Journal: Viruses

Article Title: Inhibition of MAVS Aggregation-Mediated Type-I Interferon Signaling by Foot-and-Mouth Disease Virus VP3

doi: 10.3390/v13091776

Figure Lengend Snippet: FMDV VP3 binds to the transmembrane domain of MAVS. ( A ) HEK293T cells were transfected with an IFN-β reporter plasmid, plus TK-Renilla plasmid and increasing amounts of FMDV VP3 plasmid, along with overexpression plasmids for RIG-I, MDA5, MAVS, TRIF, TRAF3, or TBK1. At 24 h post-transfection, luciferase activity was measured in a luminometer. TK-Renilla was used as transfection control to normalize firefly luciferase activity. ( B ) HEK293T cells were cotransfected with the control vector (Flag), Flag-tagged RIG-I, MDA5, MAVS, and TBK1 plasmids together with GST-tagged FMDV VP3 plasmids. Cell lysates were subjected to immunoprecipitation (IP), followed by immunoblotting with an anti-GST antibody. WCL was immunoblotted with anti-Flag and anti-GST antibodies. ( C ) HEK293T cells were cotransfected with the control vector (GST), Flag-MAVS, and GST-tagged FMDV VP3 plasmid. Cell lysates were subjected to GST pulldown (PD), followed by immunoblotting with an anti-Flag antibody. Whole-cell lysate (WCL) was immunoblotted with anti-GST and anti-Flag antibodies. Lysates of ( D ) HEK293T cells transfected with control vector (Flag) or with Flag-tagged FMDV VP3 plasmid were subjected to immunoprecipitation with Flag antibody or control IgG, followed by immunoblotting with anti-MAVS antibody. WCL was immunoblotted with anti-MAVS and anti-Flag antibodies. ( E ) HEK293T cells were transfected with Flag-tagged MAVS plasmid together with V5-tagged FMDV VP3 plasmid, followed by confocal microscopy assay with anti-Flag (red) and anti-V5 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar represents 5 μM. Arrow indicates the co-localized VP3 and MAVS protein. ( F ) GST-tagged full-length and deletion mutants of MAVS were constructed for the immunoprecipitation assay. ( G ) GST-tagged full length and amino acid 1–80, 1–180, 1–470, and 180–540 fragments of MAVS, and ( H ) GST-tagged full length and amino acid 451–470, 470–540, 451–540, and 180–540 fragments of MAVS or control vector (GST) were cotransfected to HEK293T cells together with Strep-tagged FMDV VP3 plasmid. Cell lysates were subjected to GST-PD and immunoblotted with anti-Strep antibody following immunoblotting of the WCL with both anti-Strep and anti-GST antibodies. In A, data are representative of three independent experiments, each with similar results, and all the values are expressed as mean ± SD of two biological replicates. All the immunoblot and confocal data are representative of at least two independent experiments, each with similar results. Student’s t -test; * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: The antibody for V5 (A190-220A) was purchased from BETHYL Laboratories.

Techniques: Transfection, Plasmid Preparation, Over Expression, Luciferase, Activity Assay, Control, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining, Construct

Journal: Viruses

Article Title: Inhibition of MAVS Aggregation-Mediated Type-I Interferon Signaling by Foot-and-Mouth Disease Virus VP3

doi: 10.3390/v13091776

Figure Lengend Snippet: FMDV VP3 disrupts MAVS activation. ( A , B ) Immunoblot analysis of mitochondria fraction isolated from the ( A ) PK15 and ( B ) HeLa cells which transfected with increasing amounts of V5-tagged FMDV VP3 expression plasmid, and Sendai virus (SeV) infected (1MOI). Mitochondria fraction was immunoblotted with the MAVS, Tom40, and α-tubulin antibodies. PK15 cell whole-cell lysate (WCL) was immunoblotted with MAVS and α-tubulin antibodies, while HeLa cell WCL was immunoblotted with anti-MAVS, anti-V5, and anti-α-tubulin antibodies. In PK15 cells, qRT-PCR was done to detect the expression of FMDV VP3. ( C ) HEK293T cells were cotransfected with control vector (Strep), Flag-MAVS, Strep-MAVS, and increasing doses of V5-tagged FMDV VP3 plasmid. The cell lysates were subjected to Strep-PD and subsequent immunoblotting with anti-Flag, anti-V5, and anti-Strep antibodies. Further, WCL was immunoblotted with anti-Flag, anti-Strep, and anti-V5 antibodies. ( D ) PK15 and ( E ) HeLa cells were transfected with increasing amounts of V5-tagged FMDV VP3 plasmid and infect the SeV (1MOI). Following that, crude mitochondria were isolated from the cells and subjected to the semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) and immunoblotted with anti-MAVS antibody for the MAVS aggregation detection. The same sample was used for the SDS-PAGE and immunoblotted with anti-MAVS and anti-Tom40 antibodies for PK15 cells, and anti-MAVS, anti-V5, and anti-Tom40 antibodies for HeLa cells. In PK15 cells, qRT-PCR was done to detect the expression of FMDV VP3. ( F ) HeLa cells were transiently transfected with the increasing amounts of V5-tagged FMDV VP3 plasmid and infected with SeV (1MOI). Cell lysates were immunoblotted against phosphorylated (p-) TBK1, TBK1, pIRF3, IRF3, V5, and β-actin antibodies, followed by the qRT-PCR for SeV C protein, and β-actin. All the data are representative of two independent experiments, each with similar results.

Article Snippet: The antibody for V5 (A190-220A) was purchased from BETHYL Laboratories.

Techniques: Activation Assay, Western Blot, Isolation, Transfection, Expressing, Plasmid Preparation, Virus, Infection, Quantitative RT-PCR, Control, Agarose Gel Electrophoresis, SDS Page

Journal: Cancers

Article Title: DUSP10 Is a Regulator of YAP1 Activity Promoting Cell Proliferation and Colorectal Cancer Progression

doi: 10.3390/cancers11111767

Figure Lengend Snippet: DUSP10 interacts with YAP1 through Ser397 residue. ( a ) Expression of DUSP10, YAP1, p-YAP Ser397 , and p-YAP Ser127 proteins in DUSP10-wild type (DUSP10-WT), phosphatase catalytic site mutant (DUSP10-C408S), and p38 binding site mutant (DUSP10-AA) HT29 cell line in LD and HD. TUBULIN is the control protein. (Top) A representative image of three independent experiments. (Bottom) Quantification of all blots performed (mean ± SEM; Student’s t -test; * p < 0.05, ** p < 0.01, *** p < 0.001). ( b ) Analysis of DUSP10, YAP1, and p-YAP Ser127 proteins from nuclei and cytoplasm extracts of HT29 DUSP10 mutant cell lines in HD. LAMIN A/C and TUBULIN are used as nuclear and cytoplasm control proteins, respectively. (Left) A representative image of three independent experiments. (Right) Quantification of all blots performed (mean ± SEM; Student’s t -test; * p < 0.05, ** p < 0.01). ( c ) Immunoprecipitation of YAP-FLAG and detection of DUSP10 and p38 in HCT116. DUSP10-V5 and YAP-FLAG plasmids were co-transfected into the cell line and detected by anti-FLAG and anti-V5 antibodies, respectively. YAP1 wild type (YAP1-FLAG) and mutant (S381A-FLAG, S127A-FLAG) plasmids were immunoprecipitated with anti-FLAG. Representative images of three independent experiments. ( d ) Relative luciferase activity of the 8xGTII-luc (YAP/TEAD binding element reporter) was measured in HD, responding to DUSP10 overexpression and DUSP10 mutant constructs. HT29 (Top graph) and HCT116 (Bottom graph) were transiently transfected with the indicated plasmids and its control constructs. Student’s t -test (mean ± SEM; ** p < 0.01, *** p < 0.001) and three independent experiments were performed. Completed immunoblots of a–c are in , respectively.

