G ) Proximity ligation assay ( PLA ) for the OXPHOS subunits NDUFB8 and MTCO1 in adult hippocampal sections from Bcl7afl / fl ; Baf53b - Cretg / wt ( ko ; n = 4 ) and control littermates ( ctr ; n = 4 ) . Sections were co - stained with TOM20 and NeuN to label mitochondria and neurons , respectively . The total number of PLA dots was normalized to the mitochondrial area ( panel on the lower right ) .

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G) Proximity ligation assay (PLA) for the OXPHOS subunits NDUFB8 and MTCO1 in adult hippocampal sections from Bcl7afl/fl; Baf53b-Cretg/wt (ko; n=4) and control littermates (ctr; n=4). Sections were co-stained with TOM20 and NeuN to label mitochondria and neurons, respectively. The total number of PLA dots was normalized to the mitochondrial area (panel on the lower right).


A ) OCR measurements in wt and BCL7A KO eNPCs treated with 3 μM of CHIR99021 or DMSO ( as control , ctr ) for 24 h . The average maximal respiration upon FCCP treatment is shown on the right ( n = 5 - 7 biological replicates ) .

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A) OCR measurements in wt and BCL7A KO eNPCs treated with 3 μM of CHIR99021 or DMSO (as control, ctr) for 24 h. The average maximal respiration upon FCCP treatment is shown on the right (n= 5-7 biological replicates).


B ) OCR measurements in wt and BCL7A KO eNPCs following 48h of spontaneous differentiation . BCL7A KO eNPCs were treated for 72 h ( 24 h in proliferation plus 48 h in differentiation medium ) with CHIR99021 or DMSO . The average maximal respiration upon FCCP treatment is shown on the right ( n = 4 biological replicates ) .

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B) OCR measurements in wt and BCL7A KO eNPCs following 48h of spontaneous differentiation. BCL7A KO eNPCs were treated for 72 h (24 h in proliferation plus 48 h in differentiation medium) with CHIR99021 or DMSO. The average maximal respiration upon FCCP treatment is shown on the right (n= 4 biological replicates).


C ) Immunoblot analysis of differentiated BCL7A KO eNPCs exposed to 3 μM CHIR99021 , in presence or absence of 20 nM rotenone ( rot ) . Antibodies against β - III tubulin and actin were used .

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C) Immunoblot analysis of differentiated BCL7A KO eNPCs exposed to 3 μM CHIR99021, in presence or absence of 20 nM rotenone (rot). Antibodies against β-III tubulin and actin were used.


D ) Immunofluorescence staining of spontaneously differentiated eNPCs . BCL7A KO eNPCs were treated for 72 h ( i . e . , 24 h in proliferation plus 48 h in differentiation medium ) with 3 μM CHIR99021 , in presence or absence of 20 nM rotenone . Scale bar = 50 μm . Statistics are shown on the right ( n = 3 - 6 technical replicates from 3 independent experiments ) .

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D) Immunofluorescence staining of spontaneously differentiated eNPCs. BCL7A KO eNPCs were treated for 72 h (i.e., 24 h in proliferation plus 48 h in differentiation medium) with 3 μM CHIR99021, in presence or absence of 20 nM rotenone. Scale bar= 50 μm. Statistics are shown on the right (n=3-6 technical replicates from 3 independent experiments).


E ) Sholl analysis of β - III tubulin - positive immature neurons following 7 days of spontaneous differentiation . BCL7A KO eNPCs were exposed to 3 μM CHIR99021 , in presence or absence of 20 nM rotenone ( n = 3 biological replicates ) .

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E) Sholl analysis of β-III tubulin-positive immature neurons following 7 days of spontaneous differentiation. BCL7A KO eNPCs were exposed to 3 μM CHIR99021, in presence or absence of 20 nM rotenone (n=3 biological replicates).


F ) Representative OCR measurements in BCL7A KO eNPCs treated with 5 and 10 μM of pioglitazone or DMSO ( as control , ctr ) for 24 h . The average maximal respiration upon FCCP treatment is shown on the right ( n = 3 biological replicates ) .

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F) Representative OCR measurements in BCL7A KO eNPCs treated with 5 and 10 μM of pioglitazone or DMSO (as control, ctr) for 24 h. The average maximal respiration upon FCCP treatment is shown on the right (n= 3 biological replicates).


G ) Immunofluorescence staining of spontaneously differentiated eNPCs . BCL7A KO eNPCs were treated for 72 h ( i . e . , 24 h in proliferation plus 48 h in differentiation medium ) with 5 μM pioglitazone . The percentage of β - III - tubulin - positive cells is shown on the right ( n = 3 - 8 technical replicates from 2 independent experiments ) .

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G) Immunofluorescence staining of spontaneously differentiated eNPCs. BCL7A KO eNPCs were treated for 72 h (i.e., 24 h in proliferation plus 48 h in differentiation medium) with 5 μM pioglitazone. The percentage of β-III-tubulin-positive cells is shown on the right (n=3-8 technical replicates from 2 independent experiments).


C , D , E Examples of embryos expressing the tFT - Bcd reporter with C : mCherry - sfGFP - Bcd , D : fmCherry - sfGFP - Bcd , and E : mCherry - sfGFP . For all , ( i ) Images of " shells " of embryos in early n . c . 14 . 3D images of the embryos were generated , and then the interior of the embryo was erased , leaving only the embryo cortex to improve clarity ( ii ) Mean AP intensity profile of each color for the embryo in i . Shade region represents ± 1 s . d . Inset shows same profiles after multiplication of red intensity by constant factor . Data binned into 10 µm bins ( n = 4 embryos ) . ( iii ) Ratio of green over red signal , reflecting protein age . The thin lines represent individual embryos , while the thick solid line is the mean . The solid dashed line depicts the mean green / red ratio for a line with the tFT - Bcd reporter but lacking endogenous Bcd ( BcdE1 mutant , n = 4 ) .

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C,D,E Examples of embryos expressing the tFT-Bcd reporter with C: mCherry-sfGFP-Bcd, D: fmCherry-sfGFP-Bcd, and E: mCherry-sfGFP. For all, (i) Images of "shells" of embryos in early n.c. 14. 3D images of the embryos were generated, and then the interior of the embryo was erased, leaving only the embryo cortex to improve clarity (ii) Mean AP intensity profile of each color for the embryo in i. Shade region represents ±1 s.d. Inset shows same profiles after multiplication of red intensity by constant factor. Data binned into 10 µm bins (n=4 embryos). (iii) Ratio of green over red signal, reflecting protein age. The thin lines represent individual embryos, while the thick solid line is the mean. The solid dashed line depicts the mean green/red ratio for a line with the tFT-Bcd reporter but lacking endogenous Bcd (BcdE1 mutant, n=4).


, B , C Injection of the proteasome inhibitor MG132 in embryos increases both the amount and the average age of Bcd ( gray , open squares , N = 10 ) . Control embryos were injected with DMSO ( black , filled circles , N = 9 ) . All embryos are y / w and were injected while in cycle 14 and imaged 60 min later .

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,B,C Injection of the proteasome inhibitor MG132 in embryos increases both the amount and the average age of Bcd (gray, open squares, N=10). Control embryos were injected with DMSO (black, filled circles, N=9). All embryos are y/w and were injected while in cycle 14 and imaged 60 min later.


D The age of Bcd and its gradient are unaltered in fsd mutant . Comparison of a representative y / w embryo ( left ) and loss - of - function mutation fsdKG02393 ( right ) are shown . Both embryos have the tFT - Bcd construct in the same locus , are in early n . c . 14 , were imaged side - by - side under identical conditions , and are displayed with the same settings . Embryo " shells " are displayed .

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D The age of Bcd and its gradient are unaltered in fsd mutant. Comparison of a representative y/w embryo (left) and loss-of-function mutation fsdKG02393 (right) are shown. Both embryos have the tFT-Bcd construct in the same locus, are in early n.c. 14, were imaged side-by-side under identical conditions, and are displayed with the same settings. Embryo "shells" are displayed.


E Quantification of embryos Fluorescence intensity from sfGFP ( green ) and mCherry ( magenta ) for wt ( N = 8 , filled circles ) and fsd ( N = 6 , open squares ) at early cycle 14 . Inset : tFT - Bcd ratio for wt ( black ) and FSD ( gray ) embryos .

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E Quantification of embryos Fluorescence intensity from sfGFP (green) and mCherry (magenta) for wt (N=8, filled circles) and fsd (N=6, open squares) at early cycle 14. Inset: tFT-Bcd ratio for wt (black) and FSD (gray) embryos.


