Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Lentiviral vector mediated delivery and expression of sTNFR-Fc . (A) Schematic maps of the lentiviral transfer plasmids, pHR-hTNFR-Fc-eGFP and pHR-Fc-eGFP. LTR: long terminal repeat; ψ: packaging signal; SA, SD: splice donor, splice acceptor; CMV: cytomegalovirus promoter; hTNFR-Fc: codon-optimized gene encoding the extracellular domain of the human TNF receptor type 2, fused to the hinge domain from IgG1 and the Fc domain from human IgG3; Fc: hinge domain from IgG1 and the Fc domain from human IgG3; IRES: internal ribosome entry site; GFP: green fluorescent protein.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Plasmid Preparation, Expressing

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Stable and high level secretion of sTNFR-Fc in transduced cells . (A) Immunoblot detection of sTNFR-Fc protein in transfected cells. Mock = supernatant from mock transfected 293T cells; Intracellular = transfected cell lysate; and Secreted = supernatant from the transfected 293T cells. (B) Immunoblot detection of sTNFR-Fc protein in transduced cells intracellularly (cell lysate) and extracellularly (supernatant). HTB-T = transduced HTB-11 cells; CHME-T2 = CHME-5 cells transduced twice with the vector; CHME-T1 = CHME-5 cells transduced once with the vector. (C) Stable expression of sTNFR-Fc protein in conditioned medium of transduced cells. Expression of sTNFR-Fc in supernatants from transduced cells was measured by ELISA; results shown at different cell passages represent mean values from three independent experiments and error bars denote the standard deviation. (D) Stable expression of GFP in lentivirus vector-transduced cells. Transduced cells were passed in vitro and the percentage of GFP positive cells was determined every 5 passages by calculating the percentage of GFP+ cells within the culture using an inverted fluorescent microscope (Nikon Eclipse TE2000-U) with a digital camera attachment. Data presented here represent mean values from at least three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation, In Vitro, Microscopy

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Specific binding of expressed sTNFR-Fc to TNF-α by dot immunoblot assay . One microgram of standard recombinant human TNF-α protein was spotted onto nitrocellular membrane (NCM) and then blocked and incubated with supernatants from normal and transduced cells. After three washes with TBST, a goat-anti-human IgG Fc-HRP conjugate was applied and incubated at RT for 1 h. Specific binding was visualized by color deposition on the NCM following incubation with diaminobenzidine (DAB) substrate. Lanes: HTB-NT = supernatant from non-transduced HTB-11 cells and CHME-NT = supernatant from non-transduced CHME-5 cells used as negative controls; HTB-T = supernatant collected from transduced HTB-11 cells and CHME-T2 = supernatant from transduced CHME-5 cells; and C + = purified recombinant sTNFR-Fc protein as a positive control.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Binding Assay, Western Blot, Recombinant, Membrane, Incubation, Purification, Positive Control

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: Functional antagonization of sTNFR-Fc against TNF-α . As described in materials and methods, TNF-α sensitive L929 cells were treated with TNF-α alone (80 ng/mL) or with TNF-α plus culture supernatants from vector-transduced cells; purified recombinant sTNFR-Fc protein (160 ng/mL) was used as a positive control. After incubation for 24 h, cell viability was then evaluated by MTT assay. Viability was significantly higher for the cells treated with conditioned medium from transduced cells expressing hTNFR-Fc (** p < 0.01 for HTB-T; *p < 0.05 for CHME-T) when compared to cultures that received TNF-α alone, or TNF-α plus culture supernatants from parental cells (CHME-N and HTB-N). Results shown represent mean levels of three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Functional Assay, Plasmid Preparation, Purification, Recombinant, Positive Control, Incubation, MTT Assay, Expressing, Standard Deviation

Journal: Journal of Neuroinflammation

Article Title: Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS

doi: 10.1186/1742-2094-8-48

Figure Lengend Snippet: sTNFR-Fc mediated protection of neuronal cells from TNF-α, HIV-Tat and gp120 . (A) Non-transduced human neuronal cells, HTB-11, were treated with TNF-α alone (80 ng/mL), or with TNF-α plus culture supernatants (1:10 dilution) from vector-transduced cells (as indicated). Cells were incubated at 37°C for 3 days, and viability of the cells was determined by the trypan blue exclusion assay. sTNFR-Fc = supernatant collected from HTB-11 or CHME-5 cells transduced with the hTNFR-Fc encoding vector; Fc = supernatant collected from HTB-11 or CHME-5 cells transduced with the vector encoding human Fc only; rTNFR = purified commercial recombinant TNFR-Fc protein (160 ng/mL), used as a positive control. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (B) Vector transduced human HTB-11 cells were exposed to TNF-α as described in 7A. HTB-11 cells transduced either with the sTNFR-Fc or Fc encoding vector were used in this experiment, along with non-transduced cells as controls. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (C) sTNFR-Fc protects primary rat neurons against HIV-1 Tat-mediated toxicity. Medium from vector-transduced or parental HTB-11 cells was collected at day 10, diluted and mixed with 500 nM HIV-1 Tat protein, prior to addition to primary rat neurons. Following 24 hours incubation at 37°C, these test cultures along with the control were analyzed by TUNEL assay. Cell death was significantly reduced in neuronal cultures that were treated with Tat plus conditioned medium from HTB-11 cells transduced with the sTNRF-Fc encoding lentiviral vector (HTB-T 10d) versus cells treated with Tat plus conditioned medium from parental HTB-11 cells (HTB 10d) (*p < 0.01). NT, normal neuronal cells received no Tat, and -, neuronal cells exposed to Tat as a positive control. Results shown represent mean levels of three independent experiments and error bars denote the standard deviation. (D) sTNFR-Fc mediated neuronal protection of human HTB-11 cells against HIV-1 gp120 toxicity. Conditioned media from hTNFR-Fc or Fc vector transduced HTB-11 cells were collected, diluted and mixed with 100 ng/mL (gp120A) or 250 ng/mL (gp120B) HIV-1 gp120 protein, with or without HIV-1 Tat, prior to addition to HTB-11 cells. Following 3 days incubation at 37°C, cell viability of these cultures, together with control cultures, was determined by trypan blue exclusion assay (***p < 0.001). Results shown represent mean levels of three independent experiments and error bars denote the standard deviation.

Article Snippet: The quantitation of sTNFR-Fc protein was based on the optical density values at 450 nm, compared with a standard curve of purified human sTNFR-Fc protein (R&D Systems, Recombinant Human TNF RII/TNFRSF1B/Fc Chimera), using an ELISA reader (Beckman Coulter AD340).

Techniques: Plasmid Preparation, Incubation, Trypan Blue Exclusion Assay, Transduction, Purification, Recombinant, Positive Control, Standard Deviation, TUNEL Assay

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Luciferase gene expression results of polyplexes formulated with pDNA and each of the five different polymers in HeLa cells. Luciferase expression was measured 48 hours after transfection; all data are normalized to the cells only control. Cells were transfected with JetPEI (PEI) at an N/P ratio of 5. All other polymers were used at an N/P ratio of 20. Statistical analysis for expression efficiency was performed using the Tukey-Kramer HSD method on the log of the data (n = 6). Bars with different letters are statistically significant from each other (p < 0.05). For the cell viability data, T443 is significantly lower than A442 and PEI (p < 0.05, denoted with similar asterisks), and PEI, T443 and A442 were all statistically significant from the cells only and DNA only controls (p < 0.05) according to the Tukey-Kramer HSD method (n = 6).

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Luciferase, Gene Expression, Expressing, Transfection, Control

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: (a) Mitochondrial membrane potential measured by DiIC(1)5 fluorescence normalized to the cells only control and (b) phosphatidylserine (PS) exposure measured by Annexin V binding assay in HeLa cells transfected with polyplexes formed using PEI, T4, and A442. The data in (b) is presented as the percent increase in PS exposure compared to the cells only control. Letters represent statistical analysis; bars with different letters are statistically significant (p < 0.05) according to the Tukey-Kramer HSD method (n = 3).

