This protocol can be used in the initial phase of drug discovery to identify inhibitors that abolish the activities of EGFR and RAS proteins. The main advantage of this assay is that inhibitory effects of anti-EGFR and anti-RAS therapeutics can be easily visualized in C.egelans. Begin by spotting 100 microliters of overnight grown E.coli OP50 onto the center of each NGM plate.
Allow the plates to dry for 24 hours in a laminar hood, then store them in a polystyrene container. Using a sterile worm pick, gather 10 to 12 gravid adult worms from a previously grown plate and transfer them to a fresh NGM plate seeded with E.coli OP50. Incubate the plate for 24 hours at 20 degrees Celsius.
After 24 hours, use a sterile worm pick to remove adult worms from the plates. Incubate the plates at 20 degrees Celsius for approximately three days to allow the embryos to develop into gravid adult worms. Collect gravid adult worms into a 15 milliliter conical tube by washing two to four plates with M9W.
Pellet the worms by centrifuging the tube at 450 times G for one minute, then decant the supernatant without disturbing the worm pellet. Prepare worm lysis solution by combining 400 microliters of 8.25%sodium hypochlorite and 100 microliters of five normal sodium hydroxide. Add this solution to the worm pellet and flick the tube to mix.
Observe the lysis of the worms under a dissecting microscope to prevent overbleaching of the embryos. When 70%of the adult worms have lysed, add 10 milliliters of M9W to the conical tube to dilute the lysis mix. Centrifuge the tube at 450 times G for one minute, then replace the supernatant with 10 milliliters of M9W.
After completing the washing steps, add three to five milliliters of M9W to resuspend the egg pellet. Rotate the tube overnight at a speed of 18 RPM on a tube rotator at room temperature. After overnight incubation, remove the tube from the rotator and pellet the L1 larvae by centrifuging the tube at 450 times G for one minute.
Aspirate the M9W until 250 microliters is left in the tube. Begin by spotting 100 microliters of overnight grown E.coli OP50 onto the center of each NGM plate. Allow the plates to dry for 24 hours in the laminar hood, then store them in a polystyrene container.
Grow 30 milliliters of E.coli OP50 in a 50 milliliter conical tube at 37 degrees Celsius overnight in an orbital shaker at 150 RPM. On the next day, spin the E.coli OP50 culture at 4, 000 times G for 10 minutes to pellet the cells, then remove the supernatant and resuspend the pellet in three milliliters of M9W to concentrate the culture. Prior to preparing the working solutions for the drug assay, add 0.1 milliliter of cholesterol into 100 milliliters of M9W.
Prepare working solutions of each experimental drug by diluting the drug and vehicle in 4.8 milliliters of M9W supplemented with cholesterol. Dissolve DMSO in 4.8 milliliters of M9W supplemented with cholesterol to prepare the vehicle control. Add 200 microliters of concentrated E.coli OP50 culture to each tube containing the vehicle control or drugs and vortex the tubes to mix.
Add two milliliters of each working drug solution or vehicle control to each well in a 12 well tissue culture plate. Test each drug concentration and vehicle control in duplicate. Add approximately 100 L1 larvae per well using a sterile micropipette, limiting the volume to 10 microliters.
Incubate the plates at 20 degrees Celsius. Supplement wells with 50 microliters of 10X concentrated E.coli OP50 on day three of the assay if needed. Prepare a 2%weight to volume solution of agarose by adding 0.1 gram of agarose to five milliliters of deionized water.
Heat the agarose in a microwave to dissolve it. Place strips of lab tape along two glass slides, which will act as spacers limiting the thickness of the agarose pads. Then place a third clean glass slide between the taped slides.
To make an agarose pad, spot 100 microliters of molten agarose onto the center of the clean slide. Place another clean glass slide across the top of the agarose and gently press down to form a pad. Remove the top slide when the pad has solidified.
When the appropriate stage of the lifecycle is reached, remove the plates from the incubator and collect the worms in 15 milliliter conical tubes. Centrifuge the tubes at 450 times G for one minute, then remove the M9W without disturbing the worm pellet. Wash the worms twice with five milliliters of fresh M9W.
After removing all remaining M9W, add 500 microliters of two millimolar sodium azide or two millimolar tetramisole hydrochloride to anesthetize the worms. Allow tubes to incubate at room temperature for 15 minutes. Add 10 microliters of the anesthetized worm suspension onto the center of an agarose pad and place a number 1.5 cover slip gently over the suspension.
If needed, fix the cover slip with nail polish to prevent drying. Use a DIC microscope at 10 and 20 times magnifications to observe the let-60(n1046)let-23(sa62)and lin-1(sy254)strains. For let-60(n1046)and let-23(sa62)score the adult worms based on the presence or absence of the Muv phenotype.
For the lin-1 strain, count the number of VPCs that adopted one or two degrees cell fades on the ventral side of the L4 larvae. This protocol was used to demonstrate that R-fendiline is able to suppress the Muv phenotype in let-60(n1046)mutant strain in a dose dependent manner. Non-reversal of the Muv phenotype was observed in the lin-1 no mutant strain in response to increasing concentrations of R-fendiline, suggesting that R-fendiline blocks activated let-60 signaling at the level of RAS and C.elegans.
Similarly, the Muv phenotype was significantly reduced in the let-23(sa62)strain in response to three, 10, and 30 micromolar R-fendiline treatments in comparison to the DMSO treated worms. During the bleach treatment, it is important to observe the lysis of the worms. The long incubation in the bleach mix will lead to death of the embryos, resulting in lower yields of L1 larvae.
The localization of let-23 can be determined in the presence of anti-EGFR inhibitors using transgenic worms expressing GFP fused to let-23. This allows an investigator to determine if the anti-EGFR inhibitors cause the mislocalization of let-23 to the plasma membrane.
