transfection lipid reagent  (Bio-Rad)

Journal: Cell reports

Article Title: A ribosome-bound pseudoknot in the HCV coding region stimulates viral growth by tuning viral translation

doi: 10.1016/j.celrep.2025.116739

Figure Lengend Snippet: (A) Schematic of the WT pseudoknot and structure-disrupting mutants. (B) Luciferase activity of IRES-driven constructs measured over time; construct designs shown on the left. (C) Normalized luciferase activity of various IRES-driven constructs at different time points, relative to the random control; construct designs shown on the left. (D) Workflow for full-length HCV growth assay. (E) Measurement of WT and mutant viral growth by luciferase activity at 24, 48, and 72 h post-transfection. Data are represented as mean ± SD of three biological replicates. Statistical significance was determined using an unpaired Student’s t test with equal variance (** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: 3 × 10 6 Huh7.5 cells were plated in a 10 cm dish and transfected with 15.5 μg of full-length HCV RNA or mutant RNA using Mirus mRNA lipid transfection reagent (MIR-2250) according to the manufacturer’s protocol.

Techniques: Luciferase, Activity Assay, Construct, Control, Growth Assay, Mutagenesis, Transfection

Journal: Cell reports

Article Title: A ribosome-bound pseudoknot in the HCV coding region stimulates viral growth by tuning viral translation

doi: 10.1016/j.celrep.2025.116739

Figure Lengend Snippet: (A–C) Top: Schematic of the dual luciferase constructs used to test pk1’s regulation of upstream and downstream protein expression. Bottom: Gaussia and Firefly luciferase activities for pk1 and random control constructs, measured at 2 h post-transfection and normalized to the random control. (D) qPCR quantification of Actin mRNA or luciferase construct RNA amount relative to 18S rRNA for each sample, normalized to the random control RNA. (E) Left: siRNA knockdown workflow. Right: western blot analysis of ZNF598, GIGYF2, and GAPDH confirming siRNA knockdown efficiency. (F) Gaussia and Firefly luciferase activities after siRNA treatments for pk1 and random control constructs, measured at 2 h post-transfection and normalized to the random control. The inhibitory effect in the siCtrl condition for the pk1-middle construct was weaker than in previous experiments , possibly reflecting variability in RNA input associated with the siRNA transfection setup and limited time point sampling. (G) Graphic showing the proposed pk1 inhibiting translation initiation mechanism. Data are represented as mean ± SD of three biological replicates. Statistical significance was determined using an unpaired Student’s t test with equal variance (** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: 3 × 10 6 Huh7.5 cells were plated in a 10 cm dish and transfected with 15.5 μg of full-length HCV RNA or mutant RNA using Mirus mRNA lipid transfection reagent (MIR-2250) according to the manufacturer’s protocol.

Techniques: Luciferase, Construct, Expressing, Control, Transfection, Knockdown, Western Blot, Sampling

Journal: Cell reports

Article Title: A ribosome-bound pseudoknot in the HCV coding region stimulates viral growth by tuning viral translation

doi: 10.1016/j.celrep.2025.116739

Figure Lengend Snippet: (A) Workflow of polysome fractionation and RNA quantification for HCV-transfected cells. (B) Top: chromatogram showing the separation of monosome and polysome fractions. Middle: distribution of Actin mRNA across all fractions in WT and PUR-treated control. Bottom: distribution of HCV RNA across all fractions in WT virus and PUR-treated control. (C) Top: chromatogram showing the separation of monosome and polysome fractions. Middle: distribution of Actin mRNA across all fractions in WT and unzip mutant virus. Bottom: distribution of HCV RNA across all fractions in WT virus and unzip mutant virus. (D) Graphic showing the proposed pk1 acting mechanism.

Article Snippet: 3 × 10 6 Huh7.5 cells were plated in a 10 cm dish and transfected with 15.5 μg of full-length HCV RNA or mutant RNA using Mirus mRNA lipid transfection reagent (MIR-2250) according to the manufacturer’s protocol.

Techniques: Fractionation, Transfection, Control, Virus, Mutagenesis

Journal: Cell reports

Article Title: A ribosome-bound pseudoknot in the HCV coding region stimulates viral growth by tuning viral translation

doi: 10.1016/j.celrep.2025.116739

Figure Lengend Snippet: (A) Workflow for identifying pk1-like elements across RNA virus genomes. (B) Top: pie chart summarizing the family distribution of input viruses. Middle: pie chart showing the family distribution of viruses identified after step 2. Bottom: pie chart displaying the family distribution and location of pk1-like elements after step 3. (C) Examples of high-confidence pk1-like elements located near viral protein boundaries. (D) Left: secondary structure representation of a pk1-like motif in Simian pegivirus and the design of the unzip mutant. Right: translation luciferase activity illustrating the function of pk1-like element in translation elongation, measured at 2 h post-transfection. Construct design is shown on the right. Data are represented as mean ± SD of three biological replicates. Statistical significance was determined using an unpaired Student’s t test with equal variance (** p < 0.01, *** p < 0.001, and **** p < 0.0001). (E) AlphaFold3 prediction of tertiary structures for pk1, pk1-like elements, and a canonical frameshifting pseudoknot. The corresponding secondary structures derived from the tertiary models are shown below.

Article Snippet: 3 × 10 6 Huh7.5 cells were plated in a 10 cm dish and transfected with 15.5 μg of full-length HCV RNA or mutant RNA using Mirus mRNA lipid transfection reagent (MIR-2250) according to the manufacturer’s protocol.

Techniques: Virus, Mutagenesis, Luciferase, Activity Assay, Transfection, Construct, Derivative Assay