p lyn r geco plasmid  (Addgene inc)

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PACAP-stimulated DAG production is impaired in PLCε KO cells. ( A ) A PACAP-triggered signaling cascade regulates Ca 2+ transients in chromaffin cells. PACAP binds to its high-affinity receptor, PAC1, activating Gα s . Gα s stimulates adenylate cyclase, leading to cAMP production. Elevated cAMP activates Epac and, subsequently, PLCε. PLCε hydrolyzes PIP 2 into two key signaling molecules: IP 3 and DAG. IP 3 binds to its receptors on the endoplasmic reticulum, triggering Ca 2+ release into the cytosol. This study investigates the consequences of the DAG signaling axis ( boxed ). ( B ) Representative images obtained by TIRF imaging of WT and PLCε KO cells expressing a DAG sensor during stimulation (begins at time 0) with 500 nM PACAP. The images show changes in fluorescence intensity over time, indicating DAG production. Dotted lines indicate the cell boundaries based on bright-field images. Scale bars, 5 μ m. ( C ) The percentage change in fluorescence (%ΔF/F 0 ) versus time record of the DAG sensor in WT and PLCε KO cells under basal conditions (physiological saline solution [PSS]) and during PACAP stimulation. The graph depicts the time course of DAG production after PACAP stimulation, with bold lines representing the mean response and shaded areas representing the standard error of the mean. Data were collected from two independent experiments. Sample sizes are n = 15 (WT basal), n = 11 (WT PACAP), n = 9 (KO basal), and n = 15 (KO PACAP). ( D ) Scatterplots showing the individual maximum percent change in DAG sensor fluorescence in response to PACAP in both WT and PLCε KO cells, derived from the data shown in ( C ). Data are presented as mean ± SD. Statistical significance: ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test. Not all comparisons are shown for clarity.

Article Snippet: In some shRNA knockdown experiments, chromaffin cells were electroporated with shRNA first, and cotransduced with red fluorescent Ca 2+ sensor R-GECO (Montana Molecular, Bozeman, MT, U0600R), following the manufacturer’s protocol.

Techniques: Imaging, Expressing, Fluorescence, Saline, Derivative Assay

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PACAP-stimulated Ca 2+ signals and exocytosis are inhibited by NPC 15437 in a dose-dependent manner. ( A ) Representative %ΔF/F 0 versus time trace for chromaffin cells expressing Lck-GCaMP5G and stimulated with either 500 nM PACAP alone or 500 nM PACAP + 1 μ M NPC 15437 (NPC) for 45 s. Experiments were performed on a TIRF microscope. ( B ) Time series images of cells expressing Lck-GCaMP5G during stimulation with 500 nM PACAP or 500 nM PACAP + 1 μ M NPC, in the PACAP + NPC group, NPC was also applied to the bath at least 1 min before stimulation. Scale bars, 4 μ m. ( C ) Representative images of a cell expressing NPY-pHluorin stimulated with 500 nM PACAP. The outline of the cell footprint is indicated by the white lines. Arrows show the location of individual NPY fusion events in the time series. ( D ) Maximum %ΔF/F 0 for chromaffin cells stimulated with 500 nM PACAP alone or with three different concentrations of NPC (1, 10, and 50 μ M). Sample sizes are n = 38 (PACAP), n = 24 (+1 μ M NPC), n = 28 (+10 μ M NPC), and n = 31 (+50 μ M NPC). ( E ) PACAP-stimulated Ca 2+ spike area was reduced by NPC in a dose-dependent manner. Chromaffin cells stimulated with 500 nM PACAP alone or with three different concentrations of NPC (1, 10, and 50 μ M). Sample sizes are n = 37 (PACAP), n = 24 (+1 μ M NPC), n = 28 (+10 μ M NPC), and n = 27 (+50 μ M NPC). ( F ) PACAP-stimulated exocytosis was reduced by NPC in a dose-dependent manner. Chromaffin cells stimulated with 500 nM PACAP alone or with three different concentrations of NPC (1, 10, and 50 μ M) for 45 s. Sample sizes are n = 13 (PACAP), n = 13 (+1 μ M NPC), n = 13 (+10 μ M NPC), and n = 11 (+50 μ M NPC). In all PACAP +NPC groups, NPC was also applied to the bath at least 1 min earlier. Data were collected from 3 independent experiments and are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA with Kruskal-Wallis test. Statistical significance: ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: In some shRNA knockdown experiments, chromaffin cells were electroporated with shRNA first, and cotransduced with red fluorescent Ca 2+ sensor R-GECO (Montana Molecular, Bozeman, MT, U0600R), following the manufacturer’s protocol.

