Structured Review

Millipore dids
HEK cells transfected with EYFP H148Q/I152L , KCNJ10, Clc‐kb, and barttin and exposed to different [KCl] o . Fluorescence at 2 mM KCl recorded from 1 to 200 s was adjusted to a baseline of 100%. For those wells where [KCl] o was changed, a baseline was obtained for the first 50 s in 2 mM KCl, after which the [KCl] o was increased from 2 mM through 5 mM KCl, respectively. (a) EYFP quenching at different [KCl] o . (b) Normalized response to KCl (Δ F / F o) plotted against final [Ko]. Data points are means of four different experiments. (c) Effect of different channel blockers, such as Ba 2+ (1 mM), Tertiapin Q (100 nM), <t>Valsartan</t> (100 µM), <t>DIDS</t> (200 µM) on EYFP quenching
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Images

1) Product Images from "Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K. Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K"

Article Title: Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K. Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K

Journal: Physiological Reports

doi: 10.14814/phy2.14280

HEK cells transfected with EYFP H148Q/I152L , KCNJ10, Clc‐kb, and barttin and exposed to different [KCl] o . Fluorescence at 2 mM KCl recorded from 1 to 200 s was adjusted to a baseline of 100%. For those wells where [KCl] o was changed, a baseline was obtained for the first 50 s in 2 mM KCl, after which the [KCl] o was increased from 2 mM through 5 mM KCl, respectively. (a) EYFP quenching at different [KCl] o . (b) Normalized response to KCl (Δ F / F o) plotted against final [Ko]. Data points are means of four different experiments. (c) Effect of different channel blockers, such as Ba 2+ (1 mM), Tertiapin Q (100 nM), Valsartan (100 µM), DIDS (200 µM) on EYFP quenching
Figure Legend Snippet: HEK cells transfected with EYFP H148Q/I152L , KCNJ10, Clc‐kb, and barttin and exposed to different [KCl] o . Fluorescence at 2 mM KCl recorded from 1 to 200 s was adjusted to a baseline of 100%. For those wells where [KCl] o was changed, a baseline was obtained for the first 50 s in 2 mM KCl, after which the [KCl] o was increased from 2 mM through 5 mM KCl, respectively. (a) EYFP quenching at different [KCl] o . (b) Normalized response to KCl (Δ F / F o) plotted against final [Ko]. Data points are means of four different experiments. (c) Effect of different channel blockers, such as Ba 2+ (1 mM), Tertiapin Q (100 nM), Valsartan (100 µM), DIDS (200 µM) on EYFP quenching

Techniques Used: Transfection, Fluorescence

2) Product Images from "Molecular Determinant of DIDS Analogs Targeting RAD51 Activity"

Article Title: Molecular Determinant of DIDS Analogs Targeting RAD51 Activity

Journal: Molecules

doi: 10.3390/molecules26185460

Self-association of RAD51 is inhibited in the presence of DIDS, SITS, and DAZ DS. ( A ) The polymerization of RAD51 was estimated by cross-link approach (see Material and Method section). ( B ) Oligomer and monomer forms ratio is presented as a histogram graph. Three independent experiments were performed and statistical analysis was performed using paired Student’s t -test. Statistical significance was assumed at ** p
Figure Legend Snippet: Self-association of RAD51 is inhibited in the presence of DIDS, SITS, and DAZ DS. ( A ) The polymerization of RAD51 was estimated by cross-link approach (see Material and Method section). ( B ) Oligomer and monomer forms ratio is presented as a histogram graph. Three independent experiments were performed and statistical analysis was performed using paired Student’s t -test. Statistical significance was assumed at ** p

Techniques Used:

Chemical structures of DIDS characterized by two isothiocyanate groups and its derivatives DAZDS including two azido groups, SITS including isothiocyanate and acetamide group, DADS including two amino groups and DNDS including two nitro groups.
Figure Legend Snippet: Chemical structures of DIDS characterized by two isothiocyanate groups and its derivatives DAZDS including two azido groups, SITS including isothiocyanate and acetamide group, DADS including two amino groups and DNDS including two nitro groups.

Techniques Used:

DIDS derivatives modulate the RAD51 nucleofilament formation. The association and dissociation between RAD51 and ssDNA were evaluated by Blitz interferometry. RAD51 (2 µM) was pre-incubated in phosphate buffer in absence or presence of each molecule at different concentrations before the measurement. The association phase followed until 50 s while the dissociation phase was monitored until 80 or 90 s, as indicated in graphs. ( A ) Effect of DIDS family on the ssDNA-RAD51 association was evaluated at 40 µM concentration of each molecule including DIDS, DADS, DAZDS, DNDS, and SITS. ( B ) DIDS, ( C ) DAZDS, and ( D ) SITS were incubated with RAD51 at increasing concentrations of 2, 10, and 20 µM for 5 min before the measurement by BLItz approach. ( E ) The percent of nucleofilament formation in presence of stilbene molecule was calculated in comparison to the condition in absence of drug after 50 s. This percentage was presented in function to the increasing concentration (from 2 to 20 µM) of each small molecule. ( F ) RAD51 associates with ssDNA until 50 s, and then each molecule is added at 40 µM as indicated by the narrow in the graph.
Figure Legend Snippet: DIDS derivatives modulate the RAD51 nucleofilament formation. The association and dissociation between RAD51 and ssDNA were evaluated by Blitz interferometry. RAD51 (2 µM) was pre-incubated in phosphate buffer in absence or presence of each molecule at different concentrations before the measurement. The association phase followed until 50 s while the dissociation phase was monitored until 80 or 90 s, as indicated in graphs. ( A ) Effect of DIDS family on the ssDNA-RAD51 association was evaluated at 40 µM concentration of each molecule including DIDS, DADS, DAZDS, DNDS, and SITS. ( B ) DIDS, ( C ) DAZDS, and ( D ) SITS were incubated with RAD51 at increasing concentrations of 2, 10, and 20 µM for 5 min before the measurement by BLItz approach. ( E ) The percent of nucleofilament formation in presence of stilbene molecule was calculated in comparison to the condition in absence of drug after 50 s. This percentage was presented in function to the increasing concentration (from 2 to 20 µM) of each small molecule. ( F ) RAD51 associates with ssDNA until 50 s, and then each molecule is added at 40 µM as indicated by the narrow in the graph.

Techniques Used: Incubation, Concentration Assay

3) Product Images from "DUAL AND OPPOSITE EFFECTS OF hRAD51 CHEMICAL MODULATION ON HIV-1 INTEGRATION"

Article Title: DUAL AND OPPOSITE EFFECTS OF hRAD51 CHEMICAL MODULATION ON HIV-1 INTEGRATION

Journal: Chemistry & biology

doi: 10.1016/j.chembiol.2015.04.020

Effect of hRAD51 modulators on HIV-1 integration The chemical structure of the stimulatory compounds RS-1 and P-ter ( A ) and the inhibitory compounds RI-1 and DIDS, as well as the sequence of the aptamers ( B ) are indicated. Increasing concentrations of wt-hRAD51 were added in a standard concerted integration assay in the presence of 100 µM ATP (w/o molecule or aptamer), and with 7.5 or 15 µM RS-1 ( C ), P-ter ( D ), RI-1 ( E ) or 0.1, 0.25 or 0.5 µM of A30 ( F , was normalized to 100 %.
Figure Legend Snippet: Effect of hRAD51 modulators on HIV-1 integration The chemical structure of the stimulatory compounds RS-1 and P-ter ( A ) and the inhibitory compounds RI-1 and DIDS, as well as the sequence of the aptamers ( B ) are indicated. Increasing concentrations of wt-hRAD51 were added in a standard concerted integration assay in the presence of 100 µM ATP (w/o molecule or aptamer), and with 7.5 or 15 µM RS-1 ( C ), P-ter ( D ), RI-1 ( E ) or 0.1, 0.25 or 0.5 µM of A30 ( F , was normalized to 100 %.

Techniques Used: Sequencing

4) Product Images from "Small molecule‐facilitated anion transporters in cells for a novel therapeutic approach to cystic fibrosis, et al. Small molecule‐facilitated anion transporters in cells for a novel therapeutic approach to cystic fibrosis"

Article Title: Small molecule‐facilitated anion transporters in cells for a novel therapeutic approach to cystic fibrosis, et al. Small molecule‐facilitated anion transporters in cells for a novel therapeutic approach to cystic fibrosis

Journal: British Journal of Pharmacology

doi: 10.1111/bph.14649

Intracellular pH measured during a NH 4 + pulse protocol. Perfusion of FRT (a–e) and CFTR‐FRT cells (f) with solutions saturated with 5% CO 2 are indicated at the top of the traces. (a) Perfusion with 40 mM NH 4 + causes the alkalinisation of the cell, followed by a rapid acidification when external NH 4 + is removed. Perfusion with bicarbonate immediately induced a pH i increase, correlated with the bicarbonate influx. (b) The main bicarbonate and proton transport mechanisms were inhibited by 1 mM amiloride and 300 μM DIDS and therefore, there is no pH i change. Perfusion with 2 μM EH130 (c ) or 2 μM MM3 (d), in the presence of amiloride and DIDS, induced a pH i increase correlated with bicarbonate influx. (e) When anionophore is perfused in the absence of bicarbonate, there is no pH i change. (f) Similar experiments were performed on FRT–CFTR cells. Also, in this case, no pH i increase was observed until the activation of CFTR by forskolin (20 μM Fk). Each curve is the mean of at least four different regions in the acquired images. (g) The change of the pH i , correlated with bicarbonate influx, upon perfusion of the cells with 2 μM of the prodigiosines EH130, OBX and PRG (red bars), the tambjamines MM3 and RQ363 (blue bars), and the activation of the CFTR by 20 μM forskolin. Data are the mean ± SEM of five independent experiments
Figure Legend Snippet: Intracellular pH measured during a NH 4 + pulse protocol. Perfusion of FRT (a–e) and CFTR‐FRT cells (f) with solutions saturated with 5% CO 2 are indicated at the top of the traces. (a) Perfusion with 40 mM NH 4 + causes the alkalinisation of the cell, followed by a rapid acidification when external NH 4 + is removed. Perfusion with bicarbonate immediately induced a pH i increase, correlated with the bicarbonate influx. (b) The main bicarbonate and proton transport mechanisms were inhibited by 1 mM amiloride and 300 μM DIDS and therefore, there is no pH i change. Perfusion with 2 μM EH130 (c ) or 2 μM MM3 (d), in the presence of amiloride and DIDS, induced a pH i increase correlated with bicarbonate influx. (e) When anionophore is perfused in the absence of bicarbonate, there is no pH i change. (f) Similar experiments were performed on FRT–CFTR cells. Also, in this case, no pH i increase was observed until the activation of CFTR by forskolin (20 μM Fk). Each curve is the mean of at least four different regions in the acquired images. (g) The change of the pH i , correlated with bicarbonate influx, upon perfusion of the cells with 2 μM of the prodigiosines EH130, OBX and PRG (red bars), the tambjamines MM3 and RQ363 (blue bars), and the activation of the CFTR by 20 μM forskolin. Data are the mean ± SEM of five independent experiments

Techniques Used: Activation Assay

5) Product Images from "Abscisic Acid Transport in Human Erythrocytes *"

Article Title: Abscisic Acid Transport in Human Erythrocytes *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.629501

The ABA antagonist #10 and Band 3 inhibitor DIDS inhibit the ABA-induced [cAMP] i rise and ATP release. A, RBC were preincubated at a 50% hematocrit with 250 μ m of the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine for 15 min; cells
Figure Legend Snippet: The ABA antagonist #10 and Band 3 inhibitor DIDS inhibit the ABA-induced [cAMP] i rise and ATP release. A, RBC were preincubated at a 50% hematocrit with 250 μ m of the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine for 15 min; cells

Techniques Used:

6) Product Images from "Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation"

Article Title: Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00256.2015

Cotreatment with anion-channel blockers and transient knock-down of LRRC8A abolish Cisplatin-induced but not TNFα and Hyperosmotic-induced Caspase-3 activation in wild-type A2780 cells. A2780WT and A2780CisR cell lysates were used for Caspase-3 activity assay using a commercial kit (see experimental procedures ). A : A2780CisR cells were exposed to 10 μM Cisplatin for 18 h and A2780WT cells were likewise treated with Cisplatin in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 6–8 (A2780WT) and 4 (A2780CisR) individual sets of experiments ± SE. * and #: Significantly increased by Cisplatin compared with the respective control and significantly reduced compared with Cisplatin treatment in the absence of the inhibitor, respectively (ANOVA, Fisher LSD method). B : A2780WT were treated with either 25 nM scramble siRNA or LRRC8A siRNA for 30 h and subsequently exposed to 10 μM Cisplatin for another 18 h. Data represent 4 individual experiments ± SE. * and #: Significantly increased by Cisplatin compared with the respective control and significantly reduced compared with Cisplatin treatment in Scramble siRNA-treated cells (Student's t -test). C: A2780CisR cells were exposed to 20 nM TNFα for 18 h. A2780WT cells were likewise treated with TNFα in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 6–8 (WT) and 4 (CisR) individual experiments ± SE. *Significantly increased compared with the respective control (ANOVA, Fisher LSD method). D : A2780WT cells were exposed to isotonic or hypertonic NaCl for 4.5 h in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 3 individual experiments ± SE. *Significantly increased compared with isotonic control (ANOVA, Fisher LSD method).
Figure Legend Snippet: Cotreatment with anion-channel blockers and transient knock-down of LRRC8A abolish Cisplatin-induced but not TNFα and Hyperosmotic-induced Caspase-3 activation in wild-type A2780 cells. A2780WT and A2780CisR cell lysates were used for Caspase-3 activity assay using a commercial kit (see experimental procedures ). A : A2780CisR cells were exposed to 10 μM Cisplatin for 18 h and A2780WT cells were likewise treated with Cisplatin in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 6–8 (A2780WT) and 4 (A2780CisR) individual sets of experiments ± SE. * and #: Significantly increased by Cisplatin compared with the respective control and significantly reduced compared with Cisplatin treatment in the absence of the inhibitor, respectively (ANOVA, Fisher LSD method). B : A2780WT were treated with either 25 nM scramble siRNA or LRRC8A siRNA for 30 h and subsequently exposed to 10 μM Cisplatin for another 18 h. Data represent 4 individual experiments ± SE. * and #: Significantly increased by Cisplatin compared with the respective control and significantly reduced compared with Cisplatin treatment in Scramble siRNA-treated cells (Student's t -test). C: A2780CisR cells were exposed to 20 nM TNFα for 18 h. A2780WT cells were likewise treated with TNFα in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 6–8 (WT) and 4 (CisR) individual experiments ± SE. *Significantly increased compared with the respective control (ANOVA, Fisher LSD method). D : A2780WT cells were exposed to isotonic or hypertonic NaCl for 4.5 h in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 3 individual experiments ± SE. *Significantly increased compared with isotonic control (ANOVA, Fisher LSD method).

