Fig 1: ERO1A protein levels correlate with levels of LOXL2 and LAMC2 protein levels.Levels of LAMC2 and LOXL2 are dramatically reduced in conditioned medium collected from ERO1A KO cells. Representative western blots on whole cell lysates and conditioned media in PC-9 cell line are shown (A). Concentration of LAMC2 secreted into conditioned media by ERO1A KO cell lines is reduced compared to control cells. ELISA was performed in triplicate and the average ± S.D. of n = 3 combined results are shown. ****p < 0.0001; Dunnett’s multiple comparisons test following one-way ANOVA (B–C). Representative images of immunofluorescence staining for LOXL2 and LAMC2 in HCC4006 spheroids, scale bar - 50 µm (D–E). Quantification of LOXL2 and LAMC2 immunofluorescent staining in control and ERO1A KO HCC4006 cells cultured as tumor spheres. *p = 0.0209 by two-tailed unpaired t-test, n = 5, experiment was repeated 3 times. ns – not significant (F).
Fig 2: Tumorigenic properties of ERO1A are mediated in part by a secreted factor.Soft agar colony formation assay using regular non-conditioned medium (Normal), medium conditioned by control cells (Conditioned) and conditioned medium from control cells after heat denaturation (Heat denatured), ****p < 0.0001, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 3 (A). Conditioned medium generated by ERO1A KO cells does not rescue colony formation abilities of ERO1A KO cells (B). Representative immunoblot showing level of LAMC2 and LOXL2 knockdown in PC-9 cells. LAMC2 or LOXL2 knockdown dramatically decrease levels of secreted LAMC2 and LOXL2, accordingly (C). Conditioned medium generated by shLOXL2 and shLAMC2 PC-9 cells partially rescue colony formation abilities of ERO1A KO cells in soft agar assay. GFP—empty vector control. Data are means ± SD. ****p < 0.0001, Tukey’s multiple comparisons test following two-way ANOVA. ns – not significant (D).
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