Figure 2. An IFN-like Response in HGPS Fibroblasts is Repressed by Calcitriol.

(A) Scheme of the samples analyzed by RNA-seq. NFs derived from three parents of HGPS patients were sequenced. One line of NFs was supplemented with calcitriol (100 nM) or vehicle for 4 days in three biological repeats. HGPS fibroblasts derived from four patients were subjected to prolonged (3 months) and short (4 days) treatments with calcitriol or vehicle. RNA was extracted and sequenced.

(B) Heatmap shows the Gene Ontology (GO) analysis performed with the differential gene expression analysis (FC > 1.5 and < 0.6 and p ≤ 0.05) between HGPS/NF and HGPScalcitriol/vehicle using DAVID and STRING.

(C) Heatmap shows differential expression (upregulation) of ~50 genes in the IFN/antiviral/innate immunity pathway between HGPS/NF and a decrease in HGPScalcitriol/vehicle.

(D) NF and HGPS fibroblasts were processed for immunofluorescence with antibodies recognizing STAT1 and active P-STAT1^Y701 and nuclei stained with DAPI.

(E) Total cell lysates from untreated NFs and HGPS fibroblasts treated with vehicle or calcitriol for 4 days were processed for immunoblotting to monitor global levels of progerin, STAT1, and P-STAT1^Y701. β-Tubulin was used as loading control.

(F) Untreated NFs and HGPS cells treated with calcitriol or vehicle for 4 days were subjected to subcellular fractionation to monitor localization of P-STAT1^Y701 and IRFs. Note the nuclear accumulation of P-STAT1 and IRF3 in HGPS cells and how calcitriol reduces their nuclear levels (red square).

(G) Graph shows mean densitometry analysis (a.u.) of immunoblots from (E) and (F).

* denotes p value of statistical significance (*p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001). Error bars represent SEM.