Journal: The American journal of pathology

Article Title: The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Is Critical for Corneal Endothelial Cell Protection and Graft Survival after Transplantation.

doi: 10.1016/j.ajpath.2021.10.016

Figure Lengend Snippet: Figure 1 Corneal endothelial cells (CEnC) express high levels of the receptor for a-MSH. A: Immunostaining for a-MSH receptor, melanocortin receptor 1 (MC1R), on murine (mCEnC) and human (hCEnC) CEnC lines and primary CEnCs. Blue: DAPI, purple: MC1R. B and C: Representative Western blot bands of MC1R in murine and human CEnC lines (B) and in primary murine corneal endothelium lysates (C). SIT lysate: small intestine tissue lysate used as positive control for mouse MC1R (mMC1R). A-375: human skin epithelial cell lysate used as positive control for human MC1R (hMC1R). Scale bars Z 50 mm.

Article Snippet: The mouse small intestine tissue (SIT) lysate (No. PK-AB718-1408; PromoCell, Heidelberg, Germany) and A375 human skin epithelial cell lysate (No. sc-3811; Santa Cruz Biotechnology, Dallas, TX) were used as positive controls for mouse and human MC1R, respectively.

Techniques: Immunostaining, Western Blot, Positive 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 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

Journal: Cells

Article Title: Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features

doi: 10.3390/cells10040769

Figure Lengend Snippet: Estimation of efficiency of silencing of TMSB4X expression in A375 cells. Stable clones with lowered TMSB4X expression were obtained using shRNA constructs. ( A ) qPCR analysis of TMSB4X expression level. As templates, cDNAs of the A375 scr and sh-Tβ4 clones served. The results were normalized against the HPRT1 gene and scr cells ( n = 3). The significance level was set at **** p < 0.0001. The graph indicates means ± SD. ( B ) RT-PCR analysis of TMSB4X expression level. 2% Tris-acetate-EDTA (TAE)-agarose gel was used to separate DNA fragments.

Article Snippet: The stable A375 cell clones were cultured in a medium with the addition of 1 µg/mL puromycin from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Expressing, Clone Assay, shRNA, Construct, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Cells

Article Title: Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features

doi: 10.3390/cells10040769

Figure Lengend Snippet: Characterization of WM1341D (low TMSB4X expression level) and A375 (high TMSB4X expression level) cells’ colony formation abilities in the soft agar colony formation assay and the effect of TMSB4X expression silencing on this parameter. ( A ) Representative phase-contrast images of colonies. White arrows indicate a single colony. ( B ) Quantitative analysis of the number of colonies ( n = 3^). ( C ) Analysis of colonies’ area ( n = 3^). ( D – F ) Analysis of colony formation ability of A375 control and sh-Tβ4 cells. ( D ) Representative phase-contrast images of colonies formed by the A375 clones. White arrow point at a single colony. ( E ) Quantitative analysis of colonies ( n = 3^). ( F ) Analysis of colonies’ area ( n = 3^). Measurements were performed with ImageJ software, which expressed results in arbitrary units (AU). The significance level was set at * p < 0.05, *** p < 0.001. Graphs indicate average-max-min values. ^The number of colonies formed and their area was calculated based on 21 microscopic pictures for every condition obtained from three separate experiments. The results presented separately for every clone are shown in .

Article Snippet: The stable A375 cell clones were cultured in a medium with the addition of 1 µg/mL puromycin from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Expressing, Soft Agar Assay, Clone Assay, Software

Journal: Cells

Article Title: Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features

doi: 10.3390/cells10040769

Figure Lengend Snippet: Analysis of cell proliferation rate and cell cycle progression in tested melanoma cells and evaluating the effect of TMSB4X expression silencing on these parameters. ( A , B ) Cell proliferation was measured using the BrdU assay 24, 48, and 72 h after seeding the cells ( n = 3): ( A ) WM1341D and A375 cells ( B ) scr and sh-Tβ4 clones. The graphs indicate the mean ± SD. The results presented separately for every clone are shown in . ( C – H ) Flow cytometric analysis of cell cycle with propidium iodide DNA staining ( n = 3). ( C – E ) Analysis of the cell cycle was performed on WM1341D, and A375 cells collected, respectively, 24, 48, and 72 h after seeding. ( F – H ) Analysis of the cell cycle was performed on scr and sh-Tβ4 cells collected 24, 48, and 72 h after seeding, respectively. The graphs indicate the mean ± SD. The results presented separately for every clone are shown in . The significance level was set at * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The stable A375 cell clones were cultured in a medium with the addition of 1 µg/mL puromycin from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Expressing, BrdU Staining, Clone Assay, Staining

