Fig. 2: DF-003 blocks induction of TIFAsome formation and chemokine/cytokine production by the ALPK1 agonist DF-006.
From: Discovery of a selective alpha-kinase 1 inhibitor for the rare genetic disease ROSAH syndrome
a Chemical structues of the ALPK1 agonists ADP-heptose (showing the D-isomer, ADP-D-heptose) and DF-006. b HEK-293 cells were transfected with GFP-TIFA overexpression plasmids. Cells were treated with 10 nM to 1 μM DF-003 or vehicle followed by 1 μM DF-006 stimulation for 1 h. Nuclei are labeled with DAPI (blue), while TIFA is labeled with GFP, and TIFAsomes are represented by punctate green fluorescent structures. c Quantification of the fractions of cells with TIFAsomes among total GFP positive cells as shown in (b). d, e THP-1 cells were treated with PMA for 48 h to induce macrophage differentiation, after which they were incubated for 2 h with serial dilutions of DF-003 dissolved in DMSO. DF-006 was then added for 4 h, and qPCR was used to measure TNF (d) and CXCL8 (e) mRNA expression, with GAPDH serving as a normalization control. Data were expressed as mRNA fold induction compared to THP-1 macrophages not treated with DF-006 activation. Curve fitting and IC50 value calculations were performed with GraphPad Prism 6. In (c) data represent means ± SEM of 5 technical repeats from 2 independent studies. Statistical comparisons between non-DF-003 treated cells with or without DF-006 were made using two-tailed unpaired Student’s t-tests and comparisons between DF-003-treated groups to the vehicle-treated, DF-006-stimulated group were made with one-way ANOVAs followed by Dunnett’s post hoc tests. In d, e data represent means ± SEM for the quadruplicate technical repeat wells at each treatment dose.
