EMBO/soda-nel huggingface dataset:
The structure of the MICU1-MICU2 complex unveils the regulation of the mitochondrial calcium uniporter (2020)
Figure 4

Figure 4 The characterization of interactions between EMRE peptide and MICU1 in vitro and EMRE peptide competition in cells.

A The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA.

B The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2+.

C Comparison of binding of MICU1 and EMRE peptide in both conditions.

D MICU1-MICU2 heterodimer interacts with the EMRE peptide at a 1:1 ratio.

E Carbachol (CCh)-induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt-EMRE and scramble peptides. Solid lines, mean; shaded regions, ± SEM, n = 3.

F Resting mitochondrial Ca2+ levels in mt-EMRE and scramble peptides treated cells, evaluated via ratiometric imaging of the mitochondrial targeted mt-riG6m. Mean ± s.d., student t-test, **p < 0.01, n = 3.

G Mean mitochondrial Ca2+ peaks response to 100 μM CCh. Mean ± s.d., student t-test, ***p ≤ 0.001, n = 3.

H Representative [Ca2+]c clearance traces of U2OS cells treated with PBS and mt-EMRE peptide.

I Quantification of mitochondria Ca2+ uptake rate in response to 100 μM histamine. Mean ± s.d., student t-test, n.s., not significant, n = 4.

Data information: Error bars for some points are too small to be visible.
The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity (2020)
Figure 1

Figure 1. OTUD1 stabilizes IREB2 and promotes TFRC expression.

(A) Gene set enrichment analysis (GSEA) of differentially expressed genes in colorectal cancer from the GEO database (GDS4382). ES, enrichment score; NES, normalized enrichment score. n = 3 biological samples.

(B) Kaplan-Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset (n = 590 samples, P = 0.016, Log-rank (Mantel-Cox) test). "Normal" represents the patients with unaltered expression of iron homoeostasis-related genes, including LTF, CP, FTH1, SLC30A1 and TFRC; "Dysfunctional iron homoeostasis" represents the patients with altered expression of iron homoeostasis-related genes.

(C) Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues (Left), the relative protein level of IREB2 was assessed by ImageJ software (Right) (n = 8 human samples, *P = 0.0114, two-tailed paired Student's t-test).

(D) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue, and was subsequently analyzed by anti-ubiquitin antibody for the assessment of ubiquitination.

(E) Mass spectrum analysis of IREB2-associated proteins. Mock or IREB2-FLAG was transfected into HEK293T cells and FLAG-tagged proteins were enriched by anti-FLAG M2 beads and incubated with lysates from normal mice colon tissues. Five matched peptides corresponding to OTUD1 were shown on the right panel.

(F) In vivo ubiquitination assay of IREB2. HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti-FLAG antibody followed by western blot analysis.

(G) Half-life analysis of IREB2 in wild-type (WT) and OTUD1─/─ NCM460 cells. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated times for western blot analysis (Up), the relative protein level of IREB2 was assessed by ImageJ software (Down) (n = 3 biological replicates, mean ± s.e.m., **P = 0.0049, ***P < 0.0001, two-tailed unpaired Student's t-test).

(H) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC (50 μM) and Hemin (100 μM) treatment. MFI, mean fluorescence intensity. Control uses isotype-matched control antibody (n = 4 biological replicates, mean ± s.e.m., **P = 0.0010, ***P = 0.0008, two-tailed unpaired Student's t-test).

(I) Flow cytometric analysis of TFRC expression in wild-type (WT) and OTUD1─/─ NCM460 cells with DFO (100 μM) treatment. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., ***P = 0.0001, two-tailed unpaired Student's t-test).
The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity (2020)
Figure 2

Figure 2. OTUD1 is selectively downregulated in colon cancers.

(A) RT-qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues (n = 101 human samples, ***P < 0.001, two-tailed paired Student's t-test)

(B) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer (n = 101 human samples, two-tailed Pearson correlation analysis, ****P < 0.0001).

(C) Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues (n = 4 human samples), the protein levels relative to GAPDH are marked.

(D) Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue (The scale bars represent 1000 μm).

