Figure 4A . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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.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.


Figure 5A . 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 . 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 . 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 .

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

Figure 5A. 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.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.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.


Figure 6A . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. 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).


Figure 7A . 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 . 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 % . 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 . 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 ) .

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Figure 7A. 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.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%.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.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).


Figure 1 ( A ) LncRNA CRNDE expression level was measured by qRT - PCR in cancerous tissues and adjacent normal tissues obtained from 35 cases of gastric cancer ( GC ) patients . Data information : Data are expressed as mean ± SD . Student ' s t - test * * P < 0 . 01 . ( C ) LncRNA CRNDE expression level was measured by qRT - PCR in adjacent normal tissue - derived macrophages ( NTMs ) and tumor - associated macrophages ( TAMs ) obtained from 7 cases of GC patients . Data information : Data are expressed as mean ± SD . Student ' s t - test * * P < 0 . 01 .

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Figure 1(A) LncRNA CRNDE expression level was measured by qRT-PCR in cancerous tissues and adjacent normal tissues obtained from 35 cases of gastric cancer (GC) patients. Data information: Data are expressed as mean ± SD. Student's t-test **P<0.01.(C) LncRNA CRNDE expression level was measured by qRT-PCR in adjacent normal tissue-derived macrophages (NTMs) and tumor-associated macrophages (TAMs) obtained from 7 cases of GC patients. Data information: Data are expressed as mean ± SD. Student's t-test **P<0.01.


Figure 2 ( C ) and ( D ) Left : representative electron micrograph of exosomes isolated from the medium of BMDMs , BMDM - polarized M2 macrophages , monocytes and monocyte - polarized M2 macrophages are depicted , showing the typical morphology of exosomes . Upper right : the protein levels of CD63 and CD81 were measured by Western blot . Lower left : Nanoparticle tracking analysis showed that most of the particles had a similar size of 50 - 150 nm in diameter with a peak at around 100 nm . Lower right : LncRNA CRNDE expression levels in different exosomes . Data information : Scale bar = 100 nm . The results shown are from three biological replicates . Data are expressed as mean ± SD . Student ' s t - test . * * P < 0 . 01 vs . BMDMs or monocytes or BMDMs - exo or monocytes - exo .

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

Figure 2(C) and (D) Left: representative electron micrograph of exosomes isolated from the medium of BMDMs, BMDM-polarized M2 macrophages, monocytes and monocyte-polarized M2 macrophages are depicted, showing the typical morphology of exosomes. Upper right: the protein levels of CD63 and CD81 were measured by Western blot. Lower left: Nanoparticle tracking analysis showed that most of the particles had a similar size of 50-150 nm in diameter with a peak at around 100 nm. Lower right: LncRNA CRNDE expression levels in different exosomes. Data information: Scale bar= 100 nm. The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test. **P<0.01 vs. BMDMs or monocytes or BMDMs-exo or monocytes-exo.


Figure 3 ( B ) BMDM - polarized M2 macrophages were transfected with Cy3 - labelled CRNDE and exosomes were isolated from the medium ( Cy3 - CRNDE - EXO ) . Then , MFC cells were incubated with Cy3 - CRNDE - EXO . Left : Confocal microscopy was used to detect the Cy3 - labelled CRNDE in MFC cells . The nuclei were stained with DAPI . Scale bar = 20 µm . Right : Flow cytometry was performed to quantitate the Cy3 - positive cells . * * P < 0 . 01 vs . MFC cells incubated with the control EXO . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . Student ' s t - test . ( D ) Monocyte - polarized M2 macrophages were transfected with Cy3 - labelled CRNDE and exosomes were isolated from the medium ( Cy3 - CRNDE - EXO ) . Then , SGC7901 cells were incubated with Cy3 - CRNDE - EXO . Confocal microscopy was used to detect the Cy3 - labelled CRNDE in SGC7901 cells . The nuclei were stained with DAPI . Scale bar = 20 µm . Right : Flow cytometry was performed to quantitate the Cy3 - positive cells . * * P < 0 . 01 vs . SGC7901 cells incubated with the control EXO . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . Student ' s t - test . ( E ) SGC7901 cells were incubated with chlorpromazine ( CPZ , 50 μM ) , genistein ( GEN , 200 μM ) , 5 - ( n - ethyl - n - isopropyl ) - amiloride ( EIPA , 100 μM ) , or methyl - β - cyclodextrin ( MβCD , 10 mM ) , following by the incubation of Cy3 - CRNDE - EXO . Confocal microscopy was used to detect the Cy3 - labelled CRNDE in SGC7901 cells . The nuclei were stained with DAPI . Right : Flow cytometry was performed to quantitate the Cy3 - positive cells . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . ( F ) SGC7901 cells were incubated with 0 , 25 , 50 , 100 μM CPZ , following by the incubation of Cy3 - CRNDE - EXO . Confocal microscopy was used to detect the Cy3 - labelled CRNDE in SGC7901 cells . The nuclei were stained with DAPI . Right : Flow cytometry was performed to quantitate the Cy3 - positive cells . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA .

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

Figure 3(B) BMDM-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, MFC cells were incubated with Cy3-CRNDE-EXO. Left: Confocal microscopy was used to detect the Cy3-labelled CRNDE in MFC cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. MFC cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.(D) Monocyte-polarized M2 macrophages were transfected with Cy3-labelled CRNDE and exosomes were isolated from the medium (Cy3-CRNDE-EXO). Then, SGC7901 cells were incubated with Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Scale bar= 20 µm. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. **P<0.01 vs. SGC7901 cells incubated with the control EXO. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. Student's t-test.(E) SGC7901 cells were incubated with chlorpromazine (CPZ, 50 μM), genistein (GEN, 200 μM), 5-(n-ethyl-n-isopropyl)-amiloride (EIPA, 100 μM), or methyl-β-cyclodextrin (MβCD, 10 mM), following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.(F) SGC7901 cells were incubated with 0, 25, 50, 100 μM CPZ, following by the incubation of Cy3-CRNDE-EXO. Confocal microscopy was used to detect the Cy3-labelled CRNDE in SGC7901 cells. The nuclei were stained with DAPI. Right: Flow cytometry was performed to quantitate the Cy3-positive cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA.


