tumor cell conditioned medium  (ATCC)

Journal: Nature cancer

Article Title: Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs

doi: 10.1038/s43018-020-0064-0

Figure Lengend Snippet: a. A representative immunofluorescence staining of CD11b (left) and quantification of CD11b+ cells (right) in the lung tissues from WT mice treated with tumor cell conditioned media (TCM) from B16F1, or B16F10 cells or with serum-free media (SFM) as control (100 μl i.v., 3x per week for 3 weeks). Quantitative data shown as mean±SEM (n=4 mice for SFM and F10 TCM group, n=7 mice for F1 TCM group). Two-tailed Unpaired t test was performed for the comparisons between two groups. Here and henceforth, the inset shows a zoomed (2x) area of the stained image. Scale bar: 100 μm.

Article Snippet: Cell lines and tumor-conditioned medium (TCM) preparation Mouse melanoma cell lines B16F1 (ATCC® CRL-6323TM) and B16F10 (ATCC® CRL-6475TM) were purchased from ATCC and maintained in DMEM (Gibco) supplemented with 10% FBS (HyClone) and 100 U/ml Penicillin-Streptomycin (Gibco).

Techniques: Immunofluorescence, Staining, Control, Two Tailed Test

Journal: Nature cancer

Article Title: Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs

doi: 10.1038/s43018-020-0064-0

Figure Lengend Snippet: a. The primary tumor growth in WT mice s.c injected with 1*105 B16F1 or B16F10 tumor cells prior to surgery at equivalent tumor size (~200 mm2). Data shown as mean±SEM (n=7 mice per group). Repeated-measure two-way ANOVA and Sidak’s multiple comparisons test were performed. b. A representative lung images and the corresponding H&E stained lung sections in the B16F1 and B16F10 tumor bearing mice after surgery. Scale bar: 1 mm. This experiment was repeated three times independently with similar results. c. Quantification of the number of metastatic lesions and total area in the lung tissues from B16F1 and B16F10 tumor bearing mice after surgery as shown in b. Data shown as mean±SEM (n=7 mice per group). Two-tailed Unpaired t test was performed for the comparison. d. The primary tumor growth in the B16F1 tumor bearing mice treated with SFM or B16F10 TCM prior to surgery (100 μl every other day until primary tumor removal upon reaching the size ~200 mm2). n=5 mice per group.

Article Snippet: Cell lines and tumor-conditioned medium (TCM) preparation Mouse melanoma cell lines B16F1 (ATCC® CRL-6323TM) and B16F10 (ATCC® CRL-6475TM) were purchased from ATCC and maintained in DMEM (Gibco) supplemented with 10% FBS (HyClone) and 100 U/ml Penicillin-Streptomycin (Gibco).

Techniques: Injection, Staining, Two Tailed Test, Comparison

Journal: Nature cancer

Article Title: Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs

doi: 10.1038/s43018-020-0064-0

Figure Lengend Snippet: a. A representative western blot analysis of p-p38 and total p38 in the leukocytes isolated from the peripheral blood of melanoma patients with metastasis (Met) and without metastasis (Non-Met). The ratio of p-38 to p38 was shown at bottom for each patient. This experiment was repeated three times independently with similar results. b. A representative flow cytometry histogram (left) and the quantification of surface IFNAR1 level (right) in WT lung fibroblasts 2 hr after SFM, conditioned media from normal lung fibroblasts (FCM), or TCM from different tumor cells including B16F1, B16F10, MH6499c4, and E0771. Quantitative data shown as mean±SEM (n=3 biologically independent samples). Two- tailed Unpaired t test was performed for the comparisons between two groups. c. A representative immunofluorescence staining of IFNAR1 (left) and the quantification of IFNAR1 level (right) in the lung tissues from WT mice treated with SFM, B16F1 TCM or B16F10 TCM (100 μl i.v., 3x per week for 3 weeks). Scale bar: 100 μm. Quantitative data shown as mean±SEM (n=10 mice per group). Two-tailed Unpaired t test was performed for the comparisons between two groups. d. Representative lung images and the corresponding H&E-stained lung sections in WT and Ifnar1−/− B16F1 tumor bearing mice after surgery. Lung metastases were analyzed around 30–60 days after primary tumor removal upon reaching the size ~200mm2. Scale bar: 1 mm. This experiment was repeated three times independently with similar results. e. Quantification of the number of metastatic lesions and total area in the lung tissues from WT (n=13 mice) and Ifnar1−/− (n=14 mice) B16F1 tumor bearing mice after surgery. Data shown as mean±SEM. Two-tailed Unpaired t test was performed for the comparison. f. Kaplan-Meier analysis of survival of WT (n=9 mice) and Ifnar1−/− (n=10 mice) B16F1 tumor bearing mice after surgery by Log-rank test.

Article Snippet: Cell lines and tumor-conditioned medium (TCM) preparation Mouse melanoma cell lines B16F1 (ATCC® CRL-6323TM) and B16F10 (ATCC® CRL-6475TM) were purchased from ATCC and maintained in DMEM (Gibco) supplemented with 10% FBS (HyClone) and 100 U/ml Penicillin-Streptomycin (Gibco).

