a375 human metastatic melanoma cells  (ATCC)

Journal: Scientific Reports

Article Title: First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies

doi: 10.1038/s41598-024-65137-7

Figure Lengend Snippet: Image cytometry analysis strategy of irradiated A375:NHEM co-cultures. Upon labelling with antibodies against p-γ-H2AX (red) and with Hoechst for nuclear DNA (blue), samples were automatically scanned using the TissueFAXSiPlus imaging system. After regions of interest (ROIs) reconstitution out of stitched individual fields of view (FOV), the obtained images were imported into the TissueQuest image quantification software. Each cell was identified based on nuclear DAPI signal by the segmentation algorithm. Next, scattergrams were generated based on DAPI-Eccentricity versus DAPI-Mean Intensity profiles and cell lines differentiated based on nuclear elongation (eccentricity): elongated nuclei of NHEM were gated (red gate) based on higher eccentricity values, while non-elongated A375 nuclei were gated (blue gate) based on low eccentricity values. Using forward gating, one can identify the scattergram displayed signals for each cell in a population. Here, we exemplify a NHEM nucleus encircled in red and an A375 nucleus encircled in green and their corresponding signals in the scattergrams (high vs. low elongation). In order to quantify p-γ-H2AX mean fluorescence intensity (MFI) and number of foci per nucleus, TxRed signals from the nuclear mask were quantified using 2 parameters: Texa-Mean Intensity and Texa_DOTS_ON_DAPI-Dots Count, respectively, visible on the axes. Histograms displaying frequency distribution of number of foci per nucleus are displayed in blue for each cell population. Also, scattergrams of number of foci versus p-γ-H2AX expression level can be generated. Here, red bars and dots identify the selected nuclei in the picture. Foci are indicated by white arrows (in NHEM) or arrowheads (in A375).

Article Snippet: Radiation-resistant A375 human metastatic melanoma cells and normal human epidermal melanocytes (NHEMs) were obtained from ATCC (#CRL-1619) and Lonza (#CC-2504), respectively.

Techniques: Cytometry, Irradiation, Imaging, Software, Generated, Fluorescence, Expressing

Journal: Scientific Reports

Article Title: First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies

doi: 10.1038/s41598-024-65137-7

Figure Lengend Snippet: Analysis of the A375 melanoma and NHEM co-cultures upon laser-plasma accelerated electron exposure during 2 sequentially performed irradiation experiments (PW1, PW2) versus pulsed X-ray irradiation. ( a , c ) Correspondence between the irradiated GF fingerprint and selected areas of the SlideFlasks for image cytometry analysis (5 × magnification preview mode). ( b , d , e , f ) DNA damage response revealed as p-γ-H2AX foci at 30 min post-exposure to PW or X-ray-generated radiation versus non-exposed (untreated) or mock-exposed (PW env) negative controls. Immunofluorescence microscopy images of overlapping DAPI and TxRed signals (top), as well as grayscale images of the TxRed signal (bottom) of a representative field of view (FOV) under each irradiation condition, are shown. The p-γ-H2AX foci are visible in the detailed insets ( b , d , f —bottom right). Scale bar = 20 μm ( b , d , f ) or 100 μm ( e ).

Article Snippet: Radiation-resistant A375 human metastatic melanoma cells and normal human epidermal melanocytes (NHEMs) were obtained from ATCC (#CRL-1619) and Lonza (#CC-2504), respectively.

Techniques: Irradiation, Cytometry, Generated, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies

doi: 10.1038/s41598-024-65137-7

Figure Lengend Snippet: Comparison of “number of p-γ-H2AX foci per nucleus” values for each tested irradiation condition. The bar graphs show the comparisons between treatments for the overall A375 and NHEM co-cultures ( a ), for the A375 cells in co-cultures ( b ), and for the NHEM cells in co-cultures ( c ). Sampled areas of PW-irradiated specimens and the standard pulsed X-ray-irradiated cells were compared to non-irradiated controls. Medians of each data set and 95% confidence intervals are displayed. The statistical differences were determined using the nonparametric Mann–Whitney test, (n = 4473–10,629 analysed nuclei, * p < 0.05, *** p < 0.001, **** p < 0.0001, ns = not significant).

