EMBO/SourceData huggingface dataset:
Figure 5B. Class averages of antibody-bound T4SS3-10+D4FLAG. Class averages of the aligned NS-EM images of the T4SS3-10+D4FLAG with 3-4 particles per class. Red circles indicate the position of the antibody.
Figure 1A,B Tumor growth curve (left) and summary of end-point tumor masses (right) of 6-8 week-old wildtype (WT) and Peli1-KO (KO) mice inoculated s.c. with B16F10 (A; WT, n=7; KO, n=6) or E.G7 (B; WT, n=5; KO, n=5) tumor cells.C,D Flow cytometry analysis of CD4+ and CD8+ T cells in CD45.2+ TILs and dLN cells from B16F10 tumor-bearing wildtype and Peli1-KO mice, presented as a representative plot (C) and summary graph based on multiple mice (D; WT, n=6; KO, n=6).E,F Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs, and dLN cells of B16F10 tumor-bearing (day 19) wildtype and Peli1-KO mice, presented as a representative plot (E) and summary graph (F). (WT, n=6; KO, n=6)G,H Flow cytometry analysis of IFNγ+CD8+ T cells in TILs of E.G7 tumor-bearing (day 24) wildtype and Peli1-KO mice, presented as a representative plot (G) and summary graph (H). (WT, n=3; KO, n=3)
Figure 2A,B Tumor growth curve (left) and summary graph of end-point tumor masses (right) of wildtype (WT) and Peli1-TKO (TKO) mice inoculated s.c. with B16-OVA (A; WT, n=5; KO, n=5) or E.G7 (B; WT, n=6; KO, n=4) tumor cells.C Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs and dLN cells from B16OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)D Flow cytometry analysis of GZMB+CD8+ T cells in TILs from B16-OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)E Tumor growth curve (left) and summary graph of end-point tumor masses (right) of B16-OVA tumor-bearing wildtype and the indicated Peli1 conditional KO mice. (Treg KO model: WT, n=5; Treg KO, n=6; MKO model: WT, n=3; MKO, n=5; BKO model: WT, n=9; BKO, n=9)
Figure 3A,B Seahorse analysis of basal (glucose injection) and maximal (oligomycin injection) ECAR or basal (no treatment) and maximal (FCCP injection) OCR in WT and Peli1-KO OT-I CD8 T cells that were either untreated (naïve) or activated with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) for 16 h. Data are presented as a representative plot (A) or summary graph based on 6 wildtype and 6 KO mice (B).qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h.D qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h.E Immunoblot analysis of HK2 and Glut1 in wildtype or Peli1-KO OT-I CD8 T cells that were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h.
Figure 4A Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of freshly isolated CD8 T cells from wildtype (WT) or Peli1 KO (KO) mice (6 weeks old). Data are presented as a representative blot (left) and summary graphs of densitometric quantifications of the indicated proteins (presented as phosphorylated/total protein ratio) based on three independent experiments.B,C Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were first rested on ice for 15 min and then stimulated in low-serum (1% FBS) medium with anti-CD3 and anti-CD28 for the indicated time periods.D,E Seahorse analysis of basal and maximal ECAR (D) or OCR (E) using wildtype or Peli1-KO OT-I CD8 T cells that were activated for 16 h with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence of solvent control (DMSO), rapamycin (Rapa, 10 nM), or Torin 1 (100 nM). Data are presented as a representative plot (left) and summary graphs (right).F,G Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated (+) for 1 h (F) or 24 h (G) with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence (+) or absence (-) of rapamycin (10 nM) and Torin 1 (100 nM).H qRT-PCR analysis of Ifng gene expression in naïve wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated for 6 h (+) with anti-CD3 plus anti-CD28 in the presence (+) or absence (-) or rapamycin.I-K Schematic of experimental design (I), tumor growth curve (J), and summary of day 15 tumor weight (K) of B6.SJL mice inoculated with B16-OVA melanoma cells (2 x 105) and adoptively transferred on day 7 with in vitro activated and rapamycin- or DMSO-treated wildtype or Peli1-KO OT-I CD8 T cells (4 x 105).
Figure 5Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with FBS (10%) (A)Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with insulin (B).Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with FBS (C)Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with insulin (D)Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with EGF (E).
