondria and a polyclonal antibody to HA. Panels a-f display representative images. Scale bar = 10 μm. (B) The results of quantification of (A). Results are shown as the mean±SD of seven sets of independent experiments, with 100 cells counted in each group in each independent experiment. An asterisk indicates a significant difference at P<0.01 compared with the others (Bonferroni method).


( C ) A single living cell expressing FTCDwt - HA - MAO was tracked during mitosis . Mitotic cells were collected by flushing from the HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO , and cultured in the absence or presence of DOX . The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30 - 40 cells were randomly taken at the 23 hr time point . From the 23 hr to 27 hr time point , cells were tracked in the bright field every 30 mins . At the 27 hr time point , confocal images were taken of the 21 - 38 cells that were successfully tracked . Panels a - j display representative images . Asterisks indicate the tracked cells . Scale bar = 10 μm . ( D ) The results of quantification of ( C ) . The experiment was repeated five times and each line shows the average percentage of an independent experiment . The percentages of cells that underwent mitosis are as follows : 41 . 5 % in the DOX ( - ) group and 54 . 7 % in the DOX ( + ) group .

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(C) A single living cell expressing FTCDwt-HA-MAO was tracked during mitosis. Mitotic cells were collected by flushing from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO, and cultured in the absence or presence of DOX. The cells were stained with MitoTracker at the 22 hr time point and confocal images of 30-40 cells were randomly taken at the 23 hr time point. From the 23 hr to 27 hr time point, cells were tracked in the bright field every 30 mins. At the 27 hr time point, confocal images were taken of the 21-38 cells that were successfully tracked. Panels a-j display representative images. Asterisks indicate the tracked cells. Scale bar = 10 μm. (D) The results of quantification of (C). The experiment was repeated five times and each line shows the average percentage of an independent experiment. The percentages of cells that underwent mitosis are as follows: 41.5 % in the DOX(-) group and 54.7 % in the DOX(+) group.


( E ) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis . Mitotic cells were collected from the HeLa Tet - off cells inducibly expressing FTCDwt - HA - MAO and cultured in the absence or presence of DOX as in ( C ) . Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point . Panels a - t display representative images . Scale bar = 10 μm .

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(E) FTCD located in mitochondria causes mitochondria aggregation at the end of mitosis. Mitotic cells were collected from the HeLa Tet-off cells inducibly expressing FTCDwt-HA-MAO and cultured in the absence or presence of DOX as in (C). Cells were stained with MitoTracker at the 23hr time point and observed by confocal microscopy from the 24 hr to 26 hr time point. Panels a-t display representative images. Scale bar = 10 μm.


( F ) The results of quantification of ( E ) . Cells were classified by their cell cycle phases into the following three categories : prometaphase / metaphase , anaphase / telophase and cytokinesis . Results are shown as the mean ± SD of three sets of independent experiments , with 10 - 14 cells counted in each group in each independent experiment . Asterisks indicate a significant difference at P < 0 . 01 compared with the DOX ( + ) group and with the prometaphase / metaphase category ( Bonferroni method ) .

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(F) The results of quantification of (E). Cells were classified by their cell cycle phases into the following three categories: prometaphase/metaphase, anaphase/telophase and cytokinesis. Results are shown as the mean±SD of three sets of independent experiments, with 10-14 cells counted in each group in each independent experiment. Asterisks indicate a significant difference at P<0.01 compared with the DOX(+) group and with the prometaphase/metaphase category (Bonferroni method).


Starvation triggers accumulation of nucleosides , bases and S7P . Wild ‐ type S . cerevisiae cells growing in minimal media were switched to minimal media containing no carbon , no nitrogen or no phosphate . After the indicated duration of starvation , the metabolome was quantified by LC ‐ MS . For media composition , see Supplementary Table S2 . For the list of absolute concentration of metabolties , see Supplementary Table S3 . Data are shown in heat map format , with each line reflecting the dynamics of a particular compound in a particular culture condition . Metabolite levels of biological duplicates were averaged , normalized to cells growing steadily in glucose ( time zero ) , and the resulting fold changes log2 transformed .

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

Starvation triggers accumulation of nucleosides, bases and S7P. Wild‐type S. cerevisiae cells growing in minimal media were switched to minimal media containing no carbon, no nitrogen or no phosphate. After the indicated duration of starvation, the metabolome was quantified by LC‐MS. For media composition, see Supplementary Table S2. For the list of absolute concentration of metabolties, see Supplementary Table S3. Data are shown in heat map format, with each line reflecting the dynamics of a particular compound in a particular culture condition. Metabolite levels of biological duplicates were averaged, normalized to cells growing steadily in glucose (time zero), and the resulting fold changes log2 transformed.


( A ) Experimental design for demonstrating metabolite production via macromolecule degradation . Yeast cells were grown on 2 % unlabeled glucose , and then switched to U ‐ 13C ‐ glucose for 70 min , which completely labels free metabolite but only partially labels macromolecules . Thereafter , glucose was removed and the metabolome analyzed by LC ‐ MS .

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(A) Experimental design for demonstrating metabolite production via macromolecule degradation. Yeast cells were grown on 2% unlabeled glucose, and then switched to U‐13C‐glucose for 70 min, which completely labels free metabolite but only partially labels macromolecules. Thereafter, glucose was removed and the metabolome analyzed by LC‐MS.


( B ) Fraction of unlabeled nucleosides , nucleic bases and PPP intermediates as a function of starvation time , in wild ‐ type and autophagy deficient ( atg7 deletion ) yeast . The x axis represents minutes after carbon starvation , and the y axis represents fraction of unlabeled metabolites ( mean ± range of N = 2 biological replicates ) .

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(B) Fraction of unlabeled nucleosides, nucleic bases and PPP intermediates as a function of starvation time, in wild‐type and autophagy deficient (atg7 deletion) yeast. The x axis represents minutes after carbon starvation, and the y axis represents fraction of unlabeled metabolites (mean±range of N=2 biological replicates).


( C ) Ratio of metabolite levels in atg7 strain versus wild ‐ type strain in carbon starvation .

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(C) Ratio of metabolite levels in atg7 strain versus wild‐type strain in carbon starvation.


( D ) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild ‐ type strain in carbon starvation .

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(D) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild‐type strain in carbon starvation.


( E ) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild ‐ type yeast . In ( C ) to ( E ) , all reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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(E) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild‐type yeast. In (C) to (E), all reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.


( A ) Ratio of metabolite levels in sdt1 and phm8 strains versus wild ‐ type strain in carbon starvation . All reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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(A) Ratio of metabolite levels in sdt1 and phm8 strains versus wild‐type strain in carbon starvation. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.


( B ) Summary of transcripts data of PHM8 , SDT1 and other enzymes in the pathway ( Gasch et al , 2000 ; Bradley et al , 2009 ; Klosinska et al , 2011 ) . All values are log2 transformed fold changes .

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(B) Summary of transcripts data of PHM8, SDT1 and other enzymes in the pathway (Gasch et al, 2000; Bradley et al, 2009; Klosinska et al, 2011). All values are log2 transformed fold changes.


( C ) Screening of Phm8 ' s phosphatase activity against 90 phosphorylated compounds . Phosphatase activity was measured in the presence of 0 . 5 mM substrate and 5 mM Mg2 + ( pH = 7 . 0 , 30 ° C ) . Compounds with specific activity higher than 0 . 1 μmol / mg / min are shown . The x axis represents specific phosphatase activity ( μmol of phosphate produced per minute per mg of enzyme , mean ± range of N = 2 replicates ) .

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(C) Screening of Phm8's phosphatase activity against 90 phosphorylated compounds. Phosphatase activity was measured in the presence of 0.5 mM substrate and 5 mM Mg2+ (pH=7.0, 30°C). Compounds with specific activity higher than 0.1 μmol/mg/min are shown. The x axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).


( D ) Top table : absolute intracellular concentration of nucleotide monophosphates in carbon starvation . Bottom plots : Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration . The x axis represents CMP concentration and the y axis represents specific phosphatase activity ( μmol of phosphate produced per minute per mg of enzyme , mean ± range of N = 2 replicates ) .