Techniques Used: Activation Assay, Caspase-3 Activity Assay

Impairment of swelling induced taurine release following pharmacological inhibition or transient knock-down of LRRC8A in wild-type A2780 cells. Volume-sensitive taurine release and LRRC8A protein expression were measured in ovarian A2780WT cells by tracer technique and Western blot analysis, respectively. A2780WT cells were loaded with [ 3 H]taurine for 2 h, washed, and exposed to isosmotic NaCl medium (300 mOsM) for 10 min and subsequently to hyposmotic medium (200 mOsM) for 20 min (arrow in A indicates shift to hypotonicity). Samples were taken every second minute. A : fractional rate constant (min −1 ) for taurine release was determined and plotted vs. time (min) under isosmotic and hyposmotic conditions in the absence (○) or presence of the VRAC/VSOAC blockers NS3728 (●) or DIDS (■). The anion blockers NS3728 and DIDS were used in the concentration of 10 μM (≈2.5 μM free) and 100 μM, respectively, and present during the isotonic/hypotonic release experiment. Data represent 1 of 3 sets of experiments. B : maximal rate constant for taurine release was determined 6 min after hyposmotic exposure as illustrated in A . Data represent the mean maximal rate constants determined from 3 sets of experiments ± SE. *Significantly reduced compared with control (Student's t -test). C : efficiency of siRNA-mediated LRRC8A knock-down in A2780WT cells was determined by Western blot analysis using control, scramble siRNA and LRRC8A siRNA transfected cells and specific monoclonal antibody raised against human LRRC8A and β-actin (loading control). D : LRRC8A/β-actin protein band intensity ratios were calculated from Western blots shown in C and values given relative to control cells. Data represent mean of 4 experiments ± SE. * and #: Significantly reduced compared with Control and Scramble siRNA, respectively (Student's t -test). Scramble siRNA was not significantly larger than Control ( P = 0.15, Student's t -test). E : maximal rate constant for taurine release was determined at time 6 min after hypotonic exposure in A2780WT (open bar) cells or in A2780 cells exposed to scramble siRNA (light gray bar) or siRNA directed against human LRRC8A (dark gray bar). Data represent mean of 4 experiments ± SE. *Significantly reduced compared with Control (Student's t -test).
Figure Legend Snippet: Impairment of swelling induced taurine release following pharmacological inhibition or transient knock-down of LRRC8A in wild-type A2780 cells. Volume-sensitive taurine release and LRRC8A protein expression were measured in ovarian A2780WT cells by tracer technique and Western blot analysis, respectively. A2780WT cells were loaded with [ 3 H]taurine for 2 h, washed, and exposed to isosmotic NaCl medium (300 mOsM) for 10 min and subsequently to hyposmotic medium (200 mOsM) for 20 min (arrow in A indicates shift to hypotonicity). Samples were taken every second minute. A : fractional rate constant (min −1 ) for taurine release was determined and plotted vs. time (min) under isosmotic and hyposmotic conditions in the absence (○) or presence of the VRAC/VSOAC blockers NS3728 (●) or DIDS (■). The anion blockers NS3728 and DIDS were used in the concentration of 10 μM (≈2.5 μM free) and 100 μM, respectively, and present during the isotonic/hypotonic release experiment. Data represent 1 of 3 sets of experiments. B : maximal rate constant for taurine release was determined 6 min after hyposmotic exposure as illustrated in A . Data represent the mean maximal rate constants determined from 3 sets of experiments ± SE. *Significantly reduced compared with control (Student's t -test). C : efficiency of siRNA-mediated LRRC8A knock-down in A2780WT cells was determined by Western blot analysis using control, scramble siRNA and LRRC8A siRNA transfected cells and specific monoclonal antibody raised against human LRRC8A and β-actin (loading control). D : LRRC8A/β-actin protein band intensity ratios were calculated from Western blots shown in C and values given relative to control cells. Data represent mean of 4 experiments ± SE. * and #: Significantly reduced compared with Control and Scramble siRNA, respectively (Student's t -test). Scramble siRNA was not significantly larger than Control ( P = 0.15, Student's t -test). E : maximal rate constant for taurine release was determined at time 6 min after hypotonic exposure in A2780WT (open bar) cells or in A2780 cells exposed to scramble siRNA (light gray bar) or siRNA directed against human LRRC8A (dark gray bar). Data represent mean of 4 experiments ± SE. *Significantly reduced compared with Control (Student's t -test).

Techniques Used: Inhibition, Expressing, Western Blot, Concentration Assay, Transfection

Administration of anion- and cation-channel blockers reduces Cisplatin-induced expression of p53 and p21 Waf1/Cip1 in wild-type A2780 cells (A2780WT) to the same level as observed in Cisplatin-resistant A2780 cells (A2780CisR). Whole cell protein lysates for Western blot analysis were obtained from A2780WT and A2780CisR cells exposed to 10 μM Cisplatin for 18 h in combination with the anion/amino acid channel blockers NS3728 (100 μM)/DIDS (400 μM) or the cation-channel (TASK-2) blocker clofilium (25 μM). Only A2780WT cells were exposed to channel blockers. Protein expression of LRRC8A, p53, p-p53 (Ser15), p21, and Bax was determined using specific antibodies (see experimental procedures ). β-Actin was used as loading control. A : representative Western blot. B : p53, pp53, p21, and Bax protein expression relative to β-actin in A2780WT and A2780CisR (open bars) control cells and following Cisplatin exposure (black bars). Data represent 4–9 individual sets of experiments where ratios are given relative to the untreated WT cells ± SE. * and #: Expression is significantly increased in Cisplatin-treated cell compared with control cells and significantly reduced in Cisplatin-treated A2780CisR cells compared with Cisplatin-treated A2780WT cells, respectively (ANOVA, Fisher LSD method). C : graph represents the relative effect of Cisplatin (Cisplatin-treated/respective untreated control) on LRRC8A, p53, p-p53, and p21 protein expression in A2780WT, A2780CisR (CisR), and A2780WT cells cotreated with the anion- and cation-channel blockers. Data represent mean of 4–9 individual experiments ± SE. *Significantly reduced effect of Cisplatin compared with A2780WT (ANOVA, Fisher LSD method).
Figure Legend Snippet: Administration of anion- and cation-channel blockers reduces Cisplatin-induced expression of p53 and p21 Waf1/Cip1 in wild-type A2780 cells (A2780WT) to the same level as observed in Cisplatin-resistant A2780 cells (A2780CisR). Whole cell protein lysates for Western blot analysis were obtained from A2780WT and A2780CisR cells exposed to 10 μM Cisplatin for 18 h in combination with the anion/amino acid channel blockers NS3728 (100 μM)/DIDS (400 μM) or the cation-channel (TASK-2) blocker clofilium (25 μM). Only A2780WT cells were exposed to channel blockers. Protein expression of LRRC8A, p53, p-p53 (Ser15), p21, and Bax was determined using specific antibodies (see experimental procedures ). β-Actin was used as loading control. A : representative Western blot. B : p53, pp53, p21, and Bax protein expression relative to β-actin in A2780WT and A2780CisR (open bars) control cells and following Cisplatin exposure (black bars). Data represent 4–9 individual sets of experiments where ratios are given relative to the untreated WT cells ± SE. * and #: Expression is significantly increased in Cisplatin-treated cell compared with control cells and significantly reduced in Cisplatin-treated A2780CisR cells compared with Cisplatin-treated A2780WT cells, respectively (ANOVA, Fisher LSD method). C : graph represents the relative effect of Cisplatin (Cisplatin-treated/respective untreated control) on LRRC8A, p53, p-p53, and p21 protein expression in A2780WT, A2780CisR (CisR), and A2780WT cells cotreated with the anion- and cation-channel blockers. Data represent mean of 4–9 individual experiments ± SE. *Significantly reduced effect of Cisplatin compared with A2780WT (ANOVA, Fisher LSD method).

Techniques Used: Expressing, Western Blot

7) Product Images from "The gastric H,K-ATPase in stria vascularis contributes to pH regulation of cochlear endolymph but not to K secretion"

Article Title: The gastric H,K-ATPase in stria vascularis contributes to pH regulation of cochlear endolymph but not to K secretion

Journal: BMC Physiology

doi: 10.1186/s12899-016-0024-1

K + - and H + - fluxes from stria vascularis; bumetanide and DIDS. a and c K + flux; b and d H + flux. a Summary traces ( N = 7) of effect of perfusion of bumetanide (50 μM; bumet) on K + flux from stria vascularis. b Summary traces ( N = 6) of effect of perfusion of bumetanide (50 μM) on H + flux from stria vascularis. c Concentration-response curve for bumetanide on K + flux from stria vascularis fit by the Michaelis-Menton equation ( N = 5–6). d Bar graph summaries of steady–state effects of bumetanide (50 μM; N = 6; Bumet) and DIDS (1 mM; N = 6) on H + flux from stria vascularis. *, P
Figure Legend Snippet: K + - and H + - fluxes from stria vascularis; bumetanide and DIDS. a and c K + flux; b and d H + flux. a Summary traces ( N = 7) of effect of perfusion of bumetanide (50 μM; bumet) on K + flux from stria vascularis. b Summary traces ( N = 6) of effect of perfusion of bumetanide (50 μM) on H + flux from stria vascularis. c Concentration-response curve for bumetanide on K + flux from stria vascularis fit by the Michaelis-Menton equation ( N = 5–6). d Bar graph summaries of steady–state effects of bumetanide (50 μM; N = 6; Bumet) and DIDS (1 mM; N = 6) on H + flux from stria vascularis. *, P

Techniques Used: Concentration Assay

8) Product Images from "Low pH Attenuates Apoptosis by Suppressing the Volume-Sensitive Outwardly Rectifying (VSOR) Chloride Current in Chondrocytes"

Article Title: Low pH Attenuates Apoptosis by Suppressing the Volume-Sensitive Outwardly Rectifying (VSOR) Chloride Current in Chondrocytes

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2021.804105

Pharmacological properties of VSOR Cl − channels in C28/I2 cells. (A) Mean peak VSOR Cl − current amplitudes ± SEM at +100 mV and −100 mV under hypotonic conditions in the absence and presence of different inhibitors. (B) VSOR Cl − current traces elicited by 500-ms voltage steps from −100 mV to +100 mV obtained at maximal VSOR Cl − current amplitudes under hypotonic conditions in the absence and presence of DCPIB (10 µM), niflumic acid (NFA; 500 µM), DIDS (100 µM), NPPB (100 µM) and Tamoxifen (10 µM) (n = 3).
Figure Legend Snippet: Pharmacological properties of VSOR Cl − channels in C28/I2 cells. (A) Mean peak VSOR Cl − current amplitudes ± SEM at +100 mV and −100 mV under hypotonic conditions in the absence and presence of different inhibitors. (B) VSOR Cl − current traces elicited by 500-ms voltage steps from −100 mV to +100 mV obtained at maximal VSOR Cl − current amplitudes under hypotonic conditions in the absence and presence of DCPIB (10 µM), niflumic acid (NFA; 500 µM), DIDS (100 µM), NPPB (100 µM) and Tamoxifen (10 µM) (n = 3).

Techniques Used:

Effect of different VSOR Cl − channel inhibitors on caspase 3/7 activity and cell viability. (A) Caspase 3/7 activity and (B) viability of C28/I2 cells treated for 6 h with pH 7.4, pH 5.5 and pH 7.4 + different VSOR Cl − current inhibitors in the absence and presence of staurosporine (st) (1 µM) (n = 3). (C) Caspase 3/7 activity and (D) viability of C28/I2 cells treated for 8 h with pH 7.4, pH 5.5 and pH 7.4 + DIDS (100 µM) in the absence or presence of staurosporine (st) (1 and 5 µM) (n = 4). (E) Caspase 3/7 activity and (F) viability of cells treated for 24 h with pH 7.4 and pH 6.0 with or without DCPIB (10 µM) in the absence or presence of staurosporine (st) (1 µM) (n = 2–3). Data are represented as mean ± SEM.
Figure Legend Snippet: Effect of different VSOR Cl − channel inhibitors on caspase 3/7 activity and cell viability. (A) Caspase 3/7 activity and (B) viability of C28/I2 cells treated for 6 h with pH 7.4, pH 5.5 and pH 7.4 + different VSOR Cl − current inhibitors in the absence and presence of staurosporine (st) (1 µM) (n = 3). (C) Caspase 3/7 activity and (D) viability of C28/I2 cells treated for 8 h with pH 7.4, pH 5.5 and pH 7.4 + DIDS (100 µM) in the absence or presence of staurosporine (st) (1 and 5 µM) (n = 4). (E) Caspase 3/7 activity and (F) viability of cells treated for 24 h with pH 7.4 and pH 6.0 with or without DCPIB (10 µM) in the absence or presence of staurosporine (st) (1 µM) (n = 2–3). Data are represented as mean ± SEM.

