Figure 6: EpoR expression and Epo inhibition of preadipocyte differentiation.

(a) EpoR mRNA levels in tissues from WT-mice (male, n=3) as indicated including brown adipose tissue (BAT) and WAT were quantified using SYBR green real-time RT–PCR and presented as fold changes relative to mRNA level in liver. (b) Absolute amount of EpoR mRNA in spleen, neonatal cardiomyocytes (CM) and WAT stromal vascular cells (SVC) and fat cells (FC) (n=3) was determined using Taqman PCR. (c) WT-MEF cells treated with Epo at concentrations as indicated were stained with oil red O at the end of differentiation (day 9). (d) Quantification of oil red O staining in differentiated WT-MEF and Tg-MEF cultures was presented as percentage relative to WT-MEF culture without Epo. MDI(−) indicates cultures without differentiation induction. (e) Relative PPARγ and aP2 mRNA levels in 3T3-L1 cells without and with Epo treatment (5 U ml⁻¹) during differentiation (day 6). (f) 3T3-L1 cells (1.5×10⁵/well) were cultured with Epo at 0 (circle, blue-line), 1 (square, green-line), 5 (triangle, yellow-line) and 10 U ml⁻¹ (inverted-triangle, red-line) until reaching full confluence at day 6. Cells were counted daily using Countess automated cell counter (Invitrogen) in three replicate experiments. (g) 3T3-L1 cells were cultured for 2 days after reaching confluence and then for 3 days in differentiation induction medium (MDI) with Epo at concentrations as indicated. Cells were counted at day 3 (three replicate experiments). (h) Similarly, WT- and Tg-MEF cells were cultured in MDI with or without Epo (5 U ml⁻¹), and cells were counted at day 3 (three replicate experiments). (i–j) 3T3-L1 cells were cultured as described in g and assessed with TUNEL staining at day 3. TUNEL(+) cells were counted in 20 randomly selected fields (×200) from each well (six-well plate) of three wells in total for each condition (i). The positive control were treated with DNase I before application of TUNEL staining, and the negative control was stained without terminal deoxynucleotidyl transferase (j). Scale bar=100 μm. Error bars indicate s.e.m. and significance is indicated as **P<0.01 and ***P<0.001 determined by one-way analysis of variance (Dunnet's multiple comparison test) (a,b,d,g) and Student's t-test (e,h).