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(D) Top table: absolute intracellular concentration of nucleotide monophosphates in carbon starvation. Bottom plots: Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration. The x axis represents CMP concentration and the y axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).


Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase , Urh1 is the pyrimidine hydrolase , and Prm15 ( Pgm3 ) is the phosphoribomutase . Ratio of metabolite levels in pnp1 / urh1 , pnp1 , urh1 and prm15 ( pgm3 ) strains versus wild ‐ type strain during carbon starvation . Data are shown in heat map format , with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild ‐ type strain . All reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase, Urh1 is the pyrimidine hydrolase, and Prm15 (Pgm3) is the phosphoribomutase. Ratio of metabolite levels in pnp1/urh1, pnp1, urh1 and prm15 (pgm3) strains versus wild‐type strain during carbon starvation. Data are shown in heat map format, with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild‐type strain. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.


( B ‐ E ) DHAP , SBP and S7P levels in wild type , tkl1 / tkl2 ( B ) , tal1 / nqm1 ( C ) and pfk1 ( E ) strains upon carbon starvation , and wild ‐ type strain upon abruptly switching from glucose to no carbon ( WT ) versus to dihydroxyacetone ( WT + DHA ) as the sole carbon source ( D ) . The x axis represents hours after carbon starvation , and the logarithmic y axis represents absolute intracellular concentration ( mean ± range of N = 2 biological replicates ) .

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(B‐E) DHAP, SBP and S7P levels in wild type, tkl1/tkl2 (B), tal1/nqm1 (C) and pfk1 (E) strains upon carbon starvation, and wild‐type strain upon abruptly switching from glucose to no carbon (WT) versus to dihydroxyacetone (WT+DHA) as the sole carbon source (D). The x axis represents hours after carbon starvation, and the logarithmic y axis represents absolute intracellular concentration (mean±range of N=2 biological replicates).


( A ) Level of hallmark metabolites and energy charge in wild type , phm8 and pnp1 / urh1 strains in starvation . The x axis represents hours after starvation , and the logarithmic y axis represents energy charge ( [ ATP ] + 0 . 5 [ ADP ] / ( [ ATP ] + [ ADP ] + [ AMP ] ) or absolute intracellular concentration ( mean ± range of N = 2 biological replicates ) .

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(A) Level of hallmark metabolites and energy charge in wild type, phm8 and pnp1/urh1 strains in starvation. The x axis represents hours after starvation, and the logarithmic y axis represents energy charge ([ATP]+0.5[ADP]/([ATP]+[ADP]+[AMP]) or absolute intracellular concentration (mean±range of N=2 biological replicates).


( B and C ) Growth and viability of wild type , phm8 and pnp1 / urh1 strains in starvation .

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(B and C) Growth and viability of wild type, phm8 and pnp1/urh1 strains in starvation.


( D ) Growth of wild type , phm8 and pnp1 / urh1 strains in the switch from glucose to glycerol + ethanol . The x axis represents hours after starvation and the logarithmic y axis represents optical density ( A600 ) in ( B and D ) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in ( C ) ( mean ± range of N = 2 biological replicates ) .

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(D) Growth of wild type, phm8 and pnp1/urh1 strains in the switch from glucose to glycerol+ethanol. The x axis represents hours after starvation and the logarithmic y axis represents optical density (A600) in (B and D) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in (C) (mean±range of N=2 biological replicates).


( C and D ) DHAP , S7P , G6P , and glutathione levels and NADPH / NADP + ratio in wild type , phm8 and pnp1 / urh1 strains in the experiment shown in ( A ) The x axis represents minutes after oxidative stress , and the y axis represents absolute intracellular concentration or ratio of intracellular concentration ( mean ± range of N = 2 biological replicates ) .

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(C and D) DHAP, S7P, G6P, and glutathione levels and NADPH/NADP+ ratio in wild type, phm8 and pnp1/urh1 strains in the experiment shown in (A) The x axis represents minutes after oxidative stress, and the y axis represents absolute intracellular concentration or ratio of intracellular concentration (mean±range of N=2 biological replicates).


( E ) Viability of wild type , phm8 and pnp1 / urh1 strains in oxidative stress . The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment ( mean ± range of N = 2 biological replicates ) .

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(E) Viability of wild type, phm8 and pnp1/urh1 strains in oxidative stress. The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment (mean±range of N=2 biological replicates).


Representative calcium signature enriched in P - EMT tumors ( comparing ECAD - vs . ECAD + tumor cells ) . NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA .

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Representative calcium signature enriched in P-EMT tumors (comparing ECAD- vs. ECAD+ tumor cells). NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


Representative image of tumor cells transduced with GCaMP6 ( Left ) . Yellow arrows ( mesenchymal cells ) , red arrows ( epithelial cells ) , scale bar = 50μm . GCaMP6 mean fluorescent intensity ( MFI ) in ECADHIGH or ECADLOW cells cultured in indicated medium ( 0 Ca2 + or 2 Ca2 + ) for 1h ( Right ) . Representative of 3 independent murine PDA cell lines run in triplicate . Statistical analysis by Student ' s unpaired t - test ( * , p < 0 . 05 ; non - significant ( NS ) ; ± SD ) .

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Representative image of tumor cells transduced with GCaMP6 (Left). Yellow arrows (mesenchymal cells), red arrows (epithelial cells), scale bar = 50μm. GCaMP6 mean fluorescent intensity (MFI) in ECADHIGH or ECADLOW cells cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h (Right). Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (*, p<0.05; non-significant (NS); ±SD).


Flow cytometry analysis of ECADHIGH or ECADLOW cells loaded with Indo - 1 and cultured in indicated medium ( 0 Ca2 + or 2 Ca2 + ) for 1h . Representative of 2 independent cell lines ( n indicates the number of cells analyzed in each group , ECADHIGH 0 Ca2 + n = 4963 , ECADLOW 0 Ca2 + n = 14 , ECADHIGH 2 Ca2 + n = 6575 , ECADLOW 2 Ca2 + n = 29 ) . Representative of 2 independent murine PDA cell lines run in triplicate . Statistical analysis by Student ' s unpaired t - test ( * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

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Flow cytometry analysis of ECADHIGH or ECADLOW cells loaded with Indo-1 and cultured in indicated medium (0 Ca2+ or 2 Ca2+) for 1h. Representative of 2 independent cell lines (n indicates the number of cells analyzed in each group, ECADHIGH 0 Ca2+ n=4963, ECADLOW 0 Ca2+ n=14, ECADHIGH 2 Ca2+ n=6575, ECADLOW 2 Ca2+ n=29). Representative of 2 independent murine PDA cell lines run in triplicate. Statistical analysis by Student's unpaired t-test (****, p<0.0001; non-significant (NS); ±SD).


Representative 20X brightfield and fluorescent images of murine YFP + tumor cells treated for 72h with vehicle control ( DMSO ) , 2 . 5μM Ionomycin , or 10ng / ml TGFβ and co - stained for E - cadherin ( Red ) and DAPI ( Blue ) . Scale bar is 100μm .

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Representative 20X brightfield and fluorescent images of murine YFP+ tumor cells treated for 72h with vehicle control (DMSO), 2.5μM Ionomycin, or 10ng/ml TGFβ and co-stained for E-cadherin (Red) and DAPI (Blue). Scale bar is 100μm.


Representative flow cytometry analysis of surface ECAD ( Left ) , MFI of surface ECAD ( Middle ) , and percentage of surface ECAD negative cells ( Right ) in murine PDA cells , after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , and 10ng / ml TGFβ for 72h . Representative of 3 independent murine PDA cell lines run in triplicate . Statistical analysis by ANOVA ( * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

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Representative flow cytometry analysis of surface ECAD (Left), MFI of surface ECAD (Middle), and percentage of surface ECAD negative cells (Right) in murine PDA cells, after treatment with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 72h. Representative of 3 independent murine PDA cell lines run in triplicate. Statistical analysis by ANOVA (****, p<0.0001; non-significant (NS); ±SD).


