Figure 3A ) Fragment size distribution , determined by sWGS , in CSF ( blue line ) , plasma ( red dashed line ) and urine ( black dashed line ) from patient G1 . The 3 samples were collected simultaneously before initiation of treatment . B ) Fragment size distribution of cfDNA , determined by sWGS , in CSF from 13 glioma patients . The samples contained a high fraction of reads from DNA fragments with lengths corresponding to ~ 145 bp and ~ 167 bp . The fragment size profile of cfDNA from the plasma sample from patient G1 is shown in red . All samples were collected simultaneously before initiation of treatment C ) Cumulative frequency analysis of the average density of fragment size in CSF ( blue ) and plasma ( red ) . The vertical dashed line represents 167 bpMean fragment size density in patients with detected SCNAs in the CSF ( yellow ) , and those with no detected SCNAs ( dark blue ) Log2 ratio of the difference in cfDNA fragment sizes between CSF samples with detected SCNAs and those samples in which no SCNAs were detectedThe amplitude of the 10 bp periodic peaks in fragment size is a signature for non - tumor DNA in the CSF . The amplitude of the 10 bp periodic peaks in fragment length distribution ( in the range between 75 and 150 bp , see Methods ) when compared to the mean copy number alteration calculated from sWGS revealed a significant negative correlation ( Pearson - 0 . 85 ; p = 0 . 0002 )

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Figure 3A) Fragment size distribution, determined by sWGS, in CSF (blue line), plasma (red dashed line) and urine (black dashed line) from patient G1. The 3 samples were collected simultaneously before initiation of treatment. B) Fragment size distribution of cfDNA, determined by sWGS, in CSF from 13 glioma patients. The samples contained a high fraction of reads from DNA fragments with lengths corresponding to ~145 bp and ~167 bp. The fragment size profile of cfDNA from the plasma sample from patient G1 is shown in red. All samples were collected simultaneously before initiation of treatment C) Cumulative frequency analysis of the average density of fragment size in CSF (blue) and plasma (red). The vertical dashed line represents 167 bpMean fragment size density in patients with detected SCNAs in the CSF (yellow), and those with no detected SCNAs (dark blue)Log2 ratio of the difference in cfDNA fragment sizes between CSF samples with detected SCNAs and those samples in which no SCNAs were detectedThe amplitude of the 10 bp periodic peaks in fragment size is a signature for non-tumor DNA in the CSF. The amplitude of the 10 bp periodic peaks in fragment length distribution (in the range between 75 and 150 bp, see Methods) when compared to the mean copy number alteration calculated from sWGS revealed a significant negative correlation (Pearson -0.85; p=0.0002)


Figure 1A The distribution of the GFP - tagged Hxt2 changes during the cell division . In small and medium budded cells ( white arrowheads ) , Hxt2 is mainly present in the membrane of the mother cell . In large budded cells , Hxt2 is distributed over the entire plasma membrane ( yellow arrowheads ) . B HXT2 mRNA localization correlate with that of the protein . FISH with an HXT2 - specific probe . Early in the cell cycle the mRNA is predominantly found in the mother cell ( white arrowheads ) while it is equally distributed after meta - to - anaphase transition ( MAT ) ( yellow arrowheads ) . C Quantification of ( B ) . The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated . Values > 1 indicate a stronger signal in the mother , < 1 a stronger signal in the bud and around 1 equal distribution . Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed . Boxes represent the interquartile range from the 25th to the 75th percentile with the median . Whiskers represent the 10th and 90th percentile , respectively . D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton . Cells were either treated for 15 min with 30 mg / ml latrunculin A or with 30 mg / ml benomyl . White arrowheads point to small - budded and yellow arrowheads to large budded cells . E Quantification of ( D ) of three in dependent experiments with at least 50 cells each . Values > 1 indicate a stronger signal in the mother , < 1 a stronger signal in the bud and around 1 equal distribution . Boxes represent the interquartile range from the 25th to the 75th percentile with the median . Whiskers represent the 10th and 90th percentile , respectively .

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Figure 1A The distribution of the GFP-tagged Hxt2 changes during the cell division. In small and medium budded cells (white arrowheads), Hxt2 is mainly present in the membrane of the mother cell. In large budded cells, Hxt2 is distributed over the entire plasma membrane (yellow arrowheads).B HXT2 mRNA localization correlate with that of the protein. FISH with an HXT2-specific probe. Early in the cell cycle the mRNA is predominantly found in the mother cell (white arrowheads) while it is equally distributed after meta-to-anaphase transition (MAT) (yellow arrowheads). C Quantification of (B). The fluorescence intensities from mother and bud were independently measured and the ratio of the signal from the mother over the signal from the bud was calculated. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Three independent experiments with 50 mother cells and 50 buds per experiment were analyzed. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively. D Changes of HXT2 mRNA localization appear to be independent of the cytoskeleton. Cells were either treated for 15 min with 30 mg/ml latrunculin A or with 30 mg/ml benomyl. White arrowheads point to small-budded and yellow arrowheads to large budded cells. E Quantification of (D) of three in dependent experiments with at least 50 cells each. Values > 1 indicate a stronger signal in the mother, < 1 a stronger signal in the bud and around 1 equal distribution. Boxes represent the interquartile range from the 25th to the 75th percentile with the median. Whiskers represent the 10th and 90th percentile, respectively.


Figure 2A , Inhibiting translation by either applying Cycloheximide ( CHX ) or Verrucarin A ( VerruA ) leads to the retention of HXT2 mRNA in the mother cell after MAT . Chloro : chloroform ; solvent control for VerruA ; NT : not treated ; control for CHX . FISH of CHX or VerruA - treated cells . Chloro : chloroform ; solvent control for VerruA ; NT : not treated ; control for CHX . C CHX treatment leads to increased transcript stability . Quantitative PCR of HXT2 mRNA in CHX - treated cells in comparison to prt1 - 1 mutant cells . The data represents 3 independent experiments and the standard deviation is given . D Translation of HXT2 mRNA is more active late in the cell cycle . The HXT2 mRNA level bound to either di - / trisomes and polysomes ( ≥ 4 ribosomes ) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1 / S or M - phase followed by qPCR . At G1 / S similar levels of the HXT2 mRNA were found in the different ribosome fractions , whereas in M - phase more mRNA was bound to polysomes , suggesting a more active translation in M - phase . E The localization of HXT2 mRNA is independent of the Signal Recognition Particle ( SRP ) . Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA .

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Figure 2A, Inhibiting translation by either applying Cycloheximide (CHX) or Verrucarin A (VerruA) leads to the retention of HXT2 mRNA in the mother cell after MAT. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.FISH of CHX or VerruA-treated cells. Chloro: chloroform; solvent control for VerruA; NT: not treated; control for CHX.C CHX treatment leads to increased transcript stability. Quantitative PCR of HXT2 mRNA in CHX-treated cells in comparison to prt1-1 mutant cells. The data represents 3 independent experiments and the standard deviation is given.D Translation of HXT2 mRNA is more active late in the cell cycle. The HXT2 mRNA level bound to either di-/trisomes and polysomes (≥ 4 ribosomes) over monosomes was determined by extraction of mRNA from cells elutriated and harvested at G1/S or M-phase followed by qPCR. At G1/S similar levels of the HXT2 mRNA were found in the different ribosome fractions, whereas in M-phase more mRNA was bound to polysomes, suggesting a more active translation in M-phase.E The localization of HXT2 mRNA is independent of the Signal Recognition Particle (SRP). Deletion of SRP54 does not affect the retention in the mother cell or the equal distribution of HXT2 mRNA.


Figure 3A Deletion of SCP160 , ASC1 , or BFR1 increases HXT2 mRNA signal in the bud . FISH experiment with deletions in various RNA binding proteins . B The effect of ∆ scp160 on HXT2 mRNA localization is independent of its increased ploidy . Scp160 truncations that lack either the last two ( ∆ C2 ) or the last four ( ∆ C4 ) KH domains or a Tet - off SCP160 construct , in which expression is blocked by the addition of doxycycline ( Doxy ) D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud . Treatment of cells with forskolin ( + Forsko ) or using a ∆ bcy1 strain , causes HXT2 mRNA to be bud - enriched . E HXT2 mRNA enrichment upon refeeding is Ras2 - dependent .