Techniques Used: Activity Assay

AVD in chondrocytes is impaired under acidic conditions and in the presence of VSOR Cl-channel inhibitors. (A, C, E) Mean cell volume (MCV) ± SEM in femtoliters (fl) measured (A) over 2 h under hypertonic (360 mOsm/kg) pH 7.4 conditions and under isotonic (300 mOsm/kg) conditions at pH 7.4 or pH 6.0 in the presence and absence of 5 µM staurosporine (st) (n = 4) and (C, E) MCV measured over 60 min under isotonic pH 7.4 conditions in the absence (control) or presence of the Cl − channel inhibitors NPPB (100 µM), Tamoxifen (Tamox) (10 µM), DCPIB (10 µM), niflumic acid (NFA; 500 µM) or DIDS (100 µM) ± staurosporine (1 µM) (n = 3). (B) Differences in MCV (ΔMCV in fl) calculated from data in (A) at the timepoints 60 and 120 min for matching pH conditions (pH 7.4 and 6.0) in the presence and absence of staurosporine. (D, F) ΔMCV at 60 min calculated from data in (C, E) for control (absence of blockers) and matching blocker conditions in the presence and absence of 1 µM staurosporine. (G) MCV ± SEM assessed by using flow cytometry after 5 h of incubation with pH 7.4, pH 7.4 + DIDS (100 µM) and pH 5.5 in the presence and absence of staurosporine (st) (1 µM or 5 µM) (n = 4).
Figure Legend Snippet: AVD in chondrocytes is impaired under acidic conditions and in the presence of VSOR Cl-channel inhibitors. (A, C, E) Mean cell volume (MCV) ± SEM in femtoliters (fl) measured (A) over 2 h under hypertonic (360 mOsm/kg) pH 7.4 conditions and under isotonic (300 mOsm/kg) conditions at pH 7.4 or pH 6.0 in the presence and absence of 5 µM staurosporine (st) (n = 4) and (C, E) MCV measured over 60 min under isotonic pH 7.4 conditions in the absence (control) or presence of the Cl − channel inhibitors NPPB (100 µM), Tamoxifen (Tamox) (10 µM), DCPIB (10 µM), niflumic acid (NFA; 500 µM) or DIDS (100 µM) ± staurosporine (1 µM) (n = 3). (B) Differences in MCV (ΔMCV in fl) calculated from data in (A) at the timepoints 60 and 120 min for matching pH conditions (pH 7.4 and 6.0) in the presence and absence of staurosporine. (D, F) ΔMCV at 60 min calculated from data in (C, E) for control (absence of blockers) and matching blocker conditions in the presence and absence of 1 µM staurosporine. (G) MCV ± SEM assessed by using flow cytometry after 5 h of incubation with pH 7.4, pH 7.4 + DIDS (100 µM) and pH 5.5 in the presence and absence of staurosporine (st) (1 µM or 5 µM) (n = 4).

Techniques Used: Flow Cytometry, Incubation

9) Product Images from "Chloride channel blocker 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid inhibits nitric oxide-induced apoptosis in cultured rat hippocampal neurons ★"

Article Title: Chloride channel blocker 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid inhibits nitric oxide-induced apoptosis in cultured rat hippocampal neurons ★

Journal: Neural Regeneration Research

doi: 10.3969/j.issn.1673-5374.2013.02.003

Immunofluorescence staining of apoptosis-inducing factor (AIF) in rat hippocampal neurons (inverted fluorescence microscope, × 200). The specific neuronal antibody anti-NeuN was green (FITC labeled), AIF expression was significantly increased in the SIN-1 group (Cy3 labeled), and significantly decreased after treatment with the chloride channel blocker DIDS. SIN-1: 3-morpholinosydnonimine; DIDS: 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid.
Figure Legend Snippet: Immunofluorescence staining of apoptosis-inducing factor (AIF) in rat hippocampal neurons (inverted fluorescence microscope, × 200). The specific neuronal antibody anti-NeuN was green (FITC labeled), AIF expression was significantly increased in the SIN-1 group (Cy3 labeled), and significantly decreased after treatment with the chloride channel blocker DIDS. SIN-1: 3-morpholinosydnonimine; DIDS: 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid.

Techniques Used: Immunofluorescence, Staining, Fluorescence, Microscopy, Labeling, Expressing

Apoptotic neurons in rat hippocampus. (A) Morphology of cultured rat hippocampal neurons following DNA fluorescent staining (Hoechst 33258 staining, inverted fluorescence microscope, × 200). Arrows indicate apoptotic neurons (Hoechst 33258- positive cells displayed strong blue fluorescence). Apoptotic cells were evident in the 3-morpholinosydnonimine (SIN-1; 1.0 mM) group, but fewer were visible in the SIN-1 + DIDS group. (B) Quantification of apoptotic cells. a P
Figure Legend Snippet: Apoptotic neurons in rat hippocampus. (A) Morphology of cultured rat hippocampal neurons following DNA fluorescent staining (Hoechst 33258 staining, inverted fluorescence microscope, × 200). Arrows indicate apoptotic neurons (Hoechst 33258- positive cells displayed strong blue fluorescence). Apoptotic cells were evident in the 3-morpholinosydnonimine (SIN-1; 1.0 mM) group, but fewer were visible in the SIN-1 + DIDS group. (B) Quantification of apoptotic cells. a P

Techniques Used: Cell Culture, Staining, Fluorescence, Microscopy

Expression of poly(adenosine diphosphate- ribose)polymerase-1 (PARP-1) in rat hippocampal neurons (inverted fluorescence microscope, × 200). The specific neuronal antibody anti-NeuN was green (FITC labeled) and PARP-1 was red (Cy3 labeled). PARP-1 expression was significantly increased in the SIN-1 group, and was significantly decreased after treatment with the chloride channel blocker DIDS. SIN-1: 3-morpholinosydnonimine; DIDS: 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid.
Figure Legend Snippet: Expression of poly(adenosine diphosphate- ribose)polymerase-1 (PARP-1) in rat hippocampal neurons (inverted fluorescence microscope, × 200). The specific neuronal antibody anti-NeuN was green (FITC labeled) and PARP-1 was red (Cy3 labeled). PARP-1 expression was significantly increased in the SIN-1 group, and was significantly decreased after treatment with the chloride channel blocker DIDS. SIN-1: 3-morpholinosydnonimine; DIDS: 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid.

Techniques Used: Expressing, Fluorescence, Microscopy, Labeling

Poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) and apoptosis-inducing factor (AIF) protein expression in rat hippocampal neurons. Expression levels in the SIN-1 group were significantly higher than those in the control group. DIDS downregulated PARP-1 and AIF expression. SIN-1: 3-morpholinosydnonimine; DIDS: 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid.
Figure Legend Snippet: Poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) and apoptosis-inducing factor (AIF) protein expression in rat hippocampal neurons. Expression levels in the SIN-1 group were significantly higher than those in the control group. DIDS downregulated PARP-1 and AIF expression. SIN-1: 3-morpholinosydnonimine; DIDS: 4,4-diisothiocyanatostilbene-2,2′-disulfonic acid.

Techniques Used: Expressing

10) Product Images from "The cellular chloride channels CLIC1 and CLIC4 contribute to virus-mediated cell motility"

Article Title: The cellular chloride channels CLIC1 and CLIC4 contribute to virus-mediated cell motility

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA117.001343

Cl − channel blockers inhibit the metastatic phenotypic changes associated with ST expression. A and B , in ( A ) i293-EGFP and i293-EGFP-ST cells were induced with doxycycline hyclate and in ( B ) MCC13 cells were transfected with EGFP and EGFP-ST. ( i ) cells were then treated with DMSO, DIDS, RIAA, or NPPB and after 24 h, cell motility was measured using the IncuCyte kinetic imaging system. Images were taken every 30 min for a 24-h period. ( ii ) scatter plot showing cell movement tracked using ImageJ ( n = 25). Average cell movement, as indicated by the horizontal bar , was calculated and significance tested using a two-tailed Student's t test; *, p
Figure Legend Snippet: Cl − channel blockers inhibit the metastatic phenotypic changes associated with ST expression. A and B , in ( A ) i293-EGFP and i293-EGFP-ST cells were induced with doxycycline hyclate and in ( B ) MCC13 cells were transfected with EGFP and EGFP-ST. ( i ) cells were then treated with DMSO, DIDS, RIAA, or NPPB and after 24 h, cell motility was measured using the IncuCyte kinetic imaging system. Images were taken every 30 min for a 24-h period. ( ii ) scatter plot showing cell movement tracked using ImageJ ( n = 25). Average cell movement, as indicated by the horizontal bar , was calculated and significance tested using a two-tailed Student's t test; *, p

Techniques Used: Expressing, Transfection, Imaging, Two Tailed Test

11) Product Images from "An in vitro ischemic penumbral mimic perfusate increases NADPH oxidase-mediated superoxide production in cultured hippocampal neurons"

Article Title: An in vitro ischemic penumbral mimic perfusate increases NADPH oxidase-mediated superoxide production in cultured hippocampal neurons

Journal: Brain Research

doi: 10.1016/j.brainres.2012.03.004

DIDS penetrates damaged, but not intact neuronal membranes (A) Two-photon confocal DIC and Z-stack projection images (2 microns total) of neurons treated without DIDS, or with DIDS during normoxia or IS. Upper panel: DIC images. Lower panel: DIC images (grey) overlaid with PI (red) and DIDS (blue) fluorescent Z-stack projection images. Images are representative of 4 separate experiments for each treatment. (B) Summary of PI uptake. Data are mean ± SEM from 10–20 replicates per treatment. Asterisks (*) indicate significant difference from control ( p
Figure Legend Snippet: DIDS penetrates damaged, but not intact neuronal membranes (A) Two-photon confocal DIC and Z-stack projection images (2 microns total) of neurons treated without DIDS, or with DIDS during normoxia or IS. Upper panel: DIC images. Lower panel: DIC images (grey) overlaid with PI (red) and DIDS (blue) fluorescent Z-stack projection images. Images are representative of 4 separate experiments for each treatment. (B) Summary of PI uptake. Data are mean ± SEM from 10–20 replicates per treatment. Asterisks (*) indicate significant difference from control ( p

Techniques Used:

12) Product Images from "Neuronal and Astroglial Correlates Underlying Spatiotemporal Intrinsic Optical Signal in the Rat Hippocampal Slice"

Article Title: Neuronal and Astroglial Correlates Underlying Spatiotemporal Intrinsic Optical Signal in the Rat Hippocampal Slice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057694

Neuronal K + /Cl − cotransporter, non-specific Cl − and volume-regulated anion channels contribute to IOS. A Left and Middle: Representative IOS amplitude map and field response curve under control condition and 5 mM furosemide application, respectively. The colorbar indicates the maximum change of the transmittance compared to the resting light intensity. A Right: Spatial visualization of the percentage of control changes of IOS signal caused by furosemide application. B Left and Middle: Representative IOS amplitude map and field response curve under control condition and 200 µM DIDS application, respectively. The colorbar indicates the maximum change of the transmittance compared to the resting light intensity. B Right: Spatial visualization of the percentage of control changes of IOS signal caused by DIDS application. C: The effect of 5 mM furosemide, 10 µM bumetanide, 200 µM DIDS and 40 µM DCPIB on the field response and IOS parameters in percentage of control. Asterisks indicate significant changes compared to control (P
Figure Legend Snippet: Neuronal K + /Cl − cotransporter, non-specific Cl − and volume-regulated anion channels contribute to IOS. A Left and Middle: Representative IOS amplitude map and field response curve under control condition and 5 mM furosemide application, respectively. The colorbar indicates the maximum change of the transmittance compared to the resting light intensity. A Right: Spatial visualization of the percentage of control changes of IOS signal caused by furosemide application. B Left and Middle: Representative IOS amplitude map and field response curve under control condition and 200 µM DIDS application, respectively. The colorbar indicates the maximum change of the transmittance compared to the resting light intensity. B Right: Spatial visualization of the percentage of control changes of IOS signal caused by DIDS application. C: The effect of 5 mM furosemide, 10 µM bumetanide, 200 µM DIDS and 40 µM DCPIB on the field response and IOS parameters in percentage of control. Asterisks indicate significant changes compared to control (P

Techniques Used:

13) Product Images from "Cl− channel is required for CXCL10-induced neuronal activation and itch response in a murine model of allergic contact dermatitis"

Article Title: Cl− channel is required for CXCL10-induced neuronal activation and itch response in a murine model of allergic contact dermatitis

Journal: Journal of Neurophysiology

doi: 10.1152/jn.00187.2017

Effects of Cl − channel antagonists and intracellular Ca 2+ on CXCL10-induced currents in DRG neurons innervating CHS skin. A and B : sample traces of the I CXCL10 recorded in the absence ( A ) and presence of the Cl − channel blockers DIDS (100 µM; B ) or NFA (100 µM; C ) applied to the bath or in the presence of 10 mM BAPTA in the pipette solution ( D ). The high-[Cl − ] i pipette solution was used. E : pretreatment with DIDS or NFA for 3 min significantly reduced the peak amplitude of I CXCL10 . Replacement of 11 mM EGTA with 10 mM BAPTA in the pipette solution almost abolished this inward current. The numbers of DRG neurons tested are given in parentheses. * P
Figure Legend Snippet: Effects of Cl − channel antagonists and intracellular Ca 2+ on CXCL10-induced currents in DRG neurons innervating CHS skin. A and B : sample traces of the I CXCL10 recorded in the absence ( A ) and presence of the Cl − channel blockers DIDS (100 µM; B ) or NFA (100 µM; C ) applied to the bath or in the presence of 10 mM BAPTA in the pipette solution ( D ). The high-[Cl − ] i pipette solution was used. E : pretreatment with DIDS or NFA for 3 min significantly reduced the peak amplitude of I CXCL10 . Replacement of 11 mM EGTA with 10 mM BAPTA in the pipette solution almost abolished this inward current. The numbers of DRG neurons tested are given in parentheses. * P

Techniques Used: Transferring

14) Product Images from "CO2 permeability of the rat erythrocyte membrane and its inhibition"

Article Title: CO2 permeability of the rat erythrocyte membrane and its inhibition

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

doi: 10.1080/14756366.2021.1952194

Time course of the decay of 18 O in CO 2 vs. time for rat red cells in the presence and absence of DIDS. Y-axis is log (10 7 ·(Δ[CO 2 *])), where Δ[CO 2 *] is the concentration of 18 O-labelled CO 2 minus its final value at isotope equilibrium, in the unit 10 −7 M. The Y-axis gives the logarithm of this value after it has been multiplied by 10 7 . The curve shows three phases: (1) a pre-phase representing the slow uncatalysed decay of 18 O-labelled CO 2 , (2) by adding, at the sharp bend in the curve, red cells into the measuring chamber the next phase is initiated, which we call the rapid first phase after addition of red cells and which is strongly dependent on P CO2 , (3) a second slower phase follows, which is dependent on P HCO3 − . During the mass spectrometric measurement of red cells an extracellular pH of 7.4 and a CO 2 partial pressure of 40 mmHg prevail.
Figure Legend Snippet: Time course of the decay of 18 O in CO 2 vs. time for rat red cells in the presence and absence of DIDS. Y-axis is log (10 7 ·(Δ[CO 2 *])), where Δ[CO 2 *] is the concentration of 18 O-labelled CO 2 minus its final value at isotope equilibrium, in the unit 10 −7 M. The Y-axis gives the logarithm of this value after it has been multiplied by 10 7 . The curve shows three phases: (1) a pre-phase representing the slow uncatalysed decay of 18 O-labelled CO 2 , (2) by adding, at the sharp bend in the curve, red cells into the measuring chamber the next phase is initiated, which we call the rapid first phase after addition of red cells and which is strongly dependent on P CO2 , (3) a second slower phase follows, which is dependent on P HCO3 − . During the mass spectrometric measurement of red cells an extracellular pH of 7.4 and a CO 2 partial pressure of 40 mmHg prevail.

