cell culture 3ll  (ATCC)

Journal: EMBO Reports

Article Title: Plasma fractalkine contributes to systemic myeloid diversity and PD‐L1 / PD ‐1 blockade in lung cancer

doi: 10.15252/embr.202255884

Figure Lengend Snippet: A Baseline plasma FKN concentrations in responders (R, n = 13) and progressors (PR, n = 5) in a validation cohort of NSCLC patients treated with anti‐PD‐1/PD‐L1 immunotherapy as a first‐line treatment. Error bars are shown (standard deviations, SD). Statistical significance was tested by the chi‐square test. B, C (B) Representative double lung immunostainings for Pan‐Cytokeratin (PanCK) (brown) and FKN (blue; red arrows indicate FKN positive cells) in lung tissue sections from LUAD patients and (C) adjacent healthy tissue. D, E (D) Pearson's correlation of PanCK with FKN positive areas (%) in histologies of adjacent nontumor and (E) LUAD tissues. Pearson's correlation coefficients are shown in the graphs. n = 10. F FKN mRNA expression level in tumor (T) and normal tissue (N) of clinical samples from lung adenocarcinoma (LUAD) and squamous lung carcinoma (LUSC) patients registered in the TCGA database. Distributions of gene expression levels are displayed using box plots. Box and whisker plots indicate median (central line), 25 th to 75 th percentiles (box), and minimum to maximum values (whiskers). G Top, lentivectors for the expression of cytokines of interest. SIN, self‐inactivating deleted LTR; LTR, long‐terminal repeat; SFFVp, spleen focus‐forming virus promoter; UBIp, human ubiquitin promoter; Puro R, puromycin resistance gene. Down, ELISA quantification of cytokine secretion by the indicated engineered lung cancer cell lines expressing the indicated cytokines (T). Endogenous secretion of each cytokine was also quantified in supernatants from cultures of parental unmodified controls (UT). Data are presented as mean ± SD ( n = 3 independent biological replicates). H Real‐time cell growth (RTCA) of 3LL cell lines engineered to secrete the indicated cytokines. Relevant statistical comparisons of delta‐cell indexes after 50 h of culture were carried out by ANOVA. Data are presented as mean ± SD ( n = 3 independent biological replicates). I Real‐time cell growth (RTCA) of unmodified and FKN‐producing 3LL cell lines cultured with 1 μg/ml of recombinant FKN (rFKN). J Flow cytometric assessment of propidium iodide (PI) uptake by apoptotic unmodified 3LL cells cultured in 3LL‐WT (control) or 3LL‐FKN (FKN) conditioned medium for 24, 48, or 72 h. Data information: The statistical significance computed by the Wilcoxon test is annotated by the number of stars. *, **, *** indicate significant ( P < 0.05), very significant ( P < 0.01) and highly significant ( P < 0.001) differences.

Article Snippet: The indicated 3LL cell lines (1.5 × 10 6 cells/mouse; n = 6 mice/group) were subcutaneously injected in the flanks of 10‐week‐old C57BL/6 female and male mice (Envigo).

Techniques: Clinical Proteomics, Biomarker Discovery, Expressing, Gene Expression, Whisker Assay, Virus, Ubiquitin Proteomics, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Control

Journal: EMBO Reports

Article Title: Plasma fractalkine contributes to systemic myeloid diversity and PD‐L1 / PD ‐1 blockade in lung cancer

