The involvement of HIF-2α in hypoxia-induced RECK silencing-mediated cell proliferation.

<p>(<b>A</b>) HEK293 cells were transfected with scrambled or RECK siRNA and exposed to normoxic or hypoxic conditions for 24 h. The expressions of HIF-1α HIF-2α, NFκB, and phophorylated-STAT1 were determined by western blotting. β-actin was used as an internal control. (<b>B</b>) Using the same samples as in A, the mRNA expressions of RECK and HIF-2α were determined by semi-quantitative RT-PCR. β-actin was used as an internal control. (<b>C</b>) HEK293 cells were plated onto 96-well plates and transfected with siHIF-1α and/or siHIF-2α or scrambled siRNA using HiPerFect Transfection reagent (Qiagen). After 24 h, transfected cells were incubated under normoxic or hypoxic conditions for an additional 24 h. At the indicated time points, cell proliferation assays were performed using CCK-8 solution. Data are presented as means ± SDs (n>3). Expression of HIF-1α and HIF-2α was confirmed in each siRNA transfectant by western blot analysis (right panel). RECK, HIF-1α, HIF-2α mRNA expression pattern was confirmed by semiquantitative RT-PCR at indicated time point. β-actin was used as an internal control (lower panel). *, <i>p</i><0.01 significantly different from the normoxic control group at each time point. †, <i>p</i><0.01 significantly different from the hypoxic control group at each time point. </p

Involvement of the EGFR signaling pathway in HIF-2α expression in RECK-silenced cells.

<p>(<b>A</b>) HEK293 cells were exposed hypoxia for 6, 12, and 24 h and determined time-dependent HIF-2α expression by western blot analysis (upper panel). HEK293 cells transfected with scrambled si RNA (-) or siRECK were treated with gefitinib (Ge, 1 μM, an EGFR inhibitor) or PD98059 (PD, 50 μM; an ERK MAPK inhibitor) under normoxia (N) or hypoxia (H) for 24 h, and the protein expressions of HIF-2α were determined by western blot analysis. β-actin was used for internal control. (<b>B</b>) The activations of EGFR and ERK-MAPK in scrambled (scr) or siRECK (siR) transfected cells exposed to normoxia (N) or hypoxia (H) were determined by western blotting using antibodies against total form or phosphorylated-form of EGFR or ERK. (<b>C</b>) The activations of EGFR and ERK and RECK expression in siHIF-1α (siH1) -, siHIF-2α (siH2) -, and MMP inhibitor (Mib)-treated cells under hypoxia (H) were determined by western blot analysis using antibodies against phosphorylated-EGFR and RECK antibody. Scr. Scrambled siRNA. </p

Silencing of <i>RECK</i> increased <i>in</i><i>vitro</i> soft-agar colony formation and <i>in</i><i>vivo</i> tumorigenesis.

<p>(<b>A</b>) HEK293 cells were transfected with si<i>RECK</i> and plated onto agar-coated 96-well plates 24 h after transfection. Colonies were observed one week after seeding (upper panel). Original magnification 40X. Quantitation of colony formation by HEK293 cells transfected with scrambled or RECK siRNA1 one week after seeding. Colony formation was measured using a fluorometer, as described in Materials and Methods. Data presented as means ± SDs (n=4). *, <i>p</i><0.01. (lower panel) (<b>B</b>) HEK293 cells stably transfected with shRECK were injected subcutaneously into the flanks of nude mice. One week later, tumor volumes (length x width x height, mm) were measured; animals were sacrificed 22 days after the first measurement. The upper panel shows tumors in sacrificed mice; including the largest and smallest shRECK tumors (RECK) and the largest shlamin tumor (Lamin). RECK expression is shown inside a graph with stable transfectant of shlamin and shRECK with RT-PCR. β-actin was used as an internal control. Scale bar=10 mm. Data are presented as means ± SDs (n=4). *, <i>p</i><0.01. Scale bar=100 µm. (<b>C</b>) Immunohistochemical staining analysis with anti-CD31 (a, b), anti-phosphorylated-Rb (c, d), PCNA (e, f) and anti-p16 (g, h) antibodies in the control (shlamin) and shRECK tumors shown in C. Anti-rhodamine (for CD31) and DAB (c-f) were used for visualization. Scale bar=10 µm. (<b>D</b>) Immunohistochemical staining analysis with anti-Hypoxic probe-1 (a, d), anti-CA9 (b, e), and anti-RECK (c, f) in xenofraft tumor section of stably transfected shRECK or shlamin (control). Scale bar=100 µm. (<b>E</b>) A scheme for the involvement of RECK/EGFR/HIF-2α in normal epithelial proliferation and viability in hypoxia. A dotted line represents findings in reference 8. </p

RECK silencing induced by hypoxia stimulated epithelial cell proliferation.