Time - course of a representative y / w embryo with the tFT - Bcd reporter from the division cycle 10 to the early gastrulation . A SPIM images of the embryo early development . Embryo " shells " are displayed . Left : Green fluorescence , center : Red fluorescence , right : Intensity - weighted ratiometric image of the mCherry / sfGFP ratio , reporting average protein age . The scale corresponds to 50 μm . B Quantification of the same embryo as is A . Each line represents the fluorescence profile along the anterior - posterior axis of the embryo for a time frame ( separated by 6 min ) . In the lower panel , where the mCherry / sfGFP ratio is shown , the posterior half of the embryo is not shown because the signal drops to the background levels ( due to softer imaging conditions for the long term time - course ) . C The same data is shown as a function of time for different embryo segments .

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Time-course of a representative y/w embryo with the tFT-Bcd reporter from the division cycle 10 to the early gastrulation. A SPIM images of the embryo early development. Embryo "shells" are displayed. Left: Green fluorescence, center: Red fluorescence, right: Intensity-weighted ratiometric image of the mCherry/sfGFP ratio, reporting average protein age. The scale corresponds to 50 μm. B Quantification of the same embryo as is A. Each line represents the fluorescence profile along the anterior-posterior axis of the embryo for a time frame (separated by 6 min). In the lower panel, where the mCherry/sfGFP ratio is shown, the posterior half of the embryo is not shown because the signal drops to the background levels (due to softer imaging conditions for the long term time-course). C The same data is shown as a function of time for different embryo segments.


C . Aliquots of the supernatants of the indicated HEK293T transfectants ( all expressing λ chains ) were resolved under non - reducing conditions and blotted with anti - µ antibodies . Wild type µs chains are secreted only as pentamers and hexamers , whilst mutants lacking C575 release mostly µs2L2 subunits ( indicated with a red arrow ) as does the double C414A - C575A . L566A releases instead mainly µs2L2 subunits of faster migration ( blue arrow ) . C414 mutants release no hexamers , but smaller assemblies , including fast migrating µs2L2 subunits .

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C. Aliquots of the supernatants of the indicated HEK293T transfectants (all expressing λ chains) were resolved under non-reducing conditions and blotted with anti-µ antibodies. Wild type µs chains are secreted only as pentamers and hexamers, whilst mutants lacking C575 release mostly µs2L2 subunits (indicated with a red arrow) as does the double C414A-C575A. L566A releases instead mainly µs2L2 subunits of faster migration (blue arrow). C414 mutants release no hexamers, but smaller assemblies, including fast migrating µs2L2 subunits.


E . Aliquots of the supernatants were treated with or without Endo - H and electrophoresed under reducing conditions . The blot was decorated with anti - µ antibodies . N563 - glycans acquire Endo - H resistance in µsL566A and µsC575A , but remain largely sensitive in wild type µs .

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E. Aliquots of the supernatants were treated with or without Endo-H and electrophoresed under reducing conditions. The blot was decorated with anti-µ antibodies. N563-glycans acquire Endo-H resistance in µsL566A and µsC575A, but remain largely sensitive in wild type µs.


A . Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non - reducing conditions before or after removal of N - glycans by PNGaseF . Blots were decorated with anti - µ antibodies . Blue and red arrows point to the closed ( oxidized ) and open µs2L2 species respectively .

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A. Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non-reducing conditions before or after removal of N-glycans by PNGaseF. Blots were decorated with anti-µ antibodies. Blue and red arrows point to the closed (oxidized) and open µs2L2 species respectively.


Intracellular µs2L2 species purified from the murine hybridoma B1 . 8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS / MS spectrum Y and B ions are shown in blue and red , respectively .

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Intracellular µs2L2 species purified from the murine hybridoma B1.8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS/MS spectrum Y and B ions are shown in blue and red, respectively.


A . I . 29µ + B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion . At the indicated times , cells were lysed in TritonX114 to separate soluble and integral membrane proteins ( aqueous and detergent phases , respectively ) . Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti - µ antibodies . The blue arrow points at a band likely corresponding to µs2L2C575OX , the intensity of which increases upon LPS stimulation .

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A. I.29µ+ B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion. At the indicated times, cells were lysed in TritonX114 to separate soluble and integral membrane proteins (aqueous and detergent phases, respectively). Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti-µ antibodies. The blue arrow points at a band likely corresponding to µs2L2C575OX, the intensity of which increases upon LPS stimulation.


C . µ - chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM .

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C. µ-chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM.


D . The bar graphs show the levels of C575 and C414 oxidation , expressed as the ratio between IAA and NEM modified peptides .

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D. The bar graphs show the levels of C575 and C414 oxidation, expressed as the ratio between IAA and NEM modified peptides.


ERp44KO HeLa cells expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains and Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . A . Aliquots from the spent media ( Extracellular ) were blotted under non - reducing conditions and decorated with anti - µ antibodies . The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575 - dependent retention , and wild type µs are secreted only as polymers ( black arrow ) . Traces of covalent HaloERp44 - µ2L2 complexes ( black asterisks ) are detected in the media of cells expressing HaloERp44 ∆ RDEL and µs or L566A . The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44 ∆ RDEL suggest that the latter inhibits processing of the N563 glycan , likely hindering it to the Golgi enzymes upon non - covalent binding . Data information : All membranes were decorated with anti - μ antibodies . Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition .

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ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. A. Aliquots from the spent media (Extracellular) were blotted under non-reducing conditions and decorated with anti-µ antibodies. The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575-dependent retention, and wild type µs are secreted only as polymers (black arrow). Traces of covalent HaloERp44-µ2L2 complexes (black asterisks) are detected in the media of cells expressing HaloERp44∆RDEL and µs or L566A. The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44∆RDEL suggest that the latter inhibits processing of the N563 glycan, likely hindering it to the Golgi enzymes upon non-covalent binding. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.


ERp44KO HeLa cells expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains and Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . B . The corresponding cell lysates are shown in this panel . The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44 . The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2 . Traces of intracellular µs2L2 - ERp44 ∆ RDEL complexes are visible in µs and L566A but absent from C575A expressing cells . Data information : All membranes were decorated with anti - μ antibodies . Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition .

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ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. B. The corresponding cell lysates are shown in this panel. The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44. The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2. Traces of intracellular µs2L2-ERp44∆RDEL complexes are visible in µs and L566A but absent from C575A expressing cells. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.


C . Lysates of the ERp44KO transfectants were precipitated with NP - sepharose , resolved under non reducing conditions and the blot decorated sequentially with anti - µ , anti - ERp44 or anti - Halo . The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo - tagged ERp44 .

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C. Lysates of the ERp44KO transfectants were precipitated with NP-sepharose, resolved under non reducing conditions and the blot decorated sequentially with anti-µ, anti-ERp44 or anti-Halo. The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo-tagged ERp44.


B . To selectively label intracellular HC575A , cells were treated with 1 nM Halo - tag TMR Direct Ligand for 24 hours . The left panel shows aliquots of the lysates of the indicated transfectants , resolved under non - reducing conditions and decorated with anti - λ antibodies . In the two right panels , samples were precipitated with NP - Sepharose and electrophoresed under non reducing conditions . The signal for the Halo - tag TMR was checked directly in the gel . The arrows point at the various assemblies between µ , HC575A and λ chains .

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B. To selectively label intracellular HC575A, cells were treated with 1 nM Halo-tag TMR Direct Ligand for 24 hours. The left panel shows aliquots of the lysates of the indicated transfectants, resolved under non-reducing conditions and decorated with anti-λ antibodies. In the two right panels, samples were precipitated with NP-Sepharose and electrophoresed under non reducing conditions. The signal for the Halo-tag TMR was checked directly in the gel. The arrows point at the various assemblies between µ, HC575A and λ chains.


C . The experiment shown aims to determine whether ERp44 can bind also to a single C575 , or rather to non - native bonds . Aliquots of the lysates from the indicated ERp44KO transfectants were precipitated with NP - Sepharose , resolved under non reducing conditions and the blot decorated with anti - λ antibodies or anti - ERp44 as indicated . Black asterisks point to covalent complexes of Halo - ERp44 with µs2L2 .

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C. The experiment shown aims to determine whether ERp44 can bind also to a single C575, or rather to non-native bonds. Aliquots of the lysates from the indicated ERp44KO transfectants were precipitated with NP-Sepharose, resolved under non reducing conditions and the blot decorated with anti-λ antibodies or anti-ERp44 as indicated. Black asterisks point to covalent complexes of Halo-ERp44 with µs2L2.