doi: 10.15252/embr.202255884

Figure Lengend Snippet: A Secreted FKN in cell cultures of a collection of 10 LUAD and LUSC cancer cell lines quantified by ELISA. Results are presented as mean ± SD. Samples were assayed in duplicates from a pool of three independent replicates. Statistical differences among groups were analyzed by ANOVA followed by Tukey's tests. B Tumor growth curves of 3LL tumor‐bearing mice treated with recombinant FKN (rFKN), the CX3CR1 inhibitor AZD8797, a nonrelevant protein (IgG2a) or saline buffer as vehicle control (WT). Arrows indicate the time of intraperitoneal injections of rFKN (blue) and AZD8797 (black). Data are expressed as mean ± SD ( n = 6 mice per group). Statistical comparisons by ANOVA followed by Tukey's pairwise comparison tests are provided. C Dot plot of 3LL tumor size in the indicated groups of mice at day 10 after cancer cell inoculation. Statistical comparisons by ANOVA and Tukey's pairwise comparison tests are indicated. D Proliferation of two LUAD murine cell lines overexpressing FKN and their parental unmodified counterparts (WT), as indicated. Real‐time cell growth was monitored by RTCA. Delta‐cell indexes are expressed as mean ± SD from three independent replicates. Differences in delta‐cell index proliferation data were tested by ANOVA following Tukey's pairwise comparison tests after 40 h of culture. E Bar graphs with cell growth rates for the indicated 3LL cell lines relative to the growth of unmodified 3LL cells. Error bars are shown (SD). Statistical comparisons among three independent biological replicates by ANOVA and Tukey's pairwise comparison tests are indicated. F In vivo tumor growth of engrafted 3LL cell lines producing the indicated cytokines. Data are expressed as mean ± SD ( n = 6 mice per group). Comparisons between groups were performed by ANOVA and Tukey's pairwise comparison tests. G Kaplan–Meier survival plots. Differences between the control group (WT) and the FKN group were evaluated by a two‐sided log‐rank test. Data information: Statistical comparisons are shown in the graph. *, **, ***, ****, indicate significant ( P < 0.05), very significant ( P < 0.01), highly significant ( P < 0.001), and very highly significant ( P < 0.0001) differences; ns, nonsignificant differences.

Article Snippet: The indicated 3LL cell lines (1.5 × 10 6 cells/mouse; n = 6 mice/group) were subcutaneously injected in the flanks of 10‐week‐old C57BL/6 female and male mice (Envigo).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Saline, Control, Comparison, In Vivo

Journal: EMBO Reports

Article Title: Plasma fractalkine contributes to systemic myeloid diversity and PD‐L1 / PD ‐1 blockade in lung cancer

doi: 10.15252/embr.202255884

Figure Lengend Snippet: A FKN plasma concentration at day 15 in the indicated groups of tumor‐bearing mice. Data are expressed as mean ± SD from a pool of 6 mice/group. Statistical comparisons were performed by ANOVA and Tukey's pairwise comparison tests. B Tumor volumes 15 days after tumor inoculation. Data are expressed as mean ± SD ( n = 6 mice per group), and comparisons between groups were performed by ANOVA and Tukey's pairwise comparison tests. C Relative myeloid and lymphoid composition of peripheral blood at day 15 in mice transplanted with 3LL‐expressing the indicated cytokines. WT, unmodified 3LL cells; Healthy, mice without tumors. D Percentage of the indicated peripheral immune cell populations at day 15 after injection of groups of mice ( n = 6 mice per group) with 3LL cells overexpressing the indicated myeloid‐regulating cytokines. The relevant immune populations were quantified as percentages of total leukocytes (CD45 + cells), specifying DCs (CD11c), macrophages (F4/80), Ly6C + monocytes, and Ly6C − monocytes, granulocytes (Ly6G), B cells (CD19), NKs (NK1.1), CD4 T cells (CD3 CD4) and CD8 T cells (CD3 CD8). Relevant statistical comparisons were performed by the Wilcoxon's test followed by pairwise comparisons of relevance by the Mann–Whitney U test. Box and whisker plots indicate median (central line), 25 th to 75 th percentiles (box), and minimum to maximum values (whiskers). Data information: *, **, ***, ****, indicate significant ( P < 0.05), very significant ( P < 0.01), highly significant ( P < 0.001), and very highly significant ( P < 0.0001) differences. In this last case, no exact P ‐value is provided in the figure; ns, nonsignificant differences.

Article Snippet: The indicated 3LL cell lines (1.5 × 10 6 cells/mouse; n = 6 mice/group) were subcutaneously injected in the flanks of 10‐week‐old C57BL/6 female and male mice (Envigo).

Techniques: Clinical Proteomics, Concentration Assay, Comparison, Expressing, Injection, MANN-WHITNEY, Whisker Assay

