Journal: iScience
Article Title: Prothymosin α accelerates dengue virus-induced thrombocytopenia
doi: 10.1016/j.isci.2023.108422
Figure Lengend Snippet: Overexpression of ProT promotes Nrf2 nuclear translocation and decreases ROS production, leading to reduced PLT production (A) Detection of Nrf2 and viral NS3 in Meg-01 cells infected with DENV-2 at an MOI of 1 at different time points by immunoblotting. Expression of GAPDH served as the loading control. Values shown at the bottom are ratios between the intensity of the bands corresponding to Nrf2 and those corresponding to GAPDH. (B) Nuclear translocation of Nrf2 in ProT-overexpressing cells. Meg-01 cells were transfected with pLAS2w.hProT-HA (ProT-HA), pLAs2w.hProT-Venus (ProT-Venus), or pLAS2w.Ppuro (Vector). After 48 h, nuclear and cytosolic extracts were harvested for immunoblot analysis. Lamin A/C and α-tubulin served as the nuclear and cytoplasmic markers, respectively. Values shown at the bottom are ratios between the intensity of the bands corresponding to Nrf2 and those corresponding to α-tubulin or Lamin A/C. (C and D) Quantification of HO-1 (left) and NQO1 (middle) by RT-qPCR and measurement of ROS production (right) in Meg-01 cells transduced with lentiviral vectors encoding ProT (LV.ProT) or no transgenes (LV.Null) (C) and shRNA specific to ProT (LV.shProT-17 and -20) or LacZ (LV.shLacZ) (D). ROS production in ProT overexpression (C) and knockdown (D) Meg-01 cells or control cells were measured by flow cytometry after stimulation with PMA (10 −6 M) for 7 days and staining with DCFDA. Values shown are means ± SD (n = 3, Student’s t test or one-way ANOVA). (E) Overexpression of miR-126 reduces Nrf2 levels and antioxidant gene expression. Detection and quantification of Nrf2, HO-1, and NQO1 in Meg-01 cells transduced with LV.miR-126 or LV.miR-Ctrl by RT-qPCR. Values shown are mean ± SD (n = 4, Student’s t test). (F) Quantification of GP9 and TUBB1, which are involved in PLT release, in ProT knockdown (shProT-20) or control (shLacZ) Meg-01 cells by RT-qPCR. Values shown are means ± SD (n = 3, Student’s t test). (G) Detection of the PLT activation marker CD42b in ProT knockdown (shProT-20) or control (shLacZ) Meg-01 cells by flow cytometry. Values shown are means ± SD (n = 3, Student’s t test or one-way ANOVA).
Article Snippet: PE-Cy5-conjugated anti-human CD42b antibody , BD Biosciences , Cat #551141; RRID: AB_394069.
Techniques: Over Expression, Translocation Assay, Infection, Western Blot, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Transduction, shRNA, Flow Cytometry, Staining, Activation Assay, Marker
