Figure 5 ( A - C , E ) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs , released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs . ( A - C , E ) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs , released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs . ( A - C , E ) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs , released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs . ( D ) Immunoblotting of HeLa protein extracts , treated as indicated . ( A - C , E ) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs , released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs . ( F ) Quantification of 3 independent experiments evaluating the localization of the KT factors shown in A - C , e relative to CREST . 100 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining . P values are obtained by 2 - way ANOVA with Sidak ' s multiple comparison analysis relative to localization of HEC1 , which is not lost on SNAP29KD cells .

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Figure 5(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.(D) Immunoblotting of HeLa protein extracts, treated as indicated.(A-C, E) Max projection of single control and SNAP29 KD HeLa cells treated with 2 mM thymidine for 24 hrs, released in normal medium for 8 hrs and arrested in prometaphase with Nocodazole for 2 hrs.(F) Quantification of 3 independent experiments evaluating the localization of the KT factors shown in A-C, e relative to CREST. 100 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Sidak's multiple comparison analysis relative to localization of HEC1, which is not lost on SNAP29KD cells.


Figure 6 ( A - C ) KNL1 localization analysis in control , SNAP29 KD and SNAP29 KD expressing full length human SNAP29 , CFP tagged , full length Drosophila Snap29 ( CFP - Snap29 ) , or the mutant forms schematized above the panels . Max projections of single cells in prophase are shown . ( D ) Quantification of 3 independent experiments evaluating the localization of KNL1 relative to CREST . 200 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining . P values are obtained by 2 - way ANOVA with Dunnett ' s multiple comparison analysis relative to localization of KNL1 in SNAP29KD cells . ( E - F ) Single confocal sections of HeLa cells in interphase overexpressing the indicated constructs . In cells expressing SNAP29 Q1 Q2 , SNAP29 and KNL1 accumulate in large compartments that are often positive for p62 ( arrows ) .

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Figure 6(A-C) KNL1 localization analysis in control, SNAP29 KD and SNAP29 KD expressing full length human SNAP29, CFP tagged, full length Drosophila Snap29 (CFP-Snap29), or the mutant forms schematized above the panels. Max projections of single cells in prophase are shown. (D) Quantification of 3 independent experiments evaluating the localization of KNL1 relative to CREST. 200 KTs from 10 control cells and 200 KTs from 10 SNAP29 KD cells per sample were identified by CREST staining. P values are obtained by 2-way ANOVA with Dunnett's multiple comparison analysis relative to localization of KNL1 in SNAP29KD cells.(E-F) Single confocal sections of HeLa cells in interphase overexpressing the indicated constructs. In cells expressing SNAP29 Q1 Q2, SNAP29 and KNL1 accumulate in large compartments that are often positive for p62 (arrows).


Figure 7 ( A - B ) Confocal sections of wild - type and Snap29 mutant eye - antennal imaginal discs ( eye disc portion to the left ) of L3 larvae . Discs were labeled with phalloidin and DAPI to visualize the subcortical f - actin cytoskeleton and nuclei , respectively . Arrowheads in B point to actin - rich unpolarized cells in apical , medial and basal sections of the same disc . ( C ) High magnification of a single confocal cross - section of wild - type and Snap29 mutant eye disc tissues . Two facing folds of the tissue are separated by the apical lumen . aPKC and Dlg mark the apical and lateral plasma membrane domains , respectively . In Snap29 mutant tissue the apical - basal polarity is disrupted . ( D ) Single confocal cross - sections of wild - type and Snap29 eye - antennal discs , expressing Puckered - LacZ ( Puc - lacZ ) , a reporter of JNK signaling activation . ( E - H ) Single confocal cross - sections of wild - type and Snap29 eye - antennal discs ( E - F ) , and of otherwise wild - type and Snap29 eye - antennal discs expressing the bacterial apoptosis inhibitor p35 ( G - H ) . Anti - activated - Caspase 3 ( Casp3 ) marks apoptotic cells .

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Figure 7(A-B) Confocal sections of wild-type and Snap29 mutant eye-antennal imaginal discs (eye disc portion to the left) of L3 larvae. Discs were labeled with phalloidin and DAPI to visualize the subcortical f-actin cytoskeleton and nuclei, respectively. Arrowheads in B point to actin-rich unpolarized cells in apical, medial and basal sections of the same disc.(C) High magnification of a single confocal cross-section of wild-type and Snap29 mutant eye disc tissues. Two facing folds of the tissue are separated by the apical lumen. aPKC and Dlg mark the apical and lateral plasma membrane domains, respectively. In Snap29 mutant tissue the apical-basal polarity is disrupted.(D) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs, expressing Puckered-LacZ (Puc-lacZ), a reporter of JNK signaling activation.(E-H) Single confocal cross-sections of wild-type and Snap29 eye-antennal discs (E-F), and of otherwise wild-type and Snap29 eye-antennal discs expressing the bacterial apoptosis inhibitor p35 (G-H). Anti-activated-Caspase 3 (Casp3) marks apoptotic cells.


Figure 1 ( B ) Representative images of cells released from mitosis for 1 , 2 or 3h in EdU - containing medium . Top , DNA staining with DAPI ; Bottom , EdU immunofluorescence . L , M and H indicate cells with low , medium or high levels of EdU incorporation . The barplot shows the quantification of the proportion of replicating cells at each time point .

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Figure 1(B) Representative images of cells released from mitosis for 1, 2 or 3h in EdU-containing medium. Top, DNA staining with DAPI; Bottom, EdU immunofluorescence. L, M and H indicate cells with low, medium or high levels of EdU incorporation. The barplot shows the quantification of the proportion of replicating cells at each time point.


Figure 2Y - axis , z - score ; X - axis , timepoints . A indicates asynchronous cells , M mitotic cells and the numbers represent the minutes after release from the mitotic block ) . Dots show individual replicates and lines averages ( n = 3 for A and M ; n = 2 for each post - mitotic sample ) . Pre - mRNA levels are shown for pluripotency genes ( Oct4 , Klf4 , Esrrb ) and cell cycle regulators ( E2f1 , Cdk2 , Rb ) ; mRNA levels for individual histone genes ( only averages are shown ) .

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Figure 2Y-axis, z-score; X-axis, timepoints. A indicates asynchronous cells, M mitotic cells and the numbers represent the minutes after release from the mitotic block). Dots show individual replicates and lines averages (n=3 for A and M; n=2 for each post-mitotic sample). Pre-mRNA levels are shown for pluripotency genes (Oct4, Klf4, Esrrb) and cell cycle regulators (E2f1, Cdk2, Rb); mRNA levels for individual histone genes (only averages are shown).


Figure 4 ( C ) Percentage of genes presenting at least 1 TF binding site as detected by ChIP - seq within 25kb centred on the TSS , calculated for each gene category shown in ( A ) .

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Figure 4(C) Percentage of genes presenting at least 1 TF binding site as detected by ChIP-seq within 25kb centred on the TSS, calculated for each gene category shown in (A).


Figure 5 ( B ) Heatmaps presented for untreated ( - IAA ; left ) or CTCF - depleted ( + IAA ; right ) CTCF - AID cells .

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Figure 5(B) Heatmaps presented for untreated (-IAA; left) or CTCF-depleted (+IAA; right) CTCF-AID cells.


Figure 6 ( A ) Gene expression changes ( z - score ) in asynchronous cells and during post - mitotic release of CTCF - AID cells treated or not with IAA to deplete CTCF ( blue , presence of CTCF ; red , absence ) . Samples are indicated on the X - axis ( A : asynchronous cells ; M : mitotic cells ; numbers : minutes since release from mitotic block ) . Six gene groups are shown , based on the time of their response to CTCF depletion ( asy , late , early ) and the direction of the transcriptional change ( up , down ) . The number of genes of each group is indicated . The yellow shadow areas indicate times showing expression changes . The boxplots show the median ( bar ) , 25 - 75 % percentiles ( box ) and 1 . 5 - fold inter - quartile range ( whiskers ) . ( C ) Average binding profile ( X - axis , reads per million ) of CTCF ( top ) and the Cohesin subunit SMC1 ( bottom ) , across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells ( left ) or either late or early upon mitotic release ( middle and right , respectively ) . Interphase is shown in blue and mitosis in red .