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Membrane, Fluorescence, Control, Binding Assay, Transfection

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Confocal microscopy images of HeLa cells 30 minutes after being transfected with polyplexes formed with Cy5-labeled pDNA and the following polymers: (a) pDNA only; (b) JetPEI (PEI); (c) T46; (d) T412; (e) T443; (f) A442. Colors represent the following; green = WGA AF488-labeled cytosol; blue = DAPI-labeled nucleus; magenta = Cy5-labeled pDNA. Scale bar = 20 µm. Arrows denote sites of polyplex internalization. (g) Manders coefficients between each polyplex type (formed with Cy5-pDNA) and AlexaFluor-488-conjugated Wheat Germ Agglutinin-labeled plasma membrane. Bars with different letters are statistically significant from each other (p < 0.05) according to the Tukey-Kramer HSD method (n = 3).

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Confocal Microscopy, Transfection, Labeling, Clinical Proteomics, Membrane

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Measurement of propidium iodide fluorescence in HeLa cells after transfection with each polyplexes type at time points of 30 mins and 4 hours as measured via flow cytometry. Data is presented as the percent increase in PI positive cells as normalized to the cells only control.

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Fluorescence, Transfection, Flow Cytometry, Control

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Confocal images of HeLa cells fixed 4 hours after transfection. Cells were transfected with the following: (a) DNA only; (b) JetPEI (PEI); (c) T46; (d) T412; (e) T443; (f) A442. Colors represent the following; blue = nucleus; green = nuclear lamin; magenta = pDNA. Scale bar = 20 µm. White arrows indicate sites of deep nuclear envelope indentation.

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Transfection

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Confocal microscopy images of the nuclei in whole HeLa cells transfected with polyplexes formed with Cy5-pDNA and (a) JetPEI (PEI); (b) T46; (c) T412; (d) T443; (e) A442. The nucleus is shown as blue, nuclear lamin is shown as green, and the polyplexes are shown as magenta. Scale bar = 5 µm.

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Confocal Microscopy, Transfection

Journal: Molecular pharmaceutics

Article Title: Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

doi: 10.1021/mp200368p

Figure Lengend Snippet: Trypan blue exclusion assay performed on nuclei treated with polyplexes for 4 hours. Nuclei were isolated from HeLa cells and treated with (a) pDNA only; (b) JetPEI (PEI); (c) T46; (d) T412; (e) T443; or (f) A442. Scale bar = 400 µm. (g) Nuclei were counted and analyzed for pixel intensity using ImageJ and the statistics were calculated using JMP. Roughly 300 nuclei were analyzed for each polyplex, and the Tukey-Kramer HSD test reveal each set of data points to be statistically significant from one another.

Article Snippet: Human cervix adenocarcinoma (HeLa) cells were purchased from ATCC (Rockville, MD).

Techniques: Trypan Blue Exclusion Assay, Isolation

Journal: PLoS ONE

Article Title: JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

doi: 10.1371/journal.pone.0075965

Figure Lengend Snippet: (A) Time course assay was performed on BC3 and BCBL1 cells treated with 20 nM of bortezomib (BZ) for 0, 4, 8, 16 hrs. Total cell lysates were prepared and immunoblotted with anti-BiP, anti-CHOP, anti IRE1α, anti-phospho-JNK (p-JNK 1/2) and t-JNK antibodies. Anti-tubulin was assayed as protein loading control. (B) Electron microscopy (EM) analysis was performed on BC3 and BCBL1 cells treated with Bortezomib (20 nM) for 16 hrs (BZ). Cells showed several signs of ER stress compared to untreated control (CT). More apoptotic features such as nuclear (N) condensation were observed in BCBL1. Er (Endoplasmic reticulum), G (Golgi Apparatus), N (Nucleus), Au (Autophagosomes), Ne (Nuclear Envelope), M (Mitochondria). Bars: 1 µm. Results are representative of three independent experiments.

Article Snippet: The BC3, BCBL1, JSC1 and BC1 PEL cell lines (ATCC) were cultured in RPMI 1640 10% Fetal Calf Serum (FBS) (Euroclone), glutamine and streptomycin (100 μg/ml) and penicillin (100U/ml) in 5% CO 2 at 37°C.

Techniques: Control, Electron Microscopy

Journal: PLoS ONE

Article Title: JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

doi: 10.1371/journal.pone.0075965

Figure Lengend Snippet: (A) Electron microscopy (EM) on BC3 and BCBL1 cells treated with 20 nM of bortezomib after 16 hrs shows pronounced cell vacuolization (A a, a 4 ) with autophagosome membrane-bound compartment in different stage of maturation (A a 1, a 2 , a 4, a 5 ). Small intracellular structures consistent with aggresomes were evident in BCLB1 cells (A a 6 ). N (Nucleus), Au (Autophagosomes), M (Mitochondria), ag (aggresomes). Bars 1 µm. Results are representative of three independent experiments. (B) PEL cells were treated with bortezomib (20 nM), bafilomycin A (Baf) (10 nM) or combination of both. The western blot analysis was performed to detect the two isoforms of LC3 protein. The histograms indicate LC3-II/I and LC3-II/Actin ratio based on densitometric analysis (mean ± the standard deviation, n = 3 experiments). An antibody against β-actin was used as loading control.

Article Snippet: The BC3, BCBL1, JSC1 and BC1 PEL cell lines (ATCC) were cultured in RPMI 1640 10% Fetal Calf Serum (FBS) (Euroclone), glutamine and streptomycin (100 μg/ml) and penicillin (100U/ml) in 5% CO 2 at 37°C.

Techniques: Electron Microscopy, Membrane, Western Blot, Standard Deviation, Control

Journal: PLoS ONE

Article Title: JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

doi: 10.1371/journal.pone.0075965

Figure Lengend Snippet: (A) BC3 and BCBL1 cells were treated with bortezomib (20 nM for 16 hrs) with or without pre-treatment with JNK inhibitor (SP600125) (20 µM). Total cell lysates were prepared and immunoblotted with the following antibodies: anti-LC3, anti-pJNK, anti-T-JNK and anti-p62. Anti-β-actin was used as loading control. The histograms indicate LC3-II/I and LC3-II/Actin ratio based on densitometric analysis (mean ± the standard deviation, n = 3 experiments). (B) BC3 cells were transfected with DN-JNK expression vector or with an control empty vector (CV) and after 16 hrs treated with bortezomib (20 nM) for an additional 16 hrs. Total cell lysates were prepared and immunoblotted with anti-LC3 and p62 antibodies. β-actin was used as internal control. The histograms indicate LC3-II/I and LC3-II/Actin ratio based on densitometric analysis (mean ± the standard deviation, n = 3 experiments). (C) BC3 and BCBL1 cells were treated with Bortezomib (20 nM) alone or in combination with JNK inhibitor (SP600125) (20 µM) or ERK inhibitor (PD98059) (10 µM) for 16 hrs. A western blotting was performed using the following antibodies: anti-pBcl2(S70) and anti-total Bcl2. β-actin was used as loading control.

Article Snippet: The BC3, BCBL1, JSC1 and BC1 PEL cell lines (ATCC) were cultured in RPMI 1640 10% Fetal Calf Serum (FBS) (Euroclone), glutamine and streptomycin (100 μg/ml) and penicillin (100U/ml) in 5% CO 2 at 37°C.

Techniques: Control, Standard Deviation, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: JNK and Macroautophagy Activation by Bortezomib Has a Pro-Survival Effect in Primary Effusion Lymphoma Cells

doi: 10.1371/journal.pone.0075965

Figure Lengend Snippet: (A) Viability assay evaluated by trypan blue exclusion was performed in BC3 (left panel) and BCBL1 (right panel) cells treated with bortezomib (20 nM) or 3-MA (5 mM) alone or in combination for 16 hrs. Mean ± the standard deviation was also indicated (n = 3 experiments). * p-value = 0.05, ** p-value = 0.02. Western blotting analysis was performed on BC3 cells to evaluate the expression of cleaved (cl) PARP p85 fragment (middle panel). β-actin was used as loading control. (B) BC3 and BCBL1 cells were transfected with ATG5 siRNA or scramble siRNA (siRNASc), and than a western blot was performed with the anti-ATG5 antibody. β-actin was used as loading control. Viability assay evaluated by trypan blue exclusion was performed in BC3 and BCBL1 cells ATG5 or scramble-knocked down upon bortezomib treatment (20 nM) for 16 hrs. Mean ± the standard deviation was indicated (n = 3 experiments). ♦ p-value = 0.02, ♦♦ p-value = 0.03. Western blotting analysis was also performed on BC3 cells to evaluate the expression of PARP p85 fragment (cl PARP) and β-actin was used as internal control. (C) Cells viability assay on BC3 and BCBL1 cells treated with bortezomib (20 nM) and SP600125 (20 µM) alone or in combination for 16 hrs. The percentage of live cells was evaluated by trypan blue exclusion assay. Mean ± the standard deviation was indicated (n = 3 experiments). p-value = 0.01, p-value = 0.01. Western blotting analysis was performed on BC3 cells to evaluate the expression of PARP p85 fragment (cl PARP) and β-actin was used as loading control.

