Journal: Cells

Article Title: PI3K/mTOR Dual Inhibitor PF-04691502 Is a Schedule-Dependent Radiosensitizer for Gastroenteropancreatic Neuroendocrine Tumors

doi: 10.3390/cells10051261

Figure Lengend Snippet: Analysis of pAkt (Ser473) expression in NET patient samples. ( A ) Representative pAkt (Ser473) immunohistochemistry staining of patient neuroendocrine tumor samples. Positive staining was observed in the cytoplasm. Patient GEP-NET samples’ ( n = 39) scores were evaluated by a pathologist and the staining intensity was classified using a semi-quantitative seven-tier system. ( B ) Percentage distributions of the cytoplasmic expression of pAkt (Ser473) scored by abundance and intensity is shown in the pie chart.

Article Snippet: Next, sections were blocked for 1 h with 2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA; S-2012). pAkt (Ser473) antibody was diluted in Dako background reducing antibody diluent (Agilent Dako Products, Santa Clara, CA, USA; S3022).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cells

Article Title: PI3K/mTOR Dual Inhibitor PF-04691502 Is a Schedule-Dependent Radiosensitizer for Gastroenteropancreatic Neuroendocrine Tumors

doi: 10.3390/cells10051261

Figure Lengend Snippet: pAkt (Ser473) inhibition by PKI-402 and PF-04691502 in NET cell lines. ( A ) QGP-1 and BON cells were seeded in a 6-well plate at 800,000 cells/2 mL density and treated with PKI-402 from 50 to 1000 nM, and collected at 24 h. Western blot analysis was performed to confirm PI3K/Akt pathway inhibition at 24 h. ( B ) QGP-1 and BON cells were seeded in 6-well plates at 800,000 cells/2 mL density and treated with PF-04691502 from 100 to 10,000 nM, and collected at 24 h. Western blot analysis was performed to confirm PI3K/Akt pathway inhibition. ( C ) QGP-1 and BON cells were seeded in 6-well plates at 800,000 cells/2 mL density and treated with 500 nM PF-04691502. Protein was collected at 24, 48, and 72 h. In addition to pAkt (Ser473) inhibition, downstream targets of the PI3K/Akt pathway, pS6 (Ser235/236) and p4EBP-1 (Thr37/46) inhibition, were confirmed with Western blot analysis. β-actin was used as a loading control.

Article Snippet: Next, sections were blocked for 1 h with 2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA; S-2012). pAkt (Ser473) antibody was diluted in Dako background reducing antibody diluent (Agilent Dako Products, Santa Clara, CA, USA; S3022).

Techniques: Inhibition, Western Blot

Journal: Cells

Article Title: PI3K/mTOR Dual Inhibitor PF-04691502 Is a Schedule-Dependent Radiosensitizer for Gastroenteropancreatic Neuroendocrine Tumors

doi: 10.3390/cells10051261

Figure Lengend Snippet: Patient-derived NET tumor spheroids treatment with PF-04691502. ( A ) Photographs of tumor spheroids in low-attachment plates. GEP-NET patient tumor sample M3210 was a pT3N2M1a grade 2 tumor ileal neuroendocrine tumor, and tumor sample M1893 was a pT3N2Mx small intestinal neuroendocrine tumor. ( B ) GEP-NET tumor spheroids were plated in low-attachment plates and 7 d later protein lysates were collected for GEP-NET origin confirmation by Western blot. Mouse brain and mouse adrenal glands were used as positive controls for SYP and CgA, correspondingly. ( C ) GEP-NET tumor spheroids were seeded in low-attachment plates with equal density and treated with 500 nM PF-04691502 for 24 h. Protein lysates were collected to confirm PI3K/Akt pathway inhibition by pAkt (Ser473), pS6 (Ser235/236) and p4EBP-1 (Thr37/46) by immunoblotting. β-actin was used as a loading control. GAPDH was used as a loading control. ( D ) GEP-NET tumor spheroids were seeded in a 96-well clear round-bottom ultra-low attachment microplate at 10,000 cells / 1 mL density and treated 24 h later with 1000 nM PF-04691502. CCK-8 reagent was added to each well 48 h later; CCK-8-induced colorimetric changes were measured by absorbance at 450 nM, which is directly proportional to the rate of cell proliferation, after 72 h. * denotes p -value < 0.01.

Article Snippet: Next, sections were blocked for 1 h with 2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA; S-2012). pAkt (Ser473) antibody was diluted in Dako background reducing antibody diluent (Agilent Dako Products, Santa Clara, CA, USA; S3022).

Techniques: Derivative Assay, Western Blot, Inhibition, CCK-8 Assay