Article Snippet: Antibodies: DUSP10 #3483 from Cell Signaling Technology. ( Western Blot ) DUSP10 ab140123 from Abcam. ( Immunohistochemistry ) phospho-p38 (3D7) #9215 from Cell Signaling Technology. ( Western Blot and Immunohistochemistry ) p38 #9212 from Cell Signaling Technology. ( Western Blot ) V5-Tag (D3H8Q) #13202 from Cell Signaling Technology. ( Western Blot ) YAP1 (D8H1X) #14074 from Cell Signaling Technology. (Western Blot, Immunofluorescence and Immunohistochemistry) phospho-YAP Ser397 (D1E7Y) #13619 from Cell Signaling Technology. ( Western Blot ) phospho-YAP Ser127 (D9W21) #13008 from Cell Signaling Technology. ( Western Blot ) HSP90α/β (H-114) #sc7947 from Santa Cruz Biotechnology. ( Western Blot ) phospho-JNK (G-7) #sc6254 from Santa Cruz Biotechnology. ( Western Blot ) JNK1/2/3 (FL) #sc474 from Santa Cruz Biotechnology. ( Western Blot ) C-Myc (9E10): sc-40 from Santa Cruz Biotechnology. ( Immunofluorescence ) GAPDH #MAB374 from Millipore. ( Western Blot ) LAMIN A/C (131C3) #ab8984 from Abcam. ( Western Blot ) FLAG ® M2 #F1804 from Sigma. (Western Blot and Immunoprecipitation) α-TUBULIN (DM1A) #T6199 from Sigma. ( Western Blot ) Normal mouse IgG #12-371 from EMD Millipore. ( Immunoprecipitation ) Mouse HRP secondary antibody: #7076 from Cell Signaling. ( Western Blot ) Rabbit HRP secondary antibody: #7074 from Cell Signaling. ( Western Blot ) Rabbit Biotinylated secondary antibody: E0432 from DAKO. ( Immunohistochemistry ) Mouse Biotinylated secondary antibody: E0433 from DAKO. ( Immunohistochemistry ) Alexa-conjugated secondary antibody: A-31570 from Invitrogen. ( Immunofluorescence )

Techniques: Residue, Expressing, Mutagenesis, Binding Assay, Control, Immunoprecipitation, Transfection, Luciferase, Activity Assay, Over Expression, Construct, Western Blot

Journal: Virology

Article Title: The rinderpest virus non-structural C protein blocks the induction of type 1 interferon.

doi: 10.1016/j.virol.2008.11.022

Figure Lengend Snippet: Fig. 2. The RPV C protein suppresses interferon induction. (a) Vero-SLAM cells were transfected with 0.5 μg each pIFΔ(−116)lucter, pJatLac and 1 μg of a plasmid driving the expression of the indicated viral protein (or empty vector for “none”). 24 h post-transfection the cells were transfected with dsRNA (poly(I:C)) or left untreated. After a further 24 h the cells were lysed and luciferase and beta-galactosidase assays performed as in “Materials and methods”. (b) A549 cells were transfected with 0.5 μg each pIFΔ(−116)lucter, pJatLac and 1 μg of a plasmid driving the expression of the indicated viral protein (or empty vector for “none”). 30 h post-transfection the cells were infected with NDV (moi≈1.5) or left uninfected. After a further 18 h the cells were lysed and luciferase and beta-galactosidase assays performed as in “Materials and methods”. (a,b) Results from separate experiments were combined by setting the RLUs induced by dsRNA or NDV in cells transfected with empty vector to 100% (c,d) Vero-SLAM cells were transfected with 1 μg each pIFΔ(−116)lucter and pJatLac. 6 h post-transfection cells were infected with equal amounts (moi≈0.3) of the indicated viruses or transfected with dsRNA or left untreated (“none”).18 h post-infection/transfection the cells were lysed and luciferase and beta-galactosidase activities measured; results from separate experiments were combined by setting the RLUs induced by dsRNA to 100%. Error bars show standard deviations of normalised data. (e) Vero-SLAM cells were transfected with plasmids driving the expression of the indicated viral protein, or empty vector, in the same way as for (a). Samples were harvested 48 h post-transfection into SDS-PAGE sample buffer and the expressed proteins detected by Western blot using monoclonal antibody to the V5 epitope tag.

Article Snippet: Antibodies used were mouse monoclonal antibody to RPV P ‘2-1’ (Sugiyama et al., 1989) and mouse monoclonal antibody to the V5 epitope tag (Serotec).

Techniques: Transfection, Plasmid Preparation, Expressing, Luciferase, Infection, SDS Page, Western Blot

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Effective knockdown - replace gene therapy in a novel mouse model of DNM1 developmental and epileptic encephalopathy.

doi: 10.1016/j.ymthe.2024.08.009

Figure Lengend Snippet: Figure 2. Bivalent AAV vector development design (A) Renilla-Firefly dual-luciferase assay for testing efficacy of microRNA shuttle designed to interfere with mouse (Dnm1) or human (DNM1) dynamin-1 mRNA in cell culture. Graph shows efficacy luciferase expression screen of fifteen test inserts compared with empty or miGFP vector against DNM1 expressed in HEK cells. (B) Five test inserts were evaluated against wild-type (left) mouse Dnm1, all but one of which did not target codon-optimized, RNAi-resistant mouse Dnm1 (right). (C) Design of bivalent AAV vector showing tail-to-tail configuration of SYN1-coDNM1-V5 and U6-mi1869 inserts. Error bars represent standard error of the mean.

Article Snippet: The membranes were incubated with primary antibodies: Dynamin1- 1:200 (Invitrogen #PA1-660); V5-Tag (D3H8Q), 1:1,000 (CST #13202); ACTB, 1:15,000 (Santa Cruz Biotechnology, Dallas, TX, USA: sc-47778) overnight at 4 C, followed by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit (1:10,000) (Proteintech, Rosemont, IL, USA; SA00001-2) for 1 h at room temperature.

Techniques: Plasmid Preparation, Luciferase, Cell Culture, Expressing

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Effective knockdown - replace gene therapy in a novel mouse model of DNM1 developmental and epileptic encephalopathy.

doi: 10.1016/j.ymthe.2024.08.009

Figure Lengend Snippet: Figure 5. Quantification of endogenous and viral-delivered dynamin-1 mRNA and protein in PND15-PND17 mouse pups (A) Normalized RNA-seq transcript counts (also see Table S3) in bivalent treated or control heterozygous G359A/+ and hemizygous /+ pups for endogenous Dnm1 mRNA (Generalized Linear Mixed Model: p < 0.01 genotype effect, p < 0.0001 treatment effect) and exogenous codon-optimized, RNAi resistant, V5 epitope-tagged virally transduced Dnm1 mRNA (CO-Dnm1-v5). (B and C) Western blot showing total DNM1 protein (detected with DNM1-specific antibody; least-squares regression: p < 0.05 genotype effect, p < 0.05 treatment effect) and exogenous DNM1 protein detected with antibody to the V5 epitope tag. Error bars represent standard effort of the mean.

Article Snippet: The membranes were incubated with primary antibodies: Dynamin1- 1:200 (Invitrogen #PA1-660); V5-Tag (D3H8Q), 1:1,000 (CST #13202); ACTB, 1:15,000 (Santa Cruz Biotechnology, Dallas, TX, USA: sc-47778) overnight at 4 C, followed by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit (1:10,000) (Proteintech, Rosemont, IL, USA; SA00001-2) for 1 h at room temperature.

Techniques: RNA Sequencing, Control, Western Blot

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Effective knockdown - replace gene therapy in a novel mouse model of DNM1 developmental and epileptic encephalopathy.

doi: 10.1016/j.ymthe.2024.08.009

Figure Lengend Snippet: Figure 6. Viral transduction at 2 weeks postnatal Shown at the left are z stack images from Dnm1 heterozygous G359/+ and hemizygous /+ brain from treated and control pups. scAAV9-U6-miDnm1-hSYN1-coDnm1 is visualized with an antibody to the V5 epitope tag (green). Inset is higher magnification of stippled boxes, showing examples from single layer of V5 and parvalbumin (PV, red) and colocalization (yellow-orange). Shown at the right are neuron counts of V5 (green), PV (red) and colocalized cells (yellow) and their mean values (V5, dark gray; PV, medium gray; colocalized, light gray).

Article Snippet: The membranes were incubated with primary antibodies: Dynamin1- 1:200 (Invitrogen #PA1-660); V5-Tag (D3H8Q), 1:1,000 (CST #13202); ACTB, 1:15,000 (Santa Cruz Biotechnology, Dallas, TX, USA: sc-47778) overnight at 4 C, followed by incubation with secondary horseradish peroxidase-conjugated goat anti-rabbit (1:10,000) (Proteintech, Rosemont, IL, USA; SA00001-2) for 1 h at room temperature.

