Figure 42D 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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Figure 42D 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.


Figure 5Analysis of DNA content by flow cytometry . 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 . 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 .

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

Figure 5Analysis of DNA content by flow cytometry.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.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.


Figure 6Cells 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 ) . 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 .

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

Figure 6Cells 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).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.


Figure 1 ( 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 : 30µm ( 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 . ( 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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Figure 1(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: 30µm(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.(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.


Figure 2 ( 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 . ( 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 . ( 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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Figure 2(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.(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.(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.


Figure 1A - 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 .

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Figure 1A-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.


Figure 2A - 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 .

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

Figure 2A-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.


Figure 3A 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 ) . 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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Figure 3A 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).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.


Figure 4The 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 .

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Figure 4The 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.


Figure 5B 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 . 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 .

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Figure 5B 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.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.


Figure 6A 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 ) . 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 ) .

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

Figure 6A 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).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).


Figure 7Basolateralα - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Basolateralα-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.


Figure 8A 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 ) . 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 .

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

Figure 8A 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).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.


Figure 1A . 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 cellsB . 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 ANOVAC . 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 ) D . Miro shRNAs mitigate the effect of A & O on EYFP - Parkin translocation . PC6 cells transfected with EYFP - Parkin with and without Miro shRNAs were treated with DMSO or A & O ( both 10 µM ) for 3h . The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs - expressing group when compared with the scrambled shRNA - expressing group . * * * * P < 0 . 0001 , n = 4 dishes , 20 fields per dish , one - way ANOVAE . Miro shRNAs inhibit PINK1 - induced mitophagy in PC6 cells . The figure shows the changes in the excitation spectra of mitochondrially targeted pH - sensitive Keima ( ratio of 561 nm / 458nm fluorescence intensities ) . * * * * P < 0 . 0001 , n = 80 cells from 4 dishes , Kruskal - Wallis testF . Miro shRNAs inhibit basal mitophagy in primary cortical neurons expressing mitochondrially targeted Keima . Mitochondria in lysosomes ( acidic pH ) were counted manually , by a blinded observer ( * * P < 0 . 01 , n = 9 dishes , 9 fields per dish , from 2 independent experiments , one - way ANOVA ) G . Miro shRNAs decrease the number of mitochondria co - localised with the autophagosome marker EGFP - LC3B in primary cortical neurons ( * * P < 0 . 01 , n = 5 dishes , 9 fields per dish , one - way ANOVA ) .

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

Figure 1A. 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 cellsB. 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 ANOVAC. 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)D. Miro shRNAs mitigate the effect of A&O on EYFP-Parkin translocation. PC6 cells transfected with EYFP-Parkin with and without Miro shRNAs were treated with DMSO or A&O (both 10 µM) for 3h. The figure demonstrates that the percentage of cells with Parkin translocated to mitochondria was lower in the Miro shRNAs-expressing group when compared with the scrambled shRNA-expressing group. ****P < 0.0001, n = 4 dishes, 20 fields per dish, one-way ANOVAE. Miro shRNAs inhibit PINK1-induced mitophagy in PC6 cells. The figure shows the changes in the excitation spectra of mitochondrially targeted pH-sensitive Keima (ratio of 561 nm/458nm fluorescence intensities). ****P < 0.0001, n = 80 cells from 4 dishes, Kruskal-Wallis testF. Miro shRNAs inhibit basal mitophagy in primary cortical neurons expressing mitochondrially targeted Keima. Mitochondria in lysosomes (acidic pH) were counted manually, by a blinded observer (**P < 0.01, n = 9 dishes, 9 fields per dish, from 2 independent experiments, one-way ANOVA)G. Miro shRNAs decrease the number of mitochondria co-localised with the autophagosome marker EGFP-LC3B in primary cortical neurons (**P < 0.01, n = 5 dishes, 9 fields per dish, one-way ANOVA).


Figure 2A . The pattern of Parkin translocation caused by Miro1 overexpression is different from A & O - induced translocation . The figure illustrates puncta - like EYFP - Parkin on mitochondria in antimycin and oligomycin - treated ( both 10 µM for 3h ) PC6 cells ( middle panels ) , whereas in Miro1 - overexpressing cells , EYFP - Parkin appears along rod - shaped mitochondria visualised with CFP - mito ( lower panels ) . The merged panels present EYFP - Parkin ( green ) and CFP - mito ( red ) B . Overexpression of myc - Miro1 and untagged Miro2 , but not myc - Miro2 , induce EYFP - Parkin translocation to mitochondria . * * * * P < 0 . 0001 vs control group , n = 5 - 6 dishes , 20 fields per dish , one - way ANOVA or t - testC . Quantification of EYFP - Parkin signal heterogeneity in PC6 cells demonstrates that Miro1 overexpression induces Parkin translocation from the cytosol to mitochondria , which is significantly weaker than PINK1 overexpression - or A & O ( 3 h ) - induced translocation . * * P < 0 . 01 and * * * * P < 0 . 0001 vs control group , n = 18 - 32 cells from 3 dishes per group , Kruskal - Wallis testMiro1 overexpression does not cause mitochondrial depolarization ( D ) Representative image of Miro1 and EYFP - Parkin - transfected cells stained with TMRE . The merged panels present EYFP - Parkin ( green ) and TMRE ( red ) Miro1 overexpression does not cause mitochondrial depolarization ( E ) Relative TMRE intensity quantified in neurons transfected with Miro1 ( detected by CFP - mito co - transfection ) ; the control group is non - transfected cells in the same image . The relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs - expressing neurons . Note that FCCP treatment ( 3 μM for 15 min ) almost completely abolished TMRE fluorescence . * * * * P < 0 . 0001 vs control group , n = 94 - 186 cells from 4 dishes per group , Kruskal - Wallis testF . Co - expression of HA - Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons . Overexpression of Miro1 alone increased the motion time in both directions as well as the relative mitochondrial velocity . Co - transfection of Parkin did not affect significantly the motility parameters . Data are presented as Tukey boxplot . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 , n = 689 - 846 individual mitochondria from 39 - 44 axons per group pooled from at least from 10 individual dishes from 4 independent experiments , Kruskal - Wallis testG . Parkin overexpression does not induce degradation of myc - Miro1 in control conditions . Representative Western blot image and analysis of myc - Miro1 expression in HEK cells . Treatment with A & O ( both 15 µM ) for 3h in the absence of MG132 led to slight degradation of myc - Miro1 . Co - expression of EYFP - Parkin did not induce degradation of Miro1 although an additional band above the Miro1 band was present . However , Parkin co - expression enhanced Miro1 degradation when cells were treated with A & O . * P < 0 . 05 and * * * * P < 0 . 0001 , ns : not significant , n = 4 independent experiments , one way ANOVA . Note that only the main band of myc - Miro1 at 80 kDa was analysed and that YFP was used to compensate in groups not expressing EYFP - ParkinH . EYFP - Parkin co - immunoprecipitates with overexpressed myc - Miro1 , both in DMSO - or A & O ( both 15 µM for 3h ) treated HEK cellsI . Endogenous Parkin co - immunoprecipitates with endogenous Miro1 . Miro1 was immunoprecipitated from non - transfected and non - treated HEK cells using mouse monoclonal anti - Miro1 antibody and immunoblotted for endogenous Parkin .