Article Snippet: The BC3, BCBL1, JSC1 and BC1 PEL cell lines (ATCC) were cultured in RPMI 1640 10% Fetal Calf Serum (FBS) (Euroclone), glutamine and streptomycin (100 μg/ml) and penicillin (100U/ml) in 5% CO 2 at 37°C.

Techniques: Viability Assay, Standard Deviation, Western Blot, Expressing, Control, Transfection, Trypan Blue Exclusion Assay

Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: GSKJ4 and A-485 treatments modulate Notch receptors expression and activity. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (A) TALL-1 or (C) MOLT3 cells treated for 48 h with 2 μM GSKJ4 or with DMSO. (B) Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panel) and HA and β-actin protein levels (lower panel) in TALL-1 cells transfected with HA-tagged EZH2 expression vector (HA-EZH2) or with the empty control vector. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (D) TALL-1 or (E) MOLT3 cells treated for 48 h with 5 μM A-485 or DMSO. Data represent mean values of three biological replicates ± Standard Error of the Mean (S.E.M.); ( n = 3) * P < 0.05, ** P < 0.01, *** P < 0.001. Uncropped western blots related to this figure are displayed in .

Article Snippet: To inhibit enzymatic activity of JMJD3 or of p300, cells were exposed to 2 μM GSK J4 HCL (GSKJ4) (S7070; Selleckchem, Houston, TX, USA) or to 5 μM A-485 (HY-107455; MedChemExpress, Monmouth Junction, NJ, USA ) for times indicated in figures.

Techniques: Expressing, Activity Assay, Gene Expression, Transfection, Plasmid Preparation, Control, Western Blot

Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: Enforced Notch/c-Myc axis partially shields from inhibitory effects of GSKJ4 and A-485 on T-ALL cell viability. Change in number of viable cells after 48 h of exposure to 2 μM GSKJ4 or to 5 μM A-485 in DND41 cells transduced with the mICN1 or with the empty control retroviruses ( A , upper panel), in DND41 cells transiently transfected with hICN3 or with the empty vector ( B , upper panel), and in TALL-1 cells transfected with c-Myc or with the control plasmid ( C , upper panel). Numbers of viable cells were calculated via trypan blue exclusion assay. Relative cell number reported in charts represents the mean of at least four biological replicates normalized to cell number in DMSO-treated cells transduced or transfected with empty control ± Standard Error of the Mean (S.E.M.); * P < 0.05, ** P < 0.01. Western blot analysis by using antibodies against: Notch1 in mICN1 transduced DND41 cells ( A , lower panel), Notch3 in hICN3 transfected DND41 cells ( B , lower panel) and c-Myc in TALL-1 cells transfected with the c-Myc expression vector ( C , lower panel). β-actin was used as loading control. Non-specific bands are indicated with asterisks. Uncropped western blots related to this figure are displayed in .

Article Snippet: To inhibit enzymatic activity of JMJD3 or of p300, cells were exposed to 2 μM GSK J4 HCL (GSKJ4) (S7070; Selleckchem, Houston, TX, USA) or to 5 μM A-485 (HY-107455; MedChemExpress, Monmouth Junction, NJ, USA ) for times indicated in figures.

Techniques: Transduction, Control, Transfection, Plasmid Preparation, Trypan Blue Exclusion Assay, Western Blot, Expressing

Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: GSKJ4 and A-485 treatments inhibit viability in T-ALL cell lines. Cell viability measurement by MTS assays (A,C) and protein expression levels of p27, cleaved, and full-length form of PARP (C PARP and FL PARP, respectively) and c-Myc (B,D) in TALL-1, MOLT3, P12, DND41, and KOPTK1 cells treated for times indicated in figure with 2 μM of GSKJ4 (A,B) or with 5 μM of A-485 (C,D) . β-actin was used as loading control. Non-specific bands are indicated with asterisks. Cell viability at each time point represents the mean of three biological replicates ± Standard Error of the Mean (S.E.M.); ( n = 3) * P < 0.05, *** P < 0.001. Uncropped western blots and additional exposures of films related to this figure are displayed in .

Article Snippet: To inhibit enzymatic activity of JMJD3 or of p300, cells were exposed to 2 μM GSK J4 HCL (GSKJ4) (S7070; Selleckchem, Houston, TX, USA) or to 5 μM A-485 (HY-107455; MedChemExpress, Monmouth Junction, NJ, USA ) for times indicated in figures.

Techniques: Expressing, Control, Western Blot

Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: The histone modifiers JMJD3 and p300 sustain Notch signaling and cell viability in T-ALL . (A) Notch signaling trans-activation is primed by the binding between a Notch receptor (N3R or N1R) and a ligand expressed on the surface of two neighboring cells. Ligand interaction allows two consecutive proteolytic cleavages of the receptor by ADAM metalloproteases and by the gamma-secretase complex that releases the active Notch intracellular domain (N3ICD or N1ICD) from the cell membrane leading to its nuclear translocation. In the nucleus, N3ICD or N1ICD associates with the DNA binding protein CBF1–Suppressor of Hairless(SuH)–LAG1 (CSL) and assembles a multifactorial transcriptional complex including MAML1 complex (MAM) and p300 to promote Notch target gene expression. By our study, we demonstrated that the binding of N3ICD, as well as N1ICD, to the regulatory region of NOTCH3 , is strictly associated with high levels of accumulation of the active chromatin marks H3K27ac and H3K4me3 on the NOTCH3 gene locus and combines with the co-recruitment of the histone modifiers p300 and JMJD3. Moreover, we found that the interplay between Notch and these histone modifiers is required to sustain NOTCH3 expression in T-ALL cells. (B) Indeed, pharmacological inhibition of the last step of Notch signaling activation, by the gamma-secretase inhibitor DAPT, restrains the binding of JMJD3 and p300 to the NOTCH3 gene, allows H3K27me3 accumulation and counteracts NOTCH3 expression. (C) Therefore, JMJD3 and p300 sustain the expression and the oncogenic transcriptional activity of Notch3 and Notch1 receptors in T-ALL cells. (D) Accordingly, the pharmacological inhibition of their enzymatic activity by treatment with GSKJ4 and A-485, respectively produced the following outcomes: (i) reduced NOTCH3 and NOTCH1 levels; (ii) decreased the expression of Notch target genes DELTEX1 and c-MYC; (iii) promotes the accumulation of the cell cycle-regulator factors p27 Kip1 and of the apoptosis-related cleaved form of PARP (C PARP); (ìv) impairs viability in several T-ALL cell lines.

Article Snippet: To inhibit enzymatic activity of JMJD3 or of p300, cells were exposed to 2 μM GSK J4 HCL (GSKJ4) (S7070; Selleckchem, Houston, TX, USA) or to 5 μM A-485 (HY-107455; MedChemExpress, Monmouth Junction, NJ, USA ) for times indicated in figures.

