Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: TRAIL induces the activation of apoptosis and non-apoptotic cascades leading to fractional survival. ( a ) Contour plot from flow cytometry assays assessing cleaved caspase-8 (p18) immunolabelling in populations of naïve (control) or TRAIL-treated cells (TRAIL, 3 h). Subpopulations displaying low (1) and high (2; percentage is depicted) levels of caspase-8 cleavage are indicated. ( b ) Experiment as in ( a ) but analyzing cleaved PARP immunolabelling ( c – f ) levels of cleaved PARP in clonal populations of naïve cells (control), the full population of cells treated 16 h with TRAIL (TRAIL) and subpopulations of apoptotic (apoptotic) or surviving (survivors) cells obtained after 16 h of treatment. ( a – f ) Images correspond to one representative experiment out of three independent biological replicates. ( g – h ) Percentage of cells with high levels of cleaved caspase-8 ( g ) or high levels of cleaved PARP ( h ) after TRAIL treatment (6 h, maximum rate of cell death). ( i ) Western blots displaying total and phosphorylated protein levels of Erk1/2, Akt, p38MAPK and I κ B α in naïve cells (0) or upon TRAIL treatment. Depicted images correspond to one representative experiment out of at least three independent biological replicates. α -Tubulin, loading control. ( j ) Percentage of cells with high levels of cleaved PARP in populations treated with U0126 (MEK1/2 inhibitor), LY294002 (PI3 kinase inhibitor), PD 169316 (p38 MAP kinase inhibitor), Bay-11-7082 (NF- κ B inhibitor) or vehicle (DMSO) and either left unchallenged (control) or treated with TRAIL (5 h). ( k ) Percentage of cells with high levels of cleaved PARP in populations either pretreated with pan-caspase inhibitor (zVAD.fmk) or vehicle (DMSO) and then either left untreated (control) or challenged for 6 h with TRAIL. ( g , h , j and k ) Mean±standard deviation (S.D.) from three independent biological replicates is shown. *** P- value <0.0005, ** P- value <0.005, *P- value <0.05. ( l ) Western blots displaying total and phosphorylated protein levels of Erk1/2, Akt, p38 and I κ B α in naïve cells (0) or upon TRAIL treatment in cells either pretreated with caspase-8 inhibitor Z-IETD-FMK, pan-caspase inhibitor zVAD.fmk or vehicle (DMSO). Depicted images correspond to one representative experiment out of at least three independent biological replicates. α -Tubulin, loading control

Article Snippet: Immunolabelling of cleaved PARP or cleaved caspase-8 was performed at 21 °C for 1 h when using fluorescently labeled antibodies (Cleaved caspase-8 PE (cat. # 12602), Cleaved PARP Alexa 647 (cat. # 6987) and Cleaved PARP PE (cat. # 8978) from Cell Signaling) or incubated for 1 h with primary antibodies (Cleaved caspase-8 (cat. # 9496)), washed three times with 1 × PBS and incubated 1 h with fluorescently tagged secondary antibodies anti-rabbit Alexa 488 (cat. # A21206; Life Technologies).

Techniques: Activation Assay, Flow Cytometry, Control, Western Blot, Standard Deviation

Journal: Cell Death & Disease

Article Title: Dual role of DR5 in death and survival signaling leads to TRAIL resistance in cancer cells

doi: 10.1038/cddis.2017.423

Figure Lengend Snippet: Role of TRAIL receptors and TRAIL-receptor-binding proteins in the triggering of apoptosis. ( a ) Percentage of cells with high levels of cleaved PARP in populations transfected with pooled siRNAs targeting DR4 (DR4), DR5 (DR5), DcR2 (DcR2) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged with TRAIL (6 h). ( b ) Percentage of cells with high levels of cleaved PARP in naïve (control) or TRAIL-challenged (6 h) DR5 knockout (DR5 KO) or DcR2 knockout (DcR2 KO) cells as compared with controls (ctrol). ( c ) Contour plot for cleaved PARP immunolabelling in DcR2 knockout (DcR2 KO) cells either unchallenged (control) or treated 6 h with TRAIL (TRAIL). Subpopulations displaying low (survivors, '1') and high (apoptotic, '2') levels of PARP cleavage in treated samples are depicted. Image shows one representative experiment out of three independent biological replicates. ( d ) Percentage of cells with high levels of cleaved PARP in control and DcR2 KO naïve cells (control) or control and DcR2 KO cells challenged with TRAIL for 16 h (TRAIL). ( e ) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting FADD (FADD) , caspase-8 (casp-8), RIPK1 (RIPK1), TRAF2 (TRAF2) or cFlip (cFlip) mRNAs or non-targeting scramble siRNAs (scr) further left either untreated (control) or challenged for 6 h with TRAIL (TRAIL). ( f ) Percentage of cells with high levels of cleaved PARP in populations transfected with siRNAs targeting RIPK1 (RIPK1) , TRAF2 (TRAF2) or RIPK1 and TRAF2 (RIPK1+TRAF2) mRNAs or non-targeting scramble siRNAs at concentrations corresponding to single (30 nM; scr 30) or double (60 nM; scr 60) transfections, further left either untreated (control) or challenged during 6 h with TRAIL (TRAIL). Results obtained in conditions of pan-caspase inhibition (control+ zVAD and TRAIL+zVAD) as compared with basal conditions of cell response (control and TRAIL) are shown. ( g ) Percentage of cells with high levels of cleaved PARP in cells transfected with siRNAs targeting RIPK1 (RIPK1) , TRAF2 (TRAF2) , DR5 (DR5), RIPK1 and DR5 (RIPK1+DR5), TRAF2 and DR5 (TRAF2+DR5) mRNAs or non-targeting scramble siRNAs ('scr 30' and 'scr 60') further left either untreated (control) or challenged 6 h with TRAIL (TRAIL). ( a , b and d – g ) Mean±standard deviation (S.D.) from three independent biological replicates is shown. *** P- value <0.0005, ** P- value <0.005, * P- value <0.05. NS, not significant

Article Snippet: Immunolabelling of cleaved PARP or cleaved caspase-8 was performed at 21 °C for 1 h when using fluorescently labeled antibodies (Cleaved caspase-8 PE (cat. # 12602), Cleaved PARP Alexa 647 (cat. # 6987) and Cleaved PARP PE (cat. # 8978) from Cell Signaling) or incubated for 1 h with primary antibodies (Cleaved caspase-8 (cat. # 9496)), washed three times with 1 × PBS and incubated 1 h with fluorescently tagged secondary antibodies anti-rabbit Alexa 488 (cat. # A21206; Life Technologies).

Techniques: Binding Assay, Transfection, Control, Knock-Out, Inhibition, Standard Deviation

Journal: eLife

Article Title: The mitochondrial iron transporter ABCB7 is required for B cell development, proliferation, and class switch recombination in mice

doi: 10.7554/eLife.69621

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-PARP (Rabbit monoclonal, 46D11) , Cell Signaling Technology , Cat#:9532S; RRID: AB_659884 , FC (1:100).

Techniques: Transgenic Assay, Selection, Concentration Assay, Sequencing, Recombinant, Flow Cytometry, EdU Assay, DNA Purification, Isolation, Colony-forming Unit Assay, Cell Cycle Assay, Software