Journal: Scientific Reports

Article Title: Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids

doi: 10.1038/s41598-019-56273-6

Figure Lengend Snippet: Comparative drug response profiles of 2D cell cultures and MCSs. ( a – c ) Drug response curves for LK0917, LK0902, and LK1108 for 2D and MCSs cultures treated with 170–17000 µM of doxorubicin; 3.33–333 µM of cisplatin; and 2.2–220 µM of methotrexate. ( d ) IC 50 (µM) values calculated from the drug response curves for both 2D and MCSs of all cell types for all drugs. Significantly large differences between the IC 50 values of 2D and MCSs are denoted with “ # ”. Each IC 50 value is the average of three independent experiments (n = 3) with triplicates set for each concentration.

Article Snippet: For the generation of MCSs sized 300–500 μm, 200 μL of LK0917 (MCS 17 ), LK0902 (MCS 02 ), and LK1108 (MCS 08 ) single cell suspensions were seeded in ultra-low attachment (ULA) plates (Corning Life Sciences, Massachusetts, USA) at varying cell densities in the range of 0.25–0.75 × 10 5 cells/mL.

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids

doi: 10.1038/s41598-019-56273-6

Figure Lengend Snippet: Real-time monitoring of calcein-AM uptake in 2D and MCSs. ( a ) Elucidate calcein-AM uptake in 2D cell cultures and MCSs obtained from LK0917- MCS 17 (upper panel), LK0902- MCS 02 (middle panel), and LK1108- MCS 08 (lower panel) and over a time span of 10 hours with image acquisition at 20-minute intervals (scale bar, 200 μm). ( b ) Heat map pseudo color images of MCSs for differential calcein-AM uptake for the three cell lines (scale bar, 200 μm, scale bar on the right represents the pixel intensity of green fluorescence). ( c ) Mean fluorescence intensity profile with respect to time (12 hours) in the 2D cell cultures (LK0917, LK0902, and LK1108) and MCSs (MCS 17 , MCS 02 , and MCS 08 ) respectively. ( d ) Total accumulated calcein over time (12 hours) in 2D and MCSs for all three cell lines. ( e ) Total accumulated calcein profiles of MCSs obtained from different cell densities. The data are shown as mean ± SD; ***p < 0.001, **p < 0.05, *p = 0.019, and ns = non-significant (n = 3).

Article Snippet: For the generation of MCSs sized 300–500 μm, 200 μL of LK0917 (MCS 17 ), LK0902 (MCS 02 ), and LK1108 (MCS 08 ) single cell suspensions were seeded in ultra-low attachment (ULA) plates (Corning Life Sciences, Massachusetts, USA) at varying cell densities in the range of 0.25–0.75 × 10 5 cells/mL.

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids

doi: 10.1038/s41598-019-56273-6

Figure Lengend Snippet: ROS activity of all cell lines in 2D and MCSs. ( a ) Live-cell fluorescence images of the 2D (LK0917, LK0902, and LK1108) and MCSs (MCS 17 , MCS 02 , and MCS 08 ) cultures obtained from cell lines in the presence of DCFDA over a period of 60 minutes with image acquisition at 20-minute intervals (upper, middle and lower panel respectively), scale bar, 200 μm). ( b ) Redox state in 2D and MCSs cultures. Data are presented as the mean ± SD; ***p < 0.001, **p < 0.05, *p = 0.019, and no significant differences were observed in case of 2D (n = 3). ( c ) Heat map pseudo color images of MCSs for differential redox status for the three cell lines (scale bar, 200 μm).

Article Snippet: For the generation of MCSs sized 300–500 μm, 200 μL of LK0917 (MCS 17 ), LK0902 (MCS 02 ), and LK1108 (MCS 08 ) single cell suspensions were seeded in ultra-low attachment (ULA) plates (Corning Life Sciences, Massachusetts, USA) at varying cell densities in the range of 0.25–0.75 × 10 5 cells/mL.

