Journal: Heliyon

Article Title: Median raphe glutamatergic neuron-mediated enhancement of GABAergic transmission and suppression of long-term potentiation in the hippocampus

doi: 10.1016/j.heliyon.2024.e38192

Figure Lengend Snippet: MRN VGLUT3 neuron-mediated glutamatergic transmission in SR/SLM 5-HT3aR neurons. A) A schematic of AAV injection into the MRN. The left panel shows an image of the MRN from VGLUT3 Cre/5-HT3aR-EGFP mouse which received an AAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA injection into the MRN. Scale 150 μm. B) An image of the hippocampus from MRN VGLUT3 ChR2/5-HT3aR-EGFP mouse. The inset shows EGFP-expressing SR/SLM 5-HT3aR neurons and mCherry-expressing MRN VGLUT3 axons. Scale 300 μm/25 μm (inset). C) The upper panel shows a schematic for recording MRN VGLUT3 neuron-mediated transmission in SR/SLM 5-HT3aR neurons. The bottom panel shows example traces for MRN VGLUT3 neuron-mediated EPSCs in SR/SLM 5-HT3aR neurons without drug application, in the presence of tetrodotoxin (TTX, 1 μM), TTX+4-aminopyridine (4-AP, 100 μM) and TTX+4-AP + DNQX (10 μM). Scale 50 pA/10 ms. The blue line indicates light application. D) EPSC amplitude in SR/SLM 5-HT3aR neurons before the drug application, in the presence of TTX, TTX+4-AP, and TTX+4-AP + DNQX (6 neurons from 3 female mice and 6 neurons from 3 male mice). F (1,11) = 22.01, P = 0.001. Data are presented as mean ± SEM. E) EPSC amplitude in female (17 neurons/5 mice) and male (16 neurons/5 mice) groups. Female and male groups did not show statistically significant difference in EPSC amplitude (P = 0.8). The horizontal line in each group represents the mean and the vertical line represents SEM. F) The delay between the onset of the light stimulation and the onset of EPSCs in female (17 neurons/5 mice) and male (16 neurons/5 mice) groups. Female and male groups did not show a statistically significant difference in the delay between the onset of light stimulation and the onset of EPSCs (P = 0.77). n.s. not statistically significant.

Article Snippet: The slices were then incubated in mCherry polyclonal antibody (Rockland Immunochemicals, Cat. No. 600401P16) or Anti-GFP antibody (Abcam, Cat. No. ab13970, RRID: AB_300798 ) at a dilution of 1:1000 in 0.3 % Triton-X/TBS for 24 h at 4 °C [ , ].

Techniques: Transmission Assay, Injection, Expressing

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: ( A ) Expression levels of PKA-RIIβ (top panel) and PKA-RIIα (middle panel). PKAR-FKBP-FP appears as a separate band above endogenous PKA-RIIβ due to its larger size. ( B ) Quantification of total (endogenous plus exogenous) RIIβ expression in cells transiently or stably expressing PKAR-FKBP-FP, normalized to expression in HeLa cells (endogenous only). RIIβ expression is significantly increased in HeLa PFM and PFY cell lines vs. control. ( C ) Quantification of PKA-RIIα expression normalized to expression in HeLa cells. ( D ) PKA-RI expression levels. ( E ) Quantification of RI expression normalized to expression in HeLa cells. ( F ) PKA-C expression levels. ( G ) Quantification of PKA-C expression normalized to expression in HeLa cells. GAPDH used as a loading control for all experiments. Lanes (from left to right) contain lysate from HeLa cells transiently expressing our YFP- or mCherry-tagged PKA-R translocation system (lanes 1 and 2), our HeLa PFM and PFY cell lines (lanes 3 and 4), and untransfected HeLa cells (lane 5). Each blot was repeated twice with representative images shown. Raw immunoblot images are provided in . Quantification, including exact p values, is provided in . (*p<0.05). Figure 1—figure supplement 1—source data 1. Raw immunoblot images, labeled and unlabeled. Images are divided into two folders – one for each technical replicate. Figure 1—figure supplement 1—source data 2. Immunoblot statistical analysis. One-way ANOVAs with multiple comparisons for PKAR-IIb (Sheet 1), PKAR-IIa (Sheet 2), PKA-RI (Sheet 3), and PKA-C (Sheet 4) expression data presented in . Mean, standard deviation, and number of technical replicates also shown for each condition.

