shctrl b16f10 non targeting shrna plasmid plk01  (Addgene inc)

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: ( A ) SK1 mRNA level (left panel) and enzymatic activity (right panel) were measured in B16F10 cells stably transfected with a control (shCtrl) or SK1 targeted shRNA (shSK1, shSK1#2 or shSK1#3). Data are expressed as fold-change over shCtrl B16F10 cells and are means ± sem of 3–5 independent experiments. ( B ) B16F10 murine melanoma cells (3.10 5 ) were injected in the dermis of C57BL/6 mice. After excision 10 days later, tumors were collected and weighed (left panel). Data are means ± sem ( n = 4 to 5 mice per group). The relationship between SK1 enzymatic activity and tumor weight was evaluated with a Pearson correlation analysis (right panel). ( C ) Cellular lipids were extracted from shCtrl or shSK1 tumors and sphingolipid levels were quantified by LC/MS. Levels of S1P (left panel), total ceramide (middle panel) and individual ceramide species (right panel) were normalized to protein content. Results represent means ± sem of 2 independent experiments. ( D ) Tumor volume was determined at the indicated days in the mice that were implanted with shCtrl or shSK1 B16F10 cells, as described in B. Results are from 2 independent experiments performed with 7 mice per group. Values determined for individual tumors are depicted and horizontal lines correspond to means. For all panels, significant differences were evaluated using Student t test.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Activity Assay, Stable Transfection, Transfection, Control, shRNA, Injection, Liquid Chromatography with Mass Spectroscopy

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: ( A , C ) shCtrl or shSK1 B16F10 murine melanoma cells were injected in C57BL/6 mice. Ten days after injection, mice were sacrificed, tumors were collected, and their leukocyte content was analyzed. Bars represent means ± sem of 4 mice per group. Data are representative of two independent experiments. Significant differences were evaluated using Student t test. (A) Cells were counted and the percentage of CD45 and F4/80 among total cells was determined by flow cytometry. (B) Mice ( n = 5–6/group) bearing shCtrl or shSK1 B16 tumors were treated with control liposomes (L-Control) or clodronate-containing liposomes (L-Clodronate) the day before tumor cell injection and then every 4 days for 2 weeks. Tumor volume was determined 13 days after melanoma cell implantation. Values determined for individual tumors are depicted and horizontal lines correspond to means. Data are representative of two independent experiments. Statistical analysis was performed using the Mann-Whitney U -test. (C) Representative flow cytometry density plots. Values indicate the percentages of MHC-II high CD206 low (left panel), MHC-II low CD206 high (middle panel) and iNOS + (right panel) cells among the F4/80 + cells. ( D) Ten days after injection, tumors were collected for mRNA isolation and analysis. Relative mRNA expression (fold induction relative to shCtrl) is depicted for M2 markers (YM1 and ARG-1) and M1 markers (Il12, Tnfα, Ifnγ, Ccl5, Cxcl9, Cxcl10). Significant differences were evaluated using Student t test. NS : not significant.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Injection, Flow Cytometry, Control, Liposomes, MANN-WHITNEY, Isolation, Expressing

