Journal: The Journal of Biological Chemistry

Article Title: Protein tyrosine kinase Src suppresses hepatitis C virus particle release through regulation of Ndrg1

doi: 10.1016/j.jbc.2025.111125

Figure Lengend Snippet: Ndrg1 negatively regulates HCV virion release without affecting intracellular viral production . A , RT-qPCR analysis of Ndrg1 mRNA expression in HCV-infected Huh-7.5 cells with or without Bosutinib treatment. Bosutinib significantly reduced Ndrg1 expression compared to the control group. B , a trend toward downregulation of Ndrg1 mRNA expression was observed in HCV-infected Huh-7.5 cells ( p = 0.0587). C , Immunoblot analysis confirming reduced Ndrg1 protein expression in HCV-infected cells. D , efficient knockdown of Ndrg1 in Huh-7.5 cells was validated by immunoblotting using two independent siRNAs. E , immunoblot analysis of HCV-infected cells treated with control or Ndrg1 siRNAs showed that knockdown of Ndrg1 did not alter the expression levels of NS3 and core proteins, indicating no effect on intracellular viral protein production. F , TCID 50 assay and RT-qPCR analysis to assess intra- and extracellular HCV production in Ndrg1-knockdown cells. Schematic of the experimental workflow ( top ). Intracellular viral titers remained unchanged, while extracellular viral titers and RNA copies were significantly increased in Ndrg1-knockdown cells compared to wild-type and siRNA controls ( bottom panel ). Data are presented as mean ± SD from at least three independent biological replicates. Statistical significance was analyzed using Welch’s t test ( A , B , C ) or One-way ANOVA followed by the Dunnett’s multiple comparisons test ( F ). ∗ p < 0.05, ∗∗ p < 0.01, ns, not significant.

Article Snippet: Anti-HCV core monoclonal antibody (Abcam, catalog number: ab2740, lot number: 1076091–2), anti-NS3 polyclonal antibody (GeneTex, catalog number: GTX131276, lot number: 41,836), anti-Src monoclonal antibody (Cell signaling, catalog number: 2123, lot number: 5), anti-Ndrg1 polyclonal antibody (Proteintech, catalog number: 26902-1-AP, lot number: 00050845), anti-Hif1α polyclonal antibody (Proteintech, catalog number: 20960-1-AP, lot number: 00149455), anti-Stat3 polyclonal antibody (Santa Cruz, catalog number: sc-7179, lot number: A069), anti-Stat3 monoclonal antibody (Cell signaling, catalog number: 4904P, lot number: 7), anti-phospho-Stat3 (Tyr705) monoclonal antibody (Cell signaling, catalog number: 9145, lot number: 43), anti-β-actin monoclonal antibody (Santa Cruz, catalog number: sc-47778, lot number: l2822), and anti-mouse IgG(H + L), F(ab’) 2 fragment (Cell signaling, Alexa Fluor 488 conjugate, catalog number: 4408, lot number: 24) were used in this study.

Techniques: Quantitative RT-PCR, Expressing, Infection, Control, Western Blot, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Protein tyrosine kinase Src suppresses hepatitis C virus particle release through regulation of Ndrg1