Journal: Cells

Article Title: Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features

doi: 10.3390/cells10040769

Figure Lengend Snippet: The actin cytoskeleton and organization of Nestin and Vimentin filaments in WM1341D and A375 cells. ( A – D ) Immunocytochemical stainings to detect selected cytoskeleton components in WM1341D and A375 cells. The confocal microphotographs pictures for each cell were captured at three focal planes: cell contact area to the substratum (basal—green), a cross-section of the cell nucleus (mid-height—red), and nucleus apical outer surface (apical—blue). Finally, the photos were merged into a single image. ( A ) Detection of filamentous actin (F-actin). White arrows indicate stress fibers. ( B ) Visualization of monomeric actin (G-actin). Red arrows point at the denser aggregation of G-actin in nucleoli. ( C ) Detection of subcellular localization of Nestin. Yellow arrows indicate a ring-shaped localization around the nucleus. ( D ) Immunocytochemical staining to detect Vimentin. Blue arrows indicate a ring-shaped structure around the nucleus. Separated microphotographs with additional F-actin and cell nucleus visualization are presented in for G-actin Nestin- and Vimentin-staining, respectively. In the case of , population photos are presented. We quantified our observations and presented the data in the form of graphs indicating the mean ± SD. At least 30 cells from 3 population photos per condition were analyzed. The significance level was set at *** p < 0.001, and **** p < 0.0001.

Article Snippet: The stable A375 cell clones were cultured in a medium with the addition of 1 µg/mL puromycin from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Staining

Journal: Cells

Article Title: Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features

doi: 10.3390/cells10040769

Figure Lengend Snippet: Tβ4 influences melanoma cell morphology. ( A ) 2D visualization as a grayscale map of the cell surface of tested melanoma cells using the atomic force microscopy (AFM). Yellow arrows point at filamentous structures. ( B ) WM1341D, A375, scr, and sh-Tβ4 cells’ body height measurements by AFM. The data obtained from the height measurements were visualized by a heat map (height retrace) of the cell body. The black color corresponds to 0 μm, passes through various red and yellow shades, and the highest point of the tested object is marked with white. ( C ) Histograms present the difference in the height of the cells shown in A-B in correlation to a drawn line’s position across the cell. ( D ) Graphs are illustrating the maximal height of the cells ( n = 5–10). Graphs indicate average-max-min values. The significance level was set at ** p < 0.01. The results presented separately for every clone are shown in .

Article Snippet: The stable A375 cell clones were cultured in a medium with the addition of 1 µg/mL puromycin from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Microscopy

Journal: Cells

Article Title: Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features

doi: 10.3390/cells10040769

Figure Lengend Snippet: Analysis of the role of Tβ4 in the mechanical properties of melanoma cells. Single-cell force spectroscopy (SCFS) measurements were performed to calculate Young’s modulus for tested melanoma cells. For every cell, five curves were acquired. The average values of Young’s modulus are presented in the graphs: ( A ) for WM1341D and A375 cells ( n = 30) and ( B ) for scr ( n = 30) and sh-Tβ4 clones ( n = 30 for every clone). The significance level was set at **** p < 0.0001. Graphs indicate average-max-min values. The results presented separately for every clone are shown in .

Article Snippet: The stable A375 cell clones were cultured in a medium with the addition of 1 µg/mL puromycin from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Spectroscopy, Clone Assay

Journal: Cells

Article Title: Changes in Biomechanical Properties of A375 Cells Due to the Silencing of TMSB4X Expression Are Not Directly Correlated with Alterations in Their Stemness Features

doi: 10.3390/cells10040769

Figure Lengend Snippet: A summary of obtained results. Accordingly to the results obtained by us for the A375 cells with decreased TMSB4X expression, lowering Tβ4 level leads to reorganization of focal adhesion . The cells form more focal adhesion (FA) sites but of smaller size. That leads to more pronounced stress fibers and Vimentin and Nestin’s relocalization towards the perinuclear area, where “rings” of these intermediate filaments (IF) proteins are observed around the cell nucleus. Finally, the cells are more spread on the substratum and become stiffer. Changes in the cells’ biomechanical properties with lowered TMSB4X expression influence stemness features only in a part of cell clones.

Article Snippet: The stable A375 cell clones were cultured in a medium with the addition of 1 µg/mL puromycin from Santa Cruz Biotechnology Inc. (Heidelberg, Germany).

