Fig 1.
(A) Expression of HE4 in ovarian cancer cell lines. mRNA expression of HE4 was determined by semiquantitative reverse transcription PCR. GAPDH served as a loading control. NTC: Non-template control. (B) TNFα and IL-1β promote the secretion of HE4 in ovarian cancer cell lines. Cells were subjected to TNFα (30 ng/mL) or IL-1β (10 ng/mL) treatment for 72 hours. Afterward, cell culture medium was collected. HE4 levels were determined using HE4 ELISA.
Fig 2.
TNFα promoted HE4 secretion through the NF-kB signaling pathway (A) A2780 cells were incubated with TPCA-1 (25 μM, an IKK2 inhibitor), BIRB-769 (1 μM, a p38 inhibitor), JNK-IN-8 (1 μM, a JNK inhibitor), or CI-1040 (20 μM, a MEK inhibitor) for 1 hour, after which cells were subjected to TNFα treatment (30 ng/mL) for 15 minutes. Subsequently, protein expression and phosphorylation were assessed via immunoblot analysis using target-specific antibodies, with α-tubulin serving as a loading control. (B) A2780 cells underwent the same inhibitor treatment as in (A), after which cells were treated with TNFα (30 ng/mL) for 24 hours. HE4 levels in culture medium were determined using HE4 ELISA. (C) A2780 cells were transfected with a construct encoding luciferase under the control of an NF-κB response element or HE4 promoter construct (HE4-652), and luciferase activity was assessed following TNFα (30 ng/mL) or IL-1β (10 ng/mL) treatment for 7 hours. (D) Same as (C), but HCH-1 cells were employed.
Fig 3.
(A) TNFα enhanced the nuclear localization of p65 and p50 in A2780 cells. A2780 and OVCAR-3 cells were stimulated with TNFα (30 ng/mL) for 5 hours. Cells were lysed, and cytoplasmic and nuclear fractions were isolated, followed by immunoblot analysis using antibodies against GAPDH (a cytoplasmic marker) and lamin A/C (a nuclear marker), as well as against p65, and p50. The expression levels of p65 and p50 in nuclear fraction upon TNFα stimulation were determined relative to Lamin A/C expression (top). (B) p65 knockdown abolished TNFα induced HE4 secretion in A2780 cells. A2780 cells were transfected with siRNAs against p65 or with non-targeting control. Following transfection cells were exposed to TNFα treatment (30 ng/mL) for 48 hours. Afterward, cell culture medium was collected. HE4 levels were determined using HE4 ELISA. (C) p65 expression promotes HE4 expression in OVCAR-3 cells. Cell culture medium from OVCAR-3 cells transfected for 72 hours with empty or p65 expression vector was analyzed for HE4 levels using HE4 ELISA (left). Simultaneously, the cell population for each condition was assessed via the MTS assay (right).
Fig 4.
(A) Expression of constitutively active IKK2 mutant (IKK2-EE) led to the phosphorylation of Iκβα. 2008 cells were transfected with either IKK2-EE or an empty vector for 24 hours. Cells were lysed, and protein expression was assessed via immunoblot analysis. Phosphorylated Iκβα levels were quantified relative to total Iκβα. (B) IKK2 inhibitor TPCA-1 reduced HE4 expression. 2008 cells were treated with TPCA-1 at a range of concentrations for 24 hours. HE4 levels in culture medium were determined using HE4 ELISA (left). Simultaneously, the cell population for each condition was assessed via the MTS assay (right) (C-E) Expression of p65 and IKK2-EE promoted HE4 expression in ovarian cancer cell lines. A2780, OVCAR-3, and 2008 cells were transfected with p65, IKK2-EE, or empty vector for 72 hours. Afterward, HE4 levels in culture medium were determined using HE4 ELISA.
Fig 5.
NF-kB upregulated the mRNA levels of HE4.
A2780 cells were treated with TNFα (30 ng/mL) for 24 hours. OVCAR-3 and 2008 cells were transfected with p65, IKK2-EE, or empty vector for 24 hours. Cells were lysed, and isolated RNAs were analyzed for HE4 (A) or TNF (B) via quantitative reverse transcription PCR, as described in “Materials and Methods”.
Fig 6.
NF-kB promoted α5-integrin, while HE4 knockdown blocked its expression.
(A) 2008 cells were co-transfected with IKK2-EE expression vector and HE4 siRNAs (sourced from two different vendors) for 48 hours. Cells were lysed, and protein expression of putative NF-kB targets was measured via immunoblot analysis. Heat map represents the relative expression of each NF-kB target evaluated by densitometric analysis. GAPDH served as a loading control for normalization. (B) 2008 cells were transfected with HE4 siRNAs from three different vendors (48 or 72 hours). Expression of HE4, α5-integrin, and α-tubulin was measured via immunoblot analysis. (C)The effects of HE4 knockdown on adhesion molecules. 2008 cells were transfected with HE4 siRNA. Protein extracts were prepared and subjected to immunoblot analysis against the proteins as indicated. (D) as in (C) but other ovarian (HCH-1 and Caov-3) and non-ovarian (BxPC-3, pancreatic) cancer cell lines were employed. (E) HE4 knockdown inhibited cell adhesion to fibronectin. Cell adhesion of 2008 cells transfected with HE4 siRNA (72 hours) was determined in fibronectin- or collagen-coated cell culture plate as described in “Materials and Methods.” (F) HE4 knockdown inhibited cell migration. 2008 cells were transfected with either control or HE4 siRNA and cultured until confluence. A scratch was made using a P1000 pipette tip, and images were captured at 0, 6, 12, and 18 hours (scale bar: 500 μm). Right panel: The average width of the gap (y-axis, in μm) was calculated as described in the “Materials and Methods” section.