Journal: The Journal of biological chemistry

Article Title: Uncoupling ceramide glycosylation by transfection of glucosylceramide synthase antisense reverses adriamycin resistance.

doi: 10.1074/jbc.275.10.7138

Figure Lengend Snippet: FIG. 2. Expression of GCS mRNA and protein in MCF-7-AdrR cell variants. A, mRNA expression of GCS. Isolated mRNA (5 ng) was amplified by high fidelity RT-PCR. The reverse PCR product, a 300- base pair fragment of GCS, was resolved on 1% agarose gel electro- phoresis and stained with ethidium bromide (top panel). Housekeeper gene b-actin was used as a control for even loading (bottom panel). Control, RT-PCR product without cellular mRNA; MCF-7-AdrR, MCF- 7-AdrR parental cells; AdrR/asGCS, MCF-7-AdrR GCS antisense transfected cells. B, GCS Western blot. GCS (50 mg of protein/lane) was resolved using 4–20% SDS-polyacrylamide gel electrophoresis and re- acted with GCS polyclonal antibody (1:1,000). AdrR/GCS, MCF-7-AdrR cells transfected with GCS cDNA (pcDNA 3.1/his A-GCS); MCF-7- AdrR, the parent cell line; AdrR/asGCS, GCS antisense-transfected MCF-7-AdrR cells. C, Western blots of anti-Xpress antibody. Blots were done as described above. The Xpress fused protein was reacted with Xpress antibody (1:500). Abbreviations are as in B.

Article Snippet: RNA Analysis—Cellular mRNA was purified using a mRNA isolation kit (Roche Molecular Biochemicals).

Techniques: Expressing, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Control, Transfection, Western Blot, Polyacrylamide Gel Electrophoresis

Journal: Cell

Article Title: SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses

doi: 10.1016/j.cell.2020.10.004

Figure Lengend Snippet:

Article Snippet: HiScribe T7 ARCA mRNA Kit , NEB , Cat. # E2060S.

Techniques: Virus, Recombinant, Transfection, RNA Sequencing Assay, Software

Journal: PLoS Pathogens

Article Title: The RNA helicase DDX5 promotes viral infection via regulating N 6 -methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity

doi: 10.1371/journal.ppat.1009530

Figure Lengend Snippet: (A, B): m6A methylation of transcripts was detected by m6A qRT-PCR in DDX5-knockdown or DDX5 overexpressed MEFs after VSV infection. MEFs were transfected with DDX5 siRNA (siNC) for 48hand infected with VSV for 6h (A), and MEFs were transfected with Myc-DDX5 expressed vector (DDX5) or Myc tag control vector (Con) for 24h and infected with VSV for 6h (B). After extracting total RNA, purifying mRNA, and removing ribosomal RNA, purified mRNA was fragmented and incubated with anti-rabbit m6A or anti-rabbit IgG-conjugated dynabeads for 4h. RNA was isolated from the solution with phenol-chloroform, and cDNA was subjected to qRT-PCR using GAPDH, TBK1, DHX58, IKKγ, and p65 primers. Results are presented relative to those obtained with NC or control groups, and the expression of all the indicated proteins was analyzed using western blotting. (C, D): The interaction between METTL3 and transcripts was detected through METTL3 RIP qRT-PCR in knockdown-DDX5 (C) or DDX5-expressing (D) MEFs after VSV infection. MEFs were transfected with DDX5 siRNA (siNC) for 48 h and infected with VSV for 6 h (C), and transfected with DDX5 expression plasmid (DDX5) or control vector (Con) for 24 h, infected with VSV for 6h, and subjected to METTL3 RIP qRT-PCR to detect GAPDH, TBK1, DHX58, IKKγ, and p65. Results are presented relative to those obtained with NC or control groups, and the expression of all the indicated proteins was analyzed using western blotting. (E, F): Nuclear transcript retention increased in DDX5-knockdown MEFs. MEFs were transfected with DDX5 siRNA (siNC), infected with VSV for 8h, and lysed to extract nuclear to cytoplasmic RNA fractions. Then, RNA was used to analyze m6A modified DHX58, IKKγ, and p65 mRNA by m6A qRT-PCR (E) with RNU6 and GAPDH as the nuclear and cytoplasmic controls, respectively. The quantitative distribution of m6A modified DHX58, IKKγ, and p65 mRNAs in DDX5-knockdown MEFs were detected by m6A qRT-PCR (F). (G, H): Nuclear transcript export was increased in DDX5-expressing MEFs. MEFs were transfected with DDX5 expression plasmid (control vector), infected with VSV for 8h, and lysed to extract nuclear or cytoplasmic RNA; then, RNA was used to analyze m6A modified DHX58, IKKγ, and p65 mRNA by m6A qRT-PCR (G), and the quantitative distribution of these mRNAs was detected by m6AqRT-PCR (H). (I, J) : Immunoblot analysis of DHX58, IKKγ, and p65 in DDX5-knockdownMEFs (I) or DDX5-expressing MEFs (J) after infection with VSV at 0, 4, and 6 h. All data are mean ± SEM of biologically independent samples. Data are representative of three independent experiments. ns, no significant difference. * p <0.05, ** p <0.01, and *** p <0.001 (Student’s t -test).

