pc12 cells  (Alomone Labs)


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    Structured Review

    Alomone Labs pc12 cells
    Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in <t>PC12</t> cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P
    Pc12 Cells, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation"

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    Journal: Journal of Radiation Research

    doi: 10.1093/jrr/rrx032

    Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P
    Figure Legend Snippet: Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Techniques Used: Irradiation

    Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.
    Figure Legend Snippet: Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.

    Techniques Used: Western Blot, Irradiation

    Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P
    Figure Legend Snippet: Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Techniques Used: Inhibition, Activity Assay, Irradiation

    Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P
    Figure Legend Snippet: Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P

    Techniques Used: Irradiation, Activation Assay, Activity Assay

    2) Product Images from "Caspase-cleaved tau is senescence-associated and induces a toxic gain of function by putting a brake on axonal transport"

    Article Title: Caspase-cleaved tau is senescence-associated and induces a toxic gain of function by putting a brake on axonal transport

    Journal: Molecular Psychiatry

    doi: 10.1038/s41380-022-01538-2

    The residence time of individual TauC3 molecules is increased on axonal microtubules without modifying axonal microtubule polymerization. A Single-molecule tracking of a single TauC3 molecule in an axon-like process of a differentiated PC12 cell. The position of tau at selected times is displayed; dashed lines indicate the limit of the neurite. TauC3 was fused N-terminally with a HaloTag to enable substoichiometric labeling with TMR-HTL as indicated below. Scale bar, 0.5 μm. B Pseudotrajectory generated from the time series shown in A (3.4 s = 156 frames) indicating fast and undirected movement in the longitudinal and transverse direction of the axonal process. For comparison, the thickness and density of MTs are indicated schematically by gray bars. The starting point is indicated by a black circle and the time is color-coded as shown on the right. Representative histograms of the residence times of Halo-TauC3 on MTs in PC12 cells ( C ) and in primary neurons prepared from tau knockout mice ( D ). The monoexponential fit is indicated by a red line. The time constant (residence time) is given above (mean ± SEM for n = 8 (PC12 cells) and n = 5 (primary neurons) from 2413 and 1410 temporally immobile molecules, respectively). The residence time of full-length tau was previously determined (Janning et al. [ 18 ]) and is shown in gray. E Representative micrographs of FDAP time-lapse recordings of PAGFP-α-tubulin in a process of a PC12 cell. mCherry-tagged Tau or TauC3 was co-expressed by lentiviral (LV) expression. The photoactivated segment is indicated by a white box. Scale bar, 5 µm. F FDAP diagrams of PAGFP-α-tubulin in cells co-expressing mCherry-Tau (Tau) or mCherry-TauC3 (TauC3). Mean ± SEM of 42 cells for Tau and 34 cells for TauC3 is shown. G Scatter plots of average association (avg k* on ), average dissociation (avg k off ) rate constants with mean ± SEM and percent of polymerized tubulin ( n = 42 and 34 cells for Tau and TauC3, respectively). A schematic representation is shown on the left which gives avg k* on and avg k off of the tubulin-MT equilibrium and the calculation of polymerized tubulin. All transfected tau constructs are based on the human tau sequence. Statistically significant differences determined by unpaired Student’s t test are indicated. * p
    Figure Legend Snippet: The residence time of individual TauC3 molecules is increased on axonal microtubules without modifying axonal microtubule polymerization. A Single-molecule tracking of a single TauC3 molecule in an axon-like process of a differentiated PC12 cell. The position of tau at selected times is displayed; dashed lines indicate the limit of the neurite. TauC3 was fused N-terminally with a HaloTag to enable substoichiometric labeling with TMR-HTL as indicated below. Scale bar, 0.5 μm. B Pseudotrajectory generated from the time series shown in A (3.4 s = 156 frames) indicating fast and undirected movement in the longitudinal and transverse direction of the axonal process. For comparison, the thickness and density of MTs are indicated schematically by gray bars. The starting point is indicated by a black circle and the time is color-coded as shown on the right. Representative histograms of the residence times of Halo-TauC3 on MTs in PC12 cells ( C ) and in primary neurons prepared from tau knockout mice ( D ). The monoexponential fit is indicated by a red line. The time constant (residence time) is given above (mean ± SEM for n = 8 (PC12 cells) and n = 5 (primary neurons) from 2413 and 1410 temporally immobile molecules, respectively). The residence time of full-length tau was previously determined (Janning et al. [ 18 ]) and is shown in gray. E Representative micrographs of FDAP time-lapse recordings of PAGFP-α-tubulin in a process of a PC12 cell. mCherry-tagged Tau or TauC3 was co-expressed by lentiviral (LV) expression. The photoactivated segment is indicated by a white box. Scale bar, 5 µm. F FDAP diagrams of PAGFP-α-tubulin in cells co-expressing mCherry-Tau (Tau) or mCherry-TauC3 (TauC3). Mean ± SEM of 42 cells for Tau and 34 cells for TauC3 is shown. G Scatter plots of average association (avg k* on ), average dissociation (avg k off ) rate constants with mean ± SEM and percent of polymerized tubulin ( n = 42 and 34 cells for Tau and TauC3, respectively). A schematic representation is shown on the left which gives avg k* on and avg k off of the tubulin-MT equilibrium and the calculation of polymerized tubulin. All transfected tau constructs are based on the human tau sequence. Statistically significant differences determined by unpaired Student’s t test are indicated. * p

    Techniques Used: Labeling, Generated, Knock-Out, Mouse Assay, Expressing, Transfection, Construct, Sequencing

    TauC3 slows down the microtubule-dependent transport of mitochondria and reduces the processivity of APP-vesicle transport. A Micrograph of a PC12 cell expressing eGFP-SDHD for tracking the movement of mitochondria in living cells. mCherry-tagged Tau or TauC3 was co-expressed by lentiviral (LV) expression. A moving mitochondrion is indicated by an arrow in the enlarged time-lapse images shown below. Scale bar, 10 µm (top) and 2 µm (bottom). B Bar graphs showing fractions of stationary and mobile mitochondria in the process. Velocity and speed of the mobile mitochondria are displayed in the middle and right graphs. Mean ± SEM, n = 12 cells with 672 trajectories (mCherry-Tau) and n = 22 cells with 1137 trajectories (mCherry-TauC3). C APP-vesicle tracking in a process of a PC12 cell using eGFP-tagged APP and an autoregressive motion algorithm. mCherry-tagged Tau or TauC3 was co-expressed by lentiviral (LV) expression. The images on the left show an overview of a cell that co-expresses tau and APP. The images on the right show selected points in time of the APP-vesicle movement from the part of the process that is indicated by the white box in the overview image. Arrows point to a moving (white) and a stationary vesicle (red). Scale bars, 10 µm (overview) and 2.5 µm (time lapse). D The quantification of the transport parameters is shown on the left. The bar chart shows fractions of mobile and stationary vesicles in the process. The velocity, processivity, and speed of the mobile vesicles are shown in the scatter plots on the right. Each point represents an average value for a respective cell (mean ± SEM of n = 21 cells with 470 trajectories (Tau) and n = 20 cells with 413 trajectories (TauC3)). E The definition of state changes of individual vesicle tracts is shown on the left. The box plot on the right shows the cumulative changes in state. Every dot represents one cell of the data set shown in D . The two diagrams in the middle show the phases of movement of 5 long representative trajectories for cells expressing Tau or TauC3. All transduced tau constructs are based on the human tau sequence. Statistically significant differences determined by unpaired Student’s t test are indicated. * p
    Figure Legend Snippet: TauC3 slows down the microtubule-dependent transport of mitochondria and reduces the processivity of APP-vesicle transport. A Micrograph of a PC12 cell expressing eGFP-SDHD for tracking the movement of mitochondria in living cells. mCherry-tagged Tau or TauC3 was co-expressed by lentiviral (LV) expression. A moving mitochondrion is indicated by an arrow in the enlarged time-lapse images shown below. Scale bar, 10 µm (top) and 2 µm (bottom). B Bar graphs showing fractions of stationary and mobile mitochondria in the process. Velocity and speed of the mobile mitochondria are displayed in the middle and right graphs. Mean ± SEM, n = 12 cells with 672 trajectories (mCherry-Tau) and n = 22 cells with 1137 trajectories (mCherry-TauC3). C APP-vesicle tracking in a process of a PC12 cell using eGFP-tagged APP and an autoregressive motion algorithm. mCherry-tagged Tau or TauC3 was co-expressed by lentiviral (LV) expression. The images on the left show an overview of a cell that co-expresses tau and APP. The images on the right show selected points in time of the APP-vesicle movement from the part of the process that is indicated by the white box in the overview image. Arrows point to a moving (white) and a stationary vesicle (red). Scale bars, 10 µm (overview) and 2.5 µm (time lapse). D The quantification of the transport parameters is shown on the left. The bar chart shows fractions of mobile and stationary vesicles in the process. The velocity, processivity, and speed of the mobile vesicles are shown in the scatter plots on the right. Each point represents an average value for a respective cell (mean ± SEM of n = 21 cells with 470 trajectories (Tau) and n = 20 cells with 413 trajectories (TauC3)). E The definition of state changes of individual vesicle tracts is shown on the left. The box plot on the right shows the cumulative changes in state. Every dot represents one cell of the data set shown in D . The two diagrams in the middle show the phases of movement of 5 long representative trajectories for cells expressing Tau or TauC3. All transduced tau constructs are based on the human tau sequence. Statistically significant differences determined by unpaired Student’s t test are indicated. * p

    Techniques Used: Expressing, Construct, Sequencing

    Caspase-3-cleaved tau (TauC3) is present in mice and patients and shows decreased dynamics of its microtubule (MT) interaction. A Immunoblots from lysates of hippocampal brain tissue from young (2 months (M)) and aged mice (27 M) showing caspase-3-cleaved tau (TauC3), total tau (Tau5) and GAPDH as loading control. Molecular mass standards are indicated. The relative amounts of TauC3/GAPDH, Tau5/GAPDH, and TauC3/Tau5 are shown below (mean ± SEM; n = 9). B Immunoblots from lysates of the human temporal neocortex from healthy controls (con) and AD patients (AD) showing TauC3 and total tau (panTau). Molecular mass standards are indicated. The relative amounts of TauC3/Coomassie, panTau/Coomassie and TauC3/panTau with respect to the Braak stage are shown below (mean ± SEM; n = 6 (con) and n = 7 (AD)). C Schematic representation of the expressed tau constructs. The MT-binding repeat regions (RR1–RR4) are indicated by yellow boxes, the pseudorepeat region in orange and the N-terminal PAGFP-fusion in green. Adult-specific exons in the N-terminus of tau (N1, N2) are shown in dark gray. Immunoblots of PC12 lysates after transfection with the respective tau constructs are displayed below, which shows the specific detection of the shorter tau construct with the TauC3 antibody. Molecular mass standards are given. D A schematic representation of an FDAP experiment is shown on the left. Representative time-lapse micrographs of fluorescence decay after photoactivation (FDAP) in an axon-like process are shown on the right. A 6 µm long segment (white box) in the middle of a process was photoactivated and the fluorescence decay over time within this region was monitored. E FDAP diagrams after photoactivation of PAGFP-Tau (Tau) and PAGFP-TauC3 (TauC3)-expressing cells. Mean ± SEM of 16 (Tau) and 15 (TauC3) cells is shown. Scatter plots of the effective diffusion constants (D eff ) ( F ), and association (k* on ) and dissociation rate constants (k off ) ( G ) (mean ± SEM, n = 16 (Tau) and n = 15 (TauC3)). A schematic representation indicating k* on and k off of the MT-Tau interaction is shown in G . Statistically significant differences between samples determined by an unpaired Student’s t test are indicated. * p
    Figure Legend Snippet: Caspase-3-cleaved tau (TauC3) is present in mice and patients and shows decreased dynamics of its microtubule (MT) interaction. A Immunoblots from lysates of hippocampal brain tissue from young (2 months (M)) and aged mice (27 M) showing caspase-3-cleaved tau (TauC3), total tau (Tau5) and GAPDH as loading control. Molecular mass standards are indicated. The relative amounts of TauC3/GAPDH, Tau5/GAPDH, and TauC3/Tau5 are shown below (mean ± SEM; n = 9). B Immunoblots from lysates of the human temporal neocortex from healthy controls (con) and AD patients (AD) showing TauC3 and total tau (panTau). Molecular mass standards are indicated. The relative amounts of TauC3/Coomassie, panTau/Coomassie and TauC3/panTau with respect to the Braak stage are shown below (mean ± SEM; n = 6 (con) and n = 7 (AD)). C Schematic representation of the expressed tau constructs. The MT-binding repeat regions (RR1–RR4) are indicated by yellow boxes, the pseudorepeat region in orange and the N-terminal PAGFP-fusion in green. Adult-specific exons in the N-terminus of tau (N1, N2) are shown in dark gray. Immunoblots of PC12 lysates after transfection with the respective tau constructs are displayed below, which shows the specific detection of the shorter tau construct with the TauC3 antibody. Molecular mass standards are given. D A schematic representation of an FDAP experiment is shown on the left. Representative time-lapse micrographs of fluorescence decay after photoactivation (FDAP) in an axon-like process are shown on the right. A 6 µm long segment (white box) in the middle of a process was photoactivated and the fluorescence decay over time within this region was monitored. E FDAP diagrams after photoactivation of PAGFP-Tau (Tau) and PAGFP-TauC3 (TauC3)-expressing cells. Mean ± SEM of 16 (Tau) and 15 (TauC3) cells is shown. Scatter plots of the effective diffusion constants (D eff ) ( F ), and association (k* on ) and dissociation rate constants (k off ) ( G ) (mean ± SEM, n = 16 (Tau) and n = 15 (TauC3)). A schematic representation indicating k* on and k off of the MT-Tau interaction is shown in G . Statistically significant differences between samples determined by an unpaired Student’s t test are indicated. * p

