Journal: Frontiers in Immunology

Article Title: Ex vivo expanded human regulatory T cells promote cholesterol efflux and PON1 expression in oxLDL-exposed macrophages via gap junction-mediated cAMP transfer

doi: 10.3389/fimmu.2025.1662925

Figure Lengend Snippet: Treg exp increase ABCA1 expression by transferring cAMP into M IL4 . (A) Changes in the expression of ABCA1 and ABCG1 transcripts in M IL4 co-cultured with either Teffs or Treg exp . Gene expression quantified by qRT-PCR comparing mRNA levels in M IL4 alone (RQ = 1, shown as dashed line) with the same cells co-cultured for 4h with either Teffs or Tregs. UBC was used as reference gene. (B) Intracellular concentration of cAMP in Tregs exp alone, M IL4 alone, or M IL4 co-cultured with Tregs exp for 4h. cAMP levels were normalized to total cellular protein content. Statistical analysis was performed only between M IL4 and M IL4 + Tregs exp , as the aim was to assess whether cAMP levels in M IL4 increase upon co-culture. The cAMP level in Tregs exp is shown as a reference to highlight the high concentration of this molecule in these cells but was not included in the statistical comparison due to the difference in cell type. (C) Changes in the expression of ABCA1 mRNA in M IL4 co-cultured with Treg exp (ratio 1:1) after 1h preincubation or not with GAP27 (300 µM). (D) Changes in the expression of ABCA1 mRNA in M IL4 co-cultured with Tregs (ratio 1:1) after 1h preincubation or not with PKA inhibitor H89 (5 µM). (E) Western blot analysis of ABCA1 protein level in cell lysates of M IL4 alone or M IL4 co-cultured (ratio 1:1) with either Treg exp or Teff for 4h. Data were plotted as ABCA1 protein intensity normalised to GAPDH protein intensity. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. *p<0.05, **p<0.01.

Article Snippet: Equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membrane and probed overnight at 4C with the respective antibodies: monoclonal anti-ABCA1 antibody (CellSignaling; Cat Number 96292), polyclonal anti-PON1 antibody (Proteintech; Cat Number 18155-1-AP), monoclonal anti-GAPDH antibody (CellSignaling; Cat Number 5174), recombinant monoclonal anti-ABCA1 (phosho-S2054) antibody (Abcam; Cat Number ab125064).

Techniques: Expressing, Transferring, Cell Culture, Gene Expression, Quantitative RT-PCR, Concentration Assay, Co-Culture Assay, Comparison, Western Blot

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 1. A novel NSD3–NUT fusion is identifi ed in NMC. A, histology of the NMC from which the 1221 cell line was derived reveals a very poorly differentiated tumor (magnifi cation, ×400). B, IHC of the tumor using the anti-NUT monoclonal antibody C52 (magnifi cation, ×400). C, RNA-sequencing reads spanning the junction of NSD3 and NUT . D, immunoblot of three NMC cell lines and 293T control cells stained with AX.1 polyclonal antibody to NUT. E, immunoblot of the 1221 cell line 48 hours following transfection with control (CTRL), NSD3 , and NUT siRNAs stained with the AX.1 antibody to NUT. F, NSD3–NUT dual-color bring-together FISH assay (magnifi cation, ×1,000) using bacterial artifi cial chromosome (BAC) probes telomeric (3′) to NUT (green), and BAC probes centromeric (5′) to NSD3 (red) as depicted in the chromosomes 8 and 15 ideograms. Yellow arrows, NSD3–NUT fusions. G, gel electrophoresis of PCR of TC-797 and 1221 cell lines with (+) and without (−) reverse transcriptase reaction. H, schematic of the NSD3–NUT predicted encoded protein in comparison with NSD3, NUT, and BRD4–NUT. PWWP, Pro–Trp–Trp–Pro motif; PHD, plant homeo domain; SET, Drosophila Su(var)3-9 and ‘Enhancer of zeste’; C/H, Cys-His; NES, nuclear export signal sequence; Bromo, bromodomain. Arrows, breakpoints. I, NSD3 dual-color split-apart FISH assay using BAC probes fl anking NSD3 , as depicted in the chromosome 8 ideogram, depicted in three NMCs, not including 1221, desig- nated cases 1–3. All photomicrographs are of identical magnifi cation (×1,000).

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Derivative Assay, RNA Sequencing, Western Blot, Control, Staining, Transfection, Nucleic Acid Electrophoresis, Reverse Transcription, Comparison, Sequencing

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 3. Wild-type NSD3 is required for the blockade of differentiation in BRD4–NUT-expressing NMC cells. A, immunoblots of BRD4–NUT-positive NMC cell lines TC-797, PER-403, and 8645 120 hours following transfection with control and NSD3 siRNAs stained with the terminal squamous differen- tiation marker involucrin, using GAPDH as loading control. B, representative photomicrographs of TC-797s 120 hours following transfection with either control or NSD3 siRNAs stained either with H&E for morphology, or involucrin IHC. All photographs are of identical magnifi cation (×400). C, qRT-PCR of NSD3 levels 24 hours following transfection of control or NSD3 siRNAs. Primers were either 5′ of the breakpoint (NSD3–5′ primers), or 3′ of the break- point (NSD3-3′ primers) with NUT. Results are of a single biologic replicate performed in triplicate. Error bars, the mean ± SD of the triplicate wells. D, proliferation assay (Ki-67 fraction) comparing BRD4–NUT-positive TC-797, 8645, and PER-403 NMC cells transfected with control and NSD3–6 siRNAs. Three hundred cells were counted per cell block. E, 797TRex cells induced to express FLAG-tagged NLS-ET domain of BRD4 for 120 hours. Immu- noblot was performed with anti-involucrin (Inv), anti-FLAG, or anti-GAPDH (left). Cell block preparations were H&E stained, or subjected to involucrin IHC (right). All photographs are of identical magnifi cation (×400). F, cell viability assay (CellTiter-Glo) of 797TRex, 293TRex, and U2OSTRex cells induced to express FLAG-tagged NLS-ET domain for 120 hours. Results are the average of three biologic replicates, each performed in quadruplicate and normal- ized to the negative control (ethanol vehicle control) for each cell line. Error bars, the mean ± SD of the three biologic replicates. Immunoblot demonstrat- ing NLS-FLAG-ET expression was stained with anti-FLAG or anti-GAPDH (right).

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Expressing, Western Blot, Transfection, Control, Staining, Marker, Quantitative RT-PCR, Proliferation Assay, Blocking Assay, Viability Assay, Negative Control

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 4. The N-terminus of NSD3 associates with BRD4 and BRD4–NUT. A, immunofl uorescence microscopy of 797TRex cells induced to express the HA-tagged portion of NSD3 included in NSD3–NUT (NSD3Tr) for 24 hours stained with anti-NUT monoclonal antibody (red), and anti-HA monoclonal anti- body (green). B, immunoblot of anti-HA immunoprecipitations (IP) of tet-repressor–positive C33A cell (C33A-6TR) lysates following induction of expres- sion of HA-tagged NSD3 variants: HA-NSD3 (full-length), HA-NSD3–NUT, and HA-NSD3-tr (NSD3 portion of the NSD3–NUT fusion protein). Indicated proteins were detected using anti-HA and anti-Brd4 antibodies. The smaller bands are degraded protein. C, immunoblot of anti-HA immunoprecipitations of C33A-6TR lysates following induction of expression of HA-tagged NUT, BRD4, and BRD4–NUT constructs stained with anti-HA, anti-NSD3, anti-p300, and anti-actin antibodies. To identify the NSD3-specifi c bands, lysates from TC-797s subjected to siRNA knockdown of NSD3 are shown. D, immunoblot of 797TRex lystes 120 hours following induction of expression of BioTAP-tagged NLS fusion construct of NSD3Tr stained with anti-involucrin, anti-PAP (recognizes the protein A moiety of the BioTAP tag), and anti-GAPDH antibodies.

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Microscopy, Staining, Western Blot, Expressing, Construct, Knockdown

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 5. BRD4–NUT foci are dependent on NSD3. A, immunofl uores- cence microscopy of TC-797 cells 24 hours following transfection with control or NSD3–6 siRNAs stained with monoclonal antibody to NUT. All photographs are of identical magnifi cation (×1,000). B, quantitation of BRD4–NUT foci was performed in triplicate and the average of the three experiments is shown. Error bars, the mean ± SD of triplicate experi- ments. *, P < 0.005. C, immunoblot of TC-797 lysates 24 hours following transfection with control, NSD3–6 , or NUT siRNAs stained with anti-NUT polyclonal antibody, AX.1.

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Microscopy, Transfection, Control, Staining, Quantitation Assay, Western Blot

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 6. NSD3–NUT can replace the function of BRD4–NUT to block differentiation. A, H&E and anti-involucrin IHC micrographs of 797TRex cells with tetracycline (ON), or treated with vehicle (OFF) to express NSD3–NUT 120 hours following transfection with either control or NUT 3′-UTR siRNA. All photographs are of identical magnifi cation (×400). B, immunoblots using lysates corresponding to the experiment in A were performed for the differen- tiation marker, involucrin, NSD3–NUT, and BRD4–NUT using antibodies to NUT. C, quantifi cation of immunohistochemical Ki-67 proliferation fraction of 797TRex cells induced to express NSD3–NUT 120 hours following transfection with either control or NUT 3′-UTR siRNA as in A. Results are the average of three biologic replicates performed using the 384-well high-throughput assay as in Fig. 2A , each performed in triplicate. Error bars, the mean ± SD of the three biologic replicates. *, P < 0.0001.

