Figure 7: Fluorescent microscopy on distribution of f-actin fibers and RhoA and immunoblotting analysis of RhoA for the effect of TcdA of Clostridium difficile and the effect of micronutrients. The cells were treated with TcdA at 10 ng/mL for 24 h and supplemented or not with 10 mM of Ala-Gln or Gln and incubated with rhodamine-phalloidin, FITC-RhoA, and DAPI. The IEC-6 cells were divided into 6 groups: control (a); treated with TcdA at 10 ng/mL for 24 h (b); treated with 10 mM of alanyl-glutamine without TcdA (c); treated with 10 mM of glutamine alone without TcdA (d); treated with TcdA at 10 ng/mL for 24 h and supplemented with 10 mM alanyl-glutamine (e); treated with TcdA at 10 ng/mL for 24 h and supplemented 10 mM of glutamine (f). Immunoblotting was performed to evaluate the expression of RhoA (g and h). The quantification was done comparatively defaulting protein α-tubulin. compared to control. compared to TcdA.