Rhs4348

The pTRIPZ lentiviral vectors for DUSP1 (V3THS_407291, V2THS_160994) and non-silencing shRNA control (RHS4743), and the pGIPZ lentiviral vectors for MKP2 (V3LHS_333999, V3LHS_334001) and non-silencing shRNA control (RHS4348) were purchased from Open Biosystems, Huntsville, AL. Packaging was performed using a second generation plasmid transfection system as previously described [36] (link). After infecting AsPC-1, BxPC-3, and COLO-357 cells with lentivirus in the presence of 8 µg/ml polybrene (Sigma-Aldrich, St Louis, MO), cells were selected with 6 µg/ml puromycin. For cells transduced with pTRIPZ lentivirus, DUSP1 levels were assessed by immunoblotting 72 h following the addition of 2 µg/ml doxycycline. Both puromycin and doxycycline were from Sigma-Aldrich (St Louis, MO).

A set of GIPZ Lentiviral shRNA clones directed against mouse HDAC5 (Open Biosystems Huntsville, AL, USA) was assessed by western blot for optimum knockdown efficiency in the hypothalamic cell line CLU177 (mHypoA-2/12; Cellutions Biosystem Inc., Toronto, Canada). Clone V3LMM_432051 was selected for subsequent animal studies due to a knockdown efficiency of 70%. Lentiviral particles for the HDAC5 shRNA (Open Biosystems, Cat # VGM5520–200368831) and the appropriate non-silencing control (Open Biosystems, Cat # RHS4348) were injected bilaterally into the MBH of C57BL/6J mice (10 to 12-week old) (0.5 μl, 10⁸ TU ml⁻¹) using a motorized stereotaxic system from Neurostar (Tubingen, Germany). Stereotaxic coordinates were −1.5 mm posterior and −0.3 mm lateral to bregma and −5.8 mm ventral from the dura. Surgeries were performed using a mixture of ketamine and xylazine (100 and 7 mg kg⁻¹, respectively) as anaesthetic agents and Metamizol (50 mg kg⁻¹, subcutaneous (s.c.)) followed by Meloxicam (1 mg kg⁻¹, three consecutive days s.c.) for postoperative analgesia. Three days after the surgery, the single-housed mice were switched to HFD and daily food intake and body weight were recorded. A knockdown efficiency of 40 to 60% was measured by quantitative (qPCR) from hypothalamic RNA two weeks and 10 weeks after lentivirus administration, respectively.