( 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 ) .

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-CONTROLLED_VAR 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) 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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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 - ) , 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 ) .

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

A. Representative 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 cells ( grown and phenotyped as mentioned in A ) . Analyzed were 31 clones from LSKs , 107 from GMPs and 28 from DN2 cells .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 .

O O O O O O O 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. 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (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 death within 33 ± 4 days after transplantation . Two DN2 clones were transplanted into 5 recipient mice .

O O O O O O O O O O O O O O O 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. 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 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 .

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

A. Representative 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 cells ; Graph shows n = 11 Myc - Bcl2 + LSK - , n = 14 DN2 - , and n = 8 GMP - transplanted animals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 cell engraftment in spleens ranged from 75 . 1 % - 95 . 5 % .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 cell 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the Myc / Bcl2 + DN2 - derived myeloid blasts .

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 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 .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 primary Myc / Bcl2 + LSK - , GMP - or DN2 - transplanted mice . Cell percentages in indicated gates are shown .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

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 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 .

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 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 ) .

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O 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. 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 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 .

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 and thymus in comparison to the reference .

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


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 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 protein expression , that diminishes after treatment with the Jak - inhibitor ruxolitinib .

O O O O O O O O O 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

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 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 ) .

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 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 .

O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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 and 3 . 5 0 . 4 without mitochondria , paired t - test , * * * p < 0 . 0001 .

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

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 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 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O 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 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 .

O O O O O O O O O O O O O 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 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 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 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 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 ) .

O O O O O 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 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 the presence of FCCP ( CMA , 20 μM ) or concanamycin A ( 20 μM ) , or in the absence of ATP .

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(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 ) 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 .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ∆ 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ∆ 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 .

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(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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 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 ) 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 .

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(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 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 .

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 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 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 .

O O O O O O O O O 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 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 : 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 .

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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 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 .

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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 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 .

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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 , 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 ) .

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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).


esgts midguts expressing GFP alone ( control ) , or expressing EcR - DN , and EcR - DN + pri . Samples were stained for GFP ( green ) . The graph shows quantification of the number of GFP - positive cells in the different genotypes .

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esgts midguts expressing GFP alone (control), or expressing EcR-DN, and EcR-DN + pri. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in the different genotypes.


Posterior midguts containing control and Ubr3 null MARCM clones ( GFP , green ) .

Posterior midguts containing control and Ubr3 null MARCM clones (GFP, green).


esgts midguts expressing GFP alone ( control ) , or expressing Ubr3 - RNAi , and Ubr3 - RNAi + OvoB . Samples were stained for GFP ( green ) . H ' quantification of GFP - positive cells from H .

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esgts midguts expressing GFP alone (control), or expressing Ubr3-RNAi, and Ubr3-RNAi + OvoB. Samples were stained for GFP (green). H' quantification of GFP-positive cells from H.


esgts midguts expressing GFP alone ( control ) or expressing SvbACT . Samples were stained for GFP ( green ) , and ß - catenin ( purple ) , DE - Cadherin ( white ) or Scribble ( yellow ) . Top and bottom pictures show separate channels for a same region .

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esgts midguts expressing GFP alone (control) or expressing SvbACT. Samples were stained for GFP (green), and ß-catenin (purple), DE-Cadherin (white) or Scribble (yellow). Top and bottom pictures show separate channels for a same region.


esgts midguts expressing GFP alone ( control ) , or expressing miR8 , OvoB , and mir8 + OvoB . Samples were stained for GFP ( green ) and ß - catenin ( purple ) . The graph shows quantification of GFP - positive cells for each genotype . The Y axis is drawn using a log ( 10 ) scale .

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esgts midguts expressing GFP alone (control), or expressing miR8, OvoB, and mir8 + OvoB. Samples were stained for GFP (green) and ß-catenin (purple). The graph shows quantification of GFP-positive cells for each genotype. The Y axis is drawn using a log(10) scale.


esgts midguts expressing GFP alone ( control ) , or expressing Notch Intra Cellular Domain ( NICD ) , and NICD + OvoB . In closeups ( right ) , DAPI is shown in purple for improved contrast ; the arrow highlights a cell with intermediate phenotype .

esgts midguts expressing GFP alone (control), or expressing Notch Intra Cellular Domain (NICD), and NICD + OvoB. In closeups (right), DAPI is shown in purple for improved contrast; the arrow highlights a cell with intermediate phenotype.


esgtsF / O midguts expressing GFP alone ( control ) , or expressing RasV12 , and RasV12 + svb - RNAi ( top panels ) . Bottom panels show esgts midguts expressing GFP alone ( control ) , or expressing EGFR - DN , and EGFR - DN + OvoB . Samples were stained for GFP ( green ) . The graph shows quantification of the number of GFP - positive cells in esgts midguts expressing GFP alone ( ctrl ) , or expressing EGFR - DN , EGFR - DN + OvoB , and EGFR - DN + svb - RNAi . The Y axis is plotted as log ( 10 ) .

