1. SOX4 Promotes Proliferative Signals by Regulating Glycolysis through AKT Activation in Melanoma Cells 
Wei Dai, Xinyuan Xu, Shuli Li, Jingjing Ma, Qiong Shi, Sen Guo, Lin Liu, Weinan Guo, Peng Xu, Yuanmin He, Guannan Zhu, Liwen Wang, Rui Ge, Yu Liu, Zhe Jian, Gang Wang, Lan Shen, Tianwen Gao, Chunying Li 
Journal of Investigative Dermatology 
Volume 137, Issue 11, Pages (November 2017)
DOI: /j.jid
Copyright © 2017 The Authors Terms and Conditions

2. Figure 1
SOX4 is overexpressed in melanoma. (a) SOX4 mRNA was assessed by quantitative real-time reverse transcriptase-PCR in melanocytic nevus (n = 20) and malignant melanoma (n = 25). Results were normalized against the expression level of β-actin mRNA in each sample. (b) SOX4 protein level was measured by Western blot in melanocytic nevus (n = 5) and malignant melanoma (n = 9). β-Actin served as a loading control. (c) Further confirmation of SOX4 mRNA expression in an additional six pairs of melanoma samples (Ca) and adjacent normal tissue (Nor). (d) SOX4 protein expression was assessed in the same clinical samples as described in (c). GAPDH served as a loading control. (e) Representative images of nuclear SOX4 immunohistochemical staining in melanocytic nevus and human malignant melanoma (scale bar = 100 μm). (f) Statistical analysis of SOX4 nuclear staining in human melanoma samples through the melanoma tissue microarray (n = 100). (g) SOX4 expression is increased in malignant melanoma compared with melanocytic nevus from the NCBI GEO database (GDS1375/201418_s_at and GDS1375/201416_s_at: melanocytic nevus n = 18 and malignant melanoma n = 45). (h) Kaplan-Meier survival curves were plotted for patients with melanoma on the SOX4 level. Data are presented as the mean ± standard deviation, *P < 0.05; **P < 0.01; NS means no significant difference. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SOX4, sex-determining region Y-related high-mobility group box transcription factor 4.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
SOX4 regulates apoptosis and cell cycle arrest. (a, b) Apoptosis and cell cycle analysis by flow cytometry assay after SOX4 knockdown by small interfering RNA in A2058 and WM35. (c, d) Apoptosis and cell cycle analysis by flow cytometry assay after SOX4 overexpression by plasmid in A2058 and WM35. Bar graphs represent the mean values of flow cytometry data. Data are presented as the mean ± standard deviation, *P < (e) Expressions of cyclinD1, cyclinE2, p15, Bcl2, BAX, and cleaved caspase-3 were detected by Western blot after SOX4 knockdown and overexpression in A2058 and WM35. β-Actin was used as loading control. SOX4, sex-determining region Y-related high-mobility group box transcription factor 4.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
SOX4 regulates glucose consumption and lactate production, and controls expression of the metabolic regulators HK2, PKM2, GLUT1, and LDHA. (a, b) Glucose consumption and lactate production were measured after SOX4 knockdown by shRNA. (c) Western blot analysis of GLUT1, HK2, PKM2, LDHA, and SOX4 expression in melanoma cells after SOX4 knockdown. β-Actin was used as loading control. (d, e) Glucose consumption and lactate production were measured after SOX4 overexpression by plasmid. (f) Western blot analysis of GLUT1, HK2, PKM2, LDHA, and SOX4 expression in melanoma cells after SOX4 overexpression. β-Actin was used as loading control. Data are presented as the mean ± standard deviation, *P < GLUT1, glucose transporter type 1; HK2, hexokinase 2; LDHA, lactate dehydrogenase A; PKM2, pyruvate kinase M2 isoform; shRNA, short hairpin RNA; SOX4, sex-determining region Y-related high-mobility group box transcription factor 4.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
SOX4 regulates AKT phosphorylation. (a, b) Western blot analysis of SOX4, total AKT, and phosphorylated AKT at Ser473 and Thr308 expression after SOX4 knockdown and overexpression. (c) Combined treatment of the PI3K/AKT inhibitor LY for 1 hour and SOX4 overexpression by plasmid. Western blot analysis of SOX4, total AKT, and phosphorylated AKT at Ser473 and Thr308 expression. β-Actin was used as loading control. AKT, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; PI3K, phosphatidylinositol-3-kinase; shRNA, short hairpin RNA; SOX4, sex-determining region Y-related high-mobility group box transcription factor 4.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 5
The downstream AKT effectors p70S6K and 4E-BP1, but not GSK3β, are affected by SOX4. (a, b) Western blot analysis of SOX4, total mTOR, p-mTOR, p-4E-BP1 at Thr37/46, p-p70S6K at Ser371 and Thr389, MITF, and PGC1α expression after SOX4 knockdown and overexpression. (c) Western blot analysis of GSK3β and p-GSK3β expression after SOX4 knockdown. GAPDH was used as loading control. AKT, acutely transforming retrovirus AKT8 in rodent T-cell lymphoma; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MITF, micropthalmia-associated transcription factor; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1; shRNA, short hairpin RNA; SOX4, sex-determining region Y-related high-mobility group box transcription factor 4.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 6
Knockdown of SOX4 inhibits the growth and proliferation of melanoma cells. (a) CCK8 tests were performed in A2058 SOX4 stable knockdown cells. (b) Tumor growth was monitored by caliper every 5 days after injection. (c) Tumor weight of mice was calculated 30 days after injection. Data are presented as the mean ± standard deviation, *P < 0.05; **P < (d) Representative tumor formation was photographed 30 days after injection. (e) A suggested model for the regulation of metabolic and cellular pathways in melanoma cells by SOX4. 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; GLUT1, glucose transporter type 1; LDHA, lactate dehydrogenase A; shRNA, short hairpin RNA; SOX4, sex-determining region Y-related high-mobility group box transcription factor 4.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions