Fig. 2: Lin28b expression in CAFs is induced by conditioned medium from Lin28bhigh tumor cells. | Nature Communications

Fig. 2: Lin28b expression in CAFs is induced by conditioned medium from Lin28bhigh tumor cells.

From: The Lin28b/Wnt5a axis drives pancreas cancer through crosstalk between cancer associated fibroblasts and tumor epithelium

Fig. 2

a, b Real-time qPCR analysis (a) and western blotting analysis (b) for Lin28b expression in mCAFs cultured alone or co-cultured with indicated tumor cells. Representative of n  =  3 independent experiments (b). c, d 14837CAFs were direct co-cultured with 14837T or 15376T for six days and then sorted by flow cytometric cell sorting (FACS) (c). The levels of Lin28b were measured by real-time qPCR (d). e–j 14837CAFs (e–g) or 15376CAFs (h–j) were ‍cultured with 15376T-CM for up to 6 days. CM was derived from 15376T with low density (30–40%) or high density (80–90%). Cells were collected for real-‍time qPCR (e, h) and Western blotting (f, g, i, j) at indicated time points. Lin28b levels in 15376T were included as a positive control. Representative of n  =  3 independent experiments (f, g, i, j). k, l 14837CAFs (k) or 15376CAFs (l) ‍were cultured with 15376T-CM or Lin28b-KO 15376T-‍CM for 6 days. Then, Lin28b levels were measured by western blotting. Lin28b levels in 15376T were included as a positive control. Representative of n  =  3 independent experiments. m, n The levels of LIN28B in human PDAC cell lines were measured by real-time qPCR (m) and western blotting (n). Representative of n  =  3 independent experiments (n). o human CAFs (hCAFs) were cultured with CM from human PDAC cell lines for 6 days. Then, LIN28B levels were measured by western blotting. Representative of n  =  3 independent experiments (p–r) hCAFs were cultured with PANC-1-CM (p), LIN28B-KO PANC-1-CM (p), PANC03.27-CM (q), LIN28B-KO PANC03.27-CM (q), hPDAC1#-CM (r), and LIN28B-KO hPDAC1#-CM (r) for 6 days. Then, the levels of LIN28B were measured by western blotting. LIN28B levels in PANC-1 (p), PANC03.27 (q), and hPDAC1# (r) were included as a positive control. Representative of n  =  3 independent experiments. s Orthotopic PDAC tumors generated with 15376T or Lin28b-KO 15376T were analyzed by Lin28b and α-SMA immunofluorescence staining (n = 6 mice). Representative images are shown. Scale bar: 30 μm. Three biologically independent experiments were performed (a, d, e, h, m). Data are shown as mean ± s.d. P-values were determined by one-way ANOVA with Tukey’s multiple comparison test (a, e, h, m) or two-tailed unpaired Student’s t-tests (d).

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