Figure 6A Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype (WT) and Peli1-KO (KO) CD8 T cells that were either not treated (NT) or activated for 16 h with plate-bound anti-CD3 and anti-CD28. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype and Peli1-KO CD8 T cells that were activated for 1 h with anti-CD3 and anti-CD28 and then treated with cycloheximide (CHX) for the indicated time periods. Data are presented as a representative blot (B)a summary graph of quantified TSC1 and TSC2 protein bands presented as percentage of the level in untreated (0 min) cells (C).D Immunoblot analysis of TSC1 and TSC2 in wildtype or Peli1-KO CD8 T cells stimulated with anti-CD3 plus anti-CD28 for 1 h and then incubated with (+) or without (-) cycloheximide (CHX) and/or MG132 for 2 h. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.Ubiquitination analysis of immunoprecipitated TSC2 (E) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.Ubiquitination analysis of immunoprecipitated TSC1 (F) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.Analysis of ubiquitin conjugation to TSC1 (G, H) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.Analysis of ubiquitin conjugation to TSC1 TSC2 (I, J) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.K Co-immunoprecipitaton analysis of TSC1-TSC2 interaction in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.L Co-immunoprecipitation analysis of endogenous TSC1-TSC2 interaction in wildtype and Peli1-KO CD8 T cells that were either not treated (NT) or stimulated for 2 h with anti-CD3 plus anti-CD28. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the blot from anti-TSC1 IP (the right four lanes of the top two panels).
Figure 7A Analysis of ubiquitin conjugation to wildtype (WT) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag-tagged TSC1 and its mutants along with (+) or without (-) the indicated expression vectors.B Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in lysates of TSC1-KO MEFs transfected with (+) or without (-) TSC1 wildtype or K30A expression vectors. The cells were serum starved and then left either untreated (NT) or stimulated with 10% FBS for 30 mins. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC2 (right panel) protein bands relative to the level of β-Actin.C Analysis of TSC2 ubiquitination in 293 cells transfected (for 48 h) with (+) or without (-) the indicated expression vectors.D Co-immunoprecipitation analysis of the interaction between endogenous TSC2 and exogenous TSC1 wildtype and K30A in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) the indicated expression vectors. KO MEF cells were transfected with indicated expression plasmids. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the last four lanes of the anti-Flag IP blot in left panel.E Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) TSC1 WT or K30A and treated with MG132 for 2 h before lysis.F Immunoblot analysis of endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with Myc-TSC1 wildtype or K30A and subsequently treated with cycloheximide (10 μg/ml) for the indicated time periods before lysis. The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated (0 min) lane.
Figure 1C-E. 2a and 2b ESCs maintained in 2i medium were cultured in the absence (Ctrl) or presence of OHT and subsequently used for WB (C)C-E. 2a and 2b ESCs maintained in 2i medium were cultured in the absence (Ctrl) or presence of OHT and subsequently used for growth curves and RT-qPCR (D,E).F-G. 2a and 2b ESCs were adapted to grow in serum-containing ESC medium, exposed to OHT as indicated and subsequently used for growth curves and RT-qPCR analyses.H-I. Jmjd2a(+/-); Jmjd2a(+/-) (H) or Jmjd2b(+/-); Jmjd2b(+/-) (I) mice were inter-crossed and the number of pups alive at time of weaning reported. P values were calculated using a Chi-square test.
Figure 2A-D. WB and growth curves of 2ac (A), 2abc (B), 2ab (C) and 2bc (D) ESCs cultured in 2i medium and exposed to OHT as indicated.E. Flow cytometry profiles of 2ac ESCs pulsed with BrdU in 2i medium. The upper table shows the percentages of living cells (the subG1 fraction is omitted from the analysis), which can be classified as being in S (BrdU+), G1 (2N DNA content, BrdU-) or G2/M phase (4N DNA content, BrdU-). The percentages of dead cells (subG1, events with a <2N DNA content) is shown in red.F. RT-qPCR analyses for ESCs cultured in 2i medium.G-J. 2ac and 2abc ESCs were adapted to grow in serum-containing ESC medium, exposed to OHT as indicated and subsequently used for growth curves (G-H) and RT-qPCR analyses (I-J). The RT-qPCR data are also presented in Appendix Fig S2C-D with a zoom-in on the lower region of the y-axis.
Figure 3A-B. 2ac ESCs expressing GFP were cultured in the absence or presence of OHT and subsequently used for morula injections. (A) Average volume of the GFP-positive area in chimeric embryos at 24 and 72 hrs after morula injection. Number of embryos used for quantification: Ctrl 24h n=18; +OHT 24h n=15; Ctrl 72h n=5; +OHT 72h n=5. Data are presented as mean ± SD. (B) Representative images of chimeric embryos. The white bars indicate a length of 32 µm. Relative intensities of the GFP signal are indicated for each image.C-D. Jmjd2a(+/-); Jmjd2c(+/-) mice were inter-crossed. The number of (C) pups alive at weaning or (D) embryos recovered at E6.5 is presented. P values were calculated using exact binomial test with the most conservative estimate of expected (-/-;-/-) embryos (6.25 %, assuming no linkage between the Jmjd2a and Jmjd2cloci even though they are located on the same chromosome).
