F HUVEC were grown on collagen - coated polyacrylamide gels of varying physiologic stiffness of 0 . 2 kPa and 20 kPa , and stimulated with TNFα for 17 h prior to adding HL60 - derived neutrophils for 20 min . VE - cadherin was immunoprecipitated and precipitates as well as total cell lysates were analyzed by Western blotting for indicated antigens . Relative quantifications of pY731 blot signals adjusted to the amount of precipitated VE - cadherin are shown on the

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

F HUVEC were grown on collagen-coated polyacrylamide gels of varying physiologic stiffness of 0.2 kPa and 20 kPa, and stimulated with TNFα for 17 h prior to adding HL60-derived neutrophils for 20 min. VE-cadherin was immunoprecipitated and precipitates as well as total cell lysates were analyzed by Western blotting for indicated antigens. Relative quantifications of pY731 blot signals adjusted to the amount of precipitated VE-cadherin are shown on the right


D , Scoring of the PI3Kα activation transcriptomic signature was used to cluster patients with high , medium ( both groups were combined , high + medium : red ) and low ( black ) scoring levels in the primary tumours of confirmed PDAC patients from PAAD ( TCGA ) or PACA - AU ( BH corrected p - values ) . The survival curves of each cluster were then plotted , and the statistical significance was calculated using the logrank test .

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

D, Scoring of the PI3Kα activation transcriptomic signature was used to cluster patients with high, medium (both groups were combined, high+medium: red) and low (black) scoring levels in the primary tumours of confirmed PDAC patients from PAAD (TCGA) or PACA-AU (BH corrected p-values). The survival curves of each cluster were then plotted, and the statistical significance was calculated using the logrank test.


E , Murine pancreatic tumour cells R211 were treated with the vehicle , α , β , β / δ , γ - specific or pan - PI3K inhibitors at 0 . 1 or 1 µM and simultaneously subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . Representative image of filter after Crystal violet staining is shown . Scale = 200µm . n = 3 in each group . F , Murine pancreatic tumour cells R211 were treated with vehicle , BYL - 719 1 µM , AZD4547 2 µM or both treatments and subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . n = 4 in each group . G

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

E, Murine pancreatic tumour cells R211 were treated with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors at 0.1 or 1 µM and simultaneously subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. Representative image of filter after Crystal violet staining is shown. Scale=200µm. n = 3 in each group. F, Murine pancreatic tumour cells R211 were treated with vehicle, BYL-719 1 µM, AZD4547 2 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 4 in each group. G


L , PANC - 1 - transduced cells were subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . n = 5 in each group .

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L, PANC-1-transduced cells were subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 5 in each group.


A - B A , Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle , α - specific A66 or pan - PI3K inhibitor BKM120 at 0 . 01 , 0 . 05 , 0 . 1 , 0 . 5 , 1 or 5 µM in the presence of 10 % FBS and protein levels of P - Akt ( Ser473 ) , total Akt and β Actin were observed by western blot . B , P - Akt on Ser473 was quantified and normalised with β Actin . n = 3 in each group .

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A-B A, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle, α-specific A66 or pan-PI3K inhibitor BKM120 at 0.01, 0.05, 0.1, 0.5, 1 or 5 µM in the presence of 10% FBS and protein levels of P-Akt (Ser473), total Akt and β Actin were observed by western blot. B, P-Akt on Ser473 was quantified and normalised with β Actin. n = 3 in each group.


R211 cells were treated with the vehicle , α - specific inhibitor A66 or pan - PI3K inhibitor BKM120 at 1 µM D , migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle ( DMSO ) . n = 3 in each group .

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R211 cells were treated with the vehicle, α-specific inhibitor A66 or pan-PI3K inhibitor BKM120 at 1 µM D, migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle (DMSO). n = 3 in each group.


E , Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle ( 0 . 01 % DMSO ) or α - specific A66 or pan - PI3K inhibitor BKM120 at 1µM . Phospholipids were extracted , total PIP , PIP2 and PIP3 were quantified and compared to the vehicle ( DMSO ) . n = 3 in each group .

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E, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle (0.01% DMSO) or α-specific A66 or pan-PI3K inhibitor BKM120 at 1µM. Phospholipids were extracted, total PIP, PIP2 and PIP3 were quantified and compared to the vehicle (DMSO). n = 3 in each group.


A , Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour cells ( R211 , PDAC8661 , PANC - 1 , 10593 , 10158 , R6344 , R6430 , R6065 , R6141 ) . The mean of these values was plotted against the in vitro IC50 for each class I PI3K and each inhibitor . IC50 is determined on recombinant proteins . Pearson correlation tests were performed and its p - values were presented separately for each class I PI3K isoform .

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

A, Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour cells (R211, PDAC8661, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141). The mean of these values was plotted against the in vitro IC50 for each class I PI3K and each inhibitor. IC50 is determined on recombinant proteins. Pearson correlation tests were performed and its p-values were presented separately for each class I PI3K isoform.


A , Representative images of PI3K activation ( as shown by positive pAkt substrate staining ) on the primary tumours of PDAC patients , with values of quantification of cfDNA and mutated KRAS allele frequency .

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

A, Representative images of PI3K activation (as shown by positive pAkt substrate staining) on the primary tumours of PDAC patients, with values of quantification of cfDNA and mutated KRAS allele frequency.


C , Survival curve of KPC mice presenting different levels of cfDNA . n in each group is indicated . Log rank test , * * * p < 0 .

C, Survival curve of KPC mice presenting different levels of cfDNA. n in each group is indicated. Log rank test, *** p<0.001


G , Survival curve of KPC mice treated with the vehicle or BYL - 719 . Log rank test , * p < 0 . 05 .

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G, Survival curve of KPC mice treated with the vehicle or BYL-719. Log rank test, * p<0.05.


H - J H , Representative images and i , quantification of Ki67 positive cells in pancreatic tumours and in J , metastasized liver sections after treatment with oral doses of vehicle ( 0 . 5 % methyl cellulose with 0 . 2 % Tween - 80 ) or of BYL - 719 ( 50mg / kg ) .

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H-J H, Representative images and i, quantification of Ki67 positive cells in pancreatic tumours and in J, metastasized liver sections after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).


K - L K , Treatment regimen of the tail vein injection experiment in nude mice treated with the PI3Kα inhibitor or vehicle ( n is indicated ) , quantification at two time points and L , representative images of luminescence via IVIS ® Spectrum in vivo imaging system .

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K-L K, Treatment regimen of the tail vein injection experiment in nude mice treated with the PI3Kα inhibitor or vehicle (n is indicated), quantification at two time points and L, representative images of luminescence via IVIS® Spectrum in vivo imaging system.


N , Tail vein injection experiment in C57 / B6 mice of murine syngeneic pancreatic cancer cells ( Kras mutated A338 , Kras mutated and half PI3Kα inactive , A260 ; n > 8 in each group ) , quantification of micro - and macro - metastasis at final time point .

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N, Tail vein injection experiment in C57/B6 mice of murine syngeneic pancreatic cancer cells (Kras mutated A338, Kras mutated and half PI3Kα inactive, A260; n>8 in each group), quantification of micro- and macro-metastasis at final time point.


A - D Blood count of A , white blood cells , B , monocytes , C , granulocytes and D , lymphocytes in KPC mice , with cohort size in each group .

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A-D Blood count of A, white blood cells, B, monocytes, C, granulocytes and D, lymphocytes in KPC mice, with cohort size in each group.


h - jH - J H , Representative images and quantification of I , F4 / 80 positive cells and J , CD206 positive cells after the pharmacological inhibition of PI3Kα . Black arrowheads show positive immune cells .

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

h-jH-J H, Representative images and quantification of I, F4/80 positive cells and J, CD206 positive cells after the pharmacological inhibition of PI3Kα. Black arrowheads show positive immune cells.


S , Murine pancreatic tumour cells R211 were treated with vehicle , TNF - α 20 ng / mL , BYL - 719 1 µM or both treatments and subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . n = 4 independent values .

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S, Murine pancreatic tumour cells R211 were treated with vehicle, TNF-α 20 ng/mL, BYL-719 1 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n= 4 independent values.


B . Size - exclusion chromatography ( SEC ) experiments of Mdm12 , tMmm1 , and the Mdm12 - tMmm1 complex comparing the molecular size of these proteins in solution . The proteins indicated were injected into a Superdex 200 column ( GE Healthcare ) with a buffer containing 25 mM Tris - HCl ( pH 7 . 5 ) , 150 mM NaCl , and 5 mM DTT . The standard molecular masses for the SEC experiments ( top ) are shown for relative molecular weight comparison ( blue dextran , void ; ferritin , 440 kDa ; aldolase , 158 kDa ; cobalbumin , 75 kDa ; ovalbumin , 44 kDa ; and carbonic anhydrase , 29 kDa ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Size-exclusion chromatography (SEC) experiments of Mdm12, tMmm1, and the Mdm12-tMmm1 complex comparing the molecular size of these proteins in solution. The proteins indicated were injected into a Superdex 200 column (GE Healthcare) with a buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM DTT. The standard molecular masses for the SEC experiments (top) are shown for relative molecular weight comparison (blue dextran, void; ferritin, 440 kDa; aldolase, 158 kDa; cobalbumin, 75 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa).


C . Graph indicating the molecular weights of Mdm12 , His6 - Mdm12 , and the Mdm12 - tMmm1 complex in solution as measured by analytical ultracentrifugation .

