Journal: Nature chemical biology

Article Title: Phospholipase iPLA 2 β Averts Ferroptosis By Eliminating A Redox Lipid Death Signal

doi: 10.1038/s41589-020-00734-x

Figure Lengend Snippet: a , Time course of RSL3-induced ferroptosis ( upper ) and concentration-dependent effect of RSL3 ( lower ) in WT H109 and fPD R747W cells. Data are means±s.d., **p=0.0018, ****p<0.0001 vs H109, N=3 biologically independent experiments, two-way ANOVA (Sidak’s post-hoc test). IC 50 was 28.9nM and 22.0nM for H109 and fPD R747W , respectively. b , Effect of inhibitors (z-VAD-fmk 50μM, Nec-1s 20μM, Fer-1 0.4μM, DFO 10μM, vit E 10μM, baicalein 2μM) on RSL3-induced (25nM, 14 hrs) death in H109 ( upper ) and fPD R747W ( lower ) cells. Data are means±s.d., ****p<0.001 vs Control, #### p<0.001 vs RSL3, N=3 biologically independent experiments, one-way ANOVA (Dunnett post-hoc test). c , RSL3-induced ferroptosis in WT and iPLA 2 β knock-down (KD) H109 cells. Cell death was quantified by LDH release. Data are means ± s.d., ****p<0.001 vs WT, N=3 biologically independent experiments, one-way ANOVA, (Sidak’s post-hoc test). d, e , LPCAT3 KD protects H109 cells from RSL3-induced death. d , Representative immunoblot and protein levels of LPCAT3 (quantified from three biological replicates normalized to actin). Data represent mean±s.d., *p=0.0018 vs si-NT, unpaired two-tailed t -test. e , si-NT or LPCAT3 KD cells were exposed to RSL3 (100 nM) and cell death was monitored after 20 hrs by PI staining using flow cytometry. Data are mean±s.d., N=3 biologically independent experiments; ****p<0.0001 vs si-NT control, #### p<0.0001 vs.si-LPCAT3 control, $$$$ p<0.0001 vs si-NT/RSL3, one-way ANOVA (Tukey’s post-hoc test) . f, Quantitative LC/MS-based assessments of lyso-PE (1-SA-2-OH-PE, left ) and lyso-PC (1-SA-2OH-PC, right ) in H109 cells. Data are mean±s.d., N=6 biologically independent experiments, **p=0.0018 vs si-NT, unpaired two-tailed t -test. g, The contents of oxygenated PE (1-SA-2-HpETE-PE, left ) and PC (1-SA-2-HpETE-PC, right ) in H109 cells. Cells were exposed to RSL3 (100nM) for 20 hrs. Data are mean±s.d., N=6 biologically independent experiments, *p=0.0118 vs si-NT control, ****p<0.0001 vs si-NT control, #### p<0.0001 vs si-LPCAT3 control, $$$$ p<0.0001 vs si-NT/RSL3, one-way ANOVA.

Article Snippet: Protein expression was detected using anti-iPLA 2 β (polyclonal, PA5–27945, Thermo Fisher Scientific), anti-tyrosine hydroxylase (ab112, abcam), anti- α -synuclein (SC-7011-R, Santa Cruz), anti-4-HNE (ab46545, abcam), anti-LPCAT3 (ProSci, 16–999; 1:500 dilution), anti-GAPDH (FD0063, Fude Biotech), and anti-β-actin (mouse monoclonal, A3854, clone AC-15, Sigma-Aldrich) antibodies (1% BSA in PBST) overnight at RT, washed 3 times, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody-goat anti-rabbit IgG (A0545, Sigma-Aldrich, FDR07, Fude Biotech) and goat anti-mouse IgG (FDM07, Fude Biotech) (1 hr) in blocking solution before developing with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Techniques: Concentration Assay, Western Blot, Two Tailed Test, Staining, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy

Journal: Nature chemical biology

Article Title: Phospholipase iPLA 2 β Averts Ferroptosis By Eliminating A Redox Lipid Death Signal

doi: 10.1038/s41589-020-00734-x

Figure Lengend Snippet: a , Representative immunoblots and quantification of LPCAT3 in cell treated with non-targeted siRNA (si-NT) or LPCAT3 siRNA (si-LPCAT3). LPCAT3 levels were quantified from three biological replicates and normalized to actin. Data represent mean ± s.d., *p=0.0004 vs si-NT, unpaired two-tailed t -test. b , si-NT or LPCAT3 KD cells were exposed to RSL3 (100 nM) and cell death was monitored after 20 hrs by PI staining using flow cytometry. Data are mean ± s.d., N = 3 biologically independent experiments; ****p<0.0001 vs si-NT control, ## p=0.0081 vs.si-LPCAT3 control, $$ p=0.0078 vs si-NT/RSL3, one-way ANOVA. c, Quantitative LC/MS-based assessments of lyso-PE (1-SA-2-OH-PE, left ) and lyso-PC (1-SA-2OH-PC, right ) in MEF cells. Data are mean ± s.d., N=3 biologically independent experiments, ***p=0.0008, ****p<0.0001 vs si-NT, unpaired two-tailed student’s t -test. d, The contents of oxygenated PE (1-SA-2-HpETE-PE, left ) and PC (1-SA-2-15-HpETE-PC, right ) in MEF cells. Cells were exposed to RSL3 (100nM) for 20 hrs. Data are mean ± s.d., N=3 biologically independent experiments, *p=0.0282, ****p<0.0001 vs si-NT control, one-way ANOVA, (Tukey’s post-hoc test).

Article Snippet: Protein expression was detected using anti-iPLA 2 β (polyclonal, PA5–27945, Thermo Fisher Scientific), anti-tyrosine hydroxylase (ab112, abcam), anti- α -synuclein (SC-7011-R, Santa Cruz), anti-4-HNE (ab46545, abcam), anti-LPCAT3 (ProSci, 16–999; 1:500 dilution), anti-GAPDH (FD0063, Fude Biotech), and anti-β-actin (mouse monoclonal, A3854, clone AC-15, Sigma-Aldrich) antibodies (1% BSA in PBST) overnight at RT, washed 3 times, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody-goat anti-rabbit IgG (A0545, Sigma-Aldrich, FDR07, Fude Biotech) and goat anti-mouse IgG (FDM07, Fude Biotech) (1 hr) in blocking solution before developing with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).

Techniques: Western Blot, Two Tailed Test, Staining, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy