Scrambled control sirna

AS160 siRNA (mouse Tbc1d4 siRNA) was purchased from Santa Cruz. HSL siRNA (Lipe smart pool mouse siRNA) and scrambled control siRNA were obtained from Dharmacon. On day 0, 3T3F442A cells were transfected with siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific) by following the manufacturer’s instructions. Four hr later, cells were changed to differentiation media. On day 5, differentiated 3T3F442A adipocytes were used for further experiments.

The effect of keratinocyte-conditioned media (CM) on TGFβ1 and CTGF expression in normal human dermal fibroblasts (NHDF) was performed as follows. In cases where CM was collected from transfected cells, conditioned media was collected from transfected keratinocytes 24 hours after transfection with FOXO1 siRNA or scrambled control siRNA (Dharmacon) using GenMute siRNA Transfection Reagent (SignaGen Labs). Cells were incubated with 5–10 nM siRNA with transfection reagent for six hours and transferred to standard medium for 16 hours. Cells were transferred to fresh media for the collection of conditioned media. To assess the impact of conditioned media on fibroblasts or human bone marrow-derived mesenchymal stem cells (hBMSCs) the conditioned media was diluted 1:1 with α-MEM medium supplemented with 1% FBS. Therefore, the fibroblasts were incubated in low-serum (total 0.5% FBS) when stimulated with keratinocyte conditioned media. For antibody neutralization studies, anti-TGFβ139 (link) (10 μg/ml, R&D Systems) or anti-CTGF40 (link) blocking antibody (10 μg/ml, Santa Cruz) was added 1 hour prior to incubation of keratinocyte-conditioned media with NHDF or hBMSCs for 3 days.

SMARTpool ON-TARGET plus siRNAs targeting CycA2, CDK1, CDK2, p21 or p53 as well as a scrambled control siRNA were purchased from Dharmacon and used at a concentration of 20 nM using HiPerFect transfection reagent (QIAGEN) and OptiMEM (Invitrogen) at 48 and 24 h before live-cell imaging or fixation.

Barr2 siRNA pool (SMARTpool) and scrambled control siRNA were purchased from GE Dharmacon. On day 0, 3T3-F442A cells were transfected with siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific) by following the manufacturer’s instructions. Four hour later, cells were changed to differentiation media (see previous paragraph). On day 5, differentiated 3T3-F442A adipocytes were used for further experiments.

4 × 10⁵ cells/well (6-well plates) were cultured with 0.5 ml of OptiMEM media (Life Technologies) containing 5 µl Lipofectamine 2000 (Life Technologies) and 50 nM specific siRNAs or scrambled control siRNA (Dharmacon) for 6 h. OptiMEM media were removed, and cells were cultured in 2 ml DMEM supplemented with 10% FBS for 96 h before harvesting.