Mm04277571 s1

Cellular RNA and PCR reactions were performed on the whole rat hypothalamus at 3 and 12 weeks of age as previously described in ref.³¹ . The target genes were tumor necrosis factor alpha (TNFα) (Rn99999017_m1), interleukin 1 beta (IL-1β) (Rn00580432_m1), Nuclear Factor-κB (NFκB) (Rn01399566_m1), superoxide dismutase 1 (SOD1) (Rn01477289_m1), superoxide dismutase 2 (SOD2) (Rn00690588_g1), catalase (Rn00560930_m1) and18S ribosomal RNA (Mm04277571_s1) (Applied Biosystems). Level of expression of each target gene of interest was presented as a percentage of the expression of the appropriate housekeeping gene 18S ribosomal RNA. 18S ribosomal RNA was chosen as the most appropriate reference gene for the hypothalamus as it was not affected by the treatments.

Total RNA was extracted from 100 mg of liver using the RNeasy Lipid Tissue Mini Kit (Qiagen, Tokyo, Japan). TaqMan Universal PCR Master Mix (Applied Biosystems, Tokyo, Japan) and Assay-on-Demand, Gene Expression Products (Mn01304289_m1 for ACC1, Mn00662319_m1 for FAS, Mn00550438_m1 for CPT1a, Mn00443579_m1 for ACO1, Mn00441242_m1 for MCP1, Mn00443258_m1 for TNFα, and Mm04277571_s1 for 18S rRNA, all from Applied Biosystems, Tokyo, Japan) were used for quantitative RT-PCR analysis of ACC1, FAS, CPT1a, ACO1, MCP1, TNFα, and 18S RNA expression in the liver. Amplifications were carried out using an ABI Prism 7000 RT-PCR sequence detection system (Applied Biosystems, Tokyo, Japan).

WT/BCR-ABL and Egr1^-/-/BCR-ABL GFP sorted BM, as well as dissociated spleen cells from animals transplanted with WT/BCR-ABL or Egr1^-/-/BCR-ABL BM, were washed in PBS, spun in a centrifuge at 1000 RPM for 5 minutes, then supernatant was removed and cell pellet frozen at -80°C freezer until RNA extraction. RNA was extracted from samples using RNaesy kit (Qiagen) according to manufacturer’s protocol. A reverse transcriptase polymerase chain reaction (RT-PCR) was performed to convert RNA to c-DNA using TaqMan Reverse Transcription reagents (Applied Byosistems) according to manufacturer’s protocol. c-DNA was then used to run the real time polymerase chain reaction analysis (qRT-PCR) in a StepOne Real Time PCR machine (Applied Byosistems) as described previously [38 (link)]. Probes used were the following, all purchased from Life Technologies: Mm00656724_m1 (mouse Egr1), and Mm04277571_S1, for 18, which was used as endogenous control.