Journal: Oxidative Medicine and Cellular Longevity

Article Title: GSDME Increases Chemotherapeutic Drug Sensitivity by Inducing Pyroptosis in Retinoblastoma Cells

doi: 10.1155/2022/2371807

Figure Lengend Snippet: After GSDME expression was upregulated by decitabine, pyroptosis was increased. (a and b) The toxicity of decitabine to Y79 and WERI-RB-1 cells was detected by CCK-8 assay at different concentrations of decitabine. (c and d) The mRNA expression of GSDME was detected using RT-PCR after treatment with different concentrations of decitabine for 3 days (the cells were centrifuged, and decitabine was replaced every day). (e and f) The protein level of GSDME in cells was quantitatively analysed by western blot. (g and h) The protein level of GSDME in cells was detected using immunofluorescence. (i and j) CCK-8 assay of Y79 and WERI-RB-1 cell viability after treatment with carboplatin or decitabine plus carboplatin. (k) The death morphologies of the three cell lines were observed using an Olympus microscope. Y79 and WERI-RB-1 cells were treated with decitabine plus carboplatin; ARPE-19 cells were treated with decitabine; black arrows, apoptotic cells; white arrows, pyroptotic cells. (l–p) GSDME-N and its pathway proteins were detected by WB. (q and r) Flow cytometry showed the ratio of FITC monostaining to FITC and PI double-positive staining after treatment with decitabine plus carboplatin, and quantitative maps were drawn for carboplatin-treated cells after 24 h. (s) LDH in the cell supernatant was detected by ELISA. Each experiment was repeated at least three times. Scale bar, 100 μ m.

Article Snippet: When the tumor was approximately 100 mm 3 , the second group was injected with 0.5 mg/kg decitabine (MedChemExpress, USA) 1 time/day for 7 days in total.

Techniques: Expressing, CCK-8 Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Microscopy, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: GSDME Increases Chemotherapeutic Drug Sensitivity by Inducing Pyroptosis in Retinoblastoma Cells

doi: 10.1155/2022/2371807

Figure Lengend Snippet: IC 50 values and statistical analyses of decitabine and carboplatin treatments in RB cell lines.

Article Snippet: When the tumor was approximately 100 mm 3 , the second group was injected with 0.5 mg/kg decitabine (MedChemExpress, USA) 1 time/day for 7 days in total.

Techniques:

Journal: Molecular biology reports

Article Title: DNA methylation of CRB3 is a prognostic biomarker in clear cell renal cell carcinoma.

doi: 10.1007/s11033-019-04892-7

Figure Lengend Snippet: Fig. 6 Restoration of CRB3 mRNA by DNA methylation inhibi- tor Decitabine in ccRCC cell lines. Cakl-1(a) and Cakl-2(b) cells were incubated with 1, 5, 10 µM Decitabine for 48 h or 72 h, and the mRNA levels of CRB3 were detected by Real-time PCR. All data was represented as mean ± SEM (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001)

Article Snippet: The same numbers of cells were seeded in each well of 6-well plates and cultured at 37 °C in the medium containing 1, 5, or 10 μM Decitabine (Selleck, Houston, TX, USA) or vehicle (DMSO).

Techniques: DNA Methylation Assay, Incubation, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: Neuroendocrine lineage commitment of small cell lung cancers can be leveraged into p53-independent non-cytotoxic therapy

doi: 10.1016/j.celrep.2023.113016

Figure Lengend Snippet: Human SCLC cells H82 and H146 were transfected with DNMT1 siRNA (siDNMT1) or scrambled siRNA (siCtrl). (A) DNMT1 knockdown. DNMT1 protein was measured by western blot 72 h after transfection. (B) Cell growth inhibition. Cell counts by automated counter 72 h after transfection. Mean ± SD of four independent experiments. (C) Apoptosis not activated. Apoptosis was measured by Annexin V staining and flow cytometry analysis 24 h after transfection. Fluorizoline (10 μM) was used as a positive control for apoptosis. Mean ± SD of three independent experiments. (D) Activation of pulmonary NE-lineage signature genes. Gastrin-related peptide (GRP), neuronatin (NNAT), chromogranin B (CHGB), and secretogranin II (SCG2), also known as chromogranin C (CHGC), were increased by DNMT1 knockdown. Quantitative reverse polymerase chain reaction (qPCR) 72 h after transfection. Internal control was β-ACTIN; relative gene expression was calculated by the Livak-Schmittgen method. Mean ± SD of three independent experiments. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, p > 0.05. t test two-sided vs. siCtrl. (E) Cell morphology. Giemsa-stained cell imaged by Leica Upright Microscope-Orion. Magnification ×400; scale bar, 10 μm. (F) DNMT1 depletion using decitabine (Dec) or 5-azacytidine (5Aza) decreased SCLC proliferation. SCLC cells H82 and H146 were treated with DMSO vehicle control, Dec 0.5 μM, or 5Aza 1 μM added once at time 0 h. Cell counts by automated counter. (G) The cytoreduction was not by apoptosis. Apoptosis was measured by Annexin V staining and flow cytometry at 24 h. Positive control fluorizoline (10 μM). (H) Dec and 5Aza treatment decreased DNMT1 and MYC and increased p27/CKDN1B (mediator of cell-cycle exits by terminal lineage differentiation). Western blot. (I) Dec and 5Aza treatment increased expression of pulmonary NE-lineage signature genes. Measured at 72 h in H82 cells and 96 h in H146 cells, as per (D). Experiment in triplicate. Statistics are as per (D) treated vs. vehicle. (J) Cell morphology. Giemsa-stained cytospin preparations of cells harvested after 72–96 h. Leica Upright Microscope-Orion; magnification ×400; scale bar, 10 μm.

Article Snippet: Decitabine , BOC Sciences , Cat# 2353-33-5.

Techniques: Transfection, Knockdown, Western Blot, Inhibition, Staining, Flow Cytometry, Positive Control, Activation Assay, Polymerase Chain Reaction, Control, Gene Expression, Microscopy, Expressing

Journal: Cell reports

Article Title: Neuroendocrine lineage commitment of small cell lung cancers can be leveraged into p53-independent non-cytotoxic therapy

doi: 10.1016/j.celrep.2023.113016

Figure Lengend Snippet:

Article Snippet: Decitabine , BOC Sciences , Cat# 2353-33-5.

Techniques: Control, Recombinant, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Giemsa Stain, cDNA Synthesis, Software