    Techniques Used: Mouse Assay, Western Blot, Construct, Binding Assay, Transfection, Fluorescence, Expressing, Diffusion-based Assay

    The microtubule-targeting drug Epothilone D increases MT polymer, normalizes the interaction of TauC3 with microtubules, and modulates the transport of APP-vesicles in the presence of overexpressed human tau. A Effect of EpoD on the percentage of tubulin polymerized in PC12 cell processes. The average constants for association (avg k* on ) and dissociation rate (avg k off ) are shown in the middle and on the right. The rate constants are displayed relative to the carrier control (0.01% DMSO). Mean ± SEM ( n = 15–24). B Effect of EpoD on the association (k* on ) and dissociation rate constants (k off ) of the tau-MT interaction in processes of PC12 cells. Rate constants are indicated relative to PAGFP-tau (Tau) in the presence of the carrier control (0.01% DMSO). Mean ± SEM ( n = 7–13). C Effect of EpoD on the transport of mobile APP-vesicles. mCherry-tagged Tau or TauC3 was co-expressed with eGFP-tagged APP and vesicle mobility was determined using an autoregressive motion algorithm. The velocity, processivity, and state changes are shown in the scatter plots. Each point represents an average value for a respective cell (mean ± SEM of n = 19–25 cells with 532–624 trajectories). D a, a’, a” Time series under physiological conditions: Tau (blue) interacts dynamically with microtubules by kiss-and-hop. Due to the high dynamics of the interaction of tau with microtubules, it does not stand in the way of kinesin-driven vesicle transport. D b, b’, b” Time series after caspase-3 cleavage of tau in senescent neurons. TauC3 (red) shows less dynamics in its interaction with microtubules, which can create a temporary roadblock to vesicle movement causing a change in direction. This leads to a reduced processivity of the movement. D c, c’, c” Time series after treatment with Epothilone D. EpoD modulates the structure of microtubules, resulting in increased dynamics of the TauC3-MT interaction, similar to full-length tau. However, EpoD also reduces the processivity of APP-vesicle transport in the presence of overexpressed human tau. All transfected or transduced tau constructs are based on the human tau sequence. Treatment with EpoD or carrier control was done 1 h before imaging. Statistically significant differences determined by one-way ANOVA ( A ) or two-way ANOVA ( B , C ) and post hoc Fischer LSD are indicated. * p
    Figure Legend Snippet: The microtubule-targeting drug Epothilone D increases MT polymer, normalizes the interaction of TauC3 with microtubules, and modulates the transport of APP-vesicles in the presence of overexpressed human tau. A Effect of EpoD on the percentage of tubulin polymerized in PC12 cell processes. The average constants for association (avg k* on ) and dissociation rate (avg k off ) are shown in the middle and on the right. The rate constants are displayed relative to the carrier control (0.01% DMSO). Mean ± SEM ( n = 15–24). B Effect of EpoD on the association (k* on ) and dissociation rate constants (k off ) of the tau-MT interaction in processes of PC12 cells. Rate constants are indicated relative to PAGFP-tau (Tau) in the presence of the carrier control (0.01% DMSO). Mean ± SEM ( n = 7–13). C Effect of EpoD on the transport of mobile APP-vesicles. mCherry-tagged Tau or TauC3 was co-expressed with eGFP-tagged APP and vesicle mobility was determined using an autoregressive motion algorithm. The velocity, processivity, and state changes are shown in the scatter plots. Each point represents an average value for a respective cell (mean ± SEM of n = 19–25 cells with 532–624 trajectories). D a, a’, a” Time series under physiological conditions: Tau (blue) interacts dynamically with microtubules by kiss-and-hop. Due to the high dynamics of the interaction of tau with microtubules, it does not stand in the way of kinesin-driven vesicle transport. D b, b’, b” Time series after caspase-3 cleavage of tau in senescent neurons. TauC3 (red) shows less dynamics in its interaction with microtubules, which can create a temporary roadblock to vesicle movement causing a change in direction. This leads to a reduced processivity of the movement. D c, c’, c” Time series after treatment with Epothilone D. EpoD modulates the structure of microtubules, resulting in increased dynamics of the TauC3-MT interaction, similar to full-length tau. However, EpoD also reduces the processivity of APP-vesicle transport in the presence of overexpressed human tau. All transfected or transduced tau constructs are based on the human tau sequence. Treatment with EpoD or carrier control was done 1 h before imaging. Statistically significant differences determined by one-way ANOVA ( A ) or two-way ANOVA ( B , C ) and post hoc Fischer LSD are indicated. * p

    Techniques Used: Transfection, Construct, Sequencing, Imaging

    3) Product Images from "ENT-A010, a Novel Steroid Derivative, Displays Neuroprotective Functions and Modulates Microglial Responses"

    Article Title: ENT-A010, a Novel Steroid Derivative, Displays Neuroprotective Functions and Modulates Microglial Responses

    Journal: Biomolecules

    doi: 10.3390/biom12030424

    ENT-A010 promoted survival of PC12 cells via TRKA. PC12 cells were treated for 24 h with NGF (100 ng/mL), ENT-A010 (500 nM) or vehicle control, with or without the selective TRKA inhibitor GW174456 under serum starvation. Upper panel: Cells were stained with CellTox (green) and Hoechst (blue). Scale bar: 200 μm (applies to all photos). Lower panel: Quantification of cell viability, calculated as percentage of dead (CellTox + ) cells per total number of Hoechst + cells in each image. Data are shown as mean ± SEM, n = 3–7, **: p
    Figure Legend Snippet: ENT-A010 promoted survival of PC12 cells via TRKA. PC12 cells were treated for 24 h with NGF (100 ng/mL), ENT-A010 (500 nM) or vehicle control, with or without the selective TRKA inhibitor GW174456 under serum starvation. Upper panel: Cells were stained with CellTox (green) and Hoechst (blue). Scale bar: 200 μm (applies to all photos). Lower panel: Quantification of cell viability, calculated as percentage of dead (CellTox + ) cells per total number of Hoechst + cells in each image. Data are shown as mean ± SEM, n = 3–7, **: p

    Techniques Used: Staining

    ENT-A010 induced TRKA phosphorylation. PC12 cells were treated for 30 min with NGF (100 ng/mL), ENT-A010 (500 nM), or vehicle control. TRKA was immunoprecipitated, and membranes were immunoblotted for phosphor-Tyrosine (pTyr). Whole-cell lysates were analyzed for TRKA. Representative blots are shown. The intensity of the bands was measured, the ratio pTyr/TRKA was calculated and set in each experiment as 1. Data are shown as mean ± SEM, **: p
    Figure Legend Snippet: ENT-A010 induced TRKA phosphorylation. PC12 cells were treated for 30 min with NGF (100 ng/mL), ENT-A010 (500 nM), or vehicle control. TRKA was immunoprecipitated, and membranes were immunoblotted for phosphor-Tyrosine (pTyr). Whole-cell lysates were analyzed for TRKA. Representative blots are shown. The intensity of the bands was measured, the ratio pTyr/TRKA was calculated and set in each experiment as 1. Data are shown as mean ± SEM, **: p

    Techniques Used: Immunoprecipitation

    4) Product Images from "DNA repair protein DNA-PK protects PC12 cells from oxidative stress-induced apoptosis involving AKT phosphorylation"

    Article Title: DNA repair protein DNA-PK protects PC12 cells from oxidative stress-induced apoptosis involving AKT phosphorylation

    Journal: Molecular Biology Reports

    doi: 10.1007/s11033-021-06934-5

    Left Panel. Immunofluorescence images of proliferating PC12 cells in the absence or presence of 0.3 mM H 2 O 2 treatment. Only cells exposed to H 2 O 2 treatment show γH2AX positive foci (green) in nuclei counterstained with Hoechst (red). Scale bar, 5 μm. Right Panel. After exposure with 0.3 mM H 2 O 2 for 1 h (baseline), PC12 cells were incubated for 4, 8, and 24 h with fresh H 2 O 2 -free medium (recovery condition). Cells were fixed and stained with anti-γH2AX antibody and subjected to immunofluorescence microscopy. The number of γH2AX-positive cells were counted and plotted as histograms to show the repair kinetics of damaged DNA. After 24 h recovery, DNA damages are completely repaired. Results were representative of 5 independent experiments. [*] Significant differences (p
    Figure Legend Snippet: Left Panel. Immunofluorescence images of proliferating PC12 cells in the absence or presence of 0.3 mM H 2 O 2 treatment. Only cells exposed to H 2 O 2 treatment show γH2AX positive foci (green) in nuclei counterstained with Hoechst (red). Scale bar, 5 μm. Right Panel. After exposure with 0.3 mM H 2 O 2 for 1 h (baseline), PC12 cells were incubated for 4, 8, and 24 h with fresh H 2 O 2 -free medium (recovery condition). Cells were fixed and stained with anti-γH2AX antibody and subjected to immunofluorescence microscopy. The number of γH2AX-positive cells were counted and plotted as histograms to show the repair kinetics of damaged DNA. After 24 h recovery, DNA damages are completely repaired. Results were representative of 5 independent experiments. [*] Significant differences (p

    Techniques Used: Immunofluorescence, Incubation, Staining, Microscopy

    A Proliferating PC12 cells were differentiated with NGF for 7 days to be then exposed to H 2 O 2 followed by a recovery in fresh medium. Cells were then fixed and immunolabelled for γH2AX foci detection. Upper panel. A microscopic field of proliferating PC12 cells in the absence of NGF showing a circular morphology and a field of PC12 cells after NGF treatment with a typical neuronal morphology, are shown in phase contrast images. After 7 days of NGF treatment, PC12 cells acquire neuronal features as indicated by the labelling with MAP2 (green) and DNA (red). Lower panel. Immunofluorescence of NGF-differentiated PC12 cells exposed to 0.3 mM H 2 O 2 for 4 h followed by a 24 h recovery in fresh medium. Immunolabelled cells show the presence of γH2AX foci after exposure to H 2 O 2 (green). During recovery, differentiated PC12 cells maintain γH2AX foci (green) in the presence of NU7026 as compared to control cells. Scale bar, 5 μm. B Bar chart showing that in NGF-differentiated PC12 cells exposed to 0.3 mM H 2 O 2 for 4 h followed by a 24 h recovery in fresh medium, γH2AX foci are not repaired in presence of DNA-PK inhibitor, NU7026 (+55%, vs. H 2 O 2 -treated, p = 0.015). For completeness, without recovery the effect of H 2 O 2 -damage is shown (Mdiff=61%, t(6)=40.27, p
    Figure Legend Snippet: A Proliferating PC12 cells were differentiated with NGF for 7 days to be then exposed to H 2 O 2 followed by a recovery in fresh medium. Cells were then fixed and immunolabelled for γH2AX foci detection. Upper panel. A microscopic field of proliferating PC12 cells in the absence of NGF showing a circular morphology and a field of PC12 cells after NGF treatment with a typical neuronal morphology, are shown in phase contrast images. After 7 days of NGF treatment, PC12 cells acquire neuronal features as indicated by the labelling with MAP2 (green) and DNA (red). Lower panel. Immunofluorescence of NGF-differentiated PC12 cells exposed to 0.3 mM H 2 O 2 for 4 h followed by a 24 h recovery in fresh medium. Immunolabelled cells show the presence of γH2AX foci after exposure to H 2 O 2 (green). During recovery, differentiated PC12 cells maintain γH2AX foci (green) in the presence of NU7026 as compared to control cells. Scale bar, 5 μm. B Bar chart showing that in NGF-differentiated PC12 cells exposed to 0.3 mM H 2 O 2 for 4 h followed by a 24 h recovery in fresh medium, γH2AX foci are not repaired in presence of DNA-PK inhibitor, NU7026 (+55%, vs. H 2 O 2 -treated, p = 0.015). For completeness, without recovery the effect of H 2 O 2 -damage is shown (Mdiff=61%, t(6)=40.27, p

    Techniques Used: Immunofluorescence

    PC12 cells were treated for 1 h with 0.3 mM H 2 O 2 and then incubated for 24 h with fresh medium with or without NU7026 (10 µM), a potent DNA-PK inhibitor. Cells were fixed and stained with anti-γH2AX antibody to count foci and nuclei were counterstained with Hoechst to count condensed and/or fragmented nuclei as apoptotic cells. A Bar chart showing that γH2AX foci are repaired after a 24 h recovery even if DNA-PK activity is inhibited. As supplementary information, percentage foci without recovery (grey background) are reported to show the increase after H 2 O 2 treatment. B Representative western blots of γH2AX in PC12 cells treated for 1 h with 0.3 mM of H 2 O 2 confirming the repair of DNA damage both in presence and absence of DNA-PK inhibitor during recovery. After H 2 O 2 incubation, cells were processed to obtain whole cell extracts as described in “ Materials and Methods ” section. β-actin was used as loading control. Image is representative of 3 independent experiments. C Bar chart showing that 0.3 mM H 2 O 2 treatment caused a 30% of apoptotic cells after 24 h recovery. A further increase is observed in the presence of 10 µM NU7026, as compared with H 2 O 2 -treated cells (+15%). In addition, without repair (grey background) H 2 O 2 treatment did not induce augmented apoptosis (p = 38). Bars in the plot represent mean ± S.D. of apoptotic cells expressed as percentage. D FACS analysis was conducted to confirm the occurrence of apoptosis after 0.3 mM H 2 O 2 1 h treatment followed by 24 h recovery both in the presence ad absence of 10 µM NU7026. Cells were stained with PI, according to Nicoletti’s protocol. Histograms show DNA content distribution in the different experimental conditions and indicate an increase of approx. 20% in apoptotic cells in the presence of DNA-PK inhibitor. Apoptotic cells appear with fractional DNA content before the peak of G1 cells
    Figure Legend Snippet: PC12 cells were treated for 1 h with 0.3 mM H 2 O 2 and then incubated for 24 h with fresh medium with or without NU7026 (10 µM), a potent DNA-PK inhibitor. Cells were fixed and stained with anti-γH2AX antibody to count foci and nuclei were counterstained with Hoechst to count condensed and/or fragmented nuclei as apoptotic cells. A Bar chart showing that γH2AX foci are repaired after a 24 h recovery even if DNA-PK activity is inhibited. As supplementary information, percentage foci without recovery (grey background) are reported to show the increase after H 2 O 2 treatment. B Representative western blots of γH2AX in PC12 cells treated for 1 h with 0.3 mM of H 2 O 2 confirming the repair of DNA damage both in presence and absence of DNA-PK inhibitor during recovery. After H 2 O 2 incubation, cells were processed to obtain whole cell extracts as described in “ Materials and Methods ” section. β-actin was used as loading control. Image is representative of 3 independent experiments. C Bar chart showing that 0.3 mM H 2 O 2 treatment caused a 30% of apoptotic cells after 24 h recovery. A further increase is observed in the presence of 10 µM NU7026, as compared with H 2 O 2 -treated cells (+15%). In addition, without repair (grey background) H 2 O 2 treatment did not induce augmented apoptosis (p = 38). Bars in the plot represent mean ± S.D. of apoptotic cells expressed as percentage. D FACS analysis was conducted to confirm the occurrence of apoptosis after 0.3 mM H 2 O 2 1 h treatment followed by 24 h recovery both in the presence ad absence of 10 µM NU7026. Cells were stained with PI, according to Nicoletti’s protocol. Histograms show DNA content distribution in the different experimental conditions and indicate an increase of approx. 20% in apoptotic cells in the presence of DNA-PK inhibitor. Apoptotic cells appear with fractional DNA content before the peak of G1 cells