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

Figure 2A. The pattern of Parkin translocation caused by Miro1 overexpression is different from A&O-induced translocation. The figure illustrates puncta-like EYFP-Parkin on mitochondria in antimycin and oligomycin-treated (both 10 µM for 3h) PC6 cells (middle panels), whereas in Miro1-overexpressing cells, EYFP-Parkin appears along rod-shaped mitochondria visualised with CFP-mito (lower panels). The merged panels present EYFP-Parkin (green) and CFP-mito (red)B. Overexpression of myc-Miro1 and untagged Miro2, but not myc-Miro2, induce EYFP-Parkin translocation to mitochondria. ****P < 0.0001 vs control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVA or t-testC. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that Miro1 overexpression induces Parkin translocation from the cytosol to mitochondria, which is significantly weaker than PINK1 overexpression- or A&O (3 h)-induced translocation. **P < 0.01 and ****P < 0.0001 vs control group, n = 18-32 cells from 3 dishes per group, Kruskal-Wallis testMiro1 overexpression does not cause mitochondrial depolarization (D) Representative image of Miro1 and EYFP-Parkin-transfected cells stained with TMRE. The merged panels present EYFP-Parkin (green) and TMRE (red)Miro1 overexpression does not cause mitochondrial depolarization (E) Relative TMRE intensity quantified in neurons transfected with Miro1 (detected by CFP-mito co-transfection); the control group is non-transfected cells in the same image. The relative TMRE signal was slightly increased in Miro1 overexpressing neurons but unaffected in Miro shRNAs-expressing neurons. Note that FCCP treatment (3 μM for 15 min) almost completely abolished TMRE fluorescence. ****P < 0.0001 vs control group, n = 94-186 cells from 4 dishes per group, Kruskal-Wallis testF. Co-expression of HA-Parkin with Miro1 does not suppress mitochondrial motility in axons of cortical neurons. Overexpression of Miro1 alone increased the motion time in both directions as well as the relative mitochondrial velocity. Co-transfection of Parkin did not affect significantly the motility parameters. Data are presented as Tukey boxplot. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, n = 689-846 individual mitochondria from 39-44 axons per group pooled from at least from 10 individual dishes from 4 independent experiments, Kruskal-Wallis testG. Parkin overexpression does not induce degradation of myc-Miro1 in control conditions. Representative Western blot image and analysis of myc-Miro1 expression in HEK cells. Treatment with A&O (both 15 µM) for 3h in the absence of MG132 led to slight degradation of myc-Miro1. Co-expression of EYFP-Parkin did not induce degradation of Miro1 although an additional band above the Miro1 band was present. However, Parkin co-expression enhanced Miro1 degradation when cells were treated with A&O. *P < 0.05 and ****P < 0.0001, ns: not significant, n = 4 independent experiments, one way ANOVA. Note that only the main band of myc-Miro1 at 80 kDa was analysed and that YFP was used to compensate in groups not expressing EYFP-ParkinH. EYFP-Parkin co-immunoprecipitates with overexpressed myc-Miro1, both in DMSO- or A&O (both 15 µM for 3h) treated HEK cellsI. Endogenous Parkin co-immunoprecipitates with endogenous Miro1. Miro1 was immunoprecipitated from non-transfected and non-treated HEK cells using mouse monoclonal anti-Miro1 antibody and immunoblotted for endogenous Parkin.


Figure 3A . Miro1 induces EYFP - Parkin translocation in the presence of PINK1 shRNA . PC6 cells were transfected with EYFP - Parkin and Miro1 or treated for 3h with A & O ( both 10 µM ) in the presence of scrambled shRNA or PINK1 shRNA . Miro1 induced Parkin translocation also in the presence of PINK1 shRNA while A & O treatment did not . * * * * P < 0 . 0001 compared with the respective control group , n = 5 - 6 dishes , 20 fields per dish , one - way ANOVAB . Miro1 induces EYFP - Parkin translocation in PINK1 - / - MEFs . MEFs obtained from PINK1 + / + and - / - mice were transfected with EYFP - Parkin and Miro1 or treated with A & O ( both 10 µM for 3h ) . Miro1 induced Parkin translocation in both MEF lines but A & O only in PINK1 + / + MEFs . * * * * P < 0 . 0001 compared with respective control group , n = 6 dishes , one - way ANOVAC . Mutated EYFP - Parkin is recruited to mitochondria in response to Miro1 overexpression . PC6 cells were transfected with wild type , T240R or C431N EYFP - Parkin and Miro1 or treated for 3 hours with A & O ( both 10 µM ) . Miro1 induced translocation of all EYFP - Parkin mutants whereas A & O treatment was not able to induce equivalent translocation of Parkin mutants . * * P < 0 . 01 and * * * * P < 0 . 0001 compared with respective control group , n = 3 - 6 dishes , 20 fields per dish , one - way ANOVAD . Parkin mutants T240R and C431N interact with Miro1 both in DMSO - or A & O - treated ( both 15 µM for 3h ) HEK cells . Note that the higher molecular weight bands of Miro1 were observed only in WT Parkin - expressing groups .

