Overexpression and knockdown of HIF-lα in NB cells by lentiviral vectors.

<p>(A, D) SH-SY5Y and IMR32 cells were transfected with lentiviral vectors and observed by fluorescence microscopy after 72 h. More than 90% of the cells expressed green fluorescent protein (GFP). Magnification, ×200. (B, E) Western blot analysis showed significantly higher HIF-lα expression in SH-SY5Y and IMR32 cells transfected by Lenti-HIF-lα than in cells transfected by Lenti-GFP after 72 h under normoxia. β-actin was used as a loading control. (C, F) SH-SY5Y and IMR32 cells were transfected with Lenti-scrambled siRNA and three different siRNAs targeting HIF-lα. After a 72-h-transduction, an additional 8-h-hypoxic treatment was needed before Western blotting. siRNA2 was proven to be the most effective one of the three siRNAs. β-actin was used as a loading control. N, normoxia; H, hypoxia.</p

Effect of HIF-1α on the invasion of NB cells.

<p>(A, D) The Transwell assay showed that HIF-1α overexpression enhanced invasion in SH-SY5Y and IMR32 cells under normoxia (n = 9). (B, E) HIF-1α knockdown reduced invasion in both cell lines under hypoxia (n = 9). (C, F) HIF-1α overexpression enhanced invasion in both cell lines under hypoxia (n = 9). All ×200 magnification. Data are expressed as means ± SD.</p

Effect of HIF-1α on the proliferation of NB cells.

<p>(A, B) The CCK-8 assay showed that overexpression of HIF-1α using a lentiviral vector did not promote cell proliferation in either SH-SY5Y or IMR32 cells in normoxia (n = 9). (C, D) Lentiviral vector-mediated HIF-lα knockdown inhibited cell proliferation in SH-SY5Y and IMR32 cells under hypoxia (n = 9). Data are expressed as means ± SD.</p

HIF-1α regulates the SHH signaling pathway in NB cells.

<p>(A, B) Western blotting results showed that the protein levels of SHH, PTCH1 and GLI1 were positively associated with the levels of HIF-1α in NB cells under normoxia and hypoxia. β-actin was used as a loading control. (C, D) The mRNA expression of SHH, PTCH1 and GLI1 were estimated by real-time PCR. The results indicated that the mRNA expression of SHH pathway components were also regulated by HIF-1α (n = 9). The expression of each target gene was quantified using β-actin as a normalization control. Data are expressed as means ± SD. N, normoxia; H, hypoxia.</p

Effect of GLI1 knockdown on the expression levels of HIF-1α and SHH pathway components in NB cells.

<p>(A, B) Western blotting results showed that the protein levels of HIF-1α, SHH and PTCH1 did not change in SH-SY5Y and IMR32 cells when GLI1 was knocked down under hypoxia. β-actin was used as a loading control.</p

HIF-1α regulates NB growth and the SHH pathway in vivo.

<p>(A) Representative xenografts dissected from different groups of nude mice are shown. (B) The volume of subcutaneous xenografts formed by SH-SY5Y cells stably infected with the indicated lentiviruses is illustrated by growth curves (n = 5). Compared with the control, cells overexpressing HIF-lα formed significantly larger tumors. By contrast, the tumor weight in the HIF-lα knockdown group was significantly reduced. GANT61 could reverse the pro-growth effect of HIF-lα. (C) Immunohistochemical staining of HIF-1α, SHH pathway components and CD31 in xenografts. The statistical results showed that the expression levels of SHH pathway components and angiogenesis in xenografts were correlated with HIF-lα (n = 5). In the bar graph, 4 bars were indicated for HIF-1α and PTCH1 as mean scores for both subgroups were 0. Magnification, ×400. Scale bars, 200 μm. Arrows, positive cells.</p

Expression of SHH pathway proteins in NB samples at different stages and differentiation conditions.

<p>* <i>p</i><0.05 versus stage I;</p><p>** <i>p</i><0.01 versus stage I;</p><p>^#<i>p</i><0.05 versus stage II;</p><p>^##<i>p</i><0.01 versus stage II;</p><p>^Δ<i>p</i><0.05 versus the without lymph node metastasis group;</p><p>^§<i>p</i><0.05 versus the well-differentiated group;</p><p>^§§<i>p</i><0.01 versus the well-differentiated group.</p><p>Expression of SHH pathway proteins in NB samples at different stages and differentiation conditions.</p

Representative immunohistochemical staining images of HIF-1α and SHH pathway components in human NB samples.

<p>Compared with early-stage (stage I or II) NB, advanced-stage (stage III or IV) tumors tended to show stronger staining for HIF-1α and SHH signals. Spearman rank correlation indicated that HIF-1α was positively correlated with SHH and GLI1 staining. HIF-1α, SHH and GLI1 expression levels were related with tumor stage. All ×400 magnification. Scale bars, 200 μm. Arrows, positive cells.</p

GLI1 knockdown inhibits the cell proliferation, migration and invasion abilities in NB cells under hypoxic conditions.

<p>(A) Three siRNAs targeting GLI1 and NC siRNA were transfected into SH-SY5Y cells. Western blotting indicated that siRNA2 provided the highest inhibition efficiency. (B, C) GLI1 knockdown significantly inhibited proliferation in IMR32 cells, but only led to a slight reduction in proliferation in SH-SY5Y cells (n = 9). (D, E) Decreased cell migration was observed in both cell lines transfected with GLI1 siRNA (n = 9). Magnification, ×40. (F, G) Invasion ability was reduced in both cell lines transfected with GLI1 siRNA (n = 9). Magnification, ×200. Data are expressed as means ± SD.</p

Effect of HIF-1α on the migration of NB cells.

<p>(A, D) The wound healing assay showed that HIF-1α overexpression did not increase migration in SH-SY5Y and IMR32 cells under normoxia (n = 9). (B, E) HIF-1α knockdown decreased migration in both cell lines under hypoxia (n = 9). (C, F) HIF-1α overexpression increased migration in both cell lines under hypoxia (n = 9). All ×40 magnification. Data are expressed as means ± SD.</p