Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional, Genetically Encoded, Small Molecule–Regulated Inhibition of NFκB Signaling in RPE Cells

doi: 10.1167/iovs.17-22133

Figure Lengend Snippet: Stabilization of DHFR-IκBα inhibits NFκB signaling. ARPE-19 cells were plated and pretreated with DMSO or dox/TMP (100 ng/mL/1 μM) for 48 hours followed by treatment with IL-1α (25 ng/mL) for 24 additional hours for all experiments. (A) Stabilization of DHFR-IκBα prevents NFκB nuclear translocation after IL-1α stimulation. (B) DHFR-IκBα prevents binding of a fluorescently labeled NFκB consensus oligonucleotide to nuclear NFκB as determined by an EMSA. (C, D) Stabilized DHFR-IκBα reduces transcription of classic NFκB-dependent cytokines IL-1β (C) and IL-6 (D). n = 3. **P < 0.01, unpaired, two-tailed t-test assuming equal variance. (E) Transcription of IL-18, an NFκB-independent cytokine, is not increased by IL-1α, nor is it affected by stabilization of DHFR-IκBα. (F) NFκB TaqMan array of genes that are consistently upregulated or downregulated (≥ or ≤ 2-fold, respectively, P < 0.05), one-sample t-test compared with a hypothetical mean of 1 (i.e., unchanged) in DHFR-IκBα + IL-1α + DMSO versus DHFR-IκBα + IL-1α + dox/TMP. n = 2 independent experiments. Note: TNFSF15 was amplified in only one DHFR-IκBα + IL-1α + dox/TMP sample, whereas TLR2 and VCAM1 were not amplified in either DHFR-IκBα + IL-1α + dox/TMP replicate. Representative data shown of n ≥ 3 independent experiments for (A–E), mean ± SD for all bar graphs in this figure.

Article Snippet: For array-based work, diluted cDNA was applied to a fast 96-well Human NFκB pathway TaqMan array plate (Life Technologies) and run under fast conditions using the master mix described above.

Techniques: Translocation Assay, Binding Assay, Labeling, Two Tailed Test, Amplification