Techniques Used: Concentration Assay

15) Product Images from "In Mycoplasma hominis the OppA-mediated cytoadhesion depends on its ATPase activity"

Article Title: In Mycoplasma hominis the OppA-mediated cytoadhesion depends on its ATPase activity

Journal: BMC Microbiology

doi: 10.1186/1471-2180-11-185

Adherence of OppA to HeLa cells in the presence of ATPase inhibitors . OppA (black bars) or P60/P80 as a control (white bars), (0.5 μg OppA/well and 0.3 μg P60/well) were preincubated with 200μM DIDS, suramin, ouabain or oligomycin for 20 min before analyzing in adhesion assay. ATPase activity (A) and adhesion efficiency (B) were measured and depicted in relation to the untreated OppA. OppA (0.5 μg protein) was preincubated with FSBA or MgATP for 20 min and then added to HeLa cells (C). Adherence of OppA to HeLa cells in dependence on supplement concentration was determined as described in Material and Methods. Data represent means of three independent experiments with triplicate samples in each experiment. Statistical analysis was performed by unpaired t-test and statistically significant results designated by *. *P
Figure Legend Snippet: Adherence of OppA to HeLa cells in the presence of ATPase inhibitors . OppA (black bars) or P60/P80 as a control (white bars), (0.5 μg OppA/well and 0.3 μg P60/well) were preincubated with 200μM DIDS, suramin, ouabain or oligomycin for 20 min before analyzing in adhesion assay. ATPase activity (A) and adhesion efficiency (B) were measured and depicted in relation to the untreated OppA. OppA (0.5 μg protein) was preincubated with FSBA or MgATP for 20 min and then added to HeLa cells (C). Adherence of OppA to HeLa cells in dependence on supplement concentration was determined as described in Material and Methods. Data represent means of three independent experiments with triplicate samples in each experiment. Statistical analysis was performed by unpaired t-test and statistically significant results designated by *. *P

Techniques Used: Cell Adhesion Assay, Activity Assay, Concentration Assay

16) Product Images from "Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns"

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.450

Chloride channels are associated with the release of nucleosomes and DAMPs in dying HeLa cells. ( a ) HeLa cells were incubated with staurosporine (1 μ g/ml) in combination with various ion channel inhibitors for 12 h; an inhibitor of epithelial Na + channel, amiloride hydrochloride hydrate; Cl − channel inhibitors, DIDS (200 μ M) and NPPB (200 μ M); an inhibitor of Na + /Cl − cotransporter, HCT (100 μ M); an inhibitor of Na + K + ATPase, ouabain (100 μ M); an inhibitor of non-selective cation channel, flufenamide (100 μ M); an inhibitor of stretch-activated ion channel, Gad (III) chloride hexahydrate (40 μ M); an inhibitor of K + channel, tetraethyl ammonium chloride (TEA) (5 mM); an inhibitor of Na + /K + cotransporter, bumetanide (100 μ M). The released DNA was measured with PicoGreen dye ( a ) and the released protein from cells treated with staurosporine together with solvent, amiloride, DIDS or amiloride and DIDS was western blotted for histones and DAMPs ( b ). HeLa cells were treated with solvent or ion channel inhibitors with or without staurosporine for 8 h. Viability was measured with Calcein assay ( c ) and cell death was detected by SYTOX red staining ( d ). HeLa cells were incubated in MEM, Na + -deficient medium or Cl − -deficient medium with staurosporine (1 μ g/ml) for 10 h, and the released DNA was measured by PicoGreen ( e ). HeLa cells were incubated with staurosporine in combination with control solvent or DIDS. Nuclear fragmentation was detected with confocal microscopy ( f ), TUNEL staining ( g ) or agarose gel electrophoresis after genomic DNA preparation ( h ). Cellular chloride ion contents were measured by MQAE fluorescence in cells incubated with MEM containing either control solvent or staurosporine at the indicated time periods ( i ). Cellular Cl − currents were measured in cells treated with staurosporine by halide-sensitive YFP quenching at the indicated times ( j ). HeLa cells treated with solvent or staurosporine in the presence or absence of DIDS, amiloride, DIDS and amiloride, or zVAD for 4 h and chloride ion currents were measured with halide-sensitive YFP quenching methods ( k – n ). Data performed in triplicate are presented as mean±S.D. ( a , c , d and e , right panel of g and i )
Figure Legend Snippet: Chloride channels are associated with the release of nucleosomes and DAMPs in dying HeLa cells. ( a ) HeLa cells were incubated with staurosporine (1 μ g/ml) in combination with various ion channel inhibitors for 12 h; an inhibitor of epithelial Na + channel, amiloride hydrochloride hydrate; Cl − channel inhibitors, DIDS (200 μ M) and NPPB (200 μ M); an inhibitor of Na + /Cl − cotransporter, HCT (100 μ M); an inhibitor of Na + K + ATPase, ouabain (100 μ M); an inhibitor of non-selective cation channel, flufenamide (100 μ M); an inhibitor of stretch-activated ion channel, Gad (III) chloride hexahydrate (40 μ M); an inhibitor of K + channel, tetraethyl ammonium chloride (TEA) (5 mM); an inhibitor of Na + /K + cotransporter, bumetanide (100 μ M). The released DNA was measured with PicoGreen dye ( a ) and the released protein from cells treated with staurosporine together with solvent, amiloride, DIDS or amiloride and DIDS was western blotted for histones and DAMPs ( b ). HeLa cells were treated with solvent or ion channel inhibitors with or without staurosporine for 8 h. Viability was measured with Calcein assay ( c ) and cell death was detected by SYTOX red staining ( d ). HeLa cells were incubated in MEM, Na + -deficient medium or Cl − -deficient medium with staurosporine (1 μ g/ml) for 10 h, and the released DNA was measured by PicoGreen ( e ). HeLa cells were incubated with staurosporine in combination with control solvent or DIDS. Nuclear fragmentation was detected with confocal microscopy ( f ), TUNEL staining ( g ) or agarose gel electrophoresis after genomic DNA preparation ( h ). Cellular chloride ion contents were measured by MQAE fluorescence in cells incubated with MEM containing either control solvent or staurosporine at the indicated time periods ( i ). Cellular Cl − currents were measured in cells treated with staurosporine by halide-sensitive YFP quenching at the indicated times ( j ). HeLa cells treated with solvent or staurosporine in the presence or absence of DIDS, amiloride, DIDS and amiloride, or zVAD for 4 h and chloride ion currents were measured with halide-sensitive YFP quenching methods ( k – n ). Data performed in triplicate are presented as mean±S.D. ( a , c , d and e , right panel of g and i )

Techniques Used: Incubation, Western Blot, Staining, Confocal Microscopy, TUNEL Assay, Agarose Gel Electrophoresis, Fluorescence

17) Product Images from "The cellular chloride channels CLIC1 and CLIC4 contribute to virus-mediated cell motility"

Article Title: The cellular chloride channels CLIC1 and CLIC4 contribute to virus-mediated cell motility

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA117.001343

Cl − channel blockers inhibit the metastatic phenotypic changes associated with ST expression. A and B , in ( A ) i293-EGFP and i293-EGFP-ST cells were induced with doxycycline hyclate and in ( B ) MCC13 cells were transfected with EGFP and EGFP-ST. ( i ) cells were then treated with DMSO, DIDS, RIAA, or NPPB and after 24 h, cell motility was measured using the IncuCyte kinetic imaging system. Images were taken every 30 min for a 24-h period. ( ii ) scatter plot showing cell movement tracked using ImageJ ( n = 25). Average cell movement, as indicated by the horizontal bar , was calculated and significance tested using a two-tailed Student's t test; *, p
Figure Legend Snippet: Cl − channel blockers inhibit the metastatic phenotypic changes associated with ST expression. A and B , in ( A ) i293-EGFP and i293-EGFP-ST cells were induced with doxycycline hyclate and in ( B ) MCC13 cells were transfected with EGFP and EGFP-ST. ( i ) cells were then treated with DMSO, DIDS, RIAA, or NPPB and after 24 h, cell motility was measured using the IncuCyte kinetic imaging system. Images were taken every 30 min for a 24-h period. ( ii ) scatter plot showing cell movement tracked using ImageJ ( n = 25). Average cell movement, as indicated by the horizontal bar , was calculated and significance tested using a two-tailed Student's t test; *, p

Techniques Used: Expressing, Transfection, Imaging, Two Tailed Test

18) Product Images from "PI3K and PKC contribute to membrane depolarization mediated by ?2-adrenoceptors in the canine isolated mesenteric vein"

Article Title: PI3K and PKC contribute to membrane depolarization mediated by ?2-adrenoceptors in the canine isolated mesenteric vein

Journal: BMC Physiology

doi: 10.1186/1472-6793-5-9

The EFS-induced slow membrane depolarization of canine isolated mesenteric vein is reduced by various ion channel inhibitors . Intracellular recordings of membrane potential in response to EFS (0.1 ms, 13 V, 10 s, 0.5 Hz) in canine isolated mesenteric veins preincubated for 60 min prior to and throughout the experiment with 100 μM NFA (A), 30 μM NPPB (B), 200 μM DIDS (C) and 30 μM Gd 3+ (D). The experimental data are presented as mean ± SEM, n = 3–4 (E). *, P
Figure Legend Snippet: The EFS-induced slow membrane depolarization of canine isolated mesenteric vein is reduced by various ion channel inhibitors . Intracellular recordings of membrane potential in response to EFS (0.1 ms, 13 V, 10 s, 0.5 Hz) in canine isolated mesenteric veins preincubated for 60 min prior to and throughout the experiment with 100 μM NFA (A), 30 μM NPPB (B), 200 μM DIDS (C) and 30 μM Gd 3+ (D). The experimental data are presented as mean ± SEM, n = 3–4 (E). *, P

Techniques Used: Isolation, Mass Spectrometry

19) Product Images from "Euryhaline pufferfish NBCe1 differs from nonmarine species NBCe1 physiology"

Article Title: Euryhaline pufferfish NBCe1 differs from nonmarine species NBCe1 physiology

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00233.2011

Experimental protocol used to study HCO 3 − -independent components and DIDS inhibition of NBCe1 proteins. A and B : current traces ( V h = −60 mV, n = 3) from fNBCe1 and hkNBCe1. Solution changes are indicated by horizontalbars above traces.
Figure Legend Snippet: Experimental protocol used to study HCO 3 − -independent components and DIDS inhibition of NBCe1 proteins. A and B : current traces ( V h = −60 mV, n = 3) from fNBCe1 and hkNBCe1. Solution changes are indicated by horizontalbars above traces.

Techniques Used: Inhibition

DIDS effects on CO 2 /HCO 3 − -elicited currents of NBCe1 proteins. A and B : DIDSinhibition of CO 2 /HCO 3 − -elicited currents in oocytes expressing fNBCe1 and hkNBCe1. Experimental protocolsare described in legend. I-V relationships show
Figure Legend Snippet: DIDS effects on CO 2 /HCO 3 − -elicited currents of NBCe1 proteins. A and B : DIDSinhibition of CO 2 /HCO 3 − -elicited currents in oocytes expressing fNBCe1 and hkNBCe1. Experimental protocolsare described in legend. I-V relationships show

Techniques Used: Expressing

20) Product Images from "Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation"

Article Title: Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00256.2015

Cotreatment with anion-channel blockers and transient knock-down of LRRC8A abolish Cisplatin-induced but not TNFα and Hyperosmotic-induced Caspase-3 activation in wild-type A2780 cells. A2780WT and A2780CisR cell lysates were used for Caspase-3 activity assay using a commercial kit (see experimental procedures ). A : A2780CisR cells were exposed to 10 μM Cisplatin for 18 h and A2780WT cells were likewise treated with Cisplatin in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 6–8 (A2780WT) and 4 (A2780CisR) individual sets of experiments ± SE. * and #: Significantly increased by Cisplatin compared with the respective control and significantly reduced compared with Cisplatin treatment in the absence of the inhibitor, respectively (ANOVA, Fisher LSD method). B : A2780WT were treated with either 25 nM scramble siRNA or LRRC8A siRNA for 30 h and subsequently exposed to 10 μM Cisplatin for another 18 h. Data represent 4 individual experiments ± SE. * and #: Significantly increased by Cisplatin compared with the respective control and significantly reduced compared with Cisplatin treatment in Scramble siRNA-treated cells (Student's t -test). C: A2780CisR cells were exposed to 20 nM TNFα for 18 h. A2780WT cells were likewise treated with TNFα in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 6–8 (WT) and 4 (CisR) individual experiments ± SE. *Significantly increased compared with the respective control (ANOVA, Fisher LSD method). D : A2780WT cells were exposed to isotonic or hypertonic NaCl for 4.5 h in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 3 individual experiments ± SE. *Significantly increased compared with isotonic control (ANOVA, Fisher LSD method).
Figure Legend Snippet: Cotreatment with anion-channel blockers and transient knock-down of LRRC8A abolish Cisplatin-induced but not TNFα and Hyperosmotic-induced Caspase-3 activation in wild-type A2780 cells. A2780WT and A2780CisR cell lysates were used for Caspase-3 activity assay using a commercial kit (see experimental procedures ). A : A2780CisR cells were exposed to 10 μM Cisplatin for 18 h and A2780WT cells were likewise treated with Cisplatin in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 6–8 (A2780WT) and 4 (A2780CisR) individual sets of experiments ± SE. * and #: Significantly increased by Cisplatin compared with the respective control and significantly reduced compared with Cisplatin treatment in the absence of the inhibitor, respectively (ANOVA, Fisher LSD method). B : A2780WT were treated with either 25 nM scramble siRNA or LRRC8A siRNA for 30 h and subsequently exposed to 10 μM Cisplatin for another 18 h. Data represent 4 individual experiments ± SE. * and #: Significantly increased by Cisplatin compared with the respective control and significantly reduced compared with Cisplatin treatment in Scramble siRNA-treated cells (Student's t -test). C: A2780CisR cells were exposed to 20 nM TNFα for 18 h. A2780WT cells were likewise treated with TNFα in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 6–8 (WT) and 4 (CisR) individual experiments ± SE. *Significantly increased compared with the respective control (ANOVA, Fisher LSD method). D : A2780WT cells were exposed to isotonic or hypertonic NaCl for 4.5 h in the absence or presence of 100 μM NS3728 or 400 μM DIDS. Data represent 3 individual experiments ± SE. *Significantly increased compared with isotonic control (ANOVA, Fisher LSD method).

