Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Effect of UTP and suramin on I A of small-diameter and FG-labeled TG neurons, with diameters ranging from 18 to 39 μm in control rats. (A) Fluorescence microscopic view of TG neurons from control rats. (a) Retrograde labeling of TG neurons (blue) innervating whisker pad skin. (b) P2Y 2 receptor-positive (green) TG neurons were seen in the section of TG. (c) The merged images (purple) of retrograde labeling of TG neurons and P2Y 2 receptor-positive TG neurons from the same section, indicating co-localization. (d) Retrograde labeling of TG neurons (blue) innervating whisker pad skin in cultured TG neurons. (B) Electrophysiology recording for small-diameter and FG-labeled TG neurons in control rats. (a) Representative traces showing that the application of 30 μM UTP reduced I A . Suppression of the mean peak amplitudes of I A seen after UTP application was antagonized by suramin 100 μM. (b) Current–voltage relationship for the effects of UTP and suramin on I A . Each value represents the mean ± SEM (con: 0.14 ± 0.01 nA, n = 9; UTP: 0.09 ± 0.01 nA, n = 20, p < 0.05 vs control; suramin: 0.13 ± 0.01 nA, n = 9). I A was initiated via a prepulse (100 ms) of −120 mV and test pulses (400 ms) from −60 to +60 mV in a 10 mV step.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Labeling, Fluorescence, Whisker Assay, Cell Culture

Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Effect of UTP on the expression levels of I A subunits in control TG neurons. (A) Double-immunostaining revealed the expression of Kv1.4, Kv3.4, Kv4.2 and Kv4.3 subunits in P2Y 2 receptor-positive neurons in the ION-CCI TG sections. The P2Y 2 receptor-positive TG neurons also expressed Kv1.4, Kv3.4, Kv4.2 and Kv4.3, respectively, n = 5 rats. (B) Reduction in the mRNA levels of I A subunits by UTP in cultured TG neurons from control rats. Treatment with suramin (100 μM) in the UTP-incubated (30 μM) TG neurons for 16 h in control rats reversed the decrease in the mRNA levels of Kv1.4, Kv3.4, Kv4.2, and Kv4.3 subunits. n = 10 samples in each group, *, p < 0.05, **, p < 0.01 vs control; ## , p < 0.01 vs UTP.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Expressing, Double Immunostaining, Cell Culture, Incubation

Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: The role of P2Y 2 receptors in mechanical hyperalgesia in ION-CCI rats. (A) The peripheral target injection to TG of suramin (0.3-3 μg/μl) reduced mechanical allodynia in the whisker pad. n = 6-8, **, p < 0.01 compared with injection of saline, ## , p < 0.01 compared with injection of high-dose suramin. Suramin led to a time- and dose-dependent increase in PWT, this anti-allodynia effect started 10 min after the suramin injection and remained for at least 45 min. (B) The peripheral target injection to TG of P2Y 2 antisense oligodeoxynucleotides significantly alleviated mechanical allodynia of the whisker pad. n = 5, *, p < 0.05, **, p < 0.01 compared with injection of saline. The effect started at 6 h and persisted for at least 120 h. (C) Western blots showed successful suppression of P2Y 2 receptor expression in TG by P2Y 2 receptor antisense oligodeoxynucleotides treatment n = 4 for each group, **, p < 0.01.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Injection, Whisker Assay, Western Blot, Expressing

Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Difference of I A channel expression in TG between sham and ION-CCI rats. (A) Double-immunostaining for P2Y 2 receptors and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3 on TG neurons in sham and ION-CCI sections, respectively. (B) Percentages of numbers of Kv1.4, Kv3.4, Kv4.2 and Kv4.3 subunits in P2Y 2 receptor-positive neurons are significantly decreased in TG from ION-CCI rats compared with sham rats. n = 4 rats, **, p < 0.01). (C) Changes in the mRNA levels of I A subunits in TG after P2Y 2 receptor antisense oligodeoxynucleotides treatment. The mRNA levels of Kv1.4, Kv3.4 and Kv4.2 were significantly decreased in the saline group of ION-CCI rats compared with the sham rats. They were reversed after P2Y 2 receptor antisense oligodeoxynucleotides treatment. n = 5-9 rats, *, p < 0.05, ** p < 0.01 compared with saline groups. There was no difference in the levels of Kv4.3 mRNA among the groups. n = 6-8 rats, p > 0.05.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Expressing, Double Immunostaining

Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Role of ERK pathway in activation of P2Y 2 receptors mediates an inhibition of I A channels on small-diameter TG neurons in control rats. (A) Comparison of the phosphorylation of ERK1/2 in TG from sham and ION-CCI rats. Western blot results showed that the level of ERK1/2 phosphorylation was significantly increased in the ipsilateral TG after ION-CCI, compared with that from the sham group. n = 5 for each group *, p < 0.05. (B) In TG from ION-CCI rats, treatment with P2Y 2 receptor antisense oligodeoxynucleotides (15 μg/50 μl) significantly decreased the expression of ERK protein in TG. n = 5 for each group, ** , p < 0.01.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Activation Assay, Inhibition, Western Blot, Expressing

Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Effect of UTP and suramin on I A of small-diameter and FG-labeled TG neurons, with diameters ranging from 18 to 39 μm in control rats. (A) Fluorescence microscopic view of TG neurons from control rats. (a) Retrograde labeling of TG neurons (blue) innervating whisker pad skin. (b) P2Y 2 receptor-positive (green) TG neurons were seen in the section of TG. (c) The merged images (purple) of retrograde labeling of TG neurons and P2Y 2 receptor-positive TG neurons from the same section, indicating co-localization. (d) Retrograde labeling of TG neurons (blue) innervating whisker pad skin in cultured TG neurons. (B) Electrophysiology recording for small-diameter and FG-labeled TG neurons in control rats. (a) Representative traces showing that the application of 30 μM UTP reduced I A . Suppression of the mean peak amplitudes of I A seen after UTP application was antagonized by suramin 100 μM. (b) Current–voltage relationship for the effects of UTP and suramin on I A . Each value represents the mean ± SEM (con: 0.14 ± 0.01 nA, n = 9; UTP: 0.09 ± 0.01 nA, n = 20, p < 0.05 vs control; suramin: 0.13 ± 0.01 nA, n = 9). I A was initiated via a prepulse (100 ms) of −120 mV and test pulses (400 ms) from −60 to +60 mV in a 10 mV step.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Labeling, Fluorescence, Whisker Assay, Cell Culture

Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Effect of UTP on the expression levels of I A subunits in control TG neurons. (A) Double-immunostaining revealed the expression of Kv1.4, Kv3.4, Kv4.2 and Kv4.3 subunits in P2Y 2 receptor-positive neurons in the ION-CCI TG sections. The P2Y 2 receptor-positive TG neurons also expressed Kv1.4, Kv3.4, Kv4.2 and Kv4.3, respectively, n = 5 rats. (B) Reduction in the mRNA levels of I A subunits by UTP in cultured TG neurons from control rats. Treatment with suramin (100 μM) in the UTP-incubated (30 μM) TG neurons for 16 h in control rats reversed the decrease in the mRNA levels of Kv1.4, Kv3.4, Kv4.2, and Kv4.3 subunits. n = 10 samples in each group, *, p < 0.05, **, p < 0.01 vs control; ## , p < 0.01 vs UTP.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Expressing, Double Immunostaining, Cell Culture, Incubation

Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: The role of P2Y 2 receptors in mechanical hyperalgesia in ION-CCI rats. (A) The peripheral target injection to TG of suramin (0.3-3 μg/μl) reduced mechanical allodynia in the whisker pad. n = 6-8, **, p < 0.01 compared with injection of saline, ## , p < 0.01 compared with injection of high-dose suramin. Suramin led to a time- and dose-dependent increase in PWT, this anti-allodynia effect started 10 min after the suramin injection and remained for at least 45 min. (B) The peripheral target injection to TG of P2Y 2 antisense oligodeoxynucleotides significantly alleviated mechanical allodynia of the whisker pad. n = 5, *, p < 0.05, **, p < 0.01 compared with injection of saline. The effect started at 6 h and persisted for at least 120 h. (C) Western blots showed successful suppression of P2Y 2 receptor expression in TG by P2Y 2 receptor antisense oligodeoxynucleotides treatment n = 4 for each group, **, p < 0.01.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Injection, Whisker Assay, Western Blot, Expressing

Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Difference of I A channel expression in TG between sham and ION-CCI rats. (A) Double-immunostaining for P2Y 2 receptors and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3 on TG neurons in sham and ION-CCI sections, respectively. (B) Percentages of numbers of Kv1.4, Kv3.4, Kv4.2 and Kv4.3 subunits in P2Y 2 receptor-positive neurons are significantly decreased in TG from ION-CCI rats compared with sham rats. n = 4 rats, **, p < 0.01). (C) Changes in the mRNA levels of I A subunits in TG after P2Y 2 receptor antisense oligodeoxynucleotides treatment. The mRNA levels of Kv1.4, Kv3.4 and Kv4.2 were significantly decreased in the saline group of ION-CCI rats compared with the sham rats. They were reversed after P2Y 2 receptor antisense oligodeoxynucleotides treatment. n = 5-9 rats, *, p < 0.05, ** p < 0.01 compared with saline groups. There was no difference in the levels of Kv4.3 mRNA among the groups. n = 6-8 rats, p > 0.05.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Expressing, Double Immunostaining

Journal: Molecular Pain

Article Title: Inhibition of G protein-coupled P2Y 2 receptor induced analgesia in a rat model of trigeminal neuropathic pain

doi: 10.1186/1744-8069-10-21

Figure Lengend Snippet: Role of ERK pathway in activation of P2Y 2 receptors mediates an inhibition of I A channels on small-diameter TG neurons in control rats. (A) Comparison of the phosphorylation of ERK1/2 in TG from sham and ION-CCI rats. Western blot results showed that the level of ERK1/2 phosphorylation was significantly increased in the ipsilateral TG after ION-CCI, compared with that from the sham group. n = 5 for each group *, p < 0.05. (B) In TG from ION-CCI rats, treatment with P2Y 2 receptor antisense oligodeoxynucleotides (15 μg/50 μl) significantly decreased the expression of ERK protein in TG. n = 5 for each group, ** , p < 0.01.

Article Snippet: The preparations were then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/l PBS pH 7.2) for 30 min. For double-immunostaining of P2Y 2 and Kv1.4 or Kv3.4 or Kv4.2 or Kv4.3, sections were incubated in a mixture of rabbit polyclonal P2Y 2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal Kv1.4 (1:200 dilution, Abcam, HongKong, China) or goat polyclonal KCNC4 (KV3.4) (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.2 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or goat polyclonal Kv4.3 (1:50 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight.

Techniques: Activation Assay, Inhibition, Western Blot, Expressing

Journal: BioMed Research International

Article Title: The Influence of Simulated Microgravity on Purinergic Signaling Is Different between Individual Culture and Endothelial and Smooth Muscle Cell Coculture

doi: 10.1155/2014/413708

Figure Lengend Snippet: P2 Receptor Expression in Endothelial Cells after 24 h under Normal Gravity and Simulated Microgravity. All cells on the surface of flasks were isolated for RT-PCR. P2X5, P2Y4, P2Y11, P2Y13 were up-regulated and P2X7, P2Y1 and P2Y2 were down-regulated in the ECs after 24 h in the clinostat (a). Cells grown within 6 mm of the center had the optimal simulated microgravity condition and were therefore isolated to confirm the above P2 receptor alteration on the RNA (b) and protein (c) level after 24 h simulated microgravity with and without SMC-conditioned medium collected under normal gravity. P2X5 and P2Y11 were up-regulated in ECs but P2X5 up-regulation was not significant on protein level. P2X7, P2Y1, P2Y2 were down-regulated on both gene and protein level. The SMC-conditioned medium can compensate the decrease of P2X7 expression but cause no significant effect on the alteration of P2Y1, P2Y2 and P2Y11. The fluorescent staining confirmed the protein change of P2X7, P2Y1 and P2Y11 (d).

Article Snippet: The membrane was blocked in TBST containing 5% BSA and incubated with anti-P2X7, P2Y1, P2Y2, P2Y11, VEGFR2, VE-cadherin, PECAM-1, calponin, SMA-α, MYH-11 (1 : 500), or GAPDH antibodies (1 : 5,000) (Santa Cruz Biotechnology, CA, USA) overnight at 4°C.

