( A ) Digitonin - solubilized mitochondria from doxycycline - treated 143B , COX1 ∆ COX2 ∆ cybrids were analyzed by BN - PAGE followed by CI - IGA assays ( upper panels ) or immunoblotting ( medium lower panels ) with antibodies against CI subunits NDUFA9 NDUFS1 . identities of MRC complexes SCs are as in Figure 1 .

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(A) Digitonin-solubilized mitochondria from doxycycline-treated 143B cells, COX1∆ and COX2∆ cybrids were analyzed by BN-PAGE followed by CI-IGA assays (upper panels) or immunoblotting (medium and lower panels) with antibodies against CI subunits NDUFA9 and NDUFS1. The identities of MRC complexes and SCs are as in Figure 1.


( B ) Mean incorporation rates of NDUFA9 NDUFS1 into SC I + III2 + IV1 from 143B into SCs I + III2 from CIV - KO cybrids . Signals from at least three independent experiments were quantified normalized by CII subunit SDHA . Time - point values are expressed as percentages of untreated ( SS ) indicated as mean ± SD .

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(B) Mean incorporation rates of NDUFA9 and NDUFS1 into SC I+III2+IV1 from 143B cells and into SCs I+III2 from the CIV-KO cybrids. Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels. Time-point values are expressed as percentages of untreated cells (SS) and indicated as mean±SD.


( C ) Samples were subsequently analyzed by 2D - BN / SDS - PAGE immunoblotting with indicated antibodies . identities of MRC complexes SCs are as in Figure 1 .

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(C) Samples were subsequently analyzed by 2D-BN/SDS-PAGE and immunoblotting with the indicated antibodies. The identities of MRC complexes and SCs are as in Figure 1.


( D ) Mean incorporation rates of CIII subunits CORE2 RISP into SC I + III2 + IV1 from control into SCs I + III2 from CIV - KO cybrids . Signals from at least three independent experiments were quantified normalized by CII subunit SDHA . Time - point values are expressed as percentages of untreated ( SS ) indicated as mean ± SD .

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(D) Mean incorporation rates of CIII subunits CORE2 and RISP into SC I+III2+IV1 from control cells and into SCs I+III2 from CIV-KO cybrids. Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels. Time-point values are expressed as percentages of untreated cells (SS) and indicated as mean±SD.


( A ) Digitonized mitochondria from doxycycline - treated 143B COX2 ∆ cybrids were analyzed by 2D - BN / SDS - PAGE western blot with indicated antibodies .

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(A) Digitonized mitochondria from doxycycline-treated 143B cells and COX2∆ cybrids were analyzed by 2D-BN/SDS-PAGE and western blot with the indicated antibodies.


( B ) Mean incorporation rates of COX subunits into SC I + III2plus from COX2 ∆ cybrids . Signals from three independent experiments were quantified normalized by CII . Time - point values are expressed as percentages of untreated ( SS ) indicated as mean ± SD . Control ( 143B ) values for subunits COX4 , COX5A COX6C were obtained from ( Moreno - Lastres et al . , 2012 ) .

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(B) Mean incorporation rates of COX subunits into SC I+III2plus from COX2∆ cybrids. Signals from three independent experiments were quantified and normalized by CII. Time-point values are expressed as percentages of the untreated cells (SS) and indicated as mean ± SD. Control (143B) values for subunits COX4, COX5A and COX6C were obtained from (Moreno-Lastres et al., 2012).


C - D SDS - PAGE ( C ) BN - PAGE ( D ) analyses from 143B COX2 ∆ cybrids transiently transfected either with a COX5B - Myc - DDK construct or with empty vector , followed by immunoblotting with antibodies against COX5B CII subunit SDHA ( loading control ) . identities of MRC complexes SCs are as in Figure 1 . red arrow points to accumulation of over expressed COX5B bound to SC I + III2 in control .

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C-D SDS-PAGE (C) and BN-PAGE (D) analyses from 143B cells and COX2∆ cybrids transiently transfected either with a COX5B-Myc-DDK construct or with the empty vector, followed by immunoblotting with antibodies against COX5B and CII subunit SDHA (loading control). The identities of MRC complexes and SCs are as in Figure 1. The red arrow points to the accumulation of over expressed COX5B bound to SC I+III2 in control cells.


Mitochondrial translation products were - labelled in control 143B ( left panel ) , COX1 ∆ ( middle panel ) COX2 ∆ ( right panel ) cybrids with [ 35S ] - methionine for 30 minutes in presence of anisomycin to inhibit translation . For chase , were washed incubated with fresh culture medium for indicated time points ( T0 - T24 hours ) . Radiolabeled mitochondrial ( indicated on right ) were separated by 2D - BN / SDS - PAGE visualized by autoradiography . A enlarged section of SCs region at T24 ( delimited by a square ) shows co - migration of COX1 with SC I + III2plus ( black arrow ) in COX2 ∆ cybrids relative to position of canonical SC I + III2 ( gray arrow ) . identities of MRC complexes SCs ( bottom ) are as in Figure 1 . Molecular markers are indicated on left .

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Mitochondrial translation products were pulse-labelled in control 143B cells (left panel), COX1∆ (middle panel) and COX2∆ (right panel) cybrids with [35S]-methionine for 30 minutes in the presence of anisomycin to inhibit cytosolic translation. For chase, cells were washed and incubated with fresh culture medium for the indicated time points (T0-T24 hours). Radiolabeled mitochondrial proteins (indicated on the right) were separated by 2D-BN/SDS-PAGE and visualized by autoradiography. A enlarged section of the SCs region at T24 (delimited by a square) shows the co-migration of COX1 with SC I+III2plus (black arrow) in the COX2∆ cybrids relative to the position of canonical SC I+III2 (gray arrow). The identities of MRC complexes and SCs (bottom) are as in Figure 1. Molecular weight markers are indicated on the left.


( A ) Respiratory chain enzyme activities of control ( blue ) , COX1 ∆ ( red ) COX2 ∆ ( green ) cybrids . Enzyme activities were measured in three experimental replicates . Data are expressed as percentages of control , indicated as means ± SD . Mann - Whitney U was applied for statistical analyses . Variations between controls mutants : α , p < 0 . 05 ; γ , p < 0 . 001 . Specific variations between COX1 ∆ COX2 ∆ mutants : ∗ p < 0 . 05 .

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(A) Respiratory chain enzyme activities of control (blue), COX1∆ (red) and COX2∆ (green) cybrids. Enzyme activities were measured in three experimental replicates. Data are expressed as the percentages of control cells, and indicated as means ± SD. Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05.


Colorectal outcomes in WT ( n = 7 ) , IL - 33KO ( n = 4 ) ST2KO ( n = 3 ) mice at completion of AOM / DSS carcinogenesis protocol . ( A ) volumes at endpoint ( each dot represents a , mice with no are marked as zero ) , Skin outcomes in WT ( n = 7 ) , IL - 33KO ( n = 6 ) ST2KO ( n = 7 ) mice at completion of DMBA / DNFB carcinogenesis protocol . ( D ) volumes at endpoint ( each dot represents a , mice with no are marked as zero ) ,

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Colorectal tumor outcomes in WT (n=7), IL-33KO (n=4) and ST2KO (n=3) mice at the completion of the AOM/DSS carcinogenesis protocol. (A) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero), Skin tumor outcomes in WT (n=7), IL-33KO (n=6) and ST2KO (n=7) mice at the completion of the DMBA/DNFB carcinogenesis protocol. (D) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero),


H ) of IL - 33 + epidermal dermal in DNFB - versus acetone - treated WT skin . Dots represent from three randomly selected high power field ( HPF ) per sample ( n = 5 per group ) . J ) of IL - 33 + epithelial stromal of colon treated with AOM / DSS versus no treatment . Dots represent from three randomly selected HPF per sample ( n = 4 per group ) . Data information : Graphs show mean + SD , unpaired t - .

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H) Quantification of nuclear IL-33+ epidermal and dermal cells in the DNFB- versus acetone-treated WT skin. Dots represent cell counts from three randomly selected high power field (HPF) images per sample (n=5 per group). J) Quantification of nuclear IL-33+ epithelial and stromal cells of colon treated with AOM/DSS versus no treatment. Dots represent cell counts from three randomly selected HPF images per sample (n=4 per group). Data information: Graphs show mean + SD, unpaired t-test.


( H ) ChIP - qPCR for Smad6 in presence of IL - 33 full or cytokine domain using an anti - RUNX2 antibody . After Pam212 were transfected with IL - 33 full or cytokine domain for 24 hours , lysates were subjected to - immunoprecipitation with anti - RUNX2 antibody eluted RUNX2 - bound was used for qPCR with primers against Smad6 promoter region . Anti - IgG ChIP - qPCR results are shown as negative controls ( n = 4 per group ) .

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(H) ChIP-qPCR assay for Smad6 in the presence of IL-33 full length or cytokine domain using an anti-RUNX2 antibody. After Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours, cell lysates were subjected to chromatin-immunoprecipitation with anti-RUNX2 antibody and eluted RUNX2-bound chromatin was used for qPCR with primers against Smad6 promoter region. Anti-IgG ChIP-qPCR results are shown as negative controls (n=4 per group).


( D ) Immunoblot of p - SMAD2 / 3 SMAD2 / 3 in response to TGF - β poly ( I : C ) treatment . Pam212 were treated with poly ( I : C ) or PBS followed by incubation with without TGF - β ( 5nM ) . Data represent three independent experiments with similar results . ( E ) Immunoblot of p - SMAD2 / 3 SMAD2 / 3 in response to TGF - β + poly ( I : C ) after knocking down of IL - 33 . Pam212 were transfected with siIl33 knockdown construct or siRNA control ( siCon ) for 30 hours followed by incubation with poly ( I : C ) TGF - β . Data represent three independent experiments with similar results . ( F ) Immunoblot of p - SMAD2 / 3 SMAD2 / 3 upon expression of IL - 33 full or cytokine domain . Pam212 were transfected with IL - 33 full or cytokine domain for 24 hours followed by incubation with without TGF - β . Data represent three independent experiments with similar results . Data information : GAPDH is used as control housekeeping in D - F ) . Graphs show mean + SD , NS : not significant , unpaired t - .