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Figure 6(A) Gene expression changes (z-score) in asynchronous cells and during post-mitotic release of CTCF-AID cells treated or not with IAA to deplete CTCF (blue, presence of CTCF; red, absence). Samples are indicated on the X-axis (A: asynchronous cells; M: mitotic cells; numbers: minutes since release from mitotic block). Six gene groups are shown, based on the time of their response to CTCF depletion (asy, late, early) and the direction of the transcriptional change (up, down). The number of genes of each group is indicated. The yellow shadow areas indicate times showing expression changes. The boxplots show the median (bar), 25-75% percentiles (box) and 1.5-fold inter-quartile range (whiskers).(C) Average binding profile (X-axis, reads per million) of CTCF (top) and the Cohesin subunit SMC1 (bottom), across 8kb centred on the TSS of genes responding to CTCF depletion exclusively in asynchronous cells (left) or either late or early upon mitotic release (middle and right, respectively). Interphase is shown in blue and mitosis in red.


f1 ( a ) Nox2 - specific ROS production was assessed using the Nox2 redox biosensor p47 - roGFP redox biosensor Cat : catalase , PEG - Cat : pegylated catalase . ( b ) Measurement of intracellular glutathione redox potential with Grx1 - roGFP2 . ( c ) Analysis of Rac1 and ( d ) Src . . ( e ) Immunoblot of precipitated p47phox probed with an anti - phosphoserine or anti - p47phox antibody . ( f ) Nox2 - specific intracellular ROS production was measured using p47 - roGFP redox biosensor . g ) Extracellular ROS production was assessed using Amplex - red dye . ( h ) Plasma membrane calcium influx was measured by analysing the Fura - 2 fluorescence quench rate upon addition of extracellular Mn2 + . ( i ) Intracellular RNS generation was measured using DAF - FM . Bars represent average ± s . e . m . from n = 15 individual fibres for each condition in ( a , b , f , g , i , j ) . Markers of autophagy were analysed in isolated fibres ( incubated with or without PP2 ) from FDBs . ( k ) Autophagosome formation was analysed using fluorescence microscopy ( scale bar = 100 μm ) and illustrated LC3 localization and autophagosome formation . ( l ) Confocal microscopy detected p62 - LC3 localization in single fibres from FDBs ( scale bar = 140 and 50 μm for white box areas ) . All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated . Glyceraldehyde - 3 - phosphate dehydrogenase ( GAPDH ) was detected as a loading control . Representative images are shown . Bars represent average ± s . e . m . from n = 3 independent biological experiments . Statistical differences between groups were determined using analysis of variance ( ANOVA ) with Tukey ' s post hoc test . * P0 . 05 and * * P0 . 01 .

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f1(a) Nox2-specific ROS production was assessed using the Nox2 redox biosensor p47-roGFP redox biosensor Cat: catalase, PEG-Cat: pegylated catalase. (b) Measurement of intracellular glutathione redox potential with Grx1-roGFP2.(c) Analysis of Rac1 and (d) Src.. (e) Immunoblot of precipitated p47phox probed with an anti-phosphoserine or anti-p47phox antibody.(f) Nox2-specific intracellular ROS production was measured using p47-roGFP redox biosensor.g) Extracellular ROS production was assessed using Amplex-red dye.(h) Plasma membrane calcium influx was measured by analysing the Fura-2 fluorescence quench rate upon addition of extracellular Mn2+.(i) Intracellular RNS generation was measured using DAF-FM. Bars represent average±s.e.m. from n=15 individual fibres for each condition in (a,b,f,g,i,j). Markers of autophagy were analysed in isolated fibres (incubated with or without PP2) from FDBs.(k) Autophagosome formation was analysed using fluorescence microscopy (scale bar=100 μm) and illustrated LC3 localization and autophagosome formation. (l) Confocal microscopy detected p62-LC3 localization in single fibres from FDBs (scale bar=140 and 50 μm for white box areas). All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using analysis of variance (ANOVA) with Tukey's post hoc test. *P0.05 and **P0.01.


f2 ( a ) FDB lysates were analysed using immunoblotting with antibodies as indicated . ( b ) Confocal microscopy shows the effect of Nox2 inhibition on p62 - LC3 localization in single FDB fibres . Representative images of independent biological experiments ( n = 3 ) are shown ( scale bar = 100 and 35 μm for white box areas ) . ( c ) Autophagic flux was analysed by immunoblotting with anti - LC3B and anti - p62 antibodies . ( d ) Effect of gp91 ds and PP2 on LC3 - LAMP1 colocalization was analysed using confocal microscopy . Representative images from independent biological experiments ( n = 3 ) are shown ( scale bar = 140 and 40 μm for red box areas ) . ( e ) Immunoblot analysis of LAMP1 , f ) PARP - 1 and caspase3 activity . ( g ) Immunoblot of Caspase3 activity . All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated . GAPDH was detected as a loading control . Representative images are shown . Bars represent average ± s . e . m . from n = 3 independent biological experiments . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc test . * P0 . 05 and * * P0 . 01 .

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f2(a) FDB lysates were analysed using immunoblotting with antibodies as indicated.(b) Confocal microscopy shows the effect of Nox2 inhibition on p62-LC3 localization in single FDB fibres. Representative images of independent biological experiments (n=3) are shown (scale bar=100 and 35 μm for white box areas).(c) Autophagic flux was analysed by immunoblotting with anti-LC3B and anti-p62 antibodies.(d) Effect of gp91 ds and PP2 on LC3-LAMP1 colocalization was analysed using confocal microscopy. Representative images from independent biological experiments (n=3) are shown (scale bar=140 and 40 μm for red box areas).(e) Immunoblot analysis of LAMP1,f) PARP-1 and caspase3 activity.(g) Immunoblot of Caspase3 activity. All immunoblots were performed with isolated proteins from FDBs and probed with antibodies as indicated. GAPDH was detected as a loading control. Representative images are shown. Bars represent average ±s.e.m. from n=3 independent biological experiments. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


f3 ( a ) Intracellular ROS production was assessed using DCFH - DA . ( b ) Plasma membrane calcium influx was measured by analysing the quench of Fura - 2 fluorescence upon addition of extracellular Mn2 + . ( a , b ) Were conducted in enzymatically digested single FDBs from WT , mdx and p47 − / − - mdx mice . Bars represent average ± s . e . m . from n = 15 individual fibres for each condition . ( c ) Immunoblot analysis of Src protein with anti - P - Src or anti - Src antibodies . ( d ) Autophagy marker proteins were analysed by immunoblotting with antibodies as indicated ( c ) and ( d ) were conducted in enzymatically isolated fibres from FDBs of WT , mdx and p47 − / − - mdx mice . Representative images are shown . GAPDH was detected as a loading control . Bars represent average ± s . e . m . from n = 3 independent biological experiments . ( e ) LC3 - LAMP1 colocalization in single FDBs from mdx and p47 − / − - mdx mice was analysed using confocal microscopy . Representative images from n = 3 independent biological experiments are shown ( scale bar = 100 and 40 μm for red box areas ) . f ) Immunofluorescence assay to detect lysosomes ( LAMP1 ) in TA muscle from WT , mdx and p47 − / − - mdx mice . White arrows indicate lysosome and yellow arrows indicate nucleus . ( g ) Immunohistochemistry to detect lysosome ( LAMP1 ) in TA muscle from WT , mdx and p47 − / − - mdx mice . Black arrows indicate LAMP1 positive ( brown ) structures . Histogram plot quantifying the number of immunopositive LAMP1 structures per fibre . Representative images from n = 3 independent biological experiments are shown . Scale bar represents 100 μm for f and 140 μm for g . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc test . * P0 . 05 and * * P0 . 01 .

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f3(a) Intracellular ROS production was assessed using DCFH-DA. (b) Plasma membrane calcium influx was measured by analysing the quench of Fura-2 fluorescence upon addition of extracellular Mn2+. (a,b) Were conducted in enzymatically digested single FDBs from WT, mdx and p47−/−-mdx mice. Bars represent average±s.e.m. from n=15 individual fibres for each condition.(c) Immunoblot analysis of Src protein with anti-P-Src or anti-Src antibodies.(d) Autophagy marker proteins were analysed by immunoblotting with antibodies as indicated (c) and (d) were conducted in enzymatically isolated fibres from FDBs of WT, mdx and p47−/−-mdx mice. Representative images are shown. GAPDH was detected as a loading control. Bars represent average±s.e.m. from n=3 independent biological experiments.(e) LC3-LAMP1 colocalization in single FDBs from mdx and p47−/−-mdx mice was analysed using confocal microscopy. Representative images from n=3 independent biological experiments are shown (scale bar=100 and 40 μm for red box areas).f) Immunofluorescence assay to detect lysosomes (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. White arrows indicate lysosome and yellow arrows indicate nucleus.(g) Immunohistochemistry to detect lysosome (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. Black arrows indicate LAMP1 positive (brown) structures. Histogram plot quantifying the number of immunopositive LAMP1 structures per fibre. Representative images from n=3 independent biological experiments are shown. Scale bar represents 100 μm for f and 140 μm for g. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


f4 ( a ) Haematoxylin and eosin staining of cross - sections from Dia showing central nuclei ( arrow head ) and smaller fibres ( arrow ) . ( b ) Immunoblot analysis of macrophage content ( CD68 ) . ( c ) Immunofluorescent and bright field images of Dia cross - sections showing fibre - type distribution . Type I ( red ) , IIA ( green ) , IIB / IIX ( white x , unstained and viewed from bright field overlay ) . ( d ) Serum creatine kinase activity . ( e ) Force - frequency relationship in Dia muscle strips from WT ( black ) , mdx ( red ) and p47 − / − - mdx ( blue ) mice . Scale bars represent 55 μm . For e , # P0 . 01 p47 − / − - mdx versus mdx . # # P0 . 01 p47 − / − - mdx versus WT and mdx . Mdx was statistically different than WT at all frequencies of stimulation . Statistical differences between groups were determined using ANOVA with Tukey ' s post hoc test . * P0 . 05 and * * P0 . 01 .

