Journal: Cancer Research

Article Title: Tissue Factor Regulation by Epidermal Growth Factor Receptor and Epithelial-to-Mesenchymal Transitions: Effect on Tumor Initiation and Angiogenesis

doi: 10.1158/0008-5472.can-08-2067

Figure Lengend Snippet: Figure 3. Changes in TF expression associated with multilineage differentiation of cancer cells in vivo. A, the majority of cells within the tumor mass were human cancer cells, as determined by staining with an antibody directed against human (tumor) cell nuclear antigen (green). Tumors grown in YFP/SCID mice were double-stained using anti-TF (human cancer cells, green) and anti-GFP (host, red) antibodies. Infiltration of YFP-positive host cells in A431 tumors is minimal (<10%) and many stroma-poor tumor regions remain negative for TF. Immunofluorescence staining of cytokeratin (red) reveals highly keratinized pearl structures within these tumors. B, some tumor cells have lost their epithelial phenotype and express the mesenchymal marker vimentin (green), indicative of cells undergoing EMT. H&E staining depicts which regions of the tumor are likely to express vimentin and undergo EMT (tumor periphery) and those that retain their epithelial phenotype and differentiate along an epithelial pathway (central tumor). Arrowhead, a keratin pearl. Cytokeratin immunofluorescence (red) shows regions of the tumor that have undergone epithelial differentiation. C, comparative Western blot analysis between A431 cells maintained in culture and those grown as tumors. A global decrease in TF protein levels were observed in tumors. Changes in EGFR were unremarkable whereas the expression of E-cadherin is reduced and the extracellular region is cleaved, leaving an 80 kDa protein that is unable to make cell-cell contacts. Consistent with this loss of E-cadherin and epithelial phenotype, some tumor regions express vimentin, whereas other regions of these tumors remain epithelial and express high levels of cytokeratin. Bars, 100 AM.

Article Snippet: All other tissue staining was performed on 4-Am paraffin sections which were incubated overnight at 4jC with the respective primary antibodies following heat antigen retrieval; except for cytokeratin (Novus Biological, Inc.) staining which required proteolytic digestion prior to a 60 min incubation with the prediluted antibody at room temperature.

Techniques: Expressing, In Vivo, Staining, Immunofluorescence, Marker, Western Blot

Journal: Cytometry. Part A : the journal of the International Society for Analytical Cytology

Article Title: In situ detection of CD73+ CD90+ CD105+ lineage: Mesenchymal stromal cells in human placenta and bone marrow specimens by chipcytometry.

doi: 10.1002/cyto.a.23509

Figure Lengend Snippet: Figure 3. Detection of CD73+ CD90+ CD105+ lineage- mesenchymal stromal cells in human placenta. Autofluorescence at 560 nm of a frozen human placenta 2 μm section (A) and color overlay of CD90 (red), CD73 (yellow), CD105 (cyan), panCytokeratin (magenta), CD31 (white), DNA (orange) and the dump channel (green) (B, C) are shown for one representative specimen out of three. The part figures (B) and (C) represent higher magnifications of the areas of interest, as indicated by the rectangles. Two CD90+ CD73+ CD105+ dump-MSCs are depicted in (C). The placenta derived from a normally progressing pregnancy that was legally interrupted in the 11th week of pregnancy.

Article Snippet: The monoclonal antibodies were applied in the following order: CD90 PE (5E10, Biolegend, 1:30), CD105 PE (clone 43A3, Biolegend, 1:30), CD73 PE (AD2, Biolegend, 1:30), CD45 Alexa Fluor 488 (HI30, Biolegend, 1:30), CD14 PE (RMO52, Beckman Coulter, 1:30), CD19 PE (HIB19, Biolegend, 1:30), CD34 PE (QBEnd10, Bio-Techne, 1:30), HLADR BUV395 (G46–6, BD Biosciences, 1:30), panCytokeratin PE (C11, Novus Biologicals, 1:250), and CD31 PE (WM-59, Biolegend, 1:100).

Techniques: Derivative Assay

Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA.

doi: 10.3390/cells13010077

Figure Lengend Snippet: Figure 4. Modulation of FoxP3 pre-mRNA splicing with SSO targeting only exon 7 cis-elements does not allow us to obtain Tregs expressing a single splice variant. (A) Two splicing regulator proteins, SC35 and SRp75 (shown as green ellipses), interact with their binding sites (shown in bold green font) within exon 7 and are responsible for the inhibition of exon 7 insertion in mature FoxP3 mRNA. The splicing regulator proteins SF2/ASF (shown as a red ellipse) interact with its binding site (shown in bold red font) within intron 7 and are responsible for the inhibition of exon 7 deletion from mature mRNA. (B) Treg transfection with #Ins7, a 36-mer-specific antisense SSO (presented in green italics font), blocks both SC35 and SRp75 from binding to their sensitive cis-elements and induces the insertion of exon 7 into the mature mRNA. (C) Treg transfection with #Del7, a 36-mer-specific antisense SSO (presented in red italics font), blocks SF2/ASF from binding to its sensitive cis-elements and induces the deletion of exon 7 from the mature mRNA. FoxP3 splice variant mRNA levels in cells 96 h after transfection with (D) #Ins7 or (E) #Del7 SSOs. The levels of investigated mRNAs were normalized to the mean expression of three reference genes: 18S, GAPDH, and beta-actin. N = 4. The results are shown as the mean ± SD. FL, full-length splice variant; ∆2, splice variant with deleted exon 2; ∆7, splice variant with deleted exon 7; ∆2∆7, splice variant with deleted both exon 2 and exon 7. ND, not detected.

