Journal: Journal of cell communication and signaling

Article Title: Mutant p53 gain-of-function stimulates canonical Wnt signaling via PI3K/AKT pathway in colon cancer.

doi: 10.1007/s12079-023-00793-4

Figure Lengend Snippet: Fig. 3 Mutant p53 knockdown in the SW480 cell line decreases canonical Wnt pathway. A Comparation of non-phospho- rylated β-catenin (active) and p53 protein levels measured in different colon cancer cell lines with different TP53 status, RKO (wild-type p53), SW480 (R273H p53), and SW620 (R273H p53). Densitometric analysis of immunoblots of p53 and active β-catenin was performed using GAPDH as a loading control. B Representa- tive immunoblots and graph of p53 protein in SW480 under different doses of siRNA p53 (25–100 nM); the scramble condition was used as a control (100 nM). C Luciferase activity measured with the TOP/FOP system in both scramble and siRNA p53 (100 nM) condi- tions. The relative units were normalized to Renilla activity. D Representative immunoblot and graph of c-myc normalized to GAPDH. E. Representa- tive colonies and relative area quantification corresponding to scramble and shp53 conditions of SW480-transduced cells. Graphs represent densito- metric analysis from at least three independent experiments (means ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The PI3K/AKT inhibitor wortmannin was obtained from Sigma (Cat. No. 19545-26-7), and the p53 Activator III compound RITA was from Santa Cruz Biotechnology (Cat. No. sc-202743).

Techniques: Mutagenesis, Knockdown, Western Blot, Control, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: BK channels regulate extracellular Tat-mediated HIV-1 LTR transactivation

doi: 10.1038/s41598-019-48777-y

Figure Lengend Snippet: BK channel activator NS1619 acidified endolysosomes and restricted Tat-mediated LTR transactivation. ( A ) Endolysosome pH was measured ratiometrically with LysoSensor. As shown in the representative endolysosome pH tracing and bar graph, NS1619 (20 μM), a BK channel activator, significantly decreased endolysosome pH in U87MG cells, when compared with DMSO control (n = 18 cells from 3 experimental replicates, ***p < 0.001). ( B ) As shown in the representative endolysosome pH tracing and bar graph, co-treatment of NS1619 (20 μM) with chloroquine (CQ, 100 μM) decreased endolysosome pH in U87MG cells, when compared to chloroquine (CQ, 100 μM) treatment alone (n = 12 cells from 2 experimental replicates, ***p < 0.001). ( C ) NS1619 treatment (5, 10, and 20 μM for 48 h) restricted Tat (2 μg/ml)-mediated LTR transactivation in the presence of CQ in U87MG cells stably transfected with an integrated luciferase gene under the control of an HIV-1 LTR promoter (n = 3 experimental replicates, *p < 0.05).

Article Snippet: In some experiments, cells were co-treated with TRPML1 agonist ML-SA1 (Millipore), BK channel blocker Penitrem A (Tocris), or BK channel activator NS1619 (Tocris).

Techniques: Control, Stable Transfection, Transfection, Luciferase

Journal: Scientific Reports

Article Title: BK channels regulate extracellular Tat-mediated HIV-1 LTR transactivation

doi: 10.1038/s41598-019-48777-y

Figure Lengend Snippet: BK channel knockdown blocks NS1619’s effect in restricting Tat-mediated LTR transactivation. ( A ) As shown in the representative immunoblots (GAPDH as loading control) and bar graph, U87MG cells stably transfected with BK channel shRNA exhibited decreased protein levels of BK channel, when compared with cells stably transfected with control-shRNA (n = 4 experimental replicates, **p < 0.01). ( B ) The degree of NS1619 (20 μM)-induced decreases in endolysosome pH was reduced in BK channel knockdown cells, when compared with cells stably transfected with control-shRNA (n = 21 cells from 3 experimental replicates, ***p < 0.001). ( C ) Compared with DMSO control, NS1619 (20 μM for 48 h) restricted Tat-mediated LTR transactivation (in the presence of chloroquine) in U87MG cells stably transfected with control-shRNA. However, NS1619 (20 μM) failed to restrict Tat-mediated LTR transactivation (in the presence of chloroquine) in BK channel knockdown cells (n = 3 experimental replicates, *p < 0.05, NS p > 0.05).

