Journal: Molecular Cell

Article Title: Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution

doi: 10.1016/j.molcel.2020.05.025

Figure Lengend Snippet:

Article Snippet: A375M Human melanoma cell line (male) , ATCC , ( , ) RRID:CVCL_B222 .

Techniques: Virus, Subcloning, Bacteria, Recombinant, Protease Inhibitor, Purification, Mutagenesis, Expressing, Fluorescence, Sequencing, Software

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

doi: 10.1016/j.ymthe.2023.11.021

Figure Lengend Snippet: Figure 1. Chemical changes within the OMe-miR126 sequence: a comparative analysis of its function and stability in plasma (A) OMe-miR126 consists of a double-stranded ribonucleotide and the anti-sense strand contains mature miR-126-3p sequence. All the pyrimidine residues of the sense strand and one of the uridine residues in the anti-sense strand are modified by O-methylation at the 20-O pentose (20-OMe). The sense and anti-sense strands have been modified with the addition of two 20-deoxythymidine residues at the 30 terminal of each strand. (B) Analysis of miR126 expression obtained by qRT-PCR performed 24 h after transfection of A375M-VR cell line with Pre-miR126 (Pre), PTO-miR126 (PTO), OMe-miR126 (OMe), and relative negative control. U6 snRNA was used as normalizer. The histogram columns represent the ratio ± SD of the values obtained from transfection of miR126 sequences and their respective controls. Commercial miR126-3p was used as positive reference. (C) WES analysis of p85-b protein in A375M-VR cell line after transfection with Pre-miR126, PTO-miR126, OMe-miR126 sequences or their relative negative controls. MiR126-3p and -5p were used as positive and negative references, respectively. b-Actin was utilized as internal loading control. (D) Analysis of miR126 expression in A375M-VR by qRT-PCR, performed 24, 48, and 72 h after OMe-miR126 internalization. Relative miR126 expression level was normalized on U6 snRNA. The

Article Snippet: Vemurafenib-resistant A375M (A375M-VR), dabrafenib-resistant A375M (A375M-DR), and dabrafenib-resistant SKMEL28 (SKMEL28-DR) cell lines were generated by culturing parental cells in increasing concentrations of vemurafenib or dabrafenib (from 0.5 to 10 mM; Selleckchem, Munich, Germany) for at least 2 months and subsequently maintained in full growth medium containing 5 mM vemurafenib for A375M-VR or 1.5 mM dabrafenib for A375M-DR and SKMEL28-DR. All melanoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Sequencing, Clinical Proteomics, Methylation, Expressing, Quantitative RT-PCR, Transfection, Negative Control, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

doi: 10.1016/j.ymthe.2023.11.021

Figure Lengend Snippet: Figure 2. Analysis of melanoma cell line growth following treatment with OMe-miR126, BRAF, and PI3K inhibitors (A) Cell growth inhibition mediated by OMe-miR126 sequence compared to OMe-CTRL in combination with vemurafenib, dabrafenib, PIK-75, vemurafenib + PIK-75, and dabrafenib + PIK-75 in A375M wild-type (WT) cell line (upper panel). Evaluation of excess over bliss values (EOB) for each condition in A375M WT (bottom panel). (B) Cell growth inhibition with the same sequences with dabrafenib, PIK-75, and dabrafenib + PIK-75. EOB in SKMEL28 WT. (C) Cell growth inhibition mediated by OMe-miR126 sequence and OMe-CTRL in combination with PIK-75 in A375M-DR cell line and relative EOB values. (D) Cell growth inhibition in SKMEL28-DR cell line. EOB indicates synergism >0, independent = 0, antagonism <0. Means ± SD of almost three experiments are reported.

Article Snippet: Vemurafenib-resistant A375M (A375M-VR), dabrafenib-resistant A375M (A375M-DR), and dabrafenib-resistant SKMEL28 (SKMEL28-DR) cell lines were generated by culturing parental cells in increasing concentrations of vemurafenib or dabrafenib (from 0.5 to 10 mM; Selleckchem, Munich, Germany) for at least 2 months and subsequently maintained in full growth medium containing 5 mM vemurafenib for A375M-VR or 1.5 mM dabrafenib for A375M-DR and SKMEL28-DR. All melanoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Inhibition, Sequencing

