EMBO/SourceData huggingface dataset:
Figure 1A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.B, H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.C, Lipid ROS levels were assessed by flow cytometry after C11-BODIPY staining in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.D, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown.E, Control H1299 cells (transfected with siCont. or H2BWT) and H2Bub1-depleted H1299 cells (transfected with siRNF20 or H2BK120R) were treated with erastin either with or without a ferroptosis-specific inhibitor, ferrostatin-1 (2 μM), for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.
Figure 2A, qRT-PCR (left) and Western blot (right) analyses of H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) for 24 h. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.B, Chromatin immunoprecipitation (ChIP) assay was carried out with anti-H2Bub1 antibodies in H1299 cells (left) or 293T cells either untreated or treated with 20 μM erastin for 24 h (right). The intergenic region was used as a negative control for the occupancy of H2Bub1. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.C, Intracellular GSH levels were examined in H1299 cells treated as indicated, and bar graphs are shown. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.D, H1299 cells transfected as indicated were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20). E, Surviving cells from the assay shown in (D) were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.G, Affected metal ion-binding genes were selected and subjected to cluster analysis.H, Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells.I, Labile iron levels examined were quantified. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.
Figure 3Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.Western blot analysis was performed with H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.C, Immunofluorescence (IF) assay for H1299 cells transfected with a Flag-tagged p53 (Flag-p53) or an empty plasmid (Cont.) for 48 h. Representative images are shown (magnification, ×40). Arrowhead indicates a Flag-p53 overexpressing cell.D, Western blot analysis was performed with H1299 transfected with a Flag-tagged p53 or an empty vector (Cont.) for 48 h.E, 293T cells transfected with a control siRNA (siCont.) and three p53-specific siRNAs (sip53) were subjected to Western blot analysis.F, SMMC cells were treated with 100 μM etoposide (+) or untreated (-) for 24 h, and Western blot analysis was performed., Western blot analysis of A549 cells and H1299 cells.H, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) were analyzed by micrococcal nuclease (MNase) digestion assay.I, Western blot analysis of H1299 cells transfected with a wild-type p53 (p53-WT) or two "gain-of-function" mutants (p53-R273H and p53-R175H) for 48 h.
Figure 4A, Coimmunoprecipitation (Co-IP) analysis was performed in A549 cells with antibodies against p53 or USP7.B, H1299 cells were transfected with an empty vector (Cont.), a Flag-tagged p53, a USP7-specific shRNA, or the last two plasmids together for 48 h. Western blot analysis was then performed.C, Nuclear and cytosolic extracts were differentially prepared from H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.). Western blot analysis was then performed.D, H1299 cells transfected with a Myc-tagged USP7 or a Myc-tagged USP7 together with a Flag-tagged p53 were subjected to IF analysis (upper). A549 cells transfected with a p53-specific siRNA or a control siRNA (siCont.) were examined by IF analysis, and the RNAi efficiency is shown (lower). Representative images are shown (magnification, ×40).F, IF analysis of A549 cells transfected with a Myc-tagged wild-type USP7 (Myc-USP7-WT) or three Myc-tagged truncated mutants. Representative images are shown (magnification, ×40).G, H1299 cells were transfected with a Flag-tagged p53 or an empty vector together with a USP7-specific siRNA or a control siRNA as indicated for 48 h. Cells were then harvested and subjected to a MNase digestion assay.
Figure 5A, Co-IP analysis was performed in 293T cells with antibodies against p53 or H2Bub1.Peptide pull-down assay was performed with synthesized peptidesD, Co-IP analysis for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) treated with a USP7-specific inhibitor P5091.
Figure 6A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.B, H1299 cells were transfected with Flag-tagged p53 for 24 h and then treated with 12 μM erastin or untreated for another 24 h. Co-IP analysis was performed with antibodies against Flag.C, H1299 cells transfected with Flag-tagged p53 were treated with erastin or untreated for 24 h. Nuclear and cytosolic extracts were differentially prepared and subjected to Western blot analysis.D, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA, or a control siRNA (siCont.) were treated with 20 μM erastin or untreated for 24 h as indicated. Western blot analysis was then performed., qRT-PCR (above) and Western blot (below) analyses for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.F, ChIP analysis of H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.G, H1299 cells transfected with Flag-tagged p53 were treated with 12 μM erastin or untreated for 24 h. ChIP analysis with antibodies against Flag or USP7 was performed. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.H, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h. ChIP analysis was performed with anti-H2Bub1 antibodies. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.
Figure 7A, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as were treated with 12 μM erastin (+) or untreated (-) for 24 h as indicated. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.B, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown. Arrow head indicates condensed and marginated chromatin.C, Lipid ROS levels were examined in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.
