Journal: Biomaterials

Article Title: Lipopolyplex potentiates anti-tumor immunity of mRNA-based vaccination

doi: 10.1016/j.biomaterials.2017.02.019

Figure Lengend Snippet: Stimulation of DC antigen cross-presentation by the LPP/mRNA vaccine a ) Flow cytometry analysis on H-2k b -OVA 257–264 presentation. b ) Time-dependent IL-2 secretion by OVA-specific CD4 + and CD8 + T cells after co-incubation with post-treated BMDCs. B3Z: OVA-specific CD8 + T cell; DOBW: OVA-specific CD4 + T cell. c ) Time-dependent IL-2 secretion by OVA-specific CD4 + and CD8 + T cells after co-incubation with post-treated DC2.4 cells. d ) Comparison of IL-2 secretion by OVA-specific CD8 + T cells after co-incubation with DC2.4 cells pretreated with LPP/OVA mRNA or LPP/OVA protein.

Article Snippet: DC2.4 served as the antigen presenting cells and mRNA molecules encoding the eGFP protein was applied to prepare the polyplex core.

Techniques: Flow Cytometry, Cytometry, Incubation

Journal: Biomaterials

Article Title: Lipopolyplex potentiates anti-tumor immunity of mRNA-based vaccination

doi: 10.1016/j.biomaterials.2017.02.019

Figure Lengend Snippet: Preferential uptake of the lipopolyplex mRNA vaccine by dendritic cells (a) Flow cytometry analysis on GFP-positive cells after DC2.4, MDA-MB-231 and mDMEC cells were incubated with lipopolyplex/eGFP mRNA for 24 hours. (b) Bioluminescent image to detect luciferase expression in mice 48 hours after s.c. injection of LPP/Luc. Left: control mouse; right: LPP/Luc-treated mouse.

Article Snippet: DC2.4 served as the antigen presenting cells and mRNA molecules encoding the eGFP protein was applied to prepare the polyplex core.

Techniques: Flow Cytometry, Cytometry, Multiple Displacement Amplification, Incubation, Luciferase, Expressing, Mouse Assay, Injection

Journal: Biomaterials

Article Title: Lipopolyplex potentiates anti-tumor immunity of mRNA-based vaccination

doi: 10.1016/j.biomaterials.2017.02.019

Figure Lengend Snippet: Mechanism of cell entry ( a – h ) Images of DC2.4 cells treated with LPP/0.5 μg FAM-labeled eGFP mRNA for 4 hours in presence of (a) mock control, (b) amiloride, (c) chlorpromazine, (d) chloroquine, (e) genistein, (f) pimozide, (g) nocodazole, and (h) cytochalasin D. ( i ) Image J analysis on FAM-positive cells. ( j ) Time-dependent uptake of LPP/FAM-labeled eGFP mRNA by DC2.4 cells. Error bars represent the mean ± standard deviation of triplicate experiments.

Article Snippet: DC2.4 served as the antigen presenting cells and mRNA molecules encoding the eGFP protein was applied to prepare the polyplex core.

Techniques: Labeling, Standard Deviation

Journal: Biomaterials

Article Title: Lipopolyplex potentiates anti-tumor immunity of mRNA-based vaccination

doi: 10.1016/j.biomaterials.2017.02.019

Figure Lengend Snippet: Structure and characterization of lipopolyplex mRNA vaccine ( a ) Schematic view of the lipopolyplex mRNA vaccine. The vaccine is composed of a polyplex core assembled through electrostatic interaction between the positively charged PbAE polymer and the negatively charged mRNA molecule. The polyplex is encapsulated into a lipid shell. ( b ) Gel retardation assay on polyplex-mRNA binding. Samples were loaded in the following order: free mRNA, polyplex/mRNA with 10, 20, 30 and 40 (w/w). ( c–e ) TEM images of the empty liposomal shell (c), polyplex/mRNA core (w/w=20) (d), and lipopolyplex/mRNA core-shell structure (e). ( f–k ) eGFP expression in DC2.4 cells treated with mRNA-packaged particles. DC2.4 cells were treated with (f) PBS control, g) PbAE/eGFP mRNA core, h) EDOPC/DOPE-packaged PbAE/eGFP mRNA, i) DOTAP/Chol-packaged PbAE/eGFP mRNA, j) CHEMS/DOPE/R8-packaged PbAE/eGFP mRNA or k) protamine/eGFP mRNA core, and eGFP expression was detected under a fluorescent microscope 24 hours later. l) DC2.4 viability upon treatment with the different particles.