Journal: EMBO Reports

Article Title: Plasma fractalkine contributes to systemic myeloid diversity and PD‐L1 / PD ‐1 blockade in lung cancer

doi: 10.15252/embr.202255884

Figure Lengend Snippet: A Dot plot of tumor volume in mice 22 days after engraftment of the indicated cancer cell lines and subjected to either a control treatment or anti‐PD‐1 therapy. Relevant statistical comparisons are shown in the graph by the U of Mann–Whitney test. B Unmodified and FKN‐expressing 3LL cells were subcutaneously inoculated in mice and tumors were allowed to grow for 7 days. Tumor‐engrafted mice were treated intraperitoneally with anti‐PD‐1 antibody or vehicle (days 7, 11, and 15, as indicated by arrows) following randomization into two groups. Tumor growth was monitored. Data are presented as mean ± SD ( n = 6 mice per group). C Kaplan–Meier survival plots of the indicated groups of mice. Survival differences between the control group (WT + αPD‐1) and FKN + αPD‐1 were evaluated by a two‐sided log‐rank test. D–H (D) Percentage of splenic Ly6C+ monocytes, (E) Ly6G − monocytes, (F) Ly6G + neutrophils, (G) PD‐1 + T cells (CD3 + ), and (H) PD‐1 + NK cells (NK1.1 + ). Data are presented as mean ± SD ( n = 6 mice per group). I–K (I) Percentage of tumor infiltration with total leukocytes (CD45 + ), (J) CD4 T cells, and (K) NK cells (NK1.1 + ). Infiltration data are shown as mean ± SD ( n = 8 mice per group). Data information: Relevant statistical comparisons are shown in the graphs with ANOVA and Tukey's pairwise comparisons. *, **, ***, ****, indicate significant ( P < 0.05), very significant ( P < 0.01), highly significant ( P < 0.001), and very highly significant ( P < 0.0001) differences; In this last case, no specific P ‐value is given in the figure; ns, nonsignificant differences.

Article Snippet: The indicated 3LL cell lines (1.5 × 10 6 cells/mouse; n = 6 mice/group) were subcutaneously injected in the flanks of 10‐week‐old C57BL/6 female and male mice (Envigo).

Techniques: Control, MANN-WHITNEY, Expressing

Journal: EMBO Reports

Article Title: Plasma fractalkine contributes to systemic myeloid diversity and PD‐L1 / PD ‐1 blockade in lung cancer

doi: 10.15252/embr.202255884

Figure Lengend Snippet: A The graphs represent percentages of the indicated infiltrating immune cell types as quantified by flow cytometry, in spleens obtained from mice inoculated with the indicated cell lines (parental cell line, WT; 3LL cells expressing FKN, FKN) with or without PD‐1 blockade treatment. CD4 and CD8 T lymphocytes, NK cells (NK1.1), B cells (CD19), MDSCs (Ly6G + CD115 + ), macrophages (F4/80), and DCs (CD11c) were quantified at day 14 after tumor inoculation. Data are shown as the mean of the percentage within total leukocytes (CD45 + ) ± SD ( n = 6 mice). Relevant statistical comparisons are shown in the graphs, evaluated by ANOVA and Tukey's pairwise comparisons. *, ***, indicate significant ( P < 0.05) and highly significant ( P < 0.001) differences. ns, nonsignificant differences. B Graphs represent percentages of the indicated infiltrating immune cell types as quantified by flow cytometry, in tumors excised from mice inoculated with the indicated cell lines (parental cell line, WT; 3LL cells expressing FKN, FKN) with or without PD‐1 blockade. Neutrophils (Ly6G), CD8, B cells (CD19), Ly6C + monocytes, DCs (CD11c), and macrophages (F4/80) were quantified at day 14 after tumor inoculation. Data are shown as the mean of the percentage within total leukocytes (CD45 + ) ± SD ( n = 8 mice). Relevant statistical comparisons are shown in the graphs, evaluated by ANOVA and Tukey's pairwise comparisons. ns, nonsignificant differences.

Article Snippet: The indicated 3LL cell lines (1.5 × 10 6 cells/mouse; n = 6 mice/group) were subcutaneously injected in the flanks of 10‐week‐old C57BL/6 female and male mice (Envigo).

Techniques: Flow Cytometry, Expressing

Journal: EMBO Reports

Article Title: Plasma fractalkine contributes to systemic myeloid diversity and PD‐L1 / PD ‐1 blockade in lung cancer

