Journal: Molecular Cell

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

doi: 10.1016/j.molcel.2020.05.025

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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: 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:

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: Oncology reports

Article Title: EV20‑sss‑vc/MMAF, an HER‑3 targeting antibody‑drug conjugate displays antitumor activity in liver cancer.

doi: 10.3892/or.2020.7893

Figure Lengend Snippet: Figure 2. In vitro antitumor activity of EV20‑based ADCs. (A) The cytotoxic response of LC cells to EV20‑based ADCs treatment was evaluated by MTT after 120 h of treatment with increasing doses ranging between 0.006 nM and 100 nM. The IC50 values were calculated with GraphPad Prism 5.0 software and reported. (B) A375m and HepG2 cells were maintained for 30 min on ice in the presence of 10 µg/ml EV20 and placed again in the incubator at 37˚C for 1 h. The internalization rate of the antibody was evaluated by flow cytometry and by confocal microscopy imaging. The histogram represents the percentage of MFI referred to control (cells maintained on ice). Plotted results are an average ± SD of three independent experiments. For confocal microscopy imaging, EV20 and nuclei were visualized on green and blue channels, respectively. (C) A375m and HepG2 cells were incubated for 72 h with eight increasing concentrations of free MMAF, diluted from 10 µM to 10 pM, in 1:10 dilution increments. Proliferation was evaluated by MTT assay (left panel). HepG2 cells were incubated for 72 h with the same increasing doses of free MMAF used in (C) in absence or presence of MK571 (25 µM), PSC833 (3 µM) and Reversan (15 µM) and proliferation was evaluated by MTT assay (right panel). (D) EV20‑sss‑vc/MMAF characterization. HIC was used for DAR calculation; melanoma A375m HER3+ cells were used for cell binding by flow cytometry. ELISA was performed for in vitro binding with naked EV20‑sss mAb used as control. (E) PLC/PRF/5 LC cells were incubated for 2 h or not with naked or conjugated EV20 mAb, at a dose of 10 µg/ml, before NRG‑1β stimulation (10 min, 10 ng/ml). Total and phosphorylated HER‑3 receptor was analysed by western blotting and bands were quantified using actin as loading control. Histograms represent densitometric analysis of a single experiment, expressed as arbitrary units. MFI, mean fluorescence intensity; ADCs, antibody‑drug conjugates; LC, liver cancer; MTT, 3‑(4,5‑dimethyldiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide; MMAF, monomethyl auristatin F; vc, valine‑citrulline; HIC, hydrophobic chromatography; DAR, drug antibody ratio.

Article Snippet: A375m human melanoma cell line (CRL3223) and SJSA‐1 human osteosarcoma cell line (CRL2098) were purchased from ATCC.

Techniques: In Vitro, Activity Assay, Software, Flow Cytometry, Confocal Microscopy, Imaging, Control, Incubation, MTT Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Fluorescence, Chromatography

Journal: Oncology reports

Article Title: EV20‑sss‑vc/MMAF, an HER‑3 targeting antibody‑drug conjugate displays antitumor activity in liver cancer.

doi: 10.3892/or.2020.7893

Figure Lengend Snippet: Figure 3. EV20‑sss‑vc/MMAF cell killing activity is target‑dependent and superior to T‑DM1. (A) A375m cells were incubated for 120 h with increasing doses of EV20‑sss‑vc/MMAF or EV20/MMAF ranging between 0.006 nM and 100 nM, and proliferation was evaluated by MTT assay. (B) Cytotoxic response of HER‑3 negative SJSA‑1 cells to EV20‑sss‑vc/MMAF treatment (with doses ranging between 0.006 and 100 nM) was evaluated by MTT after 120 h of drug exposure. HER‑3 expression by flow cytometric analysis is presented as inset. (C) A375m cells were incubated for 120 h with increasing doses of EV20‑sss‑vc/MMAF (ranging between 0.006 and 100 nM) alone or with 500‑fold molar excess of naked EV20 and proliferation was evaluated by MTT assay. (D) LC cells were exposed for 120 h to increasing doses of T‑DM1 or EV20‑sss‑vc/MMAF (ranging between 0.006 and 100 nM) and proliferation was evaluated by MTT assay as aforementioned. vc, valine‑citrulline; MMAF, monomethyl auristatin F; MTT, 3‑(4,5‑dimethyldiazol‑2‑yl)‑2,5‑diphenyl tetrazolium bromide; LC, liver cancer.

Article Snippet: A375m human melanoma cell line (CRL3223) and SJSA‐1 human osteosarcoma cell line (CRL2098) were purchased from ATCC.

Techniques: Activity Assay, Incubation, MTT Assay, Expressing