Top : Relative mRNA expression of Ecad after treatment with 2 . 5μM ionomycin or 10ng / ml TGFβ for denoted time . Bottom : Western blot analysis of total E - cadherin and Vimentin protein for denoted time . Experiment was run in duplicate with three different murine PDA cell lines . Statistical analysis by ANOVA ( * * * , p < 0 . 001 ; non - significant ( NS ) ; ± SEM ) .

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Top: Relative mRNA expression of Ecad after treatment with 2.5μM ionomycin or 10ng/ml TGFβ for denoted time. Bottom: Western blot analysis of total E-cadherin and Vimentin protein for denoted time. Experiment was run in duplicate with three different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; non-significant (NS); ±SEM).


Normalized MFI of surface E - cadherin in PDA cells treated with DMSO , 2 . 5μM Ionomycin ( IONO ) , and 10ng / ml TGFβ for 7 days , followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on the x - axis . Treated cells were analyzed at day 2 , 3 , and 7 . Withdrawn cells were analyzed on days 1 , 2 , and 7 . Experiment was performed in duplicate in two different murine PDA cell lines . Error bars indicate SD .

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Normalized MFI of surface E-cadherin in PDA cells treated with DMSO, 2.5μM Ionomycin (IONO), and 10ng/ml TGFβ for 7 days, followed by withdrawal of Ionomycin or TGFβ for 7 days as indicated on the x-axis. Treated cells were analyzed at day 2, 3, and 7. Withdrawn cells were analyzed on days 1, 2, and 7. Experiment was performed in duplicate in two different murine PDA cell lines. Error bars indicate SD.


Quantification of live cellular migration over 4h after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , or 10ng / ml TGFβ for 48hrs . ( n = 16 cells per condition . Experiment was run in triplicate in two murine PDA different cell lines ) ( Right ) . Quantification of transwell migration after treatment with DMSO , 2 . 5μM Ionomycin ( IONO ) , or 10ng / ml TGFβ for 72h ( n = 2 cell lines , 3 replicates per cell line with 3 20X images taken per transwell ) ( Left ) . Statistical analysis by ANOVA ( * , p < 0 . 05 ; * * * * , p < 0 . 0001 ; non - significant ( NS ) ; ± SD ) .

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

Quantification of live cellular migration over 4h after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 48hrs. (n=16 cells per condition. Experiment was run in triplicate in two murine PDA different cell lines) (Right). Quantification of transwell migration after treatment with DMSO, 2.5μM Ionomycin (IONO), or 10ng/ml TGFβ for 72h (n= 2 cell lines, 3 replicates per cell line with 3 20X images taken per transwell) (Left). Statistical analysis by ANOVA (*, p<0.05; ****, p<0.0001; non-significant (NS); ±SD).


Schematic for RNA sequencing after EMT induction for 48h . Heatmap illustration of EMT related genes in three independent murine PDA cell lines , sequenced in duplicate , treated with DMSO , 2 . 5μM Ionomycin , or 10ng / ml TGFβ . Scale indicates Z - score .

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Schematic for RNA sequencing after EMT induction for 48h. Heatmap illustration of EMT related genes in three independent murine PDA cell lines, sequenced in duplicate, treated with DMSO, 2.5μM Ionomycin, or 10ng/ml TGFβ. Scale indicates Z-score.


GSEA of ionomycin versus DMSO ( Left ) or TGFβ versus DMSO ( Right ) for EMT signatures . NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA .

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

GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for EMT signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


GSEA of ionomycin versus DMSO ( Left ) or TGFβ versus DMSO ( Right ) for calcium signatures . NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA .

GSEA of ionomycin versus DMSO (Left) or TGFβ versus DMSO (Right) for calcium signatures. NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


Leading edge plots showing enrichment of C - EMT gene signatures in TGFβ treated cells ( Left ) or P - EMT gene signatures in ionomycin ( IONO ) treated cells ( Right ) , based on GSEA . Enrichment scores ( ES ) , NES , nominal P - value , and FDR are shown . Statistical analysis performed in GSEA .

O 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 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

Leading edge plots showing enrichment of C-EMT gene signatures in TGFβ treated cells (Left) or P-EMT gene signatures in ionomycin (IONO) treated cells (Right), based on GSEA. Enrichment scores (ES), NES, nominal P-value, and FDR are shown. Statistical analysis performed in GSEA.


Fura - 2 Ca2 + measurements after addition of 5μM CNO ( Top ) or 10μM ATP ( Bottom ) in murine PDA cells expressing a Gαq - DREADD . Blue or red line indicates average of n = 22 cells . ± SD . Tracings are representative of experiments performed twice in 2 independent cell lines .

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

Fura-2 Ca2+ measurements after addition of 5μM CNO (Top) or 10μM ATP (Bottom) in murine PDA cells expressing a Gαq-DREADD. Blue or red line indicates average of n=22 cells. ±SD. Tracings are representative of experiments performed twice in 2 independent cell lines.


Representative 20X brightfield images of Gαq - DREADD - expressing cells 48h after addition of CNO ( Left ) . Scale bar = 100μm . MFI of surface E - cadherin in cells expressing a Gαq - DREADD after treatment with DMSO , 5μM CNO , or 2 . 5μM ionomycin ( Right ) . Both experiments performed in triplicate in 2 independent cell lines . Statistical analysis by ANOVA ( * * , p < 0 . 01 ; ± SD ) .

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 B-CONTROLLED_VAR 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

Representative 20X brightfield images of Gαq-DREADD-expressing cells 48h after addition of CNO (Left). Scale bar = 100μm. MFI of surface E-cadherin in cells expressing a Gαq-DREADD after treatment with DMSO, 5μM CNO, or 2.5μM ionomycin (Right). Both experiments performed in triplicate in 2 independent cell lines. Statistical analysis by ANOVA (**, p<0.01; ±SD).


MFI of surface E - cadherin in CnB knockout lines after 48h treatment with DMSO , 2 . 5μM ionomycin , or 10ng / ml TGFβ . Experiment was repeated in two different murine PDA cell lines in triplicate . Statistical analysis by ANOVA ( * * * , p < 0 . 001 ; * * * * , p < 0 . 00001 ; ± SD ) .

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MFI of surface E-cadherin in CnB knockout lines after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).


MFI of surface E - cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO , 2 . 5μM ionomycin , or 20μM W7 with 2 . 5μM ionomycin for 24h . Experiment was repeated in two different murine PDA cell lines in triplicate . Statistical analysis by ANOVA ( * * , p < 0 . 01 ; * * * , p < 0 . 001 ; non - significant ( NS ) ; ± SD ) .

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 B-CONTROLLED_VAR 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

MFI of surface E-cadherin of murine PDA cells pretreated with 20μM W7 for 24h and then DMSO, 2.5μM ionomycin, or 20μM W7 with 2.5μM ionomycin for 24h. Experiment was repeated in two different murine PDA cell lines in triplicate. Statistical analysis by ANOVA (**, p<0.01; ***, p<0.001; non-significant (NS); ±SD).


MFI of surface E - cadherin of shRNA non - targeting ( shNT ) ( Left ) or shCAMK2B ( Right ) after 48h treatment with DMSO , 2 . 5μM ionomycin , or 10ng / ml TGFβ . Experiment was repeated in two different murine PDA cell lines . Statistical analysis by ANOVA ( * * * , p < 0 . 001 ; * * * * , p < 0 . 00001 ; ± SD ) .

O O O O O O O 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

MFI of surface E-cadherin of shRNA non-targeting (shNT) (Left) or shCAMK2B (Right) after 48h treatment with DMSO, 2.5μM ionomycin, or 10ng/ml TGFβ. Experiment was repeated in two different murine PDA cell lines. Statistical analysis by ANOVA (***, p<0.001; ****, p<0.00001; ±SD).


