Quantitation of microvessels formed in empty vector and RBMS3-transfected NPC cells induced mouse tumor xenografts (n = 10 tumors/group).

a<p>Values represent mean±standard error.</p>**<p>Statistically significant as compared to control cells.</p

<i>RBMS3</i> arrests cell cycle at the G1/S checkpoint.

<p>(<b>A</b>) Representative and summary of DNA content detected by flow cytometry showed that the percentage of cells in the S phase was lower while the percentage of cells in the G1 phase was higher in SUNE1-RBMS3 cells than that in SUNE1-V1 cells. (* p<0.05, Student’s <i>t</i>-test). Values were expressed as mean ± SD of three independent experiments. (<b>B</b>) Protein expressions of cyclin D1, cyclin E, CDK2, p53, p21, Rb and Rb (Ser780) were compared between <i>RBMS3</i>- and empty vector-transfected NPC cells. β-actin was used as a loading control.</p

Downregulation of RBMS3 in nasopharyngeal carcinoma (NPC).

<p>(<b>A</b>) Expression of <i>RBMS3</i> in 15 primary NPC cases was compared using qPCR between tumor tissues (T) and their paired normal tissue (N). GAPDH was set as an internal control. (<b>B</b>) <i>RBMS3</i> expression was normalized by internal control <i>GAPDH</i>. Statistical analysis confirmed the qPCR results (p<0.001). (<b>C</b>) qPCR analysis of <i>RBMS3</i> expression in three NPC cell lines (C666, CNE2 and SUNE1). The fold changes of <i>RBMS3</i> expression were compared with the immortalized NP cell line, NP460. (<b>D</b>) Immunohistochemical detection of RBMS3 protein in NPC tissue samples and non-tumor nasopharyngeal tissues. Normal: strong positive staining for RBMS3 protein in normal nasopharyngeal epitheliums (arrowhead). NPC1: weak positive staining for RBMS3 protein in NPC tissues (arrowhead). NPC2: negative staining for RBMS3 protein in NPC tissues (arrowhead).</p

<i>RBMS3</i>-silenced NP460 cell showed malignant features.

<p>(<b>A</b>) siRNA against <i>RBMS3</i> effectively reduced the mRNA and protein expression of RBMS3 in NP460 cells compared to siScramble-transfected cells. (<b>B</b>) An MTT assay was used to compare cell growth rates between siRBMS3- and siScramble-transfected NP460 cells (* p<0.05; ** p<0.001, Student’s <i>t</i>-test). Values were expressed as mean ± SD of three independent experiments. (<b>C</b>) Foci formation assay showed that the number of foci was significantly increased in siRBMS3-transfected NP460 cells compared to control cells (* p<0.05, Student’s <i>t</i>-test). The results were expressed as mean ± SD of three independent experiments. (<b>D</b>) Representative and summary of DNA content detected by flow cytometry showed that the percentage of cells in the S phase was higher while the percentage of cells in the G1 phase was lower in siRBMS3-transfected NP460 cells than that in control cells. (* p<0.05, Student’s <i>t</i>-test). (<b>E</b>) Expression of <i>VEGF</i> in siRBMS3- and siScramble-transfected NP460 cells was confirmed by qPCR. (* p<0.05, Student’s <i>t</i>-test). The results were expressed as mean ± SD of three independent experiments. (<b>F</b>) β-catenin was significantly up-regulated while p53 was significantly down-regulated in siRBMS3-transfected NP460 cells compared to control cells by Western blot analysis. β-actin was used as a loading control.</p

A schematic diagram showing the role of <i>RBMS3</i> in malignant transformation of nasopharyngeal carcinoma cell.

<p>Activation →, inhibition →.</p

Ectopic expression of <i>RBMS3</i> induces apoptosis.

<p>(<b>A</b>) Representative images of TUNEL staining. After STS treatment, more apoptotic cells (green) were detected in SUNE1-RBMS3 cells compared to SUNE1-V1 cells (arrowhead). The apoptotic indexes of SUNE1-RBMS3 cells and SUNE1-V1 cells (before and after STS treatment) were summarized in the right panel (*p<0.05). (<b>B</b>) This event was measured by loss of mitochondrial ΔΨm using JC-1 dye. Red or orange fluorescence indicates intact mitochondria, whereas green fluorescence indicated a collapse in mitochondrial ΔΨm. Prior to STS treatment the SUNE1-RBMS3 cells reveal a significant ΔΨ_m loss (green fluorescence, ×400) and higher apoptotic index compared to SUNE1-V1 cells (red/orange fluorescence; left panel) (*p<0.05). (<b>C</b>) The cleavages of caspase-9, caspase-8, and PARP were compared between <i>RBMS3</i>-transfected and empty vector-transfected SUNE1 cells. β-actin was used as loading control.</p

Tumor suppressor function of <i>RBMS3</i> in NPC cells.

<p>Expression of <i>RBMS3</i> in <i>RBMS3</i>-transfected SUNE1 (SUNE1-R4 and R5) and CNE2 cells (CNE2-R1 and R2) was confirmed by quantitative PCR (<b>A</b>) and Western blot (<b>B</b>). Empty vector-transfected cells (SUNE1-V1, CNE2-V1) were used as control. (<b>C</b>) An MTT assay was used to compare cell growth rate between <i>RBMS3</i>- and empty vector-transfected NPC cells. The results found that cell growth rate was significantly decreased in <i>RBMS3</i>-transfected SUNE1 and CNE2 cells (* p<0.05; ** p<0.001, Student’s <i>t</i>-test). Values were expressed as mean ± SD of three independent experiments. (<b>D</b>) Foci formation assay showed that the number of foci was significantly decreased in <i>RBMS3</i>-transfected SUNE1 and CNE2 cells compared to the control cells, respectively (** p<0.001, Student’s <i>t</i>-test). The results were expressed as mean ± SD of three independent experiments. (<b>E</b>) Representatives of tumor formation in nude mice. Tumors induced by SUNE1-V1 (<i>left</i>) and SUEN1-RBMS3 (<i>right</i>) were indicated by arrows, respectively. Excised tumors were shown in the bottom. Summary of tumor growth rates in nude mice induced by <i>RBMS3</i>- and empty vector-transfected NPC cells. The average tumor volume was expressed as mean ± SD in 10 inoculated sites for each group (* p<0.05).</p