Time-dependent effects of NE/IFN-γ on cellular proliferation and invasion.

<p>The stimulatory effects of NE (10⁻⁸ M) continued to increase for 1 to 2 weeks, while the suppressive effects of NE (10⁻⁶ M, 10⁻⁵ M) were enhanced at 1 week and then were gradually reversed (A and B). The suppressive effects of IFN-γ (100 ng/mL) reached their peak at approximately 1 week and then stayed at the same level over the next 3 weeks (C and D). NE partially reversed the effects of IFN-γ (100 ng/mL) starting at 3 weeks. When NE (10⁻⁶ M), IFN-γ (100 ng/mL) or NE (10⁻⁶ M) plus IFN-γ (100 ng/mL) were removed, the suppressive effects began to decrease gradually starting at 2 weeks (E and F). The same phenomenon was also observed in the Transwell images (G), Magnification, 200×.</p

Time-dependent effects of NE/IFN-γ on MHC-I (A), B7-1 (B), IDO (C) and B7-H1 (D) mRNA expression.

<p>NE/IFN-γ had time-dependent suppressive effects on MHCI and B7-1 and stimulatory effects on IDO and B7-H1 expression. When NE (10⁻⁶ M), IFN-γ (100 ng/mL) or NE (10⁻⁶ M) plus IFN-γ (100 ng/mL) was removed after 48 hours, the residual effects decreased dramatically within 1 week.</p

The effect of NE/IFN-γ on MHC-I, B7-1, IDO and B7-H1 expression in MIA PaCa-2 (A and B) and BxPC-3 cell lines (C and D).

<p>Compared with control group, NE suppressed MHC-I and B7-1 and up-regulated IDO and B7-H1 in a dose-dependent manner. IFN-γ suppressed B7-1, and up-regulated IDO and B7-H1. Compared with NE group, NE and IFN-γ had significant synergistic effects on IDO and B7-H1 (B and D).</p

The effects of NE/IFN-γ on MHC-I, B7-1, IDO and B7-H1 protein expression.

<p>Western blot results are shown on the left and at the top; histograms depicting relative intensities are shown on the right and at the bottom (A and B). Immunohistochemical staining of MIA PaCa-2 cells is shown at 400×magnification (C).</p

Dose-dependent effects of NE/IFN-γ on the proliferation and invasion of PC cells.

<p>NE and IFN-γ affect proliferation (OD 490 nm) and invasion (OD 570 nm) of MIA PaCa-2 (A and B) and BxPC-3 (C and D) cells in a dose-dependent manner. Low concentrations of NE (10⁻⁸ M) exhibited a trend toward stimulation, while NE (10⁻⁶ M) exhibited a trend toward suppression (<i>P</i>>0.05). High concentrations of NE (10⁻⁵ M) and IFN-γ (100 ng/mL, 200 ng/mL) had significant suppressive effects (<i>P</i><0.05). A β₂-AR antagonist decreased the stimulatory effects of NE (10⁻⁸ M) but did not alter the suppressive effects of NE (10⁻⁶ M) (E and F). (* Significant difference with control group, <i>P</i><0.05).</p

Additional file 2: Table S1. of PKM2 regulates neural invasion of and predicts poor prognosis for human hilar cholangiocarcinoma

Association between expression of PKM2, PFKB, and HK1 and clinical variables in HC patients. Table S2. Multivariate analysis of variables associated with TTP and OS in HC patients. Table S3. Multivariate analysis of variables associated with OS in HC patients. Table S4. Correlation of SDC2 expression and clinicopathological factors in HC patients. Table S5. Correlation between PKM2 protein overexpression and SDC2 protein overexpression in HC patients. (DOCX 40 kb

Additional file 1: Figure S1. of PKM2 regulates neural invasion of and predicts poor prognosis for human hilar cholangiocarcinoma

PKM2 expression in HC tissues. A, Western blotting revealed the expression pattern of KK1, PFKB, and PKM2 in HC (T) and matched adjacent noncancerous normal tissue (N) (A). B, Graphical representation of the different expressions of KK1, PFKB, and PKM2 between N and T from (A) using Image J. C, Western blotting showed higher expressions of PKM2 in tumor (“T”) than normal (“N”) in another three cases of HC. Figure S2. PFKB expression and HC patient survival. Kaplan-Meier survival curves for patients with HC according to expression of PFKB. DFS (A) and OS (B) did not differ significantly between HC patients with low or high PFKB expression. Cum, cumulative. Figure S3. Subgroup survival analysis of PKM2 expression in HC patients according to TNM category. A, patients with stage I or II HC and high PKM2 expression had a shorter median time to recurrence than did those with stage I or II HC but without PKM2 overexpression. B, patients with stage III or IV HC and high PKM2 expression had a significantly shorter median time to recurrence than did patients with stage III or IV HC but with low PKM2 expression. C, the median OS duration was significantly worse in patients with stage I or II HC and PKM2 expression than in those with stage I or II HC but with low PKM2 expression. D, the median OS duration was significantly worse in patients with stage III or IV HC and PKM2 expression than in those with stage III or IV HC but with low PKM2 expression. Cum, cumulative. Figure S4. Representative MS/MS spectrum showing the peptide of PKM2 protein. A, the intensity of repots ions of precursor peptides indicating protein expression levels. N, normal bile duct tissue. B, MS/MS spectra demonstrating identified sequences of the peptide LAPITSDPTEATAVGAVEASFK leading to identification of PKM2. Figure S5. Localization of PKM2 expression in HC cells. A and B, cytoplasmic staining for PKM2 in well/moderately differentiated cancer cells. C and D, cytoplasmic and nuclear staining (arrow) for PKM2 in poorly differentiated cancer cells. Figure S6. Xenograft models of HC. A and B, tumor cells were treated with either control shRNA or PKM2 shRNA and orthotopically implanted into nude mice as described in Materials and Methods. Tumor cells with high PKM2 expression had a higher frequency of lymph node metastasis and liver lesions than did cells with low PKM2 expression (red arrows). C, tumors with downregulated PKM2 expression had lower SDC2 expression than did those with high PKM2 expression. Figure S7. Kaplan-Meier survival curves for patients with HC according to SDC2 expression. A, patients with high SDC2-expressing tumors had a shorter median DFS duration than did patients with low SDC2-expressing tumors. B, the median OS duration in patients with high SDC2 expression did not differ significantly from that in patients with low SDC2 expression. Cum, cumulative. Figure S8. Effect of metformin treatment on tumor cell growth. A, Metformin inhibited the ability of cell proliferation of RBE cells in vitro as determined by using the CCK8 assay. B, Representative photos of HC protein staining positivity and expression pattern of IHC staining were presented. (PPTX 6948 kb