( A ) Schematic representation of the SILAC - based quantitative proteomics experiment in B . subtilis . Cells overexpressing active YqgP ( BS50 , Table EV2 ) or its catalytically dead mutant YqgP . S288A ( BS51 ) , both auxotrophic for lysine , were grown in parallel in " heavy " ( containing 13C615N2 - Lysine isotope ) or " light " ( containing stable 12C614N2 - Lysine ) M9 minimal medium , respectively . After mixing the cell cultures in the 1 : 1 ratio ( based on OD600 ) , cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC - MS / MS experiment . Using bioinformatic analysis of the MS data , highest - scoring candidate substrates were further evaluated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Schematic representation of the SILAC-based quantitative proteomics experiment in B. subtilis. Cells overexpressing active YqgP (BS50, Table EV2) or its catalytically dead mutant YqgP.S288A (BS51), both auxotrophic for lysine, were grown in parallel in "heavy" (containing 13C615N2- Lysine isotope) or "light" (containing stable 12C614N2- Lysine) M9 minimal medium, respectively. After mixing the cell cultures in the 1:1 ratio (based on OD600), cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC-MS/MS experiment. Using bioinformatic analysis of the MS data, highest-scoring candidate substrates were further evaluated.


( E ) Results of MS analyses of affinity co - purification experiments in wild - type B . subtilis control ( strain BTM2 , Table EV2 ) and B . subtilis deficient in endogenous YqgP expressing the wild - type YqgP - sfGFP bait ( strain BTM84 , Table EV2 ) or the proteolytically inactive YqgP . S288A - sfGFP bait ( strain BBM1 , Table EV2 ) . Proteins were considered as potential interactors of YqgP if they were identified only in both positive co - purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts . The protein highlighted in red was the only overlapping hit between the two proteomics approaches .

(E) Results of MS analyses of affinity co-purification experiments in wild-type B. subtilis control (strain BTM2, Table EV2) and B. subtilis deficient in endogenous YqgP expressing the wild-type YqgP-sfGFP bait (strain BTM84, Table EV2) or the proteolytically inactive YqgP.S288A-sfGFP bait (strain BBM1, Table EV2). Proteins were considered as potential interactors of YqgP if they were identified only in both positive co-purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts. The protein highlighted in red was the only overlapping hit between the two proteomics approaches.


( A ) Steady - state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B . subtilis ( BTM2 and BTM501 , respectively , Table EV2 ) , in minimal medium at low magnesium concentration ( 10 µM ) . Strain lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) was used as a control . Proteins were detected by immunoblotting with chemiluminescence detection . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Steady-state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B. subtilis (BTM2 and BTM501, respectively, Table EV2), in minimal medium at low magnesium concentration (10 µM). Strain lacking YqgP (ΔyqgP, BTM78, Table EV2) was used as a control. Proteins were detected by immunoblotting with chemiluminescence detection. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE)


( B ) The cleavage is efficiently inhibited by 1 μM STS736 , a specific peptidyl ketoamide rhomboid inhibitor Ticha et al . , 2017b ( ) . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) and ectopic YqgP by anti - YqgP antibodies .

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

(B) The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor Ticha et al., 2017b(). Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.


( C ) To map the cleavage site region within endogenous MgtE ( second lane from the left , from strain BTM501 ) , MgtE - derived reference fragments encoding first 300 , 315 , 330 amino acids as well as full - length MgtE ( black arrow ) , were in vitro transcribed and translated . Mobility of the N - terminal cleavage product ( red arrow ) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments . Data information : In all panels , endogenous full - length MgtE is indicated by a black arrow , and N - terminal cleavage product by red arrows . Endogenous MgtE was visualised by anti - MgtE ( 2 - 275 ) ( α - MgtE ) and ectopic YqgP by anti - YqgP antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) To map the cleavage site region within endogenous MgtE (second lane from the left, from strain BTM501), MgtE-derived reference fragments encoding first 300, 315, 330 amino acids as well as full-length MgtE (black arrow), were in vitro transcribed and translated. Mobility of the N-terminal cleavage product (red arrow) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments. Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.


( A ) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B . subtilis cells ( BS72 , Table EV2 ) depending on the concentrations of magnesium and manganese ions . Cells were cultivated in glucose M9 minimal medium with limiting ( 0 . 01 mM ) or high ( 1 mM ) concentration of MgSO4 , in the presence or absence of 100 μM MnCl2 , and analysed by western blotting with near - infrared detection ( upper panel ) . Black arrow denotes full - length MgtE , and red arrow denotes its N - terminal cleavage product formed by YqgP . The corresponding fluorescence signals were quantified by densitometry , and are displayed as relative specific activity , which is substrate conversion normalized to enzyme expression level ( lower panel ) .

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

(A) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B. subtilis cells (BS72, Table EV2) depending on the concentrations of magnesium and manganese ions. Cells were cultivated in glucose M9 minimal medium with limiting (0.01 mM) or high (1 mM) concentration of MgSO4, in the presence or absence of 100 μM MnCl2, and analysed by western blotting with near-infrared detection (upper panel). Black arrow denotes full-length MgtE, and red arrow denotes its N-terminal cleavage product formed by YqgP. The corresponding fluorescence signals were quantified by densitometry, and are displayed as relative specific activity, which is substrate conversion normalized to enzyme expression level (lower panel).


( B ) Growth curves of wild - type ( BTM843 , Table EV2 ) , yqgP deficient ( BTM844 , Table EV2 ) and rescue ( BTM845 , Table EV2 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese stress elicited by adding 75 µM MnSO4 in mid - exponential phase ( stress - phase denoted by blueish background ) . All strains further contain a deletion in the putative manganese efflux pump MntP ( ΔywlD , Table EV2 ) . Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain , and that reintroduction of YqgP rescues fitness during manganese stress to above - wild - type level . Top panel shows no difference between the strains in the absence of manganese stress . Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time - point , which illustrates the reproducibility of the assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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-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-CONTROLLED_VAR 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

(B) Growth curves of wild-type (BTM843, Table EV2), yqgP deficient (BTM844, Table EV2) and rescue (BTM845, Table EV2) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese stress elicited by adding 75 µM MnSO4 in mid-exponential phase (stress-phase denoted by blueish background). All strains further contain a deletion in the putative manganese efflux pump MntP (ΔywlD, Table EV2). Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain, and that reintroduction of YqgP rescues fitness during manganese stress to above-wild-type level. Top panel shows no difference between the strains in the absence of manganese stress. Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time-point, which illustrates the reproducibility of the assay.


( C ) Top panel : growth curves of wild - type ( BTM843 ) , yqgP deficient ( BTM844 ) and rescue ( BTM845 ) strains of B . subtilis in M9 minimal medium with limiting magnesium ( 0 . 01 mM MgSO4 ) , exposed to manganese shock elicited by adding 75 µM MnSO4 in mid - exponential phase . Bottom panel : manganese toxicity is prevented by further adding 5 mM magnesium ( MgSO4 ) in otherwise identical conditions .

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

(C) Top panel: growth curves of wild-type (BTM843), yqgP deficient (BTM844) and rescue (BTM845) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese shock elicited by adding 75 µM MnSO4 in mid-exponential phase. Bottom panel: manganese toxicity is prevented by further adding 5 mM magnesium (MgSO4) in otherwise identical conditions.


( D ) Inhibition of YqgP by a rhomboid - specific peptidyl ketoamide inhibitor ( STS736 , i . e . compound 9 from Ticha et al . , 2017b ( ) ) abolishes the YqgP induced fitness of B . subtilis under manganese stress , in a dose - dependent manner , both with endogenous YqgP ( top panel ) and overexpressed YqgP ( bottom panel ) . Media and growth conditions were identical to those used in panel ( B ) .

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 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

(D) Inhibition of YqgP by a rhomboid-specific peptidyl ketoamide inhibitor (STS736, i.e. compound 9 from Ticha et al., 2017b()) abolishes the YqgP induced fitness of B. subtilis under manganese stress, in a dose-dependent manner, both with endogenous YqgP (top panel) and overexpressed YqgP (bottom panel). Media and growth conditions were identical to those used in panel (B).


