Journal: Cell death & disease

Article Title: Conjugated fatty acids drive ferroptosis through chaperone-mediated autophagic degradation of GPX4 by targeting mitochondria.

doi: 10.1038/s41419-024-07237-w

Figure Lengend Snippet: Fig. 3 CLA/CLNAs promote lysosomal degradation of GPX4. A, B Immunoblot of lysates from HT1080 cells treated with 10-CLA (200 µM) (A) or ESA (20 µM) (B) for indicated time using antibodies against GPX4 and β-actin. Images are cropped for clarity; full-length blots are presented in Supplementary Fig. 13A, B. C HT1080 cells were treated with 10-CLA (200 µM) or ESA (20 µM) for 4 h, and relative mRNA level of GPX4 was measured by qRT-PCR analysis and shown as mean ± SD (n = 3). D–G Immunoblot of lysates from HT1080 cells pretreated with chloroquine (CQ: 20 µM) (D, E) or MG132 (5 µM) (F, G) for 0.5 h, and treated with 10-CLA (200 µM) (D, F) or ESA (20 µM) (E, G) for indicated time, using antibodies against the indicated proteins. Images are cropped for clarity; full-length blots are presented in Supplementary Fig. 13C–F.

Article Snippet: All reagents were obtained from commercial suppliers: 1S,3R-RSL3 (#SML2234), Ferrostatin-1 (#SML0583) (Sigma, Burlington, MA, USA), mitoTEMPO (#sc-221945), Necrostatin-1 (#sc-200142), MG132 (#sc-201270) (Santa Cruz, Dallas, TX, USA), z-VAD-fmk (#3188-v) (Peptide Institute, Osaka, Japan), Necrostatin-1s (7-Cl-O-Nec-1, #S8641), Rucaparib (#S4948) (Selleck, Houston, TX, USA), Liperfluo (#L248), mitoPeDPP (#M466), FerroOrange (#F374) (Dojindo, Kumamoto, Japan), LipiRADICAL Green (#FDV-0042) (Funakoshi, Tokyo, Japan), MitoSOX (#M36008) (Invitrogen, Waltham, MA, USA), Erastin (#17754), Triacsin C (#10007448), PF-04620110 (iDGAT1, #16425), PF06424439 (iDGAT2, #17680), deferoxamine (DFO, #14595), Perhexiline (#16982), 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oate (CDDO, #81035) (Cayman, Ann Arbor, MI, USA), Chloroquine (#08660-04), Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP, #07253-74) (Nacalai Tesque, Kyoto, Japan).

Techniques: Western Blot, Quantitative RT-PCR

Journal: PLOS Pathogens

Article Title: A novel SO 2 probe inhibits lysophagy induced by Senecavirus A infection by promoting LAMP1 Cys375 sulfenylation

doi: 10.1371/journal.ppat.1013932

Figure Lengend Snippet: (A) The ubiquitination level of LAMP1 treated with CQ (10 μM) and MG132 (50 nM) before and after SVA infection. (B,C) Effect of CQ (50 μM) and MG132 (50 nM) on LAMP1 protein levels before and after SVA infection. (D,E) Effects of DLC alone or in combination with MG132 (50 nM) on LAMP1 protein levels after SVA infection. (F) Effect of DLC on the interaction between endogenous TRIM16 and Gal3 in SVA infected cells by Co-IP (After Gal protein was immunoprecipitated, Gal3 and TRIM16 proteins were respectively used for detection). (G,H) Colocalization changes of TRIM16 and Gal3 in cells with and without DLC (5 μM) after SVA infection (0.01MOI, 24 hpi) and quantitative analysis.(*p < 0.05, **p < 0.01, ***p < 0.001, n = 3).

Article Snippet: Chemical reagents LAMP1, MG132 (HY-13259), chloroquine (CQ; HY-17589A), L-Aspartic acid β-hydroxamate (HY-134450), Stretavidin Agarose 6FF (HY-K0218A), were purchased from MedChemExpress; Lysosomal protein extraction kit(Best bio, BB-31452); Cytoplasmic protein extraction kit(Best bio, BB-3113); DLC, CIJ and BTD were synthesized by the research group of Baoxiang Zhao, College of Chemistry, Shandong University.