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esgtsF/O midguts expressing GFP alone (control), or expressing RasV12, and RasV12 + svb-RNAi (top panels). Bottom panels show esgts midguts expressing GFP alone (control), or expressing EGFR-DN, and EGFR-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing EGFR-DN, EGFR-DN + OvoB, and EGFR-DN + svb-RNAi. The Y axis is plotted as log(10).


esgts midguts expressing GFP alone ( control ) , or expressing ArmS10 , ArmS10 + svb - RNAi , ArmS10 + OvoB , TCF - DN , and TCF - DN + OvoB . Samples were stained for GFP ( green ) . The graph shows quantification of the number of GFP - positive cells in esgts midguts expressing GFP alone ( ctrl ) , or expressing TCF - DN , TCF - DN + OvoB , and TCF - DN + svb - RNAi . The Y axis is plotted as log ( 10 ) .

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esgts midguts expressing GFP alone (control), or expressing ArmS10, ArmS10 + svb-RNAi, ArmS10 + OvoB ,TCF-DN, and TCF-DN + OvoB. Samples were stained for GFP (green). The graph shows quantification of the number of GFP-positive cells in esgts midguts expressing GFP alone (ctrl), or expressing TCF-DN, TCF-DN + OvoB, and TCF-DN + svb-RNAi. The Y axis is plotted as log(10).


esgts midguts expressing GFP alone ( control ) , or expressing SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . Closeups correspond to boxed regions , with DAPI shown in purple and GFP - positive cells outlined in yellow .

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esgts midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and Prospero (red). Closeups correspond to boxed regions, with DAPI shown in purple and GFP-positive cells outlined in yellow.


esgts - F / O midguts expressing GFP alone ( control ) , or expressing SvbREP . Samples were stained for GFP ( green ) and cleaved DCP1 ( red ) . E ' shows quantification of the number of GFP - positive cells in esgts guts expressing GFP alone ( ctrl ) , or DIAP , SvbREP , and SvbREP + DIAP .

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esgts-F/O midguts expressing GFP alone (control), or expressing SvbREP. Samples were stained for GFP (green) and cleaved DCP1 (red). E' shows quantification of the number of GFP-positive cells in esgts guts expressing GFP alone (ctrl), or DIAP, SvbREP, and SvbREP + DIAP.


esgts midguts expressing GFP alone ( control ) , or expressing Notch - RNAi , Notch - RNAi + svb - RNAi , and Notch - RNAi + SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . The graph shows quantification of the number of GFP - positive cells .

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esgts midguts expressing GFP alone (control), or expressing Notch-RNAi, Notch-RNAi + svb-RNAi, and Notch-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.


esgts midguts expressing GFP alone ( control ) , or expressing STAT - RNAi , STAT - RNAi + svb - RNAi and STAT - RNAi + SvbREP . Samples were stained for GFP ( green ) and Prospero ( red ) . The graph shows quantification of the number of GFP - positive cells .

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esgts midguts expressing GFP alone (control), or expressing STAT-RNAi, STAT-RNAi + svb-RNAi and STAT-RNAi + SvbREP. Samples were stained for GFP (green) and Prospero (red). The graph shows quantification of the number of GFP-positive cells.


Cross sections of control MyoIAts midguts ( expressing GFP and mCherry - RNAi ) , or expressing svb - RNAi , SvbACT , and Pri . Samples were stained for F - actin ( white ) , GFP ( green ) and Tsp2a ( yellow ) .

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Cross sections of control MyoIAts midguts (expressing GFP and mCherry-RNAi), or expressing svb-RNAi, SvbACT, and Pri. Samples were stained for F-actin (white), GFP (green) and Tsp2a (yellow).


MyoIAts midguts expressing GFP alone ( control ) , or expressing SvbACT . Samples were stained for GFP ( green ) , Scribble ( yellow ) , ß - catenin ( purple ) and DAPI ( Blue ) . A ' pictures display cross sections of the regions shown in A .