( E ) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain ( BTM610 , Table EV2 ) in rich LB medium supplemented with 75 μM MnSO4 . Cell fitness is improved by overexpression of YqgP ( BTM611 , Table EV2 ) or its catalytically dead mutant YqgP . S288A ( BTM612 , Table EV2 ) . For clarity , for panels C - E , representative experiments of 2 - 3 independent biological replicates are shown .

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(E) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain (BTM610, Table EV2) in rich LB medium supplemented with 75 μM MnSO4. Cell fitness is improved by overexpression of YqgP (BTM611, Table EV2) or its catalytically dead mutant YqgP.S288A (BTM612, Table EV2). For clarity, for panels C - E, representative experiments of 2-3 independent biological replicates are shown.


( B ) Detection ( left panel ) and quantification ( right panel ) of steady - state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA Stevenson et al . , 2007Ticha et al . , 2017aTicha et al . , 2017b ( , , ) ( schemes in the middle ) by ectopically expressed YqgP and its N - and C - terminally truncated variants ( strains BS184 - 187 , Table EV2 ) in living B . subtilis grown in rich LB medium . Black arrows indicate full - length substrates and red arrows indicate cleavage products .

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(B) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA Stevenson et al., 2007Ticha et al., 2017aTicha et al., 2017b(, , ) (schemes in the middle) by ectopically expressed YqgP and its N- and C-terminally truncated variants (strains BS184-187, Table EV2) in living B. subtilis grown in rich LB medium. Black arrows indicate full-length substrates and red arrows indicate cleavage products.


( C ) Similar analysis of the same strains but grown in minimal M9 medium ( with 10 µM MgSO4 ) supplemented with increasing concentrations of MnCl 2 . Black arrows denote full - length substrate ( MgtE ) , red arrows denote the cleavage products generated by YqgP . ( D ) Western blots of four independent experiments shown in panel ( C ) were quantified by near - infrared fluorescence detection , quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl 2 relative to 1 µM MnCl 2 activity . Average values and all fours datapoints are plotted for each indicated YqgP variant .

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

(C) Similar analysis of the same strains but grown in minimal M9 medium (with 10 µM MgSO4) supplemented with increasing concentrations of MnCl2. Black arrows denote full-length substrate (MgtE), red arrows denote the cleavage products generated by YqgP. (D) Western blots of four independent experiments shown in panel (C) were quantified by near-infrared fluorescence detection, quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl2 relative to 1 µM MnCl2 activity. Average values and all fours datapoints are plotted for each indicated YqgP variant.


( A ) Detection ( left panel ) and quantification ( right panel ) of steady - state conversion of endogenous MgtE by YqgP ( strain BS72 , Table EV2 ) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 and CaCl2 ( reference conditions ) with the additions of 10 µM or 100 μM of a given divalent cation salt solution ( MnCl2 , ZnCl2 , CoCl2 , or NiCl2 ) , or 50 µM and 500 μM for CaCl2 . Western blots were quantified by near - infrared fluorescence detection ( left ) , quantified by densitometry and displayed as relative specific activity ( graph on the right ) , which is substrate conversion normalized to enzyme expression level . Black arrow denotes full - length substrate ( MgtE ) , and red arrow denotes the N - terminal cleavage product ( s ) generated by YqgP . All bands originate from the same western blot and identical treatment series .

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

(A) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE by YqgP (strain BS72, Table EV2) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (reference conditions) with the additions of 10 µM or 100 μM of a given divalent cation salt solution (MnCl2, ZnCl2, CoCl2, or NiCl2), or 50 µM and 500 μM for CaCl2. Western blots were quantified by near-infrared fluorescence detection (left), quantified by densitometry and displayed as relative specific activity (graph on the right), which is substrate conversion normalized to enzyme expression level. Black arrow denotes full-length substrate (MgtE), and red arrow denotes the N-terminal cleavage product(s) generated by YqgP. All bands originate from the same western blot and identical treatment series.


( B ) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations .

(B) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations.


( C ) Cation toxicity assays and their relationship to YqgP activity . Wild type B . subtilis ( BTM2 , Table EV2 ) , its variant lacking YqgP ( ΔyqgP , BTM78 , Table EV2 ) , and the rescue strain ectopically expressing YqgP ( BTM501 , Table EV2 ) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2 , ZnCl2 , CoCl2 , NiCl2 and CaCl2 ( i . e . reference conditions ) , and in mid - exponential phase were stressed by the addition of either 70 µM MnCl2 , 500 µM ZnCl2 , 25 µM CoSO4 or 400 µM NiCl2 ( pale blue area ) . YqgP activity specifically improved cell fitness during manganese and zinc stress , while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations . Representative experiments of 2 - 3 independent replicates are shown .

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

(C) Cation toxicity assays and their relationship to YqgP activity. Wild type B. subtilis (BTM2, Table EV2), its variant lacking YqgP (ΔyqgP, BTM78, Table EV2), and the rescue strain ectopically expressing YqgP (BTM501, Table EV2) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (i.e. reference conditions), and in mid-exponential phase were stressed by the addition of either 70 µM MnCl2, 500 µM ZnCl2, 25 µM CoSO4 or 400 µM NiCl2 (pale blue area). YqgP activity specifically improved cell fitness during manganese and zinc stress, while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations. Representative experiments of 2-3 independent replicates are shown.


( E Detection of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 . Black arrow marks full - length MgtE , and red arrow marks its N - terminal cleavage product by YqgP .

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 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

(E Detection of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2. Black arrow marks full-length MgtE, and red arrow marks its N-terminal cleavage product by YqgP.


F ) quantification of steady - state conversions of endogenous MgtE by YqgP variants bearing single - point mutations in putative Mn - binding region ( BS184 ; 187 ; 196 - 203 , Table EV2 ) , cultivated in modified M9 minimal medium supplemented by low ( 1 μM ) or high ( 100 μM ) MnCl2 .

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 B-CONTROLLED_VAR O

F) quantification of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2.


( A ) The proteolytically dead mutant YqgP . S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH . Pattern of C - terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP ( strain BS72 , Table EV2 ) or its inactive S288A mutant ( BS73 , Table EV2 ) in rich LB medium in the presence or absence of endogenous FtsH . Black arrow denotes the full - length MgtE substrate , and red arrows denote its cleavage products generated by YqgP . While the presence of the faster migrating YqgP - dependent products ( full red arrows ) do not depend on the presence of FtsH , the slower migrating product formed in the presence of YqgP . S288A ( open red arrow ) does , indicating that FtsH protease is responsible for the alternative cleavage of MgtE . The same arrow symbolics are valid throughout this figure .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The proteolytically dead mutant YqgP.S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH. Pattern of C-terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP (strain BS72, Table EV2) or its inactive S288A mutant (BS73, Table EV2) in rich LB medium in the presence or absence of endogenous FtsH. Black arrow denotes the full-length MgtE substrate, and red arrows denote its cleavage products generated by YqgP. While the presence of the faster migrating YqgP-dependent products (full red arrows) do not depend on the presence of FtsH, the slower migrating product formed in the presence of YqgP.S288A (open red arrow) does, indicating that FtsH protease is responsible for the alternative cleavage of MgtE. The same arrow symbolics are valid throughout this figure.


( B ) Mapping of the boundary of the FtsH generated proteolytic product of MgtE . Immunoblot comparing the SDS PAGE mobility of the N - terminal fragment of endogenous MgtE generated by FtsH in the presence of the pseudoprotease version of YqgP ( catalytically dead YqgP . S288A , strains BTM78 , BTM502 , Table EV2 ) to the mobilities of in vitro translated reference fragments corresponding to MgtE 1 - 340 , - 355 and - 370 shows that YqgP . S288A acts as an FtsH adaptor , and that FtsH processing of MgtE in the presence of YqgP . S288A stops at around amino acid 355 , which is near or at the cytoplasmic end of TMH 3 of MgtE .