( A ) Quantification of CXCR4 + cells by flow cytometry and Sox17 + cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH . Average and standard deviation ( SD ) of eight independent experiments .

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(A) Quantification of CXCR4+ cells by flow cytometry and Sox17+ cells by immunostaining at day 7 following differentiation in the presence or absence of 3 µM CH. Average and standard deviation (SD) of eight independent experiments.


( B ) Gene expression profiling by RT - PCR at day 7 differentiation . Average and SD of three independent experiments .

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(B) Gene expression profiling by RT-PCR at day 7 differentiation. Average and SD of three independent experiments.


( C ) Immunostaining for Ecadherin ( left ) and FoxA2 ( right ) at day 7 of differentiation in presence of 3 µM CH .

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(C) Immunostaining for Ecadherin (left) and FoxA2 (right) at day 7 of differentiation in presence of 3 µM CH.


( D ) Quantification of CXCR4 + by flow cytometry over a seven - day differentiation time course in the presence of 3 µM CH . Average and SD of three independent experiments .

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(D) Quantification of CXCR4+ by flow cytometry over a seven-day differentiation time course in the presence of 3 µM CH. Average and SD of three independent experiments.


( E ) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH . Scale bars = 200

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(E) Immunostaining for Sox17 at day 4 to day 7 of differentiation in the continuous presence or absence of 3 µM CH. Scale bars = 200 µM


( A ) Assay of Tcf7l1 mRNA by RT - PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH . Average and SD of three independent experiments . Day2 * p = 0 . 048 , Day3 * p = 0 . 036 .

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(A) Assay of Tcf7l1 mRNA by RT-PCR for the first 4 days of differentiation in the presence or absence of 3 μM CH. Average and SD of three independent experiments. Day2 *p= 0.048, Day3 *p=0.036.


B ) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence ( + ) or absence ( - ) of 3 μM CH .

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B) Immunoblot for Tcf7l1 at day 3 of differentiation in the presence (+) or absence (-) of 3 μM CH.


( C ) Assay of FoxA2 mRNA by RT - PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . * * p = 5x10 - 7 .

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(C) Assay of FoxA2 mRNA by RT-PCR for the first 3 days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. **p=5x10-7.


( D ) ChiP - seq data from a compendium of transcription factor ChIP - seq analyses ( Sánchez - Castillo et al , 2015 ) . Gene tracks represent binding of Tcf7l1 at the FoxA2 , Nodal and Eomesodermin ( Eomes ) gene locus . Red lines indicate the regions analyzed by ChIP PCR for Tcf7l1 .

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(D) ChiP-seq data from a compendium of transcription factor ChIP-seq analyses (Sánchez-Castillo et al, 2015). Gene tracks represent binding of Tcf7l1 at the FoxA2, Nodal and Eomesodermin (Eomes) gene locus. Red lines indicate the regions analyzed by ChIP PCR for Tcf7l1.


( E ) ChIP for Tcf7l1 followed by qPCR for the regions indicated in Fig . 2D . Analysis was performed in wild - type ES cells in 2i culture conditions . Average and SD of three independent experiments . Klf2 * p = 0 . 025 , FoxA2 * p = 0 . 050 , Eomes * p = 0 . 023 .

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(E) ChIP for Tcf7l1 followed by qPCR for the regions indicated in Fig. 2D. Analysis was performed in wild-type ES cells in 2i culture conditions. Average and SD of three independent experiments. Klf2 *p=0.025, FoxA2 *p=0.050, Eomes *p=0.023.


( F ) ChIP for Tcf7l1 followed by qPCR . Analysis was performed in wild - type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments . FoxA2 * p = 0 . 049 .

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(F) ChIP for Tcf7l1 followed by qPCR. Analysis was performed in wild-type ES cells on day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments. FoxA2 *p = 0.049.


( A ) Assay of FoxA2 mRNA by RT - PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH . Average and SD of three independent experiments .

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(A) Assay of FoxA2 mRNA by RT-PCR in Tcf7l1 null cells on day 1 to 4 of differentiation without CH. Average and SD of three independent experiments.


( B ) Gene expression analysis of endodermal associated genes by RT - PCR in control wild - type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH . Average and SD of three independent experiments .

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(B) Gene expression analysis of endodermal associated genes by RT-PCR in control wild-type and Tcf7l1 null cells on day 4 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.


( C ) FoxA2 ( green ) and Oct4 ( red ) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH . DAPI staining in blue .

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(C) FoxA2 (green) and Oct4 (red) immunostaining of Tcf7l1 null ES cells on day 4 of differentiation in the presence or absence of 3 µM CH. DAPI staining in blue.


( D ) Assay of CXCR4 , Hex and Sox17 mRNA by RT - PCR in control wild - type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH . Average and SD of three independent experiments .

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(D) Assay of CXCR4, Hex and Sox17 mRNA by RT-PCR in control wild-type and Tcf7l1 null cells on day 6 of differentiation in the presence and absence of 3 µM CH. Average and SD of three independent experiments.


( A ) Assay of FoxA2 mRNA by RT - PCR in FoxA2 - Tet ES cell lines 48 hr after treatment with the indicated dose of DOX . Average and SD of two independent experiments from 2 independent lines .

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(A) Assay of FoxA2 mRNA by RT-PCR in FoxA2-Tet ES cell lines 48 hr after treatment with the indicated dose of DOX. Average and SD of two independent experiments from 2 independent lines.


B ) FoxA2 immunostaining of FoxA2 - Tet ES cells 48 hr after DOX treatment . Left , no DOX control . Middle and right , 10ng / ml DOX . DAPI staining shown in blue . Right panel shows middle image without DAPI signal .

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B) FoxA2 immunostaining of FoxA2-Tet ES cells 48 hr after DOX treatment. Left, no DOX control. Middle and right, 10ng/ml DOX. DAPI staining shown in blue. Right panel shows middle image without DAPI signal.


( C ) Assay of FoxA2 and CXCR4 mRNA by RT - PCR on day 3 of differentiation with DOX at the indicated dose . Average and SD of two experiments from two independent cell lines .

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(C) Assay of FoxA2 and CXCR4 mRNA by RT-PCR on day 3 of differentiation with DOX at the indicated dose. Average and SD of two experiments from two independent cell lines.


( D ) Assay of Sox17 mRNA by RT - PCR on day 7 of differentiation with addition of 3 µM CH and / or 10 ng / ml DOX at the indicated time points ( in days ) . Average and SD of two independent experiments from two independent cell lines .

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(D) Assay of Sox17 mRNA by RT-PCR on day 7 of differentiation with addition of 3 µM CH and/or 10 ng/ml DOX at the indicated time points (in days). Average and SD of two independent experiments from two independent cell lines.


( E ) Sox17 immunostaining of FoxA2 - Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng / ml DOX and the presence and absence of 3 µM CH as indicated . Far right panel shows image to its left without DAPI .

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(E) Sox17 immunostaining of FoxA2-Tet ES cells at day 7 of differentiation in the presence or absence of 10 ng/ml DOX and the presence and absence of 3 µM CH as indicated. Far right panel shows image to its left without DAPI.


( A ) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng / ml ( W100 ) or 200 ng / ml ( W200 ) recombinant Wnt3a plus 500 ng / ml recombinant RSpondin1 . Relative luciferase units are normalised to FOPflash . Average and SD of three independent experiments .

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(A) TOPFlash reporter activity following differentiation for 3 days in 3 µM CH or 100 ng/ml (W100) or 200 ng/ml (W200) recombinant Wnt3a plus 500 ng/ml recombinant RSpondin1. Relative luciferase units are normalised to FOPflash. Average and SD of three independent experiments.


( B ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a ( 200 ng / ml plus 500 ng / ml RSpondin1 ( WR ) ) . Average and SD of three independent experiments . * * p < 0 . 01 .

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(B) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR at day 2 of differentiation in the presence or absence of 3 µM CH or recombinant Wnt3a (200 ng/ml plus 500 ng/ml RSpondin1(WR)). Average and SD of three independent experiments. ** p<0.01.


( C ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH . Average and SD of three independent experiments .

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(C) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in Ctnnb1 null cells at day 1 and 3 of differentiation in the absence or presence of 3 µM CH. Average and SD of three independent experiments.


( D ) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH . Results from two independent , clonally - derived Ctnnb1 null ES cell lines are shown . Tubulin was used as a loading control .

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(D) Immunoblot for Tcf7l1 in Ctnnb1 null cells during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH. Results from two independent, clonally-derived Ctnnb1 null ES cell lines are shown. Tubulin was used as a loading control.


( A ) Assay of cMyc mRNA by RT - PCR in wild - type ES cells at day 1 to 3 of differentiation . Average and SD of three independent experiments .

