Journal: Scientific Reports

Article Title: 5-Fluorouracil induces apoptosis in nutritional deprived hepatocellular carcinoma through mitochondrial damage

doi: 10.1038/s41598-024-73143-y

Figure Lengend Snippet: Cellular ROS, GST, and nitric oxide determination in autophagic cells: ( a ) ROS production was determined by 2’-7’-Dichlorodihydrofluoroscein diacetate (H 2 DCFDA) dye (2 µg/ml) in presence of complete media control), PBS, H 2 O 2 , and rapamycin. (i) All the cells were observed using fluorescence microscope (Magnus MLXi, India). Images included here are representative of three independent experiments on both cell lines. (ii) Quantitative change in fluorescence intensity relative to control. ( b ) ( c ) ( d ) & ( e ) Stress response enzyme assay was performed by quantitative measurement of glutathione’s transferase, nitric oxide, nitrite and nitrate activity, respectively. Values are represented as ± SD of at least three independent experiments. p < 0.05 was considered as significant; the standard deviations of the data have been shown in the form of error bars. Scale bar = 30 µM.

Article Snippet: At last, cell suspension and trypan blue solution (0.04%) were mixed in a 1:1 ratio and the number of viable and non-viable cells were quantified using hemocytometer under a compound microscope (Olympus CK40).

Techniques: Control, Fluorescence, Microscopy, Enzymatic Assay, Activity Assay

Journal: Scientific Reports

Article Title: 5-Fluorouracil induces apoptosis in nutritional deprived hepatocellular carcinoma through mitochondrial damage

doi: 10.1038/s41598-024-73143-y

Figure Lengend Snippet: The comparative cell viability study in starved autophagic cells in presence of 5-FU: ( a ) The percentage of cell viability after 5- Fluorouracil treatment in WRL-68 and HepG2 cell by MTT assay after 24 h. IC 50 dose of 250 µM and 300 µM were determined for WRL-68 and HepG2, respectively. ( b ) Trypan blue exclusion assay was performed for the accurate quantification of viable and non-viable cells in all treated conditions – control, PBS only (without nutrient), control + 5-FU and PBS + 5-FU (starvation induced autophagic condition + 5-Fluorouracil). All the values plotted are the averages of three different experiments; the standard deviations of the data have been shown in the form of error bars. ( c ) Morphological changes of HepG2 cell line and WRL-68 were observed under phase contrast microscope at initial stage (0 h) and after successful autophagy induction (3 h). All the experiments were performed in triplicate. Scale bar = 80 µM. ( d ) Autophagolysosomes were detected by the formation of acidic vacuole organelles under following conditions– complete media(control), nutrient deficient condition (PBS), 5-FU treated cells in presence/absence of nutrient (control + 5-FU and PBS + 5-FU) and Rapamycin treated cells (as positive control). All the experiments were done in triplicate and images represented are representative of three individual treatments. Scale bar = 30 µM.

Article Snippet: At last, cell suspension and trypan blue solution (0.04%) were mixed in a 1:1 ratio and the number of viable and non-viable cells were quantified using hemocytometer under a compound microscope (Olympus CK40).

Techniques: MTT Assay, Trypan Blue Exclusion Assay, Control, Microscopy, Positive Control

Journal: Scientific Reports

Article Title: 5-Fluorouracil induces apoptosis in nutritional deprived hepatocellular carcinoma through mitochondrial damage

doi: 10.1038/s41598-024-73143-y

Figure Lengend Snippet: Detection of reactive oxygen species formation using Dihydroethidium (DHE) dye. ( a ) Superoxide formation was detected by staining the cells with DHE (2 µg/ml) in presence and absence of nutrient along with 5-Fluorouracil. (i) WRL-68 and HepG2 cells were visualized under fluorescence microscope (Magnus MLXi, India). (ii) Quantitative change in fluorescence intensity relative to control. Scale bar = 50 µM. ( b ) Qualitative measurement of cellular lipidation status under each mentioned condition –complete media(control), PBS (nutrient deficient condition), 5-Fluorouracil under nutrient sufficient (control + 5-FU) and nutrient deficient condition (PBS + 5-FU). All stained cells were observed under a fluorescence microscope (Magnus MLXi, India). Scale bar = 30 µM. ( c ) Differential lipidation pattern was observed in – Control (without treatment), PBS (Nutrient deficient condition), 5-Fluorouracil under nutrient sufficient (control + 5-FU) and nutrient deficient condition (PBS + 5-FU). Extraction of the dye was done with 100% isopropanol and absorbance was recorded at 492 nm by SPECTROstar nano plate reader (BMG Labteck, Germany). 100% isopropanol was used as background control to subtract the background signal. All the values plotted are the averages of three different experiments; the standard deviations of the data have been shown in the form of error bars.

Article Snippet: At last, cell suspension and trypan blue solution (0.04%) were mixed in a 1:1 ratio and the number of viable and non-viable cells were quantified using hemocytometer under a compound microscope (Olympus CK40).

Techniques: Staining, Fluorescence, Microscopy, Control, Extraction

Journal: Scientific Reports

Article Title: 5-Fluorouracil induces apoptosis in nutritional deprived hepatocellular carcinoma through mitochondrial damage

doi: 10.1038/s41598-024-73143-y

Figure Lengend Snippet: Detection of mitochondrial membrane damage through TMRE Assay. ( a ) Assessment of cell death was separately recorded in WRL-68 (normal liver cell line) and HepG2 (Hepatocarcinoma cell line) by Annexin V-FITC/PI conjugate assay- WRL-68 and HepG2 cells were treated with 5-FU drugs in complete media (control), PBS for 3 h and stained according to manufacturer’s protocol. (i) Slides were visualized under a fluorescence microscope (Magnus MLXi, India) and all the figures are representative of three individual experiments. (ii) Quantitative change in fluorescence intensity relative to control. Scale bar = 100 µM. ( b ) WRL-68 and HepG2 cells were treated with 5-FU drug in complete media (control) and with PBS. Cells were stained with 50 nM of tetramethylrhodamine ethyl ester (TMRE) dye. (i) Cells were observed using a fluorescence microscope (Magnus MLXi, India).and all the images included here are representative of three independent experiments on both cell lines. (ii) Quantitative change in fluorescence intensity relative to control. Scale bar = 30 µM. ( c ) Acridine Orange/Ethidium Bromide (2 µg/ml) staining was performed to detect apoptosis in cells under the combinatorial effect of PBS + 5-FU (5-Fluorouracil under nutrient deprived condition). (i) Early and late apoptotic cells were detected by staining with acridine orange and ethidium bromide dye using a fluorescence microscope (Magnus MLXi, India). Presence of apoptotic cells were compared with that of untreated control (with growth media) by three independent experiments. (ii) Quantitative changes in red and green fluorescence intensity. Scale bar = 30 µM.

Article Snippet: At last, cell suspension and trypan blue solution (0.04%) were mixed in a 1:1 ratio and the number of viable and non-viable cells were quantified using hemocytometer under a compound microscope (Olympus CK40).

Techniques: Membrane, Control, Staining, Fluorescence, Microscopy