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Expressing, Stable Transfection, Control, Translocation Assay, Western Blot, Labeling, Standard Deviation

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: ( A ) Schematic of PKA-R translocation system. Rapamycin induces heterodimerization of FKBP and FRB, resulting in translocation of PKA-R to the plasma membrane (FKBP = FK506-binding protein, FRB = FKBP-rapamycin-binding domain, R = rapamycin, PKA-R = PKA regulatory subunit, C = PKA catalytic subunit). ( B ) DNA construct design. Two versions of recombinant PKA-R were created with different fluorescent labels. ( C ) Subcellular localization of PKAR-FKBP-YFP (green) within a transiently transfected HeLa cell at 0 and 10 min after addition of 100 nM rapamycin. Scale bar, 10 µm. mCherry protein (red) co-expressed for visualization. ( D ) PKA-R translocation in HeLa PFM cells quantified as cytoplasmic intensity drop in mCherry channel following addition of DMSO or 100 nM rapamycin. p = 0.0039 at t = 24 min post-rapamycin addition; two-tailed Student’s t -test. ( E ) Catalytic subunit of the protein kinase A (PKA-C) translocation in HeLa cells transiently transfected with PKAR-FKBP-YFP, Lyn-FRB, and mCherry-PKA-C, quantified as a cytoplasmic intensity drop in mCherry channel following addition of 100 nM rapamycin. Cells transfected with mCherry protein instead of mCherry-PKA-C were used as a control. p = 0.037 at t = 24 min post-rapamycin addition; two-tailed Student’s t -test. Graphs display the mean of each data set with standard error of the mean (SEM) indicated by shaded region. Number of cells in each data set is as indicated in the figure. Data is the result of one ( D ) and three ( E ) independent experiments, respectively. Mean and SEM values for each condition and time point are provided in . Arrows indicate the timing of drug addition. Figure 1—source data 1. Characterization of regulatory subunit of the protein kinase A (PKA-R) translocation system. ( a ) Sheet 1, Time Course. Change in mCherry cytoplasmic intensity following addition of 0.1% DMSO or 100 nM rapamycin. Mean, standard error of the mean (SEM), and number of cells given for each time point and condition. ( b ) Sheet 2, Time Course. Change in mCherry cytoplasmic intensity following addition of 100 nM rapamycin. Mean, SEM, and number of cells given for each time point and condition.

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Binding Assay, Construct, Recombinant, Transfection, Two Tailed Test, Control

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: Plasmid maps for ( A ) PKAR-FKBP-YFP and ( B ) PKAR-FKBP-mCherry.

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Plasmid Preparation

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: ( A ) Schematic of the microfluidic device used to produce gradients of rapamycin across microchannels housing HeLa PFY or PFM cells. ( B ) Single cell response to 20 nM rapamycin gradient. Numbers show time in minutes. (Green = PKAR-FKBP-YFP [stably expressed], Blue = H2β-mCerulean [transiently expressed nuclear marker], Red = Alexa Fluor 594 dye). ( C ) Average nuclear position (normalized to t = 0) for HeLa PFY cells in 20 nM rapamycin gradient (0.08 nM/µm) vs. no-translocation rapamycin control (HeLa cells stably expressing PKAR-FKBP-YFP but not Lyn-FRB). Standard error of the mean (SEM) indicated by shaded regions. p = 1.34 × 10 -5 at 180 min post-rapamycin addition; two-tailed Student’s t -test. Number of cells in each data set as indicated in the figure. ( D ) Single cell nuclear position data for no-translocation rapamycin control cells in 20 nM rapamycin gradient. Data from one independent experiment. ( E ) Single cell nuclear position data for HeLa PFY cells in 20 nM rapamycin gradient. Data from three independent experiments. Data from ( D ) superimposed in light gray. ( F ) Tracking of intracellular PKA activity gradient in HeLa PFM cells using the transiently expressed FRET probe Lyn-AKAR4. Rapamycin gradient introduced at t = 0. Colorimetric FRET ratio scale as indicated by color bar. ( G ) Mean intracellular plasma membrane (PM) PKA activity tracking along the cell length from the high end of the rapamycin gradient (‘0’ in panel F) to the low end (‘1’ in panel F). Data represent the mean of n = 9 HeLa PFM cells from two independent experiments with SEM indicated by shaded region. Cells were divided into 20 bins with the average FRET ratio value taken for each. Arrows in ( C–E ) indicate addition of rapamycin. Scale bars in ( B ) and ( F ), 10 µm. Mean and SEM values for each time point and condition in ( C ) and each time point and position in ( G ) are provided in . Figure 3—source data 1. Nuclear position and Lyn-AKAR4 data in microfluidic device. ( a ) Sheet 1, and Time Course. Normalized nuclear position data for cells in 20 nM rapamycin gradient (0.08 nM/µm) with or without the membrane subunit of the regulatory subunit of the protein kinase A (PKA-R) translocation system (Lyn-FRB). Results also shown for cells in a volume equivalent DMSO gradient, as displayed in . Mean, standard error of the mean (SEM), and number of cells given for each time point and condition. ( b ) Sheet 2, Intracellular PKA Activity Distribution. Mean intracellular PM PKA activity along the cell length from the high end of the rapamycin gradient (‘0’) to the low end (‘1’) for three time points. Mean, SEM, and number of cells given for all 20 bins and each time point.