Techniques: Expressing, Clone Assay

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: A Downregulation of TGF-β1 and TGF-β2 after infection with adenoviruses expressing TGF-β shRNAs. a Schematic structure of adenoviral vectors expressing TGF-β shRNA. dl324-∆E1A-∆E1B-∆E3-IX-U6-NC (Ad-NC) is a replication-incompetent adenovirus used as the negative control. It contains the scrambled DNA sequence for shRNA, which is under the control of the U6 promoter. dl324-∆E1A-∆E1B∆E3-IX-U6-shTGF-β1 or -β2 (Ad-shTGF-β1 or -β2) is a replication-incompetent adenovirus expressing human TGF-β1 or -β2 shRNA. NC negative control. b Downregulation of human transforming growth factor β1 (TGF-β1) or β2 by adenovirus expressing shTGF-β1 or -β2. Human A375 and HPAC cells were infected with various MOIs of adenovirus expressing shRNA targeting human TGF-β1 (Ad-shTGF-β1) or human TGF-β2 (Ad-shTGF-β2) or scrambled RNA (Ad-NC). TGF-β1 or -β2 mRNA was assayed by quantitative real-time polymerase chain reaction (qRT-PCR), and c protein levels were assayed by enzyme-linked immunosorbent assay (ELISA). MOI multiplicity of infection, NC negative control. Error bars represent the standard error from three independent experiments. B Various cancer cell lines were treated with adenovirus expressing shTGF-β1 or -β2 for 48 h and incubated for an additional 14 days for clonogenic assays. The numbers indicate the relative ratio of clone numbers to those of Ad-NC. C Global transcriptome analysis of TGF-β downregulation. Expression values of differentially expressed genes are shown in heat map format. Gene expression levels are visualized as row standardized z scores ranging from green (−1) to red (+1) across all samples. The rows are organized by hierarchical clustering analysis with complete linkage and Euclidean distance as a measure of similarity from samples of A375 cells (NC, shTGF-β1, shTGF-β2) (Left) and HPAC cells (NC, shTGF-β1, shTGF-β2) (Right). NC, A375, and HPAC cells infected with adenovirus expressing nonsense shRNA as a negative control at 100 MOI; shTGF-β1 or -β2; A375 and HPAC cells infected with adenoviruexpressing TGF-β1 or -β2 shRNA at 100 MOI. D Effect of adenovirus expressing shTGF-β1 or shTGF-β2 in various cancer cell lines. Each cell line was treated with adenovirus expressing shTGF-β1 or shTGF-β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. E Pancreatic normal cell lines were treated with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. F ROS generation was induced by shTGF-β1- and shTGF-β2-expressing adenoviruses. A375 cells, HPAC cancer cells, and pancreatic normal cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively, after 48 h of incubation with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. Cell viability was tested via an MTS viability assay after 48 h of infection. Error bars represent the standard error from three independent experiments. p Values <0.01 indicate a very significantly different viability of Ad-shTGF-β1 or -β2 compared to the control (Ad-NC). G Effects of NAC treatment with adenovirus expressing TGF-β1 or -β2 shRNA in melanoma and pancreatic cancer cells. A375 (top) or HPAC (bottom) cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 6 h, infected cells were treated with NAC (10 mM) for 42 h and then incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. H A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2. After 6 h, infected cells were treated with NAC (10 mM) for 42 h, and then the expression levels of PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, JNK, Trx, and GAPDH were detected by western blot analysis

Article Snippet: After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, shRNA, Negative Control, Sequencing, Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Gene Expression, Western Blot, Microscopy, Viability Assay

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of NOX1, NOX4, 5-lipoxygenase, and GAPDH were detected by western blot analysis. b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or transfected with pCMV6-myr-Akt or infected with adenovirus expressing shTGF-β1 and subsequently transfected with pCMV6-myr-Akt. After 48 h, NOX4, phospho-Akt, Akt, and GAPDH were detected by western blot analysis. c Expression levels of NOX4 mRNA in A375 and HPAC cells were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). d A375 cells were infected with adenovirus expressing shTGF-β1. After 6 h, infected cells were treated with NAC (10 mM) for 42 h, and then the expression of NOX4 and GAPDH was detected by western blot analysis. e A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with p38 inhibitor (SB203580, 10 μM) or JNK inhibitor (SP600125, 10 μM) or both for 42 h. Then the expression of NOX4 and GAPDH was detected by western blot analysis. f A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by NAC (10 mM) or adenovirus expressing shTGF-β1 followed by p38 inhibitor (SB203580, 10 μM) or adenovirus expressing shTGF-β1 followed by transfection with pCMV6-myr-Akt or adenovirus expressing shTGF-β1 followed by transfection with siRNA of Smad4 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, the expression levels of NOX4 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). g A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of Smad4. After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). i A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by treatment with NOX4 inhibitor (GKT137831, 140 nM). After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right)