Figure 1Examples of CT scan images of several metastases (M032R1, M032R2, and M032R5).Volumetric measurements based on the PET and CT scans could be generated from M032, M032R1, and M032R5. Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment, but regressed upon vemurafenib treatment. M032R5 showed progressive disease after 4 months, whereas M032R1 still displayed stable disease. M032 was excised and no recurrence was observed.
Figure 2Hematoxylin-eosin (H&amp;amp;E) and p-ERK stainings on FFPE material of all metastases showed that all vemurafenib-resistant tumors had reactivation of the MAPK pathway. Scale bar represents 100 μmqPCR was performed on gDNA retrieved from each of the metastases, using primers for BRAF and CRAF and normalized on LINE levels. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in M032R1, M032R2, and M032R5.Staining for BRAFV600E with a mutant epitope-specific antibody confirmed the upregulation of BRAFV600E in R1, R2, and R5. Scale bar represents 100 μm.
Figure 3Immunoblotting for MAPK pathway components confirmed reactivation of p-ERK and p-MEK in all resistant metastases.Immunoblotting for M032 patient's samples using an antibody directed against either the mutant BRAFV600E epitope or an N-terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3, of approximately 80 kDa. This 80-kDa band could not be detected with the N-terminus-recognizing antibody.
Figure 4Validation of T55delinsRT was done by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used.Transfection of pQXCIP-GFP, MEK1-WT, or MEK1-T55delinsRT into HEK293T cells showed that this mutation induces p-ERK and p-RSK.Validation of expression of pQXCIP-GFP, MEK1WT, or MEK1T55delinsRT in A375 melanoma cells by immunoblotting.Dose-response curves for A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib, ERK inhibitor SCH772984, or MEK inhibitor trametinib. Error bars indicate standard deviation.Colony formation assays with A375 melanoma cells, infected with GFP, MEK1WT, or MEK1T55delinsRT-encoding lentivirus, and treated with indicated doses and inhibitors.Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO (−), 250 nM dabrafenib (D), 10 nM trametinib (T), or a combination (D+T).
Figure 5A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT were injected into immune-deficient mice (n = 8 per group), and after the tumor size of ˜100 mm3 was reached, mice were treated with 30 mg/kg dabrafenib or vehicle. Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment. Error bars indicate standard error of the mean.Average tumor weight of all groups at the end of the experiment described in (A). Error bars indicate standard deviation. Unpaired t-test was used to calculate statistical significance. P-values are indicated in the graph.Immunoblotting for MAPK pathway components performed on tumors of all groups.Immunohistochemistry for Sirius Red (staining for collagen) and H&amp;amp;E on tumors from all groups, to identify grade of demarcation and local invasion. Scale bar represents 500 (upper row) and 100 (lower row) μm.
Figure 6gDNA was isolated from another fragment of the tumor resection, and the PCR with the insertion-specific primers was repeated. Primers amplifying exon 2 of MEK1WT were used as an input control.Source data are available online for this figure.
Figure 7Staining for hematoxylin-eosin (H&amp;amp;E) and p-ERK on the PDX samples M032.X1, M032R1.X1, M032R2.X1, M032R4.X1, and M032R5.X1. Stainings showed that p-ERK is higher in the PDX derived from the resistant metastases. Scale bar represents 100 μm.Immunoblotting for components of the MAPK pathway confirmed reactivation of p-ERK in the PDX derived from the vemurafenib-resistant metastases, although the p-ERK signal is heterogeneous in the pre-treatment PDX.Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX, using primers for BRAF and CRAF and normalized on LINE expression. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5, but not from M032R1.Presence of the MEK1T55delinsRT was analyzed by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used. The insertion was found in M032R1.X1 and M032R4.X1.
Figure 1A-C Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (see Figure EV1, 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows.D Representative microscopy images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots and the nuclei are visualized by DAPI staining. Scale bar: 5 μm.E The Fab-PLA results are quantified by Blobfinder software and presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from treated samples were compared to data from resting cells, p-values were calculated by Kruskal-Wallis one-way analysis of variance (ANOVA).
Figure 2A-C Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.D Representative microscopy images showing Fab-PLA results monitoring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. Scale bar: 5 μm.E Quantified Fab-PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. p-values were calculated by Kruskal-Wallis one-way ANOVA.
Figure 3A-D Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.E-H Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.
Figure 4A-D Proximity between IgD-BCRs on the surface of 3046SD (A, C) or 3046D (B, D) cells before and after a 1-min stimulation, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.E-H Calcium flux measured by FACScan for 3046SD (E, F) and 3046D (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.
Figure 6A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan.F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.
Figure 1API delivery is blocked at low temperatures. SEY6210 cells were pulse labeled for 5 min and chased for the times indicated. Identical experiments were performed at 10°, 14°, and 30°C. API was recovered by immunoprecipitation followed by SDS-PAGE and detected by a STORM phosphorimager (Molecular Dynamics, Sunnyvale, CA). The positions of prAPI and mAPI are indicated.