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C. Graph indicating the molecular weights of Mdm12, His6-Mdm12, and the Mdm12-tMmm1 complex in solution as measured by analytical ultracentrifugation.


A . Ribbon diagram of the yeastMdm12 dimer . The crystal structure of full - length Mdm12 was determined by SAD and refined with native data to 3 . 1 Å resolution . Lipids bound to Mdm12 are drawn with black stick models .

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

A. Ribbon diagram of the yeastMdm12 dimer. The crystal structure of full-length Mdm12 was determined by SAD and refined with native data to 3.1 Å resolution. Lipids bound to Mdm12 are drawn with black stick models.


B . Schematic diagram indicating the secondary structure elements and their organization in Mdm12 . Three structural elements of Mdm12 are highlighted in different colored boxes .

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B. Schematic diagram indicating the secondary structure elements and their organization in Mdm12. Three structural elements of Mdm12 are highlighted in different colored boxes.


C , D . Surface representations of the Mdm12 dimer are shown in different orientations .

C, D. Surface representations of the Mdm12 dimer are shown in different orientations.


A . Ribbon diagram showing the 2 - fold dimerization interface of Mdm12 . Oxygen and nitrogen atoms are shown in red and blue , respectively . The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12 . The sequence alignment of yeastMdm12 orthologs is shown to highlight the sequence conservation in the N - terminus β1 - strand . Ten orthologs are aligned from residues 1 to 11 . Absolute and highly conserved residues are indicated in red and orange , respectively .

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

A. Ribbon diagram showing the 2-fold dimerization interface of Mdm12. Oxygen and nitrogen atoms are shown in red and blue, respectively. The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12. The sequence alignment of yeastMdm12 orthologs is shown to highlight the sequence conservation in the N-terminus β1-strand. Ten orthologs are aligned from residues 1 to 11. Absolute and highly conserved residues are indicated in red and orange, respectively.


B . The molecular weight of the Mdm12 ( I5P ) mutant was measured by size - exclusion chromatography ( below ) and ultracentrifugation ( top ) as in Figure 1B and C .

B. The molecular weight of the Mdm12 (I5P) mutant was measured by size-exclusion chromatography (below) and ultracentrifugation (top) as in Figure 1B and C.


C . Ribbon diagram showing the structures of the SMP domain in E - SYT2 , CETP , and BPI for the comparison of dimeric interfaces among SMP domains . Note that CETP and BPI are not dimers but monomers containing two tandem SMP domains .

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C. Ribbon diagram showing the structures of the SMP domain in E-SYT2, CETP, and BPI for the comparison of dimeric interfaces among SMP domains. Note that CETP and BPI are not dimers but monomers containing two tandem SMP domains.


A . Simulated annealing omit map ( Fo - Fc , contoured at 1 . 5σ ) showing the molecule bound to Mdm12 ( left ) . The final model for the bound PE is shown as in stick representation . The electron density ( 2Fo - Fc ) calculated in the final model is shown with the stick model of PE in the right ( 3 . 1 Å resolution , contoured at 0 . 8σ ) .

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

A. Simulated annealing omit map (Fo-Fc, contoured at 1.5σ) showing the molecule bound to Mdm12 (left). The final model for the bound PE is shown as in stick representation. The electron density (2Fo-Fc) calculated in the final model is shown with the stick model of PE in the right (3.1 Å resolution, contoured at 0.8σ).


B . Surface representation of the Mdm12 dimer . Hydrophobic amino acids lining the Mdm12 channel are indicated by a blue mesh . Lipids built in Mdm12 are in space - filling representation .

B. Surface representation of the Mdm12 dimer. Hydrophobic amino acids lining the Mdm12 channel are indicated by a blue mesh. Lipids built in Mdm12 are in space-filling representation.


C . Ribbon diagram showing lipid coordination by Mdm12 . Mdm12 residues and lipid fatty acids are colored in green and yellow , respectively .

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C. Ribbon diagram showing lipid coordination by Mdm12. Mdm12 residues and lipid fatty acids are colored in green and yellow, respectively.


D . Mdm12 binds NBD - PE . Wild - type and monomeric ( I5P mutant ) Mdm12 were incubated with NBD - PE and separated from free NBD - PE in native PAGE . Coomassie staining ( left ) and fluorescent ( right ) detection indicates that Mdm12 directly interacts with NBD - PEin vitro . E . Quantitative data showing binding affinities for NBD - PE by Mdm12 . The binding affinities of Mdm12 ( monomer / dimer shown in Native PAGE and I5P mutant ) for NBD - PE was measured with a NBD - PE concentration - dependent manner . All experiments were carried out three times and the means ± SD are given .

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D. Mdm12 binds NBD-PE. Wild-type and monomeric (I5P mutant) Mdm12 were incubated with NBD-PE and separated from free NBD-PE in native PAGE. Coomassie staining (left) and fluorescent (right) detection indicates that Mdm12 directly interacts with NBD-PEin vitro.E. Quantitative data showing binding affinities for NBD-PE by Mdm12. The binding affinities of Mdm12 (monomer/dimer shown in Native PAGE and I5P mutant) for NBD-PE was measured with a NBD-PE concentration-dependent manner. All experiments were carried out three times and the means ± SD are given.


F . Mdm12 mutants ( L256W , I262W , and L256W / I262W double mutant ) were incubated with NBD - PE , and subjected to native PAGE . Because wild - type Mdm12 separates as both monomer and dimer on native PAGE , the purely monomeric form ( I5P ) of Mdm12 was used as the wild type for clarity . The graph in the right indicates the quantities measured in the experiments . The bar shows the relative amounts of the band ratio ( fluorescence / Coomassie ) . Values represent the means and SD from three independent experiments .

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

F. Mdm12 mutants (L256W, I262W, and L256W/I262W double mutant) were incubated with NBD-PE, and subjected to native PAGE. Because wild-type Mdm12 separates as both monomer and dimer on native PAGE, the purely monomeric form (I5P) of Mdm12 was used as the wild type for clarity. The graph in the right indicates the quantities measured in the experiments. The bar shows the relative amounts of the band ratio (fluorescence/Coomassie). Values represent the means and SD from three independent experiments.


A . Structural views suggesting that Mdm12 might have a preference for certain phospholipids . The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution ( left ) and the sequence conservation ( right ) in the same orientation . Surface electrostatics and sequence conservation were calculated using an APBS program [ 31 ] with the nonlinear Poisson - Boltzmann equation and contoured at ± 3kT / e , and ConSurf website ( consurf . tau . ac . il ) [ 32 ] with 34 different yeast orthologs , respectively . The ribbon diagram shown in the middle indicates the overall orientation of Mdm12 .

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

A. Structural views suggesting that Mdm12 might have a preference for certain phospholipids. The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution (left) and the sequence conservation (right) in the same orientation. Surface electrostatics and sequence conservation were calculated using an APBS program [31] with the nonlinear Poisson-Boltzmann equation and contoured at ±3kT/e, and ConSurf website (consurf.tau.ac.il) [32] with 34 different yeast orthologs, respectively. The ribbon diagram shown in the middle indicates the overall orientation of Mdm12.


B . Lipid displacement experiments to identity the natural ligands of Mdm12in vitro ( see material and methods section for details ) . NBD - PE - preloaded Mdm12 - GST was mixed with a series of glycerophospholipids at different concentrations , and displacement of NBD - PE by non - labeled ligand was estimated from the decrease in fluorescence . Means ± SD are shown ( n = 3 independently performed experiments ) .

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B. Lipid displacement experiments to identity the natural ligands of Mdm12in vitro (see material and methods section for details). NBD-PE-preloaded Mdm12-GST was mixed with a series of glycerophospholipids at different concentrations, and displacement of NBD-PE by non-labeled ligand was estimated from the decrease in fluorescence. Means ± SD are shown (n=3 independently performed experiments).


A . Overall structure ( left ) and electron density ( right ) of Mdm12 in the asymmetric unit . Four molecules ( two Mdm12 dimers ) are organized with 2 - fold rotation symmetry . The 2 - fold axes are indicated with a black dotted line . B . Overall structure ( left ) and electron density ( right ) of ΔMdm12 in the asymmetric unit . Six ΔMdm12 molecules ( three Mdm12 dimers ) are arranged with 2 - fold rotation symmetry as shown above .

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

A. Overall structure (left) and electron density (right) of Mdm12 in the asymmetric unit. Four molecules (two Mdm12 dimers) are organized with 2-fold rotation symmetry. The 2-fold axes are indicated with a black dotted line.B. Overall structure (left) and electron density (right) of ΔMdm12 in the asymmetric unit. Six ΔMdm12 molecules (three Mdm12 dimers) are arranged with 2-fold rotation symmetry as shown above.


C . Ribbon diagram showing a 2 - fold interface ( tail - to - tail ) in two crystal structures ( See text for details ) .

C. Ribbon diagram showing a 2-fold interface (tail-to-tail) in two crystal structures (See text for details).


D . Size - exclusion chromatography revealing that the N - terminus - truncated version of Mdm12 ( residues 11 - 271 ) retains the ability to interact with Mmm1 . The experiment was performed as in Figure 1B . Eluted fractions indicated by shading were subjected to SDS - PAGE followed by Coomassie Blue staining .

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D. Size-exclusion chromatography revealing that the N-terminus-truncated version of Mdm12 (residues 11-271) retains the ability to interact with Mmm1. The experiment was performed as in Figure 1B. Eluted fractions indicated by shading were subjected to SDS-PAGE followed by Coomassie Blue staining.


