Figure 1.
Loss-of-function PPA1 analysis in N1E115 cells.
(A and B) Mouse PPA1 knock-down in N1E115 cells using siRNA targeted to mouse PPA1 (si-PPA1). si-RNA targeted to luciferase (si-Luc) was used as a control. Representative RT-PCR (A) and quantitative analysis of PPA1 expression using real time PCR (B). Representative Western blot analysis (C) and quantitative analysis of PPA1 protein (D). Values are the fold increase relative to the si-Luc, with its value arbitrarily set to 1 (n = 5). (E) Representative N1E115 cells treated with si-RNA targeted to mouse PPA1 and targeted to luciferase as a control (E). (F) Quantitative analysis of the neurite growth ratio is shown. Neurite growth is determined by morphological analysis as described in the Experimental procedure. (n = 100) (G) N1E115 cell proliferation. Cell proliferation was measured using bromodeoxyuridine (BrdU) incorporation into the cells (n = 5). *p<0.05 vs. si-Luc.
Figure 2.
Gain-of-function PPA1 analysis in N1E115 cells.
(A and B) Mouse PPA1 overexpression in N1E115 cells using adenoviral vector (AD-PPA1). An adenoviral vector containing green fluorescence protein (GFP) was used (AD-GFP) as a control. Representative RT-PCR (A) and quantitative PPA1 expression analysis using real time PCR (B). Representative Western blot analysis (C) and quantitative analysis of PPA1 protein (D). Values are the fold increase relative to the AD-GFP, with its value arbitrarily set to 1 (n = 5). (E) N1E115 cell proliferation was measured using bromodeoxyuridine (BrdU) incorporation into the cells (n = 5). (F) Typical morphological changes of in N1E115 cells treated with 1 mM valproric acid (VPA), a stimulator of neuronal differentiation in N1E115 cells, by using AD-GFP and AD-PPA1. Overexpression of AD-GFP was used as a control. (G) Quantitative analysis of the neurite growth ratio is shown. Neurite growth is determined by morphological analysis as described in the Experimental procedure (n = 100). *p<0.05 vs. AD-GFP.
Figure 3.
Role of pyrophosphatase activity on PPA1-induced inhibition of neuronal differentiation in N1E115 cells.
(A) Recombinant wild-type PPA1 (his-PPA1) and PPA1 Asp117Ala (his-PPA1 D117A) proteins. Western blot using anti-PPA1 antibody showed that the his-PPA1 and his-PPA1 D117A protein amount is similar. (B) Recombinant protein pyrophosphatase activity. Pyrophosphatase activity was seen in his-PPA1 while no activity was detected in his-PPA1 D117A (n = 5). (C) Effect of either green fluorescence protein (AD-GFP), wild-type PPA1 (AD-PPA1), or PPA1 D117A (AD-PPA1 D117A) overexpression using adenoviral vector, on PPA1 protein levels in N1E115 cells treated with 1 mM valproic acid (VPA). (D) Typical morphological changes in N1E115 cells treated with VPA, using AD-GFP overexpression as a control (VPA+GFP), AD-PPA1 (VPA+AD-PPA1) or D117A (VPA+AD-PPA1 D117A). (E) Quantitative analysis of the neurite growth ratio is shown. Neurite growth is determined by morphological analysis as described in the Experimental procedure (n = 100). *p<0.05 vs. his-PPA1, #p<0.05 vs. N1E115 cells treated with VPA+GFP, a<0.05 vs. VPA+Ad-PPA1.
Figure 4.
Phosphorylated protein kinase levels in N1E115 cells.
(A–E) Representative Western blots using anti-phospho-JNK and JNK (A), phospho-AKT and AKT (B), phospho-ERK and ERK (C), phospho p38 and p38 (D) and GAPDH (E). N1E115 cells were treated with si-RNA targeted to mouse PPA1 (si-PPA1) or treated with adenovirus containing mouse PPA1 (PPA1). Si-RNA targeted to luciferase (si-luc) was used as a si-PPA1 control and adenovirus containing GFP (GFP) was used as a control for adenovirus containing mouse PPA1. (F–I) Quantitative analysis of the phosphorylated JNK/JNK ratio (F), pAKT/AKT ratio (G), pERK/ERK ratio (H) and p-p38/p38 ratio (I) are shown. (J and K) Direct effects of recombinant PPA1 (his-PPA1) or PPA1 D117A (his-PPA1 D117A) proteins on the phosphorylated JNK level immunoprecipitated from N1E115 cells. As a control, buffer without the recombinant protein was added (buffer). (n = 5) Representative Western blot using anti-phospho-JNK and JNK (J) and quantitative analysis of the phosphorylated JNK/JNK ratio (K) (n = 5). (L and M) Phosphorylated paxillin level in N1E115 cells. Representative Western blot using anti-phospho-paxillin and paxillin (L) and quantitative analysis of the phosphorylated paxillin/paxillin ratio (M) (n = 5). *p<0.05 vs. si-luc, #p<0.05 vs. GFP, a<0.05 vs. buffer, and b<0.05 vs. his-PPA1.
Figure 5.
Modification of PPA1 in rat cortical neurons.
(A) Representative Western blot analysis (A) and quantitative analysis of PPA1 protein (B). PPA1 knock-down in rat cortical neurons using siRNA targeted to rat PPA1 (si-PPA1). As a control, si-RNA targeted to luciferase was used (si-luc) (n = 5). (C) A representative rat cortical neuron treated with si-RNA targeted to rat PPA1 and targeted to luciferase as a control. (D–E) Quantitative analysis of neurite growth in rat cortical neurons. Neurite growth is determined by morphological analysis as described in the Experimental procedure (n = 100). (F) GFP overexpression in rat cortical neurons using adenovirus containing GFP. (G and H) Representative Western blot analysis (G) and quantitative analysis of PPA1 protein (H). PPA1 overexpression in rat cortical neurons using adenovirus containing mouse PPA1 (PPA1). As a control, adenovirus containing GFP was used (GFP) (n = 5). (I) Representative rat cortical neurons treated with PPA1 adenovirus and GFP virus as a control. (J and K) Quantitative analysis of neurite growth from rat cortical neurons (n = 100). (L and M) A representative rat cortical neuron treated with 10 µM SP600126, an inhibitor of JNK (SP600125), and vehicle as a control (vehicle) (n = 5).*p<0.05 vs. si-luc, #p<0.05 vs. GFP. ap<0.05 vs. vehicle.