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

doi: 10.1016/j.ymthe.2023.11.021

Figure Lengend Snippet: Figure 5. Evaluation of tumor suppressor role played by NPs contained OMe-miR126 in in vivo experiments (A) Schematic animal model. A375M-DR stably expressing firefly luciferase and GFP (A375M-DR-LUC-GFP) were injected into the spleen of severely immunodeficient mice. The splenectomy was performed after 30 min. About 2 weeks after inoculation, when the signal was barely detectable, the mice were randomly allocated to six treatment arms. Tumor engraftment was monitored weekly by in vivo bioluminescent imaging. The DAB, PIK-75, and NPs treatments have been administered as indicated. (B)

Article Snippet: Vemurafenib-resistant A375M (A375M-VR), dabrafenib-resistant A375M (A375M-DR), and dabrafenib-resistant SKMEL28 (SKMEL28-DR) cell lines were generated by culturing parental cells in increasing concentrations of vemurafenib or dabrafenib (from 0.5 to 10 mM; Selleckchem, Munich, Germany) for at least 2 months and subsequently maintained in full growth medium containing 5 mM vemurafenib for A375M-VR or 1.5 mM dabrafenib for A375M-DR and SKMEL28-DR. All melanoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: In Vivo, Animal Model, Stable Transfection, Expressing, Luciferase, Injection, Imaging

Journal: International journal of molecular sciences

Article Title: Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance.

doi: 10.3390/ijms23179910

Figure Lengend Snippet: Figure 1. Shift in IC50 values following acquired resistance to vemurafenib in WM793B and A375M cell lines. WM793B (A) and A375M (B) parental and resistant cell lines were exposed to serial dilutions of vemurafenib (from 1 nM to 50 µM) for 72 h and cell viability was assessed by MTT assay. Vemurafenib-resistant melanoma cell lines WM793B-R1 and A375M-R1 showed 33-fold and 224-fold increased resistance to vemurafenib, respectively. Prolonged treatment of A375M cell line with vemurafenib (7 months, R2) resulted in peak viability of resistant cells at the concentration of vemurafenib at which they were grown (C). Each curve represents the mean ± SD from three to four independent experiments performed in quadruplets for each vemurafenib concentration.

Article Snippet: Primary WM793B (ATCC® CRL_2806TM) and metastatic A375M (ATCC® CRL_1619TM) human melanoma cell lines (both BRAF V600E) were used for the generation of vemurafenibresistant sublines.

Techniques: MTT Assay, Concentration Assay

Journal: International journal of molecular sciences

Article Title: Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance.

doi: 10.3390/ijms23179910

Figure Lengend Snippet: Figure 3. EMT marker expression in parental (CTRL) and resistant (R) WM793B and A375M cell lines. (A) The expression of EMT markers at the protein level was investigated by western blotting. β-actin was used as a loading control. (B) The gene expression levels of EMT markers were measured by q-PCR. The D’Agostino–Pearson test was used to confirm the normal distribution of continuous variables, while the parametric statistical test one-way ANOVA with the Tukey Kramer post hoc method was used to determine a statistically significant difference in the expression between sub- groups. Statistical analysis was performed using MedCalc (v. 18.11.3). Statistically significant results are marked by an asterisk (*). A p-value less than 0.05 is flagged with one star (*), a p-value less than 0.01 is flagged with two stars (**), and p-value less than 0.001 is flagged with three stars (***).

Article Snippet: Primary WM793B (ATCC® CRL_2806TM) and metastatic A375M (ATCC® CRL_1619TM) human melanoma cell lines (both BRAF V600E) were used for the generation of vemurafenibresistant sublines.

Techniques: Marker, Expressing, Western Blot, Control, Gene Expression

Journal: International journal of molecular sciences

Article Title: Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance.

doi: 10.3390/ijms23179910

Figure Lengend Snippet: Figure 4. Migration of parental and resistant WM793B and A375M melanoma cells. For the wound healing assay (A), parental cells (CTRL) and vemurafenib-resistant sublines (R1 and R2) were imaged at the time of causing the wound and 8 and 24 h after. The results are shown as a filled area at a given time point. The mean and standard deviation of 12 measurements for each sample and time point are shown. For the culture insert assay (B), parental cells (CTRL) and vemurafenib-resistant sublines (R1 and R2) were imaged one hour after seeding in the upper chamber. The results are presented as the number of cells passing the membrane from the upper to the lower chamber. The mean and standard deviation of six measurements per sample are shown. The Mann–Whitney test (GraphPad Prism v. 7.04) was used for statistical analysis. Statistically significant results are marked by an asterisk (*). A p-value less than 0.05 is flagged with one star (*), a p-value less than 0.01 is flagged with two stars (**), a p-value less than 0.001 is flagged with three stars (***), and if a p-value is less than 0.0001, it is flagged with four stars (****). Micrograph of the wound healing assay of A375M melanoma cells after 16 h filmed with video microscopy (C). The parental A375M cells (CTRL) close the wound by single-cell migration, whereas the vemurafenib-resistant cells (R1 and R2) exhibit collective migration.