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Figure 3A Deletion of SCP160, ASC1, or BFR1 increases HXT2 mRNA signal in the bud. FISH experiment with deletions in various RNA binding proteins.B The effect of ∆scp160 on HXT2 mRNA localization is independent of its increased ploidy. Scp160 truncations that lack either the last two (∆C2) or the last four (∆C4) KH domains or a Tet-off SCP160 construct, in which expression is blocked by the addition of doxycycline (Doxy)D Hyperactivation of PKA drives accumulation of HXT2 mRNA in the bud. Treatment of cells with forskolin (+Forsko) or using a ∆bcy1 strain, causes HXT2 mRNA to be bud-enriched.E HXT2 mRNA enrichment upon refeeding is Ras2-dependent.


Figure 4A Table of the glucose affinity of the four most important hexose transporters . B FISH of HXT1 , 3 and 4 under glucose rich conditions . White arrowheads depict cells before , yellow arrowheads cells after MAT . C Quantification of ( B ) . D Hyperactive PKA promotes enrichment of all HXTs after MAT . E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation . F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding . Daughters and mothers were separated through elutriation . qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions . G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2 - GFP transporter levels at the plasma membrane of the daughter cells . Daughter cells were enriched through elutriation . Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment . Western blot analysis confirms the higher amount of Hxt2 - GFP transporter protein present in daughter cells under glucose - shift

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Figure 4A Table of the glucose affinity of the four most important hexose transporters. B FISH of HXT1, 3 and 4 under glucose rich conditions. White arrowheads depict cells before, yellow arrowheads cells after MAT. C Quantification of (B). D Hyperactive PKA promotes enrichment of all HXTs after MAT.E HXT2 mRNA is the only HXT mRNA showing enrichment in the bud after MAT upon transient PKA activation.F Elutriation confirms enrichment of HXT2 mRNA in daughter cells upon refeeding. Daughters and mothers were separated through elutriation. qPCR was performed to determine the relative levels of different HXTs and ASH1 mRNA in both fractions.G Enrichment of HXT2 mRNA in the bud leads to higher Hxt2-GFP transporter levels at the plasma membrane of the daughter cells. Daughter cells were enriched through elutriation. Quantification of the fluorescence intensity of the plasma membrane from life cell imaging from three independent experiments in which at least 50 cells were analyzed per condition and experiment. Western blot analysis confirms the higher amount of Hxt2-GFP transporter protein present in daughter cells under glucose-shift conditions


Figure 5A RNA degradation is not essential for asymmetric HXT2 mRNA localization . FISH of wild - type and ∆ xrn1 cells . B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud . Cells were treated upon glucose addition with either 120 mg / ml benomyl for 15 min , 30 mg / ml latrunculin A for 30 min or 200 µM CK - 666 for 30 min . HXT2 mRNA was detect by FISH . C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function . HXT2 mRNA FISH with ∆ bim1 and ∆ kar9 strains . Arrows point to a nucleus with HXT2 signal . D Quantification of the daughter cell enrichment from data displayed in ( C ) . E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in ( C ) . Percentage of cells with asymmetric HXT2 mRNA staining is displayed .

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Figure 5A RNA degradation is not essential for asymmetric HXT2 mRNA localization. FISH of wild-type and ∆xrn1 cells.B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud. Cells were treated upon glucose addition with either 120 mg/ml benomyl for 15 min, 30 mg/ml latrunculin A for 30 min or 200 µM CK-666 for 30 min. HXT2 mRNA was detect by FISH.C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function. HXT2 mRNA FISH with ∆bim1 and ∆kar9 strains. Arrows point to a nucleus with HXT2 signal. D Quantification of the daughter cell enrichment from data displayed in (C). E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in (C). Percentage of cells with asymmetric HXT2 mRNA staining is displayed.


Figure 6A Deletion of NUP2 and MLP1 / 2 reduced HXT2 mRNA enrichment in the bud . FISH under glucose shift conditions with strains in which different NPC components were deleted . B Quantification of ( A ) . C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift . FISH - IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84 - HA . White arrows point to HXT2 mRNA signals on the nuclear envelope . D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift . Stills of time lapse movies . HXT2 mRNA was visualized using the MS2 - GFP system . Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus . Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus . Cyan : Nup84 - mCherry ; white arrowhead : HXT2 mRNA - MS2 + MCP - GFP .

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Figure 6A Deletion of NUP2 and MLP1/2 reduced HXT2 mRNA enrichment in the bud. FISH under glucose shift conditions with strains in which different NPC components were deleted. B Quantification of (A). C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift. FISH-IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84-HA. White arrows point to HXT2 mRNA signals on the nuclear envelope.D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift. Stills of time lapse movies. HXT2 mRNA was visualized using the MS2-GFP system. Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus. Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus. Cyan: Nup84-mCherry; white arrowhead: HXT2 mRNA-MS2 + MCP-GFP.


Figure 7HXT2 only strains grow faster than the other HXT only strain . Growth was assessed 2 - 3 days after incubation at 30 ° C . D Quantification of competition assay . qPCR with primers against either HXT1 , 2 , 3 or 4 was performed . HXT gene abundance was normalized against actin . E Strains that lack HXT2 grow slower when coming from quiescence compared to WT . F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96 % of cells independently of the cell cycle . Arrows point to examples of HXT2 - U1A mRNA in the bud early and late in the cell cycle . G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2 . H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers . Cells were re - fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points .

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Figure 7HXT2 only strains grow faster than the other HXT only strain. Growth was assessed 2-3 days after incubation at 30°C.D Quantification of competition assay. qPCR with primers against either HXT1, 2, 3 or 4 was performed. HXT gene abundance was normalized against actin.E Strains that lack HXT2 grow slower when coming from quiescence compared to WT.F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96% of cells independently of the cell cycle. Arrows point to examples of HXT2-U1A mRNA in the bud early and late in the cell cycle.G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2.H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers. Cells were re-fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points.


Figure 4 ( A ) Michaelis - Menten analysis of the reduction of 2 - hydroxyacetophenone ( 2 - HAP ) . The initial velocities ( V0 / [ Etot ] ) are given for the individual subunits ( i . e . , normalized to subunit not oligomer concentration ) . Enzyme assays were conducted at 30 ˚ C in 100 mM sodium phosphate ( pH 6 . 0 ) using 500 µM NADPH and 100 nM SCR or 1 µM SCR - his6 . Enzyme activity at each substrate concentration was assayed at least three and usually four times . The data points show the average values for these technical replicates , while the error bars show the standard deviations . Comparison of kcat values obtained from Michaelis - Menten analyses of the reduction of four different substrates . The turnover rates ( kcat ) are given for the individual subunits ( i . e . , normalized to subunit not oligomer concentration ) . Enzyme assays were conducted at 30 ˚ C in 100 mM sodium phosphate ( pH 6 . 0 ) using 500 µM NADPH and 100 nM SCR or 1 µM SCR - his6 . The data points show the average values for these technical replicates , while the error bars show the standard deviations .

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Figure 4(A) Michaelis-Menten analysis of the reduction of 2-hydroxyacetophenone (2-HAP). The initial velocities (V0/[Etot]) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH and 100 nM SCR or 1 µM SCR-his6. Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.Comparison of kcat values obtained from Michaelis-Menten analyses of the reduction of four different substrates. The turnover rates (kcat) are given for the individual subunits (i.e., normalized to subunit not oligomer concentration). Enzyme assays were conducted at 30 ˚C in 100 mM sodium phosphate (pH 6.0) using 500 µM NADPH and 100 nM SCR or 1 µM SCR-his6. The data points show the average values for these technical replicates, while the error bars show the standard deviations.


Figure 6 ( A ) Analytical gel - filtration of purified his8 - MBP - SCR2 ( black ) , strep - SCR ( yellow ) , and his8 - MBP - SCR2 / strep - SCR on a Superdex 200 Increase column at 20 ˚ C in 150 mM NaCl , 50 mM Tris - HCl , pH 8 . 0 . ( B ) Michaelis - Menten kinetic analyses of NADPH - dependent reduction of 2 - hydroxyacetophenone and methyl acetoacetate at 30 ˚ C in 100 mM sodium phosphate , pH 6 . 0 . The turnover rate ( V0 / [ Etot ] ) is given for the individual subunits Enzyme activity at each substrate concentration was assayed at least three and usually four times . The data points show the average values for these technical replicates , while the error bars show the standard deviations .

