Fig. 5. UDPG-P2Y₁₄ pathway regulates STAT1 expression.

a Stat1 siRNA transfected BMDMs were stimulated with IFN-γ/LPS for 24 h, Nos2, Tnf, Il6, and Il1b expression was determined by real-time PCR. b Pgm1, Ugp2, Pygl or P2ry₁₄ siRNA transfected BMDMs were stimulated with IFN-γ/LPS ± UDPG for 24 h, Stat1 expression was determined by real-time PCR. c–f BMDMs were pretreated with inhibitor (GPI or 6AN) for 30 min or pre-transfected with siRNA (Pygl or G6pdx) for 24 h prior to stimulation with IFN-γ/LPS for 24 or 36 h, STAT1, ZNF-148, IRF-1, and RARβ expression was determined by western blot (c, d), RARβ expression and location were analyzed by real-time PCR (e) and confocal microscope, scale bar, 10 μm (f). g–i P2ry₁₄ siRNA or P2Y₁₄-overexpression vectors (P2Y₁₄-OE) transfected BMDMs were stimulated with IFN-γ/LPS for 24 or 36 h, RARβ expression and location were determined by real-time PCR (g, h) and confocal microscope, scale bar, 10 μm (i). j, k IFN-γ/LPS stimulated BMDMs were treated with UDPG for 24 or 36 h, RARβ and STAT1 expression was determined by real-time PCR (j) and western blot (k). l–q RARβ-overexpression vectors (RARβ-OE) or Rarb siRNA transfected BMDMs were stimulated with IFN-γ/LPS for 24 or 36 h, Stat1, Nos2, Tnf, Il6, and Il1b expression was determined by real-time PCR (l, o), RARβ, STAT1, iNOS, TNF, and IL-6 expression was determined by western blot (m, p) and ELISA (n, q). r Schematic representation of the promoter region on the upstream of the transcription start site of Stat1. BMDMs were pretreated with GPI or 6AN for 30 min prior to stimulation with IFN-γ/LPS for 24 h, RARβ enrichment around the promoter of Stat1 were analyzed by ChIP-PCR and IgG was used as a negative control. ChIP-qPCR were used to Stat1 quantitative detection and total genomic DNA was used as input. Data are presented as mean ± SEM of n = 3 biologically independent experiments (a, b, e, g, j, l, n, o, q) or n = 4 biologically independent experiments (h) or n = 12 from two independent experiments (r). P values were calculated using one-way ANOVA (a, b, e, g, j, o, q, and r) and two-tailed unpaired Student’s t-tests (h, l, and n).