Article Snippet: After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Viability Assay, Incubation, Microscopy

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a ASK1 mediates TGF-β-induced cell death via p38/JNK activation. A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 (100 MOI) and subsequently transfected with siASK1. siASK1 transfection was based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested by an MTS viability assay. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with siASK1. After 48 h, morphological changes were observed using microscopy. c A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with siASK1. After 48 h, the expression levels of p-p38, p-Akt, p-HSP27, HSP27, p-ERK, p-src, p-p65, p-JNK, p-stat3, and GAPDH were detected by western blot analysis. d A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with ASK1 kinase mutant ASK1 (K709M). After 48 h, cell viability was tested with an MTS viability assay. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). e A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with ASK1 kinase mutant ASK1 (K709M). After 48 h, morphological changes were observed using microscopy. f A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with ASK1 kinase mutant ASK1 (K709M). After 48 h, the expression levels of p-p38, p-Akt, p-HSP27, HSP27, p-ERK, p-src, p-p65, p-JNK, p-stat3, and GAPDH were detected by western blot analysis. g A375 cells were infected with adenovirus expressing shTGF-β1 and subsequently transfected with siSmad4. After 48 h of incubation with DCF-DA (20 μM, 1 h), ROS were detected using a fluorescent reader and microscopy. h After 48 h, the expression of various ER stress markers in addition to survival-related molecules was detected by western blot analysis; A375 cells were infected with adenovirus expressing shTGF-β1 and subsequently transfected with pCMV6-myr-Akt. i After 48 h of incubation with DCF-DA (20 μM, 1 h), ROS were detected using a fluorescent reader and microscopy. j After 48 h, the expression levels of various ER stress markers and phospho-Akt and Akt were detected by western blot analysis. k After 48 h, the expression levels of PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, JNK, and GAPDH were detected by western blot analysis. l After 48 h, cell viability was tested via an MTS viability assay. Error bars represent the standard error from three independent experiments. Decreased p value to 0.041 from <0.001 after Akt activation followed by TGF-β1 downregulation means a significant decrease in cell death

Article Snippet: After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Activation Assay, Infection, Expressing, Transfection, Viability Assay, Control, Microscopy, Western Blot, Mutagenesis, Incubation

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of p-ASK1, ASK1, Trx, GSTM1 and GAPDH were detected by western blot analysis. b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of Trx and GSTM1 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). c A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, lysates were then subjected to immunoprecipitation with an anti‐Trx (Left) or anti‐GSTM1 (Right) antibody to identify changes in complex formation. The numbers indicate the relative band intensity to that of Ad-NC. d A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 6 h, infected cells were treated with NAC (10 mM) for 42 h. Then lysates were subjected to immunoprecipitation with an anti‐Trx (Left) or anti‐GSTM1 (Right) antibody to identify changes in complex formation. The numbers indicate the relative band intensity to that of Ad-NC after measuring band intensity using a densitometer

Article Snippet: After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a A375 and HPAC cell lines were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, Trx and GSTM1 promoter activities were analyzed by Chip assays using antibodies to AP-1 or Sp1. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of AP-1, Sp1 and GAPDH were detected by western blot analysis using whole-cell lysates. c p-Smad2, Smad2, p-Smad3, Smad3, Smad4, and GAPDH were detected by western blot analysis. d A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, lysates were then subjected to immunoprecipitation with an anti‐AP1 or anti-Sp1 antibody to identify changes in Smad4 in complex formation. e A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, Smad4 localization was examined by confocal immunofluorescence staining. Representative confocal immunofluorescence staining of each infected A375 was performed as described in the Materials and methods section to confirm the localization of Smad4. f A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, Smad4 localization was examined in nuclear or cytosolic fractionation using nuclear (histone 1) or cytosol (actin) markers. g A375 and HPAC cell lines were transfected with negative control siRNA or Smad4 siRNA. After 48 h, transfected cells were detected with GSTM1, Trx, Smad4, and GAPDH. h Effects of NAC on Trx and GSTM1 expression in melanoma and pancreatic cancer cells expressing shTGF-β1 or -β2 adenovirus. A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 6 h, infected cells were treated with NAC (10 mM) for 42 h. Then, p-Smad2, p-Smad3, Trx, GSTM1, and GAPDH were detected by western blot analysis. i A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 6 h, infected cells were treated with NAC (10 mM) for 42 h. Then the expression levels of Trx and GSTM1 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01)

