Journal: Biochemical and biophysical research communications

Article Title: Rebound pathway overactivation by cancer cells following discontinuation of PI3K or mTOR inhibition promotes cancer cell growth.

doi: 10.1016/j.bbrc.2019.04.044

Figure Lengend Snippet: Fig. 4. Blocking IGF-1R prevents drug washout mediated cancer cell growth. (A) Washout of PI3K or mTOR inhibitors increases IGF-1Rb phosphorylation. HT-29 cells were treated for six hours with DMSO, BYL719 (10 mM) (BYL), BKM-120 (500 nM) (BKM), PP242 (5 mM) or Ku0063794 (5 mM) (Ku). Following washout, cells were incubated for six hours, and cell lysates were collected and analyzed for IGF-1Rb phosphorylation and actin. (B) IGF-1R inhibition with NVP-AEW541 blocks AKT phosphorylation mediated by washout of PP242 or BKM-120. HT-29 cells were treated with DMSO, PP242 (5 mM) or BKM-120 (500 mM) for 6 h. Following drug washout, cells were treated as indicated with sapitinib (2 mM), NVP- AEW541 (1 mM) (NVP-AEW) or erlotinib (1 mM) for six hours, and AKT phosphorylation was assessed by Western blot in cell lysates. (C) NVP-AEW541 inhibits cancer cell pro- liferation induced by washout of PP242 or BKM-120. HT-29 cells were treated with DMSO, PP242 (5 mM) or BKM-120 (500 nM) for six hours. Following drug washout, cells were incubated with DMSO or NVP-AEW541 (1 mM) for an additional 48 h. Cell proliferation was assessed with an MTS proliferation assay. Columns: Mean cell proliferation of three independent experiments expressed as percentage of DMSO treated cancer cells. Bars: SD. White columns: DMSO treated cells, grey columns: cells treated with PP242, and black columns: cells treated with BKM-120. *p < 0.05 compared to control cells.

Article Snippet: Anti-phospho-AKT (#4060), anti-AKT (#2920), and anti phospho-IGF-1Rb (#3024) antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Blocking Assay, Phospho-proteomics, Incubation, Inhibition, Western Blot, Proliferation Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Silencing of Insulin-like Growth Factor-binding Protein-2 in Human Glioblastoma Cells Reduces Both Invasiveness and Expression of Progression-associated Gene CD24

doi: 10.1074/jbc.m609567200

Figure Lengend Snippet: FIGURE 2. Modulation of IGF/IGF-IR signaling caused by knockdown of IGFBP-2. The IGFBP-2-knockdown cells (IGFBP-2 KD) and control cells were treated with the indicated concentrations of IGF-I or IGF-II in serum-free con- dition, and extents of phosphorylation of IGF-IR (p-IGF-IR), Akt (p-Akt), and ERK1/2 (p-Erk1/2) were analyzed by immunoblot and a representative result of U251 was shown. The band intensity was measured and the ratio of phos- phorylated protein to the corresponding total protein was calculated.

Article Snippet: The following primary antibodies were used: anti-human IGFBP-2 mouse monoclonal antibody (C-10; Santa Cruz Biotechnology, Santa Cruz, CA), anti- -actinmousemonoclonal antibody (AC-74; Sigma), anti-phosphoERK1/2 rabbit monoclonal antibody (Thr185/Tyr187, clone AW39;Upstate Cell Signaling Solutions, Lake Placid, NY), antiphospho-IGF-IR rabbit polyclonal antibody (Tyr1131), antiIGF-IR rabbit polyclonal antibody, anti-phospho-Akt mouse monoclonal antibody (Ser473), anti-Akt rabbit polyclonal antibody, anti-phospho-ERK1/2 rabbit monoclonal antibody (Thr185/Tyr187, clone AW39; Upstate Cell Signaling Solutions, Lake Placid, NY), anti-ERK1/2 rabbit polyclonal antibody, anticaspase-3, and cleaved capase-3 (Asp175) rabbit polyclonal antibodies, and anti-heat shock protein 70 rabbit polyclonal antibody (Cell Signaling Technology, Danvers, MA).

Techniques: Knockdown, Control, Phospho-proteomics, Western Blot