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

Figure 3A. Miro1 induces EYFP-Parkin translocation in the presence of PINK1 shRNA. PC6 cells were transfected with EYFP-Parkin and Miro1 or treated for 3h with A&O (both 10 µM) in the presence of scrambled shRNA or PINK1 shRNA. Miro1 induced Parkin translocation also in the presence of PINK1 shRNA while A&O treatment did not. ****P < 0.0001 compared with the respective control group, n = 5-6 dishes, 20 fields per dish, one-way ANOVAB. Miro1 induces EYFP-Parkin translocation in PINK1 -/- MEFs. MEFs obtained from PINK1 +/+ and -/- mice were transfected with EYFP-Parkin and Miro1 or treated with A&O (both 10 µM for 3h). Miro1 induced Parkin translocation in both MEF lines but A&O only in PINK1 +/+ MEFs. ****P < 0.0001 compared with respective control group, n = 6 dishes, one-way ANOVAC. Mutated EYFP-Parkin is recruited to mitochondria in response to Miro1 overexpression. PC6 cells were transfected with wild type, T240R or C431N EYFP-Parkin and Miro1 or treated for 3 hours with A&O (both 10 µM). Miro1 induced translocation of all EYFP-Parkin mutants whereas A&O treatment was not able to induce equivalent translocation of Parkin mutants. **P < 0.01 and ****P < 0.0001 compared with respective control group, n = 3-6 dishes, 20 fields per dish, one-way ANOVAD. Parkin mutants T240R and C431N interact with Miro1 both in DMSO- or A&O-treated (both 15 µM for 3h) HEK cells. Note that the higher molecular weight bands of Miro1 were observed only in WT Parkin-expressing groups.


Figure 4A . Miro1 K572R mutant interacts with HA - Parkin similarly to Miro1 WT . HEK cells overexpressing myc - Miro1 WT or K572R mutant were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . Myc - Miro1 was immunoprecipitated and probed for myc - Miro1 and HA - Parkin . Note the diminished degradation of Miro1 K572R in input and the additional band above Miro1 WT in the IP , which is missing in the case of the K572R mutantB . Miro1 K572R ubiquitination by Parkin is reduced . HEK cells overexpressing HA - ubiquitin , myc - Miro1 or myc - Miro1 K572R and EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . HA - ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin - HA and myc - Miro1C . Representative images of PC6 cells overexpressing Miro1 K572R or Miro1 WT together with EYFP - Parkin . K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A & O ( both 7 . 5 µM ) for 3 hours . Note the spatial heterogeneity values shown in the imagesD . Quantification of EYFP - Parkin signal heterogeneity in PC6 cells demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A & O ( both 7 . 5 µM ) treatment for 3 hours , but K572R mutation of Miro1 abolishes this effect . * * * P < 0 . 001 and * * * * P < 0 . 0001 , n = 152 - 172 cells from 3 dishes per group , Kruskal - Wallis test . Data are presented as means ± SEM .

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

Figure 4A. Miro1 K572R mutant interacts with HA-Parkin similarly to Miro1 WT. HEK cells overexpressing myc-Miro1 WT or K572R mutant were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probed for myc-Miro1 and HA-Parkin. Note the diminished degradation of Miro1 K572R in input and the additional band above Miro1 WT in the IP, which is missing in the case of the K572R mutantB. Miro1 K572R ubiquitination by Parkin is reduced. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1 K572R and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin-HA and myc-Miro1C. Representative images of PC6 cells overexpressing Miro1 K572R or Miro1 WT together with EYFP-Parkin. K572R mutant induces Parkin translocation similar to WT in basal conditions but not after treatment with A&O (both 7.5 µM) for 3 hours. Note the spatial heterogeneity values shown in the imagesD. Quantification of EYFP-Parkin signal heterogeneity in PC6 cells demonstrates that WT Miro1 overexpression amplifies Parkin translocation in response A&O (both 7.5 µM) treatment for 3 hours, but K572R mutation of Miro1 abolishes this effect. ***P < 0.001 and ****P < 0.0001, n = 152-172 cells from 3 dishes per group, Kruskal-Wallis test. Data are presented as means ± SEM.


Figure 5A . A Ca2 + - insensitive mutant of Miro1 ( Miro1 - EF ) interacts with Parkin . HEK cells overexpressing myc - Miro1 or myc - Miro1 - EF and EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . Myc - Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 ( red ) and Parkin ( green ) . The high - contrast grayscale image from the green channel of this blot shows that endogenous Parkin co - immunoprecipitated with Miro1 in all conditions . The bar chart shows the quantification of co - immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP - Parkin signal ( * P < 0 . 05 and * * P < 0 . 01 , repeated measures ANOVAB . Miro1 - EF is less susceptible to ubiquitination by Parkin . HEK cells overexpressing HA - ubiquitin , myc - Miro1 or myc - Miro1 - EF and EYFP - Parkin were treated with DMSO ( control ) or A & O ( both 15 µM ) for 3 hrs . HA - ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin ( red ) , Miro1 ( green ) and Parkin ( green ; representative images are from the same experimental material on separate SDS - PAGE - WB membranes ) . The high - contrast grayscale shows the green channel of this blot , and the bar chart shows quantification of co - immunoprecipitated Miro from 3 different blots ( * * P < 0 . 01 and * * * P < 0 . 001 , repeated measures ANOVA ) . Note that EYFP - Parkin co - immunoprecipitated with ubiquitin only after A & O treatmentC . Mutation of Miro1 EF - hands diminishes Miro1 ubiquitination with S65 - phosphorylated ubiquitin . HEK cells were transfected with EYFP - Parkin with either myc - Miro1 - WT or myc - Miro1 - EF with or without PINK1 - V5 . Cells were treated with either DMSO or A & O ( both 10 µM ) for 5 hrs with the exception of the PINK1 - V5 group . Myc - Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 ( red ) , S65 - Phosphoubiquitin and PINK1 - V5 ( green ; the weak bands marked with the asterisk are nonspecific ) .