Techniques: Transduction, Control

Journal: Virus Research

Article Title: Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage

doi: 10.1016/j.virusres.2024.199430

Figure Lengend Snippet: Replication of rVSV∆G EBOV GP_wt expressing uncleaved (preGP) glycoprotein in Vero and Huh-7 cells. A) Schematic illustration of EBOV GP surface protein domain structure. The GP is a type I transmembrane protein consisting of 676 aa. The receptor binding region (RBR) is located at the N-terminal end followed by the glycan cap and the mucin-like domain (MLD). The domains responsible for viral fusion, including the hydrophobic patch (HP) and the two heptad repeat domains (HR1 and HR2), are located at the C-terminal end of the protein prior to the transmembrane domain (TM). The EBOV GP is synthesized as an inactive precursor protein (preGP) that must be proteolytically activated ("primed") in order to gain its fusion capacity. Priming of preGP is a multistep process starting with an initial cleavage of preGP at R501 into the GP 1 and GP 2 subunits (GP 1/2 ). GP 1 is then further trimmed by endosomal cysteine proteases cathepsin B and L, leading to a loss of MLD and glycan cap domain and exposure of the RBR. B) GP expression on virus particles of furin-cleaved VSV∆G EBOV GP 1/2 and uncleaved VSV∆G EBOV preGP. Both rVSV∆G EBOV GP 1/2 and preGP virus stocks were propagated in Huh-7 cells. For the generation of VSV∆G bearing uncleaved preGP on the surface, cells were incubated with 30 µM of MI-1148 for 24 h. Virus supernatants were then subjected to an 8 % SDS-PAGE and analyzed with an EBOV-specific serum by western blot analysis. C) Multicycle replication of rVSV∆G EBOV GP 1/2 or preGP in untreated VeroE6 and Huh-7 cells (left panels) or under single and combined inhibitor treatment with E64d, MI-1148 and BAPA (middle and right panels). Cells were treated with 50 µM MI-1148, 20 µM E64d and/or 50 µM BAPA for 1 h prior to rVSV∆G EBOV preGP infection or were left untreated. Afterward, the cells were inoculated with rVSV∆G EBOV preGP at a MOI of 0.005 for 1 h. Protease inhibitor treatment was continued during 72 h incubation period post infection (p.i.). Virus supernatants were collected at 16, 24, 48 and 72 h p.i. Viral titers were determined by TCID 50 endpoint dilution assay. Data shown are means (+SD) of three to five independent experiments ( n = 3–5). LoD: limit of detection. D) Comparative growth kinetics of rVSV∆G EBOV GP 1/2 or preGP in Vero-TMPRSS2 cells. Infections were performed without prior inhibitor treatment and viral titers were analyzed as described above. Data shown are means (+SD) of five independent experiments ( n = 5). E) Proteolytic cleavage of EBOV GP_wt with a C-terminal V5-tag (EBOV GP_wt-V5) by human TMPRSS2 in the absence of endogenous furin. HeLa cells were co-transfected with EBOV GP_wt-V5 and human TMPRSS2 plasmid for 24 h with or without MI-1148 (50 µM) treatment, empty vector transfection served as control. The cells were then harvested, lysed and the samples subjected to a 10% SDS-PAGE and immunoblotting. EBOV GP cleavage forms were detected with an antibody directed against the C-terminal V5 epitope of EBOV GP. ß-Actin staining was used as a loading control. preGP: uncleaved precursor; preGP ER : uncleaved precursor in endoplasmic reticulum (ER). The western blot shown is a representative immunoblot of three independent experiments ( n = 3).

Article Snippet: A monoclonal rabbit antibody directed against the C-terminal V5-tag of the EBOV GP constructs was purchased from Novus Biologicals (NB600–381).

Techniques: Expressing, Binding Assay, Glycoproteomics, Synthesized, Virus, Incubation, SDS Page, Western Blot, Infection, Protease Inhibitor, Endpoint Dilution Assay, Transfection, Plasmid Preparation, Control, Staining

Journal: Virus Research

Article Title: Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage

doi: 10.1016/j.virusres.2024.199430

Figure Lengend Snippet: Identification of trypsin-like protease cleavage site in EBOV GP_AGTAA mutant. A) Schematic illustration of C-terminally V5-tagged EBOV GP. The region of interest from aa 465 to 539 is enlarged, comprising the mutated furin cleavage site with the AGTAA sequence (cyan), the aa forming the hydrophobic patch (magenta) and the C511 (yellow) involved in the disulfide bridge to C556 forming the internal fusion loop. Underlined aa indicate deletions from aa 465 to 496 (∆1) and aa 502 to 510 (∆2). B) Proteolytic cleavage of EBOV GP_AGTAA mutants by human TMPRSS2. HeLa cells were co-transfected with plasmids encoding for EBOV GP_wt, EBOV GP_AGTAA without aa exchange ( ∅ ) or single alanine scanning mutants from aa position 502 to 509 as well as basic aa K478, K510 and H516 with human TMPRSS2 plasmid for 24 h. C) EBOV GP_AGTAA without aa exchange ( ∅ ) or multiple alanine mutants from aa position 502–505 and 506–510 were co-expressed with human TMPRSS2 or empty vector (ev) in HeLa cells for 24 h. D) HeLa cells were co-transfected with plasmids encoding for EBOV GP_AGTAA, EBOV GP_AGTAA∆1, EBOV GP_AGTAA∆2 and EBOV GP_AGTAA∆1&2 and human TMPRSS2 for 24 h. The cells were then harvested, lysed and the samples subjected to a 10 % SDS-PAGE and immunoblotting. EBOV GP and its cleavage forms were detected with a V5-specific antibody. ß-Actin staining was used as a loading control. preGP: uncleaved precursor; preGP ER : uncleaved precursor in endoplasmic reticulum (ER). Western blots shown are representative immunoblots of three independent experiments ( n = 3). Lanes are identified by gray numbers.

Article Snippet: A monoclonal rabbit antibody directed against the C-terminal V5-tag of the EBOV GP constructs was purchased from Novus Biologicals (NB600–381).

Techniques: Mutagenesis, Sequencing, Transfection, Plasmid Preparation, SDS Page, Western Blot, Staining, Control

Journal: Virus Research

Article Title: Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage

doi: 10.1016/j.virusres.2024.199430

Figure Lengend Snippet: Functional analysis of EBOV GP_AGTAA and GP_AGTAA∆1&2 in trVLP assay reveals an alternative processing by endosomal cathepsins in Huh-7 cells. A) Transcription and replication-competent virus-like particles (trVLPs) were generated in HEK293 cells. After 72 h cellular supernatants were harvested and trVLPs were concentrated on a sucrose cushion by ultracentrifugation. The trVLP pellets were resuspended in PBS, samples were subjected to a 10 % SDS-PAGE and analyzed by western blot. EBOV proteins were detected by an EBOV-specific serum while for detection of GP 2 a V5 epitope antibody was used. B) Entry and reporter genome replication of EBOV GP trVLPs. Huh-7, VeroE6 and Vero-TMPRSS2 cells were incubated with or without E64d (20 µM) for 1 h and subsequently inoculated with trVLPs bearing EBOV GP_wt, GP_AGTAA or GP_AGTAA∆1&2 for 72 h. Afterward, the cells were harvested and lysed for 30 min on ice. Renilla luciferase reporter gene signal was normalized to firefly luciferase activity and GP_wt activity was set to 1. Data shown are mean values (+SD) of three to six ( n = 3-6) independent experiments. For statistical group analysis of differences between untreated EBOV GP variants, a Kruskal-Wallis one-way ANOVA on ranks with subsequent Dunn´s multiple comparison test was performed. Further comparison of treated and untreated samples was statistically analyzed by Mann-Whitney test. Statistically significant p values are represented as followed: ≤ 0.05 (*) and ≤ 0.01 (**), whereas p values of >0.05 were considered not significant (ns). C) Alternative cleavage of overexpressed EBOV GP_AGTAA and GP_AGTAA∆1&2 by recombinant cathepsin B and L (rCatB and rCatL) in HeLa cells. HeLa cells were co-transfected with plasmids encoding for EBOV GP_AGTAA or EBOV GP_AGTAA∆1&2 and human TMPRSS2 or empty vector (ev) for 24 h. Subsequently, the cells were harvested, and cellular pellets were incubated with 25 µg/ml of rCatB or rCatL at pH 4.5 for 20 min at 37 °C. Cells were lysed, and the samples subjected to a 10 % SDS-PAGE and immunoblotting. EBOV GP cleavage products were detected using a V5-specific antibody. ß-actin was used as a loading control. Western blots shown are representative immunoblots of four independent experiments ( n = 4).

Article Snippet: A monoclonal rabbit antibody directed against the C-terminal V5-tag of the EBOV GP constructs was purchased from Novus Biologicals (NB600–381).

Techniques: Functional Assay, Virus, Generated, SDS Page, Western Blot, Incubation, Luciferase, Activity Assay, Comparison, MANN-WHITNEY, Recombinant, Transfection, Plasmid Preparation, Control

Journal: Virus Research

Article Title: Novel proteolytic activation of Ebolavirus glycoprotein GP by TMPRSS2 and cathepsin L at an uncharted position can compensate for furin cleavage

doi: 10.1016/j.virusres.2024.199430

Figure Lengend Snippet: Alternative processing of EBOV GP_AGTAA furin cleavage mutants. A) Schematic illustration of proteolytic activation of EBOV GP_wt by furin and endosomal cathepsins, leading to a fusion competent GP 2 and GP 1 with exposed receptor binding region (RBR). Abrogated furin cleavage observed for EBOV GP_AGTAA and EBOV GP_AGTAA∆1&2 (deletions are indicated as hatched areas) is substituted by other host cell proteases, including trypsin-like serine proteases as TMPRSS2 and the endosomal cysteine protease CatL. Processing of GP_AGTAA mutants is carried out at so far uncharted positions resulting in cleavage products with possible higher molecular weights, which remain fusion competent. B) EBOV GP activation model during viral replication cycle. The preGP of EBOV GP_wt is cleaved by furin within the TGN along the secretory pathway into GP 1 and GP 2 and subsequently incorporated into budding EBOV particles. Upon entry in new target cells GP 1 is trimmed by endosomal cathepsins (GP cl ) leading to NPC1 binding and fusion within the late endosomes. The preGP of EBOV GP_AGTAA furin cleavage mutants can no longer be processed by furin. However, trypsin-like proteases, like TMPRSS2, and endosomal CatL might be able to compensate for lack of furin cleavage leading to fusion competent GP 2 and free GP 1 that is further trimmed by endosomal cathepsins for NPC1 binding and subsequent fusion.

