Fig. 3

Bcl-2 modulates Sema5A expression in melanoma models. a Western blot analysis of Sema5A and Bcl-2 expression in M14 and A375SM-SC1 human melanoma control (empty) and Bcl-2 overexpressing (bcl-2) clones. β-actin was evaluated as control of equivalent transfer and loading. Reported images are representative of three independent experiments with similar results. b Analysis of Sema5A mRNA expression evaluated by qRT-PCR in the indicated cell lines. mRNA levels were normalized using β-actin. Values are expressed as means of ratio ± SEM where ratio was calculated considering Bcl-2-overexpressing versus control clones. Experiments were performed at least three times (three technical replicates). *p-values < 0.05. c Sema5A mRNA amount was evaluated by qRT-PCR in control (empty) and Bcl-2 overexpressing (bcl-2) clones after 0, 90, 180, and 360 min of treatment with Actinomycin D (10 μg/ml). The β-actin (ACTB) gene was used as a reference gene, and the ratio of Sema5A and ACTB in each sample was calculated. Data are shown as mean ± SD (n = 2). d Western blot analysis of Sema5A in A375SM-SC1 control (empty) and Bcl-2 overexpressing (bcl-2) clones treated or not with 10 μM MG132 (Sigma-Aldrich) for 6 h and e relative densitometric analysis performed using Image J software. Representative images of two experiments with similar results are reported. β-actin expression was evaluated to confirm equivalent transfer and loading. f Immunohistochemical analysis of Sema5A expression in M14 control (empty) and Bcl-2 overexpressing (bcl-2) xenografts. Representative images of Sema5A expression in empty (SCORE 1+) and bcl-2 overexpressing (SCORE 3+) xenografts are reported. Scale bar, 20 μm