EMBO/SourceData huggingface dataset:
Figure 6 A Unmodified ReNcell VM human neuronal progenitor cells were co-plated with comparable Ren-GFP or Ren-APPSL cells into 3-dimensional Matrigel matrices, and differentiated into neurons for 60 days. Cytosolic GFP is produced by both Ren-GFP and Ren-APPSL cells, and the latter also produce pathogenic human APP with the Swedish and London mutations, and secrete Aβ that becomes entrapped in the Matrige Following neuronal differentiation, 2P-FLIM was used to monitor NiMA in the ReNcell VM neurons, which were discriminated from Ren-GFP and Ren-APPSL neurons because they lack GFP. Note that NiMA was observed in ReNcell VM neurons when they were co-cultured with Ren-GFP neurons, but not when they were co-cultured with Ren-APPSL neurons. Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test B ReNcell VM cells were differentiated into neurons for 30 days in 2D cultures. Subsequent AβO exposure for 16 hours blocked NiMA, but expression of Flag-Raptor-Rheb15, which forces mTORC1 to lysosomes, prevented NiMA inhibition by AβOs. Each colored line refers to a single field of view containing 5-15 cells, and each identically colored pair of solid and dotted lines refers to the same field of view. Flag-Raptor -Rheb15 expression was confirmed by western blot­ting. Statistical analyses were performed using Student's two-tailed unpaired t-test. Western blots are representative of 3 independent assays
Figure 7 A Cortical mouse neurons from tau knockout (KO) mice were serum-starved in Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin or amino acids were immediately added, and the cells were then imaged again 30 minutes later Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test B Cortical mouse neurons from tau KO mice were treated with AβOs for 18 hours before being serum-starved in AβO-containing Hank's balanced salt solution for 2 hours. The cells were then imaged for NiMA, after which insulin was immediately added, and the cells were then imaged again 30 minutes later. Note that NiMA occurred in the absence or presence of AβOs, which inhibited NiMA in wild type human neuron Data information: Each colored lin refers to a single field of view containing 2-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Statistical analyses were performed using Student's two-tailed unpaired t-test
Figure 8Wild type mouse cortical neurons depleted of Nprl with shRNA had mitochondria that were insensitive to amino acids (R+L Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-testWild type mouse cortical neurons depleted o Tsc2 with shRNA had mitochondria tha were insensitive t insuli Each colored line refers to a single field of view containing 5-15 cells, and each pair of solid and dotted lines (same color) refers to the same field of view. Knockdown efficiency was monitored by western blotting of phosphorylated ribosomal S6 protein, a downstream target of the mTORC1-S6K pathway. Western blots are representative of 3 independent assays. Statistical analyses were performed using Student's two-tailed unpaired t-test(D Human fibroblasts deficient in Tsc2 (TSC2 Δexons1-14) were imaged before and 30 minutes after insulin addition. Lack of Tsc2 made mitochondria insensitive to insulin Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view Statistical analyses were performed using Student's two-tailed unpaired t-test(E) Re-expression of human Tsc2 rescues mitochondrial responses to insulin. Cultures were serum-starved in Hank's balanced salt solution for 2 hours before stimulation with insulin or amino acids, and were imaged before and 30 minutes after stimulation. Each colored line refers to a single field of view containing 2-5 cells, and each pair of solid and dotted lines of the same color refers to the same field of view. Re-expression of the Flag-WT Tsc2 was corroborated by indirect immun­o­fluorescence (IF). Statistical analyses were performed using Student's two-tailed unpaired t-test
Figure 1C Sequences of synthetic PrP alleles differing in the octarepeat region of mousePrP. Conserved histidines in octarepeats 2-5 are coloured blue and missense mutations are shown in red. Overproduction of C2 fragment is indicated by ++ signs to the right of the panel.