Figure 4Exosomes were isolated from the media of BMDMs ( BMDMs - exo ) , BMDM - polarized M2 macrophages ( M2 - exo ) , BMDM - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , and BMDM - polarized M2 macrophages transfected with the negative control of si - CRNDE ( M2 - si - control - exo ) . ( A ) LncRNA CRNDE expression in indicated exosomes was detected by qRT - PCR . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . : one - way ANOVA . * * P < 0 . 01 . Exosomes were isolated from the media of BMDMs ( BMDMs - exo ) , BMDM - polarized M2 macrophages ( M2 - exo ) , BMDM - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , and BMDM - polarized M2 macrophages transfected with the negative control of si - CRNDE ( M2 - si - control - exo ) . Then , MFC cells were treated with indicated exosomes and cisplatin ( CDDP ; 1μg / ml ) for 48 h . ( C ) Representative images of colony formation assay performed on MFC cells . Exosomes were isolated from the medium of monocytes ( monocytes - exo ) , monocyte - polarized M2 macrophages ( M2 - exo ) , monocyte - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , and monocyte - polarized M2 macrophages transfected with si - control ( M2 - si - control - exo ) . ( E ) LncRNA CRNDE expression were measured . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . * * P < 0 . 01 . Exosomes were isolated from the medium of monocytes ( monocytes - exo ) , monocyte - polarized M2 macrophages ( M2 - exo ) , monocyte - polarized M2 macrophages transfected with si - CRNDE ( M2 - si - CRNDE - exo ) , and monocyte - polarized M2 macrophages transfected with si - control ( M2 - si - control - exo ) . Then , SGC7901 cells were treated with indicated exosomes and CDDP ( 1μg / ml ) . ( G ) cell proliferation were measured .

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

Figure 4Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). (A) LncRNA CRNDE expression in indicated exosomes was detected by qRT-PCR. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. : one-way ANOVA. **P<0.01.Exosomes were isolated from the media of BMDMs (BMDMs-exo), BMDM-polarized M2 macrophages (M2-exo), BMDM-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and BMDM-polarized M2 macrophages transfected with the negative control of si-CRNDE (M2-si-control-exo). Then, MFC cells were treated with indicated exosomes and cisplatin (CDDP; 1μg/ml) for 48 h. (C) Representative images of colony formation assay performed on MFC cells.Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). (E) LncRNA CRNDE expression were measured. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.Exosomes were isolated from the medium of monocytes (monocytes-exo), monocyte-polarized M2 macrophages (M2-exo), monocyte-polarized M2 macrophages transfected with si-CRNDE (M2-si-CRNDE-exo), and monocyte-polarized M2 macrophages transfected with si-control (M2-si-control-exo). Then, SGC7901 cells were treated with indicated exosomes and CDDP (1μg/ml). (G) cell proliferation were measured.


Figure 53 × 105 MFC cells were subcutaneously injected into nude mice . Ten days after injection , BMDMs - exo , M2 - exo , M2 - si - control - exo , or M2 - si - CRNDE - exo ( 10 µg ) was injected into the center of the homograft tumors of mice ( n = 7 each group ) , followed by intraperitoneal injection of CDDP ( 10 mg / kg ) . Six days after CDDP treatment , all mice were sacrificed and the homograft tumors were collected . ( A ) Growth curves of MFC subcutaneous homograft tumors ( The homograft tumor sizes were recorded from the day of CDDP treatment ) . * P < 0 . 05 , * * P < 0 . 01 vs . BMDMs - exo ; # P < 0 . 05 , # # P < 0 . 01 vs . M2 - si - control - exo . Data information : Data are expressed as mean ± SD . one - way ANOVA . 3 × 105 MFC cells were subcutaneously injected into nude mice . Ten days after injection , BMDMs - exo , M2 - exo , M2 - si - control - exo , or M2 - si - CRNDE - exo ( 10 µg ) was injected into the center of the homograft tumors of mice ( n = 7 each group ) , followed by intraperitoneal injection of CDDP ( 10 mg / kg ) . Six days after CDDP treatment , all mice were sacrificed and the homograft tumors were collected . ( D ) LncRNA CRNDE expression in homograft tumors of mice . * * P < 0 . 01 . Data information : Data are expressed as mean ± SD . : one - way ANOVA .

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

Figure 53×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected. (A) Growth curves of MFC subcutaneous homograft tumors (The homograft tumor sizes were recorded from the day of CDDP treatment). *P<0.05, **P<0.01 vs. BMDMs-exo; #P<0.05, ##P<0.01 vs. M2-si-control-exo. Data information: Data are expressed as mean ± SD. one-way ANOVA.3×105 MFC cells were subcutaneously injected into nude mice. Ten days after injection, BMDMs-exo, M2-exo, M2-si-control-exo, or M2-si-CRNDE-exo (10 µg) was injected into the center of the homograft tumors of mice (n=7 each group), followed by intraperitoneal injection of CDDP (10 mg/kg). Six days after CDDP treatment, all mice were sacrificed and the homograft tumors were collected. (D) LncRNA CRNDE expression in homograft tumors of mice. **P<0.01. Data information: Data are expressed as mean ± SD. : one-way ANOVA.


Figure 6 ( C ) SGC7901 cells were transfected with pcDNA - CRNDE or its negative control ( Mock ) before MG132 treatment ( 10 µM , 6 h ) . The Co - immunoprecipitation ( Co - IP ) assay was performed to examine the combination of PTEN and NEDD4 - 1 . ( E ) The expressions of PTEN and NEDD4 - 1 were measured in SGC7901 cells transfected with pcDNA - CRNDE or Mock at four time - points after cycloheximide ( CHX , 100 μg / mL ) treatment . GAPDH was served as an internal control . Left : Representative bands . Right : Quantitative results . The results shown are from three biological replicates and are expressed as mean ± SD . Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 vs . Mock . G ) The ubiquitylation of PTEN was measured in 293T cells transfected with indicated vectors .

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

Figure 6(C) SGC7901 cells were transfected with pcDNA-CRNDE or its negative control (Mock) before MG132 treatment (10 µM, 6 h). The Co-immunoprecipitation (Co-IP) assay was performed to examine the combination of PTEN and NEDD4-1.(E) The expressions of PTEN and NEDD4-1 were measured in SGC7901 cells transfected with pcDNA-CRNDE or Mock at four time-points after cycloheximide (CHX, 100 μg/mL) treatment. GAPDH was served as an internal control. Left: Representative bands. Right: Quantitative results. The results shown are from three biological replicates and are expressed as mean ± SD. Student's t-test. *P<0.05, **P<0.01 vs. Mock.G) The ubiquitylation of PTEN was measured in 293T cells transfected with indicated vectors.