Article Snippet: A monoclonal rabbit antibody directed against the C-terminal V5-tag of the EBOV GP constructs was purchased from Novus Biologicals (NB600–381).

Techniques: Activation Assay, Binding Assay

Journal: Nature immunology

Article Title: The tumor suppressor kinase DAPK3 drives tumor-intrinsic immunity through the STING–IFN-β pathway

doi: 10.1038/s41590-021-00896-3

Figure Lengend Snippet: a - d , ( a ) qRT-PCR of Sting1 and Dapk3 , ( b ) immunoblot, ( c ) IRF3 nuclear translocation, and ( d ) p65 nuclear translocation in L929-mRuby-hIRF3 transduced with indicated shRNA stimulated with poly (dA:dT) (0.5 μg/ml) or VACV70 (2 μg/ml) for 3 h. e , Immunoblot of L929-mRuby-hIRF3 transfected with indicated siRNA. f , Immunoblot of L929-mRuby-hIRF3 transduced with indicated shRNA stimulated with VACV70 (2 μg/ml) for 2 h and 4 h. g , Immunoblot of HUVEC stably expressing V5-tagged DAPK3(D161A), DAPK3(T180A), or luciferase. Cells were infected at MOI=5, 2, or 1. h , Immunoblot of THP1-Blue ISG stably expressing V5-tagged DAPK3(WT) or DAPK3(D161A). i , qRT-PCR of Ifnb1 in L929-mRuby-hIRF3 pre-treated with DAPK inhibitors for 3 h prior to 2′,3′-cGAMP stimulation (10 μg/ml) for 4 h. j , qRT-PCR of IFNB1 in THP1-Blue ISG pre-treated with DAPK inhibitors (50 μM) for 6 h prior to 2′,3′-cGAMP or c-di-GMP stimulation (10 μg/ml for both) for 4 h. k , l , In vitro kinase assay of ( k ) GST-tagged human STING C-terminus (aa 149-379) and ( l ) GST-tagged human TBK1(K38M). Peptides were incubated with GST-tagged DAPK3 or TBK1 in the presence of [γ-32P] ATP. Data in ( b, e-h, k, l ) are representative or ( a, c, d, i, j ) mean of three independent experiments. Values represent mean ± s.d. * P <0.05, ** P <0.01, and *** P <0.001. Statistical comparisons were conducted using two-tailed t -test ( a , c , d , i , j ).

Article Snippet: Luciferase from pLenti-CMV-Puro-LUC (w168-1) (Addgene #17477) was ligated into pLX304 with or without V5 tag. pEF-BOS-huTBK1-FlagHis (#27241) and pEF-BOS-huTBK1-K38M-Flag-His (#27240) were obtained from Addgene.

Techniques: Quantitative RT-PCR, Western Blot, Translocation Assay, Transduction, shRNA, Transfection, Stable Transfection, Expressing, Luciferase, Infection, In Vitro, Kinase Assay, Incubation, Two Tailed Test

Journal: Science advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer's disease gene expression linked to neuropathology.

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: Fig. 1. Aβ42 promotes CREB3L2-ATF4 heterodimerization. (A) Analysis of nuclear CHOP protein by quantitative immunofluorescence. Axons were transfected with control or Creb3l2-targeting siRNAs before Aβ42 treatment. Box plots summarize n = 8 replicates; ***P = 0.0008, one-way analysis of variance (ANOVA) with Bonferroni correction. Here and henceforth, whiskers denote 10th to 90th percentile; “+” sign marks sample mean. Creb3l2 knockdown ranged between 77.3 and 86.5% in disso- ciated neurons. Scale bar, 15 μm. (B) TUNEL assay in hippocampal neurons. Experimental outline as in (A). Box plots summarize n = 10 to 12 replicates; *P = 0.0357, one- way ANOVA with Bonferroni correction. Scale bar, 15 μm. (C) CREB3L2-ATF4 coimmunoprecipitation using in vitro translated proteins. IP, immunoprecipitation; WB, Western blot. (D) CREB3L2-ATF4 coimmunoprecipitation analysis in HEK293 cells. (E) Coimmunoprecipitation of endogenous CREB3L2-ATF4 in neuritic extracts treated with Aβ42. Mixed cortical and hippocampal neurons were grown on transwell inserts. ATF4 levels are normalized against input βIII-tubulin. Plot shows mean of n = 3 replicates; *P = 0.0418, unpaired t test. (F) Visualization of axonal CREB3L2-ATF4 by PLA. Hippocampal neurons were cultured in microfluidic chambers, and axons were treated with Aβ42 for 12 hours. Box plots summarize n = 3 replicates; ***P < 0.0001, unpaired t test. Scale bar, 10 μm. (G) PLA visualization of CREB3L2-ATF4 in dissociated hippocampal neurons. Aβ42 was bath-applied for 12 hours. Box plots summarize n = 3 replicates. Nuclear CREB3L2-ATF4 events: ***P = 0.0007; somatic CREB3L2-ATF4 events: **P = 0.0016 (unpaired t tests). ns, not significant. Scale bar, 10 μm. (H) Subcellular distribution of CREB3L2-ATF4. This is the same dataset analyzed in (G). (I) PLAvisualization of CREB3L2-ATF4 after inhibition of axonal retrograde transport. Ciliobrevin A was delivered in the last 6 hours of an 18-hour Aβ42 protocol. Box plots summarize n = 3 replicates; ***P < 0.0001, one-way ANOVA test with Bonferroni correction. PLA signals were normalized to axon length. Scale bar, 10 μm.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Immunofluorescence, Transfection, Control, Knockdown, TUNEL Assay, In Vitro, Immunoprecipitation, Western Blot, Cell Culture, Inhibition

Journal: Science advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer's disease gene expression linked to neuropathology.

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: Fig. 2. CREB3L2-ATF4 up-regulation in β-amyloid pathology 5xFAD model. In vivo detection and quantification of CREB3L2-ATF4 dimers by PLA in the dentate gyrus of 10-week-old 5xFAD or wild-type hippocampus. ML, molecular layer; IPL, inner polymorphic layer; GCL, granule cell layer. Wild-type (WT), n = 6; 5xFAD, n = 5; ML, *P = 0.0182; GCL, P = 0.0574; IPL, *P = 0.0285; unpaired one-tailed t tests. Whis- kers extend to the smallest and largest data values, and sample means are indicat- ed by + sign. Scale bar, 10 μm.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: In Vivo, One-tailed Test

Journal: Science advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer's disease gene expression linked to neuropathology.

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: Fig. 3. The CREB3L2-ATF4 transcriptional program. (A) Experimental outline. ChIPmera uses chemically induced proximity to promote the formation of specific TF pairs. Each monomer is engineered with two unique features: a specific dimerization domain (FRB or FKBP, C-terminally fused) and an N-terminal epitope tag (HA or V5). (B) Motif analysis of CREB3L2-CREB3L2, ATF4-ATF4, and CREB3L2-ATF4 binding sites. Heterodimer-bound sequences were centrally enriched in canonical CREB3L2 or ATF4 recognition motifs. CREB3L2: CentriMo E = 4.7 × 10−39; ATF4: CentriMo E = 4.0 × 10−34. Other motifs identified were not centrally distributed. (C) Representative binding behaviors are displayed by CREB3L2-CREB3L2, ATF4-ATF4, and CREB3L2-ATF4 dimers. ENCODE-generated histone H3 lysine 27 acetylation (H3K27Ac) and de- oxyribonuclease I (DNase I) hypersensitivity profiles are shown. (D) Coincidence analysis of CREB3L2-ATF4 and CREB3L2-CREB3L2 DNA binding sites across the genome. Genomic distances were computed within a ±2-kb window around CREB3L2-CREB3L2 peaks and are plotted as a frequency histogram using a 10-bp bin size. (E) Same as in (D), except that here CREB3L2-ATF4 and ATF4-ATF4 dimers are compared. (F) Differential enrichment analysis of genomic regions bound by CREB3L2-ATF4 and ATF4- ATF4 dimers. Enrichment fold change and statistical significance are plotted along the x and y axes, respectively, from n = 2 independent replicates (each replicate includes two parallel ChIP-seq runs). Magenta data points: false discovery rate (FDR) ≤0.01; blue data points: FDR > 0.01. (G) Differential enrichment analysis of genomic regions bound by CREB3L2-ATF4 and CREB3L2-CREB3L2 dimers. Axes layout and data point codification are the same as in (F). The strong signal ascribed to RNF157 coincides with an ATF4 pseudogene found within this locus. (H) GO functional analysis of the CREB3L2-ATF4 transcriptional program (biological process).

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Binding Assay, Generated, ChIP-sequencing, Functional Assay

Journal: Science advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer's disease gene expression linked to neuropathology.

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: Fig. 4. CREB3L2-ATF4 orchestrates an AD-linked transcription network. (A) Gene expression changes triggered by CREB3L2-ATF4 in rat hippocampal neurons an- alyzed by RNA sequencing (RNA-seq). Basal expression levels were measured in cells expressing Renilla luciferase homodimers. Fold changes over baseline (log2 trans- formed) and adjusted P values (−log10 transformed), as calculated by DESeq2 from n = 5 independent replicates, are plotted along the x and y axes, respectively. Magenta data points: adjusted P < 0.05; blue data points: adjusted P > 0.05. (B) Strategy used to characterize the AD-associated transcription network regulated by CREB3L2-ATF4. First, we determined which of the differentially expressed genes (DEGs) in our RNA-seq dataset were direct DNA binding targets of CREB3L2-ATF4 as identified by ChIP- mera. Second, we evaluated the transcriptional signatures of these common hits in AD prefrontal cortex to understand which CREB3L2-ATF4–regulated targets had relevant disease-associated expression profiles (significance cutoff: P < 1 × 10−15). We found that this subset included four up-regulated TFs, NFE2L2, SOX9, NFATC1, and MXD4, as well as CREB3L2. Third, we explored the regulatory connections and functional relationships within this extended transcription network, described in (C) and (D). (C) Circos plot illustrating the regulatory relationships within the wider CREB3L2-ATF4 transcription network and their interaction with the AD transcriptome (bulk tissue level). Genes within each category (e.g., CREB3L2-ATF4 targets) are arranged along the ideogram’s arc. Inner lines link genes shared by two datasets (e.g., SOX9 and NFATC1); black-colored lines additionally identify genes with AD-associated transcriptional profiles. (D) Representative GO terms enriched across input gene lists, colored by P values (−log10-transformed). This comparative analysis integrates the DNA binding program of each TF, AD-associated gene expression changes, and the neuronal transcriptional profile promoted by CREB3L2-ATF4 measured in (A).

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Gene Expression, RNA Sequencing, Expressing, Luciferase, Transformation Assay, Binding Assay, Functional Assay

Journal: Science advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer's disease gene expression linked to neuropathology.

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: Fig. 6. CREB3L2-ATF4 interacts with β-amyloid and tau neuropathologies. (A) Top three enriched GO terms for CREB3L2-ATF4–interacting genes positively and negatively associated with β-amyloid or tau neuropathologies (−3 ≤signed −log10 P value ≥3). β-Amyloid pathology overlap: representation factor = 1.4, P < 7.54 × 10−23, hypergeometric test; tau pathology overlap: representation factor = 1.4, P < 8.15 × 10−8, hypergeometric test; cognitive decline overlap: representation factor = 1.5, P < 3.36 × 10−43, hypergeometric test. (B) Analysis of extracellular Aβ42, Aβ40, and Aβ42/Aβ40 ratios in culture supernatants from hippocampal neurons expressing CREB3L2-ATF4 (C-A), CREB3L2-CREB3L2 (C-C), or Renilla luciferase (L-L) dimers. Plots show individual measurements and mean ± SEM of n = 7 replicates; one-way ANOVA tests with Tukey’s correction. (C) Western blot analysis of full-length APP. Plots display individual measurements and mean ± SEM of n = 6 replicates. (D) Western blot quantification of tau phosphorylation in rat hippocampal neurons normalized against total tau levels. Plots show individual measurements and mean ± SEM of n = 4 replicates; one-way ANOVA tests with Tukey’s correction. Measurements shown here pertain only to tau signals around 50 kDa (see also fig. S10F). (E) Extracellular tau protein levels. Plots show individual measurements and mean ± SEM of n = 7 replicates; one-way ANOVA tests with Tukey’s correction. (F) Analysis of PP2A phosphatase activity in purified neuronal extracts. Plot shows mean ± SEM of relative cell number–normalized absorbance measurements from n = 4 replicates; **P = 0.0031, unpaired t test. (G) The findings support a model whereby CREB3L2-ATF4 promotes AD-linked gene expression changes and contributes to characteristic features of AD pathophysiology, including retromer dysfunction, altered β-amyloid metabolism, and tau hyperphosphorylation.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Expressing, Luciferase, Western Blot, Phospho-proteomics, Activity Assay, Purification, Gene Expression

Journal: Science advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer's disease gene expression linked to neuropathology.

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: Fig. 7. CREB3L2-ATF4 in the human AD brain. (A) Coimmunoprecipitation analysis of CREB3L2-ATF4 heterodimers in control and late-onset AD prefrontal cortex (immunoprecipitation with anti-ATF4 antibody). Plots show individual measurements and mean ± SEM of CREB3L2/ATF4 ratios from n = 4 controls and n = 6 AD cases; *P = 0.0133, unpaired t test. (B) PLA detection of CREB3L2-ATF4 heterodimers (green punctate signals) in AD dorsolateral prefrontal cortex costained for neurofi- lament (magenta labeling), a neuronal marker. This pseudocolored representative micrograph was produced using chromogenic detection methods. See fig. S11D for quantification and technical controls. Scale bar, 25 μm. (C) Genomic distribution of AD CREB3L2 ChIP-seq signals. Cutoff for proximal promoter/enhancer regions was defined as ±3 kb from a transcription start site. (D) Cumulative frequency distribution of CREB3L2 ChIP-seq peaks relative to known transcription start sites (TSS). (E) GO functional analysis of AD CREB3L2 transcriptional program (biological process). (F) Representative CREB3L2 ChIP-seq tracks juxtaposed with ENCODE-produced H3K27Ac and DNase I hypersensitivity profiles. SEC31A encodes a component of the COPII protein complex and participates in vesicle budding from the ER; SNX3 governs the interaction between the retromer and early endosomes; PTBP1 is a splicing regulator. (G) AD CREB3L2 ChIP-seq genome browser tracks in VPS26B locus juxtaposed with ChIPmera datasets and ENCODE-produced H3K27Ac and DNase I hypersensitivity profiles. For clarity, AD ChIP-seq and ChIPmera tracks are displayed using different viewing ranges, as the ChIPmera signals are consistently stronger. (H) GO term enrichment analysis (biological process) of targets common to both CREB3L2-ATF4 ChIP- mera and AD CREB3L2 ChIP-seq datasets.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Control, Immunoprecipitation, Labeling, Marker, Produced, ChIP-sequencing, Functional Assay

Journal: Science advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer's disease gene expression linked to neuropathology.

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: Fig. 8. Gene expression as a driver and intervention target in Aβ42 neurodegeneration. (A) Cell viability analysis of dissociated rat hippocampal neurons after 48- hour Aβ42 stimulation protocol. Plots show individual measurements and mean ± SEM of n = 3 biological replicates. For each background, data are presented as Aβ42/ vehicle ratios. ATF4 aZIP: *P = 0.0167; CREB3L2 aZIP: *P = 0.0137; unpaired t tests. (B) Gene expression changes triggered by Aβ42 in rat hippocampal neurons analyzed by RNA-seq. Basal expression levels were measured in vehicle-treated cells. DEGs identified in Aβ42 condition are compared against their expression values in neurons co- treated with dovitinib. Individual data points show fold changes over baseline (log2-transformed) of n = 2 biological replicates. Aβ42 and dovitinib were applied for 24 hours before RNA collection. A GO enrichment analysis of DEGs (n = 203; 128 up-regulated, 75 down-regulated) in Aβ42-treated neurons is also shown. FGF, fibroblast growth factor; MAPK, mitogen-activated protein kinase.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Gene Expression, RNA Sequencing, Expressing, Transformation Assay

Journal: Science Advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer’s disease gene expression linked to neuropathology

