Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: PK11000 bound to and stabilized p53 DBD. (A) Melting curve of the stabilized p53-Y220C DBD (T-p53C-Y220C) recorded via differential scanning fluorimetry in absence (blue) or in presence of 1 mM PK11000 (magenta). (B) Inhibition of T-p53C-Y220C aggregation by PK11000. Aggregation kinetics were recorded by monitoring light scattering at 500 nm with 3 μM protein at 37 °C in standard assay buffer [25 mM KPi pH 7.2, 150 mM NaCl, 1 mM TCEP, and 5% (vol/vol) DMSO]. Unlike for noncovalent binders that inhibit aggregation where the amplitude of the reaction remains unchanged and just the rate constants being lower, the amplitude of light scattering was substantially decreased at 250, 500, and 1,000 µM of PK11000, suggesting the presence of two species: unmodified p53-Y220C and a covalent adduct of PK11000 and p53. (C) Mapping of PK11000 induced peak shifts in the 15N-1H HSQC NMR spectrum onto the structure of the p53-Y220C DBD. Large peak shifts are highlighted in red; medium shifts in orange; and small shifts in yellow.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: DSF ΔTm values of different p53 mutants (8 μM) after incubation with diverse 2-sulfonylpyrimidine compounds (250 μM) for 30 min.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: PK11000 alkylated cysteines of p53 DBD. (A) SNAr reaction mechanism for PK11000 cysteine alkylation. (B) ESI (ES+) mass spectra of 50 µM T-p53C Y220C incubated for 4 h at 20 °C with no compound (black) or 250, 500, 1,000, and 5,000 µM PK11000 (red).

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: ESI (ES+) mass spectra of different cysteine to p53 core DBD serine mutants after incubation without (black) and with PK11000 (red). (A) T-p53C C124S/C182S/C277S and (D) T-p53C C182S/C277S mutants showed no cysteine modification by PK11000, whereas (B) T-p53C C124S/C182S and (C) T-p53C C124S/C277S showed one cysteine modification by PK11000.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Incubation, Modification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: 15N-1H HSQC NMR spectrum of the p53 Y220C core domain (red) with 1,000 µM (blue), 436 µM (yellow), and 218 µM (green) PK11000 at 293 K.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Alkylation of full-length p53 with 2-sulfonylpyrimdines does not compromise its DNA binding capability. Stabilized full-length p53 was incubated with 1 mM PK11000, PK11007, PK11010, and 5% DMSO for 2 h at 4 °C and then titrated into a cuvette containing 20 nM carboxyfluoresceine-labeled Gadd45a DNA. Fluorescence polarization data were recorded and fitted to the Hill equation including a linear drift term.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Binding Assay, Incubation, Labeling, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Structural effect of Cys182 modification by PK11000. Superposition of the structure of the p53 cancer mutant Y220C with (gray) and without (green) PK11000 shows that Cys182 on the surface of the L2 loop is modified by the alkylating agent, with the covalent modification pointing toward the solvent. Chain A of the asymmetric unit is shown. Alkylation fixes Cys182 in a defined conformation, but there is little interaction between the modification and the rest of the protein. The figure was generated using PyMOL (www.pymol.org).

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Modification, Mutagenesis, Solvent, Generated

