Figure 3
Figure 3. LANA and Kaposin B down-regulate miR-221 and miR-222 levels, resulting in modulation of EC motility. (A) Immunofluorescence staining for LANA proteins. HMEC1 cells with stable LANA expression or the vector control were fixed, and LANA protein was detected by anti-LANA mAb, followed by anti–rat IgG secondary antibody conjugated with rhodamine (red). An enlarged LANA staining pattern is shown in the bottom panel. (B) Immunofluorescence staining and immunoblotting for Kaposin B proteins. HMEC1 cells with stable Kaposin B expression or the vector control were fixed, and Kaposin B proteins were detected by anti-Flag mAb, followed by anti–mouse IgG secondary antibody conjugated with FITC (green). Note that Kaposin B is expressed in the nucleus. Scale bar represents 25 μm. Immunoblotting of Kaposin B proteins with anti-Flag mAb is shown. Actin was used as an internal control (bottom panel). (C) Both LANA and Kaposin B can repress the expression of miR-221 and miR-222. HMEC1 cells with stable LANA (top panel) or Kaposin B (bottom panel) expression were used to detect the expression levels of miR-221 and miR-222 by qRT-PCR. (D) Knocking down LANA expression in HK cells resulted in the restoration of the miR-221 level, but not the level of miR-222. HK cells electroporated with siLANA or siGFP control siRNA were subjected to the Transwell cell-migration assay (top panel; n = 2) and also used to detect the expression level of miR-221 and miR-222 (bottom panel). (E) LANA- or Kaposin B–induced cell migration is countered by miR-221/miR-222. HMEC1 cells with stable LANA (left panel) or Kaposin B (right panel) expression were transduced with vector or the miR-221/miR-222 cluster, and then the cells were subjected to the Transwell cell-migration assay (n = 3). (F) LANA and Kaposin B individually repress miR-221/miR-222 expression by targeting the promoter region. miR-221/miR-222 promoter reporter assays were performed by transfection with different promoter fragments inserted into luciferase reporter plasmids in stably LANA- or Kaposin B–expressing 293T cell lines. Note that LANA and Kaposin B repress miR-221/miR-222 promoter activity by targeting different regions of the promoter, namely the −150 to −200 bp and the −200 to −600 bp regions, respectively (n = 3).

LANA and Kaposin B down-regulate miR-221 and miR-222 levels, resulting in modulation of EC motility. (A) Immunofluorescence staining for LANA proteins. HMEC1 cells with stable LANA expression or the vector control were fixed, and LANA protein was detected by anti-LANA mAb, followed by anti–rat IgG secondary antibody conjugated with rhodamine (red). An enlarged LANA staining pattern is shown in the bottom panel. (B) Immunofluorescence staining and immunoblotting for Kaposin B proteins. HMEC1 cells with stable Kaposin B expression or the vector control were fixed, and Kaposin B proteins were detected by anti-Flag mAb, followed by anti–mouse IgG secondary antibody conjugated with FITC (green). Note that Kaposin B is expressed in the nucleus. Scale bar represents 25 μm. Immunoblotting of Kaposin B proteins with anti-Flag mAb is shown. Actin was used as an internal control (bottom panel). (C) Both LANA and Kaposin B can repress the expression of miR-221 and miR-222. HMEC1 cells with stable LANA (top panel) or Kaposin B (bottom panel) expression were used to detect the expression levels of miR-221 and miR-222 by qRT-PCR. (D) Knocking down LANA expression in HK cells resulted in the restoration of the miR-221 level, but not the level of miR-222. HK cells electroporated with siLANA or siGFP control siRNA were subjected to the Transwell cell-migration assay (top panel; n = 2) and also used to detect the expression level of miR-221 and miR-222 (bottom panel). (E) LANA- or Kaposin B–induced cell migration is countered by miR-221/miR-222. HMEC1 cells with stable LANA (left panel) or Kaposin B (right panel) expression were transduced with vector or the miR-221/miR-222 cluster, and then the cells were subjected to the Transwell cell-migration assay (n = 3). (F) LANA and Kaposin B individually repress miR-221/miR-222 expression by targeting the promoter region. miR-221/miR-222 promoter reporter assays were performed by transfection with different promoter fragments inserted into luciferase reporter plasmids in stably LANA- or Kaposin B–expressing 293T cell lines. Note that LANA and Kaposin B repress miR-221/miR-222 promoter activity by targeting different regions of the promoter, namely the −150 to −200 bp and the −200 to −600 bp regions, respectively (n = 3).

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