    Techniques Used: Incubation, Staining, Activity Assay, Western Blot, FACS

    Representative western blots of DNA-PKcs complex in PC12 cells treated for 1, 4, and 24 h with different mM doses of H 2 O 2 . After H 2 O 2 incubation, cells were processed to obtain whole cellular extracts as described in “ Materials and Methods ” section and DNA-PKcs, Ku86 and Ku70 protein levels were assayed by western blot analysis. β-Actin was used as loading control. Densitometric quantitation of the immunoreactive bands corresponding to DNA-PKcs, Ku70 and Ku86 are represented in plots. Values in plots represent the normalized percent changes in protein levels with respect to control (100%) after exposure to H 2 O 2 . Results were representative of 5 independent experiments. [*] Significant differences (p
    Figure Legend Snippet: Representative western blots of DNA-PKcs complex in PC12 cells treated for 1, 4, and 24 h with different mM doses of H 2 O 2 . After H 2 O 2 incubation, cells were processed to obtain whole cellular extracts as described in “ Materials and Methods ” section and DNA-PKcs, Ku86 and Ku70 protein levels were assayed by western blot analysis. β-Actin was used as loading control. Densitometric quantitation of the immunoreactive bands corresponding to DNA-PKcs, Ku70 and Ku86 are represented in plots. Values in plots represent the normalized percent changes in protein levels with respect to control (100%) after exposure to H 2 O 2 . Results were representative of 5 independent experiments. [*] Significant differences (p

    Techniques Used: Western Blot, Incubation, Quantitation Assay

    Western blot analysis of AKT phosphorylation in proliferating PC12 cells pre-exposed for 24 h with 10 µM NU7026 and then incubated for 30 min with different doses of H 2 O 2 ( A ) or for 10 and 30 min with 100 nM insulin ( B ). C Analysis of full length and cleaved Caspase-3 and cleaved-PARP-1 protein levels in PC12 cells pre-treated for 24 h with NU7026 and for 30 min with 0.5 and 1 mM H 2 O 2 . Results were representative of 5 independent experiments. [*] Significant differences (p
    Figure Legend Snippet: Western blot analysis of AKT phosphorylation in proliferating PC12 cells pre-exposed for 24 h with 10 µM NU7026 and then incubated for 30 min with different doses of H 2 O 2 ( A ) or for 10 and 30 min with 100 nM insulin ( B ). C Analysis of full length and cleaved Caspase-3 and cleaved-PARP-1 protein levels in PC12 cells pre-treated for 24 h with NU7026 and for 30 min with 0.5 and 1 mM H 2 O 2 . Results were representative of 5 independent experiments. [*] Significant differences (p

    Techniques Used: Western Blot, Incubation

    5) Product Images from "Palmitic and stearic fatty acids induce caspase-dependent and -independent cell death in nerve growth factor differentiated PC12 cells"

    Article Title: Palmitic and stearic fatty acids induce caspase-dependent and -independent cell death in nerve growth factor differentiated PC12 cells

    Journal: Journal of neurochemistry

    doi:

    Hoffmann modulation contrast and fluorescent micrographs of NGF-differentiated PC12 cells exposed to selected fatty acids complexed with BSA (2 : 1). Images show NGF-differentiated PC12 cells exposed to the indicated fatty acid for 24 h. Fluorescent nuclear staining with DAPI was performed as described in Materials and methods. Panels on the left show Hoffman modulation contrast images of cells before fixation. Panels on the right show nuclear staining with DAPI. The fields shown on the left panels are different from the fields shown on the right panels.
    Figure Legend Snippet: Hoffmann modulation contrast and fluorescent micrographs of NGF-differentiated PC12 cells exposed to selected fatty acids complexed with BSA (2 : 1). Images show NGF-differentiated PC12 cells exposed to the indicated fatty acid for 24 h. Fluorescent nuclear staining with DAPI was performed as described in Materials and methods. Panels on the left show Hoffman modulation contrast images of cells before fixation. Panels on the right show nuclear staining with DAPI. The fields shown on the left panels are different from the fields shown on the right panels.

    Techniques Used: Staining

    Induction of caspases-3 and -8, but not caspase-9, activity in cellular extracts of differentiated PC12 cells exposed to either palmitic or stearic acid complexed (2 : 1) with BSA. (a) Palmitic acid. (b) Stearic acid. Data represent the mean ± SD of three independent experiments performed in triplicate. # indicates significance of p
    Figure Legend Snippet: Induction of caspases-3 and -8, but not caspase-9, activity in cellular extracts of differentiated PC12 cells exposed to either palmitic or stearic acid complexed (2 : 1) with BSA. (a) Palmitic acid. (b) Stearic acid. Data represent the mean ± SD of three independent experiments performed in triplicate. # indicates significance of p

    Techniques Used: Activity Assay

    Induction of Fas receptor and Fas ligand mRNA in cellular extract of NGF-differentiated PC12 cells exposed to either stearic or palmitic acid complexed with BSA (2 : 1). (a) RNA blot showing the induction of Fas receptor mRNA during the course of exposure to either stearic or palmitic fatty acid. Each lane was loaded with 30 μg of RNA extracted from differentiated PC12 cells after 2, 4 and 6 h exposure to stearic or palmitic acid. Representative blot of two independent experiments. (b) RT-PCR analysis performed on total RNA extracted from cells treated for 2, 6, 9, 12 and 15 h with either palmitic or stearic acid complexed with BSA (2 : 1). The PCR products shown are representative of the results obtained from two independent experiments. C, control; SA, stearic acid; PA, palmitic acid; h, hours after initial exposure to fatty acids.
    Figure Legend Snippet: Induction of Fas receptor and Fas ligand mRNA in cellular extract of NGF-differentiated PC12 cells exposed to either stearic or palmitic acid complexed with BSA (2 : 1). (a) RNA blot showing the induction of Fas receptor mRNA during the course of exposure to either stearic or palmitic fatty acid. Each lane was loaded with 30 μg of RNA extracted from differentiated PC12 cells after 2, 4 and 6 h exposure to stearic or palmitic acid. Representative blot of two independent experiments. (b) RT-PCR analysis performed on total RNA extracted from cells treated for 2, 6, 9, 12 and 15 h with either palmitic or stearic acid complexed with BSA (2 : 1). The PCR products shown are representative of the results obtained from two independent experiments. C, control; SA, stearic acid; PA, palmitic acid; h, hours after initial exposure to fatty acids.

    Techniques Used: Northern blot, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    6) Product Images from "Role of TrkB expression in rat adrenal gland during acute immobilization stress"

    Article Title: Role of TrkB expression in rat adrenal gland during acute immobilization stress

    Journal: Journal of Neurochemistry

    doi: 10.1111/jnc.12030

    Characterization of anti-tyrosine receptor kinase B (TrkB) (clone 47/TrkB). PC12 cells were treated with anti-TrkB (clone 47/TrkB) and/or TrkB inhibitor, K252a. Levels of noradrenaline (a) and dopamine (b) in the media were then assayed by HPLC. (a) Significant differences in noradrenaline levels were seen between the agonist group and all other groups. Data were analyzed by the Mann–Whitney U -test and the Kruskal–Wallis test (* p
    Figure Legend Snippet: Characterization of anti-tyrosine receptor kinase B (TrkB) (clone 47/TrkB). PC12 cells were treated with anti-TrkB (clone 47/TrkB) and/or TrkB inhibitor, K252a. Levels of noradrenaline (a) and dopamine (b) in the media were then assayed by HPLC. (a) Significant differences in noradrenaline levels were seen between the agonist group and all other groups. Data were analyzed by the Mann–Whitney U -test and the Kruskal–Wallis test (* p

    Techniques Used: High Performance Liquid Chromatography, MANN-WHITNEY

    7) Product Images from "C-terminal Domain of Chromogranin B Regulates Intracellular Calcium Signaling *"

    Article Title: C-terminal Domain of Chromogranin B Regulates Intracellular Calcium Signaling *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.251330

    The extent of calcium release is increased and signal initation sites are altered upon expression of CGB and C-CGB in PC12 cells. A , in control cells, endogenous CGB is mostly expressed in the growth cones and neuritic branching points. Overexpressed
    Figure Legend Snippet: The extent of calcium release is increased and signal initation sites are altered upon expression of CGB and C-CGB in PC12 cells. A , in control cells, endogenous CGB is mostly expressed in the growth cones and neuritic branching points. Overexpressed

    Techniques Used: Expressing

    8) Product Images from "Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1"

    Article Title: Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.202101075

    Model. Neural development includes early-stage neural induction and late-stage neural genesis. During neural genesis, PC12 or N2A cells (dark green) respond to NGF or RA to differentiate into neuronal cells (bright green). The content of EVs exhibits dynamic changes corresponding to the fate conversion. Cyclin D1 (magenta dots inside the purple EVs) was enriched in EVs from differentiating neurons. Additional cyclin D1 enriched in EVs from the neuronal cells accelerates the commitment of mESCs (light orange) to neural progenitor cells (mNPC, light green). Exosomal communication between different development stages may contribute to commitment and conversion of mESCs to the neural lineage.
    Figure Legend Snippet: Model. Neural development includes early-stage neural induction and late-stage neural genesis. During neural genesis, PC12 or N2A cells (dark green) respond to NGF or RA to differentiate into neuronal cells (bright green). The content of EVs exhibits dynamic changes corresponding to the fate conversion. Cyclin D1 (magenta dots inside the purple EVs) was enriched in EVs from differentiating neurons. Additional cyclin D1 enriched in EVs from the neuronal cells accelerates the commitment of mESCs (light orange) to neural progenitor cells (mNPC, light green). Exosomal communication between different development stages may contribute to commitment and conversion of mESCs to the neural lineage.

    Techniques Used:

    EV production increased during neuronal differentiation. (A) Schematic of the EV purification strategy. (B) Representative electron micrographs of negatively stained samples of purified EVs at 9,300× magnification. Purified EVs from untreated PC12 cells cultured for 3 d (PC12-EV) or treated with NGF for 3, 6, and 9 d (N3-EV, N6-EV, and N9-EV). During PC12 differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with NGF were replaced every 3 d. Scale bar, 0.2 µm. (C) Nanoparticle tracking analysis of the size distribution and the number of purified EVs from 420-ml medium of untreated PC12 cells cultured for 3 d or treated with NGF for 3, 6, and 9 d. (D) The number of EVs released per PC12 cell untreated or treated with NGF for 3, 6, and 9 d. EV number was quantified by nanoparticle tracking analysis. Cell number was quantified with a hemocytometer. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (E) The number of EVs released per N2A cell cultured for 3 d or treated with RA for 3 and 6 d. During N2A differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with RA were replaced every 3 d. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (F) The number of EVs released per ESC untreated or differentiated for 8 and 12 d. During ES differentiation, EVs were collected from 2-d-cultured cells, and fresh medium was replaced every 2 d. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. (G) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 10 7 PC12 cells were untreated or treated with NGF for 3, 6, and 9 d. (H) Quantitative analysis of the immunoblots in G. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in PC12-EV group was set as 1. (I) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 10 7 N2A cells were untreated or treated with RA for 3 and 6 d. (J) Quantitative analysis of the immunoblots in I. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in N2A-EV group was set as 1.
    Figure Legend Snippet: EV production increased during neuronal differentiation. (A) Schematic of the EV purification strategy. (B) Representative electron micrographs of negatively stained samples of purified EVs at 9,300× magnification. Purified EVs from untreated PC12 cells cultured for 3 d (PC12-EV) or treated with NGF for 3, 6, and 9 d (N3-EV, N6-EV, and N9-EV). During PC12 differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with NGF were replaced every 3 d. Scale bar, 0.2 µm. (C) Nanoparticle tracking analysis of the size distribution and the number of purified EVs from 420-ml medium of untreated PC12 cells cultured for 3 d or treated with NGF for 3, 6, and 9 d. (D) The number of EVs released per PC12 cell untreated or treated with NGF for 3, 6, and 9 d. EV number was quantified by nanoparticle tracking analysis. Cell number was quantified with a hemocytometer. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (E) The number of EVs released per N2A cell cultured for 3 d or treated with RA for 3 and 6 d. During N2A differentiation, EVs were collected from 3-d-cultured cells, and fresh medium together with RA were replaced every 3 d. The values represent the mean ± SD, from three independent experiments. Error bars represent SD from independent samples. (F) The number of EVs released per ESC untreated or differentiated for 8 and 12 d. During ES differentiation, EVs were collected from 2-d-cultured cells, and fresh medium was replaced every 2 d. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. (G) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 10 7 PC12 cells were untreated or treated with NGF for 3, 6, and 9 d. (H) Quantitative analysis of the immunoblots in G. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in PC12-EV group was set as 1. (I) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix, and Tsg101 in EVs from the same number of cells. 2 × 10 7 N2A cells were untreated or treated with RA for 3 and 6 d. (J) Quantitative analysis of the immunoblots in I. The values represent the mean ± SD, from two independent experiments. Error bars represent SD from independent samples. The signal in N2A-EV group was set as 1.

    Techniques Used: Purification, Staining, Cell Culture, Western Blot

    RA-induced EVs promote mESC neural fate commitment. (A) The cellular morphology of PC12 cells untreated or treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. Scale bars, 50 µm. (B) Gene expression analysis of Tuj1 and Tau in PC12 cells untreated and treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. The values represent the mean ± SD, from two independent experiments (*, P
    Figure Legend Snippet: RA-induced EVs promote mESC neural fate commitment. (A) The cellular morphology of PC12 cells untreated or treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. Scale bars, 50 µm. (B) Gene expression analysis of Tuj1 and Tau in PC12 cells untreated and treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. The values represent the mean ± SD, from two independent experiments (*, P

    Techniques Used: Expressing

    EVs show different buoyant density distribution during PC12 neuronal differentiation. (A) The cellular morphology of PC12 cells cultured in growth medium or low-serum medium with NGF (50 ng/ml) for 3, 6, and 9 d. Scale bars, 50 µm. (B) Expression profiling of Tuj1 and Tau genes during neuronal differentiation of PC12 cells in low-serum medium without (Control) or with different doses of NGF (50 and 100 ng/ml). Expression was normalized to Gapdh in this and all others by qPCR analysis. Data plotted are from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD (*, P
    Figure Legend Snippet: EVs show different buoyant density distribution during PC12 neuronal differentiation. (A) The cellular morphology of PC12 cells cultured in growth medium or low-serum medium with NGF (50 ng/ml) for 3, 6, and 9 d. Scale bars, 50 µm. (B) Expression profiling of Tuj1 and Tau genes during neuronal differentiation of PC12 cells in low-serum medium without (Control) or with different doses of NGF (50 and 100 ng/ml). Expression was normalized to Gapdh in this and all others by qPCR analysis. Data plotted are from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD (*, P

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    Cyclin D1 enriched in EVs during neurogenesis. (A) Immunoblot analysis of cyclin D1, 2, and 3 in PC12 cells induced by NGF for different times. D0, PC12 cells without NGF treatment. D1–D9, PC12 cells incubated with NGF for 1–9 d. (B) Immunoblot analysis of cyclin D1/2 in EVs purified from PC12 cells (D0) and EVs purified from NGF-induced PC12 cells for 3, 6, and 9 d (D3, D6, and D9). (C) Quantitative immunoblot analysis of protein levels described in B. The D0 signal was set as 1. Flot2 signal was used as a internal control. The values represent the mean ± SD, from three independent experiments (*, P
    Figure Legend Snippet: Cyclin D1 enriched in EVs during neurogenesis. (A) Immunoblot analysis of cyclin D1, 2, and 3 in PC12 cells induced by NGF for different times. D0, PC12 cells without NGF treatment. D1–D9, PC12 cells incubated with NGF for 1–9 d. (B) Immunoblot analysis of cyclin D1/2 in EVs purified from PC12 cells (D0) and EVs purified from NGF-induced PC12 cells for 3, 6, and 9 d (D3, D6, and D9). (C) Quantitative immunoblot analysis of protein levels described in B. The D0 signal was set as 1. Flot2 signal was used as a internal control. The values represent the mean ± SD, from three independent experiments (*, P

    Techniques Used: Incubation, Purification

    Cyclin D1 is enriched in EVs during N2A neurogenesis. (A) Immunoblots of cyclin D, CDK4, Hsc70, Tsg101, and actin of EVs from RA-induced N2A cells for 2, 4, 6, and 8 d (D2, D4, D6, and D8). (B) Immunoblots of pRB, p57, p27, p21, pErk, and actin in differentiated PC12 cells and EVs. (C) Immunoblots of cyclin D1, CDK4, and multiple EV markers from the N6-EVs treated with different concentrations of proteinase K (PK), with or without 1% Triton X-100.
    Figure Legend Snippet: Cyclin D1 is enriched in EVs during N2A neurogenesis. (A) Immunoblots of cyclin D, CDK4, Hsc70, Tsg101, and actin of EVs from RA-induced N2A cells for 2, 4, 6, and 8 d (D2, D4, D6, and D8). (B) Immunoblots of pRB, p57, p27, p21, pErk, and actin in differentiated PC12 cells and EVs. (C) Immunoblots of cyclin D1, CDK4, and multiple EV markers from the N6-EVs treated with different concentrations of proteinase K (PK), with or without 1% Triton X-100.

    Techniques Used: Western Blot

    The chaperone protein Hsc70 facilities cyclin D1 package into EVs. (A) Characterization of APEX-mediated proximity biotinylation of cyclin D1 protein targets by blotting with streptavidin. Cyclin D1–APEX fusion gene was delivered into N2A cells by lentivirus infection. Biotinylated protein was detected by blotting with streptavidin (SA)-HRP. Ponceau S staining (left) of the same membrane served as loading control. (B) Table showing MS analysis of the unique peptides in biotin-phenol together with H 2 O 2 (B+H) or without H 2 O 2 (B). (C) CoIP analysis of Hsc70 and Hsc90 with cyclin D1 and CDK4 in N2A cells. (D) CoIP of cyclin D1 and Hsc70 in PC12 cells. (E) CoIP of cyclin D1 and Hsc70 in 5 × 10 10 RA-EVs. (F) Immunoblots of cyclin D1, Alix, and CD9 in EVs collected from the differentiated N2A cells treated with VER-155008 (VER). N2A cells pretreated with RA-containing differentiation medium for 4 d, after which cells were exposed to fresh differentiation medium with or without 5 µM VER for two more days. EVs collected from 6-d differentiation of N2A cells. (G) Immunoblots of cyclin D1, Alix, and CD9 in EVs collected from the differentiated N2A cells transfected with WT Hsc70 (WT) or D10N mutant Hsc70 (D10N; > 50% transfection efficiency). WT Hsc70 or D10N mutant Hsc70 were transfected by Lipofectamine 2000 in seven plates of 70%-confluency N2A cells in DMEM medium for 10 h, followed by a change to fresh differentiation medium for 3 d. EVs were collected from both cells. (H) Expression analysis of Pax6 , Nestin , and Six3 in differentiated mESCs treated with 2 × 10 9 EVs from RA-induced N2A cells with (VER-EV) or without (RA-EV) VER. EVs were collected as described in F. The values represent the mean ± SD, from three independent experiments (*, P
    Figure Legend Snippet: The chaperone protein Hsc70 facilities cyclin D1 package into EVs. (A) Characterization of APEX-mediated proximity biotinylation of cyclin D1 protein targets by blotting with streptavidin. Cyclin D1–APEX fusion gene was delivered into N2A cells by lentivirus infection. Biotinylated protein was detected by blotting with streptavidin (SA)-HRP. Ponceau S staining (left) of the same membrane served as loading control. (B) Table showing MS analysis of the unique peptides in biotin-phenol together with H 2 O 2 (B+H) or without H 2 O 2 (B). (C) CoIP analysis of Hsc70 and Hsc90 with cyclin D1 and CDK4 in N2A cells. (D) CoIP of cyclin D1 and Hsc70 in PC12 cells. (E) CoIP of cyclin D1 and Hsc70 in 5 × 10 10 RA-EVs. (F) Immunoblots of cyclin D1, Alix, and CD9 in EVs collected from the differentiated N2A cells treated with VER-155008 (VER). N2A cells pretreated with RA-containing differentiation medium for 4 d, after which cells were exposed to fresh differentiation medium with or without 5 µM VER for two more days. EVs collected from 6-d differentiation of N2A cells. (G) Immunoblots of cyclin D1, Alix, and CD9 in EVs collected from the differentiated N2A cells transfected with WT Hsc70 (WT) or D10N mutant Hsc70 (D10N; > 50% transfection efficiency). WT Hsc70 or D10N mutant Hsc70 were transfected by Lipofectamine 2000 in seven plates of 70%-confluency N2A cells in DMEM medium for 10 h, followed by a change to fresh differentiation medium for 3 d. EVs were collected from both cells. (H) Expression analysis of Pax6 , Nestin , and Six3 in differentiated mESCs treated with 2 × 10 9 EVs from RA-induced N2A cells with (VER-EV) or without (RA-EV) VER. EVs were collected as described in F. The values represent the mean ± SD, from three independent experiments (*, P

    Techniques Used: Infection, Staining, Co-Immunoprecipitation Assay, Western Blot, Transfection, Mutagenesis, Expressing

    9) Product Images from "Extracellular and intraneuronal HMW-AbetaOs represent a molecular basis of memory loss in Alzheimer's disease model mouse"

    Article Title: Extracellular and intraneuronal HMW-AbetaOs represent a molecular basis of memory loss in Alzheimer's disease model mouse

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-6-20

    Biophysical and structural characterization of neurotoxic Aβ assembly . (Upper half of panel A) Representative calcein AM/PI stainings of NGF-treated PC12 (PC12N) cells treated at 37°C for 48 h with: TBS alone; 0-h preincubated Aβ1-42 (0 h); 2-h preincubated Aβ1-42 (2 h); 540,000 g supernatant obtained from 2 h (2 h sup); 4-h preincubated Aβ1-42 (4 h); 540,000 g supernatant obtained from 2 h (4 h sup). Green staining for viable cells versus red staining for dead cells. Resultant cell viability for each treatment is shown in lower half of panel A. Experimental results were analyzed by one-way ANOVA, followed by Tukey's test for posthoc analysis: statistical significance compared with TBS alone (* p
    Figure Legend Snippet: Biophysical and structural characterization of neurotoxic Aβ assembly . (Upper half of panel A) Representative calcein AM/PI stainings of NGF-treated PC12 (PC12N) cells treated at 37°C for 48 h with: TBS alone; 0-h preincubated Aβ1-42 (0 h); 2-h preincubated Aβ1-42 (2 h); 540,000 g supernatant obtained from 2 h (2 h sup); 4-h preincubated Aβ1-42 (4 h); 540,000 g supernatant obtained from 2 h (4 h sup). Green staining for viable cells versus red staining for dead cells. Resultant cell viability for each treatment is shown in lower half of panel A. Experimental results were analyzed by one-way ANOVA, followed by Tukey's test for posthoc analysis: statistical significance compared with TBS alone (* p

    Techniques Used: Staining

    10) Product Images from "Identification of Centella asiatica's Effective Ingredients for Inducing the Neuronal Differentiation"

    Article Title: Identification of Centella asiatica's Effective Ingredients for Inducing the Neuronal Differentiation

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2016/9634750

    The combination of asiatic acid and madecassic acid found in 75-4 induces the expression of neurofilament of cultured PC12 cells. (a) Cultured PC12 cells were treated with asiatic acid (14.4 μ M), madecassic acid (40.8 μ M), their combination, and 75-4 (0.216 mg/mL) for 72 h. The cell lysates were collected to determine the expression of NF68 and NF200. NGF (50 ng/mL) served as the positive control. GAPDH served as loading control. (b) Quantification plot is shown in histograms. Data are expressed as the fold of change (100% of Basal) against the control, mean ± SEM, n = 4, pooled from four independent experiments. Statistical comparison was made between asiatic acid, the combination, and 75-4; ∗ p
    Figure Legend Snippet: The combination of asiatic acid and madecassic acid found in 75-4 induces the expression of neurofilament of cultured PC12 cells. (a) Cultured PC12 cells were treated with asiatic acid (14.4 μ M), madecassic acid (40.8 μ M), their combination, and 75-4 (0.216 mg/mL) for 72 h. The cell lysates were collected to determine the expression of NF68 and NF200. NGF (50 ng/mL) served as the positive control. GAPDH served as loading control. (b) Quantification plot is shown in histograms. Data are expressed as the fold of change (100% of Basal) against the control, mean ± SEM, n = 4, pooled from four independent experiments. Statistical comparison was made between asiatic acid, the combination, and 75-4; ∗ p

    Techniques Used: Expressing, Cell Culture, Positive Control

    75-4 induces the neurite outgrowth of cultured PC12 cells. 75-4 (0.216 mg/mL) was applied into cultured PC12 cells for 72 h, with fresh medium or 75-4 every 24 h. NGF (50 ng/mL) served as the positive control. Cells were fixed with ice-cold 4% paraformaldehyde, and then the neurite outgrowth was examined under microscope. To quantify the differentiation effect, length of neurite (a) and the percentage of differentiated cell numbers (b) were counted as described in Materials and Methods. Data are expressed as the percentage of cells in 100 counted cells, mean ± SEM, n = 5, pooled from five independent experiments. Statistical comparison was made with the control; ∗ p
    Figure Legend Snippet: 75-4 induces the neurite outgrowth of cultured PC12 cells. 75-4 (0.216 mg/mL) was applied into cultured PC12 cells for 72 h, with fresh medium or 75-4 every 24 h. NGF (50 ng/mL) served as the positive control. Cells were fixed with ice-cold 4% paraformaldehyde, and then the neurite outgrowth was examined under microscope. To quantify the differentiation effect, length of neurite (a) and the percentage of differentiated cell numbers (b) were counted as described in Materials and Methods. Data are expressed as the percentage of cells in 100 counted cells, mean ± SEM, n = 5, pooled from five independent experiments. Statistical comparison was made with the control; ∗ p

    Techniques Used: Cell Culture, Positive Control, Microscopy

    75-4 is the most effective fraction for inducing neurofilament expression of cultured PC12 cells. (a) Cultured PC12 cells were treated with 50-8, 50-9, 75-1, 75-2, and 75-4 at the same dilution ratio for 72 h. The cell lysates were collected to determine the expressions of NF68. NGF (50 ng/mL) served as the positive control. GAPDH served as loading control. (b) Quantification plot is shown in histograms. Values are expressed as the percentage of increase to basal reading (untreated culture), mean ± SEM. n = 5, pooled from five independent experiments.
    Figure Legend Snippet: 75-4 is the most effective fraction for inducing neurofilament expression of cultured PC12 cells. (a) Cultured PC12 cells were treated with 50-8, 50-9, 75-1, 75-2, and 75-4 at the same dilution ratio for 72 h. The cell lysates were collected to determine the expressions of NF68. NGF (50 ng/mL) served as the positive control. GAPDH served as loading control. (b) Quantification plot is shown in histograms. Values are expressed as the percentage of increase to basal reading (untreated culture), mean ± SEM. n = 5, pooled from five independent experiments.

    Techniques Used: Expressing, Cell Culture, Positive Control

    The fractions show wide range of neuronal inductive effects. After the PC12 cells were treated with NGF (50 ng/mL) and fractions (at the same dilution ratio of 1 : 50) for 72 h, randomly selected fields were observed using a camera attached to a microscope (×20). Control (A) 50-3, 50-6, and 75-3; (B) 50-2, 50-5, and 50-10; (C) 50-8, 50-9, 75-1, 75-2, 75-4, 75-5, and NGF. Bar = 50 μ m.
    Figure Legend Snippet: The fractions show wide range of neuronal inductive effects. After the PC12 cells were treated with NGF (50 ng/mL) and fractions (at the same dilution ratio of 1 : 50) for 72 h, randomly selected fields were observed using a camera attached to a microscope (×20). Control (A) 50-3, 50-6, and 75-3; (B) 50-2, 50-5, and 50-10; (C) 50-8, 50-9, 75-1, 75-2, 75-4, 75-5, and NGF. Bar = 50 μ m.

    Techniques Used: Microscopy

    Inhibition of MEK signaling suppresses the combination-induced neurofilament expression of cultured PC12 cells. (a) Cultured PC12 cells were pretreated with or without MEK inhibitor PD98059 (20 μ m) for 5 h and then cotreated with the combination of asiatic acid (14.4 μ M) and madecassic acid (40.8 μ M) and NGF (50 ng/mL) for 72 h. The cell lysates were collected to determine the expression of NF68 and NF200. GAPDH served as loading control. (b) Quantification plot is shown in histograms. Data are expressed as the fold of change (100% of Basal) against the control, mean ± SEM, n = 4, pooled from four independent experiments.
    Figure Legend Snippet: Inhibition of MEK signaling suppresses the combination-induced neurofilament expression of cultured PC12 cells. (a) Cultured PC12 cells were pretreated with or without MEK inhibitor PD98059 (20 μ m) for 5 h and then cotreated with the combination of asiatic acid (14.4 μ M) and madecassic acid (40.8 μ M) and NGF (50 ng/mL) for 72 h. The cell lysates were collected to determine the expression of NF68 and NF200. GAPDH served as loading control. (b) Quantification plot is shown in histograms. Data are expressed as the fold of change (100% of Basal) against the control, mean ± SEM, n = 4, pooled from four independent experiments.

    Techniques Used: Inhibition, Expressing, Cell Culture

    11) Product Images from "Extracellular vesicles from neuronal cells promote neural induction of mESCs through cyclinD1"

    Article Title: Extracellular vesicles from neuronal cells promote neural induction of mESCs through cyclinD1

    Journal: bioRxiv

    doi: 10.1101/2021.05.09.443321

    Model Neural development includes early-stage neural induction and late-stage neural genesis. During neural genesis, PC12 cells or N2A cells (dark green) respond to NGF or RA to differentiate into neuronal cells (bright green). The content of extracellular vesicles exhibits dynamic changes corresponding to the fate conversion. Cyclin D1 (magenta dots inside the purple EVs) was enriched in EVs from differentiating neuron. Additional cyclin D1 enriched in EVs from the neuronal cells accelerates the commitment of mouse embryonic stem cells (light orange) to neural progenitor cells (mNPC, light green). Exosomal communication between different development stages may contribute to commitment and conversion of mESCs to the neural lineage.
    Figure Legend Snippet: Model Neural development includes early-stage neural induction and late-stage neural genesis. During neural genesis, PC12 cells or N2A cells (dark green) respond to NGF or RA to differentiate into neuronal cells (bright green). The content of extracellular vesicles exhibits dynamic changes corresponding to the fate conversion. Cyclin D1 (magenta dots inside the purple EVs) was enriched in EVs from differentiating neuron. Additional cyclin D1 enriched in EVs from the neuronal cells accelerates the commitment of mouse embryonic stem cells (light orange) to neural progenitor cells (mNPC, light green). Exosomal communication between different development stages may contribute to commitment and conversion of mESCs to the neural lineage.

    Techniques Used:

    EV production increased during neuronal differentiation (A) Schematic of EV purification strategy. (B) Representative electron micrographs of negatively-stained samples of purified EVs at 9300X magnification. Purified EVs from untreated PC12 cells cultured for 3 days (PC12-EV) or PC12 cells treated with NGF for 3, 6 and 9 d (N3-EV, N6-EV, N9-EV). During PC12 differentiation, EVs were collected from 3 d cultured cells and fresh medium together with NGF were replaced every 3 d. Scale bar denotes 0.2 μm. (C) Nanoparticle tracking analysis of the size and number distribution of purified EVs from 420 ml medium of untreated PC12 cells cultured for 3 days or PC12 cells treated with NGF for 3, 6 and 9 d. (D) The number of EVs released per PC12 cell or PC12 cells treated with NGF for 3, 6 and 9 d. EV number quantified by nanoparticle tracking analysis. Cell number quantified by a hemocytometer. The values represent the mean ± SD, from three independent experiments. (E) The number of EVs released per N2A cell cultured for 3 days or N2A cells treated with RA for 3 and 6 d. During N2A differentiation, EVs were collected from 3 d cultured cells and fresh medium together with RA were replaced every 3 d. The values represent the mean ± SD, from three independent experiments. (F) The number of EVs released per ES cell or ES cell differentiated for 8 d and 12 d. During ES differentiation, EVs were collected from 2 d cultured cells and fresh medium was replaced every 2 d. The values represent the mean ± SD, from two independent experiments. (G) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix and Tsg101 in EVs from same number of cells. PC12 (2×10 7 ) cells or PC12 cells treated with NGF for 3, 6 and 9 d. (H) Quantitative analysis of the immunoblots in G. The values represent the mean ± SD, from two independent experiments. The signal in PC12-EV group was set as 1. (I) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix and Tsg101 in EVs from same number of cells. N2A (2×10 7 ) cells or N2A cells treated with RA for 3 and 6 d. (J) Quantitative analysis of the immunoblots in I. The values represent the mean ± SD, from two independent experiments. The signal in N2A-EV group was set as 1.
    Figure Legend Snippet: EV production increased during neuronal differentiation (A) Schematic of EV purification strategy. (B) Representative electron micrographs of negatively-stained samples of purified EVs at 9300X magnification. Purified EVs from untreated PC12 cells cultured for 3 days (PC12-EV) or PC12 cells treated with NGF for 3, 6 and 9 d (N3-EV, N6-EV, N9-EV). During PC12 differentiation, EVs were collected from 3 d cultured cells and fresh medium together with NGF were replaced every 3 d. Scale bar denotes 0.2 μm. (C) Nanoparticle tracking analysis of the size and number distribution of purified EVs from 420 ml medium of untreated PC12 cells cultured for 3 days or PC12 cells treated with NGF for 3, 6 and 9 d. (D) The number of EVs released per PC12 cell or PC12 cells treated with NGF for 3, 6 and 9 d. EV number quantified by nanoparticle tracking analysis. Cell number quantified by a hemocytometer. The values represent the mean ± SD, from three independent experiments. (E) The number of EVs released per N2A cell cultured for 3 days or N2A cells treated with RA for 3 and 6 d. During N2A differentiation, EVs were collected from 3 d cultured cells and fresh medium together with RA were replaced every 3 d. The values represent the mean ± SD, from three independent experiments. (F) The number of EVs released per ES cell or ES cell differentiated for 8 d and 12 d. During ES differentiation, EVs were collected from 2 d cultured cells and fresh medium was replaced every 2 d. The values represent the mean ± SD, from two independent experiments. (G) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix and Tsg101 in EVs from same number of cells. PC12 (2×10 7 ) cells or PC12 cells treated with NGF for 3, 6 and 9 d. (H) Quantitative analysis of the immunoblots in G. The values represent the mean ± SD, from two independent experiments. The signal in PC12-EV group was set as 1. (I) Immunoblots of CD9, Hsc70, Flot2, CD63, Alix and Tsg101 in EVs from same number of cells. N2A (2×10 7 ) cells or N2A cells treated with RA for 3 and 6 d. (J) Quantitative analysis of the immunoblots in I. The values represent the mean ± SD, from two independent experiments. The signal in N2A-EV group was set as 1.

    Techniques Used: Purification, Staining, Cell Culture, Western Blot

    (A) The cellular morphology of PC12 cells, PC12 cells treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. Scale bars, 50 μm. (B) Gene expression analysis of Tuj1 and Tau in PC12 cells, PC12 cells treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. The values represent the mean ± SD, from two independent experiments. (*p
    Figure Legend Snippet: (A) The cellular morphology of PC12 cells, PC12 cells treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. Scale bars, 50 μm. (B) Gene expression analysis of Tuj1 and Tau in PC12 cells, PC12 cells treated with NGF (50 ng/ml) without or with NGF neutralizing antibody (500 ng/ml) for 6 d. The values represent the mean ± SD, from two independent experiments. (*p

    Techniques Used: Expressing

    Extracellular vesicles show different buoyant density distribution during PC12 neuronal differentiation (A) The cellular morphology of PC12 cells cultured in growth medium or low serum medium with NGF (50ng/ml) for 3, 6 and 9 days (d). Scale bars, 50 μm. (B) Expression profiling of Tuj1 and Tau genes during neuronal differentiation of PC12 cells in low serum medium without (Control) or with different doses of NGF (50ng/ml, 100ng/ml). Expression was normalized to Gapdh in this and all others by qPCR analysis. Data plotted were from three independent experiments. The values represent the mean ± SD. (*p
    Figure Legend Snippet: Extracellular vesicles show different buoyant density distribution during PC12 neuronal differentiation (A) The cellular morphology of PC12 cells cultured in growth medium or low serum medium with NGF (50ng/ml) for 3, 6 and 9 days (d). Scale bars, 50 μm. (B) Expression profiling of Tuj1 and Tau genes during neuronal differentiation of PC12 cells in low serum medium without (Control) or with different doses of NGF (50ng/ml, 100ng/ml). Expression was normalized to Gapdh in this and all others by qPCR analysis. Data plotted were from three independent experiments. The values represent the mean ± SD. (*p

    Techniques Used: Cell Culture, Expressing, Real-time Polymerase Chain Reaction

    Cyclin D1 enriched in EVs during neurogenesis (A) Immunoblot analysis of cyclin D1, 2 and 3 in PC12 cells induced by NGF for different times. D0, PC12 cells without NGF treatment. D1 to D9, PC12 cells incubate with NGF for 1 to 9 d. (B) Immunoblot analysis of cyclin D1/2 in EVs purified from PC12 cells (D0), and EVs purified from NGF-induced PC12 cells for 3, 6 and 9 d (D3, D6, D9). (C) Quantitative immunoblot analysis of protein level described in B. The D0 signal was set as 1. Flot2 signal was used as a internal control. The values represent the mean ± SD, from three independent experiments. (*p
    Figure Legend Snippet: Cyclin D1 enriched in EVs during neurogenesis (A) Immunoblot analysis of cyclin D1, 2 and 3 in PC12 cells induced by NGF for different times. D0, PC12 cells without NGF treatment. D1 to D9, PC12 cells incubate with NGF for 1 to 9 d. (B) Immunoblot analysis of cyclin D1/2 in EVs purified from PC12 cells (D0), and EVs purified from NGF-induced PC12 cells for 3, 6 and 9 d (D3, D6, D9). (C) Quantitative immunoblot analysis of protein level described in B. The D0 signal was set as 1. Flot2 signal was used as a internal control. The values represent the mean ± SD, from three independent experiments. (*p

