Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: (A) (i) Use of TRPV4 channel gene specific primer amplified a PCR product of 351-bp as determined by semiquantitative RT-PCR. Shown in (ii) is the internal control GAPDH. (B) Western blot analysis using homogenates of freshly isolated FHH rat cerebral arterial myocytes lysates and the polyclonal anti-TRPV4 antibody (Alomone ACC-034 at 1:200) detected expression of the right molecular size for TRPV4 channel protein (n = 2–3 separate trials for each group).

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Isolation, Expressing

Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: (A) Characterization and differentiation by immunofluorescent staining of a population of freshly isolated cerebral arterial myocytes using the nuclei stain DAPI, the endothelial cell marker anti-CD31 (PECAM-1) antibody and anti-smooth muscle α-actin (Anti-SM-α-actin) antibody visualized with donkey anti-rabbit Alexa Fluor 488-conjugated secondary antibody. These findings revealed that population of freshly isolated cerebral arterial myocytes obtained using the method described in this study are smooth muscle α-actin positive cells that are free of endothelial cell contamination. (B) Depiction of single freshly isolated rat cerebral arterial myocyte stained with DAPI alone (nuclei stained blue) or co-stained with either anti-SM-α-actin antibody (red) or with anti-TRPV4 antibody and visualized with donkey anti-rabbit Alexa Fluor 488-conjugated secondary antibody. The image of the arterial myocyte stained with anti-TRPV4 antibody demonstrates expression of TRPV4 channel protein in the cerebral arterial myocyte positively stained with anti-SM-α-actin antibody.

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Staining, Isolation, Marker, Expressing

Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: Immunohistochemically detection of expression of TRPV4 channel protein using 4 μm sections of FHH rat cerebral arterial segment by treatment with either a secondary antibody alone (secondary antibody Donkey anti-Rabbit Alexa Fluor 488, 1:750, Thermo Fisher Scientific, upper panel) or with a primary polyclonal anti-TRPV4 antibody (Alomone ACC-034, 1:200, lower panel) revealing expression of TRPV4 protein in FHH rat cerebral arterial muscle cells as shown by punctate staining pattern both at the intima and media layers of the arterial wall. Blue: indicates nuclear staining with DAPI of the fixed arterial sections.

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: Single-channel cationic currents recorded from FHH rat cerebral arterial myocytes exhibited two slope conductances: 85 ± 2 pS at more hyperpolarizing potentials and 96 ± 3 pS at more depolarizing potentials. (A) The single-channel cationic currents recorded at patch holding potential (PP) of 60 mV were sensitive to inhibition by the TRPV4 channel antagonist RN 1734 (5 μM) (B) or by HC 067047 (300 nM) (C) (n = 5–6 cells).

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Inhibition

Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: The TRPV4-like cationic single-channel current recorded in cell-attached patches of FHH rat cerebral arterial myocytes was activated by the specific TRPV4 channel agonist GSK1016790A (100 nM) that was reduced or suppressed by the TRPV4 channel inhibitor HC 067047 (300 nM) (A and B). * denotes statistically significant difference from control at P < 0.05, n = 5–6 cells).

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Control

Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: (A) Representative single TRPV4-like single-channel cationic currents recorded at a holding potential of 60 mV from cell attached patches of cerebral arterial myocytes shown in slower (a) and faster time spans (b) under control condition and following membrane stretch by application of negative pressure of 10 mm H 2 O or 30 mm H 2 O and (c) attenuation of the 30 mm H 2 O negative pressure induced activation of the single-channel cationic current by pretreatment with the TRPV4 channel inhibitor HC 067047. (B) Summary of application of a negative pressure of 10 cm H 2 O or 30 cm H 2 O induced marked increase in the open state probability of (NPo) of the TRPV4-like single-channel cationic current and the attenuation of the negative pressure of 30 mm H20 induced increase in NPo following treatment with the TRPV4 channel inhibitor HC 067047 (300 nM) (*, † , P < 0.05, n = 5–6 cells for each group).

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Control, Membrane, Activation Assay

Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: (A) Examples of single-channel current traces recorded at a patch holding potential of 40 mV using symmetrical (145 mM KCl) recording solution under control condition and following treatment with 10 nM or 100 nM GSK1016790A. Application of GSK1016790A induced concentration-dependent increase in the opening frequency of the single-channel K Ca currents that was attenuated by treatment with the K Ca channel blocker paxilline. (B) Summary of the effects of treatment with 10 nM or 100 nM GSK1016790A on the open state probability (NPo) of the K Ca single-channel currents recorded at a patch holding potential of 40 mV. Treatment with 10 nM or 100 nM GSK1016790A significantly increased the NPo of the K Ca single-channel currents compared to the control NPo value that was attenuated by treatment with the K Ca channel inhibitor paxilline. (*,† denote P < 0.05, n = 5–6 cells for each group; c and o represent closed and open states of the channel, and 1,2,3 indicate stocked channel opening levels).

