EMBO/SourceData huggingface dataset:
Figure 2(A) Control 143B cells (left panels), COX1∆ cybrids (middle panels), and COX2∆ cybrids (right panels). The right lane highlights structural subunits of CI (yellow), CIII (light green), and CIV (orange brown), and assembly factors (orange). Intensity-based absolute quantification (iBAQ) values were normalized to the sum of all control values. Top lanes indicate gel slice numbers and their relative native masses.(B) Intensity profiles showing abundance and relative distribution of structural subunits from CI (upper graphs), CIII (middle graphs), and CIV (bottom graphs). The identities of MRC complexes and SCs are as in Figure 1.
Figure 3(A) Excerpts of complexome profiling data showing the distribution of CIV subunits and assembly factors HIGD1A, HIGD2A, CMC1 and COA3 in control (143B), COX1∆ and COX2∆ cybrids. The relative abundance and distribution of detected proteins is shown in the lower graphs. The localization of SC I+III2plus in COX2Δ cybrids is highlighted with a red line. The approximate molecular weights of CIV-containing structures are indicated on the top (in kDa).(B) 2D-BN/SDS-PAGE analysis from control (143B) and COX2Δ cybrids followed by immunoblotting with the indicated antibodies. I+III2+IV1, SC containing CI, CIII, and CIV; I+III2plus, SC containing CI, CIII and a COX1-submodule; III2+IV, SC containing CIII and CIV; IV2, CIV dimer; IV, CIV monomer. The presence of a COX1-CMC1-COA3-containing subcomplex is indicated.
Figure 4Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (A) CIV subunits COX1 and COX5B. (B) CI subunit NDUFB7. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (B) CI subunit NDUFB7. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.Mitochondrial extracts from control 143B cells (left), COX1∆ cybrids (right), and COX2∆ cybrids (middle) were immunoprecipitated using antibodies against: (C) CIII subunit CORE2. Samples were subsequently analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. In, input. IP, immuno-precipitate. FT, flow-through. Ig, unrelated immunoglobulin used as a negative control.
Figure 5(A) Digitonin-solubilized mitochondria from doxycycline-treated 143B cells, COX1∆ and COX2∆ cybrids were analyzed by BN-PAGE followed by CI-IGA assays (upper panels) or immunoblotting (medium and lower panels) with antibodies against CI subunits NDUFA9 and NDUFS1. The identities of MRC complexes and SCs are as in Figure 1.(B) Mean incorporation rates of NDUFA9 and NDUFS1 into SC I+III2+IV1 from 143B cells and into SCs I+III2 from the CIV-KO cybrids. Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels. Time-point values are expressed as percentages of untreated cells (SS) and indicated as mean±SD.(C) Samples were subsequently analyzed by 2D-BN/SDS-PAGE and immunoblotting with the indicated antibodies. The identities of MRC complexes and SCs are as in Figure 1.(D) Mean incorporation rates of CIII subunits CORE2 and RISP into SC I+III2+IV1 from control cells and into SCs I+III2 from CIV-KO cybrids. Signals from at least three independent experiments were quantified and normalized by CII subunit SDHA levels. Time-point values are expressed as percentages of untreated cells (SS) and indicated as mean±SD.
Figure 6(A) Digitonized mitochondria from doxycycline-treated 143B cells and COX2∆ cybrids were analyzed by 2D-BN/SDS-PAGE and western blot with the indicated antibodies.(B) Mean incorporation rates of COX subunits into SC I+III2plus from COX2∆ cybrids. Signals from three independent experiments were quantified and normalized by CII. Time-point values are expressed as percentages of the untreated cells (SS) and indicated as mean ± SD. Control (143B) values for subunits COX4, COX5A and COX6C were obtained from (Moreno-Lastres et al., 2012).C-D SDS-PAGE (C) and BN-PAGE (D) analyses from 143B cells and COX2∆ cybrids transiently transfected either with a COX5B-Myc-DDK construct or with the empty vector, followed by immunoblotting with antibodies against COX5B and CII subunit SDHA (loading control). The identities of MRC complexes and SCs are as in Figure 1. The red arrow points to the accumulation of over expressed COX5B bound to SC I+III2 in control cells.