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Figure 6(A) Analytical gel-filtration of purified his8-MBP-SCR2 (black), strep-SCR (yellow), and his8-MBP-SCR2/strep-SCR on a Superdex 200 Increase column at 20 ˚C in 150 mM NaCl, 50 mM Tris-HCl, pH 8.0.(B) Michaelis-Menten kinetic analyses of NADPH-dependent reduction of 2-hydroxyacetophenone and methyl acetoacetate at 30 ˚C in 100 mM sodium phosphate, pH 6.0. The turnover rate (V0/[Etot]) is given for the individual subunits Enzyme activity at each substrate concentration was assayed at least three and usually four times. The data points show the average values for these technical replicates, while the error bars show the standard deviations.


Figure 1 ( D ) Western blot analysis of ADAR1 expression in E13 . 5 immortalized MEFs of the indicated genotypes + / - interferon - β ( IFNβ ) . Data from two independent cell lines per genotype . ( G ) Survival analysis of mice of the indicated genotypes ; number as indicated for each genotype . Statistical difference determined by Log - rank ( Mantel - Cox ) test . ( H ) Total white blood cell counts ( WBC ) in peripheral blood ( PB ) . ( J - L ) Peripheral blood ( J ) red blood cell ( RBC ) , ( K ) hematocrit ( HCT ) and ( L ) platelets .

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Figure 1(D) Western blot analysis of ADAR1 expression in E13.5 immortalized MEFs of the indicated genotypes +/- interferon-β (IFNβ). Data from two independent cell lines per genotype.(G) Survival analysis of mice of the indicated genotypes; number as indicated for each genotype. Statistical difference determined by Log-rank (Mantel-Cox) test.(H) Total white blood cell counts (WBC) in peripheral blood (PB).(J-L) Peripheral blood (J) red blood cell (RBC), (K) hematocrit (HCT) and (L) platelets.


Figure 2 ( C ) Survival analysis of mice of the indicated genotypes ; number as indicated for each genotype . No statistical difference between genotypes by Log - rank ( Mantel - Cox ) test . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . ( E ) Peripheral blood leukocyte populations ( by lineage ) between genotypes at day 0 and day 28 . ; R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . All BM , spleen and thymic analysis at day 28 post tamoxifen treatment ( G ) Differential analysis of leukocyte populations in the BM . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . All BM analysis at day 28 post tamoxifen treatment ( H ) BM erythroid cells . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . All BM , analysis at day 28 post tamoxifen treatment ( J ) Differential analysis of leukocyte populations in the spleen . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . spleen analysis at day 28 post tamoxifen treatment ; ( K ) Thymic cellularity . ( L ) Differential analysis of thymocyte populations in the thymus . ( R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) . thymic analysis at day 28 post tamoxifen treatment ( N ) qPCR ( SYBR green ) based analysis of indicated gene expression in BM . Data expressed as mean + / - SEM gene expression related to Ppia expression . R26 - CreERki / + Adar1fl / + ( control ; n = 3 ) and R26 - CreERki / + Adar1fl / P195A ( P195A ; n = 4 ) .

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Figure 2(C) Survival analysis of mice of the indicated genotypes; number as indicated for each genotype. No statistical difference between genotypes by Log-rank (Mantel-Cox) test. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4).(E) Peripheral blood leukocyte populations (by lineage) between genotypes at day 0 and day 28. ; R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM, spleen and thymic analysis at day 28 post tamoxifen treatment(G) Differential analysis of leukocyte populations in the BM. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM analysis at day 28 post tamoxifen treatment(H) BM erythroid cells. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). All BM, analysis at day 28 post tamoxifen treatment(J) Differential analysis of leukocyte populations in the spleen. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). spleen analysis at day 28 post tamoxifen treatment;(K) Thymic cellularity. (L) Differential analysis of thymocyte populations in the thymus. ( R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4). thymic analysis at day 28 post tamoxifen treatment(N) qPCR (SYBR green) based analysis of indicated gene expression in BM. Data expressed as mean +/- SEM gene expression related to Ppia expression. R26-CreERki/+ Adar1fl/+ (control; n=3) and R26-CreERki/+ Adar1fl/P195A (P195A; n=4).


Figure 4 , ( C ) survival analysis of mice of the indicated genotypes . ( E ) survival of mice from inbreeding to generate P195A / - animals . ( H ) survival of pups derived from breeding of a Adar1P195A / P195A to a Adar1 + / - animal ; number as indicated for each genotype . ( K ) survival of pups derived from breeding an Adar1P195A / - to an Adar1P195A / + genotype ; number indicated for each genotype . Inset photo : 35 - day - old Adar1P195A / - male bred from Adar1P195A / P195A x Adar1 + / - parents ( data in Panel F ) ; sibling 29 - day - old Adar1P195A / + and Adar1P195A / - male bred from an Adar1P195A / - x Adar1P195A / + ( data in Panel I ) .

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Figure 4, (C) survival analysis of mice of the indicated genotypes.(E) survival of mice from inbreeding to generate P195A/- animals.(H) survival of pups derived from breeding of a Adar1P195A/P195A to a Adar1+/- animal; number as indicated for each genotype.(K) survival of pups derived from breeding an Adar1P195A/- to an Adar1P195A/+ genotype; number indicated for each genotype. Inset photo: 35-day-old Adar1P195A/- male bred from Adar1P195A/P195A x Adar1+/- parents (data in Panel F); sibling 29-day-old Adar1P195A/+ and Adar1P195A/- male bred from an Adar1P195A/- x Adar1P195A/+ (data in Panel I).


Figure 5 ( B ) survival of Adar1P195A / E861AIfih1 - / - and littermates ( all Ifih1 - / - ) . ( D ) survival of Adar1P195A / - Ifih1 - / - and littermates ( all Ifih1 - / - ) .

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Figure 5(B) survival of Adar1P195A/E861AIfih1-/- and littermates (all Ifih1-/-).(D) survival of Adar1P195A/-Ifih1-/- and littermates (all Ifih1-/-).


Figure 6 ( F ) Individual plots of the normalised expression of ISGs and ISR genes in the kidney of the indicated genotypes using Taqman - based qPCR . ( G ) Western blot of PKR , phospho - eIF2α and total eIF2α levels in whole kidney lysates derived from the indicated genotypes ; same mice as used for panel F . Relative phospho - eIF2α expression was calculated against total eIF2α normalised to the average of P195A / + samples using ImageJ Fiji . All images are from the same gel and re - probed for different antibodies .

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Figure 6(F) Individual plots of the normalised expression of ISGs and ISR genes in the kidney of the indicated genotypes using Taqman-based qPCR.(G) Western blot of PKR, phospho-eIF2α and total eIF2α levels in whole kidney lysates derived from the indicated genotypes; same mice as used for panel F. Relative phospho-eIF2α expression was calculated against total eIF2α normalised to the average of P195A/+ samples using ImageJ Fiji. All images are from the same gel and re-probed for different antibodies.


Figure 7 ( E ) Viability of cell lines with and without tamoxifen treatment ( isogenic pairs ) over 14 days of treatment ( n = 3 independent lines per genotype ; biological replicates ) . ( F ) qPCR ( SYBR green ) analysis of Ifit1 ( left ) and Irf7 ( right ) expression on day 14 of analysis . ( G ) Cell viability at IFNβ dosages 0 , 50 and 1000U / mL after 24 hours . Statistical analysis was Two - way ANOVA with multiple comparisons correction . ( H ) qPCR ( SYBR green ) of Ifit1 ( left ) and Irf7 ( right ) expression in the cell lines collected at 3 or 24 hours post - treatment with 50 or 1000 U / mL of IFNβ . No statistical difference between genotypes in Ifit1 ( left ) and Irf7 ( right ) expression .

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Figure 7(E) Viability of cell lines with and without tamoxifen treatment (isogenic pairs) over 14 days of treatment (n=3 independent lines per genotype; biological replicates).(F) qPCR (SYBR green) analysis of Ifit1 (left) and Irf7 (right) expression on day 14 of analysis.(G) Cell viability at IFNβ dosages 0, 50 and 1000U/mL after 24 hours. Statistical analysis was Two-way ANOVA with multiple comparisons correction.(H) qPCR (SYBR green) of Ifit1 (left) and Irf7 (right) expression in the cell lines collected at 3 or 24 hours post-treatment with 50 or 1000 U/mL of IFNβ. No statistical difference between genotypes in Ifit1 (left) and Irf7 (right) expression.


