Skip to main content
. 2016 Sep 23;7(46):75366–75378. doi: 10.18632/oncotarget.12207

Figure 4. Colon cancer-derived IGF-1 promotes M2-like macrophage polarization.

Figure 4

A. IGF-1 concentrations were determined by ELISA in SW620 and SW620 EGFR cell culture media. SW620 cells were cultured to 50% confluence and then transfected with human pCDNA6 vector or pCDNA6-EGFR WT plasmid. After 24 h, cells were transferred to normal medium and cultured for an additional 24 h. Conditioned medium from SW620 cells was collected, centrifuged at 3000 rpm for 10 minutes, and stored at −80°C until use. B. IGF-1 concentrations in HCT116 and HCT116 EGFR (left) and HCT116 and HCT116 KO-EGFR (right) cell culture media were measured by ELISA. C. IGF-1 mRNA levels in HCT116, HCT116 EGFR, and HCT116 siEGFR cells were measured by q-PCR. D. IGF-1 mRNA levels in normal, 2AD, and 2AD+cetu mouse tissues were measured by q-PCR. E. Arg1 protein levels were detected by Western blot in Ana-1 macrophages after treatment with mouse recombinant IGF1 (0, 50, 100, or 200 ng/mL) for 48 h. F. Levels of IGF1R signaling pathway-related proteins were detected by Western blot. Ana-1 cells were harvested after treatment with 100 ng/mL mouse recombinant IGF-1 for 5 or 10 min, or after pretreatment with AG1024 for 30 min followed by 100 ng/mL mouse recombinant IGF-1 for 5 or 10 min. G. Protein levels were detected by Western blot in Ana-1 cells. Ana-1 cells were pretreated with AG1024 for 30 min, and medium was then replaced with fresh RPMI 1640 with AG1024 (10 mM) or HCT116 CM with AG1024 (10 mM) followed by an additional 48 h of culture. Cells were harvested and levels of Arg1, iNOS, and IGF1R signaling pathway-related proteins p-IRS(Tyr632), IGF1R, p-p44/42 MAPK(T202/Y204), p-44/42 MAPK(Erk1/2), p-Akt(Thr308), and Akt were measured by Western blot. Bars represent means ± SD (n = 3) for each treatment. *p < 0.05; **p < 0.01.