Techniques: Activity Assay, Fluorescence

Journal: Scientific Reports

Article Title: Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids

doi: 10.1038/s41598-019-56273-6

Figure Lengend Snippet: ( a –c) Median fluorescence intensity (MFI) values for 2D and MCSs cultures of LK0917, LK0902, and LK1108 in presence of different ABC pump inhibitors for MDR1, MRP1, and BCRP. ( d ) MFI for MDR1 pump inhibitor (verapamil) and its corresponding live cell imaging for 2D and MCSs cultures. ( e ) Total calcein-AM uptake for the entire course of 10 hours +/− verapamil for 2D and MCSs. The data are shown as a mean of ± SD, ***p < 0.001, **p < 0.05. ( f ) Elucidate calcein-AM uptake in 2D for LK0917, LK0902 and LK1108 in presence and absence of verapamil over a time span of 10 hours with images taken after every 20 minutes (images shown here are at 2 h interval). Scale bar 200 μm.

Article Snippet: For the generation of MCSs sized 300–500 μm, 200 μL of LK0917 (MCS 17 ), LK0902 (MCS 02 ), and LK1108 (MCS 08 ) single cell suspensions were seeded in ultra-low attachment (ULA) plates (Corning Life Sciences, Massachusetts, USA) at varying cell densities in the range of 0.25–0.75 × 10 5 cells/mL.

Techniques: Fluorescence, Live Cell Imaging

Journal: Journal of Microbiology and Biotechnology

Article Title: Deinococcus radiodurans R1 Lysate Induces Tolerogenic Maturation in Lipopolysaccharide-Stimulated Dendritic Cells and Protects Dextran Sulfate Sodium-Induced Colitis in Mice

doi: 10.4014/jmb.2203.03008

Figure Lengend Snippet: Cells were treated with anti-IL-10 mAb (5 ng/ml) or rat IgG (isotype control) for 2 h prior to LPS and DeinoLys treatment. ( A ) Extracellular cytokine levels in the cell culture supernatant were measured using commercial ELISA kits. ( B ) Surface molecules were measured by flow cytometry. The mean fluorescence intensity of each surface molecule in CD11c + cells is represented by bar graphs. ( C ) CD4 + and CD8 + T cells isolated from splenocytes of BALB/c mice were stained with CellTrace™ Violet Cell Proliferation Kit and cocultured with DCs treated with PBS (Con), DeinoLys, LPS, or LPS with DeinoLys in the presence or absence of anti-IL-10 mAb or rat IgG. The proliferation of CD4 + or CD8 + T cells was analyzed by flow cytometry. The mean SD ( n = 3) is shown in all bar graphs. One-way ANOVA was used for statistics, followed by Tukey's multiple comparison. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Following the established methodology, single-cell suspensions of splenocytes were obtained from 7-week-old BALB/c mice (Orient Bio, Inc.) [ ].

Techniques: Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence, Isolation, Staining, Comparison

Journal: Pharmaceutics

Article Title: Drug-Like Small Molecule HSP27 Functional Inhibitor Sensitizes Lung Cancer Cells to Gefitinib or Cisplatin by Inducing Altered Cross-Linked Hsp27 Dimers

doi: 10.3390/pharmaceutics13050630

Figure Lengend Snippet: Characterization of non-small cell lung cancer (NSCLC) cell lines.

Article Snippet: NCI-H1650 single-cell suspension (1 × 10 7 cells) was injected subcutaneously into the hind leg of 6-week-old NOD-SCID mice (Koatech, Korea).

Techniques: Expressing, Mutagenesis

Journal: Pharmaceutics

Article Title: Drug-Like Small Molecule HSP27 Functional Inhibitor Sensitizes Lung Cancer Cells to Gefitinib or Cisplatin by Inducing Altered Cross-Linked Hsp27 Dimers

doi: 10.3390/pharmaceutics13050630

Figure Lengend Snippet: HSP27 cross-linking activities of NA49 and effect of gefitinib in EGFR mutant-type lung cancer cell lines. ( A ) NCI-H1650 and NCI-H1975 cells were treated with NA49 (0, 5, 10, and 20 μM) or gefitinib (0, 10, 20, and 30 μM) for 24 h, and cell lysates were detected by Western blot analysis. Protein levels were quantified using Image J software. The data were expressed as the fold change relative to the control and normalized to β-actin in graph. ( B ) NCI-H1650 and NCI-H1975 cells were treated with NA49 (0, 5, 10, and 20 μM) or gefitinib (0, 10, 20, and 30 μM) for 48 h, and cell death was analyzed by flow cytometry after propidium iodide (PI) staining. Results are the mean and standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated control cells).