Figure 1The reversal potential of currents was positively shifted when a long voltage ramp pulse followed the depolarizing steps . ( top panel ) The voltage protocol used for the experiment . ( bottom panel ) Representative traces of currents elicited by voltage ramp pulses are shown . ( top panel ) . The red line represents the arithmetical difference between currents elicited by 500 - ms ( 2 ) and 2 - ms pulses ( 1 ) . B . The summary bar graph represents the mean values of the reversal potentials of currents . ( 9 cells from 3 mice , paired Student t - test ; data are presented as mean values ± s . e . m ) . Tail currents elicited by the hyperpolarizing voltage steps following the depolarizing steps of variable durations . Representative traces of currents elicited by the voltage protocol with a pipette solution containing 150 mM Cl - and 0 . 2 mM EGTA ( top panel ) , 150 mM gluconate ( middle panel ) , or 2 mM BAPTA ( bottom panel ) . A summary graph of averaged tail current amplitudes as a function of the depolarizing step duration ( the numbers on the right indicate the numbers of recorded cells , data are presented as mean values ± s . e . m . ) A bar graph of averaged values of the tail current amplitudes summarizing the results of ( c ) at 500 ms . ( The numbers inside each bar indicate the numbers of recorded cells from at least 3 mice , unpaired Student t - test ; data are presented as mean values ± s . e . m . ) .

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Figure 1The reversal potential of currents was positively shifted when a long voltage ramp pulse followed the depolarizing steps. (top panel) The voltage protocol used for the experiment. (bottom panel) Representative traces of currents elicited by voltage ramp pulses are shown. (top panel). The red line represents the arithmetical difference between currents elicited by 500-ms (2) and 2-ms pulses (1). B. The summary bar graph represents the mean values of the reversal potentials of currents. (9 cells from 3 mice, paired Student t-test; data are presented as mean values ± s.e.m).Tail currents elicited by the hyperpolarizing voltage steps following the depolarizing steps of variable durations. Representative traces of currents elicited by the voltage protocol with a pipette solution containing 150 mM Cl- and 0.2 mM EGTA (top panel), 150 mM gluconate (middle panel), or 2 mM BAPTA (bottom panel).A summary graph of averaged tail current amplitudes as a function of the depolarizing step duration (the numbers on the right indicate the numbers of recorded cells, data are presented as mean values ± s.e.m.)A bar graph of averaged values of the tail current amplitudes summarizing the results of (c) at 500 ms. (The numbers inside each bar indicate the numbers of recorded cells from at least 3 mice, unpaired Student t-test; data are presented as mean values ± s.e.m.).


Figure 2A summary bar graph showing the quantitative real - time PCR data for TMEM16A , TMEM16B , and BEST1 with cDNA samples prepared from the mHb and hippocampus of adult mouse brains ( N = 3 , n = 3 each , unpaired Student t - test , data are presented as mean values ± s . e . m . ) . RNAscope ISH showed an apparent expression of TMEM16A mRNA co - localized with ChAT and Tac1 signals in the mHb compared to the ones in lHb . Enlarged images showed that signals of TMEM16A and Tac1 in the dorsal mHb or ChAT in the ventral mHb reside together at the single cell level . Immunohistochemical staining of 40 - µm coronal brain sections with anti - TMEM16A antibody . In the mHb , TMEM16A signals colocalize with the cholinergic marker ChAT ( arrows ) . High magnifications of the indicated areas are shown in the merged panel .

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Figure 2A summary bar graph showing the quantitative real-time PCR data for TMEM16A, TMEM16B, and BEST1 with cDNA samples prepared from the mHb and hippocampus of adult mouse brains (N=3, n=3 each, unpaired Student t-test, data are presented as mean values ± s.e.m.).RNAscope ISH showed an apparent expression of TMEM16A mRNA co-localized with ChAT and Tac1 signals in the mHb compared to the ones in lHb. Enlarged images showed that signals of TMEM16A and Tac1 in the dorsal mHb or ChAT in the ventral mHb reside together at the single cell level.Immunohistochemical staining of 40-µm coronal brain sections with anti-TMEM16A antibody. In the mHb, TMEM16A signals colocalize with the cholinergic marker ChAT (arrows). High magnifications of the indicated areas are shown in the merged panel.


Figure 3Immunohistochemical staining of 40 - µm coronal brain sections with an anti - TMEM16A antibody . The TMEM16A signals in the mHb of TMEM16A cKO mice were compared to the ones of littermate controls . TMEM16A colocalized with the cholinergic marker ChAT in the mHb . The specific deletion of TMEM16A in the mHb is evident . Representative traces demonstrating the spontaneous activity of the mHb neurons in a cell - attached mode from TMEM16A cKO mice compared to the one from littermate controls . D . A summary bar graph showing the frequencies of spontaneous firing of the mHb neurons in TMEM16A cKO ( 4 mice ) and littermate controls ( The numbers inside each bar indicate the numbers of recorded cells from 4 mice each , paired Student t - test , data are presented as mean values ± s . e . m . ) . Representative averaged traces of sAPs of the mHb neurons with the pipette solution ( 40 mM Cl - and 0 . 5 mM EGTA ) from TMEM16A cKO mice and littermate controls . Summary bar graphs showing the frequency , half - width , and AHP slope of the sAPs of mHb neurons from TMEM16A cKO mice and littermate controls ( The numbers inside each bar indicate the numbers of recorded cells , unpaired Student t - test , data are presented as mean values ± s . e . m . ) .

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Figure 3Immunohistochemical staining of 40-µm coronal brain sections with an anti-TMEM16A antibody. The TMEM16A signals in the mHb of TMEM16A cKO mice were compared to the ones of littermate controls. TMEM16A colocalized with the cholinergic marker ChAT in the mHb. The specific deletion of TMEM16A in the mHb is evident.Representative traces demonstrating the spontaneous activity of the mHb neurons in a cell-attached mode from TMEM16A cKO mice compared to the one from littermate controls. D. A summary bar graph showing the frequencies of spontaneous firing of the mHb neurons in TMEM16A cKO (4 mice) and littermate controls (The numbers inside each bar indicate the numbers of recorded cells from 4 mice each, paired Student t-test, data are presented as mean values ± s.e.m.).Representative averaged traces of sAPs of the mHb neurons with the pipette solution (40 mM Cl- and 0.5 mM EGTA) from TMEM16A cKO mice and littermate controls.Summary bar graphs showing the frequency, half-width, and AHP slope of the sAPs of mHb neurons from TMEM16A cKO mice and littermate controls (The numbers inside each bar indicate the numbers of recorded cells, unpaired Student t-test, data are presented as mean values ± s.e.m.).


Figure 4Immunohistochemical images of the IPN region from control and TMEM16A cKO mice . The c - Fos signal was greatly reduced in TMEM16A cKO mice . Scale bar = 200 μm . A summary bar graph showing reduced c - Fos expression in the IPN in TMEM16A cKO mice . ( NCTL = 7 ( n = 42 ) , NcKO = 6 ( n = 36 ) , unpaired Student t - test , data are presented as mean values ± s . e . m . ) . Representative traces of sEPSCs of neurons in the medial part of the IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls . Summary bar graphs of the frequency and amplitude of the sEPSCs of IPN neurons in TMEM16A cKO ( cKO ) mice and littermate controls ( CTL ) . Data are presented as mean values ± s . e . m . ( paired Student t - test ) . The numbers inside each bar indicate the numbers of recorded cells .

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Figure 4Immunohistochemical images of the IPN region from control and TMEM16A cKO mice. The c-Fos signal was greatly reduced in TMEM16A cKO mice. Scale bar = 200 μm.A summary bar graph showing reduced c-Fos expression in the IPN in TMEM16A cKO mice. (NCTL=7 (n=42), NcKO=6 (n=36), unpaired Student t-test, data are presented as mean values ± s.e.m.).Representative traces of sEPSCs of neurons in the medial part of the IPN showing a reduced frequency in TMEM16A cKO mice compared to that of littermate controls.Summary bar graphs of the frequency and amplitude of the sEPSCs of IPN neurons in TMEM16A cKO (cKO) mice and littermate controls (CTL). Data are presented as mean values ± s.e.m. (paired Student t-test). The numbers inside each bar indicate the numbers of recorded cells.