Article Snippet: Primary WM793B (ATCC® CRL_2806TM) and metastatic A375M (ATCC® CRL_1619TM) human melanoma cell lines (both BRAF V600E) were used for the generation of vemurafenibresistant sublines.

Techniques: Migration, Wound Healing Assay, Standard Deviation, Membrane, MANN-WHITNEY, Microscopy

Journal: International journal of molecular sciences

Article Title: Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance.

doi: 10.3390/ijms23179910

Figure Lengend Snippet: Figure 6. Differential sensitivity of parental and resistant WM793B and A375M cell lines to cisplatin and etoposide. WM793B and A375M parental and resistant cells were exposed to serial dilutions of cisplatin and etoposide for 72 h, and the cell viability was assessed by MTT assay. Vemurafenib- resistant WM793B cells increased tolerance to both cisplatin (A) and etoposide (C). On the other hand, vemurafenib-resistant A375M cells showed similar resistance to cisplatin (B) and etoposide (D) as parental cells. Each dose–response curve represents the mean ± SD from three to five independent experiments.

Article Snippet: Primary WM793B (ATCC® CRL_2806TM) and metastatic A375M (ATCC® CRL_1619TM) human melanoma cell lines (both BRAF V600E) were used for the generation of vemurafenibresistant sublines.

Techniques: MTT Assay

Journal: International journal of molecular sciences

Article Title: Characterization of Vemurafenib-Resistant Melanoma Cell Lines Reveals Novel Hallmarks of Targeted Therapy Resistance.

doi: 10.3390/ijms23179910

Figure Lengend Snippet: Figure 7. The cell-cycle analysis in the vemurafenib-resistant cell lines WM793B and A375M. The cell cycle phase distribution of parental (CTRL) and corresponding vemurafenib-resistant cells (R1 and R2) was analyzed by flow cytometry (A,B). The experiment was performed with untreated cells (NT) and after treatment with etoposide for 24 h. Statistical analysis was performed using MedCalc (v. 18.11.3). The normal distribution of continuous variables was confirmed using the D’Agostino–Pearson test, and the parametric statistical test one-way ANOVA with the Tukey–Kramer post hoc method was used. Asterisks indicate the statistical significance of each cell cycle phase in the resistant cell lines compared to the parental line. A p-value less than 0.01 is flagged with two stars (**), and a p-value less than 0.001 is flagged with three stars (***). Protein expression of cell cycle regulators (C) and PCNA proliferation marker (D) in parental (CTRL) and resistant (R1 and R2) cells. β-actin was used as a loading control. The proliferation rate of parental (CTRL) and resistant (R1 and R2) WM793B cells (E). The mean values of three biological replicates with standard deviations are shown.

Article Snippet: Primary WM793B (ATCC® CRL_2806TM) and metastatic A375M (ATCC® CRL_1619TM) human melanoma cell lines (both BRAF V600E) were used for the generation of vemurafenibresistant sublines.

Techniques: Cell Cycle Assay, Cytometry, Expressing, Marker, Control

Journal: Molecular Cancer

Article Title: Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)-targeted delivery of soluble TRAIL potently inhibits melanoma outgrowth in vitro and in vivo

doi: 10.1186/1476-4598-9-301

Figure Lengend Snippet: MCSP-restricted induction of apoptosis by anti-MCSP:TRAIL . A M14 and M14.MCSP cells were treated with increasing concentrations of anti-MCSP:TRAIL for 16 h and apoptosis was assessed by ∆ψ. B MCSP-negative cell line M14 and MCSP-positive cell lines M14.MCSP, SK-MEL-28, A2058 and A375M were treated with 500 ng/ml anti-MCSP:TRAIL for 16 h and apoptosis was assessed by ∆ψ. C A375M, A2058 and SK-MEL-28 cells were treated with equimolar concentrations of anti-MCSP:TRAIL, anti-EpCAM:TRAIL, anti-MCSP mAb or anti-EpCAM:TRAIL+anti-MCSP mAb for 16 h and apoptosis was assessed by ∆ψ. D A2058 cells were treated with 500 ng/mL anti-MCSP:TRAIL in the absence or presence of parental MCSP-blocking mAb 9.2.27, TRAIL-neutralizing mAb 2E5 or pan-caspase inhibitor zVAD-fmk for 16 h and apoptosis was assessed by ∆ψ. E A375M and A375M.FADD-DED cells were treated with anti-MCSP:TRAIL or rhTRAIL for 16 h and apoptosis was assessed by ∆ψ. F A375M cells were incubated with anti-TRAIL-R1 (thin line) or anti-TRAIL-R2 (thick line) mAb and expression of TRAIL-R1 and TRAIL-R2 was analyzed by flow cytometry. Shaded area indicates the fluorescence signal when cells were incubated with fluorescent-labeled secondary antibody alone.

