Fig 2: 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 Figure 1A. (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.

Fig 3: Promotion of Inflammatory Macrophage Phenotype by SREBP2 after HTNV Infection. (A) RNAi efficiency of silencing Srebf1 (si-BF1) and Srebf2 (si-BF2) in hMDMs confirmed by qRT-PCR. (B) (i) qRT-PCR analysis of M1-related genes in hMDMs at 24 hpi. (ii) qRT-PCR analysis of M2-related genes in hMDMs at 24 hpi. The hMDMs were transfected with siRNAs for 24 h and then infected with HTNV at an MOI of 5. (C) Immunoblot analysis of the indicated proteins in hMDMs from (B). (D) Immunoblot analysis of the indicated proteins in hMDMs that were electrotransfected with plasmids expressing eGFP (as a control) or N-SREBP2 for 24 h and then infected with HTNV at an MOI of 5. (E) qRT-PCR analysis of HTNV S segments in hMDMs from (D). (F) ROS detection in hMDMs from (D). (G) Cytokines/chemokines upregulated by N-SREBP overexpression in HTNV-infected hMDMs at 36 hpi. The results were acquired through BioPlex Multiplex Immunoassays. The hMDMs were acquired and differentiated from seven healthy donors and then electrotransfected with the indicated plasmids for 24 h. The hMDMs from one donor were divided into two groups for the transfection of eGFP and N-SREBP2. Concentration (Y unit), pg/ml. (H) Downregulated cytokines/chemokines as in (G). Concentration (Y unit), pg/ml. (I) Unchanged cytokines/chemokines as in (G). Concentration (Y unit), pg/ml. (J) Immunoblot analysis of HTNV NPs in HUVECs. N-SREBP-overexpressing hMDMs were cocultured with HUVECs as designed in Figure 2A-i and then infected with HTNV at an MOI of 5. Immunoblot assays were performed at various time points after HTNV infection. (K) qRT-PCR analysis of the indicated genes in HUVECs from (J). 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 except G–I). Analysis was performed using the unpaired Student’s t-test (A–F), paired Student’s t-test (G–I), or one-way ANOVA (K). *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 4: A graphical abstract demonstrating the synergistic hepatic toxic effect of doxorubicin (DOX) and small-sized gold nanoparticles (AuNPs) in rats. In the figure, DOX triggers insulin resistance (IR) and lipolysis and the white adipose tissue (WAT), which leads to an increase in the influx of free fatty acid (FFAs) to the livers and induces oxidative stress and inflammation by the generation of reactive oxygen species (ROS) and scavenging glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), suppression of Nrf2, activation of NF-κB p65, and increasing levels of inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, DOX (possibly through increasing hepatic FFAs) can directly stimulate SREBP1 and inhibit PPRAα. On the other hand, AuNPs affect liver health and lipogenesis by acting on oxidative stress and inflammatory markers (similar to the effect of DOX) as well as inhibiting SREBP1.

Fig 5: 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.