A - F ' Upper panels ( A - F ) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage , lower panels ( A ' - F ' ) show c4da neuron morphology at 18 h after puparium formation ( APF ) . Neurons were labeled by CD8 : : GFP expression under ppk - GAL4 or by tdtomato expression in MARCM clones . ( A , A ' ) C4da neurons expressing control Orco dsRNA . Scale bars are 100 μm in A ( for larval images ) and 50 μm in A ' ( for pupal images ) . ( B , B ' ) C4da neurons expressing PP2A - 29B dsRNA . ( C , C ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones . ( D , D ' ) Homozygous PP2A - 29BGE16781 mutant c4da neuron MARCM clones expressing UAS - HAPP2A - 29B . ( E , E ' ) C4da neurons expressing dominant - negative Mts . ( F , F ' ) Homozygous mtss5286 mutant c4da neuron MARCM clones . G Penetrance of pruning defects at 18 h APF in A ' - E ' . * * P < 0 . 005 , * * * P < 0 . 0005 , Fisher ' s exact test . Number of neurons for each genotype are given in the figure , the number of animals is given below in parentheses . H Severity of pruning defects at 18 h APF in A ' - E ' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

A - F' Upper panels (A - F) show morphology of c4da neurons of the indicated genotypes at the third instar larval stage, lower panels (A' - F') show c4da neuron morphology at 18 h after puparium formation (APF). Neurons were labeled by CD8::GFP expression under ppk-GAL4 or by tdtomato expression in MARCM clones. (A, A') C4da neurons expressing control Orco dsRNA. Scale bars are 100 μm in A (for larval images) and 50 μm in A' (for pupal images). (B, B') C4da neurons expressing PP2A-29B dsRNA. (C, C') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones. (D, D') Homozygous PP2A-29BGE16781 mutant c4da neuron MARCM clones expressing UAS-HAPP2A-29B. (E, E') C4da neurons expressing dominant-negative Mts. (F, F') Homozygous mtss5286 mutant c4da neuron MARCM clones. G Penetrance of pruning defects at 18 h APF in A' - E'. ** P<0.005, *** P<0.0005, Fisher's exact test. Number of neurons for each genotype are given in the figure, the number of animals is given below in parentheses. H Severity of pruning defects at 18 h APF in A' - E' as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A - J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and dendrite pruning defects were assessed at 18 h APF . Panels A - E show effects of Sox14 / Mical pathway manipulations alone , panels F - J show neurons coexpressing PP2A - 29B dsRNA . The scale bar in A is 50 μm . ( A , F ) C4da neurons ( co - ) expressing lacZ . ( B , G ) C4da neurons ( co - ) overexpressing Sox14 . ( C , H ) C4da neurons ( co - ) overexpressing Mical . ( D , I ) C4da neurons ( co - ) expressing Mical ∆ redox . ( E , J ) C4da neurons ( co - ) expressing Mical ∆ CH . K Penetrance of pruning defects in panels A - J . * P < 0 . 05 , * * * P < 0 . 0005 , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure the numbers of animals are given below in parentheses . L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

A-J The indicated Sox14 or Mical constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and dendrite pruning defects were assessed at 18 h APF. Panels A - E show effects of Sox14/Mical pathway manipulations alone, panels F - J show neurons coexpressing PP2A-29B dsRNA. The scale bar in A is 50 μm. (A, F) C4da neurons (co-)expressing lacZ. (B, G) C4da neurons (co-)overexpressing Sox14. (C, H) C4da neurons (co-)overexpressing Mical. (D, I) C4da neurons (co-)expressing Mical∆redox. (E, J) C4da neurons (co-)expressing Mical∆CH. K Penetrance of pruning defects in panels A - J. * P<0.05, *** P<0.0005, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure the numbers of animals are given below in parentheses. L Severity of pruning defects in panels A - J as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and expression of Sox14 was assessed by immunofluorescence . A - C Sox14 stainings at 2 h APF . ( A ) Control c4da neurons . ( B ) C4da neurons expressing PP2A - 29B dsRNA . ( C ) C4da neuron expressing dominant - negative Mts . D Quantification of Sox14 expression in ( A - C ) . C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons ( identified by characteristic cell body shape ) . Data are mean + / - s . d . . The numbers of neurons ( animals ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * * P < 0 . 0005 , Wilcoxon ' s test .

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

C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Sox14 was assessed by immunofluorescence. A - C Sox14 stainings at 2 h APF. (A) Control c4da neurons. (B) C4da neurons expressing PP2A-29B dsRNA. (C) C4da neuron expressing dominant-negative Mts. D Quantification of Sox14 expression in (A - C). C4da neuron Sox14 expression was normalized to Sox14 expression in neighboring ddaE c1da neurons (identified by characteristic cell body shape). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, *** P<0.0005, Wilcoxon's test.


C4da neurons were labeled by CD8 : : GFP expression under ppk - GAL4 , and expression of Mical was assessed by immunofluorescence . E - H Mical stainings at the indicated time points . ( E ) Mical expression in control c4da neurons at 2 h APF . ( F ) Mical expression in c4da neurons expressing dominant - negative Mts at 2 h APF . ( G ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 2 h APF . ( H ) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF . ( I ) Mical expression in c4da neurons expressing PP2A - 29B dsRNA at 0 h APF . J Quantification of Mical expression in ( E - I ) . C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons ( marked by asterisks ) . Data are mean + / - s . d . . The numbers of neurons ( animals ) for each genotype are given in each graph ( data were not derived from independent experiments ) . n . s . , not significant , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Data information : Scale bars in A and E are 10 μm .

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

C4da neurons were labeled by CD8::GFP expression under ppk-GAL4, and expression of Mical was assessed by immunofluorescence. E - H Mical stainings at the indicated time points. (E) Mical expression in control c4da neurons at 2 h APF. (F) Mical expression in c4da neurons expressing dominant-negative Mts at 2 h APF. (G) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 2 h APF. (H) Mical expression in c4da neurons expressing Orco dsRNA at 0 h APF. (I) Mical expression in c4da neurons expressing PP2A-29B dsRNA at 0 h APF. J Quantification of Mical expression in (E - I). C4da neuron Mical expression was normalized to Mical expression in neighboring ddaE c1da neurons (marked by asterisks). Data are mean +/- s. d.. The numbers of neurons (animals) for each genotype are given in each graph (data were not derived from independent experiments). n. s., not significant, ** P<0.005, *** P<0.0005, Wilcoxon's test. Data information: Scale bars in A and E are 10 μm.


A - G The indicated UAS - tsr constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning , panels D - G show neurons coexpressing PP2A - 29B dsRNA . ( A , E ) C4da neuron ( co - ) overexpressing wild type tsr . ( B , F ) C4da neuron ( co - ) overexpressing tsr S3A . ( C , G ) C4da neuron ( co - ) overexpressing tsr S3E . The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite . ( D ) C4da neuron co - expressing PP2A - 29B dsRNA and lacZ as a UAS titration control . H Penetrance of pruning defects in panels A - G . * P < 0 . 05 , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure . I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

A - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control. H Penetrance of pruning defects in panels A - G. * P<0.05, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. I Severity of pruning defects in panels A - G as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


A - G The indicated UAS - tsr constructs were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning , panels D - G show neurons coexpressing PP2A - 29B dsRNA . ( A , E ) C4da neuron ( co - ) overexpressing wild type tsr . ( B , F ) C4da neuron ( co - ) overexpressing tsr S3A . ( C , G ) C4da neuron ( co - ) overexpressing tsr S3E . The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite . ( D ) C4da neuron co - expressing PP2A - 29B dsRNA and lacZ as a UAS titration control .

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

A - G The indicated UAS-tsr constructs were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. Panels A - C show effects of the expression of wild type or phosphomutant tsr variants on dendrite pruning, panels D - G show neurons coexpressing PP2A-29B dsRNA. (A, E) C4da neuron (co-)overexpressing wild type tsr. (B, F) C4da neuron (co-)overexpressing tsr S3A. (C, G) C4da neuron (co-)overexpressing tsr S3E. The arrow in panel G denotes a characteristic varicosity at the tip of an unpruned dendrite. (D) C4da neuron co-expressing PP2A-29B dsRNA and lacZ as a UAS titration control.


J - L GFP - tagged Drosophila cofilin ( GFP : : tsr ) was expressed in c4da neurons under ppk - GAL4 , and its localization was assessed at the indicated developmental stages by live imaging . Panels J - L show GFP : : tsr localization in c4da neurons at the third instar stage , panels J ' - L ' show GFP : : tsr localization in c4da neurons at 6 h APF . ( J , J ' ) Control c4da neurons expressing Orco dsRNA . ( K , K ' ) C4da neurons expressing Mical dsRNA . ( L , L ' ) C4da neurons expressing PP2A - 29B dsRNA . M Quantification of GFP : : tsr punctae in samples J - L ' . Data are mean + / - s . d . , the numbers of neurons ( animals ) analyzed for each genotype are given in the figure ( data were not derived from independent experiments ) . * * P < 0 . 005 , * * * P < 0 . 0005 , n . s . , not significant , Wilcoxon ' s test .