(A) Assay of cMyc mRNA by RT-PCR in wild-type ES cells at day 1 to 3 of differentiation. Average and SD of three independent experiments.


( B ) Immunoblot for total cMyc and phospho Ser62 ( pSer62 ) cMyc during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH .

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(B) Immunoblot for total cMyc and phospho Ser62 (pSer62) cMyc during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH.


C , D ) Immunoblot for the destabilized form of cMyc protein ( pThr58 ) during the first 3 days of differentiation in the absence ( - ) or presence ( + ) of 3 µM CH in wild - type and Ctnnb1 null cells respectively .

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C,D) Immunoblot for the destabilized form of cMyc protein (pThr58) during the first 3 days of differentiation in the absence (-) or presence (+) of 3 µM CH in wild-type and Ctnnb1 null cells respectively.


( E ) Assay of Tcf7l1 mRNA by RT - PCR in wild - type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc ( cMyc Inhibitor II ; Myci ) . Treatment with Myc inhibitor 10058 - F4 ( Sigma ) generated similar results ( data not shown ) . Average and SD of three independent experiments . * * p = 0 . 0002 .

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(E) Assay of Tcf7l1 mRNA by RT-PCR in wild-type cells at day 2 of differentiation in the presence or absence of a small molecule inhibitor of cMyc (cMyc Inhibitor II; Myci). Treatment with Myc inhibitor 10058-F4 (Sigma) generated similar results (data not shown). Average and SD of three independent experiments. **p=0.0002.


( F ) Assay of Tcf7l1 and FoxA2 mRNA by RT - PCR in cMyc null / nMyc heterozygous cells ( C - / - ; N + / - ) and floxed parental cells ( cMycfl / fl ; nMycfl / fl ( control ) ) at day 3 of differentiation in the presence or absence of 3 µM CH . Average and SD of four or six independent experiments . Tcf7l1 * p = 0 . 01 , FoxA2 * p = 0 . 034 .

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(F) Assay of Tcf7l1 and FoxA2 mRNA by RT-PCR in cMyc null/nMyc heterozygous cells (C-/-;N+/-) and floxed parental cells (cMycfl/fl;nMycfl/fl (control)) at day 3 of differentiation in the presence or absence of 3 µM CH. Average and SD of four or six independent experiments. Tcf7l1 *p=0.01, FoxA2 *p=0.034.


( G ) FoxA2 immunostaining of control and cMyc null / nMyc heterozygous ( cMyc - / - ; nMyc + / - ) cells at day 3 of differentiation in the presence of 3 µM CH .

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(G) FoxA2 immunostaining of control and cMyc null/nMyc heterozygous (cMyc-/-;nMyc+/-) cells at day 3 of differentiation in the presence of 3 µM CH.


( H ) Assay of cMyc and Tcf7l1 mRNA by RT - PCR in cMycT58A inducible cell lines ( icMycT58A ) 24 hr after DOX treatment . Average of 2 independent experiments .

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(H) Assay of cMyc and Tcf7l1 mRNA by RT-PCR in cMycT58A inducible cell lines (icMycT58A) 24 hr after DOX treatment. Average of 2 independent experiments.


( I ) Assay of Tcf7l1 mRNA by RT - PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng / ml DOX . Average and SD of 3 independent experiments . * * p = 0 . 007 .

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(I) Assay of Tcf7l1 mRNA by RT-PCR in icMycT58A clone 1 cell line at day 3 of differentiation in the absence of 3 µM CH following cMyc induction with 10 ng/ml DOX. Average and SD of 3 independent experiments. **p=0.007.


( J ) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng / ml .

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(J) Immunoblot for cMyc and Tcf7l1 in icMycT58A clonal cell line 1 48 hr after DOX induction at 10ng/ml.


( B ) ChIP for cMyc performed in wild - type ES cells differentiated for 2 days in the absence or presence of 3 µM CH . qPCR was carried out for the regions indicated in Fig . 7a . Average and SD for five independent experiments , * p = 0 . 028 .

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(B) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the absence or presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, *p=0.028.


( C ) ChIP for cMyc performed in wild - type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058 - F4 . Average and SD for three independent experiments , * p = 0 . 026 .

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(C) ChIP for cMyc performed in wild-type ES cells differentiated for 2 days in the presence of 3 µM CH plus or minus Myc inhibitor 10058-F4. Average and SD for three independent experiments, *p=0.026.


( D ) Assay for Miz1 mRNA by RT - PCR for the first three days of differentiation in the presence or absence of 3 µM CH . Average and SD of three independent experiments .

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(D) Assay for Miz1 mRNA by RT-PCR for the first three days of differentiation in the presence or absence of 3 µM CH. Average and SD of three independent experiments.


( E ) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH . qPCR was carried out for the regions indicated in Fig . 7a . Average and SD for five independent experiments , * * p = 0 . 0015 .

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(E) ChIP for Miz1 or IgG control performed in E14 ES cells differentiated for 2 days in presence of 3 µM CH. qPCR was carried out for the regions indicated in Fig. 7a. Average and SD for five independent experiments, **p=0.0015.


B Trans - activation activity assays of CDF4 in Arabidopsis protoplasts . The full - length ORF of CDF4 and its four deletion constructs Dof ( gray boxes ) represent the DNA - binding domains of CDF4 . Negative control was the effector vector without gene inserts . The effector vector used is shown in the left panel . The GAL4 transient expression assays were performed using Arabidopsis protoplasts , shown in the right panel . Deletion of the CDF4 N - terminal domain compromised the transcriptional repression by CDF4 in our assay . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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B Trans-activation activity assays of CDF4 in Arabidopsis protoplasts. The full-length ORF of CDF4 and its four deletion constructs Dof (gray boxes) represent the DNA-binding domains of CDF4. Negative control was the effector vector without gene inserts. The effector vector used is shown in the left panel. The GAL4 transient expression assays were performed using Arabidopsis protoplasts, shown in the right panel. Deletion of the CDF4 N-terminal domain compromised the transcriptional repression by CDF4 in our assay. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


C Localized expression of CDF4 in a senescing rosette leaf . The ~ 50 % senesced rosette leaf was cut into three parts as indicated by the red lines . The level of CDF4 mRNA was determined in the three sections of the senescing leaf . Base ; Middle ; Tip . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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C Localized expression of CDF4 in a senescing rosette leaf. The ~50% senesced rosette leaf was cut into three parts as indicated by the red lines. The level of CDF4 mRNA was determined in the three sections of the senescing leaf. Base; Middle; Tip. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


D CDF4 expression level at various developmental stages of rosette leaves . YL , young leaf ; NS , fully expanded , non - senescent leaf ; ES , early - senescent leaf , with 1 / 4 leaf area turned yellow ; LS , late - senescent leaf , with > 50 % leaf area turned yellow . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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D CDF4 expression level at various developmental stages of rosette leaves. YL, young leaf; NS, fully expanded, non-senescent leaf; ES, early-senescent leaf, with 1/4 leaf area turned yellow; LS, late-senescent leaf, with >50% leaf area turned yellow. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


E Effect of plant growth hormones ( 1 mM SA , 1 μM ABA , 10 μM JA and 10 μM 1 - aminocyclopropane - 1 - carboxylic acid ( ACC ) on CDF4 expression . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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E Effect of plant growth hormones (1 mM SA, 1 μM ABA, 10 μM JA and 10 μM 1-aminocyclopropane -1-carboxylic acid (ACC) on CDF4 expression. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


F Effects of abiotic stress treatments on CDF4 expression . Rosette leaves were treated with salt ( 100 mM NaCl ) , drought , H2O2 ( 2mM ) or darkness . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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F Effects of abiotic stress treatments on CDF4 expression. Rosette leaves were treated with salt (100 mM NaCl), drought, H2O2 (2mM) or darkness. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


G Effects of SA , H2O2 , and dark treatment on CDF4 expression in the aba2 - 1 mutant background . AtACT2 was used as an internal control . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 .

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G Effects of SA, H2O2, and dark treatment on CDF4 expression in the aba2-1 mutant background. AtACT2 was used as an internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3.


A The age - dependent leaf senescence symptoms in the vector control and 35S : : CDF4 lines grown under long - day conditions were shown at 21 - days - old . The 35S : : CDF4 plants had a small leaf size and extremely early leaf senescent phenotype , while the control vector leaves were fully green . Scale bars indicate 1 cm .

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A The age-dependent leaf senescence symptoms in the vector control and 35S::CDF4 lines grown under long-day conditions were shown at 21-days-old. The 35S::CDF4 plants had a small leaf size and extremely early leaf senescent phenotype, while the control vector leaves were fully green. Scale bars indicate 1 cm.