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Blocking Assay, Transfection, Control, Western Blot, Marker, Immunohistochemical staining, High Throughput Screening Assay

Journal: Science (New York, N.Y.)

Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway

doi: 10.1126/science.aau0159

Figure Lengend Snippet: (A) Schematic description of the in vitro binding analysis of Flag-tagged unmodified or ubiquitinated PTEN with GST-PTEN from bacteria (left). In vitro pull-down assay with indicated Flag-tagged unmodified or ubiquitinated PTEN and GST-PTEN (right). Flagged untagged PTEN or ubiquitinated PTEN was purified from HEK293 cells transfected with Flag-PTEN or WWP1, along with individual His-ubiquitin variants, using M2 beads, whereas GST-PTEN was purified from bacteria. (B) Membrane and soluble fractions isolated from DU145 cells transfected with indicated constructs were analyzed by Western blot. Epidermal growth factor receptor (EGFR) serves as a membrane marker and actin as the internal control. (C) Analysis of AKT activation in DU145 cells. Total lysates were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and then probed with the indicated antibodies. (D) Evaluation of PTEN dimerization in Wwp1−/− MEFs with stable reconstitution of either WWP1 WT or its catalytic dead mutant (C890A) by native gel electrophoresis. Total lysates from cells transduced with indicated constructs were immunoprecipitated with a rabbit anti-PTEN antibody, and then the immunocomplexes were natively eluted from the beads. The eluted samples were immediately run on the native gel. (E) Membrane and soluble fractions isolated from Wwp1+/+ and Wwp1−/− MEFs reconstituted with the indicated constructs were analyzed by Western blot. EGFR served as a membrane marker, and actin as the internal control. pS473-AKT and pT308-AKT are the markers of AKT activation. (F) Subcelluar localization of PTEN in Wwp1+/+ or Wwp1−/− MEFs. Confocal images of Wwp1+/+ or Wwp1−/− MEFs stained with 4′,6-diamidino-2-phenylindole (DAPI, blue) and PTEN (green) (top). The white arrows indicate PTEN plasma membrane localization. Scale bar, 20 μm. The percentage of cells displaying PTEN plasma membrane localization was quantified (bottom). Data are shown as mean ± SD for triplicate experiments, with 50 cells per group per experiment (***P < 0.0005, Student’s t test). (G) Evaluation of PTEN dimerization potential in DLP tissues derived from Wwp1 WT or Wwp1 knockout mice (n = 3). (H) Analysis of PI3K-AKT-mTOR signaling cascade in DLP tissues derived from Wwp1 WT or Wwp1 knockout mice (n = 3). Actin was used as a loading control. pS6 and S6 are markers to indicate the activation of the mTOR pathway. (I) Effects of the PTEN K342/K344R mutant on PTEN dimerization in PC3 cells. PC3 cells transfected with indicated constructs were serum starved for 6 hours and then treated with 100 ng/ml insulin for 10 min. Total lysates from cells were immunoprecipitated with a rabbit anti-Myc antibody, and then the immunocomplexes were natively eluted from the beads. The eluted samples were immediately run on a native gel, and Western blot using PTEN antibody showed the monomer and dimer of PTEN, as indicated by arrows. (J) Membrane localization of PTEN K342/K344R mutant in PC3 cells as in (I). Membrane and soluble fractions isolated from PC3 cells with indicated constructs were analyzed by Western blot. EGFR serves as the marker for the membrane fraction and actin as the internal control for the soluble fraction. Vector indicates cells transfected with empty vector plasmids. (K) Subcellular localization of the PTEN K342/K344R mutant in PC3 cells. Confocal images of PC3 cells stably expressing indicated PTEN WT or the K342/K344R mutant stained with DAPI and PTEN (green) (top). White arrows indicate PTEN plasma membrane localization. Scale bar, 20 μm. The percentage of cells displaying PTEN plasma membrane localization was quantified (bottom). Data are shown as mean ± SD for triplicate experiments, with 50 cells per group per experiment (***P < 0.0005, Student’s t test). (L) Effects of indicated PTEN KR mutants on PTEN lipid phosphatase activities in PC3 cells with indicated constructs. Data are shown as mean ± SD (***P < 0.0005, **P < 0.005, triplicate experiments, Student’s t test). OD620nm, absorbance at 620 nm. (M) Effects of indicated PTEN KR mutants on AKT activation in PC3 cells with indicated constructs. Total lysates were harvested and then probed with indicated antibodies. Actin was used as a loading control. (N) Effects of the PTEN K342/K344R mutant on tumor growth of PC3 cells as used in (K) in a xenograft mouse model. Error bars represent SEM (n = 5 mice per group).

Article Snippet: For western blotting: Anti-Myc-Tag (2276), anti-PTEN (9559), Anti-MYC for western blot and ChIP assay (13987), anti-EGFR (4267), anti-Ubiquitin (3936), anti-Cleaved Capase3 (9661), anti-Phospho-AKT (pSer473, 9271; pThr308, 9275), anti-AKT (pan AKT, 4685), anti-PKM2 (4053), anti-PFKFB3 (13123), anti-pS6 (2211), anti-S6 (2217), anti-Smurf2 (2174) antibodies were all purchased from Cell Signaling Technology.

Techniques: In Vitro, Binding Assay, Pull Down Assay, Purification, Transfection, Isolation, Construct, Western Blot, Marker, Activation Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Mutagenesis, Nucleic Acid Electrophoresis, Transduction, Immunoprecipitation, Staining, Derivative Assay, Knock-Out, Plasmid Preparation, Stable Transfection, Expressing

Journal: Science (New York, N.Y.)

Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway

doi: 10.1126/science.aau0159

Figure Lengend Snippet: (A) Schematic description of the MYC responsive element on the WWP1 promoter (top). Chromatin level of MYC at the promoter of human WWP1 was measured in DU145 cells. Fold enrichment of MYC was determined by quantitative chromatin immunoprecipitation (qChIP) assays. JunB and RPL30 served as positive and negative controls, respectively. TSS, transcription start site. Data are shown as mean ± SD (***P < 0.0005, **P < 0.005, triplicate experiments, Student’s t test). (B) Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) analysis of WWP1 in DU145 cells expressing the indicated constructs. The mRNA level of WWP1 was determined by RT-qPCR and is presented as a fold increase, as compared with the vector control (−). The HA-MYC levels are indicated by the triangle, from left to right. (C) Analysis of WWP1 and PTEN expression and AKT activation in DU145 cells expressing different amounts of HA-MYC. Total lysates were resolved by SDS-PAGE and then probed with indicated antibodies. The “−” indicates the vector control. (D) Tandem mass spectrum of a peptide derived from endogenous ubiquitinated PTEN in DU145 cells stably expressing the indicated constructs showed ubiquitin conjugation at the K27 residue of ubiquitin. (E) Ratio of indicated ubiquitin linkages detected by MS analysis of endogenous ubiquitinated PTEN purified from MYC overexpression or MYC overexpression–shWWP1 cells to that from control cells (without MYC overexpression). The abundance of each ubiquitin linkage was calculated as described in the materials and methods. (F) Analysis of PTEN K27-linked polyubiquitination in DU145 cells stably expressing MYC and/or WWP1 shRNAs. pCDH-puro-MYC, lentiviral expression of MYC. (G) Analysis of WWP1 and PTEN expression and AKT activation in DU145 cells expressing indicated siRNA SMARTpool. Total lysates were resolved by SDS-PAGE and then probed with indicated antibodies. (H) Analysis of WWP1 and PTEN expression and AKT activation in DU145 cells stably expressing MYC and/or WWP1 shRNAs. (I) Effects of WWP1 with or without PTEN on MYC-induced colony-forming activity in soft agar. The colony numbers are quantified and presented as mean ± SD (***P < 0.0005, **P < 0.005, *P < 0.05, triplicate experiments, Student’s t test). sgRNA, single-guide RNA. (J) Apoptosis assay of DU145 cells stably expressing MYC and/or WWP1 shRNAs. The percentage of apoptotic cells were stained with annexin V–PTEN and 7ADD and then quantified by fluorescence-activated cell sorting. Data are shown as mean ± SD (***P < 0.0005, **P < 0.005, triplicate experiments, Student’s t test). shCon, control. (K) Effects of WWP1 on Ras and MYC-induced colony-forming activity in soft agar or signaling pathway in cells. The colony numbers are quantified and presented as mean ± SD (***P < 0.0005, **P < 0.005, triplicate experiments, Student’s t test) (left). Total lysates were resolved by SDS-PAGE and then probed with indicated antibodies (right). ERK, extracellular signal–regulated kinase; pERK, phosphorylated ERK. (L) Effects of WWP1 on PTEN lipid phosphatase activities in MEFs with indicated constructs. Data are shown as mean ± SD (**P < 0.005, triplicate experiments, Student’s t test). In (C), (F), (H), and (K), actin was used as a loading control.