doi: 10.15252/embr.202255884

Figure Lengend Snippet: A Experimental schedule for testing systemic FKN anti‐tumor activities. 3LL‐parental (WT) or 3LL‐FKN (FKN) cells were subcutaneously injected into the left flank of mice (inoculation side). Seven days later, 3LL‐parental cells were engrafted on the right flank (target tumor side). Mice were intraperitoneally treated with anti‐PD‐1 antibody at days 7, 11, and 15 after the last tumor inoculation. B Tumor growth of right and left flank engrafted tumors in the experimental schedule shown in (A). Data are presented as mean ± SD ( n = 6 mice per group). C Tumor growth in mice intraperitoneally treated with anti‐CD4, anti‐CD8, and anti‐NK1.1 depleting antibodies at days 6, 10, 14, and 18 after tumor inoculation, as indicated by arrows. Data are presented as mean ± SD ( n = 6 mice per group). D Kaplan–Meier survival plot of the indicated treatment groups. Survival differences between the control group (WT) and FKN were evaluated by a two‐sided log‐rank test. E–J (E) Percentage of splenic NKs (NK1.1 + ) expressing KLRG1, (F) TIGIT, and (G) TIM3. (H) Percentage of splenic T cells (CD3 + ) expressing KLRG1, (I) TIGIT, and (J) TIM3. Data are presented as mean ± SD ( n = 6 mice per group). Comparisons among groups were performed by ANOVA and Tukey's pairwise comparison tests. Data information: ** indicate very significant ( P < 0.01) differences; ns, nonsignificant differences.

Article Snippet: The indicated 3LL cell lines (1.5 × 10 6 cells/mouse; n = 6 mice/group) were subcutaneously injected in the flanks of 10‐week‐old C57BL/6 female and male mice (Envigo).

Techniques: Injection, Control, Expressing, Comparison

Journal: Pharmaceutics

Article Title: An Approach to Evaluate the Effective Cytoplasmic Concentration of Bioactive Agents Interacting with a Selected Intracellular Target Protein

doi: 10.3390/pharmaceutics15020324

Figure Lengend Snippet: ( a ) Melting curves averaged over Hepa1, MEF, and RAW264.7 cell lines of free Nrf2 and Nrf2 bound to Keap1 and total Nrf2 in 3LL and AML12 cell lines. The dependences are normalized to the band intensity at 37 °C; ( b ) Dependence between free Nrf2 fraction (Δ i /Δ max ) in 3LL cells and temperature. The green line shows the linear extrapolation of experimental data to 37 °C. Standard errors (SE) are shown ( n = 4–12).

Article Snippet: After incubation of 3LL cells for 2 h with 20 μM proteasome inhibitor MG-132 (Selleckchem.com), another band of about 85 kDa appeared on the Western blot ( ).

Techniques:

Journal: Pharmaceutics

Article Title: An Approach to Evaluate the Effective Cytoplasmic Concentration of Bioactive Agents Interacting with a Selected Intracellular Target Protein

doi: 10.3390/pharmaceutics15020324

Figure Lengend Snippet: Parameters estimated in this work.

Article Snippet: After incubation of 3LL cells for 2 h with 20 μM proteasome inhibitor MG-132 (Selleckchem.com), another band of about 85 kDa appeared on the Western blot ( ).

Techniques:

Journal: Pharmaceutics

Article Title: An Approach to Evaluate the Effective Cytoplasmic Concentration of Bioactive Agents Interacting with a Selected Intracellular Target Protein

doi: 10.3390/pharmaceutics15020324

Figure Lengend Snippet: ( a ) Melting curves averaged over Hepa1, MEF, and RAW264.7 cell lines of bound Keap1 and Keap1 in 3LL cell lysate and reconstructed curve of free Keap1. The dependences are normalized to the band intensity at 37 °C; ( b ) Dependence between free Keap1 fraction in 3LL cell lysate and temperature. The green line shows the linear extrapolation of experimental data to 37 °C. Standard errors (SE) are shown ( n = 4–12).

Article Snippet: After incubation of 3LL cells for 2 h with 20 μM proteasome inhibitor MG-132 (Selleckchem.com), another band of about 85 kDa appeared on the Western blot ( ).

Techniques:

Journal: International Journal of Pharmaceutics: X

Article Title: Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy ☆

doi: 10.1016/j.ijpx.2022.100138

Figure Lengend Snippet: Cell viability results obtained with 3LL cells after 24- (a), and 72-h (c) incubation; and obtained with EA.hy926 endothelial cells after 24- (b), and 72- h (d) incubation. The error bars, obtained from the standard deviation for 3 technical replicates, are smaller than the height of the square data. Data were evaluated by using GraphPad Prism version 9 with a two-way analysis of variance (ANOVA) with a Bonferroni multiple comparison analysis.

Article Snippet: EA.hy926 and 3LL cell lines were bought from American Type Culture Collection (ATCC® CRL-2922TM for EA.hy926 cell line, ATCC® CRL-1642TM for 3LL cell line, LGC Standards Ltd., Molsheim, France) and cultured at 37 °C in a 5% CO 2 -humidified atmosphere in the DMEM completed medium.

