Journal: Molecular Therapy. Nucleic Acids
Article Title: Antisense oligonucleotide targeting the E3 ligase RFFL potentiates CFTR modulator efficacy in CF primary bronchial epithelial cells
doi: 10.1016/j.omtn.2025.102756
Figure Lengend Snippet: RFFL ASO 3074B improves the efficacy of CFTR modulators in CFBE cells (A) Western blot analysis of ΔF508-CFTR-3HA in CFBE Tet-on cells transfected with 20 nM negative control ASO (NEG) or RFFL ASO 3074B (#37). Cells were treated with either 0.3% DMSO or ETI (1 μM ELX, 3 μM TEZ, 1 μM IVA) for 2 days at 37°C. The immature (B and B) and mature (B and C) forms of CFTR were quantified and normalized to NEG. Ponceau staining served as a loading control. The asterisk denotes a non-specific band. (B and C) Effects of RFFL ASO 3074B on ΔF508-CFTR PM levels (B, n = 27) and cell viability (C, n = 27) in CFBE-ΔF508-CFTR-HRP cells following lipofection with 20 nM ASOs. ΔF508-CFTR PM expression was induced by ETI treatment for 2 days at 37°C, as shown in A. (D) PM stability of ΔF508-CFTR-3HA in CFBE Tet-on cells transfected with either NEG or RFFL ASO 3074B ( n = 4). Cells were treated with ETI for 2 days at 37°C prior to analysis, as shown in A. (E) The channel function of ΔF508-CFTR-3HA in CFBE Tet-on cells transfected with 50 nM NEG or ASO 3074B was assessed using the halide-sensitive YFP quenching assay. The initial YFP quenching rate was quantified as CFTR channel activity (right, n = 6). CFTR expression was induced by Dox (1 μg/mL) treatment for 4 days, and cells were pre-treated with ETI for 2 days at 37°C prior to the assay, as described in A (+ETI). (F and G) TLR9-stimulatory activity and cell viability of RFFL ASO 3074B in HEK-Blue hTLR9 cells. HEK-Blue hTLR9 cells were treated with RFFL ASO 3074B, or the positive control SY-ODN18 (ODN18), at various concentrations for 18 h. TLR9 activity was then measured and normalized to the untreated control (F). Cell viability was assessed by the WST-8 assay and similarly normalized (G). Data are presented as mean ± SD ( n = 3). (H) Western blot analysis demonstrates sustained RFFL KD following a single transfection of RFFL ASO in CFBE-ΔF508-CFTR-3HA cells. Cells were transfected with 20 nM of the indicated ASO. Endogenous RFFL protein levels were quantified by densitometry and expressed as a percentage relative to NEG ASO-transfected cells ( n = 6). Ponceau staining served as a loading control. (I) PM levels of ΔF508-CFTR-HRP in CFBE Tet-on cells transfected with 20 nM NEG or ASO 3074B were measured at 6, 10, and 14 days post-transfection. CFTR expression was induced with Dox (1 μg/mL) for 4 days, and ETI (1 μM ELX, 3 μM TEZ, 1 μM IVA) was applied for 2 days prior to the HRP assay ( n = 10–12). Data represent mean values unless otherwise specified. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test (B and C) or a two-tailed unpaired t test (D, E, H, I). For data showing a significant effect, the corresponding p value is indicated in the figure.
Article Snippet: As previously described, HEK-Blue hTLR9 cells were seeded at a density of 5 × 10 4 cells per well onto 96-well plates (Corning, Corning, NY) in DMEM supplemented with HEK-Blue Detection (Invivogen, San Diego, CA).
Techniques: Western Blot, Transfection, Negative Control, Staining, Control, Expressing, Activity Assay, Positive Control, Comparison, Two Tailed Test
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