<p>(<b>A</b>) Site and silencing efficiency of two RECK siRNAs. The upper panel shows RECK siRNA sites (red bar). The lower panel shows that transfection of RECK siRNAs (5 nM) into HEK293 cells effectively blocked RECK expression. The experiment was performed in triplicate. (<b>B</b>) Proliferation assays were performed at 24 h post-transfection (0 time) at the indicated times after seeding siRNA-transfected cells at 5x10³ per well in 96-well plates. Fold change is the ratio of the cell population at each time point versus the start time (point 0). Data are presented as means ± SDs (n=4). *, <i>p</i><0.01 versus scrambled-transfected controls. (<b>C</b>) Total protein lysates were extracted from HEK293, PrEC, TCMK, and MCF10A cells after exposure to normoxia or hypoxia for the indicated times. RECK expressions were determined by western blotting. β-actin was used as an internal control. (<b>D</b>) HEK293, PrEC, TCMK, and MCF10A cells were plated at a density of 5x10³ in 96-well plates. After 24 h, cells were maintained under normoxic (N) or hypoxic (H) conditions for the indicated times. Cell proliferation assays were performed using a CCK-8 cell proliferation assay kit. Data are presented as means ± SDs (n=4). *, <i>p</i><0.01, **, p<0.001 versus normoxic controls. (<b>E</b>) Full-length <i>pCMV5-RECK</i> plasmids were transfected into HEK293 cells, and RECK protein levels and cell proliferation rate were determined by Western blot analysis and CCK-8 cell proliferation assay kit, respectively, after incubation under normoxic (N) or hypoxic (H) conditions for 24 h. Data are presented as means ± SDs (n=3). *, <i>p</i><0.01, versus normoxic controls, **, p<0.01 versus hypoxic controls. All experiments were performed at least three times. </p

MMP-2/-9 and MT1-MMP were also involved in hypoxia-induced epithelial cell proliferation.

<p>(<b>A</b>) Cells were transfected with scrambled siRNA or the two types of siRECKs. After 24 h, transfected cells were incubated under normoxic or hypoxic conditions for an additional 24 h. Conditioned media were collected from siRECK transfected (si1 and si2) or hypoxia-exposed HEK 293 cells, and gelatin zymography was performed. Western blotting for MT1-MMP was also performed using protein lysates obtained under each condition. (<b>B</b>) HEK293 cells were pretreated with a MMP inhibitor (5 µM) 2 h before normoxic or hypoxic incubation and cell proliferation assays were performed at the indicated times under normoxic (N) or hypoxic (H) conditions. Data are presented as means ± SDs (n=4). *, <i>p</i><0.01 versus the t = 0 control, **, <i>p</i><0.01 versus the vehicle control. </p

Hypoxia and silencing of <i>RECK</i> increased cell cycle progression.

<p>(<b>A</b>) Western blot analysis with anti-c-Myc, cyclin D1, cyclin A, phosphorylated Rb (p-pRb), p21, p27, and p16 were performed under normoxic (N) and hypoxic (H) conditions after 24 h of exposure. (<b>B</b>) After transfecting scrambled siRNA (Scr) and two kinds of siRECK (si1 and si2), levels of cell cycle proteins in cell lysates were determined by Western blotting. (<b>C</b>) Cell cycle analysis by FACS after PI staining. The diagram shows the diploid S and sub-G1 phase HEK293 cells under hypoxic conditions up to 72 h after staining and those of normoxic controls (cont). Right cytograms show the proportion of cell populations in each cell cycle stage in 12 h normoxia (12N) and hypoxia (12H). All experiments were performed at least in duplicate. </p

Nucleotide sequence of a 676 bp fragment from a common type clone (eel00-01) amplified from an adult Japanese eel (<i>Anguilla japonica</i>; eel00).

<p>This clone comprised 202 bp partial sequence of 18S rDNA, 422 bp entire sequence of ITS1, and 52 bp partial sequence of 5.8S rDNA. PCR primers are underlined.</p

Sequence alignment among a common type clone (eel00-01) and three intra-individual variant clones (eel01-01 to -03).

<p>PCR primers (18SL and 5.8S) are underlined. The positions of five PNA probes (PNA-F0 to F3 and R1) are shown by a horizontal bracket. The sequences of all PNA but one (PNA-F3 having one mismatch) were fully complementary to those of eel00-01 (top sequence). PNA-F2 was fully complementary to all types. PNA-F0 and F3 were designed to have one and two substitutions against variant types, respectively. PNA-F1 and R1 were designed to have three substitutions against variant types.</p

A result of PNA-directed PCR clamping using a mixed DNA template from a common type clone (eel00-01) and spiny lobster.

<p>Without PNA probe, eel ITS1 (c.a. 500 bp) was predominately amplified but lobster ITS1 was not (2^nd lane from the left). All PNA probes successfully inhibited amplification of eel ITS1 but did not inhibit amplification of lobster ITS1 (c.a. 680 bp; 3^rd to 6^th lane from the left).</p

Sequences and melting temperature (<i>Tm</i>) of PNA probes used in this study.

<p>Sequences and melting temperature (<i>Tm</i>) of PNA probes used in this study.</p