Figure 5.

The co-incubation or pre-incubation with either NESF or NLP block the stimulatory effects of GRL on both gh and pit-1 mRNA levels in somatotrophs. Effects of pre-incubation for 150 min with growth culture media, or either 1 nM NESF or 1 nM NLP (labeled Pre-inc), or 10 nM GRL in combination with 1 nM NESF or NLP (as Co-inc) for 2 h, on the regulation of gh (a) and pit-1 (b) gene expression. For the “pre-incubation”, cells were either incubated with growth culture media alone, or media supplemented with 1 nM NESF or 1 nM NLP for 150 min. Then, cells were again incubated for 120 min with fresh media alone (CT), or media supplemented with 1 nM NESF + 10 nM rodent GRL, or 1 nM NLP + 10 nM rodent GRL⁴⁰. Cells were then washed twice with 1X PBS before the collection of total RNA, as explained below. In the “co-incubation” study, cells were either treated with plain growth media (CT), or with 1 nM NESF + 10 nM rodent GRL or 1 nM NLP + 10 nM rodent GRL for 120 min (no pre-treatment with nesfatin-1 or NLP in this co-incubation study). Four independent experiments with triplicates (n = 3 wells/treatment/experiment) were performed for each study. Data from all four experiments were pooled to conduct statistical analyses and are shown as mean ± SEM (n = 12 wells) relative to the reference genes β-actin and rpl13. Different letters indicate significant differences (p < 0.05) between the different conditions detected by one-way ANOVA test followed by Tukey’s multiple comparison test.