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: 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: 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

Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 1 Concentration-dependent induction of AA-related cell death in GPAT2-silenced cells. scr-MDA and sh-MDA cells were grown in 10% FBS DMEM supplemented with 50 or 100 µM AA for 24–48 h before the cell proliferation rate was measured via an MTT proliferation assay. The values represent the means ± SDs of 3 independent experiments (***P< 0.001, **P< 0.01)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: Concentration Assay, Proliferation Assay

Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 2 AA induces apoptosis only in GPAT2-silenced cells. scr-MDA and sh-MDA cells were treated with 100 µM AA for 48 h, and the percentage of apoptotic cells was determined by counting the number of apop totic and nonapoptotic cells via a TUNEL assay and hematoxylin staining (***P< 0.001)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: TUNEL Assay, Staining

Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 3 AA increases the activity of several caspases in GPAT2-silenced cells. sh-MDA cells were treated with 100 µM AA for 48 h, and the activity of cas pases 1, 2, 3, 5, 6, 8 and 9 was quantified using the colorimetric assay kit described in the Materials and Methods section. Staurosporine (ST) was used as a positive control for caspase-3 activation. The results are expressed as the mean ± SD of 3 independent experiments (***P< 0.001)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: Activity Assay, Colorimetric Assay, Positive Control, Activation Assay

Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 5 GPAT2 silencing alters the mRNA expression of genes involved in arachidonic acid metabolism. Total RNA was extracted from scr-MDA and sh- MDA cells, subjected to cDNA synthesis, and amplified via qRT‒PCR with primers for human PTGS2, ALOX5, AKR1C3 and EPHX2. The values represent the means ± SDs of 3 independent experiments (***P< 0.001, **P< 0.01)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: Expressing, cDNA Synthesis, Amplification

Journal: Lipids in health and disease

Article Title: The protumorigenic enzyme GPAT2 inhibits arachidonic acid-triggered apoptosis in breast cancer.

doi: 10.1186/s12944-024-02344-1

Figure Lengend Snippet: Fig. 6 Schematic model of arachidonic acid-induced apoptosis in GPAT2-silenced cells. The cellular level of unesterified arachidonic acid is a general mechanism by which apoptosis is regulated, and GPAT2 promotes carcinogenesis by decreasing this level. In scr-MDA cells, GPAT2 esterifies arachidonoyl- CoA to glycerol−3-phosphate, allowing these cells to survive AA treatment. Instead, sh-MDA cells overexpress eicosanoid-producing enzymes, and AA increases BNIP3 expression, which leads to apoptosis in GPAT2-silenced cells. (cyt. C: cytochrome C, AA: arachidonic acid)

Article Snippet: Cell lines stably expressing a small hairpin RNA targeting GPAT2 mRNA (shRNA-GPAT2) (HuSH−29 plasmid, OriGene) or a non-silencing scrambled RNA (shRNA-scr) were generated in our laboratory, as we previously reported [1], to generate sh-MDA (reduced GPAT2 expression) and scr-MDA (retaining GPAT2 expression) cell lines.

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Tetraspanin CD63 is a regulator of HIV-1 replication

doi:

Figure Lengend Snippet: CD63 mRNA down regulation by siRNA in human MDMs which are challenged with different concentration of HIV-1. A. MDMs (5 × 105 cells/well) were plated in 24-well plates and transfected with 50 nM siRNAs (CD63, CD4, and ERBB2IP). For controls, cells were treated with AZT (1 mM) or raltegravir (20 mM). Controls also included untreated cells or cells infected with HIV-1 SX (m.o.i. = 0.02) only. Total mRNA was isolated from each well using the Qiagen RNeasy kit 48 h after transfection. Quantitative RT-PCR was used to determine relative CD63 expression levels, normalizing to GAPDH expression as an internal control. B. MDMs were challenged with different concentration of HIV-1 SX (m.o.i. = 0.6, 0.2. 0.06 and 0.02, respectively). Supernatants were harvested for p24 detection on day 7 post-infection for MDMs using p24 Capture ELISA kit (ImmunoDiagnostics, Woburn, MA).

Article Snippet: Total mRNA was isolated from each well using the Qiagen RNeasy kit 48 h after transfection.

