Journal: International Journal of Molecular Sciences

Article Title: TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons

doi: 10.3390/ijms21082735

Figure Lengend Snippet: Impaired SLC8A3 expression by loss of TGF-β signaling. ( A – D ): Immunoperoxidase light microscopy for SLC8A3 on fixed coronal sections from the mouse midbrain ( A , B ) and ventral hindbrain (vH; C , D ) of wild type ( wt) ( A , C ) and conditional knock out ( cKO) ( B , D ) at embryonic day 16 shows a decreased labelling intensity in the area of midbrain dopaminergic neurons (a1, a2, b1, and b2 are a higher magnification of the black-boxed areas in A and B ) and hindbrain dopaminergic neurons (c1, c2, d1, and d2 are a higher magnification of the black-boxed areas in C and D ) in cKO , compared to wt . ( E ) Quantification of SLC8A3-positive cells in the Engrailed 1 area/mm 2 . ** p < 0.01, using the two-tailed unpaired Student’s t -test, n = 4/genotype).

Article Snippet: Primary antibodies were diluted: Rabbit polyclonal antibody anti- SLC8A3 (NCX3 Cat# orb11106, Biozol,) 1:1000, rabbit polyclonal anti-TβRII (C-16) 1:1000 (Cat# Sc-220, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-GAPDH 1:20,000 (Cat# ab8245, Abcam, Cambridge, UK).

Techniques: Expressing, Light Microscopy, Knock-Out, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons

doi: 10.3390/ijms21082735

Figure Lengend Snippet: SLC8A3 and Bcl-xL expression correlate in developing Engrailed 1-derived midbrain and hindbrain neurons. ( A – D ) Confocal double immunofluorescent representative labelling of SLC8A3 (red) and the anti-apoptotic marker Bcl-xL (green) in primary TβRII flox/flox ::En1 cre/+ neuronal ventral midbrain/hindbrain cultures at E14 show either high or low SLC8A3 expression and high or low Bcl-xL expression. Line scan (1) is a representative image of a neuron with high SLC8A3 expression and high Bcl-xL expression (neuron 1 in the magnification of the white boxed area). Line scan (2) depicts neurons with high SLC8A3 expression while Bcl-xL is low (neuron 2 in the magnification of the white boxed area). Line scan (3) shows low cellular SLC8A3 abundance together with low Bcl-xL (neuron 3 in the magnification of the white boxed area). Arrowheads correspond to cells 1-3. ( E ) Quantification of number of cells (%) based on high/low SLC8A3 and high/low Bcl-xL expression (total: 96 cells from 3 independent experiments).

Article Snippet: Primary antibodies were diluted: Rabbit polyclonal antibody anti- SLC8A3 (NCX3 Cat# orb11106, Biozol,) 1:1000, rabbit polyclonal anti-TβRII (C-16) 1:1000 (Cat# Sc-220, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-GAPDH 1:20,000 (Cat# ab8245, Abcam, Cambridge, UK).

Techniques: Expressing, Derivative Assay, Marker

Journal: International Journal of Molecular Sciences

Article Title: TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons

doi: 10.3390/ijms21082735

Figure Lengend Snippet: Differential regulation of SLC8A3 in Nestin-positive cells and neurons by TGF-β. ( A ) Protein abundance of SLC8A3 by immunoblotting in control MN9D cells in the presence of exogenous recombinant TGF-β (2 ng/mL), 30 µg protein was loaded per lane. ( B ) Quantification, not significant after densitometric analysis of the signal ratio SLC8A3:GAPDH and two-tailed unpaired Student’s t -test, n = 3. The value of control (ctl) was set to 1. ( C – F″ ): Confocal double immunofluorescence for SLC8A3 (red) with the stem cell marker Nestin (green; C’ , D’ ) or the neuronal marker β-III tubulin (green; E’ , F’ ) in MN9D cells in the presence ( D , F ) or absence ( C , E ) of TGF-β. ( G ) Quantification of SLC8A3 immunofluorescence intensity in Nestin-positive cells and β-III-tubulin-positive cells in controls and following TGF-β treatment. ** p < 0.01, using two-tailed unpaired Student’s t -test, 30–100 cells/experimental group from 3 to 7 independent experiments. The value of control (ctl) was set to 1.

Article Snippet: Primary antibodies were diluted: Rabbit polyclonal antibody anti- SLC8A3 (NCX3 Cat# orb11106, Biozol,) 1:1000, rabbit polyclonal anti-TβRII (C-16) 1:1000 (Cat# Sc-220, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-GAPDH 1:20,000 (Cat# ab8245, Abcam, Cambridge, UK).