Article Snippet: DC2.4 served as the antigen presenting cells and mRNA molecules encoding the eGFP protein was applied to prepare the polyplex core.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Transmission Electron Microscopy, Expressing, Microscopy

Journal: Journal of Translational Medicine

Article Title: Stress hormones promote growth of B16-F10 melanoma metastases: an interleukin 6- and glutathione-dependent mechanism

doi: 10.1186/1479-5876-11-72

Figure Lengend Snippet: CRH expression and serum IL-6 levels in non-tumor- and B16-F10 (metastases)-bearing mice. Measurements were performed in non-tumor-bearing and in tumor-bearing mice (7 days after inoculation). mRNA expression of CRH in the hypothalamic PVN was evaluated as described under Materials and methods. CRH expression data (optical density arbitrary units, AU), measured at 0 h (circadian time, see Figure 1 ), are expressed as mean values + S.D. (error bars) of 6–7 different animals. For IL-6 levels determination blood was collected from the tail vein every 6 h during the 24-h period of the indicated day, and data are mean values of the peak serum cytokine concentrations + S.D. ( error bars ) (pg/ml) measured in 7–8 different animals. *P

Article Snippet: The siRNA molecules targeting IL-6 mRNA were purchased from Ambion.

Techniques: Expressing, Mouse Assay

Journal: PLoS ONE

Article Title: Resolving Cell Population Heterogeneity: Real-Time PCR for Simultaneous Multiplexed Gene Detection in Multiple Single-Cell Samples

doi: 10.1371/journal.pone.0006326

Figure Lengend Snippet: Sensitivity of detecting numbers of DNA and RNA molecules. Real-time PCR was performed over a concentration range from A) TNF DNA standard template or B) TNF mRNA standard template. cDNA synthesis was performed on the dilution series of mRNA samples and the difference in signal between the same amount of input mRNA (▪) versus input DNA (ο) indicates a reverse transcription efficiency of 54% in this experiment. For each copy number, the mean and standard deviation are shown for 12 samples in A) and 4 samples in B). The Y-axis is a log 10 rescaling of the C t values.

Article Snippet: Single samples also can be processed for real-time PCR on the Fluidigm instrument to count mRNA molecules , .

Techniques: Real-time Polymerase Chain Reaction, Concentration Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Resolving Cell Population Heterogeneity: Real-Time PCR for Simultaneous Multiplexed Gene Detection in Multiple Single-Cell Samples

doi: 10.1371/journal.pone.0006326

Figure Lengend Snippet: Sensitivity for single-cell mRNA measurements. Macrophages were activated with the bacterial stimulus, lipopolysaccharide (30 ng/ml for 2 hours), and the indicated number of cells (1, 10 or 100 cells) were sorted by flow cytometry. mRNA expression of the indicated genes was measured by real-time PCR using 1/8 th of the sample cDNA lysate per measurement. The mean and standard deviation of 12 samples are presented for each of the indicated number of cells. The C t values were arbitrarily scaled to log 10 values (y-axis).

Article Snippet: Single samples also can be processed for real-time PCR on the Fluidigm instrument to count mRNA molecules , .

Techniques: Flow Cytometry, Cytometry, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Doxorubicin cardiomyopathy is ameliorated by acacetin via Sirt1‐mediated activation of AMPK/Nrf2 signal molecules, et al. Doxorubicin cardiomyopathy is ameliorated by acacetin via Sirt1‐mediated activation of AMPK/Nrf2 signal molecules