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Stable Transfection, Marker, Translocation Assay, Control, Expressing, Two Tailed Test, Activity Assay, Clinical Proteomics, Membrane

Journal: eLife

Article Title: Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

doi: 10.7554/eLife.66869

Figure Lengend Snippet: ( A ) Single cell response to DMSO gradient (0–0.1% from ‘sink’ to ‘source’ as labeled in ). Numbers show time in minutes. Scale bar, 10 µm. (Green = PKAR-FKBP-YFP [stably expressed], Blue = H2β-mCerulean [transiently expressed nuclear marker], Red = Alexa Fluor 594 dye). ( B ) Nuclear position normalized to t = 0 min post-DMSO addition. A positive nuclear position indicates net movement of the nuclear centroid toward the DMSO source as described in Materials and methods. Arrow indicates DMSO addition. Data represent nuclear position for n = 18 cells from two independent experiments. Mean and standard error of the mean (SEM) values for each time point are provided in .

Article Snippet: The gel purified PCR product was ligated into a transient expression plasmid containing FKBP and either YFP or mCherry fluorescent protein (gifts from Dr Takanari Inoue, Johns Hopkins University) in the case of PKAR-FKBP-FP, or mCherry alone in the case of mCherry-PKA-C.

Techniques: Labeling, Stable Transfection, Marker

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA Encoding a Bispecific Single Domain Antibody Construct Protects against Influenza A Virus Infection in Mice

doi: 10.1016/j.omtn.2020.04.015

Figure Lengend Snippet: Delivery of DOTAP/Cholesterol mRNA Lipid Nanoparticles via Intratracheal Instillation (A) Particle size analysis after incubating DOTAP/cholesterol mRNA lipoplexes in HEPES buffer or bronchoalveolar lavage fluid (BALF). BALB/c mice were i.t. administered with DOTAP/cholesterol particles formulated with different mRNA sequences (mRNA dose of 5 μg). (B) Graph and representative whole-body images showing expression levels of luciferase mRNA (5-methoxyuridine) in lungs of BALB/c mice measured via bioluminescence imaging at 6 h (n = 3 mice). DOTAP/cholesterol nanoparticles containing 1 mol% of the lipophilic dye DiR and packaged with mCherry mRNA (5-methoxyuridine) were i.t. administered, after which nanoparticle uptake (DiR fluorescence) and mRNA expression (mCherry protein) were evaluated in a variety of pulmonary cells subsets (n = 5 mice) at 6 and 24 h post administration. The flow cytometry gating strategy used to discriminate between pulmonary immune cell subsets can be found in the Figure S1 and was adopted from Knight et al. PBS-instilled mice serve as negative controls. (C) Graph shows nanoparticle uptake in CD45 positive cells (immune cells) and CD45 negative cells (nonimmune cells). (D) Nanoparticle uptake in a variety of pulmonary immune cells subsets, including alveolar macrophages, CD103 + dendritic cells (CD103 + DCs), granulocytes, other subsets of monocytes and macrophages that are not encompassed by the other subsets (Mono/macrophage), interstitial macrophages, and CD11b + DCs. (E) Representative flow cytometry plots of nanoparticle uptake and mCherry mRNA expression in alveolar macrophages. The mean percentage of DiR-positive and mCherry-positive cells, together with the mean fluorescence intensity of each signal, are given in each flow plot. (F) 5 μg of FcγRIV VHH-M2e VHH or FcγRIV VHH-RSVF VHH (irrelevant mRNA) formulated in DOTAP/cholesterol particles or 50 μg FcγRIV VHH-M2e VHH protein was instilled i.t. in BALB/c mice. 6, 24, or 48 h after instillation, BALF was isolated and cells were removed from the BALF and the ability of His 6 -tagged proteins to bind to M2e was investigated in a peptide ELISA (see Figure S2 ). De absolute titers were calculated using the standard curve shown in Figure S2 . Graphs show mean ± SEM (n = 3 mice per group). Statistical analyses on datasets were performed by one-way ANOVA followed by Tukey’s post hoc test. Asterisks indicate statistical significance compared to negative control (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Article Snippet: The mRNA constructs encoding for firefly luciferase or the fluorescent mCherry protein were purchased from TriLink (San Diego, CA, USA).

Techniques: Particle Size Analysis, Expressing, Luciferase, Imaging, Fluorescence, Flow Cytometry, Isolation, Peptide ELISA, Negative Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA Encoding a Bispecific Single Domain Antibody Construct Protects against Influenza A Virus Infection in Mice

doi: 10.1016/j.omtn.2020.04.015

Figure Lengend Snippet: Inflammatory Response to Intratracheally Administered mRNA Nanoparticles (A) Graph depicts the relative distribution of each pulmonary immune cell subset as a percent of total viable cells, for PBS-treated mice, and mice that were i.t. instilled with DiR-labeled mRNA nanoparticles containing mCherry mRNA (mRNA dose of 5 μg), that were either sacrificed at 6 or 24 h post administration (n = 5 mice). (B) Serum samples collected from these mice were screened for the presence of inflammatory cytokine responses. Statistical analyses on datasets were performed by one-way ANOVA followed by Tukey’s post hoc test. Asterisks indicate statistical significance compared to negative control (∗p < 0.05; ∗∗∗p < 0.001).

Article Snippet: The mRNA constructs encoding for firefly luciferase or the fluorescent mCherry protein were purchased from TriLink (San Diego, CA, USA).

Techniques: Labeling, Negative Control