Techniques: Quantitative Proteomics, Western Blot, Control, Recombinant, Two Tailed Test, Immunofluorescence, Marker

Journal: International Journal of Molecular Sciences

Article Title: TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons

doi: 10.3390/ijms21082735

Figure Lengend Snippet: TGF-β canonical pathway and p38 pathway regulate basal SLC8A3 levels. Confocal double immunofluorescence for SLC8A3 (red) with the neuronal marker β-III tubulin (green) in control (ctl; untreated) ( A – E ) and TGF-β-treated ( F – J ) MN9D cells for 60 min following application of the inhibitor of Smad3 SIS3 (3 µM; B – B‴ , G – G‴ ), or the MAP kinase inhibitor PD98059 (25 µM; C – C‴ , H – H‴ ), or the p38 inhibitor SB239063 (10 µM; D – D‴ , I – I‴ ), or the inhibitor of JNK signaling SP600125 (10 µM; E – E‴ , J – J‴ ), ( K ) Quantification of SLC8A3 fluorescence intensity, * p < 0.05, ** p < 0.01, using the two-tailed unpaired Student’s t-test or one-way ANOVA and Bonferroni post hoc test, total 979 cells analyzed from 5 independent experiments. The values of control were set to 1.

Article Snippet: Primary antibodies were diluted: Rabbit polyclonal antibody anti- SLC8A3 (NCX3 Cat# orb11106, Biozol,) 1:1000, rabbit polyclonal anti-TβRII (C-16) 1:1000 (Cat# Sc-220, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-GAPDH 1:20,000 (Cat# ab8245, Abcam, Cambridge, UK).

Techniques: Immunofluorescence, Marker, Control, Fluorescence, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons

doi: 10.3390/ijms21082735

Figure Lengend Snippet: Smad4 specifically binds to the promoter of Slc8a3 . ( A ) Smad4 binding site(s) (red) to the Slc8a3 promoter region. Underlined sequences represent primer sequences used to detect Smad4 binding. ( B ) PCR following the chromatin immunoprecipitation assay of the control and TGF-β-treated (2 ng/mL for 30 min) Lund human mesencephalic (LUHMES) cells demonstrates increased binding of Smad4 to the predicted binding sequence in the promoter region of Slc8a3 . Predicted PCR product size 193 bp. Histone 3 (H3) and mouse IgG represent the positive and negative control, respectively. The figure is a representative of n = 2.

Article Snippet: Primary antibodies were diluted: Rabbit polyclonal antibody anti- SLC8A3 (NCX3 Cat# orb11106, Biozol,) 1:1000, rabbit polyclonal anti-TβRII (C-16) 1:1000 (Cat# Sc-220, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-GAPDH 1:20,000 (Cat# ab8245, Abcam, Cambridge, UK).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Control, Sequencing, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: TGF-β Signaling Regulates SLC8A3 Expression and Prevents Oxidative Stress in Developing Midbrain Dopaminergic and Dorsal Raphe Serotonergic Neurons

doi: 10.3390/ijms21082735

Figure Lengend Snippet: Knockdown of Slc8a3 increases oxidative stress. ( A – D ) Efficiency of SLC8A3 knockdown. SLC8A3 immunofluorescence in MN9D cells that were either non-transfected (A, untransfected), treated with the transfection agent only (B, HiPerFect), transfected with negative siRNA (C, green, Neg si ), or with a specific siRNA against Slc8a3 (green in D). SLC8A3 protein showed no differences between the controls and the cells transfected with negative siRNA but was significantly downregulated in cells transfected with specific Slc8a3 siRNA (magnification of the white boxed areas). ( E – E‴ ) Immunofluorescence for the lipid peroxidation marker malondialdehyde (MDA) was comparable between MN9D cells transfected with negative si RNA (green arrows) and non-transfected cells (white arrow). ( F – F‴ ) MDA immunofluorescence was increased in cells transfected with specific siRNA against Slc8a3 . Green arrows indicate successfully transfected cells with specific siRNA against Slc8a3, white arrow points to a cell from the same culture that failed to incorporate Slc8a3 siRNA.

Article Snippet: Primary antibodies were diluted: Rabbit polyclonal antibody anti- SLC8A3 (NCX3 Cat# orb11106, Biozol,) 1:1000, rabbit polyclonal anti-TβRII (C-16) 1:1000 (Cat# Sc-220, Santa Cruz Biotechnology, Dallas, TX, USA), and mouse monoclonal anti-GAPDH 1:20,000 (Cat# ab8245, Abcam, Cambridge, UK).

Techniques: Knockdown, Immunofluorescence, Transfection, Marker