rrm2 kit  (Thermo Fisher)

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: Effect of RRM2 knockdown on DENV RNA replication and infectious virus production (A) Schematic representation of the experimental protocol. Briefly, HuH-7, HepG2, and A549 cells were transfected with RRM2 siRNA or non-targeting control siRNA (Ctrl.si) one day before infection with DENV-1 and DENV-2 at a MOI of 0.1. Cells and culture supernatants were harvested 72 h later to measure intracellular and extracellular DENV RNA levels using RT-qPCR and quantify infectious virus titers in culture supernatants via a focus-forming assay. Created using Biorender.com. (B) SDS-PAGE and western blotting were utilized to assess the efficiency of RRM2 knockdown in HepG2, HuH-7, and A549 cells after treatment with specific RRM2 siRNA for 96 h at final concentrations of 10, 20, 5, and 5 nM, respectively. The expression levels of other RR subunits, RRM1 and p53R2, were also characterized. β-actin served as a loading control. (C–E) Intracellular and extracellular DENV RNA copy numbers were measured 72 h post-transfection (hpi) via RT-qPCR in HuH-7, HepG2, and A549 cells. Intracellular DENV RNA levels were normalized to GAPDH mRNA levels. Results are presented as mean+/-standard deviation (SD). p values were assessed using Student’s t test. (F–H) Infectious virus titers in culture supernatants from non-transfected and siRNA-transfected HuH-7, HepG2, and A549 cells were determined using a fluorescent focus assay on BHK-21 cells. Results are presented as mean +/- SD. p values were evaluated by Student’s t test. (I) Viability of HuH-7, HepG2, and A549 cells following treatment with RRM2 siRNA. Cells were seeded in 96-well plates and transfected with the indicated final concentrations of RRM2 siRNA. At 96 h post-transfection, cell viability was assessed using the 2(2-methyl-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4disulfophenyl)-2H-tetrazolium, monosodium salt (WST)-8 assay (OD 450 ). The percentage of viability was calculated and compared to the untreated control (100% viability). (J) Immunofluorescence microscopy was conducted to evaluate the infection efficiency of DENV particles released from RRM2 or control siRNA-transfected and non-treated cells. Schematic representation of the experimental procedure. Culture supernatants harvested from non-transfected and siRNA-transfected DENV-infected HuH-7 and HepG2 cells were used to infect naive cell lines at an MOI of 1 (left). Three days post-infection, cell monolayers were analyzed by immunofluorescence for DENV envelope (E) protein expression using anti-flavivirus monoclonal antibody 4G2, followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. Representative immunofluorescence images (400× magnification) depicting DENV-E protein expression in HuH-7 and HepG2 cells. Cell nuclei were stained with DAPI, and cells were observed under a BZ-X700 fluorescence microscope (Keyence Co., Osaka, Japan). White squares indicate enlarged insets. Scale bars, 50 μm (right).

Article Snippet: To evaluate the cytotoxicity of RRM2 siRNA, HuH-7, HepG2, and A549 cells were seeded in 96-well plates at 5 × 10 3 cells per well and incubated with various concentrations of RRM2 siRNA at 37°C and 5% CO 2 for 96 h. Cell viability following RRM2 siRNA treatment was assessed using the Cell counting kit-8 assay (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.

