Fig 2: RAC1 functions with BCAR1 in cell proliferation, colony formation, cell invasion and migration, and resistance to anoikis in LUAD. A. Co-IP results failed to show a direct interaction between BCAR1 and RAC1 in LUAD cells. B. Cell proliferation was inhibited after BCAR1-KO in H1975 and H1299 cells and promoted after BCAR1-OE in A549 cells. BCAR1-NC and BCAR1-OE in A549 cells reduced cell proliferation following treatment with NSC-23766. C. Colony-formation was significantly decreased and increased in H1975 and A549 cells following BCAR1-KO and -OE, respectively. Colony-formation of A549-BCAR1-OE cells was significantly antagonized by NSC-23766 treatment. D. Cell invasion was decreased after BCAR1-KO in H1975 and H1299 cells. Both invasion and migration were increased following BCAR1-OE in A549 cells. E. Enhanced invasion and migration following BCAR1-OE in A549 cells were significantly antagonized following treatment with NSC-23766. F. Survival of A549 cells following BCAR1-KO was significantly lower than that of respective controls in H1975 and H1299 cells (P < 0.05). Survival of A549 cells following BCAR1-OE was significantly higher than that of the control in A549 cells (P < 0.001). G. BCAR1 induced anoikis resistance in A549 cells following BCAR1-OE, which was antagonized by NSC-23766 treatment.

Fig 3: Statins mitigate RSV-induced increases to Rho GTPase prenylation and activity.a A click chemistry-based approach was used to isolate and visualize prenylated proteins. HEp-2 cells were treated with azide-modified isoprenoids, farnesyl alcohol azide (FAA) or geranylgeranyl alcohol azide (GGAA), and infected with RSV A2 (MOI 0.5). Lysates were collected 48 h p.i. and conjugated to biotin-PEG4-alkyne by a copper-catalyzed reaction. Prenylated proteins were then visualized by western blot using a streptavidin-HRP probe. b RSV A2 infection of HEp-2 cells (MOI 0.5) induced an increase in total protein prenylation (farnesylation and geranylgeranylation) relative to uninfected cells 48 h p.i. Notably, the virus increased the prenylation of ~20 kDa proteins corresponding to small GTPases (as outlined on the blot). Atorvastatin (3.5 µM) treatment 2 h post-viral exposure inhibited the RSV-induced increase to small GTPase prenylation (both farnesylation and geranylgeranylation). c The subcellular localization and activity of Rho GTPases is regulated by prenylation. Active, GTP-bound RhoA and Rac1 levels were measured 48 h p.i. by affinity purification of RSV A2-infected HEp-2 lysates (MOI 0.5) +/- atorvastatin (3.5 µM). Atorvastatin reduced the GTP-bound levels of both RhoA and Rac1 in infected cells. Averaged GAPDH-normalized band intensity is denoted by the numbers above each lane. Values are reported as means ± SEM (n = 2 cell lysates). GAPDH controls were processed on a parallel blot with the same samples and protein load.

Fig 4: CBX3 indirectly increases the amount of active Rac1 by repressing ARHGAP24 expression in LUAD cells.a, b A549 and H1299 cells were infected with shControl, shCBX3 #1, or shCBX3 #2 for 72 h. Cells were collected for Western blotting analysis (a) and RT-qPCR analysis (b). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. **P < 0.01. c, d A549 and H1299 cells were transfected with indicated plasmids for 24 h. Cells were collected for Western blotting analysis (c) and RT-qPCR analysis (d). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with three replicates. **P < 0.01; ***P < 0.001. e, f The ChIP-seq of CBX3 on the promoter region of ARHGAP24. g The ChIP-qPCR of CBX3 on the promoter region of ARHGAP24 in A549 and H1299 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates. NS not significant; **P < 0.01; ***P < 0.001. h The ChIP-qPCR of H3K9me3 on the promoter region of ARHGAP24 in A549 and H1299 cells. Statistical significance was determined by two-side Student t test. Data presented as Mean ± SD with three replicates. NS not significant; *P < 0.05; ***P < 0.001. i A549 cells were infected with shControl or shARHGAP24 for 48 h. Then, cells were transfected with pcDNA3.1 or Flag-CBX3 as indicated. After 24 h, cells were harvested for Western blotting analysis. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. For quantification of active Rac1, data presented as Mean ± SD with two replicates. NS not significant; *P < 0.05; **P < 0.01. j, k A549 cells were infected with indicated shRNAs for 72 h. Cells were collected for Western blotting analysis (j) and MTS assay (k). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. For quantification of active Rac1, data presented as Mean ± SD with two replicates. For MTS assay, data presented as Mean ± SD with three replicates. NS not significant; *P < 0.05; **P < 0.01; ***P < 0.001. l–n A549 cells were infected with indicated shRNAs. After 72 h puromycin selection, cells were harvested and subcutaneously injected into nude mice for xenografts assay. The image of tumor was shown in panel (l). The tumor growth curve was indicated in panel (m). The tumor mass was demonstrated in panel (n). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SD with five replicates. NS not significant; ***P < 0.001.

Fig 5: Experimental validation of functional impact of mutations in 3D clusters in MAP2K1 and RAC1. a Seven residues in a 3D cluster in MAP2K1, in the context of the domain structure of the protein. Notation as in Fig. 1: each circle is an occurrence in a sample; connecting lines (bottom) indicate cluster membership, i.e., statistically significant proximity in 3D in the protein structure. b The same cluster of mutated residues in the 3D structure of MAP2K1. The purple helix is known to negatively regulate the kinase activity of MAP2K1/MEK1. c Functional characterization of MAP2K1/MEK1 mutants in HEK-293H cells. Expression of G128D and Y130C (as well as the previously characterized F53L, Q56P, and K57N) mutants each resulted in increased expression of phosphorylated ERK compared to wild-type MAP2K1 — but not the cluster member A52V. d ERK phosphorylation was inhibited by trametinib in cells expressing the Q56P or Y130C MAP2K1 mutations in HEK-293H cells. e The four residues (two single-residue hotspots: P29 and A159, and two rarely mutated residues: G15 and C18) in the identified 3D cluster in RAC1 in the linear domain structure of the protein. f The same cluster in the 3D structure of RAC1. g Western blot analysis of RAC1 activation (GTP-bound RAC1 levels) by PAK1 pulldown (left) and of total RAC1 levels (right) in HEK-293 T cells. The RAC1 3D cluster mutations G15S and C18Y, as well as the previously characterized P29S and A159V, were associated with significant RAC1 activation, as compared to wild-type RAC1