Lung tumor spheres have high tumorigenic potential.

<p>Tumorigenic potential of tumor sphere cells is greater than monolayer cancer cells. Monolayer and tumor sphere cells from A549 were injected s.c. into athymic nude mice at concentrations of 1×10⁴ cells/ml. Mice were sacrificed at day 60, and tumor was surgically removed and measured, and the mean values of the tumor size were shown in the table.</p

Chemoresistence of monolayer and tumor sphere.

<p><b>A.</b> cisplatin, <b>B.</b> doxorubicin resistance of monolayer culture and tumor spheres were analyzed. Cells dissociated from lung tumor spheres or monolayers were plated out in 96-well plate at 2×10⁴ cells/ml, 24 h later, media containing different concentrations of cisplatin or doxorubicin was added. After 48 h culturing, 10% Alamar Blue was added and read at 544/590 nm, the percent viability was calculated relative to cells not exposed to any chemotherapeutic drugs. The results represent the means ± SD.</p

SP and NSP cells form tumor spheres with equal capacity in human adenocarcinoma cell lines.

<p><b>A.</b> Characteristic Hoechst 33342 dye staining profile demonstrating the existence of SP (polygon gated) and NSP (ellipse gated) cells in human A549 and H1299 adenocarcinoma cells. Percentage of SP cells is indicated. Cells were stained with 5 mg/ml Hoechst33342 (HO), and control cells were also co-stained with 10 mM fumitremorgin C (FTC) to specify the SP cells. Cells were sorted using a Beckman MoFlo cytometer and data were annotated using FCS Express. <b>B.</b> Phase-contrast photographs of lung tumor spheres obtained from SP and NSP cells. SP and NSP cells (1000 cell/ml) were plated onto ultra low adherent flasks in serum free media supplemented with growth factors and cultivated as described in Material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013298#s2" target="_blank">Methods</a>. <b>C.</b> Re-stain of SP and NSP cells from A549 and H1299 with Hoechst 33342 dye. Percentage of SP cells is labeled.</p

Histopathologic examination of the engrafted tumors by H&E and β-Catenin staining.

<p>Surgically removed tumor was fixed in buffered formalin and subsequently analyzed by immunohistochemistry (IHC) by standard protocol.</p

Track CSCs in tumor sphere based on their proteasome activity.

<p><b>A.</b> Frequency of ZsGreen cells in A549 and H1299 monolayer cultures. <b>B.</b> Frequency of ZsGreen cells in A549 and H12199-derived tumor spheres with accumulation of ZsGreen-cODC, and thus low proteasome activity is indicated. <b>C.</b> Number of spheres formed from the ZsGreen positive and the ZsGreen negative cells after sorting with flow cytometry into 96-well plates. ZsGree-positive and –negative cells were sorted into 96-well plate (100cells/well), after cultured in tumor sphere media for 2 weeks; number of formed spheres was counted. Student's paired, and two-tailed t-tests were performed, ***p<0.001 (three replicates per experiment).</p

Lung tumor spheres exhibit cancer stem cell features.

<p><b>A.</b> Lung tumor spheres showed increased CD133 expression. Expression of CD44 and CD133 in monolayer and tumor sphere cells, as determined by two-color flow cytometry analysis. Isotype-matched monoclonal antibodies and a preimmune second antiserum were used as negative controls. The percentage of single- and double-positive cells are indicated. <b>B.</b> Lung tumor spheres showed increased OCT3/4 expression. An average fluorescence intensity of OCT3/4 in control (grey area), monolayer cells (dotted line) and tumor spheres (solid line) is shown. Percentages of positive cells are indicated in the table.</p

Decreased proteasome activities of the 26S proteasome in A549 and H1299 tumor spheres.

