necrostatin  (TargetMol)

Journal: Genes & Diseases

Article Title: Non-canonical role of “S6K1–SGK1” pathway in neuronal necroptosis following traumatic brain injury

doi: 10.1016/j.gendis.2025.101876

Figure Lengend Snippet: TSZ induces necroptosis in HT22 cells. (A) LDH assay indicated an increased percentage of cell death following TSZ application, which was reduced by Nec-1 pretreatment before TSZ application. (B) PI staining (red) indicated the increased changes of necrotic cells after TSZ application and the decreased changes of necrotic cells after pretreatment with Nec-1 before TSZ application. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. (C) Statistical results of the PI-positive necrotic cells. (D) Western blotting showed the increased changes of p-MLKL/MLKL and p-RIP3/RIP3 expression in HT22 cells after TSZ application or pretreatment with Nec-1 before TSZ application. (E, F) Statistical results of Western blotting. (G) Representative transmission electron microscopic images showed necroptosis after TSZ treatment. The black arrowhead indicates plasma membrane rupture. The triangle indicates mitochondrial swelling in TSZ-treated HT22 cells. Scale bar: 10 μm. n = 3 or 9. ∗ P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001 versus the CTL group. ## P < 0.01 and #### P < 0.0001 versus the TSZ 3 h group. Two-tailed unpaired Student's t -test was used for Nec-1 comparison.

Article Snippet: Nec-1 (20 μM), PF4708671 (20 μM), and GSK650394 (15 μM) were all from MedChemExpress (New Jersey, USA) and were added 30 min before TSZ injury.

Techniques: Lactate Dehydrogenase Assay, Staining, Western Blot, Expressing, Transmission Assay, Clinical Proteomics, Membrane, Two Tailed Test, Comparison

Journal: Journal of Human Immunity

Article Title: Human ISG15 deficiency unveils impaired healing of ulcerations via type I interferon–mediated fibrosis

doi: 10.70962/jhi.20250011

Figure Lengend Snippet: IFNα induces apoptosis in ISG15 KO cells. (A) Healing/migration ability of WT and ISG15 KO fibroblasts was assessed with the scratch assay. Left; percent confluency was recorded every hour for a total of 93 h. Right; experimental replicates at 48, 72, and 93 h after scratch. (B) Representative images of WT and ISG15 KO fibroblasts at 1 h (left), 49 h (middle), and 92 h (right) of 1,000 IU ml −1 IFNα treatment. White arrows indicate macroscopic evidence of cellular stress. (C) WT and ISG15 KO fibroblasts were treated with IFNα2b (0, 100, 1,000 IU/ml) for 72 h, and cell death was quantified by measuring Annexin V and Propidium Iodide (PI) levels through flow cytometry. (D) Control and ISG15 KO fibroblasts were treated with IFNα2b (0, 100, 1,000 IU/ml) for 72 h following a 30-min pretreatment with necrostatin-1, and cell death was quantified by measuring Annexin V and PI levels through flow cytometry. (E) Metabolic activity of two control and one ISG15 KO fibroblast cell lines was assessed with the Deep Blue assay. Nec-1; necrostatin-1. (F) Control and ISG15-deficient fibroblasts were treated with either IFNα2b (1, 100, 1,000 IU/ml) or chloroquine (0, 25, 50, 100 μM) for 72 h. Cell lysates were analyzed by western blotting for autophagy (LC3B). (G) Control and ISG15-deficient fibroblasts were treated with either IFNα2b (100 IU/ml), or TNFα (5 ng/μl), or both for 72 h, and percent death was assessed with the Deep Blue assay as compared to the nontreated condition for each variant. (H) WT and ISG15 KO fibroblasts were treated with IFNα2b (0, 100, 1,000 IU/ml) for 72 h, and cell death was quantified by measuring cleaved (active) caspase 3 levels through flow cytometry. Source data are available for this figure: . P values were calculated with two-tailed t test. **P < 0.01; ***P < 0.001.

Article Snippet: Relevant samples were pretreated with 10 mM of the RIPK1 inhibitor, necrostatin-1 (S8037; Selleck Chemicals) for 30 min before an 48-h stimulation with 1,000 IU ml −1 IFN-α2b (Merck IntronA).

