EMBO/SourceData huggingface dataset:
Figure 5A) NADPH levels are reduced in IDHm compared to wildtype tumors, resulting in a reduced NADPH/NAD+ ratio (n=3/ sample). p-value from T test: *<0.05. NADPH, NADP: reduced, oxydized nicotinamide adenine dinucleotide phosphate, NADH, NAD: reduced, oxydized nicotinamide adenine dinucleotide.B) MALDI images for glutathione (GSH) and cystathionine on IDHwt (P3) and IDHm (E478) PDXs.C) Quantification of MSI signals for GSH and cystathionine from 6 PDXs (n=3 / group).D) LC-MS quantification of independent samples for GSH, GSSG (oxidized GSH), GSSG/GSH ratio and cystathionine from 6 PDXs (n=3/ sample / group). Statistical significance between IDH groups calculated by T test, pv: p-value.
Figure 6A) Comparative gene expression analysis of key enzymes involved in GSH and cysteine synthesis and supply in IDHwt (n=220), IDHm with 1p/19q co-deletion (n=151) or IDHm without 1p/19q co-deletion (n=226) patients based on TCGA RNA-seq data from a cohort of 597 patients. CBS: Cystathionine beta synthase, key enzyme of the transsulfuration pathway. GCLC: catalytic subunit of glutamate-cysteine ligase. SLC7A11: gene encoding xCT, the cystine-glutamate antiporter.B) Kaplan-Meier curves of CBS gene expression versus overall survival in patients with IDHwt, IDHm with or without 1p/19q co-deletion. Statistical significance for the survival analysis is shown as: pv: p-value, ns= non significant.
Figure 1B. Protein structural predictions for S. pombe Rbd2 (blue) and E. coli GlpG (red). Protein structures were predicted using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2). Rbd2 model structure was aligned to GlpG using PyMOL.E. Cells carrying a plasmid expressing rbd2-6xmCherry and either of two Golgi proteins dsc2-6xGFP(dsc2-6xGFPrbd2-6xmCherry) or Sec72-6xGFP (Sec72-6xGFPrbd2-6xmCherry) were imaged by confocal fluorescence microscopy.
Figure 2A. Wild-type (WT), rbd2Δ or sre1Δ yeast (200 cells) containing either empty vector (EV) or a plasmid expressing sre1N (Sre1amino acids 1-440) were grown on rich medium in the presence or absence of cobalt chloride (CoCl2).B. Western blot was probed with anti-Sre1 IgG of phosphatase-treated, whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen. Dsc5 serves as a loading control and was detected by chemiluminescence. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.C. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre1 cleavage. Western blot was performed using anti-Sre1 or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen.D. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre2 cleavage by western blot probed with anti-Sre2 serum or anti-FlagIgG of phosphatase-treated, whole cell lysates from cells grown in the presence of oxygen.E. Sre1 cleavage assay was performed as in (C). G128V, A127D and A186T mutant strains were obtained from our previous mutagenesis screening (Stewart et al, 2012). Dsc5 serves as a loading control.F. Sre2 cleavage assay for indicated yeast strains was performed as in (D).G. A model substrate of Sre2 containing amino acids 423-793 (Sre2MS) with an N-terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ, dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains. Whole cell lysates from indicated yeast strains were analyzed by western blot with anti-Flag antibody. Three independent isolates (A-C) are shown for each strain. Empty vector (EV) transformation serves as a negative control.
Figure 3A. Wild-type (WT) and the indicated mutant strains were grown for 3 hr at the non-permissive temperature (36°C) to inactivate Mts3. Phosphatase-treated cell lysates were analyzed by western blot with anti-Sre2 serum and imaged using chemiluminescence. P and N denote Sre2 precursor and cleaved nuclear forms, respectively.B. WT, rbd2Δ and sre2Δ cells were treated with bortezomib (Bz) for 3 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum.C. WT, dsc1Δ and rbd2Δ cells carrying either empty vector (EV) or a plasmid expressing 3xFlag-Sre2MS (MS) were treated with bortezomib (Bz) for 3 hr. Whole cell lysates were analyzed by western blot with anti-Flag antibody.D. Whole cell lysates from the indicated strains were analyzed by western blot with anti-Sre2 serum.E. Catalytically dead Dsc1 E3 ligase (dsc1-C634A) mutant and other indicated yeast strains were analyzed by western blot probed with anti-Sre2 serum and imaged using chemiluminescence.