Article Snippet: For western blotting: Anti-Myc-Tag (2276), anti-PTEN (9559), Anti-MYC for western blot and ChIP assay (13987), anti-EGFR (4267), anti-Ubiquitin (3936), anti-Cleaved Capase3 (9661), anti-Phospho-AKT (pSer473, 9271; pThr308, 9275), anti-AKT (pan AKT, 4685), anti-PKM2 (4053), anti-PFKFB3 (13123), anti-pS6 (2211), anti-S6 (2217), anti-Smurf2 (2174) antibodies were all purchased from Cell Signaling Technology.

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Construct, Plasmid Preparation, Activation Assay, SDS Page, Derivative Assay, Stable Transfection, Conjugation Assay, Purification, Over Expression, Activity Assay, Apoptosis Assay, Staining, Fluorescence, FACS

Journal: Science (New York, N.Y.)

Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway

doi: 10.1126/science.aau0159

Figure Lengend Snippet: (A) In silico modeling of predicted interactions with the HECT domain of WWP1. (B) MST analysis determined the Kd of I3C toward His-WWP1 HECT WT or the His-WWP1 HECT F577/Y656A mutant. Concentration is reported in nanomolar. (C) Analysis of the effects of I3C on prostate organoid–forming ability from WT or Hi-Myc mice treated with or without 10 or 20 μM I3C for 3 days. Scale bar, 100 μm. DMSO, dimethyl sulfoxide. (D) Gross anatomy of representative urogenital tracts from Hi-Myc mice treated with vehicle or I3C. The mice, at 5 months of age, were treated intraperitoneally with I3C (20 mg/kg), three times a week for 1 month starting on day 0. Representative pictures are shown in the pane (n = 9 mice per group). (E) H&E and IHC staining of DLPs from Hi-Myc mice treated with vehicle or I3C with indicated antibodies. Scale bar, 50 μm. (F) In vivo ubiquitination analysis of endogenous PTEN ubiquitination levels of DLPs from Hi-Myc mice treated with vehicle or I3C. (G) Western blot analysis of AP, DLP, and VP lysates from Hi-Myc mice, as shown in (D), with the indicated antibodies. Actin was used as a loading control. (H) Confocal analysis of DLPs from indicated mice stained with PTEN (green) and DAPI (blue). The white-outlined areas in the top row are enlarged 2.6-fold (bottom row) to show the subcellular localization of PTEN. Representative pictures are shown (n = 2 mice per genotype). Scale bars, 50 μm. (I) GSEA of RNA-seq data from the DLPs of (i) I3C-treated mice versus vehicle (VEH)–treated mice and (ii) BKM120-treated mice versus vehicle-treated mice using the PI3K-Akt signaling pathway gene set annotated in the KEGG. Mice from all four groups contain the same Hi-Myc genetic background unless otherwise noted. (J) H&E staining of APs from Pten+/− mice treated with vehicle or I3C. At 7.5 months of age, the mice were treated intraperitoneally with I3C (20 mg/kg), three times a week for 1 month starting on day 0. Representative pictures are shown (n = 3 mice per group). Scale bar, 100 μm. (K) Western blot analysis of AP lysates from Pten+/− mice, as shown in (J), with the indicated antibodies. Actin was used as a loading control. (L) Model for WWP1-mediated PTEN K27-linked polyubiquitination in cell growth, tumor development, and progression. Deregulated MYC overexpression or MYC amplification promotes WWP1 expression and, in turn, triggers PTEN K27-linked polyubiquitination. Aberrant K27-linked polyubiquitination suppresses PTEN dimerization, plasma membrane recruitment, and tumor-suppressive function, leading to the tumor initiation and progression. PIP2, phosphatidylinositol 4,5-bisphosphate; u, ubiquitin.

Article Snippet: For western blotting: Anti-Myc-Tag (2276), anti-PTEN (9559), Anti-MYC for western blot and ChIP assay (13987), anti-EGFR (4267), anti-Ubiquitin (3936), anti-Cleaved Capase3 (9661), anti-Phospho-AKT (pSer473, 9271; pThr308, 9275), anti-AKT (pan AKT, 4685), anti-PKM2 (4053), anti-PFKFB3 (13123), anti-pS6 (2211), anti-S6 (2217), anti-Smurf2 (2174) antibodies were all purchased from Cell Signaling Technology.

Techniques: In Silico, Mutagenesis, Concentration Assay, Immunohistochemistry, In Vivo, Western Blot, Staining, RNA Sequencing Assay, Over Expression, Amplification, Expressing

Journal: Cell Death & Disease

Article Title: Bortezomib-inducible long non-coding RNA myocardial infarction associated transcript is an oncogene in multiple myeloma that suppresses miR-29b

doi: 10.1038/s41419-019-1551-z

Figure Lengend Snippet: a–e U266 cells were pretreated with the selective pharmacological inhibitors U0126 (ERK, 50 μM), SP600125 (JNK, 50 μM), Bay-11-7082 (NF-κB, 10 μM), SB203580 (p38, 50 μM), or PF-04965842 (Jak1, 50 nM), and were then treated with BTZ at 40 nM for 12 h. MIAT expression levels were determined using qRT-PCR. f Western blotting analyses of Stat1 and phosphorylated Stat1 after overexpression or knockdown in U266 cells. g Effects of Stat1 overexpression on MIAT expression in U266 cells. h Effects of Stat1 knockdown on MIAT expression in U266 cells. i Effects of p38 overexpression on MIAT expression in U266 cells pretreated with sh-NC or sh-Stat1. j Luciferase reporter constructs containing the MIAT promoter were co-transfected into U266 cells with the internal control plasmid pRL-TK, and with sh-NC or sh-Stat1, and were then subjected to BTZ challenge (40 nM, 12 h). Relative luciferase activities are expressed as percentages of those in the control group. k Cell lysates from U266 cells were used for RIP with antibodies against stat1, stat3, or NF-κB. MIAT expression levels were detected using qRT-PCR. IgG was used as a negative control. l BTZ induced Stat1 phosphorylation; data are presented as means ± standard errors of the mean from three independent experiments; * P < 0.05; two-tailed pairwise comparisons were made with Student’s t -test

Article Snippet: Following separation on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, proteins were transferred onto polyvinylidene (PVDF) membranes and were immunoblotted with the following primary antibodies: phospho-Stat1 (Tyr701) antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 9167, Cell Signaling Technology), phospho-Stat1 (Ser727) antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 8826, Cell Signaling Technology), Stat1 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 14994, Cell Signaling Technology), p38 MAPK antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 8690, Cell Signaling Technology), Bcl-2 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 4223, Cell Signaling Technology), Bak1 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 12105, Cell Signaling Technology), cleaved PARP antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 5625, Cell Signaling Technology), total PARP antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 9532, Cell Signaling Technology), Mcl-1 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 94296, Cell Signaling Technology), CDK-6 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 13331, Cell Signaling Technology), PSME4 antibody (Rabbit polyclonal, diluted at 1:1000, cat no. ABIN5533127, Abgent Biotech. (SuZhou) Co. Ltd., Suzhou, China), SP-1 antibody (Rabbit monoclonal, diluted at 1:1000, cat no. 9389, Cell Signaling Technology), and GAPDH antibody (Rabbit polyclonal, diluted at 1:1000, cat no. 5174, Cell Signaling Technology).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Knockdown, Luciferase, Construct, Transfection, Control, Plasmid Preparation, Negative Control, Phospho-proteomics, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Extracellular UDP-Glucose Activates P2Y 14 Receptor and Induces Signal Transducer and Activator of Transcription 3 (STAT3) Tyr 705 Phosphorylation and Binding to Hyaluronan Synthase 2 ( HAS2 ) Promoter, Stimulating Hyaluronan Synthesis of Keratinocytes *

doi: 10.1074/jbc.M114.551804

Figure Lengend Snippet: UDP-Glc activates ERK and STAT3. A, dot blot images from a phosphokinase array on protein samples extracted from cells incubated for 30 min with 100 μm UDP-Glc. The phosphorylated proteins STAT-Tyr705 and ERK are encircled. Other spots in array: A3A4, p38a; A7A8, JNK1/2/3; A9A10, GSK-3a/b; B5B6, MSK1/2; B9B10, Akt1/2/3; C1C2, TOR; C3C4, CREB; C7C8, AMPKa2; C9C10, β-catenin; D1D2, Src; D7D8, STAT2; D9D10, STAT5a; E1E2, Fyn; E3E4, Yes; E9E10, STAT5b; F1F2, Hck; F3F4, Chk-2; F5F6, FAK; F7F8, PDGF Rb; F9F10, STAT5a/b (B and C). Western blots of cells incubated for 30–60 min with 100 μm UDP-Glc with antibodies against p-ERK and Tyr(P)705-STAT3. Mean ± S.D. of 4 independent experiments are shown. Statistical significance of the difference versus control, **, p = 0.002 (Dunnett's test).

Article Snippet: The antibodies against rabbit IgG (sc-2027), pSTAT3 (Tyr 705 ) (sc-7993), were obtained from Santa Cruz Biotechnologies and pSTAT (Ser 727 ) (number 9134) and total STAT3 (number 9132) were obtained from Cell Signaling.

