Kenpaullone

Bovine type II collagen (CII; Chondrex) was dissolved in 0.1 M acetic acid (final concentration, 2 mg/ml). CII was emulsified equally with complete Freund’s adjuvant (CFA; Chondrex) containing 2 mg/ml heat-killed Mycobacterium tuberculosis. Female DBA1/J mice were injected intradermally with 0.1 ml of the CII/CFA emulsion on day 0. Mice then received intradermally with 0.1 ml of 2 mg/ml CII emulsified in incomplete Freund’s adjuvant (IFA; Chondrex) (2 mg/ml) on day 21 [for mice treated with Kenpaullone (a KLF4 inhibitor; Cayman Chemical, Ann Arbor, MI, USA)] and day 28 (for mice transduced with the minicircle vector). Injection of CII/IFA into minicircle-transduced mice took place 7 days later than that into Kenpaullone-treated mice to induce lower activity of arthritis. Severity of arthritis was scored as previously described (19 (link)).

HeLa, HEK293FT, H1299, HT1080, and U2OS cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS; Biosera, Ringmer, UK) and, 50 mg/ml penicillin and streptomycin (regular medium) in a 5% CO₂ incubator. Tetracycline-On (Tet-On) cells were generated by lentiviral transduction with a pCW57.1 vector containing the single-vector Tet-On component. For drug treatment, cells were incubated for the indicated times in medium containing one or a cocktail of the following reagents: 0.2 μM bafilomycin A₁ (LC Laboratories, Woburn, MA, USA), 50 μg/ml brefeldin A (Wako, Osaka, Japan), 1 μM Torin1 (Tocris Bioscience, Ellisville, MO, USA), 10 μg/ml E64d (Peptide Institute, Osaka, Japan), 100 μM pepstatin A (Peptide Institute), 20 μg/ml leupeptin (Peptide Institute), 10 μM kenpaullone (Cayman Chemical, Ann Arbor, MI, USA), 10 μM purvalanol A (Cayman Chemical), 10 μM jervine (Wako), 10 μM BMS-345541 (Cayman Chemical), 2.5 mM HU (Wako), 20 μM etoposide (Cayman Chemical), 5 μg/ml nocodazole (Cayman Chemical), or 1 μg/ml doxycycline (Clontech, Mountain View, CA, USA).