Techniques: Knockdown, Virus, Transfection, Control, Infection, Quantitative RT-PCR, Focus Forming Assay, SDS Page, Western Blot, Expressing, Standard Deviation, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: RRM2 knockdown reduced furin protein levels in the cells, decreasing cleavage of DENV prM protein (A) RT-qPCR analysis of RRM2 mRNA levels in HuH-7 cells either mock-infected or infected with DENV-1 or DENV-2 at an MOI of 0.1. RRM2 mRNA levels were normalized to GAPDH mRNA levels. Data are expressed as mean ± standard deviation (SD) of triplicate measurements. p values were assessed using Student’s t test. n.s., not significant. (B) DENV infection increased RRM2 protein levels. HuH-7 cells were mock-infected or infected with DENV-1 or DENV-2 at an MOI of 0.1. Cells were harvested at the indicated time points (24, 48, and 72 hpi) and analyzed by western blotting using anti-RRM2, anti-p53R2, and mouse monoclonal anti-flavivirus E (4G2) antibodies. Endogenous β-actin expression served as an internal control. Protein levels were quantified using ImageJ software and normalized to β-actin levels. Densitometric p53R2/actin and RRM2/actin ratios are shown below the blots. (C) Representative western blot images depict RRM2, DENV envelope (E), DENV capsid, and DENV prM protein expression levels in HuH-7 cells that were transfected with RRM2 siRNA or control (Ctrl) siRNA (5 nM). HuH-7 cells were either non-transfected or transfected with siRNAs one day prior to infection with DENV-1 or DENV-2 at an MOI of 0.1. At 72 hpi, cells were harvested and subjected to western blot analysis under non-reducing conditions using anti-prM, anti-capsid, anti-flavivirus E (4G2), and anti-RRM2 antibodies. β-actin was utilized as a loading control. (D) Determination of viral maturation was carried out by calculating the ratio of prM to E protein expression, normalized to non-transfected DENV-infected HuH-7 cells. Detected signals of DENV E and prM proteins in (C) were quantified using ImageJ software, and prM-to-E ratios are plotted as bar graphs. Numbers indicate n -fold increase compared to non-transfected cells. (E) Western blot analysis of prM and pr proteins in culture supernatants of non-transfected, control siRNA or RRM2 siRNA-transfected DENV-1 infected HuH-7 cells (C) under reducing conditions using an anti-pr mouse mAb. (F) Analysis of DENV E protein glycosylation status. Mock infected (−) or DENV (+) infected and siRNA-transfected HuH-7 cell lysates were treated with PNGaseF (+) or buffer control (−), then subjected to non-reducing SDS-PAGE and western blotting with anti-E 4G2, anti-RRM2, and anti-actin antibodies. Arrows indicate the positions of undigested and glycosylated E (2N) protein and deglycosylated forms (1N and 0N) of E protein. 0N, 1N, and 2N correspond to the number of N-linked glycans on E protein. β-actin was utilized as a loading control. (G) Quantification of endogenous furin mRNA levels relative to GAPDH in HuH-7 cells that were non-transfected or transfected with RRM2 siRNA or control siRNA (Ctrl si) (5 nM) for 96 h. Data are expressed as mean ± SD of triplicate measurements. (H) Effect of RRM2 knockdown on furin protein expression. HuH-7 cells were either non-transfected or transfected with control siRNA or RRM2 siRNA one day before mock infection or infection with DENV-1 or DENV-2 at an MOI of 0.1. At 72 hpi, cells were harvested and analyzed by western blotting using anti-furin, anti-RRM2, and anti-DENV-E proteins. β-actin served as a loading control. Protein band intensities were analyzed using ImageJ software. Densitometric furin/actin ratios are shown below the blots. (I) Furin protein levels in HuH-7 and A549 cells with or without transfection of furin expression plasmid (myc-DDK-tagged human furin, 4 μg) and those non-transfected or transfected with control or RRM2 siRNA (5 nM). Densitometric furin/actin ratios are shown below the blots. (J) A549 cells were seeded in 60 mm dishes, and after 24 h, cells were transfected with the furin expression plasmid (4 μg) alone or co-transfected with RRM2 or control siRNA. The following day, the cells were infected with DENV-2 at an MOI of 0.1. At 72 hpi, cells were harvested and subjected to western blot analysis under non-reducing conditions using anti-furin, anti-RRM2, anti-prM, anti-flavivirus E (4G2), and anti-actin antibodies. Densitometric prM/actin ratios are shown below the blots (left). Determination of viral maturation was carried out by calculating prM to E protein expression ratios normalized to those of control siRNA-transfected A549 cells. Detected signals of DENV E and prM proteins were quantified using ImageJ software as described in . prM-to-E ratios are plotted as bar graphs. Number indicates n -fold increase compared to Ctrl siRNA-transfected cells (right).

Article Snippet: To evaluate the cytotoxicity of RRM2 siRNA, HuH-7, HepG2, and A549 cells were seeded in 96-well plates at 5 × 10 3 cells per well and incubated with various concentrations of RRM2 siRNA at 37°C and 5% CO 2 for 96 h. Cell viability following RRM2 siRNA treatment was assessed using the Cell counting kit-8 assay (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.