Figure 4A. Examples of ChIP-seq data obtained for Jmjd2a, Jmjd2c (Pedersen et al, 2014), and H3K4me3.C. Unsupervised k-means clustering of ChIP-seq tags over all TSSs (+/- 5kb).D-E. Bar graphs showing the number of Jmjd2a peaks displaying at least 1 bp overlap with (D) an H3K4me3 peak. Jmjd2a peaks were classified as TSS-associated ('TSS') if overlapping a region of +/- 1kb of a TSS.D-E. Bar graphs showing the number of Jmjd2a peaks displaying at least 1 bp overlap with (E) a Jmjd2c peak. Jmjd2a peaks were classified as TSS-associated ('TSS') if overlapping a region of +/- 1kb of a TSS.G. ChIP-qPCR validations. Graphs show mean ± SD for 3 technical replicates and are representative of results obtained in at least 2 independent experiments.
Figure 5A. Density plots showing average ChIP-seq signals with 95% confidence intervals of the mean indicated in grey. y-axes show mean Tags per Million (TPM). Data were obtained using the 2ac ESCs line #4. TSSs were classified as 'Bound' if containing binding sites for both Jmjd2a and Jmjd2c within +/- 1kb (see Fig 4F), or 'Not bound' if neither protein binds within the same region.B. Heat map showing H3K9me3 ChIP-seq data for Jmjd2a/c bound TSSs (+/- 5kb) sorted according to read number in 2abc #13+OHT.C. ChIP-qPCR validations for selected Jmjd2a/c targets (see Fig 4G). Graphs show mean ± SD for technical triplicates and are representative of results obtained in at least 2 independent experiments.
Figure 6A-B. Independent RT-qPCR validations of genes identified as being (A) down-regulated or (B) up-regulated by expression arrays. Graphs show mean ± SD for technical triplicates and are representative of results obtained with at least 2 different ESCs lines of each genotype in independent experiments.A-B. Independent RT-qPCR validations of genes identified as being (A) down-regulated or (B) up-regulated by expression arrays. Graphs show mean ± SD for technical triplicates and are representative of results obtained with at least 2 different ESCs lines of each genotype in independent experiments.C-D. Density plots showing average ChIP-seq signals for (C) H3K9me3 or (D) H3K36me3 with 95% confidence intervals of the mean indicated in grey. Plots are shown for genes, which are down- or up-regulated according to the microarray analyses (FDR<0.05, absolute fold change log2(|FC|)>0.5) for both 2ac and 2abc KO ESCs (overlaps presented in Appendix Fig S6A), or do not show strong expression changes (log2(|FC|) < 0.2). Genes are further classified as "Bound" if containing binding sites for both Jmjd2a and Jmjd2c within +/- 1kb of a TSS.E. Heat map presenting array data in the form of log2(fold change) values comparing OHT with control treated ESCs. Genes are shown for which TSS regions (+/- 1kb) show the most substantial increase in H3K9me3 levels in OHT treated cells according to the ChIP-seq analyses (defined in Fig EV4).
Figure 7A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (A) WB.A-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (B) RT-qPCR analysesA-C. 2ac clones established after transfection with an empty vector (empty) or plasmids expressing wild type Jmjd2c (2c wt) or a catalytic mutant (2c mut) were exposed to OHT as indicated and subsequently used for (C) serial plating for the generation of growth curves.
figf1a, Polychromasia (Wright-Giemsa stain, indicated by arrows) and increased reticulocytes (brilliant cresyl blue stain) revealed by staining blood smears from 6-week-old wild-type (WT) or Nix-/- mice (scale bar, 20 µm).b, c, Ter119 (b) or Mitotracker deep red (c) versus CD71 staining of RBCs from 3- or 6-week-old mice. Cell counts were also calculated (mean ± s.e.m.; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.b, c, Ter119 (b) or Mitotracker deep red (c) versus CD71 staining of RBCs from 3- or 6-week-old mice. Cell counts were also calculated (mean ± s.e.m.; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.d, Analyses of RBCs from 3- or 6-week-oldmice by immunocytochemistryAnalyses of RBCs from 3- or 6-week-oldmice by transmission electron microscopy
figf2a, Quantification of NHS-biotin-labelled RBCs or transferred CMFDA-labelled RBCs (n = 3).b, Western blot analyses of caspases in RBCs after in vitro culture. Asterisks denote processed caspases. FL, full length.c, The relative luminescence units (RLU) of caspase activities in cultured RBCs and the suppression of annexin V staining in Nix-/- RBCs after 12-h culture in the presence of qVD-oph or solvent control (DMSO) (n = 3).d, Mitotracker versus annexin V staining of RBCs after in vitro culture.e, Mean fluorescent intensity (MFI) of ROS staining in RBCs after in vitro culture, and caspase activities in Nix-/- RBCs after 24 h culture with solvent control, ROS scavengers or qVD-oph (n = 3). BHT, butylated hydroxytoluene; CAT, catalase; TEM, tempol.f, Haematoxylin and eosin (HE) staining of spleen sections of 9-week-old wild-type and Nix-/- mice. Arrows denote iron deposits within macrophagecytoplasm. Iron deposits were also stained with Prussian blue, followed by counterstain with nuclear-fast red. Scale bar, 20 µm.g, Real-time RT-PCR of erythropoietin (Epo) (n = 12). For all relevant panels, statistical significance of the data (mean ± s.e.m.) is: *P < 0.05; **P < 0.01; ***P < 0.001.