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: BioMed Research International

Article Title: The Influence of Simulated Microgravity on Purinergic Signaling Is Different between Individual Culture and Endothelial and Smooth Muscle Cell Coculture

doi: 10.1155/2014/413708

Figure Lengend Snippet: P2 Receptor Expression in Smooth Muscle Cells after 24 h under Normal Gravity and Simulated Microgravity. The experiments for SMC were performed similarly to those for the endothelial cells above. All cells on the surface of flasks were isolated for RT-PCR. P2X4, P2X7 and P2Y2 were up-regulated whereas P2X2, P2Y1 and P2Y14 were down-regulated in the SMCs after 24 h clinorotation (a). Cells grown within 6 mm of the center had the optimal simulated microgravity condition and were thus isolated to confirm the above P2 receptor alteration on both, RNA (b) and protein (c) level after 24 h clinorotation with and without EC-conditioned medium from normal gravity. P2X2 and P2X4 showed no significantly changed. P2X7 and P2Y2 was up-regulated, P2Y1 and P2Y14 were down-regulated. The EC-conditioned medium can compensate for the increase of P2X7 and P2Y2 expression, but no significant effect was observed on P2Y1 and P2Y14 (d).

Article Snippet: The membrane was blocked in TBST containing 5% BSA and incubated with anti-P2X7, P2Y1, P2Y2, P2Y11, VEGFR2, VE-cadherin, PECAM-1, calponin, SMA-α, MYH-11 (1 : 500), or GAPDH antibodies (1 : 5,000) (Santa Cruz Biotechnology, CA, USA) overnight at 4°C.

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: BioMed Research International

Article Title: The Influence of Simulated Microgravity on Purinergic Signaling Is Different between Individual Culture and Endothelial and Smooth Muscle Cell Coculture

doi: 10.1155/2014/413708

Figure Lengend Snippet: Scheme of P2 Receptor Alteration and the Postulated Paracrine Effect in ECs and SMCs under Simulated Microgravity. Several P2 receptor expressions were altered in ECs and SMCs under 24 h simulated microgravity condition using a clinostat. To point out that the expression P2X7 and P2Y2 was altered differentially between ECs and SMCs under simulated microgravity. Especially, the change of P2X7 in ECs was compensated under SMC-conditioned medium and vice versa. The conditioned medium collected under simulated microgravity showed the pathogenic influence of EC and SMC proliferation and migration if compared to condition medium from normal gravity.

Article Snippet: The membrane was blocked in TBST containing 5% BSA and incubated with anti-P2X7, P2Y1, P2Y2, P2Y11, VEGFR2, VE-cadherin, PECAM-1, calponin, SMA-α, MYH-11 (1 : 500), or GAPDH antibodies (1 : 5,000) (Santa Cruz Biotechnology, CA, USA) overnight at 4°C.

Techniques: Expressing, Migration

Journal: Molecular Vision

Article Title: Silencing of P2Y 2 receptor delays Ap 4 A - corneal re-epithelialization process

doi:

Figure Lengend Snippet: Rabbit P2Y 2 -R cDNA. A : Nucleotide sequence and deduced amino acid sequence of cloned rabbit P2Y 2 -R are shown. siRNA-target sequences are indicated with solid lines. B : Alignment of the rabbit deduced amino acid sequence with the human P2Y 2 -R sequence is displayed. Identical amino acids are shaded. Protein database searches revealed that the deduced sequence had the highest amino acid identity with human P2Y 2 -R (93%). These sequences are available under GenBank accession number EU886321 (rabbit) and NP_788086.1 (human).

Article Snippet: Cells were then washed with 1X PBS and 3% BSA and incubated with primary goat polyclonal anti-P2Y 2 -R (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) or with 1X PBS and 3% BSA for negative controls at room temperature for 1 h. Cells were washed twice in 1X PBS and 3% BSA and incubated with the secondary antibody donkey anti-goat IgG-FITC (1:200; Santa Cruz Biotechnology) for 1 h at room temperature.

Techniques: Sequencing, Clone Assay

Journal: Molecular Vision

Article Title: Silencing of P2Y 2 receptor delays Ap 4 A - corneal re-epithelialization process

doi:

Figure Lengend Snippet: P2Y 2 -R immunostaining of transfected cells and treated corneas. A : SIRC cells were incubated for 72 h with transfection reagent alone (control) or with P2Y 2 -R siRNA #2 and then processed for ICC (40X magnification). B : The chart shows P2Y 2 -R staining intensity quantification of control and P2Y 2 -R siRNA transfected cells 72 h post-transfection. C : A series of micrographs shows the P2Y 2 -R signal in corneas treated with 0.9% saline, 100 μM Ap 4 A, and siRNA+100 μM Ap 4 A while we can observed the nuclear staining for DAPI in blue (40X). D : The graph shows the P2Y 2 -R intensity signal in the three different treatments at 24 h and 36 h after wounding. Three asterisks mean p<0.001 when compared to the control. Green fluorescence (FITC) localizes P2Y 2 -R.

