Journal: Free radical biology & medicine
Article Title: Redox regulation of cell migration via Nischarin S-glutathionylation
doi: 10.1016/j.freeradbiomed.2025.11.013
Figure Lengend Snippet: ( a ) A scheme of the G-PROV approach that enables functional analysis of glutathionylation on the protein of interest (POI): dehydroglutathione (dhG) is used to introduce a non-reducible glutathione modification on the purified engineered NISCH construct (enNISCH). Subsequently, the glutathione-modified NISCH construct is delivered to cells via fusogenic liposome, where the effect of glutathionylation on NISCH can be analyzed. ( b ) dhG modification on purified enNISCH. Purified enNISCH WT and C185S were incubated with dhG. The dhG-mediated glutathione modification was analyzed by glutathione antibody (n = 2). ( c ) Analysis of enNISCH delivered to MDA-MB-231 cells via fusogenic liposome. After incubating fusogenic liposomes containing His-enNISCH constructs (modified or non-modified by dhG) for 2 h, lysates were analyzed by Western blot (n = 2). ( d-e ) The migration and invasion assays. After incubating fusogenic liposomes for 2 h, MDA-MB-231 cells were analyzed by the wound-healing migration ( d , n = 6) and transwell invasion ( e , n = 2) assays. ( f ) The colony formation by enNISCH constructs. Fusogenic liposomes containing enNISCH WT or C185S were incubated with MDA-MB-231 cells on plates, and the number of colonies formed was counted after 15 days. Cells were incubated in DMEM containing 25 mM Glc ( d – e ) or 5 mM Glc ( f ). The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( b – e ) or two-way ANOVA and Sidak’s post-hoc test ( f ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.
Article Snippet: For validation of protein delivery, cells were lysed and analyzed by western blotting using anti-HIS antibody (Cell Signaling Cat #2365).
Techniques: Functional Assay, Introduce, Modification, Purification, Construct, Incubation, Liposomes, Western Blot, Migration
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