Article Snippet: MCSP-positive/EpCAM-negative melanoma cell lines A375M, A2058 and SK-MEL-28 were obtained from and characterized (STR profiling, karyotyping, isoenzyme analysis) by the American Tissue Culture Collection (ATCC).

Techniques: Blocking Assay, Incubation, Expressing, Flow Cytometry, Fluorescence, Labeling

Journal: Molecular Cancer

Article Title: Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)-targeted delivery of soluble TRAIL potently inhibits melanoma outgrowth in vitro and in vivo

doi: 10.1186/1476-4598-9-301

Figure Lengend Snippet: Inhibition of anchorage-independent growth by anti-MCSP:TRAIL . A A375M and A2058 cells were treated with increasing concentrations of anti-MCSP:TRAIL for 21 days and colony formation was assessed. B A375M cells were treated with increasing concentrations of anti-MCSP:TRAIL, anti-EpCAM:TRAIL, anti-MCSP mAb or anti-EpCAM:TRAIL+anti-MCSP mAb for 21 days and colony formation was assessed. C A375M cells were treated with rhTRAIL, anti-EpCAM:TRAIL or anti-MCSP:TRAIL for 21 days and colony formation was assessed. D A375M cells were treated with 100 ng/ml anti-MCSP:TRAIL in the absence or presence of TRAIL-neutralizing mAb 2E5, parental anti-MCSP mAb 9.2.27 or pan-caspase inhibitor zVAD-fmk for 21 days and colony formation was assessed. E A375M and A375M.FADD-DED cells were treated with increasing concentrations of anti-MCSP:TRAIL for 21 days and colony formation was assessed. F A375M.FADD-DED cells were treated with increasing concentrations of anti-MCSP:TRAIL, anti-EpCAM:TRAIL, anti-MCSP mAb or anti-EpCAM:TRAIL+anti-MCSP mAb for 21 days and colony formation was assessed.

Article Snippet: MCSP-positive/EpCAM-negative melanoma cell lines A375M, A2058 and SK-MEL-28 were obtained from and characterized (STR profiling, karyotyping, isoenzyme analysis) by the American Tissue Culture Collection (ATCC).

Techniques: Inhibition

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

doi: 10.1016/j.ymthe.2023.11.021

Figure Lengend Snippet: Figure 1. Chemical changes within the OMe-miR126 sequence: a comparative analysis of its function and stability in plasma (A) OMe-miR126 consists of a double-stranded ribonucleotide and the anti-sense strand contains mature miR-126-3p sequence. All the pyrimidine residues of the sense strand and one of the uridine residues in the anti-sense strand are modified by O-methylation at the 20-O pentose (20-OMe). The sense and anti-sense strands have been modified with the addition of two 20-deoxythymidine residues at the 30 terminal of each strand. (B) Analysis of miR126 expression obtained by qRT-PCR performed 24 h after transfection of A375M-VR cell line with Pre-miR126 (Pre), PTO-miR126 (PTO), OMe-miR126 (OMe), and relative negative control. U6 snRNA was used as normalizer. The histogram columns represent the ratio ± SD of the values obtained from transfection of miR126 sequences and their respective controls. Commercial miR126-3p was used as positive reference. (C) WES analysis of p85-b protein in A375M-VR cell line after transfection with Pre-miR126, PTO-miR126, OMe-miR126 sequences or their relative negative controls. MiR126-3p and -5p were used as positive and negative references, respectively. b-Actin was utilized as internal loading control. (D) Analysis of miR126 expression in A375M-VR by qRT-PCR, performed 24, 48, and 72 h after OMe-miR126 internalization. Relative miR126 expression level was normalized on U6 snRNA. The