Techniques: Expressing, Microscopy

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PACAP-mediated enhancement of activity-dependent exocytosis requires PKC signaling. ( A ) An example trace of step depolarization-evoked changes in whole-cell capacitance in a chromaffin cell. Membrane capacitance was measured before and after each step depolarization and the corresponding Ca 2+ currents (I ca ) were time integrated to estimate the Ca 2+ charge entry. Cells were held at −90 mV and currents were evoked by 50 ms step depolarizations to 0 mV. ( B ) Cumulative changes in charge (pC) were plotted against cumulative membrane capacitance changes (fF) to assess the input-output relationship. ( C ) A comparison of the pC/ΔC m relationship between cells pretreated with or without the PKC inhibitor NPC (10 μ M) for 5 min, and subsequently treated with PACAP (500 nM). PACAP was bath applied for 1 min after a control train and maintained during the second pulse train ( n = 18). NPC was maintained during the treatment train ( n = 6). Ca 2+ sensitivity of exocytosis was enhanced by PACAP. The PACAP-mediated enhancement was abolished when the cells were pretreated with NPC ( p = 0.5). ( D ) There was no change in IRP size of PACAP-treated cells either in the absence ( p = 0.06) or presence of NPC ( p = 0.09). ( E ) There was a significant increase in the RRP measured for PACAP-treated cells that was eliminated by NPC treatment ( p = 0.2). Statistical significance: ∗∗ p < 0.01; ns, not significant.

Article Snippet: In some shRNA knockdown experiments, chromaffin cells were electroporated with shRNA first, and cotransduced with red fluorescent Ca 2+ sensor R-GECO (Montana Molecular, Bozeman, MT, U0600R), following the manufacturer’s protocol.

Techniques: Activity Assay, Membrane, Comparison, Control

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: PKCβ and PKCε, but not PKCμ, knockdown attenuated PACAP-evoked Ca 2+ transients in chromaffin cells. ( A ) Representative trace of %ΔF/F₀ versus time for PKCβ knockdown and scrambled shRNA-transfected cells loaded with Cal520 and stimulated with 500 nM PACAP. The bottom images illustrate the signal changes at different time points in the same cell as shown in the intensity versus time record. ( B ) Knockdown of PKCβ significantly reduced the maximum amplitude of PACAP-induced Ca 2+ transients. PKCβ knockdown cells exhibited an approximately 70% reduction in the maximum %ΔF/F₀. Data are presented as mean ± SD. ( C ) The total spike area of Ca 2+ transients was also significantly decreased in PKCβ knockdown cells compared with scrambled control cells. Chromaffin cells were loaded with 1 μ M Cal520 for 30 min after transfection with the shRNA plasmid for at least 48 h. Knockdown cells were identified by RFP expression from the shRNA vector. Data are from two independent experiments ( n = 22 scrambled, n = 16 PKCβ KD). ( D ) Representative trace of %ΔF/F₀ versus time for PKCε knockdown and scrambled shRNA-transfected cells stimulated with PACAP. The bottom images show fluorescence signal changes at different time points in the same cell as shown in the trace. ( E ) Knockdown of PKCε using shRNA significantly reduced the maximum amplitude of PACAP-induced Ca 2+ transients. PKCε knockdown cells showed an approximately 70% reduction in the maximum %ΔF/F₀ compared with scrambled shRNA control cells. Data are presented as mean ± SD. ( F ) The total spike area of Ca 2+ transients was also significantly decreased in PKCε knockdown cells compared with scrambled control cells. Chromaffin cells were cotransduced with red GECO and the shRNA plasmid. Knockdown cells were identified by GFP expression from the shRNA vector. Data are from two independent experiments ( n = 18 scrambled, n = 24 PKCε KD). ( G ) Representative trace of %ΔF/F₀ versus time for PKCμ knockdown and scrambled shRNA-transfected cells stimulated with PACAP. The bottom images illustrate fluorescence signal changes at different time points in the same cell as shown in the trace. ( H ) Knockdown of PKCμ using shRNA did not significantly affect the maximum amplitude of PACAP-induced Ca 2+ transients. Data are presented as mean ± SD. ( I ) The total spike area of Ca 2+ signals was not significantly reduced in PKCμ knockdown cells compared with scrambled control cells. Chromaffin cells were loaded with 1 μ M Cal590 for 30 min after transfection with the shRNA plasmid for at least 48 h. Knockdown cells were identified by GFP expression from the shRNA plasmid. Data are from two independent experiments ( n = 29 scrambled, n = 25 PKCμ KD). Statistical significance was determined using a two-tailed unpaired t -test. ∗∗∗∗ p < 0.0001; ns, not significant.

Article Snippet: In some shRNA knockdown experiments, chromaffin cells were electroporated with shRNA first, and cotransduced with red fluorescent Ca 2+ sensor R-GECO (Montana Molecular, Bozeman, MT, U0600R), following the manufacturer’s protocol.