Techniques: Activation Assay, Binding Assay, Membrane, Translocation Assay, Targeted Gene Expression, Expressing, Inhibition, Activity Assay, Produced

Journal: International journal of experimental pathology

Article Title: MMP7 is a target of the tumour-associated antigen EpCAM.

doi: 10.1111/j.1365-2613.2012.00826.x

Figure Lengend Snippet: Figure 1 MMP7 expression correlates with EpCAM. (a) FaDu hypopharynx carcinoma cells were transiently transfected with a control siRNA or an EpCAM-specific siRNA. Expression levels of EpCAM were assessed upon flow cytometry with specific antibodies (HO-3) in both samples. Control transfectants are represented with a solid line and EpCAM siRNA transfectants with a solid histogram. EpCAM siRNA induced a mean 43% reduction in EpCAM expression at the cell membrane. Control staining is displayed as a thin-lined graph. (b) Numbers of viable FaDu cells after transfection with control or EpCAM siRNA were determined in a standard trypan blue exclusion assay after 2 days. Shown are the mean and standard deviations of three independent experiments. Cell numbers were reduced by 35% in average after transfection of EpCAM-specific siRNA. Open graph represents mean values of FaDu cells transfected with control siRNA and solid graph EpCAM siRNA. (c) Levels of EpCAM, MMP7 and GAPDH mRNAs were determined upon RT-PCR in FaDu transfectants. Two days after transfection of control or EpCAM siRNA, cells were harvested, mRNA was isolated, and EpCAM, MMP7 and GAPDH levels were assessed with specific primer pairs. Shown are representative results from three independent experiments. H2O served as a negative control. (d) Same as in (c) except mRNA measurement was conducted via quantitative PCR. Shown are% EpCAM (left panel) and MMP7 (right panel) mRNA expression levels from two independent experiments. P-values are indicated for each subfigure.

Article Snippet: Two days after plating, cells were collected and the ELISA was performed according to the instructions of R&D Quantikine, Human MMP7 ELISA Kit manual (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Transfection, Control, Cytometry, Membrane, Staining, Trypan Blue Exclusion Assay, Reverse Transcription Polymerase Chain Reaction, Isolation, Negative Control, Real-time Polymerase Chain Reaction

Journal: International journal of experimental pathology

Article Title: MMP7 is a target of the tumour-associated antigen EpCAM.

doi: 10.1111/j.1365-2613.2012.00826.x

Figure Lengend Snippet: Figure 2 EpCAM and MMP7 expression correlates in vivo in primary human samples. Serial cryosections of normal and dysplastic mucosa, and head and neck carcinoma samples were stained with EpCAM- or MMP7- specific antibodies (red staining). Shown are representative examples. EpCAM and MMP7 pattern and expression levels tightly correlated, even at the single-cell levels (exemplified with *).

Article Snippet: Two days after plating, cells were collected and the ELISA was performed according to the instructions of R&D Quantikine, Human MMP7 ELISA Kit manual (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, In Vivo, Staining

Journal: International journal of experimental pathology

Article Title: MMP7 is a target of the tumour-associated antigen EpCAM.

doi: 10.1111/j.1365-2613.2012.00826.x

Figure Lengend Snippet: Figure 3 Coincident, non-coincident and opposing expression patterns of EpCAM and MMP7. Serial cryosections of normal and dysplastic mucosa, and head and neck carcinoma samples were stained with EpCAM- or MMP7-specific antibodies (red staining). Shown are representative examples of coincident, non-coincident and opposing expression patterns of EpCAM and MMP7.

Article Snippet: Two days after plating, cells were collected and the ELISA was performed according to the instructions of R&D Quantikine, Human MMP7 ELISA Kit manual (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Staining

Journal: International journal of experimental pathology

Article Title: MMP7 is a target of the tumour-associated antigen EpCAM.

doi: 10.1111/j.1365-2613.2012.00826.x

Figure Lengend Snippet: Figure 4 EpCAM and MMP7 expression correlates in vivo in primary human samples. Serial cryosections of normal (n = 10) and head and neck carcinoma samples (n = 20) were stained with EpCAM- or MMP7- specific antibodies. Fields of view (FOV) of normal mucosa (one FOV/ sample) and tumours (five FOV/sample) were assessed with respect to staining patterns and intensity of EpCAM and MMP7. Results are given as percentages of FOV with coincident, non-coincident or opposing patterns for tumours (a) and normal mucosas (b) Similarities in staining intensities are given in each table.

Article Snippet: Two days after plating, cells were collected and the ELISA was performed according to the instructions of R&D Quantikine, Human MMP7 ELISA Kit manual (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, In Vivo, Staining

Journal: International journal of experimental pathology

Article Title: MMP7 is a target of the tumour-associated antigen EpCAM.

doi: 10.1111/j.1365-2613.2012.00826.x

Figure Lengend Snippet: Figure 5 MMP7 is a signalling target of EpCAM. (a) FaDu cells were transiently transfected with control or EpCAM siRNA to regulate EpCAM expression. Simultaneously, cells were co-transfected with a control pCAG or pCAG-EpICD plasmid. After 2 days, cells were harvested, mRNA was isolated, and MMP7 and GAPDH levels were assessed with specific primer pairs. Shown are representative results from three independent experiments. H2O served as a negative control. (a) Same as in (a) except primer pairs specific to WISP2 have been used. (c) Same as in (a) except mRNA measurement was conducted via quantitative PCR using HPRT1 as endogenous control. Shown are% epcam (left panel) and mmp7 (right panel) mRNA expression levels from two independent experiments. (d) FaDu cells were transiently transfected with control or EpCAM siRNA to regulate EpCAM expression. Simultaneously, cells were co-transfected with luciferase reporter plasmid for the MMP7 promoter region. As a control, luciferase reporter plasmid for the MMP7 promoter region, which carried mutated Tcf consensus sequences, was transfected in parallel. Promoter activation was calculated as the ratio of non-mutated vs. mutated reporter, and values after transfection of control siRNA were set to 100%. Shown are mean values and standard deviations of three independent experiments performed in duplicates. P-values are indicated for each subfigure.

Article Snippet: Two days after plating, cells were collected and the ELISA was performed according to the instructions of R&D Quantikine, Human MMP7 ELISA Kit manual (R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Isolation, Negative Control, Real-time Polymerase Chain Reaction, Luciferase, Activation Assay

Journal: International journal of experimental pathology

Article Title: MMP7 is a target of the tumour-associated antigen EpCAM.

doi: 10.1111/j.1365-2613.2012.00826.x

Figure Lengend Snippet: Figure 6 Nuclear translocation of EpICD is essential for transcriptional induction of MMP7. (a) HeLa cells transiently transfected with EpICD-ERT were cultured in the presence or absence of 100 nM 4-hydroxytamoxifen (4-OHT). EpICD-ERT was stained with EpICD-specific antibodies (red) and the localization assessed using confocal laser scanning microscopy. Nuclei were stained with DAPI (blue). Shown are representative images of three independent experiments. (b) At the indicated time points (0 and 4 h post- treatment), mRNAs from non-induced and induced cells were isolated, and the expression of c-myc, GAPDH and IC-ERT was assessed upon RT-PCR with specific primer pairs. Shown are representative results of three independent experiments. H2O served as a negative control. (c) Transient HeLa transfectants expressing ERT or EpICD-ERT were treated with 4-OHT (+) or with solvent only (), for the indicated time periods (0, 2, 4 and 8 h). mRNA levels of MMP7 and GAPDH were assessed upon RT-PCR with specific primer pairs. Shown are representative results of three independent experiments. H2O served as a negative control.

Article Snippet: Two days after plating, cells were collected and the ELISA was performed according to the instructions of R&D Quantikine, Human MMP7 ELISA Kit manual (R&D Systems, Minneapolis, MN, USA).

Techniques: Translocation Assay, Transfection, Cell Culture, Staining, Confocal Laser Scanning Microscopy, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Solvent

Journal: International journal of experimental pathology

Article Title: MMP7 is a target of the tumour-associated antigen EpCAM.

doi: 10.1111/j.1365-2613.2012.00826.x

Figure Lengend Snippet: Figure 7 Levels of soluble MMP7 positively correlate with EpCAM expression. (a) Representative histograms of EpCAM FACS stainings in FaDu, HeLa and HEK293 cells are shown. Left panel: Expression of EpCAM was assessed upon flow cytometry with specific antibodies on FaDu, HeLa and HEK293 cells. Shown are mean fluorescence intensity ratios of EpCAM staining vs. control staining with standard deviations. Right panel: Levels of MMP7 in supernatants of FaDu, HeLa and HEK293 cells were measured with an ELISA kit. Soluble MMP7 levels are given as mean relative units (RU) with standard deviations of three independent experiments performed in duplicates. (b) Left panel: FaDu cells were transiently transfected with control siRNA or EpCAM siRNA. After 2 days, EpCAM expression at the cell surface was assessed upon flow cytometry with specific antibodies. Shown are mean fluorescence intensity ratios of EpCAM vs. control stainings with standard deviations from three independent experiments. Right panel: Supernatants from siRNA-treated FaDu cells were collected after 2 days, and levels of soluble MMP7 were measured with an ELISA kit. Soluble MMP7 levels are given as mean relative units (RU) with standard deviations of three independent experiments performed in duplicates. P-values are indicated for each subfigure.