Article Snippet: Vemurafenib-resistant A375M (A375M-VR), dabrafenib-resistant A375M (A375M-DR), and dabrafenib-resistant SKMEL28 (SKMEL28-DR) cell lines were generated by culturing parental cells in increasing concentrations of vemurafenib or dabrafenib (from 0.5 to 10 mM; Selleckchem, Munich, Germany) for at least 2 months and subsequently maintained in full growth medium containing 5 mM vemurafenib for A375M-VR or 1.5 mM dabrafenib for A375M-DR and SKMEL28-DR. All melanoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Sequencing, Clinical Proteomics, Methylation, Expressing, Quantitative RT-PCR, Transfection, Negative Control, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

doi: 10.1016/j.ymthe.2023.11.021

Figure Lengend Snippet: Figure 2. Analysis of melanoma cell line growth following treatment with OMe-miR126, BRAF, and PI3K inhibitors (A) Cell growth inhibition mediated by OMe-miR126 sequence compared to OMe-CTRL in combination with vemurafenib, dabrafenib, PIK-75, vemurafenib + PIK-75, and dabrafenib + PIK-75 in A375M wild-type (WT) cell line (upper panel). Evaluation of excess over bliss values (EOB) for each condition in A375M WT (bottom panel). (B) Cell growth inhibition with the same sequences with dabrafenib, PIK-75, and dabrafenib + PIK-75. EOB in SKMEL28 WT. (C) Cell growth inhibition mediated by OMe-miR126 sequence and OMe-CTRL in combination with PIK-75 in A375M-DR cell line and relative EOB values. (D) Cell growth inhibition in SKMEL28-DR cell line. EOB indicates synergism >0, independent = 0, antagonism <0. Means ± SD of almost three experiments are reported.

Article Snippet: Vemurafenib-resistant A375M (A375M-VR), dabrafenib-resistant A375M (A375M-DR), and dabrafenib-resistant SKMEL28 (SKMEL28-DR) cell lines were generated by culturing parental cells in increasing concentrations of vemurafenib or dabrafenib (from 0.5 to 10 mM; Selleckchem, Munich, Germany) for at least 2 months and subsequently maintained in full growth medium containing 5 mM vemurafenib for A375M-VR or 1.5 mM dabrafenib for A375M-DR and SKMEL28-DR. All melanoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: Inhibition, Sequencing

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

doi: 10.1016/j.ymthe.2023.11.021

Figure Lengend Snippet: Figure 5. Evaluation of tumor suppressor role played by NPs contained OMe-miR126 in in vivo experiments (A) Schematic animal model. A375M-DR stably expressing firefly luciferase and GFP (A375M-DR-LUC-GFP) were injected into the spleen of severely immunodeficient mice. The splenectomy was performed after 30 min. About 2 weeks after inoculation, when the signal was barely detectable, the mice were randomly allocated to six treatment arms. Tumor engraftment was monitored weekly by in vivo bioluminescent imaging. The DAB, PIK-75, and NPs treatments have been administered as indicated. (B)

Article Snippet: Vemurafenib-resistant A375M (A375M-VR), dabrafenib-resistant A375M (A375M-DR), and dabrafenib-resistant SKMEL28 (SKMEL28-DR) cell lines were generated by culturing parental cells in increasing concentrations of vemurafenib or dabrafenib (from 0.5 to 10 mM; Selleckchem, Munich, Germany) for at least 2 months and subsequently maintained in full growth medium containing 5 mM vemurafenib for A375M-VR or 1.5 mM dabrafenib for A375M-DR and SKMEL28-DR. All melanoma cell lines were cultured in Dulbecco’s modified Eagle’s medium (Euroclone, Milan, Italy) supplemented with 10% fetal bovine serum (FBS) (Euroclone).

Techniques: In Vivo, Animal Model, Stable Transfection, Expressing, Luciferase, Injection, Imaging

Journal:

Article Title: Retrovirus Targeting by Tropism Restriction to Melanoma Cells

doi:

Figure Lengend Snippet: Percentage of HMW-MAA-positive or -negative cells infected. A375m and B-1 cells were incubated with virus with no envelope (Non) or an scLPA, scLPMA, or unmodified MLV-A 4070A (A) envelope concentrated by centrifugation (see Materials and Methods) without any treatment (N) or after mixing with PB or Lipofectamine (Lip) (see Materials and Methods).

Article Snippet: BOWES (ATCC CRL-9607) and A375m (ATCC CRL-1619) are human melanoma cell lines.

