Establishment of in-vivo tumor xenograft experiments

DLD-1 cells that possessed stable integrations of GRP94-shRNA and scrambled control sequences were established. 5 weeks old male Nu/Nu mice were used as in-vivo experimental model as described previously ^{31, 32}. 10⁷cells/ml of GRP94-KD DLD-1 and scrambled control cells were suspended in PBS. 0.2ml cell suspension was injected subcutaneously (s.c.) in the flank of mouse. Total 16 mice (8 mice for each group) were used for one set experiment. The body weight and tumor dimensions were measured twice per week. We measured subcutaneous tumors with the equation (L × w²)/2 in accordance with the rules of Taipei Medical University Animal Care and Use. After sacrificing the mice, tumors were excised and weighed. The xenografts were either flash frozen on dry ice and kept at -80°C for analyzing RNA and protein; or fixed in 10% formalin and then embedded in paraffin for IHC (immunohistochemical staining) staining. All the protocols for animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University (LAC-2014-0401).