Figure 7Normal , si - control - transfected , or si - PTEN - transfected SGC7901 cells were treated with CDDP ( 1 µg / ml ) and M2 - si - control - exo or M2 - si - CRNDE - exo . Si - control , the negative control of si - PTEN . ( A ) The protein levels of PTEN , phosphorylated phosphatidylinositol 3 - kinase ( p - PI3K ) , phosphorylated protein kinase B ( p - AKT ) , p - glycoprotein ( p - gp ) , B - cell lymphoma 2 ( Bcl - 2 ) were measured . GAPDH was served as an internal control . Normal , si - control - transfected , or si - PTEN - transfected SGC7901 cells were treated with CDDP ( 1 µg / ml ) and M2 - si - control - exo or M2 - si - CRNDE - exo . Si - control , the negative control of si - PTEN . The cell apoptosis in SGC7901 cells . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . * * P < 0 . 01 . SGC7901 cells were divided into four groups : M2 - si - control - exo , M2 - si - CRNDE - exo , M2 - si - CRNDE - exo + PTEN inhibitor ( SF1670 ; 50 μmol / l ) , M2 - si - control - exo + PTEN inhibitor . All cells were treated with CDDP . The cell survival rate were measured . Data information : The results shown are from seven biological replicates . Data are expressed as mean ± SD . : one - way ANOVA . * * P < 0 . 01 . SGC7901 cells were divided into four groups : M2 - si - control - exo , M2 - si - CRNDE - exo , M2 - si - CRNDE - exo + PTEN inhibitor ( SF1670 ; 50 μmol / l ) , M2 - si - control - exo + PTEN inhibitor . All cells were treated with CDDP . The cell apoptosis were measured . Data information : The results shown are from three biological replicates . Data are expressed as mean ± SD . one - way ANOVA . * * P < 0 . 01 .

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

Figure 7Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. (A) The protein levels of PTEN, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-AKT), p-glycoprotein (p-gp), B-cell lymphoma 2 (Bcl-2) were measured. GAPDH was served as an internal control.Normal, si-control-transfected, or si-PTEN-transfected SGC7901 cells were treated with CDDP (1 µg/ml) and M2-si-control-exo or M2-si-CRNDE-exo. Si-control, the negative control of si-PTEN. The cell apoptosis in SGC7901 cells. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.SGC7901 cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell survival rate were measured. Data information: The results shown are from seven biological replicates. Data are expressed as mean ± SD. : one-way ANOVA. **P<0.01.SGC7901 cells were divided into four groups: M2-si-control-exo, M2-si-CRNDE-exo, M2-si-CRNDE-exo +PTEN inhibitor (SF1670; 50 μmol/l), M2-si-control-exo+PTEN inhibitor. All cells were treated with CDDP. The cell apoptosis were measured. Data information: The results shown are from three biological replicates. Data are expressed as mean ± SD. one-way ANOVA. **P<0.01.


Figure 1A . cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 and the wildtype , from left to right on control plates without genotoxic agent 10 days after germination . B . The wildtype , single and double mutants of cycb1 were grown on control plates without genotoxic agent . Root lengths were measured 10 days after germination . C . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 1 mM hydroxy urea ( HU ) 10 days after germination . The rightmost plant is the wee1 mutant that shows high sensitivity to HU . D . The wildtype , single and double mutants of cycb1 were grown on plates supplemented with 1 mM HU . Root lengths were measured 10 days after germination . E . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 0 , 6 μg / mL bleomycin ( BLM ) 10 days after germination . The rightmost plant is the ku70 mutant that shows high sensitivity to BLM . F . The wildtype , single and double mutants of cycb1 were grown on plates supplemented with 0 , 6 μg / mL BLM . Root lengths were measured 10 days after germination . G . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 15 μM cisplatin 6 days after germination , i . e . 3 days after transfer from control plates . H . cycb1 mutants were germinated on control plates and were transferred to new control plates three days after germination . Root lengths mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . I . cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . J . cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . K . The wildtype , cycb1 ; 1 , cycb1 ; 2 , cycb1 ; 3 , cycb1 ; 4 , cycb1 ; 1 / 1 ; 2 , cycb1 ; 1 / 1 ; 3 , cycb1 ; 1 / 1 ; 4 , cycb1 ; 2 / 1 ; 4 , cycb1 ; 3 / 1 ; 4 from left to right on plates containing 30 μM cisplatin 6 days after germination , i . e . 3 days after transfer from control plates . L . cycb1 double mutants germinated on control plates and were transferred to new control plates three days after germination . Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs . M . cycb1 mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs . N . cycb1 mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination . Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs .

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

Figure 1A. cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 and the wildtype, from left to right on control plates without genotoxic agent 10 days after germination.B. The wildtype, single and double mutants of cycb1 were grown on control plates without genotoxic agent. Root lengths were measured 10 days after germination.C. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 1 mM hydroxy urea (HU) 10 days after germination. The rightmost plant is the wee1 mutant that shows high sensitivity to HU.D. The wildtype, single and double mutants of cycb1 were grown on plates supplemented with 1 mM HU. Root lengths were measured 10 days after germination.E. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 0,6 μg/mL bleomycin (BLM) 10 days after germination. The rightmost plant is the ku70 mutant that shows high sensitivity to BLM.F. The wildtype, single and double mutants of cycb1 were grown on plates supplemented with 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.G. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 15 μM cisplatin 6 days after germination, i.e. 3 days after transfer from control plates.H. cycb1 mutants were germinated on control plates and were transferred to new control plates three days after germination. Root lengths mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.I. cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.J. cycb1 single mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.K. The wildtype, cycb1;1, cycb1;2, cycb1;3, cycb1;4, cycb1;1/1;2, cycb1;1/1;3, cycb1;1/1;4, cycb1;2/1;4, cycb1;3/1;4 from left to right on plates containing 30 μM cisplatin 6 days after germination, i.e. 3 days after transfer from control plates.L. cycb1 double mutants germinated on control plates and were transferred to new control plates three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.M. cycb1 mutants germinated on control plates and were transferred to plates supplemented with 15 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.N. cycb1 mutants germinated on control plates and were transferred to plates supplemented with 30 μM cisplatin three days after germination. Root lengths of cycb1 mutants were measured three days after transfer and the net root growth of three days is shown in the graphs.