Techniques: Derivative Assay, Activation Assay, Western Blot, Isolation, Flow Cytometry, Two Tailed Test, Immunofluorescence, Staining, Comparison

Journal: Nature cancer

Article Title: Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs

doi: 10.1038/s43018-020-0064-0

Figure Lengend Snippet: a. A representative immunofluorescence staining of IFNAR1 (left) and the quantification of IFNAR1 level (right) in the lung tissues from WT and SA mice treated with SFM or B16F10 TCM. Scale bar: 100 μm. Quantitative data shown as mean±SEM (n=5 mice per group). Two-way ANOVA and Sidak’s multiple comparisons test were performed. b. qPCR analysis of mRNA levels of indicated interferon stimulated genes in the lung tissues of WT and SA mice treated with SFM or B16F10 TCM. Data shown as mean±SEM (n=3, n=4 mice in SFM and TCM treated group respectively). Two-way ANOVA and Tukey’s multiple comparisons test were performed. c. Schematic illustration for analysis of the lung metastasis in the B16F1 tumor bearing SA mice treated with SFM or B16F10 TCM (100 μl i.v. every other day until primary tumor removal upon reaching the size ~200 mm2). d. The primary tumor growth of B16F1 in SA mice treated with SFM or B16F10 TCM prior to surgery. Data shown as mean±SEM (n=4 mice per group). Repeated-measure two-way ANOVA and Sidak’s multiple comparisons test were performed. e. Representative lung images and the corresponding H&E-stained lung sections of indicated mice as described in c. Scale bar: 1 mm. Similar results were obtained from three independent experiments. f. Quantification of the number of metastatic lesions and total area in the lung tissues from B16F1 tumor bearing SA mice treated with SFM (n=10 mice) or B16F10 TCM (n=9 mice) after surgery. Data shown as mean±SEM. Two-tailed Unpaired t test was performed for the comparison. g. Kaplan-Meier analysis of survival of B16F1 tumor bearing SA mice treated with SFM (n=9 mice) or B16F10 TCM (n=9 mice) after surgery by Log-rank test. h. Representative flow cytometry analysis of tumor cells (TdTomato+) in the lung tissues of WT, SA, and tamoxifen treated-Mapk14fl/flUbc9-CreER- (Mapk14fl/fl) and Mapk14fl/flUbc9-CreER+ (Mapk14Δ/Δ) mice pretreated with SFM or B16F10 TCM followed by intravenous injection of 5*105 B16F10-TdTomato cells. Similar results were obtained from three independent experiments.

Article Snippet: Cell lines and tumor-conditioned medium (TCM) preparation Mouse melanoma cell lines B16F1 (ATCC® CRL-6323TM) and B16F10 (ATCC® CRL-6475TM) were purchased from ATCC and maintained in DMEM (Gibco) supplemented with 10% FBS (HyClone) and 100 U/ml Penicillin-Streptomycin (Gibco).

Techniques: Immunofluorescence, Staining, Two Tailed Test, Comparison, Flow Cytometry, Injection

Journal: Nature cancer

Article Title: Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs

doi: 10.1038/s43018-020-0064-0

Figure Lengend Snippet: a. Representative flow cytometry analysis of myeloid cell subpopulations (above) and the quantification of these cell subpopulations (below) in the lung tissues of WT and SA mice treated with SFM or B16F10 TCM. Quantitative data shown as mean±SEM (n=4 mice per group). Two-way ANOVA and Sidak’s multiple comparisons test were performed. b. Representative flow cytometry analysis of the purity of the isolated granulocytes from lung tissues of WT and SA mice treated with B16F10 TCM. Similar results were obtained from three independent experiments. c. Antigen-specific proliferation of CD8+ T cells in the presence of isolated granulocytes (Ly-6G+) from the lung tissues of WT and SA mice treated with B16F10 TCM at a ratio of 1:1, 1:2 or 1:4 measured as the uptake of 3H thymidine and presented relative to that in the absence of granulocytes which set as 100%. Splenic PMN-MDSC isolated from MC38 tumor-bearing mice served as a positive control for the suppression. Data shown as mean±SEM (n=6 mice for WT and SA group, n=3 mice for PMN-MDSC). Two-way ANOVA and Sidak’s multiple comparisons test were performed. d. qPCR analysis of mRNA levels of the indicated chemokines in WT lung fibroblasts 6 hr after SFM, conditioned media from normal lung fibroblasts (FCM), or TCM from different tumor cells including B16F1, B16F10, MH6499c4, and E0771. Data shown as mean±SEM (n=3 biologically independent samples). Two-tailed Unpaired t test was performed for the comparisons between two groups. e. qPCR analysis of mRNA levels of the indicated chemokines in the lung tissues of WT mice treated with SFM (n=3 mice), B16F1 TCM (n=4 mice) or B16F10 TCM (n=4 mice). Data shown as mean±SEM. Two-way ANOVA and Tukey’s multiple comparisons test were performed. f. Representative flow cytometry analysis (left) and the quantification of percent of neutrophils (right) in the lung tissues of WT mice treated with SFM (n=5 mice), B16F10 TCM plus vehicle (n=4 mice) or CXCR2 inhibitor (n=5 mice). Quantitative data shown as mean±SEM. Two-tailed Unpaired t test was performed for the comparisons between two groups. g. qPCR analysis of mRNA level of Cxcl1 in the lung tissues of SA mice intranasally administered with control (n=3 mice) or CXCL1 (n=7 mice) expressing adenovirus. Data shown as mean±SEM. Two-tailed Unpaired t test was performed for the comparison. h. The percent of total myeloid cells in the lung tissues of SA mice intranasally administered with control (n=4 mice) or CXCL1 (n=5 mice) expressing adenovirus. Data shown as mean±SEM. Two-tailed Unpaired t test was performed for the comparison.

Article Snippet: Cell lines and tumor-conditioned medium (TCM) preparation Mouse melanoma cell lines B16F1 (ATCC® CRL-6323TM) and B16F10 (ATCC® CRL-6475TM) were purchased from ATCC and maintained in DMEM (Gibco) supplemented with 10% FBS (HyClone) and 100 U/ml Penicillin-Streptomycin (Gibco).

Techniques: Flow Cytometry, Isolation, Positive Control, Two Tailed Test, Control, Expressing, Comparison