Fig 2: Rac1 activated Src by promoting actin filament production in macrophages. (A) p-Tyr MyD88 in whole-cell lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time was detected by immunoprecipitation and western blot analysis. (B) Src and p-Src in whole-cell lysates of RAW264.7 cells incubated with vehicle (DMSO) or CytoB (20 μM), followed by the treatment with LPS (1 μg/mL) for the indicated time were detected by western blot analysis, and the p-Src/β-actin level was plotted. (C) p-Syk in whole-cell lysates of RAW264.7 cells incubated with the vehicle (DMSO) or CytoB (20 μM), followed by treatment of LPS (1 μg/mL) for the indicated time were detected by western blot analysis, and the p-Syk/β-actin level was plotted. (D) Rac1-GTP and Rac1 in whole-cell lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by western blot analysis. (E) Rac1, Src, and p-Src in whole-cell lysates of RAW264.7 cells transfected with siN.C or siRac1 for 24 h, followed by the treatment of LPS (1 μg/mL) for the indicated time, were detected by western blot analysis, and the p-Src/Src level was plotted. (F) RAW264.7 cells incubated with DMSO or NSC23766 (100 μM) were treated with LPS (1 μg/mL) for the indicated time in the presence of Texas Red-X phalloidin (200 units/mL), and fluorescence was determined with a flow cytometer and plotted. (G) Rac1-GTP, Src, and p-Src in the whole-cell lysates of RAW264.7 cells incubated with DMSO or NSC23766 (100 μM), followed by the treatment of LPS (1 μg/mL) for the indicated time were detected by western blot analysis, and the p-Src/Src level was plotted. (H) Rac1-GTP, Syk, and p-Syk in whole-cell lysates of RAW264.7 cells incubated with DMSO or NSC23766 (100 μM), followed by treatment of LPS (1 μg/mL) for the indicated time were detected by western blot analysis, and the p-Syk/Syk level was plotted.

Fig 3: miR-483-5p inhibitor inhibits TGF-β1-induced EMT by targeting RhoGDI1 via the Rac1/PI3K/AKT signaling pathway. The relative expression of RhoGDI1 in A549 cells infected with lentivirus carrying the interference sequence of RhoGDI1 was detected by (A) RT-qPCR and (B and C) western blotting. **P<0.01 vs. shRNA-NC. A549 cells or A549 cells with shRNA-RhoGDI1 silencing were transfected with miR-483-5p inhibitor for 24 h, and then treated with TGF-β1 for 48 h. The relative of expression of (D) E-cadherin, (E) vimentin and (F) α-SMA were determined by RT-qPCR and (G and H) western blotting. (I) Expression of (J) Rac1-GTP, Rac1, (K) p-PI3K, PI3K, (L) p-AKT and AKT were determined by western blot analysis. Data are presented as the mean ± SD (n=3). *P<0.05; **P<0.01. NS, not significant. 1=Ctrl, 2=TGF-β1 group, 3=TGF-β1+ miR-483-5p inhibitor+ shRNA-NC group, 4=TGF-β1+ miR-483-5p inhibitor + shRNA-RhoGDI1 group. EMT, epithelial-mesenchymal transition; RhoGDI1, Rho GDP dissociation inhibitor 1; TGF-β1, transforming growth factor-β1; α-SMA, α-smooth muscle actin; miR, microRNA; Rac1, Rac family small GTPase 1; RT-q, reverse transcription-quantitative; p-, phosphorylated; sh, short hairpin; NC, negative control; Ctrl, control.

Fig 4: Mitochondrial APE1 (mtAPE1) overexpression enhances cisplatin resistance by inhibiting ROS production. (A) U2OSscr cells, U2OS(R)scr cells, and APE1-deficient cells (U2OSshAPE1; U2OS(R)shAPE1) were treated with DMSO, cisplatin (5 mg/ml), or hydrogen peroxide (H2O2) (100 µM) for 6 h; U2OS and mtAPE1-transfected cells (U2OSmtAPE1) were treated with DMSO, cisplatin (CDDP) (1 mg/ml), or H2O2 (20 µM) for 24 h, and ROS production was then measured and analyzed by flow cytometry. (B) Western blot analysis of the cell lines was performed with an APE1 antibody, Rac1 antibody, p-Rac1 antibody, and cleaved PARP antibody. Anti-β-tubulin antibody was used as loading control. (C) Western blot analysis of U2OSmtAPE1 and SAOS2mtAPE1 cells treated with or without 10 µM CDDP for 24 h was performed with an APE1 antibody, Rac1 antibody, p-Rac1 antibody, and cleaved PARP antibody. Anti-β-tubulin antibody was used as loading control. (D) U2OSscr, U2OS(R)scr, U2OSshAPE1, U2OS(R)shAPE1, and U2OSmtAPE1 cells were treated with DMEM, cisplatin (1 mg/ml), or H2O2 (20 µM) with or without 5 mM N-acetyl cysteine (NAC) for 24 h, and ROS production was then measured and analyzed by flow cytometry. (E) After treatment as described in D, the cells were prepared and subjected to western blot analysis with an APE1 antibody, cleaved PARP antibody, and γ-H2AX antibody and probed with a β-tubulin antibody as a loading control. *P<0.05, statistically significant from the vector alone transfected control; **P<0.05, statistically significant from the untreated control. APE1, apurinic/apyrimidinic endonuclease 1; ROS, reactive oxygen species; Rac1, Ras-related C3 botulinum toxin substrate 1; p-, phosphorylated; PARP, poly ADP ribose polymerase.

Fig 5: EGB761 could prevent the degeneration of dendritic spines and downregulation of molecules and pathways related to the formation and stability of dendritic spines after CCH. The rats’ brains were fixed with formaldehyde and impregnated with Golgi staining solution to observe the dendritic spines. (A) Schematic diagram of morphological classification of dendritic spines was showed. (B) The dendritic spine were observed in 100μm sections (Bar scale=5μm) and density (C), percentage of different kinds of spines (D), and maturity (E) of dendritic spines were counted and calculated (n=3 for 4 group). The brain homogenates were investigated and relatively quantitatively analyzed by proteins blotting for Drebrin, p-cofilin, t-cofilin, Fyn, p-LIMK1, t-LIMK1, p-PAK1, t-PAK1, Rac1 and β-actin antibody (F–L).