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

J - L GFP-tagged Drosophila cofilin (GFP::tsr) was expressed in c4da neurons under ppk-GAL4, and its localization was assessed at the indicated developmental stages by live imaging. Panels J - L show GFP::tsr localization in c4da neurons at the third instar stage, panels J' - L' show GFP::tsr localization in c4da neurons at 6 h APF. (J, J') Control c4da neurons expressing Orco dsRNA. (K, K') C4da neurons expressing Mical dsRNA. (L, L') C4da neurons expressing PP2A-29B dsRNA. M Quantification of GFP::tsr punctae in samples J - L'. Data are mean +/- s. d., the numbers of neurons (animals) analyzed for each genotype are given in the figure (data were not derived from independent experiments). ** P<0.005, *** P<0.0005, n. s., not significant, Wilcoxon's test.


A - H Genetic interactions between PP2A - 29B and actin regulators during dendrite pruning . The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A - 29B dsRNA expression . Suppression tests were performed as in Figure 2 . ( A ) C4da neuron coexpressing PP2A - 29B dsRNA and tdtomato as suppression control . ( B ) C4da neuron coexpressing PP2A - 29B dsRNA and GFP : : tsr . ( C ) C4da neuron coexpressing PP2A - 29B dsRNA and RacN17 . ( D ) C4da neuron coexpressing PP2A - 29B dsRNA and RhoN19 . ( E ) C4da neuron expressing PP2A - 29B dsRNA in ena210 / + background . ( F ) C4da neuron coexpressing PP2A - 29B dsRNA and Orco dsRNA as suppression control . ( G ) C4da neuron coexpressing PP2A - 29B dsRNA and Chicadee dsRNA . ( H ) C4da neuron coexpressing PP2A - 29B dsRNA and moesin dsRNA . I Penetrance of pruning defects in panels E - L . n . s . , not significant , Fisher ' s exact test . Numbers of neurons ( animals ) for each genotype are given in the figure . J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF . Data are mean + / - s . d . , * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 , Wilcoxon ' s test . Numbers of neurons ( animals ) for each genotype are given in the figure .

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

A - H Genetic interactions between PP2A-29B and actin regulators during dendrite pruning. The indicated manipulations of actin regulators were tested for their ability to suppress pruning defects induced by PP2A-29B dsRNA expression. Suppression tests were performed as in Figure 2. (A) C4da neuron coexpressing PP2A-29B dsRNA and tdtomato as suppression control. (B) C4da neuron coexpressing PP2A-29B dsRNA and GFP::tsr. (C) C4da neuron coexpressing PP2A-29B dsRNA and RacN17. (D) C4da neuron coexpressing PP2A-29B dsRNA and RhoN19. (E) C4da neuron expressing PP2A-29B dsRNA in ena210/+ background. (F) C4da neuron coexpressing PP2A-29B dsRNA and Orco dsRNA as suppression control. (G) C4da neuron coexpressing PP2A-29B dsRNA and Chicadee dsRNA. (H) C4da neuron coexpressing PP2A-29B dsRNA and moesin dsRNA. I Penetrance of pruning defects in panels E - L. n. s., not significant, Fisher's exact test. Numbers of neurons (animals) for each genotype are given in the figure. J Severity of pruning defects in panels E - L as assessed by number of primary and secondary dendrites attached to soma at 18 h APF. Data are mean +/- s. d., * P<0.05, ** P<0.005, *** P<0.0005, Wilcoxon's test. Numbers of neurons (animals) for each genotype are given in the figure.


( A ) Sequential COX - SDH histochemical staining in muscle biopsies of Pa2 , Pa3 - 1 , Pa4 and Pa6 ( as indicated ) , indicating a mosaic pattern of COX deficiency . Scale bar represents 100 µm .

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

(A) Sequential COX-SDH histochemical staining in muscle biopsies of Pa2, Pa3-1, Pa4 and Pa6 (as indicated), indicating a mosaic pattern of COX deficiency. Scale bar represents 100 µm.


( C ) Long - range PCR assay to detect mtDNA deletions in TOP3A patient muscle ( lanes 2 - 5 ) . Samples from unaffected individuals ( lanes 6 and 7 ) and a single - deletion mitochondrial disease patient ( lane 8 ) are used as negative and positive controls , respectively . ' M ' indicates marker .

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

(C) Long-range PCR assay to detect mtDNA deletions in TOP3A patient muscle (lanes 2-5). Samples from unaffected individuals (lanes 6 and 7) and a single-deletion mitochondrial disease patient (lane 8) are used as negative and positive controls, respectively. 'M' indicates marker.


( A - B ) Analysis of mtDNA structure in muscle from individuals with TOP3A - related mitochondrial disease using Southern blotting following restriction with BamHI ( A ) or PvuII ( B ) . The black bar indicates the probe ( nt . 16 , 262 - 128 ) .

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

(A-B) Analysis of mtDNA structure in muscle from individuals with TOP3A-related mitochondrial disease using Southern blotting following restriction with BamHI (A) or PvuII (B). The black bar indicates the probe (nt. 16,262 - 128).


mtDNA phenotypes in U2OS Flp - In cells treated with TOP3A siRNA , rescued using variant - containing siRNA - resistant forms of TOP3A . ( C ) Western blot of TOP3A protein expression . Cells were untransfected ( lane 1 ) or transfected with TOP3A siRNA ( lanes 2 - 11 ) , then induced to express WT TOP3A ( lane 3 ) or pathological variants of TOP3A ( lanes 4 - 11 ) . Note that TOP3A Ser810 * does not carry a C - terminal HA tag . β - actin is used as a loading control .

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

mtDNA phenotypes in U2OS Flp-In cells treated with TOP3A siRNA, rescued using variant-containing siRNA-resistant forms of TOP3A. (C) Western blot of TOP3A protein expression. Cells were untransfected (lane 1) or transfected with TOP3A siRNA (lanes 2-11), then induced to express WT TOP3A (lane 3) or pathological variants of TOP3A (lanes 4-11). Note that TOP3A Ser810* does not carry a C-terminal HA tag. β-actin is used as a loading control.


mtDNA phenotypes in U2OS Flp - In cells treated with TOP3A siRNA , rescued using variant - containing siRNA - resistant forms of TOP3A . ( D ) mtDNA copy number for TOP3A rescue cells as in ( C ) determined using qPCR . Error bars represent SEM , n = 3 ( Ala95Val ) or n = 4 ( all other samples ) , where each data point represents the mean of 3 technical triplicates . Significance values are shown for one - way ANOVA compared to untreated wild - type cells , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

mtDNA phenotypes in U2OS Flp-In cells treated with TOP3A siRNA, rescued using variant-containing siRNA-resistant forms of TOP3A. (D) mtDNA copy number for TOP3A rescue cells as in (C) determined using qPCR. Error bars represent SEM, n = 3 (Ala95Val) or n = 4 (all other samples), where each data point represents the mean of 3 technical triplicates. Significance values are shown for one-way ANOVA compared to untreated wild-type cells, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


mtDNA phenotypes in U2OS Flp - In cells treated with TOP3A siRNA , rescued using variant - containing siRNA - resistant forms of TOP3A . ( E ) Southern blot of mtDNA structure from cells as in ( C ) . The probe ( black bar , nt . 16 , 262 - 128 ) detects both full - length ( FL ) mtDNA and 7S DNA . The presence of hemicatenated mtDNAs is indicated . ( F ) Southern blot of uncut mtDNA to visualise different topological forms , from cells as in ( C ) . Control DNA treated with BamHI ( lane 1 ) or E . coli topoisomerase I ( lane 2 ) act as markers for linear and relaxed monomeric mtDNA , respectively . Diagrams indicate the migration of different mtDNA forms .

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

mtDNA phenotypes in U2OS Flp-In cells treated with TOP3A siRNA, rescued using variant-containing siRNA-resistant forms of TOP3A. (E) Southern blot of mtDNA structure from cells as in (C). The probe (black bar, nt. 16,262 - 128) detects both full-length (FL) mtDNA and 7S DNA. The presence of hemicatenated mtDNAs is indicated. (F) Southern blot of uncut mtDNA to visualise different topological forms, from cells as in (C). Control DNA treated with BamHI (lane 1) or E. coli topoisomerase I (lane 2) act as markers for linear and relaxed monomeric mtDNA, respectively. Diagrams indicate the migration of different mtDNA forms.


( A ) Purification of TOP3A variants . Each variant ( 3 pmol ) was separated on a 4 - 20 % stain - free Criterion TGX SDS - PAGE gel ( Bio - Rad ) and imaged using a stain - free imager .

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(A) Purification of TOP3A variants. Each variant (3 pmol) was separated on a 4-20% stain-free Criterion TGX SDS-PAGE gel (Bio-Rad) and imaged using a stain-free imager.


Electrophoretic mobility shift assay ( EMSA ) to assess DNA binding activity of TOP3A variants to a 5 ' radiolabelled 80 nt - long ssDNA oligonucleotide .

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Electrophoretic mobility shift assay (EMSA) to assess DNA binding activity of TOP3A variants to a 5' radiolabelled 80 nt-long ssDNA oligonucleotide.


( K ) Quantification of DNA binding activity as in ( B - J ) , expressed as the proportion of bound DNA . Data represents mean values with at least 3 independent experiments per data point . Error bars represent SEM . Symbols represent significance values ( one - way ANOVA ) , colour - coded to the data points . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

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(K) Quantification of DNA binding activity as in (B-J), expressed as the proportion of bound DNA. Data represents mean values with at least 3 independent experiments per data point. Error bars represent SEM. Symbols represent significance values (one-way ANOVA), colour-coded to the data points. *p<0.05, **p<0.01, ****p<0.0001.


