Journal: iScience

Article Title: HMOX1 drives dihydroartemisinin-sensitized ferroptosis antagonized by mitochondrial fusion

doi: 10.1016/j.isci.2025.114382

Figure Lengend Snippet: DHA increases sensitivity to ferroptosis by modulating cellular oxidative stress (A–E) Western blot and quantifications of the GPX4, ACSL4, FSP1, DHODH, β-actin, and α-tubulin expression in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h. (F) GSH levels in N27 cells treated with different doses of DHA (0, 1.5 μM, 12.5 μM) for 12 h were detected, n = 6 wells from one representative of two independent experiments. (G) Western blot and quantifications of the 4-HNE and β-actin expression in N27 cells treated with different doses of DHA (0, 1.5, 12.5, and 25 μM) for 12 h. (H) N27 cells were treated with different doses of DHA (0, 1.5, and 12.5 μM) for 12 h and lipid ROS was detected by C11-BODIPY using flow cytometry. Representative histograms for fluorescence of oxidized C11-BODIPY and the ratio of the MFI of oxidized to reduced C11-BODIPY are shown. (I) TEM of N27 cells treated with DHA (12.5 μM) for 12 h. Red arrows indicate shrunken mitochondria. Scale bars: upper panel = 2 μm; lower panel = 500 nm, as indicated. (J) The average fluorescence intensity of FerroOrange in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h, n = 5 wells from one representative of two independent experiments. (K) The iron levels detected by ICP-MS in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h, n = 5 wells from one representative of two independent experiments. (L) Cell viability of N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) in the absence or presence of DFO (50 μM) for 48 h, n = 6 wells from one representative of two independent experiments. (M) Cell viability of N27 cells treated with DHA (12.5 μM) in the absence or presence of DFO (50 μM) for 48 h, n = 6 wells from one representative of two independent experiments. (N) The representative images of H 2 DCFDA staining in N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h, and the average fluorescence intensity are shown. Scale bars, 200 μm, as indicated. (O) N27 cells were treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h and detected by CellROX Green using flow cytometry. Representative histograms for fluorescence of CellROX Green and the average fluorescence are shown, n = 6 wells from one representative of two independent experiments. (P) The representative images of H 2 DCFDA staining in N27 cells treated with DHA (1.5 μM), RSL-3 (100 nM), and NAC (1 mM) for 12 h, and the average fluorescence intensity are shown. Scale bars, 200 μm, as indicated. (Q) Cell viability of N27 cells treated with NAC and DHA (1.5 μM) and RSL-3 (100 nM) for 48 h, n = 6 wells from one representative of two independent experiments. (R) The representative images of H 2 DCFDA staining in N27 cells treated with DHA (1.5 μM), RSL-3 (100 nM), and DFO (50 μM) for 12 h, and the average fluorescence intensity are shown. Scale bars, 200 μm, as indicated. Data are means ± SEM, n = 3 wells from one representative of two independent experiments unless specified. One-way ANOVA was performed.

Article Snippet: The cellular ROS levels were measured by H 2 DCFDA (HY-D0940, MedChemExpress).

Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, Staining

Journal: iScience

Article Title: HMOX1 drives dihydroartemisinin-sensitized ferroptosis antagonized by mitochondrial fusion

doi: 10.1016/j.isci.2025.114382

Figure Lengend Snippet: DHA promotes ferroptosis through the activation of HO-1 (A) Venn’s analysis used the data from the GSE162550 dataset and the GSE214030 dataset, and a set of genes related to oxidative stress from the Gene Ontology knowledgebase. Genes with an absolute log 2 fold change greater than 2 (log 2 FC > 2 or log 2 FC < −2) and a p value <0.05 were considered significantly differentially expressed. (B) Volcano plot of the DEGs in the GSE162550 dataset and the GSE214030 dataset. (C–E) Western blot and quantifications of the HO-1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin expression in N27 cells treated with different doses of DHA (0, 1.5, 12.5, and 25 μM) or treated with DHA (1.5 μM) and RSL-3 (100 nM) for 12 h. (F) N27 cells were transfected with two selected HO-1 siRNAs or a negative control siRNA, or FAM siRNA for 48 h, and the protein levels of HO-1 and GAPDH were detected by western blot. Cell viability of N27 cells treated with DHA (1.5 μM) and RSL-3 (100 nM) in the absence or presence of HO-1 siRNA for 48 h, n = 6 wells from one representative of two independent experiments. (G) Western blot and quantifications of the HO-1 and β-actin expression in N27 cells treated with DHA (1.5 μM), RSL-3 (100 nM), and ZnPP (5 μM) for 12 h. (H) Cell viability of N27 cells treated with ZnPP and DHA (1.5 μM) and RSL-3 (100 nM) for 48 h, n = 6 wells from one representative of two independent experiments. (I) The representative images of H 2 DCFDA staining in N27 cells treated with DHA (1.5 μM), RSL-3 (100 nM), and ZnPP (5 μM) for 12 h, and the average fluorescence intensity are shown. Scale bars, 200 μm, as indicated. Data are means ± SEM, n = 3 wells from one representative of two independent experiments unless specified. One-way ANOVA was performed.

Article Snippet: The cellular ROS levels were measured by H 2 DCFDA (HY-D0940, MedChemExpress).

Techniques: Activation Assay, Western Blot, Expressing, Transfection, Negative Control, Staining, Fluorescence