Journal: Cell Death and Differentiation

Article Title: MS4A15 drives ferroptosis resistance through calcium-restricted lipid remodeling

doi: 10.1038/s41418-021-00883-z

Figure Lengend Snippet: A Enrichment of MS4A15-FLAG co-immunoprecipitated proteins in HEK293T cells as determined by label-free proteomic quantification. Mean abundance ratios were calculated compared to GFP-expressing cells incubated with anti-FLAG as a control. Dotted horizontal line indicates significance (paired t -test, p < 0.05). B Single sample Gene Set Enrichment Analysis (GSEA) correlation analysis in primary lung tumors between MS4A15 and Ca 2+ transmembrane transporters (RSEM, RNA-Seq by Expectation-Maximization). Significance was evaluated by Pearson correlation. C Western blot of IP 3 R1 protein in Ms4a15 OE and control cells. Vinculin is given as loading control. D Schematic of calcium related processes in ( E – G ). Activation of G protein-coupled receptors (GPCRs) such as Bradykinin receptor stimulates phospholipase C (PLC) cleavage of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to yield IP 3 , triggering Ca 2+ release from the endoplasmic reticulum (ER). Thapsigargin (Tgn) blocks SERCA-mediated ER Ca 2+ uptake, while ionophore catalyzes nonspecific store release in Ca 2+ free medium. Membrane channels mediate uptake following re-addition of CaCl 2 -containing medium. E Calcium levels detected by cytosolic sensor GCaMP6s using flow cytometry (normalized fluorescence, ex488/em530 nm). Top panels: ER Ca 2+ release mediated by 50 nM Bradykinin (∆) or 5 μM Ionophore (^) in Ms4a15 OE compared to control cells in Ca 2+ -free buffer. Bottom panels: control cells pretreated with 50 nM Tgn for 3 h. Addition of 2 mM CaCl 2 (▲). Data shown are representative results of three independent repetitions performed in triplicate with similar outcomes. Fluorescent images were acquired 30 s following Bradykinin stimulation for respective genotypes. F Time-dependent (0 h–14 days) effect of Tgn pretreatment on lipid peroxidation detected by BODIPY-C11 induced by RSL3 (0.3 μM for 3 h) in control cells (left panels) compared to DMSO. A typical FACS histogram of three independent repetitions is depicted. Viability of control cells pretreated with 2.5 nM Tgn for 7 days or 14 days prior to RSL3 induction (untreated, 0 days). Fluorescent images were acquired 30 s following Bradykinin stimulation for 14 d treated cells. G Dose-dependent sensitization of Ms4a15 OE cells to RSL3 by overexpressing Serca2 ( Ms4a15 OE + Serca2 OE) or empty virus control ( Ms4a15 OE + control) in Ms4a15 OE cells (left panel). Restoration of Ca 2+ dynamics is indicated by Bradykinin (right panels). Insets show SERCA2 expression by western and viability (PI%) measurements in respective cell lines. Viability data are representative mean ± SD of n = 4 ( F ) or n = 3 ( G ) replicates for experiments repeated independently at least three times. Curve p -values of two-way ANOVA comparisons are shown. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Hybridoma supernatants were screened in a bead-based flow cytometry assay (iQue, Intellicyte; Sartorius) on his-tagged, biotinylated peptide captured on streptavidin beads (PolyAN) and incubated for 90 min with hybridoma supernatant and Atto 488-coupled isotype-specific monoclonal mouse-anti-rat IgG secondary antibodies.

Techniques: Immunoprecipitation, Expressing, Incubation, RNA Sequencing Assay, Western Blot, Activation Assay, Membrane, Flow Cytometry, Fluorescence, Virus

Journal: Cell Death and Differentiation

Article Title: MS4A15 drives ferroptosis resistance through calcium-restricted lipid remodeling

