Data_Sheet_1_Stochastic factors drive dynamics of ammonia-oxidizing archaeal and bacterial communities in aquaculture pond sediment.DOCX

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) play an important role in nitrification, which is essential in the global nitrogen cycle. However, their dynamics and the underlying community processes in agricultural ecosystems under disturbance remain largely unknown. In this study we examined the spatiotemporal dynamics of AOA and AOB communities and analyzed their community processes in the sediment of aquaculture ponds across three different areas in China. We found some significant temporal changes in AOA and AOB community diversity and abundances, but no temporal changes in community composition, despite the significant variations in sediment properties between different sampling times. Nevertheless, significant differences were found for AOA and AOB communities between different areas. Distinct area-specific taxa were detected, and they were found to be important in determining the response of AOA and AOB communities to environmental factors. In addition, geographic distance was found to be significantly correlated with AOA and AOB community composition, which demonstrates that dispersal limitation could significantly contribute to the variations in AOA and AOB communities, and stochastic processes were found to be important in structuring AOA/AOB communities in aquaculture ponds. Taken together, our study indicates that the dynamics of AOA and AOB are based on their community characteristics in aquaculture pond sediment. Our results, for the first time, provide evidence for the dynamics of AOA and AOB communities being driven by stochastic factors in a disturbed environment, and might also be of use in the management of the aquaculture environment.</p

IFNs induce DKK1 expression and have no effects on GSK3β.

<p>HepG2 and Huh7 cells were treated with or without IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ (100 ng/ml) and expression of DKK1 was measured by flow cytometry (<b>A</b>, <b>C</b>) or ELISA (<b>B</b>, <b>D</b>) at 48 hrs post-treatment. Active GSK3β level was measured in HepG2 and Huh7 cells at 48 hrs post different IFN exposure by flow cytometry (<b>E</b>, <b>F</b>). Data represent a minimum of three experiments and asterisks denote p<0.05 in comparison to untreated samples.</p

Impact of IFN-mediated β-catenin regulation on proliferation and apoptosis in HCC.

<p>HepG2 (<b>A</b>) and Huh7 (<b>D</b>) cells were left untreated or treated with IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ (100 ng/ml) for 72 hrs. Cell viability was determined at 72 hrs. The abscissa represents the types of stimulation. The ordinate represents percentage of live cells relative to the mock treated cells. Apoptosis in HepG2 (<b>B, C</b>) and Huh7 (<b>E, F</b>) was measured by TUNEL assay and flow cytometry targeting active caspase 3. The ordinate represents fold increase of fluorescence intensity relative to the untreated cells. Data represent a minimum of three experiments and asterisks denote p<0.05 in comparison to untreated samples.</p

IFNs induce activation of STAT1 and STAT3 in HCC.

<p>HepG2 cells were left untreated or treated with IFNα, IFNγ or IFNλ for 0.5 hr in the absence (black bars) or presence of STAT1 inhibitor (FLUD, grey bars) or STAT3 inhibitor (S3I, open bars), and active STAT1 (<b>A</b>) and STAT3 (<b>B</b>) levels were measured by flow cytometry. Data represent a minimum of three independent experiments. Asterisks denote p<0.05 in comparison to untreated samples.</p

IFNs down-regulate β-catenin signaling pathway.

<p>HCC cell lines, HepG2 (<b>A</b>) and Huh7 (<b>C</b>), were left untreated or treated with IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ (100 ng/ml) for 24 hrs prior to transfection with TOPflash luciferase and Renilla luciferase constructs. After resting for 4 hrs, the cells were cultured with or without initial treatment of different IFNs. Dual luciferase activity was measured 24 hrs later. Data shown is normalized to Renilla activity. HepG2 (<b>B</b>) and Huh7 (<b>D</b>) were treated with or without IFNα (100 ng/ml), IFNγ (100 ng/ml) or IFNλ (100 ng/ml) for 48 hrs and expression of hypophosphorylated/active β-catenin level was measured by conventional intracellular flow cytometry. Data represent a minimum of three experiments and asterisks denote p<0.05 in comparison to untreated samples.</p

IFN-induced apoptosis in HCC is DKK1 and STAT3-dependent.

<p>HepG2 (<b>A</b>) and Huh7 (<b>B</b>) cells were left untreated or treated with FLUD, S3I, or αDKK1 alone or in combination with IFNα, IFNγ or IFNλ, respectively for 72 hrs. The levels of induced apoptosis were measured by TUNEL assay. Data represent a minimum of three independent experiments. Asterisks denote p<0.05 in comparison to untreated samples.</p

IFN-induced up-regulation of DKK1 is dependent on STAT3 activation.

<p>HepG2 (<b>A</b>) and Huh7 (<b>B</b>) cells were left untreated or treated with FLUD, S3I, or αDKK1 (DKK1 neutralizing antibody) alone or in combination with IFNα, IFNγ or IFNλ, respectively for 48 hrs. DKK1 levels were measured by ELISA. Data represent a minimum of three independent experiments. Asterisks denote p<0.05 in comparison to untreated samples.</p

Distribution of Doxorubicin in HIFU-ablated Tumor Tissue.

<p>Zero signifies the center of the implants, and the other numerical values refer to the radial distances from the center of the implants. A: Light microscopy image of cryosectioned tumor tissue. B: Fluorescence microscopy image of the same tumor tissue section. C: High-power image of the area in the white box shown in B. D: Fluorescent intensity. The asterisk (*) indicates the PLGA implant, the white arrow indicates the edge of the PLGA implant, and the yellow arrow indicates the distance from the edge. The scale bar represents 100μm.</p

In vitro Cumulative DOX Release (%).

<p>The data are presented as the means ± STDEV (the standard deviation, n = 3).</p

Contrast-Enhanced HIFU Ablation Imaging.

<p>Representative ultrasound tumor images with and without intentionally incomplete HIFU ablation. A: Without HIFU ablation and without contrast enhancement. B: TTC-stained tumor that was not subjected to HIFU ablation. C: Contrast-enhanced ultrasound image of tumor without HIFU ablation. D: HIFU-ablated tumor without contrast enhancement. E: TTC-stained tumor that received HIFU ablation. F: Contrast-enhanced ultrasound image of tumor with HIFU ablation. The arrows indicate the ablated area. The scale bars represent 1cm.</p