Article Snippet: Two days after plating, cells were collected and the ELISA was performed according to the instructions of R&D Quantikine, Human MMP7 ELISA Kit manual (R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Cytometry, Staining, Control, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Cancers

Article Title: Dexamethasone-Induced Fatty Acid Oxidation and Autophagy/Mitophagy Are Essential for T-ALL Glucocorticoid Resistance.

doi: 10.3390/cancers15020445

Figure Lengend Snippet: Figure 5. Dex causes structural changes in mitochondria and induces mitophagy. (a) High-resolution electron micrographs of CCRF-CEM cells under control (upper panel) and after Dex treatment (lower panel, 1 µM, 24 h). N: nuclei. Green arrows: mitochondria, yellow arrows: lysosomes, purple asterisks: double-membrane autophagosomes., red squares: mitochondria undergoing the fission process, blue squares: autophagic components close to lysosomes. Scale bars: 2 µm. (b) Representative confocal images of MCF-7 and OP9 cells co-stained with MtRed and Hoechst, either untreated or treated with Dex (upper panel). Scale bars: 10 µm. Quantitative analysis of MtRed-positive particles’ size and number (lower panel). Independent t-tests were performed. (c) Confocal images of OP9 (left) or MDA-MB-231 cells (right) co-stained with MtRed (red), MDC (blue), and Lysotracker (green). OP9 micrographs (left) show different zones in Z-stacked layers of a representative cell. White arrows indicate the proximity of mitochondria to acidic organelles. Scale bars: 2 µm (OP9) and 10 µm (MDA-MB-231). (d) Experimental strategy for the mitophagy detection with mtKeima (upper panel). The excitation spectrum of mtKeima at neutral (green) and acidic (red) pH (lower panel). Dotted lines: wavelengths corresponding to lasers used for excitation in confocal microscopy. (e) Representative confocal microscopy micrographs of CCRF-CEM cells transfected with mtKeima, untreated (control), and treated either with Dex (24 h) or CCCP (positive control). Scale bars: 10 µm. (f) Quantitative analysis of mitophagy through spectrofluorometry. The level of mitophagy was calculated as the ratio between the values of the maximum fluorescence obtained upon excitation at 586 nm and 440 nm (ratio 586/440). One-way ANOVA with Dunnet’s multiple comparison test was run. (g) Cell viability by trypan blue exclusion assay in T-ALL cells exposed to Dex alone (0–10 µM) and in combination with CQ (1 µM) for 24 h. Two-way ANOVA and Sidak’s multiple comparison tests were employed. In (b,f,g), data are mean ± S.E. for at least 4 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Medium alpha-MEM supplemented with 5% of fetal bovine serum (FBS), 2 mM of GlutaMAX, 10 mM of HEPES, 100 U/mL penicillin, and 100 μg/mL of streptomycin was used for murine mesenchymal stromal cells (MSC) from bone marrow (BM), OP9 (ATCC® CRL-2749TM).

Techniques: Control, Membrane, Staining, Confocal Microscopy, Transfection, Positive Control, Comparison, Trypan Blue Exclusion Assay

Journal: Cancers

Article Title: Dexamethasone-Induced Fatty Acid Oxidation and Autophagy/Mitophagy Are Essential for T-ALL Glucocorticoid Resistance.

doi: 10.3390/cancers15020445

Figure Lengend Snippet: Figure 6. Effects of Dex on T-ALL in (CCRF-CEM) MSCs (OP9) co-cultures. (a) Effect of Dex (0–5 µM, 24 h) on the metabolism of the OP9-GFP cells. Data are mean ± S.E. of at least 3 indepen- dent experiments. Raw data were normalized to control. An independent t-test was performed. (b) Representative micrographs of 2D-CC, either untreated or exposed to Dex (24 h). Green: OP9-GFP cells, red: CCRF-CEM cells stained with Deep Red cell tracker. Scale bars: 50 µm. (c) Representa- tive morphology of CCRF-CEM cells on the surface of 2D-CC (from b, brightfield, black squares). (d) Quantification of the living CCRF-CEM cells, interacting with the MSCs monolayer, in untreated and Dex-treated (24 h) 2D-CC. (e) Quantification of the living CCRF-CEM cells in the supernatant of 2D-CC, either untreated or after exposure for 24 h to Dex. (f) Quantification of the dead CCRF-CEM cells in the supernatant of 2D-CC, untreated or after Dex exposure (24 h). In (d-f), an indepen- dent t-test was performed. (g) Experimental strategy to obtain 3D-CC. (h) Representative light microscopy images of intact 3D-CC, untreated or Dex-treated (1 and 10 µM, 24 h). Scale bars: 200 µm. (i) Quantitation of 3D-CC spheroids’ diameter, untreated or after Dex exposure (1 and 10 µM, 24 h), light microscopy. (j) Representative confocal microscopy micrographs of intact 3D-CC spheroids, un- treated and exposed to Dex (1 and 10 µM, 24 h). Green: OP9-GFP cells, red: CCRF-CEM cells stained with Deep Red. Scale bars: 200 µm. (k) Quantification of 3D-CC spheroids’ diameter, untreated or exposed to Dex (1 and 10 µM, 24 h). Confocal microscopy. (l) Distribution profiles of Deep Red fluorescence (corresponding to CCRF-CEM) intensity across 3D-CC spheroid. The histogram is the average of at least 6 independent experiments. (m) Representative high-resolution (40×) micrographs of independent 3D-CC spheroids. Green: OP9-GFP cells, red: CCRF-CEM cells stained with Deep Red. Scale bars: 50 µm. (n) Quantitative analysis of the mean fluorescence intensity (MFI) of the CCFR-CEM cells retained in the 3D-CC spheroid. One-way ANOVA with multiple comparison tests was used. (o) Effect of Dex (1 µM) treatment on the migration of CCRF-CEM cells from the upper to the lower chamber. The experimental strategy is specified under the graph: CCRF-CEM or OP9 cells were either pretreated with Dex (1 µM, 24 h, red font color) or untreated (black font). One-way ANOVA with multiple comparison tests was performed. In (a,d–f,o), data are mean ± S.E. for at least 4 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Medium alpha-MEM supplemented with 5% of fetal bovine serum (FBS), 2 mM of GlutaMAX, 10 mM of HEPES, 100 U/mL penicillin, and 100 μg/mL of streptomycin was used for murine mesenchymal stromal cells (MSC) from bone marrow (BM), OP9 (ATCC® CRL-2749TM).

Techniques: Control, Staining, Light Microscopy, Quantitation Assay, Confocal Microscopy, Comparison, Migration

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: (A) LETM1 interactome as determined by affinity purification mass spectrometry (AP-MS). All high confidence interaction partners of LETM1 are shown as nodes. Node color indicates SAINT score, a probability-based measure of interaction confidence. Data are from a single MS experiment. See also (B) Co-immunoprecipitation of MICS1 and LETM1 protein in tandem. Mitochondria were crudely isolated from HEK293 cells and used for immunoprecipitation. The input represents the mitochondrial crude lysate used as input for the co-IP, LETM1 was immunoprecipitated (left panel, IP:LETM1) using a LETM1 polyclonal antibody and Protein A/G agarose beads (ProtA/G). ProtA/G beads alone were used as a negative control for binding, immunoprecipitates were immunoblotted (IB) for the indicated proteins to demonstrate interaction. Prohibitin was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane proteins complexes. The right panel illustrates the converse experiment, MICS1 was precipitated (right panel, IP: MICS1) using a MICS1 polyclonal antibody. (C) Native immunoblot of LETM1 (left) and MICS1 (right) show that both proteins can be found in protein complexes of the same size (arrows), MICS1 additionally resides in other protein complexes. (D) Immunoblot analysis of LETM1 and MICS1 expression on conditions of reduced FBS (0.5%) in culture media. Mitochondrial complex III integral subunit UQCRC2 is used as loading control.

