Rodent holder

Unanesthetized mice were placed into a rodent holder (Kent Scientific, Torrington, CT, USA), which was placed on a motion platform (SK-L180-Pro, Scilogex, CT, USA). The platform was set to a frequency of 480 cycles per minute and Gz ± 3.0 m/s² for 1 h per day (morning 9–11 a.m.) for 8 consecutive days, as previously described (Adams 2009 [7 (link)]). The WT and db/db control groups were also placed in the rodent holder for 1 h per day for 8 consecutive days, but did not receive pGz treatment.

For subcutaneous xenograft experiments, cells were prepared at a concentration of 1 × 10⁶ cells per 100 μL in a 1:1 mix of 75 μL cell culture medium (DMEM/ F12) and 75 μL Matrigel basement membrane (BD Biosciences, San Jose, CA), for a final volume of 150 μL slurry. A 100 μL bolus of cell slurry was injected subcutaneously into the left hind flank region of each mouse and the puncture site sealed using degradable tissue adhesive (3 M Health Care, St. Paul, MN). Tumor growth was monitored weekly using a digital caliper (VWR, Radnor, PA). Tumor volume was calculated using the formula Volume = (Width × 2 × Length)/2. To investigate whether micro-environmental or paracrine factors affect tumor growth of MDA-MB-231^EP300KD cells, wild type cells expression mCherry and MDA-MB-231^EP300KD clone 1 (GFP positive) were injected as a 1:1 mix for a total of 1 × 10⁶ cells per mouse.
For tail vein injections, the mice were placed under a heating lamp for 2 min to dilate blood vessels. After subsequent immobilization in a rodent holder (Kent Scientific, Torrington, CT) 1 × 10⁶ cells in 100 μL culture medium were injected per mouse using hypodermic syringes.