Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Upregulation of P2X3R gene promoter activity by Runx1 (A) Representative tibial bone radiographs obtained from PBS- and MRMT-1-treated rats on days 14 (left) and 21 (right) after surgery. The higher magnification of the proximal third of the bones from MRMT-1-treated rats (in the square box) are shown in the bottom. Note that severe deterioration with medullary bone loss and full-thickness bicortical bone loss were observed on ipsilateral but not contralateral tibia bone in MRMT-1-treated rats. (B–E) Assessment of pain behaviors. (B) Paw withdrawal threshold (PWT) in response to von Frey filaments stimuli. (C) Paw withdrawal latency (PWL) in response to radiant heat stimulation. (D and E) Spontaneous flinching (D) and guarding (E) behaviors were video recorded to assess spontaneous pain. n = 11–15 rats per group. ∗∗ p<0.01, ∗∗∗ p<0.001, MRMT-1 versus naïve; ### p<0.001, MRMT-1 versus PBS, repeated-measures two-way ANOVA with Tukey’s post hoc test. (F and G) RT-qPCR and Western blot analyses of P2X3R mRNA (F, n = 6–7 rats per group) and protein (G, n = 4 rats per group) abundance in ipsilateral L4/5 DRGs from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery. Upper in (G): Representative blots are shown. (H–L) Immunofluorescent staining with P2X3R and Runx1 in L4/5 DRG neurons obtained from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery (n = 3 rats per group). (H) Representative images showing the immunostaining of Runx1 (red), P2X3R (green), and DAPI (blue). Right: merged images. Scale bar = 50 μm. (I–L) Violin plots show the statistical analysis for the percentage of P2X3R-positive neurons on Runx1-positive neurons (I), Runx1-positive neurons on P2X3R-positive neurons (J), mean fluorescence intensity of P2X3R immunoreactivity (K) and Runx1 immunoreactivity (L). (M−O) Western blot analyses of Runx1 and pRunx1 Ser249 protein abundance in ipsilateral L4/5 DRGs from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery (n = 4 rats per group). (M) Representative blots are shown. (N and O) Bar graphs show the statistical analysis for the relative intensity of pRunx1 Ser249 (N) Runx1(O) blots. (P) Schematic representation of luciferase reporter constructs carrying either the full-length (1,934 bp) rat P2X3R gene promoter containing 6 putative consensus-binding sites (R1-R6) for Runx1 (p1,934-P2X3-Luc), 5′ deletion of this promoter containing R1 Runx1 motif (p415-P2X3-Luc), or promoter carrying mutated R1 Runx1 motif (p415-P2X3-Mut-R1-Luc). (Q) Forced expression of Runx1 upregulates P2X3R gene promoter activity in PC12 cells (n = 4 biological replicates). (R) Mutation of Runx1-binding site R1 blocks the upregulation of P2X3R gene promoter activity by Runx1 overexpression (n = 5 biological replicates). RLU, relative luciferase units. Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, one-way ANOVA with Tukey’s post hoc test for (F), (G), (I)–(L), (N), (O), and (Q); two-way ANOVA with Sidak’s post hoc test for (R).

Article Snippet: Rabbit anti-rat P2X3R (for Western blot) , Alomone labs , Cat# APR-016.

Techniques: Activity Assay, Quantitative RT-PCR, Western Blot, Staining, Immunostaining, Fluorescence, Luciferase, Construct, Binding Assay, Expressing, Mutagenesis, Over Expression

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Runx1 knockdown reverses the increased P2X3R expression, DRG neurons hyperexcitability, and pain hypersensitivity in tumor–bearing rats (A) Western blot analysis of Runx1 protein abundance in ipsilateral L4/5 DRGs from intrathecal scramble- and Runx1 siRNA-treated rats at 14 days after tumor cells inoculation (n = 3 rats per group). Upper: Representative blots are shown. (B and C) RT-qPCR and Western blot analyses of P2X3R mRNA (B, n = 10–12 rats per group) and protein abundance (C, n = 4 rats per group) in ipsilateral L4/5 DRGs from intrathecal scramble- and Runx1 siRNA-treated rats at 14 days after tumor cells inoculation. Upper in (C): Representative blots are shown. (D and E) Electrophysiological analyses of P2X3R currents (D) and neuronal excitability (E) in ipsilateral L4/5 DRG neurons from intrathecal scramble- and Runx1 siRNA-treated rats at 14 days after tumor cells inoculation (n = 12–18 cells from six to seven rats per group). Left in (D) and (E): Representative traces of α,β-meATP-induced P2X3R-currents (D) and neuronal action potentials (E) are shown. (F and G) Assessment of ipsilateral PWT (F) and PWL (G) from intrathecal scramble- and Runx1 siRNA-treated rats from day 14 to day 20 after tumor cells inoculation (n = 8–11 rats per group). (H) Assessment of animal’s locomotor function by inclined-plate test, compared before and after drug administration (n = 8–11 rats per group). Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, unpaired t test for (A)–(E); repeated-measures two-way ANOVA with Sidak’s post hoc test for (F) and (G); two-way ANOVA with Sidak’s post hoc test for (H).

Article Snippet: Rabbit anti-rat P2X3R (for Western blot) , Alomone labs , Cat# APR-016.

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Runx1 overexpression induces the increases of P2X3R, DRG neurons hyperexcitability, and pain hypersensitivity in naïve rats (A–C) Representative images (A) and a summary for the mean fluorescence intensity of Runx1 (B) and P2X3R (C) immunostaining in L4/5 DRG neurons from naïve rats at 14 days after intrathecal LV-Runx1 or LV-GFP administration (n = 80–157 cells from three rats per group). Scale bar = 100 μm. (D) Western blot analysis of Runx1 protein abundance in L4/5 DRG neurons from naïve rats at 14 days after intrathecal LV-Runx1 or LV-GFP administration (n = 4 rats per group). Upper: Representative blots are shown. (E and F) RT-qPCR and Western blot analyses of P2X3R mRNA (E, n = 9–13 rats per group) and protein (F, n = 4 rats per group) abundance in L4/5 DRGs from naïve rats at 14 days after intrathecal LV-Runx1 or LV-GFP administration. Upper in (F): Representative blots are shown. (G and H) Electrophysiological analyses of P2X3R currents (G, n = 6–8 cells from five rats per group) and neuronal excitability (H, n = 11–15 cells from five rats per group) in L4/5 DRG neurons from naïve rats at 14 days after intrathecal LV-Runx1 or LV-GFP administration. Left in (G) and (H): Representative traces of α,β-meATP-induced P2X3R-currents (G) and neuronal action potentials (H) are shown. (I and J) Assessment of PWT (I) and PWL (J) from naïve rats before and after intrathecal LV-Runx1 or LV-GFP administration (n = 12 rats per group). (K) Assessment of animal’s locomotor function before and after virus injection (n = 12 rats per group). Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, unpaired t test for (B)–(H); repeated-measures two-way ANOVA with Sidak’s post hoc test for (I) and (J); two-way ANOVA with Sidak’s post hoc test for (K).

