Journal: Current issues in molecular biology

Article Title: Metabolic Silencing via Methionine-Based Amino Acid Restriction in Head and Neck Cancer.

doi: 10.3390/cimb45060289

Figure Lengend Snippet: Figure 1. Analysis of proliferation in different cell lines subjected to restriction of the amino acid methionine. (a–g) In each case, 10,000 cells/well were seeded on 96-well plates and stimulated in triplicate on the following day with a control medium or Met(-) medium. Absolute cell counts were analysed at 0 h, 24 h, 72 h, and 120 h using digital microscopy with the ImageXpress Pico automated cell imaging system. A summary of the results from three experimental replicates (n = 3) is shown in each case. (h) Summary of the proliferation analysis based on relative cell number counted using crystal violet staining. In each case, 10,000 cells/well were seeded on 96-well plates and stimulated the following day with a control medium (set as 100%) or Met(-) medium. Cells were analysed at 24 h, 48 h, 72 h, 96 h, and 120 h. A summary of the results from three experimental replicates (n = 3) is shown in each case (* p < 0.05, ** p < 0.01; *** p < 0.001).

Article Snippet: Measurements were made using the “Cell Scoring: 3 Channels” program of the ImageXpress Pico Automated Cell I aging System (Molecular Devices) with a 10× magnification.

Techniques: Control, Microscopy, Imaging, Staining

Journal: Current issues in molecular biology

Article Title: Metabolic Silencing via Methionine-Based Amino Acid Restriction in Head and Neck Cancer.

doi: 10.3390/cimb45060289

Figure Lengend Snippet: Figure 3. Homocysteine (Hcy)-based competition of MetR in different cell lines. (a–g) In each case, 10,000 cells/well were seeded on 96-well plates and stimulated in triplicate on the following day with control medium, Met(-) medium, or Met(-) medium to which 800 µM Hcy had been added (Met(-)/Hcy). Cells were analysed at 0 h, 72 h, and 120 h using the ImageXpress Pico automated cell imaging system to determine absolute cell counts. A summary of the results from three experimental replicates (n = 3) is shown in each case (ns; nonsignificant; * p < 0.05, ** p < 0.01; *** p < 0.001).

Article Snippet: Measurements were made using the “Cell Scoring: 3 Channels” program of the ImageXpress Pico Automated Cell I aging System (Molecular Devices) with a 10× magnification.

Techniques: Control, Imaging

Journal: Current issues in molecular biology

Article Title: Metabolic Silencing via Methionine-Based Amino Acid Restriction in Head and Neck Cancer.

doi: 10.3390/cimb45060289

Figure Lengend Snippet: Figure 4. The cell lines L929, HeLa, HaCaT, Detroit562, FaDu, SCC9, and SCC25 were seeded at 10,000 cells/well on a 96-well plate. On the following day, the medium was removed, and the cells were stimulated with full or Met(-) medium. Cells stimulated with 1 µM staurosporine served as the dead control cells. After 6 h, 24 h, and 48 h, measurements were performed with the “EarlyTox Live/Dead Assay Kit” (Molecular Devices). Measurements were made using the “Cell Scoring: 3 Channels” program of the ImageXpress Pico Automated Cell Imaging System (Molecular Devices) with a 10× magnification. The cytoplasm of leaving cells was measured in the FITC channel with CAM, and dead cells were measured in Texas Red-(TRITC)-channel with EthD-III. The results for 24 h and 48 h are shown. The 6 h time point showed no cell death for the control or MetR group. The dead

Article Snippet: Measurements were made using the “Cell Scoring: 3 Channels” program of the ImageXpress Pico Automated Cell I aging System (Molecular Devices) with a 10× magnification.

Techniques: Control, Live Dead Assay, Imaging

Journal: Current issues in molecular biology

Article Title: Metabolic Silencing via Methionine-Based Amino Acid Restriction in Head and Neck Cancer.

doi: 10.3390/cimb45060289

Figure Lengend Snippet: Figure 6. Analysis of the efficacy of cisplatin under MetR. (a–f) A total of 10,000 cells/well were seeded on 96-well plates. On the following day, cells were incubated in triplicate with control or Met(-) medium and a log2 dilution of cisplatin, with a starting concentration of 400 µM (12.5 µM for HeLa and 50 µM for FaDu). After 72 h, the cells were analysed using the ImageXpress Pico automated cell imaging system to determine absolute cell numbers. To compare the efficacy of cisplatin under different conditions, we used the IC50, which is defined as the concentration at which the drug reaches its half-maximal efficacy. For this purpose, the absolute cell counts (shown on the left side for each cell line) must be converted into percentages (shown on the right side for each cell line). The respective control value (without cisplatin) was set as 100%, and the lowest value in a measurement series was set as 0%. If one now places the two curves in a diagram, one can see whether there is a shift in the curve and, thus, a change in the IC50. For objective representation, the results of the absolute cell numbers are shown for each cell line, as well as the normalised representation in percent to the right. The dashed line indicates the inhibition of proliferation at 50% (IC50). The experiments were carried out three times. The figures show a summary of the experiments (n = 3) in one diagram.

Article Snippet: Measurements were made using the “Cell Scoring: 3 Channels” program of the ImageXpress Pico Automated Cell I aging System (Molecular Devices) with a 10× magnification.

Techniques: Incubation, Control, Concentration Assay, Imaging, Inhibition