Fig. 4: βIII tubulin and FGF12 are differentially regulated in neurites of HC and SCZ by PI3K/GSK3 inhibitors.

A–I Representative images of neurons from healthy controls (HC) and schizophrenia (SCZ) cell lines. i MAP2 (blue), ii β-III tubulin (green), iii FGF12 (red), and iv merge of all three channels with v zoom to ROI used for analysis of vi βIII tubulin and vii FGF12. Squares in panels (ii–iv) represent the ROI used for analysis based on the βIII tubulin fluorescent intensity threshold. A HC neurons treated for 24 h with DMSO control. B SCZ neurons treated for 24 h with DMSO control. C HC neurons treated for 24 h with GSK3 inhibitor CHIR99021. D SCZ neurons treated for 24 h with GSK3 inhibitor CHIR99021. E HC neurons treated for 14 h with GSK3 inhibitor CHIR99021 followed by 10-h treatment with SGK1 inhibitor GSK650394. F SCZ neurons treated for 14 h with GSK3 inhibitor CHIR99021 followed by 10-h treatment with SGK1 inhibitor GSK650394. G HC neurons treated for 24 h with SGK1 inhibitor GSK650394. H SCZ neurons treated for 24 h with SGK1 inhibitor GSK650394. Scale bars represent iv 20 μm or vii 10 μm. I βIII immunofluorescence in neurites is increased in SCZ patients compared to healthy controls when treated for 24 h with DMSO (two-way mixed model ANOVA with Sidak’s multiple comparison’s test, p = 0.0095). Twenty-four-hour treatment with 20 μm GSK3 inhibitor CHIR99021 increases fluorescent intensity in HC neurons, but not SCZ neurons, while 24 h with SGK1 inhibitor GSK650394 decreases fluorescent intensity in SCZ neurons, but not healthy controls (two-way mixed model ANOVA with Dunnett’s multiple comparison’s test, p = 0.0215 (HC GSK3) and p = 0.0124 (SCZ SGK1)). J FGF12 immunofluorescence in neurites is decreased in SCZ neurons compared to healthy controls when treated for 24 h with DMSO (two-way mixed model ANOVA with Sidak’s multiple comparison’s test, p = 0.0107). Treatment with SGK1 inhibitor GSK650394 decreases fluorescent intensity in HC neurons, while 14-h treatment with GSK3 inhibitor CHIR99021 followed by 10-h treatment with SGK1 inhibitor GSK650394 increased fluorescent intensity of FGF12 in SCZ neurons (two-way mixed model ANOVA with Dunnett’s multiple comparison’s test, p = 0.0009 (HC SGK1) and p = 0.0073 (SCZ GSK3 + SGK1)). Data are mean ± SEM. ^# indicates p < 0.05 and ^## indicates p < 0.01 by two-way ANOVA with Sidak’s multiple comparison’s test. * indicates p < 0.05 and ** indicates p < 0.01 by two-way ANOVA with Dunnett’s multiple comparison’s test. Dots represent measurements from single neurons.