Article Snippet: Rabbit anti-rat P2X3R (for Western blot) , Alomone labs , Cat# APR-016.

Techniques: Over Expression, Fluorescence, Immunostaining, Western Blot, Quantitative RT-PCR, Injection

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Exogenous GDNF upregulates P2X3R gene promoter activity in SH-SY5Y cells, increases the abundance of phosphorylated ERK, Runx1, and P2X3R mRNA and protein in cultured DRG neurons (A) Luciferase reporter assay of P2X3R gene promoter activity in SH-SY5Y cells treated with GDNF or GDNF + PD98059. (B) Mutation of Runx1 serine 249/266 sites blocks the increased P2X3R gene promoter activity by GDNF in SH-SY5Y cells. RLU, relative luciferase units. (C) RT-qPCR analysis of P2X3R mRNA abundance in cultured DRG neurons with GDNF treatment. (D–F) Western blot analysis of ERK and phosphorylated ERK abundance in cultured DRG neurons with GDNF treatment. (D) Representative blots are shown. (G–I) Western blot analysis of Runx1 and phosphorylated Runx1 abundance in cultured DRG neurons with GDNF treatment. (G) Representative blots are shown. (J and K) Western blot analysis of P2X3R protein abundance in cultured DRG neurons with GDNF treatment. (J) Representative blots are shown. (L) Protein translation inhibitor, anisomycin, blocks the increased P2X3R protein abundance in cultured DRG neurons with 6 h GDNF treatment. (L) Representative blots are shown. n = 3–5 biological replicates per group as indicated. Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, one-way ANOVA with Tukey’s post hoc test for (A) and (L); two-way ANOVA with Sidak’s post hoc test for (B); one-way ANOVA with Dunnett’s post hoc test for (C), (E)–(F), and (H)–(I), and (K). See also Figure S1 .

Article Snippet: Rabbit anti-rat P2X3R (for Western blot) , Alomone labs , Cat# APR-016.

Techniques: Activity Assay, Cell Culture, Luciferase, Reporter Assay, Mutagenesis, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Intrathecal GDNF activates ERK-Runx1 signaling, increases functional P2X3R expression and DRG neurons excitability, and induces pain hypersensitivity in naïve rats (A) Western blot analysis of phosphorylated ERK abundance in L4/5 DRGs from naïve rats received intrathecal GDNF treatment (200 ng in a 10-μL volume, once per day for three consecutive days) (n = 5 rats per group). Upper: Representative blots are shown. (B) Western blot of phosphorylated Runx1 abundance in L4/5 DRGs from naïve rats received intrathecal GDNF treatment (n = 4 rats per group). Upper: Representative blots are shown. (C and D) RT-qPCR and Western blot analyses of P2X3R mRNA (C, n = 6 rats per group) and protein (D, n = 4 rats per group) abundance in L4/5 DRGs from naïve rats with intrathecal GDNF treatment. Upper in (D): Representative blots are shown. (E and F) Electrophysiological analyses of P2X3R currents (E, n = 10–15 cells from six rats per group) and neuronal excitability (F, n = 21 cells from six rats per group) in L4/5 DRG neurons from naïve rats with intrathecal GDNF treatment. Left in (E) and (F): Representative traces of α,β-meATP-induced P2X3R-currents (E) and neuronal action potentials (F) are shown. (G–J) Assessment of pain behaviors in naïve rats with intrathecal GDNF treatment at one day after the last drug injection. (G and H) PWT of left (G) and right (H) hind paw. (I and J) PWL of left (I) and right (J) hind paw. n = 11 rats per group. (K) Assessment of animal’s locomotor function before and after drug administration (n = 11 rats per group). Data are presented as mean ± SEM. ∗∗ p<0.01, ∗∗∗ p<0.001, unpaired t test for (A)–(F); two-way ANOVA with Sidak’s post hoc test for (G)–(K). See also Figure S2 .

Article Snippet: Rabbit anti-rat P2X3R (for Western blot) , Alomone labs , Cat# APR-016.

Techniques: Functional Assay, Expressing, Western Blot, Quantitative RT-PCR, Injection

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: GFRα1 knockdown prevents the activation of ERK-Runx1 signaling, reverses the increase of P2X3R, DRG neurons hyperexcitability, and pain hypersensitivity in bone cancer–bearing rats (A) Enzyme-linked immunosorbent assay of GDNF content in ipsilateral L4/5 DRGs from PBS- and MRMT-1-treated rats at 14 days after surgery (n = 5–8 rats per group). (B) Western blot analyses of GFRα1 protein abundance in ipsilateral L4/5 DRGs from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery (n = 4 rats per group). Upper: Representative blots are shown. (C and D) RT-qPCR and Western blot analyses of GFRα1 mRNA (C) and protein (D) abundance in ipsilateral L4/5 DRGs from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 3–4 rats per group). Upper: Representative blots are shown. (E and F) Western blot analyses of phosphorylated ERK (pERK1/2, E) and phosphorylated Runx1 (pRunx1 Ser249 , F) abundance in ipsilateral L4/5 DRGs from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 4–5 rats per group). Upper: Representative blots are shown. (G and H) RT-qPCR and Western blot analyses of P2X3R mRNA (G) and protein (H) abundance in ipsilateral L4/5 DRGs from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 4–5 rats per group). Upper in (H): Representative blots are shown. (I and J) Electrophysiological analyses of P2X3R currents (I) and neuronal excitability (J) in L4/5 DRG neurons from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 18–20 cells from six to seven rats per group). Left in (I) and (J): Representative traces of α,β-meATP-induced P2X3R-currents (I) and neuronal action potentials (J) are shown. (K and L) Assessment of ipsilateral PWT (K) and PWL (L) from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 12–13 rats per group). (M) Assessment of animal’s locomotor function before and after antisense oligodeoxynucleotide administration (n = 12–13 rats per group). Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, two-way ANOVA with Sidak’s post hoc test for (A), and (K)–(M); one-way ANOVA with Tukey’s post hoc test for (B); unpaired t test for (C)–(J).