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Expressing, Variant Assay, Binding Assay, Inhibition, Transfection

Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA.

doi: 10.3390/cells13010077

Figure Lengend Snippet: Figure 5. Modulation of FoxP3 pre-mRNA splicing with SSOs targeting both exon 2 and exon 7 cis-elements allowed us to obtain Tregs expressing a single splice variant. FoxP3 splice variant mRNA levels in Treg cells 96 h after transfection with (A) control nonspecific 36-mer nucleotides #Con1 & #Con2; (B) SSOs #Ins2 & #Ins7, which could induce the expression of the FL variant only; (C) SSOs #Del2 & #Ins7, which could induce the expression of the ∆2 splice variant only; (D) SSOs #Ins2 & #Del7, which could induce the expression of the ∆7 splice variant only; and (E) SSOs #Del2 & #Del7, which could induce the expression of the ∆2∆7 splice variant only. N = 4. The results are shown as the mean ± SD. * p ≤0.001 by Mann–Whitney U test. FL, full-length splice variant; ∆2, splice variant with deleted exon 2; ∆7, splice variant with deleted exon 7; ∆2∆7, splice variant with deleted both exon 2 and exon 7. ND, not detected. (F) Western blotting results demonstrate the induction of the ∆2 splice variant. Clone 150D is exon 2 specific, while clone 259D recognizes an epitope after exon 2 common for all FoxP3 splice variants.

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Expressing, Variant Assay, Transfection, Control, MANN-WHITNEY, Western Blot

Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA.

doi: 10.3390/cells13010077

Figure Lengend Snippet: Figure 8. Immunophenotype of Tregs expressing only one FoxP3 splice variant. The expression of Treg-associated cell markers was determined by flow cytometry in Tregs four days after transfection with each of the SSOs. Cell membrane markers were (A) CD4High, (B) CD25High, (C) CD127Low, and (D) CD152High. Cell membrane markers associated with Treg suppressive activity were (E) CD39High

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Expressing, Variant Assay, Flow Cytometry, Transfection, Membrane, Activity Assay

Journal: Cells

Article Title: Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA.

doi: 10.3390/cells13010077

Figure Lengend Snippet: Figure 11. Levels of mRNA of molecules associated with Treg suppressive activity. Total mRNA was isolated from Tregs transfected with oligonucleotides and analyzed by real-time RT-PCR. The mRNA levels of (A) CTLA4, (B) LGALS9, and (C) NRP1 were normalized to the mean expression of three reference genes: 18S, GAPDH, and ACTB. n = 4. * p ≤0.05; ** p ≤0.01; *** p ≤0.005 vs. cells transfected with #Con1 & #Con2 oligonucleotides by the Mann–Whitney U test.

Article Snippet: The purity of the obtained Tregs was monitored by flow cytometry using a Treg Surface Marker Analysis Cocktail (Miltenyi Biotec, Bergisch Gladbach, Germany, #130-096-082) and by the detection of the nuclear markers FoxP3 and Helios described above.

Techniques: Activity Assay, Isolation, Transfection, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: Molecules

Article Title: Evaluation and Optimization of the Anti-Melanogenic Activity of 1-(2-Cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone Derivatives

doi: 10.3390/molecules24071372

Figure Lengend Snippet: Figure 4. Chalcone 21-21 inhibits the expression of melanogenesis-related proteins. (A,B) B16F10 cells were incubated for 24 h with vehicle (0.1% DMSO) alone or various concentrations of chalcone 21-21 in the absence or presence of α-MSH (10 nM). The levels of TRP1, TRP2, and MITF were determined by Western blot or RT-PCR analyses. GAPDH served as a loading control. (C) B16F10 cells were transiently transfected with a reporter construct of MITF together with the pRL-TK. The cells were incubated for 24 h with vehicle (0.1% DMSO) alone or with various concentrations of chalcone 21-21 in the absence or presence of α-MSH (10 nM). Luciferase activity was measured and firefly luciferase activities were normalized to that of Renilla luciferase. Results are represented as mean ±SD from three independent experiments (n = 3). p-values of # p < 0.005 compared to the vehicle-treated group and * p < 0.05 and ** p < 0.005 compared to the α-MSH-treated group were considered statistically significant.

Article Snippet: Primary antibodies specific for tyrosinase, TRP1, TRP2, and MITF were obtained from Novus Biologicals, and phospho-ERK1/2, ERK1/2, phospho-CREB, CREB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Construct, Luciferase, Activity Assay

Journal: Molecules

Article Title: Evaluation and Optimization of the Anti-Melanogenic Activity of 1-(2-Cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone Derivatives

doi: 10.3390/molecules24071372

Figure Lengend Snippet: Figure 6. A schematic diagram illustrating the proposed action mechanisms of chalcone 21-21. Chalcone 21-21 specifically targets the ERK/CREB signaling, thereby inhibiting MITF and MITF-regulated target genes including those encoding tyrosinase, TRP1, and TRP2. AC, adenylate cyclase; α-MSH, alpha-melanocyte stimulating hormone; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; ERK, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; MC1R, melanocortin receptor type 1; MITF, microphthalmia-associated transcription factor; PKA, protein kinase A; ROS, reactive oxygen species; TRP, tyrosinase-related protein; TYR, tyrosinase.

Article Snippet: Primary antibodies specific for tyrosinase, TRP1, TRP2, and MITF were obtained from Novus Biologicals, and phospho-ERK1/2, ERK1/2, phospho-CREB, CREB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Binding Assay