Supplementary Figure 5: LIFR knockdown does not alter proliferation but confers an invasive phenotype in vitro.
(a) Western blot for pSTAT3 (Y705), total Stat3, and β-actin (loading control) in MCF7NSC and MCF7shLIFR (#3 from Supplementary Fig. 1g) following 15 min treatment with PBS (vehicle control), recombinant OSM (50 ng ml−1) or recombinant LIF (50 ng ml−1). (b) SOCS3 mRNA levels in MCF7NSC and MCF7shLIFR cells following 1 h treatment with PBS, recombinant OSM (50 ng ml−1) or recombinant LIF (50 ng ml−1). 3 technical replicates from a single experiment representative of 2 independent experiments. (c) Cell counting assay to assess changes in proliferation in MCF7NSC and MCF7shLIFR cells over 3 days. 2 biological replicates/point. (d) Average tumor volume for MCF7NSC (n = 4 mice) or MCF7shLIFR (n = 3 mice) tumors inoculated into the mammary fat pad (500K cells/fat pad). Student’s unpaired t-test. (e) Images (left) and quantification (right) of scratch wound assay in MCF7NSC and MCF7shLIFR cells in vitro. Scale bar = 200 μm. Student’s unpaired t-test. 2 biological replicates, one each from 2 independent experiments. (f) 3D culture of MCF7NSC and MCF7shLIFR cells in type I rat collagen. Images taken at day 5 (scale bar = 200 μm) and day 9 (scale bar = 50 μm) after tumor cell seeding. Images representative of 3 independent experiments. Source data for Suppl. 5b-e available in Supplementary Table 1 and unprocessed blots in Supplementary Fig. 9. Graphs represent the mean/group and error bars represent standard error of the mean (SEM). ∗P < 0.05, ∗∗∗P < 0.001 and ∗∗∗∗P < 0.0001.##P < 0.01 versus MCF7NSC + LIF treatment.
