Cell lines

HCT116 cell

Preparation Method

HCT116 cells were seeded at 1 × 10⁵cells/well in a six-well plate and grown for 2 days. Cells were treated with 0.5 μg/mL of doxycycline for 24 h, and then 1 μM 5-Ph-IAA was added.

Reaction Conditions

1μM 5-Ph-IAA

Applications

The mAID-fused targets were rapidly degraded by the addition of 1μM 5-Ph-IAA.

Animal models

Balb/c-nu female(7 weeks old) weighing 16-20 g

Preparation Method

Indicated HCT116 lines (1 × 10⁵cells for mAID-BRD4 and 2 × 10⁵cells for TOP2A-mAC) were resuspended in 0.1 ml of HBSS containing 0.05 ml of Matrigel. The suspension was injected into the both sides of flank. Six or 7 days after 5-Ph-IAA injection, the mice were randomized and treated daily with the indicated dose of 5-Ph-IAA by IP injection for additional 6 or 7 days. Tumour volume was measured on the indicated days. At the end of the experiment, xenograft tumour was removed and weighed.

Dosage form

5-Ph-IAA by IP injection，0, 1, 3, 10 mg/kg

Applications

significant tumour suppression of mAID-BRD4 xenografts at all doses of 5-Ph-IAA treatment

5-phenyl-indole-3-acetic acid (5-Ph-IAA) is a derivative of IAA. 5-Ph-IAA is a ligand to establish the auxin-induced degron 2 system (AID2) together with the OsTIR1 (F74G) mutant. Using AID2, degron fusion proteins can now be more precisely controlled, enabling target protein degradation with a half-life of 10 to 45 minutes by adding low doses of 5-Ph-IAA^[1]. 5-Ph-IAA shows higher affinity to AtTIR1(F79G).

The mAID-fused targets were rapidly degraded by the addition of 1 μM 5-Ph-IAA and showed significant tumour suppression of mAID-BRD4 xenografts at all doses of 5-Ph-IAA treatment including 0, 1, 3, 10 mg/kg^[1]. The treatment with 50 mM 5-Ph-IAA caused a rapid decrease in fluorescence intensity in many embryos (78%, 18/23 embryos), but 5 mM 5-Ph-IAA did not. 5-Ph-IAA can be used for loss-of-function experiments in C. elegans embryos, although treatment with 5-Ph-IAA is not always effective, possibly because the C. elegans embryo develops within the eggshell, which blocks its permeability to many compounds^[2].

References:
[1].Yesbolatova A, Saito Y, Kitamoto N, Makino-Itou H, Ajima R, Nakano R, Nakaoka H, Fukui K, Gamo K, Tominari Y, Takeuchi H, Saga Y, Hayashi KI, Kanemaki MT. The auxin-inducible degron 2 technology provides sharp degradation control in yeast, mammalian cells, and mice. Nat Commun. 2020 Nov 11;11(1):5701.
[2].Negishi T, Kitagawa S, Horii N, Tanaka Y, Haruta N, Sugimoto A, Sawa H, Hayashi KI, Harata M, Kanemaki MT. The auxin-inducible degron 2 (AID2) system enables controlled protein knockdown during embryogenesis and development in Caenorhabditis elegans. Genetics. 2022 Feb 4;220(2):iyab218.