Journal: PLoS ONE

Article Title: Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

doi: 10.1371/journal.pone.0140471

Figure Lengend Snippet: Results from tumor cell viability measurements show samples normalized to the controls. Cells incubated without r28M served as controls, their number was considered as 1. Values are means ± SE. Significance levels: x: p ≤ 0.05; xx: p ≤ 0.01; xxx: p ≤ 0.001. PBMC from two different donors were used for the shown experiments. (A) CSPG4 positive (IPC-298) and negative cells (U-251 MG) were incubated with PBMC and monomeric r28M in concentrations ranging from 100–50000 ng/ml for 72 hrs in a final volume of 150 μl. (B) CSPG4 positive cells (A-375) were incubated with PBMC and 1000 ng/ml monomeric r28M for 72 hrs. 1000 ng/ml r28M were applied either immediately (day 1), immediately and with a repeated administration after 24 hrs (day 1/2), immediately and after 24 and 48 hrs (day 1/2/3) or immediately and after 48 hrs (day 1/3).

Article Snippet: A-375 (#300110) and U-251 MG (#300385) cell lines were purchased from CLS-cell line service (Eppelheim, Germany).

Techniques: Incubation

Journal: PLoS ONE

Article Title: Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

doi: 10.1371/journal.pone.0140471

Figure Lengend Snippet: CSPG4 positive cells (A-375) were incubated with PBMC, CD4 + , CD8 + and a CD56 + enriched cell fraction, respectively, together with 1000 ng/ml monomeric r28M in a final volume of 150 μl. The amount of viable tumor cells was determined after 72 and 168 hrs of incubation. Results show samples normalized to the controls. Cells incubated without r28M served as controls, their number was considered as 1. Values are means ± SE. Significance levels: x: p ≤ 0.05; xx: p ≤ 0.01; xxx: p ≤ 0.001.

Article Snippet: A-375 (#300110) and U-251 MG (#300385) cell lines were purchased from CLS-cell line service (Eppelheim, Germany).

Techniques: Incubation

Journal: Cancer Research

Article Title: Targeting the PREX2/RAC1/PI3Kβ Signaling Axis Confers Sensitivity to Clinically Relevant Therapeutic Approaches in Melanoma

doi: 10.1158/0008-5472.CAN-23-2814

Figure Lengend Snippet: Cotargeting of MEK1/2 and the RAC1 effector p110β suppresses growth of melanoma lines in vitro . A, Left, relative confluence of WM266.4 cells treated with indicated treatments over time. Representative of a minimum of five independent experiments and three technical replicates. Data, mean ± SEM (confluence relative to starting point). Right, relative change in confluence of WM266.4 cells over indicated 72-hour treatment. Center line, mean. P values calculated by one-way ANOVA corrected for multiple comparisons (Tukey). B, Left, relative confluence of A375 cells treated with indicated treatments over time. Representative of three independent experiments with a minimum of three technical replicates. Data, mean ± SEM (confluence relative to starting point). Right, relative change in confluence of A375 cells over indicated 72-hour treatment. Center line, mean. P values calculated by one-way ANOVA corrected for multiple comparisons (Tukey). C, Fluorescence labeled (LI-COR) immunoblotting for indicated activated components of the MAPK–PI3K–mTOR pathway in the indicated human melanoma cells treated with vehicle or the indicated targeted therapeutics. Co-labeled total protein detection for each phosphoprotein serves as a sample integrity control. The blots are representative of three repeated experiments. D, RPPA dataset comparing PTEN-deficient and PTEN-proficient melanoma cell lines treated with indicated treatments for 24 hours. Cell line names and treatments are indicated below the heatmap. Antigens, detected by RPPA antibodies, are listed vertically according to biological process/signaling pathway. Color intensity scale indicates high (red) and low (blue) log 2 FC of RPPA intensity values relative to the relevant DMSO control. Results are representative of two technical and five biological replicates per condition.

Article Snippet: WM266.4, A375, WM793, WM1158, and A2058 cells were the kind gift of Prof. Lionel Larue (Institut Curie).

Techniques: In Vitro, Fluorescence, Labeling, Western Blot, Control

Journal: Cancer Research

Article Title: Targeting the PREX2/RAC1/PI3Kβ Signaling Axis Confers Sensitivity to Clinically Relevant Therapeutic Approaches in Melanoma

