Transfection Updates and Inhibition Studies

Bhuvi M -

Hello everyone! Last week, I began another round of Axl transfection into OE33.

Here are some pictures from Day 3 of the transfection process:

We have switched from serum-free media with the transfection reagent to media with the antibiotic G418. So, some things we can observe are that the kill plate is starting to die, displaying the efficacy of the antibiotic dose, and the cells in the transfection wells are reducing, leaving only the transfected cells.

Our goal is to grow the transfected cells with the antibiotic and then test for gene expression within the cells.

Another thing we started this week was titrations of Axl and c-Met inhibitors in the resistant clone of OE33. BMS777607 targets the MET family and TP-0903 targets Axl. These markers were chosen as targets per our plan.

Here is our setup of a 6-well plate for titration and day 1 of our titration:

Most of the wells on day 1 have the same number of cells. This illustrates that we will have an accurate experiment, as the number of cells should be a control.

One of the concerns with inhibitors is their off-target effects. BMS77607, while it is meant to target specifically the MET family, also targets Axl and a few other markers. Regardless, this provides us with insight into how these markers may drive afatinib resistance.

The next step is to complete a dot blot using the protein of these cells next week.

Thank you for reading!

 

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