Figure 2A . Representative examples of comets of 21 days old wild - type plants , cdkb1 ; 1 cdkb1 ; 2 and cycb1 ; 1 cycb1 ; 3 double mutant seedlings in full spectrum view of the TriTek Comet Score software . Shown are comets of plants incubated with 50µM cisplatin for one hour and then transferred to medium without cisplatin for 30 min ( recovery ) and plants incubated without cisplatin for one hour ( control ) , respectively . B . Box plot of percentage of tail DNA of wild - type cells , cdkb1 ; 1 cdkb1 ; 2 and cycb1 ; 1 cycb1 ; 3 under cisplatin treatment . Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replications . The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software . The box represents the interquartile range , the line across the box indicates the median values and whiskers represent 5 - 95 percentile values . Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99 . 99 % calculated with Student ' s t - test . C . Immunostaining of γ - H2AX foci in wild - type plants and mutant cells after two hours of treatment with 50µM cisplatin . D . Counted number of γ - H2AX foci per cell detected after two hours of treatment with 50µM cisplatin in wild - type and mutant plants . For each sample the γ - H2AX foci of 100 cells were counted and grouped into six categories : cells with no , 1 - 2 , 3 - 5 , 6 - 10 , 11 - 20 and more than 20 foci per cell .

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

Figure 2A. Representative examples of comets of 21 days old wild-type plants, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 double mutant seedlings in full spectrum view of the TriTek Comet Score software. Shown are comets of plants incubated with 50µM cisplatin for one hour and then transferred to medium without cisplatin for 30 min (recovery) and plants incubated without cisplatin for one hour (control), respectively.B. Box plot of percentage of tail DNA of wild-type cells, cdkb1;1 cdkb1;2 and cycb1;1 cycb1;3 under cisplatin treatment. Plots are based on analyses of 200 cells per sample from random microscopic fields of three independent biological replications. The percentage of DNA fragments in the comet tail was calculated by the TriTek Comet Score software. The box represents the interquartile range, the line across the box indicates the median values and whiskers represent 5-95 percentile values. Brackets connect plots of sample groups that are significantly different with a confidence level higher than 99.99% calculated with Student's t-test.C. Immunostaining of γ-H2AX foci in wild-type plants and mutant cells after two hours of treatment with 50µM cisplatin.D. Counted number of γ-H2AX foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the γ-H2AX foci of 100 cells were counted and grouped into six categories: cells with no, 1-2, 3-5, 6-10, 11-20 and more than 20 foci per cell.


Figure 3B . Wild - type plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin . Arrows indicate representative blue sectors . C . cycb1 ; 1 plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin . Arrows indicate representative blue sectors . D . Graphs show numbers of blue sectors per plant grown without drug treatment . E . Graphs show numbers of blue sectors per plant grown after incubation on 15 μM cisplatin for three days . F . Graphs show numbers of blue sectors per plant grown after incubation on 30 μM cisplatin for three days .

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Figure 3B. Wild-type plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.C. cycb1;1 plants show blue spots on the leaves after three days of incubation on 30 μM cisplatin. Arrows indicate representative blue sectors.D. Graphs show numbers of blue sectors per plant grown without drug treatment.E. Graphs show numbers of blue sectors per plant grown after incubation on 15 μM cisplatin for three days.F. Graphs show numbers of blue sectors per plant grown after incubation on 30 μM cisplatin for three days.


Figure 4A . The wildtype and CDKA ; 1 - DE mutants were grown on control plates for 10 days . Root lengths were measured 10 days after germination . B . The wildtype and CDKA ; 1 - DE mutants were grown on plates containing 1 mM hydroxy urea for 10 days . The mutant wee1 was used as a positive control for hydroxy urea sensitivity . Root lengths were measured 10 days after germination . C . The wildtype and CDKA ; 1 - DE mutants were grown on plates containing 0 , 6 μg / mL bleomycin for 10 days . The mutant ku70 was used as a positive control for bleomycin sensitivity . Root lengths were measured 10 days after germination . D . The wildtype , cdkb1 ; 1 , cdkb1 ; 2 and the double mutant cdkb1 ; 1 cdkb1 ; 2 were grown on control plates for 10 days . Root lengths were measured 10 days after germination . E . The wildtype , cdkb1 ; 1 , cdkb1 ; 2 and the double mutant cdkb1 ; 1 cdkb1 ; 2 were grown on plates containing 1 mM hydroxy urea for 10 days The mutant wee1 was used as a positive control for hydroxy urea sensitivity . Root lengths were measured 10 days after germination . F . The wildtype , cdkb1 ; 1 , cdkb1 ; 2 and the double mutant cdkb1 ; 1 cdkb1 ; 2 were grown on plates containing 0 , 6 μg / mL bleomycin for 10 days . The mutant ku70 was used as a positive control for bleomycin sensitivity . Root lengths were measured 10 days after germination . G . The wildtype , CDKA ; 1 - DE ( grey bar ) and the double mutant cdkb1 ; 1cdkb1 ; 2 ( purple bar ) were grown on control plates and were transferred to plates containing 15 μM or 30 μM cisplatin three days after germination . Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs . H . Graphs represent the ratio of the mean growth rate on 15 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype , CDKA ; 1 - DE and cdkb1 ; 1cdkb1 ; 2 . I . Graphs represent the ratio of the mean growth rate on 30 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype , CDKA ; 1 - DE and cdkb1 ; 1cdkb1 ; 2 . J - N . Images show a wild - type plant , CDKA ; 1 - DE and the double mutant cdkb1 ; 1 cdkb1 ; 2 ( from left to right ) on the indicated day after germination and the indicated drug treatment .