Techniques: Knockdown, Quantitative RT-PCR, Infection, Standard Deviation, Western Blot, Expressing, Control, Software, Transfection, Glycoproteomics, SDS Page, Plasmid Preparation

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: RRM2 colocalizes and interacts with furin in DENV-infected cells (A) Analysis of RRM2 and furin interactions through immunoprecipitation. HuH-7 cells were either mock-transfected (non) or transfected with the expression plasmid for RRM2 (pcDNA6/myc-His-RRM2) or the empty vector pcDNA6/myc-His (5 μg) using Lipofectamine LTX (Invitrogen), followed by either mock-infection or infection with DENV-2 (MOI = 0.1). At 48 hpi, cell lysates were precipitated with anti-RRM2 antibody, and the expression levels of RRM2 and furin in the precipitates were analyzed via immunoblotting using mouse anti-RRM2 (left and right) and rabbit anti-furin (right) antibodies. ∗IgG heavy and light chains. (B) Western blot analysis of the inputs from each cell lysate. β-actin served as a loading control. (C) Representative confocal microscopy images showing the subcellular localization and expression of RRM2 and furin in RRM2 siRNA-treated cells compared to those in untreated (non) and control siRNA-treated cells. HuH-7 cells were either mock-infected or infected with DENV-2 following transfection with RRM2 or control siRNA. On day 3 post-infection, cells were stained with anti-furin and anti-RRM2 antibodies and detected using Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies, respectively. Cell nuclei were stained with DAPI. Cells were examined using a fluorescence microscope, BZ-X700 (Keyence Co., Osaka, Japan). Images were captured at 200× magnification. Scale bars, 50 μm. White squares denote enlarged insets. The z stack was set to 0.3 μm. (D) Quantification of colocalization between RRM2 and furin in mock-infected and DENV-2-infected cells was based on Pearson’s correlation coefficient using BIOP JACoP (Fiji/ImageJ software) ( n = 40 cells per group). Black lines represent mean ± SD. Statistical analysis was performed using two-way ANOVA. (E) Schematic representation of the experimental design. Female AG129 mice (5 weeks old) were subcutaneously infected with DENV-2 (10 4 focus-forming units/mouse), and at 14 days post-infection, mice were euthanized, and liver tissues were collected for immunoprecipitation and western blot analysis. Mock-infected AG129 mice were used as controls. (F) Analysis of RRM2 and furin interactions in mouse liver tissues through immunoprecipitation. Tissue lysates were precipitated with anti-RRM2 antibody, and the hepatic protein expression levels of RRM2 and furin in the precipitates were analyzed via immunoblotting using mouse anti-RRM2 and rabbit anti-furin antibodies. (G) Hepatic protein expression levels of RRM2, furin, DENV-NS1, -E, and -prM in the inputs of liver tissue lysates were determined via western blot analysis. β-actin served as a loading control.

Article Snippet: To evaluate the cytotoxicity of RRM2 siRNA, HuH-7, HepG2, and A549 cells were seeded in 96-well plates at 5 × 10 3 cells per well and incubated with various concentrations of RRM2 siRNA at 37°C and 5% CO 2 for 96 h. Cell viability following RRM2 siRNA treatment was assessed using the Cell counting kit-8 assay (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.

Techniques: Infection, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Confocal Microscopy, Staining, Fluorescence, Microscopy, Software

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: RRM2 expression is important for furin protein stability (A) RRM2 overexpression enhances furin protein stability. HuH-7 cells were plated in 60 mm dishes and transfected with the RRM2-encoding plasmid (pcDNA6/myc-His-RRM2, 5 μg) or the empty vector pcDNA6/myc-His (5 μg) as a negative control. At 72 h post-transfection, cells were treated with the protein synthesis inhibitor CHX (100 μg/mL) for 1, 3, and 6 h. Cells were harvested and analyzed by western blotting using anti-furin, anti-RRM2, and anti-actin antibodies. Protein band intensities were evaluated using ImageJ software. Densitometric furin/actin ratios are displayed below the blots. (B) Effects of specific proteasome and lysosome inhibitors on furin stability. HuH-7 cells were transfected with RRM2 or control siRNA for 72 h and then either left untreated (NT) or treated with the proteasome inhibitors lactacystin (Lac, 5 μM), MG-132 (10 μM), the lysosome inhibitor bafilomycin A1 (Baf A1, 20 nM), DMSO, or combinations of these inhibitors for 6 h prior to harvest. Cell lysates underwent western blot analysis using anti-furin and anti-RRM2 antibodies. β-actin was utilized as a loading control. An anti-LC3I/II antibody was employed to assess lysosomal inhibition by bafilomycin A1. An increase in LC3-II protein levels indicates the effect of bafilomycin A1. Densitometric furin/actin ratios are presented below the blots.

Article Snippet: To evaluate the cytotoxicity of RRM2 siRNA, HuH-7, HepG2, and A549 cells were seeded in 96-well plates at 5 × 10 3 cells per well and incubated with various concentrations of RRM2 siRNA at 37°C and 5% CO 2 for 96 h. Cell viability following RRM2 siRNA treatment was assessed using the Cell counting kit-8 assay (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.

Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, Negative Control, Western Blot, Software, Control, Inhibition

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: RRM2 regulates furin protein, DENV-prM, and intracellular viral RNA levels through mTOR-dependent pathway (A) Immunoblot analysis of furin and RRM2 protein expression in HuH-7 cells after treatment with the mTOR inhibitor rapamycin. Cells were seeded in a 12-well culture plate and treated after 24 h with rapamycin (Rapa) at escalating concentrations (50, 100, and 200 nM) or DMSO for an additional 12 h. Cells were harvested and analyzed by western blotting with anti-furin, anti-RRM2, and anti-actin antibodies (left). Densitometric analysis of RRM2 (upper) and furin (lower) relative to β-actin is expressed as a percentage of DMSO treatment (lane 1) using ImageJ software and displayed as bar graphs (right). Quantification data are expressed as mean ± standard deviation (SD) from three independent experiments. (B) The combination of RRM2 siRNA transfection and rapamycin treatment synergistically reduced intracellular furin protein levels. HuH-7 cells were transfected with RRM2 siRNA or control siRNA one day prior to infection with DENV-2 at an MOI of 0.1. At 48 hpi, cells were treated with DMSO or rapamycin (200 nM) for an additional 12 h. Cells were harvested to isolate intracellular viral RNA and total protein for RT-qPCR and western blot analysis, respectively. Intracellular DENV RNA levels were assessed via RT-qPCR (DENV NS1 ) and normalized to those of GAPDH . Significant p values were determined using Student’s t test (upper). Cell lysates were analyzed via western blotting using anti-furin, anti-RRM2, anti-E (4G2), anti-prM, and anti-actin antibodies. prM protein levels were quantified using ImageJ software and normalized to β-actin levels. Densitometric ratios of prM/actin are depicted below the blots (lower). (C) Effect of mTOR overexpression on furin expression. HuH-7 cells were transfected with RRM2 siRNA or control siRNA with or without the mTOR expression plasmid (pRP[Exp]-Neo-CAG>hMTOR, 2.5 μg), followed by infection with DENV-2 at an MOI of 0.1. At 48 hpi, cells were harvested to isolate intracellular viral RNA and total protein for RT-qPCR and western blot analysis, respectively. Intracellular DENV RNA levels were measured by RT-qPCR (DENV NS1 ) in HuH-7 cells and normalized to GAPDH mRNA levels. Significant p values were calculated using Student’s t test (upper). Expression levels of mTOR, furin, RRM2, 4E-BP1, phospho-4E-BP1 (Ser65), phospho-4E-BP1 (Thr70), and prM were assessed by immunoblotting of total cell lysates with anti-mTOR, anti-RRM2, anti-furin, anti-4E-BP1, anti-phospho-4E-BP1 (Ser65), anti-phospho-4E-BP1 (Thr70), and anti-prM antibodies. β-actin was employed as a loading control. Densitometric ratios of furin/actin are indicated below the blots (lower).

Article Snippet: To evaluate the cytotoxicity of RRM2 siRNA, HuH-7, HepG2, and A549 cells were seeded in 96-well plates at 5 × 10 3 cells per well and incubated with various concentrations of RRM2 siRNA at 37°C and 5% CO 2 for 96 h. Cell viability following RRM2 siRNA treatment was assessed using the Cell counting kit-8 assay (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.

Techniques: Western Blot, Expressing, Software, Standard Deviation, Transfection, Control, Infection, Quantitative RT-PCR, Over Expression, Plasmid Preparation

Journal: iScience

Article Title: Ribonucleotide reductase subunit M2 mediates the mTOR pathway to recruit furin endoprotease and promote maturation of dengue virus

doi: 10.1016/j.isci.2025.113998

Figure Lengend Snippet: Schematic illustration of the mechanism underlying RRM2 regulation of furin protein levels and DENV maturation Treatment with RRM2 siRNA increased intracellular DENV RNA levels and promoted furin protein degradation through an mTOR/4E-BP1-dependent pathway, leading to elevated levels of uncleaved prM protein in DENV particles and hindering viral maturation. In contrast, DENV infection induced colocalization and interaction between RRM2 and furin in cells, and RRM2 overexpression, which enhanced furin stability. Created using Biorender.com .

Article Snippet: To evaluate the cytotoxicity of RRM2 siRNA, HuH-7, HepG2, and A549 cells were seeded in 96-well plates at 5 × 10 3 cells per well and incubated with various concentrations of RRM2 siRNA at 37°C and 5% CO 2 for 96 h. Cell viability following RRM2 siRNA treatment was assessed using the Cell counting kit-8 assay (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.

Techniques: Infection, Over Expression