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: ( A ) Analysis of nuclear CHOP protein by quantitative immunofluorescence. Axons were transfected with control or Creb3l2 -targeting siRNAs before Aβ 42 treatment. Box plots summarize n = 8 replicates; *** P = 0.0008, one-way analysis of variance (ANOVA) with Bonferroni correction. Here and henceforth, whiskers denote 10th to 90th percentile; “+” sign marks sample mean. Creb3l2 knockdown ranged between 77.3 and 86.5% in dissociated neurons. Scale bar, 15 μm. ( B ) TUNEL assay in hippocampal neurons. Experimental outline as in (A). Box plots summarize n = 10 to 12 replicates; * P = 0.0357, one-way ANOVA with Bonferroni correction. Scale bar, 15 μm. ( C ) CREB3L2-ATF4 coimmunoprecipitation using in vitro translated proteins. IP, immunoprecipitation; WB, Western blot. ( D ) CREB3L2-ATF4 coimmunoprecipitation analysis in HEK293 cells. ( E ) Coimmunoprecipitation of endogenous CREB3L2-ATF4 in neuritic extracts treated with Aβ 42 . Mixed cortical and hippocampal neurons were grown on transwell inserts. ATF4 levels are normalized against input βIII-tubulin. Plot shows mean of n = 3 replicates; * P = 0.0418, unpaired t test. ( F ) Visualization of axonal CREB3L2-ATF4 by PLA. Hippocampal neurons were cultured in microfluidic chambers, and axons were treated with Aβ 42 for 12 hours. Box plots summarize n = 3 replicates; *** P < 0.0001, unpaired t test. Scale bar, 10 μm. ( G ) PLA visualization of CREB3L2-ATF4 in dissociated hippocampal neurons. Aβ 42 was bath-applied for 12 hours. Box plots summarize n = 3 replicates. Nuclear CREB3L2-ATF4 events: *** P = 0.0007; somatic CREB3L2-ATF4 events: ** P = 0.0016 (unpaired t tests). ns, not significant. Scale bar, 10 μm. ( H ) Subcellular distribution of CREB3L2-ATF4. This is the same dataset analyzed in (G). ( I ) PLA visualization of CREB3L2-ATF4 after inhibition of axonal retrograde transport. Ciliobrevin A was delivered in the last 6 hours of an 18-hour Aβ 42 protocol. Box plots summarize n = 3 replicates; *** P < 0.0001, one-way ANOVA test with Bonferroni correction. PLA signals were normalized to axon length. Scale bar, 10 μm.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Immunofluorescence, Transfection, Control, Knockdown, TUNEL Assay, In Vitro, Immunoprecipitation, Western Blot, Cell Culture, Inhibition

Journal: Science Advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer’s disease gene expression linked to neuropathology

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: In vivo detection and quantification of CREB3L2-ATF4 dimers by PLA in the dentate gyrus of 10-week-old 5xFAD or wild-type hippocampus. ML, molecular layer; IPL, inner polymorphic layer; GCL, granule cell layer. Wild-type (WT), n = 6; 5xFAD, n = 5; ML, * P = 0.0182; GCL, P = 0.0574; IPL, * P = 0.0285; unpaired one-tailed t tests. Whiskers extend to the smallest and largest data values, and sample means are indicated by + sign. Scale bar, 10 μm.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: In Vivo, One-tailed Test

Journal: Science Advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer’s disease gene expression linked to neuropathology

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: ( A ) Experimental outline. ChIPmera uses chemically induced proximity to promote the formation of specific TF pairs. Each monomer is engineered with two unique features: a specific dimerization domain (FRB or FKBP, C-terminally fused) and an N-terminal epitope tag (HA or V5). ( B ) Motif analysis of CREB3L2-CREB3L2, ATF4-ATF4, and CREB3L2-ATF4 binding sites. Heterodimer-bound sequences were centrally enriched in canonical CREB3L2 or ATF4 recognition motifs. CREB3L2: CentriMo E = 4.7 × 10 −39 ; ATF4: CentriMo E = 4.0 × 10 −34 . Other motifs identified were not centrally distributed. ( C ) Representative binding behaviors are displayed by CREB3L2-CREB3L2, ATF4-ATF4, and CREB3L2-ATF4 dimers. ENCODE-generated histone H3 lysine 27 acetylation (H3K27Ac) and deoxyribonuclease I (DNase I) hypersensitivity profiles are shown. ( D ) Coincidence analysis of CREB3L2-ATF4 and CREB3L2-CREB3L2 DNA binding sites across the genome. Genomic distances were computed within a ±2-kb window around CREB3L2-CREB3L2 peaks and are plotted as a frequency histogram using a 10-bp bin size. ( E ) Same as in (D), except that here CREB3L2-ATF4 and ATF4-ATF4 dimers are compared. ( F ) Differential enrichment analysis of genomic regions bound by CREB3L2-ATF4 and ATF4-ATF4 dimers. Enrichment fold change and statistical significance are plotted along the x and y axes, respectively, from n = 2 independent replicates (each replicate includes two parallel ChIP-seq runs). Magenta data points: false discovery rate (FDR) ≤ 0.01; blue data points: FDR > 0.01. ( G ) Differential enrichment analysis of genomic regions bound by CREB3L2-ATF4 and CREB3L2-CREB3L2 dimers. Axes layout and data point codification are the same as in (F). The strong signal ascribed to RNF157 coincides with an ATF4 pseudogene found within this locus. ( H ) GO functional analysis of the CREB3L2-ATF4 transcriptional program (biological process).

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Binding Assay, Generated, ChIP-sequencing, Functional Assay

Journal: Science Advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer’s disease gene expression linked to neuropathology

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: ( A ) Gene expression changes triggered by CREB3L2-ATF4 in rat hippocampal neurons analyzed by RNA sequencing (RNA-seq). Basal expression levels were measured in cells expressing Renilla luciferase homodimers. Fold changes over baseline (log 2 transformed) and adjusted P values (−log 10 transformed), as calculated by DESeq2 from n = 5 independent replicates, are plotted along the x and y axes, respectively. Magenta data points: adjusted P < 0.05; blue data points: adjusted P > 0.05. ( B ) Strategy used to characterize the AD-associated transcription network regulated by CREB3L2-ATF4. First, we determined which of the differentially expressed genes (DEGs) in our RNA-seq dataset were direct DNA binding targets of CREB3L2-ATF4 as identified by ChIPmera. Second, we evaluated the transcriptional signatures of these common hits in AD prefrontal cortex to understand which CREB3L2-ATF4–regulated targets had relevant disease-associated expression profiles (significance cutoff: P < 1 × 10 −15 ). We found that this subset included four up-regulated TFs, NFE2L2 , SOX9 , NFATC1 , and MXD4 , as well as CREB3L2 . Third, we explored the regulatory connections and functional relationships within this extended transcription network, described in (C) and (D). ( C ) Circos plot illustrating the regulatory relationships within the wider CREB3L2-ATF4 transcription network and their interaction with the AD transcriptome (bulk tissue level). Genes within each category (e.g., CREB3L2-ATF4 targets) are arranged along the ideogram’s arc. Inner lines link genes shared by two datasets (e.g., SOX9 and NFATC1); black-colored lines additionally identify genes with AD-associated transcriptional profiles. ( D ) Representative GO terms enriched across input gene lists, colored by P values (−log 10 -transformed). This comparative analysis integrates the DNA binding program of each TF, AD-associated gene expression changes, and the neuronal transcriptional profile promoted by CREB3L2-ATF4 measured in (A).

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Gene Expression, RNA Sequencing, Expressing, Luciferase, Transformation Assay, Binding Assay, Functional Assay

Journal: Science Advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer’s disease gene expression linked to neuropathology

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: ( A ) Retromer regulatory inputs within the CREB3L2-ATF4 transcription network. Pink-colored squares denote significant enrichments. ( B ) VPS35 ChIP-seq tracks alongside ENCODE-produced H3K27Ac marks and DNase I hypersensitivity profiles. ( C ) Retromer transcriptomic profiles, mined from Zhang et al . , in nondemented control and AD individuals (bulk prefrontal cortex). Data are log 2 -transformed. Rows denote individual cases (controls, n = 101; AD, n = 129). EHD1 : Pearson r = 0.79; VPS29 : Pearson r = −0.72; SNX1 : Pearson r = 0.70; SNX6 : Pearson r = 0.68; VPS35 : Pearson r = −0.67; VPS26B : Pearson r = −0.65. ( D ) VPS35 ( x axis) and CREB3L2 ( y axis) mRNA expression prefrontal cortex. Controls, Pearson r = −0.4481, *** P < 0.0001; AD, Pearson r = −0.5595, *** P < 0.0001. ( E ) Retromer protein levels in hippocampal neurons infected with control or Creb3l2 -targeting shRNAs. Measurements were normalized to βIII-tubulin and are presented relative to control baseline. Mean ± SEM of n = 3 to 4 replicates; unpaired t tests. ( F ) ChIP-qPCR analysis of CREB3L2 normalized to total input chromatin. Mean ± SEM of n = 3 to 5 replicates; unpaired t tests. ( G ) RT-qPCR analysis of retromer gene expression in CREB3L2-ATF4 (C-A), CREB3L2-CREB3L2 (C-C), and control (L-L) hippocampal neurons. Measurements were normalized to Tubb3 (βIII-tubulin) and are presented relative to control (L-L background). Plots show individual measurements and mean ± SEM of n = 5 to 7 replicates; one-way ANOVA with Tukey’s multiple comparison tests. ( H ) Western blot analysis of retromer protein levels in CREB3L2-ATF4 and CREB3L2-CREB3L2 neurons. Measurements were normalized to βIII-tubulin levels and are shown relative to control (L-L) baseline. Plots show individual measurements and mean ± SEM of n = 4 replicates (Vps29, n = 3); one-way ANOVA tests with Sidak’s multiple comparison correction.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: ChIP-sequencing, Produced, Control, Transformation Assay, Expressing, Infection, ChIP-qPCR, Quantitative RT-PCR, Gene Expression, Comparison, Western Blot

Journal: Science Advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer’s disease gene expression linked to neuropathology

