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. 2021 Jun 18;7(25):eabf4885. doi: 10.1126/sciadv.abf4885

Fig. 4. Secreted PLAU inhibits anoikis and promotes metastatic colonization.

Fig. 4

(A) Western blot confirmation of target gene depletion in siRNA-transfected 344SQ cells. (B to D) Lung metastases in tail vein–injected syngeneic, immunocompetent mice were imaged (B) and quantified on the basis of mean number per mouse (C) and mean size (D). (E) Colonies formed in soft agar by siRNA-transfected H1299 cells and CALU-1 cells. Values expressed relative to siCTL. (F and G) Western blot analysis of cleaved PARP1 (F) and annexin V/propidium iodide flow cytometry (G) to detect apoptotic cells under adherent (Adh.) and suspension (Susp.) conditions. (H) Western blot analysis of C-PARP1 in siRNA-transfected H1299 cells treated for 2 days with (+) or without (−) recombinant PLAU (10 ng/ml). (I) Annexin V/propidium iodide flow cytometry of H1299 cells in (H). (J and K) Western blot analysis of cleaved PARP1 (J) and annexin V/propidium iodide flow cytometry (K) to detect apoptotic cells following treatment with CM samples or fresh medium (−). (L) Annexin V/propidium iodide flow cytometry of 393P murine LUAD cells cotransfected with PAQR11 or empty (Vec) expression vectors and siPLAU or siCTL. (M) Lung metastases generated in syngeneic, immunocompetent mice by tail vein injection of shRNA-transfected 344SQ cells alone or in combination with 10 μg of recombinant PLAU (+PLAU; in 100-μl cell suspension). Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.