figf3a, Ter119+CD71+ reticulocytes sorted at day 3 or 6 after PHZ treatment were cultured for in vitro maturation for 0 (top row), 2 (middle row) or 4 (bottom row) days, followed by Mitotracker deep red and Ter119 staining.b, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were cultured and stained with new methylene blue. Scale bar, 20 µm.c, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were stained for LC3 and COX IV and analysed by deconvolution microscopy. Scale bar, 5 µm. The Pearson coefficiency for LC3 and COX IV co-localization at day 0 (mean ± s.e.m.) is: wild type, 0.81 ± 0.001; Nix-/-, 0.44 ± 0.036 (n = 35, P = 0.0006).d, CD71+Ter119+ reticulocytes sorted at day 3 after PHZ treatment were analysed by electron microscopy. Scale bar, 0.5 µm.
figf4a, Ter119+CD71+ reticulocytes were sorted from the peripheral blood of wild-type and Nix-/- mice at day 6 after PHZ treatment. The cells were cultured for in vitro maturation in the presence of 10 µM FCCP or 1 µM ABT-737, followed by staining with Mitotracker deep red and phycoerythrin-conjugated anti-Ter119.b, c, Reticulocytes were cultured with FCCP (b) or ABT-737 (c) for 24 h and analysed by transmission electron microscopy. Scale bar, 0.5 µm.
Figure 1RNAi injections schedule in starved conditions. Planarians are at 14dS when the amputation is performed (indicated by the grey cross at day 14). Live images show that cct3A(RNAi) planarians form a minimal blastema compared to controls at the time points shown. At the bottom are the number of planarians with the phenotype shown. The remaining planarians are dead by the time point of regeneration shown. dR indicates days of regeneration. RNAi injections schedule in feeding conditions. One extra feeding in respect to (B) is introduced one day prior to injections. Planarians are at 8dS when the amputation is performed. The live images show that most of the cct3A(RNAi) planarians regenerate like controls. At the bottom are the number of planarians with the phenotype shown. At 8-10dR, the remaining planarians are either dead (4/124) or have a tiny blastema (23/124) and either died or regenerated later. At 51-68dR, the remaining planarians are either dead (10/93) or have a tiny blastema and died later (5/93).
Figure 2The graph shows the mitotic numbers of trunks during different time points of regeneration in the starved condition for cct3A RNAi and controls. Error bars are s.d. from the mean and asterisks indicate P < 0.001 (three asterisks), P < 0.01 (two asterisks) and n.s. indicates not significant using two-tailed Student's test with equal variance. n ≥ 9 planarians per time point. Also shown are maximum projections of representative trunks labelled with anti-H3P at different time points of regeneration in the starved condition. On the bottom, the number of planarians with the phenotype shown from the total is displayed. Note that at 50dR planarians show few (a4) or no mitoses (a5). The dashed line delimits the body of the planarian.Maximum projections of representative trunks after FISH for smedwi-1 at different time points of regeneration under starved and feeding conditions. On the bottom, the number of planarians with the phenotype shown from the total is displayed. At 15dR the expression levels are similar. At 30dR some planarians have almost no expression (c5) and at 50dR almost all planarians show no expression (c10 and c11) in starved conditions. Under the feeding condition the expression levels at 30dS are similar. On the bottom, the number of planarians with the phenotype shown from the total is displayed. The dashed line delimits the body of the planarian.
Figure 3The cartoon represents a regenerating trunk and the squares display the region analyzed at the anterior "A" and posterior "P" blastemas. The images are TUNEL maximum projections of representative blastemas from 72 hours and 4 days regenerating trunks in starved conditions. The dashed line delimits the body of the planarian. The graph shows the number of TUNEL+ cells per mm2. The differences in cell death are not significant (n.s.) at 72hR (two-tailed Student's test with equal sample variance) and significant (***P < 0.001 using two-tailed Student's test with equal variance) at 4dR. n ≥ 5 planarians per condition.