Fig. 2

Reduction in ANGPTL4 impairs cisplatin efflux and increases in vivo tumor chemosensitivity. a A schematic illustration showing the metastatic xenograft model. MKN74_{Snai1ER:shANGPTL4} cells were injected to form primary xenograft tumors (n = 6 per experimental subgroup, two tumors per mouse). All mice were injected twice with 4-OHT to induce EMT of the MKN74_{Snai1ER:shANGPTL4} and treated with cisplatin. Mice were fed with either normal chow diet (4-OHT:chow) or doxycycline containing diet (4-OHT:dox; 625 mg/kg). Cisplatin treatment (4 mg/kg/week) was administered from week 3 (left panel). Primary xenograft tumors were measured with a pair of Vernier calipers with tumor volume set to be (Length×Width×Width)/2 (right panel). b-c Relative mRNA expression of ANGPTL4 (b) and FACS analysis (c) of cisplatin-DNA adduct-positive among CK18⁺ cells (top panel) and apoptotic (Annexin V-PI positive; bottom panel) cells from 4-OHT:chow and 4-OHT:dox primary xenograft tumors. For real-time PCR, ribosomal 18S (18S) was used as a housekeeping gene. Values are mean ± s.d. from n = 3 independent experiments with 5–6 mice per time point. d Immunofluorescence staining for laminin 332 (green) of 4-OHT:chow and 4-OHT:dox primary xenograft tumors. The nuclei were counterstained with DAPI (blue). Scale bar: 100 μm (top panel). Macroscopic images of lung and liver from mice bearing 4-OHT:chow and 4-OHT:dox primary xenograft tumors. e Relative mRNA expression of human TBP in the lung (top panel) and liver (bottom panel) tissues of cisplatin-treated xenograft mice. Human-specific TBP primers were used to identify and quantify human. Ribosomal 18S primers were used to detect both human and mouse cells for normalization. Values are mean ± s.d. of triplicate runs from 6 mice. f FACS analysis cisplatin-adduct⁺ cells among human CK18⁺ MKN74 cells that metastasized to the lung tissues of 4-OHT:chow and 4-OHT:dox mice. Analysis showed 38.6% (red) CK18-positive cells in 4-OHT:chow lungs compared with 28.61% (red) 4-OHT:dox lungs. Data are represented as mean ± s.d. from n = 3 independent experiments. *P < 0.05, ** P < 0.01