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MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green), Scribble (yellow), ß-catenin (purple) and DAPI (Blue). A' pictures display cross sections of the regions shown in A.


MyoIAts midguts expressing GFP alone ( control ) , or expressing SvbACT . Samples were stained for GFP ( green ) and PH3 ( red ) . The graph plots number of mitotic PH3 - positive cells per midgut of MyoIAts guts expressing GFP alone ( ctrl ) , or expressing SvbACT , and SvbREP + pri .

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-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

MyoIAts midguts expressing GFP alone (control), or expressing SvbACT. Samples were stained for GFP (green) and PH3 (red). The graph plots number of mitotic PH3-positive cells per midgut of MyoIAts guts expressing GFP alone (ctrl), or expressing SvbACT , and SvbREP + pri.


A . qPCR analysis shows loss of Setd1b in forebrain regions while levels in the cerebellum are not affected ( CA : Control , n = 6 ; cKO , n = 6 . DG : Control , n = 6 ; cKO , n = 6 . Cortex : Control , n = 7 ; cKO , n = 7 . Cerebellum : Control , n = 4 ; cKO , n = 4 ) . * p - value < 0 . 05 ( Student t - test ) .

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A. qPCR analysis shows loss of Setd1b in forebrain regions while levels in the cerebellum are not affected (CA: Control, n = 6; cKO, n = 6. DG: Control, n = 6; cKO, n = 6. Cortex: Control, n = 7; cKO, n = 7. Cerebellum: Control, n = 4; cKO, n = 4). * p-value < 0.05 (Student t-test).


B . Immunoblot analysis shows loss of Setd1b protein in the hippocampus of Setd1b cKO mice ( Control , n = 4 ; cKO , n = 4 ) . * * p - value < 0 . 01 ( Student t - test ) .

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B. Immunoblot analysis shows loss of Setd1b protein in the hippocampus of Setd1b cKO mice (Control, n = 4; cKO, n = 4). ** p-value < 0.01 (Student t-test).


C . Immunohistochemical staining ( upper level ) for marker proteins of neuronal integrity and quantification ( lower panel ) show no difference in control and Setd1b cKO mice ( NeuN : Control , n = 6 ; cKO , n = 6 ; Student t - test p - value = 0 . 57 . MAP2 : Control , n = 4 ; cKO , n = 4 ; Student t - test p - value = 0 . 72 . Iba1 : Control , n = 5 ; cKO , n = 5 ; Student t - test p - value = 0 . 8 . Gfap : Control , n = 5 ; cKO , n = 5 ; Student t - test p - value = 0 . 09 . ) . Scale bar : 100 µm .

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C. Immunohistochemical staining (upper level) for marker proteins of neuronal integrity and quantification (lower panel) show no difference in control and Setd1b cKO mice (NeuN: Control, n = 6; cKO, n = 6; Student t-test p-value = 0.57. MAP2: Control, n = 4; cKO, n = 4; Student t-test p-value = 0.72. Iba1: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.8. Gfap: Control, n = 5; cKO, n = 5; Student t-test p-value = 0.09.). Scale bar: 100 µm.


D . Average speed ( left panel ) and percent time spent in the center ( right panel ) during exposure to the open field test was similar in control and Setd1b cKO mice ( Average speed : Control , n = 15 ; cKO , n = 15 ; Student t - test p - value = 0 . 075 . Time spent in center : Control , n = 15 ; cKO , n = 15 ; Student t - test p - value = 0 . 96 ) .

D. Average speed (left panel) and percent time spent in the center (right panel) during exposure to the open field test was similar in control and Setd1b cKO mice (Average speed: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.075. Time spent in center: Control, n = 15; cKO, n = 15; Student t-test p-value = 0.96).


E . Short term memory was not different between control and Setd1b cKO mice as indicated by similar percent of alternations in the Y - maze test ( Control , n = 15 ; cKO , n = 15 ; Student t - test p - value = 0 . 3 ) .

E. Short term memory was not different between control and Setd1b cKO mice as indicated by similar percent of alternations in the Y-maze test (Control, n = 15; cKO, n = 15; Student t-test p-value = 0.3).


F . Depressive - like behavior was measured in the Porsolt forced swim test . There was no significant difference in the % time spent immobile between groups ( Control , n = 8 ; cKO , n = 8 ; Student t - test p - value = 0 . 5 ) .

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F. Depressive-like behavior was measured in the Porsolt forced swim test. There was no significant difference in the % time spent immobile between groups (Control, n = 8; cKO, n = 8; Student t-test p-value = 0.5).