Figure 8(A) Respiratory chain enzyme activities of control (blue), COX1∆ (red) and COX2∆ (green) cybrids. Enzyme activities were measured in three experimental replicates. Data are expressed as the percentages of control cells, and indicated as means ± SD. Mann-Whitney U test was applied for statistical analyses. Variations between controls and mutants: α, p<0.05; γ, p<0.001. Specific variations between COX1∆ and COX2∆ mutants: ∗p<0.05.
Figure 1Colorectal tumor outcomes in WT (n=7), IL-33KO (n=4) and ST2KO (n=3) mice at the completion of the AOM/DSS carcinogenesis protocol. (A) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero), Skin tumor outcomes in WT (n=7), IL-33KO (n=6) and ST2KO (n=7) mice at the completion of the DMBA/DNFB carcinogenesis protocol. (D) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero),H) Quantification of nuclear IL-33+ epidermal and dermal cells in the DNFB- versus acetone-treated WT skin. Dots represent cell counts from three randomly selected high power field (HPF) images per sample (n=5 per group). J) Quantification of nuclear IL-33+ epithelial and stromal cells of colon treated with AOM/DSS versus no treatment. Dots represent cell counts from three randomly selected HPF images per sample (n=4 per group). Data information: Graphs show mean + SD, unpaired t-test.
Figure 2(H) ChIP-qPCR assay for Smad6 in the presence of IL-33 full length or cytokine domain using an anti-RUNX2 antibody. After Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours, cell lysates were subjected to chromatin-immunoprecipitation with anti-RUNX2 antibody and eluted RUNX2-bound chromatin was used for qPCR with primers against Smad6 promoter region. Anti-IgG ChIP-qPCR results are shown as negative controls (n=4 per group).
Figure 3(D) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β and poly (I:C) treatment. Pam212 cells were treated with poly (I:C) or PBS followed by incubation with and without TGF-β (5nM). Data represent three independent experiments with similar results. (E) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β + poly (I:C) after knocking down of IL-33. Pam212 cells were transfected with siIl33 knockdown construct or siRNA control (siCon) for 30 hours followed by incubation with poly (I:C) and TGF-β. Data represent three independent experiments with similar results. (F) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins upon the expression of IL-33 full length or cytokine domain. Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours followed by incubation with and without TGF-β. Data represent three independent experiments with similar results. Data information: GAPDH is used as the control housekeeping protein in D-F). Graphs show mean + SD, NS: not significant, unpaired t-test.(G) Impact of IL-33 knockdown on cell proliferation in response to TGF-β. Pam212 cells were treated with siIl33 or siCon followed by TGF-β treatment (n=7 in each group). (H) Impact of IL-33 knockdown and SB431542 treatment on cell proliferation. Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO (carrier control, n=7 in each group).
Figure 4(I) SMAD signaling proteins levels in WT and K14-IL33tg,ST2KO skin treated with acetone (control) at the completion of the DMBA/TPA carcinogenesis protocol. Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 antibodies. GAPDH is used as the control housekeeping protein (n=5 in each group).(J) Representative images of IL-33 and p-SMAD2/3 immunostaining on the adjacent sections of human squamous cell carcinoma (SCC) and normal skin (scale bar: 100 μm). Data information: Graphs show mean + SD, NS: not significant, unpaired t-test.
Figure 5(B) Il33 and Smad6 mRNA levels in caerulein-treated WT pancreas compared with PBS-treated controls at the completion of chronic pancreatitis protocol (n=8 in caerulein and n=9 in PBS group for Il33, n=9 in caerulein and n=10 in PBS group for Smad6, unpaired t-test).(D) Pancreatic tumor-free survival of WT (n=4), IL-33KO (n=5) and ST2KO (n=6) mice at the completion of the DMBA/caerulein carcinogenesis protocol (log-rank test).(G, H) Quantification of nuclear (G) IL-33+ and (H) p-SMAD2/3+ epithelial cells per HPF in the matched human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=18 patients, paired t-test).