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Immunoprecipitation, Isolation, Co-Immunoprecipitation Assay, Negative Control, Binding Assay, Control, Membrane, Western Blot, Expressing

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: (A) Western blot analysis of LETM1 and MICS1 in HEK293 MICS1WT cell with scramble shRNA (scr) or two different MICS1 knockdowns (KD#1 or KD#2). HSP60 served as a loading control. (B) Proliferation curve of MICS1WT (WT) with a scrambled construct compared to MICS1KD cells (KD) over 4 days using a trypan blue exclusion assay to count cells. Data are means ± SEM (n=3), at 96h statistical analysis using an unpaired student’s t-test (***p<0.001). (C-F) Cellular bioenergetics of MICS1KD cells in various nutrient conditions. Oxygen consumption rate of WT cells with a scrambled control (“WT”) and MICS1KD#1 cells grown in (C) 25 mM glucose, (E) 10 mM galactose for 24 hours before measurement. Data are representative of at least 3 independent experiments. Shown are mean data of triplicate measurements ± SEM. Inhibitors as indicated: A-oligomycin (0.5 μM), B & C-FCCP (0.2 μM each), D-antimycin A/rotenone (0.5 μM). (D & F) Bar charts of XF experiment traces (C & E), data are means of multiple time points after experiment start or drug addition of at least three independent experiments ± SEM. (n=3). Statistical analysis using an unpaired student’s t-test (** p <0.01, *** p <0.001).

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Western Blot, shRNA, Control, Construct, Trypan Blue Exclusion Assay

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: (A) Western blot analysis of MICS1 in control and targeted HeLa and HEK293 clones, HSP60 served as loading control. (B) Proliferation assay of HEK293 cells in function of MICS1. Graph shows the mean of three individual counts, Two-way ANOVA with Dunnett’s multiple comparisons test performed against MICS1WT *p=0.0155. (C) Live imaging of HEK293 MICS1WT and KO cells stained with MitoTracker Green FM. Bars: 10 μm (D) Alteration of the mitochondrial ultrastructure shown by transmission electron microscopy, red arrow pointing to dilated matrix. Wider mitochondria in middle and right panel compared to controls, a middle panel showing the strongest phenotype of matrix width and cristae forms. (E) Isolated mitochondria from three independent replicates of HEK293 MICS1WT and MICS1KO#1 (#1) and KO#2 (#2) were analyzed by immunoblotting using the indicated antibodies, HSP60 and TOM40 served as mitochondrial loading controls. (F) Densitometric analysis of the bands in (E) normalized to loading control, bar graph of three individual counts, One-way ANOVA with Bonferroni’s multiple comparisons test performed against MICS1WT *p<0.05, **p<0.008, Two-way ANOVA with Bonferroni’s multiple comparisons test performed for the OPA1 statistics against MICS1WT, ***p=0.0009, ****p<0.0001.

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Western Blot, Control, Clone Assay, Proliferation Assay, Imaging, Staining, Transmission Assay, Electron Microscopy, Isolation

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: KOAc-induced swelling was measured in mitochondria derived from HEK293 MICS1WT, MICS1KO and MICS1KO cells stably re-expressing MICS1WT (A-B) , HeLa MICS1WT and MICS1KO (C-D) and HeLa LETM1 scramble and LETM1 KD (E-F) cells. MICS1WT: black traces, MICS1KO: red traces, MICS1KO + MICS1WT: blue traces, LETM1scr: black trace, LETM1KD: green trace. (B) Quantification of swelling amplitudes from independent experiments (n=3) HEK293 MICS1WT (black bar, 100 ± 19.71) and HEK293 MICS1KO (red bar, 48.08 ± 11.906). Complementation of MICS1KO with re-expression of MICS1WT restored swelling rates (blue bar, 90.55 ± 12.93). (D) Similar differences in swelling capacities were obtained between HeLa MICS1WT (black bar; 100 ± 9.47) and HeLa MICS1KO (red bar; 63.48 ± 8.60). Lower basal optical density indicates swollen matrix prior KOAc addition; Inhibition of KHE with quinine in HEK293 cells: WT grey bar, 18.14 ± 21.02; KO#1 pink bar, 9.33 ± 28.17; MICS1KO+MICS1, 15.79 ± 10.04. Statistical analysis: One-Way ANOVA with Bonferroni correction (*p <0.05, **p <0.01, ***p <0.001). See also .

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Derivative Assay, Stable Transfection, Expressing, Inhibition

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: Ca 2+ uptake/release dynamics are shown as extramitochondrial Ca 2+ changes of fluorescence intensities of Calcium Green 5N (Ca 2+ 5N) (0.24 μM) (A-L and P-Q) and membrane potential as change of fluorescence intensities of TMRM (330 nM) (M-N) corresponding to the measurement of Ca 2+ fluxes in (K) . Experiments were performed using permeabilized HEK293 MICS1WT, MICS1KO (KO#1, KO#2) ( A-D) and (G-R), MICS1KO#1 + MICS1WT cells was included in (A-B) , and HEK293 LETM1 scr and LETM1KD cells (E-F) in presence of CGP37157 (2 μM). Ca 2+ (10 μM), RR (0.2 μM) and FCCP (2 μM) or alamethicin (2.5 μM) were added when indicated. CsA was added two minutes before measurements in (G-J and Q-R) , nigericin was added 1 min before measurements in absence of thapsigargin or after Ca 2+ uptake in thapsigargin experiments to prevent slowed Ca 2+ uptake dynamics in (I-J) , thapsigargin (1 μM) was added as indicated in K-R , and ADP as indicated in (Q-R) . Quantification of Ca 2+ release rates from independent experiments (n=3) (t: 300-920 s) and statistical analysis: One-Way ANOVA with Bonferroni correction (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001). See also for quantification of Ca 2+ uptake. Quantification of TMRM performed with unpaired two-sided t-test (Welsh correction), *p <0.05. (O-P) CRCs showing that absence of MICS1 supersensitizes mitochondria to Ca 2+ -induced PTP opening by thapsigargin. See also for CRCs. In absence of thapsigargin. Permeabilized HEK293 MICS1WT (O) and MICS1KO#1 (P) cells exposed or not to CsA were subjected to sequential Ca 2+ bolus of 5 µM Ca 2+ and fluorescence intensity was recorded. (Q-R) Thapsigargin-dependent Ca 2+ uptake/release experiments repeated in presence of CsA and ADP in MICS1WT and MICS1KO#1 and KO#2 showing the suppression of Ca 2+ release, quantifications using One-Way ANOVA with Bonferroni correction.

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Fluorescence, Membrane

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: Permeabilized HEK293 MICS1 WT (black trace) and MICS1 KO (red trace) cells exposed (A) or not (B) to CsA were subjected to sequential Ca 2+ bolus of 5 µM Ca 2+ while NCLX was not inhibited and fluorescence intensity was recorded.

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Fluorescence

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: A) Nucleotide sequence of the codon optimized sequence of MICS1 (B) MICS1 expression and purification. Lane 1: page ruler prestained plus marker; lane 2: insoluble fraction of not induced cell lysate (negative control); lane 3: insoluble fraction of cell lysate after 2 hours of 0.4 mM IPTG induction at 37 °C; lane 4: purified MICS1 protein. (C) Ribbon representation of the hMICS1 protein (Q9H3K2). PDB file retrieved from AlphaFhold at https://alphafold.ebi.ac.uk/entry/Q9H3K2 . The unstructured loop containing the first 55 amino acids has been removed.