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

Figure 5A. A Ca2+-insensitive mutant of Miro1 (Miro1-EF) interacts with Parkin. HEK cells overexpressing myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red) and Parkin (green). The high-contrast grayscale image from the green channel of this blot shows that endogenous Parkin co-immunoprecipitated with Miro1 in all conditions. The bar chart shows the quantification of co-immunoprecipitated Parkin EYFP from 4 different blots normalised to total EYFP-Parkin signal (*P < 0.05 and **P < 0.01, repeated measures ANOVAB. Miro1-EF is less susceptible to ubiquitination by Parkin. HEK cells overexpressing HA-ubiquitin, myc-Miro1 or myc-Miro1-EF and EYFP-Parkin were treated with DMSO (control) or A&O (both 15 µM) for 3 hrs. HA-ubiquitin was immunoprecipitated and probes were immunoblotted for ubiquitin (red), Miro1 (green) and Parkin (green; representative images are from the same experimental material on separate SDS-PAGE-WB membranes). The high-contrast grayscale shows the green channel of this blot, and the bar chart shows quantification of co-immunoprecipitated Miro from 3 different blots (**P < 0.01 and ***P < 0.001, repeated measures ANOVA). Note that EYFP-Parkin co-immunoprecipitated with ubiquitin only after A&O treatmentC. Mutation of Miro1 EF-hands diminishes Miro1 ubiquitination with S65-phosphorylated ubiquitin. HEK cells were transfected with EYFP-Parkin with either myc-Miro1-WT or myc-Miro1-EF with or without PINK1-V5. Cells were treated with either DMSO or A&O (both 10 µM) for 5 hrs with the exception of the PINK1-V5 group. Myc-Miro1 was immunoprecipitated and probes were immunoblotted for Miro1 (red), S65-Phosphoubiquitin and PINK1-V5 (green; the weak bands marked with the asterisk are nonspecific).


Figure 6A . Kinetics of EYFP - Parkin translocation in response to A & O is different in control and Miro1 - EF - expressing PC6 cells . The inset shows how different compartments of the same dish were transfected with EYFP - Parkin and control plasmid or Miro1 - EF and followed over 3 hrs using a confocal microscope after addition of A & O ( both 10 µM ) . The graph shows the average spatial heterogeneity of EYFP - Parkin fluorescence , reflecting the ratio of mitochondrial - to - cytosolic Parkin over time ( n = 40 - 43 cells , difference between the curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed area shows SEM ) The percentage of cells where EYFP - Parkin translocates in response to A & O ( both 10 µM , B is decreased in Miro1 - EF - expressing PC6 cells . * * P < 0 . 01 , * * * * P < 0 . 0001 compared with the indicated group ( n = 3 - 6 dishes , 20 fields per dish , one way ANOVA forThe percentage of cells where EYFP - Parkin translocates in response t PINK1 overexpression ( C ) is decreased in Miro1 - EF - expressing PC6 cells . * * P < 0 . 01 , * * * * P < 0 . 0001 compared with the indicated group ( n = 3 - 6 dishes , 20 fields per dis Welch " s ANOVA fD . Time course showing laser - induced EYFP - Parkin accumulation in the irradiated area of the neuronal soma of primary rat cortical neurons . Cells were transfected with CFP - mito and EYFP - Parkin and visualized using a confocal microscope . Mitochondria were irradiated in a 0 . 9 µm x 0 . 9 µm - frame using a 405 - nm laser line ( 25 iterations using 100 % laser power , pixel time 164 µs ) E , F Miro1 - EF delays and reduces irradiation - induced EYFP - Parkin translocation . ( E ) The time course of translocation strength in control and Miro1 - EF - expressing neurons . Difference between the curves P = 0 . 0016 , two - way ANOVA . ( F ) Comparison at the 60 - min time point ( * P < 0 . 05 , n = 6 dishes , at least 9 neurons per dish , t - test ) .

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

Figure 6A. Kinetics of EYFP-Parkin translocation in response to A&O is different in control and Miro1-EF-expressing PC6 cells. The inset shows how different compartments of the same dish were transfected with EYFP-Parkin and control plasmid or Miro1-EF and followed over 3 hrs using a confocal microscope after addition of A&O (both 10 µM). The graph shows the average spatial heterogeneity of EYFP-Parkin fluorescence, reflecting the ratio of mitochondrial-to-cytosolic Parkin over time (n = 40-43 cells, difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)The percentage of cells where EYFP-Parkin translocates in response to A&O (both 10 µM, B is decreased in Miro1-EF-expressing PC6 cells. **P < 0.01, ****P < 0.0001 compared with the indicated group (n = 3-6 dishes, 20 fields per dish, one way ANOVA forThe percentage of cells where EYFP-Parkin translocates in response t PINK1 overexpression (C) is decreased in Miro1-EF-expressing PC6 cells. **P < 0.01, ****P < 0.0001 compared with the indicated group (n = 3-6 dishes, 20 fields per dis Welch"s ANOVA fD. Time course showing laser-induced EYFP-Parkin accumulation in the irradiated area of the neuronal soma of primary rat cortical neurons. Cells were transfected with CFP-mito and EYFP-Parkin and visualized using a confocal microscope. Mitochondria were irradiated in a 0.9 µm x 0.9 µm-frame using a 405-nm laser line (25 iterations using 100% laser power, pixel time 164 µs)E, F Miro1-EF delays and reduces irradiation-induced EYFP-Parkin translocation. (E) The time course of translocation strength in control and Miro1-EF-expressing neurons. Difference between the curves P = 0.0016, two-way ANOVA. (F) Comparison at the 60-min time point (*P < 0.05, n = 6 dishes, at least 9 neurons per dish, t-test).