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(D) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β and poly (I:C) treatment. Pam212 cells were treated with poly (I:C) or PBS followed by incubation with and without TGF-β (5nM). Data represent three independent experiments with similar results. (E) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β + poly (I:C) after knocking down of IL-33. Pam212 cells were transfected with siIl33 knockdown construct or siRNA control (siCon) for 30 hours followed by incubation with poly (I:C) and TGF-β. Data represent three independent experiments with similar results. (F) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins upon the expression of IL-33 full length or cytokine domain. Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours followed by incubation with and without TGF-β. Data represent three independent experiments with similar results. Data information: GAPDH is used as the control housekeeping protein in D-F). Graphs show mean + SD, NS: not significant, unpaired t-test.


( G ) Impact of IL - 33 knockdown on proliferation in response to TGF - β . Pam212 were treated with siIl33 or siCon followed by TGF - β treatment ( n = 7 in each group ) . ( H ) Impact of IL - 33 knockdown SB431542 treatment on proliferation . Pam212 were treated with siIl33 or siCon in combination with SB431542 or DMSO ( carrier control , n = 7 in each group ) .

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(G) Impact of IL-33 knockdown on cell proliferation in response to TGF-β. Pam212 cells were treated with siIl33 or siCon followed by TGF-β treatment (n=7 in each group). (H) Impact of IL-33 knockdown and SB431542 treatment on cell proliferation. Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO (carrier control, n=7 in each group).


( I ) SMAD signaling in WT K14 - IL33tg , ST2KO skin treated with acetone ( control ) at completion of DMBA / TPA carcinogenesis protocol . Tissue lysates prepared from whole back skin of were subjected to immunoblot with p - SMAD2 / 3 , p - SMAD1 / 5 , SMAD6 , SMAD2 / 3 SMAD1 antibodies . GAPDH is used as control housekeeping ( n = 5 in each group ) .

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(I) SMAD signaling proteins levels in WT and K14-IL33tg,ST2KO skin treated with acetone (control) at the completion of the DMBA/TPA carcinogenesis protocol. Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 antibodies. GAPDH is used as the control housekeeping protein (n=5 in each group).


( J ) Representative of IL - 33 p - SMAD2 / 3 immunostaining on adjacent sections of human squamous carcinoma ( SCC ) normal skin ( scale bar : 100 μm ) . Data information : Graphs show mean + SD , NS : not significant , unpaired t - .

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(J) Representative images of IL-33 and p-SMAD2/3 immunostaining on the adjacent sections of human squamous cell carcinoma (SCC) and normal skin (scale bar: 100 μm). Data information: Graphs show mean + SD, NS: not significant, unpaired t-test.


( B ) Il33 Smad6 mRNA in caerulein - treated WT pancreas compared with PBS - treated controls at completion of chronic pancreatitis protocol ( n = 8 in caerulein n = 9 in PBS group for Il33 , n = 9 in caerulein n = 10 in PBS group for Smad6 , unpaired t - ) .

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(B) Il33 and Smad6 mRNA levels in caerulein-treated WT pancreas compared with PBS-treated controls at the completion of chronic pancreatitis protocol (n=8 in caerulein and n=9 in PBS group for Il33, n=9 in caerulein and n=10 in PBS group for Smad6, unpaired t-test).


( D ) Pancreatic - free survival of WT ( n = 4 ) , IL - 33KO ( n = 5 ) ST2KO ( n = 6 ) mice at completion of DMBA / caerulein carcinogenesis protocol ( log - rank ) .

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(D) Pancreatic tumor-free survival of WT (n=4), IL-33KO (n=5) and ST2KO (n=6) mice at the completion of the DMBA/caerulein carcinogenesis protocol (log-rank test).


( G , H ) of ( G ) IL - 33 + ( H ) p - SMAD2 / 3 + epithelial per HPF in matched human pancreatic tissues . Each dot represents average across three randomly selected HPF per sample ( n = 18 , paired t - ) .

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(G, H) Quantification of nuclear (G) IL-33+ and (H) p-SMAD2/3+ epithelial cells per HPF in the matched human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=18 patients, paired t-test).


A . Representative FACS plot ( left ) showing a mixed - lineage clone with a T - lymphoid ( CD25 + / CD11b - Gr1 - ) , myeloid ( CD11b + Gr1 + / CD25 - ) , biphenotypic ( CD11b + Gr1 + / CD25 + ) fraction derived from a single Myc / Bcl2 - transformed DN2cell grown on 1 : 1 OP9 : OP9 - Dll1 co - culture with lineage - promiscuous cytokines IL - 2 , IL - 3 , IL - 6 , IL - 7 , SCF , GM - CSF , Flt3 . of indicated flow - sorted fractions was analyzed by Giemsa ( right ) .

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A. Representative FACS plot (left) showing a mixed-lineage clone with a T-lymphoid (CD25+/CD11b-Gr1-), myeloid (CD11b+Gr1+/CD25-), and biphenotypic (CD11b+Gr1+/CD25+) fraction derived from a single Myc/Bcl2-transformed DN2cell grown on 1:1 OP9:OP9-Dll1 co-culture with lineage-promiscuous cytokines IL-2, IL-3, IL-6, IL-7, SCF,GM-CSF, Flt3. Morphology of the indicated flow-sorted cell fractions was analyzed by Giemsa staining (right).


B . Phenotype frequencies of clones from Myc / Bcl2 - transformed single ( grown phenotyped as mentioned in A ) . Analyzed were 31 clones from LSKs , 107 from GMPs 28 from DN2 .

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B. Phenotype frequencies of clones from Myc/Bcl2-transformed single cells (grown and phenotyped as mentioned in A). Analyzed were 31 clones from LSKs, 107 from GMPs and 28 from DN2 cells.


C . Cumulative survival of 31 ± 2 27 ± 6 days of mice that received 5104 to 5105 T - lymphoid or myeloid , respectively , that were flow - sorted from mixed Myc / Bcl2 + DN2 - derived clones . summarized data are from 3 mice injected with T - lymphoid 4 mice injected with myeloid fraction of two different clones .

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C. Cumulative survival of 31±2 and 27±6 days of mice that received 5104 to 5105 T-lymphoid cells or myeloid cells, respectively, that were flow-sorted from mixed Myc/Bcl2+ DN2-derived clones. The summarized data are from 3 mice injected with the T-lymphoid and 4 mice injected with the myeloid fraction of two different clones.


D . Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc / Bcl2 - transformed LSK or GMPcells on OP9 : OP9 - Dll1 co - cultures .

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D. Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc/Bcl2-transformed LSK or GMPcells on OP9:OP9-Dll1 co-cultures.


E . ( top panels ) Serial replating of Myc - Bcl2 + DN2 in methylcellulose generated myeloid clones with stable genomic reassembly of Dβ1 TCR locus in vitro ( gel : left two lanes ) . Upon transplantation , same clones induced myeloid leukemia that retained initial rearrangement in vivo ( gel : right two lanes ) as assessed by nested PCR . GL = germline . ( bottom panels ) Methylcellulose - replating of Myc - Bcl2 + GMPs generated myeloid clones that exclusively displayed TCRß locus in germline configuration .

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E. (top panels) Serial replating of Myc-Bcl2+DN2 cells in methylcellulose generated myeloid clones with stable genomic reassembly of the Dβ1 TCR locus in vitro (gel: left two lanes). Upon transplantation, the same clones induced myeloid leukemia that retained the initial rearrangement in vivo (gel: right two lanes) as assessed by nested PCR. GL = germline. (bottom panels) Methylcellulose-based replating of Myc-Bcl2+GMPs generated myeloid clones that exclusively displayed the TCRß locus in germline configuration.


F . Cumulative survival of mice that received 106cells of TCRβ - rearranged myeloid DN2 - derived clones leading to within 33 ± 4 days after transplantation . Two DN2 clones were transplanted into 5 recipient mice .

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F. Cumulative survival of mice that received 106cells of TCRβ-rearranged myeloid DN2-derived clones leading to death within 33±4 days after transplantation. Two DN2 clones were transplanted into 5 recipient mice.


A . Representative FACS plots of spleens from leukemic recipient mice that had received LSK , GMP or DN2 grafts immediately after retroviral Myc / Bcl2 transduction . Numbers indicate frequencies ( % ) of CD45 . 2 donor within indicated gates summarizing mean ± of at least 8 recipients per group from a total of 10 independent experiments .

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A. Representative FACS plots of spleens from leukemic recipient mice that had received LSK, GMP or DN2 cell grafts immediately after retroviral Myc/Bcl2 transduction. Numbers indicate frequencies (%) of CD45.2 donor cells within the indicated gates summarizing the mean±SEM of at least 8 recipients per group from a total of 10 independent experiments.


B . Cumulative survival of mice that received 2104 to 2105 freshly Myc / Bcl2 - transduced LSK , GMP or DN2 ; Graph shows n = 11 Myc - Bcl2 + LSK - , n = 14 DN2 - , n = 8 GMP - transplanted .

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B. Cumulative survival of mice that received 2104 to 2105 freshly Myc/Bcl2-transduced LSK, GMP or DN2 cells; Graph shows n=11 Myc-Bcl2+LSK-, n=14 DN2-, and n=8 GMP-transplanted animals.


C . Histological spleen sections of Myc - Bcl2 + LSK - , GMP - or DN2 - transplanted mice displaying myeloperoxidasestaining . Leukemic engraftment in spleens ranged from 75 . 1 % - 95 . 5 % .

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C. Histological spleen sections of Myc-Bcl2+LSK-, GMP- or DN2-transplanted mice displaying myeloperoxidasestaining. Leukemic cell engraftment in spleens ranged from 75.1%-95.5%.


