JARID1B downregulation results in the decrease of cell invasion capability.

<p>(A) Western blots confirmed JARID1B expression was suppressed in the two shRNA clones 1 and 2 as compared to the control, and there was correlative down-regulation of Nestin and BCL-xL, while BAX was up-regulated. (B) Wound healing migration assay of SK-N-BE(2) cells show that JARID1B-shRNA infected cells were less motile than control wild type cells (C) Matrigel invasion assay and histogram representation show that JARID1B knockdown induced very significant downregulation of the invasive potential of the SK-N-BE(2) cells (**, p<0.01).</p

Silencing JARID1B downregulates Jagged/Notch signaling transduction pathway.

<p>The expression of Notch and its ligand in wild type and JARID1B-silenced SK-N-BE(2) cells were analyzed by Western blot assay. (A) JARID1B-silencing disrupted Jagged1, Notch1 and Notch 2 signaling. (B) Comparative bar diagrams demonstrate the downregulation of Notch signaling in the wild type SK-N-BE(2) cells, compared to the JARID1B-silenced SK-N-BE(2) cells. The intensity was measured relative to loading control (β-actin) from three independent experiments. *, p<0.05 (C) Immunofluorescent staining showing that JARID1B-silencing downregulated the expression of Notch1. Notch1 (green) colocalizes with JARID1B (red) in the nucleus (DAPI for nuclear staining, blue).</p

JARID1B-silencing suppresses tumorsphere formation and increases cisplatin sensitivity.

<p>(A) Tumorspheres generated from wild type and JARID1B-shRNA 1- and 2- infected SK-N-BE(2) cells. (B and C) JARID1B knockdown caused significant reduction in number and sizes of tumorspheres formed. (D) Cell viability assay show that the cytotoxicity effect of cisplatin was significantly enhanced by JARID1B silencing. All assays were repeated at least three times. * and ** indicate p<0.05 and p<0.001.</p

Neuroblastoma SP cells with higher JARID1B expression are more resistant to chemotherapeutics.

<p>(A) The morphology of SK-N-BE(2), SK-N-DZ, SK-N-AS and SK-N-SH spheroid. (B) Comparative analysis of JARID1B expression in SK-N-BE(2) and SK-N-AS cells, as well as the tumorsphere derived from either cells, SK-N-BE(2)-S and SK-N-AS-S show that the tumorspheres were more enriched in JARID1B and Nestin protein. (C) Immunofluorescence analysis shows SK-N-BE(2) and SK-N-AS tumorspheres expressing JARID1B (red) and Nestin (green) and nucleus marker, DAPI, blue. (D) SRB assay showing that the tumorspheres SK-N-BE (2)-S were more resistant to chemotherapeutics than the parental SK-N-BE (2) cells. Error bars represent the SD from three independent experiments. ** p < 0.01.</p

Silencing JARID1B decreases epithelial to mesenchymal transition (EMT).

<p>(A) Western blot analysis of epithelial and mesenchymal markers’ expression in wild type and JARID1B-silenced SK-N-BE(2) cells show downregulation of N-cadherin and vimentin in response to JARID1B knockdown, while E-cadherin was upregulated. β-actin served as loading control. (B) Bar chart quantification of (A). Expression of EMT markers are normalized against β-actin. Assay was performed three times. *, represent p<0.05.</p

Evaluation of JARID1B expression and stemness functions in <i>MYCN</i> and <i>non-MYCN</i> neuroblastoma (NB) cell lines.

<p>(A) Western blot analysis was performed to investigate the expression of JARID1B and α-MYCN in <i>MYCN</i> amplification (MNA⁺) and <i>non-MYCN</i> amplification (MNA^-) NB cells. (B) The SP percentage was analyzed in MNA⁺ and MNA^- NB cells by Hoechst staining and flow cytometry. SK-N-BE(2) and SK-N-AS cells had 9.05% and 1.45% SP cells (C) Representative Aldeflour assay result of MNA⁺ SK-N-BE(2) and MNA^- SK-N-AS NB cells showed 17.2% and 3.43% ALDH+ subpopulation, respectively. Data was collected from three independent experiments.</p