Journal: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) Western blot showing expression changes of several N-cad binding catenins following 6–12 cycles of irradiation (5 Gy) in mGS cells. (B) Fluorescence microscopy shows that β-catenin (green) selectively coaccumulates with N-cad (red) on the cell surface of mGSRR but not mGS cells. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 25 μm. (C) Wnt/β-catenin regulated transcriptional activity in mGS and mGSRR cells measured through transient transfection with a luciferase reporter driven by a WT (TOP) or mutant (FOP) TCF binding site. ***P < 0.001, 2-tailed Student’s t test. (D) TOP/FOP ratio showing Wnt/β-catenin activity in parental N-cad–overexpressing and N-cad–KO mGS cells. **P < 0.01, ***P < 0.001, Tukey’s HSD test. (E) Microarray analysis showing that mRNA expression of multiple Wnt target genes is suppressed in mGSRR compared with mGS cells. Each group contains 2 independent replicates (n = 2). (F) qRT/PCR showing that NeuroD1, Ngn1, and Brn3a mRNAs are reduced in mGSRR cells. Two-tailed Student’s t test. (G) Western blot showing expression change of β-catenin (pan and non-phospho), c-Myc, and Tuj1 by N-cad–overexpressing and N-cad–KO mGS cells. All blots show representative images (n = 3 or more).

Article Snippet: Luciferase reporter gene transfections were performed with lipofectamine using a reporter gene construct driven by a TCF binding site (M50 Super 8x TOP Flash, Addgene, #12456) and a negative control with a mutant TCF binding site (M51 Super 8x FOP Flash, Addgene, #12457), which were provided by Randall Moon (University of Washington, Seattle, Washington, USA).

Techniques: Western Blot, Expressing, Binding Assay, Irradiation, Fluorescence, Microscopy, Activity Assay, Transfection, Luciferase, Mutagenesis, Microarray, Quantitative RT-PCR, Two Tailed Test

Journal: PLOS ONE

Article Title: Recovery after human bone marrow mesenchymal stem cells (hBM-MSCs)-derived extracellular vesicles (EVs) treatment in post-MCAO rats requires repeated handling

doi: 10.1371/journal.pone.0312298

Figure Lengend Snippet: (a–c) Characterization and quantification of human-bone marrow mesenchymal stem cell (hBM-MSC)-derived extracellular vesicles (EVs). (a) Nanosight Tracking analyses (NTA) showing the size distribution pattern of EVs. (b) Western blots of hBM-MSCs marker (GM130) and EVs markers (ALIX, CD9, CD81). “Wash” lane represents the negative control. Each image comes from different blots. (c) Representative image of EVs using transmission electron microscopy (TEM). Scale bar 100 nm. (d–f) In vivo tracking of ExoGlow-labeled EVs demonstrated that EVs reach the brain and accumulate near the stroke injury 6 hours after administration at 2 dps. (d) Timeline of intranasal single dose treatment of ExoGlow-labeled EVs (~2.4 x 10 9 EVs in 200 μl), starting 2 dps and collecting the tissue 6 hours after. (e) Representative coronal section after a single intranasal administration of ExoGlow-labeled EVs (~2.4 x 10 9 EVs in 200 μl) at 2 dps. Hoechst indicates cell nuclei. Scale bars 4 mm and 100 μm. (f) Quantification of the average size of ExoGlow+ particles in the ipsilateral and contralateral hemispheres. Data was analyzed using a paired t-test (*p<0.05). Bars show mean +/− SD. Each symbol represents the average size of ExoGlow+ particles from all ROIs in each hemisphere (5 ROIs/hemisphere in each rat) of a single rat ( n = 4 ).

Article Snippet: Human bone marrow mesenchymal stem cells (hBM-MSCs) were purchased from Celprogren (frozen vial with ~1.2 x 10 6 cells; #36094–22) and plated on human mesenchymal bone marrow stem cell culture extracellular expansion matrix pre-coated T75 Flasks (Celprogen, #E36094-21-T75) following Celprogren recommendations.