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

Figure 4A. The wildtype and CDKA;1-DE mutants were grown on control plates for 10 days. Root lengths were measured 10 days after germination.B. The wildtype and CDKA;1-DE mutants were grown on plates containing 1 mM hydroxy urea for 10 days. The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.C. The wildtype and CDKA;1-DE mutants were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.D. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on control plates for 10 days. Root lengths were measured 10 days after germination.E. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 1 mM hydroxy urea for 10 days The mutant wee1 was used as a positive control for hydroxy urea sensitivity. Root lengths were measured 10 days after germination.F. The wildtype, cdkb1;1, cdkb1;2 and the double mutant cdkb1;1 cdkb1;2 were grown on plates containing 0,6 μg/mL bleomycin for 10 days. The mutant ku70 was used as a positive control for bleomycin sensitivity. Root lengths were measured 10 days after germination.G. The wildtype, CDKA;1-DE (grey bar) and the double mutant cdkb1;1cdkb1;2(purple bar) were grown on control plates and were transferred to plates containing 15 μM or 30 μM cisplatin three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graphs.H. Graphs represent the ratio of the mean growth rate on 15 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2.I. Graphs represent the ratio of the mean growth rate on 30 μM cisplatin compared to control experiments on plates lacking cisplatin for the wildtype, CDKA;1-DE and cdkb1;1cdkb1;2.J-N. Images show a wild-type plant, CDKA;1-DE and the double mutant cdkb1;1 cdkb1;2 (from left to right) on the indicated day after germination and the indicated drug treatment.


Figure 5A . WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 μM or 30 μM cisplatin , respectively , three days after germination . Root lengths were measured three days after transfer and the net root growth of three days is shown in the graph . Asterisk indicates significant differences within a 5 % confidence interval ( Student ' s t - test ) . B . Image shows a wild - type plant ( left ) and rad51 mutant ( right ) grown on control plates . Images were taken 6 days after germination . C . Image shows a wild - type plant ( left ) and rad51 mutant ( right ) germinated on control plates and transferred to plates supplemented with 15 μM three days after germination . Images were taken 6 days after germination , i . e . 3 days after transfer to cisplatin . D . Immunostaining of RAD51 foci in the wildtype and indicated mutant cells after two hours of treatment with 50µM cisplatin . E . Counted number of RAD51 foci per cell detected after two hours of treatment with 50µM cisplatin in wild - type and mutant plants . For each sample the RAD51 foci of 100 cells were counted and grouped into six categories : cells with 0 , 1 , 2 , 3 , 4 or > 5 foci per cell . F . In vitro kinase assay of purified CDK complexes phosphorylating RAD51 . RAD51 and histone H1 kinase assays were performed with [ γ - 32P ] ATP as a phosphate donor . Proteins were subjected to SDS - PAGE after the kinase reaction and stained with coomassie brilliant blue demonstrating the equal loading of the substrates ( lower panels ) . Phosphorylated proteins were detected by autoradiograph ( upper panels ) . The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK - cyclin complexes , protein blots indicate the relative amounts of the CDKs in the reaction ( bottom panel ) . Abbreviation : p - RAD51 and p - Histone H1 for [ 32P ] - phosphorylated MBP - RAD51 - His6 and recombinant human histone H1 , respectively , resulting from kinase assays with radio - labeled ATP . Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure . 1 : without kinase , 2 : CDKA ; 1 - CYCA2 ; 3 , 3 : CDKB1 ; 1 - CYCA2 ; 3 , 4 : CDKB1 ; 1 - CYCD2 ; 1 , 5 : CDKA ; 1 - CYCB1 ; 1 , 6 : CDKB1 ; 1 - CYCB1 ; 1 .

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Figure 5A. WT and rad51 mutants were grown on control plates or transferred to plates supplemented with 15 μM or 30 μM cisplatin, respectively, three days after germination. Root lengths were measured three days after transfer and the net root growth of three days is shown in the graph. Asterisk indicates significant differences within a 5% confidence interval (Student's t-test).B. Image shows a wild-type plant (left) and rad51 mutant (right) grown on control plates. Images were taken 6 days after germination.C. Image shows a wild-type plant (left) and rad51 mutant (right) germinated on control plates and transferred to plates supplemented with 15 μM three days after germination. Images were taken 6 days after germination, i.e. 3 days after transfer to cisplatin.D. Immunostaining of RAD51 foci in the wildtype and indicated mutant cells after two hours of treatment with 50µM cisplatin.E. Counted number of RAD51 foci per cell detected after two hours of treatment with 50µM cisplatin in wild-type and mutant plants. For each sample the RAD51 foci of 100 cells were counted and grouped into six categories: cells with 0, 1, 2, 3, 4 or >5 foci per cell.F. In vitro kinase assay of purified CDK complexes phosphorylating RAD51. RAD51 and histone H1 kinase assays were performed with [γ-32P]ATP as a phosphate donor. Proteins were subjected to SDS-PAGE after the kinase reaction and stained with coomassie brilliant blue demonstrating the equal loading of the substrates (lower panels). Phosphorylated proteins were detected by autoradiograph (upper panels). The reactions were normalized by using equal amounts of CDKs assuring equal levels of active CDK-cyclin complexes, protein blots indicate the relative amounts of the CDKs in the reaction (bottom panel).Abbreviation: p-RAD51 and p-Histone H1 for [32P]-phosphorylated MBP-RAD51-His6 and recombinant human histone H1, respectively, resulting from kinase assays with radio-labeled ATP. Asterisks indicate varying amounts of cyclins that can be in the reaction due to purification procedure. 1: without kinase, 2: CDKA;1-CYCA2;3, 3: CDKB1;1-CYCA2;3, 4: CDKB1;1-CYCD2;1, 5: CDKA;1-CYCB1;1, 6: CDKB1;1-CYCB1;1.


Figure 6A . Transgenic Arabidopsis plants harboring the CYCB1 ; 1 promoter and a GFP fused to the N - terminal part of CYCB1 ; 1 including the destruction box grown on control plates and imaged five days after germination . D . Transgenic Arabidopsis plants harboring the CYCB1 ; 1 promoter and a GFP fused to the N - terminal part of CYCB1 ; 1 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application . B . Transgenic Arabidopsis plants harboring the CYCB1 ; 2 promoter and a GFP fused to the N - terminal part of CYCB1 ; 2 including the destruction box grown on control plates and imaged five days after germination . E . Transgenic Arabidopsis plants harboring the CYCB1 ; 2 promoter and a GFP fused to the N - terminal part of CYCB1 ; 2 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application . C . Transgenic Arabidopsis plants harboring CDKB1 ; 1 promoter fused to GUS grown on control plates , were stained and imaged five days after germination . F . Transgenic Arabidopsis plants harboring CDKB1 ; 1 promoter fused to GUS germinated on control plates and were transferred four days after germination on plates supplemented with 50 μM cisplatin and were stained and imaged 24h after drug application . G . Chromatin immunoprecipitation ( ChIP ) of wild - type plants and PROSOG1 : SOG1 - Myc lines and anti - MYC antibody . The promoter region of CYCB1 ; 1 is enriched in SOG1 - MYC after cisplatin treatment . H . Structure of genes tested by ChIP with an antibody anti - MYC in PROSOG1 : SOG1 - Myc and wild - type plants . Red arrowheads indicate the primer binding sites for PCR . A total of four regions were tested as indicated by arrowheads . Asterisks indicates significant differences within a 5 % confidence interval ( Student ' s t - test ) .