Article Snippet: After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Control, Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Fractionation, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a A375 cells were infected with adenovirus expressing shTGF-β1 or -β2. After 48 h, various ER stress-related proteins were detected by western blot analysis. b A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then cell viability was tested using an MTS viability assay (left). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (** p < 0.01). Additionally, the expression levels of PARP and GAPDH were detected by western blot analysis (right). c A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then ER stress-related proteins that responded to TGF-β downregulation (Fig. ) were detected by western blot analysis. d A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h and then incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. e A375 cell lines were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then lysates were subjected to immunoprecipitation using an anti-Trx or anti-GSTM1 antibody to identify changes in ASK1. f A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then the expression levels of PARP, ASK1, p-ASK1, p-p38, p38, p-JNK, JNK, Trx, GSTM1, and GAPDH were detected by western blot analysis

Article Snippet: After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Western Blot, Viability Assay, Control, Incubation, Microscopy, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a BALB/c athymic nude mice were injected with 8 × 10 6 A375 or HPAC cells in 100 μL. When the tumors reached an average size of 60–80 mm 3 , the nude mice received intratumoral injections of 1 × 10 9 plaque-forming units (pfu) of various kinds of adenovirus (Ad-NC, Ad-shTGF-β1, or Ad-shTGF-β2) in 50 μL of PBS or PBS alone on days 1, 3, and 5. Tumor volume was monitored and recorded every 2 days until the end of the study. Values represent the mean ± SE (five animals per group) (top). Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). Overall survival was determined throughout a 31-day time course (bottom). b Representative immunohistochemical analysis of recombinant adenovirus-infected tumor sections was performed as follows. Three animals per group of BALB/c athymic nude mice were injected with 8 × 10 6 A375 or HPAC cells/100 μL and treated with intratumoral injections of 1 × 10 9 pfu/50 μL of various types of adenovirus (Ad-NC, Ad-shTGF-β1, or Ad-shTGF-β2) on days 1, 3, and 5. Tumors were collected on day 11 for histological analysis. Paraffin sections of tumor tissue were stained using anti-Hexon, anti-TGF-β1, and anti-TGF-β2 antibodies. c A TUNEL assay was performed on tissue sections to quantify apoptotic cell death, as described in the “Materials and methods” section. The percentage of TUNEL-positive cells was determined by counting the TUNEL-positive cells under 10 noncontinuous low-power fields. d TGF-β1 or -β2 downregulation can cause both NOX4-mediated ROS production and a reduction in Smad complexes (phospho-Smad2, 3 with Smad4) that translocate to the nucleus to bind to gene promoters for the expression of Trx/GSTM1. ROS triggered by decreased Akt activity can dissociate Trx or GSTM1 from ASK1–Trx and ASK1-GSTM1 complexes. ASK1 activation is also related to the reduction in Trx and GSTM1 gene expression, which results from decreased transcriptional activity of the Smad complex. In these ways, ASK1 was fully activated and induced cytotoxic tumor cell death via p38/JNK activation and induction of ER stress, which stimulated the escalation of ASK1 activation by completion of a sustained positive feedback loop circuit. Circled numbers indicate the chronological order of the signaling events leading to escalated cancer cell death

Article Snippet: After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Injection, Control, Immunohistochemical staining, Recombinant, Infection, Staining, TUNEL Assay, Expressing, Activity Assay, Activation Assay, Gene Expression

