Journal: Oncogene

Article Title: IGFBP-3 methylation-derived deficiency mediates the resistance to cisplatin through the activation of the IGFIR/Akt pathway in non-small cell lung cancer.

doi: 10.1038/onc.2012.146

Figure Lengend Snippet: Figure 4. Differential activation of EGF and IGFI receptors in 41S and 41R cell lines. (a) The protein levels of pEGFR, EGFR, pIGFIR and IGFIR were measured by WB. Cells were seeded and 24 h later incubated in serum-depleted medium for 24 h, then treated with the indicated CDDP doses for 6 h or with EGF or IGFI for 30 min as control stimuli. Total cell protein (20 mg) was subjected to WB and the membranes were hybridized with antibodies against pEGFR, pIGFIR, EGFR and IGFIR. (b) The 41R cell line were transiently transfected with pCMV5 (|) or with pCMV5-IGFBP-3 vectors, then cells were treated as in a. (c and d) IGFIR and EGFR cellular localization analyzed by immunofluorescence in 41S and 41R cell lines. Cells were grown on coverslips, 24 h later cells were shifted into medium containing 0.5% fetal bovine serum for 16 h, then cells were treated or not with IGFI or EGF as positive controls for 30 min, subsequently the coverslips were fixed and incubated with antibodies against IGFIR or EGFR, and then with a secondary antibody conjugate (fluorescent dye alexa fluor 488, Invitrogen, Carlsbad, CA, USA). The immunofluorescence was visualized with a confocal microscope.

Article Snippet: Antibodies used were anti-AKT (BD Biosciences, San Jose, CA, USA), pAKT-Ser473, PTEN, EGFR, pEGFR-Tyr1068 (1H12, WB), pEGFR-Tyr1068 (D7A5, IHC; Cell Signaling, Danvers, MA, USA), anti-IGFIR, anti-pIGFIRTyr1161 (Santa Cruz Biotechnology, Heidelberg, Germany), anti-phospho AKT-pS473 (Dakopatts, Glostrup, Denmark) and anti-a-tubulin (SigmaAldrich).

Techniques: Activation Assay, Incubation, Control, Transfection, Microscopy

Journal: Oncogene

Article Title: IGFBP-3 methylation-derived deficiency mediates the resistance to cisplatin through the activation of the IGFIR/Akt pathway in non-small cell lung cancer.

doi: 10.1038/onc.2012.146

Figure Lengend Snippet: Figure 5. Correlation between IGFBP-3 expression and IGFIR/AKT activation in different human cancer cell lines. The cell lines H1299, PANC-1, H727 and HT29 were transiently transfected with 1.5 mg pCMV5 (|) or with pCMV5-IGFBP-3 (IGFBP-3) vectors and 24 h later treated with CDDP at indicated doses for 6 h. Then RNA and protein extracts were obtained in parallel experiments. (a) IGFBP-3 overexpression was confirmed by RT–PCR in all cell lines and GAPDH mRNA was co-amplified as a loading control. (b and c) Total cell protein (20 mg) was subjected to WB and then membranes were hybridized with antibodies against pIGFIR, IGFIR, pEGFR, EGFR, p-AKT, AKT and a-tubulin as a loading control.

Article Snippet: Antibodies used were anti-AKT (BD Biosciences, San Jose, CA, USA), pAKT-Ser473, PTEN, EGFR, pEGFR-Tyr1068 (1H12, WB), pEGFR-Tyr1068 (D7A5, IHC; Cell Signaling, Danvers, MA, USA), anti-IGFIR, anti-pIGFIRTyr1161 (Santa Cruz Biotechnology, Heidelberg, Germany), anti-phospho AKT-pS473 (Dakopatts, Glostrup, Denmark) and anti-a-tubulin (SigmaAldrich).

Techniques: Expressing, Activation Assay, Transfection, Over Expression, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Oncogene

Article Title: IGFBP-3 methylation-derived deficiency mediates the resistance to cisplatin through the activation of the IGFIR/Akt pathway in non-small cell lung cancer.

doi: 10.1038/onc.2012.146

Figure Lengend Snippet: Figure 6. Combined use of EGFR, IGFIR and AKT protein phosphorylation together with IGFBP-3 methylation status predicts a potential response to CDDP for NSCLC. A total of 25 NSCLC tissue samples were analyzed for stain localization and intensity. Immunohistochemistry (IHCs) were performed using polyclonal antibody that specifically recognizes p-EGFR or pIGFIR or p-AKT. Same samples were previously used for the analysis of IGFBP-3 gene methylation status measured by MSP.16. (a) Example of IHC analysis in the examined tumor samples. Primary tumor 17 presents positive p-EGFR staining, without p-IGFIR and p-AKT staining, whereas patient number 19 presents positive p-IGFIR and p-AKT staining without significant p-EGFR stain. (b) Nomogram representing prior probability of positive-negative test (both of them with a 50% of chance for being positive or negative) and the post-test probability.

Article Snippet: Antibodies used were anti-AKT (BD Biosciences, San Jose, CA, USA), pAKT-Ser473, PTEN, EGFR, pEGFR-Tyr1068 (1H12, WB), pEGFR-Tyr1068 (D7A5, IHC; Cell Signaling, Danvers, MA, USA), anti-IGFIR, anti-pIGFIRTyr1161 (Santa Cruz Biotechnology, Heidelberg, Germany), anti-phospho AKT-pS473 (Dakopatts, Glostrup, Denmark) and anti-a-tubulin (SigmaAldrich).

Techniques: Phospho-proteomics, Methylation, Staining, Immunohistochemistry