Article Snippet: Rabbit anti-rat P2X3R (for Western blot) , Alomone labs , Cat# APR-016.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: ERK inhibitor prevents Runx1 activation and functional upregulation of P2X3R, and reverses DRG neurons hyperexcitability and pain hypersensitivity in bone cancer–bearing rats (A and B) Western blot analyses of ERK (A) and phosphorylated ERK (B) abundance in ipsilateral L4/5 DRGs from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery (n = 3–5 rats per group). Upper in (A): Representative blots are shown. (C) Western blot analyses of phosphorylated Runx1 (pRunx1 Ser249 ) abundance in ipsilateral L4/5 DRGs from intrathecal SCH772984- and vehicle-treated rats at 14 days after tumor cells inoculation (n = 4 rats per group). Upper: Representative blots are shown. (D and E) RT-qPCR and Western blot analyses of P2X3R mRNA (D, n = 8 rats per group) and protein (E, n = 4 rats per group) abundance in ipsilateral L4/5 DRGs from intrathecal SCH772984- and vehicle-treated rats at 14 days after tumor cells inoculation. Upper in (E): Representative blots are shown. (F–I) Electrophysiological analyses of P2X3R currents (F and G) and neuronal excitability (H and I) in L4/5 DRG neurons from intrathecal SCH772984- and vehicle-treated rats at 14 days after tumor cells inoculation (n = 14–20 cells from six rats per group). (F) and (H): Representative traces of α,β-meATP-induced P2X3R-currents (F) and neuronal action potentials (H) are shown. (J and K) Assessment of ipsilateral PWT (J) and PWL (K) from tumor-bearing rats received intrathecal SCH772984 or vehicle treatment (n = 10 rats per group). (L) Assessment of animal’s locomotor function before and after drug administration (n = 10 rats per group). Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, one-way ANOVA with Tukey’s post hoc test for (A), (B); unpaired t test for (C)–(I); repeated-measures two-way ANOVA with Sidak’s post hoc test for (J) and (K); two-way ANOVA with Sidak’s post hoc test for (L). See also Figure S3 .

Article Snippet: Rabbit anti-rat P2X3R (for Western blot) , Alomone labs , Cat# APR-016.

Techniques: Activation Assay, Functional Assay, Western Blot, Quantitative RT-PCR

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet:

Article Snippet: Rabbit anti-rat P2X3R (for Western blot) , Alomone labs , Cat# APR-016.

Techniques: Immunofluorescence, Staining, Western Blot, Recombinant, Modification, Bicinchoninic Acid Protein Assay, Mutagenesis, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Transfection, Software

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Upregulation of P2X3R gene promoter activity by Runx1 (A) Representative tibial bone radiographs obtained from PBS- and MRMT-1-treated rats on days 14 (left) and 21 (right) after surgery. The higher magnification of the proximal third of the bones from MRMT-1-treated rats (in the square box) are shown in the bottom. Note that severe deterioration with medullary bone loss and full-thickness bicortical bone loss were observed on ipsilateral but not contralateral tibia bone in MRMT-1-treated rats. (B–E) Assessment of pain behaviors. (B) Paw withdrawal threshold (PWT) in response to von Frey filaments stimuli. (C) Paw withdrawal latency (PWL) in response to radiant heat stimulation. (D and E) Spontaneous flinching (D) and guarding (E) behaviors were video recorded to assess spontaneous pain. n = 11–15 rats per group. ∗∗ p<0.01, ∗∗∗ p<0.001, MRMT-1 versus naïve; ### p<0.001, MRMT-1 versus PBS, repeated-measures two-way ANOVA with Tukey’s post hoc test. (F and G) RT-qPCR and Western blot analyses of P2X3R mRNA (F, n = 6–7 rats per group) and protein (G, n = 4 rats per group) abundance in ipsilateral L4/5 DRGs from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery. Upper in (G): Representative blots are shown. (H–L) Immunofluorescent staining with P2X3R and Runx1 in L4/5 DRG neurons obtained from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery (n = 3 rats per group). (H) Representative images showing the immunostaining of Runx1 (red), P2X3R (green), and DAPI (blue). Right: merged images. Scale bar = 50 μm. (I–L) Violin plots show the statistical analysis for the percentage of P2X3R-positive neurons on Runx1-positive neurons (I), Runx1-positive neurons on P2X3R-positive neurons (J), mean fluorescence intensity of P2X3R immunoreactivity (K) and Runx1 immunoreactivity (L). (M−O) Western blot analyses of Runx1 and pRunx1 Ser249 protein abundance in ipsilateral L4/5 DRGs from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery (n = 4 rats per group). (M) Representative blots are shown. (N and O) Bar graphs show the statistical analysis for the relative intensity of pRunx1 Ser249 (N) Runx1(O) blots. (P) Schematic representation of luciferase reporter constructs carrying either the full-length (1,934 bp) rat P2X3R gene promoter containing 6 putative consensus-binding sites (R1-R6) for Runx1 (p1,934-P2X3-Luc), 5′ deletion of this promoter containing R1 Runx1 motif (p415-P2X3-Luc), or promoter carrying mutated R1 Runx1 motif (p415-P2X3-Mut-R1-Luc). (Q) Forced expression of Runx1 upregulates P2X3R gene promoter activity in PC12 cells (n = 4 biological replicates). (R) Mutation of Runx1-binding site R1 blocks the upregulation of P2X3R gene promoter activity by Runx1 overexpression (n = 5 biological replicates). RLU, relative luciferase units. Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, one-way ANOVA with Tukey’s post hoc test for (F), (G), (I)–(L), (N), (O), and (Q); two-way ANOVA with Sidak’s post hoc test for (R).

Article Snippet: The membranes were blocked with 5% nonfat milk or bovine serum albumin in Tris-buffered saline and Tween [TBST, 20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature, and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-rat P2X3R antibody (1:1000, Alomone Labs), rabbit anti-rat pERK1/2 antibody (1:1000, Cell Signaling Technology), rabbit anti-rat ERK1/2 antibody (1:2000, Cell Signaling Technology), rabbit anti-rat pRunx1 Ser249 (1:500, Cell Signaling Technology), rabbit anti-Runx1 (1:1000, Abcam), rabbit anti-GFRα1 (1:1000, Santa Cruz), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology), or mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology).

Techniques: Activity Assay, Quantitative RT-PCR, Western Blot, Staining, Immunostaining, Fluorescence, Luciferase, Construct, Binding Assay, Expressing, Mutagenesis, Over Expression

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Runx1 knockdown reverses the increased P2X3R expression, DRG neurons hyperexcitability, and pain hypersensitivity in tumor–bearing rats (A) Western blot analysis of Runx1 protein abundance in ipsilateral L4/5 DRGs from intrathecal scramble- and Runx1 siRNA-treated rats at 14 days after tumor cells inoculation (n = 3 rats per group). Upper: Representative blots are shown. (B and C) RT-qPCR and Western blot analyses of P2X3R mRNA (B, n = 10–12 rats per group) and protein abundance (C, n = 4 rats per group) in ipsilateral L4/5 DRGs from intrathecal scramble- and Runx1 siRNA-treated rats at 14 days after tumor cells inoculation. Upper in (C): Representative blots are shown. (D and E) Electrophysiological analyses of P2X3R currents (D) and neuronal excitability (E) in ipsilateral L4/5 DRG neurons from intrathecal scramble- and Runx1 siRNA-treated rats at 14 days after tumor cells inoculation (n = 12–18 cells from six to seven rats per group). Left in (D) and (E): Representative traces of α,β-meATP-induced P2X3R-currents (D) and neuronal action potentials (E) are shown. (F and G) Assessment of ipsilateral PWT (F) and PWL (G) from intrathecal scramble- and Runx1 siRNA-treated rats from day 14 to day 20 after tumor cells inoculation (n = 8–11 rats per group). (H) Assessment of animal’s locomotor function by inclined-plate test, compared before and after drug administration (n = 8–11 rats per group). Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, unpaired t test for (A)–(E); repeated-measures two-way ANOVA with Sidak’s post hoc test for (F) and (G); two-way ANOVA with Sidak’s post hoc test for (H).

