Sirnas for

siRNAs for Fox and KLF16 genes used in this experiment were purchased from Sigma. The KLF 16 expression vector (pCMV-KLF16) was kindly provided by Dr. Urrutia. Brown preadipocytes and 3T3-L1 preadipocytes were transfected with siRNA or KLF 16 expression vector using the lipofectamine RNA iMAX reagent kit or lipofectamine LTX with PLUS Reagent kit (Invitrogen). The transfected cells were differentiated in differentiation medium 2 days after transfection.

Mouse minigene and Human minigene were transfected into Mouse fibroblasts and Neuro2a following standard protocols with a 3:1 FuGENE^® 6 Transfection Reagent:DNA ratio (Promega). For silencing, 20 nM siRNAs for PTB and nPTB27 (link) and 20 nM siRNA for hnRNP H (Sigma) were transfected separately into 28 × 10³ SK-N-SH cell line with the INTERFERin^® reagent (Polyplus transfection). Cells were grown overnight and fresh complete medium was added to repeat the siRNA assay. Cells were grown for additional 48 h for siPTB and sinPTB assays and 24 h for sihnRNP H assay. Total RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Total RNA (1 μg) was reversetranscribed using random hexamer primers (Promega) and cDNA was then amplified by PCR using the FastStart Taq DNA Polymerase (ROCHE) in a total volume of 50 μL with primers specifically designed to amplify processed transcripts derived from the minigene (95 °C for 4 minutes, followed by 95 °C for 30 seconds, 54 °C for 30 seconds, 72 °C for 45 seconds, and final extension at 72 °C for 7 minutes). Each transfection experiment was performed at least in triplicate.