Article Snippet: Biotin-labeled RNA was detected and visualized according to the instructions of the chemiluminescent nuclei acid detection module (Thermo Fisher, 89880), the biotin-unlabeled RNA was acquired according to the biotin-labeled protein–RNA complex blotting, and mRNAs were purified with the Dynabeads mRNA Purification Kit (Invitrogen, 61006).

Techniques: Methylation, Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, Purification, Incubation, Isolation, Expressing, Western Blot, Modification

Journal: PLoS Pathogens

Article Title: The RNA helicase DDX5 promotes viral infection via regulating N 6 -methyladenosine levels on the DHX58 and NFκB transcripts to dampen antiviral innate immunity

doi: 10.1371/journal.ppat.1009530

Figure Lengend Snippet: (A): m6A methylation of transcripts was detected in DDX5 +/+ or DDX5 +/- primary mouse macrophages infected for 8 h with VSV (MOI = 10). After extracting total RNA and purifying mRNA, mRNA was used to perform m6A qRT-PCR by incubating with anti-rabbit m6A or anti-rabbit IgG-conjugated dynabeads for 4 h. RNA was isolated and subjected to qRT-PCR using GAPDH, TBK1, DHX58, IKKγ, and p65 primers. Results are presented relative to those obtained in the control group, and the expression of DDX5 was analyzed bywestern blotting. (B): Immunoblot analysis of DDX5, DHX58, p65, and IKKγ in lysates of DDX5 +/+ or DDX5 +/- mouse macrophages infected for 0, 4, and 8 h with VSV (MOI = 10). (C, D): ELISA of IFN-β (C) and IL-6 (D) in cell supernatants ofDDX5 +/+ or DDX5 +/- mouse macrophages infected for 0, 4, and 8 h with VSV (MOI = 10). (E, F): ELISA of IFN-β (E) and IL-6 (F) in serum after DDX5 +/+ or DDX5 +/- mice were intraperitoneally injected with PBS or VSV (5×10 8 plaque-forming units/g body weight) for 8h (n = 6). (G, H): ELISA of IFN-β (G) and IL-6 (H) in serum after DDX5 +/+ or DDX5 +/- mice were intraperitoneally injected with PBS or SeV (1×10 8 plaque-forming units/g body weight) for 8h (n = 6). (I, J): The TCID 50 dose of VSV (I) or SeV (J) was measured in lungs, liver, and spleen of DDX5 +/+ or DDX5 +/- mice. (K): Pathological lesions in lungs, liver, and spleen of DDX5 +/+ or DDX5 +/- mice observed by hematoxylin-eosin staining with intraperitoneal injection of PBS, VSV (5×10 8 plaque-forming units/g body weight) or SeV (1×10 8 plaque-forming units/g body weight) for 12h. Scale bars, 100 μm. All data are presented as mean ± SEM of biologically independent samples. n = number of biological replicates. Data are representative of three independent experiments. NS, no significant difference. ** p <0.01, *** p <0.001 (Student’s t -test).

Article Snippet: Biotin-labeled RNA was detected and visualized according to the instructions of the chemiluminescent nuclei acid detection module (Thermo Fisher, 89880), the biotin-unlabeled RNA was acquired according to the biotin-labeled protein–RNA complex blotting, and mRNAs were purified with the Dynabeads mRNA Purification Kit (Invitrogen, 61006).

Techniques: Methylation, Infection, Quantitative RT-PCR, Isolation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Injection, Staining

Journal: Nanoscale Horizons

Article Title: A modular polymer platform for efficient mRNA delivery in cancer immunotherapy

doi: 10.1039/d5nh00299k

Figure Lengend Snippet: In vivo therapeutic efficacy and biosafety of PFHA-PEI-mRNA-HP nanoparticles. (a) Diagram of the treatment schedule for the in vivo luciferase mRNA transfection study. (b) IVIS imaging of mice 5 h post-subcutaneous injection of PFHA-PEI-mRNA-HP nanoparticles containing 15 μg luciferase mRNA, with untreated mice as controls. (c) Diagram of the treatment schedule for the in vivo therapeutic study. (d) Tumor volume measurements in mice treated with anti-PD-L1 antibody, IL12 mRNA encapsulated in PFHA-PEI-mRNA-HP nanoparticles, or combination therapy, compared to the untreated controls. (e) Representative tumor images from different treatment groups on day 14. (f) Blood chemistry analysis of untreated and PFHA-PEI-mRNA-HP-treated mice. (g) Body weight monitoring of untreated and PFHA-PEI-mRNA-HP-treated mice over the treatment period, showing no significant weight loss.

Article Snippet: The plasmid DNA was transcribed using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs Inc, Ipswich, MA), and then purified with the Monarch RNA cleanup kit (New England Biolabs Inc, Ipswich, MA).

Techniques: In Vivo, Drug discovery, Luciferase, Transfection, Imaging, Injection

Journal: Nanoscale Horizons

Article Title: A modular polymer platform for efficient mRNA delivery in cancer immunotherapy

doi: 10.1039/d5nh00299k

Figure Lengend Snippet: Schematic of the synthesis of PFHA-PEI-mRNA-HP. (a) Reaction scheme for conjugating PFHA onto PEI via EDC/NHS coupling chemistry. For clarity, a monomeric PEI unit is shown rather than the full branched structure of 2 kDa PEI used in the synthesis. The schematic depicts conjugation to a primary amine, which is favored due to its higher nucleophilicity and accessibility. The PFHA : PEI ratio is not drawn to scale; the actual substitution was determined by 19 F NMR to be approximately 4.79 : 1 (see Fig. S5). (b) Illustration of the process of mRNA being condensed by PFHA-PEI. mRNA solution was loaded into a syringe and injected into the PFHA-PEI solution at a fixed flow rate (1 μL s −1 ) while the solution was stirred by a rotor tip (500 rpm) for homogeneous mixing. (c) Illustration of the process of embellishing the surface of PFHA-PEI-mRNA with HP. HP solution was loaded into a syringe and injected into the PFHA-PEI-mRNA solution at a slow flow rate (0.5 μL s −1 ) while the solution is stirred by a rotor tip (500 rpm) for homogeneous mixing.

Article Snippet: The plasmid DNA was transcribed using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs Inc, Ipswich, MA), and then purified with the Monarch RNA cleanup kit (New England Biolabs Inc, Ipswich, MA).

Techniques: Conjugation Assay, Injection

Journal: Nanoscale Horizons

Article Title: A modular polymer platform for efficient mRNA delivery in cancer immunotherapy

doi: 10.1039/d5nh00299k

Figure Lengend Snippet: Physicochemical characterization of PFHA-PEI-mRNA-HP. (a) FTIR spectra of PFHA, PEI and PFHA-PEI. The gray dashed box marks the region of the addition of characteristic peak patterns from the spectrum of PFHA to the spectrum of PEI. The gray dashed line indicates the presence of amide bonds between PFHA and PEI. (b) X ray photoelectron spectroscopy (XPS) spectrum of PFHA-PEI with peak fitting analysis. Hydrodynamic size (c), polydispersity index (d) and zeta potential (e) measurements of PEI-mRNA, PFHA-PEI-mRNA and PFHA-PEI-mRNA-HP with various HP amounts. For the labels on the x axis of (c)–(e), PEI represents PEI-mRNA. 0, 1, 2, and 5 correspond to PFHA-PEI-mRNA + 0, 1, 2, and 5 μg HP per μg mRNA, respectively. (f) Gel retardation assay of PEI-mRNA, PFHA-PEI-mRNA and PFHA-PEI-mRNA-HP (with different HP amounts) with free mRNA as the control. (g) Serum stability data of PEI-mRNA, PFHA-PEI-mRNA and PFHA-PEI-mRNA-HP with mRNA : HP ratio of 1 : 1 wt/wt All samples were placed in PBS supplemented with 10% v/v FBS solutions and incubated at 37 °C. (h) TEM images of PFHA-PEI-mRNA-HP (with different HP amounts) at high and low magnifications. The scale bars are 400 nm and 50 nm, respectively. (i) Size distribution profiles of PFHA-PEI-mRNA (no HP) and PFHA-PEI-mRNA-HP NPs from the low-magnification TEM images in (h).