B The increase in SAG12 , SAG13 , PR1 and PR5 gene expression levels were investigated in 35S : : CDF4 plants . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 in comparison with the vector control by Student ' s t test .

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B The increase in SAG12, SAG13, PR1 and PR5 gene expression levels were investigated in 35S::CDF4 plants. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 in comparison with the vector control by Student's t test.


C Phenotypes of vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi and pER8 : : amiCDF4 plants , after treatment with 20 µM estradiol for seven days at 18 - days - old . Scale bars indicate 1 . 5 cm .

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C Phenotypes of vector control, pER8::CDF4, pER8::CDF4-RNAi and pER8::amiCDF4 plants, after treatment with 20 µM estradiol for seven days at 18-days-old. Scale bars indicate 1.5 cm.


E Analysis of the relative chlorophyll concentration in the sixth and seventh rosette leaves from the vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi and pER8 : : amiCDF4 lines shown at various development stages . Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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E Analysis of the relative chlorophyll concentration in the sixth and seventh rosette leaves from the vector control, pER8::CDF4, pER8::CDF4-RNAi and pER8::amiCDF4 lines shown at various development stages. Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.


D The leaf senescence phenotypes of detached rosette leaves in vector control , pER8 : : CDF4 , pER8 : : CDF4 - RNAi and pER8 : : amiCDF4 lines before and after treatment with 2 mΜ H2O2 in the dark for four days . Scale bars indicate 0 . 7 cm .

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D The leaf senescence phenotypes of detached rosette leaves in vector control, pER8::CDF4, pER8::CDF4 -RNAi and pER8::amiCDF4 lines before and after treatment with 2 mΜ H2O2 in the dark for four days. Scale bars indicate 0.7 cm.


F The chlorophyll concentration in leaves was analyzed . The percentage indicates the chlorophyll content relative to vector control before treatment . Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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F The chlorophyll concentration in leaves was analyzed. The percentage indicates the chlorophyll content relative to vector control before treatment. Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.


A Measurement of free ABA levels in the third and fourth rosette leaves from 21 - day - old transgenic lines with altered CDF4 expression after estradiol induction for three days ( once every day ) . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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

A Measurement of free ABA levels in the third and fourth rosette leaves from 21-day-old transgenic lines with altered CDF4 expression after estradiol induction for three days (once every day). Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


B , C qPCR analysis of ABA synthesis and signaling - related genes in Col - 0 and 35S : : CDF4 plants . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s 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

B, C qPCR analysis of ABA synthesis and signaling-related genes in Col-0 and 35S::CDF4 plants. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.


D , E Relative expression of NCED2 and NCED3 in 14 - day - old CDF4GR transgenic plants treated with 20 μM β - estradiol or mock treatment for 0 , 2 , or 4 h . The expression of the corresponding genes in mock - treated plants was set to 1 . 0 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E Relative expression of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM β-estradiol or mock treatment for 0, 2, or 4 h. The expression of the corresponding genes in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


F , G Relative expression level of NCED2 and NCED3 in 14 - day - old CDF4GR transgenic plants treated with 20 μM DEX , 100μM CHX , DEX plus 100μM CHX , or mock . The gene expression in mock - treated plants was set to 1 . 0 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, G Relative expression level of NCED2 and NCED3 in 14-day-old CDF4GR transgenic plants treated with 20 μM DEX, 100μM CHX, DEX plus 100μM CHX, or mock. The gene expression in mock-treated plants was set to 1.0. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


H Schematic diagram indicating the locations of six DOF motif clusters ( D1 to D6 ) in the NCED2 and NCED3 gene promoters . I ChIP - qPCR analysis of the ability of CDF4 to bind to the promoters of NCED2 and NCED3 . An anti - HA monoclonal antibody was used for DNA immunoprecipitation from three - week - old pER8 : : CDF4 - HA transgenic plants . Black bars indicate the enrichment fold - changes normalized to that of ACT2 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t - test . Transient dual - luciferase reporter assay . The pGreenII - 0800 LUC construct containing the CDF4 promoter and the p62 - SK construct with or without the CDF4 coding region and CDF4 - VP16 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) and Renilla luciferase ( REN ) activities were measured after culturing the protoplasts under low light conditions for 16 h . The ProNCED2 / 3 : LUC / Pro35S : REN ratio represents the relative activities of NCED2 and NCED3 transcription . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s 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 O O O 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 Schematic diagram indicating the locations of six DOF motif clusters (D1 to D6) in the NCED2 and NCED3 gene promoters. I ChIP-qPCR analysis of the ability of CDF4 to bind to the promoters of NCED2 and NCED3. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from three-week-old pER8::CDF4-HA transgenic plants. Black bars indicate the enrichment fold-changes normalized to that of ACT2. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test. Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CDF4 promoter and the p62-SK construct with or without the CDF4 coding region and CDF4-VP16 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured after culturing the protoplasts under low light conditions for 16 h. The ProNCED2/3:LUC /Pro35S:REN ratio represents the relative activities of NCED2 and NCED3 transcription. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.


K Transient dual - luciferase reporter assay . The pGreenII - 0800 LUC construct containing the CDF4 promoter and the p62 - SK construct with or without the CDF4 coding region and CDF4 - VP16 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) and Renilla luciferase ( REN ) activities were measured after culturing the protoplasts under low light conditions for 16 h . The ProNCED2 / 3 : LUC / Pro35S : REN ratio represents the relative activities of NCED2 and NCED3 transcription . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s 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

K Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CDF4 promoter and the p62-SK construct with or without the CDF4 coding region and CDF4-VP16 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured after culturing the protoplasts under low light conditions for 16 h. The ProNCED2/3:LUC /Pro35S:REN ratio represents the relative activities of NCED2 and NCED3 transcription. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t-test.


A - D Transcript levels were determined by qPCR and normalized to AtACT2 . ( A , C ) Localized NCED2 and NCED3 expression in the wild - type senescing leaf . Half - senesced leaves were split into three parts , as shown in Fig . 1A , C . ( B , D ) NCED2 and NCED3 expression during leaf senescence in the vector control and CDF4 - RNAi - 1 plants . Transgenic plant leaves were analyzed from 7 to 56 d ( d , days after planting ) . Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t - test .

A-D Transcript levels were determined by qPCR and normalized to AtACT2. (A, C) Localized NCED2 and NCED3 expression in the wild-type senescing leaf. Half-senesced leaves were split into three parts, as shown in Fig. 1A, C. (B, D) NCED2 and NCED3 expression during leaf senescence in the vector control and CDF4- RNAi-1 plants. Transgenic plant leaves were analyzed from 7 to 56 d (d, days after planting). Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.


E Observation of rosette leaf longevity from the 26 - day - old vector control , pER8 : : CDF4 , pER8 : : CDF4 & nced2 , pER8 : : CDF4 & nced3 and pER8 : : CDF4 & nced2nced3 transgenic plants after 20 μM estradiol induction for two weeks . Phenotype of detached rosette leaves arranged from oldest to youngest . Scale bars indicate 1 . 5 cm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Observation of rosette leaf longevity from the 26-day-old vector control, pER8::CDF4, pER8::CDF4 & nced2, pER8::CDF4 & nced3 and pER8::CDF4 & nced2nced3 transgenic plants after 20 μM estradiol induction for two weeks. Phenotype of detached rosette leaves arranged from oldest to youngest. Scale bars indicate 1.5 cm.


( F ) SAG12 expression level in rosette leaves at various development stages . Three independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t - test .

(F) SAG12 expression level in rosette leaves at various development stages. Three independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.


( G ) Chlorophyll concentration in rosette leaves at various development stages . Three independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t - test .

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(G) Chlorophyll concentration in rosette leaves at various development stages. Three independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t-test.


A The senescence phenotype of the vector control , pER8 : : CDF4 and pER8 : : CDF4 : : RNAi detached leaves treated with H2O2 and incubated in the dark for three days . The third leaf in rosettes of 3 - week - old plants was detached and incubated in MES buffer ( 2 mM MES , pH 5 . 8 ) treated with 2 mM H2O2 and 20 μM β - estradiol under dark conditions for three days . Picture shows the leaves before and after treatment . Scale bars indicate 1 cm .

O 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 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

A The senescence phenotype of the vector control, pER8::CDF4 and pER8::CDF4::RNAi detached leaves treated with H2O2 and incubated in the dark for three days. The third leaf in rosettes of 3-week-old plants was detached and incubated in MES buffer (2 mM MES, pH 5.8) treated with 2 mM H2O2 and 20 μM β-estradiol under dark conditions for three days. Picture shows the leaves before and after treatment. Scale bars indicate 1 cm.