Figure 4A. HumanHEK293T cells were transfected with plasmids expressing different 3xHA-tagged rhomboid proteases: S. pomberbd2, S. pomberbd2 recoded for human expression, humanRHBDL2, D. melanogaster Rho1, and P. falciparumROM4. Whole cell lysates were analyzed by western blot to detect the HA-tagged rhomboid [asterisks indicate loading control bands detected with antibody ab179726 (abcam)].B. HumanHEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S. pombe rbd2, D. melanogaster Rho1, or P. falciparumROM4. Conditioned media and cell lysates were analyzed by western blot. P and C denote precursor and cleaved products, respectively.C. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA-Flag substrate and the indicated rhomboid proteases. Whole cell lysates were analyzed by western blot. P, CN, CC denote precursor, N-terminal cleaved and C-terminal cleaved products, respectively.D. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and either wild-type or mutant recoded S. pomberbd2. Whole cell lysates were analyzed by western blot.E. HumanHEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and different rhomboid proteases. Whole cell lysates were analyzed by western blot.F. HumanHEK293T cells were transfected with plasmids expressing the GFP-TatA (A8L)-Flag and recoded S. pomberbd2. C-terminal cleavage products were purified using anti-Flag antibodies and purified protein was analyzed by mass spectrometry.
Figure 5B. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively.D-E. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively. Asterisks indicate non-specific, loading control bands, which were used to normalize the intensities of P and N in Figure 5F. Two independent isolates, A and B, are shown for each strain.F. Intensities of individual P and N bands from western blots were quantified using LI-COR software. Relative intensity of P and N to corresponding non-specific band (asterisk) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis. The ratio of N to P or relative P level was presented in bar graphs.G. The average ratio of N to P level for different Sre2MS mutants in wild-type cells (Appendix Fig S2) was plotted. Cleavage was scored as normal if the ratio was >2.H. For Sre2MS mutants showing defects in cleavage, the average fold-increase in P level in dsc1Δ cells compared to WT cells from two isolates (Appendix Fig S2) was plotted. Mutants were grouped into three phenotypic classes (intermediate, dsc1Δ, and rbd2∆) according to observed precursor accumulation. Precursor accumulation was defined as dsc1Δ if <1.5 (orange), rbd2Δ if >3 (pink), and intermediate if in between (blue).
Figure 6B. Recombinant proteins GST-fused Rbd2 C-terminus (Rbd2200-251), Dsc5 UBX (Dsc5323-425), Dsc2 UBA (Dsc2298-372), and GST-HA-V5 control were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild-type cells. Equivalent amounts of unbound and bound fractions were probed for anti-Cdc48, anti-ubiquitin and anti-GSTIgG.C. GSTpulldown assay was performed as in (B) using truncated forms of Rbd2 C-terminus; Rbd2200-251, Rbd2200-225, Rbd2225-251 and Rbd2200-240. Unbound (1x) and bound (5x) fractions were analyzed by western blottingD. Single residue mutants of conserved glycine residues in Rbd2200-251 (G244R and G246R) were tested for Cdc48 binding as in (C). Western blots (B-D) were imaged using chemiluminescence.
Figure 7A. Detergent-solubilized yeast extracts from cells containing chromosomal Flag-tagged rbd2 or rbd2-G246R in rbd2Δ background were prepared, and proteins associated with Rbd2-Flag were immunopurified using anti-Flag magnetic beads. Equal amount of total (lanes 1-3) along with 25x bound fractions (lanes 4-6) were analyzed by western blot with anti-Cdc48 serum and anti-FlagIgG.C. Biotin-phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods. WT cells or rbd2Δ cells carrying rbd2-Flag-APEX2 (rbd2-F-AX) or rbd2-G246R-Flag-APEX2 (G246R-F-AX) plasmid were lysed after biotin-labeling reaction, and proteins were denatured by heating the cells in lysis buffer containing 1%SDS. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates and 50x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Cdc48 serum, or anti-FlagIgG.
Figure 8A. Yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G244R, rbd2-G246R or rbd2-S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).B. Western blot of phosphatase-treated, whole cell lysates from strains in (A) grown for 3 hr in the presence or absence of oxygen was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.C. Sre2 cleavage was assayed by western blot using anti-Sre2 serum or anti-Flag IgG of phosphatase-treated, whole cell lysates from the indicated strains. N denotes Sre2 cleaved nuclear form.D. Whole cell lysates from indicated yeast strains carrying a plasmid expressing 3xFlag-Sre2MS were analyzed by western blot with anti-Flag antibody. Two independent isolates, A and B, are shown for each strain. Asterisk denotes non-specific band.F. Yeast strains containing Flag-tagged rbd2-SHP, rbd2-SHP*, rbd2-G246R-SHP, and rbd2-G246R-SHP* were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in (E). Indicated yeast strains were then assayed for Sre1 cleavage as in (B).G. Indicated strains were assayed for Sre2 cleavage as in (C).H. Wild-type, sre2Δ or strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130S in rbd2Δ background (lane 7-12) were treated with bortezomib (Bz) for 2 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum or anti-Flag IgG.