Figure 2Precursor API is trapped in a prevacuolar compartment in vps18 cells. (A) Yeast strain JSR18Δ1 with plasmid pJSR9 (vps18 ts) was grown at the permissive temperature of 26°C. Before labeling, the cells were incubated at either 26° or 38°C for 10 min. Pulse labeling was for 5 min, followed by the indicated chase times.(B) vps18 spheroplasts were shifted to 38°C for 5 min, pulse labeled for 5 min, and then chased for either 0 or 60 min. At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer. The Total (T) fraction either received no treatment, was treated with 50 μg/ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100. The supernatant (S0) and pellet (P0) fractions were collected after centrifugation at 10,000 g for 5 min.
Figure 3A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment. (A) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer. The Total (T) fraction (after differential lysis) either received no treatment, was treated with 50 μg/ ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100.(B) The supernatant (S200) and pellet (P200) fractions were collected after centrifugation at 5,000 g for 5 min. The P200 fraction was resuspended in 10% Ficoll, and vacuoles were isolated by flotation through 4% Ficoll. V, vacuole; I, 4/10% Ficoll interface; P, gradient pellet. Proteins were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.(C) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.
Figure 4Precursor API is trapped within a vesicle in cvt17 mutants. (A) Precursor API is in a protease-protected compartment in cvt17 mutants. THY32 (cvt17) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig 3B) Precursor API is not free in the vacuolar lumen in cvt17 mutants. Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant (S0) and pellet (P0) fractions by centrifugation at 10,000 g. The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X-100 as indicated. All fractions were precipitated with 10% TCA and resolved by SDS PAGE; proteins of interest were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.
Figure 5Precursor API is contained within subvacuolar vesicles in cvt17 mutants. (A) Isolated vacuoles (V) from cvt17 cells were lysed in PS0 buffer, loaded on top of an Optiprep step gradient, and centrifuged at 170,000 g for 60 min as described in Materials and Methods. Fractions VV, 0/1.06 interface; LD, 1.06 region; SV, 1.06/1.12 interface; HD, 1.12 region.(B) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X-100 as indicated. Because of the presence of protease inhibitors in the vesicle isolation procedure, an intermediate-sized API degradation product results from proteinase K treatment. The V fraction represents one-tenth of the load of the Optiprep gradient. Proteins were detected by Western blotting. The positions of prAPI and mALP are indicated.
Figure 6Immuno-EM of cvt17 cells. Yeast strains SEY6210 (parental wild type) and THY32 (cvt17) were grown in YPD and prepared for microscopy as described in Materials and Methods. (A) Wild-type cells probed with antibody against mature API. (B) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion. (C) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API. N, nucleus; V, vacuole; (arrows) API containing vesicles. Bars, 0.5 μm.
Figure 7Characterization of API-containing vesicles. (A) Fractionations were performed as in Fig. 5. The total (T), vacuole (V), vacuolar vesicle (VV), and subvacuolar vesicle (SV) fractions are shown. Proteins were detected by immunoblotting as indicated in the figure.(B) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager. The relative mobility of the molecular mass standards are indicated on the left. Polypeptides enriched in the subvacuolar vesicles are indicated on the right.
Figure 1C Maximal cytochrome c release for all DX/REL pairs with ≥3 biological replicates); box-whisker plots summarize data (box 25%-75%; belt=median; dot=mean; whiskers=minimum and maximum values). Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend p<0.10). Cyto c, cytochrome cD Bak oligomerization in response to escalating tBid concentration for DX/REL pairs. Relative mitochondrial protein loading per lane is assessed by densitometry, showing ration of loading control in REL lane with patient-matched DX lane at same tBid exposure (no REL lane is underloaded compared to DX lane).
Figure 2A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin. Results are shown for three DX/REL neuroblastoma cell line pairs, two with attenuated cytochrome c release (CHLA15/CHLA20 and SKNBE1/SKNBE2C) and one without (CHLA122/CHLA136). Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates dotted-line represents 50% viability.C, In vitro viability for CHLA15 (DX) and CHLA20 (REL) following ionizing radiation at 7 days Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates, dotted-line represents 50% viability.F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells, in comparison to cells cultured in escalating concentrations of crizotinib until resistant (NB1643-ALKR and SY5Y-ALKR). Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.
Figure 3A, B, C Transmission electron microscopy image analysis was used to quantify mitochondrial size, circularity and roundness in DX/REL neuroblastoma pairs (mean +/- SD shown). Data information: , n=137-241 mitochondria per cell line statistical analyses were performed using a two-tailed Mann-Whitney U test, with significance p<0.05 (trend p<0.10).