Techniques: Concentration Assay, Transfection, Infection, Isolation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Tetraspanin CD63 is a regulator of HIV-1 replication

doi:

Figure Lengend Snippet: Effects of CD63 silencing on HIV-1 replication in human PBLs and DCs. A. PBLs (1 × 105 cells/well) were plated in triplicate in 24-well plates and transfected with 50 nM siRNAs (CD63, CD4, and ERBB2IP). Controls included untreated cells or cells treated with virus infection only. Total mRNA was isolated from each well using the Qiagen RNeasy kit 48 h after transfection. Quantitative RT-PCR was used to determine relative CD63 expression levels, normalizing to GAPDH expression as an internal control. B. PBLs cells (1 × 105 cells/well) or (C) DCs cells (5 × 105 cells/well) were plated in triplicate in 24-well plates and transfected with 50 nM siRNAs (CD63, CD4, and ERBB2IP). Controls included untreated cells or cells treated with virus infection only. Forty eight hours post-transfection, cells were infected with HIV-1 89.6 (m.o.i. = 0.02). Supernatants were harvested for p24 detection on day 5 post-infection for PBLs cells, and on day 7 post-infection for DCs using p24 Capture ELISA kit (ImmunoDiagnostics, Woburn, MA). C. Cell lysates from MDMs on day 7 post-infection were also harvested for detection of intracellular p24. *P < 0.05, **P < 0.01, compared with ERBB2IP control group, respectively.

Article Snippet: Total mRNA was isolated from each well using the Qiagen RNeasy kit 48 h after transfection.

Techniques: Transfection, Infection, Isolation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor

doi: 10.1038/onc.2016.72

Figure Lengend Snippet: CD146 expression in MRT cell lines and primary tumors. ( a ) MP-MRT-AN, KP-MRT-NS, KP-MRT-RY and KP-MRT-YM cells were stained with anti-CD146 (black histograms) or isotype-matched control antibodies (gray histograms), and then analyzed by flow cytometry. ( b ) Representative flow cytometric profile of ATRT primary tumor cells stained with anti-CD146 and anti-CD133 antibodies, showing that a small CD146 + sub-population was present, whereas CD133 + cells were rarely observed.

Article Snippet: Two distinct shRNA plasmids targeting human CD146 mRNA (TG311550A, shRNA#1; TG311550D, shRNA#2) and a scrambled control shRNA plasmid (TR30013, scrambled shRNA) were purchased from OriGene (Rockville, MD, USA).

Techniques: Expressing, Staining, Flow Cytometry

Journal: Oncogene

Article Title: CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor

doi: 10.1038/onc.2016.72

Figure Lengend Snippet: CD146 + MRT cells show more enhanced self-renewal and invasive potential than CD146 − cells in vitro . ( a ) Sphere-forming potential of purified CD146 + and CD146 − MRT cells. ( b ) Representative light micrographs of spheres generated from CD146 + and CD146 − KP-MRT-NS cells. Scale bars=50 μm. ( c ) Sphere-forming potential of purified CD146 + and CD133 + MRT cells. ( d ) Invasive potential of CD146 + and CD146 − MRT cells in vitro . ( e ) Representative light micrographs of invaded CD146 + and CD146 − KP-MRT-RY cells (scale bars=500 μm). Error bars indicate s.d. All experiments were performed in at least triplicate (* P <0.05).

Article Snippet: Two distinct shRNA plasmids targeting human CD146 mRNA (TG311550A, shRNA#1; TG311550D, shRNA#2) and a scrambled control shRNA plasmid (TR30013, scrambled shRNA) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vitro, Purification, Generated

Journal: Oncogene

Article Title: CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor

doi: 10.1038/onc.2016.72

Figure Lengend Snippet: Tumor formation ability of sorted CD146 + and CD146 − MRT cells in NOG mice

Article Snippet: Two distinct shRNA plasmids targeting human CD146 mRNA (TG311550A, shRNA#1; TG311550D, shRNA#2) and a scrambled control shRNA plasmid (TR30013, scrambled shRNA) were purchased from OriGene (Rockville, MD, USA).

Techniques: Injection, Transplantation Assay

Journal: Oncogene

Article Title: CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor

doi: 10.1038/onc.2016.72

Figure Lengend Snippet: CD146 + cells replicate phenotypically heterogeneous cell populations during in vivo tumor formation. ( a ) Gating strategy for sorting live MRT cells. ( b ) Flow cytometric analysis showing the expression of CD146 in engrafted KP-MRT-NS cells and ATRT primary tumor cells during serial transplantation.