D . Multiplex quantitative RT - PCR demonstrating strong expression of PU . 1 in myeloid ( M3L : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) biphenotypic T / myeloid ( BL : CD11b + Gr1 + / CD3 + CD4 + CD8 + ) fractions but not in T - lymphoid ( TL : CD11b - Gr1 - / CD3 + CD4 + CD8 + ) fraction that were flow - sorted from diseased Myc - Bcl2 + DN2 - transplanted mice . Myeloid progeny of Myc / Bcl2 + GMP - transplanted or T - lymphoid progeny of Myc / Bcl2 + DN3 - transplanted as well as healthy thymocytes ( Thy ) BM from WT mice served as controls . Results are shown as fold over actin transcripts .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Multiplex quantitative RT-PCR demonstrating strong expression of PU.1 in the myeloid (M3L: CD11b+Gr1+/CD3-CD4-CD8-) and biphenotypic T/myeloid (BL: CD11b+Gr1+/CD3+CD4+CD8+) fractions but not in the T-lymphoid (TL: CD11b-Gr1-/CD3+CD4+CD8+) fraction that were flow-sorted from diseased Myc-Bcl2+DN2-transplanted mice. Myeloid progeny of Myc/Bcl2+GMP-transplanted or T-lymphoid progeny of Myc/Bcl2+DN3-transplanted animals as well as healthy thymocytes (Thy) and BM cells from WT mice served as controls. Results are shown as fold over actin transcripts.


E . D - J rearrangement PCR of flow - sorted T - lymphoid ( TL ) , biphenotypic ( BL ) myeloid ( ML ) fractions of donor splenocytes of a diseased Myc / Bcl2 + DN2 - transplanted mouse . three fractions revealed a stable genomic reassembly of Dβ1 TCR locus ( Dβ1Jβ1 . 5 ) demonstrating clonality in DN2 leukemia . thy = thymus .

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E. D-J rearrangement PCR of flow-sorted T-lymphoid (TL), biphenotypic (BL) and myeloid (ML) fractions of donor splenocytes of a diseased Myc/Bcl2+DN2-transplanted mouse. All three fractions revealed a stable genomic reassembly of the Dβ1 TCR locus (Dβ1Jβ1.5) demonstrating clonality in DN2 leukemia. thy = thymus.


F . FACS plot of diseased intrathymic grafts ( CD45 . 2 - gated ) , demonstrating that Myc / Bcl2 + DN2 could produce multi - lineage leukemia within a T - supporting environment ; Myc / Bcl2 + GMPs remained myeloid restricted under these conditions . Numbers indicate frequencies within gates ( mean ± ) representing six independent experiments .

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F. FACS plot of diseased intrathymic grafts (CD45.2-gated), demonstrating that Myc/Bcl2+DN2 cells could produce multi-lineage leukemia within a T-cell supporting environment; Myc/Bcl2+GMPs remained myeloid restricted under these conditions. Numbers indicate cell frequencies within the gates (mean±SEM) representing six independent experiments.


G . Intrathymic transplantation of mock - infected non - leukemic DN2 produced predominantly T - lymphoid but also myeloid progeny 23 days after injection . This differentiation outcome resembles that of freshly isolated DN2 ( Bell Bhandoola , 2008 ; Richie Ehrlich et al . , 2011 ) , indicates that short - term culture necessary for retroviral transduction did not interfere with physiological lineage potential of DN2 .

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G. Intrathymic transplantation of mock-infected non-leukemic DN2 cells produced predominantly T-lymphoid but also myeloid progeny 23 days after injection. This differentiation outcome resembles that of freshly isolated DN2 cells (Bell and Bhandoola, 2008; Richie Ehrlich et al., 2011), and indicates that the short-term culture necessary for retroviral transduction did not interfere with the physiological lineage potential of DN2 cells.


A . GSEA demonstrates that Myc / Bcl2 + DN2 - derived myeloid blasts were significantly ( FDR > 0 . 05 ) enriched for expression of a pan - dendritic gene set . Gene sets were published in ( Schonheit et al . , 2013 ) .

A. GSEA demonstrates that Myc/Bcl2+DN2-derived myeloid blasts were significantly (FDR >0.05) enriched for expression of a pan-dendritic cell gene set. Gene sets were published in (Schonheit et al., 2013).


B . Heat map of top 100 differentially expressed genes ( adjusted p - value of ≤ 0 . 05 ) in each myeloid fraction ( ML : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) from GMP - , LSK - DN2 - derived leukemias .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Heat map of the top 100 differentially expressed genes (adjusted p-value of ≤ 0.05) in each myeloid fraction (ML: CD11b+Gr1+/CD3-CD4-CD8-) from GMP-, LSK- and DN2-derived leukemias.


D . GSEA demonstrates significant enrichment of a normal DN2 - specific gene set ( see methods section ) in Myc / Bcl2 + DN2 - derived myeloid blasts .

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D. GSEA demonstrates significant enrichment of a normal DN2-specific gene set (see methods section) in the Myc/Bcl2+DN2-derived myeloid blasts.


A . Western blot illustrating abundant Bcl11b Gata3protein expression in Myc / Bcl2 + DN2 - but not LSK - or GMP - derived blasts . Thymus ( Thy ) bone marrow ( BM ) extracts from healthy WT mice served as controls . Detection of α - tubulin or valosin - containing ( VCP ) ensured comparable loading .

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A. Western blot illustrating abundant Bcl11b and Gata3protein expression in Myc/Bcl2+DN2- but not LSK- or GMP-derived blasts. Thymus (Thy) and bone marrow (BM) extracts from healthy WT mice served as controls. Detection of α-tubulin or valosin-containing protein (VCP) ensured comparable loading.


B , C . Proliferation assays illustrating growth of Myc / Bcl2 + DN2 - ( B ) or GMP - derived ( C ) blasts after lentiviral transduction with indicated shRNAs . shRNA - transduced were flow - sorted on co - expression of RFP - reporter seeded on OP9 / OP9Dll1 1 : 1 layers . Growing were RFP - sorted for 3 rounds within 21 days of culture growth was calculated over initially seeded number in relation to Luc control . Shown are results of 4 independent experiments with from 4 different DN2 - 3 different GMP - leukemic . Luc = control shRNA targeting luciferase ( Suzuki et al . , 2003 ) . At least 2 different shRNAs were used against Bcl11b or Gata3 as indicated . * = p ≤ 0 . 05 , Student ' s t - .

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

B,C. Proliferation assays illustrating growth of Myc/Bcl2+ DN2- (B) or GMP-derived (C) blasts after lentiviral transduction with the indicated shRNAs. The shRNA-transduced cells were flow-sorted based on co-expression of the RFP-reporter and seeded on OP9/OP9Dll1 1:1 layers. Growing cells were RFP-sorted for 3 rounds within 21 days of culture and percent growth was calculated over the initially seeded cell number in relation to the Luc control. Shown are results of 4 independent experiments with cells from 4 different DN2- and 3 different GMP-leukemic animals. Luc= control shRNA targeting luciferase (Suzuki et al., 2003). At least 2 different shRNAs were used against Bcl11b or Gata3 as indicated. * = p≤0.05, Student's t-test.


D . Representative FACS plots showing CD45 . 2 - donor splenocytes of a secondary ( CD45 . 1 ) recipient 23 after transplantation with 4103RFP + Myc - Bcl2 + DN2 - leukemia blasts carrying indicated shRNAs . Luc : luciferase control shRNA . data are representative of 2 Gata3 , 4 Bcl11b , 2 luciferase shRNA - transplanted .

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

D. Representative FACS plots showing CD45.2-donor splenocytes of a secondary (CD45.1) recipient 23 weeks after transplantation with 4103RFP+Myc-Bcl2+DN2-leukemia blasts carrying the indicated shRNAs. Luc: luciferase control shRNA. The data are representative of 2 Gata3, 4 Bcl11b, and 2 luciferase shRNA-transplanted animals.


A . Representative FACS plots of splenocytes from diseased secondary recipients that were transplanted with 104 to 105 flow - sorted myeloid leukemia blasts ( ML : CD11b + Gr1 + / CD3 - CD4 - CD8 - ) from sick Myc / Bcl2 + LSK - , GMP - or DN2 - transplanted mice . percentages in indicated gates are shown .

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A. Representative FACS plots of splenocytes from diseased secondary recipients that were transplanted with 104 to 105 flow-sorted myeloid leukemia blasts (ML: CD11b+Gr1+/CD3-CD4-CD8-) from sick primary Myc/Bcl2+LSK-, GMP- or DN2-transplanted mice. Cell percentages in indicated gates are shown.


B . Table summarizing phenotype frequencies within spleens of secondary recipients that were retransplanted with Myc / Bcl2 + LSK - , GMP - or DN2 - derived myeloid blasts as described in A . T - lymphoid leukemia : ( TL : CD11b - Gr1 - / CD3 + CD4 + CD8 + ) , biphenotypic leukemia : BL ( CD11b + Gr1 + / CD3 + CD4 + CD8 + ) , myeloid leukemia : ML ( CD11b + Gr1 + / CD3 - CD4 - CD8 - ) . Numbers before dash represent mice holding indicated leukemia fraction ; numbers after dash indicate total mouse analyzed .

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B. Table summarizing the phenotype frequencies within the spleens of secondary recipients that were retransplanted with Myc/Bcl2+LSK-, GMP- or DN2-derived myeloid blasts as described in A. T-lymphoid leukemia: (TL: CD11b-Gr1-/CD3+CD4+CD8+), biphenotypic leukemia: BL (CD11b+Gr1+/CD3+CD4+CD8+), myeloid leukemia: ML (CD11b+Gr1+/CD3-CD4-CD8-). Numbers before the dash represent mice holding the indicated leukemia fraction; numbers after the dash indicate the total mouse count analyzed.


C . Single myeloid blasts from 4 different Myc / bcl2 + DN2 - derived leukemias were flow - sorted from transplanted mice grown on irradiated OP9 : OP9DL1 stroma . clones were analyzed at two different time points . FACS plots of two representative clones , one with strong ( top ) one with weak ( bottom ) lineage switching potential are shown .