doi: 10.1111/jcmm.15859

Figure Lengend Snippet: Effects of acacetin on Sirt1 and the dominant signal molecules. A, Western blots and relative level of Sirt1 in rat cardiomyoblasts in the absence and presence of 0.3, 1 or 3 μM acacetin. B, Western blots and mean relative levels of Sirt1 in rat cardiomyoblasts treated with 1 μM doxorubicin in the absence and presence of 0.3, 1 or 3 μM acacetin. C, Western blots and mean relative levels of pAMPK in rat cardiomyoblasts with the same treatment as in B. D, Western blots and mean relative levels of pLKB in rat cardiomyoblasts with the same treatment as in B. E, Western blots and relative level of pLKB and total LKB (t‐LKB) in rat cardiomyoblasts transfected with control siRNA or Sirt1 siRNA in the absence (V, vehicle) or presence of 3 μM acacetin. F, Western blots and relative level of pAMPK and total AMPK (t‐AMPK) in rat cardiomyoblasts transfected with control siRNA or Sirt1 siRNA and treated as in E. G, Western blots and relative level of Nrf2 in rat cardiomyoblasts with the same treatment as in E. H, Western blots and relative level of nuclei Nrf2 in rat cardiomyoblasts with the same treatment as in E. I, Relative Nrf2 mRNA level measured with real‐time PCR in rat cardiomyoblasts with the same treatment as in E. (n = 5 individual experiments, * P

Article Snippet: Small interfering RNA (siRNA) molecules targeting rat Sirt1 mRNA (sc‐108043) was from Santa Cruz Biotechnology (Dallas, TX, USA), whereas siRNA molecules targeting rat AMPK and scrambled control siRNA were synthesized by RiboBio (Guangzhou, Guangdong, China).

Techniques: Western Blot, Transfection, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

doi: 10.3390/ijms20030659

Figure Lengend Snippet: CD200 restrains p38 signaling upon Staphylococcal infection. ( A – L ) Mouse PEMs were transfected with CD200 siRNA (siCD200) or control siRNA (NC) for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK, p-TAK1, TAK1, p-MMK3/6, MMK3/6, p-CREB, CREB, p-MSK1, MSK, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( M – Q ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods. The protein expression levels of p-p38, p38, p-c-Fos, c-Fos, p-c-Jun, and c-Jun were detected by western blotting. ( R ) RAW264.7 macrophages were pre-transfected with CD200 siRNA for 48 h and then challenged with S. aureus (MOI = 10) for 2 h. The AP-1 luciferase activity was analyzed by the dual luciferase. Representative blots from three independent experiments are shown ( A , E , J , M , and O ). Changes in the phosphorylation state of different proteins were semi-quantitated using ImageJ software ( B – D , F – I , K – L , N , P – Q ). ND means not detected; * p

Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

Techniques: Infection, Transfection, Expressing, Western Blot, Luciferase, Activity Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

doi: 10.3390/ijms20030659

Figure Lengend Snippet: The p38 inhibition largely abolished the effects of CD200 on NO synthesis induced by staphylococcal infection. ( A – D ) Mouse PEMs ( A , B ) or BMDMs ( C , D ) were pre-treated with SB239063 (10 μM, 0.5 h) or vehicle (DMSO), and then treated with S. aureus for indicated time periods (0–18 h). The mRNA level of iNOS was detected by qPCR, and the concentration of NO in culture supernatants was determined using Griess reagent. ( E , F ) Mouse PEMs were transfected with CD200 siRNA or NC for 48 h and treated with SB239063 or vehicle (DMSO) for 0.5 h. Cells were then infected with S. aureus for indicated time periods (0–12 h). The mRNA level of iNOS and the concentration of NO in culture supernatants were analyzed. Results are expressed as the mean ± SD of three independent experiments; * p

Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

Techniques: Inhibition, Infection, Real-time Polymerase Chain Reaction, Concentration Assay, Transfection

Journal: International Journal of Molecular Sciences

Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

doi: 10.3390/ijms20030659

Figure Lengend Snippet: CD200 inhibits the production of inflammatory cytokines induced by S. aureus . Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). ( A – F ) Relative mRNA expression levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. Results are expressed as the mean ± SD of three independent experiments; * p

Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

doi: 10.3390/ijms20030659

Figure Lengend Snippet: Knockdown of CD200 enhances S. aureus -induced inflammatory responses. Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). ( A – F ) Relative mRNA levels of IL-1β ( A ), IL-6 ( B ), TNF-α ( C ), IL-12p40 ( D ), IL-10 ( E ), or CXCL1 ( F ) were detected by qPCR, with β-actin as an internal control. ( G – L ) The amount of IL-1β ( G ), IL-6 ( H ), TNF-α ( I ), IL-12 ( J ), IL-10 ( K ), or CXCL1 ( L ) in the cell culture supernatant was determined by ELISA. ( M ) The CD200 knockdown efficiency was detected by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p

Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

doi: 10.3390/ijms20030659

Figure Lengend Snippet: CD200 signaling inhibits NO synthesis and bactericidal activity of S. aureus -infected macrophages. ( A – C ) Mouse BMDMs were pre-treated with CD200-Fc (2 μg/mL) or IgG1-Fc (2 μg/mL) for 1 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–12 h). Relative mRNA levels of Arg1 ( A ) or iNOS ( B ) were detected by qPCR. NO release was determined using Griess reagent system ( C ). ( D – F ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–18 h). Arg1 ( D ) and iNOS ( E ) mRNA levels and NO release ( F ) were determined. ( G ) PEMs transfected with siCD200 or NC siRNA were challenged with CFSE-labeled S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were collected, fixed, and stained with DAPI. Intracellular bacterial were observed using a fluorescence microscope. A partially enlarged view was shown in the lower left corner. Scale bars: 10 µm. Shown are representative images from three independent experiments. ( H ) Mouse PEMs were transfected with siCD200 or NC siRNA for 48 h, and then challenged with S. aureus (MOI = 10) for indicated time periods (0–18 h). Cells were lysed and the intracellular bacterial burden was determined by CFU counting. Results are expressed as the mean ± SD of three independent experiments; * p

Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

Techniques: Activity Assay, Infection, Real-time Polymerase Chain Reaction, Transfection, Labeling, Staining, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

doi: 10.3390/ijms20030659

Figure Lengend Snippet: S. aureus infection induces CD200 expression in murine macrophages. Mouse BMDMs ( A , D ), PEMs ( B , E ), or RAW264.7 cells ( C , F ) were challenged with S. aureus (MOI = 1) for the indicated time periods (0–18 h), or with S. aureus at the indicated MOIs (0–20) for 6 h. Cells were then collected and detected for CD200 mRNA level by qPCR. Results are expressed as the mean ± SD of three independent experiments; * p

Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

Techniques: Infection, Expressing, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: CD200 Modulates S. aureus-Induced Innate Immune Responses Through Suppressing p38 Signaling

doi: 10.3390/ijms20030659

Figure Lengend Snippet: CD200 mildly affects NF-κB activation triggered by Staphylococcal infection. Mouse PEMs were transfected with CD200 siRNA or NC siRNA for 48 h, and then stimulated with S. aureus (MOI = 1) for indicated time periods (0–2 h). ( A ) Cells were lysed, and the protein level of p-p65, p65, p-IκBα, or IκBα was detected by western blotting. β-Actin served as a loading control. ( B ) Immunofluorescence was performed to visualize p65 nuclear translocation. Scale bars: 10 µm. Representative images of three independent experiments are shown. The phosphorylation state of p65 was semi-quantitated using ImageJ software. ND means not detected; * p

Article Snippet: Transfection of siRNA The siRNA molecules targeting CD200 mRNA were purchased from GenePharma Corporation (Shanghai, China), and delivered into macrophages using X-tremeGENE siRNA transfection reagent (Roche, Basel, Switzerland), according to the manufacturer’s instructions.

Techniques: Activation Assay, Infection, Transfection, Western Blot, Immunofluorescence, Translocation Assay, Software

Journal: PLoS Pathogens

Article Title: The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes

doi: 10.1371/journal.ppat.1006456

Figure Lengend Snippet: RNAi depletion of ALPH1 causes growth arrest and increase in mRNA levels. RNAi depletion of TbALPH1 was induced by tetracycline (TET). Three independent clonal cell lines were analysed over a time-course of RNAi induction. (A) Reduction in the number of ALPH1 mRNA molecules. ALPH1 mRNA and DBP1 mRNA (control) were visualized by dual colour single molecule mRNA FISH (Affymetrix), using green ( ALPH1 ) and red ( DBP1 ) fluorescent Affymetrix probe sets. The number of mRNA molecules per cell was quantified after 0, 24 and 48 hours of ALPH1 RNAi induction. Data are presented as box plot (waist is median; box is IQR; whiskers are ±1.5 IQR; only the smallest and largest outliers are shown; n = 100 for each time-point); the number of mRNAs from the individual cells is also presented as green ( ALPH1 ) or red ( DBP1 ) dots. One representative cell for each time-point is shown. The data are from one RNAi clone; data of a second clone are shown in S12 Fig . (B) Growth arrest. Growth was measured over a time-course of ALPH1 RNAi induction (±TET). Averages of the three clonal cell lines are shown; error bars indicate standard deviations between the three cell lines. (C-E) Increase in mRNAs. Total RNA was isolated over a time-course of ALPH1 RNAi and as a control from bloodstream form trypanosomes (BSF) and analysed by northern blots. The blots were probed for total mRNA with an oligo antisense to the miniexon of the spliced leader RNA (C), for PGKC (D) and for GPI-PLC (E). mRNA abundances were quantified by Odyssey (total mRNA) or phosphorimager ( PGKC , GPI-PLC ). rRNA was used as a loading control and all samples were calibrated to the amount of BSF RNA (= 1). Average values of the three clones are shown, standard deviations are presented as error bars. For each probe, one representative northern blot is shown. Note that the three red bands in C) are not rRNA bands, but are caused by a squeezing of the mRNAs due to the very abundant rRNA.