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f4(a) Haematoxylin and eosin staining of cross-sections from Dia showing central nuclei (arrow head) and smaller fibres (arrow).(b) Immunoblot analysis of macrophage content (CD68).(c) Immunofluorescent and bright field images of Dia cross-sections showing fibre-type distribution. Type I (red), IIA (green), IIB/IIX ( white x, unstained and viewed from bright field overlay).(d) Serum creatine kinase activity. (e) Force-frequency relationship in Dia muscle strips from WT (black), mdx (red) and p47−/−-mdx (blue) mice. Scale bars represent 55 μm. For e, #P0.01 p47−/−-mdx versus mdx.##P0.01 p47−/−-mdx versus WT and mdx. Mdx was statistically different than WT at all frequencies of stimulation. Statistical differences between groups were determined using ANOVA with Tukey's post hoc test. *P0.05 and **P0.01.


Figure 1 ( B ) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP fluorescence ( magenta ) and the effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , fluorescence ) . Dotted lines indicate the electroporated area of the VZ . Single optical sections ; scale bars , 200 μm . ( C ) Higher magnification of the VZ and SVZ of the electroporated area shown in ( B ) , with DAPI staining ( blue ) depicted in addition to PaprikaRFP ( Cas9 ) and GFP fluorescence . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm . ( D ) Quantification of the proportion of Cas9 - positive cells in the VZ plus SVZ that are GFP - positive 48 h after control ( Con , white ) or gGFP ( black ) Cas9 plasmid electroporation . Data are the mean of 4 independent experiments ( 7 embryos per condition in total , from 4 litters ) . ( F ) Overview of electroporated areas of neocortices as revealed by mCherry fluorescence ( magenta ) showing the effects of Cas9 protein together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green , fluorescence ) . Dotted lines indicate the electroporated area of the VZ . Single optical sections ; scale bars , 200 μm . ( G ) Higher magnification of the VZ and SVZ of the electroporated area shown in ( F ) , with DAPI staining ( blue ) depicted in addition to mCherry and GFP fluorescence . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm . H ) Quantification of the proportion of mCherry - positive cells in the VZ plus SVZ that are GFP - positive 48 h after control ( Con , white ) or gGFP ( black ) Cas9 protein electroporation . Data are the mean of 4 independent experiments ( 5 embryos per condition in total , from 4 litters ) . ( I ) VZ and SVZ of the electroporated areas showing Cas9 expression as revealed by PaprikaRFP fluorescence ( magenta ) and the effects of Cas9 expression , together with control gRNA ( top ) or gGFP ( bottom ) , on GFP expression ( green , fluorescence ) ; blue , DAPI staining . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm . ( J ) VZ and SVZ of the electroporated areas showing targeted cells as revealed by mCherry fluorescence ( magenta ) and the effects of Cas9 protein together with either control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( green ) ; blue , DAPI staining . Boxes indicate areas shown at higher magnification in the insets ( 35 x 35 μm ) . Dotted lines indicate nuclei of progeny of electroporated aRGCs ; note the presence of GFP fluorescence in the control ( top ) and its absence upon Cas9 / gGFP electroporation ( bottom ) . Images are single optical sections . Scale bars , 20 μm . K ) Quantification of the proportion of Cas9 - positive cells ( left ) and mCherry - positive cells ( right ) in the VZ plus SVZ that are GFP - positive 24 h after control ( Con , white ) or gGFP ( black ) Cas9 plasmid ( left ) or protein ( right ) electroporation . Data are the mean of 3 independent experiments each ( 3 embryos per condition in total , from 3 litters ) . ( D , H , K ) Controls were set to 100 % and the gGFP conditions expressed relative to control ( D , 11 % ; H , 24 % ; K left , 40 % K right , 28 % ) . Error bars indicate SD ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Overview of electroporated neocortices showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.(C) Higher magnification of the VZ and SVZ of the electroporated area shown in (B), with DAPI staining (blue) depicted in addition to PaprikaRFP (Cas9) and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.(D) Quantification of the proportion of Cas9-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 plasmid electroporation. Data are the mean of 4 independent experiments (7 embryos per condition in total, from 4 litters).(F) Overview of electroporated areas of neocortices as revealed by mCherry fluorescence (magenta) showing the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green, fluorescence). Dotted lines indicate the electroporated area of the VZ. Single optical sections; scale bars, 200 μm.(G) Higher magnification of the VZ and SVZ of the electroporated area shown in (F), with DAPI staining (blue) depicted in addition to mCherry and GFP fluorescence. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.H) Quantification of the proportion of mCherry-positive cells in the VZ plus SVZ that are GFP-positive 48 h after control (Con, white) or gGFP (black) Cas9 protein electroporation. Data are the mean of 4 independent experiments (5 embryos per condition in total, from 4 litters).(I) VZ and SVZ of the electroporated areas showing Cas9 expression as revealed by PaprikaRFP fluorescence (magenta) and the effects of Cas9 expression, together with control gRNA (top) or gGFP (bottom), on GFP expression (green, fluorescence); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.(J) VZ and SVZ of the electroporated areas showing targeted cells as revealed by mCherry fluorescence (magenta) and the effects of Cas9 protein together with either control gRNA (top) or gGFP (bottom) on GFP expression (green); blue, DAPI staining. Boxes indicate areas shown at higher magnification in the insets (35 x 35 μm). Dotted lines indicate nuclei of progeny of electroporated aRGCs; note the presence of GFP fluorescence in the control (top) and its absence upon Cas9/gGFP electroporation (bottom). Images are single optical sections. Scale bars, 20 μm.K) Quantification of the proportion of Cas9-positive cells (left) and mCherry-positive cells (right) in the VZ plus SVZ that are GFP-positive 24 h after control (Con, white) or gGFP (black) Cas9 plasmid (left) or protein (right) electroporation. Data are the mean of 3 independent experiments each (3 embryos per condition in total, from 3 litters).(D, H, K) Controls were set to 100% and the gGFP conditions expressed relative to control (D, 11%; H, 24%; K left, 40% K right, 28%). Error bars indicate SD; *, P<0.05; **, P<0.01; ***, P<0.001.


Figure 2 ( B ) Daughter cells of single aRGCs microinjected with either Cas9 / control gRNA ( top ) or Cas9 / gGFP ( bottom ) revealed by Dx - A555immunofluorescence ( magenta ) ; Cells 1 , aRGC daughter ; Cells 2 , BP daughter . Dashed lines indicate ventricular surface . Images are maximum intensity projections of 20 ( Control ) and 24 ( gGFP ) optical sections . Scale bars , 20 µm . ( C ) Single optical sections of Cells 1 and Cells 2 depicted in ( B ) , showing the effects of Cas9 and control gRNA ( top ) or gGFP ( bottom ) on GFP expression ( as revealed by immunofluorescence , green ) . Cells 1 and Cells 2 are indicated by dotted lines ; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9 / gGFP microinjection . Scale bars , 5 µm . ( D ) Quantification of the proportion of daughter cells ( Dx - A555 + ) of microinjected cells that show GFP expression 24 h after control ( Con , white ) or gGFP ( black ) microinjection . Control is set to 100 % and the gGFP condition expressed relative to control ( 39 % ) . Data are the mean of two independent experiments ( 3 embryos and 1 litter per experiment , total number of daughter cells scored : control 142 , gGFP 88 ) ; bars indicate the variation of the two individual values from the mean ( P < 0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Daughter cells of single aRGCs microinjected with either Cas9/control gRNA (top) or Cas9/gGFP (bottom) revealed by Dx-A555immunofluorescence (magenta); Cells 1, aRGC daughter; Cells 2, BP daughter. Dashed lines indicate ventricular surface. Images are maximum intensity projections of 20 (Control) and 24 (gGFP) optical sections. Scale bars, 20 µm.(C) Single optical sections of Cells 1 and Cells 2 depicted in (B), showing the effects of Cas9 and control gRNA (top) or gGFP (bottom) on GFP expression (as revealed by immunofluorescence, green). Cells 1 and Cells 2 are indicated by dotted lines; note the presence of GFP immunofluorescence in the daughter cells in control and its absence upon Cas9/gGFP microinjection. Scale bars, 5 µm.(D) Quantification of the proportion of daughter cells (Dx-A555+) of microinjected cells that show GFP expression 24 h after control (Con, white) or gGFP (black) microinjection. Control is set to 100% and the gGFP condition expressed relative to control (39%). Data are the mean of two independent experiments (3 embryos and 1 litter per experiment, total number of daughter cells scored: control 142, gGFP 88); bars indicate the variation of the two individual values from the mean (P<0.05).