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: A Downregulation of TGF-β1 and TGF-β2 after infection with adenoviruses expressing TGF-β shRNAs. a Schematic structure of adenoviral vectors expressing TGF-β shRNA. dl324-∆E1A-∆E1B-∆E3-IX-U6-NC (Ad-NC) is a replication-incompetent adenovirus used as the negative control. It contains the scrambled DNA sequence for shRNA, which is under the control of the U6 promoter. dl324-∆E1A-∆E1B∆E3-IX-U6-shTGF-β1 or -β2 (Ad-shTGF-β1 or -β2) is a replication-incompetent adenovirus expressing human TGF-β1 or -β2 shRNA. NC negative control. b Downregulation of human transforming growth factor β1 (TGF-β1) or β2 by adenovirus expressing shTGF-β1 or -β2. Human A375 and HPAC cells were infected with various MOIs of adenovirus expressing shRNA targeting human TGF-β1 (Ad-shTGF-β1) or human TGF-β2 (Ad-shTGF-β2) or scrambled RNA (Ad-NC). TGF-β1 or -β2 mRNA was assayed by quantitative real-time polymerase chain reaction (qRT-PCR), and c protein levels were assayed by enzyme-linked immunosorbent assay (ELISA). MOI multiplicity of infection, NC negative control. Error bars represent the standard error from three independent experiments. B Various cancer cell lines were treated with adenovirus expressing shTGF-β1 or -β2 for 48 h and incubated for an additional 14 days for clonogenic assays. The numbers indicate the relative ratio of clone numbers to those of Ad-NC. C Global transcriptome analysis of TGF-β downregulation. Expression values of differentially expressed genes are shown in heat map format. Gene expression levels are visualized as row standardized z scores ranging from green (−1) to red (+1) across all samples. The rows are organized by hierarchical clustering analysis with complete linkage and Euclidean distance as a measure of similarity from samples of A375 cells (NC, shTGF-β1, shTGF-β2) (Left) and HPAC cells (NC, shTGF-β1, shTGF-β2) (Right). NC, A375, and HPAC cells infected with adenovirus expressing nonsense shRNA as a negative control at 100 MOI; shTGF-β1 or -β2; A375 and HPAC cells infected with adenoviruexpressing TGF-β1 or -β2 shRNA at 100 MOI. D Effect of adenovirus expressing shTGF-β1 or shTGF-β2 in various cancer cell lines. Each cell line was treated with adenovirus expressing shTGF-β1 or shTGF-β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. E Pancreatic normal cell lines were treated with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, the expression levels of p-p38, p38, p-HSP27, HSP27, p-ERK, ERK, p-JNK, JNK, and GAPDH were detected via western blot analysis. F ROS generation was induced by shTGF-β1- and shTGF-β2-expressing adenoviruses. A375 cells, HPAC cancer cells, and pancreatic normal cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively, after 48 h of incubation with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. Cell viability was tested via an MTS viability assay after 48 h of infection. Error bars represent the standard error from three independent experiments. p Values <0.01 indicate a very significantly different viability of Ad-shTGF-β1 or -β2 compared to the control (Ad-NC). G Effects of NAC treatment with adenovirus expressing TGF-β1 or -β2 shRNA in melanoma and pancreatic cancer cells. A375 (top) or HPAC (bottom) cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 6 h, infected cells were treated with NAC (10 mM) for 42 h and then incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. H A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2. After 6 h, infected cells were treated with NAC (10 mM) for 42 h, and then the expression levels of PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, JNK, Trx, and GAPDH were detected by western blot analysis

Article Snippet: After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, shRNA, Negative Control, Sequencing, Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Gene Expression, Western Blot, Microscopy, Viability Assay

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of NOX1, NOX4, 5-lipoxygenase, and GAPDH were detected by western blot analysis. b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or transfected with pCMV6-myr-Akt or infected with adenovirus expressing shTGF-β1 and subsequently transfected with pCMV6-myr-Akt. After 48 h, NOX4, phospho-Akt, Akt, and GAPDH were detected by western blot analysis. c Expression levels of NOX4 mRNA in A375 and HPAC cells were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). d A375 cells were infected with adenovirus expressing shTGF-β1. After 6 h, infected cells were treated with NAC (10 mM) for 42 h, and then the expression of NOX4 and GAPDH was detected by western blot analysis. e A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with p38 inhibitor (SB203580, 10 μM) or JNK inhibitor (SP600125, 10 μM) or both for 42 h. Then the expression of NOX4 and GAPDH was detected by western blot analysis. f A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by NAC (10 mM) or adenovirus expressing shTGF-β1 followed by p38 inhibitor (SB203580, 10 μM) or adenovirus expressing shTGF-β1 followed by transfection with pCMV6-myr-Akt or adenovirus expressing shTGF-β1 followed by transfection with siRNA of Smad4 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, the expression levels of NOX4 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). g A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of Smad4. After 48 h, NOX4 and GAPDH were detected by western blot analysis. h A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). i A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by treatment with NOX4 inhibitor (GKT137831, 140 nM). After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right)

Article Snippet: After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Viability Assay, Incubation, Microscopy