Techniques: Dot Blot, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Extracellular UDP-Glucose Activates P2Y 14 Receptor and Induces Signal Transducer and Activator of Transcription 3 (STAT3) Tyr 705 Phosphorylation and Binding to Hyaluronan Synthase 2 ( HAS2 ) Promoter, Stimulating Hyaluronan Synthesis of Keratinocytes *

doi: 10.1074/jbc.M114.551804

Figure Lengend Snippet: UDP-Glc-induces both Tyr(P)705 and Ser(P)727 phosphorylation of STAT3. A, Western blots of Tyr(P)705-STAT3 and Ser(P)727-STAT3 30–120 min after introduction of 100 μm UDP-Glc. B, quantitation of the changes in phosphorylation of Tyr(P)705 and C, Ser(P)727 of STAT3. Mean ± S.E. from 3 to 6 separate experiments are shown. **, p < 0.01, UDP-Glc versus control, by paired sample t test. D, verification of the effect of STAT3 inhibitor IX and JAK2 inhibitor AG490 on STAT3 Tyr705 phosphorylation after 120 min preincubation with the inhibitors followed by 60 min incubation with UDP-Glc. E, inhibition of the UDP-Glc-induced HAS2 up-regulation by STAT3 inhibitor IX. The cultures were preincubated for 120 min with 50 μm STAT3 inhibitor IX, followed by 120 min in the presence and absence of 100 μm UPD-Glc. Mean ± S.E. of five separate experiments, *, p < 0.05, Dunnett's test.

Article Snippet: The antibodies against rabbit IgG (sc-2027), pSTAT3 (Tyr 705 ) (sc-7993), were obtained from Santa Cruz Biotechnologies and pSTAT (Ser 727 ) (number 9134) and total STAT3 (number 9132) were obtained from Cell Signaling.

Techniques: Western Blot, Quantitation Assay, Incubation, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Extracellular UDP-Glucose Activates P2Y 14 Receptor and Induces Signal Transducer and Activator of Transcription 3 (STAT3) Tyr 705 Phosphorylation and Binding to Hyaluronan Synthase 2 ( HAS2 ) Promoter, Stimulating Hyaluronan Synthesis of Keratinocytes *

doi: 10.1074/jbc.M114.551804

Figure Lengend Snippet: UDP-Glc treatment enhances Tyr(P)705-STAT3 binding to the HAS2 gene promoter. HaCaT cells were incubated for 2 h with 100 μm UDP-Glc and STAT3 binding into the HAS2 promoter was studied by ChIP. Total STAT3, Tyr(P)705-STAT3 and Ser(P)727-STAT3 binding to the transcription start site region is shown in A, to region −481 to −244 in B, to region −1048 to −655 in C, and to region −1896 to −1554 in D. The data represent mean ± S.E. of five independent experiments. Statistical significance control versus UDP-Glc: *, p < 0.05 (paired sample t test).

Article Snippet: The antibodies against rabbit IgG (sc-2027), pSTAT3 (Tyr 705 ) (sc-7993), were obtained from Santa Cruz Biotechnologies and pSTAT (Ser 727 ) (number 9134) and total STAT3 (number 9132) were obtained from Cell Signaling.

Techniques: Binding Assay, Incubation

Journal: Theranostics

Article Title: Interleukin 33-mediated inhibition of A-type K + channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice

doi: 10.7150/thno.69320

Figure Lengend Snippet: IL-33 decreases I A via p38 MAPK. A, effects of 50 ng/mL IL-33 on phospho-Akt ( p -Akt) or total Akt ( t -Akt) protein abundance in the presence or absence of Akt inhibitor III (Akt-III, 10 µM) in DRG cells. Blots are representative of three independent experiments with β-tubulin serving as a loading control. ** p < 0.01 vs. control, unpaired t test. B, colabeling (white arrows) of ST2 and Akt and p38 in mouse DRG sections. Scale bar, 50 µm. C-D, time course of I A changes indicating the effects of IL-33 on I A in the presence of 10 µM Akt inhibitor III ( C ) or 20 µM LY294002 ( D ). Arabic numerals indicate the points utilized for the example current traces. E, bar graph showing the effects of 50 ng/mL IL-33 on I A in the presence of Akt inhibitor III ( n = 10 cells) or LY294002 ( n = 8 cells) as indicated in Panels B and C , respectively. Application of 10 µM Akt inhibitor III ( n = 6 cells) or 20 µM LY294002 ( n = 6 cells) alone did not significantly affect I A . F, effects of 50 ng/mL IL-33 on p -p38, p -JNK and p -ERK protein abundance in DRG cells. Blots are representative of three independent experiments with β-tubulin serving as a loading control. * p < 0.05 vs. control, unpaired t test. G, pretreatment of DRG cells with the ST2 neutralizing antibody (ST2 Ab, 2 µg/mL) or R406 (1 µM) abolished the 50 ng/mL IL-33-induced increase in p -p38 protein abundance. Blots are representative of three independent experiments with β-tubulin serving as a loading control. H, time course of I A changes indicating the effect of 50 ng/mL on I A in cells pretreated with 10 µM SB203580. I, bar graph showing that pretreating cells with SB203580 ( n = 10 cells), but not its inactive analogue SB202474 (10 µM, n = 6 cells), prevented the IL-33-induced I A decrease. ** p < 0.01 vs. control, paired t test.

Article Snippet: In brief, samples containing 25 μg of protein were separated on SDS-polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes (Merk Millipore), and probed with antibodies against ST2/IL-33R (rabbit, 1:1000; Novus Biologicals, Cat. No. NBP2-53096), Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2712), p -Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2710), JAK2 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #3230), p -JAK2 (rabbit, 1:500; Cell Signaling Technology, Cat. No. #3771), p -p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4511), p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #8690), p -ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4370), ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4695), p -Akt (rabbit, 1:800; Cell Signaling Technology, Cat. No. #4060), Akt (rabbit, 1:800; Abcam, Cat. No. ab8805), p -JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4668), JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9252), p38α (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9218), p38β (rabbit, 1:1000; ProteinTech Group, Cat. No. 17376-1-AP) and β-tubulin (rabbit, 1:1000; ProteinTech Group, Cat. No. 10094-1-AP).

Techniques: Quantitative Proteomics, Control

Journal: Theranostics

Article Title: Interleukin 33-mediated inhibition of A-type K + channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice

doi: 10.7150/thno.69320

Figure Lengend Snippet: p38β mediates the IL-33-induced I A decrease. A, immunoblot analysis of p38α and p38β protein abundance in DRGs. Mouse brains were used as positive controls. Blots are representative of three independent experiments with β-tubulin serving as a loading control. B, time course of I A changes ( left ) and bar graph ( right ) indicating the effect of 50 ng/mL IL-33 on I A in the presence of JX-401 (50 nM, n = 8 cells). The application of 50 nM JX-401 ( n = 6 cells) alone had no significant effect on I A . Arabic numerals indicate the points utilized for the example current traces. C, immunoblot analysis showing that the protein expression level of p38β was significantly reduced in the p38β-siRNA-treated groups, while the expression of p38α was not affected. Blots are representative of three independent experiments with β-tubulin serving as a loading control. ** p < 0.01 vs. NC-siRNA, unpaired t test. D, example traces ( left ) and bar graph ( right ) demonstrating the effects of 50 ng/mL IL-33 on I A in cells treated with control siRNA (NC-siRNA, n = 9 cells) or p38β-siRNA ( n = 11 cells). ** p < 0.01 vs. control + NC-siRNA group, one-way ANOVA with a Bonferroni post hoc test.

Article Snippet: In brief, samples containing 25 μg of protein were separated on SDS-polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes (Merk Millipore), and probed with antibodies against ST2/IL-33R (rabbit, 1:1000; Novus Biologicals, Cat. No. NBP2-53096), Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2712), p -Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2710), JAK2 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #3230), p -JAK2 (rabbit, 1:500; Cell Signaling Technology, Cat. No. #3771), p -p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4511), p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #8690), p -ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4370), ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4695), p -Akt (rabbit, 1:800; Cell Signaling Technology, Cat. No. #4060), Akt (rabbit, 1:800; Abcam, Cat. No. ab8805), p -JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4668), JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9252), p38α (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9218), p38β (rabbit, 1:1000; ProteinTech Group, Cat. No. 17376-1-AP) and β-tubulin (rabbit, 1:1000; ProteinTech Group, Cat. No. 10094-1-AP).

Techniques: Western Blot, Quantitative Proteomics, Control, Expressing

Journal: Theranostics

Article Title: Interleukin 33-mediated inhibition of A-type K + channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice

doi: 10.7150/thno.69320

Figure Lengend Snippet: IL-33/ST2 signaling participates in peripheral pain sensitivity. A-B, intraplantar injection (i.p.l.) of IL-33 at 30 ng, 100 ng, and 300 ng significantly decreased the mechanical paw withdrawal threshold (PWT, A ) and heat paw withdrawal latency (PWL, B ). * p < 0.05, ** p < 0.01, *** p < 0.001, vs. vehicle at the corresponding points, two-way ANOVA with a Bonferroni post hoc test. C-D, intraplantar pretreatment with 1 µg of ST2 neutralizing antibody (ST2 Ab) completely prevented the mechanical ( C ) and heat ( D ) hypersensitivity induced by 100 ng of IL-33 (i.p.l.). *** p < 0.001 vs. vehicle at 3 h, two-way ANOVA with a Bonferroni post hoc test. E-F, p38β siRNA attenuated mechanical and heat hypersensitivity induced by 100 ng of IL-33 (i.p.l.). *** p < 0.001 vs. vehicle at 3 h, # P < 0.05, ## P < 0.01 vs. vehicle in the NC-siRNA groups, two-way ANOVA with a Bonferroni post hoc test. G-H, intraplantar pretreatment with 25 nmol 4-AP occluded mechanical ( G ) and heat ( H ) hypersensitivity mediated by 100 ng of IL-33. *** p < 0.001 vs. vehicle at 3 h, two-way ANOVA with a Bonferroni post hoc test. I-J, intraplantar injection of sST2 at 2 µg attenuated the mechanical hypersensitivity ( I ) and thermal hyperalgesia ( J ) in CFA mice. The arrow indicates the injection of sST2 or vehicle. *** p < 0.001 vs. vehicle at the corresponding points, two-way ANOVA with a Bonferroni post hoc test. K-L, representative current traces ( K ) and summary data ( L ) indicating that intraplantar injection of sST2 (2 µg) abolished the CFA (2 d)-induced I A decrease in small-sized DRG neurons ( n = 11-14 neurons per group). ** p < 0.01 compared with the normal saline (NS) group, ## p < 0.01 compared with the CFA + vehicle group, one-way ANOVA with a Bonferroni post hoc test. N = at least 7 mice for all animal behavior experiments.