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Figure 6A. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box grown on control plates and imaged five days after germination.D. Transgenic Arabidopsis plants harboring the CYCB1;1 promoter and a GFP fused to the N-terminal part of CYCB1;1 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.B. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box grown on control plates and imaged five days after germination.E. Transgenic Arabidopsis plants harboring the CYCB1;2 promoter and a GFP fused to the N-terminal part of CYCB1;2 including the destruction box germinated on control plates and were transferred four days after germination to plates supplemented with 50 μM cisplatin and were imaged 24h after drug application.C. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS grown on control plates, were stained and imaged five days after germination.F. Transgenic Arabidopsis plants harboring CDKB1;1 promoter fused to GUS germinated on control plates and were transferred four days after germination on plates supplemented with 50 μM cisplatin and were stained and imaged 24h after drug application.G. Chromatin immunoprecipitation (ChIP) of wild-type plants and PROSOG1:SOG1-Myc lines and anti-MYC antibody. The promoter region of CYCB1;1 is enriched in SOG1-MYC after cisplatin treatment.H. Structure of genes tested by ChIP with an antibody anti-MYC in PROSOG1:SOG1-Myc and wild-type plants. Red arrowheads indicate the primer binding sites for PCR. A total of four regions were tested as indicated by arrowheads. Asterisks indicates significant differences within a 5% confidence interval (Student's t-test).


Figure 7A . The wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 were grown on control plates and root lengths were measured 10 days after germination . B . The wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 were grown on plates containing 0 , 6 μg / mL BLM . Root lengths were measured 10 days after germination . C . Graph represents the ratio of the mean growth rate on 0 , 6 µg / mL BLM compared to control experiments on plates lacking BLM for the wildtype , ku70 , cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 . D . Image shows the wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 ( from left to right ) grown on control plates 10 days after germination . E . Images show the wildtype , ku70 , the double mutants cycb1 ; 1 ku70 , cycb1 ; 2 ku70 and cycb1 ; 1cycb1 ; 2 and the triple mutant cycb1 ; 1cycb1 ; 2ku70 ( from left to right ) on plates containing 0 , 6 μg / mL BLM .

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Figure 7A. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on control plates and root lengths were measured 10 days after germination.B. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on plates containing 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.C. Graph represents the ratio of the mean growth rate on 0,6 µg/mL BLM compared to control experiments on plates lacking BLM for the wildtype, ku70, cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70.D. Image shows the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) grown on control plates 10 days after germination.E. Images show the wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 (from left to right) on plates containing 0,6 μg/mL BLM.


Figure 1B : CL formation was measured in the presence of 60 nmol MLCL ( 18 : 2 ) 3 , 40 nmol PC ( 18 : 2 ) 2 , 4 µg TAZ , and 0 . 2 mg MBP or 0 . 2 mg Alb . C : CL formation was measured as in B with different amounts of MBP or Alb . G : CL formation was measured in the presence of 40 nmol PC ( 18 : 2 ) 2 , 10 nmol MLCL ( 18 : 2 ) 3 , 8 µg TAZ , 1250 nmol Triton X - 100 ( TX ) , and 5 . 6 nmol bacteriorhodopsin ( BR ) . H : PC formation was measured in the presence of 180 nmol CL ( 18 : 2 ) 4 , 20 nmol LPC16 : 0 , 8 µg TAZ , 20 mM Ca2 + , and 5 . 6 nmol BR . J : Transacylations were measured as in I in the presence of 0 . 9 g BR / g lipid . The ratio of total linoleoyl transfer over total oleoyl transfer was calculated .

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Figure 1B: CL formation was measured in the presence of 60 nmol MLCL(18:2)3, 40 nmol PC(18:2)2, 4 µg TAZ, and 0.2 mg MBP or 0.2 mg Alb. C: CL formation was measured as in B with different amounts of MBP or Alb. G: CL formation was measured in the presence of 40 nmol PC(18:2)2, 10 nmol MLCL(18:2)3, 8 µg TAZ, 1250 nmol Triton X-100 (TX), and 5.6 nmol bacteriorhodopsin (BR). H: PC formation was measured in the presence of 180 nmol CL(18:2)4, 20 nmol LPC16:0, 8 µg TAZ, 20 mM Ca2+, and 5.6 nmol BR. J: Transacylations were measured as in I in the presence of 0.9 g BR/ g lipid. The ratio of total linoleoyl transfer over total oleoyl transfer was calculated.


Figure 2A : Inner ( IM ) and outer ( OM ) mitochondrial membranes were traced in randomly collected electron micrographs in order to calculate the IM / OM surface area ratio . Multiple micrographs were analyzed per biological replica .

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Figure 2A: Inner (IM) and outer (OM) mitochondrial membranes were traced in randomly collected electron micrographs in order to calculate the IM/OM surface area ratio. Multiple micrographs were analyzed per biological replica.


Figure 3A : Mitochondrial membranes were traced in electron micrographs to determine the IM / OM surface area ratio . Tracings of the IM are shown in yellow boxes . C : Protein abundances were measured in adult hearts by quantitative Western blotting with fluorescence - labeled secondary antibodies . The abundances of IM proteins ( NDUFB6 , UQCR2 , ATP5A1 ) are expressed relative to the abundance of the OM protein TOM70 .

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Figure 3A: Mitochondrial membranes were traced in electron micrographs to determine the IM/OM surface area ratio. Tracings of the IM are shown in yellow boxes.C: Protein abundances were measured in adult hearts by quantitative Western blotting with fluorescence-labeled secondary antibodies. The abundances of IM proteins (NDUFB6, UQCR2, ATP5A1) are expressed relative to the abundance of the OM protein TOM70.


