Journal: Cell & Bioscience

Article Title: Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing’s sarcoma cells

doi: 10.1186/s13578-021-00571-y

Figure Lengend Snippet: Increased apoptosis after ATRi and HSP90i combinations. p53 wild-type WE-68 and p53 null A673 cells were treated with the indicated concentrations of AUY922, VE821, KU55933 and their combinations. DMSO was used as control. a – d The loss of the mitochondrial transmembrane potential (ΔΨ M ) 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 test (* p < 0.05; ** p < 0.01; *** p < 0.001). WE-68 ( e ) and A673 ( f ) cells were treated with 15–45 nM of AUY922, 2 µM of VE821, 5 µM of KU55933 and their combinations. Analysis of indicated proteins was done by Western blot after 24 h; vinculin was used to control protein loading. Immunoblots are representative of at least two independent experiments. Quantification of selected proteins is shown in g

Article Snippet: Cells were treated with 0–50 mM AUY922 (Luminespib; S1069), 1–10 μM VE821 (S8007), 5–15 μM KU55933 (S1092), 0.4–5 μg/ml tunicamycin (S7894) (all in DMSO and from Selleck Chemicals) or their combinations for up to 72 h.

Techniques: Control, Flow Cytometry, Western Blot

Journal: Cell & Bioscience

Article Title: Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing’s sarcoma cells

doi: 10.1186/s13578-021-00571-y

Figure Lengend Snippet: Decreased cell proliferation after AUY-VE in WE-68 cells. WE-68 ( a ) and A673 ( b ) cells were treated with 15–45 nM of AUY922, 2 µM of VE821, 5 µM of KU55933 and their combinations. Cell densities at 24, 48 and 72 h after treatment were assessed by crystal violet-based cell proliferation assay. DMSO was used as control. c Representative images of a BALB/c cell transformation assay (BALB-CTA) after 72 h of treatment with 15–45 nM AUY922 ± 2 µM VE821 or 5 µM KU55933. MCA/TPA-transformed cell foci are Giemsa stained and appear in blue. Quantification of the number of type-III foci/well from BALB-CTA experiments is shown in D. All graphs show the mean ± SEM of three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Cells were treated with 0–50 mM AUY922 (Luminespib; S1069), 1–10 μM VE821 (S8007), 5–15 μM KU55933 (S1092), 0.4–5 μg/ml tunicamycin (S7894) (all in DMSO and from Selleck Chemicals) or their combinations for up to 72 h.

Techniques: Proliferation Assay, Control, Cell Transformation Assay, Transformation Assay, Staining

Journal: Cell & Bioscience

Article Title: Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing’s sarcoma cells

doi: 10.1186/s13578-021-00571-y

Figure Lengend Snippet: Increased p53 target gene expression in WE-68 cells. 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 as control. a – d The mRNA expression of indicated genes was analyzed after 24 h by qPCR. All graphs show the mean ± SEM of three independent experiments (* p < 0.05; ** p < 0.01; *** p < 0.001)

Article Snippet: Cells were treated with 0–50 mM AUY922 (Luminespib; S1069), 1–10 μM VE821 (S8007), 5–15 μM KU55933 (S1092), 0.4–5 μg/ml tunicamycin (S7894) (all in DMSO and from Selleck Chemicals) or their combinations for up to 72 h.

Techniques: Targeted Gene Expression, Control, Expressing

Journal: Cell & Bioscience

Article Title: Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing’s sarcoma cells

doi: 10.1186/s13578-021-00571-y

Figure Lengend Snippet: Increased autophagy and ER stress in A673 cells. WE-68 and A673 cells were treated with 15–45 nM of AUY922, 2 µM of VE821, 5 µM of KU55933 and their combinations. DMSO was used for control. a , b , d , e Analysis of indicated proteins was done by Western blot after 24 h; vinculin and β-actin were used to control protein loading. Immunoblots are representative of at least two independent experiments. c , f Quantifications of selected proteins

Article Snippet: Cells were treated with 0–50 mM AUY922 (Luminespib; S1069), 1–10 μM VE821 (S8007), 5–15 μM KU55933 (S1092), 0.4–5 μg/ml tunicamycin (S7894) (all in DMSO and from Selleck Chemicals) or their combinations for up to 72 h.

Techniques: Control, Western Blot

Journal: Cell & Bioscience

Article Title: Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing’s sarcoma cells

doi: 10.1186/s13578-021-00571-y

Figure Lengend Snippet: Increased intracellular damages after AUY-VE. A673 cells were treated with 45 nM AUY922 ± 2 µM VE821 for 24 h. a Intracellular structures were analyzed by transmission electron microscopy (TEM). Red arrows indicate lipid-filled vesicles; yellow arrows indicate lysosomes. b Golgi networks were analyzed by fluorescence microscopy of GM130. Red arrows indicate dispersed Golgi structures. c Endoplasmic reticulum (ER) networks were analyzed by fluorescence microscopy of GRP94. Red arrows indicate vesicular ER structures. A673 cells were treated with 1–2 µg/ml of tunicamycin for 24 h. e ER networks were analyzed by fluorescence microscopy of GRP94. c – e DAPI was used to stain cell nuclei. A shows one representative experiments; B-E are representative for at least two independent experiments

Article Snippet: Cells were treated with 0–50 mM AUY922 (Luminespib; S1069), 1–10 μM VE821 (S8007), 5–15 μM KU55933 (S1092), 0.4–5 μg/ml tunicamycin (S7894) (all in DMSO and from Selleck Chemicals) or their combinations for up to 72 h.

Techniques: Transmission Assay, Electron Microscopy, Fluorescence, Microscopy, Staining

Journal: Cell & Bioscience

Article Title: Cooperative treatment effectiveness of ATR and HSP90 inhibition in Ewing’s sarcoma cells

doi: 10.1186/s13578-021-00571-y

Figure Lengend Snippet: ATRi and HSP90i share common molecular targets. 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 (refer to Additional file : File S1). The proteome data set was further processed by ingenuity pathway analysis (IPA; cutoff: q < 0.05) for alterations among canonical pathways ( a ), diseases and bio functions ( c ) and upstream regulators ( d ). The activation z-score of individual processes/molecules is shown in yellow for activation and blue for inactivation. b Analysis of indicated proteins was done by Western blot; vinculin was used to control protein loading. Immunoblots are representative of at least two independent experiments. e IPA of toxicities after AUY922 in combination with VE821 (AUY-VE). f DEPs of individual treatments (cutoff: q < 0.05) and their overlaps

Article Snippet: Cells were treated with 0–50 mM AUY922 (Luminespib; S1069), 1–10 μM VE821 (S8007), 5–15 μM KU55933 (S1092), 0.4–5 μg/ml tunicamycin (S7894) (all in DMSO and from Selleck Chemicals) or their combinations for up to 72 h.

Techniques: Mass Spectrometry, Activation Assay, Western Blot, Control