Fig. 5.

The proliferative effect of group X sPLA₂ on Colon-26 cells is not dependent on binding to the M-type receptor. A and B, expression of the M-type receptor in Colon-26, YAMC, AJ02-nm0, and Apc(+/Min) cells. Mouse M-type receptor expression was measured by binding experiments (A) using the specific ligand ¹²⁵I-OS₁ and by Western blotting (B) of cell lysates as described under Materials and Methods. C, validation of M-type receptor silencing by siRNA. Two different commercially available siRNAs (QIAGEN) targeting the mouse M-type receptor were tested for silencing by binding assays and immunocytochemistry to measure the percentage of receptor silencing. The two siRNAs were effective, and representative results of at least two experiments obtained with siRNA1 versus an irrelevant GFP-directed siRNA are shown. Colon-26 cells were transfected with siRNAs on day 1, starved on day 2 for 24 h, and then assayed on day 3 (48 h) and day 4 (72 h) after transfection for M-type receptor expression by binding experiments using ¹²⁵I-OS₁ ligand. The decrease in binding at 48 and 72 h compared with GFP-siRNA is statistically significant (**, P < 0.01; Student's t test). Immunocytochemistry using mouse M-type receptor antiserum was performed 48 h after transfection. D, effect of M-type receptor siRNA1 versus GFP-directed siRNA on incorporation of [³H]thymidine triggered by hGX sPLA₂. Colon-26 cells were transfected with siRNAs on day 1, starved on day 2 for 24 h, and then stimulated on day 3 for 24 h in the absence (−) or presence of mGX and hGX sPLA₂s (200 nM). [³H]Thymidine was added during the last 4 h of sPLA₂ stimulation, and cells were processed as described under Materials and Methods. Under untreated conditions, the incorporation of [³H]thymidine was 42,395 dpm. Incorporation of [³H]thymidine in sPLA₂-treated cells over untreated is statistically significant (**, P < 0.05; Student's t test), but no significant difference was found between the two siRNAs. Data are representative of two experiments.