Article Snippet: Radiation-resistant A375 human metastatic melanoma cells and normal human epidermal melanocytes (NHEMs) were obtained from ATCC (#CRL-1619) and Lonza (#CC-2504), respectively.

Techniques: Comparison, Irradiation, MANN-WHITNEY

Journal: Scientific Reports

Article Title: First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies

doi: 10.1038/s41598-024-65137-7

Figure Lengend Snippet: Image cytometry analysis strategy of irradiated A375:NHEM co-cultures. Upon labelling with antibodies against p-γ-H2AX (red) and with Hoechst for nuclear DNA (blue), samples were automatically scanned using the TissueFAXSiPlus imaging system. After regions of interest (ROIs) reconstitution out of stitched individual fields of view (FOV), the obtained images were imported into the TissueQuest image quantification software. Each cell was identified based on nuclear DAPI signal by the segmentation algorithm. Next, scattergrams were generated based on DAPI-Eccentricity versus DAPI-Mean Intensity profiles and cell lines differentiated based on nuclear elongation (eccentricity): elongated nuclei of NHEM were gated (red gate) based on higher eccentricity values, while non-elongated A375 nuclei were gated (blue gate) based on low eccentricity values. Using forward gating, one can identify the scattergram displayed signals for each cell in a population. Here, we exemplify a NHEM nucleus encircled in red and an A375 nucleus encircled in green and their corresponding signals in the scattergrams (high vs. low elongation). In order to quantify p-γ-H2AX mean fluorescence intensity (MFI) and number of foci per nucleus, TxRed signals from the nuclear mask were quantified using 2 parameters: Texa-Mean Intensity and Texa_DOTS_ON_DAPI-Dots Count, respectively, visible on the axes. Histograms displaying frequency distribution of number of foci per nucleus are displayed in blue for each cell population. Also, scattergrams of number of foci versus p-γ-H2AX expression level can be generated. Here, red bars and dots identify the selected nuclei in the picture. Foci are indicated by white arrows (in NHEM) or arrowheads (in A375).

Article Snippet: Radiation-resistant A375 human metastatic melanoma cells and normal human epidermal melanocytes (NHEMs) were obtained from ATCC (#CRL-1619) and Lonza (#CC-2504), respectively.

Techniques: Cytometry, Irradiation, Imaging, Software, Generated, Fluorescence, Expressing

Journal: Scientific Reports

Article Title: First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies

doi: 10.1038/s41598-024-65137-7

Figure Lengend Snippet: Analysis of the A375 melanoma and NHEM co-cultures upon laser-plasma accelerated electron exposure during 2 sequentially performed irradiation experiments (PW1, PW2) versus pulsed X-ray irradiation. ( a , c ) Correspondence between the irradiated GF fingerprint and selected areas of the SlideFlasks for image cytometry analysis (5 × magnification preview mode). ( b , d , e , f ) DNA damage response revealed as p-γ-H2AX foci at 30 min post-exposure to PW or X-ray-generated radiation versus non-exposed (untreated) or mock-exposed (PW env) negative controls. Immunofluorescence microscopy images of overlapping DAPI and TxRed signals (top), as well as grayscale images of the TxRed signal (bottom) of a representative field of view (FOV) under each irradiation condition, are shown. The p-γ-H2AX foci are visible in the detailed insets ( b , d , f —bottom right). Scale bar = 20 μm ( b , d , f ) or 100 μm ( e ).

Article Snippet: Radiation-resistant A375 human metastatic melanoma cells and normal human epidermal melanocytes (NHEMs) were obtained from ATCC (#CRL-1619) and Lonza (#CC-2504), respectively.

Techniques: Irradiation, Cytometry, Generated, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: First in vitro cell co-culture experiments using laser-induced high-energy electron FLASH irradiation for the development of anti-cancer therapeutic strategies

doi: 10.1038/s41598-024-65137-7

Figure Lengend Snippet: Comparison of “number of p-γ-H2AX foci per nucleus” values for each tested irradiation condition. The bar graphs show the comparisons between treatments for the overall A375 and NHEM co-cultures ( a ), for the A375 cells in co-cultures ( b ), and for the NHEM cells in co-cultures ( c ). Sampled areas of PW-irradiated specimens and the standard pulsed X-ray-irradiated cells were compared to non-irradiated controls. Medians of each data set and 95% confidence intervals are displayed. The statistical differences were determined using the nonparametric Mann–Whitney test, (n = 4473–10,629 analysed nuclei, * p < 0.05, *** p < 0.001, **** p < 0.0001, ns = not significant).