(B) Mapping of the boundary of the FtsH generated proteolytic product of MgtE. Immunoblot comparing the SDS PAGE mobility of the N-terminal fragment of endogenous MgtE generated by FtsH in the presence of the pseudoprotease version of YqgP (catalytically dead YqgP.S288A, strains BTM78, BTM502, Table EV2) to the mobilities of in vitro translated reference fragments corresponding to MgtE 1-340, -355 and -370 shows that YqgP.S288A acts as an FtsH adaptor, and that FtsH processing of MgtE in the presence of YqgP.S288A stops at around amino acid 355, which is near or at the cytoplasmic end of TMH 3 of MgtE.


( D ) In vivo cleavage of endogenous MgtE in the presence of ectopically expressed full - length YqgP . S288A ( strain BS73 ) , YqgPΔNTD . S288A ( BS185 , Table EV2 ) or YqgPΔCTD . S288A ( BS186 , Table EV2 ) , in rich LB medium shows that YqgPNTD is necessary for the FtsH - dependent processing of MgtE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vivo cleavage of endogenous MgtE in the presence of ectopically expressed full-length YqgP.S288A (strain BS73), YqgPΔNTD.S288A (BS185, Table EV2) or YqgPΔCTD.S288A (BS186, Table EV2), in rich LB medium shows that YqgPNTD is necessary for the FtsH-dependent processing of MgtE.


( E ) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products . B . subtilis strains expressing YqgP or its S288A mutant ( strains BTM78 ; 501 and 502 , Table EV2 ) in the presence or absence of endogenous FtsH protease ( strains BTM795 , BTM796 and BTM797 , Table EV2 ) were cultured in LB medium and at early exponential phase , after having been expressing YqgP variants for 30 min , were either left grown untreated ( as a control with ongoing translation ) or treated with 20 μg / mL tetracycline to stop proteosynthesis ( translation shut - off ) . At given time intervals afterwards , all cultures were analysed by α - MgtE western blotting with near infrared detection . Equal cell number ( judged by OD600 ) was loaded into each lane . Black arrow marks full - length MgtE , red full arrows mark YqgP - dependent and red open arrows mark YqgP . S288A - dependent cleavage product of MgtE .

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

(E) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products. B. subtilis strains expressing YqgP or its S288A mutant (strains BTM78; 501 and 502, Table EV2) in the presence or absence of endogenous FtsH protease (strains BTM795, BTM796 and BTM797, Table EV2) were cultured in LB medium and at early exponential phase, after having been expressing YqgP variants for 30 min, were either left grown untreated (as a control with ongoing translation) or treated with 20 μg/mL tetracycline to stop proteosynthesis (translation shut-off). At given time intervals afterwards, all cultures were analysed by α-MgtE western blotting with near infrared detection. Equal cell number (judged by OD600) was loaded into each lane. Black arrow marks full-length MgtE, red full arrows mark YqgP-dependent and red open arrows mark YqgP.S288A-dependent cleavage product of MgtE.


A , B In - gel kinase assay of OST1 activity in ABA receptor mutants under cold stress . Ten - day - old wild type , ost1 - 3 , pyr1 pyl1 , 4 , pyr1 pyl1 , 2 , 4 , pyr1 pyl4 , 5 , 8 and pyr1 pyl1 , 4 , 5 , 8 mutants were treated at 4 ° C for 2 h . Total protein extracts were prepared and separated on SDS - PAGE gel containing 0 . 1 mg / mL GST - ∆ ABF2 as a substrate , and incubated with 60 μCi of [ γ - 32P ] ATP . Representative pictures are shown in ( A ) and relative kinase activity is shown in ( B ) . Data information : In ( A , Top , gel autoradiograph ; bottom , Coomassie Brilliant Blue ( CBB ) staining of RuBisCO large subunit was used as a loading control . The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In ( B , each bar represents the mean ± SEM of three biological repeats . * P < 0 . 05 ( two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B In-gel kinase assay of OST1 activity in ABA receptor mutants under cold stress. Ten-day-old wild type, ost1-3, pyr1 pyl1,4, pyr1 pyl1,2,4, pyr1 pyl4,5,8 and pyr1 pyl1,4,5,8 mutants were treated at 4°C for 2 h. Total protein extracts were prepared and separated on SDS-PAGE gel containing 0.1 mg/mL GST-∆ABF2 as a substrate, and incubated with 60 μCi of [γ-32P]ATP. Representative pictures are shown in (A) and relative kinase activity is shown in (B). Data information: In (A , Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (B , each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).


C , D In - gel kinase assay of OST1 activity in ABI1 - OE14 and abi1 abi2 hab1 mutants under cold stress . Representative pictures are shown in ( C ) and relative kinase activity is shown in ( D ) . Data information : In C ) , Top , gel autoradiograph ; bottom , Coomassie Brilliant Blue ( CBB ) staining of RuBisCO large subunit was used as a loading control . The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In D ) , each bar represents the mean ± SEM of three biological repeats . * P < 0 . 05 ( two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D In-gel kinase assay of OST1 activity in ABI1-OE14 and abi1 abi2 hab1 mutants under cold stress. Representative pictures are shown in (C) and relative kinase activity is shown in (D). Data information: In C), Top, gel autoradiograph; bottom, Coomassie Brilliant Blue (CBB) staining of RuBisCO large subunit was used as a loading control. The ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In D), each bar represents the mean ± SEM of three biological repeats. *P < 0.05 (two-tailed t-test).


A Interaction of OST1 and EGR2 in yeast . Yeast cells harboring different vector combinations were grown on SC / − Leu / − Trp medium for 3 d or SC / − Leu / − Trp / − His / − Ade medium for 6 d . OST1 - BD / AD and BD / EGR2 - AD were used as negative controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Interaction of OST1 and EGR2 in yeast. Yeast cells harboring different vector combinations were grown on SC/−Leu/−Trp medium for 3 d or SC/−Leu/−Trp/−His/−Ade medium for 6 d. OST1-BD/AD and BD/EGR2-AD were used as negative controls.


B OST1 interacts with EGR2 in vitro . Purified recombinant MBP - His - EGR2 or MBP - His proteins from E . coli were immunoprecipitated with MBP beads , and then incubated with purified recombinant GST - OST1 . Precipitated proteins were detected with anti - His and anti - GST antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 OST1 interacts with EGR2 in vitro. Purified recombinant MBP-His-EGR2 or MBP-His proteins from E. coli were immunoprecipitated with MBP beads, and then incubated with purified recombinant GST-OST1. Precipitated proteins were detected with anti-His and anti-GST antibodies.


C Co - IP assay of OST1 with EGR2 in vivo . 35S : HF - OST1 / Super : EGR2 - Myc or 35S : HF - OST1 / Super : Myc was expressed in N . benthamiana leaves . Total proteins were immunoprecipitated with anti - Myc agarose beads , and the co - immunoprecipitation products were subjected to immunoblot analysis . EGR2 - Myc and HF - OST1 were detected with anti - Myc and anti - HA antibodies , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Co-IP assay of OST1 with EGR2 in vivo. 35S:HF-OST1/Super:EGR2-Myc or 35S:HF-OST1/Super:Myc was expressed in N. benthamiana leaves. Total proteins were immunoprecipitated with anti-Myc agarose beads, and the co-immunoprecipitation products were subjected to immunoblot analysis. EGR2-Myc and HF-OST1 were detected with anti-Myc and anti-HA antibodies, respectively.


D Bimolecular fluorescence complementation ( BiFC ) assay . Wild - type Arabidopsis protoplasts transformed with OST1 - YFPC and EGR2 - YFPN proteins were incubated for 18 h . The interaction signal was detected by confocal microcopy . The combinations of OST1 - YFPC / GUS - YFPN and GUS - YFPC / EGR2 - YFPN were used as negative controls . Scale bars : 100 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

D Bimolecular fluorescence complementation (BiFC) assay. Wild-type Arabidopsis protoplasts transformed with OST1-YFPC and EGR2-YFPN proteins were incubated for 18 h. The interaction signal was detected by confocal microcopy. The combinations of OST1-YFPC/GUS-YFPN and GUS-YFPC/EGR2-YFPN were used as negative controls. Scale bars: 100 μm.