doi: 10.1158/0008-5472.CAN-23-2814

Figure Lengend Snippet: Genetic targeting of p110β and its interaction with RAC1 sensitizes to MEK1/2 inhibition in vitro and in vivo. A, Left, flow cytometry–based cell-cycle profiling of WM266.4 cells following indicated 24-hour treatment. Right, proportion of WM266.4 cells in G 1 /S at 24 hours. n = 3 independent experiments. P values calculated by one-way ANOVA corrected for multiple comparisons (Tukey). B, Left, flow cytometry–based cell-cycle profiling of A375 cells following indicated 24 hours treatment. Middle, proportion of A375 cells in G 1 /S at 24 hours. Right, proportion of subdiploid A375 cells at 24 hours. n = 4 independent experiments. P values calculated by one-way ANOVA corrected for multiple comparisons (Tukey). C, Left, Kaplan–Meier overall survival of BRAF PTEN mice treated with vehicle or AZD6244. BRAF PTEN + vehicle ( n = 8; median survival, 12.5 days) vs. BRAF PTEN + AZD6244 ( n = 8; median survival, 74.5 days), P = 0.0096; BRAF PTEN PIK3CB-mut + vehicle ( n = 4; median survival, 11.5 days) vs. BRAF PTEN PIK3CB-mut + AZD6244 ( n = 7; median survival, 83 days), P = 0.0082; BRAF PTEN + AZD6244 ( n = 8; median survival, 74.5 days) vs. BRAF PTEN PIK3CB-mut + AZD6244 ( n = 7; median survival, 83 days), P = 0.1643. P values calculated using the log-rank (Mantel–Cox) test. Middle, longitudinal growth of individual tumors from vehicle-treated ( n = 8) vs. AZD6244-treated ( n = 7) BRAF PTEN cohorts and vehicle-treated ( n = 5) vs. AZD6244-treated ( n = 7) BRAF PTEN PIK3CB-mut cohorts. Right, relative change in tumor volume over the first 7 days of indicated treatment. BRAF PTEN + vehicle ( n = 8), BRAF PTEN + AZD6244 ( n = 7), BRAF PTEN PIK3CB-mut + vehicle ( n = 5), BRAF PTEN PIK3CB-mut + AZD6244 ( n = 6); P values calculated by one-way ANOVA corrected for multiple comparisons (Tukey). Center line, mean. Note that the BRAF PTEN treatment datasets are reproduced from and . D, Left, relative confluence of WM266.4 PIK3CB KO cells treated with indicated treatments over the 96-hour period. Representative of a minimum of three independent experiments and three technical replicates. Data, mean ± SEM (confluence relative to starting point). Right, relative change in confluence of WM266.4 PIK3CB KO cells over the indicated 96-hour period. Center line, mean. P values calculated by one-way ANOVA corrected for multiple comparisons (Tukey).

Article Snippet: WM266.4, A375, WM793, WM1158, and A2058 cells were the kind gift of Prof. Lionel Larue (Institut Curie).

Techniques: Inhibition, In Vitro, In Vivo, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Effect of Tryptophan-Derived AhR Ligands, Kynurenine, Kynurenic Acid and FICZ, on Proliferation, Cell Cycle Regulation and Cell Death of Melanoma Cells—In Vitro Studies

doi: 10.3390/ijms21217946

Figure Lengend Snippet: The effect of L-KYN ( a ), KYNA ( b ) and FICZ ( c ) on proliferation (DNA synthesis) of melanocytes and melanoma A375 and RPMI7951 cells. Normal human adult primary epidermal melanocytes (HEMa) and human melanoma A375 and RPMI7951 cells were exposed to fresh medium (control, C) or serial dilutions of L-KYN, KYNA and FICZ for 24 h. The effect of tested compounds on proliferation (DNA synthesis) was determined by means of BrdU Assay. Data represent a mean value (% of control; C = 100%) ± SEM of eight independent experiments. Values significant (*) in comparison to control (100%) with p < 0.05 (one-way ANOVA with Tukey post hoc test).

Article Snippet: All A375 and RPMI 7951 cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: DNA Synthesis, BrdU Staining

Journal: International Journal of Molecular Sciences

Article Title: Effect of Tryptophan-Derived AhR Ligands, Kynurenine, Kynurenic Acid and FICZ, on Proliferation, Cell Cycle Regulation and Cell Death of Melanoma Cells—In Vitro Studies

doi: 10.3390/ijms21217946

Figure Lengend Snippet: The toxicity of L-KYN ( a ), KYNA ( b ) and FICZ ( c ) towards melanocytes and melanoma A375 and RPMI7951 cells. Normal human adult primary epidermal melanocytes (HEMa) and human melanoma A375 and RPMI7951 cells were exposed to fresh medium (control, C) or serial dilutions of L-KYN, KYNA and FICZ for 24 h. The toxicity of tested compounds was assessed by means of LDH Assay measuring LDH release. Data represent a mean value (% of control) ± SEM of eight independent experiments. Values significant (*) in comparison to control (100%) with p < 0.05 (one-way ANOVA with Tukey post hoc test). Positive control for melanoma A375 cells (Total LDH) = 1720%.

Article Snippet: All A375 and RPMI 7951 cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Lactate Dehydrogenase Assay, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Effect of Tryptophan-Derived AhR Ligands, Kynurenine, Kynurenic Acid and FICZ, on Proliferation, Cell Cycle Regulation and Cell Death of Melanoma Cells—In Vitro Studies

doi: 10.3390/ijms21217946

Figure Lengend Snippet: The effect of L-KYN ( a ), KYNA ( b ) and FICZ ( c ) on the protein level of selected cell cycle regulators in melanoma A375 and RPMI7951 cells. Western blot analysis of the protein level of cyclin D1, CDK4 and phosphorylation of Rb in A375 and RPMI7951 cells after treatment with L-KYN ( a ) and KYNA ( b ) in the range of concentrations 10 −9 –5 mM and FICZ ( c ) in the range of concentrations 10 −6 –50 µM for 24 h (C control; not treated). Western blots shown in the figure were selected as the most representative of the series of repetitions. The data were normalized relative to β-actin. The results of densitometric analysis are shown as % of control (the changes ≥30% were considered as significant (*)).