(E) Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05-118e.

(F) Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a (The scale bars represent 1000 μm).
The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity (2020)
Figure 3

Figure 3. OTUD1 promotes iron transportation.

(A and B) Intracellular iron concentration was measured in CT26 cells stably expressing mock, OTUD1 or OTUD1C320S (n = 2 biological replicates) (A), wild-type (WT) or OTUD1─/─ NCM460 cells (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) (B).

(C) The protein levels of IREB2, TFRC and OTUD1 were assessed by western blot in colon tissues from wild-type (WT) or Otud1─/─ mice.

(D) Intracellular iron concentration was measured in colon tissues from wild-type (WT) or Otud1─/─ mice with supplementation of ferrous gluconate (n = 3 biological replicates, mean ± s.e.m., ***P = 0.0007, two-tailed unpaired Student's t-test).

(E-I) Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0472, two-tailed unpaired Student's t-test) (E), mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0048, two-tailed unpaired Student's t-test) (F), mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0068, two-tailed unpaired Student's t-test) (G), red boold cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0049, two-tailed unpaired Student's t-test) (H) and hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0036, ***P = 0.0002, two-tailed unpaired Student's t-test) (I) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.
The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity (2020)
Figure 4

Figure 4. OTUD1 limits tumor growth and augments host antitumor immunity.

(A-B) Macroscopic evaluation (A) and tumor volume (mock, n = 7 biological replicates; OTUD1, n = 9 biological replicates; OTUD1C320S, n = 9 biological replicates; mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) (B) of mock, OTUD1 and OTUD1C320S expressing CT26 tumors in BALB/c mice.

(C-E) Macroscopic evaluation (C), tumor volume (n=6 biological replicates, mean ± s.e.m., **P < 0.01, ***P = 0.0007, two-tailed unpaired Student's t-test) (D) and survival analysis (n=5 biological replicates, **P = 0.0021, Log-rank (Mantel-Cox) test) (E) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.

(F) Endogenous IREB2 and TFRC protein levels in murine CT26 tumors were measured by anti-IREB2 antibody and anti-TFRC antibody.

(G) Flow cytometric analysis of TFRC expression in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors. MFI, mean fluorescence intensity (mean ± s.e.m., **P < 0.01, two-tailed unpaired Student's t-test).

(H) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors. MFI, mean fluorescence intensity. (n = 5 biological replicates, mean ± s.e.m., **P = 0.0011, two-tailed unpaired Student's t-test).

(I) Iron concentration of mock, OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry (n = 3 biological replicates, mean ± s.e.m., *P = 0.0237, **P = 0.0098, two-tailed unpaired Student's t-test).

(J)Analysis of tumor infiltrating CD8+ T cells or CD4+ T cells in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors in BALB/c mice (mean ± s.e.m., **P < 0.01, ***P < 0.001, two-tailed unpaired Student's t-test).

(K and L) Flow cytometric analysis of CD8+ T cells, CD4+ T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P = 0.0003, two-tailed unpaired Student's t-test) (K) and PD-1 expression in T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P < 0.0001, two-tailed unpaired Student's t-test) (L) isolated from mock and OTUD1 overexpressing LLC tumors. MFI, mean fluorescence intensity.
The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity (2020)
Figure 5

Figure 5. OTUD1 enhances ferroptotic cell death.

(A) Quantitative proteomics study of wild-type (WT) and OTUD1─/─ NCM460 cells. The expression of TFRC and IREB2 were highlighted with red color. Intensity indicates label-free quantification (LFQ) intensity of proteins in mass spectrometry.

(B) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., *P < 0.05, **P < 0.01, two-tailed unpaired Student's t-test).

(C) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer-1 (10 μM), Z-VAD (25 μM) and nec-1 (20 μM). Cell viability was measured by PI staining n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0119, **P = 0.0015, ***P < 0.001, two-tailed unpaired Student's t-test).

(D) CT26 cells stably expressing mock, OTUD1 or OTUD1C320S were treated with Erastin (10 μM) and RSL3 (5 μM), and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P < 0.01, two-tailed unpaired Student's t-test).

