Extended Data Fig. 7. Gain- and loss of function experiments in cultured cells.

a, Lentiviral constructs were modified from pLKO. 1, a generic vector for expressing human RNU6-1 promoter-driven short hairpin RNAs (red loop). LTR, long terminal repeat;ψ, retroviral packaging element, RRE, Rev response element; cPPT, central polypurine tract; PGK, phosphoglycerate kinase promoter; H2B-GFP, Hist2h2be fused to GFP cDNA; P2A, 2A peptide, b, Western blot shows expression of ALX3 in nuclear extracts of B16-F1 cells. Positive controls were extracts from mouse embryonic mesenchyme (MEM) or COS cells transfected with a pcDNA-ALX3 expression vector. COS cells transfected with empty pcDNA served as negative controls. ACTIN immunoreactivity is shown for the same extracts as a control. c, Quantitative PCR showing mRNA levels of cells transduced with LV-Alx3:GFP, relative to cells transduced with the LV-GFP control (LV-Alx3:GFP vs. LV-GFP, two-tailed t test; n = 3. Red bars indicate the mean), d, Quantitative PCR showing mRNA levels of cells transduced with shRNA lentiviral constructs, relative to cells transduced with a scrambled control (shRNAl, 2, 3, or 4 vs. shRNA scrambled, two-tailed t test; n = 3; statistically significant differences [P < 0.05] are indicated by different letters. Red bars indicate the mean).