Techniques: Migration, Wound Healing Assay, Flow Cytometry, Control, Activity Assay, Western Blot, Variant Assay, Two Tailed Test

Journal: Journal of Human Immunity

Article Title: Human ISG15 deficiency unveils impaired healing of ulcerations via type I interferon–mediated fibrosis

doi: 10.70962/jhi.20250011

Figure Lengend Snippet: Cell death markers identified through ST. (A) Left: percentage of spots expressing apoptosis markers ( CASP3 , CASP8 , BAX , BAK1 , and CYCS ) out of total biopsy. Middle and right: percentage of spots expressing either two or five apoptosis markers in total biopsy, epidermis only, or dermis only (respectively). (B) Left: percentage of spots expressing necroptosis markers ( RIPK1 and MLKL ) out of total biopsy. Middle and right: percentage of spots expressing either RIPK1 or MLKL in total biopsy, epidermis only, or dermis only (respectively). (C) Percentage of spots expressing at least one apoptotic marker ( CASP3 , CASP8 , BAX , BAK1 , or CYCS ) that also express or not at least one of the ISGs ( IFIT1 , USP18 , MX1 , IFI27 , IFI44L , or OAS1 ). (D) Percentage of spots out of total biopsy, epidermis only, and dermis only that express ZBP1 . P values were calculated with a two-tailed t test. *P < 0.05; **P < 0.01.

Article Snippet: Relevant samples were pretreated with 10 mM of the RIPK1 inhibitor, necrostatin-1 (S8037; Selleck Chemicals) for 30 min before an 48-h stimulation with 1,000 IU ml −1 IFN-α2b (Merck IntronA).

Techniques: Expressing, Marker, Two Tailed Test

Journal: Redox Biology

Article Title: USP20 governs tyrosine kinase inhibitors resistance through ferroptosis evasion by targeting GPX4 in cancers

doi: 10.1016/j.redox.2026.104086

Figure Lengend Snippet: TKI-resistant cancer cells exhibit reduced ferroptosis susceptibility due to increased GPX4 stability. ( A ) Development of sorafenib-resistant (Sora-R) cell models. Renal carcinoma 769-P cells were subjected to chronic sorafenib exposure (initial 0.5 μM for 5 days, followed by 1 μM dose escalation every 5 days until reaching 10 μM, then cultured in medium with 10 μM sorafenib for 3 month). Surviving populations were designated as Sora-R. The validity of this treatment was assessed in parental and Sora-R cells after 24 h sorafenib treatment using MTT assays. ( B ) The validity of sorafenib-resistant cells with colony formation assay. Sorafenib-resistant and parental cells was cultured for 7 days while treated with indicated concentration of sorafenib. The colony number was measured with Image J. ( C-D ) Parental and Sora-R 769-P cells were treated with 30 μM sorafenib for 6 h after pretreated with Tempol (50 μM), EUK-143 (50 μM), DFO (50 μM), and Fer-1 (1 μM) for 2 h, followed by quantification of total ROS using fluorescence microscope with DCFH-DA probe. Scale bar: 50 μm. ( E ) ROS generation and clearance. ( F ) Parental and Sora-R 769-P cells were treated with 30 μM sorafenib for 6 h, then quantified of GPX4 activity. ( G-H ) Parental and Sora-R 769-P cells were treated with 30 μM sorafenib for 6 h after pretreated with Tempol (50 μM), EUK-143 (50 μM), DFO (50 μM), and Fer-1 (1 μM) for 2 h, followed by quantification of lipid ROS using fluorescence microscope with C11-BODIPY 581/591 probe. Scale bar: 50 μm. ( I ) Parental and Sora-R 769-P cells were treated with 30 μM sorafenib for 6 h after pretreated with DFO (50 μM) and Fer-1 (1 μM) for 2 h, followed by quantification of MDA via ELISA kits. ( J-K ) Parental and Sora-R cells were treated with 30 μM IKE for 6 h, followed by quantification of lipid ROS using fluorescence microscope with C11-BODIPY 581/591 probe. Scale bar: 50 μm. ( L ) 769-P cells were treated with 20 μM sorafenib for 24 h after pretreated with 1 μM Fer-1, 2 μM Lip-1, 50 μM DFO, 10 μM Nec-1, 25 μM Z-VAD-FMK, 25 μM 3-MA, 50 μM CQ, and 10 μM TTM, cell viability was measured via MTT assay. ( M-N ) Immunoblot analysis of key antioxidant and ferroptosis-related proteins in parental and Sora-R cell lines. For A-D and F-L , data are presented as mean ± s.d. of n = 3 biological replicates.

Article Snippet: T2490), Afatinib (cat. no. T21312 ), GSK2643943A (cat. no. T11485 ), Tempol (cat. no. T6699), EUK-134(cat. no. T6495), DFO (cat. no. T18971 ), Nec-1 (cat. no. T1847), Z-VAD-FMK (cat. no. T 7020), 3-MA (cat. no. T1879), TTM (cat. no. T77774 ), Lip-1 (cat. no. T2376), and MTT (cat. no. T19029 ) were purchased from TargetMol.

Techniques: Cell Culture, Colony Assay, Concentration Assay, Fluorescence, Microscopy, Activity Assay, Enzyme-linked Immunosorbent Assay, MTT Assay, Western Blot