Techniques: Incubation, Standard Deviation, Comparison

Journal: International Journal of Pharmaceutics: X

Article Title: Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy ☆

doi: 10.1016/j.ijpx.2022.100138

Figure Lengend Snippet: The maximal inhibitory concentration in μM for killing 50% (IC50), 85% (IC85) of 3LL and EAhy926 incubated with Fisetin NCs, * p < 0.05, n = 3.

Article Snippet: EA.hy926 and 3LL cell lines were bought from American Type Culture Collection (ATCC® CRL-2922TM for EA.hy926 cell line, ATCC® CRL-1642TM for 3LL cell line, LGC Standards Ltd., Molsheim, France) and cultured at 37 °C in a 5% CO 2 -humidified atmosphere in the DMEM completed medium.

Techniques: Concentration Assay, Incubation

Journal: International Journal of Pharmaceutics: X

Article Title: Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy ☆

doi: 10.1016/j.ijpx.2022.100138

Figure Lengend Snippet: Phase contrast morphology micrographs of 3LL (left column) and EA.hy926 (right column) cell lines after 24-h incubation with 10 and 25 μM Fisetin NCs (a, e), and free Fisetin at the same respective concentration (b, f). Control groups: (c, g) P407, and (d, h) DMSO at the same ratio as the Fisetin NCs and the free Fisetin, respectively.

Article Snippet: EA.hy926 and 3LL cell lines were bought from American Type Culture Collection (ATCC® CRL-2922TM for EA.hy926 cell line, ATCC® CRL-1642TM for 3LL cell line, LGC Standards Ltd., Molsheim, France) and cultured at 37 °C in a 5% CO 2 -humidified atmosphere in the DMEM completed medium.

Techniques: Incubation, Concentration Assay, Control

Journal: International Journal of Pharmaceutics: X

Article Title: Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy ☆

doi: 10.1016/j.ijpx.2022.100138

Figure Lengend Snippet: Immunofluorescence micrographs of 3LL (left column) and EA.hy926 cell lines (right column) after 24-h incubation with 10 and 25 μM Fisetin NCs (a, e), and free Fisetin at the same respective concentration (b, f). Control groups: (c, g) P407, and (d, h) DMSO at the same ratio as the Fisetin NCs and the free Fisetin, respectively. FITC (green) and DAPI (blue) indicate the tubulins and nuclei, respectively. (i) The corresponding form factor for each system, calculated as percentage of the control group, i.e. the endothelial cells grown in the culture medium only. The data were processed with GraphPad Prism version 9 from immunofluorescence micrographs. Data are mean of triplicate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: EA.hy926 and 3LL cell lines were bought from American Type Culture Collection (ATCC® CRL-2922TM for EA.hy926 cell line, ATCC® CRL-1642TM for 3LL cell line, LGC Standards Ltd., Molsheim, France) and cultured at 37 °C in a 5% CO 2 -humidified atmosphere in the DMEM completed medium.

Techniques: Immunofluorescence, Incubation, Concentration Assay, Control

Journal: International Journal of Pharmaceutics: X

Article Title: Designing fisetin nanocrystals for enhanced in cellulo anti-angiogenic and anticancer efficacy ☆

doi: 10.1016/j.ijpx.2022.100138

Figure Lengend Snippet: Apoptosis obtained with 3LL cells after 24-h incubation (a); and obtained with EA.hy926 endothelial cells after 24-h incubation (b) with 50 μM Fisetin NCs, free Fisetin, P407, and DMSO at the same respective concentration. Colour code: (black) late apoptosis, (dark gray) early apoptosis, (light gray) necrosis, and (white) viable cells. Control group: untreated cell groups were added to the mixture (Annexin V-FITC, PI and binding buffer). The figures were treated with GraphPad Prism version 9 from flow cytometry device. Data are mean of triplicate (value ± SD).

Article Snippet: EA.hy926 and 3LL cell lines were bought from American Type Culture Collection (ATCC® CRL-2922TM for EA.hy926 cell line, ATCC® CRL-1642TM for 3LL cell line, LGC Standards Ltd., Molsheim, France) and cultured at 37 °C in a 5% CO 2 -humidified atmosphere in the DMEM completed medium.

Techniques: Incubation, Concentration Assay, Control, Binding Assay, Flow Cytometry