Figure 1A. Representative FACS plot (left) showing a mixed-lineage clone with a T-lymphoid (CD25+/CD11b-Gr1-), myeloid (CD11b+Gr1+/CD25-), and biphenotypic (CD11b+Gr1+/CD25+) fraction derived from a single Myc/Bcl2-transformed DN2cell grown on 1:1 OP9:OP9-Dll1 co-culture with lineage-promiscuous cytokines IL-2, IL-3, IL-6, IL-7, SCF,GM-CSF, Flt3. Morphology of the indicated flow-sorted cell fractions was analyzed by Giemsa staining (right).B. Phenotype frequencies of clones from Myc/Bcl2-transformed single cells (grown and phenotyped as mentioned in A). Analyzed were 31 clones from LSKs, 107 from GMPs and 28 from DN2 cells.C. Cumulative survival of 31±2 and 27±6 days of mice that received 5104 to 5105 T-lymphoid cells or myeloid cells, respectively, that were flow-sorted from mixed Myc/Bcl2+ DN2-derived clones. The summarized data are from 3 mice injected with the T-lymphoid and 4 mice injected with the myeloid fraction of two different clones.D. Representative FACS plots demonstrating predominant outgrowth of myeloid clones from single Myc/Bcl2-transformed LSK or GMPcells on OP9:OP9-Dll1 co-cultures.E. (top panels) Serial replating of Myc-Bcl2+DN2 cells in methylcellulose generated myeloid clones with stable genomic reassembly of the Dβ1 TCR locus in vitro (gel: left two lanes). Upon transplantation, the same clones induced myeloid leukemia that retained the initial rearrangement in vivo (gel: right two lanes) as assessed by nested PCR. GL = germline. (bottom panels) Methylcellulose-based replating of Myc-Bcl2+GMPs generated myeloid clones that exclusively displayed the TCRß locus in germline configuration.F. Cumulative survival of mice that received 106cells of TCRβ-rearranged myeloid DN2-derived clones leading to death within 33±4 days after transplantation. Two DN2 clones were transplanted into 5 recipient mice.
Figure 2A. Representative FACS plots of spleens from leukemic recipient mice that had received LSK, GMP or DN2 cell grafts immediately after retroviral Myc/Bcl2 transduction. Numbers indicate frequencies (%) of CD45.2 donor cells within the indicated gates summarizing the mean±SEM of at least 8 recipients per group from a total of 10 independent experiments.B. Cumulative survival of mice that received 2104 to 2105 freshly Myc/Bcl2-transduced LSK, GMP or DN2 cells; Graph shows n=11 Myc-Bcl2+LSK-, n=14 DN2-, and n=8 GMP-transplanted animals.C. Histological spleen sections of Myc-Bcl2+LSK-, GMP- or DN2-transplanted mice displaying myeloperoxidasestaining. Leukemic cell engraftment in spleens ranged from 75.1%-95.5%.D. Multiplex quantitative RT-PCR demonstrating strong expression of PU.1 in the myeloid (M3L: CD11b+Gr1+/CD3-CD4-CD8-) and biphenotypic T/myeloid (BL: CD11b+Gr1+/CD3+CD4+CD8+) fractions but not in the T-lymphoid (TL: CD11b-Gr1-/CD3+CD4+CD8+) fraction that were flow-sorted from diseased Myc-Bcl2+DN2-transplanted mice. Myeloid progeny of Myc/Bcl2+GMP-transplanted or T-lymphoid progeny of Myc/Bcl2+DN3-transplanted animals as well as healthy thymocytes (Thy) and BM cells from WT mice served as controls. Results are shown as fold over actin transcripts.E. D-J rearrangement PCR of flow-sorted T-lymphoid (TL), biphenotypic (BL) and myeloid (ML) fractions of donor splenocytes of a diseased Myc/Bcl2+DN2-transplanted mouse. All three fractions revealed a stable genomic reassembly of the Dβ1 TCR locus (Dβ1Jβ1.5) demonstrating clonality in DN2 leukemia. thy = thymus.F. FACS plot of diseased intrathymic grafts (CD45.2-gated), demonstrating that Myc/Bcl2+DN2 cells could produce multi-lineage leukemia within a T-cell supporting environment; Myc/Bcl2+GMPs remained myeloid restricted under these conditions. Numbers indicate cell frequencies within the gates (mean±SEM) representing six independent experiments.G. Intrathymic transplantation of mock-infected non-leukemic DN2 cells produced predominantly T-lymphoid but also myeloid progeny 23 days after injection. This differentiation outcome resembles that of freshly isolated DN2 cells (Bell and Bhandoola, 2008; Richie Ehrlich et al., 2011), and indicates that the short-term culture necessary for retroviral transduction did not interfere with the physiological lineage potential of DN2 cells.
