stable transfection human melanoma cell lines a375  (ATCC)

Journal: Annals of Translational Medicine

Article Title: LncRNA SNHG12 promotes the malignant progression of melanoma by targeting miR-199b

doi: 10.21037/atm-22-214

Figure Lengend Snippet: SNHG12 regulates the proliferation, migration, and invasion of melanoma cells through miR-199b. Transfection of miR-199b mimics in A375 cells overexpressing SNHG12, and transfection of an miR-199b inhibitor in SNHG12-silenced CHL-1 cells. (A,B) The CCK-8 experiment and (C,D) EdU experiment (red: EdU, blue: DAPI, purple: merge; 100×) were carried out to study the roles of SNHG12 and miR-199b on cell proliferation, (E,F) the scratch experiment (40×) was performed to study the effects of SNHG12 and miR-199b on cell migration, and (G,H) the transwell experiment (crystal violet; 100×) was conducted to study the roles of SNHG12 and miR-199b on cell invasion. *, P<0.05. SNHG12, small nucleolar RNA host gene 12; CCK-8, Cell-Counting-Kit-8; EdU, 5-ethynyl-2'-deoxyuridine; NC, normal control.

Article Snippet: Cell culture and transfection Human melanoma cell lines (A2058, SK-MEL-28, CHL-1, and A375) and the human epidermal melanocyte cell line HEMa-LP were all from ATCC (ATCC, USA).

Techniques: Migration, Transfection, CCK-8 Assay, Cell Counting, Control

Journal: Oncology Letters

Article Title: Ubiquitination of P53 by E3 ligase MKRN2 promotes melanoma cell proliferation

doi: 10.3892/ol.2020.11261

Figure Lengend Snippet: Expression profile of MKRN2 in normal human epithelial melanocyte or keratinocyte and human malignant melanoma cell lines. (A) Protein expression profiles of MKRN2, P53, P21, MDM2 and P65 in normal human epithelial melanocyte (A375, SK-MEL-28 and WM-115), keratinocyte (HaCaT) and human malignant melanoma (NHEM) cell lines detected by immunoblotting. The protein expression levels of MKRN2, MDM2, P53, P21 and P65 in different cell lines were compared with those of GAPDH. n=3. (B) mRNA expression levels of MKRN2 in normal human epithelial melanocyte or keratinocyte cell lines and human malignant melanoma cell lines detected by reverse transcription-quantitative PCR. n=3. Data are expressed as the mean ± standard deviation and were analysed using one-way ANOVA with Tukey's post hoc test. *P<0.05, **P<0.01. MDM2, MDM2 proto-oncogene; MKRN2, makorin ring finger protein 2; P21, cyclin dependent kinase inhibitor 1A; P65, golgi reassembly stacking protein 1; IB, immunoblot.

Article Snippet: Cell culture and transfection Human malignant melanoma cell lines A375, SK-MEL-28 and WM-115 were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Oncology Letters

Article Title: Ubiquitination of P53 by E3 ligase MKRN2 promotes melanoma cell proliferation

doi: 10.3892/ol.2020.11261

Figure Lengend Snippet: MKRN2-knockdown induces arrest of melanoma cell growth. (A) Test of the knockdown efficiency of shRNAs targeting MKRN2. A375 and SK-MEL-28 cells were transfected with MKRN2 shRNAs and then subjected to screening for puromycin resistance to establish stably expressed cell lines. (B) MKRN2-knockdown inhibited melanoma cell proliferation. A375 and SK-MEL-28 cells that stably expressed MKRN2 shRNAs were seeded into a 96-well plate and detected using an MTT assay at the indicated time points. The 0 h time point was defined as 6 h after the cells were seeded. n=3. (C) MKRN2-knockdown inhibited melanoma cell colony formation. A375 and SK-MEL-28 cells that stably expressed MKRN2 shRNAs were seeded into a 6-well plate; colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet 7 days later. The images were obtained using a camera, colony numbers were counted and the average number of colonies was calculated. Three samples were analysed for each group. Data are expressed as the mean ± standard deviation and were analysed using one-way ANOVA with Tukey's post hoc test. *P<0.05. MKRN2, makorin ring finger protein 2; sh, short hairpin RNA; shRNA, short hairpin RNA; IB, immunoblot.

