Journal: Frontiers in Microbiology

Article Title: LncRNA NEAT1 Potentiates SREBP2 Activity to Promote Inflammatory Macrophage Activation and Limit Hantaan Virus Propagation

doi: 10.3389/fmicb.2022.849020

Figure Lengend Snippet: Regulation of the SREBP2 Pathway by NEAT1-2 after HTNV Infection. (A) Immunoblot analysis of total and phosphorylated Stat1/p65 in hMDMs treated with RNAi (MOI = 5). (B) Detection of the transcriptional activity of Stat1 and p65 in hMDMs from (A) . (C) Heatmap of genes involved in cholesterol metabolism of mBMDMs from . (D) Immunoblot analysis of the indicated proteins in hMDMs at 12 hpi with an MOI of 5. (E) Immunofluorescence assays for SREBP2 and HTNV NP in hMDMs at 12 hpi with an MOI of 5. (F) Immunoblot analysis of the indicated proteins in hMDMs treated with RNAi (MOI = 5). (G) qRT-PCR analysis of Srebf1 and Srebf2 in hMDMs from (F) . (H) qRT-PCR analysis of the indicated genes associated with cholesterol synthesis from (F) . Data are shown as the mean ± SEM and are representative of three independent experiments. Each point represents a single sample ( n = 4 in each group). Analysis was performed using the unpaired Student’s t -test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Briefly, HEK 293T cells were transfected with plasmids encoding flag-Stat1 (Sino Biological, HG12766-NF), flag-p65 (Sino Biological, HG12054-NF), flag-SREBP1 (Sino Biological, HG17512- CF ), or flag-SREBP2 (OriGene, RC208942) for 24 h and infected with HTNV at an MOI of 5.

Techniques: Infection, Western Blot, Activity Assay, Immunofluorescence, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: LncRNA NEAT1 Potentiates SREBP2 Activity to Promote Inflammatory Macrophage Activation and Limit Hantaan Virus Propagation

doi: 10.3389/fmicb.2022.849020

Figure Lengend Snippet: NEAT1-2 Promotes SREBP-2-Dependent Inflammation in HTNV-infected Macrophages. (A) qRT-PCR analysis of proinflammatory genes in hMDMs with the indicated treatments. The hMDMs were electrotransfected with pCMV-NEAT1-2 or vectors for 24 h and then infected with HTNV at an MOI of 5 with or without fatostatin (20 μM) treatment. Cells were collected for qRT-PCR at 36 hpi. (B) qRT-PCR analysis of proinflammatory genes in hMDMs with the indicated treatments. The hMDMs were electrotransfected with si-NEAT1-2 and/or plasmids coding N-SREBP2 and then infected with HTNV at an MOI of 5. Cells were collected for qRT-PCR at 36 hpi. (C) Detection of the transcriptional activity of SREBP1 in hMDMs from 0 to 36 hpi. (D) Detection of the transcriptional activity of SREBP2 in hMDMs from 0 to 36 hpi. (E) RIP assays to measure the enrichment of NEAT1-2 by different transcription factors. HEK 293T cells were transfected with plasmids expressing Stat1, p65, SREBP1 or SREBP2 and then infected with HTNV at an MOI of 5. Cells at various time points after HTNV infection were collected for RIP analysis. Data are shown as the mean ± SEM and are representative of three independent experiments. Each point represents a single sample ( n = 4 in each group. Analysis was performed using the unpaired Student’s t -test (A–D) or one-way ANOVA (E) . * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Briefly, HEK 293T cells were transfected with plasmids encoding flag-Stat1 (Sino Biological, HG12766-NF), flag-p65 (Sino Biological, HG12054-NF), flag-SREBP1 (Sino Biological, HG17512- CF ), or flag-SREBP2 (OriGene, RC208942) for 24 h and infected with HTNV at an MOI of 5.

Techniques: Infection, Quantitative RT-PCR, Activity Assay, Transfection, Expressing

Journal: Molecular cancer

Article Title: The homeoprotein DLX4 controls inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer.

doi: 10.1186/s12943-015-0368-3

Figure Lengend Snippet: Figure 3 DLX4 stimulates STAT1 activity and induces iNOS expression in a STAT1-dependent manner. (A) qRT-PCR analysis of relative NOS2 mRNA levels in vector-control ES2 cells and in ES2 cells that expressed wild-type DLX4 or mutant DLX4 (DLX4-TA) with or without dominant-negative STAT1 (STAT1-dn). (B) Vector-control ES2 cells and ES2 cells that expressed wild-type or mutant DLX4 were transfected with a firefly luciferase reporter construct driven by GAS elements (GAS-LUC), stimulated without or with IFN-γ (10 ng/mL) for 16 h and then assayed for luciferase activity. (C) Activity of the GAS-LUC reporter construct was assayed in 2008 cells that expressed non-targeting and DLX4 shRNAs as described in (B). (D) Western blot analysis of levels of total STAT1 and phosphorylated STAT1 in vector-control and +DLX4 ES2 cells that were stimulated with IFN-γ (10 ng/mL) for 0, 1, 6 and 18 h. (E) Lysates of U3A cell lines that lacked or stably expressed GFP-STAT1 fusion protein and/or FLAG-tagged DLX4 fused to GFP were assayed by Western blot using Abs to STAT1 and DLX4. (F) Activity of the GAS-LUC reporter construct in STAT1-deficient U3A cells and in U3A cells reconstituted with STAT1 that lacked or expressed DLX4. Transfected cells were stimulated with or without IFN-γ (10 ng/mL) for 16 h and then assayed for luciferase activity. (G) FLAG Ab was used to pull down FLAG-tagged DLX4 in U3A cells that were stimulated with IFN-γ (10 ng/mL) for 1 h. Immunoprecipitates were analyzed by Western blot using Ab to STAT1. Pulldown using control Ig was included as a negative control. Shown in B, C and F are relative firefly luciferase activities in three independent experiments.

Article Snippet: Other plasmids were as follows: pGFP-V-RS plasmids containing non-targeting and DLX4 shRNAs (OriGene Technologies), pGIPZ plasmids containing NOS2 shRNAs (GE Healthcare), eGFP-STAT1 (provided by Alan Perantoni, National Cancer Institute, Frederick, MD; Addgene plasmid 12301) [48], pRc/CMV-Flag STAT1α Y701F [28] (provided by James Darnell, Rockefeller University, New York, NY; Addgene plasmid 8702).

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Mutagenesis, Dominant Negative Mutation, Transfection, Luciferase, Construct, Western Blot, Stable Transfection, Negative Control