Figure 5In the EPM task , the time spent in the open arms was significantly decreased in cKO mice compared to that in the control ( CTL ) group ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . B . In the LDB task , the time spent in the light zone by cKO mice was significantly decreased compared to that spent by the CTL mice ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . C . In the PA task , the entry latency of cKO mice was significantly decreased compared to that of the CTL mice ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . . D . Nestlet shredding behaviors of the cKO group were also decreased compared to those of the CTL group . The weight of the shredded nestlets of the cKO group was significantly lower than that of the CTL group ( nCTL = 20 , ncKO = 12 , unpaired Student t - test ) . In the 3 - chamber test , the cKO mice exhibited a decreased interest for the stranger mouse ( S1 ) against objects ( O ) compared to the CTL group ( nCTL = 12 , ncKO = 14 , unpaired Student t - test ) . F . When cKO mice were exposed to a new stranger mouse ( S2 ) against the familiar S1 mouse , they also showed a decreased interest for the S2 mouse compared to that of the CTL group . The preference index showed the decreased sociability of the cKO group in both sessions ( nCTL = 12 , ncKO = 14 , unpaired Student t - test ) . Data information : All data are presented as mean values ± s . e . m .

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Figure 5In the EPM task, the time spent in the open arms was significantly decreased in cKO mice compared to that in the control (CTL) group (nCTL=20, ncKO=12, unpaired Student t-test). B. In the LDB task, the time spent in the light zone by cKO mice was significantly decreased compared to that spent by the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test). C. In the PA task, the entry latency of cKO mice was significantly decreased compared to that of the CTL mice (nCTL=20, ncKO=12, unpaired Student t-test)..D. Nestlet shredding behaviors of the cKO group were also decreased compared to those of the CTL group. The weight of the shredded nestlets of the cKO group was significantly lower than that of the CTL group (nCTL=20, ncKO=12, unpaired Student t-test).In the 3-chamber test, the cKO mice exhibited a decreased interest for the stranger mouse (S1) against objects (O) compared to the CTL group (nCTL=12, ncKO=14, unpaired Student t-test). F. When cKO mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to that of the CTL group. The preference index showed the decreased sociability of the cKO group in both sessions (nCTL=12, ncKO=14, unpaired Student t-test). Data information: All data are presented as mean values ± s.e.m.


Figure 6Representative traces showing that the spontaneous firing of mHb neurons from ChAT ( + ) - cre : : hM4D ( Gi ) mice were reduced when CNO ( 10 μM ) was applied in the ACSF . A summary bar graph showing the reduction of normalized frequencies of spontaneous firing of the mHb neurons transfected with AAV - hM4Di - mCherry before and during CNO treatment ( The numbers inside each bar indicate the numbers of recorded cells ) . In the EPM task , the time spent in the open arms of CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice was significantly decreased compared to that of the PBS - treated controls ( CTL ) . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . E . In the LDB task , the time spent in the light zone by CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice was significantly decreased compared to that spent by the PBS - treated control mice . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . F . In the PA task , the entry latency of CNO - treated mice was significantly decreased compared to that of the CTL group . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . G . Nestlet shredding behaviors of the CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice were also decreased . ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ; ) . In the 3 - chamber test , the subject mouse of the PBS - treated mice and the CNO - treated mice explored freely the three test chambers . The time spent in each chamber during two sessions was measured . The CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice exhibited a decreased interest for a stranger mouse ( S1 ) against objects ( O ) compared to the PBS - treated group ( left two panels ) ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ) . I . When CNO - treated ChAT ( + ) - cre : : hM4D ( Gi ) mice were exposed to a new stranger mouse ( S2 ) against the familiar S1 mouse , they also showed a decreased interest for the S2 mouse compared to the PBS - treated control group ( right two panels ) . The preference index showed decreased sociability of the CNO - treated group in both sessions ( nCTL = 6 , ncKO = 9 , unpaired Student t - test ; ) .

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 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 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 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

Figure 6Representative traces showing that the spontaneous firing of mHb neurons from ChAT(+)-cre::hM4D(Gi) mice were reduced when CNO (10 μM) was applied in the ACSF.A summary bar graph showing the reduction of normalized frequencies of spontaneous firing of the mHb neurons transfected with AAV-hM4Di-mCherry before and during CNO treatment (The numbers inside each bar indicate the numbers of recorded cells).In the EPM task, the time spent in the open arms of CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that of the PBS-treated controls (CTL). (nCTL=6, ncKO=9, unpaired Student t-test). E. In the LDB task, the time spent in the light zone by CNO-treated ChAT(+)-cre::hM4D(Gi) mice was significantly decreased compared to that spent by the PBS-treated control mice. (nCTL=6, ncKO=9, unpaired Student t-test). F. In the PA task, the entry latency of CNO-treated mice was significantly decreased compared to that of the CTL group. (nCTL=6, ncKO=9, unpaired Student t-test). G. Nestlet shredding behaviors of the CNO-treated ChAT(+)-cre::hM4D(Gi) mice were also decreased. (nCTL=6, ncKO=9, unpaired Student t-test;).In the 3-chamber test, the subject mouse of the PBS-treated mice and the CNO-treated mice explored freely the three test chambers. The time spent in each chamber during two sessions was measured. The CNO-treated ChAT(+)-cre::hM4D(Gi) mice exhibited a decreased interest for a stranger mouse (S1) against objects (O) compared to the PBS-treated group (left two panels) (nCTL=6, ncKO=9, unpaired Student t-test). I. When CNO-treated ChAT(+)-cre::hM4D(Gi) mice were exposed to a new stranger mouse (S2) against the familiar S1 mouse, they also showed a decreased interest for the S2 mouse compared to the PBS-treated control group (right two panels). The preference index showed decreased sociability of the CNO-treated group in both sessions (nCTL=6, ncKO=9, unpaired Student t-test;).


Figure 2A . Western blot analysis of reporter HslO - FLAG protein produced with a custom - made PURExpress ® coupled in vitro transcription - translation system supplemented with the indicated EF - G proteins and incubated for the indicated times . Blot was developed using anti - FLAG antibodies . Shown is a representative from at least two independent experiments . Eco EF - G : E . coli EF - G .

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

Figure 2A. Western blot analysis of reporter HslO-FLAG protein produced with a custom-made PURExpress® coupled in vitro transcription-translation system supplemented with the indicated EF-G proteins and incubated for the indicated times. Blot was developed using anti-FLAG antibodies. Shown is a representative from at least two independent experiments. Eco EF-G: E. coli EF-G.


Figure 4B . Western blot analysis of reporter HslO - FLAG protein synthesized in vitro using a custom - made PURExpress ® system with EF - G1 , EF - G2 , and two engineered EF - G2 variants : one missing the 26 - amino acid insert , EF - G2 ( ∆ 514 - 539 ) and one missing the beta hairpin in the insert , EF - G2 ( ∆ 516 - 527 ) . Shown is a representative experiment from at least two independent experiments . D . SDS - PAGE analysis of co - sedimentation assays monitoring factor ( EF - G1 EF - G2 or EF - G2 variant EF - G2 ( ∆ 516 - 527 ) or EF - G2 ( ∆ 514 - 539 ) ) binding to E . coli 70S ribosomes . Binding reactions were centrifuged through a sucrose cushion to recover ribosome and associated factor in the pellets . The positions of EF - G proteins and ribosomal proteins and molecular weight markers are indicated . E . Western blot analysis of reporter HisC - FLAG protein synthesized in vitro using a custom - made PURExpress ® system with the EF - G1 , EF - G2 or EF - G2 ( S84H ) proteins . Shown is a representative from at least two 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 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 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

Figure 4B. Western blot analysis of reporter HslO-FLAG protein synthesized in vitro using a custom-made PURExpress® system with EF-G1, EF-G2, and two engineered EF-G2 variants: one missing the 26-amino acid insert, EF-G2 (∆514-539) and one missing the beta hairpin in the insert, EF-G2 (∆516-527). Shown is a representative experiment from at least two independent experiments.D. SDS-PAGE analysis of co-sedimentation assays monitoring factor (EF-G1 EF-G2 or EF-G2 variant EF-G2 (∆516-527) or EF-G2 (∆514-539)) binding to E. coli 70S ribosomes. Binding reactions were centrifuged through a sucrose cushion to recover ribosome and associated factor in the pellets. The positions of EF-G proteins and ribosomal proteins and molecular weight markers are indicated.E. Western blot analysis of reporter HisC-FLAG protein synthesized in vitro using a custom-made PURExpress® system with the EF-G1, EF-G2 or EF-G2(S84H) proteins. Shown is a representative from at least two independent experiments.


