Journal: Frontiers in Physiology

Article Title: A sex-dependent role of Kv1.3 channels from macrophages in metabolic syndrome

doi: 10.3389/fphys.2024.1487775

Figure Lengend Snippet: Electrophysiological characterization of P2X 7 and inward rectifier K + currents from BMDM. (A) Representative currents obtained from a female control BMDM upon application of either BzATP (750 µM) or 2 mM ATP during the indicated time to show the kinetics and amplitude of the P2X7-mediated currents. Holding potential was kept at 0 mV. (B) The right panels show de average current amplitude elicited by 750 µM BzATP in BMDM from the indicated groups obtained from female (red scale) or male (gray scale) mice. Each column is mean ± SEM of 12–20 cells in each group, obtained from 4 to 6 different animals. A two-way ANOVA did not show significant differences. (C) Representative recordings of the current elicited in one control and one LPS-treated BMDM SD male with voltage ramps from a holding potential of −80 mV with control solution (black traces) or in the presence of a baths solution containing 100 nM PAP-1 and 100 µM BaCl 2 to block Kv1.3 and Kir2.1 currents respectively (red traces). (D) The amplitude of the BaCl 2 -sensitive current was measured at −120 mV, and the averaged values obtained in all experimental conditions are plotted in the right graphs, both in female (red color scale) and male (gray colors) BMDM. Statistical analyses were carried out with a two-way ANOVA followed by Tukey´s post-hoc test (in the females group) and with a Kruskal-Wallis analysis followed by Dunn’s test in the males group. Mean ± SEM, n = 8-12 cells per group from at least 4 different cultures.

Article Snippet: Purinergic currents were obtained in continuous recording from a holding potential of 0 mV, by perfusing macrophages with an external solution containing 2 mM ATP (Sigma # A-2383) or the specific P2X 7 activator-BzATP (700 μM; Alomone #: A-385).

Techniques: Control, Blocking Assay

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by GFAP + ependymo-radial glial cells. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of GFAP and P2X 7 labelling in naïve fish (A-C′), at 7 dpi (D-F′), and at 14 dpi (G-I′). Colocalization (arrows) was seen in radial processes and cell bodies (C′,F′,I′). GFAP + cells that did not colocalize with P2X 7 were also detected (open arrowheads) (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by PCNA + proliferating cells. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of PCNA + and P2X 7 labelling in naïve fish (A-C′), at 7 dpi rostral (D-F′), and at 7 dpi caudal (G-I′). Colocalization (arrows) was seen in some cells (C′,F′,I′). PCNA + cells that did not colocalize with P2X 7 were also detected (open arrowheads) (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by HuC/D + neurons. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of HuC/D + and P2X 7 labelling in naïve fish (A-C′), at 14 dpi rostral (D-F′), and at 14 dpi caudal (G-I′). Colocalization (arrows) was seen in larger mature neurons and smaller immature neurons (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: Temporal changes in P2X 7 protein expression following SCI in adult zebrafish. Schematic representations of naïve and injured spinal cord areas of analysis (A-B), as well as tissue collection timeline (C). Representative western blot and corresponding total protein in naïve zebrafish (Zf) spinal cord tissue, mouse (Ms) hippocampal tissue as a positive control (+), and pre-absorption of naïve zebrafish spinal cord tissue with a blocking peptide as a negative control (-) (D). Representative western blot and corresponding total protein at 1 dpi (E), 7 dpi (G), and 14 dpi (I). Quantitative analysis of the 50 kDa isoform showed significant downregulation within the injury (In, n =10, one-way ANOVA with Dunnett's post hoc test, P =0.0079) and caudal to the lesion (Ca, n =9, P =0.0046) at 7 dpi when normalized to naïve tissue ( n =9) (H). Protein expression of the 50 kDa isoform showed basal levels of expression at 1 dpi (F) and 14 dpi (J). Data presented as means±s.d. **, significant differences, P <0.01. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques: Expressing, Western Blot, Positive Control, Blocking Assay, Negative Control