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a ASK1 mediates TGF-β-induced cell death via p38/JNK activation. A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 (100 MOI) and subsequently transfected with siASK1. siASK1 transfection was based on the siRNA transfection protocol (Santa Cruz, CA, USA). After 48 h, cell viability was tested by an MTS viability assay. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with siASK1. After 48 h, morphological changes were observed using microscopy. c A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with siASK1. After 48 h, the expression levels of p-p38, p-Akt, p-HSP27, HSP27, p-ERK, p-src, p-p65, p-JNK, p-stat3, and GAPDH were detected by western blot analysis. d A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with ASK1 kinase mutant ASK1 (K709M). After 48 h, cell viability was tested with an MTS viability assay. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). e A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with ASK1 kinase mutant ASK1 (K709M). After 48 h, morphological changes were observed using microscopy. f A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 and subsequently transfected with ASK1 kinase mutant ASK1 (K709M). After 48 h, the expression levels of p-p38, p-Akt, p-HSP27, HSP27, p-ERK, p-src, p-p65, p-JNK, p-stat3, and GAPDH were detected by western blot analysis. g A375 cells were infected with adenovirus expressing shTGF-β1 and subsequently transfected with siSmad4. After 48 h of incubation with DCF-DA (20 μM, 1 h), ROS were detected using a fluorescent reader and microscopy. h After 48 h, the expression of various ER stress markers in addition to survival-related molecules was detected by western blot analysis; A375 cells were infected with adenovirus expressing shTGF-β1 and subsequently transfected with pCMV6-myr-Akt. i After 48 h of incubation with DCF-DA (20 μM, 1 h), ROS were detected using a fluorescent reader and microscopy. j After 48 h, the expression levels of various ER stress markers and phospho-Akt and Akt were detected by western blot analysis. k After 48 h, the expression levels of PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, JNK, and GAPDH were detected by western blot analysis. l After 48 h, cell viability was tested via an MTS viability assay. Error bars represent the standard error from three independent experiments. Decreased p value to 0.041 from <0.001 after Akt activation followed by TGF-β1 downregulation means a significant decrease in cell death

Article Snippet: After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Activation Assay, Infection, Expressing, Transfection, Viability Assay, Control, Microscopy, Western Blot, Mutagenesis, Incubation

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of p-ASK1, ASK1, Trx, GSTM1 and GAPDH were detected by western blot analysis. b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of Trx and GSTM1 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). c A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, lysates were then subjected to immunoprecipitation with an anti‐Trx (Left) or anti‐GSTM1 (Right) antibody to identify changes in complex formation. The numbers indicate the relative band intensity to that of Ad-NC. d A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 6 h, infected cells were treated with NAC (10 mM) for 42 h. Then lysates were subjected to immunoprecipitation with an anti‐Trx (Left) or anti‐GSTM1 (Right) antibody to identify changes in complex formation. The numbers indicate the relative band intensity to that of Ad-NC after measuring band intensity using a densitometer

Article Snippet: After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a A375 and HPAC cell lines were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, Trx and GSTM1 promoter activities were analyzed by Chip assays using antibodies to AP-1 or Sp1. Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). b A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, the expression levels of AP-1, Sp1 and GAPDH were detected by western blot analysis using whole-cell lysates. c p-Smad2, Smad2, p-Smad3, Smad3, Smad4, and GAPDH were detected by western blot analysis. d A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, lysates were then subjected to immunoprecipitation with an anti‐AP1 or anti-Sp1 antibody to identify changes in Smad4 in complex formation. e A375 cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI. After 48 h, Smad4 localization was examined by confocal immunofluorescence staining. Representative confocal immunofluorescence staining of each infected A375 was performed as described in the Materials and methods section to confirm the localization of Smad4. f A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 48 h, Smad4 localization was examined in nuclear or cytosolic fractionation using nuclear (histone 1) or cytosol (actin) markers. g A375 and HPAC cell lines were transfected with negative control siRNA or Smad4 siRNA. After 48 h, transfected cells were detected with GSTM1, Trx, Smad4, and GAPDH. h Effects of NAC on Trx and GSTM1 expression in melanoma and pancreatic cancer cells expressing shTGF-β1 or -β2 adenovirus. A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 6 h, infected cells were treated with NAC (10 mM) for 42 h. Then, p-Smad2, p-Smad3, Trx, GSTM1, and GAPDH were detected by western blot analysis. i A375 and HPAC cells were infected with adenovirus expressing shTGF-β1 or -β2 at 100 MOI, respectively. After 6 h, infected cells were treated with NAC (10 mM) for 42 h. Then the expression levels of Trx and GSTM1 mRNA were assayed by quantitative real-time polymerase chain reaction (qRT-PCR). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01)

