Fig 2: 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 3: Overexpression of sFRP1 in gastric cancer cells activates Rac1 and GSK3β. (A) Overexpression of sFRP1 in SGC-7901 cells activates Rac1. The morphologies of SGC-7901/vector and SGC-7901/sFRP1 were visualized by confocal microscopy (scale bar, 20 µM) using F-actin with Alexa Fluor 555®-labeled phalloidin. DAPI was used to visualize cell nuclei. White arrows indicate the lamellipodia structures. (B) The activity of Rac1 was investigated by an in vitro activity assay. Equal amounts of lysates from SGC-7901/vector and SGC-7901/sFRP1 cells were used (left). Equal amounts of the lysates from BGC823/vector and BGC823/sFRP1-KD cells were used (right). The Rac1 activated kinase-Rac/Cdc42 (p21) binding domain beads were used for precipitation of activated Rac1. Total cell lysates were loaded for input control. (C) Western blotting assays were performed to visualize the inactivated form (p-Rac1 S71) of the Rac1 protein. GAPDH was used as a loading control. Quantification of the intensity of the bands was normalized relative to the SGC-7901/vector, which is depicted on top of the bands. sFRP1, secreted frizzled-related protein 1; Rac1, Rac family small GTPase 1; GSK3β, glycogen synthase kinase 3β; KD, knockdown; p-, phosphorylated.

Fig 4: CDH12 regulates neuronal growth via the PKA/Rac1/Cdc42 pathway. (A,B) PKA agonist (8-Bro) could rescue the CDH12 knockdown phenotype when its concentration reached 5 μM. The scale bar represents 75 μm. The statistical data show the mean ± SE. n = 20, ∗∗P < 0.01. (C) Representative images of Tuj1 immunostaining in neurons treated with control (Ctrl) or H89 (3 μM) for 12 h. The scale bar represents 75 μm. (D) The statistical result of axonal length after treatment with the negative control or H89 (3 μM) in E18 neurons. The data shown are the mean ± SE. n = 30, ∗∗∗P < 0.001. (E) CDH12 regulates neuronal growth via the PKA/Rac1/Cdc42 pathway. (a) Representative western blotting results show the levels of p-CREB and p-Rac1/Cdc42 proteins with or without CDH12 knockdown. (b) Statistical result (mean ± SE) of relative p-CREB levels. The value of p-CREB/total CREB in the control treatment was normalized as 1. (c) Statistical result (mean ± SE) of relative p-Rac1/Cdc42 levels. The value of p-Rac1/Cdc42/total Rac1/Cdc42 in the control treatment was normalized as 1. Panel d. Statistical result (mean ± SE) of relative CDH12 levels. The value of CDH12/β-Actin in the control treatment was normalized as 1. n = 3, ∗∗P < 0.01. (F) CDH12 siRNA-mediated inhibition of the Rac1/Cdc42 pathway is rescued by 8-Bro. (a) Representative western blotting results show the levels of p-CREB and p-Rac1/Cdc42 proteins with or without 8-Bro treatment. (b) Statistical results (mean ± SE) of relative p-CREB levels. The value of p-CREB/total CREB in the control treatment was normalized as 1. (c) Statistical results (mean ± SE) of relative p-Rac1/Cdc42 levels. The value of p-Rac1/Cdc42/total Rac1/Cdc42 in the control treatment was normalized as 1. The statistical significance of the difference was determined using ANOVA (∗∗∗P < 0.001, ∗∗P < 0.01 vs. control).

Fig 5: Rac1 and GSK3β participate in the regulation of the TGFβ pathway in sFRP1-overexpressing gastric cancer cells. (A) The expression of TGFβ signaling proteins were examined using immunoblotting. Cytoplasm and nuclear extracts from SGC-7901/vector and SGC-7901/sFRP1 cells were collected using cells cultured for 24 h under normal conditions. Actin and Lamin A/C were used as loading controls for cytoplasm and nuclear proteins, respectively. (B) Nuclear or cytoplasm extracts were collected from SGC-7901/sFRP1 cells treated with IM-12 or NSC23766. TGFβ-signaling downstream targets were examined by immunoblotting. (C) Reverse transcription-quantitative polymerase chain reaction analysis was performed to examine the expression of Smad3-responsive genes in SGC-7901/sFRP1 cells. The cells were treated with dimethyl sulfoxide (vehicle), Rac1 inhibitor (NSC23766) or GSK3β inhibitor (IM-12) in normal culture conditions for 24 h. Relative expression levels, compared with vehicle, are plotted. The data are presented as the mean ± standard deviation of three independent experiments. Statistical analysis was performed for each inhibitor (NSC23766 or IM-12) compared to vehicle individually (ANOVA test). #P<0.05 vs. vehicle. (D) Immunoblotting analysis of protein expression levels in vector-only SGC-7901 cells transfected with vector, WT Flag-Smad3 or Ser204 mutant Flag-Smad3 (the phosphorylation site modulated by GSK3β). (E) Immunoblotting analysis of protein expression levels in vector-only SGC-7901 cells transfected with vector and WT HA-Smad2. GAPDH was used as a loading control. Quantification of the intensity of the bands was normalized in relative to vehicle or vector, which are depicted on top of the bands. sFRP1, secreted frizzled-related protein 1; Rac1, Rac family small GTPase 1; GSK3β, glycogen synthase kinase 3β; TGFβ, transforming growth factor β; WT, wild-type.