Fig 1: Effect of HIF-1a siRNA TRAIL-induced neuronal apoptosis in vitro. (A) Representative Western blot images of different DcR1 peptide dosage groups. (B) Densitometric quantification of DcR1 of different DcR1 peptide dosage groups. (C) Cell viability after siRNA or peptide treatment. (D) Cell total lactate dehydrogenase (LDH) level after siRNA or peptide treatment. (E) Representative Western blot images of HIF-1a, DcR1, and cleaved caspase-3 after different treatments. (F) Densitometric quantification of HIF-1a. (G) Densitometric quantification of DcR1. (H) Densitometric quantification of cleaved caspase-3. (I) Flow-cytometric analysis of HT22 neuronal cells exposed to siRNA or peptide treatment. A surviving cell was defined as PI-/FITC-. (J) Statistical analysis of cell survival in different groups. N = 3–6 per group. Data are represented as mean ± SD. *p < 0.05 vs. Control; #p < 0.05 vs. Injury + Vehicle; &p < 0.05 vs. Injury + TRAIL; \textdollarp < 0.05 vs. Injury + TRAIL + HIF-1a siRNA. One-way ANOVA, Tukey’s post hoc test.
Fig 2: Exogenous rTRAIL inhibits HTNV infection by enhancing caspase-8-dependent apoptosis. HUVECs were infected with HTNV76-118 (MOI = 1) for 2 h, treated with/without rTRAIL (40 ng/mL), and then followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors and HTNV. (A) HTNV S, TRAIL DR4, DR5, caspase-8, and caspase-3 mRNA expression. (B) HTNV NP and TRAIL-related apoptosis proteins. (C) DcR1/DcR2 mRNA and protein. The mRNA results shown are the average of three replicates and values represented the mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001). The protein result represents one of three similar experiments. Numbers represent the relative density of the bands relative to the internal control. (D) Immunofluorescence images and (E) statistical analysis of TUNEL assay on HTNV-infected HUVECs with rTRAIL treatment. Cells were stained for virus infection [NP (sred)], for apoptosis [TUNEL (green)], and for nucleus [Hoechst (blue)]. Images data showed one of three independent experiments with similar results.
Fig 3: Proposed signaling pathway underlying the effect of HIF-1a-mediated TRAIL-induced neuronal apoptosis after TBI. Microglia increased TRAIL expression after TBI, which activated neuronal DR5 receptor and initiated caspase cascade to apoptosis. Meanwhile, TBI also upregulated HIF-1a expression and further inhibited DcR1 expression. This indirectly increased DR5 function by dismissing the competitive effect of DcR1 to DR5 and then promoted TRAIL-induced neuronal apoptosis.
Fig 4: shRNA-mediated knockdown and rescue of TRAIL influence HTNV replication and apoptosis in HUVECs. HUVECTRAIL-KD cells were infected with HTNV76-118 (MOI = 1) for 2 h, treated with/without rTRAIL (40 ng/mL), and then followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors and HTNV. The mRNA results shown were the average of three replicates. Values represent the mean ± SD (*p < 0.05; **p < 0.01; ***, p 0.001). (A) HTNV S mRNA, (B) TRAIL mRNA, (C) DR4 mRNA, (D) DR5 mRNA, (E) DcR1 mRNA, (F) DcR2 mRNA, (G) caspase-8 mRNA, and (H) caspase-3 mRNA. (I) HTNV NP and TRAIL-related apoptosis proteins in HTNV-infected HUVECTRAIL-KD cells at 2, 3, and 4 dpi. (J) HTNV NP and TRAIL-related apoptosis proteins of HUVECTRAIL-KD cells with rTRAIL rescue. Proteins were quantified using carestream software and compared with the mock group of 2 dpi; the data shown represents one of three similar independent experiments. The numbers represent the relative density of the bands relative to the corresponding control.
Fig 5: HTNV induces TRAIL-dependent apoptosis in primary HUVECs. HUVECs were infected with/without HTNV 76-118 (MOI = 1) for 2 h and followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors. (A) HTNV S, TRAIL, DcR1, DcR2, caspase-8, and caspase-3 mRNA in HTNV-infected HUVECs. (B) HTNV NP and TRAIL-related apoptosis proteins expression in HTNV-infected HUVECs. (C) TRAIL-DR4/DR5 and (D) caspase-9 mRNA and protein expression during HTNV infection. The mRNA results shown are the average of three replicates; values represent the mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001). Proteins were quantified using carestream software and compared with the normal control group of 2 dpi. Data were from one of three experiments with similar results. The numbers represented the relative density of the bands relative to the corresponding control. (E) Histogram showing DR4, DR5, DcR1, and DcR2 surface expression on HTNV-infected and uninfected HUVECs at 2 dpi. White histograms indicate fluorescent labeling with receptor-specific monoclonal antibodies, and gray histograms show background labeling with isotype-matched control antibody in the same group. The experiment was repeated three times and data represent one of three separate experiments. (F) Data showing the MFI of DR4, DR5, DcR1, and DcR2 surface expression on HTNV-infected relative to the uninfected HUVECs at 2 dpi. The MFI of TRAIL receptors was assessed by FlowJo software. The experiment was repeated three times. The dots represented the average of each experiment and data were analyzed by a two-tailed, two-sample student t-test (p-values were indicated).
Supplier Page from Abcam for Anti-DcR1 antibody [EPR6162]