Journal: The Journal of Cell Biology

Article Title: Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

doi: 10.1083/jcb.201506048

Figure Lengend Snippet: TRIM3 polyubiquitylates ACTG1. (A–D) TPA stimulation induces ACTG1 polyubiquitylation by TRIM3 in HEK293 cells. HEK293 cells were transfected with TRIM3 or ΔRBCC-TRIM3. After incubation for 4 h in the presence of MG132, cells were lysed and ACTG1 was immunoprecipitated from the lysates, resolved on SDS-PAGE, and immunoblotted. Blots were first stained for polyubiquitin (A) and then stripped and restained for ACTG1 (B). Under these basal conditions, only unmodified ACTG1 was detected. However, when cells were incubated in the presence of MG132 and TPA, a significant increase in high-molecular-weight polyubiquitylated forms of ACTG1 (indicated with *) was observed specifically in TRIM3-transfected cells (C–E; means ± SEM, two-tailed t test, *, P < 0.05, n = 3 cultures per condition). (F) TPA induces ACTG1 polyubiquitylation by TRIM3 in hippocampal neurons. Cultured hippocampal neurons from wild-type and Trim3 −/− mice were treated with MG132 and TPA for 4 h. Lysates were immunoblotted and stained for ACTG1. TPA induced the appearance of multiple high molecular weight (50–100 kD) bands (indicated with *) consistent with polyubiquitylation and specifically in neurons from wild-type mice and not from Trim3 −/− mice. (G) Actg1 mRNA is present in TRIM3/PURA containing mRNP granules. mRNP granules were immunoprecipitated from hippocampal lysates with antibodies against TRIM3 or PURA. mRNA was isolated from immunoprecipitates, reverse transcribed into cDNA, and used for real-time quantitative PCR. Actg1 and Actb mRNA was detected at equal levels in all precipitates, whereas the negative control mRNA Slc1a3 was ∼10-fold lower detected (means ± SEM, n = 3 immunoprecipitations per condition).

Article Snippet: After 14 d in culture, neurons were either lysed in SDS loading buffer directly or first incubated with 20 μM MG132 (Tocris Bioscience) and 500 nM TPA (Sigma-Aldrich) for 4 h and then lysed.

Techniques: Transfection, Incubation, Immunoprecipitation, SDS Page, Staining, High Molecular Weight, Two Tailed Test, Cell Culture, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

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: 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

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: HPV E6/E7-Induced Acetylation of a Peptide Encoded by a Long Non-Coding RNA Inhibits Ferroptosis to Promote the Malignancy of Cervical Cancer.

doi: 10.1002/advs.202414018

Figure Lengend Snippet: Figure 7. Acetylated TUBORF promotes IRGQ protein degradation to suppress ferroptosis and enhance the malignant proliferation of cervical cancer cells. A) Mass spectrometry analysis of the differential binding proteins in TUBORF-WT- and TUBORF-K(10/16)R-overexpressing HeLa cells. B) IP assay of the interaction between TUBORF-K(10/16)R and ZNF324A, LRRFIP2, or IRGQ with the anti-Flag antibody in HeLa cells. C) IP assay of the interaction between IRGQ and TUBORF-K(10/16)R or TUBORF-K(10/16)Q with anti-Flag antibody in HeLa cells. D) IRGQ-Myc-overexpressing cells were transduced with lentiviral TUBORF-K(10/16)R-Flag (TUBORF-KR-Flag) or pHAGE. An immunoprecipitation assay was performed to examine the interaction between IRGQ and TUBORF-KR with the anti-Flag antibody in HeLa cells. E) TUBORF-K(10/16)R-Flag-expression cells (TUBORF-KR-Flag) were co-transduced with IRGQ-Myc or its control pHAGE. An immunoprecipitation assay was performed to examine the interaction between IRGQ and TUBORF-KR with the anti-Myc antibody in HeLa cells. F) HeLa cells transduced with wild-type TUBORF (TUBORF-WT-Flag) or K10/16R mutant TUBORF (TUBORF-KR- Flag) were treated with CHX (20 μg mL−1) for 0, 3, 6 and 9 h, respectively. Western blotting was performed to examine IRGQ expression. G) HeLa cells transduced with wild-type TUBORF (TUBORF-WT-Flag) or K10/16R mutant TUBORF (TUBORF-KR-Flag) were treated with MG132 (10 μm) for 8 h. Western blotting was performed to detect IRGQ expression. H) HeLa cells treated as in (G) were co-transfected with the HA-Ub and IRGQ-Myc constructs. An immunoprecipitation assay was performed to examine the level of IRGQ ubiquitination with the anti-Myc antibody. I) Western blotting analysis of the IRGQ protein level in lentiviral sgHPV E6-1 (sgHPV E6-1), sgHPV E6-2 (sgHPV E6-2), or its control (Lenti-V2) transduced HeLa cells. J) Western blotting analysis of the IRGQ protein level in lentiviral sgHPV E7-1 (sgHPV E7-1), sgHPV E7-2 (sgHPV E7-2), or its control (Lenti-V2) transduced HeLa cells. K) Western blotting was performed to examine the IRGQ protein level in C-33A cells transfected with HPV E6 construct (HPV E6) or its control

Article Snippet: The cells were respectively treated with 20 μg mL−1 Cycloheximide (CHX) (Cell Signal Technology) for different time-points, proteasome inhibitor MG132 (Selleck) at 10 μm for 6 h, ferroptosis inducer Erastin (MCE) for 24 h, or CBP/p300 catalytic inhibitor A-485 at 10 μm for 24 h. siRNA oligonucleotides targeting HPVs E6/E7 were purchased from GenePharma as previously described.

Techniques: Mass Spectrometry, Binding Assay, Transduction, Immunoprecipitation, Expressing, Control, Mutagenesis, Western Blot, Transfection, Construct, Ubiquitin Proteomics