Article Snippet: Radiation-resistant A375 human metastatic melanoma cells and normal human epidermal melanocytes (NHEMs) were obtained from ATCC (#CRL-1619) and Lonza (#CC-2504), respectively.

Techniques: Comparison, Irradiation, MANN-WHITNEY

Journal: Pharmaceuticals

Article Title: Unveiling the Role of Tryptophan 2,3-Dioxygenase in the Angiogenic Process

doi: 10.3390/ph17050558

Figure Lengend Snippet: HUVEC proliferation in response to A375 or SK-Mel-28 conditioned media (C.M.). Melanoma cells were treated with 680C91 (40 µM) or epacadostat (1 µM) for 24 h, and then media were recovered, clarified and added to HUVECs. After 2 days, HUVEC proliferation was assessed. Mean ± SEM, n = 4. * p < 0.05, *** p < 0.001 vs. untreated C.M. § p < 0.05 vs. SK-Mel-28 C.M.

Article Snippet: Human metastatic melanoma cell line A375 (ATCC, Manassas, VA, USA) was grown in high D-glucose DMEM, with 10% ( v / v ) heat-inactivated fetal bovine serum (Euroclone Milan, Italy), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mmol/L glutamine in a humidified atmosphere with 5% CO 2 in air.

Techniques:

Journal: Pharmaceuticals

Article Title: Unveiling the Role of Tryptophan 2,3-Dioxygenase in the Angiogenic Process

doi: 10.3390/ph17050558

Figure Lengend Snippet: ( A , B ) RT-PCR for TDO2 expression and C) Kyn release in A375 HUVECs and co-cultures. ( A ) RT-PCR in A375 alone and in A375 co-cultured with HUVECs (A375-co). Time course. Mean ± SEM, n = 5. ** p < 0.01 vs. A375 alone; ° p < 0.05, °° p < 0.01 vs. HUVEC alone; ( B ) RT-PCR in HUVECs alone and in HUVECs co-cultured with A375 (HUVEC-co). Mean ± SEM, n = 3. §§§ p < 0.001 vs. HUVECs alone; ^^ p < 0.01 vs. A375 alone. ( C ) ELISA assay for Kyn production in HUVECs, A375 and in the co-cultures (Huvec + A375) for 6, 9 and 24 h. Mean ± SEM. n = 5. * p < 0.05, ** p < 0.01 vs. HUVECs alone. The Kruskal–Wallis test and Dunn’s post hoc test were performed.

Article Snippet: Human metastatic melanoma cell line A375 (ATCC, Manassas, VA, USA) was grown in high D-glucose DMEM, with 10% ( v / v ) heat-inactivated fetal bovine serum (Euroclone Milan, Italy), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mmol/L glutamine in a humidified atmosphere with 5% CO 2 in air.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceuticals

Article Title: Unveiling the Role of Tryptophan 2,3-Dioxygenase in the Angiogenic Process

doi: 10.3390/ph17050558

Figure Lengend Snippet: ( A ) Relative mRNA expression levels of stemness markers in A375 and in A375 co-cultured for 24 h with HUVECs in the presence or absence of the TDO inhibitor. After 24 h, total RNA was extracted to perform real-time PCR. Data are shown as Mean ± SEM, n = 3. * p < 0.05 vs. A375 alone, § p < 0.05, §§ p < 0.01 vs. A375 co-cultured. ( B ) Protein expression for Myc and Klf4 in A375 and in A375 co-cultured (A375-co) for 24 h with HUVECs in the presence of the TDO inhibitor 680C91. Mean ± SEM, n = 3.