( B ) DAB staining of H2O2 before and after 2 mM H2O2 plus 20 µM β - estradiol treatment under dark conditions for two days . The brown color represents H2O2 accumulation . Scale bars indicate 1 cm .

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(B) DAB staining of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. The brown color represents H2O2 accumulation. Scale bars indicate 1 cm.


( C ) measurement of H2O2 before and after 2 mM H2O2 plus 20 µM β - estradiol treatment under dark conditions for two days . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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(C) measurement of H2O2 before and after 2 mM H2O2 plus 20 µM β-estradiol treatment under dark conditions for two days. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


D Chlorophyll concentrations in the leaves shown in A . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

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D Chlorophyll concentrations in the leaves shown in A. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


A Measurement of catalase activity in vector control and CDF4RNAi plants . Ten - day - old green seedlings ( " Young " ) and the third and fourth rosette leaves from 36 - day - old plants ( " Old " ) were used , respectively . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t test , * p < 0 . 05 .

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A Measurement of catalase activity in vector control and CDF4RNAi plants. Ten-day-old green seedlings ("Young") and the third and fourth rosette leaves from 36-day-old plants ("Old") were used, respectively. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.


B qPCR analysis of CAT genes expression in the third and fourth rosette leaves from 36 - day - old plants . The expression of CAT genes in the wild - type plant is given as 1 . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qPCR analysis of CAT genes expression in the third and fourth rosette leaves from 36-day-old plants. The expression of CAT genes in the wild-type plant is given as 1. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.


D ChIP - qPCR analysis of the relative binding of CDF4 to the promoter of CAT2 . An anti - HA monoclonal antibody was used for DNA immunoprecipitation from 32 - d - old pER8 : : CDF4 - HA transgenic plants . Black columns indicate the enrichment fold - changes normalized to that of ACT2 . Three independent experiments were conducted . Data are represented as means ± SD , n = 3 . Student ' s t test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ChIP-qPCR analysis of the relative binding of CDF4 to the promoter of CAT2. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from 32-d-old pER8::CDF4-HA transgenic plants. Black columns indicate the enrichment fold-changes normalized to that of ACT2. Three independent experiments were conducted. Data are represented as means ± SD, n=3. Student's t test, *p<0.05.


E Transient dual - luciferase reporter assay . The pGreenII - 0800 LUC construct containing the CAT2 promoter and the p62 - SK construct with or without CDF4 were transiently co - transformed into Col - 0 protoplasts . Firefly luciferase ( LUC ) and Renilla luciferase ( REN ) activity were measured after culturing the protoplasts under low light conditions for 16 h . six independent experiments were conducted . Values are given as mean ± SD , n = 6 . * p < 0 . 05 by Student ' s 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

E Transient dual-luciferase reporter assay. The pGreenII-0800 LUC construct containing the CAT2 promoter and the p62-SK construct with or without CDF4 were transiently co-transformed into Col-0 protoplasts. Firefly luciferase (LUC) and Renilla luciferase (REN) activity were measured after culturing the protoplasts under low light conditions for 16 h. six independent experiments were conducted. Values are given as mean ± SD, n=6. *p<0.05 by Student's t test.


A The senescence phenotypes of five - week - old vector control , proSAG12 : : CDF4 and proSAG12 : : CDF4 & 35S : : CAT2 plants , from old to young . Scale bars indicate 1 . 5 cm .

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A The senescence phenotypes of five-week-old vector control, proSAG12::CDF4 and proSAG12::CDF4 & 35S::CAT2 plants, from old to young. Scale bars indicate 1.5 cm.


B qPCR analysis of the expression of CDF4 and CAT2 in the leaves Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s 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

B qPCR analysis of the expression of CDF4 and CAT2 in the leaves Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.


qPCR analysis of the expression of CAT2 in the leaves Four independent experiments were conducted . Values are given as mean ± SD , n = 4 . * p < 0 . 05 by Student ' s 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

qPCR analysis of the expression of CAT2 in the leaves Four independent experiments were conducted. Values are given as mean ± SD, n=4. *p<0.05 by Student's t test.


( D ) Measurement of chlorophyll in the leaves The third leaves in the rosette were used . Four independent experiments were conducted . Data are presented as the mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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(D) Measurement of chlorophyll in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test.


( E ) H2O2 concentrations in the leaves The third leaves in the rosette were used . Four independent experiments were conducted . Data are presented as the mean ± SD , n = 4 . * p < 0 . 05 by Student ' s t test .

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(E) H2O2 concentrations in the leaves The third leaves in the rosette were used. Four independent experiments were conducted. Data are presented as the mean ± SD, n=4. *p<0.05 by Student's t test.


( A ) Observation of inflorescences of 7 - week - old wild - type ( WT ) Arabidopsis and the CDF4 transgenic plants . The asterisk represents the flower organ attached along the inflorescence . Scale bar indicates 1 . 5 cm .

O O O O O O O O O O O O O O O O O O O O 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) Observation of inflorescences of 7-week-old wild-type (WT) Arabidopsis and the CDF4 transgenic plants. The asterisk represents the flower organ attached along the inflorescence. Scale bar indicates 1.5 cm.


Selected abscission - related gene expression levels in ( B ) WT and proIDA : : CDF4 lines by using qPCR analysis . The expression of these selected genes in the wild - type plant is given as 1 . The relative expression level represents only the level of expression of the gene relative to the wild type . ACTIN2 was used as the internal control . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s 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

Selected abscission-related gene expression levels in (B) WT and proIDA::CDF4 lines by using qPCR analysis. The expression of these selected genes in the wild-type plant is given as 1. The relative expression level represents only the level of expression of the gene relative to the wild type. ACTIN2 was used as the internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


Selected abscission - related gene expression levels in ( C ) WT and proIDA : : CDF4RNAi lines by using qPCR analysis . The expression of these selected genes in the wild - type plant is given as 1 . The relative expression level represents only the level of expression of the gene relative to the wild type . ACTIN2 was used as the internal control . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s t test .

Selected abscission-related gene expression levels in (C) WT and proIDA::CDF4RNAi lines by using qPCR analysis. The expression of these selected genes in the wild-type plant is given as 1. The relative expression level represents only the level of expression of the gene relative to the wild type. ACTIN2 was used as the internal control. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


E Observation of GUS staining activity in the proPGAZAT : : GUS and proPGAZAT : : GUS & proIDA : : CDF4 . Scale bar indicates 0 . 5 cm .

O O O O O O O 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 Observation of GUS staining activity in the proPGAZAT::GUS and proPGAZAT::GUS & proIDA::CDF4. Scale bar indicates 0.5 cm.


F ChIP - qPCR analysis of the relative binding of CDF4 to the promoter regions of PGAZAT . An anti - HA monoclonal antibody was used for DNA immunoprecipitation from 6 - week - old pER8 : : CDF4 - HA transgenic plants after estradiol induction . Black columns indicate the enrichment fold changes normalized to ACT2 . Three independent experiments were conducted . Values are given as mean ± SD , n = 3 . * p < 0 . 05 by Student ' s 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 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

F ChIP-qPCR analysis of the relative binding of CDF4 to the promoter regions of PGAZAT. An anti-HA monoclonal antibody was used for DNA immunoprecipitation from 6-week-old pER8::CDF4-HA transgenic plants after estradiol induction. Black columns indicate the enrichment fold changes normalized to ACT2. Three independent experiments were conducted. Values are given as mean ± SD, n=3. *p<0.05 by Student's t test.


( A ) CXCL10 assay characteristics . Standard curves of the short , long and total CXCL10Simoa assays are shown . For each curve , limit of detection ( LOD ) defined as blank + 3SD , are shown as horizontal lines . The LOD was 1 . 7pg / ml for long and short CXCL10 assays and 0 . 22pg / ml for total CXCL10 . Standard curves were fitted using the 4 Parameter Logistic non - linear regression model . Samples reporting a signal below the LOD were replaced with 1pg / ml for short and long CXCL10 .

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(A) CXCL10 assay characteristics. Standard curves of the short, long and total CXCL10Simoa assays are shown. For each curve, limit of detection (LOD) defined as blank+3SD, are shown as horizontal lines. The LOD was 1.7pg/ml for long and short CXCL10 assays and 0.22pg/ml for total CXCL10. Standard curves were fitted using the 4 Parameter Logistic non-linear regression model. Samples reporting a signal below the LOD were replaced with 1pg/ml for short and long CXCL10.


