Supplementary figures

Supplementary figures

Files in this Data Supplement:

• Supplementary figure 1: Induction and half life of c-myc mRNA in G0/G1-arrested Y1 adrenocortical cells maintained in SFM. - (A) cells were treated with FGF2 (1nM) and/ or ACTH (1nM) for the indicated times, lysed and processed to extract total RNA used to assay for c-myc-mRNA levels by Northern blot (15µg total RNA per lane). (B) Cells pre-treated or not with actinomycin D (5µg/mL, 0.5h before hormones) were treated for 3h with FGF2 (1nM) and/or ACTH (1nM) and processed for RNA extraction and Northern blot. (C) Cells maintained in SFM were, first, treated with FGF2 (1nM) and/or ACTH (1nM) for 3h and then submitted to actinomycin D (5µg/mL). At the indicated times cells were lysed and processed to extract RNA and assay for c-myc-mRNA by Northern blot. Hybridization of the same membranes with GAPDH probe was used for normalization. Northern blots were quantitated by densitometry (see Table 1).
• Supplementary figure 2: - Western blot analysis to estimate c-Myc protein steady state levels for determination of c-Myc half life under several conditions. G0/G1-arrested Y1 cells were first treated for 4 hours with 10% fetal calf serum (10% FCS), 1nM FGF2, 1nM ACTH, or left in serum free medium (SFM). Second, cycloheximide (10µg/ml) was added to the medium and cells were harvested and lysed, after the indicated periods of time. Cell lysates were used for Western blotting and cMyc steady state levels were estimated by densitometry. The same membranes were probed with anti-JunD for normalization. Relative values of c-Myc levels fitted well into a straight in semi-log plots of log c-Myc levels in function of time, allowing graphic determination of half life from the slope of the straight line. 10% FCS, % FCS+ACTH and SFM+FGF2 yielded reliable determinations of half life, whereas SFM and SFM+ACTH with fewer time points for c-Myc levels led to poor determinations of half life (see Table 2).
• Supplementary figure 3 - ACTH and FGF2 have no effect on levels of Max and Mad proteins in Y1 adrenocortical cells. G0/G1-arrested Y1 cells were treated with ACTH (1nM) and/or FGF2 (1nM). (A) Max protein levels assayed by immunocytochemistry, after ACTH and/or FGF2 for 4h. (B) Mad proteins levels assayed by Western blot analysis, after ACTH and/or FGF2 for the indicated times.