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C. Single myeloid blasts from 4 different Myc/bcl2+ DN2-derived leukemias were flow-sorted from transplanted mice and grown on irradiated OP9:OP9DL1 stroma. The clones were analyzed at two different time points. FACS plots of two representative clones, one with strong (top) and one with weak (bottom) lineage switching potential are shown.


D . Table summarizing lineage switching outcome of 20 independent clones that were grown from single DN2 - derived myeloid blasts of 4 different leukemias . Percentages at right indicate relative amounts of with T - lymphoid ( TL ) , biphenotypic ( BL ) or myeloid ( ML ) phenotype as analyzed by FACS at end of experiment .

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D. Table summarizing the lineage switching outcome of 20 independent clones that were grown from single DN2-derived myeloid blasts of 4 different leukemias. Percentages at the right indicate the relative amounts of cells with T-lymphoid (TL), biphenotypic (BL) or myeloid (ML) phenotype as analyzed by FACS at the end of the experiment.


E . FACS plot of DN2 - derived leukemic transduced with a retrovirus carrying GFP alone or GFP together with PU . 1 as a bicistronic . transduced were grown on OP9 / OP9Dll1 1 : 1 layer for 8 - 13 days analyzed using a GFP - gate . data are representative of 3 different experiments with similar outcome .

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E. FACS plot of DN2-derived primary leukemic cells transduced with a retrovirus carrying GFP alone or GFP together with PU.1 as a bicistronic RNA. The transduced cells were grown on OP9/OP9Dll1 1:1 layer for 8-13 days and analyzed using a GFP-gate. The data are representative of 3 different experiments with similar outcome.


A . Heat map depicting human AML samples from Haferlach study ( Haferlach et al . , 2010 ) whose transcriptomes resembled DN2 - leukemia by a Spearman ' s rank correlation coefficient ≥ 0 . 5 . top 100 up - downregulated gene signature of Myc / Bcl2 + DN2 - derived myeloid blasts was applied ( see also Figure 3B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Heat map depicting all human AML samples from the Haferlach study (Haferlach et al., 2010) whose transcriptomes resembled DN2-leukemia by a Spearman's rank correlation coefficient ≥ 0.5. The top 100 up- and downregulated gene signature of Myc/Bcl2+DN2-derived myeloid blasts was applied (see also Figure 3B).


C , E . GSEA confirming enrichment of Myc / Bcl2 + DN2 - derived myeloid blast top 100 UP ( C ) pan - DC ( Schonheit et al . , 2013 ) ( E ) gene signatures in human FAB M2 DN2 - like AML samples as compared to residual M2 AMLs from Verhaak study ( Verhaak et al . , 2009 ) . D . Relative transcript of T receptor alpha locus ( TRAC ) in human DN2 - like M2 AML compared to residual M2 AML samples from Verhaak study ( Wilcoxon rank - sum , * * = p ≤ 0 . 01 ) .

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

C,E. GSEA confirming enrichment of the Myc/Bcl2+DN2-derived myeloid blast top 100 UP (C) and pan-DC (Schonheit et al., 2013) (E) gene signatures in the human FAB M2 DN2-like AML samples as compared to all residual M2 AMLs from the Verhaak study (Verhaak et al., 2009). D. Relative transcript levels of the T cell receptor alpha locus (TRAC) in human DN2-like M2 AML compared to residual M2 AML patient samples from the Verhaak study (Wilcoxon rank-sum test, **=p≤0.01).


A . Survival of Myc / Bcl2 + GMP - DN2 - derived leukemia that were isolated from diseased mice treated for 24 h with 0 . 15 μM daunorubicine or 1 μM doxorubicine in liquid culture . was measured by FACS of 7 - AAD + was normalized to solvent controls . graphs show mean + of three different DN2 - five GMP - leukemias . * * = p ≤ 0 . 01 .

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A. Survival assay of Myc/Bcl2+GMP- and DN2-derived leukemia cells that were isolated from diseased mice and treated for 24 h with 0.15 μM daunorubicine or 1 μM doxorubicine in liquid culture. Cell death was measured by FACS of 7-AAD+cells and was normalized to the solvent controls. The graphs show the mean+SEM of three different DN2- and five GMP-leukemias. **=p≤0.01.


C . Scatterplot depicting averaged quantitative changes ( log10 ) of each shRNA within library in T1 spleen BM samples in reference to T0 . changes in both organs were concordant ( R2 = 0 . 72 ) . T1 samples from 11 mice were compared to 3 T0 reference samples . Blue dots represent shRNAs with a significant depletion by ≥ 10 - fold .

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C. Scatterplot depicting the averaged quantitative changes (log10) of each shRNA within the library in the T1 spleen and BM samples in reference to T0. The changes in both organs were concordant (R2=0.72). T1 samples from 11 mice were compared to 3 T0 reference samples. Blue dots represent shRNAs with a significant depletion by ≥10-fold.


D . Top 5 genes with ≥ 10 - fold shRNA depletion in BM , spleen thymus in comparison to reference .

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D. Top 5 genes with ≥10-fold shRNA depletion in BM, spleen and thymus in comparison to the reference.


E . growth of 5 different DN2 - derived leukemic lines transduced with 2 different shRNA constructs targeting Jak2 . were sorted for GFP or YFP reporter normalized to control shRNA targeting lacZ gene ( lacZ ) . Four of five DN2 - derived leukemias show reduced growth with both shRNAs after second round of sorting within 8 days in culture . lacZ control was set to 100 % .

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E. Percent cell growth of 5 different DN2-derived leukemic cell lines transduced with 2 different shRNA constructs targeting Jak2. The cells were sorted for the GFP or YFP reporter and normalized to the control shRNA targeting the lacZ gene (lacZ). Four of five DN2-derived leukemias show reduced growth with both shRNAs after the second round of sorting within 8 days in culture. The lacZ control was set to 100%.


F . Proliferation curves of 3 Myc / Bcl2 + DN2 - 3 GMP - derived leukemia lines during treatment with 0 . 5 μM ruxolitinib . 1 . 5105 were seeded in culture counted at indicated time points . Results are shown as fold growth of starting number which was set to 1 . * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , Student ' s t - .

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F. Proliferation curves of 3 Myc/Bcl2+ DN2- and 3 GMP-derived leukemia lines during treatment with 0.5 μM ruxolitinib. 1.5105 cells were seeded in culture and counted at the indicated time points. Results are shown as fold growth of the starting cell number which was set to 1. *=p≤0.05, **=p≤0.01, Student's t-test.


G . Western blot indicating active Jak / Stat signaling in DN2 - derived leukemia with Stat3 as well as pStat3 expression , that diminishes after treatment with Jak - inhibitor ruxolitinib .

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G. Western blot indicating active Jak/Stat signaling in DN2-derived leukemia with Stat3 as well as pStat3 protein expression, that diminishes after treatment with the Jak-inhibitor ruxolitinib.


H . GSEA indicating significant enrichment of genes of JAK / STAT signaling pathway of KEGG pathway database in DN2 - like M2 AML samples as compared to residual M2 AML samples using Verhaak AML data set ( Verhaak et al . , 2009 ) .

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H. GSEA indicating significant enrichment of genes of the JAK/STAT signaling pathway of the KEGG pathway database in the DN2-like M2 AML samples as compared to the residual M2 AML samples using the Verhaak AML data set (Verhaak et al., 2009).


A , Live of neurons co - transfected with SyGCaMP5 MtDsRed before during 10 Hz field stimulation . full arrow indicates a mitochondrially occupied terminal , empty arrow a terminal without a mitochondrion . Scale bar , 10 m .

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A, Live images of neurons co-transfected with SyGCaMP5 and MtDsRed before and during 10 Hz field stimulation. The full arrow indicates a mitochondrially occupied terminal, and the empty arrow a terminal without a mitochondrion. Scale bar, 10 m.


B , Average ΔF / F0 SyGCaMP5 traces from n = 11 neurons ( 98 terminals ) plotting average of terminals without a mitochondrion ( black trace ) with a mitochondrion ( red trace ) . Stimulation occurred for 10s ( t = 20 - 30 ) at 10 Hz .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Average ΔF/F0 SyGCaMP5 traces from n=11 neurons (98 terminals) plotting the average of terminals without a mitochondrion (black trace) and with a mitochondrion (red trace). Stimulation occurred for 10s (t=20-30) at 10 Hz.


C , Average Ca2 + response following stimulation in terminals with or without a mitochondrion ( average of ΔF measurement taken for a t = 20 - 30s of B ) , ΔF / F0 = 1 . 9 0 . 3 with 3 . 5 0 . 4 without mitochondria , paired t - , * * * p < 0 . 0001 .

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C, Average Ca2+ response following stimulation in terminals with or without a mitochondrion (average of ΔF measurement taken for a t=20-30s of B), ΔF/F0 = 1.9 0.3 with and 3.5 0.4 without mitochondria, paired t-test, ***p<0.0001.


D E , However , there is no significant difference in presynaptic Ca2 + response in terminals with without a mitochondrion in response to single action potentials . D , Example trace of hippocampal neurons transfected with MtDsRed SyGCaMP5 . 1 ms stimulation pulses were applied every 5 s . red trace represents an average of terminals occupied with a mitochondrion in this neuron , whereas black trace represents averaged response of terminals without a mitochondrion . E , Maximal in terminals without a mitochondrion ( left ) or with a mitochondrion ( right ) within same axon ( dots connected by a ) ( n = 8 neurons , 44 terminals , paired t - , p = 0 . 55 ) .

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D E, However, there is no significant difference in the presynaptic Ca2+ response in terminals with and without a mitochondrion in response to single action potentials. D, Example trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5. 1 ms stimulation pulses were applied every 5 s. The red trace represents an average of the terminals occupied with a mitochondrion in this neuron, whereas the black trace represents the averaged response of terminals without a mitochondrion. E, Maximal fluorescence intensity in terminals without a mitochondrion (left) or with a mitochondrion (right) within the same axon (dots connected by a line) (n=8 neurons, 44 terminals, paired t-test, p=0.55).