Techniques Used: Activation Assay, Caspase-3 Activity Assay

Impairment of swelling induced taurine release following pharmacological inhibition or transient knock-down of LRRC8A in wild-type A2780 cells. Volume-sensitive taurine release and LRRC8A protein expression were measured in ovarian A2780WT cells by tracer technique and Western blot analysis, respectively. A2780WT cells were loaded with [ 3 H]taurine for 2 h, washed, and exposed to isosmotic NaCl medium (300 mOsM) for 10 min and subsequently to hyposmotic medium (200 mOsM) for 20 min (arrow in A indicates shift to hypotonicity). Samples were taken every second minute. A : fractional rate constant (min −1 ) for taurine release was determined and plotted vs. time (min) under isosmotic and hyposmotic conditions in the absence (○) or presence of the VRAC/VSOAC blockers NS3728 (●) or DIDS (■). The anion blockers NS3728 and DIDS were used in the concentration of 10 μM (≈2.5 μM free) and 100 μM, respectively, and present during the isotonic/hypotonic release experiment. Data represent 1 of 3 sets of experiments. B : maximal rate constant for taurine release was determined 6 min after hyposmotic exposure as illustrated in A . Data represent the mean maximal rate constants determined from 3 sets of experiments ± SE. *Significantly reduced compared with control (Student's t -test). C : efficiency of siRNA-mediated LRRC8A knock-down in A2780WT cells was determined by Western blot analysis using control, scramble siRNA and LRRC8A siRNA transfected cells and specific monoclonal antibody raised against human LRRC8A and β-actin (loading control). D : LRRC8A/β-actin protein band intensity ratios were calculated from Western blots shown in C and values given relative to control cells. Data represent mean of 4 experiments ± SE. * and #: Significantly reduced compared with Control and Scramble siRNA, respectively (Student's t -test). Scramble siRNA was not significantly larger than Control ( P = 0.15, Student's t -test). E : maximal rate constant for taurine release was determined at time 6 min after hypotonic exposure in A2780WT (open bar) cells or in A2780 cells exposed to scramble siRNA (light gray bar) or siRNA directed against human LRRC8A (dark gray bar). Data represent mean of 4 experiments ± SE. *Significantly reduced compared with Control (Student's t -test).
Figure Legend Snippet: Impairment of swelling induced taurine release following pharmacological inhibition or transient knock-down of LRRC8A in wild-type A2780 cells. Volume-sensitive taurine release and LRRC8A protein expression were measured in ovarian A2780WT cells by tracer technique and Western blot analysis, respectively. A2780WT cells were loaded with [ 3 H]taurine for 2 h, washed, and exposed to isosmotic NaCl medium (300 mOsM) for 10 min and subsequently to hyposmotic medium (200 mOsM) for 20 min (arrow in A indicates shift to hypotonicity). Samples were taken every second minute. A : fractional rate constant (min −1 ) for taurine release was determined and plotted vs. time (min) under isosmotic and hyposmotic conditions in the absence (○) or presence of the VRAC/VSOAC blockers NS3728 (●) or DIDS (■). The anion blockers NS3728 and DIDS were used in the concentration of 10 μM (≈2.5 μM free) and 100 μM, respectively, and present during the isotonic/hypotonic release experiment. Data represent 1 of 3 sets of experiments. B : maximal rate constant for taurine release was determined 6 min after hyposmotic exposure as illustrated in A . Data represent the mean maximal rate constants determined from 3 sets of experiments ± SE. *Significantly reduced compared with control (Student's t -test). C : efficiency of siRNA-mediated LRRC8A knock-down in A2780WT cells was determined by Western blot analysis using control, scramble siRNA and LRRC8A siRNA transfected cells and specific monoclonal antibody raised against human LRRC8A and β-actin (loading control). D : LRRC8A/β-actin protein band intensity ratios were calculated from Western blots shown in C and values given relative to control cells. Data represent mean of 4 experiments ± SE. * and #: Significantly reduced compared with Control and Scramble siRNA, respectively (Student's t -test). Scramble siRNA was not significantly larger than Control ( P = 0.15, Student's t -test). E : maximal rate constant for taurine release was determined at time 6 min after hypotonic exposure in A2780WT (open bar) cells or in A2780 cells exposed to scramble siRNA (light gray bar) or siRNA directed against human LRRC8A (dark gray bar). Data represent mean of 4 experiments ± SE. *Significantly reduced compared with Control (Student's t -test).

Techniques Used: Inhibition, Expressing, Western Blot, Concentration Assay, Transfection

Administration of anion- and cation-channel blockers reduces Cisplatin-induced expression of p53 and p21 Waf1/Cip1 in wild-type A2780 cells (A2780WT) to the same level as observed in Cisplatin-resistant A2780 cells (A2780CisR). Whole cell protein lysates for Western blot analysis were obtained from A2780WT and A2780CisR cells exposed to 10 μM Cisplatin for 18 h in combination with the anion/amino acid channel blockers NS3728 (100 μM)/DIDS (400 μM) or the cation-channel (TASK-2) blocker clofilium (25 μM). Only A2780WT cells were exposed to channel blockers. Protein expression of LRRC8A, p53, p-p53 (Ser15), p21, and Bax was determined using specific antibodies (see experimental procedures ). β-Actin was used as loading control. A : representative Western blot. B : p53, pp53, p21, and Bax protein expression relative to β-actin in A2780WT and A2780CisR (open bars) control cells and following Cisplatin exposure (black bars). Data represent 4–9 individual sets of experiments where ratios are given relative to the untreated WT cells ± SE. * and #: Expression is significantly increased in Cisplatin-treated cell compared with control cells and significantly reduced in Cisplatin-treated A2780CisR cells compared with Cisplatin-treated A2780WT cells, respectively (ANOVA, Fisher LSD method). C : graph represents the relative effect of Cisplatin (Cisplatin-treated/respective untreated control) on LRRC8A, p53, p-p53, and p21 protein expression in A2780WT, A2780CisR (CisR), and A2780WT cells cotreated with the anion- and cation-channel blockers. Data represent mean of 4–9 individual experiments ± SE. *Significantly reduced effect of Cisplatin compared with A2780WT (ANOVA, Fisher LSD method).
Figure Legend Snippet: Administration of anion- and cation-channel blockers reduces Cisplatin-induced expression of p53 and p21 Waf1/Cip1 in wild-type A2780 cells (A2780WT) to the same level as observed in Cisplatin-resistant A2780 cells (A2780CisR). Whole cell protein lysates for Western blot analysis were obtained from A2780WT and A2780CisR cells exposed to 10 μM Cisplatin for 18 h in combination with the anion/amino acid channel blockers NS3728 (100 μM)/DIDS (400 μM) or the cation-channel (TASK-2) blocker clofilium (25 μM). Only A2780WT cells were exposed to channel blockers. Protein expression of LRRC8A, p53, p-p53 (Ser15), p21, and Bax was determined using specific antibodies (see experimental procedures ). β-Actin was used as loading control. A : representative Western blot. B : p53, pp53, p21, and Bax protein expression relative to β-actin in A2780WT and A2780CisR (open bars) control cells and following Cisplatin exposure (black bars). Data represent 4–9 individual sets of experiments where ratios are given relative to the untreated WT cells ± SE. * and #: Expression is significantly increased in Cisplatin-treated cell compared with control cells and significantly reduced in Cisplatin-treated A2780CisR cells compared with Cisplatin-treated A2780WT cells, respectively (ANOVA, Fisher LSD method). C : graph represents the relative effect of Cisplatin (Cisplatin-treated/respective untreated control) on LRRC8A, p53, p-p53, and p21 protein expression in A2780WT, A2780CisR (CisR), and A2780WT cells cotreated with the anion- and cation-channel blockers. Data represent mean of 4–9 individual experiments ± SE. *Significantly reduced effect of Cisplatin compared with A2780WT (ANOVA, Fisher LSD method).

Techniques Used: Expressing, Western Blot

21) Product Images from "Acidic pH-activated Cl- Current and Intracellular Ca2+ Response in Human Keratinocytes"

Article Title: Acidic pH-activated Cl- Current and Intracellular Ca2+ Response in Human Keratinocytes

Journal:

doi: 10.4196/kjpp.2008.12.4.177

Effects of anion channel blockers on I Cl,pH . (A, B) Representative current traces obtained by step-like pulses, same as in . I Cl,pH was activated by pH e 5.0, and DIDS (1 µM) or niflumic acid (NFA, 100 µM) was added (right panels).
Figure Legend Snippet: Effects of anion channel blockers on I Cl,pH . (A, B) Representative current traces obtained by step-like pulses, same as in . I Cl,pH was activated by pH e 5.0, and DIDS (1 µM) or niflumic acid (NFA, 100 µM) was added (right panels).

Techniques Used:

22) Product Images from "Molecular determinants of differential pore blocking of kidney CLC-K chloride channels"

Article Title: Molecular determinants of differential pore blocking of kidney CLC-K chloride channels

Journal: EMBO Reports

doi: 10.1038/sj.embor.7400169

Effects of 3-phenyl-CPP and DIDS on CLC-Kb. Voltage-clamp traces of CLC-Kb currents before and during application of 200 μM 3-phenyl-CPP ( A ) and 200 μM DIDS ( B ), and immediately after wash-out. Horizontal scale bars indicate 100 ms. Vertical scale bars indicate 2 μA ( A ) and 1 μA ( B ).
Figure Legend Snippet: Effects of 3-phenyl-CPP and DIDS on CLC-Kb. Voltage-clamp traces of CLC-Kb currents before and during application of 200 μM 3-phenyl-CPP ( A ) and 200 μM DIDS ( B ), and immediately after wash-out. Horizontal scale bars indicate 100 ms. Vertical scale bars indicate 2 μA ( A ) and 1 μA ( B ).

Techniques Used: Conditioned Place Preference, Mass Spectrometry

Effects of 3-phenyl-CPP and DIDS on CLC-Ka. ( A,B ) Voltage-clamp traces of CLC-Ka currents before and during application of 200 μM 3-phenyl-CPP ( A ) and 200 μM DIDS ( B ), and immediately after wash-out. The pulse protocol is shown in the inset. Horizontal scale bars indicate 50 ms ( A,B ). Vertical scale bars indicate 5 μA ( A,B ). ( C ) Chemical structures of 3-phenyl-CPP and DIDS. ( D ) Slow recovery from DIDS block for a representative oocyte. Arrows indicate application of DIDS and wash-out. A pulse to 60 mV was applied every 2 s and the current is shown as a function of the elapsed time.
Figure Legend Snippet: Effects of 3-phenyl-CPP and DIDS on CLC-Ka. ( A,B ) Voltage-clamp traces of CLC-Ka currents before and during application of 200 μM 3-phenyl-CPP ( A ) and 200 μM DIDS ( B ), and immediately after wash-out. The pulse protocol is shown in the inset. Horizontal scale bars indicate 50 ms ( A,B ). Vertical scale bars indicate 5 μA ( A,B ). ( C ) Chemical structures of 3-phenyl-CPP and DIDS. ( D ) Slow recovery from DIDS block for a representative oocyte. Arrows indicate application of DIDS and wash-out. A pulse to 60 mV was applied every 2 s and the current is shown as a function of the elapsed time.

Techniques Used: Conditioned Place Preference, Mass Spectrometry, Blocking Assay

23) Product Images from "DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism"

Article Title: DIDS inhibits Na-K-ATPase activity in porcine nonpigmented ciliary epithelial cells by a Src family kinase-dependent mechanism

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00057.2013

The influence of DIDS on ERK1/2 phosphorylation. A : a typical time course study in cells exposed to DIDS (100 μM). B : effect of U0126 (10 μM) on DIDS-induced ERK1/2 phosphorylation. Cells were preincubated with U0126 (10 μM) for
Figure Legend Snippet: The influence of DIDS on ERK1/2 phosphorylation. A : a typical time course study in cells exposed to DIDS (100 μM). B : effect of U0126 (10 μM) on DIDS-induced ERK1/2 phosphorylation. Cells were preincubated with U0126 (10 μM) for

Techniques Used:

The influence of U0126 on Na-K-ATPase activity in cells exposed to DIDS. Na-K-ATPase activity (ouabain-sensitive ATP hydrolysis) was measured in samples obtained by homogenizing cells that had been preincubated with U0126 (10 μM) for 20 min before
Figure Legend Snippet: The influence of U0126 on Na-K-ATPase activity in cells exposed to DIDS. Na-K-ATPase activity (ouabain-sensitive ATP hydrolysis) was measured in samples obtained by homogenizing cells that had been preincubated with U0126 (10 μM) for 20 min before

Techniques Used: Activity Assay

Comparison of Na-K-ATPase activity inhibition elicited by DIDS and SITS. Na-K-ATPase activity (ouabain-sensitive ATP hydrolysis) was measured in samples obtained by homogenizing cells that had been exposed to DIDS (100 μM) or SITS (100 μM)
Figure Legend Snippet: Comparison of Na-K-ATPase activity inhibition elicited by DIDS and SITS. Na-K-ATPase activity (ouabain-sensitive ATP hydrolysis) was measured in samples obtained by homogenizing cells that had been exposed to DIDS (100 μM) or SITS (100 μM)

Techniques Used: Activity Assay, Inhibition

24) Product Images from "The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle"

Article Title: The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle

Journal: The Journal of Physiology

doi: 10.1111/j.1469-7793.2000.00541.x

Effects of chloride ion channel blockers on single-channel NCC27 currents The top panel demonstrates the effect of IAA-94. Current blockade begins within 30 s and is complete at 3 min, but recovers fully and rapidly. A9C (middle panel) produces rapid, complete but irreversible block. DIDS had no effect on the NCC27 single-channel current (bottom panel).
Figure Legend Snippet: Effects of chloride ion channel blockers on single-channel NCC27 currents The top panel demonstrates the effect of IAA-94. Current blockade begins within 30 s and is complete at 3 min, but recovers fully and rapidly. A9C (middle panel) produces rapid, complete but irreversible block. DIDS had no effect on the NCC27 single-channel current (bottom panel).

