Journal: Antioxidants

Article Title: Inhibition of Cell Proliferation and Cell Viability by Sinecatechins in Cutaneous SCC Cells Is Related to an Imbalance of ROS and Loss of Mitochondrial Membrane Potential

doi: 10.3390/antiox11071416

Figure Lengend Snippet: Reduced cell viability by PE. ( A – C ) SCL-I, SCL-II, SCC-12, and SCC-13 were seeded in 24-well plates and were treated with PE (100, 200 µg/mL) for 24 h and 48 h, respectively. For control, cells were also treated with 100 µg EGCG for 48 h. ( A ) Cell viability was determined by calcein-AM staining and flow cytometry. Values represent the percentage of cells with high calcein staining (= viable cells). ( B ) Examples of flow cytometry reading are shown for the cell lines at 24 h and 200 µg/mL PE treatment (overlays of treated cells vs. Ctr, logarithmic scale). Non-viable and viable cell populations are indicated. For control, cells are also shown without calcein staining (PE-200-treated, w/o). ( C ) Apoptosis was quantified by propidium iodide staining and flow cytometry (cell cycle analyses). Values represent the percentage of sub-G1 cells (=apoptotic cells). ( A , C ) Each one of at least two independent experiments is shown; each independent experiment consisted of triplicate values. Statistical significance for PE treatments was calculated from all individual values (#6) and is indicated by asterisks ( p < 0.05, as compared to Ctr). ( D ) For SCL-I and SCL-II, induced cell death by PE-100 and PE-200 was determined by AnnV/PI staining. Treatment with the indirubin derivative DKP-071 in combination with TRAIL was used as control (DKP/TR). Representative flow cytometry histograms are shown of treated and control cells. ( E ) Mean values and SDs of AnnV(+)/PI(+) cells and of AnnV(+)/PI(−) cells are shown (in %). Mean values and SDs correspond to each six individual values obtained in two independent experiments with triplicates.

Article Snippet: For further apoptosis control, cells were treated with an indirubin derivative (DKP-071, 10 μM) and/or TRAIL (TNF-related apoptosis inducing ligand; KillerTRAIL, Adipogen, San Diego, CA, AG-40T-0001; 50 ng/mL).

Techniques: Control, Staining, Flow Cytometry

Journal: Antioxidants

Article Title: Inhibition of Cell Proliferation and Cell Viability by Sinecatechins in Cutaneous SCC Cells Is Related to an Imbalance of ROS and Loss of Mitochondrial Membrane Potential

doi: 10.3390/antiox11071416

Figure Lengend Snippet: Weak caspase activation. Expression of caspase-3, caspase-8, caspase-9, p19, and p21 is shown by Western blotting in cell lines SCL-I, SCL-II, and SCC-12. Cells were treated for 24 h with PE (100, 200 µg/mL). Protein size (in kDa) is indicated on the right side, as determined in comparison to a protein size marker separated in parallel. Caspase activation is seen either by characteristic cleavage products, as 18/16 kDa for caspase-3 and 35/37 kDa for caspase-9 or by loss of the caspase-8 proform (55 kDa). For demonstrating full caspase-3 activation, a positive control is shown, (+)Ctr, consisting of SCC-12 cells treated with an indirubin derivative in combination with the death ligand TRAIL (TNF-related apoptosis-inducing ligand . Expression of β-actin is shown as loading control. Largely similar results were obtained in two independent Western blot experiments using independent series of cell extracts.

Article Snippet: For further apoptosis control, cells were treated with an indirubin derivative (DKP-071, 10 μM) and/or TRAIL (TNF-related apoptosis inducing ligand; KillerTRAIL, Adipogen, San Diego, CA, AG-40T-0001; 50 ng/mL).

Techniques: Activation Assay, Expressing, Western Blot, Comparison, Marker, Positive Control, Control

Journal: Antioxidants

Article Title: Inhibition of Cell Proliferation and Cell Viability by Sinecatechins in Cutaneous SCC Cells Is Related to an Imbalance of ROS and Loss of Mitochondrial Membrane Potential

doi: 10.3390/antiox11071416

Figure Lengend Snippet: Less effects of caspase inhibition and Bax/Bak knockdown. ( A ) SCL-I and SCL-II cells were treated with 200 µg/mL PE (PE-200) or with an indirubin derivative DKP-071 in combination with TRAIL (DKP/TR, positive control). In addition, cells received the pancaspase inhibitor Q-VD-Oph (10 µM), when indicated. ( B , C ) HCT-116 WT cells and HCT-116 double knockout cells for Bax and Bak (KO) were treated with PE (100 or 200 µg/mL) or with TRAIL (positive control). ( A , B ) Cell death analysis by AnnV/PI staining and flow cytometry was performed after 24 h. Representative flow cytometry histograms of treated and control cells are shown on the right side or below. Mean values and SDs of two cell death fractions, namely AnnV(+)/PI(−) and AnnV(+)/PI(+) cells are shown (in %). ( C ) For microscopic visualization of low MMP and of morphological changes, cells were double stained with JC-1/Hoechst-33342 at 24 h. Blue, nuclear staining; red, mitochondria with high (normal) MMP; faint green, JC-1-stained cytosol; bright green or turquoise, rounded and detached cells.

Article Snippet: For further apoptosis control, cells were treated with an indirubin derivative (DKP-071, 10 μM) and/or TRAIL (TNF-related apoptosis inducing ligand; KillerTRAIL, Adipogen, San Diego, CA, AG-40T-0001; 50 ng/mL).

Techniques: Inhibition, Knockdown, Positive Control, Double Knockout, Staining, Flow Cytometry, Control