human neuroblastoma cell lines  (ATCC)

Figure Legend Snippet: (a) AOH1160 structure (patent pending). The sole substitution of an ether oxygen in AOH1160 for the corresponding methylene group in AOH39 is indicated by a red dashed box. The indicated (b) human neuroblastoma cell lines, (c) breast cancer cell lines, and (d) small cell lung cancer cell lines were cultured in the presence of various concentrations of AOH1160. The non-malignant (b) 7SM0032 cells and human PBMCs, (c) human mammary epithelial cells (hMEC), and (d) human small airway epithelial cells (SAEC) were also cultured under the same AOH1160 treatment. Cells cultured in the absence of AOH1160 were used as control. Cell growth was measured by a CellTitor Glo assay (Promega). The average of luminescence signals in triplicates normalized to the control for each cell line was graphed ± S.D. e) Three normal neural stem cell lines (NSC005-007) and three glioblastoma stem cell (GSC) lines, PBT003, PBT707, and PBT017, respectively representing three of the four glioblastoma subtypes (classical, proneural, and mesenchymal) (50) were treated with DMSO or 1 µM of AOH1160 for 72 hours. Shown are cell growth related to the control cells ± S.D. f) TRβ reporter cells were treated with various concentrations of T3, AOH39, or AOH1160 for 24 h. The effect of compounds on TRβ activity was examined by measuring the relative luminescence units (RLU) in a luminescence plate reader. Grey: Signals from T3-treated cells and black: overlapping signals from AOH39 or AOH1160-treated cells. g) Computer modeling of small molecule binding to PCNA. The model was initially built using the AAD methodology (see text) and further refined by 50ns metadynamics simulation. Shown are small molecules (in stick) and PCNA surface around the binding pocket. The loop residues of L126-Y133 of PCNA are colored in blue to green. Top panel: AOH39 is shown as colored sticks and T3 as grey sticks. Bottom panel: AOH1160 is shown as colored sticks and AOH39 as grey sticks. h) STD NMR experiments using 1 µM of PCNA. The T3 compound structure is shown on top along with proton labels. The inserted table shows the reduction in STD ± errors for each of the T3 proton positions in the presence of AOH1160 (see Methods).

Techniques Used: Cell Culture, Control, Glo Assay, Activity Assay, Microplate Reader Luminescence Measurement, Binding Assay

Figure Legend Snippet: (a) Chemical structure of AOH39 (patent pending). The indicated human (b) neuroblastoma cell lines and (c) human breast cancer cell lines were cultured in the presence of various concentrations of AOH39 for 72 hours. The non-malignant 7SM0032 human embryonic progenitor cell line, (black diamonds in panel b), the immortalized, but non-transformed MCF10A cells (black circles in panel c), and PBMCs (black circles and black squares in panels b and c respectively) isolated from a healthy human donor were also cultured in the presence of the same AOH39 treatment. Cells cultured in the absence of AOH39 were used as control. Cell growth was measured by a CellTitor Glo assay (Promega). The average of luminescence signals from triplicate samples normalized to the control for each cell line was graphed plus/minus standard deviations. (d) The SK-N-DZ cells were treated with 3 µM AOH39 for 0, 6, or 24 h. Cells were fixed and stained with PI. The cellular PI fluorescence intensity was analyzed by flow cytometry. The flow cytometry data were analyzed by the FlowJo program to model various cell populations. (e) The SK-N-DZ cells were treated with 3 µM AOH39 for 0, 6, or 24 h. Total cell extracts were analyzed by western blot, using antibodies specific to γH2A.X or total H2A.X. (f) The DR-GFP and EJ5-GFP cell lines were transiently transfected with the pCBASce plasmid that expresses the I-SceI meganuclease. Three hours after transfection, cells were treated with or without 4 µM AOH39 in fresh growth medium. The HR and EJ-mediated DSB repair events, indicated by the restoration of a functional GFP gene in the respective cell lines, were quantified by measuring the relative abundance of GFP-positive cells by flow cytometry. Results from triplicates for each cell line with (light bars) or without (dark bars) AOH39 treatment were averaged and graphed plus/minus standard deviations.

Techniques Used: Cell Culture, Transformation Assay, Isolation, Control, Glo Assay, Staining, Fluorescence, Flow Cytometry, Western Blot, Transfection, Plasmid Preparation, Functional Assay

Figure Legend Snippet: Synchronized (a) SK-N-BE(2)c neuroblastoma cells and (b) small cell lung cancer cells (H82 and H526) were sequentially incubated in the presence of CldU (green) and IdU (red) before and after AOH1160 treatment, respectively. Cells sequentially incubated with the same two nucleotide analogues but without AOH1160 were used as control. Shown in the left of panel (a) are representative images of the labeled DNA strands from cells treated with or without AOH1160. The lengths of CldU (green) and IdU (red) incorporated DNA segments measured from more than 30 independent DNA strands in the indicated cells treated with or without AOH1160 were averaged and graphed with standard deviations in the right of panel (a) and in panel (b). The p values were determined by the Student’s t-test on the lengths of IdU labeled DNA segments (red bars) between each AOH1160 treatment and the untreated control.

Techniques Used: Incubation, Analogues, Control, Labeling