Fig. 6
From: Mutations in LRRK2 impair NF-κB pathway in iPSC-derived neurons
TNFα-induced NF-κB activation in neurons derived from iPSCs with LRRK2 mutations. a TNFα (10 ng/ml, 8 h) induced a significant NF-κB activation (two-way ANOVA, ** P < 0.01) but only in control WT neurons (* P < 0.05, post hoc test) and not in LRRK2G2019S or LRRK2R1441G neurons (n.s.). Bars represent the mean ± SEM of two to four determinations, including two different lines per genotype and expressed as fold values over untreated. In addition, basal NF-κB activities are normalized to the activity in the control WT neurons. b Schematic representation of NF-κB pathway activation. c Time-course of IκBα protein levels after treatment with TNFα (10 ng/ml). Immunoblots were quantified and normalized to non-stimulated (NS) samples. Each point in the curve is the mean ± SEM of four to five independent experiments, including two different cell lines per genotype. α-tubulin was used as the loading control. Statistical analysis showed a significant effect of time on IκBα protein levels (two-way RT-ANOVA, *** P < 0.0001). d For comparison, the same experiment is shown in fibroblasts. Points represent the mean ± SEM of three to six independent experiments, including at least two different cell lines per genotype. Sam68 was used as the loading control. Statistical analysis showed a significant effect of time on IκBα protein levels (two-way ANOVA, *** P < 0.0001). e Representative immunofluorescence staining of p65 (green) and βIII-tubulin (blue) in mature neuronal cultures incubated with TNFα (15 ng/ml, 0.5 and 2 h). Nuclei were counterstained with Hoechst 33342 (red). Scale bar, 20 μm. f Quantification of p65 immunoreactivity at baseline and g after TNFα incubation showing a significant effect of genotype (two-way ANOVA, *** P < 0.001). Bars represent the mean ± SEM of counts from two different lines per group in two to three independent experiments. NS non-stimulated. See Additional file 5 for uncropped blots