( A - I ) Relaxation of negatively supercoiled pUC19 substrate by recombinant TOP3A variants . ' M ' indicates marker .

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(A-I) Relaxation of negatively supercoiled pUC19 substrate by recombinant TOP3A variants. 'M' indicates marker.


( J ) Quantification of DNA relaxation data as in ( A - I ) . Data represents the percentage of substrate released from negatively supercoiled form , expressed as the mean of 3 independent experiments ( except WT , for which n = 4 ) . Error bars represent SEM . Symbols represent significance values ( one - way ANOVA ) , colour - coded to the data points . * * * * p < 0 . 0001 .

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(J) Quantification of DNA relaxation data as in (A-I). Data represents the percentage of substrate released from negatively supercoiled form, expressed as the mean of 3 independent experiments (except WT, for which n = 4). Error bars represent SEM. Symbols represent significance values (one-way ANOVA), colour-coded to the data points. ****p<0.0001.


( B - J ) ssDNA decatenation assays using TOP3A variants , using substrates constructed as in ( A ) .

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(B-J) ssDNA decatenation assays using TOP3A variants, using substrates constructed as in (A).


( K ) Quantification of ssDNA decatenation activity as in ( B - J ) . Data represents the percentage of decatenated substrate expressed as mean values , ± SEM , from independent experiments , n = 3 ( WT , p . Ala95Val , p . Met100Val , p . Arg558Trp ) , n = 4 ( p . Ala176Val ) , n = 5 ( p . Met575Val ) , n = 2 ( p . Leu37Val ) . Symbols represent significance values ( one - way ANOVA ) , colour - coded to the data points . * p < 0 . 05 , * * p < 0 . 01 .

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(K) Quantification of ssDNA decatenation activity as in (B-J). Data represents the percentage of decatenated substrate expressed as mean values, ±SEM, from independent experiments, n = 3 (WT, p.Ala95Val, p.Met100Val, p.Arg558Trp), n = 4 (p.Ala176Val), n = 5 (p.Met575Val), n = 2 (p.Leu37Val). Symbols represent significance values (one-way ANOVA), colour-coded to the data points. *p<0.05, **p<0.01.


( M ) Quantifications of ssDNA decatenation assays as in ( L ) . Data represents the mean percentage of decatenated substrate from 3 independent experiments . Significance values are shown for one - way ANOVA compared to the WT protein . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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(M) Quantifications of ssDNA decatenation assays as in (L). Data represents the mean percentage of decatenated substrate from 3 independent experiments. Significance values are shown for one-way ANOVA compared to the WT protein. *p<0.05, **p<0.01, ***p<0.001.


Analysis of DNA content by flow cytometry .

Analysis of DNA content by flow cytometry.


Analysis of BrdU incorporation by BrdU - IP - chip 45 minutes after release into S phase in cells treated with 0 . 1 % MMS as described in ( A ) . A representative region on chromosome VII is shown . The position of replication origins is indicated above the map . Asterisks : late origins firing in mrc1 ∆ cells .

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Analysis of BrdU incorporation by BrdU-IP-chip 45 minutes after release into S phase in cells treated with 0.1% MMS as described in (A). A representative region on chromosome VII is shown. The position of replication origins is indicated above the map. Asterisks: late origins firing in mrc1∆ cells.


Quantification of the BrdU incorporation ( signal log ratio ) at 384 early and late replication origins in wild type , rad9 ∆ and mrc1 ∆ strains exposed to 0 . 1 or 0 . 033 % MMS . Red lines indicate median values . Median replication times ( Trep ) are indicated for bins of 96 origins .

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Quantification of the BrdU incorporation (signal log ratio) at 384 early and late replication origins in wild type, rad9∆ and mrc1∆ strains exposed to 0.1 or 0.033% MMS. Red lines indicate median values. Median replication times (Trep) are indicated for bins of 96 origins.


Wild type , mrc1 ∆ and rad9 ∆ cells were released into S phase in the presence of BrdU and 0 . 033 % MMS , as described in Fig . 1A . DNA combing analysis was performed for samples collected 45 and 60 min after release into S phase . Representative DNA fibers are shown . Red : DNA , green : BrdU . Bar is 20 kb .

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Wild type, mrc1∆ and rad9∆ cells were released into S phase in the presence of BrdU and 0.033% MMS, as described in Fig. 1A. DNA combing analysis was performed for samples collected 45 and 60 min after release into S phase. Representative DNA fibers are shown. Red: DNA, green: BrdU. Bar is 20 kb.


Distribution of center - to - center distances in cells released for 45 minutes into S phase .

Distribution of center-to-center distances in cells released for 45 minutes into S phase.


Distribution of BrdU track lengths in cells released for 45 or 60 minutes into S phase , respectively .

Distribution of BrdU track lengths in cells released for 45 or 60 minutes into S phase, respectively.


Wild type , mrc1 ∆ and rad9 ∆ cells were synchronized in G1 with α - factor prior to release with pronase in the presence of 0 . 033 % MMS . Cells were collected at the indicated times and Rad53 phosphorylation was monitored by western blot The asterisk indicates a non - specific band detected by the anti - Rad53 antibody .

Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored by western blot The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.


Wild type , mrc1 ∆ and rad9 ∆ cells were synchronized in G1 with α - factor prior to release with pronase in the presence of 0 . 033 % MMS . Cells were collected at the indicated times and Rad53 phosphorylation was monitored with an in situ phosphorylation assay - ISA ( F ) .

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Wild type, mrc1∆ and rad9∆ cells were synchronized in G1 with α-factor prior to release with pronase in the presence of 0.033% MMS. Cells were collected at the indicated times and Rad53 phosphorylation was monitored with an in situ phosphorylation assay - ISA (F).


Analysis of Rad53 phosphorylation by Western blot .

Analysis of Rad53 phosphorylation by Western blot.


Analysis of DNA replication by DNA combing in cells released for 45 and 60 minutes into S phase in the presence of 100 μg / ml Zeocin . IdU tracks ( red ) correspond to initiation events occurring during the HU treatment . Elongation events after HU removal appear as CldU tracks ( yellow ) flanking IdU tracks . New initiation events as detected as isolated CldU tracks ( asterisks ) . Bar is 20 kb .

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Analysis of DNA replication by DNA combing in cells released for 45 and 60 minutes into S phase in the presence of 100 μg/ml Zeocin. IdU tracks (red) correspond to initiation events occurring during the HU treatment. Elongation events after HU removal appear as CldU tracks (yellow) flanking IdU tracks. New initiation events as detected as isolated CldU tracks (asterisks). Bar is 20 kb.


Number of new initiation events per DNA fibers detected at 60 minutes in the experiment shown in ( D ) . For each sample , at least 65 individual DNA fiber larger than 200 kb were analyzed , for a total of > 17 Mb .

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Number of new initiation events per DNA fibers detected at 60 minutes in the experiment shown in (D). For each sample, at least 65 individual DNA fiber larger than 200 kb were analyzed, for a total of >17 Mb.


2D gel analysis of the initiation of DNA replication at the early origin ARS305 and the late origin ARS1212 . The experiment was performed as in ( A ) for the indicated times . Arrows indicate late origin firing .

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2D gel analysis of the initiation of DNA replication at the early origin ARS305 and the late origin ARS1212. The experiment was performed as in (A) for the indicated times. Arrows indicate late origin firing.


Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in ( D ) . Median values are indicated in red . For each sample , at least 250 tracks were analyzed in two biological replicates .

Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in (D). Median values are indicated in red. For each sample, at least 250 tracks were analyzed in two biological replicates.


2D gel analysis of initiation and elongation in wild type and rad9 ∆ cells at the early origin ARS719 ( probe 1 ) and at a locus 7 kb away of the origin ( probe 2 ) . Time in Zeocin after the HU wash is indicated . Arrows point to Y arcs ( fork signals ) with the strongest intensity .

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2D gel analysis of initiation and elongation in wild type and rad9∆ cells at the early origin ARS719 (probe 1) and at a locus 7 kb away of the origin (probe 2). Time in Zeocin after the HU wash is indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.


2D gel analysis of fork progression in wild type and rad9 ∆ cells through a 70 - kb region of chromosome VI for which all active origins downstream of ARS607 have been deleted . The position of probes and the time after release from HU are indicated . Arrows point to Y arcs ( fork signals ) with the strongest intensity .

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2D gel analysis of fork progression in wild type and rad9∆ cells through a 70-kb region of chromosome VI for which all active origins downstream of ARS607 have been deleted. The position of probes and the time after release from HU are indicated. Arrows point to Y arcs (fork signals) with the strongest intensity.


Analysis of Rad53 phosphorylation in G1 prior to Zeocin addition ( G1 ) and at indicated times . The asterisk indicates a non - specific band detected by the anti - Rad53 antibody .

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Analysis of Rad53 phosphorylation in G1 prior to Zeocin addition (G1) and at indicated times. The asterisk indicates a non-specific band detected by the anti-Rad53 antibody.


DNA combing analysis of replication profiles 60 minutes after release into S phase in the presence of Zeocin . Representative DNA fibers are shown . Red : DNA , green : BrdU . The green channel is also shown separately . Bar : 20 kb .

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DNA combing analysis of replication profiles 60 minutes after release into S phase in the presence of Zeocin. Representative DNA fibers are shown. Red: DNA, green: BrdU. The green channel is also shown separately. Bar: 20 kb.