F , Average ΔF / F0 SyGCaMP5 traces for 80 stimuli ( n = 7 neurons , 21 terminals , t = 20 - 28s , ΔF / F0 = 1 . 3 0 . 2 with 2 . 2 0 . 5 without mitochondria , paired t - , * p < 0 . 05 ) , 40 stimuli ( n = 5 neurons , 25 terminals , t = 20 - 24s , ΔF / F0 = 0 . 9 0 . 3 with 2 . 0 0 . 4 without mitochondria , paired t - , * p < 0 . 05 ) , 20 stimuli ( n = 10 neurons , 55 terminals , t = 20 - 22s , ΔF / F0 = 0 . 8 0 . 4 with 1 . 8 0 . 4 without mitochondria , paired t - , * p < 0 . 05 ) 10 stimuli ( n = 4 neurons , 21 terminals , t = 20 - 21s , ΔF / F0 = 0 . 4 0 . 3 with 0 . 4 0 . 1 without mitochondria , paired t - , p = 0 . 90 ) , delivered at 10 Hz . Experiments performed in E18 or P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Average ΔF/F0 SyGCaMP5 traces for 80 stimuli (n=7 neurons, 21 terminals, t= 20-28s, ΔF/F0 = 1.3 0.2 with and 2.2 0.5 without mitochondria, paired t-test, *p<0.05), 40 stimuli (n=5 neurons, 25 terminals, t= 20-24s, ΔF/F0 = 0.9 0.3 with and 2.0 0.4 without mitochondria, paired t-test, *p<0.05), 20 stimuli (n=10 neurons, 55 terminals, t= 20-22s, ΔF/F0 = 0.8 0.4 with and 1.8 0.4 without mitochondria, paired t-test, *p<0.05) and 10 stimuli (n=4 neurons, 21 terminals, t= 20-21s, ΔF/F0 = 0.4 0.3 with and 0.4 0.1 without mitochondria, paired t-test, p=0.90), all delivered at 10 Hz. Experiments performed in E18 or P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


A , Average trace of hippocampal neurons transfected with MtDsRed SyGCaMP5 imaged after incubation in ACSF for 30 min . Neurons were stimulated using field stimulation for 10s at 10 Hz . Red trace = average of terminals occupied with a mitochondrion , black trace = averaged response of terminals without a mitochondrion . B , as in A , but after 30 min treatment with 10 µM Ru360 .

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

A, Average trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 imaged after incubation in ACSF for 30 min. Neurons were stimulated using field stimulation for 10s at 10 Hz. Red trace = average of the terminals occupied with a mitochondrion, black trace = averaged response of terminals without a mitochondrion. B, as in A, but after 30 min treatment with 10 µM Ru360.


C , Summary bar graph of control neurons ( corresponds to traces shown in panel A ) . Average Ca2 + response ( t = 20 - 30s ) following stimulation for terminals occupied by a mitochondrion ( ΔF / F0 = 3 . 5 0 . 4 ) unoccupied terminals ( ΔF / F0 = 4 . 9 0 . 5 , n = 15 neurons , 96 terminals , * * * p < 0 . 001 , paired t - ) . D , Summary bar graph of Ru360 - treated neurons ( corresponds to traces shown in panel B ) . Average Ca2 + response ( t = 20 - 30s ) following stimulation for terminals occupied by a mitochondrion ( ΔF / F0 = 4 . 1 0 . 5 ) unoccupied terminals ( ΔF / F0 = 3 . 8 0 . 9 , n = 13 neurons , 79 terminals , p = 0 . 53 , paired t - ) . Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent .

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

C, Summary bar graph of the control neurons (corresponds to the traces shown in panel A). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 3.5 0.4) and unoccupied terminals (ΔF/F0 = 4.9 0.5, n=15 neurons, 96 terminals, ***p<0.001, paired t-test). D, Summary bar graph of the Ru360-treated neurons (corresponds to the traces shown in panel B). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 4.1 0.5) and unoccupied terminals (ΔF/F0 = 3.8 0.9, n=13 neurons, 79 terminals, p=0.53, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


A , Example of hippocampal neurons co - transfected with Mito - mKate2 VGlut1pHluorin before during field stimulation . arrow indicates a terminal occupied with a mitochondrion , while empty arrow indicates a terminal without a mitochondrion . B , Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin Mito - mKate2 stimulated using field stimulation at 10 Hz for 20s ( 40 - 60s ) . Terminals with a mitochondrion are represented by red trace terminals without are represented by black trace . C , Summary graph corresponds to traces shown in panel B ) . Mean stimulation ( time points 60 - 70s ) comparing terminals of same axon which contain mitochondria ( right , ΔF / F0 = 2 . 3 0 . 9 ) compared to terminals without mitochondria ( left , ΔF / F0 = 3 . 2 0 . 7 ) ( n = 9 neurons , 66 terminals , * p < 0 . 05 , paired t - ) . Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Example images of hippocampal neurons co-transfected with Mito-mKate2 and VGlut1pHluorin before and during field stimulation. The white arrow indicates a terminal occupied with a mitochondrion, while the empty arrow indicates a terminal without a mitochondrion. B, Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin and Mito-mKate2 and stimulated using field stimulation at 10 Hz for 20s (40-60s). Terminals with a mitochondrion are represented by the red trace and terminals without are represented by the black trace. C, Summary graph corresponds to the traces shown in panel B). Mean stimulation fluorescence (time points 60-70s) comparing terminals of the same axon which contain mitochondria (right, ΔF/F0 = 2.3 0.9) compared to terminals without mitochondria (left, ΔF/F0 = 3.2 0.7) (n=9 neurons, 66 terminals, *p<0.05, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


A , C E , Confocal of neuronal processes of fixed neurons transfected with MtDsRed SYN - GFP C , myc - Miro1 ( Miro OE ) or E , myc - Miro1ΔEF ( Miro ΔEF ) . Neurons are either non - treated ( NT ) , TTX treated ( 1 M , 48h ) , PTX treated ( 100 M , 48h ) , or DMSO treated ( 1 : 2000 , as PTX ) . Scale bar , 10 m . B , D F , Fraction of SYN - GFP clusters co - localising with mitochondria in cultures in B , control conditions , DMSO 28 . 7 % 3 . 9 , TTX 16 . 6 % 3 . 1 , PTX 50 . 0 % 7 . 2 , ANOVA * * p0 . 001 , n = 8 - 9 neurons , D , with expression of myc - Miro1 , DMSO 33 . 9 % 4 . 7 , TTX 35 . 9 % 5 . 4 , PTX 46 . 7 % 4 . 0 , ANOVA , * p0 . 05 , n = 8 - 9 , F , with expression of myc - Miro1ΔEF , ANOVA p0 . 05 , n = 9 - 12 . Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent .

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

A, C E, Confocal images of neuronal processes of fixed neurons transfected with MtDsRed and SYN-GFP and C, myc-Miro1 (Miro OE) or E, myc-Miro1ΔEF (Miro ΔEF). Neurons are either non-treated (NT), TTX treated (1 M, 48h), PTX treated (100 M, 48h), or DMSO treated (1:2000, as PTX). Scale bar, 10 m. B, D F, Fraction of SYN-GFP clusters co-localising with mitochondria in cultures in B, control conditions, DMSO 28.7% 3.9, TTX 16.6% 3.1, PTX 50.0% 7.2, ANOVA **p0.001, n=8-9 neurons, D, with the expression of myc-Miro1, DMSO 33.9% 4.7, TTX 35.9% 5.4, PTX 46.7% 4.0, ANOVA, *p0.05, n=8-9, F, with the expression of myc-Miro1ΔEF, ANOVA p0.05, n=9-12. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


A , Live of neurons co - transfected with SyGCaMP5 MtDsRed or myc - ΔEF - Miro1 - IRES - MtDsRed ( ΔEF Miro ) before during 10 Hz field stimulation with without TTX treatments . Scale bar , 10 m . B , Average ΔF / F0 SyGCaMP5 traces from terminals treated with TTX ( red trace , ΔF / F0 = 2 . 5 0 . 5 ) , n = 10 neurons ( 62 terminals ) non - treated terminals ( black trace , ΔF / F0 = 1 . 3 0 . 6 ) , n = 11 neurons ( 69 terminals ) co - transfected with MtDsRed , * p < 0 . 05 , t - . C , Average ΔF / F0 SyGCaMP5 traces from terminals treated with TTX ( red trace , ΔF / F0 = 1 . 8 0 . 3 ) in n = 7 neurons ( 33 terminals ) non - treated terminals ( black trace , ΔF / F0 = 2 . 3 0 . 6 ) in n = 7 neurons ( 32 terminals ) co - transfected with myc - ΔEF - Miro1 - IRES - MtDsRed ; p = 0 . 5 , t - . Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent .

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

A, Live images of neurons co-transfected with SyGCaMP5 and MtDsRed or myc-ΔEF-Miro1-IRES-MtDsRed (ΔEF Miro) before and during 10 Hz field stimulation with and without TTX treatments. Scale bar, 10 m. B, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 2.5 0.5), n=10 neurons (62 terminals) and non-treated terminals (black trace, ΔF/F0 = 1.3 0.6), n=11 neurons (69 terminals) co-transfected with MtDsRed, *p<0.05, t-test. C, Average ΔF/F0 SyGCaMP5 traces from terminals treated with TTX (red trace, ΔF/F0 = 1.8 0.3) in n=7 neurons (33 terminals) and non-treated terminals (black trace, ΔF/F0 = 2.3 0.6) in n=7 neurons (32 terminals) co-transfected with myc-ΔEF-Miro1-IRES-MtDsRed; p=0.5, t-test. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


Luciferase sequestration as an for in vitro uptake . ( A ) Luciferase cofractionating with vacuoles is proteinase K protected . In vitro uptake reactions of three times standard volume were performed ( 60 min , 27 ° C ) in presence or absence of ATP - regenerating system . vacuoles were reisolated , washed , resuspended in 150 mM KCl in PS buffer to a of 0 . 23 mg / ml , split into aliquots . After addition of 1 % Triton X - 100 or buffer , mixture was digested with indicated concentrations of proteinase K ( 15 min , 0 ° C ) . Proteinase treatment was stopped by adding one volume of 0 . 5 mM PMSF in 150 mM KCl in PS buffer . Then , luciferase activity was assayed .

