Journal: Biomolecules

Article Title: Mechanistic Target of Rapamycin Complex 1 Signaling Links Hypoxia to Increased IGFBP-1 Phosphorylation in Primary Human Decidualized Endometrial Stromal Cells

doi: 10.3390/biom11091382

Figure Lengend Snippet: Hypoxia treatment inhibits IGF-1R activity in decidualized primary HESCs. CM was collected from decidualized primary HESCs treated under normoxic (N) (incubator air) or hypoxic (H) (1% O 2 ) conditions for 72 h and buffer exchanged with high glucose DMEM media. Equal total IGFBP-1 (50 ng) in the buffer exchanged media were incubated with recombinant human IGF-1 (1 ng) and used to treat P6 cells in culture. P6 cells were lysed and Western blotting was performed (equal loading, 30 µg protein) to determine changes in IGF-1-induced IGF-1R autophosphorylation (Tyr1135) compared to IGF-1 Control 1 (CTR-1). Media with IGFBP-1 (50 ng) from primary decidualized (HESCs) cells under normoxia was considered as Control 2 (CTR-2). The Western blot data are summarized in the bar graphs ( n = 4). Data was first normalized to total IGF-1R followed by β-actin. Values are given as mean + SEM. *, p < 0.05; ** or ## , p = 0.001–0.05 versus control as per one-way ANOVA. Tukey’s multiple comparisons test. * = significant difference compared to IGF-1 positive control (CTR-1); # = significant difference compared to conditioned sample control (CTR-2).

Article Snippet: The treatment was terminated by aspirating the P6 CM and, subsequently, attached P6 cells were lysed using cell lysis buffer (Cell Signaling Technologies) containing protease and phosphatase inhibitors (Sigma) as described earlier [ ].

Techniques: Activity Assay, Incubation, Recombinant, Western Blot, Control, Positive Control