Article Snippet: The membranes were blocked with 5% nonfat milk or bovine serum albumin in Tris-buffered saline and Tween [TBST, 20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature, and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-rat P2X3R antibody (1:1000, Alomone Labs), rabbit anti-rat pERK1/2 antibody (1:1000, Cell Signaling Technology), rabbit anti-rat ERK1/2 antibody (1:2000, Cell Signaling Technology), rabbit anti-rat pRunx1 Ser249 (1:500, Cell Signaling Technology), rabbit anti-Runx1 (1:1000, Abcam), rabbit anti-GFRα1 (1:1000, Santa Cruz), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology), or mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Runx1 overexpression induces the increases of P2X3R, DRG neurons hyperexcitability, and pain hypersensitivity in naïve rats (A–C) Representative images (A) and a summary for the mean fluorescence intensity of Runx1 (B) and P2X3R (C) immunostaining in L4/5 DRG neurons from naïve rats at 14 days after intrathecal LV-Runx1 or LV-GFP administration (n = 80–157 cells from three rats per group). Scale bar = 100 μm. (D) Western blot analysis of Runx1 protein abundance in L4/5 DRG neurons from naïve rats at 14 days after intrathecal LV-Runx1 or LV-GFP administration (n = 4 rats per group). Upper: Representative blots are shown. (E and F) RT-qPCR and Western blot analyses of P2X3R mRNA (E, n = 9–13 rats per group) and protein (F, n = 4 rats per group) abundance in L4/5 DRGs from naïve rats at 14 days after intrathecal LV-Runx1 or LV-GFP administration. Upper in (F): Representative blots are shown. (G and H) Electrophysiological analyses of P2X3R currents (G, n = 6–8 cells from five rats per group) and neuronal excitability (H, n = 11–15 cells from five rats per group) in L4/5 DRG neurons from naïve rats at 14 days after intrathecal LV-Runx1 or LV-GFP administration. Left in (G) and (H): Representative traces of α,β-meATP-induced P2X3R-currents (G) and neuronal action potentials (H) are shown. (I and J) Assessment of PWT (I) and PWL (J) from naïve rats before and after intrathecal LV-Runx1 or LV-GFP administration (n = 12 rats per group). (K) Assessment of animal’s locomotor function before and after virus injection (n = 12 rats per group). Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, unpaired t test for (B)–(H); repeated-measures two-way ANOVA with Sidak’s post hoc test for (I) and (J); two-way ANOVA with Sidak’s post hoc test for (K).

Article Snippet: The membranes were blocked with 5% nonfat milk or bovine serum albumin in Tris-buffered saline and Tween [TBST, 20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature, and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-rat P2X3R antibody (1:1000, Alomone Labs), rabbit anti-rat pERK1/2 antibody (1:1000, Cell Signaling Technology), rabbit anti-rat ERK1/2 antibody (1:2000, Cell Signaling Technology), rabbit anti-rat pRunx1 Ser249 (1:500, Cell Signaling Technology), rabbit anti-Runx1 (1:1000, Abcam), rabbit anti-GFRα1 (1:1000, Santa Cruz), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology), or mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology).

Techniques: Over Expression, Fluorescence, Immunostaining, Western Blot, Quantitative RT-PCR, Injection

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Exogenous GDNF upregulates P2X3R gene promoter activity in SH-SY5Y cells, increases the abundance of phosphorylated ERK, Runx1, and P2X3R mRNA and protein in cultured DRG neurons (A) Luciferase reporter assay of P2X3R gene promoter activity in SH-SY5Y cells treated with GDNF or GDNF + PD98059. (B) Mutation of Runx1 serine 249/266 sites blocks the increased P2X3R gene promoter activity by GDNF in SH-SY5Y cells. RLU, relative luciferase units. (C) RT-qPCR analysis of P2X3R mRNA abundance in cultured DRG neurons with GDNF treatment. (D–F) Western blot analysis of ERK and phosphorylated ERK abundance in cultured DRG neurons with GDNF treatment. (D) Representative blots are shown. (G–I) Western blot analysis of Runx1 and phosphorylated Runx1 abundance in cultured DRG neurons with GDNF treatment. (G) Representative blots are shown. (J and K) Western blot analysis of P2X3R protein abundance in cultured DRG neurons with GDNF treatment. (J) Representative blots are shown. (L) Protein translation inhibitor, anisomycin, blocks the increased P2X3R protein abundance in cultured DRG neurons with 6 h GDNF treatment. (L) Representative blots are shown. n = 3–5 biological replicates per group as indicated. Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, one-way ANOVA with Tukey’s post hoc test for (A) and (L); two-way ANOVA with Sidak’s post hoc test for (B); one-way ANOVA with Dunnett’s post hoc test for (C), (E)–(F), and (H)–(I), and (K). See also Figure S1 .

Article Snippet: The membranes were blocked with 5% nonfat milk or bovine serum albumin in Tris-buffered saline and Tween [TBST, 20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature, and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-rat P2X3R antibody (1:1000, Alomone Labs), rabbit anti-rat pERK1/2 antibody (1:1000, Cell Signaling Technology), rabbit anti-rat ERK1/2 antibody (1:2000, Cell Signaling Technology), rabbit anti-rat pRunx1 Ser249 (1:500, Cell Signaling Technology), rabbit anti-Runx1 (1:1000, Abcam), rabbit anti-GFRα1 (1:1000, Santa Cruz), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology), or mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology).

Techniques: Activity Assay, Cell Culture, Luciferase, Reporter Assay, Mutagenesis, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: Intrathecal GDNF activates ERK-Runx1 signaling, increases functional P2X3R expression and DRG neurons excitability, and induces pain hypersensitivity in naïve rats (A) Western blot analysis of phosphorylated ERK abundance in L4/5 DRGs from naïve rats received intrathecal GDNF treatment (200 ng in a 10-μL volume, once per day for three consecutive days) (n = 5 rats per group). Upper: Representative blots are shown. (B) Western blot of phosphorylated Runx1 abundance in L4/5 DRGs from naïve rats received intrathecal GDNF treatment (n = 4 rats per group). Upper: Representative blots are shown. (C and D) RT-qPCR and Western blot analyses of P2X3R mRNA (C, n = 6 rats per group) and protein (D, n = 4 rats per group) abundance in L4/5 DRGs from naïve rats with intrathecal GDNF treatment. Upper in (D): Representative blots are shown. (E and F) Electrophysiological analyses of P2X3R currents (E, n = 10–15 cells from six rats per group) and neuronal excitability (F, n = 21 cells from six rats per group) in L4/5 DRG neurons from naïve rats with intrathecal GDNF treatment. Left in (E) and (F): Representative traces of α,β-meATP-induced P2X3R-currents (E) and neuronal action potentials (F) are shown. (G–J) Assessment of pain behaviors in naïve rats with intrathecal GDNF treatment at one day after the last drug injection. (G and H) PWT of left (G) and right (H) hind paw. (I and J) PWL of left (I) and right (J) hind paw. n = 11 rats per group. (K) Assessment of animal’s locomotor function before and after drug administration (n = 11 rats per group). Data are presented as mean ± SEM. ∗∗ p<0.01, ∗∗∗ p<0.001, unpaired t test for (A)–(F); two-way ANOVA with Sidak’s post hoc test for (G)–(K). See also Figure S2 .