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

Luciferase sequestration as an assay for in vitro uptake. (A) Luciferase cofractionating with vacuoles is proteinase K protected. In vitro uptake reactions of three times the standard volume were performed (60 min, 27°C) in the presence or absence of the ATP-regenerating system. The vacuoles were reisolated, washed, resuspended in 150 mM KCl in PS buffer to a concentration of 0.23 mg/ml, and split into aliquots. After addition of 1% Triton X-100 or buffer, the mixture was digested with the indicated concentrations of proteinase K (15 min, 0°C). Proteinase treatment was stopped by adding one volume of 0.5 mM PMSF in 150 mM KCl in PS buffer. Then, luciferase activity was assayed.


( B ) Luciferase can be released from vacuolar lumen by freeze - thaw treatment . An uptake reaction was performed ( 60 min , 27 ° C ) . vacuoles were reisolated washed as described in Materials Methods section . final pellet was resuspended in 90 μl PS buffer with 150 mM KCl split into two aliquots . One was frozen at − 80 ° C thawed again slowly , whereas other remained on ice . samples were centrifuged ( 15 min , 145 , 000 g , 4 ° C ) . supernatants ( Sup ) were recovered pellets were resuspended in an equal volume of PS with 150 mM KCl . Luciferase activity in both fractions was determined . reaction tubes for this experiment had been coated with BSA ( 0 . 5 mg / ml , 15 min at room ) to reduce unspecific binding of luciferase to surface .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Luciferase can be released from the vacuolar lumen by freeze-thaw treatment. An uptake reaction was performed (60 min, 27°C). The vacuoles were reisolated and washed as described in the Materials and Methods section. The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots. One was frozen at −80°C and thawed again slowly, whereas the other remained on ice. The samples were centrifuged (15 min, 145,000 g, 4°C). The supernatants (Sup)were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl. Luciferase activity in both fractions was determined. The reaction tubes for this experiment had been coated with BSA (0.5 mg/ml, 15 min at room temperature) to reduce unspecific binding of luciferase to the surface.


time course dependence of uptake . ( A ) Standard uptake reactions were performed in presence or absence of ATP - regenerating system . After indicated times at 27 ° C , vacuoles were reisolated , washed , treated with protease , luciferaseuptake activity was assayed .

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

time course and temperature dependence of uptake. (A) Standard uptake reactions were performed in the presence or absence of the ATP-regenerating system. After the indicated times at 27°C, the vacuoles were reisolated, washed, treated with protease, and luciferaseuptake activity was assayed.


( B ) A standard uptake reaction was performed . After 70 min of incubation , vacuoles were reisolated , washed , treated with protease . vacuoles were reisolated again , resuspended in reaction buffer without luciferase , incubated for indicated times at 27 ° C . Aliquots were withdrawn to total luciferase remaining . As a control , luciferase was incubated in reaction buffer for same periods of time assayed for activity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) A standard uptake reaction was performed. After 70 min of incubation, the vacuoles were reisolated, washed, and treated with protease. The vacuoles were reisolated again, resuspended in reaction buffer without luciferase, and incubated for the indicated times at 27°C. Aliquots were withdrawn to assay the total luciferase remaining. As a control, luciferase was incubated in reaction buffer for the same periods of time and assayed for activity.


Requirements for in vitro uptake . Standard uptake reactions were performed ( 60 min ) ( A ) at different temperatures , or in presence of different concentrations of ( B ) cytosol , ( C ) ATP , or ( D ) MgCl2 . Luciferase uptake was determined as described in legend to Fig . 3 A . In B , titration curves for four independent cytosol preparations are shown . maximal uptake signal of each titration curve was set at 100 % . In D , sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in reaction .

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

Requirements for in vitro uptake. Standard uptake reactions were performed (60 min) (A) at different temperatures, or in the presence of different concentrations of (B) cytosol, (C) ATP, or (D) MgCl2. Luciferase uptake was determined as described in the legend to Fig. 3 A. In B, titration curves for four independent cytosol preparations are shown. The maximal uptake signal of each titration curve was set at 100%. In D, the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction.


In vitro uptake is independent of components involved in homotypic vacuolar fusion . ( A ) Standard uptake reactions were performed ( 60 min , 27 ° C ) in presence of indicated reagents using a mixture of vacuoles from strains DBY5734 - 16 ( Δpep4 ) DBY5734 - 19 ( Δpho8 ) . Therefore , homotypic vacuolar fusion luciferase uptake could be assayed simultaneously from same sample . Four independent experiments were averaged . Error bars indicate SD . For averaging , uptake fusion activities of control sample without inhibitor were set at 100 % . Inhibitors were used at following concentrations : anti - Sec17p , 1 μM ; anti - Sec18p , 1 μM ; Gdi1p , 2 . 5 μM ; anti - Vam3p , 1 μM ; anti - Vam7p , 1 μM ; anti - Nyv1p , 1 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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O

In vitro uptake is independent of components involved in homotypic vacuolar fusion. (A) Standard uptake reactions were performed (60 min, 27°C) in the presence of the indicated reagents using a mixture of vacuoles from strains DBY5734-16 (Δpep4) and DBY5734-19 (Δpho8). Therefore, homotypic vacuolar fusion and luciferase uptake could be assayed simultaneously from the same sample. Four independent experiments were averaged. Error bars indicate SD. For averaging, uptake and fusion activities of the control sample without inhibitor were set at 100%. Inhibitors were used at the following concentrations: anti-Sec17p, 1 μM; anti-Sec18p, 1 μM; Gdi1p, 2.5 μM; anti-Vam3p, 1 μM; anti-Vam7p, 1 μM; and anti-Nyv1p, 1 μM.


( B ) Vacuoles cytosols were prepared from sec18 - 1 from corresponding wild - type ( Wt ) grown spheroplasted at 23 ° C . isolated vacuoles cytosols were exposed to heat treatment ( 10 min , 37 ° C ) . Standard uptake reactions were performed with vacuoles cytosols in indicated combinations . Control reactions were performed in absence of ATP . Five independent experiments were averaged ; sample with vacuoles cytosol from wild - type was used as 100 % reference . Error bars indicate SD . Mut , mutant .

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

(B) Vacuoles and cytosols were prepared from sec18-1 and from corresponding wild-type (Wt) cells grown and spheroplasted at 23°C. The isolated vacuoles and the cytosols were exposed to heat treatment (10 min, 37°C). Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations. Control reactions were performed in the absence of ATP. Five independent experiments were averaged; the sample with vacuoles and cytosol from wild-type cells was used as the 100% reference. Error bars indicate SD. Mut, mutant.


In vitro uptake depends on GTP hydrolysis on V - ATPase . ( A ) Standard uptake reactions were performed in presence of different concentrations of GTPγS . Luciferase uptake was assayed as in legend to Fig . 3 A . Four independent experiments were averaged , using sample without inhibitor as 100 % reference . Error bars indicate SD . GTPγS did not influence luciferase activity by itself ( not shown ) .

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

In vitro uptake depends on GTP hydrolysis and on the V-ATPase. (A) Standard uptake reactions were performed in the presence of different concentrations of GTPγS. Luciferase uptake was assayed as in the legend to Fig. 3 A. Four independent experiments were averaged, using the sample without inhibitor as the 100% reference. Error bars indicate SD. GTPγS did not influence luciferase activity by itself (not shown).


( B ) Standard uptake reactions were performed in presence of FCCP ( CMA , 20 μM ) or concanamycin A ( 20 μM ) , or in absence of ATP .

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

(B) Standard uptake reactions were performed in the presence of FCCP (CMA, 20 μM) or concanamycin A (20 μM), or in the absence of ATP.


( H ) bur1 - ΔC mutant exhibits a synthetic growth defect with vac17Δ mutant . Plasmids were transformed into a bur1Δ or bur1Δ vac17Δ mutant containing YCp50 [ URA3 ] BUR1 . Plasmids tested were pRS415 [ LEU2 ] ( mock ) , pRS415 BUR1 , or pRS415 bur1 - ΔC . Transformed colonies were cultured in liquid medium serial dilutions spotted onto SC + 5 - FOA or SC - Leu - Ura plates . Plates were incubated at 24 ̊ C for 3 days .

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

(H) The bur1-ΔC mutant exhibits a synthetic growth defect with the vac17Δ mutant. Plasmids were transformed into a bur1Δ or bur1Δ vac17Δ mutant containing YCp50 [URA3] BUR1. Plasmids tested were pRS415 [LEU2] (mock), pRS415 BUR1, or pRS415 bur1-ΔC. Transformed colonies were cultured in liquid medium and serial dilutions spotted onto SC+5-FOA or SC-Leu-Ura plates. Plates were incubated at 24 ̊C for 3 days.


( A , B ) bur1 - ΔC vac17Δ double mutant exhibits a delay in progression through G1 phase of cycle . Yeast were grown in YPD medium collected in log phase growth . content was measured using propidium iodide ( PI ) assessed by flow cytometry . p - value determined with a one - way ANOVA Tukey post hoc . * * ( p - value < 1 x 10 - 2 ) . Error bars ; SD calculated from at least four independent experiments with at least 100 counted in each / experiment . ( C , D ) A bur1 - 267 mutant also exhibits a delay in progression through G1 phase of cycle . content was measured using PI assessed by flow cytometry . p - value determined with a one - way ANOVA Tukey post hoc . * * * * ( p - value < 1 x 10 - 4 ) . Error bars ; SD calculated from at least four independent experiments with at least 100 counted in each / experiment .