Techniques: Derivative Assay, Western Blot, Marker, Negative Control, Transmission Assay, Electron Microscopy, In Vivo, Labeling

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: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

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 has p53-dependent growth inhibitory effect in melanoma cells with enhancement of p53 transcriptional activity. ( A – C ) Colony formation assay for silenced p53 (sip53) and control (CTRL) A375 cells treated with SLMP53-2, allowed to grow for 11 days. In ( A ), silencing efficacy of p53 by siRNA is shown; immunoblots are representative of five independent experiments and GAPDH was used as loading control; data plotted were normalized to CTRL and correspond to mean ± SEM, n = 5; values are significantly different from CTRL: * p < 0.05, unpaired Student’s t -test. In ( B ), images are representative of five independent experiments. In ( C ), data are normalized to DMSO and correspond to mean ± SEM, n = 5; values of sip53 cells significantly different from CTRL cells: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( D , E ) Protein levels of p53 transcriptional targets in A375 cells treated with SLMP53-2 for 24 h (p53, MDM2, PTEN, Cyclin D1, p21 and KILLER) or 48 h (GADD45, PUMA, BCL-2, BCL-xL and BAX). In ( D ), immunoblots are representative of five independent experiments; GAPDH was used as loading control. In ( E ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( F ) mRNA levels of p53 target genes were determined by RT-qPCR in A375 cells after 24 h treatment with SLMP53-2; fold change is relative to DMSO; data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, two-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Activity Assay, Colony Assay, Control, Western Blot, Expressing, Quantitative RT-PCR, Comparison

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 enhances p53 stabilization by disrupting the p53–MDM2 interaction and interferes with the miRNA network in melanoma cells. ( A ) p53 protein levels in A375 melanoma cells treated for 24 h with 12 µM SLMP53-2 or solvent followed by cycloheximide treatment from 0 to 2 h (CHX; 150 μg/mL). ( B ) Quantification of p53 protein expression levels; immunoblots are representative of five independent experiments; GAPDH was used as loading control. Values for cells nontreated with cycloheximide (0 h) were set as 1; data are mean ± SEM, n = 5. ( C , D ) Coimmunoprecipitation (Co-IP) was performed in A375 cells treated with SLMP53-2 for 4 h. In C, representative immunoblots of five independent experiments are shown—whole-cell lysate (Input). p53 from IP was used as loading control. In D, quantification of protein expression levels relative to DMSO is shown (set as 1). Data shown are mean ± SEM, n = 5. ( E ) Expression levels of miR-145 and miR-23a in A375 cells after 24 h of treatment with SLMP53-2 were determined by RT-qPCR; fold of change is relative to DMSO; 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 ) Protein levels of miR-145 target genes, in A375 cells treated with SLMP53-2 for 24 h. In ( F ), immunoblots are representative of five independent experiments; GAPDH was used as loading control. In ( G ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Solvent, Expressing, Western Blot, Control, Co-Immunoprecipitation Assay, Quantitative RT-PCR

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: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

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: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Expressing, Western Blot, Control

