T24 cells were allowed to form a monolayer, after which trypsin digestion was used for form a single-cell suspension. The cell density was adjusted to 4 × 107 in 100 μl of PBS and then subcutaneously injected into the right flank of each mouse. Once the T24 cells developed a tumor (tumor volume about 100 mm3), the mice were randomly assigned to four treatment groups (n = 5 mice in each group). The tumors of the mice in each group were injected directly with HNTs/siRIPK4 (20 μg of siRIPK4 per injection, 50:1 mass ratio) or an equal amount of unloaded HNTs, siRIPK4 only, or PBS, respectively. The injections were repeated once a week for 5 weeks. The long and short axial lengths of the tumors were measured every other day. The tumor xenografts were harvested, weighed, and snap frozen for cryosectioning or formalin fixed for paraffin sectioning. H&E was used to stain the histological sections of the tumors. Tumor apoptosis was examined histologically using the TUNEL assay on primary tumor sections using a Colorimetric TUNEL Apoptosis Assay Kit (Beyotime, Haimen, China) according to the manufacturer’s protocol. A Dako Real Envision Kit (K5007, Dako) was then used for immunohistochemical staining with primary antibodies to detect the protein levels.

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