Article Snippet: All A375 and RPMI 7951 cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Effect of Tryptophan-Derived AhR Ligands, Kynurenine, Kynurenic Acid and FICZ, on Proliferation, Cell Cycle Regulation and Cell Death of Melanoma Cells—In Vitro Studies

doi: 10.3390/ijms21217946

Figure Lengend Snippet: The effect of L-KYN on the protein level and cellular localization of cyclin D1 ( a ) and CDK4 ( b ) in melanoma A375 cells. Immunofluorescent staining of cyclin D1 ( a ) and CDK4 ( b ) in A375 cells treated with L-KYN 5 mM for 24 h (control; not treated). Cell nuclei were labeled with cell permeable fluorescent DNA dye DraQ5. Magnification 40×.

Article Snippet: All A375 and RPMI 7951 cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Effect of Tryptophan-Derived AhR Ligands, Kynurenine, Kynurenic Acid and FICZ, on Proliferation, Cell Cycle Regulation and Cell Death of Melanoma Cells—In Vitro Studies

doi: 10.3390/ijms21217946

Figure Lengend Snippet: The effect of L-KYN, KYNA and FICZ on induction of apoptosis and necrosis in melanoma A375 ( a , b ) and RPMI7951 ( c , d ) cells. Human melanoma A375 and RPMI7951 cells were exposed to fresh medium (control, C) or selected tryptophan-derived AhR ligands: L-KYN (1 mM), KYNA (5 mM) and FICZ (50 µM) for 24 h. The effect of tested compounds on induction of apoptosis ( a , c ) and necrotic cell death ( b , d ) was determined by means of Cell Death Detection ELISA. Data represent a mean value ± SEM of three independent experiments. Values significant (*) in comparison to control with p < 0.05 (one-way ANOVA with Tukey post hoc test).

Article Snippet: All A375 and RPMI 7951 cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Effect of Tryptophan-Derived AhR Ligands, Kynurenine, Kynurenic Acid and FICZ, on Proliferation, Cell Cycle Regulation and Cell Death of Melanoma Cells—In Vitro Studies

doi: 10.3390/ijms21217946

Figure Lengend Snippet: The effect of L-KYN, KYNA and FICZ on induction of cell death in melanoma A375 and RPMI7951 cells. Human melanoma A375 and RPMI7951 cells were exposed to fresh medium (control, C) or selected tryptophan-derived AhR ligands: L-KYN (1 mM) ( a ), KYNA (5 mM) ( b ) and FICZ (50 µM) ( c ) for 24 h. The effect of tested compounds on induction of apoptosis and necrotic cell death was determined by means of staining with Hoechst 33342 and propidium iodide. Cells with fragmented nuclei stained in an intense blue color were considered apoptotic cells while the pink staining of the nuclei was necrotic cells. Magnification 10×.

Article Snippet: All A375 and RPMI 7951 cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Derivative Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Effect of Tryptophan-Derived AhR Ligands, Kynurenine, Kynurenic Acid and FICZ, on Proliferation, Cell Cycle Regulation and Cell Death of Melanoma Cells—In Vitro Studies

doi: 10.3390/ijms21217946

Figure Lengend Snippet: The effect of L-KYN, KYN A and FICZ on AhR protein level in melanoma A375 and RPMI7951 cells. Western blot analysis of protein level of AhR in A375 and RPMI7951 cells after treatment with L-KYN ( a ) and KYNA ( b ) in the range of concentrations 10 −9 –5 mM and FICZ ( c ) in the range of concentrations 10 −6 –50 µM for 24 h (C control; not treated). Western blots shown in the figure were selected as the most representative of the series of repetitions. The data were normalized relative to β-actin. The results of densitometric analysis are shown as % of control (changes ≥30% were considered as significant (*)).

Article Snippet: All A375 and RPMI 7951 cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Effect of Tryptophan-Derived AhR Ligands, Kynurenine, Kynurenic Acid and FICZ, on Proliferation, Cell Cycle Regulation and Cell Death of Melanoma Cells—In Vitro Studies

doi: 10.3390/ijms21217946

Figure Lengend Snippet: The effect of L-KYN, KYNA and FICZ on AHR gene expression in melanoma A375 ( a ) and RPMI7951 ( b ) cells. Real-Time PCR analyses were used to evaluate AHR gene expression in A375 and RPMI7951 cells after 24 h treatment with L-KYN, KYNA and FICZ at the concentration of 1mM, 5 mM and 50 µM, respectively (C, control–not treated). ACTB was used as the reference gene. For more reliable results, RQ values were analyzed after log transform to log10RQ. Data represent a mean value ± SEM of three independent experiments. The differences in AHR gene expression were not statistically significant in comparison to control (C) (values significant with p < 0.05, unpaired t -test).

Article Snippet: All A375 and RPMI 7951 cell culture reagents were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Concentration Assay