Techniques: Knockdown, shRNA, Transfection, Control, Plasmid Preparation, Expressing, Fluorescence, Two Tailed Test

Journal: Biophysical Journal

Article Title: Roles for PKC signaling in chromaffin cell exocytosis

doi: 10.1016/j.bpj.2024.12.005

Figure Lengend Snippet: Roles for PKC signaling in the PACAP secretory pathway. Activated PLCε hydrolyzes PIP 2 into DAG and IP3. DAG activates PKCs. Activated PKCs translocate from the cytosol to the plasma membrane, where they interact with target proteins, resulting in several downstream effects: 1) enhancement of the size of the readily releasable pool of secretory granules, 2) increased Ca 2+ sensitivity of fusion, and 3) increased Ca 2+ current at negative potentials.

Article Snippet: In some shRNA knockdown experiments, chromaffin cells were electroporated with shRNA first, and cotransduced with red fluorescent Ca 2+ sensor R-GECO (Montana Molecular, Bozeman, MT, U0600R), following the manufacturer’s protocol.

Techniques: Clinical Proteomics, Membrane

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A , B ) BHK cells infected with ZIKV were co-stained with anti-TRPC4 (green) and anti-ZIKV-E-protein (red) antibodies. The scale bar represents 10 μm. The regions of interest (ROI), where correlation analyses are conducted, are depicted in the white boxes. Pearson’s Coefficient ( r = 0.46) was determined to assess the co-localization of the TRPC4 protein and the viral E protein. ( C ) the Co-IP of TRPCs protein and ZIKV-E protein in BHK cells. The anti-flag antibody was used to immunoprecipitate TRPC proteins tagged with flag from cell lysates. The immunoprecipitated complexes were subsequently analyzed using Western blots and probed using the anti-E-protein antibody. The data indicates that there was no association between TRPC and ZIKV-E proteins under our experimental conditions. ( D ) BHK cells were transfected with R-GECO Ca 2+ sensor plasmid to monitor the intracellular Ca 2+ levels in mock or ZIKV-infected cells ( n = 4–5 biological replicates). ΔRFU represents the change in fluorescence intensity relative to the baseline. ( E , F ) A representative immunoblot and the results of densitometry analyses comparing relative TRPC4 protein levels in control, ZIKV-NS3, or ZIKV-NS3 + TRPC4 expressing BHK cells. GAPDH was used as a loading control. Co-expression of ZIKV-NS3 and TRPC4 cDNAs in BHK cells augmented the expression rate of the TRPC4 protein. ( G ) Normalized fluorescence intensity changes (F/F o ) in R-GECO cells co-expressing NS3 and/or TRPC4 in the presence or absence of 10 μM HC-070 ( n = 9–12; 3–4 wells per each experiment; 3 biological replicates). The MANOVA test with Bonferroni correction was employed to determine if there was a significant difference among multiple groups. Data information: In ( D , F , G ), data are presented as mean ± SEM.

Article Snippet: The R-GECO Red Fluorescent Ca 2+ Assay was purchased from Montana Molecular (#U0600R, Bozeman, MT, USA), and the BHK cells were transfected with the R-GECO sensor plasmid according to the manufacturer’s instructions.

Techniques: Infection, Staining, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Fluorescence, Control, Expressing

