Fig 2: Single or dual inhibition of AMPK and CHK1 is selective against BL-CL cell lines. Cell lines were treated and the bar graphs (with standard deviation) with all data displayed as scatter dot diagrams of treatment EC50 values were plotted. Every dot represents the EC50 for a cell line. Luminal/HER2+ and BL-CL cell lines were treated with (A) the AMPK inhibitor, dorsomorphin, (B) the CHK1 inhibitor, Ly2603618, or (C) the concurrent treatment with dorsomorphin and Ly2603618 for 72 h. The differences were assessed using an un-paired t-test (p < 0.05 was considered significant).

Fig 3: Revisiting the sealed heating method–heating cells in a low volume decreases nuclear Chk1.(A) Thermal response profile of Chk1 in HT29 cells in 0, 25 or 50 μL media detected by immunofluorescent imaging with the EP691Y antibody following heating at 37 to 58°C by floating in a pre-heated water bath and fixation with formaldehyde or formaldehyde then methanol fixation. Values are the mean of three wells ± SD. (B) Nuclei parameters were determined in Harmony software. Values represent the individual results of 3 different wells.

Fig 4: Revisiting the sealed heating method–V158411 decreases Chk1 nuclear immunofluorescence in HT29 cells heated in a low well volume.(A) HT29 cells were treated with 0–20 μM V158411 for 10 minutes in 25 μL media before being heated at the indicated temperature for 3 minutes followed by room temperature for 5 minutes. Nuclear Chk1 intensity was determined by immunofluorescent staining using the EP691Y antibody and high content imaging. Values are the mean of three wells ± SD. (B) HT29 cells were treated with 0–20 μM V158411 for 10 minutes in 25 μL media before being heated to 49°C. For 37°C, mean of n = 4 ± SD; for 49°C, dose response curves of 3 independent experiments are shown (mean of 3 wells ± SD). (C) U2OS cells were treated with 0–20 μM V158411 for 10 minutes in 25 μL media before being heated to 49°C. Mean of 3 determinations ± SD. (D) HT29 cells were treated with 0–20 μM V158411 for 10 or 30 minutes in 50 μL media before being heated to 51°C. Mean of 3 wells ± SD. (E) HT29 or U2OS cells were treated with 0–10 μM V158411 under identical conditions of cell number to media volume as (B) above for 10 minutes. Protein expression levels were determined by immunoblotting and quantified by densitometry.

Fig 5: Gemcitabine impairs the growth of Ewing sarcoma cells(A) Analysis of Genomics of Drug Sensitivity in Cancer data shows that Ewing sarcoma cell lines are more sensitive to gemcitabine than other cancer cell lines. A Mann-Whitney test was performed to compare the drug sensitivity of Ewing sarcoma cell lines versus other cell lines. (B) Quantification of BrdU incorporation into the DNA of EW8 cells at different time points after the addition of gemcitabine (100 nM). (C) EW8 cells were treated with gemcitabine (100 nM) for 6 hours. BrdU incorporation was then quantified using flow cytometry at different time points after the removal of the drug. (D) Dose-response curves for four Ewing sarcoma cell lines and three non-Ewing sarcoma cell lines (U2OS, HEK-293T, and HT1080) treated with different concentrations of gemcitabine for 6 hours. Cell viability was then assessed 42 hours after drug removal using the AlamarBlue Fluorescence Assay. The results are representative of two independent experiments. Error bars represent mean ± SD of three technical replicates. (E) Representative flow cytometry plot for Annexin-V and PI staining of EW8 cells treated with DMSO or gemcitabine (100 nM) for 6 hours followed by a 42-hour recovery period. (F) Percentage of Annexin-V positive cells for two Ewing sarcoma cell lines and an osteosarcoma cell line (U2OS) treated with gemcitabine as described in (E). Results are representative of two independent experiments. Error bars represent mean ± SD of two technical replicates. (G) Western blot showing that treatment of EW8 cells with gemcitabine for 6 hours results in the dose-dependent phosphorylation of CHK1-345. (H) Western blot showing that treatment of EW8 cells with gemcitabine for 6 hours results in the dose-dependent phosphorylation of H2AX. (I) EW8 and U2OS cells were treated with 100 nM gemcitabine for 6 hours. Cell lysates were then collected and blotted for γH2AX at 0 hours and 18 hours after drug removal.