Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Dose- and time-dependent effect of BAY and medium refreshment on cell viability. ( a ) Dose-dependent effect of BAY ( N =2, n =4) on the viability of G361 and SK-MEL-28 cells (at 72 h). A Boltzmann equation was used to determine the IC 50 ( x 0 ) value: y =( A 2 +( A 1 − A 2 )/(1+exp(( x − x 0 )/d x ))). ( b ) G361 and SK-MEL-28 melanoma cells were treated with 10 nM BAY ( N =3, n =6) and their viability was measured at different time points. A sigmoidal (logistic) equation was used to determine the T 1/2 ( x 0 ) value: y =([( A 1 − A 2 )/(1+( x/x 0 )]+ A 2 ). Statistics: In panel b, significant differences with the SK-MEL-28 cell line are indicated by * P <0.05 and *** P <0.001

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques:

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of TRAP1 knockdown/overexpression on the BAY-induced reduction in cell viability. ( a ) Detection of photoinduced mitochondrial membrane potential (Δ ψ ) depolarizations (‘Δ ψ -flickering') in a typical TMRM-stained G361 cells (see Results for details). ( b ) Effect of BAY (2 min), CsA (2 h), empty vector and TRAP1-OE on Δ ψ -flickering ( N =3, n ≥20). ( c ) Effect of BAY on reactive oxygen species (ROS) levels (at 24 h; N=3, n =3) in cells transfected with the empty or TRAP1-OE vector. ( d ) Effect of vehicle ( N =5, n =15), BAY ( N =5, n =15) and CsA ( N =3, n =6) on cell death (G361: at 48 h; SK-MEL-28: at 72 h). ( e ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siTRAP1, empty vector and TRAP1-OE. Statistics: Significant differences relative to the vehicle condition (‘Veh' in panel b) and between treatments (a, b, c, d in panels c and d ) are indicated by * P <0.05, ** P <0.01 and *** P <0.001. In panel ( e ), significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Over Expression, Membrane, Staining, Plasmid Preparation, Transfection

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of ATG5 knockdown on the BAY-induced stimulation of autophagy, reactive oxygen species (ROS) increase and reduction in cell viability. ( a ) Effect of BAY in the absence and presence of BafA1, TOC and ATG5 knockdown on the number of green puncta in G361 and SK-MEL-28 cells (at 24 h; N =3, n =30). ( b ) Effect of BAY on ROS levels (at 24 h; N =3, n =9) in cells transfected with an empty or GPX4 overexpression vector (GPX-OE). ( c ) Effect of BAY on cellular MG fluorescence (G361: at 24 h; SK-MEL-28: at 24 h; N =3, n =9) in cells transfected with siCTRL, siATG5-no. 1 and siATG5-no. 2. ( d ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siATG5-no. 1 and siATG5-no. 2. Statistics: Significant differences relative to the marked conditions are indicated by * P <0.05, ** P <0.01 and *** P <0.001. NS indicates nonsignificant. In panel ( d ), significant differences with the (siCTRL+10 nM BAY condition) are marked by ‘&'. In panel ( b ) significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Transfection, Over Expression, Plasmid Preparation, Fluorescence

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of PINK1 knockdown on the BAY-induced stimulation of mitophagy, Δ ψ depolarization, reactive oxygen species (ROS) levels and reduction in cell viability. ( a ) Effect of BAY treatment, siPINK1-no. 1 and siPINK1-no. 2 on the number of green puncta colocalizing with mitochondria in G361 and SK-MEL-28 cells (at 24 h; N =3, n ≥14). ( b ) Similar to panel a, but now for the effect on mitochondrial membrane potential (i.e. the JC-1 red/green ratio signal; N =3, n =6). ( c ) Similar to panel a, but now for the effect on cellular ROS levels ( N =3, n =6). ( d ) Similar to panel a, but now for the effect on cellular MG fluorescence intensity (G361: at 24 h; SK-MEL-28: at 24 h; N =3, n =9). ( e ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siPINK1-no. 1 and siPINK1-no. 2. Statistics: Significant differences relative to the indicated conditions are marked by * P <0.05, ** P <0.01 and *** P <0.001. In panel c statistical analysis was performed using a one-sample t-test against a value of 100. In panel d, NS indicates nonsignificant and significant differences with the (siCTRL+10 nM BAY condition) are marked by ‘&'. In panel ( e ) significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Membrane, Fluorescence, Transfection

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of Drp1 knockdown on mitochondrial morphology and the BAY-induced reduction in cell viability. ( a ) Typical examples visualizing mitochondria morphology in MG-stained cells treated with siCTRL, siDrp1-no. 1 and siDrp1-no. 2 in the absence and presence of 10 nM BAY (G361: at 16 h; SK-MEL-28: at 24 h). ( b ) Quantification of mitochondrial morphology in multiple cells ( N =2; numerals indicate the number of cells analyzed) for the conditions in panel ( a ). ( c ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siDrp1-no. 1 and siDrp1-no. 2. Statistics: In panel ( c ), statistically significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Staining, Transfection

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of z-VAD-FMK, Nec-1, Nec-1s, Fer-1 and knockdown of RIPK1 and MLKL on the BAY-induced reduction in cell viability. ( a ) Effect of vehicle ( N =5, n =15), BAY ( N =5, n =15), the pancaspase inhibitor z-VAD-FMK ( N =3, n =9), Nec-1 ( N =3, n =9) and Fer-1 ( N =3, n =9) on cell death (G361: at 48 h; SK-MEL-28: at 72 h). ( b ) Effect of BAY on viability (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) in the absence and presence of the ferroptosis inhibitor Fer-1 and the RIPK1 inhibitors Nec-1 and Nec-1s. ( c ) Effect of BAY on cell viability (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) in cells transfected with siCTRL, siRIPK1-no. 1 and siRIPK1-no. 2. ( d ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N=3, n =6) transfected with siCTRL, siMLKl-no. 1 and siMLKL-no. 2. Statistics: Significant differences relative to the marked conditions are indicated by * P <0.05 and *** P <0.001. In panels b–d, significant differences ( P <0.05) between conditions are marked by symbols (a, b, &, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Transfection

Journal: Cell Death & Disease

Article Title: Mitochondrial complex I inhibition triggers a mitophagy-dependent ROS increase leading to necroptosis and ferroptosis in melanoma cells

doi: 10.1038/cddis.2017.133

Figure Lengend Snippet: Effect of GPX4 knockdown/overexpression on the BAY-induced reduction in cell viability. ( a ) Effect of BAY on the number of MBB-negative (GSH-depleted) cells in the vehicle- and BAY-treated condition (G361: at 12 h; SK-MEL-28: at 24 h; N =3, n =9). Higher bars reflect a reduction in the number of MBB-positive cells. ( b ) Effect of BAY on reactive oxygen species (ROS) levels (at 24 h; N =3, n =3) in cells transfected with the empty or GPX4-OE vector. ( c ) Cellular lipid peroxidation (at 24 h) in vehicle-treated cells (G361: N =3, n =7; SK-MEL-28: N =3, n =6), TOC-treated cells (G361: N =3, n =7; SK-MEL-28: N =3, n =6), BAY-treated cells (G361: N =3, n =7; SK-MEL-28: N =3, n =6), BAY+TOC-treated cells (G361: N =3, n =7; SK-MEL-28: N =3, n =6), empty vector-transfected cells (G361: N =3, n =9; SK-MEL-28: N =3, n =8), GPX4-OE-transfected cells (G361: N =3, n =9; SK-MEL-28: N =3, n =8), empty vector-transfected-+BAY-treated cells (G361: N =3, n =9; SK-MEL-28: N =3, n =8) and GPX-OE transfected-+BAY-treated cells (G361: N =3, n =8; SK-MEL-28: N =3, n =8). ( d ) Effect of BAY on the viability of cells (G361: at 48 h; SK-MEL-28: at 72 h; N =3, n =6) transfected with siCTRL, siGPX4, empty vector and GPX4-OE. Statistics: Significant differences relative to vehicle (panel a), empty vector (panel b) and the marked conditions (panel c) are indicated by * P <0.05, ** P <0.01, * P <0.01 and *** P <0.001. In panel d, significant differences ( P <0.05) between conditions are marked by symbols (&, $)

Article Snippet: BRAF V600E human melanoma cell lines G361 (no. ATCC-CRL-1424) and SK-MEL-28 (no. ATCC-HTB-72) were derived from a skin melanoma site and obtained from American Type Culture Collection (ATCC; LGC Standards GmbH, Wesel, Germany).

Techniques: Knockdown, Over Expression, Transfection, Plasmid Preparation