Media matrigel

All in vivo experiments were performed with female athymic nude CrTac:NCr-Fo mice were purchased from Taconic Laboratories (Hudson, NY) at age 6–8 weeks. During subcutaneous injections, mice were anesthetized using 2% isoflurane gas in 2 L/min medical air. 1×10⁶ TS543 cells were injected in the right shoulder subcutaneously in 150 μL volume of 50% media/matrigel (BD Biosciences, San Jose, CA) and allowed to grow for approximately two weeks until the tumors reached about 8 mm in diameter (100 ± 8 mm³).
For intravenous injections, the lateral tail vein was used. Mice were warmed with a heat lamp, placed in a restrainer, and the tail was sterilized with alcohol pads before injection.
For orthotopic injections, TS543 cells (5×10⁵ cells in 2 μL growth media) were injected 2 mm lateral and 1 mm posterior to the bregma using a Stoelting Digital New Standard Stereotactic Device and a 5 μL Hamilton syringe and allowed to grow for three weeks before treatment. All mouse experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of MSK and followed National Institutes of Health (NIH) guidelines for animal welfare.

Following University of Iowa IACUC approval, twelve male and twelve female nu/J mice (RRID:IMSR_JAX:002019, Jackson Laboratory, Bar Harbor, ME, USA) were flank injected with 1×10⁶ cells of either parental HCT116 or sub-clone KO-3 suspended in media/Matrigel (BD Biosciences) in a 1:1 ratio. As previously described (29 (link)), tumors were allowed to establish for seven days prior to starting treatments. Mice were randomized to a treatment group prior to flank injection so that each condition and respective treatment group had an equal number of male and female mice.
They received either 40 mg/kg NVP-BKM120 (Selleck Chemicals, catalog no. S2247) or control (water) for ten consecutive days by oral gavage. Tumor growth was monitored by calipers and tumor volume was calculated in mm³ using the width, length and height of tumors (16 (link)). Animals were euthanized when tumors reached 1000 mm³. Tumors were harvested, fixed in formalin and embedded in paraffin. Immunohistochemistry staining for pHH3 (Cell Marque, catalog no. 369A-14) was performed and quantified on all harvested tumors.