LRIG2 and LRIG2ecto overexpression promote the growth of glioblastoma cells <i>in vitro</i>.

<p>(<b>A</b>) Time-course effects of LRIG2 or LRIG2ecto overexpression on the growth of glioblastoma cells. The proliferation rates of cells were measured by CCK8 assay at the times indicated, which revealed that both LRIG2 and LRIG2ecto overexpression cells proliferate more rapidly than control cells (*<i>P</i><0.05 vs con). (<b>B</b>) Representative images of immunofluorescent staining of Ki-67 were shown in the upper panel (scale bars, 50 µm). The percentages of Ki-67-positive cells in the LRIG2 and LRIG2ecto group were compared with that of the control group, respectively, shown in the lower panel (*<i>P</i><0.05 vs con). (<b>C</b>) Soft agar colony formation assay was used to detect the anchorage-independent proliferation in the forementioned cells. The representative high-resolution images were shown (scale bars, 100 µm) and the quantitative analysis of colony numbers per microscopic field and colony sizes were present in the lower panel (compared with control, *<i>P</i><0.05 vs con). All experiments were performed at least three times with consistent and repeatable results.</p

Establishment of glioblastoma cell lines stably expressing LRIG2 or LRIG2ecto.

<p>(<b>A</b>) Schematic drawing of the domain organization of the Flag tagged full-length LRIG2 and LRIG2 ectodomain. Indicated are the signal peptides (SP), Flag tag (Flag), the leucine-rich repeat domains, consisting of cysteine-rich N-flanking domain (NF), 15 leucine-rich repeats (LRR) and cysteine-rich C-flanking domain (CF), three immunoglobulin-like domains (Ig-C2), the transmembrane domain (TM) and the cytoplasmic tail (Cyto). (<b>B</b>) The total cell lysates of stably transduced cells, cultured in complete medium for 48 h, were subjected to immunoblotting using an anti-Flag antibody. β-Actin served as an internal loading control. (<b>C</b>) After maintained in complete medium for 48 h, the cells were subjected to total RNA extraction, followed by quantitative RT-PCR to measure the LRIG2 and LRIG2ecto mRNA expression levels. Expressions are shown as the fold changes of control cells (**<i>P</i><0.01, vs con). (<b>D</b>) After cultured in complete medium for 48 h, stably transduced cells were subjected to immunofluorescence analysis. Flag staining is green, and nuclear stain is blue. Representative images of three independent experiments were shown. Scale bars, 50 µm.</p

LRIG2 or LRIG2ecto overexpression enhances EGFR signaling and regulates the cell cycle and apoptosis related proteins.

<p>(<b>A</b>) After synchronization for 24 h, cells were cultured in DMEM with 10% FBS for 48 h and cell lysates were subjected to western blotting for the levels of EGFR signaling related proteins. Analysis of quantification of the bands intensity was shown in the lower panel (*<i>P</i><0.05 vs con). (<b>B</b>) The indicated cells were starved for 24 h and cultured in complete medium for 48 h. The cell lysates were exacted and examined by western blotting for the cell cycle and apoptosis associated proteins. Analysis of quantification of the bands intensity was shown in the lower panel (*<i>P</i><0.05 vs con).</p

LRIG2 expression positively correlates with the WHO grade of glioma.

<p>(<b>A</b>) Microarray-based gene expression data of brain low grade gliomas (LGGs) and glioblastoma multiform (GBMs) were downloaded from public TCGA website. The expression level of LRIG2 gene in GBM was much higher compared with that in LGG (<i>P</i><0.001). (<b>B</b>) Human gliomas of different grade were immunostained for the LRIG2 protein. Representative images of each grade of glioma were present (scale bar, 100 µm).</p

LRIG2 or LRIG2ecto overexpression inhibits the spontaneous apoptosis of glioblastoma cells <i>in vitro</i>.