Figure 7A . EYFP - Parkin translocation requires Ca2 + . PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2 + or in Ca2 + free Krebs containing 5 μM BAPTA - AM , were treated with A & O ( both 10 μM ) . Images ( left panels ) demonstrate that EYFP - Parkin translocation was significantly weaker at zero Ca2 + at 195 min after A & O treatment ( compared with 15 min ; note the spatial heterogeneity values shown in photos ) . Spatial heterogeneity analysis ( right panel ) demonstrated that Parkin translocation was significantly decelerated in zero Ca2 + conditions ( n = 25 - 27 cells ; difference between the curves P < 0 . 0001 , two - way ANOVA ; lines show mean , dashed area shows SEM ) B . EYFP - Parkin translocation accelerates in the presence of the L - type Ca2 + channel agonist Bay K 8644 ( 5 μM ; n = 19 - 21 cells ; difference between the curves p < 0 . 0001 , two - way ANOVA ; lines show mean , dashed area shows SEM ) Treatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into the cytosol , visualised by increased cytosolic GCaMP7b fluorescenceTreatment of neurons for 10 min with 100 μM glutamate / 10 μM glycine led to excessive Ca2 + entry into the cytosol Glutamate - induced Ca2 + entry was not affected by Miro1 WT or Miro1 EF overexpression ; n = 31 - 48 neurons per group ; ns , Kruskal - Wallis test ) E , F Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) led to the appearance of a few Parkin - positive strong puncta co - localising with mitochondrial marker ( white arrow ) , imaged 2 . 5 h after treatment ( E ) . Further analysis demonstrated that the expression of Miro1 - EF mutant decreased and Miro1 WT increased the number of neurons with EYFP - Parkin - positive mitochondria following glutamate treatment ( F ; n = 6 - 10 ; * * P < 0 . 01 and * * * * P < 0 . 0001 , one - way ANOVA ) G . Glutamate treatment ( 100 μM glutamate / 10 μM glycine for 10 min ) was associated with an increased number of mitochondrial co - localisation with the autophagosome marker EGFP - LC3B in neuronal soma at 5 - 6 h later , which was not observed in Miro1 - EF - expressing neurons ( n = 8 dishes ; * P < 0 . 05 , one - way ANOVA ) H . Glutamate treatment ( 30 μM glutamate and 1 μM glycine for 10 min ) was also associated with a significant loss of mitochondrial density in axons at 6 h later , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 60 - 90 axons from 6 - 9 dishes ; * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 , Kruskal - Wallis test ) I . Glutamate treatment ( 100 μM glutamate and 10 μM glycine for 10 min ) led to a decline , at 6 - 8 h , in cytosolic ATP measured using ATP / ADP sensor Perceval HR , which was reversed by Miro1 - EF but not by Miro1 WT ( n = 59 - 60 neurons from 6 dishes ; * * P < 0 . 01 , Welch " s AJ . Glutamate ( 100 μM glutamate and 10 μM glycine for 10 min ) induced excitotoxicity ( 24 h after treatment ) that was partially reversed by Miro1 - EF but not by Miro1 WT . Data is presented as a percentage of control neuron count ( n = 10 dishes ; * * * P < 0 . 001 , repeated measures ANOVA ) .

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

Figure 7A. EYFP-Parkin translocation requires Ca2+. PC6 cells plated in separated compartments of the same dish maintained during the experiment in Krebs containing 1 mM Ca2+ or in Ca2+ free Krebs containing 5 μM BAPTA-AM, were treated with A&O (both 10 μM). Images (left panels) demonstrate that EYFP-Parkin translocation was significantly weaker at zero Ca2+ at 195 min after A&O treatment (compared with 15 min; note the spatial heterogeneity values shown in photos). Spatial heterogeneity analysis (right panel) demonstrated that Parkin translocation was significantly decelerated in zero Ca2+ conditions (n = 25-27 cells; difference between the curves P < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)B. EYFP-Parkin translocation accelerates in the presence of the L-type Ca2+ channel agonist Bay K 8644 (5 μM; n = 19-21 cells; difference between the curves p < 0.0001, two-way ANOVA; lines show mean, dashed area shows SEM)Treatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol, visualised by increased cytosolic GCaMP7b fluorescenceTreatment of neurons for 10 min with 100 μM glutamate/10 μM glycine led to excessive Ca2+ entry into the cytosol Glutamate-induced Ca2+ entry was not affected by Miro1 WT or Miro1 EF overexpression; n = 31-48 neurons per group; ns, Kruskal-Wallis test)E, F Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) led to the appearance of a few Parkin-positive strong puncta co-localising with mitochondrial marker (white arrow), imaged 2.5 h after treatment (E). Further analysis demonstrated that the expression of Miro1-EF mutant decreased and Miro1 WT increased the number of neurons with EYFP-Parkin-positive mitochondria following glutamate treatment (F; n = 6-10; **P < 0.01 and ****P < 0.0001, one-way ANOVA)G. Glutamate treatment (100 μM glutamate/10 μM glycine for 10 min) was associated with an increased number of mitochondrial co-localisation with the autophagosome marker EGFP-LC3B in neuronal soma at 5-6 h later, which was not observed in Miro1-EF-expressing neurons (n = 8 dishes; *P < 0.05, one-way ANOVA)H. Glutamate treatment (30 μM glutamate and 1 μM glycine for 10 min) was also associated with a significant loss of mitochondrial density in axons at 6 h later, which was reversed by Miro1-EF but not by Miro1 WT (n = 60-90 axons from 6-9 dishes; **P < 0.01, ***P < 0.001 and ****P < 0.0001, Kruskal-Wallis test)I. Glutamate treatment (100 μM glutamate and 10 μM glycine for 10 min) led to a decline, at 6-8 h, in cytosolic ATP measured using ATP/ADP sensor Perceval HR, which was reversed by Miro1-EF but not by Miro1 WT (n = 59-60 neurons from 6 dishes; **P < 0.01, Welch"s AJ. Glutamate (100 μM glutamate and 10 μM glycine for 10 min) induced excitotoxicity (24 h after treatment) that was partially reversed by Miro1-EF but not by Miro1 WT. Data is presented as a percentage of control neuron count (n = 10 dishes; ***P < 0.001, repeated measures ANOVA).


