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In o u r continuing efforts to dem o n strate the m olecular target o f azadirachtin, we now provide evidence o f the activity o f azadirachtin A and sev eral related com pounds on the Spodoptera fru gi perda ovarian cell line (Sf 9), which is currently in wide use as an in vitro eukaryotic p ro tein expres sion system. Materials and Methods Insect cell cultureS f9 cells used in this study were derived from exponentially grow ing m onolayer cultures (A TC A C at. N o. C R L 1711). A b o u t 2 -2 .5 * 106 cells were seeded into sterile 25 cm 3 cell culture flasks co n taining 4 ml o f fresh TC 100 insect culture m edium (B iochrom , G erm any) and 10% fetal calf serum. T he cells were allowed to a tta c h to the b o tto m o f the flask for 1 h. Incubation with botanicalsA zadirachtin A (I), ohchinin (II), salannin (III), and volkensin (IV), w ith their structures presented in Fig. 1, were purified using stan d ard protocols [7], The com pounds were dissolved in 30% e th a nol/w ater and added to the culture flasks at a con centration o f 1 jig/ml o f the m edium . A control flask was m aintained containing only the solvents. T he flasks were kept at 27 ± 1 °C for altogether 48 h and the cells m onitored at every 12 h interval u n der an inverted m icroscope. P hotom icrographs o f the treated and control cells were m ade after 48 h. Processing o f the cellsThe cells were harvested, w ashed in p h osphate buffered saline and used for 2 D protein analysis or

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