Article Snippet: Two distinct shRNA plasmids targeting human CD146 mRNA (TG311550A, shRNA#1; TG311550D, shRNA#2) and a scrambled control shRNA plasmid (TR30013, scrambled shRNA) were purchased from OriGene (Rockville, MD, USA).

Techniques: In Vivo, Expressing, Transplantation Assay

Journal: Oncogene

Article Title: CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor

doi: 10.1038/onc.2016.72

Figure Lengend Snippet: Knockdown of CD146 suppresses self-renewal potential and survival of MRT cells by inducing apoptosis. ( a ) CD146 mRNA levels in shRNA-treated KP-MRT-NS cells were analyzed by qRT–PCR. ACTB mRNA served as an internal control. ( b ) Western blotting of CD146 and β-actin after shRNA transduction (lane 1, purified CD146 + KP-MRT-NS cells; lane 2, Scramble shRNA; lane 3, CD146 shRNA#1; lane 4, CD146 shRNA#2; lane 5, purified CD146 − KP-MRT-NS cells). ( c ) WST-8 assays after transduction of CD146-specific shRNA (closed squares and triangles) or scrambled shRNA (closed circles). OD, optical density. ( d ) Sphere-forming assays after shRNA transduction. ( e ) Apoptosis assays after shRNA transduction. ( f ) Proliferation assays after shRNA transduction. Error bars indicate s.d. All experiments were performed in triplicate (* P <0.05). NS, not significant.

Article Snippet: Two distinct shRNA plasmids targeting human CD146 mRNA (TG311550A, shRNA#1; TG311550D, shRNA#2) and a scrambled control shRNA plasmid (TR30013, scrambled shRNA) were purchased from OriGene (Rockville, MD, USA).

Techniques: shRNA, Quantitative RT-PCR, Western Blot, Transduction, Purification

Journal: Oncogene

Article Title: CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor

doi: 10.1038/onc.2016.72

Figure Lengend Snippet: Antitumor activity of anti-CD146 polyclonal antibody against KP-MRT-NS cells. ( a ) WST-8 assays after treatment with normal rabbit serum (5% open circles, 10% open triangles), anti-CD146 antiserum (5% closed circles, 10% closed triangles), and control culture medium (open squares). P -values were calculated by comparing viable cells at the same serum concentrations. OD, optical density. ( b ) Apoptosis assays after anti-CD146 antiserum treatment. ( c ) Proliferation assays after anti-CD146 antiserum treatment. ( d ) In vivo antitumor effects of purified anti-CD146 antibody (Ab) (triangles), normal rabbit IgG (circles), and phosphate-buffered saline (squares) on the volume of tumor cell xenograft tumors in NOG mice. ( e ) Effect on tumor weights. ( f ) Macroscopic appearance of tumor tissues. Error bars indicate s.d. Results shown are representatives of three independent experiments in vitro and four independent experiments in vivo (* P <0.05). NS, not significant.

Article Snippet: Two distinct shRNA plasmids targeting human CD146 mRNA (TG311550A, shRNA#1; TG311550D, shRNA#2) and a scrambled control shRNA plasmid (TR30013, scrambled shRNA) were purchased from OriGene (Rockville, MD, USA).

Techniques: Activity Assay, In Vivo, Purification, In Vitro

Journal: Oncogene

Article Title: CD146 is a novel marker for highly tumorigenic cells and a potential therapeutic target in malignant rhabdoid tumor

doi: 10.1038/onc.2016.72

Figure Lengend Snippet: Clinical characteristics, outcome and the CD146 staining pattern in 11 patients with MRT

Article Snippet: Two distinct shRNA plasmids targeting human CD146 mRNA (TG311550A, shRNA#1; TG311550D, shRNA#2) and a scrambled control shRNA plasmid (TR30013, scrambled shRNA) were purchased from OriGene (Rockville, MD, USA).