Figure 3A. GSEA demonstrates that Myc/Bcl2+DN2-derived myeloid blasts were significantly (FDR >0.05) enriched for expression of a pan-dendritic cell gene set. Gene sets were published in (Schonheit et al., 2013).B. Heat map of the top 100 differentially expressed genes (adjusted p-value of ≤ 0.05) in each myeloid fraction (ML: CD11b+Gr1+/CD3-CD4-CD8-) from GMP-, LSK- and DN2-derived leukemias.D. GSEA demonstrates significant enrichment of a normal DN2-specific gene set (see methods section) in the Myc/Bcl2+DN2-derived myeloid blasts.
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.
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).
Figure 1A, Live images of neurons co-transfected with SyGCaMP5 and MtDsRed before and during 10 Hz field stimulation. The full arrow indicates a mitochondrially occupied terminal, and the empty arrow a terminal without a mitochondrion. Scale bar, 10 m.B, Average ΔF/F0 SyGCaMP5 traces from n=11 neurons (98 terminals) plotting the average of terminals without a mitochondrion (black trace) and with a mitochondrion (red trace). Stimulation occurred for 10s (t=20-30) at 10 Hz.C, Average Ca2+ response following stimulation in terminals with or without a mitochondrion (average of ΔF measurement taken for a t=20-30s of B), ΔF/F0 = 1.9 0.3 with and 3.5 0.4 without mitochondria, paired t-test, ***p<0.0001.D E, However, there is no significant difference in the presynaptic Ca2+ response in terminals with and without a mitochondrion in response to single action potentials. D, Example trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5. 1 ms stimulation pulses were applied every 5 s. The red trace represents an average of the terminals occupied with a mitochondrion in this neuron, whereas the black trace represents the averaged response of terminals without a mitochondrion. E, Maximal fluorescence intensity in terminals without a mitochondrion (left) or with a mitochondrion (right) within the same axon (dots connected by a line) (n=8 neurons, 44 terminals, paired t-test, p=0.55).F, Average ΔF/F0 SyGCaMP5 traces for 80 stimuli (n=7 neurons, 21 terminals, t= 20-28s, ΔF/F0 = 1.3 0.2 with and 2.2 0.5 without mitochondria, paired t-test, *p<0.05), 40 stimuli (n=5 neurons, 25 terminals, t= 20-24s, ΔF/F0 = 0.9 0.3 with and 2.0 0.4 without mitochondria, paired t-test, *p<0.05), 20 stimuli (n=10 neurons, 55 terminals, t= 20-22s, ΔF/F0 = 0.8 0.4 with and 1.8 0.4 without mitochondria, paired t-test, *p<0.05) and 10 stimuli (n=4 neurons, 21 terminals, t= 20-21s, ΔF/F0 = 0.4 0.3 with and 0.4 0.1 without mitochondria, paired t-test, p=0.90), all delivered at 10 Hz. Experiments performed in E18 or P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.
Figure 2A, Average trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 imaged after incubation in ACSF for 30 min. Neurons were stimulated using field stimulation for 10s at 10 Hz. Red trace = average of the terminals occupied with a mitochondrion, black trace = averaged response of terminals without a mitochondrion. B, as in A, but after 30 min treatment with 10 µM Ru360.C, Summary bar graph of the control neurons (corresponds to the traces shown in panel A). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 3.5 0.4) and unoccupied terminals (ΔF/F0 = 4.9 0.5, n=15 neurons, 96 terminals, ***p<0.001, paired t-test). D, Summary bar graph of the Ru360-treated neurons (corresponds to the traces shown in panel B). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 4.1 0.5) and unoccupied terminals (ΔF/F0 = 3.8 0.9, n=13 neurons, 79 terminals, p=0.53, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.