Figure 2A Lysates from RK13 cells transfected with WT, S1, S3 and S3.F88W PrP were probed with antibody Sha31, 9A2 and 12B2 after digestion with PNGaseF.B, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).B, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).D Lysates of N2a, HEK, SMB-PS and SH-SY5Y cells not transfected (−) and transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF and analysed by Western blot using Sha31.E Analysis of lysates from RK13 cells transfected with plasmids incorporating Phe substitutions into the hydrophobic domain of WT PrP, S1 PrP and S3 PrP. Lysates were PNGaseF-digested before Western blot using the Sha31 antibody. F1: A114F, F2: G118F, F3: G122F, F4: G126F
Figure 3A Brain homogenates of TgPrP(WT), TgPrP(S1)-17 and TgPrP(S3.F88W)-35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31, 12B2 and 9A2. Prnp0/0, and WT brain homogenates were used as controls.B The ratio of C2 to full-length PrP in TgPrP(S3.F88W)-35 and WT mice (left panel, n = 7, P = 1.46E-06) and in RK13 cells expressing WT and S3 PrP (right panel, n = 5, P = 4.01E-05) is shown. Values are presented as mean ± SEM. Unpaired, two-tailed t-test, ****P < 0.0001.C Different tissue homogenates from a TgPrP(S3.F88W)-35 mouse were PNGaseF-digested and analysed for PrP using the antibody 1A6. FL, full-length PrP; ns, non-specific signal detected in the immunoblotting procedure.
Figure 4A Sections of sciatic nerve from TgPrP(S3.F88W)-14 female mice (all 610 days old) and TgPrP(S3.F88W)-35 female mice (614, 570 and 614 days old, respectively) were analysed using toluidine blue staining (upper row), with the third panels for each line showing a hypermyelinated phenotype. Scale bar in left panel represents 50 μm. The lower rows show EM analyses of mice of the indicated genotypes. Scale bar is 10 μm for panels 1-4, with the fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3.F88W PrP (scale bar, 5 μm).B Western blot analysis of sciatic nerve protein extracts (10 μg) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain (see Fig3). The asterisk indicates a greater than full-length fragment present in TgPrP(S3.F88W) mice, possibly corresponding to incomplete removal of the N-terminal signal peptide.C Quantification of fibre morphology in aged animals (492-608 days old), representing the percentage of the nerve occupied by fibres versus the total cross-sectional area of the nerve. The S1 and S3.F88W alleles rescue Prnp0/0 nerves to a similar percentage as the WT allele, but the variance and P-value is greater for the two Tg lines expressing the TgPrP(S3.F88W) allele. Unpaired, two-tailed t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Sample sizes and exact P-values compared with Prnp0/0 (n = 9) were as follows: TgPrP(WT) n = 6, P = 3.63E-05; Prnp0/0, n = 9; TgPrP(S1)-17 n = 5, P = 3.54E-05; TgPrP(S1)-19 n = 3, P = 6.35E-06; TgPrP(S3.F88W)-14 n = 3, P = 3.70E-03; and TgPrP(S3.F88W)-35 n = 7, P = 7.18E-03.
Figure 5A, B Infected brain homogenates were PK-digested and analysed by Western blot using (A) Sha31 antibody and (B) after PK and PNGaseF digestion using Sha31.C Conformational stability assay on RML-infected brain homogenates incubated with increasing concentrations of GdnHCl. The best-fit alignments of the data are presented. Genotypes are noted on the individual panels.Source data are available online for this figure.
Figure 6Pathology of RML-infected miceA-H The pattern of PrPSc deposition (A-D) and GFAP immunostaining (E-H) is presented.I-P A higher power view of H&amp;amp;E stained hippocampus (I-L) and thalamus (M-P) to reveal vacuolation is presented.Data information: Animals of the same genotype are shown within a column, and incubation times of the presented animals with the RML prion isolate are shown (days). Mice expressing PrP S1 and S3 alleles (central columns) exhibit attenuated PrPSc deposition, gliosis and spongy change. Pathological features of the RML isolate are not altered upon sequential passage through TgPrP(S3.88W)-35 mice (see schematic at bottom of the diagram indicating sequential passages and finally, passage back into WT mice, compare the data in first and fourth columns).
