Figure 1.
Preparation of WRN CM by Lentiviral Infection
(A and B) Wnt activities of recombinant hWNT3A or mWNT3A (A) and recombinant hRSPO1 or mRSPO1 (B). Activities in the absence of the hRSPO1 expression plasmid and recombinant proteins were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. n.s., not significant (Student’s t test).
(C–H) Mouse L cells stably expressing WRN were generated by lentiviral infection of each gene. (C–E) Wnt signaling activities of CM from L-WRN cells at densities of 2.8, 1.4, 0.7, and 0.35 × 106 cells/35-mm dish after 48 hr of culture (C); at a density of 1.4 × 106 cells/dish cultured between 24 and 96 hr (D); and at 1.4 × 106 cells/dish cultured for 72 hr in diluted CM or 300 ng/mL recombinant mWNT3A, with or without an hRSPO1 expression plasmid (E). Activities in the absence of WRN CM were considered as “1.” The assays were performed in three independent biological replicates. Data expressed as mean ± SEM. (F) Western blot analysis of the supernatants used in (C) undiluted (8 μL) and 4-fold diluted, and the recombinant proteins mWNT3A (1 and 0.3 ng), hRSPO1 (30 and 10 ng), and hNOG (10 and 3 ng) using anti-WNT3A, anti-RSPO1, and anti-NOG antibodies. (G) Images of human primary ileal organoids cultured in 25% WRN CM or the corresponding recombinant proteins for 7 days after a passage. Scale bars, 200 μm. (H) Wnt signaling activities of WRN CM (×1, ×1/3, ×1/10) stored in the deep freezer for 0, 6, or 12 months. The activities in the absence of CM were considered as “1.” The assay was performed in three independent biological replicates. Data expressed as mean ± SEM.