Fig. 9: NAIA-5 enables the identification of new covalent ligand CL1 to mediate G1-phase cell cycle arrest in HepG2 cells.
a Competitive MS-based ABPP experiment using NAIA-5 as the probe reveals promiscuous binding of CL1 onto 79 ligandable cysteines. b Analysis of the 79 liganded cysteines by CL1, revealing a number of new cysteines and proteins identified by using NAIA-5 but not DBIA as the cysteine probe. c Gene Ontology analysis of the biological processes involving the 79 proteins which are liganded by CL1. d Flow cytometry analysis of the percentage of HepG2 cells in the Go/G1, S, and G2/M phase after incubation with the indicated concentration of CL1 for 6 and 24 h respectively. e Representative images of the cell cycle analysis by flow cytometry. f Co-immunoprecipitation of GST-Rac1 shows disruption of interactions between Rac1 and TIAM1 upon incubation with CL1. g Rac1 activation assay indicates a decrease in Rac1-GTP level in CL1-treated HepG2 cells. h Western blots show decreases in pPAK levels in HepG2 cells after incubation with CL1. i FRET-Rac1 biosensor reveals a decrease in Rac1 activity in CL1-treated HepG2 cells. Quantified data were shown on average ± SD from n = 8 individual cells from 3 different biological replicates/groups. j CL1 treatment leads to decreases in pRb, cyclin D, and E2F1 levels in HepG2 cells. k Flow cytometry experiment shows no significant changes in the cell cycle of CL1-treated HepG2 cells with genetic knockdown (KD) of Rac1 by siRNA. l Representative images of the cell cycle analysis in (k) by flow cytometry. m pRb level in HepG2 cells with Rac1 KD is not sensitive to CL1 treatment, suggesting that Rac1 is one of the protein targets of CL1 to regulate the cell cycle. Quantified data were shown on average ± SD from n = 3 replicates/group. Statistical analyses were performed with unpaired two-tailed Student’s t-tests. Statistical significance is expressed as *P = 1.56 × 10−2, #P = 3.30 × 10−4 and ^P = 7.88 × 10−3 respectively as compared to the % of cells in G0/G1 in solvent control in (d); *P = 1.83 × 10−2 and #P = 7.67 × 10−3 respectively in (h); *P = 3.14 × 10−3 and #P = 5.23 × 10−3 respectively in (i); *P = 1.17 × 10−3 and #P = 3.93 × 10−5 respectively as compared to the % of cells in G0/G1 in solvent control in (k); n.s. not significant.