Figure 7A Spot counts in the scrapie cell assay using WT, TgPrP(S1)-17 and TgPrP(S3.F88W)-14 brain homogenates (0.1% or 0.01%) from mice infected with RML are shown in the upper panel (WT vs S1 P = 2.09E-05 and vs S3 P = 4.62E-08) with numbers corrected for PrPC expression levels as per Table1 presented in the lower panel (WT vs S1 P = 1.04E-13 and vs S3 P = 3.39E-20; two-tailed t-test, n = 3).B A comparison of incubation time vs PrPC:PrPSc ratio for mice expressing different levels of WT PrP (WT, TgPrP(WT), tga20), S1 PrP (TgPrP(S1)-17, TgPrP(S1)-19, TgPrP(S1)-39) and S3 PrP (TgPrP(S3.F88W)-14, TgPrP(S3.F88W)-35).
Figure 1B) Co-immunoprecipitation analysis to elucidate the interaction between Suv39h1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Suv39h1 or 3xFlag-ΔN-Suv39h1 were subjected to immunoprecipitation with the anti-Flag tag antibody followed by immunoblotting with the indicated antibodies. IgG represents the heavy or light chainF) Co-immunoprecipitation analysis to elucidate the interaction between Setdb1 and HP1. Whole-cell extracts of HEK293T cells expressing 3xFlag-Setdb1 or 3xFlag-Setdb1 mutants were subjected to immunoprecipitation with the anti-HP1β antibody The immunocomplex was analyzed by immunoblotting with the indicated antibodies.G) Protein decay analysis of HP1 interaction-defective Setdb1 mutant. mESC lines stably expressing 3xFlag-Setdb1 or 3xFlag-Setdb1-3A were treated with CHX and MG132 for the indicated times. Stacked bar plots show the summarized protein amounts of Setdb1 (right). Data represented as mean ± SD (n = 4, biological replicates). **p < 0.01 (Tukey's honestly significant difference test).
Figure 2B) Protein expression levels of H3K9 MTs and histone modification levels in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and means of the experimental replicates (n ≥ 3, biological replicates), respectively.C) Comparison of H3K9me3 levels between HP1-deficient cells and Suv39h/Setdb1-deficient cells. H3K9me levels were quantified using ImageJ software and are summarized at the bottom; the horizontal line represents the mean intensity of each cell. More than 75 nuclei were counted for each staining. **p < 0.01 (Tukey's honestly significant difference test).D) Electron microscopy images of HP1-cTKO cells in the absence of 4OHT (left) and of cells treated with 4OHT for 4 days (right). Boxes represent enlarged images.
Figure 3B) Subcellular distribution of H3K9 MTs in the HP1-cTKO cells was examined by immunoblotting. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction. Stacked bar plots show the abundance ratios of H3K9 MTs among the three fractions (right).
Figure 4B, Elucidation of protein-protein interaction between HP1 and H3K9 MTs by co-immunoprecipitation analysis. Whole-cell extracts of HEK293T cells co-expressing HA-HP1β mutants and epitope-tagged proteins of interest were subjected to immunoprecipitation with the anti-HA tag antibody. The immunocomplex was analyzed by immunoblotting with the indicated antibodies. The intensities were quantified using ImageJ software, normalized, and shown on the top.E) Evaluation of HP1β mutants for maintaining H3K9me2/3 and ensuring protein stability of H3K9 MTs. H3K9me2/3 levels and protein levels of H3K9 MTs in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).
Figure 5B) Protein expression levels of Jmjd1a/b in HP1-cTKO cells were monitored by immunoblotting. The intensities were quantified using ImageJ software and summarized on the right. The thin gray line and bold colored line show individual data and the mean of experimental replicates, respectively.C) Evaluation of HP1β mutants for ensuring protein stability of Jmjd1a/b. Protein levels of Jmjd1a/b in HP1-cTKO lines expressing 3xFlag-hHP1β mutants were compared before and after 4OHT treatment. The intensities were quantified with ImageJ software and are summarized on the right. Data represented as mean ± SD (n > 3, biological replicates). *p < 0.05, **p < 0.01 (Welch's test).
Figure 6C) Impact of Suv39h/Setdb1/G9a depletion on HP1-mediated protein stability regulation of Jmjd1a/b. Jmjd1a/b were destabilized in mESCs lacking Suv39h/Setdb1/G9a, even though HP1 was present.D) Functional evaluation of H3K9me2/3 for confining HP1 to chromatin. Subcellular localizations of HP1α, β and γ in TT2 and mESCs lacking Suv39h/Setdb1/G9a were compared. α-Tubulin, Ezh2, and histone H3 were used as representative markers for each fraction.