Techniques: Staining

Journal: eLife

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.7554/elife.70948

Figure Lengend Snippet: Figure 1. T6B fusion protein prevents miRNA-induced silencing complex (miRISC) assembly and impairs microRNA (miRNA) activity in vitro. (A) Schematics of T6B action: T6B competes with TNRC6 for binding to AGO proteins preventing miRISC assembly. (B) Schematics of the size-exclusion chromatography (SEC) assay for the fractionation of AGO-containing complexes according to their molecular weight. (C) SEC profiling of miRISC components upon T6B expression: total lysates from HCT116 cells expressing no fusion protein (upper panel), T6B (middle panel), or T6BMut (lower panel) were fractionated as described in (B) and immunoblotted to detect AGO2, TNRC6A, T6B, and PABP1. For each blot, the relative signal intensity was assessed by densitometric analysis. (D) RNAseq analysis of total and small RNAs isolated from mouse embryo fibroblasts (MEFs) cell lines expressing either no fusion protein, T6B, or T6BMut (n = 3 for each cell line). Upper panel: bubble plot of target de-repression against miRNA abundance. The mean log2-fold change (T6B or T6BMut vs. control) of predicted targets for each conserved miRNA family was calculated, converted to a z-score and is plotted on the x-axis against the miRNA family abundance (log of the sum of read counts for each member of the family). The size of each circle is proportional to the number of predicted targets. A positive z-score indicates that the targets for that family are preferentially upregulated upon T6B expression, while a negative score would indicate preferential downregulation. Expression of T6B, but not of T6BMut, causes preferential upregulation of miRNA targets of the most miRNA families and the effect is roughly proportional to each miRNA family abundance. Lower panel: cumulative distribution plot of predicted let-7 targets compared to background in T6B-expressing MEFs. (E) Scatter plots of miRNA abundance as determined by small-RNAseq of total RNA extracted from MEFs expressing either T6B or T6BMut (n = 3 for each cell line). Each dot represents a miRNA in miRbase. (F) Effect of T6B expression on AGO2 slicing activity. MEFs expressing either T6B or T6BMut were transfected with siRNAs targeting GAPDH mRNA (siGAPDH) or with scramble siRNA (siCTL). Levels of GAPDH, T6B, and tubulin were assessed by immunoblot 72 hr post-transfection. T6B and T6BMut have slightly different migration on PAGE, as previously observed by Hauptmann et al., 2015.

Article Snippet: DOI: https://doi.org/10.7554/eLife.70948 17 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence- based reagent Col1a1 common _F This paper PCR primers AATCATCCCAGGTG CACAGCATTGCGG Sequence- based reagent Col1a1 wildtype _R This paper PCR primers CTTTGAGGGCTCAT GAACCTCCCAGG Sequence- based reagent Col1a1 mutant _R This paper PCR primers ATCAAGGAAACCC TGGACTACTGCG Sequence- based reagent R26_F This paper PCR primers AAAGTCGCTCT GAGTTGTTAT Sequence- based reagent R26a_R This paper PCR primers GCGAAGAGTTTG TCCTCAACC Sequence- based reagent R26b_R This paper PCR primers CCTC CAAT TTTA CACC TGTTC Sequence- based reagent T6B- YFP_F This paper PCR primers GACTACAAGGACG ACGATGACAAG Sequence- based reagent T6B- YFP_R This paper PCR primers GTTACTTGTACAG CTCGTCCATG Commercial assay or kit RNAscope 2.5 HD Detection Reagent, BROWN ACD #320771 Commercial assay or kit RNAScope Igfbp5 Probe ACD #425738 Commercial assay or kit Superose 6 10/300 GL Cytiva #GE17- 5172- 01 Now available as Increase 10/300 GL, Cytiva #GE29- 0915- 96 Commercial assay or kit Novex NuPAGE SDS/ PAGE gel system Thermo Fisher #NP0321 Commercial assay or kit EnVision + HRP DAKO, Glostrup, Denmark #K401111- 2, RRID:AB_2827819 Commercial assay or kit GFP- trap Chromotek #gtma- 10 RRID:AB_2827592 Commercial assay or kit TruSeq Stranded mRNA LT Kit, Illumina #RS- 122- 2102 Software, algorithm OMERO PMID:22373911 RRID:SCR_002629 Software, algorithm STAR v2.5.3a PMID:23104886 Software, algorithm DESeq2 PMID:25516281 RRID:SCR_015687 Software, algorithm miRbase version 21 https://www.mirbase.org/ Software, algorithm TargetScan PMID:26267216 RRID:SCR_010845 Chemical compound, drug Doxycyline- containing Rodent diet Envigo #TD01306 625 mg/kg doxycycline Chemical compound, drug Dextran sulfate sodium (DSS) Cayman Chemical #23250 Chemical compound, drug Surgipath Decalcifier I Leica Biosystems #3800400 Formic acid solution Other EDTA- free complete protease inhibitors Sigma- Aldrich #11836170001 Other KnockOut DMEM GIBCO #10829018 Continued Continued on next page La Rocca, King, et al. eLife 2021;10:e70948.