Figure 9A. Western blot of phosphatase-treated whole cell lysates from yeast strains carrying rbd2-Flag-APEX2 (rbd2-F), rbd2-G246R-Flag-APEX2 (G246R) or rbd2-S130A-Flag-APEX2 (S130A) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.B. Indicated yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background (lane 3-5) or yeast strains (200 cells) carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background (lane 6-8) were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).C. Comparison of Rbd2 protein levels from overexpression system to endogenous expression. Western blot of whole cell lysates from yeast strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background and yeast strains carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background was probed with anti-FlagIgG. For quantification, the intensity of Flag band in each lane was normalized to the non-specific band (asterisk).D. rbd2Δ cells expressing indicated plasmids of GFP-Anp1 or GFP-Sre2MS together with rbd2-Flag-APEX2, rbd2-S130A-Flag-APEX2 or rbd2-S130A/G246R-Flag-APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin-labeling reaction. After labeling termination, cells were lysed and denatured as described in Materials and Methods. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates (1x input) and 100x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Flag IgG, anti-Cdc48 serum, and anti-GFP IgG. Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample, then normalized to the Flag intensity in enriched sample. Relative intensities of biotinylated GFP level are shown in bar graph. Error bars denote standard deviation from three biological replicates.
Figure 1A, B Representative time-lapse images (A) and kymograph (B) of mouse zygotes expressing H2B-RFP (magenta) and labelled with SiR-Tubulin (green). Shown is an example of marked chromosomal displacement to the cortex. C Changes in chromosome-to-cell centre distances (d) over time relative to anaphase-onset (n = 50 blastomeres). Data information: In (A, time is shown as h post-metaphase and scale bar = 20 µm. In (C, data are shown as mean ± SEM.
Figure 2B, C Representative time-lapse images of embryos in which DMSO (B) or nocodazole (C) was added after anaphase completion. D Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition following DMSO- (n = 22 blastomeres) or nocodazole-treatment (n = 26 blastomeres) at anaphase completion. Data information: In (B,C, time is shown as h post-drug addition and scale bar = 20 µm. In (D, data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).E-G Representative time-lapse images of embryos in which DMSO (E), Nocodazole (F) or Cytochalasin D (G) was added when nuclei had travelled to the cortex. H Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition DMSO- (n = 20 blastomeres), nocodazole- (n = 12 blastomeres) or cytochalasin D-treatment (n = 14 blastomeres) after nuclei reached the cortex. Data information: In E - G), time is shown as h post-drug addition and scale bar = 20 µm. In H), data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).
Figure 3A, B Representative time-lapse images of embryos that have been washed out of either nocodazole after nuclei were prevented from travelling to the cortex after anaphase (A, n = 15 embryos) or cytochalasin D after nuclei had been trapped at the cortex by disrupting actin (B, n = 22 embryos). C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst stage or not. Each data point represents the mean maximal d value of the two blastomeres from the same embryo (n = 45 embryos). D Graph of time taken to develop to the blastocyst stage in relation to maximal d values (n = 27 embryos). E Graph comparing the distribution of times taken for nuclei to re-centre in embryos that developed to the blastocyst stage or not (n = 45 embryos). F Graph of time taken to develop to the blastocyst stage in relation to times taken for nuclei to re-centre (n = 27 embryos). Data information: In (A, B), time is shown as h post-drug washout and scale bar = 20 µm. In (C - F), data are shown as mean ± SEM. In (C, E), statistical significance was assessed by Welch's t-test, ns: not significant. In (D, F), the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test, ns: not significant.