Figure 3 ( A - D ) Neocortex of mouseE13 . 5embryos was in utero electroporated with a plasmid encoding , under constitutive promoters , Cas9 _ T2A _ PaprikaRFP and gRNA targeting either LacZ ( Control , Con ) or Tbr2 ( Tbr2 ) , followed by analysis at E15 . 5 . ( A ) Electroporated area showing the effects of Cas9 expression ( revealed by PaprikaRFPDAPI , magenta ) together with either control gRNA ( top ) or gTbr2 ( bottom ) on aRGCs expression ( green , immunofluorescence ) ; blue , Cas9staining . Boxes indicate areas shown at higher magnification in the insets ( 45 x 45 μm ) . Dotted lines indicate nuclei of progeny of electroporated Cas9 ; note the presence of Tbr2 immunoreactivity in the control ( top ) and its absence upon Cas9 / gTbr2 plasmid electroporation ( bottom ) . Scale bars , 20 μm . ( B ) Quantification of the proportion of Cas9 + cells that are Tbr2 + 48 h after control ( Con , white ) or gTbr2 ( black ) plasmid electroporation . Data are the mean of 4 independent experiments ( 6 embryos per condition , from 4 litters ) . ( C ) Electroporated area showing the effects of Cas9 expression ( revealed by PaprikaRFP fluorescence , magenta ) together with either control gRNA ( top ) or gTbr2 ( bottom ) on the abundance of mitotic progenitors as revealed by phosphohistone H3 ( PH3 ) immunofluorescence ( green ) . Apical and basal mitoses are indicated by arrows and arrowheads , respectively ; note the reduction in mitotic Cas9 + BPs upon Cas9 / gTbr2 plasmid electroporation . Scale bars , 20 μm . ( D ) Quantification of apical and basal Cas9 + mitoses ( as revealed by PH3 immunofluorescence ) in a unit area 48 h after Cas9 / gTbr2 plasmid electroporation . Data are expressed as percentage of the data obtained upon control plasmid electroporation and are the mean of 3 independent experiments ( 3 embryos per condition in total , from 3 litters ) ; note the reduction in basal ( black , 68 % ) but not apical ( white ) mitoses . ( E - H ) Single aRGCs in neocortex in organotypic slices of telencephalon of E14 . 5 mouse embryos were microinjected with recombinant Cas9 protein together with gRNA targeting either LacZ ( Control , Con ) or Tbr2 ( gTbr2 ) and with Dextran 10 , 000 - Alexa 555 ( Dx - A555 ) , followed by analysis after 48 h of culture . ( E ) Progeny of single microinjected aRGCs ( revealed by Dx - A555immunofluorescence , magenta , dotted lines ) showing the effects of Cas9 together with either control gRNA ( top ) or gTbr2 ( bottom ) on Tbr2 expression ( green , immunofluorescence ) ; blue , DAPI staining . Note the presence of Tbr2 immunoreactivity in the control ( top ) and its absence upon Cas9 / gTbr2 ( bottom ) protein microinjection . Images are single optical sections . Scale bars , 5 µm . ( F ) Quantification of the proportion of the progeny ( Dx - A555 + ) of microinjected aRGCs that show Tbr2 expression 48 h after control ( Con , white ) or gTbr2 ( black ) microinjection . Data are the mean of 3 independent experiments ( 3 embryos per condition ; total number of cells scored : control 45 , gTbr2 41 ) . ( G ) Distribution across the cortical wall of the progeny ( revealed by Dx - A555immunofluorescence , magenta ; arrows , aRGC ; arrowheads , BPs ; open arrow , neuron ) of single control ( left ) or gTbr2 ( right ) microinjected aRGCs . Dashed lines indicate ventricular surface ( bottom ) and basal lamina ( top ) . Images are maximum intensity projections of 67 ( Control ) and 94 ( gTbr2 ) single optical sections . Scale bars , 20 µm . ( H ) Quantification of the distribution of the progeny ( Dx - A555 + ) of microinjected aRGCs in the VZ , SVZ and intermediate zone / cortical plate ( CP + IZ ) 48 h after control ( Con , white ) or gTbr2 ( black ) microinjection ( total number of cells scored : control 37 , gTbr2 51 , pooled from at least 3 independent experiments ) . Note that upon gTbr2 microinjection the distribution of cells across the various cortical zones is significantly different ( VZ : control 77 % , gTbr2 39 : SVZ : control 20 % , gTbr2 37 % ; IZ / CP : control 3 % , gTbr2 24 % , * , P < 0 . 05 ) . ( B , F ) Controls were set to 100 % and the gTbr2 conditions expressed relative to control ( B , 48 % ; F , 50 % ) . ( B , D , F ) Error bars indicate SD ( B , D , F ) ; * , P < 0 . 05 ; * * , P < 0 . 01 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A-D) Neocortex of mouseE13.5embryos was in utero electroporated with a plasmid encoding, under constitutive promoters, Cas9_T2A_PaprikaRFP and gRNA targeting either LacZ (Control, Con) or Tbr2 (Tbr2), followed by analysis at E15.5.(A) Electroporated area showing the effects of Cas9 expression (revealed by PaprikaRFPDAPI, magenta) together with either control gRNA (top) or gTbr2 (bottom) on aRGCs expression (green, immunofluorescence); blue, Cas9staining. Boxes indicate areas shown at higher magnification in the insets (45 x 45 μm). Dotted lines indicate nuclei of progeny of electroporated Cas9; note the presence of Tbr2 immunoreactivity in the control (top) and its absence upon Cas9/gTbr2 plasmid electroporation (bottom). Scale bars, 20 μm.(B) Quantification of the proportion of Cas9+ cells that are Tbr2+ 48 h after control (Con, white) or gTbr2 (black) plasmid electroporation. Data are the mean of 4 independent experiments (6 embryos per condition, from 4 litters).(C) Electroporated area showing the effects of Cas9 expression (revealed by PaprikaRFP fluorescence, magenta) together with either control gRNA (top) or gTbr2 (bottom) on the abundance of mitotic progenitors as revealed by phosphohistone H3 (PH3) immunofluorescence (green). Apical and basal mitoses are indicated by arrows and arrowheads, respectively; note the reduction in mitotic Cas9+ BPs upon Cas9/gTbr2 plasmid electroporation. Scale bars, 20 μm.(D) Quantification of apical and basal Cas9+ mitoses (as revealed by PH3 immunofluorescence) in a unit area 48 h after Cas9/gTbr2 plasmid electroporation. Data are expressed as percentage of the data obtained upon control plasmid electroporation and are the mean of 3 independent experiments (3 embryos per condition in total, from 3 litters); note the reduction in basal (black, 68%) but not apical (white) mitoses.(E-H) Single aRGCs in neocortex in organotypic slices of telencephalon of E14.5 mouse embryos were microinjected with recombinant Cas9 protein together with gRNA targeting either LacZ (Control, Con) or Tbr2 (gTbr2) and with Dextran 10,000-Alexa 555 (Dx-A555), followed by analysis after 48 h of culture.(E) Progeny of single microinjected aRGCs (revealed by Dx-A555immunofluorescence, magenta, dotted lines) showing the effects of Cas9 together with either control gRNA (top) or gTbr2 (bottom) on Tbr2 expression (green, immunofluorescence); blue, DAPI staining. Note the presence of Tbr2 immunoreactivity in the control (top) and its absence upon Cas9/gTbr2 (bottom) protein microinjection. Images are single optical sections. Scale bars, 5 µm.(F) Quantification of the proportion of the progeny (Dx-A555+) of microinjected aRGCs that show Tbr2 expression 48 h after control (Con, white) or gTbr2 (black) microinjection. Data are the mean of 3 independent experiments (3 embryos per condition; total number of cells scored: control 45, gTbr2 41).(G) Distribution across the cortical wall of the progeny (revealed by Dx-A555immunofluorescence, magenta; arrows, aRGC; arrowheads, BPs; open arrow, neuron) of single control (left) or gTbr2 (right) microinjected aRGCs. Dashed lines indicate ventricular surface (bottom) and basal lamina (top). Images are maximum intensity projections of 67 (Control) and 94 (gTbr2) single optical sections. Scale bars, 20 µm.(H) Quantification of the distribution of the progeny (Dx-A555+) of microinjected aRGCs in the VZ, SVZ and intermediate zone/cortical plate (CP+IZ) 48 h after control (Con, white) or gTbr2 (black) microinjection (total number of cells scored: control 37, gTbr2 51, pooled from at least 3 independent experiments). Note that upon gTbr2 microinjection the distribution of cells across the various cortical zones is significantly different (VZ: control 77%, gTbr2 39: SVZ: control 20%, gTbr2 37%; IZ/CP: control 3%, gTbr2 24%, *, P<0.05).(B, F) Controls were set to 100% and the gTbr2 conditions expressed relative to control (B, 48%; F, 50%).(B, D, F) Error bars indicate SD (B, D, F); *, P<0.05; **, P<0.01.