Article Snippet: The plasmid DNA was transcribed using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs Inc, Ipswich, MA), and then purified with the Monarch RNA cleanup kit (New England Biolabs Inc, Ipswich, MA).

Techniques: Spectroscopy, Zeta Potential Analyzer, Electrophoretic Mobility Shift Assay, Control, Incubation

Journal: Nanoscale Horizons

Article Title: A modular polymer platform for efficient mRNA delivery in cancer immunotherapy

doi: 10.1039/d5nh00299k

Figure Lengend Snippet: Cell uptake and endosomal escape studies of PFHA-PEI-mRNA-HP on 3 different cancer cell types. All treatments were applied to cells at 37 °C for 12 h at an mRNA concentration of 2 μg mL −1 and 3D Z stacked confocal images were taken with a z-resolution of 0.5 μm. The blue color represents cell nuclei; green represents LysoTracker and red represents mRNA. (a) Z-stacked 3D images (top panel) and 3D-rendered models (bottom panel) of three cancer cell lines treated with PFHA-PEI-mRNA-HP. In the 3D rendered models, cell nuclei are presented as blue surface, and green and red spots represent endo-lysosomes and mRNA, respectively. (b) Cross-sectional images of the z-stacked 3D images in (a) viewing from coronal, sagittal and transverse planes with bright field image as the background. (c) Top-down view of the 3D-rendered model (a) excluding cell nuclei. 3D viewing, model rendering and colocalization analysis were performed on the IMARIS image analysis software (Oxford Instruments).

Article Snippet: The plasmid DNA was transcribed using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs Inc, Ipswich, MA), and then purified with the Monarch RNA cleanup kit (New England Biolabs Inc, Ipswich, MA).

Techniques: Concentration Assay, Software

Journal: Nanoscale Horizons

Article Title: A modular polymer platform for efficient mRNA delivery in cancer immunotherapy

doi: 10.1039/d5nh00299k

Figure Lengend Snippet: Cell viability test results of PFHA-PEI-mRNA-HP. (a) Quantitative Alamar Blue cell viability assay results on 4T1, HepG2 and M6 cells. Each cell type was treated with PEI-mRNA, PFHA-PEI-mRNA, PFHA-PEI-mRNA-HP and Lipofectamine 2000-mRNA at 0, 5, 1, 2 and 3 μg mL −1 for 24 h. The untreated cell viability was normalized to 100% for all cell lines. Statistical analysis was performed to determine if the difference between the data points from the Lipo-mRNA-treated cells and the data points from other treated cells was significant. (b) Representative bright field images of untreated, Lipofectamine 2000-mRNA-treated and PFHA-PEI-mRNA-HP-treated cells at 2 μg mL −1 mRNA concentration. Scale bar is 50 μm.

Article Snippet: The plasmid DNA was transcribed using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs Inc, Ipswich, MA), and then purified with the Monarch RNA cleanup kit (New England Biolabs Inc, Ipswich, MA).

Techniques: Viability Assay, Concentration Assay

Journal: Nanoscale Horizons

Article Title: A modular polymer platform for efficient mRNA delivery in cancer immunotherapy

doi: 10.1039/d5nh00299k

Figure Lengend Snippet: Transfection results on three different cancer cell lines. (a) Transfection images of PEI-mRNA, PFHA-PEI-mRNA and PFHA-PEI-mRNA-HP, with Lipofectamine 2000-mRNA as the positive control on 4T1, HepG2 and M6 cells. Scale bar is 100 μm. (b) Quantitative analysis of the transfection results presented in (a). Statistical analysis was performed by comparing each treatment groups to the positive control Lipo2000-mRNA group. (c) Flow cytometric quantitative analysis of the transfection efficiency of PFHA-PEI-mRNA-HP with Lipofectamine 2000-mRNA as the positive control on three cancer cell lines.