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 sensitizes melanoma cells to clinically used chemotherapeutic agents. ( A – C ) Cells were treated with a concentration range of vemurafenib ( A ), dacarbazine ( B ) and cisplatin ( C ) alone and in combination with 2 μM SLMP53-2, for 48 h, and the growth was analysed by SBR assay. Growth with DMSO was set as 100%. For each combination, the combination index (C.I.) and dose reduction index (D.R.I.) values were obtained. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from chemotherapeutic drug alone: * p < 0.05; two-way ANOVA followed by Sidak’s test. ( D ) Apoptosis (Annexin V-positive cells) was evaluated in A375 cells after 48 h of treatment with 2 μM SLMP53-2 (SLMP) and 5 µM cisplatin and 0.03 µM vemurafenib. Data are mean ± SEM, n = 5; values are significantly different from drug alone: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( E , F ) Protein expression levels of BCL-2 after 48 h of treatment of SLMP53-2 with cisplatin (cisp) and with vemurafenib (vem). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In F , quantification of protein expression levels; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( G ) Cell cycle analysis in A375 cells was determined after 48 h of treatment with 2 μM SLMP53-2 and 2 μM dacarbazine (Dac). Data are mean ± SEM, n = 5; values are significantly different from drug alone: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( H , I ) Protein expression levels of p21 after 48 h treatment of SLMP53-2 with dacarbazine (Dac). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In I , quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( J , K ) Effect of 2 μM SLMP53-2 in combination with 0.027 μM Vemurafenib (Vem) on three-day-old A375 spheroids for up to 8 days of treatment. For the combination, the C.I. value was obtained. Images are representative of five independent experiments; scale bar = 100 μm; magnification = 100×. In ( K ), data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Concentration Assay, Expressing, Western Blot, Control, 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: Melanoma cells do not develop resistance to SLMP53-2: vemurafenib-resistant melanoma cells show no cross-resistance to SLMP53-2 and are resensitized to vemurafenib by SLMP53-2. ( A ) A375 cells were exposed to six rounds of treatment with 6, 9, 12, 18, 24 and 30 μM of SLMP53-2. IC 50 values were determined at the end of each round by SRB assay after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 5 (two replicates each); values not significantly different from parental cells: p > 0.05, two-way ANOVA followed by Sidak’s test. ( B ) Representative images of parental, vemurafenib-resistant (Vem-res) A375 cells; scale bar = 100 μm; magnification = 100×. ( C ) Concentration–response curves for vemurafenib in parental and Vem-res A375 cells after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 6 (two replicates each); values of Vem-res cells significantly different from parental cells: * p < 0.05; two-way ANOVA followed by Sidak’s test. ( D , E ) Protein levels of p-AKT/AKT, p-ERK/ERK, PTEN and MDR-1 in untreated parental and Vem-res A375 cells. In D, immunoblots are representative of five independent experiments; GAPDH was used as loading control. In E, quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( F ) Concentration–response curves for SLMP53-2 in parental and Vem-res A375 cells after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 6 (two replicates each); values of Vem-res cells are not significantly different from parental cells: two-way ANOVA followed by Sidak’s test. ( G ) Vem-res A375 cells were treated with a concentration range of vemurafenib alone and in combination with 2 μM of SLMP53-2. Cell growth was evaluated for 48 h of treatment; growth obtained with DMSO was set as 100%. For each combination, the C.I. and D.R.I. values were obtained. Data are mean ± SEM, n = 6 (two replicates each); values are significantly different from vemurafenib alone: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( H ) Representative images of Vem-res A375 cells treated with DMSO, 2 μM SLMP53-2, 1.3 μM vemurafenib (Vem) and the combination (SLMP53-2 + Vem) for 48 h; images are representative of five treatments; scale bar = 100 μm; magnificatio n = 100×. ( I , J ) Protein levels of PTEN, BCL-2, MDR-1 and p-AKT/AKT, in Vem-res cells after 48 h of treatment with 2 µM SLMP53-2. In I, immunoblots are representative of five independent experiments; GAPDH was used as loading control. In J, quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Sulforhodamine B Assay, Concentration Assay, Western Blot, Control, Expressing

Journal: Cancers

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

doi: 10.3390/cancers13071648

Figure Lengend Snippet: In vivo melanoma antitumour activity of SLMP53-2. C57BL/6-Rag2 −/− IL2rg −/− mice carrying A375 xenografts were treated with 50 mg∙kg −1 SLMP53-2 or vehicle by intraperitoneal injection twice a week for a total of six administrations. ( A ) Tumour volume curves of mice carrying A375 xenografts treated with SLMP53-2 or vehicle. Fold change is relative to the start of treatments; data are mean ± SEM, n = 7; values are significantly different from vehicle: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( B ) Tumour weights measured at the end of the in vivo experiment; data are mean ± SEM, n = 7; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test. Representative images of the tumours treated with SLMP53-2 or vehicle at the end of the experiment. ( C ) Body weight of the mice registered during the course of the experiment. Data are mean ± SEM, n = 7; values are not significantly different from vehicle: p > 0.05, two-way ANOVA followed by Sidak’s test. ( D ) Weight of heart, spleen, kidney and livers from animals treated with SLMP53-2 or vehicle. Data are mean ± SEM, n = 7; values are not significantly different from vehicle: p > 0.05, two-way ANOVA followed by Sidak’s test. ( E ) Representative images of p53, Ki-67, BAX, BCL-2, TUNEL, β-catenin, Vimentin, and Slug detection in tumour tissues of A375 xenografts treated with SLMP53-2 or vehicle, collected at the end of treatment (scale bar = 5 μm; magnificatio n = 200×); haematoxylin and eosin (H&E). ( F – H ) Quantification of immunohistochemistry of A375 xenograft tumour tissues treated with SLMP53-2 or vehicle. In F, quantification of the number of Ki-67-positive and -negative cells; values are significantly different from vehicle: * p < 0.05, two-way ANOVA followed by Sidak’s test. In G, quantification of the percentage of positive-staining cells with TUNEL, n = 5; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test. In H, quantification of the p53, Vimentin, BAX, BCL-2, β-catenin and Slug staining by evaluation of 3,3′-diaminobenzidine (DAB) intensity is shown, n = 5; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: In Vivo, Activity Assay, Injection, TUNEL Assay, Immunohistochemistry, Staining