doi: 10.1038/s41418-021-00883-z

Figure Lengend Snippet: A Antioxidant activity of plasmalogens (50 parts per million, ppm) “e MUFA” (P-18:0/18:1) PC or “e PUFA” (P-16:0/20:4) PE and 3 ppm ferrostatin-1 (Fer-1) on BODIPY-C11 oxidation in the presence of 50 ppm ester lipids (PE 18:0/22:6 and PE 16:0/20:4) in 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH). Fer-1 is given as control. Significance was evaluated by two-tailed t -test. B Peak area stability (LC-MS²) of PUFA ester lipids (PE 16:0/20:4) in presence of plasmalogens in 2,2’-Azobis(2-amidinopropane) dihydrochloride (AAPH). C Cell viability of control cells incubated with 25 µM plasmalogens (e MUFA and e PUFA) or EtOH for 8 h then challenged with 0.3 µM RSL3 in the presence of αToc in a dose-dependent manner. D Viability of Ms4a15 OE cells pretreated with PUFAs eicosapentaenoic acid (C20:5, EPA), docasapentaenoic acid (C22:5, DPA), and doxosahexaenoic acid (C22:6, DHA) with ferroptosis induction by 2 μM RSL3 and αToc rescue. Significance was evaluated by two-tailed t -test. E RSL3 treatment of 72 h siRNA knockdown of Scd1 , Fads2 , or Hsd17b12 compared to siGFP in control cells as individual experiments (left panel) or all three siRNAs together (3x siRNA, right panel). Inset shows relative gene expression by qPCR (rel. mRNA). F Heatmap showing dysregulation of genes involved in lipid droplet formation. G BODIPY 493/503 staining of lipid droplets of Ms4a15 OE, control and 14 d Tgn-treated cells. High-content images (upper) showing lipid droplet dispersion. Quantification of lipid droplet number (LDs/cell) and area (μm 2 /cell) was performed by Harmony software (PerkinElmer). H Analysis of average lipid droplet number and area (left) and fluorescence intensity (right). Data were obtained from three independent experiments and a representative experiment shown with analysis by Harmony software. Lipid droplet intensity is depicted via a flow cytometry histogram of a representative experiment of three independent repetitions. Significance was evaluated by two-tailed t -test. Cell-free assay and viability assays are reported as mean ± SD of n = 3 ( A , C , E ) or n = 4 ( D ) technical replicates of three independent experiments with similar outcomes. Curve statistics, p -values of two-way ANOVA, shown above comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Hybridoma supernatants were screened in a bead-based flow cytometry assay (iQue, Intellicyte; Sartorius) on his-tagged, biotinylated peptide captured on streptavidin beads (PolyAN) and incubated for 90 min with hybridoma supernatant and Atto 488-coupled isotype-specific monoclonal mouse-anti-rat IgG secondary antibodies.

Techniques: Antioxidant Activity Assay, Two Tailed Test, Liquid Chromatography with Mass Spectroscopy, Incubation, Expressing, Staining, Dispersion, Software, Fluorescence, Flow Cytometry, Cell-Free Assay

Journal: Molecular oncology

Article Title: The miR-200 family differentially regulates sensitivity to paclitaxel and carboplatin in human ovarian carcinoma OVCAR-3 and MES-OV cells.

doi: 10.1016/j.molonc.2015.04.015

Figure Lengend Snippet: Figure 1 e Characterization of OVCAR-3 GFP and OVCAR-3/TP GFP cell lines. (A) The cells were treated with different concentrations of paclitaxel (left panel) and carboplatin (right panel) during 72 h. Cells survival was measured by SRB assay. All data are expressed as the average percentage of survival values relative to an untreated control ± SD with significance determined between the indicated cell lines per paclitaxel or carboplatin concentration tested (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) The constitutive expression of miR-200 family members was determined by real time PCR 48 h after cell seeding, using the RNU6 gene as an internal loading control, and calculating the ratio of parental to resistant cells. (C) The constitutive expression of CDH1, FN1 and VIM was measured in cells by using real time PCR 48 h after seeding. All data are expressed as the average of at least three measurements. Significance was determined between the OVCAR-3/TP GFP compared to the OVCAR-3 GFP cell line (*, P < 0.05; **, P < 0.01; ***, P < 0.001), (B and C). (D) EMT marker proteins were measured in cells 48 h after seeding using flow cytometry, and representative histograms of 10,000 events per cell line for each channel (E-cadherin-PE, Vimentin-Brilliant Violet 421, and Fibronectin-APC) are shown. Results for OVCAR-3 GFP cells are shown in orange and OVCAR-3/TP-GFP cells in blue.

Article Snippet: For intracellular staining, cells were exposed to Flow Cytometry Fixation Buffer (R & D Systems) for 20 min at 4 C in the dark, followed by the addition of Flow Cytometry Permeabilization buffer for 5 min at room temperature (R & D Systems).

Techniques: Sulforhodamine B Assay, Control, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Marker, Cytometry