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Control, Concentration Assay

Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: (A) Representative K Ca single-channel current traces recorded at a patch holding potential of 40 mV from cell-attached patches under control condition and following treatment with 100 nM GSK1016790A before and after addition of 1 μM nifedipine or the K Ca channel inhibitor 1 μM paxilline to the bath. Treatment with 100 nM GSK1016790A increased the opening frequency of the K Ca single-channel currents that was not influenced by treatment with the L-type Ca 2+ channel inhibitor 1 μM nifedipine, but attenuated by the K Ca channel inhibitor 1 μM paxilline. (B) Summary of the effects of treatment with 100 nM GSK1016790A on the NPo of the K Ca single-channel currents in the absence and presence of the L-type Ca 2+ channel inhibitor 1 μM nifedipine or the K Ca channel inhibitor paxilline (1 μM). Treatment with 100 nM GSK1016790A induced a significant increase in NPo of the K Ca single-channel current that was not affected by pretreatment with the L-type Ca 2+ channel inhibitor 1 μM nifedipine, but attenuated in the presence of the K Ca channel inhibitor 1 μM paxilline. (*,† denote P < 0.05, **denotes P > 0.05, n = 5–6 cells for each group, c and o represent closed and open states of the channel and 1,2,3 indicate stocked channel opening levels).

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Control

Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: (A) Representative single-channel current traces recorded at a patch holding potential of 40 mV from cell attached patches under control condition and following treatment with 100 nM GSK1016790A before and after addition of the T-type Ca 2+ channel inhibitor 50 μM nickel (Ni 2+ ) or the K Ca channel inhibitor 1 μM paxilline to the bath. Treatment with 100 nM GSK1016790A increased the opening frequency of the K Ca single-channel currents that was not influenced by pretreatment with the T-type Ca 2+ channel inhibitor 50 μM Ni 2+ but attenuated by the K Ca channel inhibitor 1 μM paxilline. (B) Summary of the effects of treatment with 100 nM GSK1016790A on the NPo of single-channel K Ca currents in the absence and presence of the T-type Ca 2+ channel inhibitor 50 μM Ni 2+ or the K Ca channel inhibitor paxilline (1 μM). Treatment with 100 nM GSK1016790A induced significant increase in NPo of the K Ca single-channel current that was not affected by pretreatment with the T-type Ca 2+ channel inhibitor 50 μM Ni 2+ , but attenuated in the presence of the K Ca channel inhibitor 1 μM paxilline. (*,† denote P < 0.05, ** denotes P > 0.05, n = 5–6 cells for each group; c and o represent closed and open states of the channel, and 1,2,3 indicate stocked channel opening levels).

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Control

Journal: PLoS ONE

Article Title: Detection of TRPV4 channel current-like activity in Fawn Hooded hypertensive (FHH) rat cerebral arterial muscle cells

doi: 10.1371/journal.pone.0176796

Figure Lengend Snippet: Pretreatment with the specific TRPV4 channel inhibitor HC067047 (1 μM) had no significant effect on increases in intraluminal pressure induced reductions in diameter of the cerebral arterial segments of SD rat (A) and the FHH rat (B) (n = 5). In Ca 2+ -free PSS bath solution the cannulated cerebral arterial segments of FHH rat exhibited an average passive dilation of 11.2 ± 2.2% whereas the Sprague Dawley rats elicited a mean passive dilation of 31.8 ± 5% (n = 5, < 0.05). Presented in (C) is Western blot showing the level of expression of TRPV4 channel protein with bar graphs depicting summary of the normalized TRPV4 channel protein with the internal control β-actin determined using brain homogenates of SD and FHH rats which are not significantly different (n = 3 independent trials).

Article Snippet: The patch potential (60 mV) at which moderate single-channel cationic current opening was observed was chosen to study the effects of specific pharmacological antagonist of the TRPV4 channel HC 067047 or RN 1734 [ – ] (Tocris Biosciences) and the TRPV4 channel agonist GSK1016790A [ ] (Sigma-Aldrich, MO) that have been previously shown to selectively inhibit or activate TRPV4 channel current, respectively, in expression systems [ , , ].