Article Snippet: In brief, samples containing 25 μg of protein were separated on SDS-polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes (Merk Millipore), and probed with antibodies against ST2/IL-33R (rabbit, 1:1000; Novus Biologicals, Cat. No. NBP2-53096), Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2712), p -Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2710), JAK2 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #3230), p -JAK2 (rabbit, 1:500; Cell Signaling Technology, Cat. No. #3771), p -p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4511), p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #8690), p -ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4370), ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4695), p -Akt (rabbit, 1:800; Cell Signaling Technology, Cat. No. #4060), Akt (rabbit, 1:800; Abcam, Cat. No. ab8805), p -JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4668), JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9252), p38α (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9218), p38β (rabbit, 1:1000; ProteinTech Group, Cat. No. 17376-1-AP) and β-tubulin (rabbit, 1:1000; ProteinTech Group, Cat. No. 10094-1-AP).

Techniques: Injection, Saline

Journal: Theranostics

Article Title: Interleukin 33-mediated inhibition of A-type K + channels induces sensory neuronal hyperexcitability and nociceptive behaviors in mice

doi: 10.7150/thno.69320

Figure Lengend Snippet: Schematic showing the molecular mechanism of IL-33-induced neuronal hyperexcitability of DRG neurons and pain hypersensitivity in mice. IL-33 acting through ST2 receptors does not affect the activity of JAK2 but leads to the activation of Syk. The increased level of p -Syk stimulates downstream p38β signaling, which in turn regulates A-type channel activity and results in I A reduction. IL-33/ST2-mediated signaling enhances neuronal excitability of DRG neurons and nociceptive behaviors in mice. Neither PKA nor PI3K/Akt was necessary for the IL-33-induced I A response in this study. Whether p38β directly phosphorylates the channels encoding I A or stimulates intermediate molecules in small DRG neurons needs to be investigated further. Created with BioRender.com.

Article Snippet: In brief, samples containing 25 μg of protein were separated on SDS-polyacrylamide gel electrophoresis, electroblotted onto polyvinylidene difluoride membranes (Merk Millipore), and probed with antibodies against ST2/IL-33R (rabbit, 1:1000; Novus Biologicals, Cat. No. NBP2-53096), Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2712), p -Syk (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #2710), JAK2 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #3230), p -JAK2 (rabbit, 1:500; Cell Signaling Technology, Cat. No. #3771), p -p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4511), p38 (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #8690), p -ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4370), ERK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4695), p -Akt (rabbit, 1:800; Cell Signaling Technology, Cat. No. #4060), Akt (rabbit, 1:800; Abcam, Cat. No. ab8805), p -JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #4668), JNK (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9252), p38α (rabbit, 1:1000; Cell Signaling Technology, Cat. No. #9218), p38β (rabbit, 1:1000; ProteinTech Group, Cat. No. 17376-1-AP) and β-tubulin (rabbit, 1:1000; ProteinTech Group, Cat. No. 10094-1-AP).

Techniques: Activity Assay, Activation Assay

Journal: Science (New York, N.Y.)

Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway

doi: 10.1126/science.aau0159

Figure Lengend Snippet: (A) Schematic description of the in vitro binding analysis of Flag-tagged unmodified or ubiquitinated PTEN with GST-PTEN from bacteria (left). In vitro pull-down assay with indicated Flag-tagged unmodified or ubiquitinated PTEN and GST-PTEN (right). Flagged untagged PTEN or ubiquitinated PTEN was purified from HEK293 cells transfected with Flag-PTEN or WWP1, along with individual His-ubiquitin variants, using M2 beads, whereas GST-PTEN was purified from bacteria. (B) Membrane and soluble fractions isolated from DU145 cells transfected with indicated constructs were analyzed by Western blot. Epidermal growth factor receptor (EGFR) serves as a membrane marker and actin as the internal control. (C) Analysis of AKT activation in DU145 cells. Total lysates were resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and then probed with the indicated antibodies. (D) Evaluation of PTEN dimerization in Wwp1−/− MEFs with stable reconstitution of either WWP1 WT or its catalytic dead mutant (C890A) by native gel electrophoresis. Total lysates from cells transduced with indicated constructs were immunoprecipitated with a rabbit anti-PTEN antibody, and then the immunocomplexes were natively eluted from the beads. The eluted samples were immediately run on the native gel. (E) Membrane and soluble fractions isolated from Wwp1+/+ and Wwp1−/− MEFs reconstituted with the indicated constructs were analyzed by Western blot. EGFR served as a membrane marker, and actin as the internal control. pS473-AKT and pT308-AKT are the markers of AKT activation. (F) Subcelluar localization of PTEN in Wwp1+/+ or Wwp1−/− MEFs. Confocal images of Wwp1+/+ or Wwp1−/− MEFs stained with 4′,6-diamidino-2-phenylindole (DAPI, blue) and PTEN (green) (top). The white arrows indicate PTEN plasma membrane localization. Scale bar, 20 μm. The percentage of cells displaying PTEN plasma membrane localization was quantified (bottom). Data are shown as mean ± SD for triplicate experiments, with 50 cells per group per experiment (***P < 0.0005, Student’s t test). (G) Evaluation of PTEN dimerization potential in DLP tissues derived from Wwp1 WT or Wwp1 knockout mice (n = 3). (H) Analysis of PI3K-AKT-mTOR signaling cascade in DLP tissues derived from Wwp1 WT or Wwp1 knockout mice (n = 3). Actin was used as a loading control. pS6 and S6 are markers to indicate the activation of the mTOR pathway. (I) Effects of the PTEN K342/K344R mutant on PTEN dimerization in PC3 cells. PC3 cells transfected with indicated constructs were serum starved for 6 hours and then treated with 100 ng/ml insulin for 10 min. Total lysates from cells were immunoprecipitated with a rabbit anti-Myc antibody, and then the immunocomplexes were natively eluted from the beads. The eluted samples were immediately run on a native gel, and Western blot using PTEN antibody showed the monomer and dimer of PTEN, as indicated by arrows. (J) Membrane localization of PTEN K342/K344R mutant in PC3 cells as in (I). Membrane and soluble fractions isolated from PC3 cells with indicated constructs were analyzed by Western blot. EGFR serves as the marker for the membrane fraction and actin as the internal control for the soluble fraction. Vector indicates cells transfected with empty vector plasmids. (K) Subcellular localization of the PTEN K342/K344R mutant in PC3 cells. Confocal images of PC3 cells stably expressing indicated PTEN WT or the K342/K344R mutant stained with DAPI and PTEN (green) (top). White arrows indicate PTEN plasma membrane localization. Scale bar, 20 μm. The percentage of cells displaying PTEN plasma membrane localization was quantified (bottom). Data are shown as mean ± SD for triplicate experiments, with 50 cells per group per experiment (***P < 0.0005, Student’s t test). (L) Effects of indicated PTEN KR mutants on PTEN lipid phosphatase activities in PC3 cells with indicated constructs. Data are shown as mean ± SD (***P < 0.0005, **P < 0.005, triplicate experiments, Student’s t test). OD620nm, absorbance at 620 nm. (M) Effects of indicated PTEN KR mutants on AKT activation in PC3 cells with indicated constructs. Total lysates were harvested and then probed with indicated antibodies. Actin was used as a loading control. (N) Effects of the PTEN K342/K344R mutant on tumor growth of PC3 cells as used in (K) in a xenograft mouse model. Error bars represent SEM (n = 5 mice per group).

Article Snippet: The chromatin fractions were incubated in each case with 10 mg of antibodies to one of the following: MYC (Cell Signaling, #13987), human RPL30 or normal rabbit IgG (both provided by Cell Signaling kit, Cell Signaling) at 4°C overnight with Magnetic Protein G Beads.

Techniques: In Vitro, Binding Assay, Pull Down Assay, Purification, Transfection, Isolation, Construct, Western Blot, Marker, Activation Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Mutagenesis, Nucleic Acid Electrophoresis, Transduction, Immunoprecipitation, Staining, Derivative Assay, Knock-Out, Plasmid Preparation, Stable Transfection, Expressing

Journal: Science (New York, N.Y.)

Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway

doi: 10.1126/science.aau0159

Figure Lengend Snippet: (A) Schematic description of the MYC responsive element on the WWP1 promoter (top). Chromatin level of MYC at the promoter of human WWP1 was measured in DU145 cells. Fold enrichment of MYC was determined by quantitative chromatin immunoprecipitation (qChIP) assays. JunB and RPL30 served as positive and negative controls, respectively. TSS, transcription start site. Data are shown as mean ± SD (***P < 0.0005, **P < 0.005, triplicate experiments, Student’s t test). (B) Reverse transcription–quantitative polymerase chain reaction (RT-qPCR) analysis of WWP1 in DU145 cells expressing the indicated constructs. The mRNA level of WWP1 was determined by RT-qPCR and is presented as a fold increase, as compared with the vector control (−). The HA-MYC levels are indicated by the triangle, from left to right. (C) Analysis of WWP1 and PTEN expression and AKT activation in DU145 cells expressing different amounts of HA-MYC. Total lysates were resolved by SDS-PAGE and then probed with indicated antibodies. The “−” indicates the vector control. (D) Tandem mass spectrum of a peptide derived from endogenous ubiquitinated PTEN in DU145 cells stably expressing the indicated constructs showed ubiquitin conjugation at the K27 residue of ubiquitin. (E) Ratio of indicated ubiquitin linkages detected by MS analysis of endogenous ubiquitinated PTEN purified from MYC overexpression or MYC overexpression–shWWP1 cells to that from control cells (without MYC overexpression). The abundance of each ubiquitin linkage was calculated as described in the materials and methods. (F) Analysis of PTEN K27-linked polyubiquitination in DU145 cells stably expressing MYC and/or WWP1 shRNAs. pCDH-puro-MYC, lentiviral expression of MYC. (G) Analysis of WWP1 and PTEN expression and AKT activation in DU145 cells expressing indicated siRNA SMARTpool. Total lysates were resolved by SDS-PAGE and then probed with indicated antibodies. (H) Analysis of WWP1 and PTEN expression and AKT activation in DU145 cells stably expressing MYC and/or WWP1 shRNAs. (I) Effects of WWP1 with or without PTEN on MYC-induced colony-forming activity in soft agar. The colony numbers are quantified and presented as mean ± SD (***P < 0.0005, **P < 0.005, *P < 0.05, triplicate experiments, Student’s t test). sgRNA, single-guide RNA. (J) Apoptosis assay of DU145 cells stably expressing MYC and/or WWP1 shRNAs. The percentage of apoptotic cells were stained with annexin V–PTEN and 7ADD and then quantified by fluorescence-activated cell sorting. Data are shown as mean ± SD (***P < 0.0005, **P < 0.005, triplicate experiments, Student’s t test). shCon, control. (K) Effects of WWP1 on Ras and MYC-induced colony-forming activity in soft agar or signaling pathway in cells. The colony numbers are quantified and presented as mean ± SD (***P < 0.0005, **P < 0.005, triplicate experiments, Student’s t test) (left). Total lysates were resolved by SDS-PAGE and then probed with indicated antibodies (right). ERK, extracellular signal–regulated kinase; pERK, phosphorylated ERK. (L) Effects of WWP1 on PTEN lipid phosphatase activities in MEFs with indicated constructs. Data are shown as mean ± SD (**P < 0.005, triplicate experiments, Student’s t test). In (C), (F), (H), and (K), actin was used as a loading control.

Article Snippet: The chromatin fractions were incubated in each case with 10 mg of antibodies to one of the following: MYC (Cell Signaling, #13987), human RPL30 or normal rabbit IgG (both provided by Cell Signaling kit, Cell Signaling) at 4°C overnight with Magnetic Protein G Beads.

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Construct, Plasmid Preparation, Activation Assay, SDS Page, Derivative Assay, Stable Transfection, Conjugation Assay, Purification, Over Expression, Activity Assay, Apoptosis Assay, Staining, Fluorescence, FACS

Journal: Science (New York, N.Y.)

Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway

doi: 10.1126/science.aau0159

Figure Lengend Snippet: (A) Gross anatomy of representative urogenital tracts from Hi-Myc; Wwp1+/+ or Hi-Myc; Wwp1−/− mice. The mice were analyzed at 5 months of age. (B) Analysis of the weight of the prostate lobes derived from Hi-Myc; Wwp1+/+ or Hi-Myc; Wwp1−/− mice. The mice were analyzed at 5 months of age (n = 5 mice per genotype). Data are shown as mean ± SD (***P < 0.0005, **P < 0.005, Student’s t test). KO, knockout. (C) Hematoxylin and eosin (H&E) and IHC staining of DLPs from Hi-Myc; Wwp1+/+ or Hi-Myc; Wwp1−/− mice with indicated antibodies. The mice were analyzed at 5 months of age. Scale bar, 50 μm. (D) Western blot analysis of DLP lysates from Hi-Myc; Wwp1+/+ or Hi-Myc; Wwp1−/− mice. The mice were analyzed at 5 months of age (n = 3 mice per genotype). Actin was used as a loading control. CC3, cleaved caspase-3. (E) Confocal analysis of DLPs from indicated mice stained with PTEN (green) and DAPI (blue). The white-outlined areas in the top row are enlarged 2.6-fold (bottom row) to show the subcellular localization of PTEN. Representative pictures are shown (n = 2 mice per genotype). Scale bars, 50 μm. (F) GSEA of RNA-seq data from the DLPs of Wwp1 knockout versus Wwp1 WT mice using the PI3K-Akt signaling pathway gene set annotated in the KEGG. Mice from all four groups contain the same Hi-Myc genetic background unless otherwise noted. NES, normalized enrichment score.

Article Snippet: The chromatin fractions were incubated in each case with 10 mg of antibodies to one of the following: MYC (Cell Signaling, #13987), human RPL30 or normal rabbit IgG (both provided by Cell Signaling kit, Cell Signaling) at 4°C overnight with Magnetic Protein G Beads.

Techniques: Derivative Assay, Knock-Out, Immunohistochemistry, Western Blot, Staining, RNA Sequencing Assay

Journal: Science (New York, N.Y.)

Article Title: Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway

doi: 10.1126/science.aau0159

Figure Lengend Snippet: (A) In silico modeling of predicted interactions with the HECT domain of WWP1. (B) MST analysis determined the Kd of I3C toward His-WWP1 HECT WT or the His-WWP1 HECT F577/Y656A mutant. Concentration is reported in nanomolar. (C) Analysis of the effects of I3C on prostate organoid–forming ability from WT or Hi-Myc mice treated with or without 10 or 20 μM I3C for 3 days. Scale bar, 100 μm. DMSO, dimethyl sulfoxide. (D) Gross anatomy of representative urogenital tracts from Hi-Myc mice treated with vehicle or I3C. The mice, at 5 months of age, were treated intraperitoneally with I3C (20 mg/kg), three times a week for 1 month starting on day 0. Representative pictures are shown in the pane (n = 9 mice per group). (E) H&E and IHC staining of DLPs from Hi-Myc mice treated with vehicle or I3C with indicated antibodies. Scale bar, 50 μm. (F) In vivo ubiquitination analysis of endogenous PTEN ubiquitination levels of DLPs from Hi-Myc mice treated with vehicle or I3C. (G) Western blot analysis of AP, DLP, and VP lysates from Hi-Myc mice, as shown in (D), with the indicated antibodies. Actin was used as a loading control. (H) Confocal analysis of DLPs from indicated mice stained with PTEN (green) and DAPI (blue). The white-outlined areas in the top row are enlarged 2.6-fold (bottom row) to show the subcellular localization of PTEN. Representative pictures are shown (n = 2 mice per genotype). Scale bars, 50 μm. (I) GSEA of RNA-seq data from the DLPs of (i) I3C-treated mice versus vehicle (VEH)–treated mice and (ii) BKM120-treated mice versus vehicle-treated mice using the PI3K-Akt signaling pathway gene set annotated in the KEGG. Mice from all four groups contain the same Hi-Myc genetic background unless otherwise noted. (J) H&E staining of APs from Pten+/− mice treated with vehicle or I3C. At 7.5 months of age, the mice were treated intraperitoneally with I3C (20 mg/kg), three times a week for 1 month starting on day 0. Representative pictures are shown (n = 3 mice per group). Scale bar, 100 μm. (K) Western blot analysis of AP lysates from Pten+/− mice, as shown in (J), with the indicated antibodies. Actin was used as a loading control. (L) Model for WWP1-mediated PTEN K27-linked polyubiquitination in cell growth, tumor development, and progression. Deregulated MYC overexpression or MYC amplification promotes WWP1 expression and, in turn, triggers PTEN K27-linked polyubiquitination. Aberrant K27-linked polyubiquitination suppresses PTEN dimerization, plasma membrane recruitment, and tumor-suppressive function, leading to the tumor initiation and progression. PIP2, phosphatidylinositol 4,5-bisphosphate; u, ubiquitin.

Article Snippet: The chromatin fractions were incubated in each case with 10 mg of antibodies to one of the following: MYC (Cell Signaling, #13987), human RPL30 or normal rabbit IgG (both provided by Cell Signaling kit, Cell Signaling) at 4°C overnight with Magnetic Protein G Beads.