Techniques: Activity Assay, In Vitro, Binding Assay, Size-exclusion Chromatography, Fractionation, Molecular Weight, Expressing, Isolation, Control, Transfection, Western Blot, Migration

Journal: eLife

Article Title: Inducible and reversible inhibition of miRNA-mediated gene repression in vivo

doi: 10.7554/elife.70948

Figure Lengend Snippet: Figure 4. T6B-induced block of miRNA-induced silencing complex (miRISC) assembly leads to impaired intestinal regeneration. (A) R26T6B and R26CTL mice (n = 6 for each genotype) kept on doxycycline diet were treated with dextran sulfate sodium (DSS) for 5 days to induce inflammatory colitis and their weight was monitored daily. Data are presented as mean ± SD. p-Values (from left to right): *p=0.034, *p=0.005, *p=0.029, *p=0.024, *p=0.011, from unpaired t-test. (B) Kaplan–Meier curves of animals treated with DSS as described in panel (A). p-Value from log-rank test (C) Representative hematoxylin-eosin-stained sections of intestine of R26T6B and R26CTL mice (n = 3 for each genotype) at different time points pre- and post-DSS treatment. (D) Ki67 immunostaining of section of intestine at the indicated time points. (E) Sections from the large intestine of control and T6B mice euthanized at day 13 were subjected to RNA in situ hybridization with a probe against the IGFBP5 transcript. The results show increased levels of IGFBP5 mRNA in ulcerated areas of R26T6B as compared to controls (n = 4 for each genotype).

Article Snippet: DOI: https://doi.org/10.7554/eLife.70948 17 of 30 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Sequence- based reagent Col1a1 common _F This paper PCR primers AATCATCCCAGGTG CACAGCATTGCGG Sequence- based reagent Col1a1 wildtype _R This paper PCR primers CTTTGAGGGCTCAT GAACCTCCCAGG Sequence- based reagent Col1a1 mutant _R This paper PCR primers ATCAAGGAAACCC TGGACTACTGCG Sequence- based reagent R26_F This paper PCR primers AAAGTCGCTCT GAGTTGTTAT Sequence- based reagent R26a_R This paper PCR primers GCGAAGAGTTTG TCCTCAACC Sequence- based reagent R26b_R This paper PCR primers CCTC CAAT TTTA CACC TGTTC Sequence- based reagent T6B- YFP_F This paper PCR primers GACTACAAGGACG ACGATGACAAG Sequence- based reagent T6B- YFP_R This paper PCR primers GTTACTTGTACAG CTCGTCCATG Commercial assay or kit RNAscope 2.5 HD Detection Reagent, BROWN ACD #320771 Commercial assay or kit RNAScope Igfbp5 Probe ACD #425738 Commercial assay or kit Superose 6 10/300 GL Cytiva #GE17- 5172- 01 Now available as Increase 10/300 GL, Cytiva #GE29- 0915- 96 Commercial assay or kit Novex NuPAGE SDS/ PAGE gel system Thermo Fisher #NP0321 Commercial assay or kit EnVision + HRP DAKO, Glostrup, Denmark #K401111- 2, RRID:AB_2827819 Commercial assay or kit GFP- trap Chromotek #gtma- 10 RRID:AB_2827592 Commercial assay or kit TruSeq Stranded mRNA LT Kit, Illumina #RS- 122- 2102 Software, algorithm OMERO PMID:22373911 RRID:SCR_002629 Software, algorithm STAR v2.5.3a PMID:23104886 Software, algorithm DESeq2 PMID:25516281 RRID:SCR_015687 Software, algorithm miRbase version 21 https://www.mirbase.org/ Software, algorithm TargetScan PMID:26267216 RRID:SCR_010845 Chemical compound, drug Doxycyline- containing Rodent diet Envigo #TD01306 625 mg/kg doxycycline Chemical compound, drug Dextran sulfate sodium (DSS) Cayman Chemical #23250 Chemical compound, drug Surgipath Decalcifier I Leica Biosystems #3800400 Formic acid solution Other EDTA- free complete protease inhibitors Sigma- Aldrich #11836170001 Other KnockOut DMEM GIBCO #10829018 Continued Continued on next page La Rocca, King, et al. eLife 2021;10:e70948.

Techniques: Blocking Assay, Staining, Immunostaining, Control, RNA In Situ Hybridization