Figure 4Figure 4 . Sequencing analysis of the CRISPR / Cas9 - induced disruption of the Tbr2 locus . ( A - C ) Next - generation DNA sequencing analysis of amplicons containing the Tbr2 locus and 4 off - target loci generated from PaprikaRFP - expressing cells isolated at E15 . 5 from neocortex of mouse embryos in utero electroporated at E13 . 5 with plasmids encoding Cas9 _ T2A _ PaprikaRFP and either gTbr2 ( black ) or control gRNA ( Con , white ) as in Fig 3 A - D . ( A ) Quantification of the frequency of mismatches to the mouse genome sequence spanning 30 bp upstream and downstream of the Tbr2 target site and the off - target sites ( OT1 - 4 ) . Data are expressed as percentage of the total number of reads and are the mean of two independent experiments ( 8 and 5 embryos per condition , respectively ) . ( B , C ) Analysis of one of the experiments of ( A ) for deletions ( B ) and insertions ( C ) in the region spanning 80 bp upstream and downstream of the Tbr2 target site upon Cas9 / gTbr2 plasmid electroporation .

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Figure 4Figure 4. Sequencing analysis of the CRISPR/Cas9-induced disruption of the Tbr2 locus.(A-C) Next-generation DNA sequencing analysis of amplicons containing the Tbr2 locus and 4 off-target loci generated from PaprikaRFP-expressing cells isolated at E15.5 from neocortex of mouse embryos in utero electroporated at E13.5 with plasmids encoding Cas9_T2A_PaprikaRFP and either gTbr2 (black) or control gRNA (Con, white) as in Fig 3 A-D.(A) Quantification of the frequency of mismatches to the mouse genome sequence spanning 30 bp upstream and downstream of the Tbr2 target site and the off-target sites (OT1-4). Data are expressed as percentage of the total number of reads and are the mean of two independent experiments (8 and 5 embryos per condition, respectively).(B, C) Analysis of one of the experiments of (A) for deletions (B) and insertions (C) in the region spanning 80 bp upstream and downstream of the Tbr2 target site upon Cas9/gTbr2 plasmid electroporation.


Figure 2C Genomic PCR analysis indicating the existence of MpFGMYB in both male [ Y ] and female [ X ] genomes of M . polymorpha . Two biological replicates were analyzed . Constitutively expressed MpEF1α was used as a control . D Real - time RT - PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes . MpEF1α was used for normalization . Measurements of six biological replicates for thalli and sporophyte , and three biological replicates for gametangiophore are plotted . Bars represent mean ± SD . Symbols above the bars indicate grouping by p < 0 . 05 in a Tukey - Kramer test . E A transcriptional reporter with 5 ′ - and 3 ′ - flanking sequences revealed transcription of MpFGMYB throughout mature archegonia . Scale bar , 10 µm . Magenta , chlorophyll autofluorescence ; green , Citrine fluorescence . F MpFGMYB - Citrine fusion proteins expressed using the 5 ′ - and 3 ′ - flanking sequences rescued the Mpfgmybge - 1 mutant and accumulated in the nuclei of the egg and the ventral canal cell ( VCC ) Scale bar , 10 µm . Magenta , chlorophyll autofluorescence ; green , Citrine fluorescence . G A transcriptional AtMYB64 reporter ( AtMYB64 - NYG ) is specifically expressed in all four cell types of the A . thaliana embryo sac Scale bar : 25 µm . Green , YFP fluorescence ; white , cell walls . H Expression of AtMYB64 - Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells ( CC ) and egg cells ( EC ) of the A . thaliana embryo sac Scale bar : 25 µm . Green , Citrine fluorescence ; white , cell walls .

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Figure 2C Genomic PCR analysis indicating the existence of MpFGMYB in both male [Y] and female [X] genomes of M. polymorpha. Two biological replicates were analyzed. Constitutively expressed MpEF1α was used as a control.D Real-time RT-PCR analyses indicating preferential accumulation of MpFGMYB transcripts in female sexual organs and the sporophytes. MpEF1α was used for normalization. Measurements of six biological replicates for thalli and sporophyte, and three biological replicates for gametangiophore are plotted. Bars represent mean ± SD. Symbols above the bars indicate grouping by p <0.05 in a Tukey-Kramer test.E A transcriptional reporter with 5′- and 3′-flanking sequences revealed transcription of MpFGMYB throughout mature archegonia. Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.F MpFGMYB-Citrine fusion proteins expressed using the 5′- and 3′-flanking sequences rescued the Mpfgmybge-1 mutant and accumulated in the nuclei of the egg and the ventral canal cell (VCC) Scale bar, 10 µm. Magenta, chlorophyll autofluorescence; green, Citrine fluorescence.G A transcriptional AtMYB64 reporter (AtMYB64-NYG) is specifically expressed in all four cell types of the A. thaliana embryo sac Scale bar: 25 µm. Green, YFP fluorescence; white, cell walls.H Expression of AtMYB64-Citrine fusion proteins under the AtMYB64 promoter was detected in the central cells (CC) and egg cells (EC) of the A. thaliana embryo sac Scale bar: 25 µm. Green, Citrine fluorescence; white, cell walls.


Figure 3B Diagnosis of genetic sex using Y - and X - chromosome - linked rbm27 and rhf73 markers , respectively . Two biological replicates were analyzed for each genotype . C - L Gametangiophore morphology ( C - G ) and gamete development ( H - L ) of wild - type and mutant plants . Scale bars , 5 mm ( C - G ) , 10 µm ( H and J ) , 100 µm ( I , K , and L ) .

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Figure 3B Diagnosis of genetic sex using Y- and X-chromosome-linked rbm27 and rhf73 markers, respectively. Two biological replicates were analyzed for each genotype.C-L Gametangiophore morphology (C-G) and gamete development (H-L) of wild-type and mutant plants. Scale bars, 5 mm (C-G), 10 µm (H and J), 100 µm (I, K, and L).


Figure 4A - C DAPI - staining visualization of sperm formation in wild - type male ( A ) , Mpfgmyb [ X ] ( B ) , and Mpfgmyb [ Y ] ( C ) plants . Note that background DAPI staining visualizes flagella ( arrows ) in addition to nuclei ( arrowheads ) . ( B ′ ) is an enlarged image of the boxed region in ( B ) , visualizing an incompletely condensed nucleus . Scale bars , 5 µm . D RT - PCR analysis indicating acquisition of male - like autosomal gene expression patterns in Mpfgmyb [ X ] antheridiophores . Two independent Mpfgmyb [ X ] mutant alleles were analyzed . E , F TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [ X ] sperm ( F ) , as compared with those of wild - type males ( E ) . Scale bars , 100 nm .

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Figure 4A-C DAPI-staining visualization of sperm formation in wild-type male (A), Mpfgmyb [X] (B), and Mpfgmyb [Y] (C) plants. Note that background DAPI staining visualizes flagella (arrows) in addition to nuclei (arrowheads). (B′) is an enlarged image of the boxed region in (B), visualizing an incompletely condensed nucleus. Scale bars, 5 µm.D RT-PCR analysis indicating acquisition of male-like autosomal gene expression patterns in Mpfgmyb [X] antheridiophores. Two independent Mpfgmyb [X] mutant alleles were analyzed.E, F TEM analyses visualizing the abnormal arrangement of axonemal microtubules in Mpfgmyb [X] sperm (F), as compared with those of wild-type males (E). Scale bars, 100 nm.


