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. 2013 Jun 13;8(6):e66501. doi: 10.1371/journal.pone.0066501

Figure 2. NLGP normalizes protumor angiogenic and hypoxic TME.

Figure 2

Sarcoma 180 tumor tissue (100 mg) harvested from Swiss albino mice was lysed by freeze-thaw technique in 1 ml PBS supplemented with a cocktail of protease inhibitors. A. Tumor tissue lysates, representing TME from either PBS or NLGP treated mice (n = 6 in each case) were assessed for TGFβ and VEGF by ELISA. Cytokines were quantitated as pg/mg of tissue ± SE. *p<0.001, **p<0.01, in comparison to PBS treated tumor on day 15 and 20. B. Total protein was isolated from PBS and NLGP treated tumors (n = 3 in each case) to assess TGFβ, VEGF, HIF1α, VEGFR1, VEGFR2 and β-actin by Western blot analysis C.1. Total RNA was isolated from tumors of PBS and NLGP treated mice (n = 3 in each case) to analyze genes, like, VEGF, HIF1α, VEGFR1, VEGFR2 and TGFβ at transcriptional level by RT-PCR C.2. Densitometric analysis was performed in each case. Frozen sections of tumors from either PBS or NLGP treated mice were stained D.1. immunohistochemically with monoclonal antibodies, specific for VEGF, VEGFR1, VEGFR2, HIF1α and TGFβ and D.2. with fluorescence tagged anti-CD31 antibody E.1. Total RNA from tumors of PBS and NLGP treated mice was used to determine the status of perforin, granzyme B on day 15 and 20 of tumor inoculation. E.2. Densitometric analysis was performed in each case.