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: ( A ) S1P release from shCtrl or shSK1 B16F10 melanoma cells as determined after the conversion of [ 3 H]sphingosine to [ 3 H]S1P. Concentrations of radiolabeled S1P in the medium are expressed as mean ± sem of three independent experiments. Significant differences were evaluated using Student t test. Transwell migration assays were performed to evaluate migration of BMDM ( B and C ) or THP-1 cells ( E – G ). Data are expressed as percent increase or decrease over migration in serum-free medium, and are means ± sem of 2–4 independent experiments. For panels B, C and E-G, significant differences were evaluated using one-way ANOVA with post hoc Tukey test. (B and C) BMDM were pre-treated (+) or not (−) with 2 μM VPC23019 (VPC) or 5 μM W146 for 1 hour. Then, BMDM were incubated for 5 hours in serum-free medium alone (Medium), the conditioned medium (CM) from shCtrl or shSK1 B16F10 melanoma cells containing or not S1P (B) or in serum-free medium containing S1P at the indicated concentrations (C). ( D ) SK1 mRNA expression (left panel), enzymatic activity (middle panel) and S1P release (right panel) were measured in COLO829 melanoma cells transfected either with an empty vector (Vector) or a plasmid encoding human SK1 (SK1). Data are means ± sem of 3 independent experiments. Significant differences were evaluated using Student t test. E and G, THP-1 cells were pre-treated or not with 2 μM VPC23019, 2 μM FTY720 (FTY) or 5 μM W146 for 1 hour. Then, THP-1 cells were incubated for 6 hours in the CM from control (Vector) or SK1-overexpressing (SK1) COLO829 melanoma cells (E) or in serum-free medium containing S1P at the indicated concentrations (G). F, Melanoma CM-induced cell migration was evaluated, as described in E, on THP-1 cells 48 hours after transfection with control (Ctrl) or S1PR1 siRNA (20 nM). Silencing of S1PR1 in THP-1 cells by siRNA was assessed by Western blot.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Migration, Incubation, Expressing, Activity Assay, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: BMDM were incubated for 24 hours in the conditioned medium (CM) from B16F10 melanoma cells ( A and B ) or serum-free medium containing or not 1 μM S1P ( C ). Relative mRNA expression is depicted for M2 markers ( Mrc1 (CD206), Chi3l3 (YM1) and Arg1 ) and M1 markers ( iNos , Il12 , Tnfα , Ifnγ , Ccl5 , Cxcl9 , Cxcl10 ). Data are plotted against the SK1 activity of B16F10 melanoma cells submitted to distinct treatments. (A) CM was prepared from shCtrl or shSK1 B16F10 melanoma cells treated or not with 3 μM SKI-I for 48 hours. (B) BMDM were pre-treated with 2 μM VPC23019 before incubation with the CM from shCtrl B16F10 melanoma cells. Data are expressed as fold-increase over shCtrl B16F10 cells and are means ± sem of 2–6 independent experiments. (C) Data are expressed as fold-change over migration in serum-free medium, and are means ± sem of 3–4 independent experiments. For all panels, significant differences were evaluated using Student t test.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Incubation, Expressing, Activity Assay, Migration

Journal: Oncotarget

Article Title: Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

doi: 10.18632/oncotarget.12380

Figure Lengend Snippet: ( A ) Analysis of SK1 and TGF-β1 expression in melanoma tumors was carried out using the TCGA database. ( B ) SK1 enzymatic activity (left), TGF-β1 mRNA expression (middle) and TGF-β1 secreted protein level (right) were measured in shCtrl or shSK1 B16F10 melanoma cells treated or not with 3 μM SKI-I for 48 hours. ( C ) TGF-β1 mRNA level was measured in shCtrl B16F10 melanoma cells pretreated or not with 2 μM VPC23019, 2 μM FTY720 or 10 μM JTE013 and incubated with 1 μM S1P for 24 hours. Data are expressed as fold-increase over shCtrl B16F10 cells and are means ± sem of 2–3 independent experiments. Significant differences were evaluated using Student t test. ( D and E ) BMDM were incubated for 48 hours in the conditioned medium (CM) from shCtrl or shSK1 B16F10 melanoma cells. Relative mRNA level is depicted for M2 markers (CD206, YM1 and Arg-1) and M1 markers (iNos, Il12, Tnfα, Ifnγ, Ccl5, Cxcl9, Cxcl10). D, Recombinant murine TGF-β1 (50 ng/ml) was added to the CM from shSK1 B16F10 melanoma cells before incubation with BMDM. E, Anti-TGF-β1 (1 μg/ml) was added to the CM from shCtrl B16F10 melanoma cells before incubation with BMDM. Results represent means ± sem of 2–3 independent experiments. Significant differences were evaluated using Student t test. ( F ) Mice ( n = 6/group) bearing shCtrl B16 tumors were treated with TGF-b-neutralizing antibody or PBS, one day after tumor cell injection and then three times per week for 2 weeks. Tumor volume was determined 13 days after implantation. Values determined for individual tumors are depicted and horizontal lines correspond to means. Data are representative of two independent experiments. Statistical analysis was performed using the Mann-Whitney U -test.

Article Snippet: B16F10 cells were co-transfected, in a 1:10 ratio, with the pEGFP-C1 empty vector plus one SK1 shRNA plasmid (shSK1 B16F10, 3 different shRNA from Thermoscientific were used) or a control (shCtrl B16F10) non-targeting shRNA plasmid (pLK01, Addgene).

Techniques: Expressing, Activity Assay, Incubation, Recombinant, Injection, MANN-WHITNEY