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) Survival rates of ZIKV-infected BHK cells were increased in the HC-070 (10 μM), EGTA (1 mM, the extracellular calcium chelating agent), and KN-93 (10 μM, CaMKII inhibitor) treatment groups ( n = 5–7 biological replicates). ( B ) Representative images (left) and summary data (right) of ZIKV- or mock-infected BHK cells immunoassayed for the ZIKV E protein (red). The cells were pretreated with DMSO, HC-070 (10 μM), EGTA (1 mM), or KN-93 (10 μM). The fluorescence intensity (F) was quantified using ImageJ software, and the obtained data were compared in the right panel ( n = 4–6 biological replicates). The scale bar represents 50 μm. ( C ) Western blots were performed to detect and quantify the production of ZIKV-NS1 proteins (NS1) in KN-93 (10 μM) or HC-070 (10 μM) pretreated BHK cells infected with ZIKV ( n = 4 biological replicates). ( D ) Viral replication was quantified by counting infectious viral particles isolated from the supernatants of BHK cells infected with ZIKV and treated with either DMSO (vehicle control), HC-070 (10 μM), or KN-93 (10 μM). Infectious viral particles were detected using the viral plaque-forming unit assay ( n = 4 biological replicates). ( E ) Representative immunoblot images showing a decrease of TRPC4 protein levels in KN-93-treated BHK cells without ZIKV infection. ( F ) Co-immunoprecipitation (Co-IP) assays are shown. Whole-cell extracts from ZIKV-NS3 overexpressed BHK cells were subjected to immunoprecipitation (IP) using either anti-NS3 or anti-CaMKII antibody. The co-immunoprecipitated proteins were subsequently detected by western blotting using specific antibodies against CaMKII or NS3. ( G ) The graph shows that ZIKV-infected neonatal mouse brains exhibit an enhanced immunofluorescence staining of pCREB ( n = 4 mice) compared to mock-infected mice. ( H ) Western blot analysis was performed to detect (left) and quantify (right) the phosphorylation level of CREB (pCREB) relative to total CREB in KN-93 (10 μM) or HC-070 (10 μM) pretreated BHK cells infected with ZIKV ( n = 5 biological replicates). ( I – L ) Representative immunoblot images show the efficacy of siRNA-mediated knockdown of the ZIKV-NS1 protein production in ZIKV-infected BHK cells ( n = 4 biological replicates). Prior to ZIKV challenge (MOI 0.01), cells were transfected with scRNAs or siRNAs targeting CaMKII ( I , J ) or CREB ( K , L ) for a duration of 48 h ( n = 4 biological replicates). ( M ) Shown are the changes in normalized R-GECO fluorescence (F/F o ) induced by Gd 3+ (100 μM) in TRPC4 (black line), NS3 (green line), or TRPC4 + NS3 (red line)-expressing cells. Intracellular Ca 2+ levels were monitored using the R-GECO biosensor. Cells were transfected with the indicated cDNAs. The horizontal bar shows the times when Gd 3+ was added to the wells with cells. The right panel displays a comparison of normalized R-GECO fluorescence increases in each tested group ( n = 9–12, 3–4 wells were for each experiment, with three biological replicates). The unpaired T test (two-tailed) and one-way ANOVA followed by the Dunnett’s test as the post hoc were employed to determine if there was a significant difference between two groups or among multiple groups, respectively. Data information: In ( A – D , G , H , J , L , M ), data are presented as mean ± SEM, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. .

Article Snippet: The R-GECO Red Fluorescent Ca 2+ Assay was purchased from Montana Molecular (#U0600R, Bozeman, MT, USA), and the BHK cells were transfected with the R-GECO sensor plasmid according to the manufacturer’s instructions.

Techniques: Infection, Fluorescence, Software, Western Blot, Isolation, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Phospho-proteomics, Knockdown, Transfection, Expressing, Comparison, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: In vitro and in vivo inhibition of the host TRPC4 channel attenuates Zika virus infection

doi: 10.1038/s44321-024-00103-4

Figure Lengend Snippet: ( A ) ZIKV infection of the neonatal mouse brain elevates the protein level of pCREB. 1-day-old ICR mice were infected with ZIKV, and then the brains were collected at 12 dpi. Tissues were fixed and stained with anti-CREB (green), and anti-pCREB (red) antibodies. The scale bar represents 100 μm. ( B , C ) Representative Western blot images demonstrate the effectiveness of siRNA-induced reduction of ZIKV-NS1 protein production in HT22 cells ( n = 4 biological replicates). Prior to exposing the cells to ZIKV (MOI 0.01), a 48-h transfection was performed using scRNAs or siRNAs that specifically target CaMKII ( n = 4 biological replicates) or CREB ( n = 3 biological replicates). ( D ) cells were transfected with R-GECO Ca 2+ sensor plasmid to monitor the intracellular Ca 2+ levels. No difference was observed between the Gd 3+ (100 μM)-induced fluorescence increases in the presence of the vehicle (DMSO) or 10 μM KN-93 in R-GECO + TRPC4 expressing cells ( n = 6 biological replicates). Gd 3+ was applied at the times indicated with the horizontal bar. ( E ) The averaged normalized fluorescence increases induced by histamine (10 μM) in H1R (histamine receptor, green line), H1R + NS3 (orange line), TRPC4 + H1R (black line), or NS3 + TRPC4 + H1R (red line) expressing cells are shown (n = 6 biological replicates). Histamine was added at the times indicated by the horizontal bar. ( E ) Right panel, a comparison of averaged peak values for data shown in the left panel. The unpaired T test (two-tailed) was employed to determine if there was a significant difference between two groups. Data information: In ( B – E ), data are presented as mean ± SEM, ** P ≤ 0.001.

Article Snippet: The R-GECO Red Fluorescent Ca 2+ Assay was purchased from Montana Molecular (#U0600R, Bozeman, MT, USA), and the BHK cells were transfected with the R-GECO sensor plasmid according to the manufacturer’s instructions.

Techniques: Infection, Staining, Western Blot, Transfection, Plasmid Preparation, Fluorescence, Expressing, Comparison, Two Tailed Test