Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The anti-cancer activity of a first-in-class small molecule targeting PCNA

doi: 10.1158/1078-0432.CCR-18-0592

Figure Lengend 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).

Article Snippet: The human neuroblastoma cell lines: SK-N-DZ, SK-N-BE(2)c, SK-N-AS, and LAN-5, breast cancer cell lines: MDA-MB-436, MDA-MB-468, Hs578t, MCF7, HCC1937, and small cell lung cancer cell lines: H82, H524, and H526 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in DMEM with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin.

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

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The anti-cancer activity of a first-in-class small molecule targeting PCNA

doi: 10.1158/1078-0432.CCR-18-0592

Figure Lengend 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.

Article Snippet: The human neuroblastoma cell lines: SK-N-DZ, SK-N-BE(2)c, SK-N-AS, and LAN-5, breast cancer cell lines: MDA-MB-436, MDA-MB-468, Hs578t, MCF7, HCC1937, and small cell lung cancer cell lines: H82, H524, and H526 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in DMEM with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin.

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

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The anti-cancer activity of a first-in-class small molecule targeting PCNA

doi: 10.1158/1078-0432.CCR-18-0592

Figure Lengend 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.

Article Snippet: The human neuroblastoma cell lines: SK-N-DZ, SK-N-BE(2)c, SK-N-AS, and LAN-5, breast cancer cell lines: MDA-MB-436, MDA-MB-468, Hs578t, MCF7, HCC1937, and small cell lung cancer cell lines: H82, H524, and H526 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were cultured in DMEM with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin.

Techniques: Incubation, Analogues, Control, Labeling

Journal: Cell

Article Title: Illuminating G-Protein-Coupling Selectivity of GPCRs

doi: 10.1016/j.cell.2019.04.044

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Gα11 antibody (mouse monoclonal, clone D-6; recognizing Gαq and Gα11) , Santa Cruz Biotechnologies , Cat# sc-390382; RRID: AB_2715558.

Techniques: Virus, Recombinant, Electrophoresis, Plasmid Preparation, Reverse Transcription, Activation Assay, DC Protein Assay, Sequencing, Control, Construct, Software, Microplate Reader Absorbance Measurement, Flow Cytometry

Journal: Advanced Science

Article Title: DDRGK1 Enhances Osteosarcoma Chemoresistance via Inhibiting KEAP1‐Mediated NRF2 Ubiquitination

doi: 10.1002/advs.202204438

Figure Lengend Snippet: DDRGK1 regulates NRF2 in a UFMylation‐independent pathway. A) Interaction between NRF2 and UFM1. The NRF2‐HA and ufm1‐HA plasmids were transfected into HEK293T cells. The interaction between NRF2 and ufm1 was detected by co‐immunoprecipitation with HA antibody followed by western blot with NRF2 and ufm1 antibody. B) Interaction between NRF2 and DDRGK1. The NRF2‐HA and DDRGK1‐Flag plasmids were transfected into HEK293T cells. The interaction between NRF2 and DDRGK1 was detected by co‐immunoprecipitation using anti‐HA immunomagnetic beads and anti‐Flag immunomagnetic beads. C) NRF2 levels of heart, lung, kidney, and cartilage in WT and K268R mouse detected by western blot. D) Re‐express of WT‐DDRGK1 and K267R‐DDRGK1 in DDRGK1 deficient cells rescued level of NRF2. E) Mitochondrial metabolism analysis. Cells were seeded in an XF microplate at an optimal density and cultured for 24 h. The cellular oxygen consumption rate (OCR) was measured using a Seahorse XF Analyzer. F) ROS level measurement. The controlled, DDRGK1‐knockout cells, and WT‐DDRGK1/K267R‐DDRGK1 re‐expressed cells were subjected to H 2 O 2 , then loaded with a DHE probe for 30 min and detected by flow cemetery. (MFI, mean fluorescence intensity; Three samples for each statistical analysis **** p < 0.0001).

Article Snippet: Then, 10 μL of CCK8 reagent was added, maintained at 37 °C for 1 h. The optical density was then measured at 450 nm using an automatic i‐control microplate reader (Tecan Life Sciences, Switzerland).

Techniques: Transfection, Immunoprecipitation, Western Blot, Cell Culture, Knock-Out, Fluorescence

Journal: Cancers

Article Title: EZH2 Inhibition as New Epigenetic Treatment Option for Pancreatic Neuroendocrine Neoplasms (PanNENs)

doi: 10.3390/cancers13195014

Figure Lengend Snippet: ( A ) In vitro viability curves using the metabolic surrogate assay RealTime-Glo (RTG) in 3D human primary PanNET culture treated with DMSO (control (Ctrl)) and GSK126 for 7 days. Data were first normalized per-well using a RTG baseline measurement for each individual well and then normalized to the average of the corresponding DMSO control of the respective day. Data represent means ± SEM ( n = 1 per patient, three technical replicates). RLU, relative luminescence unit. ( B ) Micro-cell-block of two representative samples. IHC of synaptophysin and H&E staining of samples from the day of isolation and DMSO-treated samples 12 days post-isolation.

Article Snippet: The RTG assay was performed according to the manufacturer’s instructions, and luminescence was measured in an Infinite ® 200 PRO plate reader (Tecan, Switzerland).

Techniques: In Vitro, Surrogate Assay, Control, Blocking Assay, Staining, Isolation