Article Snippet: Human metastatic melanoma cell line A375 (ATCC, Manassas, VA, USA) was grown in high D-glucose DMEM, with 10% ( v / v ) heat-inactivated fetal bovine serum (Euroclone Milan, Italy), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mmol/L glutamine in a humidified atmosphere with 5% CO 2 in air.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Pharmaceuticals

Article Title: Unveiling the Role of Tryptophan 2,3-Dioxygenase in the Angiogenic Process

doi: 10.3390/ph17050558

Figure Lengend Snippet: Gelatin zymography for MMP2 and MMP9 activity in A375, HUVECs and in A375 co-cultured with HUVECs (co-culture) for 6–24 h. In some experiments, A375 was pretreated overnight with 680C91 or epacadostat and then co-cultures were set up. Densitometric analysis shows the effect of co-culture on latent and activated (aMMP2) MMP2 and on latent and activated (aMMP9) MMP9 at 24 h representative zymograms. Mean ± SEM of 5 experiments. § p < 0.05 vs. activated MMP9 of HUVECs; * p < 0.05, *** p < 0.001 vs. activated MMP9 of co-culture; °°° p < 0.001 vs. latent MMP9 of co-culture.

Article Snippet: Human metastatic melanoma cell line A375 (ATCC, Manassas, VA, USA) was grown in high D-glucose DMEM, with 10% ( v / v ) heat-inactivated fetal bovine serum (Euroclone Milan, Italy), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mmol/L glutamine in a humidified atmosphere with 5% CO 2 in air.

Techniques: Zymography, Activity Assay, Cell Culture, Co-Culture Assay

Journal: PLoS ONE

Article Title: The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

doi: 10.1371/journal.pone.0076550

Figure Lengend Snippet: A) Representative examples of FACS density plots of Hoechst-incubated cells from primary melanoma (cutaneous malignant form; top left), in-transit metastasis (top right), metastasis to a regional lymph node (bottom left) and visceral metastasis (bottom right) with indication of SP percentage, and the corresponding controls with verapamil. B) Boxplot of the SP proportions from all human melanoma samples taken together (All, n=38), and from the different progression phases as indicated (primary melanomas, n=13; in-transit metastases, n=8; regional lymph-node metastases, n=14; visceral metastases, n=3). C) SP proportion in primary (cutaneous malignant) melanomas as outlined against tumor’s thickness (Breslow’s depth). A significant correlation is observed (Spearman r=0.68; p=0.01; dashed line, 95% confidence interval of the best-fit line). D) Representative example of FACS density plots of Hoechst-incubated cells from the human malignant melanoma cell line A375 with indication of SP percentage, and the corresponding verapamil control (n=18).

Article Snippet: The malignant (metastatic) human melanoma cell line A375 [ ] was obtained from Dr. L. Van Kempen (Department of Pathology and Oncology, McGill University, Montreal, Canada).

Techniques: Incubation

Journal: PLoS ONE

Article Title: The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

doi: 10.1371/journal.pone.0076550

Figure Lengend Snippet: A) Sections (H&E staining) of the xenograft tumors developed in SCID mice 18 weeks after sc injection of SP and MP cells from a primary melanoma (pigmented, see arrows) (upper row) and from a lymph-node metastasis (lower row), together with sections of the original tumors (scale bar, 150µm). B) Volume of the xenograft tumors grown in SCID mice 18 weeks after sc injection of the melanoma SP and MP cells (10,000-50,000 cells). Bars represent mean ± SEM (n=6). *, p<0.05. C) Photographs and sections (H&E staining) of the xenograft tumors developed in SCID mice 18-32 weeks after sc injection of SP and MP cells from the SP-derived first-generation xenograft tumors (F1, see A), together with sections of the original tumors (scale bar, 150µm). Again note the pigmentation (upper row) as clear from the picture of the dissected tumor. D) Volume of the F2 xenograft tumors grown in SCID mice 32 weeks after sc injection of the SP and MP cells from the SP-derived F1 tumors (10,000 SP cells, 50,000 MP cells). Bars represent mean ± SEM (n=2). E) Tumors grown in SCID mice 5-7 weeks after sc injection of 2500, 1000 and 500 SP cells (upper row) or MP cells (lower row) from the A375 melanoma cell line (n=3). Representative examples are shown. F) Summary of tumor volume (relative to volume of the tumors grown from 2500 SP cells) after sc injection of the indicated number of A375 SP cells (black bar) and MP cells (white bar). Bars represent mean ± SEM (n=3). *, p<0.05; ***, p<0.001.

Article Snippet: The malignant (metastatic) human melanoma cell line A375 [ ] was obtained from Dr. L. Van Kempen (Department of Pathology and Oncology, McGill University, Montreal, Canada).

Techniques: Staining, Injection, Derivative Assay

Journal: PLoS ONE

Article Title: The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

doi: 10.1371/journal.pone.0076550

Figure Lengend Snippet: A) Clonogenic capacity of A375 SP cells (relative to MP cells) as assessed at day 9 after seeding at low density. Representative examples of colony-forming activity by SP (bottom left) and MP (bottom right) are shown. Bars represent mean ± SEM (n=3). ***, p<0.001 versus MP. B) SP analysis (representative FACS density plots) 5, 10 and 11 days after seeding A375 SP cells (upper row) and MP cells (lower row) in standard culture medium (n=4 except for day 11 where n=2; numbers indicate the mean SP proportion).

Article Snippet: The malignant (metastatic) human melanoma cell line A375 [ ] was obtained from Dr. L. Van Kempen (Department of Pathology and Oncology, McGill University, Montreal, Canada).

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

doi: 10.1371/journal.pone.0076550

Figure Lengend Snippet: A) Expression ratios of the indicated genes in the SP versus the MP from patient melanomas. Bars represent mean ± SEM (n= 3 primary melanomas and 4 melanoma metastases). *, p<0.05; **, p<0.01. B) Expression ratios of the indicated genes in the SP versus the MP from the A375 cell line. Bars represent mean ± SEM (n=4). *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: The malignant (metastatic) human melanoma cell line A375 [ ] was obtained from Dr. L. Van Kempen (Department of Pathology and Oncology, McGill University, Montreal, Canada).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The Human Melanoma Side Population Displays Molecular and Functional Characteristics of Enriched Chemoresistance and Tumorigenesis

doi: 10.1371/journal.pone.0076550

Figure Lengend Snippet: A) Representative example of FACS density plots of Hoechst-incubated A375 cells treated with dacarbazine (+DTIC; 150µg/ml for 3 days) (left; verapamil control not shown) and summary of the SP proportions (right). Bars represent mean ± SEM (n=3). *, p<0.05 versus control (–DTIC). B) Representative example of FACS density plots of Hoechst-incubated A375 cells cultured in hypoxic conditions (1.5% O 2 3 days) (left; verapamil control not shown) and summary of the SP proportions (right). Bars represent mean ± SEM (n=3). *, p<0.05 versus standard cell-culture condition (20% O 2 ).

Article Snippet: The malignant (metastatic) human melanoma cell line A375 [ ] was obtained from Dr. L. Van Kempen (Department of Pathology and Oncology, McGill University, Montreal, Canada).

Techniques: Incubation, Cell Culture

Journal: PLoS ONE

Article Title: MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor

doi: 10.1371/journal.pone.0115919

Figure Lengend Snippet: Following transfection with control pre-miR or pre-miR-21, cells were plated on Radius Migration Assay ECM-coated plates that have uniform wounds. Photographs were taken of the wound immediately following migration initiation and 14 hours later. Photographs of representative experiments for each cell line on the collagen-coated wells are shown ( A ). Migration was measured as the percent of the original wound area on the collagen-coated wells (n = 4) ( B ). Boyden chamber assays were used to evaluate invasive activity of melanoma cells transfected with control pre‑miR or pre-miR-21. Photographs of representative experiments for each cell line are shown ( C ). Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel (n = 4) ( D ). Following transfection with control pre-miR or pre-miR-21 at 3.125, 6.25, or 12.5 nM, cells were plated on Boyden chamber assays to evaluate invasive activity of A375 melanoma cells at lower doseage of oligonucleotide. Data are represented as the ratio of the average number of cells migrating through control inserts to the average number of cells invading through inserts coated with matrigel ( E ). Error bars represent standard error. * p < 0.05.