<p>(<b>A</b>) Cells were cultured in DMEM with 10% FBS for 48 h, stained by Annexin V/PI and subjected to flow cytometry analysis. Early apoptotic populations were in the lower-right quadrant and late apoptotic cells were in the upper-right quadrant. Representative images were shown in the left panel and percentages of early and late apoptotic cells were expressed as the mean±SD of three independent experiments, shown in the right panel (*<i>P</i><0.05 vs con). (<b>B</b>) Cells were maintained in complete medium for 48 h, stained with JC-1 and analyzed by flow cytometry. Representative flow cytometric images of mitochondrial membrane potential were shown. The shift of JC-1 fluorescence from red (FL2) to green (FL-1) indicates a collapse of the mitochondrial membrane potential. Change in mitochondrial membrane potential was determined by the ratio of JC-1 red fluorescence intensity to JC-1 green fluorescence intensity, shown in the right panel. Ratio was normalized to the control cells. (*<i>P</i><0.05 vs con).</p

The detection of soluble LRIG2 ectodomain in the supernatants and its proliferative effects on glioblastoma cells.

<p>(<b>A</b>) Cells of U87-Con, U87-LRIG2 and U87-LRIG2ecto were cultured in DMEM for 48 h, the conditional cell culture supernatants were harvested, concentrated and subjected to western blot using an anti-Flag antibody. A representative blot from three independent experiments is shown. (<b>B</b>) After cells were cultured in DMEM for 48 h, the supernatants were harvested, filtered through 0.45 µm filter and subjected to immunoprecipitation to eliminate the expression of Flag-fusion proteins. The supernatants with or without immunoprecipitation were concentrated and subjected to western blotting. Representative image was shown. (<b>C</b>) The conditioned mediums after or before immnoprecipitation were subjected to proliferation assay. Statistical analysis was present (*<i>P</i><0.05 vs con).</p

Overexpresson of LRIG2 or LRIG2ecto promotes tumor xenograft growth <i>in vivo</i>.

<p>(<b>A</b>) Photographs obtained on postimplantation Day 50 showing representative mice bearing U87-con, U87-LRIG2 and U87-LRIG2ecto xenografts. The corresponding surgically removed tumor xenografts were shown in the lower panel. (<b>B</b>) Tumor size was measured every five days from postimplantation day 15 and the growth curves of xenografts of each group were present (five mice per group). Data were shown as means±SD (**<i>P</i><0.01 vs con). (<b>C</b>) The surgically removed tumor xenografts were fixed in 4% paraformaldehyde and subjected to immunohistochemistry staining of Ki-67, PCNA and Caspase3. The representative images were shown (scale bars, 100 µm). The percentages of immunostaining positive cells of Ki-67, PCNA and Caspase3 in the LRIG2 and LRIG2ecto group were compared with that of the control group, respectively, shown in the lower panel (*<i>P</i><0.05 vs con).</p

LRIG2 as well as LRIG2ecto interacts with EGFR in glioblastoma cells.

<p>(<b>A</b>) Confocal immunofluorescence laser microsopy showed colocalization of LRIG2 and EGFR in LRIG2 overexpresing U87 and U251 glioblastoma cells. Cells were stained with anti-LRIG2 and anti-EGFR antibodies, followed by using Cy3- and FITC-conjugated secondary antibodies. Yellow of merge panel indicates regions of colocalization. Scale bar, 50 µm. (<b>B</b>) Lysates from cells overexprssing LRIG2 or LRIG2ecto were immunoprecipitated with purified mouse anti-Flag monoclonal antibodies or control mouse IgG, and immunoprecipitates were blotted with antibodies to EGFR or Flag.</p

Overexpresson of LRIG2 or LRIG2ecto enhances the EGFR signaling in serum-free medium.

<p>(<b>A</b>) The stably tranduced U87 cells were synchronized in DMEM for 24 h and then stimulated with EGF (100 ng/ml) in serum-free medium for the times indicated. The expression levels of EGFR signaling proteins were analyzed by western blotting. The representative western blotting images of three independent experiments were shown and analysis of quantification of the bands intensity was shown in the lower panel (*<i>P</i><0.05 vs con). (<b>B</b>) The stably tranduced U87 cells were cultured with 10% FBS addition with gefitinib (10 µM) or DMSO for 48 h. The total lysates were extracted and subjected to western blotting. The representative images of three independent experiments were present and quantification of the bands intensity was shown in the right panel (*<i>P</i><0.05 vs con).</p