Techniques Used: Blocking Assay

Line graphs from 3 independent experiments demonstrating the effects of increasing concentrations of Cl − channel blockers on the proportion of CHO-K1 cells in G2/M phase (expressed as % change from control) DIDS had no effect at any concentration (left). A9C significantly increased the fraction in G2/M at 2 mM ( P
Figure Legend Snippet: Line graphs from 3 independent experiments demonstrating the effects of increasing concentrations of Cl − channel blockers on the proportion of CHO-K1 cells in G2/M phase (expressed as % change from control) DIDS had no effect at any concentration (left). A9C significantly increased the fraction in G2/M at 2 mM ( P

Techniques Used: Concentration Assay

25) Product Images from "Patch Clamp on the Luminal Membrane of Exocrine Gland Acini from Frog Skin (Rana esculenta) Reveals the Presence of Cystic Fibrosis Transmembrane Conductance Regulator-like Cl− Channels Activated by Cyclic AMP "

Article Title: Patch Clamp on the Luminal Membrane of Exocrine Gland Acini from Frog Skin (Rana esculenta) Reveals the Presence of Cystic Fibrosis Transmembrane Conductance Regulator-like Cl− Channels Activated by Cyclic AMP

Journal: The Journal of General Physiology

doi:

Pharmacology. Inside-out patch with two active Cl − channels held at −V p = 50 mV. In “Control” periods 1–5, the patch was washed with I-28 + 1.5 mM ATP on the inside. Sequential addition of putative blockers to I-28 + 1.5 mM ATP are indicated. In Control period 5, the patch was washed for 6 min without DIDS. Note the appearance of channel transitions with depressed amplitude.
Figure Legend Snippet: Pharmacology. Inside-out patch with two active Cl − channels held at −V p = 50 mV. In “Control” periods 1–5, the patch was washed with I-28 + 1.5 mM ATP on the inside. Sequential addition of putative blockers to I-28 + 1.5 mM ATP are indicated. In Control period 5, the patch was washed for 6 min without DIDS. Note the appearance of channel transitions with depressed amplitude.

Techniques Used:

26) Product Images from "Assessment of Methods for the Intracellular Blockade of GABAA Receptors"

Article Title: Assessment of Methods for the Intracellular Blockade of GABAA Receptors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0160900

Effect of DIDS, the absence of nucleotides and KF on evoked IPSCs. Upper panels of A-D: Example traces during the diffusion of three intracellular pipette solutions (K-Gluconate +DIDS (A), K-Gluconate without ATP or GTP (B), and K-Fluoride at -80mV (C) and -50mV (D)). Each trace shows the IPSC at the start of recording (black, time point i in middle panels), in the last minute before bath application of picrotoxin (magenta, time point ii in middle panels) and at the end of the bath application of picrotoxin (blue, time point iii in middle panels). Middle panels of A-D: Normalised IPSC amplitudes. Blue bar indicates the presence of bath picrotoxin. Lower panels of A-D: Corresponding series resistance during experiments. NBQX and AP5 were present throughout the experiments. E) Group data of the normalised IPSC amplitudes, taken at time point ii, individual data points marked with magenta dots. Summary statistics represent tests comparing the normalised evoked IPSC amplitudes at time point ii with the normalised baseline amplitude for each data set. Data are plotted as mean ± SEM.
Figure Legend Snippet: Effect of DIDS, the absence of nucleotides and KF on evoked IPSCs. Upper panels of A-D: Example traces during the diffusion of three intracellular pipette solutions (K-Gluconate +DIDS (A), K-Gluconate without ATP or GTP (B), and K-Fluoride at -80mV (C) and -50mV (D)). Each trace shows the IPSC at the start of recording (black, time point i in middle panels), in the last minute before bath application of picrotoxin (magenta, time point ii in middle panels) and at the end of the bath application of picrotoxin (blue, time point iii in middle panels). Middle panels of A-D: Normalised IPSC amplitudes. Blue bar indicates the presence of bath picrotoxin. Lower panels of A-D: Corresponding series resistance during experiments. NBQX and AP5 were present throughout the experiments. E) Group data of the normalised IPSC amplitudes, taken at time point ii, individual data points marked with magenta dots. Summary statistics represent tests comparing the normalised evoked IPSC amplitudes at time point ii with the normalised baseline amplitude for each data set. Data are plotted as mean ± SEM.

Techniques Used: Diffusion-based Assay, Transferring

Effect of DNDS, picrotoxin and CsF-DIDS on evoked IPSCs. Upper panels of A-E: Example traces during the diffusion of K-Gluconate (A), K-Gluconate +DNDS (B), K-Gluconate +DNDS CaCl 2 (C), K-Gluconate + picrotoxin (D) and Cs-Fluoride +DIDS (E) intracellular pipette solutions. Each trace shows the IPSC at the start of recording (black, time point i in middle panels), in the last minute before bath application of picrotoxin (magenta, time point ii in middle panels) and at the end of the bath application of picrotoxin (blue, time point iii in middle panels). Middle panels of A-E: Normalised IPSC amplitudes. Blue bar indicates the presence of bath picrotoxin (PTX). Lower panels of A-D: Corresponding series resistance during experiments. NBQX and AP5 were present throughout the experiments. E) Group data of the normalised evoked IPSC amplitudes for the four intracellular pipette solutions, taken at time point ii, individual data points marked as magenta dots. Summary statistics represent tests comparing the normalised evoked IPSC amplitudes at time point ii with the normalised baseline amplitude for each data set. Data are plotted as mean ± SEM.
Figure Legend Snippet: Effect of DNDS, picrotoxin and CsF-DIDS on evoked IPSCs. Upper panels of A-E: Example traces during the diffusion of K-Gluconate (A), K-Gluconate +DNDS (B), K-Gluconate +DNDS CaCl 2 (C), K-Gluconate + picrotoxin (D) and Cs-Fluoride +DIDS (E) intracellular pipette solutions. Each trace shows the IPSC at the start of recording (black, time point i in middle panels), in the last minute before bath application of picrotoxin (magenta, time point ii in middle panels) and at the end of the bath application of picrotoxin (blue, time point iii in middle panels). Middle panels of A-E: Normalised IPSC amplitudes. Blue bar indicates the presence of bath picrotoxin (PTX). Lower panels of A-D: Corresponding series resistance during experiments. NBQX and AP5 were present throughout the experiments. E) Group data of the normalised evoked IPSC amplitudes for the four intracellular pipette solutions, taken at time point ii, individual data points marked as magenta dots. Summary statistics represent tests comparing the normalised evoked IPSC amplitudes at time point ii with the normalised baseline amplitude for each data set. Data are plotted as mean ± SEM.

Techniques Used: Diffusion-based Assay, Transferring

27) Product Images from "DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonate) directly inhibits caspase activity in HeLa cell lysates"

Article Title: DIDS (4,4'-Diisothiocyanatostilbene-2,2'-disulfonate) directly inhibits caspase activity in HeLa cell lysates

Journal: Cell Death Discovery

doi: 10.1038/cddiscovery.2015.37

DIDS totally eliminates caspase activities in a cell-free extract. HeLa cells that were previously treated with or without STS (1 μ M) for 4 h were lysed and these extracts were incubated with DIDS (50 nM), 2-APB (50 nM) or flufenamic acid (50 nM) for 45 min at 37 °C followed by the corresponding caspase activity procedure. Note that DIDS but not the other ion channel inhibitors abolished caspase-3 ( a , n =5), caspase-9 (LEHDase) and caspase-8 (lETDase) activities ( b , n =3). Neither 2-APB nor flufenamic acid induced any DEVDase activity (not shown). * P
Figure Legend Snippet: DIDS totally eliminates caspase activities in a cell-free extract. HeLa cells that were previously treated with or without STS (1 μ M) for 4 h were lysed and these extracts were incubated with DIDS (50 nM), 2-APB (50 nM) or flufenamic acid (50 nM) for 45 min at 37 °C followed by the corresponding caspase activity procedure. Note that DIDS but not the other ion channel inhibitors abolished caspase-3 ( a , n =5), caspase-9 (LEHDase) and caspase-8 (lETDase) activities ( b , n =3). Neither 2-APB nor flufenamic acid induced any DEVDase activity (not shown). * P

Techniques Used: Incubation, Activity Assay

DIDS inhibits caspase-3 processing, PARP degradation and apoptotic nuclei features. HeLa cells preincubated with or without DIDS (50 or 500 μ M) for 30 min before incubation with STS (1 μ M) for 4 h were lysed and ( a ) pro-caspase 3 was detected with antibody (clone 3G2, n =3) and ( b ) PARP fragmentation with antibody (clone 4C10-5, n =5). DIDS totally abolished procaspase-3 proteolytic processing and strongly diminished STS-induced PARP fragmentation. ( c ) Apoptotic nuclei were induced by STS (4 h incubation). However, neither the vehicle (DMSO, 0.1 or 1%) nor DIDS alone induced apoptotic nuclei. Preincubation with DIDS (50 μ M) strongly inhibited STS-induced apoptotic nuclei features. Interestingly, this inhibitory effect was a little bit smaller for a 10 times higher concentration of DIDS (500 μ M) * , P
Figure Legend Snippet: DIDS inhibits caspase-3 processing, PARP degradation and apoptotic nuclei features. HeLa cells preincubated with or without DIDS (50 or 500 μ M) for 30 min before incubation with STS (1 μ M) for 4 h were lysed and ( a ) pro-caspase 3 was detected with antibody (clone 3G2, n =3) and ( b ) PARP fragmentation with antibody (clone 4C10-5, n =5). DIDS totally abolished procaspase-3 proteolytic processing and strongly diminished STS-induced PARP fragmentation. ( c ) Apoptotic nuclei were induced by STS (4 h incubation). However, neither the vehicle (DMSO, 0.1 or 1%) nor DIDS alone induced apoptotic nuclei. Preincubation with DIDS (50 μ M) strongly inhibited STS-induced apoptotic nuclei features. Interestingly, this inhibitory effect was a little bit smaller for a 10 times higher concentration of DIDS (500 μ M) * , P

Techniques Used: Incubation, Concentration Assay

DIDS inhibits STS-induced caspase activation in HeLa cells. Cells that were preincubated with or without DIDS (50 or 500 μ M) for 30 min were incubated with STS 1 μ M for 4 h and lysed to determine caspase activity, which was normalized based on the response obtained with STS. Panels show caspase-3 ( a , n =7), capase-9 ( b , n =4) and caspase-8 ( c , n =4) activities without (total activity, solid columns) and with caspase inhibitors (open columns), which were Ac-DEVD-CHO for caspase-3 ( a ), Ac-LEHD-CHO for caspase-9 ( b ) and Ac-IETD-CHO for capase-8 ( c ).ln all cases, STS induced a significant caspase activity with respect to control (** P
Figure Legend Snippet: DIDS inhibits STS-induced caspase activation in HeLa cells. Cells that were preincubated with or without DIDS (50 or 500 μ M) for 30 min were incubated with STS 1 μ M for 4 h and lysed to determine caspase activity, which was normalized based on the response obtained with STS. Panels show caspase-3 ( a , n =7), capase-9 ( b , n =4) and caspase-8 ( c , n =4) activities without (total activity, solid columns) and with caspase inhibitors (open columns), which were Ac-DEVD-CHO for caspase-3 ( a ), Ac-LEHD-CHO for caspase-9 ( b ) and Ac-IETD-CHO for capase-8 ( c ).ln all cases, STS induced a significant caspase activity with respect to control (** P

Techniques Used: Activation Assay, Incubation, Activity Assay

DIDS does not abolish STS-induced cyt c release. HeLa cells that were in the absence of serum for 19.5 h were preincubated with or without DIDS (50 or 500 μ M) for 30 min followed by STS (1 μ M) incubation for 4 h and lysed to determine cytochrome c release from mitochondria. ( a ) Representative western blots of cytochrome c (cyt c) and p tubulin in supernatants of cell lysates. Note that DIDS 500 μ M release some cyt c by itself. ( b ) The optical density ratio between signals of cyt c and p-tubulin was normalized using the STS-induced ratio as 100%. DIDS did not inhibit cytochrome c release, only reduced it by 20% at both concentrations of DIDS ( n =7). * P
Figure Legend Snippet: DIDS does not abolish STS-induced cyt c release. HeLa cells that were in the absence of serum for 19.5 h were preincubated with or without DIDS (50 or 500 μ M) for 30 min followed by STS (1 μ M) incubation for 4 h and lysed to determine cytochrome c release from mitochondria. ( a ) Representative western blots of cytochrome c (cyt c) and p tubulin in supernatants of cell lysates. Note that DIDS 500 μ M release some cyt c by itself. ( b ) The optical density ratio between signals of cyt c and p-tubulin was normalized using the STS-induced ratio as 100%. DIDS did not inhibit cytochrome c release, only reduced it by 20% at both concentrations of DIDS ( n =7). * P

Techniques Used: Incubation, Western Blot

28) Product Images from "Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism"

Article Title: Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism

Journal: Scientific Reports

doi: 10.1038/srep31433

Effects of DIDS (400 μM) ( A ), AZ (200 μM) ( B ), EIPA (400 μM) ( C ), and bumetanide (Bumex; 400 μM) and/or DIDS (400 μM) ( D ) on Cl − flux at ionocytes of larval skin. Data are presented as the means ± SEM. * p
Figure Legend Snippet: Effects of DIDS (400 μM) ( A ), AZ (200 μM) ( B ), EIPA (400 μM) ( C ), and bumetanide (Bumex; 400 μM) and/or DIDS (400 μM) ( D ) on Cl − flux at ionocytes of larval skin. Data are presented as the means ± SEM. * p

Techniques Used:

29) Product Images from "Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism"