doi: 10.1016/j.jbc.2025.111125

Figure Lengend Snippet: Src kinase regulates Ndrg1 expression and HCV particle release . A , RT-qPCR analysis of Ndrg1 mRNA expression in Abl-KO#1 Huh-7.5 cells. No significant differences in Ndrg1 transcript levels were observed between Abl-KO#1 and wild-type cells under either uninfected ( left ) or HCV-infected ( right ) conditions. B , immunoblot analysis confirmed that Ndrg1 protein expression remained unchanged in Abl-KO#1 cells compared to normal Huh-7.5 cells. C , RT-qPCR analysis of uninfected Src-KO#1 and Src-KO#2 clones showed significantly reduced Ndrg1 mRNA expression relative to Huh-7.5 cells. D , immunoblotting confirmed a marked reduction in Ndrg1 protein levels in uninfected Src-KO clones. E , RT-qPCR analysis of HCV-infected cells revealed significantly decreased Ndrg1 mRNA expression in Src-KO#1 and Src-KO#2 clones. F , immunoblot analysis further demonstrated suppression of Ndrg1 protein in Src-KO clones following HCV infection. G , immunoblot analysis of cells transfected with Ndrg1-targeting siRNAs confirmed that Ndrg1 knockdown did not affect Src protein levels. H , immunoblot analysis showing Ndrg1 expression in Src-KO#1 and Src-KO#2 cells following transient transfection with an Ndrg1-overexpressing plasmid (OE) or an empty vector (Mock). Quantification of Ndrg1 protein levels showed that relative Ndrg1 expression increased by 2.3-fold in Src-KO#1 and by 4.1-fold in Src-KO#2 compared with the corresponding Mock controls. I and J . Extracellular HCV RNA copies and viral titers in Src-KO#1 ( I ) and Src-KO#2 ( J ) cells, with or without Ndrg1 overexpression. Data represent the mean ± SD from three or more independent biological replicates. Statistical significance was analyzed using Welch’s t test ( A , C , E , H ) and two-tailed unpaired Student’s t test ( I and J ). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns, not significant.

Article Snippet: Anti-HCV core monoclonal antibody (Abcam, catalog number: ab2740, lot number: 1076091–2), anti-NS3 polyclonal antibody (GeneTex, catalog number: GTX131276, lot number: 41,836), anti-Src monoclonal antibody (Cell signaling, catalog number: 2123, lot number: 5), anti-Ndrg1 polyclonal antibody (Proteintech, catalog number: 26902-1-AP, lot number: 00050845), anti-Hif1α polyclonal antibody (Proteintech, catalog number: 20960-1-AP, lot number: 00149455), anti-Stat3 polyclonal antibody (Santa Cruz, catalog number: sc-7179, lot number: A069), anti-Stat3 monoclonal antibody (Cell signaling, catalog number: 4904P, lot number: 7), anti-phospho-Stat3 (Tyr705) monoclonal antibody (Cell signaling, catalog number: 9145, lot number: 43), anti-β-actin monoclonal antibody (Santa Cruz, catalog number: sc-47778, lot number: l2822), and anti-mouse IgG(H + L), F(ab’) 2 fragment (Cell signaling, Alexa Fluor 488 conjugate, catalog number: 4408, lot number: 24) were used in this study.

Techniques: Expressing, Quantitative RT-PCR, Infection, Western Blot, Clone Assay, Transfection, Knockdown, Plasmid Preparation, Over Expression, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Protein tyrosine kinase Src suppresses hepatitis C virus particle release through regulation of Ndrg1

doi: 10.1016/j.jbc.2025.111125

Figure Lengend Snippet: Src kinase regulates Ndrg1 transcription via the Stat3-Hif1α signaling pathway . A , silencing Hif1α decreases Ndrg1 expression. B , stabilization of Hif1α by CoCl 2 upregulates Ndrg1 expression. Huh-7.5 cells were treated with CoCl 2 (150 μM, 24 h). Right: quantification of Ndrg1 protein levels relative to control. C , Hif1α expression is not reduced in Src-KO cells. Huh-7.5, Src-KO#1, and Src-KO#2 cells were either infected or left uninfected with HCV (MOI = 5) for 72 h, followed by immunoblot analysis. Hif1α levels remained comparable across all cell lines. CoCl 2 treatment (150 μM) served as a positive control for Hif1α stabilization. D , Src knockout reduces Stat3 Tyr705 phosphorylation. Densitometric ratios normalized to Huh-7.5 control are indicated below each blot. E . Loss of Src kinase disrupts the interaction between Hif1α and Stat3. The Stat3-Hif1α interaction was markedly reduced in both Src-KO clones. F . Ndrg1 expression is decreased in Stat3-KO cells. Loss of Stat3 significantly reduced Ndrg1 levels without altering Hif1α abundance. G , Stat3 is required for CoCl 2 -induced upregulation of Ndrg1. Stat3-KO#1, and Stat3-KO#2 cells were treated with or without CoCl 2 (150 μM, 24 h). CoCl 2 -mediated induction of Ndrg1 was abolished in Stat3-KO cells. Data represent the mean ± SD of three independent biological replicates. Statistical significance was analyzed using Welch’s t test. ∗ p < 0.05, ns, not significant.