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Increased WIP1 Expression With Aging Suppresses the Capacity of Oocytes to Respond to and Repair DNA Damage

doi: 10.3389/fcell.2021.810928

Figure Lengend Snippet: WIP1 inhibition promotes DNA damage repair in aged oocytes. (A–C) After treatment with etoposide (ETP) or DMSO as a control (CTL), oocytes aged in vitro for 24 h were cultured in ETP-free medium for 1 h with GSK2830371 (GSK) or DMSO. The intensities of γ -H2AX (A) , MDC1 (B) , and TUNEL (C) signals were normalized to the mean DAPI intensity, and representative images are shown. DNA was counterstained with DAPI. Scale bar, 10 μm ** p < 0.001; *** p < 0.0001, calculated using one-way ANOVA with Tukey’s post hoc test. (D–F) Oocytes aged in vitro were co-treated with an ATM inhibitor (ATMi) and GSK2830371 (GSK) during recovery from etoposide (ETP). The intensities of γ -H2AX (D) , MDC1 (E) and TUNEL (F) signals were normalized to the mean DAPI intensity, and representative images are shown. DNA was counterstained with DAPI. Scale bar, 10 μm. Data are presented as the mean ± SEM of three independent experiments. *** p < 0.0001.

Article Snippet: For in vitro maturation, GV oocytes were cultured in IBMX-free M2 medium covered with mineral oil at 37°C in a 5% CO 2 atmosphere for 16 h. To induce DNA damage, MII oocytes were exposed to 50 μg/ml etoposide for 15 min. For recovery, the etoposide-treated oocytes were washed and cultured in fresh M2 medium for 1 h with or without 5 μM GSK2830371 (Tocris).

Techniques: Inhibition, Control, In Vitro, Cell Culture, TUNEL Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Increased WIP1 Expression With Aging Suppresses the Capacity of Oocytes to Respond to and Repair DNA Damage

doi: 10.3389/fcell.2021.810928

Figure Lengend Snippet: Prolonged inhibition of WIP1 during in vitro aging improves oocyte quality. MII oocytes were cultured in vitro for 24 h with GSK2830371 (GSK) or DMSO. (A,B) The intensities of TUNEL and γ -H2AX signals normalized to the mean DAPI intensity are shown using representative images. DNA was counterstained with DAPI. Scale bar, 10 μm. (C) Oocyte fragmentation was scored, and representative images are shown. Scale bar, 100 μm. (D) The intensity of Annexin V signals was scored, and representative images are shown. Scale bar, 20 μm. (E) The intensity of mitochondrial staining was scored, and representative images are shown. Scale bar, 20 μm. (F) The rate of 2-cell and blastocyst development is shown using representative images taken at 24 h (E2.5) and 72 h (E4.5) of culture after ICSI, respectively. Scale bar, 100 μm. Data are presented as the mean ± SEM from three independent experiments. * p < 0.05; ** p < 0.001; *** p < 0.0001.

Article Snippet: For in vitro maturation, GV oocytes were cultured in IBMX-free M2 medium covered with mineral oil at 37°C in a 5% CO 2 atmosphere for 16 h. To induce DNA damage, MII oocytes were exposed to 50 μg/ml etoposide for 15 min. For recovery, the etoposide-treated oocytes were washed and cultured in fresh M2 medium for 1 h with or without 5 μM GSK2830371 (Tocris).

Techniques: Inhibition, In Vitro, Cell Culture, TUNEL Assay, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Increased WIP1 Expression With Aging Suppresses the Capacity of Oocytes to Respond to and Repair DNA Damage

doi: 10.3389/fcell.2021.810928

Figure Lengend Snippet: WIP1 inhibition increases the DNA repair capacity of oocytes from aged mice. (A) Increased WIP1 levels in maternally aged oocytes. MII oocytes were collected from young (8-week-old) and old (12-month-old) mice and subjected to immunoblot analysis using the WIP1 antibody, with ß -actin as a loading control. Each lane contains 50 oocytes. (B,C) Oocytes from young and old mice were treated with etoposide (ETP) or DMSO as a control (CTL) for 15 min. For recovery, the aged oocytes were cultured in ETP-free medium with GSK2830371 (GSK) or DMSO for 1 h after ETP exposure. The intensities of γ -H2AX and TUNEL, normalized to the mean DAPI intensity, are shown using representative images. DNA was counterstained with DAPI. Scale bar, 10 μm. Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test and are expressed as the mean ± SEM of two independent experiments. ** p < 0.001; *** p < 0.0001; ns, not significant. (D) A diagram showing the relationship between the aging-associated increase in WIP1 levels and the decline in DNA repair capacity in aged oocytes.

Article Snippet: For in vitro maturation, GV oocytes were cultured in IBMX-free M2 medium covered with mineral oil at 37°C in a 5% CO 2 atmosphere for 16 h. To induce DNA damage, MII oocytes were exposed to 50 μg/ml etoposide for 15 min. For recovery, the etoposide-treated oocytes were washed and cultured in fresh M2 medium for 1 h with or without 5 μM GSK2830371 (Tocris).

Techniques: Inhibition, Western Blot, Control, Cell Culture, TUNEL Assay