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Effects of diverse 2-sulfonylpyrimidines on p53 stabilization. (A) Library of diverse 2-sulfonylpyrimidines and structurally related compounds. (B and C) Time-dependent stabilization (DSF ΔTm) of T-p53C-Y220C after 15-, 30-, 60-, and 120-min incubation at room temperature with stabilizing or destabilizing/nonreactive 2-sulfonylpyrimidine compounds, respectively.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Biological effects of PK11007 on diverse cancer cell lines and one human fibroblast cell line. (A) Concentration-dependent viability reduction of WI-38 (fibroblast), NUGC-4 (p53 WT), HUH-6 (p53 WT), SJSA-1 (p53 WT), SW480 (p53 R273H/P309S), NUGC-3 (p53 Y220C), HUH-7 (p53 Y220C), and MKN1 (p53 V143A) cells after treatment with PK11007 for 24 h. The mutant p53 cells HUH-7, MKN1, and NUGC-3 were more sensitive to PK11007 treatment as indicated by strong viability reduction at low compound concentrations. Cell viability was measured in quadruplicate and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM. (B) Incubation of the isogenic H1299 (p53−/−), H1299 (p53 H175), HCT116, and HCT116 p53−/− cancer cells with PK11007 yielded a comparable viability reduction after 24 h. (C) Cell viability of HUH-6, HUH-7, and MKN1 after p53 knockdown via siRNA. Cells were treated with PK11007 for 24 h (48 h for 15 µM PK11007 MKN1 sample). In HUH-6 and HUH-7, cell death was independent of p53, whereas it was partially dependent on p53 in MKN1 cells. (D) Western blots of NUGC-4, NUGC-3, MKN1, HUH-6, and HUH-7 cancer cells after 3 h (6 h for MKN1) treatment with PK11007. p53 target genes p21, PUMA, and MDM2 were up-regulated not only in MKN1 (p53-V143A), NUGC-3, and HUH-7 (both p53-Y220C), but also in HUH-6 and NUGC-4 (both p53 WT) cells. With increasing PK11007 concentration, the molecular weight of p53 gradually increased to ∼3 kDa for the mutant p53 cell lines (HUH-7, MKN1, and NUGC-3), suggesting hyperalkylation of unfolded/aggregated p53. The protein level of the antioxidative gene GSTP1 was higher in NUGC-4 (p53 WT) than in NUGC-3. The asterisks at the p53 target gene levels of HUH-6 and HUH-7 cells highlight the genes for which a different β-actin control was used. (E) Quantification of relative mRNA levels of p53 target genes via real-time PCR. Cells were treated with DMSO or 15 µM (MKN1, HUH-7) or 20 µM (NUGC-3, HUH-6) PK11007 for 6 h (4.5 h for HUH-7). p21 and PUMA mRNA levels were especially up-regulated in mutant p53 cells, whereas in p53 WT cells (HUH-6) no increased transcription was observed. Significance levels were calculated with the Student t test (***P < 0.001; **P < 0.01; *P < 0.05). (F) Western blots of protein levels of UPR key markers in MKN1, HUH-6, and HUH-7 cells. PK11007 treatment for 3 h (6 h for MKN1) increased levels of spliced XBP-1 and CHOP (especially in HUH-7). (G) Inhibition of cellular glutathione synthesis by buthionine sulfoximine strongly potentiated cell viability reduction by PK11007 in HUH-7, NUGC-3, and MKN1 mutant p53 cancer cells. (H) Determination of relative intracellular ROS levels via CellROX Deep Red fluorescence after incubating four cancer cell lines with 30 or 60 µM PK11007 for 2 h. PK11007 caused increase of ROS in all tested cell lines. However, at high doses, the increase of relative ROS levels was higher in HUH-7, NUGC-3, and MKN1 cells. Median fluorescence levels were determined in triplicate with error bars depicting the SE. Significance levels were calculated using a one-way ANOVA with the Bonferroni post hoc test for mean comparison (***P < 0.001; **P < 0.01; *P < 0.05).