Figure 4Tissues were analyzed by quantitative electron microscopy . Multiple micrographs were analyzed per biological replica F , G : Tissues were analyzed by label - free quantitative proteomics . C I - V , complex I - V ; CAC , citric acid cycle enzymes ; MICOS , contact site complex ; mt TA , mitochondrial transaminases ; NS , not significant ; PDH pyruvate dehydrogenase ; VDAC , voltage - dependent anion channel

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Figure 4Tissues were analyzed by quantitative electron microscopy. Multiple micrographs were analyzed per biological replica F, G: Tissues were analyzed by label-free quantitative proteomics. C I-V, complex I-V; CAC, citric acid cycle enzymes; MICOS, contact site complex; mt TA, mitochondrial transaminases; NS, not significant; PDH pyruvate dehydrogenase; VDAC, voltage-dependent anion channel


Figure 2Glycolytic flux of wildtype ( WT ) and TM6 strains strongly correlates with fructose - 1 , 6 - bisphosphate ( FBP ) concentration . The glycolytic flux is reported here as the flux between the metabolites fructose 6 - phosphate ( F6P ) and FBP . Glycolytic fluxes were obtained on the basis of physiological and metabolome data , and via a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) . While on high glucose , the WT strain accomplishes a high glucose uptake rate ( and thus glycolytic flux ) , the mutant strain ( TM6 ) only generates a low glucose uptake ( and thus glycolytic flux ) . On maltose , also the mutant strain achieves a high glycolytic flux , since maltose is transported by a separate transporter ( Chang et al , 1989 ) . To assess the linear correlation between the FBP concentration and the glycolytic flux across the studied conditions , we implemented Pearson ' s correlation analysis assisted by bootstrapping . Specifically , we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions ( combinations of strains and carbon sources ) , biological and technical replicates . We paired each of these FBP measurements with the mean and standard deviation of the model - derived glycolytic flux ( A ) in the corresponding metabolic condition . We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition , and implemented ordinary nonparametric bootstrapping with 100 , 000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth - rate distributions to calculate the correlation statistics . FBP concentration as a function of cellular growth rate shows weaker correlation . For metabolite levels and growth rates , error bars correspond to the standard deviation between three independent experiments ; for glycolytic fluxes to the mean and standard deviations of the sampled flux solution space ( cf . Methods ) . The carbon sources were used at a final concentration of 10 gL - 1 and are indicated : Glucose ( GLU ) ; Galactose ( GAL ) ; Maltose ( MAL ) and Pyruvate ( PYR ) . To assess the linear correlation between the FBP concentration and the growth rate ( B ) across the studied conditions , we implemented Pearson ' s correlation analysis assisted by bootstrapping . Specifically , we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions ( combinations of strains and carbon sources ) , biological and technical replicates . We paired each of these FBP measurements with the mean and standard deviation of the growth rate ( B ) in the corresponding metabolic condition . We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition , and implemented ordinary nonparametric bootstrapping with 100 , 000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth - rate distributions to calculate the correlation statistics .

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

Figure 2Glycolytic flux of wildtype (WT) and TM6 strains strongly correlates with fructose-1,6-bisphosphate (FBP) concentration. The glycolytic flux is reported here as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were obtained on the basis of physiological and metabolome data, and via a novel method to estimate intracellular fluxes (Niebel et al, 2019). While on high glucose, the WT strain accomplishes a high glucose uptake rate (and thus glycolytic flux), the mutant strain (TM6) only generates a low glucose uptake (and thus glycolytic flux). On maltose, also the mutant strain achieves a high glycolytic flux, since maltose is transported by a separate transporter (Chang et al, 1989). To assess the linear correlation between the FBP concentration and the glycolytic flux across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the model-derived glycolytic flux (A) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.FBP concentration as a function of cellular growth rate shows weaker correlation. For metabolite levels and growth rates, error bars correspond to the standard deviation between three independent experiments; for glycolytic fluxes to the mean and standard deviations of the sampled flux solution space (cf. Methods). The carbon sources were used at a final concentration of 10 gL-1 and are indicated: Glucose (GLU); Galactose (GAL); Maltose (MAL) and Pyruvate (PYR). To assess the linear correlation between the FBP concentration and the growth rate (B) across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the growth rate (B) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.


Figure 4CggR intracellular abundance in the wildtype ( WT ) strain on glucose strongly varies with the promoter used . The CggR intracellular levels were quantified by proteomics in steady - state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL - 1 . Error bars represent the standard deviation of at least three replicate experiments . The relative abundance of CggR ( normalized to the abundance measured on glucose and the same promoter ) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells , but not with PTEFmut2 and PCMV . The CggR intracellular levels were quantified by proteomics in steady - state cultures grown in minimal media with glucose , galactose , maltose or pyruvate as carbon sources at a final concentration of 10 gL - 1 . WT data includes all three promoters , whereas TM6 only includes the PTEFmut2 and PTEFmut7 data . Error bars represent the standard deviation of at least three replicate experiments .

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Figure 4CggR intracellular abundance in the wildtype (WT) strain on glucose strongly varies with the promoter used. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL-1. Error bars represent the standard deviation of at least three replicate experiments.The relative abundance of CggR (normalized to the abundance measured on glucose and the same promoter) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells, but not with PTEFmut2 and PCMV. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose, galactose, maltose or pyruvate as carbon sources at a final concentration of 10 gL-1. WT data includes all three promoters, whereas TM6 only includes the PTEFmut2 and PTEFmut7 data. Error bars represent the standard deviation of at least three replicate experiments.


Figure 5Thermal shift assays were used to determine the melting curves of wild - type CggR and mutants . Error bars correspond to the standard deviation of at least five replicates . CggR - FBP affinity constants ( KD ' s ) of the wildtype and mutant variants , determined by fitting a simple cooperative binding model to the melting curves data . Error bars indicate the 95 % confidence intervals . Quantification of CggR binding to DNA . The CggR bound to DNA fraction was calculated by dividing the intensity of the protein - DNA complex band by the total DNA . The background - subtracted total intensities of the CggR - DNA complex and the free - DNA bands were assessed with ImageJ . Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype .

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Figure 5Thermal shift assays were used to determine the melting curves of wild-type CggR and mutants. Error bars correspond to the standard deviation of at least five replicates.CggR-FBP affinity constants (KD's) of the wildtype and mutant variants, determined by fitting a simple cooperative binding model to the melting curves data. Error bars indicate the 95% confidence intervals.Quantification of CggR binding to DNA. The CggR bound to DNA fraction was calculated by dividing the intensity of the protein-DNA complex band by the total DNA. The background-subtracted total intensities of the CggR-DNA complex and the free-DNA bands were assessed with ImageJ. Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype.