Techniques: In Silico, Mutagenesis, Concentration Assay, Immunohistochemistry, In Vivo, Western Blot, Staining, RNA Sequencing Assay, Over Expression, Amplification, Expressing

Journal: Nutrients

Article Title: Effect of Konjac Glucomannan (KGM) on the Reconstitution of the Dermal Environment against UVB-Induced Condition

doi: 10.3390/nu12092779

Figure Lengend Snippet: The acceleration of hyperpigmentation in UVB-induced senescent HEMns was regulated by KGM in a dose-dependent manner. The senescence of HEMns was induced by UVB irradiation twice followed by cell culturing with various concentrations of KGM. Proteins and melanins were extracted by lysis from each group of HEMns. ( A ) The melanin content was visualized (upper) or quantified by measuring the absorbance (O.D, optical density) at 450 nm and normalized by total cell count numbers (lower) ( n = 3). ( B ) mRNA expression levels were determined by Real-time quantitative polymerase chain reaction (RT-qPCR) using specific Taqman probes for tyrosinase ( TYR) , ( C ) tyrosinase- related protein 1 ( TRP1 ), and TRP2 . The data are presented as mean ± SD (* p < 0.05, ** p < 0.01 compared to CTL ( n = 3): †/‡ p < 0.05, ††/‡‡ p < 0.01 compared to 0 group ( n = 3) [untreated after UVB exposure] for each gene; unpaired Student’s t -test). ( D , E ) Protein expression levels were analyzed by Western blotting using specific antibodies for aging-related factors, p53 and p21CIP, and pigmentation-related factors, TYR, TRP1, and TRP2 and DNA damage marker, gamma-H2A histone family member X (γ-H2AX). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control ( n = 3).

Article Snippet: Total protein (15 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted with anti-H2A histone family member X (H2AX), anti-γ-H2AX, anti-p53, anti-phospho-p53 (Ser15), anti-acetyl-p53 (Lys382), and anti-p21CIP1 antibodies (Cell Signaling Technology, Danvers, MA, USA), and with anti-TYR, anti-TYRP1, anti-TYRP2, and anti-GAPDH antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), respectively.

Techniques: Irradiation, Cell Culture, Lysis, Cell Counting, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Marker, Control

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: GenomeGraph of SKAR as a splice target of TDP-43. HEK293E cells were transfected with control siRNA (scrambled) or treated with siRNA against TDP-43 (siRNA TDP-43 ). Four biological replicates of each group were hybridized on a Human Exon 1.0-ST Gene Chip. Intensity values of microarray hybridizations, single values (gray), mean group intensities of scrambled siRNA (blue) and siRNA TDP-43 (green), are shown as normalized background-corrected logarithmic intensities ( A ) and RMA corrected probe-level data ( B ). Vertical lines separate the 18 individual probe sets covering the POLDIP3/SKAR gene. ( C ) Depicted are the mean group values of the FIRMA score. The fold change of the FIRMA score (FC(F)) is shown in red. ( D ) Genomic representation of the POLDIP3/SKAR gene in orange. Gray lines at the top of this panel indicate localization of the individual probe sets within the genomic coordinates. ( E ) The two Ensembl annotated alternative splice isoforms SKAR α and SKAR β are depicted in blue. SKAR exon 3 is highlighted by a box. ( F ) The SKAR α protein isoform is shown in pink, the RRM domain is shown in dark blue. Highlighted in green is the exon 3 derived part. At the bottom the amino acid sequence of exon 3 is given.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Transfection, Microarray, Derivative Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: Validation of SKAR alternative splicing upon transient silencing of TDP-43. TDP-43 was either silenced transiently by siRNA treatment ( A , C , E and G ) or stably by use of lentiviral particles encoding for a TDP-43-specific shRNA followed by the selection of single cell clones ( B , D and F ). For transient silencing, HEK293E cells were either mock treated (m) or transiently transfected with scrambled control siRNA (scr), with one of four different TDP-43-specific siRNAs (siRNA TDP-43 A-D) or with one of five specific siRNAs against FUS (siRNA FUS A-E), as indicated. (A–D) Total RNA was extracted and analyzed by RT–PCR. (A and B) Semi-quantitative RT–PCR was performed with primer pairs specific for TDP-43, SKAR (ex2–ex4), SKAR α (ex2|3–ex4) and SKAR β (ex2|4–ex4). (C and D) Real-time PCR was performed with primer pairs against SKAR α (ex2|3–ex4) (white bars), SKAR β (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKARα/PBGD, SKARβ/PBGD and total SKAR/PBGD ratios were recalculated into absolute copy values and normalized to total SKAR values. Shown are the mean values of five independent experiments ± SEM. * P < 0.05; ** P < 0.005; *** P < 0.0005; ns = not significant. Original qRT–PCR data is presented in Supplementary Figure S1A and S1B , respectively. (E–G) Protein was extracted, electrophoresed and resulting western blots probed with antibodies specific for TDP-43, SKAR (both isoforms) and SKAR α. GAPDH was used as a loading control. FUS silencing efficiency was controlled by use of an anti-FUS antibody. Note, that, depending on the primer pair and antibody used, SKAR RNA and protein isoforms, respectively, are visualized as two bands with different molecular weights. The upper band represents SKAR α, the lower corresponds to SKAR β, as indicated.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Stable Transfection, shRNA, Selection, Clone Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: SKAR alternative splicing is dependent on RRM1 of TDP-43. ( A ) Stably silenced HEK293E cells (shRNA TDP-43 ) or transiently silenced HEK293 cells (siRNA TDP-43 ) were transiently transfected with either control vector (−) or Flag-TDP-43 variants (wt, ΔRRM1, ΔRRM2, ΔRRM1/2, FFLL and ΔGRD or disease-associated mutations, as indicated). Parental HEK293E cells or cells treated with a scrambled siRNA (−) were used as an internal control. (A) Total RNA was extracted and subjected to semi-quantitative RT–PCR using primer pairs amplifying total TDP-43, endogenous TDP-43, total SKAR (ex2–ex4), SKAR α (ex2|3–ex4), SKAR β (ex2|4–ex4) and PBGD as a housekeeping gene. ( B and E ) RNA was extracted and real-time PCR performed with primer pairs against SKAR α (ex2|3–ex4) (white bars), SKAR β (ex2|4–ex4) (gray bars) and total SKAR (ex5|6–ex7). PBGD was used as a housekeeping gene. Resulting relative SKAR α/PBGD, SKAR β/PBGD and total SKAR/PBGD ratios were re-calculated into absolute copy values and normalized to total SKAR values. Original qRT data is presented in Supplementary Figure S1C and S1D , respectively. * P < 0.05; ** P < 0.005; *** P < 0.0005; ns = not significant. ( C and D ) Protein was extracted, electrophoresed and resulting western blots probed with anti-TDP-43, anti-Flag and anti-SKAR antibodies. GAPDH was used as a loading control. (D) Shown are the mean values ± SEM of densitometric analysis of three independent experiments. * P < 0.05; ** P < 0.005; ns = not significant.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Stable Transfection, shRNA, Transfection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: A repeat containing RNA stretch 3′ of exon 3 is necessary for TDP-43 and SKAR splicing. ( A ) Schematic representation of constructs used for refined RNA crosslinking assays. ( B ) Indicated fragments of SKAR DNA were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt or FFLL. No RNA was added to control samples. Samples were UV crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates (left panel) were probed with anti-TDP-43 and anti-Flag to visualize co-precipitated endogenous and exogenous TDP-43. Biotinylated SKAR RNAs pulled down transfected as well as endogenous TDP-43 wt but not FFLL. Protein inputs (right panel) of HEK293E lysates confirmed even transfection efficiencies. ( C ) Schematic representation of the three repeat motifs and mutagenized variants within the SKAR pre-RNA 3′ of exon 3. ( D ) Non-mutated or mutagenized variants of SKAR DNA part-5 were in vitro transcribed/biotinylated and mixed with lysates form HEK293E cells transiently transfected with Flag-TDP-43 wt. No RNA was added to control samples. Samples were UV-crosslinked and precipitated with streptavidin-agarose. Western blots of streptavidin precipitates were probed with anti-TDP-43 and anti-Flag to visualize coprecipitated endogenous and exogenous TDP-43. ( E ) Schematic representation of the used SKAR minigene construct pTB SKAR part-3/4/5. Primer annealing sites are indicated by arrows. ( F and G ) HEK293E cells were transfected with pTB SKAR part-3/4/5 variants, as indicated. RNA was extracted and used for RT–PCR using primers for pTB and PBGD as a housekeeping gene. (F) Representative RT–PCR is shown. (G) Shown are the results (mean values ± SEM) of densitometric analysis of seven independent experiments calculated as the ratio of SKAR α to SKAR β. * P < 0.05; *** P < 0.0005.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Construct, In Vitro, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: TDP-43 regulates global translational yield by splicing of exon junction complex component SKAR