Article Snippet: The extreme 5’ and 3’ ends of this mRNA are simultaneously probed with a red and green fluorescent single molecule mRNA FISH probe (Affymetrix), respectively.

Techniques: Fluorescence In Situ Hybridization, Isolation, Northern Blot, Planar Chromatography, Clone Assay

Journal: PLoS Pathogens

Article Title: The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes

doi: 10.1371/journal.ppat.1006456

Figure Lengend Snippet: RNAi depletion of ALPH1 causes an increase in intact mRNA molecules, but no increase in 5’-3’ decay intermediates. (A) Experimental design: The endogenous very long and short-lived mRNA Tb427.01.1740 was used as a reporter for mRNA metabolism [ 55 ]. Simultaneous probing of the extreme 5’ and 3’ ends with red and green fluorescent single mRNA FISH probes results in yellow, green or red fluorescent mRNA molecules, corresponding to intact mRNAs, mRNAs in 5’-3’ decay and mRNAs in transcription or 3’-5’ decay, respectively. (B) The numbers of yellow, green and red fluorescent spots were quantified from cells after induction of ALPH1 RNAi (0, 24 or 48 hours TET) from at least 130 cells per time-point, for three RNAi clones. Average data are shown; error bars represent the standard deviations between the different RNAi clones. The numbers of the differently coloured spots per total cell are shown (left), but also the numbers of spots in the nucleus (middle) or in the cytoplasm (right). If the centre of a spot was overlapping with the DAPI stained nucleus on a Z-stack projection image, the spot was defined as nuclear, otherwise as cytoplasmic. (C) Representative Z-stack projection images of untreated cells (no TET) and cells after 48 hours of ALPH1 RNAi (48 h TET).

Article Snippet: The extreme 5’ and 3’ ends of this mRNA are simultaneously probed with a red and green fluorescent single molecule mRNA FISH probe (Affymetrix), respectively.

Techniques: Fluorescence In Situ Hybridization, Clone Assay, Staining

Journal: Nucleic Acids Research

Article Title: Simultaneous detection of mRNA transcription and decay intermediates by dual colour single mRNA FISH on subcellular resolution

doi: 10.1093/nar/gkw1245

Figure Lengend Snippet: Technical controls and the definition of a yellow spot. ( A ) Red and green fluorescence in situ hybridization (FISH) probes were designed to anneal to the same mRNA sequence of an average-sized mRNA (Tb427.10.14550). The signals from the two probes showed a strong overlap. One representative cell is shown; more cells are shown in Supplementary Figure S2 . ( B ) Red and green FISH probes were designed to anneal to adjacent mRNA sequences of the same, average-sized mRNA. The signals from the two probes again showed a strong overlap. One representative cell is shown; more cells are shown in Supplementary Figure S3 . ( C–E ) Trypanosome cells were simultaneously probed with the 5΄ probe (red) and the 3΄ probe (green) specific for the GB4 mRNA (Tb427tmp.160.1200) (details on top left of C). Together, these probes cover the entire 24 645 nucleotides of the GB4 open reading frame: the 5΄ probe binds to the 2256 nucleotides at the 5΄ end and the 3΄ probe to the remaining 22 389 nucleotides. ( C ) One representative Z-stack projection image with merged fluorescence channels is shown. ( D ) The length of 100 yellow GB4 mRNA molecules was measured from Z-stack projection images. In total, 97% are less than 0.5 μm in length (D). ( E ) Example images of very extended GB4 mRNA molecules; the length is indicated on top and the blue line shows how the length measurement was performed (from the middle of the ‘red circle’ to the middle of the most distant ‘green circle’ at the end of the green string). These very long mRNA molecules are rare and never longer than 1.0 μm. ( F ) Trans-probing: dual colour mRNA FISH using probes antisense to two different mRNAs (details on top left). The percentage of red, green and yellow spots was quantified from 152 cells (top right). The average cell had 7.3 ± 2.8 mRNA molecules. A representative image is shown (bottom).

