Fig 1: Sohlh2 regulates IL-8 expression(A) Supervised hierarchical clustering of differentially expressed genes after sohlh2 knockdown in MCF-7 cells. (B, C) The mRNA (B) and protein (C) expression of IL-8 and sohlh2 in breast cancer cell lines. ß-actin was used as a loading control. (D) Measurement of IL-8 secretion in sohlh2-overexpressing MDA-MB-231 and sohlh2-ablated MCF-7 cells by ELISA. E. Inhibition of pGL4-IL-8 luciferase activity by sohlh2 overexpression in MDA-MB-231 cells, and activation of pGL4-IL-8 luciferase activity by sohlh2 silencing in MCF-7 cells. (F) Sohlh2 partially repressed the activation of IL-8 promoter caused by TNFa. ??P < 0.01. (G) ChIP assay was carried out in sohlh2-overexpressing MDA-MB-231 cells. qPCR was performed to determine sohlh2 occupied abundance on IL-8 promoter with 2 primer pairs spanning E-Motifs and one control primer. qPCR results showed that sohlh2 bound to the specific regions of IL-8 promoter. Results represent mean ± SD from three independent experiments. *P < 0.05; **P < 0.01.
Fig 2: IL-8 is required for sohlh2-mediated EMT, migration and invasion(A–C) IL-8 decreased E-cadherin expression but enhanced vimentin expression in sohlh2-overexpressing MDA-MB-231 cells. IL-8 Silencing induced the opposite effects in sohlh2-ablated MCF-7 cells. (D) IL-8 enhanced transwell migration and Matrigel invasion in sohlh2-overexpressing MDA-MB-231 cells. IL-8 silencing inhibited transwell migration and Matrigel invasion in sohlh2-ablated MCF-7 cells. Scale bar = 50 µm. All results are obtained from three independent experiments. *P < 0.05; **P < 0.01.
Fig 3: Sohlh2 suppresses EMT in breast cancer cells(A) Phase contrast photographs show that pShS1-transfected MCF7 cells display fibroblastic morphology with increased cell spreading, while pSohlh2-transfected MDA-MB-231 cells display epithelial morphology. Scale bar = 100 µm. (B) Immunostaining of E-cadherin and vimentin in MDA-MB-231 and MCF-7 cells. The nuclei were stained by DAPI. Scale bar = 50 µm. (C) The mRNA expression of EMT markers including E-cadherin, N-cadherin, Fibronectin and vimentin in sohlh2-overexpressing MDA-MB-231 cells or sohlh2-ablated MCF-7 cells was examined by qPCR. (D) Cell lysates were prepared and the protein expression of EMT markers was analyzed by immunoblotting. ß-actin was used as an internal control. All results are obtained from three independent experiments. *P < 0.05; **P < 0.01.
Fig 4: Sohlh2 inhibits breast cancer metastasis in miceSohlh2–expressing (sohlh2-silencing) or parental cells were injected into BALB/c nude mice via tail vein. The mice were sacrificed after 8 weeks, and tumor colonies in the lung and liver were examined. (A) Representative images of lung samples and sections with H&E staining (right), metastasis nodules formed by parental and sohlh2–expressing MDA-MB-231 cells were quantified (left). (B) Representative images of liver samples and sections with H&E staining at 8 wk after injection were shown (right), metastasis nodules formed by parental and sohlh2–expressing MDA-MB-231 tumors were quantified (left). (C) Metastasis nodules in lungs and livers formed by parental and sohlh2–silencing MCF-7 cells were quantified. (D) Sohlh2–expressing or parental cells were injected in mammary pad of BALB/c nude mice. Spontaneous metastasis nodules in lungs formed by parental and sohlh2–expressing MDA-MB-231cells were quantified. Arrows show metastasis nodules. Scale bars indicate 50 µm.
Fig 5: Reduced expression of sohlh2 is correlated with the metastasis of breast cancer(A) Immunohistochemical staining of Sohlh2 in adjacent tissues of breast cancer (a), intraductal carcinoma in situ (b), breast cancer without or with metastasis (c, d). A representative image from each group is shown. Scale bar = 50 µm. (B) Intensity of sohlh2 staining was scored from 0 to 12. Graph representing the average intensity of sohlh2 staining for adjacent tissues of breast cancer (a) versus intraductal carcinoma in situ (b), breast cancer without or with metastasis (c, d). (C, D) The mRNA and protein expression of sohlh2 were analyzed by qPCR (C) and Western blot (D) in the transformed mammary epithelial cell lines (HBL-100, MCF-10a) and breast cancer cell lines (MDA-MB-231, MCF-7). ß-actin was used as a loading control. *P < 0.05; **P < 0.01.
Supplier Page from Abcam for Anti-Sohlh2 antibody