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

(A, B) The bur1-ΔC vac17Δ double mutant exhibits a delay in progression through G1 phase of the cell cycle. Yeast were grown in YPD medium and collected in log phase growth. DNA content was measured using propidium iodide (PI) staining and assessed by flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. (C, D) A bur1-267 mutant also exhibits a delay in progression through G1 phase of the cell cycle. DNA content was measured using PI staining and assessed by flow cytometry. p-value determined with a one-way ANOVA and Tukey post hoc test. **** (p-value < 1 x 10-4). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment.


( E , F ) bur1 - ΔC vac17Δ double mutant shows accumulation of Whi5 in nucleus . WT , vac17Δ , bur1 - ΔC , bur1 - ΔC vac17Δ , which express Whi5 - 3xGFP Nup188 - mCherry from endogenous loci , were incubated at 24 ̊ C . were scored for presence of Whi5 - 3xGFP in within nucleus ( Nup188 - mCherry ) . Error bars ; SD calculated from at least four independent experiments with at least 100 counted in each / experiment . p - value determined with a one - way ANOVA Tukey post hoc . * * ( p - value < 1 x 10 - 2 ) . Error bars ; SD calculated from at least four independent experiments with at least 100 counted in each / experiment . Scale bar , 2 μm .

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

(E, F) The bur1-ΔC vac17Δ double mutant shows accumulation of Whi5 in the nucleus. WT, vac17Δ, bur1-ΔC, and bur1-ΔC vac17Δ cells, which express Whi5-3xGFP and Nup188-mCherry from endogenous loci, were incubated at 24 ̊C. Cells were scored for the presence of Whi5-3xGFP in within the nucleus (Nup188-mCherry). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. p-value determined with a one-way ANOVA and Tukey post hoc test. ** (p-value < 1 x 10-2). Error bars; SD calculated from at least four independent experiments with at least 100 cells counted in each strain/experiment. Scale bar, 2 μm.


( B ) bur1 - ∆ C vac17 ∆ double mutant have ability to generate a vacuole de novo , which suggests that - cycle delay is due to a defect in Bur1 - dependent signaling . WT , vac17Δ , bur1 - ∆ C , bur1 - ∆ C vac17 ∆ which express Vph1 - GFP from its endogenous locus , were labeled with vacuole specific dye FM4 - 64 . WT bur1 - ∆ C have both FM4 - 64 Vph1 - GFP signals in both mother daughter . vac17Δ bur1 - ∆ C vac17 ∆ have both Vph1 - GFP FM4 - 64 on vacuole in mother , however daughter solely have a Vph1 - GFP labeled vacuole , indicating that vacuole was generated de novo , rather than inherited from mother vacuole . Arrowheads ; new vacuoles in daughter . Scale bar , 2 μm .

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

(B) The bur1-∆C vac17∆ double mutant cells have the ability to generate a vacuole de novo, which suggests that the cell-cycle delay is due to a defect in Bur1-dependent signaling. WT, vac17Δ, bur1-∆C, and bur1-∆C vac17∆ cells which express Vph1-GFP from its endogenous locus, were pulse labeled with the vacuole specific dye FM4-64. WT and bur1-∆C cells have both FM4-64 and Vph1-GFP signals in both mother and daughter cells. vac17Δ and bur1-∆C vac17∆ cells have both Vph1-GFP and FM4-64 on the vacuole in mother cells, however the daughter cells solely have a Vph1-GFP labeled vacuole, indicating that the vacuole was generated de novo, rather than inherited from the mother vacuole. Arrowheads; new vacuoles in daughter cells. Scale bar, 2 μm.


( A ) In vivo phosphorylation of Sch9 is reduced in bur1 - ∆ C vac17 ∆ . WT , bur1 - ∆ C , vac17 ∆ , or bur1 - ∆ C vac17 ∆ expressing SCH9 - HA from URA3 locus were grown in YPD . lysates were analyzed by immunoblot with antibodies directed against HA ( top ) Pgk1 ( loading control ) . ( B ) In vivo phosphorylation of Sch9 is reduced in bur1 - 267 . WT , vac17 ∆ , bur1 - 267 , or bur1 - 267 vac17 ∆ expressing SCH9 - HA from URA3 locus were grown in YPD . lysates were analyzed by immunoblot with antibodies directed against HA ( top ) Pgk1 ( loading control ) .

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

(A) In vivo phosphorylation of Sch9 is reduced in bur1-∆C vac17∆ cells. WT, bur1-∆C, vac17∆, or bur1-∆C vac17∆ cells expressing SCH9-HA from the URA3 locus were grown in YPD. Cell lysates were analyzed by immunoblot with antibodies directed against HA (top) and Pgk1 (loading control). (B) In vivo phosphorylation of Sch9 is reduced in bur1-267 cells. WT, vac17∆, bur1-267, or bur1-267 vac17∆ cells expressing SCH9-HA from the URA3 locus were grown in YPD. Cell lysates were analyzed by immunoblot with antibodies directed against HA (top) and Pgk1 (loading control).


( C ) bur1 - ∆ C shows high rapamycin sensitivity . WT , tor1 ∆ , bur1 - ∆ C , tor1 ∆ bur1 - ∆ C or vac17 ∆ were cultured in liquid YPD medium serial dilutions were spotted onto YPD plates with 0 , 2 , 4 or 8 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 days .

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

(C) bur1-∆C shows high rapamycin sensitivity. WT, tor1∆, bur1-∆C, tor1∆ bur1-∆C or vac17∆ cells were cultured in liquid YPD medium and serial dilutions were spotted onto YPD plates with 0, 2, 4 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.


( B ) In vitro , Bur1 directly phosphorylates kinase domain of Sch9 as well as Bur1 itself . pRS425 BUR1 - HA pVT102 - Ura BUR2 , essential cyclin for BUR1 , were co - expressed in a bur1Δ mutant , immunoprecipitated using anti - HA antibody A beads , then incubated with [ γ - 32P ] ATP GST , GST - N - Sch9 ( 1 - 390 ) or GST - C - Sch9 ( 391 - 824 ) . were eluted with SDS sample buffer , separated by SDS - PAGE , transferred to nitrocellulose membranes . Membranes exposed to x - ray film ( top ) or stained with Ponceau S ( bottom ) . Red arrowhead ; phosphorylated GST - C - Sch9 . Gray arrowhead ; auto - phosphorylated Bur1 .

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

(B) In vitro, Bur1 directly phosphorylates the kinase domain of Sch9 as well as Bur1 itself. pRS425 BUR1-HA and pVT102-Ura BUR2, the essential cyclin for BUR1, were co-expressed in a bur1Δ mutant, immunoprecipitated using anti-HA antibody and protein A beads, and then incubated with [γ-32P]ATP and GST, GST-N-Sch9 (1-390) or GST-C-Sch9 (391-824). Proteins were eluted with SDS sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes exposed to x-ray film (top) or stained with Ponceau S (bottom). Red arrowhead; phosphorylated GST-C-Sch9. Gray arrowhead; auto-phosphorylated Bur1.


( D ) Sch9 - 2D3E , but not Sch9 - 3A - 2D3E , partially suppressed rapamycin sensitivity of tor1 ∆ . tor1 ∆ which expresses pRS426 ( mock ) , pRS425 TOR1 , pVT102 - Ura ( mock ) , pVT102 - Ura Sch9 , pVT102 - Ura Sch9 - 2D3E , or pVT102 - Ura Sch9 - 3A - 2D3E , were cultured in liquid SC - Ura medium serial dilutions spotted onto SC - Ura plates with 0 or 8 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 - 3 days .

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

(D) Sch9-2D3E, but not Sch9-3A-2D3E, partially suppressed the rapamycin sensitivity of tor1∆ cells. tor1∆ which expresses pRS426 (mock), pRS425 TOR1, pVT102-Ura (mock), pVT102-Ura Sch9, pVT102-Ura Sch9-2D3E, or pVT102-Ura Sch9-3A-2D3E, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2-3 days.


( E ) Sch9 - 2D3E mutant did not suppress rapamycin sensitivity of bur1 - ∆ C . bur1 - ∆ C which expresses pRS426 ( mock ) , pRS426 BUR1 , pVT102 - Ura ( mock ) , pVT102 - Ura SCH9 , pVT102 - Ura sch9 - 2D3E , pVT102 - Ura sch9 - 3A - 2D3E , pVT102 - Ura sch9 - 3D / E , or pVT102 - Ura sch9 - 3D / E - 2D3E were cultured in liquid SC - Ura medium serial dilutions spotted onto SC - Ura plates with 0 or 8 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 3 days .

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

(E) The Sch9-2D3E mutant did not suppress the rapamycin sensitivity of bur1-∆C cells. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pVT102-Ura (mock), pVT102-Ura SCH9, pVT102-Ura sch9-2D3E, pVT102-Ura sch9-3A-2D3E, pVT102-Ura sch9-3D/E, or pVT102-Ura sch9-3D/E-2D3E were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0 or 8 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 3 days.


( A ) Bur1 fused SV40 - NLS does not rescue rapamycin sensitivity of bur1 - ∆ C compared to Bur1 without NLS . bur1 - ∆ C which expresses pRS416 ( mock ) , pRS416 BUR1 , pRS416 BUR1 - mNG , pRS416 BUR1 - mNG - NLS , pRS416 bur1 - ∆ C , pRS416 bur1 - ∆ C - mNG - NLS or pRS416 bur1 - ∆ N , were cultured in liquid SC - Ura medium serial dilutions spotted onto SC - Ura plates with 0 , 4 , 8 , or 16 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 days .

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

(A) Bur1 fused SV40-NLS does not rescue the rapamycin sensitivity of bur1-∆C compared to Bur1 without the NLS. bur1-∆C which expresses pRS416 (mock), pRS416 BUR1, pRS416 BUR1-mNG, pRS416 BUR1-mNG-NLS, pRS416 bur1-∆C, pRS416 bur1-∆C-mNG-NLS or pRS416 bur1-∆N, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.


( B ) Adding - export signal ( NES ) of PKI to Bur1 / or deletion of importin α - dependent NLS from Bur1 resulted in higher sensitivity to rapamycin compared to Bur1 without this NES . bur1 - ∆ C which expresses pRS426 ( mock ) , pRS426 BUR1 , pRS426 bur1 - ∆ N , pRS426 BUR1 - mNG , pRS426 BUR1 - mNG - NES , or pRS426 bur1 - ∆ N - mNG - NES , were cultured in liquid SC - Ura medium serial dilutions spotted onto SC - Ura plates with 0 , 4 , 8 , or 16 ng / ml of rapamycin added . Plates were incubated at 24 ̊ C for 2 days .

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

(B) Adding nuclear-export signal (NES) of PKI to Bur1 and/or deletion of the importin α-dependent NLS from Bur1 resulted in higher sensitivity to rapamycin compared to Bur1 without this NES. bur1-∆C which expresses pRS426 (mock), pRS426 BUR1, pRS426 bur1-∆N, pRS426 BUR1-mNG, pRS426 BUR1-mNG-NES, or pRS426 bur1-∆N-mNG-NES, were cultured in liquid SC-Ura medium and serial dilutions spotted onto SC-Ura plates with 0, 4, 8, or 16 ng/ml of rapamycin added. Plates were incubated at 24 ̊C for 2 days.


C , Immunoblot of lysates from MEF treated with EBSS , 250 nM Torin - 1 or 100 μM NaAsO2 for 1h run under non - reducing conditions . For refeed condition , starved were incubated in complete medium for indicated times .

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

C, Immunoblot analysis of protein lysates from MEF cells treated with EBSS, 250 nM Torin-1 or 100 μM NaAsO2 for 1h and run under non-reducing conditions. For refeed condition, starved cells were incubated in complete medium for the indicated times.


C , Immunoblot of lysates from ARPE - 19 expressing TFEB - FLAG - WT or mutants treated with 250 μM NaAsO2 or EBSS for 1h run under non - reducing condition .

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

C, Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-FLAG-WT or mutants treated with 250 μM NaAsO2 or EBSS for 1h and run under non-reducing condition.


D , Immunoblot of lysates from ARPE - 19 overexpressing TFEB - FLAG or TFE3 - MYC , treated with 250 nM Torin - 1 for 1 h run under non - reducing condition . E , of immunoblot data shown in ( D ) . Data are presented as mean ± SD using two - t - ( * ) p < 0 . 05 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 O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, Immunoblot analysis of protein lysates from ARPE-19 cells overexpressing TFEB-FLAG or TFE3-MYC, treated with 250 nM Torin-1 for 1 h and run under non-reducing condition. E, Quantification of immunoblot data shown in (D). Data are presented as mean ± SD using two-tailed t-test (*)p<0.05 from three independent experiments.


D , Relative quantitative RT - PCR of mRNA expression of autophagy - ( UVRAG ) , mitochondria - ( PGC - 1α ) lysosome ( MCOLN1 , ATPV1C1 HEXA ) - related genes in ARPE - 19 overexpressing TFEB - FLAG - WT or TFEB - FLAG - C212A . Data are presented as mean ± SD using two - way ANOVA , ( ns ) not significant as compared to same gene in TFEB - WT overexpressing , from three independent experiments .

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

D, Relative quantitative RT-PCR analysis of the mRNA expression of autophagy- (UVRAG), mitochondria- (PGC-1α) and lysosome (MCOLN1, ATPV1C1 and HEXA)-related genes in ARPE-19 cells overexpressing TFEB-FLAG-WT or TFEB-FLAG-C212A. Data are presented as mean ± SD using two-way ANOVA, (ns) not significant as compared to the same gene in TFEB-WT overexpressing cells, from three independent experiments.


G , Relative quantitative RT - PCR of mRNA expression of lysosome - related genes ( MCOLN1 ATPV61C1 ) in ARPE - 19 HeLa treated with 100 μM 50 μM NaAsO2 respectively for indicated times . Data are presented as mean ± SD using one - way ANOVA ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 from two independent experiments .

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

G, Relative quantitative RT-PCR analysis of the mRNA expression of lysosome-related genes (MCOLN1 and ATPV61C1) in ARPE-19 and HeLa cells treated with 100 μM and 50 μM NaAsO2 respectively for the indicated times. Data are presented as mean ± SD using one-way ANOVA (*)p<0.05, (**)p< 0.01, (***)p<0.001 from two independent experiments.


D , Relative quantitative RT - PCR of mRNA expression of autophagy - ( UVRAG ) , lysosome - ( ATP6V1C1 ) , oxidative stress response - ( TXNRD1 HMOX1 ) , cycle control - ( CDKN1a ) cytokine production ( IL6 ) - related genes in TFEB / TFE3 - double knockout MEFs stably expressing TFEB - FLAG - WT or TFEB - FLAG - C212A treated with 5 μM arsenic trioxide ( ATO ) or 12 . 5 μM NaAsO2 for 12 h . Data are presented as mean ± SD using two - way ANOVA ( * ) p < 0 . 05 , ( * * ) p < 0 . 01 , ( * * * ) p < 0 . 001 , ( * * * * ) p < 0 . 0001 from three independent experiments .

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

D, Relative quantitative RT-PCR analysis of the mRNA expression of autophagy- (UVRAG), lysosome- (ATP6V1C1), oxidative stress response- (TXNRD1 and HMOX1), cell cycle control- (CDKN1a) and cytokine production (IL6) -related genes in TFEB/TFE3-double knockout MEFs cells stably expressing TFEB-FLAG-WT or TFEB-FLAG-C212A and treated with 5 μM arsenic trioxide (ATO) or 12.5 μM NaAsO2 for 12 h. Data are presented as mean ± SD using two-way ANOVA (*)p<0.05, (**)p<0.01, (***)p<0.001, and (****)p<0.0001 from three independent experiments.


C , Representative examples of HLH - 30 : : 3xFLAG : : eGFP subcellular localization : , diffuse GFP distribution along whole of worm ; Weak , some in posterior part of worm display GFP labeling but no labeling is found in head ; Strong , distinct GFP labeling in along worm , including neurons in head . Arrowheads denote localization of HLH - 30 : : 3xFLAG : : eGFP . Scale bar 200 µm 50 µm . D , of subcellular distribution of HLH - 30 : : 3xFLAG : : eGFP HLH - 30 ( C284A ) : : 3xFLAG : : eGFP upon treatment with different stimuli . Data are from three independent experiments with at least 50 per experiment .

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

C, Representative examples of HLH-30::3xFLAG::eGFP subcellular localization: Cytoplasmic, diffuse GFP distribution along the whole body of the worm; Weak nuclear, some cells in the posterior part of the worm display nuclear GFP labeling but no nuclear labeling is found in the head cells; Strong nuclear, distinct nuclear GFP labeling in cells along the worm body, including neurons in the head area. Arrowheads denote nuclear localization of HLH-30::3xFLAG::eGFP. Scale bar 200 µm and 50 µm. D, Quantification of the subcellular distribution of HLH-30::3xFLAG::eGFP and HLH-30(C284A)::3xFLAG::eGFP upon treatment with different stimuli. Data are from three independent experiments with at least 50 animals per experiment.


A , Lifespan of hlh - 30 mutants at 25 ° C . Graph represents Kaplan - Meier survival plots of two independent experiments initiated with 100 per . ( * * ) p < 0 . 01 ( * * * * ) p < 0 . 0001 by Log - rank ( Mantel - Cox ) compared to wild type control . B , Survival of hlh - 30 mutants on 7 . 5 mM NaAsO2 . Data are mean ± SD of three independent experiments initiated with 120 per . ( * * * * ) p < 0 . 0001 by two - way ANOVA compared to wild type control . C , Survival of hlh - 30 mutants to infection by Staphylococcus aureus . Data are mean ± SD of three independent experiments initiated with 100 per . ( * * * * ) p < 0 . 0001 by two - way ANOVA compared to wild type control .

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

A, Lifespan assay of hlh-30 mutants at 25°C. Graph represents Kaplan-Meier survival plots of two independent experiments initiated with 100 animals per strain. (**)p<0.01 and (****)p<0.0001 by Log-rank (Mantel-Cox) test compared to wild type control. B, Survival of hlh-30 mutants on 7.5 mM NaAsO2. Data are the mean ± SD of three independent experiments initiated with 120 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control. C, Survival of hlh-30 mutants to infection by Staphylococcus aureus. Data are the mean ± SD of three independent experiments initiated with 100 animals per strain. (****)p<0.0001 by two-way ANOVA compared to wild type control.


E , Synthetic developmental delay phenotype of eat - 2 ( ad1116 ) worms carrying mutations in hlh - 30 gene . Data are mean ± SD of three independent experiments with at least 20 per . ( * * * * ) p < 0 . 0001 by One - way ANOVA with multiple comparison . F , Representative of worms quantified in ( E ) . Scale bars 200 µm .

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

E, Synthetic developmental delay phenotype of eat-2(ad1116) worms carrying mutations in the hlh-30 gene. Data are the mean ± SD of three independent experiments with at least 20 animals per assay. (****)p<0.0001 by One-way ANOVA with multiple comparison test. F, Representative images of worms quantified in (E). Scale bars 200 µm.


ISCts midguts expressing YFP alone ( control ) , or expressing two non - overlapping EcR - RNAi , of number of YFP - positive ( yellow ) . graph also plots number of GFP - positive in esgts midgut expressing GFP alone ( ctrl ) , or expressing EcR - RNAi # 1 , EcR - RNAi # 2 , mcherry - RNAi as an additional negative control ( see EV2B ) .

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

ISCts midguts expressing YFP alone (control), or expressing two non-overlapping EcR-RNAi, and quantification of the number of YFP-positive cells (yellow). The graph also plots the number of GFP-positive cells in esgts midgut expressing GFP alone (ctrl), or expressing EcR-RNAi#1, EcR-RNAi#2, and mcherry-RNAi as an additional negative control (see EV2B).