Article Snippet: The membranes were blocked with 5% nonfat milk or bovine serum albumin in Tris-buffered saline and Tween [TBST, 20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature, and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-rat P2X3R antibody (1:1000, Alomone Labs), rabbit anti-rat pERK1/2 antibody (1:1000, Cell Signaling Technology), rabbit anti-rat ERK1/2 antibody (1:2000, Cell Signaling Technology), rabbit anti-rat pRunx1 Ser249 (1:500, Cell Signaling Technology), rabbit anti-Runx1 (1:1000, Abcam), rabbit anti-GFRα1 (1:1000, Santa Cruz), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology), or mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology).

Techniques: Functional Assay, Expressing, Western Blot, Quantitative RT-PCR, Injection

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: GFRα1 knockdown prevents the activation of ERK-Runx1 signaling, reverses the increase of P2X3R, DRG neurons hyperexcitability, and pain hypersensitivity in bone cancer–bearing rats (A) Enzyme-linked immunosorbent assay of GDNF content in ipsilateral L4/5 DRGs from PBS- and MRMT-1-treated rats at 14 days after surgery (n = 5–8 rats per group). (B) Western blot analyses of GFRα1 protein abundance in ipsilateral L4/5 DRGs from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery (n = 4 rats per group). Upper: Representative blots are shown. (C and D) RT-qPCR and Western blot analyses of GFRα1 mRNA (C) and protein (D) abundance in ipsilateral L4/5 DRGs from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 3–4 rats per group). Upper: Representative blots are shown. (E and F) Western blot analyses of phosphorylated ERK (pERK1/2, E) and phosphorylated Runx1 (pRunx1 Ser249 , F) abundance in ipsilateral L4/5 DRGs from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 4–5 rats per group). Upper: Representative blots are shown. (G and H) RT-qPCR and Western blot analyses of P2X3R mRNA (G) and protein (H) abundance in ipsilateral L4/5 DRGs from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 4–5 rats per group). Upper in (H): Representative blots are shown. (I and J) Electrophysiological analyses of P2X3R currents (I) and neuronal excitability (J) in L4/5 DRG neurons from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 18–20 cells from six to seven rats per group). Left in (I) and (J): Representative traces of α,β-meATP-induced P2X3R-currents (I) and neuronal action potentials (J) are shown. (K and L) Assessment of ipsilateral PWT (K) and PWL (L) from intrathecal GFRα1-MM- and GFRα1-AS-treated rats at 14 days after tumor cells inoculation (n = 12–13 rats per group). (M) Assessment of animal’s locomotor function before and after antisense oligodeoxynucleotide administration (n = 12–13 rats per group). Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, two-way ANOVA with Sidak’s post hoc test for (A), and (K)–(M); one-way ANOVA with Tukey’s post hoc test for (B); unpaired t test for (C)–(J).

Article Snippet: The membranes were blocked with 5% nonfat milk or bovine serum albumin in Tris-buffered saline and Tween [TBST, 20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature, and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-rat P2X3R antibody (1:1000, Alomone Labs), rabbit anti-rat pERK1/2 antibody (1:1000, Cell Signaling Technology), rabbit anti-rat ERK1/2 antibody (1:2000, Cell Signaling Technology), rabbit anti-rat pRunx1 Ser249 (1:500, Cell Signaling Technology), rabbit anti-Runx1 (1:1000, Abcam), rabbit anti-GFRα1 (1:1000, Santa Cruz), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology), or mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet: ERK inhibitor prevents Runx1 activation and functional upregulation of P2X3R, and reverses DRG neurons hyperexcitability and pain hypersensitivity in bone cancer–bearing rats (A and B) Western blot analyses of ERK (A) and phosphorylated ERK (B) abundance in ipsilateral L4/5 DRGs from naïve, PBS-, and MRMT-1-treated rats at 14 days after surgery (n = 3–5 rats per group). Upper in (A): Representative blots are shown. (C) Western blot analyses of phosphorylated Runx1 (pRunx1 Ser249 ) abundance in ipsilateral L4/5 DRGs from intrathecal SCH772984- and vehicle-treated rats at 14 days after tumor cells inoculation (n = 4 rats per group). Upper: Representative blots are shown. (D and E) RT-qPCR and Western blot analyses of P2X3R mRNA (D, n = 8 rats per group) and protein (E, n = 4 rats per group) abundance in ipsilateral L4/5 DRGs from intrathecal SCH772984- and vehicle-treated rats at 14 days after tumor cells inoculation. Upper in (E): Representative blots are shown. (F–I) Electrophysiological analyses of P2X3R currents (F and G) and neuronal excitability (H and I) in L4/5 DRG neurons from intrathecal SCH772984- and vehicle-treated rats at 14 days after tumor cells inoculation (n = 14–20 cells from six rats per group). (F) and (H): Representative traces of α,β-meATP-induced P2X3R-currents (F) and neuronal action potentials (H) are shown. (J and K) Assessment of ipsilateral PWT (J) and PWL (K) from tumor-bearing rats received intrathecal SCH772984 or vehicle treatment (n = 10 rats per group). (L) Assessment of animal’s locomotor function before and after drug administration (n = 10 rats per group). Data are presented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.001, one-way ANOVA with Tukey’s post hoc test for (A), (B); unpaired t test for (C)–(I); repeated-measures two-way ANOVA with Sidak’s post hoc test for (J) and (K); two-way ANOVA with Sidak’s post hoc test for (L). See also Figure S3 .

Article Snippet: The membranes were blocked with 5% nonfat milk or bovine serum albumin in Tris-buffered saline and Tween [TBST, 20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature, and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-rat P2X3R antibody (1:1000, Alomone Labs), rabbit anti-rat pERK1/2 antibody (1:1000, Cell Signaling Technology), rabbit anti-rat ERK1/2 antibody (1:2000, Cell Signaling Technology), rabbit anti-rat pRunx1 Ser249 (1:500, Cell Signaling Technology), rabbit anti-Runx1 (1:1000, Abcam), rabbit anti-GFRα1 (1:1000, Santa Cruz), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology), or mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology).

