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

Journal: Scientific Reports

Article Title: Luman contributes to brefeldin A-induced prion protein gene expression by interacting with the ERSE26 element

doi: 10.1038/srep42285

Figure Lengend Snippet: Primer sequences used for PCR.

Article Snippet: LUMAN , F , OriGene-qStar Primers-H.sapiens CREB3 (Luman/LZIP) #HP209330.

Techniques: Sequencing, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration showing the localization of CREB3-FL and CREB3-CF and changes in nuclear morphology. b Western blotting showing the localization of endogenous CREB3-FL and CREB3-CF in the cytosolic and nuclear fractions. c Illustration showing the nuclear membrane localization of CREB3-FL and CREB3-FL-mtS1P. d Left panels: Diagram showing the experimental strategies used to obtain the extracts of the cytosolic and nuclear fractions. Right panels, Western blotting showing nuclear DNA bound to CREB3-FL, CREB3-CF, and CREB3-dTM. e Confocal microscopy image illustrating the nuclear membrane localization of the Myc-CREB3-FL-HA double-tagged protein. f Diagram showing the experimental strategy used to determine the extent of nuclear DNA binding to CREB3-FL, CREB3-CF, and CREB3-FL-mtS1P. g Western blotting showing nuclear DNA binding of CREB3-FL, CREB3-CF, and CREB3-FL-mtS1P in the presence of NaCl. h Illustration of the localization and orientation of CREB3 at the nuclear membrane.

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Western Blot, Membrane, Confocal Microscopy, Binding Assay

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration showing the relationship between the localization of CREB3-FL, CREB3-dTM and CREB3-CF and changes in nuclear morphology. b ICF images showing the relationship between domain deletion of CREB3-CF and changes in nuclear morphology. c ICF images showing the relationship between the serial deletion of CREB3-CF and changes in nuclear morphology. d ICF images showing the effects of CREB3-FL, CREB3-CF, and CREB3-dTM on DDR, DNA herniation, and microsatellite formation. e Illustration showing Myc-CREB-FL-HA at the inner nuclear membrane and nuclear membrane ripping and aggregation via karyoptosis. f Illustration of the frequency of nuclear abnormalities in relation to CREB3-CF expression. The error bars indicate the SEMs. *** P < 0.0001 (Student’s t test).

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Membrane, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration of nuclear membrane deformation-induced nuclear DNA loss. Left panels, Illustration of CREB3-CF-induced nuclear membrane deformity and loss of nuclear DNA. Central panels. Magnification of the boxed area in the left panels. Graphs, Comparison of DAPI intensity between CREB3-CF-negative and CREB3-CF-positive cells ( n = 56–87 cells). The error bars indicate the SEMs. * P < 0.05, *** p < 0.0001 (Student’s t test). b IP/Western blots showing the interaction of Lamin A and B1 with CREB3-CF. c Illustration of the interaction between the bZIP domain of CREB3-CF and Lamin B1. d Transmission electron microscopy images showing nuclear membrane abnormalities in terms of shape and integrity in mock- and CREB3-CF-overexpressing cells. Arrows indicate nuclear membrane invagination and membrane integrity loss. e Illustration of explosive nuclear membrane rupture during karyoptosis. Frame 1, image showing normal nuclear morphology before karyoptosis initiation; Frame 2, image showing initiation of karyoptosis; Frame 3, image of early-stage karyoptosis; Frame 4, image of the moment of karyoptotic nucleus explosion (karyoptosis); Frame 5, image after karyoptotic nucleus explosion. The live and time-lapse videos are in Supplementary Live Images, Supplementary Live Image – .

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Membrane, Comparison, Western Blot, Transmission Assay, Electron Microscopy

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration showing that CREB3-CF-induced karyoptosis evokes DDR, indicating an increase in γH2AX. b Illustration showing that CREB3-CF-induced karyoptosis is decoupled from the p53-dependent DDR signaling pathway. c ICF images showing that loss of CREB3 anchoring at the nuclear membrane induces nuclear lobulation, invagination, and nuclear DNA herniation. d Western blots indicating that CREB3-CF-induced karyoptosis does not induce the cleavage of apoptosis markers. e Western blots indicating that CREB3-CF-induced karyoptosis inhibits autophagy. f ICF analysis showing that CREB3-CF overexpression increased the nuclear accumulation of LC3. g ICF image showing that CREB3-Cf increases the formation of extracellular vesicles containing CREB3-CF, lamin, and genomic DNA. Right panels, TEM images illustrating that CREB3-CF overexpression induces abnormal nuclear morphology and increases extracellular vesicle formation. h Western blots showing that CREB3-CF-induced karyoptosis differs from necroptosis. i Western blots showing that CREB3-CF-induced karyoptosis is distinguishable from pyroptosis.