(E) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with Erastin (10 μM) and RSL3 (5 μM) for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., **P = 0.0082, ***P < 0.001, two-tailed unpaired Student's t-test).

(F) Intracellular ROS levels in mock, OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin (10 μM), AFC (50 μM) or H2O2 (100 μM) were detected by DCFDA staining. MFI, mean fluorescence intensity (n = 2 biological replicates).

(G) Intracellular ROS levels in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) or OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors (mean ± s.e.m., *P = 0.0189, **P = 0.0086, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.

(H) Schematic diagram of subcutaneous tumor treatment with nanoparticles-loading Vitamin E (NP-VE). BALB/c mice were transplanted with 1×106 mock or OTUD1 expressing CT26 cells. 50uL of 5M NP-VE was intratumorally injected every other day as shown.

(I) Tumor volume (mock, n = 4 biological replicates; mock + NP-VE, n = 6 biological replicates; OTUD1, n = 4 biological replicates; OTUD1 + NP-VE, n = 6 biological replicates; mean ± s.e.m., *P = 0.0325, ***P = 0.0003, two-tailed unpaired Student's t-test)

(J-K) Flow cytometric analysis of DCFDA (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) (J) and the percentage of tumor infiltrating CD8+ T cells (n = 4 biological replicates, mean ± s.e.m., *P = 0.0433, ***P = 0.0001, two-tailed unpaired Student's t-test) (K) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. MFI, mean fluorescence intensity.
The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity (2020)
Figure 6

Figure 6. OTUD1 triggers immunogenic cell death.

(A and B) CT26 cells stably expressing mock or OTUD1 (A) and wild-type (WT) or OTUD1─/─ NCM460 cells (B) were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test).

(C) Volcano plots of genes differentially expressed in tumor interstitial fluid (TIF) of mock and OTUD1 expressing CT26 tumors. Left, example of genes downregulated in OTUD1 overexpressing tumor interstitial fluid; right, example of genes upregulated in OTUD1 overexpressing tumor interstitial fluid.

(D) Immunoblot analysis of protein levels of HMGB1, HSP70 and HSP90 in tumor interstitial fluid of mock, OTUD1 and OTUD1C320S expressing CT26 tumors.

(E) ATP level in tumor interstitial fluid (TIF) of mock or OTUD1 overexpressing CT26 cells (n = 5 mice, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test).

(F and G) BALB/c mice were immunized with 1×106 OTUD1 expressing or mock CT26 cells in the left flank and challenged with 2×106 wild-type CT26 cells in the right flank 10 days later. Macroscopic evaluation (F) and tumor volume (n=7 mice, mean ± s.e.m., **P = 0.0053, two-tailed unpaired Student's t-test) (G) of tumors in the right flank were shown.
The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity (2020)
Figure 7

Figure 7. Loss of OTUD1 enhances susceptibility to colon carcinogenesis.

(A) Representative pictures of colon tumors in AOM/DSS-induced Colitis-Associated Cancer (CAC) model (n = 6 biological replicates).

(B and C) The tumor load (B) or tumor number (C) in the whole colon was measured (n = 12 biological replicates, mean ± s.e.m., *P = 0.0431, **P = 0.003, two-tailed unpaired Student's t-test).

(D) Representative H&E (Hematoxylin-eosin) staining pictures of colon tumors (The scale bars represent 1000 μm).

(E) Flow cytometric analysis of TFRC expression in intestinal epithelial cells (IECs) from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., **P = 0.0062, two-tailed unpaired Student's t-test).

(F) GSEA of genes expressed in colon tissues from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 3 biological replicates). ES, enrichment score; NES, normalized enrichment score.

(G) Representative immunohistochemistry staining pictures of CD3+ T cells in colon tumors (The scale bars represent 400 μm ).

(H) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells (LPMC) isolated from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 5 biological replicates, mean ± s.e.m., **P = 0.0025, ***P = 0.0005, two-tailed unpaired Student's t-test).

(I) Intracellular ROS levels in colon tissues from wild-type (WT) or Otud1─/─ mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., **P = 0.0072, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.