    Techniques Used: Purification

    The chaperone protein Hsc70 facilities cyclin D1 package into EVs (A) Characterization of APEX-mediated proximity biotinylation of cyclin D1 protein targets by blotting with streptavidin. cyclin D1-APEX fusion gene was delivered into N2A cells by lentivirus infection. Biotinylated protein was detected by blotting with streptavidin-horseradish peroxidase (SA-HRP). Ponceau S staining (left panel) of the same membrane served as loading control. (B) Table showing Mass Spectrometry (MS) analysis of the unique peptides in biotin-phenol together with H 2 O 2 (B+H) or without H 2 O 2 (B). (C) Co-immunoprecipitation analysis of Hsc70 and Hsc90 with cyclin D1 and CDK4 in N2A cells. (D) Co-immunoprecipitation of cyclin D1 and Hsc70 in PC12 cells. (E) Co-immunoprecipitation of cyclin D1 and Hsc70 in 5×10 10 RA-EVs. (F) Immunoblots of cyclin D1, Alix and CD9 in EVs collected from the differentiated N2A cells treated with VER-155008 (VER). N2A cells pretreated with RA-containing differentiation medium for 4 d, after which cells were exposed to fresh differentiation medium with or without 5 μM VER for 2 more d. EVs collected from 6 d differentiation of N2A cells. (G) Immunoblots of cyclin D1, Alix and CD9 in EVs collected from the differentiated N2A cells transfected with wild type Hsc70 (WT) or D10N mutant Hsc70 (D10N) ( > 50% transfection efficiency). Wild type Hsc70 or D10N mutant Hsc70 were transfected by Lipofectamine™ 2000 in 7 plates of 70% confluency N2A cells in DMEM medium for 10 h, followed by a change to fresh differentiation medium for 3 d. EVs were collected from both cells. (H) The expression analysis of Pax6 , Nestin and Six3 in differentiated mESCs treated with 2×10 9 EVs from RA-induced N2A cells with (VER-EV) or without VER (RA-EV). EVs were collected as described in F. The values represent the mean ± SD, from three independent experiments. (*p
    Figure Legend Snippet: The chaperone protein Hsc70 facilities cyclin D1 package into EVs (A) Characterization of APEX-mediated proximity biotinylation of cyclin D1 protein targets by blotting with streptavidin. cyclin D1-APEX fusion gene was delivered into N2A cells by lentivirus infection. Biotinylated protein was detected by blotting with streptavidin-horseradish peroxidase (SA-HRP). Ponceau S staining (left panel) of the same membrane served as loading control. (B) Table showing Mass Spectrometry (MS) analysis of the unique peptides in biotin-phenol together with H 2 O 2 (B+H) or without H 2 O 2 (B). (C) Co-immunoprecipitation analysis of Hsc70 and Hsc90 with cyclin D1 and CDK4 in N2A cells. (D) Co-immunoprecipitation of cyclin D1 and Hsc70 in PC12 cells. (E) Co-immunoprecipitation of cyclin D1 and Hsc70 in 5×10 10 RA-EVs. (F) Immunoblots of cyclin D1, Alix and CD9 in EVs collected from the differentiated N2A cells treated with VER-155008 (VER). N2A cells pretreated with RA-containing differentiation medium for 4 d, after which cells were exposed to fresh differentiation medium with or without 5 μM VER for 2 more d. EVs collected from 6 d differentiation of N2A cells. (G) Immunoblots of cyclin D1, Alix and CD9 in EVs collected from the differentiated N2A cells transfected with wild type Hsc70 (WT) or D10N mutant Hsc70 (D10N) ( > 50% transfection efficiency). Wild type Hsc70 or D10N mutant Hsc70 were transfected by Lipofectamine™ 2000 in 7 plates of 70% confluency N2A cells in DMEM medium for 10 h, followed by a change to fresh differentiation medium for 3 d. EVs were collected from both cells. (H) The expression analysis of Pax6 , Nestin and Six3 in differentiated mESCs treated with 2×10 9 EVs from RA-induced N2A cells with (VER-EV) or without VER (RA-EV). EVs were collected as described in F. The values represent the mean ± SD, from three independent experiments. (*p

    Techniques Used: Infection, Staining, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Expressing

    (A) Immunoblots of cyclin D, CDK4, Hsc70, Tsg101 and actin of EVs from RA-induced N2A cells for 2, 4, 6 and 8 d (D2, D4, D6, D8). (B) Immunoblots of pRB, p57, p27, p21, pErk and actin in differentiated PC12 cells and EVs. (C) Immunoblots of cyclin D1, CDK4, and multiple EV markers from the N6-EVs treated with different concentrations of proteinase K (PK), with or without 1% Triton.
    Figure Legend Snippet: (A) Immunoblots of cyclin D, CDK4, Hsc70, Tsg101 and actin of EVs from RA-induced N2A cells for 2, 4, 6 and 8 d (D2, D4, D6, D8). (B) Immunoblots of pRB, p57, p27, p21, pErk and actin in differentiated PC12 cells and EVs. (C) Immunoblots of cyclin D1, CDK4, and multiple EV markers from the N6-EVs treated with different concentrations of proteinase K (PK), with or without 1% Triton.

    Techniques Used: Western Blot

    12) Product Images from "Neuroprotective effects of VCP modulators in mouse models of glaucoma"

    Article Title: Neuroprotective effects of VCP modulators in mouse models of glaucoma

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2016.e00096

    Effects of KUS121 and KUS187 on neuronal PC12 cells treated with mitochondrial respiratory chain inhibitors. (A) ATP levels per cell were measured with luciferase assays. Neuronally differentiated PC12 cells were treated with 100 nM antimycin or 0.01 μg/mL oligomycin for 20 h in the presence of 50 μM KUSs (KUS121 or KUS187) or vehicle (DMSO, −), and were harvested. Total ATP amounts and live cell numbers were measured, and then ATP amounts per cell were calculated (see “Materials and methods”). *** P
    Figure Legend Snippet: Effects of KUS121 and KUS187 on neuronal PC12 cells treated with mitochondrial respiratory chain inhibitors. (A) ATP levels per cell were measured with luciferase assays. Neuronally differentiated PC12 cells were treated with 100 nM antimycin or 0.01 μg/mL oligomycin for 20 h in the presence of 50 μM KUSs (KUS121 or KUS187) or vehicle (DMSO, −), and were harvested. Total ATP amounts and live cell numbers were measured, and then ATP amounts per cell were calculated (see “Materials and methods”). *** P

    Techniques Used: Luciferase

    13) Product Images from "p62/SQSTM1 Differentially Removes the Toxic Mutant Androgen Receptor via Autophagy and Inclusion Formation in a Spinal and Bulbar Muscular Atrophy Mouse Model"

    Article Title: p62/SQSTM1 Differentially Removes the Toxic Mutant Androgen Receptor via Autophagy and Inclusion Formation in a Spinal and Bulbar Muscular Atrophy Mouse Model

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3021-12.2013

    Depletion of p62 promotes the accumulation of AR. A , PC12 cells expressing wild-type (10Q) and mutant (112Q) AR were transfected with either control or p62 siRNA. The cells were treated with DHT for 3 or 7 d. B , Neuro2A cells transfected with wild-type (24Q) and mutant (97Q) AR were cotransfected with control or p62 siRNA. C , Pulse-chase analysis of two forms of AR in PC12 cells. Data from one representative experiment for wild-type and mutant AR. D , Pulse-chase assessment of half-life of the wild-type (left) and mutant AR (right). The percentages of wild-type (10Q) and mutant (112Q) remaining in the presence (●) and absence (○) of p62 siRNA are indicated. E , Real-time RT-PCR for wild-type (10Q) and mutant (112Q) AR mRNA normalized to GAPDH levels in PC12 cells. F , Real-time RT-PCR for wild-type (24Q) and mutant (97Q) AR mRNA normalized to GAPDH levels in Neuro2A cells. These experiments were repeated in five sets of cells, and equivalent results were obtained. All of the values are expressed as the means ± SEM ( n = 5). * p
    Figure Legend Snippet: Depletion of p62 promotes the accumulation of AR. A , PC12 cells expressing wild-type (10Q) and mutant (112Q) AR were transfected with either control or p62 siRNA. The cells were treated with DHT for 3 or 7 d. B , Neuro2A cells transfected with wild-type (24Q) and mutant (97Q) AR were cotransfected with control or p62 siRNA. C , Pulse-chase analysis of two forms of AR in PC12 cells. Data from one representative experiment for wild-type and mutant AR. D , Pulse-chase assessment of half-life of the wild-type (left) and mutant AR (right). The percentages of wild-type (10Q) and mutant (112Q) remaining in the presence (●) and absence (○) of p62 siRNA are indicated. E , Real-time RT-PCR for wild-type (10Q) and mutant (112Q) AR mRNA normalized to GAPDH levels in PC12 cells. F , Real-time RT-PCR for wild-type (24Q) and mutant (97Q) AR mRNA normalized to GAPDH levels in Neuro2A cells. These experiments were repeated in five sets of cells, and equivalent results were obtained. All of the values are expressed as the means ± SEM ( n = 5). * p

    Techniques Used: Expressing, Mutagenesis, Transfection, Pulse Chase, Quantitative RT-PCR

    14) Product Images from "Expression and Endocytosis of Lysosomal Aspartylglucosaminidase in Mouse Primary Neurons"

    Article Title: Expression and Endocytosis of Lysosomal Aspartylglucosaminidase in Mouse Primary Neurons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.18-19-07750.1998

    Endocytosis of AGA by different neuronal cells. A , Immunoprecipitation of endocytosed labeled AGA from E14 primary neurons cultured for 2 d ( n2 ) or 16 d ( n16 ), fibroblasts ( F ), PC12 cells (+/− NGF induction), and N18 cells. The recombinant CHO-AGA given to the cells is shown on the right ( M ). B , Immunofluorescence staining of endocytosed AGA ( red ) in a cultured primary neuron co-stained with neurofilament antibody ( green ). A background control is not presented, because the AGA antibody does not detect any endogenous AGA in AGU mouse neurons. Magnification 600×. C , Mannose-6-phosphate-mediated endocytosis of AGA by primary neurons ( n2 ) and fibroblasts ( F ). The presence or absence of mannose-6-phosphate is indicated (+/−).
    Figure Legend Snippet: Endocytosis of AGA by different neuronal cells. A , Immunoprecipitation of endocytosed labeled AGA from E14 primary neurons cultured for 2 d ( n2 ) or 16 d ( n16 ), fibroblasts ( F ), PC12 cells (+/− NGF induction), and N18 cells. The recombinant CHO-AGA given to the cells is shown on the right ( M ). B , Immunofluorescence staining of endocytosed AGA ( red ) in a cultured primary neuron co-stained with neurofilament antibody ( green ). A background control is not presented, because the AGA antibody does not detect any endogenous AGA in AGU mouse neurons. Magnification 600×. C , Mannose-6-phosphate-mediated endocytosis of AGA by primary neurons ( n2 ) and fibroblasts ( F ). The presence or absence of mannose-6-phosphate is indicated (+/−).

    Techniques Used: Immunoprecipitation, Labeling, Cell Culture, Recombinant, Immunofluorescence, Staining

    Expression of CI-M6PR in neuronal cells. CI-M6PR was analyzed from mouse fibroblasts ( A ) and cultured primary neurons 2 ( B ), 8 ( C ), and 13 ( D ) d after plating. CI-M6PR was immunostained with goat anti-human M6PR 300 followed by HRP-conjugated anti-goat IgG. M6PR immunostaining was also performed in PC12 cells ( E ) and N18 cells ( F ).
    Figure Legend Snippet: Expression of CI-M6PR in neuronal cells. CI-M6PR was analyzed from mouse fibroblasts ( A ) and cultured primary neurons 2 ( B ), 8 ( C ), and 13 ( D ) d after plating. CI-M6PR was immunostained with goat anti-human M6PR 300 followed by HRP-conjugated anti-goat IgG. M6PR immunostaining was also performed in PC12 cells ( E ) and N18 cells ( F ).

    Techniques Used: Expressing, Cell Culture, Immunostaining

    15) Product Images from "Effects of Genistein and Exercise Training on Brain Damage Induced by a High-Fat High-Sucrose Diet in Female C57BL/6 Mice"

    Article Title: Effects of Genistein and Exercise Training on Brain Damage Induced by a High-Fat High-Sucrose Diet in Female C57BL/6 Mice

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2022/1560435

    (a) Effect of high glucose, palmitate and combined high glucose and palmitate on PC12 cell viability. (b) Effect of genistein on PC12 cell viability. HG: high glucose; PA: palmitate; HG + PA: high glucose + palmitate. Data are presented as mean ± SEM. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001.
    Figure Legend Snippet: (a) Effect of high glucose, palmitate and combined high glucose and palmitate on PC12 cell viability. (b) Effect of genistein on PC12 cell viability. HG: high glucose; PA: palmitate; HG + PA: high glucose + palmitate. Data are presented as mean ± SEM. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001.

    Techniques Used:

    Effect of genistein alone or with tamoxifen on high glucose and palmitate-induced apoptosis in PC12 cells. (a) Representative blot images with the corresponding densitometry measurement of caspase-3. (b) Representative blot images with the corresponding densitometry measurement of Bcl-2. HG + PA: high glucose + palmitate; HG + PA + G: high glucose + palmitate + 5 mM genistein; HG + PA + G + T: high glucose + palmitate + 5 mM genistein + tamoxifen. Data are presented as mean ± SEM for 3 mice per group. ∗∗ p ≤ 0.01 and ∗∗∗ p ≤ 0.001.
    Figure Legend Snippet: Effect of genistein alone or with tamoxifen on high glucose and palmitate-induced apoptosis in PC12 cells. (a) Representative blot images with the corresponding densitometry measurement of caspase-3. (b) Representative blot images with the corresponding densitometry measurement of Bcl-2. HG + PA: high glucose + palmitate; HG + PA + G: high glucose + palmitate + 5 mM genistein; HG + PA + G + T: high glucose + palmitate + 5 mM genistein + tamoxifen. Data are presented as mean ± SEM for 3 mice per group. ∗∗ p ≤ 0.01 and ∗∗∗ p ≤ 0.001.