Article Snippet: Affymetrix® single molecule mRNA FISH probe sets were designed antisense to the 1100 most 5΄ nucleotides (red fluorescence) and the 1100 most 3΄ nucleotides (green fluorescence) of Tb427.01.1740 and Tb427.04.310.

Techniques: Fluorescence, In Situ Hybridization, Fluorescence In Situ Hybridization, Sequencing

Journal: Oncotarget

Article Title: Small molecule/ML327 mediated transcriptional de-repression of E-cadherin and inhibition of epithelial-to-mesenchymal transition

doi:

Figure Lengend Snippet: Treatment with ML327 partially reverses TGF-β-induced EMT A. Bright field microcopy images (200x magnification) showing the cell morphology after 72 hours TGF- β1 treatment. B. Immunofluorescence images showing E-cadherin (red) expression and localization in NMuMg cells following treatment without or with 5ng/mL TGF-β1 for 72 hours, then adding either DMSO, or 10 μM ML327 for an additional 48 hours. Nuclei (blue) are labeled with DAPI. Bar = 100 μM. C. Relative levels of E-cadherin, Occludin and Vimentin specific mRNA species in NMuMg cells following treatments shown in B. , the graph shows mean values with standard error bars from 4 replicate wells in a representative experiment. Statistical significance was calculated using unpaired t test **** indicates p

Article Snippet: Small-molecule ML327 modulates E-cadherin mRNA levels independently of de novo protein synthesis We previously reported that treatment of SW620 cells with first generation molecules resulted in significant induction of E-cadherin mRNA by 24 hours after exposure [ ].

Techniques: Immunofluorescence, Expressing, Labeling

Journal: Oncotarget

Article Title: Small molecule/ML327 mediated transcriptional de-repression of E-cadherin and inhibition of epithelial-to-mesenchymal transition

doi:

Figure Lengend Snippet: Treatment with ML327 inhibits tumor cell migration in vivo A. Relative E-cadherin mRNA expression in HEp3 cells following 6 hours treatment with either 266Y or ML327, the graph shows mean values with standard error bars from 3 replicate wells in a representative experiment, statistical significance was calculated using unpaired t test, *** indicates p

Article Snippet: Small-molecule ML327 modulates E-cadherin mRNA levels independently of de novo protein synthesis We previously reported that treatment of SW620 cells with first generation molecules resulted in significant induction of E-cadherin mRNA by 24 hours after exposure [ ].

Techniques: Migration, In Vivo, Expressing

Journal: Oncotarget

Article Title: Small molecule/ML327 mediated transcriptional de-repression of E-cadherin and inhibition of epithelial-to-mesenchymal transition

doi:

Figure Lengend Snippet: ML327 increases E-cadherin expression in SW620inv colon cancer cells A. Quantitative PCR results for E-cadherin specific mRNA in SW620inv cells following treatment with ML327 at the indicated time periods (data points represent results from3 biological replicates). Fold change relative to DMSO treatment is determined by the formula log2 −ΔΔCp . B. Western blot shows time dependent changes in SW620inv E-cadherin (E-cad) protein expression relative to β-actin following treatment with DMSO, 10 μM ML327, or 266Y. C. Quantitative PCR analysis of E-cadherin mRNA in SW620inv cells following 1 hour with or without CHX pretreatment, then adding DMSO, ML327, or 266Y for another 6 hours. Fold change in E-cadherin mRNA, relative to DMSO without CHX is calculated by the formula log2 −ΔΔCp . Graphed data represent four independent experiments. D. ICW plate matched to C. showing quantification of cyclin D1 protein (green) and β-actin (red). E. The graph shows quantification of relative cyclin D1 signal matched to D. .

Article Snippet: Small-molecule ML327 modulates E-cadherin mRNA levels independently of de novo protein synthesis We previously reported that treatment of SW620 cells with first generation molecules resulted in significant induction of E-cadherin mRNA by 24 hours after exposure [ ].

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot