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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: SOX9 is a dose-dependent metastatic fate determinant in melanoma

Fig. 3

Upregulated or low level of SOX9 expression contributes to the anti-metastatic/anti-oncogenic activities of SOX10 knockdown (KD) melanoma cells. a Expression of SOX10, SOX9, and NEDD9 in human melanocytes (HEMa-LP), and a panel of metastatic melanoma cell lines. GAPDH was used as a loading control. The yellow box indicates protein bands corresponding to the size of SOX9. The red arrow indicates a phosphorylated form of NEDD9. The intensity of protein bands in arbitrary units for SOX10, SOX9, and NEDD9 in each melanoma cell line is relative to HEMa-LP which is set to 1 as a reference. b Line plots represent the intensity of protein bands shown in (a). c qRT-PCR analysis of SOX10, SOX9 and NEDD9 transcript levels in A375M and WM266–4 cells treated with scramble control, SOX10 KD and NEDD9 KD. Data represent fold change normalized to scramble control and the average of three independent assays. d Western blot analysis of SOX9, SOX10 and NEDD9 protein levels in each cell line transduced with scramble control, SOX10 KD and SOX10 KD + SOX9 KD. GAPDH serves as a loading control. The red arrow indicates a phosphorylated form of NEDD9. AlamarBlue (e), transwell invasion (f, g) and colony formation assays (h, i) of each cell line treated with scramble control, SOX10 KD and SOX10 KD + SOX9 KD. g DAPI images of transwell invasion of melanoma cells treated with the indicated constructs. Scale bars: 100 μM (i) Representative images showing crystal violet stained colonies formed from A375M and WM266–4 cells treated with scramble control, SOX10 KD and SOX10 KD + SOX9 KD. j Western blot analysis of SOX10, SOX9 and p21 protein levels in each cell line transduced with scramble control, SOX10 KD alone, SOX10 KD + SOX9 KD, two different volume (100 μL, 200 μL) of lentiviruses encoding SOX9 gene (SOX9 OE) in SOX10 KD and maximum dose of SOX9 OE in parental cells. GAPDH serves as a loading control. Error bars represent mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

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