Article Snippet: In some experiments, cells were co-treated with TRPML1 agonist ML-SA1 (Millipore), BK channel blocker Penitrem A (Tocris), or BK channel activator NS1619 (Tocris).

Techniques: Knockdown, Western Blot, Control, Stable Transfection, Transfection, shRNA

Journal: Scientific Reports

Article Title: BK channels regulate extracellular Tat-mediated HIV-1 LTR transactivation

doi: 10.1038/s41598-019-48777-y

Figure Lengend Snippet: ML-SA1 restriction of Tat-mediated LTR transactivation is dependent on activation of BK channels. ( A ) The degree of ML-SA1 (20 μM)-induced decreases in endolysosome pH was reduced in BK channel knockdown cells, when compared with that of U87MG cells stably transfected with control-shRNA (n = 15 cells from 2 experimental replicates, **p < 0.01). ( B ) Compared with DMSO control, ML-SA1 (20 μM for 48 h) restricted Tat-mediated LTR transactivation (in the presence of chloroquine) in U87MG cells stably transfected with control-shRNA (n = 3 experimental replicates, **p < 0.01). However, ML-SA1 (20 μM) failed to restrict Tat-mediated LTR transactivation (in the presence of chloroquine) in BK channel knockdown cells (n = 3 experimental replicates, *p < 0.05, NS p > 0.05). ( C ) The degree of NS1619 (20 μM)-induced decreases in endolysosome pH was similar in TRPML1 knockdown cells as that of U87MG cells stably transfected with control-shRNA (n = 17 cells from 2 experimental replicates, ns p > 0.05). ( D ) Compared with DMSO control, NS1619 (20 μM for 48 h) restricted Tat-mediated LTR transactivation (in the presence of chloroquine) in U87MG cells stably transfected with control-shRNA (n = 3 experimental replicates, **p < 0.01). NS1619 (20 μM) was still able to restrict Tat-mediated LTR transactivation (in the presence of chloroquine) in TRPML1 knockdown cells (n = 3 experimental replicates, *p < 0.05, NS p > 0.05).

Article Snippet: In some experiments, cells were co-treated with TRPML1 agonist ML-SA1 (Millipore), BK channel blocker Penitrem A (Tocris), or BK channel activator NS1619 (Tocris).

Techniques: Activation Assay, Knockdown, Stable Transfection, Transfection, Control, shRNA

Journal: Scientific Reports

Article Title: BK channels regulate extracellular Tat-mediated HIV-1 LTR transactivation

doi: 10.1038/s41598-019-48777-y

Figure Lengend Snippet: ML-SA1 and NS1619 enhanced cellular degradation of extracellularly added Tat. U87MG cells were treated with DMSO, NS1619 (20 μM), or ML-SA1(20 μM) in the presence of high levels of Tat (5.0 μg/ml) and chloroquine (100 μM) for 24 h. Quantitative immunoblotting showed that co-treatment of Tat with NS1619 or ML-SA1 significantly decreased cellular levels of exogenous Tat, when compared with co-treatment of Tat with DMSO (n = 3 experimental replicates, ***p < 0.001).

Article Snippet: In some experiments, cells were co-treated with TRPML1 agonist ML-SA1 (Millipore), BK channel blocker Penitrem A (Tocris), or BK channel activator NS1619 (Tocris).

Techniques: Western Blot

Journal: Journal of Neuroscience

Article Title: Presynaptic 7 Nicotinic Acetylcholine Receptors Enhance Hippocampal Mossy Fiber Glutamatergic Transmission via PKA Activation

doi: 10.1523/jneurosci.2973-13.2014

Figure Lengend Snippet: Figure1. NicotineenhancedtheamplitudeofmossyfiberEPSCswiththeadditionofan7 nAChR PAM. A, Representative traces of eEPSCs recorded from a CA3 pyramidal neuron before (left),during(middle),andafter(right)applicationofnicotine(10M)andthe7nAChRPAM NS1738 (5 M). B, Normalized EPSC amplitude was plotted against the time of nicotine appli- cation (from 0 to 10 min) with (E) and without (F) NS1738 (5 M). C, Normalized EPSC amplitude was plotted against the time of nicotine and NS1738 application (from 0 to 10 min) with(Œ)andwithout(E)thepresenceofMLA(20nM),an7nAChR-selectiveantagonist.D, The histogram shows nicotine-induced net change of normalized EPSC amplitude in listed conditions.DatashownaremeanSEM;statisticalsignificancewasdeterminedbyStudent’s t test (*p 0.05).

