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. 2019 Apr 18;10:850. doi: 10.3389/fimmu.2019.00850

Figure 1.

Figure 1

TonEBP reduces expression of HO-1 both in human and murine macrophages. (A) Peritoneal macrophages (PM) were obtained from non-diabetic (Veh, n = 5) and streptozotocin-induced diabetic (STZ, n = 8–9) TonEBP+/Δ and TonEBP+/+ mice (34). The abundance of HO-1 mRNA was measured by quantitative RT-PCR. Mean + SEM. (B,C) PM (B) and bone marrow-derived macrophages (BMDM) (C) obtained from TonEBP+/+ or TonEBP+/Δ mice were cultured in normal glucose (5.5 mM), high glucose (25 mM), or 5.5 mM glucose + 19.5 mM mannitol (osmotic control for high glucose) for 24 h and then treated with LPS (100 ng/ml) for 6 h. Quantitative RT-PCR was performed to measure expression of mRNA encoding TonEBP and HO-1. (D) RAW264.7 cells were transfected with scrambled [Scr (-)] or two siRNAs (Ton #1 or Ton #2) targeting different regions of mouse TonEBP mRNA for 24 h. Immunoblotting to detect TonEBP, HO-1 and Hsc70 was performed. (E–G) RAW264.7 cells transfected with scrambled (Scr) siRNA or siRNA targeting TonEBP (Ton) for 24 h. (E) Transfected cells were further cultured for 24 or 72 h, followed by immunoblotting to detect TonEBP, HO-1, and Hsc70. (F) Transfected cells were treated with vehicle (Con) or LPS (100 or 1,000 ng/ml) for 24 h and immunoblotted with antibodies specific for TonEBP, HO-1, HO-2, and Hsc70. (G) Transfected cells were treated with LPS (100 ng/ml) for 6 or 18 h, and abundance of HO-1 mRNA was measured by quantitative RT-PCR. (H) RAW264.7 cells infected with adenovirus expressing TonEBP (Ad-TonEBP) or with empty vector (Ad-EV) at an MOI of 50 for 24 h and then treated with LPS for 6 h, followed by immunoblotting to detect TonEBP and quantitative RT-PCR to detect HO-1 mRNA. (I) Human peripheral blood monocyte-derived macrophages were transfected for 48 h with Scr siRNA or siRNA targeting TonEBP and then treated with LPS (100 ng/ml) for 6 h. Quantitative RT-PCR to measure HO-1 mRNA was performed. (J,L) Human PMA-differentiated THP-1 cells transfected for 24 h with Scr siRNA or siRNA targeting TonEBP. (J,K) Transfected cells were treated with vehicle (Con) or LPS as indicated. The abundance of mRNA encoding HO-1 was measured by quantitative RT-PCR. (L) Transfected cells were treated with vehicle (Con) or LPS (100 ng/ml) for 24 h, followed by immunoblotting to detect TonEBP, HO-1, and Hsc70. (A–C,G–K) Two-way ANOVA with Tukey's post-hoc test was used for multiple comparisons. Different letters indicate statistical differences at P < 0.05. (B,C,G–K) Data (mean + SD) were from three independent experiments (n = 3) each with more than three replicates. (D–F,L) Data are representative of three independent experiments. AU, arbitrary units.