Article Snippet: Cell culture and transfection Human malignant melanoma cell lines A375, SK-MEL-28 and WM-115 were purchased from the American Type Culture Collection (ATCC).

Techniques: Knockdown, Transfection, Stable Transfection, MTT Assay, Staining, Standard Deviation, shRNA, Western Blot

Journal: Oncology Letters

Article Title: Ubiquitination of P53 by E3 ligase MKRN2 promotes melanoma cell proliferation

doi: 10.3892/ol.2020.11261

Figure Lengend Snippet: MKRN2-knockdown induces the arrest of melanoma cell growth by upregulating P53. (A) Protein expression levels of P53 and P21 were upregulated in MKRN2-knockdown cells. The protein levels of P53 and P21 were detected by immunoblotting in melanoma cell lines (A375 and SK-MEL-28) that stably expressed MKRN2 shRNAs. (B) mRNA levels of P21 but not P53 were upregulated in MKRN2-knockdown cells. The mRNA levels of TP53 and p21 were detected by reverse transcription-quantitative PCR in melanoma cells (A375 and SK-MEL-28) that stably expressed MKRN2 shRNAs. Data are expressed as the mean ± standard deviation and were analysed using one-way ANOVA with Tukey's post hoc test. n=3. **P<0.01. MKRN2, makorin ring finger protein 2; P21, cyclin dependent kinase inhibitor 1A; sh, short hairpin; sh, short hairpin RNA; shRNA, short hairpin RNA; IB, immunoblot.

Article Snippet: Cell culture and transfection Human malignant melanoma cell lines A375, SK-MEL-28 and WM-115 were purchased from the American Type Culture Collection (ATCC).

Techniques: Knockdown, Expressing, Western Blot, Stable Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, shRNA

Journal: Oncology Letters

Article Title: Ubiquitination of P53 by E3 ligase MKRN2 promotes melanoma cell proliferation

doi: 10.3892/ol.2020.11261

Figure Lengend Snippet: MKRN2 interacts with and ubiquitylates P53. (A) Endogenous MKRN2 and P53 formed a complex in A375 cells. A375 cell lysates were immunoprecipitated with anti-P53 antibody and subjected to immunoblotting analysis. (B) MKRN2 interacted with P53 in vitro. Recombinant GST-tagged MKRN2 and His6-tagged P53 were purified, and then GST pull-down assays were performed and subjected to immunoblotting analysis. (C) MKRN2 ubiquitylated P53 in vitro. An in vitro ubiquitylation assay was carried out with the recombinant proteins P53-His6, E1 (UBA1-His6), E2 (UBCH5A-His6), His6-Ub and Gst-MKRN2, together with the indicated components, and subjected to immunoblotting analysis. (D) MKRN2 ubiquitylated P53 in vivo. A375 cells stably transfected with MKRN2 shRNAs were lysed in RIPA buffer and immunoprecipitated with anti-P53 antibody and subjected to immunoblotting analysis using the indicated antibodies. COIP, co-immunoprecipitation; GST, glutathione S-transferase; His, histidine; IgG, immunoglobulin G; MKRN2, makorin ring finger protein 2; sh, short hairpin RNA; shRNA, short hairpin RNA; UBA1, ubiquitin like modifier activating enzyme 1; UBCH5A, ubiquitin conjugating enzyme E2 D1; USP2, ubiquitin specific peptidase 2; IB, immunoblot.

Article Snippet: Cell culture and transfection Human malignant melanoma cell lines A375, SK-MEL-28 and WM-115 were purchased from the American Type Culture Collection (ATCC).

Techniques: Immunoprecipitation, Western Blot, In Vitro, Recombinant, Purification, Ubiquitin Assay, In Vivo, Stable Transfection, Transfection, shRNA, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: Ubiquitination of P53 by E3 ligase MKRN2 promotes melanoma cell proliferation

doi: 10.3892/ol.2020.11261

Figure Lengend Snippet: MKRN2 regulates the proliferation of melanoma cells in a P53-dependent manner. (A) A TP53−/− A375 cell line was established using the CRISPR-Cas9 technique. A375 cells were transfected with CRISPR-Cas9-based sgRNA indicated in red (right panel), monoclonal cells were picked and detected by immunoblotting analysis (left panel), and then genetic ablation of TP53 with a 5-bp deletion was confirmed by sequencing. (B) MKRN2 knockdown induced melanoma cell growth arrest in a P53-dependent manner. WT or TP53−/− A375 cells that stably expressed MKRN2 shRNAs were seeded into a 96-well plate and detected by MTT assay at the indicated time points (0 and 48 h). The 0 h time point was defined as 6 h after the cells were seeded. Data are expressed as the mean ± standard deviation and were analysed using one-way ANOVA with Tukey's post hoc test. n=3. *P<0.05, **P<0.01. KO, knockout; MKRN2, makorin ring finger protein 2; sgRNA, single guide RNA; sh, short hairpin RNA; shRNA, short hairpin RNA; WT, wild-type; IB, immunoblot.

