plc5 ms2 gfp  (Addgene inc)

Journal: Journal of Virology

Article Title: circCBL and its host gene CBL collaboratively enhance the antiviral immunity and antibacterial immunity by targeting MITA in fish

doi: 10.1128/jvi.01046-23

Figure Lengend Snippet: circCBL promotes the antiviral and antibacterial innate immunity. (A, B) The schematic diagram of siRNAs (A) and oe-circ structure (B). (C) qPCR analysis of circCBL and linear CBL mRNA in MIC treated with siRNAs (left) and in MKC stably overexpressing circCBL (right). (D) qPCR assays were performed to determine the expression levels of ISG15, Viperin, and Mx1 in MKC or MIC transfected with overexpression plasmid (oe-circ) or control vector (pLC5-ciR) and with (si-circCBL-1) si-circ or NC after SRCV infected. (E) qPCR assays were performed to determine the expression levels of TNF-α, IL-1β, and IL-8 in MKC or MIC transfected with oe-circ or control vector and with si-circ or NC after V. anguillarum infection. (F) MIC or MKC seeded in 48-well plates overnight were treated with SCRV at the dose indicated for 48 h. Then, cell monolayers were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (si-NC) NC, si-circ, vector, or oe-circ was transfected. (G) circCBL suppresses SCRV replication. MIC or MKC were transfected with NC or si-circ and vector or oe-circ plasmid for 24 h and then infected with SCRV for 24 h. The qPCR analysis was conducted for SCRV RNA levels. (H) MKC were transfected with oe-circ or control vector, and MIC were transfected with si-circ or NC, infected with FITC-D-Lys-labeled V. anguillarum, and then examined by using a fluorescence microscope. Scale bars, 20 µm; original magnification ×200. (I) MIC were transfected with si-circ or NC, and MKC were transfected with oe-circ or control vector and then infected with V. anguillarum. After 6 h of infection, the intracellular bacterial number was measured and displayed as CFUs. (J) Cell proliferation was assessed by EdU assays in MIC transfected with si-circ or NC after SCRV infected 24 h or V. anguillarum infected 6 h and MKC transfected with oe-circ or pLC5-ciR vector after SCRV infected 24 h or V. anguillarum infected 6 h. Scale bars, 20 µm; original magnification ×200. All data represented the mean ± SE from three independent triplicated experiments. *P < 0.05; **P < 0.01.

Article Snippet: PLC5-MS2-GFP plasmid can express GFP protein and can be detected by anti-GFP antibody (Abcam).

Techniques: Stable Transfection, Expressing, Transfection, Over Expression, Plasmid Preparation, Infection, Staining, Labeling, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: circCBL and its host gene CBL collaboratively enhance the antiviral immunity and antibacterial immunity by targeting MITA in fish

doi: 10.1128/jvi.01046-23

Figure Lengend Snippet: circCBL functions as a miRNA sponge of miR-125a-1-3p. (A) Schematic illustration of circCBL-wt and circCBL-mut luciferase reporter vectors. (B) The relative luciferase activities were detected in MIC after cotransfection with circCBL-wt or circCBL-mut and miR-125a-1-3p or NC. (C) The concentration gradient experiment of miR-125a-1-3p mimics was conducted in MIC. (D, E) circCBL downregulated GFP expression. Epithelioma papulosum cyprini cells (EPC) were cotransfected with circCBL-wt or circCBL-mut and miR-125a-1-3p or NC. The fluorescence intensity and the GFP expression were evaluated by enzyme-labeled instrument, fluorescence microscope, and western blot. Scale bars, 20 µm; original magnification ×200. (F) The Ago2-RIP assay was executed in MIC after transfection with miR-125a-1-3p, and NC, followed by qPCR to detect cirCBL expression levels. (G) RNA pull-down assay was executed in MKC, followed by qPCR to detect the enrichment of circCBL. (H) The MS2-RIP assay was executed in MKC cells after transfection with pLC5-circ-MS2, pLC5-MS2-circCBL, and pLC5-MS2-circCBL-mut, followed by qPCR to detect the enrichment of miR-125a-1-3p. All data represented the mean ± SE from three independent triplicated experiments. *P < 0.05; **P < 0.01.

Article Snippet: PLC5-MS2-GFP plasmid can express GFP protein and can be detected by anti-GFP antibody (Abcam).

Techniques: Luciferase, Cotransfection, Concentration Assay, Expressing, Fluorescence, Labeling, Microscopy, Western Blot, Transfection, Pull Down Assay

Journal: Journal of Virology

Article Title: circCBL and its host gene CBL collaboratively enhance the antiviral immunity and antibacterial immunity by targeting MITA in fish

doi: 10.1128/jvi.01046-23

Figure Lengend Snippet: circCBL acts as a sponge of miR-125a-1-3p to enhance MITA expression. (A) The protein levels of MITA in MIC and MKC after cotransfected with si-NC or si-circ, and vector (pLC5-ciR) or oe-circ by western blot, respectively. (B) Relative mRNA levels of MITA in MIC and MKC after cotransfected with si-NC or si-circ, and vector or oe-circ by qPCR assay, respectively. (C) The relative luciferase activities were detected in MIC cells after cotransfection with MITA-3′UTR luciferase reporter vector, NC, miR-125a-1-3p, oe-circ, or oe-circ-mut. (D) Western blot assays were detected in MIC after cotransfection with MITA overexpression plasmid, NC, mimics, or oe-circ. (E) oe-circ counteracts the negative effect of miR-125a-1-3p. The relative luciferase activities were detected in MIC after cotransfection with MITA expression plasmid, phRL-TK Renilla luciferase plasmid, luciferase reporters, NC, miR-125a-1-3p, or oe-circ. (F) MIC seeded in 48-well plates overnight were treated with SCRV at the dose indicated for 48 h. The cell monolayers then were fixed with 4% paraformaldehyde and stained with 1% crystal violet. MIC were transfected with NC, miR-125a-1-3p, or oe-circ for 24 h and then infected with SCRV for 24 h. (G) miR-125a-1-3p promotes SCRV replication, whereas circCBL suppresses SCRV replication. MIC were transfected with NC, miR-125a-1-3p, or oe-circ for 24 h and then infected with SCRV for 24 h. The qPCR analysis was conducted for SCRV RNA levels. (H) MIC were cotransfected with NC, miR-125a-1-3p, or oe-circ for 24 h; infected with FITC-D-Lys-labeled V. anguillarum for 6 h; and then examined by fluorescence microscope. Scale bars, 20 µm; original magnification ×200. (I) MIC were cotransfected with NC, miR-125a-1-3p mimics, or oe-circ for 24 h and then infected with V. anguillarum. After 6 h of infection, the intracellular bacterial number was measured and displayed as CFUs. (J) Cell proliferation was assessed by EdU assays in MIC cells after cotransfected with NC, mimics, or oe-circ. Scale bars, 20 µm; original magnification ×200. All data represented the mean ± SE from three independent triplicated experiments. **P < 0.01.

Article Snippet: PLC5-MS2-GFP plasmid can express GFP protein and can be detected by anti-GFP antibody (Abcam).

Techniques: Expressing, Plasmid Preparation, Western Blot, Luciferase, Cotransfection, Over Expression, Staining, Transfection, Infection, Labeling, Fluorescence, Microscopy