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by GFAP + ependymo-radial glial cells. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of GFAP and P2X 7 labelling in naïve fish (A-C′), at 7 dpi (D-F′), and at 14 dpi (G-I′). Colocalization (arrows) was seen in radial processes and cell bodies (C′,F′,I′). GFAP + cells that did not colocalize with P2X 7 were also detected (open arrowheads) (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: The blocking agent was then removed and replaced with one of the following primary antibodies diluted in 0.1% Triton X-100/0.01 M PBS: P2X 7 (rabbit polyclonal; 1:500, Alomone Labs, APR-004, RRID: AB_2040068), GFAP (chicken polyclonal, 1:2000, ThermoFisher Scientific, PA1-10004, RRID: AB_1074620), PCNA (mouse monoclonal, 1:1000, Abcam, Cambridge, UK, ab29, RRID: AB_303394), or HuC/D (mouse monoclonal, 5 μg/ml, ThermoFisher Scientific, A-21271, RRID: AB_221448).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by PCNA + proliferating cells. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of PCNA + and P2X 7 labelling in naïve fish (A-C′), at 7 dpi rostral (D-F′), and at 7 dpi caudal (G-I′). Colocalization (arrows) was seen in some cells (C′,F′,I′). PCNA + cells that did not colocalize with P2X 7 were also detected (open arrowheads) (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: The blocking agent was then removed and replaced with one of the following primary antibodies diluted in 0.1% Triton X-100/0.01 M PBS: P2X 7 (rabbit polyclonal; 1:500, Alomone Labs, APR-004, RRID: AB_2040068), GFAP (chicken polyclonal, 1:2000, ThermoFisher Scientific, PA1-10004, RRID: AB_1074620), PCNA (mouse monoclonal, 1:1000, Abcam, Cambridge, UK, ab29, RRID: AB_303394), or HuC/D (mouse monoclonal, 5 μg/ml, ThermoFisher Scientific, A-21271, RRID: AB_221448).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by HuC/D + neurons. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of HuC/D + and P2X 7 labelling in naïve fish (A-C′), at 14 dpi rostral (D-F′), and at 14 dpi caudal (G-I′). Colocalization (arrows) was seen in larger mature neurons and smaller immature neurons (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: The blocking agent was then removed and replaced with one of the following primary antibodies diluted in 0.1% Triton X-100/0.01 M PBS: P2X 7 (rabbit polyclonal; 1:500, Alomone Labs, APR-004, RRID: AB_2040068), GFAP (chicken polyclonal, 1:2000, ThermoFisher Scientific, PA1-10004, RRID: AB_1074620), PCNA (mouse monoclonal, 1:1000, Abcam, Cambridge, UK, ab29, RRID: AB_303394), or HuC/D (mouse monoclonal, 5 μg/ml, ThermoFisher Scientific, A-21271, RRID: AB_221448).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: Temporal changes in P2X 7 protein expression following SCI in adult zebrafish. Schematic representations of naïve and injured spinal cord areas of analysis (A-B), as well as tissue collection timeline (C). Representative western blot and corresponding total protein in naïve zebrafish (Zf) spinal cord tissue, mouse (Ms) hippocampal tissue as a positive control (+), and pre-absorption of naïve zebrafish spinal cord tissue with a blocking peptide as a negative control (-) (D). Representative western blot and corresponding total protein at 1 dpi (E), 7 dpi (G), and 14 dpi (I). Quantitative analysis of the 50 kDa isoform showed significant downregulation within the injury (In, n =10, one-way ANOVA with Dunnett's post hoc test, P =0.0079) and caudal to the lesion (Ca, n =9, P =0.0046) at 7 dpi when normalized to naïve tissue ( n =9) (H). Protein expression of the 50 kDa isoform showed basal levels of expression at 1 dpi (F) and 14 dpi (J). Data presented as means±s.d. **, significant differences, P <0.01. Created with BioRender.com.

Article Snippet: The blocking agent was then removed and replaced with one of the following primary antibodies diluted in 0.1% Triton X-100/0.01 M PBS: P2X 7 (rabbit polyclonal; 1:500, Alomone Labs, APR-004, RRID: AB_2040068), GFAP (chicken polyclonal, 1:2000, ThermoFisher Scientific, PA1-10004, RRID: AB_1074620), PCNA (mouse monoclonal, 1:1000, Abcam, Cambridge, UK, ab29, RRID: AB_303394), or HuC/D (mouse monoclonal, 5 μg/ml, ThermoFisher Scientific, A-21271, RRID: AB_221448).

Techniques: Expressing, Western Blot, Positive Control, Blocking Assay, Negative Control