Techniques: Activation Assay, Functional Assay, Western Blot, Quantitative RT-PCR

Journal: iScience

Article Title: Activation of GDNF-ERK-Runx1 signaling contributes to P2X3R gene transcription and bone cancer pain

doi: 10.1016/j.isci.2022.104936

Figure Lengend Snippet:

Article Snippet: The membranes were blocked with 5% nonfat milk or bovine serum albumin in Tris-buffered saline and Tween [TBST, 20 mM tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] for 60 min at room temperature, and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-rat P2X3R antibody (1:1000, Alomone Labs), rabbit anti-rat pERK1/2 antibody (1:1000, Cell Signaling Technology), rabbit anti-rat ERK1/2 antibody (1:2000, Cell Signaling Technology), rabbit anti-rat pRunx1 Ser249 (1:500, Cell Signaling Technology), rabbit anti-Runx1 (1:1000, Abcam), rabbit anti-GFRα1 (1:1000, Santa Cruz), mouse anti-β-actin (1:2000, Santa Cruz Biotechnology), or mouse anti-GAPDH (1:2000, Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Staining, Western Blot, Recombinant, Modification, Bicinchoninic Acid Protein Assay, Mutagenesis, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Transfection, Software

Journal: Neurobiology of Pain

Article Title: The naked mole-rat has a functional purinergic pain pathway despite having a non-functional peptidergic pain pathway

doi: 10.1016/j.ynpai.2020.100047

Figure Lengend Snippet: Inhibiting P2X3R Reduces Naked Mole-Rat Pain Response to Formalin. Inhibition of P2X3r decreases pain responses induced by formalin in naked mole-rats in Phase I (A) and Phase II (B) but has no effect on those responses in mice. P2X3r Ant = Antagonist A-317491. (n = 8 mice and NMR saline control; 4 mice and 5 NMR with antagonist; * p < .05, *** p < .001, Statistical analysis was performed using a paired T-test).

Article Snippet: Primary antibodies used were rabbit anti-P2X3r polyclonal (1:1000, Alomone) and guinea pig anti-TRPV1 polyclonal (1:500, Alomone).

Techniques: Inhibition

Journal: Neurobiology of Pain

Article Title: The naked mole-rat has a functional purinergic pain pathway despite having a non-functional peptidergic pain pathway

doi: 10.1016/j.ynpai.2020.100047

Figure Lengend Snippet: P2X3R is Functional in Cultured Naked Mole-Rat Dorsal Root Ganglion Neurons. Functionality of P2X3r was assessed with IHC and Ca 2+ -imaging. Example images of P2X3r and TRPV1 expression in DRG neurons of mice (A, n = 3) and naked mole-rats (C, n = 3). Quantification of neurons by cell area in mice (B) and naked mole-rats (D) shows a similar distribution of P2X3r and TRPV1 expression/co-expression but note the enhanced peak of small diameter fibers in mouse compared to naked mole-rat. Quantification of P2X3r expression in mouse and naked mole-rat DRG reveals no significant difference (E, n = 3). Responsiveness of mouse and naked mole-rat DRG neurons to the P2X3r agonist αβ-meATP is similar across species (F, n = 4). Statistical analysis of data in E and F was performed using Mann Whitney U test.

Article Snippet: Primary antibodies used were rabbit anti-P2X3r polyclonal (1:1000, Alomone) and guinea pig anti-TRPV1 polyclonal (1:500, Alomone).

Techniques: Functional Assay, Cell Culture, Imaging, Expressing, MANN-WHITNEY

Journal: Neurobiology of Pain

Article Title: The naked mole-rat has a functional purinergic pain pathway despite having a non-functional peptidergic pain pathway

doi: 10.1016/j.ynpai.2020.100047

Figure Lengend Snippet: CB1r and P2X3r are Co-expressed in Lamina II of the Dorsal Horn of Mice and Naked Mole-Rat Spinal Cords Example confocal images of CB1r and P2X3r expression in the dorsal horn of the spinal cord in mice (A) and naked mole-rats (B). Scale bar 25 µm.

Article Snippet: Primary antibodies used were rabbit anti-P2X3r polyclonal (1:1000, Alomone) and guinea pig anti-TRPV1 polyclonal (1:500, Alomone).

Techniques: Expressing

Journal: Molecular Pain

Article Title: Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat

doi: 10.1177/1744806919847810

Figure Lengend Snippet: SNL-induced changes in P2X3R expression in ipsilateral L4 and L5 DRG were altered by EA stimulation. (a) Examples of P2X3R expressions in different rat groups. (b) EA-induced changes in P2X3R protein expression in L4 DRGs. Following SNL+ s-EA stimulation, P2X3R expression was 1.4 times (= 1.1/0.77) higher than that in control rats. The expression was retuned close to control level (0.7/0.77 = 0.9) after EA stimulation. (c) EA-induced changes P2X3R protein expressions in L5 DRGs. The expression of P2X3R was much reduced after SNL+s-EA treatment (0.2/0.97 = 0.21) or after SNL+EA treatment (0.16/0.97 = 0.17). (n = 6 for each rat group, * P < 0.01). DRG: dorsal root ganglion; EA: electroacupuncture; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: The samples were then electro-transferred to a polyvinylidene difluoride membrane, which was subsequently incubated in a Tris-buffered saline blocking buffer containing 5% w/v fat-free dry milk at room temperature (RT) for 1 h. The membrane was then immunoblotted with rabbit anti-P2X3R primary antibody (1:1000, Alomone Labs) at 4°C, overnight followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (1:10000, Abcam) at RT for 1 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), probed with rabbit anti-GAPDH antibody (1:1000, Cell Signaling Technilogy), was used as loading control.

Techniques: Expressing

Journal: Molecular Pain

Article Title: Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat

doi: 10.1177/1744806919847810

Figure Lengend Snippet: The effect of immunohistochemical study of P2X3R-ir and ATF3-ir expression in L4 DRG neurons. (a) P2X3R-ir (red) + ATF3-ir (green) double labeled DRG neurons in Control (Con), SNL+ s-EA (s-EA) and SNL+EA (EA) rats. Bar =100 µm. (b) Percentages of P2X3R+ expressing neurons in Control and SNL+ s-EA rats were similar (28.24 ± 1.69% in Con vs. 26.83 ± 0.9% in s-EA rats), The percentage of P2X3R+ neurons in SNL+ EA rats was significantly reduced (15.37 ± 1.75%). (c) Integrated optical density measured in SNL+s-EA rats ((9.21 ± 1.08) × 10 5 ) was higher than that in control rats ((6.64 ± 0.75) ×10 5 ) and much reduced ((2.81 ± 0.39) × 10 5 ) in SNL+EA rats. (d) The percentage of ATF+ neurons in SNL+ s-EA and in SNL+EA rats were not significantly different (6.7 ± 1.7 vs. 6.1 ± 0.6%, P > 0.05). Both were higher than control rats (0%). (e) Almost no P2X3+/ATF3+ neurons were expressed in the three rat groups. Thus, all of the P2X3R+ were expressed in un-injured neurons (Con: n = 7, SNL+ s-EA: n = 3, SNL+EA: n = 3). * P < 0.01. EA: electroacupuncture; ATF3: activating transcription factor 3; IOD: integrated optical density.

