The overexpression of dominant negative mutants of Rho GTPases and the expression silencing of Rho GTPases in host cells diminished the invasiveness of T. gondii RH tachyzoites. (A-B) RhoA or Rac1 overexpression: When compared with the untransfected cells (mock group), RhoA-WT or Rac1-WT overexpressed cells showed the almost same infection rate, while dominant-negative mutant RhoA-N19 or Rac1 N17 overexpressed cells showed a significantly lower infection rate (P = 0.001 and P = 0.005), proximately 60% of the Mock. (C) Silencing of RhoA or Rac1: When compared with the untransfected cells (mock group) and negative control siRNA transfected groups, cells transfected with RhoA siRNA, Rac1 siRNA or RhoA + Rac1 siRNA showed a significantly lower infection rate (P < 0.001). It was about 65% of the Mock in the two single knockdown groups and about 50% of the Mock in the double knockdown group. (D-E) Detection of RhoA or Rac1 RNAi efficiency: anti-actin panel showed the same amount of total protein was loaded for detection in different cell lysates including mock, negative control siRNA, RhoA or RAC1 siRNA, and RhoA + Rac1 siRNA transfected groups. Anti-RhoA panel showed the apparent inhibition of RhoA expression in RhoA silenced and RhoA + Rac1 silenced cells; anti-Rac1 panel showed the apparent inhibition of Rac1 expression in Rac1 and RhoA + Rac1 silenced cells.