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: ( A ) Top three enriched GO terms for CREB3L2-ATF4–interacting genes positively and negatively associated with β-amyloid or tau neuropathologies (−3 ≤ signed −log 10 P value ≥ 3). β-Amyloid pathology overlap: representation factor = 1.4, P < 7.54 × 10 −23 , hypergeometric test; tau pathology overlap: representation factor = 1.4, P < 8.15 × 10 −8 , hypergeometric test; cognitive decline overlap: representation factor = 1.5, P < 3.36 × 10 −43 , hypergeometric test. ( B ) Analysis of extracellular Aβ 42 , Aβ 40 , and Aβ 42 /Aβ 40 ratios in culture supernatants from hippocampal neurons expressing CREB3L2-ATF4 (C-A), CREB3L2-CREB3L2 (C-C), or Renilla luciferase (L-L) dimers. Plots show individual measurements and mean ± SEM of n = 7 replicates; one-way ANOVA tests with Tukey’s correction. ( C ) Western blot analysis of full-length APP. Plots display individual measurements and mean ± SEM of n = 6 replicates. ( D ) Western blot quantification of tau phosphorylation in rat hippocampal neurons normalized against total tau levels. Plots show individual measurements and mean ± SEM of n = 4 replicates; one-way ANOVA tests with Tukey’s correction. Measurements shown here pertain only to tau signals around 50 kDa (see also fig. S10F). ( E ) Extracellular tau protein levels. Plots show individual measurements and mean ± SEM of n = 7 replicates; one-way ANOVA tests with Tukey’s correction. ( F ) Analysis of PP2A phosphatase activity in purified neuronal extracts. Plot shows mean ± SEM of relative cell number–normalized absorbance measurements from n = 4 replicates; ** P = 0.0031, unpaired t test. ( G ) The findings support a model whereby CREB3L2-ATF4 promotes AD-linked gene expression changes and contributes to characteristic features of AD pathophysiology, including retromer dysfunction, altered β-amyloid metabolism, and tau hyperphosphorylation.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Expressing, Luciferase, Western Blot, Phospho-proteomics, Activity Assay, Purification, Gene Expression

Journal: Science Advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer’s disease gene expression linked to neuropathology

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: ( A ) Coimmunoprecipitation analysis of CREB3L2-ATF4 heterodimers in control and late-onset AD prefrontal cortex (immunoprecipitation with anti-ATF4 antibody). Plots show individual measurements and mean ± SEM of CREB3L2/ATF4 ratios from n = 4 controls and n = 6 AD cases; * P = 0.0133, unpaired t test. ( B ) PLA detection of CREB3L2-ATF4 heterodimers (green punctate signals) in AD dorsolateral prefrontal cortex costained for neurofilament (magenta labeling), a neuronal marker. This pseudocolored representative micrograph was produced using chromogenic detection methods. See fig. S11D for quantification and technical controls. Scale bar, 25 μm. ( C ) Genomic distribution of AD CREB3L2 ChIP-seq signals. Cutoff for proximal promoter/enhancer regions was defined as ±3 kb from a transcription start site. ( D ) Cumulative frequency distribution of CREB3L2 ChIP-seq peaks relative to known transcription start sites (TSS). ( E ) GO functional analysis of AD CREB3L2 transcriptional program (biological process). ( F ) Representative CREB3L2 ChIP-seq tracks juxtaposed with ENCODE-produced H3K27Ac and DNase I hypersensitivity profiles. SEC31A encodes a component of the COPII protein complex and participates in vesicle budding from the ER; SNX3 governs the interaction between the retromer and early endosomes; PTBP1 is a splicing regulator. ( G ) AD CREB3L2 ChIP-seq genome browser tracks in VPS26B locus juxtaposed with ChIPmera datasets and ENCODE-produced H3K27Ac and DNase I hypersensitivity profiles. For clarity, AD ChIP-seq and ChIPmera tracks are displayed using different viewing ranges, as the ChIPmera signals are consistently stronger. ( H ) GO term enrichment analysis (biological process) of targets common to both CREB3L2-ATF4 ChIPmera and AD CREB3L2 ChIP-seq datasets.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Control, Immunoprecipitation, Labeling, Marker, Produced, ChIP-sequencing, Functional Assay

Journal: Science Advances

Article Title: CREB3L2-ATF4 heterodimerization defines a transcriptional hub of Alzheimer’s disease gene expression linked to neuropathology

doi: 10.1126/sciadv.add2671

Figure Lengend Snippet: ( A ) Cell viability analysis of dissociated rat hippocampal neurons after 48-hour Aβ 42 stimulation protocol. Plots show individual measurements and mean ± SEM of n = 3 biological replicates. For each background, data are presented as Aβ 42 /vehicle ratios. ATF4 aZIP: * P = 0.0167; CREB3L2 aZIP: * P = 0.0137; unpaired t tests. ( B ) Gene expression changes triggered by Aβ 42 in rat hippocampal neurons analyzed by RNA-seq. Basal expression levels were measured in vehicle-treated cells. DEGs identified in Aβ 42 condition are compared against their expression values in neurons cotreated with dovitinib. Individual data points show fold changes over baseline (log 2 -transformed) of n = 2 biological replicates. Aβ 42 and dovitinib were applied for 24 hours before RNA collection. A GO enrichment analysis of DEGs ( n = 203; 128 up-regulated, 75 down-regulated) in Aβ 42 -treated neurons is also shown. FGF, fibroblast growth factor; MAPK, mitogen-activated protein kinase.

Article Snippet: Capped, poly(A)-tailed Homo sapiens CREB3L2 (HA- or V5-tagged, cleaved form) and ATF4 (HA- or V5-tagged; engineered from Addgene plasmid #26114, gifted by Y. Ye) transcripts were synthesized using the mMESSAGE mMACHINE SP6 and Poly(A) Tailing kits from Thermo Fisher Scientific.

Techniques: Gene Expression, RNA Sequencing, Expressing, Transformation Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

doi: 10.1038/sigtrans.2017.51

Figure Lengend Snippet: STAT3 is a potential therapeutic target in CLL. ( a ) STAT3 is overexpressed in CLL cell lines and patient cells. (i) JVM-3 cells, Mec-2 cells, PBMCs from two different normal blood donors and PBMCs from four CLL patients were lysed and western blot analysis was performed. The final image was created by grouping different parts of the same film of the same gel as indicated by the black dividing line. (ii) CD19+ B cells were isolated from blood donated by healthy donors and protein levels were compared to JVM-3 cells by western blot analysis. ( b ) Knockdown of STAT3 induces cell death in CLL cells. JVM-3 cells were transfected with several clones of STAT3 shRNA. (i) Flow cytometric analysis was performed to determine % dead cells 24–96 h after doxycycline induction and (ii) western blot analysis done. Cells nucleofected with TE buffer containing no plasmid were used as a control. An aliquot of 1 μg ml −1 doxycycline was used to induce the expression of STAT3 shRNA 24 h after nucleofection and doxycycline level was maintained during the assay period. The graph represents two independent experiments. Student’s t -test was used for statistical analysis, *** P <0.0001, ** P <0.05. The final western blot image was created by grouping different parts of the same film of the same gel as indicated by the black dividing line. ( c ) STAT3 inhibition reduces viability of CLL cell lines and patient cells. (i) PBMCs from three normal donors and three CLL patients, (ii) JVM-3 cells and (iii) Mec-2 cells were treated with Stattic for 24 h and cell viability was determined by the MTS assay. (ii) Western blotting analysis in JVM-3 confirms the effectiveness of Stattic treatment. The graphs represent results from three independent experiments.

Article Snippet: Human EF.STAT3C.Ubc.GFP vector from Addgene (Cambridge, MA, USA) was used for expressing STAT3-C in JVM-3 cells.

Techniques: Western Blot, Isolation, Transfection, Clone Assay, shRNA, Plasmid Preparation, Expressing, Inhibition, MTS Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

doi: 10.1038/sigtrans.2017.51

Figure Lengend Snippet: CNL suppresses the phosphorylation of STAT3 at both Y705 and S727 residues. CNL suppresses phosphorylation of STAT3 in ( a ) CLL patient cells; ( b ) JVM-3 cells; ( c ) Mec-2 cells; ( d ) ex vivo xenograft tumors. Cells were treated with 20 μM and/or 40 μM of ghost nanoliposomes or CNL as indicated in the figure for 24 h (CLL patient cells and JVM-3 cells) or 48 and 72 h (Mec-2 cells). Western blotting analysis was performed. The graphs represent the quantification of western blotting from: ( a ) 7 CLL patient cells; and ( b ) three independent experiments. The final western blot image was created by grouping different parts of the same film of the same gel as indicated by the black dividing line. Statistical analysis was performed using Student’s t -test, * P <0.05, ** P <0.01. ( d ) JVM-3 xenograft tumors were obtained from a subcutaneous CLL mouse model in Balb/c Nu/nu mice that were injected with ghost nanoliposomes or CNL (from Ryland et al. ). Western blotting was performed for one tumor treated with ghost nanoliposomes and two CNL-treated tumors obtained from two separate mice. ( e ) CNL does affect cell viability and STAT3 phosphorylation in HEK293 cells. (i) Cell viability of HEK293 cells was determined by MTS assay after 24 h treatment and (ii) western blotting analysis was performed. The results are representative of three independent experiments.