(J) ATP level in tumor interstitial fluid (TIF) of mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., *P = 0.0292, two-tailed unpaired Student's t-test).

(K) Model for the role of OTUD1 in iron absorption and tumor suppression. OTUD1 facilitates TFRC-mediated iron absorption through stabilization of IREB2 under steady condition. However, during colorectal cancer development, downregulation of OTUD1 restricts iron absorption and promotes resistance to ferroptosis, thereby subverting host immune surveillance against cancer.

Table 1. Information on microarrays of samples from patients with colon cancer.

Expanded View Figure legends
Beyond the seed: structural basis for supplementary microRNA targeting by human Argonaute2 (2019)
Figure 4

Fig. 4. Impact of seed-supplementary bridging region on target affinity.

A. Overview of central cleft shows path of target RNA. Seed, bridge, and supplemental regions indicated.

B. Schematic of pairing between miRNAs (loaded into Ago2) and targets used in equilibrium binding experiments.

C. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an AU-rich supplementary region) binding to target RNAs with bridging regions of various lengths.

D. Dissociation constants (Kd) of Ago2-miRNA complexes (containing an GC-rich supplementary region) binding to target RNAs with bridging regions of various lengths.

E. Fold repression of a luciferase reporter (relative to no target site control) plotted versus affinity (1/Kd) values measured for the same sites in vitro.

Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.
Beyond the seed: structural basis for supplementary microRNA targeting by human Argonaute2 (2019)
Figure 5

Fig. 5. Stabilization of supplementary interactions with increasing 3' miRNA length.

A. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed region only. Bottom: release of the seed-paired target from Ago2-miR complexes was assessed by monitoring the fraction of the total target RNA bound as a function of time.

B. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and supplementary (g13-g17) regions. Bottom: fraction target bound to the Ago2-miR complexes as a function of time.

C. Top: schematic of miR-122 isomiRs paired with a target complementary to the seed and shifted supplementary (g15-g19) regions. . Bottom: fraction target bound to the Ago2-miR complexes as a function of time.

D. Structure of the miRNA 3' tail suggests tension in the miRNA may restrict PAZ domain movments and opening of the supplementary chamber (target RNA not shown for clarity).

E. Release half times (t1/2) of target RNAs in panels (A-C) plotted against isomiR length.

Data information: All plotted data are the means of at least three independent replicates. Error bars indicate SEM.
Image-based genome-wide siRNA screen identifies selective autophagy factors (2011)
f1

a, Correlative light and electron microscopy of HeLa/GFP–LC3 cell infected with SIN/mCherry.capsid virus. Top left, deconvolved image of red and green fluorescence channels; middle, three-dimensional surface reconstruction of red and green channels; right, yellow (red + green) colocalization channel. Arrows denote yellow puncta that correspond to ‘1’ and ‘2’ in electron microscopy images below. Bottom left, electron microscopy of identical cell; middle and right, high magnification images of insets ‘1’ and ‘2’. Scale bars, left, 10 &mgr;m; middle and right, 200 nm. b, Ranked distribution of median Z-scores for each siRNA pool in primary colocalization (virophagy) screen. Red, decreased colocalization; green, increased colocalization; blue, insufficient numbers of green or red puncta per cell or total number of cells per well for analysis. c, Maps of protein interactions in enriched network modules (see Supplementary Fig. 3b). d, Association of siRNA hits with autophagy network.
Image-based genome-wide siRNA screen identifies selective autophagy factors (2011)
f3