Figure 6Western blot analysis of B . thetaiotaomicron strains expressing C - terminally FLAG - tagged EF - G1 ( WH405 ) ( A ) , or EF - G2 ( GT1301 ) ( B ) , from their normal promoters and chromosomal locations , grown in Tryptone Yeast Extract Glucose liquid medium ( TYG ) and sampled at the indicated times . E - F . Western blot of crude extracts from wild - type B . thetaiotaomicron harvested from the ceca of mice ( representative blot for 2 out of 4 mice tested ) or corresponding to stationary phase cultures harvested following growth in TYG for 24 h or purified EF - G1 and EF - G2 proteins at the indicated amounts ( E ) . Blots were developed with anti - EF - G1 or EF - G2 polyclonal antibodies ; anti - GroEL antibodies were used as loading controls . Western blot quantifications of all four biological replicates and their average are shown in ( F ) , where EF - G1 and EF - G2 abundances were estimated by normalizing the signal of bacterial sample to the closest signal of known amount of purified protein , and the derived absolute amount ( pmol ) was normalized to the signal of GroEL .

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 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

Figure 6Western blot analysis of B. thetaiotaomicron strains expressing C-terminally FLAG-tagged EF-G1 (WH405) (A), or EF-G2 (GT1301) (B), from their normal promoters and chromosomal locations, grown in Tryptone Yeast Extract Glucose liquid medium (TYG) and sampled at the indicated times.E-F. Western blot of crude extracts from wild-type B. thetaiotaomicron harvested from the ceca of mice (representative blot for 2 out of 4 mice tested) or corresponding to stationary phase cultures harvested following growth in TYG for 24 h or purified EF-G1 and EF-G2 proteins at the indicated amounts (E). Blots were developed with anti-EF-G1 or EF-G2 polyclonal antibodies; anti-GroEL antibodies were used as loading controls. Western blot quantifications of all four biological replicates and their average are shown in (F), where EF-G1 and EF-G2 abundances were estimated by normalizing the signal of bacterial sample to the closest signal of known amount of purified protein, and the derived absolute amount (pmol) was normalized to the signal of GroEL.


Figure 1 ( A ) Dynamic regulation of fluorescently tagged Beta - catenin / Armadillo ( Arm - GFP ) during mitosis in the growing fly wing epithelium . Notice down - regulation of Arm - GFP as cells round up for mitosis ( arrow ) . Scale bar ~ 2µm . n > 10 independent biological replicates . ( B ) Dynamic regulation of fluorescently tagged E - cadherin ( E - cad - GFP ) during mitosis in the growing fly wing epithelium . Notice down - regulation of E - cad - GFP as cells round up for mitosis ( arrow ) . Scale bar ~ 2µm . n > 10 independent biological replicates . ( C ) E - cad - GFP remains downregulated in cells arrested in mitosis with Colchicine . Scale bar ~ 2µm . n > 10 independent biological replicates . ( D ) Quantification of the degree of down - regulation of Arm - GFP and E - cad - GFP at the junction between mitotic cells and their interphase neighbours ( n > 10 independent samples per cell cycle stage ) . Mean + / - 1 S . D . shown . ( E ) Two adjacent mitotic cells show no detectable Arm - GFP at the junction between them . Scale bar ~ 2µm . ( F - I ) Electron microscopy cross - sections of wing epithelial cells at interphase , prophase , prometaphase and telophase . Notice adherens junctions ( dark staining , highlighted in insets ) are downregulated as cells round up for mitosis . Scale bars ~ 1µm ( low mag . ) and ~ 10nm ( high mag . ) . n > 10 independent biological replicates . Asterisks indicate a single mitotic cell . Green arrowheads indicate adherens junctions .

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 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 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 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 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 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

Figure 1(A) Dynamic regulation of fluorescently tagged Beta-catenin/Armadillo (Arm-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of Arm-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates.(B) Dynamic regulation of fluorescently tagged E-cadherin (E-cad-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of E-cad-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates.(C) E-cad-GFP remains downregulated in cells arrested in mitosis with Colchicine. Scale bar ~2µm. n>10 independent biological replicates.(D) Quantification of the degree of down-regulation of Arm-GFP and E-cad-GFP at the junction between mitotic cells and their interphase neighbours (n > 10 independent samples per cell cycle stage). Mean +/- 1 S.D. shown.(E) Two adjacent mitotic cells show no detectable Arm-GFP at the junction between them. Scale bar ~2µm.(F-I) Electron microscopy cross-sections of wing epithelial cells at interphase, prophase, prometaphase and telophase. Notice adherens junctions (dark staining, highlighted in insets) are downregulated as cells round up for mitosis. Scale bars ~1µm (low mag.) and ~10nm (high mag.). n>10 independent biological replicates. Asterisks indicate a single mitotic cell. Green arrowheads indicate adherens junctions.


Figure 2 ( A ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; purple ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild - type wing epithelial cells in the anterior ( A ) compartment , but not in Rho - kinase ( Rok ) RNAi expressing cells of the posterior ( P ) compartment ( GFP - positive ; green ) . Scale bar ~ 10µm . n > 10 independent biological replicates . ( B ) High - magnification view of single mitotic cell stained for p - MLC ( purple ) and E - cadherin ( green ) , with metaphase plate chromosomes stained by DAPI ( blue ) . Scale bar ~ 1µm . n > 10 independent biological replicates . ( C ) High - magnification view of single UAS . Rok - RNAi expressing mitotic cell stained for p - MLC ( purple ) and E - cadherin ( green ) , with metaphase plate chromosomes stained by DAPI ( blue ) . Note loss of cortical p - MLC staining ( purple ) . Scale bar ~ 1µm . n > 10 independent biological replicates . ( D ) Expression of constitutively active Rho - kinase ( UAS . Rok - CA ) in the posterior compartment of the wing epithelium is sufficient to elevate p - MLC and reduce E - cadherin immunostaining . Zoom ( right ) shows high p - MLC and low E - cad in both a mitotic cell and its neighbours . Scale bar ~ 10µm . n > 10 independent biological replicates . ( E ) MyoII RLC - GFP ( MyoII - GFP ) accumulates at the cortex as cells round up for mitosis and later appears at the cleavage furrow during cytokinesis ( basal section , diagrammed left ) . Treatment with the Rho - kinase inhibitor Y - 27632 inhibits mitotic accumulation of MyoII - GFP . Quantification shown right ( n = 8 independent samples per genotype ) . Scale bar ~ 1µm . Mean + / - 1 S . D . shown . ( F ) E - cad - GFP is downregulated as cells round up for mitosis ( apical section , diagrammed left ) . Treatment with the Rho - kinase inhibitor Y - 27632 inhibits mitotic downregulation of E - cad - GFP . Extended treatment with Y - 27632 leads to a general defect in all interphase and mitotic cells in which apical surface areas enlarge and E - cad - GFP is generally lost . Arrow indicates mitotic cell cortex at maximal rounding . Quantification shown right ( n = 6 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bar ~ 1µm .