Plasma from healthy individuals receiving ( B ) placebo ( n = 9 ) or ( C ) sitagliptin ( n = 27 ) were analyzed by Simoa . Blood samples were collected at screening visit ( SV ) and day 0 ( D0 ) before treatment , at day 3 ( D3 ) , day 14 ( W2 ) and day 28 ( W4 ) under treatment and 5 weeks after treatment interruption ( W9 ) . ( C ) Plasma from chronic HCVpatients receiving sitagliptin ( n = 3 ) were collected before ( D0 ) and weekly during sitagliptin treatment ( W1 W3 ) . Antagonist CXCL103 - 77 ( short CXCL10 , in blue ) , agonist CXCL101 - 77 ( long CXCL10 , in red ) and total CXCL10 ( in black ) levels are shown . Each dot represents a donor and bars are at the median . Grey areas highlight the period under placebo or sitagliptin treatment . Statistical analysis of ( B ) and ( C ) was performed using non parametric Friedman ' s test , ns : non significant , * * : P0 . 01 , * * * * : P0 . 001 . For ( C ) , additional size effect analysis was performed and Cohen ' s d values are reported . No statistical analysis was performed in ( D ) due to sample size , nd : non - determined .

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 B-CONTROLLED_VAR 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 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

Plasma from healthy individuals receiving (B) placebo (n=9) or (C) sitagliptin (n=27) were analyzed by Simoa. Blood samples were collected at screening visit (SV) and day 0 (D0) before treatment, at day 3 (D3), day 14 (W2) and day 28 (W4) under treatment and 5 weeks after treatment interruption (W9). (C) Plasma from chronic HCVpatients receiving sitagliptin (n=3) were collected before (D0) and weekly during sitagliptin treatment (W1 W3). Antagonist CXCL103-77 (short CXCL10, in blue), agonist CXCL101-77 (long CXCL10, in red) and total CXCL10 (in black) levels are shown. Each dot represents a donor and bars are at the median. Grey areas highlight the period under placebo or sitagliptin treatment. Statistical analysis of (B) and (C) was performed using non parametric Friedman's test, ns: non significant, **: P0.01, ****: P0.001. For (C), additional size effect analysis was performed and Cohen's d values are reported. No statistical analysis was performed in (D) due to sample size, nd: non-determined.


C GST pulldown of transiently expressed AdipoR1 ( residues 1 - 375 ) with immobilized GST - P5 or GST - P10 fusion proteins . Pulldown fractions were subjected to western blotting using Flag and 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

C GST pulldown of transiently expressed AdipoR1 (residues 1-375) with immobilized GST-P5 or GST-P10 fusion proteins. Pulldown fractions were subjected to western blotting using Flag and GST antibodies.


C AdipoR1 siRNA - transfected ORS or DP cells were treated with APN or P5 . The cell lysates were analyzed for p - AMPK .

O O 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 AdipoR1 siRNA-transfected ORS or DP cells were treated with APN or P5. The cell lysates were analyzed for p-AMPK.


D - H The relative gene expression levels of growth factors in P5 - treated DP cells ( n = 4 ( VEGF , PDGFA ) , 5 ( IGF1 ) , 6 ( HGF , FGF7 ) biological replicates / group )

D-H The relative gene expression levels of growth factors in P5-treated DP cells (n = 4 (VEGF, PDGFA), 5 (IGF1), 6 (HGF, FGF7) biological replicates / group)


C IF staining ( p - AMPK ) of the HF bulb area . Yellow arrow indicates p - AMPK signal ; scale bar : 100 µm . D IF staining ( Ki - 67 ) of the HF bulb area ; scale bar : 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

C IF staining (p-AMPK) of the HF bulb area. Yellow arrow indicates p-AMPK signal; scale bar: 100 µm. D IF staining (Ki-67) of the HF bulb area; scale bar: 100 µm.


C H & E and IF for versican of skin tissue ; scale bars : 100 µm .

C H&E and IF for versican of skin tissue; scale bars: 100 µm.


G H & E and IF for versican of skin tissue ; scale bars : 100 µm . H Anagen induction scores of vehicle - , or P5 - treated Adipoq - / - mice ( n = 6 tissues / mice , 4 mice / group ) ; Unpaired 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

G H&E and IF for versican of skin tissue; scale bars: 100 µm. H Anagen induction scores of vehicle-, or P5- treated Adipoq-/- mice (n = 6 tissues / mice, 4 mice / group); Unpaired t test


K . H & E and IF for versican of skin tissue ; scale bars : 100 µm . L Anagen induction scores of vehicle - , or P5 - treated Adipor1 - / - mice ( n = 6 tissues / mice , 5 mice / group ) ; Unpaired 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

K. H&E and IF for versican of skin tissue; scale bars: 100 µm. L Anagen induction scores of vehicle-, or P5- treated Adipor1-/-mice (n = 6 tissues / mice, 5 mice / group); Unpaired t test


Pulldown of Flag - fused AdipoR1 input ( residues 89 - 375 ) with amino acid mutation ( s ) , as indicated , with GST - P5 . The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 ( residue 89 - 375 ) inputs , as indicated ( n = 3 technical replicates / group ) .

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

Pulldown of Flag-fused AdipoR1 input (residues 89-375) with amino acid mutation(s), as indicated, with GST-P5. The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).


F Pulldown of AdipoR1 ( residues 89 - 375 ) with GST - P5 or GST fused to two mutant peptides ( GLYDF and GLYAF ) . The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 ( residue 89 - 375 ) inputs , as indicated ( n = 3 technical replicates / group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Pulldown of AdipoR1 (residues 89-375) with GST-P5 or GST fused to two mutant peptides (GLYDF and GLYAF). The relative pulldown efficiency is expressed as the ratio of the band intensities of bound AdipoR1 to those of AdipoR1 (residue 89-375) inputs, as indicated (n = 3 technical replicates / group).


B Fatty acid composition of B . subtilis WT cells grown in PF medium , and Δbkd grown with MB , IB or depleted for precursor for 90 min ( IBPF ) . For detailed analyses , see Appendix Fig S1B .

O B-MEASURED_VAR I-MEASURED_VAR 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

B Fatty acid composition of B. subtilis WT cells grown in PF medium, and Δbkd grown with MB, IB or depleted for precursor for 90 min (IBPF). For detailed analyses, see Appendix Fig S1B.


E Fatty acid composition of E . coli WT and fabA ( Ts ) cells grown at 30 ° C and shifted to different temperatures for 120 min . For detailed analyses , see Appendix Fig S2B and C .

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

E Fatty acid composition of E. coli WT and fabA(Ts) cells grown at 30°C and shifted to different temperatures for 120 min. For detailed analyses, see Appendix Fig S2B and C.


A DPH anisotropy of B . subtilis WT or Δbkd cells supplemented either with MB , IB or depleted for precursor ( IBPF ) for the times indicated . High DPH anisotropy indicates low membrane fluidity . B DPH anisotropy of E . coli WT cells grown steady state at 30 ° C , 37 ° C or shifted from 30 ° C to 37 ° C followed by immediate measurement . In addition , DPH anisotropy of fabA ( Ts ) cells grown steady state at 30 ° C or shifted from 30 ° C to 37 ° C followed by measurement at the times indicated .

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

A DPH anisotropy of B. subtilis WT or Δbkd cells supplemented either with MB, IB or depleted for precursor (IBPF) for the times indicated. High DPH anisotropy indicates low membrane fluidity. B DPH anisotropy of E. coli WT cells grown steady state at 30°C, 37°C or shifted from 30°C to 37°C followed by immediate measurement. In addition, DPH anisotropy of fabA(Ts) cells grown steady state at 30°C or shifted from 30°C to 37°C followed by measurement at the times indicated. D


A Images of B . subtilis WT and Δbkd cells co - labelled with the membrane potential - sensitive dye DISC3 ( 5 ) and the membrane permeability indicator Sytox Green . Membrane properties were assessed for Δbkd cells grown in the presence of MB , IB or washed precursor - free ( IBPF ) for the times indicated . As controls , WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC ( gABC ) or pore - forming lantibiotic Nisin . For cross - correlation between membrane depolarisation and membrane permeabilisation , see Appendix Fig S3A - C . B Quantification of DISC3 ( 5 ) fluorescence for cells ( n = 100 - 142 ) depicted in panel A . Median represented by red line . C Images of E . coli WT and fabA ( Ts ) cells co - labelled with the same indicator dyes as in panel A . Membrane properties were assessed for fabA ( Ts ) at 30 ° C and upon transfer to non - permissive 37 ° C for the times indicated . As controls , WT cells were incubated with the pore - forming antibiotic Polymyxin B ( PolyB ) . For cross - correlation between membrane depolarisation and membrane permeabilisation , see Appendix Fig S3D and E . The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background ( see Fig EV1 ) . D Quantification of DISC3 ( 5 ) fluorescence for cells ( n = 76 - 141 ) depicted in panel C . Median represented by red line .

O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Images of B. subtilis WT and Δbkd cells co-labelled with the membrane potential-sensitive dye DISC3(5) and the membrane permeability indicator Sytox Green. Membrane properties were assessed for Δbkd cells grown in the presence of MB, IB or washed precursor-free (IBPF) for the times indicated. As controls, WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC (gABC) or pore-forming lantibiotic Nisin. For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3A-C. B Quantification of DISC3(5) fluorescence for cells (n=100-142) depicted in panel A. Median represented by red line. C Images of E. coli WT and fabA(Ts) cells co-labelled with the same indicator dyes as in panel A. Membrane properties were assessed for fabA(Ts) at 30°C and upon transfer to non-permissive 37°C for the times indicated. As controls, WT cells were incubated with the pore-forming antibiotic Polymyxin B (PolyB). For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3D and E. The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background (see Fig EV1). D Quantification of DISC3(5) fluorescence for cells (n=76-141) depicted in panel C. Median represented by red line. Dat


A Images of B . subtilis Δbkd cells stained with membrane dye FM 5 - 95 and expressing GFP fusions of DNA - binding protein HBsu ( top ) , cell division protein FtsZ ( middle ) or cell elongation protein MreB ( bottom ) . Cells were grown with IB or depleted for precursors for 90 min ( IBPF ) . For further examples and additional time points , see Appendix Fig S4A . B Images of E . coli fabA ( Ts ) cells stained with FM 5 - 95 for the outer membrane and with DNA - intercalating dye DAPI ( top ) , or expressing GFP sandwich ( SW ) fusions to the cell division protein FtsZ ( middle ) and the cell elongation protein MreB ( bottom ) , respectively . Depicted are cells grown at 30 ° C or with a temperature shift to 37 ° C for 120 min . For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery , see Figs EV2 , EV3 , EV4 , and S5 . For further examples and additional time points , see Appendix Fig S4B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Images of B. subtilis Δbkd cells stained with membrane dye FM 5-95 and expressing GFP fusions of DNA-binding protein HBsu (top), cell division protein FtsZ (middle) or cell elongation protein MreB (bottom). Cells were grown with IB or depleted for precursors for 90 min (IBPF). For further examples and additional time points, see Appendix Fig S4A. B Images of E. coli fabA(Ts) cells stained with FM 5-95 for the outer membrane and with DNA-intercalating dye DAPI (top), or expressing GFP sandwich (SW) fusions to the cell division protein FtsZ (middle) and the cell elongation protein MreB (bottom), respectively. Depicted are cells grown at 30°C or with a temperature shift to 37°C for 120 min. For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery, see Figs EV2, EV3, EV4, and S5. For further examples and additional time points, see Appendix Fig S4B. D


B Images of B . subtilis Δbkd cells grown with IB or without precursor ( IBPF ) . Cells were stained with membrane fluidity - sensitive dye Laurdan and imaged at 460 nm , 520 nm and as the corresponding colour - coded Laurdan GP map . C Images of E . coli fabA ( Ts ) cells grown at 30 ° C or shifted to non - permissive 37 ° C for 120 min . Cells were stained and imaged as described in panel B . For a more pronounced view on domain formation associated with local differences in membrane fluidity , see Appendix Fig S7 , showing E . coli fabA ( Ts ) grown in LB instead of M9 minimal medium with glucose / casamino acids . D Images of B . subtilis Δbkd cells grown and stained with Laurdan ( imaged at 460 nm ) as in panel B , but additionally expressing WALP23 - mCherry . For corresponding fluorescence intensity correlation between images , see Appendix Fig S8A . Co - localisation of membrane dye FM 5 - 95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C . E Images of E . coli fabA ( Ts ) cells grown and stained as in panel C , but additionally expressing WALP23 - mScarlet - I . For corresponding fluorescence intensity correlation between images , see Appendix Fig S8B .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Images of B. subtilis Δbkd cells grown with IB or without precursor (IBPF). Cells were stained with membrane fluidity-sensitive dye Laurdan and imaged at 460 nm, 520 nm and as the corresponding colour-coded Laurdan GP map. C Images of E. coli fabA(Ts) cells grown at 30°C or shifted to non-permissive 37°C for 120 min. Cells were stained and imaged as described in panel B. For a more pronounced view on domain formation associated with local differences in membrane fluidity, see Appendix Fig S7, showing E. coli fabA(Ts) grown in LB instead of M9 minimal medium with glucose/casamino acids. D Images of B. subtilis Δbkd cells grown and stained with Laurdan (imaged at 460 nm) as in panel B, but additionally expressing WALP23-mCherry. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8A. Co-localisation of membrane dye FM 5-95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C. E Images of E. coli fabA(Ts) cells grown and stained as in panel C, but additionally expressing WALP23-mScarlet-I. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8B. Dat


A Time lapse images of E . coli fabA ( Ts ) cells expressing FOF1 a - mNG . For corresponding controls , see Appendix Fig S10 . Cells were grown at 30 ° C and shifted from 30 ° C to non - permissive 37 ° C . See Movie EV2 for full time lapse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Time lapse images of E. coli fabA(Ts) cells expressing FOF1 a-mNG. For corresponding controls, see Appendix Fig S10. Cells were grown at 30°C and shifted from 30°C to non-permissive 37°C. See Movie EV2 for full time lapse.


E Fatty acid composition of E . coli fabA ( Ts ) cells ( same cell batch as in D ) upon growth at 30 ° C , upon depletion of UFA by incubation at 37 ° C for 120 min and upon recovery by oleate supplementation for additional 120 min . For detailed analyses , see Appendix Fig S11B - D .

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 B-MEASURED_VAR 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

E Fatty acid composition of E. coli fabA(Ts) cells (same cell batch as in D) upon growth at 30°C, upon depletion of UFA by incubation at 37°C for 120 min and upon recovery by oleate supplementation for additional 120 min. For detailed analyses, see Appendix Fig S11B-D.


A Representative trajectory maps of individual FOF1 a - mNG molecules in E . coli WT and fabA ( Ts ) cells grown at 30 ° C and upon shift to non - permissive 37 ° C for 120 min . See Movie EV3 ( WT ) and Movie EV4 ( fabA ( Ts ) ) for accumulating trajectory maps of FOF1 a - mNG molecules .

A Representative trajectory maps of individual FOF1 a-mNG molecules in E. coli WT and fabA(Ts) cells grown at 30°C and upon shift to non-permissive 37°C for 120 min. See Movie EV3 (WT) and Movie EV4 (fabA(Ts)) for accumulating trajectory maps of FOF1 a-mNG molecules.


D Apparent lateral diffusion coefficients ( Dapp ) of FOF1 a - mNG analysed in panel B .

D Apparent lateral diffusion coefficients (Dapp) of FOF1 a-mNG analysed in panel B.


( A ) Negative stain electron micrograph showing the tendency of S . cerevisiae prApe1 ( sc - prApe1 ) dodecamers to form higher - order assemblies or chains ( black arrowheads ) . ( B ) Negative stain electron micrograph of S . cerevisiae mApe1 ( sc - mApe1 ) showing monodisperse dodecameric particles ( circles ) as well as hexameric particles ( white arrowheads ) . ( C ) Bar plot showing the occurrence of single , double , triple or multiple aggregates of sc - prApe1 in comparison with sc - mApe1 . Insets show single , double , triple or multiple dodadecamers . Data information : n = 3 , 186 particles were counted in the case of sc - prApe1 and n = 3 , 107 particles were counted in the case of sc - mApe1 . Particles were picked from 20 different micrographs . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared 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

(A) Negative stain electron micrograph showing the tendency of S. cerevisiae prApe1 (sc-prApe1) dodecamers to form higher-order assemblies or chains (black arrowheads). (B) Negative stain electron micrograph of S. cerevisiae mApe1 (sc-mApe1) showing monodisperse dodecameric particles (circles) as well as hexameric particles (white arrowheads). (C) Bar plot showing the occurrence of single, double, triple or multiple aggregates of sc-prApe1 in comparison with sc-mApe1. Insets show single, double, triple or multiple dodadecamers. Data information: n=3,186 particles were counted in the case of sc-prApe1 and n=3,107 particles were counted in the case of sc-mApe1. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).