Article Snippet: QX314, CNQX, DCG-IV, NS1738, KT5720, PKI 14 –22 amide peptide, KN62, PNU-282987, and PNU-120596 were purchased from Tocris Bioscience.

Techniques:

Journal: Journal of Neuroscience

Article Title: Presynaptic 7 Nicotinic Acetylcholine Receptors Enhance Hippocampal Mossy Fiber Glutamatergic Transmission via PKA Activation

doi: 10.1523/jneurosci.2973-13.2014

Figure Lengend Snippet: Figure 3. Presynaptic nAChRs mediated nicotine’s action on mossy fiber EPSC amplitude. A, Representative traces of paired EPSCs evoked with 50 ms interval were shown before and during application of nicotine and the 7 nAChR PAM NS1738. B, PPRs of EPSCs were plotted for each individual experiment. Summary of data showed a significant reduction in PPR for wild-type mice (C), but not 7 nAChR knock-out mice (D) after 10 min application of nicotine and the 7 nAChR PAM. E, Normalized EPSC amplitude was plotted against the application time of nicotine and 7 nAChR PAM (from 0 to 10 min)withstandard(E)andBAPTA(10mM)-containinginternalsolution().F,Thehistogramindicatesthatthenicotine and the 7 nAChR PAM-induced net change of normalized EPSC amplitude remains the same after postsynaptic dialysis of BAPTA (10 mM). Data shown are mean SEM; statistical significance was determined by Student’s t test (*p 0.05; NS, p 0.05).

Article Snippet: QX314, CNQX, DCG-IV, NS1738, KT5720, PKI 14 –22 amide peptide, KN62, PNU-282987, and PNU-120596 were purchased from Tocris Bioscience.

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm biogenesis

doi: 10.1101/2023.06.05.543669

Figure Lengend Snippet: (A) Graphical representation of respiratory epithelial co-culture biofilm assay utilized for all imaging studies. (B) CFBE41o - cells on glass coverslips were infected with GFP-producing P. aeruginosa (green) while stimulated with NS19504 (25 µM), a BK Ca channel potentiator, or 0.05% DMSO in MEM without phenol red and imaged by fluorescent microscopy at 1-, 3-, and 6-hours. CFBE41o - cell nuclei are stained with Hoescht33342 (blue). Scale bar represents 10 μm. (C) Biomass (μm 3 /μm 2 ) measurements at 1-, 3-, and 6-hours post-inoculation from three independent experiments. Line in bar represents mean value and error bars represent standard error of the mean. Statistical significance was tested by two-way ANOVA with multiple comparisons (* p<0.05). (D) P. aeruginosa biofilms grown in 96-well plates in LB and (E) SCFM with or without NS19504 measured using crystal violet absorbance at 550 nm. (F) Planktonic growth kinetics of P. aeruginosa grown in LB Lennox broth (LB), minimal essential media (MEM), and synthetic cystic fibrosis sputum media (SCFM) with or without NS19504.