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Membrane, Western Blot, Over Expression

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Western blot showing that UVB exposure increases the endogenous protein levels of CREB3-FL and CREB3-CF. b Western blot analyses revealing that ER stress, induced by vesicle trafficking inhibitors such as brefeldin A and golgicide A, leads to increased cleavage of CREB3-FL and subsequent production of CREB3-CF. c Western blot results indicating that ER stress, triggered by elevated levels of Ca 2+ and ROS, moderately increases the protein levels of CREB3-FL and CREB3-CF, while the inhibition of glycosylation does not have this effect. d Western blot analyses showing that direct DNA damage caused by cisplatin and doxorubicin, but not oxaliplatin, slightly increases CREB3-CF protein levels without affecting CREB3-FL protein levels. e Western blot analyses illustrating the changes in the protein levels of CREB3-FL and CREB3-CF over time following washout of BFA. f TEM images demonstrating that BFA damages the nuclear membrane. g ICF image revealing that both overexpressed and endogenous S1P are localized not only to the cytoplasm but also to the nuclear membrane.

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Western Blot, Inhibition, Glycoproteomics, Membrane

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Illustration showing the sampling strategies used for proteomic analysis of CREB3-CF-induced karyoptosis. The comparisons used were as follows: analysis 1, mock and CREB3-FL; analysis 2, mock and CREB3-CF; analysis 3 CREB3-CF and mock+UVB, mock+UVB and mock, CREB3-CF and mock, and CREB3-CF/mock+UVB and mock; and analysis 4, mock+UVB and CREB3-FL + UVB. b Illustration of the distances of the protein sets in 5 different cells in Fig. . c Hit map of CREB3-CF-induced karyoptosis according to analysis 2. Red, increase; green, decrease. d Protein frequency of the Cluster 2 proteins in CREB3-CF-induced karyoptotic cells according to analysis 2. e GO analysis of Cluster 2 proteins in CREB3-CF-induced karyoptotic cells according to analysis 2. A detailed list is provided in Supplementary Table . f Hit maps of CREB3-CF, mock+UVB, and mock to compare CREB3-CF and mock+UVB; mock+UVB and mock CREB3-CF and mock, and CREB3-CF/mock+UVB and mock according to analysis 3. Red, increase; green, decrease. g Illustration showing the protein frequency of Cluster 1 and 2 proteins in CREB3-CF-induced karyoptotic cells according to analysis 3. h GO analysis of Cluster 1 and 2 proteins in CREB3-CF-induced karyoptotic cells according to analysis 3. A detailed list is provided in Supplementary Table .

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Sampling

Journal: Experimental & Molecular Medicine

Article Title: Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death

doi: 10.1038/s12276-024-01195-1

Figure Lengend Snippet: a Graphs showing cellular senescence induction by the expression of CREB3-FL or CREB3-CF. Left, percentage of β-gal-positive cells among more than 200 cells. Right, Fold increase in β-gal-positive cells among cancer cells. The cells stained with β-gal are shown in Supplementary Fig. . b CREB3-CF induces p21 protein expression in SK-MEL-2 and HeLa cells. c Graphs showing that the expression of CREB3-CF or CREB3-dTM, but not CREB3-FL, suppresses the proliferation of SK-MEL-2 cells. The cells stained with crystal violet are shown in Supplementary Fig. . d hat UVB increases CREB3-FL and CREB3-CF expression. e UVB induces DDR via the detection of nuclear puncta of γH2AX. f Illustration of the increase in nuclear DNA bound to endogenous CREB3-FL and -CF by UVB irradiation. g Cell cycle analysis data showed that the increase in UVB dose was not associated with sub-G1 enhancement. h Flow cytometry data showing that the early necrosis/necroptosis population is not associated with the late apoptosis/necrosis/necroptosis population. In contrast, UVB irradiation continuously decreased the live cell population in a dose-dependent manner. i Images of confocal microscopy images showing nuclear membrane ripping and rupture in CREB3-FL-overexpressing cells after UVB treatment.

Article Snippet: Anti-CREB3 antibodies were obtained from CUSABIO (cat #: CSB-PA005948, Houston, TX, USA), FineTest (cat #: FNab01962, Wuhan, Hubei, China) or Proteintech (cat #:11275-1-AP, Rosemont, IL, USA).

Techniques: Expressing, Staining, Irradiation, Cell Cycle Assay, Flow Cytometry, Confocal Microscopy, Membrane