Article Title: Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism

Journal: Scientific Reports

doi: 10.1038/srep31433

Effects of DIDS (400 μM) ( A ), AZ (200 μM) ( B ), EIPA (400 μM) ( C ), and bumetanide (Bumex; 400 μM) and/or DIDS (400 μM) ( D ) on Cl − flux at ionocytes of larval skin. Data are presented as the means ± SEM. * p
Figure Legend Snippet: Effects of DIDS (400 μM) ( A ), AZ (200 μM) ( B ), EIPA (400 μM) ( C ), and bumetanide (Bumex; 400 μM) and/or DIDS (400 μM) ( D ) on Cl − flux at ionocytes of larval skin. Data are presented as the means ± SEM. * p

Techniques Used:

30) Product Images from "Exploring the role of stromal osmoregulation in cancer and disease using executable modelling"

Article Title: Exploring the role of stromal osmoregulation in cancer and disease using executable modelling

Journal: Nature Communications

doi: 10.1038/s41467-018-05414-y

Application of the qualitative network to murine embryonic fibroblasts. Expansion of the QN to p53 −/− , Kras G12D/+ (HET) and p53 −/− , Kras G12D/G12D (HOM) MEFs. a Transport proteins deregulated within HOM MEFs when compared to HET MEFs. Proteins are upregulated (red arrows), downregulated (blue arrows), or unchanged (grey). b Phenotype change for MEFS when HOM MEFs are compared to HET MEFs. The model output represents the physiological behaviour predicted by the model when proteins from ( a , where it is known. The model predicts that attachment will be significantly different between the two cell types, but viability and cell size will remain unchanged. c Effects of application of channel inhibitors to HOM MEFs in vitro showing application of 10 µM DIDs for 72 h, resulting in decreased cellular attachment (left), and application of 10 nM EIPA ± 10 µM DIDs (right). Data are technical replicates. Dotted lines represent calculated Bliss independence values. d Effects of application of 10 µM DIDs ± 10 nM EIPA on viability in HOM MEFs. e Effects of application of 10 µM DIDs ± 10 nM AHCL on attachment in HOM MEFs. f Effects of application of 10 µM DIDs ± 10 nM AHCL on viability in HOM MEFs.* P
Figure Legend Snippet: Application of the qualitative network to murine embryonic fibroblasts. Expansion of the QN to p53 −/− , Kras G12D/+ (HET) and p53 −/− , Kras G12D/G12D (HOM) MEFs. a Transport proteins deregulated within HOM MEFs when compared to HET MEFs. Proteins are upregulated (red arrows), downregulated (blue arrows), or unchanged (grey). b Phenotype change for MEFS when HOM MEFs are compared to HET MEFs. The model output represents the physiological behaviour predicted by the model when proteins from ( a , where it is known. The model predicts that attachment will be significantly different between the two cell types, but viability and cell size will remain unchanged. c Effects of application of channel inhibitors to HOM MEFs in vitro showing application of 10 µM DIDs for 72 h, resulting in decreased cellular attachment (left), and application of 10 nM EIPA ± 10 µM DIDs (right). Data are technical replicates. Dotted lines represent calculated Bliss independence values. d Effects of application of 10 µM DIDs ± 10 nM EIPA on viability in HOM MEFs. e Effects of application of 10 µM DIDs ± 10 nM AHCL on attachment in HOM MEFs. f Effects of application of 10 µM DIDs ± 10 nM AHCL on viability in HOM MEFs.* P

Techniques Used: In Vitro, Cell Attachment Assay

31) Product Images from "The Interaction between Trolox and 4,4’-diisothiocyanatostilbene-2,2’-disulfonic Acid on Hypoxic Pulmonary Vasoconstriction in the Isolated Rabbit Lung"

Article Title: The Interaction between Trolox and 4,4’-diisothiocyanatostilbene-2,2’-disulfonic Acid on Hypoxic Pulmonary Vasoconstriction in the Isolated Rabbit Lung

Journal: Iranian Journal of Medical Sciences

doi:

Pulmonary vascular resistance (∆PVR) in Trolox-treated and Trolox+DIDS-treated groups were lower than other groups at 30 minutes. Data are presented as mean±SE (n=5 in each group). **P
Figure Legend Snippet: Pulmonary vascular resistance (∆PVR) in Trolox-treated and Trolox+DIDS-treated groups were lower than other groups at 30 minutes. Data are presented as mean±SE (n=5 in each group). **P

Techniques Used:

Pulmonary vascular resistance (∆PVR) in Trolox-treated and Trolox+DIDS-treated groups were lower than other groups at 5 minutes. Data are presented as mean±SE (n=5 in each group). *P
Figure Legend Snippet: Pulmonary vascular resistance (∆PVR) in Trolox-treated and Trolox+DIDS-treated groups were lower than other groups at 5 minutes. Data are presented as mean±SE (n=5 in each group). *P

Techniques Used:

Concentrations of NO metabolites of the perfusate in Trolox+DIDS-treated was more than other groups at 30 minutes. Data are presented as mean±SE (n=5 in each group). **P
Figure Legend Snippet: Concentrations of NO metabolites of the perfusate in Trolox+DIDS-treated was more than other groups at 30 minutes. Data are presented as mean±SE (n=5 in each group). **P

Techniques Used:

Pulmonary vascular resistance (∆PVR) in Trolox-treated and Trolox+DIDS-treated groups at 30 minutes were lower than their values at 5 minutes. Data are presented as mean±SE (n=5 in each group). *P
Figure Legend Snippet: Pulmonary vascular resistance (∆PVR) in Trolox-treated and Trolox+DIDS-treated groups at 30 minutes were lower than their values at 5 minutes. Data are presented as mean±SE (n=5 in each group). *P

Techniques Used:

32) Product Images from "HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase"

Article Title: HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

Journal: Cell Death & Disease

doi: 10.1038/cddis.2012.21

Tat-induced swelling in liver isolated mitochondria. ( a ) Sequence of full-length Tat[1-86] (HIV-1 Lai) and Tat derived peptides. ( b ) Dose/time response of Tat[1-86]-induced swelling. Isolated mouse liver mitochondria were exposed to full-length Tat at the indicated concentrations and mitochondrial swelling (measured as 90° light scattering at 545 nm) was monitored continuously. ( c ) Comparative analysis of the effect of Tat-derived peptides on mitochondrial swelling. Isolated mouse liver mitochondria were exposed to the indicated concentrations of Tat-derived peptides. Mitochondrial swelling was monitored for 30 min. Percentages of mitochondrial swelling were calculated as described under Materials and Methods. Data are means (±S.D.) of three independent experiments. ( d ) Evaluation of PTP-related inhibitors on mitochondrial swelling. Liver mitochondria were exposed to Tat[1-86] (0.3 μ M; 30 min) in the presence or absence (Co.) of the following compounds (added 5 min before Tat): cyclosporin A (CsA; 30 μ M), ADP (1 mM), bongkrekic acid (BA; 50 μ M), Bcl-2 (400 nM), Bcl-XL (400 nM), or DIDS (5 μ M). Histograms represent mean values (±S.D.) of five independent experiments. * P
Figure Legend Snippet: Tat-induced swelling in liver isolated mitochondria. ( a ) Sequence of full-length Tat[1-86] (HIV-1 Lai) and Tat derived peptides. ( b ) Dose/time response of Tat[1-86]-induced swelling. Isolated mouse liver mitochondria were exposed to full-length Tat at the indicated concentrations and mitochondrial swelling (measured as 90° light scattering at 545 nm) was monitored continuously. ( c ) Comparative analysis of the effect of Tat-derived peptides on mitochondrial swelling. Isolated mouse liver mitochondria were exposed to the indicated concentrations of Tat-derived peptides. Mitochondrial swelling was monitored for 30 min. Percentages of mitochondrial swelling were calculated as described under Materials and Methods. Data are means (±S.D.) of three independent experiments. ( d ) Evaluation of PTP-related inhibitors on mitochondrial swelling. Liver mitochondria were exposed to Tat[1-86] (0.3 μ M; 30 min) in the presence or absence (Co.) of the following compounds (added 5 min before Tat): cyclosporin A (CsA; 30 μ M), ADP (1 mM), bongkrekic acid (BA; 50 μ M), Bcl-2 (400 nM), Bcl-XL (400 nM), or DIDS (5 μ M). Histograms represent mean values (±S.D.) of five independent experiments. * P

Techniques Used: Isolation, Sequencing, Derivative Assay

Tat-induced swelling in heart-isolated mitochondria. Isolated mouse heart mitochondria were exposed to full-length Tat[1-86] 0.6 μ M and mitochondrial swelling (measured as 90° light scattering at 545 nm) was monitored for 30 min. When indicated, mitochondria were preexposed for 5 min in the presence or absence of the following compounds: cyclosporin A (CsA; 30 μ M), ADP (1 mM), bongkrekic acid (BA; 50 μ M), Bcl-2 (400 nM), or DIDS (5 μ M). Then, mitochondria were incubated with Tat[1-86] (0.3 μ M; 30 min). Percentages of mitochondrial swelling (left panel) were calculated as described under Materials and Methods. Positive control was defined by the addition of 50 μ M CaCl 2 . Histograms represent mean values (±S.D.) of three independent experiments
Figure Legend Snippet: Tat-induced swelling in heart-isolated mitochondria. Isolated mouse heart mitochondria were exposed to full-length Tat[1-86] 0.6 μ M and mitochondrial swelling (measured as 90° light scattering at 545 nm) was monitored for 30 min. When indicated, mitochondria were preexposed for 5 min in the presence or absence of the following compounds: cyclosporin A (CsA; 30 μ M), ADP (1 mM), bongkrekic acid (BA; 50 μ M), Bcl-2 (400 nM), or DIDS (5 μ M). Then, mitochondria were incubated with Tat[1-86] (0.3 μ M; 30 min). Percentages of mitochondrial swelling (left panel) were calculated as described under Materials and Methods. Positive control was defined by the addition of 50 μ M CaCl 2 . Histograms represent mean values (±S.D.) of three independent experiments

Techniques Used: Isolation, Incubation, Positive Control

33) Product Images from "Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine"

Article Title: Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine

Journal: The Journal of Physiology

doi: 10.1111/j.1469-7793.1998.203by.x

Pharmacology of slow wave oscillations in cultured ICC A , lack of effect of nisoldipine (1 μm) on slow waves. B , inhibition of slow waves when cells were exposed to a bathing solution in which Ca 2+ was replaced by Mn 2+ (in physiological saline solution, MnPSS). C , effects of cyclopiazonic acid (CPA). CPA increased frequency and produced a small hyperpolarization in MDP. D , gadolinium (Gd 3+ ) blocked slow wave activity. E , DIDS reduced the amplitude of slow waves, caused a small hyperpolarization in MDP, but did not block spontaneous rhythmicity.
Figure Legend Snippet: Pharmacology of slow wave oscillations in cultured ICC A , lack of effect of nisoldipine (1 μm) on slow waves. B , inhibition of slow waves when cells were exposed to a bathing solution in which Ca 2+ was replaced by Mn 2+ (in physiological saline solution, MnPSS). C , effects of cyclopiazonic acid (CPA). CPA increased frequency and produced a small hyperpolarization in MDP. D , gadolinium (Gd 3+ ) blocked slow wave activity. E , DIDS reduced the amplitude of slow waves, caused a small hyperpolarization in MDP, but did not block spontaneous rhythmicity.

Techniques Used: Cell Culture, Immunocytochemistry, Inhibition, Produced, Activity Assay, Blocking Assay

34) Product Images from "Intracellular Ca2+-Mediated AE2 Is Involved in the Vectorial Movement of HaCaT Keratinocyte"

Article Title: Intracellular Ca2+-Mediated AE2 Is Involved in the Vectorial Movement of HaCaT Keratinocyte

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21228429

Inhibition of transporters by DIDS attenuated HaCaT migration. ( A ) Time-dependent representative images of HaCaT keratinocytes migrating (4 and 48 h) towards agarose spots containing PBS (pH 7.4) with and without 500 nM His and in the absence or presence of 500 μM DIDS-containing media. The direction of migration across the boundary of the agarose spot is shown as a dashed curve (blue dotted lines). The red dotted lines indicate the lineage of keratinocytes that moved into the spots. ( B ) CBE activity of HaCaT keratinocytes with 500 μM DIDS (orange open square) and without (control, black open square) at 48 h. Averaged traces were represented. ( C ) Analysis of CBE activity. Bars indicate the means ± SEM of the number of experiments (* p
Figure Legend Snippet: Inhibition of transporters by DIDS attenuated HaCaT migration. ( A ) Time-dependent representative images of HaCaT keratinocytes migrating (4 and 48 h) towards agarose spots containing PBS (pH 7.4) with and without 500 nM His and in the absence or presence of 500 μM DIDS-containing media. The direction of migration across the boundary of the agarose spot is shown as a dashed curve (blue dotted lines). The red dotted lines indicate the lineage of keratinocytes that moved into the spots. ( B ) CBE activity of HaCaT keratinocytes with 500 μM DIDS (orange open square) and without (control, black open square) at 48 h. Averaged traces were represented. ( C ) Analysis of CBE activity. Bars indicate the means ± SEM of the number of experiments (* p

Techniques Used: Inhibition, Migration, Activity Assay

35) Product Images from "Chloride channels are involved in sperm motility and are downregulated in spermatozoa from patients with asthenozoospermia"

Article Title: Chloride channels are involved in sperm motility and are downregulated in spermatozoa from patients with asthenozoospermia

Journal: Asian Journal of Andrology

doi: 10.4103/1008-682X.181816

Inhibitory effects of the chloride channel blockers NPPB and DIDS (both at 100 μmol l −1 ) on the motility of spermatozoa from the normozoospermic and asthenozoospermic semen samples in hypotonic BWW 290 . The inhibitory effects of NPPB and DIDS on the (a) progressive motility (PR), (b) straight-line velocity (VSL), (c) curved-line velocity (VCL) and (d) average path velocity (VAP), respectively, when incubated for 30 min. Data are shown as the mean ± s.e. (semen samples, n from 8 to 22). ** P
Figure Legend Snippet: Inhibitory effects of the chloride channel blockers NPPB and DIDS (both at 100 μmol l −1 ) on the motility of spermatozoa from the normozoospermic and asthenozoospermic semen samples in hypotonic BWW 290 . The inhibitory effects of NPPB and DIDS on the (a) progressive motility (PR), (b) straight-line velocity (VSL), (c) curved-line velocity (VCL) and (d) average path velocity (VAP), respectively, when incubated for 30 min. Data are shown as the mean ± s.e. (semen samples, n from 8 to 22). ** P

