Fig. 3: RSV-induced cholesterol accumulation in lysosomes promotes LDL uptake by activating the SREBP2–LDLR axis.

HEK293T cells transiently expressing pGL3-SRE-LUC and pRL-TK, HEp-2, 16HBE, or HBECs cells were either mock-infected or infected with RSV (MOI = 1) in the presence or absence of U18666A (10 μM) for the indicated durations. a–c Western blotting analysis of SREBP2 in HEp-2 cells 0 h, 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h after RSV infection (n = 3 independent experiments). d The transcriptional activity of SREBP2 in HEK293T cells 0 h, 6 h, 12 h, 24 h, and 48 h after RSV infection was measured via the dual-luciferase reporter assay system (n = 6 independent experiments). e, f Western blotting analysis of LDLR in HEp-2 cells 0 h, 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h after RSV infection (n = 3 independent experiments). g The mRNA level of the LDLR gene in HEp-2 cells 24 h after RSV infection was measured using RT-PCR (n = 3 independent experiments). h, i Exogenous cholesterol (Dil-LDL, 30 μg/mL) uptake in HEp-2 cells 0 h, 1 h, 2 h, 6 h, 12 h, 24 h, and 48 h after RSV infection was determined using flow cytometry (n = 3 independent experiments). j–l Immunocolocalization of Dil-LDL and LAMP-1 in mock-infected, RSV-infected (24 h post-infection), and U18666A-treated cells (HEp-2 (j); 16HBE (k); HBECs (l)). Scale bar: 2 μm. Data are one representative of three independent experiments. Image parameters: pixel size (j, l: 20 nm; k: 25 nm); image size (j, l: 20 × 20 μm², 1000 × 1000 px²; k: 20 × 20 μm², 800 × 800 px²); Objective (j, k, l: 100×, 1.45). Data are shown as the mean ± SD, statistical analysis using two-sided Student’s t-test (d, g) or one-way ANOVA (b, c, f, i) (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared to the blank control group).