Article Snippet: Anti-HCV core monoclonal antibody (Abcam, catalog number: ab2740, lot number: 1076091–2), anti-NS3 polyclonal antibody (GeneTex, catalog number: GTX131276, lot number: 41,836), anti-Src monoclonal antibody (Cell signaling, catalog number: 2123, lot number: 5), anti-Ndrg1 polyclonal antibody (Proteintech, catalog number: 26902-1-AP, lot number: 00050845), anti-Hif1α polyclonal antibody (Proteintech, catalog number: 20960-1-AP, lot number: 00149455), anti-Stat3 polyclonal antibody (Santa Cruz, catalog number: sc-7179, lot number: A069), anti-Stat3 monoclonal antibody (Cell signaling, catalog number: 4904P, lot number: 7), anti-phospho-Stat3 (Tyr705) monoclonal antibody (Cell signaling, catalog number: 9145, lot number: 43), anti-β-actin monoclonal antibody (Santa Cruz, catalog number: sc-47778, lot number: l2822), and anti-mouse IgG(H + L), F(ab’) 2 fragment (Cell signaling, Alexa Fluor 488 conjugate, catalog number: 4408, lot number: 24) were used in this study.

Techniques: Expressing, Control, Infection, Western Blot, Positive Control, Knock-Out, Phospho-proteomics, Clone Assay

Journal: Molecular microbiology

Article Title: The Regulation of Antimicrobial Peptide Resistance in the Transition to Insect Symbiosis

doi: 10.1111/mmi.13598

Figure Lengend Snippet: Distribution and sequence coverage of Tn5 mutants in the S. praecaptivus genome are depicted as a histogram (A, Upper). Note that mutations affecting Sant_4061 are the most abundant in the library, following growth in LB media. The zoomed in regions of the histogram (A, Lower) reveal the distribution and abundance of Tn5 mutants in the genomic regions encoding phoPQ and Sant_4061 (in red), with the mutants recovered in the screen for polymyxin B sensitivity highlighted in orange, with the number of recovered mutants shown above each column. Growth curves for WT S. praecaptivus and Sant_4061 and phoP mutants grown in either LB or LB + Polymyxin B (PB) for 10 hours (B). Standard errors are shown above and below the average of three replicates at each time point. ΔSant_4061, ΔphoQ, and ΔphoP are unable to grow on LB supplemented with polymyxin B (C).

Article Snippet: Construction of phoP and phoQ knockouts A plasmid construct consisting of approximately 500 bp of homologous sequence up and downstream of phoP with an internal kanamycin cassette was commercially produced (Genscript).

Techniques: Sequencing

Journal: Molecular microbiology

Article Title: The Regulation of Antimicrobial Peptide Resistance in the Transition to Insect Symbiosis

doi: 10.1111/mmi.13598

Figure Lengend Snippet: Phylogeny of S. praecaptivus and related Sodalis-allied endosymbionts and free-living bacteria based on maximum likelihood analyses of the phoQ coding sequence (1.45 kbp) and 16S rRNA (1.46 kbp). Sequences below strain names on the phoQ phylogeny show the amino acids sequences of the Mg2+-binding site within PhoQ, with acidic residues highlighted in bold. The numbers adjacent to nodes indicate maximum likelihood bootstrap values shown for nodes with bootstrap support > 80%.

Article Snippet: Construction of phoP and phoQ knockouts A plasmid construct consisting of approximately 500 bp of homologous sequence up and downstream of phoP with an internal kanamycin cassette was commercially produced (Genscript).

Techniques: Bacteria, Sequencing, Binding Assay

Journal: Molecular microbiology

Article Title: The Regulation of Antimicrobial Peptide Resistance in the Transition to Insect Symbiosis

doi: 10.1111/mmi.13598

Figure Lengend Snippet: Bacterial strains used in this study

Article Snippet: Construction of phoP and phoQ knockouts A plasmid construct consisting of approximately 500 bp of homologous sequence up and downstream of phoP with an internal kanamycin cassette was commercially produced (Genscript).

Techniques: