Journal: bioRxiv
Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation
doi: 10.64898/2026.05.01.722049
Figure Lengend Snippet: ( a ) ARHGEF7 (human isoform-a, 646 residues, NM_001113513) domains, structure, and selected residues. The structure is an AlphaFold model (AF-Q14155-1-F1-model_v6_isoform_a) of ARHGEF7 showing the structured SH3, DH, and PH domains with cysteines and nearby residues. ( b ) Accessible surface areas (ASA) of cysteines in ARHGEF7. ( c ) Sequence alignments around C312 in ARHGEF7 orthologs. ( d ) Clickable glutathione approach. Cells expressing a glutathione synthetase mutant (GS M4) synthesize and produce clickable glutathione (N 3 -GSH) using endogenous γGlu-Cys and exogenous azido-Ala. Clickable glutathione forms S-glutathionylation with proteins, which are analyzed after click reactions. ( e - f ) Glutathionylation of ARHGEF7 WT and cysteine mutants in MDA-MB-231 cells in response to high glucose (HG, 25 mM) or low glucose (LG, 5 mM) conditions: cells were incubated for 20 h (n=3). ( g - h ) Glutathionylation of ARHGEF7 constructs in MDA-MB-231 cells upon adding EGF (n=3). EGF was incubated for 20 h. The lysates were used for click reactions with biotin-alkyne and analyzed by western blot with FLAG antibody before (input) and after (eluted) pull-down with streptavidin-agarose. Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( e - h ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.
Article Snippet: Breast cancer cell line MDA-MB-231 (ATCC, HTB-26TM) and breast cancer cell line MDA-MB-468 (ATCC, HTB-132TM) were maintained in Dulbecco’s Modified Eagle Medium with high glucose (DMEM, Cytiva, HycloneTM) supplemented with 10% Fetal Bovine Serum (FBS, Cytiva, HycloneTM) and 1% (100 U/mL) Penicillin-Streptomycin (Pen-strep, Thermo Scientific) in a humified incubator at 37°C in a 5% CO 2 environment.
Techniques: Sequencing, Expressing, Mutagenesis, Incubation, Construct, Western Blot