Figure 1Growth suppression in cancer cells after 72h of treatment with APR-246 +/- MRP1 inhibitor MK-571 as shown by the WST-1 assay (n ≥ 3, n shown in Table S1). Cross in heatmap indicates no data available.Growth suppression in human dermal fibroblasts (HDF) by WST-1 assay after 72h of with APR-246 +/- MK-571 (n = 3). Mean IC50 values (µM) for APR-246 +/- 20 μM MK-571 of 21 cell lines. *p < 0.0001, Wilcoxon test. Each dot represents one cell line. Mean IC50 values and n shown in Table S1. ZIP synergy scores of most synergistic area sub-grouped according to TP53 gene status for 21 cell lines. Each dot represents one cell line. Scores and n are shown in Table S1. Sub-G1 DNA content of OVCAR-3 cells (R248Q) after APR-246 +/- MK-571 treatment as assessed by propidium iodide staining at 48h (n = 3).OVCAR-3 cell confluency by IncuCyte® during 72h of treatment with APR-246 +/- MK-571 (n = 3).Growth suppression in HCT116 WT (n = 4) and R248W cells (n = 3) after 72h treatment with APR-246 +/- MRP1 inhibitor Reversan, as shown by WST-1.Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 48h after transfection with negative control siRNAs or siRNAs targeting MRP1 or p53.Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with MRP1 siRNA (n ≥ 5 and n ≥ 2 respectively) after 48h APR-246 treatment. Indicated values are average of four individual siRNAs targeting MRP1 and two individual negative control siRNAs. Data and n of individual siRNAs are shown in Fig S1J, data is also part of Fig 7B.Western blot analysis of MRP1 (Cell Signaling.), p53 (DO-1) and GAPDH of HCT116 WT and R248W cells 72h after transfection with empty vector (EV) and MRP1 expression vector.Growth suppression (WST-1 assay) of HCT116 WT and R248W cells transfected with empty vector (EV) and MRP1 vector after 72h APR-246 treatment (n = 4). Data is also part of Fig S7E.
Figure 2Growth of Eso26 xenografts (R248W mutant TP53) in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. p values determined by one-way ANOVA with Tukey's correction at day 18, n is indicated in the figure.   Kaplan-Meier plot of time to reach 1400 mm3 tumor volume in mice treated with vehicle, APR-246 (50mg/kg), MK-571 (50mg/kg) or APR-246 and MK-571 for 16 days. Mice that were culled at ethical endpoints prior to reaching a tumor volume of >1400mm3 are marked as censored observations. p values were determined by Log-rank (Mantell-Cox) test between different treatment groups. n is indicated in the figure.   Quantification of Ki67 immunohistochemistry staining of Eso26 xenograft tumors at an early timepoint (5 days) or post treatment / late timepoint (>22 days) after treatment initiation with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). Dots indicate individual tumors.Quantification of cleaved caspase 3 immunohistochemistry staining of Eso26 xenograft tumors in same tumors as shown in Fig. 2C. Dots indicate individual tumors. Representative images of hematoxylin/eosin (H&E) staining and immunostaining of Ki67, cleaved caspase 3 and p53, post treatment / late timepoint (>22 days after treatment initiation) with APR-246 (50mg/kg) +/- MK-571 (50mg/kg). Growth suppression determined by the ATP-based CTG assay in colorectal cancer patient-derived organoids (colo-PDO) after treatment with APR-246 +/- MK-571 (n = 3).IC50 values (µM) APR-246 +/- 20 μM MK-571 in indicated PDOs. *p = 0.04, paired t test (n = 3 except colo-PDO2 and eso-PDO3 where n = 2).Organoid area (in black, left y-axis) and PI intensity (in red, right y-axis) of colo-PDO1 and colo-PDO2 as determined by image analysis 72h after treatment with APR-246 +/- MK-571 (n = 3).
