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Additional file 6: Figure S6. Accumulation of intracellular defects in WE-68 cells. WE-68 cells were treated with 45 nM AUY922 (B), 2 µM VE821 (C) and their combination (D). DMSO was used for control (A). Intracellular structures were analyzed by transmission electron microscopy (TEM) and are labeled in red: lysosome (lyso), mitochondria (mito), endoplasmic reticulum (ER), nucleus (nuc), autophagosomes (auto), β-glycagon granule (glycagon), lipid droplets, vesicles and lipid-filled vesicles. Red arrows indicate sites of cell rupture. TEM pictures show one representative experiment.

Additional file 4: Figure S4. Analysis of molecular alterations. (A) Otherwise isogenic p53 wild-type (wt) and p53 null (p53-/-) HCT116 cells were treated with 45 nM AUY922 ± 2 µM VE821 for 24 h. Analysis of indicated proteins was done by Western blot. ⍺-tubulin and vinculin were used to control protein loading. Immunoblots are representative for at least two independent experiments. (B) Schematic workflow of the BALB/c cell transformation assay (BALB-CTA). WE-68 cells were treated with 30 nM of AUY922, 1 µM of VE821, 7.5 µM of KU55933 and their combinations; A673 cells were treated with 15 nM of AUY922, 1 µM of VE821, 5 µM of KU55933 and their combinations. DMSO was used for control. (C-D) The mRNA expression of indicated gene was analyzed after 24 h by qPCR. All graphs show the mean ± SEM of three independent experiments (* p

Additional file 3: Figure S3: Analysis of cell cycle distribution. (A) WE-68 and (B) A673 cells were treated with 15–45 nM of AUY922, 2 µM of VE821, 5 µM of KU55933 and their combinations for 24 h. (C-E) WE-68 and A673 cells were treated with indicated concentrations of AUY922 (C), VE821 (D) or KU55933 (E) for 48 h. Cell cycle distributions were determined by flow-cytometric analysis of PI-stained ethanol-fixed cells. All graphs show the mean ± SEM of at least two independent experiments.

Additional file 7: Figure S7. Accumulation of intracellular defects in A673 cells. A673 cells were treated with 45 nM AUY922 (B), 2 µM VE821 (C) and their combination (D). DMSO was used for control (A). Intracellular structures were analyzed by transmission electron microscopy (TEM) and are labeled in red: lysosome (lyso), mitochondria (mito), endoplasmic reticulum (ER), nucleus (nuc), autophagosomes (auto), β-glycagon granule (glycagon), lipid droplets, vesicles and lipid-filled vesicles. A673 cells were treated with 0.4–5 µg/ml of tunicamycin for 24 h. (E) Analysis of indicated proteins was done by Western blot. α-tubulin was used to control protein loading. TEM pictures show one representative experiment; immunoblots are representative for at least two independent experiments.

Additional file 10: Figure S10. Ingenuity pathway analysis of the proteome data set. A673 cells were treated with 45 nM AUY922 ± 2 µM VE821 for 24 h, and a quantitative whole proteome analysis was done by mass spectrometry from three individual experiments. The proteome data set was further processed by ingenuity pathway analysis (IPA; cutoff: q

Additional file 5: Figure S5. Analysis of ER stress. WE-68 and A673 cells were treated with 15–45 nM of AUY922, 2 µM of VE821, 5 µM of KU55933 and their combinations for 24 h. (A) Intracellular reactive oxygen species (ROS) level were analyzed by flow cytometry using CM-H2DCFDA. Both graphs show the mean ± SEM of three independent experiments. (B) Quantification of LAMP1 analyzed by Western blot (see Fig. 4) is shown representing the mean ± SEM of two independent experiments. (C) Otherwise isogenic p53 wild-type (wt) and p53 null (p53-/-) HCT116 cells were treated with 45 nM AUY922 ± 2 µM VE821 for 24 h. Analysis of indicated proteins was done by Western blot. ⍺-tubulin and vinculin were used to control protein loading. Immunoblots are representative for at least two independent experiments.

Additional file 1: Figure S1: Analysis of apoptosis and DDR signaling in ES cells. (A) Analysis p53 expression level by Western blot. β-actin was used to control protein loading. p53 null SaOS-2 cells were treated with the indicated concentrations of AUY922, VE821, KU55933 and their combinations. DMSO was used as control. (A-C) The loss of the mitochondrial transmembrane potential (ΔΨM) and cell death were assessed by flow cytometry after 48 h. All graphs show the mean ± SEM of three independent experiments. Statistical analysis was done using two-way ANOVA tests with GraphPad (*p

Additional file 2: Figure S2: Analysis of cell proliferation. (A) WE-68 and (B) A673 cells were treated with 15–45 nM of AUY922, 5 µM of VE821 and their combinations for up to 72 h. Cell densities were analyzed and quantified using crystal violet staining. A-B show the mean ± SEM of three independent experiments (*p

Introduction: Ewing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells.Methods: Effects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis.Results: AUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis. p53 null cells, however, accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821 in p53 null cells. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect.Conclusion: Our study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status.

Introduction Ewing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells. Methods Effects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution as well as fluorescence and transmission electron microscopy. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis. Results AUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis, while p53 null cells accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect. Conclusion Our study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status. Supplementary Information The online version contains supplementary material available at 10.1186/s13578-021-00571-y.

We introduce Replica, a dataset of 18 highly photo-realistic 3D indoor scene reconstructions at room and building scale. Each scene consists of a dense mesh, high-resolution high-dynamic-range (HDR) textures, per-primitive semantic class and instance information, and planar mirror and glass reflectors. The goal of Replica is to enable machine learning (ML) research that relies on visually, geometrically, and semantically realistic generative models of the world - for instance, egocentric computer vision, semantic segmentation in 2D and 3D, geometric inference, and the development of embodied agents (virtual robots) performing navigation, instruction following, and question answering. Due to the high level of realism of the renderings from Replica, there is hope that ML systems trained on Replica may transfer directly to real world image and video data. Together with the data, we are releasing a minimal C++ SDK as a starting point for working with the Replica dataset. In addition, Replica is `Habitat-compatible', i.e. can be natively used with AI Habitat for training and testing embodied agents.