Techniques: Infection, Incubation, Virus, Centrifugation

Journal:

Article Title: Retrovirus Targeting by Tropism Restriction to Melanoma Cells

doi:

Figure Lengend Snippet: Infection by targeted virosomes (A) The graph at the top shows titers of scLPMA-enveloped virus (best clone virus; see Materials and Methods) and unmodified MLV-A 4070A on HMW-MAA-negative (B-1 and Ecv304) cells and HMW-MAA-positive melanoma (A375m and BOWES) cells. Infections were performed after complexing of unconcentrated viruses with DOSPA-DOPE liposomes to produce virosomes. Titers are expressed as the mean infectious units (iu) per milliliter (± the standard error) of triplicate determinations. The lower graph shows cleavage of the PLGLWA peptide by B-1, Ecv304, BOWES, and A375m cells (see Materials and Methods). (B) Inhibition of scLPMA virosome infection of BOWES cells by competition for HMW-MAA or inhibition of MMP activity. scLPMA-enveloped virus and MLV-A 4070A were used to infect BOWES cells with LMH2 antibody, TIMP-2, or a combination of both as described in Materials and Methods. Data are expressed as mean percentages of the titer of untreated viruses (triplicate determinations ± the standard error).

Article Snippet: BOWES (ATCC CRL-9607) and A375m (ATCC CRL-1619) are human melanoma cell lines.

Techniques: Infection, Virus, Liposomes, Inhibition, Activity Assay

Journal:

Article Title: Retrovirus Targeting by Tropism Restriction to Melanoma Cells

doi:

Figure Lengend Snippet: Infection of mixed target populations. (A) Mixed populations were infected with unconcentrated MLV-A 4070A or scLPMA-enveloped virus complexed with DOSPA-DOPE liposomes. Two days after infection, cells were stained for β-galactosidase (red) and HMW-MAA (green) expression as described in Materials and Methods. (B) Percentage of HMW-MAA-positive and -negative cells infected in cocultures using unmodified MLV-A 4070A or scLPMA-enveloped virus. Final cell ratios at confluency were 40:1 Ecv304 to A375m cells and 60:1 B-1 to A375m cells. Data are means (± the standard errors) from two separate experiments in which 20 randomized fields (∼3,000 cells) were counted for each infection. To determine the infection of B-1 or Ecv304 cells by scLPMA-enveloped virus, 200 fields (∼30,000 cells) were counted.

Article Snippet: BOWES (ATCC CRL-9607) and A375m (ATCC CRL-1619) are human melanoma cell lines.

Techniques: Infection, Virus, Liposomes, Staining, Expressing

Journal: International journal of molecular sciences

Article Title: BRAF and MEK Inhibitors Influence the Function of Reprogrammed T Cells: Consequences for Adoptive T-Cell Therapy.

doi: 10.3390/ijms19010289

Figure Lengend Snippet: Figure 1. CSPG4-specific CAR and CSPG4 expression on effector T cells and target cells, respectively. (a,b) Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors through density gradient centrifugation and CD8+ T cells were isolated with magnetic activated cell sorting (MACS) beads. These cells were either mock electroporated (EP) or were transfected with RNA encoding the CSPG4-specific CAR. CAR expression was flow-cytometrically determined 4 h after electroporation by staining with a PE-labeled goat-F(ab′)2 anti-human IgG antibody. (a) Percentage of CAR-positive cells (average of 8 independent experiments + SEM; original data see Table S1) and (b) Histograms of one typical experiment (grey histograms: unstained cells, and blue histograms: goat-F(ab′)2 anti-human IgG-PE stained cells) are shown. (c) The CSPG4 antigen expression on the target cell lines T2 and A375M was determined by flow cytometry after primary staining with a CSPG4-specific antibody and secondary staining PE-labeled with goat-anti-mouse. Grey histograms: unstained cells; black histograms: secondary only staining; red histograms: primary anti-CSPG4 staining + secondary staining.

Article Snippet: The CSPG4+ A375M melanoma cell line (ATCC® CRL-3223TM) was described previously by Kozlowski et al. [46].

Techniques: Expressing, Isolation, Gradient Centrifugation, FACS, Transfection, Electroporation, Staining, Labeling, Cytometry

Journal: International journal of molecular sciences

Article Title: BRAF and MEK Inhibitors Influence the Function of Reprogrammed T Cells: Consequences for Adoptive T-Cell Therapy.

