Journal: Frontiers in Oncology

Article Title: Mutant NPM1-Regulated FTO-Mediated m 6 A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis

doi: 10.3389/fonc.2022.817584

Figure Lengend Snippet: FTO Promotes Leukemic Cell Proliferation by Facilitating Cell Cycle and Suppressing Cell Apoptosis. (A) CCK-8 analysis of cell growth in the FTO-silenced OCI-AML3. (B) EdU analysis of cell proliferation in the FTO-silenced OCI-AML3 cells. The bar graph showed the percentage of EdU positive cells. Scale bar: 50 μm. (C) Flow cytometric analysis of cell cycle distribution in FTO-silenced OCI-AML3 cells. The bar graph showed the percentage of G0/G1, S, and G2/M phase cells. (D) Cell apoptosis was measured by flow cytometric analysis of the Annexin V/DAPI stained cells. The bar graph showed the cell apoptosis rate. (E) Western blot analysis of Cyclin A2, t-Caspase 9, and c-Caspase 9 protein level in the FTO-silenced OCI-AML3. β-actin was used as the internal control. The bar graph showed the relative level of protein. Data were presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The cell apoptosis was determined by an Annexin V-APC/DAPI Apoptosis Detection Kit (Elabscience, Wuhan, China, #E-CK-A258).

Techniques: CCK-8 Assay, Staining, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Mutant NPM1-Regulated FTO-Mediated m 6 A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis

doi: 10.3389/fonc.2022.817584

Figure Lengend Snippet: The Oncogenic Function of FTO is Dependent on m 6 A RNA Demethylase Activity. (A) CCK-8 analysis of cell proliferation in OCI-AML3 cells following transfection of the HA-FTO-WT and HA-FTO-MUT plasmids. (B) m 6 A dot blot analysis of the m 6 A level of global RNAs in OCI-AML3 cells with MA or DMSO treatment for 24 h. Methylene blue staining served as a loading control. (C) Dose-dependent effect of MA on cell viability. CCK-8 analysis of cell proliferation in OCI-AML3 cells exposed to 0, 10, 25, 50, and 100 μM MA for 24 h. (D) Time-dependent effect of MA on cell viability. CCK-8 analysis of cell proliferation in OCI-AML3 cells exposed to 100 μM MA for 0, 24, and 48 h. (E) EdU analysis of cell proliferation in OCI-AML3 cells upon 50 μM and 100 μM MA treatments of 24 h. The bar graph showed the percentage of EdU positive cells. Scale bar: 50 μm. (F) Flow cytometric analysis of the cell cycle distribution in OCI-AML3 cells upon 50 μM and 100 μM MA treatments of 24 h. The bar graph showed the percentage of G0/G1, S, and G2/M phase cells. (G) Cell apoptosis was measured by flow cytometric analysis of the Annexin V/DAPI stained cells. The bar graph showed the cell apoptosis rate. Data were presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The cell apoptosis was determined by an Annexin V-APC/DAPI Apoptosis Detection Kit (Elabscience, Wuhan, China, #E-CK-A258).

Techniques: Activity Assay, CCK-8 Assay, Transfection, Dot Blot, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Lidocaine Modulates Cytokine Production and Reprograms the Tumor Immune Microenvironment to Enhance Anti-Tumor Immune Responses in Gastric Cancer

doi: 10.3390/ijms26073236

Figure Lengend Snippet: Apoptotic effects of lidocaine on TIICs, PBMCs, PGCCs, and CD8 + TIICs co-cultured with PGCCs assessed by flow cytometry. Concentrations of 5 mM and 10 mM lidocaine induced apoptosis of TIICs and PBMCs, but concentrations of 0.5 and 1.5 mM lidocaine did not induce PGCCs apoptosis. TIICs ( A ), PBMCs ( C ), and PGCCs ( E ) were assessed by flow cytometric analysis using propidium iodide-stained cells. Firstly, 10 4 cells were incubated in 96-well plates in the presence or absence of the indicated concentrations of lidocaine. After 72 h treatment, cells were washed with PBS and fixed with 70% ethanol for 1 h on ice. Pelleted cells were incubated with RNaseA (0.1 mg/mL) and propidium iodide (40 μg/mL) for 1 h with shaking and protected from light. The percentage of subG1 population was determined by flow cytometry. ( G ) Flow cytometry assessment of cell death of lidocaine (1.5 m M)-treated CD8 + TIICs, -treated PGCCs, or -treated CD8 + TIICs were co-cultured with PGGCs. We gated on CD8 + TIICs and GRN + PGGCs, as described in our previously published paper . ( I ) CD8 + TIICs-, PGCCs-, and CD8 + TIICs-treated with lidocaine at a concentration of 1.5 mM were co-cultured with PGGCs at the 5:1 E:T ratios. Target cell cytotoxicity was determined at 2 h by a DELFIA EuTDA assay. MFI: mean fluorescence intensity. Data are representative of three independent experiments; n >= 3. ( B , D , F , H , I ) Data are from distinct samples and presented as the mean ± SEM in three different experiments, each performed in duplicate. ** p < 0.01.

Article Snippet: Apoptosis was assessed by flow cytometric analysis of cells stained with annexin V-PE according to the manufacturer’s instructions (Annexin V-PE Apoptosis Kit, Elabscience, Houston, TX, USA).

Techniques: Cell Culture, Flow Cytometry, Staining, Incubation, Concentration Assay, Fluorescence