doi: 10.1093/nar/gkr1082

Figure Lengend Snippet: SKAR β is more active than SKAR α and leads to enhanced translation and increased cell size. ( A ) HEK293E cells were treated with control siRNA or transfected with siRNA against SKAR or TDP-43 as indicated. Stably silenced siRNA TDP-43 and transiently transfected HEK293E cells were transfected with either control vector (−) or plasmids encoding for Myc-SKAR α, Myc-SKAR β or Flag-TDP-43 wt, as indicated. Cells were serum starved for 16 h. After 6 h serum-stimulation cells were harvested, protein extracted and electrophoresed. Resulting western blots were probed with anti-SKAR, anti-phospho S6K1 (Thr389), anti-S6K1, anti-phospho S6 (Ser235/236), anti-S6, anti-phospho Akt substrate (RXRXXS/T) and anti-TDP-43 antibodies. GAPDH was used as a loading control. Transfection of SKAR β or depletion of TDP-43 results in overall stronger phospho-signal compared to SKAR α. ( B ) Schematic representation of luciferase constructs used for analysis of translation. ( C–G ) HEK293E cells were transfected with either Myc-SKAR α or Myc-SKAR β (C) or with control siRNA (scr) and individual siRNA TDP-43 A–D, as indicated (D–G). (C–E) Before DNA/siRNA transfection, cells were transfected with firefly control vector plus either intron-containing or intron-less Renilla luciferase constructs. (C and D) Luciferase activity was measured and normalized to control treated HEK293E cells. Shown are the mean values ± SEM of five independent experiments. * P < 0.05. Western blotting confirmed equal expression of Myc-SKAR α and Myc-SKAR β (C, right panel). (E) qRT–PCR confirmed equal RNA levels of Renilla and firefly luciferase in non-silenced and silenced HEK293E cells. (F) Cells were counted and equal numbers of cells was collected. Protein amount was determined using BCA protein assay. Shown are the mean values ± SEM of five independent experiments. * P < 0.05. (G) Cell size was analyzed by flow cytometry, monitoring the forward scatter parameter. Shown are the mean values ± SEM of five independent experiments. * P < 0.05; ** P < 0.005.

Article Snippet: Moreover, while both isoforms are detected with a total SKAR antibody (CST #3794), isoform β is not recognized by an antibody that has been produced with a synthetic peptide corresponding to human SKAR α (CST #3235).

Techniques: Transfection, Stable Transfection, Plasmid Preparation, Western Blot, Luciferase, Construct, Activity Assay, Expressing, Quantitative RT-PCR, Bicinchoninic Acid Protein Assay, Flow Cytometry

Journal: Journal of Virology

Article Title: The Virion Host Shutoff Protein of Herpes Simplex Virus 1 Blocks the Replication-Independent Activation of NF-?B in Dendritic Cells in the Absence of Type I Interferon Signaling ^▿

doi: 10.1128/JVI.05557-11

Figure Lengend Snippet: HSV-induced DC maturation has type I IFN signaling-dependent and -independent requirements. (A) BM-DCs were generated from C57BL/6 mice and infected with the following: wild-type HSV-1 (strain KOS) (MOI = 5), vhs− mutant virus (MOI = 5), and SeV-HD (strain Cantel) (MOI = 5). At 6 h p.i., cells were harvested, and the quantities of IFN-β and IFN-α in the supernatants were measured by ELISA. The error bars represent the differences between duplicate assays. (B) At 12 h p.i., RNA was extracted from infected cells, reverse transcribed, and assayed for IFN-λ (IL-28a) by qRT-PCR. The error bars represent the differences between triplicate assays. (C) BM-DCs were generated from Sv129 control and type I IFNR KO mice and infected with the following: KOS (MOI = 5) and vhs− virus (MOI = 5). At 24 h p.i., cells were harvested, and the quantities of IL-6, TNF-α, IL-12p40, and IFN-β in the supernatants were measured by ELISA. The error bars represent the differences between duplicate assays. (D) At 6 h p.i., RNA was extracted from infected cells, reverse transcribed, and assayed for IRF7 and Mx1 by qRT-PCR. The error bars represent the differences between triplicate assays. (E) At 24 h p.i., infected cells were assayed for cell surface expression of CD86, CD80, and MHC II by flow cytometry. (F) BM-DCs were generated from Sv129, IFNR KO, and STAT1 KO mice and infected with the following: UV-inactivated KOS and vhs− viruses at an MOI of 5. At 12 h p.i., IL-6 was measured by ELISA. The error bars represent the differences between duplicate assays. *, P < 0.05; **, P < 0.005. NI, not infected.

Article Snippet: Denatured samples were resolved on 10% Tris-Bis gels, transferred to polyvinylidene difluoride (PVDF) membranes, and immunoblotted with antibodies to ΙκΒ-α, phospho-ΙκB-α (Ser32/36), IRF3, and phospho-IRF3 (Ser396) (all obtained from Cell Signaling).

Techniques: Generated, Infection, Mutagenesis, Virus, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Quantitative RT-PCR, Control, Expressing, Flow Cytometry

Journal: Journal of Virology

Article Title: The Virion Host Shutoff Protein of Herpes Simplex Virus 1 Blocks the Replication-Independent Activation of NF-?B in Dendritic Cells in the Absence of Type I Interferon Signaling ^▿

doi: 10.1128/JVI.05557-11

Figure Lengend Snippet: Virion-associated vhs blocks the early replication-independent activation of NF-κB during HSV infection of DCs. BM-DCs were generated from C57BL/6 mice and infected with the following: live and UV-inactivated KOS and vhs− virus (MOI = 5), SeV-LD (LD particle) (MOI = 1.5), SeV-HD (HD particle) (MOI = 1.5), and LPS (300 ng/ml). (A and B) Infected cells were harvested at 1, 3, and 5 h p.i. Denatured protein extracts were resolved by gel electrophoresis and immunoblotted for phospho- and total ΙκΒ-α (A) or phospho- and total IRF3 and GAPDH (B). (C) Infected cell supernatants were assayed for IL-6, TNF-α, and IFN-β by ELISA at the indicated time points. The error bars represent the differences between duplicate assays. (D) BM-DCs were generated from C57BL/6 mice and infected with the following: UV-inactivated KOS virus (MOI = 5), UV-inactivated KOS virus (2×, MOI = 10), UV vhs− virus and SeV-HD (MOI = 1.5), and LPS-treated virus (300 ng/ml). Where noted, UV-inactivated KOS and UV-inactivated vhs− virus-infected cells were also coinfected with SeV or cotreated with LPS. SeV and LPS were added sequentially immediately after HSV. Cells were harvested at 3 h p.i., and denatured protein extracts were resolved by gel electrophoresis. Expression of phospho-ΙκΒ-α and GAPDH was determined by immunoblotting. (E) The intensities of the phospho-ΙκΒ-α bands from three independent experiments were determined by densitometry using the AlphaImager3000. Values were normalized to 100% and graphed. The error bars represent triplicate readings from each of the three experiments. (F) BM-DCs were generated from IFNR KO mice and infected with the following: live and UV-inactivated KOS and vhs− viruses (MOI = 5), NDV (MOI = 1.5), SeV-HD (MOI = 1.5), and LPS-treated virus (300 ng/ml). Cells were harvested at 1 h p.i., and denatured protein extracts were resolved by gel electrophoresis. The expression levels of phospho-IκB-α and GAPDH were determined by immunoblotting.

Article Snippet: Denatured samples were resolved on 10% Tris-Bis gels, transferred to polyvinylidene difluoride (PVDF) membranes, and immunoblotted with antibodies to ΙκΒ-α, phospho-ΙκB-α (Ser32/36), IRF3, and phospho-IRF3 (Ser396) (all obtained from Cell Signaling).

Techniques: Activation Assay, Infection, Generated, Virus, Nucleic Acid Electrophoresis, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Journal of Virology

Article Title: The Virion Host Shutoff Protein of Herpes Simplex Virus 1 Blocks the Replication-Independent Activation of NF-?B in Dendritic Cells in the Absence of Type I Interferon Signaling ^▿

doi: 10.1128/JVI.05557-11

Figure Lengend Snippet: Model describing the modulation of HSV-1-induced DC maturation by the vhs protein. HSV-1 infection of DCs begins with binding of the virus to the necessary receptors expressed on the surface of the cell. The binding of HSV-1 gD to the HVEM receptor has been reported to drive NF-κB activation in response to UV-inactivated virus (step 1). A second mechanistic explanation for the early replication-independent activation of NF-κB has been reported to occur through the virion-associated protein UL37. Whatever the nature of the stimulus, the virion-associated form of vhs can block this signaling event during the first 3 h of the infection. Between 3 and 5 h p.i., a second wave of NF-κB signaling begins. IRF3 is also activated and detectable by 3 h p.i. We have shown that replication-competent HSV-1 is not detected by either TLRs (7) or RLRs/MAVS during infection. A recent report described a DNA sensor for HSV-1 in DCs. If IFI16, present in DCs, detects viral DNA, this would potentially result in the activation of IRF3 and an additional wave of NF-κB (step 2) (62). The release of IFN-β by 3 h p.i. results in signaling through the type I IFN receptor, and this has important implications for the establishment of the antiviral state in the infected cell. The surface expression of costimulatory markers and MHC molecules during HSV-1 infection is dependent on type I IFN signaling (step 3). However, stimulation with the vhs− virus circumvents the requirement for type I IFN signaling for the production of proinflammatory cytokines. We propose that the increase in the stoichiometric availability of NF-κB subunit proteins early during infection with the vhs− virus can explain this phenotype.

Article Snippet: Denatured samples were resolved on 10% Tris-Bis gels, transferred to polyvinylidene difluoride (PVDF) membranes, and immunoblotted with antibodies to ΙκΒ-α, phospho-ΙκB-α (Ser32/36), IRF3, and phospho-IRF3 (Ser396) (all obtained from Cell Signaling).

Techniques: Infection, Binding Assay, Virus, Activation Assay, Blocking Assay, Expressing