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Concentration Assay, Mutagenesis, Incubation, Knockdown, Western Blot, Molecular Weight, Control, Real-time Polymerase Chain Reaction, Inhibition, Fluorescence, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Cell viability time course for (A) HUH-6 (p53 WT), HUH-7 (p53 Y220C), and (B) MKN1 cancer cells at 30 or 60 µM PK11007 (10 and 30 µM for MKN1). Cell viability was measured in quadruplicates and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Biological effects of 2-sulfonylpyrimidines on diverse cell lines. Concentration-dependent viability reduction of WI-38 (p53 WT, noncancer), NUGC-4 (p53 WT), HUH-6 (p53 WT), SJSA-1 (p53 WT), SW480 (p53 R273H/P309S), NUGC-3 (p53 Y220C), HUH-7 (p53 Y220C), and MKN1 (p53 V143A) cells after compound treatment for 24 h. The strongest effects on cell viability were observed for PK11007 and PK11012. Mutant p53 cell lines were mostly significantly more sensitive for these compounds than p53 WT and noncancer cell lines. This behavior was also observed for PK11010 and PK11029, however only at higher compound concentrations. PK11003 led to a strong viability decrease; however, it did not distinguish clearly between mutant and WT p53 cell lines. Compounds that contain a negatively charged carboxyl group, such as PK11000 and PK11009, did not lead to a significant cell viability decrease in the tested cell lines at concentrations below 120 µM. Mutant p53 cell lines were also extra sensitive to compounds that destabilized the p53 core domain in vitro, such as PK11012 and PK11015. Cell viability was measured in quadruplicate and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Concentration Assay, Mutagenesis, In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Viability reduction of APR-246 in NUGC-3 (p53 WT), HUH-6 (p53 WT), NUGC-4 (p53 Y220C), and HUH-7 (p53 Y220C) cancer cells after treatment for 24 h. Cell viability was measured in quadruplicates and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: NAC prevents PK11007-mediated ROS formation. (A) Determination of intracellular ROS levels via CellROX Deep Red fluorescence after incubating HUH-6 and HUH-7 cancer cells with 5 mM NAC, 400 µM tert-Butyl hydroperoxide (TBHP), or a combination of 30 µM PK11007 and 5 mM NAC for 1.25 h. NAC does not only decrease basal ROS levels, but also completely prevents PK11007 from increasing ROS levels. (B) Relative intracellular ROS levels via CellROX Deep Red fluorescence after incubating four cancer cell lines with 30 or 60 µM PK11007 for 2 h. PK11007 led to an increase of ROS in all tested cell lines; however, at high doses the relative ROS level was significantly higher in HUH-7 and NUGC-3 cells, whereas the ROS levels in MKN1 cells remained on the level of the p53 WT cell lines. Median fluorescence levels were determined in triplicates. Error bars depict SEM values.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: PK11007 changes the folding state of mutant p53 (Y220C) in NUGC-3 cells. PK11007 treatment led to a significant reduction of unfolded p53 (Pab 240) and a slight increase in folded p53 (Pab 1620). Hoechst 33342 dye was used to stain the nucleus. To facilitate comparison of Pab 1620 and Pab 240 fluorescence levels, we increased exposure of all raw pictures by 2 eV.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques: Mutagenesis, Staining, Comparison, Fluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: 2-Sulfonylpyrimidines: Mild alkylating agents with anticancer activity toward p53-compromised cells

doi: 10.1073/pnas.1610421113

Figure Lengend Snippet: Protein levels of p53 after treatment with nontarget or p53 siRNA in MKN1, HUH-6, and HUH-7 cancer cells.

Article Snippet: Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung — CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes’ type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non–small cell lung cancer CRL-5803 Open in a separate window ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources.

Techniques:

Journal: Nature Metabolism

Article Title: Genetic architecture of oral glucose-stimulated insulin release provides biological insights into type 2 diabetes aetiology

