Fig 2: P-Rex1 is an essential mediator of Rac1 activation in response to AngII. A Inhibition of P-Rex1 activity in NRCFs impaired AngII-induced Rac1 activation. Serum starved NRCFs were pre-treated with 30 μM 1A-116 for 30 min and then were stimulated with AngII (1 μM, 90 s). The fraction of Rac1-GTP was isolated by pulldown. Both GAPDH and Ponceau staining were used as loading controls. B Densitometric values of Rac-GTP levels (normalized to Rac1-GDP) are presented as mean±SD (n = 4). *P < 0.05 versus control group, #P < 0.05 versus AngII treatment group. C Stable depletion of P-Rex1 in NRCFs impairs AngII-induced Rac1 activation. NRCFs were transfected with validated P-Rex1 siRNA or control duplexes. After 24 h, cells were serum starved for 24 h and then stimulated with AngII (1 μM, 90 s). The fraction of Rac1-GTP was isolated by pulldown. Both GAPDH and Ponceau staining were used as loading controls. DFold induction in Rac1-GTP levels normalized to Rac1-GDP, as determined by densitometry, is expressed as mean±SD (n = 4). *P < 0.05 versus control group, #P < 0.05 versus AngII treatment group

Fig 3: RABV P downregulates Rac1 activity. (A–C) N2a cells were infected by CVS-11 at MOI of 1. At 24 and 48 h p.i., total cell lysis was collected. Rac1 activity was measured by G-lisa assay (A). GTP-bound Rac1 was precipitated using Pak1 PBD-Agarose Beads in pull-down assay and processed for western blot analysis. The density of GTP-bound Rac1, total Rac1, and RABV N was quantified compared with GAPDH by ImageJ (B). The phosphorylation and total protein level of Rac1 signaling downstream molecules (Pak1, Limk1, and Cofilin1) were detected by western blot (C). (D–F) Flag-P was transfected into N2a cells for 48 h as an empty vector used as a control. Total cell lysis was collected. G-lisa assay was performed to measure Rac1 activity (D), and a pull-down assay was performed to analyze the amount of GTP-bound Rac1 compared with that of total Rac1 (E). The p-Pak1, p-Limk1, and p-Cofilin1 were immunoblotted and analyzed by the mean density compared with total amounts of Pak1, Limk1, and Cofilin1, respectively (F). Flag expression was measured as a control of transfection. (G and H) After being inoculated with CVS-11 (MOI = 0.1) for 24 h, N2a cells were transfected with RABV P-siRNA-1, RABV P-siRNA-2, and nonspecific control siRNA (NC), respectively, for 24 h, and total protein was collected from the lysed cells at 24 h p.i. Glutathione S-transferase (GST) pull-down assay was proceeded by contrasting the amount of GTP-bound Rac1 to total Rac1 (G), and the phosphorylation and total protein level of Pak1, Limk1, and Cofilin1 were determined by western blot (H). GAPDH was used as a loading control. Results are represented as mean ± SD of three independent experiments. Statistical significances of the differences are indicated. Student’s t test, P < 0.05 (*); P < 0.01 (**); P < 0.001 (***).

Fig 4: Rho GTPase Rac1 is involved in RABV infection. (A and B) N2a cells were inoculated with CVS-11 (MOI = 1) and harvested at 0, 3, 12, and 24 h p.i.; the mRNA and protein levels of RhoA, Cdc42, and Rac1 were detected by RT-qPCR (A) and western blot (B). (C) N2a cells were transfected with WT or the DN mutant forms of RhoA, Rac1, or Cdc42 for 24 h. After being infected with CVS-11 (MOI = 1) for 24 h, cells were harvested for mRNA quantification of RABV N and P. (D–L) Following ruling out the toxic effects of drugs, CCG-1423, ML141, and NSC23766, by MTT assays, N2a cells were disposed with increasing concentrations of CCG-1423 (0, 1.25, 2.5, 5, and 10 µM) (D), ML141 (0, 1, 2, 4, and 8 µM) (G), or NSC23766 (0, 12.5, 25, 50, and 100 µM) (J) for 1 h and then infected with CVS-11 (MOI = 1) for 24 h p.i. Cells were collected to detect the RNA copy numbers of RABV N and P by RT-qPCR. The protein levels of RABV N and P were examined by western blot and analyzed by densitometric analysis using ImageJ normalized to GAPDH (E, H, and K). With treatment of CCG-1423 (10 µM) (F), ML141 (8 µM) (I), or NSC23766 (100 µM) (L), the viral titer in the cell culture supernatant was quantified by TCID50 assay at 24 h p.i. Results are represented as mean ± SD of three independent experiments. Statistical significances of the differences are indicated. Student’s t test, P < 0.05 (*); P < 0.01 (**); P < 0.001 (***).

Fig 5: Perturbation of Rac1 activity interferes with RABV infection. (A–C) N2a cells were transfected with Rac1-siRNA-1, Rac1-siRNA-2, and nonspecific control siRNA (NC), respectively, followed by infection with CVS-11 at MOI of 1 at 24 h post-transfection. At 24 h p.i., the cells were lysed to determine the RNA copy numbers of RABV N, P, and Rac1 (A). (B) The protein levels of RABV N, P, and Rac1 were determined by western blot and quantitated by densitometric analysis using ImageJ. GAPDH was used as a loading control. (C) Cell culture supernatant was harvested at 24 h p.i., and virus titer was measured by TCID50. (D–F) N2a cells were transfected with either constitutively active Rac1 (Rac1 CA) or dominant-negative Rac1 (Rac1 DN) or wild-type (Rac1 WT) forms followed by CVS-11 infection (MOI = 1) at 24 h posttransfection. At 24 h p.i., relative RNA copies and protein level of RABV N were examined by RT-qPCR (D) and western blot (E), respectively, normalized to that of GAPDH. (F) TCID50 assay was also performed to quantify the viral titer. (G–I) N2a cells were pretreated with the indicated concentration of IPA-3 (0, 12.5, 25, 50, and 100 µM) for 1 h and subsequently infected with CVS-11 (MOI = 1). Cytotoxicity of IPA-3 on N2a cells was analyzed by MTT, and RNA copy numbers of RABV (N and P) were detected by RT-qPCR (G). The protein levels of RABV N and P were measured by western blot and were quantified by densitometric analysis using ImageJ, with GAPDH used as a loading control (H). (I) The viral titer was quantified by TCID50 assay with pretreatment of IPA-3 at 100 µM. (J–L) Cells were pretreated with NSC23766, IPA-3, or both, respectively, for 1 h and infected with CVS-11 at MOI of 1. At 24 h p.i., N2a cells were harvested and lysed. The expression levels of RABV N and P were determined by RT-qPCR (J) and western blot (K). GAPDH was used as a loading control. (L) The viral titer in the cell culture supernatant was also measured at 24 h p.i. Results are represented as mean ± SD of three independent experiments. Statistical significances of the differences are indicated. Student’s t test, P < 0.05 (*); P < 0.01 (**); P < 0.001 (***).