Figure 314C accumulation (cpm/mg x ml-1) in 11 cell lines after 24h 14C -APR-246 +/- MK-571 treatment (n ≥ 3). *p = 0.0006, paired t-test. 14C-accumulation and n of individual cell lines as shown in Table S3.14C accumulation (cpm/mg x ml-1) in HCT116 (WT and R248W) cells after 24h +treatment with 14C -APR-246 +/- MK-571 or Reversan (n ≥ 3, each dots in figure).14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells (n ≥ 3 and ≥ 2) transfected with MRP1 siRNAs after 24h of 14C-APR-246 treatment. Indicated values represent four individual MRP1 siRNAs and two individual negative control (Ctrl) siRNAs, where each dot is an individual siRNA. Mean of 14C-accumulation after treatment and n with individual siRNA sequences is shown in Table S4. *p = 0.04 and for R248W p = 0.06, Wilcoxon matched pairs signed rank test.
Figure 4APR-246 content in OVCAR-3 cells (TP53 R248Q) 24h after treatment with APR-246 +/- MK-571, (n = 3). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.Glutathione-conjugated MQ (GS-MQ) in OVCAR-3 cells after 24h treatment with APR-246 +/- MK-571 (n = 2). Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.Amount of GS-MQ at indicated timepoints after incubation of GS-MQ +/- NAC (n = 2). See Fig. EV4 for chemical reaction. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.Amount of GS-MQ by LC-MS (green line, left axis) and NAC-MQ (purple line, right axis) over time after incubation of GS-MQ with NAC (n = 2). Values on y-axes are not comparable because GS-MQ and NAC-MQ have different response signals on MS. Data information: Indicated values are assessed by LC-MS. Data are represented as mean ± SEM. See also Appendix Figure S4.
Figure 6 Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24h MK-571 treatment in six different cell lines (n ≥ 3, except HCT116 WT n = 2). *p = 0.014, Paired t test. Each dot represents one cell line, see Fig. EV6A for individual cell lines and n. Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24h treatment with APR-246 +/- MK-571 (n = 3). Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).  Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24h. Western blot analysis of MRP1 (Cell Signaling), xCT and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24h. Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).  Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24h treatment with APR-246 +/- MK-571 (n = 3).
Figure 7 14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W mutant TP53 cells +-transfected with MRP1 or xCT siRNA following 24h of 14C -APR-246 treatment +/- MK-571 . Indicated values are mean values from four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n = 1-2) and two control siRNAs (n ≥ 3). Mean 14C -accumulation after treatment and n of individual siRNAs is shown in Table S4 . Data for control and MRP1 siRNA after 14C -APR-246 treatment is also shown in Fig 3D.  Growth suppression in HCT116 WT and R248W cells transfected with MRP1 or xCT siRNA after 48h of APR-246 treatment as shown by WST-1 assay. Indicated values are mean values of four MRP1 siRNAs (n ≥ 3), three xCT siRNAs (n =1-2) and two control siRNAs (n ≥ 3). Data for control and MRP1 siRNA after APR-246 single treatment are also included in Fig 1I. Values and n for individual siRNA shown in Fig. S7C.  Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT cells as shown by GR re-cycling assay 48h post transfection of siRNA. Indicated values are means from four MRP1 siRNAs (n = 3), two xCT siRNA (n = 2-3) and two control siRNA (n = 2-3).  Total intracellular glutathione (GSH + GSSG per 106 cells) in HCT116 WT and R248W cells measured by GR re-cycling assay after 24h of MK-571 or sulfasalazine (SSZ) treatment (n ≥ 3, except SSZ where n = 2, n indicated by dots).  14C accumulation (cpm/mg x ml-1) in HCT116 WT and R248W cells at 24h treatment with 14C -APR-246, combined with MK-571 or sulfasalazine (SSZ), (n ≥ 4, except SSZ where n = 2, n indicated by dots). Data for 14C -APR-246 +/- MK-571 is also shown in Fig 3C and S3D.  Protein concentration of HCT116 WT and R248W assessed by DCTM Protein assay at 24h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ), or untreated (n ≥ 4, except SSZ where n = 2, n indicated by dots)  OVCAR-3 (R248Q TP53) confluence by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalzine (SSZ) (n = 3). Part of the data are shown in Fig 1G. Growth suppression in OVCAR-3 cells as determined by IncuCyte® during 72h treatment with APR-246 +/- MK-571 or sulfasalazine (SSZ) depicted as heatmaps (n = 3).