doi: 10.1038/s42255-024-01140-6

Figure Lengend Snippet: a , Regional signal plot at the ACSL1 locus, showing P values on a −log 10 scale ( y axis) in hg19 locations ( x axis) from DIBIG single-variant GWAS meta-analyses. Variants are coloured by their LD correlation ( r 2 ) with the lead variant ( rs10022124 ). Below, epigenomic datasets are shown, including ATAC-seq and ChIP-seq data in human pancreatic islets. Enhancer-gene assignments from pcHi-C data are represented as pink arcs; those inferred using pcHi-C data are in purple. Fine-mapped DI and DIBIG signals located in islet active enhancers connected to ACSL1 and CENPU , or identified as T2D-colocalizing islet ACSL1 eQTLs, are highlighted. b , Forest plot of the strongest functionally prioritized signal at the ACSL1 locus ( rs4862423 , DIBIG). For each of the BCF traits, glycaemic levels and T2D risk, the square represents the β estimate with 95% CI error bars. The square size reflects precision. The effect allele and variant RSID are provided at the top. Summary statistics data were generated in this study or obtained from previous publications , and FinnGen (release v.8). OR, odds ratio. c , d , Insulin secretion in human EndoC-β H1 cells following ACSL1 siRNA silencing in response to high glucose ( c ) and KCl stimuli ( d ). e – g , Insulin secretion in INS-1 832/13 cells upon Acsl1 gene silencing in response to high glucose ( e ), KCl ( f ) and pyruvate ( g ). Bar plots show the means of three (in c and d ) and four (in e – g ) independent replicates; error bars, s.e.m. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test of each condition against the negative control group (scrambled siRNA).

Article Snippet: For qPCR, 10 ng of cDNA per well (in duplicates) was used with human ACSL1 and FAM46C , or rat Acsl1 , Fam46c and Cenpu Taqman gene expression assays (Thermo Fisher Scientific; code: Rn01488229_m1, Rn01534138_m1, Rn01488384_m1, Hs00960561_m1, Hs01933465_s1). qPCR assays were performed using the Applied Biosystems QuantStudio7 Flex Real-Time PCR System (Thermo Fisher).

Techniques: Variant Assay, ChIP-sequencing, Generated, Negative Control

Journal: Nature Metabolism

Article Title: Genetic architecture of oral glucose-stimulated insulin release provides biological insights into type 2 diabetes aetiology

doi: 10.1038/s42255-024-01140-6

Figure Lengend Snippet: (a) ACSL1 mRNA expression levels assessed by qPCR after 72h transfection with scramble (control) and ACSL1 siRNA 1-2 in human EndoC-βH1 cells. (b) Acsl1 mRNA expression levels after 72h transfection with scramble (control) and Acsl1 siRNA 1-2 in INS-1 832/13 rat insulinoma cells, measured by qPCR. (c) Measurement of insulin content relative to total protein level in INS-1 832/13 cells. (d) Insulin secretion levels normalized by total insulin content in response to high glucose (16.7 mM) in INS-1 832/13 cells. (e) Cenpu mRNA expression levels assessed by qPCR after 72h transfection with scramble (control) and Cenpu siRNA 1 in INS-1 832/13 cells. (f) Insulin secretion levels in response to high glucose (16.7 mM) concentration in INS-1 832/13 cells. (g) Measurement of total insulin content in INS-1 832/13 cells. (h) Insulin secretion normalization for total insulin content in response to high glucose (16.7 mM) concentration. Bar plots in panels (a-h) depict mean +/- SE from three (a) and four (b-h) independent replicates. Two-sided Student’s t-test was used for panels (c,e,g) . One-way analysis of variance (ANOVA) followed by Tukey post-hoc test was used for panels (a,b,d,f,h) . Panels (i) and (j) show morphology changes in INS-1 832/13 cells after transfection with scramble and Cenpu siRNA 1 , respectively. Images from a basic optical microscope, where scale bars are not available.

Article Snippet: For qPCR, 10 ng of cDNA per well (in duplicates) was used with human ACSL1 and FAM46C , or rat Acsl1 , Fam46c and Cenpu Taqman gene expression assays (Thermo Fisher Scientific; code: Rn01488229_m1, Rn01534138_m1, Rn01488384_m1, Hs00960561_m1, Hs01933465_s1). qPCR assays were performed using the Applied Biosystems QuantStudio7 Flex Real-Time PCR System (Thermo Fisher).

Techniques: Expressing, Transfection, Control, Concentration Assay, Microscopy