Article Snippet: Cells were then washed with 1X PBS and 3% BSA and incubated with primary goat polyclonal anti-P2Y 2 -R (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) or with 1X PBS and 3% BSA for negative controls at room temperature for 1 h. Cells were washed twice in 1X PBS and 3% BSA and incubated with the secondary antibody donkey anti-goat IgG-FITC (1:200; Santa Cruz Biotechnology) for 1 h at room temperature.

Techniques: Immunostaining, Transfection, Incubation, Staining, Fluorescence

Journal: Molecular Vision

Article Title: Silencing of P2Y 2 receptor delays Ap 4 A - corneal re-epithelialization process

doi:

Figure Lengend Snippet: Quantification of P2Y 2 -R mRNA levels by qRT–PCR analysis. Corneal impression cytology samples were collected at 48, 72, and 96 h after the first siRNA instillation and then submitted to total RNA isolation. After retrotranscription, we measured P2Y 2 -R mRNA knockdown in corneas treated with siRNA #2. Values derived from qRT–PCR results from untreated corneas are set as 100%, and the relative expressions of corneas treated with siRNA are indicated. Three asterisks indicate p<0.001 when compared to the control.

Article Snippet: Cells were then washed with 1X PBS and 3% BSA and incubated with primary goat polyclonal anti-P2Y 2 -R (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) or with 1X PBS and 3% BSA for negative controls at room temperature for 1 h. Cells were washed twice in 1X PBS and 3% BSA and incubated with the secondary antibody donkey anti-goat IgG-FITC (1:200; Santa Cruz Biotechnology) for 1 h at room temperature.

Techniques: Quantitative RT-PCR, Isolation, Derivative Assay

Journal: Molecular Vision

Article Title: Silencing of P2Y 2 receptor delays Ap 4 A - corneal re-epithelialization process

doi:

Figure Lengend Snippet: Effect of P2Y 2 -R siRNA on Ap 4 A-induced migration of corneal epithelial cells. SIRC cells were incubated for 72 h with transfection reagent alone (control), P2Y 2 -R siRNA #1, or P2Y 2 -R siRNA #2 and then wounded with a pipette tip in the presence or absence of Ap 4 A. The graphs ( A and B ) show the variation of the wounded area versus time and the estimated migration rates (EMR) of cells transfected with siRNA 1 and siRNA 2, respectively, in the presence of Ap 4 A ( P2Y 2 -R siRNA+Ap 4 A). Ap 4 A accelerated the rate of healing in cells transfected with transfection reagent alone (control+Ap 4 A) compared with the rate of both P2Y 2 -R siRNA-transfected cells in the presence of Ap 4 A ( P2Y 2 -R siRNA+Ap 4 A). Three asterisks indicate p<0.0001 when compared to the control, and two asterisks indicate p<0.001 when compared to Ap 4 A alone.

Article Snippet: Cells were then washed with 1X PBS and 3% BSA and incubated with primary goat polyclonal anti-P2Y 2 -R (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) or with 1X PBS and 3% BSA for negative controls at room temperature for 1 h. Cells were washed twice in 1X PBS and 3% BSA and incubated with the secondary antibody donkey anti-goat IgG-FITC (1:200; Santa Cruz Biotechnology) for 1 h at room temperature.

Techniques: Migration, Incubation, Transfection, Transferring

Journal: Molecular Vision

Article Title: Silencing of P2Y 2 receptor delays Ap 4 A - corneal re-epithelialization process

doi:

Figure Lengend Snippet: Effect of P2Y 2 -R siRNA on Ap 4 A-mediated corneal wound healing. A : A representative sequence of the progress in the corneal wound in the three different treatments (0.9% saline, 100 μM Ap 4 A, and siRNA+100 μM Ap 4 A) is shown. B : The graphs show the variation of the wounded area versus time and the estimated migration rates (EMR) in the three treatments. An asterisk means p<0.05 when compared to the control, and a double asterisk indicates p<0.01 when compared to the control.