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Sequencing, Expressing, Purification, Marker, Negative Control

Journal: bioRxiv

Article Title: MICS1 is the Ca 2+ /H + antiporter of mammalian mitochondria

doi: 10.1101/2021.11.11.468204

Figure Lengend Snippet: (A ) Sketch illustrating the reconstitution of hMICS1 (red) in proteoliposomes and the empty liposomes (blue) for transport measurements. (B ) Western blot analysis of purified and reconstituted hMICS1 for evaluating the incorporation of hMICS1 into proteoliposomes prepared as described in materials and methods. Transport of Ca 2+ (C-E) or H + (F) by hMICS1 reconstituted in proteoliposomes. Purified hMICS1 was reconstituted in proteoliposomes containing 10 μM Calcium Green 5N at the pH indicated in the panels (C-E) or 20 μM pyranine at pH 7.0 (F) . After reconstitution, the fluorescence measurement was started by diluting 200 µL proteoliposomes (red trace) up to 3 mL with transport buffer prepared as described in materials and methods at the indicated pH (C) or at pH 7.0 (D) . After 100 sec, as indicated by the arrow, 7 mM Ca 2+ was added to the sample and fluorescence change was recorded. As a control, the same measurement was performed diluting 200 µL liposomes (without incorporated protein, blue trace) up to 3 mL with the same transport buffer. The fluorescence intensity is indicated as Arbitrary Units (AU). Results are representative of three independent experiments. See also for MICS1 optimization, induction and structure overview.

Article Snippet: LETM1 (Abnova, #H00003954), 1:1000, LETM1 (Santa Cruz Biotechnology, #sc-163013), 1:1000, LETM1 C-terminal region (Aviva, Systems Biology, #OAAB12878), 1:1000, MICS1 (Abcam, #ab106754), 1:1000, MICS1 (Aviva systems biology, #OAAF06415), 1:1000, OPA1 (BD Biosciences, #612606), 1:1000, SIRT3 (Cell Signaling Technology, #5490), 1:1000, DRP1 (Santa Cruz Biotechnology, #sc-271583), 1:1000, HSP60 (Santa Cruz Biotechnology, #sc-1052), 1:1000, TOM20 (Cell Signaling Technology, #42406), 1:1000, TOM40 (Santa Cruz Biotechnology, #sc-365467), 1:1000, OMA1 (Santa Cruz Biotechnology, #sc-515788), 1:1000, Prohibitin (Abcam, #ab210082), 1:1000, β-Actin (Invitrogen, #MA5-11869), 1:1000, PolyHistidine-Peroxidase (Sigma Aldrich, #a7058), 1:10000, Goat-α-mouse (Jackson ImmunoResearch, #115-035-003) 1:5000, Rabbit-α-goat (Jackson ImmunoResearch, #305-035-003) 1:5000, Goat-α-rabbit (Jackson ImmunoResearch, #111-035-144) 1:5000.

Techniques: Liposomes, Western Blot, Purification, Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Aloysia Citrodora Essential Oil Inhibits Melanoma Cell Growth and Migration by Targeting HB-EGF-EGFR Signaling

doi: 10.3390/ijms22158151

Figure Lengend Snippet: ALOC-EO targets EGFR and ERK1/2. ( A ) Immunoblot for phosphorylated murine EGFR in B16F10 cells treated with either DMSO or ALOC-EO after 12 h. Right panel: The histogram represents the signal intensity of the indicated protein bands in arbitrary units after normalization with the signal intensity of β-actin internal control for each sample. Values are means and SEM of two independent experiments. ( B ) Representative immunoblot for phosphorylated murine EGFR and loading control β-actin in tumor cell lysates from control and ALOC-EO-treated mice. Right panel: The histogram represents the signal intensity of the indicated protein bands in arbitrary units after normalization with the signal intensity of β-actin internal control for each sample. Values are means and SEM of two independent experiments. ( C ) EGFR overexpression (EGFR OE) was confirmed in B16F10 cells and mock controls by qPCR, and the fold change in EGFR expression compared to expression of β-actin is given (n = 3). ( D ) EGFR phosphorylation was performed in cell lysates of EGFR OE and mock cells by Western blotting. Right panel: The histogram represents the signal intensity of the indicated protein bands in arbitrary units after normalization with the signal intensity of β-actin internal control for each sample. Values are means and SEM of two independent experiments. ( E ) EGFR OE and mock cells were treated with/without ALOC-EO. Cells were counted after 24 h in culture (n = 6/group). ( F ) B16F10 cells were treated with/without rec. HB-EGF in the presence or absence of ALOC-OC. Cells were counted 24 h later (n = 6/group). ( G ) Murine MMP7 , MMP9 , ADAM9 , and HB-EGF expression in EGFR OE and mock B16F10 cells treated with or without HB-EGF in the presence or absence of ALOC-EO by qPCR. Transcript levels were normalized to β-actin. +/− SEM. * p < 0.05; ** p < 0.01 (Student’s t -test).

Article Snippet: Cell lysates (2–50 μg proteins) were applied on 12% acrylamide gel, transferred to PVDF membrane (Millipore, Immobilon), and then probed with one of the following primary antibodies (all mouse IgG, 1 μg/mL) overnight at 4 °C: C-terminal cytoplasmic domain of HB-EGF of mouse origin (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-1414), MMP7 (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-515702), β-actin (Cell Signaling, #4967), total p44/42 MAPK (Erk1/2)(Cell Signaling, #9102), p-EGFR (Santa Cruz Biotech, sc-57545), MMP9 (Chemicom, AB19047)), ERK1/2 (Cell signaling, 4370S), BAX2 (Cell signaling, Danvers, MA, USA, #2772S).

Techniques: Western Blot, Control, Over Expression, Expressing, Phospho-proteomics

Journal: International Journal of Molecular Sciences

Article Title: Aloysia Citrodora Essential Oil Inhibits Melanoma Cell Growth and Migration by Targeting HB-EGF-EGFR Signaling

doi: 10.3390/ijms22158151

Figure Lengend Snippet: Chemotherapy-induced HB-EGF-EGFR upregulation is blocked by ALOC-EO and improves chemo-sensitivity. ( A ) Fold change expression of murine HB-EGF and ( B ) EGFR in B16F10 cells treated with indicated concentrations of the antitumor drugs bortezomib or doxorubicin, as a carrier by qPCR (A; n = 3/group). ( C – E ) HB-EGF expression was determined after ALOC-EO treatment for 24 h in B16F10 cells and tumor tissues retrieved from tumor-bearing mice treated with or without ALOC-EO at day 12 by qPCR (C; n = 3/group) by Western blotting analysis ( D , E ). Right panel: The histograms represent the signal intensity of the indicated protein bands in arbitrary units after normalization with the signal intensity of β-actin internal control for each sample. Values are means and SEM of two independent experiments. ( F – H ) B16F10 cells were treated with BTZ ( G ) or doxorubicin ( F , H ) at indicated concentration in the presence or absence of 100 µg/mL ALOC-EO. ( F ) Fold expression of MMP7/9 , and ADAM9 in cells treated with indicated drug combinations as determined by qPCR. ( G , H ) Viable cells were counted after 24 h, using the trypan blue exclusion assay (n = 6). The results of three independent experiments performed in triplicates are expressed as fold change according to 2-ΔΔCT method using β-actin as calibrator. Transcript levels were normalized to β-actin (n = 3/group). ( I ) Proposed mode of action for ALOC-EC in melanoma: ALOC-EO can block EGFR and ERK1/2 phosphorylation, leading to the reduced expression of MMP7/9 and ADAM9. Not shown in this study is that these proteases are known to enhance the shedding of the EGFR ligand HB-EGF. Abbreviations: ALOC-EO, Aloysia citrodora essential oil; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal-regulated kinase; MMP7, matrix metalloproteinase-7, matrix metalloproteinase9 (MMP9), a disintegrin and metalloproteinase domain-containing protein 9 (ADAM9). * p < 0.05; ** p < 0.01.

Article Snippet: Cell lysates (2–50 μg proteins) were applied on 12% acrylamide gel, transferred to PVDF membrane (Millipore, Immobilon), and then probed with one of the following primary antibodies (all mouse IgG, 1 μg/mL) overnight at 4 °C: C-terminal cytoplasmic domain of HB-EGF of mouse origin (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-1414), MMP7 (Santa Cruz Biotech, Santa Cruz, CA, USA, sc-515702), β-actin (Cell Signaling, #4967), total p44/42 MAPK (Erk1/2)(Cell Signaling, #9102), p-EGFR (Santa Cruz Biotech, sc-57545), MMP9 (Chemicom, AB19047)), ERK1/2 (Cell signaling, 4370S), BAX2 (Cell signaling, Danvers, MA, USA, #2772S).