Article Snippet: The samples were then electro-transferred to a polyvinylidene difluoride membrane, which was subsequently incubated in a Tris-buffered saline blocking buffer containing 5% w/v fat-free dry milk at room temperature (RT) for 1 h. The membrane was then immunoblotted with rabbit anti-P2X3R primary antibody (1:1000, Alomone Labs) at 4°C, overnight followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (1:10000, Abcam) at RT for 1 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), probed with rabbit anti-GAPDH antibody (1:1000, Cell Signaling Technilogy), was used as loading control.

Techniques: Immunohistochemical staining, Expressing, Labeling

Journal: Molecular Pain

Article Title: Electroacupuncture downregulates P2X3 receptor expression in dorsal root ganglia of the spinal nerve-ligated rat

doi: 10.1177/1744806919847810

Figure Lengend Snippet: EA stimulation did not affect P2X3R or ATF3 expression in injured neurons ipsilateral to L5 DRGs. (a) Examples of P2X3R-ir + ATF3-ir double labeled neurons in L5 DRGs of three rat groups, Bar = 100 µm. (b) Compared to control rats, L5 nerve ligation induced a substantial reduction in P2X3R+ expression in DRGs of SNL+ s-EA or SNL+EA rats (from 29.0 ± 2.1% to 13.7 ± 4.9% and to 11.0 ± 3.3% respectively). (c) Unlike the minimal expression of ATF3+ cells (0.4 ± 0.2%) in L5 DRG of control rats, a majority of L5 DRG neurons in SNL+ s-EA rat group (85.7 ± 1.6%) and in SNL+EA group (81.7 ± 3.1%) were ATF3+. (d) Most P2X3Rs in injured L5 DRGs were expressed in ATF3+ neurons (P2X3R/ATF3+ =10.6% in SNL+ s-EA rats and =8.8% in SNL+EA rats). (e) The cell count of P2X3R+/ATF3− cells was reduced from 30.0 ± 2.0% in control L5 DRGs to 2.7 ± 0.8% in SNL+ s-EA and 1.8 ± 0.5% in SNL+EA rats. EA stimulation did not reverse the reduction in P2X3R expression induced by SNL (Con: n = 7, SNL+ s-EA: n = 3, SNL+EA: n = 3). * P < 0.01. EA: electroacupuncture; ATF3: activating transcription factor 3.

Article Snippet: The samples were then electro-transferred to a polyvinylidene difluoride membrane, which was subsequently incubated in a Tris-buffered saline blocking buffer containing 5% w/v fat-free dry milk at room temperature (RT) for 1 h. The membrane was then immunoblotted with rabbit anti-P2X3R primary antibody (1:1000, Alomone Labs) at 4°C, overnight followed by horse radish peroxidase-conjugated goat anti-rabbit IgG (1:10000, Abcam) at RT for 1 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), probed with rabbit anti-GAPDH antibody (1:1000, Cell Signaling Technilogy), was used as loading control.

Techniques: Expressing, Labeling, Ligation, Cell Counting

Journal: Pain

Article Title: Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

doi: 10.1097/j.pain.0000000000001021

Figure Lengend Snippet: Confocal immunofluorescence of primary cultured neurons of 2 DIV from mouse TG (A and C) and DRG (B and D), expressing endogenous levels of P2X2R (green), p35 and Cdk5 (Red). Neurons were immunostained with βIII-tubulin or Map1B (white). Arrows show TG or DRG neurons positive for P2X2R staining. E. Magnification of representative neurites from cultured TG neurons stained with P2X2R (green) and p35 (red). F. Magnification of representative neurites from cultured TG neurons stained with P2X2R (green) and Cdk5 (red). G. Representative RT-PCR from total RNA obtained from mouse TG and DRG tissues. P2X2aR, P2X2bR, P2X2eR, P2X3R, p35 and Cdk5 mRNA levels were evaluated. S29 levels were used as housekeeping control. H. Representative Western blots of protein extract from mouse TG, DRG, and brain tissues showing P2X2R, P2X3R, p35, Cdk5 and βIII-tubulin expression. I. Immunoprecipitation experiments of mouse TG protein extracts with Cdk5 antibody and detected by Western blot for P2X2R. As a positive control, the corresponding Cdk5 bands were detected after stripping the membrane. For a control with no antibody, we only used Protein A/G agarose beads and we did not find the P2X2R in those immunocomplexes.

Article Snippet: Western blots were performed using P2X2R rabbit antibody #APR-003 and P2X3R rabbit antibody #APR-016 from Alomone Labs (Jerusalem, Israel); Cdk5 rabbit antibody C8, Cdk5 mouse antibody DC17, p35 rabbit antibody C19, and p35 goat antibody A18 from Santa Cruz Biotechnology (Dallas, TX); Phospho-(Thr) MAPK/CDK substrate mouse antibody #2321, GFP rabbit antibody #2555, and p35 #2680 were obtained from Cell Signaling Technology; and α-tubulin antibody from Sigma-Aldrich (San Louis, MO) was used as loading control.

Techniques: Immunofluorescence, Cell Culture, Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Positive Control, Stripping Membranes

Journal: Pain

Article Title: Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

doi: 10.1097/j.pain.0000000000001021

Figure Lengend Snippet: A. Representative recordings evoked by 100 μM ATP on PC12 cells in the absence (black tracing), after 6 h (gray tracing) or 24 h (red tracing) incubation with 30 μM roscovitine. Membrane voltage was held at −60 mV. B. Summary of desensitization constants (τdes) calculated for PC12 cells in control conditions (C) and after 6 and 24 h of incubation with roscovitine (Rosc). * p<0.05, Man-Whitney test, n=5. C. Summary of current densities obtained in control (C) and roscovitine-treated (Rosc) PC12 cells. n=5. D. Representative recordings of a HEK293 cell expressing the P2X2/3R alone or co-transfected with p35 and the typical slow-desensitizing current (as compared to the fast-desensitizing P2X3R-mediated current) evoked by 10 μM α,β-meATP. E. Summary of the desensitization constants (τdes) of the currents mediated by the P2X2/3R alone (C) or co-transfected with p35 (+p35). n=4–8; *p<0.05, Mann-Whitney test. F. Summary of current densities obtained in the absence (C) and in the presence of p35 (+p35) in P2X2a/3R-expressing HEK293 cells. n=3–5.

Article Snippet: Western blots were performed using P2X2R rabbit antibody #APR-003 and P2X3R rabbit antibody #APR-016 from Alomone Labs (Jerusalem, Israel); Cdk5 rabbit antibody C8, Cdk5 mouse antibody DC17, p35 rabbit antibody C19, and p35 goat antibody A18 from Santa Cruz Biotechnology (Dallas, TX); Phospho-(Thr) MAPK/CDK substrate mouse antibody #2321, GFP rabbit antibody #2555, and p35 #2680 were obtained from Cell Signaling Technology; and α-tubulin antibody from Sigma-Aldrich (San Louis, MO) was used as loading control.

