Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 inhibits melanoma cell growth through induction of cell cycle arrest and apoptosis. ( A ) IC 50 values of SLMP53-2 in A375, G361, MEWO and SK-MEL-5 melanoma cells obtained by colony formation assay for 11 days; data were normalized to DMSO and correspond to mean ± SEM, n = 5 (two replicates each). ( B ) Colony formation assay for A375, G361, MEWO and SK-MEL-5 melanoma cells treated with SLMP53-2 for the indicated concentrations. Images are representative of five independent experiments. ( C ) Effect of SLMP53-2 on growth and morphology of A375 cells for the indicated time points; images are representative of five independent experiments (scale bar = 100 μm, magnification = ×100). ( D ) Apoptosis (Annexin V-positive cells) was evaluated in A375 cells after 24, 48 and 72 h of treatment with 12 μM SLMP53-2. ( E ) Cell cycle analysis in A375 cells was determined after 24, 48 and 72 h of treatment with 12 μM SLMP53-2. In ( D , E ), data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( F , G ) Effect of SLMP53-2 on three-day-old A375 spheroids, for up to 8 days of treatment. In G , data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( H , I ) Evaluation of spheroid formation after 10 days of treatment with SLMP53-2; treatment was performed at the seeding time of A375 cells. In I , data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. In ( F , H ), images are representative of five independent experiments; scale bar = 100 μm; magnification = 100×.

Article Snippet: Human melanoma A375 (CLS Cat# 300110/p852_A-375, RRID:CVCL_0132) and SK-MEL-5 (CLS Cat# 300157/p634_SK-MEL-5, RRID:CVCL_0527) cells were purchased from CLS Cell lines service (Eppelheim, Germany).

Techniques: Colony Assay, Cell Cycle Assay

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 inhibits melanoma cell migration and invasion. ( A ) A375 and SK-MEL-5 confluent cells were treated with 2 or 4 μM SLMP53-2, respectively; cells were observed at 24 and 32 h (A375) and 30 and 48 h (SK-MEL-5) in the wound-healing assay. Images are representative of five independent experiments; scale bar = 100 μM; magnification = 100×. ( B ) Quantification of wound closure using randomly selected microscopic fields (six fields per sample). Data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( C ) Effect of 2 μM SLMP53-2 on migration of A375 and SK-MEL-5 cells after 24 h of treatment. The relative number of migratory cells was determined by analysis of fluorescence signal intensity; values with DMSO were set as 1. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05, Student’s t -test. ( D ) Effect of 2 μM SLMP53-2 on the invasion of A375 and SK-MEL-5 cells after 24 h of treatment. Cells able to invade through an ECMatrix layer were quantified by fluorescence signal; values with DMSO were set as 1. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05, Student’s t -test. ( E ) Effect of SLMP53-2 on lactate secretion by A375 and SK-MEL-5 cells after 8 h of treatment. Cell density for each sample was used to normalize relative luminescence units (RLU) signal. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05; unpaired Student’s t -test.

Article Snippet: Human melanoma A375 (CLS Cat# 300110/p852_A-375, RRID:CVCL_0132) and SK-MEL-5 (CLS Cat# 300157/p634_SK-MEL-5, RRID:CVCL_0527) cells were purchased from CLS Cell lines service (Eppelheim, Germany).

Techniques: Migration, Wound Healing Assay, Fluorescence

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 interferes with key molecular players in epithelial-to-mesenchymal transition (EMT) and angiogenesis. ( A – D ) Protein expression levels of crucial regulators of EMT and angiogenesis in A375 ( A , B ) and SK-MEL-5 ( C , D ) melanoma cells after 48 h of treatment with SLMP53-2 (in A375 cells, β-catenin was detected for 8 h and E-cadherin and TWIST for 24 h of treatment). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In ( B , D ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are means ± SEM, n = 5.

Article Snippet: Human melanoma A375 (CLS Cat# 300110/p852_A-375, RRID:CVCL_0132) and SK-MEL-5 (CLS Cat# 300157/p634_SK-MEL-5, RRID:CVCL_0527) cells were purchased from CLS Cell lines service (Eppelheim, Germany).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Deficits in N-Methyl-D-Aspartate Receptor Function and Synaptic Plasticity in Hippocampal CA1 in APP/PS1 Mouse Model of Alzheimer’s Disease

doi: 10.3389/fnagi.2021.772980

Figure Lengend Snippet: The expression of synapse-associated proteins and glutamate receptor subunit proteins in the CA1 area of 8-month-old WT and APP/PS1 mice. (A) Representative western blots of glutamate receptor subunits. (B–D) Quantitative of NMDAR subunits expression showed a significant decrease of NR2A and NR2B expression ( n = 6 mice/group, * p < 0.05, ** p < 0.01, unpaired t -test) in the hippocampus in 8-month-old APP/PS1 mice compared with WT mice, but there was no difference on the expression of NR1 from two groups. (E,F) Quantitative of AMPA receptors subunits (GluR1, GluR2) expression showed no change in hippocampus from 8-month-old APP/PS1 mice and WT mice. (G) Representative western blots of PSD95 and SNAP25. (H,I) A significant decrease of the levels of PSD95 and SNAP25 in the hippocampus from 8-month-old APP/PS1 mice compared with WT mice ( n = 4 mice/group, * p < 0.05, unpaired t -test).

Article Snippet: The membrane was blocked in 5% milk-TBST at RT and probed with rabbit anti-NR2A (ab133265, Abcam, United Kingdom; 1:1,000), rabbit anti-NR2B (ab183942, Abcam, United Kingdom; 1:1,000), rabbit anti-NR1 (ab109182, Abcam, United Kingdom; 1:1,000), rabbit anti-GluR1 (04-855, Millipore, Germany; 1:1,000), mouse anti-GluR2 (MAB397, Millipore, Germany; 1:1,000), PSD95 antibody (sc2290, Santa Cruz Biotechnology Inc., United States; 1:500), SNAP25 antibody (A2234, Abclonal, China; 1:500) at 4°C overnight and then reacted with the Alexa Fluor 800 conjugated antibody (1:5,000) for 60 min.

Techniques: Expressing, Western Blot