<p><b>A.</b> caspase-like, <b>B.</b> chymotrypsin-like, and <b>C.</b> trypsin-like activity of monolayer culture and tumor spheres from A549 and H1299 cells was measued over the indicated times. <b>D.</b> End point analysis (120 min) of 26S proteasome activities in monolayer cultures and tumor spheres. Mean ± SD is plotted and derived from three independent experiments. Student's paired, and two-tailed t-tests were performed, *p<0.05, **p<0.01, and ***p<0.001 (three replicates per experiment).</p

Insights into the Effects of Co-Regulated Factors on Li_1.3Al_0.3Ti_1.7(PO₄)₃ Solid Electrolyte Preparation: Sources, Calcination Temperatures, and Sintering Temperatures

The ionic conductivity, phase components, and microstructures of LATP depend on its synthesis process. However, their relative importance and their interactions with synthesis process parameters (such as source materials, calcination temperature, and sintering temperature) remain unclear. In this work, different source materials were used to prepare LATP via the solid-state reaction method under different calcination and sintering temperatures, and an analysis via orthogonal experiments and machine learning was used to systematically study the effects of the process parameters. Sintering temperatures had the greatest effect on the total ionic conductivity of LATP pellets, followed by the sources and calcination temperatures. Sources, as the foundational factors, directly determine the composition of a major secondary phase of LATP pellets, which influences the whole process. The calcination temperature had limited impact on the ion conductivity of LATP pellets if pellets were sintered under the optimal temperature. The sintering temperature is the most important factor that influences the ion conductivity by eliminating most secondary phases and altering the microstructure of LATP, including the intergranular contact, grain size, relative densities, etc. This work offers a novel perspective to comprehend the synthesis of solid-state electrolytes beyond LATP

<i>In Situ</i> Infrared Spectroscopic Evidence of Enhanced Electrochemical CO₂ Reduction and C–C Coupling on Oxide-Derived Copper

The reaction mechanism of CO2 electroreduction on oxide-derived copper has not yet been unraveled even though high C2+ Faradaic efficiencies are commonly observed on these surfaces. In this study, we aim to explore the effects of copper anodization on the adsorption of various CO2RR intermediates using in situ surface-enhanced infrared absorption spectroscopy (SEIRAS) on metallic and mildly anodized copper thin films. The in situ SEIRAS results show that the preoxidation process can significantly improve the overall CO2 reduction activity by (1) enhancing CO2 activation, (2) increasing CO uptake, and (3) promoting C–C coupling. First, the strong *COO– redshift indicates that the preoxidation process significantly enhances the first elementary step of CO2 adsorption and activation. The rapid uptake of adsorbed *COatop also illustrates how a high *CO coverage can be achieved in oxide-derived copper electrocatalysts. Finally, for the first time, we observed the formation of the *COCHO dimer on the anodized copper thin film. Using DFT calculations, we show how the presence of subsurface oxygen within the Cu lattice can improve the thermodynamics of C2 product formation via the coupling of adsorbed *CO and *CHO intermediates. This study advances our understanding of the role of surface and subsurface conditions in improving the catalytic reaction kinetics and product selectivity of CO2 reduction

<i>In Situ</i> Infrared Spectroscopic Evidence of Enhanced Electrochemical CO₂ Reduction and C–C Coupling on Oxide-Derived Copper

The reaction mechanism of CO2 electroreduction on oxide-derived copper has not yet been unraveled even though high C2+ Faradaic efficiencies are commonly observed on these surfaces. In this study, we aim to explore the effects of copper anodization on the adsorption of various CO2RR intermediates using in situ surface-enhanced infrared absorption spectroscopy (SEIRAS) on metallic and mildly anodized copper thin films. The in situ SEIRAS results show that the preoxidation process can significantly improve the overall CO2 reduction activity by (1) enhancing CO2 activation, (2) increasing CO uptake, and (3) promoting C–C coupling. First, the strong *COO– redshift indicates that the preoxidation process significantly enhances the first elementary step of CO2 adsorption and activation. The rapid uptake of adsorbed *COatop also illustrates how a high *CO coverage can be achieved in oxide-derived copper electrocatalysts. Finally, for the first time, we observed the formation of the *COCHO dimer on the anodized copper thin film. Using DFT calculations, we show how the presence of subsurface oxygen within the Cu lattice can improve the thermodynamics of C2 product formation via the coupling of adsorbed *CO and *CHO intermediates. This study advances our understanding of the role of surface and subsurface conditions in improving the catalytic reaction kinetics and product selectivity of CO2 reduction