Article Snippet: Cells were then washed with 1X PBS and 3% BSA and incubated with primary goat polyclonal anti-P2Y 2 -R (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) or with 1X PBS and 3% BSA for negative controls at room temperature for 1 h. Cells were washed twice in 1X PBS and 3% BSA and incubated with the secondary antibody donkey anti-goat IgG-FITC (1:200; Santa Cruz Biotechnology) for 1 h at room temperature.

Techniques: Sequencing, Migration

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The inhibitory effects of extracellular ATP on the growth of nasopharyngeal carcinoma cells via P2Y2 receptor and osteopontin

doi: 10.1186/1756-9966-33-53

Figure Lengend Snippet: The role of P2Y2 in mediating the effects of ATP on NPC cells. A : Schematic representation of the constructed vectors. CMV, CMV promoter; EGFP, enhanced green fluorescent protein gene; U6, U6 promoter. B : P2Y2 and OPN regulate the growth inhibitory effects of ATP on NPC cells. The levels of P2Y2 and OPN were significantly decreased after transfection with the shRNA expressing vector or siRNA. P2Y2 overexpression enhanced the growth inhibitory effect of ATP on NPC cells, whereas OPN overexpression reversed this effect. There were statistically significant differences between the control and treated conditions: *P < 0.05. C : The role of P2Y2 expression in the effects of ATP on the cell cycle and apoptosis of CNE-2 cells. P2Y2 expression increased the percentage of G1-phase cells and decreased the proportion of G2/M-phase cells. The overexpression of P2Y2 increased the level of apoptotic cells in ATP treated cells.

Article Snippet: The antibodies used were anti-OPN (Sigma, USA), anti-P2Y2 (Santa Cruz, USA), and anti-cleaved caspase-3 (Cell Signal Technology, USA); other antibodies were purchased from Beyotime (Nantong, China).

Techniques: Construct, Transfection, shRNA, Expressing, Plasmid Preparation, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The inhibitory effects of extracellular ATP on the growth of nasopharyngeal carcinoma cells via P2Y2 receptor and osteopontin

doi: 10.1186/1756-9966-33-53

Figure Lengend Snippet: Effects of ATP and P2Y2 on the regulation of OPN by p65. A : Overexpression of P2Y2 downregulated the levels of p65 and OPN, and p65 knockdown with shRNA decreased the level of OPN in 5-8 F and CNE-2 cells. B : ATP and P2Y2 reduced the transcriptional activity of OPN promoter. C : ATP lowered the binding of p65 to the promoter region of OPN in 5-8 F and CNE-2 cells. The data represent the means ± SD of 3 independent experiments. Statistical significance between the control and treated conditions: *P < 0.05 (100 μM).

Article Snippet: The antibodies used were anti-OPN (Sigma, USA), anti-P2Y2 (Santa Cruz, USA), and anti-cleaved caspase-3 (Cell Signal Technology, USA); other antibodies were purchased from Beyotime (Nantong, China).

Techniques: Over Expression, shRNA, Activity Assay, Binding Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The inhibitory effects of extracellular ATP on the growth of nasopharyngeal carcinoma cells via P2Y2 receptor and osteopontin

doi: 10.1186/1756-9966-33-53

Figure Lengend Snippet: P-AKT was involved in the effects of ATP and P2Y2 in NPC cells. A : The Akt pathway was involved in the effect of ATP on NPC cells. ATP decreased the level of p-Akt in CNE-2 cells, and OPN downregulation decreased the level of p-Akt and enhanced the extent of the downregulation of p-Akt by ATP. The overexpression of OPN increased the level of p-Akt and mitigated the effect of ATP on the p-Akt level. B : The overexpression of P2Y2 could decrease the p-AKT level in CNE-2 cells. The level of p-Akt was detected by western blotting, with quantification of the band intensity. Statistical significance between the control and treated conditions: *P < 0.05.

Article Snippet: The antibodies used were anti-OPN (Sigma, USA), anti-P2Y2 (Santa Cruz, USA), and anti-cleaved caspase-3 (Cell Signal Technology, USA); other antibodies were purchased from Beyotime (Nantong, China).

Techniques: Over Expression, Western Blot