G . Escape latency during water maze training indicated severe learning impairment in Setd1b cKO mice ( Control : n = 15 , cKO : n = 15 . Repeated measures ANOVA , genotype effect : F ( 1 , 28 ) = 82 . 34 , * * * * p - value < 0 . 0001 ) .

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G. Escape latency during water maze training indicated severe learning impairment in Setd1b cKO mice (Control: n = 15, cKO: n = 15. Repeated measures ANOVA, genotype effect: F (1,28) = 82.34, **** p-value < 0.0001).


H . Plots showing the specific search strategies during water maze training . Note the failure of Setd1b cKO mice to adopt hippocampus - dependent search strategies . I . The cognitive score calculated on the basis of the hippocampal search strategies reflects severe memory impairment in Setd1b cKO mice ( Student t - test : * * * p - value < 0 . 001 ) .

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H. Plots showing the specific search strategies during water maze training. Note the failure of Setd1b cKO mice to adopt hippocampus-dependent search strategies. I. The cognitive score calculated on the basis of the hippocampal search strategies reflects severe memory impairment in Setd1b cKO mice (Student t-test: *** p-value < 0.001).


F . Left panel shows representative images of RNAscope performed for Kmt2a , Kmt2b and Setd1b . Right panel shows a bar chart quantifying the dots / cell ( n = 1500 cells from 2 mice ) indicative of the corresponding expression level . Kmt2a expression was significantly higher when compared to Kmt2b or Setd1b ( * * * p - value < 0 , 0001 , Student t - test ) .

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F. Left panel shows representative images of RNAscope performed for Kmt2a, Kmt2b and Setd1b. Right panel shows a bar chart quantifying the dots/cell (n = 1500 cells from 2 mice) indicative of the corresponding expression level. Kmt2a expression was significantly higher when compared to Kmt2b or Setd1b (*** p-value < 0,0001, Student t-test).


( A ) The transcript level of solo LTR in the wild type and the epcr1 mutant . Error bars are standard deviations of three technical replicates . The experiment was independently performed three times and similar results were obtained .

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(A) The transcript level of solo LTR in the wild type and the epcr1 mutant. Error bars are standard deviations of three technical replicates. The experiment was independently performed three times and similar results were obtained.


( B ) The developmental phenotype of the epcr1 and epcr2 single mutants and the epcr1 / 2 double mutant relative to the wild type . Two - week - old seedlings grown on vertical MS medium plates are shown .

(B) The developmental phenotype of the epcr1 and epcr2 single mutants and the epcr1/2 double mutant relative to the wild type. Two-week-old seedlings grown on vertical MS medium plates are shown.


( C ) The transcript levels of solo LTR , SDC , AtSN1 , AtGP1 and AtCOPIA28 in the wild type , epcr1 and epcr2 single mutants , epcr1 / 2 double mutant , and two individual EPCR1 transgenic lines in the epcr1 / 2 double mutant background . Error bars are standard deviations of three biological replicates .

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(C) The transcript levels of solo LTR, SDC, AtSN1, AtGP1 and AtCOPIA28 in the wild type, epcr1 and epcr2 single mutants, epcr1/2 double mutant, and two individual EPCR1 transgenic lines in the epcr1/2 double mutant background. Error bars are standard deviations of three biological replicates.


( A ) The interaction between EPCR1 and ARID2 or ARID3 . Arabidopsis plants carrying EPCR1 - Flag and ARID2 - Myc or ARID3 - Myc transgenes were used for co - IP .

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(A) The interaction between EPCR1 and ARID2 or ARID3. Arabidopsis plants carrying EPCR1-Flag and ARID2-Myc or ARID3-Myc transgenes were used for co-IP.


( B ) The interaction between TRB1 and ARID2 . Arabidopsis plants carrying TRB1 - Flag and / or ARID2 - Myc transgenes were used for co - IP .

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(B) The interaction between TRB1 and ARID2. Arabidopsis plants carrying TRB1-Flag and/or ARID2-Myc transgenes were used for co-IP.


( C ) The interaction between TRB1 and EPCR1 . Arabidopsis plants carrying TRB1 - Flag and / or EPCR1 - Myc transgenes were used for co - IP .

(C) The interaction between TRB1 and EPCR1. Arabidopsis plants carrying TRB1-Flag and/or EPCR1-Myc transgenes were used for co-IP.


( E ) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull - down assay .

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(E) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull-down assay.