E EGR2 inhibits OST1 kinase activity in vitro . Proteins were incubated with 1 μCi [ γ - 32P ] ATP in kinase reaction buffer for 30 min at 30 ° C , and then separated by SDS - PAGE . Top , autoradiograph ; bottom , CBB staining .

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

E EGR2 inhibits OST1 kinase activity in vitro. Proteins were incubated with 1 μCi [γ-32P]ATP in kinase reaction buffer for 30 min at 30°C, and then separated by SDS-PAGE. Top, autoradiograph; bottom, CBB staining.


F , G In - gel assay of OST1 activity in 10 - d - old wild type , egr1 and egr2 single mutants , and the egr1 egr2 double mutant under cold stress . Data information : the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In ( F representative pictures are shown : Top , autoradiograph ; bottom , CBB staining . In ( G relative kinase activity is shown . Each bar represents the mean ± SEM of three biological repeats . * * P < 0 . 01 ( two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G In-gel assay of OST1 activity in 10-d-old wild type, egr1 and egr2 single mutants, and the egr1 egr2 double mutant under cold stress. Data information: the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In (F representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In (G relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


H , I In - gel assay of OST1 activity in 10 - d - old wild - type and EGR2 - overexpressing plants under cold stress . Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1 . 00 . In H ) , representative pictures are shown : Top , autoradiograph ; bottom , CBB staining . In I ) , relative kinase activity is shown . Each bar represents the mean ± SEM of three biological repeats . * * P < 0 . 01 ( two - tailed t - test ) .

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

H, I In-gel assay of OST1 activity in 10-d-old wild-type and EGR2-overexpressing plants under cold stress. Data information the ratio of band intensity of OST1 to RuBisCO in the wild type with cold treatment for 2 h was set to 1.00. In H), representative pictures are shown: Top, autoradiograph; bottom, CBB staining. In I), relative kinase activity is shown. Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


Freezing phenotype ( A ) of the egr2 - 3 mutant and egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) and acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) .

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

Freezing phenotype (A) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA).


survival rate ( B ) of the egr2 - 3 mutant and egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) and acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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

survival rate (B) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA). Data information each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


ion leakage ( C ) of the egr2 - 3 mutant and egr2 / EGR2 : EGR2 - Myc complementation lines under non - acclimated ( NA ) and acclimated ( CA ; 4 d at 4 ° C ) conditions . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar were treated at − 5 ° C for 0 . 5 h ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . Data information each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

ion leakage (C) of the egr2-3 mutant and egr2/EGR2:EGR2-Myc complementation lines under non-acclimated (NA) and acclimated (CA; 4 d at 4°C) conditions. Two-week-old seedlings grown on MS medium containing 0.8% agar were treated at −5°C for 0.5 h (NA) or −8°C for 0.5 h (CA). Data information each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Freezing phenotype of EGR2 - overexpressing plants . Two - week - old Super : EGR2 - Myc

Freezing phenotype of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants


survival rate ( E ) of EGR2 - overexpressing plants . Two - week - old Super : EGR2 - Myc plants Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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

survival rate (E) of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


ion leakage ( F ) of EGR2 - overexpressing plants . Two - week - old Super : EGR2 - Myc plants Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

ion leakage (F) of EGR2-overexpressing plants. Two-week-old Super:EGR2-Myc plants Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Expression of CBFs ( G in egr2 - 3 mutant plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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Expression of CBFs (G in egr2-3 mutant plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Expression of CBFs target genes ( H in egr2 - 3 mutant plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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

Expression of CBFs target genes (H in egr2-3 mutant plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Expression of CBFs in EGR2 - overexpressing plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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

Expression of CBFs in EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


Expression of CBFs target genes J ) in EGR2 - overexpressing plants . Two - week - old seedlings were treated at 4 ° C for the indicated period and subjected to qRT - PCR analysis . Data information : each bar represents the mean ± SEM of three independent experiments , each of which has three technical repeats . Asterisks indicate significant differences compared to the wild type with the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) .

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Expression of CBFs target genes J) in EGR2-overexpressing plants. Two-week-old seedlings were treated at 4°C for the indicated period and subjected to qRT-PCR analysis. Data information: each bar represents the mean ± SEM of three independent experiments, each of which has three technical repeats. Asterisks indicate significant differences compared to the wild type with the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test).


N - myristoylation of EGR2 and EGR2G2A in N . benthamiana leaves . Protein extracts prepared from N . benthamiana leaves expressing EGR2 - Myc or EGR2G2A - Myc were immunoprecipated by anti - Myc agarose beads . Precipitated proteins were detected by anti - Myc and anti - myristic acid antibodies .

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N-myristoylation of EGR2 and EGR2G2A in N. benthamiana leaves. Protein extracts prepared from N. benthamiana leaves expressing EGR2-Myc or EGR2G2A-Myc were immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies.


D Immunoblot analysis showing that the total level of myristoylated proteins in wild type plants . Total proteins were prepared from two - week - old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22 ° C for 72 h and then subjected to click - reaction with the biotin azide . The reaction was protected from light at room temperature for 3 h . The products were detected by anti - streptavidin - HRP antibody .

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D Immunoblot analysis showing that the total level of myristoylated proteins in wild type plants. Total proteins were prepared from two-week-old wild type seedlings treated with or without 80 μM myristic acid alkyne at 22°C for 72 h and then subjected to click-reaction with the biotin azide. The reaction was protected from light at room temperature for 3 h. The products were detected by anti-streptavidin-HRP antibody.


E N - myristoylation of EGR2 in plants . Total proteins were prepared from two - week - old EGR2 - Myc seedlings The products were immunoprecipated by anti - Myc agarose beads and detected by anti - streptavidin - HRP antibody . EGR2 - Myc detected by anti - Myc antibody was used as a loading control .

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

E N-myristoylation of EGR2 in plants. Total proteins were prepared from two-week-old EGR2-Myc seedlings The products were immunoprecipated by anti-Myc agarose beads and detected by anti-streptavidin-HRP antibody. EGR2-Myc detected by anti-Myc antibody was used as a loading control.


F Immunoblot assay of myristoylated EGR2 under cold stress . Total proteins were prepared form EGR2 - Myc plants treated with 4 ° C for the indicated period , and immunoprecipated by anti - Myc agarose beads . Precipitated proteins were detected by anti - Myc and anti - myristic acid antibodies . The ratio of band intensity detected by anti - myristic acid antibody to anti - Myc antibody without cold treatment was set to 1 . 00 . Representative pictures are shown ( left ) and quantitative analysis is shown ( right ) . Each bar represents the mean ± SEM of three biological repeats . * * P < 0 . 01 ( two - tailed t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot assay of myristoylated EGR2 under cold stress. Total proteins were prepared form EGR2-Myc plants treated with 4°C for the indicated period, and immunoprecipated by anti-Myc agarose beads. Precipitated proteins were detected by anti-Myc and anti-myristic acid antibodies. The ratio of band intensity detected by anti-myristic acid antibody to anti-Myc antibody without cold treatment was set to 1.00. Representative pictures are shown (left) and quantitative analysis is shown (right). Each bar represents the mean ± SEM of three biological repeats. **P < 0.01 (two-tailed t-test).


G Expression of EGR2 in egr2 - 3 EGR2 : EGR2G2A - Myc complementation transgenic plants . Total RNAs were extracted from 2 - week - old seedlings and subjected to RT - PCR analysis . EF1α was used as a loading control .

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G Expression of EGR2 in egr2-3 EGR2:EGR2G2A-Myc complementation transgenic plants. Total RNAs were extracted from 2-week-old seedlings and subjected to RT-PCR analysis. EF1α was used as a loading control.


