Journal: Nucleic Acids Research

Article Title: Arxes: retrotransposed genes required for adipogenesis

doi: 10.1093/nar/gkq1289

Figure Lengend Snippet: Multiple alignments of Arxes1, Arxes2 and Spcs3 promoters and transcripts

Article Snippet: Lentiviral silencing particles targeting Spcs3 were obtained from Santa Cruz Biotechnologies (TCR-5).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Arxes: retrotransposed genes required for adipogenesis

doi: 10.1093/nar/gkq1289

Figure Lengend Snippet: mRNA expression patterns of Arxes1, Arxes2 and Spcs3 during in vitro adipogenesis, in mouse tissues and upon in vitro and in vivo rosiglitazone treatment. ( A–C ) Shown are qPCR measurements during the course of adipogenesis in three different cell models. Cells were induced with DMI cocktails 2 days post confluence (d0) as specified in ‘Materials and Methods’ section. Values are expressed relative to d0 measurements. Data are presented as mean ± SEM from two independent MEF isolations or two and three experiments for OP9 and 3T3-L1 cells, respectively. ( D ) Distribution of mRNA expression in metabolically relevant tissues in wild-type mice. Expression is shown relative to heart as fold-expression. Inset represents a scale-up of the Spcs3 expression values. Data are presented as mean ± SEM ( n = 3). Abbreviations: WAT, white adipose tissue (epididymal); BAT, brown adipose tissue (interscapular); L, liver; SM, skeletal muscle; H. heart. ( E ) 3T3-L1 adipocytes were treated with 1 µM rosiglitazone at Day 7 of differentiation. RNA was harvested 24 h after rosiglitazone treatment or medium change (DMEM) and measured with qPCR using the indicated primers. Data are represented as mean ± SEM from three independent experiments. Student’s t -test: ** P < 0.01. ( F ) Male mice were kept on a rosiglitazone-containing chow diet (0.01% w/w) or on normal chow for 7 weeks post-weaning. Prior to harvesting epididymal fat pads mice were fasted for ∼4 h to synchronize nutritional states. Data are represented as mean ± SEM ( n = 5–6). Student’s t -test: ** P < 0.01.

Article Snippet: Lentiviral silencing particles targeting Spcs3 were obtained from Santa Cruz Biotechnologies (TCR-5).

Techniques: Expressing, In Vitro, In Vivo, Metabolic Labelling

Journal: Nucleic Acids Research

Article Title: Arxes: retrotransposed genes required for adipogenesis

doi: 10.1093/nar/gkq1289

Figure Lengend Snippet: Silencing of the Arxes abrogates adipogenesis, while Spcs3 knockdown does not change adipogenic marker gene expression. 3T3-L1 cells were transduced with silencing constructs (siArxes or siSpcs3) or nontargeting control (ntc), and induced to undergo adipogenesis with standard DMI treatment unless indicated otherwise. ( A ) qPCR mRNA measurement shows that Arxes knockdown using the siArxes silencing constructs is specific to the Arxes, while Spcs3 expression is not reduced. Data are presented as mean ± SEM from three independent transduction experiments. ( B ) Lipid droplets were visualized by oil red O staining of 7 days differentiated 3T3-L1 cells. Lower panel of the oil red O staining shows a partial rescue of the differentiation deficiency if 1 µM rosiglitazone is present during differentiation procedure. ( C ) Expression of marker genes of adipogenesis was measured with qPCR on Day 7 of adipogenesis in Arxes-silenced and control cells. Data are presented as mean ± SEM from three independent transduction experiments. ( D ) qPCR mRNA measurement shows that Spcs3 knockdown using the siSpcs3 silencing construct is specific to the Spcs3 while Arxes mRNA is not reduced. Data are presented as mean ± SEM from two independent transduction experiments. ( E ) Spcs3 knockdown does not influence adipogenic marker gene expression as measured with qPCR on Day 7 in DMI induced cells. Data are presented as mean ± SEM from two independent transduction experiments.

Article Snippet: Lentiviral silencing particles targeting Spcs3 were obtained from Santa Cruz Biotechnologies (TCR-5).

Techniques: Knockdown, Marker, Gene Expression, Transduction, Construct, Control, Expressing, Staining