Journal: Viruses

Article Title: CREB3 Plays an Important Role in HPSE-Facilitated HSV-1 Release in Human Corneal Epithelial Cells

doi: 10.3390/v14061171

Figure Lengend Snippet: Upregulation of CREB3 causes an increase in extracellular virus: Human corneal epithelial cells were transfected with a control plasmid or increasing concentrations of CREB3 plasmid for 24 h followed by infection with HSV-1 at 0.1 MOI. At 24 h post infection, samples were collected for analysis. ( A ) Representative immunoblot images confirming the upregulation of CREB3 protein with increasing plasmid concentrations ( B ) Cellular supernatants were collected and centrifuged to remove any cellular components prior to overlaying on Vero cells to titrate extracellular infectious virion particles. One-way ANOVA was performed between the datasets to determine statistical significance. ** p -value < 0.01.

Article Snippet: The membranes were then blocked overnight with antibodies specific for CREB3 (ProteinTech 11275-1-AP), HSV-1 gB (Abcam, 6506), syndecan-1 (Santa Cruz Biotechnology, sc-6532), and HPSE (Advanced Targeting Systems, HP130) followed by the corresponding species-specific secondary antibodies.

Techniques: Virus, Transfection, Control, Plasmid Preparation, Infection, Western Blot

Journal: Viruses

Article Title: CREB3 Plays an Important Role in HPSE-Facilitated HSV-1 Release in Human Corneal Epithelial Cells

doi: 10.3390/v14061171

Figure Lengend Snippet: Upregulation of HPSE increases CREB3 expression: ( A ) Human corneal epithelial cells were transfected with a control plasmid, HPSE-GS3 (constitutively active form of HPSE), or full-length HPSE plasmid for 24 h followed by infection with HSV-1 at 0.1 MOI. At 24 h post infection, samples were collected for immunoblotting analysis. ( B ) Human corneal epithelial cells were transfected with a control plasmid or CREB3 plasmid for 24 h followed by infection with HSV-1 at 0.1 MOI. At 24 h post infection, samples were collected for immunoblotting analysis. ( C ) Heparanase wild-type or knockout mouse embryonic fibroblasts were infected with 0.1 MOI HSV-1 and cell lysates were collected at the indicated time points for immunoblotting analysis.

Article Snippet: The membranes were then blocked overnight with antibodies specific for CREB3 (ProteinTech 11275-1-AP), HSV-1 gB (Abcam, 6506), syndecan-1 (Santa Cruz Biotechnology, sc-6532), and HPSE (Advanced Targeting Systems, HP130) followed by the corresponding species-specific secondary antibodies.

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Infection, Western Blot, Knock-Out

Journal: Viruses

Article Title: CREB3 Plays an Important Role in HPSE-Facilitated HSV-1 Release in Human Corneal Epithelial Cells

doi: 10.3390/v14061171

Figure Lengend Snippet: Upregulation of CREB3 increases extracellular HPSE: Human corneal epithelial cells were not transfected (lipofectamine only—L2K) or transfected with a control plasmid, CREB3 plasmid, or full-length HPSE plasmid for 48 h with or without HSV-1 infection. Cellular supernatants were collected and analyzed via a slot blot technique. ( A ) HPSE secretion, ( B ) Syndecan-1 secretion, and ( C ) HSV-1 envelope glycoprotein-B secretion were detected using the slot blot technique. ( D , E ) Experiments mentioned in ( A , B ) were performed in triplicate and the quantification of the slot blot bands was performed using image J and represented as bar charts. * p -value < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The membranes were then blocked overnight with antibodies specific for CREB3 (ProteinTech 11275-1-AP), HSV-1 gB (Abcam, 6506), syndecan-1 (Santa Cruz Biotechnology, sc-6532), and HPSE (Advanced Targeting Systems, HP130) followed by the corresponding species-specific secondary antibodies.

Techniques: Transfection, Control, Plasmid Preparation, Infection, Dot Blot

Journal: Viruses

Article Title: CREB3 Plays an Important Role in HPSE-Facilitated HSV-1 Release in Human Corneal Epithelial Cells

doi: 10.3390/v14061171

Figure Lengend Snippet: Upregulation of CREB3 increases COPII vesicle transcripts: ( A ) Human corneal epithelial cells were transfected with a control plasmid, CREB3 plasmid, or full-length HPSE plasmid for 48 h. Total cellular RNA was extracted from the samples using the TriZol technique, reverse transcribed to form cDNA, and analyzed via RT-qPCR. ( A ) qPCR confirmation of CREB3 upregulation in transfected cells. ( B – H ) COPII vesicle machinery and HPSE mRNA transcripts shown as a fold-change relative to non-transfected samples. Two-way ANOVA was used to determine statistical differences between rows (treatment groups) within a given a column (mRNA transcript expression level). * p -value < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were then blocked overnight with antibodies specific for CREB3 (ProteinTech 11275-1-AP), HSV-1 gB (Abcam, 6506), syndecan-1 (Santa Cruz Biotechnology, sc-6532), and HPSE (Advanced Targeting Systems, HP130) followed by the corresponding species-specific secondary antibodies.

Techniques: Transfection, Control, Plasmid Preparation, Reverse Transcription, Quantitative RT-PCR, Expressing

Journal: Viruses

Article Title: CREB3 Plays an Important Role in HPSE-Facilitated HSV-1 Release in Human Corneal Epithelial Cells

doi: 10.3390/v14061171

Figure Lengend Snippet: Co-expression of CREB3 and HPSE increased extracellular virus release: ( A ) Human corneal epithelial cells were left non-transfected, treated with lipofectamine alone, or transfected with a control plasmid, CREB3 plasmid, or full-length HPSE plasmid for 24 h followed by infection with HSV-1 for 24 h. ( A ) Cell lysates were immunoblotted to confirm co-transfection of CREB3 and HPSE. ( B ) Cellular supernatants were collected, centrifuged, and overlayed on Vero cells to titrate infectious titer present in the extracellular space via a plaque assay. ( C ) Cell lysates were collected, ultrasonicated, and overlaid on Vero cells to titrate intracellular infectious titer via a plaque assay. One-way ANOVA was performed between the sample sets to determine statistically significant differences. ns-non-significant ** p -value < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The membranes were then blocked overnight with antibodies specific for CREB3 (ProteinTech 11275-1-AP), HSV-1 gB (Abcam, 6506), syndecan-1 (Santa Cruz Biotechnology, sc-6532), and HPSE (Advanced Targeting Systems, HP130) followed by the corresponding species-specific secondary antibodies.

Techniques: Expressing, Virus, Transfection, Control, Plasmid Preparation, Infection, Cotransfection, Plaque Assay

Journal: iScience

Article Title: CREB3L4 promotes hepatocellular carcinoma progression and decreases sorafenib chemosensitivity by promoting RHEB-mTORC1 signaling pathway

doi: 10.1016/j.isci.2024.108843

Figure Lengend Snippet:

Article Snippet: CREB3L4 , Proteintech , Cat No. 13630-1-AP; RRID: AB_2276550.

Techniques: Virus, Recombinant, CCK-8 Assay, Chromatin Immunoprecipitation, Reporter Assay, Expressing, Sequencing, Plasmid Preparation, Software