doi: 10.3390/ijms19010289

Figure Lengend Snippet: Figure 2. BRAF and MEK inhibitor treatment affects CAR-T-cell activation after antigen-specific stimulation. CAR-T cells were generated as described in Figure 1. Four hours after electroporation, these cells were co-incubated overnight with CSPG4-negative T2 cells and the CSPG4+ melanoma cell line A375M at a 1:1 ratio. Mock-transfected T cells were used as control. Co-incubations were performed in the absence of inhibitors (no inhibitor), in the presence of DMSO only (solvent control), or in the presence of the different kinase inhibitors, either alone or in combination. The used kinase inhibitors vemurafenib (Vem, V), dabrafenib (Dabra, D), cobimetinib (Cobi, C), and trametinib (Tram, T) were used in final concentrations listed in Table 1. Mock-transfected T cells (mock T) stimulated with T2 or A375M, and mock-transfected T cells and CAR-T cells incubated without target cells served as negative controls. After 16 h of co-incubation, the cells were harvested and stained for the activation markers CD25 (a) and CD69 (b) and measured by flow cytometry. The specific mean fluorescence intensity (MFI) of the respective activation markers on cells in the T-cell gate is depicted. The MFI was calculated by subtracting the value of the respective isotype control. Data are presented as mean + SEM derived from four independent experiments (original data see Table S2; for statistical analyses see Tables S3 and S4). Flow-cytometric data of a representative donor is shown in Supplemental Figure S1.

Article Snippet: The CSPG4+ A375M melanoma cell line (ATCC® CRL-3223TM) was described previously by Kozlowski et al. [46].

Techniques: Activation Assay, Generated, Electroporation, Incubation, Transfection, Control, Solvent, Staining, Cytometry, Derivative Assay

Journal: International journal of molecular sciences

Article Title: BRAF and MEK Inhibitors Influence the Function of Reprogrammed T Cells: Consequences for Adoptive T-Cell Therapy.

doi: 10.3390/ijms19010289

Figure Lengend Snippet: Figure 3. BRAF and MEK inhibitor treatment affects CAR-T-cell cytokine secretion after antigen-specific stimulation. CAR-T cells were generated as described in Figure 1. Four hours after electroporation, these cells were co-incubated overnight with CSPG4-negative T2 cells and the CSPG4+ melanoma cell line A375M at a 1:1 ratio. Mock-transfected T cells (mock T) were used as control. Co-incubations were performed in the absence of inhibitors (no inhibitor), in the presence of DMSO only (solvent control), or in the presence of the different kinase inhibitors, either alone or in combination. The used kinase inhibitors vemurafenib (Vem, V), dabrafenib (Dabra, D), cobimetinib (Cobi, C), and trametinib (Tram, T) were added in final concentrations listed in Table 1. T2 and A375M cells without T cells and mock-transfected T cells stimulated with T2 or A375M served as negative controls. Concentrations of interleukin (IL)-2 (a), tumor necrosis factor (TNF) (b), and interferon gamma (IFNγ) (c) were determined after overnight co-incubation with a Cytometric Bead Array (CBA), and are depicted in [ng/mL]. Data are presented as mean + SEM of four independent experiments (original data, see Table S5; for statistical analyses see Tables S6–S8).

Article Snippet: The CSPG4+ A375M melanoma cell line (ATCC® CRL-3223TM) was described previously by Kozlowski et al. [46].

Techniques: Generated, Electroporation, Incubation, Transfection, Control, Solvent

Journal: International journal of molecular sciences

Article Title: BRAF and MEK Inhibitors Influence the Function of Reprogrammed T Cells: Consequences for Adoptive T-Cell Therapy.

doi: 10.3390/ijms19010289

Figure Lengend Snippet: Figure 4. BRAF and MEK inhibitor treatment affects CAR-T-cell cytokine secretion after antigen-specific stimulation. CAR-T cells were generated as described in Figure 1. Four hours after electroporation, these cells were co-incubated overnight with CSPG4-negative T2 cells and the CSPG4+ melanoma cell line A375M at a 1:1 ratio. Mock-transfected T cells (mock T) were used as control. Co-incubations were performed in the absence of inhibitors (no inhibitor), in the presence of DMSO only (solvent control), or in the presence of the different kinase inhibitors, either alone or in combination. The used kinase inhibitors vemurafenib (Vem, V), dabrafenib (Dabra, D), cobimetinib (Cobi, C), and trametinib (Tram, T) were added in final concentrations listed in Table 1. T2 and A375M cells without T cells and mock-transfected T cells stimulated with T2 or A375M served as negative controls. Concentrations of IL-6 (a) and IL-10 (b) were determined after overnight co-incubation with a CBA, and are depicted in [ng/mL]. Data are presented as mean + SEM of four independent experiments (original data, see Table S9; for statistical analyses see Tables S10 and S11).

Article Snippet: The CSPG4+ A375M melanoma cell line (ATCC® CRL-3223TM) was described previously by Kozlowski et al. [46].