Article Snippet: NCI-H1650 single-cell suspension (1 × 10 7 cells) was injected subcutaneously into the hind leg of 6-week-old NOD-SCID mice (Koatech, Korea).

Techniques: Mutagenesis, Western Blot, Software, Flow Cytometry, Staining, Standard Deviation

Journal: Pharmaceutics

Article Title: Drug-Like Small Molecule HSP27 Functional Inhibitor Sensitizes Lung Cancer Cells to Gefitinib or Cisplatin by Inducing Altered Cross-Linked Hsp27 Dimers

doi: 10.3390/pharmaceutics13050630

Figure Lengend Snippet: J2 and NA49 showed sensitization effects on EGFR mutant-type lung cancer cells in combination with gefitinib. ( A ) HCC827 and PC9 cells were treated with J2 (10 μM) and NA49 (10 μM) for 24 h, with or without gefitinib (10 nM), and cell lysates were detected by Western blot analysis. Protein levels were quantified using Image J software. The data were expressed as the fold change relative to the control and normalized to β-actin in the graph. ( B ) Cell death was analyzed by flow cytometry after propidium iodide (PI) staining. Results are the mean and standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated-control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. gefitinib alone). ( C ) NCI-H1650 cells were treated with J2 (10 μM) or NA49 (10 μM) for 24 h, with or without gefitinib (10 or 20 μM) and NCI-H1975 cells were treated with J2 (10 μM) or NA49 (10 μM) for 24 h, with or without gefitinib (10 μM), and cell lysates were detected by Western blot. Protein levels were quantified using Image J software. The data were expressed as the fold change relative to the control and normalized to β-actin in the graph. ( D ) Cell death was analyzed by flow cytometry after propidium iodide (PI) staining. Results are the mean and standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated-control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. gefitinib alone 10 μM; + p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. gefitinib alone 20 μM). ( E ) NCI-H1650 cells transfected with control (siCont) or siRNA of HSP27 (siHSP27) were treated with NA49 (10 μM) with or without gefitinib (10 μM), cell lysates were detected by Western blot analysis, and cell death was analyzed by flow cytometry after propidium iodide (PI) staining. Protein levels were quantified using Image J software. The data were expressed as the fold change relative to the control and normalized to β-actin in the graph. Results are the mean and standard deviation of three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. untreated-control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. gefitinib alone).

Article Snippet: NCI-H1650 single-cell suspension (1 × 10 7 cells) was injected subcutaneously into the hind leg of 6-week-old NOD-SCID mice (Koatech, Korea).

Techniques: Mutagenesis, Western Blot, Software, Flow Cytometry, Staining, Standard Deviation, Transfection

Journal: Pharmaceutics

Article Title: Drug-Like Small Molecule HSP27 Functional Inhibitor Sensitizes Lung Cancer Cells to Gefitinib or Cisplatin by Inducing Altered Cross-Linked Hsp27 Dimers

doi: 10.3390/pharmaceutics13050630

Figure Lengend Snippet: NA49 sensitized tumors in an NCI-H460 and NCI-H1650 cells-injected xenograft mouse model with cisplatin and ge-fitinib. ( A ) NCI-H460 and NCI-H1650 cells were injected subcutaneously into BALB/c nude mice ( n = 6/group). Xenografted mice were treated 7 times with NA49 (20 mg/kg) intraperitoneal treatment in combination with four injections of cisplatin (2 mg/kg) or gefitinib (5 mg/kg). ( B ) Tumor size was measured every other day. Results are the mean and standard error. ( C ) Ki67 staining ( D ) and immunohistochemistry of HSP27 were performed using tumor tissues. Results are the mean and standard error (* p < 0.05, *** p < 0.001 vs. untreated-control; # p < 0.05, ## p < 0.01, ### p < 0.001 ### vs. cisplatin or gefitinib alone).

Article Snippet: NCI-H1650 single-cell suspension (1 × 10 7 cells) was injected subcutaneously into the hind leg of 6-week-old NOD-SCID mice (Koatech, Korea).

Techniques: Injection, Staining, Immunohistochemistry