Article Snippet: Potassium channel modulating reagents, NS19504 (Cat No. 5276) and paxilline (Cat No. 2006), were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques: Co-Culture Assay, Biofilm Production Assay, Imaging, Infection, Microscopy, Staining

Journal: bioRxiv

Article Title: Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm biogenesis

doi: 10.1101/2023.06.05.543669

Figure Lengend Snippet: (A, B, C) Bacterial attachment at 1 hour and average aggregate area and number at 6 hours grown on CFBE41o - cells in co-culture experiments were measured using Nikon Elements. (A) Number of bacteria attached per 20x field for epithelial cells treated with 0.05% DMSO or NS19504 (25 µM) during respiratory epithelial co-culture biofilm experiments at 1 hour. Line represents mean and error bars represent standard error of the mean. (B) Average aggregate area per 20x field measured at 6-hour time point for epithelial cells treated with 0.05% DMSO or NS19504 (25 µM) during live-cell co-culture experiments. Line represents mean and error bars represent standard error of the mean. (C) Average aggregate number per 20x field measured at 6-hour time point for epithelial cells treated with 0.05% DMSO or NS19504 (25 µM) during live-cell co-culture experiments. Line represents mean and error bars represent standard error of the mean. Line connecting data points indicates data points from same biologic replicate. Statistical significance was tested by unpaired t-test (* p<0.05) for all panels.

Article Snippet: Potassium channel modulating reagents, NS19504 (Cat No. 5276) and paxilline (Cat No. 2006), were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques: Co-Culture Assay, Bacteria

Journal: bioRxiv

Article Title: Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm biogenesis

doi: 10.1101/2023.06.05.543669

Figure Lengend Snippet: (A and B) CFBE41o - cells on glass coverslips are infected with a 1:1:1 mixture of TFP-, YFP-, and tdTomato-producing P. aeruginosa while treated with NS19504 (25 µM) or 0.05% DMSO in respiratory epithelial co-culture biofilm assay and imaged by fluorescent microscopy at 1-, 3-, and 6-hours post-infection. (A) Representative images of bacterial coalescence at 1-, 3-, and 6-hours growth. Scale bar represents 10 μm. White box indicates area of magnification in panel B. (B) Magnification of one quarter of panel A images demonstrating multicolor aggregates (polyclonal) compared to single color aggregates (monoclonal). (C) Proportion of colocalized bacteria at 6 hours measured with Nikon Elements Software. Bar represents mean with dots representing each of the four biologic replicates and error bars represent standard error of the mean. Statistical significance was determined by unpaired t-test (* p<0.05).

Article Snippet: Potassium channel modulating reagents, NS19504 (Cat No. 5276) and paxilline (Cat No. 2006), were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques: Infection, Co-Culture Assay, Biofilm Production Assay, Microscopy, Bacteria, Software

Journal: bioRxiv

Article Title: Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm biogenesis

doi: 10.1101/2023.06.05.543669

Figure Lengend Snippet: (A) CFBE41o - cells on glass coverslips were infected with wild type (WT) PAO1 or ΔkdpFABCDE GFP-producing P. aeruginosa , grown with continuous flow of MEM with either DMSO or NS19504 and imaged by fluorescent microscopy at 1-, 3-, and 6-hours. CFBE41o - cell nuclei are stained with Hoescht33342 (blue). Scale bar represents 10 μm. (B) Biomass (μm 3 /μm 2 ) (measured with Nikon Elements) at 1-, 3-, and 6-hours post-inoculation from four independent experiments. Statistical significance was tested by 2-way ANOVA with multiple comparisons (** p<0.01, *** p<0.001). (C-E) Bacterial attachment at 1 hour and average aggregate area and number at 6 hours grown on CFBE41o - cells in live-cell co-culture experiments were measured using Nikon Elements. (C) Number of bacteria attached per 20x field with WT or ΔkdpFABCDE GFP-producing P. aeruginosa during live-cell co-culture experiments at 1 hour. Line represents mean and error bars represent standard error of the mean. (D) Average aggregate area per 20x field measured at 6-hour time point when infected WT or ΔkdpFABCDE GFP-producing P. aeruginosa . Line represents mean and error bars represent standard error of the mean. (E) Average aggregate number per 20x field measured at 6-hour time point with WT or ΔkdpFABCDE GFP-producing P. aeruginosa . Line represents mean of four biologic replicates and error bars represent standard error of the mean. Statistical significance was tested by unpaired t-test (* p<0.05).

Article Snippet: Potassium channel modulating reagents, NS19504 (Cat No. 5276) and paxilline (Cat No. 2006), were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques: Infection, Microscopy, Staining, Co-Culture Assay, Bacteria