Fig. 5: Forced overexpression of IRG1 activates nuclear factor E2-related factor (2Nrf2) signaling and protects osteoblasts from H₂O₂.

Stable OB-6 cells with the lentiviral IRG1 expression construct (“OE-IRG1” cells) or the empty vector (“Vec” cells) were established, expression of listed genes was tested by qPCR and western blotting analyses (a, c). The itaconate contents (b) and relative ARE reporter luciferase activity (d) are shown. “OE-IRG1” cells or “Vec” control OB-6 cell were treated with H₂O₂ (400 μM) and cultured for applied time periods; reactive oxygen species (ROS) contents (CellROX intensity, e), cell viability (CCK-8 OD, f), cell apoptosis (nuclear TUNEL staining assay, g) were tested; Mitochondrial depolarization was tested by JC-1 green monomers (h), and cell necrosis examined by quantifying medium LDH release (i). Expression of the listed proteins was quantified and normalized to the loading control (a, c). Quantified values were mean ± standard deviation (SD, n = 5). “C” stands for the untreated control cells. *P < 0.05 vs. “C” cells. ^#P < 0.05 vs. H₂O₂ stimulation in “Vec” cells. Experiments were repeated three times, with similar results obtained. Bar = 100 μm (e, g, h).