Journal: Journal of Virology

Article Title: The white spot syndrome virus wsv156 protein hijacks Parkin-dependent mitophagy to promote viral infection

doi: 10.1128/jvi.00418-26

Figure Lengend Snippet: Wsv156 targets mitochondria via Cq TOMM70 and triggers mito-aggresome formation. ( A ) Wsv156 expression increased following WSSV infection. Temporal expression of wsv156 at the mRNA (left panel) and protein level (right panel) was assessed by RT-qPCR and western blot, respectively, in Hpt cells after WSSV infection. ( B ) Wsv156 localized to mitochondria. Endogenous wsv156 protein in Hpt cells (left panel) and overexpressed wsv156 in HEK 293T cells (right panel) both predominantly co-fractionated with mitochondria. ( C ) Identification of host mitochondrial proteins interacting with wsv156 by affinity purification-mass spectrometry (AP-MS). ( D ) Wsv156 interacted with Cq TOMM70. Co-IP with anti-Myc beads was performed in HEK 293T cells co-transfected with wsv156-Myc and Cq TOMM70-HA. ( E ) Wsv156 expression induced mitochondrial aggregation. Confocal microscopy of HEK 293T cells showed co-localization of wsv156-GFP (green) with the mitochondrial marker Hs COX IV (red). Cells expressing GFP alone or wsv152-GFP served as controls. ( F ) Wsv156 knockdown attenuated WSSV-induced mitochondrial aggregation. Gene silencing of wsv156 in Hpt cells reduced mitochondrial clustering (red) compared to control cells at 12 hpi. NT, non-targeting dsRNA (GFP dsRNA) control; RNAi wsv156, wsv156-specific dsRNA. The right panel quantifies mitochondrial aggregation using the CI and the percentage of cells containing mito-aggresomes. Nuclei were stained with DAPI. **, P < 0.01.

Article Snippet: Co-IP was performed using anti-Myc magnetic beads (MedChemExpress, HY-K0206) as previously described ( ).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Co-Immunoprecipitation Assay, Transfection, Confocal Microscopy, Marker, Knockdown, Control, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: SPARC Promotes Corneal Epithelial Wound Healing Through β-catenin Nuclear Translocation and c-Met Activation

doi: 10.1167/iovs.67.10.23

Figure Lengend Snippet: SPARC knockdown disrupts Wnt signaling and β-catenin nuclear translocation. ( A , B ) Western blot and RT-qPCR analysis showing reduced β-catenin and c-Myc protein and mRNA expression in HCECs following SPARC knockdown ( n = 5 independent experiments per group). ( C , D ) Western blot and RT-qPCR analysis of β-catenin protein and mRNA expression in corneal epithelial cells from WT and Sparc −/− mice at the indicated postinjury time points ( n = 3 independent pooled biological replicates per group). ( E , F ) Immunofluorescence staining of β-catenin ( red ) and DAPI ( blue ) in HCECs after SPARC knockdown ( n = 5 independent experiments per group). ( G ) Immunofluorescence images and quantification of β-catenin ( green ) in corneal sections from WT and Sparc −/− mice at different postinjury time points; nuclei were counterstained with DAPI ( blue ) ( n = 5 mice per group). ( H , I ) Scratch-wound assay and quantification of wound closure in HCECs after β-catenin knockdown with or without exogenous SPARC supplementation (the knockdown efficiency of β-catenin in HCECs is shown in B; n = 5 independent experiments per group). Scale bars are indicated in the images. Data are presented as mean ± SD. * P < 0.05, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibodies used were anti-SPARC ( AB290636 ; Abcam), anti–β-catenin (A19657; ABclonal), anti–p-Met (3077; CST), anti–c-Met (25869-1-AP; Proteintech, Rosemont, IL, USA), anti–cellular myelocytomatosis oncogene (c-Myc) (A1309; ABclonal), anti–T-cell factor (TCF) 4 (A1141; ABclonal), and anti-GAPDH (A19056; ABclonal).

Techniques: Knockdown, Translocation Assay, Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Scratch Wound Assay Assay