Article Snippet: Cell culture and transfection Human malignant melanoma cell lines A375, SK-MEL-28 and WM-115 were purchased from the American Type Culture Collection (ATCC).

Techniques: CRISPR, Transfection, Western Blot, Sequencing, Knockdown, Stable Transfection, MTT Assay, Standard Deviation, Knock-Out, shRNA

Journal: Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques

Article Title: Design and Evaluation of Gemini Surfactant-Based Lipoplexes Modified with Cell-Binding Peptide for Targeted Gene Therapy in Melanoma Model.

doi: 10.18433/jpps30010

Figure Lengend Snippet: Figure 2. SAXS profile of [P.G.L] lipoplexes constructed at 1:10 -/+ charge ratio and with helper lipid DOPE (L) in all formulation. P: pDNA, G:12-7N(GK)-12, L: DOPE, p18-4-G : 12-7N( p18-4)-12, p18-4: p18-4 linear peptide. In vitro evaluation Transfection efficiency and cellular toxicity To determine the optimum formulation strategy and amount of targeting peptide leading to the highest gene expression and lowest cellular toxicity, the transfection efficiency of the p18-4-modified lipoplexes was evaluated in human malignant melanoma (A375) cell line. Different concentrations of p18-4 modified gemini surfactant or free p18-4 (5, 10, 15, 20% and 100% of the total amount of cationic lipid) were tested. The highest levels of gene expression (IFN-γ level by ELISA) were observed when 10% of p18-4 conjugated gemini surfactant (p18-4-G) or physical co-formulation of free p18-4 was used [Figure 3 A]. ]. Lipoplexes formulated with

Article Snippet: Cell culture and in vitro transfection Human malignant melanoma (A375) cell line (ATCC® CRL-1619TM) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic and incubated at 37 °C under an atmosphere of 5% CO2/95% air.

Techniques: Construct, Formulation, In Vitro, Transfection, Gene Expression, Modification, Enzyme-linked Immunosorbent Assay

Journal: Journal of pharmacy & pharmaceutical sciences : a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques

Article Title: Design and Evaluation of Gemini Surfactant-Based Lipoplexes Modified with Cell-Binding Peptide for Targeted Gene Therapy in Melanoma Model.

doi: 10.18433/jpps30010

Figure Lengend Snippet: Figure 3 (A) IFN-γ expression in A375 cells at 48-h post-treatment with lipoplexes constructed at 1:10 -/+ charge ratio. P: pDNA, G:12-7N(GK)-12, L: DOPE, p18-4-G : 12-7N( p18-4)-12, p18-4: p18-4 linear peptide. (Ch) indicates lipoplexes chemically conjugated p18-4-G and (Ph) lipoplexes physically co-formulated with free p18-4. IFN-γ level determined by ELISA. Significant increases (* p<0.01, one-way ANOVA) in IFN- expression were observed in all p18-4 modified lipoplexes compared to non-modified lipoplexes (L1:[P.G.L]). (B) Cell viability in the same A375 cells after a 48-h treatment with p18-4-modified lipoplex formulations as determined by MTT assay. Cell viability values are given as % relative to non- transfected cells. Four wells of each formulations were loaded on three different plates. The results are expressed as the mean of the three plates (n = 3). Bars represent standard deviation. * Indicates significance at p < 0.05 in comparison to standard formulation [P.G.L] (L1)

Article Snippet: Cell culture and in vitro transfection Human malignant melanoma (A375) cell line (ATCC® CRL-1619TM) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic and incubated at 37 °C under an atmosphere of 5% CO2/95% air.

Techniques: Expressing, Construct, Enzyme-linked Immunosorbent Assay, Modification, MTT Assay, Transfection, Standard Deviation, Comparison, Formulation