Freezing tolerance assays of the egr2 - 3 mutant and EGR2 : EGR2G2A - Myc transgenic plants in egr2 - 3 mutant . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar at 22 ° C were treated at − 5 ° C for 0 - 1 . 5 h ( NA ) or − 8 ° C for 0 - 1 . 5 h ( CA ) . Representative photographs ( - 5oC , 0 . 5 h ; - 8oC , 0 . 5 h ) are shown in ( H )

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Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). Representative photographs (-5oC, 0.5 h; -8oC, 0.5 h) are shown in (H)


Freezing tolerance assays of the egr2 - 3 mutant and EGR2 : EGR2G2A - Myc transgenic plants in egr2 - 3 mutant . Two - week - old seedlings grown on MS medium containing 0 . 8 % agar at 22 ° C were treated at − 5 ° C for 0 - 1 . 5 h ( NA ) or − 8 ° C for 0 - 1 . 5 h ( CA ) . survival rate is shown in ( I ) . Data shown in ( I ) are mean values ± SEM of three biological replicates , each of which has three technical replicates . Asterisks indicate significant differences compared with wild type for the same treatment ( * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test ) ; n . s . , not significant .

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Freezing tolerance assays of the egr2-3 mutant and EGR2:EGR2G2A-Myc transgenic plants in egr2-3 mutant. Two-week-old seedlings grown on MS medium containing 0.8% agar at 22°C were treated at −5°C for 0-1.5 h (NA) or −8°C for 0-1.5 h (CA). survival rate is shown in (I). Data shown in (I) are mean values ± SEM of three biological replicates, each of which has three technical replicates. Asterisks indicate significant differences compared with wild type for the same treatment (*P < 0.05, **P < 0.01, two-tailed t-test); n. s., not significant.


A In vitro pull - down assay showing the interaction of NMT1 and EGR2 . Recombinant GST - NMT1 was incubated with immunoprecipated MBP - His - EGR2 or MBP - His at 4 ° C for 2 h . Anti - GST and anti - MBP were used to detect NMT1 , EGR2 and MBP - His , respectively .

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A In vitro pull-down assay showing the interaction of NMT1 and EGR2. Recombinant GST-NMT1 was incubated with immunoprecipated MBP-His-EGR2 or MBP-His at 4°C for 2 h. Anti-GST and anti-MBP were used to detect NMT1, EGR2 and MBP-His, respectively.


Co - localization of NMT1 and EGR2 in N . benthamiana leaves . Scale bar : 50

Co-localization of NMT1 and EGR2 in N. benthamiana leaves. Scale bar: 50 μm


C , D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co - IP assay . Arabidopsis protoplasts expressing HF / NMT1 - Myc and HF - EGR2 / NMT1 - Myc were treated with or without 4 ° C for 2 h . Total proteins were immunoprecipated by anti - HA agarose beads . Anti - Myc and anti - HA were used to detect NMT1 - Myc and HF - EGR2 , respectively . In ( C ) , relative band intensity is shown below the co - IP blot . In ( D ) , each bar represents the mean ± SEM of three biological experiments ( * * P < 0 . 01 , two - tailed t - test ) .

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C, D Effect of cold stress on the interaction of EGR2 and NMT1 determined by co-IP assay. Arabidopsis protoplasts expressing HF/NMT1-Myc and HF-EGR2/NMT1-Myc were treated with or without 4°C for 2 h. Total proteins were immunoprecipated by anti-HA agarose beads. Anti-Myc and anti-HA were used to detect NMT1-Myc and HF-EGR2, respectively. In (C), relative band intensity is shown below the co-IP blot. In (D), each bar represents the mean ± SEM of three biological experiments (**P < 0.01, two-tailed t-test).


E In vitro pull - down assay showing decreased interaction intensity of EGR2 ( 4 ° C ) and NMT1 compared with that of EGR2 ( 22 ° C ) and NMT1 . Total proteins prepared from two - week - old EGR2 - Myc plants grown at 4 ° C for 0 h and 6 h were immunoprecipated by anti - Myc agarose beads and incubated with GST - NMT1 in pull down buffer for 2 h at 4 ° C . Anti - Myc and anti - GST antibodies were used to detect EGR2 and NMT1 proteins . Relative band intensity is shown below the output blot .

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E In vitro pull-down assay showing decreased interaction intensity of EGR2 (4°C) and NMT1 compared with that of EGR2 (22°C) and NMT1. Total proteins prepared from two-week-old EGR2-Myc plants grown at 4°C for 0 h and 6 h were immunoprecipated by anti-Myc agarose beads and incubated with GST-NMT1 in pull down buffer for 2 h at 4°C. Anti-Myc and anti-GST antibodies were used to detect EGR2 and NMT1 proteins. Relative band intensity is shown below the output blot.


F Expression of NMT1 in NMT1 - Myc overexpression plants . EF1α was used as a loading control .

F Expression of NMT1 in NMT1-Myc overexpression plants. EF1α was used as a loading control.


Freezing phenotype ( G ) of NMT1 - Myc overexpression transgenic plants . Two - week - old Super : NMT1 - Myc plants were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) .

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Freezing phenotype (G) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA).


survival rate ( H ) of NMT1 - Myc overexpression transgenic plants . Two - week - old Super : NMT1 - Myc plants were treated at − 5 ° C ( NA ) or − 8 ° C for 0 . 5 h ( CA ) . In ( H ) , each bar represents the mean ± SEM of three biological experiments , and asterisks indicate significant differences compared with the wild type for the same treatment ( * P < 0 . 05 , two tailed t - test ) .

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survival rate (H) of NMT1-Myc overexpression transgenic plants. Two-week-old Super:NMT1-Myc plants were treated at −5°C (NA) or −8°C for 0.5 h (CA). In (H), each bar represents the mean ± SEM of three biological experiments, and asterisks indicate significant differences compared with the wild type for the same treatment (*P < 0.05, two tailed t-test).


OST1 kinase activity in NMT1 - Myc overexpression plants under cold stress . Top , autoradiograph ; bottom , CBB staining . Representative pictures are shown in ( I ) In ( I ) : the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1 . 00 .

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OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. Top, autoradiograph; bottom, CBB staining. Representative pictures are shown in (I) In (I): the ratio of band intensity of OST1 to RuBisCO in wild type with cold treatment for 2 h was set to 1.00.


OST1 kinase activity in NMT1 - Myc overexpression plants under cold stress . the relative kinase activity is shown in ( J ) . In ( J ) : each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two tailed t - test ) .

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OST1 kinase activity in NMT1-Myc overexpression plants under cold stress. the relative kinase activity is shown in (J). In (J): each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two tailed t-test).


A , B Pull - down assay . Total proteins prepared from EGR2 - Myc or EGR2G2A - Myc transgenic plants was immunoprecipated with anti - Myc agarose beads , and then incubated with GST - OST1 . Anti - Myc and anti - GST antibody were used to detect GST - OST1 and EGE2 - Myc . Representative pictures are shown in ( A ) . Relative interaction is shown in ( B ) . Data information : In ( A Relative band intensities are shown . In ( B each bar represents the mean ± SEM of three independent experiments ( * * P < 0 . 01 , two - tailed t - test ) .

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A, B Pull-down assay. Total proteins prepared from EGR2-Myc or EGR2G2A-Myc transgenic plants was immunoprecipated with anti-Myc agarose beads, and then incubated with GST-OST1. Anti-Myc and anti-GST antibody were used to detect GST-OST1 and EGE2-Myc. Representative pictures are shown in (A). Relative interaction is shown in (B). Data information: In (A Relative band intensities are shown. In (B each bar represents the mean ± SEM of three independent experiments (**P < 0.01, two-tailed t-test).


C LCI of EGR2 and EGR2G2A with OST1 in N . benthamiana leaves . EGR2 - cLuc / OST1 - nLuc and EGR2G2A - cLuc / OST1 - nLuc were expressed in N . benthamiana leaves for 48 h . EGR2 - cLuc / nLuc , EGR2G2A - cLuc / nLuc , and cLuc / OST1 - nLuc were used as negative controls . Scale bar : 2 cm .

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C LCI of EGR2 and EGR2G2A with OST1 in N. benthamiana leaves. EGR2-cLuc/OST1-nLuc and EGR2G2A-cLuc/OST1-nLuc were expressed in N. benthamiana leaves for 48 h. EGR2-cLuc/nLuc, EGR2G2A-cLuc/nLuc, and cLuc/OST1-nLuc were used as negative controls. Scale bar: 2 cm.