a, Electron microscopy analysis of wild-type (WT) and Smurf1−/− (KO) MEFs grown in normal media or EBSS (starvation) for 4 h. Arrowheads, representative autophagosomes; arrows, representative autolysosomes. Scale bars, 500 nm. b, Western blot analyses of LC3-I/II (non-lipidated and lipidated forms of MAP1LC3, respectively) and p62 levels in MEFs of indicated genotype. c, Quantification of colocalization of SIN/mCherry.capsid and GFP–LC3 in indicated MEFs 16 h after SIN/mCherry.capsid/GFP–LC3 infection. Data shown represent mean ± s.e.m. number of colocalized red and green puncta per cell for 50 cells per well in triplicate samples. *P  0.001 against wild-type, Students t-test. d, Representative confocal micrographs of images used for quantification in c. Arrows, colocalized red and green puncta. Scale bar, 15 &mgr;m. e, Representative electron microscopy images of indicated MEFs infected with HSV-1 (strain 17termA). White arrowheads, partially degraded viral nucleocapsids inside autolysosomes; black arrowheads, intact viral nucleocapsids inside viral vesicles. Scale bar, 0.5 &mgr;m. For a–e, similar results were obtained in 3–5 independent experiments.
Image-based genome-wide siRNA screen identifies selective autophagy factors (2011)
f4

a, Representative mitochondrial morphology in Smurf1+/+ (wild-type) and Smurf1−/− (KO) MEFs transfected with indicated construct and treated with DMSO or 10 &mgr;M CCCP for 24 h. b, Quantification of percentage of total cells with a diffuse accumulation of abnormal fragmented mitochondria and lack of mitochondrial clearance. Results shown represent combined data from 3–5 experiments per condition with triplicate wells (of at least 100 cells per well) analysed for each condition per experiment. Shown are mean ± s.e.m. for average values from each experiment. Similar results were observed in each independent experiment. *P  0.001, Students t-test. c, Measurement of mitochondrial fractional area (percentage of total cellular area) in MEFs treated as in a. Results shown represent mean ± s.e.m. for 50 cells per condition. d, Representative confocal micrographs of KO MEFs transfected with YFP–SMURF1 wild-type or YFP–SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with DMSO or CCCP. e, Representative confocal micrographs of KO MEFs transfected with GFP–LC3 and wild-type mCherry–SMURF1 (WT) or mCherry–SMURF1Dgr;C2 (Dgr;C2) and treated for 4 h with CCCP. Inset, upper right, formation of completed autophagosome around a damaged mitochondrion associated with wild-type SMURF1; insets, lower right, incomplete autophagosomes or absence of LC3 signal around mitochondria associated with SMURF1Dgr;C2. See also Supplementary Figs 10 and 11 for enlarged images. f, Representative electron microscopy images of indicated tissues from 10-month-old Smurf1+/+ (WT) or Smurf1−/− (KO) mice. Arrows, representative abnormal mitochondria. Scale bars, 1 &mgr;m. Similar abnormalities were observed throughout entire electron microscopy tissue section for each mouse and in tissue samples from three different mice for each genotype.
Blocking STAT3/5 through direct or upstream kinase targeting in leukemic cutaneous T-cell lymphoma (2022)
Figure 1

Figure 1. L-CTCL patients present with STAT3/5 and MYC copy number gains, as well as TP53, STAT1 and SOCS1 copy number loss

A sWGS performed on sorted CD3+/Vβ+ malignant T-cells isolated from the blood of P1, shown as a representative example. Copy number profiles and ploidy estimates were calculated using ichorCNA. Blue represents balanced genomic regions, green indicates loss/µdeletion, and red indicates gain/amplification of regions on chromosome 17. Darker color shades indicate higher log-2 ratios. Copy number profiles from all six patients (P1-P6) are shown in Appendix Figure S2.

B Heatmap of extracted CNA [log2 ratios] of STAT genes on chr17 (i.e., STAT3, STAT5A and STAT5B), and other relevant tumor suppressors (i.e, STAT1, SOCS1 and TP53) and oncogenes (MYC). Red indicates gain/amplification, blue indicates loss/deletion. The balance threshold was set to +/- 0.1. P33 presented with two malignant clones in the blood and both were sequenced (P33_1 (Clone 1), P33_2 (Clone 2)). *Tetraploid malignant clone.

C-E Pie charts representing the identified genetic alterations in the patient cohort based on the CNA log2 ratios. (C) STA3/5 copy number gain (n = 17/23), STAT1 copy number loss (n = 7/17), SOCS1 loss (n = 3/17), (D) TP53 loss (n = 18/23), (E) MYC gain (n = 13/23).