Techniques Used: Incubation

Effects of hypotonic stimulation and the chloride channel blockers NPPB and DIDS on the volumes of spermatozoa from normozoospermic and asthenozoospermic semen samples. Sperm volume was detected by monitoring the forward scatter signals (FSCs) using flow cytometry. (a) Relative sperm volumes expressed as FSC signals of the in the hypotonic (BWW 290 ) and isotonic (BWW 330 ) solutions; (b) and (c) the effects of NPPB and DIDS (both at 100 μmol l −1 ) on the relative sperm volume (expressed as the FSC signal ratio of blockers/BWW 290 control) in the normozoospermic and asthenozoospermic semen samples, respectively; and the percentage sperm volume increases caused by (d) DIDS and (e) NPPB in the hypotonic solution, respectively. ** P
Figure Legend Snippet: Effects of hypotonic stimulation and the chloride channel blockers NPPB and DIDS on the volumes of spermatozoa from normozoospermic and asthenozoospermic semen samples. Sperm volume was detected by monitoring the forward scatter signals (FSCs) using flow cytometry. (a) Relative sperm volumes expressed as FSC signals of the in the hypotonic (BWW 290 ) and isotonic (BWW 330 ) solutions; (b) and (c) the effects of NPPB and DIDS (both at 100 μmol l −1 ) on the relative sperm volume (expressed as the FSC signal ratio of blockers/BWW 290 control) in the normozoospermic and asthenozoospermic semen samples, respectively; and the percentage sperm volume increases caused by (d) DIDS and (e) NPPB in the hypotonic solution, respectively. ** P

Techniques Used: Flow Cytometry, Cytometry

36) Product Images from "Quantitative phase imaging to study transmembrane water fluxes regulated by CFTR and AQP3 in living human airway epithelial CFBE cells and CHO cells"

Article Title: Quantitative phase imaging to study transmembrane water fluxes regulated by CFTR and AQP3 in living human airway epithelial CFBE cells and CHO cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0233439

Effect of chloride currents inhibition on the forskolin-activated OPD increase in CHO WT-CFTR cells. (A) Time course of the OPD shift after forskolin application without (blue) or with (red) preincubation of CHO WT-CFTR with CFTR(inh)-172 (10 μM). (B) Quantification of the forskolin-activated OPD increase in the presence of HgCl 2 (5 μM), CFTR(inh)-172 (10 μM) or DIDS (200 μM).
Figure Legend Snippet: Effect of chloride currents inhibition on the forskolin-activated OPD increase in CHO WT-CFTR cells. (A) Time course of the OPD shift after forskolin application without (blue) or with (red) preincubation of CHO WT-CFTR with CFTR(inh)-172 (10 μM). (B) Quantification of the forskolin-activated OPD increase in the presence of HgCl 2 (5 μM), CFTR(inh)-172 (10 μM) or DIDS (200 μM).

Techniques Used: Inhibition

37) Product Images from "Depolarization-induced depression of inhibitory transmission in cerebellar Purkinje cells"

Article Title: Depolarization-induced depression of inhibitory transmission in cerebellar Purkinje cells

Journal: Physiological Reports

doi: 10.1002/phy2.61

CaCC blockers disturbed DDI occurrence. (A) The experiments were performed using a pipette filled with NFA (10 μmol/L, n = 7), (B) DIDS (10 μmol/L, n = 8), and (C) vehicle (DMSO; 0.01%, n = 6). The left panel (a) represents the time course of the experiments. The right panel (b) shows representative traces from each experiment. The traces of a (before, at t = 0) and b (after, at t = 10) correspond to the timings shown on the left in the figures. Depolarizing trains were applied at t = 5, arrow. (D) a. Current-voltage relationships of calcium currents in the presence of NFA (filled circles, n = 8) and DMSO (open circles, n = 8). Data obtained from vehicle experiments with DMSO were used as a control. Inset; representative trace of voltage-dependent calcium current was recorded at a holding voltage of −70.5 mV to 20.5 mV with NFA in the pipette. b. Representative traces of chloride tail currents, elicited by holding voltages of −80.5 mV and −60.5 mV. NFA blocked the tail current of CaCCs ( n = 8). Bar graph shows the effect of NFA on the charge of the tail current ( P
Figure Legend Snippet: CaCC blockers disturbed DDI occurrence. (A) The experiments were performed using a pipette filled with NFA (10 μmol/L, n = 7), (B) DIDS (10 μmol/L, n = 8), and (C) vehicle (DMSO; 0.01%, n = 6). The left panel (a) represents the time course of the experiments. The right panel (b) shows representative traces from each experiment. The traces of a (before, at t = 0) and b (after, at t = 10) correspond to the timings shown on the left in the figures. Depolarizing trains were applied at t = 5, arrow. (D) a. Current-voltage relationships of calcium currents in the presence of NFA (filled circles, n = 8) and DMSO (open circles, n = 8). Data obtained from vehicle experiments with DMSO were used as a control. Inset; representative trace of voltage-dependent calcium current was recorded at a holding voltage of −70.5 mV to 20.5 mV with NFA in the pipette. b. Representative traces of chloride tail currents, elicited by holding voltages of −80.5 mV and −60.5 mV. NFA blocked the tail current of CaCCs ( n = 8). Bar graph shows the effect of NFA on the charge of the tail current ( P

Techniques Used: Transferring

38) Product Images from "Mechanisms of U46619-induced contraction of rat pulmonary arteries in the presence and absence of the endothelium"

Article Title: Mechanisms of U46619-induced contraction of rat pulmonary arteries in the presence and absence of the endothelium

Journal: British Journal of Pharmacology

doi: 10.1111/j.1476-5381.2008.00084.x

The effect of the potassium channel blockers PNU37883 (KATP ), ChTx (BKCa and IKCa ), TRAM 34 (IKCa ) and 4-AP (KV ) on basal tone and on the concentration–response curve to U46619 in E+ rings in the absence and presence of the VOCC blockers nifedipine and mibefradil, the chloride channel blockers NFA and DIDS or the putative IP3 receptor antagonist 2-APB
Figure Legend Snippet: The effect of the potassium channel blockers PNU37883 (KATP ), ChTx (BKCa and IKCa ), TRAM 34 (IKCa ) and 4-AP (KV ) on basal tone and on the concentration–response curve to U46619 in E+ rings in the absence and presence of the VOCC blockers nifedipine and mibefradil, the chloride channel blockers NFA and DIDS or the putative IP3 receptor antagonist 2-APB

Techniques Used: Concentration Assay

(A) The effect of VOCC and chloride channel blockade on KCl-induced constriction and (B) relaxation of U46619-induced tone by the chloride channel blockers DIDS and NFA compared with the BK Ca activator NS1619. (A) E+, the effect of nifedipine (1 µmol·L
Figure Legend Snippet: (A) The effect of VOCC and chloride channel blockade on KCl-induced constriction and (B) relaxation of U46619-induced tone by the chloride channel blockers DIDS and NFA compared with the BK Ca activator NS1619. (A) E+, the effect of nifedipine (1 µmol·L

Techniques Used:

The contractile response to the SERCA inhibitor CPA (10 µmol·L −1 ) alone in (E+) rat pulmonary arteries and in the presence of NPPB (30 µmol·L −1 ), NFA (30 µmol·L −1 ), DIDS (100 µmol·L
Figure Legend Snippet: The contractile response to the SERCA inhibitor CPA (10 µmol·L −1 ) alone in (E+) rat pulmonary arteries and in the presence of NPPB (30 µmol·L −1 ), NFA (30 µmol·L −1 ), DIDS (100 µmol·L

Techniques Used:

Effect of the chloride channel blockers NPPB, NFA and DIDS on the concentration–response curve for U46619-induced contraction of (E+) rat pulmonary arteries. (A) Response to U46619 in the absence and presence of NPPB at 10 µmol·L
Figure Legend Snippet: Effect of the chloride channel blockers NPPB, NFA and DIDS on the concentration–response curve for U46619-induced contraction of (E+) rat pulmonary arteries. (A) Response to U46619 in the absence and presence of NPPB at 10 µmol·L

Techniques Used: Concentration Assay

39) Product Images from "Allicin Facilitates Airway Surface Liquid Hydration by Activation of CFTR"

Article Title: Allicin Facilitates Airway Surface Liquid Hydration by Activation of CFTR

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2022.890284

The short-circuit current ( I SC ) response stimulated by allicin was mediated by cystic fibrosis transmembrane conductance regulator (CFTR). (A–C) Representative traces of the I SC responses induced by the allicin (200 μM) (A) without or with pretreatment of (B) CFTRinh-172 (20 μM) or (C) DIDS (100 μM) in 16HBE14o- cells, with (D) the corresponding statistical analysis. (E) Representative trace of the I SC responses induced by the allicin in CFBE41o- cells, with (F) the corresponding statistical analysis. Each column and error bar indicate the mean ± SD. *** p
Figure Legend Snippet: The short-circuit current ( I SC ) response stimulated by allicin was mediated by cystic fibrosis transmembrane conductance regulator (CFTR). (A–C) Representative traces of the I SC responses induced by the allicin (200 μM) (A) without or with pretreatment of (B) CFTRinh-172 (20 μM) or (C) DIDS (100 μM) in 16HBE14o- cells, with (D) the corresponding statistical analysis. (E) Representative trace of the I SC responses induced by the allicin in CFBE41o- cells, with (F) the corresponding statistical analysis. Each column and error bar indicate the mean ± SD. *** p

Techniques Used:

40) Product Images from "SCRAMBLING OF PHOSPHOLIPIDS ACTIVATES RED CELL MEMBRANE CHOLESTEROL"

Article Title: SCRAMBLING OF PHOSPHOLIPIDS ACTIVATES RED CELL MEMBRANE CHOLESTEROL

Journal: Biochemistry

doi: 10.1021/bi6023397

Effect of scramblase inhibitors on ionomycin-Ca ++ promoted oxidation of red cell cholesterol. Washed cells (80 μl) were incubated for 30 min at 37 °C in 0.72 ml PBS, 25 μM DIDS or 1.5 mM TNBS, all containing 0.1 mM EGTA. The cells
Figure Legend Snippet: Effect of scramblase inhibitors on ionomycin-Ca ++ promoted oxidation of red cell cholesterol. Washed cells (80 μl) were incubated for 30 min at 37 °C in 0.72 ml PBS, 25 μM DIDS or 1.5 mM TNBS, all containing 0.1 mM EGTA. The cells

Techniques Used: Incubation

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    HEK cells transfected with EYFP H148Q/I152L , KCNJ10, Clc‐kb, and barttin and exposed to different [KCl] o . Fluorescence at 2 mM KCl recorded from 1 to 200 s was adjusted to a baseline of 100%. For those wells where [KCl] o was changed, a baseline was obtained for the first 50 s in 2 mM KCl, after which the [KCl] o was increased from 2 mM through 5 mM KCl, respectively. (a) EYFP quenching at different [KCl] o . (b) Normalized response to KCl (Δ F / F o) plotted against final [Ko]. Data points are means of four different experiments. (c) Effect of different channel blockers, such as Ba 2+ (1 mM), Tertiapin Q (100 nM), <t>Valsartan</t> (100 µM), <t>DIDS</t> (200 µM) on EYFP quenching
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    HEK cells transfected with EYFP H148Q/I152L , KCNJ10, Clc‐kb, and barttin and exposed to different [KCl] o . Fluorescence at 2 mM KCl recorded from 1 to 200 s was adjusted to a baseline of 100%. For those wells where [KCl] o was changed, a baseline was obtained for the first 50 s in 2 mM KCl, after which the [KCl] o was increased from 2 mM through 5 mM KCl, respectively. (a) EYFP quenching at different [KCl] o . (b) Normalized response to KCl (Δ F / F o) plotted against final [Ko]. Data points are means of four different experiments. (c) Effect of different channel blockers, such as Ba 2+ (1 mM), Tertiapin Q (100 nM), Valsartan (100 µM), DIDS (200 µM) on EYFP quenching

    Journal: Physiological Reports

    Article Title: Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K. Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K

    doi: 10.14814/phy2.14280

    Figure Lengend Snippet: HEK cells transfected with EYFP H148Q/I152L , KCNJ10, Clc‐kb, and barttin and exposed to different [KCl] o . Fluorescence at 2 mM KCl recorded from 1 to 200 s was adjusted to a baseline of 100%. For those wells where [KCl] o was changed, a baseline was obtained for the first 50 s in 2 mM KCl, after which the [KCl] o was increased from 2 mM through 5 mM KCl, respectively. (a) EYFP quenching at different [KCl] o . (b) Normalized response to KCl (Δ F / F o) plotted against final [Ko]. Data points are means of four different experiments. (c) Effect of different channel blockers, such as Ba 2+ (1 mM), Tertiapin Q (100 nM), Valsartan (100 µM), DIDS (200 µM) on EYFP quenching

    Article Snippet: Appropriate negative controls were employed, and selective blockers including DIDS (Sigma) (L'Hoste et al., ), Valsartan (Sigma) (Imbrici et al., ), Tertiapin Q (Abcam) (Doupnik, ), and Ba (Sigma) were used as indicated.

    Techniques: Transfection, Fluorescence

    The ABA antagonist #10 and Band 3 inhibitor DIDS inhibit the ABA-induced [cAMP] i rise and ATP release. A, RBC were preincubated at a 50% hematocrit with 250 μ m of the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine for 15 min; cells

    Journal: The Journal of Biological Chemistry

    Article Title: Abscisic Acid Transport in Human Erythrocytes *

    doi: 10.1074/jbc.M114.629501

    Figure Lengend Snippet: The ABA antagonist #10 and Band 3 inhibitor DIDS inhibit the ABA-induced [cAMP] i rise and ATP release. A, RBC were preincubated at a 50% hematocrit with 250 μ m of the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine for 15 min; cells

    Article Snippet: To inhibit Band 3 transport, ghosts were preincubated without (control) or with 100 μ m DIDS (Sigma) that was dissolved at 1 m m concentration with 0.1 m KHCO3 for 30 min at 37 °C and then centrifuged once at 100,000 × g .

    Techniques: