The mutation frequency in Mtb to CGI 17341 resistance was low enough to allow t

Confocal Microscopy Confocal fluorescence images were obtained by using ?20 or ?40 objectives on a laser scanning microscope equipped with a motorized Axioplan microscope, argon laser, HeNe laser, LSM 510 control and image acquisition software, and appropriate filters. Confocal images were exported to Adobe Photoshop software, and montages were prepared. Statistical Analysis We used the Mann Cabozantinib Whitney U test to compare the body weight of mice, tumor weight, the number of Ki 67?positive cells, the MVD, and the number of TUNEL positive cells. Expression of TGF and EGFR in SW620CE2 Parent, SW620CE2 Nontargeting shRNA, and SW620CE2 TGF shRNA Human Colon Carcinoma Cells In Vitro In the first set of experiments, we examined the expression of TGF in SW620CE2 parent, SW620CE2 nontargeting shRNA, and SW620CE2 TGF shRNA cells growing in culture by RTPCR and ELISA. SW620CE2 parent cells and SW620CE2 nontargeting shRNA cells expressed high levels of TGF. The expression of TGF Retroperitoneal lymph node dissection by SW620CE2 TGF shRNA cells was reduced by more than 80%. Because immunohistochemistry as a single parameter may not determine absolute presence or absence of the EGFR on colon cancer cells, we also examined the in vitro expression of EGFR by RT PCR and Western blot analysis. SW620CE2 parent, SW620CE2 nontargeting shRNA, and SW620CE2 TGF shRNA cells expressed minimal levels of EGFR protein or mRNA. HT29 human colon carcinoma cells used as a positive control expressed high levels of EGFR. The SW620CE2 cells do not express the VEGFR2 but do express VEGFA. Transduction with nontargeting shRNA or TGF shRNA did not change these properties. Treatment of SW620CE2 WT, SW620CE2 Nontargeting shRNA, or SW620CE2 TGF shRNA Human Colon Cancer Cells Growing in the Cecum of Nude Mice In the next set of experiments, AG-1478 we determined the therapeutic effects of PKI166, irinotecan, or the combination of PKI166 and irinotecan, and the growth and metastasis of SW620CE2 WT, SW620CE2 nontargeting shRNA, or SW620CE2 TGF shRNA human colon cancer cells growing in the cecum of nude mice. Tumor cells were injected into the cecal wall of nude mice. Treatment began 2 weeks later when the tumors were established. After 5 weeks of treatment, all mice were euthanized and necropsied. All three cell lines produced cecal tumors in all injected mice, suggesting that autocrine paracrine loops of TGF /EGFR are not required for tumor growth. None of the treatments significantly affected body weight. In mice injected with SW620CE2 WT tumors, control mice had the largest tumors. Mesenteric lymph node metastasis was found in 7 of 10 mice. Treatment with only PKI166 significantly reduced the weight of cecal tumors. Three of 10 mice had lymph node metastasis. Treatment with only irinotecan also inhibited tumor growth. Lymph node metastasis was found in 4 of 10 mice. Treatment with oral administration of PKI166 and i. p.