Distribution of BrdU tracks length in wild type , mrc1 ∆ and mrc1AQ cells . DNA fibers are fully labeled in the case of rad9 ∆ cells . Median values are indicated in red and correspond to the analysis of at least 150 tracks from two biological replicates . ns : non - significant Mann - Whitney rank sum test .

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Distribution of BrdU tracks length in wild type, mrc1∆ and mrc1AQ cells. DNA fibers are fully labeled in the case of rad9∆ cells. Median values are indicated in red and correspond to the analysis of at least 150 tracks from two biological replicates. ns: non-significant Mann-Whitney rank sum test.


Cells from the indicated strains were released synchronously into S phase in the presence of Zeocin as described in Fig . 5A and DNA content was analyzed by flow cytometry .

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Cells from the indicated strains were released synchronously into S phase in the presence of Zeocin as described in Fig. 5A and DNA content was analyzed by flow cytometry.


Analysis of chromosome mobility by Pulse Field Gel Electrophoresis ( PFGE ) after staining of DNA with ethidium bromide . In G1 , the chromosomes are linear and are sorted according to their length . Upon entry into S phase ( 30 min ) , they cannot enter the gel due to the presence of replication intermediates . Quantitation of chromosomes re - entering the gel shows completion of DNA replication in 40 % of rad9 ∆ , rad53 - 11 and rad9 ∆ rad53 - 11 mutants , but not in wild type cells and mrc1 mutants as determined for a representative experiment . Error bars indicate SD between the signal intensity determined for the three larger chromosomes ( arrowheads ) .

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Analysis of chromosome mobility by Pulse Field Gel Electrophoresis (PFGE) after staining of DNA with ethidium bromide. In G1, the chromosomes are linear and are sorted according to their length. Upon entry into S phase (30 min), they cannot enter the gel due to the presence of replication intermediates. Quantitation of chromosomes re-entering the gel shows completion of DNA replication in 40% of rad9∆, rad53-11 and rad9∆ rad53-11 mutants, but not in wild type cells and mrc1 mutants as determined for a representative experiment. Error bars indicate SD between the signal intensity determined for the three larger chromosomes (arrowheads).


DNA combing analysis of replication profiles 30 minutes after release into S phase in the presence of Zeocin . Representative images are shown . Red : DNA , green : BrdU . Bar is 20 kb .

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DNA combing analysis of replication profiles 30 minutes after release into S phase in the presence of Zeocin. Representative images are shown. Red: DNA, green: BrdU. Bar is 20 kb.


Length distribution of BrdU tracks measured at that time for wild type , rad9 ∆ , rad53 - 11 and rad53 - 11 rad9 ∆ cells . Median lengths are indicated in red . For each sample , at least 200 tracks were measured from two biological replicates .

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Length distribution of BrdU tracks measured at that time for wild type, rad9∆, rad53-11 and rad53-11 rad9∆ cells. Median lengths are indicated in red. For each sample, at least 200 tracks were measured from two biological replicates.


( A ) Western blot analysis with representative blot including lamin A / C , NLRP3 , caspase 1 , and actin levels in skin fibroblats from patients with HGPS , n = 2 controls and 2 patients . Positive control correspond to THP1 cells stimulated with LPS + ATP .

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(A) Western blot analysis with representative blot including lamin A/C, NLRP3, caspase 1, and actin levels in skin fibroblats from patients with HGPS, n = 2 controls and 2 patients. Positive control correspond to THP1 cells stimulated with LPS+ATP.


( B ) Immunofluorescence ( IF ) visualization of NLRP3 ( green ) and nuclei ( blue ) in skin fibroblasts from a representative patient and control . Scale bar :

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(B) Immunofluorescence (IF) visualization of NLRP3 (green) and nuclei (blue) in skin fibroblasts from a representative patient and control. Scale bar: 30µm


( C ) Protein expression of NLRP3 and caspase 1 in lymphoblasts from control and one patient after stimulation with uric acid and cholesterol crystal .

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(C) Protein expression of NLRP3 and caspase 1 in lymphoblasts from control and one patient after stimulation with uric acid and cholesterol crystal.


( D ) IL - 1β and IL - 18 medium release from lymphoblasts which were assessed after a 24 hr incubation with uric acid and cholesterol . * * * P < 0 . 001 , * * P < 0 . 005 , * treatment vs no treatment ; aaaP < 0 . 001 ; aaP < 0 . 01 control cells vs patient cells .

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(D) IL-1β and IL-18 medium release from lymphoblasts which were assessed after a 24 hr incubation with uric acid and cholesterol. ***P < 0.001, **P < 0.005, * treatment vs no treatment; aaaP < 0.001; aaP < 0.01 control cells vs patient cells.


( E ) Western blot analysis with representative blot including NLRP3 , caspase 1 , IL - 1β and actin levels in heart and liver tissues from wild - type and Zmpste24 - / - mice .

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(E) Western blot analysis with representative blot including NLRP3, caspase 1, IL-1β and actin levels in heart and liver tissues from wild-type and Zmpste24-/- mice.


( F ) NLRP3 and Caspase 1 transcript expression levels were determined in heart and liver tissues by real - time quantitative RT - PCR . n = 5 for Zmpste24 + / + and n = 5 for Zmpste24 - / - groups respectively . * * * P < 0 . 001 , * * P < 0 . 005 , wild - type vs Zmpste24 - / - mice . Data are showed means ± SD , n = 4 mice per group .

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(F) NLRP3 and Caspase 1 transcript expression levels were determined in heart and liver tissues by real-time quantitative RT-PCR. n=5 for Zmpste24+/+ and n=5 for Zmpste24-/- groups respectively. ***P < 0.001, **P < 0.005, wild-type vs Zmpste24-/- mice. Data are showed means ± SD, n = 4 mice per group.


( A ) Cell growthn ( lower subpanel left patient 1 and right patient 2 ) and morphological aspect ( upper subpanel ) with MCC950 determined in healthy and representative HGPS fibroblasts . * * P < 0 . 01 , * P < 0 . 05 patient cells no treatment vs treatment . Results are presented as the mean ± SD of three independent experiments .

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(A) Cell growthn (lower subpanel left patient 1 and right patient 2) and morphological aspect (upper subpanel) with MCC950 determined in healthy and representative HGPS fibroblasts. **P < 0.01, *P < 0.05 patient cells no treatment vs treatment. Results are presented as the mean ± SD of three independent experiments.


( B ) Western blot analysis with representative blots including lamin A / C , NLRP3 , IL - 1β and actin levels in skin fibroblats from control and HGPS patient after 48 hr of vehicle and MCC950 treatment .

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(B) Western blot analysis with representative blots including lamin A/C, NLRP3, IL-1β and actin levels in skin fibroblats from control and HGPS patient after 48 hr of vehicle and MCC950 treatment.


( C - E ) Representative fluorescence images of HGPS and control fibroblasts to evaluate the effect of the MCC950 in the nuclear morphology ( D ) and NLRP3 expression ( E ) . Scale bar : 30µm . Results are presented as the mean ± SD of three independent experiments . * * * P < 0 . 01 patient vs control cells , aaaP < 0 . 01 patient cells no treatment vs treatment . One - way ANOVA test was used for statistical analysis .

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(C-E) Representative fluorescence images of HGPS and control fibroblasts to evaluate the effect of the MCC950 in the nuclear morphology (D) and NLRP3 expression (E). Scale bar: 30µm. Results are presented as the mean ± SD of three independent experiments. ***P < 0.01 patient vs control cells, aaaP < 0.01 patient cells no treatment vs treatment. One-way ANOVA test was used for statistical analysis.


( F ) Kaplan - Meier graph showing a significant increase in the maximum lifespan in WT mice compared with Zmpste24 - / - mice . N = 7 per group .

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(F) Kaplan-Meier graph showing a significant increase in the maximum lifespan in WT mice compared with Zmpste24-/- mice. N=7 per group.


( G ) Body weights evolution of the groups over time . Data are showed means ± SD , n = 6 mice per group .

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(G) Body weights evolution of the groups over time. Data are showed means ± SD, n = 6 mice per group.


( H ) Analysis of serum concentrations of IL - 1β measured by ELISA . N = 6 per group . Data are shown as means ± SD . * * * P < 0 . 001 , wild - type vs Zmpste24 - / - mice ; aaaP < 0 . 001 ; vehicle vs MCC950 .

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(H) Analysis of serum concentrations of IL-1β measured by ELISA. N=6 per group. Data are shown as means ± SD. ***P < 0.001, wild-type vs Zmpste24-/- mice; aaaP < 0.001; vehicle vs MCC950.


A - O UAS . RNAi lines were driven with eyeless . Gal4gmr . Gal4 for expression during eye development or nubbin - Gal4 for expression during wing development . ( A , B ) Control adult Drosophilaeye ( A ) and wing ( B ) . ( C , D ) α - spectrin RNAi results in overgrowth of the eye ( C ) and wing ( D ) . ( E , F ) βH - spectrin / karst RNAi results in overgrowth of the eye ( E ) and wing ( F ) . ( G , H ) β - spectrin RNAi does not affect eye size ( G ) or wing size ( H ) . ( I , J ) kibra RNAi results in overgrowth of the eye ( I ) and wing ( J ) . ( K , L ) α - spectrin , kibra double RNAi results in stronger overgrowth of the eye ( K ) and wing ( L ) . ( M , N ) βH - spectrin / wing , kibra double RNAi results in stronger overgrowth of the eye ( M ) and wing ( N ) . ( O ) Quantification of female wing sizes by pixel area , 5 wings per genotype were measured . Error bars show standard deviation . P - RThe eyeless FLP MARCM system was used to generate clonally mutant flyeyes . eyes mutant eyes ( Q ) overgrow slightly compared to controls ( P ) , while α - spectrin mutant eyes expressing α - spectrin RNAi ( R ) overgrow strongly compared to controls ( P ) . Data information : Scale bars , 250 μm .

