stat1 alpha flag prc cmv  (Addgene inc)

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