Fig 2: Quantitative PCR and immunoblotting of five candidate gene products in DLBCL and LCL lines. (A) Reverse transcriptase and real-time PCR analysis of mRNAs for BMP8A, CCR6, HOXA9, NANOG and S100A8 in LCL and DLBCL (HBL-2, HT, SU-DHL-5, U2932 and U2940) cell lines. Data were normalized by the amount of GAPDH mRNA, expressed relative to the corresponding value for LCL, and are shown as means ± SD from triplicate data. (B) After quantification of Western blot intensity, the relative expression levels of each protein are shown with normalization of GAPDH. U2940 cells have the highest expression of the stem cell genes, HOXA9 and NANOG. Molecular weight listed in parenthesis. Error bars represent the standard error of the mean of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student t-test.

Fig 3: Pathologic findings of DLBCL with expression of five candidate genes. (A) BLS-type DLBCL shows patchy or interstitial infiltration by tumor cells in the bone marrow without sinusoidal involvement (HE stain, 100X). The centroblast-like tumor cells show large, vesicular nuclei with one to several prominent nucleoli (inset, 400X). (B) Immunohistochemical analysis showed that the lymphoma cells involving bone marrow express NANOG in the cytoplasm in contrast to the negative hematopoietic elements (100X; inset, 400X). (C) Lymphoma cells in this nodal case are positive for HOXA9 in nuclei (100X; inset, 400X). (D) S100A8 (MRP8) is positive in the cytoplasm of tumor cells of a nodal case (400X). (E) This case of BLS-type DLBCL involving bone marrow expresses cytoplasmic BMP8B (400X). (F) This nodal case expresses CCR6 in cytoplasm (400X).

Fig 4: Administration of siCCL20 or siCCR6 significantly inhibits CTCL cell metastasis in vivoA. q-RT-PCR for CCL20 in My-La or HH cells transiently transduced with siCCL20 (181), siCCL20 (232), and control siRNA (Scr) for 24 h. The expression observed was as follows: 40.0% and 42.1% in siCCL20 (181)-transduced My-La and HH cells, respectively; 66.7% and 57.4% in siCCL20 (232)-transduced My-La or HH cells, respectively. B. q-RT-PCR for CCR6 in My-La or HH cells transiently transduced with siCCR6 (1058), siCCR6 (705), and control siRNA (Scr) for 24 h. The expression observed was as follows: 32.4% and 32.0% in siCCR6 (1058)-transduced My-La and HH cells, respectively; 43.0% and 57.1% in siCCR6 (705)-transduced My-La and HH cells, respectively. C. Schematic illustration of the migration assay using siRNAs. HS: human serum. D. Migration assay of My-La and HH cells transiently transduced with siSTAT3, siCCL20, siCCR6, and control (Scr). Changes in the migration of the cells were determined relative to that observed with RFU (480 nm/520 nm, relative fluorescent unit); the RFU of cells treated with scrambled (control) was standardized as 1.0. Student's t test was used for examining significance. Asterisks (*) indicate statistical significance: *0.01 = P < 0.05, **0.001 = P < 0.01, n.s.: not significant. Bars are means ± 95% CI (confidence interval) of three independent experiments. E. Schematic illustration of the in vivo protocol of siCCL20 or siCCR6 injection in NOG mice inoculated with My-La cells. F. Kaplan-Meier survival curve of the mouse model of human CTCL inoculated with siCCL20 or siCCR6 are also shown. Log-rank test was used for examining significance. Asterisks (*) indicate statistical significance: *0.01 = P < 0.05, **0.001 = P < 0.01, ***P < 0.001.

Fig 5: Migration inhibition by neutralizing CCL20 antibody in CTCL cellsA. q-RT-PCR for CCR6 and CCL20 in My-La and HH cell lines treated with neutralizing CCL20 antibody (described “CCL20 Ab” in this Figure) for 24 h. n.s.: not significant. B. Migration of the CTCL cells treated with neutralizing CCL20 antibody against My-La or HH cells stably transduced with siCCR6 or miR-150 or control (GFP-empty). The RFU of cells treated with GFP-empty was standardized as 1.0. Student's t test was used for examining significance. Asterisks (*) indicate statistical significance: *0.01 = P < 0.05, **0.001 = P < 0.01. n.s.: not significant. Bars are means ± 95% CI (confidence interval) of three independent experiments. C. Schematic illustration of the migration assay using neutralizing CCL20 antibody is shown. HS: human serum. D. Schematic illustration of the in vivo protocol of neutralizing CCL20 antibody injection in NOG mice inoculated with My-La cells. E. Kaplan-Meier survival curve of the mouse model of human CTCL metastasis. NOG mice were inoculated with neutralizing CCL20 antibody. Log-rank test was used for examining significance. Asterisks (*) indicate statistical significance: ***P < 0.001.