Techniques: Expressing, Western Blot, Control, Concentration Assay, Trypan Blue Exclusion Assay, Blocking Assay, Phospho-proteomics

Journal: Experimental & Molecular Medicine

Article Title: Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

doi: 10.1038/emm.2014.28

Figure Lengend Snippet: Primer sequences for RT–PCR amplification of target genes

Article Snippet: The membrane was incubated for 1 h at room temperature in blocking buffer (TBS-T containing 5% skim milk) to block nonspecific protein binding and then incubated overnight at 4 °C with a primary antibody against Oct4 (1:1000), Sox2 (1:500), cyclin D1 (1:1000) or β-actin (1:1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted in blocking buffer.

Techniques: Amplification

Journal: Experimental & Molecular Medicine

Article Title: Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

doi: 10.1038/emm.2014.28

Figure Lengend Snippet: Expression analysis of Oct4 and Sox2 in Oct4/Sox2-ATMSCs. ( a ) In RT–PCR analysis, the mRNA expression levels of Oct4 and Sox2 in Oct4/Sox2-ATMSCs were significantly higher than those of RFP-ATMSCs at 24 h post-transfection. Band densities in RT–PCR were evaluated semi-quantitatively by densitometry. Results are the ratio of Oct4 and Sox2 expression normalized to GAPDH mRNA levels. ( b ) Western blots show high levels of Oct4 and Sox2 expression in Oct4/Sox2-ATMSCs. Band densities in western blot analysis were evaluated semi-quantitatively by densitometry. Results are the ratio of Oct4 and Sox2 expression normalized to beta-actin protein levels. Data are representative of three independent experiments, with similar results. Data are expressed as the means+s.d. **P< 0.01 compared with the control (RFP-ATMSCs) value as determined by Student's t -test.

Article Snippet: The membrane was incubated for 1 h at room temperature in blocking buffer (TBS-T containing 5% skim milk) to block nonspecific protein binding and then incubated overnight at 4 °C with a primary antibody against Oct4 (1:1000), Sox2 (1:500), cyclin D1 (1:1000) or β-actin (1:1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted in blocking buffer.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Control

Journal: Experimental & Molecular Medicine

Article Title: Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

doi: 10.1038/emm.2014.28

Figure Lengend Snippet: Immunophenotyping of RFP- and Oct4/Sox2-transfected ATMSCs. Non-transfected MSCs at passage 3, RFP-transfected ATMSCs at passage 5 and Oct4/Sox2-transfected ATMSCs at passage 5 were immunophenotyped for CD29, CD31, CD34, CD44, CD45, CD73, CD90 and CD105 by flow cytometry. The expression of surface markers of ATMSCs transduced with Oct4/Sox2 or RFP were not different from those of non-transfected ATMSCs. Data are representative of three independent experiments, with similar results.

Article Snippet: The membrane was incubated for 1 h at room temperature in blocking buffer (TBS-T containing 5% skim milk) to block nonspecific protein binding and then incubated overnight at 4 °C with a primary antibody against Oct4 (1:1000), Sox2 (1:500), cyclin D1 (1:1000) or β-actin (1:1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted in blocking buffer.

Techniques: Transfection, Flow Cytometry, Expressing, Transduction

Journal: Experimental & Molecular Medicine

Article Title: Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

doi: 10.1038/emm.2014.28

Figure Lengend Snippet: Proliferation assay using Oct4/Sox2-ATMSCs. ( a ) WST-1 assay showed that Oct4/Sox2-ATMSCs have higher cell metabolic activity than RFP-ATMSCs at 1, 2 and 3 days. ( b ) In the trypan blue exclusion assay, viable cell numbers were increased significantly in Oct4/Sox2-ATMSCs cultured for 9 days compared with RFP-ATMSCs. Data are expressed as the mean±s.d. ** P <0.01 compared with the corresponding control value as determined by Student's t -test.

Article Snippet: The membrane was incubated for 1 h at room temperature in blocking buffer (TBS-T containing 5% skim milk) to block nonspecific protein binding and then incubated overnight at 4 °C with a primary antibody against Oct4 (1:1000), Sox2 (1:500), cyclin D1 (1:1000) or β-actin (1:1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted in blocking buffer.

Techniques: Proliferation Assay, WST-1 Assay, Activity Assay, Trypan Blue Exclusion Assay, Cell Culture, Control

Journal: Experimental & Molecular Medicine

Article Title: Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

doi: 10.1038/emm.2014.28

Figure Lengend Snippet: Cell cycle analysis by flow cytometry, and western blot analysis of cyclin D1 in Oct4/Sox2-ATMSCs. ( a ) Representative flow cytometry results of RFP- and Oct4/Sox2-ATMSCs ( b ) The percentage of cells at each cell cycle stage as determined by flow cytometry analysis. Compared with the control (RFP-ATMSCs), cell cycle analysis showed a decrease in the proportion of Oct4/Sox2-ATMSCs in the G0/G1 phase, whereas an increase was observed in the proportion of those in the S phase. ( c ) The expression of cyclin D1, which is a major regulator, was increased in Oct4/Sox2-ATMSCs compared with RFP-ATMSCs. Data are expressed as the mean+s.d. ** P <0.01 compared with the corresponding control value as determined by Student's t -test.

Article Snippet: The membrane was incubated for 1 h at room temperature in blocking buffer (TBS-T containing 5% skim milk) to block nonspecific protein binding and then incubated overnight at 4 °C with a primary antibody against Oct4 (1:1000), Sox2 (1:500), cyclin D1 (1:1000) or β-actin (1:1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted in blocking buffer.

Techniques: Cell Cycle Assay, Flow Cytometry, Western Blot, Control, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

doi: 10.1038/emm.2014.28

Figure Lengend Snippet: Adipogenic differentiation assay using Oct4/Sox2-ATMSCs and RT–PCR analysis for adipogenic markers. ( a ) Oil red O staining for lipid droplets in Oct4/Sox2-transfected ATMSCs was strong at 7, 14 and 21 days during adipogenic differentiation compared with RFP-ATMSCs. ( b ) RT–PCR results show a significant increase in PPARγ and lipoprotein lipase mRNA expression at 7, 14 and 21 days during adipogenic differentiation. Band densities of PPARγ in RT–PCR were evaluated semi-quantitatively by densitometry. Results are the ratio of PPARγ expression normalized to GAPDH mRNA levels. Data are representative of three independent experiments with similar results. (1) RFP-ATMSCs, (2) Oct4/Sox2-ATMSCs.

Article Snippet: The membrane was incubated for 1 h at room temperature in blocking buffer (TBS-T containing 5% skim milk) to block nonspecific protein binding and then incubated overnight at 4 °C with a primary antibody against Oct4 (1:1000), Sox2 (1:500), cyclin D1 (1:1000) or β-actin (1:1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted in blocking buffer.

Techniques: Differentiation Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Transfection, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Enhanced proliferation and differentiation of Oct4- and Sox2-overexpressing human adipose tissue mesenchymal stem cells

doi: 10.1038/emm.2014.28

Figure Lengend Snippet: Osteogenic differentiation assay using Oct4/Sox2-ATMSCs and RT–PCR analysis for osteogenic markers. ( a ) Alizarin red S staining for mineralization in Oct4/Sox2-ATMSCs was strong at 7, 14 and 21 days during osteogenic differentiation compared with RFP-ATMSCs. ( b ) RT–PCR results show upregulation of collagen I and osteocalcin mRNA levels in Oct4/Sox2-ATMSCs at 7, 14 and 21 days during osteogenic differentiation compared with RFP-ATMSCs. Band densities of collagen I and osteocalcin in RT–PCR were evaluated semi-quantitatively by densitometry. Results are the ratio of collagen I and osteocalcin expression normalized to GAPDH mRNA levels. Data are representative of three independent experiments with similar results. (1) RFP-ATMSCs, (2) Oct4/Sox2-ATMSCs.

Article Snippet: The membrane was incubated for 1 h at room temperature in blocking buffer (TBS-T containing 5% skim milk) to block nonspecific protein binding and then incubated overnight at 4 °C with a primary antibody against Oct4 (1:1000), Sox2 (1:500), cyclin D1 (1:1000) or β-actin (1:1000) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted in blocking buffer.

Techniques: Differentiation Assay, Reverse Transcription Polymerase Chain Reaction, Staining, Expressing