Article Snippet: The plasmid DNA was transcribed using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs Inc, Ipswich, MA), and then purified with the Monarch RNA cleanup kit (New England Biolabs Inc, Ipswich, MA).

Techniques: Transfection, Positive Control

Journal: Nanoscale Horizons

Article Title: A modular polymer platform for efficient mRNA delivery in cancer immunotherapy

doi: 10.1039/d5nh00299k

Figure Lengend Snippet: Transfection results on three additional cancer cell lines. (a) Transfection images of PEI-mRNA, PFHA-PEI-mRNA and PFHA-PEI-mRNA-HP, with Lipofectamine 2000-mRNA as the positive control on C6, SF763 and MCF7 cells. Scale bar is 100 μm. (b) Quantitative analysis of the transfection results presented in (a). Statistical analysis was performed by comparing each of the treatment groups to the positive control Lipo2000-mRNA group. (c) Flow cytometric quantitative analysis of transfection efficiency of PFHA-PEI-mRNA-HP, with Lipofectamine 2000-mRNA as the positive control on the additional three cancer cell lines.

Article Snippet: The plasmid DNA was transcribed using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs Inc, Ipswich, MA), and then purified with the Monarch RNA cleanup kit (New England Biolabs Inc, Ipswich, MA).

Techniques: Transfection, Positive Control

Journal: Nanoscale Horizons

Article Title: A modular polymer platform for efficient mRNA delivery in cancer immunotherapy

doi: 10.1039/d5nh00299k

Figure Lengend Snippet: Storage stability test above 0 °C on 4T1 and HepG2 cells. PFHA-PEI-mRNA-HP and Lipofectamine 2000-mRNA were prepared on day 0 and refrigerated at 4 °C throughout the course of the study. PFHA-PEI-mRNA-HP and Lipofectamine 2000-mRNA were allowed to equilibrate to room temperature before they were added to the 4T1 and HepG2 cell cultures at 2 μg mL −1 mRNA concentration on day 0, 1, 2, 3, 4, 7 and 15. (a) Fluorescent images of transfected cells. Images were collected 24 h after PFHA-PEI-mRNA-HP and Lipofectamine 2000-mRNA were added on each day. Scale bar is 100 μm. (b) Quantification of the fluorescence intensities shown in the images. Fluorescence intensities in each panel were normalized against the intensity at day 0, which was assigned as 100%.

Article Snippet: The plasmid DNA was transcribed using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs Inc, Ipswich, MA), and then purified with the Monarch RNA cleanup kit (New England Biolabs Inc, Ipswich, MA).

Techniques: Concentration Assay, Transfection, Fluorescence

Journal: Nanoscale Horizons

Article Title: A modular polymer platform for efficient mRNA delivery in cancer immunotherapy

doi: 10.1039/d5nh00299k

Figure Lengend Snippet: IL12 mRNA delivery to 4T1 cells. (a) Immunofluorescence images showing IL-12 protein intracellular expression in 4T1 cell 24 h after treatment with PFHA-PEI-IL12 mRNA-HP nanoparticles (bottom) compared to untreated cells (top). Cells were stained with DAPI (blue, nuclei), and IL-12 protein was detected using an anti-IL-12 antibody (red). Scale bar is 100 μm. (b) Quantification of secreted IL-12 protein in culture medium via ELISA. PFHA-PEI-IL12 mRNA-HP nanoparticle-treated (NP treated) cells exhibited ∼55-fold higher IL-12 expression compared to untreated controls. *** p < 0.001.

Article Snippet: The plasmid DNA was transcribed using the HiScribe® T7 ARCA mRNA Kit (New England Biolabs Inc, Ipswich, MA), and then purified with the Monarch RNA cleanup kit (New England Biolabs Inc, Ipswich, MA).

Techniques: Immunofluorescence, Expressing, Staining, Enzyme-linked Immunosorbent Assay