Figure 5A RNA - seq analysis showing male - specific accumulation of lncRNAs derived from the MpFGMYB 3 ′ region . Diagrams illustrate wild - type and mutant MpFGMYB / SUF loci . Folded lines with a ∆ symbol indicate a deletion . B RT - PCR analysis of wild - type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far - red irradiation . Two independent suf mutant alleles were analyzed . The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms . C - H Gametangiophore morphology ( C , E and G ) and gametangium development ( D , F and H ) of plants with the designated genotypes . Scale bars , 1 mm ( C , E and G ) , 20 µm ( D ) , 50 µm ( F ) , 100 µm ( H ) .

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Figure 5A RNA-seq analysis showing male-specific accumulation of lncRNAs derived from the MpFGMYB 3′ region. Diagrams illustrate wild-type and mutant MpFGMYB/SUF loci. Folded lines with a ∆ symbol indicate a deletion.B RT-PCR analysis of wild-type and genetically male suf mutants revealed loss of SUF expression and gain of MpFGMYB expression after induction of reproductive growth by far-red irradiation. Two independent suf mutant alleles were analyzed. The SUF primer pair used here flanked an intron and the duplicated bands of SUF likely represent spliced and unspliced forms.C-H Gametangiophore morphology (C, E and G) and gametangium development (D, F and H) of plants with the designated genotypes. Scale bars, 1 mm (C, E and G), 20 µm (D), 50 µm (F), 100 µm (H).


Figure 6A gametangiophore of MpEF1pro : SUF / suf - 30ge [ Y ] , indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [ Y ] . Scale bar , 2mm . B Real - time RT - PCR analyses confirming SUF transcript accumulation in SUF - overexpressing lines . Constitutively expressed MpEF1α was used as control . Measurements of six biological replicates for WT [ Y ] , and three biological replicates for each SUF - overexpressing line are plotted . Data information : bars represent mean ± SD . Asterisks indicate significant differences from WT [ Y ] ( B ) ( p < 0 . 05 , two - tailed Student ' s t - test ) . D Real - time RT - PCR measurement of MpFGMYB transcript levels . Three biological replicates are analyzed for each line . Data information bars represent mean ± SD . Asterisks indicate significant differences from WT [ X ] ( D ) ( p < 0 . 05 , two - tailed Student ' s t - test ) . E - H Gametangiophores ( E , G ) and gametangia ( F , H ) of gMpFGMYB - S [ Y ] ( E , F ) and gMpFGMYB - L [ Y ] ( G , H ) . Scale bars , 1 mm ( E , G ) , 50 μm ( F ) , 100 μm ( H ) .

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Figure 6A gametangiophore of MpEF1pro:SUF/suf-30ge [Y], indicating the inability of transgenic SUF overexpression in rescuing the feminization phenotype of sufge [Y]. Scale bar, 2mm.B Real-time RT-PCR analyses confirming SUF transcript accumulation in SUF-overexpressing lines. Constitutively expressed MpEF1α was used as control. Measurements of six biological replicates for WT [Y], and three biological replicates for each SUF-overexpressing line are plotted. Data information: bars represent mean ± SD. Asterisks indicate significant differences from WT [Y] (B) (p <0.05, two-tailed Student's t-test).D Real-time RT-PCR measurement of MpFGMYB transcript levels. Three biological replicates are analyzed for each line. Data information bars represent mean ± SD. Asterisks indicate significant differences from WT [X] (D) (p <0.05, two-tailed Student's t-test).E-H Gametangiophores (E, G) and gametangia (F, H) of gMpFGMYB-S [Y] (E, F) and gMpFGMYB-L [Y] (G, H). Scale bars, 1 mm (E, G), 50 μm (F), 100 μm (H).


figf1 ( a ) Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP . Where indicated , cells were starved for 2 . 5 h . Scale bar , 20 μm . ( b ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in a . Data represent mean ± s . e . m . of three independent experiments ( n = 100 cells per condition in each experiment ) . ( c ) Top left , 48 h after transfection with the indicated siRNA , MEFs were lysed and 25 μg samples of proteins were separated by DS - polyacrylamide gel electrophoresis ( PAGE ) and immunoblotted with the indicated antibodies . Top right and bottom , representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP . After a further 24 h , confocal micrographs were acquired . Scale bar , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S8 . ( d ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in c . Data represent mean ± s . e . m . of five independent experiments ( n = 100 cells per condition in each experiment ) . ( e ) Representative images of mitochondrial fusion over time ( indicated in min ) . MEFs were co - transfected with mtPAGFP and mtRFP and after 24 h , mtPAGFP was photoactivated in a region of interest and cells were imaged by real - time confocal microscopy . Where indicated , MEFs were starved for 2 . 5 h . Scale bar , 20 μm . See also Supplementary Movies S1 and S2 . ( f , g ) Quantification of mitochondrial fusion in MEFs of the indicated genotype . Experiments were carried out as in e . Data represent mean ± s . e . m . of four independent experiments . ( h ) Representative electron micrographs of muscle ( longitudinal sections ; four top micrographs ) and liver ( four bottom micrographs ) from CD1 mice . Where indicated , mice were fasted for 12 h . The outlined regions of micrographs from muscle samples are magnified × 7 in the images below . Scale bars , 2 μm .

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figf1(a) Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP. Where indicated, cells were starved for 2.5 h. Scale bar, 20 μm. (b) Morphometric analysis of mitochondrial shape. Experiments were carried out as in a. Data represent mean ± s.e.m. of three independent experiments (n=100 cells per condition in each experiment).(c) Top left, 48 h after transfection with the indicated siRNA, MEFs were lysed and 25 μg samples of proteins were separated by DS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. Top right and bottom, representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP. After a further 24 h, confocal micrographs were acquired. Scale bar, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S8. (d) Morphometric analysis of mitochondrial shape. Experiments were carried out as in c. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition in each experiment).(e) Representative images of mitochondrial fusion over time (indicated in min). MEFs were co-transfected with mtPAGFP and mtRFP and after 24 h, mtPAGFP was photoactivated in a region of interest and cells were imaged by real-time confocal microscopy. Where indicated, MEFs were starved for 2.5 h. Scale bar, 20 μm. See also Supplementary Movies S1 and S2. (f,g) Quantification of mitochondrial fusion in MEFs of the indicated genotype. Experiments were carried out as in e. Data represent mean ± s.e.m. of four independent experiments.(h) Representative electron micrographs of muscle (longitudinal sections; four top micrographs) and liver (four bottom micrographs) from CD1 mice. Where indicated, mice were fasted for 12 h. The outlined regions of micrographs from muscle samples are magnified ×7 in the images below. Scale bars, 2 μm.


figf2 ( a - d ) Levels of mitochondria - shaping proteins during starvation . Protein samples ( 20 μg ) from MEFs of the indicated genotype were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Cells were starved for the indicated times . ( e ) Association of DRP1 with mitochondria during starvation . Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS - PAGE and immunoblotted with the indicated antibodies . ( f ) Levels of Ser 637 phosphorylation of DRP1 during starvation . Equal amounts of cell lysates from wild - type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS - PAGE and immunoblotted with the indicated antibodies . ( g ) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation . Experiments were carried out as in f . Data are normalized to total levels of DRP1 and represent the mean ± s . e . m . of three independent experiments . ( h ) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin ( FRSK ) for 0 . 5 h , lysed and equal amounts ( 50 μg ) of proteins were separated by SDS - PAGE and immunoblotted using the indicated antibodies . ( i ) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0 . 5 h and lysed . Equal amounts ( 50 μg ) of proteins were separated by SDS - PAGE and immunoblotted using the indicated antibodies . Uncropped images of all blots in this figure are shown in Supplementary Fig . S8 .

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figf2(a-d) Levels of mitochondria-shaping proteins during starvation. Protein samples (20 μg) from MEFs of the indicated genotype were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Cells were starved for the indicated times.(e) Association of DRP1 with mitochondria during starvation. Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (f) Levels of Ser 637 phosphorylation of DRP1 during starvation. Equal amounts of cell lysates from wild-type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (g) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation. Experiments were carried out as in f. Data are normalized to total levels of DRP1 and represent the mean ± s.e.m. of three independent experiments.(h) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin (FRSK) for 0.5 h, lysed and equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies.(i) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0.5 h and lysed. Equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.


figf3 ( a ) Pseudocolour - coded images of EPACI - camps FRET from real - time imaging of wild - type MEFs transfected with EPACI - camps . Where indicated , cells were perfused with the starvation solution for the indicated times ( in min ) or with 25 μM forskolin ( FRSK ) . Scale bar , 20 μm . Colour scale indicates F1 / F0 ratio . See also Supplementary Movie S3 . ( b ) Quantitative analysis of CFP / YFP FRET ratio . Experiments were carried out as in a . Where indicated , cells were perfused with the starvation solution or with 25 μM forskolin . Data represent mean ± s . e . m . of 13 independent experiments . ( c ) Lysate samples ( 50 μg ) from MEFs of the indicated genotypes were analysed by SDS - PAGE and immunoblotting using the indicated antibodies . Where indicated , MEFs were starved , or treated with 25 μM forskolin . Where indicated , 20 μM H89 was added during starvation . Uncropped images of all blots in this figure are shown in Supplementary Fig . S8 . ( d ) Representative images of the effect of H89 on mitochondrial morphology on starvation . Wild - type and Mfn2 − / − MEFs were transfected with mtYFP , and after 24 h confocal micrographs were acquired . Where indicated , cells were starved for 2 . 5 h and 20 μM H89 was added . Scale bar , 20 μm . ( e ) Morphometric analysis . Experiments were carried out as in d . Data represent mean ± s . e . m . of five independent experiments ( n = 100 cells per condition ) . ( f ) Starvation - induced mitochondrial elongation depends on Ser 637 of DRP1 . Representative confocal micrographs of mitochondrial morphology of wild - type MEFs co - transfected with mtRFP and the indicated plasmids . At 24 h after transfection , where indicated cells were starved for 2 . 5 h and imaged . Where indicated , 20 μM H89 was present during starvation . Scale bar , 20 μm . ( g ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in f . Data represent mean ± s . e . m . of five independent experiments ( n = 50 cells per condition ) . ( h ) Representative confocal micrographs of mitochondrial morphology of Drp1 − / − MEFs co - transfected with mtRFP and the indicated plasmids . At 24 h after transfection , where indicated cells were starved for 2 . 5 h and imaged . Where indicated , 20 μM H89 was present during starvation . Scale bar , 20 μm . ( i ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in h . Data represent mean ± s . e . m . of five independent experiments ( n = 50 cells per condition ) .

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figf3(a) Pseudocolour-coded images of EPACI-camps FRET from real-time imaging of wild-type MEFs transfected with EPACI-camps. Where indicated, cells were perfused with the starvation solution for the indicated times (in min) or with 25 μM forskolin (FRSK). Scale bar, 20 μm. Colour scale indicates F1/F0 ratio. See also Supplementary Movie S3. (b) Quantitative analysis of CFP/YFP FRET ratio. Experiments were carried out as in a. Where indicated, cells were perfused with the starvation solution or with 25 μM forskolin. Data represent mean ± s.e.m. of 13 independent experiments.(c) Lysate samples (50 μg) from MEFs of the indicated genotypes were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Where indicated, MEFs were starved, or treated with 25 μM forskolin. Where indicated, 20 μM H89 was added during starvation. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.(d) Representative images of the effect of H89 on mitochondrial morphology on starvation. Wild-type and Mfn2−/−MEFs were transfected with mtYFP, and after 24 h confocal micrographs were acquired. Where indicated, cells were starved for 2.5 h and 20 μM H89 was added. Scale bar, 20 μm. (e) Morphometric analysis. Experiments were carried out as in d. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition).(f) Starvation-induced mitochondrial elongation depends on Ser 637 of DRP1. Representative confocal micrographs of mitochondrial morphology of wild-type MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (g) Morphometric analysis of mitochondrial shape. Experiments were carried out as in f. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).(h) Representative confocal micrographs of mitochondrial morphology of Drp1−/− MEFs co-transfected with mtRFP and the indicated plasmids. At 24 h after transfection, where indicated cells were starved for 2.5 h and imaged. Where indicated, 20 μM H89 was present during starvation. Scale bar, 20 μm. (i) Morphometric analysis of mitochondrial shape. Experiments were carried out as in h. Data represent mean ± s.e.m. of five independent experiments (n=50 cells per condition).


figf4 ( a - c ) MEFs of the indicated genotype were treated as indicated , counted and 2 . 7 × 105 cells were lysed . Lysates were separated by SDS - PAGE and immunoblotted using the indicated antibodies . ( d ) Ratio between the densitometric levels of cyclophilin D and those of PMP70 in MEFs of the indicated genotype . One representative experiment of five independent repetitions performed as in a - c is shown . ( e ) MEFs of the indicated genotype starved for 5 h were treated where indicated with 0 . 5 μM wortmannin ( Wortm . ) . Lysates from 2 . 7 × 105 cells were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Uncropped images of all blots in this figure are shown in Supplementary Fig . S8 .

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figf4(a-c) MEFs of the indicated genotype were treated as indicated, counted and 2.7×105 cells were lysed. Lysates were separated by SDS-PAGE and immunoblotted using the indicated antibodies. (d) Ratio between the densitometric levels of cyclophilin D and those of PMP70 in MEFs of the indicated genotype. One representative experiment of five independent repetitions performed as in a-c is shown.(e) MEFs of the indicated genotype starved for 5 h were treated where indicated with 0.5 μM wortmannin (Wortm.). Lysates from 2.7×105 cells were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.


figf5 ( a , b ) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype . Where indicated , cells were starved for 5 h before TMRM loading . Where indicated ( arrows ) , 2 . 5 μg ml − 1 oligomycin and 2 μM carbonyl cyanide - p - trifluoromethoxyphenylhydrazone ( FCCP ) were added . Data represent mean ± s . e . m . of seven independent experiments . ( c ) Total cellular ATP levels were measured in cells of the indicated genotype starved for the indicated times . Data represent mean ± s . e . m . of five independent experiments . ( d ) Mitochondrial ATP measured in situ by mitochondrially targeted luciferase in cells of the indicated genotype starved for the indicated times . Data represent mean ± s . e . m . of five independent experiments and are normalized to the initial value . ( e , f ) Cells of the indicated genotype were starved for the indicated times . Viability was determined by flow cytometry as the percentage of annexin - V - and propidium iodide ( PI ) - negative events . Data represent mean ± s . e . m . of five independent experiments . ( g ) MEFs of the indicated genotype were starved for 2 . 5 h . Where indicated , cells were treated with 20 μM H89 . Viability was determined as in e , f . Data represent mean ± s . e . m . of five independent experiments . ( h ) Cells of the indicated genotype were starved for the indicated times . Viability was determined as in e , f . Data represent mean ± s . e . m . of five independent experiments . ( i ) Drp1 − / − MEFs were transfected with the indicated plasmids and after 24 h starved for 5 h where indicated . Cell death was determined by flow cytometry as the percentage of YFP - and propidium - iodide - positive events . Data represent mean ± s . e . m . of four independent experiments . ( j , k ) MEFs of the indicated genotype were starved for 5 h in the presence of 2 . 5 μg ml − 1 oligomycin where indicated . Viability was determined as in e , f . Data represent mean ± s . e . m . of five independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a,b) Quantitative analysis of TMRM fluorescence changes over mitochondrial regions in MEFs of the indicated genotype. Where indicated, cells were starved for 5 h before TMRM loading. Where indicated (arrows), 2.5 μg ml−1 oligomycin and 2 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were added. Data represent mean ± s.e.m. of seven independent experiments.(c) Total cellular ATP levels were measured in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments.(d) Mitochondrial ATP measured in situ by mitochondrially targeted luciferase in cells of the indicated genotype starved for the indicated times. Data represent mean ± s.e.m. of five independent experiments and are normalized to the initial value.(e,f) Cells of the indicated genotype were starved for the indicated times. Viability was determined by flow cytometry as the percentage of annexin-V- and propidium iodide (PI)-negative events. Data represent mean ± s.e.m. of five independent experiments.(g) MEFs of the indicated genotype were starved for 2.5 h. Where indicated, cells were treated with 20 μM H89. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.(h) Cells of the indicated genotype were starved for the indicated times. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.(i) Drp1−/− MEFs were transfected with the indicated plasmids and after 24 h starved for 5 h where indicated. Cell death was determined by flow cytometry as the percentage of YFP- and propidium-iodide-positive events. Data represent mean ± s.e.m. of four independent experiments.(j,k) MEFs of the indicated genotype were starved for 5 h in the presence of 2.5 μg ml−1 oligomycin where indicated. Viability was determined as in e,f. Data represent mean ± s.e.m. of five independent experiments.


figf6 ( a ) Blue - native electrophoresis analysis of ATPase dimerization and activity . Cells of the indicated genotype were treated as indicated and 500 μg of total cell extract was solubilized with 4 % digitonin and proteins were separated by blue - native PAGE . ATPase activity was measured in - gel ( top ) and ATPase levels were measured by immunoblotting for the indicated antibody ( bottom ) . ( b , c ) Quantitative analysis of the ratio of levels ( b ) and activity ( c ) between dimeric and monomeric forms of ATPase , assessed by densitometric analysis of gels from experiments performed as in a . Data represent mean ± s . e . m . of five independent experiments . ( d ) Experiments were carried out as in a , but with cells of the indicated genotype .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) Blue-native electrophoresis analysis of ATPase dimerization and activity. Cells of the indicated genotype were treated as indicated and 500 μg of total cell extract was solubilized with 4% digitonin and proteins were separated by blue-native PAGE. ATPase activity was measured in-gel (top) and ATPase levels were measured by immunoblotting for the indicated antibody (bottom). (b,c) Quantitative analysis of the ratio of levels (b) and activity (c) between dimeric and monomeric forms of ATPase, assessed by densitometric analysis of gels from experiments performed as in a. Data represent mean ± s.e.m. of five independent experiments. (d) Experiments were carried out as in a, but with cells of the indicated genotype.


figf7 ( a ) Representative electron micrographs of cells of the indicated genotype starved where indicated for 5 h , fixed and processed for electron microscopy . Scale bar , 2 μm . ( b ) Representative electron micrographs of randomly selected mitochondria from cells of the indicated genotype . Where indicated , cells were starved for 5 h . Scale bars , 0 . 5 μm . ( c ) Morphometric analysis of cristae density in cells of the indicated genotype . Experiments were carried out as in a . The number of cristae in 50 randomly selected mitochondria of the indicated genotype was normalized for the calculated surface of the organelle . Data represent mean ± s . e . m . of five independent experiments .