Article Snippet: After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Control, Western Blot, Immunoprecipitation, Immunofluorescence, Staining, Fractionation, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a A375 cells were infected with adenovirus expressing shTGF-β1 or -β2. After 48 h, various ER stress-related proteins were detected by western blot analysis. b A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then cell viability was tested using an MTS viability assay (left). Error bars represent the standard error from three independent experiments. Asterisks indicate a significant difference compared to each given control (** p < 0.01). Additionally, the expression levels of PARP and GAPDH were detected by western blot analysis (right). c A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then ER stress-related proteins that responded to TGF-β downregulation (Fig. ) were detected by western blot analysis. d A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h and then incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy. e A375 cell lines were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then lysates were subjected to immunoprecipitation using an anti-Trx or anti-GSTM1 antibody to identify changes in ASK1. f A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI, and after 6 h, infected cells were treated with a JNK inhibitor (SP600125, 10 μM) or a p38 inhibitor (SB203580, 10 μM) for 42 h. Then the expression levels of PARP, ASK1, p-ASK1, p-p38, p38, p-JNK, JNK, Trx, GSTM1, and GAPDH were detected by western blot analysis

Article Snippet: After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Infection, Expressing, Western Blot, Viability Assay, Control, Incubation, Microscopy, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: TGF-β downregulation-induced cancer cell death is finely regulated by the SAPK signaling cascade

doi: 10.1038/s12276-018-0189-8

Figure Lengend Snippet: a BALB/c athymic nude mice were injected with 8 × 10 6 A375 or HPAC cells in 100 μL. When the tumors reached an average size of 60–80 mm 3 , the nude mice received intratumoral injections of 1 × 10 9 plaque-forming units (pfu) of various kinds of adenovirus (Ad-NC, Ad-shTGF-β1, or Ad-shTGF-β2) in 50 μL of PBS or PBS alone on days 1, 3, and 5. Tumor volume was monitored and recorded every 2 days until the end of the study. Values represent the mean ± SE (five animals per group) (top). Asterisks indicate a significant difference compared to each given control (* p < 0.05; ** p < 0.01). Overall survival was determined throughout a 31-day time course (bottom). b Representative immunohistochemical analysis of recombinant adenovirus-infected tumor sections was performed as follows. Three animals per group of BALB/c athymic nude mice were injected with 8 × 10 6 A375 or HPAC cells/100 μL and treated with intratumoral injections of 1 × 10 9 pfu/50 μL of various types of adenovirus (Ad-NC, Ad-shTGF-β1, or Ad-shTGF-β2) on days 1, 3, and 5. Tumors were collected on day 11 for histological analysis. Paraffin sections of tumor tissue were stained using anti-Hexon, anti-TGF-β1, and anti-TGF-β2 antibodies. c A TUNEL assay was performed on tissue sections to quantify apoptotic cell death, as described in the “Materials and methods” section. The percentage of TUNEL-positive cells was determined by counting the TUNEL-positive cells under 10 noncontinuous low-power fields. d TGF-β1 or -β2 downregulation can cause both NOX4-mediated ROS production and a reduction in Smad complexes (phospho-Smad2, 3 with Smad4) that translocate to the nucleus to bind to gene promoters for the expression of Trx/GSTM1. ROS triggered by decreased Akt activity can dissociate Trx or GSTM1 from ASK1–Trx and ASK1-GSTM1 complexes. ASK1 activation is also related to the reduction in Trx and GSTM1 gene expression, which results from decreased transcriptional activity of the Smad complex. In these ways, ASK1 was fully activated and induced cytotoxic tumor cell death via p38/JNK activation and induction of ER stress, which stimulated the escalation of ASK1 activation by completion of a sustained positive feedback loop circuit. Circled numbers indicate the chronological order of the signaling events leading to escalated cancer cell death

Article Snippet: After 48 h, cell viability was tested via an MTS viability assay (upper left), or cells were incubated with DCF-DA (20 μM, 1 h) for the detection of ROS using a fluorescent reader and microscopy (upper right, lower left) or the expression levels of various ER stress markers; in addition, PARP, p-ASK1, ASK1, p-p38, p38, p-JNK, and JNK were detected by western blot analysis (right). j A375 cells were infected with adenovirus expressing shTGF-β1 at 100 MOI or adenovirus expressing shTGF-β1 followed by transfection with siRNA of NOX4 or NOX1 based on the siRNA transfection protocol (Santa Cruz, CA, USA).

Techniques: Injection, Control, Immunohistochemical staining, Recombinant, Infection, Staining, TUNEL Assay, Expressing, Activity Assay, Activation Assay, Gene Expression