    Techniques Used: Mouse Assay

    Effect of genistein alone or with tamoxifen on cell viability in high glucose and palmitate-treated PC12 cells. HG + P: high glucose + palmitate; HG + P + G1: high glucose + palmitate +2 μ M genistein; HG + P + G2: high glucose + palmitate + 5 μ M genistein; HG + P + G1 + T: high glucose + palmitate + 2 μ M genistein + tamoxifen; HG + P + G2 + T: high glucose + palmitate + 5 μ M genistein + tamoxifen. Data are presented as mean ± SEM. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001.
    Figure Legend Snippet: Effect of genistein alone or with tamoxifen on cell viability in high glucose and palmitate-treated PC12 cells. HG + P: high glucose + palmitate; HG + P + G1: high glucose + palmitate +2 μ M genistein; HG + P + G2: high glucose + palmitate + 5 μ M genistein; HG + P + G1 + T: high glucose + palmitate + 2 μ M genistein + tamoxifen; HG + P + G2 + T: high glucose + palmitate + 5 μ M genistein + tamoxifen. Data are presented as mean ± SEM. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, and ∗∗∗ p ≤ 0.001.

    Techniques Used:

    16) Product Images from "Sevoflurane Induces Learning and Memory Impairment in Young Mice Through a Reduction in Neuronal Glucose Transporter 3"

    Article Title: Sevoflurane Induces Learning and Memory Impairment in Young Mice Through a Reduction in Neuronal Glucose Transporter 3

    Journal: Cellular and Molecular Neurobiology

    doi: 10.1007/s10571-019-00779-0

    Sevoflurane inhibits GLUT3 surface expression and GLUT3 mRNA expression in neurons. a PC12 cells were transfected with LV5-GLUT3. The transfection efficiency was examined by GFP expression (green) 36 h post infection, which was analysed by phase contrast (left panel) and fluorescence microscopy (right panel). b The expression levels of GLUT3 protein in PC12 cells infected with LV5-GLUT3 were determined by WB analysis. PC12 cells were normally cultured (the Con group), transfected with LV5 empty vectors (the LV5 group), or transfected with LV5-GLUT3 (the LV5-GLUT3 group). c Flow cytometry analysis of GLUT3 surface expression in PC12 cells 24 h after the last exposure to sevoflurane. Four groups: the Con group, the Sev group (PC12 cells only exposed to sevoflurane), the LV5-GLUT3 group and the LV5-GLUT3 + Sev group (PC12 cells transfected with LV5-GLUT3 and then exposed to sevoflurane). n = 5 for each group. * P
    Figure Legend Snippet: Sevoflurane inhibits GLUT3 surface expression and GLUT3 mRNA expression in neurons. a PC12 cells were transfected with LV5-GLUT3. The transfection efficiency was examined by GFP expression (green) 36 h post infection, which was analysed by phase contrast (left panel) and fluorescence microscopy (right panel). b The expression levels of GLUT3 protein in PC12 cells infected with LV5-GLUT3 were determined by WB analysis. PC12 cells were normally cultured (the Con group), transfected with LV5 empty vectors (the LV5 group), or transfected with LV5-GLUT3 (the LV5-GLUT3 group). c Flow cytometry analysis of GLUT3 surface expression in PC12 cells 24 h after the last exposure to sevoflurane. Four groups: the Con group, the Sev group (PC12 cells only exposed to sevoflurane), the LV5-GLUT3 group and the LV5-GLUT3 + Sev group (PC12 cells transfected with LV5-GLUT3 and then exposed to sevoflurane). n = 5 for each group. * P

    Techniques Used: Expressing, Transfection, Infection, Fluorescence, Microscopy, Western Blot, Cell Culture, Flow Cytometry

    17) Product Images from "Expression and Role of the BDNF Receptor-TrkB in Rat Adrenal Gland under Acute Immobilization Stress"

    Article Title: Expression and Role of the BDNF Receptor-TrkB in Rat Adrenal Gland under Acute Immobilization Stress

    Journal: Acta Histochemica et Cytochemica

    doi: 10.1267/ahc.10027

    Immunofluorescence analysis of the effect of BDNF on intracellular dopamine in PC12 cells. PC12 cells were either not treated ( A ) or were treated ( B ) with 100 ng of BDNF for 5 min. Intracellular dopamine was then assayed using immunofluorescence. The lack of intracellular dopamine expression in ( B ) indicated degranulation of the PC12 cells. Bar=30 µm.
    Figure Legend Snippet: Immunofluorescence analysis of the effect of BDNF on intracellular dopamine in PC12 cells. PC12 cells were either not treated ( A ) or were treated ( B ) with 100 ng of BDNF for 5 min. Intracellular dopamine was then assayed using immunofluorescence. The lack of intracellular dopamine expression in ( B ) indicated degranulation of the PC12 cells. Bar=30 µm.

    Techniques Used: Immunofluorescence, Expressing

    Effect of BDNF-TrkB signaling on catecholamine secretion from PC12 cells. PC12 cells were treated with recombinant BDNF (rBDNF) and/or the Trk inhibitor, K252a. The levels of dopamine ( A ) and adrenaline ( B ) in the media were then assayed using a fluorescent assay following their purification from the media. Dopamine levels were 2.96×10 2 ±0.39×10 2 pg/ml in the K252a, 3.44×10 3 ±1.18×10 3 pg/ml in the rBDNF and 2.83×10 2 ±1.22×10 2 pg/ml in the K252a+ rBDNF groups and were 3.80×10 2 ±1.02×10 2 pg/ml in the control non-treated group (error bars, SD). Noradrenaline levels were 4.00±1.58 pg/ml in the K252a, 76.4±18.4 pg/ml in the rBDNF and 4.60±2.30 pg/ml in the K252a+rBDNF groups, and were 5.00±2.76 pg/ml in the control non-treated group (error bars, SD). Significant differences were observed between the dopamine and the noradrenaline levels of the rBDNF group and those of each of the other groups (p
    Figure Legend Snippet: Effect of BDNF-TrkB signaling on catecholamine secretion from PC12 cells. PC12 cells were treated with recombinant BDNF (rBDNF) and/or the Trk inhibitor, K252a. The levels of dopamine ( A ) and adrenaline ( B ) in the media were then assayed using a fluorescent assay following their purification from the media. Dopamine levels were 2.96×10 2 ±0.39×10 2 pg/ml in the K252a, 3.44×10 3 ±1.18×10 3 pg/ml in the rBDNF and 2.83×10 2 ±1.22×10 2 pg/ml in the K252a+ rBDNF groups and were 3.80×10 2 ±1.02×10 2 pg/ml in the control non-treated group (error bars, SD). Noradrenaline levels were 4.00±1.58 pg/ml in the K252a, 76.4±18.4 pg/ml in the rBDNF and 4.60±2.30 pg/ml in the K252a+rBDNF groups, and were 5.00±2.76 pg/ml in the control non-treated group (error bars, SD). Significant differences were observed between the dopamine and the noradrenaline levels of the rBDNF group and those of each of the other groups (p

    Techniques Used: Recombinant, Fluorescence, Purification

    RT-PCR analysis of the mRNA expression of TrkB isoforms in PC12 cells. RT-PCR analysis detected mRNA expression of the truncated TrkB-T1 isoform in PC12 cells but did not detect TrkB-full length (TrkB-FL) mRNA. GAPDH mRNA was assayed as an internal control.
    Figure Legend Snippet: RT-PCR analysis of the mRNA expression of TrkB isoforms in PC12 cells. RT-PCR analysis detected mRNA expression of the truncated TrkB-T1 isoform in PC12 cells but did not detect TrkB-full length (TrkB-FL) mRNA. GAPDH mRNA was assayed as an internal control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    18) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1091/mbc.E06-05-0453

    KIM overexpression impairs Kidins220/ARMS trafficking in PC12 cells. (A) EGFP-Kidins220/ARMS is transported along neurites in PC12 cells. (a) Phase image of a microinjected cell. (b) Time series of the boxed area. Arrowheads indicate an EGFP-Kidins220/ARMS–positive
    Figure Legend Snippet: KIM overexpression impairs Kidins220/ARMS trafficking in PC12 cells. (A) EGFP-Kidins220/ARMS is transported along neurites in PC12 cells. (a) Phase image of a microinjected cell. (b) Time series of the boxed area. Arrowheads indicate an EGFP-Kidins220/ARMS–positive

    Techniques Used: Over Expression

    Kidins220/ARMS and kinesin-1 colocalize in PC12 cells. (A) PC12 cells treated for 3 d with NGF were stained with polyclonal (b) or monoclonal (n) anti-Kidins220/ARMS, polyclonal anti-SyD/JIP-3 (f), polyclonal anti-Syt (j), monoclonal anti-KHC (c, g, and
    Figure Legend Snippet: Kidins220/ARMS and kinesin-1 colocalize in PC12 cells. (A) PC12 cells treated for 3 d with NGF were stained with polyclonal (b) or monoclonal (n) anti-Kidins220/ARMS, polyclonal anti-SyD/JIP-3 (f), polyclonal anti-Syt (j), monoclonal anti-KHC (c, g, and

    Techniques Used: Staining

    Overexpression of KIM reduces the phosphorylation of MAPK in PC12 cells. (A) PC12 cells were transfected with mRFP-KIM (a–c) or mRFP-KIM(Y24A) (d–f) and then fixed and immunostained for P-MAPK (c and f). mRFP-KIM–expressing cells
    Figure Legend Snippet: Overexpression of KIM reduces the phosphorylation of MAPK in PC12 cells. (A) PC12 cells were transfected with mRFP-KIM (a–c) or mRFP-KIM(Y24A) (d–f) and then fixed and immunostained for P-MAPK (c and f). mRFP-KIM–expressing cells

    Techniques Used: Over Expression, Transfection, Expressing

    KIM overexpression inhibits neurite outgrowth in PC12 cells. (A) Undifferentiated PC12 cells were transfected with mRFP (a and b), mRFP-KIM (c and d), or mRFP-KIM(Y24A) (e and f); treated for 3 d with NGF; and analyzed by confocal microscopy. Bars, 20
    Figure Legend Snippet: KIM overexpression inhibits neurite outgrowth in PC12 cells. (A) Undifferentiated PC12 cells were transfected with mRFP (a and b), mRFP-KIM (c and d), or mRFP-KIM(Y24A) (e and f); treated for 3 d with NGF; and analyzed by confocal microscopy. Bars, 20

    Techniques Used: Over Expression, Transfection, Confocal Microscopy

    19) Product Images from "NGF-mediated photoablation of nociceptors reduces pain behavior in mice"

    Article Title: NGF-mediated photoablation of nociceptors reduces pain behavior in mice

    Journal: bioRxiv

    doi: 10.1101/575274

    Characterization of NGF R121W-SNAP . ( a ) NGF R121W-SNAP was coupled with BG-549 in vitro, run on an SDS-Page, stained with Coomassie blue, and in-gel fluorescence visualized under a UV-light. ( b-d ) Confocal images of Hek293T cells transiently transfected with TrkA, TrkB or TrkC (panel b, c and d respectively) and p75 stained with 100 nM NGF R121W-SNAP conjugated to surface BG-546. NGF R121W-SNAP selectively binds only to TrkA/p75 transfected cells, with virtually no binding to TrkB or TrkC transfected cells. Scale bar 20 μm. (e) Representative western blot of three independent experiments, showing the expression level of MAPK, phospho MAPK, AKT, phospho AKT and actin (loading control) in untreated PC12 cells (lane 1), treated with NGF (lane 2) or NGF R121W-SNAP (lane 3). (f) Levels of each protein were expressed as ratio between the phosphorylated form and the total counterpart and then normalized to the NGF-treated sample. ( g-j ) Neuron differentiation in untreated (g) , NGF-treated (h) and NGF R121W-SNAP -treated (i) PC12 cells, after 6 days of treatment. Arrows indicate differentiated cells. Scale bar 20 µm. (j) Quantitation of neuron-like differentiated PC12 cells, expressed as percentage (%); for the analysis, 241 untreated cells, 246 NGF-treated-cells and 285 NGF R121W-SNAP -treated cells were considered. Error bars indicate SEM. (k) Von Frey thresholds after injection of NGF (shaded box) or NGF R121W-SNAP (open box) into the hind paw (ipsi) of mice (n=4 animals). Box plots represent the force expressed in grams (g) required to trigger a 50% response. * p=0.05 (two-tailed t-Test). (l) Hotplate test after injection of NGF (n=6 animals, red box) and NGF R121W-SNAP (n=5 animals, grey box) into the hind paw of the mice. Box plots represent the latency expressed in seconds (s) of the paw withdrawal in response to heat (52°C). * p=0.009 (two-tailed t-Test).
    Figure Legend Snippet: Characterization of NGF R121W-SNAP . ( a ) NGF R121W-SNAP was coupled with BG-549 in vitro, run on an SDS-Page, stained with Coomassie blue, and in-gel fluorescence visualized under a UV-light. ( b-d ) Confocal images of Hek293T cells transiently transfected with TrkA, TrkB or TrkC (panel b, c and d respectively) and p75 stained with 100 nM NGF R121W-SNAP conjugated to surface BG-546. NGF R121W-SNAP selectively binds only to TrkA/p75 transfected cells, with virtually no binding to TrkB or TrkC transfected cells. Scale bar 20 μm. (e) Representative western blot of three independent experiments, showing the expression level of MAPK, phospho MAPK, AKT, phospho AKT and actin (loading control) in untreated PC12 cells (lane 1), treated with NGF (lane 2) or NGF R121W-SNAP (lane 3). (f) Levels of each protein were expressed as ratio between the phosphorylated form and the total counterpart and then normalized to the NGF-treated sample. ( g-j ) Neuron differentiation in untreated (g) , NGF-treated (h) and NGF R121W-SNAP -treated (i) PC12 cells, after 6 days of treatment. Arrows indicate differentiated cells. Scale bar 20 µm. (j) Quantitation of neuron-like differentiated PC12 cells, expressed as percentage (%); for the analysis, 241 untreated cells, 246 NGF-treated-cells and 285 NGF R121W-SNAP -treated cells were considered. Error bars indicate SEM. (k) Von Frey thresholds after injection of NGF (shaded box) or NGF R121W-SNAP (open box) into the hind paw (ipsi) of mice (n=4 animals). Box plots represent the force expressed in grams (g) required to trigger a 50% response. * p=0.05 (two-tailed t-Test). (l) Hotplate test after injection of NGF (n=6 animals, red box) and NGF R121W-SNAP (n=5 animals, grey box) into the hind paw of the mice. Box plots represent the latency expressed in seconds (s) of the paw withdrawal in response to heat (52°C). * p=0.009 (two-tailed t-Test).

