Fig 2: SLX4IP Is Lost in a Subset of ALT-Positive Osteosarcomas(A) Relative expression of hTERT from RNA sequencing preformed on a panel of osteosarcoma cell lines and patient-derived osteosarcoma xenografts. RNA sequencing was performed in triplicate, and each dot represents a separate experiment.(B) Quantification of C-circle abundance in the osteosarcoma PDX samples. DNA was extracted from three separate tissue sections taken from each tumor. DNA extracted from HUO9 cells was used as a positive control, and DNA extracted from SJSA1 xenografts was used as a negative control. Data are represented as mean ± SD; n = 3. Dotted line represents 5-fold change in C-circle abundance.(C) Relative expression of SLX4IP from RNA sequencing preformed on a panel of osteosarcoma cell lines and patient-derived osteosarcoma xenografts. RNA sequencing was performed in triplicate, and each dot represents a separate experiment.(D) Whole-cell extracts of the indicated cell lines were analysed by SLX4IP immunoblotting. a-Tubulin was used as loading control.(E) PDX tumor samples were analysed by SLX4IP, ATRX, SMARCAL1, DAXX and H3.3 immunoblotting. a-Tubulin was used as loading control. Arrow indicates SLX4IP band.See also Figure S7.

Fig 3: DAXX deletion does not affect Mdm2/p53 stability or p53-mediated gene expression. (A) Mdm2 and p53 protein stability was examined in control U2OS cells, 2 independent DAXX+/+ clones (0–4, 0–18), 3 independent DAXX-/- clones (17-7, 17-18, 17–42) and in 17-7 DAXX-/- clone stably transduced with pCDH empty vector (EV) or pCDH-DAXXWT. Cells were collected after the treatment with 50 µl/ml CHX at the indicated time and subjected to protein gel blotting analysis using labeled antibodies. THIIF was used as a loading control. (B) DAXX+/+ (clone 0–18) and DAXX-/- (clones 17-18) cells were exposed to 4 nM NCS for the specified time points. Lysed cells were then separated by SDS–PAGE and immunoblotted with indicated antibodies. GAPDH was used as a loading control. (C) RNA expression of p53-dependent genes 8 hours after the treatment with 10 µM VP16 (expressed as fold change after VP16) in U2OS clones. RNA was analyzed by quantitative RT-PCR and the expression values were normalized to the average of 3 reference genes (ß-actin, SDH and ALAS). (D) U2OS cells transfected with control non-targeting siRNA and siRNA against p53 were treated with 10 µM VP16 for 8 hours and RNA expression of p53 and indicated p53-dependent genes was analyzed by quantitative RT-PCR. The expression values were normalized to the average of 3 reference genes (ß-actin, SDH and ALAS).

Fig 4: Wip1 affects DNA damage-induced phosphorylation of endogenous DAXX. MCF7 or U2OS cells were transfected with control (siLuc) or siRNA targeting Wip1 and 3 d later exposed to 5 Gy of IR or 4 nM NCS. Cells were lysed at the indicated time points after DNA damage and analyzed by protein gel blotting with the indicated antibodies (* unspecific band). a-tubulin was used as a loading control.

Fig 5: (See previous page) DAXX is phosphorylated on S564 rapidly after DNA damage. (A) HEK 293T cells were transfected with a vector expressing FLAG-DAXXWT and treated or not, as indicated, with 8 nM NCS or10 µM VP16 for 1 hour. Cells were lysed and FLAG-tagged proteins immunoprecipitated using anti-FLAG M2 beads, prior to analysis by SDS-PAGE and western blotting with antibodies against phospho-(S/T) ATM/ATR substrate, FLAG M2, phospho-Chk2 (T68) and DAXX. (B) HEK 293T cells were transfected with expression constructs encoding FLAG-tagged wild-type DAXX, single (DAXXS564A), triple or quadruple mutant as indicated and treated with 10 µM VP16 for 1 hour. Cells were lysed, immunoprecipitated using anti-FLAG M2 beads and analyzed by protein gel blotting with indicated antibodies. (C) BJ fibroblasts were exposed to 10 Gy of IR, collected at the indicated time points and subjected to western blotting analysis using specific phospho-DAXX (S564), DAXX, phospho-p53 (S15) and phospho-Chk2 (T68) antibodies. GAPDH was used as a loading control. (D) BJ fibroblasts were transfected with control (siCTRL) or DAXX siRNA and 3 d later treated or not as indicated with 10 µM VP16 for 1 hour. Immunofluorescence analysis using a-PML (red) and a-P-DAXX (S564) (green) showed that upon DNA damage DAXX is preferentially phosphorylated at PML nuclear bodies.