Figure 4A Representative time-lapse image of DNA and SiR-tubulin in DMSO- and BFA-treated embryos during the first mitotic division and subsequent interphase. Time is shown as h relative to the first mitotic anaphase. B Changes in nucleus-to-cell centre distances (d) over time in BFA-treated embryos, relative to maximal nuclear displacement to the cortex (n = 14 blastomeres). Data information: In (A, scale bar = 20 µm.D Representative time-lapse images of the second mitotic division in DMSO- and BFA-treated embryos. Dashed white lines highlight position of nuclei shortly before NEBD. Dashed yellow lines outline daughter-cells. Time is shown as h relative to the second mitotic NEBD. E Dot plot of the duration between first mitotic division anaphase-onset and second mitotic division NEBD for DMSO- (n = 47 blastomeres) and BFA-treated (n = 47 blastomeres) embryos. F Plot of the duration between NEBD and anaphase of the second mitotic division for DMSO- (n = 44 blastomeres) and BFA-treated (n = 43 blastomeres) embryos. G Plot of the spindle-to-cell centre distances against pre-NEBD nucleus-to-cell centre distance (n = 34 blastomeres). Line indicates mathematically regressed fit. Data information: In D), scale bar = 20 µm. In E - G, data are shown as mean ± SEM. In (E, F, statistical significance was assessed by Mann-Whitney test, ****P < 0.0001, ns: not significant. In (G, the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test. Two-tailed p values are reported (**P < 0.01).
Figure 5A Representative time-lapse images of 2-cell embryos cultured with SiR-DNA to label nuclei (white circles) and microinjected with oil droplets (yellow circles). Time is shown as h:min relative to maximal nuclear displacement. B Plot of the distances between oil droplets and nuclei to the cell-centre over time (n = 14 oil-nucleus pairs). Data information: Scale bar = 20 µm in A, In (B), data are shown as mean ± SEM.E Top panel: an example of fluorescent bead distribution in a 2-cell embryo. Lower panel: an example of region imaged for bead tracking at high temporal resolution. F, G Trajectories of bead movement over a 20 s-duration in oocytes (F, n = 217 beads from 23 oocytes) and 2-cell embryos (n = 277 beads from 26 embryos). Each coloured line represents a single bead track. Data information: Scale bar = 20 µm in E, except 2 µm in the lower panel of E.
Figure 7B Shown is a representative 2-cell embryo immunostained for β-tubulin (green) and DNA (magenta) at anaphase completion (n = 8 embryos). C Dot plot of mean β-tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus (cortex-facing aspect, dashed white triangle in B) or behind the nucleus (cleavage plane-facing aspect, dashed yellow triangle in B) (n = 14 embryos). Data information: In (B scale bar = 20 µm. In (C, data are shown as mean ± SEM. In (C, statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.D Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of dynarrestin at anaphase completion (n = 14 embryos). Data information: In (D, time is shown as h post-drug addition. In D, scale bar = 20 µm.E Dot plot of maximal d values between DMSO- and dynarrestin-treated embryos (n = 28 blastomeres per group). Data information: In E, data are shown as mean ± SEM. In E), statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.G Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of taxol at maximal nuclear displacement. Data information: In G, scale bar = 20 µm.H Changes in nucleus-to-cell centre distances (d) over time relative to the time of drug addition in DMSO- (n = 19) and taxol- (n = 19) treated 2-cell blastomeres (mean ± SEM). Data information: In H), data are shown as mean ± SEM. In (H), statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, unremarked datapoints = not significant).I Representative images of embryos immunostained for β-tubulin and Hoechst following the completion of live imaging (shown in E) in DMSO- and taxol-treated embryos (n = 9 embryos per group). Time is shown as h post-drug addition. Scale bar = 20 µm. Data information: In I), scale bar = 20 µm.
Figure 3A. Hematoxylin and eosin (H&E)-stained cross section of a human liver slice. Scale bar is representative of A-F. Image is also used in Fig. S3A. B. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from A. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant) which is representative of B, D, and F. C. H&E-stained cross section of a human lung slice. Image is also used in Fig. S3D. D. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from C. E.H&E-stained cross section of frontal cortical region of a human brain tissue. Image is also used in Fig. S3G. F. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from E. G. Quantification of relative abundance of total glycogen calculated from the sum of CL3-CL18 in human liver, lung, and brain. Values are presented as mean +/- standard error (n=3 technical replicates per region). *0.01 < P < 0.05; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison for total glycogen. H. Glycogen structure represented by normalized chain length distribution (ratio to CL3) of each human liver, lung and brain from G. Values are presented as mean +/- standard error (n=3 technical replicates per region).
Figure 4A. Hematoxylin and eosin (H&E)-stained cross section of adenocarcinoma of the prostate, lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), and Ewing sarcoma (ES) of the abdomen tumor. Annotated regions are T-tumor, S-stroma, D/G-duct/glandular, and A-adipose tissue. Scale bar is representative of A and B. Images are also used in Fig. S4A, 5D and S6G. B. Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Images are also used in Fig. S4E, 5D, and S6G. C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from tumor regions shown in A. Values are presented as mean +/- standard error (n=3 technical replicates per region).