Figure 1HSC ( 100 cells ) were untreated or cultured under pH 7 . 4 and pH 6 . 9 for 40 h and transplanted with competitor cells . n = at least 18 mice per group out of at least 7 experiments ( biological repeats ) Representative FACS dot plots and quantitative and statistical analysis of LT - HSC , ST - HSC , and LMPP distribution among donor derived LSKs in primary ( I ) recipients . * p < 0 . 05 using two - tailed unpaired Student ' s t - test . HSC ( 100 cells ) were untreated or cultured under pH 7 . 4 and pH 6 . 9 for 40 h and transplanted with competitor cells . n = at least 18 mice per group out of at least 7 experiments ( biological repeats ) Representative FACS dot plots and quantitative and statistical analysis of LT - HSC , ST - HSC , and LMPP distribution among donor derived LSKs secondary ( J ) recipients . * p < 0 . 05 using two - tailed unpaired Student ' s t - test .

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Figure 1HSC (100 cells) were untreated or cultured under pH 7.4 and pH 6.9 for 40 h and transplanted with competitor cells. n = at least 18 mice per group out of at least 7 experiments (biological repeats) Representative FACS dot plots and quantitative and statistical analysis of LT-HSC, ST-HSC, and LMPP distribution among donor derived LSKs in primary (I) recipients. *p < 0.05 using two-tailed unpaired Student's t-test.HSC (100 cells) were untreated or cultured under pH 7.4 and pH 6.9 for 40 h and transplanted with competitor cells. n = at least 18 mice per group out of at least 7 experiments (biological repeats) Representative FACS dot plots and quantitative and statistical analysis of LT-HSC, ST-HSC, and LMPP distribution among donor derived LSKs secondary (J) recipients. *p < 0.05 using two-tailed unpaired Student's t-test.


Figure 2 ( C ) Representative flow cytometric analyses of EdU incorporation and DNA content in HSCs after culture under pH 7 . 4 and pH 6 . 9 conditions for 40 h . Percentage of cells in G1 , S and G2 / M phase of cell cycle under pH 7 . 4 or pH 6 . 9 conditions . ( n = 4 biological replicates , * * * p < 0 . 001 ; using two - tailed unpaired Student ' s t - test ) ( E ) Relative levels of expression of the Hif1α transcriptional targets Pdk - 1 and Vegfa ( n = 4 biological replicates , * p < 0 . 05 ; using two - tailed unpaired Student ' s t - test ) . ( F ) Representative image analyses of autophagy markers LC3 and LAMP2 with DAPI in HSCs after culture under pH 7 . 4 or pH 6 . 9 conditions ( Scale bar = 5μm ) ( H ) Representative ultra - structural transmission electron microscopy images of untreated and cultured HSCs under pH 7 . 4 or pH 6 . 9 conditions . Data represent n = 20 cells from 2 biological replicates . Arrows label mitochondria , while arrowheads label vacuolar structures ( Scale bar = 1μm ) .

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Figure 2(C) Representative flow cytometric analyses of EdU incorporation and DNA content in HSCs after culture under pH 7.4 and pH 6.9 conditions for 40 h. Percentage of cells in G1, S and G2/M phase of cell cycle under pH 7.4 or pH 6.9 conditions. (n=4 biological replicates, ***p < 0.001; using two-tailed unpaired Student's t-test)(E) Relative levels of expression of the Hif1α transcriptional targets Pdk-1 and Vegfa (n=4 biological replicates, *p < 0.05; using two-tailed unpaired Student's t-test).(F) Representative image analyses of autophagy markers LC3 and LAMP2 with DAPI in HSCs after culture under pH 7.4 or pH 6.9 conditions (Scale bar = 5μm)(H) Representative ultra-structural transmission electron microscopy images of untreated and cultured HSCs under pH 7.4 or pH 6.9 conditions. Data represent n=20 cells from 2 biological replicates. Arrows label mitochondria, while arrowheads label vacuolar structures (Scale bar = 1μm).


Figure 3 ( B ) Hierarchical clustering and heatmap representing global visualization of all the DEGs in HSCs present at pH 6 . 9 or pH 7 . 4 for 40 h ( n = 6 biological replicates ) . Results were visualized using Treeview .

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Figure 3(B) Hierarchical clustering and heatmap representing global visualization of all the DEGs in HSCs present at pH 6.9 or pH 7.4 for 40 h (n=6 biological replicates). Results were visualized using Treeview.


Figure 4 ( C ) Relative levels of spermidine and spermine in pH 6 . 9 and pH 7 . 4 conditions with respect to untreated samples . n = 3 biological replicates . * p < 0 . 05 and exact p values are mentioned between groups . ( E ) Effect of DFMO treatment on pHi of HSCs . , n = 5 biological replicates . * * * p < 0 . 001 using two - tailed unpaired Student ' s t - test . ( G ) Pictures of HSCs after culture at pH 7 . 4 or pH 6 . 9 and in presence or absence of DFMO and SPN for 40 h . Data are representative of 6 biological replicates . Scale bar = 100μm ( H ) Cell volume of HSCs cultured at pH 7 . 4 in the presence of DFMO or in combination with SPN . Data are from > 80 cells per group , representative of 3 biological replicates . * * * p < 0 . 001 using two - tailed unpaired Student ' s t - test . ( I , J ) Reconstitution level of donor derived - Ly5 . 2 + cells , - lineage distribution in peripheral blood and BM in primary ( I ) and secondary ( J ) recipients from 100 HSCs cultured at pH 7 . 4 in presence of DFMO or in combination of SPN . Data in figure I , J are from n = 9 - 12 recipient mice from 2 independent biological transplantation experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 using two - tailed unpaired Student ' s t - test . ( K ) Representative images of autophagy markers LAMP2 with DAPI in untreated HSCs or after culture under pH 6 . 9 , pH 7 . 4 or pH 7 . 4 in presence of DFMO ( Scale bar = 5μm ) . Bar graphs represent quantification of LAMP2 levels in cells . Data are from > 50 cell representing 3 biological replicates . * * * p < 0 . 001 , * * * * p < 0 . 0001 using two - tailed unpaired Student ' s t - test . ( L ) Reconstitution level of donor derived Ly5 . 2 + cells or human derived cells in peripheral blood in primary recipients from either untreated 100 murine or human HSCs or after culture in serum free medium STEMSpan under pH 6 . 9 , pH 7 . 4 or pH 7 . 4 in presence of DFMO . n = 7 - 9 mice from 2 biological transplantation experiments for murine HSC data . For human HSPC transplants , n = 6 recipient per condition from 3 biological transplantation experiments . * p < 0 . 05 , or exact p value mentioned between groups . Statistic significance was calculated using two - tailed unpaired Student ' s t - test .