Article Snippet: The A375 human metastatic melanoma cell line was obtained from American Type Cell Culture Collection (ATCC, Manassas, VA).

Techniques: Transfection, Migration, Activity Assay

Journal: PLoS ONE

Article Title: MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor

doi: 10.1371/journal.pone.0115919

Figure Lengend Snippet: A375 cells were transfected in vitro with an LNA oligonucleotide targeting miR-21 or a control LNA oligonucleotide. Cells were collected 24 hours post‑transfection for total RNA isolation. The RNA was converted to cDNA, and Real‑Time PCR for miR-21 confirmed reduced miR-21 expression ( A ). Transfected cells were then injected subcutaneously into athymic nude mice, and tumor growth was followed. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( B ). At the completion of the study, tumors were harvested, fixed and embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). N = 4 for control LNA tumors and n = 6 for anti‑miR‑21 LNA tumors. Error bars represent standard error. * p < 0.05.

Article Snippet: The A375 human metastatic melanoma cell line was obtained from American Type Cell Culture Collection (ATCC, Manassas, VA).

Techniques: Transfection, In Vitro, Isolation, Real-time Polymerase Chain Reaction, Expressing, Injection, Immunohistochemistry

Journal: PLoS ONE

Article Title: MiR-21 Enhances Melanoma Invasiveness via Inhibition of Tissue Inhibitor of Metalloproteinases 3 Expression: In Vivo Effects of MiR-21 Inhibitor

doi: 10.1371/journal.pone.0115919

Figure Lengend Snippet: A375 cells were subcutaneously injected into the right flank of athymic nude mice and tumor development was measured. Each tumor was treated with four 50 µL injections of PBS, control LNA, or anti-miR-21 LNA at 500 nM at days 0, 4, 7, 11 (arrows). Day 0 is the day of the first treatment injection. Tumor volume was calculated as V = L × l 2 × 0.5, where L and l represent the larger and the smaller tumor diameter, respectively. Mean tumor volumes are represented ( A ). Following the completion of the study, tumors were harvested. Total RNA was extracted from the tumors. The RNA was converted to cDNA, and Real-Time PCR for miR-21 confirmed reduced miR-21 expression ( B ). Tumors were also fixed, embedded, and evaluated for TIMP3 protein by immunohistochemistry, where TIMP3 immunoreactivity is in brown ( C ). Tumors were evaluated for mean areas of confluent necrosis ( D ) and mean mitotic indices ( E ). N = 5 for each group. Error bars represent standard error.

Article Snippet: The A375 human metastatic melanoma cell line was obtained from American Type Cell Culture Collection (ATCC, Manassas, VA).

Techniques: Injection, Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry

Journal: Molecules

Article Title: Application of Pharmacokinetic Prediction Platforms in the Design of Optimized Anti-Cancer Drugs

doi: 10.3390/molecules27123678

Figure Lengend Snippet: In vitro screening of novel NL compounds: ( A ) hit compounds were screened at 10 micromolar concentrations for 24 h. All but one compound demonstrated significant activity and were selected for a dose response analysis; ( B ) compounds were screened at increasing concentrations to assay for activity. NL221-75 and NL350-02 were as potent as FDA-approved controls, demonstrating low nanomolar range activity; ( C ) MTT A375 cells were treated with increasing concentrations of test items and cell proliferation was determined at 24 h using the MTT method. All compounds demonstrated dose-dependent activity in preventing cell proliferation. Experimental compounds NL221-75 and NL350-02 were as effective as FDA-approved controls in preventing cell proliferation. Data are plotted as percent inhibition of proliferation; and ( D ) hERG inhibition experiments performed on CHO-cells. Cobimetinib demonstrated low nanomolar inhibition of hERG. NL34-113, NL221-75, and NL350-02 did not inhibit hERG at the concentrations tested.

Article Snippet: Human A375 metastatic melanoma cells (ATCC (Manassas, VA, USA); No. CRL-1619IG-2) were cultured in Gibco Dulbecco’s Modified Eagle Medium (DMEM) (ATCC; No. 30-2002) using 10% fetal bovine serum and 1% Penicillin-Streptomycin.

Techniques: In Vitro, Activity Assay, Inhibition