Techniques: Incubation, Expressing, Transfection, MANN-WHITNEY

Journal: Pain

Article Title: Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372

doi: 10.1097/j.pain.0000000000001021

Figure Lengend Snippet: Confocal immunofluorescence of primary cultured neurons of 2 DIV from mouse TG (A) and DRG (B), expressing endogenous levels of P2X2R (red) and P2X3R (green). Neurons were immunostained with βIII-tubulin (white) and Dapi was used as nuclear staining. Arrows indicates neurons expressing heteromeric P2X2/3R subtype. C. Representative fields of cultured TG neurons loaded with Fluo-4 before (left image) and after (right image) the addition of 100 μM ATP. D. Summary of the percentage of responding cells to ATP and α,β-meATP (α,β). n=3, *p<0.05, student’s t test. E. Typical Ca2+ spikes induced by 100 μM ATP. F. Representative Ca2+ spikes induced by 100 μM α,β-meATP without (control) or with a 6-hour roscovitine treatment (rosc). G–I. Summary of the results obtained in TG neurons without (C) or with the roscovitine treatment (rosc) for decay constant (G), percentage of responding cells (H) and relative amplitude (I). n=20–71; *p<0.05, Mann-Whitney test.

Article Snippet: Western blots were performed using P2X2R rabbit antibody #APR-003 and P2X3R rabbit antibody #APR-016 from Alomone Labs (Jerusalem, Israel); Cdk5 rabbit antibody C8, Cdk5 mouse antibody DC17, p35 rabbit antibody C19, and p35 goat antibody A18 from Santa Cruz Biotechnology (Dallas, TX); Phospho-(Thr) MAPK/CDK substrate mouse antibody #2321, GFP rabbit antibody #2555, and p35 #2680 were obtained from Cell Signaling Technology; and α-tubulin antibody from Sigma-Aldrich (San Louis, MO) was used as loading control.

Techniques: Immunofluorescence, Cell Culture, Expressing, Staining, MANN-WHITNEY

Journal: Molecular Pain

Article Title: F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

doi: 10.1177/1744806916660557

Figure Lengend Snippet: CPT increases the membrane expression of P2X3Rs in cultured DRGs. The P2X3R levels in total protein (Total), membrane protein (Mem), and cytoplasmic protein samples (Cytosol) before (Con) and after CPT (1 µM, 10 min) treatment were determined. Total, membrane, and cytosol protein samples were loaded onto gels. The expression of the ubiquitous membrane protein Na+/K+ ATPase was probed to indicate the membrane expressed protein levels in our samples. The expressions of β-tubulin used as sample loading controls were also indicated. CPT significantly increased P2X3Rs expressed in the cell membrane (total protein: CPT/Con = 1.19; membrane protein: CPT/Con = 1.93 and cytoplasmic protein: CPT/Con = 0.75). N = 3. * P < 0.05.

Article Snippet: Immediately after treatment, DRG cells were fixed with 4% paraformaldehyde at room temperature for 20 min, washed with phosphate-buffered saline (PBS), and blocked with PBS containing 5% normal goat serum for 30 min. To determine the membrane expression of P2X3Rs in DRG cells, the primary antibody rabbit anti-P2X3R extracellular epitope, (ext-P2X3R), (1:100, Alomone Lab, Jerusalem), was used.

Techniques: Expressing, Cell Culture

Journal: Molecular Pain

Article Title: F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

doi: 10.1177/1744806916660557

Figure Lengend Snippet: Epac control of the membrane expression of P2X3Rs in DRG neurons is F-actin mediated. (a) The membrane expression of P2X3Rs was examined in cultured DRG neurons using anti-ext-P2X3R antibody. The enlarged views of neurons (indicated by red arrows) are shown on the right side of each panel. Bars = 25 µm. (b) In the presence of CPT (1 µM), the percentage of cells expressing ext-P2X3R increased by 1.80 fold and the intensity of ext-P2X3R labels increased by 5.57 fold. The CPT-induced increase in membrane expressed P2X3Rs was significantly reduced by pre-incubating cells with the F-actin disrupter, LaA (1 µM, 60 min). A total of 1192 cells obtained from three experiments were used for the analyses. * P < 0.05, # P < 0.01. NS: not significant.

Article Snippet: Immediately after treatment, DRG cells were fixed with 4% paraformaldehyde at room temperature for 20 min, washed with phosphate-buffered saline (PBS), and blocked with PBS containing 5% normal goat serum for 30 min. To determine the membrane expression of P2X3Rs in DRG cells, the primary antibody rabbit anti-P2X3R extracellular epitope, (ext-P2X3R), (1:100, Alomone Lab, Jerusalem), was used.

Techniques: Expressing, Cell Culture

Journal: Molecular Pain

Article Title: F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

doi: 10.1177/1744806916660557

Figure Lengend Snippet: CPT increases the membrane expression of P2X3Rs in cultured DRGs. The P2X3R levels in total protein (Total), membrane protein (Mem), and cytoplasmic protein samples (Cytosol) before (Con) and after CPT (1 µM, 10 min) treatment were determined. Total, membrane, and cytosol protein samples were loaded onto gels. The expression of the ubiquitous membrane protein Na+/K+ ATPase was probed to indicate the membrane expressed protein levels in our samples. The expressions of β-tubulin used as sample loading controls were also indicated. CPT significantly increased P2X3Rs expressed in the cell membrane (total protein: CPT/Con = 1.19; membrane protein: CPT/Con = 1.93 and cytoplasmic protein: CPT/Con = 0.75). N = 3. * P < 0.05.

Article Snippet: Antibodies used were rabbit anti-pPKC ɛ (1:1000, Santa Cruz, Dallas, TX) and rabbit anti-P2X3R (1:2000, Alomone Labs).

Techniques: Expressing, Cell Culture

Journal: Molecular Pain

Article Title: F-actin links Epac-PKC signaling to purinergic P2X3 receptor sensitization in dorsal root ganglia following inflammation

doi: 10.1177/1744806916660557

Figure Lengend Snippet: Epac control of the membrane expression of P2X3Rs in DRG neurons is F-actin mediated. (a) The membrane expression of P2X3Rs was examined in cultured DRG neurons using anti-ext-P2X3R antibody. The enlarged views of neurons (indicated by red arrows) are shown on the right side of each panel. Bars = 25 µm. (b) In the presence of CPT (1 µM), the percentage of cells expressing ext-P2X3R increased by 1.80 fold and the intensity of ext-P2X3R labels increased by 5.57 fold. The CPT-induced increase in membrane expressed P2X3Rs was significantly reduced by pre-incubating cells with the F-actin disrupter, LaA (1 µM, 60 min). A total of 1192 cells obtained from three experiments were used for the analyses. * P < 0.05, # P < 0.01. NS: not significant.

Article Snippet: Antibodies used were rabbit anti-pPKC ɛ (1:1000, Santa Cruz, Dallas, TX) and rabbit anti-P2X3R (1:2000, Alomone Labs).

Techniques: Expressing, Cell Culture