Fig. 4: Increased Nox4 levels in Parp3-deficient astrocytes cause uncomplete differentiation.

a qPCR for Nox4 and Duox1 Nadph oxidases in Parp3^+/+ and Parp3^–/– NPSCs and d4 astrocytes. Data are expressed relative to Gapdh. Values represent the means ±s.e.m. of three independent experiments and two independent clones. **P < 0.01, ***P < 0.001. b Western blot analysis for the levels of Nox4 and Duox1 relative to β actin in Parp3^+/+ and Parp3^–/– NPSCs and d4 astrocytes. The expression of Gfap relative to β actin confirms impaired astrocyte differentiation in the Parp3^–/– versus the Parp3^+/+ cultures. c The bar graph depicts the relative fold increase of Nox4 levels in astrocytes relative to NPSCs in the Parp3^+/+ and Parp3^–/– genotypes. Values represent means ± s.d. of three independent experiments and two independent clones. **P < 0.01. d The depletion of Nox4 re-establishes astrocyte differentiation in the Parp3^–/– cultures. Western blot analysis for the expression levels of Nox4 relative to β actin and Gfap relative to β actin in sicontrol and siNox4-treated Parp3^+/+ and Parp3^–/– NPSCs and astrocytes d4. e The bar graph depicts the fold increase in the expression of Nox4 (left) and Gfap (right) in the Nox4 silenced (siNox4) relative to the control (sicontrol) Parp3^+/+ and Parp3^–/– NPSC and astrocytes d4. Values represent means ± s.d. of three independent experiments. **P < 0.01. f The depletion of Nox4 restaures the increased levels of mitochondrial ROS observed in the Parp3^–/– astrocytes d6 to the levels detected in Parp3^+/+ astrocytes d6. Measurements of mitochondrial ROS production in sicontrol and siNOX4-treated Parp3^+/+ versus Parp3^–/– NPSC and astrocytes (d4, d6). Values represent means ± s.d. of three biological replicates. **P < 0.01. g Parp3^–/– astrocytes display enhanced nuclear translocation of NF-κB p65. Western blot analysis for the levels of NF-κB p65 subunit in cytoplasmic (CE) and nuclear (NE) extracts of Parp3^+/+ and Parp3^–/– NPSCs and astrocytes d2. Lamin B1 and α tubulin were used as loading controls of NE and CE, respectively. h The bar graph depicts the fold increase in the levels of NF-κB p65 in NE versus CE and relative to Parp3^+/+ NPSC set to 1. Values represent means ± s.d. of three independent experiments. ***P < 0.001. i The depletion of NF-kB p65 restores the expression of GFAP in the Parp3^–/– astrocytes d4 ssuggesting improved differentiation. Western blot analysis for the expression levels of NF-kB p65 and GFAP relative to β actin in sicontrol and sip65^RelA-treated Parp3^+/+ and Parp3^–/– NPSCs and astrocytes d4. j The bar graph depicts the fold increase in the levels of GFAP relative to β actin in sicontrol and sip65^RelA-treated Parp3^+/+ and Parp3^–/– astrocytes d4 and relative to Parp3^+/+ sicontrol set to 1. Values represent means ± s.d. of three independent experiments. **P < 0.01; *P < 0.05.