Techniques: Ubiquitin Proteomics, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: The Journal of Immunology Author Choice

Article Title: Resolvin D1 Improves the Resolution of Inflammation via Activating NF-κB p50/p50–Mediated Cyclooxygenase-2 Expression in Acute Respiratory Distress Syndrome

doi: 10.4049/jimmunol.1700315

Figure Lengend Snippet: RvD1 facilitates the resolution of inflammation by promoting NF-κB p50–derived COX-2 expression in the ARDS model. Rats were i.v. injected with LPS (3 mg/kg) 12 h before they received tail vein injections with RvD1 (5 μg/kg) or an equivalent volume of ethanol. For treatment, rats were i.v. administered MG-132 at 1 h prior to RvD1 injection, whereas other groups received an equal volume of a DMSO saline solution. Rats were sacrificed 24 h after LPS exposure. Lung tissue samples were collected for morphological evaluation, Western blot, and EMSA supershift assays. (A) Representative photomicrographs of pulmonary histology, as shown by H&E staining. Original magnification, ×100 (inset, ×400). Black arrows indicate LPS-induced thickening of the alveolar walls and red arrows show neutrophil infiltration. (B) Acute lung injury score. (C) COX-2 protein expression was assessed via Western blot and analyzed by densitometry. Values were compared with β-actin expression. (D) NF-κB activation was determined via EMSA supershift assays with the nuclear protein extracts prepared from the lung tissues. EMSAs were used to quantify NF-κB activation in each group. Supershift assays were used to identify the activated NF-κB dimers in the LPS plus RvD1 group. EMSA supershift assays were performed as detailed in Materials and Methods. The NF-κB DNA–protein complexes (open arrow) were separated from the free probe (closed arrow) by electrophoresis in the EMSA. A supershift analysis was conducted with Abs directed against p50 and p65 to characterize specific NF-κB complexes in the LPS plus RvD1 group. Supershifted bands with an asterisk indicate binding of the p50 subunit. CK, negative control; CN, control group; L, LPS group; L+M, LPS plus MG-132 group; L+R, LPS plus RvD1 group; L+R+M, LPS plus RvD1 plus MG-132 group; M, MG-132 group; R, RvD1 group; 100×, competition group. Data are shown as mean ± SEM, n = 5–7 rats per treatment per group, and are representative of at least three independent experiments. *p < 0.05 compared with the control group; &p < 0.05 compared with the LPS group; #p < 0.05 compared with the LPS plus RvD1 group. **p < 0.01, ****p < 0.0001.

Article Snippet: After 24 h of culture with LPS (1 μg/ml) or a control medium, fibroblasts were treated with 100 nM RvD1 or a vehicle solution (0.1% ethanol, as the RvD1 was supplied in ethanol) for an additional 24 h. SP600125 (10 μM), SB603580 (10 μM), MG-132 (10 μM), and BOC-2 (10 μM) were added 30 min prior to RvD1 administration.

Techniques: Derivative Assay, Expressing, Injection, Western Blot, Staining, Activation Assay, Electrophoresis, Binding Assay, Negative Control

Journal: The Journal of Immunology Author Choice

Article Title: Resolvin D1 Improves the Resolution of Inflammation via Activating NF-κB p50/p50–Mediated Cyclooxygenase-2 Expression in Acute Respiratory Distress Syndrome

doi: 10.4049/jimmunol.1700315

Figure Lengend Snippet: NF-κB is responsible for promoting the proresolving COX-2 protein expression in pulmonary fibroblasts. Primary pulmonary fibroblasts were incubated with 1 μg/ml LPS (L) for 24 h followed by administration of 100 nM RvD1 or vehicle (0.1% ethanol) for an additional 24 h, and 10 μM SP600125 (a JNK inhibitor), 10 μM SB203580 (a p38 MAPK inhibitor), and 10 μM MG-132 (an NF-κB inhibitor) were added 30 min prior to RvD1 administration. After incubation, the cells were harvested and sonicated. The expression of COX-2 was determined via Western blot. Cells were differentiated from lung tissues harvested from six rats per condition; n = 8 per treatment per group. Data are shown as mean ± SEM and are representative of at least four independent experiments. **p < 0.01, ***p < 0.001.

Article Snippet: After 24 h of culture with LPS (1 μg/ml) or a control medium, fibroblasts were treated with 100 nM RvD1 or a vehicle solution (0.1% ethanol, as the RvD1 was supplied in ethanol) for an additional 24 h. SP600125 (10 μM), SB603580 (10 μM), MG-132 (10 μM), and BOC-2 (10 μM) were added 30 min prior to RvD1 administration.

Techniques: Expressing, Incubation, Sonication, Western Blot