Techniques: Generated, Electroporation, Incubation, Transfection, Control, Solvent

Journal: International journal of molecular sciences

Article Title: BRAF and MEK Inhibitors Influence the Function of Reprogrammed T Cells: Consequences for Adoptive T-Cell Therapy.

doi: 10.3390/ijms19010289

Figure Lengend Snippet: Figure 5. BRAF and MEK inhibitor treatment affects CAR-T-cell lytic capacity after antigen-specific stimulation. T cells were generated as described in Figure 1, and electroporated without RNA (mock T; (a,c)) or with CAR-RNA (CAR T; (b,d)). Twenty-four hours after electroporation, the cytolytic capacity of these cells toward the CSPG4-negative T2 cells (a,d) and the CSPG4+ melanoma cell line A375M (b,d) was examined at indicated effector to target ratios in a standard 4 to 6 h chromium release assay. Co-incubations were performed in the absence of inhibitors (no inhibitor), in the presence of DMSO only (solvent control), or in the presence of the different kinase inhibitors, either alone or in combination. The used kinase inhibitors vemurafenib (Vem, V), dabrafenib (Dabra, D), cobimetinib (Cobi, C), and trametinib (Tram, T) were supplemented in final concentrations listed in Table 1. The release of chromium into the supernatant was determined and lysis was calculated as described in materials and methods. Data are presented as mean + SEM derived from three independent experiments, each performed in technical triplicates (for original data see Table S12; for statistical analyses see Table S13).

Article Snippet: The CSPG4+ A375M melanoma cell line (ATCC® CRL-3223TM) was described previously by Kozlowski et al. [46].

Techniques: Generated, Electroporation, Release Assay, Solvent, Control, Lysis, Derivative Assay

Journal: Cancers

Article Title: In Vitro Setup for Determination of Nanoparticle-Mediated Magnetic Cell and Drug Accumulation in Tumor Spheroids under Flow Conditions

doi: 10.3390/cancers14235978

Figure Lengend Snippet: Accumulation of MTO and induction of cell death in spheroids. Spheroids were cultured from A375M cells and fibroblasts in agarose-coated wells for 72 h, then mitoxantrone was added. ( A ) Fluorescence microscopy of cryosections of the spheroids on day 7. ( B ) Single-cell suspensions of the spheroids were analyzed in flow cytometry for intracellular MTO fluorescence on day 7. ( C ) Transmission microscopy and determination of spheroid size by Image J software. ( D ) Transmission microscopy after 96 h of incubation with MTO and ( E ) size determination. ( F ) AxA5-PI staining of single cell-suspensions on day 7. The experiment was performed four times with n > 18 ( C – E ), or three times in triplicates ( B , F ). The mean values with standard deviations are shown. Significances (treatment versus control) were calculated using one-way ANOVA with * p ≤ 0.01 and ** p ≤ 0.05.

Article Snippet: A375M melanoma cells (ATCC, Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal calf serum (FCS, Gibco, Life Technologies Corporation, New York, NY, USA), 1% penicillin/streptomycin, 1% L-Glutamine, 0.04% Beta-Mercapto-Ethanol (all from Thermo Fisher Scientific, Waltham, MA, USA) and 0.2% Hepes Puffer (Biowest, Nuaillé, France).

Techniques: Cell Culture, Fluorescence, Microscopy, Flow Cytometry, Transmission Assay, Software, Incubation, Staining, Control

Journal: Cancers

Article Title: In Vitro Setup for Determination of Nanoparticle-Mediated Magnetic Cell and Drug Accumulation in Tumor Spheroids under Flow Conditions

doi: 10.3390/cancers14235978

Figure Lengend Snippet: Impact of free and nanoparticle-bound MTO on the growth of spheroids. Spheroids were cultured from A375M and fibroblasts. After 72 h, free MTO, SPION MTO or unloaded SPIONs were added. ( A ) Transmission microscopy of spheroids on day 10. ( B ) The growth of the spheroids was observed with IncuCyte, and analyzed with ImageJ over 10 days. ( C ) Comparison of spheroid sizes on day 10. The experiment was performed in quadruplicates. The mean values with standard deviations are shown. Significances (treatment versus control) were calculated using one-way ANOVA, with * p ≤ 0.05, ** p ≤ 0.01 ( B ).

Article Snippet: A375M melanoma cells (ATCC, Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% fetal calf serum (FCS, Gibco, Life Technologies Corporation, New York, NY, USA), 1% penicillin/streptomycin, 1% L-Glutamine, 0.04% Beta-Mercapto-Ethanol (all from Thermo Fisher Scientific, Waltham, MA, USA) and 0.2% Hepes Puffer (Biowest, Nuaillé, France).

Techniques: Cell Culture, Transmission Assay, Microscopy, Comparison, Control