Fig 2: (A) Details of the study cohort. (B and C) Kaplan–Meier survival curves for ATRX- and DAXX-negative cases (n=17) as compared to ATRX/DAXX-positive cases (n=17) and ATRX-negative (n=8) and DAXX-negative (n=9) cases analysed independently and compared to ATRX/DAXX-positives cases respectively. The 5-year PFS was 85% for positive cases, 52% for ATRX-negative cases and 16% for DAXX-negative cases. (D) Unsupervised cluster analysis using the top 1000 MVPs that showed segregation of ATRX-negative (in green) and DAXX-negative (in orange) tumours as compared to normal pancreatic tissue. Of the six ATRX/DAXX-negative tumour samples (from six cases) that clustered with the normal control pancreatic samples, only one case (G1, DAXX-negative) progressed (PFS 51 months), and two cases had no follow-up data. Of the six metastatic samples included in this study, two of them (both DAXX-negative) had a matched G1 tumour sample. One of them grouped tightly with the G1 primary tumour. DNA methylation values (β-value 0–1) are represented using a colour scale, where yellow=low methylation and blue=high methylation. Samples are shown on the x-axis, and probes are shown on the y-axis. (E) Confirmation of tumour segregation primarily by grade and then by ATRX or DAXX status using the top 1000 MVPs. (F) Copy number variation (CNV) profiles associated with low and intermediate PNETs. The top panel demonstrates increasing CNV across the genome for low-grade (G1, ki-67 <2%) tumours, with the least CNV occurring in the ATRX/DAXX-positive primary (left). Increasing CNV alterations are seen in the ATRX/DAXX-negative tumour (middle) and most CNV alterations occur in the ATRX/DAXX-negative G1 liver metastasis (right). The same is observed in intermediate grade (G2, ki-67 3–20%) tumours in the bottom panel. Segmented copy numbers are shown as a red line, and sequential chromosomes are shown in green and black (chr. 1–22).

Fig 3: Daxx interacts through its helical domain with Slug and HDAC1.(a) Mapping the Daxx-binding domain of Slug. Upper panel: schematic diagram of Slug and Slug deletion mutants. Lower panel: lysates from H1299 cells co-transfected with HA-Daxx and different Flag-Slug constructs or an empty vector for 24 h were co-immunoprecipitated (co-IP) with anti-HA antibodies and fractionated by SDS–polyacrylamide gel electrophoresis (PAGE). Immunoblots were probed with anti-HA or anti-Flag antibodies. (b) Mapping the Slug-binding domain of Daxx. Upper panel: schematic diagram of Daxx and Daxx deletion mutants. SPE, Ser/Pro/Glu-rich domain; SPT, Ser/Pro/Thr-rich domain. Lower panel: lysates from H1299 cells co-transfected with HA-Slug and different Flag-Daxx constructs or Flag-Twist for 24 h were immunoprecipitated with anti-Flag antibody and fractionated by SDS–PAGE. Immunoblots were probed with anti-Slug or anti-Flag antibodies. (c) ChIP–PCR analyses of HDAC1 on the E-cadherin promoter in CL1–5 cells. CL1–5 cells were transfected with control vector (Ctrl), Slug or both Slug and Daxx plasmids for 30 h, and then ChIP assays were performed using anti-HDAC1 antibody. The precipitated DNA fragments were analysed by PCR to detect the E-cadherin promoter region. The Input and IgG control are same as Fig. 2f which came from identical experiment. (d) The effect of Daxx on Slug–HDAC1 interactions was assayed by co-IP. H1299 cells were co-transfected with the indicated plasmids for 30 h. Lysates were immunoprecipitated with anti-HA antibody and fractionated by SDS–PAGE. Immunoblots were probed with the indicated antibodies. (e) Daxx binding to HDAC1 through its helical domain was assayed by co-IP. H1299 cells were co-transfected with the indicated plasmids for 30 h. Lysates were immunoprecipitated with anti-Flag antibody and fractionated by SDS–PAGE. Immunoblots were probed with anti-Flag or anti-HA antibodies.

Fig 4: Immunostaining patterns of DAXX in different histotypes of endometrial cancers: (A) Serous type with low power at 4× and (inlet, 40×), (B) clear cell type with low power at 10× (inlet, 40×), (C) FIGO 3 endometroid type with low power at 4× (inlet 40×), and (D) low-grade endometroid type with low power at 4×and (inlet, 40×). At high power (40×), high-grade endometrial carcinomas (A, B, C) all show strongly retained nuclear staining of DAXX in tumor cells while low-grade endometrial carcinomas (D) mainly show loss of nuclear DAXX in tumor cells.Abbreviation: DAXX, death domain-associated protein 6; FIGO, The International Federation of Gynecology and Obstetrics.

Fig 5: Representative examples of non-functional pancreatic neuroendocrine tumours (NF-PanNETs) that have been assessed by immunolabelling for ARX, PDX1, ATRX and DAXX, and telomere-specific fluorescence in situ hybridisation (FISH) for ALT. (A) NF-PanNET with positive expression for ARX (B) and negative expression for PDX1 (C), while both ATRX (D) and DAXX (E) exhibited preserved nuclear expression and an absence of alternative lengthening of telomeres (ALT) (F). (G) NF-PanNET with negative expression for ARX (H) and positive expression for PDX1 (I), while both ATRX (J) and DAXX (K) had preserved expression and an absence of ALT (L). (M) NF-PanNET with expression for both ARX (N) and PDX1 (O), but loss of nuclear ATRX (P) and preserved nuclear expression for DAXX (Q). the loss of ATRX expression correlated with the presence of large, ultrabright intranuclear foci by telomere-specific FISH, consistent with ALT (R). (S) NF-PanNET with positive ARX expression (T), negative PDX1 expression (U), preserved ATRX expression (V) and loss of DDAXX expression (W). Similar to loss of ATRX, DAXX-negative NF-PanNETs were associated with large, ultrabright telomere signals, consistent with ALT (X). ARX, aristaless-related homeobox gene; ATRX, alpha-thalassemia/mental retardation X-linked; DAXX, death domain-associated protein; PDX1, pancreatic and duodenal homeobox 1.