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 O O O O O O O O O O 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 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 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

Figure 2(A) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells in the anterior (A) compartment, but not in Rho-kinase (Rok) RNAi expressing cells of the posterior (P) compartment (GFP-positive; green). Scale bar ~10µm. n>10 independent biological replicates.(B) High-magnification view of single mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Scale bar ~1µm. n>10 independent biological replicates. (C) High-magnification view of single UAS.Rok-RNAi expressing mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Note loss of cortical p-MLC staining (purple). Scale bar ~1µm. n>10 independent biological replicates. (D) Expression of constitutively active Rho-kinase (UAS.Rok-CA) in the posterior compartment of the wing epithelium is sufficient to elevate p-MLC and reduce E-cadherin immunostaining. Zoom (right) shows high p-MLC and low E-cad in both a mitotic cell and its neighbours. Scale bar ~10µm. n>10 independent biological replicates.(E) MyoII RLC-GFP (MyoII-GFP) accumulates at the cortex as cells round up for mitosis and later appears at the cleavage furrow during cytokinesis (basal section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic accumulation of MyoII-GFP. Quantification shown right (n = 8 independent samples per genotype). Scale bar ~1µm. Mean +/- 1 S.D. shown.(F) E-cad-GFP is downregulated as cells round up for mitosis (apical section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic downregulation of E-cad-GFP. Extended treatment with Y-27632 leads to a general defect in all interphase and mitotic cells in which apical surface areas enlarge and E-cad-GFP is generally lost. Arrow indicates mitotic cell cortex at maximal rounding. Quantification shown right (n = 6 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.


Figure 3 ( A ) Nuclear localised Pbl - GFP becomes cytoplasmic in mitotic cells , co - incident with Rok - GFP relocalisation to the plasma membrane . Scale bar ~ 1µm . n > 5 independent biological replicates . ( B ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; purple ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild - type wing epithelial cells , but not those expressing UAS . pbl - RNAi . Phospho - Histone H3 ( pH3 ) staining ( green ) marks mitotic cell chromosomes . Quantification shown right ( n > 10 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bar ~ 1µm . ( C ) A stripe of expression of ptc . Gal4 UAS . pbl - RNAi in the wing disc leads to both enlarged cells and many basally extruded cells that undergo apoptosis , marked by pyknotic nuclei and loss of aPKC / Dlg staining . Scale bars ~ 20µm . n > 6 independent biological replicates .

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 O O O O O O O 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

Figure 3(A) Nuclear localised Pbl-GFP becomes cytoplasmic in mitotic cells, co-incident with Rok-GFP relocalisation to the plasma membrane. Scale bar ~1µm. n > 5 independent biological replicates.(B) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those expressing UAS.pbl-RNAi. Phospho-Histone H3 (pH3) staining (green) marks mitotic cell chromosomes. Quantification shown right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.(C) A stripe of expression of ptc.Gal4 UAS.pbl-RNAi in the wing disc leads to both enlarged cells and many basally extruded cells that undergo apoptosis, marked by pyknotic nuclei and loss of aPKC/Dlg staining. Scale bars ~20µm. n > 6 independent biological replicates.


Figure 4 ( A ) Nuclear localised Tum - GFP becomes cytoplasmic in mitotic cells , including a distinct plasma membrane localisation ( arrows ) . Note also localisation to the cytokinetic ring ( asterisk ) and remnant mid - bodies / ring canals in interphase cells . Quantification shown on the right ( n > 7 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 2µm . ( B ) A stripe of expression of ptc . Gal4 UAS . tum - RNAi in the wing disc leads to basally extruded cells that undergo apoptosis , marked by Dcp1 immunostaining , similar to UAS . pbl - RNAi . Quantification shown on the right ( n > 10 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 20µm . ( C ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; red ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild - type wing epithelial cells , but not those cells homozygous mutant for tum347 , marked by expression of GFP in single - celled clones ( MARCM system ) . pH3 staining ( blue ) marks mitotic cell chromosomes . Arrow indicates a GFP - positive single cell tum347 mutant clone . Quantification shown on the right ( n > 3 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 20µm .

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 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 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

Figure 4(A) Nuclear localised Tum-GFP becomes cytoplasmic in mitotic cells, including a distinct plasma membrane localisation (arrows). Note also localisation to the cytokinetic ring (asterisk) and remnant mid-bodies/ring canals in interphase cells. Quantification shown on the right (n > 7 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~2µm.(B) A stripe of expression of ptc.Gal4 UAS.tum-RNAi in the wing disc leads to basally extruded cells that undergo apoptosis, marked by Dcp1 immunostaining, similar to UAS.pbl-RNAi. Quantification shown on the right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.(C) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; red) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those cells homozygous mutant for tum347, marked by expression of GFP in single-celled clones (MARCM system). pH3 staining (blue) marks mitotic cell chromosomes. Arrow indicates a GFP-positive single cell tum347 mutant clone. Quantification shown on the right (n > 3 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.


Figure 5 ( A ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; purple ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells ( pH3 , red ) in wild - type wing discs , and aurA homozygous mutant wing discs , but not upon inhibition of both AurA and AurB by treatment with VX - 680 . Note increased numbers of mitotic cells in aurA homozygous mutant wing discs , due to delayed mitotic progression . Scale bars ~ 20µm ( low mag . ) and ~ 1µm ( high mag . ) . n > 15 independent biological replicates . ( B ) Live - imaging of MyoII - GFP reveals delayed mitosis in aurA homozygous mutant wing discs , while inhibition of both AurA and AurB by treatment with VX - 680 leads to complete loss of mitotic rounding and MyoII - GFP membrane localisation . Asterisks indicate a single mitotic cell . Scale bars ~ 1µm . Quantification shown on the bottom right ( n = 5 independent samples per genotype ) . ( C ) Live - imaging of E - cad - GFP reveals delayed mitosis in aurA homozygous mutant wing discs , cytokinesis failure in UAS . aurB - RNAi expressing wing discs , and complete failure of mitotic rounding and E - cad - GFP downregulation upon inhibition of both AurA and AurB by treatment with VX - 680 . Asterisks indicate a single mitotic cell . Scale bars ~ 1µm . Quantification shown on the bottom right ( n = 9 independent samples per genotype ) .

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 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 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

Figure 5(A) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells (pH3, red) in wild-type wing discs, and aurA homozygous mutant wing discs, but not upon inhibition of both AurA and AurB by treatment with VX-680. Note increased numbers of mitotic cells in aurA homozygous mutant wing discs, due to delayed mitotic progression. Scale bars ~20µm (low mag.) and ~1µm (high mag.). n > 15 independent biological replicates.(B) Live-imaging of MyoII-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, while inhibition of both AurA and AurB by treatment with VX-680 leads to complete loss of mitotic rounding and MyoII-GFP membrane localisation. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 5 independent samples per genotype).(C) Live-imaging of E-cad-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, cytokinesis failure in UAS.aurB-RNAi expressing wing discs, and complete failure of mitotic rounding and E-cad-GFP downregulation upon inhibition of both AurA and AurB by treatment with VX-680. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 9 independent samples per genotype).


Figure 6 ( A ) Striped ptc . Gal4 - driven expression of UAS . GFP in a control wing imaginal disc . Scale bars ~ 20µm . n > 6 independent biological replicates . ( B ) Striped ptc . Gal4 - driven expression of UAS . GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic cells . n > 7 independent biological replicates . ( C ) Striped ptc . Gal4 - driven expression of UAS . GFP and UAS . aurB - RNAi in a control wing imaginal disc leads to enlarged cells with large nuclei ( marked by DAPI ; white arrowhead ) . n > 6 independent biological replicates . ( D ) Striped ptc . Gal4 - driven expression of UAS . GFP and UAS . aurB - RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion and apoptosis of cells ( marked by Dcp1 and pyknotic nuclei ) . n > 8 independent biological replicates . ( E ) Striped ptc . Gal4 - driven expression of UAS . GFP and UAS . pbl - RNAi in the wing imaginal disc leads to extrusion and apoptosis of cells ( marked by Dcp1 and pyknotic nuclei ) . n > 8 independent biological replicates . ( F ) ptc . Gal4 - driven expression of UAS . aurB - RNAi in an aurA homozygous mutant endoreplicating salivary gland has no phenotypic consequence . n > 6 independent biological replicates . ( G ) MARCM clones ( GFP - positive ) expressing UAS . aurB - RNAi in an aurA homozygous mutant cells result in single cell clones ( i . e . : no cell division ) , while individual loss of AurA or AurB allows some cell proliferation . Quantification shown on the right ( n > 7 independent samples per genotype ; Mean + / - 1 S . D . shown ) . Scale bar ~ 20µm .