Article Snippet: Human EF.STAT3C.Ubc.GFP vector from Addgene (Cambridge, MA, USA) was used for expressing STAT3-C in JVM-3 cells.

Techniques: Ex Vivo, Western Blot, Injection, MTS Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

doi: 10.1038/sigtrans.2017.51

Figure Lengend Snippet: Suppression of STAT3 phosphorylation is specific to STAT3 and C6-ceramide. ( a ) CNL-induced suppression of phosphorylation is specific to STAT3. JVM-3 cells were treated with 40 μM ghost nanoliposomes or CNL for 24 h and western blotting analysis was done. A positive control of STAT2 phosphorylation at Y690 was also used. The images are representative of three independent experiments. ( b ) Only C6-ceramide sphingolipid suppresses STAT3 phosphorylation. JVM-3 cells were treated with dihrdro-C6-ceramide nanoliposomes or BSA:sphingosine complex or BSA:S1P complex for 24 h. Western blotting analysis was performed. The images are representative of three independent experiments.

Article Snippet: Human EF.STAT3C.Ubc.GFP vector from Addgene (Cambridge, MA, USA) was used for expressing STAT3-C in JVM-3 cells.

Techniques: Western Blot, Positive Control

Journal: Signal Transduction and Targeted Therapy

Article Title: STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

doi: 10.1038/sigtrans.2017.51

Figure Lengend Snippet: CNL induces necrotic cell death in CLL cells. CNL induces necrotic cell death in ( a ) CLL patient cells and ( b ) CLL cell lines JVM-3 and Mec-2. Cells were treated with 20 μM and/or 40 μM ghost nanoliposomes or CNL for indicated time periods. Flow cytometric analysis using Annexin-V and 7AAD staining was performed to determine the effect on cell death. ( a ) The graph represents the quantification of all seven CLL patient samples. Student’s t -test was used to perform statistical analysis ** P <0.01. ( b ) The graphs represent the quantification of results from three independent experiments. Two-way ANOVA with Tukey’s multiple comparisons test was used to perform statistical analysis * P <0.01. ( c ) CNL-induced suppression of p-STAT3 precedes induction of cell death (i) JVM-3 cells were treated with ghost nanoliposomes or CNL for indicated time periods and flow cytometric analysis was performed to determine % cell death. The graph is a quantification of three independent experiments. Statistical analysis was done using Student’s t -test * P <0.05 (ii) JVM-3 cells were treated with 40 μM ghost nanoliposomes or CNL for indicated time periods and western blotting was performed. (iii) and (iv) Graphical representation of western blotting. The graph is a quantification of three independent experiments. Statistical analysis was done using Student’s t -test * P <0.05. ( d ) CNL induces early suppression of p-STAT3 in CLL patient cells. Cells from three CLL patients were treated for 12 h with 40 μM ghost nanoliposomes or CNL and western blotting was done.

Article Snippet: Human EF.STAT3C.Ubc.GFP vector from Addgene (Cambridge, MA, USA) was used for expressing STAT3-C in JVM-3 cells.

Techniques: Staining, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

doi: 10.1038/sigtrans.2017.51

Figure Lengend Snippet: CNL suppresses STAT3 phosphorylation via multiple kinases including BTK. ( a ) (i) CNL suppresses the activity of BTK. JVM-3 cells were treated with 40 μM ghost liposomes or CNL for indicated time periods and western blotting was performed. The blots are a representative of three independent experiments. (ii) and (iii) BTK inhibitors suppress phosphorylation of STAT3 in JVM-3 cells and CLL patient cells. Cells were treated with varying concentrations of ibrutinib for indicated time periods and western blotting was performed. Graphical representation of the western blot is also shown. The blots and graphs are representative of three independent experiments or three CLL patient samples. Student’s t -test was used to perform statistical analysis, * P <0.05. The final western blot image was created by grouping different parts of the same film of the same gel as indicated by the black dividing line. (iv) Synergism analysis of CNL and ibrutinib treatments. JVM-3 cells were treated with single agents and co-treated with different doses of CNL (1–10 μM) and ibrutinib (1–2.5 μM) for 24 h and MTS assay was performed. The cell viability data were analyzed for synergism using Compusyn software. No synergism was observed with ghost nanoliposomes. ( b ) (i) CNL suppresses the activity of MEK1/2 kinase. JVM-3 and Mec-2 cells were treated with 40 μM ghost liposomes or CNL for indicated time periods and western blotting was performed. The blots are a representative of three independent experiments. (ii) JVM-3 cells were treated with 10 μM U0126 for indicated time periods and p-Erk levels were evaluated to confirm the effectiveness of U0126 as a MEK inhibitor. (iii) and (iv) MEK1/2 inhibitor suppresses phosphorylation of STAT3 in JVM-3 cells and CLL patient cells. Cells were treated with 10 μM U0126 for indicated time periods and western blotting was performed. The blots and graphs are representative of three independent experiments or three CLL patient samples. Student’s t -test was used for statistical analysis, * P <0.05. ( c ) (i) CNL suppresses the activity of PKC. JVM-3 cells were treated with 40 μM ghost liposomes or CNL for indicated time periods and western blotting was performed. The blots are a representative of three independent experiments. (ii) and (iii) PKC inhibitor suppresses phosphorylation of STAT3 in JVM-3 cells and CLL patient cells. Cells were treated with 5 μM Bis-I for indicated time periods and western blotting was performed. The blots and graphs are representative of three independent experiments or three CLL patient samples. Student’s t -test was used for statistical analysis, * P <0.05. ( d ) CNL does not activate phosphatases. JVM-3 cells were pretreated for 2 h with: (i) 5 nM OA; (ii) 50 μM PV, followed by 12 h of treatment with 40 μM ghost nanoliposomes or CNL. Both the inhibitors were non-toxic to cells at the specific concentration. The blots are a representative of two independent experiments. The final western blot image was created by grouping different parts of the same film of the same gel as indicated by the black dividing line.

Article Snippet: Human EF.STAT3C.Ubc.GFP vector from Addgene (Cambridge, MA, USA) was used for expressing STAT3-C in JVM-3 cells.

Techniques: Activity Assay, Western Blot, MTS Assay, Software, Concentration Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

doi: 10.1038/sigtrans.2017.51

Figure Lengend Snippet: CNL suppresses the transcriptional activity of STAT3. ( a ) CNL reduces levels of STAT3-regulated genes. JVM-3 cells and Mec-2 cells were treated with 20 μM or 40 μM ghost liposomes or CNL and western blotting was performed. JVM3 cells were treated for 24 h and Mec-2 cells were treated with 48 h. The images are representative of three independent experiments. ( b ) Reduction of STAT3 phosphorylation precedes reduction of Mcl-1 levels following CNL treatment. JVM-3 cells were treated with 40 μM ghost liposomes or CNL for indicated time periods and western blotting was performed. ( c ) CNL inhibits expression of luciferase in a STAT3 luciferase reporter assay. JVM-3 cells were transfected with different luciferase constructs. Twelve hours after transfection, cells were treated with ghost nanoliposomes or CNL for 12 h and luciferase assay was performed. The graphs are representative of three independent experiments.

Article Snippet: Human EF.STAT3C.Ubc.GFP vector from Addgene (Cambridge, MA, USA) was used for expressing STAT3-C in JVM-3 cells.

Techniques: Activity Assay, Western Blot, Expressing, Luciferase, Reporter Assay, Transfection, Construct

Journal: Signal Transduction and Targeted Therapy

Article Title: STAT3 mediates C6-ceramide-induced cell death in chronic lymphocytic leukemia

doi: 10.1038/sigtrans.2017.51

Figure Lengend Snippet: Overexpression of STAT3-C rescues CNL-induced cell death. ( a ) STAT3-C-expressing cells are resistant to CNL-induced cell death. Lentiviral transduction was performed to express STAT3-C in JVM-3 cells. Seventy-two hours after the last transduction, FACS was performed to obtain a pure population of cells expressing STAT3-C and the treatments were done. An overexpression construct expressing RFP was used as a negative control. Seventy-two hours after the last transduction cycle, cells were treated with ghost liposomes and CNL for 24 h. (i) Expression of STAT3-C was confirmed by western blotting and probing for Flag-tag. (ii) Flow cytometric analysis for Annexin-V and 7AAD was performed to quantitate % necrotic cells. Student’s t -test was used for statistical analysis, * P <0.05.

Article Snippet: Human EF.STAT3C.Ubc.GFP vector from Addgene (Cambridge, MA, USA) was used for expressing STAT3-C in JVM-3 cells.

Techniques: Over Expression, Expressing, Transduction, Construct, Negative Control, Western Blot, FLAG-tag