Figure 6The four reporter plasmids were transferred to the wildtype strain containing the CggR ( R250A ) under the control of the PTEFmut7 . The strength of the four promoters was assessed by quantifying YFP ( FL1 ) and mCherry ( FL3 ) fluorescence in exponentially growing wildtype cells in minimal medium with 10 g l - 1 glucose . The FL1 and FL3 fluorescence shown is the non - background corrected median of 100 000 cell events . The non - FL control is the signal from a wildtype strain grown under the same conditions . The background fluorescence , assessed by the wildtype harboring the YCplac33 plasmid , was substracted from FL1 and FL3 . The final reporter activity is the ratio of the background - corrected YFP and mCherry values . Error bars represent the standard deviation of three independently determined ratios from three replicate experiments . Reporter activity of the sensor across ( D ) multiple FBP levels Reporter activity is given by the YFP / mCherry ratio , calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry . Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid ( Appendix Table S8 ) . The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR . Error bars represent the standard deviation of at least three replicate experiments . Reporter activity of the sensor across glycolytic fluxes . The glycolytic flux is reported as the flux between the metabolites fructose 6 - phosphate ( F6P ) and fructose - 1 , 6 - bisphosphate ( FBP ) . Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) . Reporter activity is given by the YFP / mCherry ratio , calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry . Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid ( Appendix Table S8 ) . The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR . Error bars represent the standard deviation of at least three replicate experiments . Fraction of CggR bound to FBP across FBP concentrations . The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant . The percentage of CggR molecules bound to FBP was calculated after normalizing the Tm values for unbound / bound state using the Tm at 0 mM FBP as unbound and at 36 mM ( corresponding to maximum FBP concentration used ) as total bound states . The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one - site specific binding model in GraphPad . The solid line corresponds to the wildtype CggR and the dashed line to the R250A variant . Vertical lines delimit the physiological FBP range .

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

Figure 6The four reporter plasmids were transferred to the wildtype strain containing the CggR (R250A) under the control of the PTEFmut7. The strength of the four promoters was assessed by quantifying YFP (FL1) and mCherry (FL3) fluorescence in exponentially growing wildtype cells in minimal medium with 10 g l-1 glucose. The FL1 and FL3 fluorescence shown is the non-background corrected median of 100 000 cell events. The non-FL control is the signal from a wildtype strain grown under the same conditions. The background fluorescence, assessed by the wildtype harboring the YCplac33 plasmid, was substracted from FL1 and FL3. The final reporter activity is the ratio of the background-corrected YFP and mCherry values. Error bars represent the standard deviation of three independently determined ratios from three replicate experiments.Reporter activity of the sensor across (D) multiple FBP levels Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.Reporter activity of the sensor across glycolytic fluxes. The glycolytic flux is reported as the flux between the metabolites fructose 6-phosphate (F6P) and fructose-1,6-bisphosphate (FBP). Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019). Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.Fraction of CggR bound to FBP across FBP concentrations. The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant. The percentage of CggR molecules bound to FBP was calculated after normalizing the Tm values for unbound/bound state using the Tm at 0 mM FBP as unbound and at 36 mM (corresponding to maximum FBP concentration used) as total bound states. The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one-site specific binding model in GraphPad. The solid line corresponds to the wildtype CggR and the dashed line to the R250A variant. Vertical lines delimit the physiological FBP range.


Figure 7Sub - populations of WT and TM6 cells , grown separately and mixed in different fractions as indicated in percentages , can easily be distinguished by flow cytometry , FL1 YFP channel , FL3 mCherry channel . Histogram of single - cell ratios of FL1 / FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry . Here , a sub - population of minimally 5 % can be distinguished . Tukey boxplots showing the YFP / mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux . At least 35 cells were analyzed in each condition . The glycolytic flux is here reported as the flux between the metabolites fructose 6 - phosphate ( F6P ) and FBP . Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) . YFP / mCherry ratio measured by microscopy in co - existing dividing ( high - flux ) versus non - dividing ( low - flux ) isogenic TM6 cells on 10 gL - 1 glucose . Brightfield ( BF ) , YFP and mCherry microscopy images for a co - existing dividing ( high - flux ) and a non - dividing ( low - flux ) TM6 cell expressing the flux sensor in 10g L - 1 glucose minimal medium . The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor - expressing strain ( F ) , which reflects the cell - cycle dynamics of intracellular FBP concentration and glycolytic flux . In a control strain , lacking CggR , the production rates of YFP and mCherry are coupled ( G ) . The uncoupling was calculated for individual cell - cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale ( see more details in Materials and methods ; Appendix Figure S10 ) . Each curve represents the mean across the indicated number of cell cycles in a replicate experiment . The corresponding shaded areas denote the 95 % confidence intervals of the means ( bootstrapping with 5 000 iterations ) . We smoothed the single - cell - cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression , and used these trajectories to derive the YFP and mCherry production rates , accounting for fluorescent - protein maturation in a first - order kinetics model . To align the cell - cycle trajectories and to calculate the phase , we used the array of three cell cycle events E { cytokinesis ( cyt ) , budding , next cyt } as reference points . The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes , with the mean duration presented in parentheses for each replicate experiment . The cells belonged to the TM6 strain and were cultivated on 20 gL - 1 glucose in the microfluidic device .

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

Figure 7Sub-populations of WT and TM6 cells, grown separately and mixed in different fractions as indicated in percentages, can easily be distinguished by flow cytometry, FL1 YFP channel, FL3 mCherry channel. Histogram of single-cell ratios of FL1/FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry. Here, a sub-population of minimally 5% can be distinguished.Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019).YFP/mCherry ratio measured by microscopy in co-existing dividing (high-flux) versus non-dividing (low-flux) isogenic TM6 cells on 10 gL-1 glucose.Brightfield (BF), YFP and mCherry microscopy images for a co-existing dividing (high-flux) and a non-dividing (low-flux) TM6 cell expressing the flux sensor in 10g L-1 glucose minimal medium.The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor-expressing strain (F), which reflects the cell-cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell-cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale (see more details in Materials and methods; Appendix Figure S10). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5 000 iterations). We smoothed the single-cell-cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent-protein maturation in a first-order kinetics model. To align the cell-cycle trajectories and to calculate the phase, we used the array of three cell cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 gL-1 glucose in the microfluidic device.