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

A-O UAS.RNAi lines were driven with eyeless.Gal4gmr.Gal4 for expression during eye development or nubbin-Gal4 for expression during wing development. (A, B) Control adult Drosophilaeye (A) and wing (B). (C, D) α-spectrin RNAi results in overgrowth of the eye (C) and wing (D). (E, F) βH-spectrin/karst RNAi results in overgrowth of the eye (E) and wing (F). (G, H) β-spectrin RNAi does not affect eye size (G) or wing size (H). (I, J) kibra RNAi results in overgrowth of the eye (I) and wing (J). (K, L) α-spectrin, kibra double RNAi results in stronger overgrowth of the eye (K) and wing (L). (M, N) βH-spectrin/wing, kibra double RNAi results in stronger overgrowth of the eye (M) and wing (N). (O) Quantification of female wing sizes by pixel area, 5 wings per genotype were measured. Error bars show standard deviation.P-RThe eyeless FLP MARCM system was used to generate clonally mutant flyeyes. eyes mutant eyes (Q) overgrow slightly compared to controls (P), while α-spectrin mutant eyes expressing α-spectrin RNAi (R) overgrow strongly compared to controls (P).Data information: Scale bars, 250 μm.


A - F The eyeless FLP MARCM system was used to generate mutant eyes that also express UAS . RNAi targeting the Spectrin cytoskeleton . Pupalretinas were examined at 42 - 46 h after puparium formation ( APF ) . Cellmembranes were marked with Dlg staining . ( A ) Control pupalretina showing cone cells surrounded by interommatidial cells . ( B ) kibra mutant pupalretina displaying additional interommatidial cells . ( C ) Pupalretina expressing α - spectrin RNAi showing additional interommatidial cells . ( D ) kibra mutant pupalretinas expressing α - spectrin RNAi showing many additional interommatidial cells . ( E , F ) Clones of α - spectrin mutant cells ( GFP negative ) ( E ) and βH - spectrin / karst mutant cells ( GFP negative ) ( F ) show extra interommatidial cells . Scale bars , 20 μm .

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A-F The eyeless FLP MARCM system was used to generate mutant eyes that also express UAS.RNAi targeting the Spectrin cytoskeleton. Pupalretinas were examined at 42-46 h after puparium formation (APF). Cellmembranes were marked with Dlg staining. (A) Control pupalretina showing cone cells surrounded by interommatidial cells. (B) kibra mutant pupalretina displaying additional interommatidial cells. (C) Pupalretina expressing α-spectrin RNAi showing additional interommatidial cells. (D) kibra mutant pupalretinas expressing α-spectrin RNAi showing many additional interommatidial cells. (E, F) Clones of α-spectrin mutant cells (GFP negative) (E) and βH-spectrin/karst mutant cells (GFP negative) (F) show extra interommatidial cells. Scale bars, 20 μm.


G - K UAS . RNAi lines were driven with hh . Gal4 UAS . GFP for expression in the posterior compartment and contained the ex . lacZ reporter transgene . ( G ) Control wing showing a normal ex . lacZ expression pattern , which is low at the dorsal - ventral boundary but high in the proximal regions of the wing disc . ( H ) RNAi knock - down of α - Spectrin results in a mild up - regulation of ex . lacZ in the posterior compartment . ( I ) RNAi knock - down of kibra results in a mild up - regulation of ex . lacZ in the posterior compartment . ( J ) Dual RNAi knock - down of both α - spectrin and kibra results in enhanced up - regulation of ex . lacZ in the posterior compartment . ( K ) RNAi knock - down of hippo results in an up - regulation of ex . lacZ in the posterior compartment . Scale bars , 50 μm .

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G-K UAS.RNAi lines were driven with hh.Gal4 UAS.GFP for expression in the posterior compartment and contained the ex.lacZ reporter transgene. (G) Control wing showing a normal ex.lacZ expression pattern, which is low at the dorsal-ventral boundary but high in the proximal regions of the wing disc. (H) RNAi knock-down of α-Spectrin results in a mild up-regulation of ex.lacZ in the posterior compartment. (I) RNAi knock-down of kibra results in a mild up-regulation of ex.lacZ in the posterior compartment. (J) Dual RNAi knock-down of both α-spectrin and kibra results in enhanced up-regulation of ex.lacZ in the posterior compartment. (K) RNAi knock-down of hippo results in an up-regulation of ex.lacZ in the posterior compartment. Scale bars, 50 μm.


A Co - IP of V5 - tagged Expanded with the FLAG - tagged N - terminal region of Karst / βH - Spectrin , but not other Karst truncation constructs .

A Co-IP of V5-tagged Expanded with the FLAG-tagged N-terminal region of Karst/βH-Spectrin, but not other Karst truncation constructs.


B , C Expanded co - localises with Karst at the apical membrane of wing imaginal disc cells ( apical view , B ; cross section , C ) .

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B, C Expanded co-localises with Karst at the apical membrane of wing imaginal disc cells (apical view, B; cross section, C).


D Control nubbin . Gal4 expressing wing . E Overexpression of Ex activates Hippo signalling to produce a small wing . F RNAi knock - down of α - Spectrin results in an overgrown wing . G Overexpression of Ex blocks the effect of α - Spectrin RNAi . H RNAi knock - down of βH - spectrin / karst results in an overgrown wing . I Overexpression of Ex blocks the effect of βH - spectrin / karst RNAi .

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D Control nubbin.Gal4 expressing wing.E Overexpression of Ex activates Hippo signalling to produce a small wing.F RNAi knock-down of α-Spectrin results in an overgrown wing.G Overexpression of Ex blocks the effect of α-Spectrin RNAi.H RNAi knock-down of βH-spectrin/karst results in an overgrown wing.I Overexpression of Ex blocks the effect of βH-spectrin/karst RNAi.


The apical α - βHSpectrincytoskeleton may be mechanosensory in the wing imaginal discA Schematic diagram of central compression and circumferencial stretching in the third instarwing pouch . B - D Third instarwing imaginal disc stained for beta - galactosidase expressed from the expanded . lacZ reporter gene ( B ) , Crb ( C ) and Kst - YFP ( D ) . Quantification of line intensity shown below . Note inverse correlation of Crb and Kst - YFP with ex . lacZ . Scale bars , 50 μm . E , F Zoom of Kst - YFP cells under compression ( E ) and under stretch ( F ) . Scale bars , 10 μm . G Schematic diagram of the effect of force on the apical spectrin cytoskeleton leading to declustering of Crumbs complexes and reduced Hippo signalling .

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

The apical α-βHSpectrincytoskeleton may be mechanosensory in the wing imaginal discA Schematic diagram of central compression and circumferencial stretching in the third instarwing pouch.B-D Third instarwing imaginal disc stained for beta-galactosidase expressed from the expanded.lacZ reporter gene (B), Crb (C) and Kst-YFP (D). Quantification of line intensity shown below. Note inverse correlation of Crb and Kst-YFP with ex.lacZ. Scale bars, 50 μm.E, F Zoom of Kst-YFP cells under compression (E) and under stretch (F). Scale bars, 10 μm.G Schematic diagram of the effect of force on the apical spectrin cytoskeleton leading to declustering of Crumbs complexes and reduced Hippo signalling.


B Control nub . Gal4 wing . C , D Expression of UAS . CrbExTM - GFP with Gal4 . Gal4 ( C ) or UAS . Wts with nub . Gal4 ( D ) results in a small wing . E RNAi knock - down of α - Spectrin results in an overgrown wing . F , G Expression of UAS . CrbExTM - GFP ( F ) or UAS . Wts ( G ) blocks the effect of α - Spectrin RNAi . H RNAi knock - down of βH - spectrin / karst results in an overgrown wing . I , J Expression of UAS . CrbExTM - GFP ( I ) or UAS . Wts ( J ) blocks the effect of α - Spectrin RNAi . K , L RNAi knock - down of Wts results in a strongly overgrown wing ( K ) and expression of UAS . CrbExTM - GFP does not block the effect of wts RNAi ( L ) . M , N RNAi knock - down of Ajuba results in a small wing ( M ) , and expression of UAS . CrbExTM - GFP enhances the effect of ajuba RNAi ( N ) . O Quantification of various wing sizes . Error bars show standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Control nub.Gal4 wing.C, D Expression of UAS.CrbExTM-GFP with Gal4.Gal4 (C) or UAS.Wts with nub.Gal4 (D) results in a small wing.E RNAi knock-down of α-Spectrin results in an overgrown wing.F, G Expression of UAS.CrbExTM-GFP (F) or UAS.Wts (G) blocks the effect of α-Spectrin RNAi.H RNAi knock-down of βH-spectrin/karst results in an overgrown wing.I, J Expression of UAS.CrbExTM-GFP (I) or UAS.Wts (J) blocks the effect of α-Spectrin RNAi.K, L RNAi knock-down of Wts results in a strongly overgrown wing (K) and expression of UAS.CrbExTM-GFP does not block the effect of wts RNAi (L).M, N RNAi knock-down of Ajuba results in a small wing (M), and expression of UAS.CrbExTM-GFP enhances the effect of ajuba RNAi (N).O Quantification of various wing sizes. Error bars show standard deviation.


