Silencing of HAS2 in tumor cells attenuated their ability to cause dysfunction of monocytes/Mφ. (A) Expression of HAS family in cells from different cells. The levels of mRNA for HAS2 and HAS3 were determined by reverse-transcription PCR using specific primers. The hypoxanthine guanine phosphoribosyl transferase gene (Hprt) was served as an internal control. (B) Reverse-transcription PCR analysis showing that the level of HAS2 was dramatically reduced in stably transfected pSi-HAS2-U251 and pSi-HAS2-SK-Hep-1 clones, compared with the parental and mock-transfected cells. The hypoxanthine guanine phosphoribosyl transferase gene served as an internal control. (C) Western blot analysis demonstrating that the expression of HAS2 protein was markedly reduced in both of the stably pSi-HAS2–transfected clones. The blots were stripped and reprobed with antiactin to confirm equal protein loading. (D) Silencing of HAS2 attenuated the ability of the tumor cells to activate monocytes. Monocytes were cultured with TSN from parental, mock-transfected, or pSi-HAS2-transfected cells for 18 hours, and the release of TNF-α from the monocytes was determined by enzyme-linked immunosorbent assay. (E) Monocytes were cultured with TSN from the indicated tumor cells for 7 days, and the Mφ were subsequently stimulated with 10 ng/mL LPS for 18 hours. Values represent the mean (± SE) of 4 separate experiments; P < .05 and ** P < .01 indicate significantly different from parental and mock cells.