My lab has been studying the role of Hippo pathway in breast and lung cancer, and recently, we have developed a novel biosensor to monitor Hippo signaling activity. This method can be used to answer key questions about cellular signaling networks, such as how the Hippo pathway responds to factors and how it interacts with other signaling pathways. The main advantage of this technique over existing methods is its great sensitivity and also the large number of the samples can be processed quickly and simultaneously.
This technique has diverse applications in basic and translational research to investigate regulators of the Hippo signaling pathway both in vitro and in vivo. To begin, mini-prep LATS-BS plasmids fresh from DH5-Alpha bacteria liquid culture. An hour prior to transfection, aspirate the medium from the plate, and add 500 microliters of growth medium to each well.
One important point is that LATS biosensor must be prepared fresh in order to get maximum expression and activity. Also, it is recommended to always use an upstream regulator of the Hippo pathway, such as MSD, as a positive control when performing luciferase assay. After this, return the cells to the incubator.
Next, prepare solution A, a DNA solution, and solution B, a diluted polymer-based transfection reagent, as described in the text protocol. After this, immediately add solution B to solution A, and gently pipette up and down to mix. Incubate the sample at room temperature for 15 minutes to allow the transfection complexes to form.
Then add the total volume of transfection complexes drop-wise to each well of the plate with gentle swirling. Incubate the cells overnight at 37 degrees Celsius. The next day, remove the transfection complex-containing media and replace it with fresh complete media.
Then culture the cells at 37 degrees Celsius for one more day. Dilute an appropriate amount of 5x Passive lysis buffer with distilled water. Then, completely aspirate the media from the plate.
Add one milliliter of PBS to each well, and swirl the plate gently to remove detached cells and residual growth media. Then aspirate the PBS completely. Next, add 250 microliters of 1xPLB to each well.
Place the culture plate on a rocking platform for 15 minutes to ensure even coverage of the cell monolayer with PLB. After this, transfer the lysate to a 1.5 milliliter microcentrifuge tube. Remove the LAR-2 from 80 degrees Celsius storage, and equilibrate it to room temperature.
Then, prepare Renilla detection reagent for an appropriate quantity of assays. Add the Renilla detection reagent to 50x substrate and its buffer. Next, dispense 10 microliters of each cell lysate into 1.5 milliliter tubes.
Then, program a luminometer to perform a two second pre-measurement delay, followed by a 10 second measurement period. Carefully, transfer 20 microliters of LAR-2 reagent into one tube, and quickly pipette up and down to mix. Then, immediately place the tube in the luminometer and initiate the reading.
After the reading, remove the sample tube from the luminometer, add 20 microliters of Renilla reagent, and pipette up and down to mix. Then, place the sample in the luminometer and initiate the next reading. First, culture, detach, count, and plate HEK293 Ad-cells as described in the text protocol.
Then, one hour before transfection, aspirate the media from the plate and replace it with five milliliters of fresh complete growth media. Finally, add diluted solution B to solution A, and pipette up and down to mix. Then, incubate the sample at room temperature for 15 minutes before adding the total volume of the transfection complexes to the plate, while swirling gently.
Incubate the cells overnight at 37 degrees Celsius. The next day, trypsinize and count the cells. Plate 10, 000 to 20, 000 cells into each well of a 96-well plate in a total volume of 100 microliters of media per well.
After this, incubate the cells for one more day. One to four hours prior to performing the luciferase assay, add agents potentially regulating the Hippo pathway, at a final concentration of 10 micromolar per well. Next, aspirate the media completely from the cells and wash the cells in each well with 100 microliters of PBS.
Then, add 20 microliters of PLB to each well. Place the culture plate on a rocking platform for 15 minutes. Then, transfer 20 microliters of LAR-2 reagent to each well of the plate, and pipette up and down to mix.
Immediately after this, place the plate into a plate-reading luminometer and initiate the reading. In the first part of this protocol, the LATS-BS was cotransfected with different genes to evaluate their effect on LATS kinase activity. In the second protocol, LATS-BS was transfected into HEK293-Ad, and the cells were passed into 96-well plates.
40 hours after transfection, the cells were treated with different known small molecule regulators of Hippo signaling, followed by a luciferase assay. When interpreting the results of these assays, it should be noted that proteins and drug treatments may activate feedback pathways that influence biosensor activity. Further experiments will be necessary to confirm a relationship between the candidate and the Hippo signaling pathway.
Following this procedure, other methods like quantitative Real-Time PCR, or Western Blot, can be used to answer additional questions about LATS kinase activity. After its development, this technique paved the way for the researchers in the field of cancer biology to explore the dynamics and activity of LATS kinase and Hippo signaling pathway in many biological and biochemical processes. Don't forget that drugs for screening can be extremely hazardous, and appropriate precautions should always be taken while performing this procedure.