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figf7(a) Representative electron micrographs of cells of the indicated genotype starved where indicated for 5 h, fixed and processed for electron microscopy. Scale bar, 2 μm. (b) Representative electron micrographs of randomly selected mitochondria from cells of the indicated genotype. Where indicated, cells were starved for 5 h. Scale bars, 0.5 μm. (c) Morphometric analysis of cristae density in cells of the indicated genotype. Experiments were carried out as in a. The number of cristae in 50 randomly selected mitochondria of the indicated genotype was normalized for the calculated surface of the organelle. Data represent mean ± s.e.m. of five independent experiments.


Figure 1p53 has been show to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs . Schematic representations of p53 dynamics in both cellular conditions are shown . Induction of selected p53 target genes after DNA damage induction in A549 wild - type and p53 knockdown cells . RNA levels were measured by qRT - PCR before and 3h after treatment with 10 Gy R . Fold changes relative to basal levels are shown for each cell line . Fluorescence microscopy images of smFISH probes labelled with CAL - Fluor 610 ( grey ) overlayed with Hoechst 33342 stainings ( blue ) for the indicated target genes in untreated A549 cells . Scale bar corresponds to 10 µm distance , images were contrast and brightness enhanced for better visualization . Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage ( basal ) . smFISH staining and quantitative analysis of p53 targets shows broad variability of RNA counts per cell for all genes in basal conditions .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1p53 has been show to response with a series of undamped pulse to ionizing irradiation leading to cell cycle arrest while intrinsic DNA damage during cell cycle does not induce regular pulsatile p53 and subsequent gene expression programs. Schematic representations of p53 dynamics in both cellular conditions are shown.Induction of selected p53 target genes after DNA damage induction in A549 wild-type and p53 knockdown cells. RNA levels were measured by qRT-PCR before and 3h after treatment with 10 Gy R. Fold changes relative to basal levels are shown for each cell line.Fluorescence microscopy images of smFISH probes labelled with CAL-Fluor 610 (grey) overlayed with Hoechst 33342 stainings (blue) for the indicated target genes in untreated A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization.Histograms of quantitative analysis of RNAs per cell for each target gene in the absence of DNA damage (basal). smFISH staining and quantitative analysis of p53 targets shows broad variability of RNA counts per cell for all genes in basal conditions.


Figure 2Fluorescence microscopy images of smFISH probes CAL - Fluor 610 ( grey ) overlayed with Hoechst 33342 staining ( blue ) at 3 h after 10 Gy IR or the indicated target genes in A549 cells . Scale bar corresponds to 10 µm distance , images were contrast and brightness enhanced for better visualization . We quantified RNAs per cell for the indicated target genes before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . smFISH based single cell analysis of gene expression patterns highlights distinct RNA counts for p53 targets . RNA counts per cell are displayed as boxplots ( see Data Visualization section ) ; n : number of analyzed cells , fc : median fold of induction relative to time point basal ( indicated by grey lines ) Distributions of RNAs per cell for the indicated target genes before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . Despite a clear change in median levels ( m : median ) , single cell analysis reveals a strong dispersion that overlaps for the different conditions , as shown by the strongly overlapping distributions of RNA counts per cells . For better visualization , probability density estimates ( PDF ) based on a normal kernel are

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Fluorescence microscopy images of smFISH probes CAL-Fluor 610 (grey) overlayed with Hoechst 33342 staining (blue) at 3 h after 10 Gy IR or the indicated target genes in A549 cells. Scale bar corresponds to 10 µm distance, images were contrast and brightness enhanced for better visualization.We quantified RNAs per cell for the indicated target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). smFISH based single cell analysis of gene expression patterns highlights distinct RNA counts for p53 targets. RNA counts per cell are displayed as boxplots (see Data Visualization section); n: number of analyzed cells, fc: median fold of induction relative to time point basal (indicated by grey lines) Distributions of RNAs per cell for the indicated target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). Despite a clear change in median levels (m: median), single cell analysis reveals a strong dispersion that overlaps for the different conditions, as shown by the strongly overlapping distributions of RNA counts per cells. For better visualization, probability density estimates (PDF) based on a normal kernel are shown


Figure 3Quantification of stochastic gene expression for the indicated p53 target genes before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) . The fraction ( f ) of active promoters ( proxy for burst frequency ) increases , while the transcription rate ( µ ) ( proxy for burst size ) at active TSS remains similar upon DNA damage for all time points . Left panel : The percentage of cells with active TSS is shown as stacked bar graphs . We subdivided the population in cells with strong TSS activity ( > 75 % of TSS active , solid colors ) and those with partial TSS activity ( at least one , but less than 75 % of TSS active , shaded colors ) . The mean fraction of active promoters ( ratio of all active TSS to the total number of genomic loci analyzed ) is indicated above each bar . Right panel : Distributions of calculated transcription rates µ [ RNAs / h ] at active TSS are presented for each time point as probability density estimates ( PDF , see Data Visualization section ) . The number of TSS analyzed is indicated in each plot ( compare Fig EV2C ) . Mean degradation rates of indicated RNAs in transcriptionally active cells before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) as calculated from smFISH data . RNA stability is not changing in the measured time frame upon DNA damage . The plot displays the average RNA degradation rate per cell [ 1 / h ] over time after DNA damage , calculated from model ( C ) in actively transcribing cells for each gene . Amount of p53 bound to indicated target gene promoters before ( basal , grey ) and 3 h ( red ) , 6 h ( blue ) and 9 h ( orange ) after DNA damage ( 10 Gy IR ) as measured by ChIP . The amount of bound p53 was calculated as percentage of input and normalized to the time - point of the first p53 peak at 3 h . Individual data points ( mean values of triplicate quantification in qRT - PCR measurements ) from 3 - 4 biological repeats are shown as dots , mean values are displayed as black horizontal lines . Dashed lines serve as guide to the eyes . We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter ( indicated by n . d . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Quantification of stochastic gene expression for the indicated p53 target genes before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR). The fraction (f) of active promoters (proxy for burst frequency) increases, while the transcription rate (µ) (proxy for burst size) at active TSS remains similar upon DNA damage for all time points. Left panel: The percentage of cells with active TSS is shown as stacked bar graphs. We subdivided the population in cells with strong TSS activity (> 75% of TSS active, solid colors) and those with partial TSS activity (at least one, but less than 75% of TSS active, shaded colors). The mean fraction of active promoters (ratio of all active TSS to the total number of genomic loci analyzed) is indicated above each bar. Right panel: Distributions of calculated transcription rates µ [RNAs/h] at active TSS are presented for each time point as probability density estimates (PDF, see Data Visualization section). The number of TSS analyzed is indicated in each plot (compare Fig EV2C).Mean degradation rates of indicated RNAs in transcriptionally active cells before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR) as calculated from smFISH data. RNA stability is not changing in the measured time frame upon DNA damage. The plot displays the average RNA degradation rate per cell [1/h] over time after DNA damage, calculated from model (C) in actively transcribing cells for each gene.Amount of p53 bound to indicated target gene promoters before (basal, grey) and 3 h (red), 6 h (blue) and 9 h (orange) after DNA damage (10 Gy IR) as measured by ChIP. The amount of bound p53 was calculated as percentage of input and normalized to the time-point of the first p53 peak at 3 h. Individual data points (mean values of triplicate quantification in qRT-PCR measurements) from 3-4 biological repeats are shown as dots, mean values are displayed as black horizontal lines. Dashed lines serve as guide to the eyes. We could not detect p53 binding above IgG controls at the published p53 response element in the PPM1D promoter (indicated by n.d.)