Extended Data Fig. 4: FBXL7 silencing promotes cell migration.

a, MCF-7 cells were transfected with two different siRNAs to FBXL7 or a non-targeting (NT) siRNA oligo (left panels). PL45 cells were transfected with an siRNA oligo to FBXL7 or NT siRNA (right panels). Twenty-four hours after transfection, whole-cell extracts were immunoblotted as indicated.

b, PNT1A cells were transfected with an siRNA oligo to FBXL7 (FBXL7 si#1) or a non-targeting (NT) siRNA. Twenty-four hours after transfection, whole-cell extracts (WCE) were denatured and subjected to immunoprecipitation (IP) with anti-ETS1 antibody followed by immunoblotting. ETS1 phosphorylated on Tyr was detected with an anti-phospho-Tyr antibody (PY20).

a, b: Two independent experiments were performed with similar results.

c-d, PNT1A cells were transfected with an siRNA oligo to FBXL7 (FBXL7 si#1) or a non-targeting (NT) siRNA. Twenty-four hours after, cells were treated with either vehicle alone or two c-SRC kinase inhibitors [SU6656 or dasatinib] for an additional twenty-four hours, and either re-plated (c) or seeded on collagen type I-coated transwells (d). In c, after 18 hours, a wound-healing assay was performed up to 48 h in presence or absence of SU6656 or dasatinib. The graph shows a representative experiment out of two, each performed in triplicate. Mean ± s.d. is shown; 24 h, *** P = 0.00093 (NT vs. FBXL7 siRNA), 48 h, *** P = 0.00032 (NT vs. FBXL7 siRNA). P values are from unpaired, two-tailed t-test. In d, after 5 h, cells that migrated on the bottom of the transwells were counted in 10 different fields/well. The graph shows a representative experiment out of two performed for each condition. Mean ± s.d. is shown. n = 10. P values are from unpaired, two-tailed t-test.

e, PNT1A cells were transfected with an siRNA oligo to FBXL7 (FBXL7 si#1) or a non-targeting (NT) siRNA. Twenty-four hours after, cells were re-plated in 6-weel plates in a soft agar layer. After three weeks, the colonies were stained with nitro-blue-tetrazolium and photographed. PC-3 cancer cells were used as positive control.

f, PNT1A cells treated as in c-d, were analyzed by immunoblotting.

g, PC-3 prostate carcinoma cells were transfected with FLAG-tagged FBXL7 or an empty vector (EV). Twenty-four hours after transfection, cells were plated on 96-well plate in triplicates, allowed to adhere and, after 18 hours, assayed for cell motility through a wound-healing assay. The graph shows quantification from two independent experiments. e-g, n = 3 independent experiments. In g, mean ± s.d. is shown. P values are from unpaired, two-tailed t-test.