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

Figure 6(A) Striped ptc.Gal4-driven expression of UAS.GFP in a control wing imaginal disc. Scale bars ~20µm. n > 6 independent biological replicates. (B) Striped ptc.Gal4-driven expression of UAS.GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic cells. n > 7 independent biological replicates. (C) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB-RNAi in a control wing imaginal disc leads to enlarged cells with large nuclei (marked by DAPI; white arrowhead). n > 6 independent biological replicates. (D) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB -RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (E) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.pbl-RNAi in the wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (F) ptc.Gal4-driven expression of UAS.aurB-RNAi in an aurA homozygous mutant endoreplicating salivary gland has no phenotypic consequence. n > 6 independent biological replicates. (G) MARCM clones (GFP-positive) expressing UAS.aurB-RNAi in an aurA homozygous mutant cells result in single cell clones (i.e.: no cell division), while individual loss of AurA or AurB allows some cell proliferation. Quantification shown on the right (n > 7 independent samples per genotype; Mean +/- 1 S.D. shown). Scale bar ~20µm.


Figure 7 ( A ) Live - imaging of MyoII - GFP in a control wing disc epithelium . Asterisk indicates the location of a single mitotic cell about to initiate rounding . Scale bar ~ 1µm . n > 5 independent biological replicates . ( B ) Live - imaging of MyoII - GFP upon treatment with the dual AurA / B inhibitor VX - 680 after initiation of mitotic rounding , which rapidly arrests progression of rounding and prevents cytokinesis . Scale bar ~ 1µm . n > 9 independent biological replicates . ( C ) Live - imaging of E - cad - GFP upon treatment with the dual AurA / B inhibitor VX - 680 after initiation of mitotic rounding , which rapidly arrests progression of rounding and prevents cytokinesis . Scale bar ~ 1µm . n > 6 independent biological replicates .

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 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

Figure 7(A) Live-imaging of MyoII-GFP in a control wing disc epithelium. Asterisk indicates the location of a single mitotic cell about to initiate rounding. Scale bar ~1µm. n > 5 independent biological replicates. (B) Live-imaging of MyoII-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 9 independent biological replicates. (C) Live-imaging of E-cad-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 6 independent biological replicates.


Figure 8 ( A ) Early stage Drosophila egg chambers showing giant germ - line nurse cells surrounded by E - cadherin - positive follicle cell epithelium . A follicle cell undergoing mitotic rounding is highlighted with an asterisk ( * ) . Scale bar ~ 3µm . n > 3 independent biological replicates . ( B ) High resolution imaging of the F - actin cortex , E - cadherin , and phospho - MyoII in a mitotically rounded follicle cell . Note maintenance of adherens junctions including both E - cad and the contractile actomyosin ring during mitotic rounding . Asterisks or pH3 staining indicate a single mitotic cell . Scale bar ~ 3µm . n > 4 independent biological replicates .

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Figure 8(A) Early stage Drosophila egg chambers showing giant germ-line nurse cells surrounded by E-cadherin-positive follicle cell epithelium. A follicle cell undergoing mitotic rounding is highlighted with an asterisk (*). Scale bar ~3µm. n > 3 independent biological replicates.(B) High resolution imaging of the F-actin cortex, E-cadherin, and phospho-MyoII in a mitotically rounded follicle cell. Note maintenance of adherens junctions including both E-cad and the contractile actomyosin ring during mitotic rounding. Asterisks or pH3 staining indicate a single mitotic cell. Scale bar ~3µm. n > 4 independent biological replicates.


Figure 1 ( B ) RAW 264 . 7 cells transfected for 48 h with control ( sc ) , MTMR6 or Jumpy siRNA , were pretreated for 30 min with 100 nM Bafilomycin A1 ( BafA1 ) , 10 μg / ml E64d and 10 μg / ml pepstatin , then incubated for 30 min in full or starvation media in the presence of BafA1 , E64d and pepstatin . Cells were lysed and analysed by immunoblotting with anti - LC3 or anti ‐ actin . Densitometric LC3 ‐ II / actin ratios are shown underneath the blot . Graph : LC3 ‐ II / actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3 ‐ II / actin ratio for control siRNA ( sc ) in starvation medium . ( C , D ) RAW 264 . 7 cells were transfected for 36 h with control ( scramble ) or Jumpy siRNA , transfected once more with corresponding siRNA and mRFP ‐ GFP ‐ LC3 DNA construct overnight and incubated for 2 h in full or starvation media . Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy . ( C ) Representative confocal images of RAW 264 . 7 cells in full or starvation media after transfection with control ( scramble ) or Jumpy siRNA ( siJumpy ) . Red and yellow arrows indicate GFP − RFP + and GFP + RFP + puncta , respectively . ( D ) Quantitation of number of LC3 puncta per cell , total puncta per cell ( GFP + RFP + and GFP − RFP + puncta ) GFP + RFP + puncta per cell and GFP − RFP + puncta per cell . Data , mean ± s . e . m . n = 5 independent experiments , 30 cells per experiments . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( t ‐ test ) . Scale bars , 5 μm . ( E , F ) C2C12 myoblasts were transfected for 48 h with control ( scramble ) or Jumpy siRNA , followed by a second transfection overnight with corresponding siRNA and mRFP ‐ GFP ‐ LC3 DNA construct . Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy . ( E ) Representative confocal images of C2C12 cells in full media after transfection with control ( scramble ) or Jumpy siRNA ( siJumpy ) . ( F ) Quantification of the number of LC3 puncta per cell . Data , mean ± SEM for n = 3 ( independent experiments ) , 30 cells per experiments . * P 0 . 05 ( t - test ) . Scale bars , 10 μm . ( G , H ) C2C12 cells were transfected for 48 h with control ( sc ) ( lanes 1 , 2 , 5 , 6 ) or Atg5 siRNA ( lanes 3 , 4 , 6 , 7 ) followed by a second transfection for 48 h with same siRNA and control ( lanes 1 , 3 , 5 , 7 ) or Jumpy siRNA ( lanes 2 , 4 , 6 , 8 ) . Cells were incubated for 1 h with or without 100 nM Baf A1 , 10 μg / ml E64d and 10 μg / ml pepstatin in full media , lysed and analysed by immunoblotting with anti ‐ LC3 or anti ‐ actin ( G ) . ( H ) Densitometric LC3 ‐ II / actin ratios for samples treated with BafA1 and protease inhibitors from G ( lanes 5 - 8 ) . Inset shows Atg5 knock ‐ down by immunoblotting .

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 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 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 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

Figure 1(B) RAW 264.7 cells transfected for 48 h with control (sc), MTMR6 or Jumpy siRNA, were pretreated for 30 min with 100 nM Bafilomycin A1 (BafA1), 10 μg/ml E64d and 10 μg/ml pepstatin, then incubated for 30 min in full or starvation media in the presence of BafA1, E64d and pepstatin. Cells were lysed and analysed by immunoblotting with anti-LC3 or anti‐actin. Densitometric LC3‐II/actin ratios are shown underneath the blot. Graph: LC3‐II/actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3‐II/actin ratio for control siRNA (sc) in starvation medium.(C, D) RAW 264.7 cells were transfected for 36 h with control (scramble) or Jumpy siRNA, transfected once more with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct overnight and incubated for 2 h in full or starvation media. Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy. (C) Representative confocal images of RAW 264.7 cells in full or starvation media after transfection with control (scramble) or Jumpy siRNA (siJumpy). Red and yellow arrows indicate GFP−RFP+ and GFP+RFP+ puncta, respectively. (D) Quantitation of number of LC3 puncta per cell, total puncta per cell (GFP+RFP+ and GFP−RFP+ puncta) GFP+RFP+ puncta per cell and GFP−RFP+ puncta per cell. Data, mean±s.e.m. n=5 independent experiments, 30 cells per experiments. *P0.05, **P0.01, ***P0.001 (t‐test). Scale bars, 5 μm.(E, F) C2C12 myoblasts were transfected for 48 h with control (scramble) or Jumpy siRNA, followed by a second transfection overnight with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct. Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy. (E) Representative confocal images of C2C12 cells in full media after transfection with control (scramble) or Jumpy siRNA (siJumpy). (F) Quantification of the number of LC3 puncta per cell. Data, mean±SEM for n=3 (independent experiments), 30 cells per experiments. *P 0.05 (t-test). Scale bars, 10 μm.(G, H) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 μg/ml E64d and 10 μg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin (G). (H) Densitometric LC3‐II/actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5-8). Inset shows Atg5 knock‐down by immunoblotting.