Figure 3. siRNA-mediated silencing of HAS2, CD44 or RHAMM reduced NSCLC cell viability and the level of cell proliferation- and apoptosis-related proteins and these effects were modulated by exogenous HA.

(A) Effects of HAS2, CD44 and RHAMM siRNA on the viability of NSCLC cells. Each cell line was transfected with the individual siRNAs and cell viability was determined by MTT assay as described in the Materials and Methods section. (B) HAS2, CD44 and RHAMM siRNAs modulated the expression of cell proliferation-and survival-related proteins. NSCLC cells were transfected with the siRNAs and Western immunoblotting was performed as described in the Materials and Methods section. (C) Effect of HAS2 siRNA on HA synthesis. A549 cells were transfected with HAS2 siRNA and accumulation of HA in the culture media was determined as described in the Materials and Methods section. (D, E) Exogenous HA (2.5 mg/mL) rescued cells from the cytotoxic effects of siRNAs targeting HAS2 (D), CD44, RHAMM or CD44 + RHAMM (E). A549 cells were treated with HAS2, CD44, RHAMM or CD44 + RHAMM siRNAs or HA alone or siRNA + HA and cell viability determined by MTT assay. (F) Exogenous HA attenuated the effects of triptolide on cell proliferation-and apoptosis-related proteins. A549 cells grown in RPMI media supplemented with 2.5% FBS were treated with HAS2, CD44, RHAMM or CD44 + RHAMM siRNAs or HA alone or siRNA + HA and expression of the proteins determined by Western immunoblotting. For all experiments, at least three independent assays were carried out. *P < 0.05, compared to treatment with DMSO-treated cells; Δ, compared to DMSO-treated cells; ♯, compared to treatment with siRNA only.