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

Figure 4(C) Relative levels of spermidine and spermine in pH 6.9 and pH 7.4 conditions with respect to untreated samples. n=3 biological replicates. *p < 0.05 and exact p values are mentioned between groups.(E) Effect of DFMO treatment on pHi of HSCs., n= 5 biological replicates. ***p < 0.001 using two-tailed unpaired Student's t-test.(G) Pictures of HSCs after culture at pH 7.4 or pH 6.9 and in presence or absence of DFMO and SPN for 40 h. Data are representative of 6 biological replicates. Scale bar = 100μm (H) Cell volume of HSCs cultured at pH 7.4 in the presence of DFMO or in combination with SPN. Data are from >80 cells per group, representative of 3 biological replicates. ***p < 0.001 using two-tailed unpaired Student's t-test.(I,J) Reconstitution level of donor derived -Ly5.2+ cells, -lineage distribution in peripheral blood and BM in primary (I) and secondary (J) recipients from 100 HSCs cultured at pH 7.4 in presence of DFMO or in combination of SPN. Data in figure I,J are from n= 9-12 recipient mice from 2 independent biological transplantation experiments. *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed unpaired Student's t-test.(K) Representative images of autophagy markers LAMP2 with DAPI in untreated HSCs or after culture under pH 6.9, pH 7.4 or pH 7.4 in presence of DFMO (Scale bar = 5μm). Bar graphs represent quantification of LAMP2 levels in cells. Data are from > 50 cell representing 3 biological replicates. ***p < 0.001, ****p < 0.0001 using two-tailed unpaired Student's t-test.(L) Reconstitution level of donor derived Ly5.2+ cells or human derived cells in peripheral blood in primary recipients from either untreated 100 murine or human HSCs or after culture in serum free medium STEMSpan under pH 6.9, pH 7.4 or pH 7.4 in presence of DFMO. n= 7-9 mice from 2 biological transplantation experiments for murine HSC data. For human HSPC transplants, n= 6 recipient per condition from 3 biological transplantation experiments. *p < 0.05, or exact p value mentioned between groups. Statistic significance was calculated using two-tailed unpaired Student's t-test.


Figure 1 ( C ) Period length decreases with continual differentiation of PSCs into HIOs . Maturation of HIOs into kcHIEs leads to 24 - hour rhythms with greater reproducibility . ( D ) The amplitude of oscillations are significantly higher in kcHIEs / bHIEs compared to HIOs . Bmal1 - luc traces in B were generated by plotting the standard deviation of n ≥ 3 biological replicates .

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Figure 1(C) Period length decreases with continual differentiation of PSCs into HIOs. Maturation of HIOs into kcHIEs leads to 24-hour rhythms with greater reproducibility. (D) The amplitude of oscillations are significantly higher in kcHIEs/bHIEs compared to HIOs. Bmal1-luc traces in B were generated by plotting the standard deviation of n≥3 biological replicates.


Figure 2 ( A - C ) mRNA expression of Bmal1 ( black ) , Rev - erbα ( grey ) and Per2 ( red ) in HIOs ( A ) , kcHIEs ( B ) and bHIEs ( C ) .

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Figure 2(A-C) mRNA expression of Bmal1 (black), Rev-erbα (grey) and Per2 (red) in HIOs (A), kcHIEs (B) and bHIEs (C).


Figure 3 ( A ) Representative images of necrotic cell death ( SYTOX orange - red fluorescence ) at 2 - , 24 - and 48 - hours post exposure to 10ng / mL TcdB in control Bmal1f / f - EsrCRE enteroids . Scale bar = 250μm . ( B ) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f / f - EsrCRE enteroids without tamoxifen treatment . ( C ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 10ng / mL TcdB in tamoxifen - treated circadian arrhythmic Bmal1f / f - EsrCRE enteroids . Scale bar = 250μm . ( D ) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen - treated circadian arrhythmic Bmal1f / f - EsrCRE enteroids .

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Figure 3(A) Representative images of necrotic cell death (SYTOX orange - red fluorescence) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in control Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in control Bmal1f/f-EsrCRE enteroids without tamoxifen treatment. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 10ng/mL TcdB in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids. Scale bar = 250μm. (D) Quantitative analysis of fluorescent intensity from SYTOX orange in tamoxifen-treated circadian arrhythmic Bmal1f/f-EsrCRE enteroids.


Figure 4 ( A ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 1μg / mL TcdB in HIOs . Scale bar = 250μm . ( B ) Quantification of necrotic cell death in HIOs . ( C ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 5μg / mL TcdB in kcHIEs . Scale bar = 250μm . ( D ) Quantification of necrotic cell death in kcHIEs . ( E ) Representative images of necrotic cell death ( SYTOX orange ) at 2 - , 24 - and 48 - hours post exposure to 0 . 5μg / mL TcdB in bHIEs . Scale bar = 250μm . ( F ) Quantification of necrotic cell death in bHIEs .

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Figure 4(A) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.1μg/mL TcdB in HIOs. Scale bar = 250μm. (B) Quantification of necrotic cell death in HIOs. (C) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in kcHIEs. Scale bar = 250μm. (D) Quantification of necrotic cell death in kcHIEs. (E) Representative images of necrotic cell death (SYTOX orange) at 2-, 24- and 48-hours post exposure to 0.5μg/mL TcdB in bHIEs. Scale bar = 250μm. (F) Quantification of necrotic cell death in bHIEs.


