Male nod scid mice

MM.1S-Luc-GFP cells stably expressing the corresponding shRNA (shKDM3A, shKLF2, shIRF4 or control shRFP; 15 × 10⁶ viable cells) were injected via tail vein into 300-cGy-irradiated NOD-SCID male mice (Charles River Laboratories). Twenty-four hours after injection, mice were killed, and the bilateral femurs of each mouse were collected. After crunching femurs, bone marrow cells are sieved through a 70-μm filter, and red blood cells were lysed with ACK Lysing Buffer (Lonza). The bone marrow cells were then resuspended in PBS with 2% (v/v) fetal bovine serum, and GFP-positive cells were detected with flow cytometry (BD FACSCanto II, BD Biosciences). Data are shown as relative homing efficacy compared with control cells expressing shRFP. Animal studies were performed under a protocol approved by the Dana-Farber Institutional Animal Care and Use Committee.

All animal studies were performed in compliance with institutional guidelines under an IACUC approved protocol. To analyze IRS1 S1101 (S1097 in mouse) phosphorylation levels in various tissues including kidney, liver, abdominal fat and muscle tissues were obtained from WT and TKO FVB mice. Tissue extracts were prepared by homogenizing the tissues in RIPA lysis buffer, as described previously [46 (link)]. To examine the effects of a Pim kinase inhibitor on IRS1 S1101 phosphorylation levels in in vivo tumors, mouse xenograft model of T-cell leukemia, H-SB2 cells stably expressing luciferase (2 × 10⁵ cells/mouse) were injected via tail vein into irradiated (2.5 Gy) 4–6 week old NOD/SCID male mice (Charles River Laboratories). Starting on day 3, the mice were treated with AZD1208 (30 mg/kg twice daily by oral gavage) or control vehicle (0.5% HPMC, 0.1% Tween-80) daily for ~17 days. Tumor growth was followed by bioluminescence imaging at the time-points indicated.

All animal experiments were approved by and performed in compliance with the guidelines and regulations approved by the Institutional Animal Care and Use Committee of the Albert Einstein College of Medicine (protocols 20161008, 20180707). Animals were housed in a controlled environment, target conditions: temperature 19–25 °C, relative humidity 30 to 60%. An electronic time-controlled lighting system was used to provide a 12 h light/12 h dark cycle. For toxicity studies 6–8 weeks old CD1-IGS male and female mice were purchased from Charles River. For xenograft studies and pharmacodynamics analysis experiments, 6–8 weeks old Nu/Nu nude mice and NOD SCID male mice were purchased from Charles River. All mice were kept under standard conditions and diet and had a weight > 20 grs.

A total of 6–8 weeks old NOD SCID male mice were purchased from Charles River. Approximately, 1.0 × 10⁶ COLO-1 or COLO-2 cells were suspended in a 1:1 DMEM:matrigel and injected subcutaneously into the right flanks of mice. PDX characterization: mice were divided into two groups COLO-1 and COLO-2 (n = 3) were divided into two groups. Tumor was collected once tumor reached a volume of ~1,000 mm³. COLO-1 efficacy study: Mice were divided into four groups (vehicle, BTSA1.2, Navitoclax and combination n = 4) and treated with vehicle, 200 mg/kg BTSA1.2, 50 mg/kg Navitoclax or BTSA1.2 and Navitoclax combination by oral gavage daily. Mice in the combination group were first administered with 50 mg/kg Navitoclax and after 6–8 h were administered 200 mg/kg BTSA1.2. Treatments started once tumors reached a volume of ~200 mm³. Tumor volume was monitored every 3–4 days by caliper measurements until the cessation of the experiment when tumors reached an ethically unacceptable size or after 18 days of daily treatment (whichever came first). Body weight of mice were monitored during treatment.