Fig 2: Rnd3 knockout in mice. (A) Rnd3 expression in the brain of wild-type mice according to western blot analysis. Rnd3 expression was completely knocked out in Rnd3- mice, which were generated by gene trapping. (B) Immunohistochemical staining of Rnd3 in the brain of Rnd3+/+ and Rnd3- mice using an anti-RND3 antibody. Enlarged regions in the boxes on the left are shown on the right. Brown color indicates positive signals. Scale bar, 200 µm (left) or 100 µm (right). (C) Immunofluorescence analysis of Rnd3 protein in the brain of Rnd3+/+ and Rnd3- mice using an anti-RND3 antibody (red). Nuclei were stained with DAPI (blue). Scale bar, 100 µm. (D) Immunohistochemical staining of RND3 in human brain-adjacent GBM tissue (para-GBM) using an anti-RND3 antibody. An enlarged region in the box on the left is shown on the right. Brown color indicates positive signals. Scale bar, 100 µm. GBM, glioblastoma; Rnd3, Rho family GTPase 3.

Fig 3: RND3 interacts with P65. (A) A Rnd3 positive signal in immunoblotting of the mouse brain lysates which was immunoprecipitated with the antibody against P65. (B) Lysates immunoprecipitated with anti-RND3 were incubated with an antibody against P65, revealing a positive signal of P65. Arrows indicate the immunoprecipitated band. (C) Compared with that in the myc group, mRNA expression of P65 was not significantly different in the myc-RND3 group (P>0.05). (D) Co-localization of RND3 with P65 protein in the brain of adult mice. Immunofluorescence assays were performed in brain sections using anti-RND3 (green) and anti-P65 (red) antibodies. Merged images are shown in the lower right panel. Nuclei were stained with DAPI (blue). Scale bar, 50 µm. (E) Immunofluorescence staining revealing the co-localization of endogenous RND3 (green) and p65 (red) proteins in PC12 cells. Nuclei were stained with DAPI (blue). Scale bar, 10 µm. RND3, Rho family GTPase 3; IB, immunoblotting; IP, immunoprecipitation.

Fig 4: RND3 increases apoptosis in PC12 cells. (A and B) Overexpression of RND3 upregulated c-caspase-3 protein expression. Protein levels of (A) western blots were (B) semi-quantified and normalized to GAPDH. *P<0.05. n=3. (C) Percentage of apoptotic cells assessed by flow cytometry. (D) RND3 overexpression promoted apoptosis in PC12 cells. *P<0.05. n=3. (E) Knockdown of RND3 decreased c-caspase-3 protein expression in PC12 cells treated with siRND3. siCtrl was used as a control. (F) Relative protein expression levels in the siRND3 and siCtrl groups were semi-quantified and normalized to GAPDH. *P<0.05. n=3. c-, cleaved; Ctrl, control; RND3, Rho family GTPase 3; si, small interfering RNA.

Fig 5: RND3 blocks the anti-apoptotic role of NF-?B P65. (A and B) Knockdown of P65 promoted apoptosis in PC12 cells. c-caspase-3 protein expression was upregulated following siRNA knockdown of P65. Protein levels in (A) western blots were (B) semi-quantified. *P<0.05. n=3. (C) Flow cytometry assays demonstrated that the percentage of apoptotic cells was increased following P65 knockdown. (D) The percentage of apoptotic cells was quantified. *P<0.05. n=3. (E and F) Western blot analysis of c-caspase-3 expression following knockdown of RND3, P65 or both. Protein levels of (E) western blots were (F) semi-quantified. *P<0.05. n=3. c-, cleaved; Ctrl, control; RND3, Rho family GTPase 3; si, small interfering RNA.