P , Q Localisation of Wts - GFP ( P ) and E - cadherin ( Q ) in the third instar wing imaginal disc .

P, Q Localisation of Wts-GFP (P) and E-cadherin (Q) in the third instar wing imaginal disc.


R Quantification of line intensity in ( P ) and ( Q ) . No increased Wts - GFP intensity is observed in proximal regions where cells are subject to stretching .

R Quantification of line intensity in (P) and (Q). No increased Wts-GFP intensity is observed in proximal regions where cells are subject to stretching.


A Control egg chamber stained for DAPI to mark nuclei . B Overexpression of Yki3SA stimulates follicle cell proliferation ( MARCM clone , GFP positive ) . C - F Mutation of βH - spectrin / karst ( C ) or crb ( D ) does not increase follicle cell proliferation ( GFP negative clone ) , while mutation of α - spectrin ( E ) or β - spectrin ( F ) stimulates follicle cell proliferation ( GFP negative clone ) .

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A Control egg chamber stained for DAPI to mark nuclei.B Overexpression of Yki3SA stimulates follicle cell proliferation (MARCM clone, GFP positive).C-F Mutation of βH-spectrin/karst (C) or crb (D) does not increase follicle cell proliferation (GFP negative clone), while mutation of α-spectrin (E) or β-spectrin (F) stimulates follicle cell proliferation (GFP negative clone).


G Control egg chamber stained for Cut , a marker of proliferating cells that is normally down - regulated after stage 6 of oogenesis . H - K Mutation of βH - spectrin / karst ( H ) or crb ( I ) does not increase Cut expression in follicle cells ( GFP negative clone ) , while mutation of α - spectrin ( J ) or β - spectrin ( K ) stimulates Cut expression in posterior follicle cells ( GFP negative clone ) . L Control egg chamber showing ex . lacZ reporter gene expression . M - P Mutation of βH - spectrin / karst ( M ) or crb ( N ) does not increase ex . lacZ expression in follicle cells ( GFP negative clone ) , while mutation of α - spectrin ( O ) or β - spectrin ( P ) stimulates ex . lacZ expression in posterior follicle cells ( GFP negative clone ) .

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

G Control egg chamber stained for Cut, a marker of proliferating cells that is normally down-regulated after stage 6 of oogenesis.H-K Mutation of βH-spectrin/karst (H) or crb (I) does not increase Cut expression in follicle cells (GFP negative clone), while mutation of α-spectrin (J) or β-spectrin (K) stimulates Cut expression in posterior follicle cells (GFP negative clone).L Control egg chamber showing ex.lacZ reporter gene expression.M-P Mutation of βH-spectrin/karst (M) or crb (N) does not increase ex.lacZ expression in follicle cells (GFP negative clone), while mutation of α-spectrin (O) or β-spectrin (P) stimulates ex.lacZ expression in posterior follicle cells (GFP negative clone).


Q Wild - type follicle cells showing normal hexagonal packing of epithelial membranes . R Mutation of β - spectrin disrupts the normal hexagonal packing , suggesting a role in maintaining membrane tension . S Clone of β - spectrin mutant cells ( GFP negative ) showing differential membrane tension at the interface with wild - type cells ( GFP positive ) .

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Q Wild-type follicle cells showing normal hexagonal packing of epithelial membranes.R Mutation of β-spectrin disrupts the normal hexagonal packing, suggesting a role in maintaining membrane tension.S Clone of β-spectrin mutant cells (GFP negative) showing differential membrane tension at the interface with wild-type cells (GFP positive).


Basolateralα - β Spectrins are required to restrict cellproliferation in theDrosophila intestinal epitheliumA Control myo1A . Gal4UAS . GFP adult midgut stained for phospho - histone H3 to mark mitotic stem cells . B - E RNAi knock - down of βH - spectrin / karst ( B ) or crumbs ( C ) does not increase stem cellproliferation , while RNAi knock - down of β - spectrin ( D ) or α - spectrin ( E ) strongly stimulates stem cellproliferation . F Overexpression of Yki strongly stimulates stem cellproliferation . G Quantification of the number of PH3 - positive mitotic cells . N > 13 samples . Statistical analysis performed using the two - tailed Student ' s t - test . Error bars show standard deviation . * * * P - value ≤ 0 . 001 . H - K A Hippo reporter DIAP1 - HRE - GFP is normally expressed in sporadic stem cells . Its expression is not affected by RNAi knock - down of βH - spectrin / karst ( I ) , but is up - regulated in many stem cells by RNAi knock - down of α - spectrin ( J ) or by overexpression of Yki ( K ) . Data information : Scale bars , 100 μm .

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

Basolateralα-β Spectrins are required to restrict cellproliferation in theDrosophila intestinal epitheliumA Control myo1A.Gal4UAS.GFP adult midgut stained for phospho-histone H3 to mark mitotic stem cells.B-E RNAi knock-down of βH-spectrin/karst (B) or crumbs (C) does not increase stem cellproliferation, while RNAi knock-down of β-spectrin (D) or α-spectrin (E) strongly stimulates stem cellproliferation.F Overexpression of Yki strongly stimulates stem cellproliferation.G Quantification of the number of PH3-positive mitotic cells. N > 13 samples. Statistical analysis performed using the two-tailed Student's t-test. Error bars show standard deviation. ***P-value ≤ 0.001.H-K A Hippo reporter DIAP1-HRE-GFP is normally expressed in sporadic stem cells. Its expression is not affected by RNAi knock-down of βH-spectrin/karst (I), but is up-regulated in many stem cells by RNAi knock-down of α-spectrin (J) or by overexpression of Yki (K).Data information: Scale bars, 100 μm.


A Control Caco - 2 epithelial cells plated a low density show strong nuclear YAP localisation . B Control Caco - 2 epithelial cells plated at high density show relocalisation of YAP to the cytoplasm . C , D siRNA knock - down of SPTAN1 ( C ) or SPTBN1 ( D ) prevents relocalisation of YAP to the cytoplasm .

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A Control Caco-2 epithelial cells plated a low density show strong nuclear YAP localisation.B Control Caco-2 epithelial cells plated at high density show relocalisation of YAP to the cytoplasm.C, D siRNA knock-down of SPTAN1 (C) or SPTBN1 (D) prevents relocalisation of YAP to the cytoplasm.


E Quantification of YAP localisation in ( B - D ) .

E Quantification of YAP localisation in (B-D).


F Western blotting reveals that SPTAN1 or SPTBN1 are required for normal levels of YAP phosphorylation , and of LATS1 phosphorylation .

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

F Western blotting reveals that SPTAN1 or SPTBN1 are required for normal levels of YAP phosphorylation, and of LATS1 phosphorylation.


G siRNAs against SPTAN1 or SPTBN1 efficiently deplete their target proteins .

G siRNAs against SPTAN1 or SPTBN1 efficiently deplete their target proteins.


A . Representative pseudocolor images of PC6 cells transfected with EYFP - Parkin ( green ) and mitochondrially targeted CFP ( red ) with and without PINK1 and Miro1 shRNA and Miro2 shRNA . Zoomed images show translocation of EYFP - Parkin to mitochondria in PINK1 - overexpressing PC6

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

A. Representative pseudocolor images of PC6 cells transfected with EYFP-Parkin (green) and mitochondrially targeted CFP (red) with and without PINK1 and Miro1 shRNA and Miro2 shRNA. Zoomed images show translocation of EYFP-Parkin to mitochondria in PINK1-overexpressing PC6 cells


B . Quantification of EYFP - Parkin translocation to mitochondria . The percentage of PC6 cells with EYFP - Parkin translocated to mitochondria in response to PINK1 overexpression is lower in the Miro shRNAs - expressing groups when compared with the scrambled shRNA - expressing group ( scr shRNA ) . * * * * P < 0 . 0001 , n = 6 dishes per group , 20 fields per dish , one - way

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of EYFP-Parkin translocation to mitochondria. The percentage of PC6 cells with EYFP-Parkin translocated to mitochondria in response to PINK1 overexpression is lower in the Miro shRNAs-expressing groups when compared with the scrambled shRNA-expressing group (scr shRNA). ****P < 0.0001, n = 6 dishes per group, 20 fields per dish, one-way ANOVA


C . The kinetics of EYFP - Parkin translocation is different in PC6 cells transfected with scrambled or Miro shRNAs . Cells were plated in separate compartments of the same dish , enabling us to visualize Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment ( A & O , both 10 µM ) . The spatial heterogeneity of EYFP - Parkin ( coefficient of variation of the intensity of individual pixels ) was estimated for individual cells for each time point ( n = 39 - 47 cells ; P < 0 . 0001 difference between the curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed area shows SEM )

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

C. The kinetics of EYFP-Parkin translocation is different in PC6 cells transfected with scrambled or Miro shRNAs. Cells were plated in separate compartments of the same dish, enabling us to visualize Parkin translocation simultaneously over 3 h under similar conditions in response to antimycin with oligomycin treatment (A&O, both 10 µM). The spatial heterogeneity of EYFP-Parkin (coefficient of variation of the intensity of individual pixels) was estimated for individual cells for each time point (n = 39-47 cells; P < 0.0001 difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)