Figure 5 ( A ) Heatmap of circadian transcripts over 48 hours in mouse and human enteroids . ( B ) Spearman ' s rho for circadian clock and clock - associated genes .

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Figure 5(A) Heatmap of circadian transcripts over 48 hours in mouse and human enteroids.(B) Spearman's rho for circadian clock and clock-associated genes.


Figure 6 ( A ) Validation of anti - phasic Rac1 expression in mouse ( mRac1 , red ) and human enteroids ( hRac1 , black ) by qRT - PCR . ( B ) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1 - KD bHIEs . ( C - D ) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f / f ; CAGGCre - ERTM without tamoxifen ( C ) or Rac1 f / f ; CAGGCre - ERTM with tamoxifen - treated ( D ) enteroids .

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Figure 6(A) Validation of anti-phasic Rac1 expression in mouse (mRac1, red) and human enteroids (hRac1, black) by qRT-PCR. (B) Quantitative analysis of fluorescent intensity from SYTOX orange in Rac1-KD bHIEs. (C-D) Quantitative analysis of fluorescent intensity from SYTOX orange in control Rac1 f/f;CAGGCre-ERTM without tamoxifen (C) or Rac1 f/f;CAGGCre-ERTM with tamoxifen-treated (D) enteroids.


Figure 1B . Top : representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 as indexes of mTOR activity in PFC of adult WT mice are illustrated . Bottom : data represent the ratios of immunoreactive signals of the anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to the immunoreactive signal of the anti - β - actin antibody and are expressed in % of values in vehicle - injected WT mice . They are the means ± S . E . M . of results obtained in five mice per group . * p < 0 . 05 ; * * p < 0 . 01 , one - way ANOVA followed by Newman - Keuls test . C . Top : schemas illustrating the behavioral tasks examined in WT mice . Bottom : the plots represent the discrimination index for the novel object recognition task ( Vehicle N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) , the sociability index ( Vehicle : N = 11 , THC : N = 11 , THC + SB : N = 11 , THC + Rapa : N = 12 ) and the discrimination index for the social discrimination task ( N = 8 for each group ) , measured in each condition . * p < 0 . 05 , * * p < 0 . 01 , one - way ANOVA followed by Bonferroni test ( error bars correspond to the mean ± SEM , the dotted line to a discrimination index ( exploration of novel object - exploration of familiar object / total object exploration ) equal to zero . D . Top : representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 in PFC of adult 5 - HT6 - / - mice are illustrated . Bottom : data represent the ratios of immunoreactive signals of the anti phospho - mTOR ( S2448 ) or anti phospho - p70S6K ( T389 ) antibodies to the immunoreactive signal of the anti - β - actin antibody and are expressed in % of values in vehicle - injected 5 - HT6 - / - mice . They are the means ± S . E . M . of results obtained in four mice per group . n . s . p > 0 . 05 , one - way ANOVA followed by Newman - Keuls test . E . Top : schemas illustrating the behavioral tasks in 5 - HT6 - / - mice . Bottom : the plots represent the discrimination index for the novel object recognition test ( discrimination index : 0 . 30 ± 0 . 05 , N = 10 and 0 . 31 ± 0 . 03 , N = 11 , for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , the 3 - chamber social preference test ( sociability index : 0 . 49 ± 0 . 04 , N = 10 and 0 . 48 ± 0 . 07 , N = 11 for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) and the social discrimination test ( discrimination index : 0 . 15 ± 0 . 06 , N = 8 and 0 . 21 ± 0 . 06 , N = 8 for THC + Vehicle and Vehicle + Vehicle conditions , respectively , p > 0 . 05 ) , measured in each condition . One - way ANOVA followed by Bonferroni test ( error bars correspond to the mean ± SEM , the dotted line corresponds to a discrimination index equal to zero ) .

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

Figure 1B. Top: representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 as indexes of mTOR activity in PFC of adult WT mice are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected WT mice. They are the means ± S.E.M. of results obtained in five mice per group. * p < 0.05; ** p < 0.01, one-way ANOVA followed by Newman-Keuls test.C. Top: schemas illustrating the behavioral tasks examined in WT mice. Bottom: the plots represent the discrimination index for the novel object recognition task (Vehicle N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12), the sociability index (Vehicle: N=11, THC: N=11, THC+SB: N=11, THC+Rapa: N=12) and the discrimination index for the social discrimination task (N=8 for each group), measured in each condition. * p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line to a discrimination index (exploration of novel object - exploration of familiar object/total object exploration) equal to zero.D. Top: representative Western blots assessing mTOR phosphorylation at S2448 and p70S6K phosphorylation at T389 in PFC of adult 5-HT6-/- mice are illustrated. Bottom: data represent the ratios of immunoreactive signals of the anti phospho-mTOR (S2448) or anti phospho-p70S6K (T389) antibodies to the immunoreactive signal of the anti-β-actin antibody and are expressed in % of values in vehicle-injected 5-HT6-/- mice. They are the means ± S.E.M. of results obtained in four mice per group. n.s. p > 0.05, one-way ANOVA followed by Newman-Keuls test.E. Top: schemas illustrating the behavioral tasks in 5-HT6-/- mice. Bottom: the plots represent the discrimination index for the novel object recognition test (discrimination index: 0.30 ± 0.05, N=10 and 0.31 ± 0.03, N=11, for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), the 3-chamber social preference test (sociability index: 0.49 ± 0.04, N=10 and 0.48 ± 0.07, N=11 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05) and the social discrimination test (discrimination index: 0.15 ± 0.06, N=8 and 0.21 ± 0.06, N=8 for THC+Vehicle and Vehicle+Vehicle conditions, respectively, p > 0.05), measured in each condition. One-way ANOVA followed by Bonferroni test (error bars correspond to the mean ± SEM, the dotted line corresponds to a discrimination index equal to zero).