    Techniques Used: In Vitro, SDS Page, Staining, Fluorescence, Transfection, Binding Assay, Western Blot, Expressing, Quantitation Assay, Injection, Mouse Assay, Two Tailed Test

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    Alomone Labs pc12 cells
    Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in <t>PC12</t> cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P
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    Effect of BDNF-TrkB signaling on catecholamine secretion from PC12 cells. PC12 cells were treated with recombinant BDNF (rBDNF) and/or the Trk inhibitor, <t>K252a.</t> The levels of dopamine ( A ) and adrenaline ( B ) in the media were then assayed using a fluorescent assay following their purification from the media. Dopamine levels were 2.96×10 2 ±0.39×10 2 pg/ml in the K252a, 3.44×10 3 ±1.18×10 3 pg/ml in the rBDNF and 2.83×10 2 ±1.22×10 2 pg/ml in the K252a+ rBDNF groups and were 3.80×10 2 ±1.02×10 2 pg/ml in the control non-treated group (error bars, SD). Noradrenaline levels were 4.00±1.58 pg/ml in the K252a, 76.4±18.4 pg/ml in the rBDNF and 4.60±2.30 pg/ml in the K252a+rBDNF groups, and were 5.00±2.76 pg/ml in the control non-treated group (error bars, SD). Significant differences were observed between the dopamine and the noradrenaline levels of the rBDNF group and those of each of the other groups (p
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    Effect of BDNF-TrkB signaling on catecholamine secretion from PC12 cells. PC12 cells were treated with recombinant BDNF (rBDNF) and/or the Trk inhibitor, <t>K252a.</t> The levels of dopamine ( A ) and adrenaline ( B ) in the media were then assayed using a fluorescent assay following their purification from the media. Dopamine levels were 2.96×10 2 ±0.39×10 2 pg/ml in the K252a, 3.44×10 3 ±1.18×10 3 pg/ml in the rBDNF and 2.83×10 2 ±1.22×10 2 pg/ml in the K252a+ rBDNF groups and were 3.80×10 2 ±1.02×10 2 pg/ml in the control non-treated group (error bars, SD). Noradrenaline levels were 4.00±1.58 pg/ml in the K252a, 76.4±18.4 pg/ml in the rBDNF and 4.60±2.30 pg/ml in the K252a+rBDNF groups, and were 5.00±2.76 pg/ml in the control non-treated group (error bars, SD). Significant differences were observed between the dopamine and the noradrenaline levels of the rBDNF group and those of each of the other groups (p
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    Effect of BDNF-TrkB signaling on catecholamine secretion from PC12 cells. PC12 cells were treated with recombinant BDNF (rBDNF) and/or the Trk inhibitor, <t>K252a.</t> The levels of dopamine ( A ) and adrenaline ( B ) in the media were then assayed using a fluorescent assay following their purification from the media. Dopamine levels were 2.96×10 2 ±0.39×10 2 pg/ml in the K252a, 3.44×10 3 ±1.18×10 3 pg/ml in the rBDNF and 2.83×10 2 ±1.22×10 2 pg/ml in the K252a+ rBDNF groups and were 3.80×10 2 ±1.02×10 2 pg/ml in the control non-treated group (error bars, SD). Noradrenaline levels were 4.00±1.58 pg/ml in the K252a, 76.4±18.4 pg/ml in the rBDNF and 4.60±2.30 pg/ml in the K252a+rBDNF groups, and were 5.00±2.76 pg/ml in the control non-treated group (error bars, SD). Significant differences were observed between the dopamine and the noradrenaline levels of the rBDNF group and those of each of the other groups (p
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    Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Journal: Journal of Radiation Research

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    doi: 10.1093/jrr/rrx032

    Figure Lengend Snippet: Irradiation with 137 Cs γ-rays depresses NGF-induced neurite extension in PC12 cells. (a) Phase-contrast micrographs, (b) lengths of neurites and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Article Snippet: Reagents We utilized the following reagents to investigate the effects of chronic, low-dose-rate 137 Cs-γ radiation on NGF-induced neurite extension in PC12 cells: NGF 2.5S (N-100, Alomone Labs, Jerusalem, Israel); anti-ERK1/ERK2 mouse-monoclonal antibody (MAB1576) and anti-phosphorylated ERK1/ERK2 rabbit-polyclonal antibody (MAB1018, both R & D systems, Minneapolis, MN, USA); anti-Trk A rabbit polyclonal antibody (sc-118) and anti-phosphorylated Trk A mouse-monoclonal antibody (sc-8058, both Santa Cruz Biotechnology, Dallas, TX, USA)—Trk A is a high-affinity nerve growth factor receptor; anti-tyrosine hydroxylase rabbit-monoclonal antibody (ab137869); anti-Akt rabbit polyclonal antibody (ab8805) and anti-phosphorylated Akt rabbit monoclonal antibody (ab81283); anti-Ca2+/calmodulin-dependent kinase II (CaMKII) rabbit polyclonal antibody (ab131468) and anti-phosphorylated CaMKII rabbit polyclonal antibody (ab5683, all Abcam plc, Cambridge, UK); KN-62 (I2142, Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Irradiation

    Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.

    Journal: Journal of Radiation Research

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    doi: 10.1093/jrr/rrx032

    Figure Lengend Snippet: Western blot analysis of NGF stimulation–related proteins in PC12 cells. Immunoblot showing varying levels of (a) phosphorylated NGF receptor (P-NGFR, upper) and NGF receptor (NGFR, lower), (b) phosphorylated ERK (P-ERK, upper) and ERK (lower), (c) tyrosine hydroxylase (TH) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Similar results were obtained in three separate experiments. NGF = nerve growth factor, ERK = extracellular signal–regulated kinase.

    Article Snippet: Reagents We utilized the following reagents to investigate the effects of chronic, low-dose-rate 137 Cs-γ radiation on NGF-induced neurite extension in PC12 cells: NGF 2.5S (N-100, Alomone Labs, Jerusalem, Israel); anti-ERK1/ERK2 mouse-monoclonal antibody (MAB1576) and anti-phosphorylated ERK1/ERK2 rabbit-polyclonal antibody (MAB1018, both R & D systems, Minneapolis, MN, USA); anti-Trk A rabbit polyclonal antibody (sc-118) and anti-phosphorylated Trk A mouse-monoclonal antibody (sc-8058, both Santa Cruz Biotechnology, Dallas, TX, USA)—Trk A is a high-affinity nerve growth factor receptor; anti-tyrosine hydroxylase rabbit-monoclonal antibody (ab137869); anti-Akt rabbit polyclonal antibody (ab8805) and anti-phosphorylated Akt rabbit monoclonal antibody (ab81283); anti-Ca2+/calmodulin-dependent kinase II (CaMKII) rabbit polyclonal antibody (ab131468) and anti-phosphorylated CaMKII rabbit polyclonal antibody (ab5683, all Abcam plc, Cambridge, UK); KN-62 (I2142, Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Western Blot, Irradiation

    Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Journal: Journal of Radiation Research

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    doi: 10.1093/jrr/rrx032

    Figure Lengend Snippet: Inhibition of CaMKII activity results in 137 Csγ-ray irradiation-induced depression of NGF-induced neurite extension in PC12 cells. (a) Immunoblot showing varying levels of phosphorylated CaMKII (P-CaMKII, upper) and CaMKII (lower) observed in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Similar results were obtained in three separate experiments. (b) Lengths and (c) numbers of neurites in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation in the presence or absence of KN-62. Lengths or numbers of neurites are expressed as the relative ratio to the non-irradiated group. Data are presented as the mean ± standard error of triplicate samples. * P

    Article Snippet: Reagents We utilized the following reagents to investigate the effects of chronic, low-dose-rate 137 Cs-γ radiation on NGF-induced neurite extension in PC12 cells: NGF 2.5S (N-100, Alomone Labs, Jerusalem, Israel); anti-ERK1/ERK2 mouse-monoclonal antibody (MAB1576) and anti-phosphorylated ERK1/ERK2 rabbit-polyclonal antibody (MAB1018, both R & D systems, Minneapolis, MN, USA); anti-Trk A rabbit polyclonal antibody (sc-118) and anti-phosphorylated Trk A mouse-monoclonal antibody (sc-8058, both Santa Cruz Biotechnology, Dallas, TX, USA)—Trk A is a high-affinity nerve growth factor receptor; anti-tyrosine hydroxylase rabbit-monoclonal antibody (ab137869); anti-Akt rabbit polyclonal antibody (ab8805) and anti-phosphorylated Akt rabbit monoclonal antibody (ab81283); anti-Ca2+/calmodulin-dependent kinase II (CaMKII) rabbit polyclonal antibody (ab131468) and anti-phosphorylated CaMKII rabbit polyclonal antibody (ab5683, all Abcam plc, Cambridge, UK); KN-62 (I2142, Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Inhibition, Activity Assay, Irradiation

    Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P

    Journal: Journal of Radiation Research

    Article Title: Chronic irradiation with low-dose-rate 137Cs-γ rays inhibits NGF-induced neurite extension of PC12 cells via Ca2+/calmodulin-dependent kinase II activation

    doi: 10.1093/jrr/rrx032

    Figure Lengend Snippet: Irradiation with 137 Cs γ-rays attenuates NGF-induced Rac1 activation without increasing phosphorylation of Akt in PC12 cells. (a) The activity of Rac1 in PC12 cells after 5 days of NGF stimulation or non-stimulation, with or without 137 Csγ-ray irradiation. Rac1 activity is expressed as the relative ratio to the NGF non-stimulated group without irradiation. Data are presented as the mean ± standard error of triplicate samples. ** P

    Article Snippet: Reagents We utilized the following reagents to investigate the effects of chronic, low-dose-rate 137 Cs-γ radiation on NGF-induced neurite extension in PC12 cells: NGF 2.5S (N-100, Alomone Labs, Jerusalem, Israel); anti-ERK1/ERK2 mouse-monoclonal antibody (MAB1576) and anti-phosphorylated ERK1/ERK2 rabbit-polyclonal antibody (MAB1018, both R & D systems, Minneapolis, MN, USA); anti-Trk A rabbit polyclonal antibody (sc-118) and anti-phosphorylated Trk A mouse-monoclonal antibody (sc-8058, both Santa Cruz Biotechnology, Dallas, TX, USA)—Trk A is a high-affinity nerve growth factor receptor; anti-tyrosine hydroxylase rabbit-monoclonal antibody (ab137869); anti-Akt rabbit polyclonal antibody (ab8805) and anti-phosphorylated Akt rabbit monoclonal antibody (ab81283); anti-Ca2+/calmodulin-dependent kinase II (CaMKII) rabbit polyclonal antibody (ab131468) and anti-phosphorylated CaMKII rabbit polyclonal antibody (ab5683, all Abcam plc, Cambridge, UK); KN-62 (I2142, Sigma-Aldrich, St Louis, MO, USA).

    Techniques: Irradiation, Activation Assay, Activity Assay

    Effect of BDNF-TrkB signaling on catecholamine secretion from PC12 cells. PC12 cells were treated with recombinant BDNF (rBDNF) and/or the Trk inhibitor, K252a. The levels of dopamine ( A ) and adrenaline ( B ) in the media were then assayed using a fluorescent assay following their purification from the media. Dopamine levels were 2.96×10 2 ±0.39×10 2 pg/ml in the K252a, 3.44×10 3 ±1.18×10 3 pg/ml in the rBDNF and 2.83×10 2 ±1.22×10 2 pg/ml in the K252a+ rBDNF groups and were 3.80×10 2 ±1.02×10 2 pg/ml in the control non-treated group (error bars, SD). Noradrenaline levels were 4.00±1.58 pg/ml in the K252a, 76.4±18.4 pg/ml in the rBDNF and 4.60±2.30 pg/ml in the K252a+rBDNF groups, and were 5.00±2.76 pg/ml in the control non-treated group (error bars, SD). Significant differences were observed between the dopamine and the noradrenaline levels of the rBDNF group and those of each of the other groups (p

    Journal: Acta Histochemica et Cytochemica

    Article Title: Expression and Role of the BDNF Receptor-TrkB in Rat Adrenal Gland under Acute Immobilization Stress

    doi: 10.1267/ahc.10027

    Figure Lengend Snippet: Effect of BDNF-TrkB signaling on catecholamine secretion from PC12 cells. PC12 cells were treated with recombinant BDNF (rBDNF) and/or the Trk inhibitor, K252a. The levels of dopamine ( A ) and adrenaline ( B ) in the media were then assayed using a fluorescent assay following their purification from the media. Dopamine levels were 2.96×10 2 ±0.39×10 2 pg/ml in the K252a, 3.44×10 3 ±1.18×10 3 pg/ml in the rBDNF and 2.83×10 2 ±1.22×10 2 pg/ml in the K252a+ rBDNF groups and were 3.80×10 2 ±1.02×10 2 pg/ml in the control non-treated group (error bars, SD). Noradrenaline levels were 4.00±1.58 pg/ml in the K252a, 76.4±18.4 pg/ml in the rBDNF and 4.60±2.30 pg/ml in the K252a+rBDNF groups, and were 5.00±2.76 pg/ml in the control non-treated group (error bars, SD). Significant differences were observed between the dopamine and the noradrenaline levels of the rBDNF group and those of each of the other groups (p

    Article Snippet: In other experiments, PC 12 cells were stimulated with culture medium containing 10 µg/ml K252a (K252a group) or 100 ng/ml rBDNF (rBDNF group).

    Techniques: Recombinant, Fluorescence, Purification