treatment with chx  (MedChemExpress)

Journal: Cell Discovery

Article Title: TGM2-mediated serotonylation of GPX4 confers ferroptosis resistance to promote gastric tumorigenesis

doi: 10.1038/s41421-026-00885-6

Figure Lengend Snippet: a HEK293T cells transfected with GPX4 WT or GPX4 2A were treated with 5-HT (100 μM) and analyzed using CHX (50 μM) chase experiments at different time points. CHX chase assays were performed to analyze the protein stability of GPX4 WT and GPX4 2A in the absence (left) or presence (right) of a ferroptosis inducer (RSL3). b Quantification of GPX4 protein levels (Flag) from Panel ( a ). c Western blot showing the stability of HEK293T cells transfected with GPX4 WT or GPX4 2A . Cells were treated with 50 μg/mL CHX for the indicated duration (0 h or 4 h) in the absence or presence of the proteasome inhibitor MG132 (10 μM), the lysosomal acidification inhibitor NH₄Cl, or the autophagy inhibitor chloroquine (CQ). d Relative levels of Flag-GPX4 and its mutants in the CHX chase assay ( c ). e HEK293T cells transfected with WT or the GPX4 2A mutant were co-transfected with HA-Ub for 24 h before being harvested. Ubiquitinated GPX4 was detected with an anti-Flag antibody. f Myc-TRIM25 overexpression in HEK293T cells co-transfected with Flag-tagged GPX4 WT or GPX4 2A mutants. In the absence (left) or presence (right) of RSL3, the interaction between TRIM25 and GPX4 was detected by immunoprecipitation (IP) with an anti-Flag antibody and western blotting for Myc. g Western blot validation of exogenous GPX4 transfection efficiency in GPX4-KO AGS cells. Immunofluorescence images (bottom) of AGS cells stably expressing GPX4 WT or GPX4 2A . Cells were stained with DAPI (blue), GFP (green), and Flag-GPX4 (red). Scale bars, 10 μm. h The relative viability of AGS cells transfected with GPX4 WT or GPX4 2A was measured following treatment with 5-HT (10 μM) and RSL3 (500 nM) or ML-210 (5 μM) for 24 h ( n = 3–5). i Western blot analysis validating the expression of exogenous Flag-tagged GPX4 (WT) and the Q2A mutant in GPX4-silenced MFC cells (shGPX4). The specific expression of exogenous and endogenous proteins was detected using an anti-GPX4 antibody. j Schematic diagram of the workflow for analyzing subcutaneous MFC tumors (5 × 10 6 cells) in nude mice treated with or without 5-HT (10 mg/kg) for 12 days. Insert: Representative images of subcutaneous tumors are shown on the bottom. k Weights of subcutaneous MFC tumors in nude mice ( n = 4 per group). Data are presented as mean ± SD from two or three independent experiments. Statistical analysis was performed using two-way ANOVA ( b , d ) or ordinary one-way ANOVA ( h , k ).

Article Snippet: The GPX4 (WT) and GPX4 (mutant) expression plasmids were transfected into HEK293T cells for 48 h, followed by treatment with CHX (MCE, HY-12320) at the indicated time points.

Techniques: Transfection, Western Blot, Mutagenesis, Over Expression, Immunoprecipitation, Biomarker Discovery, Immunofluorescence, Stable Transfection, Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: A Coordinated Translational Control Mediated by eEF2 Phosphorylation Safeguards Erythroid Differentiation

doi: 10.3390/ijms26104801

Figure Lengend Snippet: Inhibition of protein synthesis restrains erythroid differentiation. ( A ) Experimental scheme. Isolated BM cells were cultured in vitro for 30 h in a medium containing erythropoietin (EPO) and stem cell factor (SCF) to induce erythroid differentiation, with cycloheximide (CHX) treatment applied during the final 6 h. ( B ) Protein synthesis rates in differentiated CD71 + Ter119 + cells were measured by OP-Puro intensity after CHX treatment as described in ( A ) at the indicated concentrations ( n = 4). ( C , D ) Representative flow cytometric profiles ( C ) and quantification of the percentages ( D ) of CD71 + Ter119 + and enucleated erythroblasts cultured as described in ( A ) at the indicated CHX concentrations ( n = 3). ( E ) Ex vivo detection of protein synthesis rates in BM CD71 + Ter119 + cells from WT and eEF2 +/− mice ( n = 4). ( F ) Representative flow cytometric profiles (left) and quantification of the percentage (right) of BM CD71 + Ter119 + erythroblasts from WT and eEF2 +/− mice ( n = 4). ( G ) Quantification of the absolute numbers of BM CD71 + Ter119 + and CD71 − Ter119 + cells from WT and eEF2 +/− mice ( n = 4). ( H ) Quantification of the percentage of BM CD71 + Ter119 + cells from WT and eEF2 +/− mice following in vitro differentiation for 24 or 48 h ( n = 5). Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001. Statistical significance was assessed relative to the control group (CHX: 0 µg/mL) in ( B , D ).

Article Snippet: Murine bone marrow cells were subjected to cycloheximide (CHX) treatment (0.5 μg/mL; Selleck, Houston, TX, USA) during the final 6 h of a 30 h in vitro culture with EPO and SCF.

Techniques: Inhibition, Isolation, Cell Culture, In Vitro, Ex Vivo, Control